Clinical Immunology

and Serology
A Laboratory Perspective
Clinical Immunology
and Serology
A Laboratory Perspective

EdD, MT(ASCP)
Professor Emeritus of Clinical Laboratory Science
Western Carolina University
Cullowhee, North Carolina

PhD, I, MBCM(ASCP)SI
Professor of Clinical Laboratory Science
SUNY Upstate Medical University
Syracuse, New York
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Library of Congress Cataloging-in-Publication Data

Names: Stevens, Christine Dorresteyn, author. | Miller, Linda E., author.

Title: Clinical immunology & serology : a laboratory perspective / Christine
Dorresteyn Stevens and Linda E. Miller.
Other titles: Clinical immunology and serology
Description: Fourth edition. | Philadelphia : F.A. Davis Company, [2017] |
Includes bibliographical references and index.
Identifiers: LCCN 2016023350 | ISBN 9780803644663
Subjects: | MESH: Immunity—physiology | Immune System Diseases—diagnosis |
Immunologic Techniques | Immunologic Tests | Serologic Tests
Classification: LCC RB46.5 | NLM QW 540 | DDC 616.07/56—dc23 LC record available at https://lccn.loc.gov/2016023350

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 To my wonderful family: Eric, Kathy, Hannah, and Matthew, and Kevin,
Melissa, Turner, and Avery for their love and encouragement.
— C.D.S.

To my wonderful family, for their love and support; to the Clinical

Laboratory Science faculty and the Clinical Immunology laboratory staff at
SUNY Upstate Medical University, in appreciation of their expertise and
collegiality; and especially to my students, who have inspired me to share
my passion for immunology over the years.
— L.E.M.
 Preface
The fourth edition of Clinical Immunology and Serology: A these areas. The chapter on autoimmunity (Chapter 15) has
Laboratory Perspective is built on the success of the first three been expanded to include some diseases that are increasing in
editions. This text is tailored to meet the needs of clinical importance. Additionally, Molecular Diagnostic Techniques
laboratory students on both the 2- and 4-year levels. It com- (Chapter 12) and Tumor Immunology (Chapter 17) have been
bines practical information about laboratory testing with a con- expanded to help bring readers up-to-date on new developments
cise discussion of the theory behind the testing. For practicing in the field. Information on quality assessment, regulatory issues,
laboratorians and other allied health professionals, the book and quality management systems has been added to the chapter
may serve as a valuable reference about new developments in on laboratory safety. Perhaps the most exciting new change,
the field of immunology. however, is the addition of full color illustrations. Not only does
The organization of the chapters is based on the experi- this increase the visual appeal of the book, but full color is help-
ence of many years of teaching immunology to clinical labo- ful to students in promoting a better understanding of principles
ratory science students. The book is divided into four major and techniques discussed in the chapters.
sections: I. Nature of the Immune System; II. Basic Immuno- The book remains a practical introduction to the field of
logic Procedures; III. Immune Disorders; and IV. Serological clinical immunology that combines essential theoretical prin-
and Molecular Diagnosis of Infectious Disease. Sections build ciples with serological techniques commonly used in the clin-
upon one another, and chapters relate previous material to ical laboratory. The theory is comprehensive but concise, and
new material by means of boxes titled Connections and the emphasis is on direct application to the clinical laboratory.
Clinical Correlations. These new features help the students The text is readable and user-friendly, with learning outcomes,
to recall information from previous chapters and to bridge chapter outlines, and a glossary of all key terms. Each chapter
theory with actual clinical diagnosis and testing. Information is a complete learning module that contains theoretical princi-
in the chapters is related to real world events in order to make ples, illustrations, definitions of relevant terminology, and
it more interesting for the student and to show the important questions and case studies that help to evaluate learning.
role that immunology plays in people’s daily lives. New to this For the instructor, there are many new online resources at
edition are the Study Guide Tables at the end of many of the DavisPlus to assist in course development. These resources in-
chapters, which can be used as study tools by the students. clude PowerPoint slides, suggested laboratory exercises, addi-
All chapters have been updated to include new information tional case studies, and a large bank of test questions that can
about the immune system as well as new treatments for im- be used for review or test preparation. Because the field of im-
munologic diseases. With this edition comes added emphasis munology is expanding so rapidly, the challenge in writing this
on the basic immune mechanisms. Three new chapters have book has been to ensure adequate coverage but to keep it on
been added: Innate Immunity (Chapter 3), Adaptive Immunity an introductory level. Every chapter has been revised to in-
(Chapter 4), and Immunization and Vaccines (Chapter 25). clude current practices as of the time of writing. It is hoped
These chapters have been added in response to comments from that this book will kindle an interest in both students and lab-
reviewers and readers, as well as the burgeoning information in oratory professionals in this exciting and dynamic field.
 Art Consultant
Joseph G. Cannon, PhD
Professor and Kellett Chair of Allied Health Sciences
Clinical Laboratory Sciences Program
Augusta University
Augusta, Georgia
 Contributors
Thomas S. Alexander, PhD, D(ABMLI) Marc Golightly, PhD, SI(ASCP)
Immunologist, Summa Health System, Akron, Ohio Head of Clinical Immunology Laboratories
Professor of Clinical Immunology Stony Brook Medicine
Northeast Ohio Medical University Stony Brook, New York
Rootstown, Ohio
Jeannie Guglielmo, MS, MAT, MT(ASCP)
Lela Buckingham, PhD, MB(ASCP), DLM(ASCP) Clinical Assistant Professor
Director, Medical Oncology Department of Clinical Laboratory Sciences
Rush Medical Laboratories Stony Brook University
Rush University Medical Center Stony Brook, New York
Chicago, Illinois
Rita M. Heuertz, PhD, MT(ASCP)
Marjorie Schaub Di Lorenzo, MT(ASCP)SH Director of Research
Program Coordinator Department of Biomedical Laboratory Science
Phlebotomy Technician Program St. Louis University
Nebraska Methodist College St. Louis, Missouri
Omaha, Nebraska
Donald C. Lehman, EdD
Uthayashanker R. Ezekiel, PhD, MB(ASCP)CM Associate Professor
Assistant Professor Department of Medical Laboratory Sciences
Department of Biomedical Laboratory Science University of Delaware
St. Louis University Newark, Delaware
St. Louis, Missouri
John L. Schmitz, PhD, D(ABMLI, ABHI)
Ashley Frazer-Able, PhD Professor
Director, Complement Laboratory Department of Pathology and Laboratory Medicine
National Jewish Health University of North Carolina at Chapel Hill
Denver, Colorado Chapel Hill, North Carolina

Candace Golightly, MS, MLT(ASCP) James L. Vossler, MS, MLSCM(ASCP)SMCM

Director of Clinical Education Assistant Professor
Department of Clinical Laboratory Sciences Department of Clinical Laboratory Science
Stony Brook University SUNY Upstate Medical University
Stony Brook, New York Syracuse, New York
 Reviewers
Laura Ahonen, MS, MT(ASCP) Karen Golemboski, Ph.D., MLS(ASCP)CM
MLT Program Director Associate Professor
Medical Laboratory Technician Medical Laboratory Science
Northcentral Technical College Bellarmine University
Wausau, Wisconsin Louisville, Kentucky

Leah Ames, MS, MLS(ASCP)CM Karen R. Gordon, MS, MLS(ASCP)CMSLS

Adjunct Faculty Associate Professor
Northern Illinois University Medical Laboratory Technology Program
Clinical Laboratory Sciences Northern Virginia Community College
DeKalb, Illinois Springfield, Virginia

Denise E. Anamani, MA, I(ASCP)MBCM Risa Grimme, MTASCP, MA

Lecturer Director
Allied Health Sciences Clinical Laboratory Technician Program
University of Connecticut Calhoun Community College
Storrs, Connecticut Decatur, Alabama

Joseph G. Cannon, PhD Liz Johnson, MS, MLS(ASCP)CM, CLS(NCA), MT(AMT)

Professor PT Instructor
Clinical Laboratory Sciences Program HWPS–MLT Program
Georgia Regents University Central New Mexico Community College
Augusta, Georgia Albuquerque, New Mexico

Demetra Castillo, MAdEd, MLS(ASCP)CM Amy R. Kapanka, MS, MT(ASCP)SC

Assistant Professor MLT Program Director
Medical Laboratory Science Health Sciences
Rush University Hawkeye Community College
Chicago, Illinois Cedar Falls, Iowa

Hencelyn G. Chu, PhD, MLS(ASCP) Nadine M. Lerret, PhD, MLS(ASCP)CM

Program Director Assistant Professor
Medical Laboratory Technician Medical Laboratory Sciences
Saddleback College Rush University Medical Center
Mission Viejo, California Chicago, Illinois

Shirley F. Cruzada, EdD, MS, MT(AMT) Louise Millis, MS, MLS(ASCP)CM

Professor Associate Professor Biology & MLS Program Director
Clinical Laboratory Sciences Biology & School of Health & Human Services
College of Southern Nevada St. Cloud State University
Las Vegas, Nevada St. Cloud, Minnesota

Daniel P. deRegnier, MS, MT(ASCP) Hamida Nusrat, PhD, PHM

Associate Professor/Program Director Lecturer
Clinical Laboratory Sciences Clinical Lab Sciences Internship Program
Ferris State University San Francisco State University
Big Rapids, Michigan San Francisco, California
Amy L. Raugh, MS, MT(ASCP) Rujin Tian, PhD
Program Director, Phlebotomy Technician Program Associate Professor
Faculty Medical, Laboratory Technician Program Biological Science
Health and Public Service Bronx Community College, City University of New York
Harrisburg Area Community College Bronx, New York
Harrisburg, Pennsylvania
Ifor Williams, MD, PhD
Karen A. Reiner, MS, MT(ASCP) Professor of Pathology
Associate Professor Department of Pathology
Medical Laboratory Sciences Emory University School of Medicine
Andrews University Atlanta, Georgia
Berrien Springs, Michigan

Terri Talbot, MHA, MT(ASCP)

Assistant Professor
Clinical Laboratory Science
Our Lady of the Lake College
Baton Rouge, Louisiana
 Acknowledgments
We are grateful for the assistance we received from a number of Christa Fratantoro, you were a gentle taskmaster who kept us
sources during the preparation of this fourth edition. I am de- going, and you were a wonderful listener and problem solver
lighted to have had the help of Linda Miller, my co-editor for this when we needed help. We also appreciate the efforts of Dean
edition. Her input and sharing of ideas has been invaluable. Our DeChambeau, our developmental editor, whose thoroughness
chapter contributors, whose expertise enriched this book, are and promptness were invaluable in keeping us on track.
Thomas Alexander, Lela Buckingham, Marjorie Di Lorenzo, Thanks to George Lang and all the others behind the scenes
Uthayashanker Ezekiel, Ashley Frazer-Able, Candace Golightly, who helped this book come to life.
Marc Golightly, Jeannie Guglielmo, Rita Heuertz, Donald Our immunology students—past, present, and future—are
Lehman, John Schmitz, and James Vossler. A special thank you the reason for writing this book. We hope that this text will
to Joe Cannon, who changed our ideas into workable full color help make a very complex subject a little easier to understand.
illustrations for this edition. We would also like to thank the re- A thank you to friends, especially Bayard, for their encour-
viewers whose additional sets of eyes and thoughtful suggestions agement and interest.
helped to strengthen the chapters. Finally, to my two wonderful sons Eric and Kevin, their
Finally, we would like to acknowledge all the folks at wives Kathy and Melissa, and my grandchildren, Hannah,
F.A. Davis for their hard work in making this fourth edition a Turner, Matthew, and Avery, you are my inspiration and my
reality. To the Senior Acquisitions Editor for Health Professions, source of strength.
 Contents
SECTION I Hinge Region 64
Three-Dimensional Structure of Antibodies 65
Nature of the Immune System 1 Theories to Explain Antibody Diversity 69
Introduction to Immunity Genes Coding for Immunoglobulins 70
and the Immune System 2 Monoclonal Antibody 72

Cytokines 77
Immunity and Immunization 3
Innate Versus Adaptive Immunity 4
Cells of the Innate Immune System 4 Introduction to Cytokines 78
Cells of the Adaptive Immune System 7 Cytokines in the Innate Immune Response 80
Organs of the Immune System 8 Cytokines in the Adaptive Immune Response 83
Th17 Cytokines in Innate and Adaptive Immune
Nature of Antigens and the Major Responses 85
Histocompatibility Complex 16 Hematopoietic Growth Factors 85
Cytokine and Anticytokine Therapies 86
Factors Influencing the Immune Response 17 Clinical Assays for Cytokines 87
Traits of Immunogens 17
Complement System 91
Epitopes 18
Haptens 19
Pathways of the Complement System 92
Adjuvants 20
System Controls 97
Relationship of Antigens to the Host 21
Complement Receptors and Their Biological Roles 100
Major Histocompatibility Complex 21
Biological Manifestations of Complement
Innate Immunity 31 Activation 102
Complement and Disease States 103
External Defense System 32 Complement Deficiencies 103
Internal Defense System 33 Laboratory Detection of Complement
Abnormalities 104
Adaptive Immunity 45
SECTION II
T-Cell Differentiation 46
Basic Immunologic Procedures 111
Stages in B-Cell Differentiation 50
The Role of T Cells in the Adaptive Immune
Safety and Quality Management 112
Response 53
The Role of B Cells in the Adaptive Immune Laboratory Hazards 113
Response 54 Quality Management 121
Laboratory Identification of Lymphocytes 58 Regulatory Issues 126
Antibody Structure and Function 61 Quality Management Systems 127

Principles of Serological Testing 132

Tetrapeptide Structure of Immunoglobulins 62
The Nature of Light Chains 64 Blood Specimen Preparation and Measuring 133
Heavy-Chain Sequencing 64 Dilutions 134
 Precipitation and Agglutination Type III Hypersensitivity 225
Reactions 140 Type IV Hypersensitivity 226

Autoimmunity 233
Antigen–Antibody Binding 141
Precipitation Curve 142
Etiology of Autoimmune Disease 234
Measurement of Precipitation by Light
Systemic Autoimmune Diseases 237
Scattering 143
Organ-Specific Autoimmune Diseases 250
Passive Immunodiffusion Techniques 143
Electrophoretic Techniques 144 Transplantation Immunology 263
Comparison of Precipitation Techniques 145
Principles of Agglutination Reactions 145 Histocompatibility Systems 264
Types of Agglutination Reactions 147 Allorecognition 266
Instrumentation 149 Transplant Rejection 267
Quality Control and Quality Assurance 150 Graft-Versus-Host Disease (GVHD) 268
Labeled Immunoassays 155 Immunosuppressive Agents 268
Clinical Histocompatibility Testing 269

Formats for Labeled Assays 156 Tumor Immunology 278

Heterogeneous Versus Homogeneous Assays 157
Radioimmunoassay 157 Introduction to Tumor Biology 279
Enzyme Immunoassays 158 Tumor Antigens 280
Fluorescent Immunoassays 161 Clinically Relevant Tumor Markers 283
Chemiluminescent Immunoassays 163 Laboratory Detection of Tumors 290
Molecular Diagnostic Techniques 168 Interactions Between the Immune System
and Tumors 294
Immunoediting and Tumor Escape 296
Characteristics of Deoxyribonucleic Acid
Immunotherapy 297
and Ribonucleic Acid 169
Molecular Analysis 175 Immunoproliferative Diseases 306
DNA Sequencing 185

Flow Cytometry and Laboratory Malignant Transformation of Hematologic Cells 307

Automation 193 Classification of Hematologic Malignancies 308
Leukemias 308
Cell Flow Cytometry 194 Lymphomas 310
Immunoassay Automation 203 Plasma Cell Dyscrasias 312
Role of the Laboratory in Evaluating
SECTION III Immunoproliferative Diseases 315

Immune Disorders 211 Immunodeficiency Diseases 326

Hypersensitivity 212
Clinical Effects of Primary Immunodeficiencies 327
The Nine Categories of Primary
Type I Hypersensitivity 213 Immunodeficiencies 329
Type II Hypersensitivity 221 Laboratory Evaluation of Immune Dysfunction 336
SECTION IV Laboratory Testing for Viral Infections 411
Hepatitis Viruses 411
Serological and Molecular Herpes Virus Infections 419
Diagnosis of Infectious Disease 345 Other Viral Infections 425
Serological and Molecular
Detection of Bacterial Infections 346 Laboratory Diagnosis
of HIV Infection 433
Human–Microbe Relationships 347
HIV Transmission 434
Bacterial Virulence Factors 348
Characteristics of HIV 435
Immune Defenses Against Bacterial Infections
and Mechanisms of Evasion 351 Immunologic Manifestations 437
Laboratory Detection and Diagnosis of Bacterial Clinical Symptoms of HIV Infection 438
Infections 353 Treatment and Prevention 439
Group A Streptococci (Streptococcus Pyogenes) 354 Laboratory Testing for HIV Infection 441
Helicobacter Pylori 359 Screening and Diagnosis 441
Mycoplasma Pneumoniae 361 Disease Monitoring 446
Rickettsial Infections 362 Testing of Infants Younger Than 18 Months 450

Spirochete Diseases 370 Immunization and Vaccines 454

Vaccines 455
Syphilis 371 Passive Immunization 467
Lyme Disease 379 Adoptive Immunotherapy 470
Relapsing Fever Group—Borrelia Miyamotoi 382
Glossary 477
Serological and Molecular
Diagnosis of Parasitic and Fungal References 491
Infections 388 Answer Key 525

Parasitic Immunology 389

Index 535
Fungal Immunology 397

Serology and Molecular

Detection of Viral Infections 408

Immune Defenses Against Viral Infections 409

Viral Escape Mechanisms 411
 Nature of the
Immune System
 Introduction to
Immunity and
the Immune System

After finishing this chapter, you should be able to: IMMUNITY AND IMMUNIZATION
1. Discuss how immunology as a science began with the study INNATE VERSUS ADAPTIVE IMMUNITY
of immunity. CELLS OF THE INNATE IMMUNE
2. Describe what is meant by an attenuated vaccine. SYSTEM
3. Explain how the controversy over humoral versus cellular immunity Leukocytes in Peripheral Blood
contributed to expanding knowledge in the field of immunology. Tissue Cells
4. Distinguish innate from adaptive immunity. CELLS OF THE ADAPTIVE IMMUNE
5. Describe the types of white blood cells (WBCs) capable of SYSTEM
phagocytosis. B Cells
6. Explain the role of tissue cells in immunity. T Cells
7. Discuss how natural killer (NK) cells differ from T lymphocytes. Natural Killer (NK) Cells
8. Identify the two primary lymphoid organs and discuss the main ORGANS OF THE IMMUNE SYSTEM
functions of each. Primary Lymphoid Organs
9. List four secondary lymphoid organs and discuss their overall Secondary Lymphoid Organs
importance to immunity.
SUMMARY
10. Describe the function and architecture of a lymph node.
CASE STUDIES
11. Compare a primary and a secondary follicle.
REVIEW QUESTIONS
12. Explain the makeup of a cluster of differentiation.
13. Differentiate the roles of T cells and B cells in the immune response.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Adaptive immunity Dendritic cells Lymphocyte Plasma cells
Antibodies Diapedesis Macrophages Primary follicles
Antigens Eosinophils Mast cells Primary lymphoid organs
Attenuation Germinal center Memory cells Secondary follicles
Basophils Humoral immunity Monocytes Secondary lymphoid organs
Bone marrow Immunity Natural killer (NK) cells Spleen
Cell-mediated immunity Immunology Neutrophil Thymocytes
Chemotaxins Innate (natural) immunity Periarteriolar lymphoid Thymus
Clusters of differentiation (CD) Leukocytes sheath (PALS)
Cytokines Lymph nodes Phagocytosis

Although humans have been trying for many centuries to un- The next major development in disease prevention did not
ravel the secrets of preventing disease, the field of immunol- occur until almost a hundred years later when Louis Pasteur,
ogy is a relatively new science. Immunology can be defined often called the father of immunology, observed by chance that
as the study of a host’s reactions when foreign substances are older bacterial cultures would not cause disease in chickens
introduced into the body. Such foreign substances that induce (Fig. 1–1).2,3 Subsequent injections of more virulent organisms
a host response are called antigens. Antigens are all around had no effect on the birds that had been previously exposed to
us in nature and they vary from substances such as pollen that the older cultures. In this manner, the first attenuated vaccine
may make us sneeze to serious bacterial pathogens such as was discovered; this event can be considered the birth of im-
Staphylococcus aureus or Group A Streptococcus that can cause munology.3 Attenuation, or change, means to make a pathogen
life-threatening illnesses. The study of immunology has given less virulent; it takes place through heat, aging, or chemical
us the ability to prevent diseases such as smallpox, polio, means. Attenuation remains the basis for many of the immuniza-
diphtheria, and measles through the development of vaccines. tions that are used today. Pasteur applied this same principle of
In addition, understanding how the immune system works attenuation to the prevention of rabies in affected individuals.
has made successful organ transplantation possible and has
given us new tools to treat diseases such as cancer and certain
autoimmune diseases. Immunological techniques have affected
testing in many areas of the clinical laboratory and allowed for
such testing to be more precise and automated. Thus, the
study of immunology is important to many areas of medicine.
In this chapter, we will provide a brief look at the history of
the field and then introduce the cells and tissues of the immune
system to form a basis for understanding how the immune sys-
tem works. In later chapters we will apply this knowledge to
principles of testing for specific diseases.

Immunity and Immunization

Immunology as a science has its roots in the study of immunity:
the condition of being resistant to infection. The first recorded
attempts to deliberately induce immunity date back to the
1500s when the Chinese inhaled powder made from smallpox
scabs in order to produce protection against this dreaded dis-
ease. The hypothesis was that if a healthy individual was ex-
posed as a child or young adult the effects of the disease would
be minimized. However, the early exposure did not always
work. Further refinements did not occur until the late 1700s
when an English country doctor by the name of Edward Jenner
was able to successfully prevent infection with smallpox by
injecting a more harmless substance—cowpox—from a disease
affecting cows.1 Details of the development of this first vaccine Louis Pasteur. (Courtesy of the National Library
can be found in Chapter 25. of Medicine.)
Innate Versus Adaptive Immunity There are five principal types of leukocytes in peripheral
blood: neutrophils, eosinophils, basophils, monocytes, and
In the late 1800s, scientists turned to identifying the actual lymphocytes. The first four types are all part of innate im-
mechanisms that produce immunity in a host.2 Elie Metchnikoff, munity. Because lymphocytes are considered part of adaptive
a Russian scientist, observed under a microscope that foreign immunity, they will be considered in a separate section.
objects introduced into transparent starfish larvae became sur- Several cell lines that are found in the tissues, namely mast
rounded by motile amoeboid-like cells that attempted to destroy cells, macrophages, and dendritic cells, will also be discussed
the penetrating objects. This process was later termed phagocy- in this chapter because they all contribute to the process of
tosis, meaning cells that eat cells.2,4 He hypothesized that im- immunity.
munity to disease was based on the action of these scavenger All blood cells arise from a type of cell called a hematopoi-
cells and was a natural, or innate, host defense.4 etic stem cell (HSC). To form WBCs, the HSC gives rise to
Other researchers contended that noncellular elements in two distinct types of precursor cells: common myeloid pre-
the blood were responsible for protection from microorgan- cursors (CMP) and common lymphoid precursors (CLP).
isms. Emil von Behring demonstrated that diphtheria and CMPs give rise to the WBCs that participate in phagocytosis,
tetanus toxins, which are produced by specific microorganisms which are known as the myeloid line. Phagocytic cells are
as they grow, could be neutralized by the noncellular portion key to innate immunity, but they are also important in pro-
of the blood of animals previously exposed to the microorgan- cessing antigens for the adaptive response. Lymphocytes
isms. The theory of humoral immunity was thus born and arise from CLPs and form the basis of the adaptive immune
sparked a long-lasting dispute over the relative importance of response. Mature lymphocytes are found in the tissues
cellular versus humoral immunity. as well as in peripheral blood. Refer to Figure 1–2 for a
In 1903, an English physician named Almroth Wright simplified scheme of blood cell development, known as
linked the two theories by showing that the immune response hematopoiesis.
involved both cellular and humoral elements. He observed that
certain humoral, or circulating, factors called opsonins acted to The neutrophil, or polymorphonuclear neutrophilic (PMN)
coat bacteria so that they became more susceptible to ingestion leukocyte, represents approximately 50% to 75% of the total
by phagocytic cells.2 These serum factors include specific pro- peripheral WBCs in adults.5 These are around 10 to 15 µm in
teins known as antibodies, as well as other factors called acute- diameter with a nucleus that has between two and five lobes
phase reactants that increase nonspecifically in any infection. (Fig. 1–3). Hence, they are often called segmented neu-
Antibodies are serum proteins produced by certain lympho- trophils, or “segs.” They contain a large number of neutral
cytes when exposed to a foreign substance and they react staining granules when stained with Wright stain, two-thirds
specifically with that foreign substance (see Chapter 5). of which are specific granules; the remaining one-third are
These discoveries showed that there were two major called azurophilic granules.6 Azurophilic or primary granules
branches of immunity, currently referred to as innate immunity contain antimicrobial products such as myeloperoxidase,
and adaptive immunity. Innate, or natural immunity, is the in- lysozyme, elastase, proteinase-3, cathepsin G, and defensins,
dividual’s ability to resist infection by means of normally present which are small proteins that have antibacterial activity.5 Spe-
body functions. These are considered nonadaptive or nonspe- cific granules, also known as secondary granules, contain
cific and are the same for all pathogens or foreign substances to lysozyme, lactoferrin, collagenase, gelatinase, and respiratory
which one is exposed. No prior exposure is required and the burst components.5,7 See Chapter 3 for a discussion of the
response lacks memory and specificity. Many of these mecha- oxidative burst, which takes place during phagocytosis. The
nisms are subject to influence by such factors as nutrition, age, main function of neutrophils is phagocytosis, resulting in
fatigue, stress, and genetic determinants. Adaptive immunity, in the destruction of foreign particles.6
contrast, is a type of resistance that is characterized by speci- Normally, half of the total neutrophil population in periph-
ficity for each individual pathogen, or microbial agent, and the eral blood is found in a marginating pool adhering to blood
ability to remember a prior exposure. Memory and specificity vessel walls, whereas the rest circulate freely for approximately
result in an increased response to that pathogen upon repeated 6 to 8 hours.5 There is a continuous interchange, however, be-
exposure, something that does not occur in innate immunity. tween the marginating and the circulating pools. Margination
Both systems are necessary to maintain good health. In fact, occurs to allow neutrophils to move from the circulating blood
they operate in combination and are dependent upon one an- to the tissues through a process known as diapedesis, or
other for maximal effectiveness. Certain key cells are considered movement through blood vessel walls. They are attracted to a
essential to both systems and they will be discussed next. specific area by chemotactic factors. Chemotaxins are chemi-
cal messengers that cause cells to migrate in a particular direc-
tion. Once in the tissues, neutrophils have a life span of up to
Cells of the Innate Immune System several days. Normally, the influx of neutrophils from the bone
marrow equals the output from the blood to the tissues to
Leukocytes in Peripheral Blood maintain a steady state. However, in the case of acute infection
White blood cells (WBCs), or leukocytes, in the peripheral an increase of neutrophils in the circulating blood can occur
blood play a key role in both innate and adaptive immunity. almost immediately.8
 T cells

NK cell

B cell
Neutrophils. (From Harmening D. Clinical Hematology
and Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis;
2009. Fig. 1–4.)
CLP

Dendritic cell

Monocyte

HSC

Neutrophil

Eosinophil
Eosinophil. (From Harmening D. Clinical Hematology
and Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis;
2009. Fig. 1–6.)
CMP
Basophil
reddish-orange granules. Granules in eosinophils, which are
spherical and evenly distributed throughout the cell, contain
a large number of previously synthesized proteins including
Erythrocytes catalase, lysozyme, cytokines (chemical messengers), growth
factors, and cationic proteins.5,9
Eosinophils are capable of phagocytosis but are much less
Platelets efficient than neutrophils because they are present in smaller
numbers and they lack digestive enzymes. Eosinophils are able
Simplified scheme of hematopoiesis. In the marrow, to neutralize basophil and mast cell products. In addition, they
hematopoietic stem cells (HSC) give rise to two different lines—a can use cationic proteins to damage cell membranes and kill
common lymphoid precursor (CLP) and a common myeloid precur- larger parasites that cannot be phagocytized. (See Chapter 22
sor (CMP). CLPs give rise to T/NK progenitors, which differentiate for details.) However, the most important role of eosinophils
into T and NK cells, and to B-cell progenitors, which become is regulation of the immune response, including regulation of
B cells and dendritic cells. The CMP differentiates into neutrophils,
mast cell function.5
monocytes/macrophages, eosinophils, basophils, erythrocytes,
and platelets.
Basophils are the least numerous of WBCs found in periph-
eral blood, representing less than 1% of all circulating WBCs.
The smallest of the granulocytes, basophils are slightly larger
Eosinophils are approximately 12 to 15 µm in diameter and than RBCs (between 10 to 15 µm in diameter) and contain
normally make up between 1% and 3% of the circulating coarse, densely staining deep-bluish-purple granules that
WBCs in a nonallergic person. Their number increases in an often obscure the nucleus5,9 (Fig. 1–5). Constituents of
allergic reaction or in response to certain parasitic infections. these granules include histamine, cytokines, growth factors,
The nucleus is usually bilobed or ellipsoidal and is often ec- and a small amount of heparin, all of which have an impor-
centrically located (Fig. 1–4). Eosinophils take up the acid tant function in inducing and maintaining allergic reac-
eosin dye and the cytoplasm is filled with large orange to tions.5,8 Histamine contracts smooth muscle and heparin is
 Tissue Cells

All macrophages arise from monocytes, which can be thought

of as macrophage precursors because additional differentiation
and cell division takes place in the tissues. The transition from
monocyte to macrophage in the tissues is characterized by
progressive cellular enlargement to between 25 and 80 µm.8
Unlike monocytes, macrophages contain no peroxidase.8
Tissue distribution appears to be a random phenomenon.
Macrophages have specific names according to their partic-
ular tissue location. Macrophages in the lung are alveolar
macrophages; in the liver, Kupffer cells; in the brain, microglial
Basophil. (From Harmening D. Clinical Hematology and cells; in the bone, osteoclasts; and in connective tissue, histio-
Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis; 2009. cytes. Macrophages may not be as efficient as neutrophils in
Fig. 1–7.) phagocytosis because their motility is slow compared with that
of the neutrophils. Some macrophages progress through the
tissues by means of amoeboid action, whereas others are im-
an anticoagulant. In addition, basophils regulate some mobile. However, their life span appears to be in the range of
T helper (Th) cell responses and stimulate B cells to produce months rather than days.
the antibody IgE.5,10 Basophils have a short life span of only Macrophages play an important role in initiating and regu-
a few hours in the bloodstream; they are then pulled out and lating both innate and adaptive immune responses. Their innate
destroyed by macrophages in the spleen. immune functions include microbial killing, anti-tumor activity,
intracellular parasite eradication, phagocytosis, and secretion of
cell mediators. Killing activity is enhanced when macrophages
Monocytes are the largest cells in the peripheral blood with a become “activated” by contact with microorganisms or with
diameter that can vary from 12 to 22 µm (the average is chemical messengers called cytokines, which are released by
18 µm).9 One distinguishing feature is an irregularly folded or T lymphocytes during the immune response. (See Chapter 6 for
horseshoe-shaped nucleus that occupies almost one-half of the a complete discussion of cytokines.) Macrophages play a major
entire cell’s volume (Fig. 1–6). The abundant cytoplasm stains role in the adaptive immune response by presenting antigens to
a dull grayish blue and has a ground-glass appearance because T and B cells.
of the presence of fine dustlike granules. These granules are
actually of two types. The first type contains peroxidase, acid
phosphatase, and arylsulfatase, indicating that these granules Tissue mast cells resemble basophils, but they come from a dif-
are similar to the lysosomes of neutrophils.8 The second type ferent lineage. Mast cells are distributed throughout the body in
of granule may contain β-glucuronidase, lysozyme, and lipase, a wide variety of tissues such as skin, connective tissue, and the
but no alkaline phosphatase. Digestive vacuoles may also be mucosal epithelial tissue of the respiratory, genitourinary, and
observed in the cytoplasm. Monocytes make up between 4% digestive tracts.5 Mast cells are larger than basophils with a small
and 10% of total circulating WBCs; however, they do not re- round nucleus and more granules (Fig. 1–7). Unlike basophils,
main in the circulation for long. They stay in peripheral blood they have a long life span of between 9 and 18 months.11 The
for up to 30 hours; they then migrate to the tissues and become
known as macrophages.5

Two monocytes. (From Harmening D. Clinical Mast cell. (From Harmening D. Clinical Hematology and
Hematology and Fundamentals of Hemostasis. 5th ed. Philadelphia, Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis; 2009.
PA: F.A. Davis; 2009. Fig. 1–13.) Fig. 1–44.)
enzyme content of the granules in mast cells helps to distinguish to 20% of lymphocytes are B cells, 61% to 80% are T cells, and
them from basophils because they contain acid phosphatase, 10% to 15% are NK cells.13
alkaline phosphatase, and protease, as well as histamine.5,7,8 The three types of cells are difficult to distinguish visually.
Mast cells play a role in allergic reactions, but they can also func- In the laboratory, proteins, or antigens, on cell surfaces can be
tion as antigen-presenting cells (APCs). They can both enhance used to identify each lymphocyte subpopulation. In order to
and suppress the adaptive immune response. standardize the nomenclature, scientists set up the Human
Leukocyte Differentiation Antigens Workshops to relate re-
search findings.15 Panels of antibodies from different laborato-
Dendritic cells are so named because they are covered with
ries were used for analysis and antibodies reacting similarly
long membranous extensions that make them resemble nerve
with standard cell lines were said to define clusters of differ-
cell dendrites. They were discovered by Steinman and Cohn
entiation (CD). As each antigen, or CD, was found, it was as-
in 1973.7 Progenitors in the bone marrow give rise to dendritic
signed a number. The list of CD designations currently
cell precursors that travel to lymphoid as well as nonlymphoid
numbers more than 500.16 Table 1–1 lists some of the most
tissue.12 They are classified according to their tissue location
important CD numbers used to identify lymphocytes.
in a similar manner to macrophages. After capturing an antigen
in the tissue by phagocytosis or endocytosis, dendritic cells
present the antigen to T lymphocytes to initiate the adaptive B Cells
immune response in a similar way as macrophages. Dendritic B cells are derived from a lymphoid precursor that differenti-
cells, however, are considered the most effective APC in the ates to become either a T cell, B cell, or NK cell depending on
body, as well as the most potent phagocytic cell.13,14 exposure to different cytokines. B cells remain in the environ-
ment provided by bone marrow stromal cells. B-cell precursors
Cells of the Adaptive go through a developmental process that prepares them for
their role in antibody production and, at the same time, re-
Immune System stricts the types of antigens to which any one cell can respond.
The end result is a B lymphocyte programmed to produce a
The key cell involved in the adaptive immune response is the unique antibody molecule. B cells can be recognized by the
lymphocyte. Lymphocytes represent between 20% and 40% of presence of membrane-bound antibodies of two types, namely
the circulating WBCs. The typical small lymphocyte is similar immunoglobulin M (IgM) and immunoglobulin (IgD). Other
in size to RBCs (7–10 µm in diameter) and has a large rounded surface proteins that appear on the B cell include CD19, CD21,
nucleus that may be somewhat indented. The nuclear chro- and class II major histocompatibility complex (MHC) mole-
matin is dense and tends to stain a deep blue (Fig. 1–8).9 Cy- cules (see Chapter 2).10
toplasm is sparse, containing few organelles and no specific
granules, and consists of a narrow ring surrounding the nu-
cleus.6 The cytoplasm stains a lighter blue. These cells are T Cells
unique because they arise from an HSC and then are further T cells are so named because they differentiate in the thymus.
differentiated in the primary lymphoid organs, namely the bone Lymphocyte precursors called thymocytes enter the thymus
marrow and the thymus. Lymphocytes can be divided into three from the bone marrow through the bloodstream. As they ma-
major populations—T cells, B cells, and natural killer (NK) ture, the T cells express unique surface markers that allow
cells—based on specific functions and the proteins on their cell them to recognize foreign antigens bound to cell membrane
surfaces. In the peripheral blood of adults, approximately 10% proteins called MHC molecules. The role of T cells is to pro-
duce cytokines that contribute to immunity by stimulating
B cells to produce antibodies, assisting in killing tumor cells
or infected target cells, and helping to regulate both the innate
and adaptive immune response. The process is known as cell-
mediated immunity.
Three main subtypes of T cells can be distinguished accord-
ing to their unique functions: helper, cytolytic, and regulatory
T cells. The subtypes can be identified by the presence of the
CD3 marker on their cell surface, and either CD4, or CD8.
T cells bearing the CD4 receptor are mainly either helper or
regulatory cells, whereas the CD8-positive (CD8+) population
consists of cytotoxic T cells. The ratio of CD4+ to CD8+ cells
is approximately 2:1 in peripheral blood.

Natural Killer (NK) Cells

Typical lymphocyte found in peripheral blood.
(From Harr R. Clinical Laboratory Science Review. 4th ed. Philadelphia, A small percentage of lymphocytes do not express the mark-
PA: F.A. Davis; 2013. Color Plate 31.) ers of either T cells or B cells. They are named natural killer
 Table 1–1 Surface Markers on T, B, and NK Cells
ANTIGEN MOL WT (KD) CELL TYPE FUNCTION
CD3 20–28 Thymocytes, T cells Found on all T cells; associated with
T-cell antigen receptor
CD4 55 T helper cells, monocytes, macrophages Identifies T helper cells; also found on
most T regulatory cells
CD8 60–76 Thymocyte subsets, cytotoxic T cells Identifies cytotoxic T cells
CD16 50–80 Macrophages, NK cells, neutrophils Low affinity Fc receptor for antibody;
mediates phagocytosis
CD19 >120 B cells, follicular dendritic cells Part of B-cell coreceptor; regulates B-cell
development and activation
CD21 145 B cells, follicular dendritic cells Receptor for complement component
C3d; part of B-cell coreceptor with CD19
CD 56 175–220 NK cells, subsets of T cells Not known

(NK) cells because they have the ability to kill target cells antigens can occur (Fig. 1–9). Secondary lymphoid organs
without prior exposure to them. NK cells do not require the include the spleen, lymph nodes, and various types of
thymus for development but appear to mature in the bone mucosal-associated lymphoid tissues (MALT). The primary and
marrow itself.17,18 NK cells are generally larger than T cells secondary organs are differentiated according to their function
and B cells at approximately 15 µm in diameter and contain in both adaptive and innate immunity.
kidney-shaped nuclei with condensed chromatin and promi-
nent nucleoli. Described as large granular lymphocytes, NK Primary Lymphoid Organs
cells make up 10% to 15% of the circulating lymphoid pool
and are found mainly in the liver, spleen, and peripheral
blood.5,10 Bone marrow is considered one of the largest tissues in the
There are no surface markers that are unique to NK cells, body and it fills the core of all long flat bones. It is the main
but they express a specific combination of antigens that can be source of hematopoietic stem cells, which develop into ery-
used for identification. Two such antigens are CD16 and CD56. throcytes, granulocytes, monocytes, platelets, and lympho-
CD16 is a receptor for the nonspecific end of antibodies. (See cytes. Each of these lines has specific precursors that originate
Chapter 5 for more details.) Because of the presence of CD16, from the pleuripotential stem cells.
NK cells are able to make contact with and then lyse any cell Some lymphocyte precursors remain in the marrow to ma-
coated with antibodies.10 NK cells are also capable of recog- ture and become NK and B cells. B cells received their name
nizing any foreign cell and represent the first line of defense because they were originally found to mature in birds in an
against virally infected cells and tumor cells.19 organ called the bursa of Fabricius, which is similar to the ap-
Although NK cells have traditionally been considered part pendix in humans. After searching for such an organ in hu-
of the innate immune system because they can respond to a mans, it was discovered that B-cell maturation takes place
variety of antigens, it appears that they also have the capability within the bone marrow itself. Thus, the naming of these cells
to develop memory to specific antigens in a similar manner to was appropriate. Other lymphocyte precursors go to the thy-
T cells.19 Normally, NK cells have a half-life of 7 to 10 days, mus and develop into T cells, so named because of where they
but new evidence suggests that they are able to survive for a mature.7 Immature T cells appear in the fetus as early as
longer time because they can generate highly specific memory 8 weeks in the gestational period.21 Thus, differentiation of
cells.19,20 Thus, they play an important role as a transitional lymphocytes appears to take place very early in fetal develop-
cell bridging the innate and the adaptive immune response ment and is essential to acquisition of immunocompetence by
against pathogens.17 the time the infant is born.

Organs of the Immune System T cells develop their identifying characteristics in the thymus,
which is a small, flat, bilobed organ found in the thorax, or
Just as the cells of the immune system have diverse functions, chest cavity, right below the thyroid gland and overlying the
so, too, do key organs that are involved in the development of heart. In humans, the thymus reaches a weight of 30 to 40 g
the immune response. The bone marrow and thymus are con- by puberty and then gradually shrinks in size.22 It was first
sidered the primary lymphoid organs where maturation of thought that the thymus produces enough virgin T lympho-
B lymphocytes and T lymphocytes takes place, respectively. The cytes early in life to seed the entire immune system, making
secondary organs provide a location where contact with foreign the organ unnecessary later on. However, it now appears that
 lymphoid tissue (CALT), and MALT in the respiratory, gastroin-
testinal, and urogenital tracts. It is within these secondary
Adenoids
organs that the main contact with foreign antigens takes place.
Tonsils Lymphocyte circulation between the secondary organs is com-
plex and is regulated by different cell surface adhesion mole-
Thymus
cules and by cytokines.
Lymph nodes
Heart Each lymphocyte spends most of its life span in solid tissue,
Lungs entering the circulation only periodically to go from one sec-
ondary organ to another. Lymphocytes in these organs travel
Liver
through the tissue and return to the bloodstream by way of the
Spleen
thoracic duct. The thoracic duct is the largest lymphatic vessel
in the body. It collects most of the body’s lymph fluid and emp-
Small intestine ties it into the left subclavian vein. The majority of circulating
Peyer’s patches lymphocytes are T cells.5 Continuous recirculation increases
the likelihood of a T lymphocyte coming into contact with the
Bone marrow specific antigen with which it can react.
Lymphocytes are segregated within the secondary organs
according to their particular functions. T lymphocytes are
effector cells that serve a regulatory role, whereas B lymphocytes
produce antibodies. It is in the secondary organs that contact
Tissue with foreign antigens is most likely to take place.
lymphatics Lymphopoiesis, or multiplication of lymphocytes, occurs in
the secondary lymphoid tissue and is strictly dependent on
antigenic stimulation. Formation of lymphocytes in the bone
marrow, however, is antigen-independent, meaning that lym-
phocytes are constantly being produced without the presence
of specific antigens. Most naïve or resting lymphocytes die
within a few days after leaving the primary lymphoid organs
unless activated by the presence of a specific foreign antigen.
Antigen activation gives rise to long-lived memory cells and
shorter-lived effector cells that are responsible for the genera-
tion of the immune response.

Sites of lymphoreticular tissue. Primary organs The spleen, the largest secondary lymphoid organ, has a length
include the bone marrow and the thymus. Secondary organs are of approximately 12 cm and weighs 150 g in the adult. It is lo-
distributed throughout the body and include the spleen, lymph cated in the upper-left quadrant of the abdomen just below the
nodes, and mucosal-associated lymphoid tissue (MALT). The diaphragm and is surrounded by a thin connective tissue cap-
spleen filters antigens in the blood, whereas the lymphatic system
sule. The organ can be characterized as a large discriminating
filters fluid from the tissues.
filter as it removes old and damaged cells and foreign antigens
from the blood.
although the thymus diminishes in size as humans age, it is Splenic tissue can be divided into two main types: red pulp
still capable of producing T lymphocytes, although at a dimin- and white pulp. The red pulp makes up more than one-half of
ished rate.22,23 the total volume and its function is to destroy old red blood
Each lobe of the thymus is divided into smaller lobules filled cells (RBCs). Blood flows from the arterioles into the red pulp
with epithelial cells that play a central role in the differentiation and then exits by way of the splenic vein. The white pulp com-
process. Maturation of T cells takes place over a 3-week period prises approximately 20% of the total weight of the spleen and
as cells filter through the thymic cortex to the medulla. Differ- contains the lymphoid tissue, which is arranged around arteri-
ent surface antigens are expressed as T cells mature. In this oles in a periarteriolar lymphoid sheath (PALS) (Fig. 1–10).
manner, a repertoire of T cells is created to protect the body This sheath contains mainly T cells. Attached to the sheath are
from foreign invaders. Mature T lymphocytes are then released primary follicles, which contain B cells that are not yet stim-
from the medulla. ulated by antigens. Surrounding the PALS is a marginal zone
containing dendritic cells that trap antigens. Lymphocytes enter
and leave this area by means of the many capillary branches
Secondary Lymphoid Organs that connect to the arterioles. The spleen receives a blood
Once lymphocytes mature in the primary organs, they are re- volume of approximately 350 mL/minute, which allows lym-
leased and make their way to secondary lymphoid organs, phocytes and macrophages to constantly survey for infectious
which include the spleen, lymph nodes, cutaneous-associated agents or other foreign matter.22
 Lymphocytes and any foreign antigens present enter nodes
via afferent lymphatic vessels. Numerous lymphocytes also
enter the nodes from the bloodstream by means of specialized
venules called high endothelial venules, which are located in the
paracortical areas of the node tissues.24 The outermost layer,
the cortex, contains macrophages and aggregations of B cells
in primary follicles similar to those found in the spleen. These
are the mature, resting B cells that have not yet been exposed
to antigens. Specialized cells called follicular dendritic cells are
also located here. These cells exhibit a large number of recep-
Capsule tors for antibodies and help to capture antigens to present to
T and B cells.
Central Secondary follicles consist of antigen-stimulated prolifer-
artery
ating B cells. The interior of a secondary follicle is known as the
Germinal germinal center because it is here that transformation of the
center B cells takes place. When exposed to an antigen, plasma cells
(Fig. 1–12), which actively secrete antibodies, and memory
Periarteriolar cells, which are just a step away from forming plasma cells, are
lymphoid formed. Thus, the lymph nodes provide an ideal environment
sheath (PALS) for the generation of B-cell memory.
(T cells)
T lymphocytes are mainly localized in the paracortex, the
region between the follicles and the medulla. T lymphocytes
Trabecular are in close proximity to APCs called interdigitating cells. The
vein
medulla is less densely populated than the cortex but con-
tains some T cells (in addition to B cells), macrophages, and
Sinuses in
red pulp
numerous plasma cells.
Primary
follicle
Particulate antigens are removed from the fluid as it travels
(B cells)
Marginal across the node from cortex to medulla. Fluid and lymphocytes
zone
exit by way of the efferent lymph vessels. Such vessels form a
Cross-section of the spleen showing organization of larger duct that eventually connects with the thoracic duct and
the lymphoid tissue. T cells surround arterioles in the PALS. B cells
the venous system. As a result, lymphocytes are able to recir-
are just beyond in follicles. When stimulated by antigens, the B cells
culate continuously between lymph nodes and the peripheral
form germinal centers. All of the lymphoid tissue is referred to as the
white pulp. blood.
If contact with an antigen takes place, lymphocyte traffic
shuts down. Lymphocytes able to respond to a particular antigen
proliferate in the node. Accumulation of lymphocytes and other
Lymph nodes serve as central collecting points for lymph fluid cells causes the lymph nodes to become enlarged, a condition
from adjacent tissues. Lymph fluid is a filtrate of the blood and known as lymphadenopathy. As lymphocyte traffic resumes, re-
arises from passage of water and low-molecular-weight solutes circulation of expanded numbers of lymphocytes occurs.
out of blood vessel walls and into the interstitial spaces be-
tween cells. Some of this interstitial fluid returns to the blood- Additional areas of lymphoid tissue include the mucosal-
stream through venules, but a portion flows through the tissues associated tissue known as MALT. MALT is found in the gas-
and is eventually collected in thin-walled vessels known as lym- trointestinal, respiratory, and urogenital tracts. Some examples
phatic vessels. Lymph nodes are located along lymphatic ducts include the tonsils; appendix; and Peyer’s patches, a specialized
and are especially numerous near joints and where the arms type of MALT located at the lower ileum of the intestinal tract.
and legs join the body (see Fig. 1–9). These mucosal surfaces represent some of the main ports of
Filtration of interstitial fluid from around cells in the tissues entry for foreign antigens, and thus, numerous macrophages
is an important function of these organs because it allows con- and lymphocytes are localized here.
tact between lymphocytes and foreign antigens from the tissues The skin is considered the largest organ in the body and the
to take place. Whereas the spleen helps to protect us from for- epidermis contains a number of intraepidermal lymphocytes.
eign antigens in the blood, the lymph nodes provide the ideal Most of these are T cells, which are uniquely positioned to
environment for contact with foreign antigens that have pene- combat any antigens that enter through the skin. In addition,
trated into the tissues. The lymph fluid flows slowly through monocytes, macrophages, and dendritic cells are found here.
spaces called sinuses, which are lined with macrophages, cre- The collective term for these cells is the cutaneous-associated
ating an ideal location where phagocytosis can take place. The lymphoid tissue, or CALT.
node tissue is organized into an outer cortex, a paracortex, and All of these secondary organs function as potential sites for
an inner medulla (Fig. 1–11). contact with foreign antigens and they increase the probability
 Afferent
lymphatic

Germinal
Capsule center

Cortex
(B-cell area) Subcapsular
sinus
Afferent
Afferent
lymphatic
lymphatic

Structure of a lymph node. A

lymph node is surrounded by a tough outer
capsule. Right underneath is the subcapsular
Primary
sinus, where lymph fluid drains from afferent follicle Sinusoid
lymphatic vessels. The outer cortex contains
collections of B cells in primary follicles.
When stimulated by antigens, secondary Secondary
follicle with
follicles are formed. T cells are found in
germinal center
the paracortical area. Fluid drains slowly Medulla
through sinusoids to the medullary region Paracortex
(T-cell area)
and out the efferent lymphatic vessel to Efferent
the thoracic duct. lymphatic

SUMMARY
• Immunology has its roots in the study of immunity—the
condition of being resistant to disease.
• Jenner performed the first vaccination against smallpox by
using cowpox.
• Louis Pasteur is considered the father of immunology for
his use of attenuated vaccines.
• Metchnikoff was the first to observe phagocytosis—meaning
cells that eat cells.
• Immunity has two branches. Innate immunity is the ability
of the body to resist infection through means of normally
present nonspecific body functions. Adaptive immunity is
characterized by specificity, memory, and dependence
A typical plasma cell. (From Harmening D. Clinical upon lymphocytes.
Hematology and Fundamentals of Hemostasis. 5th ed. Philadelphia,
• All blood cells arise in the bone marrow from hematopoi-
PA: F.A. Davis 2009. Fig. 1–47.)
etic stem cells.
• The five principal types of leukocytes are neutrophils,
of an immune response. Within each of these secondary eosinophils, basophils, monocytes, and lymphocytes.
organs, T and B cells are segregated and perform specialized • Tissue cells involved in immunity include mast
functions. B cells differentiate into memory cells and plasma cells, dendritic cells, and macrophages that arise from
cells and are responsible for humoral immunity or antibody monocytes.
formation. T cells play a role in cell-mediated immunity; as • Cells that are involved in the innate immune response and
such, they produce sensitized lymphocytes that secrete cytokines. are actively phagocytic include neutrophils, monocytes,
Both cell-mediated immunity and humoral immunity are part macrophages, and dendritic cells.
of the overall adaptive immune response.
• Lymphocytes are the key cells involved in the adaptive • Natural killer (NK) cells are types of lymphocyte that
immune response. arise from a lymphocyte precursor but do not develop
• CD stands for clusters of differentiation, which are types in the thymus. They can kill virally infected or cancerous
of proteins found on cell surfaces that can be used for target cells without previous exposure to them.
identification of specific cells and cell stages. • The bone marrow and the thymus are considered primary
• B cells are a type of lymphocyte that develop in the bone lymphoid organs. B cells remain in the bone marrow to
marrow and are capable of secreting antibody when ma- mature, whereas the thymus is where T cells develop their
ture. They can be identified by the presence of CD19 and specific characteristics.
surface antibody. • Secondary lymphoid organs include the spleen,
• T cells acquire their specificity in the thymus and consist lymph nodes, mucosal-associated lymphoid tissue
of two subtypes: CD4+, which are mainly helper or reg- (MALT), and cutaneous-associated lymphoid tissue
ulatory T cells, and CD8+, which are cytotoxic T cells. (CALT).

Study Guide: Comparison of T, B, and NK Cells

T CELLS B CELLS NK CELLS
Develop in the thymus Develop in the bone marrow Develop in the bone marrow
Found in lymph nodes, Found in bone marrow, spleen, lymph nodes, Found in spleen, liver
thoracic duct fluid 10–15% of circulating lymphocyte pool in blood 5–15% of circulating lymphocyte
60–80% of circulating pool in blood
lymphocyte pool in blood
Adaptive immunity: end Adaptive immunity: end product of activation is Innate immunity: lysis of virally
products of activation antibody infected cells and tumor cells;
are cytokines production of cytokines
Antigens include CD2, Antigens include CD19, CD20, CD21 surface Antigens include CD16, CD56
CD3, CD4, or CD8 antibody

Study Guide: Primary and Secondary Lymphoid Organs

LYMPHOID ORGAN
CATEGORY ORGANS INVOLVED FUNCTION
Primary Bone marrow Produces hematopoietic stem cells;
maturation of B and NK cells
Thymus Maturation of T cells
Secondary Spleen Filters blood
Lymph nodes Places where contact between T cells,
Mucosal associated lymphoid tissue (MALT) antigens, and B cells occur
Cutaneous-associated lymphoid tissue (CALT)
Study Guide: Cells of the Immune System
CELL TYPE WHERE FOUND FUNCTION
Neutrophil 50–75% of circulating First responder to infection, phagocytosis
WBCs, also in tissue

Eosinophil 1–3% of circulating WBCs Kill parasites, neutralize basophil and mast
cell products, regulate mast cells

Basophil < 1% of circulating WBCs Induce and maintain allergic reactions,

stimulate production of IgE

Mast cell Found in skin, connective Antigen presentation to T and B cells;

tissue, mucosal epithelium enhancement and suppression of the
adaptive immune response

Monocyte 4–10% of circulating WBCs Phagocytosis; migrate to tissues to become

macrophages

Macrophage In lungs, liver, brain, bone, Phagocytosis; kill intracellular parasites;

connective tissue, other tumoricidal activity; antigen presentation
tissue to T and B cells

Dendritic cell In skin, mucous membranes, Most potent phagocytic cell; most effective
heart, lungs, liver, kidney, at antigen presentation
other tissue

Lymphocyte 20–40% of circulating Subtypes are T cells, B cells, and NK cells;

WBCs; also found in lymph T cells produce cytokines, B cells produce
nodes, spleen, other sec- antibody in adaptive immune response, and
ondary lymphoid organs NK cells are involved in innate immunity
CASE STUDIES
1. A 13-year-old girl had her ears pierced at a small jewelry 2. You and a friend are discussing the relative merits of
store in a mall. Although she was instructed to clean the immunizations. Your friend says that he doesn’t want to
area around the earrings with alcohol, she forgot for the get a tetanus booster shot because he has a good immune
first 2 days. On the third day she noticed that the area system and his natural defenses will take care of any pos-
around one earlobe was red and slightly swollen. sible infection. You have just been studying this subject
in your immunology class.
Questions
a. Which branch of the immune system is likely the Question
cause of the symptoms? a. What argument could you make to convince him that
b. What type of cell would you expect to see in the tissue? a tetanus booster is a good idea?

REVIEW QUESTIONS
1. Which of the following can be attributed to Pasteur? 7. The ability of an individual to resist infection by
a. Discovery of opsonins means of normally present body functions is called
b. Observation of phagocytosis a. innate immunity.
c. First attenuated vaccines b. humoral immunity.
d. Theory of humoral immunity c. adaptive immunity.
d. cross-immunity.
2. Which WBC is capable of further differentiation in
tissues? 8. A cell characterized by a nucleus with two to five
a. Neutrophil lobes, a diameter of 10 to 15 µm, and a large number
b. Eosinophil of neutral staining granules is identified as a(n)
c. Basophil a. eosinophil.
d. Monocyte b. monocyte.
c. basophil.
3. The cells that Metchnikoff first observed are associated d. neutrophil.
with which phenomenon?
a. Innate immunity 9. Which of the following is a primary lymphoid organ?
b. Adaptive immunity a. Lymph node
c. Humoral immunity b. Spleen
d. Specific immunity c. Thymus
d. MALT
4. Where are all undifferentiated lymphocytes made?
a. Bone marrow 10. What type of cells would be found in a primary follicle?
b. Spleen a. Unstimulated B cells
c. Thymus b. Germinal centers
d. Lymph nodes c. Plasma cells
d. Memory cells
5. Which of the following statements is true of
NK cells? 11. Which of the following is a distinguishing feature
a. They rely upon memory for antigen recognition. of B cells?
b. They have the same CD groups as B cells. a. Act as helper cells
c. They are found mainly in lymph nodes. b. Presence of surface antibody
d. They kill target cells without prior exposure to c. Able to kill target cells without prior exposure
them. d. Active in phagocytosis

6. Which cell is the most potent phagocytic cell in the 12. Where do lymphocytes mainly come in contact
tissue? with antigens?
a. Neutrophil a. Secondary lymphoid organs
b. Dendritic cell b. Bloodstream
c. Eosinophil c. Bone marrow
d. Basophil d. Thymus
13. Which of the following is found on the T cell subset 17. Immunity can be defined as
known as helpers? a. the study of medicines used to treat diseases.
a. CD19 b. a specific population at risk for a disease.
b. CD4 c. the condition of being resistant to disease.
c. CD8 d. the study of the noncellular portion of the blood.
d. CD56
18. A blood cell that has reddish staining granules and is
14. Which of the following statements best characterizes able to kill large parasites describes
adaptive immunity? a. basophils.
a. Relies on normally present body functions b. monocytes.
b. Response is similar for each exposure c. neutrophils.
c. Specificity for each individual pathogen d. eosinophils.
d. Involves only cellular immunity
19. Which of the following statements best describes a
15. The main function of T cells in the immune response lymph node?
is to a. It is considered a primary lymphoid organ.
a. produce cytokines that regulate both innate and b. It removes old RBCs.
adaptive immunity. c. It collects fluid from the tissues.
b. produce antibodies. d. It is where B cells mature.
c. participate actively in phagocytosis.
d. respond to target cells without prior exposure. 20. Antigenic groups identified by different sets of
antibodies reacting in a similar manner to certain
16. Which of the following is a part of humoral immunity? standard cell lines best describes
a. Cells involved in phagocytosis a. cytokines.
b. Neutralization of toxins by serum b. clusters of differentiation (CD).
c. Macrophages and mast cells in the tissue c. neutrophilic granules.
d. T and B cells in lymph nodes d. opsonins.
 Nature of Antigens
and the Major
Histocompatibility
Complex

After finishing this chapter, you should be able to: FACTORS INFLUENCING THE IMMUNE
RESPONSE
1. Define and characterize the nature of immunogens.
TRAITS OF IMMUNOGENS
2. Differentiate an immunogen from an antigen.
EPITOPES
3. Discuss several biological properties of individuals that influence the
nature of the immune response. HAPTENS
4. Describe four important characteristics of immunogens that affect the ADJUVANTS
ability to stimulate a host response. RELATIONSHIP OF ANTIGENS
5. Identify the characteristics of a hapten. TO THE HOST
6. Describe how an epitope relates to an immunogen. MAJOR HISTOCOMPATIBILITY
COMPLEX
7. Discuss the role of adjuvants.
Genes Coding for MHC Molecules
8. Differentiate heterophile antigens from alloantigens and autoantigens.
(HLA Antigens)
9. Explain what a haplotype is in regard to inheritance of major
Structure of Class I and II MHC
histocompatibility complex (MHC) antigens.
Molecules
10. Describe the differences in the structure of class I and class II
Role of Class I and II Molecules in the
molecules.
Immune Response
11. Compare the transport of antigen to cellular surfaces by class I and
Clinical Significance of MHC
class II molecules.
SUMMARY
12. Describe the role of transporters associated with antigen processing
(TAP) in selecting peptides for binding to class I molecules. CASE STUDY
13. Discuss the differences in the source and types of antigen processed REVIEW QUESTIONS
by class I and class II molecules.
14. Explain the clinical significance of the class I and class II molecules.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Adjuvant Class I MHC (HLA) molecules Haptens Linear epitopes
Alleles Class II MHC (HLA) Heteroantigens Major histocompatibility
Alloantigens molecules Heterophile antigens complex (MHC)
Antigen Conformational epitope Immunogenicity Transporters associated
Epitope with antigen processing
Antigen presentation Immunogens
(TAP1 and TAP2)
Autoantigens Haplotype Invariant chain (Ii)

Whereas the innate immune system responds nonspecifically to larger quantity required for the response allows the innate im-
certain patterns found on pathogens, the adaptive immune mune response to take care of small amounts of pathogens and
system is characterized by specific recognition of individual leave the adaptive response for pathogens that are present in
pathogens. Lymphocytes are the key cells that are responsible large numbers. Generally, the larger the amount of an immuno-
for the specificity, diversity, and memory that characterize adap- gen one is exposed to, the greater the immune response. How-
tive immunity. The immune response of lymphocytes is triggered ever, very large amounts can result in T- and B-cell tolerance,
by materials called immunogens, which are macromolecules ca- a phenomenon that is not well understood.
pable of triggering an adaptive immune response by inducing There are many ways that we come in contact with im-
the formation of antibodies or sensitized T cells in an immuno- munogens in nature. How we are exposed to them and where
competent host. Immunogens can then specifically react with they get into our bodies determines the actual amount of
such antibodies or sensitized T cells. The term antigen refers to immunogen needed to generate an immune response. Such
a substance that reacts with an antibody or sensitized T cells but routes include intravenous (into a vein), intradermal (into the
may not be able to evoke an immune response in the first place. skin), subcutaneous (beneath the skin), and oral contact. The
Thus, all immunogens are antigens, but the converse is not true. route where the immunogen enters the body also determines
However, many times the terms are used synonymously and the which cell populations will be involved in the response.1 For
distinction between them is not made. In discussing serological example, if an immunogen enters the body via an intravenous
reactions or particular names of substances such as blood route, as might occur with a deep puncture wound, the im-
groups, the term antigen is still more commonly used; hence, munogen goes directly to the spleen, where a response is
both terms are used in this chapter. mounted. On the other hand, if an immunogen enters subcu-
One of the most exciting areas of immunological research taneously, such as through a cut or scratch, local lymph nodes
focuses on how and why we respond to particular immuno- are involved.
gens. This response is actually caused by a combination of fac- Finally, a genetic predisposition may be involved that allows
tors: unique biological properties of the individual, the nature individuals to respond to particular immunogens. This predis-
of the immunogen itself, genetic coding of major histocom- position is linked to the MHC and to the receptors generated
patibility complex (MHC) molecules that must combine with during T- and B-lymphocyte development. The MHC is a sys-
an immunogen before T cells are able to respond, and im- tem of genes that code for cell-surface molecules that play an
munogen processing and presentation. This chapter focuses important role in antigen recognition. Further details are found
on all these areas and discusses future clinical implications of in a later section in this chapter.
some recent findings.
Traits of Immunogens
Factors Influencing In general, immunogenicity—the ability of an immunogen
the Immune Response to stimulate a host response—depends on the following char-
acteristics: (1) macromolecular size, (2) foreignness, (3) chem-
Biological properties of the individual that influence the nature ical composition and molecular complexity, and (4) the ability
of the immune response include several factors such as age, to be processed and presented with MHC molecules.1 Usually,
overall health, dose, route of inoculation, and genetic capacity. an immunogen must have a molecular weight of at least
In general, older individuals are more likely to have a decreased 10,000 to be recognized by the immune system and the most
response to antigenic stimulation. At the other end of the age active immunogens typically have a molecular weight of over
scale, neonates do not fully respond to immunogens because 100,000 daltons.1 However, there are exceptions because
their immune systems are not completely developed. Overall a few substances with a molecular weight of lower than
health plays a role because individuals who are malnourished, 1,000 have been known to induce an immune response. For
fatigued, or stressed are less likely to mount a successful im- the most part, the rule of thumb is that the greater the molecular
mune response. weight, the more potent the molecule is as an immunogen.
A significant quantity of an immunogen must be present in Another characteristic that all immunogens share is foreign-
order for an adaptive immune response to take place. The ness. The immune system is normally able to distinguish
between self and nonself; those substances recognized as non- Levels of
self are immunogenic. This ability is acquired as lymphocytes Protein Organization
mature in the primary lymphoid organs. Any lymphocyte Amino acids
capable of reacting with self-antigen is normally eliminated.
Typically, the more distant taxonomically the source of the im- Primary
protein structure
munogen is from the host, the more successful it is as a stim-
ulus. For example, plant protein is a better immunogen for an
animal than is material from a related animal. Occasionally,
however, autoantibodies, or antibodies to self-antigens, exist.
This is the exception rather than the rule; this phenomenon is
discussed in Chapter 15.
Immunogenicity is also determined by a substance’s chemi-
cal composition and molecular complexity. Proteins and poly- Secondary
Alpha helix
protein structure
saccharides are the most effective immunogens. Proteins are
powerful immunogens because they are made up of a variety
of units known as amino acids. The particular sequential arrange-
Pleated sheet
ment of amino acids, the primary structure, determines the sec-
ondary structure, which is the relative orientation of amino
acids within the chain. Chains are usually arranged in an alpha
helix or a beta pleated sheet. The tertiary structure embodies Pleated sheet
the spatial or three-dimensional orientation of the entire mole-
cule and is based on folding of particular regions of the mole-
cule; the quaternary structure is based on the association of two Tertiary Alpha helix
or more chains into a single polymeric unit (Fig. 2–1). Because protein structure
of the variations in subunits, proteins may have an enormous
variety of three-dimensional shapes. In contrast, synthetic poly-
mers such as nylon or Teflon are made up of a few simple
repeating units with no bending or folding within the molecule,
which means these materials are nonimmunogenic. For this
reason, they are used in making artificial heart valves, elbow
replacements, and other medical appliances.
Carbohydrates are less immunogenic than protein because
they are smaller than proteins and have a limited number of Quaternary
sugars available to create their structures. As immunogens, protein structure
carbohydrates most often occur in the form of glycolipids or
glycoproteins. Many of the blood group antigens are composed
of such carbohydrate complexes. For example, the A, B, and
H blood group antigens are glycolipids and the Rh and Lewis Levels of protein organization.
antigens are glycoproteins.2,3 Other carbohydrates that are im-
portant immunogens are the capsular polysaccharides of bac-
teria such as Streptococcus pneumoniae. Pure nucleic acids and Epitopes
lipids are not immunogenic by themselves, although a re-
sponse can be generated when they are attached to a suitable Although an immunogen must have a molecular weight of
carrier molecule.3 This is the case for autoantibodies to DNA at least 10,000, only a small part of the immunogen is actu-
that are formed in systemic lupus erythematosus (SLE). These ally recognized in the immune response. This key portion of
autoantibodies are actually stimulated by a DNA-protein com- the immunogen is known as the determinant site or epitope.
plex rather than by DNA itself. Epitopes are molecular shapes or configurations that are rec-
Finally, for a substance to elicit an immune response it must ognized by B or T cells. There is evidence that for proteins,
be subject to antigen processing, which involves enzymatic di- epitopes recognized by B cells may consist of as few as 6 to
gestion to create small peptides or pieces that can be complexed 15 amino acids.4 Large molecules may have numerous epi-
to MHC molecules to present to responsive lymphocytes. If a topes and each one may be capable of triggering specific an-
macromolecule cannot be degraded and presented with MHC tibody production or a T-cell response. Epitopes may be
molecules, then it would be a poor immunogen. The particular repeating copies or they may have differing specificities.
MHC molecules produced also determine responsiveness to in- They may also be sequential or linear epitopes (i.e., amino
dividual antigens. Each individual inherits the ability to produce acids following one another on a single chain) or they may
a certain limited repertoire of MHC molecules, discussed in the be conformational. A conformational epitope results from
section on the genes coding for MHC molecules. the folding of one chain or multiple chains, bringing certain
 Connections
The Florida Panther
Polymorphism of the MHC genes in a species is thought to serve
as a protection against infectious diseases because diverse genes
allow for a response to a wide variety of antigens. The fate of the
Florida panther is a good example of what happens when there
is a lack of genetic diversity in a particular population 1 1
In the early 1990s, only about 20 to 25 adult panthers remained
in Florida because of a combination of factors, including de-
struction of their habitat and inbreeding. The latter severely
decreased the genetic pool of the population. It has been pos-
tulated that the panthers became increasingly susceptible to
viral diseases because of the limited polymorphism of the MHC 2 2
antigens. Conservationists decided to increase the strength of
the gene pool by moving eight females from the Texas popula-
tion to Florida.
Now, a decade and a half after introducing new females into
the population, the Florida panthers have exhibited a marked
improvement in health and fitness. The increased diversity of the
3
MHC genes allowed for a response to diverse pathogens. The
Florida panthers are able to withstand disease better because of
the introduction of these new genes into the population.
A B
Linear epitopes Conformational epitopes
Linear versus conformational epitopes. (A) Linear
epitopes consist of sequential amino acids on a single polypep-
tide chain. There may be several different types on one chain.
(B) Conformational epitopes result from the folding of a polypep-
tide chain or chains, and nonsequential amino acids are brought
into close proximity.

linear and conformational epitopes present on the surface of an

immunogen. Anything that is capable of cross-linking surface im-
munoglobulin molecules is able to trigger B-cell activation. The
immunogen does not necessarily have to be degraded first. How-
ever, for T cells to be able to recognize an immunogen it must
first be degraded into small peptides by an antigen-presenting
cell (APC). Then the peptides form a complex with MHC proteins
and are carried to the surface of the APC. (See Role of Class I and
II Molecules in the Immune Response later in this chapter.)

Haptens
Some substances are too small to be recognized, but if they are
combined with larger molecules they are then able to stimulate
a response. Haptens are nonimmunogenic materials that,
when combined with a carrier, create new antigenic determi-
nants. Thus, by themselves haptens are antigens but not im-
munogens. Once antibody production is initiated, the hapten
The Florida panther. (John Hollingsworth/U.S. Fish
is capable of reaction with antibody even when the hapten is
and Wildlife Service.)
not complexed to a carrier molecule. However, precipitation
or agglutination reactions will not occur because a hapten has
amino acids from different segments of a linear sequence or a single determinant site and cannot form the cross-links with
sequences into close proximity with each other so they can more than one antibody molecule that are necessary for pre-
be recognized together (Fig. 2–3). cipitation or agglutination (Fig. 2–4).
Epitopes recognized by B cells may differ from those recog- Haptens may be artificially joined to carrier molecules in a
nized by T cells.1 Surface antibody on B cells may react with both laboratory setting or this may occur naturally within a host and
 Immunogenicity Antigenicity Another example of haptens coupling with normal proteins in the
B cell
body to provoke an immune response occurs with certain drug-
receptor protein conjugates. The best known example of this occurs with
penicillin, which can result in a life-threatening allergic response.
Karl Landsteiner, an Austrian scientist perhaps best known
Hapten
for his discovery of the ABO blood groups, conducted the most
famous study of haptens. In his book The Specificity of Serolog-
B cell surface ical Reactions, published in 1917, he detailed the results of an
A C exhaustive study of haptens that has contributed greatly to our
knowledge of antigen–antibody reactions. Landsteiner immu-
nized rabbits with haptens attached to a carrier molecule and
then tested the serum to measure how the antibodies produced
Carrier reacted with different haptens. He discovered that antibodies
not only recognize chemical features such as polarity, hydropho-
bicity, and ionic charge, but the overall three-dimensional con-
figuration is also important.1 The spatial orientation and the
chemical complementarity are responsible for the lock-and-
key relationship that allows for tight binding between antibody
and epitope (Fig. 2–5). Today, it is known that many thera-
B D peutic drugs and hormones can function as haptens.2
Characteristics of haptens. (A) Haptens bind B-cell
receptors, but the receptors remain independent: no activation (not
immunogenic). (B) Hapten/carrier complex cross-links receptors: Adjuvants
B cells are activated and begin producing antibodies (immunogenic).
(C) Although haptens can bind to the antibody binding sites (anti- The power of immunogens to generate an immune response
genic), all of the antibody-hapten complexes remain independent: can be increased through the use of adjuvants. An adjuvant
The reaction cannot be visualized. (D) When bound to carriers, the is a substance administered with an immunogen that in-
haptens contribute to the formation of an interconnected lattice, creases the immune response in order to provide immunity
which is the basis for the precipitation and agglutination reactions.
to a particular disease. Addition of an adjuvant to a substance
used for an immunization helps to make the immunization
set off an immune response. An example of the latter is an allergic more effective. Adjuvants actually work by targeting APCs,
reaction to poison ivy. Poison ivy (Rhus radicans) contains which are key to the adaptive immune response.5 Substances
chemical substances called catechols, which are haptens. Once used as adjuvants protect immunogens from degradation and
in contact with the skin, these can couple with tissue proteins allow a longer response time that attracts a large number of
to form the immunogens that give rise to contact dermatitis. immune system cells to the injection site, which helps to

Reactivity with

NH2 NH2 NH2 NH2

COOH

COOH

COOH

Antiserum against Aminobenzene (aniline) o -Aminobenzoic acid m -Aminobenzoic acid p -Aminobenzoic acid

Aminobenzene +++ 0 0 0
o -Aminobenzoic acid 0 +++ 0 0
m -Aminobenzoic acid 0 0 ++++ 0
p -Aminobenzoic acid 0 0 0 + + + /+ + + +

Landsteiner’s study of the specificity of haptens. Spatial orientation of small groups is recognized, because antibodies made
against aminobenzene coupled with a carrier will not react with other similar haptens. The same is true for antiserum to o-aminobenzoic acid,
m-aminobenzoic acid, and p-aminobenzoic acid. Antibody to a carboxyl group in one location would not react with a hapten, which has the
carboxyl group in a different location. (From Landsteiner K. The Specificity of Serological Reactions. Revised Edition. New York, NY: Dover Press; 1962.)
boost the strength of the response.5,6 Aluminum salts are the indicates that the genetic capability to mount an immune re-
only ones currently approved for clinical use in the United sponse is linked to a group of molecules originally referred to
States and are used to complex with the immunogen to in- as human leukocyte antigens (HLA). The French scientist
crease its size and to prevent a rapid escape from the tissues.6 Dausset gave them this name because they were first defined
It must be injected into the muscle to work. The hepatitis B by discovering an antibody response to circulating white blood
vaccination is an example of using this type of adjuvant. cells (WBCs).7 These molecules are now known as MHC mol-
Adjuvants are used to accelerate the immune response and ecules because they determine whether transplanted tissue is
increase the duration of protection, thus reducing the need for histocompatible and thus accepted or recognized as foreign
booster immunizations.6 In addition to successfully enhancing and rejected. MHC molecules are actually found on all nucle-
the immune response, they must meet the criterion of causing ated cells in the body and they play a pivotal role in the devel-
minimal toxicity to humans, which is why more substances are opment of both humoral and cellular immunity. Although
not approved for use at this time. MHC molecules themselves can function as antigens when
transplanted from one individual to another, their main func-
tion is to bring antigen in the body to the surface of cells for
Relationship of Antigens to the Host recognition by T cells. T-cell activation will occur only when
antigen is combined with MHC molecules on the surface of
Antigens can be placed in broad categories according to their other cells. The genes that encode these cell-surface molecules
relationship to the host. Autoantigens are those antigens that are the system of genes known as the MHC.
belong to the host. These do not evoke an immune response
under normal circumstances. However, if an immune response
does occur to autoantigens, it may result in an autoimmune dis- Genes Coding for MHC Molecules
ease. Refer to Chapter 15 for a discussion of some of the most (HLA Antigens)
well-known autoimmune diseases. Alloantigens are from other
members of the host’s species and are capable of eliciting an im- The MHC system is the most polymorphic system found in hu-
mune response. They are important to consider in tissue trans- mans.7,8 MHC genes code for proteins that play a pivotal role
plantation and in blood transfusions. Heteroantigens are from in immune recognition and it is thought that this polymor-
other species, such as other animals, plants, or microorganisms. phism is essential to our survival because it allows for an im-
Heterophile antigens are heteroantigens that exist in unre- mune response to diverse immunogens.7 Genes coding for the
lated plants or animals but are either identical or closely related MHC molecules in humans are found on the short arm of chro-
in structure so that antibody to one will cross-react with antigen mosome 6 and are divided into three categories or classes.
of the other. An example of this is the human blood group A and Class I genes are found at three different locations or loci,
B antigens, which are related to bacterial polysaccharides.3 It is termed A, B, and C. Class II genes are situated in the D region,
believed that anti-A antibody, which is normally found in individ- and there are several different loci, known as DR, DQ, and DP.
uals with blood types other than A (e.g., type B and type O), is For class I molecules, there is only one gene coding for each
originally formed after exposure to pneumococci or other similar particular molecule. Class II molecules, in contrast, have one
bacteria. Naturally occurring anti-B antibody is formed after gene that codes for the α chain and one or more genes that
exposure to a similar bacterial cell wall product. The presence of code for the β chain. The area of class III genes lies between
naturally occurring antibodies is an important consideration in the class I and class II regions on chromosome 6. Class III
selecting the correct blood type for transfusion purposes. genes code for the C4A, C4B, C2, and B complement proteins,
Normally in serological reactions, the ideal is to use a reaction as well as cytokines such as tumor necrosis factor (TNF)
that is completely specific, but the fact that cross-reactivity (Fig. 2–6). The class I and II molecules are involved in antigen
exists can be helpful for certain diagnostic purposes. Indeed, the recognition; in this role, they influence the repertoire of anti-
first test for infectious mononucleosis (IM) was based on a gens to which T cells can respond. In contrast, class III mole-
heterophile antibody reaction. During the early states of IM, a cules are secreted proteins that have an immune function, but
heterophile antibody is formed, stimulated by an unknown anti- they are not expressed on cell surfaces, as are class I and II.
gen. This antibody was found to react with sheep red blood cells Class III molecules also have a completely different structure
(SRBCs), which formed the basis of the Paul-Bunnell screening compared with the other two classes.
test for mononucleosis (see Chapter 23). This procedure was a At each of these loci, or locations, there is the possibility
useful screening test when the causative agent of IM had not yet of multiple alleles. Alleles are alternate forms of a gene that
been identified. Current rapid screening tests for IM are based code for slightly different varieties of the same product. The
on the principle of detection of heterophile antibody. MHC system is described as polymorphic because there are
so many possible alleles at each location. There are more
than 2,013 different alleles of HLA-A, over 2,605 alleles of
Major Histocompatibility Complex HLA-B, and 1,551 alleles of HLA-C that have been identified
at this time.1,8
For years, scientists searched to identify possible immune re- The probability that any two individuals will express the
sponse genes that would account for differences in how indi- same MHC molecules is very low. One individual inherits
viduals respond to particular immunogens. Evidence now two copies of chromosome 6; thus, there is a possibility of
 Chromosome 6
Tel Long arm Cen Short arm Tel

Class II Class III Class I

C4B
C4A
C2
2
1

1
2
1

3
1

C
B

A
Centromere Telomere

DP DQ DR
region region region
The major histocompatibility complex. Location of the class I, II, and III genes on chromosome 6. Class I consists of loci A, B, and
C, whereas class II has at least three loci: DR, DQ, and DP.

two different alleles for each gene on the chromosome unless structural similarities and are found on the same types of cells
that person is homozygous (has the same alleles) at a given (Fig. 2–7). Whereas class I MHC (HLA) molecules are ex-
location. These genes are described as codominant, meaning pressed on all nucleated cells, class II MHC (HLA) molecules
that all alleles that an individual inherits code for products are found primarily on APCs. Their differing structures, as
that are expressed on cells. Because the MHC genes are explained in the text that follows, are tailored to their specific
closely linked, they are inherited together as a package called functions in the adaptive immune response.
a haplotype. Thus, each inherited chromosomal region con-
sists of a package of genes for A, B, C, DR, DP, and DQ. The
full genotype would consist of two of each gene at a particular Although class I MHC molecules are expressed on all nucle-
locus (see Chapter 16). ated cells, they differ in the level of expression. They are
One haplotype is inherited from each parent. Because there highest on lymphocytes and myeloid cells and low or unde-
are numerous alleles or variant forms at each locus, an indi- tected on liver hepatocytes, neural cells, muscle cells, and
vidual’s MHC type is about as unique as a fingerprint. Because sperm.4,7 This may explain why HLA matching is not done
of the tremendous diversity of alleles, more than 1.7 billion in the case of liver transplants. Additionally, HLA-C antigens
different class I haplotypes alone are possible in the human are expressed at a much lower level than HLA-A and HLA-B
population.1 The frequency of particular HLA alleles differs sig- antigens, so the latter two are the most important to match
nificantly among different ethnic populations and can some- for transplantation.7
times be used to trace one’s ancestry or ethnic origins.7 Each class I antigen is a glycoprotein dimer made up of two
Traditionally, HLA nomenclature had been defined sero- noncovalently linked polypeptide chains. The α chain has a
logically through the use of a battery of antibodies. Advances molecular weight of 44,000.4,7,8 A lighter chain associated
in DNA analysis have made identification of actual genes pos- with it, called a β2–microglobulin, has a molecular weight of
sible. The nomenclature has become correspondingly more 12,000 and is encoded by a single gene on chromosome 15
complex. For instance, the notation HLA DRB1*1301 indi- that is not polymorphic.8 This means that every class I mole-
cates the actual gene involved in coding for the β chain of an cule contains the same β2–microglobulin. The α chain is
HLA DR1 antigen is the number 13 and the 01 is a specific folded into three domains—α1, α2, and α3—and it is in-
subtype. The uniqueness of the HLA antigens creates a major serted into the cell membrane via a transmembrane segment
problem in matching organ donors to recipients because that is hydrophobic.4,7 The three external domains consist of
these antigens are highly immunogenic. However, in cases of about 90 amino acids each; the transmembrane domain has
disputed paternity, polymorphisms can be used as a helpful about 25 hydrophobic amino acids along with a short stretch
identification tool. of about 5 hydrophilic amino acids, as well as an anchor of
30 amino acids.1
Structure of Class I and II β2–microglobulin does not penetrate the cell membrane,
but it is essential for proper folding of the α chain. X-ray
MHC Molecules crystallographic studies indicate that the α1 and α2 domains
Each of the MHC genes codes for a protein molecule that ap- each form an alpha helix and that these serve as the walls of
pears on cell surfaces. All the proteins of a particular class share a deep groove at the top of the molecule that functions as
 Class I molecule Class II molecule

Peptide-binding Peptide-binding
cleft cleft
Membrane-distal
domains (many 1 Membrane-distal
polymorphisms) domains 1 S S 1
2

2-microglobulin
(coded by a gene CD4 binds here
CD8 binds here S in chromosome 15) Membrane-proximal
S
domains
S

S S S
Membrane-proximal 2
S

domains 3 S 2
(constant regions)

Transmembrane Transmembrane
segment segment

Cytoplasmic Cytoplasmic
A B
tail tail
Structure of (A) class I and (B) class II MHC products. Class I MHC molecules consist of an α chain with three domains and a
shorter chain, β2–microglobulin, common to all class I molecules. Class II MHC molecules consist of an α chain and a β chain, each of which
has two domains.

the peptide-binding site in antigen recognition.4,7 This bind- called heterodimers because they contain two different chains.
ing site is able to hold peptides that are between 8 and DR is expressed at the highest level because it accounts for
11 amino acids long. Most of the polymorphism resides in about one-half of all the class II molecules on a particular
the α1 and α2 regions, whereas the α3 and β2 regions cell.7 The DRβ gene is the most highly polymorphic; close to
are relatively constant.4,7 The α3 region reacts with CD8 on 2000 different alleles are known at this time.9 DP molecules
cytotoxic T cells. Binding of peptides to class I and class II are found in the shortest supply.7
molecules is not as specific as the binding of peptides to Both the α chain, with a molecular weight of 34,000, and
T-cell receptors (TCRs) because numerous different peptides the β chain, with a molecular weight of 29,000, are anchored
can bind to a particular MHC molecule.1 to the cell membrane.4,8 Each chain has two domains and
Another group of molecules called the nonclassical class I the α1 and β1 domains come together to form the peptide-
antigens are designated E, F, and G. This group of molecules, binding site, similar to the one found on class I molecules7
except for G, are not expressed on cell surfaces and do not (see Fig. 2–7). However, both ends of the peptide-binding
function in antigen recognition but may play other roles in cleft are open and thus allow class II molecules to capture
the immune response. G antigens are expressed on fetal tro- longer peptides than class I molecules. The α1 domains and
phoblast cells during the first trimester of pregnancy, where the β2 domains are highly conserved in a similar manner to
they come in direct contact with maternal tissue. There the the class I molecules. At least three other class II genes have
G antigens are thought to help ensure tolerance for the fetus been described—DM, DN, and DO, the so-called nonclassical
by protecting placental tissue from the action of NK cells. The class II genes. Products of these genes play a regulatory role
G antigens bind to NK inhibitory receptors and turn off the in antigen processing.7
NK cytotoxic response.6 (See Chapter 3 for the action of DM helps to load peptides onto class II molecules, whereas
NK cells.) E molecules play a similar role. The function of the DO modulates antigen binding. The function of DN is not
F antigens is unknown at this time.8 known at this time.10

Class II MHC molecules are found on the APCs that include Role of Class I and II Molecules
B lymphocytes, monocytes, macrophages, dendritic cells, and
thymic epithelium4,7 Because dendritic cells are the most
in the Immune Response
effective APCs, they have the highest levels of class II molecules The main role of the class I and II MHC molecules is in antigen
on their surface.1 presentation, the process by which degraded peptides within
The major class II molecules—DP, DQ, and DR—consist of cells are transported to the plasma membrane where T cells
two noncovalently bound polypeptide chains that are encoded can then recognize them. T cells can only “see” and respond
by separate genes in the MHC complex. These molecules are to antigens when they are combined with MHC molecules.
 Whereas one individual can express only a small number newly made cellular proteins that fail to fold correctly and
of MHC molecules, each molecule can present a large number hence are defective.
of different antigenic peptides to T cells.1 This allows the Digestion of these intracellular proteins is carried out by
body to respond to many different antigens, which is impor- proteases that reside in large cytoplasmic complexes called
tant for survival of the species. It is thought that the two main proteasomes.1,11 Proteasomes are packets of enzymes formed
classes of these molecules have evolved to deal with two types into a cylindrical shape through which peptides pass and are
of infectious agents: those that attack cells from the outside cleaved. Peptides must be unfolded before entering the cylin-
(such as bacteria) and those that attack from the inside drical chamber of the proteasome; they are then cleaved into
(viruses and other intracellular pathogens). Class I molecules the proper size for delivery to class I molecules. Once cleaved,
mainly present peptides synthesized within the cell to CD8 the peptides must then be pumped from the cytoplasm to the
(cytotoxic) T cells, whereas class II molecules present exoge- lumen of the endoplasmic reticulum by specialized trans-
nous antigen to CD4 (helper) T cells. Exogenous proteins porter proteins.13 Two proteins, transporters associated
presented by class II molecules are those taken into the with antigen processing (TAP1 and TAP2), are responsible
cell from the outside and degraded.11,12 Class I molecules are for the adenosine triphosphate-dependent transport of pep-
thus the watchdogs of viral, tumor, and certain parasitic tides suitable for binding to class I molecules.1,14,16,17
antigens that are synthesized within the cell, whereas class II TAP1 and TAP2 are most efficient at transporting peptides
molecules help to mount an immune response to bacterial that are between 8 to 16 amino acids in size.13,17 Tapasin
infections or other pathogens found outside cells.11,13 In brings the TAP transporters into close proximity to the newly
either case, for a T-cell response to be triggered, peptides formed MHC molecules and mediates interaction with them
must be available in adequate supply for MHC molecules to so that peptides can be loaded onto the class I molecules.11,13
bind, they must be able to be bound effectively, and they Once the α chain has bound the peptide, the class I MHC
must be recognized by a TCR.2 Some viruses, such as herpes peptide complex is rapidly transported to the cell surface8
simplex and adenovirus, have managed to block the immune (see Fig. 2–8).
response by interfering with one or more processes involved Of the thousands of peptides that may be processed in this
in antigen presentation.2 These viruses are able to maintain a manner, only a small fraction of them (1% or less) actually in-
lifelong presence in the host. duce a T-cell response.13 Binding is based on interaction of only
The difference in functioning of the two molecules is tied two or three amino acid residues with the class I binding
to the mechanisms by which processed antigen is transported groove. Different class I molecules will have slightly different
to the surface. Both types of molecules, however, must be binding affinities and these small differences determine to
capable of presenting an enormous array of different antigenic which particular antigens one individual will respond.
peptides to T cells. The chemistry of the MHC antigens con- It is estimated that a single cell may express between
trols what sorts of peptides fit in the binding pockets. These 100,000 and 200,000 copies of each class I molecule, so many
two pathways are discussed here. different peptides can be captured and expressed in this man-
ner.8 As few as 10 to 100 identical antigen class I MHC com-
plexes can induce a cytotoxic response.13 In healthy cells, most
Class I molecules are synthesized in the rough endoplasmic
of these class I MHC complexes contain self-peptides that are
reticulum and for a time they remain anchored in the endo-
ignored by the T cells, whereas in diseased cells peptides are
plasmic reticulum membrane. It is here that these molecules
derived from viral proteins or proteins associated with cancer-
bind peptides. This is known as the endogenous pathway of
ous states. Display of hundreds of class I molecules complexed
antigen presentation because antigens that bind to class I pro-
to antigen allows CD8+ T cells to continuously check cell sur-
teins are actually synthesized in the same cell as the class I mol-
faces for the presence of nonself antigen. If it recognizes an
ecules.14,15 In fact, binding of the newly synthesized proteins
antigen as being foreign, the CD8+ T cell produces cytokines
helps to stabilize the association of the α chain of class I with
that cause lysis of the entire cell (Fig. 2–9).
the β2–microglobulin.10 However, before binding with antigen
occurs, newly synthesized α chains freely bind a molecule
called calnexin. This 88-kd molecule is membrane-bound in Class II MHC molecules participate in the exogenous pathway
the endoplasmic reticulum and keeps the α chain in a partially of antigen presentation. This means that antigen is taken into
folded state while it awaits binding to β2–microglobulin.11,14 the cell from the outside by means of either phagocytosis or
Another molecule, ERp57, binds to this complex also. When endocytosis, processes by which cells ingest extracellular mol-
β2–microglobulin binds, calnexin and ERp57 are released and ecules by enclosing them in a small portion of the plasma
two other chaperone molecules—calreticulin and tapasin— membrane. Dendritic cells, the most potent activators of
associate with the complex and help to stabilize it for peptide T cells, are excellent at capturing and digesting exogenous anti-
binding14,16 (Fig. 2–8). gens such as bacteria. Hydrolytic enzymes within the endo-
Peptides that bind with the class I molecules are approxi- somes digest antigen into peptides of 13 to 18 amino acids in
mately 8 to 11 amino acids in length and are derived from par- length.1
tial digestion of proteins synthesized in the cytoplasm. These Class II molecules are synthesized in the endoplasmic reticu-
intracellular peptides may include viral, tumor, or even bacte- lum and associate with a protein called the invariant chain (Ii).
rial antigens.8 Other peptides that may also bind are from Because the open structure of class II molecules would permit
 1. Endogenous antigen within cytosol is
degraded by proteasome.

2. Peptides transported into endoplasmic

reticulum by TAP.

3. Alpha chain of class I MHC binds

T
2-microglobulin.

4. Alpha chain of class I MHC binds peptide.

CD8
5. Peptide-class I MHC transported to Golgi
complex and then to cell surface.
6
6. Class I MHC peptide binds to CD8! T cell.

Class I MHC 2-microglobulin

Golgi complex

Ribosomes

4
2 Tap Rough endoplasmic
Protein reticulum
3

Antigen-processing
1 Proteosome
pathway for endogenous antigens.

CD8
together in the ER lumen and then moving them out through
the Golgi complex to the endocytic vesicles where digested
antigen is found.15 Unlike class I molecules, class II molecules
T must be transported from the endoplasmic reticulum (ER)
to an endosomal compartment where they can then bind
peptides17 (Fig. 2–10).
Perforins Once transported to an endosomal compartment, class II
granzymes molecules encounter peptides derived from endocytosed, ex-
Apoptosis
ogenous proteins. The invariant chain is gradually degraded
The CD8+ T cell recognizes antigen in association with by a protease, leaving just a small fragment called class II in-
class I MHC. If the antigen is recognized as being foreign, cytokines variant chain peptide (CLIP) attached to the peptide-binding
are released, causing destruction of the target cell.
cleft.1,15 CLIP is then exchanged for exogenous peptides.
Selective binding of peptides is favored by the low pH of
binding of segments of endogenous peptides within the ER, the endosomal compartment.14 HLA-DM molecules help to
Ii serves to protect the binding site.8,15 This chain is a 31-kd mediate the reaction by removing the CLIP fragment and
protein that is made in excess so that enough is available to helping to load peptides into the binding groove.1,15 Gener-
bind with all class II molecules shortly after they are synthe- ally, peptides of approximately 13 to 18 amino acid residues
sized. Ii may be responsible for helping to bring α and β chains can bind because the groove is open on both ends, unlike
 1. Class II MHC binds invariant chain to block
binding of endogenous antigen.
TH
2. MHC complex goes through Golgi complex.

3. Invariant chain is degraded, leaving

CLIP fragment.
CD4
Antigen 4. Exogenous antigen taken in and degraded
7 and routed to intracellular vesicle.

5. CLIP fragment exchanged for antigenic

peptide.

6 6. Class II MHC antigenic peptide is

transported to cell surface.

7. Class II MHC peptide complex binds

4 to CD4! T cell.

Class II MHC Invarient chain

2 Golgi complex

Ribosomes

1 Rough endoplasmic
reticulum

Antigen-processing pathway for exogenous antigen. The binding site of class II MHC molecules is first occupied by an invariant
chain (Ii). This is degraded and exchanged for short exogenous peptides in an endosomal compartment. The exogenous peptide class II MHC
complex is then transported to the cell surface.

class I molecules, which have a closed end.12,18,19 Within a T helper (Th) cell recruits and triggers a B-cell response, re-
central core of the bound peptide, 7 to 10 residues provide sulting in antibody formation (Fig. 2–11).
the major contact points.1
Hydrogen bonding takes place along the length of the cap-
tured peptide; this is in contrast to class I molecules, which
Clinical Significance of MHC
only bond at the amino and carboxy-terminal ends.19,20 There Testing for MHC antigens has typically been carried out be-
are also several pockets in the class II proteins that easily fore tissue transplant procedures because both class I and
accommodate amino acid side chains. This gives class II pro- class II molecules can induce a response that leads to graft
teins more flexibility in the types of peptides that can be rejection. Testing methodology is transitioning from sero-
bound.19,20 Once binding has occurred, the class II protein- logical principles to molecular methods because they are
peptide complex is stabilized and is transported to the cell more accurate. The role of the laboratory in transplantation
surface (see Fig. 2–10). On the cell surface, class II molecules is presented in Chapter 16. MHC antigens also appear to play
are responsible for forming a trimolecular complex that oc- a role in the development of autoimmune diseases. Inheri-
curs between antigen, class II molecule, and an appropriate tance of certain HLA antigens appears to predispose a person
TCR. If binding occurs with a TCR on a CD4+ T cell, the to certain autoimmune diseases. The closest link known is
 between inheritance of HLA B27 and the disease called anky-
APC losing spondylitis, a progressive chronic inflammatory dis-
order affecting the vertebrae of the spine.21 See Table 2–1
for other links between HLA antigens and diseases. This
MHCII topic is discussed more fully in Chapter 15.
CD4
However, the evidence that both class I and class II mol-
ecules play a major role in antigen presentation has more
Th
far-reaching consequences. They essentially determine the
types of peptides to which an individual can mount an im-
mune response. Although the MHC molecules typically
have a broad binding capacity, small biochemical differences
in these proteins are responsible for differences seen in the
Cytokine
secretion
ability to react to a specific antigen. It is possible that non-
responders to a particular vaccine such as hepatitis B do not
have the genetic capacity to respond. On the other hand,
the presence of a particular MHC protein may confer addi-
Proliferation B tional protection, as the example of HLA B8 and increased
resistance to HIV infection shows.1 Therefore, it will be im-
portant to know an individual’s MHC type for numerous
reasons.
Proliferation
Much of the recent research has focused on the types
of peptides that can be bound by particular MHC mole-
cules.19,22,23 Future developments may include tailoring vac-
cines to certain groups of such molecules. As more is learned
about antigen processing, researchers can specifically develop
vaccines containing certain amino acid sequences that serve as
immunodominant epitopes. These vaccines may avoid the risk
associated with using live organisms. Additionally, if an indi-
vidual suffers from allergies, knowing a person’s MHC type
may also help predict the types of allergens to which he or she
may respond.24 Certain drug hypersensitivities have also been
CD4+ T cells recognize exogenous antigen on linked to particular HLA alleles and knowledge of one’s HLA
phagocytic antigen-presenting cells along with class II MHC. genes might prevent severe reactions to these drugs.25 Another
CD4+ helper T cells are stimulated by contact with antigen and area for future research is the development of possible tumor
clonal expansion takes place. These cells secrete cytokines that vaccines based on an individual’s MHC type.17 It is likely that
cause an antigen-activated B cell to proliferate and produce plasma knowledge of the MHC molecules will affect many areas of
cells, which make antibody. patient care in the future.

Table 2–1 Association of HLA Alleles and Disease

STRENGTH OF
DISEASE SYMPTOMS HLA ALLELE ASSOCIATION
Ankylosing spondylitis Inflammation of the vertebrae of the spine B27 +++
Celiac disease Diarrhea, weight loss, intolerance to gluten DQ2 +++
DQ8 +
Rheumatoid arthritis Inflammation of multiple joints DR4 +
Type 1 diabetes Increase in blood glucose because of DQ8 ++
destruction of insulin-producing cells DQ2 +
+++ = very strong association, ++ = strong association, + = clear association, – = negative association. (From Margulies DH, Natarajan K, Rossjohn J, and
McCluskey J. Major histocompatibility complex (MHC) molecules: structure, function, and genetics. In: Paul WE ed. Fundamental Immunology. 6th ed.
Philadelphia, PA: Wolters Kluwer, Lippincott Williams & Wilkins; 2012; Ch 21:487–523.)
SUMMARY • Heterophile antigens exist in unrelated species, but
their structure is so similar that antibody formed to
• Immunogens are macromolecules that elicit formation of one will cross-react with antigen from a different
immunoglobulins or sensitized cells in an immunocom- species.
petent host. • The major histocompatibility complex (MHC) encodes
• The term antigen is sometimes used to denote a substance class I and class II molecules, which play a major role in
that does not elicit a host response but reacts with anti- antigen presentation to T cells.
body once it has been formed. • The MHC has so many different alleles that it is consid-
• Immunogenicity is influenced by factors such as age, ered the most polymorphic system found in humans.
health, route of inoculation, and genetic capacity. • Class I and class II molecules bind peptides within cells
• Most immunogens have a molecular weight of at least and transport them to the plasma membrane, where the
100,000, molecular complexity, and are foreign to the peptides can be recognized by T cells.
host. Although immunogens are fairly large molecules, the • Class I MHC molecules are found on all nucleated cells;
immune response is keyed to only small portions of these these molecules associate with foreign antigens, such as
molecules, or epitopes, and very small differences in these viral proteins, synthesized within a host cell. This is known
epitopes can be detected by the immune system. as the endogenous pathway for antigen presentation.
• Haptens are nonimmunogenic substances that must be com- • Class II molecules are only found on B cells, monocytes,
bined with a carrier in order to provoke an immune response. macrophages, dendritic cells, and thymic epithelium.
They are too small to initiate a response by themselves. These molecules associate with foreign antigens taken into
• Once an antibody response is generated, haptens are ca- the cell from the outside, in the pathway known as exoge-
pable of reacting with that antibody, but precipitation or nous antigen presentation.
agglutination will not occur. • Class I MHC molecules consist of an α chain encoded by
• Adjuvants are substances that can be mixed with antigen to the MHC complex, as well as a second lighter chain called
enhance the immune response. Most adjuvants work by β2–microglobulin, encoded by a gene on chromosome 15.
keeping the antigen in the area and by increasing the num- • Class II MHC molecules have an α and a β chain, both of
ber of cells involved in the immune response. which are encoded by genes in the MHC complex.
• Antigens can be characterized by their relationship to the • Class I molecules present antigen to CD8+ T cells, trigger-
host. Autoantigens are those that belong to the host; al- ing a cytotoxic reaction.
loantigens are from the same species as the host but are not • Class II molecules present antigen to CD4+ T cells, which
identical to the host; heteroantigens are from other species. are helper cells involved in antibody production.

Study Guide: A Comparison of Class I and Class II MHC Molecules

CLASS I MHC MOLECULES CLASS II MHC MOLECULES
Cellular All nucleated cells B cells, monocytes, macrophages, dendritic cells,
Distribution thymic epithelial cells
Structure One α chain and β2–microglobulin An α chain and a β chain
Classes A, B, C DP, DQ, DR
Size of Peptides 8 to 11 amino acids 13 to 18 amino acids
Bound
Nature of Peptide Closed at both ends Open at both ends
Binding Cleft
Interaction with Presents endogenous antigen to CD8+ T cells Presents exogenous antigen to CD4+ T cells
T Cells
CASE STUDY
A 15-year-old boy needs to have a kidney transplant be- Questions
cause of the effects of severe diabetes. His family members a. How many alleles are shared by mother and son?
consist of his father, mother, and two sisters. All of them Father and son?
are willing to donate a kidney so that he can come off dial- b. What are the chances that one of the sisters would
ysis. He is also on a list for a cadaver kidney. His physician be an exact match?
suggests that the family be tested first for the best HLA c. Is there a possibility that a cadaver kidney might be
match. a better match than any of the family members’?

REVIEW QUESTIONS
1. All of the following are characteristics of an effective 7. A heterophile antigen is one that
immunogen except a. is a self-antigen.
a. internal complexity. b. exists in unrelated plants or animals.
b. large molecular weight. c. has been used previously to stimulate antibody
c. the presence of numerous epitopes. response.
d. found on host cells. d. is from the same species but is different from
the host.
2. Which of the following best describes a hapten?
a. Cannot react with antibody 8. Which of the following is true of class II MHC (HLA)
b. Antigenic only when coupled to a carrier antigens?
c. Has multiple determinant sites a. They are found on B cells and macrophages.
d. A large chemically complex molecule b. They are found on all nucleated cells.
c. They all originate at one locus.
3. Which would be the most effective immunogen? d. They are coded for on chromosome 9.
a. Protein with a molecular weight of 200,000
b. Nylon polymer with a molecular weight of 250,000 9. Class II MHC molecules are recognized by which of
c. Polysaccharide with a molecular weight of 220,000 the following?
d. Protein with a molecular weight of 175,000 a. CD4+ T cells
b. CD8+ T cells
4. Which of the following individuals would likely c. Natural killer cells
respond most strongly to a bacterial infection? d. Neutrophils
a. An adult who is 75 years of age
b. A malnourished 40-year-old 10. Which of the following best describes the role of TAP?
c. A weightlifter who is 35 years old a. They bind to class II molecules to help block the
d. A newborn baby antigen-binding site.
b. They bind to class I proteins in proteasomes.
5. Which best describes an epitope? c. They transport peptides into the lumen of the
a. A peptide that must be at least 10,000 MW endoplasmic reticulum.
b. An area of an immunogen recognized only by T cells d. They help cleave peptides for transport to
c. A segment of sequential amino acids only endosomes.
d. A key portion of the immunogen
11. What is the purpose of the invariant chain in antigen
6. Adjuvants act by which of the following methods? processing associated with class II MHC molecules?
a. Protects antigen from being degraded a. Helps transport peptides to the binding site
b. Facilitates rapid escape from the tissues b. Blocks binding of endogenous peptides
c. Limits the area of the immune response c. Binds to CD8+ T cells
d. Decreases number of APCs d. Cleaves peptides into the proper size for binding
12. An individual is recovering from a bacterial infection 15. Class I MHC antigens E and G serve which function?
and tests positive for antibodies to a protein normally a. Enhance the response by macrophages
found in the cytoplasm of this bacterium. Which of b. Transport antigen for recognition by CD4+ T cells
the following statements is true of this situation? c. Bind to A, B, and C antigens to protect the
a. Class I molecules have presented bacterial antigen binding site
to CD8+ T cells. d. Protect fetal tissue from destruction by NK cells
b. Class I molecules have presented bacterial antigen
to CD4+ T cells. 16. Which best explains the difference between
c. Class II molecules have presented bacterial antigen immunogens and antigens?
to CD4+ T cells. a. Only antigens are large enough to be recognized
d. B cells have recognized bacterial antigen without by T cells.
help from T cells. b. Only immunogens can react with antibody.
c. Only immunogens can trigger an immune response.
13. In relation to a human, alloantigens would need to be d. Only antigens are recognized as foreign.
considered in which of the following events?
a. Transplantation of a kidney from one individual to 17. When a child inherits one set of six HLA genes
another together from one parent, this is called a(n)
b. Vaccination with the polysaccharide coat of a a. genotype.
bacterial cell b. haplotype.
c. Oral administration of a live but heat-killed virus c. phenotype.
particle d. allotype.
d. Grafting skin from one area of the body to another
18. HLA molecules A, B, and C belong to which MHC
14. Which is characteristic of class I MHC molecules? class?
a. Consists of one α and one β chain a. Class I
b. Binds peptides made within the cell b. Class II
c. Able to bind whole proteins c. Class III
d. Coded for by DR, DP, and DQ genes d. Class IV
 Innate Immunity

After finishing this chapter, you should be able to: EXTERNAL DEFENSE SYSTEM
1. Differentiate between the external and internal defense systems. INTERNAL DEFENSE SYSTEM
2. Give examples of several external defense mechanisms. Pathogen Recognition Receptors
3. Describe how normal flora act as a defense against pathogens. Acute-Phase Reactants
4. Explain what a pathogen-associated molecular pattern (PAMP) is and Inflammation
give some examples. Phagocytosis
5. Discuss the role of pathogen recognition receptors (PRRs) in both the Action of Natural Killer Cells
innate and adaptive immune responses. SUMMARY
6. Describe the function of Toll-like receptors (TLRs). CASE STUDIES
7. Discuss the role of acute-phase reactants in the innate immune REVIEW QUESTIONS
response.
8. Explain how each of the following acute-phase reactants contributes
to innate immunity: C-reactive protein (CRP), serum amyloid A,
complement, alpha1-antitrypsin, haptoglobin, fibrinogen, and
ceruloplasmin.
9. Determine the significance of abnormal levels of acute-phase
reactants.
10. Describe the process of inflammation.
11. List the steps in the process of phagocytosis.
12. Discuss the intracellular mechanism for destruction of foreign
particles during the process of phagocytosis.
13. Explain the importance of phagocytosis in both innate and adaptive
immunity.
14. Explain how natural killer (NK) cells recognize target cells.
15. Describe two methods that NK cells use to kill target cells.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Acute-phase reactants C-reactive protein (CRP) Internal defense system Pathogen recognition
Alpha1-antitrypsin (AAT) Diapedesis Opsonins receptors (PRRs)
Antibody-dependent cell External defense system Oxidative burst Phagocytosis
cytotoxicity (ADCC) Fibrinogen Pathogen-associated Phagolysosome
Ceruloplasmin Haptoglobin molecular patterns Phagosome
Chemotaxis (PAMPs) Serum amyloid A (SAA)
Inflammation
Complement Innate immunity Toll-like receptor (TLR)

Humans are protected by two systems of immunity—innate within minutes and clears invaders as quickly as possible. Inter-
and adaptive—as discussed in Chapter 1. Innate immunity nal defenses include cellular responses that recognize specific
consists of the defenses against infection that are ready for im- molecular components of pathogens. Both of these systems work
mediate action when a host is attacked by a pathogen. If a together to promote phagocytosis. The process of phagocytosis,
pathogen manages to evade these defenses, there is a coordi- as defined in Chapter 1, is the engulfment and destruction of
nated series of interactions between various cells and molecules foreign cells or particulates by leukocytes, macrophages, and
to destroy any invading pathogens before disease can occur.1 other cells. The process of inflammation brings cells and hu-
These defenses are considered nonadaptive or nonspecific; re- moral factors to the injured area. If the healing process is begun
gardless of the infectious agent to which the body is exposed, and resolved as quickly as possible, the tissues are less likely to
innate immunity produces the same response. Components of be damaged. The innate immune system is so efficient that most
innate immunity can be thought of as the first responders be- pathogens are destroyed before they ever encounter cells that
cause they react immediately to infectious agents. Adaptive im- are part of the adaptive immune response.
munity, in contrast, is a more tailored response. It takes a
longer time to be activated, but it is more specific and longer External Defense System
lasting. The two systems, however, are highly interactive and
interdependent; innate immunity actually sets the stage for the The external defense system is composed of physical, chemi-
more specific and longer lasting adaptive immune response. cal, and biological barriers that function together to prevent most
The innate immune system is composed of two parts: the ex- infectious agents from entering the body (Fig. 3–1). First and
ternal defense system and the internal defense system. The ex- foremost are the unbroken skin and the mucosal membrane sur-
ternal defense system consists of anatomical barriers designed faces. The outer layer of the skin, the epidermis, contains several
to keep microorganisms from entering the body. If these defenses layers of tightly packed epithelial cells. These cells are coated
are overcome, then the internal defense system is triggered with a protein called keratin, making the skin impermeable to

Lacrimal glands
Lysozyme
Salivary glands
Cough and
sneeze
reflexes expel
microbes
Airways
Mucus traps microbes.
Cilia expel mucus.

Skin
Keratinocytes (physical barrier)
Sweat glands: lactic acid
Sebaceous glands: fatty acids

Stomach
Gastric acid

The external defense system.

most infectious agents. The outer skin layer is renewed every system is composed of both cells and soluble factors that have
few days to keep it intact.1 The dermis is a thicker layer just un- specific and essential functions. Cells that are capable of phago-
derneath the epidermis that is composed of connective tissue cytosis play a major role (see Chapter 1). Phagocytic cells en-
with blood vessels, hair follicles, sebaceous glands, sweat glands, gulf and destroy most of the foreign cells or particles that enter
and white blood cells (WBCs) including macrophages, dendritic the body; this is the most important function of the internal
cells, and mast cells. To understand how important a role the defense system. Phagocytosis is enhanced by specific receptors
skin plays, one has only to consider how vulnerable victims of on cells that capture invaders through identification of unique
severe burns are to infection. microbial substances. In addition, soluble factors called acute-
Not only does the skin serve as a major structural barrier, phase reactants act by several different methods to either facil-
but the presence of several secretions on it discourages the itate contact between microbes and phagocytic cells or mop
growth of microorganisms. Lactic acid in sweat, for instance, up and recycle important proteins after the process of phago-
and fatty acids from sebaceous glands maintain the skin at a cytosis has taken place. Both cellular receptors and soluble
pH of approximately 5.6. This acidic pH keeps most microor- factors are described in more detail here.
ganisms from growing. In addition, human skin cells produce
psoriasin, a small protein that has antibacterial effects, espe- Pathogen Recognition Receptors
cially against gram-negative organisms such as Escherichia coli. The internal defense system is designed to recognize mole-
Additionally, each of the various organ systems in the body cules that are unique to infectious organisms.3 Macrophages
has its own unique mechanisms. In the respiratory tract, mucous and dendritic cells constituting between 10% and 15% of the
secretions block the adherence of bacteria to epithelial cells. total cellular population in the tissues are the most important
These secretions contain small proteins called surfactants that cells involved in pathogen recognition.2 They are able to dis-
are produced by the epithelial cells and bind to microorganisms tinguish pathogens from normally present molecules in the
to help move pathogens out. The motion of the cilia that line body by means of receptors known as pathogen recognition
the nasopharyngeal passages clears away almost 90% of the de- receptors (PRRs), which are also found on neutrophils,
posited material. The simple acts of coughing and sneezing also eosinophils, monocytes, mast cells, T cells, and epithelial
help to move pathogens out of the respiratory tract. The flushing cells.3-6 These receptors are encoded by the host’s genomic
action of urine, plus its slight acidity, helps to remove many po- DNA and act as sensors for extracellular infection. PRRs thus
tential pathogens from the genitourinary tract. Lactic acid pro- play a pivotal role as a second line of defense if microorgan-
duction in the female genital tract keeps the vagina at a pH of isms penetrate the external barriers.6 Once these receptors
about 5, which is another means of preventing invasion of bind to a pathogen, phagocytic cells become activated and are
pathogens. In the digestive tract, the stomach’s hydrochloric acid better able to engulf and eliminate any microorganisms.2,6 Ac-
keeps the pH as low as 1. We take in many microorganisms with tivated cells proceed to secrete proinflammatory cytokines and
food and drink and the low pH serves to halt microbial growth. chemokines, chemical messengers that make capillaries more
Lysozyme—an enzyme found in many bodily secretions such as permeable and recruit additional phagocytic cell types to the
tears and saliva—attacks the cell walls of microorganisms, espe- area of infection. In addition, cytokines and chemokines also
cially those that are gram-positive. trigger the adaptive immune response.
In many locations of the human body, the presence of nor- PRRs are able to distinguish self from nonself by recognizing
mal flora (also called microbiota) helps to keep pathogens from substances, known as pathogen-associated molecular pat-
establishing themselves in these areas. Normal flora is the mix terns (PAMPs), that are only found in microorganisms. Some
of bacteria that are normally found at specific body sites and examples include peptidoglycan in gram-positive bacteria,
do not typically cause disease. Resident microorganisms in the lipoproteins in gram-negative bacteria, zymosan in yeast, and
gut, for example, may produce colicins, a type of protein that flagellin in bacteria with flagellae.2
binds to the negatively charged surface of certain bacteria and Charles Janeway’s discovery of the first receptor in humans,
kills them by penetrating the membrane.2 The significance of the Toll-like receptor (TLR), had a major impact on the un-
the presence of normal flora is readily demonstrated by looking derstanding of innate immunity.7 Toll is a protein originally
at the side effects of antimicrobial therapy. For example, discovered in the fruit fly Drosophila, where it plays an impor-
women who take an antibiotic for a urinary tract infection tant role in antifungal immunity in the adult fly. Very similar
(UTI) frequently develop a yeast infection because of the pres- molecules were found on human leukocytes and some other
ence of Candida albicans. In this case, antimicrobial therapy cell types. The highest concentration of these TLRs occurs on
wipes out not only the pathogenic bacteria but also the normal monocytes, macrophages, and neutrophils5 (Fig. 3–2).
flora that would ordinarily compete with such opportunists Ten different TLRs have been identified in humans; some
that are usually present in very small numbers. are found on cell surfaces, whereas others are found in the cy-
toplasm.2,5 (Table 3–1). TLR1, TLR2, TLR4, TLR5, and TLR6
Internal Defense System are found on cell surfaces, whereas TLR3, TLR7, TLR8, and
TLR9 are found in the endosomal compartment of a cell.4
If microorganisms do penetrate the barriers of the external de- Each of these receptors recognizes a different microbial prod-
fense system, the innate immune system has additional mech- uct. For example, TLR2 recognizes teichoic acid and peptido-
anisms to destroy foreign invaders. The internal defense glycan found in gram-positive bacteria; TLR4 recognizes
 Gram-positive bacteria responses are rapidly activated by production of cytokines and
Mycobacteria Mycoplasma Gram- Motile
chemokines. Neutrophils are recruited to the area because of
negative bacteria increased capillary permeability; in addition, macrophages and
Fungi bacteria dendritic cells become more efficient because of increased
expression of adhesion molecules on their cell surfaces. These
Extracellular processes enhance phagocytosis and destroy most pathogens
that humans are exposed to before disease sets in.
In addition to TLRs, there are several other families of re-
Plasma ceptors that activate innate immune responses. One such
membrane family is the C-type lectin receptor (CLR). CLRs are plasma
membrane receptors found on monocytes, macrophages, den-
dritic cells, neutrophils, B cells, and T-cell subsets. These re-
1 2 6 2 4 4 5 5
ceptors bind to mannan and β-glucans found in fungal cell
Cytoplasm walls.5 Although the initial signaling pathway differs from
TLRs, the end result is the same—production of cytokines and
3 3 7 7 8 8 9 9 chemokines to eliminate microbes.
Other families of receptors that recognize pathogens in-
Endosomal clude retinoic acid-inducible gene-I-like receptors (RLRs) and
membrane nucleotide-binding oligomerization domain receptors (NOD).
The RLR family recognizes RNA from RNA viruses in the cy-
toplasm of infected cells and induces inflammatory cytokines
Endosomal and type I interferons. Type I interferons inhibit viral replica-
lumen
tion and induce apoptosis (cell death) in infected cells. NOD
Viral Viral Viral Bacterial/viral receptors bind peptidoglycans found in bacterial cell walls and
dsRNA ssRNA ssRNA DNA also help to protect against intracellular protozoan parasites.1,5
Toll-like receptors on a WBC membrane. Each of the Mutations in NOD receptors may result in Crohn’s disease, a
10 different TLRs recognizes a different pathogenic product. TLRs painful inflammatory disease of the bowel.1
found on the cell surface tend to form dimers to increase chances of
binding to a foreign substance.
Acute-Phase Reactants
lipopolysaccharide, which is found in gram-negative bacteria; In addition to the cells and receptors that enhance the destruc-
and TLR5 recognizes bacterial flagellin (Fig. 3–3). The func- tion of pathogens, the internal defense system also consists of
tion of TLR10 is not yet known. soluble factors called acute-phase reactants that contribute to
TLRs are membrane-spanning glycoproteins that share a the innate immune response. Acute-phase reactants are nor-
common structural element called leucine-rich repeats (LRRs).5 mal serum constituents that increase rapidly because of infec-
Once TLRs bind to their particular substances, host immune tion, injury, or trauma to the tissues. Many act by binding to

Table 3–1 The 10 Toll-Like Receptors

RECEPTOR SUBSTANCE RECOGNIZED TARGET MICROORGANISM
TLR Receptors Found on Cell Surfaces
TLR1 Lipopeptides Mycobacteria
TLR2 Peptidoglycan, lipoproteins, zymosan Gram-positive bacteria, mycobacteria, yeasts
TLR4 Lipopolysaccharide, fusion proteins, mannan Gram-negative bacteria, RSV fungi
TLR5 Flagellin Bacteria with flagellae
TLR6 Lipopeptides, lipoteichoic acid, zymosan Mycobacteria, gram-positive bacteria, yeasts
TLR Receptors Found in Endosomal Compartments
TLR3 Double-stranded RNA RNA viruses
TLR7 Single-stranded RNA RNA viruses
TLR8 Single-stranded RNA RNA viruses
TLR9 Double-stranded DNA DNA viruses, bacterial DNA
TLR10 Unknown Unknown
 microorganisms and promoting adherence, the first step in
phagocytosis. Others help to limit destruction caused by the
release of proteolytic enzymes from WBCs as the process of
phagocytosis takes place. Some of the most important ones are
C-reactive protein, serum amyloid A, complement compo-
nents, alpha1-antitrypsin, haptoglobin, fibrinogen, and ceru-
loplasmin.8 They are produced primarily by hepatocytes (liver
parenchymal cells) within 12 to 24 hours in response to an in-
crease in cytokines (see Chapter 6 for a complete discussion
of cytokines). The particular cytokines involved are inter-
leukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis
factor-α (TNF-α), all of which are produced by monocytes and
macrophages at the sites of inflammation.1,9 Table 3–2 sum-
marizes characteristics of the main acute-phase reactants.

A C-reactive protein (CRP) is a trace constituent of serum orig-

inally thought to be an antibody to the C-polysaccharide of
pneumococci. It was discovered by Tillet and Francis in 1930
when they observed that serum from patients with Streptococcus
pneumoniae infection precipitated with a soluble extract of the
bacteria.10 Now CRP is known to have a more generalized role
in innate immunity.
The molecular weight of CRP is between 118,000 and
144,000 daltons8 and has a structure that consists of five iden-
tical subunits held together by noncovalent bonds. It is a mem-
ber of the family known as the pentraxins, all of which are
proteins with five subunits. CRP acts somewhat like an anti-
body because it is capable of opsonization (the coating of for-
eign particles), agglutination, precipitation, and activation of
complement by the classical pathway. However, binding is
calcium-dependent and nonspecific. The main substrate is phos-
phocholine, a common constituent of microbial membranes. It
B also binds to small ribonuclear proteins; phospholipids; pep-
tidoglycan; and other constituents of bacteria, fungi, and para-
A. Digitally colored and negative stained transmission
sites.11 In addition, CRP promotes phagocytosis by binding to
electron micrograph of the influenza A virus, an RNA virus that
TLR3, TLR7, and TLR8 recognize as foreign. B. Scanning electron specific receptors found on monocytes, macrophages, and neu-
micrograph of Staphylococcus aureus; gram-positive bacteria that are trophils. Thus, CRP can be thought of as a primitive, nonspecific
recognized by the TLR2 receptor. (A. Courtesy of the CDC/FA Murphy, form of an antibody molecule that is able to act as a defense
Public Health Image Library. B. Courtesy of the CDC/Matthew Arduino, against microorganisms or foreign cells until specific antibodies
Public Health Image Library, DRPH.) can be produced.

Table 3–2 Characteristics of Acute-Phase Reactants

NORMAL
RESPONSE CONCENTRATION
PROTEIN TIME (HR) (MG/DL) INCREASE FUNCTION
C-reactive protein 4–6 0.5 1000X Opsonization, complement activation
Serum amyloid A 24 5 1000X Activates monocytes and macrophages
Alpha1-antitrypsin 24 200–400 2–5X Protease inhibitor
Fibrinogen 24 200–400 2–5X Clot formation
Haptoglobin 24 40–290 2–10X Binds hemoglobin
Ceruloplasmin 48–72 20–40 2X Binds copper and oxidizes iron
Complement C3 48–72 60–140 2x Opsonization, lysis
 CRP is a relatively stable serum protein with a half-life of
about 18 hours.10,11 It increases rapidly within 4 to 6 hours Serum amyloid A (SAA) is the other major protein besides CRP
following infection, surgery, or other trauma to the body. Levels whose concentration can increase almost a thousandfold in
increase dramatically as much as a hundredfold to a thousand- response to infection or injury. It is an apolipoprotein that is
fold, reaching a peak value within 48 hours.10,11 CRP also de- synthesized in the liver and has a molecular weight of
clines rapidly with cessation of the stimuli.10 Elevated levels 11,685 daltons. Normal circulating levels are approximately 5 to
are found in conditions such as bacterial infections, rheumatic 8 ug/mL.22 In plasma, SAA has a high affinity for HDL cholesterol
fever, viral infections, malignant diseases, tuberculosis, and and is transported by HDL to the site of infection. SAA appears
after a heart attack. The median CRP value for an individual to act as a chemical messenger, similar to a cytokine, and it acti-
increases with age, reflecting an increase in subclinical inflam- vates monocytes and macrophages to then produce products that
matory conditions.12 increase inflammation.22 It has been found to increase signifi-
Because the levels rise and then decline so rapidly, CRP is cantly more in bacterial infections than in viral infections.22 Levels
the most widely used indicator of acute inflammation. Al- reach a peak between 24 to 48 hours after an acute infection.23
though CRP is a nonspecific indicator of disease or trauma, SAA can also be increased because of chronic inflammation, ath-
monitoring of its levels can be useful clinically to follow a dis- erosclerosis, and cancer.23 Because SAA has been found in ather-
ease process and observe the response to treatment of inflam- osclerotic lesions, it is now thought to contribute to localized
mation and infection.11 It is a nonsurgical means of following inflammation in coronary artery disease.9 Elevated levels may
the course of malignancy and organ transplantation because a predict a worse outcome for the patient.9
rise in the level may mean a return of the malignancy or, in the
case of transplantation, the beginning of organ rejection. CRP
levels can also be used to monitor the progression or remission Complement refers to a series of serum proteins that are nor-
of autoimmune diseases. Assays for CRP are sensitive, repro- mally present and whose overall function is mediation of
ducible, and relatively inexpensive.10 inflammation. Nine such proteins are activated by bound
CRP has lately received attention as a risk marker for car- antibodies in a sequence known as the classical cascade; an
diovascular disease. In accord with the finding that athero- additional number are involved in the alternate pathway that is
sclerosis (coronary artery disease) may be the result of a triggered by the presence of microorganisms. The major func-
chronic inflammatory process,13 an increased level of CRP tions of complement are opsonization, chemotaxis, and lysis of
has been shown to be a significant risk factor for myocardial cells. Complement is discussed more fully in Chapter 7.
infarction, ischemic stroke, and peripheral vascular disease
in men and women who have no previous history of cardio-
vascular disease.14–19 When inflammation is chronic, in- Alpha1-antitrypsin (AAT) is a 52-kD protein that is primarily
creased amounts of CRP react with endothelial cells lining synthesized in the liver. It is the major component of the alpha
vessel walls and predispose these walls to vasoconstriction, band when serum is electrophoresed. Although the name im-
platelet activation, thrombosis (clot formation), and vascular plies that it acts against trypsin, it is a general plasma inhibitor
inflammation.10 Monitoring of CRP, therefore, is now an es- of proteases released from leukocytes.8 Elastase, one such pro-
tablished clinical tool to evaluate subtle chronic systemic in- tease, is an enzyme secreted by neutrophils during inflam-
flammation and predict cardiovascular or cerebrovascular mation that can degrade elastin and collagen. In chronic
disease.8,14 The ability to monitor CRP is significant because pulmonary inflammation, elastase activity damages lung tissue.
cardiovascular disease is a major cause of mortality in the Thus, AAT acts to “mop up” or counteract the effects of neu-
world today.8,14,15 trophil invasion during an inflammatory response. It also reg-
The Centers for Disease Control and Prevention (CDC) has ulates expression of proinflammatory cytokines such as TNF-α,
recommended that a CRP concentration of less than 1 mg/L interleukin-1β, and interleukin-6, mentioned previously. As a
is associated with a low risk for cardiovascular disease; 1 to result, activation of monocytes and neutrophils is inhibited,
3 mg/L is associated with an average risk; and greater than limiting the harmful side effects of inflammation.24
3 mg/L is associated with a high risk.20,21 Normal levels in AAT deficiency can result in premature emphysema, espe-
adults range from approximately 0.47 to 1.34 mg/L.8 A mean cially in individuals who smoke or who have frequent exposure
for people with no coronary artery disease is 0.87 mg/L.14 to noxious chemicals.8 In such a deficiency, uninhibited pro-
Thus, monitoring CRP may be an important preventative teases remain in the lower respiratory tract, leading to destruc-
measure in determining the potential risk of heart attack or tion of parenchymal cells in the lungs and to development of
stroke. High-sensitivity CRP testing has the necessary lower emphysema or idiopathic pulmonary fibrosis. It has been esti-
level of detection of 0.01 mg/L, which enables measurement mated that as many as 100,000 Americans suffer from this de-
of much smaller increases than the traditional latex aggluti- ficiency, although many of them are undiagnosed.25,26 There
nation screening test.11 are at least 75 alleles of the gene coding for AAT and 17 of these
CRP is easily destroyed by heating serum to 56°C for are associated with low production of the enzyme.8 One par-
30 minutes. The destruction of CRP is often necessary in the ticular variant gene for AAT is responsible for a complete lack
laboratory because it interferes with some testing for the pres- of production of the enzyme; individuals who inherit this gene
ence of antibodies. are at risk of developing liver disease and emphysema.22
Homozygous inheritance of this particular gene may lead to Inflammation
development of cirrhosis, hepatitis, or hepatoma in early child-
hood. The only treatment is a liver transplant.8 When pathogens breach the outer barriers of innate immunity,
AAT can also react with any serine protease, such as pro- both cellular and humoral mechanisms are involved in a com-
teases generated by the triggering of the complement cascade plex, highly orchestrated process known as inflammation.
or fibrinolysis. Once bound to AAT, the protease is completely Inflammation can be defined as the body’s overall reaction to
inactivated and is subsequently removed from the area of tissue injury or invasion by an infectious agent. Each individual re-
damage. actant plays a role in initiating, amplifying, or sustaining the
reaction and a delicate balance must be maintained for the
process to be speedily resolved. The four cardinal signs or clin-
Haptoglobin is an alpha2-globulin with a molecular weight of ical symptoms of inflammation are redness (erythema),
100,000 daltons. It binds irreversibly to free hemoglobin re- swelling (edema), heat, and pain. Major events that occur
leased by intravascular hemolysis. Haptoglobin thus acts as an rapidly after tissue injury are:
antioxidant to provide protection against oxidative damage me- 1. Increased blood supply to the affected area. Dilation of
diated by free hemoglobin.27,28 Once bound, the complex is the blood vessels caused by the release of chemical me-
cleared rapidly by macrophages in the liver.29,30 A two- to ten- diators such as histamine from injured mast cells brings
fold increase in haptoglobin can be seen following inflamma- additional blood flow to the affected area, resulting in
tion, stress, or tissue necrosis.8 Early in the inflammatory redness and heat.
response, however, haptoglobin levels may drop because of in- 2. Increased capillary permeability caused by contraction
travascular hemolysis, consequently masking the protein’s be- of the endothelial cells lining the vessels. The increased
havior as an acute-phase reactant.28 Thus, plasma levels must permeability of the vessels allows fluids in the plasma to
be interpreted in light of other acute-phase reactants. Normal leak into the tissues, resulting in the swelling and pain
plasma concentrations range from 40 to 290 mg/dL.8 associated with inflammation.
3. Migration of WBCs, mainly neutrophils, from the capil-
Fibrinogen is an acute-phase protein involved in the coagu- laries to the surrounding tissue in a process called dia-
lation pathway. A small portion is cleaved by thrombin to form pedesis. As the endothelial cells of the vessels contract,
fibrils that make up a fibrin clot.8 The molecule is a dimer neutrophils move through the endothelial cells of the
with a molecular weight of 340,000 daltons. Normal levels vessel and out into the tissues. Soluble mediators, which
range from 200 to 400 mg/dL.8,31 The clot increases the include acute-phase reactants, chemokines, and cy-
strength of a wound and stimulates endothelial cell adhesion tokines, act as chemoattractants to initiate and control
and proliferation, which are critical to the healing process. the response. Neutrophils are mobilized within 30 to
Formation of a clot creates a barrier that helps prevent the 60 minutes after the injury and their emigration may last
spread of microorganisms further into the body. Fibrinogen 24 to 48 hours.
makes blood more viscous and serves to promote aggregation 4. Migration of macrophages to the injured area.27 Migra-
of red blood cells (RBCs) and platelets. Increased levels may tion of macrophages and dendritic cells from surround-
contribute to an increased risk for developing coronary artery ing tissue occurs several hours later and peaks at 16 to
disease.8,15,18,31 48 hours.
5. Acute-phase reactants stimulate phagocytosis of mi-
croorganisms. Macrophages, neutrophils, and dendritic
Ceruloplasmin consists of a single polypeptide chain with cells all attempt to clear the area through phagocytosis;
a molecular weight of 132,000 daltons.8 It is the principal in most cases, the healing process is completed with a
copper-transporting protein in human plasma, binding more return of normal tissue structure (Fig. 3–4).
than 70% of the copper found in plasma by attaching six The acute inflammatory response acts to combat the early
cupric ions per molecule.32Additionally, ceruloplasmin acts as stages of infection and also begins a process that repairs tissue
an enzyme, converting the toxic ferrous ion (Fe2+) to the non- damage. However, when the inflammatory process becomes
toxic ferric form (Fe3+).32 The normal plasma concentration prolonged, it is said to be chronic. The failure to remove mi-
for adults is 20 to 40 mg/dL. croorganisms or injured tissue may result in continued tissue
A depletion of ceruloplasmin is found in Wilson’s disease, damage and loss of function.
an autosomal recessive genetic disorder characterized by a
massive increase of copper in the tissues. Normally, circulat-
ing copper is absorbed out by the liver and either combined
Phagocytosis
with ceruloplasmin and returned to the plasma or excreted The main purpose of the inflammatory response is to attract
into the bile duct. In Wilson’s disease, copper accumulates cells to the site of infection and remove foreign cells or
in the liver and subsequently in other tissues such as the pathogens by means of phagocytosis. Although the acute-
brain, corneas, kidneys, and bones.8,33 Treatment is either phase reactants enhance the process of phagocytosis, it is the
long-term chelation therapy to remove the copper or a liver cellular elements of the internal defense system that play the
transplant.8 major role. The cells that are most active in phagocytosis are
 1. Resident macrophages and mast cells at the
site of infection release chemokines that cause
vasodilation and induce selectins.

2. Selectins loosely bind circulating leukocytes

and cause them to roll along the vascular wall.

3. Chemokine-induced integrins on the leukocytes

bind firmly to the endothelial cells and
1
4. Integrins enable the leukocytes to crawl between
endothelial cells (diapedesis).

5. Leukocytes then follow the chemokine

concentration gradient to the site of infection
2 (chemotaxis).

Site of
infection

Local inflammatory events.

neutrophils, monocytes, macrophages, and dendritic cells, as meaning “to prepare for eating.” Opsonins are serum proteins
discussed in Chapter 1. that attach to a foreign cell or pathogen and help prepare it for
Once the WBCs are attracted to the area, the actual process phagocytosis. CRP, complement components, and antibodies
of phagocytosis consists of seven main steps (Fig. 3–5): are all important opsonins. Opsonins may act by neutralizing
the surface charge on the foreign particle, making it easier for
1. Physical contact between the WBC and the foreign cell
the cells to approach one another. In addition to receptors
2. Outflowing of the cytoplasm to surround the
for pathogens themselves, phagocytic cells also have receptors
microorganism
for immunoglobulins and complement components, which aid
3. Formation of a phagosome
in contact and in initiating ingestion.
4. Fusion with lysosomal granules with the phagosome
Once contact with surface receptors occurs, phagocytic
5. Formation of the phagolysosome with release of lysosomal
cells secrete chemoattractants such as cytokines and
contents
chemokines; these recruit additional cells to the site of in-
6. Digestion of microorganisms by hydrolytic enzymes
fection. Neutrophils are followed by monocytes, after which
7. Release of debris to the outside by exocytosis
macrophages and dendritic cells arrive at the site. 1
Physical contact occurs as neutrophils roll along in the Macrophages and dendritic cells are not only able to ingest
bloodstream in a random pattern until they encounter the site whole microorganisms, but they can also clean up injured
of injury or infection.34 They adhere to receptors on the en- or dead host cells.
dothelial cell wall of the blood vessels and penetrate through After attachment to a foreign cell or pathogen has occurred,
to the tissue by means of diapedesis. This adhering process is the cell membrane invaginates and pseudopodia (outflowing
aided by chemotaxis, whereby cells are attracted to the site of of cytoplasm) surround the pathogen. The pseudopodia fuse
inflammation by chemical substances such as soluble bacterial to completely enclose the pathogen, forming a structure known
factors or acute-phase reactants including complement com- as a phagosome. The phagosome is moved toward the center
ponents and CRP. Macrophages and dendritic cells already re- of the cell. Lysosomal granules quickly migrate to the phago-
side in the tissues. Receptors on neutrophils, macrophages, and some and fusion between granules and the phagosome occurs.
dendritic cells bind to certain molecular patterns on a foreign At this point, the fused elements are known as a phagolyso-
particle surface as discussed previously. This binding process some. The granules contain lysozyme, myeloperoxidase, and
is enhanced by opsonins, a term derived from the Greek word other proteolytic enzymes. The contents of the granules are
 1
2
1. Adherence: physical contact between the
phagocytic cell and the microorganism occurs,
aided by opsonins.
3
2. Engulfment: outflowing of cytoplasm to surround
the microorganism.

3. Formation of phagosome: microorganism is

completely surrounded by a part of the cell
4 membrane.

4. Granule contact: lysosomal granules contact

and fuse with the phagosome.

5. Formation of the phagolysosome: contents of the

lysosome are emptied into this membrane-bound
space.
5
6. Digestion of the microorganism by hydrolytic
enzymes.

7. Excretion: contents of phagolysosome are expelled

to the outside by exocytosis.
6

7
Steps involved in phagocytosis.

released into the phagolysosome and digestion occurs. Any when fusion with the phagosome occurs, allowing hydrogen and
undigested material is excreted from the cells by exocytosis. potassium ions to enter the vacuole. This alters the pH, which in
Heavily opsonized particles are taken up in as little as 20 seconds turn activates proteases that contribute to microbial elimination.
and killing is almost immediate.2,35 Some of these lytic enzymes include small cationic proteins called
The elimination of pathogens actually occurs by two differ- defensins. When defensins are released from lysosomal granules,
ent processes: an oxygen-dependent pathway and an oxygen- they are able to cleave segments of bacterial cell walls without the
independent pathway. In the oxygen-dependent process, an benefit of oxygen. Defensins kill a wide spectrum of organisms,
increase in oxygen consumption, known as the oxidative burst, including both gram-positive and gram-negative bacteria, many
occurs within the cell as the pseudopodia enclose the particle fungi, and some viruses. Cathepsin G is another example of
within a vacuole. This mechanism generates considerable energy a protein that is able to damage bacterial cell membranes.2,34
via oxidative metabolism. The hexose monophosphate shunt is Chapter 1 lists some of the contents of granules in neutrophils.
used to change nicotinamide adenine dinucleotide phosphate The importance of NADPH oxidase in the elimination of
(NADP) to its reduced form by adding a hydrogen. Electrons microbes is demonstrated by the fact that a lack of it may lead
then pass from NADPH to oxygen in the presence of NADPH to an increased susceptibility to infection. Patients with chronic
oxidase, a membrane-bound enzyme that is only activated granulomatous disease have a gene mutation that causes a defect
through conformational change triggered by microbes them- in NADPH oxidase, resulting in an inability to kill bacteria dur-
selves.36 A radical known as O2– (superoxide) is then formed. ing the process of phagocytosis. Individuals with this disease suf-
Superoxide is highly toxic but can be rapidly converted to even fer from recurring, severe bacterial infections (see Chapter 19).34
more lethal products. By adding hydrogen ions, the enzyme Following phagocytosis, macrophages and dendritic cells
superoxide dismutase (SOD) converts superoxide to hydrogen process peptides from pathogens for presentation to T cells.
peroxide or the hydroxyl radical OH. T cells then interact with B cells to produce antibodies (see
Hydrogen peroxide has long been considered an important Chapter 5 for details). Because T cells are not able to respond
bactericidal agent and is more stable than any of the free radi- to intact pathogens, phagocytosis is a crucial link between the
cals. Its antimicrobial effect is further enhanced by the forma- innate and the adaptive immune systems.
tion of hypochlorite ions through the action of the enzyme
myeloperoxidase in the presence of chloride ions. Hypochlorite
is a powerful oxidizing agent and is highly toxic for microor-
Action of Natural Killer Cells
ganisms. It is the main component of household bleach used Another important cellular defense that is part of innate im-
to disinfect surfaces (Fig. 3–6). munity is the action of natural killer (NK) cells. Although
NADPH oxidase also plays a major role in the oxygen- phagocytosis is important in eliminating infectious agents,
independent pathway. NADPH oxidase depolarizes the membrane NK cells represent the first line of defense against cells that are
 Anti-
microbial Phagolysosome
enzymes

Cytoplasm
•OH ! OH"
Fe
SD MPO
202•" ! 2H! H2O2 ! Cl" OCl" ! H2O
Pentose-5-P NADPH
e"

HMP shunt
Start here NOC 202 Pathogen

Glucose G-6-phosphate NADP!

ATP ADP

H! MPO

Creation of oxygen radicals in the phagocytic cell. The hexose monophosphate (HMP) shunt reduces NADP to NADPH. NADPH
_
reduces oxygen to superoxide (2O2• ) when the NADPH oxidase complex (NOC) is assembled in the membrane of the phagolysosome. Super-
oxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide (H2O2). Myeloperoxidase (MPO) catalyzes formation of
_
hypochlorite (OCl ), a very powerful oxidizing agent. Hydroxyl radicals (•OH), which are also powerful oxidizing agents, may also be formed if
iron ions are present.

virally infected, cells infected with other intracellular inhibitory signals, and activating receptors, which deliver sig-
pathogens, and tumor cells.37–39 NK cells have the ability to nals to activate the cytotoxic mechanisms.37 It appears that
recognize any damaged cell and to eliminate such target cells there is a balance between activating and inhibitory signals that
without prior exposure to them. The fact that they lack speci- enables NK cells to distinguish healthy cells from infected or
ficity in their response is essential to their function as early de- cancerous ones.
fenders against pathogens.37,38 By quickly engaging infected The inhibitory signal is based on recognition of class I major
target cells, NK cells give the immune system time to activate histocompatibility complex (MHC) proteins, which are ex-
the adaptive response of specific T and B cells. pressed on all healthy cells (see Chapter 2 for details). If NK cells
NK cell activity is stimulated by exposure to cytokines such react with class I MHC proteins, then inhibition of natural killing
as interleukin-12, interferon-α, and interferon-β. Because these occurs. Examples of this type of inhibitory receptor include killer
cytokines rise rapidly during a viral infection, NK cells are able cell immunoglobulin-like receptors (KIRs)37 and CD94/NKG2A
to respond early during an infection and their activity peaks receptors, both of which bind class I MHC molecules.
in about 3 days, well before antibody production or a cytotoxic Diseased and cancerous cells tend to lose their ability to pro-
T-cell response. They localize in the tissues in areas where in- duce MHC proteins. NK cells are thus triggered by a lack of
flammation is occurring and where dendritic cells are found.37 MHC antigens, sometimes referred to as recognition of “miss-
Once activated, NK cells themselves become major producers ing self.”37,39 This lack of inhibition appears to be combined
of cytokines such as interferon-gamma (IFN-γ) and TNF-α with an activating signal switched on by the presence of pro-
that help to recruit T cells.37,39 In addition, NK cells release teins produced by cells under stress, namely those cells that
various colony stimulating factors that act on developing gran- are infected or cancerous.39
ulocytes and macrophages. Actions of NK cells, therefore, have Examples of activating receptors that bind stress proteins are
a major influence on both innate and adaptive immunity. CD16 and NKG2D.37 If an inhibitory signal is not received when
binding to activating receptors occurs, then NK cells release
substances called perforins and granzymes (Fig. 3–7). These
For years it had been a mystery how NK cells tell the difference substances are released into the space between the NK cell and
between normal and abnormal cells. However, the mechanism is the target cell. Perforins are proteins that form channels (pores)
now beginning to be understood. NK cells are constantly moni- in the target cell membrane.40 Granzymes are packets of
toring potential target cells through two main classes of binding enzymes that may enter through the channels and mediate
receptors on NK cells: inhibitory receptors, which deliver cell lysis. The elimination of target cells can occur in as little
 Inhibition
SUMMARY
• Innate immunity encompasses all the body’s normally
present defense mechanisms for resisting disease. It is
A characterized by lack of specificity, no need for a prior
NK cell Target cell No killing exposure, and a similar response with each exposure.
• External defenses are structural barriers such as skin,
mucous membranes, cilia, and secretions such as lactic
acid and lysozyme that keep microorganisms from enter-
ing the body.
• Internal defenses include both cells capable of phagocy-
tosis and acute-phase reactants that enhance the process
of phagocytosis. Cells that are most active in phagocyto-
sis include neutrophils, monocytes, macrophages, and
dendritic cells.
NK cell Antibody-coated Apoptosis • Pathogen recognition receptors (PRRs) are molecules on
B
target cell
host cells that recognize substances found only on
pathogens. They are found on neutrophils, monocytes,
eosinophils, mast cells, and dendritic cells. Once recep-
tors bind a pathogen, phagocytosis can take place.
• Pathogen-associated molecular patterns (PAMPS) are
molecules found only on pathogens, which allow host
cells to distinguish them from self. They are recognized
Activation by the PRRs.
• Acute-phase reactants are serum proteins that increase
rapidly in response to infection or injury and include
C NK cell Virally infected Apoptosis
target cell
C-reactive protein (CRP), serum amyloid A, complement
components, alpha1-antitrypsin, haptoglobin, fibrinogen,
Actions of NK cells. NK cells are constantly surveying
and ceruloplasmin.
cells. (A) If class I MHC protein is present and there are no foreign or
stress proteins, then an inhibitory signal is sent to the NK cell, no
• CRP is the most widely monitored acute-phase reactant;
killing occurs, and the normal cell is released. (B) Alternatively, it increases 100 to 1000 times in response to infection
infected cells may express foreign proteins on their surface that are or trauma, acts as an opsonin, and is able to fix
recognized by antibody. NK cells express CD16 receptors that bind complement.
the immobilized antibody and activate the release of perforins and • All acute-phase reactants increase the likelihood of phago-
granzymes (antibody-dependent cell-mediated cytotoxicity). (C) If an cytosis of pathogens and help healing occur.
activating receptor is engaged by a foreign or stress protein and • The first step in phagocytosis is physical contact between
class I MHC is altered or missing (“missing self”), then no inhibitory the phagocytic cell and the foreign particle.
signal is given, granzymes and perforins are released, and the • Chemotaxis is the process that attracts cells to the area of
infected or diseased cell is eliminated by apoptosis. infection.
• Cytoplasm flows around the foreign particle to form a
as 30 to 60 minutes.2 Thus, depending on the signals, the NK cell phagosome. Fusion of the phagosome with lysosomal
either proceeds to activate cell destruction or detaches and moves granules creates a phagolysosome. Inside this structure,
on to search for another target cell. enzymes such as lysozyme and myeloperoxidase are re-
leased and the foreign particle is digested.
• Creation of hypochlorite and hydroxyl ions, which dam-
A second method of destroying target cells is also available to age protein irreversibly, occur in the oxygen-dependent
NK cells. They recognize and lyse antibody-coated cells phase of phagocytosis.
through a process called antibody-dependent cell cytotoxic- • Phagocytosis must occur before the specific immune re-
ity (ADCC). Binding occurs through the CD16 receptor for sponse can be initiated, so this process is essential to both
the Fc portion of immunoglobulin G (IgG). Any target cell innate and adaptive immunity.
coated with IgG can be bound and destroyed. This method is • Inflammation is the body’s response to injury or invasion
not unique to NK cells, as monocytes, macrophages, and neu- by a pathogen. It is characterized by increased blood
trophils also exhibit such a receptor and act in a similar man- supply to the affected area, increased capillary perme-
ner. Nonetheless, the overall importance of NK cells as a ability, migration of neutrophils to the surrounding
defense mechanism is demonstrated by the fact that patients tissue, and migration of macrophages to the injured
who lack these cells have recurring, serious viral infections and area.
an increased incidence of tumors.41
• Natural killer (NK) cells are able to kill target cells that are protein found on normal cells. This capability is called
infected with a virus or other intracellular pathogen. They recognition of missing self.
also recognize malignant cells. • NK cells bind to and kill any antibody-coated target cells.
• The action of NK cells does not require prior exposure • NK cells represent an important link between the innate
and is nonspecific. They recognize a lack of class I MHC and adaptive immune systems.

Study Guide: Mechanisms of Innate Immunity

TYPE OF DEFENSE EXAMPLE FUNCTION
External Skin and mucous membranes Biological barriers
Lactic acid Keeps down growth of microorganisms
Cilia Move pathogens out of respiratory tract
Stomach acid Low pH keeps pathogens from growing
Urine Flushes out pathogens from the body
Lysozyme Attacks cell walls of pathogens
Normal flora Compete with pathogens
Produce antimicrobial peptides
Internal Cells Participate in phagocytosis
NK cells destroy target cells using granzymes and perforins
Pathogen recognition receptors Help phagocytic cells recognize pathogens
(e.g., Toll-like receptors)
Acute-phase reactants Recruit WBCs for phagocytosis
Coat pathogens to enhance phagocytosis
Mop up debris

CASE STUDIES
1. A 45-year-old male named Rick went to his physician for a slight fever. A complete blood count (CBC) was per-
an annual checkup. Although he was slightly overweight, formed and both the RBC and WBC count were within
his laboratory results indicated that both his total choles- normal limits. A normal WBC count ruled out the possi-
terol and his HDL cholesterol were within normal limits. bility of a bacterial infection. A rapid strep test was per-
His fibrinogen level was 450 mg/dL and his CRP level was formed, which was negative. A slide agglutination test for
3.5 mg/dL. His physical examination was perfectly nor- infectious mononucleosis was indeterminate (neither pos-
mal. The physician cautioned Rick that he might be at itive or negative), whereas a slide agglutination test for
risk for a future heart attack and he counseled him to be CRP was positive. Results of a semiquantitative CRP de-
sure to exercise and eat a healthy, low-fat diet. Rick’s wife termination indicated an increased level of approximately
told him that as long as his cholesterol level was normal, 20 mg/dL. The student was advised to return in a
he didn’t have anything to worry about. few days for a repeat mono test.
Question Questions
a. Who is correct? Explain your answer. a. What conditions might cause a rise in CRP?
b. Would an increase in CRP be consistent with the
2. A 20-year-old female college student went to the infir-
possibility of infectious mononucleosis?
mary with symptoms of malaise, fatigue, sore throat, and
REVIEW QUESTIONS
1. The term for enhancement of phagocytosis by coating 8. Pathogen recognition receptors act by
of foreign particles with serum proteins is a. recognizing molecules common to both host cells
a. opsonization. and pathogens.
b. agglutination. b. recognizing molecules that are unique to pathogens.
c. solubilization. c. helping to spread infection because they are found
d. chemotaxis. on pathogens.
d. all recognizing the same pathogens.
2. Which of the following plays an important role as an
external defense mechanism? 9. Which of the following are characteristics of
a. Phagocytosis acute-phase reactants?
b. C-reactive protein a. Rapid increase following infection
c. Lysozyme b. Enhancement of phagocytosis
d. Complement c. Nonspecific indicators of inflammation
d. All of the above
3. The process of inflammation is characterized by all
of the following except 10. Which is the most significant agent formed in the
a. increased blood supply to the area. phagolysosome for the elimination of microorganisms?
b. migration of WBCs. a. Proteolytic enzymes
c. decreased capillary permeability. b. Hydrogen ions
d. appearance of acute-phase reactants. c. Hypochlorite ions
d. Superoxides
4. Skin, lactic acid secretions, stomach acidity, and
the motion of cilia represent which type of 11. Which acute-phase reactant helps to prevent formation
immunity? of peroxides and free radicals that may damage tissues?
a. Innate a. Haptoglobin
b. Cross b. Fibrinogen
c. Adaptive c. Ceruloplasmin
d. Auto d. Serum amyloid A

5. The structure formed by the fusion of engulfed mate- 12. Which statement best describes Toll-like receptors
rial and enzymatic granules within the phagocytic cell (TLRs)?
is called a a. They protect adult flies from infection.
a. phagosome. b. They are found on all host cells.
b. lysosome. c. They only play a role in adaptive immunity.
c. vacuole. d. They enhance phagocytosis.
d. phagolysosome
13. The action of CRP can be distinguished from that of
6. The presence of human microbiota (normal flora) acts an antibody because
as a defense mechanism by which of the following a. CRP acts before the antibody appears.
methods? b. only the antibody triggers the complement cascade.
a. Maintaining an acid environment c. binding of the antibody is calcium-dependent.
b. Competing with potential pathogens d. only CRP acts as an opsonin.
c. Keeping phagocytes in the area
d. Coating mucosal surfaces 14. How does innate immunity differ from adaptive
immunity?
7. Measurement of CRP levels can be used for all of the a. Innate immunity requires prior exposure to a
following except pathogen.
a. monitoring drug therapy with anti-inflammatory b. Innate immunity depends upon normally present
agents. body functions.
b. tracking the progress of an organ transplant. c. Innate immunity develops later than adaptive
c. diagnosis of a specific bacterial infection. immunity.
d. determining active phases of rheumatoid d. Innate immunity is more specific than adaptive
arthritis. immunity.
15. A 40-year-old male who is a smoker develops symp- 16. Which statement best describes NK cells?
toms of premature emphysema. The symptoms may a. Their response against pathogens is very specific.
be caused by a deficiency of which of the following b. They only react when an abundance of MHC
acute-phase reactants? antigens is present.
a. Haptoglobin c. They react when both an inhibitory and activating
b. Alpha1-antitrypsin signal is triggered.
c. Fibrinogen d. They are able to kill target cells without previous
d. Ceruloplasmin exposure to them.
 Adaptive Immunity

After finishing this chapter, you should be able to: TCELL DIFFERENTIATION
1. Compare and contrast adaptive immunity and innate immunity. Double-Negative Stage
2. Discuss the role of the thymus in T-cell maturation. Double-Positive Stage
3. Describe the CD3 receptor for antigen on a T cell. Mature T Cells
4. Explain how positive and negative selection contribute to the devel- STAGES IN BCELL DIFFERENTIATION
opment of immunocompetent T cells. Pro-B Cells
5. List and describe five different subsets of T cells that bear the CD4 Pre-B Cells
marker. Immature B Cells
6. Describe maturation of a B cell from the pro-B cell to a plasma cell. Mature B Cells
7. Contrast the antigen-independent and antigen-dependent phases of Plasma Cells
B-cell development.
THE ROLE OF T CELLS IN THE
8. Explain how cytotoxic T cells recognize and kill target cells. ADAPTIVE IMMUNE RESPONSE
9. Discuss the role of class I MHC and class II MHC molecules in the Action of T Helper Cells
presentation of antigens to T cells.
Action of Cytotoxic T Cells
10. Differentiate T-dependent antigens from T-independent antigens on
THE ROLE OF B CELLS IN THE
the basis of how each activates B cells.
ADAPTIVE IMMUNE RESPONSE
11. Discuss how T helper (Th) cells stimulate B cells to transform into
Response to T-Dependent Antigens
plasma cells.
Response to T-Independent Antigens
12. Explain the importance of both T and B memory cells to the adaptive
immune response. LABORATORY IDENTIFICATION OF
LYMPHOCYTES
13. Apply knowledge of T- and B-cell function to immunologically based
disease states. SUMMARY
14. Describe current testing used to identify T and B cells. CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Adaptive immunity Chemokines Immature B cells Surrogate light chain
Allelic exclusion Clonal deletion MHC restriction T-dependent antigens
Antigen-dependent phase Cytotoxic T cells Negative selection T helper (Th) cells
Antigen-independent Double-negative (DN) Plasma cell T-independent antigens
phase thymocytes Positive selection T regulatory (Treg) cells
Cell flow cytometry Double-positive (DP) Pre-B cells Thymocytes
Cell-mediated immunity thymocytes
Pro-B cells Variable regions
Central tolerance Humoral immunity

As you learned in Chapter 3, innate immunity is based on ex- T-Cell Differentiation

ternal defenses that keep pathogens from entering the body. If
these external defenses are breached, there are internal defenses, Between 60% to 80% of circulating lymphocytes in the periph-
including phagocytic cells found in the blood and tissues, that eral blood are T cells (Fig. 4–1) that become differentiated in
respond similarly to any pathogens they encounter and attempt the thymus. Lymphocyte precursors enter the thymus from the
to destroy them. Adaptive immunity, in contrast, is a type of bone marrow. Within the lobules of the thymus are two main
resistance characterized by: zones, the outer cortex and the inner medulla. Early precursors
• Specificity for each individual pathogen or microbial enter the thymus at the cortico-medullary junction and migrate
agent to the outer cortex. Migration occurs in waves and is driven by
• The ability to remember a prior exposure chemical messengers called chemokines.3,4 Chemokines are a
• An increased response to that pathogen upon repeated large family of cytokines that have the ability to recruit specific
exposure cells to a particular site. Once in the thymus, precursors that are
Adaptive immunity is a more tailored response. It takes a committed to becoming T cells are known as thymocytes.
longer time to be activated, but it is more specific and longer As thymocytes travel through the thymus, there is an orderly
lasting. rearrangement of the genes coding for the antigen receptor. At
The key cell involved in the adaptive immune response is the same time, distinct surface markers appear during specific
the lymphocyte. The two main types of lymphocytes are T cells stages of development. Maturation is an elaborate process that
and B cells. T lymphocytes mature in the thymus and serve a takes place over a 3-week period as cells filter through the cortex
regulatory role by providing help to B cells in responding to to the medulla. Thymic stromal cells include epithelial cells,
antigens as well as by killing virally infected target cells. B lym- macrophages, fibroblasts, and dendritic cells, all of which play
phocytes mature in the bone marrow and differentiate into a role in T-cell development.5 Interaction with stromal cells
plasma cells that produce antibodies. Immunologic memory is under the influence of cytokines, especially interleukin-7 (IL-7),
based on clonal selection, expansion, and differentiation of is critical for growth and differentiation.6 A significant selection
antigen-specific T and B cells.1 The result is an ability to re- process occurs as maturation takes place because an estimated
spond with greater speed and intensity to a re-encounter with
the same pathogen, thus protecting the host from reinfection.1
B and T cells go through an elaborate maturation process in
which this specificity is developed while possible self-reactive
cells are destroyed. Each stage is marked by well-orchestrated
signaling mechanisms that help to develop specific receptors
for antigens while at the same time only selecting cells that will
be helpful and not harmful to the host. During this process, cre-
ation of a wide variety of antigen-specific receptors must occur,
enough to recognize any harmful antigens to which we may be
exposed.
Differentiation of lymphocytes appears to take place very early
in fetal development and is essential to the acquisition of im-
munocompetence by the time the infant is born. Progenitors of
T and B cells appear in the fetal liver as early as 8 weeks of preg-
nancy.2 Later in fetal development, production of lymphocyte
progenitors shifts to the bone marrow, which becomes the pri-
mary producer of hematopoietic cells at birth. This chapter will
discuss developmental stages of both T and B cells and will then Scanning electron micrograph of a typical T cell. (Cour-
relate this to their function in the adaptive immune response. tesy of National Institute of Allergy and Infectious Diseases [(NIAID].)
97% of the cortical cells die intrathymically before becoming thymocytes actively proliferate in the outer cortex under the
mature T cells.7 This selection process includes both positive influence of IL-7.
and negative selection; each is discussed in more detail in the Rearrangement of the genes that code for the antigen recep-
next section. tor known as the T-cell receptor (TCR) begins at this stage3,7
This random gene rearrangement is what builds in the diversity
that allows T cells to respond to the myriads of different anti-
Double-Negative Stage gens that the body might encounter in a lifetime. The TCR con-
Early thymocytes lack CD4 and CD8 markers, which are im- sists of two specific chains, called alpha (α) and beta (β) chains,
portant to their later function; hence, they are known as which both contain variable regions that recognize specific
double-negative (DN) thymocytes (Fig. 4–2). These large DN antigens (Fig. 4–3). These two chains occur in a complex with

Lobule

Cortex

Medulla

Gamma delta
CD3/TCR
CD4
CD8

Cortex DP
DN
Positive
selection

CD8 CD4

TP
T-cell maturation in
the thymus. T-lymphocyte precur-
sors (TP) enter the thymus at the
corticomedullary junction. They Singly
positive
migrate upward in the cortex and
begin development of the T-cell
receptor. A small percentage of
precursors develop gamma-delta
chains, whereas the majority
develop alpha-beta chains and Apoptosis
become double-positive (DP)
(both CD4 and CD8 are present).
Medulla
Positive and negative selection
takes place through the CD3/T-cell
Negative
receptor for antigen. If positively selection
selected, the T cell becomes
single-positive (SP); that is, either
CD4+ or CD8+. Further interac-
tions with macrophages or den-
CD8! CD4!
dritic cells take place to weed out
any T cells able to respond to
self-antigen. Surviving CD4+ and
CD8+ cells exit the thymus to the Mature T cells
peripheral blood. exit the thymus
 of the epithelium.3,7 They are capable of recognizing antigens
without being presented by major histocompatibility complex
(MHC) proteins, so they may represent an important bridge
between innate and adaptive immunity.
# $
Double-Positive Stage
! " % !
At this second stage, when thymocytes express both CD4 and
CD8 antigens, they are called double-positive (DP) thymo-
cytes. Young DP thymocytes begin to rearrange the genes cod-
ing for the α chain.1 When the CD3-αβ receptor complex
(TCR) is complete and expressed on the cell surface, a positive
selection process takes place that allows only DP cells with func-
Signal Signal
activation activation
tional TCR receptors to survive. T cells must recognize foreign
antigen in association with class I or class II MHC molecules
Signal
activation (Fig. 4–4). When thymocytes bind to self-MHC antigens in
the cortex by means of the newly formed TCR receptors, an
enzyme cascade involving a group of enzymes called kinases
& &
is activated. Enzyme activity causes changes in cell shape and
The CD3:T-cell receptor complex. The TCR that recog- motility that lead to increased cell survival.3 The selection of
nizes antigen consists of two chains, α and β, which have constant thymocytes that will only interact with the MHC antigens
and variable regions. Four other types of chains are collectively known found on host cells is known as MHC restriction. Any thy-
as CD3. These are ε, γ, δ, and ζ. They take part in signaling to the inte-
mocytes that have either a very low or a very high affinity for
rior of the cell when antigen binding occurs. Note that the γ and δ
chains found here differ from those found on CD4–/CD8– T cells.
self-MHC antigens die by apoptosis.5 This weeding out is im-
portant, because functioning T cells must be able to recognize
foreign antigen along with MHC molecules.
A second selection process, known as negative selection,
six other chains that are common to all T cells. The combina- takes place among the surviving DP T cells. This second selec-
tion of the eight chains is known as the CD3/TCR complex. tion process takes place in the corticomedullary region and the
The six chains of the nonspecific CD3 portion of the complex medulla of the thymus as medullary epithelial cells express a
assist in signaling when an antigen binds to the T cells.3,7,8 wide variety of self-antigens3,7 (Fig. 4–5). Strong reactions with
These chains occur in three pairs: delta-epsilon (δ–ε), gamma- self-peptides other than MHC antigens triggers apopotosis.7 The
epsilon (γ–ε), and a tau-tau (ζ–ζ) chain that is in the cytoplasm process of elimination of clones of T cells that would be capable
of the cell.8 of an autoimmune response is called clonal deletion. This
The α and β chains of the TCR are coded for by the selec- selection process is very rigorous as evidenced by the fact that
tion of certain gene segments and deletion of others in a ran- only 1% to 3% of the DP thymocytes in the cortex survive.4
dom fashion.3 Rearrangement of the β chain coded for on
chromosome 7 occurs first; then, the α chain coded for on
chromosome 14 is rearranged afterward.3,7,8 Three different
Mature T Cells
gene segments—V, D, and J—are rearranged and combined Survivors of selection exhibit only one type of marker, either
with a constant region to code for the β chain; in contrast, there CD4 or CD8. It is not certain why one marker is downregu-
are only two gene segments combined with a constant region lated, but it may depend on which MHC protein the cell in-
for the β chain. The appearance of a functional α chain on the teracts with, how strongly they react, and to which cytokines
cell surface sends a signal to suppress any further β chain gene they are exposed.7 CD4+ T cells recognize antigen along with
rearrangements. The selection of an allele on one chromosome class II MHC protein, whereas CD8+ T cells interact with anti-
only is known as allelic exclusion. The combination of the gen and class I MHC proteins. The two separate mature T-cell
β chain with the rest of CD3 forms the pre-TRC receptor. The populations created differ greatly in function. T cells bearing
appearance of the β chain also triggers the thymocyte to the CD4 receptor are mainly T helper (Th) cells, whereas the
become CD4–positive (CD4+) and CD8–positive (CD8+).3,4 CD8+ population consists of cytotoxic T cells. Approximately
Early on, some thymocytes, representing 10% or less of the two-thirds of peripheral T cells express CD4 antigen, whereas
total number, rearrange and express two other chains— the remaining one-third express CD8 antigen.
gamma (γ) and delta (δ)—when there is not a productive re- Several subsets of Th cells exist, of which the most promi-
arrangement of DNA coding for a β chain.3,7 These cells nent are termed Th1 and Th2 cells. All Th cells have a different
proceed down a different developmental pathway and they role to play in the immune response (Fig. 4–6). Th1 cells pro-
typically remain negative for both CD4 and CD8. However, as duce interferon gamma (IFN-γ), interleukin-2 (IL-2), and
mature T cells, they appear to represent the dominant T-cell tumor necrosis factor-β (TNF-β), which protect cells against
population in the skin, intestinal epithelium, and pulmonary intracellular pathogens by activating cytotoxic lymphocytes
epithelium. Their tasks include wound healing and protection and macrophages.1 Th2 cells produce a variety of interleukins,
 MHC Thymic
DP I or II epithelial cell
CD4
Strong

binding

CD8
Apoptosis
of thymocyte CD4
MHC Thymic
DP I or II epithelial cell DP
CD4
Intermediate
Positive
selection
binding

CD8

CD8
MHC Thymic
DP I or II epithelial cell Single-
CD4 positive
No

binding
CD8
Apoptosis
of thymocyte
Positive selection of thymocytes in the cortex. Double-positive (CD4+ and CD8+) thymocytes interact with thymic epithelial
cells. If very strong bonding occurs, cells are eliminated by apoptosis. If very weak or no bonding occurs, cells are also eliminated.

CD4 or 8
Binding

to self-
TCR antigen
SP
Apoptosis

CD4 or 8 CD4 or 8

No binding Exit

to self- thymus
TCR TCR
antigen
Negative selection of thymocytes in the SP
medulla. When self-antigen is presented by a macrophage, Macrophage,
dendritic cell, or thymic epithelial cell to a thymocyte, if dendritic cell,
the T-cell receptor (TCR) binds, the thymocyte is or thymic
eliminated by apoptosis. epithelial cell

including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. The essential play an important role in suppressing the immune response to
role of the Th2 cells is to help B cells produce antibodies self-antigens. They inhibit proliferation of other T-cell popula-
against extracellular pathogens and to generally regulate B-cell tions by secreting inhibitory cytokines and the response is
activity.1 antigen-specific.1
An additional T-cell subpopulation, called T regulatory Two other T-cell subpopulations have been identified. These
(Treg) cells, possess the CD4 antigen as well as CD25.3 These cells are called Th9 and Th17, based on the type of cytokines
cells comprise approximately 5% of all CD4+ T cells.3,9, 10 Tregs they produce. Th9 cells produce interleukin-9 (IL-9) and appear
 Extracellular pathogens
Parasites

Fungi

TGF$ IL-4
Bacteria TGF"
viruses
IL-6
TGF$ IL-4 Polarizing
cytokines
IL-12
IFN%

Naìve
CD4+ TH

Differentiation

TH1 TH17 TReg TH9 TH2

T helper (Th) subsets. Depending TGF$ IL-4

IFN% IL-17 IL-10 IL-9 Effector
IL-5
upon the type of pathogen encountered, antigen- TGF$ IL-22 IL-13
cytokines
presenting cells (APCs) secrete a specific combina-
tion of polarizing cytokines that direct naïve CD4+
Th cells to further differentiate into one of five
subsets: Th1, Th2, Treg, Th9, or Th17. These special- Cytotoxic T cells Mast cells, B cells,
(#) (!$#)
ized T cells release different types of cytokines to Macrophages Eosinophils
coordinate an appropriate immune response Cell-mediated immunity Humoral immunity
against the pathogen. inflammation antiparasitic

to have a proinflammatory effect. They may play a role at ep- When antigen recognition occurs in the secondary lymphoid
ithelial surfaces by warding off fungi and extracellular bacteria.9 tissue, T lymphocytes are activated and differentiate into func-
However, in the process, they stimulate growth of hematopoietic tionally active small lymphocytes that produce cytokines. Activ-
cells, especially mast cells; as such, they may promote autoim- ities of specific cytokines include assisting B cells in commencing
mune inflammation.11 Th17 cells produce interleukin-17 (IL-17) antibody production, eliminating tumor and other target cells,
and interleukin-22 (IL-22).12 Both of these cytokines can increase rejecting grafts, stimulating hematopoiesis in the bone marrow,
inflammation and joint destruction. They have been associated and initiating delayed hypersensitivity allergic reactions. This
with autoimmune diseases such as rheumatoid arthritis, multiple type of immune response is known as cell-mediated immunity.
sclerosis, and inflammatory bowel disease.11 These activities are discussed more fully in The Role of T Cells in
All single-positive T cells spend approximately 12 days in the Adaptive Immune Response later.
the medulla.4 Additional proliferation of these carefully screened
T cells occurs; they are then released from the thymus to seed
peripheral lymphoid organs and recirculate through the blood-
Stages in B-Cell Differentiation
stream and peripheral organs approximately once every 12 to
24 hours.13 Recirculation is important for ensuring that T cells
Pro-B Cells
make contact with antigen. Resting T cells have a life span of up B cells are derived from a hematopoietic stem cell that develops
to several years in these peripheral organs. into an early lymphocyte progenitor in the bone marrow. Unlike
T cells, which leave the bone marrow and travel to the thymus
and mature B cells.16 Figure 4–7 depicts the changes that
to mature, B cells remain and mature in the bone marrow itself.
occur as B cells mature from the pro-B stage to become mem-
Bone marrow stromal cells form special niches where stem cells
ory cells or plasma cells.
and B-cell precursors reside.14 The niches keep B-cell precur-
At the earliest developmental stage, B-cell progenitors re-
sors localized in order to receive signals for differentiation.15
quire direct contact with bone marrow stromal cells.15 Several
B-cell precursors go through a developmental process that pre-
transcription, or growth, factors are necessary to differentiate
pares them for their role in antibody production and, at the
common lymphoid precursors into pro-B cells. Some of these
same time, restricts the types of antigens to which any one cell
factors are E2A, EBF (early B-cell factor), interferon regulatory
can respond. This process can be divided into three distinct
factor (IFR8), and paired box protein 5 (PAX5).14,15,17 In
phases:
addition, a cytokine called interleukin-7 (IL-7) is also necessary
• Development of mature immunocompetent B cells at this early developmental stage. All of these factors are
• Activation of B cells by antigen produced in the microenvironment of the bone marrow.
• Differentiation of activated B cells into plasma cells, which During this maturation process, the first step in the pro-B
produce antibodies phase is the rearrangement of genes that code for the heavy and
The first phase of B-cell development in the bone marrow, light chains of an antibody molecule. Although portions of each
which results in mature B cells that have not yet been ex- chain are identical for every antibody molecule, it is the so-called
posed to antigen, is known as the antigen-independent variable regions that make each antibody molecule specific for a
phase. This phase can be divided according to formation of certain antigen or group of antigens. Rearrangement of the DNA
several distinct subpopulations: pro-B cells (progenitor by cutting out certain regions is similar to the process that occurs
B cells), pre-B cells (precursor B cells), immature B cells, in T cells, where antigen specificity is built into the α and β chains

A. Pro-B Cell
B. Pre-B Cell

%
Stem cell "

C-kit C. Immature B Cell

mu chains in
cytoplasm
Bone marrow
mu and surrogate
stromal cell
light chains

Self-antigens
Apoptosis give negative
IgM
signals

Spleen Lymph
nodes

"%
Self-antigen CD1 CD23
' %
" "

IgD
IgM IgM

D. Marginal zone D. Follicular

B cells B cells

Remain Enter
in spleen circulation
B-cell development in the bone marrow. Selected markers are shown for the various stages in the differentiation of B cells.
Stages up to the formation of mature B cells occur in the bone marrow. (A) Pro-B cell. (B) Pre-B cell. (C) Immature B cell. (D) Mature B cell.
of the TCR for antigen. Heavy chains of antibody molecules are heavy chains, determine the specificity for antigen. Preexisting
coded for on chromosome 14 and light chains are coded for on diversity of receptors for antigen is a hallmark of the adaptive
chromosomes 2 and 22. immune system. It means that the capability to respond to a
Gene rearrangement of the DNA that codes for antibody specific antigen is built in before a B cell ever encounters an
production occurs in a strict developmental sequence. Re- antigen. Once surface immunoglobulins appear, µ chains are
arrangement of genes on chromosome 14, which code for the no longer detectable in the cytoplasm.
heavy-chain part of the antibody molecule, takes place first in Other surface proteins that appear on the immature B cell
a random fashion (see Chapter 5 for details). C-Kit, a receptor include CD21, CD40, and class II MHC molecules. CD21 acts
on the pro-B cell, interacts with a cell surface molecule called as a receptor for a breakdown product of the complement
stem cell factor found on stromal cells.15,16 This interaction trig- component C3, known as C3d (see Chapter 6 for details on
gers the activation process. The DNA is cleaved randomly at complement). Presence of the CD21 receptor enhances the
certain possible recombination sites and the enzyme terminal likelihood of contact between B cells and antigens because
deoxyribonucleotidyl transferase (TdT) helps to join the pieces antigens frequently become coated with complement frag-
back together by incorporating additional nucleotides in the ments during the immune response. CD40 and class II MHC
joining areas.2,15 are important for interaction of B cells with T cells.
Differentiation of pro-B cells into pre-B cells occurs upon suc- At this stage, there is evidence that self-antigens give a
cessful rearrangement of heavy-chain genes on one of the num- negative signal to immature B cells.14,15 Immature B cells that
ber 14 chromosomes.17,18 If rearrangement of genes on the first tightly bind self-antigens through cross-linking of surface
chromosome 14 is not successful, then rearrangement of genes IgM molecules receive a signal to halt development, resulting
on the second chromosome 14 occurs. If neither rearrangement in arrested maturation and cell death.15 Thus, many B cells
is successful, development of the cell is halted. Only pro-B cells capable of producing antibody to self-antigens are deleted
that successfully rearrange one set of heavy-chain genes go on from the marrow by the process of apoptosis. It is estimated
to become pre-B cells. that more than 90% of B cells die in this manner without
leaving the bone marrow.15 The elimination of B cells that
Pre-B Cells bear self-reactive receptors is known as central tolerance.
Immature B cells that survive this selection process leave the
Once heavy-chain genes are rearranged, then these genes are bone marrow and proceed to the spleen, where they become
transcribed to make the protein that will be part of an antibody mature B cells.
molecule. When synthesis of the heavy-chain part of the anti-
body molecule occurs, the pre-B stage begins.15 The first heavy
chains synthesized are the µ chains, which belong to the class
Mature B Cells
of immunoglobulins called immunoglobulin M (IgM). The In the spleen, immature B cells develop into mature cells
µ chains accumulate in the cytoplasm.15 Pre-B cells may also known as either marginal zone B cells or follicular B cells.2 Mar-
express µ chains on the cell surface, accompanied by an un- ginal B cells remain in the spleen in order to respond quickly
usual light chain molecule called a surrogate light chain.15 to any blood-borne pathogens they may come into contact
Surrogate light chains consist of two short polypeptide chains with.2 Follicular B cells migrate to lymph nodes and other
that are noncovalently associated with each other, along with secondary organs. Unlike marginal B cells that remain in the
two shorter chains, Ig-α and Ig-β, which are signal-transducing spleen, follicular B cells are constantly recirculating through-
subunits14 (see Fig. 4–7B). The combination of the two heavy out the secondary lymphoid organs. In addition to IgM, all
chains along with Ig-α, Ig-β and the surrogate light chains form mature B cells exhibit immunoglobulin D (IgD), another
the pre-B cell receptor (pre-BCR). It appears that only pre-B class of antibody molecule, on their surface (see Fig. 4–7D).
cells expressing the µ heavy chains in association with surro- Both IgM and IgD have the same specificity for a particular
gate light chains survive and proceed to further differentia- antigen or group of antigens. These surface immunoglobu-
tion.14,15 Signaling through the pre-B receptors formed stimulates lins provide the primary activating signal to B cells when
a burst of clonal expansion. If, however, gene rearrangement contact with antigen takes place.19,20 IgD is not required for
does not work, then B-cell development is halted and cells are B-cell function, but it may prolong the life span of mature
destroyed by apoptosis.15 B cells in the periphery. Unless contact with antigen occurs,
the life span of a mature B cell is typically only a few days.2
Immature B Cells If, however, a B cell is stimulated by antigen, it undergoes
transformation to a blast stage that eventually forms memory
Immature B cells are distinguished by the appearance of cells and antibody-secreting plasma cells (Fig. 4–8). This
complete IgM antibody molecules on the cell surface 2,16 (see process is known as the antigen-dependent phase of B-cell
Fig. 4–7C). This indicates that rearrangement of the genetic development. The production of antibodies by plasma cells
sequence coding for light chains on either chromosome 2 or is called humoral immunity.
22 has taken place by this time. Completion of light chain
rearrangement commits a cell to produce an antibody mol-
ecule with specificity for a particular antigen or group of re-
Plasma Cells
lated antigens. IgM molecules thus serve as the receptor for Plasma cells are spherical or ellipsoidal cells between 10 and
antigen. Variable regions, which occur on both the light and 20 µm in size that are characterized by the presence of
 Plasma cells

Cytokines

Immature B Cell Mature B Cell

CD25
Antigen

Immunoglobulins

IgM IgM IgD IgM IgD

Exit bone Spleen and

marrow secondary lymphoid IgM
organs (capping)

Memory cells

B-cell activation in peripheral lymph nodes. B cells capture specific antigen by means of immunoglobulin receptors. The activity
of cytokines produced by Th cells produces transformation of naïve B cells into antibody-producing plasma cells and memory cells.

abundant cytoplasmic immunoglobulin and little to no sur- the most fully differentiated lymphocyte and their main
face immunoglobulin21 (Fig. 4–9). The nucleus is eccentric function is antibody production. They are not normally
or oval with heavily clumped chromatin that stains darkly. found in the blood; rather. they are located in germinal cen-
An abundant endoplasmic reticulum and a clear well-defined ters in the peripheral lymphoid organs or they reside in
Golgi zone are present in the cytoplasm. Plasma cells represent the bone marrow. In the bone marrow, plasma cells can
survive in niches surrounded by stromal cells. Stromal cells
provide chemical stimulation by cytokines, which allow
plasma cells to be long-lived and continually produce anti-
bodies.1 Plasma cells found elsewhere are nondividing and,
after several days of antibody production, they die without
further proliferation.17

The Role of T Cells in the Adaptive

Immune Response
Interaction between T cells and antigen-presenting cells (APCs)
is the initiating event in the adaptive immune response.9 APCs
such as macrophages and dendritic cells are first activated
during the innate immune response through their pattern
recognition receptors (see Chapter 3). Cells displaying
A typical plasma cell. (From Harmening DM. Clinical processed antigen on the outside along with either class I or
Hematology and Fundamentals of Hemostasis. 5th ed. Philadelphia, class II MHC proteins are transported to the T-cell zones of the
PA: F.A. Davis; 2009.) lymph node or spleen. As you recall from Chapter 2, class I
MHC presents antigen that is from intracellular pathogens such Action of Cytotoxic T Cells
as viruses to CD8+ T cells, whereas class II MHC presents anti-
gen from extracellular pathogens such as bacteria processed by T cells that bear the CD8 marker have a different role to play than
a phagocytic cell to CD4+ T cells. that of the Th cells. Once activated by antigen in the lymph nodes
Each mature T cell has a unique receptor for antigen or spleen, cytotoxic T cells leave the secondary lymphoid tissue
(TCR). T cells circulate continuously through the blood- and circulate to sites of infection. Here they bind and kill infected
stream, lymph nodes, and secondary lymphoid tissue search- cells by triggering apoptosis. This action differs from that of NK
ing for antigen. It is estimated that each naïve or unstimulated cells because it is antigen-specific. Cytotoxic T cells recognize
T cell circulates from the lymph nodes to the blood and back antigen in association with class I MHC complexes, which were
again within 12 to 24 hours.9 Only about 1 in 105 naïve described in Chapter 2. Because all nucleated host cells express
T cells are specific for any given antigen, so recirculating in- class I antigens on their surfaces, cytotoxic T cells act as a primary
creases the chances that a T cell will make contact with the defense against intracellular pathogens such as viruses, as well as
appropriate antigen.9 other altered host cells such as tumor cells that exhibit new anti-
As discussed in the section T-Cell Differentiation earlier, gens. The cytotoxic T cells bind to altered host cells by using the
T cells have either CD4 or CD8 on their cell surface. CD4+ TCR receptor for antigen and by CD8, which recognizes class I
T cells are known as Th cells and CD8+ T cells are called cy- MHC molecules (Fig. 4–11).
totoxic T cells (Tc). Each of these two main types of T cells has Naïve CD8+ T cells require 5 to 8 days after antigen activa-
a different role to play in the adaptive immune response. The tion to differentiate into cytotoxic lymphocytes. During this
function of the Th cells will be discussed first. time, they proliferate to increase the numbers that can respond
to the same antigen. After differentiation, they migrate to the
Action of T Helper Cells affected tissue to find and destroy target cells.19
Cytotoxic T cells can kill target cells in two major ways.
The first activating signal to induce transformation of a T cell Once antigen recognition occurs, the cytotoxic T cells either
occurs when a CD4+ T cell encounters an antigen along release the contents of granules that damage the cell or they
with a class II MHC molecule and binds by using its antigen bind to the host cell and, using intracellular signaling, in-
receptor. CD4 acts as a co-receptor to stabilize binding. duce apoptosis. In either case, the target cell is induced to
A second signal is provided by the binding of CD28 on the undergo apoptosis, usually within 30 minutes of contact19,23
T cell with CD80 and CD86 found on APCs.19 Cytokines, (see Fig. 4–11).
chemical messengers, provide an additional signal (see Granules within cytotoxic T cells contain two different types
Chapter 6). of toxins: granzymes and perforins. Granzymes are a class of en-
Within 1 to 2 days after antigen recognition has occurred, zymes called serine proteases and perforins are pore-forming
T lymphocytes are transformed into large activated blast cells proteins that insert themselves into the target cell membrane.
that are characterized by polyribosome-filled cytoplasm. Once a cytotoxic T cell has bound to a target cell, the granules
They then begin cell division and generate progeny with move within the T cell to the site where the target cell has bound.
an antigen receptor that is identical to that of the parent Granules fuse with the T-cell membrane and the perforins and
T cell. These functionally active small lymphocytes produce granzymes are released into the space between the target cell and
cytokines. The two main subsets of Th cells, Th1 and Th2, the T cell. Perforins insert themselves into the target cell mem-
secrete different types of cytokines and affect different classes brane and polymerize, forming pores in the membrane.
of cells. The Th1 subset secretes IL-2, interferon-gamma Granzymes enter through the pores. Once the granzymes are in
(IFN-γ) and TNF-β, which are responsible for the activation the cytoplasm of the target cell, a cascade of events is initiated
of cytotoxic T lymphocytes and macrophages. Th2 cells se- that fragment the target cell DNA into small pieces.23,24
crete interleukins that regulate B-cell activity. Most antigens Granzymes themselves do not directly break down DNA , but
encountered in the body are so-called T-dependent anti- they activate a nuclease that destroys the target cell DNA as well
gens, meaning that T-cell help is required in order for B cells as any viral DNA that may be contained inside it. Granzymes
to respond to antigen (Fig. 4–10). can also activate enzymes in the target cell that disrupt the cell’s
In addition to effector cells that immediately secrete inter- mitochondria.24 The end result in either case is apoptosis or
leukins, T memory cells are also generated. T memory cells cell death.
arise early in the course of an immune response, but the actual
cells of origin are not yet known. They may arise independently
from effector cells or they may arise as soon as the original The Role of B Cells in the Adaptive
T cell is stimulated.9 T memory cells have a higher affinity for Immune Response
antigen than unstimulated T cells and are capable of immediate
cytokine production when they reencounter the initiating Antigen-dependent activation of B cells takes place in the pri-
antigen.22 T memory cells are able to proliferate sooner than mary follicles of peripheral lymphoid tissue. Follicular den-
naïve T cells, express a broader array of cytokines, and appear dritic cells present antigen to B cells and thus play a key role
to persist for years.22 in the immune response.1,2
 Macrophage

Bacterial
antigen

CD4 MHC
TCR class II

TH cell

Cytokines

IL-2 receptor

Activated TH cell

Memory
Clonal expansion of effector CD4! T cells CD4! T cells

Cytokines

CD8

Neutrophils Macrophages Cytotoxic T cell B cell

Inflammation Cellular immunity Humoral immunity
Activation of Th cells. Exposure to antigen presented by macrophages causes production of CD25 receptors for interleukin-2
(IL-2). IL-2 causes sensitized CD4+ T cells to secrete cytokines, resulting in CD4+ effector cells that have various functions. Some CD4+ cells
secrete interleukins that recruit macrophages and neutrophils, whereas others activate CD8+ T cells to increase cytotoxicity against virally
infected cells. Activated Th cells also enhance antibody production by B cells.

intracellular signaling pathways that allow stimulated B cells to

Response to T-Dependent Antigens interact with T cells.16 The second signal is provided by Th cells
When B cells respond to T-dependent antigens, the B cells re- themselves, which bind to the B cell both through its antigen
quire two signals to be activated. The first occurs when antigen receptor and through CD40 on the B cell and CD40L on the
binds to membrane immunoglobulin receptors on the B-cell sur- activated Th cell.13,16 The bound T cell then delivers cytokines
face and cross-links them. Cross-linking leads to activation of and other signals to fully activate the B cell (Fig. 4–12).
 TD antigen
Connections
Mutation of CD40L Gene Causes Cytokines
3
Immunodeficiency
1
When the delicate balance between T- and B-cell interaction is
disrupted, immune deficiencies may result. An X-linked mutation
causing a CD40L deficiency on Th cells was found to affect cer-
tain immunoglobulin classes. Normal or increased levels of IgM
are present, but B cells are unable to switch to producing other B cell CD40/CD40L TH cell
antibody classes having a higher affinity for the initiating antigen. 2
A
IgG and IgA levels are decreased, leading to an increased sus-
ceptibility to infection in individuals with this mutation. See TI-1 antigen
Chapter 17 for further discussion on immunodeficiency diseases.
1

CD8 MHC 2
class I TLR

TCR
Antigen
B B cell
TC cell Target cell Activating signals for T-dependent and T-independent
antigens. (A) T-dependent antigens bind to immunoglobulin recep-
tors on B cells. The antigen is processed and delivered to CD4+
T cells. The Th cell binds by means of its CD3-TCR complex and
delivers further activating signals through binding of CD40 on the
B cell to the CD40L receptor on the Th cell. Cytokines are released
from the T cell, which enhance B-cell transformation to plasma
cells. (B) T-independent antigens can bind to B cells through im-
munoglobulin receptors and trigger B-cell transformation directly.
Vesicle
Several antigen receptors must be cross-linked in order to activate a
Perforin B cell directly. Antigens can also be bound to B cells’ innate immune
Granzyme receptors such as TLRs.
Intercellular
Cytotoxic Cytotoxic T cell space
T cell membrane

Activated B cells exhibit identifying markers that include

CD25, which is found on both activated T and B cells and
Target cell which acts as a receptor for IL-2, a growth factor produced by
Perforin pore T cells. Additional receptors that appear at this time are specific
partially Completed pore;
for other growth factors produced by T cells. Once a B cell has
Intact target assembled granzyme passing become activated, it will replicate. All the daughter cells will
cell membrane
through have the same receptor as the parent cell for the initiating anti-
gen. The daughter cells of proliferating B cells either migrate
into the T-cell zone in lymph nodes and differentiate into
plasma cells that secrete IgM in about 4 days or they enter
B-cell follicles to form germinal centers.1,13
Within the follicles, however, B cells undergo further dif-
ferentiation under the influence of follicular Th cells. The
CD40 on B cells must interact with CD40L on Th cells in order
for germinal center formation to occur.16 Here, both plasma
Target cell death
cells and memory cells are formed (Fig. 4–13). Memory cells
are progeny of B cells that have been exposed to antigen; they
Activation of cytotoxic T cells. The CD8+ T cell recog-
nizes foreign antigen along with class I MHC. When binding occurs,
are characterized by a long life span and a rapid response to
granules move toward the point of contact with the target cell. second exposure to the triggering antigen.25,26 They are similar
Granules fuse with the membrane and release perforin. Perforin in- in appearance to unstimulated B cells, but they remain in an
serts itself into the target cell membrane and polymerizes to form a activated state for months or years, ready to respond to the ini-
pore. Contents of the granules are released, triggering apoptosis of tial antigen. CD27 is used as a marker to identify memory cells
the target cell. because they are similar in appearance to mature B cells.2 It is
 APC

B7 MHC class II
CD28 Antigen
TCR CD4

TH cell

CD40L CD40

CD4 CD4

Clonal TH cell B cell

expansion
Cytokine IL-2
production

Proliferation and differentiation

Plasma
cell
Memory
B cells
Cytokines

T- and B-cell cooperation in the immune re-

sponse. CD4+ T cells recognize exogenous antigen on a
macrophage along with class II MHC. Binding between CD28
and B7 enhances interaction between the cells. Th cells go IgM
through clonal expansion and produce cytokines, including
interleukin-2 (IL-2). B cells capable of responding to the same
antigen present antigen to Th cells through the class II MHC
receptor. The TCR binds antigen and CD4 binds to class II
MHC. CD40L bind to CD40, enhancing the reaction. Cytokine
production by the T cell causes B cells to proliferate and pro-
duce plasma cells, which secrete antibody. IgG

this built-in memory that distinguishes the adaptive immune have a relatively short life span. Some plasma cells migrate
response from innate immunity. to the bone marrow, as mentioned previously, where they
Gene rearrangement in B cells in germinal centers allows for provide long-lasting memory. Others locate to the gastroin-
expression of different classes of immunoglobulins. Cytokines testinal tract, where they secrete an antibody type called IgA.
acting on B cells determine the class of antibody that will be The IgA serves as a protection against organisms that may
expressed. The variable regions remain the same, so the antigen reach the gut.13
specificity does not change. Rearrangement only occurs in the
constant region, which determines the particular class of anti-
body that will be expressed. These altered B cells with rearranged
Response to T-Independent Antigens
DNA then differentiate into plasma cells capable of making Some antigens are able to elicit antibody formation in the absence
antibody other than IgM. Most class switching results in of T cells. These antigens are called T-independent antigens.
IgG-producing plasma cells. Therefore, the antibody found in the Such antigens are able to interact with multiple immunoglobulin
greatest concentration in the serum is IgG. (See Chapter 5 for a receptors on a B cell to cross-link them and induce proliferation
complete discussion on types of immunoglobulins.) and antibody production2,17 (see Fig. 4-12B). Examples of these
Once formed, plasma cells leave the lymph nodes and cir- include plant lectins, polymerized proteins with repeating
culate in the peripheral tissues. They secrete antibodies and molecular patterns, and lipopolysaccharides found in bacterial
cell walls.17,25 Typically, these antigens produce IgM only
because the induction of memory cells does not occur to any • Adaptive immunity is characterized by specificity, the
great extent.25 ability to remember a prior exposure to an antigen, and
an increased response upon re-exposure to that same
antigen.
Laboratory Identification • B cells mature in the bone marrow itself, whereas T cells
of Lymphocytes acquire their specificity in the thymus.
• B cells can be recognized by the presence of surface anti-
Identification of lymphocytes as either T cells or B cells may be gens, or CDs, that are detected by monoclonal antibodies.
useful in diagnosis of any of the following states: malignancies B-cell markers include CD19, class II MHC proteins, and
such as leukemias and lymphomas; immunodeficiency diseases surface immunoglobulins.
involving either T or B cells or both; and acquired immunodefi- • Surface immunoglobulins on B cells are receptors for
ciency disease (AIDS). In some immunodeficiency diseases such antigen. Their specificity is built in as the B cell matures.
as X-linked hypogammaglobulinemia, B cells are frequently ab- • Antigen-independent development of B cells occurs in the
sent, whereas in severe combined immunodeficiency disease bone marrow, whereas the antigen-dependent phase takes
(SCID) both T and B cells are either absent or present in very low place in the secondary lymphoid organs.
numbers. Because the human immunodeficiency virus (HIV) in- • Class II MHC proteins allow B cells to interact with
fects and progressively kills CD4+ T cells, assays for CD4+ T cells T helper cells in the production of antibodies.
are useful in evaluating the stage of infection. Laboratory analysis • When contact with a specific antigen occurs, B cells dif-
usually involves distinguishing the following lymphocyte subsets: ferentiate into plasma cells that produce antibodies and
T cytotoxic cells (CD8), Th cells (CD4), and B cells. The gold memory cells that are able to respond more quickly the
standard for testing is cell flow cytometry.27 next time the same antigen is encountered.
Cell flow cytometry is an automated system for identifying • Production of antibodies is known as humoral immunity.
cells based on the scattering of light as cells in a stream of fluid • T cells are distinguished by the presence of CD3, CD2,
flow in single file by a laser beam. Flow cytometry is able to and either CD4 or CD8.
segregate lymphocytes into subsets using a technique that relies • Cells that express CD4 belong to a T-cell subset that
on labeled monoclonal antibodies against specific surface anti- includes helper/inducer cells.
gens. Monoclonal antibodies are highly specific antibodies. • CD8+ T cells are cytotoxic cells that are able to destroy
Some of the more common antigens tested for include CD2, cancer cells or virally infected host cells by producing
CD3, CD4, and CD8 on T cells and CD19, CD20, CD22, and perforins and granzymes.
surface immunoglobulin on B cells. Fluorescent antibodies are • CD3/TCR is the T-cell receptor for antigen. The major por-
used to screen for subpopulations, such as B cells, Th cells, tion of it is common to all T cells, but two chains—alpha
and T cytotoxic cells. Each antibody has a different fluorescent and beta—contain variable regions that can bind to only
tag. The principles of flow cytometry are discussed more fully certain antigens.
in Chapter 12. • Positive selection of immature T cells is based on interac-
Recently, some point-of-care testing has been developed tion with the unique MHC antigens of the host.
using either fluorescent or antibody-labeled beads. These • Negative selection in T-cell maturation is based on inter-
technologies measure the CD4 count and report it as a per- action with self-antigens of the host. If a T cell recognizes
centage of the total T-cell count. Although not as accurate as self-antigens, then it is destroyed by apoptosis.
flow cytometry, it can be used in areas of the world where • T cells are responsible for cell-mediated immunity, which
there is limited access to laboratories with sophisticated involves production of cytokines that serve as regulatory
equipment.28,29 factors for the immune response.
• Laboratory determination of individual lymphocyte
populations is essential in diagnosis of such conditions as
SUMMARY lymphomas, immunodeficiency diseases, unexplained
infections, or acquired immune diseases such as AIDS.
• All undifferentiated lymphocytes arise in the bone marrow • Lymphocytes are identified using monoclonal antibodies
from hematopoietic stem cells. They mature in the primary directed against specific surface antigens. They are enu-
lymphoid organs and are the key cell involved in the adap- merated through the use of cell flow cytometry, which
tive immune response. categorizes cells on the basis of light scattering.
Study Guide: Comparison of T and B Cells
T CELLS B CELLS
Develop in the thymus Develop in the bone marrow
Found in blood (60–80% of circulating lymphocytes), Found in bone marrow, spleen, lymph nodes
thoracic duct fluid, lymph nodes
Identified by rosette formation with SRBCs Identified by surface immunoglobulin
End products of activation are cytokines End product of activation is antibody
Antigens include CD2, CD3, CD4, CD8 Antigens include CD19, CD20, CD21, CD40, class II MHC
Located in paracortical region of lymph nodes Located in cortical region of lymph nodes

SRBC = Sheep red blood cells

CASE STUDIES
1. A 2-year-old boy is sent for immunologic testing because 2. You and a friend of yours in your immunology class are
of recurring respiratory infections, including several bouts discussing how the body is able to fight infection. Your
of pneumonia. The results show decreased immunoglob- friend states that as long as you have a good innate
ulin levels, especially of IgG. Although his WBC count immune system and you can make antibodies, then a
was within the normal range, the lymphocyte count was decrease in CD8+ T cells is not really important.
low. Flow cytometry was performed to determine the lev-
Question
els of different classes of lymphocytes. The result showed
a decrease in CD4+ cells. The CD19+ lymphocyte popu- a. How do you answer your friend?
lation was normal.
Questions
a. How can these findings be interpreted?
b. How can this account for his recurring infections?

REVIEW QUESTIONS
1. Which MHC molecule is necessary for antigen recog- 3. Humoral immunity refers to which of the following?
nition by CD4+ T cells? a. Production of antibody by plasma cells
a. Class I b. Production of cytokines by T cells
b. Class II c. Elimination of virally infected cells by
c. Class III cytotoxic cells
d. No MHC molecule is necessary. d. Downregulation of the immune response

2. Which would be characteristic of a T-independent 4. Where does antigen-independent maturation of

antigen? B lymphocytes take place?
a. The IgG antibody is produced exclusively. a. Bone marrow
b. A large number of memory cells are produced. b. Thymus
c. Antigens bind only one receptor on B cells. c. Spleen
d. It consists of a limited number of repeating d. Lymph nodes
determinants.
 5. In the thymus, positive selection of immature T cells is 13. Which of the following would represent a double-
based upon recognition of which of the following? negative thymocyte?
a. Self-antigens a. CD2–CD3+CD4–CD8+
b. Stress proteins b. CD2–CD3–CD4+CD8–
c. MHC antigens c. CD2+CD3+CD4–CD8–
d. µ chains d. CD2–CD3–CD4+CD8–

6. Which of these are found on a mature B cell? 14. Which of the following best describes the T-cell
a. IgG and IgD receptor for antigen?
b. IgM and IgD a. It consists of IgM and IgD molecules.
c. Alpha and beta chains b. It is the same for all T cells.
d. CD3 c. It is present in the double-negative stage.
d. Alpha and beta chains are unique for each antigen.
7. How do cytotoxic T cells kill target cells?
a. They produce antibodies that bind to the cell. 15. A cell flow cytometry pattern belonging to a 3-year-
b. They engulf the cell by phagocytosis. old patient showed the following: normal CD4+ T-cell
c. They stop protein synthesis in the target cell. count, normal CD19+ B-cell count, low CD8+ T-cell
d. They produce granzymes that stimulate apoptosis. count. Which type of immunity would be affected?
a. Production of antibody
8. Which of the following can be attributed to b. Formation of plasma cells
antigen-stimulated T cells? c. Elimination of virally infected cells
a. Humoral response d. Downregulation of the immune response
b. Plasma cells
c. Cytokines 16. Which of the following is a unique characteristic of
d. Antibody adaptive immunity?
a. Ability to fight infection
9. Which is a distinguishing feature of a pre-B cell? b. Ability to remember a prior exposure to a pathogen
a. µ chains in the cytoplasm c. A similar response to all pathogens encountered
b. Complete IgM on the surface d. Process of phagocytosis to destroy a pathogen
c. Presence of CD21 antigen
d. Presence of CD25 antigen 17. Clonal deletion of T cells as they mature is important
in which of the following processes?
10. When does genetic rearrangement for coding of a. Elimination of autoimmune responses
antibody light chains take place during B-cell b. Positive selection of CD3/TCR receptors
development? c. Allelic exclusion of chromosomes
a. Before the pre-B cell stage d. Elimination of cells unable to bind to MHC
b. As the cell becomes an immature B cell antigens
c. Not until the cell becomes a mature B cell
d. When the B cell becomes a plasma cell 18. Where do germinal centers occur?
a. In the thymus
11. Which of the following antigens are found on the b. In the bone marrow
T-cell subset known as helper/inducers? c. In peripheral blood
a. CD3 d. In lymph nodes
b. CD4
c. CD8
d. CD11

12. Where does the major portion of antibody production

occur?
a. Peripheral blood
b. Bone marrow
c. Thymus
d. Lymph nodes
 Antibody Structure
and Function

After finishing this chapter, you should be able to: TETRAPEPTIDE STRUCTURE OF
IMMUNOGLOBULINS
1. Describe the structure of a typical immunoglobulin.
THE NATURE OF LIGHT CHAINS
2. Identify the electrophoretic fraction of serum that contains the majority
of immunoglobulins. HEAVYCHAIN SEQUENCING
3. Differentiate between isotypes, allotypes, and idiotypes. HINGE REGION
4. Characterize the five immunoglobulin types found in humans. THREEDIMENSIONAL STRUCTURE
OF ANTIBODIES
5. Differentiate between light and heavy chains of immunoglobulins.
Immunoglobulin G (IgG)
6. Describe experimental evidence for the structure of IgG.
Immunoglobulin M (IgM)
7. Discuss how the IgG subclasses differ in functional capability.
Immunoglobulin A (IgA)
8. Explain how the structure of IgM differs from that of IgG.
Immunoglobulin D (IgD)
9. Relate differences in structure of the five immunoglobulin classes to
their function. Immunoglobulin E (IgE)
10. Describe the secretory component of IgA. THEORIES TO EXPLAIN ANTIBODY
DIVERSITY
11. Discuss how IgD differs from other immunoglobulin types.
Ehrlich’s Side-Chain Theory
12. Identify the types of cells that IgE binds to in allergic reactions.
Clonal Selection Hypothesis
13. Compare the primary and secondary responses to antigen.
GENES CODING FOR
14. Describe how recent knowledge about immunoglobulin genes supports
IMMUNOGLOBULINS
the clonal selection hypothesis.
Rearrangement of Heavy-Chain
15. Discuss the process of monoclonal antibody production.
Genes
16. Relate the influence of monoclonal antibodies to current laboratory
Light Chain Rearrangement
testing practices.
MONOCLONAL ANTIBODY
Hybridomas
Clinical Applications
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Allelic exclusion Domains and neutrophils Idiotype Monoclonal antibodies
Allotypes F(ab')2 Immunoglobulin Primary response
Antibody Fab fragments Isotype Secondary response
Bence Jones proteins FC fragment Joining (J) chain Secretory component (SC)
Class switching Heavy (H) chains Kappa (κ) chains Tetrapeptide
Clonal selection hypothesis Hinge region Lambda (λ) chains Variable region
Constant region Hybridoma Light (L) chains

When B lymphocytes are stimulated by antigen and undergo involving over- or underproduction of antibodies, so it will
differentiation, the end product is an antibody, also known be discussed in later chapters in relation to specific diseases.
as an immunoglobulin. Immunoglobulins, glycoproteins Immunoglobulins are considered to be the main humoral
found in the serum portion of the blood, constitute approx- element of the adaptive immune response. They play an essential
imately 20% of plasma proteins in healthy individuals. All role in antigen recognition and in biological activities related to
immunoglobulins are composed of 86% to 98% polypeptide the immune response such as opsonization and complement ac-
and 2% to 14% carbohydrate.1 Some of the early experiments tivation. Immunoglobulins are divided into five major classes on
attempting to determine the structure of immunoglobulins the basis of a part of the molecule called the heavy chain. These
involved serum protein electrophoresis. In electrophoresis, classes are designated as IgG, IgM, IgA, IgD, and IgE (with
serum is placed on an agarose gel and an electrical current is Ig being the abbreviation for immunoglobulin). The heavy
applied to separate out the proteins. If electrophoresis is car- chains are γ, µ, α, δ, and ε, respectively. Although each class has
ried out at a pH of 8.6, most serum proteins can be separated distinct properties, all immunoglobulin molecules share many
out on the basis of size and charge. Five distinct bands are common features and their basic structure is similar.
obtained in this manner. Immunoglobulins are the slowest The immunoglobulin structure is also similar to other
moving proteins and appear primarily in the gamma (γ) band molecules belonging to the immunoglobulin superfamily, a
(Fig. 5–1). It was discovered that the gamma band contained group of glycoproteins that share a common ancestral gene.
most of the antibody activity. Hence, an early name for anti- This gene originally coded for 110 amino acids, but it has
body was gamma globulin. Serum protein electrophoresis been duplicated and mutated over time. However, each type
is used today as a screening tool for some clinical diseases of immunoglobulin is made up of a number of regions called
domains, which consist of approximately 110 amino acids
each. Although the different immunoglobulin classes may
have differing numbers of domains, the three-dimensional
structure of each is essentially the same.
This chapter presents the nature of this generalized struc-
ture and discusses the characteristics of each immunoglobulin
type. Specific functions for each of the classes are examined in
relation to structural differences. Knowing the type of im-
munoglobulin produced is important in the diagnosis of many
infectious diseases, allergies, and autoimmune diseases. There
Normal pattern
are different laboratory tests for particular antibodies based on
the immunoglobulin class. This is especially important in
blood banking because some antibodies are more harmful than
!1 !2 " #
others when considering giving blood to a patient. Thus,
knowing the particular type of antibody produced is extremely
helpful in clinical diagnosis.

! " Tetrapeptide Structure

of Immunoglobulins
alb !1 !2 " #
Serum electrophoresis. Serum is subjected to an elec- All immunoglobulin molecules are made up of a basic four-
trical charge, and proteins are separated out on the basis of size and chain tetrapeptide unit that consists of two large chains
charge. Antibodies are found in the gamma region because they are called heavy or H chains and two smaller chains called light
the slowest proteins and have a charge that is close to neutral. or L chains. Each chain has a single variable region and one
Hence, they do not move far from the origin. or more constant regions. The variable region is unique to
each specific antibody. These chains are held together by non- VH
covalent forces and disulfide interchain bridges. The basic * *
structure of immunoglobulins was first discovered in the CH1
1950s and 1960s because of the efforts of two men: Gerald Fab region
VL
Edelman, working at the Rockefeller Institute in the United
S S
States, and Rodney Porter, working at Oxford University in
CL
England. They chose to work with immunoglobulin G (IgG), S
S S
S Light chain
Hinge region S S
the most abundant of all the antibodies. For their contribu-
tions, these men shared the Nobel Prize in physiology and
medicine in 1972. CH2
Edelman’s work centered on using the analytic ultracen- Complement and Fc region Heavy chain
trifuge, a very high speed centrifuge, to separate out im- Fc receptor binding
munoglobulins on the basis of molecular weight.2 He found CH3
that intact IgG molecules had a sedimentation coefficient of A
7 S (the Svedberg unit [S] indicates the sedimentation rate in
an analytical ultracentrifuge). Larger molecules will travel
farther and thus have a larger sedimentation coefficient. On ob-
taining a purified preparation of IgG, Edelman used 7 M urea Fab Fab
to unfold the molecule. Once unfolded, the exposed sulfhydryl
bonds could be broken by a reducing agent such as mercap-
toethanol. After such treatment, the material was subjected
again to ultracentrifugation, and two separate fractions, one at
3.5 S and one at 2.2 S, were obtained.
The 3.5 S fraction, with a molecular weight of approxi-
mately 50,000, was designated the H chain; the 2.2 S fraction, Fc
with a molecular weight of 22,000, was named the L chain.3
These two pieces occurred in equal amounts, indicating that
the formula for IgG had to be H2L2. This is the generalized for-
mula for all immunoglobulins (Fig. 5–2). In any one im-
munoglobulin molecule, the two H chains are identical as are Papain
the two L chains. B digestion
Porter’s work was based on the use of the proteolytic en-
zyme papain, which was used to cleave IgG into three pieces
of about equal size, each having a sedimentation coefficient of
3.5 S and representing a molecular weight of approximately F(ab’)2
45,000 to 50,000 d.3,4 Carboxymethyl cellulose ion exchange
chromatography separated this material into two types of frag-
ments, one of which spontaneously crystallized at 4°C. This
fragment, known as the Fc fragment (for “fragment crystalliz-
able”), had no antigen-binding ability and is now known to
represent the carboxy-terminal halves of two H chains that are
held together by S–S bonding.3 The Fc fragment is important Fc'
in effector functions of immunoglobulin molecules, which
include opsonization and complement fixation.
The remaining two identical fragments were found to have
antigen-binding capacity and were named Fab fragments (frag-
ment antigen binding). Because precipitation would not occur
if Fab fragments were allowed to react with antigen, it was Pepsin
guessed that each fragment represented one antigen-binding site C digestion

and that two such fragments were present in an intact antibody Basic structure of an immunoglobulin molecule, as
discovered by Gerald Edelman. (A) Immunoglobulin G is made up of
molecule. Such a molecule would be able to form a cross-linked
two H chains (50,000 MW each) and two L chains (22,000 MW each),
complex with antigen and the complex would precipitate. Each
held together by disulfide bonds. Intrachain disulfide bonds create
Fab fragment thus consists of one L chain and one-half of an looped regions or domains. The amino-terminal end of each chain
H chain held together by disulfide bonding4,5 (see Fig. 5–2). is a variable region, whereas the carboxy-terminal end consists of
Alfred Nisonoff used pepsin to obtain additional evidence one or more constant regions. (B) Papain digestion yields two Fab
for the structure of immunoglobulins.3 This proteolytic enzyme fragments and an Fc portion. (C) Pepsin digestion yields an F(ab’)2
was found to cleave IgG at the carboxy-terminal side of the fragment with all the antibody activity, as well as an Fc′ portion.
interchain disulfide bonds, yielding one single fragment with amino-terminal end constitute the variable domain; the remain-
a molecular weight of 100,000 d and all the antigen-binding ing amino acids can typically be divided up into three or more
ability, known as F(ab')2. An additional fragment called FC' constant regions with very similar sequences, designated CH1,
was similar to FC except that it disintegrated into several CH2, and CH3. Constant regions of the H chain are unique
smaller pieces. to each class and give each immunoglobulin type its name.
The combined work of the three researchers provided a Hence, IgG has an γ H chain, IgM a µ chain, IgA an α chain,
basic picture of the four-chain unit of the immunoglobulin IgD a δ chain, and IgE an ε chain. Each of these represents an
molecule, which indicated that each L chain was bonded to an isotype, a unique amino acid sequence that is common to all
H chain by means of an S–S bond and the H chains were joined immunoglobulin molecules of a given class in a given species.
to each other by one or more S–S bonds (see Fig. 5–2). The Minor variations of these sequences that are present in some
exact number of disulfide bonds differs among antibody classes individuals but not others are known as allotypes (Fig. 5–3).
and subclasses. Allotypes occur in the four IgG subclasses, in one IgA subclass,
Once the overall structure of immunoglobulins was discov- and in the κ L chain.3 These genetic markers are found in the
ered, scientists wanted to look more closely at the makeup of constant region and are inherited in simple Mendelian fashion.
the individual chains. In order to perform amino acid analysis Some of the best-known examples of allotypes are variations of
of individual immunoglobulin chains, it was necessary to have the γ chain known as G1m3 and G1m17.
a source for pure antibody molecules. Immunologic reactions The variable portions of each chain are unique to a specific
in both animals and humans normally produce a mixture of antibody molecule, and they constitute what is known as the
antibodies, making it difficult to isolate a single antibody type. idiotype of the molecule. The amino-terminal ends of both
This proved to be an obstacle to early research on the nature L and H chains contain these regions, which are essential to
of immunoglobulins. the formation of the antigen-binding site. Together they serve
as the antigen-recognition unit.

The Nature of Light Chains

Hinge Region
The difficulty in obtaining a significant amount of a specific im-
munoglobulin for amino acid analysis was overcome by the The segment of H chain located between the CH1 and CH2
chance discovery that Bence Jones proteins, found in the urine regions is known as the hinge region. It has a high content
of patients with multiple myeloma, were in fact L chains of proline and hydrophobic residues; the high proline content
that were being secreted by the malignant plasma cells.3 allows for flexibility.3 This ability to bend lets the two antigen-
Dr. Henry Bence Jones discovered the proteins in 1845 when binding sites operate independently and engage in an angular
he noted their peculiar behavior: When heated to 60°C, they motion relative to each other and to the FC stem. Thus, two
precipitate from urine, but on further heating to 80°C, they Fab arms can cover a good bit of territory.5 Such flexibility also
redissolve. These characteristics made it possible to isolate the assists in effector functions including initiation of the comple-
L chains and obtain the amino acid sequence. ment cascade (see Chapter 7 for details) and binding to cells
Analysis of several Bence Jones proteins revealed that there with specific receptors for the Fc portion of the molecule.5
were two main types of L chains, which were designated Gamma, delta, and alpha chains all have a hinge region, but
kappa (κ) chains and lambda (λ) chains. Each contained mu and epsilon chains do not. However, the CH2 domains of
between 200 and 220 amino acids; from position number 111 these latter two chains are paired in such a way as to confer
onward (the amino terminus is position number 1), it was dis- flexibility to the Fab arms.3
covered that each type had essentially the same sequence. This In addition to the four polypeptide chains, all types of im-
region was called the constant region and the amino-terminal munoglobulins contain a carbohydrate portion, which is local-
end was called the variable region. Thus, all κ L chains have ized between the CH2 domains of the two H chains. Functions
an almost identical carboxy-terminal end; the same is true of of the carbohydrate include (1) increasing the solubility of im-
λ chains. Sixty percent of L chains are κ chains because they are munoglobulin, (2) providing protection against degradation,
coded for first in DNA transcription of genes coding for antibody and (3) enhancing functional activity of the FC domains. This
molecules.3 The difference between the κ and λ chains lies in
the amino acid substitutions at a few locations along the chain. Isotype Allotype Idiotype
There are no functional differences between the two types. Both
κ and λ L chains are found in all five classes of immunoglobu-
lins, but only one type is present in a given molecule.

Heavy-Chain Sequencing
Antibody variations (shown in black). (A) Isotype—the
Heavy-chain sequencing demonstrates the presence of domains H chain that is unique to each immunoglobulin class. (B) Allotype—
similar to those in the L chains—that is, variable and constant genetic variations in the constant regions. (C) Idiotype—variations in
regions. The first approximately 110 amino acids at the variable regions that give individual antibody molecules specificity.
latter function may be the most important because glycosylation fold by binding to a small number of amino acids at strategic
appears to be critical for recognition by FC receptors that are locations on each chain known as hypervariable regions.
found on phagocytic cells.5 Three small hypervariable regions consisting of approxi-
mately 30 amino acid residues are found within the variable
regions of both H and L chains. Each of these regions, called
Three-Dimensional Structure complementarity-determining regions (CDRs), is between 9 and
of Antibodies 12 residues long.3 They occur as loops in the folds of the vari-
able regions of both L and H chains. The antigen-binding site
The basic four-chain structure of all immunoglobulin mole- is actually determined by the apposition of the six hypervari-
cules does not actually exist as a straight Y shape but is in fact able loops, three from each chain (see Fig. 5–4). Antigen binds
folded into compact globular subunits based on the formation in the middle of the CDRs, with at least four of the CDRs
of balloon-shaped loops at each of the domains.6 Intrachain involved in the binding.3 Thus, a small number of amino acids
disulfide bonds stabilize these globular regions. Within each can create an immense diversity of antigen-binding sites.
of these regions or domains, the polypeptide chain is folded Properties of individual antibody classes are considered in the
back and forth on itself to form what is called a β-pleated sheet. following sections.
The folded domains of the H chains line up with those of
the L chains to produce a cylindrical structure called an im-
munoglobulin fold (Fig. 5–4).6 Antigen is captured within the
Immunoglobulin G (IgG)
IgG is the predominant immunoglobulin in humans, com-
prising approximately 70% to 75% of the total serum im-
CDR1 munoglobulins. As seen in Table 5–1, IgG has the longest
half-life of any immunoglobulin class, approximately 23 days,
CDR3
CDR2 which may help to account for its predominance in serum.
There are four major subclasses with the following distribu-
tion: IgG1, 66%; IgG2, 23%; IgG3, 7%; and IgG4, 4%.6 These
subclasses differ mainly in the number and position of the
disulfide bridges between the γ chains, as seen in Figure 5–5.
Variability in the hinge region affects the ability to reach for
Variable antigen and the ability to initiate important biological func-
domain tions such as complement activation.1,7 IgG3 has the largest
hinge region and the largest number of interchain disulfide
bonds; therefore, it is the most efficient at binding comple-
ment, followed by IgG1.1 IgG2 and IgG4 have shorter hinge
segments, which tend to make them poor mediators of com-
plement activation.1,7 All subclasses have the ability to cross
the placenta except IgG2.
Major functions of IgG include the following:
• Providing immunity for the newborn because IgG is the
only antibody that can cross the placenta
• Fixing complement
• Coating antigen for enhanced phagocytosis (opsonization)
• Neutralizing toxins and viruses
• Participating in agglutination and precipitation reactions
All subclasses are able to participate in the secondary im-
mune response, an enhanced and quicker response to anti-
Constant
domain gen, although their appearance depends on the triggering
antigen. IgG1and IgG3 are induced in response to protein
antigens, whereas IgG2 and IgG4 are associated with poly-
saccharide antigens.7
Macrophages, monocytes, and neutrophils have receptors
Three-dimensional structure of an L chain. In this
on their surfaces that are specific for the FC region of IgG. This
ribbon diagram tracing the polypeptide backbone, β strands
enhances contact between antigen and phagocytic cells and
(polypeptide chains) are shown as wide ribbons, with other regions
as narrow strings. Each of the two globular domains consists of a generally increases the efficiency of phagocytosis. IgG1 and
barrel-shaped assembly of seven to nine antiparallel β strands IgG3 are particularly good at initiating phagocytosis, because
(polypeptide chains). The three hypervariable regions (CDR1, they bind most strongly to FC receptors.7
CDR2, and CDR3) are flexible loops that project outward from the IgG has a high diffusion coefficient that allows it to enter
amino-terminal end of the VL domain. extravascular spaces more readily than other immunoglobulin
 Table 5–1 Properties of Immunoglobulins
IgG IgM IgA IgD IgE
Molecular weight 150,000 900,000 160,000 180,000 190,000
monomer
Sedimentation coefficient 7S 19 S 7S 7S 8S
Serum half-life (days) 23 6 5 1–3 2–3
Serum concentration (mg/dL) 800–1600 120–150 70–350 1–3 0.005
Percent of total immunoglobulin 70–75 10 10–15 <1 0.02
H chain γ µ α δ ε
H chain subclasses γ1, γ2, γ3, γ4 None α1, α2 None None
H chain molecular weight 50,000–60,000 70,000 55,000–60,000 62,000 70,000–75,000
Constant domains (H chain) 3 4 3 3 4
Carbohydrate content 2–3 12 7–11 9–14 12
(weight percent)-
Electrophoretic migration γ2–α1 γ1–β12 γ2–β2 γ1 γ1
Complement fixation Yes Yes No No No
Crosses placenta Yes No No No No

IgG1 IgG2 IgG3 IgG4

The four IgG subclasses are IgG1, IgG2, IgG3, and IgG4. These differ in the number and linkages of the disulfide bonds.

types. In fact, it is distributed almost equally between the in-

travascular and extravascular spaces.1 Thus, it plays a major
Immunoglobulin M (IgM)
role in neutralizing toxins and viruses. IgM is known as a macroglobulin because it has a sedimentation
Agglutination and precipitation reactions take place in rate of 19 S, which represents a molecular weight of approxi-
vitro, although it is not known how significant a role these mately 900,000.6 The half-life of IgM is about 6 days, much
reactions play in vivo. IgG is better at precipitation reactions shorter than that of IgG (see Table 5–1). It accounts for between
than at agglutination because precipitation involves small 5% and 10% of all serum immunoglobulins.
soluble particles, which are more easily brought together by If IgM is treated with mercaptoethanol, it dissociates into
the relatively small IgG molecule. Agglutination is the clump- five 7 S units, each having a molecular weight of 190,000 and
ing together of larger particles such as red blood cells (RBCs); a four-chain structure that resembles IgG. The molecular
because it is a larger molecule, IgM is much more efficient at weight of the H or µ chain is approximately 70,000. It consists
this than IgG. of about 576 amino acids and includes one more constant
domain than is found on the γ chain. The pentamer form is Primary response Secondary response
IgG
found in serum, whereas the monomer form occurs on the
surface of B cells.1,5

Serum antibody level

The five monomeric units are held together by a J or joining
chain, which is a glycoprotein made in plasma cells that con-
tains several cysteine residues.6 These serve as linkage points
for disulfide bonds between two adjacent monomers. Linkage
occurs at the carboxy-terminal end of two of the µ chains, and
it appears that the J chain may initiate polymerization by sta- IgG
IgM IgM
bilizing FC sulfhydryl groups so that cross-linking can occur.5
The J chain also facilitates secretion at mucosal surfaces.7 The Lag phase
lag
molecular weight of the J chain is approximately 15,000. One
J chain is present per pentamer. 10 20 30 60 70 80 90
IgM thus configured assumes a starlike shape (Fig. 5–6) with Time (Days)
10 functional binding sites. The Fab arms can bend out of the First exposure Subsequent
plane to bind two or more separate antigens or multivalent anti- to immunogen contact with
immunogen
gens.7 The high valency of IgM antibodies helps to overcome
the fact that they tend to have a low affinity for antigen. A comparison of the primary and secondary responses
to an immunogen. The primary response is characterized by a long
Because of its large size, IgM is found mainly in the
lag phase, whereas the secondary or anamnestic response has a
intravascular pool and not in other body fluids or tissues. It can-
shortened lag phase and a much more rapid increase in antibody
not cross the placenta. IgM is known as the primary response titer, mainly IgG.
antibody; it is the first to appear after antigenic stimulation and
the first to appear in the maturing infant. It is synthesized only
as long as antigen remains present because there are no memory
the classical complement pathway (see Chapter 7) because a
cells for IgM. Thus, IgM can be used to diagnose an acute infec-
single molecule can initiate the reaction as a result of its multiple
tion, as its presence indicates a primary exposure to antigen.7
binding sites. This probably represents the most important
Figure 5–7 depicts the difference between the primary re-
function of IgM. The larger number of binding sites also makes
sponse, which is predominantly IgM, and the secondary re-
IgM more efficient at agglutination reactions, especially with
sponse, which is mainly IgG. The primary response is
multivalent antigens. Thus, IgM forms a potent defense against
characterized by a long lag phase, a slow increase in antibody,
many bacterial diseases.
and a short-lived response. The second or anamnestic response
IgM also serves as a surface receptor for antigen. Mu
is distinguished by a shortened lag period, a much more rapid
chains first appear in the cytoplasm of the pre-B cell. When
rise in antibody, and higher serum levels for a longer period of
they associate with the early surrogate L chains, rearrange-
time. The secondary response is the result of the larger num-
ment of the genes controlling L chain synthesis is begun.5,8
ber of antigen-specific memory T and B cells generated during
Later, as L chains are synthesized, IgM monomers are formed
the primary response.
and become inserted into the plasma membrane. The pres-
The functions of IgM include (1) complement fixation,
ence of membrane IgM classifies lymphocytes as immature
(2) agglutination, (3) opsonization, and (4) toxin neutralization.
B cells. (See Chapter 4 for a complete discussion of B-cell
IgM is the most efficient of all immunoglobulins at triggering
development.)

Immunoglobulin A (IgA)
In the serum, IgA represents 10% to 15% of all circulating
immunoglobulin. It appears as a monomer with a molecular
weight of approximately 160,000, has a sedimentation coef-
ficient of 7 S, and migrates between the β and γ regions on
electrophoresis. The H chain, called the α chain, has a mo-
lecular weight between 55,000 and 60,000 and consists of
about 472 amino acids. These amino acids comprise one
variable and three constant regions. There are two sub-
classes, designated IgA1 and IgA2. They differ in content by
22 amino acids, 13 of which are located in the hinge region
and are deleted in IgA2. The lack of this region appears to
make IgA2 more resistant to some bacterial proteinases that
The pentameric structure of IgM is linked by a J chain are able to cleave IgA1.7,9 Hence, IgA2 is the predominant
(shown in red). Each arm can bend out of the plane to capture form in secretions at mucosal surfaces, whereas IgA1 is
antigen. mainly found in serum. The major role of serum IgA is as an
anti-inflammatory agent.10 Serum IgA appears to downreg- the plasma cells.3,12,14 This homing, or seeking out the
ulate IgG-mediated phagocytosis, chemotaxis, bactericidal subepithelial tissue by activated lymphocytes, depends on a
activity, and cytokine release. high level of certain adhesion molecules that allow binding
IgA2 is found as a dimer along the respiratory, urogenital, to epithelial cells.9 Once binding takes place, IgA and
and intestinal mucosa; it also appears in breast milk, colostrum, SC precursor are taken inside the cell and then released to
saliva, tears, and sweat.7,9,11 Because mucosal surfaces are a the opposite surface by a process known as transcytosis. The
major point of entry for pathogens, IgA2 serves to keep antigens vesicle carrying IgA and the SC receptor fuses with the mem-
from penetrating farther into the body. brane on the cell’s opposite side; a small fragment of SC is
The IgA dimer consists of two monomers held together by then cleaved to liberate the IgA dimer with the remaining
a J chain that has a molecular weight of about 15,000. The SC.9,12 The SC may thus act to facilitate transport of IgA to
J chain is essential for the polymerization and secretion of IgA.7 mucosal surfaces.12 It also makes the dimer more resistant
Secretory IgA is synthesized in plasma cells found mainly in to enzymatic digestion by masking sites that would be sus-
mucosal-associated lymphoid tissue and is released in dimeric ceptible to protease cleavage.3,6
form. IgA is synthesized at a much greater rate than that of The main function of secretory IgA is to patrol mucosal
IgG—approximately 3 grams per day in the average adult— surfaces and act as a first line of defense. It plays an important
but because it is mainly in secretory form, the serum concen- role in neutralizing toxins produced by microorganisms and
tration is much lower.9,10,12 helps to prevent bacterial and viral adherence to mucosal sur-
A secretory component (SC), which has a molecular faces.1,13 Complexes of IgA and antigen are easily trapped in
weight of about 70,000, is later attached to the FC region mucus and then eliminated by the ciliated epithelial cells of
around the hinge portion of the α chains.3,6,9,13 This protein, the respiratory or intestinal tract. This prevents pathogens
consisting of five immunoglobulin-like domains, is derived from colonizing the mucosal epithelium.11 Because IgA is
from epithelial cells found in close proximity to the plasma found in breast milk, breastfeeding helps to maintain the
cells.1,3 As Figure 5–8 indicates, SC precursor, with a health of newborns by passively transferring antibodies
molecular weight of 100,000, is actually found on the sur- and greatly decreasing infant death from both respiratory and
face of epithelial cells and serves as a specific receptor for gastrointestinal infections.12
IgA. Plasma cells that secrete IgA are attracted to subepithe- It appears that IgA is not capable of fixing complement by
lial tissue, where IgA can bind as soon as it is released from the classical pathway, although aggregation of immune com-
plexes may trigger the alternate complement pathway9,14 (see
Chapter 7). Lack of complement activation may actually assist
in clearing antigen without triggering an inflammatory re-
sponse, thus minimizing tissue damage.11,13
Epithelial
cell
Additionally, neutrophils, monocytes, and macrophages
possess specific receptors for IgA. Binding to these sites triggers
a respiratory burst and degranulation.7,11 This occurs for both
Plasma cell IgA Poly-lg
receptor serum and secretory IgA, indicating that they are capable of
acting as opsonins. The success of oral immunizations such as
the Sabin vaccine, which induces IgA almost exclusively,
demonstrates the effectiveness of IgA’s protective role on
Endocytosis mucosal surfaces.

Immunoglobulin D (IgD)
IgD was not discovered until 1965, when it was found in a
Transcytosis patient with multiple myeloma, a cancer of the plasma cells.
It is extremely scarce in the serum, representing less than
0.001% of total immunoglobulins. It is synthesized at a low
level and has a half-life of only 1 to 3 days. The molecule has
a molecular weight of approximately 180,000 and migrates
as a fast γ protein. The delta (δ) H chain has a molecular
Exocytosis
weight of 62,000 and appears to have an extended hinge re-
gion consisting of 58 amino acids.1
IgA + secretory
Most of the IgD is found on the surface of immunocompe-
component tent but unstimulated B lymphocytes. It is the second type of
Formation of secretory IgA. IgA is secreted as a dimer immunoglobulin to appear (IgM being the first) and it may
from plasma cells and is captured by specific receptors on epithelial play a role in B-cell activation, although its function is not
cells. The receptor is actually an SC, which binds to IgA and exits the completely understood.7 The high level of surface expression
cell along with it. and its intrinsic flexibility make it an ideal early responder to
antigen.5,6 Those cells bearing only IgM receptors appear in- have several hundred thousand receptors, each capable of
capable of an IgG response, whereas those with both IgM and binding an IgE molecule. When two adjacent IgE molecules
IgD receptors are capable of responding to T-cell help and on a mast cell bind specific antigen, a cascade of cellular
switching to synthesis of IgG, IgA, or IgE.5 Thus, IgD may play events is initiated that results in degranulation of the mast
a role in regulating B-cell maturation and differentiation.1 cells with release of vasoactive amines such as histamine and
Because of its unusually long hinge region, IgD is more heparin. Release of these mediators induces what is known
susceptible to proteolysis than other immunoglobulins. This as a type I immediate hypersensitivity or allergic reaction (see
may be the main reason for its short half-life. In the secreted Chapter 14). Typical reactions include hay fever, asthma, vom-
form in the serum, IgD does not appear to serve a protective iting and diarrhea, hives, and life-threatening anaphylactic
function because it does not bind complement, it does not shock.5 Recently developed anti-IgE antibody that targets free
bind to neutrophils or macrophages, and it does not cross the IgE has been used as therapy for allergies and asthma.7
placenta.1 IgE appears to be a nuisance antibody; however, it may
serve a protective role by triggering an acute inflammatory
Immunoglobulin E (IgE) reaction that recruits neutrophils and eosinophils to the area
to help destroy invading antigens that have penetrated IgA
IgE is best known for its very low concentration in serum defenses.14 Eosinophils, especially, play a major part in the
and the fact that it has the ability to activate mast cells and destruction of large antigens such as parasitic worms that can-
basophils. It is the least abundant immunoglobulin in the not be easily phagocytized (see Chapter 22 for details).
serum, accounting for only 0.0005% of total serum im-
munoglobulins.1 The molecular weight of IgE is approxi-
mately 190,000, making it an 8 S molecule, and it has a Theories to Explain Antibody
carbohydrate content of 12%.15 The epsilon (ε) or H chain is Diversity
composed of around 550 amino acids that are distributed
over one variable and four constant domains. A single disul- It is estimated that humans can make at least 106 to 109 dif-
fide bond joins each ε chain to an L chain and two disulfide ferent antibody molecules.17 Attempts to explain how this
bonds link the H chains to one another. many possible combinations with exquisite specificity for a
IgE is the most heat-labile of all immunoglobulins; heating particular antigen can occur began long before the actual
to 56°C for between 30 minutes and 3 hours results in con- structure of immunoglobulins was discovered. The central
formational changes and loss of ability to bind to target cells. issue was whether an antigen selected lymphocytes with the
IgE does not participate in typical immunoglobulin reactions inherent capability of producing specific antibody to it or
such as complement fixation, agglutination, or opsonization. whether the presence of antigen added a new specificity to a
Additionally, it is incapable of crossing the placenta. Instead, generalized type of antibody.
shortly after synthesis it attaches to basophils, Langerhans
cells, eosinophils, and tissue mast cells by means of specific
surface proteins, termed high-affinity FC ε RI receptors, which
Ehrlich’s Side-Chain Theory
are found exclusively on these cells.3,5 The molecule binds One of the first theories to be formulated was that of Paul
at the CH3 domain on the FC region,16 leaving the antigen- Ehrlich in the early 1900s, termed the side-chain theory. Ehrlich
binding sites free to interact with specific antigen (Fig. 5–9). postulated that certain cells had specific surface receptors for
Plasma cells that produce IgE are located primarily in the antigen that were present before contact with antigen occurred.
lungs and in the skin.5 Once antigen was introduced, it would select the cell with the
Mast cells are also found mainly in the skin and in the lin- proper receptors, combination would take place, and then re-
ing of the respiratory and alimentary tracts. One such cell may ceptors would break off and enter the circulation as antibody

Antigen specific
IgE

Fc$R1 Antigen

Action of IgE on mast

cells. (A) IgE binds to specific ε recep-
tors on mast cells. (B) When antigen
bridges two nearby IgE molecules, the
membrane is disturbed and degranu- Degranulation
lation results. Chemical mediators are
released.
molecules. New receptors would form in place of those broken rearrangement involves a cutting and splicing process that gets
off, after which this process could be repeated. Although rid of much of the intervening DNA, resulting in a functional
this represented a rather simplistic explanation for antibody gene that codes for a specific antibody. Once this rearrange-
synthesis, two key premises emerged: first, the lock-and-key ment occurs, it permanently changes the DNA of the particular
concept of the fit of antibody for antigen, and second, the idea lymphocyte.
that an antigen selected cells with the built-in capacity to re-
spond to it. Although this theory did not explain the kinetics Rearrangement of Heavy-Chain Genes
of the immune response or the idea of immunologic memory,
it laid the foundation for further hypotheses. The selection process begins with rearrangement of the genes
for the H chains. All H chains are derived from a single region
on the long arm of chromosome 14. The genes that code for the
Clonal Selection Hypothesis variable region are divided into three groups—VH, D, and J.
In the 1950s, Niels Jerne and Macfarlane Burnet indepen- There are at least 39 VH (variable) genes, approximately
dently supported the idea of a clonal selection process for an- 27 functional D (diversity) genes, and 6 J (joining) genes.6,7,22
tibody formation.18,19,20 The key premise of the clonal In addition, there is a set of genes (C) that codes for the con-
selection hypothesis is that individual lymphocytes are stant region. This set includes one gene for each H chain iso-
genetically preprogrammed to produce one type of im- type. They are located in the following order: Cµ, Cδ, Cγ3,
munoglobulin and that a specific antigen finds or selects those Cγ1, Cα1, Cγ2, Cγ4, Cε, and Cα2, as shown in Figure 5–10.
particular cells capable of responding to it, causing them to Only one of these constant regions is selected at any one time.
proliferate. The receptors Ehrlich originally postulated are the For synthesis of the entire H chain, a choice is made from each
surface immunoglobulins IgM and IgD, which are found on of the sections so as to include one VH gene, one D gene, one
unstimulated B lymphocytes. Repeated contact with antigen J gene, and one constant region. During the process of B-cell
would continually increase a specific lymphocyte pool. Such maturation, the pieces are spliced together to commit that
a model provides an explanation for the kinetics of the im- B lymphocyte to making antibody of a single specificity.
mune response. Joining of these segments occurs in two steps: First, at
The main drawback to the clonal selection hypothesis was the DNA level, one D and one J are randomly chosen and are
consideration of the genetic basis for the diversity of antibody joined by means of a recombinase enzyme with deletion of the
molecules. If separate genes were present to code for antibody intervening DNA (see Fig. 5–10). Next, a V gene is joined to
to every possible antigen, an overwhelming amount of DNA the DJ complex, resulting in a rearranged V(D)J gene. The VJD
would be needed. In 1965, Dreyer and Bennett proposed a gene combination codes for the entire variable region of the
solution to this dilemma by suggesting that the constant and H chain. This rearrangement occurs early in B-cell develop-
variable portions of immunoglobulin chains are actually ment in pro-B cells.22–24 (See Chapter 4 for additional details.)
coded for by separate genes.21 There could be a small number The recombinase enzymes RAG-1 and RAG-2, which are
coding for the constant region and a larger number coding distinctive markers of this stage, are essential for initiating
for the variable region. This would considerably simplify the this process. The recombinase enzymes recognize specific tar-
task of coding for such variability. This notion implied that get sequences called recombination signal sequences that flank
although all lymphocytes start out with identical genetic all immunoglobulin gene segments.21 However, joining of the
germline DNA, diversity is created by a series of recombina- V, J, and D segments doesn’t always occur at a fixed position,
tion events that occur as the B cell matures. Scientific evi- so each sequence can vary by a small number of nucleotides.
dence now indicates that this is exactly what happens, as This variation is called junctional diversity, which is a major
explained in the following discussion. source of additional diversity.7,17 If a successful rearrangement
of DNA on one chromosome 14 occurs, then the genes on the
second chromosome are not rearranged; this phenomenon is
Genes Coding for Immunoglobulins known as allelic exclusion. If the first rearrangement is non-
productive, then rearrangement of the second set of genes on
Tonegawa’s pioneering experiments with DNA revealed that the other chromosome 14 occurs.
chromosomes contain no intact immunoglobulin genes, only The variable and constant regions are joined at the ribonu-
building blocks from which genes can be assembled. This con- cleic acid (RNA) level, thus conserving the DNA of the constant
firmed the hypothesis of Dreyer and Bennett.17 Tonegawa was regions and allowing for a later phenomenon called class
awarded the Nobel Prize in 1987 for this monumental discov- switching, whereby daughter plasma cells can produce anti-
ery. Human immunoglobulin genes are found in three un- body of another type. During transcription and synthesis of
linked clusters: H chain genes are located on chromosome 14, messenger ribonucleic acid (mRNA), a constant region is
κ chain genes are on chromosome 2, and λ chain genes are on spliced to the VDJ complex.25 Because Cµ is the region closest
chromosome 22. Within each of these clusters, a selection to the J region, µ H chains are the first to be synthesized; these
process occurs. The genes cannot be transcribed and translated are the markers of the pre-B lymphocytes. The Cδ region,
into functional antibody molecules until this rearrangement, which lies closest to the Cµ region, is often transcribed along
assisted by special recombinase enzymes, takes place. Gene with Cµ. The presence of DNA for both the Cµ and Cδ regions
 V1 V2 V3 Vn D1 D2 D3 Dn J1 J2 J3 Jn C% C& C#3 C#1 C#2 C#4 C$ C!
DNA

Excised D/J joining

V1 V2 V3 Vn D1 D2 J3 Jn C% C& C#3 C#1 C#2 C#4 C$ C!

DNA

Excised V/D/J joining

V1 V2 D2 J3 Jn C% C& C#3 C#1 C#2 C#4 C$ C!

Coding for im- DNA

munoglobulin H chains. Four
separate regions on chromosome Transcription and splicing
14 code for H chains. DJ regions are
spliced first, and then this segment V2 D2 J3 C%
is joined to a variable region. When mRNA
RNA synthesis occurs, one constant
region is attached to the VDJ combi- Translation
nation. µ H chains are made first,
but the cell retains its capacity V2 D2 J3 C%
to produce immunoglobulin of % Heavy chain protein for IgM
another class.

allows for RNA for IgD and IgM to be transcribed at the same produced by these rearrangements, then the particular B cell
time. Thus, a B cell could express IgD and IgM with the same dies by apoptosis.
variable domain on its surface at the same time. The process L chains are then joined with µ chains to form a complete
of switching to other immunoglobulin classes occurs later as a IgM antibody, which first appears in immature B cells. Once
result of the looping out and deletion of other constant regions. IgM and IgD are present on the surface membrane, the B lym-
This allows the same VDJ region to be coupled with a different phocyte is fully mature and capable of responding to antigen
C region to produce antibody of a different class (i.e., IgA, IgG, (see Chapter 4). The large variety of V, J, D, and C combinations
or IgE) but with the identical specificity for antigen.7,24 Contact
with T cells and with cytokines provides the signal for switch-
ing to take place. V1 V2 V3 Vn J1 J2 J3 Jn C'
DNA
Light Chain Rearrangement
Because L chain rearrangement occurs only after µ chains ap- Excised V/J joining
pear, µ-chain synthesis represents a pivotal step in the process.
V1 V2 J2 Jn C'
L chains exhibit a similar genetic rearrangement, except they
lack a D region. Recombination of segments on chromosome 2, DNA
coding for κ chains, occurs before that on chromosome 22,
which codes for λ chains.7,22 The process of VJ joining is Transcription and splicing
accomplished by cutting out intervening DNA. This results in
Vκ and Jκ segments becoming permanently joined to one an- V2 J2 C'
other on the rearranged chromosome. Transcription begins at mRNA
one end of the Vκ segment and proceeds through the Jκ and
Cκ segments. Unrearranged J segments are removed during Translation
RNA splicing, which occurs in the translation (Fig. 5–11). V2 J2 C'
A productive rearrangement of the κ genes with subsequent
protein production keeps the other chromosome 2 from rear- ' Light chain protein
ranging and shuts down any recombination of the λ-chain Assembly and expression of the κ L chain locus. A
locus on chromosome 22. Only if a nonfunctioning gene prod- DNA rearrangement fuses one V segment to one J segment. The VJ
uct arises from κ rearrangement does λ chain synthesis occur. segment is then transcribed along with a unique C region to form
The λ locus contains approximately 30 to 36 Vλ, 7 Jλ, and four mature κ mRNA. Unarranged J segments are removed during RNA
functional Cλ segments.7 If functional H and L chains are not splicing.
for each type of chain, plus the different possibilities for L and The other pathway, the de novo pathway, which makes
H chain combination, make for more than enough configura- DNA from new nucleotides, is blocked by the presence of
tions to allow us to respond to any antigen in the environment. aminopterin. Consequently, the myeloma cells die out. Nor-
mal B cells cannot be maintained continuously in cell culture,
so these die out as well. This leaves only the fused hybridoma
Monoclonal Antibody cells, which have the ability (acquired from the myeloma cell)
to reproduce indefinitely in culture and the ability (acquired
The knowledge that B cells are genetically preprogrammed to
from the normal B cell) to synthesize nucleotides by the
synthesize very specific antibody has been used in developing
HGPRT and thymidine kinase pathway (Fig. 5–12).
monoclonal antibodies for diagnostic testing. Monoclonal
antibodies are so called because they are derived from a single
parent antibody-producing cell that has reproduced many times, The remaining hybridoma cells are diluted out and placed in
thus forming a clone. Every cell in the clone is just like every microtiter wells, where they are allowed to grow. Each well,
other cell; the antibody produced by each cell is exactly the same containing one clone, is then screened for the presence of the
as that of every other cell. This differs from the normal response desired antibody by removing the supernatant. Once identi-
to an antigen, which is heterogeneous, because even a purified fied, a hybridoma is capable of being maintained in cell culture
antigen has multiple epitopes that stimulate a variety of B cells. indefinitely and produces a permanent and uniform supply of
In 1975, Georges Kohler and Cesar Milstein discovered a tech- monoclonal antibody that reacts with a single epitope.26,27
nique to produce antibody arising from a single B cell, which
has revolutionized serological testing. They were awarded the
Nobel Prize in 1984 for their pioneering research.
Clinical Applications
Monoclonal antibodies were initially used for in vitro diagnos-
Hybridomas tic testing. A familiar example is pregnancy testing, which uses
antibody specific for the β chain of human chorionic go-
Kohler and Milstein’s technique fuses an activated B cell with a
nadotropin, thereby eliminating many false-positive reactions.
myeloma cell that can be grown indefinitely in the laboratory.
Other examples include detection of tumor antigens and mea-
This fusion of two different types of cells is called a hybridoma.
surement of hormone levels.
Myeloma cells are cancerous plasma cells. Normally, plasma
Recently, however, there has been an emphasis on the use
cells produce antibody, so a particular cell line that is not capa-
of monoclonal antibodies as therapeutic agents. Because mouse
ble of producing antibody is chosen. In addition, this cell line
monoclonal antibodies are highly immunogenic for humans,
has a deficiency of the enzyme hypoxanthine-guanine phos-
several techniques have been used to “humanize” monoclonal
phoribosyltransferase (HGPRT) that makes it incapable of syn-
antibodies to prevent reactions to the mouse antibodies. One
thesizing nucleotides from hypoxanthine and thymidine, which
of these techniques grafts the antibody-combining site from
are needed for DNA synthesis. The fact that these myeloma cells
antibodies grown in mice onto the rest of a human im-
cannot make their own DNA means that they will die out unless
munoglobulin.28 Another technique injects human DNA that
they are fused to a plasma cell that has the enzymes necessary
codes for antibody molecules into bacteriophage. Bacterio-
to synthesize DNA. This deficiency keeps the myeloma cells
phage are viruses that only grow inside bacteria. Bacteriophage
from reproducing on their own.
with human genes are used to infect Escherichia coli; as E coli
colonies grow, they produce human antibodies.27,28
The production of hybridomas begins by immunizing a One of the major breakthroughs in the treatment of cancer
mouse with a certain antigen. After a time, the mouse’s spleen involves the use of monoclonal antibodies. There are now a
cells are harvested. Spleen cells are combined with myeloma number of such antibodies that have been approved for cancer
cells in the presence of polyethylene glycol (PEG), a surfac- treatment. In the case of metastatic breast cancer, trastuzumab
tant. The PEG brings about fusion of plasma cells with (Herceptin), an antibody directed against HER-2 protein, has
myeloma cells, producing a hybridoma. Only a small percent- been helpful in slowing the disease’s progress.28,29 HER-2 pro-
age of cells actually fuse, and some of these are like cells— tein is present in large numbers on tumor cells and regulates
that is, two myeloma cells or two spleen cells. After fusion, growth. Another example is rituximab (Rituxan), the first mon-
all cells are placed in culture using a medium containing hy- oclonal antibody to be approved by the FDA for the treatment
poxanthine, aminopterin, and thymidine (HAT). This culture of malignancies.30 Rituximab targets the B-cell marker CD20,
medium separates out the hybridoma cells by allowing them leading to depletion of peripheral B cells. The antibody is used
to grow selectively and not allowing fused myeloma cells or to treat non-Hodgkin lymphoma and other B-cell malignan-
fused spleen cells to survive. Myeloma cells are normally able cies. Other monoclonal antibodies approved by the FDA in-
to grow indefinitely in tissue culture, but in this case they clude cetuximab (Erbitux) to treat colorectal cancer and head
cannot because both pathways for the synthesis of nu- and neck cancers and bevacizumab (Avastin) to treat colorectal,
cleotides are blocked. One pathway, the salvage pathway, non-small lung, and breast cancers.26,28
which builds DNA from degradation of old nucleic acids, is In some therapies, monoclonal antibodies are linked to cy-
blocked because the myeloma cell line employed is deficient totoxic agents, such as radioactive substances and other drugs
in the required enzymes HGPRT and thymidine kinase.26,27 that are delivered directly to cancerous cells. These monoclonal
 and Crohn’s disease (a progressive inflammatory colitis). Both
of these diseases have been treated with a monoclonal antibody
Antigen called infliximab that blocks the action of tumor necrosis fac-
Primary and
secondary mouse tor-alpha (TNF-α.29,32,33 Another TNF blocker, adalimumab
immunization (Humira), has also proven effective in decreasing symptoms of
these two diseases.29 Rituximab is also used in the treatment
Cell culture of several autoimmune diseases, including RA and systemic
myeloma cells lupus erythematosis.34 (See Chapter 15 for a discussion of
autoimmune diseases.)
Use of monoclonal antibodies in several areas of medicine
Mouse
continues to grow. This represents a developing area of
spleen cells
research in pharmacology; as it rapidly expands, it is likely
to change future treatment options for numerous diseases.
(See Chapter 25 for additional information about the use of
monoclonal antibodies.)
Fused in
presence of PEG

SUMMARY
Placed in HAT • The basic structural unit for all immunoglobulins is a
restrictive medium tetrapeptide composed of two L and two H chains joined
together by disulfide bonds.
• The five classes of antibodies are IgM, IgG, IgA, IgD, and
IgE. IgG, IgD, and IgE exist as monomers. IgA has a
dimeric form, whereas IgM is a pentamer whose subunits
are held together by a J chain.
• Kappa and lambda (L chains) are found in all types of im-
munoglobulins, but the H chains differ for each im-
munoglobulin class.
• Each immunoglobulin molecule has constant and variable
Hybridoma Spleen Myeloma regions. The variable region is at the amino-terminal end,
cells grow cells die cells die called the Fab fragment; this determines the specificity of
that molecule for a particular antigen.
• The constant region, located at the carboxy-terminal end
of the molecule and named the FC fragment, is responsible
for binding to effector cells such as neutrophils, basophils,
eosinophils, and mast cells.
• The five different types of heavy chains are called isotypes.
• Minor variations in a particular type of heavy chain are
called allotypes.
• The variable portion of the light and heavy chains unique
Desired antibody
secreted to a particular immunoglobulin molecule is known as the
idiotype.
Formation of a hybridoma in monoclonal antibody
production. A mouse is immunized and spleen cells are removed. • IgG is relatively small and easily penetrates into tissues,
These cells are fused with nonsecreting myeloma cells and then whereas IgM is much larger and excels at complement
plated in a restrictive medium. Only the hybridoma cells will grow fixation.
in this medium where they synthesize and secrete a monoclonal • IgA has an SC that protects it from enzymatic digestion
immunoglobulin specific for a single determinant on an antigen. while it patrols mucosal surfaces.
• An extended hinge region gives IgD an advantage as a
surface receptor for antigen.
antibodies are called antibody-drug conjugates. Drugs in this • IgE binds to mast cells to initiate a local inflammatory
category include ibritumomab tiuxetan (Zevalin) for cancerous reaction.
B lymphocytes and tositumomab (Bexxar) to treat some non- • The primary response to an antigen takes 5 to 7 days
Hodgkin lymphomas that no longer respond to rituximab.26,31 before antibody can be detected.
Monoclonal antibodies are also useful in the treatment of • The primary response consists of approximately equal
autoimmune diseases. One of the biggest success stories is in amounts of IgM and IgG.
the treatment of two such diseases: rheumatoid arthritis (RA)
• The secondary response to antigen occurs in a shorter segments are joined to make antibody of a single
time; the amount of IgM is similar to that of the primary specificity.
response, whereas IgG may be up to one hundred times • Monoclonal antibodies are made when a cancerous cell or
greater than that of the primary response. myeloma is fused with an antibody-producing cell to form
• Ehrlich’s side-chain theory is based on the antigen select- a hybridoma.
ing the correctly programmed B lymphocyte. • Hybridomas formed by fusion of one of each cell type
• The clonal selection hypothesis postulated that lympho- (e.g., myeloma and B cell) are identified by using HAT, a
cytes are generally pre-endowed to respond to one antigen selective medium.
or a group of antigens. • Monoclonal antibodies are used both in diagnosis and
• Several genes code for a particular immunoglobulin; treatment of disease.
through a random selection process, these individual

Study Guide: The Five Classes of Immunoglobulins

IgG IgM IgA IgD IgE

Most abundant Primary response Monomer and Present on Binds to mast

in serum antibody dimer B cells cells
Able to cross Pentamer with 10 antibody- Protects mucosal Role in B-cell Triggers allergic
placenta combining sites surfaces activation response
Increases with Indicates acute infection Has secretory Identifies mature Role in response
second exposure component B cells to parasites

CASE STUDIES
1. A 15-year-old male exhibited symptoms of fever, fatigue, 2. A 10-year-old female experienced one cold after another
nausea, and sore throat. He went to his primary care in the springtime. She had missed several days of school
physician who ordered a rapid strep test and a test for in- and her mother was greatly concerned. The mother took
fectious mononucleosis to be performed in the office. The her daughter to the pediatrician, worried that her
rapid strep test result was negative, but the test result for daughter might be immunocompromised because she
infectious mononucleosis was faintly positive. The patient couldn’t seem to fight off infections. A blood sample was
mentioned that he thought he had mononucleosis about obtained and sent to a reference laboratory for a deter-
2 years earlier, but it was never officially diagnosed. His mination of antibody levels, including an IgE level. The
serum was sent to a reference laboratory to test with spe- patient’s IgM, IgG, and IgA levels were all normal for
cific Epstein-Barr viral antigens. The results indicated the her age, but the IgE level was greatly increased.
presence of IgM only.
Questions
Questions a. What does the increase in IgE signify?
a. Is this a new or reactivated case of mononucleosis? b. Should there be a concern about the patient being
Explain your answer. immunocompromised?
b. How do the results relate to the difference between the
primary and a secondary response to exposure to the
same antigen?
REVIEW QUESTIONS
1. Which of the following is characteristic of variable 9. The structure of a typical immunoglobulin consists of
domains of immunoglobulins? which of the following?
a. They occur on both the H and L chains. a. 2L and 2H chains
b. They represent the complement-binding site. b. 4L and 2H chains
c. They are at the carboxy-terminal ends of the c. 4L and 4H chains
molecules. d. 2L and 4 H chains
d. They are found only on H chains.
10. Which of the following are L chains of antibody
2. All of the following are true of IgM except that it molecules?
a. can cross the placenta. a. Kappa
b. fixes complement. b. Gamma
c. has a J chain. c. Mu
d. is a primary response antibody. d. Alpha

3. How does the structure of IgE differ from that of IgG? 11. If the results of serum protein electrophoresis show a
a. IgG has a secretory component and IgE does not. significant decrease in the gamma band, which of the
b. IgE has one more constant region than IgG. following is a likely possibility?
c. IgG has more antigen-binding sites than IgE. a. Normal response to active infection
d. IgG has more light chains than IgE. b. Multiple myeloma
c. Immunodeficiency disorder
4. How many antigen-binding sites does a typical IgM d. Monoclonal gammopathy
molecule have?
a. 2 12. The subclasses of IgG differ mainly in
b. 4 a. the type of L chain.
c. 6 b. the arrangement of disulfide bonds.
d. 10 c. the ability to act as opsonins.
d. molecular weight.
5. Bence Jones proteins are identical to which of the
following? 13. Which best describes the role of the secretory compo-
a. H chains nent of IgA?
b. L chains a. A transport mechanism across endothelial cells
c. IgM molecules b. A means of joining two IgA monomers together
d. IgG molecules c. An aid to trapping antigen
d. Enhancement of complement fixation by the
6. A Fab fragment consists of classical pathway
a. two H chains.
b. two L chains. 14. Which represents the main function of IgD?
c. one L chain and one-half of an H chain. a. Protection of the mucous membranes
d. one L chain and an entire H chain. b. Removal of antigens by complement fixation
c. Enhancing proliferation of B cells
7. Which antibody best protects mucosal surfaces? d. Destruction of parasitic worms
a. IgA
b. IgG 15. Which antibody is best at agglutination and comple-
c. IgD ment fixation?
d. IgM a. IgA
b. IgG
8. Which of the following pairs represents two different c. IgD
immunoglobulin allotypes? d. IgM
a. IgM and IgG
b. IgM1 and IgM2
c. Anti-human IgM and anti-human IgG
d. IgG1m3 and IgG1m17
16. Which of the following can be attributed to the clonal 20. Papain digestion of an IgG molecule results in which
selection hypothesis of antibody formation? of the following?
a. Plasma cells make generalized antibody. a. 2 Fab' and 1 Fc' fragment
b. B cells are preprogrammed for specific antibody b. F(ab')2 and 1 Fc' fragment
synthesis. c. 2 Fab and 2 Fc fragments
c. Proteins can alter their shape to conform to antigen. d. 2 Fab and 1 Fc fragment
d. Cell receptors break off and become circulating
antibody. 21. Which antibody provides protection to the growing
fetus because it is able to cross the placenta?
17. All of the following are true of IgE except that it a. IgG
a. fails to fix complement. b. IgA
b. is heat stable. c. IgM
c. attaches to tissue mast cells. d. IgD
d. is found in the serum of allergic persons.
22. Which best characterizes the secondary response?
18. Which best describes coding for immunoglobulin a. Equal amounts of IgM and IgG are produced.
molecules? b. There is an increase in IgM only.
a. All genes are located on the same chromosome. c. There is a large increase in IgG but not IgM.
b. L chain rearrangement occurs before H chain d. The lag phase is the same as in the primary
rearrangement. response.
c. Four different regions are involved in coding of
H chains.
d. Lambda rearrangement occurs before kappa
rearrangement.

19. What is the purpose of HAT medium in the prepara-

tion of monoclonal antibody?
a. Fusion of the two cell types
b. Restricting the growth of myeloma cells
c. Restricting the growth of spleen cells
d. Restricting antibody production to the IgM class
 Cytokines

After finishing this chapter, you should be able to: INTRODUCTION TO CYTOKINES
1. Define cytokine. CYTOKINES IN THE INNATE IMMUNE
RESPONSE
2. Define and describe the term cytokine storm and relate its medical
importance. Interleukin-1 (IL-1)
3. Distinguish between autocrine, paracrine, and endocrine effects of Tumor Necrosis Factors
cytokines. Interleukin-6
4. Define pleiotropy as it relates to cytokine activities. Chemokines
5. Explain the functions of interleukin-1 (IL-1) in mediating the Transforming Growth Factor-β
immune response. Interferon-α and Interferon-β
6. Explain the effects of tumor necrosis factor (TNF). CYTOKINES IN THE ADAPTIVE IMMUNE
7. Discuss how interleukin-6 (IL-6) affects inflammation and other RESPONSE
activities of the immune system. Th1 Cytokines
8. Determine the role of chemokines in chemotaxis of white blood cells Th2 Cytokines
(WBCs).
Cytokines Associated With
9. Compare the functions of type 1 and type 2 interferons (IFN). T Regulatory Cells
10. Describe the actions of interleukin-2 (IL-2) on target cells. TH17 CYTOKINES IN INNATE AND
11. Discuss the biological roles of the hematopoietic growth factors. ADAPTIVE IMMUNE RESPONSES
12. Discuss cytokines involved in differentiation of T helper (Th) cell HEMATOPOIETIC GROWTH FACTORS
subpopulations: Th1, Th2, Th17, and T regulatory. CYTOKINE AND ANTICYTOKINE
13. Describe the biological role of colony stimulating factors. THERAPIES
14. Describe the current types of anticytokine therapies. CLINICAL ASSAYS FOR CYTOKINES
15. Describe clinical assays for cytokines. SUMMARY
CASE STUDY
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Adaptive T regulatory 1 (Tr1) Endocrine Integrins Synergistic
cells Endogenous pyrogen Interferons (IFN) T helper 1 cells (Th1)
Antagonism Erythropoietin (EPO) Interleukins (IL) T helper 2 cells (Th2)
Autocrine Granulocyte colony Macrophage colony T helper 17 cells (Th17)
Cascade induction stimulating factor (G-CSF) stimulating factors T regulatory (Treg) cells
Chemokines Granulocyte-macrophage (M-CSF)
Transforming growth
Colony stimulating factors colony stimulating factor Paracrine factor-β (TGF-β)
(CSF) (GM-CSF) Pleiotropy Tumor necrosis factors (TNF)
Cytokines Hypercytokinemia Redundancy

Introduction to Cytokines
The cells of the immune system are spread throughout the body
and need a communication system to coordinate an immune
response. Cytokines are the chemical messengers that regulate
the immune system, orchestrating both innate immunity and IL-1!
the adaptive response to infection. They are small proteins pro-
duced by several different types of cells that influence the
hematopoietic and immune systems through activation of cell-
bound receptors.1 Cytokines are induced in response to the
binding of stimuli, such as bacterial lipopolysaccharides, fla-
gellin, or other bacterial products, to specific cell receptors or
through the recognition of foreign antigens by host lympho-
cytes. The effects of cytokines in vivo include regulation of IL-1!
growth, differentiation, and gene expression by many different
cell types, including leukocytes. These effects are achieved
through both autocrine stimulation (i.e., affecting the same cell Autocrine
IL-1!
that secreted it) and paracrine (i.e., affecting a target cell in
close proximity) activities. Occasionally, cytokines will also exert IL-1!
endocrine (i.e., systemic) activities (Fig. 6–1). Thus, individual
cytokines do not act alone but in conjunction with many other Endocrine
cytokines that are induced during the process of immune acti- Paracrine
APC
vation. The resulting network of cytokine expression regulates
leukocyte activity and leads to elimination of the infection.
Initially, cytokines were named based on their activities and
IL-1!
types of cells from which they were first isolated. For example,
cytokines released from lymphocytes were called lymphokines,
cytokines released from monocytes and macrophages were
called monokines, and cytokines secreted by leukocytes that
mainly act on other leukocytes were called interleukins. Now, Th2
cytokines are grouped into families, which include tumor Range of cytokine actions. Autocrine: Cytokine acts
necrosis factor (TNF), interferon (IFN), chemokine, transform- on the cell that secreted it (e.g., IL-1β increases activation of APC).
ing growth factor (TGF), and colony stimulating factor (CSF). Paracrine: Cytokine acts on nearby cells (e.g., IL-1β stimulates
There are also the interleukins (IL), which currently number Th cells, activates neutrophils, depolarizes neurons). Endocrine:
from IL-1 to IL-38.2 The major functions of these groups will Cytokine travels through blood vessels to distant cells (e.g., IL-1β
be discussed in this chapter. stimulates acute-phase protein synthesis in the liver, as well as fever
Cytokines were originally thought to act solely on cells of induction in the hypothalamus).
the immune system, but it soon became apparent that many
also act on cells outside the immune system. Functionally, cy- fact that many cytokines share receptor subunits. For instance,
tokines may exhibit pleiotrophy, redundancy, synergy, antago- IL-6 and IL-11 use the gp130 subunit as part of their
nism, and cascade induction. Pleiotropy means that a single receptors.3 Thus, some cytokines may have overlapping effects
cytokine can have many different actions. When different cy- and may alter the activity of many of the same genes.
tokines activate some of the same pathways and genes, it is Cytokines often act in networks; if the effects complement
called redundancy. The redundancy may be explained by the and enhance each other, these are called synergistic interactions
(Fig. 6–2). In certain instances, one cytokine may counteract activates macrophages to secrete IL-12, which then activates
the action of another cytokine, causing antagonism as an action Th cells to produce other cytokines.
(see Fig 6–2B). Cascade induction may also occur, in which a A cytokine cascade produces a spectrum of activities that lead
cytokine secreted by a specific type of cell can activate target cells to the rapid generation of both innate and adaptive immune re-
to produce additional cytokines (see Fig. 6–2C). For example, sponses. In fact, the ability or inability to generate certain cytokine
activated T helper (Th) cells secrete IFN-gamma, which in turn patterns often determines the outcome and clinical course of

X
A B
C alone

B
th
A 11

wi
s
A

ze
an

gi
tag

er
on
n
Target cell response

ize
sC sy
A

B alone e
A alon

0
0 Cytokine A concentration

1000

IL-6
Plasma conc. (pg/mL)

100

IL-1!

10

TNF"

0
0 6 12 18
C Time (hours)
(A) Cytokine characteristics of synergy, antagonism, and cascade induction. Synergy: Neither cytokine A nor B induce a strong
response individually; however, when combined, the net response is much greater than the sum of the individual responses. Antagonism:
individually, cytokine C induces a strong response and cytokine A induces a weak (but positive) response. When combined, cytokine A
diminishes the action of cytokine C. In this illustration, the concentrations of cytokines B and C are held constant, whereas cytokine A increases
along the horizontal axis. (B) Synergistic and antagonistic interactions may be the result of cytokine A (1) altering the expression or function of
the receptor for cytokine X, (2) altering the activity of a key enzyme in the signaling pathway for cytokine X, or (3) altering the stability and/or
translation of the mRNA induced by cytokine X. (C) Cytokines can induce release of other cytokines in an amplifying cascade. For example,
following intravenous injection of a bacterial toxin, TNF-α is first released by monocytes and macrophages. Both the toxin and TNF-α induce
subsequent release of IL-1β. Then all three induce IL-6 release from a wide variety of cell types.
infection. In extreme circumstances, massive overproduction and infiltrates into damaged tissues. The innate immune re-
dysregulation of cytokines produce hyperstimulation of the im- sponse is nonspecific but occurs within hours of first contact
mune response or hypercytokinemia, a condition commonly re- with microorganisms (see Chapter 1). It may play a crucial
ferred to as cytokine storm. Cytokine storms may lead to shock, part in recovery from infection. The main function of the innate
multiorgan failure, or even death, thus contributing to pathogen- immune response is to recruit effector cells to the area. Cytokines
esis.4,5 Select pathogens have been known to induce cytokine involved in triggering this response are interleukin-1 (IL-1),
storms. For example, pathogenic viruses (e.g., influenza A) and TNF-α, interleukin-6 (IL-6), chemokines, transforming
bacteria (e.g., Francisella tularensis) disrupt the delicate balance growth factor-β, and interferons α and (β. The function of
of a suitable inflammatory response, tipping it from beneficial to each is discussed here.
destructive by causing the release of large amounts of cytokines
such as tumor necrosis factor-α (TNF-α) and interleukin-1β Interleukin-1 (IL-1)
(IL-1β). Indeed, cytokine storms were considered a main cause
of death in the 1918 Spanish flu pandemic. Historical documents The IL-1 family consists of IL-1α, IL-1β, and IL-1RA (IL-1 recep-
that describe the symptoms suggest that the massive fatalities tor antagonist).8 IL-1α and IL-1β are proinflammatory cytokines
likely resulted from the production of extremely high levels of produced by monocytes, macrophages, and dendritic cells early
cytokines.1 Hypotension, fever, and edema follow infection and on in the immune response. IL-1 production may be induced by
eventually lead to organ dysfunction and death. It is evident that the presence of microbial pathogens, bacterial lipopolysaccha-
the inflammatory response requires regulation to prevent the rides, or other cytokines. IL-1α and IL-1β exhibit the same ac-
damaging systemic inflammation of a cytokine storm. Anti- tivities in many test systems and share about 25% sequence
inflammatory cytokines can resolve the inflammation and asso- homology.8 However, IL-1α remains within the cells that produce
ciated collateral damage to host cells.6 Table 6–1 displays major it and is rarely found outside these cells. IL-1α can be released
proinflammatory and anti-inflammatory cytokines. after cell death and can help attract inflammatory cells to areas
The increasing clinical usage of cytokines, cytokine antago- where cells and tissues are being killed or damaged.
nists, and cytokine receptor antagonists in conditions such as IL-1β is responsible for most of the systemic activity attrib-
rheumatoid arthritis (RA), asthma, and Crohn’s disease drives uted to IL-1, including fever, activation of phagocytes, and pro-
demand for cytokine assays in the clinical laboratory. In addition, duction of acute-phase proteins. It is cleaved intracellularly to
the clinical laboratory plays an important role in assessing treat- an active form that is then secreted by monocytes. IL-1 acts as
ment modalities, effectiveness, and potential gene-replacement an endogenous pyrogen (induces fever) in the acute-phase re-
therapies for numerous immunodeficiency syndromes and sponse through its actions on the hypothalamus.9 The hypo-
leukemias caused by defects in cytokines or their receptors/signal thalamus functions as the thermostat for the human body; IL-1
transduction circuits.7 sets the thermostat at a higher level. Elevated body temperatures
The following sections will discuss cytokines according to may serve to inhibit growth of pathogenic bacteria and viruses
their participation in either the innate immune response or the and also increase lymphocyte activity. Additionally, IL-1 induces
adaptive immune response. The major cytokines and their the production of vascular cell-adhesion molecules as well as
functions are summarized in the Study Guides at the end of chemokines and IL-6. These chemokines and cell-adhesion
this chapter. molecules attract and assist leukocytes to enter the inflamed
area through a process known as diapedesis, which is the passage
of leukocytes through the walls of the blood vessels into the tis-
Cytokines in the Innate sues (see Chapter 1). IL-1 also induces the production of CSFs
Immune Response in the bone marrow, thereby increasing the available number of
phagocytic cells that can respond to the damaged tissues.10
Cytokines involved in the innate immune response are re- IL-1RA, which is also produced by monocytes and
sponsible for many of the physical symptoms attributed to macrophages, is the best characterized cytokine inhibitor.
inflammation, such as fever, swelling, pain, and cellular IL-1RA acts as an antagonist to IL-1 by blocking the IL-1 re-
ceptor, which helps to regulate the physiological response to
IL-1 and turn off the response when no longer needed.
Table 6–1 Examples of Major Proinflammatory
and Anti-Inflammatory Cytokines Tumor Necrosis Factors
MAJOR MAJOR ANTI- Tumor necrosis factors (TNF) were first isolated from lym-
PROINFLAMMATORY INFLAMMATORY phocytes and macrophages and were so named because they
CYTOKINES CYTOKINES induced lysis in tumor cells. TNF-α is the most prominent
TNF-α TGF-β member of the TNF superfamily, which consists of at least
19 different peptides that have diverse biological functions.11
IL-1 IL-10
TNF-α exists in both membrane-bound and soluble forms and
IL-6 IL-13 causes vasodilation and increased vasopermeability. The main
trigger for TNF-α production is the presence of lipopolysac-
IFN- γ IL-35
charide, which is found in gram-negative bacteria.
 TNF-α secreted by activated monocytes and macrophages TNFR1 (TNF receptor 1) is constitutively expressed on most
can activate T cells through its ability to induce expression of tissues and binds soluble TNF-α. It is the primary mediator of
class II MHC molecules, vascular adhesion molecules, and TNF-α signal transduction in most cell types. TNFR2 is usually
chemokines in a manner similar to IL-1. These actions enhance expressed in epithelial cells and cells of the immune system and
antigen presentation and activate T cells to respond to the is activated by the membrane-bound form of TNF-α. Overall,
pathogen that triggered the initial inflammatory response. How- TNF-α activity is at least partially regulated by soluble forms of
ever, when secreted at higher levels, TNF can have deleterious both TNF receptors. These receptors act to bind excess TNF-α
systemic effects, leading to septic shock. This condition results and, combined with the short half-life of the soluble form, serve
from large amounts of TNF secreted in response to gram- to limit the cytokine’s signaling activity.
negative bacterial infections, causing a decrease in blood pres- TNF-α as well as IL-1 are present in rheumatoid synovial
sure, reduced tissue perfusion, and disseminated intravascular fluids and synovial membranes of patients with rheumatoid
coagulation. The latter may lead to uncontrolled bleeding. arthritis (RA). Studies with anti-TNF-α and anti-IL-1 demon-
strate that TNF-α is the central mediator of pathological
processes in RA and other inflammatory illnesses such as
Crohn’s disease, ulcerative colitis, and juvenile arthritis.12
Clinical Correlation Given the profound therapeutic potential of TNF-α inhi-
bition, it has been suggested that different chronic inflamma-
The Role of Cytokine Storm
tory diseases may share common pathophysiology and that
in Ebola Virus Infection
several different cytokine-targeted therapies may emerge as
The Ebola virus causes one of the most serious and fatal diseases feasible tools to disrupt the cytokine network, leading to
known to humans This single-stranded RNA virus
TNF-α activation.
begins its attack on the body within 2 to 21 days after exposure.
Early symptoms include severe headache, fever, muscle pain,
fatigue, diarrhea, vomiting, abdominal pain, and unexplained
Interleukin-6
bruising or hemorrhaging. It is extremely infectious and can IL-6 is a single protein produced by both lymphoid and non-
spread quickly through a population. lymphoid cell types. It is part of the cytokine cascade released
As the immune system tries to halt the spread of the virus, a in response to lipopolysaccharide and plays an important role
profuse release of proinflammatory cytokines occurs, often called in acute-phase reactions. IL-6 is expressed by a variety of nor-
a cytokine storm. It is actually the overabundance of cytokines that
mal and transformed cells, including T cells, B cells, monocytes
is responsible for the devastating effects on the body. TNF-α, in
particular, causes the blood vessels to become more permeable, and macrophages, vascular endothelial cells, and various tumor
resulting in dangerously low blood pressure. As the platelet count cells. IL-1 primarily triggers its secretion.
drops, excessive bleeding occurs from every orifice in the body. This pleiotropic cytokine affects inflammation, acute-phase
Death typically results in 1 to 2 weeks after infection. reactions, immunoglobulin synthesis, and the activation states of
The cytokine storm is a profound example of the importance B cells and T cells. IL-6 stimulates B cells to proliferate and dif-
of regulation of the immune system. If left unchecked, the ferentiate into plasma cells and induces CD4+ T cells to produce
cytokines that normally help to overcome infection can have a greater quantities of both pro- and anti-inflammatory cytokines.3
deleterious effect. Only one IL-6 receptor has been identified that consists of
IL-6Rα (the IL-6-specific receptor) and gp130 (the common
signal-transducing receptor subunit used by several cytokines).
Binding of IL-6 to the IL-6Rα induces dimerization of gp130
with the α-subunit (Fig. 6–4). Homodimerization following
IL-6 binding causes conformational changes in gp130 that ex-
pose tyrosine residues in the intracellular portion of the mole-
cule. Through a series of phosphorylation reactions, genes for
acute-phase proteins such as C-reactive protein (CRP), the
third component of complement (C3), and fibrinogen are
activated, as is interferon regulatory factor-1 (IRF-1). B- and
T-cell genes are turned on in the same manner.
Studies have demonstrated that chronic psychosocial stress
increases the risk of atherosclerotic cardiovascular disease,
which may involve many mediators and pathways. The hy-
pothesis has been formulated that stress promotes atherogen-
esis by activation of vascular inflammation via elevated
circulating proinflammatory cytokine levels (e.g., TNF-α,
False-colored micrograph of the Ebola virus virion. IL-6). Although it is clear that circulating cytokine levels serve
(Courtesy of the CDC/Frederick A. Murphy, Public Health Image as reliable biomarkers of systemic inflammation, further studies
Library.) are needed to better define the role of cytokines in mediating
inflammatory reactions.13
 IL-6 Table 6–2 Select Chemokines and Their
Receptors
! ! CHEMOKINE CHEMOKINE CHEMOKINE
! ! GROUP NAMES RECEPTORS
IL-6
CC Chemokines CCL2 CCR2
! ! CCL3 CCR1
CCR5
CCL4 CCR5
CCL5 CCR1
CCR3
JAK JAK CCR5

JAK JAK CCL11 CCR3

CCL19 CCR7
STAT CCL20 CCR6
CCL25 CCR9
CXC Chemokines CXCL1 CXCR2
CXCL2
CXCL3
CXCL5
CXCL7
IL-6 binding to the IL-6R subunit recruits two gp130
subunits to the receptor complex. Signal transduction takes place CXCL8 CXCR1
through tyrosine phosphorylation of the gp130 subunits by one of CXCR2
the Janus kinases (J). CXCL9 CXCR3-A
CXCL10 CXCR3-B
CXCL11
Chemokines CXCL12 CXCR4
Chemokines are a family of cytokines that enhance motility
and promote migration of many types of white blood cells
(WBCs) toward the chemokine source via a process known as chemokine receptors among different types of leukocytes allows
chemotaxis.14 Most of the chemotactic activity of leukocytes is for the co-localization of multiple cell types to the damaged tis-
regulated by the activities of chemokines, including the re- sue and helps to broaden the response to tissue damage. The
sponse to infectious diseases, autoimmune inflammation, can- gradient of chemokine concentration enables the leukocytes to
cer, and the homing of lymphocytes to all the lymphoid tissues. migrate between the endothelial cells into the tissue in the
The chemokines are classified into four families based on the direction of increasing chemokine concentration.
position of N-terminal cysteine residues. The first group—the The spectrum of chemokines and cytokines expressed in the
alpha, or CXC, chemokines—contains a single amino acid inflammatory response determines the types of cells that re-
between the first and second cysteines. The second group— spond and the genes that are turned on in response to the stim-
the beta, or CC, chemokines—has adjacent cysteine residues. uli. The types of cell surface receptors expressed by leukocytes
The third group—the C chemokines—lacks one of the cys- are often developmentally regulated—for example, immature
teines. CX3C, the last major group, has three amino acids T cells possess only the chemokine receptors related to lym-
between the cysteines. Chemokines play key roles in the initi- phoid tissue homing. Only mature T cells express the receptors
ation and development of inflammatory responses in numer- that allow them to participate in an ongoing immune reaction.
ous disease processes. Currently, over 40 chemokines and HIV uses the chemokine receptors CXCR4 and CCR5 as co-
20 chemokine receptors have been identified: select chemokines receptors for infection of CD4+ T lymphocytes and
and receptors are displayed (Table 6–2). macrophages.15 Individuals with certain polymorphisms in these
Both TNF-α and IL-6 are among the many cytokines that chemokine receptors are long-term nonprogressors. They remain
induce chemokine production in the inflammatory response. asymptomatic with normal CD4+ T-cell counts and immune
Combined with cell adhesion molecules, the chemokines facil- function as well as low or undetectable viral loads. The altered
itate the extravasation of leukocytes into the tissues. Leukocytes protein sequences of the receptors block or diminish the virus’s
rolling on capillary endothelial cells activate their chemokine ability to enter the cells and thereby increase the infected indi-
receptors in the presence of chemokines. Integrins, or cell ad- vidual’s chances of survival. The CCR5-δ32 polymorphism is a
hesion molecules on leukocytes, are then activated, leading to 32 bp deletion in the CCR5 gene and is the most important of
firm adhesion to the endothelial cells. Shared expression of the host resistance factors. Homozygous individuals are protected
from HIV infection, whereas heterozygous persons exhibit longer area of infection. In contrast, cytokines involved in the adaptive
periods between HIV infection and AIDS development. immune response are mainly secreted by T cells, especially
Th cells, and affect T- and B-cell function more directly. There
Transforming Growth Factor-β are three main subclasses of Th cells: T helper 1 (Th1),
T helper 2 (Th2), and T regulatory (Treg) cells. Each has
The transforming growth factor-β (TGF-β) superfamily is a specific function and produces a different set of cytokines.
composed of three isoforms: TGF-β1, TGF-β2, and TGF-β3. T helper 17 (Th17) cells are a fourth subset of Th cells.
TGF-β was originally characterized as a factor that induced Because Th17 cells affect both the innate and adaptive immune
growth arrest in tumor cells. Later, it was identified as a factor response, these will be discussed separately.
that induces antiproliferative activity in a wide variety of cell Once the T-cell receptor (TCR) captures antigen, clonal
types. Active TGF-β is primarily a regulator of cell growth, dif- expansion of those particular CD4+ Th cells occurs.20 Dif-
ferentiation, apoptosis, migration, and the inflammatory re- ferentiation into Th1, Th2, or Treg cell lineages is influenced
sponse. Thus, it acts as a control to help downregulate the by the spectrum of cytokines expressed in the initial re-
inflammatory response when no longer needed. sponse (Fig. 6–5).21 The Th1 lineage is driven by the ex-
In the immune response, TGF-β functions as both an activa- pression of IL-12 by dendritic cells and is primarily
tor and an inhibitor of proliferation, depending on the develop- responsible for cell-mediated immunity, whereas Th2 cells
mental stage of the affected cells.16 TGF-β regulates the drive antibody-mediated immunity and are developmentally
expression of CD8 in CD4–CD8– thymocytes and acts as an au-
tocrine inhibitory factor for immature thymocytes. It inhibits the
activation of macrophages and the growth of many different so- 2. Extracellular pathogens
parasites
matic cell types and functions as an anti-inflammatory factor for 3. Fungi
mature T cells. TGF-β blocks the production of IL-12 and
strongly inhibits the induction of IFN-gamma. In addition, the 1. Bacteria 1 IL-12, IFN#
production of TGF-β by T helper 2 (Th2) cells is now recognized viruses
as an important factor in the establishment of oral tolerance 3 IL-6, IL-23
to bacteria normally found in the mouth. In activated B cells, TGF"
TGF-β typically inhibits proliferation and may function as an 2 IL-4*
autocrine regulator to limit the expansion of activated cells.
Naive
CD4"
Interferon-α and Interferon-β
Interferons (IFN) were originally so named because they in-
terfere with viral replication. However, it is the type I IFNs con-
sisting of IFN-α and IFN-β that function primarily in this
manner. These IFNs are produced by dendritic cells and in-
duce production of proteins and pathways that directly inter-
fere with viral replication and cell division. In most cases, this TH1 TH17 TReg TH2
helps limit the infection to one relatively small area of the body.
Type I IFN activates natural killer (NK) cells and enhances the
expression of class I MHC proteins, thus increasing the recog-
nition and killing of virus-infected cells.
The type I IFNs are also active against certain malignancies
and other inflammatory processes. For instance, IFN-β is effi-
cacious in treating multiple sclerosis, although the exact mech- IFN# IL-17 TGF" IL-4
anism of action remains unclear.17 A clue has been gained by TNF" IL-22 IL-10 IL-5
IL-13
recent genome-wide association studies that discovered a rela-
tionship between IFN-β response and ion receptor channel (#) ("$#)
proteins; however, the role of these proteins is not elucidated.18
IFN-α has been used to treat hepatitis C and Kaposi’s sarcoma, Cytotoxic T cells Mast cells, B cells,
Macrophages Eosinophils
as well as certain leukemias and lymphomas.19
Cell-mediated immunity Humoral immunity
inflammation antiparasitic
Cytokines in the Adaptive Subpopulations of T cells. Depending upon the
pathogen encountered, antigen-presenting cells (APCs) secrete
Immune Response a specific combination of polarizing cytokines that direct naïve
T helper (Th) cells to differentiate into specialized subsets. Each
Cytokines involved in the innate immune response are pro- T-cell subpopulation exerts different types of immune functions.
duced by many different cell types. Cytokines function mainly The release of effector cytokines coordinates an appropriate
to increase acute-phase reactants and to recruit WBCs to the immune response against the pathogen.
regulated by IL-4. Treg cells are derived from naïve T cells is demonstrated in individuals who have mutations in this
in response to TGF-β. Tregs help to regulate the activities of chain. These persons have X-linked severe combined immu-
Th1 and Th2 cells. nodeficiency syndrome and lack functional T and B cells.26

Th1 Cytokines Th2 Cytokines

Dendritic cells in damaged tissues produce IL-12 in response As mentioned previously, Th2 cells are primarily responsible
to certain stimuli such as mycobacteria, intracellular bacteria, for antibody-mediated immunity. Among the cytokines pro-
and viruses. It is also produced by macrophages and B cells duced are IL-4 and IL-10. Both of these cytokines are impor-
and has multiple effects on both T cells and NK cells. IL-12 tant regulators of the immune response but have opposite
binds to its receptor on naïve T cells and causes the expression effects.
of a new set of genes, including those that determine matura-
tion into the Th1 lineage. Activation of Th1 cells induces high-
IL-4 is one of the key cytokines regulating Th2 immune activ-
level expression of IFN-gamma.22 IL-12 also increases the
ities and helps drive antibody responses in a variety of
cytolytic ability of NK cells; therefore, it serves as an important
diseases.26 The IL-4 receptor is expressed on lymphocytes and
link between the innate and adaptive immune responses by
on numerous nonhematopoietic cell types. IL-4 activity on
enhancing defenses against intracellular pathogens.
naïve T cells turns on the genes that generate Th2 cells and
γ γ turns off the genes that promote Th1 cells.
IFN-γ, the principal molecule produced by Th1 cells, affects Th2 cells are responsible for regulating many aspects of the
the RNA expression levels of more than 200 genes.23 A major immune response, including those related to allergies, autoim-
function of IFN-γ is stimulation of antigen presentation by mune diseases, and parasites. IL-4 induces production of
class I MHC and class II MHC molecules. Increased expression MHC-I, IL-4, IL-5, IL-13, and the costimulatory molecules
of class I and II MHC molecules on antigen-presenting cells CD80 and CD86. IL-4 also stimulates the production of IgG2a
(APCs) increases the likelihood of antigen capture and involve- and IgE and, along with IL-5, drives the differentiation and ac-
ment of additional lymphocytes. IFN-γ is also the most potent tivation of eosinophils in both allergic immune responses and
activator of macrophages and boosts their tumoricidal activ- response to parasitic infections.26 IL-13 is a cytokine with
ity.24 In this capacity, it stimulates the phagocytic and cyto- many of the same properties as IL-4; both cytokines induce
toxic abilities of these cells, creating activated or “super” worm expulsion and favor IgE-class switching. IL-13, however,
macrophages. In addition, IFN-γ is involved in regulation and differs from IL-4 because it also plays an anti-inflammatory role
activation of CD4+ Th1 cells, CD8+ cytotoxic lymphocytes, by inhibiting activation and cytokine secretion by monocytes.
and NK cells. Thus, IFN-γ influences the immune response in
a number of key ways.
In contrast to the cytokines previously discussed, IL-10 has
primarily inhibitory effects on the immune system. It is pro-
Th1 cells also secrete IL-2 in addition to IFN-γ. IL-2, also duced by monocytes, macrophages, CD8+ T cells, and Th2
known as the T-cell growth factor, drives the growth and dif- CD4+ T cells and has anti-inflammatory and suppressive ef-
ferentiation of both T and B cells and induces lytic activity in fects on Th1 cells. It also inhibits antigen presentation by
NK cells. IL-2 and IFN-γ induce the development of Th1 macrophages and dendritic cells. In addition, one of the major
cells, which, in turn, induces macrophage activation and de- effects of IL-10 is the inhibition of IFN-gamma production via
layed type hypersensitivity. Th1 cells stimulate the produc- the suppression of IL-12 synthesis by accessory cells and the
tion of IgG1 and IgG3 opsonizing and complement fixing promotion of a Th2 cytokine pattern.27 Thus, IL-10 serves as
antibodies by antigen-activated B cells. These isotypes assist an antagonist to IFN-gamma—it is a downregulator of the
in cell-mediated immune responses driven by Th1 cells. immune response.
IL-2 alone can activate proliferation of Th2 cells and helps
to generate IgG1- and IgE-producing cells. Clonal expansion Cytokines Associated
of activated Th cells is a necessary part of mounting an ade-
quate immune response to any immunologic challenge. The
With T Regulatory Cells
cytokine network that develops will continue to regulate T-cell The third major subclass of CD4+ T cells are the T regulatory
growth and differentiation until the challenge is gone and the (Treg) cells. Tregs are CD4+ CD25+ T cells that are selected in
response subsides. the thymus.28 An additional population of Tregs, called in-
Transcription of the gene for IL-2 and IL-2R begins within duced Tregs (iTregs), can develop from mature T cells in the
1 hour of binding to the TCR. The functional IL-2R consists periphery.26 Tregs play a key role in establishing peripheral tol-
of α, β, and γ subunits or just β and γ subunits. The β and γ erance to a wide variety of self-antigens, allergens, tumor anti-
subunits increase the affinity of the receptor for IL-2 and are gens, transplant antigens, and infectious agents. CD4+ CD25+
responsible for most of the signal transduction through the re- Tregs affect T-cell activity primarily through the actions of
ceptor. The γ chain is also shared by the receptors for IL-4, TGF-β. TGF-β induces expression of Foxp3, a transcription
IL-7, IL-9, IL-15, and IL-21.25 The importance of the γ chain factor that causes Treg cells to suppress the activity of other
T cells.29 Tregs may be found in transplanted tissue and help defense. Dysregulation of Th17 cell subsets and secreted cy-
to establish tolerance to the graft by the host immune system tokines has been implicated in pathogenesis of multiple in-
through the alteration of antigen presentation. flammatory diseases and several autoimmune conditions,
Tregs are also responsible for inducing IL-10 and TGF-β ex- including RA, MS, inflammatory bowel disease (IBD), and
pression in adaptive T regulatory 1 (Tr1) cells in the periph- psoriasis.32,33
eral circulation. Tr1 cells are CD4+ T cells that are induced from IL-17 can produce proinflammatory mediators from
antigen-activated naïve T cells in the presence of IL-10. They myeloid and synovial fibroblasts and perpetuate the inflamma-
exert their suppressive activities on both Th1 and Th2 cells by tory process in RA.33 Increased numbers of Th17 cells and
producing more IL-10, TGF-β, or IL-35. T-cell suppression oc- IL-17A have also been observed in asthmatic and allergic
curs through IL-10 inhibition of proinflammatory cytokines and patients.33 IL-17A directly induces IgE production by B cells33;
inhibition of costimulatory molecule expression on APCs. higher amounts of IL-17A and IL-17F in patient lungs have
TGF-β downregulates the function of APCs and blocks prolif- been associated with more severe asthma.34 Interestingly, re-
eration and cytokine production by CD4+ T cells. IL-35 is a re- moval of Th17 cells from peripheral blood mononuclear cells
cently identified cytokine that has immunosuppressive effects of allergic asthma patients has led to decreased levels of IgE.33
on Th1, Th2, and Th17 cells while promoting growth of
Tregs.20 All of these activities lead to downregulation of the Hematopoietic Growth Factors
immune response and the prevention of chronic inflammation.
Both Tregs and Tr1 cells operate through a network of cy- A number of cytokines produced during innate and adaptive
tokines to establish peripheral tolerance to certain antigens; immune responses stimulate the proliferation and differentia-
therefore, they play a key role in limiting autoimmunity. The tion of bone marrow progenitor cells. Thus, the responses that
relative importance of each phenotype depends largely on require a supply of leukocytes produce mediators to provide
the antigen involved, the context of antigen presentation, and those cells. The primary mediators of hematopoiesis are called
the biology of the tissue. colony stimulating factors (CSFs) because they stimulate the
formation of colonies of cells in the bone marrow. The CSFs
include IL-3, erythropoietin (EPO), granulocyte colony
Th17 Cytokines in Innate stimulating factor (G-CSF), macrophage colony stimulating
and Adaptive Immune Responses factor (M-CSF), and granulocyte-macrophage colony stim-
ulating factor (GM-CSF).20,35–37 In response to inflammatory
The Th17 subset secretes the IL-17 family of cytokines and cytokines such as IL-1, the different CSFs act on bone marrow
plays critical roles in both innate and adaptive immune re- cells at different developmental stages and promote specific
sponses. Key cytokines that differentiate T cells to maintain colony formation for the various cell lineages. IL-3 is a multi-
them as Th17 cells are TGF-β and IL-6.30 Interleukin-23, pro- lineage CSF that induces bone marrow stem cells to form
duced by macrophages and dendritic cells, also plays a role in T and B cells. In conjunction with IL-3, the CSFs direct imma-
finalizing the commitment to Th17 cells.31 ture bone marrow stem cells to develop into red blood cells
IL-17A, the first IL-17 identified, is the most studied IL-17 (RBCs), platelets, and the various types of WBCs (Fig. 6–6).
cytokine. Other IL-17 family members include IL-17B, IL-17C, IL-3 acts on bone marrow stem cells to begin the differentiation
IL-17D, IL-17E, and IL-17F. Most of the IL-17 cytokine family cycle; the activity of IL-3 alone drives the stem cells into the
members are potent proinflammatory cytokines and induce ex- lymphocyte differentiation pathway.
pression of TNF-α, IL-1β, and IL-6 in epithelial, endothelial, GM-CSF acts to drive differentiation toward other WBC
keratinocyte, fibroblast, and macrophage cells. types. If M-CSF is activated, the cells become macrophages.
Th17 cells play an important role in host defense against M-CSF also increases phagocytosis, chemotaxis, and additional
bacterial and fungal infections at mucosal surfaces. Upon en- cytokine production in monocytes and macrophages. If G-CSF
counter with bacteria or fungus, APCs secrete cytokines, which is activated, the cells become neutrophils. G-CSF enhances the
differentiate Th17 subsets of cells. Th17 cells invade the in- function of mature neutrophils and affects the survival, prolif-
fected area and secrete IL-17 cytokines necessary for continuous eration, and differentiation of all cell types in the neutrophil
recruitment of neutrophils.31 Th17 cells in the local tissue may lineage. It decreases IFN-gamma production, increases IL-4
be important for long-term maintenance of antimicrobial re- production in T cells, and mobilizes multipotential stem cells
sponse during chronic bacterial infections. Mucosal surfaces, in from the bone marrow. These stem cells are used to repair dam-
response to IL-17 cytokine stimulation, secrete antimicrobial aged tissues and create new vasculature to reconstruct the tis-
peptides. IL-17A and IL-17F induce epithelial cells, endothelial sues following an infection. However, IL-3 in conjunction with
cells, and fibroblasts to produce CXC ligand 8 (CXCL-8), which GM-CSF drives the development of basophils and mast cells,
is crucial for recruitment of neutrophils to the site of inflamma- whereas the addition of IL-5 to IL-3 and GM-CSF drives the
tion. IL-17A and IL-17F also act together with granulocyte- cells to develop into eosinophils. The net effect is an increase
macrophage CSF to produce CXCL-8 in macrophages, which in WBCs to respond to the ongoing inflammatory processes.
also signals neutrophils to the site.31 EPO regulates RBC production in the bone marrow but is pri-
The fine regulation of APC cytokines (IL-23 or IL-12) and marily produced in the kidneys. EPO-α is the form licensed for
Th17 development may also be important for antimicrobial clinical use by the FDA. EPO-α is often prescribed to improve
 HSC

TPO SCF IL-7

CMP CLP

EPO IL-3 GM-CSF

Influence of colony stimulating factors on growth

and differentiation of blood cells. Growth of hematopoietic
stem cells (HSC) requires stem cell factor (SCF) with differentia-
tion determined by IL-7 or thrombopoietin (TPO). Growth of EB GMP
IL-3 M-CSF
common myeloid progenitors (CMP) depends upon IL-3.
G-CSF
Differentiation is driven by granulocyte-macrophage colony EPO G-CSF IL-5
stimulating factor (GM-CSF) or erythropoietin (EPO). Common
granulocyte/monocyte precursors (GMP) differentiate into
granulocytes in response to granulocyte-CSF. Further specificity
is provided by IL-3 or IL-5. Macrophage-CSF (M-CSF) promotes
development of monocytes. CLP = common lymphoid
progenitor, EB = erythroblast. Erythrocytes Basophil Eosinophil Neutrophil Monocyte

RBC counts for individuals with anemia and for those with cancer Another approach to anticytokine therapy is the develop-
who have undergone radiation and chemotherapy. RBC prolifer- ment of a class of hybrid proteins containing cytokine receptor
ation induced by EPO improves oxygenation of the tissues and binding sites attached to immunoglobulin constant regions.
eventually switches off EPO production. The normal serum EPO Etanercept (Enbrel), for example, is a member of this class that
values range from 5 to 28 U/L but must be interpreted in relation has been approved for use in humans.39 Enbrel consists of the
to the hematocrit because levels can increase up to a thousand- extracellular domains of the type 2 TNF receptor fused to the
fold during anemia. heavy-chain constant region of IgG1. The fusion protein can
bind TNF-α and block its activity. It has a 4.8-day half-life in
serum. Etanercept remains an important cost-effective treat-
Cytokine and Anticytokine ment option in patients with RA, ankylosing spondylitis, pso-
Therapies riatic arthritis or psoriasis, and pediatric arthritis and psoriasis.
Etanercept effectively reduces signs and symptoms, disease
Cytokine-inhibiting biologics disrupt the interaction between activity, and disability and improves health-related quality of
cytokines and their cognate receptors. Initial studies used life, with these benefits sustained during long-term treatment.
murine monoclonal antibodies that were able to function only The blockade of IL-17 function has also been a therapeutic
for short periods of time before the host mounted an immune target. Researchers are currently developing an IL-17 blocking
response against them. Recombinant DNA techniques have al- antibody to prevent IL-17 mediated pathogenesis, such as in
lowed for the production of humanized monoclonal antibodies psoriasis.40 RA patients treated with the IL-17A blocking anti-
that are much less immunogenic and that function as cytokine body known as ixekizumab (formerly LY2439821 from
antagonists. An example is infliximab (Remicade), a chimeric Eli Lilly) in clinical trials showed significantly improved signs
antibody containing human constant regions and murine antigen- and symptoms with no significant safety concern.41 Blockade of
specific arms that bind human TNF-α.38 Infliximab is a valuable IL-17 receptors is another approach under therapeutic assessment
treatment option for patients with Crohn’s disease or RA be- for specific subgroups of asthma patients.42 An additional cy-
cause it blocks activity of TNF-α in RA and Crohn’s disease with tokine receiving attention is IL-23, which enhances the differ-
rapid onset of therapeutic action after administration. entiation of Th17 lymphocytes. Blocking of this interleukin has
become another focus of research in the treatment of asthma cytokine, allows for the detection of up to 100 different
and may have an application for other autoimmune diseases.43 analytes in one tube. The use of a multiplexed bead array
Preclinical studies, such as those supporting the central role enables simultaneous measurement of a multitude of
of IL-17 in models of inflammatory bowel disease, have not biomarkers; these include acute-phase reactants such as
only reinforced the concept of common pathophysiology of CRP; proinflammatory cytokines; Thl/Th2 distinguishing
several autoimmune diseases such as RA, but have also raised cytokines such as IFN-gamma, IL-2, IL-4, IL-5, and IL-10;
hopes that blockade of new cytokines may show even superior other nonspecific acting cytokines; and CSFs.
efficacy over TNF-α inhibition. These lines of evidence suggest One drawback of protein-based technologies is the short
that there is a shared cytokine framework that defines highly half-life of certain cytokines. This can be overcome by looking
conserved mechanisms of inflammation in certain human dis- at RNA expression in cells using reverse transcription poly-
eases and indicates multiple vulnerable nodes.12 Neutralization merase chain reaction (PCR). The PCR product is made using
of one of these nodes may therefore suffice to disrupt the in- a fluorescent-labeled primer and can be hybridized to either
flammatory process in a large variety of human inflammatory solid-phase or liquid microarrays. Solid-phase arrays have up
diseases. to 40,000 spots containing specific ssDNA oligonucleotides
representing individual genes. Clinically useful arrays generally
have substantially fewer genes represented. Fluorescence of a
Clinical Assays for Cytokines spot indicates that the gene was expressed in the cell and that
the cell was producing the cytokine.
Clinical evaluation of the cytokine profile in the patient could
The liquid arrays use the same beads as the antibody mi-
be of prognostic and diagnostic value to the physician when
crobead arrays discussed earlier. However, instead of anti-
treating autoimmune diseases such as the ones mentioned in
bodies, these beads have oligonucleotides on their surfaces
the previous sections. Several cytokine assay formats are avail-
and allow up to 100 different cDNAs to be identified. The
able for basic and clinical research use, including ELISpot
combination of the bead fluorescence and the fluorescence
assays, multiplexed ELISAs, and microbead assays.
of the labeled cDNA produces an emission spectrum that
The ELISpot assay employs the enzyme-linked immunosor-
identifies the cytokine gene that was expressed in the cells.
bent assay (ELISA) technique on in vitro-activated peripheral
Real time PCR has emerged as a means of detecting cytokine
WBCs. In the ELISpot process, either a monoclonal or poly-
response at the level of gene expression.46 This type of testing
clonal antibody specific for the chosen cytokine is precoated
is much faster than traditional PCR. See Chapter 12 for a
onto a microplate. Antigen-stimulated, mitogen-stimulated
discussion of molecular techniques.
(positive control), or saline-stimulated (negative control) WBCs
are pipetted into the wells and the microplate is placed into a
humidified CO2 incubator at 37°C for a specified period of
SUMMARY
time. During the incubation period, the immobilized antibody
in the immediate vicinity of the secreting cells binds the se- • Cytokines are small, soluble proteins secreted by white
creted cytokine. After any cells and unbound substances are blood cells and a variety of other cells. They act as chem-
washed away, a biotinylated polyclonal antibody specific for ical messengers to regulate the immune system.
the chosen cytokine is added to the wells. Following a wash • Cytokines are induced in response to specific stimuli such
to remove any unbound biotinylated antibody, alkaline- as bacterial lipopolysaccharides, flagellin, or other bacterial
phosphatase conjugated to streptavidin is added. Unbound en- products.
zyme is subsequently washed away and a substrate solution is • The effects of cytokines in vivo include regulation of
added. A colored precipitate forms and appears as spots at the growth, differentiation, and gene expression by many
sites of cytokine localization, with each spot representing an different cell types.
individual cytokine-secreting cell. The spots can be counted • If a cytokine has an autocrine effect, it affects the same cell
with a stereomicroscope or with an automated ELISpot reader. that secreted it.
Multiplexed ELISAs use several detector antibodies bound • A cytokine can have a paracrine effect when it affects a
to individual microwells or antibody microarrays and allow target cell in close proximity.
for simultaneous detection of several cytokines from serum • Occasionally, cytokines will exert systemic or endocrine
or plasma.44 Current formulations allow for the detection of activities.
12 to 25 pro- and anti-inflammatory cytokines in one reaction. • Individual cytokines do not act alone but in conjunction
In the microarray format, each well on the slide contains a mi- with many other cytokines that are induced during the
croarray of spotted antibodies, with “spots” for each of the process of immune activation.
cytokines plus additional “spots” for positive and negative • The combined cytokines produce a spectrum of activities
controls. The replicate spots allow for acquisition of reliable that lead to the rapid generation of innate and adaptive
quantitative data from a single sample. immune responses.
New microbead assays allow for the simultaneous detec- • Massive overproduction is called a “cytokine storm” that
tion of multiple cytokines in a single tube.45 Each bead type leads to shock, multi-organ failure, or even death, thus
has its own fluorescent wavelength, which, when combined contributing to pathogenesis.
with the fluorescent secondary antibody bound to a specific
• The pleiotropic nature of cytokine activity relates to • Treg cells are derived from naïve T cells in response to
the ability of a cytokine to affect many different types IL-10 and TGF-β and help to regulate the activities of
of cells. Th1 and Th2 cells.
• If several different cytokines activate some of the same • Th17 cells produce IL-17, a proinflammatory cytokine
cells, it is termed redundancy. that induces expression of TNF-α, IL- 1β, and IL-6. Forms
• The major cytokines involved in the initial stages of the of IL-17 also recruit neutrophils to an infected area.
inflammatory response are IL-1, IL-6, TNF-α, and the • Colony stimulating factors (CSFs) are responsible for
chemokines. These cytokines are responsible for many of inducing differentiation and growth of all WBC types.
the physical symptoms attributed to inflammation such • Anticytokine therapies are aimed at disrupting the inter-
as fever, swelling, pain, and cellular infiltrates into dam- action between cytokines and their specific receptors in
aged tissues. diseases such as rheumatoid arthritis and Crohn’s disease.
• Naïve T cells can differentiate into Th1, Th2, or Treg cell • Cytokine assay formats available for basic and clinical
lineages, with the help of cytokines involved in the adap- research use include ELISpot assays, multiplexed ELISAs,
tive immune response. and microbead assays.
• The Th1 lineage is driven by the expression of IL-12 • ELISpot assays are enzyme-linked immunosorbent assays
by dendritic cells and is primarily responsible for cell- on in vitro activated peripheral WBCs that detect one
mediated immunity. cytokine at a time.
• Th2 cells drive antibody-mediated immunity and are • Multiplexed ELISAs and microbead assays can detect
regulated by IL-4. several cytokines in serum at a time.

Study Guide: Cytokines Associated With Innate Immunity

CYTOKINE SECRETED BY ACTIONS
Interleukin-1β Monocytes, macrophages, dendritic cells Inflammation, fever, initiation of the
(IL-1β) acute-phase response
Tumor necrosis Monocytes, macrophages, neutrophils, Inflammation, initiation of the acute-phase
factor-α (TNF-α) NK cells, activated T cells response, death of tumor cells
Interleukin-6 (IL-6) Monocytes, macrophages, endothelial cells, Initiation of the acute-phase response,
Th2 cells activation of B and T cells
Transforming growth T cells, macrophages, other cell types Inhibition of both T- and B-cell proliferation,
factor-β (TGF-β) induction of IgA, inhibition of macrophages
Interferon-α (IFN-α) Macrophages, dendritic cells, virally infected Protects cells against viruses, increases
Interferon-β (IFN-β) cells class I MHC expression, activates NK cells

Study Guide: Cytokines Associated With Adaptive Immunity

CYTOKINE SECRETED BY ACTIONS
Interleukin-2 (IL-2) T cells Growth and proliferation of T and B cells
NK activation and proliferation
Interleukin-4 (IL-4) Th2 cells, mast cells Promotion of Th2 differentiation, stimulation of B cells to
switch to IgE production
Interleukin-5 (IL-5) Th2 cells Eosinophil generation and activation, B-cell differentiation
Interleukin-10 (IL-10) Th2 cells, monocytes, macrophages Suppression of Th2 cells, inhibition of antigen
presentation, inhibition of interferon-gamma
Interferon-γ (IFN- γ ) Th1 cells, CD8+ T cells, NK cells Activation of macrophages, increased expression of class
I and II MHC molecules, increased antigen presentation
CASE STUDY
A 55-year-old woman being treated for acute lymphocytic a liquid bead array that included the CSFs and the cytokines
leukemia (ALL) was not responding well to chemotherapy. typically seen in the innate immune response and in
Her physicians felt that increasing the dosage of her Th1 and Th2 responses.
chemotherapy drugs was necessary to eliminate the cancer.
Questions
However, laboratory results showed the patient was severely
neutropenic (477 neutrophils/mL) as a result of the drugs. a. What colony stimulating factor should the physi-
The medical team could not risk further lowering of the neu- cians prescribe to overcome the neutropenia?
trophil count because of the increased risk of infection. b. What are some of the cytokines that might be
Therefore, it was necessary to treat the patient for neutrope- detected in a Th1 type response?
nia in order to continue with chemotherapy. The patient was c. What are some of the cytokines that might be
also enrolled in a research study designed to look at cytokine detected in a Th2 type response?
expression in ALL patients with neutropenia. The study used

REVIEW QUESTIONS
1. The ability of a single cytokine to alter the expression 6. Which of the following represents an autocrine effect
of several genes is called of IL-2?
a. redundancy. a. Increased IL-2 receptor expression by the Th cell
b. pleiotropy. producing it
c. autocrine stimulation. b. Macrophages signaled to the area of antigen
d. endocrine effect. stimulation
c. Proliferation of antigen-stimulated B cells
2. Which of the following effects can be attributed to IL-1? d. Increased synthesis of acute-phase proteins
a. Mediation of the innate immune response throughout the body
b. Differentiation of stem cells
c. Halted growth of virally infected cells 7. IFN-α and IFN-β differ in which way from
d. Stimulation of mast cells IFN-gamma?
a. IFN-α and IFN-β are called immune interferons,
3. Which of the following precursors are target cells and IFN-gamma is not.
for IL-3? b. IFN-α and IFN-β primarily activate macrophages,
a. Myeloid precursors whereas IFN-gamma halts viral activity.
b. Lymphoid precursors c. IFN-α and IFN-β are made primarily by
c. Erythroid precursors activated T cells, whereas IFN-gamma is made
d. All of the above by fibroblasts.
d. IFN-α and IFN-β inhibit cell proliferation, whereas
4. A lack of IL-4 may result in which of the following IFN-gamma stimulates antigen presentation by
effects? class II MHC molecules.
a. Inability to fight off viral infections
b. Increased risk of tumors 8. A patient in septic shock caused by a gram-negative
c. Lack of IgM bacterial infection exhibits the following symptoms:
d. Decreased eosinophil count high fever, very low blood pressure, and disseminated
intravascular coagulation. Which cytokine is the most
5. Which of the following cytokines is also known as the likely contributor to these symptoms?
T-cell growth factor? a. IL-2
a. IFN-γ b. TNF
b. IL-12 c. IL-12
c. IL-2 d. IL-7
d. IL-10
 9. IL-10 acts as an antagonist to what cytokine? 14. Which cytokine acts to promote differentiation of
a. IL-4 T cells to the Th1 subclass?
b. TNF-α a. IL-4
c. IFN-gamma b. IFN-α
d. TGF-β c. IL-12
d. IL-10
10. Which would be the best assay to measure a specific
cytokine? 15. What is the major function of T regulatory cells?
a. Blast formation a. Suppression of the immune response by
b. T-cell proliferation producing TNF
c. Measurement of leukocyte chemotaxis b. Suppression of the immune response by
d. ELISA testing inducing IL-10
c. Proliferation of the immune response by
11. Selective destruction of Th cells by the human producing IL-2
immunodeficiency virus contributes to immune d. Proliferation of the immune response by
suppression by which means? inducing IL-4
a. Decrease in IL-1
b. Decrease in IL-2 16. Th17 cells affect the innate immune response by
c. Decrease in IL-8 inducing production of which cytokines?
d. Decrease in IL-10 a. IFN-γ and IL-2
b. IL-4 and IL-10
12. Why might a colony stimulating factor be given to a c. IL-2 and IL-4
cancer patient? d. TNF-α and IL-6
a. Stimulate activity of NK cells
b. Increase production of certain types of leukocytes
c. Decrease the production of TNF
d. Increase production of mast cells

13. Which of the following would result from a

lack of TNF?
a. Decreased ability to fight gram-negative bacterial
infections
b. Increased expression of class II MHC molecules
c. Decreased survival of cancer cells
d. Increased risk of septic shock
 Complement System

After finishing this chapter, you should be able to: PATHWAYS OF THE COMPLEMENT
SYSTEM
1. Describe the roles of the complement system.
The Classical Pathway
2. Differentiate between the classical, alternative, and lectin pathways
and indicate proteins and activators involved in each. The Lectin Pathway
3. Discuss the formation of the three principal units of the classical The Alternative Pathway
pathway: recognition, activation, and membrane attack units. SYSTEM CONTROLS
4. Describe how initiation of the lectin pathway occurs. Regulation of the Classical and Lectin
5. Explain how C3 plays a key role in all pathways. Pathways
6. Describe regulators of the complement system and their role in the Regulation of the Alternative Pathway
complement system. Regulation of Terminal Components
7. Discuss the complement-related kidney disorders and applicable COMPLEMENT RECEPTORS AND THEIR
complement testing. BIOLOGICAL ROLES
8. Relate biological manifestations of complement activation to BIOLOGICAL MANIFESTATIONS
generation of specific complement products. OF COMPLEMENT ACTIVATION
9. Describe the deficiencies of complement components and the diseases COMPLEMENT AND DISEASE STATES
they cause. COMPLEMENT DEFICIENCIES
10. Differentiate tests for functional activity of complement from Major Pathway Components
measurement of individual complement components.
Regulatory Factor Components
11. Analyze laboratory findings and indicate disease implications in
LABORATORY DETECTION
relation to complement abnormalities.
OF COMPLEMENT ABNORMALITIES
Immunologic Assays of Individual
Components
Assays for the Classical Pathway
Alternative Pathway Assays
Interpretation of Laboratory Findings
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Activation unit Classical pathway Hemolytic uremic syndrome Membrane cofactor protein
Alternative pathway Complement receptor type (HUS) (MCP)
Anaphylatoxin 1 (CR1) Hereditary angioedema (HAE) Paroxysmal nocturnal
Decay-accelerating factor Immune adherence hemoglobinuria (PNH)
Bystander lysis
(DAF) Lectin pathway Properdin
C1 inhibitor (C1-INH)
Factor H Mannose-binding lectin Recognition unit
C3 glomerulopathies
(C3G) Factor I (MBL) S protein
C4-binding protein Hemolytic titration (CH50) Membrane attack complex
(C4BP) assay (MAC)

As described in Chapter 3, complement is a complex series of cell walls or outer coating of bacteria, viruses, yeast, and protozoa.
more than 30 proteins that play a major part in amplifying the Although each of these pathways will be considered separately,
inflammatory response to destroy and clear foreign antigens. activation seldom involves only one pathway.
These soluble and cell-bound proteins interact in a very spe- Most plasma complement proteins are synthesized in the
cific way and have powerful abilities. They can lyse foreign liver with the exception of C1 components; these are mainly
cells, opsonize and tag the invaders for clearance, and direct produced by intestinal epithelial cells and Factor D, which is
the adaptive immune system to the site of infection.1 Comple- made in adipose tissue.1,6 Other cells, such as monocytes and
ment activation is also proinflammatory in its ability to increase macrophages, are additional sources of early complement com-
vascular permeability, recruit monocytes and neutrophils to the ponents, including C1, C2, C3, and C4.6,7 Most of these pro-
area of antigen concentration, and trigger secretion of im- teins are inactive precursors, or zymogens, which are converted
munoregulatory molecules that amplify the immune response.2 to active enzymes in a very precise order. Table 7–1 lists the
In their proinflammatory role, complement proteins serve as characteristics of the main complement proteins.
an important link between innate and adaptive immunity.
Complement has important “housekeeping” roles as well. The Classical Pathway
Complement recognizes cellular debris such as apoptotic cells
and immune complexes, tagging them for removal by innate The classical pathway, the first activation cascade described, is
immune cells.3 Because of its potential for far reaching effects, the main antibody-directed mechanism for triggering comple-
complement activation needs to be carefully regulated. Chronic ment activation. However, not all immunoglobulins are able
activation can lead to inflammation and tissue damage to the to activate this pathway. The immunoglobulin classes that can
host. Any deficiencies to the complement system can result in activate the classical pathway include IgM, IgG1, IgG2, and
an increased susceptibility to infection or the accumulation of IgG3, but not IgG4, IgA, or IgE. IgM is the most efficient of
immune complexes resulting in possible autoimmune disor- the activating immunoglobulins because it has multiple bind-
ders.4 However, numerous proteins act as controls or regulators ing sites; thus, it takes only one molecule attached to two
of the system. These controls, as well as the major proteins adjacent antigenic determinants to initiate the cascade. Two
involved in activation, will be discussed in detail. IgG molecules must attach to antigen within 30 to 40 nm of
each other before complement can bind; it may take at least
1,000 IgG molecules to ensure that there are two close enough
Pathways of the Complement System to initiate such binding.1,8 Some epitopes, notably the Rh group,

The complement system can be activated in three different ways.

The first pathway described, the classical pathway, involves Connections
nine proteins that are triggered primarily by antigen–antibody
combination. Pillemer and colleagues discovered an antibody- Jules Bordet
independent pathway in the 1950s that plays a major role as a Jules Bordet was awarded the Nobel Prize in 1919 for his role in
natural defense system.5 This second pathway, the alternative elucidating the nature of complement. Complement was origi-
pathway, was originally called the properdin system because the nally recognized in the 1890s; Paul Ehrlich coined the term com-
protein properdin was thought to be the main initiator of this plement because the substance complements the action of
antibody in destroying microorganisms.1 Present as a substance
pathway. Now it is known that properdin’s major function is to
in normal nonimmune serum, complement is part of the innate
stabilize a key enzyme complex formed along the pathway and immune system. Complement is considered an acute phase
that the other forms of activation are more prominent. The third reactant because levels rise during an infection. It also has a
pathway, likely the most ancient of the three, is the lectin path- unique property of being easily inactivated (i.e., heat-labile) by
way, another antibody-independent means of activating comple- heating serum to 56°C for 30 minutes. Refer to Chapter 3 for a
ment proteins. Its prototypic constituent, mannose- (or mannan-) further description of the innate immune system.
binding lectin (MBL), adheres to mannose found mainly in the
 Table 7–1 Proteins of the Complement System
SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (µG/ML) FUNCTION
Classical Pathway
C1q 410 150 Binds to Fc region of IgM and IgG
C1r 85 50 Activates C1s
C1s 85 50 Cleaves C4 and C2
C4 205 300–600 Part of C3 convertase (C4b)
C2 102 25 Binds to C4b—forms C3 convertase
C3 190 1,200 Key intermediate in all pathways
C5 190 80 Initiates membrane attack complex
C6 110 45 Binds to C5b in MAC
C7 100 90 Binds to C5bC6 in MAC
C8 150 55 Starts pore formation on membrane
C9 70 60 Polymerizes to cause cell lysis
Alternative Pathway
Factor B 93 200 Binds to C3b to form C3 convertase
Factor D 24 2 Cleaves Factor B
Properdin 55 15–25 Stabilizes C3bBb–C3 convertase
MBL Pathway
MBL 200–600 0.0002–10 Binds to mannose
MASP-1 93 1.5–12 Unknown
MASP-2 76 Unknown Cleaves C4 and C2
Fc = Fragment crystallizable; Ig = immunoglobulin; MAC = membrane attack complex; MBL = mannose-binding lectin; MASP = MBL-associated serine protease.

are too far apart on the cell for this to occur; therefore, they three subunits—C1q, C1r, and C1s—which require the pres-
are unable to fix complement. Within the IgG group, IgG3 is ence of calcium to maintain structure.1,8 The complex is made
the most effective, followed by IgG1 and then IgG2.8 up of one C1q subunit and two each of the C1r and C1s sub-
In addition to antibodies, a few substances can bind com- units (Fig. 7–2). Although the C1q unit is the part that binds
plement directly to initiate the classical cascade. These include to antibody molecules, the C1r and C1s subunits generate
C-reactive protein (CRP), several viruses, mycoplasmas, some enzyme activity to begin the cascade.
protozoa, and certain gram-negative bacteria such as Escherichia C1q has a molecular weight of 410,000 and is composed
coli.8 However, most infectious agents can directly activate only of six strands that form six globular heads with a collagen-like
the alternative or lectin pathways. tail portion. This structure has been likened to a bouquet of
Complement activation can be divided into three main tulips with six blossoms extending outward (see Fig. 7–2). As
stages, each of which is dependent on the grouping of certain long as calcium is present in the serum, C1r and C1s remain
reactants as a unit. The first stage involves the recognition associated with C1q.
unit, which in the case of the classical pathway is C1. Once C1q “recognizes” the fragment crystallizable (Fc) region of
C1 is fixed, the next components activated are C4, C2, and C3, two adjacent antibody molecules, but at least two of the glob-
known collectively as the activation unit of the classical path- ular heads of C1q must be bound to initiate the classical path-
way (and the lectin pathway). C5 through C9 comprise the way. C1r and C1s are serine protease proenzymes, also called
membrane attack complex (MAC); this last unit completes zymogens. As binding of C1q occurs, both are converted into
the lysis of foreign particles. Each of these is discussed in detail active enzymes. Autoactivation of C1r results from a confor-
in the following sections. Figure 7–1 depicts a simplified mational change that takes place as C1q is bound. Once acti-
scheme of the entire pathway. vated, C1r cleaves a thioester bond on C1s which, in turn,
activates it. Activated C1r is extremely specific because its only
known substrate is C1s. Likewise, C1s has a limited specificity,
The first complement component of the classical pathway to with its only substrates being C4 and C2. Once C1s is activated
bind is C1, a molecular complex of 740,000 d. It consists of the recognition stage ends.
 Recognition
C1qrs
C4
C2
C4a Inactive C1qrs
complex
C2b

C4b2a
Activation
C3

C3a

C4b2a3b
Activated C1s
C5 C1q binds Fc

C5a

C5b C1r
Membrane
attack complex C6
C7
C8
C9

C5b6789 Structure of C1qrs. When two or more globular heads

of C1q attach to bound immunoglobulin molecules, the collagen-
The classical complement cascade. C1qrs is the recog- like stalks change their configuration. The resulting shape change
nition unit that binds to the FC portion of two antibody molecules. causes C1r to become a serine protease, which cleaves a small frag-
C1s is activated and cleaves C4 and C2 to form C4b2a, also known as ment of C1s, uncovering the C1s protease, whose only targets are
C3 convertase. C3 convertase cleaves C3 to form C4b2a3b, known as C4 and C2.
C5 convertase. The combination of C4b2a3b is the activation unit.
C5 convertase cleaves C5. C5b attracts C6, C7, C8, and C9, which
bind together, forming the membrane attack complex (MAC).
histocompatibility complex (MHC). Each serves a similar
C9 polymerizes to cause lysis of the target cell.
purpose in its particular pathway.8
When combined with C4b in the presence of magnesium
ions, C2 is cleaved by C1s to form C2a (which has a molecular
weight of 70,000) and C2b (which has a molecular weight of
Phase two, the formation of the activation unit, begins when 34,000) (see Fig. 7–3A). This is the only case for the designa-
C1s cleaves C4 and ends with the production of the enzyme tion “a” to be given to the cleavage piece with enzyme activity,
C5 convertase (Fig. 7–3). C4 is the second most abundant though there are discussions to make the nomenclature of C2
complement protein, with a serum concentration of approx- match that of the other components of complement with the
imately 600 µg/mL.8 C1s cleaves C4 to split off a 77-amino “a” fragment being the smaller fragment. The short life of these
acid fragment called C4a. In the process, it opens a thioester- reactive species serves as a mechanism of control, keeping the
containing active site on the remaining part, C4b. C4b must reaction localized.
bind to protein or carbohydrate within a few seconds or it The combination of C4b and C2a is known as C3 conver-
will react with water molecules to form iC4b, which is rap- tase (see Fig. 7–3B). This is written as C4b2a to indicate that
idly degraded. Thus, C4b binds mainly to antigen in clusters the complex is an active enzyme. This complex is not very
that are within a 40-nm radius of C1. This represents the stable. The half-life is estimated to be between 15 seconds and
first amplification step in the cascade because for every C1 3 minutes, so C3 must be bound quickly. If binding does occur,
attached approximately 30 molecules of C4 are split and C3 is cleaved into two parts, C3a and C3b.
attached.1 C3, the major and central constituent of the complement
C2 is the next component to be activated. Complement pro- system, is present in the plasma at a concentration of 1 mg/mL
teins were named as they were isolated before the sequence of to 1.5 mg/mL.8 It serves as the pivotal point for all three path-
activation was known—hence the irregularity in the number- ways. The cleavage of C3 to C3b represents the most signifi-
ing system. The C2 gene is closely associated with the gene for cant step in the entire process of complement activation.9 The
Factor B (alternative pathway) on chromosome 6 in the major molecule has a molecular weight of 190,000 and consists of
 C4b
two polypeptide chains, alpha (α) and beta (β). The α chain
contains a highly reactive thioester group. When C3a is
C2 removed by cleavage of a single bond in the α chain, the
C4a thioester is exposed; the remaining piece, C3b, is then capable
of binding to hydroxyl groups on carbohydrates and proteins
in the immediate vicinity.2,5,9 C3b is estimated to have a half-
life of 60 microseconds if not bound to antigen. Therefore,
C2b only a small percentage of cleaved C3 molecules bind to anti-
C4
gen; most are hydrolyzed by water molecules and decay in the
fluid phase.8,10
The cleavage of C3 represents a second and major amplifi-
cation process because about 200 molecules are split for every
molecule of C4b2a.10 In addition to being required for the for-
mation of the MAC, C3b also serves as a powerful opsonin.
Macrophages have specific receptors for it (discussed later in
C4b2a the chapter) and make a major contribution to the process of
(C3 convertase) phagocytosis. A large number of molecules are needed for this
A
to occur; hence, the need for amplification.
If C3b is bound within 40 nm of the C4b2a, this creates a
new enzyme known as C5 convertase. Figure 7–3C depicts
C3a this last step in the formation of the activation unit. The cleav-
C3
ing of C5 with deposition of C5b at another site on the cell
membrane constitutes the beginning of the MAC.

~200x
C3b C5 consists of two polypeptide chains, α and β, which are linked
by disulfide bonds to form a molecule with a molecular weight
of about 190,000. C5 convertase, consisting of C4b2b3b, splits
off a 74-amino acid piece known as C5a that is released into cir-
C3 Convertase culation, whereas C5b attaches to the cell membrane, forming
the beginning of the MAC. The splitting of C5 and the cleavage
B of C3 represent the most significant biological consequences of
the complement system as explained in the section on biological
C5a
C5 manifestations of complement activation. However, C5b is
extremely labile and rapidly inactivated unless binding to C6
occurs.1
Once C6 is bound to C5b, subsequent binding involves
C7, C8, and C9. None of these proteins has enzymatic activ-
C5b ity; they are all present in much smaller amounts in serum
than the preceding components. C6 and C7 each have mo-
lecular weights of approximately 110,000 and have similar
physical and chemical properties. C8 is made up of three dis-
C4b2a3b
(C5 convertase)
similar chains joined by disulfide bonds and has a total mo-
lecular weight of about 150,000.6 C9 is a single polypeptide
C chain with a molecular weight of 70,000. The carboxy-
Formation of the activation unit. (A) Activated C1qrs terminal end is hydrophobic, whereas the amino-terminal
cleaves C4 and C2, with the larger pieces, C4b and C2a, binding to end is hydrophilic. The hydrophobic part serves to anchor
the target cell surface and forming the enzyme C3 convertase. the MAC within the target membrane. Formation of the mem-
(B) Each C3 convertase cleaves ~200 C3 molecules into C3a and C3b. brane attack unit is pictured in Figure 7–4. The complex
C3b is a powerful opsonin that binds to the target in many places. of C5b-C6-C7-C8 and C9 is known as C5b-9 or MAC. If the
(C) Some C3b associates with C4b2a, forming C4b2a3b, also complex is soluble in circulation, it is known as sC5b-9.
known as the C5 convertase. This convertase cleaves C5 into Measurement of the level of sC5b-9 is an indicator of the
the anaphylatoxin C5a and C5b, which binds to the target cell.
amount of terminal pathway activation that is occurring.
When formed, the MAC presents a pore of 70 to 100Å that
allows ions to pass in and out of the membrane.1,10 Destruc-
tion of target cells actually occurs through an influx of water
and a corresponding loss of electrolytes. The presence of C9
 acute phase protein because it is produced in the liver and is
normally present in the serum but increases during an initial
C8 C9 inflammatory response.13 The enzymatic role played by C1r
and C1s in the classical pathway is played in the lectin pathway
by serine proteases called MBL-associated serine proteases
(MASPs). There are currently three MASPs identified, labeled
C7 MASP-1, MASP-2, and MASP-3. The lectin pathway plays an
important role as a defense mechanism in infancy, during the
interval between the loss of maternal antibody and the acqui-
C6 sition of a full-fledged antibody response to pathogens.15

MAC
C5b The Alternative Pathway
First described by Pillemer and his associates in the early 1950s,
the alternative pathway was originally named for the protein
properdin, a constituent of normal serum with a concentration
of approximately 5 to 15 µg/mL.5 Although the alternative path-
way can be activated on its own, it appears that it functions
mainly as an amplification loop for activation started from the
Formation of the membrane attack unit. C5b binds classical or lectin pathways. Although properdin has been con-
to the target cell, whereas C6 and C7 attach to it. C8 binds to these firmed to bind and initiate activation, the primary function of
associated molecules and begins (along with C7) to penetrate the properdin is to stabilize the C3 convertase formed from activation
cell membrane. Multiple C9 molecules bind to C5b678 and polymer- of other factors. In addition to properdin, the serum proteins Fac-
ize to form a transmembrane channel, the membrane attack tor B and Factor D are unique to this pathway. C3 is a key com-
complex, which causes lysis of the cell. ponent of this pathway as well as the two other pathways. The
alternative pathway is summarized in Figure 7–5.
Triggering substances for the alternative pathway include
greatly speeds this lysis. However, sufficient perturbation of bacterial cell walls, especially those containing lipopolysaccha-
the membrane can occur in the absence of C9 so that defi- ride, fungal cell walls, yeast, viruses, virally infected cells,
ciencies in C9 appear largely benign. tumor cell lines, and some parasites, especially trypanosomes.1
All of these can serve as sites for binding the complex C3bBb,
one of the end products of this pathway. The conversion of C3
The Lectin Pathway is the first step in this pathway.
The lectin pathway represents another means of activating Native C3 is not stable in plasma. Water is able to hydrolyze
complement. Instead of activation through antibody binding, a thioester bond, thus spontaneously activating a small number
the lectin pathway is activated by recognition of surface moi- of these molecules.16 C3b, sometimes called iC3, is formed by
eties that are found on pathogens.11 This pathway provides an this spontaneous hydrolysis, from activation, or from the clas-
additional link between the innate and acquired immune re- sical or lectin pathways. It acts as the seed of activation of the
sponse because it involves nonspecific recognition of carbohy- alternative pathway. The C3b binds to Factor B, which has a
drates that are common constituents of microbial cell walls and molecular weight of 93,000 and is fairly abundant in the serum,
that are distinct from those found on human cell surfaces.12,13 at a level of 200 µg/ mL.8,17 Once bound to C3b, Factor B
Although this pathway is the most recently described of the can be cleaved by Factor D. The role of Factor B is thus anal-
three activation pathways of complement, it is probably the ogous to that of C2 in the classical pathway because it forms
most ancient. The lectin pathway molecules are structurally an integral part of a C3 convertase.
similar to those of the classical; the classical and lectin path- Factor D is a plasma protein that goes through a conforma-
ways even share the components C4 and C2. Once C4 and C2 tional change when it binds to Factor B.17,18 It is a serine pro-
are cleaved, the rest of the pathway is identical to the classical tease with a molecular weight of 24,000; its only substrate is
pathway. The role C1q serves in the classical pathway is filled bound Factor B. The concentration of Factor D in the plasma
by three classes of recognition molecules in the lectin pathway: is the lowest of all the complement proteins, approximately
lectins, ficolins, and CL-K1.11 The structure of all three classes 2 µg/mL.10 It cleaves Factor B into two pieces: Ba (with a molec-
of recognition molecules is similar to that of C1q because they ular weight of 33,000) and Bb (with a molecular weight of ap-
are all classed as collectins. One key lectin, called mannose- proximately 60,000). Bb remains attached to C3b, forming the
binding, or mannan-binding, lectin (MBL), binds to mannose initial C3 convertase of the alternative pathway. Bb is rapidly
or related sugars in a calcium-dependent manner to initiate inactivated unless it becomes bound to a site on one of the trig-
this pathway.14 These sugars are found in glycoproteins or car- gering cellular antigens.
bohydrates of a wide variety of microorganisms such as bacte- As the alternative pathway convertase, C3bBb is then capable
ria, yeasts, viruses, and some parasites. MBL is considered an of cleaving additional C3 into C3a and C3b. Some C3b attaches
 C3a
1 H2O 1. C3 is hydrolyzed by water to produce C3b, which binds
Factor B and together they attach to target cell surface.
C3b B
C3 C3b B 2. B is cleaved by Factor D into the fragments Ba and Bb.
Bb combines with C3b to form C3bBb, an enzyme with
C3 convertase activity.

3. More C3 is cleaved, forming more C3bBb. This enzyme

is stabilized by properdin, and it continues to cleave
additional C3.
2 D Ba 4. If a molecule of C3 remains attached to the C3bBbP
enzyme, the convertase now has the capability to
C3b cleave C5. The C5 convertase thus consists of
C3bBbP3b. After C5 is cleaved, the pathway is identical
Bb to the classical pathway.

C3 C3a
3

C3b Bb P C3b

C5 C5a
4 C5b

C3b Bb P C3b

The alternative pathway.

to cellular surfaces and acts as a binding site for more Factor B, self-antigens are destroyed and that the reaction remains
resulting in an amplification loop; activation initiated by the clas- localized, several plasma proteins act as system regulators.
sical or lectin pathways is amplified to levels of biological con- In addition, there are specific receptors on certain cells that
sequence. All C3 present in plasma would be rapidly converted also exert a controlling influence on the activation process.
by this method were it not for the fact that the enzyme C3bBb In fact, approximately one-half of the complement compo-
is extremely unstable unless properdin binds to the complex. nents serve as controls for critical steps in the activation
Binding of properdin increases the half-life of C3bBb from process. Because activation of C3 is the pivotal step in all
90 seconds to several minutes.8,17 In this manner, optimal rates pathways, the majority of the control proteins are aimed at
of alternative pathway activation are achieved.14 halting accumulation of C3b. However, there are controls at
C3bBb can also cleave C5, but it is much more efficient at all crucial steps along the way. Regulators will be discussed
cleaving C3.19 If, however, some of the C3b produced remains according to their order of appearance in each of the three
bound to the C3 convertase, the enzyme is altered to form pathways. A brief summary of these is found in Table 7–2.
C3bBb3bP, which has a high affinity for C5 and exhibits C5
convertase activity.16,19 C5 is cleaved to produce C5b, the first
part of the membrane attack unit. From this point on the alter- Regulation of the Classical
native, lectin, and classical pathways are identical. Figure 7–6 and Lectin Pathways
shows the convergence of all three pathways. C1 inhibitor (C1-INH) inhibits activation at the first stages of
both the classical and lectin pathways. Its main role is to inacti-
System Controls vate C1 by binding to the active sites of C1r and C1s. Clr and
Cls become instantly and irreversibly dissociated from C1q.6,10
Activation of complement could cause tissue damage and C1q remains bound to antibody, but all enzymatic activity ceases.
have devastating systemic effects if it were allowed to pro- C1-INH10 also inactivates MASP-2 binding to the MBL-MASP
ceed uncontrolled. To ensure that infectious agents and not complex, thus halting the lectin pathway.12,18 C1-INH is a
 Classical Pathway Lectin Pathway Alternative Pathway
C1qrs
Teichoic acid
MBL
Zymosan
MASP-1 LPS

MASP-2
MASP-3

C3b ! B

Mannose
Factor D

C4

C2 C3

C4b C2a C3b Bb

C3 convertase
C3 convertase

Properdin stabilizes

C2a

C4b C3b C5 C3b Bb P C3b

C5 convertase
C5 convertase

C5a

C7

C5b
C6 C8

Membrane attack complex

Convergence of the classical, alternative, and lectin pathways. The binding of C1qrs to two antibody molecules activates
the classical pathway, whereas the alternative pathway is started by hydrolysis of C3. The lectin pathway is triggered by binding of MBP to
mannose on bacterial cell walls. MASP-1, MASP-2, and MASP-3 bind to form an activated C1-like complex. MASP-2 cleaves C2 and C4 and
proceeds like the classical pathway. Factor B and Factor D operate in the alternative pathway. Although C3 convertase is formed differently
in each pathway, C3 is a key component in each one. The C5 convertase in the alternative pathway consists of C3bBb3bP. In the classical
and lectin pathways, C5 convertase is made up of C4b2a3b. After C5 is cleaved, the pathway is common to all.
 Table 7–2 Plasma Complement Regulators
SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (MG/ML) FUNCTION
C1 inhibitor 105 240 Dissociates C1r and C1s from C1q
(C1-INH)
Factor I 88 35 Cleaves C3b and C4b
Factor H 150 300–450 Cofactor with I to inactivate C3b;
prevents binding of B to C3b
C4-binding protein 520 250 Acts as a cofactor with I to
(C4BP) inactivate C4b
S protein 84 500 Prevents attachment of the C5b67
(vitronectin) complex to cell membranes

glycoprotein with a molecular weight of 105,000. Like most fibroblasts, and on numerous types of epithelial cells.6–8 DAF
of the other complement proteins, it is mainly synthesized in is capable of dissociating both classical and alternative path-
the liver; however, monocytes also may be involved to some way C3 convertases. It can bind to both C3b and C4b in a
extent in its manufacture. manner similar to CR1.4 It does not prevent initial binding
Further formation of C3 convertase in the classical and of either C2 or Factor B to the cell but can rapidly dissociate
lectin pathways is inhibited by four main regulators: soluble both from their binding sites, thus preventing the assembly
C4-binding protein (C4BP) and three cell-bound receptors, of an active C3 convertase.
complement receptor type 1 (CR1), membrane cofactor The carboxy-terminal portion of DAF is covalently attached
protein (MCP), and decay-accelerating factor (DAF).1,18 All to a glycophospholipid anchor that is inserted into the outer
of these act in concert with Factor I, a serine protease that in- layer of the membrane lipid bilayer. This arrangement allows
activates C3b and C4b when bound to one of these regulators. DAF mobility within the membrane so it can reach C3 conver-
C4BP is abundant in the plasma and has a molecular weight tase sites that are not immediately adjacent to it (Fig. 7–7).2
of about 520,000. It is capable of combining with either fluid- The presence of DAF on host cells protects them from by-
phase or bound C4b; therefore, C4b cannot bind to C2 and is stander lysis. It is one of the main mechanisms used in dis-
made available for degradation by Factor I. If C4BP attaches to crimination of self from nonself because foreign cells do not
cell-bound C4b, it can dissociate it from C4b2a complexes, possess this substance. However, it does not permanently mod-
causing the cessation of the classical pathway. ify C3b or C4b; they are capable of re-forming elsewhere as
CR1, also known as CD35, is a large polymorphic glycopro- active convertases.
tein with a molecular weight between 165,000 and 280,000.7
It is found mainly on peripheral blood cells, including neu-
trophils, monocytes, macrophages, erythrocytes, eosinophils,
Regulation of the Alternative Pathway
B lymphocytes, some T lymphocytes, and follicular dendritic The principal soluble regulator of the alternative pathway is
cells.3 It binds C3b and C4b but has the greatest affinity for Factor H, which has a molecular weight of 160,000.18 It acts
C3b.6,20 Once bound to CR1, both C4b and C3b can then be by binding to C3b, preventing the binding of Factor B. C3b in
degraded by Factor I. the fluid phase has a hundredfold greater affinity for Factor H
A main function of CR1 is as a receptor on platelets and red than for Factor B, but on cell surfaces C3b preferentially binds
blood cells (RBCs), which helps to mediate transport of C3b- to Factor B. Factor H also accelerates the dissociation of the
coated immune complexes to the liver and spleen.7,20 It is there C3bBb complex on cell surfaces. When Factor H binds to
that fixed tissue macrophages strip the immune complexes C3bBb, Bb becomes displaced. In this manner, C3 convertase
from the RBCs, process the complexes, and return the RBCs activity is curtailed in plasma and on cell surfaces.
intact to circulation. The ability of cells to bind complement- Additionally, Factor H acts as a cofactor that allows Factor I
coated particles is referred to as immune adherence. to break down C3b. It appears that only those molecules with
MCP, or CD46, has a molecular weight between 50,000 and tightly bound Factor H acquire high-affinity binding sites for
70,000 and is found on virtually all epithelial and endothelial Factor I.21 When Factor I binds, a conformational change takes
cells except erythrocytes.7 MCP is the most efficient cofactor place that allows it to cleave C3b.21 On cellular surfaces, C3b
for Factor I-mediated cleavage of C3b. It can serve as a cofactor is cleaved into C3f, which is released into the plasma, and iC3b,
for cleavage of C4b, but it is not as effective as C4BP. MCP also which remains attached but is no longer an active enzyme.
helps to control the alternative pathway because binding of iC3b is further broken down to C3c and C3dg by Factor I in con-
Factor B to C3b is inhibited. junction with another cofactor: the CR1 receptor (Fig. 7–8).10
DAF or CD55, a 70,000 d membrane glycoprotein, is the With this key role in complement regulation, it should not be
third main receptor and has a wide tissue distribution. It surprising that Factor H has recently been shown to play a role
is found on peripheral blood cells, on endothelial cells and in a variety of disorders (discussed in the text that follows).
 DAF DAF

C4b C2a C4b

Classical
pathway

A
DAF DAF

C3b C3b

Bb

Alternative
pathway

B
A. In the classical pathway, DAF dissociates C2a from C4b. B. Inhibitory effects of DAF. In the alternative pathway, when C3b binds
to cell surfaces that have DAF present, DAF helps dissociate Bb from binding to C3b.

Regulation of Terminal Components include degradation products of C3b, such as C3dg, C3d, and
iC3b. In addition, the Epstein-Barr virus gains entry to B cells
S protein is a soluble control protein that acts at a deeper level by binding to this receptor. CR2 is present only on mature
of complement activation. Also known as vitronectin, S protein B cells and is lost when conversion to plasma cells occurs. CR2
interacts with the C5b-7 complex as it forms in the fluid phase plays an important role as part of the B-cell co-receptor for
and prevents it from binding to cell membranes.8 Binding of antigen. Acting in concert with CD19, it binds complement-
C8 and C9 still proceeds, but polymerization of C9 does not coated antigen and cross-links it to membrane immunoglobu-
occur; therefore, the complex is unable to insert itself into the lin to activate B cells. In this manner, immune complexes are
cell membrane or to produce lysis.4 more effective at enhancing B-cell differentiation and produc-
A receptor, known by various terms, including membrane tion of memory cells than is antigen by itself.2,22
inhibitor of reactive lysis (MIRL) or CD59, also acts to block Another receptor, CR3 (CD11b/CD18), found on mono-
formation of the MAC. MIRL is widely distributed on the cell cytes, macrophages, neutrophils, and natural killer (NK)
membranes of all circulating blood cells, including RBCs, and cells, specifically binds particles opsonized with iC3b, a C3b
on endothelial, epithelial, and many other types of cells.8,10 degradation product.7 It does this in a calcium-dependent
Table 7–3 lists the receptors and indicates the types of cells manner. The CR3 receptor plays a key role in mediating
on which they are found. phagocytosis of particles coated with these complement frag-
ments (Fig. 7–9). These proteins trigger surface adhesion and
increased activity of phagocytic cells.2 Patients whose white
Complement Receptors blood cells (WBCs) lack these receptors fail to exhibit func-
and Their Biological Roles tions such as chemotaxis, surface adherence, and aggregation.
Deficiencies in phagocytosis are also noted. These individuals
Some complement receptors found on host cells amplify and have an impaired capacity to bind iC3b-coated particles and
enhance the immune response by augmenting phagocytosis are subject to recurrent infections.
and stimulating accessory cells rather than acting as regulators The CR4 (CD11c/CD18) receptor is very similar to CR3 in
(see Table 7–3). CR1 has been discussed in the previous sec- that it also binds iC3b fragments in a calcium-dependent fash-
tion. A second receptor, CR2 (or CD21), is found mainly on ion. CR4 proteins are found on neutrophils, monocytes, tissue
B lymphocytes and follicular dendritic cells.22 Ligands for CR2 macrophages, activated T cells, dendritic cells, NK cells, and
 1 1. Factor H (FH) competes with factor B (B) for binding
C3 FH
Blocks to spontaneously (hydrolytically) activated C3b.

2. Factor H dissociates any C3bBb complexes that form

C3a C3b B
on self-cell surfaces.

3. Factor H is a cofactor with factor I (FI), enabling

cleavage of C3b. The resuting C3bi loses enzymatic
activity, but is still an opsonin.
2
4. CR1 is a cofactor with FI, enabling cleavage of
FH
Displaces C3bi. The resulting C3dg is an opsonin and a
cofactor in B cell stimulation.
C3b Bb

Self-cell surface

Enables cleavage
3 FI
FH

C3bi C3f

4 Enables
cleavage
FI
CR1

C3dg C3c

Complement controls. CR1 receptor acts as a cofactor in the inactivation of C3b. Factor I cleaves C3b to form C3dg and C3c.
C3dg is not an effective opsonin and is not capable of further participation in the complement cascade.

Table 7–3 Receptors on Cell Membranes for Complement Components

RECEPTOR LIGAND CELL TYPE FUNCTION
CR1 (CD35) C3b, iC3b, C4b RBCs, neutrophils, monocytes, Cofactor for Factor I;
macrophages, eosinophils, B and T cells, mediates transport of
follicular dendritic cells immune complexes
CR2 (CD21) C3dg, C3d, iC3b B cells, follicular dendritic cells, epithelial B-cell co-receptor for
cells antigen with CD19
CR3 (CD11b/CD18) iC3b, C3d, C3b Monocytes, macrophages, neutrophils, NK Adhesion and increased
cells activity of phagocytic
cells
CR4 (CD11c/CD18) iC3b, C3b Monocytes, macrophages, neutrophils, NK Adhesion and increased
cells, activated T and B cells, dendritic activity of phagocytic
cells cells

Continued
 Table 7–3 Receptors on Cell Membranes for Complement Components—cont’d
RECEPTOR LIGAND CELL TYPE FUNCTION
DAF (CD55) C3b, C4b RBCs, neutrophils, platelets, monocytes, Dissociates C2b or Bb
endothelial cells, fibroblasts, T cells, from binding sites, thus
B cells, epithelial cells preventing formation of C3
convertase
MIRL (CD59) C8 RBCs, neutrophils, platelets, monocytes, Prevents insertion of C9
endothelial cells, epithelial cells into cell membrane
MCP (CD46) C3b, C4b Neutrophils, monocytes, macrophages, Cofactor for Factor I
platelets, T cells, B cells, endothelial cells cleavage of C3b and C4b

RBC = red blood cell; NK = natural killer.

MAC. Complement proteins also serve as a means of linking

innate and adaptive immunity. They act as opsonins to facil-
itate recognition and subsequent destruction by phagocytic
cells; in addition, they play a major role in the uptake and
Antigen
presentation of antigens so a specific immune response can
occur. They also facilitate B-cell activation. Recent work
demonstrates that complement is necessary for maintaining
immunologic memory.24 Effector molecules generated earlier
in the cascade play a major role in all these areas. Such mol-
FcR ecules can be classified into three main categories: anaphy-
CR3 CRP-R latoxins, chemotaxins, and opsonins.
An anaphylatoxin is a small peptide that causes increased
vascular permeability, contraction of smooth muscle, and re-
lease of histamine from basophils and mast cells. Proteins that
Role of C3b in opsonization. C3b, antibody, and play such a part are C3a and C5a. Both of these have molecular
C-reactive protein are all important opsonins that coat antigens and weights between 9000 and 11,000 and are formed as cleavage
accelerate phagocytosis. Receptors for C3b (CR3), antibody (FcR), products from larger complement components. Of these mol-
and C-reactive protein (CRP-R) on the phagocytic cell membrane ecules, C5a is the most potent; it is at least 200 times more
bind the opsonins and pull the membrane around to envelop the
powerful than C3a.25
antigen.
C3a and C5a attach to specific receptors on neutrophils, ba-
sophils, mast cells, eosinophils, smooth muscle cells, and vas-
activated B cells.2,7 Neutrophils and monocytes, however, pos- cular endothelium.24,25 C3a attaches to the C3a receptor (C3aR),
sess smaller amounts of CR4 than of CR3. Their function ap- whereas C5a attaches to the C5a receptor (C5aR). When binding
pears to be similar to that of CR3 and they may assist occurs on basophils and mast cells, histamine is released, in-
neutrophil adhesion to the endothelium during inflammation. creasing vascular permeability and causing contraction of
Receptors specific for C1q are found on neutrophils, mono- smooth muscles. C5a causes neutrophils to release hydrolytic
cytes, macrophages, B cells, platelets, and endothelial cells.7,23 enzymes, oxygen radicals, and prostaglandins, which aid in the
These receptors, known as collectin receptors, bind the collagen destruction of foreign antigens.25
portion of C1q and generally enhance the binding of C1q to FC C5a also serves as a chemotaxin for neutrophils, basophils,
receptors. Interacting only with bound C1q, the receptors appear eosinophils, mast cells, monocytes, and dendritic cells. In this
to increase phagocytic cells’ uptake of immune complexes op- manner, these cells are directed to the source of antigen concen-
sonized with C1q. On neutrophils, they may also act to enhance tration. Because of increased vascular permeability, neutrophils
the respiratory burst triggered by IgG binding to FC receptors. migrate from blood vessels to the tissues and tend to aggregate.
Binding of C5a to monocytes causes them to undergo
an oxidative burst that includes increased production of
Biological Manifestations hydrolytic enzymes, neutrophil chemotactic factor, platelet-
of Complement Activation activating factors, interleukin-1 (IL-1), and toxic oxygen
metabolites.13,19 IL-1 is a protein that enhances T-cell acti-
Activation of complement is a very effective means of ampli- vation. The activation may produce fever and lead to an in-
fying the inflammatory response to destroy and clear foreign crease in acute-phase reactants, both of which are characteristic
antigens. The cycle does not always have to proceed to lysis of an inflammatory response.
for this to be accomplished; hence, some of the initiating C3a and C5a are rapidly inactivated by an enzyme in the
proteins are much more plentiful than proteins that form the plasma called carboxypeptidase N to localize and control their
effects. C3a is cleaved in seconds, whereas conversion of C5a B locus is nearby, C2-deficient persons are often reported to have
occurs more slowly. decreases in Factor B also. Other types of complement deficien-
The last major effect of complement-derived peptides is cies are less common.
opsonization. C4b, C3b, iC3b, and C3dg, which accumulate A second deficiency that occurs with some frequency is that
on cell membranes as complement activation proceeds, bind of MBL. Deficiencies and polymorphisms in MBL occur in about
to specific receptors on erythrocytes, neutrophils, monocytes, 30% of the population. The health consequences of such varia-
and macrophages as previously discussed. This binding facili- tion in MBL levels remain unclear, however. Lack of MBL has
tates phagocytosis and clearance of foreign substances or been associated with pneumonia, sepsis, and meningococcal dis-
cellular debris, which is one of the key functions of the com- ease in infants.13,15,31,32 Low MBL has also been associated with
plement system. In addition, attachment of C3 products to an the risk of some cancers, infection during chemotherapy, and
antigen has been found to enhance the B-cell response. certain autoimmune disorders such as systemic lupus erythe-
matosus (SLE), but these connections are not yet well defined.33
The most serious deficiency is that of C3 because it is the
Complement and Disease States key mediator in all pathways. C3 deficiencies are, however, ex-
tremely rare.28 Individuals with a C3 deficiency are prone to
Although complement acts as a powerful weapon to combat
developing severe, recurrent life-threatening infections with en-
infection by amplifying phagocytosis, in some cases it can ac-
capsulated bacteria such as Streptococcus pneumoniae and may
tually contribute to tissue damage or death. Complement can
also be subject to immune complex diseases.16 Such complexes
be harmful if
can lodge in the kidney and result in glomerulonephritis.26,34
• It is activated systemically on a large scale as in gram- It appears that a deficiency of any of the terminal components
negative septicemia of the complement cascade (C5–C8) causes increased suscepti-
• It is activated by tissue necrosis such as myocardial bility to systemic Neisseria infections, including meningococcal
infarction meningitis and disseminated gonorrheal disease.10,28 Table 7–4
• Lysis of red blood cells occurs lists the complement components and the disease states associ-
In the case of septicemia caused by a gram-negative organism, ated with the absence of each individual factor.
large quantities of C3a and C5a are generated, leading to neu-
trophil aggregation and clotting. Damage to the tiny pulmonary
capillaries and interstitial pulmonary edema may result.2
Table 7–4 Deficiencies of Complement
Tissue injury following obstruction of the blood supply,
Components
such as occurs in a myocardial infarction or heart attack, can
cause complement activation and deposition of MACs on cell DEFICIENT
surfaces. Receptors for C3a and C5a have been found in coro- COMPONENT ASSOCIATED DISEASE
nary plaques, indicating that complement components may in- C1 (q, r, or s) Lupuslike syndrome; recurrent infections
crease the damage to heart tissue.25,26 C2 Lupuslike syndrome; recurrent
Lysis may be another end result of complement activation. infections; atherosclerosis
Hemolytic diseases such as cold autoimmune hemolytic anemia
are characterized by the presence of an autoantibody that binds C3 Severe recurrent infections;
at low temperatures. When these cells warm up, complement glomerulonephritis
fixation results in lysis. (See Chapter 15 for a more complete C4 Lupuslike syndrome
discussion of complement-mediated autoimmune diseases.)
C5–C8 Neisseria infections
C9 No known disease association
Complement Deficiencies Hereditary angioedema
C1-INH

Major Pathway Components DAF Paroxysmal nocturnal hemoglobinuria

Although excess activation of the complement system can result MIRL Paroxysmal nocturnal hemoglobinuria
in disease states, lack of individual components also has a dele- Factor H Recurrent pyogenic infections
terious effect. Hereditary deficiency of any complement protein, or Factor I
with the exception of C9, usually manifests itself in increased
MBL Pneumococcal diseases, sepsis,
susceptibility to infection and delayed clearance of immune Neisseria infections
complexes. Most of these conditions are inherited on an auto-
somal recessive gene and are quite rare, occurring in 0.03% of Properdin Neisseria infections
the general population.26 A lack of C2, the most common defi- MASP-2 Pneumococcal diseases
ciency, is found in 1 in 20,000 individuals.4,27–29 Recent evidence
indicates that atherosclerosis may be related to a C2 deficiency.28 C1-INH = C1 inhibitor; DAF = decay-accelerating factor; MASP-2 =
mannose-associated serine protease; MBL = mannose-binding lectin;
C2-deficient individuals may also be more prone to recurrent
MIRL = membrane inhibitor of reactive lysis.
streptococcal and staphylococcal infections.30 Because the Factor
Regulatory Factor Components connected to deficiencies, polymorphisms, or autoantibodies
involving one or multiple complement components. Key
A prime example of a disease caused by a missing or defective among these is a set of rare kidney disorders associated with
regulatory component is paroxysmal nocturnal hemoglo- complement. Hemolytic uremic syndrome (HUS) is the
binuria (PNH). Individuals with this disease have RBCs that most common cause of renal failure in children and is char-
are deficient in DAF. Hence, the RBCs are subject to lysis by acterized by hemolytic anemia, low platelet count, and acute
means of the bystander effect once the complement system renal failure.36 The primary cause of HUS is a Shiga toxin re-
has been triggered. These individuals appear to have a defi- lated to infection that is associated with acute diarrhea. The
ciency in the glycophospholipid anchor of the DAF molecule atypical form of HUS (aHUS) occurs because of complement
that prevents its insertion into the cell membrane.10,22 When dysregulation caused by genetic polymorphisms. The atypical
C3b is deposited on erythrocytes through activation of either HUS has a less acute onset and may not be associated with di-
pathway, the result is complement-mediated intravascular arrhea; otherwise, the clinical presentation is similar. The ge-
and extravascular hemolysis, resulting in a chronic hemolytic netic mutations associated with aHUS include those of Factor
anemia. H, MCP, Factor I, Factor B, and thrombomodulin, as well as
Some studies indicate that a DAF deficiency is associated autoantibodies to Factor H and Factor I.36
with a lack of CD59 (MIRL) and both are implicated in PNH.32 Complement has also been implicated in C3 glomeru-
CD59 has the same glycophospholipid anchor found in DAF; lopathies (C3G), which are diseases involving the glomeruli
therefore, the gene deficiency affects both molecules. As men- of the kidneys. Recently several forms of rare glomerulonephri-
tioned previously, CD59 prevents insertion of C9 into the cell tis (GN) were reclassified based on immunofluorescence and
membrane by binding to the C5b-8 complex, inhibiting for- the presence or absence of C3 and immunoglobulins. For the
mation of transmembrane channels.25 Both DAF and CD59 are C3 glomerulopathies, it is only C3 that is found in the deposits
important in protecting RBCs against bystander lysis. on the kidney. Analysis of these patients has shown that 71%
Another complement deficiency disorder that has recently to 100% of these patients have mutation in a complement pro-
received considerable attention is hereditary angioedema tein, specifically C3, Factor B, Factor H, or Factor I. Other pa-
(HAE). HAE involves recurrent attacks of swelling that affect tients have an acquired autoantibody. These autoantibodies are
the extremities, the skin, the gastrointestinal tract, and other known as C3 nephritic factors (C3NeF).
mucosal surfaces. This disease is caused by a deficiency or lack A C3NeF is an antibody that binds the C3-convertase from
of C1-INH, which occurs with a population frequency of 2 in the alternative pathway, C3bBb, holding it together and making
10,000.32 Although C1-INH was named for its role in control- it impervious to the normal control mechanisms. In this way,
ling complement, it is the function of C1-INH in controlling a C3NeF leads to uncontrolled cleavage of C3 with concomi-
the contact pathway of the coagulation system that is critical tant uncontrolled deposition of C3 on the kidneys. C3G caused
in this disease. C1-INH is a serpin (serine protease inhibitor) by C3NeF is clinically indistinguishable from the hereditary
that controls many of the serine proteases on contact. The lack form of the disorder.37 It is only with laboratory measurement
of C1-INH results in localized swelling that can be either sub- of the presence or absence of a C3NeF that the nature of the
cutaneous or found within the bowel or upper-respiratory disorder can be differentiated. In addition, an investigation of
tract.35 Normally, this spontaneously subsides in 48 to 72 hours, a possible complement deficiency can be complicated by de-
but if the edema occurs in the area of the oropharynx, life- pletion of complement components because of consumption
threatening upper-airway obstruction may develop.26 These through activation. For both the autoantibodies and activation-
attacks can be quite debilitating even when they are not life related consumption, laboratory testing is the key way to tell
threatening, so there is a need for proper diagnosis for these the acquired forms from the hereditary forms of complement
patients. disorders.
HAE is separated into two types, type I and type II. Type I
is characterized by a decrease in the C1-INH protein; type II
has normal levels of C1-INH, but the function is decreased. Laboratory Detection
The genetic cause of either type is an autosomal dominant gene of Complement Abnormalities
that codes for either a dysfunctional or an inactive protein. In
addition to the hereditary forms of the disorder, there are ac- Determining the levels of complement components can be use-
quired forms that result from either consumption of C1-INH ful in diagnosing disease. Hereditary deficiencies can be iden-
or from autoantibodies blocking the function of C1-INH. To tified and much can be learned about inflammatory or
differentiate the acquired and hereditary forms, measurement autoimmune states by following the consumption of comple-
of C1q can be helpful; C1q will be low in the acquired forms, ment proteins. Techniques to determine complement abnor-
but not in the hereditary forms. Measurement of C4 can also malities generally fall into two categories: (1) measurement of
be a very helpful screen for HAE, particularly during an attack, components as antigens in serum and (2) measurement of
because patients who do not exhibit a drop in C4 at that time functional activity.8 Many assays that are unavailable in routine
are rare.4,35 clinical laboratories are available in specialized laboratories.
In addition to these well-described diseases of comple- Some of the more common assays will be discussed in the text
ment, there are a growing number of disorders that are being that follows.
Immunologic Assays of Individual Assays for the Classical Pathway
Components Assays that measure lysis, the end point of complement activa-
The methods most frequently used to measure individual com- tion, are functional tests that are frequently run in conjunction
ponents include radial immunodiffusion (RID) and nephelom- with testing of individual components. The hemolytic titration
etry.26,34 C3 and C4 levels are routinely measured in most (CH50) assay is most commonly used for this purpose.26,32 This
clinical laboratories by nephelometry or by turbidity measure- assay measures the amount of patient serum required to lyse
ments that are often automated. Both of these types of meas- 50% of a standardized concentration of antibody-sensitized
urements and the methods used for the other components rely sheep erythrocytes. Because all proteins from C1 to C9 are nec-
on the precipitation of antigen (the complement component essary for this to occur, absence of any one component will result
being measured) and antibody. Nephelometry measures con- in an abnormal CH50, essentially reducing this number to zero.
centration according to the amount of light scattered by a solu- The titer is expressed in CH50 units, which is the reciprocal
tion containing a reagent antibody and a measured patient of the dilution that is able to lyse 50% of the sensitized
sample (refer to Chapter 10 for more details on nephelometry). cells.32,38 The 50% point is used because this is when the
Generally, the more antigen–antibody complexes that are pres- change in lytic activity per unit change in complement is at
ent, the more a beam of light will scatter as it passes through maximum (Fig. 7–11). Most laboratories need to establish
the solution. Such systems have a high degree of accuracy; re- their own normal values.
sults are available quickly and processing is easy because of the An additional CH50 test has also been developed based on
use of automation. However, they involve expensive equipment. lysis of liposomes that release an enzyme when lysed. This lysis
Components for which there are standardized reagents in- can be read on an analyzer and is more accurate than tradi-
clude C1q, C4, C3, C5, Factor B, Factor H, and Factor I. RID tional CH50 testing.25 However, lytic assays in general are com-
uses agarose gel into which specific antibody is incorporated. plicated to perform and lack sensitivity. Individual laboratories
Serum serves as the antigen and is placed in wells that are cut in must establish their own normal values. When the results are
the gel. Diffusion of the antigen from the well occurs in a circular abnormal, the reasons cannot be determined. Such procedures
pattern (Fig. 7–10). The radius of the resulting circle can be re- are useful in establishing functional activity or lack thereof.
lated to antigen concentration (see Chapter 10 for further details Additional testing for individual components should be
on RID). This is a sensitive technique when performed correctly, performed to follow up on any abnormality.
but at least 24 hours are needed before test results are available. Lytic activity can also be measured by radial hemolysis in
None of the assays for individual components are able to agarose plates. Rabbit RBCs that have been sensitized with an-
distinguish whether the molecules are functionally active. tibody are implanted in agarose and patient serum is added to
Thus, although the preceding techniques give quantitative re- wells punched in the gel. Lysis appears as a clear zone around
sults and are relatively easy to perform, test results must be in- each well; if complement standards are run, the size of the zone
terpreted carefully. Nephelometry and RID are both sensitive can be related to complement concentration.26,32,39 Solid-phase
tests.32 Enzyme-linked immunosorbent assay (ELISA) methods IgM attached to the walls of microtiter plates is used to initiate
are available for the measurement of the inhibitor C1-INH. complement activation. Anti-human antibody to C9 conju-
gated to alkaline phosphatase is the indicator of complement
activation. When a substrate is added, if any C9 is present and
the antibody conjugate has attached, a color change will be ev-
Unloaded gel ident. (Refer to Chapter 11 for a complete discussion of the
Calculations principle of ELISA techniques.) This type of testing, which is
Measure blue
precipitation ring very sensitive, is probably the best screen for complement ab-
normalities.25 The same method can detect split products that
Internal result from complement activation. These products include
diameter C4a, C4d, C3a, iC3b, C5a, and the soluble form of the MAC
sC5b-9, all of which are generated only if complement activa-
tion has occurred.
Gel after precipitating and staining

Outer
Alternative Pathway Assays
diameter
Alternative pathway activation can be measured by several
different means. An AH50 can be performed in the same
manner as the CH50, except magnesium chloride and ethyl-
ene glycol tetraacetic acid (EGTA) are added to the buffer and
calcium is left out.32 This buffer chelates calcium, which
Patient with low level blocks classical pathway activation. Rabbit RBCs are used as
Radioimmunodiffusion for measurement of comple- the indicator because they provide an ideal surface for alter-
ment component C5. native pathway activation.
 pathway are coated with mannose. Such testing is easy to per-
form and is not dependent upon the use of animal erythro-
cytes, which may be hard to obtain. Deficiencies can be
detected using the combined test results.

Interpretation of Laboratory Findings

Decreased levels of complement components or activity may
be caused by decreased production, consumption, or in vitro
consumption. The third condition must be ruled out before
either of the other two is considered. Specimen handling is
extremely important. Blood should be collected in a clot tube
with no serum separator.39 The tube should be spun down and
Antibody-coated
the serum should be frozen or placed on dry ice if it is not
red blood cells tested within 1 to 2 hours. If a specimen has been inadequately
refrigerated, been subjected to multiple freeze–thaws, or been
Patient serum Complement activation in prolonged storage, the results may be invalid and the test
A Red blood cell lysis needs to be repeated with a fresh specimen. Control serum
Serum to be tested is diluted serially and added to sensitized should also be included with each batch of test sera.
sheep red blood cells. The tubes are incubated at 37°C and then If a complement deficiency is suspected, it is possible to
centrifuged to pellet the unlysed cells.
narrow down the possible candidate components with a CH50
The CH50 is defined as the reciprocal of the dilution that causes and an AH50 assay. If the CH50 is low but the AH50 is normal,
lysis of 50% of the cells used in the assay. the components unique to the classical pathway should be in-
In the example shown, the CH50 would be about 80 U/mL.
vestigated. If the CH50 is normal but the AH50 is low, the al-
ternative pathway components need to be investigated. If both
the CH50 and AH50 are low, suspicion should be on the com-
100% ponents of the terminal pathway because that pathway is
shared by both the classical and alternative pathways. Although
this analysis will be true for most patients, it is also possible, if
there is sufficient activation of complement through any one
pathway, that the activation could consume enough compo-
Percent lysis

nents to lower the function of the other pathways. As previ-

ously stated, complement activation is rarely limited to just one
pathway. Such consumption can result from loss of a control
protein, presence of an autoantibody, an ongoing infection, or
other activation circumstances.
Once a CH50 and AH50 hemolytic assay have been per-
0% formed, it is appropriate to move to testing the levels or func-
1/320 1/160 1/80 1/40 1/20 tion of the components as directed by the relative results of the
B Serial dilutions CH50 and AH50. For many of the components, an antigen
CH50 testing. (A) Antibody-coated RBCs are added to level is sufficient to determine where the deficiency lies; how-
patient serum. This activates the complement in the sample and the ever, there are a few instances in which measurement of the
RBCs are lysed. Although only C1qrs is shown for simplicity, this test function would be more informative. For C8, it is necessary to
requires C1 through C9 to be present in order for lysis to occur. The measure function because it is a three-subunit protein. The loss
degree of lysis indicates the functional capacity of the complete of one of the three subunits would not put the level out of nor-
classical pathway. (B) CH50 methodology. mal range, but it would render the remaining two subunits
nonfunctional. C2 type II deficiency results from a genetic mu-
tation in C2 that renders the protein nonfunctional but does
An additional means of testing for alternative pathway func- not decrease the level of expression, which is also true for
tion is via ELISA. One such test can detect C3bBbP or C3bP C1-INH in type II HAE.
complexes in very small quantities. Microtiter wells are typi- A typical screening test for complement abnormalities usu-
cally coated with bacterial polysaccharide to trigger activation ally includes determination of the following: C3 and C4, as
of the alternative pathway. well as hemolytic content. Testing for products of complement
One test system has been developed that can determine the activation such as C3a, C4a, C5a, and Ba (as well as breakdown
activity of all three pathways.32,39 Strips used for the classical products including iC3b and C4d) can also be performed as a
pathway are coated with IgM, strips for the alternative pathway means of monitoring inflammatory processes such as rheuma-
are coated with lipopolysaccharide, and strips for the MBL toid arthritis and SLE. Table 7–5 presents some of the possible
 Table 7–5 Diagnosis of Complement activation unit consisting of C2, C4, and C3; and the
Abnormalities MAC, consisting of C5, C6, C7, C8, and C9.
IMPAIRED • The lectin pathway is activated by carbohydrates pres-
FUNCTION OR CLASSICAL LECTIN ALTERNATIVE ent in microbial cell walls and serves as an important
DEFICIENCY PATHWAY PATHWAY PATHWAY link between the innate and adaptive immune re-
C1q, C1r, C1s Low Normal Normal sponses. Molecules distinct to the lectin pathway include
C4, C2 Low Low Normal
mannose-binding lectin (MBL), MASP-1, MASP-2, and
MASP-3.
MBL, MASP2 Normal Low Normal • The alternative pathway is triggered by bacterial and fun-
B, D, P Normal Normal Low gal cell walls, yeast, viruses, tumor cells, and certain par-
asites. Factors unique to the alternative pathway include
C3, C5, C6, Low Low Low Factor B, Factor D, and properdin.
C7, C8, C9
• The MAC is common to all three pathways.
C1-INH Low Low Low • Plasma protein regulators of the complement system
Factor H and I Low Low Low
play an extremely important role because if uncon-
trolled, complement activation could have devastating
Improperly Low Low Low systemic effects.
handled • Soluble regulators include C1 inhibitor (C1-INH),
sera C4-binding protein (C4BP), Factor H, Factor I, and
Adapted from Seelen MA, et al. An enzyme-linked immunosorbent assay- S protein.
based method for functional analysis of the three pathways of the comple- • Examples of cell-bound regulators are complement recep-
ment system. In: Detrick B, Hamilton RG, and Folds JD, eds. Manual of tor type 1 (CR1), membrane cofactor protein (MCP), and
Molecular and Clinical Laboratory Immunology. 7th ed. Washington, DC: decay-accelerating factor (DAF).
ASM Press; 2006:124. • Specific complement receptors found on host cells am-
plify the immune response by enhancing phagocytosis
screening results from ELISA testing and correlates these with and stimulating other accessory cells. Some of these
deficiencies of individual factors. An understanding of these receptors include CR1, CR2, CR3, CR4, and collectin
patterns may be helpful in differentiating hereditary deficien- receptors.
cies from activational states that consume available comple- • Effector molecules generated during complement activa-
ment components. Additional testing would be necessary, tion play a major role in recognition and presentation of
however, to actually pinpoint hereditary deficiencies. antigens, activation of B cells, and maintenance of im-
munologic memory. They are classified as anaphylatoxins,
chemotaxins, and opsonins.
SUMMARY • Anaphylatoxins increase vascular permeability, whereas
chemotaxins attract phagocytic cells to a specific area
• The complement system is a series of more than 30 soluble and opsonins coat damaged or foreign cells to enhance
and cell-bound proteins that interact with both the innate phagocytosis.
and adaptive immune systems to enhance host defenses • Deficiencies of complement components can place
against infection. an individual at risk for certain infections. Missing or
• Activities of complement include lysis of foreign or dam- deficient regulators are the cause of diseases such as
aged cells, opsonization, increase in vascular permeability, paroxysmal nocturnal hemoglobinuria and hereditary
and attraction of monocytes and macrophages to areas angioedema.
where needed. • Laboratory assays for individual complement components
• The classical complement pathway is triggered by IgG or include radial immunodiffusion and nephelometry.
IgM binding to the surface of pathogens. Nine major pro- • The hemolytic titration or CH50 assay is a measure of
teins are involved in this pathway. lysis, the end point of complement activation in the clas-
• Three distinct units are involved in the classical pathway. sical pathway. The AH50 assay is a similar test for mea-
They are the recognition unit consisting of C1qrs; the suring the activity of the alternative pathway.
CASE STUDIES
1. A 3-year-old child has a history of serious infections and hands. She stated that she has had these symptoms on
is currently hospitalized with meningitis. The doctor sus- several previous occasions. After ruling out appendicitis,
pects that he may have a complement deficiency and or- the physician ordered a battery of tests, including some
ders testing. The following results are obtained: decreased for abnormalities of complement components. The
CH50, decreased AH50, and normal C4 and C3 levels. following results were obtained: red and white blood
cell count normal, total serum protein normal, CH50
Questions
decreased, alternative pathway function normal, C3 level
a. What do the results indicate about the possible normal, and C4 and C2 levels decreased.
pathway(s) affected?
b. Which component(s) are likely to be lacking? Questions
c. What sort of additional follow-up would be a. What symptoms led physicians to consider a possible
recommended? complement abnormality?
2. A 25-year-old female appeared at the local hospital’s b. What are possible reasons for a decrease in both C4
emergency department with symptoms of abdominal pain and C2?
as well as severe vomiting and swelling of the legs and c. What other testing would confirm your suspicions?

REVIEW QUESTIONS
1. The classical complement pathway is activated by 6. Which of the following describes the role of properdin
a. most viruses. in the alternative pathway?
b. antigen–antibody complexes. a. Stabilization of C3/C5 convertase
c. fungal cell walls. b. Conversion of B to Bb
d. mannose in bacterial cell walls. c. Inhibition of C3 convertase formation
d. Binding and cleavage of Factor B
2. Which of the following is characteristic of complement
components? 7. Which best characterizes the membrane attack
a. Normally present in serum complex (MAC)?
b. Mainly synthesized by B cells a. Each pathway uses different factors to form it.
c. Present as active enzymes b. C5 through C9 are not added in any particular
d. Heat stable order.
c. One MAC unit is sufficient to lyse any type
3. All of the following are true of the recognition unit except of cell.
a. it consists of C1q, C1r, and C1s. d. C9 polymerizes to form the transmembrane
b. the subunits require calcium for binding together. channel.
c. binding occurs at the FC region of antibody
molecules. 8. All of the following represent functions of the
d. C1q becomes an active esterase. complement system except
a. decreased clearance of antigen–antibody
4. Which of the following is referred to as C3 convertase? complexes.
a. C1qrs b. lysis of foreign cells.
b. C3bD c. increase in vascular permeability.
c. C3bBb d. migration of neutrophils to the tissues.
d. C4b5a
9. Which of the following is true of the amplification
5. Mannose-binding protein in the lectin pathway is loop in complement activation?
most similar to which classical pathway component? a. It is only found in the alternative pathway.
a. C3 b. The membrane attack unit is amplified.
b. C1rs c. C3b is the product that is increased.
c. C1q d. Increasing amounts of C1qrs are produced.
d. C4
10. Factor H acts by competing with which of the 16. A decreased CH50 level and a normal AH50 level
following for the same binding site? indicate which deficiency?
a. Factor B a. Decrease in components in the lectin pathway only
b. Factor D b. Decrease in components in the alternative pathway
c. C3B only
d. Factor I c. Decrease in components of both classical and
alternative pathways
11. A lack of CR1 receptors on RBCs would result in d. Decrease in components of the classical pathway
which of the following? only
a. Decreased binding of C3b to RBCs
b. Decreased clearance of immune complexes by the 17. Which best describes the role of an anaphylatoxin?
spleen a. Coats cells to increase phagocytosis
c. Decreased breakdown of C1qrs b. Attracts WBCs to the area of antigen concentration
d. Decreased binding of Factor H c. Increases production of interleukin-1
d. Increases permeability of blood vessels
12. Which best describes the role of CR2 on cell
membranes? 18. Which best describes the role of Factor H?
a. Binds C1qrs to inactivate it a. Acts with DAF to break down C3b
b. Acts as co-receptor on B cells for antigen b. Prevents binding of Factor B to C3b
c. Increases clearance of immune complexes c. Binds to the C5C6C7 complex
d. Binds particles opsonized with C3b d. Binds to C1q to shut down the classical pathway

13. Which of the following best characterizes hemolytic 19. A lack of C1-INH might result in which of the
uremic syndrome? following conditions?
a. It is a rare cause of renal failure in children. a. Paroxysmal nocturnal hemoglobinuria
b. It can be associated with deficiencies in Factor H. b. Hemolytic uremic syndrome
c. The major cause is lack of DAF on RBCs. c. Hereditary angioedema
d. It is associated with antibody-to-C3 convertase. d. Increased bacterial infections

14. The CH50 test measures which of the following? 20. Which would be most effective in measuring an
a. Patient serum required to lyse 50% of sensitized individual complement component?
sheep RBCs a. CH50 assay
b. Functioning of both the classical and alternative b. Radial immunodiffusion
pathways c. AH50 assay
c. Genetic deficiencies of any of the complement d. Lytic assay with liposome
components
d. Functioning of the lectin pathway only

15. Which of the following would be most effective in

preventing bystander lysis of RBCs?
a. C1-INH
b. Factor B
c. DAF
d. Factor H
Basic Immunologic
Procedures
 Safety and Quality
Management

After finishing this chapter, you should be able to: LABORATORY HAZARDS
1. List the six components of the chain of infection and the safety pre- Biological Hazards
cautions that break the chain. Sharps Hazards
2. Correctly perform hand hygiene procedures following CDC guidelines. Chemical Hazards
3. Describe the types of personal protective equipment used by laboratory Radioactive Hazards
personnel. Electrical Hazards
4. Differentiate between universal precautions, body substance isolation, Fire and Explosive Hazards
and standard precautions.
Physical Hazards
5. State the acceptable methods for disposal of biological waste and
QUALITY MANAGEMENT
sharp objects in the laboratory.
Procedure Manual
6. Discuss the federal regulations and guidelines for preparing and ship-
ping patient samples from the laboratory. Preexamination Variables
7. Explain the components of the Occupational Exposure to Bloodborne Examination Variables
Pathogens Compliance Directive. Postexamination Variables
8. Describe safety precautions utilized when handling chemicals. REGULATORY ISSUES
9. Discuss the components of Chemical Hygiene Plans and Safety Data Clinical Laboratory Improvement
Sheets. Amendments (CLIA)
10. State and interpret the components of the National Fire Protection Clinical and Laboratory Standards
Association hazardous material labeling system. Institute (CLSI)
11. Describe precautions that laboratory personnel should take with The Joint Commission (TJC)
regard to radioactive, electrical, fire, and physical hazards. College of American Pathologists
12. Explain the RACE and PASS actions to be taken when a fire is discovered. (CAP)
13. Recognize standard hazard warning symbols. QUALITY MANAGEMENT SYSTEMS
14. Define the preexamination (preanalytical), examination (analytical), and Quality System Essentials
postexamination (postanalytical) components of quality management. The Lean System
15. Distinguish between the components of internal quality control, ex- Six Sigma
ternal quality control, electronic quality control, and external quality
SUMMARY
assessment (proficiency testing).
CASE STUDIES
16. Discuss the roles of the Clinical Laboratory Improvement Amend-
ments (CLIA), Clinical and Laboratory Standards Institute (CLSI), the REVIEW QUESTIONS
The Joint Commission (TJC), and the College of American Patholo-
gists (CAP) in the regulation of health care.
17. State and describe the 12 quality essentials used in a quality manage-
ment system.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 18. Describe the purpose of quality indicators.
19. List the six areas of the Lean system and describe how it can benefit
the laboratory.
20. State the purpose of the Six Sigma methodology in a management system.

Accuracy External quality assessment Postexposure prophylaxis Quality system essentials

Biohazardous material (EQA) (PEP) (QSEs)
Chain of infection Infection control Precision Reliability
Chemical Hygiene Plan The Joint Commission Preexamination variables Safety data sheet (SDS)
(TJC) Proficiency testing Shift
Clinical Laboratory
Improvement Lean system Quality assessment Six Sigma
Amendments (CLIA) Occupational Safety and (QA) Standard deviation (SD)
Clinical and Laboratory Health Administration Quality control (QC) Standard of care
Standards Institute (CLSI) (OSHA)
Quality indicators Standard precautions (SP)
Coefficient of variation (CV) Personal protective
Quality management Trend
equipment (PPE)
Control mean (QM)
Postexamination Turnaround time (TAT)
Delta check Quality management
variables Variable
Examination variables system (QMS)

Laboratory Hazards necessary to prevent infection. The chain of infection requires

a continuous link between six elements: an infectious agent, a
The clinical laboratory contains a wide variety of safety hazards, reservoir, a portal of exit, a means of transmission, a portal of
many capable of producing serious injury or life-threatening dis- entry, and a susceptible source.1
ease. To work safely in this environment, clinical laboratorians Infectious agents consist of bacteria, fungi, parasites, and
must learn what hazards exist and the basic safety precautions viruses. A reservoir is a place where the infectious agent can live
associated with them. They must apply the basic rules of com- and multiply, such as a contaminated clinical specimen or an
mon sense required for everyday safety. Some hazards are unique infected patient. Humans and animals (hosts) or contaminated
to the health-care environment and others are encountered rou- inanimate objects (fomites) that contain blood, urine, or other
tinely throughout life (Table 8–1). It is essential that laboratory body fluids make ideal reservoirs. The infectious agent leaves
personnel know where all safety equipment is located and be the reservoir through a portal of exit such as through the nose,
trained in all aspects of its use on a yearly basis. mouth, and mucous membranes, as well as in blood or other
body fluids, and is transmitted to a susceptible source to con-
tinue the chain of infection.
Biological Hazards Means of transmission include direct contact (the unpro-
In the immunology laboratory, the most significant hazard ex- tected host touches the patient, specimen, or a contaminated
ists in obtaining and testing patient specimens. Understanding object); droplet (the host inhales infected aerosol droplets from
how microorganisms are transmitted (chain of infection) is a patient or specimen); airborne (the host inhales dried aerosol

Table 8–1 Types of Safety Hazards

TYPE SOURCE POSSIBLE INJURY
Biological Infectious agents Bacterial, fungal, viral, or parasitic infections
Sharp Needles, lancets, and broken glass Cuts, punctures, or bloodborne pathogen exposure
Chemical Preservatives and reagents Exposure to toxic, carcinogenic, or caustic agents
Radioactive Equipment and radioisotopes Damage to a fetus or generalized overexposure to radiation
Electrical Ungrounded or wet equipment Burns or shock
and frayed cords
Fire or explosive Open flames and organic chemicals Burns or dismemberment
Physical Wet floors, heavy boxes, and patients Falls, sprains, or strains
particles circulating on the air currents or dust particles); vehicle shields. Gloves are worn to protect the health-care worker’s hands
(the host ingests contaminated food or water); or by a vector from contamination by patient body substances and to protect
(from an animal or mosquito bite). The infectious agent now the patient from possible microorganisms on the health-care
must enter a new reservoir through a portal of entry, which can worker’s hands. However, wearing gloves is not a substitute for
be the same as the portal of exit and include mucous membranes hand sanitizing. Hands must always be sanitized when gloves are
of the nose, mouth, and eyes; breaks in the skin; and open removed. A variety of gloves are available, including sterile and
wounds to complete the chain of infection. A susceptible source nonsterile, powdered and unpowdered, and latex and nonlatex.
can be another patient, health-care personnel, or visitors. Patients Allergy to latex is decreasing among health-care workers
receiving chemotherapy, the elderly, and immunocompromised due to the availabliity of other types of gloves. However, labo-
patients are susceptible hosts. The immune system is still devel- ratorians should be alert for symptoms of reactions associated
oping in newborns and infants and begins to weaken as people with latex contact, including irritant contact dermatitis that
age, making these groups of patients more susceptible to infec- produces patches of dry, itchy irritation on the hands; delayed
tion. The immune system also is depressed by stress, fatigue, and hypersensitivity reactions resembling poison ivy that appear
lack of proper nutrition, which contribute to the susceptibility of 24 to 48 hours following exposure; and true immediate hyper-
patients and health-care personnel.1 Once the chain of infection sensitivity reactions often characterized by facial flushing and
is complete, the infected host then becomes another source able respiratory difficulty (see Chapter 14). Hand sanitizing imme-
to transmit the microorganisms to others.1 diately after removal of gloves and avoiding powdered gloves
The most likely source of infection in serological testing is may aid in preventing the development of latex allergy. Any
through contact with patient specimens; the main concern is signs of a latex reaction should be reported to a supervisor
exposure to viruses such as the hepatitis viruses and human because a true latex allergy can be life threatening.4
immunodeficiency virus (HIV). Therefore, safety precautions In the immunology laboratory, fluid-resistant laboratory
are designed to protect health-care workers from exposure to coats with wrist cuffs are worn at all times to protect skin and
potentially harmful infectious agents. The ultimate goal of bi- clothing from contamination by patient specimens. They must
ological safety is to prevent completion of the chain by pre- be completely buttoned with gloves pulled over the cuffs. Both
venting transmission. The infection control team develops gloves and laboratory coats should be changed as soon as pos-
procedures to control and monitor infections occurring within sible if they become visibly soiled and must be removed when
health-care facilities. Figure 8–1 contains the universal symbol leaving the laboratory.
for biohazardous material and illustrates the chain of infec- The mucous membranes of the eyes, nose, and mouth must
tion and how it can be broken by following safety practices.1 be protected from specimen splashes and aerosols. A variety of
Preventing the transmission of microorganisms from infected protective equipment is available, including goggles, full-face
sources to susceptible hosts is critical in controlling the spread plastic shields, and Plexiglas countertop shields (Fig. 8–3).
of infection. Procedures used to prevent microorganism trans- Particular care should be taken to avoid splashes and aerosols
mission include hand hygiene, wearing personal protective when removing container tops and when transferring and cen-
equipment (PPE), isolating highly infective or highly suscep- trifuging specimens. Never centrifuge specimens in uncapped
tible patients, and properly disposing of contaminated materi- tubes or in uncovered centrifuges. When specimens are re-
als. Strict adherence to guidelines published by the Centers for ceived in containers with contaminated exteriors, the exterior
Disease Control and Prevention (CDC) and the Occupational of the container must be disinfected; if necessary, a new spec-
Safety and Health Administration (OSHA) is essential.2 imen may be requested.

Hand contact represents the number one method of infection The CDC developed standard precautions (SP) by combining
transmission. Hands should always be sanitized before patient recommendations of universal precautions (UP) and body substance
contact, after gloves are removed, before leaving the work area, isolation (BSI) procedures. Under UP, all patients were assumed
whenever the hands have been knowingly contaminated, be- to be potential carriers of bloodborne pathogens. The BSI mod-
fore going to designated break areas, and before and after using ified UP by requiring that gloves be worn when encountering
bathroom facilities. Hand hygiene includes both hand washing blood or any other body substance. The CDC continually mod-
and using alcohol-based antiseptic cleansers. Alcohol-based ifies SP as changes occur in the health-care environment. SP as-
cleansers are not recommended after contact with spore-forming sumes every person in the health-care setting is potentially
bacteria, including Clostridium difficile and Bacillus sp. infected or colonized by an organism that could be transmitted.
The CDC’s guidelines for the correct hand washing technique SP applies to all blood and body fluids, mucous membranes, and
are pictured in Figure 8–2.3 If using alcohol-based cleansers, nonintact skin and stresses hand washing.5
apply the cleanser to the palm of one hand. Rub your hands to- Standard precautions that apply directly to the laboratory
gether and over the entire cleansing area, including between the are as follows:
fingers and thumbs. Continue rubbing until the alcohol dries.
• Hand hygiene—Hand hygiene includes both hand washing
and the use of alcohol-based antiseptic cleansers. Sanitize
PPE used by laboratorians includes gloves, gowns or laboratory hands after touching blood, body fluids, secretions, excre-
coats, masks, goggles, face shields, and Plexiglas countertop tions, and contaminated items, whether or not gloves are
 Break the link Break the link
• Immunizations • Disinfection
• Patient isolation Infectious agent • Hand hygiene
• Nursery • Bacteria
precautions • Fungi
• Healthy lifestyle • Parasites
Susceptible • Viruses
host Reservoir
• Patients • Humans
• Elderly • Animals
• Newborns • Insects
• Immuno- • Fomites
compromised • Blood/body
• Health-care fluids
workers

Portal of exit
Portal of
entry • Nose
• Mouth
• Nose
• Mucous
• Mouth
membranes
• Mucous
• Specimen
membranes
collection
• Skin
• Unsterile
equipment

Means of transmission
• Droplet
• Airborne
Break the link • Contact Break the link
• Hand hygiene • Vector • Sealed biohazardous
• Standard precautions • Vehicle waste containers
• PPE • Sealed specimen
• Sterile equipment containers
• Hand hygiene
• Standard precautions
Break the link
• Hand hygiene
• Standard precautions
• PPE
• Patient isolation

Chain of infection and safety practices related to the biohazard symbol. (From Strasinger SK, DiLorenzo MS. The Phlebotomy Text-
book. 3rd ed. Philadelphia, PA: F.A. Davis, Philadelphia; 2011, with permission.)

worn. Sanitize hands immediately after gloves are removed, patient-care activities that are likely to generate splashes
between patient contacts, and when otherwise indicated to or sprays of blood, body fluids, secretions, and excretions.
avoid transfer of microorganisms to other environments. • Gown—Wear a gown (a clean, nonsterile gown is ade-
• Gloves—Wear gloves (clean, nonsterile gloves are adequate) quate) to protect skin and to prevent soiling of clothing
when touching blood, body fluids, secretions, excretions, during procedures that are likely to generate splashes of
and contaminated items. Remove gloves promptly after use, blood, body fluids, secretions, or excretions. Select a
before touching noncontaminated items and environmental gown that is appropriate for the activity and amount of
surfaces, and before going to another patient. Sanitize hands fluid likely to be encountered (e.g., fluid-resistant in
immediately to avoid transfer of microorganisms to other the laboratory). Remove a soiled gown before leaving the
patients or environments. laboratory environment and sanitize hands to avoid trans-
• Mask, nose, and eye protection—Wear a mask and eye fer of microorganisms to other environments.
protection or a face shield to protect mucous membranes • Respiratory hygiene and cough etiquette—Educate
of the eyes, nose, and mouth during procedures and health-care personnel, patients, and visitors to contain
 1. Wet hands with warm water. Do not allow any part of the 2. Apply soap.
body to touch the sink.

3. Rub to form a lather, create friction, and loosen debris. Thor- 4. Rinse hands in a downward position to prevent recontamina-
oughly clean between the fingers and under the fingernails for tion of hands and wrists.
at least 20 seconds; include thumbs and wrists in the cleaning.

5. Dry hands with paper towel. 6. Turn off faucets with a clean paper towel to prevent
contamination.
Hand hygiene. (From Strasinger SK, DiLorenzo MS. Urinalysis and Body Fluids. 6th ed. Philadelphia, PA: F.A. Davis; 2014, with
permission.)

respiratory secretions to prevent droplet and fomite trans- any part of the body; rather, use self-sheathing needles or a
mission of respiratory pathogens. Offer masks to coughing mechanical device designed to conceal the needle. Do not
patients, distance symptomatic patients from others, and remove unsheathed needles from disposable syringes by
practice good hand hygiene to prevent the transmission hand; use a mechanical device. Do not bend, break, or
of respiratory pathogens. otherwise manipulate used needles by hand. Place used
• Needles—Never recap used needles or otherwise manipu- disposable syringes and needles, scalpel blades, and other
late them using both hands; in addition, never use any tech- sharp items in appropriate puncture-resistant containers.
nique that involves directing the point of a needle toward Place reusable syringes and needles in a puncture-resistant
 In the Laboratory
Components of the OSHA Bloodborne Pathogen
Exposure Control Plan

Providing sharps disposal containers and needles with safety

devices
Requiring discarding of needles with the safety device acti-
vated and the holder attached
Labeling all biohazardous materials and containers

Requiring all employees to practice standard precautions

Prohibiting eating, drinking, smoking, and applying cosmet-
ics in the work area
Establishing a daily work surface disinfection protocol

Personal protective equipment using a plastic shield. Providing laboratory coats, gowns, face shields, and gloves
(From Strasinger SK, DiLorenzo MS. Urinalysis and Body Fluids. 6th ed. to employees and laundry facilities for nondisposable pro-
Philadelphia, PA: F.A. Davis; 2014, with permission.) tective clothing

Providing immunization for the hepatitis B virus free of

container for transport to the reprocessing area. (See Sharps charge
Hazards later for additional information.) Providing medical follow-up to employees who have been
accidentally exposed to bloodborne pathogens

Documenting annual training of employees in safety

The federal government has enacted regulations to protect
standards
health-care workers from exposure to bloodborne pathogens. Documenting evaluations and implementation of safer
These regulations are monitored and enforced by OSHA. The needle devices
Occupational Exposure to Bloodborne Pathogens Standard6 Involving employees in the selection and evaluation of new
requires all employers to have a written Bloodborne Pathogen devices and maintaining a list of those employees and the
Exposure Control Plan and to provide necessary protection, evaluations
free of charge, for employees. A later compliance directive Maintaining a sharps injury log including the type and brand
called Enforcement Procedures for the Occupational Exposure of safety device, location and description of the incident,
to Bloodborne Pathogens Standard placed more emphasis on and confidential employee follow-up
using engineering controls to prevent accidental exposure to From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia,
bloodborne pathogens.7 The components of the current Blood- PA: F.A. Davis; 2011, with permission.
borne Pathogens Exposure Control Plan that is required of all
institutions are shown in the In the Laboratory: Components
of the OSHA Bloodborne Pathogen Exposure Control Plan
box. Each health-care institution is responsible for designing or yellow color coding. These supplies include alcohol pads,
and implementing its own exposure control plan.8 gauze, bandages, disposable tourniquets, gloves, masks, gowns,
Any accidental exposure to blood through needlestick, mu- and plastic tubes and pipettes. Disposal of needles and other
cous membranes, or nonintact skin must be reported to a sharp objects is discussed in the next section.
supervisor and a confidential medical examination must be Contaminated nondisposable equipment, blood spills, and
immediately started. Evaluation of the incident must begin right blood and body fluid processing areas must be disinfected. The
away to ensure appropriate postexposure prophylaxis (PEP) most commonly used disinfectant is a 1:10 dilution of sodium
is initiated within 24 hours. Needlesticks are the most fre- hypochlorite (household bleach) prepared daily and stored in
quently encountered exposure and place the laboratorian in a plastic, not a glass, bottle. The bleach should be allowed to
danger of contracting HIV, HBV, and hepatitis C virus (HCV). air-dry on the contaminated area before being wiped off.9
The CDC has recommended procedures to prevent these (see
In the Laboratory: Postexposure Prophylaxis).
If a laboratory accepts specimens from other health-care insti-
tutions, then it is important to know the regulations for pack-
All biological waste, except urine, must be placed in appropri- aging, transporting, and receiving these specimens. The U.S.
ate containers labeled with the biohazard symbol. This waste Department of Transportation (DOT), the International Air
includes not only specimens but also the materials with which Transport Association (IATA), and the United Nations (UN)
the specimens come in contact. Any supplies contaminated have stringent regulations that must be followed if a laboratory
with blood and body fluids must also be disposed of in con- is going to be involved in transporting or receiving patient
tainers clearly marked with the biohazard symbol or with red specimens from another institution.10,11
 In the Laboratory then all DOT rules apply. The transport vehicle should be
used exclusively for transport of specimens and should be
Postexposure Prophylaxis equipped to secure the transport containers.12 Minimum
Draw a baseline blood sample from the employee and test it shipping standards for this type of transportation include13
for HBV, HCV, and HIV. • Leakproof, watertight specimen containers
If possible, identify the source patient, collect a blood sample, • Tightly capped tubes placed in a rack to maintain an up-
and test it for HBV, HCV, and HIV. Patients must usually give right position
informed consent for these tests and they do not become
• Leakproof inner packaging surrounded by enough ab-
part of the patient’s record. In some states, a physician’s order
sorbent material to completely absorb all the liquid present
or court order can replace patient consent because a needle-
stick is considered a significant exposure. • A leakproof plastic or metal transport box with a secure,
Testing must be completed within 24 hours for maximum tight-fitting cover
benefit from PEP. • Properly labeled transport boxes accompanied by speci-
Source patient tests positive for HIV: men data and identification forms
Employee is counseled about receiving PEP using zidovudine Specimens picked up by a courier that are to be shipped to an
(ZDV) and one or two additional anti-HIV medications. out-of-the-area laboratory, such as a reference laboratory, must
Medications are started within 24 hours.
follow DOT regulations. Many of these laboratories supply
Employee is retested at intervals of 6 weeks, 12 weeks, and
6 months.
shipping containers to their clients.
Additional evaluation and counseling is needed if the source
patient is unidentified or untested. Sharps Hazards
Source patient tests positive for HBV: Sharp objects in the laboratory, including needles, lancets, and
Unvaccinated employees can be given hepatitis B immune broken glassware, present a serious biological hazard for pos-
globulin (HBIG) and HBV vaccine.
sible exposure to bloodborne pathogens caused by accidental
Vaccinated employees are tested for immunity and receive
PEP, if necessary.
puncture. Although bloodborne pathogens are also transmitted
Source patient tests positive for HCV: through contact with mucous membranes and nonintact skin,
No PEP is available. a needle or lancet used to collect blood has the capability to
Employee is monitored for early detection of HCV infection produce a very significant exposure to bloodborne pathogens.
and treated appropriately. It is essential that safety precautions be followed at all times
Any exposed employee should be counseled to report any when sharp hazards are present.
symptoms related to viral infection that occur within 12 weeks The number one personal safety rule when handling needles
of the exposure. is to never recap a needle. Many safety devices are available for
From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia,
needle disposal that provide a variety of safeguards. These include
PA: F.A. Davis; 2011, with permission. needle holders that become a sheath, needles that automatically
resheath or become blunt, and needles with attached sheaths. All
sharps must be disposed of in puncture-resistant, leakproof con-
tainers labeled with the biohazard symbol (Fig. 8–5). Containers
Under DOT and IATA should be located in close proximity to the work area and must
regulations, all diagnostic specimens require triple packaging always be replaced when the safe capacity mark is reached. Never
(Fig. 8–4). This includes the following: use any technique that involves directing the point of a needle
• The primary container (glass, metal, or plastic) must be toward any part of the body.
watertight with a positive (screw-on) cap. The Needlestick Safety and Prevention Act was signed into
• The primary container must be wrapped with enough ab- law in 2001.14 In June 2002, OSHA issued a revision to the
sorbent material to be capable of absorbing all of its con- Bloodborne Pathogens Standard compliance directive men-
tents. Multiple specimens must be wrapped individually tioned previously.15 In the revised directive, the agency requires
before placing them in the leakproof secondary container. that all blood holders with needles attached be immediately dis-
• The secondary container must be placed in a sturdy outer carded into a sharps container after the device’s safety feature is
container made of corrugated fiberboard, wood, metal, or activated. The rationale for the new directive was based on the
rigid plastic. An itemized list of contents in a sealed plastic exposure of workers to the unprotected stopper-puncturing end
bag is also placed in the outer container. Ice packs are of evacuated tube needles, the increased needle manipulation
placed between the secondary and the outer container. required to remove it from the holder, and the possible worker
Additional measures must be taken when using ice and exposure from the use of contaminated holders.
dry ice.
Specimens trans-
Chemical Hazards
ported by a hospital courier among clinics, physicians’ offices, Serological testing may involve use of chemical reagents that
and the hospital laboratory are exempt from most DOT must be handled in a safe manner to avoid injury. The general
rules, unless they are suspected of containing an infectious rules for safe handling of chemicals include taking precautions
substance. If specimens may contain an infectious substance, to avoid getting chemicals on the body, clothes, and work
 Infectious substance
Absorbent packing
Primary receptacle material (for liquids)
leakproof or siftproof
Cross section of closed package

Secondary packing
leakproof or siftproof Primary receptacle
(e.g., sealed plastic bag) leakproof or siftproof

Secondary packing
leakproof or siftproof
Bio
(e.g., sealed plastic bag
Su logica or other intermediate
Catbstanc l
erg
ory e packaging)
B Rigid outer
Rigid outer packaging packaging
UN3
373 Absorbent material
Cushioning
Package mark material

Name and telephone number of a

person responsible. (This information
may instead be provided on a written
document such as an air waybill.)
Packing and labeling of category B infectious substances. If multiple fragile primary receptacles are placed in a single secondary
package, they must be either individually wrapped or separated to prevent contact. (From U.S. Department of Transportation. Pipeline and
Hazardous Materials Safety Administration.)

then seek medical attention. Laboratorians must know the lo-

cation of the emergency shower and eyewash station in the lab-
oratory. Do not try to neutralize chemicals spilled on the skin.

All chemicals and reagents containing hazardous ingredients in

a concentration greater than 1% are required by OSHA to have a
safety data sheet (SDS) on file in the work area. By law, vendors
must provide these sheets to purchasers; however, it is the re-
sponsibility of the facility to obtain and keep them available to
employees. A SDS contains information on physical and chemical
characteristics, fire, explosion reactivity, health hazards, primary
routes of entry, exposure limits and carcinogenic potential, pre-
cautions for safe handling, spill cleanup, and emergency first aid.
Examples of puncture–resistant containers. (From Containers of chemicals that pose a high risk must be labeled
Strasinger SK, DiLorenzo MS. The Phlebotomy Textbook. 3rd ed. with a chemical hazard symbol representing the possible hazard,
Philadelphia, PA: F.A. Davis; 2011, with permission.) such as flammable, poisonous, corrosive, and so on. State and
federal regulations should be consulted for the disposal of chem-
area; wearing PPE such as safety goggles when pouring chem- icals. Containers of chemicals that pose a high risk must be
icals; observing strict labeling practices; and following instruc- labeled with a chemical hazard symbol representing the possible
tions carefully. Preparing reagents under a fume hood is also a hazard, such as flammable, poison, or corrosive (Fig. 8–6).
recommended safety precaution. Chemicals should never be
mixed together unless specific instructions are followed; in
addition, they must be added in the order specified. This is OSHA requires that all facilities that use hazardous chemicals
particularly important when combining acid and water because have a written Chemical Hygiene Plan available to employ-
acid should always be added to water, rather than adding water ees.16,17 The purpose of the plan is to detail the following:
to acid, to avoid the possibility of sudden splashing. • Appropriate work practices
When skin or eye contact occurs, the best first aid is to im- • Standard operating procedures
mediately flush the area with water for at least 15 minutes and • Personal protective equipment
 Chemical hazard symbols. (From Strasinger SK,
DiLorenzo MS. The Phlebotomy Textbook. 3rd ed. Philadelphia,
PA: F.A. Davis; 2011, with permission.)

• Engineering controls, such as fume hoods and safety cab-

inets for flammables
• Employee training requirements RADIATION
• Medical consultation guidelines
Radioactive symbol. (From Strasinger SK, DiLorenzo MS.
Each facility must appoint a chemical hygiene officer who is The Phlebotomy Textbook. 3rd ed. Philadelphia, PA: F.A. Davis; 2011,
responsible for implementing and documenting compliance with permission.)
with the plan.

Any hazardous chemical waste should be disposed of per cur- laboratories use radioactivity in testing anymore, mainly be-
rent EPA regulations. Most reagents used in the laboratory cause of the problem of disposing of waste. If radioactivity is
come with a SDS, mentioned previously. The SDS gives specific used, the laboratory typically contracts with a waste disposal
information for disposal of particular chemicals. All chemicals service that picks up the radioactive waste material.
used should be disposed of by following SDS directions. Many
kits used in immunologic testing often contain sodium azide Electrical Hazards
as a preservative, which can be disposed of by flushing down
The laboratory setting contains electrical equipment with
the drain with plenty of water. Using large amounts of water
which laboratorians have frequent contact. The same general
helps to avoid buildup in plumbing.
rules of electrical safety observed outside the workplace apply
in the laboratory, such as checking for frayed cords or over-
Radioactive Hazards loaded circuits. Laboratorians also have frequent contact with
Laboratorians can be exposed to radioactivity in the clinical water, fluids, and chemical agents; therefore, the danger of
laboratory when performing procedures using radioisotopes, water or fluid coming in contact with equipment is greater in
such as radioimmunoassay. The amount of radioactivity pres- the laboratory setting. Equipment should not be operated with
ent in most medical situations is very small and represents little wet hands. Designated hospital personnel closely monitor elec-
danger. However, the effects of radiation are related to the trical equipment. However, laboratory personnel should be ob-
length of exposure and are cumulative. Exposure to radiation servant for any dangerous conditions and report them to the
is dependent on the combination of time, distance, and shield- appropriate persons.
ing. Persons working in a radioactive environment are required When an accident involving electrical shock occurs, the
to wear measuring devices to determine the amount of radia- electrical source must be removed immediately without touch-
tion they are accumulating. ing the person or the equipment involved. Persons responding
Laboratorians should be familiar with the radioactive sym- to the accident must avoid transferring the current to them-
bol shown in Figure 8–7. This symbol must be displayed on selves by turning off the circuit breaker before unplugging the
the doors of all areas where radioactive material is present. Ex- equipment or moving the equipment using a nonconductive
posure to radiation during pregnancy presents a danger to the glass or wood object. The victim should receive immediate
fetus; personnel who are or who think they may be pregnant medical assistance following discontinuation of the electricity.
should avoid areas with this symbol. Cardiopulmonary resuscitation (CPR) may be necessary.

Disposal of medical radioactive waste is regulated by the Nu-

Fire and Explosive Hazards
clear Regulatory Commission (NRC) and is also subject to local Clinical laboratory work involves the use of potentially volatile
regulations. Such waste must be separated from other waste or explosive chemicals that require special procedures for han-
materials in the laboratory and placed in containers marked dling and storage. Flammable chemicals should be stored in
with the radioactive symbol. Disposal varies with the type safety cabinets and explosion–proof refrigerators. Cylinders of
of material (solid, liquid, or volatile chemical) and depends compressed gas should be located away from heat and securely
upon the amount of radioactivity present. Very few immunology fastened to a stationary device to prevent accidental tipping.
 The Joint Commission (TJC), an independent body that cer- HAZARDOUS MATERIALS
tifies and accredits health-care organizations in the United States, CLASSIFICATION
requires that all health-care facilities post evacuation routes and
detailed plans to follow in the event of a fire. Laboratory person- HEALTH HAZARD FIRE HAZARD
nel should be familiar with these routes. When a fire is discov- Flash Point
4 Deadly 4 Below 73 F
ered, all employees are expected to take the actions described by 3 Extreme Danger 3 Below 100 F
the acronym RACE: 2 Hazardous 2 Below 200 F
Rescue—rescue anyone in immediate danger 1 Slightly Hazardous 1 Above 200 F

2
0 Normal Material 0 Will not burn
Alarm—activate the institutional fire alarm system
Contain—close all doors to potentially affected areas
Extinguish or Evacuate—attempt to extinguish the fire if
possible, or evacuate, closing the door
Fire blankets should be present in the laboratory. Persons
whose clothes are on fire should be wrapped in the blanket to
smother the flames. The acronym PASS can be used to remem-
3 1
ber the steps in operating a fire extinguisher:
1. Pull pin
2. Aim at the base of the fire
3. Squeeze handles
SPECIFIC
HAZARD
W REACTIVITY

4 May deteriorate
4. Sweep nozzle side to side
3 Shock and heat
The Standard System for the Identification of the Fire Hazard Oxidizer OXY may deteriorate
of Materials, NFPA 704, is a symbol system used to inform fire- Acid ACID 2 Violent chemical
Alkali ALK change
fighters of the hazards they may encounter when fighting a fire Corrosive COR 1 Unstable if
in a particular area. The color-coded areas contain information Use No Water W heated
relating to health hazards, flammability, reactivity, use of water, Radiation 0 Stable
and personal protection. These symbols are placed on doors,
cabinets, and reagent bottles. An example of the hazardous
material symbol and information is shown in Figure 8–8. NFPA hazardous material symbol and classification.
(From Strasinger SK, DiLorenzo MS. The Phlebotomy Textbook. 3rd ed.
Physical Hazards Philadelphia, PA: F.A. Davis; 2014.)

Physical hazards are not unique to the laboratory; routine pre-

cautions observed outside the workplace apply. Maintaining a
clean and organized work area is essential for minimizing the test results, relationship of patient information to patient test re-
hazards. General precautions to consider include not running sults, patient confidentiality, specimen identification and integrity,
in rooms and hallways, watching for wet floors, bending the personnel competency, personnel qualifications and evaluations,
knees when lifting heavy objects, keeping long hair pulled back, communication protocols, complaint investigations, QA review
and avoiding dangling jewelry. Closed-toed shoes that provide with staff, and maintenance of QA records for 2 years.18
maximum support are essential for safety and comfort. Documentation of QA procedures is required by all labora-
tory accreditation agencies, including TJC, College of American
Pathologists (CAP), American Association of Blood Banks
Quality Management (AABB), American Osteopathic Association (AOA), American
Society of Histocompatibility and Immunogenetics (ASHI), and
The term quality management (QM) refers to the overall the Commission on Laboratory Assessment (COLA); it is also
process of guaranteeing quality patient care. As it relates to the required for Medicare and Medicaid reimbursement. Guide-
clinical laboratory, QM is the continual monitoring of the entire lines published by CAP and the Clinical and Laboratory
test process from test ordering and specimen collection Standards Institute (CLSI) provide very complete instruc-
through reporting and interpreting results. Written policies and tions for documentation and are used as a reference for the
documented actions as they pertain to the patient, the labora- ensuing discussion of the specific areas of immunology QA.1
tory, ancillary personnel, and the health-care provider are re- Documentation in the form of a procedure manual is required
quired. In addition, written remedial actions mandating the in all laboratories; this format is used as the basis for the
steps to take when any part of the system fails is essential to a following discussion.
QA program.
The Clinical Laboratory Improvements Amendments
(CLIA) are regulations that specify required components for QA
Procedure Manual
to include patient test management assessment, quality control A procedure manual (paper or digital) containing all the pro-
(QC) assessment, proficiency testing assessment, comparison of cedures performed in the immunology section of the laboratory
must be available for reference in the working area and must Preexamination Variables
comply with the CLSI guidelines. For each test performed, the
procedure manual provides In a clinical laboratory, a QM program encompasses preex-
amination variables (e.g., specimen collection, handling, and
• The principle or purpose of the test
storage), examination variables (e.g., reagent and test per-
• Clinical significance
formance, instrument calibration and maintenance, personnel
• Patient identification and preparation
requirements, and technical competence), postexamination
• Specimen type
variables (e.g., reporting of results and interpretations), and
• Method of collection
documentation that the program is being meticulously followed.
• Specimen labeling
A variable is defined as anything that can be changed or al-
• Specimen preservation
tered. Identification of variables throughout the testing process
• Conditions of transport and storage before testing
provides the basis for development of procedures and policies
• Specimen acceptability and criteria for rejection
within the immunology department that are located in the
• Reagents
procedure manual.
• Standards and controls acceptability and expiration policy
Preexamination variables occur before the actual testing of
• Instrument calibration and maintenance protocols and
the specimen and include test requests, patient preparation,
schedules
timing, specimen collection, handling, and storage. Health-
• Step-by-step procedure
care personnel outside the immunology department control
• Calculations
many of these factors, such as ordering tests and collecting
• Frequency and tolerance limits for controls and corrective
specimens; however, communication between departments
actions
and adequate training on the correct procedures for ordering
• Reference values and critical values
a test, collecting a specimen, and transporting the specimen
• Interpretation of results
improves the turnaround time (TAT) of results, avoids dupli-
• Common interferences
cation of test orders, and ensures a high-quality specimen. TAT
• Specific procedure notes
is defined as the amount of time required between the point at
• Limitations of the method
which a test is ordered by the health-care provider and the re-
• Method validation
sults are reported to the health-care provider. The laboratory
• Confirmatory testing
can monitor the TATs for both stat and routine tests to deter-
• Recording of results
mine areas in the process that need improvement.
• References
• Effective date
• Author Specific guidelines for specimen collection and handling should
• Review schedule19 be stated at the beginning of each procedure listed in the manual.
Current package inserts for all test kits used should be re- In addition to following the guidelines for specimen collection
viewed and included in the manual. The laboratory must also for each specific procedure, requisition forms and electronic entry
have a documented procedure for the correction of erroneous forms should be used to document the type of specimen to be
results.19 collected and the time and date of collection. The form should
The printed procedural manuals and electronic procedural have space for documenting (1) the patient’s first and last name,
manuals are subjected to proper document control. Only au- (2) the patient’s gender, (3) the patient’s age or date of birth,
thorized persons may make changes; these are dated and (4) the name of the person requesting the test, (5) the name of
signed (manually or digitally). The manuals must undergo the person to contact with critical results, (6) the name of the test
periodic review; documentation (i.e., proof) of the review is ordered, (7) any special handling requirements, (8) the time and
included in the manual. date of specimen collection, (9) the time the specimen was
Evaluating procedures and adopting new methodologies is delivered to the laboratory, and (10) any additional information
an ongoing process in the clinical laboratory. Whenever pertinent to laboratory interpretation.20 Information regarding
changes are made, the written procedure in the manual should patient preparation (e.g., fasting or elimination of interfering
be reviewed, referenced, and signed by a person with desig- medications) and the type and volume of specimen required must
nated authority, such as the laboratory director or section su- be included in the specific procedure.
pervisor, and personnel should be notified of the changes. An The criteria for specimen rejection for both physical char-
annual review of all procedures by the designated authority acteristics and labeling errors must be present. If a specimen
must also be documented. is rejected, the criteria for rejecting that specimen must be doc-
The procedure manual provides the basis for all testing in umented and available to the health-care provider and nursing
the immunology laboratory. Quality care in testing relies on staff. Laboratory personnel must determine the suitability of a
strictly following procedures as written. Documentation in- specimen and document any problems and corrective actions
cludes every step from specimen collection to the reporting of taken using an internal laboratory quality improvement form
results. A well-documented QM program ensures quality test (see In the Laboratory: An Example of an Internal Labora-
results and patient care. tory Quality Improvement Form). This report enables the
 In the Laboratory
The name and chemical formula of each reagent used, any
An Example of an Internal Laboratory Quality
necessary instructions for preparation or company source of
Improvement Form
prepared materials, storage requirements, and procedures for
CONFIDENTIAL
reagent QC are all found in the procedure manual. The type
Instructions: Section I should be completed by the individual identifying the event. of water used for preparing reagents and controls must be spec-
ified. Distilled or deionized water or clinical laboratory reagent
Date of report: Reported by:
Date of incident: Date/time of discovery: water (CLRW) must be available. A bold-type statement of any
Patient MR#: Patient accession #: safety or health precautions associated with reagents should be
present.
Describe what happened: All reagents must be properly labeled with the date of prepa-
ration or opening, purchase and received date, expiration date,
What immediate corrective action was taken? and appropriate safety information. Reagents should be checked
against two levels of commercial control solutions on each shift,
or at a minimum once a day and whenever a new reagent is
Provide the ORIGINAL to team leader or technical specialist within 24 hours of inci- opened. Results of all reagent checks are properly recorded.
dent discovery.
Date:
To:
The procedure manual must clearly provide instructions regard-
Forwarded for follow-up: ing the operation, performance, frequency of calibration, and
Date:
To: limitations of the instrumentation and equipment. The proce-
dures to follow when limitations or linearity are exceeded, such
Tracking #:
Instructions: Section II should be completed by laboratory management within as dilution procedures, must be included in the manual, as well
72 hours. as instructions detailing the appropriate recording procedures.
Check the appropriate problem category.
! Unacceptable patient samples ! Wrong tube type
Two levels of commercial controls must be run and
(Caused by hemolysis, QNS, or contamination) recorded. Evidence of corrective action for any failed QC tests
! Equipment-related event ! Misidentified sample must be documented. No patient’s testing may be performed
! Standard operating procedure deviation ! Wrong location
! Communication problem or complaint ! Other (explain) until QC is acceptable. A routine PM schedule for instruments
! Accident and equipment should be prepared as mandated by the TJC
Explain answers: or CAP guidelines and records kept of all routine and nonrou-
tine maintenance performed.
Preventive or corrective action recommendations:
Deionized water used for reagent preparation is quality con-
trolled by checking its pH and purity meter resistance on a
weekly basis, as well as the bacterial count on a monthly sched-
Technical specialist or team leader: Date: ule. All results must be recorded on the appropriate forms.
Medical director review: Date:
Quality assurance review: Date:
FDA reportable: Yes or no Date reported:
Detailed and concise testing instructions are written in a step-
Adapted from Danville Regional Medical Center Laboratory, Danville, VA, with by-step manner. Instructions should begin with specimen
permission. preparation, such as time and speed of centrifugation, and in-
clude types of glassware needed, time limitations and stability
of specimens and reagents, calculation formulas and a sample
laboratory director to capture the information to determine the calculation, health and safety precautions, and procedures. Ad-
root cause and develop a preventive or corrective action plan. ditional procedure information including reasons for special
Laboratory information systems have the capability to electron- precautions, sources of error and interfering substances, helpful
ically generate these forms for review. An acceptable specimen hints, clinical situations that influence the test, alternative pro-
requires verification of the patient’s identification information on cedures, and acceptable TATs for stat tests are listed under the
the requisition form and the tube label, proper collection and title of Procedure Notes following the step-by-step procedure.
processing procedures, and timely transport to the laboratory. Reference sources should be listed. The manufacturer’s
package inserts may be included but cannot replace the writ-
Examination Variables ten procedure. The laboratory director must sign and date
new procedures and all modifications of procedures before
The examination variables are the processes that directly affect they are used.21
the testing of specimens. They include reagents, instrumentation
and equipment, testing procedure, QC, preventive maintenance
(PM), access to procedure manuals, and the competency of per- Quality control (QC) refers to the materials, procedures, and
sonnel performing the tests. techniques that monitor the accuracy, precision, and reliability
of a laboratory test. QC procedures are performed to ensure that
Day
acceptable standards are met during the process of patient testing.
2 4 6 8 10 12 14 16 18 20
Specific QC information regarding the type of control specimen,
preparation and handling, frequency of use, tolerance levels, and + 2 S.D.
methods of recording should be included in the step-by-step + 1 S.D.
instructions in the procedures manual for each test. X
– 1 S.D.
QC is performed at scheduled times, such as at the begin-
– 2 S.D.
ning of each shift or before testing patient samples, and it must
In control
always be performed if reagents are changed, an instrument
malfunction has occurred, or if test results are questioned by
the health-care provider. Control results must be recorded in a + 2 S.D.
paper or electronic log. Patient test results may not be reported + 1 S.D.
X
until the QC is verified.
– 1 S.D.
External controls are used to verify the ac- – 2 S.D.
curacy (ability to obtain the expected result) and precision Shift
(ability to obtain the same result on the same specimen) of a
test. The control material is exposed to the same conditions as + 2 S.D.
the patient samples. Reliability is the ability to maintain both + 1 S.D.
precision and accuracy. Analysis of two levels of control mate- X
rial is required. One of these is a high level control and the – 1 S.D.
– 2 S.D.
other is a low level control. The concentration of controls
Trend
should be at medically significant levels and should be as much
like the human specimen as possible. Documentation of QC
Levey–Jennings charts showing in–control, shift, and
includes dating and initialing the material when it is first trend results. (From Strasinger SK, DiLorenzo MS. Urinalysis and Body
opened and recording the manufacturer’s lot number and the Fluids. 6th ed. Philadelphia, PA: F.A. Davis; 2014.)
expiration date each time a control is run and the test result is
obtained. Food and Drug Administration (FDA) standards
require that control material test negative for HIV and HBV.
External controls are tested and interpreted in the laboratory
of scatter about the mean and an uneven distribution above
by the same person performing the patient testing.
and below the mean that are most often caused by errors in
The control data are evaluated before releasing patient
technique.
results. Data obtained from repeated measurements have a
When control values are outside the tolerance limits, cor-
Gaussian distribution or spread in the values that indicate the
rective action—including the use of new reagents, or controls,
ability to repeat the analysis and obtain the same value. The
and the verification of lot numbers and expiration dates—must
laboratory, after repeated testing, establishes the value for each
be taken and documented. A protocol for corrective action is
analyte and calculates the control mean (the average of all data
shown in Figure 8–10. A designated supervisor reviews all of
points) and the standard deviation (SD) (a measurement sta-
the QC results.
tistic that describes the average distance each data point in a
Some laboratories may participate in a commercial QC
normal distribution is from the mean). The coefficient of vari-
program run by the manufacturer of the QC material. The
ation (CV) is the SD expressed as a percentage of the mean.
results from the same lot of QC material are returned to the
The CV indicates whether the distribution of values about the
manufacturer for statistical analysis and comparison with
mean is in a narrow versus broad range and should be less than
other laboratories using the same methodology.
5%. Confidence intervals are the limits between which the spec-
ified proportion or percentage of results will lie. Control ranges Internal controls, also called procedural
are determined by setting confidence limits that are within controls, consists of internal monitoring systems built into the
±2 SD or ±3 SD of the mean, which indicates that 95.5% to test system. Internal controls monitor the sufficient addition
99.7% of the values are expected to be within that range. of a patient specimen or reagent, the instrument’s and reagent’s
Values are plotted on Levey-Jennings control charts to visu- interaction, and, for lateral flow test methods, whether the
ally monitor control values. Immediate decisions about patient sample migrated through the test strip properly.22
results are based on the ability of control values to remain Electronic controls use a mechanical or
within a preestablished limit. Changes in accuracy of results electrical device in place of a liquid QC specimen. This type of
are indicated by either a trend, a gradual changing in the mean QC can be an internal or an external component inserted into
in one direction that may be caused by a gradual deterioration a point-of-care (POC) instrument. Electronic controls verify
of reagents or deterioration of instrument performance, or a the functional ability of a testing device, but it does not verify
shift, an abrupt change in the mean that may be caused by a the integrity of the testing supplies. Many test systems use a
malfunction of the instrument or a new lot number of reagents combination of external and internal controls to verify that the
(Fig. 8–9). Changes in precision are shown by a large amount entire test system is working properly.
 1. Run control

In control Out of control

QC is only as good as the personnel performing and monitor-
ing it. Personnel assessment includes education and training,
continuing education, competency assessment, and perfor-
Proceed with Go to step 2
testing mance appraisals. Each new employee must have documentation
of training during his or her orientation to the laboratory. This
documentation is a checklist of procedures and must include
2. Inspect control for: Outdate (age), proper storage, the date and initials of the person doing the training as well as
correct lot number, signs of contamination
the employee being trained. Up-to-date reference materials and
Yes there is a problem No obvious explanation atlases should be readily available and documentation of con-
tinuing education must be maintained.22
Make new control Retest
An adequate, uncluttered, safe working area is also essen-
and retest tial for both quality work and personnel morale. Standard
In control Out of control precautions for handling body fluids must be followed at
all times.
Proceed with Go to step 3
testing
Postexamination Variables
Postexamination variables are processes that affect the report-
3. Make up new bottle of control
ing of results and correct interpretation of data.
In control Out of control

Standardized reporting methods minimize health-care provider

Discard old control Go to step 4 confusion when interpreting results. The forms for reporting
and proceed with testing
results should be designed so that they present the information
in a logical sequence and provide adequate space for writing.
4. Open new can of reagent strips and test with new control A standardized reporting format and, when applicable, refer-
ence ranges should be included with each procedure in the
In control Out of control
procedure manual.
Electronic transmission is now the most common method
Discard bad reagent strips Switch lot numbers for reporting results. Many automated instruments have the
and proceed with testing and retest
capability for the laboratorian to transmit results directly from
In control Out of control the instrument to the designated health-care provider. It is
essential that the laboratorian carefully review results before
Discard entire lot, Notify supervisor or transmittal. Results may also be manually entered into the
notify manufacturer, resource person: do not
and proceed with testing proceed with testing laboratory computer system and then transmitted to the
health-care providers.
Procedure for “out of control” results. (Adapted from Documentation of the reporting of results is essential and
Schweitzer SC, Schumann JL, Schumann GB. Quality assurance guide-
required by accrediting agencies. In addition, permanent
lines for the urinalysis laboratory. J Med Technol. 1986; 3(11):567–572.
records of all reported results must be available. A method to
verify the actual reporting of results also must be available and
used by all employees.
Profi- The telephone is frequently used to transmit results of stat
ciency testing, or external quality assessment (EQA), is the tests and critical values. Personnel on hospital units and from
testing of unknown samples received from an outside agency. health-care providers often call requesting additional results.
It provides unbiased validation of the accuracy, and thus qual- When telephoning results, confirm that the results are being
ity, of patient test results. Several commercial vendors, such reported to the appropriate person. The time of the call and
as the CAP, provide proficiency testing. Laboratories subscrib- the name of the person receiving the results must be docu-
ing to these programs receive lyophilized or ready-to-use spec- mented according to the facility’s policy. The Joint Commis-
imens. The results are returned to the proficiency testing sion Patient Safety Goals require that when verbally reporting
vendors, where they are statistically analyzed with those from test results the information must be repeated by the person
all participating laboratories. The laboratory director receives receiving the information and documented by the person giv-
a report from the vendor, which enables the director to eval- ing the report. Written procedures should be available for the
uate the laboratory’s accuracy and compare it with other lab- reporting of critical values.
oratories using the same method of analysis. The director
ensures that the laboratory takes action to correct unaccept-
able results.16 The CLIA mandate comparison testing for lab- Errors may be discovered in the laboratory through a QM proce-
oratory accreditation.23 dure known as the delta check that compares a patient’s test results
with the previous results. Variation outside the established param- Regulatory Issues
eters alerts laboratory personnel to the possibility of an error
that occurred during the testing procedure or in patient identifica- QM is regulated throughout the total testing system. The
tion. Autoverification is often programmed into many laboratory health-care regulation systems include both governmental and
analyzers.20 public agencies. All agencies have the same goal, which is to
Erroneous results must be corrected in a timely manner provide safe and effective health care.
to assure that the patient does not receive treatment based
on incorrect results. Errors can occur in patient identifica-
tion, specimen labeling, or result transcription. The patient’s
Clinical Laboratory Improvement
record should be corrected as soon as the error is detected. Amendments (CLIA)
However, the original result must not be erased in the event The CLIA is a governmental regulatory agency administered
that the health-care provider treated the patient based on the by the Centers for Medicare and Medicaid Services (CMS) and
erroneous results. Appropriate documentation of erroneous the Food and Drug Administration (FDA). CLIA stipulates that
results should follow institutional protocol. The manual all laboratories that perform testing on human specimens for
must contain a written procedure for reporting, reviewing, the purposes of diagnosis, treatment, monitoring, or screening
and correcting errors. must be licensed and obtain a certificate from the CMS. Labo-
The In the Laboratory: Summary of Quality Manage- ratories with CLIA certification are inspected to document
ment Errors box summarizes QM errors in each phase of lab- compliance with the regulations. The inspections may be
oratory testing. performed by CMS personnel or an accrediting agency recog-
nized by CMS such as the CAP, the TJC, or the Commission
The specificity and the sensitivity for each test should be in- on Laboratory Assessment (COLA).
cluded in the procedure manual for correct interpretation of CLIA classifies laboratory tests into three categories:
results. Sensitivity and specificity vary among manufacturers. waived, provider-performed microscopy procedures (PPMP),
All known interfering substances should be listed for evalua- and nonwaived testing (See In the Laboratory: CLIA Test
tion of patient test data. Classifications). Nonwaived testing is separated into the
categories of moderate and high complexity with regard to
requirements for personnel performing the tests. Laboratories
must obtain the correct certification for the level of testing
In the Laboratory complexity performed. For each category, there is a descrip-
tion of the educational level necessary for personnel who
Summary of Quality Management Errors

• Patient misidentification
• Wrong test ordered In the Laboratory
• Incorrect specimen type collected
• Insufficient specimen volume CLIA Test Classifications
• Delayed transport of specimen to the laboratory
• Inadequate processing of specimen • Tests considered easy to perform by following the manufac-
• Delayed separation of serum or plasma from cells turer’s instructions that have little risk of error. No special train-
• Incorrect storage of specimen ing or education is required.
• Example: Urine pregnancy test
• Sample misidentification
• Erroneous instrument calibration • Microscopy tests performed by a physician, midlevel practi-
• Reagent deterioration tioner, or a dentist.
• Poor testing technique • Example: Microscopic urinalysis
• Instrument malfunction
• Interfering substances present • Moderate complexity tests
• Misinterpretation of quality control data Tests that require documentation of training in test princi-
ples, instrument calibration, periodic proficiency testing, and on-
• Patient misidentification site inspections.
• Poor handwriting Example: Automated complete blood count (CBC)
• Transcription error • High complexity tests
• Poor quality of instrument printer Tests that require sophisticated instrumentation and a high
• Failure to send report degree of interpretation.
• Failure to call critical values • Proficiency testing and on-site inspections are required.
• Inability to identify interfering substances Example: Urine culture and susceptibility

From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia, From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia,
PA: F.A. Davis; 2011, with permission. PA: F.A. Davis; 2011, with permission.
may perform the test, as well as the type of quality assurance Quality Management Systems
procedures that must be in place.
A quality management system (QMS) incorporates many of
Clinical and Laboratory Standards the objectives of total quality management and continuous
Institute (CLSI) quality improvement to ensure quality results, staff compe-
tence, and efficiency within an organization. In addition, QMS
The CLSI is a nonprofit organization that publishes recommen- also utilizes the concepts of the International Organization for
dations by nationally recognized experts for the performance Standardization (ISO 151189) and the Lean and Six sigma
of laboratory testing. CLSI standards are considered the stan- methods. The requirements of TJC and the CAP accreditation
dard of care for laboratory procedures. The standard of care organizations are included in QMS.
is the attention, caution, and prudence that a reasonable person A QMS is designed to coordinate activities to direct and
in the same circumstances would exercise. In a legal situation, control an organization with regard to quality and the reduc-
the CLSI standards would be considered the standard of care tion of medical errors. The first step in a laboratory QMS is to
that should have been met. determine the pathway of workflow through the laboratory as
discussed previously under the preexamination, examination,
The Joint Commission (TJC) and postexamination phases of testing. In each area of the path-
way, all the processes and procedures that occur are deter-
TJC is an independent, not-for-profit organization that accred-
mined and analyzed so everyone knows what they are
its and certifies more than 15,000 health-care organizations
supposed to do, how they are supposed to do it, and when
and programs in the United States. The mission of TJC is to
they are supposed to do it.
continuously improve the safety and quality of care provided
to the public through the provision of health-care accreditation
and related services that support performance improvement in Quality System Essentials
health-care organizations. Quality system essentials (QSEs) form the basis of a QMS.
TJC has recently published the Joint Commission Patient The 12 QSEs contain the management information needed
Safety Goals. It is essential that health-care organizations adhere for a laboratory to perform quality work (Table 8–2). They
to these goals to maintain their accreditation. Goals pertaining were developed by the former National Committee for Clinical
to the laboratory are: Laboratory Standards and the current CLSI and include the
Goal 1: Improving the accuracy of patient identification methods to meet the requirements of regulatory, accreditation,
Goal 2: Improving the effectiveness of communication and standard setting organizations. Quality indicators are the
among health-care givers measurements developed by each laboratory to determine if
Goal 7: Reduce the risk of health care-associated infections the quality system essentials are being met. They may include
(HAIs) such items as appropriateness of the testing, correct patient
Goal 13: Encourage patients’ active involvement in their identification, timely reporting of laboratory results, and correct
own care as a patient safety strategy proficiency testing results.

College of American Pathologists (CAP)

The Lean System
The CAP is an organization of board-certified pathologists that
advocates high-quality and cost-effective medical care. CAP The Lean system originated with the automobile manufactur-
provides laboratory accreditation and proficiency testing for ing industry in Japan. Its concepts have been adopted by many
laboratories. American industries, including the health-care industry. Lean
For accreditation purposes, CAP-trained pathologists and utilizes a tool called “6S,” which stands for: sort, straighten,
laboratory managers and technologists perform on-site labo- scrub, safety, standardize, and sustain. The focus is on the elim-
ratory inspections on a biennial basis. Inspectors examine the ination of waste to allow a facility to do more with less and at
laboratory’s records and QC of procedures for the preceding the same time increase customer and employee satisfaction. In
2 years. Also examined are the qualifications of the laboratory the health-care environment, the ability to decrease costs while
staff including continuing education attendance, the labora- providing quality health care is of primary importance.
tory’s equipment, facilities, safety program, and laboratory
management. CAP accreditation is accepted by both the CMS
and the TJC and fulfills Medicare and Medicaid requirements.
Six Sigma
As previously described, laboratories that subscribe to this Six Sigma is a statistical modification of the original Plan-Do-
proficiency program receive periodic samples to analyze and Check-Act (PDCA) method adopted by the TJC as a guideline
return their results to the CAP. The laboratory receives a report for health-care organizations. The primary goal of Six Sigma is
on how its results compared with other laboratories performing to reduce variables and decrease errors to a level of 3.4 defects
the procedures in a similar manner. Failure to perform satis- per 1 million opportunities. Attaining this goal indicates that
factorily on a proficiency test can result in a laboratory losing the laboratory is addressing factors critical to customer satis-
its CLIA certificate to perform the failed test. faction and quality care.
Table 8–2 The 12 Laboratory Quality System Essentials
QUALITY SYSTEM ESSENTIALS PROCESSES AND PROCEDURES
The Laboratory QSEs
1. Organization Personnel roles, responsibilities, and reporting relationships
Quality planning and risk assessment
Allocation of personnel and material resources
Review and assessment of meeting goals
2. Facilities and safety Space designed for efficiency
Adequate storage space
Required safety precautions and equipment availability
Housekeeping
Safety training
3. Personnel Qualifications
Current job descriptions
Orientation of new employees
Competency assessment
Continuing education
4. Equipment Selection criteria
Space needed and special instrument requirements
Ongoing preventive maintenance
Service and repair records
5. Purchasing and inventory Inventory of initial materials and reagents
Service contracts
Availability of reagents, supplies, and service
The Work System QSEs
6. Process control Identification of all laboratory processes
Procedure manuals and instructions for tasks
Test method verification
Verification that manufacturer specifications are in procedure manuals
Quality control and statistics
7. Documents and records Availability of all process and procedure documents
Periodic review of all process and procedure documents
Access to quality control records
Monitoring of record storage and retention
8. Information management Availability of patient records
Security of patient records
Methods for providing patient information
Processes to prevent Medicare and Medicaid fraud
The Measurement QSEs
9. Occurrence management Identification and reporting of all events
and nonconforming event Remedial actions taken
management Plans to eliminate future events
Initiation of changes
10. Assessments: external Obtaining external licensing and accreditation
and internal Participation in external proficiency testing
Periodic on-site auditing by accrediting agencies
Development of quality indicators for each phase of testing
11. Customer service Feedback from customers including patients, patients’ families, and health-care
providers
Feedback from employees
Feedback from offsite referral laboratories and health-care providers
12. Process improvement Monitoring of the above QSEs results
Determination of the root cause of problems
Utilize the Lean system tools
Utilize Six Sigma methodology
 The Six Sigma methodology is represented by the acronym
DMAIC: • Dispose of radioactive material following NRC guidelines.
• Be observant for frayed cords, overloaded circuits, and im-
Define goals and current processes properly grounded equipment. Avoid working with electri-
Measure current processes and collect data cal equipment when you, or the equipment, is wet.
Analyze the data for cause-and-effect information • Follow routine safety protocols and maintain a clean,
Improve the process using the data collected organized work area to avoid physical hazards.
Control the correction of concerns displayed in the data • The acronym RACE outlines the steps to follow when a
By instituting quality improvement methodologies, a health- fire is discovered: (R) rescue anyone in danger, (A) activate
care institution can develop a structured standardized format the fire alarm, (C) contain the fire, (E) extinguish the fire
to systematically assess and document the quality of services if possible or evacuate, closing the door.
to the customer. • Quality management is the overall process of guaranteeing
quality throughout the entire testing system.
• Quality control involves performing individual procedures
SUMMARY using acceptable standards and control material at various
medically significant levels.
• Transmission of biological hazards that are encountered • Documentation includes a procedure manual, policies to
when testing patient specimens requires a chain of infec- control and monitor procedure variables, and records of
tion, which consists of an infectious agent, reservoir, por- competency assessment and continuing education.
tal of exit, mode of transmission, portal of entry, and a • Preexamination variables occur before sample testing.
susceptible host. Examination variables occur during the specimen testing.
• Hand hygiene and wearing PPE are essential actions to Postexamination variables occur during interpretation and
prevent transmission of infectious organisms. Standard reporting of test results.
precautions should be followed at all times. • Agencies regulating the laboratory include:
• Specimens, except urine, and contaminated supplies must • CLIA—provides requirements for persons performing
be disposed of in a biohazard container. waived, provider-performed microscopy, moderate-
• All sharps, including needles and holders, must be disposed complexity, and high-complexity testing
of in puncture-proof containers. Recapping of needles is • TJC—provides accreditation and certification of health-
prohibited. care organizations
• The Occupational Exposure to Bloodborne Pathogens Stan- • CAP—provides laboratory accreditation and provision
dards are a means of providing protection from accidental of proficiency testing
exposure to bloodborne pathogens through the use of engi- • CLSI—develops written standards and guidelines for
neering controls, work practice controls, and use of PPE. sample collection, handling and processing, and labo-
• When transporting biological specimens, Department of ratory testing and reporting
Transportation and International Air Transit Association • The 12 quality essentials provide the management docu-
regulations must be followed. They include placing spec- mentation needed to demonstrate quality work. Quality
imens in screw cap containers, wrapping them in ab- indicators are developed to monitor each phase of testing.
sorbent material, and placing them in a sturdy leakproof • The Lean system utilizes the “6S” tools (sort, straighten,
container. scrub, safety, standardize, and sustain) to enhance effi-
• Follow specific directions when mixing chemicals and ciency and proficiency.
always add acid to water, rather than water to acid. • The goal of the statistical Six Sigma method is to reduce
• When chemical contact with the skin or eyes occurs, variables and decrease errors to a level of 3.4 defects per
immediately flush the area with water for 15 minutes. 1 million opportunities.
• A SDS and a Chemical Hygiene Plan must be available to
employees. Dispose of chemicals per EPA guidelines.
CASE STUDIES
1. The immunology supervisor who has been working for the 2. As the supervisor of the immunology section, you en-
last 20 years in a small rural hospital is training a new em- counter the following situations. Explain whether you
ployee. A dilution of a patient’s serum must be made to run would accept them or take corrective action.
a particular test. The supervisor is having difficulty using a
Questions
serological pipette so she removes one glove. In uncapping
the serum tube, a small amount of serum splashes onto the a. You are told that only the supervisor performs the CAP
workbench. She cleans this up with a paper towel, which proficiency survey.
she discards in the regular paper trash. She also spills a b. QC is not performed daily on the Centaur instrument.
small amount onto her disposable lab coat. She tells the c. The Streptozyme test reporting procedure has been re-
new employee that, because it is such a small amount, she cently revised.
isn’t going to worry about it and continues on to pipette d. Opened, unlabeled commercial quality control bottles
the specimen. She then replaces the glove onto her un- are in the refrigerator.
gloved hand and says that, because it is almost break time,
she will wait to wash her hands until then.
Questions
a. Please identify all the safety violations involved.

REVIEW QUESTIONS
1. A technologist who observes a red rash on her hands 5. A technician places tightly capped noninfectious
after removing her gloves serum tubes in a rack and places the rack and the
a. should apply antimicrobial lotion to the hands. specimen data in a labeled leakproof metal courier
b. may be washing the hands too frequently. box. Is there anything wrong with this scenario?
c. may have developed a latex allergy. a. Yes, DOT requirements are not met.
d. should not create friction when washing the hands. b. No, the tubes are placed in a rack.
c. Yes, absorbent material is missing.
2. In the chain of infection, a contaminated work area d. No, the box contains the specimen data.
would serve as which of the following?
a. Reservoir 6. The Occupational Exposure to Bloodborne Pathogens
b. Means of transmission Standard developed by OSHA requires employers to
c. Portal of entry provide all of the following except
d. Portal of exit a. hepatitis B immunization.
b. safety training.
3. The only biological waste that does not have to be dis- c. hepatitis C immunization.
carded in a container with a biohazard symbol is d. laundry facilities for nondisposable lab coats.
a. urine.
b. serum. 7. An employee who receives an accidental needlestick
c. feces. should immediately
d. serum tubes. a. apply sodium hypochlorite to the area.
b. notify a supervisor.
4. Patient specimens transported by the Department of c. receive HIV prophylaxis.
Transportation must be labeled as a d. receive a hepatitis B booster shot.
a. diagnostic specimen.
b. clinical specimen.
c. biological specimen, category b.
d. laboratory specimen.
 8. The first thing to do when acid is spilled on the skin is to 15. When external quality control is run, what informa-
a. notify a supervisor. tion must be documented?
b. neutralize the area with a base. a. The lot number
c. apply burn ointment. b. Expiration date of the control
d. flush the area with water. c. The test results
d. All of the above
9. When combining acid and water,
a. acid is added to water. 16. What steps are taken when the results of the quality
b. water is added to acid. control testing are outside of the stated confidence
c. water is slowly added to acid. limits?
d. both solutions are combined simultaneously. a. Check the expiration date of the control material
b. Run a new control
10. To determine the chemical characteristics of sodium c. Open a new control bottle
azide, an employee would consult the d. All of the above
a. Chemical Hygiene Plan.
b. Merck manual. 17. When a new bottle of QC material is opened, what in-
c. SDS. formation is placed on the label?
d. NRC guidelines. a. The time the bottle was opened
b. The supervisor’s initials
11. A technician who is pregnant should avoid working c. The lot number
with d. The date and the laboratory worker’s initials
a. organic chemicals.
b. radioisotopes. 18. What is the primary goal of TQM?
c. HIV-positive serum. a. Precise test results
d. needles and lancets. b. Increased laboratory productivity
c. Improved patient outcomes
12. Which of the following laboratory regulatory agencies d. Reproducible test results
classifies laboratory tests by their complexity?
a. OSHA 19. Would a control sample that has accidentally
b. CAP become diluted produce a trend or a shift in the
c. TJC Levey-Jennings plot?
d. CMS a. Trend
b. Shift
13. Which of the following organizations publishes guide-
lines that are considered the standard of care for labo- Fill in the Blank
ratory procedures? 20. Indicate whether each of the following would be
a. CLIA considered a (1) preexamination, (2) examination, or
b. CLSI (3) postexamination variable by placing the appropri-
c. TJC ate number in the space.
d. CAP Reagent expiration date
Rejection of a hemolyzed specimen
14. Quality managment refers to Construction of a Levey-Jennings chart
a. performance of two levels of testing controls. Telephoning a critical result to the nurse
b. reliable control results. Calibrating the centrifuge
c. increased productivity. Pipetting the diluent
d. quality of specimens and patient care.
 Principles of Serological
Testing

After finishing this chapter, you should be able to: BLOOD SPECIMEN PREPARATION
AND MEASURING
1. Describe how whole blood is processed in order to obtain serum for
serological testing. DILUTIONS
2. Explain the difference between a volumetric and a graduated pipette. Simple Dilutions
3. Define the following: serial dilution, solute, diluent, compound Compound Dilutions
dilution. Test Parameters
4. Describe how an accurate measurement is made using a serological SUMMARY
pipette that is marked TD. CASE STUDY
5. Calculate the final dilution of a sample, given the initial dilution REVIEW QUESTIONS
and all subsequent dilutions.
6. Explain how an antibody titer is determined.
7. Calculate the amount of diluent needed to prepare a specific dilution
of a serum specimen.
8. Determine how to make a specific percent solution from a
concentrate.
9. Differentiate sensitivity and specificity as it relates to serological
testing.
10. Discuss how positive and negative predictive values determine the
chance that an individual has a truly positive or truly negative test
result.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Blowout pipette Negative predictive value Serological pipette Specificity
Diluent Positive predictive value Serology Titer
Graduated pipettes Sensitivity Serum Volumetric pipettes
Micropipette Serial dilution Solute

Serology is the study of the fluid components in the blood, used with some sort of suctioning device such as a rubber bulb.
especially antibodies. Serum, the liquid portion of the blood, The bulb is squeezed to draw the measured amount of liquid
minus the coagulation factors is the most frequently encoun- into the pipette. Excess fluid is wiped off the outside of the
tered specimen in immunologic testing. Knowledge of speci- pipette and then the pipette is held vertically with the tip
men preparation and dilutions is essential to understanding all against the surface of a container. The suction is released and
serological testing in the clinical laboratory. the liquid is allowed to flow by gravity into the container.
Graduated pipettes have markings that allow for varying
amounts of liquid to be measured (Fig. 9–2). A graduated or
Blood Specimen Preparation measuring pipette has marks all along its length. If it is a sero-
and Measuring logical pipette, the marks go all the way down to the tip. Some
serological pipettes have a frosted band around the opening;
Blood is collected aseptically by venipuncture into a clean, dry, this type is called a blowout pipette. These pipettes may be
sterile tube. Care must be taken to avoid hemolysis as this may labeled TC, meaning to contain. If they are filled to the end, the
produce a false-positive test. The blood specimen is allowed to last drop of liquid must be forced out using a pipetting bulb
clot at room temperature or at 4°C, depending upon the pro- or other device to deliver an accurate volume.1 Alternatively, a
tocol for the specific procedure. It is then centrifuged, after specified amount of liquid can be measured from point to point
which serum should be promptly separated into another tube in the pipette. For example, if 0.3 mL is measured using a
without transferring any cellular elements. Fresh serum that 1-mL TC pipette, the liquid can be dispensed by going from
has not been inactivated by heating is usually recommended 0.0 to the 0.3 mL mark, rather than filling the pipette to the
for testing. For some testing, however, complement must be 0.7 mL mark and dispensing down to the end.
inactivated because it interferes with test results. In this case, Generally, a measuring pipette is held in a vertical position
the serum is heated to 56°C for 30 minutes to destroy any com- when drawing up a liquid (Fig. 9–3). The bottom of the
plement present. In either circumstance, if testing cannot be meniscus should be level with the calibration line on the
performed immediately, serum may be stored between 2°C and pipette. This is sighted at eye level (see Fig. 9–3B). In practice,
8°C for up to 72 hours. If there is any additional delay in test- measuring pipettes are more typically used to prepare reagents
ing, the serum should be frozen at –20°C or below. rather than for measurement of patient specimens and controls.
Pipettes are commonly used to measure either serum for Often, an automatic pipette filler is used with measuring
testing or liquid for making reagents and dilutions. The pipettes to increase the accuracy (Fig. 9–4). For patient spec-
pipettes are calibrated to transfer or deliver specific volumes imens and control calibrators, micropipettes are much more
as marked on their surfaces. They can be categorized as either accurate.2
volumetric or graduated. Micropipettes deliver volumes in the microliter (µL) range
Volumetric pipettes are marked and calibrated to deliver and can be used when very small volumes are needed. Mi-
only one volume of the specified liquid (Fig. 9–1).1 The design cropipettes are mechanical pipettes that draw up and then re-
enables the user to dispense the exact measure of liquid with lease a certain volume by depressing a plunger (Fig. 9–5). A
a small drop left behind. Pipettes are usually labeled TD, mean- one-time use disposable tip is used for each different specimen.
ing to deliver. The volumetric pipettes have an oval bulb in the Some micropipettes are adjustable and can hold a small range
center and a tapered dispensing end. Volumetric pipettes are
10

Volumetric pipette. Four different sizes of graduated pipettes.

 1 2

3 4
1. Use mechanical suction

2. Wipe off outside of pipette with gauze

3. Adjust the meniscus

4. Drain into receiving vessel

Meniscus

Calibration
mark
Eye level

(A) Steps in accurately measuring a liquid with a serological pipette. (B) How to read a meniscus.

of volumes, whereas others only hold a fixed volume. Mi- If the relative proportions of antigen and antibody present are
cropipettes are easier to use than pipettes with safety bulbs, as not similar, the reaction cannot be detected. When too much
well as more accurate. antibody is present, an end point may not be reached. In this
case, the serum that contains antibody must be diluted. There-
fore, knowledge of dilutions is essential to understanding all
Dilutions serological testing.

In the clinical laboratory, it is often necessary to make a less

concentrated solution from a reagent such as an acid or a buffer
Simple Dilutions
in order to use the reagent in a particular procedure. In this A dilution involves two entities: the solute, which is the ma-
case, either water or saline is added to the concentrate to make terial being diluted, and the diluent, which is the medium
the reagent the proper strength for testing. For many serology making up the rest of the solution. The relationship between
tests, it is the serum that is concentrated; it may be necessary these two is a ratio that can also be expressed as a fraction.
to dilute it with saline in order for a visible reaction to occur. For example, a 1:20 dilution can be set up as a fraction,
 The equation is set up using the fraction for the dilution, indi-
cating the relationship between the total volume and the solute,
or the amount of serum needed:
1/20 = x/2 mL
Note that the 20 represents the total number of parts in the
solution and that 2 mL is the total volume desired.
Cross-multiplying to solve this equation for x gives 0.1 mL
for the amount of serum needed to make this dilution. The
amount of diluent is obtained by subtracting 0.1 mL from
2.0 mL to give 1.9 mL of diluent. To check the answer, set up
a proportion between the amount of solute over the total
volume. This should equal the dilution desired.
0.1 mL/(1.9 mL diluent + 0.1 mL serum) = 1/20
Thus, the correct answer has been obtained.
If, on the other hand, one knows the amount of serum to
be used, a problem can be set up in the following manner:
A 1:5 dilution of patient serum is necessary to run a sero-
logical test. There is 0.1 mL of serum that can be used.
What amount of diluent is necessary to make this dilution
using all of the serum?

A pipette filler for use with serological pipettes ranging A slightly different formula can be used to solve this problem:
in size from 1 to 100 mL. 1/Dilution – 1 = Amount of Solute/Amount of Diluent
1
/4 = 0.1 mL/x
x = 0.4 mL of diluent
Note that the final volume is obtained by adding 0.1 mL of
solute to the 0.4 mL of diluent. Dividing the volume of
the solute by the total volume of 0.5 mL yields the desired
1:5 ratio.
Depending on the unknown being solved for, either of
these formulas can be used. To calculate the total volume, the
total dilution factor must be used. If, however, the amount of
diluent is to be calculated, the formula using dilution–1 can
be used.
Consider a further example:
Instructions that come with a buffer indicate that it must be
mixed with 19 parts of water for use in a serological test.
The volume of the buffer concentrate is 50 mL. How would
Three different sizes of micropipettes. we find out the amount of water to add and what would be
the final dilution factor?

The equation would be set up as follows:

1/20. This dilution implies 1 part of solute and 19 parts of 1/Dilution – 1 = 50 mL/ Amount of Diluent
diluent. The number on the bottom of the fraction is the
1/19 = 50/x
total volume, reached by adding the volumes of the solute
and diluent together. x = 50 19
1/Dilution = Amount of Solute/Total Volume x = 950 mL of diluent
To create a certain volume of a specified dilution, it is helpful First, we multiply 50 mL by the 19 parts to give us 950 mL,
to know how to manipulate this relationship. An algebraic which is the amount of water that needs to be added to the
equation can be set up to find the total volume, the amount of buffer concentrate. To give the final volume, we add the 50 mL
solute, or the amount of diluent needed to make a dilution. of concentrate to the 950 mL of water to get 1,000 mL total.
Consider the following example: Sometimes it is necessary to dilute a concentrate to a spe-
2 mL of a 1:20 dilution is needed to run a specific serological cific percentage. A percentage is simply a different way of ex-
test. How much serum and how much diluent are needed pressing a dilution. For instance, a 10% solution can also be
to make this dilution? expressed as 1/10. In dealing with volumes, the relationship
is volume/volume. It can also be expressed as weight/volume
if a solid is to be dissolved in a liquid. Let’s take the example
of diluting glacial acetic acid to make a 10% solution for a
testing procedure. A volume of 500 mL of a 10% solution of
acetic acid is needed. How much glacial acetic acid is needed
and how much diluent should be used?
We know the final volume and we know the percentage, so
then we are solving for the amount of the concentrated acetic
acid needed.
1/10 = x/500 mL
Cross-multiplying and solving for x gives us 50 mL of glacial
acetic acid needed. Then the amount of diluent is the total
volume minus the concentrate, or 500 mL – 50 mL = 450 mL
of diluent to make up the 10% solution. Additional problems
which give more practice with dilution calculations can be Serial dilution. Each tube contains the same amount
found in the Review Questions section at the end of this of diluent and a blue dye. Each time a dilution is made, the amount
chapter. of dye is cut in half in each successive tube. The final volume of each
tube should be exactly the same. The color becomes visibly lighter
with each dilution.
Compound Dilutions
The previous examples represent simple dilutions. Occasion- most common serial dilution is a doubling dilution in which
ally in the laboratory it is necessary to make a very large di- the amount of serum is cut in half with each dilution. For
lution; if so, it is more accurate and less costly to do this in example, six test tubes can be set up with 0.2 mL of diluent
several steps rather than all at once. Such a process is known in each. If 0.2 mL of serum is added to the first tube, this
as a compound dilution. The same approach is used, but the becomes a 1:2 dilution.
dilution occurs in several stages. For example, if a 1:500 di-
0.2 mL Serum/(0.2 mL Serum + 0.2 mL Diluent) =
lution is necessary, it would take 49.9 mL of diluent to ac-
0.2 mL/0.4 mL = 1/2
complish this in one step with 0.1 mL of serum. If only a
small amount of solution is needed to run the test, this is When 0.2 mL of the 1:2 dilution is added to 0.2 mL of dilu-
wasteful; furthermore, inaccuracy may occur if the solution ent, a 1:4 dilution is obtained. The final dilution is obtained
is not properly mixed. Therefore, it is helpful to make several by counting the number of tubes and setting up a multipli-
smaller dilutions. cation series in which the original dilution factor is raised to
To calculate a compound dilution problem, the first step is a power equal to the number of tubes. In this example, if the
to plan the number and sizes of simple dilutions necessary to first tube contains a 1:2 dilution, the dilution in tube num-
reach the desired end point. To use the preceding example, a ber six is
1:500 dilution can be achieved by making a 1:5 dilution of the 1
/2 ! 1/2 ! 1/2 ! 1/2 ! 1/2 ! 1/2 = 1/64
original serum, a 1:10 dilution from the first dilution, and If, in this instance, an end point was reached at tube number
another 1:10 dilution. This can be shown as follows: five, the actual titer would be 1:32. To avoid confusion, this is
Serum: customarily written as the reciprocal of the dilution—that is, 32.
1:5 dilution 1:10 dilution 1:10 dilution The titer is the last tube in which a positive reaction is visible.
0.1 mL serum 0.1 mL of 0.1 mL of The tubes are read all the way to the end of the dilution; then,
1:5 dilution 1:10 dilution after finding the first negative tube, the positive tube before it
0.4 mL diluent 0.9 mL diluent 0.9 mL diluent is reported out as the titer.
Multiplying 5 10 10 equals 500, or the total dilution. Serial dilutions do not always have to be doubling dilutions.
Each of the simple dilutions is calculated individually by doing Consider the following set of test tube dilutions:
mental arithmetic or by using the formula given for simple di- 1:5 1:25 1:125 1:625 1:3125
lutions. In this example, the 1:500 dilution was made using For each successive tube, the dilution is increased by a factor
very little diluent in a series of test tubes, rather than having of 5, so this would indeed be considered a serial dilution.
to use a larger volume in a flask. The volumes were kept small Having the ability to work with simple and compound dilu-
enough so that mixing could take place easily. The final volume tions and interpret serial dilutions is a necessary skill for lab-
of 1.0 mL is all that is necessary to perform a test. oratory work.
If, in each step of the dilution, the dilution factor is ex-
actly the same, this is known as a serial dilution. Serial di-
lutions are often used to obtain a titer, or indicator of an
Test Parameters
antibody’s strength. A series of test tubes is set up with ex- A question sometimes arises as to whether or not the patient
actly the same amount of diluent in each (Fig. 9–6). The actually has the condition or disease when results are
obtained in a laboratory test. In order for the clinician to In the example given earlier, out of 200 patients, 160 were
make such a judgment, it is important to know the sensitiv- truly positive and 8 were falsely positive. Using these values in
ity and specificity of a particular test. The sensitivity can be the equation,
defined as the proportion of people who have a specific 160
disease or condition and who have a positive test.3 It is Positive Predictive Value (%) = 100
described by the following ratio: 160 + 8
True Positives Positive predictive value (%) = 95%
Sensitivity (%) = 100 Thus, in this case, if an individual tests positive for a certain
True Positives and
disease, it is highly likely that the individual has the disease.
False Negatives
Now let’s look at negative predictive value for the same test
The sensitivity thus indicates how small an amount can be results.
measured and still produce a positive test result. The speci-
True Negatives
ficity refers to the proportion of people who do not have the Negative Predictive Value (%) = 100
disease or condition and who have a negative test.3 It can be True Negatives +
expressed as the following: False Negatives
True Negatives 20
Specificity (%) = 100 Negative Predictive Value (%) = 100
True Negatives and 20 + 12
False Positives
Negative predictive value (%) = 62.5%
Let’s take an example of test results to calculate the sensitivity
In this case, because there are a number of false-negative results,
and specificity. A certain new laboratory test was used with a
the negative predictive value is not as high as the positive pre-
particular population. The results were as follows: 200 patients
dictive value. These results would help the clinician make a de-
were tested and there were 160 true positives, 20 true negatives,
cision about an individual patient based on his or her test results
8 false positives, and 12 false negatives. The sensitivity is cal-
and the prevalence of the disease in that particular population.
culated by taking the true positives and dividing by the true
positives added to the false negatives:
Sensitivity (%) = 160/(160 + 12) × 100 SUMMARY
Sensitivity = 93%
• Serum for serological testing is obtained by allowing a
To calculate the specificity, the following formula is
sterile tube to clot at either room temperature or 4°C and
used:
then carefully removing the serum from the clot after
Specificity (%) = 20/(20 + 8) × 100 centrifugation has taken place.
Specificity = 71.4% • Volumetric pipettes hold a specified amount of liquid and
Thus, this particular test is highly sensitive, but it is not very are calibrated to deliver (TD) that exact amount.
specific. A test that is highly specific means that it measures • Serological pipettes are calibrated all the way to the
only the substance that it is designed to measure and does not bottom of the pipette and must be blown out to deliver
measure any interfering substances. If a test is both highly sen- the exact amount of liquid required.
sitive and highly specific, a positive result indicates that the • A dilution is the addition of a liquid to make a weaker
patient likely has the disease or condition. solution of either a reagent or a patient specimen. In order
Sensitivity and specificity are characteristics of the test it- for a visible end point to occur in antigen–antibody reac-
self. However, in order for a clinician to determine whether tions, often a dilution needs to be made.
a person with a positive test actually has the disease, it is im- • Patient serum, the solute, is made weaker by adding diluent
portant to look at how often the disease occurs in the par- so that the antibody present is not as concentrated. The rela-
ticular population. The positive predictive value is the tionship between the serum and the total volume can be ex-
probability that a person with a positive screening test actu- pressed as a ratio, 1:20, or as a fraction—for example, 1/ 20.
ally has the disease. The negative predictive value is the • When several dilutions are made in which the dilution fac-
probability that a person with a negative screening test does tor is the same in each case, this is called a serial dilution.
not have the disease. Let’s see how this is calculated with • Serial dilutions are used to determine the titer, or strength,
some actual test results. The positive predictive value is de- of an antibody. The last tube in which a visible reaction is
termined by dividing the true positive results by the true seen is considered the end point.
positive and the false positive results added together. Let’s • Sensitivity is defined as the proportion of people who have
use the example with the sensitivity of 93% calculated a specific disease or condition and have a positive test for
earlier. that disease or condition.
True Positives • Specificity refers to the proportion of people who do not
Positive Predictive Value (%) = 100 have the disease or condition and who have a negative test
True Positives and for that disease or condition.
False Positives
• If a test is highly sensitive and highly specific, it is a good • The negative predictive value is the probability that a
indicator that a patient has the disease or condition if pos- person with a negative screening test does not have the
itive results are obtained. disease.
• The positive predictive value is the likelihood that a • Positive and negative predictive values help the clinician
person with a positive screening test actually has the to determine whether a positive or a negative test is likely
disease. to be a true result based on a specific test population.

CASE STUDY
The serology supervisor who has been working for the last of diluent to the tube with the serum. She needs a
20 years in a small rural hospital is training a new employee. 1:40 dilution of the serum to run the test.
A dilution of a patient’s serum must be made to run a par-
Questions
ticular test. The supervisor is showing the new employee
how to pipette the serum specimen. The amount needed is a. Explain any mistakes the supervisor may have made
0.1 mL. Using a serological pipette, she draws up the patient during her demonstration.
specimen to the 0.9 mL mark. She then lets it drain out. b. Was the dilution correct? If necessary, correct the
There is a tiny bit left in the pipette, but she explains to the dilution.
new person that this is close enough. She then adds 1.9 mL

REVIEW QUESTIONS
1. If serum is not tested immediately, how should it be 4. If glacial acetic acid needs to be diluted with water to
treated? make a 10% solution, what does the glacial acetic acid
a. It can be left at room temperature for 24 hours. represent?
b. It can be stored in the refrigerator for up to a. Solute
72 hours. b. Diluent
c. It can be stored in the refrigerator for up to c. Titer
48 hours. d. Serial dilution
d. It needs to be frozen immediately.
5. A pipette that has markings all the way down to its tip
2. A 1:750 dilution of serum is needed to perform a is called a
serological test. Which of the following series of a. volumetric pipette.
dilutions would be correct to use in this situation? b. serial pipette.
a. 1:5, 1:15, 1:10 c. graduated pipette.
b. 1:5, 1:10, 1:5 d. micropipette.
c. 1:15, 1:10, 1:3
d. 1:15, 1:3, 1:5 6. A serological test requires 5 mL of a 1:50 dilution.
How much serum is required to make this
3. How much diluent needs to be added to 0.2 mL dilution?
of serum to make a 1:20 dilution? a. 0.5 mL
a. 19.8 mL b. 0.01 mL
b. 4.0 mL c. 1.0 mL
c. 3.8 mL d. 0.1 mL
d. 10.0 mL
 7. If 0.02 mL of serum is diluted with 0.08 mL of 14. Which of the following would be the correct way
diluent, what dilution of serum does this represent? to make a 5% solution of hydrochloric acid from
a. 1:4 concentrated hydrochloric acid?
b. 1:5 a. 0.5 mL of acid and 9.5 mL of water
c. 1:10 b. 0.5 mL of acid and 95 mL of water
d. 1:20 c. 0.1 mL of acid and 9.9 mL of water
d. 0.1 mL of acid and 4.9 mL of water
8. A tube containing a 1:40 dilution is accidently
dropped. A 1:2 dilution of the specimen is still 15. What is the final dilution of serum obtained from
available. A volume of 4 mL is needed to run the the following serial dilutions: 1:4, 1:4, 1:4, 1:4,
test. How much of the 1:2 dilution is needed to 1:4, 1:4?
remake 4 mL of a 1:40 dilution? a. 1:24
a. 0.2 mL b. 1:256
b. 0.4 mL c. 1:1,024
c. 0.5 mL d. 1:4,096
d. 1.0 mL
16. A new laboratory assay gave the following results:
9. If 0.4 mL of serum is mixed with 15.6 mL of diluent, number of patients tested = 100; number of true
what dilution of serum does this represent? positives = 54, number of true negatives = 42;
a. 1:4 number of false positives = 2; number of false
b. 1:40 negatives = 2. What is the specificity of this assay
c. 2:70 in whole numbers?
d. 1:80 a. 75%
b. 85%
10. How much diluent needs to be added to 0.1 mL c. 95%
of serum to make a 1:15 dilution? d. 98%
a. 1.4 mL
b. 1.5 mL 17. What is the sensitivity of the assay in Question 16?
c. 5.0 mL a. 84%
d. 15 mL b. 90%
c. 92%
11. Which of the following choices would be considered a d. 96%
serial dilution?
a. 1:5, 1:15, 1:20 18. A screening test gave the following results: number of
b. 1:2, 1:10, 1:25 patients tested = 150; number of true positives = 50;
c. 1:15, 1:30, 1:40 number of true negatives = 85; number of false
d. 1:5, 1:15, 1:45 positives = 5; number of false negatives = 10. What
is the positive predictive value rounded off to a
12. The following dilutions were set up to titer an whole number for a patient whose test is positive?
antibody. The following results were obtained: 1:4 +, a. 91%
1:8 +, 1:16 +, 1:32 +, 1:64 –. How should the titer b. 83%
be reported out? c. 89%
a. 4 d. 56%
b. 16
c. 32
d. 64

13. If a serological test is positive for an individual who

does not have a particular disease, the result was
caused by a problem with
a. sensitivity.
b. specificity.
c. accuracy.
d. poor pipetting.
 Precipitation
and Agglutination
Reactions

After finishing this chapter, you should be able to: ANTIGENANTIBODY BINDING
1. Discuss affinity and avidity and their influence on antigen–antibody Affinity
reactions. Avidity
2. Describe how the law of mass action relates to antigen–antibody Law of Mass Action
binding. PRECIPITATION CURVE
3. Distinguish between precipitation and agglutination. Zone of Equivalence
4. Explain how the zone of equivalence is related to the lattice hypothesis. Prozone and Postzone
5. Differentiate between turbidimetry and nephelometry and discuss the MEASUREMENT OF PRECIPITATION
role of each in measurement of precipitation reactions. BY LIGHT SCATTERING
6. Compare single diffusion to double diffusion. PASSIVE IMMUNODIFFUSION
7. Summarize the principle of the end-point method of radial TECHNIQUES
immunodiffusion. Radial Immunodiffusion
8. Determine the relationship between two antigens by looking at the Ouchterlony Double Diffusion
pattern of precipitation resulting from Ouchterlony immunodiffusion.
ELECTROPHORETIC TECHNIQUES
9. Describe immunofixation electrophoresis and explain how it differs
COMPARISON OF PRECIPITATION
from passive diffusion.
TECHNIQUES
10. Recognize how immunoglobulin M (IgM) and immunoglobulin G
PRINCIPLES OF AGGLUTINATION
(IgG) differ in their ability to participate in agglutination reactions.
REACTIONS
11. Describe physiological conditions that can be altered to enhance
TYPES OF AGGLUTINATION
agglutination.
REACTIONS
12. Define and give an example of each of the following:
Direct Agglutination
a. Direct agglutination
Passive Agglutination
b. Passive agglutination
Reverse Passive Agglutination
c. Reverse passive agglutination
Agglutination Inhibition
d. Agglutination inhibition
INSTRUMENTATION
e. Hemagglutination inhibition
QUALITY CONTROL AND QUALITY
13. Describe the principle of measurement used in particle-counting ASSURANCE
immunoassay (PACIA).
SUMMARY
14. Identify conditions that must be met for optimal results in
CASE STUDIES
agglutination testing.
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Affinity Hemagglutination Particle-counting Rate nephelometry
Agglutination Hemagglutination inhibition immunoassay (PACIA) Reverse passive
Agglutination inhibition Immunofixation Passive agglutination agglutination
Agglutinins electrophoresis Passive immunodiffusion Sensitization
Avidity Lattice Postzone phenomenon Turbidimetry
Direct agglutination Law of mass action Precipitation Zone of equivalence
Electrophoresis Nephelometry Prozone phenomenon
End-point method Ouchterlony double diffusion Radial immunodiffusion (RID)

The combination of an antigen with a specific antibody plays antigens resembling the original antigen that induced anti-
an important role in the laboratory in diagnosing many differ- body production, which is known as cross-reactivity. The
ent diseases. Immunoassays have been developed to detect ei- more the cross-reacting antigen resembles the original anti-
ther antigen or antibody and vary from easily performed gen, the stronger the bond will be between the antigen and
manual tests to highly complex automated assays. The first the binding site. However, if the epitope and the binding site
such assays were based on the principles of precipitation or ag- have a perfect lock-and-key fit, as is the case with the original
glutination. Precipitation involves combining soluble antigen antigen, the affinity will be maximal (Fig. 10–1). When the
with soluble antibody to produce insoluble complexes that are affinity is higher, the assay reaction is more sensitive because
visible. Agglutination is the process by which particulate anti- more antigen–antibody complexes will be formed and visu-
gens such as cells aggregate to form larger complexes when a alized more easily.
specific antibody is present. Precipitation and agglutination are
considered unlabeled assays because a marker label is not Avidity
needed to detect the reaction. Labeled assays, which were de-
veloped much later, will be considered in Chapter 11. Avidity represents the overall strength of antigen–antibody
Precipitation was first noted in 1897 by Kraus, who found binding and is the sum of the affinities of all the individual
that culture filtrates of enteric bacteria would precipitate when antibody–antigen combining sites.1,3 Avidity refers to the
they were mixed with specific antibodies. For such reactions strength with which a multivalent antibody binds a multi-
to occur, both the antigen and antibody must have multiple valent antigen and is a measure of the overall stability of an
binding sites for one another and the relative concentration of antigen–antibody complex.2 In other words, once binding
each must be equal. Binding characteristics of antibodies, has occurred, it is the force that keeps the molecules to-
called affinity and avidity, also play a major role. gether. A high avidity can actually compensate for a low
affinity. Different classes of antibodies actually differ in avidi-
ties. The more bonds that form between antigen and anti-
Antigen–Antibody Binding body, the higher the avidity is. IgM, for instance, has a higher
avidity than IgG because IgM has the potential to bind
The primary union of binding sites on an antibody with spe- 10 different antigens (Fig. 10–2). Both affinity and avidity
cific epitopes on an antigen depends on two characteristics of contribute to the stability of the antigen–antibody complexes,
antibody known as affinity and avidity. Such characteristics are
important because they relate to the sensitivity and specificity
of testing in the clinical laboratory. Higher affinity Lower affinity

! !
Affinity " " "
! ! ! " "
Affinity is the initial force of attraction that exists between a
" "
single Fab site on an antibody molecule and a single epitope
or determinant site on the corresponding antigen.1,2 As the epi-
tope and binding site come into close proximity to each other,
they are held together by rather weak bonds occurring only
over a short distance of approximately 1 ! 10–7 mm.1
The strength of attraction depends on the specificity of an-
tibody for a particular antigen. One antibody molecule may Affinity is determined by the three-dimensional fit
initially attract numerous different antigens, but it is the epi- and molecular attractions between one antigenic determinant and
tope’s shape and the way it fits together with the binding sites one antibody-binding site. The antigenic determinant on the left has
on an antibody molecule that determines whether the bond- a better fit and charge distribution than the epitope on the right and
ing will be stable. Antibodies are capable of reacting with hence will have a higher affinity.
 Zone of
Antigen Prozone equivalence Postzone

Antigen/antibody complexes
Individual
epitopes Antigen concentration
Avidity is the sum of the forces binding multivalent Precipitin curve. The precipitin curve shows how the
antigens to multivalent antibodies. In a comparison between IgG amount of precipitation varies with varying antigen concentrations
and IgM, IgM has the most potential binding sites for antigen and when the amount of antibody is kept constant. Excess antibody is
thus the higher avidity. Note that the monomers in IgM can swing called the prozone and excess antigen concentration is called the
up or down in order to bind more effectively. postzone.

which is essential to detecting the presence of an unknown, precipitation is the result of random, reversible reactions
whether it is antigen or antibody. whereby each antibody binds to more than one antigen and
vice versa, forming a stable network or lattice. The lattice hy-
Law of Mass Action pothesis, as formulated by Marrack, is based on the assump-
tions that each antibody molecule must have at least two
All antigen–antibody binding is reversible and is governed by binding sites and the antigen must be multivalent. As they
the law of mass action. This law states that free reactants are combine, this arrangement results in a multimolecular lattice
in equilibrium with bound reactants.3 The equilibrium con- that increases in size until it precipitates out of solution.4
stant K represents the difference in the rates of the forward and As illustrated by the precipitin curve shown in Figure 10–3,
reverse reactions according to the following equation: when the same amount of soluble antigen is added to increasing
K = [AgAb]/[Ab][Ag] dilutions of antibody, the amount of precipitation increases up
to the zone of equivalence. When the amount of antigen over-
where [AgAb] = concentration of the antigen–antibody
whelms the number of antibody-combining sites present, pre-
complex (mol/L)
cipitation begins to decline because fewer lattice networks are
[Ab] = concentration of free antibody (mol/L)
formed.
[Ag] = concentration of free antigen (mol/L)
The value of K depends on the strength of binding between
antibody and antigen. As the strength of binding, or avidity, in-
Prozone and Postzone
creases, the tendency of the antigen–antibody complexes to dis- As can be seen on the precipitin curve, precipitation declines
sociate decreases, which increases the value of K. When the on either side of the equivalence zone because of an excess of
value of K is higher, the amount of antigen–antibody complex either antigen or antibody. In the case of antibody excess, the
is larger and the assay reaction is more visible or easily de- prozone phenomenon occurs, in which antigen combines
tectable. The ideal conditions in the clinical laboratory would with only one or two antibody molecules and no cross-linkages
be to have an antibody with a high affinity, or initial force of at- are formed. In the prozone, usually only one site on an anti-
traction, and a high avidity, or strength of binding. The higher body molecule is used and many free antibody molecules re-
the values are for both of these and the more antigen–antibody main in solution.
complexes that are formed, the more sensitive the test. At the other side of the zone, where there is antigen excess,
the postzone phenomenon occurs in which small aggregates
Precipitation Curve are surrounded by excess antigen. Again, no lattice network is
formed.3 In this case, every available antibody site is bound to
In addition to the affinity and avidity of the antibody involved, a single antigen and no cross-links are formed. Thus, for pre-
precipitation depends on the relative proportions of antigen cipitation reactions to be detectable, they must be carried out
and antibody present. Optimum precipitation occurs in the in the zone of equivalence.
zone of equivalence. The prozone and postzone phenomena must be considered
in the clinical setting because negative reactions occur in both.
A false-negative reaction may take place in the prozone because
Zone of Equivalence of high antibody concentration. If it is suspected that the reac-
In the zone of equivalence, the number of multivalent sites tion is a false negative, diluting out antibody and performing
of antigen and antibody are approximately equal. In this zone, the test again may produce a positive result. In the postzone,
excess antigen may obscure the presence of a small amount of light scattering. The relationship between antigen concentra-
antibody. Typically, such a test is repeated with an additional tion, as indicated by antigen–antibody complex formation, and
patient specimen taken about a week later. The extra time the amount of light scattering will form a straight line if plotted
would allow for the further production of antibody. If the re- on a graph.4 Light scatter may be directly extrapolated by a
peated test is negative, it is unlikely that the patient has that computer to give actual concentrations in milligrams per
particular antibody. deciliter (mg/dL) or international units per milliliter (IU/mL),
based on established values of standards. Nephelometers typ-
ically measure light scatter at angles ranging from 10 degrees
Measurement of Precipitation to about 90 degrees. If a laser beam is used, light deflected only
by Light Scattering a few degrees from the original path can be measured. Al-
though the sensitivity of turbidity has increased, nephelometry
Precipitation is one of the simplest methods of detecting is more sensitive, with a lower limit of detection of 1 to 10 mg/L
antigen–antibody reactions because most antigens are multiva- for serum proteins.3
lent and thus capable of forming aggregates in the presence of Nephelometry can be used to detect either antigen or anti-
the corresponding antibody. When antigen and antibody solu- body, but typically it is run with antibody as the reagent and
tions are mixed, the antigen cross-links with numerous anti- patient antigen as the unknown. Many automated instruments
body molecules and the lattice networks become so large that use a technique called rate nephelometry for the measure-
they precipitate out of solution.5 Precipitates in fluids can be ment of serum proteins. In this instance, the rate of scattering
measured by means of turbidimetry or nephelometry. increase is measured immediately after the reagent antibody is
Turbidimetry is a measure of the turbidity or cloudiness of added. This rate change is directly related to antigen concen-
a solution. A detection device is placed in direct line with an tration if the concentration of antibody is kept constant.4
incident light, collecting the light after it has passed through Quantification of immunoglobulins such as IgG, IgA, IgM, and
the solution. This device measures the reduction in light in- IgE, as well as kappa and lambda light chains, is mainly done
tensity caused by reflection, absorption, or scatter.6 Scattering by rate nephelometry because other methods are more labor
occurs when a beam of light passes through a solution and en- intensive.4 Other serum proteins quantified by this method in-
counters molecules in its path.6 Light then bounces off the clude complement components, C-reactive protein, haptoglo-
molecules and travels in all directions. The amount of scatter bin, and ceruloplasmin.4 Nephelometry provides accurate and
is proportional to the size, shape, and concentration of mole- precise quantitation of serum proteins, and because of automa-
cules present in solution. It is recorded in absorbance units, a tion the cost per test is typically lower than other methods.
measure of the ratio of incident light to that of transmitted Additionally, very small samples can be analyzed.
light. The measurements are made using a spectrophotometer
or an automated clinical chemistry analyzer.
Nephelometry measures the light that is scattered at a par-
Passive Immunodiffusion
ticular angle from the incident beam as it passes through a sus- Techniques
pension6 (Fig. 10–4). The amount of light scattered is an index
of the solution’s concentration. If a solution has excess anti- The precipitation of antigen–antibody complexes can also be
body, adding increasing amounts of antigen results in an in- determined in a support medium such as a gel. Agarose, a pu-
crease in antigen–antibody complexes and thus an increase in rified high-molecular-weight complex polysaccharide derived
from seaweed, is used for this purpose. When antigen and an-
Cuvette tibody diffuse toward one another in a gel matrix, visible lines
of precipitation will form.5 Agarose helps stabilize the diffusion
process and allow visualization of the precipitin bands.
Light detector
turbidimetry Antigen and antibody are added to wells in the gel and
antigen–antibody combination occurs by means of diffusion.
When no electrical current is used to speed up this process, it
is known as passive immunodiffusion. The rate of diffusion
is affected by the size of the particles, the temperature, the gel
viscosity, and the amount of hydration. Immunodiffusion re-
Light source actions can be classified according to the number of reactants
diffusing and the direction of diffusion.

Radial Immunodiffusion
Nephelometry A single-diffusion technique, called radial immunodiffusion
90° light scatter (RID), has been used in the clinical laboratory. In this tech-
Principles of nephelometry. The light detection nique, antibody is uniformly distributed in the support gel and
device is at an angle to the incident light, in contrast to turbidity, antigen is applied to a well cut into the gel. As the antigen dif-
which measures light rays passing directly through the solution. fuses out from the well, antigen– antibody combination occurs
in changing proportions until the zone of equivalence is Ouchterlony Double Diffusion
reached and a stable lattice network is formed in the gel. The
area of the ring obtained is a measure of antigen concentration One of the older, classic immunochemical techniques is
that can be compared with a standard curve obtained by using Ouchterlony double diffusion. In this technique, both anti-
antigens of known concentration.3 Figure 10–5 depicts some gen and antibody diffuse independently through a semisolid
typical results. medium in two dimensions, horizontally and vertically. Wells
One technique for the measurement of radial immunodif- are cut in a gel and reactants are added to the wells. Most
fusion was developed by Mancini and is known as the end- Ouchterlony plates are set up with a central well surrounded
point method. In this technique, antigen is allowed to diffuse by four to six equidistant outer wells. Antibody that is multi-
to completion; when equivalence is reached, there is no further specific is placed in the central well and different antigens are
change in the ring diameter.7 Equivalence occurs between placed in the surrounding wells to determine if the antigens
24 and 72 hours. The square of the diameter is then directly share identical epitopes. Diffusion takes place radially from the
proportional to the concentration of the antigen. A graph is wells. After an incubation period of between 12 and 48 hours
obtained by plotting concentrations of standards on the x axis in a moist chamber, precipitin lines form where the moving
versus the diameter squared on the y axis, creating a smooth front of antigen meets that of antibody and the point of equiv-
curve to fit the points. The major drawback to this method is alence is reached. The density of the lines reflects the amount
the time it takes to obtain results. Another method, the kinetic of immune complex formed.5
or Fahey method, uses ring diameter readings taken at about The position of the precipitin bands between wells allows
19 hours before equivalence is reached.8 The diameter is then for the antigens to be compared with one another. Several pat-
proportional to the log of the concentration and a graph is plot- terns are possible: (1) Fusion of the lines at their junction to
ted using semi-log paper. The diameter is plotted on the x axis form an arc represents serological identity or the presence of a
and the concentration is on the y axis, which automatically common epitope, (2) a pattern of crossed lines demonstrates
gives a log value. Many kits also contain a chart provided by two separate reactions and indicates that the compared anti-
the manufacturer that indicates concentration relative to the gens share no common epitopes, and (3) fusion of two lines
ring diameter. with a spur indicates partial identity. In this last case, the two
The precision of the assay is directly related to accurate mea- antigens share a common epitope, but some antibody mole-
surement of samples and standards. Sources of error include cules are not captured by antigen and travel through the initial
overfilling or underfilling the wells, nicking the side of the wells precipitin line to combine with additional epitopes found in
when filling, spilling sample outside the wells, improper incu- the more complex antigen. Therefore, the spur always points
bation time and temperature, and incorrect measurement. Radial to the simpler antigen9 (Fig. 10–6). Although of more limited
immunodiffusion has been used to measure IgG and IgA sub- use because it is labor intensive and requires experience to
classes as well as complement components. Immunodiffusion is read, Ouchterlony double diffusion is still used to identify fun-
simple to perform and requires no instrumentation, but it has gal antigens such as Aspergillus, Blastomyces, Coccidioides, and
largely been replaced by more sensitive methods such as neph- Candida.9
elometry and enzyme-linked immunoassays.
Electrophoretic Techniques
Std 1 Std 2 Std 3 Unknown Diffusion can be combined with electrophoresis to speed up
or sharpen the results. Electrophoresis separates molecules
Glass slide coated according to differences in their electric charge when they are
with agar containing placed in an electric field. A direct current is forced through
specific antibody
the gel, causing antigen, antibody, or both to migrate. As dif-
fusion takes place, distinct precipitin bands are formed.
Immunoelectrophoresis is a double-diffusion technique that
incorporates electrophoresis to enhance results. Typically, the
precipitin ring (mm)2

30 source of antigen is serum, which is electrophoresed to separate

out the main proteins. A trough is then cut in the gel parallel
Diameter of

25
to the line of separation. Antiserum is placed in the trough and
20 std 3
std 2
the gel is incubated for 18 to 24 hours. Double diffusion occurs
15 std 1
at right angles to the electrophoretic separation and precipitin
lines develop where specific antigen–antibody combination
0 10 20 30 40 50 60
takes place. Interpretation is difficult; therefore, it has largely
Concentration of test reactant (mg/dL) been replaced by immunofixation electrophoresis, which gives
Radial immunodiffusion. The amount of precipitate faster results and is easier to interpret.3
formed is in proportion to the antigen present in the sample. In the Immunofixation electrophoresis, as first described by
Mancini end-point method, concentration is in proportion to the di- Alper and Johnson,10 is similar to immunoelectrophoresis
ameter squared. except that after electrophoresis takes place, antiserum is
 to gamma, alpha, or mu heavy chains and to kappa or lambda
light chains.3 The sixth lane is overlaid with antibody to all
1 1 serum proteins and serves as the reference lane. Reactions in
each of the five lanes are compared with the reference lane. Hy-
pogammaglobulinemias, which are characterized by low anti-
body production, will exhibit faintly staining bands, whereas
Ab polyclonal hypergammaglobulinemias (overproduction of an-
tibody) show darkly staining bands in the gamma region. The
presence of monoclonal antibody, such as is found in certain
A Serological identity malignancies of the immune system, will result in dark and nar-
row bands in specific lanes11 (Fig. 10–7).
Immunofixation electrophoresis is especially useful in
1 2 1 3 demonstrating those antigens present in serum, urine, or spinal
fluid in low concentrations.11,12 This method has great versa-
tility and is easy to perform at a relatively low cost, but it re-
Ab Ab quires more manual manipulation than other methods.12

Comparison of Precipitation
B Nonidentity C Partial identity Techniques
Antibody Key Each type of precipitation technique has its own distinct ad-
Reacts with 1
vantages and disadvantages. Some techniques are technically
more demanding, whereas others are more automated. Each
Reacts with 2
type of precipitation testing has particular applications for
Reacts with 1 and 3 which it is best suited. Table 10–1 presents a comparison of
the precipitation techniques discussed in this chapter.
Ouchterlony diffusion patterns. An antibody mixture
is placed in the central well. Unknown antigens are placed in the
outside wells. The antibodies and antigens all diffuse radially out of Principles of Agglutination Reactions
the wells. (A) Serological identity. If the antigens are identical, they
will react with the same antibody and the precipitate line forms a Whereas precipitation reactions involve soluble antigens, ag-
continuous arc. (B) Nonidentity. If the antigens share no identical glutination is the visible aggregation of particles caused by
determinants, they will react with different antibodies and two combination with specific antibody. Antibodies that produce
crossed lines are formed. (C) If antigen 3 has a determinant in such reactions are often called agglutinins. Because this reac-
common with antigen 1, one of the antibodies reacts with both tion takes place on the surface of the particle, antigen must be
antigens. Another antibody that reacts with different determinants
exposed and able to bind with antibody. Types of particles par-
on antigen 1 (absent on antigen 3) passes through one precipitation
ticipating in such reactions include erythrocytes, bacterial cells,
line and forms the spur on the other line. The spur formed always
points to the simpler antigen with fewer antigenic determinants. and inert carriers such as latex particles. Each particle must

applied directly to the gel’s surface rather than placed in a

trough. Agarose or cellulose acetate can be used for this pur-
pose. Immunodiffusion takes place in a shorter time and re-
sults in a higher resolution than when antibody diffuses from
a trough. Because diffusion is only across the thickness of
the gel, approximately 1 mm, the reaction usually takes place
in less than 1 hour.
Most often, an antibody of known specificity is used to de-
termine whether patient antigen is present. The unknown anti-
gen is placed on the gel, electrophoretic separation takes place, Immunofixation electrophoresis. A complex antigen
mixture such as serum proteins is separated by electrophoresis. An
and then the reagent antibody is applied. Immunoprecipitates
antiserum template is aligned over the gel. Then protein fixative and
form only where specific antigen–antibody combination has
monospecific antisera, IgG, IgA, IgM, κ, and λ are applied to the gel.
taken place and the complexes have become trapped in the gel. After incubating for 30 minutes, the gel is stained and examined
The gel is washed to remove any nonprecipitating proteins and for the presence of paraproteins. Precipitates form where specific
can then be stained for easier visibility. Typically, patient serum antigen–antibody combination has taken place. In this case, the
is applied to six lanes of the gel; after electrophoresis, five lanes patient has an IgG monoclonal antibody with λ chains. (Courtesy of
are overlaid with one each of the following antibodies: antibody Helena Laboratories, Beaumont, TX.)
 Table 10–1 Comparison of Precipitation Techniques
SENSITIVITY
TECHNIQUE APPLICATION ("G AB/ML) PRINCIPLE
Nephelometry Immunoglobulins, comple- 1–10 Light that is scattered at an angle is mea-
ment, C-reactive protein, sured, indicating the amount of antigen
other serum proteins or antibody present.
Radial immunodiffusion Immunoglobulins, 10–50 Antigen diffuses out into gel that is infused
complement with antibody. Measurement of the radius
indicates concentration of antigen.
Ouchterlony double Complex antigens such as 20–200 Both antigen and antibody diffuse out from
diffusion fungal antigens wells in a gel. Lines of precipitate formed
indicate the relationship of antigens.
Immunoelectrophoresis Differentiation of serum 20–200 Electrophoresis of serum followed by
proteins diffusion of antibody from wells.
Immunofixation Over- or underproduction Variable Electrophoresis of serum followed by
electrophoresis of antibody direct application of antibody to the gel.

have multiple antigenic or determinant sites, which are cross- and is rapid and reversible.14 The second step, or lattice for-
linked to sites on other particles through the formation of mation, is the formation of cross-links that form the visible ag-
antibody bridges.4 gregates. This represents the stabilization of antigen–antibody
In 1896, Gruber and Durham published the first report complexes with the binding together of multiple antigenic
about the ability of antibody to clump cells, based on observa- determinants14 (Fig. 10–8).
tions of agglutination of bacterial cells by serum.13 This finding Sensitization is affected by the nature of the antigens on the
gave rise to the use of serology as a tool in the diagnosis of dis- agglutinating particles. If epitopes are sparse or if they are ob-
ease and also led to the discovery of the ABO blood groups. scured by other surface molecules, they are less likely to inter-
Widal and Sicard developed one of the earliest diagnostic tests act with antibody. Additionally, red blood cells (RBCs) and
in 1896 for the detection of antibodies occurring in typhoid bacterial cells have a slight negative surface charge; because
fever, brucellosis, and tularemia. Agglutination reactions now like charges tend to repel one another, it is sometimes difficult
have a wide variety of applications in the detection of both anti- to bring such cells together into a lattice formation.4
gens and antibodies. Such testing is simple to perform and the The class of immunoglobulin is also important; IgM with a
end points can easily be read visually. potential valence of 10 is over 700 times more efficient in ag-
Agglutination, like precipitation, is a two-step process that glutination than is IgG with a valence of 2.4 (See Fig. 10–2 for
results in the formation of a stable lattice network. The first re- a comparison of IgG versus IgM.) Antibodies of the IgG class
action, called sensitization, involves antigen–antibody com- often cannot bridge the distance between particles because
bination through single antigenic determinants on the particle their small size and restricted flexibility at the hinge region may

Antibody Antigen with Sensitization Lattice formation

multiple determinants (no visible reaction) (visible agglutination)
Phases of agglutination. Sensitization: Antigen and antibody unite through antigenic determinant sites. Lattice formation:
Rearrangement of antigen and antibody bonds to form a stable lattice.
prohibit multivalent binding.14 IgM antibodies, on the other employed serological test; they can be used to identify either
hand, are strong agglutinins because of their larger size. antigen or antibody. Typically, most agglutination tests are qual-
Achieving visible reactions with IgG often requires the use itative, simply indicating the absence or presence of antigen or
of enhancement techniques that vary physicochemical condi- antibody, but dilutions can be made to obtain semiquantitative
tions such as the ionic strength of the solution, the pH, and results. Many variations exist that can be categorized according
the temperature. Antibodies belonging to the IgG class agglu- to the type of particle used in the reaction and whether antigen
tinate best at 30°C to 37°C, whereas IgM antibodies react best or antibody is attached to it.
at temperatures between 4°C and 27°C. Because naturally oc-
curring antibodies against the ABO blood groups belong to the Direct Agglutination
IgM class, these reactions are best run at room temperature.
Antibodies to other human blood groups usually belong to the Direct agglutination occurs when antigens are found naturally
IgG class; reactions involving these must be run at 37°C. These on a particle. One of the best examples of direct agglutination
latter reactions are the most important to consider in selecting testing involves known bacterial antigens used to test for the
compatible blood for a transfusion because these are the ones presence of unknown antibodies in the patient. Typically, pa-
that will actually occur in the body. tient serum is diluted into a series of tubes or wells on a slide
In addition to temperature considerations, detection of IgG and reacted with bacterial antigens specific for the suspected
antibodies often requires the use of a second antibody, anti- disease. Detection of antibodies is primarily used in diagnosis
human immunoglobulin, to visualize a reaction. Anti-human of diseases for which the bacterial agents are extremely difficult
immunoglobulin is also known as Coombs reagent and is used to cultivate. One such example is the Widal test, a rapid screen-
frequently in blood bank testing. Coombs reagent will attach to ing test used to help determine the possibility of typhoid fever.
the Fc portion of IgG and help to bridge the gap between RBCs A significant finding is a fourfold increase in antibody titer over
so a visible agglutination reaction will occur. Figure 10–9 time when paired dilutions of serum samples are tested with
demonstrates how Coombs reagent works. any of these antigens. Although more specific tests are now
available, the Widal test is still considered useful in diagnosing
typhoid fever in developing countries and remains in use in
Types of Agglutination Reactions many areas throughout the world.15
If an agglutination reaction involves RBCs, then it is called
Agglutination reactions are easy to carry out, require no com- hemagglutination. The best example of this occurs in ABO
plicated equipment, and can be performed as needed in the blood group typing of human RBCs, one of the world’s most
laboratory without having to batch specimens. Batching spec- frequently used immunoassays.5 Patient RBCs mixed with an-
imens is done if a test is expensive or complicated; in this case, tisera of the IgM type can be used to determine the presence
a large number are run at one time, which may result in a time or absence of the A and B antigens; this reaction is usually per-
delay. Many kits are available for standard testing, so reagent formed at room temperature without the need for any enhance-
preparation is minimal. Agglutination reactions are a frequently ment techniques. Group A RBCs will agglutinate with anti-A
antibody and Group B RBCs will agglutinate with anti-B anti-
body. This type of agglutination reaction is simple to perform,
is relatively sensitive, and is easy to read (Fig. 10–10).
A titer that yields semiquantitative results can be performed
in test tubes or microtiter plates by making serial dilutions of
the antibody. The reciprocal of the last dilution still exhibiting
a visible reaction is the titer, indicating the antibody’s strength.
Interpretation of the test is done on the basis of the cell sedi-
mentation pattern. If there is a dark red, smooth button at the
! bottom of the microtiter well, the result is negative. A positive
result will have cells that are spread across the well’s bottom,
usually in a jagged pattern with an irregular edge. Test tubes
also can be centrifuged and then shaken to see if the cell button
Add reagent can be evenly resuspended. If it is resuspended with no visible
antibody
(antihuman IgG)
clumping, then the result is negative. Positive reactions can be
graded to indicate the strength of the reaction (Fig. 10–11).

Antibody-coated Agglutination Passive Agglutination

patient cells
Coombs reagent. Coombs reagent is anti-human Passive, or indirect, agglutination employs particles that are
immunoglobulin used to enhance agglutination reactions by coated with antigens not normally found on their surfaces. A va-
attaching to the Fc portion of IgG found on antibody-coated RBCs. riety of particles, including erythrocytes, latex, and gelatin, are
Coombs reagent helps to bridge the gap between RBCs so a visible used for passive agglutination.4 The use of synthetic beads or
agglutination reaction will occur. particles provides the advantages of consistency and uniformity.
 antibodies to viruses such as rotavirus, cytomegalovirus,
rubella, and varicella-zoster.4 Hemagglutination kits are avail-
able for detection of antibodies to hepatitis B virus (HBV), hep-
atitis C virus (HCV), and human immunodeficiency virus
(HIV) I and II, to cite just a few examples.2
Because many of these kits are designed to detect IgM anti-
body and there is always the risk of nonspecific agglutination
caused by the presence of other IgM antibodies, reactions must
be carefully controlled and interpreted. Commercial tests are
usually performed on disposable plastic, cardboard cards, or
glass slides. Kits contain positive and negative controls; if the
controls do not give the expected results, the test is not valid.
Such tests are typically used as screening tools, which are fol-
lowed by more extensive testing if the results are positive.

Reverse Passive Agglutination

In reverse passive agglutination, antibody rather than antigen
is attached to a carrier particle. The antibody must still be re-
Red blood cell agglutination. The tube on the left active and is joined in such a manner that the active sites are
is a positive test for RBC agglutination, whereas the tube on the facing outward. Adsorption may be spontaneous, or it may re-
right is a negative test showing that the RBCs have remained in a quire some of the same manipulation as is used for antigen at-
smooth suspension. tachment. This type of testing is often used to detect microbial
antigens. Figure 10–12 shows the differences between passive
Reactions are easy to read visually and give quick results. and reverse passive agglutination.
Many antigens, especially polysaccharides, adsorb to RBCs Numerous kits are available for the rapid identification of
spontaneously, so they are also relatively easy to manipulate. antigens from such infectious agents as Group B Streptococ-
Particle sizes vary from 7 µm for RBCs down to 0.8 µm for cus, Staphylococcus aureus, streptococcal groups A and B, ro-
fine latex particles.16 tavirus, and Cryptococcus neoformans.16 Rapid agglutination
In 1955, Singer and Plotz found by happenstance that IgG tests have found the widest application in detecting soluble
was naturally adsorbed to the surface of polystyrene latex par- antigens in urine, spinal fluid, and serum.8 The principle is
ticles. Latex particles are inexpensive, are relatively stable, and the same for all these tests: Latex particles coated with anti-
are not subject to cross-reactivity with other antibodies. A large body are reacted with a patient sample containing the sus-
number of antibody molecules can be bound to the surface of pected antigen. In some cases, an extraction step is necessary
latex particles, so the number of antigen-binding sites is large.16 to isolate antigen before the reagent latex particles are added.
Additionally, the large particle size facilitates reading of the test. Organisms can be identified in a few minutes with fairly high
Latex agglutination tests have been used to detect rheuma- sensitivity and specificity, although this varies for different
toid factor, antibodies to Group A Streptococcus antigens, and organisms. The use of monoclonal antibodies has greatly cut

Shake

Red cell 4+ 3+ 2+ 1+ Negative

button after One solid Several Numerous Barely smooth
centrifugation clump large smaller discernable suspension
A clumps clumps clumps

Grading of agglutina-
tion reactions: A. tube method. If
tubes are centrifuged and shaken to
resuspend the button, reactions can
Antigen and antibody Strong agglutination— Weak agglutination— Negative—even
be graded from negative to 4+, are mixed and rotated large clumps and small clumps and suspension and
depending on the size of clumps clear background cloudy background cloudy background
observed. B. Rapid slide method. B
 A is specific for the hapten being tested. Indicator particles that
contain the same hapten one wishes to measure in the patient
are then added. If the patient sample has no free hapten, the
reagent antibody is able to combine with the carrier particles
and produce a visible agglutination. In this case, however, ag-
glutination is a negative reaction, indicating that the patient
! did not have sufficient hapten to inhibit the secondary reaction
(Fig. 10–13). Either antigen or antibody can be attached to
the particles. The sensitivity of the reaction is governed by the
avidity of the antibody itself. It can be a sensitive assay capable
of detecting small quantities of antigen. Tests used to detect il-
Carrier particles Coated Patient Agglutination
licit drugs such as cocaine or heroin are examples of aggluti-
mixed with particles sample nation inhibition tests.
soluble antigen Hemagglutination inhibition reactions use the same prin-
ciple, except RBCs are the indicator particles. This type of test-
B
ing has been used to detect antibodies to certain viruses, such
as rubella, influenza, and respiratory syncytial virus (RSV).5,9
RBCs have naturally occurring viral receptors. When virus is
present, spontaneous agglutination occurs because the virus
particles link the RBCs together. Presence of patient antibody
! inhibits the agglutination reaction (Fig. 10–14).
To perform a hemagglutination inhibition test, dilutions of
patient serum are incubated with a viral preparation. Then
RBCs that the virus is known to agglutinate are added to the
mixture. If antibody is present, it will attach to the viral parti-
cles and prevent agglutination, so a lack of or reduction in ag-
Carrier particles Coated Patient Agglutination glutination indicates the presence of patient antibody.5
mixed with particles sample
reagent antibody
Controls are necessary because there may be a factor in the
serum that causes agglutination or the virus may have lost its
Passive and reverse passive agglutination. (A) Pas-
ability to agglutinate.
sive agglutination. Antigen is attached to the carrier particle; aggluti-
nation occurs if patient antibody is present. (B) Reverse passive
agglutination. Antibody is attached to the carrier particle; agglutina- Instrumentation
tion occurs if patient antigen is present.
Although agglutination reactions require no complicated in-
down on cross-reactivity, but there is still the possibility of strumentation to read, several chemistry analyzers have been
interference or nonspecific agglutination. developed using automation to increase sensitivity.2 Neph-
Such tests are most often used for organisms that are diffi- elometry has been applied to the reading of agglutination re-
cult to grow in the laboratory or for instances when rapid iden- actions and the term particle-enhanced immunoassay is used to
tification will allow treatment to be initiated more promptly. describe such reactions. When particles are used, the sensitivity
Direct testing of specimens for the presence of viral antigens can be increased to nanograms/mL.2 For this type of reaction,
has still not reached the sensitivity of enzyme immunoassays; small latex particles with a diameter of smaller than 1 µm
however, for infections in which a large amount of viral antigen are used. One such type of instrumentation system is called
is present, such as rotavirus and enteric adenovirus in infants, particle-counting immunoassay (PACIA).
latex agglutination tests are very useful. Reverse passive agglu- PACIA involves measuring the number of residual nonag-
tination testing has also been used to measure levels of certain glutinating particles in a specimen. These particles are counted
therapeutic drugs, hormones, and plasma proteins such as by means of a laser beam in an optical particle counter similar
haptoglobin and C-reactive protein. to the one that is designed to count blood cells. Nephelometric
methods are used to measure forward light scatter. Very large
and very small particles are excluded by setting certain thresh-
Agglutination Inhibition olds on the instrument.
Agglutination inhibition reactions are based on competition Latex particles are coated with whole antibody molecules
between particulate and soluble antigens for limited antibody- or with F(ab')2 fragments. Use of the latter reduces interference
combining sites. A lack of agglutination is an indicator of a and nonspecific agglutination. If antigen is present, complexes
positive reaction. Typically, this type of reaction involves hap- will form and will be screened out by the counter because of
tens that are complexed to proteins; the hapten–protein con- their large size. An inverse relationship exists between the
jugate is then attached to a carrier particle. The patient sample number of unagglutinated particles counted and the amount
is first reacted with a limited amount of reagent antibody that of unknown antigen in the patient specimen. Measurements
 ! !

Antigen Yes No: positive test

Patient Add reagent Immune Add antigen- Agglutination

sample antibody complexes form coated particles

No antigen No Yes: negative test

! !

Agglutination inhibition. Reagent antibody is added to the patient sample. If patient antigen is present, antigen–antibody
combination results. When antigen-coated latex particles are added, no agglutination occurs, which is a positive test. If no patient antigen is
there, the reagent antibody combines with latex particles and agglutination results, which is a negative test.

are made by looking at the rate at which the number of unag- Other considerations include proper storage of reagents and
glutinated particles decrease, called a rate assay, or the total close attention to expiration dates. Reagents should never be
number of unagglutinated particles left at the end, known as used beyond the expiration date. Each new lot should be eval-
an end-point assay.2,3 PACIAs have been used to measure several uated before use and the manufacturer’s instructions for each
serum proteins, therapeutic drugs, tumor markers, and certain kit should always be followed. The sensitivity and specificity
viral antigens. of different kits may vary and thus must be taken into account.
Advantages of agglutination reactions include rapidity;
relative sensitivity; and the fact that if the sample contains a
Quality Control and Quality microorganism, the organism does not need to be viable. In
Assurance addition, most tests are simple to perform and require no ex-
pensive equipment. Tests are conducted on cards, tubes, and
Although agglutination reactions are simple to perform, inter- microtiter plates, all of which are extremely portable. A wide
pretation must be carefully done. Techniques must be stan- variety of antigens and antibodies can be tested for in this man-
dardized regarding the concentration of antigen, incubation ner. It must be kept in mind, however, that agglutination tests
time, temperature, diluent, and the method of reading. The are screening tools only and that a negative result does not rule
possibility of cross-reactivity and interfering antibody should out presence of the disease or the antigen. The quantity of anti-
always be considered. Cross-reactivity is caused by the pres- gen or antibody may be below the sensitivity of the test system.
ence of antigenic determinants that resemble one another so Although the number of agglutination tests have decreased in
closely that antibody formed against one will react with the recent years, they continue to play an important role in the
other. Most cross-reactivity can be avoided through the use of identification of rare pathogens such as Francisella and Brucella
monoclonal antibody directed against an antigenic determinant and more common organisms such as rotavirus and Cryptococ-
that is unique to a particular antigen. cus, for which other testing is complex or unavailable.
 ! !

Antibody Yes No: positive test

Patient Add viral Immune Add red Agglutination

sample antigen complexes form blood cells

No antibody No Yes: negative test

! !

Hemagglutination inhibition. In the presence of certain viruses, RBCs spontaneously agglutinate. However, if patient antibody
is present, then agglutination is inhibited. Thus, a lack of agglutination indicates the presence of antibody.

SUMMARY combination such as precipitation and agglutination are

present to a much lesser degree.
• Precipitation involves the combination of soluble antigen • If light scatter produced by immune complexes in solution
with soluble antibody to produce insoluble complexes that is measured as a reduction in light intensity, this is called
are visible. turbidimetry.
• Union of antigen and antibody depends on affinity, or the • Nephelometry is the technique that measures the amount
force of attraction that exists between one antibody-binding of light scattered at a particular angle. Several automated
site and a single epitope on an antigen. instruments are based on these principles.
• Avidity is the sum of all attractive forces occurring between • When precipitation reactions take place in a gel, this is
multiple binding sites on antigen and antibody. known as passive diffusion. In single diffusion, only one
• Maximum binding of antigen and antibody occurs when of the reactants travels, whereas the other is incorporated
the aggregate number of multivalent sites of antigen and in the gel.
antibody are approximately equal. • In radial immunodiffusion, antibody is incorporated in a
• The concentrations of antigen and antibody that yield gel. Antigen is placed in wells in the gel and diffuses out.
maximum binding represent the zone of equivalence. This The amount of precipitate formed is directly related to the
is where the multivalent sites of antigen and antibody are amount of antigen present.
approximately equal. • In Ouchterlony diffusion, both antigen and antibody dif-
• In the prozone, when antibody is being tested for against fuse from wells and travel toward each other. Precipitin
a standard concentration of antigen, antibody is in excess lines may indicate identity, nonidentity, or partial identity,
and precipitation or agglutination cannot be detected. depending on the pattern formed.
• In the postzone, antibody has been diluted out and anti- • In immunofixation electrophoresis, antibody is applied di-
gen is in excess so that manifestations of antigen–antibody rectly to the gel after electrophoresis of antigens has taken
 place. Compared with immunoelectrophoresis, precipita- • Reverse passive agglutination is so called because antibody
tion occurs in a shorter time and bands with higher reso- is attached to the indicator particle.
lution are obtained. • Agglutination inhibition is based on competition between
• The process of agglutination can be divided into two steps: antigen-coated particles and soluble patient antigen for a
(1) sensitization or initial binding, which depends on the limited number of antibody sites. It is the only instance
nature of the antibody and the antigen-bearing surface, in which agglutination represents a negative test.
and (2) lattice formation, which is governed by such • PACIA looks at residual nonagglutinating particles by
factors as pH, ionic strength, and temperature. means of nephelometry. As agglutination occurs, clumps
• Because of its larger size, IgM is usually able to effect lattice of antigens increase in size; these large clumps are not
formation without additional enhancement, whereas for counted. The amount of unknown antigen in a patient
IgG measures are necessary to see a visible reaction. specimen is therefore indirectly proportional to the num-
• In direct agglutination, antigens are found naturally on ber of unagglutinated particles.
the indicator particle. • Agglutination reactions are typically used as screening
• In passive agglutination, antigens are artificially attached tests; they are fast and sensitive and can yield valuable
to such a particle. information when interpreted correctly.

Study Guide: Comparison of Agglutination Reactions

TYPE OF REACTION PRINCIPLE RESULTS
Direct agglutination Antigen is naturally found on a particle. Agglutination indicates the presence of
patient antibody.
Indirect (passive) agglutination Particles coated with antigens not Agglutination indicates the presence of
normally found on their surfaces. patient antibody.
Reverse passive Particles are coated with reagent Agglutination indicates the presence of
antibody. patient antibody.
Agglutination inhibition Haptens attached to carrier particles. Lack of agglutination is a positive test,
Particles compete with patient antigens indicating the presence of patient antigen.
for a limited number of antibody sites.
Hemagglutination inhibition Red blood cells spontaneously agglutinate Lack of agglutination is a positive test,
if viral particles are present. indicating the presence of patient antibody.

CASE STUDIES
1. A 4-year-old female was hospitalized for pneumonia. c. How do nephelometry measurements compare with
She has had a history of upper-respiratory tract infec- the use of RID?
tions and several bouts of diarrhea since infancy. 2. A 25-year-old female who was 2 months pregnant went
Because of her recurring infections, the physician to her physician for a prenatal workup. She had been
decided to measure her immunoglobulin levels. The vaccinated against rubella, but her titer was never
following results were obtained by nephelometry: established. She was concerned because a friend of hers
who had never been vaccinated for rubella thought she
NORMAL might have the disease. The patient had been on an
LEVEL PATIENT all-day shopping trip with her friend 2 days before she
(3–5 YRS) LEVEL
saw her doctor. The physician ordered a latex aggluti-
IMMUNOGLOBULIN (MG/DL) (MG/DL)
nation test to screen for rubella as a part of the prenatal
IgG 550–1,700 800 workup. The results on an undiluted serum specimen
IgA 50–280 20 were positive, indicating that at least 10 IU/mL of
antibody was present.
IgM 25–120 75
Questions
Questions a. What does the positive rubella test indicate?
a. What do these results indicate? b. How should this be interpreted in the light of the
b. How do they explain the symptoms? (You may want patient’s condition?
to refer back to Chapter 5 for a discussion of the
function of different classes of antibody.)
REVIEW QUESTIONS
1. In a precipitation reaction, how can the ideal antibody 7. How does measurement of turbidity differ from
be characterized? nephelometry?
a. Low affinity and low avidity a. Turbidity measures the increase in light after it
b. High affinity and low avidity passes through a solution.
c. High affinity and high avidity b. Nephelometry measures light that is scattered at
d. Low affinity and high avidity an angle.
c. Turbidity deals with univalent antigens only.
2. Precipitation differs from agglutination in d. Nephelometry is not affected by large particles
which way? falling out of solution.
a. Precipitation can only be measured by an automated
instrument. 8. Which of the following refers to the force of attraction
b. Precipitation occurs with univalent antigen, between an antibody and a single antigenic determinant?
whereas agglutination requires multivalent a. Affinity
antigen. b. Avidity
c. Precipitation does not readily occur because c. Van der Waals attraction
few antibodies can form aggregates with d. Covalence
antigen.
d. Precipitation involves a soluble antigen, whereas 9. Immunofixation electrophoresis differs from
agglutination involves a particulate antigen. immunoelectrophoresis in which way?
a. Electrophoresis takes place after diffusion has
3. When soluble antigens diffuse in a gel that contains occurred in immunofixation electrophoresis.
antibody, in which zone does optimum precipitation b. Better separation of proteins with the same
occur? electrophoretic mobilities is obtained in
a. Prozone immunoelectrophoresis.
b. Zone of equivalence c. In immunofixation electrophoresis, antibody is
c. Postzone directly applied to the gel instead of being placed
d. Prezone in a trough.
d. Immunoelectrophoresis is a much faster procedure.
4. Which of the following statements apply to rate
nephelometry? 10. If crossed lines result in an Ouchterlony immunodiffu-
a. Readings are taken before equivalence is sion reaction with antigens 1 and 2, what does this
reached. indicate?
b. It is more sensitive than turbidity. a. Antigens 1 and 2 are identical.
c. Measurements are time dependent. b. Antigen 2 is simpler than antigen 1.
d. All of the above. c. Antigen 2 is more complex than antigen 1.
d. The two antigens are unrelated.
5. Which of the following is characteristic of the
end-point method of RID? 11. Which technique represents a single-diffusion reaction?
a. Readings are taken before equivalence. a. Radial immunodiffusion
b. Concentration is directly in proportion to the b. Ouchterlony diffusion
square of the diameter. c. Immunoelectrophoresis
c. The diameter is plotted against the log of the d. Immunofixation electrophoresis
concentration.
d. It is primarily a qualitative rather than a 12. Which best describes the law of mass action?
quantitative method. a. Once antigen–antibody binding takes place, it is
irreversible.
6. In which zone might an antibody-screening test be b. The equilibrium constant depends only on the
false negative? forward reaction.
a. Prozone c. The equilibrium constant is related to strength of
b. Zone of equivalence antigen–antibody binding.
c. Postzone d. If an antibody has a high avidity, it will dissociate
d. None of the above from antigen easily.
13. Agglutination of dyed bacterial cells represents which 17. Reactions involving IgG may need to be enhanced for
type of reaction? which reason?
a. Direct agglutination a. It is only active at 25°C.
b. Passive agglutination b. It may be too small to produce lattice formation.
c. Reverse passive agglutination c. It has only one antigen-binding site.
d. Agglutination inhibition d. It is only able to produce visible precipitation
reactions.
14. If a single IgM molecule can bind many more antigens
than a molecule of IgG, which of the following is 18. For which of the following tests is a lack of agglutination
higher? a positive reaction?
a. Affinity a. Hemagglutination
b. Initial force of attraction b. Passive agglutination
c. Avidity c. Reverse passive agglutination
d. Initial sensitization d. Agglutination inhibition

15. Agglutination inhibition could best be used for which 19. Typing of RBCs with reagent antiserum represents
of the following types of antigens? which type of reaction?
a. Large cellular antigens such as erythrocytes a. Direct hemagglutination
b. Soluble haptens b. Passive hemagglutination
c. Bacterial cells c. Hemagglutination inhibition
d. Coated latex particles d. Reverse passive hemagglutination

16. Which of the following correctly describes reverse 20. In a particle-counting immunoassay using reagent an-
passive agglutination? tibody attached to latex particles, if the particle count
a. It is a negative test. in solution is very low, what does this mean about the
b. It can be used to detect autoantibodies. presence of patient antigen?
c. It is used for identification of antigens. a. The patient has no antigen present.
d. It is used to detect sensitization of red blood b. The patient has a very small amount of antigen.
cells. c. The patient has a large amount of antigen present.
d. The test is invalid.
 Labeled
Immunoassays

After finishing this chapter, you should be able to: FORMATS FOR LABELED ASSAYS
1. Describe the difference between competitive and noncompetitive Competitive Immunoassays
immunoassays. Noncompetitive Immunoassays
2. Distinguish between heterogeneous and homogeneous immunoassays. HETEROGENEOUS VERSUS
3. Explain how the principle of competitive binding is used in radioim- HOMOGENEOUS ASSAYS
munoassays. RADIOIMMUNOASSAY
4. Discuss criteria for selection of an enzyme for enzyme immunoassay. ENZYME IMMUNOASSAYS
5. Explain the principle of sandwich or capture immunoassays. Heterogeneous Enzyme
6. Describe applications for homogeneous enzyme immunoassay. Immunoassays
7. Describe uses for rapid immunoassays. Homogeneous Enzyme
Immunoassays
8. Compare and contrast enzyme immunoassay and radioimmunoassay
regarding ease of performance, sensitivity, and clinical application. Rapid Immunoassays
9. Describe the difference between direct and indirect immunofluores- FLUORESCENT IMMUNOASSAYS
cence techniques. Direct Immunofluorescent Assays
10. Relate the principle of fluorescence polarization immunoassay. Indirect Immunofluorescent Assays
11. Explain how chemiluminescent assays are used to identify analytes. Fluorescence Polarization
12. Discuss advantages and disadvantages of each type of immunoassay. Immunoassays
13. Choose an appropriate immunoassay for a particular analyte. CHEMILUMINESCENT IMMUNOASSAYS
SUMMARY
CASE STUDY
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Analyte Enzyme-linked Homogeneous enzyme Noncompetitive
Capture assays immunosorbent assays immunoassay immunoassay
(ELISA) Immunochromatography Radioimmunoassay (RIA)
Chemiluminescent
immunoassay Fluorescence polarization Immunofluorescent Sandwich immunoassays
immunoassay (FPIA) assay
Competitive immunoassay
Heterogeneous enzyme Indirect immunofluorescent
Direct immunofluorescent
immunoassay assays
assay

Unlabeled immunoassays, such as the precipitation and ag- to determine the amount of patient antigen present. If patient
glutination reactions that were discussed in Chapter 10, are antigen is present, some of the binding sites will be filled with
fairly simple techniques to perform and require little in the unlabeled analyte, thus decreasing the amount of bound label
way of sophisticated equipment. However, they are relatively (Fig. 11–1). Therefore, the amount of bound label is inversely
insensitive because they rely on a high enough concentration proportional to the concentration of the labeled antigen, which
of the unknown to visualize the reaction. In contrast, labeled means that the more labeled antigen that is detected, the less
immunoassays are designed for antigens and antibodies that there is of patient antigen. This ratio can be illustrated by the
may be small in size or present in very low concentrations. following equation:
The presence of such antigens or antibodies is determined 6Ag* + 2Ag + 4Ab → 3Ag*Ab + 1AgAb + 3Ag* + 1Ag
indirectly by using a labeled reactant to detect whether or
In this example, labeled and unlabeled antigens occur in a
not specific binding has taken place.
3:1 ratio. Binding to a limited number of antibody sites will
The substance to be measured, often called the analyte, typ-
take place in the same ratio. Thus, on the right side of the equa-
ically is a protein. Examples include bacterial antigens, hor-
tion, three of the four binding sites are occupied by labeled
mones, drugs, tumor markers, specific immunoglobulins, and
antigen, whereas one site is filled by unlabeled antigen. As the
many other substances. Analytes are bound to molecules that
amount of patient antigen increases, fewer binding sites will
react specifically with them. Typically, this is antibody. One
be occupied by labeled antigen, as demonstrated by the next
reactant, either the antigen or the antibody, is labeled with a
equation:
marker so that the amount of binding can be monitored. The
sensitivity and specificity of the antibody used is the key to 6Ag* + 18Ag + 4Ab →1Ag*Ab + 3AgAb + 5Ag*+ 15Ag
successful results. In this case, the ratio of labeled to unlabeled antigen is
The development of rapid, specific, and sensitive assays to 1:3. Binding to antibody sites takes place in the same ratio
determine the presence of important biologically active mole- and the amount of bound label is greatly decreased in com-
cules ushered in a new era of testing in the clinical laboratory. parison to the first equation. A standard curve using known
Labeled immunoassays have made possible rapid quantitative amounts of unlabeled antigen can be used to extrapolate the
measurement of many important entities such as viral antigens concentration of the unknown patient antigen.1 The detec-
in patients infected with HIV. This ability to detect very small tion limits of competitive assays are largely determined by
quantities of antigen or antibody has revolutionized the diag- the affinity of the antibody.2 The higher the affinity of the
nosis and monitoring of numerous diseases and has led to antibody for the antigen, the more sensitive the assay will be.
more prompt treatment for many such conditions. Refer back to Chapter 10 for a discussion of how affinity
affects antigen–antibody binding.
Formats for Labeled Assays
Noncompetitive Immunoassays
Current techniques include the use of fluorescent, radioactive, In a typical noncompetitive immunoassay, antibody, often
chemiluminescent, and enzyme labels. The underlying princi- called capture antibody, is first passively absorbed to a solid
ples of all these techniques are essentially the same. There phase such as microtiter plates, nitrocellulose membranes, or
are two major formats for all labeled assays: competitive and plastic beads.2 Excess antibody is present so that any patient
noncompetitive. antigen present can be captured. Unknown patient antigen is
then allowed to react with and be captured by the solid-phase
Competitive Immunoassays antibody. After washing to remove unbound antigen, a second
In a competitive immunoassay, all the reactants are mixed to- antibody with a label is added to the reaction (Fig. 11–2). In
gether simultaneously; labeled antigen competes with unla- this case, the amount of label measured is directly proportional
beled patient antigen for a limited number of antibody-binding to the amount of patient antigen. This type of assay is more
sites. The concentration of the labeled analyte is in excess, sensitive than competitive immunoassays.
so all binding sites on the antibody will be occupied. After In both types of assays, the label must not alter the reactivity
separation, the amount of bound label is measured and used of the molecule and should remain stable for the reagent’s shelf
 Sample A Sample B

1 1. Unknown concentration of analyte in patient

sample (red dots) competes with labeled analyte
(yellow stars) for binding sites on immobilized
antibody. Sample A is a negative control.

2. Wash to remove unbound materials.

3. After substrate is added, a colored product

(signal) is generated with an intensity proportional
to the amount of enzyme-labeled analyte bound
to the antibody.

The signal strength is usually inversely related to

the analyte concentration.

Signal
Analyte
concentration
3

Principle of a competitive immunoassay.

life. Techniques using each different type of label will be dis- antigen occurs. Typically, homogeneous assays involve an en-
cussed in a separate section. zyme label, chosen so that the enzyme is inactivated when
bound to an antibody. This type of assay is simpler to perform
because there is no washing step. The sample containing
Heterogeneous Versus patient antigen is incubated with the labeled antigen and the
Homogeneous Assays antibody; the amount of activity then can be measured directly
(Fig. 11–3).1 Homogeneous assays are less sensitive than het-
Immunoassays can also be categorized according to whether erogeneous assays, however. Immunoassays using different
or not it is necessary to separate the bound reactants from the types of labels will now be discussed according to these two
free ones. Heterogeneous enzyme immunoassays require a categories.
step to physically separate free from bound analyte. Antigen
or antibody is attached by physical adsorption; when specific
binding takes place, complexes remain attached to the solid Radioimmunoassay
phase. This step provides a simple way to separate bound and
free reactants. The sample is then thoroughly washed and the The first type of immunoassay developed was radioimmunoas-
remaining activity is determined. say (RIA), pioneered by Yalow and Berson in the late 1950s. It
Homogeneous enzyme immunoassays, on the other hand, was used to determine the level of insulin–anti-insulin com-
do not need a separation step. The activity of the label attached plexes in diabetic patients.3,4 The technique proved to be valu-
to the antigen is diminished when binding of antibody and able in measuring a number of substances such as hormones,
 Sample A Sample B Antibody capture

1
1. For Sample B, immobilized reagent
antibodies capture analyte in patient
sample (red dots) while in the Ab capture
technique, immobilized reagent antigen
captures patient antibodies. Sample A is
a negative control.

2. Wash to remove unbound materials.

3. Add enzyme-labeled (detection) antibody

2 that binds to a different epitope on analyte
for Sample B (or binds Fc region on human
Ig for Antibody capture).

4. Wash to remove unbound materials.

5. Add substrate and measure color change.

Color intensity is directly related to analyte
(Sample B) or human antibody (Antibody
capture).

Signal
B

A Analyte
4 concentration

Noncompetitive immunoassay.

serum proteins, and vitamins that either occur at very low levels However, the chief disadvantage is the health hazard involved
in blood plasma or are so small that they could not be detected in working with radioactive substances. Disposal problems,
otherwise. Yalow was honored with the Nobel Prize in 1977 for short shelf life, and the need for expensive equipment has
her groundbreaking work. caused laboratorians to utilize other techniques for identifying
The assay uses a radioactive substance as a label. Radioactive analytes in low concentration.1,2
elements have nuclei that decay spontaneously, emitting matter
and energy. Several radioactive labels have been used, but 125I
has been the most popular.1,4 It is easily incorporated into pro- Enzyme Immunoassays
tein molecules and emits gamma radiation, which is detected
by a gamma counter. Very low quantities of radioactivity can Enzyme immunoassays, using enzymes as labels, were devel-
be easily measured.2 oped as alternatives to RIAs.2,4 Enzymes are naturally occurring
RIA is an extremely sensitive and precise technique for molecules that catalyze certain biochemical reactions. They
determining trace amounts of analytes that are small in size. react with suitable substrates to produce breakdown products
 Sample A diverse settings as clinical laboratories, doctors’ offices, and
at-home testing.
Enzymes used as labels for immunoassays are typically cho-
sen according to the number of substrate molecules converted
per molecule of enzyme, ease and speed of detection, and
stability.4 In addition, availability and cost of enzyme and sub-
strate play a role in the choice of a particular enzyme as
reagent. Typical enzymes that have been used as labels in col-
Enzyme-labeled orimetric reactions include horseradish peroxidase, alkaline
antigen phosphatase, and β-D-galactosidase.1,4 Alkaline phosphatase
and horseradish peroxidase have the highest turnover (conver-
sion of substrate) rates, high sensitivity, and are easy to detect,
so they are most often used in such assays.4,5 Enzyme assays
Patient
antigen are classified as either heterogeneous or homogeneous on the
basis of whether a separation step is necessary, as previously
mentioned.

Sample B
Heterogeneous Enzyme Immunoassays

The first enzyme immunoassays were competitive assays based

on the principles of RIA. Enzyme-labeled antigen competes
with unlabeled patient antigen for a limited number of binding
sites on antibody molecules that are attached to a solid phase.
After carefully washing to remove any nonspecifically bound
Enzyme-labeled antigen, enzyme activity is determined. Enzyme activity is in-
antigen versely proportional to the concentration of the test substance.
This method is typically used for measuring small antigens that
are relatively pure, such as drugs and hormones.5
Patient
antigen
Although competitive tests have a high specificity, the ten-
dency in the laboratory today is toward the use of noncom-
petitive assays because they have a higher sensitivity than their
competitive counterparts.1 Most noncompetitive assays are
Homogeneous immunoassay. Reagent antibody is in indirect immunoassays, or so-called indirect enzyme-linked
solution. Patient antigen and enzyme-labeled antigen are added to immunosorbent assays (ELISA), because the enzyme-labeled
the test tube. Patient antigen and enzyme-labeled antigen compete reagent does not participate in the initial antigen–antibody
for a limited number of binding sites on the antibodies. When patient binding reaction. The enzyme-labeled reagent only binds after
antigen is present, the enzyme label on the reagent antigen is not the initial antigen–antibody reaction has taken place. This type
blocked, so color development is observed. Sample A has a low of assay is one of the most frequently used immunoassays in
concentration of patient antigen, whereas Sample B contains more the clinical laboratory because of its sensitivity, specificity, sim-
patient antigen and has stronger color development. plicity, and low cost.5 Antigen is typically bound to solid
phase. A variety of solid-phase supports are used, including
microtiter plates, nitrocellulose membranes, and magnetic
that may be chromogenic, fluorogenic, or luminescent. Some latex or plastic beads.1,2 When antigen is bound to solid
type of spectroscopy can then be used to measure the changes phase, patient serum with unknown antibody is added and
involved. As labels for immunoassay, enzymes are cheap and given time to react. After a wash step, an enzyme-labeled
readily available, have a long shelf life, are easily adapted to antiglobulin, or secondary antibody, is added. This second an-
automation, and cause changes that can be measured using in- tibody reacts with any patient antibody that is bound to solid
expensive equipment.2 Sensitivity can be achieved without dis- phase. If no patient antibody is bound to the solid phase, the
posal problems or the health hazards of radiation. Because one second labeled antibody will not be bound. After a second
molecule of enzyme can generate many molecules of product, wash step, the enzyme substrate is added. The amount of
little reagent is necessary to produce high sensitivity.1 Enzyme color, fluorescence, or luminescence is measured using a de-
labels can either be used qualitatively to determine the pres- tection device and is compared with the amount of product
ence of an antigen or antibody or quantitatively to determine according to a standard curve.1,4 The amount of color, fluo-
the actual concentration of an analyte in an unknown speci- rescence, or luminescence detected is directly proportional to
men. Assays based on the use of enzymes can be found in such the amount of antibody in the specimen.
 This type of assay is used to measure antibody production Homogeneous Enzyme Immunoassays
to infectious agents that are difficult to isolate in the laboratory
and for autoantibody testing.2 Viral infections especially are Homogeneous enzyme immunoassays are generally less sen-
more easily diagnosed by this method than by other types of sitive than heterogeneous assays, but they are rapid, simple to
testing.4 This technique is especially useful as a screening tool perform, and adapt easily to automation.1,2 No washing or
for detecting antibody to HIV, hepatitis B, and hepatitis C.2,4 It separation steps are necessary. Their chief use has been in the
is best employed where quantitation is not necessary and is determination of low-molecular-weight analytes such as hor-
easily applied to point-of-care and home testing. mones, therapeutic drugs, and drugs of abuse in both serum
and urine.1,2,6 An example of a homogeneous immunoassay
is the enzyme-multiplied immunoassay technique (EMIT) de-
If antibody, rather than antigen, is bound to the solid phase, veloped by the Syva Corporation.6
these assays are often called sandwich immunoassays or Homogeneous assays are based on the principle of change
capture assays. Antigens captured in these assays must have in enzyme activity as specific antigen–antibody combination
multiple epitopes. Excess antibody attached to solid phase is occurs. Reagent antigen is labeled with an enzyme tag. When
allowed to combine with the test sample to capture any anti- antibody binds to specific determinant sites on the antigen,
gen present. After an appropriate incubation period, enzyme- the active site on the enzyme is blocked, resulting in a mea-
labeled antibody is added. This second antibody recognizes surable loss of activity.2 Free analyte (antigen) competes with
a different epitope or binding site than the solid-phase anti- enzyme-labeled analyte for a limited number of antibody-
body and completes the “sandwich.” Depending upon the binding sites, so this is a competitive assay. Enzyme activity
particular enzyme used, either a colored or chemiluminescent is directly in proportion to the concentration of patient anti-
reaction product is detected (see Fig. 11–2). Enzymatic gen or hapten present in the test solution (see Fig 11-3).
activity is directly proportional to the amount of antigen in A physical separation of bound and free analyte is thus not
the test sample. necessary.
Capture assays are best suited to antigens that have mul- The sensitivity of homogeneous assays is determined by the
tiple determinants, such as antibodies, cytokines, proteins, following: (1) detectability of enzymatic activity; (2) change in
tumor markers, and microorganisms, especially viruses.2,5 that activity when antibody binds to antigen; (3) strength of
When used with microorganisms, the epitope must be unique the antibody’s binding; and (4) susceptibility of the assay to in-
to the organism being tested and must be present in all strains terference from endogenous enzyme activity, cross-reacting
of that organism. The use of monoclonal antibodies has made antigens, or enzyme inhibitors. This technique is usually ap-
this a very sensitive test system. A major use of capture assays plied only to detection of small molecules that could not be
is in the measurement of immunoglobulins, especially those easily measured by other means because it is far less sensitive
of certain classes. For instance, the presence of IgM can than heterogeneous assays.2
be specifically determined, thus indicating an acute infec- Homogeneous assays have a number of limitations. First,
tion. When capture assays are used to measure immunoglob- only certain enzymes are inhibited in this manner. Addition-
ulins, the specific immunoglobulin class being detected is ally, enzymatic activity may be altered by steric exclusion of
actually acting as antigen and the antibody is anti-human the substrate or there may also be changes in the conforma-
immunoglobulin. Such indirect ELISA tests are quite sensi- tion structure of the enzyme, especially in the region of the
tive because all patient antigen has a chance to participate active site. Two enzymes that are frequently used in this type
in the reaction. of assay are malate dehydrogenase and glucose-6-phosphate
Heterogeneous enzyme assays, in general, achieve a sen- dehydrogenase.2
sitivity similar to that of RIA.1 In sandwich assays, capture Enzyme immunoassays in general have achieved sensitivity
antibody on solid phase must have both a high affinity and a similar to that of RIA without creating a health hazard or caus-
high specificity for this test system to be effective. However, ing disposal problems. Expensive instrumentation is not
there may be problems with nonspecific protein binding or needed because most assays can be read by spectrophotometry
the presence of antibodies to various components of the test- or by simply noting the presence or absence of color. Reagents
ing system.1 If IgG is present, rheumatoid factor can cause are inexpensive and have a long shelf life.
false-positive results. Rheumatoid factor is an IgM antibody Disadvantages include the fact that some specimens may
that reacts with IgG. See Chapter 14 for a discussion of the contain natural inhibitors. Additionally, the size of the enzyme
diseases in which rheumatoid factor may be found. If this is label may be a limiting factor in the design of some assays.
suspected, serum can be pretreated to avoid this problem. Nonspecific protein binding is another difficulty encountered
Sandwich assays are also subject to the hook effect, an unex- with the use of enzyme labels.2 However, this technique has
pected fall in the amount of measured analyte when an ex- been successfully applied to a wide range of assays.
tremely high concentration is present1. This typically occurs
in antigen excess, where the majority of binding sites are
filled and the remainder of patient antigen has no place to
Rapid Immunoassays
bind. If this condition is suspected, serum dilutions must be Rapid immunoassays are membrane based, easy to perform,
made and then retested. and give reproducible results.2 Although designed primarily
for point-of-care or home testing, many of these have been type of test device has been used to identify microorganisms
modified for increased sensitivity and can be made semiquan- such as Streptococcus pyogenes, the cause of strep throat,
titative for use in a clinical laboratory. Typically, these are and has been used for pregnancy testing as well as testing for
designed as single-use, disposable assays in a plastic cartridge. cardiac troponin after a heart attack, to name just a few ex-
The membrane is usually made of microporous nylon, which amples.1,2 Test results are most often qualitative rather than
is able to easily immobilize proteins.1 The rapid flow through quantitative.
the membrane and its large surface area enhance the speed and
sensitivity of ELISA reactions.1 Either antigen or antibody can Fluorescent Immunoassays
be coupled to the membrane; the reaction is then read by look-
ing for the presence of a colored reaction product. Some test In 1941, Albert Coons demonstrated that antibodies could be
devices require the separate addition of a patient sample, wash labeled with molecules that fluoresce.7 These fluorescent com-
reagent, labeled antigen or antibody, and the substrate. pounds, called fluorophores or fluorochromes, can absorb energy
Another type of rapid assay, called immunochromatography, from an incident light source and convert that energy into light
combines all the previously mentioned steps into one. The of a longer wavelength and lower energy as the excited elec-
analyte is applied at one end of the strip and migrates toward trons return to the ground state. Fluorophores are typically or-
the distal end where there is an absorbent pad to maintain a ganic molecules with a ring structure; each has a characteristic
constant capillary flow rate.2 The labeling and detection zones optimum absorption range. The time interval between absorp-
are set between the two ends. As the sample is loaded, it re- tion of energy and emission of fluorescence is very short and
constitutes the labeled antigen or antibody and the two form can be measured in nanoseconds.
a complex that migrates toward the detection zone. An antigen Ideally, a fluorescent probe should exhibit high intensity,
or antibody immobilized in the detection zone captures the which can be distinguished easily from background fluores-
immune complex and forms a colored line for a positive test, cence.1,2 It should also be stable. The two compounds most
which may be in the form of a plus sign (Fig. 11–4). Excess often used are fluorescein and rhodamine, usually in the form
labeled immunoreactant migrates to the absorbent pad. This of isothiocyanates, because these can be readily coupled with

mAbs to analyte
Clinical sample
with colloidal gold
with analyte

A
Conjugate pad Capillary flow

Sample Wicking
pad pad

B
Capillary flow Key:
mAbs Anti-IgG Analyte
to analyte mAbs
Antibody
to analyte
Immunochro- with colloidal
matographic assay. (A) Patient gold
sample is added to a cassette con-
taining antibody labeled with col- C Anti-IgG
loidal gold. (B) Sample combines Test line Control line antibody
(positive)
with antibody and is moved along
by capillary flow. (C) Monoclonal
antibody to the analyte captures
the patient antigen attached to
gold-labeled antibody. (D) Control
line has antibody that captures
colloidal gold-labeled antibody. D
(Courtesy of University of Nevada Test line Control line
School of Medicine.) (positive) (valid test)
antigen or antibody. Fluorescein absorbs maximally at 490 to been the standard for detecting treponemal and antinuclear
495 nm and emits a green color at 520 nm. It has a high inten- antibodies, as well as antibodies to a number of viruses.11–13
sity, good photostability, and a high quantum yield. Tetramethyl- Figure 11–5 depicts the difference between the two tech-
rhodamine absorbs at 550 nm and emits red light at 585 nm. niques. Reading immunofluorescent assays on slides may be
Because their absorbance and emission patterns differ, fluorescein open to interpretation and only experienced clinical laborato-
and rhodamine can be used together. Other compounds used are rians should be responsible for reporting out slide results.
phycobiliprotein, europium (β-naphthyl trifluoroacetone), and
lucifer yellow VS.1 Fluorescence Polarization Immunoassays
Fluorescent tags or labels on antibodies were first used for
localization of antigen in cells or tissues.5 Antibodies used to A number of quantitative heterogeneous fluorescent im-
identify such antigens are highly specific; when bound to anti- munoassays (FIAs) have been developed that correspond to
gen in the tissue, the fluorescent probe attached to the antibody similar types of enzyme immunoassays except that a fluores-
is detected under ultraviolet light using a fluorescent micro- cent tag is used. However, many of the newer developments
scope.8 This technique is called immunofluorescent assay. In in FIAs are related to homogeneous immunoassays. Homoge-
this manner, many types of antigens can be detected either in neous FIA, just like the corresponding enzyme immunoassay,
fixed tissue sections or in live cell suspensions with a high de- requires no separation procedure, so it is rapid and simple to
gree of sensitivity and specificity. perform. There is only one incubation step and no wash step;
The presence of a specific antigen is determined by the competitive binding is usually involved. The basis for this
appearance of localized color against a dark background. This technique is the change that occurs in the fluorescent label on
method is used for rapid identification of microorganisms in antigen when it binds to specific antibody. Such changes may
cell culture or infected tissue, tumor-specific antigens on neo- be related to wavelength emission, rotation freedom, polarity,
plastic tissue, transplantation antigens, and CD antigens or dielectric strength. The amount of fluorescence measured
on T and B cells through the use of cell flow cytometry. (See
Chapter 13 for a more complete discussion of the principles
of cell flow cytometry.) Fluorescently-labeled
antibody

Direct Immunofluorescent Assays

Fluorescent staining can be categorized as direct or indirect,
depending on whether the original antibody has a fluorescent
tag attached. In a direct immunofluorescent assay, antibody Patient antigen
that is conjugated with a fluorescent tag is added directly to
unknown antigen that is fixed to a microscope slide. After in-
A
cubation and a wash step, the slide is read using a fluorescence
microscope. Antigens are typically visualized as bright apple
green or orange-yellow objects against a dark background.
Examples of antigens detected by the direct method include Anti-human
Legionella pneumophila and Chlamydia trachomatis.9,10 Direct im- immunoglobin
munofluorescent assay is best suited to antigen detection in
tissue or body fluids, whereas indirect assays, described in the
text that follows, can be used for both antigen and antibody
identification.5 Patient antibody

Antigen
Indirect Immunofluorescent Assays B

Indirect immunofluorescent assays, which are more com-

monly used than direct assays, involve two steps. In the first step,
patient serum is incubated with a known antigen attached to Direct versus indirect immunofluorescent assays.
a solid phase. The slide is then washed and an anti-human im- (A) In direct fluorescent assay, the patient antigen is fixed to a micro-
munoglobulin containing a fluorescent tag is added. This im- scope slide and incubated directly with a fluorescent-labeled anti-
munoglobulin combines with the first antibody to form a body. The slide is washed to remove unbound antibody. If specific
antigen is present in the patient sample, fluorescence will be ob-
sandwich, which localizes the fluorescence. In this manner,
served. (B) In indirect fluorescence, well-characterized tissues or cells
one antibody conjugate can be used for many different types are fixed to slides. Specific antibody in patient serum (in red) binds
of reactions, eliminating the need for numerous purified, to the antigens on the slides. A wash step is performed and a labeled
labeled reagent antibodies. Indirect assays result in increased anti-human immunoglobulin is added. After a second wash step to
staining because multiple molecules can bind to each primary remove any uncombined anti-immunoglobulin, fluorescence of the
molecule, thus making this a more sensitive technique.5 Such sample is determined. The amount of fluorescence is directly in
assays are especially useful in antibody identification and have proportion to the amount of patient antibody present.
is directly related to the amount of antigen in the patient sam-
Incident
ple. As binding of patient antigen increases, binding of the (excitation) light
fluorescent analyte decreases; hence, more fluorescence is
observed.
One of the most popular techniques developed is
fluorescence polarization immunoassay (FPIA), which is
based on the change in polarization of fluorescent light emitted F
from a labeled molecule when it is bound by antibody.1,9 Inci-
dent light directed at the specimen is polarized with a lens or F
prism so that the waves are aligned in one plane. If a molecule
is small and rotates quickly enough, the emitted light is unpo- F
larized after it is excited by polarized light.1,2,8 If, however, the
labeled molecule is bound to antibody, the molecule is unable
to tumble as rapidly and it emits an increased amount of polar-
ized light (Fig. 11–6). Thus, the degree of polarized light reflects
the amount of labeled analyte that is bound. A polarization Emitted
analyzer is used to measure the amount of polarized light. (fluorescent) light
In FPIA, labeled antigens compete with unlabeled antigen A
in the patient sample for a limited number of antibody-binding
sites. The more antigen that is present in the patient sample,
the less the fluorescence-labeled antigen is bound and the less
the polarization that will be detected. Hence, the degree of
fluorescence polarization is inversely proportional to concen-
tration of the analyte.8
FPIA is limited to small molecules that tumble freely in
solution, usually low molecular weight analytes.2,8 The tech- F
nique has been used mainly to determine concentrations of
F
therapeutic drugs and hormones. It requires sophisticated in-
strumentation and is the basis for several automated analyzers
on the market today. F
The use of fluorescence has the potential for high sensitivity
and versatility. The main problem, however, has been separa-
tion of the signal on the label from autofluorescence produced
by different organic substances normally present in serum. An
additional consideration is nonspecific binding of the labeled
conjugate to other proteins in serum. Binding to these mole- B
cules would increase polarization, thus falsely decreasing
Fluorescence polarization immunoassay. Reagent
values. Fluorescence polarization has overcome some of these
antibody and fluorescently labeled analyte are added to the patient
problems and has seen more widespread use. However, it re- sample. (A) If sample analyte concentration is low, the antibody will
quires expensive, dedicated equipment, which may limit its bind most of the labeled analyte, restricting its rotation. When
use in smaller laboratories. excited by polarized light, the emitted fluorescence will remain
polarized (the light waves all oscillate with the same orientation).
Chemiluminescent Immunoassays (B) If the patient sample has a high concentration of analyte, this
unlabeled analyte will occupy most of the antibody-binding
sites, leaving the labeled analyte free to rotate. The light waves
Chemiluminescent immunoassay is another technique em- emitted by the rotating labels will not be uniformly oriented
ployed to visualize antigen–antibody combination. Chemilumi- (less polarization).
nescence is the emission of light caused by a chemical reaction,
typically an oxidation reaction, producing an excited molecule
that decays back to its original ground state.8 A large number of esters are oxidized by hydrogen peroxide under alkaline condi-
molecules are capable of chemiluminescence, but some of the tions, they emit a quick burst or flash of light. The light remains
most common substances used are luminol, acridinium esters, for a longer time with luminol.2 Different types of instrumenta-
ruthenium derivatives, and nitrophenyl oxalates.2 When these tion are necessary for each kind of emission.
substances are oxidized, intermediates are produced that are of This type of labeling can be used for heterogeneous and
a higher energy state. These intermediates spontaneously return homogeneous assays because labels can be attached to either
to their original state, giving off energy in the form of light. Light antigen or antibody. In heterogeneous assays, competitive and
emissions range from a rapid flash of light to a more continuous sandwich formats are the ones most often used. Smaller analytes
glow that can last for hours. For example, when acridinium such as therapeutic drugs and steroid hormones are measured
using competitive assays, whereas the sandwich format is used
for larger analytes such as protein hormones. • In noncompetitive assays, the amount of the label detected
Chemiluminescent assays have an excellent sensitivity; the is directly proportional to the amount of patient antigen
reagents are stable and relatively nontoxic.2,8 Because very little present.
reagent is used, they are also quite inexpensive to perform. The • Antibodies used in immunoassays must be very specific
relatively high speed of detection also means a faster turn- and have a high affinity for the antigen in question. Speci-
around time. Detection systems basically consist of photomul- ficity helps to cut down on cross-reactivity and the affin-
tiplier tubes, which are simple and relatively inexpensive. ity determines how stable the binding is between antigen
Chemiluminescent technology is the basis for several types of and antibody. These two factors help to determine the
automated immunoassays, which are used to detect a wide sensitivity of such assays.
range of substances including cardiac markers, hormones, • Radioimmunoassay was the first type of immunoassay to
vitamin D levels, and total IgE.14,15 be applied to quantitative measurements of analytes in the
A newer technique called electrochemiluminescence im- clinical laboratory. The original technique was based on
munoassay uses electrochemical compounds that generate light competition between labeled and unlabeled antigen for a
when an oxidation-reduction reaction occurs. Ruthenium, one limited number of antibody-binding sites.
of the common chemical substances used as an indicator, can • Indirect enzyme immunoassays (ELISA) are a type of non-
be conjugated with antibody and applied to sandwich type as- competitive assay in which antigen is bound to solid phase
says. It undergoes an electrochemiluminescent reaction with and patient antibody is detected. After allowing sufficient
another chemical substance, tripropylamine (TPA), at the sur- time for binding to occur, a second enzyme-labeled anti-
face of an electrode. When the ruthenium is oxidized and then body is added.
returned to its reduced state through interaction with TPA, it • In capture or sandwich assays, the antibody is bound to
gives off light that can be measured by a photomultiplier tube.1 solid phase and any patient antigen is allowed to bind or
Magnetic beads are often used as solid phase to capture the be captured. A second labeled antibody is added, which
labeled antibody. also binds to patient antigen to make a “sandwich.”
False negative results may be obtained if some biological • Homogeneous enzyme assays require no separation step.
materials such as urine or plasma cause quenching of the light They are based on the principle that enzyme activity
emission. Electrochemiluminescence assays have begun to be changes as specific antigen–antibody binding occurs.
more widely applied to immunologic testing and have great When antibody binds to enzyme-labeled antigen, steric
potential for the future. hindrance results in a loss in enzyme activity.
• Homogeneous assays are used for detection of low molec-
ular weight analytes such as hormones, therapeutic drugs,
and drugs of abuse.
• Simple one-step formats have been developed for hetero-
SUMMARY geneous enzyme assays. Rapid flow-through test devices
are able to capture antigen or antibody in a certain spot
• Labeled immunoassays were developed to measure anti- on a membrane. The results are easy to interpret.
gens and antibodies that may be small in size or present • Fluorochromes are fluorescent compounds that absorb
in very low concentrations. energy from an incident light source and convert that en-
• The substance that is being measured is called the ergy to light of a longer wavelength.
analyte. • Direct immunofluorescent assays involve antigen detec-
• Labeling techniques include the use of radioactivity, tion through a specific antibody that is labeled with a flu-
enzymes, fluorescence, and chemiluminescence. orescent tag. The presence of fluorescence is detected with
• There are two major types of immunoassays: competitive a fluorescent microscope that utilizes ultraviolet light.
and noncompetitive. In competitive assays, all the reac- • In indirect immunofluorescent assays, the original antibody
tants are added at the same time and labeled antigen com- is unlabeled. Incubation with antigen is followed by addi-
petes with patient antigen for a limited number of tion of a second fluorescent-labeled anti-immunoglobulin
antibody-binding sites. that detects antigen–antibody complexes.
• In competitive immunoassays, the concentration of pa- • Fluorescence polarization immunoassay (FPIA) is a type
tient antigen is inversely proportional to the amount of of homogeneous fluorescent immunoassay. It is based on
bound analyte. In other words, the greater the amount the principle that when an antigen is bound to antibody,
of patient antigen there is, the less the labeled antigen polarization of light increases.
will be bound. • Chemiluminescence is produced by certain compounds
• Noncompetitive assays allow any antigen present to com- when they are oxidized and emit light as they return to
bine with an excess of antibody attached to a solid phase. their original ground state. Substances that do this can be
A second antibody bearing a label is added and binds used as markers in reactions that are similar to RIA and
wherever there is patient antigen. enzyme immunoassays.
Study Guide: Labeled Immunoassays
TYPE OF ASSAY PRINCIPLE RESULTS
Competitive Patient antigen competes with labeled antigen Inverse ratio: The more patient antigen is
for limited antibody-binding sites. present, the less the label detected.
Noncompetitive or Excess solid-phase antigen binds patient All patient antibody is allowed to bind.
indirect ELISA antibody and a second labeled antibody is Amount of label is directly proportional to
added. the amount of patient antibody present.
Capture or sandwich Excess solid-phase antibody binds patient All patient antigen is allowed to bind.
antigen and a second labeled antibody is Amount of label is directly proportional to
added. the amount of patient antigen present.
Homogeneous Patient antigen and enzyme-labeled antigen No separation step.
react with reagent antibody in solution. Antibody in solution.
Enzyme label is inactivated when reagent Inverse ratio between patient antigen
antigen binds to antibody. and amount of label detected.
Direct fluorescent Patient antigen is attached to a slide. Specific If fluorescence is detected, patient anti-
fluorescent-labeled antibody is added. gen is present and the test is positive.
Indirect fluorescent Reagent antigen is attached to a slide. Patient If fluorescence is detected, patient anti-
antibody is allowed to react. A second body is present and the test is positive.
fluorescent-labeled antibody is added.
Fluorescent polarization Fluorescent-labeled antigen competes with When patient antigen binds, less reagent
patient antigen for a limited number of antigen binds and less polarization will
soluble antibody-binding sites. be detected.
Inverse ratio between patient antigen
and amount of polarization.
Immunochromatographic Patient sample is added to a test strip and If test is positive, a line or plus sign will
migrates through the strip. Labeled antigen form on the test strip where patient
or antibody binds and is captured by a antigen or antibody is captured.
second reagent in the detection zone.

CASE STUDY
A 2-year-old male child has symptoms that include fatigue, Questions
nausea, vomiting, and diarrhea. These symptoms have a. Does a negative finding rule out the presence of a
persisted for several days. Stool cultures for bacterial parasite?
pathogens such as Salmonella and Shigella were negative. b. What other type of testing could be done?
The stool was also checked for ova and parasites and the c. How does the sensitivity of testing such as enzyme
results were negative. The day-care center that the child immunoassay compare with visual inspection of
attends has had a previous problem with contaminated stained slides?
water; therefore, the physician is suspicious that this in- d. What are other advantages of enzyme immunoassay
fection might be caused by Cryptosporidium, a waterborne tests?
pathogen. However, because no parasites were found, he
is not certain how to proceed.
REVIEW QUESTIONS
1. Which of the following statements accurately 7. Which of the following is true of fluorescence polar-
describes competitive binding assays? ization immunoassay?
a. Excess binding sites for the analyte are provided. a. Both antigen and antibody are labeled.
b. Labeled and unlabeled analyte are present in b. Large molecules polarize more light than smaller
equal amounts. molecules.
c. The concentration of patient analyte is inversely c. When binding occurs, there is quenching of the
proportional to bound label. fluorescent tag.
d. All the patient analyte is bound in the reaction. d. The amount of fluorescence is directly proportional
to concentration of the analyte.
2. How do heterogeneous assays differ from homoge-
neous assays? 8. A fluorescent substance is best described as one in
a. Heterogeneous assays require a separation step. which
b. Heterogeneous assays are easier to perform than a. light energy is absorbed and converted to a longer
homogeneous assays. wavelength.
c. The concentration of patient analyte is indirectly b. the emitted wavelength can be seen under normal
proportional to bound label in heterogeneous white light.
assays. c. there is a long time between the absorption and
d. Homogeneous assays are more sensitive than emission of light.
heterogeneous ones. d. it spontaneously decays and emits light.

3. In the following equation, what is the ratio of 9. In a noncompetitive enzyme immunoassay, if a nega-
bound radioactive antigen (Ag*) to bound patient tive control shows the presence of color, which of the
antigen (Ag)? following might be a possible explanation?
12Ag* + 4Ag + 4Ab → :___Ag* a. No reagent was added.
Ab + ___AgAb + Ag* +___Ag b. Washing steps were incomplete.
c. The enzyme was inactivated.
a. 1:4
d. No substrate was present.
b. 1:3
c. 3:1 10. Which of the following best characterizes chemilumi-
d. 8:4 nescent assays?
4. Which of the following responses characterizes a a. Only the antigen can be labeled.
capture or sandwich enzyme assay? b. Tests can be read manually.
c. These are only homogeneous assays.
a. Less sensitive than competitive enzyme assays
d. A chemical is oxidized to produce light.
b. Requires two wash steps
c. Best for small antigens with a single determinant 11. Immunofluorescent assays may be difficult to interpret
d. A limited number of antibody sites on solid phase for which reason?
5. Which of the following is an advantage of enzyme a. Autofluorescence of substances in serum
immunoassay over RIA? b. Nonspecific binding to serum proteins
c. Subjectivity in reading results
a. Decrease in hazardous waste
d. Any of the above
b. Shorter shelf life of kit
c. Natural inhibitors do not affect results 12. Which statement best describes flow-through
d. Needs to be read manually immunoassays?
6. Which of the following is characteristic of direct a. Results are quantitative.
fluorescent assays? b. They are designed for point-of-care testing.
c. Reagents must be added separately.
a. The anti-immunoglobulin has the fluorescent tag.
d. They are difficult to interpret.
b. Antibody is attached to a solid phase.
c. Microbial antigens can be rapidly identified by
this method.
d. The amount of color is in inverse proportion to the
amount of antigen present.
13. Which of the following is characteristic of an indirect 15. In an indirect immunofluorescent assay, what
enzyme immunoassay? would be the outcome of an improper wash after
a. The first antibody has the enzyme label. the antibody-enzyme conjugate is added?
b. All reagents are added together. a. Results will be falsely decreased.
c. Color is directly proportional to the amount of b. Results will be falsely increased.
patient antigen present. c. Results will be unaffected.
d. Enzyme specificity is not essential. d. It would be difficult to determine the effect.

14. In a homogeneous enzyme immunoassay, which best 16. In a heterogeneous enzyme immunoassay, if the
describes the enzyme? patient sample produces more color than the highest
a. Enzyme activity is altered when binding to anti- positive control, what action should be taken?
body occurs. a. Report the results out as determined.
b. The enzyme label is on the antibody. b. Dilute the patient sample.
c. Enzyme activity is directly proportional to the c. Repeat the assay using one-half the volume of the
amount of patient antigen present. patient sample.
d. Most enzymes can be used in this type of assay. d. Report the results as falsely positive.
 Molecular Diagnostic
Techniques

After finishing this chapter, you should be able to: CHARACTERISTICS OF

DEOXYRIBONUCLEIC ACID
1. Describe the structure of deoxyribonucleic acid (DNA) and
AND RIBONUCLEIC ACID
ribonucleic acid (RNA).
Nucleotides
2. Identify complementary sequences.
The Nucleic Acid Polymer
3. Define hybridization.
DNA Replication
4. Describe restriction enzyme mapping of DNA.
RNA Synthesis
5. Discuss the principles of the polymerase chain reaction (PCR).
Protein Synthesis
6. Apply dye and probe signal detection to PCR.
Mutations
7. Assess template quantity by qPCR.
Polymorphisms
8. Discuss DNA and RNA amplification methods.
MOLECULAR ANALYSIS
9. Differentiate target amplification from probe amplification and give
examples of each. Strand Cleavage Methods
10. Explain the basis of DNA chain termination sequencing. Hybridization Methods
11. List technologies for next generation sequencing (NGS). Amplification Methods
DNA SEQUENCING
Sanger (Chain Termination)
Sequencing
Pyrosequencing
Next Generation Sequencing (NGS)
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Amplicons Gel electrophoresis Palindromic sites Strand cleavage
Amplification Genetic code Polymerase chain reaction Strand displacement
Branched DNA (bDNA) Hybridization (PCR) amplification (SDA)
Chain termination Immunogenetics Polymorphisms Stringency
sequencing Internal amplification Primers Threshold cycle
Complementary or copy control Probe Transcription
DNA (cDNA) Isothermal Quantitative PCR (qPCR) Transcription-mediated
Denatured Mutation Restriction endonucleases amplification (TMA)
Deoxyribonucleic acid Next generation sequencing Restriction fragment length Translation
(DNA) (NGS) polymorphisms (RFLPs) Variants
Electropherogram Nick Reverse transcriptase
Flanking Nucleic acid sequences Ribonucleic acid (RNA)
Fluorescence in situ Nucleotide Southern blot
hybridization (FISH)

Molecular diagnostic assays are powerful tools used to gain Nucleotides

information to aid in diagnosis and monitoring of disease and
now play a central role in medicine. These techniques are In DNA, the nitrogen base of a nucleotide—guanine, adenine,
based on the detection of specific nucleic acid sequences in cytosine, or thymine—is attached to the 1′ carbon of the de-
microorganisms or particular cells. Such techniques may be oxyribose sugar. The ribose 5′ carbon is mono-, di-, or triphos-
an advantage in clinical diagnosis because detection of nu- phorylated. The ribose 3′ carbon carries a hydroxyl group
cleic acids can be accomplished long before antibody (OH). Positions in the deoxyribose and nitrogen base rings are
detection is possible. Immunodeficiency and autoimmune numbered as shown in Figure 12–2.
diseases that have a genetic basis can also be identified more In contrast to the deoxyribose sugar in DNA, ribose is the
quickly. Molecular diagnostics also contribute to therapeutic primary sugar in RNA. Unlike deoxyribonucleotides, which
decisions, transplant selection, drug efficacy, forensics, and are hydroxylated on the 3′ carbon, the phosphorylated ribose
disease prognosis.1 Molecular techniques used in the clinical sugar in RNA carries hydroxyl groups on carbons 2′ and 3′
laboratory to identify unique nucleic acid sequences include (Fig. 12–3). Furthermore, thymine is not present in RNA and
enzymatic cleavage of nucleic acids, gel electrophoresis, en- is replaced by uracil.
zymatic amplification of target sequences, and hybridization Natural modifications of the nucleotide structure may occur
with nucleic acid probes. All of these techniques are dis- through additions, substitutions, and other chemical modifi-
cussed in this chapter in addition to an overview of the cations. These modifications may be enzymatically catalyzed
structure and functions of deoxyribonucleic acid (DNA) and in the cell or spontaneous. Enzymatic alteration of nucleotides
ribonucleic acid (RNA). in the cell is what brings about antibody diversity.

Characteristics of Deoxyribonucleic The Nucleic Acid Polymer

Acid and Ribonucleic Acid Nucleotides are polymerized by attachment of the sugar 3′
hydroxyl groups to the 5′ phosphate groups, forming a phos-
The two main types of nucleic acids are deoxyribonucleic acid phodiester bond. A chain of nucleotides makes up one strand
(DNA) and ribonucleic acid (RNA). DNA carries the primary of RNA or DNA. Most RNA exists without a partner or com-
genetic information within chromosomes found in each cell. plementary strand; they are single stranded. In contrast, DNA
RNA, in contrast, is an intermediate nucleic acid structure is mostly double stranded and arranged in a double helix
that helps convert the genetic information encoded within (Fig. 12–4). The double helix is formed by two single DNA
DNA into proteins that are the primary cellular component. chains wrapped around each other. There is a complementary
Both DNA and RNA are macromolecules of nucleotides. A relationship between the two linear polymers (strands) of the
nucleotide is a unit composed of a phosphorylated ribose double helix. All C’s are across from G’s and all A’s are across
sugar and a nitrogen base. DNA and RNA have the same two from T’s in the two corresponding strands.
purine bases, adenine and guanine, but the pyrimidine bases The hydroxyl group on the 3′ carbon and the phosphate
differ. DNA uses cytosine and thymine, whereas in RNA, cy- group on the 5′ carbon that participate in formation of the
tosine is present, but uracil replaces thymine (Fig. 12–1). DNA polymer through phosphodiester bonds give the DNA
 Purines DNA RNA
O O
Guanine Adenine
C C
H3C C NH HC NH
T U
O NH2
HC C O HC C O
N N
N O! O!
N C C
HC C NH HC C N O! P O O! P O

O O
N C C N C HC O O

N NH2 N H2C CH CH H2C CH CH

HC CH2 HC CH

OH OH OH
Pyrimidines
The ribose sugar in RNA is hydroxylated on the 2′
Cytosine Thymine and 3′ carbons (right), whereas in DNA it’s only hydroxylated at
the 3′ carbon (left). The nitrogen base, uracil, found in RNA (right)
NH2 O is analogous to thymine found in DNA (left). (Adapted from Buck-
ingham L. Molecular Diagnostics. 2nd ed. Philadelphia, PA: F.A.
C C
Davis; 2011, with permission.)
HC N H3C C NH

HC C O HC C O
The two chains (strands) of the DNA double helix are held
N N
together by hydrogen bonds between their nucleotide bases.
DNA is made up of nucleotides, each of which is
Guanine (G) and cytosine (C) are complementary, that is, they
composed of a phophorylated sugar and a nitrogen base. Adenine
and guanine are the purine bases, and cytosine and thymine are
will only hydrogen bond with each other. Similarly, adenine
the pyrimidine bases. (Adapted from Buckingham L. Molecular Diag- (A) and thymine (T) are complementary to each other. G pairs
nostics. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, with permission.) with C by three hydrogen bonds and A pairs with T by two
hydrogen bonds. Two bases paired together in this way are
called a base pair (bp). The length of a double-stranded DNA
Guanine
(nitrogenous base) macromolecule is measured in bp. The length of a single strand
Phosphate
of RNA (or DNA) is measured in bases (b). Metric prefixes are
O
O! used to describe long strands of DNA or RNA, for example,
7
6 1,000 bp or b comprise a kilobase pair (kbp) or kilobase (kb),
N C 1
O! P O
8 5 respectively. One million bp or b comprise a megabase pair
HC C NH
(Mbp) or megabase (Mb), respectively.
O 9 N C C 2 In humans, each chromosome is a double helix of DNA. There
O
4 are 46 chromosomes per nucleus—two copies of each of the
1" N NH2
H2C CH CH 3 22 autosomes (nonsex chromosomes 1 to 22) and two copies
5" 4" of the X chromosome in females (46,XX) or one copy each of
HC CH2 Deoxyribose
3" 2" the X and Y chromosome in males (46,XY). The autosomes are
(carbohydrate)
numbered according to size from the largest, chromosome 1
OH
(246 Mbp), to chromosome 22 (47 Mbp). The X chromosome,
Nucleoside # carbohydrate $ base
154 Mbp, is much larger than the Y chromosome (57 Mbp).
Genes are locations in chromosomes carrying information for
Nucleotide # phosphate $ carbohydrate $ base a gene product, either protein or RNA (noncoding RNA). The
23 chromosomes carry fewer than 30,000 protein-coding
Nucleotide structure. Nitrogen base ring positions
are numbered ordinally and the ribose ring positions are numbered
genes, with two copies of each gene making up the diploid
with prime numbers. The 1′ ribose carbon carries the nitrogen base. genome (two copies of each of the 23 chromosomes per cell
The 3′ ribose carbon hydroxyl group and the 5′ ribose carbon phos- or 46 chromosomes).
phate group are important for connecting nucleotides together into With the development of array and sequencing technol-
a DNA chain. A nucleoside is an unphosphorylated ribose sugar car- ogy, it is possible to investigate the entirety of DNA in the
rying a nitrogen base. (Adapted from Buckingham L. Molecular Diag- cell nucleus (genome). In contrast to tests that examine one
nostics. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, with permission.) gene at a time, these genomic studies are designed to analyze
all of the genes simultaneously to assess the overall genetic
status of the cell.
strands polarity, that is, a 5′ phosphate end and a 3′ hydroxyl
end. Sequences are by convention ordered in the 5′ to 3′ di-
rection. Complementary strands hydrogen bond together in
DNA Replication
an antiparallel arrangement with their 5′ phosphates on Replication of DNA is semiconservative, that is, the two strands
opposite ends of the double helix (Fig. 12–5). of the DNA duplex are separated and each single strand serves
 OH

A A A T
5" 3"

G G G C

T U T A

3" 5"
C C C G

OH OH OH

Single-stranded DNA RNA Double-stranded DNA

Nitrogen bases Sugar-phosphate backbone

3" 5"

C C C C
C G A
G G A G
DNA A G T T A
C A G C T G C A G
double helix A C
G C A C T G
C G C
T T G T

5" 3"
Nucleic acids are chains of nucleotides covalently attached, forming a sugar–phosphate backbone of phosphodiester bonds.
RNA has uracil nucleotides rather than thymines and does not always have a complementary partner strand as does double-stranded DNA. In
double-stranded DNA, the double helix is coiled such that areas of the double helix, the major and minor grooves, can interact with proteins
and other molecules. (Adapted from Buckingham L. Molecular Diagnostics. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, with permission.)

5" G T C A T C T G C C C T G A 3"
as a template for a newly synthesized complementary strand.
Two daughter strands result, each of which is an exact copy of
the original molecule. DNA replication proceeds with the for-
3" C A G T A G A C G G G A C T 5" mation of phosphodiester bonds between the 5′ phosphate of
an incoming nucleotide and the 3′ hydroxyl of the previously
Complementary sequences are not identical. In added nucleotide (Fig. 12–6). This reaction is catalyzed by
each nucleotide chain, G nucleotides always hydrogen bond to DNA polymerase. The parental template strand is read from
C nucleotides and A nucleotides bond with T nucleotides in the the 3′ to 5′ direction, whereas synthesis of the new strand of
complementary chain. Complementary sequences hybridize in an DNA proceeds 5′ to 3′, making the completely replicated
antiparallel arrangement. strand and its parent strand antiparallel.
 DNA Growing strand
Leading strand polymerase
3"
3"
5" 5" O! P O
DNA ligase
3" 3" Template strand
5" Replication fork O
5"
O
A T
Lagging strand Primer RNA H2C CH CH2
primase
HC CH2
A Overall direction of replication

O! P O

H2C O
G C
CH CH2
HC CH2

(A) DNA is replicated in a semiconserva-

tive manner where each parent strand of the double
helix serves as a template or guide for addition of new
nucleotides to the newly synthesized strand. Because
DNA polymerization proceeds in the 5′ to 3′ direction O O O
and the template is read in the 3′ to 5′ direction, one
strand (the lagging strand) has to be read discontinu- O! P O P O P O!
ously. RNA, synthesized by the primase enzyme, sup-
plies the 3′OH (priming) required by DNA polymerase. C G
O! O! O
Priming occurs once on the leading strand and repeat-
edly on the lagging strand. The RNA is then removed by O
RNase H and the missing bases filled in with a final liga- H2C CH CH
tion by DNA ligase. (B) DNA polymerase forms a phos-
phodiester bond between the 3′ hydroxyl group of the HC CH2
previous nucleotide and the phosphate of incoming
B
nucleotides. OH

DNA synthesis cannot begin without a preexisting 3′ hy- other is copied continuously (leading strand—3′ to 5′; see
droxyl group. To begin synthesis in vivo, a short (~60 b) RNA Fig. 12–6).
molecule is synthesized by an RNA polymerase (primase) en-
zyme. The RNA molecule is complementary to one strand of
the DNA to be copied and will hydrogen bond to the DNA
RNA Synthesis
template. There, it provides the preexisting 3′ hydroxyl group RNA synthesis proceeds in a manner that is chemically similar
required by DNA polymerase to copy the DNA. This aspect to that of DNA synthesis with some exceptions. Unlike DNA
of DNA synthesis is useful for directing synthesis to a specific synthesis, RNA synthesis can start de novo without a primer.
place in DNA. In the laboratory, short synthetic DNA mole- RNA synthesis is catalyzed by RNA polymerase, a more error
cules called primers are used routinely to direct copying of prone, slower polymerase (50 to 100 bases/second) than DNA
specific regions of DNA in vitro. The polymerase chain re- polymerase (1,000 bases/second). There are more start sites for
action (PCR) is an example of such targeted DNA synthesis. RNA polymerization than for DNA synthesis in the cell. The
The double helix is copied in a single pass such that DNA bulk of DNA synthesis takes place in the S phase of the cell
undergoing replication can be observed by electron mi- cycle, whereas RNA synthesis occurs throughout the cell cycle
croscopy as a replication fork. DNA synthesis on the template and varies depending on the cellular requirements.
strand in the 3′ to 5′ direction is not simultaneously consis- RNA is copied from almost all of the genome; however, only
tent with that on the 5′ to 3′ direction., Thus, one strand is about 2% of the RNA-coding regions are translated into pro-
copied discontinuously (lagging strand—3′ to 5′) whereas the tein. Some genes code for transfer RNA (tRNA) and ribosomal
RNA (rRNA), which is required for protein synthesis. Other 1 amino acid as shown in Table 12–1) is redundant. All amino
RNA products referred to as long- and short-noncoding RNA acids except methionine and tryptophan have more than one
make up the remainder of the total RNA transcribed in the cell. three-nucleotide codon. There are three stop codons that
terminate protein synthesis.
Protein Synthesis The codons are carried from the nucleus to the cytoplasm
in mRNA to be translated into protein. The tRNA serves as an
After DNA is transcribed by RNA polymerase into RNA, the adaptor between the nucleotide sequence in the RNA and the
RNA transcripts of protein-coding genes (messenger RNA or amino acid sequence in proteins, thus completing the transfer
mRNA) are translated into protein. Each mRNA is marked by a of genetic information from DNA to RNA to protein. This flow
guanidine nucleotide covalently attached to its 5′ end in an un- of information from DNA to mRNA to protein is known as the
usual 5′–5′ bond (cap) and 2 to 20 adenines at the 3′ end central dogma of molecular biology (Fig. 12–7).
(polyadenylation). These structures maintain the stability of the
mRNA and allow its recognition by the ribosomes. Ribosomes
are composed of ribosomal proteins and ribosomal RNA. Ribo-
Mutations
somes assemble on the mRNA for protein synthesis. A change in the nucleotide sequence is a mutation (Table 12–2).
Translation means converting information from one lan- Depending on how frequently they occur, changes in the nu-
guage to another. The language held in the order or sequence cleotide sequence may also be referred to as variants or
of the four nucleotides in the DNA chains must be translated polymorphisms. These alterations may be single bps up to
into the order or sequence of the 20 amino acids making up chromosomal structural abnormalities involving millions of
a protein chain. The nucleotide sequence of mRNA contains bps. Molecular technology is part of immunogenetics, the
a three-base recognition sequence (codon) for each of the analysis of gene mutations and polymorphisms that affect
20 amino acids, that is, the genetic code (Table 12–1). Each immune function. Diseases such as immunodeficiency result
amino acid has at least one associated three-base sequence from mutations in genes encoding proteins that bring about
(codon) carried in mRNA. The genetic code (3 nucleotides/ the immune response.2,3

Table 12–1 The Genetic Code Connects the Four-Base Nucleotide Sequence to the 20 Amino Acids
AMINO ACID ABBREVIATION SINGLE-LETTER CODE CODONS
Alanine Ala A GCU, GCG, GCC, GCA
Arginine Arg R AGA, AGG
Asparagine Asn N AAC, AAU
Aspartic acid Asp D GAU, GAC
Cysteine Cys C UGU, UGC
Glutamine Gln Q CAA, CAG
Glutamic acid Glu E GAA, GAG
Glycine Gly G GGU, GGA, GGC, GGG
Histidine His H CAU, CAC
Isoleucine Ile I AUU, AUC, AUA
Leucine Leu L UUA, UUG, CUA, CUC, CUG, CUU
Lysine Lys K AAA, AAG
Methionine Met M AUG
Phenylalanine Phe F UUU, UUC
Proline Pro P CCU, CCA, CCC, CCG
Serine Ser S UCU, UCC, UCA, UCG
Threonine Thr T ACU, ACA, ACG, ACC
Tryptophan Trp W UGG
Tyrosine Tyr Y UAU, UAC
Valine Val V GUU, GUA, GUC, GUG
(Stop codons) X UAA, UAG, UGA
DNA double helix Polymorphisms are defined by their frequency in a population.
Alterations in DNA or protein sequences shared by at least 2%
of a population are considered polymorphisms. The different
versions of the affected sequences are referred to as alleles. Poly-
morphic changes may or may not have phenotypic effects. Dele-
terious phenotypic changes are usually limited so that they do
Transcription not reach the required frequency in a population; however, there
(DNA RNA) are balanced polymorphisms that are maintained by an associ-
ated beneficial effect. A well-known example of this is the A to
RNA T base substitution in the beta-globin gene on chromosome 11
polymerase
that causes sickle cell anemia. This DNA substitution results in
3" the replacement of glutamic acid (E) with valine (V) at position
5" 5" 6 in the protein sequence. The mutation results in abnormal
red blood cells (RBCs) that do not circulate efficiently. The dele-
3" 3" terious effect is balanced by a beneficial phenotype of resistance
5" to Plasmodium species that cause malaria.
Translation Polymorphisms are found all over the genome. Like muta-
(RNA protein) tions, polymorphisms can involve a single bp (single nucleotide
Polypeptide polymorphisms or SNPs) or millions of bps. There are about
10 million SNPs in the human genome.5 Larger polymorphic
differences are less frequent in the genome.
Ribosome The most highly polymorphic region in the human
3" genome is the major histocompatibility complex (MHC) locus
5" on chromosome 6. The different nucleotide sequences result
in multiple versions or alleles of the human leukocyte antigen
Messenger RNA (mRNA) is transcribed from DNA (HLA) genes in the human population. These alleles differ by
using RNA polymerase. The mRNA delivers the information to ribo- nucleotide sequence at the DNA level (polymorphisms) and
somes where protein synthesis takes place. As each amino acid is by amino acid sequence. Each person will have a particular
added, the peptide chain continues to grow. This process, known as
group of HLA alleles that are inherited from his or her par-
translation, is accomplished with the help of tRNA, which brings in
ents. “Nonself” proteins are identified by the differences in
individual amino acids.
the HLA alleles. The recommended nomenclature identifies
Mutations can also affect the response to therapy; for exam- HLA alleles by type, subtype, whether the allele is silent, if
ple, cells expressing a mutation in the JAK1 gene are hypersen- the allele differs in an untranslated region, and if the protein
sitive to the antiproliferative and antitumorigenic effect of is secreted or not highly expressed.6
interferon chemotherapy, suggesting that this type of interferon Other highly polymorphic areas of the genome include
should be considered as a potential therapy for acute leukemia.4 the genes coding for the antibody proteins and antigen-
As technology advances, immunogenetic testing will become receptor proteins in B cells and T cells, respectively. Polymor-
increasingly routine in patient care. phisms are introduced in each cell through genetic events
(gene rearrangements) followed by enzymatically catalyzed
sequence changes (somatic hypermutation). Thus, these se-
Polymorphisms quences differ from cell to cell, allowing the generation of a
Structurally, mutations and polymorphisms are the same thing: large repertoire of antibodies and antigen receptors to better
a change from a reference amino acid or nucleotide sequence. match any foreign antigen.

Table 12–2 DNA Changes Affect Amino Acid Sequences

MUTATION TYPE EXAMPLE DNA CHANGE EXAMPLE RNA CHANGE EXAMPLE AMINO ACID CHANGE
Conservative ATC → CTC AUC → CUC Isoleucine → Leucine
substitution
Nonconservative CAT → CCT CAU → CCU Histidine → Proline
substitution
Silent TAT → TAC UAU → UAC Tyrosine → Tyrosine
Nonsense CAA → TAA CAA → UAA Glutamine → Stop
Frameshift TGT AAC CAG → UGU AAC CAG → Tyr Asp Glu → Tyr Lys Pro …
TGT AAA CCA G … UGU AAA CCA G …
 Polymorphisms that create, destroy, or otherwise affect re- particular DNA sequence and is used to study a small section
striction enzyme sites are detected as restriction fragment of the genome.
length polymorphisms (RFLPs) or variations that differ There are hundreds of restriction enzymes with unique bind-
among individuals. RFLPs are caused by variations in nu- ing and cleavage sites. The restriction enzymes used in the clinical
cleotides within genes that change where restriction enzymes laboratory (type II restriction enzymes) recognize palindromic
cleave the DNA. Repeat sequence polymorphisms, such sites. These sites are nucleotide sequences that read the same 5′
as short tandem repeats (STR) and variable number tandem to 3′ on both strands of the DNA. For example,
repeats (VNTR), are head-to-tail repeats of single bp to over 5′-GAATTC-3′
100 bp repeat units. STR has replaced RFLP for human iden- 3′-CTTAAG-5′
tification (DNA fingerprinting) and HLA typing for parentage
is the recognition site for the restriction enzyme, EcoRI. Re-
testing. STR and VNTR are also commonly used markers
striction enzymes are named for the organisms from which
for engraftment monitoring after allogeneic bone marrow
they were isolated. EcoRI was the first enzyme isolated from
transplantation.
Escherichia coli, strain R. HindIII is the third enzyme isolated
In addition to the nuclear genome, mitochondria located
from Haemophilus influenzae, strain d. In a bacterium, restric-
in the cytoplasm of eukaryotic cells carry their own genome.
tion enzymes serve as part of a primitive immune system that
The mitochondrial genome is circular, containing about
allows it to recognize its own DNA and degrade any incoming
16,500 bps. Polymorphisms are also found in two regions of
foreign DNA.
mitochondrial DNA sequences (hypervariable regions). These
DNA can be characterized by incubation with several
polymorphisms are not transcribed into RNA and do not
restriction enzymes, separating the products by electrophore-
affect proteins. They are used for maternal inheritance testing
sis, and observing the size and pattern of the resulting frag-
because all maternal relatives share the same mitochondria
and therefore have the same mitochondrial polymorphisms. ments. DNA with a different sequence will yield different-sized
bands characteristic of that DNA. These different band pat-
Mitochondria provide cellular energy. Mutations in the mito-
terns are RFLPs. Figure 12–8 shows the band patterns
chondrial genome affect high energy activities in muscle,
expected from digestion of two different DNA fragments, A
brain and in the immune system. When an infection occurs,
and B. Fragment A has two sites for the endonuclease to cut,
the immune response requires millions of cells and increased
resulting in three bands on the gel. Fragment B has only one
energy demands. Loss of mitochondrial function can result
site, resulting in two gel bands. The loss of the second restric-
in increases in the frequency and length of infections. In the
tion site in fragment B is caused by a mutation in the recogni-
1000 Genomes Project, an initiative to investigate the DNA of
tion site for the endonuclease. The presence of the mutation,
many diverse individuals, as many as 20% of healthy study par-
therefore, is inferred from the two band, rather than the three
ticipants had mitochondrial gene mutations.7 Mitochondrial
band pattern. Early work in recombinant DNA technology
DNA mutations can cause other diseases, including epilepsy,
relied on these types of studies. RFLP analysis is applied to
as well as complex conditions such as type 2 diabetes, aging,
epidemiological studies of microorganisms and identification
and cancer.
of resistance factors carried on extrachromosomal DNA (plas-
mids) in the cell.8
Molecular Analysis
Hybridization Methods
Nucleic acid tests are designed to detect changes (mutations
and polymorphisms) in the DNA sequence. There are four For analysis of complex genomes, such as human DNA, re-
main approaches to nucleic acid analysis: strand cleavage striction enzyme cleavage methods are not practical because
methods, hybridization methods, sequencing methods, and
amplification methods.
M A B
Restriction enzyme cut sites
Loading edge
Strand Cleavage Methods
One means of characterizing DNA is by cleaving it at specific A Migration
locations through the use of enzymes and then separating the
resulting fragments on the basis of size and charge through
gel electrophoresis. This is referred to as strand cleavage. B
Restriction endonucleases are enzymes that recognize and
Restriction enzyme mapping characterizes DNA
bind to specific nucleotide sequences in the DNA so that they
by the pattern of fragments generated when the DNA is cut with
will only separate the DNA at those locations. Restriction en- restriction enzymes. DNA double helix A has two restriction sites
donuclease (restriction enzyme) cleavage methods are highly (arrows), whereas the DNA B has only one. When these two DNAs
informative for investigating small genomes, such as those of are digested with the restriction enzyme, the DNA A fragment will
microorganisms, or plasmids. They are also used for mutation yield three fragments and DNA B will yield two. These band patterns
and polymorphism detection on small amplified regions are a characteristic of these DNAs (M = molecular weight standard,
of human DNA. Amplification makes many copies of a used for sizing the DNA fragments).
the DNA is too large and complex to generate readable frag- can either be emission of radioactivity, development of color,
ment patterns. This problem was addressed by Edwin South- or light. Radioactive nucleotides provide the radioactive sig-
ern in the mid-1970s.9,10 Using the specificity of nucleic acid nals, whereas the nonradioactive signals are generated by an
hybridization, he developed the Southern blot. Hybridization antigen–antibody or biotin–avidin binding with the antibody
is the binding of two specific complementary nucleic acid or avidin conjugated to alkaline phosphatase or horseradish
strands together. With this method, the very informative RFLP peroxidase enzymes that produce color or light when exposed
studies could be performed directly on large and complex to the color or light-emitting substrates (Fig. 12–11).
genomes. Results of a Southern blot reveal the fragmented regions
of the test DNA that are complementary to the probe. Any
locus or gene can thus be analyzed for RFLP using this
Southern blot analysis starts with restriction enzyme digestion
method. Positive human identification by DNA fingerprinting
of DNA isolated from the test organism or cells. The restriction
was first performed by Southern blot.11 Southern blots can
fragments are then separated by agarose gel electrophoresis.
also be used for detecting deletions in mitochondrial DNA
The separated double-stranded fragments are then denatured,
and gene rearrangements in nuclear DNA.12,13
that is, separated into single strands in the gel. The treated DNA
A variation of the Southern blot known as northern blot is
is transferred or blotted from the gel onto a nitrocellulose-based
used to analyze RNA structure and expression.14 Northern
membrane (Fig. 12–9). At this stage, all of the millions of frag-
blots are mostly research tools and not used routinely for
ments from complex genomes will be present on the membrane,
diagnostic purposes.
whereas only one particular gene or locus is of interest.
The key to the specificity of the Southern blot technique is
the use of a probe. A probe is generally a nucleic acid, several Although Southern blotting was helpful to analyze a single or
hundred to a few thousand bases in length, with a known se- a very few DNA sequences, as knowledge of genetic networks
quence that is used to identify the presence of a complementary and pathways grew it became apparent that there was a need
DNA or RNA sequence in an unknown sample. Probes typically to analyze hundreds or thousands of genes at the same time.
have an attached and detectable signal (Fig. 12–10). The signal Variations of the Southern blot led to the development of array
technology, where multiple targets or multiple patient samples
could be investigated simultaneously. Such techniques are used
for the prognosis in leukemia or diagnosis of complex diseases
such as autoimmune states, where multiple nucleic acid or
protein targets have to be detected.
Dry paper The first array methodology used dot blot hybridization. In
Nitrocellulose this method, instead of cutting and resolving DNA or RNA on
membrane a gel and blotting it to a membrane for probe hybridization, a
Gel highly specific unlabeled probe is spotted directly on a solid
support. Then the test sample (nucleic acids or proteins
isolated from cultures, cells, or body fluids) is labeled and
Soaked paper
hybridized to the many immobilized probes.
Buffer To assess the abnormal presence or amounts of target
molecules, a normal reference is required. The use of fluo-
Capillary transfer of DNA after restriction digestion
and electrophoresis from the gel to a nitrocellulose membrane was rescent labels provides a number of signals that can be dis-
the original method of Southern blotting. Other blotting formats tinguished from one another. This allows dual detection of
include vacuum transfer and electrophoretic transfer. the test sample and a reference sample that is hybridized to
the array along with it (Fig. 12–12). Measurement of test
material can be compared with the normal reference. Arrays
are used to detect amplifications or deletions in DNA, gene
expression (RNA synthesis), and SNP, where there is a
change in a single base.
Arrays were initially performed by manually spotting probes
on nitrocellulose membranes and hybridizing labeled test
DNA. This macroarray method was soon replaced by auto-
mated array printers that could deposit tens of thousands of
probes on a glass slide. These microarrays, also known as gene
chips, are used for a variety of applications including analysis
of genetic mutations in malignancies, detection of microdele-
tions,15,16 detection of viral resistance mutations, and gene
The labeled probe binds only to fragments on the expression profiling.17,18
membrane that are complementary to the nucleotide sequence of Another configuration of array technology is bead arrays.
the probe. These assays are based on preparations of 100 to 400 fluorescent
 Antidigoxigenin or
Radioactive isotope probe streptavidin conjugated
to alkaline phosphatase
Nitrocellulose
membrane Digoxigenin
or biotin
Probe

Nitrocellulose
membrane

Autoradiograph

X-ray film
Light-sensitive film

A B
Probes can be either (A) radioactive or (B) nonradioactive. Nonradioactive probes produce signal through enzymatic production
of color or light.

bead populations that differ by the relative amounts of two antibody directed against RNA:DNA hybrids or a covalently
distinct dyes that are incorporated into the beads. Each bead attached digoxigenin molecule used to generate chromogenic
population is attached to an antibody or other molecule that or chemiluminescent signal. This type of solution hybridiza-
will bind specifically to the target molecules or sequences. tion is the basis of the Hybrid Capture assays used to detect
A secondary antibody conjugated to a fluorescent signal will oncogenic variants of the human papilloma virus (HPV)22,23
detect the presence of the target (Fig. 12–13). Bead assays (see Fig. 12–15B). This method uses RNA probes comple-
are used for multiplex detection of proteins and nucleic acids mentary to the HPV DNA. The resulting RNA:DNA hybrid is
(Fig. 12–14). Applications include tissue typing for stem captured and detected by antibodies to the hybrid. By altering
cell and organ transplantation19 and respiratory virus the probe nucleotide sequences, HPV variants at low risk for
panels.20 causing cervical cancer can be distinguished from high-risk
HPV types.
The ability to distinguish nucleotide sequence variants by
In solution hybridization, the probe and the target are both in
solution hybridization has been applied to gene mutations and
solution. The target must be single stranded in order for hy-
polymorphism analysis. Some automated systems use electrical
bridization to work. After probes and denatured targets are
changes in a ferrocene label to indicate the presence of a mu-
mixed in solution, the bound products are resolved by gel or
tation or polymorphism recognized by the signal probe (see
capillary electrophoresis.
Fig. 12–15C). These systems can be used to assess respiratory
In variations of solution hybridization, DNA probe and
viruses, cystic fibrosis, warfarin sensitivity, and thrombophilic
RNA targets form hybrids, indicated by DNA probe:RNA. The
mutations.
target hybrids formed in solution are detected by capture on
a solid support.21 Two probes are used, both hybridizing to
the target RNA. One probe, the capture probe, has an at- In situ hybridization is an additional technique that utilizes
tached molecule of biotin that will bind specifically to strep- nucleic acid probes to identify DNA. However, in this case,
tavidin immobilized on a solid support (Fig. 12–15A). The the target DNA is found in intact cells. In this method,
other probe, which is the detection probe, is detected by an probes ranging in size from a few thousand to hundreds of
 Sample DNA
Labeled probe

Test DNA + Reference DNA

Beads with specific oligos

For nucleic acid analysis, bead array antibodies are
replaced with single-stranded oligonucleotides complementary
to the test nucleic acid. If present, biotinylated sample DNA will
hybridize to the sequences and the biotin-specific conjugate will
generate a signal.

microscope instead of expensive fluorescent microscopes

Normal = color ratio of 1.0 = yellow/orange and their special filters.24,25
Gains/amplifications = ratio > 1.0 = green In addition to identifying many different chromosomal
Losses = ratio < 1.0 = red abnormalities, FISH has been used to identify T-cell lym-
For array analysis, unlabeled probes are immobi- phomas, B-cell malignancies, and graft versus host disease
lized and hybridized to labeled sample material (green). A reference after transplants. However, FISH is sensitive to the buffer
material (red) is hybridized to the same array. The results of the array and temperature conditions of hybridization (stringency).
are relative: test/reference. Stringency refers to the conditions that affect the ability
of a probe to correctly bind to a specific DNA target se-
quence. Array methods (comparative genome hybridization)
complement FISH testing in cases of multiple or complex
Analyte genetic abnormalities as well as deletions and amplification
of genes.26
Labeled antibody
Amplification Methods
The most frequently used methods in molecular diagnostics
involve some aspect of amplification, that is, copying of nu-
Beads with specific antibodies
cleic acids. The development of the in vitro PCR reaction
by Kerry Mullis27 greatly facilitated and broadened the po-
Three of 100 to 400 bead shades, each with anti-
tential applications of gene amplification. PCR was quickly
body to a different analyte. The presence of multiple targets can be
detected by which bead has associated fluorescence from the followed by other target amplification methods, such as re-
secondary antibody. Flow cytometry is used to assay each bead verse transcriptase PCR (RT-PCR), transcription-mediated
separately for associated fluorescence. amplification (TMA), and strand displacement amplifi-
cation (SDA).
thousands of bases long are covalently attached to a fluores-
cent dye. When fluorescence is used to visualize the hy- PCR is an in vitro replication procedure that results in am-
bridization reaction, this is called fluorescence in situ plification of the target DNA. A PCR reaction includes all of
hybridization, or FISH. For FISH, thin sections of solid tis- the necessary components required for DNA replication: tem-
sue or deposits of cells are deposited on glass slides. The cell plate (sample) DNA to be copied; deoxyribonucleotides,
membranes are permeabilized to allow entry of the probe. which will be joined together; oligonucleotide primers to
The probes are applied to the prepared slides of cells where prime the synthesis of the copies; DNA polymerase to cova-
they hybridize to their complementary sequences. The re- lently join the deoxyribonucleotides; and a buffer with a pH
sulting signals will show the state of the targeted gene or re- optimal for the polymerase activity. The oligonucleotide
gion. The probes are used with reference probes to centromeric primers are key components to the specificity of the PCR
genes to assess deletion or amplification (Fig. 12–16). A vari- reaction. Primers are synthetic single-stranded nucleic acids
ation of FISH using chemiluminescent labeling (chemilumi- usually 18 to 30 bases in length. They are complementary
nescent in situ hybridization or CISH) is performed in the to sequences flanking (on either side of and including) the
same way, except the slides can be read on a regular light region of the template DNA to be copied (Fig. 12–17).
 Test RNA or DNA
Test RNA Detection probe

Test RNA or DNA

DNA or RNA
detection probe
Biotinylated
capture probe

Immobilized
capture probe

Gold electrode
Streptavidin-coated support Antibody-coated support

A B C
Variations of methods designed to detect specific DNA or RNA sequences by probe hybridization in solution. (A) Test RNA
detected by a DNA capture probe with covalently attached biotin. The biotinylated probe binds to a streptavidin-coated support, whereas un-
bound probe and other RNAs are washed away. The bound hybrids are detected with anti DNA:RNA hybrid antibody conjugates. (B) RNA:DNA
hybrids are captured by immobilized antibodies and detected with antibody conjugates or by signal protection assays. (C) Signal and capture
probes hybridize to test RNA or DNA followed by electrochemical detection of ferrocene labels on the signal probe.

For many procedures, premixed PCR reagents (deoxyri-

bonucleotides, primers, and buffer) are supplied from manu-
facturers to which only the template DNA and, in some cases,
enzyme are added. The PCR reaction mix is subjected to a
computerized amplification program consisting of a designated
number of cycles. A sequence of temperature changes com-
prises a cycle. An example of a three-step PCR cycle is (1) a
denaturation step (94°C to 96°C), (2) an annealing step (50°C
to 70°C), and (3) an extension step (68°C to 72°C). In one cycle,
each of these temperatures will be held for 30 to 60 seconds.
PCR cycles vary depending on the target DNA and the proto-
Normal col. Two-step cycles are commonly used, as well as more
complicated multi-step cycles. The cycle is repeated 20 to
50 times depending on the assay. The set of repeated cycles
make up the PCR program. Because the amplification programs
Amplified differ among assays, the program may be entered and stored in
the thermal cycler for each assay once it is validated. Alterna-
FISH analysis of the epidermal growth factor recep-
tor gene. The gene probe is labeled orange, whereas a probe com-
tively, prevalidated commercial assays may include preloaded
plementary to the centromere of chromosome 7 is labeled green. thermal cyclers. For convenience, a hold at 4°C is usually added
Normally there are two chromosomes, each carrying one gene in to the end of the amplification program.
each nucleus (left). The image on the right shows gene amplification The PCR amplification produces millions of copies or
with multiple orange signals associated with single green signals. amplicons of the region of interest. At 100% PCR efficiency,
 Region under
investigation

Template DNA
5! 3!
G A A T C G T C G A G C T G C T A G C T T T G T T C G A
G A A A C A A

Forward primer
Reverse primer

A T C G T C
C T T A G C A G C T C G A C G A T C G A A A C A A G C T
3! 5!
Template DNA
PCR

5! 3!
A T C G T C G A G C T G C T A G C T T T G T T

T A G C A G C T C G A C G A T C G A A A C A A

A T C G T C G A G C T G C T A G C T T T G T T

T A G C A G C T C G A C G A T C G A A A C A A
3! 5!

In the PCR reaction, short, single-stranded primers hydrogen hybridize to complementary sequences flanking the region of
interest. The PCR reaction will produce millions of copies of the desired sequences. Note: The image is shortened. Primers are usually 18 to
30 bases long and the region of interest (and subsequent PCR products) range from 50 to more than 1,000 base pairs.

the number of copies will be approximately 2n, where n is the also called amplification refractory mutation system PCR or
number of cycles (20 to 50) in the amplification program. The ARMS-PCR) primers are designed so that they will end on a
products of the PCR reaction can be visualized by gel elec- potentially mutated or polymorphic bp.29 Annealing of the
trophoresis (Fig. 12–18), capillary electrophoresis, or other de- last base at the 3′ end of the primer is critical for the poly-
tection methods. For some applications, the presence, absence, merase activity. If the last base of the primer is not comple-
or size of a PCR product is the test result. mentary to the template, DNA polymerase will not recognize
A variety of modifications have been made to the PCR re- the primer as a substrate for extension and no PCR product
action. RT-PCR is a method of analysis for cellular RNA will be produced.
or qualitative detection of RNA viruses such as HIV and SSP-PCR is a common approach used to detect mutations
HCV. Infection with either of these viruses can be diagnosed and polymorphism, such as in HLA typing by SSP-PCR.30
by this method long before a positive antibody screen Unlike the 3′ end of primers, the 5′ end does not have to be
would occur. RT-PCR starts with an RNA template. Reverse complementary to the template. This allows attachment of
transcriptase is a DNA polymerase that makes double-stranded noncomplementary sequences containing restriction enzyme
DNA from RNA by reverse transcription (DNA-dependent recognition sites or RNA polymerase-binding sites to PCR
RNA polymerase). The double-stranded complementary or products. The amplicons can then be conveniently inserted
copy DNA (cDNA) is thus synthesized from the RNA using into plasmids for biological analyses or transcribed into RNA
reverse transcriptase in a separate step. Alternatively, en- and translated into in vitro transcription or translation sys-
zymes that copy both RNA and DNA are used in simultane- tems. Labels in the form of fluorescent molecules, biotin, or
ous RT and PCR reactions that do not require a separate other molecules may also be covalently attached to the 5′
RT step.28 The cDNA serves as the template for the PCR end of primers. This allows capture immobilization of the
reaction. PCR products or detection in capillary electrophoresis sys-
PCR primer design introduces additional flexibility into tems (Fig. 12–19). Fluorescently labeled primers are used
the PCR reaction. In sequence-specific primer PCR (SSP-PCR, in PCR analysis of DNA fingerprinting,31,32 for cancer tests
 Molecular weight Reagent blank achieved with probes. These probes generate fluorescent sig-
markers nals from the accumulating PCR products. Probes are se-
quence specific and only bind to the targeted region within
the product to provide higher specificity for the intended
product than SYBR Green.
Accumulation of PCR product detected using TaqMan
probes through 50 PCR cycles is shown in Figure 12–20. The
fluorescence depicted on the y axis is the strength or brightness
of the fluorescence signal from the dye or probe. The fluores-
cence plotted versus cycle number generates a curve similar to
a bacterial growth curve with a lag phase, a log phase, a linear
phase, and a stationary phase (see Fig. 12–20). The length of
the lag phase is assessed by the number of cycles required to
(Misprime) reach a threshold level of fluorescence as determined by the
instrument. The cycle at which the sample fluorescence reaches
this value is the threshold cycle or Ct.
A relationship between the amount of target and the Ct
PCR
product values is established by generating a standard curve of dilutions
of known target nucleic acid (Fig. 12–21). For test samples,
the target is quantified relative to an internal amplification
(Primer
dimers) control. The internal amplification control is a gene target
that is always present at a constant level. Internal controls also
confirm negative results as true negatives and not PCR failure.
Test results are quantified using the standard curve followed
by normalization with the internal control.34
Detection of PCR products by gel electrophoresis.
Just as PCR generated a wide variety of test methods and
The first lane (left) contains molecular weight markers (fragments
of known size). The next six lanes are PCR products, followed by a
applications, qPCR has also been modified to address a variety
reagent blank control for contamination (lane 8). (From Buckingham of clinical questions. Both DNA and RNA targets are measured
L. Molecular Diagnostics. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, by qPCR. For RNA, cDNA made from the RNA using reverse
with permission.) transcriptase is the input material.
Widely used applications of qPCR and RT-qPCR include
detection of microorganisms, especially viruses and other
such as microsatellite instability testing in colon cancer for pathogens that are difficult or dangerous to culture in the lab-
Lynch syndrome,33 diagnosis of endometrial cancer,34 and oratory.39,40 Assessment of tumor-associated gene expression
for genetic testing of Fragile X syndrome and Huntington using qPCR and RT-qPCR may predict cancer recurrence.41,42
disease.35,36 Researchers have developed methods using qPCR for HLA
typing and chimerism analysis.43–45 Multiplex qPCR methods
Standard PCR results are interpreted as
are performed to assess multiple targets simultaneously, such
the presence, absence, or size of the PCR product at the com-
as the expression of various cytokine genes during different
pletion of the PCR program, but quantification of starting
stages of infections.
material is not easily measured. In 1993, Higuchi et al.
demonstrated that target quantification could be achieved
by observing the accumulation of PCR product in real time Kwoh and colleagues developed the first transcription amplifi-
during amplification.37,38 Although originally termed real- cation system in 1989.46 Commercial variations of this process
time PCR or RT-PCR, the preferred term is now quantitative include transcription-mediated amplification (TMA) (Gen-
PCR or qPCR to avoid confusion with reverse transcriptase Probe), nucleic acid sequence-based amplification (NASBA)
PCR (also RT-PCR). In qPCR, results can be seen at the end (Organon Teknika), and self-sustaining sequence replication
of each cycle, as opposed to PCR where measurement only (3SR) (Baxter Diagnostics). These three methods are similar but
occurs after all cycles are completed. have variations in enzyme systems.
The qPCR product was followed initially by using a fluo- For TMA, RNA is the usual target instead of DNA. A cDNA
rescent dye specific for double-stranded DNA (ethidium bro- copy is synthesized from the target RNA, after which transcription
mide). Product labeling is currently done using a less toxic of the cDNA produces millions of copies of RNA products. In
dye, SYBR Green. SYBR Green is not specific to sequence, so this technology, RNA is the primary product as well as the target.
any product, including artifacts of the PCR reaction (mis- The RNA products transcribed from the cDNA can also serve as
primes and primer dimers from primer self-amplification), target RNA for synthesis of more cDNA (Fig. 12–22). The RNA
will produce a signal. More specific detection of product is products are detected by chemiluminescence with acridinium
 Template DNA
5! 3!
G A A T C G T C C T T T G T T C G A
G A A A C A A

Forward primer
Reverse primer

A T C G T C
C T T A G C A G G A A A C A A G C T
3! 5!
Template DNA
PCR

Amplification
labeled primer incorporated

Denature Capillary

Electropherogram

5! 3!
G A A T C G T A C T T T G T T C G A
G A A A C A A
Mismatch

A T C G T C
C T T A G C A T G A A A C A A G C T
3! 5!

PCR

No amplification
no label incorporated

One PCR primer (forward or reverse) is covalently attached to a fluorescent dye, such as fluorescein, to allow detection of PCR
products by capillary gel electrophoresis. The double-stranded products are denatured and diluted (left). Only the single strand of the PCR
product with labeled primer will be detected (center). The output from the capillary instrument is an electropherogram (right) showing peaks
of fluorescence, which are analogous to band patterns on gel electrophoresis.

ester (Gen-Probe)47 or, in the case of NASBA, Molecular Beacon transcriptase derived from avian myeloblastosis virus (AMV)
probes.48 with inherent RNase H activity. Thus, the reaction can be run
The TMA process has been simplified with the addition with only two enzymes, AMV reverse transcriptase and
of RNase H derived from E coli to degrade the RNA from the T7 RNA polymerase. These procedures have been marketed
DNA and RNA hybrid, eliminating a heating step required as TMA, NASBA, and 3SR.
for denaturation of the DNA and RNA hybrid to complete In contrast to PCR, TMA is an isothermal process, mean-
the cDNA synthesis. An additional modification and simpli- ing that the reaction proceeds at a single temperature. This
fication of the procedure was the application of the reverse process is much simpler to perform compared with PCR
 Tailed primer
107 copies
106 copies
105 copies RNA
104 copies Reverse transcriptase
100 103 copies
102 copies cDNA
101 copies RNA
RNase II
Rn

10

ssDNA
Threshold 2nd primer
1

ssDNA
0.1 Reverse transcriptase
1 5 9 13 17 21 23 25 27 29 33 35 37 39 41 43 45 47 49
Cycle DNA
DNA
Real-time quantitative PCR signal generated from
a TaqMan probe. The normalized fluorescence (∆Rn) is plotted RNA Binding site for
against PCR cycles 1 to 50. The threshold cycle is indicated by the polymerase RNA polymerase
green line. The length of the lag phase (number of cycles required
to reach a threshold level of fluorescence) is inversely correlated
with the amount of starting material.

Multiple copies of RNA

40
Transcription-mediated amplification (TMA) targets
35 RNA. In the first step, a cDNA:RNA hybrid is synthesized by reverse
transcriptase using a primer with a tail (that will ultimately form the
Threshold cycle (C)

30
binding site for a later enzyme). Hybridized (but not single-stranded)
25
RNA is degraded by RNase II, leaving single-stranded DNA. This
20 ssDNA serves as a template for reverse transcriptase to synthesize a
15 complementary strand of DNA, including the primer tail to complete
the binding site for RNA polymerase. The RNA polymerase uses
10
dsDNA as a template to synthesize many copies of RNA. This new
5 RNA can cycle back to step one and repeat the process, resulting in
0 a large amplification of product.
10 102 103 104 105 106 107
Starting quantity (copies/rxn)
Ct values (y axis) were determined for serial 10-fold may have to be confirmed by a more specific method (which
dilutions of a synthetic target of known concentration (x axis). The may be too labor intensive or expensive for screening large
resulting standard curve is used to convert Ct values of test samples
numbers of samples).
to concentration.

The number of target nucleic acid sequences in a sample is

cycling, which requires repeated heating and cooling. Target- not changed in probe amplification. Rather, primers are ex-
ing RNA allows for the direct detection of RNA viruses, such tended or ligated into many copies of detectable probes. Ex-
as hepatitis C virus, and HIV.49,50 Targeting the RNA of organ- amples of probe amplification are ligase chain reaction (LCR)
isms with DNA genomes, such as Mycobacterium tuberculosis, and SDA.
is more sensitive than targeting the DNA because each mi- The LCR amplifies synthetic probes
croorganism makes multiple copies of RNA, whereas it has complementary to target nucleic acid. In order for LCR to
only one copy of DNA.51 Detection of Chlamydia trachomatis work, the entire target sequence must be known. Instead of
in genital specimens and cytomegalovirus (CMV) quantifica- a polymerase, LCR uses DNA ligase, an enzyme that forms
tion in blood are additional applications for TMA. The high one phosphodiester bond between two preexisting DNA
sensitivity of TMA also makes it suitable for blood screening.52 chains—in this case, two primers. In LCR, the two primers
Screening assays require high sensitivity, even at the expense bind immediately adjacent to each other on the target se-
of specificity to assure detection of low levels of the target an- quences and DNA ligase covalently attaches the adjacent
alyte, such as anti-HBV antibodies. Positive screening results primers together. These joined, or ligated, primers essentially
become the probe. There is no amplification of the product 1 Primer
because the ligated primers then serve as a template for the
annealing and ligation of additional primers. The probes (lig- Target DNA or RNA
ated primers) are detected by capture and signal detection
(Fig. 12–23).
2
LCR requires temperature changes to drive the denaturation
of the template and ligated primers. LCR was used to detect
point mutations in a target sequence. The DNA mutation that
occurs in the beta-globulin of patients with sickle cell disease,
as compared with normal beta-globulin, was one of the first 3 Probe
applications of LCR.53 LCR was used at one time for detection
of Neisseria gonorrhoeae and C trachomatis, but has been re-
placed by other amplification methods.
SDA is another isother-
4 Nick
mal amplification process. After an initial denaturation step,
the reaction proceeds at one temperature. In SDA, the ampli-
fication products are the probes. After the target DNA is
denatured by heating to 95°C, two primers bind close to
each other, an outer and an inner primer (containing a probe)
(Fig. 12–24). As the outer primer is extended by DNA poly- Probe
merase, it displaces the product formed by the simultaneous 5
extension of the inner primer (probe). The probe becomes
the target DNA for the next stage of the process. The second
stage of the reaction is the exponential probe amplification Strand displacement
phase by extension from a nick (breaking one phosphodiester
bond on one strand of a double-stranded DNA) formed by a 6
restriction endonuclease enzyme. Methods using fluorescence
polarization or fluorogenic probes to detect the amplified

…GTACTCTAGCT… …GTACTCTAGCT… Probe

A
T
Strand displacement amplification showing one
A
target strand. 1. Primers bind to single-stranded DNA at a comple-
C
…CATGAGATCGA… …CATGAGATCGA…
mentary sequence. 2. A polymerase extends the primer from the
3′ end. 3. The extended primer forms a double-stranded DNA
Ligase Ligase segment containing a site for a restriction enzyme at each end.
4. The enzyme binds double-stranded DNA at the restriction site
A
C A and forms a nick. 5. The DNA polymerase recognizes the nick
T
and extends the strand from that site, displacing the previously
A
C created strand. 6. Each strand can then anneal and continue the
A
A process.
C
T
A
C
probes have been designed to test for M tuberculosis,54 C tra-
In the ligase chain reaction, primers hybridize on
adjacent nucleotide sequences. If the primers are complementary chomatis,55 and N gonorrhoeae.56
to the template, DNA ligase will join them together to form a probe.
The probe serves as a hybridization substrate for more primers to In signal amplification, large amounts of signal are bound to
bind. Four probes are used for each reaction. One of the primers
the target sequences that are present in the sample.
carries a capture molecule (circle) and another a signal molecule
Branched DNA (bDNA) is an example of a commercially
(square). When these primers are ligated, the probe will be captured,
unbound primers removed, and signal detected from the joined available signal amplification method. In bDNA amplifica-
primers. If the primers are not complementary to the template, tion, a series of short single-stranded DNA probes are used
no ligation will occur and no signal will result. (Adapted from to capture the target nucleic acid and to bind to multiple re-
Buckingham L. Molecular Diagnostics. 2nd ed. Philadelphia, PA: porter molecules, loading the target nucleic acid with signal
F.A. Davis; 2011, with permission.) (Fig. 12–25). Because multiple probes hybridize to the
 However, newer versions of qPCR methods may have better
Amplifiers performance compared with bDNA assays. By replacing the
plate support with beads, the bDNA assay has been combined
with the bead array technology to provide a multiplex system
that can detect 100 different targets in a single sample.57
Detection of HPV by hybrid capture, as described in the
Solution Hybridization section, is also a probe amplification
Extender probes
method. Multiple antibodies bind to the DNA:RNA hybrids,
producing multiple signals from a single target molecule.
Target RNA or DNA
Capture
probes DNA Sequencing
Solid support The function of DNA is to store genetic information in the
A order or sequence of the four nucleotide bases in the DNA
chain. Early in the history of recombinant DNA technology, re-
Amplifiers
searchers actively pursued the idea of sequencing or detecting
the nucleotide order of the nucleic acids. Two sequencing
methods emerged in the early to mid-1970s: the Maxam-
Gilbert chain breakage method58 and the Sanger chain termi-
nation sequencing method.59 Sanger sequencing quickly
gained popularity because it was not subject to the toxic chem-
icals and complex interpretation required by the Maxam-
Gilbert method. Researchers have since developed alternative
sequencing methods including pyrosequencing and next
generation sequencing (NGS) methods.

Sanger (Chain Termination) Sequencing

Preamplifier Direct determination of the order, or sequence, of nucleotides
in a DNA chain is the most explicit method for identifying ge-
Extender probes netic lesions (mutations) or polymorphisms, especially when
looking for changes affecting only one or two nucleotides.
Chain termination (Sanger) sequencing is a modification of the
Target RNA or DNA
Capture DNA replication process. It utilizes modified nucleotide bases
probes called dideoxynucleotide triphosphates (ddNTP) (Fig. 12–26).
The ddNTP are incorporated into the growing DNA chain, just
Solid support as dNTP are. The ddNTP, however, cannot provide a 3′OH for
B
the addition of the next nucleotide; the chain synthesis then
In branched DNA, signal is amplified through stops (chain termination).
hybridization of complementary probes to the target DNA or RNA. In a standard sequencing reaction, a short, synthetic single-
(A) Branched DNA molecules carry multiple signals for each target stranded DNA fragment (primer) complementary to sequences
molecule. (B) A second generation of the method has increased
sensitivity because of the binding of additional signals. (Adapted
from Buckingham L. Molecular Diagnostics. 2nd ed. Philadelphia, PA:
F.A. Davis; 2011, with permission.) Nitrogen base Nitrogen base

HOCH2 O HOCH2 O
target sequences in bDNA, its sensitivity is enhanced over
C C C C
methods using a single probe or primer to bind to the target.
C C C C
This allows for multiple genotypes of the same virus to be
detected by incorporating different probes that recognize OH H H H
slightly different sequences. Because probes are amplified dNTP ddNTP
and not the target, this method can be used to quantify the Dideoxyribonucleotides lack the 3′ hydroxyl group
amount of target actually present. necessary for formation of the phosphodiester bond during DNA
The bDNA signal amplification assay has been applied to the replication. (Adapted from Buckingham L. Molecular Diagnostics.
qualitative and quantitative detection of HBV, HCV, and HIV-1. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, with permission.)
flanking the region of DNA to be sequenced primes DNA syn- identify important variants.60 HLA sequence-based typing
thesis (Fig. 12–27). Unlike PCR and other methods that re- uses this method.61,62
quire two primers, sequencing proceeds on only one DNA Germline or inherited variations in the DNA sequence are
strand, thus using a single primer. The primer is covalently readily detected, usually from blood specimens. Somatic
attached to a radioactive or fluorescent dye-labeled nucleotide (noninherited) mutations in clinical specimens, such as can-
at the 5′ end. cerous tumors, are sometimes difficult to detect as they may
All other components required for DNA synthesis are added be diluted by normal sequences that mask the somatic
to the primer and single-stranded template, including the four change.
dNTP bases. This DNA synthesis reaction results in polymer-
ization of deoxyribonucleotides to make full-length copies of Pyrosequencing
the DNA template. For sequencing, the reactions mixture is
aliquoted to four tubes and a different ddNTP is added to each Pyrosequencing is an alternate sequencing method developed
of the four reaction aliquots. DNA synthesis will stop upon in the 1980s.63,64 The procedure relies on the generation of
incorporation of a ddNTP into the growing DNA chain light (luminescence) when nucleotides are added to a growing
(chain termination) because without the hydroxyl group at the strand of DNA. With this system, there are no gels, fluorescent
3′ sugar carbon, the 5′–3′ phosphodiester bond cannot be dyes, or ddNTPs.
made. The newly synthesized chain therefore terminates when In the pyrosequencing reaction mix, a single-stranded DNA
it encounters the ddNTP (Fig. 12–28). The result of the template is mixed with a sequencing primer, enzyme, and sub-
sequencing reaction is a collection of fragments termed the strate mixes. The pyrosequencer introduces dNTPs sequentially
DNA ladder. The terminated chains can be resolved by gel elec- to the reaction. If the introduced nucleotide is complementary
trophoresis to yield the band pattern. to the base in the template strand next to the 3′ end of the
In the current sequencing methods (cycle sequencing, primer, DNA polymerase forms a phosphodiester bond between
dye-terminator sequencing), reactions with all four of the the primer and the nucleotide, releasing pyrophosphate (PPi)
ddNTPs take place in a single tube. The region of the sample (Fig. 12–30, left panels; also see DNA replication). The PPi is
DNA to be sequenced is first amplified by PCR. The double- converted to ATP to energize generation of a luminescent signal.
stranded PCR product, cleaned of residual PCR reaction This signal indicates that the introduced nucleotide is the
components, is the sequencing template. In dye-terminator correct base in the sequence. The pyrosequencing reaction gen-
sequencing, each ddNTP is labeled with a different fluores- erates a pyrogram of luminescent peaks associated with the ad-
cent dye (ddATP green, ddCTP blue, ddGTP black, ddTTP dition of the complementary nucleotide (see Fig. 12–30, right
red) so that the products of the sequencing reaction are dis- panel).
tinguished by color. Because pyrosequencing produces short- to moderate-
The fluorescently labeled DNA ladder is resolved by gel or length sequence information (up to 100 bases), it is not as ver-
capillary gel electrophoresis. Gel-based resolution will result satile as Sanger sequencing, which can produce reads longer
in a series of bands of different sizes. The DNA sequence is than 1,000 bases, especially for de novo sequencing. Two
read from the bottom to the top of the gel (smallest to largest) factors have kept pyrosequencing in use. First, because pyrose-
by which ddNTP terminated each fragment. Sequencing results quencing is less labor intensive than Sanger sequencing, it is
from capillary gel electrophoresis are a series of fluorescent more convenient for these types of short sequence analyses.
peaks, or an electropherogram (Fig. 12–29). The nucleotide Second, new instruments developed with the introduction of
sequences are read automatically by sequence analysis software genomic sequencing or NGS use the pyrosequencing chemistry
and supplied in textual form (ACGT). because NGS also relies on repeated sequencing of short tem-
Software programs interpret and apply sequence data plates. Pyrosequencing is currently used in both of these
from automatic sequencers. These programs collect the raw capacities.
data and interpret data quality. The programs report the
certainty of each nucleotide base in the sequence and com- Next Generation Sequencing (NGS)
pare the sequence with a reference sequence or database to
The first human genome sequence was performed by chain
termination (Sanger) sequencing. The 7-year Human Genome
Project involved hundreds of sequencers and bioinformatics ex-
Primer perts and cost billions of dollars. Using NGS, a human genome
5! –3! OH
now can be sequenced by a single sequencer in a few hours for
3! … T C G A C G G G C … 5!
Template
fewer than $1,000. NGS is designed to sequence large numbers
Area to be of templates simultaneously, yielding hundreds of thousands of
sequenced short sequences in a single run. These short sequences are then
The labeled sequencing primer hybridizes to the assembled into a complete genome. The development of NGS
template to provide a 3′OH group for formation of a phosphodiester was stimulated in part by a goal to sequence the human
bond by DNA polymerase. (Adapted from Buckingham L. Molecular genome for a minimal cost (less than $1,000) in order to bring
Diagnostics. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, with permission.) what were once expensive genomic studies into the realm of
 Growing strand

O" P O O" P O
Template strand
O O
O O
A T A T
H2C CH CH2 H2C CH CH2
HC CH2 HC CH2

O O

O" P O O" P O

O O

H2C O H2C O
G C G C
CH CH2 CH CH2
HC CH2 HC CH2

OH
Chain termination

O O O O O O

O" P O P O P O" O" P O P O P O"

C G C G
O" O" O O" O" O

O O
H2C CH CH H2C CH CH
HC CH2 HC CH2

A OH B OH
(A) DNA synthesis proceeds by formation of a phosphodiester bond between the ribose 3′OH of a previous nucleotide and the ribose 5′ phosphate group of the incoming
nucleotide. (B) If the 3′OH is missing, as in ddNTP, synthesis terminates (right). (Adapted from Buckingham L. Molecular Diagnostics. 2nd ed. Philadelphia, PA: F.A. Davis; 2011, with permission.)
 G A T C 3! 3! A G T C T G 5!

G
ddA
T
dAddG

dAdGddT C

T
dAdGdTddC

G
dAdGdTdCddT
A
dAdGdTdCdTddG
5!
Gel

Capillary
A sequencing ladder (left) resolved by gel electrophoresis is read from the bottom of the gel to the top of the gel (shortest
fragment to the longest). The terminating base is determined by its tube (gel lane). In dye-terminator sequencing, the sequence is read
automatically as fluorescently labeled fragments migrate through the gel or capillary. Each fragment passes a detector that will generate
an electropherogram of fluorescent peaks (right). Sequencing software will produce a text report of the DNA sequence.

Step 1
Polymerase
(DNA)n # dNTP (DNA)n#1 # PPi

Step 2 Luciferin Oxyluciferin

Pyrosequencing
detects nucleotide sequence by Sulfurylase Luciferase
introducing dNTPs in a predeter-
mined order to the sequencing
reaction. If the nucleotide is not APS # PPi
Light

complementary to the template ATP

sequence, no reaction occurs Light
and the nucleotide is then
degraded. If the nucleotide is Nucleotide sequence
complementary to the sequence,
G C " A GG CC T Time
polymerase forms a phosphodi-
ester bond extending the primer
and releasing pyrophosphate
(PPi, Step 1). The pyrophosphate
then goes through a series of
reactions to ultimately produce a
chemiluminescent signal (Step 2).
The pyrosequencing output is a
pyrogram, as shown on bottom.
(Adapted from Buckingham L.
Molecular Diagnostics. 2nd ed. G C T A G C T
Philadelphia, PA: F.A. Davis; 2011,
with permission.) Nucleotide added
clinical analysis. Thousands of genomes have been sequenced for amplification. Hundreds of thousands of products from
by NGS as part of the 1,000 Genome Project. The goal of this the library can be sequenced simultaneously.
project was to provide reference and variant information from For all NGS varieties, labeled nucleotides are used. Then
ethnically diverse genomes.65 The sequencing cost does not, after amplification, instrument software collects the sequence
however, include the expenses associated with interpretation, data in the form of processed images. The sequence quality is
reporting, and data storage. These issues are currently being assessed and then the sequence is provided as a textual report.
addressed to enable broader implementation of NGS in clinical After a successful NGS run, areas of interest will have been
analysis. sequenced from 30 to hundreds of times.
With NGS technology, DNA must be cut to create frag- NGS has a variety of clinical applications including mito-
ment libraries. Then these short fragments, usually 100 to chondrial genome sequencing, sequencing for inherited disease,
300 bp, are amplified so they can be analyzed. Adaptor se- and gene panel sequencing for known somatic mutations. One
quences are added to these fragments and serve as primers of its most important uses is in HLA high-resolution typing.66,67

SUMMARY sequence, is used to detect an unknown nucleic acid

sequence.
• The two main types of nucleic acids are deoxyribonucleic • Hybridization techniques include Southern blot analysis,
acid (DNA) and ribonucleic acid (RNA). They are poly- microarray technology for simultaneous assessment of
mers made up of chains of nucleotides. multiple genes, and fluorescent in situ hybridization of
• Nucleotides of DNA contain a deoxyribose sugar with specific genetic regions.
one of the following bases: adenine, guanine, thymine, or • Amplification involves copying of a specific nucleic acid
cytosine. sequence in order to obtain enough to identify it.
• RNA is made up of nucleotides containing a ribose sugar • Amplification methods include polymerase chain reac-
bonded to a similar nitrogen base as in DNA, except that tion (PCR), reverse transcriptase PCR (RT-PCR), and
instead of thymine, uracil is used. quantitative PCR (qPCR). All of these methods amplify
• DNA is double stranded and arranged in a double helix, the target DNA.
whereas RNA is typically single stranded. • In transcription-mediated amplification, the target is RNA
• In a DNA molecule, specific base pairing occurs: adenine instead of DNA. A cDNA copy is made of the original RNA
pairs with thymine and guanine pairs with cytosine. and used to produce millions of RNA copies.
• When a DNA molecule replicates, the two daughter • The ligase chain reaction amplifies probes rather
strands separate; each is a template for a newly synthesized than the target DNA. Two primers attach to the target
complementary strand. DNA sequence and then the primers are joined and
• The high specificity of detection of nucleic acid sequences amplified.
through complementary base pairing is the basis of all ap- • Strand displacement amplification (SDA) also involves
plications in clinical laboratory science. amplification of a probe rather than the original target
• The central dogma of molecular biology refers to the fact DNA.
that DNA serves as the template for messenger RNA, • Branched DNA represents a signal amplification method
which in turn codes for proteins. in which multiple probes attach to the original target se-
• Mutations and polymorphisms are changes in nucleotide quence DNA.
sequences that may affect specific protein sequences. • DNA sequencing allows for detection of single nucleotide
• Restriction fragment length polymorphisms are changes in polymorphisms and mutations not easily detectable with
DNA that result in different size pieces when cleaved by re- restriction enzyme mapping.
striction enzymes. • Next generation sequencing methods allow for sequencing
• Hybridization is the very specific binding of two com- of a large number of small DNA sequences at one time.
plementary DNA strands or a DNA and an RNA strand. Whole genomes can be quickly and easily sequenced to
Often a probe, which has a short known nucleic acid look for inherited diseases.
CASE STUDIES
1. The FMS-like tyrosine kinase 3 (FLT3) gene represents one c. Suppose the normal control yielded three products:
of the most frequently encountered mutations in acute 80 bp, 59 bp, and 11 bp. How would this affect the
myeloid leukemia. The FLT3 mutation status aids in clin- interpretation?
ical decisions for treatment strategy. One of the FLT3 mu- d. Suppose the internal control yielded a 150 bp product.
tations falls in the active enzyme region of the gene (kinase How would this affect the interpretation?
domain). A test for the FLT3 kinase domain gene mutation 2. A blood sample from a patient tested positive for the
is performed using PCR followed by digestion with the re- presence of HIV antibodies. A molecular test for the
striction enzyme, EcoRV. The gene mutation changes the presence of HIV by qPCR was performed. The test can
recognition site of EcoRV (GATATC) such that the enzyme detect 50 to 1,000,000 viral copies per mL plasma.
will not cut the DNA. For the test, DNA is isolated and the Previous results had shown the presence of the virus at
region containing the mutation is amplified by PCR using levels of 1,500, 600, 500, 220, and 100 copies per mL
primers that specifically hybridize and allow amplification over the course of treatment. The results of the qPCR
of that region. The resulting PCR product is 150 bp. The test for the current specimen were negative. However,
restriction enzyme digestion will separate the product into the internal amplification control for the qPCR test was
three pieces—80 bp, 59 bp, and 11 bp—in the normal also negative.
DNA sequence. If the mutation is present, the restriction
Questions
enzyme digestion will produce only two fragments, 139 bp
and 11 bp. After digestion, the products of the reaction are a. How would you interpret these results?
separated by gel electrophoresis and the size is determined b. The test was repeated; this time, the target (HIV) am-
by the distance the fragments travel in the gel under the plification was negative whereas the amplification
force of the electric current. control was positive. How would you interpret these
results?
Questions c. To prepare the test report, the results are entered
a. List the controls that would be used for the PCR reac- along with the sensitivity of the test (50 copies/mL).
tion and restriction enzyme reactions. Should these results be reported as 0 copies/mL
b. The 11 bp fragment may not be detectable on some because nothing was detected by this qPCR test?
resolution systems. Would this preclude performance
of the test by those methods?

REVIEW QUESTIONS
1. What holds two single strands of DNA together in a 3. How are DNA and RNA different?
double helix? a. Only RNA contains uracil.
a. 2′ carbon of deoxyribose attached to a hydroxyl b. Only DNA contains cytosine.
group c. DNA is usually single stranded.
b. Hydrogen bonds between A and T and C d. DNA is less stable than RNA.
and G
c. Ribose 3′ carbon hydroxyl attached to ribose 4. What is the function of restriction endonucleases?
5′ carbon phosphate a. They splice short pieces of DNA together.
d. Phosphorylation of nitrogen bases b. They cleave DNA at specific sites.
c. They make RNA copies of DNA.
2. What is the complement to the following DNA d. They make DNA copies from RNA.
sequence?
5′-GATCGATTCG-3′ 5. What is the purpose of somatic hypermutation
in genes that code for antibodies?
a. 3′-CTAGCTAAGC-5′
b. 3′-CGAATCGATC-5′ a. To increase diversity of the immunoglobulin
c. 3′-GCTTAGCTAG-5′ repertoire
d. 3′-GATCGATTCG-5′ b. To prevent further antibody formation
c. To switch antibodies from IgM to IgG
d. To prevent further VDJ recombination
 6. What characteristic distinguishes restriction enzymes 13. Which method is a signal amplification system?
from one another? a. bDNA
a. Diverse binding and cutting sites b. qPCR
b. Ability to quickly digest DNA c. PCR
c. RNA degradation capability d. Digital PCR
d. Ability of one enzyme to recognize several different
binding sites 14. Which of the following amplifications is isothermal?
a. PCR
7. Which of the following is used in a Southern blot b. qPCR
procedure? c. NASBA
a. A ligase joins two adjacent probes. d. LCR
b. Radiolabeled nucleotides are used to synthesize
DNA.
c. DNA is cleaved by enzymes and electrophoresed.
d. Many probes are placed on a small piece of glass. 15. Consider the following results for a qPCR test for the
presence of herpes virus:
8. Which best describes the PCR? Sample Ct/Sample Ct/Amp Control
a. Two probes are joined by a ligating enzyme. A 22.10 21.06
b. RNA copies of the original DNA are made.
B 35.02. 20.99
c. Extender probes are used to create a visible
product. Which of the following statements is true?
d. Primers are used to make multiple DNA a. The HSV viral load in sample A is greater than in
copies. sample B.
b. The HSV viral load in sample B is greater than in
9. What takes place during in situ hybridization? sample A.
a. RNA polymerase copies messenger RNA. c. The HSV viral load in sample A is about the same
b. Hybridization takes place in solution. as in sample B.
c. Nucleic acid probes react with intact cells in d. The absolute number of viral particles in sample B
tissues. is greater than in sample A.
d. Probes are protected from degradation if
hybridized. 16. Which terminates chains when added to a DNA
replication reaction?
10. What determines the specificity for PCR? a. dNTPs
a. Nucleotide mix ratios and concentrations b. ddNTPs
b. Mono- and divalent cation concentrations c. Sequencing primer
c. Primers and their annealing temperature d. DNA polymerase
d. DNA polymerase
17. Which technique involves probe amplification rather
11. An antibody test for HIV within 3 months of exposure than target amplification?
is negative. Does this guarantee a negative PCR test? a. Southern blot
a. Yes, because if no antibodies are present, no virus b. PCR
is present. c. Transcription-mediated amplification
b. No, PCR-detectable virus may be present before d. Ligase chain reaction
generation of detectable antibodies.
c. Yes, it has been 3 months since exposure. 18. How does next generation sequencing (NGS) technol-
d. No, but the PCR test will be less sensitive than the ogy differ from the original Sanger chain displacement
antibody test. sequencing?
a. NGS is more expensive to conduct than chain
12. How do PCR and qPCR differ? displacement sequencing.
a. In qPCR, the results can be seen at the end of b. NGS can sequence thousands of DNA pieces faster
each cycle. than Sanger sequencing.
b. SYBR Green is only used in PCR. c. Sanger sequencing involves ligation and NGS
c. PCR is an isothermal process and qPCR is not. does not.
d. Internal amplification controls are not necessary d. Only Sanger sequencing has direct clinical
in qPCR. applications.
19. Which of the following methods best describes a 21. What is the difference between a polymorphism and
nucleic acid probe? a mutation?
a. It attaches to double-stranded DNA. a. Mutations only affect A and T bases.
b. It is used in transcription-mediated amplification. b. Mutations are more frequently present in a
c. It is used to detect specific single-stranded DNA population.
sequences. c. Polymorphisms are more frequently present in a
d. It plays a key role in DNA chain termination population.
sequencing. d. Polymorphisms are easier to detect than mutations.

20. A hybridization reaction involves which of the

following?
a. Separating DNA strands by heating
b. Binding of two complementary DNA strands
c. Increasing the number of DNA copies
d. Cleaving DNA into smaller segments
 Flow Cytometry and
Laboratory Automation

After finishing this chapter, you should be able to: CELL FLOW CYTOMETRY
1. List and describe the function of each of the major components of a Instrumentation
flow cytometer. Data Acquisition and Analysis
2. Compare intrinsic and extrinsic parameters in flow cytometry. Sample Preparation
3. List the advantages and disadvantages of automated testing in a Clinical Applications
clinical immunology laboratory. IMMUNOASSAY AUTOMATION
4. Summarize the principle of hydrodynamic focusing within the flow Validation
cytometer.
SUMMARY
5. Define the concept of fluorescence in flow cytometry.
CASE STUDIES
6. Explain the difference between forward-angle light scatter (FSC) and
REVIEW QUESTIONS
side scatter (SSC).
7. Describe the difference between analyzing flow cytometry data using
single-parameter histograms and dual-parameter dot plots.
8. List several clinical applications for flow cytometry.
9. Apply knowledge of various T- and B-cell surface antigens to identify
various cell populations.
10. Compare advantages and disadvantages of automated immunoassay
analyzers.
11. Describe the difference between a random access and a batch analyzer.
12. Define accuracy, precision, reportable range, analytic sensitivity, analytic
specificity, and reference interval.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Accuracy Dual-parameter dot plot Gate Reference interval
Analytic sensitivity Extrinsic parameters Immunophenotyping Reportable range
Analytic specificity Flow cytometry Intrinsic parameters Side (right angle)
Automatic sampling Fluorochrome Precision scatter (SSC)
Batch analyzers Forward-angle light Random access analyzers Single-parameter histogram
scatter (FSC)

Cell Flow Cytometry

For cellular parameters to be accurately measured in the flow
Cell flow cytometry is an automated system in which single cytometer, it is crucial that cells pass through the laser one
cells (or beads) in a fluid suspension are analyzed in terms cell at a time. Cells are processed into a suspension; the cy-
of their intrinsic light-scattering characteristics. The cells are tometer draws up the cell suspension and injects the sample
simultaneously evaluated for their extrinsic properties (i.e., inside a carrier stream of isotonic saline (sheath fluid) to form
the presence of specific surface or cytoplasmic proteins) a laminar flow. The sample stream is constrained by the car-
using fluorescent-labeled antibodies or probes. Flow cytome- rier stream and is thus hydrodynamically focused so that the
ters, originally developed in the 1960s, did not make their cells pass single file through the intersection of the laser light
way into the clinical laboratory until the early 1980s. At that source (Fig. 13–1). Each time a cell passes in front of a laser
point, physicians started seeing patients with a new myste- beam, light is scattered and the interruption of the laser signal
rious disease characterized by a drop in circulating T helper is recorded.
(Th) cells. Since that time, flow cytometry has been routinely
used for identifying infection with HIV, as well as immunophe-
notyping cells—identifying both their surface and cytoplas- Solid-state diode lasers are typically used as light sources. The
mic antigen expression. Cytometers can simultaneously wavelength of monochromatic light emitted by a laser in turn
measure multiple cellular or bead properties by using several dictates which fluorochromes can be used in an assay. Not all
different fluorochromes. A fluorochrome, or fluorescent fluorochromes can be used with all lasers because each fluo-
molecule, is one that absorbs light across a spectrum of wave- rochrome has distinct spectral characteristics. The newer clin-
lengths and emits light of lower energy across a spectrum of ical instruments have three lasers—red, blue, and violet—each
longer wavelengths. Each fluorochrome has a distinctive spec- of which produces different colors when exciting a particular
tral pattern of absorption (excitation) and emission. By using fluorochrome. This allows for as many as 10 fluorochromes,
laser light, different populations of cells or particles can be or colors, to be analyzed in a single tube at one time.
analyzed and identified on the basis of their size, shape, and As a result of a cell passing through the laser, light is scat-
antigenic properties. tered in many directions. The amount and type of light scatter
Flow cytometry is frequently used in leukemia and lym- (LS) can provide valuable information about a cell’s physical
phoma diagnostics. In addition, flow cytometry has been properties. Light at two specific angles is measured by the
used in functional assays for chronic granulomatous disease flow cytometer: forward-angle light scatter (FSC) and side
(CGD) and leukocyte adhesion deficiency, fetal red blood cell scatter (SSC), also called right angle light scatter (SSC). FSC
(RBC) and F-cell identification in maternal blood (replacing is considered an indicator of size, whereas the SSC signal is
the Kleihauer-Betke assay), and identification of paroxysmal indicative of granularity or the intracellular complexity of the
nocturnal hemoglobinuria (PNH), to give just a few exam- cell. Thus, these two values, which are considered intrinsic
ples.1,2 Flow cytometry has now become the platform for parameters, can be used to characterize different cell types.
detection of various genetic disease mutations such as cystic If one looks at a sample of whole blood on a flow cytometer
fibrosis, as well as tissue typing for transplantation and where all the RBCs have been lysed, the three major popula-
autoimmune and infectious disease detection. A significant tions of white blood cells (WBCs)—lymphocytes, monocytes,
advantage of flow cytometry is that because the flow rate of and neutrophils—can be roughly differentiated from each
cells within the cytometer is so rapid, thousands of events other based solely on their intrinsic parameters (FSC and SSC)
can be analyzed in seconds, allowing for the accurate detec- (Fig. 13–2).
tion of cells that are present in very small numbers. Unlike FSC and SSC, which represent light-scattering
properties that are intrinsic to the cell, extrinsic parameters
require the addition of a fluorescent probe for their detection.
Instrumentation Fluorescent-labeled antibodies bound to the cell can be de-
The major components of a flow cytometer include the fluidics, tected by the laser. By using fluorescent molecules with various
the laser light source, and the optics and photodetectors. The emission wavelengths, the laboratorian can simultaneously
data analysis and management is done by computer.3 evaluate an individual cell for several extrinsic properties. The
 Computer

Printer

Electrical
signal

Air pressure

Cells in
liquid suspension Dichroic Filter
mirror

Sheath fluid Fluorescence

detectors

Lens
Filter Side scatter
detector
Laser
Lens

Forward angle
light scatter detector
Charged plates can
deflect drops to
separate population
Flow cytometry. Components of a laser-based flow cytometer include the fluidics system for cell transportation, a laser for cell
illumination, photodetectors for signal detection, and a computer-based management system. Both forward and 90-degree LS are measured,
indicating cell size and type.

clinical utility of such multicolor analysis is enhanced when used to stain the cells. The newer flow cytometers actually use
the fluorescent data are analyzed in conjunction with FSC and fiber-optic cables to direct light to the detectors.
SSC.4 The combination of data allows for characterization of When fluorescent light from fluorescently tagged antibod-
cells according to size, granularity, DNA and RNA content, ies bound to cell surfaces reaches the photomultiplier tubes,
antigens, total protein, and cell receptors.3 it creates an electrical current that is converted into a voltage
pulse. The voltage pulse is then converted (using various
methods, depending on the manufacturer) into a digital signal.
The various signals (light scatter and fluorescence) generated by
The digital signals are proportional to the intensity of light
the cells’ interaction with the laser are detected by photodiodes
detected. The intensity of these converted signals is measured
for FSC and by photomultiplier tubes for fluorescence. The
on a relative scale that is generally set into 1 to 256 channels,
number of fluorochromes capable of being measured simulta-
from lowest energy level or pulse to the highest level.
neously depends upon the number of photomultiplier tubes in
the flow cytometer. The specificity of each photomultiplier tube
for a given band length of wavelengths is achieved through the
Data Acquisition and Analysis
arrangement of a series of optical filters that are designed to max- Once the intrinsic and extrinsic cellular properties of many cells
imize collection of light derived from a specific fluorochrome (typically 10,000 to 20,000 “events” are collected for each sam-
while minimizing collection of light from other fluorochromes ple) have been collected and the data digitalized, it is ready for
 interest and analyze various parameters (extrinsic and intrinsic)
of the cells contained within the gated region (Fig. 13–4). The
gate allows the operator to screen out debris and isolate sub-
populations of cells of interest. Gates can be thought of as a set
of filtering rules for analyzing a very large database. The oper-
ator can basically filter the data in any way and set multiple or
sequential filters (or gates).
When analyzing a population of cells using a dual-parameter
dot plot, the operator chooses which parameters to analyze on
both the x and y axes. He or she then divides the dot plot into
four quadrants, separating the positives from the negatives in
each axis (Fig. 13–5). Quadrant 1 consists of cells that are pos-
itive for fluorescence on the y axis and negative for fluorescence
on the x axis. Quadrant 2 consists of cells that are positive for
Peripheral blood leukocyte analysis by simultaneous fluorescence on both the x and y axes. Quadrant 3 consists of
evaluation of forward-angle light scatter (FSC) and 90-degree LS cells that are negative for fluorescence on both the x and y axes.
(SSC). Based on the intrinsic characteristics of size (FSC) and granu-
Quadrant 4 consists of cells that are positive for fluorescence
larity (SSC) only, the three main populations of WBCs (lymphocytes,
monocytes, and neutrophils) can be discriminated into individual
on the x axis and negative for fluorescence on the y axis. The
populations. computer will then calculate the percentage of cells in each
quadrant based on the total number of events counted (typically
10,000 to 20,000 events per sample). For example, a gate can
analysis by the operator. Each parameter can be analyzed inde- be drawn around a population of cells based on their FSC versus
pendently or in any combination. Graphics of the data can be SSC characteristics and the extrinsic characteristics of the gated
represented in multiple ways. The first level of representation is population can be analyzed—that is, lymphocytes can be gated,
the single-parameter histogram, which plots a chosen parame- after which the subpopulations of T cells (CD3+ and CD4+ or
ter (generally fluorescence) on the x axis versus the number of CD8+) and B cells (CD38+, CD3–) can be analyzed (Fig. 13–6).
events on the y axis; thus, only a single parameter is analyzed The absolute count of a particular cell type—for instance, CD4+
using this type of graph (Fig. 13–3). The operator can then set T lymphocytes—is obtained by multiplying the absolute cell
a marker to differentiate between cells that have low levels of flu- count of the population of interest (e.g., lymphocytes) derived
orescence (negative) from cells that have high levels of fluores- from a hematology analyzer by the percentage of the population
cence (positive) for a particular fluorochrome-labeled antibody. of interest in the sample (CD3+ and CD4+ lymphocytes).3,4 This
The computer will then calculate the percentage of “negative” and method is considered a dual-platform analysis. The disadvantage
“positive” events from the total number of events collected. to this type of analysis is the greater potential for added error as-
The next level of representation is the bivariate histogram, sociated with using two distinct methods to derive the absolute
or dual-parameter dot plot, where each dot represents an in- count. The single platform is now the method of choice to elim-
dividual cell or event. Two parameters, one on each axis, are inate this type of error. In this method, a known quantity of
plotted against each other. Each parameter to be analyzed is beads is added to the flow cytometry tubes and a simple math
then determined by the operator. Using dual-parameter dot calculation allows the direct calculation of absolute numbers
plots, the operator can draw a “gate” around a population of from the individual flow cytometry tubes.
80 120 160 200

M1
Counts
40
0

100 101 102 103 104

Fluorescent intensity
Example of a single-parameter flow histogram. The
y axis consists of the number of events. The x axis is the parameter to
be analyzed, which is chosen by the operator; it is usually an extrin- A dual-parameter dot plot. Both parameters on the
sic parameter, such as a fluorescent-labeled antibody. The operator x and y axes are chosen by the operator. In this case, lysed whole
can then set a marker to isolate the positive events. The computer blood is analyzed on CD45 (x axis) and SSC (y axis). The operator
will then calculate the percentage of positive events within the then draws a “gate” or isolates the population of interest (e.g.,
designated markers. granulocytes) for further analysis.
 1 2 collection. Heparin can also be used for whole blood and bone
marrow and can provide improved stability in samples over
24 hours old. Blood should be stored at room temperature
(20°C to 25°C) before processing and should be well mixed
before being pipetted into staining tubes.4,6 Hemolyzed or clot-
ted specimens should be rejected. Peripheral blood, bone mar-
row, and other samples with large numbers of RBCs require
erythrocyte removal to allow for efficient analysis of WBCs.
3 4 Historically, density gradient centrifugation with Ficoll-
Hypaque (Sigma, St. Louis, MO) was used to generate a cell
suspension enriched for lymphocytes or lymphoblasts. How-
ever, this method results in selective loss of some cell popula-
FL2 tions and is time consuming.7 Density gradient centrifugation
FL1 has mainly been replaced by erythrocyte lysis techniques, both
commercial and noncommercial.7
Tissue specimens should be collected and transported in
tissue culture medium (RPMI 1640) at either room tempera-
ture (if analysis is imminent) or 4°C (if analysis will be de-
layed). The specimen is then disaggregated to form a single cell
suspension, either by mechanical dissociation or enzymatic
digestion. Mechanical disaggregation, or “teasing,” is preferred
and is accomplished by the use of a scalpel and forceps, a nee-
dle and syringe, or wire mesh screen.8 Antibodies are then
added to the resulting cellular preparation and processed for
analysis. The antibodies used are typically monoclonal, each
with a different fluorescent tag.

Clinical Applications
Routine applications of flow cytometry in the clinical laboratory
include immunophenotyping of peripheral blood lymphocytes,
enumeration of CD34+ stem cells in peripheral blood and bone
Quadrant analysis of a dual-parameter dot plot. The marrow for use in stem cell transplantation, and immunophe-
operator chooses which parameters to analyze on each axis. (A) On
notypic characterization of acute leukemias, non-Hodgkin’s
each axis there are positive (fluorescence positive) and negative
lymphomas, and other lymphoproliferative disorders.
(fluorescence negative) cells. (B) Example of a dual-parameter dot
plot to identify CD4+ T cells: CD3 on the x axis and CD4 on the y axis. Immunophenotyping by flow cytometry has become an
The cells in quadrant 2 that are positive for both CD3 and CD4 are important component of initial evaluation and subsequent
true CD4+ T cells. post-therapeutic monitoring in leukemia and lymphoma man-
agement. Flow cytometric findings have been incorporated
into current leukemia and lymphoma classifications, begin-
Detailed phenotypic analysis can determine the lineage and ning with the Revised European-American Lymphoma (REAL)
clonality, as well as the degree of differentiation and activation classification in 1994 and, more recently, in the proposed
of a specific cell population. This information is useful for World Health Organization (WHO) classifications.9,10 One of
differential diagnosis or clarification of closely related lympho- the most important components of flow cytometric analysis
proliferative disorders. Immunophenotyping requires careful is the stratification of hematopoietic malignancies by their
selection of combinations of individual markers based on a lineage (i.e., B cell, T cell, or myeloid) and the degree of dif-
given cell lineage and maturation.4 Attempts to standardize in- ferentiation. Some of the more common cell-differentiation
dividual marker panels, especially by European laboratory antigens are listed in Table 13–1.11,12
groups, are ongoing; however, multiple consensus panels vary Knowing unique characteristics of leukemias and lymphomas
from institution to institution.5 and pairing those particular markers that identify these charac-
teristics can be useful in making a more reliable diagnosis. For
example, in chronic lymphocytic leukemia (CLL), typically CD5
Sample Preparation (T-cell lymphocyte marker) is paired with CD20 (B-cell lympho-
Samples commonly used for analysis include whole blood, cyte marker). The presence of a significant number of cells that
bone marrow, and fluid aspirates. Whole blood should be col- are both CD5+ and CD20+ is an indication of CLL or mantle
lected into ethylenediaminetetraacetic acid (EDTA), the anti- cell lymphoma. Common markers and fluorochrome conjugate
coagulant of choice for samples processed within 30 hours of combinations are demonstrated in Table 13–2.
 Gating strategy to analyze lymphocyte subsets in a sample of whole blood. Whole blood is incubated with fluorescent-labeled
antibodies specific for CD3, CD4, CD8, and HLA-DR. The sample is washed, RBCs are lysed, and the sample is analyzed on the flow cytometer.
To analyze using gating strategies, the sample is first plotted on FSC versus SSC. (A) A gate, or region, is drawn around the lymphocyte popula-
tion. (B) On the subsequent plots of fluorescent markers, only the lymphocyte population is analyzed. The dot plot is divided into four quad-
rants to isolate positive from negative populations. The computer calculates the percentage of positive cells in each quadrant. The three flow
contour plots are analyzing two different cell surface markers. In the first dot plot, quadrant 2 (upper right) identifies CD4+, CD3+ T helper cell
lymphocytes. Quadrant 3 (lower left) identifies B lymphocyte and NK cells. Quadrant 4 (lower right) identifies CD3+ CD4– T-cell lymphocytes.
In the second dot plot, quadrant 1 (upper left) identifies low intensity CD8+ CD3– NK cells. Quadrant 2 identifies CD3+ CD8+ T-cytotoxic
lymphocytes. Quadrant 3 identifies any lymphocyte that is not a CD8+, CD3+, or B-cell lymphocyte. Quadrant 4 identifies CD3+ CD8–
T helper cell lymphocytes. In the third contour plot, quadrant 1 identifies CD38+ CD3– B cells, quadrant 2 identifies CD38+ CD3+ activated
T cells, quadrant 3 identifies CD38– CD3– cells, and quadrant 4 identifies CD3+ CD38– T cells, not activated.

Table 13–1 Surface Markers on Leukocytes

ANTIGEN CELL TYPE FUNCTION
CD2 Thymocytes, T cells, NK cells Involved in T-cell activation
CD3 Thymocytes, T cells Associated with T-cell antigen receptor; role in TCR
signal transduction
CD4 T helper cells, monocytes, macrophages Co-receptor for class II MHC; receptor for HIV
CD5 Mature T cells, thymocytes, subset of Positive or negative modulation of T- and B-cell
B cells (B1) receptor signaling
CD7 T cells, thymocytes, NK cells, pre-B cells Regulates peripheral T-cell and NK cell cytokine
production
CD8 Thymocyte subsets, cytotoxic T cells Co-receptor for class I MHC
Table 13–1 Surface Markers on Leukocytes—cont’d
ANTIGEN CELL TYPE FUNCTION
CD10 B- and T-cell precursors, bone marrow Protease; marker for pre-B CALLA
stromal cells
CD11b Myeloid and NK cells αM subunit of integrin CR3, binds complement
component iC3b
CD13 Myelomonocytic cells Zinc metalloproteinase
CD14 Monocytic cells Lipopolysaccharide receptor
CD15 Neutrophils, eosinophils, monocytes Terminal trisaccharide expressed on glycolipids
CD16 Macrophages, NK cells, neutrophils Low affinity FC receptor, mediates phagocytosis
and ADCC
CD19 B cells, follicular dendritic cells Part of B-cell co-receptor, signal transduction molecule
that regulates B-cell development and activation
CD20 B cells, T-cell subsets Binding activates signaling pathways, regulates B-cell
activation
CD21 B cells, follicular dendritic cells, subset of Receptor for complement component C3d; part of
immature thymocytes B-cell co-receptor with CD19
CD22 B cells B, B-ALL (surface and cytoplasmic), B-CLL, HCL, PLL
CD23 B cells, monocytes, follicular dendritic Regulation of IgE synthesis; triggers release of IL-1,
cells IL-6, and GM-CSF from monocytes
CD25 Activated T cells, B cells, monocytes Receptor for IL-2
CD33 Myeloid cell Monocytes, macrophages, granulocytes (weak),
myeloid-Pro, myeloid leukemia, AML
CD34 Hematopoietic progenitor cell Hematopoietic-Pre, endothelial cells, immature
leukemias
CD38 Plasma cell Plasma cells, thymocytes, NK lymphocytes, early B,
monocytes, multiple myelomas, ALL, acute
myeloblastic leukemia
CD44 Most leukocytes Adhesion molecule mediating homing to peripheral
lymphoid organs
CD45 All hematopoietic cells Tyrosine phosphatase, augments signaling
CD45R Different forms on all hematopoietic cells Essential in T- and B-cell antigen-stimulated activation
CD56 NK cells, subsets of T cells Not known
CD57 NK cell sub, T-cell sub, small cell lung carcinoma
CD94 NK cells, subsets of T cells Subunit of NKG2-A complex involved in inhibition of
NK cell cytotoxicity
CD103 Intraepithelial lymphocytes HCL, adult T-cell leukemia
CD138 Plasma cell, pre-B (weak)
HLA-DR B lymphocytes, activated T lymphocytes, Lack of expression diagnostic of M3 myeloid
monocytes leukemia, T-cell activation
FCM7 B-cell subset, mantle lymphoma Expressed in mantle cell lymphoma
Kappa chains B cells Light chain part of antibody molecule on B cells
Lambda chains B cells Light chain part of antibody molecule on B cells

ADCC = antibody-dependent cell cytotoxicity; ALL= acute lymphocytic leukemia; AML= acute myeloid leukemia; CALLA = common acute lymphoblastic leukemia
antigen; CLL= chronic lymphocytic leukemia; FC = fragment crystallizable; GM-CSF = granulocyte-macrophage colony-stimulating factor; HCL = hairy cell leukemia;
HIV = human immunodeficiency virus; MHC = major histocompatibility class; NK = natural killer; PLL= prolymphocytic leukemia; TCR = CD3-αβreceptor complex.
Table 13–2 Common Markers Used for Lymphoproliferative and Myeloproliferative
Studies in Clinical Flow Cytometry
DISEASE ANTIBODIES PAIRED INTERPRETATION
Chronic lymphocytic FMC7 with CD23 FMC7 negative and CD23 positive in CLL, CD23 negative in
leukemia (CLL) and mantle cell lymphoma and FMC7 is positive
prolymphocytic CD5 with CD20 When a cell is both CD5 positive and CD20 positive: characteris-
leukemia tic of CLL and well-differentiated lymphocytic lymphoma
(WDLL), as well as mantle cell lymphoma
CD19 with kappa CD19 positive with only one light chain (kappa or lambda) is
CD19 with lambda expressed in low intensity; occasionally light chains are not
detected
CD45 with CD14 CD45 fluorescence is brightly expressed and CD14 negative
(Antiglycophorin added Used to determine erythroid component to the specimen
to bone marrows)
Hairy cell leukemia CD3 with CD23 CD3 negative and CD23 negative
(HCL) CD11c with CD22 Brightly expressed CD11c and CD22, unlike CLL
CD20 with CD5 CD20 positive and CD5 negative (occasionally weak expression
of CD5)
CD19 with kappa CD19 positive with one monoclonal light chain expressed
CD103 with CD25 CD103 is highly specific for HCL, and CD25 is usually expressed;
hairy cell variants may be negative for these two markers
CD21 with HLA-DR CD21 negative and DR positive
CD45 with CD14 CD45 is brightly expressed and CD14 negative
(Antiglycophorin added To determine erythroid component to the specimen, CD10
to bone marrows) (Calla) is weakly expressed in 26% of cases
B cell CD3 with HLA-DR CD3 (T-cell receptor) negative and DR positive
Acute lymphocytic CD5 with CD20 CD5 negative and CD20 variably positive (low intensity or
leukemia (ALL) negative on precursor B-cell ALL, positive on more mature
B-cell ALL)
CD19 with kappa CD19 positive (stem cell is negative) and surface Ig negative
CD19 with lambda (a mature B-cell ALL may have surface immunoglobulin)
CD34 with CD38 CD34 positive ALL correlates with good prognosis in pediatric
patients and poor prognosis in adults; CD38 is positive from
stem cell to pre-B
TdT with CD10 TdT and CD10 (Calla) positive in common ALL and pre-B ALL;
CD10 positivity is associated with favorable complete treatment
response and disease-free survival; CD10 is usually high
intensity
TdT with CD33 CD33 negative; however, very early B-ALL may be positive
CD45 with CD14 CD45 dimly expressed and CD14 negative
(Antiglycophorin added To determine erythroid component to the specimen
to bone marrows)
Myeloma plasmacytoid CD3 with HLA-DR CD3 negative; most are HLA-DR negative, although some early
leukemia or lymphoma plasmacytoid cells may be DR positive
CD5 with CD20 CD5 and CD20 negative
CD19 with kappa CD19 and surface Ig negative; occasionally surface Ig is positive;
CD19 with lambda cytoplasmic Ig positive
CD45 with CD38 CD45 negative or low intensity; CD38 is high-intensity positive
CD40 with CD56 Usually CD40 positive; CD56 has been reported to be positive on
myeloma cells but negative on normal plasma cells
CD10 CD10 positivity indicates poor prognosis
CD138 Syndecan-1 positive in mature plasma cells
CD45 with CD14 CD14 negative
T-cell acute lymphoblastic CD1a with CD3 CD1a positivity associated with longer disease-free survival in
leukemia (ALL) adult T-cell ALL; CD3 is negative in 99% (exception is mature
medullary thymocyte T-cell ALL)
CD2 with CD25 CD2 variably expressed and CD25 negative
CD38 with CD7 CD38 and CD7 are positive
Table 13–2 Common Markers Used for Lymphoproliferative and Myeloproliferative
Studies in Clinical Flow Cytometry—cont’d
DISEASE ANTIBODIES PAIRED INTERPRETATION
CD4 with CD8 CD4 and CD8 variably expressed depending on maturity; dual
expression is common
CD5 with CD20 CD5 positive except for prothymocyte stage T-cell ALL, CD20
negative
CD45 with CD14 CD45 positive and CD14 negative
HLA-DR with CD34 DR positive T-cell ALL associated with a worse prognosis; CD34
in pediatric patients associated with CNS involvement and poor
prognosis and predicts myeloid expression
TdT with CD10 TdT is positive; CD10 positive T-cell ALL associated with prolonged
disease-free survival
CD19 and kappa Negative
CD19 with lambda Negative
(Antiglycophorin added To determine erythroid component to the specimen
to bone marrows)
Post-thymic T-cell CD1a with CD3 CD1a negative, CD3 positive; note: peripheral T-cell lymphomas
leukemia or lymphoma lack 1 or more pan-T cell antigen (CD3, CD2, CD5, or CD7) 75%
of the time
Peripheral T-cell CD2 with CD25 CD2 positive; CD25 positive in adult T-cell leukemias and some
leukemia peripheral T-cell lymphomas
Adult T-cell leukemia CD5 with CD7 CD5 positive; CD7 positive except in adult T-cell leukemia
CD4 with CD8 Variable expression
CD19 with kappa Negative
CD19 with lambda Negative
CD45 with CD14 CD45 positive and CD14 negative
TdT with CD10 Negative
(Antiglycophorin added To determine erythroid component to the specimen
to bone marrows)
Tγ proliferative disease CD2 with CD57 NK-like T-cell lymphoma tends to be CD56 positive, CD57 nega-
(NK-like T-cell leukemia) tive and is usually clinically aggressive; NK cell leukemias tend
NK-like T-cell lymphoma to be CD56 or CD16 positive, CD57 negative and are usually
NK cell leukemia clinically aggressive; Tγ proliferative disease is CD56 negative,
CD57 positive and exhibits a chronic indolent course; CD2 is
usually positive for all, but there are variants
CD3 with CD56, CD16 Surface CD3 is positive in Tγ proliferative disease and NK-like
T-cell lymphoma; CD3 is negative in NK cell leukemia; for
CD56 and CD57, see above
CD11c with CD11b Usually positive
CD4 with CD8 Usually CD8 positive, CD4 negative; however, dual staining and
CD4 positivity has been reported
CD19 Negative
CD45 with CD14 CD45 positive and CD14 negative
Acute myelogenous CD11c with CD11b CD11c positive on mature myeloid cells; CD11b positive on
leukemia (AML) myelomonocytic cells, eosinophilic myelocytes, eosinophils,
and neutrophils; differentiated AML usually expresses mature
markers
CD13 with CD15 Poorly differentiated AML usually lacks CD15, CD11c, CD11b,
but positive for CD13
CD33 with TdT CD33 in the absence of CD34, HLA-DR, or CD13 suggests
immature acute basophilic or mast cell leukemia; TdT is often
expressed in low intensity in poorly differentiated AML
CD14 with CD64 CD14 on early promonocytes to mature monocytes; high expres-
sion of CD14 and CD11b predicts poor outcome; CD64 on
immature and mature monocytes
Continued
 Table 13–2 Common Markers Used for Lymphoproliferative and Myeloproliferative
Studies in Clinical Flow Cytometry—cont’d
DISEASE ANTIBODIES PAIRED INTERPRETATION
CD3 with CD7 CD3 negative; some immature AML expresses CD7
CD19 with kappa CD19 occasionally expressed on some primitive AML
CD19 with lambda Surface Ig negative
CD34 with HLA-DR Poorly differentiated AML often expresses CD34; high-intensity
CD34 has worse prognosis; CD34 coexpressed with HLA-DR
has worse prognosis; CD34 coexpressed with CD has worse
prognosis than CD34 alone; lack of HLA-DR indicates either
APL or very immature AML
CD10 with TdT CD10 is present on neutrophils
(Antiglycophorin added To determine erythroid component present in the specimen
to bone marrows)
MPO with CD117 Myeloperoxidase (MPO) is found on fairly mature AMLs,
whereas CD117 is a myeloid blast marker

ADCC = antibody-dependent cell cytotoxicity; CALLA = common acute lymphoblastic leukemia antigen; FC = fragment crystallizable; GM-CSF = granulocyte-
macrophage colony-stimulating factor; HIV = human immunodeficiency virus; IgE = immunoglobulin E; MHC = major histocompatibility class; NK = natural killer;
TCR = CD3-αβ receptor complex; TdT=terminal deoxynucleotidyl transferase

CD45 is a pan-leukocyte marker present on all WBCs but 500 CD4 cells/mm3, or 14% to 28% CD4 cells; and fewer than
with varying levels of expression based on the cell’s maturity 200 CD4 cells/mm3, or fewer than 14% CD4 cells.13
as well as lineage. This variance in expression results in varying Additional examples of flow cytometry use include the
levels of fluorescence. Blasts express lower levels of CD45 (low determination of DNA content or ploidy status of tumor cells.
fluorescence) but show an increase of CD45 expression as the This analysis can provide the physician with important prog-
cell matures; therefore, mature WBCs have much brighter nostic information.17 Ploidy analysis is also useful in examining
fluorescence compared with their earlier progenitor stages. The products of conception for molar pregnancies.
analysis of CD45 expression levels is useful in differentiating Finding a small number of abnormal cells in a particular
various populations of WBCs and in combination with SSC cell population can easily be accomplished by flow cytometry.
has replaced the FSC and SSC gating in many laboratories. Patients who have been treated for leukemia or lymphoma can
However, it is always a good idea to examine the FSC and SSC be monitored for “minimal residual disease” because statisti-
plot to be sure a population is not being missed. cally significant rare cell events can be easily detected. In the
Immunophenotyping of WBC populations is also essential case of a fetal–maternal hemorrhage, using flow cytometry to
when an immunodeficiency is suspected. Enumeration of detect hemoglobin F-positive cells is much more sensitive than
peripheral blood CD4+ T cells in HIV-infected patients remains the traditional Kleihauer-Betke method.18,19
the highest volume test performed in the flow cytometry lab- Characterization of normal cell populations is another use
oratory because it is used in classifying stages of HIV disease for flow cytometry. Human leukocyte antigen typing and cross-
and determining treatment options.13 HIV type 1 (HIV-1) in- matching for solid organ transplantation can be performed in
fections cause a rapid, profound decrease in CD4+ T-cell num- a much faster and more accurate way than formerly utilized
bers and an expansion of CD8+ T-cell levels during the early serological methods.3,17
course (12 to 18 months) of the illness.14,15 Some individuals Flow cytometry is also the method of choice for the diagnosis
continue to rapidly lose CD4+ T cells and progress to AIDS, of several inherited chronic diseases. CGD is an X-linked and
whereas others maintain relatively stable CD4+ T-cell counts autosomal recessive disease in which neutrophils are defective
and remain AIDS-free for years. During this chronic phase of in their oxidative burst. In this case, neutrophils are exposed to
HIV-1 disease, the decline in CD4+ T cells can be slow over the dye Dihydrorhodamine 123, which is not fluorescent until
many years because of maintenance of homeostatic mecha- oxidized. When normal neutrophils are stimulated in vitro, the
nisms. However, as these homeostatic mechanisms start to fail, dye is oxidized and becomes intensely fluorescent. Neutrophils
there is a further decline in CD4+ T and CD8+ T cells, which from CGD patients are unable to oxidize the dye and do not be-
eventually leads to the development of AIDS.16 CD4+T-cell come fluorescent after stimulation.
levels are used to stage HIV disease progression, are prognostic Paroxysmal nocturnal hemoglobinuria (PNH) is an inher-
for the development of AIDS, and are used to monitor response ited disease characterized by a hemolytic anemia. The RBCs,
to antiretroviral therapy. The Centers for Disease Control and neutrophils, monocytes, and other cells are lacking the glyco-
Prevention (CDC) guidelines stage HIV-1 disease into three sylphosphatidylinositol (GPI) anchors by which many surface
groups by CD4+ T-cell level: greater than 500 CD4 cells/mm3, proteins are attached to the cell membrane. As a result, the
or greater than 28% CD4 cells within lymphocytes; 200 to RBCs are fragile. Hemolysis occurs during pH changes in the
blood, typically at night.1 Several flow cytometry tests are avail- well. Other potential benefits of immunoassay automation in-
able to detect this defect. One test looks for the antigens that clude the ability to provide more services with fewer staff; sav-
use this anchor because these antigens will be missing or ing on controls, duplicates, dilutions, and repeats; longer shelf
reduced in these patients. The other test detects the anchor; life of reagents and less disposal because of outdating; and the
for example, fluorescent aerolysin (FLAER) will bind to the GPI potential for automation of sample delivery with bar codes for
anchor itself. Thus, normal granulocytes and monocytes will better sample identification.24
be fluorescent, whereas the fluorescence in defective ones will Because of the wide variety of automated immunoassay
be reduced or absent. analyzers available, it can be difficult to determine the best
Finally, another important use for flow technology in clini- instrument for any given laboratory. Table 13–3 offers a par-
cal diagnosis is in cytometric bead arrays. In this technology, tial list of the many factors to consider in determining what
various sizes and different fluorescent beads are used to deter- type of analyzer will fulfill a laboratory’s needs. It is important
mine multiple analytes at the same time. For example, six dif- for all those involved in the instrument’s selection to prioritize
ferent sizes or colors of beads could be coated with six different the properties of any analyzer to meet the demands of the
autoantigens. Patient sera is then added, followed by fluores- laboratory.
cent anti-IgG. If the patient has any autoantibodies, the respec- There are currently two main types of immunoassay ana-
tive bead will be fluorescent and can be identified and lyzers on the market: batch analyzers and random access ana-
quantitated. Similarly, polymerase chain reaction (PCR) and lyzers (Table 13–4). Batch analyzers can examine multiple
hybridization techniques can be performed on these beads. samples and provide access to the test samples for the forma-
(See Chapter 12 for a discussion of molecular techniques used tion of subsequent reaction mixtures. However, such batch an-
to detect viral nucleic acids and various genetic mutations.) alyzers permit only one type of analysis at a time. In some
Theoretically, this technology has the potential to detect over cases, this may be considered a drawback; stat samples cannot
500 analytes from one sample of blood. be loaded randomly and there cannot be multiple analyses on
Immunophenotyping by flow cytometry relies on the use any one sample. Partially for those reasons, the next generation
of fluorescent-labeled monoclonal and polyclonal antibodies. of analyzers was designed in a modular system that could
Monoclonal antibodies specific for various surface antigens are be configured to measure numerous analytes from multiple
preferable to using polyclonal antibodies. The ability to pro- samples. These types of analyzers are called random access
duce monoclonal antibodies through hybridoma and recom- analyzers; in these analyzers, many test samples can be ana-
binant DNA techniques has contributed greatly to the accuracy lyzed and a number of different tests can be performed on any
of flow cytometry and has widened its use. (See Chapter 5 for one sample.25
a discussion of monoclonal antibody production.) Automation can and does occur in all three stages of labo-
ratory testing: the preanalytical, analytical, and postanalytical
stages. For the purposes of this section, discussion is limited
Immunoassay Automation to automation within the analytical stage of testing.
The tasks of the analytical stage include introducing a sam-
In addition to flow cytometry, the use of automated technology ple, adding reagent, mixing the reagent and sample, incubat-
has become more prevalent in clinical immunology laborato- ing, detecting, calculating, and reporting the readout or
ries with the advent of immunoassay analyzers. Reliable im- results. All or some of these tasks may be automated on
munoassay instrumentation, excluding radioimmunoassay, was various immunoassay analyzers. Automatic sampling can be
first available in the early 1990s. Using a solid support for sep- accomplished by several different methods: peristaltic pumps
arating free and bound analytes, these instruments have made (older technology) and positive-liquid displacement pipettes
it possible to automate heterogeneous immunoassays even for (newer technology) are two examples. In most systems, sam-
low-level peptides such as peptide hormones.20 Currently, ples are pipetted using thin, stainless-steel probes. Such
there are more than 60 different automated immunoassay an- probes have clot detectors that will automatically reject a
alyzers that are capable of performing almost all common di- sample if a clot is detected. They also have a liquid level
agnostic immunoassays21; they have largely replaced manual sensor that can detect the lack of sample in a tube, usually
testing, especially in larger laboratories. The driving motivation because of a short draw. Samples without the proper amount
for the development of immunoassay analyzers has been the of liquid in them are also rejected. Another issue associated
need to create an automated system capable of reducing or with reusable pipette probes is carryover or contamination of
eliminating the many manual tasks required to perform ana- one sample with material from the previous samples. Various
lytical procedures and the demand to handle large volumes of methods have been developed to reduce carryover, including
samples.22 Eliminating manual steps decreases the likelihood the use of disposable pipette tips to initially transfer samples
of error because the potential error caused by fatigue or erro- and flushing the internal and external surfaces of sample
neous sampling is reduced.23 Laboratory professionals are also probes with diluent.
trying to streamline test performance to reduce turnaround Reagent use in automated immunoassay analyzers requires
time and the cost per test. Automation, in some cases, is much consideration of the following factors: handling, preparing and
more accurate and precise compared with manual methods; storing, and dispensing. Some reagents come ready for use; if
depending on the assay platform, it may be more sensitive as they do not, the analyzer must be able to properly dilute
 Table 13–3 Factors for Consideration refrigerators; however, in larger systems, there is a reagent stor-
in Selecting an Automated age compartment within the analyzer itself.
Immunoassay Instrument After reagents have been added to the samples, the next
concern is proper mixing to obtain reliable results. Analyzers
CATEGORY FACTOR
use different methods for mixing, including magnetic stirring,
Analytical Sensitivity rotation paddles, forceful dispensing, and vigorous lateral shak-
Precision ing. Whichever method is used, it is imperative that there
Accuracy and test standardization be no splashing between sample wells in order to prevent
Linearity
erroneous results.
Interferences
Carryover effects
Timed incubation is then carried out at ambient tempera-
tures. Some analyzers have built-in incubators for temperature-
Economic Purchase cost controlled incubation. Heated metal blocks are widely used to
Lease options incubate reagent wells or cuvettes.
Shipping and installation fees Detection of the final analyte depends on the chemistry in-
Maintenance costs
volved in the immunoassay. In the past, colorimetric absorption
Reagent costs
Operator time and costs
spectroscopy has been the principal means of measurement
Disposable costs because of its ability to measure a wide variety of compounds.
Training for personnel Other methods of detection include fluorescence and chemilu-
minescence, both of which require fluorescence detectors. With
Instrument Maintenance requirements the growing trend to offer flexibility in an automated analyzer,
Automation compatibility
several companies have developed analyzers that have the abil-
Space requirements
Utility requirements
ity to combine chemistry and immunoassay testing on a single
Reliability platform. Two examples are Beckman Coulter’s UniCel DXC
Clot error detection chemistry systems and Roche Diagnostics’ COBAS analyzer
Hardware costs series.23
Nonwarranty service Instrumentation can decrease turnaround time for testing,
remove the possibility of manual errors, and allow for greater
Manufacturer Future product plans
Speed of service response
sensitivity in determining the presence of low-level analytes.
Reputation Batch analyzers may work best if only one type of testing is
Technical support performed on a large scale. Random access analyzers allow for
Menu expansion plans more flexibility and include rapid processing of stat samples.
Purchase of a warranty In either case, any new instrument requires extensive valida-
tion before patient results can be reported with confidence.
Operational Test menu
Throughput
Reagent capacity Validation
Reagent stability
STAT capability
Regardless of the instrumentation considered, proper validation
Reflex testing ability of new instrumentation or methodology must always be per-
Reagent kit size formed. The laboratory needs to determine how it will meet
Training requirements Clinical Laboratory Improvement Amendment (CLIA) regula-
Operating complexity tions for verifying the manufacturer’s performance specifications.
Waste requirements The regulations apply to each nonwaived test or test system
Reagent storage requirements brought into the laboratory for the first time. Validation of the
Reagent performance new instrument or method must be completed before patient
Downtime plans results using that instrument can be reported. There are multiple
Adapted from Remaley AT, Hortin GL. Protein analysis for diagnostic applica- resources available on the topic of method validation. The
tions. In: Detrick B, Hamilton RG, Folds JD, et al., eds. Manual of Molecular Centers for Medicare and Medicaid Services has an overview of
and Clinical Laboratory Immunology. 7th ed. Washington, DC: American the CLIA (available at http://www.cms.gov/CLIA). The specific
Society for Microbiology; 2006:15 and Appold K. Checklist for buying a requirements for method validation for nonwaived and modi-
chemistry analyzer. Clin Lab Products. Nov. 2013:12–15. fied tests can be found at http://www.cms.gov/Regulations-
and-Guidance/Legislation/CLIA/Categorization-of–Tests.html.
Table 13–5 lists other government websites with information
reagents before they can be used. Most reagents come with bar regarding method and instrument validation.26
codes that are read by the analyzer to reduce operator error; if As designated by CLIA, the required verifications to be de-
the wrong reagent is loaded into the analyzer by mistake, the termined for a new method are accuracy, precision, analytic
analyzer will detect the error and generate an error message. sensitivity, and analytic specificity to include interfering sub-
For many analyzers, reagents must be stored in laboratory stances, reportable range, and reference intervals. Accuracy
 Table 13–4 Automated Immunoassay Analyzers
MANUFACTURER INSTRUMENT OPERATIONAL TYPE ASSAY PRINCIPLE
Abbott Diagnostics AxSYM Continuous random access FPIA, MEIA
Stat processing
Abbott Diagnostics Architect Series: Batch, random access, con- Enhanced
Ci4100, Ci8200, Ci6200, tinuous random access chemiluminescence
i1000SR, i2000SR, i4000SR
Alere Agility DS2 DSX Batch EIA
Awareness Technology ChemWell Batch, random access EIA
Beckman Coulter Access Continuous random access, Chemiluminescence, EIA
STAT capability
UniCel DXI 800 Access (Up to 400 tests/hr)
UniCel DXI 600 Access (Up to 200 tests/hr)
BioMérieux VIDAS Batch, random access FEIA-coated SPR
miniVIDAS STAT capability Solid-phase receptacle
(multiparametric IA) SPR pipetting device
Bio-Rad Laboratories, BioPlex® 2200 Continuous random access Bead flow cytometric
Clinical Diagnosis Multiplex Testing (multiplex)
Group PhD System Batch EIA
Diamedix Corporation New Mago 4S Automated Batch, random access EIA
Immunoassay System ELISA and IFA
Mago Plus Automated
EIA Processor
DiaSorin, Inc. ETI-MAX (Germany/Italy) Batch, random access EIA
Dynex DS2 Batch EIA
Hycor Biomedical, Inc. HYTEC 288 Plus Random batches EIA
Inova BIO-FLASH Ds2 Batch, continued random Chemiluminescence
access
Inverness Medical AIMS (Automated IA Batch EIA, multiplexing or bead
Professional Diagnostics Multiplexing System) diagnostics
Ortho Clinical Diagnostics, VITROS 3600 Continuous random access Chemiluminescence
a J&J Co. (enhanced)
Phadia ImmunoCAP Continuous random access FEIA
Thermo Fisher Phadia Laboratory System
Scientific-Phadia
Siemens Medical ADVIA Centaur Continuous random access Chemiluminescence
Solutions Diagnostics Dimension EXL
Dimension Vista 1500 Batch, random access, con- Chemiluminescence, EIA
tinuous random access
IMMULITE Continuous random access Chemiluminescence
TOSOH Bioscience, Inc. AIA Continuous random access Fluorescence, EIA
EIA = enzyme immunoassay; FEIA = fluoroenzyme immunoassay; FPIA = fluorescent polarization immunoassay; MEIA = microparticle enzyme immunoassay;
SPR = solid-phase receptacle.

refers to the test’s ability to actually measure what it claims accuracy testing. Precision refers to the ability to consistently
to measure. For example, the assay may be tested using pre- reproduce the same result on repeated testing of the same
viously known positives or negatives as provided by profi- sample. See Figure 13–7 for a pictorial representation of the
ciency testing or interlaboratory exchange. Also, parallel difference between accuracy and precision. CLIA '88 specifies
testing with an alternative method or technology is a form of that the standard deviation and coefficient of variation should
 Table 13–5 CLIA-Related Governmental Websites
AGENCY WEBSITE
Centers for Disease Control and Prevention http://www.cdc.gov
Centers for Medicare and Medicaid Services www.cms.gov/Regulations-and-Guidance/Legislation/CLIA/
Categorization-of-Tests.html
U.S. Food and Drug Administration www.fda.gov
The College of American Pathologists Laboratory www.cap.org/web/home?
Accreditation Program Inspection Checklist for Chemistry
Clinical Laboratory Standards Institute Evaluation Standards www.clsi.org

• Scattered light in a forward direction is a measure of cell

size, whereas the side scatter determines a cell’s internal
complexity, or granularity.
• A single-parameter histogram shows a chosen parameter
(on the x axis) versus the number of events (on the
y axis).
• A dual-parameter dot plots two parameters against each
Imprecise Precise but Accurate and other.
and inaccurate inaccurate precise
• A gate blocks off a particular region of cells for further
Pictorial representation of the difference between analysis.
accuracy and precision. • Determining an individual’s lymphocyte population is
essential in diagnosing such conditions as lymphomas,
be calculated from 10 to 20 day-to-day quality control immunodeficiency diseases, unexplained infections, or
results.27 At least one normal and one abnormal control acquired immune diseases such as AIDS.
should be included in the analysis. Analytic sensitivity is • Lymphoid and myeloid cells are identified using
defined as the lowest measureable amount of an analyte, monoclonal antibodies directed against specific surface
whereas analytic specificity is the assay’s ability to generate antigens. Flow cytometry is the most commonly used
a negative result when the analyte is not present. The re- method for immunophenotyping of lymphoid and
portable range is defined as the range of values that will gen- myeloid populations.
erate a positive result for the specimens assayed by the test • Clinical immunology laboratories are now utilizing a
procedure. Note that this may not include the entire dynamic variety of automated immunoassay analyzers to replace
range of the analytic instrument used to produce the result. manual immunoassay procedures, allowing for more
Finally, the reference interval is the range of values found accurate, precise, and sensitive analysis of many analytes.
in healthy individuals who do not have the condition that is • There are many factors to consider in determining which
detected by the assay, which is used to define the expected analyzer will fill the needs of a particular laboratory,
value of a negative test. including deciding whether a batch or a random access
analyzer can best serve testing needs.
• Automation is incorporated in all stages of laboratory
SUMMARY testing: preanalytical, analytical, and postanalytical.
• Once an analyzer has been purchased, a thorough valida-
• Flow cytometry, a powerful tool to identify and enumerate tion of all assays to be performed must be done to ensure
various cell populations, was first used in clinical labora- quality; they should include at least a determination of ac-
tories to perform CD4+ T-cell counts in HIV-infected curacy, precision, reportable range, reference range, analytic
individuals. sensitivity, and analytic specificity.
• A flow cytometer measures multiple properties of cells • Accuracy refers to a test’s ability to measure what it
suspended in a moving fluid medium. actually claims to measure.
• As each cell or particle passes single file through a laser • Precision is the ability to consistently reproduce the same
light source, it produces a characteristic light pattern that result on a particular sample.
is measured by multiple detectors for scattered light (for- • Automated analyzers are costly; however, they can reduce
ward and 90 degrees) and fluorescent emissions if the cell turnaround time and hands-on time by the technical staff
is stained with a fluorochrome. and provide sensitive and precise results.
CASE STUDIES
1. A laboratory has just purchased a new immunoassay bouts of pneumonia. The results show decreased im-
analyzer and validation is being done before patient munoglobulin levels, especially of IgG. Although her
results can be reported out. Twenty random patient WBC count was within the normal range, her lympho-
samples are run by both the old and new methodology. cyte count was low. Flow cytometry was performed to
According to the newer instrumentation, three samples determine if a particular subset of lymphocytes was low
that were negative by the old method are positive by or missing. Figure 13–8 shows the flow cytometry re-
the new instrument. sults obtained.
Questions Questions
a. What sort of possible error—that is, sensitivity, speci- a. What do the flow cytometry patterns indicate about
ficity, accuracy, or precision—does this represent? the population of lymphocytes affected?
b. What steps should be taken to resolve this b. How can this account for the child’s recurring
discrepancy? infections?
c. What further type of testing might be indicated?
2. A 3-year-old female is sent for immunologic testing be-
cause of recurring respiratory infections, including several
CD3

A CD19

Normal donor Patient

104 104
40% 23%

103 103
CD4-APC

CD4-APC

102 102

101 101

100 100
100 101 102 103 104 100 101 102 103 104
CD3-FITC CD3-FITC
B
Flow cytometry patterns for case study. (A) Plot of CD3 versus CD19. (B) Plot of CD3 versus CD4.
REVIEW QUESTIONS
1. Flow cytometry characterizes cells on the basis of 7. All of the following are clinical applications for flow
which of the following? cytometry except
a. Forward and 90-degree side scatter of an inter- a. fetal hemoglobin.
rupted beam of light b. immunophenotyping of lymphocyte subpopulations.
b. Front-angle scatter only of an interrupted light c. HIV viral load analysis.
beam d. enumeration of stem cells in a peripheral blood
c. Absorbance of light by different types of cells mononuclear cell product.
d. Transmittance of light by cells in solution
8. The various signals generated by cells intersecting
2. Forward-angle light scatter is an indicator of cell with a flow cytometry laser are captured by
a. granularity. a. bandwidth waves.
b. density. b. wave channels.
c. size. c. photomultiplier tubes.
d. number. d. flow cells.

3. What is the single most important requirement for 9. Analysis of flow cytometer data of cells can be filtered
samples to be analyzed on a flow cytometer? in many ways by using a method of
a. Whole blood is collected into a serum-separator a. “gating” in a dot plot.
tube. b. banding of a histogram.
b. Cells must be in a single-cell suspension. c. single-parameter histogram monitoring.
c. Samples must be fixed in formaldehyde before d. automatic sampling.
processing.
d. Blood must be kept refrigerated while processing. 10. A newer flow cytometry technology that has the
potential to detect over 500 analytes from one
4. Which statement represents the best explanation for a sample of blood is called a/an
flow cytometer’s ability to detect several cell surface a. RBC fragmentation assay.
markers at the same time? b. Dihydrorhodamine 123.
a. The forward scatter can separate out cells on the c. sucrose test.
basis of complexity. d. cytometric bead array.
b. One detector can be used to detect many different
wavelengths. 11. Many flow cytometry laboratories now use the CD45
c. For each marker, a specific fluorochrome–antibody marker in combination with SSC in differentiating
combination is used. various populations of WBCs to replace which of the
d. Intrinsic parameters are separated out on the basis following combinations?
of the amount of side scatter. a. CD4 + SSC
b. CD4 + FSC
5. Which of the following cell surface markers would be c. FSC + SSC
present on a population of T helper (Th) cells? d. FSC + CD45
a. CD3 and CD4
b. CD3 and CD8 12. Which cell surface marker is present on cells seen in
c. CD3 only hairy cell leukemia?
d. CD4 only a. CD138
b. CD33
6. If an analyzer consistently indicates a positive test c. CD103
when the analyte in question is not present, this d. CD34
represents a problem with
a. sensitivity.
b. specificity.
c. reportable range.
d. precision.
13. CD45 is a pan-leukocyte marker expressed on WBCs 17. Operational considerations when selecting automated
in varying levels or amounts of expression, based on analyzers for your laboratory include all of the following
a. size of a cell. except
b. granularity of a cell. a. reagent stability.
c. maturity and lineage of a cell. b. test menu.
d. malignancy of a cell. c. STAT capability.
d. purchase cost.
14. Which of the following statements best describes a
single-parameter histogram? 18. Analyzers use different methods for mixing, including
a. Each event is represented by a dot. magnetic stirring, rotation paddles, forceful dispens-
b. Data is distributed in four quadrants. ing, and vigorous lateral shaking. Whichever method
c. Positive and negative events are plotted on the used, it is imperative that
x and y axis. a. reagents always be kept refrigerated.
d. A chosen parameter is plotted versus the number b. there is no splashing or carry-over between samples.
of events. c. samples are kept at room temperature.
d. multiple methods are not used simultaneously.
15. How many fluorochromes (colors) are current clinical
flow cytometers capable of detecting? 19. All of the following are benefits of automation except
a. 2 a. greater accuracy.
b. 6 b. increased turnaround time.
c. 8 c. savings on controls.
d. 10 d. less disposal of outdated reagents.

16. Which type of analyzer allows one to measure multi- 20. If an analyzer gets different results each time the
ple analytes from numerous samples, loaded at any same sample is tested, what type of problem does
time? this represent?
a. Batch analyzer a. Sensitivity
b. Random access analyzer b. Specificity
c. Front-end loaded analyzer c. Accuracy
d. Sequential access analyzer d. Precision
Immune Disorders
 Hypersensitivity

After finishing this chapter, you should be able to: TYPE I HYPERSENSITIVITY
1. Explain the concept of hypersensitivity. Immunologic Mechanism
2. Differentiate between the four types of hypersensitivity reactions in Genetic and Environmental
terms of antibody involvement, complement involvement, antigen Influences on Type I
triggers, and timing of the response. Hypersensitivity
3. Associate specific examples of clinical manifestations with each type Clinical Manifestations of Type I
of hypersensitivity. Hypersensitivity
4. Discuss the immunologic mechanisms involved in each of the four Treatment of Type I Hypersensitivity
types of hypersensitivity reactions. Testing for Type I Hypersensitivity
5. Provide examples of preformed and newly synthesized mediators TYPE II HYPERSENSITIVITY
released from IgE-sensitized mast cells and basophils and discuss Clinical Examples of Type II
their effects. Hypersensitivity
6. Discuss the influence of genetic and environmental factors on Testing for Type II Hypersensitivity
susceptibility to type I hypersensitivity responses.
TYPE III HYPERSENSITIVITY
7. Discuss the types of reactions that can result from latex sensitivity
Arthus Reaction
and their clinical manifestations.
Serum Sickness
8. Explain the underlying mechanisms of pharmacological therapy,
monoclonal anti-IgE therapy, and allergy immunotherapy in the Autoimmune Diseases and Other
treatment of allergies. Causes of Type III Hypersensitivity
9. Discuss the procedure, clinical applications, advantages, and Testing for Type III Hypersensitivity
limitations of skin testing for type I hypersensitivity. TYPE IV HYPERSENSITIVITY
10. Discuss the principles and clinical applications of allergen-specific Contact Dermatitis
and total-IgE testing. Hypersensitivity Pneumonitis
11. Explain how hemolytic disease of the newborn (HDN) arises. Skin Testing for Delayed
12. Explain the significance of a positive direct antiglobulin test. Hypersensitivity
13. Discuss the principle of cold agglutinins testing, and associate the SUMMARY
presence of a positive result with specific disorders. CASE STUDIES
14. Discuss how skin testing for delayed hypersensitivity is performed, its REVIEW QUESTIONS
clinical applications, and how to interpret the results.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Allergen Autoimmune hemolytic Hemolytic disease of the Paroxysmal cold
Allergy immunotherapy anemia newborn (HDN) hemoglobinuria
(AIT) Cold agglutinins Histamine Serum sickness
Anaphylaxis Contact dermatitis Hypersensitivity Type I hypersensitivity
Anergy Delayed hypersensitivity Immediate hypersensitivity Type II hypersensitivity
Arthus reaction Direct antiglobulin test (DAT) Isohemagglutinins Type III hypersensitivity
Atopy Granulomas Leukotrienes (LT) Type IV hypersensitivity

In previous chapters, the immune response has been de- the general characteristics of each type will help you under-
scribed as a defense mechanism by which the body rids itself stand the immune processes that trigger such tissue damage.
of potentially harmful antigens. However, in some cases, the For each of the four types of hypersensitivity, the nature of the
antigen can persist, and the immune response can cause dam- immune reactants is discussed, clinical examples are provided,
age to the host. This type of reaction is termed hypersensi- and relevant testing is reviewed.
tivity, which is defined as an exaggerated response to a
typically harmless antigen that results in injury to the tissue,
disease, or even death. British immunologists P. G. H. Gell
Type I Hypersensitivity
and R. R. A. Coombs devised a classification system for these The type I hypersensitivity reactions are commonly thought
reactions based on four different categories. These categories,
of as allergies. Some examples of these reactions are hay fever,
described briefly in the text that follows, are illustrated in allergic asthma, hives, and systemic anaphylaxis (see the fol-
Figure 14–1. lowing discussion). The antigens that trigger type I hypersen-
Type I hypersensitivity reactions are also known as ana- sitivity are called allergens. Examples of common allergens
phylactic hypersensitivity. In these reactions, exposure to an anti- include peanuts, eggs, and pollen. A distinguishing feature
gen induces production of specific immunoglobulin E (IgE) of type I hypersensitivity is the short time lag, usually min-
antibody, which binds to receptors on mast cells and basophils. utes, between exposure to allergen and the onset of clinical
Subsequent attachment of the antigen to adjacent cell-bound symptoms.
IgE results in degranulation with release of chemical mediators The first clue about the cause of type I hypersensitivity
that produce characteristic allergy symptoms. In type II was provided by Carl Wilhelm Prausnitz and Heinz Küstner,
hypersensitivity, also known as antibody-mediated cytotoxic who showed that a serum factor was responsible. In their
hypersensitivity, Immunoglobulin G (IgG) or immunoglobulin historic experiment, serum from Küstner, who was allergic
M (IgM) antibodies react with antigens on the surface of host to fish, was injected into Prausnitz. A later exposure to fish
cells. This can lead to cell damage by complement-mediated antigen at the same site resulted in redness and swelling.1
lysis or other mechanisms, dysfunction of the cell by blocking This type of reaction is known as passive cutaneous anaphy-
the binding of a ligand to a surface receptor, or overstimula- laxis. It occurs when serum is transferred from an allergic
tion of a cell’s function. Type III hypersensitivity is also re-
individual to a non-allergic individual, and the second indi-
ferred to as complex-mediated hypersensitivity. In this process, vidual is challenged at a later time with the specific allergen.
IgG or IgM antibodies react with soluble antigens to form Although this experiment was conducted in 1921, it was not
small complexes that precipitate in the tissues and activate until 1967 that the serum factor responsible for this reaction
complement. Recruitment of neutrophils to the site results in was identified as IgE.
an inflammatory response that causes injury to the tissues.
Type IV hypersensitivity differs from the other three types
because sensitized T cells, rather than antibody, are responsi- Connections
ble for the symptoms that develop. This cell-mediated hyper-
sensitivity involves the release of cytokines that induce Immunoglobulin E
inflammation and tissue damage. Recall from Chapter 5 that IgE is the least abundant antibody
Types I through III are classified as immediate hypersen- class in the serum, normally accounting for less than 1% of all
sitivity reactions because symptoms develop within a few min- the immunoglobulins. This is likely because IgE is not involved
utes to a few hours after exposure to the antigen. Type IV in typical immune responses such as complement fixation and
opsonization. IgE is unique in its ability to bind to specific recep-
hypersensitivity is sometimes referred to as delayed hypersen-
tors on mast cells and basophils. This property enables IgE to
sitivity because its manifestations are not seen until 24 to play a major role in type I hypersensitivity allergic reactions and
48 hours after contact with the antigen. in defense against parasites (see Chapter 22). Patients with these
The four main types of hypersensitivity are discussed in conditions typically have increased concentrations of IgE in their
more detail in the sections that follow. Although some disease bloodstream.
manifestations may overlap among these types, knowledge of
 Type II

Type I

Antigen

Degranulation

Type III

Tissue
Type IV

APC

Antigen

1
4

Th1

3
Blood vessel
lumen Tissue
damage
Immune mechanisms of hypersensitivity types I to IV.

Typically, patients who exhibit allergic hypersensitivity re-

actions produce a large amount of IgE antibody in response to
Immunologic Mechanism
a small concentration of allergen. IgE levels appear to depend The key immunologic components involved in type I hyper-
on the interaction of both genetic and environmental factors. sensitivity reactions are IgE antibody, mast cells, basophils, and
Atopy, a term derived from the Greek word atopos (meaning eosinophils. The response begins when a susceptible individual
“out of place”), refers to an inherited tendency to develop clas- is exposed to an allergen and produces specific IgE antibody.
sic allergic responses to naturally occurring inhaled or ingested IgE is primarily synthesized by B cells and plasma cells in the
allergens.1 The section that follows will provide details about lymphoid tissue of the respiratory and gastrointestinal tracts, as
the immunologic mechanism which occurs in susceptible in- well as the lymph nodes. The regulation of IgE production ap-
dividuals to produce the symptoms of type I hypersensitivity pears to be a function of a subset of T cells called type 2 helper
reactions. cells (Th2).1-3 In a normal immune response to microorganisms
and other antigens, there is an appropriate balance between the but distinct, from mast cells in terms of their appearance and
activity of the Th2 cells and the type 1 helper cells (Th1), which function. They are present in the peripheral blood, where they
results in protective immunity that does not harm the host. represent less than 1% of the total white blood cells (WBCs).
However, in people with allergies, the immune response is They have fewer, but larger granules than mast cells, and the
shifted so that Th2 cells predominate. This Th2 type of response concentrations of inflammatory substances in the basophil
results in production of several cytokines, including IL-4 and granules differ from those of the mast cell.5 Basophils respond
IL-13. These cytokines are responsible for the final differentia- to chemotactic stimuli during inflammation and accumulate
tion that occurs in B cells, initiating the transcription of the gene in the tissues, where they can persist for a few days. As you
that codes for the epsilon-heavy chain of immunoglobulin mol- will see in the next section, subsequent binding of allergen to
ecules belonging to the IgE class.1-3 IgE-sensitized mast cells and basophils triggers degranulation
with release of inflammatory mediators.
Type I hypersensitivity occurs in two major phases: sensitiza-
tion and activation (Fig. 14–2). A late-phase reaction may also In the activation phase of the response, adjacent cell-bound IgE
occur in some individuals. In the sensitization phase, the IgE molecules are cross-linked by a bivalent or multivalent antigen,
antibody attaches to high-affinity receptors called FcεRI, causing aggregation of the surface FcεRI receptors. This action,
which bind the fragment crystallizable (Fc) region of the in turn, initiates complex intracellular signaling events involving
epsilon-heavy chain. Large numbers of these receptors are found multiple phosphorylation reactions, an influx of calcium, and se-
on mast cells and basophils, with a single cell having as many cretion of cytokines.5,7 The increase in intracellular calcium trig-
as 200,000 such receptors.2,4 Langerhans and dendritic cells gers rapid degranulation of the mast cells and basophils, which
internalize and process allergens from the environment and release chemical mediators that have been previously made and
transport the allergen-MHC class II complex to local lymphoid stored in the granules.1 The most abundant preformed mediator
tissue where synthesis of IgE occurs.2 Binding of IgE to cell is histamine, which comprises approximately 10% of the total
membranes increases the half-life of the antibody from 2 or weight of the granules in mast cells.8 These preformed substances
3 days to at least 10 days. Once bound, IgE serves as an antigen are referred to as primary mediators. Other primary mediators
receptor on mast cells and basophils. include heparin, eosinophil chemotactic factor of anaphylaxis
Mast cells are the principal effector cells of immediate hy- (ECF-A), neutrophil chemotactic factor, and proteases.2,9 The
persensitivity.5 These cells are found throughout the body and effects of these mediators are summarized in Table 14–1. Release
in most organs tend to be concentrated around the small blood of these substances is responsible for the early-phase symptoms
vessels, the lymphatics, the nerves, and the glandular tissue.6 seen in allergic reactions, which occur within 30 to 60 minutes
Mast cells have abundant cytoplasmic granules that store nu- after exposure to the allergen. The chemical mediators bind to
merous preformed inflammatory mediators. They are long- receptors on target organs, most notably the skin, respiratory
lived, residing for months in the tissues. Basophils are similar, tract, and gastrointestinal tract, producing symptoms charac-
teristic of an allergic response. The clinical manifestations de-
pend on the target tissue and type of receptors activated. For
example, in the skin, local swelling and redness, sometimes re-
F!"R1
ferred to as a wheal-and-flare reaction, can develop. Contraction
B
of the smooth muscle in the bronchioles may result in airflow
obstruction. Increased vascular permeability can cause hypoten-
Allergen # sion or shock. Depending on the route by which an individual
IL-13 is exposed to the triggering allergen, one or more of these effects
IL-4 may be seen.
A. Sensitization

In addition to immediate release of preformed mediators, mast

APC Th2 B. Activation cells and basophils are triggered to synthesize other reactants
from the breakdown of phospholipids in the cell membrane.
These newly formed, or secondary, mediators include platelet-
activating factor (PAF); prostaglandin (PG) D2; leukotrienes
(LT) B4, C4, D4, and E4; and cytokines1,2,9 (see Table 14–1).
These products are more potent than the primary mediators
and are responsible for a late-phase allergic reaction that can
be seen in some individuals 6 to 8 hours after exposure to the
antigen. In this phase of the reaction, numerous cells, including
Type I hypersensitivity. (A) Sensitization: Formation eosinophils, neutrophils, Th2 cells, mast cells, basophils, and
of antigen-specific IgE that attaches to mast cells. (B) Activation: macrophages, exit the circulation and infiltrate the allergen-
Reexposure to the same antigen, causing degranulation of mast filled tissue. They release additional mediators that prolong the
cells and release of mediators. hyperactivity and may lead to tissue damage.4,9,10
 Table 14–1 Mediators of Type I Hypersensitivity
MEDIATOR ACTIONS
Primary Histamine Smooth muscle contraction, vasodilation,
(Preformed) increased vascular permeability
Heparin Smooth muscle contraction, vasodilation,
increased vascular permeability
Eosinophil chemotactic factor of anaphylaxis (ECF-A) Chemotactic for eosinophils
Neutrophil chemotactic factor of anaphylaxis (NCF-A) Chemotactic for neutrophils
Proteases (e.g., tryptase, chymase) Convert C3 to C3b, stimulate mucus
production, activate cytokines
Secondary Prostaglandin PGD2 Vasodilation, increased vascular permeability
(Newly
Synthesized)
Leukotriene LTB4 Chemotactic for neutrophils, eosinophils
Leukotrienes LTC4, LTD4, LTE4 Increased vascular permeability, bronchocon-
striction, mucus secretion
Platelet activating factor (PAF) Platelet aggregation
Cytokines IL-1, IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, IL-14, Increase inflammatory cells in area, and
IL-16, TNF-α, GM-CSF increase IgE production

Eosinophils play an important role in the late-phase reac- microbes and potential allergens. Another group of genes play
tion. These cells normally compose 1% to 3% of the circulating a role in recognition of the antigen by the innate immune sys-
WBCs. During allergic reactions, IL-5 and other cytokines re- tem once it has entered through the epithelial barriers. Defects
leased from the Th2 cells stimulate the bone marrow to in- in these genes, which code for pattern recognition receptors
crease production of eosinophils, and the number in the such as CD14 and the Toll-like receptors (TLRs), can affect cell
peripheral blood increases, producing eosinophilia.4,10 The interactions with antigens in the initial phases of immune de-
number of FcεRI receptors on eosinophils increases during the fense. A third group of genes can influence susceptibility to al-
allergic response, and the eosinophil is stimulated to release a lergic disorders by affecting aspects of the adaptive immune
variety of toxic molecules and inflammatory mediators from response, such as cytokine production and the ability of T cells
its granules. These mediators are believed to contribute to the to differentiate into Th1 cells, Th2 cells, and T regulatory cells.
ongoing damage that occurs during chronic allergic conditions. Aberrations in these genes can result in dysregulation of the im-
In individuals with persistent inflammation resulting from mune response, inducing production of cytokines that promote
the late-phase reaction, such as those with chronic asthma, tis- IgE synthesis, such as IL-4 and IL-13. In addition, allergy and
sue remodeling can result. This involves structural changes, asthma appear to be associated with certain HLA class II
such as thickening of smooth muscle, as well as changes in genes.13,14 The HLA-D molecules coded for by these histocom-
connective tissue, blood and lymphatic vessels, mucus glands, patibility genes are known to play a role in antigen presentation
and nerves.3,4 and may influence the tendency to respond to specific allergens
(see Chapter 3). Finally, genes that play a role in modulating
Genetic and Environmental Influences the inflammatory response can influence the long-term conse-
quences of allergies by affecting the process of tissue remodeling
on Type I Hypersensitivity and repair.
The development of IgE responses and allergy appears to de- Many environmental influences on the allergic response
pend on complex interactions between genetic factors and en- have also been identified. Exposure to infectious organisms ap-
vironmental triggers. Several hundred genes associated with pears to play a key role in the development of allergic disease.
susceptibility to developing allergies have been identified.11-13 The increased prevalence of allergy in industrialized regions
These genes affect different aspects of the immune response that may be due, in part, to increased hygiene practices and use of
contribute to the pathogenesis of the type I hypersensitivity antibiotics, with a consequent decrease in exposure to mi-
response.4,12,13 Some of these genes affect the structure of the crobes.4 This, in turn, could have significant effects on the im-
epithelium lining in places where allergens can enter the body, mune system by altering the microbial constituent of the
such as the skin, gastrointestinal tract, and respiratory tract. gut. Multiple studies in Europe, the United States, and South
Certain polymorphisms in these genes can result in altered abil- America have provided evidence for a protective “farm effect.”15
ity of the body’s protective barriers to prevent penetration of These studies indicate that in utero or early life exposure to the
diverse microbial populations in a farming environment pro- allergies are caused by cow’s milk, eggs, nuts, soy, wheat, fish,
vides protection against allergies by inducing development of and shellfish.4,10 Symptoms limited to the gastrointestinal
regulatory T cells and by directing the immune system toward tract include cramping, vomiting, and diarrhea, whereas
beneficial Th1 responses and away from Th2 atopic reac- spread of antigen through the bloodstream may cause hives
tions.15,16 In addition, exposure to stress, variations in physical and angioedema on the skin, asthma, rhinitis, or anaphylaxis
factors such as temperature, and contact with environmental (see the text that follows).
pollutants such as cigarette smoke and diesel exhaust fumes Local inflammation of the skin, or dermatitis, can also be
can intensify clinical manifestations of allergy in susceptible caused by type I immediate hypersensitivity reactions. These
individuals.4 reactions manifest as either acute urticaria or eczema.1,4,10
Urticaria, or hives, appear within minutes after exposure to the
Clinical Manifestations of Type I allergen and are characterized by severe itching, erythema (red-
ness) caused by local vasodilation, leakage of fluid into the sur-
Hypersensitivity rounding area, and a spreading area of redness around the
Clinical manifestations of type I hypersensitivity, or anaphy- center of the lesion (Fig. 14–3). Commonly called a wheal-
lactic hypersensitivity, are common. The prevalence of allergic and-flare reaction, this reaction is caused by release of vasoac-
diseases has increased greatly in developed countries in the last tive mediators from mast cells in the skin following contact
50 years, and it is estimated that 40% of the world’s population with allergens such as pet dander or insect venom. When these
has allergic sensitization to common environmental antigens reactions occur deeper in the dermal tissues, they are known
such as pollen or peanuts.17 Millions of people are affected in as angioedema (Fig. 14–4). Urticaria can also appear as a result
the United States alone, where allergies are the fifth leading of other clinical manifestations such as anaphylaxis and food
cause of chronic disease in all age-groups as well as the third allergies. Atopic eczema can take on a variety of forms, from
leading cause of chronic disease in children.18 erythematous, oozing vesicles to thickened, scaly skin, depend-
The clinical manifestations caused by release of inflamma- ing on the stage of activity and age of the individual. It is a
tory mediators from mast cells and basophils vary from a
localized skin reaction to a severe systemic response known as
anaphylaxis. Symptoms depend on such variables as route of
antigen exposure, dose of allergen, and frequency of exposure.
If an allergen is inhaled, it is most likely to cause respiratory
symptoms such as asthma or rhinitis. Ingestion of an allergen
may result in gastrointestinal symptoms, whereas injection into
the bloodstream can trigger a systemic response.
Rhinitis is the most common form of atopy, or allergy; it
affects between 10% and 30% of populations worldwide.17
Symptoms include paroxysmal sneezing; rhinorrhea, or runny
nose; nasal congestion; and itching of the nose and eyes.4,10
Although the condition itself is merely annoying, complica-
tions such as sinusitis, otitis media (ear infection), eustachian Urticaria (hives) caused by an immediate hypersensi-
tube dysfunction, and sleep disturbances may result. Pollen, tivity reaction to a medication. (From Barankin B, Freiman A. Derm
mold spores, animal dander, and particulate matter from house Notes. Philadelphia, PA: F.A. Davis; 2006, with permission.)
dust mites are examples of airborne foreign particles that act
directly on the mast cells in the conjunctiva and respiratory
mucous membranes to trigger rhinitis. Seasonal allergic rhini-
tis, triggered by tree and grass pollens in the air during the
spring in temperate climates, is called “hay fever.”
Asthma, derived from the Greek word for “panting” or
“breathlessness,” is caused by inhalation of small particles such
as pollen, dust, or fumes that reach the lower respiratory
tract.1,4,10 It can be defined clinically as recurrent airflow ob-
struction that leads to intermittent sneezing, breathlessness,
and, occasionally, a cough with sputum production. The air-
flow obstruction is caused by bronchial smooth muscle con-
traction, mucosal edema, and heavy mucus secretion. All of
these changes lead to an increase in airway resistance, making
it difficult for inspired air to leave the lungs. This trapped air
creates the sense of breathlessness. Angioedema caused by a yellow jacket sting on the
Food allergies are another example of type I immediate right hand just above the middle finger. (Courtesy of CDC/Margaret A.
hypersensitivity reactions. Some of the most common food Parsons, Public Health Image Library.)
chronic, itchy skin rash that usually develops during infancy, Treatment of Type I Hypersensitivity
persists during childhood, and is strongly associated with
allergic rhinitis and asthma. Avoidance of known allergens is the first line of defense. Individ-
Anaphylaxis is the most severe type of allergic response be- uals can employ environmental interventions such as encasing
cause it is an acute reaction that simultaneously involves mul- mattresses and pillows in allergen-proof covers and removing a
tiple organs. It may be fatal if not treated promptly. Coined by harmful food from the diet.4 However, it is not always possible
biologists Paul Portier and Charles Richet in 1902, the term to completely eliminate contact with allergens. In these cases,
literally means “without protection.” Anaphylactic reactions are pharmacological therapy is necessary to relieve acute symptoms,
typically triggered by glycoproteins or large polypeptides. control chronic allergy manifestations, and, in some cases, mod-
Smaller molecules, such as penicillin, can trigger anaphylaxis ulate the immune response to the allergen.4 Drugs used to treat
by acting as haptens that may become immunogenic by com- immediate hypersensitivity vary with the severity of the reaction.
bining with host cells or proteins. Typical agents that induce Localized allergic reactions, such as hay fever, hives, or rhinitis,
anaphylaxis include venom from bees, wasps, and hornets; can be treated with antihistamines and decongestants. Asthma is
drugs such as penicillin; and foods such as shellfish, peanuts, often treated with a combination of therapeutic reagents, includ-
and dairy products.1,4,10 Clinical signs of anaphylaxis begin ing antihistamines and bronchodilators. In cases of persistent
within minutes after antigenic challenge and may include bron- asthma, leukotriene receptor antagonists and mast cell stabilizers
chospasm and laryngeal edema, vascular congestion, skin man- are also used; in severe cases, corticosteroids can be added to
ifestations such as urticaria (hives) and angioedema, diarrhea block recruitment of inflammatory cells and their ability to cause
or vomiting, and intractable shock because of the effect on tissue damage.4 Systemic anaphylaxis is a medical emergency that
blood vessels and smooth muscle of the circulatory sys- requires timely injection of epinephrine, a powerful vasoconstric-
tem.1,4,10 The severity of the reaction depends on the number tor, to quickly reverse symptoms that could potentially be fatal.4
of previous exposures to the antigen. This is because multiple Another treatment approach is aimed at modulating the
exposures result in additional accumulation of IgE on the sur- type I hypersensitivity response through use of an anti-IgE
face of the mast cells and basophils. Massive release of reac- monoclonal antibody called omalizumab. Omalizumab is a re-
tants, especially histamine, from the granules is responsible for combinant humanized antibody that is composed of human
the ensuing symptoms. Death may result from asphyxiation IgG framework genes recombined with complementarity-
because of upper-airway edema and congestion, irreversible determining region genes from mouse anti-human IgE. This
shock, or a combination of these symptoms. antibody binds to the Cε3 domain of human IgE, which is the
Latex sensitivity has been a significant problem since the late site that IgE normally uses to bind to FcεRI receptors.4,21
1980s after implementation of Universal Precautions by the Blocking of this site prevents circulating IgE from binding to
Centers for Disease Control and Prevention and Occupational mast cells and basophils and sensitizing them. In addition,
Safety and Health regulations requiring health-care workers to treatment with omalizumab has been shown to downregulate
wear gloves when performing laboratory procedures and work- cellular expression of FcεRI receptors.21 Omalizumab has been
ing with patients.19,20 Reactions to antigens in natural rubber used successfully to treat patients with moderate to severe
latex include type I hypersensitivity and contact dermatitis asthma when added to conventional drug therapy. Its effective-
caused by skin irritation or type IV hypersensitivity.19,20 Type I ness for other allergic disorders is also being studied.22
hypersensitivity reactions include urticaria, rhinoconjunctivitis, If environmental control measures and pharmacotherapy are
asthma, angioedema, and anaphylaxis. Sensitization to latex can not successful in managing the symptoms in an individual with
occur as a result of direct skin contact or inhalation of airborne allergies, allergy immunotherapy (AIT) may be considered. The
latex particles released when gloves are donned and removed. goal of AIT is to induce immune tolerance to a specific allergen
The risk of the latter occurring is increased when cornstarch by administering gradually increasing doses of the allergen over
powder is used in gloves because residual latex proteins can time.4 This therapy is believed to shift the patient’s immune re-
bind to the powder particles. Groups at particular risk for latex sponse to the allergen to a Th1-type of response and to induce the
allergy include health-care workers; rubber industry workers; development of T regulatory cells (Tregs) that release IL-10. This
patients who have had multiple surgeries, such as children with cytokine redirects the immune system to produce allergen-specific
spina bifida; and atopic individuals, particularly those who are IgG4 “blocking” antibodies that combine with the antigen before
allergic to certain foods that cross-react with latex allergens.19,20 it can attach to IgE-coated cells and trigger degranulation.23,24
Prevalence of latex allergies in the general population is esti-
mated to be 5% to 10%, whereas incidence is thought to range
from 10% to 17% in health-care workers and more than 60% Connections
in patients who have undergone multiple surgeries early in Immune Tolerance
life.19 The incidence of latex sensitization has decreased in
As we will discuss in Chapter 15, immune tolerance is defined as
recent years to as low as 1% in countries where policies to avoid
a state of immune unresponsiveness directed against a specific
contact with latex have been implemented. These policies may antigen. Development of immune tolerance to an allergen
include use of low-protein, powder-free gloves or gloves made means that the type I hypersensitivity response to the allergen
from non-latex materials such as nitrile, neoprene, vinyl, or syn- is inhibited. This is achieved by AIT.
thetic polyisoprene rubber.19
 The standard practice for AIT has been to administer aller-
gens subcutaneously (i.e., under the skin) over 3 to 5 years.
This practice has been shown to significantly reduce symptoms
in patients with allergic rhinitis or asthma; however, it has the
potential to induce anaphylaxis and must be administered in
a physician’s office.25 More recently, other routes of adminis-
tration that pose decreased risk of severe adverse reactions have
been used, namely oral and sublingual (placement of allergen
extract under the tongue). These methods of delivery have
been shown to reduce symptoms associated with allergic
asthma and rhinitis and to significantly decrease or eliminate
allergic reactions to certain food allergens such as peanuts.23,25
Researchers also are investigating the use of purified, recom-
binant allergens and allergoids, which have been chemically
altered to reduce IgE epitopes.23,24 These approaches may fur-
ther increase the effectiveness of AIT while decreasing the This individual is undergoing an allergen sensitivity
associated risk of severe reactions. test. The strongest positive reactions are to spider, moth, scorpion,
caterpillar, and tick allergens as indicated by the wheal-and-flare
Testing for Type I Hypersensitivity reactions at the sites of injection. (Courtesy of the CDC/Dr. Frank
Perlman and M.A. Parsons. Public Health Image Library.)
Evaluation of patients with an allergy begins with a medical
history and physical examination to assess clinical symptoms.
These assessments are followed by specific in vivo skin tests tourniquet can be applied to the arm to help stop the reaction.
and in vitro tests for IgE antibodies to confirm the presence After 15 to 20 minutes, the site is inspected for erythema and
of an allergy and to determine which allergens the patient is wheal formation, and the wheal diameter is measured to de-
sensitized to. termine a score.8,26,27 Intradermal tests can be used to test for
sensitivity to many allergens but have shown no benefit in the
diagnosis of food allergies.26,27
Testing for allergies typically begins with direct skin testing
Although skin testing is sensitive as well as relatively sim-
because this procedure is less expensive and more sensitive
ple and inexpensive to perform, it has some important limi-
than serological testing and provides immediate results.26 Two
tations.26,27 Antihistamines must be discontinued a few days
types of skin tests are used in clinical practice: percutaneous
before testing because they can decrease or inhibit the skin
tests (also known as prick or puncture tests) and intradermal
reaction. Improper technique or use of an inappropriate di-
tests. Percutaneous tests can detect hypersensitivity to a wide
lution or improperly stored allergen extract can also lead to
variety of inhaled or food allergens. In these tests, the clinician
false-negative results.29 False-positive results can also occur;
uses a needle or pricking device to introduce a small drop of
these may be caused by the patient’s reaction to the diluent,
allergen extract into the upper layers of the individual’s skin
preservative, or contaminants in the allergen extract or to
in the inner forearm or the back. A panel of allergens is rou-
physical trauma to the skin in patients with severe skin der-
tinely used, with each applied to separate sites 2 to 2.5 cm
matographism or eczema.29 In addition, there is the danger
apart. A negative control consisting of the diluent used for the
that a systemic reaction can be triggered. In cases where the
allergy extract and a positive control of histamine are also in-
risk of a harmful reaction is too large, skin disorders are pres-
cluded. After 15 to 20 minutes, the clinician examines the
ent, or patients cannot discontinue medications before test-
testing spots and records the reaction. In a positive test, a
ing, serological testing for allergen-specific IgE antibodies is
wheal-and-flare reaction will appear at the site where the al-
indicated.
lergen was applied (Fig. 14–5). Scoring of the reaction is
based on the presence or absence of erythema and the diam-
eter of the wheal, with a diameter larger than 3 to 4 mm Allergen-specific IgE tests are safer to perform than skin testing;
correlating best with the presence of allergy.26-28 are easier on some patients, especially children or apprehensive
Intradermal tests use a greater amount of antigen and are adults; and have excellent analytical sensitivity.30,31 These tests
more sensitive than cutaneous tests. However, they are usually are useful in detecting allergies to a number of common trig-
performed only if prick tests are negative and allergy is still gers, including ragweed, trees, grasses, molds, animal dander,
suspected because they carry a larger risk (0.05%) for anaphy- foods, and insect venom.
lactic reaction than prick tests (0.03%).27 In intradermal test- The original commercial testing method for determining
ing, a 1-mL tuberculin syringe is used to administer 0.01 to specific IgE, the radioallergosorbent test (RAST), was introduced
0.05 mL of test solution between layers of the skin. The test in 1972. In this radioimmunoassay, patient serum was incu-
allergen is diluted 100 to 1,000 times more than the solution bated with a paper disk to which various allergens were cova-
used for cutaneous testing. This test is performed on the inner lently linked. Following a washing step to remove unbound
forearm or upper arm so that if a systemic reaction occurs, a antibody, bound IgE was detected by adding a radiolabeled
anti-IgE. After a second wash step, the amount of radioactivity interchangeable; they are likely caused by differences in the
detected was measured by a gamma counter and was propor- composition of the allergen reagents.30 Because crude allergen
tional to the amount of allergen-specific IgE in the patient’s extracts are typically used in these tests, standardization is
sample.30,31 difficult and cross-reactivity to similar but clinically insignif-
The principles of current immunoassays for serum IgE re- icant antigens may be detected. This limitation has stimulated
main the same, but the newer testing methods use enzyme la- the development of recombinant allergen components pro-
bels that react with substrates to produce fluorescence, duced by cloning the genes coding for these proteins and pu-
chemiluminescence, or colorimetric reactions rather than ra- rifying the allergenic substances produced by the genetically
dioactive labels. Figure 14-6 illustrates the principle of these modified cells.30,31 Recombinant allergens are being incorpo-
tests. All of these methods are automated immunoassays that rated into existing assay formats and should significantly
have a high level of specificity and sensitivity. The tests can be increase the diagnostic specificity of allergy testing.
run with a single allergen or as a multiallergen screen using a Using advanced biochemical and molecular techniques, sci-
panel of allergens in a single run. A commercial noncompeti- entists have been able to characterize more than 1,780 aller-
tive fluoroimmunoassay is considered by most allergy special- gens.35 This knowledge has led to the development of a
ists to be the method of choice.32,33 In this assay, patient serum microarray format that allows for parallel detection of IgE an-
is incubated with an allergen-coated cellulose sponge that has tibodies to more than 100 potential allergens using only 20 µL
a high binding capacity for IgE antibody. After a wash step, of patient serum.35 In this system, patient serum is incubated
an enzyme-labeled anti-IgE reagent is added; following incu- with a biochip containing miniature spots to which the purified
bation, another washing step is performed to remove un- allergenic components have been applied. The chip contains a
bound materials. The corresponding substrate is added, and wide variety of antigens from foods, pollens, molds, fungi,
fluorescence is produced in proportion to the amount of latex, and insect venoms. An internal control is provided be-
allergen-specific IgE in the sample. The results are derived from cause all allergens are spotted in triplicate. If allergen-specific
a standard calibration curve that is linked to the World Health IgE is present, it will bind to the appropriate spots. The chip
Organization (WHO) IgE standard. Allergen-specific IgE val- is scanned by a laser for fluorescence following addition of a
ues are reported in kilo international units (IU) of allergen- fluorescent-labeled anti-IgE.32 This advanced technology is
specific antibody per liter (kUa/L), where one unit is equal to highly sensitive and specific. It can theoretically use an unlim-
2.42 ng/mL of IgE.30 The method can detect IgE antibodies ited number of natural and recombinant allergens. The tech-
in the range of 0 to 100 kU/L, and 0.35 kU/L is commonly nology will significantly enhance the diagnostic value of
used as the cut-off for a positive test.8,34 specific IgE testing.
Other immunoassays for allergen-specific IgE have com- Another development is a point-of-care lateral flow assay
parable sensitivity and specificity, but the results are not that can be used by primary care physicians to screen for

Total
serum
IgE

Anti-IgE

Capture Wash Labeled anti-IgE Wash Substrate

Allergen-
specific
IgE

Antigen
Comparison of noncompetitive immunoassays for total serum IgE (formerly known as RIST) and allergen-specific IgE (formerly
known as RAST). IgE in the patient serum is shown in red for both tests. Total IgE is measured by capturing the antibody with solid-phase
anti-IgE. A second anti-IgE immunoglobulin with an enzyme label is used to produce a visible reaction. Antigen-specific IgE is measured by
using solid-phase antigen to capture patient antibody. Then a second antibody, enzyme-labeled anti-IgE immunoglobulin, is added. This
combines with any bound IgE to produce a visible reaction in the presence of substrate.
reactivity to a few common allergens.31,32 In this assay, the al- greater than 1,000 kU/L, whereas patients with hyper-IgE syn-
lergen extracts are coated onto nitrocellulose strips encased in drome have extremely high IgE levels (2,000–50,000 kU/L).
a cassette. A drop of blood from a finger prick is added to one Measurement of total serum IgE is also helpful in monitoring
well in the cassette and a color-developing solution is added patients undergoing allergen immunotherapy or treatment with
to a second well, driving the blood toward the antigen zone. the monoclonal anti-IgE antibody, omalizumab. Successful
In this semiquantitative test, the presence of allergen-specific treatment results in significant reductions in total serum IgE
IgE is indicated by a colored line in the corresponding allergen levels and in the ratio of allergen-specific IgE to total IgE.31
position. Patients with positive results can be referred to an
allergist for further evaluation.
Regardless of the format of the specific IgE test used, the re-
Type II Hypersensitivity
sults should always be interpreted in light of the patient’s med-
Type II hypersensitivity is also known as antibody-mediated cy-
ical history and clinical symptoms. This is because the presence
totoxic hypersensitivity. The underlying mechanism involves IgG
of allergen-specific IgE antibody indicates sensitization to the
and IgM antibodies directed against antigens found on cell sur-
allergen but not necessarily the presence of a clinical allergy.30,34
faces. These antigens may be altered self-antigens or heteroanti-
gens. Binding of the antibody to a cell can have one of three major
Tests have also been developed to measure total serum IgE. effects, depending on the situation (see Fig. 14–1): (1) The cell
The first test developed for the measurement of total IgE was can be destroyed; (2) the function of the cell can be inhibited; or
the competitive radioimmunosorbent test (RIST). The RIST used (3) the function of the cell can be increased above normal.
radiolabeled IgE to compete with patient IgE for binding sites Cell damage can occur by several different mechanisms,
on a solid phase coated with anti-IgE. Because of the expense some of which involve complement as well as antibodies:
and difficulty of working with radioactivity, RIST has largely (1) Activation of the classical pathway of complement can lead
been replaced by noncompetitive solid-phase immunoassays to the formation of the membrane attack complex and cell
or nephelometry assays with enhanced sensitivity.8,28 lysis. (2) Coating of the cell surface by antibodies can promote
In the noncompetitive solid-phase immunoassays, anti- opsonization and subsequent phagocytosis of the cells. Op-
human IgE is bound to a solid phase such as cellulose, a paper sonization can occur either through binding of IgG antibody
disk, or a microtiter well. Patient serum is added and allowed to Fc receptors on macrophages and neutrophils or binding of
to react and then an enzyme-labeled anti-IgE is added to detect cell surface C3b to complement receptors on phagocytic cells.
the bound patient IgE. The second anti-IgE antibody recog- (3) Cell damage can result from the mechanism of antibody-
nizes a different epitope than that recognized by the first anti- dependent cellular cytotoxicity (ADCC). ADCC is mediated
body. The resulting “sandwich” of solid-phase anti-IgE, serum through binding of IgG antibody to its corresponding antigen
IgE, and labeled anti-IgE is washed; a colorimetric, fluoromet- on the target cell and to Fc receptors on macrophages or nat-
ric, or chemiluminescent substrate is then added. The amount ural killer cells. This binding stimulates the release of cytotoxic
of reactivity detected is directly proportional to the IgE content enzymes that destroy the cell. Clinical examples that involve
of the serum (see Fig. 14–6).33 destruction of cells by type II hypersensitivity include blood
Total IgE values are reported in kilo international units (IU) transfusion reactions, hemolytic disease of the newborn, and
per liter. One IU is equal to a concentration of 2.4 ng of protein autoimmune hemolytic anemia. These conditions are discussed
per milliliter. IgE concentration varies with the individual’s age in the sections that follow.
and exposure to allergens. The total IgE concentration is typi- A second possible effect of type II hypersensitivity is that
cally lower than 1 kU/L in cord blood, and serum IgE usually the cell surface antibody can inhibit the function of a cell. This
reaches adult levels at about 10 years of age. In adults, a cutoff can occur when antibody blocks the binding of a physiological
value of 100 kU/L is considered the upper limit of normal. Lev- ligand to its receptor, resulting in dysfunction of the cell. An
els above 100 kU/L are common in individuals with allergies.8 example of this effect occurs in the autoimmune disease myas-
However, measurement of total serum IgE is not recommended thenia gravis, which affects the neuromuscular junctions. Pa-
for the routine clinical evaluation of patients with suspected tients with this disease produce autoantibodies to receptors on
allergies8,26 because values vary widely among patients and do muscle cells for the neurotransmitter acetylcholine (ACH).
not necessarily correlate with the presence of allergy. Patients Normally, ACH is released from the nerve endings and binds
with slightly elevated IgE levels may not have an allergy, to its corresponding receptors on muscle cells, stimulating con-
whereas many patients with allergies have a total serum traction in the muscle fibers and muscle movement. However,
IgE concentration that falls within the reference range. Thus, in myasthenia gravis, attachment of the autoantibody to the
allergen-specific IgE tests are considered to have more value in ACH receptor blocks the binding of ACH, leading to muscle
the diagnosis of allergies. weakness (see Chapter 15).
Total serum IgE testing is more beneficial in evaluating pa- Sometimes, binding of an antibody to a self-antigen can
tients with other conditions in which IgE levels may be elevated, have the opposite effect, stimulating the cell instead of inhibit-
such as helminth infections, and certain immunodeficiencies, ing its function. This results in overproduction of the cell’s
such as Wiskott-Aldrich syndrome, DiGeorge syndrome, and product, such as a hormone. The classic example of this effect
hyper-IgE syndrome.8 Children living in areas where parasitic is an autoimmune disorder of the thyroid gland called Graves
infections are endemic typically have serum IgE concentrations disease. Patients with Graves disease produce antibodies
against the receptor for the thyroid-stimulating hormone (TSH) most often associated with ABO blood group incompatibilities
on thyroid cells. TSH, a hormone produced by the pituitary and the antibodies are of the IgM class.1,40,41 As soon as cells
gland in the brain, binds to the TSH receptors and stimulates bearing the antigen are introduced into the patient, intravas-
the thyroid cells to produce hormones that increase metabo- cular hemolysis occurs because of complement activation, re-
lism. Normally, this process is carefully regulated by a feedback sulting in the release of hemoglobin and vasoactive and
loop that signals the pituitary gland to make less TSH in the procoagulant substances into the plasma. This may induce
presence of high levels of thyroid hormones.36 However, in disseminated intravascular coagulation (DIC), vascular col-
Graves disease, the autoantibody binds to the TSH receptor, lapse, and renal failure. Symptoms in the patient may include
resulting in unregulated production of thyroid hormones. This fever, chills, nausea, lower back pain, tachycardia, shock, and
leads to symptoms associated with increased metabolism, hemoglobin in the urine.39,40
known as hyperthyroidism (see Chapter 15). Delayed hemolytic reactions occur within the first 2 weeks
following a transfusion and are caused by an anamnestic re-
sponse to the antigen to which the patient has previously been
Clinical Examples of Type II exposed.41 The type of antibody responsible is IgG, which was
Hypersensitivity initially present in such low titer that it was not detectable with
an antibody screen. Antigens most involved in delayed reac-
tions include those in the Rh, Kell, Duffy, and Kidd blood
Transfusion reactions are examples of cell destruction that re- groups.1,42 Rh, Kell, and Duffy antigens may also be involved
sults from antibodies combining with heteroantigens. There in immediate transfusion reactions. In a delayed reaction,
are more than 29 different blood group systems with more than antibody-coated RBCs are removed extravascularly in the spleen
700 different RBC antigens.37,38 Some antigens are stronger or in the liver. The patient may experience a mild fever, low
than others and are more likely to stimulate antibody produc- hemoglobin, mild jaundice, and anemia. Intravascular hemol-
tion. Major groups involved in transfusion reactions include ysis does not take place to any great extent because IgG is not
the ABO, Rh, Kell, Duffy, and Kidd systems.37,39 Certain anti- as efficient as IgM in activating complement. (See Chapter 5 for
bodies are produced naturally with no prior exposure to RBCs, further details.)
whereas other antibodies are produced only after contact with
cells carrying that antigen.
Hemolytic disease of the newborn (HDN) appears in infants
The ABO blood group is of primary importance in consid-
whose mothers have been exposed to blood-group antigens on
ering transfusions. Anti-A and anti-B antibodies are naturally
the baby’s cells that differ from their own. The mother makes IgG
occurring antibodies, or isohemagglutinins, which are prob-
antibodies in response to these antigens that cross the placenta
ably triggered by contact with identical antigenic determinants
to destroy the fetal RBCs. Severe HDN is called erythroblastosis
on microorganisms. Individuals do not make these antibodies
fetalis. The most common antigen involved in severe reactions is
to their own RBCs. Thus, a person who has type A blood has
the D antigen, a member of the Rh blood group. HDN caused by
anti-B in the serum and a person with type B blood has anti-A
ABO incompatibility is actually more common; however, the dis-
antibodies. An individual with type O blood has both anti-A
ease is milder, possibly because the antibodies are neutralized by
and anti-B in the serum because O cells have neither of these
A or B antigens found in fetal tissues or because the A and B anti-
two antigens. The antibody formed typically belongs to the IgM
gens on the fetus’ RBCs are more poorly developed or reduced
class, but IgG may also be made.
in number.43 Other antibodies associated with HDN include
If a patient is given blood for which antibodies are already
anti-c, anti-C, anti-E, anti-e, and less commonly those associated
present, a transfusion reaction occurs. This reaction can range
with the Kell, Duffy, and Kidd blood groups.44
from acute massive intravascular hemolysis to an undetected
Exposure usually occurs during the birth process when fetal
decrease in RBC survival. The extent of the reaction depends
cells leak into the mother’s circulation. Typically, the first child
on the following factors:
is unaffected; however, the second and later children have an
• The temperature at which the antibody is most active increased risk of the disease because of an anamnestic re-
• The plasma concentration of the antibody sponse. The extent of the first fetal–maternal bleed influences
• The immunoglobulin class involved whether antibodies will be produced. If enough of the baby’s
• The extent of complement activation RBCs enter the mother’s circulation, memory B cells develop.
• The density of the antigen on the RBC These become activated upon re-exposure to the same RBC
• The number of RBCs transfused40 antigen; IgG is then produced. This antibody crosses the pla-
It is most important to detect antibodies that react at 37°C. centa and attaches to the fetal RBCs in a subsequent pregnancy.
If a reaction occurs only below 30°C, it can be disregarded be- Depending on the degree of antibody production in the
cause antigen–antibody complexes formed at colder tempera- mother, the fetus may be aborted, stillborn, or born with ev-
tures tend to dissociate at 37°C. idence of hemolytic disease as indicated by jaundice. As
Acute hemolytic transfusion reactions may occur within min- RBCs are lysed and free hemoglobin released, this is con-
utes or hours after receipt of incompatible blood. In this case, verted to bilirubin, which builds up in the plasma. There is
the individual has been exposed to the antigen before and has too much of it to be conjugated in the liver, so it accumulates
preformed antibodies to it. Reactions that begin immediately are in the tissues. Bilirubin levels above 20 mg/dL are associated
with deposition in tissues such as the brain and result in a Warm autoimmune hemolytic anemia, which accounts for
condition known as kernicterus. Treatment for severe HDN more than 70% of autoimmune anemias, is characterized by for-
involves an exchange transfusion to replace antibody-coated mation of IgG antibody; this reacts most strongly at 37°C.42,46
RBCs. If serum antibody titrations during the pregnancy in- Some of these antibodies may be primary with no other disease
dicate a high level of circulating antibody, intrauterine trans- association; others may be secondary to another disease process.
fusions can be performed.1,43,44 Associated diseases may include viral or respiratory infections—
To prevent the consequences of HDN, all women should be such as infectious mononucleosis, cytomegalovirus, or chronic
screened at the onset of pregnancy. If they are Rh-negative, they active hepatitis—or immunoproliferative diseases—such as
should be tested for the presence of anti-D antibodies on a chronic lymphocytic leukemia and lymphomas.46,47 Often, the
monthly basis. In current practice, anti-D immune globulin, underlying cause of antibody production is unknown; this is re-
called Rhogam, is administered prophylactically at 28 weeks of ferred to as idiopathic autoimmune hemolytic anemia.
gestation and within 72 hours following delivery.43 The mecha- In addition, certain drugs can induce production of anti-
nism by which Rhogam works is not completely known, but it is bodies that can cause hemolytic anemia. These drugs are ca-
thought to facilitate clearance of the fetal RBCs through opsoniza- pable of attaching to the RBCs directly or of forming immune
tion and therefore suppress production of maternal antibody complexes that attach to the RBCs. Damage to the RBCs is be-
(Fig. 14–7).44 This practice has dramatically reduced the number lieved to occur through several mechanisms.42,46 Some drugs,
of women who form anti-D antibodies to slightly over 1%.45 such as the penicillins and cephalosporins, can act as haptens
after binding to proteins on the RBC membrane. These drugs
stimulate the production of anti-drug antibodies that destroy
Autoimmune hemolytic anemia is an example of a type II the RBCs, primarily through extravascular hemolysis. The
hypersensitivity reaction directed against self-antigens because cephalosporins are also thought to modify the RBC membrane
individuals with this disease form antibodies to their own RBCs. by facilitating binding of immunoglobulins and complement.
Symptoms include malaise, lightheadedness, weakness, unex- Other drugs, such as quinidine and phenacetin, can stimulate
plained fever, pallor, and possibly mild jaundice.46 Such antibod- the production of anti-drug antibodies that bind to the drug
ies can be categorized into two groups: warm reactive antibodies, to form soluble immune complexes. The complexes attach
which react at 37°C, and cold reactive antibodies, which only loosely to the surface of the RBCs, which are cleared by in-
react below 30°C. Autoimmune hemolytic anemia has been travascular hemolysis after binding of complement. Other
estimated to occur in 1 in 50,000 to 80,000 individuals.42 drugs, such as methyldopa, can induce hemolytic anemia by

Maternal Rhogam

NB MB

NB

Rh$ Rh$ Rh$

Placenta
Fetal
Rh# Rh# Rh#

A1. First pregnancy A2. Second pregnancy B. First pregnancy with

Rhogam treatment
Hemolytic disease of the newborn (HDN) can develop in Rh-positive babies who are born to Rh-negative mothers if no
treatment is administered. (A1) An Rh-negative mother may produce anti-Rh antibodies if an Rh-positive baby’s RBCs enter her circulation late
in gestation or during the birth process. In this primary immune response, naïve B cells (NB) differentiate and the plasma cells secrete mainly
anti-Rh IgM, which does not cross the placenta; therefore, a healthy baby is delivered. However, memory B cells (MB) are also produced and
(A2) if the mother becomes pregnant with another Rh-positive baby, these memory cells produce a stronger secondary immune response
and secrete mainly anti-Rh IgG, which crosses the placenta and destroys the baby’s RBCs, resulting in HDN. (B) Alternatively, passive vaccina-
tion of the mother with an anti-Rh IgG called Rhogam during pregnancy and upon delivery prevents the mother from mounting an active
immune response, allowing her future Rh-positive children to be born normally.
stimulating production of autoantibodies against the RBC macrophages and rapid clearance of the cells in the liver. Less
membrane. commonly, the entire classical pathway is activated and in-
Typically, patients exhibit symptoms of anemia because of travascular hemolysis occurs.42 Both processes lead to de-
clearance of antibody-coated RBCs by macrophages in the struction of the RBCs and corresponding symptoms of
liver and spleen. Hemolysis is primarily extravascular because autoimmune hemolytic anemia.46 Patients with this disease
IgG is not as efficient as IgM in activating complement; how- are usually treated by simply avoiding cold temperatures and
ever, intravascular hemolysis can also occur if complement keeping the extremities warm; drug therapy is only used if
does become activated. Severity of the hemolysis is affected these measures are ineffective.46,50
by the subclass of IgG involved, with IgG3 and IgG1 being A rare condition known as paroxysmal cold hemoglobin-
most destructive to the RBCs because they are efficient at uria can also cause autoimmune hemolytic anemia. This con-
binding complement.46 Patients with warm autoimmune he- dition occurs most often after infection with certain viral
molytic anemia are usually treated with corticosteroids to re- illnesses, including measles, mumps, chickenpox, and infec-
duce antibody synthesis or, in more serious cases, with a tious mononucleosis. Patients produce a biphasic autoantibody
splenectomy to decrease RBC clearance.46,47 Treatment with that binds to the RBCs at cold temperatures and activates com-
anti-CD 20 (rituximab) can be used for cases that are refrac- plement at 37°C to produce an intermittent hemolysis. Conse-
tory to corticosteroids.47,48 This antibody attaches to B cells quently, an acute, rapidly progressing anemia with hemoglobin
and causes a decrease in antibody production. in the urine is seen. The condition occurs most often as a tran-
Cold agglutinins are a less frequent cause of immune he- sient disorder in children and young adults.42,46
molytic anemias. By definition, cold agglutinins are autoanti-
bodies that react with antigens on the RBC membrane at cold
All the reactions that have been discussed so far deal with indi-
temperatures.49 The reaction is reversible upon exposure to a
vidual cells that are destroyed when a specific antigen–antibody
warm temperature. These cold-reacting antibodies belong to
combination takes place. Some type II reactions involve destruc-
the IgM class and most are specific for the Ii blood groups on
tion of tissues because of their combination with antibody.
RBCs.42,46,47 Cold agglutinins may be transient or chronic, de-
Organ-specific autoimmune diseases in which antibody is di-
pending on the cause. Polyclonal cold agglutinins can be pro-
rected against a particular tissue are in this category. Goodpas-
duced secondary to certain infections, most notably Mycoplasma
ture’s syndrome is an example of such a disease (see Chapter 15
pneumonia and infectious mononucleosis but also respiratory
for details). The antibody produced during the course of this
viruses and HIV.47,48,50 In most cases, cold agglutinin produc-
disease reacts with basement membrane protein. Usually the
tion is transient and resolves in 2 to 3 weeks.46 Persistent, high
glomeruli in the kidney and pulmonary alveolar membranes are
titer, monoclonal cold agglutinins have been associated with
affected.1 Antibody binds to glomerular and alveolar capillaries;
B- or plasma cell-lymphoproliferative disorders including B-cell
this triggers the complement cascade, which provokes inflam-
chronic lymphocytic leukemia (CLL), B-cell lymphomas,
mation. An evenly bound linear deposition of IgG in the
Hodgkin disease, and Waldenstrom’s macroglobulinemia as well
glomerular basement membrane, which is detected with fluo-
as autoimmune diseases such as systemic lupus erythematosus
rescent-labeled anti-IgG, is indicative of Goodpasture’s syn-
(SLE). Chronic cold agglutinins can also be produced as a pri-
drome. Treatment usually involves the use of corticosteroids or
mary characteristic of a disease entity of unknown origin, which
other drugs to suppress the immune response.
is known as chronic cold agglutinin disease (CCAD) or chronic
Other examples of type II hypersensitivity reactions to tissue
hemagglutinin disease (CHD).46,49 CHD typically occurs in per-
antigens include some of the organ-specific autoimmune dis-
sons over the age of 50 and is responsible for about 20% of au-
eases such as Hashimoto’s disease, myasthenia gravis, and in-
toimmune hemolytic anemia cases.42,46
sulin-dependent diabetes mellitus. Immunologic manifestations
Cold agglutinins do not cause clinical symptoms unless the
and detection of these diseases are presented in Chapter 15.
individual is exposed to the cold and usually have their maxi-
mal effect when the temperature in the peripheral circulation
falls below 30°C.42,50 Under these conditions, the antibodies
Testing for Type II Hypersensitivity
can bind to the RBCs to form lattices, which can obstruct the Coombs’ discovery of the antiglobulin test in 1945 made
small capillaries in the skin.49 The areas of the body most af- possible the detection of antibody or complement on RBCs.
fected are those having greatest exposure to the cold, most no- The direct antiglobulin test (DAT) is performed to detect
tably the fingers, toes, earlobes, and nose. These areas develop transfusion reactions, HDN, and autoimmune hemolytic ane-
a blue coloration known as acrocyanosis and become numb, mia. (Refer to the exercise in this text’s DavisPlus website at
stiff, and slightly painful.50 The symptoms are quickly re- davisplus.fadavis.com keyword Stevens for details.) Polyspe-
versible when the patient returns to warm surroundings; how- cific anti-human globulin, which is a mixture of antibodies to
ever, in severe cases, peripheral necrosis may result. IgG and complement components such as C3b and C3d, is
Another consequence of cold agglutinins results from the used for initial testing. If the test is positive, it should be re-
fixation of complement. Although complement activation peated using monospecific anti-IgG, anti-C3b, and anti-C3d
cannot be completed in the cold, it can proceed once the cells to determine which of these is present.42 If an autoimmune
recirculate and reach body temperature. If the RBCs become hemolytic anemia is caused by IgM antibody, only the tests for
coated with C3b, opsonization can facilitate binding to complement components would be positive.
 The indirect Coombs’ test is used in the crossmatching of process results in the damage to host tissue that is typified
blood to prevent a transfusion reaction. It can either determine by type III reactions. Long-term changes include loss of tis-
the presence of a particular antibody in a patient or type pa- sue elements that cannot regenerate and accumulation of
tient RBCs for specific blood group antigens. The method de- scar tissue. Type III hypersensitivity reactions can be local
tects in vitro binding of antibody to RBCs rather than in vivo or systemic, depending on where the immune complexes
binding. This method is a two-step process in which RBCs deposit in the body.
and antibody are allowed to combine at 37°C and then the
cells are carefully washed to remove any unbound antibody. Arthus Reaction
Anti-human globulin is added to cause a visible reaction if
antibody has been specifically bound. Any negative tests are The classic example of a localized type III reaction is the
confirmed by quality-control cells, which are coated with Arthus reaction, demonstrated by Maurice Arthus in 1903.
antibody. Using rabbits that had been immunized to produce an abun-
To determine the titer of a cold agglutinin antibody, the dance of circulating antibodies, Arthus showed that when
patient serum can be serially diluted and incubated overnight these rabbits were challenged with an intradermal injection of
at 4°C with a dilute suspension of washed human type O the antigen, a localized inflammatory reaction resulted. The
RBCs.49 The tubes are then gently shaken and observed for reaction which is characterized by erythema and edema, peaks
agglutination. The last tube with agglutination represents the within 3 to 8 hours and is followed by a hemorrhagic necrotic
titer. Titers of 64 or higher are considered to be clinically sig- lesion that may ulcerate.1 The inflammatory response is
nificant.42,49 The agglutination should disappear after warm- caused by an antigen–antibody combination and subsequent
ing the tubes briefly in a 37°C water bath. Before testing, it is formation of immune complexes that deposit in small dermal
important to use prewarmed blood to separate the serum or blood vessels1,51 (Fig. 14–8). Complement is fixed, attracting
plasma from the patient’s RBCs.49 Failure to do so can result neutrophils and causing aggregation of platelets. Neutrophils
in binding of the cold agglutinins to the patient’s own RBCs, release toxic products such as oxygen-containing free radicals
producing false-negative results when the patient’s serum is and proteolytic enzymes. Activation of complement is essen-
assayed for cold agglutinin reactivity against the reagent type tial for the Arthus reaction because the C3a and C5a that are
O cells.

Type III Hypersensitivity

Type III hypersensitivity reactions are similar to type II reactions
in that IgG or IgM is involved and destruction is complement-
mediated. However, in the case of type III-associated diseases,
the antigen is soluble. When soluble antigen combines with Epidermis
antibody, complexes are formed that precipitate out of the
serum. Normally such complexes are cleared by phagocytic
cells; however, if the immune system is overwhelmed, these Dermis
complexes deposit in the tissues. There, they bind comple-
Subcutaneous
ment, causing damage to the particular tissue. Deposition of
antigen–antibody complexes is influenced by the relative con-
centration of both components. If a large excess of antigen is
present, sites on antibody molecules become filled before
cross-links can be formed. In antibody excess a lattice cannot
be formed because of the relative scarcity of antigenic deter-
minant sites (see Fig. 10–3). The small complexes that result
in either of the preceding cases remain suspended or may pass
directly into the urine. Precipitating complexes, on the other
hand, occur in mild antigen excess and are the ones most likely
to deposit in the tissues. Sites in which this typically occurs in-
Endothelial
clude the glomerular basement membrane, vascular endothe- cells
lium, joint linings, and pulmonary alveolar membranes.1
Blood vessel
Complement binds to these complexes in the tissues, lumen
causing the release of mediators that increase vasodilation
The Arthus phenomenon. Antigen is injected into
and vasopermeability, attract macrophages and neutrophils, the skin of an individual who has circulating antibody of that
and enhance binding of phagocytic cells by C3b-mediated specificity. Immune complexes are formed and deposit on the walls
opsonization. If the target cells are large and cannot be en- of blood vessels, activating complement. Complement-generated
gulfed for phagocytosis to take place, granule and lysosome anaphylatoxins cause vasodilation, increased vascular permeability,
contents are released by a process known as exocytosis. This edema, and accumulation of neutrophils.
generated activate mast cells to release permeability factors; Testing for Type III Hypersensitivity
consequently, immune complexes localize along the endothe-
lial cell basement membrane. The Arthus reaction can some- In autoimmune diseases such as SLE and RA, the presence of
times be seen in humans following booster injections with antinuclear antibodies can be detected by a variety of methods,
tetanus, diphtheria, or measles vaccines.52 including indirect immunofluorescence, enzyme-linked im-
munosorbent assay, and fluorescent microsphere multiplex im-
munoassays (see Chapter 15 for details). Fluorescent staining
Serum Sickness of tissue sections has also been used to determine deposition
Serum sickness is a generalized type III hypersensitivity re- of immune complexes in the tissues. The staining pattern seen
action that results from passive immunization of humans with and the particular tissue affected help to identify the disease
animal serum. Before the advent of antibiotics and vaccines, and determine its severity. Rheumatoid factor can be detected
serum sickness was observed more often because horse anti- by latex agglutination, nephelometry, or other immunoassays
serums were used to treat infections such as diphtheria, (see Chapter 15).
tetanus, and pneumonia.51 These antisera were produced by A more general method of evaluating immune complex dis-
immunizing horses with the corresponding antigen and pro- eases is by measuring complement levels. During periods of
vided immediate immunity to the individuals receiving them. high disease activity, complement levels in the serum may be
Today, horse antivenoms are still used to treat people who decreased because of binding of some of the complement to
have been bitten by poisonous snakes. Serum sickness can the antigen–antibody complexes. The results should be inter-
also occur after treatment of patients with mouse monoclonal preted in conjunction with other clinical findings. Refer to
antibodies for diseases such as cancer or autoimmune disor- Chapter 7 for a discussion of complement testing.
ders.1,51 Patients exposed to such animal sera can produce
antibodies against the foreign animal proteins. These can Type IV Hypersensitivity
combine with their corresponding antigen to form immune
complexes that can deposit in the tissues and trigger the type Type IV hypersensitivity was first described in 1890 by
III hypersensitivity response. Robert Koch. He observed that individuals infected with My-
Generalized symptoms of serum sickness appear 7 to 21 days cobacterium tuberculosis (Mtb) developed a localized inflam-
after injection of the animal serum and include headache, fever, matory response after receiving intradermal injections of a
nausea, vomiting, joint pain, rashes, and lymphadenopathy.1 filtrate from the organism.1 Type IV hypersensitivity differs
Usually this is a self-limiting disease and recovery takes a few from the other three types of hypersensitivity in that sensi-
weeks after the offending antigen is eliminated.51 However, pre- tized T cells, rather than antibodies, play the major role in
vious exposure to animal serum can result in a more rapid reac- its manifestations. Because symptoms peak between 48 to
tion with increased severity.1 72 hours after exposure to antigen, this reaction is also
known as delayed hypersensitivity. Although there are dif-
Autoimmune Diseases and Other Causes ferent mechanisms involved in type IV hypersensitivity, the
classic type IV response involves the Th1 subclass of
of Type III Hypersensitivity T helper (Th) cells (see Fig. 14–1). There is an initial sensi-
Type III hypersensitivity reactions can also be triggered by tization phase of 1 to 2 weeks that takes place after the first
autologous antigens as seen in several of the autoimmune contact with antigen. During this phase, Langerhans cells in
diseases. SLE and rheumatoid arthritis (RA) are two such the skin and macrophages in the tissues capture antigen and
examples. Patients with these diseases commonly produce migrate to nearby lymph nodes, where they present the anti-
antibodies against nuclear constituents such as DNA and gen to naïve Th cells. The antigen-presenting cells (APCs)
histones. They may also produce an antibody against IgG also release cytokines that promote differentiation of the naïve
called rheumatoid factor. The autoantibodies combine with T cells into Th1 cells and other T-cell subsets and induce their
their corresponding antigen to produce immune complexes proliferation. The expanded Th1 cells then migrate to the site
that trigger the type III hypersensitivity response. In SLE, where the antigen is located and the effector phase of the re-
immune complex deposition involves multiple organs; how- sponse begins. At the site, the activated Th1 cells release
ever, the main damage occurs to the joints, skin, and cytokines. IL-3 and GM-CSF induce hematopoiesis of cells
glomerular basement membrane in the kidneys. In rheuma- of the granulocyte-macrophage lineage and chemokines such
toid arthritis, immune complexes primarily cause damage to as monocyte chemotactic protein (MCP-1/CCL2) recruit
the joints. Complement enhances tissue destruction in both macrophages to the site. In the tissues, the monocytes differ-
diseases. See Chapter 15 for a more detailed discussion of entiate into macrophages and are activated by IFN-γ and
these two conditions. TNF-β These activated macrophages release reactive oxygen
Type III hypersensitivity can also be caused by a number of species, nitric oxide, and proinflammatory mediators that re-
other factors.1,51 These include components of vaccines, bee cruit more macrophages to the site and stimulate them to be-
stings, treatment with certain drugs (for example, penicillin come effective APCs, thus perpetuating the response.1,51,53,54
and sulfonamides), and infections, such as viral hepatitis and Chronic persistence of antigen leads to the development of
Group A Streptococcus. organized clusters of cells called granulomas, which consist
of epithelioid-shaped and multinucleated fused macrophages
with an infiltrate of lymphocytes or other WBCs. Many of these
macrophages contain engulfed antigens such as intracellular
bacteria. Thus, the granuloma can function to wall off the or-
ganism in a contained area, preventing its spread to other parts
of the body. However, cells in the granuloma can release large
amounts of lytic enzymes that can destroy surrounding tissue
and promote fibrin deposition.1 Cytotoxic T cells are also re-
cruited and they bind with antigen-coated target cells to cause
further tissue destruction.51
The antigens that can trigger the type IV hypersensitivity
are generally one of two types: intracellular pathogens or con-
tact antigens. The intracellular pathogens can be bacteria,
fungi, parasites, or viruses. Pathogens that commonly induce
delayed hypersensitivity include Mycobacterium tuberculosis, Contact hypersensitivity. Formation of papules occurs
Mycobacterium leprae, Pneumocystis carinii, Leishmania species, after exposure to poison ivy. (Courtesy of Yong Choi/Thinkstock.)
and herpes simplex virus.1 Contact antigens are those that
come into direct contact with the skin. They include plants been removed.54 Simple redness may fade of its own accord
such as poison ivy and poison oak, metals such as nickel salts, within several days. If the area is small and localized, a topical
and components of hair dyes and cosmetics.1,55 Clinical man- steroid may be used for treatment. Otherwise, systemic cortico-
ifestations of type IV hypersensitivity induced by these antigens steroids may be administered. The patient also needs to avoid
are discussed in the sections that follow. contact with the offending allergen.

Contact Dermatitis Hypersensitivity Pneumonitis

Contact dermatitis is among the most prevalent skin disorders, Evidence shows that hypersensitivity pneumonitis is mediated
affecting an estimated 15% to 20% of the general popula- predominantly by sensitized T lymphocytes that respond to
tion.53,55 Reactions are usually caused by low-molecular-weight inhaled allergens. IgG and IgM antibodies are formed, but
compounds that touch the skin. The most common causes in- these are thought to play only a minor role in the pathogenesis
clude poison ivy, poison oak, and poison sumac, all of which of this disorder. Hypersensitivity pneumonitis is an allergic dis-
release the chemical urushiol in the plant sap and on the leaves. ease of the lung parenchyma characterized by inflammation of
Allergic dermatitis caused by contact with these plants affects the alveoli and interstitial spaces. It is caused by chronic in-
millions of Americans every year.56 Other common compounds halation of a wide variety of antigens and is most often seen in
that produce allergic skin manifestations include nickel; rubber; individuals who are engaged in work or hobbies involving ex-
formaldehyde; hair dyes and fabric finishes; cosmetics; and posure to the implicated antigen.59,60 Depending on the occu-
medications applied to the skin, such as topical anesthetics, an- pation and the particular antigen, the disease goes by several
tiseptics, and antibiotics.55,57,58 In addition, latex sensitization names: farmer’s lung, bird breeder’s lung disease, and humidifier
has been reported as a cause of contact dermatitis in a signifi- or air conditioner lung disease. The reaction is most likely caused
cant number of health-care workers (see the previous text). by microorganisms, especially bacterial and fungal spores,
Most of these substances probably function as haptens that bind which individuals are exposed to from working with moldy
to glycoproteins on skin cells. Langerhans cells in the skin are hay, pigeon droppings, compost, moldy tobacco, infested flour,
thought to process the hapten–protein complexes and migrate and moldy cheese, to name just a few examples. Symptoms in-
to the regional lymph nodes where they present the antigen to clude a dry cough, shortness of breath, fever, chills, weight
Th1 cells.53,54 Sensitization of the Th cells takes several days; loss, and general malaise, which may begin 6 to 8 hours after
however, once it occurs, its effects can last for years because of exposure to a high dose of the offending antigen.59,60 Alveolar
immunologic memory.1 Cytokine production by the Th1 cells macrophages and lymphocytes trigger a chronic condition
causes macrophages to accumulate and release cytokines and characterized by interstitial fibrosis with alveolar inflammation.
other substances that produce a local inflammatory response. Systemic corticosteroid therapy is used for treatment.
Contact dermatitis produces a skin eruption characterized by
erythema, swelling, and the formation of papules that appears Skin Testing for Delayed
from 6 hours to several days after the exposure (Fig. 14–9). The
papules may become vesicular with blistering, peeling, and
Hypersensitivity
weeping. The site usually itches. The dermatitis is first limited Skin testing is used clinically to detect delayed hypersensitivity
to skin sites exposed to the antigen, but then it spreads to ad- responses to a variety of antigens. The tests are based on a
joining areas. The duration of the reaction depends upon the T-cell–mediated memory response. When antigen is injected
degree of sensitization and the concentration of antigen ab- intradermally or applied to the surface of the skin, previously
sorbed. Dermatitis can last for 3 to 4 weeks after the antigen has sensitized individuals develop a reaction at the application site.
This reaction results from infiltration of T lymphocytes and
macrophages into the area. Blood vessels in the vicinity become
lined with mononuclear cells and the reaction reaches a peak
by 72 hours after exposure. Skin testing has been used to
determine allergen sensitivity in contact dermatitis, to assess
exposure to Mycobacterium tuberculosis, and to evaluate com-
petency of cell-mediated immune responses in patients with
immune deficiency diseases. Each of these applications is dis-
cussed in the text that follows.
The patch test is considered the gold standard in testing for
contact dermatitis.54,55 This test must be done when the patient
is free of symptoms or at least has a clear test site. A nonab-
sorbent adhesive patch containing the suspected contact aller- A
gen is applied on the patient’s back and the skin is checked for
a reaction over the next 48 hours. Redness with papules or tiny
blisters is considered a positive test. Final evaluation is con-
ducted at 96 to 120 hours. All readings should be done by a
skilled evaluator. False negatives can result from inadequate
contact with the skin.
Skin testing for exposure to tuberculosis is a classic example
of a delayed hypersensitivity reaction. The test is based on the
principle that soluble antigens from Mycobacterium tuberculosis
induce a reaction in people who currently have tuberculosis or
have been exposed to M tuberculosis in the past. The tuberculin
skin test uses an M tuberculosis antigen extract prepared from a
purified filtrate of the organism’s cell wall, called a purified pro-
tein derivative (PPD). The test is routinely performed by the B
Mantoux method in which 0.1 mL of 5 tuberculin units (TU) The Mantoux test. (A) Purified protein derivative
of PPD is injected intradermally into the inner surface of the (PPD) is injected into an individual’s forearm, which causes a wheal
forearm using a fine needle and syringe (Fig. 14–10).61,62 The (i.e., a raised area of the skin surface) to form at the injection site.
test site is examined between 48 and 72 hours for the presence (B) After 48 hours, the individual presented with an induration
of a hardened, raised area called induration. Interpretation of (hardened, raised area) indicating a positive reaction. (A. Courtesy of the
the reaction depends on the particular group in which the in- CDC/Gabrielle Benenson and Greg Knobloch, Public Health Image Library.
dividual is categorized. An induration reaction of 15 mm or B. Courtesy of the CDC/Donald Kopanoff, Public Health Image Library.)
more is considered a positive test in individuals with no risk
factors, whereas a reaction of 10 mm or greater is considered tetanus toxoid, Streptococcus bacteria, and fungal antigens such
positive in recent immigrants of high prevalence countries, in- as trichophyton and histoplasmin.61,64 The antigens are injected
travenous drug users, employees of health-care and other high- intradermally by the Mantoux method; the injection sites are then
risk facilities, persons with certain clinical conditions, and examined at 48 hours for redness and induration. Normal indi-
children younger than 5 years of age.62 An induration reaction viduals should mount a memory response and develop a positive
of 5 mm or more is considered positive in persons who have skin reaction to at least one of the antigens tested. Those with de-
HIV infection or other forms of immunosuppression, features ficient cell-mediated immunity will display anergy, the absence
on a chest x-ray consistent with tuberculosis, or recent contact of positive reactions for all of the common antigens used in the
with tuberculosis patients. A positive PPD test indicates that the skin test.64
individual has previously been exposed to M tuberculosis or a
related organism, but it does not necessarily mean that he or
she has an active tuberculosis infection. Positive test results also
occur in persons who have previously received the BCG (Bacil-
SUMMARY
lus Calmette Guerin) vaccine for tuberculosis. The test has been • Hypersensitivity is an exaggerated immune response
an important screening tool to detect exposure in health-care to antigens that are usually not harmful. It results in cell
workers and other individuals at risk for the infection.63 destruction and tissue injury.
Skin testing can also be used to determine whether the cell- • Gell and Coombs devised a system for classifying hyper-
mediated arm of the immune system is functioning properly in sensitivity reactions into four types based on the immuno-
individuals suspected of having immunodeficiency disorders. logic mechanism involved and the nature of the triggering
Antigens typically used for testing are from sources to which in- antigen.
dividuals have been commonly exposed such as Candida albicans,
• Hypersensitivity types I, II, and III are antibody-mediated. • The preferred method of screening for allergies is an in
Because they occur within minutes to hours after exposure vivo skin prick test, in which very small amounts of po-
to antigen, they are referred to as immediate reactions. tential allergens are injected under the skin. A positive test
Type IV hypersensitivity is a cell-mediated response in- produces a wheal-and-flare reaction within 20 minutes.
volving T lymphocytes. Because the clinical manifestations • In patients unable to tolerate skin testing, in vitro testing
do not appear until 24 to 72 hours after contact with the by noncompetitive solid-phase immunoassays for allergen-
antigen, the type IV response is also referred to as delayed specific IgE can be performed. In these assays, patient
hypersensitivity. serum is incubated with a solid phase to which a specific
• Type I hypersensitivity is a Th2 polarized immune re- allergen has been attached. Binding is detected with an
sponse that involves production of IgE antibody to the in- enzyme-labeled anti-human IgE antibody and a colorimet-
ducing antigen or allergen. In the sensitization phase of ric, fluorescent, or chemiluminescent substrate.
this response, IgE antibody binds to high-affinity FcεRI • Solid-phase immunoassays for total serum IgE can be used
receptors on mast cells and basophils. In the activation to monitor patients undergoing treatment with AIT or
phase, the receptors become cross-linked when the aller- omalizumab or to detect patients with certain diseases
gen binds to adjacent IgE molecules. This cross-linking characterized by elevated IgE levels. The principle of these
stimulates the cells to degranulate and release preformed tests is the same as that for allergen-specific IgE tests ex-
and newly synthesized chemical mediators that cause an cept that anti-IgE, rather than allergen, is attached to the
inflammatory response. The reaction occurs very quickly, solid phase.
within minutes of exposure to the inducing antigen. Cy- • Type II hypersensitivity involves production of IgG or IgM
tokines produced during the response can cause a late- antibodies to antigens on the surface of host cells. These an-
phase response of prolonged inflammation. tibodies can destroy the cells through complement-mediated
• Preformed mediators that are released from mast cells and cytolysis, opsonization and phagocytosis, or antibody-
basophils include histamine, eosinophil chemotactic fac- dependent cellular cytotoxicity (ADCC). In other cases,
tor of anaphylaxis, neutrophil chemotactic factor, and pro- binding of the antibody to the cell surface antigen can result
teolytic enzymes such as tryptase. These factors cause in dysfunction or overstimulation of the cell.
contraction of smooth muscle in the bronchioles, blood • Examples of type II reactions that involve cell damage in-
vessels, and intestines; increased capillary permeability; clude autoimmune hemolytic anemia, transfusion reac-
chemotaxis of eosinophils and neutrophils; and decreased tions, and hemolytic disease of the newborn (HDN).
blood coagulability. Newly synthesized mediators such as Myasthenia gravis is an example of a type II disorder in
prostaglandins, leukotrienes, and PAF potentiate the which the antibody blocks binding of a ligand to cell re-
effects of histamine and other preformed mediators. ceptors, causing dysfunction of the cells. In contrast, in
• Clinical manifestations of type I hypersensitivity include Graves disease the antibody produced stimulates cells after
localized wheal-and-flare skin reactions (urticaria or binding to cell receptors.
hives); rhinitis; allergic asthma; and systemic anaphylaxis, • The direct antiglobulin test (DAT) is used to screen for trans-
which can be life-threatening. fusion reactions, autoimmune hemolytic anemia, and HDN.
• Susceptibility to allergies is based on a combination of In this test, washed patient RBCs are combined with anti-
genetic factors and environmental influences. Genes affect human globulin and observed for agglutination, indicating
different aspects of the immune response that contribute the presence of IgG or complement components on the cells.
to the pathogenesis of type I hypersensitivity. Some genes • Cold agglutinin antibodies bind to RBCs at temperatures
affect anatomical structures. Exposure to infectious organ- below 30°C and cause microocclusions of small blood ves-
isms appears to play a key role in the development of sels or destruction of the RBCs, mainly through opsoniza-
allergic disease. Stress, variations in physical factors such tion and extravascular clearance by macrophages in the
as temperature, and contact with environmental pollutants liver. Production of cold agglutinins may be from un-
can intensify clinical manifestations of allergy in suscepti- known causes or may be associated with certain infections
ble individuals. or B cell/plasma cell lymphoproliferative disorders. Cold
• Allergies can be treated with drugs such as antihistamines, agglutinin titers can be determined by incubating patient
decongestants, and corticosteroids. The monoclonal anti- serum with a dilute suspension of human type O RBCs
IgE antibody omalizumab has been used to block the overnight at 4°C and observing for agglutination.
binding of IgE to mast cells and basophils in patients with • Type III hypersensitivity involves formation of IgG or IgM
moderate to severe asthma. antibody that reacts with soluble antigen under conditions
• Allergen immunotherapy (AIT) can be administered to pa- of slight antigen excess to form small complexes that pre-
tients for whom drug therapy and environmental control cipitate in the tissues. These complexes activate comple-
measures are not successful. The goal of AIT is to induce ment, resulting in migration of neutrophils to the site with
immune tolerance by administering gradually increasing subsequent release of lysosomal enzymes that produce
doses of the allergen over time. damage to the surrounding tissues.
• The Arthus reaction, characterized by deposition of as haptens when bound to self-proteins. Hypersensitivity
antigen–antibody complexes in the blood vessels of the pneumonitis is a type IV hypersensitivity response that re-
skin, is a classic example of a type III reaction. Other sults mainly from occupational exposure to inhaled antigens.
examples include serum sickness and autoimmune dis- • Skin testing is used to detect the type IV hypersensitivity
eases such as SLE and RA. responses in contact dermatitis and tuberculin (PPD) test-
• Type IV hypersensitivity is a cell-mediated mechanism that ing. It is also used to test for functional cell-mediated
involves the activation of Th1 cells to release cytokines. immunity to common antigens in patients suspected of
As a result, macrophages and other immune cells are re- having immunodeficiency diseases. Positive test results
cruited to the area, where they induce an inflammatory appear in 48 to 72 hours and indicate sensitization to the
reaction. Cytotoxic T cells may also cause damage to the antigen(s) used in the test.
target cells involved. • All four types of hypersensitivity represent defense mech-
• Contact dermatitis is an example of a type IV hypersensitiv- anisms that stimulate an inflammatory response to cope
ity reaction. It results from exposure to chemicals released with and react to an antigen that is seen as foreign. In
by plants such as poison ivy and poison oak, metals such as many cases, the antigen is not harmful, but the response
nickel, or components of hair dyes and cosmetics that act to it results in tissue damage.

Study Guide: Comparison of Hypersensitivity Reactions

TYPE I TYPE II TYPE III TYPE IV
Immune IgE IgG or IgM IgG or IgM T cells
Mediators
Synonym Anaphylactic Antibody-mediated cytotoxic Complex-mediated Cell-mediated or
delayed type
Timing Immediate Immediate Immediate Delayed
Antigen Heterologous Cell surface: autologous or Soluble: autologous Autologous or
heterologous or heterologous heterologous
Complement No Yes Yes No
Involvement
Immune Release of Cell destruction caused by Antigen–antibody Antigen-sensitized
Mechanism mediators from antibody and complement, complexes activate Th1 cells release
IgE-sensitized opsonization, or ADCC complement pro- cytokines that re-
mast cells and Cell function inhibited by teins. Neutrophils are cruit macrophages
basophils antibody binding recruited and release and induce inflam-
Cell function stimulated by lysosomal enzymes mation or activate
antibody binding that cause tissue cytotoxic T cells to
damage. cause direct cell
damage.
Clinical Anaphylaxis, allergic Transfusion reactions, Serum sickness, Contact dermatitis,
Examples rhinitis, allergic autoimmune hemolytic Arthus reaction, tuberculin and
asthma, food anemia, hemolytic disease lupus erythematosus, anergy skin tests,
allergies, urticaria of the newborn, drug rheumatoid arthritis, hypersensitivity
reactions, myasthenia drug reactions pneumonitis
gravis, Goodpasture’s
syndrome, Graves disease
CASE STUDIES
1. A 13-year-old male had numerous absences from school he had pneumonia and was concerned that he might not
in the spring because of cold symptoms that included have completely recovered. He indicated that his symp-
head congestion and cough. He had received antibiotic toms only become noticeable if he goes out in the cold.
treatment twice, but he seemed to get one cold after an- A CBC count was performed, showing that his WBC
other. A complete blood count (CBC) showed no overall count was within normal limits; however, his RBC count
increase in WBCs, but a mild eosinophilia was present. was just below normal. A DAT performed on RBCs was
Because he had no fever or other signs of infection, his weakly positive after incubating at room temperature for
physician suggested that allergy testing be run. 5 minutes. When the DAT was repeated with monospe-
cific reagents, the tube with anti-C3d was the only one
Questions
positive.
a. What would account for the eosinophilia noted?
b. What tests should be run for this patient? Questions
c. If the patient was treated with allergy immunother- a. What does a positive DAT indicate?
apy, what test could be used to monitor his response b. What is the most likely class of the antibody causing
over time? the reaction?
2. A 55-year-old male went to his physician complaining c. Why was the DAT positive only with anti-C3d when
of feeling tired and run down. Two months previously, monospecific reagents were used?

REVIEW QUESTIONS
1. Which of the following is a general characteristic of 5. Which of the following is associated with anaphylaxis?
hypersensitivity reactions? a. Buildup of IgE on mast cells
a. The immune responsiveness is depressed. b. Activation of complement
b. Antibodies are involved in all reactions. c. Increase in cytotoxic T cells
c. An exaggerated immune response to an antigen d. Large amount of circulating IgG
occurs.
d. The antigen triggering the reaction is a harmful one. 6. To determine if a patient is allergic to rye grass, the
best test to perform is the
2. Which of the following is associated with an increase a. total IgE test.
in IgE production? b. skin prick test.
a. Transfusion reaction c. DAT.
b. Activation of Th2 cells d. complement fixation.
c. Reaction to poison ivy
d. HDN 7. Which condition would result in HDN?
a. Buildup of IgE on mother’s cells
3. Which of the following would cause a positive DAT test? b. Sensitization of cytotoxic T cells
a. Presence of IgG on RBCs c. Exposure to antigen found on both mother and
b. Presence of C3b or C3d on RBCs baby RBCs
c. A transfusion reaction caused by preformed antibody d. Prior exposure to foreign RBC antigen
d. Any of the above
8. What is the immune mechanism involved in type III
4. All of the following are associated with type I hyper- hypersensitivity reactions?
sensitivity except a. Cellular antigens are involved.
a. release of preformed mediators from mast cells. b. Deposition of immune complexes occurs in anti-
b. activation of complement. body excess.
c. cell-bound antibody bridged by antigen. c. Only heterologous antigens are involved.
d. an inherited tendency to respond to allergens. d. Tissue damage results from exocytosis.
 9. What is the immune phenomenon associated with the 13. A young woman developed red, itchy papules on her
Arthus reaction? wrist 2 days after wearing a new bracelet. This
a. Tissue destruction by cytotoxic T cells reaction was caused by
b. Removal of antibody-coated RBCs a. IgE-sensitized mast cells in the skin.
c. Deposition of immune complexes in blood vessels b. antigen-antibody complexes in the skin.
d. Release of histamine from mast cells c. damage to the skin cells by antibodies and
complement.
10. Which of the following conclusions can be drawn d. an inflammatory response induced by cytokines
about a patient whose total IgE level was determined released from Th1 cells.
to be 150 IU/mL?
a. The patient definitely has allergic tendencies. 14. Reactions to latex are caused by
b. The patient may be subject to anaphylactic shock. a. type I hypersensitivity.
c. Antigen-specific testing should be done. b. type IV hypersensitivity.
d. The patient will never have an allergic reaction. c. skin irritation.
d. all of the above.
11. Which of the following explains the difference between
type II and type III hypersensitivity reactions? 15. To determine a cold agglutinin titer
a. Type II involves cellular antigens. a. patient serum should be separated from whole
b. Type III involves IgE. blood at 4°C and tested at 4°C.
c. IgG is involved only in type III reactions. b. patient serum should be separated from whole
d. Type II reactions involve no antibody. blood at 4°C and tested at 37°C.
c. patient serum should be separated from whole
12. Two days after administration of the PPD test, a female blood at 37°C and tested at 4°C.
health-care worker developed an area of redness and d. patient serum should be separated from whole
induration 12 mm in size at the injection site. This blood at 37°C and tested at 37°C.
result means that she has
a. an active case of tuberculosis.
b. been exposed to M tuberculosis.
c. developed protective immunity against tuberculosis.
d. a result in the normal range for her risk group.
 Autoimmunity

After finishing this chapter, you should be able to: ETIOLOGY OF AUTOIMMUNE DISEASE
1. Explain the mechanisms of central and peripheral tolerance that are Self-Tolerance
essential in preventing the development of autoimmunity. Genetics
2. Discuss genetic and environmental factors that are thought to Other Endogenous and
contribute to the development of autoimmunity. Environmental Factors
3. Explain the relationship between microbial infections and the SYSTEMIC AUTOIMMUNE DISEASES
development of autoimmune disease. Systemic Lupus Erythematosus (SLE)
4. Distinguish between organ-specific and systemic autoimmune Antinuclear Antibodies (ANAs)
diseases and give examples of each and their associated target
Antiphospholipid Antibodies
tissues.
Rheumatoid Arthritis (RA)
5. Discuss the immunopathology and clinical manifestations of each
of the following diseases: systemic lupus erythematosus (SLE), Granulomatosis With Polyangiitis
rheumatoid arthritis (RA), granulomatosis with polyangiitis (Wegener’s Granulomatosis)
(Wegener’s granulomatosis), Graves disease, Hashimoto’s thyroiditis, Antineutrophil Cytoplasmic
type 1 diabetes mellitus, celiac disease, autoimmune hepatitis, Antibodies (ANCAs)
primary biliary cirrhosis, multiple sclerosis (MS), myasthenia gravis ORGANSPECIFIC AUTOIMMUNE
(MG), and Goodpasture’s syndrome. DISEASES
6. Associate each of the diseases listed in Learning Outcome 5 with its Autoimmune Thyroid Diseases (AITDs)
corresponding autoantibodies and laboratory findings.
Type 1 Diabetes Mellitus (T1D)
7. Explain the principles of laboratory methods used to screen for and
Celiac Disease
confirm the presence of antinuclear antibodies (ANA).
Autoimmune Liver Diseases
8. Describe common immunofluorescence patterns seen in the
indirect immunofluorescence (IIF) test for ANAs and their clinical Multiple Sclerosis (MS)
significance. Myasthenia Gravis (MG)
9. Describe the c-ANCA and p-ANCA patterns seen in the IIF test for Goodpasture’s Syndrome
ANCAs and their clinical significance. SUMMARY
10. Discuss the clinical significance of rheumatoid factor (RF) and CASE STUDIES
anti-CCP.
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Anergy Celiac disease Molecular mimicry Thyroglobulin (Tg)
Anticentromere antibodies Central tolerance Multiple sclerosis (MS) Thyroid peroxidase (TPO)
Anticyclic citrullinated CREST syndrome Myasthenia gravis (MG) Thyroid-stimulating
peptide (anti-CCP or Double-stranded DNA Nucleolus hormone ( TSH)
ACPA) (dsDNA) antibodies Thyroid-stimulating
Nucleosome antibodies
Antihistone antibodies Epigenetics hormone receptor
Peripheral tolerance
Antineutrophil cytoplasmic antibodies ( TRAbs)
Epitope spreading Primary biliary cirrhosis (PBC)
antibody (ANCA) Thyrotoxicosis
Extractable nuclear antigens Rheumatoid arthritis (RA)
Antinuclear antibodies (ANA) (ENAs) Thyrotropin-releasing
Rheumatoid factor (RF) hormone ( TRH)
Antiphospholipid antibodies Fluorescent antinuclear
Self-tolerance Tissue transglutaminase
Anti-RNP antibody antibody (FANA) testing
Sm antigen (tTG)
Autoantibodies Goodpasture’s syndrome
Superantigens Type 1 diabetes mellitus
Autoimmune diseases Granulomatosis with
SS-A/Ro (T1D)
Autoimmune hepatitis (AIH) polyangiitis (PGA)
SS-B/La Wegener’s granulomatosis
Autoimmune liver disease Graves disease
(WG)
Hashimoto’s thyroiditis Systemic lupus
Autoimmune thyroid
erythematosus (SLE)
diseases (AITDs) Immunologic tolerance

In the early 1900s, Paul Ehrlich noted that the immune system specific antigen, in this case, a self-antigen. In order for self-
could attack the very host it was intended to protect, a phenom- tolerance to develop, lymphocytes must be “educated” so
enon he referred to as “horror autotoxicus,” or “fear of self- they can distinguish between self-antigens and foreign anti-
poisoning.” Conditions in which this phenomenon occurred gens. This education takes place at two levels: central and
later became known as autoimmune diseases. Autoimmune dis- peripheral.
eases are disorders in which immune responses are targeted to- Central tolerance occurs in the central or primary lym-
ward self-antigens and result in damage to organs and tissues in phoid organs, the thymus, and the bone marrow.1,4 As T cells
the body. These harmful effects can be caused by T-cell–mediated mature in the thymus, they encounter self-antigens that are
immune responses or autoantibodies that are directed against normally present on the surface of the thymic epithelial cells.
host antigens. More than 100 autoimmune diseases have been In a process called negative selection, T cells that express
discovered, and these can involve various organ systems. T-cell receptors (TCRs) with a strong affinity for these self-
Autoimmune diseases are a leading cause of chronic illness and antigens are deleted by apoptosis, a physiological form of cell
death, affecting about 5% of the world’s population, including death (see Chapters 4 and 17 and Figure 15–1). Negative se-
50 million people in the United States alone.1-3 This chapter will lection occurs with both the immature, double-positive
begin by discussing the factors that are thought to contribute to CD4+/CD8+ cells in the cortex and with the more mature,
the development of autoimmunity so that you can gain a better single-positive CD4+ or CD8+ cells in the medulla. During
understanding of the underlying pathology of autoimmune dis- this process, some of the self-reactive CD4+ T cells are not
ease. The discussion will then proceed to the clinical manifesta- deleted, but instead differentiate into T regulatory (Treg)
tions and immunopathology of specific autoimmune diseases, cells that can specifically inhibit immune responses to self-
as well as the laboratory tests that are used in their diagnosis. antigens. Similarly, as B cells mature in the bone marrow,
those with receptors having a strong affinity for self-antigens
Etiology of Autoimmune Disease are eliminated by apoptosis. Some self-reactive B cells are not
deleted: rather, they are stimulated to rearrange their im-
munoglobulin genes so that their B-cell receptors are no
Self-Tolerance longer antigen specific. This process is known as receptor ed-
Under normal circumstances, the immune system is able to iting. B cells that possess receptors that only weakly recognize
differentiate between “self” and “nonself” or “foreign,” so that self-antigens are induced to downregulate the expression of
self-antigens are not destroyed. This fundamental concept was their receptors and develop a specific state of unresponsive-
introduced in Chapter 2. Central to this phenomenon is self- ness to the antigens known as anergy.
tolerance, or the ability of the immune system to accept self- Thus, there are several ways in which the central tolerance
antigens and not initiate a response against them. Autoimmune of T and B cells can be achieved. This process is not totally ef-
disease is thought to result from a loss of self-tolerance. fective, however, and some self-reactive lymphocytes manage
Self-tolerance is a type of immunologic tolerance, or a to escape to the secondary lymphoid organs such as the lymph
state of immune unresponsiveness that is directed against a nodes and spleen. Therefore, a second level of protection is
 Th Central
(bone marrow)

B
Tc
Central
(thymus)

Receptor editing
B

Negative selection

Anergy
Differentiation to Treg B
Apoptosis

Peripheral Peripheral
(lymph nodes, spleen) Treg (lymph nodes, spleen)

Tc Th
Anergy
B

Inhibition by Treg or
lack of costimulation
Apoptosis
Apoptosis

Th

Escape of Tc B
self-reactive cells

Autoimmune disease
Mechanisms of central and peripheral tolerance.

needed. In peripheral tolerance, lymphocytes that recognize be caused by complex interactions between genetics, exposure
self-antigens in the secondary lymphoid organs are rendered to environmental factors, and defects in immune regulation.
incapable of reacting with those antigens.1,4 Peripheral toler- Some of the major factors that are believed to contribute to
ance of T cells can result from anergy caused by the absence autoimmunity will be discussed in the text that follows.
of a costimulatory signal from an antigen-presenting cell
(APC) or binding of inhibitory receptors such as CTLA-4
(a molecule that prevents T-cell activation). Peripheral T-cell
Genetics
tolerance can also result from inhibition by Tregs or death by There is much evidence supporting a genetic basis for autoim-
apoptosis. Self-reactive B cells in the periphery can be deleted mune disease.5,6 Autoimmune diseases are often more preva-
by apoptosis, be rendered anergic after repeated stimulation lent among family members than among unrelated individuals
with self-antigens, or receive inhibitory signals through recep- and are more prevalent among monozygotic (genetically iden-
tors such as CD22. tical) twins than dizygotic (non-identical) twins or siblings. Re-
In some individuals, self-tolerance can fail even after this searchers conducting molecular studies continue to identify
second layer of protection; if this happens, autoimmunity can specific genetic polymorphisms or mutations that are associ-
arise. The development of autoimmune disease is thought to ated with autoimmune diseases.
 Most of the research concerning humans has focused on autoimmunity at an earlier age and have a higher risk for ac-
genes in the major histocompatibility complex (MHC) (see quiring more than one autoimmune disease as compared with
Chapter 3). Investigators have found that there is an association men. Furthermore, females have been found to have higher ab-
between the presence of certain human leukocyte antigen solute CD4+ T-cell counts and higher levels of circulating anti-
(HLA) types and the risk of developing a particular autoim- bodies than men.8 These observations suggest that there is a
mune disorder. The strongest link found is between the HLA- hormonal influence on the development of autoimmunity. Stud-
B27 allele and the development of ankylosing spondylitis, an ies on the effects of hormones have shown that estrogens tend
autoinflammatory disease that affects the spine. Individuals to direct the immune system in favor of a type 2 helper cell
who possess HLA-B27 have about a 100 times greater chance (Th2) response, resulting in more B-cell activation and antibody
of developing the disease than individuals who do not have production, whereas androgens favor a type 1 helper cell
that allele.4,6 Other associations with specific MHC genes are (Th1) response with activation of CD8+ T cells. Prolactin, a
discussed in the sections on particular autoimmune diseases hormone that stimulates production of breast milk in pregnant
in this chapter. Differences in MHC genes are thought to in- and nursing women, can stimulate both humoral and cell-
fluence the development of autoimmune disease because the mediated immune responses.8 The stimulatory effects of female
specific structure of the MHC molecule can determine whether hormones may place women at a greater risk for developing
or not a self-antigen can attach to the peptide-binding cleft of autoimmune disease.
the molecule and subsequently be processed and presented to
T cells.4 In addition, class II MHC molecules can sometimes
be abnormally expressed on cells where they are not typically When immunologic tolerance to self-antigens occurs during
found, resulting in the presentation of self-antigens for which the early development of lymphocytes in the thymus and bone
no tolerance has been established. marrow, some self-antigens may be cryptic, or hidden within
Genome-wide association studies are revealing that poly- the tissues of the host. T and B lymphocytes are shielded from
morphisms in some non-MHC genes can also be associated these sequestered antigens and are not educated to become tol-
with development of autoimmune disease. Many of these genes erant to them. At a later time in life, inflammation or tissue
influence the development and regulation of immune re- trauma could cause the cryptic antigens to be released and to
sponses. Examples include the PTPN22 gene, which has a role suddenly be accessible to the uneducated lymphocytes, trig-
in T- and B-cell receptor signaling; the IL2RA gene, which is gering an immune response.1,4,9 Tissue damage could be
involved in T-cell activation and maintenance of Tregs; the caused by factors such as infections, contact with environmen-
CTLA4 gene, which has an inhibitory effect on T-cell activation; tal toxins, or physical injury from exposure to ultraviolet (UV)
the BLK gene, which is involved in B-cell activation and devel- radiation. This concept has also been referred to as immunologic
opment; and the AIRE (autoimmune regulator) gene, which ignorance and may be responsible for the production of autoan-
promotes the development of T-cell tolerance in the thymus.4 tibodies to the lens of the eye following an ocular injury, au-
Although most autoimmune diseases involve multiple genes toantibodies to sperm after a vasectomy, and autoantibodies to
(~20 to 30), single-gene mutations that can be inherited in a DNA following damage to skin cells by overexposure to UV
Mendelian fashion have been associated with rare autoimmune rays from the sun.
disorders.
Inheritance of specific genes may make an individual more
Scientists have been very interested in the association of mi-
susceptible to a particular autoimmune disease, but genetic
crobial infections with the development of autoimmune dis-
makeup is not totally responsible because the majority of peo-
ease. Bacteria, viruses, and other infectious pathogens may be
ple with a particular gene will not develop autoimmunity. In
able to trigger autoimmune responses in a variety of ways. A
addition, the concordance rate among monozygotic twins
principal means by which microbes are thought to accomplish
(i.e., the presence of autoimmune disease in both members of
this is through molecular mimicry. Molecular mimicry refers
a pair of identical twins) is only between 20% and 30% for most
to the fact that many bacterial or viral agents contain antigens
autoimmune diseases.7 Furthermore, the prevalence and sever-
that closely resemble the structure or amino acid sequence of
ity of many autoimmune diseases vary in different geographic
self-antigens. Exposure to such foreign antigens may trigger
locations.5 This variance suggests that environmental and other
immune responses that cross-react with similar self-antigens.
factors also play a role in the development of autoimmune dis-
Molecular mimicry has been postulated as a mechanism for a
ease. A discussion of some of the major factors that are believed
number of human autoimmune diseases.9-11 The best known
to trigger autoimmune responses follows.
example involves the association between the gram-positive
bacterium Streptococcus pyogenes and rheumatic fever, an au-
Other Endogenous and Environmental toimmune disorder that primarily affects the joints and the
Factors heart. Some patients who have acquired scarlet fever or
pharyngitis as a result of infection with S pyogenes will proceed
to develop rheumatic fever if they are not treated adequately
Women are 2.7 times more likely to acquire an autoimmune dis- with antibiotics (see Chapter 20). Symptoms develop 2 to
ease than men; in fact, about 78% of patients with autoimmune 4 weeks after the infection and are thought to be caused by
diseases are of female gender.8 Women also tend to develop the production of antibodies to the M protein and N-acetyl
glucosamine components of the bacteria, which cross-react underexpression of certain genes in the immune system may
with cardiac myosin, causing damage to the heart.10 result in homeostatic imbalances and a breakdown of self-
A second way that microbes might trigger autoimmunity is tolerance, leading to autoimmunity.15,16
through a bystander effect.4,9,11 In this mechanism, the microbial Sometimes, exposure to environmental factors can lead
organism does not have to share structurally similar antigens to changes at the protein level. The changes are known as
with the host. Instead, the microorganism can induce a local post-translational modifications and may involve biochemical
inflammatory response that recruits leukocytes and stimulates processes such as acetylation, lipidation, citrullination, and
APCs to release cytokines that nonspecifically activate T cells. glycosylation.6 These modifications can alter the immuno-
Some of the T cells that are activated may have specificity for genicity of an antigen, affecting its ability to be processed
self-antigens. This expansion of the immune response to unre- by APCs and presented to T cells. Such alterations of self-
lated antigens has also been termed “epitope spreading.” antigens can make them more immunogenic, leading to au-
A third way that microorganisms might induce autoim- toimmune responses. For example, citrullination of collagen
munity is through superantigens. Superantigens are pro- might play a role in the pathogenesis of rheumatoid arthri-
teins that are produced by various microbes that have the tis (RA) and glycosylation of myelin may be involved in the
ability to bind to both class II MHC molecules and TCRs, re- pathology of multiple sclerosis (see the Rheumatoid Arthritis
gardless of their antigen specificity.12 Examples are the and Multiple Sclerosis sections in the text that follows).
staphylococcal enterotoxins that cause food poisoning and
toxic shock syndrome. These superantigens can act as potent
T-cell mitogens by activating a large number of T cells with Although the precise etiology of autoimmunity is unknown,
different antigen specificities. If some of these T cells possess there is much evidence that suggests that this heterogeneous
specificity for a self-antigen, autoimmune responses might disease entity is caused by complex interactions between
result.9 Likewise, some viruses, including the Epstein-Barr genetic and environmental factors (Fig. 15–2).1,4,6,15 Certain
virus (EBV) and cytomegalovirus (CMV), can cause poly- genes are thought to make individuals more susceptible to
clonal activation of B cells.1 immune responses against self-antigens, but are not suffi-
Scientists have also been very interested in the complex re- cient by themselves to cause autoimmune disease. Gender
lationship between microbiota, or normal flora, and the im- of the individual, tissue injury, and exposure to infectious
mune system. Research has shown that the presence of certain microorganisms or other environmental agents are all be-
strains of endogenous bacteria may be associated with a greater lieved to have significant effects on the immune system that
risk for autoimmune disease.11 These strains, as well as path- can trigger autoimmune responses in susceptible individuals.
ogenic microorganisms, may stimulate innate immune re- As a result of this break in immunologic tolerance, autore-
sponses through interaction with pattern recognition receptors active T cells recognize and proliferate in response to self-
such as the Toll-like receptors (TLRs). This interaction triggers antigens and B cells develop into plasma cells that secrete
cell-signaling pathways that result in the production of cy- autoantibodies. This can result in the release of proinflam-
tokines such as IFN α, which can stimulate cells of the adaptive matory cytokines, which, when coupled with dysfunctions
immune system, some of which are directed toward self- in immune-regulatory cells, perpetuate the autoimmune re-
antigens. A decrease in the number and function of Tregs can sponses. If these responses are not held in check, they can
perpetuate the activity of autoreactive cytotoxic T cells and culminate in autoimmune disease.1,7,14 Tissue injury in these
hyperactive B cells that produce autoantibodies. The activated disorders results from hypersensitivity reactions that involve
lymphocytes, in turn, produce proinflammatory cytokines that autoantibodies to cell-surface receptors, deposition of im-
provide signals to stimulate cells of the innate system, thus pro- mune complexes that contain self-antigens, and cell-mediated
ducing a vicious cycle that amplifies the immune response and cytotoxicity.1 The immunopathological mechanisms vary
sustains autoimmunity.7 with specific autoimmune diseases and will be discussed in
the text that follows.

Investigators have done much research in the area of epige-

netics and how it may relate to the development of autoim- Systemic Autoimmune Diseases
munity. Epigenetics refers to modifications in gene expression
that are not caused by changes in the original DNA se- Autoimmune diseases can be classified as systemic or organ-
quence.13,14 These alterations are stable and can be inherited. specific, depending on the extent of the pathology. There
They are thought to be triggered by exposure to environmen- is often a good bit of overlap between the two categories
tal toxins, ingestion of harmful foods or drugs, or the aging because some diseases may start out affecting a single
process. These factors can induce epigenetic changes by in- organ, but progress later to affect other locations in the body.
creasing or decreasing methylation of cytosine bases, modify- Table 15–1 lists some of the systemic autoimmune diseases
ing histones, and causing abnormal regulation by microRNAs. along with their corresponding target tissues and associated
These modifications can result in changes in the level at which autoantibodies, whereas Table 15–3 later provides this in-
genes are expressed by affecting their ability to be transcribed formation for some of the organ-specific diseases. The sec-
into mRNA, which is subsequently translated into proteins tions that follow will discuss three systemic autoimmune
that will influence the phenotype of an individual. Over- or diseases and the laboratory tests that are essential to their
 Environmental and Genetically Loss of Immunologic Autoimmune disease
Endogenous Triggers susceptible Tolerance
individual • Activation of self-reactive T
• Female hormones (possesses cells and B cells and
• Tissue injury and release of certain HLA or development of immune
self-antigens other genes) responses to self-antigens
• Microbial infections
(molecular mimicry, epitope
spreading, superantigens,
stimulation of immune
responses)
• Epigenetic factors (toxins,
foods, drugs, aging) and
post-translational modifications

Interactions between genetic and environmental factors in the development of autoimmunity.

Table 15–1 Systemic Autoimmune Diseases

DISEASE TARGET CELLS AND TISSUES ASSOCIATED AUTOANTIBODIES
Systemic lupus Multiple cells and organs through- Antibodies to double-stranded DNA and other nuclear
erythematosus out the body, including the skin, components, such as Sm (ANAs)
(SLE) joints, kidneys, brain, heart, lungs Phospholipid antibodies
Antibody to RBCs
Antibody to platelets
Antibody to lymphocytes
Antibody to ribosomal components
Antibody to endothelium
Rheumatoid factor
Rheumatoid Joints, bone; other tissues in Anti-CCP (cyclic citrullinated proteins)
arthritis (RA) some cases Rheumatoid factor
Antinuclear antibodies (ANAs)
Granulomatosis Upper respiratory system, lungs, Antineutrophil cytoplasmic antibodies (ANCA); c-ANCA pattern
with polyangiitis blood vessels Rheumatoid factor
(Wegener’s ANAs
granulomatosis)

diagnosis: systemic lupus erythematosus, RA, and granulo- within the immune system.21 Environmental factors thought
matosis with polyangiitis (Wegener’s granulomatosis). to play a role in SLE include UV light, certain medications, and
possibly infectious agents.17,21,22 Exposure to sunlight is a well-
Systemic Lupus Erythematosus (SLE) known trigger of the photosensitive skin rashes seen in many
lupus patients. Certain drugs, such as procainamide (used to
Systemic lupus erythematosus (SLE) is a chronic systemic
treat abnormal heart rhythms), hydralazine (used for high
inflammatory disease that affects between 40 and more than
blood pressure), and the tuberculosis drug isoniazid, can in-
200 persons per 100,000, depending on the population.17 The
duce a transient lupus-like syndrome that resolves once the
peak age of onset is usually between 20 and 40 years. Women
drug is stopped.23 Hormones are also important as indicated
are much more likely to be affected than men, by a ratio of
by the significantly higher incidence of lupus in females and
about 9 to 1.18 SLE is also more common in African Americans
an increased risk of developing lupus in women that have used
and Hispanics than in Caucasians.17,19 With earlier diagnosis
estrogen-containing contraceptives or hormone replacement
and improved treatments, the 5-year survival rate has increased
therapy.19 Hormones may be important because they may help
from 50% in the 1950s to greater than 90% today.17,18,20
regulate the transcription of genes that are central to the ex-
pression of SLE.22
SLE appears to originate from complex interactions between The majority of individuals exposed to the environmental
environmental factors, genetic susceptibility, and abnormalities factors mentioned previously do not develop lupus, and genetic
makeup is believed to play an important role in susceptibility Connections
to SLE. More than 20 genetic loci associated with lupus in
humans have been reported.18,21 People with certain HLA Type III Hypersensitivity
types, especially HLA-A1, B8, and DR3—have an increased The immune complexes generated in SLE activate complement,
chance of developing lupus.17 Another group of genes that inducing the generation of chemotaxins such as C5a. The
have been associated with increased susceptibility to SLE plays activation of complement results in recruitment of neutrophils,
a role in the clearance of immune complexes (see the text that which release lysosomal enzymes that cause injury to the sur-
follows).21,24 Other lupus-associated genes include polymor- rounding tissues. This is an example of a type III hypersensitivity
phisms in genes associated with immune function, genes cod- response, which was discussed in Chapter 14.
ing for various cytokines, and genes involved in signaling of
innate immune responses.25 These defects are thought to result
in uncontrolled autoreactivity of T and B cells, which leads to antibodies to red blood cells (RBCs) can cause hemolytic ane-
the production of numerous autoantibodies. mia and antibodies to platelets can cause thrombocytopenia
by antibody-mediated cytotoxic (type II) hypersensitivity.17 An-
tibodies to endothelial cells can cause inflammation of the
Over 100 autoantibodies associated with SLE have been dis-
blood vessels and vascular damage in lupus, which may be re-
covered.25 These include antibodies to double-stranded DNA
sponsible for the vasculitis and neuropsychiatric symptoms
(dsDNA), histones, and other nuclear components, as well
seen in some SLE patients.26 Phospholipid antibodies are as-
as autoantibodies to lymphocytes, erythrocytes, platelets,
sociated with increased miscarriage, stillbirth, and preterm de-
phospholipids, ribosomal components, and endothelium.25,26
livery in pregnant women with lupus.30,31 Neonatal lupus,
The typical patient has an average of three circulating
which occurs in up to 8% of babies born to pregnant women
autoantibodies.19
with SLE, is associated with antibodies to the nuclear antigens,
B cells and the autoantibodies they produce are believed
SS-A/Ro and SS-B/La.30 Symptoms are transient and resolve
to play a central role in the pathogenic mechanisms that are
at 6 to 8 months of age when the maternal antibodies have
responsible for this complex disease. In fact, the presence
cleared from the infant’s circulation. In utero heart block is a
of autoantibodies can precede the onset of disease by 9 to
serious complication that occurs in 2% of fetuses whose moth-
10 years.26 Abnormal apoptosis of certain types of cells may
ers have anti–SS-A antibodies.30
occur, releasing excess amounts of cellular constituents such
as DNA and ribonucleic acid (RNA). Dysfunctional removal
of cellular debris by phagocytes may allow these cellular com- The clinical signs of SLE are extremely diverse; nonspecific
ponents to persist, increasing the chances for autoantibody symptoms such as fatigue, weight loss, malaise, fever, and
production.17,27 anorexia are often the first to appear.31 The disease is marked
Antibodies to dsDNA are present in 70% of patients with by alternating relapses or flares and periods of remission.18 Joint
lupus and are highly specific for the disease.17 Anti-dsDNA involvement seems to be the most frequently reported manifes-
and complement proteins have been found in immune com- tation because over 90% of patients with SLE are subject to pol-
plexes that are deposited in organs such as the kidneys and yarthralgias or arthritis.19,32 Typically, the arthritis is symmetric
skin and are thought to play a major role in the pathogenesis and involves the small joints of the hands, wrists, and knees.
of SLE. It is not clear whether preformed immune complexes After joint involvement, the next most common signs are
circulate in the bloodstream and settle in various organs in the skin manifestations. These can present in various forms and are
body, whether the autoantibodies cross-react with protein experienced by about 80% of patients with lupus.19 An erythe-
components of the tissues, or whether nuclear antigens are at- matous rash may appear on any area of the body exposed to
tracted to the tissues by charge–charge interactions and then UV light. Less common but perhaps more dramatic is the classic
stimulate the production of autoantibodies.17,28,29 Accumula- butterfly rash across the nose and cheeks that appears in some
tion of IgG to dsDNA seems to be the most pathogenic be- SLE patients (Fig. 15–3). This rash is responsible for the name
cause it forms complexes of an intermediate size that become lupus, derived from the Latin term meaning “wolf-like.” In dis-
deposited in the glomerular basement membrane (GBM). coid lupus, skin lesions have central atrophy and scarring.
Once immune complexes are formed, they cannot be Evidence of renal involvement is present in about half of all
cleared efficiently because of other possible deficiencies in patients with lupus; nephritis is a major cause of illness and
lupus patients. These include defects in complement receptors death.28 There are several types of lesions, but the most dan-
on phagocytic cells; defects in receptors for the FC portion of gerous is diffuse proliferative glomerulonephritis, characterized
immunoglobulins; or rarely, deficiencies of early complement by cell proliferation in the glomeruli that can lead to end-stage
components such as C1q, C2, or C4 (see Chapter 7).17,19,22 renal disease.19,28,33 Other conditions involving the kidneys
The immune complexes activate complement and initiate an may include deposition of immune complexes in the suben-
inflammatory response. Leukocytes are attracted to the sites of dothelial tissue and thickening of the basement membrane, all
inflammation and release cytokines that perpetuate the re- of which can lead to renal failure.
sponse, resulting in tissue damage.17,28 Other systemic effects may include cardiac involvement with
Autoantibodies to nuclear and nonnuclear antigens can also pericarditis, tachycardia, or ventricular enlargement; pleuritis
cause cellular destruction by other mechanisms. For example, with chest pain; and neuropsychiatric manifestations such as
 The type of treatment used depends on the severity of the dis-
ease. For mild symptoms, a high dose of aspirin or other anti-
inflammatory drug may bring relief. For skin manifestations,
antimalarials such as hydroxychloroquine or chloroquine and
topical steroids are often prescribed.19 The antimalarial drugs
are thought to inhibit signaling of TLR 7, 8, and 9. Systemic
corticosteroids are used for acute fulminant (severe and sudden)
lupus, lupus nephritis, or central nervous system (CNS) com-
plications because these suppress the immune response and
lower antibody titers.19,35 Other drugs have also been used;
however, any immunosuppressive drug may have serious side
effects and patients must be monitored closely. Monoclonal an-
tibodies and other biological agents that target components of
the immune system thought to be central to pathogenesis of
lupus are being evaluated for their clinical effectiveness.17,35
The most common cause of death in lupus patients is in-
Butterfly rash in SLE. Characteristic rash over the fection, followed by heart disease.31 Renal involvement is also
cheekbones and forehead is diagnostic of SLE. The disease often a source of significant morbidity and mortality in this patient
begins in young adulthood and may eventually involve many organ group. The key to successful treatment is to prevent organ
systems. (From Steinman L. Autoimmune disease. Sci Am. 1993;269:107, damage and achieve remission. Overall, the treatments of today
with permission.) have come a long way in achieving this goal, as the 5-year
survival rate has increased to more than 90%.17,18,20
seizures, mild cognitive dysfunction, psychoses, or depression.
Hematologic abnormalities such as anemia, leukopenia, throm-
bocytopenia, or lymphopenia can also be present.32,33 General laboratory tests that can be used in the initial evalua-
Drug-induced lupus differs from the more chronic form of tion of patients include a complete blood count (CBC), a
the disease, in that symptoms usually disappear once the drug platelet count, and urinalysis.19 Some of the first laboratory
is discontinued. The most common drugs implicated are pro- findings in lupus patients are leukopenia and possible anemia
cainamide, hydralazine, chlorpromazine, isoniazid, quinidine, and thrombocytopenia.31 In addition, the erythrocyte sedimen-
anticonvulsants such as methyldopa, and possibly oral contra- tation rate (ESR) may be elevated even though the C-reactive
ceptives.19,31 Typically, this is a milder form of the disease and protein (CRP) level tends to be low or normal.
is usually manifested as fever, arthritis, or rashes; rarely are the More specific laboratory tests include the quantification of
kidneys involved.19,23 complement proteins and the detection of specific autoanti-
In 1982, the American College of Rheumatology (ACR) es- bodies. C3 is the most commonly measured complement
tablished a set of clinical and immunologic criteria that could protein.19 Serum complement levels may be low during disease
be used to define SLE for the purposes of research and surveil- flares as a result of complement consumption by immune com-
lance; an update was published in 1997.18 In 2012, the Sys- plexes. Thus, complement levels can be helpful not only in the
temic Lupus International Collaborating Clinics validated and initial diagnosis, but also for monitoring patients over time.19,31
further revised these criteria.34 There are now 11 clinical cri- When SLE is suspected, the first test typically done is a
teria and 6 immunologic criteria. The clinical criteria are acute screening test for antinuclear antibodies (ANAs) because
cutaneous lupus, chronic cutaneous lupus, oral ulcers, non- these are present in the majority of patients with the disease.
scarring alopecia (thinning or fragility of the hair), synovitis, These antibodies and the methods used to detect them are dis-
serositis, renal involvement, neurological symptoms, hemolytic cussed in more detail in the section that follows. Phospholipid
anemia, leukopenia, and thrombocytopenia34 The six immuno- antibodies are present in some patients with lupus and are
logic criteria are elevated antinuclear antibody titer, elevated discussed in a later section.
anti-dsDNA titer, presence of antibody to the Sm nuclear anti-
gen, presence of antiphospholipid antibody, low complement Antinuclear Antibodies (ANAs)
levels, and positive direct Coombs’ test in the absence of he-
molytic anemia.34 In most cases, a patient must satisfy at least
4 of the 17 criteria, including at least one clinical criterion and ANAs are autoantibodies that are directed against antigens in
one immunologic criterion, to be classified as having SLE. Al- the nuclei of mammalian cells. ANAs are present in over 95%
though these criteria are not meant to be used for diagnosis, of patients with active lupus and are used as a major marker for
they reflect the major clinical and laboratory features that are the disease.19,25,29,36 However, ANAs are not specific for SLE
associated with SLE. Some of the main laboratory tests that are because they can also be detected in a significant percentage of
helpful in diagnosis are discussed in Laboratory Diagnosis of patients with other connective tissue diseases, including mixed
Systemic Lupus Erythematosus section later in this chapter. connective tissue disease, Sjögren’s syndrome, scleroderma,
polymyositis-dermatomyositis, and RA.37 They can also be presence of these antibodies is considered diagnostic for SLE,
found in some individuals with other conditions, including especially when they are found in combination with low lev-
chronic infections, cancer, and pregnancy.36 Furthermore, up els of the complement component C3.39,40 Antibodies to
to 5% of healthy persons and up to 30% of elderly individuals dsDNA typically produce a peripheral or a homogeneous
are ANA-positive.36 staining pattern on indirect immunofluorescence (IIF).25,36,41
ANAs are a heterogeneous group of antibodies that have dif- See the Indirect Immunofluorescence (IIF) section for additional
ferent antigen specificities. The nuclear antigens they are directed information.
against include double-stranded (ds) and single-stranded (ss) Antihistone antibodies can also be found in lupus patients.
DNA (deoxyribonucleic acid), histones, nucleosomes (DNA- Histones are nucleoproteins that are essential components of
histone complexes), centromere proteins, and extractable nu- chromatin. There are five major classes of histones: H1, H2A,
clear antigens (ENAs).26 ENAs are a group of nuclear antigens H2B, H3, and H4. Antibodies to H2A and H2B can be detected
that were so named because they were isolated in saline extracts in almost all patients with drug-induced lupus. Presence of an-
of mammalian tissues.38 These antigens represent a family of tihistone antibody alone or combined with antibody to ssDNA
small nuclear proteins that are associated with uridine-rich RNA. supports the diagnosis of drug-induced lupus.32,36 About 70%
The ENAs include ribonucleoproteins (RNP), the Sm antigen, of other patients with SLE have elevated levels of antihistone
the SS-A/Ro and SS-B/La antigens, Scl-70, Jo-1, and PM-1.38 antibodies, but the titers are usually fairly low.19,25 High levels
Some of the more common ANAs and their associated features of antihistone antibodies tend to be associated with more active
are discussed in the text that follows and listed in Table 15–2. and severe SLE.32 Antihistone antibodies are also found in RA,
Double-stranded DNA (dsDNA) antibodies are the most Felty’s syndrome, Sjögren’s syndrome, systemic sclerosis, and
specific for SLE because they are mainly seen in patients with primary biliary cirrhosis, but the levels are usually lower.25
lupus and their levels correlate with disease activity.25,32,36 Antihistone antibodies typically produce a homogeneous pattern
Although they are found in only 40% to 70% of patients, the in the IIF assay.25,32,41

Table 15–2 Common Antinuclear Antibodies

CHARACTERISTICS IMMUNOFLUORESCENT
AUTOANTIBODY OF ANTIGEN PATTERN DISEASE ASSOCIATION
Anti-dsDNA dsDNA Peripheral or homogeneous SLE
Anti-ssDNA Related to purines and pyrimidines Not detected on routine SLE, many other diseases
screen
Antihistone Different classes of histones Homogeneous Drug-induced SLE, other
diseases
Anti-DNP DNA-histone complex Homogeneous SLE, drug-induced SLE
(nucleosomes)
Anti-Sm Extractable nuclear antigen Coarse speckled Diagnostic for SLE
(uridine-rich RNA component)
Anti-RNP Proteins complexed with small Coarse speckled SLE, mixed connective
nuclear RNA tissue diseases
Anti–SS-A/Ro Proteins complexed to RNA Finely speckled SLE, Sjögren’s syndrome,
others
Anti–SS-B/La Phosphoprotein complexed to Finely speckled SLE, Sjögren’s syndrome,
RNA polymerase others
Antinucleolar RNA polymerase, fibrillarin, PM-1 Prominent staining of SLE, systemic sclerosis
nucleoli (can be smooth,
clumpy, or speckled)
Anti–Scl-70 DNA topoisomerase I Atypical speckled Systemic sclerosis,
scleroderma
Anti–Jo-1 Histidyl-tRNA synthetase Fine cytoplasmic speckling Polymyositis
Autoantibody– Characteristics of Antigen-Antigens Immunofluorescent Disease Association-
Anti-Centromere in the chromosome centromeres pattern-Discrete speckled CREST syndrome
Adapted from Bradwell AR, Hughes RG, Karim AR. Immunofluorescent antinuclear antibody tests. In: Detrick B, Hamilton RG, Folds, JD, eds. Manual of Molecular
and Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM Press; 2006:996–997.
DNA = deoxyribonucleic acid; DNP = deoxyribonucleoprotein; RNA = ribonucleic acid; RNP = ribonucleoprotein; SLE = systemic lupus erythematosus.
 Nucleosome antibodies are stimulated by DNA-histone components: fibrillarin, RNA polymerase I, and PM-1.43 Anti-
complexes, known as nucleosomes, or deoxyribonucleoprotein body to fibrillarin is common in systemic sclerosis (also known
(DNP). These antibodies are directed only against the com- as scleroderma) and is indicated by clumpy nucleolar fluores-
plexes and not against DNA or the individual histones. Nucle- cence in the IIF assay.36,40,43 Scleroderma is an autoimmune
osome antibodies are found in about 85% of patients with SLE disease that primarily involves the skin and the blood vessels.
and their levels correlate with disease severity.25 They typically Antibodies to RNA polymerase are also associated with sclero-
produce a homogeneous pattern in the IIF assay.41 derma, but produce a speckled nucleolar pattern in IIF.
Antibody to the Sm antigen is specific for lupus because it Homogeneous staining of the nucleolus is associated with
is not found in other autoimmune diseases. However, it is antibodies to the PM-1 antigen (also known as PM/Scl) and is
found in only 20% to 40% of patients with SLE, depending on found in polymyositis and systemic sclerosis.36,43
the race of the population.25 It is unclear whether titers corre- Anticentromere antibodies bind to proteins in the middle
late with disease activity. Antibody to a preparation of this ENA region of a chromosome where the sister chromatids are joined.
was first described in a patient named Smith, hence the name These antibodies are directed against three centromere antigens
anti-Sm antibody. The anti-Sm antibody produces a coarse of molecular weights 16kDa, 80kDa, and 120kDa.32 They are
speckled pattern of nuclear fluorescence on IIF.41 found in 50% to 80% of patients with the CREST syndrome,
Anti-RNP antibody is directed against RNP, which consists a subset of scleroderma named after its five major features: cal-
of several nonhistone proteins complexed to a small nuclear cinosis, Raynaud’s phenomenon, esophageal dysmotility, scle-
RNA called U1-nRNP (U for “uridine-rich”). RNP forms com- rodactyly, and telangiectasia.32 In the IIF assay, centromere
plexes with the Sm antigen in the nucleus, and antisera to these antibodies produce discrete speckled staining in the nuclei of
antigens produce a pattern of partial identity when they are the cells.42,43
reacted in the Ouchterlony double immunodiffusion test (see
discussion and Figure 15–7 in the text that follows). In the IIF
A variety of methods have been developed to detect ANAs in
assay, anti-nRNP produces a coarse speckled pattern.41 Anti-
patient serum. These include IIF, immunoperoxidase staining,
bodies to RNP are detected in 20% to 30% of patients with
enzyme-linked immunosorbent assay (ELISA), microsphere
SLE, but are also found at a high titer in individuals with mixed
multiplex immunoassays (MIA), radioimmunoassay (RIA), im-
connective tissue disease and in lower levels in patients with
munodiffusion, immunoblotting (Western blot), dot blot, im-
other autoimmune rheumatic diseases such as systemic scle-
munoelectrophoresis, and microarray.32,38 Some of the more
rosis, Sjögren’s syndrome, and RA.25,32,36
commonly used assays are discussed in the text that follows.
Lupus patients can also produce antibodies to another fam-
ily of ENAs called SS-A/Ro and SS-B/La. These antigens consist
of small, uridine-rich RNAs complexed to cellular proteins and Fluorescent antinuclear antibody (FANA) testing has been
were given the prefix of SS- because a large percentage of pa- the most widely used and accepted test because it is highly sen-
tients with Sjögren’s syndrome (~70%) possess antibodies to sitive, detects a wide range of antibodies, and is inexpensive
the antigens.36 Anti–SS-A/Ro also appears in approximately and easy to perform.36,38 In addition, the antigens are in their
24% to 60% of patients with SLE and has been closely associ- original form and location within the cells used in the test.25
ated with the presence of nephritis, vasculitis, lymphadenopa- This test has several applications. It has been used as a screen-
thy, photosensitivity, and hematologic manifestations such as ing test to identify patients who have ANAs as well as patients
leukopenia.25,32 Antibodies to SS-B/La are found in only 9% to who are negative for ANAs to provide guidance in selection of
35% of patients with SLE and all of these have anti–SS-A/Ro.40 follow-up assays based on immunofluorescence patterns and
The SS-B/La antibody is most often found in patients who have to monitor ANA titers in patients during treatment.36
cutaneous manifestations of SLE, especially photosensitivity The IIF test uses a commercially prepared microscope slide
dermatitis.32 Antibodies to both SS-A/Ro and SS-B/La can cross onto which nucleated cells have been fixed. The human ep-
the placenta and have been associated with neonatal lupus.25 ithelial cell line, HEp-2, is the standard substrate for clinical
Newborns that have anti–SS-A/Ro are more likely to develop laboratories worldwide.25,36,38 HEp-2 cells are used because
cardiac manifestations, whereas those who have anti–SS-B/La they have large nuclei with high antigen expression, allowing
are more likely to have other symptoms such as skin lesions. for high sensitivity and facilitating visualization of results.36
To detect the presence of these antibodies on IIF, human tissue Patient serum is incubated with the HEp-2 cell-coated slide,
culture cells such as HEp-2 (human epithelial) must be used washed to remove unbound antibodies, and then allowed to
because SS-A/Ro and SS-B/La antigens are not found in mouse react with an anti-human immunoglobulin labeled with a flu-
or rat liver and kidney. A finely speckled pattern is evident.41-43 orescent tag to detect bound IgG or total immunoglobulins.
Antibodies to the SS-A/Ro antigen are best detected on IIF if Following a second incubation and wash, the slide is mounted
a special cell line, HEp-2000®, is used; these cells have been and viewed under a fluorescent microscope using 400X mag-
genetically transfected so that they hyperexpress the antigen. nification (40X objective and 10X eyepiece). The principle of
The nucleolus is a prominent structure within the nucleus this assay is shown in Figure 15–4.
where transcription and processing of ribosomal RNA and as- The screening test is commonly performed with a 1:40 or
sembly of the ribosomes takes place. Staining of the nucleolus 1:80 dilution of patient serum in order to avoid detecting low
in IIF is mainly caused by antibodies to one of three nucleolar positive titers that may be seen in healthy persons, although
 Homogeneous

Rim

B Speckled

Fluorescent ANA test principle.

(A) Microscope slide coated with HEp-2 cells (the Nucleolar
plasma and nuclear membranes have been perme-
abilized). (B) Patient serum (containing antinuclear
antibodies) is applied. (C) After washing, fluorescent-
labeled anti-human Ig is added to detect bound C
Centromere
autoantibodies. After a final wash, the slide is read
under a fluorescent microscope. Five typical
patterns in nondividing cells are shown on the right.

the exact dilution used for screening may vary with the labo- cells. Staining is absent in the nucleolus and in the chro-
ratory and population being tested.36 A titer of 160 is gener- matin region in dividing cells. The specks can be fine or
ally considered to be clinically significant.38 Patient samples coarse, depending on the autoantibody present. The
that are positive on the ANA screen are serially diluted and speckled pattern is associated with antibodies to ENAs
tested to determine the antibody titer, specified as the highest and can be found in patients with SLE, Sjögren’s syn-
dilution to show nuclear fluorescence. Inclusion of a 1+ end- drome, systemic sclerosis, and other systemic autoim-
point control serum can help to standardize the readings by mune rheumatic diseases.
setting the minimum level of fluorescence that is considered • Nucleolar—Prominent staining of the nucleoli within the
positive. nuclei of interphase cells is seen in this pattern. The size,
In addition to the antibody titer, the pattern of fluorescence shape, and number of the nucleoli per cell are variable and
is also reported because it can provide clues about the autoan- staining can be smooth, clumpy, or speckled, depending
tibody present and associated diseases. Fluorescence can be on the type of antibody present. Staining may or may not
within the nucleus, cytoplasm, or mitotic structures of the be present in the dividing cells. The nucleolar pattern is
cell.38 Although about 40 patterns of immunofluorescence are primarily caused by antibodies to RNA and RNP and is
possible, some of the most common nuclear patterns are ho- seen mainly in patients with scleroderma, but can also be
mogeneous, peripheral, speckled, nucleolar, and centromere present in patients with other connective tissue diseases.
(Fig. 15–5).36,42-44 • Centromere—Numerous discrete speckles are seen in the
• Homogeneous (also known as diffuse)—This pattern is nuclei of interphase cells and the chromatin of dividing
characterized by uniform staining of the entire nucleus cells. Most cells have 46 speckles, representing the num-
in interphase cells and of the condensed chromosomal ber of chromosomes. This pattern is caused by antibodies
region in metaphase cells. It is associated with antibodies to proteins in the centromeres of the chromosomes and
to dsDNA (also known as native or nDNA), histones, and is found mainly in patients with the CREST syndrome.
deoxyribonuclear protein (DNP). The homogenous pat- Mixed patterns can also be observed; in some cases, one pat-
tern is found in patients with SLE, drug-induced lupus, tern may totally or partially obscure another (for example, a
and many other autoimmune diseases. homogeneous pattern might cover up a speckled pattern). In
• Peripheral (rim or outline)—In this pattern, diffuse stain- these cases, titration of the patient serum can help to distin-
ing is seen throughout the nucleus, but there is a greater guish between the separate patterns and an antibody titer
staining intensity around the outer circle surrounding the would be reported for each one. If the FANA test is negative,
nucleus in interphase cells. Dividing cells show strong no clearly discernable fluorescent pattern is observed in the
staining of the condensed chromatin. This pattern is nuclei of the cells. Up to 5% of SLE patients test negative, so
primarily caused by antibodies to dsDNA and is highly this test cannot be used to absolutely rule out SLE.
specific for SLE. Although the IIF method is considered the gold standard
• Speckled—This pattern is characterized by discrete, flu- for ANA testing, it also has some important limitations. The
orescent specks throughout the nuclei of interphase test is time-consuming and requires a significant amount of
 volume testing laboratories, because they are automated, easy
to perform, and yield objective results.38,45 These assays can
test for a broad range of antibodies if multiple nuclear antigens
are coated onto a single test well, or for specific ANAs if each
well is coated with a single antigen. The antigens used in com-
mercial kits are derived from tissue extracts or are produced
by recombinant technology. Because of their advantages, many
laboratories are using ELISA methods to screen for the pres-
ence of ANAs in addition to identifying specific ANAs. How-
ever, there is a large variation in the performance of tests
produced by different manufacturers, which is influenced by
Homogeneous pattern
the antigen preparation used. For example, one study found
sensitivities of ELISA assays ranging from 69% to 98%, and
specificities from 81% to 98% when they were compared with
the IIF ANA method.38 These findings suggest that immunoas-
says may miss a significant proportion of ANA-positive patients
and also yield a significant number of false-positive results.
Based on such studies, the ACR has recommended that the IIF
test remain the gold standard for ANA testing and that clinical
laboratories should specify the method they used when they
are reporting results.46
MIAs are also
very popular because they are amenable to automated, high
throughput testing with objective results. In these assays, the
Speckled pattern
patient serum is incubated in a microtiter plate well containing
a suspension of polystyrene microspheres that are coated with
individual nuclear antigens or with a HEp-2 extract. Beads con-
taining specific antigens can be differentiated by their unique
shade of red created by a specific combination of infrared and
red fluorescent dyes. Antibodies in the patient serum will bind
only to the beads containing their specified antigens. Following
a washing step to remove unbound antibodies, a phycoerythrin-
labeled anti-human IgG is added. The conjugate will bind only
to the beads that have bound patient antibodies and excess con-
jugate is removed by washing. The bead suspension is analyzed
for fluorescence by a flow cytometer that has two lasers, one
Nucleolar pattern that identifies each bead and another that detects the amount
Photomicrographs showing three patterns of of fluorescent conjugate attached. MIAs are more efficient than
immunofluorescent staining for antinuclear antibodies. Examples ELISAs because they allow testing for multiple antibody speci-
of predominant staining patterns obtained are homogeneous— ficities to be performed in a single tube. Studies have shown
staining of the entire nucleus; speckled pattern—staining through- that MIAs, like ELISAs can be variable in terms of their sensi-
out the nucleus; and nucleolar pattern—staining of the nucleolus. tivity and specificity when compared with IIF and that test per-
(Courtesy of DiaSorin, Inc., with permission.) formance varies with the specific ANA detected.47,48 Hopefully,
the future will bring improvement in assays such as the MIA
and ELISA that will allow laboratories to report accurate results
technical expertise to correctly identify the fluorescent pat- while reaping the full benefits of automation.
terns. Automated IIF ANA assays have been developed that
This IIF assay
may help to reduce these problems and also allow for storage
is used to detect antibodies to dsDNA. In testing for these anti-
and retrieval of the fluorescent images.41 These assays need to
bodies, a purified antigen preparation that is free from single-
be validated further but hold promise for the future. However,
stranded DNA (ssDNA) must be used because antibodies to
the autoantibodies present cannot be precisely identified on
ssDNA occur in many individuals with other autoimmune or
the basis of the fluorescent patterns and additional tests are
inflammatory diseases. One particularly sensitive assay for
needed.38,41 Some of the more common tests used to charac-
dsDNA is an IIF test using a hemoflagellate organism called
terize ANAs are described in the section that follows.
Crithidia luciliae as the substrate.49 This trypanosome has a cir-
cular organelle called a kinetoplast that is composed mainly of
ELISAs and chemiluminescence immunoas- dsDNA (Fig. 15–6). In this test, patient serum is incubated on
says for ANAs have become very popular, especially for high a microscope slide coated with C luciliae organisms and binding
 Anti-Sm antibody
Nucleus Kinetoplast

Flagellum

A ENA B
An illustration of Crithidia luciliae fixed to a microscope
slide. A positive test for dsDNA will show green fluorescence in the
nucleus and kinetoplast.

A B

is detected with a fluorescent-labeled anti-Ig conjugate. Wash-

ing of the slide is performed after each step to remove unbound
antibody. A positive test is indicated by a brightly stained kine-
toplast. This test has a high degree of specificity, although it is
less sensitive than other FANA tests.36,45
Anti-RNP antibody
ANAs can also be detected by immunodif-
Extractable nuclear antibody immunodiffusion pat-
fusion. Typically, this method is used to determine the immuno- tern. A mixture of extractable nuclear antigens (ENA), including RNP,
logic specificity of a positive FANA test, particularly when a Sm, and other soluble nuclear antigens, is placed in a central well in
speckled pattern is observed. Ouchterlony double diffusion de- an agarose gel. Sm antibody and RNP antibody are run as positive
tects antibody to several of the small nuclear proteins, or ENA. controls and patient samples are placed between the controls. The
These include antibodies to Sm, RNP, SS-A/Ro, SS-B/La, Scl-70 pattern of precipitin lines formed indicates the antibodies present in
(DNA topoisomerase I), and Jo-1 (see the previous descriptions patient serum. The arc of serological identity formed between Sm
and Table 15–2). A solution containing ENA antigens is placed and patient A indicates that serum A contains anti-Sm antibodies.
in a central well of an agarose plate and patient samples and The arc of partial identity formed between serum A and RNP occurs
controls are placed in the surrounding wells, as indicated in because RNP is always found complexed to Sm antigen. RNP
Figure 15–7. A visible precipitate is formed between the ENA antibodies are not present. Serum B contains neither Sm nor RNP
antibody.
well and each surrounding well that contains antibodies to any
of the ENAs present (e.g., anti-Sm, anti-RNP, or antibodies to
other ENAs). If an outer well does not contain antibodies to any
of the ENAs, no precipitate is formed between that well and the The lupus anticoagulant, one of several types of antiphos-
center well. Samples in the outer wells are identified as contain- pholipid antibodies, was so named because it produces a pro-
ing antibody to a particular ENA by comparing their reactivity longed activated partial thromboplastin time (APTT) and
patterns of identity, nonidentity, or partial identity to control prothrombin time (PT). In patients with antiphospholipid an-
sera containing specific ENA antibodies. A positive reaction is tibodies, the APTT may be prolonged, but it is not corrected
indicated by immunoprecipitation lines of serological identity. by mixing with normal plasma.31 Ironically, patients with this
(Refer to Chapter 10 for a discussion of Ouchterlony test prin- antibody have an increased risk of clotting and spontaneous
ciples.) Although this type of testing is not as sensitive as some abortion. Platelet function may also be affected and thrombo-
other techniques, it is highly specific.40 cytopenia may be present. In addition to determining the APTT
and PT, there are several EIAs for antiphospholipid antibodies
Antiphospholipid Antibodies that are sensitive and relatively simple to perform.25,50-52 If
these antibodies are suspected, factor assays may also need
Antiphospholipid antibodies are a heterogeneous group of to be performed to rule out any factor deficiencies or factor-
antibodies that bind to phospholipids alone or phospholipids specific inhibitors.
complexed with protein. They can affect every organ in the
body, but they are especially associated with deep-vein and ar-
terial thrombosis and with recurrent pregnancy loss.25,50,51 An-
Rheumatoid Arthritis (RA)
tiphospholipid antibodies have been found in up to 60% of RA is another example of a systemic autoimmune disorder. It
patients with lupus, but they are also associated with several affects about 0.5% to 1.0% of the adult population, but preva-
other disease states.25,32,52 They can be identified by their abil- lence varies with ethnicity and geographic location.53,54 Typi-
ity to cause false-positive results in nontreponemal tests for cally, it strikes individuals between the ages of 25 and 55.54
syphilis, the lupus anticoagulant assay, and immunoassays for Women are three times as likely to be affected as men; in ad-
antibodies to cardiolipin or other phospholipids.32 dition, the prevalence of the disease is highest in women who
are more than 65 years of age.53,55 RA can be characterized as osteoclasts and inhibits bone formation. Significant local bone
a chronic, symmetric, and erosive arthritis of the peripheral destruction occurs and there is also generalized osteoporosis
joints that can also affect multiple organs such as the heart and throughout the body.54,58
the lungs.32,54 Progress of the disease varies because there may It is not known what role autoantibodies play in the ini-
be spontaneous remissions or an increasingly active disease in tiation of the inflammatory response. Two key antibodies
some individuals that rapidly progresses to joint deformity and found in the disease are RF and anti-CCP. RF is an antibody
disability.56 In addition to a decline in functional ability, there that is most often of the IgM class and is directed against
is a reduced life expectancy.54,57 In recent years, RA patients the FC portion of IgG. It has been postulated that RFs may
have experienced less disability and a better quality of life be- play a role in the pathogenesis of RA by increasing
cause of advances in therapy and earlier treatment (see the macrophage activity and enhancing antigen presentation to
Treatment section in the text that follows). T cells by APCs.59
Antibodies to cyclic citrullinated proteins (anticyclic cit-
rullinated peptide antibody [anti-CCP or ACPA]) are a sec-
Associations of RA with more than 30 genetic regions have ond major type of antibody associated with RA. Citrullinated
been discovered. The strongest associations have been between proteins contain an atypical amino acid called citrulline, which
a subset of patients with RA and specific HLA-DRB1 alleles or is generated when the enzyme peptidyl arginine deiminase
PTPN22 gene polymorphisms.53,54,58 These patients are posi- (PAD) modifies the amino acid arginine by replacing an NH2
tive for rheumatoid factor (RF) or antibodies to CCP (see the group with a neutral oxygen.60,61 This enzyme is associated
section on Laboratory Diagnosis). The strongest environmental with granulocytes, monocytes, and macrophages, as well as
risk factor for RA is believed to be cigarette smoking, which other types of cells. Death of granulocytes and macrophages
doubles the risk of developing the disease.53,55 Other factors triggers production of citrullinated proteins; overexpression of
have also been implicated, but their associations are these antigens may provoke an immune response in individu-
weaker.53,54 Numerous infectious agents have been proposed als with certain HLA-DRB1 alleles.61,62 These antibodies can
as possible triggering antigens for RA, but a cause-effect rela- react with citrulline-containing components of the matrix, in-
tionship has not been proven.54,55 cluding filaggrin, keratin, fibrinogen, and vimentin, and are
thought to correlate with the pathogenesis of RA.54 Antinuclear
antibodies are also present in some RA patients (see the previ-
The pathology of RA is caused by an inflammatory process
ous Antinuclear Antibodies section).
that results in the destruction of bone and cartilage. The le-
In RA, autoantibodies such as RF and anti-CCP are
sions in rheumatoid joints show an increase in cells lining the
thought to combine with their specified antigen, and the re-
synovial membrane and formation of a pannus, a sheet of in-
sulting immune complexes become deposited in the joints,
flammatory granulation tissue that grows into the joint space
resulting in a type III (or immune complex) hypersensitivity
and invades the cartilage. Infiltration of the inflamed syn-
reaction. The complement protein C1 binds to the immune
ovium with T and B lymphocytes, plasma cells, dendritic cells,
complexes, activating the classical complement cascade.
mast cells, and granulocytes is evidence of immunologic ac-
During this process, C3a and C5a are generated, which act
tivity within the joint.54
as chemotactic factors for neutrophils and macrophages. The
The balance between proinflammatory and anti-inflammatory
continual presence of these cells and their associated cy-
cytokines in RA appears to be tipped toward continual inflam-
tokines leads to chronic inflammation, which damages the
mation. Proinflammatory cytokines found in synovial fluid
synovium itself.54
that contribute to inflammation include IL-1, IL-6, IL-17, and
TNF-α (see Chapter 6).56,58 TNF-α plays a key role in the
inflammatory process by stimulating the production of other The initial symptoms of RA involve the joints, tendons, and
cytokines and facilitating the transport of white blood cells bursae.54 The RA patient commonly experiences nonspecific
(WBCs) to the affected areas.54,56 The proinflammatory cy- symptoms such as malaise, fatigue, fever, weight loss, and tran-
tokines trigger the release of matrix metalloproteinases from sient joint pain that begin in the small joints of the hands and
fibroblasts and macrophages; these enzymes degrade important feet. The joints are typically affected in a symmetric fashion.
structural proteins in the cartilage.58 Joint stiffness and pain are usually present in the morning and
Local bone erosion is another feature that is characteristic improve during the day. The disease can progress to the larger
of the pathology in RA. Multinucleated giant cells called osteo- joints, often affecting the knees, hips, elbows, shoulders, and
clasts are central to the structural damage that is seen in the cervical spine. Joint pain can lead to muscle spasms and limi-
bones. Osteoclasts absorb bone as part of the normal bone re- tation of motion. The ongoing inflammation, if left untreated,
modeling process that occurs in the body in response to growth results in permanent joint dysfunction and deformity. Osteo-
and repair of damaged bone. Normally, there is a good balance porosis (bone erosion) occurs in about 20% to 30% of RA pa-
between bone production and destruction. However, in RA, tients because of the inflammatory environment of the joints
the osteoclasts become overly activated in the inflammatory and activation of osteoclasts.54
environment of the joints. TNF-α, in conjunction with other Some patients with RA develop extra-articular manifesta-
cytokines and a molecule called RANKL (receptor activator of tions, which occur outside of the joints.54 These patients are
nuclear factor kappa-B ligand), induces the differentiation of most likely to have had a history of smoking, early disease
onset, and test positive for anti-CCP or RF. Extra-articular effective in treating RA, patients must be monitored closely
manifestations include the formation of subcutaneous nod- because they are at greater risk for infection.
ules, pericarditis, lymphadenopathy, splenomegaly, intersti-
tial lung disease, or vasculitis. Some patients have small
Diagnosis of RA is based on a combination of clinical manifes-
masses of tissue called nodules over the bones. Nodules can
tations, radiographic findings, and laboratory testing. RF is the
also be found in the myocardium, pericardium, heart valves,
antibody that is most often tested to aid in making the initial
pleura, lungs, spleen, and larynx. About 10% of patients
diagnosis. The importance of testing for the presence of RF is
with RA develop secondary Sjögren’s syndrome, an autoim-
also reflected in the fact that it is one of the classification crite-
mune disorder characterized by the presence of dry eyes and
ria for RA.64,65 Recall that RF is an autoantibody, usually of the
dry mouth in addition to connective tissue disease. A small
IgM class, that reacts with the Fc portion of IgG. Approxi-
percent develop Felty’s syndrome, a combination of chronic,
mately 70% to 90% of patients with RA test positive for RF.54,59
nodular RA coupled with neutropenia and splenomegaly.
Thus, a negative result does not rule out the presence of RA.
The most common cause of death in RA is cardiovascular
Conversely, a positive test result is not specific for RA because
disease, presumably because of the acceleration of arte-
RF is also present in about 5% of healthy individuals and in
riosclerosis by proinflammatory cytokines released during
10% to 25% of those over the age of 65.59 In addition, RF can
the disease process.55
be found in patients with other connective tissue diseases such
Diagnostic classification criteria for RA were established
as SLE, Sjögren’s syndrome, scleroderma, and mixed connec-
by the ACR in 1987 to standardize identification of RA pa-
tive tissue disease, as well as in people with some chronic
tients for clinical trials.63 These criteria were revised in 2010
infections.54,59
in collaboration with the European League Against Rheuma-
Manual agglutination tests using charcoal or latex particles
tism in order to improve the identification of patients in the
coated with IgG have been used for many years to detect
early stages of disease who could benefit from new treat-
RF.59,61 These tests, however, only detect the IgM isotype,
ments.64,65 The criteria are based on the number of joints in-
found in about 75% of patients, and have been largely replaced
volved, duration of symptoms, serology results for RF and
by ELISA, chemiluminescence immunoassay, and nephelomet-
anti-CCP, serum level of the acute-phase reactant, CRP, and
ric methods, which are automated, have greater precision and
the ESR. These important laboratory tests will be discussed
sensitivity, and can also detect other RF isotypes.54,59 Testing
in more detail.
for IgG and IgA RFs increases test specificity. The presence of
both IgM and IgA, or of all three isotypes, rarely occurs in other
In the past, therapy for RA was primarily based on nonsteroidal disease states, which may help in making a differential diag-
anti-inflammatory drugs (NSAIDs) such as salicylates (aspirin) nosis. In addition, testing for specific RF isotypes may have
and ibuprofen. Although these agents can be used to reduce local some prognostic value. Some studies have found that IgM RF
swelling and pain, they are no longer the dominant treatment levels decrease with clinical response to therapy and that ele-
for RA. The discovery that joint destruction occurs early in the vation of IgA early in the disease appears to be associated with
disease has prompted more aggressive treatment and the de- a poor response to TNF-α inhibitors and a worse prognosis.59
velopment of new drugs to prevent disease progression.54,55,66 Laboratory testing for antibody to cyclic citrullinated pep-
Disease-modifying anti-rheumatic drugs (DMARDs), most no- tides (anti-CCP) has been added to the classification criteria to
tably methotrexate, are now prescribed at the time of diagnosis. increase the specificity for RA.64,65 These assays are performed
Methotrexate is thought to act by inhibiting adenosine metabo- mainly by ELISA and use a circular synthetic form of citrulli-
lism and T-cell activation.58 If methotrexate alone does not work, nated peptides to increase test sensitivity, which ranges from
it can be combined with other DMARDs or with biological agents. 39% to 50%.61,62,68 About 20% to 30% of RF-negative patients
Biological agents have revolutionized the way that RA are positive for anti-CCP and the presence of anti-CCP pre-
is treated because they target components of the immune cedes the onset of RA by several years, making it a better
system that are central to the pathogenesis of the dis- marker for early disease.61,62,68 The presence of anti-CCP is
ease.53,54,66,67 Several key therapies for RA block the activity also associated with an increased likelihood to develop clini-
of the cytokine, TNF-α. Agents that act against TNF-α are cally significant disease activity with a worse prognosis.62,68
classified into two categories: (1) monoclonal antibodies to Importantly, specificity of anti-CCP for RA is higher than RF,
TNF-α (e.g., infliximab, adalimumab, certolizumab, goli- ranging from 91% to 93%.68 Furthermore, the combination of
mumab) and (2) TNF-α receptors fused to an IgG molecule anti-CCP and RF testing has a specificity of 98% to 100%, pro-
(etanercept). All of these agents specifically target and neu- viding for a more accurate diagnosis of RA by allowing for bet-
tralize TNF-α and have demonstrated effectiveness in halting ter differentiation from other forms of arthritis.68 For these
joint damage.67 DMARDs and biological agents have become reasons, most rheumatologists now recommend testing for
the mainstay of RA treatment because they act on specific both RF and anti-CCP.62
components of the inflammatory response and are effective Low titers of ANAs are present in about 40% of patients.
in slowing the progression of joint erosion. Use of these treat- The pattern most frequently identified is the speckled pattern
ments has resulted in higher rates of disease-free periods (re- directed against RNP. The significance of this group of autoan-
mission) and has dramatically improved the quality of life tibodies remains unclear because they do not appear to be
and prognosis of RA patients.54,66 Although these agents are directly related to pathogenesis.
 Once a diagnosis of RA is made, the most helpful tests for silica or to certain drugs such as hydralazine and penicillamine
following the progress of the disease are general indicators of may also be risk factors.72
inflammation, such as measurement of ESR, CRP, and comple- Most patients with GPA have antibodies to neutrophil cy-
ment components.56 Typically, CRP and ESR are elevated and toplasmic antigens; in 80% of these, the antibody is directed
the levels of serum complement components are normal or in- against an enzyme found in the azurophilic granules of neu-
creased because of increased acute-phase reactivity. CRP levels trophils called PR3.70,71 Antibody to the PR3 antigen is
correlate well with disease activity because levels reflect the thought to play a role in the pathophysiology of the systemic
intensity of the inflammatory response.56 vasculitis seen in GPA.70 Events such as chronic infections
can result in the release of the proinflammatory cytokine
Granulomatosis With Polyangiitis TNF-α, which stimulates neutrophils and results in migration
of the PR3 antigen from the granules to the neutrophil mem-
(Wegener’s Granulomatosis) brane. Binding of PR3 antibodies to the PR3 antigen and Fcγ-
Granulomatosis with polyangiitis (GPA), also known as receptors on the cell surface result in activation of the
Wegener’s granulomatosis or WG, is a rare autoimmune dis- neutrophils, which adhere to the endothelial cells lining the
ease involving inflammation of the small- to medium-sized blood vessels. There, they release reactive oxygen species and
blood vessels, or vasculitis. The disease usually begins with a proteolytic enzymes that damage the vascular endothelium.
localized inflammation of the upper and lower respiratory A Th1 response follows, with release of cytokines that induce
tract. General symptoms include fever, malaise, arthralgias, macrophage maturation and the formation of granulomatous
anorexia, and weight loss. The majority of patients progress lesions. Chronic activation of T cells within these lesions is
to develop a more systemic form of the disease that can affect thought to induce differentiation of autoreactive B cells into
any organ system.69-71 plasma cells that produce antibodies to PR3, thus perpetuat-
Virtually all patients have symptoms that affect the upper ing the response.
respiratory system and lungs.69,71 Symptoms of the upper air- Therapy for GPA is directed toward suppression of this in-
way include severe or persistent rhinorrhea (“runny nose”), flammatory response. Patients with severe forms of GPA are
rhinitis, sinusitis, oral or nasal ulcers, and gingivitis. Damage treated with a combination of prednisone and cyclophos-
to the nasal mucosa leads to drying of the nasal membranes, phamide, which produces remission with resolution of the in-
which become susceptible to infection and frequent bleeding. flammatory lesions in most patients.69,75 Biological agents,
Chronic otitis media can cause perforation and scarring of the such as the anti-CD20 monoclonal antibody rituximab, show
eardrums, leading to hearing loss. Tissue damage can lead to promise in providing an effective alternative to cyclophos-
perforation of the nasal septum or collapse of the bridge of the phamide with reduced toxicity. Patients must then be main-
nose. Pulmonary infiltrates are commonly observed upon x-ray tained on a less potent immunosuppressive drug regimen, but
of the lungs. Although patients can experience coughing, short- infections are the main cause of death in about half of GPA pa-
ness of breath, chest pain, or hemoptysis (coughing up blood), tients.75 Nonetheless, immunosuppressive therapy has greatly
they may be asymptomatic. improved patient outcomes, with survival rates as high as 80%
As the disease progresses, other organ systems become in- at 10 years after diagnosis.71
volved.69,71 The majority of patients have renal involvement,
which can range from mild glomerulonephritis with little
functional impairment to severe glomerulonephritis that can According to the criteria published by the ACR, patients are
rapidly lead to kidney failure. Most patients experience pain classified as having WG/GPA if two of the following conditions
and arthritis of the large joints, which is usually symmetric are met: (1) nasal or oral inflammation with oral ulcers or pu-
but not deforming. Skin lesions may occur in patients with rulent or bloody nasal discharge; (2) abnormal chest x-ray,
WG. Many patients have ocular manifestations that can po- showing presence of nodules, fixed infiltrates, or cavities;
tentially lead to vision loss. Other areas of the body that can (3) urinary sediment with microhematuria or RBC casts; and
be affected include the nervous system, heart, and thyroid (4) granulomatous inflammation on biopsy.76 Since then, clini-
gland. Without treatment, more than 90% of patients will die cians have found it useful to include a positive antineutrophil
within 2 years of diagnosis.70 cytoplasmic antibody (ANCA) result (specifically, antibody to
The etiology of GPA is unknown, but multiple genes are proteinase 3); see the Antineutrophil Cytoplasmic Antibodies
thought to be involved. The HLA-DPB1*0401 allele has been (ANCA) section in the text that follows.71
found to have a strong association with GPA in Caucasian General laboratory findings include a normochromic, nor-
patients, whereas the HLA-DRB1*0901 and HLA-DRB*1501 mocytic anemia; leukocytosis; eosinophilia; and an elevated ESR.
alleles are associated with increased risk in Asian and African In addition, there may be a decreased concentration of albumin
American populations, respectively.72-74 Although environmental in the blood and mild to severe renal insufficiency. Urinalysis
factors have not been definitively identified, chronic nasal infec- typically shows microhematuria, proteinuria, and cellular casts.
tion with Staphylococcus aureus bacteria has been associated with Serological findings include an elevated CRP, elevated im-
a greater rate of relapse in patients with WG.71 S aureus may in- munoglobulin levels, positive ANCAs (c-ANCA pattern; see the
duce molecular mimicry because it contains peptides that bear text that follows), and possibly other autoantibodies, such as RF
similarity to the proteinase 3 (PR3) autoantigen.72 Exposure to and ANAs.69
Antineutrophil Cytoplasmic
Antibodies (ANCAs)
ANCAs are autoantibodies that are produced against proteins
that are present in the neutrophil granules. These antibodies are
strongly associated with three syndromes involving vascular in-
flammation: GPA or WG, microscopic polyangiitis (MPA), and
eosinophilic granulomatosis with polyangiitis (EGPA; formerly
known as Churg-Strauss syndrome); these syndromes are col-
lectively known as ANCA-associated vasculitides (AAV).72,77 In
patients with GPA, ANCAs are mainly directed against the PR3
antigen, whereas in MPA and EGPA they are usually specific for
myeloperoxidase (MPO).72
International consensus guidelines require that patient sera
be screened for ANCAs by IIF using ethanol-fixed leukocytes A
as the cellular substrate.78,79 Ethanol treatment permeabilizes
the granule membranes, allowing for migration of the con-
tents.80 In this method, patient serum is incubated with a mi-
croscope slide containing ethanol-fixed leukocytes. Following
incubation, the slide is washed to remove unbound serum and
an anti-human IgG, FITC-labeled conjugate is added. Follow-
ing a second incubation and wash step, the slide is viewed
under a fluorescent microscope for staining of the neutrophils.
Lymphocyte staining should not be present; if it is, it should
be minimal.
Two patterns of fluorescence can be observed: cytoplasmic,
also known as c-ANCA, and perinuclear, or p-ANCA.80,81 The
c-ANCA pattern is primarily caused by PR3-ANCA and ap-
pears as a diffuse, granular staining in the cytoplasm of the
neutrophils (Fig. 15–8A). Staining is most intense in the cen-
ter of the cell between the nuclear lobes and gradually fades at B
the outer edges of the cytoplasm. In the p-ANCA pattern, flu-
orescence surrounds the lobes of the nucleus, blending them c-ANCA and p-ANCA fluorescent patterns.
(A) c-ANCA (cytoplasmic pattern). Note the granular staining of the
together so that individual lobes cannot be distinguished
primary granules in the cytoplasm of the neutrophils and the more
(Fig. 15–8B). This is because the p-ANCA pattern is caused by
intense fluorescence between the lobes of each nucleus. The
antibodies against positively charged antigens such as MPO lymphocytes have negative staining. This pattern can be seen with
that migrate out of the granules after ethanol fixation and are either ethanol-fixed or formalin-fixed neutrophils. (B) p-ANCA
attracted toward the negatively charged nucleus. Table 15–3 (fluorescent pattern). This pattern is characterized by smooth or
summarizes the main features of ANCAs. homogeneous staining of the multi-lobed nuclei in ethanol-fixed
It is important to distinguish the p-ANCA pattern from neutrophils as shown. (On formalin-fixed neutrophils, these
ANAs, which can also stain the nuclei of the neutrophils. With antibodies would produce a granular cytoplasmic staining.) The
ANAs, the nuclear lobes may be more clearly separated, speck- lymphocytes are not stained. (Reprinted with permission from
led staining may be present, and the nuclei of the lymphocytes Immuno Concepts.)
will also be stained. In addition, if a true p-ANCA pattern is
present, ANA testing using HEp-2 cells as the substrate in IIF through initial testing with IIF should be confirmed by PR3–
should be negative.81 For more definitive differentiation, the and MPO–specific immunoassays whenever possible.79 These
test can be repeated using microscope slides with formalin- assays are available in automated ELISA, chemiluminescence im-
fixed leukocytes. Formalin is a cross-linking fixative that pre- munoassay, and fluorescent EIA formats.77 By using a combina-
vents the migration of antigens out of the granules. Therefore, tion of automated immunoassays and IIF, ANCAs can be
in the presence of antibodies to MPO or other positively detected in nearly 100% of patients with active, systemic GPA.73
charged proteins, perinuclear staining will be prevented and However, failure to detect ANCAs does not rule out the presence
intense, granular staining of the cytoplasm will be seen that of AAV.80 ANCA titers are useful in monitoring disease activity,
resembles c-ANCA. but are of limited value in predicting relapses in patients who
ANCA detection by IIF has a sensitivity of more than 90% are in remission.77,78,80
for the AAV, but has a lower specificity (80% or less) because It is important to understand that ANCAs are not diagnostic
ANCAs, especially those producing the p-ANCA pattern, can be for AAV and can be found in a variety of other disorders, in-
found in other conditions.80 Therefore, samples that are positive cluding other types of vasculitis, connective tissue diseases
 Table 15–3 Antineutrophil Cytoplasmic Antibodies (ANCAs)
PATTERN ON INDIRECT
IMMUNOFLUORESCENCE
WITH ETHANOL-FIXED
LEUKOCYTES APPEARANCE AUTOANTIGENS ASSOCIATED DISEASES
c-ANCA Diffuse, granular staining PR3 antigen Granulomatosis with polyangiitis
in the cytoplasm of the (GPA; Wegener’s granulomatosis)
neutrophils, fading
toward the outer edges
of the cells
p-ANCA Fluorescence surround- Positively charged Microscopic polyangiitis (MPA)
ing the lobes of the antigens, including Eosinophilic granulomatosis with
neutrophil nuclei, myeloperoxidase polyangiitis (EGPA; Churg-Strauss
blending them together (MPO) syndrome)

such as SLE and RA, autoimmune gastrointestinal and liver dis- of thyroglobulin to produce the building blocks for the
eases, certain infections including HIV and hepatitis C, malig- hormones.83,84
nancy, and other diseases.82 Patients with these conditions Under normal conditions, the synthesis of T3 and T4 is
often have ANCAs directed toward neutrophil antigens other tightly regulated by an endocrine feedback loop called the thy-
than PR3 or MPO, which can also be detected by IIF. A positive roid axis (Fig. 15–9). Thyrotropin-releasing hormone (TRH)
ANCA test must therefore be combined with clinical manifes- is secreted by the hypothalamus to initiate the process that even-
tations and histological findings of biopsy tissue to make an tually causes release of hormones from the thyroid. TRH acts on
accurate diagnosis. the pituitary gland to induce release of thyroid-stimulating hor-
mone (TSH). TSH, in turn, binds to receptors on the cells of
the thyroid gland, causing thyroglobulin to be broken down into
Organ-Specific Autoimmune secretable T3 and T4. If the levels of T3 and T4 become too high,
Diseases they feed back to the hypothalamus and pituitary to inhibit re-
lease of TRH and TSH, resulting in decreased production of the
This chapter will refer to organ-specific autoimmune diseases thyroid hormones. The presence of autoantibodies to compo-
as those diseases in which the immune response is directed nents of the thyroid gland interferes with this process and causes
against self-antigens that are mainly found in a single organ under- or overactivity of the gland.83,84
or gland. Although the clinical manifestations are largely re-
lated to the target area, systemic effects may sometimes also
occur. Examples of organ-specific autoimmune diseases are The genes thought to be associated with a predisposition to thy-
listed in Table 15–4; many of these will be discussed in more roid autoimmunity are related to immune function or are thyroid
detail here. specific. A strong association between HLA-DR3 and Graves dis-
ease has been observed.85,86 Association of Hashimoto’s thyroidi-
tis with inheritance of HLA antigens DR3, DR4, DR5, and DQ7
Autoimmune Thyroid Diseases (AITDs) has been reported, but this is not consistent among different eth-
Autoimmune thyroid diseases (AITDs) encompass several nic populations.85,86 A unique feature of both Graves and
different clinical conditions, the most notable of which are Hashimoto’s diseases is that HLA-DR antigens are expressed on
Hashimoto’s thyroiditis and Graves disease. Although these the surface of thyroid epithelial cells, perhaps increasing the au-
conditions have distinctly different symptoms, they do share toimmune response. Mutations in the thyroglobulin gene may
some antibodies in common; in addition, both interfere with allow for interaction of the protein with HLA-DR antigens, re-
thyroid function. The thyroid gland is located in the anterior sulting in antithyroglobulin antibodies. These can be found in
region of the neck and is normally between 12 and 20 grams both Graves and Hashimoto’s disease. Additionally, in Graves
in size. It consists of units called follicles that are spherical disease, modifications in the TSH receptor gene may allow the
in shape and lined with cuboidal epithelial cells. Follicles immune system to recognize the receptor and produce antibod-
are filled with material called colloid. The primary constituent ies against it.86,87
of colloid is thyroglobulin (Tg), a large iodinated glycopro- Possible environmental triggers of AITDs include infections,
tein from which the active thyroid hormone triiodothyronine certain medications, smoking, psychological stress, and preg-
(T3) and its precursor, thyroxine (T4), are synthesized. The nancy, but the strongest link is thought to be between high
enzyme thyroid peroxidase (TPO) plays an important role iodine intake and development of Hashimoto’s disease. Highly
in the synthesis of these hormones by oxidizing iodine ions, iodinated thyroglobulin is thought to be more immunogenic,
allowing for their incorporation into the tyrosine residues possibly creating or exposing more epitopes and facilitating the
 Table 15–4 Organ-Specific Autoimmune Diseases
DISEASE TARGET CELLS OR TISSUES ASSOCIATED AUTOANTIBODIES
Addison’s disease Adrenal glands Antibody to adrenal cells
Autoimmune Red blood cells (RBCs) Antibody to RBCs
hemolytic
anemia
Autoimmune Liver AIH-1—smooth muscle antibodies; ANAs
hepatitis (AIH) AIH-2—anti-liver kidney microsomal antibody (anti-LKM-1);
anti-liver cytosol type 1 antibody (anti-LC-1)
Autoimmune Platelets Antiplatelet antibody
thrombocytopenic
purpura
Celiac disease Small intestine and other organs Antitransglutaminase (tTG)
Antibodies to deamidated gliadin peptides (DGPs)
Endomysial antibodies
Goodpasture’s Kidneys, lungs Antibody to an antigen in the renal and pulmonary
syndrome basement membranes
Graves disease Thyroid gland Thyroid-stimulating hormone receptor antibodies (TRAbs)
Antithyroglobulin
Antithyroid peroxidase (TPO)
Hashimoto’s Thyroid gland Antithyroglobulin
thyroiditis Antithyroid peroxidase (TPO)
Multiple sclerosis Myelin sheath of nerves Antibodies to myelin basic protein
Myasthenia gravis Nerve-muscle synapses Antibodies to acetylcholine receptors (AChR)
Anti-muscle-specific kinase (MuSK)
Antibody to the lipoprotein LRP4
Pernicious anemia Stomach Parietal cell antibody, intrinsic factor antibody
Poststreptococcal Kidneys Streptococcal antibodies that cross-react with kidney tissue
glomerulonephritis
Primary biliary Intrahepatic bile ducts Antimitochondrial antibodies (AMA)
cirrhosis
Rheumatic fever Heart Streptococcal antibodies that cross-react with cardiac tissue
Scleroderma Connective tissue Antinuclear antibodies: anti-Scl-70, anticentromere antibody
Sjögren’s syndrome Eyes, mouth Antinuclear antibodies, rheumatoid factor, antisalivary duct
antibodies, antilacrimal gland antibodies
Type 1 diabetes Pancreas Anti-insulin
mellitus Islet cell antibodies
Anti–IA-2 and anti–IA-2βA
Antibody to glutamic acid phosphatase (GAD)

antigen uptake and processing step of the adaptive immune women; in addition, women are 5 to 10 times more likely to
response (see Chapter 4).88 develop the disease than men.87,88 Patients develop an en-
larged thyroid called a goiter, which is irregular and rubbery.
Patients also produce thyroid-specific autoantibodies and
cytotoxic T cells. Immune destruction of the thyroid gland
Hashimoto’s thyroiditis, also known as chronic lymphocytic occurs, which results in a state of decreased thyroid function
thyroiditis, was discovered in Japan in 1912 by Dr. Hakaru called hypothyroidism. Symptoms of hypothyroidism include
Hashimoto. It is now considered to be the most common au- dry skin, decreased sweating, puffy face with edematous eye-
toimmune disease, affecting about 8 out of every 1,000 in- lids, pallor with a yellow tinge, weight gain, fatigue, and dry
dividuals.89 The disease is most often seen in middle-aged and brittle hair.84,89
 Hypothalamus (!) insomnia, depression, weight loss, heat intolerance, sweating,
rapid heartbeat, palpitations, breathlessness, fatigue, cardiac
TRH dysrhythmias, and restlessness.84,90 Another sign present in ap-
proximately 35% of patients is exophthalmos, in which hyper-
trophy of the eye muscles and increased connective tissue in
Pituitary (!) the orbit cause the eyeball to bulge out so that the patient has
Stimulatory a large-eyed staring expression (Fig. 15–10).91,92 There is ev-
Graves TSH Inhibitory idence that orbital fibroblasts express TSH receptor-like pro-
teins that are affected by thyroid-stimulating immunoglobulin
just as the thyroid is.92,93 Localized edema in the lower legs
can also occur.
TG T3/T4 The thyroid shows uniform hyperplasia with a patchy lym-
phocytic infiltration. The follicles have little colloid but are filled
TPO
Hashimoto’s with hyperplastic epithelium. A large number of these cells ex-
(and Graves)
press HLA-DR antigens on their surface in response to IFN-γ
I! produced by infiltrating T cells.94 This allows presentation of
Thyroid
self-antigens such as the thyrotropin receptor to activated
T cells. B cells, in turn, are stimulated to produce antibody.
The major antibodies involved in the pathogenesis of Graves
disease are the thyroid-stimulating hormone receptor antibod-
Hashimoto’s Tc ies (TRAbs). When TRAbs bind to the TSH receptor they mimic
the action of TSH, resulting in uncontrolled receptor stimulation
with excessive release of thyroid hormones (Fig. 15–9). In the
Autoimmune disorders in thyroid hormone synthesis end, symptoms of hyperthyroidism are produced.83,84 Other
and regulation. The actions of autoantibodies and cytotoxic T cells in antibodies present include anti-Tg, anti-TPO, and thyrotropin
the pathogenesis of Graves disease and Hashimoto’s disease is shown. receptor-blocking antibody. The thyrotropin receptor-blocking
Solid arrows = stimulation; dotted arrows = feedback inhibition.
antibody may coexist with thyroid-stimulating antibody in a
number of patients.86,93 Depending on the relative activity of
blocking and stimulating autoantibodies, patient symptoms may
Six forms of Hashimoto’s disease have been described, each vary from hyperthyroidism to euthyroidism (the state of normal
with distinct pathological features.89 In the classic form of the thyroid function) to hypothyroidism, which may confound the
disease, the thyroid shows hyperplasia with an increased num- patient’s diagnosis.
ber of lymphocytes. Cellular types present include activated T
and B cells (with T cells predominating), macrophages, and
plasma cells. Pathology to the thyroid gland is mediated pri- Treatment for Hashimoto’s disease consists of daily oral thyroid
marily by CD8+ cytotoxic T cells, which bind to the thyroid hormone replacement therapy, with levothyroxine (T4) being
cells and destroy them by releasing enzymes that cause apop- the preferred drug. TSH levels should be monitored through-
tosis or necrosis.84 The immune response also results in the out treatment and are used in adjusting the dose of the drug
development of germinal centers that almost completely re- so that normal TSH levels are maintained.83,84
place the normal glandular architecture of the thyroid and pro- Several different protocols are used in the treatment of
gressively destroy the thyroid gland.89 Antibodies to Tg and Graves disease. In the United States, the first line of treatment
TPO are produced that have the ability to fix complement; this involves radioactive iodine, which emits beta particles that are
may result in an inflammatory response that perpetuates the locally destructive within an area of a few millimeters. The io-
tissue damage.84,86 Injury to the thyroid gland results in the dine is administered for 1 to 2 years and results in a 30% to
symptoms associated with hypothyroidism.

Graves disease, in contrast to Hashimoto’s thyroiditis, is char-

acterized by hyperthyroidism, a state of excessive thyroid func-
tion. Graves disease is, in fact, one of the most frequently
occurring autoimmune diseases and the most common cause
of hyperthyroidism.84 Women exhibit greater susceptibility to
Graves disease by a margin of about 5 to 1 and most often pres-
ent with the disease in the fifth and sixth decades of life.90
The disease is manifested as thyrotoxicosis, or an excess of
thyroid hormones, with a diffusely enlarged goiter that is firm Exophthalmos indicative of Graves disease. (Courtesy
instead of rubbery. Clinical symptoms include nervousness, of CDC/Dr. Sellers/Emory University.)
50% long-term remission rate.84,93 Some patients, however, are found in the majority of patients, they are generally not
become hypothyroidal within 5 years; therefore, continued useful in making the diagnosis. TRAbs, on the other hand, are
monitoring is essential. In Europe and Japan, the patient is first highly indicative of Graves disease because they are present in
placed on antithyroid medications with beta blockers as adju- 98% to 100% of patients; TRAbs are therefore included as one
vant therapy.84,90,93 This initial course is followed by continued component of the diagnostic criteria for Graves disease.90
drug treatment, radioactive iodine therapy, or surgery to re- There are two types of tests for TRAbs: binding assays and
move part of the thyroid. Surgery is recommended for patients bioassays. With binding assays, such as automated solid-phase
resistant to drug treatment, but can damage the laryngeal ELISA or chemiluminescent immunoassays, a labeled TRAb
nerves and cause permanent hoarseness.83,84 reagent competes with the patient antibody for TSH receptor
bound to a solid phase.90,96 Bioassays require tissue culture
and thus are difficult to perform.95,96 However, these assays
can distinguish between TRAbs with stimulatory activity versus
Initial screening for AITDs involves measurement of circulating those with inhibitory activity because they are functional as-
TSH levels. Recall that because of the thyroid axis, the TSH says. Current bioassays detect the ability of TRAbs to bind to
level is inversely related to the levels of T3 and T4. TSH is rou- TSH receptors on live cells and trigger cAMP-dependent lu-
tinely measured with highly sensitive chemiluminescent im- ciferase activity. These assays show promise for use in clinical
munoassays that can detect fewer than 0.1 mU/L.83,84 A normal settings to evaluate patient response to therapy and individuals
TSH level indicates normal thyroid function, with rare excep- with alternating episodes of hyper- and hypothyroidism.96
tions.84 If the TSH level is abnormally high or low, laboratory
tests for circulating thyroid hormone levels must be performed.
Although immunoassays for total serum T3 and T4 are avail-
Type 1 Diabetes Mellitus (T1D)
able, the majority of these hormones are protein bound and Diabetes mellitus is a group of common endocrine disorders
alterations in these hormone-binding proteins can affect test that are characterized by hyperglycemia (a high level of glucose
results. Therefore, it is more useful to measure unbound thy- in the blood).97 The American Diabetes Association (ADA) has
roid hormone, usually free T4 (FT4).83,84 Table 15–5 summa- classified diabetes into three main categories based on the eti-
rizes some of the main laboratory findings in Hashimoto’s ology of the disease: type 1 diabetes, type 2 diabetes, and ges-
disease and Graves disease. tational diabetes. The majority of patients have type 2 diabetes,
Patients with Hashimoto’s thyroiditis will have normal or which is characterized by insulin resistance and occurs most
high TSH levels and low FT4 levels.83,84 To establish an autoim- commonly in obese individuals over the age of 40.98 About 5%
mune etiology for the hypothyroidism, it is necessary to follow to 10% of patients with diabetes mellitus are classified as hav-
these findings by testing for thyroid antibodies. Today, these an- ing type 1 diabetes mellitus (T1D), which is characterized
tibodies are most commonly detected by sensitive ELISA and by a complete or nearly complete deficiency in insulin. Of
chemiluminescent immunoassays.83,95 Antibodies to TPO are these patients, 90% have an immune-mediated form of the
the best indicator of the disease because they are found in about disease known as type 1A diabetes, whereas the remaining
95% of patients with Hashimoto’s disease, but only 10% to 15% 10% are idiopathic cases with no identifiable cause (type 1B
of the general population. Antibodies to Tg are less sensitive diabetes).98-100 Gestational diabetes develops in some women
and specific because they are detected in only 60% to 80% of during pregnancy. This section will focus on type 1A diabetes,
patients with the disease and are found more frequently than which will be referred to as T1D. T1D was previously known
TPO antibodies in healthy persons.83,89 Because anti-Tg anti- as juvenile onset diabetes because it usually develops in children
bodies are not found in all patients, a negative test result does or in young adults before the age of 30.97
not necessarily rule out Hashimoto’s disease. T1D is a chronic autoimmune disease that involves selective
In contrast, patients with Graves disease characteristically destruction of the beta cells of the pancreas. These cells are lo-
have low or undetectable levels of TSH and increased levels of cated in clusters called the islets of Langerhans and are respon-
FT4.83,84 Increased uptake of radioactive iodine also helps to sible for the production and secretion of the hormone, insulin.
confirm the diagnosis.90 Although antibodies to TPO and Tg Insulin plays a vital role in regulating the amount of glucose

Table 15–5 Typical Laboratory Findings in Autoimmune Thyroid Diseases

DISEASE TSH LEVEL FREE T4 (FT4) LEVEL AUTOANTIBODIES
Hashimoto’s thyroiditis Normal or elevated Decreased Antithyroglobulin
Antithyroid peroxidase (TPO)
Graves disease Decreased or undetectable Elevated Thyroid-stimulating hormone
receptor antibodies (TRAbs)*
Antithyroglobulin
Antithyroid peroxidase (TPO)
*diagnostic
in the circulation by promoting its absorption by skeletal mus- in order to prevent rejection and the number of suitable donors
cles and fat tissue so that it can be converted into energy needed is limited. New technologies, such as the use of stem cells to pro-
by our cells. The autoimmune destruction of beta cells in T1D duce islet cells in vitro and methods to induce immunologic tol-
results in insufficient insulin production, hyperglycemia, and erance in recipients, offer hope that islet cell transplantation may
toxic effects on the body. Long-term effects include cardiovas- encounter fewer obstacles in the future.105
cular complications, renal disease, nerve damage, blindness,
and infections of the lower extremities, which can lead to am-
According to the ADA, a person is considered to have diabetes
putation.97 Patients require lifelong insulin injections to control
if he or she meets one of four criteria: (1) a fasting glucose
glucose levels and lower the risk of these complications.
greater than 126 mg/dL on more than one occasion (normal
Family studies indicate that there is an inherited genetic sus-
value is lower than 100 mg/dL); (2) a random plasma glucose
ceptibility to the disease, probably attributable to multiple
level of 200 mg/dL or more with classic symptoms of diabetes;
genes. Most people with T1D carry the HLA-DR3 or DR4 gene,
(3) an oral glucose tolerance test of 200 mg/dL or more in a
and there is an increased risk when both of these genes are
2-hour sample with a 75 g glucose load; or (4) a hemoglobin
present.97 There is also a strong correlation between certain
A1c value (HbA1c) greater than 6.5%.98 HbA1c is a glycated
HLA-DQ haplotypes and type 1 diabetes mellitus.97,101 Possi-
form of hemoglobin that is made when the RBC protein com-
ble environmental influences include certain viral infections
bines with glucose in the blood. The HbA1c plasma level
and early exposure to cow’s milk.97
is proportional to the life span of the circulating RBCs (up to
120 days) and reflects the average plasma glucose concentra-
Progressive inflammation of the islets of Langerhans in the pan- tion over the previous 2 to 3 months.
creas leads to fibrosis and destruction of most beta cells. The Although T1D is usually diagnosed by the prime charac-
subclinical period may last for years. Hyperglycemia does not teristic of hyperglycemia, it may be useful to perform serolog-
become evident until 80% or more of the beta cells are de- ical tests for diabetes before beta-cell destruction occurs to the
stroyed. Immunohistochemical staining of inflamed islets shows extent necessary to cause symptoms. When T1D is suspected,
a preponderance of CD8+ lymphocytes, along with plasma cells tests for antibodies to GAD and IA-2A can be done to confirm
and macrophages.97 B cells themselves may act as APCs, stim- the diagnosis. If these results are negative, they can be fol-
ulating activation of CD4+ lymphocytes.102 A shift to a Th1 re- lowed by testing for ICA in children and for insulin antibodies
sponse causes production of certain cytokines, including in adults.99 ICAs have been reported in the sera of more than
TNF-α, IFN-γ, and IL-1. The generalized inflammation that re- 80% of patients newly diagnosed with T1D.97 Antibodies to
sults is responsible for the destruction of the beta cells. Al- islet cells have traditionally been detected by IIF using frozen
though islet autoantibodies trigger the immune response, it is sections of human pancreas.106 However, such assays are
not known what role they play in cell destruction. There is in- rather cumbersome to perform and other methods are avail-
creasing evidence that autoimmunity to insulin itself may be able, including radio-immunoprecipitation assays, Western
central to disease pathogenesis.102 Cell death, however, is likely blotting, ELISA, and mass spectrometry.103 These methods can
caused by apoptosis and attack by cytotoxic lymphocytes. also be used to detect antibodies to other pancreatic antigens
It is apparent that autoantibody production precedes the such as insulin, GAD, and IA-2.103,107 Combined screening
development of T1D by months to several years.99 Autoanti- for IA-2A, ICA, and GAD antibodies appears to have the most
bodies are present in newly diagnosed patients and in predia- sensitivity and best positive predictive value for T1D in high-
betic individuals who are being monitored because they have risk populations.99,107
a high risk of developing diabetes. Antibody production di-
minishes with time, however. Among the antibodies found are Celiac Disease
antibodies to two tyrosine phosphatase-like transmembrane
Celiac disease is an autoimmune disease affecting the small in-
proteins called insulinoma antigen 2 (IA-2) and IA-2β A (phogrin);
testine and other organs. It affects 0.6% to 1.0% of the world’s
anti-insulin antibodies; antibodies to the enzyme glutamic acid
population, but this number is thought to be an underestimate
decarboxylase (GAD-65); antibodies to zinc transporter 8 (ZnT8);
because many cases go undiagnosed.108 Celiac disease is unique
and antibodies to various other islet cell proteins, called islet
in that it is associated with a known environmental trigger—
cell antibodies (ICAs).99,100,103 Many of these antigens are com-
dietary gluten.109 Gluten is a protein complex found in wheat,
ponents of the regulated pathway that is essential for the se-
barley, and rye that is poorly digested by the upper gastroin-
cretion of insulin.103
testinal system. It contains an alcohol-soluble component called
gliadin that is rich in the amino acids glutamine and proline.
Daily injectable insulin has been the mainstay of therapy for T1D. Gliadin is resistant to digestive enzymes in the stomach, pan-
Clinical trials are investigating the use of immunosuppressive creas, and small intestine and therefore remains intact in the
drugs and biological agents to inhibit the autoimmune responses lumen, or space within the intestines, after ingestion. If there is
that lead to beta cell destruction and prevent disease progression an increase in the permeability of the intestinal walls, possibly
in T1D patients.104 Transplantation of pancreatic beta islet cells as a result of an infection, undigested gliadin is able to pass
has been used for T1D patients who have poor glucose control, through the epithelial barrier of the intestine and trigger an in-
but this treatment requires continual immunosuppressive therapy appropriate immune response. The immunogenicity of gliadin
is enhanced when it is acted on by tissue transglutaminase diet before serological testing because false-negative antibody re-
(tTG), an intestinal enzyme that converts the glutamine sults can occur in individuals on a gluten-free diet.108,112
residues in gliadin to glutamic acid.109,110 The first serological test for celiac disease was produced in
As a result, immunogenic peptides are generated that specif- the 1980s and measured antibodies to gliadin; however, this
ically react with HLA-DQ2 or HLA-DQ8 molecules on APCs. test is not commonly used today because it is associated with
In fact, the presence of one of these two HLA haplotypes is a a significant number of false-positive results.113 Currently, de-
necessary condition for developing celiac disease. The majority tection of IgA antibodies to tTG is the serological method of
of patients (90% to 95%) possess an HLA-DQ2 allele, whereas choice for initial testing.108,112,113 This is because automated,
almost all of the remaining patients have HLA-DQ8.108,109 The ELISA-based assays using purified human or recombinant tTG
gliadin peptides that are picked up by the APC are presented antigen have a high sensitivity (91% to 95%) and specificity
to antigen-specific CD4+ T cells, which produce cytokines that (95% to 97%) for celiac disease. Rapid EIA tests are also avail-
activate CD8+ T cells and trigger an inflammatory response able for detection of antibodies to tTG, but are not as sensitive
that damages the architecture of the intestinal mucosa and or specific as the ELISA tests.113 Serum IgA levels should be
causes injury to the villi.109,110 In addition, B cells are stimu- concurrently measured in these individuals because a signifi-
lated to produce antibodies to the deamidated gliadin peptides cant number also have selective IgA deficiency and will there-
(DGPs), tTG, and endomysium (a layer of connective tissue fore test negative in IgA-based assays.108,112 In patients who
surrounding the intestinal muscles). are IgA deficient, testing for IgG anti-tTG or for IgG antibodies
Environmental factors believed to play a role in the devel- to DGPs can be performed. Automated ELISA tests for anti-
opment of celiac disease include administration of gluten in DGPs are a more recent development and show an especially
the diet of an infant younger than 4 months in the absence of high sensitivity in children under the age of 2 to 3 years, who
breastfeeding, rotavirus infection, and overgrowth of patho- may test negative for other antibodies. False-positive results
genic bacteria in the gut.109,110 In addition, the disease is found for anti-tTG can occur in patients with other autoimmune dis-
more often in women than in men (ratio, 2-3:1) and among eases such as type 1 diabetes. Positive anti-tTG results can be
those who have selective IgA deficiency, Down syndrome, followed by testing for endomysial antibodies (EMA). EMA
Turner’s syndrome, or type 1 diabetes.109,110 Several non-HLA tests are highly specific for celiac disease, but are more costly
genes involved in immune function are also thought to con- because they are based on IIF assays using monkey esophagus
tribute to this autoimmune response.111 or human umbilical cord tissue as the substrate.113
Because serology results are not absolute, biopsy of the
small intestine should be performed to confirm the diagnosis.
The clinical symptoms of celiac disease vary with age.109,110 In-
Initial biopsy results can also provide a baseline for comparing
fants typically present with diarrhea, abdominal distention, and
future samples for intestinal injury. Histological examination
failure to thrive, but may also experience vomiting, irritability,
of biopsy tissue characteristically shows an increase in the
anorexia, and constipation. Older children, teenagers, and
number of intraepithelial lymphocytes, elongation of the in-
adults may have the classic symptoms of diarrhea and abdom-
testinal crypts, and partial to total atrophy of the villi.108 In
inal pain or discomfort, but often have extraintestinal manifes-
order to minimize the occurrence of false-negative results, mul-
tations that make the condition difficult to diagnose. These
tiple biopsies should be obtained from different parts of the
include short stature, arthritis or arthralgia, osteoporosis, neu-
duodenum because mucosal injury may be patchy.108,112
rological symptoms, iron-deficiency anemia, and dermatitis
HLA typing is also useful in differentiating celiac disease
herpetiformis (a skin disorder with itchy blistering).
from other conditions, especially when serological tests and
Treatment of celiac disease involves placing patients on a
biopsy results are borderline. Absence of HLA-DQ2 or HLA-
gluten-free diet. Elimination of gluten from the diet usually re-
DQ8 virtually excludes a diagnosis of celiac disease because
sults in improvement of clinical symptoms within days or weeks
individuals who are negative for these haplotypes are highly
and healing of intestinal damage in 6 months to 2 years.108,109
unlikely to have the disease.108
However, a significant number of patients do not adhere to a
Following proper diagnosis and maintenance of patients
gluten-free diet because of expense, inconvenience, or social
with celiac disease on a gluten-free diet, clinical symptoms usu-
stigma; in addition, some patients have persistent symptoms
ally improve, antibody titers revert to negative, and histology
despite adherence to the diet. Alternative treatments, such as
can return to normal. However, about 5% of patients fail to
the use of recombinant enzymes to digest the toxic gliadin, are
improve, usually because of nonadherence to the diet (which
being investigated.
can sometimes be unintentional), but sometimes caused by re-
fractory disease, incorrect diagnosis, complications of celiac
Diagnosis of celiac disease is based on clinical symptoms, sero- disease, or simultaneous gastrointestinal disorders.112
logical findings, duodenal biopsy, and presence of the HLA-DQ2
or HLA-DQ8 haplotype. Serological testing is the initial ap-
proach taken to evaluate patients suspected of having celiac dis-
Autoimmune Liver Diseases
ease and helps to differentiate these patients from those having There are three major forms of autoimmune liver disease: au-
conditions with similar symptoms, such as gluten sensitivity or toimmune hepatitis (AIH), primary biliary cirrhosis
wheat allergy.108 It is recommended that patients follow a regular (PBC), and primary sclerosing cholangitis (PSC). In AIH, the
autoimmune process targets the hepatocytes; in PBC, it affects substrates, anti-LKM-1 stains the cytoplasm of the hepatocytes
the small, interlobular bile ducts; and in PSC, it affects the and the P3 portion of the kidney tubules. The resulting im-
medium-sized, intra- and extrahepatic bile ducts.114 There munofluorescent pattern appears similar to that produced by
can also be overlap syndromes that combine features of these mitochondrial antibodies, but can be distinguished by experts
diseases; many patients also have other autoimmune disor- when testing is performed on multiple tissue substrates and
ders such as autoimmune thyroiditis or ulcerative colitis. This the intensity of fluorescent staining of different components of
section will discuss two of these diseases, AIH and PBC, and the substrates is carefully examined. Clinically significant titers
the serological tests that are used in their diagnosis in more are considered to be 1:40 in adults and 1:10 in children; anti-
detail. body titers correlate with disease activity.116,117 Antibodies to
LC-1 are directed against a folate-metabolizing enzyme in the
liver and stain the cytoplasm of liver cells in IIF. These anti-
AIH, formerly known as chronic active hepatitis, is an immune-
bodies can be masked by anti-LKM-1 if they are also present
mediated liver disease that can lead to end-stage liver failure if
and other methods, such as immunodiffusion, may be required
left untreated. It can affect children and adults of all ages and
to detect them.116,117
is more common in females than in males. The clinical features
Liver biopsy is necessary to confirm the diagnosis of AIH
of AIH can be quite variable.115-117 About 25% of individuals
and to assess the extent of liver damage. Inflammation at the
are asymptomatic and are diagnosed only after abnormal liver
portal-parenchymal boundary, known as interface hepatitis, is
function tests are found coincidentally when blood work is
typical of AIH and is characterized by an infiltrate of lympho-
performed. Adults usually present with an unexpected onset
cytes, plasma cells, and histiocytes surrounding dying hepato-
of vague symptoms, including fatigue, nausea, weight loss, ab-
cytes.116,117 Histological findings, along with detection of
dominal pain, itching, and maculopapular rashes. Less often,
autoantibodies, elevated IgG, and exclusion of viral hepatitis,
patients have symptoms of portal hypertension such as gas-
comprise the criteria recommended by the International Au-
trointestinal bleeding or hypersplenism. Jaundice may also be
toimmune Hepatitis Group for the diagnosis of AIH.115,117
present. Rarely, the initial presentation is fulminant liver failure
These criteria have a sensitivity greater than 80% and a speci-
requiring liver transplantation.
ficity greater than 95%.115
Two types of AIH can be differentiated on the basis of its
Laboratory findings are essential for early diagnosis, and treat-
autoantibody specificity (see the text that follows); these are
ment should be started promptly once the diagnosis is made. Most
referred to as AIH-1 and AIH-2. AIH-1 accounts for two-thirds
patients respond to the standard immunosuppressive treatment
of all AIH cases and has a female:male ratio of 4:1. AIH-2 has
of prednisolone (+/–azathioprine) to induce remission, followed
a female:male ratio of 10:1 and is seen mostly in children.115,117
by azathioprine alone to maintain remission. However, patients
Certain HLA-DRB1 and HLA-DQB1 alleles are associated
must be monitored carefully because they are at increased risk
with a higher risk of developing AIH; these vary in different eth-
of developing cirrhosis and possibly hepatocellular carcinoma.
nic populations.115,118 Exposure to certain viruses or drugs has
If untreated, AIH usually progresses to liver failure, at which
been suggested to play a role in triggering AIH, possibly through
point liver transplantation is required.116,117
molecular mimicry and cross-reactivity between their epitopes
and liver antigens, the most notable virus being hepatitis C.115
Common laboratory findings include elevated levels of the PBC, the most common autoimmune liver disease, occurs
liver enzymes, aspartate aminotransferase (AST) and alanine about 10 times as often in females as in males.118 There is a
aminotransferase (ALT), with less prominent increases in serum genetic link with certain HLA-DRB1, HLA-DQA1, HLA-DPB1,
bilirubin and alkaline phosphatase. Serum immunoglobulin and HLA-DQB1 haplotypes.118,119 PBC is an autoimmune dis-
levels, particularly IgG, are high; in adults, various autoanti- ease that involves progressive destruction of the intrahepatic
bodies are present, including ANAs, ANCAs, smooth muscle bile ducts.118, 120-121 The destruction leads to chronic cholestasis
antibodies (SMA), anti-liver kidney microsomal antibody (anti- (a condition in which the flow of bile is slowed or blocked),
LKM-1), anti-liver cytosol type 1 antibody (anti-LC-1), and an- inflammation of the portal vein in the liver, and accumulation
timitochondrial antibodies (AMAs).116,117 of scar tissue that can ultimately lead to cirrhosis and liver fail-
Autoantibody profiles can distinguish between AIH-1 and ure. Individual patients can be asymptomatic or have slowly
AIH-2. AIH-1 patients are characteristically positive for SMA or rapidly progressing disease. Symptoms include fatigue, pru-
or ANA. ANAs most commonly produce a homogeneous pat- ritis (itchy skin), abdominal pain, and dry eyes and mouth; in
tern on IIF, but can sometimes produce a speckled pat- the later stages, symptoms include jaundice, ascites, and greasy
tern.116,117 The SMAs are directed against actin and other stools. The only established therapy for PBC is ursodeoxycholic
components of the cytoskeleton. They can be detected by IIF acid (UDCA), a bile acid that helps move bile through the liver.
on rodent kidney, stomach, or liver sections, where they pro- Use of UDCA has helped to slow down disease progression and
duce fluorescent staining of the smooth muscle in the artery increase patient survival. Liver transplantation is the only ef-
walls and other components, such as the glomeruli and tubules fective treatment for patients who have reached end-stage liver
of the kidneys. Antibody titers are usually 1:80 or higher in disease.118,120-121
adults, but can be as low as 1:20 in children.116,117 Anti-mitochondrial antibodies (AMAs) are found in the ma-
In contrast, patients with AIH-2 characteristically produce jority of patients with PBC and their presence is one of three di-
antibodies against LKM-1 or LC-1. On IIF using rodent tissue agnostic criteria for the disease.118,120-121 The other two criteria
are a serum alkaline phosphatase level elevated at least 1.5 times MS is characterized by the formation of lesions called
the upper limit of normal for 6 months or more and liver biopsy plaques in the white matter of the brain and spinal cord, result-
showing nonsuppurative destructive cholangitis and interlobular ing in the progressive destruction of the myelin sheath of
bile duct injury. A diagnosis of PBC can be made if at least two axons. Plaques vary in size from 1 or 2 mm up to several cen-
out of three of these criteria are met. In addition, patients com- timeters.123 Within the plaques, T cells and macrophages pre-
monly have elevated levels of aminotransferases and serum im- dominate; these are believed to orchestrate demyelination.123
munoglobulins, especially IgM, because of polyclonal activation Antibody binds to the myelin membrane and may initiate the
of B cells. ANAs giving perinuclear or rimlike, nuclear dot, or immune response, stimulating macrophages and specialized
centromere patterns may also be present. phagocytes called microglial cells.125 The cascade of immuno-
Traditionally, AMAs have been detected by IIF with logic events results in acute inflammation, injury to axons and
mitochondria-rich tissue substrates such as rodent liver, kidney, glia, structural repair with recovery of some function, and then
or stomach sections. These antibodies produce a bright, uni- postinflammatory neurodegeneration.125 The Th1 cytokines
form granular cytoplasmic fluorescence in the distal renal IL-1, TNF-α, and IFN-γ are believed to be central to the patho-
tubules, gastric parietal cells, thyroid epithelial cells, and car- genesis of the disease, promoting changes in the endothelial
diac muscle.122 IIF uses antigens in their natural configuration cells that facilitate adherence of activated T cells and their mi-
and has fairly high levels of sensitivity and specificity; how- gration across the blood–brain barrier.124 Th17 cells are also
ever, the method is manual, time-consuming, and requires a thought to play an important role in the inflammatory response
high level of expertise to correctly interpret the pattern. In of the CNS. A Th2 response, characterized by production of
addition, about 5% to 10% of PBC patients test negative for IL-4, IL-5, and IL-10, may also contribute to pathogenesis.123
AMAs on IIF or may give atypical staining patterns that are
difficult to interpret.121
Since the development of IIF, the target antigens of AMAs Damage to the tissue of the CNS can cause visual disturbances,
have been identified as components of the 2-oxo-acid de- weakness or diminished dexterity in one or more limbs, loco-
hydrogenase complexes that are involved in mitochondrial motor incoordination, dizziness, facial palsy, and numerous
energy-producing pathways. Identification of these antigens al- sensory abnormalities such as tingling or “pins and needles”
lowed for the development of solid-phase ELISA assays. The that run down the spine or extremities, as well as flashes of
mitochondrial antigens employed in these ELISAs consist of light seen on eye movement.125 The disease most often begins
preparations of porcine or bovine heart, mixtures of recombi- in young and middle-aged adults between the ages of 20 and
nant subunits of pertinent antigens, designer antigens com- 50 and is twice as common in women as in men.124 MS can be
posed of particular subunits, or mixtures of native and designer classified into four major subtypes based on the clinical course
antigens, depending on the commercial manufacturer. ELISAs of the disease.123,124 More than 80% of patients fall into the
have the advantages of automation and provision of objective first subtype, relapsing-remitting MS, which is characterized
results, but their performance can vary because these assays by clearly defined episodes of neurological attacks with periods
generally do not include the full spectrum of antigenic epitopes of full or partial recovery in between. Most patients with MS
available by IIF.122 eventually develop progressive deterioration of the CNS and
A cost-effective approach to testing may be to screen sam- functional disability.
ples for AMAs and other autoantibodies by IIF, then follow-up Treatment for MS is aimed at easing recovery from acute at-
with an AMA ELISA assay for confirmation. This approach tacks and reducing the risk of future relapses.123,126 Acute
would also be helpful in identifying AMAs in those patients exacerbations are treated with corticosteroids to reduce inflam-
who test negative through IIF.122 This testing strategy would mation. Disease-modifying agents such as IFN-β1a and IFN-β1b
facilitate an earlier diagnosis for PBC patients, allowing the ini- have been approved to treat MS for the long term.123,126 These
tiation of treatment that could slow down disease progression agents are believed to work by downregulating MHC molecules
and improve patient survival. It is important to note that a di- on APCs, decreasing production of proinflammatory cytokines,
agnosis of PBC cannot be based on the presence of AMAs alone and upregulating anti-inflammatory cytokines.123,126 The
because these antibodies have also been observed in patients severity of MS can be greatly reduced by therapy with natal-
with other conditions such as SLE, RA, and graft-vs.-host dis- izumab, a humanized monoclonal antibody directed against
ease, as well as a small percentage of healthy persons.121 an adhesion molecule of lymphocytes, preventing them from
binding to endothelial cells and crossing the blood–brain bar-
Multiple Sclerosis (MS) rier.123,126 Agents such as these are improving the long-term
prognosis of MS.123
Multiple sclerosis (MS) is an autoimmune disorder involving
inflammation and destruction of the CNS. It affects approxi-
mately 350,000 Americans and 2.5 million individuals world- The diagnosis of MS is based primarily on clinical symp-
wide.123 MS is most closely associated with inheritance of a toms, demonstration of disseminated lesions in the white
particular HLA molecule coding for the beta chain of the DR matter of the brain and spinal cord by magnetic resonance
subregion, namely DRB1*1501.123 Environmental factors that imaging (MRI), and exclusion of other possible causes. Sev-
appear to be associated with MS include reduced exposure to eral laboratory tests in combination are used to support the
sunlight, vitamin D deficiency, and cigarette smoking.124 diagnosis.123,124 Immunoglobulins are increased in the
spinal fluid in 75% to 90% of patients with MS, producing Axon
two or more distinct bands on protein electrophoresis that
are not seen in the serum. These bands are referred to as
oligoclonal and can be identified by isoelectric focusing with
immunoblotting, a more sensitive technique than protein
electrophoresis.127 The IgG index, a calculated ratio of cere-
bral spinal fluid (CSF) IgG/albumin ÷ serum IgG/albumin,
is typically elevated and may also be used in making a di-
agnosis, even though it is not specific for MS. Although
there is not one specific antibody that is diagnostic for MS,
a large percentage of patients produce antibody directed
against a myelin basic protein peptide. Other antibodies are
directed against components of oligodendrocytes and
against myelin membranes.125

Myasthenia Gravis (MG) ACh

Myasthenia gravis (MG) is an autoimmune disease that affects

B
the neuromuscular junction. It is characterized by weakness
and fatigability of skeletal muscles. It is fairly common, with a AChR
A
prevalence of 2 to 7 people per 10,000.128 The disease is het-
erogeneous in terms of its age of onset and gender involvement. Na"
Early-onset MG (EOMG) occurs before the age of 40 and af- Postsynaptic membrane
fects predominantly females, whereas the late-onset form of Mechanism of immunologic injury in MG.
the disease (LOMG) occurs after the age of 40 and is seen more (A) Normal nerve impulse transmission: Acetylcholine (Ach) is
often in males.129,130 released from axon vesicles and binds to receptors (AchR) on the
In MG, antibody-mediated damage to the acetylcholine re- postsynaptic membrane, opening channels, allowing sodium ions
ceptors in skeletal muscle or to other proteins in the neuro- to enter. (B) In MG, antibodies to the AChRs are formed, blocking
muscular junction leads to progressive muscle weakness. Early transmission of nerve impulses.
signs are ptosis (drooping of the eyelids), diplopia (double vi-
sion), and the inability to retract the corners of the mouth,
often resulting in a snarling appearance.128,131 In the majority and in the clustering of ACHRs on the muscle cell membrane,
of patients, the disease progresses to a generalized form that which enhances the transmission of the signals from the nerve
involves more muscle groups.131 In patients with generalized cells.132 Consequently, there is fragmentation of the postsynap-
MG, muscle weakness is most noticeable in the upper limbs.131 tic ACHR clusters, resulting in muscle weakness and atrophy.
They can also experience difficulty in speaking, chewing, and Symptoms are usually severe, involving the facial, bulbar, and
swallowing and may be unable to maintain support of the respiratory muscles. About 2% of patients with generalized MG
trunk, neck, or head.106 If respiratory muscle weakness occurs, have antibodies against LRP4, a lipoprotein involved in the ac-
it can be life threatening.131 Onset of symptoms can be acute tivation of MuSK. These patients are typically young females
or they may develop and worsen over time. that have a mild form of the disease.132
The thymus also appears to play a role in the autoimmune
process of MG. Thymic hyperplasia is common in EOMG pa-
Approximately 80% to 85% of patients have antibody to tients with ACHR antibodies.129,132 The follicles in the thymus
acetylcholine (ACH) receptors (ACHR), which appears to con- expand and contain ectopic germinal centers with autoantibody-
tribute to the pathogenesis of the disease by three mecha- producing B cells. In addition, about 10% to 15% of patients
nisms.128 Normally, ACH is released from nerve endings to with LOMG have a thymoma, a tumor of the thymus that may
generate an action potential that causes the muscle fiber to contain autoreactive T cells. It is thought that inflammation of
contract. When the antibody combines with the receptor site, the thymus gland may be triggered by persistent activation of
binding of ACH is thought to be blocked (Fig. 15–11). In ad- TLRs by viruses such as EBV.129 The autoreactive response is
dition, the antibody can interact with complement to damage thought to be perpetuated by defective immunoregulatory
the postsynaptic muscle membrane and can promote rapid mechanisms involving an imbalance between Th17 cells and
endocytosis of the ACHRs, resulting in reduced numbers on Tregs, resulting in increased production of proinflammatory
the muscle cell membranes.128,129 cytokines and B-cell growth factors.
Patients lacking anti-ACHR may produce antibodies to Genetic factors have been implicated in susceptibility to
other proteins involved in the neuromuscular junction. About MG. The HLA haplotype, A1, B8, DR3 has a strong association
4% of patients, mostly young females, have antibodies against with EOMG, whereas the HLA antigens B7 and DR2 are more
muscle-specific kinase (MuSK), an enzyme that plays an im- likely to appear in LOMG, and HLA-DR14-DQ5 may increase
portant role in the development of the neuromuscular junction susceptibility to MuSK antibody production in MG.129
 primarily affects two age groups—men in their 30s and men
Most patients with MG can have a good quality of life with and women in their 60s and 70s.135
appropriate treatment. Anticholinesterase agents to prevent The clinical presentation of patients varies, but most pa-
destruction of the neurotransmitter, acetylcholine, are used as tients initially experience fatigue and malaise followed by
the main therapy.128 Thymectomy should be performed on clinical signs of kidney involvement such as edema and hy-
patients who have a thymoma.131 If these treatments are not pertension, which can rapidly progress to acute renal failure
effective, immunosuppression is recommended. Treatment if left untreated.135,136 Some patients develop chronic renal
generally begins with high doses of corticosteroid drugs fol- failure that requires lifetime hemodialysis or kidney trans-
lowed by other immunosuppressive drugs such as azathio- plantation. About 60% to 70% of patients with Goodpasture’s
prine or mycophenolate mofetil, to maintain the response.133 syndrome have pulmonary involvement and exhibit symp-
Plasmapheresis or intravenous immunoglobulin can be ad- toms such as cough, shortness of breath, and hemoptysis
ministered to patients in crisis. Biological agents such as mon- (coughing up blood).135
oclonal antibodies or fusion proteins targeted to specific Standard treatment of Goodpasture’s syndrome involves ad-
components of the immune system involved in the pathogen- ministration of high dose corticosteroids to stop inflammation,
esis of MG offer hope to MG patients who are unresponsive followed by immunosuppressive drugs such as cyclophos-
to conventional therapies.133 phamide to inhibit further production of autoantibodies. In
addition, plasmapheresis is performed to remove circulating
autoantibodies. Prompt initiation of therapy is important, be-
Laboratory tests are available to detect autoantibodies in sera cause symptoms can be life threatening. If started early, this
from patients with MG. The most commonly used procedure therapy is successful in preventing acute renal failure in most
is a radioimmunoprecipitation (RIPA) assay for antibody to the patients.136,137 However, the long-term prognosis is poor, with
ACHR, which is based on precipitation of the patient’s antibody many patients developing end-stage renal disease and more
with ACHRs isolated from human muscle.134 The complex is than half of patients dying within 2 years of diagnosis.136
detected with a radio-labeled snake venom called α-bungaro-
toxin, which binds with high affinity to a different site on the
receptors. This assay is sensitive and can be used to determine Although little is known about the circumstances that trigger
antibody titers. A similar RIPA method using 125I-labeled the autoimmune response in Goodpasture’s syndrome, there is
human MuSK is used to detect antibody to MuSK. Sensitivity a strong genetic association, as 70% to 80% of patients carry
of the method has been increased by using a two-step RIPA in the HLA-DRB1-15 antigen.137 Exposure to cigarette smoke and
which antibodies are isolated by an affinity purification proce- organic solvents has also been implicated in disease pathogen-
dure using Sepharose beads containing immobilized antigen esis.136,137 The autoantibodies produced in Goodpasture’s
before performing the steps of the RIPA.134 This method in- syndrome are known to be specifically directed against the
creases the likelihood of detecting antibodies present in low noncollagenous domain of the alpha-3 chain of type IV colla-
concentration. gen.136,137 This autoantibody reacts with collagen in the
Because assays using radioactive materials are not conve- glomerular or alveolar basement membranes and causes dam-
niently used in the routine clinical laboratory, there has been age by type II hypersensitivity.136,137 Complement binding to
an attempt to develop alternate methods. One approach is to the immune deposits attracts neutrophils, which mediate in-
use immunofluorescence cell-based assays in which patient jury to the membranes by releasing chemically reactive oxygen-
serum is incubated with HEK293 cells expressing all four containing molecules and proteolytic enzymes. These immune
ACHR subunits. This highly sensitive assay can detect antibod- reactants progressively destroy the renal tubular, glomerular,
ies directed toward ACHR clusters in patients that were previ- and pulmonary alveolar basement membranes. Loss of mem-
ously classified as seronegative by RIPA.134 Other assays that brane integrity results in leakage of blood and proteins into
have been developed include ELISA, luciferase immunopre- the urine.
cipitation, and fluorescence immunoprecipitation assays
(FIPA). FIPA uses ACHR subunits or MuSK antigens labeled
with green fluorescent protein to detect patient antibodies and
Laboratory evidence of renal involvement includes gross or mi-
has a sensitivity that is similar to RIPA.134
croscopic hematuria, proteinuria, a decreased 24-hour creati-
nine clearance, and elevated blood urea and serum creatinine
Goodpasture’s Syndrome levels.136 Abnormally shaped RBCs and casts can be found in
Goodpasture’s syndrome is characterized by the presence of the urine sediment. In those patients with pulmonary involve-
autoantibody to an antigen in the basement membranes in the ment, decreased total lung capacity and increased uptake of
glomeruli of the kidneys and alveoli of the lungs. The basement carbon monoxide is evident. An iron deficiency anemia with
membranes are composed of a thin, fibrous matrix that sepa- decreased hemoglobin and hematocrit can develop if pul-
rates the epithelial cell layer within these organs from under- monary hemorrhage is severe. The ESR and CRP level may be
lying connective tissue. Originally identified by Ernest normal or increased.
Goodpasture in 1919, Goodpasture’s syndrome is a rare disor- Circulating antibodies to the GBM can be detected in about
der that is found mainly in Caucasians of European origin. It 87% of patients.136 These antibodies can be identified by IIF,
ELISA, or Western blot. The IIF assay, long held as the stan-
dard, uses frozen kidney sections that are incubated with pa- immune response have been observed for several autoim-
tient serum and then overlaid with a fluorescein-labeled mune diseases. Other factors, including sex hormones, tis-
anti-IgG. Results are often hard to interpret and there is a high sue injury, and exposure to microbial infections, are
percentage of false positives and false negatives.138 Commer- thought to trigger the development of autoimmunity in
cially developed ELISAs, which use the alpha-3 subunit to de- genetically susceptible individuals.
tect antibody, are also available and have a sensitivity between • Infectious microorganisms are believed to trigger autoim-
70% and 100%, depending on the source of antigen substrate. mune responses in a variety of ways, including molecular
High antibody titers are usually associated with rapidly pro- mimicry (a resemblance to self-antigens), epitope spread-
gressing disease.135 The Western blot technique detects anti- ing (induction of a local inflammatory response that affects
bodies to the Goodpasture basement membrane antigen as well immune reactivity to unrelated antigens), and presence of
as other human α chain proteins that have been separated by superantigens that can bind to class II MHC molecules
polyacrylamide gel electrophoresis, followed by transfer to ni- and several T-cell receptors, regardless of their antigen
trocellulose paper for immunoblotting. Western blot is a highly specificity.
specific test that can be used for confirmation and minimiza- • Autoimmune diseases can be classified as organ specific
tion of false-positive results.135 Between 20% and 35% of pa- or systemic, depending on whether tissue destruction is
tients are also positive for ANCAs, which are usually specific localized or affects multiple organs. SLE, RA, and GPA are
for myeloperoxidase and exhibit the perinuclear staining pat- examples of systemic diseases, whereas Hashimoto’s thy-
tern on IIF.135,137 ANCAs may be detectable months to years roiditis, Graves disease, type 1 diabetes mellitus, celiac
before anti-GBM and symptoms are evident. disease, autoimmune hepatitis, primary biliary cirrhosis,
Histological analysis is important for confirmation of the multiple sclerosis, myasthenia gravis, and Goodpasture’s
diagnosis and for assessment of tissue damage.139 Tissue- syndrome are considered organ-specific diseases.
bound anti-GBM can be detected by performing direct im- • Specific autoantibodies are strongly associated with the pres-
munofluorescence on biopsy sections from kidney or lung ence of certain autoimmune diseases and are useful in their
specimens.136,137,139 In patients with renal disease, these an- diagnosis. For example, anti-dsDNA antibodies are found
tibodies are indicated by formation of a smooth, linear, rib- in SLE, anti-CCP (cyclic citrullinated proteins) antibodies
bonlike pattern of fluorescence along the GBM. In contrast, are seen in RA, and antibodies against the thyroid-stimulat-
glomerulonephritis caused by other autoimmune diseases ing hormone receptor are specific for Graves disease.
shows a granular pattern of immunofluorescence caused by • Antinuclear antibodies (ANAs) are found in the majority
nonspecific deposition of immune complexes in the glomeruli. of patients with SLE and in a significant number of pa-
Examination of renal biopsy tissue also reveals crescent for- tients with other connective tissue diseases. The gold stan-
mations of inflammatory macrophages in the glomeruli. In pa- dard in ANA testing is an IIF test using the human
tients with pulmonary involvement, linear IgG staining of the epithelial cell line HEp-2 as the substrate. Some of the
alveolar cell walls can be seen on direct immunofluorescence main fluorescent patterns observed in this test are homo-
of lung biopsy tissue or bronchial washings. Thus, laboratory geneous, peripheral, speckled, nucleolar, and centromere.
testing plays a key role in differentiating Goodpasture’s syn- Each pattern is correlated with the presence of certain
drome from other diseases that can cause similar symptoms ANAs and should be followed up by confirmatory tests to
and in facilitating an early diagnosis that can lead to prompt more specifically characterize the antibodies.
treatment and better clinical outcomes. • Rheumatoid factor is an autoantibody directed against the
Fc portion of IgG molecules. It is found in the majority of
patients with rheumatoid arthritis, but is not specific for
SUMMARY the disease because it is also present in a significant num-
ber of patients with other autoimmune diseases involving
• Autoimmune diseases result from a loss of self-tolerance, a the connective tissues.
delicate balance set up in the body to restrict the activity of • Antineutrophil cytoplasmic antibodies (ANCAs) are
T and B lymphocytes. Immunologic tolerance is achieved strongly associated with autoimmune syndromes involv-
at two levels. Central tolerance affects potentially reactive ing vasculitis. ANCAs are routinely detected by IIF using
B and T cells as they mature in the bone marrow and thymus, ethanol- or formalin-fixed leukocytes as the substrate.
respectively, whereas peripheral tolerance occurs in the sec- Two patterns of fluorescence can result: c-ANCA, a diffuse,
ondary lymphoid organs. granular staining of the cytoplasm of the neutrophils,
• Autoimmune disease is thought to result from complex mainly caused by antibodies against PR3 and seen in the
interactions between the genetic makeup of an individ- vast majority of patients with active systemic GPA; and
ual, exposure to environmental factors, and defects in im- p-ANCA, characterized by fluorescence surrounding the
mune regulation. Associations between certain HLA types nuclear lobes of ethanol-fixed neutrophils, caused by
or polymorphisms in non-MHC genes involved in the antibodies to positively charged antigens such as MPO.
CASE STUDIES
1. A 25-year-old female consulted her physician because 2. A 40-year-old female went to her doctor because she was
she had been experiencing symptoms of weight loss, feeling tired all the time. She had gained about 10 pounds
joint pain in the hands, and extreme fatigue. Her labo- in the last few months and exhibited some facial puffi-
ratory results were as follows: RF rapid slide test positive ness. Her thyroid gland was enlarged and rubbery. Labo-
at 1:10; ANA rapid slide test positive at 1:40; RBC 3.5 ratory results indicated a normal RBC and WBC count,
1012 per L (normal is 4.1 to 5.1 1012 per L); WBC but her FT4 level was decreased and an assay for antithy-
count 5.8 109 per L (normal is 4.5 to 11 109 per L). roglobulin antibody was positive.
Questions Questions
a. What is a possible explanation for positive results on a. What condition do these results likely indicate?
both the RF test and the ANA test? b. What effect do antithyroglobulin antibodies have on
b. What is the most likely cause of the decreased RBC thyroid function?
count? c. How can this condition be differentiated from Graves
c. What further testing would help the physician distin- disease?
guish between RA and SLE?

REVIEW QUESTIONS
1. All of the following may contribute to autoimmunity 5. A peripheral pattern of staining of the nucleus on IIF is
except caused by which of the following antibodies?
a. clonal deletion of self-reactive T cells. a. Anti-Sm antibody
b. molecular mimicry. b. Anti-SSA/Ro antibody
c. increased expression of class II MHC antigens. c. Centromere antibody
d. polyclonal activation of B cells. d. Anti-dsDNA

2. Which of the following would be considered an 6. Which of the following would be considered a signifi-
organ-specific autoimmune disease? cant finding in Graves disease?
a. SLE a. Increased TSH levels
b. RA b. Antibody to TSHR
c. GPA c. Decreased T3 and T4
d. Hashimoto’s thyroiditis d. Antithyroglobulin antibody

3. SLE can be distinguished from RA on the basis 7. Destruction of the myelin sheath of axons caused by the
of which of the following? presence of antibody is characteristic of which disease?
a. Joint pain a. MS
b. Presence of antinuclear antibodies b. MG
c. Immune complex formation with activation c. Graves disease
of complement d. Goodpasture’s syndrome
d. Presence of anti-dsDNA antibodies
8. Blood was drawn from a 25-year-old woman with
4. Which of the following would support a diagnosis suspected SLE. A FANA screen was performed and
of drug-induced lupus? a speckled pattern resulted. Which of the following
a. Antihistone antibodies actions should be taken next?
b. Antibodies to Smith antigen a. Report out as diagnostic for SLE
c. Presence of RF b. Report out as drug-induced lupus
d. Antibodies to SS-A and SS-B antigens c. Perform an assay for specific ANAs
d. Repeat the test
 9. Which of the following is a mechanism used to 13. A technologist performs an IIF test for ANCAs and
achieve peripheral tolerance? observes that there is an intense fluorescent staining
a. Negative selection of autoreactive T cells in the of the nuclear lobes of the neutrophils. How can this
thymus type of staining be differentiated from a peripheral
b. Apoptosis of autoreactive B cells in the bone marrow ANA pattern?
c. Editing of B-cell receptors that weakly recognize a. Perform the test on formalin-fixed leukocytes
self-antigens in the bone marrow b. Perform IIF with HEp-2 cells
d. Lack of a costimulatory signal to autoreactive c. Perform an ELISA for ANCAs
T cells in the lymph nodes d. All of the above

10. Epitope spreading refers to 14. A 20-year-old woman made an appointment to see her
a. post-translational modifications to self-antigens. physician because she was experiencing intermittent
b. modifications in gene expression that are not diarrhea. Laboratory testing revealed that she also had
caused by changes in DNA sequence. an iron deficiency anemia. To determine if the patient
c. expansion of the immune response to unrelated has celiac disease, her doctor should order which of
antigens. the following laboratory tests?
d. cross-reaction of the immune response to a a. Anti-tTG
pathogen with a similar self-antigen. b. Antigliadin
c. Antigluten
11. Anti-CCP (cyclic citrullinated proteins) is specifically d. All of the above
associated with which autoimmune disease?
a. RA 15. Antimitochondrial antibodies are strongly associated
b. MG with which disease?
c. Autoimmune hepatitis a. Autoimmune hepatitis
d. Goodpasture’s syndrome b. Celiac disease
c. Primary biliary cirrhosis
12. Which autoantibodies are strongly associated with gran- d. Goodpasture’s syndrome
ulomatosis with polyangiitis (Wegener’s granulomatosis)?
a. ANA
b. ANCA
c. AMA
d. SMA
 Transplantation
Immunology

After finishing this chapter, you should be able to: HISTOCOMPATIBILITY SYSTEMS
1. List the histocompatibility systems relevant to clinical transplantation. Major Histocompatibility Complex
(MHC) Antigens
2. Compare the mechanisms of direct and indirect alloantigen recognition.
Minor Histocompatibility Antigens
3. Distinguish between an allograft, autograft, xenograft, and syngeneic
(mHAs)
graft (isograft).
MHC Class I-Related Chain A (MICA)
4. Compare the immunologic mechanisms involved in hyperacute,
Antigens
acute, and chronic graft rejection.
ABO Blood Group Antigens
5. Identify risk factors for graft-versus-host disease (GVHD) and the
types of grafts in which this mechanism of rejection could occur. Killer Immunoglobulin-Like Receptors
(KIRs)
6. List the major classes of immunosuppressive agents and their effects
on the immune system. Self-Antigens
7. Explain the principles of laboratory methods for human leukocyte ALLORECOGNITION
antigen (HLA) typing. TRANSPLANT REJECTION
8. Describe laboratory methods for detecting and identifying Hyperacute Rejection
HLA antibodies (i.e., antibody screening, identification, and Acute Rejection
crossmatching).
Chronic Rejection
9. Deduce the suitability of a possible donor for a transplant recipient,
GRAFTVERSUSHOST DISEASE GVHD
based on results of HLA typing and antibody identification.
IMMUNOSUPPRESSIVE AGENTS
10. Describe the nomenclature used for HLA antigens and alleles.
CLINICAL HISTOCOMPATIBILITY
TESTING
HLA Typing
HLA Phenotyping
HLA Genotyping
HLA Antibody Screening,
Identification, and Crossmatching
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Accelerated rejection Crossmatch HLA typing Minor histocompatibility
Acute graft-versus-host Direct allorecognition Human leukocyte antigens antigens (mHAs)
disease (GVHD) Graft-versus-host disease (HLAs) Mixed lymphocyte reaction
Acute rejection (AR) (GVHD) Hyperacute rejection Percent panel reactive
Allograft Haplotypes Immunosuppressive agents antibody (%PRA)
Autograft HLA antibody screen Indirect allorecognition Polymorphism
Chronic rejection HLA genotype Isograft Syngeneic graft
Complement-dependent HLA matching Major histocompatibility Xenograft
cytotoxicity (CDC) HLA phenotype complex (MHC)

Transplantation is a potentially lifesaving treatment for end-stage closely linked alleles on a single chromosome; for example,
organ failure, cancers, autoimmune diseases, immune deficien- HLA-A1, HLA-Cw7, HLA-B8, HLA-DR3, and HLA-DQ2. Off-
cies, and a variety of other diseases. In 2015, 30,900 solid- spring receive one maternal and one paternal HLA haplotype.
organ (kidney, pancreas, liver, heart, lung, small intestine) Based on Mendelian inheritance, there is a 25% chance that any
transplants were performed in the United States.1 Today, more two siblings will inherit the same two haplotypes (i.e., are HLA
than 50,000 hematopoietic stem cell (HSC) transplants are identical), a 50% chance of them being HLA haploidentical
performed worldwide each year for a variety of indications, (i.e., share one of two HLA haplotypes), and a 25% chance of
including cancer, autoimmune disease, immunodeficiencies, and them being HLA nonidentical (i.e., share neither HLA haplotype).
other diseases.2 The number of transplants performed is a testa- Recombination within the HLA region can take place, resulting
ment to the numerous developments over the past few decades in the inheritance of unexpected haplotypic combinations; how-
in patient management pre- and post-transplant and in the tech- ever, this occurs in less than 1% of families that are HLA typed.
nologies for organ or stem cell acquisition and sharing. Of critical HLA proteins are heterodimeric molecules consisting of two
importance has been the growing knowledge of the immuno- different polypeptide chains chemically bound to each other (see
logic mechanisms of graft rejection and graft-versus-host dis- Chapter 2). They are also members of the immunoglobulin su-
ease (GVHD), in particular the role of human leukocyte perfamily, which share structural similarities with immunoglob-
antigens (HLAs) and the development of pharmacological ulin molecules.
agents that promote graft survival by interfering with various Class I proteins are the product of the HLA-A, HLA-B,
components of the immune system. and HLA-C genes and are expressed on the cell surface
The HLA system is the strongest immunologic barrier to
successful allogeneic organ and HSC transplantation (see
Chapter 2 for details). It consists of cell surface proteins that
play a central role in thymic education of T lymphocytes,
initiation of adaptive immune responses, and regulation of
other immune system components. HLA proteins are found A1 A2 A3 A24
Cw7 Cw5 Cw8 Cw6
on the surface of almost all nucleated cells and are antigeni-
B8 B44 B14 B17
cally very diverse in the human population. Therefore, trans-
DR3 DR15 ! DR7 DR4
plantation of a solid organ or HSCs into an allogeneic host DQ2 DQ6 DQ2 DQ7
is likely to result in graft rejection in the absence of immuno- a b c d
suppressive therapy.

Histocompatibility Systems
A1 A3 A1 A24 A2 A3 A2 A24
Major Histocompatibility Complex Cw7 Cw8 Cw7 Cw6 Cw5 Cw8 Cw5 Cw6
(MHC) Antigens B8 B14 B8 B17 B44 B14 B44 B17
DR3 DR7 DR3 DR4 DR15 DR7 DR15 DR4
The classical (transplant) antigens, also known as major histo- DQ2 DQ2 DQ2 DQ7 DQ6 DQ2 DQ6 DQ7
compatibility complex (MHC) antigens, are composed of the a c a d b c b d
class I and class II proteins. Class I proteins include HLA-A,
HLA genes are linked and inherited in Mendelian
HLA-B, and HLA-C; class II proteins consist of HLA-DR, HLA-DQ, fashion as haplotypes. One paternal (a or b) and one maternal
and HLA-DP. HLA proteins are encoded by a set of closely (c or d) haplotype is passed to each offspring. Four different combi-
linked genes located on the short arm of chromosome 6 within nations of haplotypes are possible in offspring. Elucidation of haplo-
the MHC region. HLA genes are inherited as haplotypes from type sharing between siblings is an important assessment in the
parental chromosomes (Fig. 16–1). A haplotype is a group of search for a transplant donor. (Figure courtesy of John Schmitz.)
covalently bound to β2–microglobulin. Class I heterodimers Table 16–2 Listing of Individual HLA Antigens*
are codominantly expressed on virtually all nucleated cells. HLA-A HLA-B HLA-C HLA-DR HLA-DQ
Class II heterodimers are the products of the HLA-D region
1 66 5 44 64 1 1 1
genes. Class II proteins are codominantly expressed prima-
rily on antigen-presenting cells (APCs; e.g., dendritic cells, 2 68 7 45 65 2 2 2
monocytes, macrophages, B lymphocytes).
3 69 8 46 67 3 3 3
As discussed in Chapter 2, HLA proteins have critical roles
in the development and functioning of the innate and adaptive 9 74 12 47 70 4 4 4
immune systems. They serve as recognition elements for anti- 10 80 13 48 71 5 5 5
gen receptors on T lymphocytes, thus initiating adaptive im-
mune responses. In addition, they serve as ligands for 11 14 49 72 6 6 6
regulatory receptors on natural killer (NK) cells in the innate 19 15 50 73 7 7 7
immune response. The CD8 molecule on cytotoxic T lympho-
23 16 51 75 8 8 8
cytes interacts with class I HLA proteins, whereas the CD4 mol-
ecule on the T helper (Th) cell subset interacts with class II 24 17 52 76 9 9 9
HLA proteins. 25 18 53 77 10 10
A cardinal feature of the genes encoding HLA proteins is the
extensive degree of allelic polymorphism. Polymorphism refers 26 21 54 78 11
to the presence of two or more different genetic compositions 28 22 55 81 12
among individuals in a population. The HLA system is the most
29 27 56 82 13
polymorphic genetic system in humans; many HLA genes exist
in the population and numerous combinations of these genes 30 35 57 14
are possible in individuals (see Tables 16–1 and 16–2). This
31 37 58 15
degree of polymorphism is believed to have resulted from the
survival benefit of initiating an immune response to a broad 32 38 59 16
array of peptides from an innumerable array of pathogenic mi- 33 39 60 17
crobes that populations encounter. Although this has success-
fully enabled populations to survive infectious challenges, it 34 40 61 18
severely restricts the ability to transplant foreign tissues or cells 36 41 62
between any two individuals because the HLA proteins are im-
43 42 63
munogenic and elicit robust allogeneic immune responses.
*Note: HLA antigen names begin with the prefix “HLA-”, followed by the

Minor Histocompatibility gene locus (A, B, C, or D) and the antigen number (e.g., HLA-A2). A “w” was
originally placed after newly identified antigens at International Histocompati-
Antigens (mHAs) bility Workshops. The “w” has been retained for HLA-C antigen names to
differentiate them from complement proteins (e.g., HLA-Cw1).
Researchers identified a second set of transplantation anti-
gens based on studies in mice and humans. These studies
demonstrated tissue rejection in MHC-identical transplants
and development of GVHD in HLA-identical sibling HSC
transplants.3 The scientists conducting these studies also ob-
Table 16–1 Approximate Number of HLA served that a “slower” rejection pace was mediated by these
Antigens and Alleles Defined transplantation antigens, thus their name—minor histo-
at the Classical Transplant Loci* compatibility antigens (mHAs).
HLA LOCUS # ANTIGENS # ALLELES
mHAs are non-HLA proteins that demonstrate variation in
the amino acid sequence between individuals. Both X-linked
A 28 3,356 and autosomally encoded mHAs have been identified. Trans-
B 62 4,179 planting one individual’s tissue or cells containing a polymor-
phic variant of one of these proteins into another individual
C 10 2,902
possessing a different polymorphic variant can induce a recip-
DRB1 24 1,860 ient’s immune response to the donor variant. CD4 or CD8
DQB1 9 900 T cells recognize the variant protein in an MHC-restricted fash-
ion and mediate the immune response. This response is anal-
*Note: The number of HLA alleles identified has increased tremendously in ogous to the reaction to a microbial antigen. Several mHAs
recent years because of the availability of improved molecular techniques. have been identified, including proteins encoded by the male
The numbers in this table were current as of January 2016. To view the
Y chromosome, proteins for which the recipient has a homozy-
most up-to-date information about the HLA alleles discovered, access the
gous gene deletion, proteins that are autosomally encoded, and
website: http://hla.alleles.org/nomenclature/stats.html.
proteins that are encoded by mitochondrial DNA.3
MHC Class I-Related Chain receptors are not engaged and a loss of negative regulatory ac-
tivity occurs, resulting in NK cell activation.
A (MICA) Antigens The regulatory role of KIRs has been exploited in haploiden-
The MHC class I-related chain A (MICA) gene encodes a cell tical stem cell transplantation.8 Stem cell donors have been
surface protein that is involved in gamma or delta T-cell re- selected for recipients who lack a corresponding class I MHC
sponses. MICA is polymorphic, with over 50 allelic variants. protein for the donor’s inhibitory KIR type. This results in al-
MICA proteins are expressed on endothelial cells, keratinocytes, loreactivity by NK cells that repopulate the recipient after trans-
fibroblasts, epithelial cells, dendritic cells, and monocytes, but plant. These alloreactive NK cells have been shown to mediate
they are not expressed on T or B lymphocytes.4 As such, MICA a graft-versus-leukemia (GVL) reaction and prevent relapse after
proteins could serve as targets for allograft immune responses. transplantation for certain types of hematologic malignancies.
Antibodies to MICA antigens have been detected in as many
as 11% of kidney-transplant patients and are associated with Self-Antigens
rejection episodes and decreased graft survival.5
In addition to these well-described alloantigen systems, hu-
moral immune responses to self-antigens in transplant recipi-
ABO Blood Group Antigens ents have been associated with poor transplant outcomes,
The ABO system is the only blood group system that affects clin- although a direct causal relationship has yet to be firmly estab-
ical transplantation. ABO blood group incompatibility is a bar- lished. Among the several proteins to which antibody has been
rier to solid-organ transplantation because these antibodies can described post-transplantation are angiotensin II type-1 recep-
bind to the corresponding antigens that are expressed on the tor,9 vimentin, K-alpha1 tubulin, collagen-v, and myosin.10
vascular endothelium. Binding activates the complement cas-
cade, which can lead to very rapid rejection of the transplanted Allorecognition
organ. This phenomenon, known as hyperacute rejection, oc-
curs within minutes to hours after the vascular supply to the Transplantation of cells or tissues is classified by the genetic
transplanted organ is established (see Transplant Rejection). Anti- relatedness of the donor and the recipient. An autograft is the
A or anti-B antibodies develop in individuals lacking the corre- transfer of tissue from one area of the body to another of the
sponding blood group antigens. As such, recipient–donor pairs same individual. A syngeneic graft (also known as an iso-
must be ABO identical or compatible to avoid this adverse out- graft) is the transfer of cells or tissues between individuals of
come. For example, an individual of blood group A will possess the same species who are genetically identical, for example,
anti-B antibodies and can thus receive an organ only from an identical twins. An allograft is the transfer of cells or tissue
ABO type A or type O donor. Likewise, a B-expressing individual between two genetically disparate individuals of the same
has anti-A antibodies and can receive an organ only from an ABO species. Finally, a xenograft is the transfer of tissue between
type B or type O donor. two individuals of different species. Most transplants fall into
Transplantation approaches using plasma exchange and in- the category of allografts. The HLA disparity between donor
travenous immunoglobulin administration have allowed suc- and recipient that occurs with allografts and xenografts will
cessful transplantation of kidneys from ABO-incompatible result in a vigorous cellular and humoral immune response to
donors. The procedures reduce the ABO antibody to levels that the foreign MHC antigens. This response is the primary stim-
significantly lower the risk of hyperacute rejection.6 ulus of graft rejection.
The recipient’s immune system recognizes foreign HLA pro-
Killer Immunoglobulin-Like teins via two distinct mechanisms—direct and indirect al-
lorecognition (Fig. 16–2).11 In direct allorecognition, recipient
Receptors (KIRs) T cells bind and respond directly to foreign (allo) HLA proteins
Another polymorphic genetic system that affects allogeneic on graft cells. Although an individual T lymphocyte can recog-
transplantation is the killer immunoglobulin-like receptor nize self-HLA + peptide, foreign HLA proteins may mimic a self-
(KIR) system.7 KIRs are one of several types of cell surface mol- HLA + peptide complex because of similarities in the structure
ecules that regulate the activity of NK lymphocytes. The KIRs of the allo-HLA protein itself or to structural similarities of allo-
contain activating and inhibitory receptors that vary in number HLA protein + peptide. Evidence suggests that virus-specific
and type on any individual NK cell. The balance of signals re- T cells may be an important source of alloreactive cells.12 Either
ceived by the activating and inhibitory receptors regulates the way, direct allorecognition is characterized by a high frequency
activity of the NK cell (see Fig. 3–7). (up to 10%) of responding T cells11,13 compared with the re-
The ligands for the inhibitory KIRs have been defined as sev- sponder frequency in a typical T-cell response to a foreign anti-
eral of the class I MHC molecules, including specific HLA-A, gen. The mixed lymphocyte reaction (MLR) is an in vitro
HLA-B, and HLA-C proteins. Normally, an NK cell encounters correlate of direct allorecognition. In this assay, lymphocytes
self class I MHC proteins as it circulates in the body. This inter- from an individual needing a transplant are incubated with
action between MHC protein and the inhibitory KIR maintains lymphocytes from a potential donor that have been inactivated
the NK cells in a quiescent state. If an NK cell encounters a cell so they cannot proliferate. A disparity in the HLA-D antigens
with absent or decreased HLA class I expression, inhibitory found on the two populations of lymphocytes results in the
 A. Direct B. Indirect

Host Tc Host APC

Graft MHC

Perforins TCR Host MHC II or

granzymes
CD8 TCR CD4

Graft Host Th
cell

Cytokines

Direct versus indirect allorecognition.

(A) In direct allorecognition, cytotoxic T cells from the host
bind directly to foreign HLA (MHC I) proteins on graft cells.
Cell-mediated cytotoxicity
(B) In indirect allorecognition, APCs from the host present antibody-mediated reactions
foreign MHC I or MHC II antigens to CD4+ Th cells, which
produce cytokines that stimulate graft rejection. Apoptosis

proliferation of the recipient cells, which can be quantitated by hyperacute rejection. Antibodies to these antigens may be
incorporation of radio-labeled (3H) thymidine into the DNA of present as a result of blood transfusion, prior transplantation,
the proliferating cells. The amount of radioactivity taken up by or exposure of a pregnant woman to fetal antigens of paternal
the cells increases in proportion to the amount of cell prolifera- origin. Binding of preformed antibodies to the alloantigens
tion. Thus, a high level of radioactivity indicates that the recip- activates the complement cascade and clotting mechanisms
ient’s T cells have divided in response to different HLA-D and leads to thrombus formation. The result is ischemia and
antigens on a potential donor’s cells and that such a donor would necrosis of the transplanted tissue.14
be more likely to stimulate graft rejection. Hyperacute rejection is seldom encountered in clinical
Indirect allorecognition is the second pathway by which transplantation. Donor–recipient pairs are chosen to be ABO
the immune system recognizes foreign HLA proteins.13 Indirect identical or compatible and patients awaiting transplantation
allorecognition involves the uptake, processing, and presenta- are screened for the presence of preformed HLA antibodies. In
tion of foreign HLA proteins by recipient APCs to recipient addition, the absence of donor HLA-specific antibodies is con-
T cells. It is analogous to the normal mechanism of recognition firmed before transplant by the performance of a crossmatch
of foreign antigens. Indirect allorecognition may play a predom- test (see HLA Antibody Screening, Identification, and Crossmatch-
inant role in induction of alloantibody and chronic rejection.12 ing). These approaches have virtually eliminated hyperacute
The effector responses against transplanted allogeneic tissue rejection episodes.
include direct cytotoxicity, delayed-type hypersensitivity re- Some individuals possess very low levels of donor-specific
sponses, and antibody-mediated mechanisms. Antibody can antibody in the pretransplant period. In these cases, antibody-
mediate antibody-dependent cellular cytotoxicity reactions and mediated rejection may take place over several days. This has
fix complement, resulting in cell death. Rejection episodes vary been termed accelerated rejection.11,15 Similar to hyperacute
in the time of occurrence and the effector mechanism that is rejection, accelerated rejection involves intravascular throm-
involved. The next section will cover three types of rejection: bosis and necrosis of donor tissue.
hyperacute, acute, and chronic.
Acute Rejection
Transplant Rejection Days to months after transplant, individuals may develop acute
rejection (AR). AR can be mediated by a cellular alloresponse
(ACR) or by donor-specific antibody (also known as antibody-
Hyperacute Rejection mediated response; AMR).14,16
As previously discussed, hyperacute rejection occurs within ACR is characterized by parenchymal and vascular injury.
minutes to hours after the vascular supply to the transplanted Interstitial cellular infiltrates contain a predominance of CD8+
organ is established. This type of rejection is mediated by pre- T cells as well as CD4 T cells and macrophages. CD8 cells
formed antibody that reacts with donor vascular endothe- likely mediate cytotoxic reactions to foreign MHC-expressing
lium. ABO, HLA, and certain endothelial antigens may elicit cells, whereas CD4 cells likely produce cytokines and induce
delayed-type hypersensitivity (DTH) reactions. Antibody may mHAs are targeted. The infused T cells can mediate GVHD in
also be involved in acute graft rejection by binding to vessel several ways, including a massive release of cytokines because
walls and activating complement. The antibody induces trans- of large-scale activation of the donor cells by MHC-mismatched
mural necrosis and inflammation as opposed to the thrombo- proteins and by infiltration and destruction of tissue.
sis typical of hyperacute rejection. Diagnostic criteria include The incidence and severity of GVHD is related to the match
characteristic histological findings, deposition of the comple- status of the donor and recipient as well as other factors.20 The
ment protein C4d in the peritubular capillaries, and detection recipient’s medical team can take several approaches to reduce
of donor-specific HLA antibody.16 The development and ap- the incidence and severity of GVHD including immunosuppres-
plication of potent immunosuppressive drugs that target mul- sive therapy in the early post-transplant period and removal of
tiple pathways in the immune response to alloantigens has T lymphocytes from the graft. T-cell reduction, achieved via pu-
improved early graft survival of solid-organ transplants by rification of donor HSCs from the peripheral blood or bone
reducing the incidence of AR and by providing approaches for marrow collection, is very effective in lowering the incidence of
its effective treatment. GVHD, but it can also reduce the GVL effect of the infused cells
and increase the incidence of graft failure. The GVL effect is
Chronic Rejection similar to the GVH response but targets the recipient’s malignant
cells as opposed to healthy tissues of the recipients such as the
Chronic rejection results from a process of graft arteriosclerosis skin and gastrointestinal tract.
characterized by progressive fibrosis and scarring with narrow- Beyond 100 days post-transplant, patients may experience
ing of the vessel lumen caused by proliferation of smooth mus- chronic GVHD. This condition resembles autoimmune disease,
cle cells.17 Chronic rejection remains the most significant cause with fibrosis affecting the skin, eyes, mouth, and other mucosal
of graft loss after the first year post-transplant because it is not surfaces.
readily amenable to treatment. Several predisposing factors
affect the development of chronic rejection, including pro-
longed cold ischemia, reperfusion injury, AR episodes, and tox- Immunosuppressive Agents
icity from immunosuppressive drugs. Chronic rejection is also
thought to have an immunologic component, presumably a The list of agents employed to suppress antigraft immune re-
delayed-type hypersensitivity reaction to foreign HLA proteins.11 sponses in solid-organ and HSC transplantation is growing.
This is indicated in studies employing animal models of graft Immunosuppressive agents are used in several ways, includ-
arteriosclerosis in which mice lacking IFN gamma do not de- ing induction and maintenance of immune suppression and
velop graft arteriosclerosis. In addition, similar studies support treatment of rejection. Combinations of different agents are fre-
an important role for CD4 T cells and B cells in this process. quently used to prevent graft rejection. However, the immuno-
Cytokines and growth factors—secreted by endothelial cells, suppressed state (and graft survival) induced by these agents
smooth muscle cells, and macrophages activated by IFN comes at a price of increased susceptibility to infection, malig-
gamma—stimulate smooth muscle cell accumulation in the nancies, and other associated toxic side effects. There are sev-
graft vasculature. Alloantibody production likely contributes to eral classes of immunosuppressive agents:
the development of chronic rejection as well.18 Corticosteroids—Corticosteroids are potent anti-inflammatory
and immunosuppressive agents used for immunosup-
Graft-Versus-Host Disease (GVHD) pression maintenance. At higher doses, they are used to
treat AR episodes. Steroids act by blocking production
HSC transplants (and less commonly lung and liver transplants) and secretion of cytokines, inflammatory mediators,
are complicated by a unique allogeneic response—GVHD. In chemoattractants, and adhesion molecules. These activ-
this condition, lymphoid cells in the graft mount an immune ities decrease macrophage function and alter leukocyte-
response against the host’s histocompatibility antigens. Recipi- trafficking patterns. However, long-term use is associated
ents of HSC transplants for hematologic malignancies typically with several complications, including hypertension and
have depleted bone marrow before transplantation as a result diabetes mellitus.
of the chemotherapy used to treat the malignancy. The individ- Antimetabolites—Antimetabolites interfere with the matu-
ual receives an infusion of donor bone marrow or, more com- ration of lymphocytes and kill proliferating cells.21
monly, peripheral blood stem cells. The infused products often Azathioprine was the first such agent employed. It has
contain some mature T cells. These cells have several beneficial been replaced in large part by mycophenolate mofetil,
effects, including promotion of engraftment, reconstitution of which has a more selective effect on lymphocytes com-
immunity, and mediation of a graft-versus-leukemia effect. pared with azathioprine and thus fewer side effects.
However, these mature T cells may also mediate GVHD. Calcineurin inhibitors—Cyclosporine and FK-506 (tacrolimus)
Acute graft-versus-host disease (GVHD) occurs during are compounds that block signal transduction in T lympho-
the first 100 days postinfusion and manifests in the skin, gas- cytes, resulting in impaired synthesis of cytokines such
trointestinal tract, and liver.19 In mismatched allogeneic stem as IL-2, IL-3, IL-4, and interferon-gamma.21 Inhibition
cell transplantation, the targets of GVHD are the mismatched of cytokine synthesis blocks the growth and differentiation
HLA proteins, whereas in matched stem cell transplantation of T cells, impairing the antigraft response. Rapamycin
 (sirolimus)22 is an agent that inhibits T-cell proliferation by must also be considered when choosing a particular donor for
binding to specific intracellular proteins, including mam- any given patient, be it a solid-organ or stem cell transplant.
malian target of rapamycin (mTOR). mTOR is an intracel- For example, ABO compatibility and infectious disease status
lular molecule involved in cellular functions such as are important considerations in donor selection.
proliferation and motility.
Monoclonal antibodies—Several monoclonal antibodies HLA Phenotyping
that bind to cell surface molecules on lymphocytes are
The classic procedure for determining the HLA phenotype is
used at the time of organ transplant and to treat severe
the complement-dependent cytotoxicity (CDC) test. Panels
rejection episodes after transplantation. Two anti-CD25
of antisera or monoclonal antibodies that define individual or
monoclonal antibodies are available for use in transplant
groups of immunologically related HLA antigens are incubated
patients.23 Basiliximab and daclizumab both bind the
with lymphocytes from the person to be HLA typed in separate
CD25 (IL-2 receptor) and thus interfere with IL-2–mediated
wells of a microtiter plate. Each well of the plate contains a
T-cell activation. They may also deplete CD25-expressing
different antibody. It is important to note that multiple antisera
cells. An additional monoclonal antibody that targets the
are used for HLA typing. This requirement is based on the
CD52 receptor found on T and B lymphocytes is alem-
presence of both unique epitopes on HLA molecules (those
tuzumab, which may be used for induction therapy at
that define the phenotypic specificity of a specific HLA antigen)
the time of transplantation.23 A problem with some mon-
and public epitopes (epitopes that are present on more than
oclonal antibody preparations is that patients can de-
one unique HLA protein). Because responses to public epitopes
velop anti-mouse antibody that may interfere with the
are common, many sera must be used to define a pattern of
effectiveness of these agents. The potential for this prob-
reactivity that correlates with a specific HLA antigen. T and
lem to occur is being reduced with increased use of
B lymphocytes are used for HLA class I typing, whereas puri-
humanized and fully human monoclonal antibodies (see
fied B lymphocytes are used for HLA class II typing because
Chapters 5 and 25).
class II antigens are not found on most T cells. After incubation
Polyclonal antibodies—Two polyclonal anti–T-cell antibody
with the antisera, complement is added. Binding of antibody
preparations have been used as induction agents or to
occurs only if the lymphocytes express the HLA antigen tar-
treat severe rejection. Thymoglobulin is an antithymo-
geted by the antisera. A complement reagent derived from rab-
cyte antibody prepared in rabbits and ATGAM is a poly-
bit serum is added to each well. If the cells possess the HLA
clonal antiserum prepared from the immunization of
antigen defined by the antibody in that well, complement is
horses. Both are potent immunosuppressive agents that
activated and the cells are killed. A vital dye such as eosin red
deplete lymphocytes from the circulation. A drawback
or trypan blue is added to distinguish live cells from dead cells
associated with administration of polyclonal antibody
when they are viewed microscopically. The dead cells, whose
preparations is the development of serum sickness be-
membranes have been made more permeable, are able to take
cause of antibody responses to the foreign immunoglob-
up the dye and appear colored, whereas the live cells, whose
ulin (see Chapter 14).
membranes remain intact, cannot take up the dye and remain
colorless. The proportion of dead cells is estimated by micro-
Clinical Histocompatibility Testing scopic examination and scored according to the following
scale, established by the American Society for Histocompati-
Appreciation of the beneficial role of HLA matching11,24 and bility and Genetics (ASHI):
the detrimental role of antibody to HLA proteins15 on graft sur- • 1 = 0% to 10% cell death; negative
vival provided the impetus for development and application • 2 = 11% to 20% cell death; doubtful negative
of specialized testing to aid in the selection of the most appro- • 4 = 21% to 50% cell death; weak positive
priate donors for patients needing transplantation. Histocom- • 6 = 51% to 80% cell death; positive
patibility laboratories provide specialized testing for both • 8 = 81% to 100% cell death; strong positive
solid-organ and stem cell transplantation programs. Two main • 0 = unreadable
activities are carried out by these laboratories in support of
transplantation: HLA typing and HLA antibody screening and The principle of the CDC is illustrated in Figure 16–3. Using
identification. this assay, an extensive array of HLA antigens can be defined
(see Table 16–2).
The CDC method has several limitations for HLA typing.
HLA Typing Viable lymphocytes must be used, which demands timely per-
HLA typing is the phenotypic or genotypic identification of formance of the assay. Separation of T and B lymphocytes is
the HLA antigens or genes in a transplant candidate or donor. required for differentiation of class I versus class II antigens.
For clinical HLA testing, phenotypes or genotypes of the clas- In addition, the source of antisera for HLA typing is not always
sical transplant antigens or genes are determined (HLA-A, consistent or reliable. Thus, reagents can vary in quality or
HLA-B, HLA-Cw, HLA-DR, HLA-DQ). This information is used quantity over time. Finally, the level of resolution (i.e., the abil-
to find the most suitable donor–recipient combination from an ity to distinguish two closely related yet distinct HLA antigens)
immunologic standpoint. It must be stressed that other factors is limited. The limits of resolution don’t significantly affect the
 role of this technology for matching solid-organ donors and
recipients. However, for allogeneic stem cell transplantation, a
higher level of resolution is required. DNA-based (molecular)
HLA typing methods are now commonly employed in histo-
compatibility laboratories because their higher resolution,
reagent quality, and amenability to higher throughput formats
overcome the limitations of CDC-based methods.

HLA Genotyping
Molecular-based HLA genotyping methods use polymerase
chain reaction (PCR)-based amplification of HLA genes
followed by analysis of the amplified DNA to identify the
specific HLA allele or allele group (see Chapter 12).25 Three
DNA-based HLA typing methods are in use: polymerase
chain reaction with sequence-specific PCR (PCR-SSP),
PCR-sequence-specific oligonucleotide probe hybridization
(PCR-SSOP), and sequence-based typing (SBT). The most
common approaches for analysis involve PCR amplification
of HLA genes with panels of primer pairs, each of which
amplifies specific alleles or related allele groups (PCR-SSP).
Only those primer pairs that bind perfectly to the target gene
result in detection of an amplification product (Fig. 16–4).
Amplification is detected by agarose gel electrophoresis. The
HLA genotype is then identified by determining which
primers resulted in amplification.
A second common approach for HLA genotyping is to per-
form PCR-SSOP. This involves a single PCR reaction that will
amplify all HLA gene variants at a specific locus (referred to as
a generic amplification). The amplified gene is then subjected
to hybridization with a panel of DNA probes, each specific for
a unique HLA allele or allele group. Only those probes that
specifically hybridize to the amplified DNA will be detected.
The HLA genotype is determined by assessing which probes
hybridized (Fig. 16–5).
A third common method for HLA genotyping is SBT, which
involves sequencing of PCR-amplified HLA genes. SBT is typ-
ically carried out using Sanger dideoxy chain terminator se-
quencing. A generic amplification of the HLA gene of interest
is conducted, followed by a sequencing reaction using dideoxy
nucleotides. The dideoxy terminators are fluorescently labeled.
Incorporation into the synthesized DNA molecule is detected
using automated DNA sequencers with fluorescent detectors.
The sequence of the target gene is compared with an HLA se-
quence database to determine the specific HLA allele for the
patient. SBT for HLA typing is considered the gold standard
and is able to detect new allelic variants because it interrogates
all nucleotides in the amplified target region as opposed to
PCR-SSP and PCR-SSOP that target small stretches of previ-
ously defined nucleotides.
HLA genotyping overcomes the limitations of CDC-based
1 4 8 HLA phenotyping. Cells do not need to be viable in order to
Principle of the complement-dependent cytotoxicity obtain DNA for HLA genotyping. Typing reagents are chemi-
(CDC) test. cally synthesized; thus, there is no reliance on human donors
of antisera. HLA genotyping can provide varying levels of
resolution that can be tailored to the specific clinical need.
DNA-based typing can provide results at a level of resolution
 Amplification Allele-specific
controls product

TCATGA...

TGACTTGCATCGTGCATCT AGCTAGCTACAGTACTACATC
ACTGAACGTAGCACGTAGA TCGATCGATGTCATGATGTAG

...CTTGCAT

Amplication

TCATGA...

TGACTTGCATCGTGCATCT AGCTAGCTACCGTACTACATC
ACTGAACGTAGCACGTAGA TCGATCGATGGCATGATGTAG

...CTTGCAT

No amplification
Principle of PCR-SSP. The sequence-specific primer ending in 3’TCATGA...5’ will be extended only from a template carrying the
polymorphism shown.

comparable to CDC-based typing (antigen equivalent) or can

provide results at the allele level, which are required for match-
ing of unrelated HSC donors and recipients. Allele-level HLA
typing has demonstrated the incredible extent of polymor-
phism within the HLA loci (see Table 16–1). The nomenclature
Color used for HLA typing is explained in Figure 16–6.
signal

Amplicon
HLA-A*03:01:01:02N
Probe

Principle of PCR-SSOP. Patient DNA is amplified HLA locus

using primers labeled with biotin or digoxigenin at the 5’ end. The
amplicons are then hybridized to panels of probes immobilized on a Allele group
membrane. If the sequence of the amplicon matches and hybridizes
to that of the probe, a secondary reaction with enzyme-conjugated Allele number
avidin or antidigoxigenin will produce a color or light signal when
exposed to substrate (bottom left). If the sequence of the amplicon
Synonymous variation
differs from that of the probe, no signal is generated (bottom right).

Noncoding variation

Connections
Variation in expression
Polymerase Chain Reaction
Recall from Chapter 12 that the PCR is a molecular method that The nomenclature used for naming HLA alleles
is used to produce millions of copies of a DNA segment. It is com- includes identification of the HLA locus, which is separated from
monly used to amplify a piece of DNA to levels that can be de- the allele number by an “*”. The first 2 digits of the allele number
tected in the laboratory. Amplification is achieved by a three-step typically correspond to the phenotypic group. The next position
cycle: (1) heat denaturation of the target DNA (in this case, DNA after the “:” identifies the specific allele within the allele group. This
coding for a specific HLA allele) to form single strands; (2) cooling position can accommodate up to 3 digits. The next position identi-
the reaction and binding of short, complementary nucleotide se- fies allelic variants resulting from synonymous nucleotide substitu-
quences called primers to the 3’ ends of the target strands to ini- tions (i.e., they do not result in a change in amino acid). The next
tiate replication; and (3) heating the reaction in the presence of position identifies allelic variants resulting from polymorphisms in
DNA polymerase and the 4 deoxynucleotide triphosphates (i.e., noncoding regions of the gene (i.e., introns). Lastly, a letter may be
the DNA building blocks) to extend the strands. These heating- added that indicates unique features of the allele such as “N” indicat-
cooling cycles are performed by an automated thermocycler. ing the allele is null (i.e., not expressed on the cell surface). (Figure
courtesy of John Schmitz.)
HLA Antibody Screening, Identification, unacceptable antigens is determined and reported as the
cPRA value. For example, a recipient with an HLA-A2 anti-
and Crossmatching body would have a cPRA value of 47% if 47% of potential
Antibodies to HLA antigens can be detected in candidates donors are projected to be HLA-A2 expressing, based on his-
and recipients of solid-organ transplants by performance of toric HLA typing data.
a crossmatch test. These antibodies can develop in response Enzyme-linked immunosorbent assay (ELISA) has been
to multiple blood transfusions or to prior HLA-mismatched developed as a substitute for CDC-based HLA antibody test-
transplants. They can also be produced by women who have ing.26 ELISA assays utilize purified HLA antigens bound
had multiple pregnancies in response to paternally derived to the wells of microtiter plates. Patient serum is added to
fetal antigens. Because of the potential adverse impact HLA the wells of the plate; if HLA-specific antibody is present, it
antibodies can have on graft survival, patients awaiting solid- will bind. Bound antibody is detected by the addition of an
organ transplantation are screened periodically for their enzyme-labeled anti-immunoglobulin reagent. Addition of
presence through an HLA antibody screen.15 If detected, substrate results in a color change in the wells that have
the HLA specificity of the antibodies is then determined so bound antibody. The wells of the ELISA plate may contain
that donors possessing those HLA antigens can be eliminated a pool of HLA antigens, thus serving as a qualitative screen
from consideration for donation to that patient. Patients are for the presence of HLA antibody in a serum. Alternatively,
tested monthly for the presence of HLA antibodies while each well may contain HLA antigens representing a single
they are waiting for an organ offer. Antibody screening and donor and thus can be used in a fashion analogous to a
identification is also performed post-transplantation to aid CDC-based analysis, allowing %PRA and specificity to be
in the diagnosis of antibody-mediated rejection and to assess determined.
the effectiveness of therapy for antibody-mediated rejection. Another approach for antibody detection and identifica-
Crossmatching is performed before transplant to confirm the tion is flow cytometry.27 Antibody in patient serum can be
absence of donor-specific antibody. incubated with beads that are coated with purified HLA anti-
The methods used for antibody detection, identification, gens, either from a pool of donors, an individual donor, or
and crossmatching have changed significantly in recent a single purified or recombinant HLA protein. Beads coated
years. The CDC method used for HLA typing is also used for with pooled HLA proteins are a sensitive qualitative screen
HLA antibody detection and identification. In this case, pan- for the presence of HLA antibody because they will detect
els of lymphocytes with defined HLA phenotypes are incu- antibodies to the majority of common HLA antigens. Beads
bated with the patient’s serum. If the serum contains HLA coated with purified HLA proteins from individual donors
antibodies, they will bind to those lymphocytes in the panel or with a single HLA type (referred to as single-antigen
that express the corresponding HLA antigen. Binding is de- beads) are used to determine the specificity of the HLA an-
tected by addition of a complement reagent derived from tibodies in a patient’s serum; this information is used to de-
rabbit serum and a vital dye to assess cell death microscop- termine the cPRA. Patient serum is incubated with the beads
ically (see Fig. 16–3). and bound antibody is detected by adding a FITC-labeled
In some scenarios, the level of antibody in a serum sample anti-IgG reagent (Fig. 16–7). A more recent version of flow
may be below the level detectable by the CDC assay. In these cytometry-based antibody detection is the multiplex bead
cases, anti-human globulin (AHG) can be added to the CDC array system that can assess binding of patient antibodies to
assay to increase the test’s sensitivity. The AHG-CDC assay up to 100 different HLA antigens in a single tube using a
can detect lower levels of antibody as well as isotypes of dedicated flow-based detection system such as a Luminex
bound antibody that don’t activate complement and thus bead array.28 Flow cytometry-based methods are the most
wouldn’t normally be detected in the standard CDC assay. sensitive technology for detecting HLA antibodies. In addi-
Usually 30 to 60 unique lymphocyte preparations are in- tion, they can provide the most specific determination of the
cluded in the panel; the proportion of lymphocytes in the specificity of HLA antibodies when beads coated with a sin-
panel that are killed by the patient’s serum is referred to as gle HLA antigenic type are used.
the percent panel reactive antibody (%PRA). In addition, Once a suitable donor has been identified for a particular
the specificity of the antibodies can be determined by evalu- patient, a donor–recipient crossmatch test is performed to
ating the phenotype of the panel cells. confirm the absence of donor-specific antibody. Donor T and
More recently, the determination of PRA for solid-organ B lymphocytes are incubated with recipient serum in a CDC
allocation has been modified. Currently, a calculated PRA assay. Microscopic analysis is used to verify a lack of binding
(cPRA) is determined for organ allocation. In this approach, after the addition of complement and a vital dye to differentiate
the HLA antigens to which a candidate has HLA antibody live from dead cells. Cell death is an indication of recipient an-
are determined using solid-phase assays. These antigens are tibody binding to donor HLA antigen(s). Alternatively, binding
then classified as unacceptable antigens for that candidate. of antibody can be detected by flow cytometry using an FITC-
Accordingly, donors expressing those antigens are excluded labeled anti-IgG reagent. As for antibody screening and iden-
from donation for that candidate. The proportion of poten- tification, the flow cytometric crossmatch is the most sensitive
tial donors in the donor pool possessing one or more of the method for detecting donor-specific antibody.15
 80
70
60
50

Counts
40
30
20
10
0
HLA-A3 HLA-A7 HLA-A11 0 200 400 600 800 1000
A (low red) (mid red) (high red)
B Anti-IgG FITC

1000

HLA-A11
800
Red bead intensity

600
HLA-A7

400
HLA-A3

200

0
0 200 400 600 800 1000
C Anti-lgG FITC
Flow cytometric detection and identification of antibodies. (A) Patient serum is incubated with a mixture of polystyrene beads
coated with different HLA proteins (e.g., HLA-A3, HLA-A7, HLA-A11). (The beads have been labeled with various amounts of a red dye for
identification.) Any HLA antibodies present in the serum will bind to the corresponding beads. After washing, patient antibodies bound to the
beads are detected by the addition of a FITC-labeled anti-IgG reagent. After a second wash, the beads are analyzed for fluorescence on a flow
cytometer. (B) Single-parameter histogram display of an HLA class I antibody screen using the three types of HLA-coated beads. The large
peak represents beads with no bound antibody, whereas the smaller peak to the right indicates the presence of HLA antibody bound to
approximately 29% of the HLA class I coated beads. This represents a positive HLA class I antibody screen. (C) The individual bead populations
are identified in the dual-parameter dot plot. The beads coated with HLA-A11 have shifted to the right relative to the other beads, indicating
that the HLA antibodies in this patient’s serum are specific for the A11 antigen.

SUMMARY individual’s immune system develops to respond to the

foreign proteins presented on its own MHC antigens, it
• The immune system’s ability to recognize and respond to responds intensely to foreign MHC proteins. Both the hu-
the myriad of infectious agents that humans encounter has moral and cellular branches of the immune system con-
obvious benefits. However, the mechanisms that impart tribute to this allogeneic immune response and mediate
this ability make the transplantation of organs and cells graft rejection.
between allogeneic individuals difficult. • The likelihood of developing an immune response to a
• The targets of the response to transplanted tissues are HLA graft depends on the genetic relatedness of the transplant
proteins and, to a lesser extent, minor histocompatibility donor and recipient. Autografts involve transfer of tissue
antigens. These proteins play critical roles as antigen- from one area of the body to another in the same indi-
presenting molecules for CD4 and CD8 T cells, resulting vidual. Syngeneic grafts (isografts) involve transfer of tis-
in the phenomenon of MHC restriction. Although an sue between two genetically identical members of the
 same species. In allografts, tissue is transplanted between the transplanted tissue. The classes of immunosuppressive
genetically nonidentical members of the same species, agents include corticosteroids, antimetabolites, calcineurin
whereas in xenografts, tissue is transplanted between inhibitors, monoclonal antibodies, and polyclonal anti-
members of different species. bodies. Unfortunately, these agents also interfere with im-
• Because of the disparity in HLA antigens, allografts and mune responses to infectious organisms and tumors.
xenografts have the potential to induce strong immune • Identifying the MHC antigens of donors and recipients
responses in the transplant recipient. and monitoring the allospecific immune response are
• Hyperacute rejection occurs within minutes to hours after critical components of clinical transplantation. Greatly
transplantation and is mediated by preformed HLA anti- improved outcomes of transplantation are seen when
bodies that react with donor vascular epithelium, resulting donor and recipient are matched for HLA types as much
in activation of the complement cascade and clotting as possible and when the recipient does not have pre-
mechanisms. Preformed antibodies can also cause accel- formed antibodies to the donor’s HLA antigens.
erated rejection, which occurs over several days. • The classic method of HLA phenotyping is the complement-
• Acute rejection develops days to months after transplan- dependent cytotoxicity test (CDC), in which HLA antigens
tation and is mediated primarily by a cellular response to are typed by incubating the individual’s lymphocytes with
foreign HLA antigens involving cytokine production by a panel of antisera and reagent complement. Positive cells
CD4+ T cells and cytotoxic activity of CD8+ T cells. are identified by their ability to bind specific antisera and
• Chronic rejection can occur after the first year of transplan- complement, resulting in membrane permeabilization and
tation through a delayed type of hypersensitivity reaction uptake of vital stains.
to foreign HLA proteins, resulting in graft arteriosclerosis, • Molecular methods such as PCR-SSP, PCR-SSOP, and SBT
fibrosis, and scarring. provide greater resolution than HLA antigen typing by
• Graft-versus-host disease can occur in HLA-mismatched determining the HLA genotype at the allele level.
recipients of HSC transplants or other transplants that • Screening and identification of preformed HLA antibodies
contain lymphoid cells. In this condition, the donor’s in transplant recipients can be performed by the CDC
T lymphocytes destroy the recipient’s cells, primarily in method through incubation of patient serum with panels
the skin, gastrointestinal tract, and liver. of lymphocytes with known HLA antigens, ELISA, tradi-
• Even in the face of intense immune responses, transplan- tional flow cytometry, or a flow cytometry-based multiplex
tation has become an effective treatment for a variety of bead array.
diseases because of the development of immunosuppres- • Crossmatching is performed by incubation of recipient
sive agents that, in various ways, inhibit the immune sys- serum with donor lymphocytes in a CDC assay to confirm
tem from responding to the allogeneic MHC proteins in the absence of donor-specific antibody.

Study Guide: Classification of Grafts

TYPE OF GRAFT DEFINITION EXAMPLES
Autograft Transfer of tissue within the same Skin graft from leg to face of a burn patient
individual Transfer of a saphenous vein from the leg of a cardiac
bypass patient to his heart
Syngeneic (Iso) Transfer of cells or tissues to a genetically Transplant between identical human twins
graft identical individual Grafts between genetically identical strains of mice
Allograft Transfer of cells or tissues to a genetically Human transplant from a cadaver donor or family
nonidentical member of the same member other than an identical twin
species Grafts between genetically nonidentical strains of mice
Xenograft Transfer of cells or tissues to a member of Transplant of a pig valve into a human heart
a different species
Study Guide: Types of Graft Rejection
TIMING (AFTER
TYPE TRANSPLANT) IMMUNOLOGIC MECHANISM
Hyperacute Minutes to hours Preformed antibodies to ABO, HLA, and certain endothelial antigens bind to
donor vascular endothelium, activating complement and clotting factors. This
leads to thrombus formation, ischemia, and necrosis of transplanted tissue.
Accelerated Days Same as for hyperacute rejection.
Acute Days to months Cell-mediated response to foreign MHC-expressing cells. CD4+ T cells
produce cytokines and induce delayed type hypersensitivity. CD8+ T cells
mediate cytotoxic reactions.
Antibodies produced against HLA antigens bind to vessel walls, activate
complement, and induce transmural necrosis and inflammation.
Chronic 1 year or more Delayed type hypersensitivity response, and possibly antibodies, to foreign
HLA antigens on graft. Graft arteriosclerosis and smooth muscle proliferation
occur, resulting in fibrosis, scarring, and narrowing of vessel lumen.
Graft-versus-host 100 days or more T cells in HSC, lung, or liver transplants react against foreign HLA proteins in
disease (GVHD) the recipient’s cells, causing massive cytokine release, inflammation, and
tissue destruction in various locations throughout the body.

CASE STUDIES
1. A 40-year-old mother of three needs to have a second patient’s single sibling might be a suitable stem cell
kidney transplant. Her first transplant was lost because donor. However, the sibling was determined to be med-
of chronic rejection. The mother’s HLA type, HLA an- ically unsuitable for donation. As such, the transplant
tibodies, and ABO blood group status was determined. center conducted an unrelated donor search for this pa-
The patient was found to have antibodies to HLA-B35 tient and a potential donor was identified. The trans-
by flow cytometric testing with HLA-B35 coated beads. plant registry provided the HLA type for the donor that
The HLA type and blood group were also determined was determined using CDC-based testing (phenotyp-
for two of her siblings and two close friends who are ing). The patient’s HLA type was determined at high
interested in donating a kidney to the patient. resolution by SBT.
Question
a. From the available donors, who would likely be the HLA- HLA- HLA- HLA- HLA-
most compatible to this patient? ID A* B* C* DRB1* DQB1*
Patient 01:01 08:01 07:02 03:01 02:01
BLOOD
02:01 44:02 02:01 15:01 06:02
IDENTIFICATION GROUP A B C DR DQ
Donor 1 8 7 3 2
Recipient O 1,2 8,44 7,5 17,4 2,7
2 44 2 15 6
Sibling 1 O 1,11 8,35 7,4 17,1 2,5
Sibling 2 A 3,11 7,35 7,4 15,1 6,5
Friend 1 B 2,24 57,7 6,7 7,15 2,6 Questions
Friend 2 O 2,24 57,7 6,7 7,15 2,6 a. Is this donor–recipient pair HLA identical?
Yes / No / Maybe
2. A 59-year-old male with leukemia needed a HSC trans- b. The transplant physician requested high-resolution
plant for his disease. Clinicians were hopeful that the HLA typing for the donor. Why?
REVIEW QUESTIONS
1. Which of the following responses is the type of allograft 7. The indirect allorecognition pathway involves which
rejection associated with vascular and parenchymal one of the following mechanisms?
injury with lymphocyte infiltrates? a. Processed peptides from polymorphic donor
a. Hyperacute rejection proteins restricted by recipient HLA class II
b. Acute cellular rejection molecules
c. Acute humoral rejection b. Processed peptides from polymorphic recipient
d. Chronic rejection proteins restricted by donor HLA class I molecules
c. Intact polymorphic donor protein molecules
2. Antigen receptors on T lymphocytes bind HLA class II recognized by recipient HLA class I molecules
+ peptide complexes with the help of which accessory d. Intact polymorphic donor protein molecules
molecule? recognized by recipient HLA class II molecules
a. CD2
b. CD3 8. Which immunosuppressive agent selectively
c. CD4 inhibits IL-2 receptor-mediated activation of T cells
d. CD8 and causes clearance of activated T cells from the
circulation?
3. Patients who have received the following types of a. Mycophenolate mofetil
grafts are at risk for graft-versus-host disease (GVHD) b. Cyclosporine mofetil
except for recipients of c. Corticosteroids
a. bone marrow transplants. d. Daclizumab
b. lung transplants.
c. liver transplants. 9. Phenotyping for HLA class II antigens requires
d. irradiated leukocytes. B lymphocytes because
a. B lymphocytes express HLA class II antigens.
4. Which of the following properties are not exhibited b. B lymphocytes do not express HLA class I
by HLA molecules? antigens.
a. They belong to the immunoglobulin superfamily. c. B lymphocytes are exquisitely sensitive to
b. They are heterodimeric. complement-mediated lysis.
c. They are integral cell membrane glycoproteins. d. B lymphocytes represent the majority of lymphocytes
d. They are monomorphic. in the peripheral blood.

5. Kidney allograft loss from intravascular thrombosis 10. A renal transplant candidate was crossmatched with a
without cellular infiltration 5 days post-transplant donor that was mismatched for only the HLA-B35
may indicate which primary rejection mechanism? antigen. The candidate was known to have an anti-
a. Hyperacute rejection body specific for HLA-B35. Which of the following
b. Accelerated humoral rejection combinations of T- and B-cell flow cytometric
c. Acute humoral rejection crossmatch results would be expected?
d. Acute cellular rejection a. T cell negative, B cell negative
b. T cell positive, B cell positive
6. Which reagents would be used in a direct (forward) c. T cell negative, B cell positive
donor–recipient crossmatch test? d. T cell positive, B cell negative
a. Donor serum and recipient lymphocytes + rabbit
serum complement 11. Which of the following HLA alleles differs from
b. Recipient serum and donor lymphocytes + rabbit A*02:01:02 by a synonymous nucleotide
serum complement substitution?
c. Donor stimulator cells + recipient responder cells + a. A*01:01:01:01
complete culture medium b. A*02:01:03
d. Recipient stimulator cells + donor responder cells + c. A*02:02
complete culture medium d. A*02:03:01
12. Which one of the following donors would be expected 14. Which of the following HLA antigens would be ex-
to elicit a positive mixed lymphocyte response in lym- pected to elicit an HLA antibody response in a kidney
phocytes from a patient who has the HLA-DRB1*01:01, transplant recipient with the following HLA type:
01:03 alleles? HLA-A*01,03; B*07,14; C*01,04N; DRB1*16,07?
a. DRB1*01:01, 01:03 a. HLA-A*01
b. DRB1*01:01, 01:01 b. HLA-B*14
c. DRB1*01:03, 01:03 c. HLA-C*04
d. DRB1*01:01, 01:05 d. HLA-DRB1*16

13. Which of the following donors would be the most 15. Suppose a 30-year-old man was found to be a suitable
appropriate, based on ABO compatibility, for a renal donor for a kidney transplant to his younger sister.
transplant candidate with the ABO type = O? This transplant would be an example of a(an)
a. O a. autograft.
b. A b. allograft.
c. B c. isograft.
d. AB d. xenograft.
 Tumor Immunology

After finishing this chapter, you should be able to: INTRODUCTION TO TUMOR BIOLOGY
1. Describe the characteristics that differentiate cancer cells from normal TUMOR ANTIGENS
cells and the process by which malignant cells are thought to develop. Tumor-Specific Antigens (TSAs)
2. Differentiate between tumor-specific antigens and different categories Tumor-Associated Antigens (TAAs)
of tumor-associated antigens and recognize examples of each. CLINICALLY RELEVANT TUMOR
3. Summarize the uses of tumor markers in screening for cancer, MARKERS
diagnosing malignancy, detecting prognosis, and monitoring patient Clinical Uses of Tumor Markers:
responses to treatment. Benefits and Limitations
4. Identify the characteristics that should be possessed by an ideal tumor Serum Tumor Markers
marker and explain how nonideal features can affect the clinical
LABORATORY DETECTION OF TUMORS
utility of a marker.
Tumor Morphology
5. Explain the principles of immunohistochemistry as they apply to
tumor marker detection. Immunohistochemistry
6. Distinguish the clinical applications of each of the following tumor Immunoassays for Circulating Tumor
markers: alpha-fetoprotein (AFP), cancer antigen 125 (CA 125), Markers
carcinoembryonic antigen (CEA), human chorionic gonadotropin Molecular Methods in Cancer
(hCG), and prostate-specific antigen (PSA). Diagnosis
7. Contrast the advantages and limitations of immunoassays for tumor INTERACTIONS BETWEEN THE IMMUNE
markers. SYSTEM AND TUMORS
8. Summarize key principles of molecular and proteomic testing for Immune Defenses Against Tumor
tumor markers. Cells
9. Describe the innate and adaptive immune responses that play a role in Innate Immune Responses
defense against tumors and how they contribute to immunosurveillance. Adaptive Immune Responses
10. Discuss the process of immunoediting and how this relates to IMMUNOEDITING AND TUMOR
mechanisms of tumor escape from the immune system. ESCAPE
11. Recognize the overall goal of immunotherapy and specific examples Elimination
of active, passive, and adoptive immunotherapy for cancer.
Equilibrium
12. Discuss principles and applications of molecular tests for cancer
Escape
diagnosis.
IMMUNOTHERAPY
Active Immunotherapy and Cancer
Vaccines
Passive Immunotherapy
Adoptive Immunotherapy
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Adoptive immunotherapy Carcinomas Immunosurveillance Proteomics
Alpha-fetoprotein (AFP) CD45 Immunotherapy Proto-oncogenes
Antibody arrays Colony stimulating factors Immunotoxins Sarcomas
Antibody–drug conjugates Cytogenetics Karyotype analysis Tumor
Apoptosis Hematopoietic growth Malignant Tumor-associated antigens
Benign factors Metastasis (TAAs)
Biomarker profiling Heterophile Microarray Tumor-infiltrating
High-dose hook effect lymphocytes ( TILs)
Cancer Mutations
Human chorionic Tumor markers
Cancer antigen 125 Neoplasm
(CA 125) gonadotropin (hCG) Tumor-specific antigens
Oncofetal antigen
Human epithelial growth (TSAs)
Cancer vaccines Passive immunotherapy
factor receptor 2 (HER2) Tumor suppressor genes
Carcinoembryonic antigen Postzone
(CEA) Immunoediting
Prostate-specific antigen (PSA)
Carcinogenesis Immunohistochemistry

Introduction to Tumor Biology Connections

Tumor immunology is the study of the relationship between Apoptosis

the immune system and cancer cells. The field encompasses Recall that apoptosis is a normal homeostatic mechanism that
several areas, including the antigens associated with tumor is necessary for maintaining normal cell numbers. This process
cells, the host’s immune responses to tumors, mechanisms by occurs through a series of biochemical reactions within a cell
that lead to chromatin condensation, DNA fragmentation, cell
which tumors are thought to escape these responses, and ther-
shrinkage, and membrane blebbing. Cell death occurs in the
apeutic use of the immune system in an attempt to eradicate
absence of cell lysis and does not provoke an inflammatory
tumors. Tumor immunology is a major area of interest to im- reaction that could damage neighboring cells. Genetic changes
munologists because cancer is a leading cause of mortality within a cell that cause it to become resistant to apoptosis are
worldwide, responsible for more than 8 million deaths each important in the development of cancer.
year.1 The American Cancer Society states that cancer is the
second most common cause of fatality in the United States, ac-
counting for almost 1 out of every 4 deaths.2
Tumor immunology is best understood with a background normal architecture. Cancer, named after the Latin word for
on the origin of cancer cells and their differences from normal “crab,” derives its name from this property of invasiveness, which
cells. Normally, cell growth and division are carefully regulated can resemble the legs of a crab when viewed in microscopic tissue
processes designed to rapidly produce new cells when neces- sections. In addition, malignant tumors commonly exhibit
sary, inhibit cell division when enough cells are present, and metastasis, or the ability of cells to break away from the original
limit cell life span through a normal physiological process of tumor mass and spread through the blood to nearby or distant
cell death called apoptosis. These activities make it possible sites in the body. Malignant tumors can vary in their degree of
for the human body to carry out functions necessary for life, differentiation, from completely differentiated, or mature, to com-
such as generation of new skin cells to replace those that are pletely undifferentiated tumors that tend to grow more aggres-
dying, proliferation of lymphocytes in immune responses, and sively and have a poorer prognosis.
regeneration of tissue during wound healing and repair. How- Malignant tumors are classified according to their tissue of
ever, if these processes are allowed to continue unchecked, origin. Approximately 80% of cancers are carcinomas, derived
they can lead to excessive cell growth and division, resulting from the skin or epithelial linings of internal organs or glands;
in the development of an abnormal cell mass called a tumor about 9% are leukemias or lymphomas, malignant white blood
(from the Latin, meaning “to swell”) or neoplasm (from the cells (WBCs) present in the circulation or lymphatic system;
Greek, meaning “new growth”). and about 1% are sarcomas, derived from bone or soft tissues
Tumors can be classified as benign or malignant. Benign such as fat, muscles, tendons, cartilage, nerves, and blood
tumors are composed of slowly growing cells that are well- vessels.4 To make a specific diagnosis and guide treatment
differentiated and organized, similar to the normal tissue from decisions, physicians use staging systems based on the site and
which they originated.3 These tumors are surrounded by a cap- type of the primary tumor, tumor size, involvement of regional
sule, which secures them in place and prevents them from circu- lymph nodes, presence or absence of metastasis, and degree of
lating to other parts of the body. In contrast, malignant tumors, resemblance to normal tissue.5 The most widely used staging
or cancer cells, are disorganized masses that are rarely encapsu- scheme is the TNM system of the American Joint Committee
lated, allowing them to invade nearby organs and destroy their on Cancer (AJCC).6
 Clinical Correlations (Fig. 17–1). In 2000, Hanahan and Weinberg defined a can-
cerous cell as one that exhibits six characteristics:
The TNM System • Sustained signaling of proliferation
In the TNM system, tumors are classified according to the size • Resistance to cell death
and extent of the primary tumor (T0 to T4), the degree of • Ability to induce angiogenesis (development of new
spread to adjacent lymph nodes (N0 to N3), and the presence blood vessels to provide oxygen and nutrients to the
or absence of distant metastases (M0 or M1). For example, tumor)
breast cancer that is classified as T2N1M0 would involve a
• Immortality in terms of cell division
tumor between 2 to 5 cm in diameter that has spread to one
• Invasion and metastasis
to three regional lymph nodes but has not spread to distant
sites.6 For many types of cancer, the TNM criteria also corre- • Ability to avoid suppressors of cell growth9
spond to one of five stages (stage 0, I, II, III, or IV). Although the Today, it is recognized that cancer is not a single disease, but
specific criteria vary according to the type of cancer, in general, rather a heterogeneous group of diseases that show variability
stage 0 represents noninvasive (in situ) carcinoma; stages I, II, in these characteristics. In fact, heterogeneity is commonly
and III are tumors that are larger or that have spread beyond found among different cancer cells in the same patient. The
the organ in which they first developed to nearby lymph nodes extent to which a tumor exhibits these characteristics will
or tissues; and stage IV tumors are those that have metasta-
determine its ability to survive, grow, and metastasize. This, in
sized to distant sites.5
turn, has important implications that influence disease aggres-
siveness, patient prognosis, and choice of therapy.
In 2011, Hanahan and Weinberg proposed four additional
The transformation of a cell into a malignant tumor is hallmark characteristics of cancer cells:10
thought to be a multistep process involving a series of genetic • Reprogramming of energy metabolism to support malig-
mutations that cause the phenotype of a cell to be changed nant proliferation
over time. This process, called carcinogenesis, is thought to • Ability to evade destruction by the immune system
be initiated by exposure of the host to factors in the environ- • Genomic instability and mutations
ment that induce genetic changes in the cell. These factors in- • Inflammatory responses that promote tumor growth
clude chemical carcinogens such as asbestos and cigarette
smoke;7 radiation such as ultraviolet rays from the sun and These additions to the original hallmarks recognize the im-
ionizing radiation from x-rays; and certain pathogenic viruses portant contributions of genetics and immunology to the de-
that have been linked to specific types of cancer.3 These agents velopment of cancer. In this chapter, we will discuss the
are all believed to create genetic changes, or mutations, in the interactions between tumors and the immune system in
DNA of our cells that affect the body’s mechanisms that nor- more detail.
mally control cell growth.
Two major types of genes are involved in malignant trans- Tumor Antigens
formation: proto-oncogenes and tumor suppressor genes.3,8
Proto-oncogenes are normal genes that have a positive influ- The concept of tumor immunology is based on the premise
ence on cell proliferation and development. Sometimes, mu- that tumors possess antigens that are recognized as foreign by
tations in proto-oncogenes can convert them to oncogene-like the immune system. Tumor antigens can be broadly classified
genes, which have DNA sequences similar to those found in the into two groups: tumor-specific antigens (TSAs) and tumor-
oncogenes of transforming viruses. These genetic alterations— associated antigens (TAAs) (Table 17–1). TSAs are unique
which include point mutations, chromosomal translocations, to tumor cells, whereas TAAs are also found on normal cells.
and gene amplifications—cause constitutive activation of the
proto-oncogenes, resulting in changes in the cell that produce
continuous cell division.
Tumor-Specific Antigens (TSAs)
Cell division is normally inhibited by the action of tumor TSAs are unique to the tumor of an individual patient or shared
suppressor genes. These genes have both “gatekeeper” and by a limited number of patients with the same type of
“caretaker” functions.8 The gatekeeper genes exert their ef- tumor.11,12 They are coded for by viral oncogenes or by host
fects by controlling the entry of cells into the cell cycle and proto-oncogenes or tumor suppressor genes that have under-
preventing cells from completing the cell cycle if they con- gone genetic mutations.3 A well-known example of a TSA is
tain damaged DNA. The caretaker genes are important in a fusion protein that is produced in chronic myelogenous
maintaining genetic stability by recognizing and repairing leukemia (CML) cells. This protein is a result of a reciprocal
damaged DNA in a cell. If a mutation occurs in which the chromosome translocation commonly known as the Philadelphia
normal function of a tumor suppressor gene is lost, growth chromosome, which involves the BCR (breakage cluster region)
inhibitory signals are removed, resulting in dysregulated cell on chromosome 9 and the c-ABL gene on chromosome 22.
growth and genetic instability. C-ABL is a cellular proto-oncogene that codes for tyrosine
In most cases, the development of a cancerous tumor is be- kinase, a key enzyme in cell-signaling pathways that promote
lieved to result from a series of mutations in proto-oncogenes cell division. During the translocation, the two chromosomes
and tumor suppressor genes that accumulate over a lifetime break and exchange parts so that the c-ABL gene is combined
 1st

2nd

3rd
Unrepaired

4th

Resists
apoptosis

Uncontrolled
cell division

Induces
angiogenesis

Evades and/or
subverts immune system

Metastatic/invasive
Genetic mutations in the evolution of cancer. As cells acquire an increasing number of mutations over time, they develop
more of the characteristics that allow them to evolve into invasive cancer cells (numbers indicate mutations).

with part of the BCR to produce a hybrid gene that is constantly TSAs can also be produced by mutations induced by car-
expressed (Fig. 17–2). The BCR/ABL gene rearrangements cinogenic chemicals and radiation. Researchers are continually
result in uncontrolled cell proliferation and are found in the discovering new TSAs by using molecular techniques.11,12
majority of CML patients. Some of these antigens can serve as targets for specifically di-
Other TSAs originate from point mutations (i.e., genetic rected therapies (see Immunotherapy later).
changes involving a single base pair) in key genes involved
in cell proliferation, such as the tumor suppressor gene p53,
and the gene coding for caspase 8, an enzyme important for
Tumor-Associated Antigens (TAAs)
apoptosis (both of which are associated with head and neck In contrast to TSAs, TAAs are expressed in normal cells
tumors and squamous cell carcinoma).13 An updated list of as well as in tumor cells. Tumor cells abnormally express
tumor antigens resulting from mutations can be found at these protein or carbohydrate antigens in terms of their con-
http://cancerimmunity.org/peptide/mutations/.14 TSAs also in- centration, location, or stage of differentiation.3 Classifica-
clude protein antigens encoded by cancer-causing viruses tion of these antigens may vary according to their source.
(Table 17–2). These antigens can be found in the nucleus, This section will focus on the major categories of peptide
cytoplasm, or plasma membrane of the associated tumor cells. TAAs that have been identified by the Cancer Research
 Table 17–1 Tumor-Specific and Tumor-Associated Antigens*
CATEGORY DESCRIPTION EXAMPLES*
Tumor-specific Antigens that are unique to a tumor or shared by tumors of BCR/ABL fusion protein (CML)
antigens (TSAs) the same type
Tumor-associated Antigens that are expressed in normal cells as well as tumor
antigens (TAAs) cells
Shared TSAs Expressed in many tumors but not in most normal tissues MAGE (melanoma)
(cancer/testis
antigens)
Differentiation Expressed on immature cells of a particular lineage CD10 (ALL)
antigens CEA (mainly in colorectal cancer)
AFP (HCC)
PSA (prostate cancer)
Overexpressed Found in higher levels on malignant cells than on normal cells HER2 (mainly in some breast
antigens cancers)
*Primary cancer associations are shown in parentheses.
AFP = alpha-fetoprotein; CD10 = cluster of differentiation 10; CEA = carcinoembryonic antigen; CML = chronic myelogenous leukemia; HER2 = human
epidermal growth factor 2; MAGE = melanoma antigen gene; PSA = prostate-specific antigen.

Normal Chromosomes Changed

chromosome 9 break chromosome 9
Changed
Normal chromosome 22
chromosome 22 (Philadelphia
chromosome)

BCR BCR-ABL
gene fusion gene

ABL gene

The BCR/ABL gene rearrange-

ment characteristic of CML.

Institute: shared TSAs, differentiation antigens, and overex- not on mature B cells. This category of antigens also includes
pressed antigens.12 the oncofetal or embryonic antigens that are normally ex-
Shared TSAs are expressed in many tumors, but not in most pressed on developing cells of the fetus but not on cells in the
normal tissues.3,12,13 The only normal cells in which they have adult.3,11,12 It is thought that the genes coding for these anti-
been detected are testicular germ cells (i.e., spermatogonia and gens are silenced during development of the embryo, but that
spermatocytes) and, to a lesser extent, placental trophoblasts the process of malignant transformation allows them to be re-
and ovaries. Hence, these antigens have also been called can- expressed.11 Examples of oncofetal antigens include carci-
cer/testis antigens. They become TAAs when the transformation noembryonic protein (CEA), alpha-fetoprotein (AFP), and
process causes them to be expressed on tumors originating prostate-specific antigen (PSA). Many of these antigens are
from other cell types. These antigens have been identified on tumor markers that can be detected in the clinical laboratory
many tumors of epithelial or mesenchymal origin. The best (see the next section).
known examples of TAAs in this category are the melanoma The third category of TAAs is composed of the overexpressed
antigen gene (MAGE) proteins that are expressed by melanoma antigens, which are found in higher levels on malignant cells
tumors. than on normal cells. Genetic mutations that occur during
A second group of TAAs contains the differentiation antigens, transformation are thought to deregulate expression of these
which are expressed on immature cells of a particular lineage. proteins, resulting in levels up to 100 times greater than
An example of a TAA in this group is the CD10 antigen (pre- normal.3 A well-known example of a TAA in this category is the
viously known as the CALLA, or common acute lymphoblastic human epithelial growth factor receptor 2 (HER2) protein,
leukemia antigen), which is normally found on pre-B cells but a transmembrane receptor that binds human epidermal growth
 Table 17–2 Human Viruses Associated Clinically Relevant Tumor Markers
With Cancer
VIRUS CANCER ASSOCIATIONS Tumor markers can be defined as biological substances that are
found in increased amounts in the blood, body fluids, or tissues
Epstein-Barr virus (EBV) Burkitt lymphoma
Hodgkin lymphoma
of patients with a specific type of cancer. These substances can
Leiomyosarcomas be produced by the tumor itself or by the patient’s body in re-
Post-transplant lymphoprolif- sponse to the tumor or related benign conditions. The concen-
erative disease tration of a tumor marker in the serum depends on the degree
Nasopharyngeal carcinoma of tumor proliferation, the size of the tumor mass, the proteolytic
activities of the tumor, or release of the marker from dying tumor
Hepatitis B virus (HBV) Hepatocellular carcinoma
cells.15 An elevated level of a tumor marker suggests that a sig-
Hepatitis C virus (HCV) Hepatocellular carcinoma nificant amount of a particular type of tumor is present.
Human herpes virus 8 Kaposi sarcoma Tumor markers can be proteins, carbohydrates, oncofetal
(HHV-8) antigens, hormones, metabolites, receptors, or enzymes.16
Table 17–3 lists some examples of clinically relevant tumor
Human papilloma virus Cervical cancer markers in each category, along with their disease associations.
(HPV) Other genital and anal cancers
An ideal tumor marker should have seven characteristics.15-17
Head and neck cancer
A marker should:
Human T-lymphotropic Adult T-cell leukemia or • Be produced by the tumor itself or by the patient’s body
virus I (HTLV-1) lymphoma
in response to the tumor
Merkel cell polyomavirus Merkel cell carcinoma (a type • Be secreted into a biological fluid, where it can be inex-
of skin cancer) pensively and easily quantified
• Have a circulating half-life long enough to permit its
concentration to rise with increasing tumor load
factor. Gene amplification in a certain type of breast cancer can • Increase to clinically significant levels above the reference
result in overexpression of this protein, which serves as a level while the disease is still treatable
marker for detection and therapy (see Immunotherapy later). In • Have a high sensitivity; in other words, it should easily
addition to peptide TAAs, glycolipid and glycoprotein antigens detect the majority of individuals in the population who
may also be overexpressed in some tumors.11 Examples of these have a particular cancer
antigens include cancer antigen 125 (CA 125), which is associ- • Have a high specificity; in other words, the marker should
ated with ovarian cancer, and cancer antigen 19-9 (CA 19-9), be absent from, or present at background levels in all
which is associated with pancreatic cancer. In the next section, individuals without the malignant disease in question to
we will discuss clinical applications of some of these markers. minimize false-positive test results

Table 17–3 Categories of Clinically Relevant Tumor Markers15-17

TUMOR MARKER
CLASS EXAMPLES DISEASE ASSOCIATIONS
Cell surface Estrogen or progesterone receptors Prognosis for hormone therapy in breast cancer
markers CD markers on white blood cells (WBCs) Clonality and lineage of WBC neoplasms
Proteins Thyroglobulin (TG) Well-differentiated papillary or follicular thyroid
carcinoma
Immunoglobulins (Ig) and Ig light chains Multiple myeloma and lymphoid malignancies
(Bence Jones proteins)
Oncofetal Alpha-fetoprotein (AFP) Germ cell carcinomas, hepatocellular carcinoma
antigens Carcinoembryonic antigen (CEA) Colorectal, breast, or lung cancer
Carbohydrate CA 125 Ovarian cancer
antigens CA 15-3 Breast cancer
CA 19-9 Pancreatic and gastrointestinal cancers
Enzymes and Prostate-specific antigen (PSA) Prostate cancer
isoenzymes Alkaline phosphatase (ALKP) Bone and liver cancer
Neuron-specific enolase Neural tissue neoplasms
Hormones Human chorionic gonadotropin (hCG) Germ cell carcinoma, trophoblastic tumors
Calcitonin Medullary thyroid cancer
Gastrin Pancreatic gastrinoma
Unfortunately, very few of the tumor markers in clinical use increased in men with a harmless enlargement of the prostate
are ideal because they are not tumor specific.16 This limitation known as benign prostatic hypertrophy (BPH) (see section on
affects the performance of tumor markers in each of the four PSA). In these cases, the finding of an elevated PSA value can
applications introduced in the next section. lead to unnecessary testing and potentially harmful treatments,
such as a prostate biopsy.17,20
Clinical Uses of Tumor Markers: It is important to remember that the effectiveness of a tumor
Benefits and Limitations marker in screening for cancer depends on the sensitivity and
specificity of the marker, as well as the cancer’s prevalence in
In general, tumor markers have four major clinical applica- the population. Screening is most effective when it is con-
tions: screening, diagnosis, prognosis, and monitoring. It is a ducted in populations at a high risk for developing the disease,
well-established fact that patient survival rates are usually such as certain ethnic groups or those with a family history for
higher when cancer is diagnosed in its earliest stages. Detecting a particular type of cancer. For example, alpha-fetoprotein
cancer early, before it begins to invade normal tissues and is (AFP), a tumor marker for hepatocellular carcinoma, is not
difficult to target, requires good screening methods. Tumor used for screening in the United States, but is used to screen
markers can be used to screen asymptomatic individuals in a people in China, where the incidence of liver cancer is high.
population for the presence of cancer. The individuals screened In high-risk populations, the predictive value of the tumor
may belong to a healthy population or to a high-risk popula- marker will be highest. In other words, a positive test result is
tion. For example, PSA has been routinely used to screen men most likely to be found in a person who truly has the disease,
aged 50 or older for the presence of prostate cancer. Secondly, whereas a negative result is most likely to occur in a person
tumor markers can be helpful in making an initial diagnosis of who truly does not have the disease.
cancer. For example, an elevated level of PSA might suggest
the presence of prostate cancer. A third use of tumor markers
is in predicting prognosis of a cancer patient. In general, an A second application of tumor markers is to help physicians
initial high concentration of a tumor marker or an increasing distinguish between different diseases with similar symptoms,
level of a tumor marker over time points to a poorer prognosis. a process called differential diagnosis. For example, if a comput-
A fourth use of tumor markers is to monitor known cancer pa- erized tomography (CT) scan revealed the presence of a lung
tients to determine whether their treatment is working and to nodule, histological examination of a lung biopsy could help
check for recurrence of their tumor. Decreasing levels indicate differentiate whether the nodule was caused by cancer or an-
that the treatment is effective in reducing the amount of tumor other disease process, such as an infection. Follow-up staining
harbored by the patient, whereas increasing levels indicate that of the biopsy for tumor markers could help determine the neo-
the treatment is ineffective and that the number of tumor cells plasm’s tissue origin.21 In order to improve the sensitivity and
in the patient is increasing. Each of these applications will be specificity of the testing, multiple tumor markers could be ex-
discussed in more detail in the text that follows. The second amined or testing for tumor markers could be combined with
half of this section will focus on traditional tumor markers that other laboratory tests.15 It is important to recognize that tumor
can be detected in the serum. Some of the more commonly de- markers can serve as a valuable aid in making a cancer diag-
tected markers will be discussed. This section will also intro- nosis when they are used in conjunction with clinical findings
duce the role of molecular and proteomic methods in detecting and other tests, but that they are not diagnostic by themselves.
newer tumor markers that are being increasingly evaluated in
the clinical laboratory. A third application of tumor markers is to assess prognosis in a
known cancer patient. A high concentration of a tumor marker
The detection of tumor markers provides an ideal way to at the time of diagnosis or increasing levels of a tumor marker
screen for tumors because the markers can be detected by a
simple blood test. However, the benefits and limitations of Connections
using a particular marker to screen a population must be con-
sidered before testing is implemented. On the positive side, Specificity
screening asymptomatic individuals can lead to earlier detec- A problem with many tumor markers is that they lack speci-
tion of tumors with a need for less aggressive treatment. In ficity for the disease they are intended to detect. Recall from
addition, many people who have been screened can receive Chapter 9 that specificity is the ability of a test to be negative
reassurance from true negative results. However, there are also for a person who does not have a particular disease. For
significant disadvantages associated with screening. Besides the example, suppose a tumor marker has a specificity of 90%.
actual dollar costs of the screening test, harm to the individuals Although this may sound like a good attribute because the
test will give a true negative result in every 90 out of 100 peo-
tested may also occur.18,19 For example, misleading reassurance
ple tested, 10% of those tested will have a false-positive result.
can be experienced by individuals with false-negative results.
If 100,000 people in a population were tested, 10,000 would
In contrast, false-positive results can lead to patient anxiety; have a positive result even though they did not have the
possible harm from more invasive, unnecessary follow-up intended disease and would likely be recommended to have
testing; and overtreatment of questionable diagnoses. For ex- additional unnecessary testing or invasive procedures.
ample, PSA, a marker elevated in prostate cancer, can also be
over time can indicate the presence of an aggressive tumor that was unresponsive to Chemotherapy #1, as reflected by the sus-
has metastasized and requires rigorous treatment.15,16 Tumor tained elevation in the marker after treatment with that drug.
markers can also be used to determine the type of therapy that This prompted a change in treatment to Chemotherapy #2,
would be most effective for a patient. For example, in breast which was successful in decreasing the amount of tumor pres-
cancer, anti-HER2 agents such as trastuzumab work best in pa- ent in the patient, as indicated by the decline in the tumor
tients whose tumors overexpress the HER2 protein or gene; marker to an undetectable level.
antiendocrine therapies such as tamoxifen are suitable for pa-
tients whose tumors overexpress the estrogen receptor.21 Serum Tumor Markers
Although there are scores of possible tumor markers in the lit-
erature, only a few have FDA approval.17 However, many tests
One of the most important uses of tumor markers is to monitor for non-FDA-approved markers are available to clinicians, with
known cancer patients to determine whether their treatment a notation on the laboratory report stating that results are for
is effective and to check for recurrence of the tumor. This can research use only. The National Academy of Clinical Biochem-
be done because the level of a serum tumor marker often cor- istry (NACB) has published consensus guidelines regarding the
relates with the amount of tumor in the patient. The laboratory clinical use of tumor markers.22,23 Some of the most commonly
typically determines a baseline concentration of the marker be- used markers are listed in Table 17–4, along with their clinical
fore treatment begins, followed by serial measurements over applications and important noncancerous conditions that can
time. A significant decrease in the concentration of a tumor cause elevations.
marker after surgery, chemotherapy, or other treatment indi-
cates that the therapy has been effective in shrinking the tumor.
In contrast, an increasing level of the marker after a return to Alpha-fetoprotein (AFP) is a 70,000 MW glycoprotein that
normal indicates that the tumor has recurred and that more is similar to albumin in its physical and chemical proper-
aggressive treatment may be needed. Elevation in a tumor ties.15,16 It is classified as an oncofetal antigen because it is
marker can become evident several months before the cancer synthesized by the fetal liver and yolk sac and is abundant
is detected by other methods.16 in fetal serum, but declines to low levels (10–20 ug/L) by
The clinical significance of monitoring a tumor marker is 12 months of age.24 AFP is frequently elevated in patients
illustrated in Figure 17–3. This figure shows tumor marker with primary hepatocellular carcinoma (HCC) and nonsemi-
levels from a hypothetical cancer patient who has been treated nomatous testicular cancer, but can also be elevated in other
with surgery and two chemotherapy drugs. As expected, the types of cancers and in nonmalignant conditions such as
level of the tumor marker in the patient’s serum declined after pregnancy and hepatitis.15,16,23,24
the initial tumor mass was removed by surgery. However, after AFP is the most widely used tumor marker for HCC, serving
a few months, the concentration of the tumor marker began as a tool in diagnosis, staging, prognosis, and monitoring pa-
to increase, indicating that the tumor had recurred. The tumor tients undergoing therapy.16,24 The sensitivity of AFP for HCC

Using a tumor marker to monitor cancer

160
Surgery Chemotherapy #2

140

120

Decline of marker
Tumor marker level

100 consistent with

biological half-life Therapy
unsuccessful
80 Therapy seems
Chemotherapy #1 successful
60
Upper limit of
Relapse reference interval
40

20
Tumor marker analysis. A curve No evidence of disease No evidence of disease
showing a sample scenario monitoring a cancer 0
patient for tumor recurrence and for therapy effi- Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar
cacy using levels of a tumor-associated antigen. 2008 2009
Table 17–4 Common Tumor Markers15-17, 22-25
NONCANCEROUS
NORMAL CONDITIONS WITH
MARKER CANCER(S) USES SOURCES ELEVATIONS COMMENTS
AFP Nonseminomatous Screening Fetal liver and Pregnancy, Screening conducted
testicular germ Diagnosis yolk sac, adult nonneoplastic in high-risk popula-
cell and staging liver liver disease tions for liver cancer
Liver Prognosis such as those with
Monitoring liver cirrhosis and
chronic hepatitis.
In germ cell tumors,
both AFP and hCG are
elevated.
β2– B-lymphocyte Prognosis Part of class I Inflammatory and Higher levels imply
microglobulin malignancies Monitoring MHC high cell turnover poor prognosis in
molecule conditions multiple myeloma.
Calcitonin and Familial medullary Diagnosis Thyroid In hypercalcemia, Can be elevated in
Ca++ thyroid carcinoma Monitoring increased calci- other forms of cancer.
tonin is expected.
Serum Ca++ may
be low when
calcitonin is ele-
vated in medullary
carcinoma.
CD markers WBC Diagnosis All WBCs WBC increase Different CD markers
Monitoring such as infection are associated with
specific WBC
malignancies.
CEA Colorectal Prognosis Tissues of Renal failure, Values increased with
Breast Monitoring endodermal nonneoplastic age and in smokers.
Lung origin liver and intestinal
disease, age
CA 125 Ovarian Screening Ovaries and Endometriosis, Increases can occur
adenocarcinoma Diagnosis various other pelvic inflamma- during menstruation.
Prognosis tissues tory disease, Screening is only
Monitoring uterine fibroids, recommended for
and pregnancy women with a family
history of ovarian
cancer.
CA 15-3 Breast cancer Prognosis Mammary Benign breast CA 15-3 is a mono-
Can also be Monitoring tissue disease, benign clonal antibody
increased in liver disease directed against an
pancreatic, epitope of episialin.
lung, colorectal,
ovarian, and liver
cancers
CA 19-9 Pancreatic Diagnosis Sialylated Benign hepatobil- Can be elevated in
Prognosis Lewisa blood iary and pancreatic some nonpancreatic
Monitoring group antigen conditions malignancies.
Subjects who are
Lewis a and b negative
persons cannot syn-
thesize CA 19-9.
ER/PR Breast Prognosis Breast N/A ER/PR+ breast cancers
adenocarcinoma benefit from estrogen
or progesterone
reduction therapy.
Table 17–4 Common Tumor Markers15-17, 22-25—cont’d
NONCANCEROUS
NORMAL CONDITIONS WITH
MARKER CANCER(S) USES SOURCES ELEVATIONS COMMENTS
hCG Nonseminomatous Diagnosis Placenta Pregnancy hCG has a high homol-
testicular cancer Prognosis ogy with luteinizing
germ cell tro- Monitoring hormone (LH). Malig-
phoblastic (hyda- nancies can produce
tidiform mole, free α and β chains as
choriocarcinoma) well as intact α and
β dimer. Immunoas-
says that detect only
intact hCG should not
be used for tumor
marker detection.
In germ cell tumors,
both AFP and hCG
are elevated.
HER2 (neu) Breast Prognosis Growth factor N/A Cancers associated
or neu gene in all with overexpression
cells of HER2 (neu) or neu
have a good response
to monoclonal antibody
therapy (trastuzumab).
Monoclonal Plasma cell, Diagnosis Normal Igs are Monoclonal Bence Jones proteins
free Ig light B lymphocytes Monitoring polyclonal. gammopathy of are free Ig light chains
chains Prognosis Few free light undetermined sig- in urine detected by
chains exist. nificance (MGUS), urine immunofixation
amyloidosis, electrophoresis.
nonsecretory
myeloma
Monoclonal Plasma cell, Diagnosis Normal Igs are Monoclonal Monoclonal IgG/IgA—
Igs B lymphocytes Monitoring polyclonal gammopathy of multiple myeloma.
Prognosis undetermined Monoclonal IgM—
significance Waldenström’s
macroglobulinemia.
PSA Prostate Screening No tissues UTI or prostatitis, Levels directly propor-
Diagnosis other than benign prostatic tional to prostate size.
Monitoring prostate hypertrophy Many elevations are
Prognosis benign or not clinically
significant.
Screening is routinely
conducted in men
aged 50 and older,
but is controversial.
Decreased percent of
free PSA and PSA
velocity greater than
0.75 ng/mL/year are
more strongly associ-
ated with prostate
cancer. Collect
specimen before
ejaculation, digital
rectal examination, or
prostate manipulation.

Continued
 Table 17–4 Common Tumor Markers15-17, 22-25—cont’d
NONCANCEROUS
NORMAL CONDITIONS WITH
MARKER CANCER(S) USES SOURCES ELEVATIONS COMMENTS
PTH and Parathyroid Diagnosis Parathyroid In hypocalcemia, PTH has a short
Ca++ carcinoma Prognosis glands increased PTH is half-life, so levels are
Monitoring expected. Serum done intraoperatively
Ca++ may be to ensure complete
high when PTH parathyroid tumor
is elevated in removal.
parathyroid
carcinoma.
TG Thyroid Monitoring Thyroid TG reflects thyroid Assays must simulta-
mass, injury, neously test for
and TSH levels. thyroglobulin antibod-
Thyroid markers ies because these
(T4, TSH) are can cause falsely
generally normal decreased results.
in thyroid cancer. Often tested after
TSH stimulation (or
less often, by with-
holding thyroid med-
ication) to see if TG
production by residual
tumor cells occurs.

AFP = alpha-fetoprotein; CA = carbohydrate antigen; Ca++ = serum calcium; CD = clusters of differentiation in WBC; CEA = carcinoembryonic antigen; ER or
PR = estrogen or progesterone receptors; hCG = human chorionic gonadotropin; Ig = immunoglobulins; PTH = parathyroid hormone; PSA = prostate-specific
antigen; TG = thyroglobulin; UTI = urinary tract infection.

is 41% to 65% and its specificity ranges between 80% to In addition to its applications as a tumor marker, AFP is
94%.25 As a result, the utility of AFP in screening for HCC has widely used as a marker to detect abnormalities in the fetus.
been a matter of debate. However, screening for HCC with AFP An increased level of AFP in the serum or amniotic fluid of a
is routinely performed in high prevalence areas of the world pregnant woman is seen with open neural tube defects such as
such as China and Southeast Asia. In the United States, AFP spina bifida, whereas low levels of AFP are associated with
screening is usually conducted in patients with a high risk for Down syndrome.28
HCC, along with liver ultrasound.24 Many of these patients
have liver cirrhosis because of hepatitis B or hepatitis C infec-
tion. Studies have shown that the diagnostic utility of AFP may Cancer antigen 125 (CA 125) is a large, heavily glycosylated,
be improved by testing specifically for the isoform AFP-L3, mucinlike protein that is a marker for ovarian cancer. This
which has a stronger correlation with HCC, and by combining marker is not unique to ovarian tumors because it is also found
AFP with other laboratory markers, such as DCP (des-γ- in the normal ovary as well as other tissues, including the en-
carboxy-prothrombin), the liver enzyme ALT (alanine amino- docervix, endometrium, fallopian tubes, pleura, pericardium,
transferase), and platelet count.24,26 In addition, high levels of peritoneum, and epithelial tissues of the colon, pancreas, lung,
AFP are associated with a poor prognosis in patients with HCC, kidney, prostate, breast, stomach, and gallbladder.27,29
whereas decreasing levels over time indicate a good response CA 125 is considered the best marker for ovarian can-
to therapy.16,24 cer.29,30 It has multiple applications to the disease, ranging
AFP is also an established tumor marker for nonsemino- from screening and diagnosis, to prognosis and monitoring
matous germ cell cancers of the testes (NSGCT).16,27 This response to therapy.23,27 Serum CA 125 levels greater than
marker, along with other markers such as human chorionic 35 kU/L are considered to be above normal.27,29 Although 90%
gonadotropin (hCG; see section later in this chapter) and lac- or more of women with ovarian cancer in stages II to IV have
tate dehydrogenase (LDH), plays an important role in patient elevated CA 125, the marker is not recommended for screening
diagnosis, tumor staging, therapeutic monitoring, and detec- of the general population because it lacks sensitivity and speci-
tion of relapse. AFP is elevated in 10% to 20% of patients with ficity. Elevated CA 125 levels are only seen in 50% to 60% of
stage I NSGCT and in nearly all patients in later stages of the women with stage I ovarian cancer; therefore, generalized
disease.16 As in HCC, increased concentrations are associated screening would miss about half of the women during the pe-
with a poor prognosis, whereas declining levels reflect respon- riod when the disease is most treatable.27,30 In addition, CA
siveness to therapy.27 125 is not specific because it can increase during pregnancy or
menstruation, or as a result of benign gynecological conditions cells that contribute to development of the placenta and promote
such as endometriosis, nongynecological conditions involving implantation of the embryo. Accordingly, it rises during the first
inflammation, and other malignancies.16,23,29 However, annual few weeks of gestation, when it can be detected in the blood and
CA 125 testing, together with transvaginal ultrasound, is rec- urine of pregnant women. In addition, hCG can be produced
ommended for women with a family history of ovarian cancer by certain malignant tumors; elevations are associated with germ
because early detection and intervention is likely to be benefi- cell tumors of the ovary and testes as well as choriocarcinoma,
cial in this population.27,31 a rare type of cancer that is caused by malignant transformation
The value of CA 125 can also be seen in clinical applications of the trophoblast cells.16,27 In these tumors, testing for hCG is
other than screening. For example, an elevated CA 125 con- recommended as an aid to diagnosis, prognosis, monitoring re-
centration combined with imaging has been shown to be sponse to therapy, and detection of recurrence.23
highly sensitive and specific for a differential diagnosis of ovar- hCG is a 45,000 MW glycoprotein that is composed of
ian cancer from benign pelvic masses, especially in post- an α subunit, which is shared by luteinizing hormone (LH),
menopausal women.27,29 Serial CA 125 testing is beneficial in follicle-stimulating hormone (FSH), and thyroid-stimulating
monitoring a patient’s response to chemotherapy and is rec- hormone (TSH), and a β subunit that is unique to hCG.15,16,27
ommended before treatment is initiated, during treatment, and Serological tests can measure either intact hCG or the hCG β
during patient follow-up. Rising concentrations of CA 125 over subunit; the presence for both should be tested to monitor pa-
time can predict recurrence of the disease. Persistent elevations tients with testicular cancer. This is because some patients may
of CA 125 or an initial CA 125 value greater than 65 kU/L only produce the β subunit and detection of only the intact
point to a poor prognosis.27 form can result in false-negative results.27 Because hCG levels
can also increase in men as the result of malfunction of the
testes, it is important to observe rising values in sequential
Carcinoembryonic antigen (CEA) is a glycoprotein with a tests before making a diagnosis of testicular cancer. Elevations
molecular weight of 180,000 to 200,000, depending on the of hCG can also occur as a result of gonadal suppression
exact structure of its carbohydrate side chains.32 CEA was the caused by chemotherapy and do not necessarily indicate
first oncofetal antigen to be discovered. In 1965, Gold and tumor recurrence.27
Freedman described its presence in tissues from the fetal colon
and colon adenocarcinoma, as well as its absence in colon
tissues from healthy adults.32 Prostate-specific antigen (PSA) is the most widely used
Increased serum concentrations of CEA are associated with marker for prostate cancer. It is a 28,000 MW glycoprotein that
colorectal cancer because CEA is the most widely used marker is produced specifically by epithelial cells in the prostate gland.
for that cancer.16,32 The main application of CEA is in moni- PSA was first discovered in semen, where its function is to reg-
toring patients undergoing therapy for colorectal cancer.27 It ulate the viscosity of the seminal fluid to facilitate mobility of
is recommended that the medical team obtain a baseline CEA the sperm cells.20 Its presence was subsequently noted in
value from the laboratory just before therapy, followed by CEA serum, where it is frequently elevated in patients with prostate
testing every 1 to 3 months during active treatment.27 Increas- cancer.
ing CEA levels are a highly sensitive indicator of liver metastasis The specificity of PSA for the prostate gland led to its routine
and can detect recurrent colorectal cancer by an average of use as a screening test for prostate cancer. Since its approval by
5 months before clinical symptoms appear.32 CEA measure- the FDA in 1994, PSA testing has resulted in a dramatic increase
ment should be used in conjunction with clinical examination, in the detection rate of early-stage prostate cancer and in the
radiological testing, and histological confirmation to maximize rate of 5-year patient survival.20 Despite these successes, general
its sensitivity in detecting disease recurrence.33 CEA levels can screening for prostate cancer has been a controversial issue be-
also be used in determining the most appropriate treatment for cause it may potentially lead to unnecessary testing and treat-
colorectal cancer patients because those with higher baseline ment. There are several reasons for this concern. Although PSA
levels before surgery tend to have a poorer prognosis.19,27 is specific for prostate tissue, it is not specific for prostate cancer.
However, CEA is not recommended for colon cancer screen- PSA can also be elevated in other conditions affecting the
ing because of its low sensitivity and specificity in this situa- prostate gland, such as benign prostatic hyperplasia (BPH), an en-
tion.27 CEA is not increased in all patients with colorectal largement of the prostate gland that commonly occurs as men
cancer and elevated CEA levels can be present as a result of age, or prostatitis, an inflammation of the gland occurring as a
other conditions, including colitis, diverticulitis, irritable bowel result of infection or irritation.16,20 Transient increases in PSA
syndrome, and nonmalignant liver disease.27,32 Cigarette levels can also occur if samples are collected shortly after ejac-
smoking can cause an increase in CEA level to nearly twice that ulation, digital rectal examination (DRE), or prostate manipu-
of nonsmokers.32 CEA levels can also be elevated in other can- lation.16,29 As such, there is concern that general PSA screening
cers, notably those of the breast, gastrointestinal tract, pan- can lead to the performance of unnecessary prostate biopsies
creas, and lung.23 and risk of infection and other complications.34 In addition,
many prostate cancers are slow growing and would be unlikely
to cause death during an older man’s remaining life span. Fur-
Human chorionic gonadotropin (hCG) is best known as the thermore, the conventional treatments for prostate cancer, rad-
“pregnancy hormone” because it is synthesized by trophoblasts, ical prostatectomy, and radiotherapy can be associated with
significant side effects, most notably urinary incontinence, erec- likely to be caused by the occurrence of cancer in a man with
tile dysfunction, and problems with bowel function.34,35 There- a small prostate gland versus a large prostate gland.20 PSAD is
fore, some clinicians believe that it may be better to carefully calculated as the ratio of total PSA to the prostate gland vol-
monitor the condition over time, by active surveillance and ob- ume.15 Although this approach appears to increase the speci-
servation (“watchful waiting”), than to initiate treatment for ficity of PSA for prostate cancer, it requires performance of
early-stage prostate cancer that could decrease quality of life.35 transrectal ultrasonography, which can be time consuming,
Large clinical trials that tested thousands of men to deter- costly, and yield a less-than-perfect measurement of the
mine the benefits and harms of PSA screening have produced prostate gland volume.20
conflicting results.34,36,37 As a result, there is disagreement PSA testing also plays an important role in the management
among major agencies about whether PSA screening should be of patients known to have prostate cancer.27 PSA values, in
performed and how it should be implemented.19,34 For example, conjunction with histological observation of prostate biopsy
the American Cancer Society recommends annual PSA screening tissue, can be used to predict the stage of prostate cancer and
in conjunction with DRE for all men over the age of 50 and the to guide physicians in determining optimal treatment. In ad-
American Urological Association recommends that routine dition, a rapid rise in PSAV or in the amount of time it takes
screening be conducted from ages 55 to 69 and age 70+ if life for the PSA level to double are indicators of more aggressive
expectancy is greater than 10 years, whereas the U.S. Preven- disease.20,27 A persistently high level of PSA after radical prosta-
tative Services Task Force does not recommend screening at tectomy indicates that residual disease is present. When sur-
all. Earlier and more frequent screening may be recommended gery is successful in removing the tumor, PSA will decrease to
for men at higher risk for prostate cancer, notably those with undetectable levels. Rising PSA levels after surgery are a sign
a family history of the condition, those who have a known or that the malignancy is recurring and can precede other indica-
suspected related genetic mutation, or those of African American tors of disease recurrence by many years.27 PSA testing is also
ancestry.16,34 There is lack of agreement regarding what should a sensitive indicator of disease recurrence in men who have
be considered a cut-off value to distinguish between positive undergone hormonal therapy, but is less sensitive in detecting
and negative results, but a total PSA value of 0 to 4.0 ng/mL recurrence after radiation therapy because circulating PSA
is generally considered to be normal.15,16,20 Prostate biopsy levels decline more slowly after that type of treatment.27
is recommended for men with a total PSA value greater than
4.0 ng/mL to determine whether the elevation is caused by
malignancy.
Laboratory Detection of Tumors
Another application of PSA testing is to assist in the diag- Three types of laboratory methods are routinely used for
nosis of prostate cancer. Great effort has been expended to dis- cancer screening and diagnosis: gross and microscopic mor-
tinguish between BPH, weakly aggressive prostate cancers, and phology of tumors, detection of tumor markers by immuno-
highly aggressive cancers using PSA. Modifications in PSA test- histochemistry or automated immunoassays, and molecular
ing may be helpful in making this differentiation. One modifi- diagnostics to detect genetic mutations in the malignant
cation involves testing for free PSA and the naturally occurring cells. These techniques are complementary in that many of
PSA-α-1-antichymotrypsin complex, in addition to total serum the changes in DNA and subsequent mRNA expression result
PSA.15,20,35 This combination increases the specificity of testing in altered protein antigens or morphology. More recently, ge-
because the proportion of free PSA is higher in benign condi- netic markers have been introduced into the clinical labora-
tions, whereas the proportion of complexed PSA is greater in tory. The use of protein expression patterns of cancer cells
prostate cancer. Because free PSA quickly degrades at temper- is being evaluated by scientists.
atures above 4°C, it is important to perform testing within
3 hours of sample collection or to store the sample at –70°C if
a longer time interval is required.15 Tumor Morphology
Another approach is to calculate the PSA velocity (PSAV), Pathologists and histology laboratories process suspected
or the rate of increase in PSA values over time. PSAV is calcu- tumor tissue with gross dissection and preparation of slides for
lated as the difference in PSA concentration divided by the microscopic analysis. Tumor marker antibodies, special stains,
number of years spanning the interval between sequential tests and nucleic acid probes can be applied to the slides to enhance
(reported as ng/mL/year). The rationale for this approach is visible features. Even so, evaluation of morphology and stain-
that PSA will increase more rapidly if a growing tumor is pres- ing patterns can be very subjective and classification categories
ent. A PSAV greater than 0.75 ng/mL/year has been shown to can be rather broad. Considerable skill is required to accurately
be strongly associated with the presence of prostate can- diagnose cancer on the basis of morphology; the final diagnosis
cer.15,20,35 To rule out the possibility that an increase in PSAV is generally made with supplemental clinical information and
is because of an infection of the prostate gland, a repeat mea- additional testing.
surement of PSAV can be conducted after a course of antibiotics
is administered.15,20
Another proposed strategy to increase the performance of
Immunohistochemistry
PSA testing is to calculate the PSA density (PSAD). The ration- Immunohistochemistry detection uses labeled antibodies to
ale behind this concept is that an increase in serum PSA is more detect tumor antigens in formalin-fixed or frozen tissue sections
of tumor biopsy material.38 Before testing, formalin-fixed sec- In the Laboratory
tions must be treated with heat to make the antigen epitopes
accessible. The indirect staining method is used because larger Detecting Immune Complexes With Avidin
immune complexes are formed, providing more sensitive am- Immune complexes can be more easily detected if an additional
plification of the signal than is achieved through direct staining. layer is added to the reaction. A common way to add this layer
An unlabeled primary antibody specific for the antigen to be is to take advantage of avidin-biotin binding. Avidin, a protein
detected is applied to the tissue section on a slide. Following found in egg whites, has a strong affinity for the B vitamin, biotin.
an incubation and wash, a labeled secondary antibody directed Therefore, in some enzyme-labeling assays, the secondary anti-
against the Fc portion of the primary antibody is applied. The body is labeled with biotin and an avidin-labeled enzyme is
label can be an enzyme such as peroxidase, alkaline phos- added in the next step. This forms a larger complex that contains
more enzyme molecules, increasing the signal intensity and
phatase, or glucose oxidase; or a fluorescent dye such as FITC,
making the assay more sensitive.
rhodamine, or Texas red. Immunofluorescence provides a
greater dynamic range than immunochromatographic staining
and is particularly useful for the identification and quantifica-
tion of co-localized proteins.39 Binding is visualized by light Immunoassays for Circulating
microscopy after the addition of the appropriate chromogen in
the case of enzyme labels and by fluorescent microscopy when
Tumor Markers
fluorescent labels are used. Serum tumor markers are most commonly measured by im-
Use of positive and negative control tissues is essential for munoassays because they are highly sensitive, lend themselves
accurate results.38 Negative controls are necessary to ensure to automation, and are relatively easy to use.16 Despite their
that the staining observed is because of antibody binding and advantages, immunoassays can be affected by several factors
not the background (i.e., nonspecific reactivity), whereas pos- that need to be considered when results are interpreted. These
itive controls confirm that the antibody reagents are working factors are related to the use of antibodies as reagents.15,16
properly. Normal tissue on the same slide can serve as an ex- First, antibody reagents from different manufacturers can
cellent internal control. Accuracy of the results is also increased vary greatly in terms of what they detect, particularly if mono-
when a broad panel of antibodies is used. clonal antibodies are used. Thus, it is important to use the same
Clinically, immunohistochemistry is used as an effective method for monitoring patients over time because results can
technique of classifying tumors of uncertain origin because be affected if patients change clinics or laboratories. It also
many tumors can have a similar appearance histologically.38 To means that if laboratories switch methods, they must provide
be helpful in pathological diagnosis, the marker must be dif- a transition period during which samples are measured by both
ferentially expressed in the tumor of origin as compared with methods and specimens are archived until new data is estab-
other tumors. The first step in immunohistochemistry is to lished for each patient.23
broadly classify the tumor into one of three major lineages: Second, although antibodies are employed for their speci-
epithelial, mesenchymal, or hematopoietic. This can be accom- ficity, it is not absolute. Antibodies will cross-react with similar
plished through the use of stains for specific markers that are structures, which is particularly problematic when the cross-
characteristic of each type of tissue. Routinely, cytokeratins, reacting substances are present in excessive amounts, as can
which are intermediate filaments found in all types of epithelial occur with cancer. For example, the α subunit of hCG is vir-
cells, are used as markers for tumors of epithelial lineage. There tually identical to the α subunit of luteinizing hormone (LH),
are 20 different cytokeratin subtypes, whose expression is often and the β subunits of the two hormones are 80% homologous,
organ and tissue specific and dependent on the stage of differ- so epitope choice is quite important. Furthermore, assay con-
entiation of the cells. Vimentin, an intermediate filament that figuration influences what is measured. Tumors may produce
is found in most mesenchymal cells, is used as a marker to in- free α and free β chains in addition to intact hCG, so an im-
dicate the presence of a sarcoma or melanoma, but its speci- munochemical method that detects only β chain epitopes to
ficity is low because it is expressed in some other tumor types minimize LH interference will measure something completely
as well. CD45, a WBC marker (see Chapter 1), can be used to different than a method that measures intact hCG.16
identify hematopoietic malignancies. A third factor that can affect immunoassay results is antigen
Once these broad differentiations are made in terms of cell excess. By virtue of their unchecked growth and aggressive me-
lineage, antibodies for more specific markers can be used for tabolism, some neoplasms may produce massive amounts of
more precise classification of the neoplasm. For example, an- tumor marker molecules. The excess of tumor antigen as com-
tibodies to numerous CD antigens can be used to identify the pared with reagent antibody can result in a postzone effect.
various lymphoid and myeloid cell types (see Chapter 18). In Recall from Chapter 10 that postzone is a well-known limita-
addition, antibodies to estrogen receptors, progesterone re- tion of immunoassays in which saturation of the antibodies
ceptors, and the HER2 antigen can be used to classify breast with antigen inhibits the cross-linkage required to visualize the
cancer and guide decisions about appropriate therapy. Auto- reaction. When the measurements exceed the linear range of
mated immunohistochemistry assays have been developed reportable results, this phenomenon is called the high-dose
that allow for quantitation of the proteins expressed by differ- hook effect16 because of the shape of the curve that depicts
ent cell populations.39 the relationship of the concentration of the analyte (in this case,
the tumor marker) and the intensity of the reaction signal
(Fig. 17–4). This effect can result in a falsely decreased mea-
surement in the area of antigen excess; therefore, the sample
must be diluted to determine its value within the reportable
range. It is critical that criteria be developed to identify situa-
tions in which the hook effect may be present so that specimens
can be systematically diluted and accurate results obtained. A
related problem of antigen excess in automated systems is sam-
ple carryover, so testing of the sample adjacent to the specimen
with excessive antigen may also need to be repeated.
A final problem with immunoassays is that interference can
be caused by the presence of endogenous heterophile, anti-
animal, or autoantibodies in the patient sample.16,40 Autoanti-
bodies are produced in response to self-antigens (see Chapter 15).
Heterophile antibodies are capable of reacting with similar
antigens from two or more unrelated species. These antibodies Interference by heterophile or anti-animal antibod-
usually have low avidity, but can react with a broad range of anti- ies. Heterophile or anti-animal antibodies (red) can cause both false
gens. Anti-animal antibodies are species-specific, higher avidity decreases and false increases, depending on their reactivity against
antibodies that are produced by patients as a result of passive the antibody species used in an assay. However, false increases
caused by linking the capture (blue) and detection (black) antibod-
immunotherapy with mouse monoclonal immunoglobulins or
ies together are most likely, as shown.
polyclonal antibodies of animal origin (see Chapter 25). Because
the antibodies used in immunoassays are of animal origin, en-
dogenous anti-animal antibodies in the patient sample can in- in Figure 17–5, false decreases are also possible. In these situ-
terfere profoundly with test results. These antibodies can affect ations, the patient antibody binds to the marker of interest or
immunoassays in more than one way, causing either falsely ele- blocks the binding of the labeled detector antibody, preventing
vated (most commonly) or falsely decreased test results.16,40,41 the formation of the immune complex “sandwich” and leading
The principle of interference resulting in a false-positive result to a falsely low or negative result.
is illustrated in Figure 17–5. In this situation, the antibody in To confirm the presence of interfering antibodies, the sam-
the patient sample binds to both the capture antibody on the ple can be diluted and the linearity of the results can be ana-
solid phase and the labeled detector antibody, forming a bridge lyzed.16 Specimens with interfering antibodies tend to exhibit
between the two antibodies in the same way the tumor marker nonlinear behavior. The laboratory can also test directly for the
would if it was actually present. This interference can have pro- antibodies themselves. The likelihood of interference by en-
found consequences on patient care. For example, a tragic case dogenous antibodies can be reduced by pretreatment of the
involving false-positive hCG results reported from an automated sample with commercial blocking reagents. These reagents are
analyzer led to several women having unnecessary chemother- typically mouse or rabbit immunoglobulins that bind to the
apy or hysterectomies for a cancer that was suspected but not interfering antibodies and neutralize them.16,40,41 Interference
actually present.42 with tumor marker tests can also be caused by factors that can
Although endogenous antibodies are mostly associated with affect other immunoassays, such as icterus, lipemia, and he-
falsely increased results by mechanisms similar to those shown molysis. In any case, patient results should not be reported
until the interference issue is resolved.
Some additional recommendations should be considered in
The hook effect the performance of tumor marker tests.15 Cut-off values for
Area of screening tests are typically selected under the presumption
Optimum
antigen excess that a relatively low number of people being screened actually
antigen
area have cancer and that it would be worse to miss a cancer than
to do further testing on a normal person to exclude cancer.
Thus, a very high number of false positives is expected because
Signal

Same signal,
different
of low disease prevalence. Establishment of a baseline level at
concentrations initial diagnosis followed by serial testing over time can provide
valuable information when a patient is being monitored for re-
sponse to treatment or tumor recurrence. In this case, it is not
a single absolute value of the tumor marker that is important,
but rather the upward or downward trend when the marker’s
Antigen concentration biological half-life is considered. When performing serial test-
The high-dose hook effect. Antigen excess can ing, each test should be performed by the same laboratory with
saturate antibodies and the intended “sandwich” configurations the same test kit to minimize variations in results. Testing for
cannot form, leading to a false decrease in signal. multiple markers, if possible, will increase sensitivity and
specificity. The limitations of immunoassays have prompted a acid amplification techniques (NAAT), fluorescent in situ hy-
search for more specific and sensitive markers using molecular bridization (FISH), microarray, and DNA sequencing. In NAAT,
and proteomic technologies. such as the polymerase chain reaction (PCR), millions of identical
copies of a specific target sequence within a nucleic acid are
Molecular Methods in Cancer Diagnosis synthesized in the laboratory from an original DNA template
derived from the cancer cell population (see Chapter 12).
Because cancer is a disease process that involves many genetic These methods are used to amplify the sequence that poten-
alterations, scientists have searched for changes in the genome tially contains the genetic mutation of interest, allowing tiny
that characterize the various types and subtypes of cancer. changes in the sequence to be detected by the differences in
Identification of genetic mutations has become an important fragment sizes that can be visualized by gel electrophoresis.49
tool in cancer diagnosis and determination of prognosis.

Cytogenetics studies play a large role in the diagnosis and

Molecular characterization is an essential component of testing management of cancer. Karyotype analysis (arrangement of
for hematologic malignancies (see Chapter 18). For example, a person’s chromosomes by their visual characteristics) has
as previously mentioned, the chromosome 9/22 [BCR/ABL] been used for many years to detect the chromosomal abnor-
gene rearrangement is a well-established biomarker for CML.43 malities associated with many cancers. The number of these
Rearrangements in immunoglobulin genes and T-cell receptor aberrations can increase as the disease advances. One type of
(TCR) genes are used to identify malignant clones of B cells or abnormality that can be detected by karyotyping is aneuploidy,
T cells, respectively, in leukemias and lymphomas. Genetic a condition in which individual chromosomes are gained or
analysis is also helpful in the diagnosis of solid tumors. For ex- lost. Another type of abnormality that can be present in cancer
ample, mutations in the MSH genes of the DNA mismatch re- cells is a deletion, in which a portion of a chromosome has been
pair system are helpful in the diagnosis of hereditary colorectal lost. A third type of abnormality is a rearrangement, involving
cancer; these alterations create microsatellite instability, an al- breakage of two different chromosomes and translocation of
teration in the length of repetitive DNA sequences that can be the fragments onto the opposite chromosomes (as discussed
visualized by molecular testing.44 Molecular analysis of gene in Chapter 18).
expression for the estrogen receptor (ER), progesterone recep- The development of FISH has allowed chromosomal abnor-
tor (PR), and HER2 can create a genetic profile that is used to malities to be characterized more precisely at a molecular level.
classify breast cancer patients into subtypes that provide prog- In FISH, interphase cells from the patient’s tumor are incubated
nostic information and guide physicians in choosing therapeu- with fluorescent-labeled nucleic acid probes that are comple-
tic plans that have the best chances of success.45,46 mentary to the sequence of interest. Cells containing the se-
Genetic biomarkers can also be used for prospective and quence will bind the probes and can be visualized with a
postdiagnostic evaluation of malignancies. Prospective markers fluorescent microscope. In oncology, FISH is most often used to
can provide valuable information regarding the risk for an detect chromosome rearrangements and gene amplification. To
asymptomatic person to develop a particular type of cancer, detect a chromosome translocation, such as the one seen in the
the growth rate of the cancer, or the development of metastatic BCR/ABL rearrangement characteristic of CML, two single probes
disease. For example, women with hereditary mutations in the are used, each specific for one of the two chromosomes and each
BRCA1 or BRCA2 genes carry a 40% to 80% lifetime risk for labeled with a different fluorochrome (for example, red and
developing breast cancer, as well as an 11% to 40% lifetime green).49 Normally, each cell should have two red signals and
risk for ovarian cancer.47 two green signals. If a translocation has occurred, a fusion probe
Postdiagnostic genetic markers are used to guide clinicians signal is generated, in which the red signal is adjacent to the
in making appropriate treatment decisions for known cancer green signal, producing a yellow color (see Figure 18–11B). To
patients. By mid-2014, the FDA had approved over 40 targeted detect gene amplification, the probe hybridizes to the gene of
cancer drugs for administration in cancer patients with the ap- interest. If that gene is overexpressed, multiple copies of the flu-
propriate genetic biomarkers.48 The list of approved therapies orescent signal representing the probe will be observed. This ap-
includes drugs that target tumors with alterations in the BRAF plication of FISH is commonly used to detect the HER2 gene
gene (metastatic melanoma), KRAS gene (colorectal cancer), amplification seen in some cases of breast cancer. Although FISH
EGFR gene (non-small cell lung cancer; NSCLC), ALK gene is a highly specific method to detect molecular abnormalities in
(NSCLC), HER2 gene (breast cancer), and ESR1 and PGR genes tumor cells, it can only detect gene sequences that are comple-
(breast cancer). Testing for these and other genetic markers is mentary to the probes used; as such, it may not detect rare, tiny
incorporated into the clinical practice guidelines published by deletions.
the National Comprehensive Cancer Network and the College
of American Pathologists. Molecular testing plays an important
role in identifying responders and nonresponders to the tar- As more clinically significant genetic abnormalities associated
geted medications, so that the drugs can be given to patients with cancer have been identified, microarray technology has
who would most likely benefit from them. been developed to test for panels of markers, rather than in-
Testing for biomarkers has been made possible through sci- dividual mutations.49 In this method, single-stranded DNA
entific advances in molecular techniques, including nucleic or RNA from the tumor is tagged with a fluorescent label and
incubated with known nucleic acid sequences that have been arrays are that they do not require fractionation or depletion of
spotted onto different areas of a membrane. The sample will high abundance proteins to detect proteins that are present in
hybridize to any complementary sequences on the tiny spots, lower concentrations. Proteomic methods may allow laborato-
allowing for simultaneous testing of the specimen for multi- ries to identify unique patterns of proteins and their metabolites
ple genes. Microarrays can also be used to compare the levels that are characteristic of particular types of cancer.53,54 This
of gene expression in cancer cells with those of normal cells process is called biomarker profiling. In the future, data gen-
by using two different colors of fluorescence to tag nucleic erated from proteomic methods may help clinicians diagnose
acid from each cell type (Fig 12-12).49 The microarray is cancer earlier and lead to the development of personalized ther-
interpreted with a fluorescent reader and analysis software. apies that can effectively target the underlying biology of this
Microarrays can screen tens of thousands of gene sequences highly complex disease entity.
at the same time for their ability to bind to the sample nucleic
acid, thus generating an enormous volume of information.
Sophisticated mathematics and computer software are nec-
Interactions Between the Immune
essary to analyze the vast amount of biological data. The col- System and Tumors
lection and analysis of such data is referred to as bioinformatics.
It is hoped that this approach will help uncover molecular sig- Paul Ehrlich proposed the idea over 100 years ago that the im-
natures that can characterize specific tumor types and subtypes mune system plays an important role in eliminating tumor
to provide better information regarding patient diagnosis and cells.55 Ehrlich reasoned that, if this were not the case, we
prognosis. Gene expression arrays, such as the MammaPrint would see cancer much more frequently in animals of ad-
and Oncotype Dx for breast cancer, are being commercially de- vanced age.56 In the 1950s, after scientists learned more about
veloped to aid in predicting the likelihood of cancer recurrence how the immune system works, F. MacFarlane Burnet and
and guiding treatment decisions in cancer patients.23,50 Lewis Thomas independently proposed the theory of immuno-
surveillance to address this issue. This theory states that the
immune system continually patrols the body for the presence
Another molecular method that provides a large amount of of cancerous or precancerous cells and eliminates them before
data is next generation sequencing (NGS). With NGS, thousands they become clinically evident.13,57 Burnet and Thomas based
of genes within the tumor can be sequenced simultaneously in their hypotheses on early studies that involved transplantation
just a few hours to identify genetic variations (see Chapter 12).51 of tumor cells in genetically inbred mice.58-60 The protective
NGS can also be used to detect metastases by analyzing DNA role of the immune system against tumors has also been sup-
from tumor cells circulating in the peripheral blood.52 ported by clinical evidence in humans. Notably, a significantly
NGS is playing a major role in generating an enormous vol- higher incidence of cancer has been observed in transplant pa-
ume of data for The Cancer Genome Atlas (TCGA).52 TCGA is tients who received immunosuppressive therapy and patients
conducting a large-scale collaborative project funded by the with immunodeficiency diseases than in the general popula-
National Cancer Institute and the National Human Genome tion.13,61,62 In addition, the incidence of cancer rises greatly
Research Institute to catalogue and characterize the genomic after the age of 60, at which point there is a decline in immune
changes that occur in cancer. The goal of the project is to iden- function.60 Although the validity of the immunosurveillance
tify all of the pertinent genomic alterations in several tumor theory was challenged in the 1970s and 1980s, technical ad-
types. This information is helping oncologists to better under- vances in genetics and monoclonal antibody technology have
stand and interpret molecular information related to cancer di- increased our knowledge in this area and there is renewed en-
agnostics. As a result of the ongoing studies, molecular thusiasm among scientists that the immune system indeed
techniques have evolved from esoteric research tools into rou- plays an important role in defense against tumors.13,55,60
tine laboratory assays that have revolutionized cancer diagnosis
and therapy. Immune Defenses Against
Tumor Cells
Researchers are also analyzing the proteins produced by cancer Both innate and adaptive immune responses are thought to
cells. Analysis of the entire protein complement of a cell popu- contribute to protection of the host from cancer.3,11,60,63 The
lation is referred to as proteomics. This analysis is being done main components of the immune system that participate in de-
through the use of two-dimensional electrophoresis coupled fense against tumor cells are NK cells, macrophages, cytotoxic
with tandem mass spectrometry (MS/MS), surface-enhanced T cells (CTLs), cytokines, and possibly T helper (Th) cells and
laser desorption/ionization mass spectrometry (SELDI-TOF), or antibodies. The mechanisms by which each of these compo-
more recently, antibody arrays.39,53 The latter are typically nents functions are the same as those that are involved in the
based on a principle of a double sandwich enzyme-linked im- elimination of pathogenic organisms (Fig. 17–6).
munoassay and are available in several different multiplex
formats. The most common format uses beads that are coated
with specific capture antibodies to bind the target proteins and
Innate Immune Responses
streptavidin- or fluorescent-labeled detection antibodies that can The key cells involved in innate immune responses to tumors
be detected by flow cytometry.39 The advantages of antibody are thought to be NK cells and possibly macrophages. As we
 Lysis

MAC
formation

ADCC
NK
Lytic enzymes
ROS, TNF!

MHC-unR APC

Tumor
Granzymes Antigens
cell
Cytokines perforins
MHC-R
from Th

Cytokines

Tc
Th

Apoptosis
Immune defenses against tumor cells (MHC-R = MHC-restricted killing, MHC-unR =MHC unrestricted killing).

discussed in Chapter 3, NK cells act in a mechanism that is Macrophages may also play a role in innate immunity
similar to CTLs (see the text that follows), but can kill tumor against tumors.11,60 Macrophages activated in vitro by IFNγ
cells without prior sensitization to tumor antigens. In addition, have been shown to possess tumoricidal capabilities. They ap-
they are activated to kill cells that lack class I MHC molecules, pear to kill tumor cells by the same mechanisms they use to
a property that is often seen in transformed cells (see Immu- kill infectious organisms, including release of lysosomal en-
noediting and Tumor Escape later). Activating receptors on NK zymes, reactive oxygen species, and nitric oxide. In addition,
cells bind to tumor antigens or substances released from they produce TNF-α, a cytokine that is thought to cause necro-
stressed tumor cells, initiating intracellular signals that promote sis of tumors by inducing local inflammation and thrombosis
degranulation and the release of perforin and granzymes, in the blood vessels within the cancerous mass.11 However, the
which ultimately kill the cells by inducing apoptosis. NK cells significance of macrophages in anti-tumor responses in the
may also participate in antibody-dependent cellular cytotoxi- body is unclear.
city (ADCC) in the presence of tumor-specific antibodies (see
the text that follows). NK cells are thought to be most effective
against malignant cells circulating in the bloodstream during
Adaptive Immune Responses
the early stages of tumor development. Their effectiveness in The primary mechanism of adaptive immunity against tumors
eliminating well-established solid tumors is questionable, how- is mediated by cytotoxic T lymphocytes (CTLs).3,11,60 CTL re-
ever, and requires further study.3,11,60 sponses are thought to be initiated by dendritic cells, which
The activity of NK cells can be increased by incubation act as antigen-presenting cells (APCs) by processing tumor
with IL-2. In vitro culture of peripheral blood cells or tumor- antigens and displaying the peptides derived from these anti-
infiltrating lymphocytes (TILs) with IL-2 results in the gen- gens on their surface in conjunction with class I MHC mole-
eration of lymphokine-activated killer (LAK) cells, which cules. (This mechanism is known as cross-presentation because
destroy tumor cells by the same mechanism as NK cells but the exogenous tumor antigens are presented in context with
are much more potent.11,60 The effectiveness of cytokine-ac- class I MHC molecules rather than class II MHC molecules.)
tivated TILs in adoptive immunotherapy for cancer patients The APCs present the tumor peptide-antigen complexes to spe-
is an active area of investigation (see Chapter 25 and Im- cific TCRs on the surface of the CTLs and provide costimula-
munotherapy later). tory signals that promote maturation of the CTLs. The mature
CTLs use their antigen-specific TCRs to bind class I MHC- altered cells under control so that they are not clinically evi-
associated tumor antigens on the surface of the tumor cell. dent. During this period, tumor cells may remain dormant or
Within minutes, their granules migrate toward the plasma evolve slowly over time. The dynamic interactions between the
membrane and release cytotoxic proteins within the synapse tumor and the immune system are thought to shape the phe-
formed between the CTL and the tumor cell. Among these pro- notype of the tumor and its ultimate outcome, hence the term
teins are perforin, which creates pores in the membrane of the immunoediting. As a result, the tumor may eventually be elim-
tumor cell, and granzymes, which enter through the pores and inated by the body, establish permanent residence in the equi-
cause apoptosis of the tumor target. NKT cells, which express librium phase, or evolve into a phenotype that can escape the
surface antigens of both NK cells and T cells, are able to destroy immune system and cause disease.
tumor cells in a mechanism that is similar to the CTLs, but During the equilibrium phase, mutations can occur in the
they have a unique type of TCR that recognizes glycolipid anti- genetically unstable transformed cells. Under selective pres-
gens instead of peptide antigens.3 sure from immunologic forces of attack by cells in the tumor
Dendritic cells are also thought to activate CD4+ Th cells microenvironment, some of the tumor cells may develop into
through presentation of tumor antigens in conjunction with genetic variants that are resistant to immune defenses.64
class II MHC molecules.11,60 The activated Th cells may play a These cells move past the equilibrium phase and enter the
role in tumor immunity by secreting cytokines such as IL-2, escape phase.
which can promote CTL development and enhance NK cell
activity, and IFNγ, which activates macrophages and increases Escape
class I MHC expression on the tumor cell surface.
Tumor-bearing individuals can also produce antibodies During this phase, the balance between immunologic control
against tumor antigens. In vitro studies have demonstrated that and tumor development is tipped in favor of the neoplasm and
these antibodies can kill tumor cells by inducing complement- tumor growth progresses, even in the presence of anti-tumor
mediated lysis or ADCC.11,60 The latter mechanism occurs immune responses.13 Cancer is a heterogeneous disease and
when the antibodies coat the tumor cells and bind to Fc recep- tumors have developed a variety of strategies for evading the
tors on the surface of macrophages, NK cells, or neutrophils, immune system (see Fig. 17–7).11,13,55,64 Some of the escape
stimulating them to release enzymes that can destroy the tumor mechanisms employed by tumors are a result of changes in the
targets. However, the relevance of these antibodies in vivo is edited tumors themselves, which lead to reduced immuno-
unclear. genicity. For example, some tumors downregulate the expres-
sion of tumor antigens or MHC molecules on the cell surface,
making them less likely to be recognized by T cells. Other
Immunoediting and Tumor Escape modifications may involve defects in components of the antigen-
processing machinery associated with class I MHC molecules.
Despite the diverse array of mechanisms employed by the im-
Tumor antigens may also be masked by glycoproteins and gly-
mune system to attack tumors, cancer occurs at a high fre-
colipids on the cell surface, making them inaccessible to the
quency in individuals who appear to be immunocompetent.
immune system.
Medical scientists have long been trying to find the answer to
Other alterations can result in tumor resistance to immune
this intriguing puzzle. As the field of tumor immunology ad-
defenses. For example, impaired cell surface binding to per-
vances, scientists are recognizing that immunosurveillance is
forin or defective apoptosis-inducing receptors such as Fas
only part of a broader process that explains the complex rela-
have been noted in some tumors. Another way that tumors can
tionship between the immune system and cancer. This process,
escape the immune system is to suppress anti-tumor immune
termed immunoediting, is thought to consist of three phases:
responses. Tumors can do this directly by secreting immuno-
elimination, equilibrium, and escape (Fig. 17–7).13,55,62
suppressive substances or indirectly by recruiting T regulatory
(Treg) cells, myeloid-derived suppressor cells, or macrophages
Elimination that produce cytokines such as transforming growth factor-β
The elimination phase of the cancer immunoediting process is and IL-10, which can inhibit protective immune responses.
essentially the same as the immunosurveillance concept that Another factor that may contribute to tumor progression is
was just discussed. If the immunologic mechanisms involved inflammation. Although acute inflammation may be protective
in immunosurveillance are highly effective, they will likely to the host, chronic inflammation is believed to modify the cel-
result in complete elimination of the tumor. If the immune lular microenvironment in ways that promote the development
responses are not completely effective, some of the tumor cells of tumors.56,64 Chronic inflammation and its associated proin-
will remain in the body. The immunoediting hypothesis sug- flammatory cytokines may contribute to tumorigenesis in a
gests that these cells will then enter the equilibrium phase. number of ways, including generation of cellular stress and free
radicals, production of growth factors that induce cell prolif-
eration, enhancement of angiogenesis and tissue invasion, and
Equilibrium suppression of adaptive immune responses.13,56
In this phase, tumor cells are thought to enter a state of dy- A clear understanding of the evasion mechanisms used by
namic equilibrium with the immune system, which keeps the tumors and of the immune responses they suppress will be
 Elimination phase

Robust innate and Tumor cells identified by

adaptive immune antigen and MHC I;
responses kill most induced to apoptosis by
tumor cells FAS ligand

FAS
FAS

Equilibrium
FAS
Immune system Tumor cells dormant;
controls limited number begin to lose or mask
of altered cells identifying features

Escape

Immune system Tumor cells highly

suppressed; chronic altered and more
inflammation promotes resistant to immune
tumor growth responses; apoptosis
impaired

Relationship between the immune system and cancer: Immunoediting and mechanisms of tumor escape.

critical in developing rational approaches to targeted im- hormone therapy to target residual tumor cells and metastases.
munotherapy. The section that follows presents specific ap- Advances in medical science have allowed immunotherapy to
proaches that have been taken in an attempt to influence gain a position among these conventional types of treatment
interactions between tumors and the immune system in order in the management of cancer. The goal of immunotherapy,
to promote tumor rejection. which is also known as biological therapy or biological response
modifier therapy, is to harness the ability of the immune system
Immunotherapy to destroy tumor cells.
Immunotherapeutic methods can be classified into three
Many different types of treatments can be used for cancer pa- major types: active, passive, and adoptive. With active im-
tients. The type of therapy used for a particular patient de- munotherapy, patients are treated in a manner that stimulates
pends on the type of tumor present and the stage of disease. them to mount an immune response against their tumors. Passive
Traditional therapies include surgery to remove solid tumors, immunotherapy involves administration of tumor-specific anti-
radiation therapy to reduce tumor size, and chemotherapy and bodies or cytokines to patients who may not be able to develop
an adequate immune response. In adoptive immunotherapy, cells (HPV) vaccine is effective in preventing cervical cancer and the
from the immune system are provided to patients. Some treat- hepatitis B virus (HBV) vaccine has been successful in reducing
ments may combine different types of immunotherapy. Although the incidence of HBV infection of the liver and its associated
it is well beyond the scope of this chapter to cover all the different complications, including hepatocellular carcinoma. These vac-
protocols, the following sections present the major types of im- cines are routinely used and are incorporated into standard vac-
munotherapy that have been developed and their applications to cination schedules.
specific kinds of cancer. Other cancer vaccines are being administered to patients
who have already developed cancer. These vaccines are de-
Active Immunotherapy signed to induce an immune response against tumor antigens
with the hope of eliminating existing tumor cells and produc-
and Cancer Vaccines ing long-lasting immunity. Early cancer vaccines used tumor
The possibility of stimulating a patient’s own immune system cell lysates or whole tumor cells that were inactivated by treat-
to respond to TAAs has long intrigued scientists. In 1891, the ments such as irradiation.67 The advantages of this approach
bone sarcoma surgeon, William Coley, began the first system- were that no knowledge was required about the tumor antigens
atic study of immunotherapy.65 In his review of the literature, themselves; the vaccine could theoretically contain all the anti-
he noted that cancer patients who developed an infection after genic components of the tumor. Unfortunately, clinical trials
surgery experienced tumor regression and had a better prog- found that most killed tumor cell vaccines did not have a sig-
nosis than patients who did not acquire an infection. Inspired nificant effect on patient survival; as a result, this approach is
by this knowledge, he decided to inject one of his cancer pa- generally not used today.68
tients with Streptococcus pyogenes bacteria. To his amazement, The focus of more recent research is on the development of
the patient’s tumor shrank and the patient became cancer free. antigen-specific vaccines for cancer.11,61,67-69 In one approach
He went on to treat additional patients and observed shrinkage to developing these vaccines, the genes that code for TSAs are
of their malignant tumors, although some of the patients died identified and cloned in recombinant vectors such as viruses
from infection because the bacteria were alive and virulent. or bacterial plasmids. The vectors can be genetically engineered
Coley then developed a less dangerous version of his treatment, so that they produce peptides that are recognized by CTLs,
which consisted of a mixture of killed S pyogenes and killed which, as we discussed, are important effectors of tumor im-
Serratia marcescens bacteria. This formulation later became munity. Another approach is to use synthetic peptides or full-
known as “Coley’s toxins” and was widely used by Dr. Coley length tumor proteins in the presence of a vaccine adjuvant.
and other physicians for the next 30 years to treat patients with Examples of such vaccines include the HER2 antigen to treat
inoperable bone and soft-tissue sarcomas. a subset of breast cancer patients, the tumor immunoglobulin
However, Coley’s treatment was controversial and many idiotype to target B-cell lymphoma, and the MAGE-3 antigen
doctors who used the toxins did not get good results, particu- for some patients with melanoma or NSCLC.61,69
larly with other types of cancer. In 1962, the FDA refused to An interesting strategy that has gained much attention is the
recognize the formulation as an effective cancer therapy and it use of dendritic cells to immunize patients against their own
became illegal to use Coley’s toxins to treat cancer. Despite the tumors. In this approach, dendritic cells (DCs) are isolated
criticism of Coley’s work, the medical community later recog- from the cancer patient and incubated with the pertinent
nized that his premise of stimulating the immune system to ef- tumor antigen or transfected with the gene that codes for the
fectively treat cancer bore some merit. Today we acknowledge antigen. The antigen-loaded DCs are then readministered to
Coley as the “Father of Immunotherapy.”65 the patient, where they are believed to function as potent APCs.
Although Coley’s toxins are no longer in use today, other Sipuleucel-T (Provenge®), the only FDA-approved cancer vac-
agents have been used to nonspecifically boost the immune sys- cine at the time of this writing, is based on this technology. The
tem. For example, Bacillus Calmette Guerin (BCG), a live but vaccine, which is designed to treat patients with advanced
weakened strain of Mycobacterium bovis bacteria, is considered prostate cancer, is produced by incubating the patient’s own
to be the treatment of choice for noninvasive bladder cancer.66 peripheral blood cells with a fusion protein composed of the
Serial treatments in which BCG is directly delivered into the antigen, prostatic acid phosphatase (PAP), and the cytokine
urinary bladder through a catheter have been shown to be ef- GM-CSF, which is thought to promote DC activation and in-
fective in reducing the progression of tumor growth and in de- duce a PAP-specific T-cell response. Phase III clinical trials with
laying recurrence of the cancer in these patients. The exact Provenge have shown improvement in median overall patient
mechanism by which BCG works is unknown, but it is believed survival.70
to stimulate cytokine production by macrophages and activated Although cancer vaccines show much promise for the fu-
lymphocytes, increase anti-tumor effects of macrophages and ture, they also have some important limitations. As previously
other cells of the innate defense system, and increase T-cell– mentioned, tumor cells can evade the immune response by
mediated immune responses. creating an immunosuppressive microenvironment in which
The development of prophylactic cancer vaccines offers an T cells are unable to fully exert their tumoricidal potential. It
effective approach to active immunotherapy. These vaccines have may therefore be necessary to return the tumor environment
been generated for the purpose of preventing virus-associated into an immunosupportive tissue before a cancer vaccine can
cancers (see Chapter 25). Notably, the human papilloma virus be fully effective.69,71 Scientists are attempting to do this by
promoting local production of certain cytokines (e.g., IL-12 Two examples of cytokines that have been widely studied are
and IFN-α) and using antibodies to eliminate Treg cells or IFN-α and IL-2.
block molecules that inhibit immune responses (see Passive Interferons were the first cytokines that were used as bio-
Immunotherapy later).71 logical response modifiers. IFN-α has been the most com-
Unlike vaccines for infectious diseases, which are used to monly used IFN in cancer therapy and has been approved by
prevent infection, most cancer vaccines are immunotherapeutic, the FDA for the treatment of several types of cancer, including
being administered after the disease has occurred. They are fre- malignant melanoma, hairy cell leukemia, chronic myeloid
quently given to patients in the advanced stages of disease when leukemia, and Kaposi’s sarcoma.72 IFN-α is thought to promote
other treatment options have been exhausted. In this situation, anti-tumor effects by increasing tumor immunogenicity, en-
the patient’s immune system has often been compromised be- hancing dendritic cell responses to the tumor, enhancing Th1
cause of the disease process or the effects of chemotherapy; responses and cell-mediated cytotoxicity, promoting tumor
therefore, response to the vaccine may be suboptimal. In these apoptosis, and inhibiting angiogenesis.77 Although high doses
cases, it may be more beneficial to provide the patient with of IFN-α are associated with better clinical responses than low
components of the immune system through passive or adoptive doses of the cytokine, they also generate strong adverse effects,
immunotherapy to more effectively target destruction of the including fever, asthenia (loss of muscle strength), neutropenia,
tumor.67 These approaches to cancer immunotherapy will be and nausea and vomiting.11,78
discussed in the sections that follow. Of all the interleukins, interleukin-2 (IL-2) has been the
most extensively studied. IL-2 induces T-cell proliferation and
Passive Immunotherapy enhancement of CTL and NK cell function (see Chapter 6).
However, clinical trials revealed that systemic administration
Passive immunotherapy, as previously mentioned, involves
of IL-2 as immunotherapy was limited because of its short half-
the administration of soluble components of the immune sys-
life (fewer than 10 minutes) and serious adverse effects, includ-
tem to boost the immune response. Two approaches to passive
ing vascular leakage syndrome, marked fluid retention, and
immunotherapy in cancer patients involve the administration
shock.78 Although this cytokine is still used to treat metastatic
of cytokines to nonspecifically enhance the immune response
melanoma and advanced renal cancer, it is rapidly cleared from
and treatment with monoclonal antibodies to target specific
the body and its most effective use may be to activate immuno-
tumor antigens.
competent cells in vitro for adoptive immunotherapy (see
Adoptive Immunotherapy later).4,11,78
As discussed in Chapter 6, cytokines are small proteins that Although cytokines continue to be incorporated in im-
play an important role in regulating immune responses by serv- munotherapy, their use has been limited because of the serious
ing as chemical messengers that affect the interactions between and sometimes life-threatening side effects associated with
cells of the immune system. There have been two main appli- high-dose systemic treatment as previously discussed. The
cations of cytokines in cancer treatment: use as hematopoietic cytokine network is very complicated and administration of a
growth factors and use as therapeutic agents. cytokine can have multiple, and sometimes unwanted, effects.4
Because chemotherapy drugs inhibit cell division, they often For example, in addition to its immunostimulatory effects,
adversely affect the development of hematopoietic stem cells in IL-2 is also thought to be necessary for the generation and
the bone marrow, resulting in decreased production of WBCs, maintenance of Treg cells, which can be involved in enhancing
red blood cells (RBCs), and platelets. Hematopoietic growth tumor growth. Studies are now underway to see if some of
factors, also known as colony stimulating factors, can be ad- these obstacles can be overcome through more localized ad-
ministered to patients to help them recover from or prevent these ministration of cytokines, use of cells that are genetically engi-
toxicities. Some of the main colony stimulating factors that have neered to express specific cytokine genes, or therapies using
been used to treat cancer patients are granulocyte colony stim- small doses of cytokines combined with each other or with
ulating factor (G-CSF), granulocyte-macrophage colony stimu- chemotherapy drugs.72,78,79
lating factor (GM-CSF), erythropoietin, and interleukin 11
(IL-11) (see Chapter 6).72 G-CSF stimulates hematopoietic stem
cells to develop into granulocytes, whereas GM-CSF stimulates Monoclonal antibodies take a more specific approach to im-
hematopoietic stem cells to develop into granulocytes and munotherapy. As we discussed in Chapter 5, these antibodies
monocytes, thus reducing the patient’s risk for severe are derived from a single clone of cells, providing for an abun-
infections.73,74 Erythropoietin stimulates production of RBCs dant source of highly specific antibodies directed toward one
from the bone marrow and can be used to treat patients with particular epitope of an antigen. Monoclonal antibodies in can-
severe anemia.75 IL-11 stimulates the maturation of megakary- cer immunotherapy have been directed against seven major
ocytes, helping patients to recover from chemotherapy-induced categories of antigens: CD antigens, glycoproteins, glycolipids,
thrombocytopenia.76 carbohydrates, vascular targets, stromal and extracellular anti-
The therapeutic application of cytokines is aimed at enhanc- gens, and growth factors.80 These antibodies have different
ing patients’ immune responses to their tumors. Preclinical and mechanisms of action, depending on their target.81 The major
clinical investigations have been conducted for the interferons approaches to monoclonal antibody therapy are discussed in
(IFNs), tumor necrosis factors (TNFs), and several interleukins. the text that follows and summarized in Table 17–5.
 Table 17–5 Approaches to Cancer Immunotherapy Using Monoclonal Antibodies
TARGET MECHANISM
OF THERAPY OF ACTION EXAMPLES
Surface Opsonization Rituximab, a MAb* directed against the CD20 antigen on B cells; used
antigens Complement- to treat B-cell neoplasms
on tumor mediated Alemtuzumab, a MAb directed against mature lymphocyte antigen,
cells cytotoxicity CD52; used to treat chronic lymphocytic leukemia and T-cell
ADCC lymphomas
Cell surface Block signaling Panitumumab, a MAb directed against epidermal growth factor receptor
receptors pathways involved (EGFR), used to treat colorectal cancer
in cell proliferation Trastuzumab, a MAb directed against HER2, used to treat breast and
and survival gastroesophageal tumors with overexpressed HER2
Antigens Inhibit formation of Bevacizumab, a MAb directed against vascular endothelial growth factor
involved in blood vessels nec- (VEGF); for treatment of glioblastoma, colon, lung, and renal cancers
angiogenesis essary for delivery
of oxygen and
nutrients to the
tumor
Molecules Enhance anti- Ipilimumab, a MAb directed against CTLA-4 (cytotoxic T-lymphocyte
that block tumor-specific antigen 4); for treatment of metastatic melanoma
T-cell activation T-cell responses by Nivolumab and Lambrolizumab, MAbs directed against PD-1
and proliferation preventing T-cell (programmed death-1); used to treat melanoma, colon cancer.
by binding to inhibition and other tumors
molecules on
antigen-presenting
cells
Antibody–drug Deliver potent toxic Brentuximab vedotin, an immunotoxin directed against the CD30
conjugates molecules directly to antigen; used to treat Hodgkin lymphoma and systemic anaplastic
(immunotoxins) tumor cells large cell lymphoma
directed against Trastuzumab-DM1, an immunotoxin directed against the HER2 antigen;
TSAs for treatment of HER2-positive metastatic breast cancer
*MAb = monoclonal antibody.

Some monoclonal antibodies are directed against antigens such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), which
found on the surface of the tumor cells. These antibodies are prevents T-cell activation when bound to the CD80 (B7-1) or
believed to destroy the tumor through the same mechanisms CD86 (B7-2) proteins on APCs, and PD-1, a receptor on T cells
that are used to attack infectious organisms, namely opsoniza- that inhibits T-cell proliferation when it is bound to pro-
tion, complement-mediated cytotoxicity, and ADCC.80,81 A sec- grammed death ligand 1 (PD-L1).81,82
ond group of immunotherapeutic monoclonal antibodies target One strategy to increase the effectiveness of monoclonal an-
surface receptors involved in intracellular pathways that lead tibodies involves linking them to potent cytotoxic drugs that
to the growth and immortality of cancer cells. These receptors can be taken up by the tumor cells. These products are known
are expressed at higher-than-normal levels on epithelial cancers as antibody–drug conjugates or immunotoxins. They reduce
of the colon, breast, lung, head, and neck. Therapeutic anti- the systemic side effects of the toxins by localizing a small
bodies bind to these receptors and block cell signals that are number of toxic molecules directly to the tumor cells using
necessary for activation of molecular pathways involved in cell a tumor-specific antibody.81 After binding to an antigen on
growth and survival.80,81 the tumor surface, the conjugate is quickly internalized by the
A third group of monoclonal antibodies target antigens in- cancer cell through receptor-mediated endocytosis and trans-
volved in angiogenesis or the formation of blood vessels that ported to the lysosomes. The cytotoxic drug is released from
are necessary to provide the oxygen and nutrients needed for its antibody into the cytoplasm of the tumor cell, where it
tumor growth.80,81 Many of the antibodies in this category are exerts potent toxic effects.83,84
directed against the vascular endothelial growth factor (VEGF) The first immunotoxins, derived from the plant toxin
family of proteins or their receptors. A fourth group of mono- ricin, or toxins from diphtheria-causing or Pseudomonas bac-
clonal antibodies boost the immune response to the tumor by teria, had some effectiveness against tumors, but produced
blocking inhibitory pathways that inactivate T cells.80 This ap- toxic side effects.85 Improvements in linker technology and
proach uses monoclonal antibodies to inhibitory receptors conjugate design have led to the development of products
that are more effective and have fewer side effects.84,85 Newer researching several new approaches to monoclonal antibody
generation antibody–drug conjugates are made from modi- therapy that may overcome these limitations in the future.86
fied toxins that can be genetically engineered by cloning
genes for antibody fragments with genes for the adapted
toxin.83 Two FDA-approved preparations that have shown
Adoptive Immunotherapy
promise are brentuximab vedotin, an immunotoxin directed Scientists have reasoned that because cell-mediated immunity
against the CD30 antigen that is used to treat Hodgkin lym- is so important in defense against tumors, transfer of cells of
phoma (HL) and systemic anaplastic large cell lymphoma the immune system to cancer patients may effectively assist
(sALCL); and trastuzumab-DM1, which has specificity for them in eliminating tumor cells. This approach, known as
the HER2 antigen and is used to treat patients with HER2- adoptive immunotherapy, is discussed in detail in Chapter 25.
positive metastatic breast cancer.81,83,84 Early experiments conducted in mice in the 1960s showed
Monoclonal antibodies have been used to treat almost all that lymphoid cells from mice immunized with certain tumors
major subtypes of cancer81; as a result, this treatment modality were able to protect against tumor growth when they were
has been established as one of the most successful therapeutic transplanted into genetically identical mice; this response was
strategies for cancer in the last 20 years.80 A listing of FDA- enhanced in the presence of IL-2.58,59,87 In pioneering studies
approved monoclonal antibodies and other drugs in oncology conducted in the late 1980s by Dr. Steven Rosenberg and his
can be found at http://www.centerwatch.com/drug-information colleagues, it was discovered that adoptive immunotherapy
by searching in the FDA-approved-drugs category for the could be applied to the treatment of human cancer.88-90 These
term oncology. scientists isolated lymphocytes from surgically removed tumors
Although monoclonal antibodies are making their way of patients with metastatic melanoma and grew them in the
into the clinic, they have limitations.81 Some patients de- laboratory in the presence of IL-2. They found that these cells,
velop hypersensitivity reactions to the antibody proteins. referred to as tumor-infiltrating lymphocytes (TILs), demon-
This problem has been substantially reduced as monoclonal strated potent cytolytic activity against autologous melanoma
antibody technology has evolved from making purely mouse cells. Taking these findings a step further, they prepared ex-
antibody products to manufacturing fully human products panded populations of TILs from melanoma patients in the
(see Chapters 5 and 25).82 Monoclonal antibody treatment laboratory and infused them back into the same patients in the
can also cause toxicity if the target antigen is expressed on presence or absence of IL-2. These early studies found only a
normal cells. Therapy with monoclonal antibodies may be slight improvement in patient response, but demonstrated the
ineffective if the antibodies are unable to permeate through potential value of adoptive immunotherapy for human cancer.
tumor tissues or bind their target antigen molecules with Subsequent modifications of technique resulted in signifi-
high affinity. Finally, cancer cells can develop resistance to cantly improved patient outcomes. Instead of administering
monoclonal antibodies, analogous to the way that bacteria the entire population of TILs, cells are subcultured and indi-
can develop resistance to antibiotics. Cancer scientists are vidually tested for their reactivity to the tumor (Fig. 17–8).

Excise Reinfuse after

tumor lymphodepletion

Select and
expand cells
Plate
fragments
" " #
Adoptive immunotherapy with
" # "
tumor-infiltrating lymphocytes (TILs). The
patient’s tumor is surgically removed and cut into
fragments, which are cultured in vitro with IL-2.
The cultures are screened for lymphocytes with
potent anti-tumor activity. Positive cultures are Assay for specific
tumor recognition
expanded further in the presence of IL-2 and are
infused into the cancer patient. Before infusion,
the patient has been treated with high-dose Culture with IL-2
chemotherapy or radiation to deplete immuno-
suppressive cells.
Only the cultures that show potent anti-tumor activity are se-
lected for further expansion and infusion into patients. In ad- • Malignant tumors consist of immortal cells that resist
dition, the effectiveness of the therapy has been improved by apoptosis and proliferate in an unregulated manner. They
pretreating patients with total body irradiation or high-dose can also induce angiogenesis, invade nearby tissues, and
chemotherapy before conducting the adoptive cell transfer. spread to distant sites in the body. These characteristics
This pretreatment is thought to eliminate cells that could exert are influenced by mutations, genomic instability, and in-
immunosuppressive effects before the treatment begins.91,92 flammatory responses of the immune system.
Adoptive therapy with autologous TILs has shown good clin- • The concept of tumor immunology is based on the
ical response rates in patients with malignant melanoma but premise that tumor cells possess antigens that can be
has not yet been successful with other types of cancer, which recognized as foreign by the immune system. Some anti-
are probably less immunogenic.93 gens are tumor specific, or unique to a particular type of
Alternative treatments being investigated involve the use of tumor cell. However, most are TAAs that are expressed
genetically engineered T cells. These approaches may be ad- by normal cells as well as tumor cells. The latter can be
vantageous because they allow for the design of T cells that are classified as shared TSAs, which are expressed in many
targeted toward specific tumor antigens. One method to con- tumors, but not in most normal tissues; differentiation
struct these genetically engineered cells involves isolating antigens, which are expressed on immature cells of a
T cells from patients with good anti-tumor responses and particular lineage; or overexpressed antigens, which are
cloning the genes for their TCRs into viral vectors that can be found in higher levels on malignant cells than on nor-
used to infect T cells from the patient to be treated.94 mal cells.
A second approach involves isolating TCR genes from hu- • Tumor markers are biological substances that are increased
manized mice that have been immunized with the tumor anti- in the blood, body fluids, or tissues of patients with a par-
gen of interest and cloning these into recombinant vectors to ticular type of cancer. They can be detected by immuno-
deliver the sequences to T cells from the cancer patient. Hu- histochemistry, automated immunoassays, or molecular
manized mice are mice into which human cells or tissues have methods, and are used in cancer screening and diagnosis,
been transplanted. Immunodeficient strains of mice are used determining patient prognosis, and monitoring patient re-
so that they do not reject the human transplant. These mice sponse to therapy.
are widely used models for studying the effects of therapeutic • The ideal tumor marker should be produced by the
agents on human cells before they are allowed to be adminis- tumor or by the patient’s body and be secreted into a
tered to humans. biological fluid, where it can be easily and inexpensively
A third method is to generate chimeric antigen receptors quantified. It should rise with increasing tumor
(CAR). CARs are most often constructed by combining the load and have a high sensitivity and specificity. Most
antigen-binding variable fragment of a monoclonal antibody tumor markers do not possess these characteristics
to a tumor antigen with intracellular domains of the TCR that and are therefore not suitable for screening the general
provide activating signals to the T cells. The CAR approach population.
has become very popular because CARs can target tumor anti- • Tumor markers are best used to monitor patient response
gens in an MHC-independent manner, so that the product can to therapy by performing serial measurements over time.
be universally used instead of being restricted for patients with If therapy is effective, the amount of tumor marker will
a particular HLA type.67,91,94 decrease. Ineffective therapy and recurrence of cancer is
Although adoptive immunotherapy looks promising, some indicated by an increase in the tumor marker level. Ideally,
toxicities have been reported.95 In addition, adoptive-based these increases would precede other signs of disease re-
therapies are expensive, personalized treatments that are currence by several months.
available in only a limited number of sites worldwide. It is • Some commonly used serum tumor markers and their pri-
hoped that advances in technology will make it possible to mary cancer associations are presented in Table 17–4.
mass-produce tumor-specific T cells for use in adoptive im- • Tests for circulating tumor markers are most commonly
munotherapy in the future.91 performed by highly sensitive, automated immunoassays.
Their results can be affected by reagent variability among
manufacturers, cross-reactivity with similar antigens,
SUMMARY tumor antigen excess (producing a postzone, or high-dose
hook effect), or presence of heterophile, anti-animal, or
• Cancer arises from exposure of the host to environmental autoantibodies in the patient sample. Therefore, results
factors that induce mutations in proto-oncogenes and should always be considered in conjunction with clinical
tumor suppressor genes. The former influence cell prolif- factors and other laboratory tests.
eration and the latter regulate entry of cells into the cell • Molecular techniques such as PCR, FISH, and microarray
cycle, maintain genetic stability, and repair damaged DNA. are commonly used in clinical testing for tumor markers.
 DNA sequencing is being used increasingly to identify involve avoiding immune recognition by downregulation
mutations associated with cancer. of surface class I MHC molecules, resistance to apopto-
• The hypothesis of immunosurveillance states that the sis, suppression of anti-tumor immune responses, and
immune system continually patrols the body for cancer chronic inflammation.
cells and eliminates them before they become clinically • An understanding of the interactions between the im-
evident. mune system and the tumor can help in the development
• There are innate and adaptive immune responses against of rational immunotherapies, whose purpose is to harness
tumor cells. Innate responses are mediated by NK cells the ability of the immune system to destroy tumor cells.
and macrophages. Adaptive immune responses are medi- Immunotherapeutic approaches include stimulation of
ated by CTLs, antibodies, activated Th cells and cytokines. the immune system with bacterial products or cancer
• Tumor cells are thought to escape these responses vaccines, passive therapy with cytokines, monoclonal
through the process of immunoediting, which allows antibodies, or antibody–drug conjugates, and adoptive
some of the tumor cells to develop into genetic variants immunotherapy with tumor-infiltrating lymphocytes or
that are resistant to immune defenses. This process can genetically engineered T cells.

Study Guide: Clinical Applications of Tumor Markers

CLINICAL
APPLICATION PURPOSE AND COMMENTS BENEFITS LIMITATIONS EXAMPLE
Screening To identify cancer in asymptomatic indi- Detection of False-positive results PSA
viduals in a population. cancer at an can lead to patient
early stage. anxiety, unnecessary
testing, and
overtreatment.
False-negative results
can give misleading
reassurance.
Diagnosis To identify cancer in a particular patient. Helps distinguish Tests by themselves PSA
Presence of the marker or an elevation between are not diagnostic
of the marker above normal levels diseases with and should be used
suggests the presence of the cancer. similar clinical in conjunction with
manifestations. other tests and
clinical findings.
Prognosis To predict the clinical outcome of a Used to identify HER2
cancer patient and aid in therapeutic the level (mild
decision making. to aggressive)
An initial high concentration or an increas- and type of
ing level of the tumor marker over time therapy that
indicates a worse prognosis. is best for
a particular
patient.
Monitoring To observe the response of a cancer Elevations can The cancer must be CA 125
patient to treatment. indicate tumor positive for the
Effective treatment is indicated by recurrence marker before
decreasing levels of the marker over before other treatment begins.
time. signs become
Increasing levels of the tumor marker evident.
indicate that the therapy is not
effective.
CASE STUDIES
1. A 45-year-old woman went to her physician’s office after an enlarged prostate with no distinct nodules or abnor-
noticing a lump during her breast self-examination. She mal areas. The patient’s serum level of prostate-specific
had a strong family history of breast cancer. The lump antigen (PSA) was determined and compared with the
was detected on mammography and was found to be a level from the previous year. The physician also asked
0.5-cm mass that was adherent to her skin. Analysis that the bound-to-total PSA ratio be determined. The test
found her CA 15-3 levels to be 60 IU/mL, which is dou- results are shown in the data that follows.
ble the upper limit of the reference interval. After sur-
gery, the levels of CA 15-3 dropped, but at a rate that Laboratory Results
was slower than the biological half-life. They remained PATIENT REFERENCE
above 30 IU/mL. The tumor morphology indicated ma- TEST RESULTS INTERVAL
lignancy, so it was tested for HER2 expression, which
PSA October 2015 3.8 ng/mL 0–3.5 ng/mL
was elevated, and estrogen and progesterone receptors,
which were negative. PSA October 2014 3.5 ng/mL 0–3.5 ng/mL
Bound/free PSA 25.8% 23.4%
Questions
a. Do you think that there is a residual tumor? If
so, why? Questions
b. In addition to chemotherapy, what other therapy a. Do any tissues other than the prostate produce PSA?
would you recommend? Why? Could there be another source of the PSA in this case?
c. What type of therapy is unlikely to be successful? b. What is PSA velocity?
Why? c. Should this man have a biopsy? Do you think this
2. A 66-year-old male went to his urologist complaining of man has cancer? Why?
frequent urination with only small volumes of urine, cre- d. At what point would PSA testing no longer be
ating great urgency. During the DRE, the urologist felt recommended for this patient?

REVIEW QUESTIONS
1. How can normal cells become malignant? 3. Which of the following is an example of a tumor-
a. Overexpression of oncogenes specific antigen?
b. Underexpression of tumor suppressor genes a. BCR/ABL fusion protein
c. Viral infection b. CEA
d. All of the above c. CA 125
d. PSA
2. Which of the following best summarizes the concept
of tumor development via immunoediting? 4. Most tumor markers are not used to screen the general
a. Tumor cells produce cytokines that are toxic to population because they
T cells. a. cannot be inexpensively quantified.
b. Tumor cells that can escape the immune system b. do not rise to high enough levels in the presence
have a growth advantage over tumor cells that are of cancer.
destroyed during immunosurveillance. c. can also be elevated in conditions other than the
c. T-cell activity causes an increase in MHC expres- cancer.
sion on tumor cells that allows them to escape the d. vary too much between patients belonging to
immune system. different ethnic populations.
d. Secreted tumor-associated antigen saturates T-cell
receptors and makes T cells incapable of binding to
tumor cells.
5. Both AFP and hCG exhibit serum elevations in 10. Which of the following markers could be elevated in
a. pregnancy. nonmalignant liver disease?
b. ovarian germ cell carcinoma. a. AFP
c. nonseminomatous testicular cancer. b. CEA
d. all of the above. c. CA 15-3
d. All of the above
6. Suppose a patient with ovarian cancer had a serum
CA 125 level of 50 kU/L at initial diagnosis. After her 11. Each of the following markers is correctly paired
tumor was surgically removed, her CA 125 level de- with a disease in which it can be used for patient
clined to 25 kU/L. She received chemotherapy drug monitoring except
#1; after 1 year, her CA 125 level was 40 kU/L. She a. CEA/choriocarcinoma.
was then given chemotherapy drug #2 and her CA b. CA 15-3/breast adenocarcinoma.
125 level rose to 60 kU/L. These results indicate that c. CA 125/ovarian adenocarcinoma.
a. surgery was effective in removing the patient’s d. CA 19-9/pancreatic adenocarcinoma.
tumor.
b. chemotherapy drug #1 was more effective than 12. Which of the following is a marker used in immuno-
chemotherapy drug #2. histochemical staining to identify tumors of epithelial
c. both chemotherapy drug #1 and chemotherapy origin?
drug #2 were effective. a. Cytokeratins
d. neither chemotherapy drug #1 nor chemotherapy b. Vimentin
drug #2 were effective. c. CD45
d. CD10
7. All of the following are recommended for cancer
screening in the groups indicated except 13. Which of the following assays would you recommend
a. CA 125/women of reproductive age. to test for chromosomal rearrangements such as the
b. AFP/subjects at high risk for liver cancer. BCR/ABL translocation seen in CML?
c. PSA/men over 50 with at least 10 years of life a. PCR
expectancy. b. FISH
d. none of the above. c. Microarray
d. Next generation sequencing
8. The best use of serum tumor markers is considered
to be in 14. Innate immune responses thought to be involved in
a. screening for cancer. defense against tumors include
b. initial diagnosis of cancer. a. NK cell-mediated apoptosis.
c. monitoring patients undergoing cancer treatment. b. MHC I-restricted T-cell–mediated destruction.
d. determining patient prognosis. c. ADCC.
d. all of the above.
9. In order to use a tumor marker to monitor the course
of the disease, which of the following must be true? 15. A woman with breast cancer is treated with a mono-
a. The laboratory measures the marker with the same clonal antibody to HER2. This is an example of
method over the entire course of the patient’s a. a cancer vaccine.
treatment. b. an immunotoxin.
b. The marker must be released from the tumor or, c. passive immunotherapy.
because of the tumor, into a body fluid that can d. active immunotherapy.
be obtained and tested.
c. The marker’s half-life is such that the marker
persists long enough to reflect tumor burden but
clears fast enough to identify successful therapy.
d. All of the above.
 Immunoproliferative
Diseases

After finishing this chapter, you should be able to: MALIGNANT TRANSFORMATION
OF HEMATOLOGIC CELLS
1. Compare and contrast leukemias and lymphomas.
Cell Properties
2. Describe some of the cellular properties and genetic changes that
occur during malignant transformation of hematologic cells. Genetic Changes
3. Cite the cellular characteristics used in the classification scheme CLASSIFICATION OF HEMATOLOGIC
recommended by the World Health Organization for identification MALIGNANCIES
of the hematopoietic neoplasms. LEUKEMIAS
4. Differentiate between Hodgkin lymphoma (HL) and non-Hodgkin Acute Lymphocytic Leukemia (ALL)
lymphoma (NHL). Chronic Lymphocytic Leukemia or
5. Differentiate between acute leukemias and chronic leukemias and Lymphoma
discuss an example of each type. Hairy Cell Leukemia
6. Associate specific CD markers with selected hematologic malignancies. LYMPHOMAS
7. Differentiate between monoclonal gammopathy of undetermined Hodgkin Lymphoma (HL)
significance (MGUS) and other plasma cell dyscrasias. Non-Hodgkin Lymphoma (NHL)
8. Correlate specified clinical manifestations and laboratory results PLASMA CELL DYSCRASIAS
with multiple myeloma or Waldenström macroglobulinemia. Monoclonal Gammopathy of
9. Indicate the ways in which laboratory tests can be used to diagnose Undetermined Significance (MGUS)
and follow the progression of immunoproliferative disorders. Multiple Myeloma
10. Explain the underlying principles of serum and urine protein Waldenström Macroglobulinemia
electrophoresis (UPE), immunofixation electrophoresis (IFE),
Heavy-Chain Diseases
immunosubtraction, and serum free light chain (sFLC) analysis.
ROLE OF THE LABORATORY IN
11. Contrast serum protein electrophoresis (SPE) and IFE results seen
EVALUATING IMMUNOPROLIFERATIVE
in monoclonal gammopathies with those observed in polyclonal
DISEASES
increases in immunoglobulins.
Immunophenotyping by Flow
12. Discuss the types of genetic abnormalities that are frequently seen
Cytometry
in hematologic malignancies and the laboratory methods used to
detect them. Evaluation of Immunoglobulins
Serum Protein Electrophoresis (SPE)
Immunofixation Electrophoresis (IFE)
Serum Free Light Chain Analysis (sFLC)
Evaluation of Genetic and
Chromosomal Abnormalities
SUMMARY
CASE STUDIES
REVIEW QUESTIONS
You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the
laboratory exercises that accompany this text.
 Bence Jones proteins Immunophenotyping Monoclonal gammopathy Paraprotein (M protein)
Cryoglobulins Immunosubtraction of undetermined Plasma cell dyscrasias
(Immunotyping) significance (MGUS)
Fluorescence in situ Polyclonal
hybridization (FISH) Leukemias Multiple myeloma
Proto-oncogene
Hairy cell leukemia Lymphomas Non-Hodgkin or lymphocytic
Waldenström
lymphomas (NHLs)
Heavy-chain diseases Monoclonal macroglobulinemia
gammopathies Oncogene
Hodgkin lymphoma (HL)

This chapter focuses on malignancies of the immune system growth regulation, mutations can result in arrested maturation
that involve cells of lymphoid lineage. The lymphoid malig- of a cell. Thus, some malignant hematopoietic cells may not
nancies can be broadly classified as leukemias, lymphomas, develop into properly functioning mature cells but may remain
and plasma cell dyscrasias. In leukemias, the malignant cells at an earlier stage of differentiation and continue to replicate.
are primarily present in the bone marrow and peripheral Malignant and premalignant proliferation of cells can occur at
blood. In lymphomas, the malignant cells arise in lymphoid any stage in the differentiation of the lymphoid lineages.
tissues, such as the lymph nodes, tonsils, or spleen. There can Cells of the immune system are at great risk for malignant
be an overlap between the sites affected by leukemias and lym- transformation because the features that characterize the de-
phomas, especially when the malignancy is far advanced. How- velopment of malignancy are also a normal part of the im-
ever, it is generally most useful to classify the malignancy mune response. For example, as we discussed in Chapter 4,
according to the site where it first arose, rather than the sites it proliferation of T and B lymphocytes is an integral part of the
can ultimately involve. immune response to an antigenic stimulus. In addition, gene
The plasma cell dyscrasias (disorders) primarily include rearrangements are a normal part of lymphocyte maturation
multiple myeloma and Waldenström macroglobulinemia. and somatic hypermutations occur in the immunoglobulin
These commonly involve the bone marrow, lymphoid organs, genes of the B cells during immunoglobulin class-switching
and other nonlymphoid sites. They are considered biologically and the generation of high-affinity antigen receptors. Despite
distinct and are not classified as either leukemias or lym- being affected by abnormal regulation, malignant lymphoid
phomas. However, plasma cells may be found in the blood late cells generally retain some or all of the morphological and
in the course of myeloma. This phenomenon is sometimes re- functional characteristics of their normal counterpart—for
ferred to as plasma cell leukemia. Monoclonal gammopathy of example, their characteristic cell surface antigens or secretion
undetermined significance (MGUS) is a premalignant con- of immunoglobulin. These characteristics are often used to
dition that can develop into multiple myeloma, Waldenström classify lymphoid malignancies.
macroglobulinemia, or other lymphoproliferative disorders The immune system is naturally diverse and heterogeneous
over time. in its response against a wide range of potential pathogens.
This chapter presents an introduction to some of the lym- Normal immune responses are polyclonal (i.e., cells with dif-
phoid malignancies that are commonly evaluated by the clin- ferent features such as antigen specificity all proliferate in re-
ical laboratory. It is not intended to provide a comprehensive sponse to an immune stimulus). In contrast, malignancies are
discussion of hematopoietic malignancies. The chapter will thought to arise from excessive proliferation of a single mutant
also cover key principles and applications of some of the lab- parent cell to form a clone of genetically identical cells that are
oratory tests that are essential to the diagnosis and monitoring similar in their appearance and surface markers. Suspicion of
of lymphoid malignancies. a hematologic malignancy is raised when there are elevated
numbers of a specific population of lymphocytic cells in the
bloodstream, bone marrow, or lymphoid tissues.
Malignant Transformation
of Hematologic Cells Genetic Changes
Malignancies are generally multifactorial in origin. As we dis-
Cell Properties cussed in Chapter 17, malignant transformation is thought
Hematologic malignancies are characterized by excessive ac- to be a multistep process involving exposures to environmen-
cumulation of cells in the blood, bone marrow, or other lym- tal agents such as chemical carcinogens and radiation, which
phoid organs. This accumulation may occur because of rapid induce a series of genetic mutations. The key genes involved
proliferation of the cells (i.e., excess production) or failure of are proto-oncogenes, which are involved in normal cell
the cells to undergo apoptosis (a normal physiological process growth and division, and tumor suppressor genes, which con-
of cell death). Malignancy may reflect the result of an initially trol cell division by regulating the progression of cells through
normal process in which regulatory steps to control the level the cell cycle and maintaining genetic stability of the cells by
of cell proliferation have failed. In addition to a failure of repairing damaged DNA. Changes in these genes can result
in uncontrolled cell proliferation. Alterations in proto-oncogenes Classification of Hematologic
can convert them into oncogenes, which are involved in
malignant transformation. Aneuploidy (an abnormal number Malignancies
of chromosomes) and deletions of specific chromosome re-
gions are common secondary events that are rarely specific The classification of hematologic malignancies has undergone
to a particular type of lymphoma but provide valuable prog- many changes over the years, as new laboratory techniques
nostic information. have been developed and more knowledge has been gained
The genetic alterations in malignant cells of hematopoietic about these diseases. In the 1950s and 1960s, classification
origin include point mutations involving a change in a single and diagnosis of hematologic malignancies was based primarily
nucleotide base, duplications or deletions of specific genes, on abnormalities in the morphological features of the malig-
and chromosome translocations in which two different chro- nant cells, which were viewed on peripheral blood or bone
mosomes break apart and exchange genetic material. The de- marrow samples fixed onto microscope slides and treated with
tection of translocations is of particular value in the diagnosis stains such as Wright-Giemsa. Advances in understanding
of disease. basic lymphocyte biology led to major rethinking of the use of
Many of the translocations involve an exchange between the classification schemes based solely on cell morphology. Dis-
immunoglobulin or T-cell receptor (TCR) loci with various covery of surface markers on T and B lymphocytes in the
partner chromosomes, leading to abnormal proto-oncogene 1970s and 1980s allowed this information to be incorporated
expression. For example, some of the hematologic malignan- into the diagnosis of hematologic malignancies. The 1990s and
cies are characterized by translocations involving the proto- 2000s witnessed a tremendous expansion of knowledge about
oncogene c-MYC, which stimulates the transcription of several the molecular changes of the tumors. Thus, today, investigators
other genes involved in cell proliferation.1 Overexpression of and clinicians are using a combination of morphological, im-
the c-MYC gene can occur as a result of a rearrangement in munologic, cytogenetic, and molecular techniques to assist in
which c-MYC is placed under the control of a different gene the classification of hematologic malignancies.
promoter sequence. For example, a translocation involving the A number of schemes have been used over the years to classify
c-MYC gene on chromosome 8 and the immunoglobulin µ the hematologic malignancies, including the French-American-
gene on chromosome 14 [t(8;14)] is believed to be involved British (FAB) Cooperative Group consensus criteria for leukemias
in the pathogenesis of some cases of Burkitt lymphoma, a and myelodysplastic syndromes5,6 and the Revised European
B-cell malignancy. As a result of persistent c-MYC expression, American Lymphoma (REAL) classification for leukemias and
several genes that are involved in cell proliferation are activated lymphomas.7
beyond normal levels. The resulting high levels of c-MYC pro- The REAL scheme became the basis for the classification
tein drive the affected cells to continually proliferate. Many scheme for all types of hematologic malignancies which was
small noncleaved cell lymphomas are also associated with the adopted in 2001 and updated in 2008 by the World Health
t(8;14) translocation. Organization (WHO)8,9 This widely accepted system is consid-
Other hematologic malignancies are associated with genes ered the “gold standard” in the classification of tumors for diag-
that affect apoptosis. For example, most cases of follicular lym- nosis and determination of appropriate therapy. The 2008 WHO
phoma have a t(14;18) gene translocation, in which portions update classifies hematologic malignancies into 12 major groups
of chromosome 14 (which contains the Ig heavy-chain genes) and numerous subgroups. These groupings are based on cell lin-
and chromosome 18 (which contains an anti-apoptotic gene eage; specific cancers are further defined by their immunologic
called BCL-2) are exchanged.2 This exchange results in the markers and genetic features, as well as their morphological and
rearrangement and constitutive overexpression of BCL-2. The cytochemical staining properties. Some of the recognized types
BCL-2 gene induces production of an inner mitochondrial of lymphomas, leukemias, and plasma cell dyscrasias are dis-
membrane protein that blocks apoptosis. Therefore, the cells cussed in more detail in this chapter. The 12-group classification
affected by this translocation do not die normally. Even though scheme will continue to evolve as new knowledge is gained
the altered cells do not proliferate at an increased rate, an ex- about the characteristics that typify the various disease entities.
cessive number of cells accumulate because their survival is
enhanced compared with normal cells. Leukemias
Other characteristic translocations result in the production
of a novel fusion protein. For example, chronic myelogenous Leukemias can be broadly divided into two groups based on the
leukemia is characterized by a translocation between the BCR cell type from which they originated: myelogenous and lympho-
(breakage cluster region) on chromosome 9 and the c-ABL cytic. The myelogenous leukemias are derived from the common
proto-oncogene on chromosome 22 (see Fig. 17–2). This re- myeloid precursor and encompass the granulocytic, monocytic,
sults in a BCR/ABL fusion protein, which codes for a continu- megakaryocytic, and erythrocytic leukemias. This section will
ously activated tyrosine kinase enzyme, causing unregulated not cover this group in detail, but will briefly present a classic
cell division. Scientists have developed the anticancer drug genetic change associated with chronic myelogenous leukemia
Gleevec to specifically target the abnormal protein produced and how it is identified in the clinical laboratory. The focus of
by this gene translocation.3,4 Gleevec slows cell growth by this section will be the lymphocytic leukemias, which originate
inhibiting the activity of the altered kinase. from mature lymphocytes or their precursors.
 Each of the two groups of leukemias can be further divided soft tissues, leading to organ dysfunction. ALL is usually seen
into acute or chronic types. Chronic leukemias are usually in children between 2 and 5 years of age and is the most com-
slowly progressive and compatible with extended survival. mon form of leukemia in this age group. ALL is a treatable
However, they are generally not curable with chemotherapy. disease with a remission rate of 90% and a cure rate of 80%
By contrast, acute leukemias are generally much more rapidly in children.10 Remission and cure rates are lower in adults
progressive but have a higher response rate to therapy. Acute with ALL.
lymphoblastic leukemias are characterized by the presence of Immunologically, there are four types of ALL: CALLA
lymphoblasts in the peripheral blood. Lymphoblasts are im- (CD10)-expressing precursor B-cell ALL, pre–B-cell ALL with-
mature lymphocyte precursors. They are small to medium-sized out CALLA (CD10), T-cell ALL, and mature B-cell ALL. CALLA
cells that contain little cytoplasm, dense nuclear chromatin, and (CD10)-expressing precursor B-cell ALL is the most common
indistinct nucleoli (Fig. 18–1). This section will discuss two ALL, whereas pre-B cell ALL is the second most common.
major types of lymphocytic leukemias: acute lymphocytic Mature B-cell ALL is rare.
leukemia and chronic lymphocytic leukemia. Cytogenetics studies provide information that aids in the
diagnosis and prognosis of patients with ALL. In patients with
Acute Lymphocytic Leukemia (ALL) precursor B-cell ALL, hyperdiploidy, in which the malignant
cells contain more than 46 chromosomes, is associated with a
Acute lymphocytic leukemia (ALL) (also known as acute lym- good prognosis, whereas hypodiploidy is associated with a
phoblastic leukemia) is characterized by the presence of very poorer prognosis. The most common translocation in ALL of
poorly differentiated precursor cells (blast cells) in the bone B-cell origin, t(12:21)(p13;q22), also known as TEL-AML-1, is
marrow and peripheral blood. These cells can also infiltrate associated with an excellent prognosis in children.11,12

Connections
Chronic Lymphocytic Leukemia
or Lymphoma
Molecular and Cytogenetic Analysis
The chronic lymphocytic leukemias or lymphomas are a group
Compare the lymphoblasts in Figure 18–1 to the normal lym-
of diseases almost exclusively of B-cell origin.13 They include
phocyte shown in Figure 1–8. Although distinct differences
chronic lymphocytic leukemia (CLL) and small lymphocytic
can be seen in the morphology of the two cell types, not
much else can be determined from simply observing the cells. lymphoma (SLL). The WHO considers CLL and SLL a single
Flow cytometry studies have added much detail to the tradi- disease with different clinical presentations. Both reveal the
tional microscope-based laboratory evaluation of hematologic B-cell marker CD19 but weakly express CD20.
malignancies. Detection of cell surface markers provides CLL is a common hematopoietic malignancy that involves
insight into the lineage and maturation stage of the malignant the expansion of a clone of B cells that have the appearance of
cell type, which can be used to make a more accurate diag- small mature lymphocytes. In about 5% of cases, the malignant
nosis. Molecular and cytogenetic analysis detect genetic clone is T-cell derived. The cytologically normal, but dysfunc-
mutations and chromosomal abnormalities in the cells, provid- tional, lymphocytes accumulate in the bone marrow and blood
ing doctors with a more precise diagnosis and information as well as in the spleen, lymph nodes, and other organs. CLL
about the patient’s prognosis, which they can use to select the
primarily occurs in patients over 50 years of age with a two-
most effective therapy for the patient.
to-one male-to-female predominance. It is the most common
leukemia in adults. Patients usually present with an increase
in the peripheral blood lymphocyte count, which may be an
incidental finding on a routine physical examination. Anemia
and thrombocytopenia are usually absent at the time of diag-
nosis. However, as the malignant lymphocytes continue to in-
crease in number, replacement of normal elements in the bone
marrow leads to anemia and thrombocytopenia. Lymph node
enlargement is prominent early in the disease. CLL is compat-
ible with a long survival. Various treatments can help control
or reduce symptoms but are not curative.

Hairy Cell Leukemia

Hairy cell leukemia is a rare, slowly progressive disease
characterized by infiltration of the bone marrow and spleen
by leukemic cells without the involvement of lymph nodes.
Lymphoblasts in the peripheral blood. (From It has a four-to-one male predominance and is seen in indi-
Harmening D. Clinical Hematology and Fundamentals of viduals over 20 years of age. Patients usually present with
Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis; 2009.) bone marrow disease and pancytopenia because of bone
marrow infiltration; however, the blood lymphocyte count is marrow can be affected. The disease is divided into nodular
usually not very high. Splenomegaly is striking, whereas lym- lymphocytic-predominant HL (NLPHL) and classic HL, which
phadenopathy is generally absent. The clinical presentation includes nodular sclerosis, mixed cellularity, lymphocyte-rich,
can resemble several other B-cell neoplasms including CLL, and lymphocyte-depleted HL. Classic HL is characterized by
SLL, and splenic marginal zone lymphoma. the presence of Hodgkin and Reed-Sternberg (RS) cells in af-
The malignant lymphocytes are round and often have ir- fected lymph nodes and lymphoid organs (Fig. 18–3). RS cells
regular “hairy” cytoplasmic projections from their surfaces are typically large with a bilobed nucleus and two prominent
(Fig. 18–2). They typically are not evident on bone marrow nucleoli. This gives the cell an “owl’s-eyes” appearance. Hodgkin
or spleen preparations. The nuclei are often oval and occupy cells resemble RS cells except that the nuclei are not bilobed
a large percentage of the cell volume. The malignant cells ex- and have a single nucleolus. NLPHL is characterized by large
press mature B-cell markers—CD19, CD20, and CD22—and lymphocyte-predominant cells. The malignant Hodgkin and RS
the IL-2 receptor, CD25, which are not specific for hairy cell cells are generally of B-cell lineage.
leukemia. In contrast, CD103 is highly specific and sensitive The REAL/WHO classification recognizes a basic distinction
for hairy cell leukemia.14 Although dim staining of CD123 is between NLPHL and classic HL, reflecting the differences in
found in other disorders, bright staining is seen in hairy cell clinical presentation, morphology, phenotype, and molecular
leukemia. These cells characteristically contain acid phos- features. RS cells in all subtypes of classic HL have a similar
phatase isoenzyme 5, which can be detected histochemically antigenic profile. They are all CD30+ and about 80% of the
and provides resistance to tartrate treatment. An accurate di- cases are CD15+. Expression of the B-cell marker CD20 is
agnosis is made in most cases because of the cytomorpholog- weak or absent. Some cells also express T-cell markers (e.g.,
ical and immunophenotypic characteristics. Polymerase chain CD3). Hodgkin RS cells were confirmed as originating from
reaction (PCR) for the detection of mutated gene BRAF-V600E B cells with the discovery of the presence of clonal and somat-
has been shown to be a sensitive and specific test for the ically mutated immunoglobulin heavy and light chain gene re-
diagnosis of hairy cell leukemia.15 arrangements. Despite their origin as B cells, Hodgkin RS cells
exhibit a decrease in typical B-cell gene products. The extent
Lymphomas of this gene downregulation is unique among B-cell lym-
phomas.17 RS cells of NLPHL have a different morphology
The lymphomas have commonly been referred to as Hodgkin compared with those found in classic HL; they are referred to
lymphoma (HL) and the non-Hodgkin, or lymphocytic lym- as lymphocytic and histiocytic cells. These cells rarely express
phomas (NHLs). Each of these entities will be discussed in CD30 or CD15 but instead express the B-cell antigens CD19
more detail in the text that follows. and CD20, which are typically absent on RS cells of classic HL.
The lymphocyte-rich form of HL represents about 5% of the
cases of HL and tends to occur in slightly older individuals.
Hodgkin Lymphoma (HL) Nodular sclerosis HL is the most common subtype, representing
HL is one of the most common lymphomas, with a reported about 70% of cases and having the best prognosis. It is charac-
incidence of about 3 cases per 100,000 people in the Western terized by infiltration of a mixture of normal macrophages, lym-
world.16 This often-curable disease occurs both in young adults phocytes, and granulocytes in affected tissues along with small
and the elderly. Peripheral lymph nodes are primarily involved, numbers of RS cells. There is also marked fibrosis, dividing af-
although numerous organs such as the liver, lung, and bone fected lymph nodes into nodules. Mixed cellularity HL also has

Hairy cell leukemia. (From Harmening D. Clinical Reed-Sternberg cell (arrows). (From Harmening D.
Hematology and Fundamentals of Hemostasis. 5th ed. Philadelphia, Clinical Hematology and Fundamentals of Hemostasis. 5th ed.
PA: F.A. Davis; 2009.) Philadelphia, PA: F.A. Davis; 2009.)
a mixed infiltrate of normal cells but with less fibrosis and progressive and compatible with long-term survival, whereas
greater numbers of RS cells. It accounts for about 25% of cases. others are typically highly aggressive and rapidly fatal if not
Lymphocyte-depleted HL has diffuse fibrosis, few infiltrating treated. The various B-cell lymphoma types can be divided
normal cells, the greatest number of RS cells, and the worst into three broad groups for prognostic purposes. The low-
prognosis compared with other HL subtypes.18 risk first group includes CLL, follicular lymphomas, and
Hodgkin RS cells interact with numerous cells including mucosa-associated lymphoid tissue (MALT) lymphomas. The
CD4+ and CD8+ T cells, B cells, plasma cells, macrophages, intermediate-risk second group includes DLBCL and Burkitt
and others. RS cells secrete cytokines and chemokines, some lymphoma. The high-risk third group includes mantle cell
of which attract these cells to the tumor. The presence of this lymphoma and lymphoblastic lymphoma. MALT lymphoma
mixed population of cells is unique among lymphomas. The is often associated with autoimmune conditions such as
nontumor cells often account for 99% of the cells in the Sjögren’s syndrome and Hashimoto’s thyroiditis.
tumor. Hodgkin RS cells are not usually found in the periph- Some lymphomas, such as SLL, originate from small lym-
eral bloodstream. phocytes that are awaiting their first encounter with an im-
Epidemiological studies suggest that HL has an infectious eti- munogen. Small B-cell lymphoma is primarily a disease of the
ology. Patients with HL often have elevated levels of antibody to elderly; the median age is 72 years.24 These lymphomas are in-
Epstein-Barr virus (EBV), the causative agent of infectious dolent but inexorable diseases that are compatible with survival
mononucleosis; EBV nucleic acid has been demonstrated in for up to a decade. They progress to prolymphocytic leukemia
Hodgkin RS cells.19,20 A history of infectious mononucleosis has in 10% to 30% of cases and to large-cell lymphoma or other
been associated with an increased risk of HL, particularly in EBV- aggressive lymphoid malignancies in 10% to 15% of cases.
positive HL in younger adults, whereas no increase of risk be- Other B-cell lymphomas, such as diffuse large-cell lym-
tween infectious mononucleosis and EBV-negative HL has been phoma or lymphoblastic lymphoma, derive from rapidly di-
found.21 Although the specific mechanism of tumorigenesis is viding cells. Lymphoid cells undergo proliferation at two stages
unknown, EBV is known to preferentially infect B cells and im- in their development: an early cycle as they first emerge from
mortalize them in vitro. In addition, viral proteins can induce the bone marrow and a later cycle in response to immunogen
activation of key signaling pathways, producing phenotypic exposure. Thus, rapidly proliferative lymphomas can corre-
changes seen in EBV-infected B cells. spond to either early or late stages of normal development.
These lymphomas behave aggressively; if untreated, they can
cause death in less than a year.
Non-Hodgkin Lymphoma (NHL) Three characteristics usually identify lymphomas as having
NHL includes a wide range of neoplasms. Over two-thirds of a B-cell origin: (1) surface immunoglobulin, which is found on
the patients are greater than 60 years of age and the incidence no other cell type; (2) other cell surface proteins such as CD19
is greater in men than women.22,23 Immunosuppression seems and CD20 that are both sensitive and specific for B cells; and
to be the greatest risk factor for NHL; in fact, an increase in (3) rearranged immunoglobulin genes. In almost all cases, both
cases corresponded to the emergence of human immunodefi- the surface immunoglobulin and the rearranged immunoglob-
ciency virus (HIV). Other conditions associated with increased ulin genes have features of clonality.
risk for NHL include certain autoimmune diseases, congenital The T-cell and NK-cell lymphomas are more difficult to
immunodeficiency disorders, organ transplantation, and expo- characterize than B-cell lymphomas because in cases that are
sure to certain infectious agents. morphologically not clearly malignant, no easy way exists to
Overall, B-cell lymphomas represent about 85% to 90% assay their clonality. Also, a number of T-cell syndromes
of all NHL cases; the remainder involves T cells and NK cells. progress stealthily from atypical but nonclonal proliferations
B-cell lymphomas generally begin in the germinal centers of into clonal malignancies. In cases that are not clearly malignant
lymph nodes. Although the lymphoma can begin in any tis- based on their morphology, two ancillary methods of establish-
sue, the gastrointestinal tract is the most common extranodal ing clonality are available. The first is to use molecular tech-
site for NHL. The most common NHL is diffuse large B-cell niques to detect a clonal rearrangement of the TCR gene. In
lymphoma (DLBCL), which accounts for 30% to 40% of benign populations, each cell exhibits a slightly different re-
NHL. DLBCL is a heterogeneous group of diseases character- arrangement, but in malignant proliferations, the population
ized by diffuse growth of large atypical cells without a hall- of cells uniformly expresses the same rearrangement. A second
mark pattern of surface markers. method is to demonstrate by flow cytometry that the suspi-
The next most common type of B-cell lymphoma, account- cious population of T cells uniformly fails to express an antigen
ing for about 20%, is follicular lymphoma, which originates in that is normally expressed on all T cells.
the follicles of the lymphoid organs and is characterized by a The clinical presentation of NHL varies and depends on the
much more aggressive course than DLBCL. Follicular lym- patient’s age, lymphoma subtype, and site of involvement. Most
phoma is often disseminated at the time of diagnosis; the spleen, individuals present with painless lymphadenopathy. More
liver, and bone marrow are frequently involved. Marginal-zone aggressive forms cause fulminant symptoms such as weight
B cell, peripheral T cell, small B lymphocytic, and mantle loss, fever, and night chills. NHLs are staged, I through IV,
cell lymphoma each constitute between 5% and 10% of all using the Ann Arbor classification system. Staging is based
lymphoma cases. Some of these lymphomas tend to be slowly on the number of lymph nodes affected, their location, and if
extranodal organs are involved. Computed tomography (CT) MGUS.26 Lifelong follow-up of MGUS patients with medical
scan, magnetic resonance imaging (MRI), and positron emis- examinations and pertinent laboratory testing (e.g., SPE, com-
sion tomography (PET) are commonly used for disease assess- plete blood count (CBC), kidney function tests, serum calcium
ment. A number of new treatments have improved the survival levels) is recommended to identify the development of malig-
rate; however, outcomes are variable. Elderly patients in gen- nancy before serious complications occur.26,28 Treatment is not
eral have a poor outcome response to therapy. In addition, cur- recommended unless symptomatic disease develops.
rent smokers with NHL have a greater mortality rate compared
with those who never smoked.25
Multiple Myeloma
Plasma Cell Dyscrasias Multiple myeloma, sometimes called plasma cell myeloma, is a
malignancy of mature plasma cells that accounts for about 10%
The plasma cell dyscrasias include several related syndromes: of all hematologic cancers.31 It is the most serious and common
multiple myeloma, Waldenström macroglobulinemia, and the of the plasma cell dyscrasias. It is usually diagnosed in persons
premalignant conditions MGUS and smoldering multiple between 40 and 70 years of age with a peak age of 65 years.
myeloma (SMM). These conditions are characterized by the Men are slightly more likely (56%) than women to develop
overproduction of a single immunoglobulin component called multiple myeloma. The American Cancer Society estimates
a myeloma protein (M protein), or paraprotein, by a clone of there are 30,330 new cases of multiple myeloma diagnosed each
identical plasma cells. M protein may also be rarely associated year in the United States and 12,650 myeloma-related deaths
with other lymphoproliferative disorders, such as NHL or each year.32 Patients progress from asymptomatic MGUS to
primary amyloidosis. Laboratory evaluation is important in SMM to the symptomatic disease, multiple myeloma. In fact,
the diagnosis and differentiation of these conditions. Diagnosis all cases of multiple myeloma are thought to be preceded by
and monitoring of the plasma cell dyscrasias depend heavily MGUS or smoldering multiple myeloma (SMM).33 Patients with
on detecting and quantitating the M protein. multiple myeloma typically have excess plasma cells in the bone
marrow, a monoclonal immunoglobulin component in the
Monoclonal Gammopathy of plasma or urine, and lytic bone lesions. The plasma cells infil-
trating the bone marrow may be morphologically normal or
Undetermined Significance (MGUS) may show atypical or bizarre cytological features. Malignant
MGUS is a common premalignant condition that is present in plasma cells phenotypically express CD38, CD56, and CD138.
about 3.5% of individuals aged 50 or older.26 People with Approximately 20% of myeloma cells express CD20. Unlike
MGUS produce a monoclonal immunoglobulin but do not normal plasma cells, multiple myeloma cells have the ability to
have symptoms of organ damage or other laboratory findings divide at a slow rate.
that are associated with multiple myeloma or the other plasma The immunoglobulin produced by the malignant clone can
cell dyscrasias. MGUS is usually diagnosed incidentally when be of any type, with IgG being the most common (50%), fol-
patients with various nonspecific symptoms have laboratory lowed by IgA and light chains only. Very often, the production
testing such as serum protein electrophoresis (SPE).27,28 The of heavy and light chains by the malignant plasma cells is not
International Myeloma Working Group (IMWG) has identified well synchronized and an excess of kappa or lambda light
three criteria that define the presence of MGUS: (1) a serum chains may be produced. In about 10% of cases, the myeloma
monoclonal protein concentration of less than 3 g/dL; (2) a cells exclusively produce light chains. These monoclonal light
plasma cell count of lower than 10% of the total cells in the chains can be found in the blood, but are rapidly excreted in
bone marrow; and (3) the absence of signs or symptoms asso- the urine, where they are known as Bence Jones proteins.
ciated with multiple myeloma, known as the CRAB features Rarely do myelomas produce IgM, IgD, IgE, or heavy chains
(increased serum calcium, renal failure, anemia, lytic bone only. Very rarely, two or more distinct M proteins are produced
lesions).29 or a myeloma might not produce a detectable secretory prod-
Research studies have found that patients with MGUS have uct. The level of normal immunoglobulin is often decreased
an average lifetime risk of developing multiple myeloma or in proportion to the amount of abnormal immunoglobulin
other related disorders of about 1% per year.26 MGUS patients (M protein) present in the serum because of the large number
who produce an IgG or IgA monoclonal Ig typically progress of myeloma cells.
to multiple myeloma, patients who produce an IgM mono- The clinical manifestations of multiple myeloma are prima-
clonal Ig can develop Waldenström macroglobulinemia or rily skeletal, hematologic, and immunologic. Hematologic
other lymphoproliferative disorders, and patients with mono- problems are often related to the failure of the bone marrow
clonal light chains can develop light chain multiple myeloma, to produce a normal number of hematopoietic cells because
amyloidosis, or light chain deposition diseases.27 Greater risk myeloma cells progressively replace them (Fig. 18–4). The low
of disease progression has been associated with production of number of hematopoietic precursors in the bone marrow leads
an M protein that is not IgG, a monoclonal Ig concentration of to anemia, thrombocytopenia, and neutropenia. High levels of M
1.5 g/dL or greater, and an abnormal free light chain (κ:λ) ratio protein can interfere with coagulation factors, leading to ab-
(see the text that follows).28,30 Currently, no treatments have normal platelet aggregation and abnormal platelet function.
been discovered to prevent or delay the progression of These abnormalities, coupled with thrombocytopenia, make
 Bone marrow biopsy sample, showing replacement
A
of marrow by plasma cells. (From Harmening D. Clinical Hematology
and Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis;
2009.)

hemorrhaging, bruising, and purpura, common complications

of multiple myeloma.
Multiple myeloma preferentially involves bone, producing
multiple lytic lesions, that often lead to bone pain and patho-
logical fractures (Fig. 18–5). Bone loss is caused by a complex
interaction between the myeloma cells and normal cells of the
bone. The myeloma cells trigger increased osteoclast activity
and decreased osteoblast activity. Morbidity is generally caused
by bone disease. The multiple myeloma cells are dependent on
the bone marrow microenvironment and interaction with sev-
eral cells including osteoclasts, osteoblasts, and dendritic cells.
Bone pain, usually involving the spine or chest, is the most
common presenting symptom of multiple myeloma. Hypercal-
cemia is very common because the myeloma promotes bone
reabsorption. In advanced disease, the hypercalcemia itself can
reach life-threatening levels. Despite the dependence of multi-
ple myeloma cells on the bone marrow, tumors occasionally
are found in the spleen and liver. These tumors are typically
more aggressive.34
When immunoglobulin levels in the blood are sufficiently
high, they may cause the formation of rouleaux, stacklike for-
mations of red blood cells (RBCs) that can be seen on exam-
ination of a peripheral blood smear. The excess production
of the abnormal immunoglobulin is accompanied by a pro-
gressive decrease in the normal immunoglobulins. This leads B
to a deficiency of normal antibody responses and a higher
incidence of infectious diseases. Hyperviscosity can develop Bone marrow lesions seen in a patient with multiple
myeloma. (A) Lytic skull lesions. (B) Left humerus. (From Harmening
when the level of M protein in the plasma is high. Because
D. Clinical Hematology and Fundamentals of Hemostasis. 5th ed.
viscosity depends on the concentration and size of the mole-
Philadelphia, PA: F.A. Davis; 2009.)
cule in solution and IgM is the largest of the immunoglobu-
lins, hyperviscosity is most often seen with IgM-producing
tumors. Hyperviscosity syndrome is also sometimes seen with in which free light chains or fragments of immunoglobulin are
an IgG3-producing myeloma because IgG3 is the largest of deposited in the tissues. Amyloid fibers stain with the dye
the IgG subclasses. Congo red and show apple-green birefringence when viewed
The type and severity of clinical manifestations depend on with a polarizing microscope. Light chains can be identified
the type of immunoglobulin component produced. Up to 15% in tissue sections by immunofluorescence or immunohisto-
of patients with multiple myeloma develop light-chain depo- chemical staining with specific antibodies. The deposition of
sition disease or amyloidosis. These are two related disorders antibody-derived material results in organ dysfunction. The
kidneys are most often affected, but every tissue in the body presence of MGUS increases the risk of Waldenström
can develop amyloid deposits. Cardiomyopathy, peripheral macroglobulinemia 200-fold over the general population.39
neuropathy, hepatosplenomegaly, and ecchymoses (areas of The malignant cells in Waldenström macroglobulinemia are
skin discoloration caused by blood in the tissues) are the most B cells or plasma cells. The B cells produce the B-cell markers
common manifestations. CD19, CD20, CD22, and CD79a. The markers CD3, CD5,
Patients with multiple myeloma can develop either acute or CD10, and CD103 are not expressed.39 The tumor cells always
chronic renal failure. As many as two-thirds of patients with infiltrate the bone marrow and are sometimes found in the
multiple myeloma exhibit some degree of renal insufficiency. spleen and lymph nodes. In bone marrow aspirates, the lym-
Patients with myelomas that produce light chains or IgD are phocyte count can be within the reference range or be severely
much more likely to develop renal failure than those with other elevated. The tumor cells can have a variety of presentations,
types. Renal insufficiency caused by Bence Jones proteins is described as small lymphocytes, plasmacytoid lymphocytes,
seen in about 50% of patients. After infection, this is the second and cells resembling mature plasma cells. Typically one mor-
leading cause of death. Bence Jones proteins are thought to be photype will predominate.
directly toxic to tubular epithelial cells and can damage the The clinical signs and symptoms of Waldenström macroglob-
kidneys by precipitating in the tubules, causing intrarenal ulinemia are variable and are caused by infiltration of the malig-
obstruction. The median survival for patients with multiple nant cells into the bone marrow, spleen, and lymph nodes with
myeloma is approximately 6 years. Prognosis is generally best the overproduction of monoclonal IgM. Signs and symptoms
with IgM disease and worst with IgG disease. Evidence of a often include weakness, fatigue, anemia, bleeding, and occasion-
deletion in chromosome 13 has a significant negative impact ally plasma hyperviscosity. Anemia is attributed to overgrowth
on outcome.35,36 Although increased treatment options have of tumor cells in the bone marrow, displacing erythropoiesis.
improved the median mortality rate, multiple myeloma is still Thrombocytopenia and leukopenia are less commonly seen. At
an incurable disease. Almost all patients will relapse. diagnosis, the median hemoglobin concentration is 100 g/L.40
Criteria for the diagnosis of multiple myeloma include Bleeding can be caused by a combination of thrombocytopenia
plasma cells comprising greater than 10% of bone marrow cells and interference of platelet function by monoclonal IgM. The
and the evidence of end-organ damage such as bone marrow monoclonal IgM can accumulate in any tissue, forming deposits
lesions (detectable by radiographs), hypercalcemia, renal in- that lead to inflammation and tissue damage. Because early
sufficiency, and anemia. Serum M protein of 3 g/dL or more diagnosis is possible, hyperviscosity is uncommon. Lytic bone
and urinary M protein of 200 mg/day or more are indicative lesions, hypercalcemia, and renal tubular abnormalities are rare,
of multiple myeloma.37,38 Serum M proteins can be detected differentiating this disease from multiple myeloma.
by protein electrophoresis, immunofixation, and free light All individuals with Waldenström macroglobulinemia
chain assays. It is important to differentiate among monoclonal have elevated serum monoclonal protein, referred to as
free light chains, heavy chains, and gamma globulins. Patients macroprotein or IgM paraprotein, that migrates in the gamma
with monoclonal gammopathy can have MGUS, SMM, multi- region during SPE. However, the concentration varies widely
ple myeloma, or one of several other clonal expansions of and it is not possible to define a concentration that differen-
plasma cells or B cells. Fluorescence in situ hybridization tiates this disease from other B-cell lymphoproliferative dis-
(FISH) of bone marrow for the detection of translocation orders. IgM levels do not affect survival rate or correlate with
events associated with multiple myeloma can be performed to symptoms. Patients with serum IgM concentrations over
assess the risk-stratification of the disease.38 5,000 mg/dL can be asymptomatic, whereas patients with
An important feature supporting the diagnosis of multiple levels of 500 mg/dL can have significant bone marrow infil-
myeloma is the presence of Bence Jones protein in the urine. tration and pancytopenia.41 The presence of IgM paraprotein
About 60% to 70% of the patients with myeloma excrete Bence is not specific for Waldenström macroglobulinemia. SPE
Jones protein in the urine, which can be detected by specific should be used to evaluate the amount of the monoclonal
techniques, such as immunofixation electrophoresis (IFE), or protein; the presence of IgM should be confirmed by IFE (see
nonspecific techniques, such as heat precipitation. the section in the text that follows). In 70% to 80% of the
patients, the light chain is κ. Bence Jones proteinuria is pres-
ent in about 10% of the cases.25 Serum β2–microglobulin
Waldenström Macroglobulinemia levels are generally above the reference range’s upper limit
Waldenström macroglobulinemia is a malignant proliferation of 3.0 mg/dL. Because of the variability in IgM concentra-
of IgM-producing lymphocytes which is also known as a lym- tion, the WHO describes Waldenström macroglobulinemia
phoplasmacytic lymphoma. It is a rare condition; only about as a lymphoproliferative lymphoma involving the bone
1,500 cases are reported annually in the United States.8 More marrow with IgM paraprotein at any concentration.42
cases occur in males than females and the median age at onset In 10% to 20% of patients, the IgM paraproteins behave as
is about 70 years. Waldenström macroglobulinemia is only cryoglobulins. Cryoglobulins precipitate at cold temperatures
about 10% to 20% as common as multiple myeloma and the and can occlude small vessels in the extremities in cold
etiology of this disease is unknown. However, because approx- weather. Occlusion of small vessels can lead to hypoxia and
imately 20% of the cases exhibit familial clustering, genetic fac- the development of skin sores or even necrosis of portions of
tors are thought to be involved in at least some cases.39 The the fingers or toes. Cryoglobulins can also contribute to
plasma hyperviscosity. Cryoglobulins can be detected when a unresponsive to antibiotics or those who have progressed to later
blood or plasma sample is refrigerated in the clinical labora- stages of the disease are treated with chemotherapy.
tory. The precipitate forms at low temperatures and dissolves Gamma chain disease is a very rare disorder that has been
upon warming. found in people around the world, usually appearing between
Some of the clinical symptoms are caused by autoantibody the ages of 60 and 70.44 One-fourth of the patients also have an
activity of the monoclonal IgM antibody. Some IgM parapro- autoimmune disease such as rheumatoid arthritis (RA).46 The
teins have specificity for the i or I antigens and will agglutinate disease is heterogeneous and can present in one of three forms:
RBCs in the cold (cold agglutinins). Antibodies can bind to RBCs disseminated lymphoma with lymphadenopathy and general-
producing an autoimmune hemolytic anemia. Coating of the ized symptoms such as fever and weight loss; localized disease
RBCs can produce rouleaux, which can be demonstrated on with lymphoma limited to the bone marrow; or localized disease
peripheral blood smears. A thrombocytopenic purpura-like involving areas outside of the lymph nodes, such as the skin.46
syndrome can develop from paraprotein binding to thrombo- The abnormal gamma chains tend to migrate to the β region on
cytes. In addition, IgM can be demonstrated against polyclonal SPE, where they may be masked by other proteins. Serum IgG
IgG. This results in immune complex disease characterized by is elevated and the abnormal protein can be seen by IFE, which
vasculitis, affecting small vessels of the skin, kidneys, liver, and demonstrates a monoclonal γ band in the absence of monoclonal
peripheral nerves. light chains. Immunohistochemistry studies reveal the presence
Approximately 20% of the patients with Waldenström of malignant B cells and plasma cells in the bone marrow, spleen,
macroglobulinemia will present with peripheral neuropathy.43 lymph nodes, or other involved areas such as the skin. Patients
It appears that the monoclonal IgM in these cases is directed are treated with chemotherapy and the prognosis is highly vari-
against glycoproteins or glycolipids of the peripheral nerves, able, ranging from 1 month to more than 20 years.46
causing symptoms of neuropathology. Mu heavy-chain disease is an extremely rare disorder that
Asymptomatic patients do not require treatment, but they has only been diagnosed in about 40 people throughout the
should be monitored. Treatment includes anti-tumor chemother- world, mainly Caucasian males aged 50 to 60 years.44,46 The
apy and plasmapheresis to reduce blood viscosity. Survival time majority of patients also have a lymphoid malignancy that re-
depends on the disease stage. Patients with low-stage disease have sembles CLL or SLL.46 Thus, patients have symptoms that are
a median survival time of about 12 years compared with 3.5 years related to the associated lymphoma, such as splenomegaly (en-
for those with high-stage disease.42 largement of the spleen), hepatomegaly (enlargement of the
liver), and anemia. More than half of patients have a normal
SPE pattern, but IFE typically reveals µ polymers of various
Heavy-Chain Diseases sizes that are not associated with κ or λ light chains.46 The urine
The heavy-chain diseases are rare B-cell lymphomas that are does not usually contain µ heavy chains, but demonstrates the
characterized by the production of a monoclonal immunoglob- presence of free monoclonal light chains (Bence Jones proteins)
ulin (Ig) heavy chain.44,45 Genetic mutations in the affected in more than half of patients.45 Bone marrow aspirates reveal a
B cells result in the production of abnormal heavy chains that mixture of plasma cells containing prominent cytoplasmic vac-
have lost part of their CH1 or variable domain so they are in- uoles and small round lymphocytes resembling those of CLL.46
capable of binding to Ig light chains.46 These diseases are clas- Patients are treated with chemotherapy and overall survival is
sified according to the type of heavy chain produced, which variable, ranging from under 1 month to more than 10 years.46
can be alpha (α), gamma (γ), or mu (µ).
Alpha heavy-chain disease is the most common of the three
types and is seen most often in young adults in their 20s or 30s Role of the Laboratory in Evaluating
who live in the Mediterranean region, including northern Africa Immunoproliferative Diseases
and the Middle East.44,45 It has been associated with poor hy-
giene, poor nutrition, and chronic bacterial and parasitic infec- Diagnosis of a hematologic malignancy is usually suggested by
tions. The disease is a lymphoma that involves the MALT and a patient’s medical history and clinical symptoms and confirmed
can occur as one of three forms: gastrointestinal, respiratory, or by laboratory testing. Laboratory evaluation of a patient sus-
lymphomatous. Most patients have the gastrointestinal form, pected of having an immunoproliferative disorder begins with
which is characterized by intestinal malabsorption with diarrhea, performance of a CBC and differential and examination of the
abdominal pain, and weight loss. The monoclonal α chains can cell populations on a peripheral blood smear. Blood samples
be identified in patient serum through reaction with anti-IgA in from some patients with hematologic malignancies may show a
IFE (see the text that follows). Because of their abnormal struc- decrease in RBCs (anemia) or platelets (thrombocytopenia) be-
ture and tendency to polymerize, they may not be evident by cause of crowding out of normal hematopoietic precursors by
SPE, which can appear normal or demonstrate a broad band that the malignant cell population. A decrease in normal white blood
migrates to the α-2 or β region.46 Histological testing of biopsy cells (WBCs) may also be evident and can result in increased
tissue obtained from the small intestine or other affected areas susceptibility to infections. Microscopic examination of cell mor-
demonstrates an infiltration of plasma cells and mature B cells. phology in the peripheral blood smear can provide important
Early diagnosis is important because treatment with antibiotics clues about the lineage of the malignant cell population. Differ-
in the early stage of the disease can improve prognosis.45 Patients entiation between cells of monocytic or granulocytic origin and
those of lymphoid origin can also be accomplished by the use Table 18–1 Markers Commonly Detected
of various cytochemical stains such as peroxidase and Sudan by Flow Cytometry in the Analysis
Black B. Once abnormalities are detected in the CBC and differ- of Hematologic Malignancies*
ential, a bone marrow aspirate and biopsy are obtained to con-
CELL TYPE ASSOCIATED MARKERS
firm the diagnosis. Although microscopic examination of the
bone marrow cells can confirm the presence of a malignant pop- T cells CD1, CD2, CD3, CD4, CD5, CD7, CD8,
ulation, specialized tests are required to more precisely identify TCR alpha-beta, TCR gamma-delta
the cells of interest. B cells CD10, CD19, CD20, CD22, CD23,
This section will discuss three types of specialized tests that CD79a, CD103, kappa (surface and
are used in the diagnosis and monitoring of patients with lym- cytoplasmic), lambda (surface and
phoproliferative disorders: immunophenotyping by flow cy- cytoplasmic )
tometry, evaluation of immunoglobulins, and genetic testing. Myeloid cells CD11b, CD13, CD14, CD15, CD33,
The laboratory can assess the immunophenotype of hematopoi- and monocytes CD64, CD117, myeloperoxidase
etic cells in the blood, bone marrow, or lymphoid tissues by
flow cytometry. This is done by detecting cell surface antigens Miscellaneous CD11c, CD16, CD25, CD26, CD30,
CD34, CD38, CD41, CD42b, CD45,
that are characteristic of a specific lineage and stage of differen-
CD56, CD57, CD61, HLA-DR,
tiation. This technology serves as an excellent complement to glycophorin, TdT, CD123, CD138,
microscope-based traditional diagnostic methods and adds dis- CD200
tinctive, discriminatory capabilities that are unmatched by any
other diagnostic technique. By performing immunophenotyp- *Modified from ARUP Laboratories. Laboratory Test Directory. Leukemia/

ing, the laboratory can determine whether the malignant cell Lymphoma Phenotyping by Flow Cytometry. http://ltd.aruplab.com/Tests/
Pub/2008003. Accessed July 2, 2015.
population consists of B cells, T cells, NK cells, plasma cells, or
cells of myeloid origin.
A second role of the laboratory is in evaluating the amount
Thus, cells that express a specific antigen are bound by the cor-
and characteristics of the immunoglobulins produced by
responding antibody and emit fluorescence of a particular
malignant B cells or plasma cells. Because the B-cell lineage
color. The fluorescence, along with cell size and other cell char-
develops into plasma cells that produce antibody, malignancies
acteristics, is analyzed by flow cytometry (see Chapter 13). This
of B cells are sometimes associated with excessive or abnormal
allows the antigenic profile, or immunophenotype, of the cell
antibody production. The concentrations and characteristics
population to be determined. Table 18–2 lists the CD markers
of the immunoglobulins in the patient’s serum or urine can be
typically found on selected hematologic malignancies of lym-
used to diagnose and evaluate the plasma cell dyscrasias.
phoid origin.
Third, the laboratory is involved in the assessment of genetic
Flow cytometry is ideal for fluids such as blood, in which
and chromosomal abnormalities in hematopoietic malignancies.
cells are naturally suspended, but it is also useful for lymphoid
Genetic techniques play an important role in routine clinical prac-
tissues, from which single-cell suspensions can be easily made.
tice. Cytogenetic analyses are used to detect chromosomal abnor-
The advantages of flow cytometry are largely based on its ability
malities such as translocations. Molecular techniques such as
to very rapidly and simultaneously analyze multiple-cell prop-
microarray and the PCR can be used to detect mutant sequences
erties, including size, granularity, and surface and intracellular
within genes that have been linked to particular diseases.
antigens, even in small samples. The quantitative nature of the
data produced, both with regard to cell population distributions
Immunophenotyping by Flow Cytometry and to expression of individual cell antigens, offers objective
Immunophenotyping, or the analysis of cell surface marker criteria for the interpretation of results.
expression, is commonly used in the diagnosis and classifica- However, laboratorians and clinicians must recognize that
tion of leukemias and lymphomas.8,47 Because the malignant malignant cells can differ from their normal counterparts (e.g.,
cells express markers that often correspond to those of their B-cell ALL versus normal B cells) in terms of the antigens that
normal precursors, insight into their lineage of origin and stage they characteristically express. This difference can occur in any
of maturation can often be determined by this technique. of the following ways:
The presence of cluster of differentiation (CD) antigens on 1. There may be a gain of antigens not usually expressed
the surface of hematopoietic cells is routinely detected by flow by the normal cell type or lineage.
cytometry. Table 18–1 lists some of the clinically relevant 2. There may be abnormally increased or decreased levels
markers. In immunophenotyping, clinical samples containing of the antigens expressed by the malignant cells, or in
cells that are potentially neoplastic are incubated with panels some cases a complete loss of normal antigens.
of antibodies that are specific for the relevant antigens. The 3. The malignant cells may express antigens at inappropriate
clinical laboratory determines the specific antibodies used for times during the maturation process.
testing on the basis of the suspected disease, the type of sam- 4. There may be a homogeneous expression of antigens that
ple, and the amount of sample available. Each antibody in a are typically heterogeneously expressed by the normal
single reaction tube is labeled with a different fluorescent dye. counterpart.47,48
 Table 18–2 Surface Markers Characteristic B cells differentiate into antibody-producing plasma cells
of Selected Leukemias by maturation through several stages. Each B cell recognizes
and Lymphomas only a single antigenic site or epitope. An early B-cell precur-
sor is stimulated to proliferate and mature when it encounters
HEMATOPOIETIC CHARACTERISTIC
an immunogen that it recognizes. When a foreign molecule
MALIGNANCY SURFACE MARKERS*
enters the body, the many different epitopes on it each stim-
Classic Hodgkin lymphoma CD15+ (most), CD30+, ulate a B-cell response, leading to the production of an array
CD3 +/–, CD20 – or weak, of different antibodies. However, in disorders such as multi-
CD45–
ple myeloma, proliferation of one clone of transformed
Nodular lymphocytic CD19+, CD20+, CD45+, plasma cells leads to overproduction of an immunoglobulin
predominant Hodgkin CD15–, CD30– of a single class and antigen specificity. These disorders are
lymphoma (NLPHL) called monoclonal gammopathies, because the diseases in-
B-cell acute lymphocytic CD10+, CD19+, CD22+, volve proteins that are produced by a single clone of plasma
leukemia (B-ALL) CD34+, TdT+ cells; these proteins are found mainly in the gamma region
of a SPE analysis. The antibody produced by the malignant
T-cell acute lymphocytic CD1a+, CD2+, CD5+,
plasma cells is referred to as an M (monoclonal) protein or
leukemia (T-ALL) CD7+, TdT+
paraprotein (i.e., an abnormal protein). All of the antibody
Chronic lymphocytic CD5+, CD19+, CD20 proteins produced by the clone of plasma cells are identical
leukemia (CLL) (weak+), CD23+ in terms of their heavy chains, light chains, and idiotypes.
Hairy cell leukemia CD19+, CD20+, CD22+, The initial tests used to screen for the presence of a mono-
CD25+, CD103+, clonal gammopathy are serum immunoglobulin levels and SPE
CD123+ (see the text that follows). Quantitative measurement of im-
munoglobulin levels in the serum is routinely performed by
Multiple myeloma CD38+, CD56+, CD138+,
~20% are CD20+
nephelometric methods, or in smaller laboratories, by radial
immunodiffusion (RID) (see Chapter 10). Because each plasma
Waldenström CD19+, CD20+, CD22+, cell produces only one type of immunoglobulin, the persistent
macroglobulinemia CD79a+, CD3–, CD5–, presence of an elevated amount of a single immunoglobulin
CD10–, CD103– class suggests malignancy. In contrast, an increase in the
* CD = cluster of differentiation, + = positive, – = negative, TdT = Terminal amount of total immunoglobulin, without an increase in any
deoxynucleotidyl transferase. one specific class, is characteristic of nonmalignant conditions
such as infections or autoimmune diseases.

Serum Protein Electrophoresis (SPE)

Because there is no single surface marker that is specific for a
particular hematologic malignancy, the laboratory must use a SPE is a technique in which serum proteins are separated
panel of carefully selected antibodies to identify the markers on the basis of their size and electric charge, as discussed in
necessary for making an accurate diagnosis.8 Chapter 5. SPE results in five regions: albumin, as well as the
alpha 1, alpha 2, beta, and gamma globulins. IgG, IgM, IgD,
and IgE migrate in the gamma globulin region, whereas
Evaluation of Immunoglobulins IgA migrates as a broad band in the beta and gamma regions.
As we discussed in Chapter 5, the basic immunoglobulin unit Figure 18–6, panel A, shows a stylized drawing of the protein
consists of two identical heavy chains and two identical light distribution in normal serum. As can be seen, immunoglobu-
chains, covalently linked by disulfide bonds. The structure lins normally show a range of mobilities because they are de-
of the heavy chain defines the class, or isotype, of the anti- rived from many clones of plasma cells and have different
body (e.g., γ heavy chain in IgG, µ heavy chain in IgM, etc.). variable region sequences. The SPE pattern for a polyclonal in-
The two types of light chains (κ and λ) can each occur in crease in serum immunoglobulins is shown in panel B. Note
combination with any of the heavy-chain types. The heavy the broad mobility, but increased height of the gamma globulin
and light chains each contain constant and variable regions. peak. Polyclonal increases in serum immunoglobulins are seen
The constant region contains the sites of immunoglobulin in a variety of disorders, including infections, autoimmune dis-
that bind to cell receptors and sites involved in complement eases, liver diseases, and some immunodeficiency states (e.g.,
fixation. The variable regions contain the idiotypes, which are hyper-IgM syndrome). The SPE result in panel C depicts a
responsible for the antigen specificity of the antibody. Nor- monoclonal immunoglobulin, which is increased in concen-
mally, immunoglobulins in plasma are heterogeneous, be- tration and has limited mobility because it is produced by an
cause they have a variety of idiotypes that recognize a variety identical clone of plasma cells. This is illustrated by the tall,
of different antigens. The variability in the isotype means narrow peak in the gamma region.
that they also vary in their physical characteristics, such as Additional evaluation of serum immunoglobulins by IFE is
molecular weight and charge. performed if the SPE shows a monoclonal component, if there
 Albumin Immunofixation Electrophoresis (IFE)
A
The performance of IFE is typically the next step in evaluating
!1 !2 " # a monoclonal gammopathy. IFE is a highly sensitive and spe-
cific assay that is used to identify the type of monoclonal pro-
tein present in a sample. It can be performed by manual or
automated capillary electrophoresis systems. In IFE, serum
samples are electrophoresed in six separate lanes on an agarose
Normal gel and specific antisera are applied directly to the lanes. The
antisera used are selected to detect the most common M pro-
teins and are directed against:
B C
• Whole human serum (lane 1)
• Anti-γ (to detect IgG) (lane 2)
• Anti-α (to detect IgA) (lane 3)
• Anti-µ (to detect IgM) (lane 4)
• Anti-κ (to detect kappa light chains) (lane 5)
• Anti-λ (to detect lambda light chains) (lane 6)
Polyclonal gammopathy Monoclonal gammopathy The antibodies combine with the immunoglobulin proteins
Serum protein electrophoresis of normal and in the sample to form complexes that are visualized by stain-
abnormal samples. The lower portion of each panel is a represen- ing. Figure 18–7 illustrates the principle of IFE. Areas of dif-
tation of a stained agarose electrophoresis gel. The intensity of fuse staining indicate polyclonal immunoglobulins, whereas
staining corresponds to the amount of protein in each region of monoclonal bands produce narrow, intensely stained bands
the gel. In the upper portion of each panel is a densitometer (Fig. 18–8).
tracing of a gel similar to the one beneath it. In the upper panel Interpretation of IFE results requires a high level of exper-
showing a normal serum sample, the largest peak is albumin. The tise. When the monoclonal protein is in high concentration
globulin regions are as indicated.
and the amount of polyclonal immunoglobulin of the same
class is low, the bands produced in IFE gels are usually clear
is a significant quantitative abnormality of serum immunoglob- and easy to identify, as shown in Figure 18–8. However, the
ulins, or if the clinical picture strongly suggests a plasma cell presence of higher levels of normal immunoglobulins can
dyscrasia. Myeloma in which only light chains are produced make it difficult to distinguish a minor monoclonal band
may not be detected on SPE because the light chains are rapidly against a background of normal proteins.49 These M proteins
cleared in the urine. Therefore, additional studies on a random can sometimes be identified by a technique called immunosub-
or 24-hour urine sample may be indicated even in the presence traction (see section that follows). Poorly resolved bands can
of a normal SPE. also be caused by poor technique during the electrophoresis

Step 2: Protein
Addition of fixative Anti-IgG Anti-IgA Anti-IgM Anti-$ Anti-%
Antisera
Anode (&)

Albumin

!1 globulins Step 1:
Electrophoresis
!2 globulins
of Sample
" globulins

# globulins
Patient sample

Cathode (')
Principle of immunofixation electrophoresis (IFE). IFE involves two major steps. In step 1, proteins in the clinical sample (serum,
urine, or CSF) are applied to an agarose gel and separated according to their surface charge under the influence of an externally applied
electrical field. At a pH of 8.6, the proteins acquire a negative charge and move toward the positively charged anode. Five major protein
fractions result: γ globulins, β globulins, α1 globulins, α2 globulins, and albumin. Immunoglobulins are located primarily in the γ globulin
fraction, but can also migrate into the β globulin fraction. In step 2, specific antisera are added to each one of the lanes and react with their
corresponding protein to produce a precipitin band. A protein fixative is added to lane 1, which binds to all of the major protein fractions. The
precipitin bands can be visualized after staining the gel with a protein stain and destaining with acetic acid to remove background color.
 1 1

2 2

3 3

4 4

5 5

6 6

7 7

A SPE IgG IgA IgM $ % SPE IgG IgA IgM $ %

Postzone effect in immunofixation electrophoresis
1 1 (IFE). IFE was performed on a 24-hour urine sample that was concen-
trated 50x by manual ultrafiltration (original protein concentration =
2 2 22 mg/dL). Note the clear area in the center of the κ band on the
3 3 gel, reflecting decreased immunoprecipitation caused by an excess
of κ chains relative to the amount of anti-κ reagent used in the κ
4 4 lane. (Linda Miller.)
5 5

6 6 Connections
7 7
Postzone Effect in IFE
B SPE IgG IgA IgM $ % Recall from Chapter 10 that equivalent amounts of antigen and
antibody are required for optimal formation of immune com-
1 1 plexes and precipitation. Extreme antigen excess results in the
formation of small immune complexes that cannot be visualized.
2 2
This is called a postzone effect. When a sample with a very large
3 3 concentration of an M protein is reacted with antisera on an IFE
gel, precipitation will be visible on the outer edges of the band,
4 4 where the reaction is in equivalence, but will not appear in the
5 5
center of the band, where the reaction is in the postzone area.
The problem can be corrected by diluting the sample and
6 6 repeating the IFE procedure.
7 7

C SPE IgG IgA IgM $ %

Sample IFEs from normal serum and sera from patients
with monoclonal gammopathies. (A) Normal serum. Note the broad, A variation of immunofixation, called immunosubtraction or
diffuse bands in all the immunoglobulin lanes. These represent immunotyping, is a sensitive procedure that uses capillary elec-
polyclonal antibodies that are heterogeneous in terms of their antigen trophoresis to identify monoclonal immunoglobulin compo-
specificity and structure and therefore migrate to slightly different nents. In this technique, antibodies to each heavy or light chain
positions on the gel. (B) Serum from a patient with a monoclonal IgG, isotype are added to separate capillary runs of the patient’s spec-
λ antibody. Note the discrete, narrow bands in the IgG and lambda imen. The binding of the antibody to its heavy or light chain
lanes. The monoclonal antibody molecules produced by the patient antigenic target changes the electrophoretic mobility of the pa-
are all identical and migrate to the same position on the gel.
tient’s immunoglobulin molecule. Monoclonal peaks thus “dis-
(C) Serum from a patient with monoclonal IgM, κ antibody. Note
the discrete, narrow bands in the IgM and kappa lanes. (Linda Miller.)
appear” in the presence of antibodies to their components,
allowing typing to occur by subtraction of peaks. Figure 18–10
shows an example of a monoclonal IgG kappa protein detected
or antisera application steps. Other situations can also affect
by immunotyping.
the quality of the results.49,50 For example, plasma is not rec-
ommended because fibrinogen can adhere to the β region of
the gel and cause monoclonal proteins to stick nonspecifically, Urine protein electrophoresis (UPE) and urine IFE also play an
resulting in distinct bands in all of the antisera tracks. Samples important role in the diagnosis of multiple myeloma and other
with large amounts of rheumatoid factor or immune complexes plasma cell dyscrasias. As previously discussed, some patients
can also produce unusual results, such as precipitin bands at with these disorders produce an excessive amount of free mon-
the place of application. An extreme excess of monoclonal oclonal Ig light chains (i.e., Bence Jones proteins). Because
immunoglobulin can cause a postzone effect that produces a these proteins are rapidly cleared from the circulation, they
clear zone in the center of the band (Fig. 18–9). may not be detectable on serum IFE. However, the excess light
 D
A

ELP IgG

E
B

IgA IgM

C F

K L

Immunotyping of a monoclonal IgG, kappa protein. Panel A: Serum protein electrophoresis. The arrow points to the M-spike in
the gamma region. Panel B: Electrophoresis with anti-alpha heavy chain. This pattern is overlaid with the original serum protein electrophoresis.
The patterns are identical. Panel C: Electrophoresis with anti-kappa light chain. The M-spike has disappeared, indicating the peak contains a
kappa light chain (see arrow). Panel D: Electrophoresis with anti-gamma heavy chain. The M-spike has disappeared, indicating the peak contains
IgG (see arrow). Panel E: Electrophoresis with anti-mu heavy chain. This pattern is overlaid with the original serum protein electrophoresis. The
patterns are identical. Panel F: Electrophoresis with anti-lambda light chain. This pattern is overlaid with the original serum protein electrophore-
sis. The patterns are identical. (Courtesy of Dr. Thomas Alexander.)

chains are excreted into the urine and can be identified using gel. Concentration can be accomplished by one of two ways.
UPE and urine IFE. The free light chains are believed to con- The first way involves the use of urine concentrators with ul-
tribute to renal disease by depositing in the glomeruli and trafiltration membranes that can retain large proteins (usually
tubules of the kidneys and by their involvement in the devel- 10,000 daltons or more), but allow water, salts, and other
opment of renal casts.51 Therefore, the IMWG has recom- small molecules to pass through, thus reducing the sample
mended that patients with plasma cell dyscrasias be routinely volume. In the process, the sample flows through a chamber
monitored by UPE and urine IFE.52 These tests can also be containing the absorbent membranes and is collected when it
used to assess the effect of therapy on the production of free reaches the desired volume and concentration. The second
monoclonal light chains and can indicate more extensive renal way involves the use of centrifugal concentrators that concen-
damage when large proteins such as intact immunoglobulins, trate the sample by high-speed filtration. Centrifugal concen-
which are normally retained in the blood, are demonstrated in trators are faster than ultrafiltration membranes and can
the urine. produce higher concentration factors. They are recommended
Urine samples for UPE and IFE are typically collected over for the preparation of urine samples in capillary electrophore-
a 24-hour period.53 The total protein concentration is deter- sis systems.
mined and the sample is treated by filtration or centrifugation In both systems, the desired concentration factor is based on
to remove any sediment that could interfere with performance the amount of protein in the urine sample and is determined
of the tests. In addition, the samples must be concentrated so by dividing the starting sample volume by the final volume.53
that enough protein is present to produce visible bands on the The concentrated sample is then applied to the electrophoresis
gel and the tests are completed as previously described for SPE Table 18–3 Cytogenetics Characteristic
and serum IFE. of Selected Leukemias
and Lymphomas
Serum Free Light Chain Analysis (sFLC) HEMATOPOIETIC CHARACTERISTIC
Although UPE and urine IFE are sensitive methods for the de- MALIGNANCY CYTOGENETICS*
tection of free monoclonal light chains, they have some limi- Burkitt lymphoma Most commonly t(8;14) [IgH/myc];
tations.54 For example, the requirement for a 24-hour urine also t(2;8) and t(8;22)
collection can delay testing. In addition, interpretation of the Follicular lymphoma t(14;18) [IgH/Bcl2]
results is subjective and can be difficult when a low level of
free light chains is present with a high level of proteinuria, Mantle cell lymphoma t(11;14) [IgH/cyclin D1]
which can create a high degree of background staining. Auto- B-cell acute t(12;21) [TEL-AML-1]
mated tests for serum free light chains (sFLC) became com- lymphoblastic
mercially available in 2001 and offer advantages over the leukemia (B-ALL)
traditional methods for urine testing described previously. Chronic myelogenous t(9;22) [bcr/abl]
The sFLC assays are latex-enhanced immunoassays that leukemia (CML)
measure free kappa and lambda light chains in the serum. The
assays employ polyclonal antibody reagents that recognize a *t = translocation; IgH = immunoglobulin heavy-chain gene.

diverse range of FLC epitopes that are normally hidden when

the light chains are bound to heavy chains in intact im-
munoglobulins. This allows for quantitative measurement of in vitro and their metaphase chromosomes can be examined for
free κ and free λ chain concentrations, as well as calculation grossly visible abnormalities that correspond to characteristic
of a κ/λ ratio (normally, 0.26–1.65).54 An abnormal κ/λ ratio translocations. Traditional cytogenetic evaluation by karyotyp-
outside of the reference range, along with an increase of either ing has been supplemented by a technique known as FISH. This
κ and λ, is a sensitive indicator for the presence of a malignant technique is used to directly identify a specific region of DNA
plasma cell clone, which is characteristic of a monoclonal in a cell. It involves the preparation of short sequences of
gammopathy. single-stranded DNA, called probes, which are complementary
The sFLC assays are highly sensitive, being capable of de- to the DNA sequences of interest. These probes bind to the
tecting concentrations less than 1 mg/L.53 This sensitivity allows complementary chromosomal DNA and, because they contain
them to detect monoclonal FLCs in patients previously thought a fluorescent label, allow the location of those DNA sequences
to be negative for monoclonal immunoglobulin production by in the chromosomes to be visualized. Figure 18–11 shows
serum or urine IFE.54 Based on the quantitative nature and high an example of a FISH result obtained from a patient with a
level of sensitivity of sFLC testing and clinical studies, the hematologic malignancy. Probes can be used on chromosomes,
IMWG developed consensus guidelines that recommend the interphase nuclei, or tissue biopsies. FISH is rapid and quite
use of the sFLC assay along with SPE and serum IFE to screen sensitive and does not require cell culture because interphase
for multiple myeloma and related disorders.49,53,54,55 In addi- chromosomes are used. However, it only provides information
tion, the group stated that the sFLC can replace the 24 h about the specific DNA sequence being detected by the probe
urine IFE to screen for most plasma dyscrasias (except for used in the test.
light chain amyloidosis). The experts also concluded that Lymphoid malignancies can also be evaluated by molecular
sFLC measurements are valuable in monitoring patients with techniques to identify abnormalities that are too subtle or di-
plasma cell disorders and in helping to determine patient verse to be detected by karyotyping or FISH. Laboratorians can
prognosis. use molecular methods to find microdeletions or clonal re-
arrangements of the immunoglobulin genes in B-cell malig-
Evaluation of Genetic and Chromosomal nancies or of the TCR genes in T-cell malignancies. The PCR is
the most widely used technique to detect these gene rearrange-
Abnormalities ments.56 To detect B-cell clonality, for example, the PCR am-
As previously discussed, researchers have identified the specific plifies the immunoglobulin heavy-chain gene sequence
mutations associated with malignant transformation for many containing the variable and joining genes (see Chapter 5). The
of the hematologic malignancies. These genetic alterations are primers used in the assay are designed to detect conserved se-
an integral part of the classification system outlined by the quences so that most of the immunoglobulin genes in the sam-
WHO.8 The detection of chromosome translocations is of ple will be amplified to detectable amounts, which can be seen
particular value in diagnosis of hematologic malignancies. on gel electrophoresis. If a malignant B-cell clone is present, a
Table 18–3 lists chromosome translocations that are charac- sharp band will migrate to a specific position on the gel, rep-
teristic of specific leukemias and lymphomas. resenting the unique V-D-J IgH gene rearrangement contained
Often, these translocations can be detected by cytogenetic in the cells of that clone (Figure 18–12, lanes 3, 6, 9, and 10).
techniques. Malignant lymphoid cells can be made to proliferate In contrast, normal polyclonal B cells produce a diverse array
 A B

FISH (fluorescence in situ hybridization) demonstrating a chromosomal translocation. In this assay, interphase cells were
hybridized with molecular probes complementary to specific regions on chromosomes 9 and 22. The green signal represents the BCR (break-
point cluster region) on chromosome 22 and the red signal represents the ABL1 proto-oncogene on chromosome 9. A yellow signal repre-
sents the BCR/ABL1 fusion caused by a 9;22 chromosome rearrangement. (A) A normal test result, showing two green signals representing
two copies of chromosome 22 and two red signals representing two copies of chromosome 9. (B) Result from a patient with a translocation
between chromosomes 9 and 22 [t(9;22)(q34.1;q11.2)]. The yellow signal indicates the fusion between the BCR and ABL1 loci on the rearranged
chromosome 22, which is also known as the Philadelphia chromosome. The smaller red signal detects the residual ABL1 sequences present on
the rearranged chromosome 9. This translocation is found in patients with CML (chronic myelogenous leukemia) as well as some individuals
with AML (acute myeloid leukemia) and ALL (acute lymphoblastic leukemia). (Courtesy of the Cytogenetics Laboratory, SUNY Upstate Medical
University.)

of V-D-J IgH gene rearrangements, which migrate to different portions of specific genes or chromosome regions and are spot-
positions on the gel, producing a smear (see Fig. 18–12, lanes ted onto separate locations on a small glass slide or nylon
4, 8, and 11). Similarly, unique rearrangements in the T-cell membrane. Genomic DNA is isolated from a clinical sample,
receptor genes can be amplified by PCR to detect a malignant labeled with a fluorescent dye, and incubated with the microar-
T-cell clone. ray. Fluorescent spots will be visible in the locations where the
Although PCR and FISH are used to detect abnormalities in sample DNA has bound. This technique is being used clinically
targeted genes, other molecular techniques can assay larger to detect small genetic changes called single nucleotide poly-
areas of the genome. DNA microarray technology enables effi- morphisms (SNPs), as well as larger chromosome deletions or
cient analysis of thousands of genes in the human genome in additions that result in copy number variations (CNVs) that
a single hybridization experiment by using a panel of molecular may occur in hematologic malignancies.57 Next generation se-
probes (see Chapter 12). The probes are complementary to quencing (NGS) of tumor cells is also making its way into rou-
tine clinical practice. This technique is being used to analyze
the nucleotide sequence of all of the genes (i.e., whole genome
Connections sequencing), the coding regions of the genome (referred to as
exome sequencing), or the transcriptome (i.e., messenger RNA)
B-Cell Maturation and Rearrangements
in samples from cancer patients, including those with hema-
Recall from Chapter 5 that rearrangements in the immunoglob- tologic malignancies.58,59 The tremendous amount of data re-
ulin heavy and light chain genes occur during B-cell maturation. sulting from these analyses is allowing clinicians to identify
The rearrangements occur randomly so that each B cell pos- genetic profiles associated with specific hematologic malignan-
sesses a unique sequence of variable-heavy (V), diversity (D), and
cies and is revealing new genetic alterations that are associated
joining (J) gene segments. This unique sequence is retained as
the B cell proliferates. Thus, the sequence can be used as a
with the pathobiology of lymphoid neoplasms. Microarrays
marker for the B-cell clonality that is characteristic of B-cell and NGS are enabling a more comprehensive analysis of the
malignancies. It is visualized as a band of a distinct size when it neoplastic genome which is being translated into better patient
is amplified by PCR and run on gel electrophoresis. diagnosis and more precise, targeted therapies for the hema-
tologic malignancies.
 L V DJ C
The WHO classification of these disorders depends on their
morphological features, cytochemical staining, immunophe-
notype as determined by flow cytometry, and cytogenetics.
• Examples of hematologic malignancies include Hodgkin
FR1 CDR1 FR2 CDR2 FR3 lymphoma, non-Hodgkin lymphomas, acute lymphocytic
leukemias (ALL), chronic lymphocytic leukemias or
lymphomas (CLL), and hairy cell leukemia.
Amplification
• Plasma cell dyscrasias result in abnormal immunoglobulin
secretion. These disorders are malignancies of the plasma
cells that are characterized by production of a monoclonal
immunoglobulin (M protein or paraprotein).
• In multiple myeloma, the M protein is usually IgG or IgA,
Amplification products but can be of any immunoglobulin class. Waldenström
macroglobulinemia is a malignancy of plasmacytoid lym-
phocytes that produces an IgM paraprotein. Some patients
produce free monoclonal light chains and excrete the ex-
cess in the urine, where they are known as Bence Jones
proteins.
• Identification and quantification of the paraprotein are
central to the diagnosis and monitoring of these condi-
tions. SPE is used to detect the presence of an M protein,
which is then characterized by immunofixation elec-
trophoresis (IFE). Presence of a monoclonal immunoglob-
Immunoglobulin heavy-chain gene rearrangement
by PCR with amplification from the variable region. Forward primers ulin is indicated by a discrete, narrow band that migrates
complementary to the variable region and reverse primers comple- to a restricted position on the gel, whereas polyclonal
mentary to the joining region are used to amplify the diversity immunoglobulins are diverse and produce broad, diffuse
region (top). In a polyclonal specimen, amplification products in a bands.
range of sizes will result. These products produce a dispersed pat- • Analysis of urine by UPE and IFE is important in the de-
tern on an ethidium bromide-stained agarose gel (lanes 4, 8, 11). If tection of Bence Jones proteins, which can contribute to
at least 1% of the sample is representative of a monoclonal gene renal damage. Before testing, the urine must be concen-
rearrangement, that product will be amplified preferentially and trated by manual filtration or high-speed centrifugal filtra-
revealed as a sharp band by gel electrophoresis (lanes 3, 6, 9, and 10). tion to yield enough protein to produce visible banding on
(From Buckingham L. Molecular Diagnostics. 2nd ed. Philadelphia,
the gel.
PA: F.A. Davis; 2012.)
• Serum free light chain assays are latex-enhanced immunoas-
says that measure free κ and λ light chains in patient serum.
These are sensitive assays that can rapidly detect low levels
SUMMARY of free light chains. An increase in κ or λ, along with an
abnormal κ:λ ratio, indicates the presence of a malignant
• Cells of the immune system can undergo malignant trans- plasma cell clone.
formation because of exposure to environmental factors • The cellular origin of a lymphoid malignancy is deter-
or genetic mutations that result in excessive cell prolifer- mined in the laboratory by flow cytometry. Fluorescent-
ation, failure to undergo apoptosis, or arrested maturation. labeled antibodies are used to identify CD markers on
• Proto-oncogenes are normal genes that are involved in cell the surface of the malignant cells to determine their lin-
growth and division. Alterations in these genes can convert eage and stage of maturation. This procedure is called
them into oncogenes, which are involved in malignant trans- “immunophenotyping.”
formation. Several of the hematologic malignancies involve • Evaluation of genetic and chromosomal abnormalities is
chromosome translocations in which two chromosomes a rapidly evolving area of laboratory practice. These alter-
break apart and exchange portions. These gene rearrange- ations can be detected by a variety of cytogenetic and mo-
ments result in the overexpression of proto-oncogenes, caus- lecular techniques, including FISH, PCR, microarray, and
ing excessive cell proliferation or inhibition of normal cell NGS. These methods allow for the detection of abnormal-
death. ities such as chromosome translocations, nucleotide dele-
• Malignancies of lymphocytes, both lymphomas and tions, and unique V-D-J gene rearrangements that are
leukemias, are commonly encountered in clinical practice. characteristic of specific hematologic malignancies.
CASE STUDIES
1. A 63-year-old male visits his primary care physician 2. A 47-year-old man presented with fever, pneumonia, and
complaining of fatigue and shortness of breath, upper splenomegaly. His hemoglobin was 11.5 g/dL, the WBC
back pain, and a cough that has become productive the count was 2,700/mm3, and the platelet count was
last 2 days. The patient was febrile and appeared acutely 70,000/mm3. A bone marrow biopsy revealed, among the
ill. A chest x-ray revealed pneumonia and the following normal bone marrow cells, numerous diffuse cells 10 to
significant laboratory results were found: RBC count of 14 µm in diameter with abundant, clear to lightly basophilic
4.1 1012/L (reference range 4.6 to 6.0 1012/L), he- or eosinophilic cytoplasm. The surface of the cells exhibited
moglobin 13 g/dL (reference range 14.0 to 18.0 g/dL), delicate broad projections. The nuclei were oval and in-
WBC count 4.8 109/L (reference range 4.5 to 11.0 dented with variable chromatin and no prominent nucleoli.
109/L), and an erythrocyte sedimentation rate of 12 mm/hr Immunohistochemical analysis revealed that the leukemic
(reference range 0 to 9 mm/hr). Based on these results, the cells were positive for CD20, DBA44 (a B cell marker),
physician ordered serum immunoglobulin levels. The CD68, and annexin A1. Expression of CD20, CD11c,
following results were reported: IgG 3,250 mg/dL (refer- CD25, and CD103 was demonstrated by flow cytometry.
ence range 600 to 1,500 mg/dL), IgM 48 mg/dL (reference
Questions
range 75 to 150 mg/dL), and IgA 102 mg/dL (reference
range 150 to 250 mg/dL). a. What disease(s) should be considered in the differential
diagnosis?
Questions b. What is the significance of the immunophenotyping
a. What disease(s) should you suspect? Why? results?
b. What additional tests could help confirm the diagnosis
and what results would you expect to find?

REVIEW QUESTIONS
1. Bence Jones proteins consist of 5. Hodgkin lymphoma is characterized by
a. monoclonal IgG. a. proliferation of T cells.
b. IgG–IgM complexes. b. excess immunoglobulin production.
c. free κ or λ light chains. c. an incurable, rapidly progressive course.
d. free µ heavy chains. d. the presence of Reed-Sternberg cells in lymph
nodes.
2. Which of the following would be the best indicator
of a malignant clone of cells? 6. Chronic leukemias are characterized as
a. Overall increase in antibody production a. usually being of B-cell origin.
b. Increase in IgG and IgM only b. being curable with chemotherapy.
c. Increase in antibody directed against a specific c. usually occurring in children.
epitope d. following a rapidly progressive course.
d. Decrease in overall antibody production
7. Which of the following is characteristic of heavy-chain
3. All of the following are features of malignancy except diseases?
a. excess apoptosis. a. Usually of B-cell origin
b. rapid proliferation. b. Rare lymphomas
c. clonal proliferation. c. Production of abnormal Ig heavy chains
d. chromosomal mutations. d. All of the above

4. All of the following features are commonly used to 8. Flow cytometry results on a patient reveal a decrease
classify lymphoid neoplasms except of cells with CD2 and CD3. What does this indicate?
a. cell of origin. a. Lack of B cells
b. presence of gene translocations. b. Lack of T cells
c. exposure of the patient to carcinogens. c. Lack of monocytes
d. morphology or cytology of the malignant cells. d. Lack of natural killer cells
 9. Which of the following is true of Waldenström 13. Which of the following is not a requirement for urine
macroglobulinemia but not multiple myeloma? testing by IFE?
a. Hyperviscosity syndrome is often present. a. Collection of a 24-hour sample
b. A single protein-producing clone is elevated. b. Concentration of the sample
c. The cancerous cell is a preplasma cell. c. Dilution of the sample
d. Bence Jones proteins are present in the urine. d. Removal of sediment

10. The presence of anemia, bone pain, thrombocytopenia, 14. Multiple myeloma is characteristically preceded by
and lytic bone lesions is suggestive of a. chronic hypogammaglobulinemia.
a. Hodgkin lymphoma. b. Helicobacter pylori infection.
b. hairy cell leukemia. c. non-Hodgkin lymphoma.
c. chronic lymphocytic leukemia. d. monoclonal gammopathy of undetermined
d. multiple myeloma. significance.

11. The presence of an M protein on immunofixation 15. Which serum free light chain (sFLC) assay result
electrophoresis (IFE) is indicated by indicates presence of a malignant plasma cell
a. broad, diffuse banding. clone?
b. a narrow, discrete band. a. An abnormal κ:λ ratio
c. a few well-defined bands in the IgG lane. b. A decrease in κ and λ concentrations
d. a single band at the point of application in all c. A decrease in IgG, IgA, and IgM concentrations
of the lanes. d. An increase in immunoglobulin concentrations
over a 24-hour period
12. Surface immunoglobulin on a leukemic cell
indicates a(n)
a. B cell.
b. T cell.
c. macrophage.
d. autoimmune disease.
 Immunodeficiency
Diseases

After finishing this chapter, you should be able to: CLINICAL EFFECTS OF PRIMARY
IMMUNODEFICIENCIES
1. Differentiate between primary immunodeficiency diseases and
secondary immunodeficiency diseases. THE NINE CATEGORIES OF PRIMARY
IMMUNODEFICIENCIES
2. Indicate the general immunologic defects associated with each of the
nine categories of primary immunodeficiency diseases. Category 3: Predominantly Antibody
Deficiencies
3. Associate examples of specific immunodeficiencies with each category.
Category 1: Combined
4. Describe the types of infections typically associated with defects in the
Immunodeficiencies
B-cell, T-cell, myeloid, or complement systems.
Category 2: Combined
5. Recognize the association between immunodeficiency states and the
Immunodeficiencies With
risk of developing malignancy.
Associated or Syndromic Features
6. Explain the immunologic defects and clinical manifestations
Category 4: Diseases of Immune
associated with selected primary immunodeficiency diseases.
Dysregulation
7. Select appropriate laboratory tests to screen for and confirm the
Category 5: Congenital Defects of
presence of specific congenital immunodeficiencies.
Phagocyte Number, Function,
8. Correlate laboratory results with the presence of different types of or Both
primary immunodeficiencies.
Category 6: Defects in Innate Immunity
Category 7: Autoinflammatory
Disorders
Category 8: Complement Deficiencies
Category 9: Phenocopies of Primary
Immunodeficiencies
LABORATORY EVALUATION OF
IMMUNE DYSFUNCTION
Screening Tests
Confirmatory Tests
Newborn Screening for
Immunodeficiencies
Evaluation of Immunoglobulins
Bone Marrow Biopsy
Family History
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Immunodeficiency Diseases

Agammaglobulinemias DiGeorge anomaly Oxidative burst Severe combined

Ataxia-telangiectasia (AT) Immunodeficiencies Primary immunodeficiencies immunodeficiency (SCID)
Bruton’s tyrosine kinase (Btk) Immunofixation (PIDs) Transient
deficiency electrophoresis Purine-nucleoside hypogammaglobulinemia
Chronic granulomatous (IFE) phosphorylase (PNP) Wiskott-Aldrich syndrome
disease (CGD) Inflammasome deficiency (WAS)
Common variable Mitogen Secondary
immunodeficiency (CVI) immunodeficiency

Immunodeficiencies are disorders in which a part of the system such as the phagocytic cells, complement, or NK cells.
body’s immune system is missing or dysfunctional. People with Figure 19–1 illustrates points in the development of the
these conditions have a decreased ability to defend themselves immune system at which some PIDs exert their main effects.
against infectious organisms and are more susceptible to de- The types of infection or symptoms displayed by a patient
veloping certain types of cancer. The clinical symptoms asso- can give important clues regarding the specific immunodefi-
ciated with immunodeficiencies range from very mild or ciency present. In general, defects in humoral immunity (anti-
subclinical to severe, recurrent infections or failure to thrive. body production) result in pyogenic (i.e., pus-forming) bacterial
Immunodeficiencies can be inherited or acquired secondary to infections, particularly of the upper and lower respiratory tract.
other conditions such as certain infections, malignancies, au- Recurrent sinusitis and otitis media (i.e., ear infections) are
toimmune disorders, and immunosuppressive therapies. An common. The clinical course of viral infections in patients with
example of a secondary immunodeficiency is the acquired predominantly antibody deficiencies is not significantly differ-
immunodeficiency syndrome (AIDS), which is caused by the ent from that in normal hosts, with the exception of hepatitis
human immunodeficiency virus (HIV). AIDS is discussed in B, which may have a fulminant course in patients with agam-
detail in Chapter 24. This chapter focuses on the primary im- maglobulinemias, conditions in which antibody levels in the
munodeficiencies (PIDs), which are inherited dysfunctions of blood are significantly decreased.
the immune system. Several of the most important immuno- Defects in T-cell–mediated immunity result in recurrent in-
deficiency syndromes show X-linked inheritance and, therefore, fections with intracellular pathogens such as viruses, fungi, and
affect primarily males. Others show autosomal recessive or intracellular bacteria. Differentiating a primary cellular defi-
autosomal dominant inheritance.1 ciency from HIV-induced immunodeficiencies is essential for
More than 200 different congenital forms of immunodefi- proper treatment. Patients with congenital T-cell deficiencies
ciency have been reported, including defects in lymphoid cells, almost always develop mucocutaneous candidiasis, a yeast
phagocytic cells, regulatory molecules, and complement pro- infection that involves the skin, nails, and mucous membranes.
teins.1 With the exception of immunoglobulin A (IgA) defi- They are also prone to disseminated viral infections, especially
ciency, the PIDs are rare disorders with a combined incidence with latent viruses such as herpes simplex, varicella zoster, and
of about 1 in 1,200 live births.2 In spite of their rarity, it is im- cytomegalovirus. Because T cells also play an important role
portant for physicians to consider the possibility of PID in chil- in tumor immunity, patients with these conditions are more
dren with recurrent infections because early detection and susceptible to developing certain types of cancer. Age-adjusted
treatment can help prevent the development of serious, long- rates of malignancy in patients with immunodeficiency disease
term tissue damage or overwhelming sepsis. Early diagnosis are 10 to 200 times greater than those observed in immuno-
can also provide the opportunity for appropriate genetic coun- competent individuals.2 Most of the malignancies are lymphoid
seling, carrier detection, and prenatal diagnosis for other family and may be related to persistent stimulation of the remaining
members.3 The clinical laboratory plays an essential role in immune cells, coupled with defective immune regulation.
identifying these important diseases. This chapter serves as an Defects in other components of the immune system also
introduction to the PIDs and the laboratory methods that are have significant consequences. For example, neutrophils are the
used to detect the presence of these disorders. first line of defense against invading organisms; defects in neu-
trophil function are usually reflected in recurrent pyogenic bac-
terial infections or impaired wound healing. Abnormalities in
Clinical Effects of Primary macrophage function will have effects on both the innate and
Immunodeficiencies the adaptive defenses because macrophages are involved in the
nonspecific phagocytosis of microorganisms during inflamma-
The PIDs can affect one or more parts of the immune system, tion as well as in the processing of antigens and their presenta-
depending on the specific disease. Some PIDs have their primary tion to T cells in humoral and cell-mediated immune responses.
effect on B cells and humoral immunity, whereas others mainly Reduction in the macrophage population by splenectomy is
affect the cell-mediated branch of the adaptive immune system. associated with an increased risk of overwhelming bacterial
Other conditions involve components of the innate defense infection accompanied by septicemia. The complement system,
 PS

CMP

CGD
LAD
DiGeorge
PNP
WAS
IT
C-H
NK

Tc
AT
CLP ADA
Jak3
xSCID
KEY TO IMMUNODEFICIENCIES BTK
ADA: adenosine deaminase deficiency
AT: ataxia-telangiectasia
BTK: Bruton’s tyrosine kinase deficiency
CGD: chronic granulomatous disease Th
C-H: Chediak-Higashi syndrome CD40 CD40L
CVI: common variable immunodeficiency
DiGeorge: DiGeorge anomaly
IgA def: selective IgA deficiency
IgG def: IgG subclass deficiency
JAK3: Janus kinase-3 deficiency IB
Cytokines
LAD: leukocyte adhesion deficiency
PNP: purine nucleoside phosphorylase deficiency CVI
WAS: Wiskott-Aldrich syndrome PS
xSCID: X-linked severe combined
immunodeficiency disease
PS

IgG def

PS

IgA def

KEY TO CELLS PS
PS: pluripotent stem cell
CMP: common myeloid precursor
CLP: common lymphoid precursor
NK: natural killer cell
IB: immature B cell
IT: immature T cell
Th: helper T cell
Tc: cytotoxic T cell
PC: plasma cell

Examples of primary immunodeficiency diseases and their effects on the immune system.

as discussed in Chapter 7, is activated directly by antigens or The components of the immune system play unique, but
by antigen–antibody complexes to produce biologically active overlapping, roles in the host defense process. Therefore, defects
molecules that enhance inflammation and promote lysis of in any one of the cellular or humoral components result in dis-
microorganisms. Deficiencies of complement components result tinct clinical manifestations, as previously discussed. However,
in recurrent bacterial infections and autoimmune-type mani- because the components of the immune system interact exten-
festations. The severity of the conditions varies with the partic- sively through many regulatory and effector networks, a defect
ular complement component that is deficient. in one branch of the system may affect other aspects of immune
 Immunodeficiency Diseases

function as well. In many cases, it appears that deficiency of • Category 3: Predominantly Antibody Deficiencies
one component of the immune system is accompanied by hy- • Category 4: Diseases of Immune Dysregulation
peractivity of other components. This may occur because per- • Category 5: Congenital Defects of Phagocyte Number,
sistent infections continuously stimulate the available immune Function, or Both
cells or because a compensatory mechanism has been activated • Category 6: Defects in Innate Immunity
to correct for the deficient immune function. • Category 7: Autoinflammatory Disorders
In addition, the deficiency may involve a component that nor- • Category 8: Complement Deficiencies
mally exerts regulatory control over other components of the im- • Category 9: Phenocopies of Primary Immunodeficiencies
mune system—control that is lacking in the deficiency state. For Although the PID diseases are separated into these categories,
instance, T helper (Th2) cells secrete cytokines that regulate the some diseases are listed in more than one category because they
development of B cells into plasma cells. A defect in Th2 cell possess overlapping features. The following sections describe the
function, such as a deficiency in CD40L (a molecule involved in main characteristics of each category and examples of specific
binding to cell receptors during T-dependent immune responses), PIDs that have been included in each category. Category 3,
removes or creates an imbalance in the regulation of those im- Predominantly Antibody Deficiencies, is discussed first because
mune responses. Whatever the mechanism, many partial immun- the conditions in this category are the most common immun-
odeficiency states are associated with allergic or autoimmune odeficiencies, representing about 50% of the PIDs.2
manifestations, currently referred to as autoinflammatory disorders
(see discussion in the text that follows).
Category 3: Predominantly Antibody
Deficiencies
The Nine Categories of Primary
This category encompasses conditions in which the main
Immunodeficiencies characteristic is low levels of serum immunoglobulins. Im-
munoglobulins migrate in the “gamma region” of the serum
In the past, the immunodeficiencies have been broadly classified protein electrophoretic profile (discussed in Chapter 5).
as defects in T cells, B cells, phagocytes, complement proteins, Therefore, deficiencies of immunoglobulins have been termed
and other components of the innate immune system. As scientific agammaglobulinemias. The mechanisms of the agammaglob-
knowledge has been gained about the complexity of these disor- ulinemias include genetic defects in B-cell maturation or
ders, experts have recognized that such a broad classification is mutations leading to defective interactions between B and
overly simplistic.4 In 2014, the International Union of Immuno- T cells.1 A wide range of immunoglobulin deficiency states
logic Societies (IUIS) updated their classification of PIDs by have been reported and involve virtually all combinations of
grouping them into nine different categories based on their char- immunoglobulins and all degrees of severity. In some cases,
acteristic clinical features, immunologic defects, and genetic ab- only a single isotype of one immunoglobulin class is deficient,
normalities.1 The IUIS has also published diagnostic flow charts whereas all of the other isotypes are normal. Only the more
to aid in classifying patients into a disease entity based on clinical common and well-characterized syndromes are described
symptoms and laboratory results.5 The nine categories are: here. These are summarized in Table 19–1.
• Category 1: Combined Immunodeficiencies In evaluating immunoglobulin deficiency states, it is impor-
• Category 2: Combined Immunodeficiencies With Associ- tant to remember that blood levels of immunoglobulins change
ated or Syndromic Features with age. The blood level of IgG at birth is about the same as

Table 19–1 Characteristics of Selected Predominantly Antibody Deficiencies (Category 3)

CONDITION DEFICIENCY LEVEL OF DEFECT PRESENTATION
Transient All antibodies; especially IgG Slow development of helper 2–6 months; resolves by
hypogammaglobulinemia function in some patients 2 years
of infancy
Selective IgA deficiency IgA; some also with reduced IgA-B cell differentiation Often asymptomatic
IgG2
Btk deficiency All antibody isotypes reduced Pre–B-cell differentiation Infancy
Common variable Reduced antibody; many dif- B cell; excess T suppression Usually 20–30 years
immunodeficiency ferent combinations of age
Isolated IgG subclass Reduced IgG1, IgG2, IgG3, or Defect of isotype Variable with the class
deficiency IgG4 differentiation and degree of deficiency
CD154 deficiency Reduced IgG, IgA, IgE, with B-cell switching Variable
elevated IgM
the adult level, reflecting transfer of maternal IgG across the is a deficiency of an enzyme called the Btk in B-cell progenitor
placenta. The IgG level declines over the first 6 months of life cells.9,10 Lack of the enzyme apparently causes a failure of
as maternal antibody is catabolized. Levels of IgA and IgM are immunoglobulin VH gene rearrangement (see Chapter 5). The
very low at birth. The concentrations of all immunoglobulins syndrome can be effectively treated by administration of intra-
gradually rise when the infant begins to produce antibodies at muscular or intravenous immunoglobulin preparations and
a few months of age in response to environmental stimuli. IgM vigorous antimicrobial treatment of infections. The syndrome
reaches normal adult levels first, around 1 year of age, followed can be differentiated from transient hypogammaglobulinemia
by IgG at about 5 to 6 years of age. In some normal children, of infancy by the absence of CD19+ B cells in the peripheral
IgA levels do not reach normal adult values until adolescence. blood, the abnormal histology of lymphoid tissues, and its
Therefore, it is important to compare a child’s immunoglobulin persistence beyond 2 years of age. Immunologists have also de-
levels to age-matched reference ranges. scribed patients with a similar clinical presentation to Btk who
have a genetic defect that is inherited in an autosomal recessive
manner.1,6,8

All infants experience low levels of immunoglobulins at ap-

proximately 5 to 6 months of age; however, in some babies the Selective IgA deficiency is the most common congenital
low levels persist for a longer time. Because these children do immunodeficiency, occurring in about 1 in 500 persons of
not begin synthesizing immunoglobulins promptly, they can American or European descent.6 Most patients with a defi-
experience severe pyogenic sinopulmonary and skin infections ciency of IgA are asymptomatic. Those with symptoms usu-
as protective maternal IgG is cleared. Cell-mediated immunity ally have infections of the respiratory and gastrointestinal
is normal and there may be normal levels of IgA and IgM.1,6 tract and an increased tendency to develop autoimmune dis-
IgG appears to be the most affected, dropping to at least 2 stan- eases such as systemic lupus erythematosus (SLE), rheuma-
dard deviations (SDs) below the age-adjusted mean with or toid arthritis (RA), celiac disease, and thyroiditis. Allergic
without a depression of IgM and IgA.6 Immunoglobulin levels disorders and malignancy are also more common.7 About
in infants with this condition usually normalize spontaneously, 20% of the IgA-deficient patients who develop infections also
often by 9 to 15 months of age. The mechanism of this tran- have an IgG2 subclass deficiency. If the serum IgA is lower
sient hypogammaglobulinemia is not known. These patients than 5 mg/mL, the deficiency is considered severe. If the IgA
have normal numbers of circulating CD19+ B cells. This con- level is two SDs below the age-adjusted mean but greater than
dition does not appear to be X-linked, although it is more com- 50 mg/dL, the deficiency is partial. Although the genetic de-
mon in males. The cause may be related to a delayed maturation fect has not been established, it is hypothesized that lack of
of one or more components of the immune system, possibly IgA is caused by impaired differentiation of lymphocytes to
Th cells.6 become IgA-producing plasma cells.6
IgE antibodies specifically directed against IgA are pro-
duced by 30% to 40% of patients with severe IgA deficiency.
These antibodies can cause anaphylactic reactions when
Bruton’s tyrosine kinase (Btk) deficiency, first described in blood products containing IgA are transfused.7 Because many
1952, is X chromosome linked, so this syndrome affects males patients with severe IgA deficiency have no other symptoms,
almost exclusively. Patients with X-linked agammaglobulinemia the IgA deficiency may not be detected until the patient ex-
lack circulating mature CD19+ B cells and exhibit a deficiency periences a transfusion reaction, resulting in the production
or lack of immunoglobulins of all classes.1,7,8 Furthermore, they of anti-IgA antibodies. Therefore, products for transfusion to
have no plasma cells in their lymphoid tissues. The patients do, known IgA-deficient patients should be collected from IgA-
however, have pre-B cells in their bone marrow.8 Because of the deficient donors or cellular products should be washed to
lack of B cells, the tonsils and adenoids are small or entirely ab- remove as much donor plasma as possible.
sent and lymph nodes lack normal germinal centers. T cells are Most gamma globulin preparations contain significant
normal in number and function. About half of the patients have amounts of IgA. However, replacement IgA therapy is not useful
a family history of the syndrome. They develop recurrent bac- because the half-life of IgA is short (around 7 days) and intra-
terial infections beginning in infancy as maternal antibody is venously or intramuscularly administered IgA is not transported
cleared. The patients most commonly develop sinopulmonary to its normal site of secretion at mucosal surfaces. Furthermore,
infections caused by encapsulated organisms such as strepto- administration of IgA-containing products can induce the
cocci, meningococci, and Haemophilus influenzae. Other infec- development of anti-IgA antibodies or provoke anaphylaxis in
tions seen include bacterial otitis media, bronchitis, pneumonia, patients who already have antibodies.
meningitis, and dermatitis.6 Some patients also have a suscep-
tibility to certain types of viral infections, including vaccine-
associated poliomyelitis. In general, live virus vaccines should Common variable immunodeficiency (CVI) is a heteroge-
not be administered to immunodeficient patients. neous group of disorders with a prevalence of about 1 in
X-linked hypogammaglobulinemia results from arrested dif- 25,000.11 Although this is a low incidence, it does make CVI
ferentiation at the pre–B-cell stage, leading to a complete absence the most common PID with a severe clinical syndrome.12
of B cells and plasma cells. The underlying genetic mechanism Patients usually begin to have symptoms in their 20s and 30s,
 Immunodeficiency Diseases

but age at onset ranges from 7 to 71 years of age. The disorder heavy-chain gene deletions and transcriptional defects. The
can be congenital or acquired, or familial or sporadic, and it most common subclass deficiency is IgG4, with IgG1 defi-
occurs with equal frequency in men and women. CVI is char- ciency being the least common, although IgG4 subclass defi-
acterized by hypogammaglobulinemia that leads to recurrent ciency may have the least clinical significance.6
bacterial infections, particularly sinusitis and pneumonia. In
addition, up to 20% of CVI patients develop herpes zoster Category 1: Combined
(shingles), a much higher incidence than in immunologically
normal young adults. There is usually a deficiency of both IgA
Immunodeficiencies
and IgG, but selective IgG deficiency may occur. CVI is often This category contains diseases in which there are defects in
associated with a spruelike syndrome characterized by malab- both humoral (B cell) and cell-mediated (T cell) immunity.
sorption and diarrhea. CVI is also associated with an increased These deficiencies result from mutations that affect develop-
risk of lymphoproliferative disorders, gastric carcinomas, and ment of both types of lymphocytes or cause defective inter-
autoimmune disorders.7 The most common autoimmune man- action between the two antigen-specific limbs of the adaptive
ifestations of CVI are immune thrombocytopenia and autoim- immune system. Because helper T-cell functions are necessary
mune hemolytic anemia. Other symptoms may include for normal differentiation and antibody secretion by B cells,
lymphadenopathy, splenomegaly, and intestinal hyperplasia.7 a severe defect of T-cell function will have effects on im-
CVI is diagnosed by demonstrating a low serum IgG level munoglobulin levels as well. Combined deficiencies are
in patients with recurrent bacterial infections. Additionally, referred to using a shorthand notation of T+/–B+/–NK+/– with
blood group isohemagglutinins, or the so-called naturally the + or – superscript denoting whether or not each cell type
occurring antibodies, are typically absent or low. In contrast to is present in the deficiency.7
X-linked agammaglobulinemia, most patients with CVI have
normal numbers of mature B cells. However, these B cells do
The most serious of the congenital immunodeficiencies is
not differentiate normally into immunoglobulin-producing
severe combined immunodeficiency (SCID). SCID is actually
plasma cells. Three major types of cellular defects have been
a group of related diseases that all affect T- and B-cell function
found in CVI patients. In some cases, T cells or their products
but with differing causes. A mutation in the interleukin-2 re-
appear to suppress differentiation of B cells into plasma cells.
ceptor gamma (IL2RG) gene located on the X-chromosome is
Secondly, T cells may fail to provide adequate help to support
the most common form of the disease, accounting for approx-
terminal differentiation of B cells. Finally, there appears to be
imately 46% of the cases in the United States today.2,14 The mu-
a primary defect in the B-cell line in some patients. CVI is often
tation occurs with a frequency of about 1 in 50,000 births.15
a diagnosis of exclusion, where an immunodeficiency is pres-
The IL2RG gene codes for a protein chain called the common
ent with no specific genetic defect defined.8
gamma chain that is common to receptors for interleukins-2,
CVI can usually be effectively treated with intramuscular or
4, 7, 9, 15, and 21.14 Normal signaling cannot occur in cells
intravenous immunoglobulin preparations.13 However, because
with defective receptors, thus halting natural maturation.2,15
of their low levels of secretory IgA, patients are still susceptible
This may result in either a T-B+NK+ or a T-B+NK- phenotype,
to respiratory and gastrointestinal infections; the clinician
depending on whether or not there is an additional defect in
should be vigilant for these infections and treat them vigorously
the JAK3 gene.7 JAK3 is required for processing an interleukin-
with antibiotics.
binding signal from the cell membrane to the nucleus. No an-
tibody production or lymphocyte proliferative response follows
IgG subclass deficiencies are conditions where the level(s) of an antigen or mitogen challenge in such cases.
one or more of the four IgG subclasses is (are) more than two Several autosomal recessive forms of SCID, which affect both
SDs below the mean age-appropriate level.6 Normally, about males and females, have also been discovered.7 For example, a
70% of the total IgG is IgG1, 20% IgG2, 6% IgG3, and 4% JAK3 deficiency may be found without the common gamma
IgG4. Therefore, a deficiency of a single subclass may not result chain deletion. The lack of the intracellular kinase JAK3 means
in a total IgG level below the normal range. In patients with that that lymphocytes are unable to transmit signals from IL-2
recurrent infections, levels of the different subclasses should and IL-4.2,11 These patients have a T-B+NK- phenotype and
be measured if the total IgG level is normal but the clinical symptoms are similar to the X-linked form of the disease.7 The
picture suggests immunoglobulin deficiency.6 JAK3 gene is located on chromosome 19, region p12. Other au-
Most IgG antibodies directed against protein antigens are of tosomal recessive forms of SCID are discussed in the paragraphs
the IgG1 and IgG3 sub-classes, whereas most IgG antibodies that follow.
against carbohydrate antigens are IgG2 or IgG4. Thus, defi- About 15% to 20% of the patients with SCID have an adeno-
ciencies involving IgG1 or IgG3 lead to a reduced capability of sine deaminase (ADA) deficiency, leading to a T-B-NK- pheno-
responding to protein antigens such as toxins, whereas selec- type.7 The ADA gene is located on chromosome 1, region q21.
tive deficiencies of IgG2 can result in impaired responses to ADA deficiency affects an enzyme involved in the metabolism
polysaccharide antigens, which cause recurrent infections with of purines, similar to another form of PID, the PNP deficiency.
polysaccharide-encapsulated bacteria such as Streptococcus In ADA deficiency, toxic metabolites of purines accumulate in
pneumoniae and H influenzae.6 A variety of genetic defects have lymphoid cells and impair proliferation of both B and T cells.
been associated with IgG subclass deficiency. These include In both ADA and PNP deficiencies, there is a progressive
decrease in lymphocyte numbers. A number of different muta- metabolism of purines. It produces a moderate to severe defect
tions have been found to lead to ADA deficiency and the degree in cell-mediated immunity, with normal or only mildly im-
of immunodeficiency correlates with the degree of ADA defi- paired humoral immunity.11 The number of T cells progres-
ciency. Patients with only mildly reduced ADA activity may have sively decreases because of the accumulation of deoxyguanosine
only a slight impairment of immune function.7 triphosphate, a toxic purine metabolite. The levels of im-
Other molecular defects have also been identified as causes munoglobulins are generally normal or increased. About two
of SCID. Infants with a lack of both T and B cells but with func- thirds of PNP-deficient patients also have neurological disor-
tioning NK cells were found to have a mutation in a recombinase ders, but no characteristic physical abnormalities have been
activating gene (RAG-1 or RAG-2).14 These mutations cause a described. Because of the relatively selective defect in cell-
profound lymphocytopenia because of the inability of T and mediated immunity, PNP deficiency can be confused with
B cells to rearrange deoxyribonucleic acid (DNA), a process that neonatal HIV infection. The two conditions can usually be
is necessary to produce functional immunoglobulins or T-cell distinguished by specific tests for HIV (see Chapter 24) and by
receptors (TCRs).11 Patients with RAG-1 or RAG-2 deficiencies assays for PNP activity.
have decreased class II MHC molecule expression. HLA class II
molecules are intimately involved in antigen presentation; thus,
this defect profoundly impairs the immune response.7,14 Another
Category 2: Combined
molecular defect that has been identified is a mutation in the Immunodeficiencies With
gene encoding a common leukocyte protein called CD45. It is a Associated or Syndromic Features
transmembrane phosphatase that regulates signal transduction
This category differs from Category 1 in that the diseases in
of T- and B-cell receptors.11,14
Category 2 are characterized by nonimmunologic features in
Patients with SCID generally present early in infancy with
addition to the combined immunodeficiency. Diseases in this
infection by nearly any type of organism. Oral candidal yeast
category are typically caused by defects in cell-mediated im-
infections, pneumonia, and diarrhea are the most common
munity, which indirectly lead to problems with the other
manifestations. The administration of live vaccines can cause
branches of the immune response. Often, these diseases can
severe illness. Unless immune reconstitution can be achieved
result from abnormalities at different stages of T-cell develop-
by bone marrow transplantation or by specifically replacing
ment.1 Many different molecular defects can result in a similar
a deficient enzyme, patients with SCID die before they are
clinical picture (as in SCID). This is because T cells provide
2 years old.14
helper functions that are necessary for normal B-cell develop-
ADA deficiency is a special case because it presents a good
ment and differentiation. Some of the more common defects
opportunity for enzyme replacement therapy or somatic cell
of cellular and combined cellular and humoral immunity are
gene therapy. Although ADA is normally located within cells,
summarized in Table 19–2.
its deficiency can be treated by maintaining high plasma levels
In general, defects in cellular immunity are more difficult to
of ADA. For some patients, red blood cell (RBC) transfusion
manage than defects in humoral immunity. When immunoglob-
can raise ADA to near normal levels. However, bovine ADA con-
ulin production is deficient, replacement therapy is often very
jugated with polyethylene glycol (PEG) has a longer half-life
effective. However, there is usually no soluble product that can
than native ADA and can raise the ADA level up to three times
be administered to treat a deficiency of cell-mediated immunity.
higher than normal. This treatment increases T-cell production
Transplantation of immunologically intact cells, usually in the
and specific antibody responses. Side effects of ADA–PEG
form of allogenic bone marrow, is often required to reconstitute
appear to be minimal. However, this therapy is very expensive
immune function.
and is primarily used in patients for whom a suitable bone mar-
Patients with severe defects in cell-mediated immunity may
row donor cannot be found or for those patients who are too
develop graft-versus-host disease (GVHD; see Chapter 16).
sick to undergo marrow transplantation.16 Human cord blood
Transfused lymphocytes are normally destroyed by the recipi-
stem cell transplantation has been used with moderate suc-
ent’s T-cell system. However, a severe defect in the T-cell system
cess.17 Studies are currently underway to attempt to treat ADA
allows the donor lymphocytes to survive, proliferate, and at-
deficiency by transfecting a normal ADA gene into patients’
tack the tissues of the recipient as foreign. GVHD can occur in
T cells or stem cells. These cells could then be reinfused into
any patient with a severe defect in cell-mediated immunity
the patient. The gene therapy has actually been curative in some
(e.g., in bone marrow transplant recipients) and can be fatal.
cases, but many obstacles remain to be overcome.13
Irradiation of cell-containing blood products (platelet concen-
trates, packed RBCs, and whole blood) before transfusion de-
stroys the ability of the donor lymphocytes to proliferate and
Another immunodeficiency state for which a specific enzymatic prevents development of GVHD in immunodeficient recipi-
basis has been defined is purine-nucleoside phosphorylase ents. It should be noted that a defect in humoral immunity
(PNP) deficiency. PNP deficiency is a rare autosomal recessive does not predispose an individual to GVHD.
trait.11 The condition presents in infancy with recurrent or GVHD also occurs in patients who have received a bone
chronic pulmonary infections, oral or cutaneous candidiasis, marrow transplant as therapy for a congenital immunodefi-
diarrhea, skin infections, urinary tract infections, and failure ciency. The closer the match between the genetic constitution
to thrive. PNP deficiency affects an enzyme involved in the of the patient and the graft donor, the less severe the GVHD
 Immunodeficiency Diseases

Table 19–2 Characteristics of Selected Combined Immunodeficiencies (Category 1) and Combined

Immunodeficiencies With Associated or Syndromic Features (Category 2)
CONDITION DEFICIENCY LEVEL OF DEFECT PRESENTATION
Category 1
CD40 ligand T cells with effects on Defective isotype switching 1–2 years of age
deficiency antibody production with increased or normal
IgM but decreased concen-
trations of other isotypes
SCID Both T and B cells ADA, purine metabolism; Infancy
RAG-1/RAG-2; JAK3;
common gamma chain
receptor; others
PNP deficiency T cells; some secondary effects PNP, purine metabolism Infancy
on antibody production
Category 2
DiGeorge anomaly T cells; some secondary effects Embryologic development Neonatal, with hypocalcemia
on antibody production of the thymus or cardiac defects if severe;
abnormal mental delay; devel-
opment; incomplete forms may
present later with infection
WAS Reduced IgM and T-cell defect CD43 expression Usually infancy; with thrombo-
cytopenia, small platelets, and
eczema
AT Reduced IgG2, IgA, IgE, and T DNA instability Infancy, with involuntary
lymphocytes muscle movements and
capillary swelling

ADA = adenosine deaminase; AT = ataxia-telangiectasia; PNP = purine-nucleoside phosphorylase; SCID = severe combined immunodeficiency;
WAS = Wiskott-Aldrich syndrome.

is likely to be. Thus, although bone marrow transplantation and IgG, and increased levels of IgE.7 These patients also have
can potentially cure the immune defect, it can also have seri- persistently increased levels of serum alpha-fetoprotein, which
ous, lifelong complications of its own. Examples of specific can also be a useful diagnostic feature.
immunodeficiencies in this category are discussed in the text The primary molecular defect in the syndrome appears to
that follows. be an abnormality of the integral membrane protein CD43,
which is involved in the regulation of protein glycosylation.8
The gene responsible for the defect, called the WAS gene, is
Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive
located on the X chromosome, region p11. Abnormalities
syndrome that is defined by the triad of immunodeficiency,
cause defective actin polymerization and affect its signal trans-
eczema, and thrombocytopenia.18 WAS is usually lethal in
duction in lymphocytes and platelets.8
childhood because of infection, hemorrhage, or malignancy.
Platelets have a shortened half-life and T lymphocytes are
Milder variants have also been described such as an X-linked
also affected, although B lymphocytes appear to function nor-
form of thrombocytopenia.
mally. Splenectomy can be very valuable in controlling the
The laboratory features of WAS include a decrease in platelet
thrombocytopenia. Current treatment for this immunodefi-
number and size with a prolonged bleeding time.7 The bone
ciency is transplantation of bone marrow or cord blood stem
marrow contains a normal or somewhat increased number of
cells from an HLA identical sibling.13
megakaryocytes. There are abnormalities in both the cellular
and humoral branches of the immune system related to a gen-
eral defect in antigen processing. As a result, patients display a DiGeorge anomaly is a developmental abnormality of the third
severe deficiency of the naturally occurring IgM antibodies to and fourth pharyngeal pouches that affects thymus development
ABO blood group antigens (isohemagglutinins). Absence of iso- in the embryo. All organs derived from these embryonic struc-
hemagglutinins is the most consistent laboratory finding in tures can be affected. Associated abnormalities include mental
WAS and is often used diagnostically. Patients with WAS can retardation, absence of ossification of the hyoid bone, cardiac
have a variety of different patterns of immunoglobulin levels, anomalies, abnormal facial development, and thymic hypopla-
but they usually have low levels of IgM, normal levels of IgA sia.7 The severity and extent of the developmental defect can
be quite variable. Many patients with a partial DiGeorge to properly repair DNA damage leads to the accumulation
anomaly have only a minimal thymic defect and, thus, near of mutations. The only effective therapy for AT is allogeneic
normal immune function.7 However, about 20% of children bone marrow transplantation.
with a defect of the third and fourth pharyngeal pouches have
a severe and persistent decrease in T-cell numbers.11 These Category 4: Diseases of Immune
children tend to have severe, recurrent viral and fungal in-
fections. Severely affected children usually present in the
Dysregulation
neonatal period with tetany (caused by hypocalcemia result- Category 4 includes many diseases with normal numbers of
ing from hypoparathyroidism) or manifestations of cardiac T or B cells but with reduced control over their functions. Many
defects. of the diseases in the category also have features of autoimmu-
The possibility of immunodeficiency can be overlooked if nity.1 The autoimmune lymphoproliferative syndrome (ALPS),
the association between the presenting abnormality and a pos- for example, may involve mutations in genes coding for caspase
sible thymic defect is not recognized. The immunodeficiency enzymes involved in apoptosis. Defective apoptosis in the thy-
associated with the DiGeorge anomaly is a quantitative defect mus may lead to autoreactive cells in the circulation. The CD25
in thymocytes. Not enough mature T cells are made, but those deficiency is manifested by a lack of T regulatory (Treg) cells,
that are present are functionally normal. The immunodefi- which leads to lymphoproliferation and autoimmunity. Muta-
ciency of DiGeorge syndrome can be treated with fetal thymus tions in the FoxP3 gene, which is required for Treg differentia-
transplantation. Bone marrow transplantation has also been tion, may show a similar clinical presentation. Chediak-Higashi
successful in some patients, as has administration of thymic syndrome, an immunodeficiency with hypopigmentation (loss
hormones. of skin color) caused by a mutation in the LYST gene, is char-
Most patients with DiGeorge syndrome show a deletion in acterized by a reduced number of natural killer (NK) cells and
chromosome 22, region q11,1,19 although this anomaly is not neutrophils, as well as an increased production of inflammatory
required for diagnosis.7 The q11 region of chromosome 22 proteins. Peripheral blood smears from patients with Chediak-
deletion is also associated with velocardiofacial syndrome Higashi syndrome show granulocytic inclusions attributed to
(VFS) and other syndromes.20. enlarged lysosomes.1

Ataxia-telangiectasia (AT) is a rare autosomal recessive syn- Category 5: Congenital Defects

drome characterized by cerebellar ataxia (involuntary muscle of Phagocyte Number, Function, or Both
movements) and telangiectasias (capillary swelling resulting in
Category 5 classifies the PIDs which are characterized by ab-
red blotches on the skin), especially on the earlobes and con-
normalities in phagocytic cells. Recall from Chapter 1 that the
junctiva. Blood vessels in the sclera of the eyes may be dilated
majority of cells that perform phagocytosis are neutrophils.
and there may also be a reddish butterfly area on the face and
Neutrophils play a crucial role in the immediate and nonspe-
ears. Ninety-five percent of patients exhibit increased levels of
cific response to invading organisms by responding before
serum alpha-fetoprotein.11 The incidence of this disease is be-
specific antibody and cell-mediated immune responses can
tween 1:10,000 to 1:100,000, although as much as 1% of the
be mounted. In addition, neutrophils are even more effective
population is heterozygous for the gene.21 Abnormal genes
at ingesting and killing organisms coated with specific antibody
produce a combined defect of both humoral and cellular im-
and thus continue to play an important role in host defense
munity.7,21 Antibody response to antigens, especially polysac-
even after an adaptive immune response is established. To
charides, is blunted. The levels of IgG2, IgA, and IgE are often
destroy invading organisms, neutrophils must adhere to vas-
low or absent, although the pattern can be quite variable. In
cular endothelial lining cells, migrate through the capillary wall
addition, the number of circulating T cells is often decreased.
to a site of infection, and ingest and kill the microbes (see
Death usually occurs in early adult life from either pulmonary
Chapter 3). Defects affecting each of these steps can lead to an
disease or malignancy.22
increased susceptibility to pyogenic infections.
Patients with AT have a defect in a gene that is apparently
essential to the recombination process for genes in the im-
munoglobulin superfamily. The AT gene is located on chro- Chronic granulomatous disease (CGD) is a group of disorders
mosome 11, region q22. This abnormality results in a involving inheritance of either an X-linked or autosomal recessive
defective kinase involved in DNA repair and in cell cycle con- gene that affects neutrophil microbiocidal function. The X-linked
trol.15 Rearrangement of TCR and immunoglobulin genes disease accounts for 70% of the cases and tends to be more
does not occur normally.11 Patients’ lymphocytes often exhibit severe.7 Symptoms of CGD include recurrent suppurative infec-
chromosomal breaks and other abnormalities involving the tions, pneumonia, osteomyelitis, draining adenopathy, liver
TCR genes in T cells and immunoglobulin genes in B cells. abscesses, dermatitis, and hypergammaglobulinemia. Typically,
These are sites of high levels of chromosomal recombination catalase-positive organisms such as Staphylococcus aureus,
and errors that occur during gene rearrangements may not Burkholderia cepacia, and Chromobacterium violaceum are involved
be repaired properly. The syndrome is associated with an in addition to fungi such as Aspergillus and Nocardia.7,21 Infections
even greater risk of lymphoid malignancy than other im- usually begin before 1 year of age and the syndrome is often fatal
munodeficiency syndromes, presumably because the failure in childhood.
 Immunodeficiency Diseases

CGD is the most common and best characterized of the neu- may have recurrent candidal infections. Defects of neutrophil
trophil abnormalities. Several specific molecular defects have secondary granules have been described also. However, the
been described in this syndrome, all of which result in the inabil- molecular nature of the defects is unknown.13
ity of the patient’s neutrophils to produce the reactive forms of
oxygen necessary for normal bacterial killing. Three different
Even if microbiocidal activity is normal, neutrophils cannot per-
autosomal recessive genes are involved and all affect subunits
form their functions properly if they fail to leave the vasculature
of nicotinamide adenine dinucleotide phosphate (NADPH) oxi-
and migrate to a site of incipient infection. Adhesion receptors
dase.23 Normally, neutrophil stimulation leads to the production
on leukocytes and their counterreceptors on endothelial cells
of reactive oxygen molecules, such as hydrogen peroxide (H2O2),
and the extracellular matrix play important roles in these activ-
by NADPH oxidase reactivity on the plasma membrane. The
ities. In leukocyte adhesion deficiency (LAD), there is a defi-
plasma membrane enfolds an organism as it is phagocytized and
ciency in a protein called CD18, which is a component of
hydrogen peroxide is generated in close proximity to the target
adhesion receptors on neutrophils and monocytes (CD11b
microbe. Neutrophil granules fuse with and release the enzyme
or CD11c) and on T cells (CD11a).7,23 The CD18 deficiency
myeloperoxidase into the forming phagosome. The myeloperox-
is transmitted through autosomal recessive inheritance and
idase uses the hydrogen peroxide to generate the potent micro-
has variable expression. This defect leads to abnormal adhe-
bicidal agent, hypochlorous acid (see Chapter 3).23
sion, motility, aggregation, chemotaxis, and endocytosis by
The process of generating partially reduced forms of oxygen
the affected leukocytes. The defects are clinically manifest as
by stimulated neutrophils was first detected as an increase in
delayed wound healing, chronic skin infections, intestinal and
oxygen consumption. Therefore, this response was originally
respiratory tract infections, and periodontitis. A defect in CD18
termed the neutrophil “respiratory burst.” A more correct term
can be diagnosed by detecting a decreased amount of the
is oxidative burst. A genetic defect in any of the several com-
CD11/18 antigen on patient leukocytes by flow cytometry.23
ponents of the NADPH oxidase system can result in the CGD
Another type of adhesion molecule deficiency (LAD II) has
phenotype by making the neutrophil incapable of generating
also been characterized. In this disorder, a carbohydrate mole-
an oxidative burst.
cule involved in adhesive interactions, CD15s, or sialyl-Lewis X,
CGD was historically diagnosed by measuring the ability of
is deficient.23
a patient’s neutrophils to reduce the dye nitroblue tetrazolium
(NBT). NBT reduction is caused by the production of hydrogen
peroxide and other reactive forms of oxygen. Reduction con- Category 6: Defects in Innate Immunity
verts the nearly colorless NBT into a blue precipitate that can
Category 6 represents a new part of the PID classification, mir-
be assessed visually on a microscope slide.23 More recently, a
roring the explosive increase in knowledge and understanding
flow cytometric assay has been used. In this assay, neutrophils
of the innate immune system. At least one disease, chronic
are labeled with dihydrorhodamine (DHR). DHR will fluoresce
mucocutaneous candidiasis, which was previously classified as
when it is reduced. The neutrophils are then activated using
a T-cell defect, is now included in this classification.1 Researchers
phorbol myristate acetate (PMA), which is mitogenic for neu-
identified two forms of this entity involving mutations in genes
trophils. The resultant oxidative burst will reduce the DHR,
coding for IL-17. Other rare entities classified under this heading
resulting in fluorescence that may be quantitated on a flow
include mutations in Toll-like receptors (TLRs). For example,
cytometer. Neutrophils from CGD patients will be unable to
TLR3 deficiency results in herpes simplex encephalitis. Defects
undergo the oxidative burst and show less fluorescence than
in TLR signaling pathways, such as IRAK4 deficiency, can also
normal neutrophils.23 This technique is more objective and
occur. Both types of defects can lead to bacterial infections.
quantitative than the traditional NBT technique.
Few clinical laboratory assays are currently available for assess-
Although therapy with granulocyte transfusions may allow
ing innate immune system functional capabilities. Diagnosing
resolution of an acute infectious episode, it is impossible to
these entities is based upon clinical presentation, which leads
provide enough granulocytes to treat the chronic condition.
to molecular analyses to identify specific genetic mutations.
Administration of cytokines, such as interferon, may increase
the oxidative burst activity in some patients. Continuous use
of antibiotics can greatly reduce the occurrence of severe in- Category 7: Autoinflammatory Disorders
fections.15 Bone marrow transplantation or use of peripheral
Autoinflammatory disorders are subdivided into two classifi-
blood stem cells may result in a permanent cure.23
cations: those involving the inflammasome and noninflamma-
some conditions. The inflammasome is a protein oligomer
Several other recognized defects can result in impaired neu- that contains caspase enzymes and other proteins associated
trophil microbiocidal activity. Neutrophil glucose-6-phosphate with apoptosis. The inflammasome is located primarily in
dehydrogenase deficiency leads to an inability to generate myeloid cells and may be activated by various microbial sub-
enough NADPH to supply reducing equivalents to the NADPH stances. Once activated, the inflammasome stimulates the pro-
oxidase system. This shortfall leads to a defect in hydrogen per- duction of the proinflammatory cytokines IL-1 and IL-18.24
oxide production and a clinical picture similar to that of CGD. Genetic defects involving the inflammasome include the
Myeloperoxidase deficiency is relatively common, occurring in Hyper IgD syndrome, also referred to as periodic fever syn-
about 1 in 3,000 persons in the United States. Deficient patients drome, and Muckle-Wells syndrome. Hyper IgD is caused by
a deficiency of mevalonate kinase, an enzyme involved in a Laboratory Evaluation of Immune
sterol synthesis pathway. The syndrome has been seen prima-
rily in northern European populations. Diagnosis includes clin- Dysfunction
ical presentation of recurrent fevers, followed by IgD testing.
Muckle-Wells syndrome is caused by a mutation in the CIAS1 When performing diagnostic testing for immunodeficiency, it
gene coding for cryopyrin, a component of the inflammasome. is important for laboratorians to compare the results for a
Patients may present with urticaria and amyloidosis. Molecular patient with appropriate age-matched controls. In tests of
assays are necessary to confirm the diagnosis.1 cellular function, the patient’s cells need to be tested in parallel
Defects not involving the inflammasome include tumor with cells from a normal control. If an abnormal test result is
necrosis factor (TNF) receptor-associated periodic syndrome obtained, it should be confirmed by repeat testing.
(TRAPS) and early-onset inflammatory bowel disease (IBD).
TRAPS is caused by a mutation in the TNFRSF1A gene, which
codes for a TNF receptor and may result in recurrent fevers,
Screening Tests
as well as ocular and joint inflammation. Early-onset IBD is Screening tests are used for the initial evaluation of a suspected
caused by mutations in genes coding for IL-10 or its receptor.1 immunodeficiency state. Most of these tests can be performed
Family history may be helpful in diagnosing diseases of Cat- routinely in any hospital laboratory. The evaluation of possible
egory 7. Muckle-Wells syndrome and TRAPS show autosomal immunodeficiency starts with a patient history, followed by a
dominant inheritance, whereas Hyper IgD and early-onset IBD complete blood count (CBC) and white blood cell (WBC) dif-
are autosomal recessive. ferential, which may reveal a reduced lymphocyte count.
Thrombocytopenia with small platelets can be detected in WAS.
Category 8: Complement Deficiencies Measurement of the levels of serum IgG, IgM, and IgA and
levels of the subclasses of IgG are used to screen for defects in
Complement consists of a series of proteins that work in a cas- antibody production. Assay for isohemagglutinins is easily per-
cade to assist in antibody destruction of cells, as described in formed by the transfusion service. By the age of 2, a child should
Chapter 7. The complement system is also part of the innate have naturally occurring IgM antibodies against ABO blood
immune system and can work as part of the inflammatory group antigens. The absence of these antibodies suggests an
system to directly eliminate a potential pathogen. Deficiencies abnormal IgM response.
in each of the major complement components have been An overall assessment of antibody-mediated immunity can
described, leading to various clinical sequalae.25 be made by measuring antibody responses to antigens to which
Deficiencies in the early complement components, C1q, C4, the population is exposed normally or following vaccination.
and C2, are usually associated with a lupuslike syndrome. De- This can be easily done by measuring the titer of the specific
ficiency of C2 is believed to be the most common complement antibody produced in response to immunization with a com-
component deficiency. A C3 deficiency may also have a lupus- mercial vaccine such as diphtheria/tetanus. In an unimmunized
like clinical presentation, but is more likely to involve recurrent child, the development of tetanus or diphtheria antibodies is
infections with encapsulated organisms. Deficiencies of the determined 2 weeks after immunization. In a previously im-
later components of complement (C5–C9) are often associated munized patient, the response to a booster injection can be
with recurrent Neisseria meningitidis infections. A deficiency of evaluated, normally 4 to 6 weeks post vaccination. A wide
C1 esterase inhibitor has been found in patients with heredi- range of other protein and polysaccharide antigens can also be
tary neuroangioedema. Most complement deficiencies appear used in these tests. This technique is often used to evaluate a
to be inherited in an autosomal recessive manner and are likely possible IgG subclass deficiency. IgG1 and IgG3 isotypes nor-
caused by random changes in DNA nucleotide sequence as mally respond to protein antigens, such as tetanus and diph-
opposed to genetic deletions.6 theria. IgG2 normally responds to polysaccharide antigens,
such as those in the H influenzae and S pneumoniae vaccines.6
Category 9: Phenocopies of Primary Delayed hypersensitivity-type skin reactions can be used to
screen for defects in cell-mediated immunity (see Chapter 14).
Immunodeficiencies These tests are generally performed by the clinician and not by
This category comprises a new classification of PIDs. Disorders laboratory personnel. Delayed cutaneous hypersensitivity is a
that fall into this category have an inherited genetic component localized cell-mediated reaction to a specific antigen. The pro-
but also include an acquired component, such as somatic muta- totype is the tuberculin skin test. An antigen to which most of
tions or autoantibody production.1 New knowledge of these fac- the population has been exposed, such as candida, mumps, or
tors has led to reclassification of some PIDs. For example, chronic tetanus toxoid, is injected intradermally. The presence of in-
mucocutaneous candidiasis, a disease that was once classified as duration 48 to 72 hours later indicates a cell-mediated immune
a cell-mediated deficiency, is now included in Category 9. This response. A negative test is not always informative because the
disease is induced by a genetic mutation in the AIRE gene, but patient may not have been previously exposed to the test anti-
also involves an antibody to either (or both) IL-17 and IL-22.1 gen. Researchers have recently developed in vitro assays to
Mutations in the nRAS or kRAS genes are also associated with measure cell-mediated immunity, which are detailed in the text
diseases that fall into this category. that follows.
 Immunodeficiency Diseases

Screening for complement deficiencies usually begins with different types of lymphocytes are labeled with a fluorescent
a CH50 assay.25 This procedure determines the level of func- probe. These antigens are generally referred to by a cluster of
tional complement in an individual. Undetectable CH50 levels differentiation (CD) number (see Chapters 1 and 13). The anti-
may indicate a deficiency of a specific component. Based upon bodies are allowed to react with the patient’s peripheral blood
the clinical history, individual component assays would be in- mononuclear cells in a direct immunofluorescence assay and
dicated. The laboratorian should be aware, however, that low RBCs in the sample are lysed. The flow cytometer is used to
CH50 levels may be caused by complement consumption and count the WBCs that are labeled with each fluorescent antibody.
do not, by themselves, indicate a complement deficiency. Lymphocytes can then be assigned to specific types based on
Defects in neutrophil oxidative burst activity may be de- antigen expression: B cells (CD19), T cells (CD3), T helper (Th)
tected by a flow cytometric assay as previously mentioned. cells (CD3/CD4), cytotoxic T cells (CD3/CD8), and NK cells
Neutrophils labeled with DHR are stimulated to undergo an (CD16 or CD56). Flow cytometry is objective and quite reliable
oxidative burst by exposure to a mitogen. The oxidative burst in detecting those defects that result in a decrease in one or more
will reduce the DHR, producing fluorescence, which can be types of lymphocytes. For example, an absence or profound
measured objectively by flow cytometry. Flow cytometry can decrease in the number of CD3 cells would be consistent with
also be used to confirm a diagnosis of LAD type 1 by looking DiGeorge syndrome. An absence of CD19+ B cells suggests Btk
for the expression of the CD18 antigen. deficiency. One should remember, however, that in the actual
clinical arena, secondary immunodeficiencies are more com-
Confirmatory Tests mon than PIDs. For example, in the laboratory of the author
(TA), the most common cause of absent B cells is not Btk defi-
If the screening tests detect an abnormality or the clinical sus- ciency, but patients treated with rituximab, a monoclonal anti-
picion is high, more specialized testing will probably be nec- CD20 antibody. This antibody, used to treat leukemic,
essary to precisely identify an immune abnormality. Some of transplant, and autoimmune patients, destroys B cells, resulting
the tests used for confirming an immunodeficiency state are in no detectable CD19+ or CD20+ cells. Figure 19–2 is a flow
listed in Table 19–3. cytometry histogram from a patient treated with rituximab.
Enumeration of classes of lymphocytes in the peripheral Note that no CD19+ B cells are detectable.
blood is performed by flow cytometry. Even though types of Most of the genes associated with PIDs have been identified
lymphocytes cannot be distinguished morphologically, they ex- and localized. Thus, genetic testing is available for many con-
hibit different patterns of antigen or surface immunoglobulin ditions, including the DiGeorge deletion, the Wiskott-Aldrich
expression that correlate with functional characteristics. Before gene, and the IL2RG mutations. Although genetic testing is
flow cytometric analysis, antibodies to antigens specific for useful to understand the pathology of the disease, it is often
not required for making a diagnosis. Genetic testing of family
members of affected patients may be helpful in determining
Table 19–3 Specialized Confirmatory Tests
who may be at risk of developing the disease or passing it on
for Immunodeficiencies
to offspring.
SUSPECTED T-cell function can be measured by assessing the ability of
DISORDER SPECIALIZED TESTS isolated T cells to proliferate in response to an antigenic stim-
Humoral B-cell counts by flow cytometry ulus or to T-cell mitogens in culture, such as phytohemagglu-
immunity B-cell proliferation in vitro (e.g., mitogen tinin (PHA) or Concanavalin A (Con A). A mitogen is a
assays) substance that stimulates mitosis in all T cells or all B cells, re-
Histology of lymphoid tissues gardless of antigen specificity. Classically, the T-cell response
Cell-mediated T-cell counts by flow cytometry (total may be measured by quantitating the uptake of radioactive
immunity and T-cell subsets) thymidine, a precursor of DNA. Increased thymidine uptake
T-cell function in vitro (e.g., mitogen suggests cell division and activation. This assay requires expe-
assays) rienced technologists, a radioactive materials license, and
Quantiferon TB assay laboratory-determined reference ranges.
Cylex ImmuKnow assay More recently, antigen- or mitogen-stimulated T-cell activa-
Enzyme assays (ADA, PNP) tion has been measured without the use of radioactive materials.
Phagocyte Leukocyte adhesion molecule analysis The FDA has cleared three such assays for diagnostic use. The
defects (CD11a, CD11b, CD11c, CD18) Quantiferon TB assay and the T-Spot assay measure an individ-
Phagocytosis and bacterial killing assays ual’s response to Mycobacterium tuberculosis antigens. Following
Chemotaxis assay overnight whole blood activation with TB antigens, gamma in-
Enzyme assays (myeloperoxidase, terferon secreted by activated Th1 cells, is quantitated by either
glucose-6-phosphate dehydrogenase, an enzyme-linked immunosorbent assay (ELISA) or ELISPOT
components of NADPH oxidase)
procedure. Either of these assays may be used as an in vitro
Complement Specific component assays assessment of exposure to M tuberculosis.
The third assay, the Cylex ImmuKnow assay, measures total
NADPH = nicotinamide adenine dinucleotide phosphate.
T-cell activity. This test uses the mitogen PHA to activate T cells.
 Flow cytometry histogram of peripheral blood stained with antibodies to CD3, CD19, and CD56. Note that no CD19+ cells are
detected in this patient sample. (Courtesy of Dr. Thomas Alexander.)

Following incubation, ATP production is measured by a fluo- performed to look for the presence of TCR excision circles
rescent immunoassay technique. This test is a general measure- (TRECs).27 TRECs, identified by quantitative PCR (see
ment of T-cell function and is often used to monitor individuals Chapter 12), are present in T cells that have undergone
receiving immunosuppressive therapy. The assay may be used alpha-beta receptor gene rearrangements. They are the ge-
to determine overall T-cell functional capabilities in an individ- netic material that has been removed from the germline DNA
ual suspected of a PID. PHA is also used as a positive control in during alpha VJ and beta VDJ recombination (Fig. 19–3).
the Quantiferon and T-Spot procedures. Their absence indicates a lack of functional T cells, allowing
early identification of T-cell related defects leading to SCID.
Newborn Screening DiGeorge and other non-SCID diseases, such as Omenn syn-
drome, have also been detected using this method. Another
for Immunodeficiencies technique not normally applied to newborn screening,
Several states have begun to include PID testing as part of whole exome sequencing, has been used to identify AT in
their newborn screening programs.26 This testing is typically two infants and will likely see increased use in the future.28
 Immunodeficiency Diseases

Thymus with maturing T lymphocytes

T cell receptor gene arrangement

TCR! TCR" TCR!

Rearranged TCR!

TCR" excision circle

Newborn testing for T-cell receptor excision

circles (TRECs) formed by normal T-cell receptor gene
rearrangement.

Connections by assays involving nephelometry, but may also be per-

formed by radial immunodiffusion (RID). In addition, serum
T-Cell Receptors (TCRs) protein electrophoresis (SPE) can be quantitative if the total
Recall from Chapter 12 that the TCR for antigen is composed of serum protein is determined and the results are read using a
two different chains: an α chain and a β chain. During T-cell mat- densitometer.
uration, rearrangement of specific V and J genes that code for
the variable region of the α chain and specific V, D, and J genes
that code for the variable region of the β chain occur to produce SPE is a technique in which molecules are separated on the
a TCR that is specific for a particular antigen (Fig. 19–3). In the basis of their size and electrical charge. SPE allows repro-
process, the intervening sequences are cut out to form circles ducible separation of the major plasma proteins. See Chapter 5
called TRECs. for details. The serum protein electrophoretic profile is tradi-
tionally divided into five regions: albumin, alpha1 globulins,
alpha2 globulins, beta globulins, and gamma globulins. Some
laboratories now use six regions, dividing the beta region into
Evaluation of Immunoglobulins the beta1 (transferrin) and beta2 (complement component C3)
Quantitative measurement of serum immunoglobulins is used regions. IgG, IgD, and IgE migrate in the gamma globulin re-
in the workup of both immunodeficiency states and some gion, whereas IgM and IgA overlap the beta and gamma re-
lymphoproliferative disorders. This is usually performed gions. Immunoglobulins normally show a range of mobilities.
Additional evaluation of serum immunoglobulin is performed
if the SPE shows a monoclonal component or if there is a sig- SUMMARY
nificant quantitative abnormality of serum immunoglobulins.
Classically, SPE has been performed using agarose gels. More • Immunodeficiencies are disorders in which part of the
recently, SPE may be performed using capillary electrophoresis body’s immune system is missing or dysfunctional. There
(described in Chapter 12), which eliminates the need for gels are two types of immunodeficiencies—primary (i.e., in-
and the drying and staining processes associated with the herited), and secondary (acquired because of other factors
agarose-based technique. such as infections, malignancies, or immunosuppressive
drugs).
• Defects in the development and regulation of individual
Another method of characterizing immunodeficiencies is im- parts of the immune system can affect the humoral or cell-
munofixation electrophoresis (IFE) (see Chapter 18). In IFE, mediated branches of the adaptive immune system or var-
serum samples are electrophoresed, just as for SPE, and then ious components of the innate defense system, such as the
specific antibody is applied directly to the separating gel. The phagocytic cells or the complement system.
antibody–antigen complexes form and are visualized by stain- • The primary immunodeficiencies (PIDs) have been classi-
ing. Polyclonal immunoglobulins are indicated by areas of dif- fied into nine categories based on their clinical features,
fuse staining, whereas monoclonal immunoglobulins produce immunologic defects, and genetic abnormalities.
narrow, intensely stained bands (see Fig. 19–4 later in the Case • Defects in antibody-mediated immunity typically lead to
Studies). Lack of bands indicates immunodeficiency of one or recurrent infections with pyogenic bacteria, particularly of
more immunoglobulin classes. For accurate interpretations, the respiratory and intestinal tracts.
specific immunoglobulin isotype levels, determined by neph- • Defects in T-cell–mediated immunity generally lead to
elometry, are necessary. recurrent infections with intracellular pathogens, such as
viruses, fungi, and intracellular bacteria.
Bone Marrow Biopsy • Defects in phagocyte function generally lead to pyogenic
bacterial infections, often of the skin. Immunodeficiency
A bone marrow aspirate and biopsy is indicated in any eval- states range from quite mild to lethal.
uation of a monoclonal gammopathy or immunodeficiency • Therapy to reconstitute the deficient immune compo-
state. It is important in establishing the diagnosis of such nent must begin as soon as possible in an attempt to
disorders and for excluding other diseases. The bone marrow prevent permanent organ damage or death caused by
specimen will be analyzed microscopically and by flow cy- infection.
tometry. Cytogenetic analysis may also be performed to de- • PIDs must be suspected clinically and are diagnosed with
tect the specific genetic anomalies associated with the PID the help of screening tests to measure leukocyte counts
diseases.9,14,29 and immunoglobulin levels, followed by specialized lab-
oratory testing, such as flow cytometry, to determine num-
Family History bers of specific lymphocyte subsets.
• Laboratory results correlate with the presence of a specific
The importance of obtaining a full family history as part of the PID. For example, patients with Bruton’s tyrosine kinase
PID diagnosis process cannot be overemphasized. The mode of (Btk) deficiency typically have decreased serum concen-
inheritance, if known, can rule in or rule out many of the PIDs. trations of all the immunoglobulins and decreased num-
The disease may be X-linked, such as in the common gamma bers of CD19+ B cells, whereas patients with chronic
chain mutation, WAS, or the CD154 deficiency; autosomal granulomatous disease (CGD) would be expected to have
dominant, such as in Hyper IgE or Muckle-Wells syndrome; or decreased fluorescence in the DHR assay, which measures
autosomal recessive, such as in AT or LAD. CGD may be either oxidative burst in neutrophils.
X-linked or autosomal recessive. Although obtaining a family • Newborn screening is available to detect SCID, a com-
history is not the responsibility of the clinical immunology lab- bined immunodeficiency disease involving defective T-cell
oratory, these diseases are not identified solely by laboratory function. The screening assay looks for the absence of
testing. A team approach among the laboratory, clinicians, med- TRECs, T-cell receptor excision circles that have been
ical geneticists, and the family is required for a final diagnosis. removed during normal gene rearrangements that occur
Laboratory results are most useful when integrated into the en- during T-cell development.
tire clinical picture. Laboratories should not test in a vacuum!
 Immunodeficiency Diseases

Study Guide: Classification of Primary Immunodeficiencies

CATEGORY DESCRIPTION PROTOTYPIC DISEASES
1. Combined immunodeficiencies Low to absent humoral and SCID (common gamma chain deficiency, JAK3
cellular capabilities deficiency, ADA deficiency, MHC deficiencies)
PNP deficiency
2. Combined immunodeficiencies Low to absent humoral and Wiskott-Aldrich syndrome, ataxia-telangiectasia,
with associated or syndromic cellular capabilities with DiGeorge syndrome
features additional, nonimmunologic
anomalies
3. Predominantly antibody One or more immunoglobulin Bruton’s thymidine kinase (Btk) deficiency,
deficiencies isotypes are decreased transient hypogammaglobulinemia of infancy,
common variable immunodeficiency, selective
IgA deficiency, IgG subclass deficiencies
4. Diseases of immune Loss of T regulatory or other Chediak-Higashi syndrome, autoimmune lympho-
dysregulation controlling mechanisms proliferative syndrome, CD25 deficiency
5. Congenital defects of phagocyte Reduction in phagocytic Chronic granulomatous disease, cyclic
number, function, or both function, number,or both neutropenia, leukocyte adhesion deficiency
6. Defects in innate immunity Interruption in signaling path- Chronic mucocutaneous candidiasis, TLR3
ways of innate immune cells deficiency, IRAK4 deficiency
7. Autoinflammatory disorders Diseases usually associated Hyper IgD syndrome, familial Mediterranean
with recurrent fevers with or fever, TNF receptor associated periodic
without infections syndrome
8. Complement deficiencies Loss of individual C2 deficiency, C3 deficiency, C6 deficiency,
components hereditary angioedema
9. Phenocopies of PID PID associated with an Autoimmune lymphoproliferative syndrome,
acquired mutation or chronic mucocutaneous candidiasis
autoantibody

Adapted from Al-Herz W, Bousfiha A, Casanova J-L, et al. Primary immunodeficiency diseases: an update on classification from the International Union of
Immunological Societies Expert Committee for Primary Immunodeficiency. Front. Immunol. 2014;5(162):1–33.

Study Guide: Screening Tests for Immunodeficiencies

SUSPECTED DISORDER SCREENING TESTS
All immunodeficiencies Complete blood cell count, white blood cell differential count
Humoral immunity Serum IgG, IgA, IgM levels, IgG subclass levels, isohemagglutinin titers (IgM), IgG
antibody response to protein and polysaccharide antigens
Cell-mediated immunity Delayed hypersensitivity skin tests (i.e., Candida, diphtheria, tetanus, PPD)
Chest x-ray (thymus shadow)
TREC screening
Phagocyte defect DHR reduction test
NBT dye test
Complement CH50 (classical pathway)
Serum complement levels (e.g., C3, C4)

NBT = nitroblue tetrazolium; PPD = purified protein derivative.

CASE STUDIES
1. A 7-month-old male child was diagnosed with bacterial
meningitis. Previously he had been hospitalized with bac-
terial pneumonia. Laboratory testing results were as fol-
lows: RBC count: normal; WBC count: 22 109/L (normal
is 5–24 109/L); differential: 70% neutrophils, 15% mono-
cytes, 5% eosinophils, and 10% lymphocytes; and SPE: no
gamma band present.
Questions
a. What possible conditions do these results indicate?
b. How are these conditions inherited?
c. What type of further testing do you recommend?

2. A 37-year-old female presents with a history of recurrent Four different immunofixation patterns.
upper respiratory infections. She states that she was always
a sick child, usually with respiratory infections, but occa-
sional diarrhea would also occur. She has received countless
antibiotic regimens over the years. The physician orders a
SPE and immunoglobulin levels. The SPE is read as a low
gamma level with no monoclonal proteins detected. Levels
of IgG, IgM, and IgA are below the reference ranges. The
physician then orders an immunofixation assay.
Question
a. Figure 19–4 contains four patient immunofixations.
Which pattern would be most representative of the
expected pattern for this patient?
b. Explain why you chose this answer.

REVIEW QUESTIONS
1. Patients with which immunodeficiency syndrome 3. What clinical manifestation would be seen in a patient
should receive irradiated blood products to protect with myeloperoxidase deficiency?
against the development of GVHD? a. Defective T-cell function
a. Bruton’s thymidine kinase (Btk) deficiency b. Inability to produce IgG
b. Selective IgA deficiency c. Defective NK cell function
c. SCID d. Defective neutrophil function
d. CGD
4. Defects in which branch of the immune system are
2. T-cell subset enumeration by flow cytometry would be most commonly associated with severe illness after
most useful in making the diagnosis of which disorder? administration of live virus vaccines?
a. Bruton’s thymidine kinase (Btk) deficiency a. Cell-mediated immunity
b. Selective IgA deficiency b. Humoral immunity
c. SCID c. Complement
d. Multiple myeloma d. Phagocytic cells
 Immunodeficiency Diseases

5. Which of the following statements applies to Bruton’s 10. A patient with a deficiency in complement component
thymidine kinase (Btk) deficiency? C7 would likely present with
a. It typically appears in females. a. recurrent Staphylococcal infections.
b. There is a lack of circulating CD19+ B cells. b. recurrent Neisserial infections.
c. T cells are abnormal. c. recurrent E coli infections.
d. There is a lack of pre-B cells in the bone marrow. d. recurrent Nocardia infections.

6. DiGeorge anomaly may be characterized by all of the 11. A FoxP3 gene mutation may lead to a deficiency of
following except what cell type?
a. autosomal recessive inheritance. a. T helper cells
b. cardiac abnormalities. b. T cytotoxic cells
c. parathyroid hypoplasia. c. B cells
d. decreased number of mature T cells. d. T regulatory cells

7. A 3-year-old boy is hospitalized because of recurrent 12. The Cylex ImmunoKnow assay is useful in determining
bouts of pneumonia. Laboratory tests are run and the functional capabilities of which cell type?
following findings are noted: prolonged bleeding time, a. T cells
decreased platelet count, increased level of serum b. B cells
alpha-fetoprotein, and a deficiency of naturally occur- c. NK cells
ring isohemagglutinins. Based on these results, which d. Neutrophils
is the most likely diagnosis?
a. PNP deficiency 13. Recurrent, periodic fevers may be associated with
b. Selective IgA deficiency increased production of which immunoglobulin?
c. SCID a. IgG
d. Wiskott-Aldrich syndrome b. IgM
c. IgD
8. Which of the following is (are) associated with d. IgE
ataxia-telangiectasia?
a. Inherited as an autosomal recessive 14. Chronic mucocutaneous candidiasis, a PID that was
b. Defect in both cellular and humoral immunity previously thought to be a cell-mediated deficiency, is
c. Chromosomal breaks in lymphocytes now classified as which type of PID?
d. All of the above a. Autoinflammatory disorder
b. Complement deficiency
9. A 4-year-old boy presents with recurrent wound c. Predominantly antibody deficiency
and soft-tissue infections. Which of the following d. Innate immunity deficiency
assays should be considered for diagnosing his
presumed PID? 15. Prenatal screening for SCID involves detecting
a. DHR reduction a. Tregs.
b. CD4 quantitation b. TRECS.
c. CD19 quantitation c. THELPS.
d. CD56 quantitation d. TCYTOS.
 Serological and
Molecular Diagnosis
of Infectious Disease
 Serological and
Molecular Detection
of Bacterial Infections

After finishing this chapter, you should be able to: HUMANMICROBE RELATIONSHIPS
1. Differentiate between commensalistic, mutualistic, and parasitic BACTERIAL VIRULENCE FACTORS
relationships. Structural Virulence Features
2. Differentiate between pathogenicity, virulence, and infectivity. Extracellular Virulence Factors
3. Cite structural features of bacteria that contribute to increased Endotoxin and Exotoxins
virulence. IMMUNE DEFENSES AGAINST
4. Describe host defenses against bacteria and the means by which bacteria BACTERIAL INFECTIONS AND
can evade the immune system. MECHANISMS OF EVASION
5. Compare and contrast endotoxins and exotoxins with respect to Immune Defense Mechanisms
biological activity, immunogenicity, and the genetic encoding for the Bacterial Evasion Mechanisms
production of the two toxin categories.
LABORATORY DETECTION AND
6. Cite the five general laboratory means of detecting the causative agent DIAGNOSIS OF BACTERIAL
of a bacterial infection. INFECTIONS
7. Explain the principle of lateral flow immunochromatographic assays. Bacterial Culture
8. List the exotoxins produced by Group A streptococci and the roles Microscopic Visualization
they play in contributing to the virulence of Streptococcus pyogenes.
Antigen Detection
9. Describe the symptoms and pathogenesis of acute rheumatic fever
Molecular Detection
and poststreptococcal glomerulonephritis.
Serological Diagnosis
10. Explain the principle, interpretation, and clinical significance of the
antistreptolysin O (ASO), antideoxyribonuclease B (anti-DNase B), GROUP A STREPTOCOCCI
and streptozyme tests. STREPTOCOCCUS PYOGENES
11. Recognize the role Helicobacter pylori plays in gastrointestinal Classification and Structure
ulcers and the virulence factors that contribute to infection by Virulence Factors
this organism. Clinical Manifestations of Group A
12. Discuss the various types of tests that may be performed to detect Streptococcal Infection
H pylori infection. Group A Streptococcal Sequelae
13. Describe the respiratory and dermatological manifestations of Laboratory Diagnosis
Mycoplasma pneumoniae infections.
HELICOBACTER PYLORI
14. Discuss the use of serology for the diagnosis of M pneumoniae
Helicobacter H pylori Virulence Factors
infections, including the clinical value of detecting cold agglutinins.
Pathology and Pathogenesis
Diagnosis of H pylori Infection

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 15. Describe the epidemiology of Rocky Mountain spotted fever with MYCOPLASMA PNEUMONIAE
respect to etiologic agent, transmission, and pathogenesis. Mycoplasma Pneumoniae
16. Select the appropriate serological and molecular techniques to Pathogenesis
diagnose Rocky Mountain spotted fever. Dermatological Manifestations
Immunology of Mycoplasma
Pneumoniae Infection
Laboratory Diagnosis of Mycoplasma
Pneumoniae Infection
RICKETTSIAL INFECTIONS
Agents of Rickettsia-Related Disease
Rocky Mountain Spotted Fever
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

Acute rheumatic fever Indigenous microbiota Parasitic Streptococcus pyogenes

Anti-DNase B Infectivity Pathogenicity Streptolysin O
ASO titer Lancefield groups Plasmids Streptozyme
Commensalistic Lateral flow Poststreptococcal Symbiotic
Endotoxin immunochromatographic glomerulonephritis Typhus
assay (LFA) Rickettsiae
Exotoxin Urease
Microbiome Rocky Mountain spotted
Group A streptococci (GAS) Virulence
Mutualistic fever (RMSF)
Helicobacter pylori Virulence factors
Mycoplasma pneumoniae Scarlet fever
Impetigo

The collection of microorganisms that exists on the body— Human–Microbe Relationships

bacteria, viruses, and single-celled prokaryotic organisms (e.g.,
yeast and fungi)—is referred to as the human microbiome. The When an individual is born, a dynamic relationship begins be-
bacteria comprising the human microbiome keep us healthy tween the human host and the bacteria in the environment.
in many ways. They protect us against disease-causing bacteria, Very quickly, various bacteria establish themselves on the
aid in digesting our food, produce certain vitamins, and stim- surfaces of an individual, including the gastrointestinal tract,
ulate both the innate and adaptive immune systems. The es- creating a symbiotic relationship. The bacteria and the host
tablishment of an organism that leads to host injury is referred “live together,” often maintaining a long-term interaction. Sym-
to as an “infection.” When a microbe causes damage to host biotic bacteria that reside on and colonize these surfaces are
cells or altered physiology that results in clinical signs and referred to as the indigenous microbiota (previously known
symptoms of disease, the phrase “infectious disease” applies. as “normal flora”). The bacterial populations that exist on the
Traditional means of determining the cause of a bacterial in- body are not homogenous; they vary in composition and num-
fection have relied largely on growing the organism in culture bers, depending on the area of the body.
and using stains to view the organism under the microscope. The microbial populations outnumber human cells by ten
These infections can also be identified by immunoassays that to one.1 Collectively, they may account for 2 to 6 pounds of an
detect bacterial antigens or antibodies and by sophisticated individual’s body weight.2 Although it was previously thought
molecular techniques that detect nucleic acid from the organisms. that a relatively limited number of bacteria make up the human
This chapter will begin with an introduction to the human– microbiome, we now know that the microbial community is
microbe relationship and factors that influence the interactions actually very diverse. Furthermore, over 90% of the bacteria
between bacteria and the immune system. The chapter will that comprise the human microbiome cannot be cultured in
then discuss laboratory methods that are commonly used to vitro, most likely because their growth depends on specific
detect bacterial infections in the context of selected patho- conditions or substances that have not been duplicated in the
genic bacteria. laboratory.3
 Our relationship with our indigenous microbiota exists to a majority of individuals that have intact immune systems.
through co-evolution, co-adaptation, and codependency between Although an organism may be pathogenic in nature, it may
the bacteria and the host. For a microorganism to survive, not always cause disease. The outcome of the host–pathogen
the organism needs to colonize the host and acquire nutrients. interaction is determined by several factors, including the host’s
Importantly, it must not stimulate the host’s immune response immunologic status. Some microorganisms may only cause
(in the case of the indigenous microbiota) or it must avoid or disease or infection in individuals who have compromised
circumvent the immune responses. Once established, it needs immune systems because of factors such as chemotherapy,
to be able to replicate and disseminate to a preferred site in radiation therapy, or various chronic diseases. These organisms
the body for survival and eventually be transmitted to a new are referred to as “opportunistic pathogens.”
susceptible host. Virulence is a quantitative trait that refers to the extent of
Three types of symbiotic relationships can exist between damage, or pathology, caused by the organism. For example,
humans and bacteria. In commensalistic relationships, there Yersinia pestis, the causative agent of bubonic and pneumonic
is no apparent benefit or harm to either organism. The indige- plague, is considered to be extremely virulent and is likely to
nous microbiota is often referred to as “commensals” or the cause severe illness and death upon infection unless antibiotics
“commensalistic bacteria,” describing bacteria that are recovered are administered. If the bacterial strain is not capable of causing
in culture that do not represent a pathogen. An example of a disease, the organism is said to be “avirulent.” Not all members
commensalistic organism is Staphylococcus epidermidis, which of a bacterial species are necessarily capable of causing disease.
colonizes and inhabits the human skin. In a mutualistic rela- For example, Escherichia coli resides as commensal bacteria in
tionship, both humans and the bacteria benefit. One example the gastrointestinal tract but only some strains of E coli are
is the Lactobacillus species that colonizes the epithelial surfaces capable of causing diarrheal disease.
of the vaginal canal. The human host provides conditions The degree of damage is mediated by specific virulence
(temperature, atmosphere, nutrients) that allow the bacteria to factors. Virulence factors may increase an organism’s patho-
grow and multiply; in exchange, the bacteria produce lactic genicity by contributing to the organism’s ability to establish
acid, which prevents colonization of bacteria and yeast that may itself on or in the host, invade or damage host tissue, or evade
cause disease. Although the vast majority of the interactions the host immune response.
between the host and members of the microbiome are harmless,
the encounter with specific organisms or viruses occasionally Bacterial Virulence Factors
results in harm to the host. In this case, a parasitic relationship
exists between the other organisms and the host. Bacterial properties or features that determine whether an or-
As previously mentioned, the establishment of an organism ganism is pathogenic and able to cause disease are referred to
that leads to host injury is referred to as an infection. Although as “virulence factors.” Factors that increase a bacterium’s viru-
there is harm to the host, not all infections are symptomatic. In lence may be classified as either structural components (e.g.,
many instances, the infection may be subclinical; in other words, endotoxin is a component of the cell walls of certain bacteria)
there are no signs or symptoms. An example is infection with or as extracellular substances produced by the bacteria, such
Chlamydia trachomatis, a sexually transmitted organism. Only as exotoxins. Various types of bacterial virulence factors are
about 10% of infected males and 5% to 30% of infected females discussed in the sections that follow.
will show symptoms when infected with C trachomatis.4,5 The For an organism to be pathogenic, it needs to possess genetic
organism may then be transmitted to other individuals or may determinants that allow for production of either the structural
result in complications such as pelvic inflammatory disease in components or the extracellular products that contribute to its
women. virulence. Genetic determinants located on the bacterial chro-
Several terms are used to describe the interaction between mosome are generally responsible for the production of struc-
the infecting organisms and the host. The terms infectivity, tural or surface molecules, which help the organism to attach
pathogenicity, and virulence are often used interchangeably; to and colonize the host. The genetic information needed to
however, each term has different meanings when discussing produce the extracellular substances that are virulence factors
an organism’s ability to cause an infection. Infectivity refers to is most frequently located on independent genetic elements
an organism’s ability to establish an infection. More specifically, called plasmids. Plasmids are self-replicating genetic elements
infectivity describes the proportion of individuals exposed to that are located in the bacteria’s cytoplasm and contain a lim-
a pathogen through horizontal transmission (i.e., person-to- ited number of genes. In addition, plasmids are mobile genetic
person spread) who will become infected. Another term that elements that can be transferred between bacteria through
is used with similar meaning is contagious. For example, the various mechanisms. Acquisition of exogenous DNA that codes
measles virus is extremely contagious and has a high degree of for the production of virulence factors can convert an avirulent
infectivity. strain into a virulent strain.
Pathogenicity refers to the inherent capacity of an organism
to cause disease. This is a qualitative trait of the organism
determined by its genetic makeup. Some organisms, such as
Structural Virulence Features
the human immunodeficiency virus (HIV), are considered to Bacterial cells are classified as prokaryotic cells, whereas human
be primary or true pathogens that are capable of causing harm cells are classified as eukaryotic cells. Although prokaryotic
bacterial cells are relatively simple, they have a well-developed dying cell, a portion of LPS called lipid A is exposed. Lipid A,
cell structure and contain a number of structural and genetic also referred to as endotoxin, is a powerful stimulator of the
features that are not found in eukaryotic cells. One significant cytokine system that leads to a variety of systemic manifesta-
difference is that the bacterial chromosome is not enclosed tions and potentially fatal endotoxic (gram-negative) shock
inside of a membrane-bound nucleus, but instead resides (discussed later in this chapter).
inside the bacterial cytoplasm. Also housed in the cytoplasm To be successful in establishing an infection, bacteria must
are internal cellular structures, such as ribosomes, mesosomes, first adhere to and colonize the host tissue. Furthermore, to
and potentially plasmids, as well as other cytoplasmic inclusion persist in the host, they must be capable of evading the immune
bodies. Not present in bacterial cells are membrane-bound system. Structural features are generally involved in the adher-
organelles such as the mitochondria found in eukaryotic cells ence of bacteria or the evasion of the immune system. Whiplike
(Fig. 20–1). structures called flagella can facilitate adherence as well as
Other significant differences between eukaryotic and movement of the bacteria toward a host cell.
prokaryotic cells are features of the cell membrane and the Bacteria have surface structures or molecules that can bind
presence of a cell wall in bacteria. Similar to the plasma mem- specifically to complementary receptors on specific cells of
brane in eukaryotic cells, the bacterial plasma membrane host tissues. The most common surface structures involved in
plays a regulatory role in the transport of molecules in and attachment are fimbriae, which are composed of protein. The
out of the cell. The cell membrane in bacteria also contains fimbriae involved in specific attachment to prokaryotic surface
various enzyme systems responsible for energy generation. In ligands are referred to as the common pili. Other pili used to
eukaryotic cells (except for plants and fungi) the plasma exchange genetic information, called the F or sex pili, are fewer
membrane is the outer surface of the cell. In contrast, bacteria in number than common pili. The sex pili may also function
have a cell wall as their outermost feature. The bacterial in the attachment to host cells.
cell wall prevents osmotic lysis and confers shape and rigidity In addition to allowing the organism to attach to and colo-
to the bacteria. Peptidoglycan is the primary component nize tissues, pili play a role in resisting phagocytosis by white
providing the shape and rigidity. The majority of antibiotics blood cells (WBCs) and may undergo antigenic variation to
used to treat bacterial infections target the production of evade the adaptive immune response. For example, the ability
peptidoglycan in the cell wall. of Neisseria gonorrhoeae to rearrange portions of its genome
The bacterial cell wall structure has two different variations, can change the antigenic makeup of its pili, allowing them to
which are classified as either gram-positive or gram-negative adhere specifically to human mucosal epithelial cells and resist
depending on their staining characteristics. Both gram-positive phagocytosis. Another example is the ability of Streptococcus
and gram-negative cell walls have features that increase an pyogenes-associated pili to attach to host cells because of its
organism’s virulence, as we will discuss later. One of the fea- production of F protein, which attaches to fibronectin. S pyogenes
tures found in the cell wall of gram-negative bacteria is the also can resist phagocytosis because of the production of
lipopolysaccharide (LPS) layer. There are three components M protein. Most strains of E coli that colonize the gastrointesti-
that make up LPS—an outer core polysaccharide, an inner core nal tract do not adhere to epithelial cells of the small intestine.
polysaccharide, and lipid A. When released from a dead or The ability of certain strains of E coli to cause gastrointestinal
disease is associated with the expression of specific pili.
Enterotoxigenic E coli (ETEC) strains have factor antigen I (CFA)
pili that adhere to the cells in the small intestine; the organism
Cell Cytoplasm Cell then produces the toxins that cause diarrhea.
membrane wall Capsule Attachment may also occur via adhesions or surface mol-
Pili (fimbriae) ecules on the bacterial cell. Attachment caused by the pres-
ence of surface molecules is referred to as afimbrial (non-pili
dependent) attachment. Host cell receptors recognized by
Plasmid
bacterial surface molecules include proteoglycans, laminins,
collagens, elastin, and hyaluronan. Glycoproteins such as
fibrinogen, fibronectin, and vitronectin are also potential
receptors for bacteria.
A major structural feature that plays an important role in
increasing an organism’s virulence is the presence of a capsule,
Flagellum Chromosome (DNA) which is usually polysaccharide in nature. Capsules contribute
to the organism’s ability to resist innate and adaptive immune
responses through a variety of means. They can block the at-
tachment of antibodies, inhibit activation of complement, or
act as a decoy when capsular material is released into the
Mesosome surrounding host environment. One of the most important
Ribosome features of a capsule is its role in blocking phagocytosis
Cross section of a bacterial cell. by WBCs. For example, Streptococcus pneumoniae’s primary
determinant of virulence is a polysaccharide capsule that pre- protein molecules that are released from living bacteria
vents the ingestion of pneumococci by alveolar macrophages (mostly gram-positive bacteria) and are considered to be some
and elaborates capsular antigens to which immunoglobulins of the most potent molecules known to harm living organ-
bind. Strains of bacteria that do not possess a capsule are, in isms. Exotoxins may be classified as neurotoxins, cytotoxins,
most cases, avirulent and not able to produce disease. or enterotoxins, according to their effect on cells. Exotoxins
bind to specific receptors on host cells. Most exotoxins have
Extracellular Virulence Factors several subunits that bind to the receptor (B subunits) and
a subunit that is actually responsible for the specific activity
Extracellular substances produced by bacteria also contribute to of the toxin (A subunit). An example of a cytotoxin is the
an organism’s virulence by breaking down primary or secondary diphtheria toxin, which interferes with protein synthesis in
defenses of the body, damaging the host tissue and cells, or epithelial cells. Neurotoxins include the tetanus toxin, which
facilitating the growth and spread of the organism. Substances prevents the release of inhibitory transmitters from the presy-
that perform the latter function are called invasins. Several of the naptic membrane of neuromuscular cells, leading to continu-
invasins include hyaluronidase, collagenase, phospholipases, ous excitement of muscle cells and spasms. Botulism toxin
lecithinases, coagulase, and various kinases. works in a reverse manner—the toxin prevents the release of
acetylcholine (ACh), resulting in paralysis of the motor sys-
Endotoxin and Exotoxins tem. Examples of enterotoxins are toxins A and B produced
by Clostridium difficile, which cause fluid secretion (diarrhea),
Bacteria may also produce two types of toxins—endotoxin and
mucosal injury, and inflammation (Fig. 20–2). Exotoxins are
exotoxins. Endotoxin (lipid A) is found in the LPS layer of the
extremely immunogenic and induce production of protective
cell walls of all gram-negative bacteria. When the cell dies, the
antibodies. Inactivated exotoxins called “toxoids” are used for
LPS layer is removed from the cell, releasing endotoxin (lipid A)
some vaccines. For example, the toxin of Corynebacterium
in the host. The effects of endotoxin are complex. Endotoxin
diphtheria is used in the vaccine to prevent diphtheria.
induces a variety of host responses, including the production
Some exotoxins may act as “superantigens.” Unlike other
of cytokines such as IL-1, IL-6, IL-8, tumor necrosis factor
antigens, these “superantigens” are not processed by antigen-
(TNF), and platelet-activating factor, which stimulate production
presenting cells (APCs). Instead, they bind directly to class II
of prostaglandins and leukotrienes. This results in inflamma-
tion, increased heart rate, increased body temperature (fever),
and a decrease in blood pressure. In addition, endotoxin acti-
vates the complement cascade, resulting in the formation of
the anaphylatoxins C3a and C5a, which cause vasodilation
and increase vascular permeability. The alternative coagulation
cascade is also activated, producing coagulation, thrombosis,
and acute disseminated intravascular coagulation (DIC), lead-
ing to hemorrhage and shock. A potential consequence of
endotoxin release is septic shock, a life-threatening illness that
is usually the result of a gram-negative bacteremia, or presence
of bacteria in the blood. With septic shock, there is a large scale
release of inflammatory mediators that results in massive va-
sodilation and hypotension. Some refer to this as a “cytokine
storm.” If the condition worsens, there is widespread organ
damage including renal failure, liver dysfunction, heart dam-
age, and eventual death. Although immunogenic, endotoxin
does not elicit a protective immune response, so no vaccine is
available against this bacterial component.
Unlike endotoxin, which has multiple effects on the body,
exotoxins have a very specific and targeted activity. They are

Clinical Correlations
Capsular Antigens and Vaccine Development
Most capsules evoke a strong humoral immune response and
are used for the development of vaccines. For example, the
vaccines for Haemophilus influenzae type b, S pneumonia, and
certain types of Neisseria meningitidis consist of antigens derived A three-dimensional (3D) computer-generated
from bacterial capsules (see Chapter 25). image of a single gram-positive Clostridium difficile bacillus. (Courtesy
of the CDC/James Archer. Public Health Image Library.)
major histocompatibility complexes (MHC II) on APCs outside Acute phase reactants also play important roles. For example,
of the normal antigen-binding groove. The MHC II receptor C-reactive protein (CRP) activates the complement system and
binds to the T-cell receptor (TCR) with the whole antigen promotes phagocytosis by macrophages. Haptoglobin binds
(toxin) attached. Normally, only 0.0001% of T cells are acti- free plasma hemoglobin, which deprives the bacteria of iron.
vated in an immune response. However, a “superantigen” Ceruloplasmin is a glycoprotein with bactericidal activities.
induces activation of up to 20% of T cells, resulting in a Bacteria, fungi, and viruses possess pathogen-associated
massive release of cytokines.6 The systemic events brought on molecular patterns (PAMPs), which are structural patterns
by the release of large amounts of cytokines leads to what is consisting of carbohydrates, nucleic acids, or bacterial pep-
referred to as “toxic shock syndrome.” Examples include the tides. PAMPs are recognized by pattern recognition receptors
TSST-1 toxin produced by Staphylococcus aureus and the super- (PRRs) expressed on the cells of the innate immune system.
antigens produced by S pyogenes, the cause of streptococcal The engagement of the PRR with the appropriate PAMP trig-
toxic shock syndrome (STSS). gers the release of immune mediators such as cytokines and
chemokines, boosts production of various defensins and pro-
teins, and initiates phagocytosis. The phagocytic process is
Immune Defenses Against Bacterial enhanced by the activation of the alternative complement
Infections and Mechanisms cascade, which is triggered by microbial cell walls or other
products of microbial metabolism.
of Evasion Adaptive immune responses include the production of
antibodies directed against bacterial antigens or extracellular
Immune Defense Mechanisms products produced by bacteria such as exotoxin. Antibody
Although bacteria may possess various features that increase formation is the main defense against extracellular bacteria.
their virulence, they must be able to circumvent or overcome The binding of antibodies to invading bacteria is referred to as
the host’s defense mechanisms. As discussed in previous opsonization (see Chapter 4).
chapters, both innate and adaptive responses may occur after Cell-mediated immunity (CMI), the other branch of the
an encounter with foreign antigens. adaptive immune response, is helpful in attacking intracellu-
The first line of defense against potential pathogens is lar bacteria, such as Mycobacterium tuberculosis, Legionella
intact skin and mucosal surfaces that serve as structural bar- pneumophila, Listeria monocytogenes, and Rickettsia species.
riers. In addition, the epithelial surface may have enzymes Through the mechanism of delayed type hypersensitivity,
and nonspecific antimicrobial defense peptides (ADPs) and CD4 T cells produce cytokines, which activate macrophages
proteins that have antimicrobial activity. One example of an to release enzymes that destroy the bacteria (see Chapter 14).
enzyme with specific antimicrobial activity is lysozyme, The recruitment of inflammatory cells results in the formation
which is found in many secretions, including tears and saliva. of granulomas that surround the bacteria-infected cells to
Lysozyme destroys the peptidoglycan found in the cell wall help prevent the spread of infection. Cytotoxic T cells are also
of bacteria, especially gram-positive bacteria. Other enzymes recruited to the site of infection and mount an antigen-specific
include ribonucleases, which destroy RNA and have antimi- attack on the infected cells.
crobial and antiviral activities. The body excretes a wide
variety of ADPs and proteins that play a role in the innate Bacterial Evasion Mechanisms
defenses of the body. Some of the ADPs and proteins are Bacteria have developed several ways to inhibit the immune
only secreted by specific tissues or cells. One group of soluble system or make it more difficult for immune responses to
peptides is the defensin peptides. Defensins are produced occur. Three main mechanisms used by bacteria involve avoid-
constitutively by the cells in the body. ing antibody, blocking phagocytosis, and inactivating the com-
The three main classes of defensins are alpha, beta, and plement cascade (Fig. 20–3). Bacteria can evade antibodies
theta. Alpha defensins are produced by neutrophils, certain by altering their bacterial antigens, a process called antigenic
macrophage populations, and Paneth cells of the small intes-
tine. This class of defensins is believed to disrupt the microbial
membrane. Beta defensins are produced by neutrophils as well
as epithelial cells lining the various organs, including the Connections
bronchial tree and genitourinary system. They are believed to
Toll-Like Receptors (TLRs)
increase resistance of epithelial cells to colonization. Theta
defensins are not found in humans. Recall from Chapter 3 that one of the main groups of PRRs are
Many antimicrobial proteins contribute to the innate im- the Toll-like receptors (TLRs). TLRs are expressed on key cells of
the innate immune system such as macrophages and dendritic
mune response. For example, complement proteins can pro-
cells. They recognize molecules that are commonly found in
mote chemotaxis. Interleukins are involved in the regulation of
microbial pathogens but not on host cells. Once TLRs have
immune responses and inflammatory reactions. Prostaglandins bound to their ligands, cell-signaling pathways are triggered that
are involved in the dilation and constriction of blood vessels result in the production of cytokines that enhance the inflamma-
and modulation of inflammation. Leukotrienes are involved in tory response, resulting in more efficient pathogen destruction.
inflammation and fever.
 E

Epithelium

Endothelium

The strategies bacteria use to evade host defenses. (A) Inhibiting chemotaxis. (B) Blocking adherence of phagocytes to the
bacterial cells. (C) Blocking digestion. (D) Inhibiting complement C3b binding. (E) Cleaving IgA.

variation. They can also coat themselves with host proteins capsule found in such organisms as N meningitidis, S pneumoniae,
such as fibrin or immunoglobulin molecules (S aureus) or Y pestis, and H influenzae inhibits the binding of neutrophils
fibronectin (Treponema pallidum), or hide their surface molecules and macrophages needed to initiate phagocytosis.9
through antigenic disguise (the hyaluronic acid capsule of Microorganisms use several different mechanisms to resist
S pyogenes). Bacteria can also evade the specific immune response digestion. Some bacteria can block fusion of lysosomal gran-
through downregulation of MHC molecules and production of ules with phagosomes after being engulfed by the phagocyte.
proteases that degrade IgA.7 N gonorrhoeae, H influenzae, and Salmonella species are able to do this, as can M tuberculosis
Streptococcus sanguinis are all examples of bacteria that can and Mycobacterium leprae. In M tuberculosis and M leprae
cleave IgA.8,9 infection, each bacillus is contained in a membrane-enclosed
Most of the evasion mechanisms target the process of fluid compartment called a pristiophorus vacuole (PV), which
phagocytosis. Bacteria can mount a defense at several stages does not fuse with the lysosomes because of the complexity
in the phagocytic process, including chemotaxis, adhesion, of the acid-fast cell walls.
and digestion.7,8 Some pathogens such as N gonorrhoeae, for An additional mechanism of resisting digestion involves the
example, inhibit the release of chemotactic factors that would production of extracellular products after the bacteria are phago-
bring phagocytic cells to the area. The cell walls of S pyogenes cytized. The primary effect is the release of lysosomal contents
produce an M protein that interferes with adhesion to the into the cytoplasm of the phagocytic cells, subsequently killing
phagocytic cell. Additionally, the presence of a polysaccharide the WBC. Examples include leukocidin, produced by S aureus;
listeriolysin O, produced by L monocytogenes; and streptolysin, Microscopic Visualization
produced by S pyogenes.8
The last major defense some bacteria use is to block the Visualization of the causative agents using microscopic tech-
action of complement. Organisms mentioned previously that niques is most often done using differential or fluorescent
produce a capsule do not bind the complement component stains. Examples include the Gram stain for differentiating
C3b, which is important in enhancing phagocytosis.9 Such gram-positive and gram-negative bacteria (Fig. 20–4), the
organisms cannot easily be phagocytized unless coated by acid-fast stain for the detection of Mycobacteria tuberculosis
opsonins. Additionally, some organisms express molecules that (Fig. 20–5), and the Giemsa stain for the detection of the
disrupt one or more of the complement pathways. Protein H, causative agents of malaria. Direct fluorescent antibody assay,
produced by S pyogenes, binds to C1 but does not allow the or DFA, involves the use of antibody conjugated with a fluores-
complement cascade to proceed further.9 Another example is cent label to detect specific bacteria in a sample. Currently,
Streptococcus agalactiae, also known as Group B streptococcus many DFAs are being replaced by molecular tests because they
or GBS. GBS has a capsule that is rich in sialic acid (a common lack sensitivity or because the reagents are not widely available.
component of host cell glycoproteins), causing degradation Although the various staining methods are not difficult to
of C3b and making the organism resistant to complement- perform, a trained microscopist is necessary for proper inter-
mediated phagocytosis. pretation. Another limitation of microscopy is that not all organ-
isms may be visualized through microscopic means.
Laboratory Detection and Diagnosis
of Bacterial Infections
Five general ways can be used to detect the causative agent of
a bacterial infection: (1) culture or growth of the causative
agent, (2) microscopy, (3) detection of bacterial antigens in the
clinical sample, (4) molecular detection of bacterial DNA or
RNA, and (5) serology, or detection of antibodies produced in
response to the infection.

Bacterial Culture
Traditional means of determining the cause of a bacterial infec-
tion rely largely on growing the organism in culture. Various
broth and solid media may be used to recover the organism.
Some media may contain substances that enhance the growth
of certain organisms and are referred to as enriched media. Photomicrograph of spherical (cocci) gram-positive
Selective media contain substances or antibiotics that suppress S aureus bacteria magnified 320X. (Courtesy of the CDC/Dr. Richard
the growth of commensalistic bacteria and support the growth Facklam, Public Health Image Library.)
of other bacteria. Differential media contain substrates that
allow for the differentiation of bacteria based on their ability
to use the substrate. For example, MacConkey agar selects for
gram-negative bacteria and differentiates between lactose and
nonlactose fermenting bacteria. Some organisms, such as
Bordetella pertussis, the causative agent of whooping cough,
have very specific growth requirements for which specialized
media must be used.
Although culture is the primary laboratory means of diag-
nosing bacterial infections, the culturing of bacterial pathogens
has limitations. There are a number of bacterial pathogens
for which clinically useful culture systems are not available.
For other organisms, recovery in culture may take too long
to be clinically useful. For example, although Mycoplasma
pneumoniae, a leading cause of community acquired pneu-
monia, can be cultured, culturing is a challenge. The organ-
ism is extremely fastidious (difficult to grow) and may take Photomicrograph of Mycobacterium tuberculosis bac-
weeks to recover in the laboratory. Other organisms present teria using acid-fast Ziehl-Neelsen stain; magnified 1000X. The acid-
a danger to the laboratory technologist if they are not grown fast stains depend on the ability of mycobacteria to retain dye when
and handled using the most rigorous safety precautions (e.g., treated with mineral acid or an acid–alcohol solution. (Courtesy of the
Y pestis). CDC/Dr. George P. Kubica, Public Health Image Library.)
Antigen Detection Chlamydophila psittaci, Coxiella burnetii, Leptospira, Rickettia
spp., and T pallidum), serological testing remains useful and,
Antigen detection assays are available for a wide variety of bac- in some cases, is the best means of detecting exposure to or
teria, viruses, parasites, and fungi in clinical samples. Testing infection with an organism. Detection of either IgM or IgG
methodologies include latex agglutination (LA), enzyme-linked antibodies may indicate recent or previous exposure to an
immunosorbent assay (ELISA), and lateral flow immunochro- organism and antibody titers may be used to assess reactivation
matographic (LFA) assays (discussed later in this chapter). The or reexposure to an infectious agent.
LA and LFA assays are advantageous because of the relative ease The primary disadvantage of using serology in diagnosis is
by which the tests can be performed, their low cost, and the that there is usually a delay between the start of the infection
rapid turnaround time. Many of the LA and LFA assays are and the production of antibodies to the infecting microorgan-
classified as “CLIA waived” tests. Under the Clinical Laboratory ism. Although IgM antibodies may appear relatively early
Improvement Amendments of 1988 (CLIA), simple, low-risk (7 to 10 days after exposure), some infections have a rapid
tests can be “waived” (i.e., laboratories performing the testing course and the need to initiate therapy limits the detection of
are not subject to routine inspection) and may be performed IgM antibodies as a diagnostic tool in those instances. In some
in physicians’ offices and various other locations. Bacteria cases, demonstration of a high IgG antibody titer in the initial
and viruses for which antigen detection is widely used include stage of infection is diagnostic; however, the high titer may be
S pyogenes (strep throat), L pneumophila (Legionnaire’s disease), caused by a past infection and the patient’s symptoms may have
rotavirus (pediatric diarrheal disease), respiratory syncytial virus, an entirely different cause. When testing for the presence of
and influenza A and B. Antigen detection assays are highly IgG antibodies, it is ideal to collect serum samples during both
specific, and because of advances in technology, sensitivities of the acute and convalescent phases of the illness so that a rising
the assays have improved dramatically. In many cases, antigen titer to the suspected pathogen can be observed. Another lim-
detection assays, particularly LFA, have replaced other methods itation of serology is that immunosuppressed patients may be
used to detect infections with bacteria, viruses, and fungi. unable to mount an antibody response.
The rest of this chapter addresses the immunologic response
Molecular Detection to several important bacteria that cause invasive disease. Sero-
logical and molecular testing play a major role in detecting and
The rapid developments in the field of molecular diagnostics
diagnosing the cause of these common pathogens.
have allowed for the increased availability and use of nucleic
acid-based assays in the clinical laboratory to detect pathogenic
microorganisms. Compact hybridization and gene amplification Group A Streptococci
assays that are easy to perform have made their way into physi- (Streptococcus Pyogenes)
cian office laboratories. The most widely known molecular tech-
nology is polymerase chain reaction (PCR) in which specific
genetic sequences are amplified and detected. The development
Classification and Structure
of real-time PCR or quantitative PCR (qPCR) has allowed for Streptococci are gram-positive cocci that are spherical, ovoid, or
results to be obtained in a few hours, as compared with several lancet-shaped organisms often arranged in pairs or chains when
days for traditional PCR. For some infectious agents, such as observed on Gram stain (Fig. 20–6).10 The streptococci are
N gonorrhoeae and C trachomatis, nucleic-acid–based assays are initially identified by their effect on sheep red blood cells (RBCs)
widely available and clearly the choice for detection. when grown in culture. Those that completely lyse or hemolyze
Although these assays are more sensitive than other methods, the blood cells are classified as being β-hemolytic. If the organ-
there are limitations associated with nucleic acid-based testing. isms only partially hemolyze the cells, causing them to appear
At the time of this writing, there are relatively few FDA-approved green, they are classified as being α-hemolytic. If the organisms
assays on the market and the cost of the instrumentation and exhibit no effect on the cells, they are referred to as being
the disposables are prohibitive to many organizations. As more γ-hemolytic. The β-hemolytic streptococci are further classified
assays receive FDA approval and additional technological ad- according to a group-specific carbohydrate composition that
vances occur, the use of molecular-based assays for the detection
of agents responsible for various infectious diseases will become
even more widespread.
Connections
Serological Diagnosis Antibody Response Curve
Serology has historically been used to detect and confirm in- A serological pattern of IgM+, IgG– generally indicates an early
stage, acute infection, whereas an IgM–, IgG+ pattern usually
fections from organisms that are difficult to grow or for which
signifies a past exposure. The best indication of a current infec-
other laboratory methods of diagnosing the infection are not tion is a four-fold rise in antibody titer when comparing two
available. Serology may also be useful in determining the serum samples collected from a patient during the beginning
causative agent when the clinical symptoms are not specific and later stages of the infection. This is a good time to review
enough to identify the cause of the infection. For certain or- the typical antibody response curve shown in Chapter 5.
ganisms (e.g., Anaplasma, Ehrlichia, Chlamydophila pneumoniae,
 molecule consisting of two alpha-helical chains twisted into a
ropelike structure that extends out from the cell surface. Some
strains possess a hyaluronic acid capsule outside the cell wall
that contributes to the bacterium’s antiphagocytic properties.
Serotyping and molecular techniques can be used to identify
a particular strain of GAS. Serotyping involves identification of
the M protein antigens by precipitation with type-specific anti-
sera. More than 80 different serotypes have been identified by
this method. However, serotyping has limitations, including
limited availability of typing sera, new M protein types that do
not react with the antisera, and difficulty in interpreting the
results.11 Genotyping techniques involving PCR amplification
of a portion of the emm gene, which codes for the M protein,
followed by sequence analysis circumvents these problems.12
Pulsed field gel electrophoresis (PFGE) has also been used for
epidemiological studies. In PFGE, DNA from Group A strepto-
coccus is separated by using an alternating current to obtain a
unique pattern or “fingerprint.” The patterns from multiple
sources may be compared when there is a Group A streptococ-
cal outbreak.13
A three-dimensional (3D) computer-generated
image of a group of erythromycin-resistant Group A streptococcus Virulence Factors
(GAS), also known as S pyogenes bacteria. (Courtesy of the CDC/James GAS is one of the most common and ubiquitous pathogenic
Archer, Public Health Image Library.)
bacteria and causes a variety of infections. The M protein is the
major virulence factor of GAS and has a net-negative charge at
divides these bacteria into 20 groups designated A through H the amino-terminal end that helps to inhibit phagocytosis.
and K through V. These are known as the Lancefield groups, In addition, the presence of the M protein limits deposition of
based on the pioneering work of Dr. Rebecca Lancefield. A mem- C3 on the bacterial surface, thereby diminishing complement
ber of the β-hemolytic streptococci is S pyogenes, which includes activation.14,15 The M protein, along with lipoteichoic acid and
the Group A carbohydrate. Group A streptococci (GAS) are a protein F, help GAS attach to host cells. Immunity to GAS
major cause of bacterial pharyngitis (a throat infection) and appears to be associated with antibodies to the M protein.
childhood impetigo (a skin infection). Additional cell wall com- There are more than 100 serotypes of this protein and immu-
ponents, the M and T proteins, allow for further classification nity is serotype-specific.15,16 Therefore, infections with one
and differentiation (Fig. 20–7). The M protein is a filamentous strain will not provide protection against another strain.
Additional virulence factors include various exotoxins that
Capsule M protein
may be produced during the course of an infection. Pyrogenic
exotoxins A, B, and C are responsible for the rash seen in
T protein scarlet fever and also appear to contribute to pathogenicity.16,17
Lancefield group Additional extracellular substances include the enzymes
carbohydrate streptolysin O, deoxyribonuclease B (DNase B), hyaluronidase,
nicotinamide adenine dinucleotide (NAD), and streptokinase.
Cell wall Antibodies produced against these substances are useful in
the diagnosis of infection and for the potential development
of complications or sequelae associated with GAS infection
(discussed later in the chapter).
Membrane

Clinical Manifestations of Group A

DNase
NADase Streptococcal Infection
Streptokinase
Exotoxins
Hyaluronidase S pyogenes can be responsible for infections ranging from
Streptolysin O pharyngitis (a throat infection) to life-threatening illnesses
SpeA and SpeC such as necrotizing fasciitis and streptococcal toxic shock syn-
drome. The two major sites of infections in humans are the
upper respiratory tract and the skin, with pharyngitis (“strep
throat”) and streptococcal pyoderma (a skin infection) being
Diagram of antigenic components of Streptococcus the most common clinical manifestations.18 Symptoms of
pyogenes. pharyngitis include fever, chills, severe sore throat, headache,
tonsillar exudates, petechial rash on the soft palate, and ante- body. Scarlet fever results from infection with a GAS that elab-
rior cervical lymphadenopathy (Fig. 20–8).18 The most com- orates streptococcal pyrogenic exotoxins (erythrogenic toxins).
mon skin infection is Streptococcal pyoderma (also known as Streptococcal pyrogenic exotoxin A (SpeA) and Streptococcal
impetigo), characterized by vesicular lesions on the extremities pyrogenic exotoxin C (SpeC) can act as superantigens that may
that become pustular and crusted (Fig. 20–9). Such infections induce toxic shock syndrome. Toxic shock syndrome is a life-
tend to occur in young children.16 Other complications include threatening multisystem disease that often initiates as a skin or
otitis media, erysipelas, cellulitis, puerperal sepsis, and sinusi- soft-tissue infection and may proceed to shock and renal failure
tis. Septic arthritis, acute bacterial endocarditis, and meningitis because of overproduction of cytokines.10,18
also can result from a pharyngeal infection.14,16 Humans are Necrotizing fasciitis may occur when a GAS skin infection
the primary reservoir for GAS and transmission of GAS is from invades the muscles in the extremities or the trunk.19 The
person to person. onset is quite acute and is a medical emergency. Exotoxins
A small percentage of individuals develop scarlet fever. produced by S pyogenes cause a rapidly spreading infection
Although usually associated with pharyngeal infections, scarlet deep in the fascia, resulting in ischemia, tissue necrosis, and
fever may occur with streptococcal infections at other sites. septicemia if not treated promptly. The disease may be asso-
Symptoms include a fever of over 101°F, nausea, vomiting, ciated with predisposing conditions such as chronic illness
headache, and abdominal pain. A distinct scarlet rash initially in the elderly or varicella in children, but healthy persons can
appears on the neck and chest and then spreads all over the be affected as well.19,20 Reporting of necrotizing fasciitis and
toxic shock syndrome is part of a surveillance program con-
ducted by the Centers for Disease Control and Prevention.
Although the incidence of this syndrome has declined in the
United States, a significant number of cases are still reported
each year.11

Group A Streptococcal Sequelae

The reason GAS receives so much attention is the potential for
the development of two serious sequelae, acute rheumatic
fever and poststreptococcal glomerulonephritis.10 The se-
quelae result from the host response to infection. Serological
testing plays a major role in the diagnosis of these two diseases,
because the organism itself may no longer be present by the
time symptoms appear.
Acute rheumatic fever develops as a sequela to pharyngitis
or tonsillitis in 2% to 3% of infected individuals. It does not
Pharyngitis, or sore throat, is characterized by occur as a result of skin infection. The latency period is typi-
swelling and reddening of the pharynx. Note the inflammation of cally 1 to 3 weeks after onset of the sore throat. Characteristic
the oropharynx and petechiae, or small red spots on the soft palate features of acute rheumatic fever include fever, pain caused by
caused by Streptococcal pharyngitis. (Courtesy of the CDC/Henry F.
inflammation in the joints, and inflammation of the heart. The
Eichenwald, Public Health Image Library.)
disease is most likely caused by antibodies or CMI originally
produced against streptococcal antigens that cross-react with
antigens present in human heart tissue.10,21
Chief among the antibodies thought to be involved are
those directed toward the M proteins, which have at least three
epitopes that resemble antigens in heart tissue, permitting
cross-reactivity to occur. Titers of some antibodies may remain
high for several years following infection.11
The second main complication following a streptococcal
infection is acute glomerulonephritis, a condition characterized
by damage to the glomeruli in the kidneys. This condition may
follow infection of either the skin or the pharynx, whereas
rheumatic fever follows only upper respiratory tract infec-
tions.18 Glomerulonephritis caused by a streptococcal infection
is most common in children between the ages of 2 and 12 and
is especially prevalent in the winter.22
Impetigo is a dermatological streptococcal infection Symptoms of glomerulonephritis may include hematuria,
that is characterized by thick, golden-yellow discharge that dries, proteinuria, edema, and hypertension. Patients may also expe-
crusts, and sticks to the skin. It is also caused by the S aureus bacteria. rience malaise, backache, and abdominal discomfort.23 Renal
(Courtesy of the CDC/Dr. Herman Miranda, Public Health Image Library.) function is usually impaired because the glomerular filtration
rate is reduced, but renal failure is not typical. The most widely replaced enzyme immunoassay (EIA) and LA assays to detect
accepted theory for the pathogenesis of poststreptococcal the antigens. FAs are widely used in outpatient clinics, physician
glomerulonephritis is that it results from deposition of immune offices, and urgent care facilities for the rapid diagnosis of
complexes containing streptococcal antigens in the glomeruli. streptococcal pharyngitis.
These immune complexes stimulate an inflammatory response The assays are technically easy to perform, allow for single
that damages the kidneys and impairs function because of re- sample testing because of the incorporation of an internal
lease of the lysosomal contents of leukocytes and activation of control, and in many cases are more sensitive than traditional
complement.10,22,23 laboratory methods and other antigen detection methods (see
Fig. 11–4).24 In the LFA, strep A antigen extracted from a
Laboratory Diagnosis throat swab reacts with an enzyme-labeled antibody on a test
membrane. The antigen–antibody complex is captured by
another antibody at a specific location on the membrane,
Diagnosis of acute streptococcal infections typically is made by where a colored line is produced if the sample is positive for
culture of the organism from the infected site. The specimen is the antigen. An example of an LFA used for the detection of
plated on sheep blood agar and incubated. If Group A strepto- GAS from a throat swab is shown in Figure 20–11.
coccus is present, small translucent colonies surrounded by a Many of the assays require no more than 2 to 5 minutes of
clear zone of β hemolysis will be visible (Fig. 20–10). Identifi- hands-on time. The specificity and sensitivity of the assays
cation is made on the basis of susceptibility to bacitracin, testing in many instances are higher than other methodologies.25
for L-pyrrolidonyl-β-naphthylamide (PYR) activity, or through Although the assays have high sensitivities, cultures should be
Lancefield typing.18 performed when rapid test results are negative.14,17 Molecular
methods, including hybridization of specific rRNA sequences
and real-time PCR, have also been developed as a means to
As an alternative to culture, rapid assays have been commer-
rapidly detect Group A streptococcal infections.12
cially developed to detect Group A streptococcal antigens
directly from throat swabs. The Group A antigens are extracted
by either enzymatic or chemical means and the process takes Culture or rapid screening methods are extremely useful for
anywhere from 2 to 30 minutes, depending on the particular diagnosis of acute pharyngitis. However, serological diagnosis
technique. must be used to identify acute rheumatic fever and post-
Lateral flow immunochromatographic assays (LFA) are streptococcal glomerulonephritis because the organism is
increasingly being used for the detection of bacterial, viral, fun- unlikely to be present in the pharynx or on the skin at the
gal, and parasitic antigens in clinical samples. LFA have largely time symptoms appear.18 Group A streptococci elaborate
more than 20 exotoxins and it is the antibody response to
one or more of these that is used as documentation of non-
suppurative disease. Some of the exotoxin products include
streptolysins O and S; deoxyribonucleases A, B, C, and D;
streptokinase; NADase; hyaluronidase; diphosphopyridine
nucleotidase; and pyrogenic exotoxins.25
The antibody response to these streptococcal products is
variable because of several factors, such as age of onset, site of
infection, and timeliness of antibiotic treatment. The most diag-
nostically important antibodies are the following: anti-streptolysin
O (ASO), anti-DNase B, anti-NADase, and anti-hyaluronidase
(AHase). Assays for detection of these antibodies can be per-
formed individually or through use of the streptozyme test
which detects antibodies to all these products (see Streptozyme
Testing later). During Group A streptococcal infections, other
antibodies are made to cellular antigens, such as the Group A
carbohydrate and the M protein.13 Generally, detection of these
antibodies is done in research or reference laboratories because
commercial reagents are not available.
Serological evidence of disease is based on an elevated or
rising titer of streptococcal antibodies. The onset of clinical
symptoms of rheumatic fever or glomerulonephritis typically
coincides with the peak of antibody response. If acute and
Throat culture plate showing a positive result for convalescent phase sera are tested in parallel, a four-fold rise
beta hemolytic Group A streptococci (S pyogenes). Bacterial colonies in titer is considered significant. The use of at least two tests
not producing beta hemolysis represent indigenous microbiota of for antibodies to different exotoxins is recommended be-
the oropharynx. (James Vossler.) cause production of detectable ASO does not occur in all
 BinaxNOW® lateral flow assay. The BinaxNOW® Strep A Test immunochromatographic assay for the qualitative detection
of Streptococcus pyogenes Group A antigen from throat swab specimens. To perform the test, a throat swab is inserted into the test card.
Extraction reagents are added from dropper bottles. The test card is closed to bring the extracted sample in contact with the test strip. Strep
A antigen captured by immobilized anti-strep A reacts to bind conjugated antibody. Immobilized species antibody captures the second visu-
alizing conjugate. The test is interpreted by the presence or absence of visually detectable pink-to-purple colored lines. A positive result is
indicated by production of both a Sample Line and a Control Line (shown on left), whereas a negative assay will produce only the Control
Line (shown on right). (BinaxNOW® is a trademark of the Alere Scarborough, Inc. Used with permission.)

patients. The most commonly used tests are those for ASO for specific populations.13 Typically, however, a single ASO titer
and anti-DNase B.18 is considered to be moderately elevated if the titer is at least
240 Todd units in an adult and 320 Todd units in a child.13
Because of the labor-intensive nature of the traditional
ASO tests detect antibodies to the streptolysin O enzyme pro-
ASO titer test and because the streptolysin O reagent and
duced by Group A streptococcus. This enzyme is able to lyse
the RBCs used are not stable, ASO testing is currently per-
RBCs. Presence of antibodies to streptolysin O indicates recent
formed by nephelometric methods. Nephelometry has the
streptococcal infection in patients suspected of having acute
advantage of being an automated procedure that provides
rheumatic fever or poststreptococcal glomerulonephritis fol-
rapid, quantitative measurement of ASO titers.13 The antigen
lowing a throat infection.
used in this technique is purified recombinant streptolysin.
The classic hemolytic method for determining the ASO
When antibody-positive patient serum combines with the
titer was the first test developed to measure streptococcal
antigen reagent, immune complexes are formed, resulting
antibodies. This test was based on the ability of antibodies in
in an increased light scatter that the instrument converts to
the patient’s serum to neutralize the hemolytic activity of
a peak rate signal. All results are reported in international
streptolysin O. The traditional ASO titer involved preparing
units, which are extrapolated from the classic hemolytic
dilutions of patient serum to which a measured amount of
method described previously. When using the nephelometric
streptolysin O reagent was added. These were allowed to
method, individual laboratories must establish their own
combine during an incubation period after which reagent
upper limits of normal for populations of different ages.
RBCs were added as an indicator. If enough antibodies were
ASO titers typically increase within 1 to 2 weeks after in-
present, the streptolysin O was neutralized and no hemolysis
fection and peak between 3 to 6 weeks following the initial
occurred. The titer was reported as the reciprocal of the high-
symptoms (e.g., sore throat).21 However, an antibody response
est dilution demonstrating no hemolysis. This titer could be
occurs in only about 85% of acute rheumatic fever patients
expressed in either Todd units (when the streptolysin reagent
within this period. Additionally, ASO titers usually do not
standard is used) or in international units (when the World
increase in individuals with skin infections.26
Health Organization international standard is used).13
The range of expected normal values varies, depending on
the patient’s age, geographic location, and season of the year. Testing for the presence of anti-DNase B is clinically useful in
ASO titers tend to be highest in school-age children and young patients suspected of having glomerulonephritis preceded by
adults. Thus, the upper limits of normal must be established streptococcal skin infections because ASO antibodies often are
not stimulated by this type of disease.17 In addition, antibodies are obtained. (See the streptozyme test laboratory exercise
to DNase B may be detected in patients with acute rheumatic online at DavisPlus.)
fever who have a negative ASO test result.
DNase B is mainly produced by Group A streptococci, so
testing for anti-DNase B is highly specific for Group A strepto-
Helicobacter Pylori
coccal sequelae.14 Macrotiter, microtiter, EIA, and nephelomet- First isolated from humans in 1982, Helicobacter pylori is now
ric methods have been developed for anti-DNase testing. The recognized as a major cause of both gastric and duodenal ul-
classic test for the measurement of anti-DNase B activity is cers.27,28 The organism resides in the mucus layer, the gastric
based on a neutralization methodology. If anti-DNase B anti- epithelium, and occasionally the duodenal epithelium.29 This
bodies are present, they will neutralize reagent DNase B, pre- gram-negative, microaerophilic spiral bacterium is observed
venting it from depolymerizing DNA. Presence of DNase is in 30% of the population in developed countries and more
measured by its effect on a DNA-methyl-green conjugate. This than 90% of the population in developing countries. In devel-
complex is green in its intact form; however, when hydrolyzed oping countries, more than 70% carry H pylori by age 10, with
by DNase, the methyl green is reduced and becomes colorless. carriage being nearly universal by the age of 20. Conversely in
An overnight incubation at 37°C is required in some testing the United States, there is little colonization during childhood.
methodologies to permit antibodies to inactivate the enzyme. The rates gradually increase during adulthood, reaching a
Tubes are graded for color, with a 4+ indicating that the inten- prevalence of 50% among persons older than 60 years.30 The
sity of color is unchanged and a 0 indicating a total loss of high incidence of colonization in developing countries where
color. The result is reported as the reciprocal of the highest living conditions are more crowded and sanitation conditions
dilution demonstrating a color intensity of between 2+ and 4+. are suboptimal suggests that fecal–oral transmission is the
Normal titers for children between the ages of 2 and 12 years most likely route.30 Since 1994, the National Institutes of
range from 240 to 640 units.21 Health has recommended that individuals with gastric or
Nephelometry provides an automated means of testing duodenal ulcers caused by H pylori be given antibiotic treat-
that can be used for rapid quantitation of anti-DNase B. ment along with anti-ulcer medications. If untreated, H pylori
In this method, immune complexes formed between anti- infection will last for the patient’s life and may lead to gastric
bodies in patient serum and DNase B reagent generate an in- carcinoma.31
crease in light scatter. Results are extrapolated from values
from the classic method and are reported in international
units per mL.13 H Pylori Virulence Factors
A major virulence factor of H pylori is the production of the
protein CagA, which is highly immunogenic. The organism has
The streptozyme test is a slide agglutination screening test for the the ability to inject the CagA protein into the gastric epithelial
detection of antibodies against streptococcal antigens. The strep- cells. Once the CagA protein is in the epithelial cells, changes
tozyme test measures antibodies against five extracellular strep- occur in the function of the cell’s signal transduction pathways
tococcal antigens: anti-streptolysin (ASO), anti-hyaluronidase and in the structure of the cytoskeleton.32,33
(AHase), anti-streptokinase (ASKase), anti-nicotinamide-adenine A second virulence factor is vacuolating cytotoxin, or VacA.
dinucleotide (anti-NAD), and anti-DNAse B antibodies. The The VacA gene codes for a toxin precursor. Epidemiological
streptozyme test is positive in 95% of patients with acute post- studies have shown that if the CagA and VacA genes are present
streptococcal glomerulonephritis because of GAS pharyngitis.18 in the strain of bacteria infecting the individual, there is a
In this test, sheep RBCs are coated with streptolysin, strep- higher risk of developing gastric or peptic ulcers or gastric
tokinase, hyaluronidase, DNase, and NADase so that antibod- carcinoma.33,34
ies to any of the streptococcal antigens can be detected.
Reagent RBCs are mixed with a 1:100 dilution of patient
serum. Hemagglutination represents a positive test, indicating
Pathology and Pathogenesis
that antibodies to one or more of these antigens are present. Unlike many other bacteria, H pylori is able to survive and
The test is rapid and simple to perform, but it appears to be multiply in the gastric environment. This occurs because of
less reproducible than other antibody tests. In addition, more several characteristics of the bacteria. Its spiral shape and
false positives and false negatives have been reported for this flagella help the organism to be highly motile and to penetrate
test than for the ASO and anti-DNase B assays.18 Because a the viscous mucus layer in the stomach. The organism pro-
larger variety of antibodies are included in this test, the poten- duces large amounts of urease, providing a buffering zone
tial is higher for detection of streptococcal antibodies. How- around the bacteria that protects it from the effects of the
ever, single-titer determinations are not as significant as several stomach acid. In addition, the acid-labile flagella are coated
titrations performed at weekly or biweekly intervals following with a flagellar sheath, protecting them from the acidic envi-
the onset of symptoms.12 The streptozyme test should be ronment of the stomach.
used in conjunction with the ASO or anti-DNase B tests when The pathology and mechanism of action leading to tissue
sequelae of Group A streptococcal infection are suspected and damage is not clearly understood. Neutrophil-induced mucosal
is especially useful when negative or borderline ASO results damage may be the result of the ammonia produced by urease.
The ammonia has been shown to induce the release of chlo-
rinated toxic oxidants from the neutrophils, which causes
inflammation and damage to the mucosal cells. Once estab-
lished below the mucosal layer, the organism does not invade
the tissue. More likely, the pathology represents the host’s
response to extracellular products produced by the bacteria.
Antibodies are formed against the signaling molecules, CagA
and VacA. Strains from individuals with stomach ulcers pro-
duce higher levels of VacA than strains from individuals
without stomach ulcers.35
The outcomes associated with H pylori exposure vary.36
Not all individuals harboring the organism go on to develop
disease, suggesting that the interaction between the host (per-
haps because of genetic predisposition) and the bacteria play
a role.37-41 In some hosts, the organism is not able to establish
itself. In others, asymptomatic colonization may persist or
hyperacidity may result in duodenal ulceration. Treatment of
H pylori consists of triple therapy with bismuth salts, metron-
idazole, and amoxicillin. Although highly effective, 10% to
40% of individuals will have treatment failure because of
CLOtest. The CLOtest rapid urease method uses a
poor patient compliance or resistance to metronidazole.42 An
tissue biopsy to detect Helicobacter pylori. The test consists of a well
increased risk of developing gastric carcinoma and mucosa- of indicator gel sealed inside a plastic cassette. The gel contains
associated lymphoid tumors (MALTomas) has also been urea, phenol red, buffers, and a bacterial static agent to prevent the
shown.43,44 growth of contaminating urease-positive organisms. If the urease
from H pylori is present in the tissue sample, it changes the gel from
Diagnosis of H Pylori Infection yellow (bottom cassette) to bright magenta (top cassette). A majority
of positive tests change color within 20 minutes. The test is reviewed
Detection of H pylori may be achieved by the invasive techniques after 24 hours, because a low-level positive infection may not
of endoscopy or biopsy, or through noninvasive techniques become detectable until then. (Courtesy of Halyard Health, Inc.,
including serological analysis, fecal antigen detection, and Irvine, CA. Used with permission.)
demonstration of urease production with urea breath tests. The
most specific test to detect H pylori infection is culture, but the
sensitivity is usually lower than other methods because the or-
ganism is not evenly distributed throughout the gastric tissue.45
Procedures for detecting H pylori that do not require the use
of endoscopy include urea breath testing, enzyme or lateral
Endoscopy and biopsy are the most expensive and invasive flow immunoassays for the detection of bacterial antigens in
methods for diagnosing an infection with H pylori. However, the feces, molecular tests for H pylori DNA, and serological
histological examination of the tissue may reveal a great deal testing. In the urea breath test, the patient ingests urea labeled
of information regarding the lesion. One method of testing for with radioactive carbon (14C) or a nonradioactive 13C. Urea is
H pylori involves the detection of urease from a biopsy taken metabolized to ammonia and bicarbonate. The bicarbonate is
from the antrum of the stomach. The antrum is the portion of excreted in the breath and the labeled carbon dioxide is meas-
the stomach before the pyloric sphincter or valve responsible ured by detection of radioactivity for 14C or mass spectrometry
for releasing stomach contents into the intestines. An example analysis for 13C. This breath technique has excellent sensitivity
of a test that detects urease in a tissue biopsy is the CLOtest. and specificity and is helpful in determining if the bacteria
The CLOtest (for Campylobacter-like organisms) detects urease have been eradicated by antimicrobial therapy; however, in
activity in gastric mucosal biopsies. During the endoscopy, a most cases it involves the use of radioactivity.10,45
small biopsy is taken (1–3 mm). The specimen is placed in the Because of the potential for treatment failure, analysis of stool
test cassette, resealed following the manufacturer’s instructions, samples before and after antimicrobial therapy for H pylori
and sent to the laboratory. If urease is present, the yellow gel antigens is done to determine if the bacteria have been elimi-
will turn a hot pink color because of an increase in pH in the nated following treatment.45 ELISA tests as well as LFA methods
presence of urease. If urease is not present, the gel will remain are available. Because of the potential for asymptomatic carriage
yellow (Fig. 20–12). A majority of the tests will turn positive of the organism, stool antigen testing for initial diagnosis of
within 20 minutes; however, the test should be held and reex- H pylori infection is not recommended.
amined after 24 hours to allow time for detection of a low-level Researchers also have developed molecular testing to detect
infection. The test is easy to use and results can be detected in H pylori DNA.45,46 However, PCR-based methods, which detect
a short period of time, making it ideal for rapid diagnosis of the presence of the organism in fecal samples, cannot distinguish
H pylori infections.46 between living and dead H pylori. Real-time PCR technology has
been developed to determine the patient’s bacterial load and has smallest known free-living life forms (150–250 nm) and have
shown good correlation with the urea breath test. At the time of a small genome. Various members colonize plants, animals,
this writing, FDA-approved molecular assays for H pylori are not and insects in addition to being human pathogens.48 These
available for clinical use. extracellular parasites attach to and exist on the surface of host
cells using attachment organelles and adhesion molecules
specific for their host cells. They absorb their nutrients from
Serological testing is a primary screening method of determining the host cells to which they are attached.49 The organisms lack
infection with H pylori. Infections from this organism result in cell walls (thus lacking peptidoglycan), have sterols in their
production of IgG, IgA, and IgM antibodies. Most serological cell membrane, and have complex growth requirements, mak-
tests in clinical use detect H pylori-specific antibodies of the IgG ing culture difficult and time consuming.
class. Although IgM antibody is produced in H pylori infections,
testing for its presence lacks clinical value because most infec-
tions have become chronic before diagnosis. Thus, IgG is the
Mycoplasma Pneumoniae Pathogenesis
primary antibody found. IgA testing has a lower sensitivity and The best-known Mycoplasma is M pneumoniae, which is a lead-
specificity than IgG testing, but it may increase sensitivity of ing cause of respiratory infections worldwide.50 M pneumoniae
detection when used in conjunction with IgG testing.45 infections are found in all age groups, with a majority of the
The presence of antibodies in the blood of an untreated infections involving the upper respiratory tract.51 M pneumoniae
patient indicates an active infection because the bacterium does is spread from one person to another by respiratory droplets.
not spontaneously clear. Antibody levels in untreated individ- Relatively close association with an infected individual appears
uals remain elevated for years. In treated individuals, the anti- to be necessary for transmission of the organism.51,52 Unlike
body concentrations decrease after about 6 months to about most respiratory infections, the incubation period is 1 to 3 weeks.
50% of the level the patient had during the active infection. The infection has an insidious onset which differs from the
Therefore, convalescent testing should be performed 6 months acute onset observed with respiratory viruses such as influenza
to a year after treatment, which requires that the acute serum and adenovirus. Typically, there is development of a fever, along
sample be stored for up to a year.47 A decrease in antibody titer with headache, malaise, and a cough—the clinical hallmark of
of more than 25% must occur for treatment to be considered a M pneumoniae infection. Depending on the age, approximately
successful.47 5% to 10% of individuals progress to tracheobronchitis or
Measurement of the antibodies may be done with several pneumonia.51
techniques, including ELISA, immunoblot, and rapid tests Originally, pneumonia caused by M pneumoniae was re-
using LA or LFA. Several LFAs are approved for CLIA-waived ferred to as “atypical pneumonia” because the infection could
testing by physician office laboratories. The method of choice not be treated with penicillin. This is because the organism
for the detection of H pylori antibodies is the ELISA technique, lacks the cell wall to which penicillin is directed against. In
which is reliable and accurate.47 Tests employing antigens from many cases, the pneumonia is mild, oftentimes appearing as
a pooled extract from multiple and genetically diverse strains a cold, and symptoms are generally mild enough that bed rest
yield the best sensitivity because H pylori is so heterogeneous. or hospitalization is not required. The infection is often re-
Very few, if any, patients produce antibodies to all of the H pylori ferred to as “walking pneumonia” because individuals often
antigens; most patients produce antibodies against the CagA do not stay home from work or school and still participate
and VacA proteins. Antibodies to these two proteins indicate a in their daily activities. Although many infections are mild,
more severe case of gastritis or an increased risk of developing M pneumoniae accounts for 20% of all hospitalizations for
gastric carcinoma.47 pneumonia in the United States.52,53 M pneumoniae may remain
When compared with other techniques for antibody detection in the respiratory tract for several months after resolution of
of H pylori, ELISA tests are sensitive, specific, and cost effective the infection, causing chronic inflammation and a lingering
for determining the organism’s presence in untreated individu- cough.50 Based on nucleic acid detection of the organism,
als.45 However, because antibodies are not rapidly cleared after there is increasing evidence that M pneumoniae may initiate or
treatment, antibody testing is not as well suited for determining exacerbate asthma.54
eradication of infection as are other methods. Additionally, indi-
viduals who are immunocompromised (the elderly or immuno- Dermatological Manifestations
suppressed individuals) may have a false-negative result with
antibody testing. Up to 7% of individuals with M pneumoniae develop Stevens–
Rapid assays for the detection of H pylori antibodies are also Johnson syndrome, or erythema multiforme major, a condition
available. It is recommended that samples with positive rapid in which the top layer of the skin dies and sheds. The syndrome
test results be tested by an ELISA method for correlation.45 is considered a medical emergency that usually requires hospi-
talization.55 The conjunctivae as well as the joints and various
organs in the genitourinary and gastrointestinal tract may also
Mycoplasma Pneumoniae be involved. The cause of Stevens–Johnson syndrome is not
clearly known, but it may be caused by the immune response
Mycoplasma is a member of a unique group of organisms that of the host or to augmented sensitivity to antibiotics while
belong to the class Mollicutes. Mycoplasmas represent the being treated for M pneumoniae.54-56
 Another manifestation of M pneumoniae infection is Ray- infection. Enzyme-linked immunoassays have been the most
naud syndrome, which is a transient vasospasm of the digits widely used methods for antibodies and can detect IgM or IgG
in which the fingers turn white when exposed to the cold. directed against M pneumoniae.61 Although IgM is the primary
Although the exact cause is unclear, it may be related to the immunoglobulin response to infection, testing for the presence
development and action of cold agglutinins in the body (see of IgG antibodies is necessary; the reason is that adults may
Chapter 14).57 Other extrapulmonary manifestations of Ray- only elicit an IgG response because of reinfection with the
naud syndrome include arthritis, meningoencephalitis, peri- organism. ELISA methods have a specificity of more than 99%
carditis, and peripheral neuropathy.58 and a sensitivity of 98%.61

Immunology of Mycoplasma For many years, before the development of antigen-specific

Pneumoniae Infection serological tests, laboratory diagnosis of M pneumoniae involved
testing for cold agglutinins. The agglutinins are capable of
In addition to stimulating the production of many proinflam-
clumping RBCs at 4°C. The reaction is reversible when the
matory and anti-inflammatory cytokines and chemokines,
samples are warmed to 37°C. Cold agglutinins develop in
several classes of antibodies are produced in the course of a
about 50% of patients with M pneumoniae infection.60 These
M pneumoniae infection. As with any infection in which there
antibodies are produced early in the disease (7–10 days) and
is a humoral response, the body produces antibodies that
can typically be detected at the time the patient seeks medical
neutralize the microorganism. M pneumoniae also induces the
attention. The titer peaks at 2 to 3 weeks and antibodies are
production of autoantibodies, including agglutinins directed
present for 2 to 3 months after infection.60
against the lungs, brain, cardiolipins, and smooth muscle.58,59
Although once considered the primary means of diagnosing
The cold isoagglutinins observed with M pneumoniae infec-
Mycoplasma infection, the assay is not very specific (50% to 70%)
tion are among the most studied agglutinins by researchers.
nor is it very sensitive.62 Testing for cold agglutinins is no
They are oligoclonal IgM antibodies directed against the altered
longer recommended because the development of cold agglu-
I antigens found on the surface of human RBCs.60 These anti-
tinins occurs in other circumstances, including some viral
bodies can agglutinate the RBCs at temperatures below 37°C.
infections and collagen vascular diseases.63 However, a titer
Development of the antibodies is thought to result from cross-
of 1:64 or greater, along with the clinical presentation of the
reaction of antibodies formed against M pneumoniae and the
patient, is suggestive of infection with M pneumoniae. It should
I antigen on human RBCs or from alteration of the RBC antigen
be noted that cold agglutinins may be found in patients with
by the organism.60
infections whose clinical presentations resemble M pneumoniae
infection such as mononucleosis caused by Epstein-Barr virus
Laboratory Diagnosis of Mycoplasma (anti-i) and cytomegalovirus (anti-I).64,65
Pneumoniae Infection
Laboratory means of detecting Mycoplasma infection may involve Molecular methods will, in all likelihood, become the gold
culturing of the organism, detection of M pneumoniae-specific standard for the diagnosis of Mycoplasma infections. Although
antibodies in serum, and detection of M pneumoniae-specific some laboratories offer “home brew” assays, very few labora-
antigens or nucleotide sequences directly in patient specimens. tories offer a molecular assay for the detection of Mycoplasma
in clinical samples. Before 2012, there were no FDA-approved
assays available for the detection of M pneumoniae. BioFire
Although culturing has been considered the gold standard for Diagnostics, Inc. (Salt Lake City, UT), now part of the Bio-
diagnosis, culturing for the organism is rarely carried out in Merieux corporation, received FDA approval in 2012 for its
the clinical laboratory because of the fastidious nature of the FilmArray® Respiratory Panel. Using nested multiplex PCR,
organism. Collection and transport of the specimen differs the assay is able to detect 20 respiratory viruses and bacteria
from traditional methods used for culturing other microorgan- including B pertussis, C pneumoniae, and M pneumoniae.66 As
isms.53 The transport media may be trypticase soy broth with additional assays are developed and receive FDA approval, the
0.5% albumin, SP4 medium, or a viral transport medium. If molecular diagnosis of Mycoplasma infection will become more
the sample cannot be plated immediately, it should be frozen widespread and will likely replace serology as the primary
at –70°C. Culturing requires the use of specialized media de- means for the diagnosis of M pneumoniae infections.
signed for the recovery of Mycoplasma. Growth of the organism
takes several weeks in most cases. If the culture is successfully
performed, the growth produces a “mulberry” colony with a Rickettsial Infections
typical “fried egg” appearance.
Members of the Rickettsiaceae family cause a variety of infections
in man and animals. Because of recent advances in molecular
technology, various members originally belonging to the genus
Detection of M pneumoniae-specific IgM immunoglobulin is the Rickettsiae have now been reclassified. These obligate intracel-
most useful diagnostic test because it likely indicates a recent lular, gram-negative bacteria now include the genera Rickettsia
(Rickettsiosis) and Orientia (Orientia tsutsugamushi causing scrub Epidemic typhus, caused by Rickettsia prowazekii (TG), is the
typhus). Additional members are the Ehrlichia group including most prevalent member of the TG globally. Typhus fever (also
Ehrlichia (Ehrlichiosis), Anaplasma (Anaplasmosis), Neorickettsia known as epidemic typhus) occurs in conditions of poor
(associated with helminths), and Neoehrlichia.67 hygiene and overcrowding such as in prisons and refugee
camps. Epidemic typhus was responsible for over 3 million
deaths in World War I.69 Once prevalent throughout the
Agents of Rickettsia-Related Disease globe in the first half of the 20th century, only a few foci of
The genus Rickettsia is made up of two distinct groups: the epidemic typhus still exist in the world today (Ethiopia,
spotted fever group (SFG) and the typhus group (TG). Each Burundi, Rwanda, part of Mexico).70 Individuals traveling to
is responsible for a different set of diseases (Table 20–1). areas with large homeless populations and regions that have
In the United States, the main Rickettsial disease is Rocky recently experienced war or natural disasters leading to poor
Mountain spotted fever (RMSF), caused by R rickettsia hygiene and crowded conditions (such as refugee camps) are
(SFG), with approximately 2,500 cases reported each year.68 at risk of acquiring typhus fever.

Table 20–1 Classification of Select Rickettsiae Known to Cause Disease in Humans

ANTIGENIC ANIMAL GEOGRAPHIC
GROUP DISEASE SPECIES VECTOR RESERVOIR(S) DISTRIBUTION
Anaplasma Human Anaplasma Tick Small mammals, Primarily United
granulocytic phagocytophilum rodents, and deer States, worldwide
anaplasmosis
Ehrlichia Human Ehrlichia Tick Deer, wild and Common in United
monocytic chaffeensis domestic dogs, States, probably
ehrlichiosis domestic rumi- worldwide
nants, and rodents
Ehrlichiosis E muris Tick Deer and rodents North America,
Europe, Asia
Ehrlichiosis E ewingii Tick Deer, wild and North America,
domestic dogs, Cameroon, Korea
and rodents
Neoehrlichia Human Neoehrlichia Tick Rodents Europe, Asia
neoehrlichiosis mikurensis
Neorickettsia Sennetsu fever Neorickettsia Trematode Fish Japan, Malaysia,
sennetsu possibly other
parts of Asia
Scrub typhus Scrub typhus Orientia Larval mite Rodents Asia-Pacific region
tsutsugamushi (chigger) from maritime
Russia and China
to Indonesia and
North Australia to
Afghanistan
Spotted fever Rocky Mountain Rickettsia Tick Rodents North, Central, and
spotted fever rickettsii South America
Rickettsiosis Rickettsia Tick Unknown South Africa,
aeschlimannii Morocco,
Mediterranean
littoral
Queensland tick Rickettsia Tick Rodents Australia, Tasmania
typhus australis
Boutonneuse Rickettsia conorii Tick Dogs, rodents Southern Europe,
fever or southern and
Mediterranean western Asia,
spotted fever Africa, India

Continued
 Table 20–1 Classification of Select Rickettsiae Known to Cause Disease in Humans—cont’d
ANTIGENIC ANIMAL GEOGRAPHIC
GROUP DISEASE SPECIES VECTOR RESERVOIR(S) DISTRIBUTION
Typhus fever Epidemic Rickettsia Human body Humans, flying Central Africa,
typhus, sylvatic prowazekii louse, flying squirrels Asia, Central
typhus squirrel ec- America, North
toparasites, America, and
Amblyomma South America
ticks
Murine typhus Rickettsia typhi Flea Rodents Tropical and sub-
tropical areas
worldwide

Adapted from Centers for Disease Control and Prevention. CDC Health Information for International Travel, 2016. New York, NY: Oxford University Press; 2016.
Online version accessed November 17, 2015.

Each of the species responsible for the various Rickettsial dis- Rocky Mountain Spotted Fever
eases has a variety of animal reservoirs. The vectors responsible
for the transmission are arthropods (ticks, mites, lice, or fleas)
which transmit the organism through its bite after feeding on an RMSF is caused by R rickettsii. The organism is transmitted to
infected animal (Fig. 20–13).71 The one exception is typhus fever the human host by the bite of a tick. In the United States, the
(epidemic louse-borne typhus) which is transmitted when an organism is transmitted by the American dog tick (Dermacentor
infected human body louse excretes R prowazekii onto the skin variabilis), the Rocky Mountain wood tick (Dermacentor ander-
while feeding and the individual becomes infected by rubbing soni), and the brown dog tick (Rhipicephalus sanguineus). Although
louse fecal matter or crushed lice into the bite wound. Except called Rocky Mountain spotted fever, five states—North Carolina,
for R prowazekii (epidemic typhus), humans are accidental hosts Oklahoma, Arkansas, Tennessee, and Missouri—account for
for Rickettsia and Rickettsia-related organisms.71 Rickettsia and over 60% of RMSF cases in the continental United States.73 The
Rickettsia-related organisms have worldwide distribution; how- occurrence of the disease has seasonal variation corresponding
ever, certain members have a specific geographic distribution. to tick activity, being most prevalent between May and Septem-
For example, Rickettsia japonica is found only in Japan, whereas ber.70 The organism is transmitted transstadially in the tick (i.e.,
R rickettsii is found in the Western hemisphere. Some species, it remains present in the tick as the tick progresses from the
such as Rickettsia typhi, are found everywhere in the world.72 nymph state to the adult) and is transmitted transovarially (from
The members of the Rickettsia genus and Anaplasma and generation to generation through the eggs of the tick), allowing
Ehrlichia cause a number of clinical diseases. Because of the for maintenance of the organism in the tick population. Trans-
prevalence of RMSF in North and South America, this chapter mission occurs when the tick bites the host for a blood meal.
will focus on RMSF. When the tick has fed after 6 to 10 hours, the organism is in-
jected into the host from the salivary glands.

Once introduced into the skin, the organisms spread via the
lymphatic and circulatory system, where they attach to and
invade their target cells, the vascular endothelium, by means
of the OmpA and OmpB ligands.74-77 The organisms multiply
by binary fission inside the endothelial cells, are released, and
infect adjacent cells. This leads to hundreds of contiguous
infected cells, producing the lesions and skin rash associated
with the infection. The main pathophysiological event caused
by the infection is endothelial cell damage, which leads to
increased vascular permeability, resulting in edema, hypovolemia,
hypotension, and hypoalbuminemia.78,79

The Rocky Mountain wood tick, Dermacentor ander- The symptoms observed with RMSF occur approximately 2 to
soni, is a known North American vector of Rickettsia rickettsii. (Cour- 14 days (median 7 days) after a tick bite. Before the development
tesy of the CDC/Dr. Christopher Paddock and James Gathany, Public of the hallmark rash, a large percentage of patients will experi-
Health Image Library.) ence a quite severe headache, nausea, vomiting, abdominal pain,
diarrhea, and abdominal tenderness. The fever usually begins antigen, performed on two paired serum samples to demon-
within the first 3 to 5 days after the onset of symptoms, and strate a significant (four-fold) rise in antibody titers.81 For
the rash usually appears 3 to 5 days after the onset of the fever. many years, antibodies produced in patients with Rickettsial
The rash typically starts on the hands and soles of the feet and infections were detected by an agglutination test known as
proceeds to the trunk, although it may start on the trunk in the Weil-Felix test, which was based on cross-reactivity of the
some individuals (Fig. 20–14). With the classic form of RMSF, patient’s antibodies with polysaccharide antigens present on
death occurs 7 to 15 days after the onset of symptoms if ap- the OX-19 and OX-2 strains of Proteus vulgaris and the OX-K
propriate therapy is not provided. With the fulminant (i.e., strain of Proteus mirabilis. This method lacks sensitivity and
severe and sudden onset) form of RMSF, death occurs within specificity and should not be relied on.
the first 5 days. The resolution or fatal outcome of the disease
is largely related to the timeliness of initiating appropriate
therapy. Immediate treatment with doxycycline reduces the
severity of the infection.80,81 SUMMARY
• The indigenous microbiota varies at different sites of the
Initial diagnosis is often made clinically after ruling out a large body.
variety of other conditions, including typhoid fever, measles, • The symbiotic relationship that exists between bacteria
rubella, enteroviral infection, and respiratory tract infection. and humans is beneficial in protecting against infection,
The overlapping symptoms, or clinical presentation, during stimulating the immune system, aiding in digestion of
the initial stages of the disease can make the diagnosis of RMSF food, and producing various vitamins.
extremely difficult. The diagnosis of fulminant RMSF is even • Humans exist in a commensalistic relationship with the bac-
more difficult because of its rapid course. The rash develops teria that comprise the human microbiome. The encounter
shortly before death, if at all; therefore, antibodies to R rickettsii with some microbial organisms results in a parasitic rela-
do not have time to develop. tionship in which there is harm to the host that may result
in an infection.
The organism infects the endothelial cells and does not circulate • Pathogenicity refers to the ability of an organism to cause
until the disease has severely progressed. Therefore, culturing disease, virulence refers to the extent that a pathogen
for the organism and molecular methods is not always useful. causes damage to the host, and infectivity refers to the
If the patient has a rash, molecular diagnosis using DNA from ability of an organism to spread from one host to another.
the skin lesions is of value. (Note: At the time of this writing • To be virulent, an organism must possess structural fea-
there are no FDA-approved assays.) Serology is the usual method tures or produce extracellular substances that allow it to
for confirming the diagnosis of RMSF, but this is a retrospective invade or cause damage to the host. These are referred to
diagnosis. Antibodies to R rickettsia develop 7 to 10 days after as virulence factors.
the onset of symptoms and a majority of patients do not show • Live bacteria may produce exotoxins that are generally
antibodies during the first week of illness. For a successful out- specific to a particular bacterial organism and have specific
come, therapy needs to be initiated before that time. modes of action on the host. Exotoxins are highly immuno-
The gold standard for the serological diagnosis of RMSF is genic and antibodies formed against them are protective.
the indirect immunofluorescence assay (IFA) with R rickettsii • Endotoxin, or lipid A, is part of the gram-negative bacte-
rial cell wall that is released from dead bacteria. Endotoxin
has a broad range of systemic effects on the body because
it induces the release of cytokines that can lead to septic
shock. Endotoxin, although immunogenic, does not result
in the production of protective antibodies.
• Nonspecific immune defenses (phagocytosis, production
of antimicrobial defense peptides, various proteins) con-
tribute heavily to the body’s ability to overcome a bacterial
infection.
• Laboratory detection of the causative agent of a bacterial in-
fection includes culturing of the organism, visualization of
the bacteria in clinical specimens, detection of bacterial anti-
gens in the clinical specimen, detection of the pathogen’s
DNA or RNA, and demonstration of antibodies formed
against the agent through serological means.
• Lateral flow immunochromatographic assays (LFAs) are
The characteristic spotted rash of Rocky Mountain increasingly being used for the detection of bacterial, viral,
spotted fever, the most severe and most frequently reported rickettsial fungal, and parasitic antigens. Many LFAs have sensitivities
illness in the United States. The disease is caused by Rickettsia rickettsii. that exceed other testing methods. The principle behind
(Courtesy of the CDC, Public Health Image Library.)
 LFAs is the movement of a liquid sample containing the • Serological testing is the primary screening method of
analyte of interest along a strip that passes through various detecting H pylori infection. Testing for urease is also used
zones containing labeled antibodies specific to the analyte. to detect and diagnose an infection with H pylori. Detection
If the antigen–antibody complex is present, it is captured of H pylori antigen in stool samples can be used to monitor
by another antibody at the end of the strip, resulting in the the effectiveness of treatment for H pylori infections.
development of a visible line. • Mycoplasma pneumoniae is a leading cause of community
• Streptococcus pyogenes (Group A streptococci or GAS) is acquired pneumonia. Infection with M pneumoniae may
the primary cause of bacterial pharyngitis. It is also a pri- not require bed rest or hospitalization and oftentimes is
mary cause of a skin infection called impetigo. Untreated referred to as “walking pneumonia.”
GAS infections may result in sequelae: acute glomeru- • Infection with M pneumoniae may result in dermatological
lonephritis or rheumatic heart disease. manifestations causing Stevens–Johnson syndrome. Ray-
• The production of exotoxins contributes to the infections naud syndrome, another manifestation that may be observed
caused by GAS. Streptolysin O, hyaluronidase, deoxyri- with M pneumoniae, is a reversible variable vasospasm of
bonuclease B (DNase B), and streptokinase all play a role the digits in which the fingers turn white when exposed to
in the infections associated with GAS. the cold.
• Scarlet fever occurs in a small percentage of individuals • Production of cold agglutinins is observed in 50% of
infected with GAS and is caused by the production of ery- individuals with M pneumoniae. Demonstration of cold
throgenic exotoxins that may also result in toxic shock agglutinins is neither specific nor sensitive when detecting
syndrome. infection by the organism. Detection of M pneumoniae-
• The laboratory diagnosis of GAS infection includes cul- specific IgM immunoglobulin is the most useful diagnostic
ture and antigen detection for acute infection, and anti- assay because it likely indicates a recent infection.
streptolysin O and anti-DNase B antibody detection for • Rocky Mountain spotted fever (RMSF) is caused by
GAS sequelae. Rickettsia rickettsii and is transmitted to the human host
• The streptozyme test measures antibodies against five by the bite of a tick.
extracellular streptococcal antigens–anti-streptolysin • The main pathophysiological event caused by RMSF is
(ASO), anti-hyaluronidase (AHase), anti-streptokinase the endothelial cell damage leading to increased vascular
(ASKase), anti-nicotinamide-adenine dinucleotide (anti- permeability, which then results in edema, hypovolemia,
NAD), and anti-DNAse B antibodies. The streptozyme test and hypotension. Various cytokines are released and dam-
is positive in 95% of patients with acute poststreptococcal age to the host may have a fatal outcome if therapy is not
glomerulonephritis caused by GAS pharyngitis. initiated in a timely fashion.
• Helicobacter pylori is the leading cause of gastric and duo- • The gold standard for the serological diagnosis of RMSF
denal ulcers and is associated with gastric carcinoma is the indirect immunofluorescence assay (IFA) with
(stomach cancer). H pylori produces a large amount of ure- R rickettsii antigen performed on two paired serum samples
ase that protects the organism from the acidic environment to demonstrate a significant (four-fold) rise in antibody
in the stomach. titers.

Study Guide: Immune Defenses Against Bacterial Infection

INNATE DEFENSES ADAPTIVE IMMUNE RESPONSES
Skin and mucosal surfaces Antibodies produced against bacterial antigens promote opsonization
and complement binding
Antimicrobial defense peptides and proteins on Antibodies produced against bacterial exotoxins have neutralizing
epithelial surfaces (e.g., lysozyme, defensins) activity
Other proteins (e.g., complement proteins, T-cell–mediated immune responses attack intracellular bacteria
interleukins, prostaglandins, leukotrienes)
Acute phase reactants (e.g., CRP, haptoglobin)
Pattern recognition receptors (e.g., TLRs on
macrophages and dendritic cells)
Phagocytosis by neutrophils and macrophages
Study Guide: Bacterial Virulence Factors
VIRULENCE
FACTOR DESCRIPTION MECHANISM OF PATHOGENESIS EXAMPLE(S)
Pili Hairlike structures on the Adherence to and colonization of Enterotoxigenic E coli pili adhere to
surface of bacteria host tissue cells in small intestine
Resistance to phagocytosis
Transfer of genetic material
Adhesion Molecules on surface of Attach to a variety of host cell Fibronectin binding proteins of
molecules bacteria receptors such as proteoglycans, S pyogenes facilitate attachment
collagen, fibrinogen to host cells
Capsule A polysaccharide layer Blocks phagocytosis Capsule of S pneumoniae bacteria
surrounding the cell wall Blocks attachment of antibodies prevents phagocytosis by alveolar
of some bacteria for opsonization macrophages
Inhibits complement activation
Acts as a decoy when released
Endotoxin Lipid A component of Powerful stimulator of cytokine Gram-negative bacterial infection
lipopolysaccharide on cell production involving bacteremia can cause
walls of gram-negative septic shock
bacteria; released when
bacteria die
Exotoxins Neurotoxins, cytotoxins, Bind to specific receptors on host Tetanus neurotoxin prevents trans-
and enterotoxins released cells mitter release from neuromuscular
from live bacteria Some can act as superantigens that cells, resulting in continuous
activate numerous T cells muscle spasms
Exotoxins from S pyogenes can
cause toxic shock syndrome

CASE STUDIES
1. A 6-year-old boy was brought to the pediatric clinic. His Protein: 2+ (normal = negative/trace)
mother indicated he had been ill for several days with Blood: large (normal = none)
fever and general lethargy. The morning of the visit, the Rapid GAS Antigen Test
boy told his mother that his back hurt and she had ob- Negative
served what appeared to be blood in his urine. History
Streptozyme
and physical examination indicated a well-nourished
Positive 1:600
child with an unremarkable health history other than a
severe sore throat with fever 3 weeks prior that was med- Questions
icated with aspirin and throat lozenges. This child’s tem- a. What disorder is indicated by the child’s history,
perature was 101.5°F and the physician noted edema in physical examination, and laboratory test results?
the child’s hands and feet. Blood and urine specimens b. What was the most likely causative agent of the sore
were collected for a rapid GAS antigen test, streptozyme throat preceding the current symptoms?
test, complete blood cell count, and urinalysis. Labora- c. Discuss the most widely accepted theory explaining
tory test results were as follows: the physiological basis for this disease. Why didn’t
Complete Blood Count the physician order a throat culture?
RBC count: normal d. What is the significance of the urinalysis results?
Platelet count: normal e. What is the significance of the streptozyme test results?
WBC count: 12.7 109/L (normal = 4.8–10.8 109/L)
Urinalysis 2. A 36-year-old female was seen by her physician because
Color: red (normal = straw) she had been experiencing flu-like symptoms along
Clarity: cloudy (normal = clear) with a sore throat and chills for the past 3 days. She
 was also having difficulty breathing. The patient had a Mycoplasma Titers
temperature of 100.2°F and was producing a moderate IgM: none detected
amount of sputum. Her physician decided that the IgG: 1:16
probable diagnosis was some type of pneumonia and Cold Agglutinin Titer
ordered the following laboratory tests to be performed: Positive 1:128
complete blood count, sputum culture, tests for influenza
virus, and M pneumoniae and cold agglutinin titers. The Questions
results were as follows: a. What is the most probable cause of the pneumonia?
b. What is the significance of the Mycoplasma titer
Complete Blood Count
results?
RBC: normal
WBC: 11.7 109/L (normal = 4.8–10.8 109/L) [somewhat
c. Should additional Mycoplasma titers be ordered as a
elevated] follow up?
d. What is the significance of the cold agglutinin titer?
Sputum Culture
Negative

REVIEW QUESTIONS
1. All of the following are protective mechanisms against 5. Which of the following indicates the presence of anti-
bacteria except DNase B activity in serum?
a. production of antimicrobial defense peptides. a. Reduction of methyl green from green to colorless
b. phagocytosis. b. Clot formation when acetic acid is added
c. activation of complement. c. Inhibition of red blood cell hemolysis
d. release of lipid A from the bacterial cell. d. Lack of change in the color indicator

2. All of the following are characteristics of streptococcal 6. Which of the following is considered to be a nonsup-
M proteins except purative complication of streptococcal infection?
a. it is the chief virulence factor of Group A a. Acute rheumatic fever
streptococci. b. Scarlet fever
b. it provokes an immune response. c. Impetigo
c. antibodies to one serotype protect against other d. Pharyngitis
serotypes.
d. it limits phagocytosis of the organism. 7. All of the following are ways that bacteria can evade
host defenses except
3. An ASO titer and a streptozyme test are performed on a. presence of a capsule.
a patient’s serum. The ASO titer is negative, the strep- b. stimulation of chemotaxis.
tozyme test is positive, and both the positive and neg- c. production of toxins.
ative controls react appropriately. What can you d. lack of adhesion to phagocytic cells.
conclude from these test results?
a. The ASO is falsely negative. 8. Antibody testing for Rocky Mountain spotted fever
b. The patient has an antibody to a streptococcal may not be helpful for which reason?
exoenzyme other than streptolysin O. a. It is not specific.
c. The patient has not had a previous streptococcal b. It is too complicated to perform.
infection. c. It is difficult to obtain a blood specimen.
d. The patient has scarlet fever. d. Antibody production takes at least a week before
detection.
4. Which of the following applies to acute rheumatic fever?
a. Symptoms begin after S. pyogenes infection of the 9. Which of the following enzymes is used to detect the
throat or the skin. presence of H pylori infections?
b. Antibodies to Group A streptococci are believed to a. DNase
cross-react with heart tissue. b. Hyaluronidase
c. Diagnosis is usually made by culture of the organism. c. Urease
d. All patients suffer permanent disability. d. Peptidase
10. Which of the following reasons make serological iden- 13. Which of the following is true regarding exotoxins
tification of a current infection with Helicobacter pylori and endotoxins?
difficult? a. Both endotoxin and exotoxins are highly immuno-
a. No antibodies appear in the blood. genic allowing for the development of protective
b. Only IgM is produced. antibodies and vaccines.
c. Antibodies remain after initial treatment. b. Endotoxin has targeted activity whereas exotoxins
d. No ELISA tests have been developed. have systemic effects when released.
c. Endotoxin is released from the cell wall of dead
11. M pneumoniae infections are associated with the bacteria, whereas exotoxin is released from live
production of which antibodies? bacteria.
a. Cold agglutinins d. Both endotoxin and exotoxins bind to specific
b. Antibodies to ATPase receptors on a bacterial cell leading to cell lysis.
c. Antibodies to DNase
d. Antibodies to Proteus bacteria 14. Characteristics of a bacterial capsule include which of
the following?
12. Which of the following best describes the principle of a. It cannot be used for vaccine development.
the IFA test for detection of antibodies produced in b. It is composed of peptidoglycan.
Rocky Mountain spotted fever? c. It is an important mechanism for protecting a
a. Patient serum is applied to a microtiter plate coated bacterium against ingestion by PMNs.
with a monoclonal antibody directed against the d. It is what causes bacteria to stain as gram-negative.
target antigen. A detection antibody labeled with
biotin and directed against the target antigen is 15. Which of the following statements regarding Helicobacter
added. After addition of a substrate, a color reac- pylori is not true?
tion develops indicating presence of the antigen. a. It is associated with an increased risk of gastric
b. Specific antibodies in the serum sample attach to carcinoma.
the antigens fixed to a microscope slide. In a sec- b. It is the cause of most cases of acute food poisoning
ond step, the attached antibodies are stained with in the United States.
fluorescein-labeled anti-human immunoglobulin c. It is a major cause of peptic ulcers in the United
and visualized with the fluorescence microscope. States.
c. The serum sample is treated chemically to link the d. It is positive for urease.
target antibodies to a fluorophore. The labeled
sample is applied to a microscope slide to which
the antigen has been attached. Following a wash
step, the slide is examined for fluorescence.
d. Patient serum is applied to a slide to which a
specific antigen is bound. Following a wash step,
a chromogenic dye is applied that binds to the Fc
region of IgG and IgM antibodies. After a second
wash step, the slide is examined for fluorescence.
 Spirochete Diseases

After finishing this chapter, you should be able to: SYPHILIS

1. Describe identifying characteristics of Treponema pallidum and Borrelia Characteristics of the Organism
burgdorferi. Mode of Transmission
2. Explain how syphilis and Lyme disease are transmitted. Stages of the Disease
3. Discuss the different stages of syphilis. Congenital Syphilis
4. Discuss the advantages of direct fluorescent staining for T pallidum Nature of the Immune Response
over dark-field examination without staining. Laboratory Diagnosis
5. Define reagin. LYME DISEASE
6. Distinguish treponemal from nontreponemal tests for syphilis. Characteristics of the Organism
7. Describe the principle of the following tests for syphilis: Venereal Stages of the Disease
Disease Research Laboratory (VDRL), rapid plasma reagin (RPR),
Nature of the Immune Response
fluorescent treponemal antibody absorption (FTA-ABS), and
T pallidum particle agglutination (TP-PA). Laboratory Diagnosis
8. Provide reasons for false-positive nontreponemal test results. Treatment
9. Compare and contrast typical results of treponemal and RELAPSING FEVER GROUPBORRELIA
nontreponemal testing during the various stages of syphilis. MIYAMOTOI
10. Discuss the advantages and limitations of polymerase chain reaction Characteristics of the Organism
(PCR) and enzyme immunoassay (EIA) testing for syphilis. Stages of the Disease
11. Explain the traditional and reverse algorithms for syphilis and discuss Laboratory Diagnosis
their advantages and limitations. SUMMARY
12. Select appropriate laboratory methods for testing of cerebrospinal CASE STUDIES
fluid (CSF) for neurosyphilis and testing for congenital syphilis.
REVIEW QUESTIONS
13. Describe early and late manifestations of Lyme disease.
14. Relate various aspects of the immune response to the stages of Lyme
disease.
15. Compare immunofluorescence assay (IFA), EIA, and immunoblot
testing for Lyme disease as to sensitivity and ease of performance.
16. Discuss causes of false positives and false negatives in serological
testing for Lyme disease.
17. Discuss the clinical manifestations and laboratory testing associated
with Borrelia miyamotoi infections.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Borrelia burgdorferi Gummas Rapid plasma reagin (RPR) Treponema pallidum
Borrelia miyamotoi Immunoblotting test Treponemal test
Chancre Lyme disease Reagin Venereal Disease Research
Congenital syphilis Nontreponemal tests Syphilis Laboratory (VDRL) test
Flocculation Particle agglutination (PA) T pallidum particle
tests agglutination
Fluorescent treponemal
(TP-PA) test
antibody absorption Prozone
(FTA-ABS) test

Spirochetes are long, slender, helically coiled bacteria contain- other pathogens in this group are so morphologically and anti-
ing axial filaments, or periplasmic flagella, which wind around genically similar to T pallidum that all but one are classified as
the bacterial cell wall and are enclosed by an outer sheath.1 subspecies.8 These other organisms are T pallidum subspecies
These gram-negative, microaerophilic bacteria exhibit a char- pertenue, the agent of yaws; T pallidum subspecies endemicum,
acteristic corkscrew flexion or motility. Diseases caused by the cause of nonvenereal endemic syphilis; and Treponema cara-
these organisms have many similarities, including a localized teum, the agent of pinta. Yaws is found in the tropics, pinta is
skin infection that disseminates to numerous organs as the dis- found in Central and South America, and endemic syphilis is
ease progresses, a latent stage, and cardiac and neurological in- found in desert regions.
volvement if the disease remains untreated. This chapter T pallidum (which will hereafter be used to refer to the sub-
discusses clinical manifestations and laboratory testing for the species pallidum) varies in length from 6 to 20 mm and in width
two major spirochete diseases, syphilis and Lyme disease, and from 0.1 to 0.2 mm, with 6 to 14 coils (Fig. 21–1).1,9 The outer
provides an introduction to relapsing fever, a more recently membrane of T pallidum is a phospholipid bilayer with very few
recognized spirochete infection. Serological testing plays a key exposed proteins. Several identified membrane proteins, called
role in diagnosis of these diseases because isolation of the or- treponemal rare outer membrane proteins (TROMPs), have been
ganisms themselves is difficult to accomplish in the laboratory characterized.8 It appears that the scarcity of such proteins
and clinical symptoms are not always apparent. delays the host immune response.

Syphilis Mode of Transmission

Pathogenic treponemes are rapidly destroyed by heat, cold,
Syphilis is the most commonly acquired spirochete disease in and drying, so they are almost always spread by direct contact.
the United States.2,3 It is typically spread through sexual trans- Sexual transmission is the primary mode of dissemination;
mission. Although the incidence of syphilis in the United States this occurs through contact of abraded skin or mucous mem-
reached an all-time low of 2.2 cases per 100,000 individuals branes with an open lesion. Approximately 30% to 50% of
in 2000,4 it slowly increased to 6.3 cases/100,000 people in the individuals who are exposed to a sexual partner with ac-
2014.3 Homosexual transmission between men was responsi- tive lesions will acquire the disease.9 Congenital infections
ble for much of this increase.3,5,6 In fact, in 2000 this group can also occur during pregnancy. Transmission to the fetus is
accounted for 6% of the syphilis cases in this country; in 2005, possible in mothers with clinically latent disease.
over 60% of syphilis cases occurred in this group. By 2012,
more than 80% of cases were within this group. Despite the
current emphasis on safe sexual practices, syphilis remains a
major health problem in many areas of the world; more than
10 million cases have been reported globally.7 Early detection
of syphilis is of major importance because treatment with an-
tibiotics in the early stages of the disease can usually provide a
cure. This section discusses characteristics of the organism that
causes syphilis, its clinical manifestations, and laboratory
methods essential to its diagnosis.

Characteristics of the Organism

The causative agent of syphilis is Treponema pallidum, sub-
species pallidum, a member of the family Spirochaetaceae. Or-
ganisms in this family have no natural reservoir in the Treponema pallidum. Electron micrograph showing
environment and must multiply within a living host. Three the coils and periplasmic flagella. (Courtesy of CDC.)
 Other potential means of transmission include parenteral Clinical Correlations
exposure through contaminated needles or blood, but this is
extremely rare. For the past 30 years, the lack of transfusion- The Great Imitator
transmitted syphilis in the United States has actually called into Patients with syphilis can be difficult to diagnose because their
question the necessity of testing potential donors for presence clinical presentations can vary widely. Because the symptoms
of the disease.10 However, current guidelines by the American of syphilis can mimic those of many other diseases or condi-
Red Cross require that people wait 12 months after treatment tions, the disease has often been referred to as “The Great
for syphilis before donating blood.11 Because syphilis can only Imitator.”
be transmitted by means of fresh blood products, the use of
stored blood components has virtually eliminated the possi-
bility of transfusion-associated syphilis.9
with secondary syphilis may exhibit neurological signs such as
visual disturbances, hearing loss, tinnitus, and facial weakness.9
Stages of the Disease Lesions persist from a few days up to 8 weeks and spontaneous
Untreated syphilis can progress through four stages: primary, healing occurs, as in the primary stage.
secondary, latent, and tertiary. Once contact has been made with The latent stage follows the disappearance of secondary
a susceptible skin site, endothelial cell thickening occurs with syphilis. This stage is characterized by a lack of clinical symp-
aggregation of lymphocytes, plasma cells, and macrophages.12 toms. It is arbitrarily divided into early latent (fewer than
The initial lesion, called a chancre, develops between 10 and 1 year’s duration) and late latent, in which the primary infec-
90 days after infection, with an average of 21 days.13 A chancre tion has occurred more than 1 year previously. Patients are
is a painless, solitary lesion characterized by raised and well- noninfectious at this time, with the exception of pregnant
defined borders (Fig. 21–2). In men these usually occur on the women, who can pass the disease on to the fetus even if they
outside of the penis, but in women they may appear in the exhibit no symptoms.
vagina or on the cervix and thus may go undetected. This pri- About one-third of the individuals who remain untreated
mary stage lasts from 1 to 6 weeks, during which time the lesion develop tertiary syphilis.9,13 This stage occurs most often
heals spontaneously. between 10 and 30 years following the secondary stage.9,13
About 25% of patients who are untreated in the primary Tertiary syphilis has three major manifestations: gummatous
stage progress to the secondary stage, in which systemic dissem- syphilis, cardiovascular disease, and neurosyphilis.
ination of the organism occurs. This stage is usually observed Gummas are localized areas of granulomatous inflamma-
about 1 to 2 months after the primary chancre disappears; tion that are most often found on bones, skin, or subcutaneous
however, in up to 15% of reported cases, the primary lesion tissue. Such lesions contain lymphocytes, epithelioid cells, and
may still be present.14 Symptoms of the secondary stage fibroblastic cells.14 They may heal spontaneously with scarring
include generalized lymphadenopathy, or enlargement of or they may remain destructive areas of chronic inflammation.
the lymph nodes; malaise; fever; pharyngitis; and a rash on the It is likely that they represent the host response to infection.
skin and mucous membranes.9,14 The rash may appear on the Cardiovascular complications usually involve the ascending
palms of the hands and the soles of the feet.9 Involvement of aorta and symptoms are caused by destruction of elastic tissue
the central nervous system (CNS) may occur earlier than pre- in the aortic arch.14 The destruction may result in aortic
viously suspected because viable organisms have been found aneurysm, thickening of the valve leaflets causing aortic regur-
in the cerebrospinal fluid (CSF) of several patients with pri- gitation, or narrowing of the ostia, producing angina pectoris.9
mary or secondary syphilis.2 Approximately 40% of patients Neurosyphilis is the complication most often associated with
the tertiary stage, but it actually can occur any time after the
primary stage and can span all stages of the disease. Immunod-
eficient individuals such as HIV patients are susceptible to early
neurosyphilis.15 During the first 2 years following infection, CNS

Connections
Gummas
A gumma is a form of granuloma characteristic of tertiary syphilis.
As discussed in Chapter 14, granulomas are organized clusters
of white blood cells (WBCs) and epithelial cells that are formed
as a result of a type IV hypersensitivity reaction. This cell-
mediated mechanism develops in response to chronic persist-
ence of the antigen. Granulomas can form in patients with
various infectious diseases, including leprosy, tuberculosis,
Primary chancre in the early stage of syphilis. cutaneous leishmaniasis, yaws, and syphilis.
(Courtesy of the CDC/Dr. N. J. Fiumara, Public Health Image Library.)
involvement often takes the form of acute meningitis. Late man- serological tests, and treponemal serological tests. These vary
ifestations of neurosyphilis include degeneration of the lower in their ability to detect syphilis at different stages of the
spinal cord with partial paralysis and chronic progressive de- disease. Principles and procedures of each type of testing are
mentia. It usually takes more than 10 years for these to occur; discussed in the text that follows. Special considerations in lab-
both are the result of structural CNS damage that cannot be oratory testing for neurosyphilis and congenital syphilis are
reversed. Fortunately, symptoms of tertiary syphilis are now also introduced.
very rare because of early detection and effective treatment with
antibiotics such as penicillin.9,13
Direct detection of spirochetes can be accomplished by dark-
field microscopy or fluorescent antibody testing. The perfor-
Congenital Syphilis mance of either test requires that the patient have active lesions.
Congenital syphilis occurs when a woman who has early Primary and secondary syphilis can
syphilis or early latent syphilis transmits treponemes to the be diagnosed by demonstrating the presence of T pallidum in
fetus. Due in large measure to a national plan launched by the exudates from skin lesions.2 In dark-field microscopy, a dark-
Centers for Disease Control and Prevention (CDC), the occur- field condenser is used to keep all incidental light out of the
rence of congenital syphilis dropped from 529 cases in the field except for that captured by the organisms themselves. It
year 2000 to 322 cases in 2012,16,17 despite increases in pri- is essential to have a good specimen in the form of serous fluid
mary and secondary syphilis. Although the disease can be from a lesion. The serous fluid is usually obtained by cleaning
transmitted at any stage of pregnancy, typically the fetus is the lesion with sterile saline and rubbing it with clean gauze.
most affected during the second or third trimester. Fetal or Pathogenic treponemes are identified on the basis of charac-
perinatal death occurs in approximately 10% of the cases.14 teristic corkscrew morphology and flexing motility.9
Infants who are liveborn often have no clinical signs of dis- Because observation of motility is the key to identification,
ease during the first few weeks of life. Some may remain specimens must be examined as quickly as possible before they
asymptomatic, but between 60% and 90% of these infants de- dry out. False-negative results can occur when there is a delay
velop later symptoms if not treated at birth.18 Such infants may in evaluating the slides, an insufficient specimen, or pretreat-
exhibit clear or hemorrhagic rhinitis, or runny nose. Skin erup- ment of the patient with antibiotics.9 Thus, a negative test does
tions, in the form of a maculopapular rash that is especially not exclude a diagnosis of syphilis. In addition, an experienced
prominent around the mouth, the palms of the hands, and the microscopist should perform testing. If a specimen is obtained
soles of the feet, are also common.19 Other symptoms include from the mouth or the rectal area, morphologically identical
generalized lymphadenopathy, hepatosplenomegaly, jaundice, nonpathogenic microbes can be found that must be differenti-
anemia, painful limbs, and bone abnormalities.2,9,16 Neu- ated from the true pathogens.
rosyphilis may occur in up to 60% of infants with congenital The use of a fluorescent-labeled
disease.20 antibody is a sensitive and highly specific alternative to dark-
field microscopy. Testing can be performed by either a direct
Nature of the Immune Response method, which uses a fluorescent-labeled antibody conjugate to
T pallidum, or an indirect method using antibody specific for
The primary body defenses against treponemal invasion are in-
T pallidum and a second labeled anti-immunoglobulin antibody.2
tact skin and mucous membranes. Once the skin is penetrated,
An advantage of these methods is that live specimens are not
T cells and macrophages play a key role in the immune
required. A specimen can be brought to the laboratory in a
response. Primary lesions show the presence of both CD4+
capillary tube and fixed slides can be prepared for later viewing.
and CD8+ T cells. Cytokines produced by these cells activate
Treponemes can be washed off the slide even after fixing; there-
macrophages; it is ultimately macrophage phagocytosis that
fore, each slide must be handled individually and rinsing must
heals the primary chancre.14 The protective role of antibodies is
be carefully done.2 The use of monoclonal antibodies has made
uncertain, however, as coating the treponemes with antibodies
fluorescent antibody testing very sensitive and specific.2 How-
does not necessarily bring about their destruction.8 T pallidum
ever, monoclonal antibodies can still cross-react with other sub-
is also capable of coating itself with host proteins, which delays
species of T pallidum, which must be taken into account when
the immune system’s recognition of the pathogen.9 The rare tre-
making a diagnosis.
ponemal proteins, or TROMPS, are important in triggering the
activation of complement, which ultimately kills the organism.8
However, the chronic nature of the disease is an indicator If a patient does not have active lesions, as may be the case in
that the organisms are able to evade the immune response. secondary or tertiary syphilis, then serological testing for anti-
Treponemes may persist in the host for years if antibiotic therapy bodies is the key to diagnosis. Serological tests can be classified
is not obtained. as nontreponemal or treponemal, depending on the reactivity
of the antibody that is detected. Nontreponemal tests have tra-
ditionally been used to screen for syphilis because of their high
Laboratory Diagnosis sensitivity and ease of performance. However, false-positive re-
Traditional laboratory tests for syphilis can be classified into sults are common because of the nonspecific nature of the anti-
three main types: direct detection of spirochetes, nontreponemal gen. Therefore, any positive results must be confirmed by
 Connections plasma reagin (RPR) test. These tests are based on floccula-
tion reactions in which patient antibody complexes with the
Complement Fixation cardiolipin antigen. Flocculation is a specific type of precipi-
The first nontreponemal serological test for syphilis was devel- tation that occurs over a narrow range of antigen concentra-
oped in 1906 by the bacteriologist August Paul von Wasser- tions. The antigen consists of very fine particles that clump
mann. This test used a crude liver extract from a fetus that was together in a positive reaction.
infected with syphilis as the source of the lipid antigen. The Typical serological results for nontreponemal tests during
Wasserman test was based on the principle of complement fix- the course of untreated and treated syphilis are shown in
ation. Patient serum was incubated with cardiolipin antigen in Figure 21–3. In general, nontreponemal tests are positive within
the presence of rabbit serum as the source of complement; this 1 to 4 weeks after the appearance of the primary chancre.2
was followed by a detection system consisting of antibody-
Titers usually peak during the secondary or early latent stages.
coated sheep red blood cells (RBCs). If the patient serum con-
In primary disease, between 13% and 41% of individuals ap-
tained cardiolipin antibody, complexes were formed that bound
the reagent complement and the indicator RBCs were not lysed. pear nonreactive; however, by the secondary stage almost all
In contrast, if cardiolipin antibody was not present in the patient patients have reactive test results.2 However, testing of sera
serum, the reagent complement was free to react with the from patients in the secondary stage is subject to false negatives
antibody-sensitized sheep RBCs to cause hemolysis. because of the prozone phenomenon (antibody excess). In this
case, a nonreactive pattern that is typically granular or rough
in appearance is seen.2 If a prozone is suspected, serial two-
fold dilutions of the patient’s sera should be made to obtain
a more specific treponemal test, which detects antibodies to a titer.
T pallidum. Cardiolipin antibody titers tend to decline in the later stages
Nontreponemal tests determine the of the disease, even if the patient remains untreated. After several
presence of an antibody that forms against cardiolipin, a lipid years, about 25% of untreated syphilis cases become nonreactive
material released from damaged cells. This antibody has some- for reagin.9 This decline occurs more rapidly in individuals who
times been referred to as reagin. It is found in the sera of patients have received treatment. A first-time infection, if in the primary
with syphilis and several other disease states. An antigen com- or secondary stage, should show a four-fold decrease in titer by
plex consisting of cardiolipin, lecithin, and cholesterol is used the third month following treatment and an eight-fold decrease
in the reaction to detect the nontreponemal reagin antibodies, by 6 to 8 months.14 Following successful treatment, tests typi-
which are either of the IgG or IgM class. cally become completely nonreactive within 1 to 2 years.
The term reagin as it applies to syphilis should not be con- The VDRL test, which was designed by the Venereal Disease
fused with the same word that was originally used to describe Research Laboratories, is both a qualitative and quantitative
IgE. They are not the same. Fortunately, the term reagin in slide flocculation test for serum that includes a modification
reference to IgE is rarely used today. for use on spinal fluid.21 Antigen for all tests must be prepared
The most widely used nontreponemal tests are the Venereal fresh daily and in a highly regulated fashion. The antigen is an
Disease Research Laboratory (VDRL) test and the rapid alcoholic solution of 0.03% cardiolipin, 0.9% cholesterol, and

Treponemal antibody test

(TP-PA, FTA-ABS, EIA/CLIA)
Percentage of patients who test positive

100

80

60

Untreated
40 Nontreponemal
antibody test
(RPR, VDRL)
20
Treated

2 4 6 8 10 12 2 10 20
Typical nontreponemal and tre- Weeks Years
Stage of Syphilis
ponemal antibody patterns in syphilis. Adapted
from Peeling RW, Ye H. Bulletin of the World Time of Primary Secondary Latent Tertiary
Health Organization 2004; 82(6): 439-446. infection (asymptomatic)
0.21% lecithin. The antigen suspension is prepared by adding NO. Test Card 18mm Circle
the VDRL antigen with a dropper to a buffered saline solution
while continuously rotating the mixture on a flat surface; at-
tention must be paid to required rotation speed and timing. A
daily calibrated Hamilton syringe is used to deliver one drop
1 2 3 4 5
of antigen for the slide test. If the delivery is off by more than Reactive Weak reac. Nonreactive
2 drops out of 60, the syringe must be cleaned with alcohol
and recalibrated.
The serum specimens to be tested are heated at 56°C for
30 minutes to inactivate complement, after which 0.05 mL is
pipetted into a ceramic ring of a glass slide. Three control 6 7 8 9 10
Patient-undil 1:2 1:4 1:8 1:16
sera—nonreactive, minimally reactive, and reactive—are pipet- RPR test results. Well 1: reactive control, showing
ted into separate rings on the glass slide in the same manner. large clumps. Well 2: weakly reactive control, showing small clumps.
Sera and patient samples are spread out to fill the entire ring. Well 3: nonreactive control, showing slight roughness and a “tail”
One drop (1/60 mL) of the VDRL antigen is then added to each upon swirling. Wells 6 to 10 show results of a serially diluted sample
ring. The slide is rotated for 4 minutes on a rotator at 180 rpm. of patient serum with a titer of 8. (Linda Miller.)
It is read microscopically to determine the presence of floccu-
lation, or small clumps. The results are recorded as reactive
Two main types of manual treponemal tests are the indirect
(medium to large clumps), weakly reactive (small clumps), or
fluorescent treponemal antibody absorption (FTA-ABS) test
nonreactive (no clumps or slight roughness).21 Tests must be
and agglutination tests. Because these tests are highly specific
performed at room temperature within the range of 23°C to
for syphilis, they have been used to confirm positive nontre-
29°C (73°F to 85°F) because results may be affected by tem-
ponemal test results. More recently, automated immunoassays
perature changes. All sera with reactive or weakly reactive
for treponemal antibodies have been developed. Their applica-
results must be tested using the quantitative slide test, in which
tions will be discussed later.
two-fold dilutions of serum ranging from 1:2 to 1:32 are
The FTA-ABS test is one of the earliest confirmatory tests. In
initially used. Sera yielding positive results at the 1:32 dilution
this test, a dilution of heat-inactivated patient serum is incubated
are titered further.
with a sorbent consisting of an extract of nonpathogenic tre-
The RPR test is a modified VDRL test involving macro-
ponemes (Reiter strain), which removes antibodies that cross-
scopic agglutination. The cardiolipin-containing antigen sus-
react with treponemes other than T pallidum. Diluted patient
pension is bound to charcoal particles; this makes the test
samples and controls are applied to individual wells on a test slide
easier to read. The suspension is contained in small glass vials,
fixed with the Nichols strain of T pallidum. Following a 30-minute
which are stable for up to 3 months after opening. The antigen
incubation at 37°C, the slides are washed and air-dried and
is similar to the VDRL antigen with the addition of ethylene-
antibody conjugate (anti-human immunoglobulin conjugated
diaminetetraacetic acid (EDTA), thimerosal, and choline chlo-
with fluorescein) is added to each well. Slides are re-incubated
ride, which stabilize the antigen and inactivate complement
as before and washed to remove excess conjugate. Mounting
so that serum does not have to be heat-inactivated before
medium is applied and coverslips are placed on the slides. They
use. Patient serum (approximately 0.05 mL) is placed in an
are then examined under a fluorescence microscope.
18-mm circle on a plastic-coated disposable card using a
If specific patient antibody is present, it will bind to the T pal-
capillary tube or Dispenstir device. Antigen is dispensed from
lidum antigens. The antibody conjugate will, in turn, only bind
a small plastic dispensing bottle with a calibrated 20-gauge
where patient immunoglobulin is present and bound to the
needle. One free-falling drop is placed onto each test area and
spirochetes. When slides are read under a fluorescence micro-
the card is mechanically rotated under humid conditions.2
scope, the intensity of the green color is reported on a scale of
Cards are read under a high-intensity light source; if floccula-
0 to 4+. No fluorescence indicates a negative test, whereas a re-
tion is evident, the test is positive (Fig. 21–4). All reactive
sult of 2+ or above is considered reactive.2 A result of 1+ means
tests should be confirmed by retesting using doubling dilu-
that the specimen was minimally reactive and the test must be
tions in a quantitative procedure. The RPR test appears to be
repeated with a second specimen drawn in 1 to 2 weeks.2 Expe-
more sensitive than the VDRL in primary syphilis.9
rienced personnel are needed to read and interpret fluorescent
Treponemal tests detect antibody directed test results. The FTA-ABS is highly sensitive and specific, but it
against the T pallidum organism or against specific treponemal is time consuming to perform and has been replaced in many
antigens. (See Figure 21–3 for typical treponemal antibody results laboratories with particle agglutination methods.
during various stages of syphilis.) Treponemal tests usually be- The particle agglutination (PA) tests originally used
come positive before nontreponemal tests, although patients with sheep RBCs coated with T pallidum antigen and were referred
early primary syphilis may be nonreactive.14 In secondary and to as MHA-TP (microhemagglutination assay for T pallidum
latent syphilis, tests are usually 100% reactive. Once a patient is antibody). Current PA tests for T pallidum, such as the Serodia
reactive, that individual remains so for life. Although there are T pallidum particle agglutination (TP-PA) test, use colored
fewer false positives compared with reagin tests, reactivity is seen gelatin particles coated with treponemal antigens and are more
with other treponemal diseases, notably yaws and pinta.9 sensitive in detecting primary syphilis.14,22 In the Serodia TP-PA
test, patient serum or plasma is diluted in microtiter plates
and incubated with either T pallidum-sensitized gel particles
or unsensitized gel particles as a control. Presence of T pal-
lidum antibodies is indicated by agglutination of the sensitized
gel particles, which form a latticelike structure that spreads to
produce a smooth mat covering the surface of the well. If a sam-
Patient serum with anti-treponemal antibodies
ple is negative for the antibody, the gel particles settle to the
bottom of the well and form a compact button (Fig. 21–5).
A variety of automated immunoassays have been developed
for the detection of antibodies to T pallidum. These include en-
zyme immunoassays (EIAs), chemiluminescent immunoassays
(CLIA), and multiplex flow immunoassays (MFI). EIAs have
been manufactured in a variety of formats, including one- or
two-step sandwich assays, one-step competitive assays, and
immune capture assays.23,24 In the sandwich assays, antibodies
in the patient sample bind to recombinant T pallidum antigens
coated onto microtiter plate wells. An enzyme-labeled antibody
or antigen conjugate and substrate are added to detect bind- Anti-human IgG/M
ing. In the immune capture format, microtiter wells are coated
with antibody to IgM or IgG and are reacted with patient
serum. Antigens that are labeled with an enzyme are then
added (Fig. 21–6). The capture EIA tests are especially useful
in diagnosing congenital syphilis in infants because they look
for the presence of IgM, which cannot cross the placenta. They
can also be used in monitoring response to therapy in the early Enzyme-labeled treponemal antigen
stages of syphilis because many patients are negative for IgM
treponemal antibodies 6 to 12 months after treatment.23 In
competitive EIAs, treponemal antibody in the patient sample
competes with an enzyme-labeled treponemal antibody con-
jugate for T pallidum antigens bound to microtiter plate wells.24
In a comparative study of several EIAs, test sensitivities ranged
from 94.7% to 99.1% and test specificities were determined to
be 100% for all of the assays evaluated.23,24
CLIAs are available as a one-step sandwich technique in
which the patient sample is incubated with paramagnetic mi-
croparticles that have been coated with T pallidum antigens

Substrate

TP-PA test results. Row A: positive control. Row B:

negative control. Row C: serum from a positive patient. T pallidum-
sensitized gel particles were placed in column 3 of each row and
unsensitized gel particles were pipetted into column 4 of each row.
Positive wells (A3 and C3) are indicated by a diffuse mat of particles Antibody capture enzyme-linked immunosorbent
that spread over the surface of the well, whereas negative results are assay (ELISA) test. Only specific anti-treponemal antibody will react
indicated by a compact button. (Linda Miller.) with enzyme-labeled antigen.
linked to a chemiluminescent derivative.23,24 After a wash step used as confirmatory tests to distinguish false-positive from
to remove unbound material, a catalyst is added and a chemical true-positive nontreponemal results. They also help establish a
reaction occurs, producing emissions of light if the test sample diagnosis in late latent syphilis or late syphilis because they are
is positive. The number of relative light units (RLUs) is propor- more sensitive than nontreponemal tests in these stages.2
tional to the amount of treponemal antibody in the sample. The traditional testing algorithm for
CLIAs have many advantages as compared with EIAs, including syphilis involves screening the sample with a nontreponemal
a higher sensitivity in the early stages of syphilis, faster perfor- test and confirming any positive results with a more specific
mance, and more stable reagents.23 treponemal test (Fig. 21–7). This testing strategy is recom-
MFI involves incubation of the patient sample with micro- mended by the CDC.27
spheres coated with recombinant T pallidum antigens. Micro- Because of the development of sensitive, automated meth-
spheres that have bound immune complexes are detected after ods for treponemal antibodies that can be easily performed in
addition of a phycoerythrin-labeled reporter antibody and are the clinical laboratory, a change in the testing strategy for
analyzed by flow cytometry. This method can simultaneously syphilis has been proposed. The newer reverse sequence algo-
detect antibodies to multiple T pallidum antigens in a small vol- rithm method is becoming increasingly popular, especially
ume of sample and has a rapid turn-around time.25 A study among large reference laboratories.28 Under this scheme, the
comparing MFI and several treponemal EIAs to the FTA-ABS testing order is reversed from the traditional algorithm in that
found a 95.4% to 98.4% agreement in test performance.26 patient samples are screened by an automated treponemal im-
Nontreponemal tests are sensitive, in- munoassay and positive results are confirmed by a nontrepone-
expensive, and simple to perform. Thus, they have been very mal test. This algorithm has several advantages over the
useful as a screening tool for syphilis. In addition, because an- traditional algorithm. The first advantage is cost. Automated
tibody titers can be determined by testing serial dilutions of testing can be performed on LIS-interfaced high-throughput
the patient sample, these methods have also been useful in analyzers as opposed to labor-intensive manual methods, sav-
monitoring the progress of the disease and in determining the ing time and reducing errors.29 Secondly, this algorithm can
outcome of treatment. Their main disadvantage is that they are potentially detect more early, late, and treated syphilis cases
subject to false positives. Transient false positives occur in dis- because of the higher sensitivity of the specific treponemal
eases such as hepatitis, infectious mononucleosis, varicella, tests. In the traditional algorithm, these may be missed because
herpes, measles, malaria, and tuberculosis, as well as during testing stops with a negative nontreponemal test result and the
pregnancy.2 Chronic conditions causing sustained false-positive treponemal specific test is not run.
results include systemic lupus erythematosus (SLE), leprosy, In the reverse algorithm, if the initial assay is negative, no
intravenous drug use, autoimmune arthritis, advanced age, and further testing is done unless early syphilis is suspected
advanced malignancy.2,14 (i.e., before seroconversion). If the automated assay result is
A reactive nontreponemal test should be confirmed by a positive and the subsequent RPR is positive, then the results are
more specific treponemal test. In pregnancy, this is especially considered positive for syphilis. However, discrepant results can
important because nontreponemal titers from a previous be obtained in some cases, with the initial automated test result
syphilis infection may increase nonspecifically.2,9 Titers can be being positive and the RPR that follows giving a negative result.
considered to be nonspecifically increased if lesions are absent, This combination of results can be problematic because it could
the increase in titer is less than four-fold, and documentation be caused by a false-positive treponemal antibody test result,
of previous treatment is available.2 a past syphilis infection, or early primary syphilis, in which
Although treponemal tests are usually reactive before non- patients have not yet produced nontreponemal antibodies.
treponemal tests in primary syphilis, they suffer from a lack of To help distinguish between these possibilities, the CDC rec-
sensitivity in congenital syphilis and neurosyphilis. Nontre- ommends that if laboratories choose to use the reverse algo-
ponemal tests should be used for these purposes.2,9 Treponemal rithm, all discrepant results should be tested reflexively using
tests are more difficult to perform, and have been traditionally the TP-PA test as a secondary confirmatory treponemal

Result: nonreactive
Interpretation: negative for syphilis
Action: no further testing required
Initial screen with a Result: nonreactive
nontreponemal test Interpretation: biological false positive;
(e.g., RPR, VDRL) negative for syphilis
Result: reactive
Interpretation: possible syphilis
Action: perform a treponemal test for
confirmation (e.g., TP-PA, FTA-ABS)
Result: reactive
Interpretation: positive for syphilis
(untreated or recently
treated case)

Traditional testing algorithm for syphilis.

test.29,30 If the TP-PA is positive, then late or latent syphilis or Chapter 12). One variation is real-time PCR, which is auto-
previous history of syphilis would be considered. If negative, mated, faster, and more sensitive than traditional PCR.
then it would be considered negative for syphilis at the time PCR is an extremely sensitive technique capable of detecting
of testing. Careful evaluation of the patient’s history should as little as one treponeme in some clinical samples.31 Sensitivity
be considered regarding possible reevaluation at a later date is highest in patients with primary syphilis, but is greatly re-
(Fig. 21–8). duced in detecting disease in secondary syphilis. 29,32,33 Clinical
availability of PCR is currently limited; however, in the future
PCR could be a useful tool for diagnosis when serological test-
ing is inconclusive and may provide a viable alternative to
PCR technology, which involves isolating and amplifying a spe-
dark-field microscopy in directly detecting the organism in ul-
cific sequence of DNA, has been used to test for the presence
cers from patients with primary disease.29,32,33 PCR may also
of treponemes in whole blood, spinal fluid, amniotic fluid, var-
be helpful in detecting treponemes in the blood of neonates
ious tissues, and swab samples from syphilis lesions. Although
with symptoms of congenital syphilis and in the CSF of
there are many variations of this procedure, DNA is basically
patients suspected of having neurosyphilis.33 Better standard-
extracted from the sample and then replicated using a DNA
ization of PCR may help the method to gain more widespread
polymerase enzyme and a primer pair to start the reaction (see
use in testing for syphilis in the future.

EIA or CIA Nontreponemal tests for congenital

syphilis performed on cord blood or neonatal serum detect the
IgG class of antibody in addition to IgM.34 It is difficult to dif-
ferentiate passively transferred IgG maternal antibodies from
those produced by the neonate, so there are problems in es-
EIA or CIA EIA or CIA tablishing a definitive diagnosis. Late maternal infection may
! " result in a nonreactive test because of low levels of fetal anti-
body. Additionally, testing the infant’s spinal fluid for the pres-
ence of treponemes often lacks sensitivity.35 Nontreponemal
titers in the infant that are higher than those in the mother may
Quantitative RPR be a good indicator of congenital disease, but this does not
or other always occur.2
nontreponemal test
Several approaches have focused on detecting IgM antibod-
ies in the infant. An FTA-ABS test for IgM alone lacks sensitivity
and the test is subject to interference because of the presence
of rheumatoid factor.35 However, an IgM capture assay is more
sensitive and a Western blot assay (see Chapter 24 for details)
RPR RPR
! "
using four major treponemal antigens has demonstrated a high
Syphilis sensitivity and specificity.2
(past or present) Currently, it is recommended that in high-risk populations,
nontreponemal tests be performed on both the mother and in-
TP-PA fant at birth, regardless of previously negative maternal tests.
Because symptoms are not always present at birth, if congenital
syphilis is suspected because of maternal history, tests should
be repeated on infant serum within a few weeks.18 If infection
is present in the infant, the titer will remain the same or will
increase. The Western blot test is recommended to confirm
TP-PA TP-PA
! " congenital syphilis.9
Syphilis Syphilis unlikely
(past or present) CSF is typically tested to determine
whether treponemes have invaded the CNS. Such testing is
usually more reliable if CNS symptoms are present. The VDRL
EIA/CIA = enzyme immunoassay/chemiluminescence immunoassay;
TP-PA = treponema pallidum particle agglutination.
test and some of the newer ELISA tests are the only ones rou-
tinely used for the testing of spinal fluid.2,9 For VDRL spinal
CDC-recommended algorithm for reverse sequence
syphilis screening (treponemal test screening followed by nontre-
fluid testing, the antigen volume used is less than the serum
ponemal test confirmation). Despite these recommendations for test and is at a different concentration. In addition, different
reverse sequence screening, CDC continues to recommend the slides are used (Boerner agglutination slides). The test is read
traditional algorithm with reactive nontreponemal tests confirmed microscopically as in the VDRL serum test. If a test is reactive,
by treponemal testing. (Adapted from Centers for Disease Control and two-fold dilutions are made and retested following the same
Prevention, reference [33].) protocol.
 A positive VDRL test on spinal fluid is diagnostic of neu- 41 kDa protein.43 Although this is usually not a problem in the
rosyphilis because false positives are extremely rare.9 However, current diagnostic scheme of testing, it can become an issue
sensitivity is lacking because samples from fewer than 70% of when these individuals become ill with certain viruses that are
patients with active neurosyphilis give positive results.9,14 If a known polyclonal B-cell activators (such as Epstein-Barr virus).
negative test is obtained, other indicators such as increased In these cases, this normally low-level antibody becomes high
lymphocyte count and elevated total protein (45 mg/dL) are enough to cause biological false positivity.
used as signs of active disease.12 PCR has been advocated in The organism divides by binary fission approximately every
diagnosing neurosyphilis and may play an important role in 12 hours. It can be cultured in the laboratory in a complex liq-
CSF testing in the future.9,36 uid medium (Barbour-Stoenner-Kelly) at 33°C, but it is difficult
to isolate from patients. The spirochetemia is short-lived and
generally found only early on in illness.40 Cultures often must
Lyme Disease be incubated for 6 weeks or longer to detect growth and are
therefore of little diagnostic utility.1
Lyme disease was first described in the United States in 1975
The main reservoir host is the white-footed mouse (Peromyscus
when an unusually large number of cases of juvenile arthritis
leucopus), although in California and Oregon the spirochete is
appeared in a geographically clustered rural area around Old
also harbored by the dusky-footed woodrat.41 Vectors are sev-
Lyme, Connecticut (hence the disease name), in the summer and
eral types of Ixodes ticks: Ixodes scapularis in the Northeast and
fall. Two mothers recognized this and brought it to the
Midwest United States (Fig. 21–9), Ixodes pacificus in the West,
attention of health officials. Because of the epidemiological
Ixodes ricinus in Europe, and Ixodes persulcatus in Asia. White-
features of this newly described “Lyme arthritis,” transmission
tailed deer are the main host for the tick’s adult stage. Nymphs
by an arthropod vector was suggested.37 In 1982, the agent was
and adult stages of the tick can transmit the disease. The peak
isolated and identified as a new spirochete. It was given the name
feeding is in the late spring, early summer, and the fall, which
Borrelia burgdorferi after Willy Burgdorfer (first author in the
corresponds to the peak biphasic occurrence of Lyme disease.1
original description).38 The clinical features of Lyme disease were
The tick must feed for a period of time before the spirochete
soon recognized to extend beyond arthritis; it is now known to
can be transmitted. Most agree that the risk for transmission is
be a multisystem illness involving the skin, nervous system,
very low when ticks have fed for fewer than 36 hours; one
heart, and joints. Lyme disease is the most common vector-borne
study found that transmission is still low at 72 hours.42
disease in the United States; over 36,307 confirmed or probable
cases were reported in 2013. The number of cases being re-
ported per year has doubled since 1991. According to the CDC, Stages of the Disease
this reflects both a true increase in the frequency of the disease Lyme disease resembles syphilis in that manifestations occur
as well as better recognition and reporting.39 in several stages. These have been characterized as (1) localized
rash, (2) early dissemination to multiple organ systems, and
Characteristics of the Organism (3) a late disseminated stage often including arthritic symp-
toms.44,45 These stages are not always sharply delineated; there-
Several species of Borrelia are known to be the causative agents fore, it may be easier to view Lyme disease as a progressive
of Lyme disease. In North America, it is exclusively B burgdorferi infectious disease that involves diverse organ systems.
sensu stricto, whereas in Europe several species are known to cause The clinical hallmark of early infection is the rash known
Lyme disease (Borrelia afzelii, Borrelia garinii, Borrelia sensu stricto, as erythema migrans (EM), which appears between 2 days and
and occasionally other Borrelia species).40,41 All share similar char-
acteristics; for simplicity, they will be referred to as B burgdorferi.
The organism is a loosely coiled spirochete, 5 to 25 mm long and
0.2 to 0.5 mm in diameter.1,40 The outer membrane, which con-
sists of glycolipid and protein, is extremely fluid and only loosely
associated with the organism. Several important lipoprotein anti-
gens, labeled OSP-A through OSP-F, are located within this struc-
ture and are actually encoded by plasmids.41 Surface proteins
allow the spirochetes to attach to mammalian cells.
Just underneath the outer envelope are 7 to 11 endoflagella
or periplasmic flagella. These run parallel to the long axis of the
organism and are made up of 41 kDa subunits that elicit a
strong antibody response. This immunodominant characteris-
tic is of diagnostic importance because the response is not only
strong but is also very early. Unfortunately, the flagellin subunit
has homology to that of other nonpathogenic and pathogenic
spirochetes, notably B recurrentis and T pallidum, causing cross- Adult tick I scapularis, which transmits Lyme disease.
reactivity in serological testing.1,42 Because of this, a large num- (Courtesy of the CDC/Michael L. Levin, PhD, and Jim Gathany, Public
ber of uninfected people have low levels of antibodies to the Health Image Library.)
2 weeks after a tick bite.46 EM begins as a small red papule arthritis has been associated with particular HLA–DRB alleles.41,44
where the bite occurred, then rapidly expands to form a large Despite resolution of objective manifestations of Borrelia infec-
ringlike erythema and often a central area that exhibits partial tion after antibiotic treatment, a small percentage of patients
clearing (Fig. 21–10). The clinical diagnosis of early Lyme dis- develop chronic fatigue, concentration and short-term memory
ease relies on the recognition of this characteristic rash, which problems, and musculoskeletal pain.52 These symptoms can last
should be at least 5 cm in diameter. At this stage, the patient may longer than 6 months in some cases.
be asymptomatic or have nonspecific flu-like symptoms.47-49
The EM usually continues to expand for over a week; even if Nature of the Immune Response
untreated, it gradually fades within 3 to 4 weeks. Unfortunately,
approximately 20% of patients do not develop the rash.45,47,48 The immune response in Lyme disease is highly variable and
At this early stage, the antibody response is minimal and most complex. A well-documented humoral and cellular response
serologies are negative. is known to exist. Spirochete lipoproteins also trigger produc-
Early dissemination occurs via the bloodstream in the days tion of macrophage-derived cytokines, which further enhance
to weeks following the EM rash. The skin, nervous system, heart, the immune response.41 However, the clinical effectiveness of
or joints may be affected. Approximately 10% to 15% of patients these responses is certainly questionable and not necessarily
will display multiple skin lesions.41 Migratory pain often occurs protective, because late Lyme disease occurs despite high levels
in the joints, tendons, muscles, and bones. If treatment is not of circulating antibody and cellular responses.
obtained, neurological or cardiac involvement develops in about
15% of patients within 4 to 6 weeks after the onset of infec- Laboratory Diagnosis
tion.41,50 The most prevalent neurological sign is facial palsy, a Diagnosis of Lyme disease is a clinical one, with laboratory testing
peripheral neuritis that usually involves one side of the face.41,50 used as supporting evidence. Unfortunately, the clinical diagnosis
Pain and weakness can occur in the limb that was bitten. Some is often difficult for the reasons previously discussed. If the
patients develop sleep disturbances, mild chronic confusional characteristic rash is present, this can be used as a presumptive
states, or difficulty with memory and intellectual functioning.45 finding, but as many as 20% of patients do not get or do not
An aseptic meningitis can also be seen.49 recognize the rash.45,46,48 Direct isolation of the organism from
Late Lyme disease may develop in some untreated patients skin scrapings, spinal fluid, or blood is possible, but the yield of
months to years after acquiring the infection.51 The major man- positive cultures is extremely low. Therefore, culture is not used
ifestations of late Lyme disease are arthritis, peripheral neuropa- as a routine diagnostic tool.
thy, and encephalomyelitis. These symptoms usually respond The antibody response is variable and may not be detectable
well to conventional antibiotic treatment, but treatment-resistant until 3 to 6 weeks after the tick bite. The IgM response occurs
first followed by the IgG response. The IgG response does not
peak until the third and fourth weeks of infection.46,47 These
antibody responses are also not mutually exclusive and can be
variable (e.g., an IgM response can occur in late Lyme disease).
In most cases of acute early Lyme disease (first 2 weeks), sero-
logical testing is too insensitive to be diagnostically helpful.51
If patients with symptoms are tested in fewer than 7 days after
infection, seropositivity is only about 30%.47,53 Therefore, the
decision to start treatment for early Lyme disease must be made
before seroconversion, similar to many acute infectious dis-
eases. However, untreated seronegative patients having symp-
toms for 6 to 8 weeks are unlikely to have Lyme disease and
other possible diagnoses should be pursued.44 Antibiotic ther-
apy begun shortly after the appearance of EM may delay or ab-
rogate the antibody response. In chronic Lyme disease, negative
serologies have been attributed to previous antibiotic therapy;
however, the scientific support of this theory is not strong.52
The CDC recommends a two-tiered approach to providing
laboratory support for the diagnosis of Lyme disease.54,55 It is rec-
ommended that patients with clinical evidence of Lyme disease
be screened with an IFA or EIA test. If this serology is positive or
borderline, a Western blot should be performed on that specimen
as supplemental testing. Lyme testing should not be performed
in the absence of supporting clinical evidence. A positive test per-
Erythema chronicum migrans rash, which appears formed under these circumstances has only a 6% positive pre-
after a tick bite in Lyme disease. (Courtesy of the CDC/Jim Gathany, dictive value (even when done in an endemic area), whereas it
Public Health Image Library.) rises to greater than 97% when clinical symptoms and history
are present and consistent with Lyme disease.55 Some of the cur- in high enough sensitivity and specificity to replace the two-
rent testing procedures are discussed next. tier method of testing.51 The specificity issues are generally
similar to the IFA assay; however, there are no clues (beaded
pattern) that a particular sample may be a false positive. As
The IFA was the first test used to evaluate the antibody re-
with IFA, false positives occur with syphilis and other trepone-
sponse in Lyme disease, followed by various forms of EIAs
mal diseases such as yaws and periodontal disease, as well as
shortly thereafter. The IFA assay is fairly easy to develop, which
relapsing fever and leptospirosis.1,57 If serum is absorbed to
is why it is usually the first assay on the market in many infec-
decrease cross-reactivity, this also decreases specific Lyme an-
tious disease arenas. Basically, doubling dilutions of patient
tibody titers. Additionally, patients with infectious mononu-
serum are incubated with commercially prepared microscope
cleosis, Rocky Mountain spotted fever, and other autoimmune
slides coated with antigen from whole or processed spiro-
diseases also have been known to be positive with EIA.53 Lyme
chetes. Following a wash step to remove unbound material, an
disease patients do not test positive with RPR, so this may be
anti-human globulin with a fluorescent tag attached is added
helpful if syphilis is in the differential diagnosis.53
and reacts with any specific antibody bound to the spirochetes
on the slide. After a second wash step, the slide is viewed under
a fluorescent microscope. Typically, a test result is only consid- Immunoblotting, or Western blotting, is used as a confirma-
ered positive if a titer of 1:256 or higher is obtained,53 although tory test for samples that initially test positive or equivocal by
this varies between manufacturers. As previously mentioned, EIA or IFA. It is the second test in the CDC-recommended two-
specimens obtained in the first few weeks are usually negative be- tier testing scheme for Lyme disease.46,57 Current CDC recom-
cause the level of antibody present is below the detection limit of mendations do not advise testing seropositive or borderline
this (and other) assays.53 As might be expected, other closely re- patients for IgM antibodies if they have had symptoms for
lated organisms such as B recurrentis (relapsing fever), T denticola more than 4 weeks for the reasons previously outlined. Sero-
and others (associated with periodontal disease), and T pal- logical evidence of Lyme disease in these patients is indicated
lidum (syphilis) may cross-react and cause biological false- by a positive result in the IgG immunoblot.46,53
positive results.42 Autoimmune connective tissue diseases such The Lyme disease immunoblot is very complex (Fig. 21–11)
as rheumatoid arthritis (RA) and SLE can also produce false and does not provide the same level of confidence as simpler
positives in the IFA assay for Lyme disease and the FTA assay systems such as HIV.58,59 In Lyme disease, the immunoblot is
for syphilis.56 An astute technologist can recognize a false pos- generally referred to as supplemental testing. The technique con-
itive by the beaded fluorescent pattern it produces. Reading of sists of electrophoresis of Borrelia antigens in an acrylamide gel
fluorescent patterns tends to be very subjective and requires
highly trained individuals. However, if performed correctly by
experienced personnel, the test can provide sensitive and ac-
curate results. This test is best suited for low-volume testing.

EIA testing is quick, reproducible (not subjective), relatively in-

expensive, and lends itself well to automation and high-volume
testing.53 Antigen preparations used in the assay include crude
sonicates of the organism, purified proteins, synthetic proteins,
and recombinant proteins. The manufacturer’s selected antigen
is then coated onto 96-well microtiter plates or strips by various
proprietary methods. Patient sera is added and allowed to incu-
bate with the antigen. After a washing step, anti-human globulin
conjugated with an enzyme tag such as alkaline phosphatase is
added to each well. Adding specific substrate produces a color
change. Plates are read in a spectrophotometer and the antibody
is quantitated based on color intensity. EIAs provide objective
results and the titer is based on a continuum range rather than
serial dilutions of patient sera. Thus, a more accurate measure-
ment of the specific antibody is possible.53
Similar to the IFA, drawbacks of EIA include a lack of sen-
sitivity during the early stages of Lyme disease and specificity
problems. The sensitivity for early serum specimens has been
reported to be anywhere from 58% to 92%.57 The differences
in using various antigens is usually manifested in trade-offs be-
tween increasing sensitivity at the expense of specificity versus
increasing specificity at the expense of sensitivity. Unfortu-
nately, the technical adaptations of the EIA have not resulted Immunoblot for Lyme disease.
and then transfer of the resulting pattern to nitrocellulose paper. of age when the tick can be reliably identified and treatment
This step is performed by the manufacturer and nitrocellulose can begin within 72 hours of tick removal. Macrolides are not
antigen strips are provided in the test kit. These strips are reacted recommended as a first-line therapy for early Lyme disease
with patient serum and developed with an anti-human globulin because they are less effective. Under rare circumstances, an
(either anti-IgG or anti-IgM) to which an enzyme label is individual with a tick bite may be fully treated: (1) if the tick
attached. The further incubation with the enzyme’s substrate was identified as a nymph or adult I scapularis; (2) if the tick
allows for visualization of any antibody that has bound to a par- was attached for more than 36 hours; (3) if treatment can be
ticular antigen. The reactivity is then scored and interpreted. started within 72 hours of tick removal; (4) if the local infection
Ten proteins are used in the CDC-recommended interpre- rate of ticks is over 20%; or (5) if doxycycline is not contraindi-
tation of this test. They are designated by their molecular cated.44 Neuroborreliosis requires the use of intravenous
weights: 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa.46 For antibiotic therapy.
a result to be considered positive for the presence of specific Currently there are no effective vaccines for humans. A
IgM antibody, two of the following bands must be present: human vaccine made with the OSP-A surface antigen has had
23(Osp C), 39, and 41 (flagellin) kDa.60,61 An IgG immunoblot limited usefulness, has been associated with side effects, and
is considered positive if any 5 of the 10 bands previously listed has been recalled from the market.64,65 There are renewed
are positive.60,61 Because of the complexity of the Lyme im- efforts to create a new vaccine, but as of this writing no
munoblots, testing and interpretation of blots should be done vaccines have been approved for clinical use.
only in qualified laboratories that follow CDC-recommended
evidence-based guidelines on immunoblot interpretations.62
Relapsing Fever Group—
In testing for Lyme disease, the PCR has found a niche in cer- Borrelia Miyamotoi
tain scenarios. Although only a few organisms need to be pres-
ent for detection under optimal conditions, the number of Relapsing fever is a relatively new disease, which was identified
spirochetes in infected tissues and body fluids is low, making by a reversal of the typical disease discovery pathway. Typically,
specimen collection, transport, and preparation of DNA critical discoveries of new infectious diseases start with the association
to the accuracy of the test results.51 of a group of patients with a similar illness for which there is
Several probes for target DNA that is present only in no known etiology. These findings, in turn, lead to efforts
strains of B burgdorferi have been made and used in PCR test- to isolate and define the causative organism. In the case of
ing.51,63 The procedure involves extracting DNA from the Borrelia miyamotoi, the exact opposite occurred. In 1995, a
patient sample followed by amplification using specific novel Borrelia species was isolated from an Ixodes tick in Japan.
primers, DNA polymerase, and nucleotides. Once a sufficient This new species subsequently was found in North America in
amount of the unknown DNA is made, this is combined with 2001 and in Eurasia in 2002. The discoveries confirmed the
a known DNA probe to see if hybridization takes place. The wide geographical distribution and the ecological niche of this
single-stranded Borrelia DNA probe will bind only to an new Borrelia species within the vectors of Lyme disease. These
exact complementary strand, thus positively identifying the findings then led to efforts using specific assays for B miyamotoi
presence of the organism’s DNA in the patient sample. This to identify patients hospitalized with flu-like illness and a his-
is much more specific than testing for antibody because there tory of a recent tick bite. Using these methods, a Russian group
is little cross-reactivity. Specificity of recent PCR studies has identified a group of 46 patients with B miyamotoi infection in
ranged from 93% to 100%. However, sensitivity remains 2011.66,67 This finding was confirmed in the United States by
problematic. In a series of studies, the median sensitivity of multiple studies in 2013.68,69
PCR on skin biopsies was 69%; of blood components, 14%;
of CSF, 38%; and of synovial fluid, 78%. However, the range Characteristics of the Organism
of sensitivities in any one type of specimen is quite large,
suggesting that testing remains to be standardized.9,51 Fur- B miyamotoi is closer phylogenetically to the relapsing fever
thermore, it would be hard to clinically justify a skin biopsy Borrelia group than it is to B burgdorferi. However, similar
for PCR as a diagnostic method for an EM rash in most cases. to B burgdorferi, it has at least one mammalian reservoir host;
However, in difficult diagnostic neurological and arthritic like B burgdorferi, the white-footed mouse is the preferred host
cases, PCR on CSF and synovial fluid is often employed. PCR in the Eastern United States. The infection prevalence of quest-
for Borrelia still has limited availability. ing nymphs in endemic areas is quite different from B burgdor-
feri. It is 20% to 50% in B burgdorferi and only 1% to 5% for
B miyamotoi. Unlike B burgdorferi, but similar to several other
Treatment relapsing fever Borrelia species, vertical transmission from the
Borrelia is sensitive to several orally administered antibiotics, female adult tick to its offspring occurs in addition to the hor-
including penicillins, tetracyclines, and macrolides.44,45 Pro- izontal transmission from the reservoir host to the tick.70 This
phylaxis, full-course treatment, or serological testing of all pa- transmission to offspring has possible implications for trans-
tients with tick bites is not recommended. A single dose of mission to humans because the extremely small tick larvae are
doxycycline may be offered to adults and children over 8 years potentially infectious.
Stages of the Disease • Syphilis can be separated into four main clinical stages:
Because this is a new disease, the disease characteristics are likely 1. The primary stage is characterized by the presence of a
to change as more experience with the disease is gained. Initial painless ulcer called a chancre at the site of initial contact.
studies suggest this is a flu-like illness. Unlike Lyme disease, an 2. An untreated patient may progress from the primary
EM rash, arthritis, and facial palsies are uncommon.67 The lack stage to the secondary stage, in which systemic dissem-
of a rash may, in fact, explain some of the documented “Lyme ination of the organism occurs and symptoms such as
cases” where no rash was observed. Upon infection of the generalized lymphadenopathy, malaise, sore throat, and
host, both B burgdorferi and B miyamotoi exhibit a short-lived skin rash appear.
bacteremia; however, B burgdorferi is at low density, whereas B 3. Disappearance of the secondary stage is followed by a
miyamotoi is a very high density bacteremia. The opposite occurs lengthy latent stage in which patients are usually free
in the skin infection: B burgdorferi is at high density and of clinical symptoms.
B miyamotoi is at low density.70 The conclusions drawn from this 4. About one-third of the individuals who remain un-
are that the infectious period for B miyamotoi appears to be re- treated develop tertiary syphilis. This late stage disease
stricted to the bacteremia stage, whereas this period for B burgdor- is characterized by three major clinical manifestations:
feri extends to the much longer skin involvement period. This granulomatous inflammation (gummas), cardiovascu-
may explain the lower infectivity rate of B miyamotoi in ticks. lar disease, and neurosyphilis. Early diagnosis and
treatment help to prevent later complications.
Laboratory Diagnosis • Direct laboratory diagnosis involves detecting the organ-
ism from a lesion and using dark-field microscopy, fluo-
The laboratory diagnosis of B miyamotoi infection is still in its rescence microscopy, or PCR. If an active lesion is not
infancy. At the time of this writing, all diagnostics are basically present, diagnosis must be made on the basis of serologi-
research and investigational only and there are no commercial cal tests.
FDA-approved kits. What follows is a brief description of some • Nontreponemal serological tests determine the presence
of the assays used to date; if history repeats, the commercial of antibody to cardiolipin, also known as reagin (e.g.,
counterparts will follow shortly. VDRL and RPR tests). These tests are fairly sensitive and
Direct detection by culture or PCR—Although B burgdorferi simple to perform; however, they lack specificity, so spec-
can be cultured, it is not useful as a diagnostic tool because imens with positive results must be confirmed with a more
it is a low yield procedure. Attempts to culture B miyamotoi specific treponemal antibody test.
in media have not been successful to date. However, if im- • Traditional treponemal antibody tests include the FTA-ABS
proved culture techniques are developed, it may be a viable and particle agglutination (TP-PA). These tests detect
diagnostic option because of the high density bacteremia. antibody formed against the organism itself. Treponemal
It is also possible that PCR testing of blood may become a tests are more specific and sensitive in early stages of the
feasible diagnostic method in the future.66,67 disease.
Antibody detection—The Russian study, which first found • Titers of treponemal antibodies remain detectable for life,
the disease associated with B miyamotoi infection, demon- whereas nontreponemal titers decline after successful
strated, not surprisingly, that these patients were positive treatment.
in the Lyme antibody assays.67 Thus, it is possible that • New developments in testing include EIA and CLIA tech-
some of the diagnosed Lyme patients could actually be nology and PCR. EIA and CLIA tests for antibody to spe-
B miyamotoi patients instead. The 2013 U.S. study that cific treponemal antigens and separation of antibodies by
confirmed B miyamotoi infection used an ELISA assay, class is possible. For large-volume testing, the EIA or CLIA
which detected antibodies against GlpQ proteins (made is commonly used as a screening test, followed by con-
from B hermsii). These antibodies are specific for the firmation with the RPR or VDRL (reverse screening
relapsing fever Borrelia and not B burgdorferi.69 algorithm).
• Lyme disease is the most common vector-borne infection
in the United States. The organism responsible is the
SUMMARY spirochete Borrelia burgdorferi, which is transmitted by the
deer tick.
• Syphilis and Lyme disease are the two major diseases • Although an expanding red rash is often the first symptom
caused by spirochetes. Spirochetes are distinguished by noted in Lyme disease, the disease can be characterized as
the presence of axial filaments that wrap around the cell a progressive infectious syndrome involving diverse organ
wall inside a sheath and give the organisms their charac- systems. Despite antibiotic treatment, a small percentage
teristic motility. of patients continue to have fatigue; concentration and
• Syphilis is caused by the organism Treponema pallidum, short-term memory problems; and musculoskeletal pain,
subspecies pallidum. The disease is acquired by direct con- which may last 6 months or longer. These symptoms may
tact, usually through sexual transmission. be caused by persistence of infection.
• The presence of IgM and IgG to B burgdorferi cannot usually • Borrelia miyamotoi relapsing fever group is the newest tick-
be detected until 3 to 6 weeks after symptoms initially borne disease. Clinical features are similar to Lyme disease
appear. Current testing protocol involves screening with and may explain some of the variations seen in “Lyme”
IFA or EIA and follow-up of equivocal or positive tests with disease. Antibodies present in this disease cross-react with
immunoblotting. All serological findings must be inter- some of the Lyme diagnostic tests. Much is still to be elu-
preted carefully and in conjunction with clinical diagnosis. cidated regarding this disease.

Study Guide: Comparison of Tests Used for the Diagnosis of Syphilis

TEST ANTIGEN ANTIBODY COMMENTS
Direct Microscopic
Dark-field T pallidum from patient None Requires active lesion; must have
good specimen, experienced
technologist; inexpensive
Fluorescent antibody T pallidum from patient Anti-treponemal Requires active lesion; more
antibody with specific than dark-field; speci-
fluorescent tag men does not have to be live
Nontreponemal
VDRL Cardiolipin Anti-cardiolipin Flocculation; good for screening
(Reagin) tests, treatment monitoring,
spinal fluid testing; false
positives are common
RPR Cardiolipin Anti-cardiolipin Modified VDRL with charcoal
(Reagin) particles; more sensitive than
VDRL in primary syphilis
Treponemal
FTA-ABS Nichols strain of T pallidum Anti-treponemal Confirmatory; specific, sensitive;
may be negative in primary
stage
Serodia TP-PA Gel particles sensitized with Anti-treponemal Not as sensitive as FTA-ABS
(formerly, MHA-TP) T pallidum sonicate (formerly
sensitized sheep RBCs)
EIA, CLIA, MFI Treponemal antigen Anti-treponemal Sensitive, automated testing pro-
vides objective results; used to
screen for syphilis in some
large laboratories; EIAs have
been developed as competitive,
sandwich, or capture im-
munoassays that can detect
IgM or IgG antibodies
PCR Nontreponemal DNA in None Highest sensitivity is in primary
patient sample is amplified stage syphilis; availability is
limited
CLIA = chemiluminescent immunoassay; DNA = deoxyribonucleic acid; EIA = enzyme immunoassay; FTA-ABS = fluorescent treponemal antibody absorption;
MFI = multiplex flow immunoassay; MHA-TP = microhemagglutination assay for antibodies to Treponema pallidum; PCR = polymerase chain reaction;
RPR = rapid plasma reagin; TP-PA = T pallidum particle agglutination; VDRL = Venereal Disease Research Laboratory.
Study Guide: Comparison of Tests for the Diagnosis of Lyme Disease
TEST ANTIGEN ANTIBODY COMMENTS
IFA Whole or processed Anti-Borrelia antibody from Initial test for Lyme disease;
B burgdorferi patient, anti-human globulin labor intensive to perform; false
with fluorescent tag positives; subjective;
EIA Sonicated B burgdorferi Anti-Borrelia antibody from Initial test for Lyme disease;
patient, anti-human globulin easy to perform; false positives;
with enzyme tag more sensitive than IFA
Purified flagellin protein Anti-flagellin antibody from Initial test for Lyme disease;
patient, anti-human globulin easy to perform; highly specific;
with enzyme tag sensitive in early Lyme disease
C6 peptide Conserved region of surface Easy to perform; highly specific;
lipoprotein (VlsE) sensitive in early and late Lyme
disease
Western blot or Antigens of B burgdor- Detects antibodies (IgG Technically difficult to perform;
immunoblot feri separated by or IgM) to individual scoring the blot can be
molecular weight B burgdorferi antigens challenging
PCR None. B burgdorferi None Availability is limited
DNA in patient sample
is amplified
DNA = deoxyribonucleic acid; EIA = enzyme immunoassay; IFA = immunofluorescence assay; PCR = polymerase chain reaction.

CASE STUDIES
1. A 30-year-old woman saw her physician to complain a baby boy who appeared to be normal. The physician
about repeated episodes of arthritislike pain in the knees obtained a blood sample from the mother for routine
and hip joints. She recalled having seen a very small tick screening. An RPR test performed on the mother’s serum
on her arm about 6 months before the development of was positive. The mother had no obvious signs of syphilis
symptoms. No rash was ever seen, however. Laboratory and denied any past history of the disease. She indicated
tests for RA and SLE were negative. An EIA test con- that she had never received any treatment for a possible
ducted on the patient’s serum for Lyme disease was syphilis infection. Cord blood from the baby also exhib-
indeterminate. ited a positive RPR result.
Questions Questions
a. Does the absence of a rash rule out the possibility of a. Is the baby at risk for congenital syphilis?
Lyme disease? b. What is the significance of a positive RPR on a cord
b. What might cause an indeterminate EIA test? blood test?
c. What confirmatory testing would help determine the c. How should these results be handled?
cause of the patient’s condition?

2. A mother who had no prenatal care appeared at the emer-

gency department in labor. The physician safely delivered
REVIEW QUESTIONS
1. False-positive nontreponemal tests for syphilis may 7. Which of the following is true of treponemal tests for
occur because of which of the following? syphilis?
a. Infectious mononucleosis a. They are usually negative in the primary stage.
b. Systemic lupus b. Titers decrease with successful treatment.
c. Pregnancy c. In large-volume testing, they are often used as
d. All of the above screening tests.
d. They are subject to a greater number of false posi-
2. In the fluorescent treponemal antibody absorption tives than nontreponemal tests.
(FTA-ABS) test, what is the purpose of absorption
with Reiter treponemes? 8. An RPR test done on a 19-year-old woman as part of a
a. It removes reactivity with lupus antibody. prenatal workup was negative but exhibited a rough
b. It prevents cross-reactivity with antibody to other appearance. What should the technologist do next?
T pallidum subspecies. a. Report the result out as negative.
c. It prevents cross-reactivity with antibody to b. Do a VDRL test.
nonpathogenic treponemes. c. Send the sample for confirmatory testing.
d. All of the above. d. Make serial dilutions and do a titer.

3. Which test is recommended for testing cerebrospinal 9. Treponemal EIA tests for syphilis are characterized by
fluid for detection of neurosyphilis? all of the following except
a. RPR a. they are adaptable to automation.
b. VDRL b. they are useful in monitoring antibody titers in
c. FTA-ABS syphilis patients undergoing therapy.
d. Enzyme immunoassay c. subjectivity in reading is eliminated.
d. they can be used to distinguish between IgG and
4. Advantages of direct fluorescent antibody testing to IgM antibodies.
T pallidum include all of the following except
a. reading is less subjective than with dark-field 10. Which of the following tests is the most specific
testing. during the early phase of Lyme disease?
b. monoclonal antibody makes the reaction very a. IFA
specific. b. EIA
c. slides can be prepared for later reading. c. Immunoblotting
d. careful specimen collection is less important than d. detection of B burgdorferi DNA by PCR
in dark-field testing.
11. False-positive serological tests for Lyme disease may
5. Which of the following is true of nontreponemal be caused by all of the following except
antibodies? a. shared antigens between Borrelia groups.
a. They can be detected in all patients with primary b. cross-reactivity of antibodies.
syphilis. c. resemblance of flagellar antigen to that of
b. These antibodies are directed against cardiolipin. Treponema organisms.
c. Nontreponemal tests remain positive after d. a patient in the early stage of the disease.
successful treatment.
d. The antibodies are only found in patients with 12. A 24-year-old man who had just recovered from
syphilis. infectious mononucleosis had evidence of a genital
lesion. His RPR test was positive. What should the
6. Which syphilis test detects specific treponemal technologist do next?
antibodies? a. Report out as false positive.
a. RPR b. Do a confirmatory treponemal test.
b. VDRL c. Do a VDRL.
c. FTA-ABS d. Have the patient return in 2 weeks for a repeat test.
d. Agglutination
13. A 15-year-old girl returned from a camping trip. 14. The reverse screening algorithm for syphilis testing
Approximately a week after her return, she a. is the CDC preferred algorithm.
discovered a small red area on her leg that had b. is more labor intensive than the “traditional” method.
a larger red ring around it. Her physician had her c. has a high number of false positives that must be
tested for Lyme disease, but the serological test resolved by doing a TP-PA test.
was negative. What is the best explanation for d. is more prone to transcription errors in reporting.
these results?
a. She definitely does not have Lyme disease. 15. Borrelia miyamotoi infection
b. The test was not performed correctly. a. may explain some cases of supposed Lyme disease
c. Antibody response is often below the level of where no rash was found.
detection in early stages. b. is a new lethal tick-borne disease.
d. Too much antibody was present, causing a false c. is carried by the common dog tick.
negative. d. is another name for Southern Tick Associated
Illness (STARI).
 Serological and
Molecular Diagnosis
of Parasitic and Fungal
Infections

After finishing this chapter, you should be able to: PARASITIC IMMUNOLOGY
1. Explain why a host has more difficulty overcoming parasitic diseases Immune Responses to Parasites
than those caused by bacteria or viruses. Parasite Survival Strategies
2. Discuss potential outcomes of host and parasite interactions. Serodiagnosis of Parasitic Diseases
3. Cite strategies used by parasites to evade host defenses. Molecular-Based Diagnosis of
4. Discuss the role of immunoglobulin E (IgE) antibody and eosinophils Parasitic Disease
in parasitic infections. FUNGAL IMMUNOLOGY
5. Discuss the roles of serological and molecular assays in the diagnosis Characteristics of Fungi
of parasitic infections. Classification of Mycotic Infections
6. Discuss the role serology plays in the diagnosis of Toxoplasma gondii (Mycoses)
and cite the limitations of serological testing for toxoplasmosis in the Immune Responses to Fungi
newborn.
Laboratory Diagnosis of Fungal
7. List possible limitations associated with parasitic serology. Infections
8. Cite the role that rapid antigen detection systems (RDTS) play in the Fungal Pathogens
detection of parasitic diseases.
SUMMARY
9. Briefly describe the principle of lateral flow assays.
CASE STUDIES
10. List factors that have led to a notable increase in fungal infections in
REVIEW QUESTIONS
the past 25 years.
11. Describe the etiological and physiological factors to be examined
when a mycosis is suspected.
12. Cite the four types of clinical manifestations that fungi can produce.
13. Describe the types of immune defenses mounted by the host in
response to fungal infections and identify the immune response that
plays the most important role in responding to a fungal infection.
14. Discuss the role of serological and molecular testing in the diagnosis
of fungal infections.
15. Recognize the clinical diseases and epidemiology of aspergillosis,
candidiasis, cryptococcosis, histoplasmosis, and coccidioidomycosis.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Antigenic concealment Candidiasis Histoplasmosis Parasites
Antigenic mimicry Coccidioidomycosis Hyphae Protozoa
Antigenic shedding Conidia Immunologic diversion Rapid antigen detection
Antigen switching Cryptococcosis Immunologic subversion systems (RDTS)
Antigenic variation Ectoparasites Lateral flow assays Toll-like receptors (TLRs)
Aspergillosis Fungi Mycelial fungi Yeast
C-type lectin receptors (CLRs) Helminths Mycosis (mycoses) Toxoplasma gondii

The detection and diagnosis of fungal and parasitic infections water infected with the parasites. Helminths, or parasitic
relies on laboratory methods as well as the patient’s clinical worms, are multicelled organisms that can live either alone or
symptoms, medical history, geographic location, and travel his- in humans. They include flatworms, tapeworms, and round-
tory. Traditional laboratory diagnosis is based on microscopic worms. Ectoparasites are multicelled organisms that live on the
observation of the causative agent in clinical material and, in skin. Common ectoparasites infecting humans include Pediculus
the case of fungi, recovery of the agent in culture. Technolog- humanus capitis (head lice), Pthirus pubis (pubic lice or crabs),
ical advancements have provided newer methodologies that and Sarcoptes scabiei (scabies). Other ectoparasites include fleas,
are an improvement over conventional methods for the detec- ticks, and mites.
tion of fungal and parasitic infections. Direct antigen detection, Mortality from infectious diseases declined substantially in
molecular assays, and newer serological assays have emerged. the United States during the first eight decades of the 20th cen-
Although the sensitivity and specificity of serological assays for tury because of improvements in living conditions, medical
fungal and parasitic infections have improved over the years, care, and sanitation. Beginning in 1981 that trend reversed,
the antigens used in parasitic and fungal assays are often cruder largely because of the AIDS epidemic. With the decline of AIDS
than those used in tests for viral and bacterial diagnosis, thus in the United States, that trend is again heading downward. In
decreasing their specificity and sensitivity. As of 2014, there 1996, a 7% drop in infectious disease deaths was recorded.1,2
have been relatively few FDA-approved serological-based However, in many areas of the globe, parasitic diseases are on
assays, limiting their use. the rise, particularly in the tropic and subtropic regions, be-
Despite the development of newer assays, the diagnosis of cause of rapid and unplanned growth in the cities. The World
parasitic and fungal infections still remains a challenge. In Health Organization (WHO) reported that globally, one-third
many cases, the clinical presentations overlap or are nonspe- of all deaths are caused by infectious and parasitic diseases.
cific. Because of the shared antigenicity of several genera and In 2010, malaria alone was the underlying cause of death for
species, the ability to distinguish a specific causative agent 1.2 million people, including more than 700,000 children
based on serology is sometimes not possible. Many fungal in- younger than 5 years and over 520,000 people aged 5 years or
fections are opportunistic infections that occur in individuals older.3 Whereas malaria, tuberculosis, and HIV are well known
who are immunocompromised. As such, those individuals may for their impact in the developing world, there are a number
not mount a humoral antibody response, limiting the utility of of neglected tropical diseases (NTDs) that cause significant
serologically based assays. In addition, many of the parasitic morbidity and mortality around the globe. A vast majority of
diseases occur in impoverished countries. Because of the lack the NTDs are caused by parasites. These diseases include leish-
of resources and expertise in these countries, the development maniasis, schistosomiasis (snail fever), African trypanosomiasis
and use of serological assays has been hindered. Finally, for (sleeping sickness), onchocerciasis (river blindness), and lym-
many of the agents, diagnostic assays (serological or molecular) phatic filariasis (elephantiasis).4,5 In the United States, trichomo-
are not available. This chapter provides information on the im- niasis, giardiasis, cryptosporidiosis, and toxoplamosis are the
munologic aspects of parasitic and fungal infections along with most common parasitic infections.
a discussion of available serological and molecular assays for Although effective vaccines have been developed for bacte-
the detection of the organisms that cause these infections. rial and viral agents, the development of vaccines for parasitic
diseases has remained elusive. A variety of roadblocks have
Parasitic Immunology hindered the development of vaccines against parasitic agents.
Parasites are complex organisms, many with elaborate life
Parasites are microorganisms that survive by living off of other cycles; over time, they have developed well-honed mechanisms
organisms, referred to as hosts. Three types of organisms may for immune evasion. In addition, the immune responses to par-
cause parasitic infections: protozoa, helminths, and ectopara- asitic organisms are not fully understood, which also con-
sites. Protozoa are a diverse group of single-celled organisms tributes to the lack of vaccine development in this area. This
that can live and multiply inside of human hosts. Giardiasis is chapter covers the various strategies used by parasites to evade
an example of a protozoal infection that can occur from drinking the immune response and survive in a host, as well as the use
of serological and molecular techniques in the diagnosis of The most severe outcome is that the host is killed. Death of
parasitic diseases. the host may be caused by a variety of reasons, ranging from
The immune responses to bacterial infections are more the parasite overwhelming the host defenses to specific features
clearly understood than the immune responses to parasitic of the parasite that contribute to the death of the host. One
infections. Bacteria are unicellular organisms with relatively sim- such example is Plasmodium falciparum. P falciparum rapidly
ple life cycles. In addition, there is limited epitope variation in multiplies in the host and produces an erythrocyte membrane
bacteria, allowing for the immune system to mount a response protein-1 (PfEMP1) that binds to the endothelium in the blood
more easily. The immune response to parasitic infections differs vessels, resulting in the small blood vessels becoming clogged.
from that associated with bacterial infections, mostly because When this occurs in the brain, the result is cerebral malaria,
of the multicellular nature of parasites. Chandra6 described gen- which can be fatal.
eral concepts that need to be considered in relation to host Death of the host is not the best strategy for a parasite. If
immune responses to parasites: (1) Heterogeneity with respect the parasite were to totally elude the host immune system and
to life cycles and antigenic expression is a key feature of para- was sufficiently virulent, the parasite would kill the host on
sitic agents. (2) Many parasitic infections are chronic in nature. which its survival depends. If the host dies, then so does the
(3) The mechanisms of immune evasion are significantly dif- parasite. Any parasite’s survival depends on its ability to live
ferent from those of bacterial infections. (4) Many parasites in a peaceful manner with its host while living and feeding off
develop significant genetic and antigenic variation in a rela- the host.
tively short period. (5) The innate immunity in the natural Defenses to parasitic infection involve both innate and ac-
hosts may be genetically determined. (6) Humans, as well as quired (adaptive) immune mechanisms. The innate or nonspe-
animals, differ widely in their ability to handle the complex cific immune response may result in the destruction and removal
antigens found in parasites. of the parasite, thus preventing establishment of an infection.
The nonspecific immune defenses can include activation of cells
that may destroy the parasite by phagocytosis, release of cy-
Immune Responses to Parasites tokines (e.g., TNF-α, IL-1, IL-10, IL-12, type I interferons, and
When an organism encounters a host, the eventual outcome chemokines) that enhance the immune response, or activation
depends on a variety of factors. These include the number of of the complement system, resulting in enhanced recognition by
organisms or size of the inoculum, the multiplication rate of the immune system (see Chapters 3, 4, and 7). Similar to other
the organism, and the virulence factors possessed by the organisms that may cause infection or disease, parasites have
organism. The degree to which the organism establishes an evolved strategies to evade natural nonadaptive host defenses.
infection or is eradicated by the host depends on the organism’s These include killing or avoiding being killed by phagocytes, in-
ability to mobilize sufficient host defenses for removal or the terfering with complement’s alternate pathway, production of
organism’s ability to overcome those defense mechanisms. iron-binding molecules, and blocking interferons.
If the parasite is able to establish itself, one of several pos- If the innate immunity is unsuccessful in eliminating the par-
sible outcomes may occur: death of the host, eradication of asite, the parasite may be eliminated through activation of the
the parasite, or, as is the case of the majority of parasitic in- adaptive immune responses. This results in either a humoral or
fections, establishment of a persistent infection. The interac- a cell-mediated response to the parasite (see Chapter 4). In
tion and outcomes that parasites have within their hosts have some parasites, complete immunity can be achieved through
been categorized into six different levels7 (Table 22–1). the humoral immune response. Specific antibody can damage

Table 22–1 Potential Outcomes of Host and Parasite Interactions

LEVEL HOST AND PARASITE INTERACTION DESCRIPTION
1 Natural resistance No invasion of host by parasite
2 Symbiosis Colonization of host with parasite with benefit to both
3 Commensalism Colonization of host with parasite with no benefit or harm
4 Sterilizing immunity Parasite invades host and causes disease; host develops
immunity and is cured
5 Concomitant immunity Parasite invades host and causes disease; host develops an
immune response and has some resistance to the parasite but
is not cured
6 Ineffective immunity Parasite invades host and causes disease; host does not
develop resistance to the parasite and is not cured

Adapted from Playfair JHL. Effective and ineffective immune responses to parasites: evidence from experimental models. Curr Top Microbiol Immunol.
1978;80:37–64.
protozoa, neutralize parasites by blocking attachment to the specific target cell. Once the parasite reaches its target cell,
host cell, prevent the spread of the parasite, promote comple- some parasites have developed strategies for entering and sur-
ment lysis, enhance phagocytosis, and destroy the parasite viving within the host cell. The host cannot recognize the
through antibody-dependent cellular cytotoxicity (ADCC). parasites while they reside inside cells.13 Examples of parasites
Many parasitic infections are characterized by eosinophilia and that have an intracellular phase in their life cycle include
high levels of immunoglobulin E (IgE). The IgE antibody binds Plasmodium species, Trypanosoma cruzi, Leishmania, and
to mast cells and basophils in the host (see Chapter 14). When Cryptosporidium parvum.
specific antigen–antibody combinations occur, mast cells degran- Another process of evasion some parasites employ is anti-
ulate and release chemotactic factors, histamine, prostaglandins, genic variation. Evasion from the immune response depends
and other mediators. One of the most important mediators on variation occurring in the parasite antigens that are recog-
released is eosinophil chemotactic factor, which attracts nized by the host’s immune system. There are three main
eosinophils to the infected area. Eosinophils can destroy some mechanisms for antigenic variation. The first mechanism in-
parasites by degranulation or through ADCC. Scientists believe volves the parasite’s ability to generate novel antigens by ran-
that the ability to produce IgE evolved mainly to protect the dom mutation. Some parasites have evolved mechanisms by
host from parasitic infections.8 Studies indicate that high levels which random mutations occur with a frequency sufficient to
of anti-parasitic IgE correlate with resistance to reinfection by evade the immune system on an ongoing basis. An example of
the parasite.9,10 this is the antigenic variation of the malaria parasite through
single nucleotide point replacement. The second mechanism
Parasite Survival Strategies of antigenic variation may occur through genetic recombina-
tion. Rearrangement of genes within an organism allows for
In many host and parasite relationships, the host’s adaptive de- the development and expression of new epitopes on the surface
fenses reduce the parasite load to low levels; however, they fail of the parasite for which a previous immune response has not
to eliminate the parasite completely and transmission contin- taken place. The ability to rearrange genes and rapidly change
ues. For example, schistosomes (a type of helminth) have surface molecules contributes to the virulence of P falciparum
mechanisms that can downregulate the host’s immune system. and T cruzi.14 The third mechanism is gene switching. Gene
This immune modulation promotes the parasite’s survival but switching is the most dramatic form of antigenic variation ob-
also limits severe damage to the host. As a result, adult schis- served in parasites. Organisms may carry upwards of one thou-
tosomes can live in the human host for up to 40 years before sand different genes, allowing for the expression of distinct
finally being eliminated.11,12 surface molecules. An organism employing this mechanism can
Parasites have developed a variety of strategies to evade adap- switch from the use of one gene to another, thus persisting
tive defenses that are more complicated than those for evading while the immune system is trying to catch up with it. Exam-
innate defenses. Survival strategies include antigenic conceal- ples of parasites that use this mechanism are the trypanosomes,
ment, antigenic variation, antigenic shedding, antigenic mimicry, Trypanosoma gambiense, and Trypanosoma rhodesiense. These or-
immunologic diversion, and immunologic subversion. ganisms are able to alter their surface glycoproteins to produce
In antigenic concealment, parasites hide their antigens an unlimited group of variable antigen types.15,16 The process
from the host. One way in which parasites can conceal their begins when the host produces antibody, mainly IgM, to the
antigens is by remaining inside of the host’s cells without their one antigen, thereby reducing the infection. The parasite re-
antigens being displayed. If a parasite becomes sequestered sponds by swapping genes, thus changing its antigen and mak-
within host cells, the parasite is hidden from the immune sys- ing the current antibody ineffective. Gene switching may occur
tem and protected. Most parasites infect only a few cell types very rapidly, within 5 to 6 days. The host must then produce
in their host. To infect a host, the parasite must reach its a new antibody. This process of antigen switching can
continue for long periods of time.
A factor that must be considered with respect to antigenic
variation is the parasite’s life cycle. Parasites are large organisms
Connections compared with bacteria and viruses. Furthermore, they have
complex life cycles and are antigenically diverse. The parasite’s
IgE Antibodies and Parasites development and progression through its life cycle are adapted
IgE antibodies are best known for their role in allergic reactions. to the physiology and behavior of the host. Not all of the
However, they also play an important role in the defense against phases of a parasite’s life cycle necessarily occur in one host. A
parasites such as helminths, which are too large to be phagocy- particular host may be the definitive host that harbors the adult
tized. Killing of the parasites is accomplished by ADCC. In this or sexual stage of the parasite (e.g., Taenia saginata and Taenia
mechanism, the Fc portions of the parasite-specific IgE antibod-
solium—the beef and pork tapeworms, respectively—where
ies bind to specific receptors on the surface of eosinophils, which
are then stimulated to release enzymes from their granules that
humans are the only definitive host), an intermediate host in
destroy the parasite. The concentration of IgE and the number which the parasite lives during the larval or asexual stage (e.g.,
of eosinophils in the peripheral blood are increased, indicating the malarial parasites where humans are the intermediate host),
their importance in defense against parasitic infections. or an accidental or dead-end host in which a relationship is
not required for propagation or continuation of the parasite
(e.g., Echinococcus granulosus, the causative agent of hydatid the parasite causes the immune system to produce proteins that
cysts, where humans are the dead-end host). divert the attention of the immune system. An example is the
Many parasites have complex life cycles that involve several ability of some parasites to cause an increase in production of
hosts. Different antigens may be expressed, depending on the beta interferon (IFN-β), which allows for increased parasite sur-
life cycle stage in the different hosts. The parasite often under- vival. For example, IFN-β has been shown to decrease the ability
goes complex growth cycles and differentiation in preparation of macrophages to kill Leishmania by significantly reducing
for transmission to its next host. In doing so, an organism’s the release of superoxide from these cells.22 Another example
surface antigens vary considerably while in a single host. An by which parasites can divert the immune system is seen with
example is Leishmania. The parasite enters the host through P falciparum-infected erythrocytes (IE), which have the potential
the bite of an insect vector as a trypomastigote in the blood- to interact with B cells in different parts of the body, inducing
stream, which is then phagocytized by macrophages, where it them to divide and differentiate into antibody-secreting cells.
transforms into an amastigote. The amastigotes differentiate The malaria parasite, as well as some protozoa, viruses, and bac-
into nonreplicating trypomastigotes and the cells rupture, teria, produce immunoglobulin-binding proteins (IBPs) that act
releasing them into the bloodstream. Additional host cells of as polyclonal B-cell activators. This results in the expansion of
various types are then infected and the trypomastigotes once numerous B-cell clones and in antibody production not specific
again form amastigotes inside these cells. Trypomastigotes cir- for the parasite. Thus, the IBPs may act as an evasion mechanism
culating in the blood are acquired by an insect vector, where to divert specific antibody responses.23
they differentiate to form epimastigotes and, finally, trypo-
mastigotes. The two forms of trypomastigotes, as well as the Serodiagnosis of Parasitic Diseases
amastigotes that occur in the human host, express very differ-
In instances where demonstration of the parasite in biological
ent antigens, thus making an immune response difficult.17
or tissue samples is not possible, serological testing is the gold
In addition to variation of antigen expression, parasites may
standard for diagnosis. Serological-based assays can be divided
evade the immune system through antigen shedding. Similar
into those that detect parasitic antigens and those that detect an-
to bacteria that shed capsular material into the host environ-
tibodies against the parasite. These tests include enzyme-linked
ment to evade the immune system, some parasites also exhibit
immunosorbent assays (ELISA), indirect immunofluorescence,
antigenic shedding. Entamoeba histolytica is one example of a
indirect hemagglutination, whole protozoan or antigen-coated
parasite that can shed antigens from its cell surface.13 Although
particle agglutination, radioimmunoassays (now rarely used),
antibody is formed against the parasite, the antibody attaches to
and newer rapid diagnostic tests (RDTs). Previously, many of
the shed antigen rather than the parasite, allowing the parasite
these assays used relatively crude antigens, making their sensi-
to escape the immune response.
tivity and specificity unpredictable. Advances in immunochem-
Antigenic mimicry may occur when the parasite expresses
istry and molecular biology have allowed for the development
epitopes that are similar, if not identical, to host molecules.
of assays that have markedly improved sensitivity and specificity.
The similarity between host and parasitic antigens may sup-
However, assays utilizing these newer technologies are costly
press the immune response and protect the invading parasite
and not widely available in those countries that have the highest
from being recognized and eliminated by the immune sys-
occurrence of parasitic diseases.24
tem.18 An example is the antigenic similarity between human
ELISA-based assays have been used to detect antigens of
host antigens and those of the Schistosoma species.19 The im-
parasites that cause human and animal infections, such as
mune response may result in host and parasite cross-reactivity,
amebiasis, babesiosis, fascioliasis, cutaneous and visceral
which may lead to autoimmunity, manifested by the presence
leishmaniasis, cysticercosis, echinococcosis, malaria, schis-
of autoantibodies or T cells with autoreactivity.20,21 An autoim-
tosomiasis, toxocariasis, toxoplasmosis, trichinosis, and
mune response has been linked to the cardiac and intestinal
trypanosomiasis.25,26
symptoms that occur in the late stages of Chagas disease that
Whereas some parasitic diseases are readily diagnosed by
may occur because of an infection with T cruzi, a bloodborne
demonstrating the causative agent in clinical material, such as
parasite.13
infection with intestinal helminths, in which the worm’s eggs are
Some organisms enhance their survival by modulating the
easily detected in fecal specimens, demonstration of other par-
immune system. The ability to modulate the immune response
asitic agents is difficult or not possible (e.g., toxoplasmosis). In
is an important strategy employed by some parasites to enable
those cases, serological assays can be very useful. Table 22–2
their survival in the host. Some parasites can subvert the im-
indicates the usefulness of serological testing for the diagnosis
mune system. Immunologic subversion is achieved by avoiding
of various parasitic diseases.25 Although serology may not be
the effector mechanisms of the immune response. Effector mol-
helpful in the diagnosis of some parasitic diseases, serology can
ecules include complement and cytokines. Effector cells include
play a role in epidemiological investigations.
plasma cells, T helper (Th) cells, and cytotoxic T cells. For ex-
ample, some parasites can subvert cytotoxic T cells by producing
decoy HLA molecules. Parasites may also subvert the Fc function Although advances have been made in the serological assays
of antibodies by making Fc receptor homologues or they can for some parasites, commercially available ELISA assays still
subvert complement by making homologues of complement vary considerably in their sensitivity and specificity. One such
control proteins (CCPs). Immunologic diversion occurs when example is the detection of antibodies against the protozoan
 Table 22–2 Usefulness of Antibody Detection for Parasitic Diseases
SEROLOGY INDICATED SEROLOGY MAY BE USEFUL SEROLOGY NOT INDICATED
Amebiasis (extraintestinal) Amebiasis Anisakiasis
Chagas disease Amebic meningoencephalitis (caused by free- Ascariasis
Clonorchiasis living amebae) Capillariasis
Cysticercosis Anaplasmosis/Ehrlichiosis Cryptosporidiosis
Hydatidosis Babesiosis Hookworm
Filariasis (lymphatic; suspect Lyme disease Malaria
cases when microfilariae Paragonimiasis (eggs not detectable in sputum Trichuriasis
cannot be identified in or feces)
blood)
Leishmaniasis (cutaneous
and visceral)
Schistosomiasis (ectopic
cases; chronic cases
when eggs cannot be
demonstrated in feces
or urine)
Toxocariasis (visceral
and ocular)
Toxoplasmosis
Trichinellosis
Adapted from Maddison SE. Serodiagnosis of parasitic diseases. Clin Microbiol Rev. 1991;4(4):457–469.

Toxoplasma gondii. T gondii has a high prevalence around the If the individual becomes immunosuppressed because of
world and can infect all species of animals and birds. Nearly HIV infection, malignancy, or immunosuppressive drugs, re-
24% of the U.S. population over age 12 is infected with this activated toxoplasmosis can occur. The tissue cysts rupture and
parasite, which usually remains dormant.27 Reactivation may release the active bradyzoite, which results in clinical disease.29
occur if the individual becomes immunosuppressed. Members T gondii infection in the immunocompromised individual can
of the cat family (Felidae) serve as the definitive hosts for be severe or even fatal.30 In immunosuppressed individuals,
T gondii. the organism can invade the central nervous system (CNS),
The life cycle of T gondii has three stages: the tachyzoite, leading to toxoplasma encephalitis. Over 95% of the cases are
which rapidly multiplies in the intermediate host (e.g., hu- caused by reactivation of a latent infection.31
mans) and in nonintestinal epithelial cells of the definitive host Another concern is the organism’s ability to be passed to the
(e.g., the cat); the bradyzoite, which forms the tissue cysts; and fetus during pregnancy. If a mother has been infected before
the sporozoite, which is found in the oocyst28 (Fig. 22–1). pregnancy occurs, then the fetus is protected because of
There are three routes by which T gondii is potentially trans- maternal antibodies. However, if the woman becomes infected
mitted to humans. Humans can become infected with T gondii just before or during pregnancy, congenital transmission of the
by eating raw or insufficiently cooked infected meat (e.g., pork, organism can occur. Congenital toxoplasmosis may result in a
mutton, or wild game) that contains the cysts or uncooked miscarriage, a stillborn child, or mental deficits later on in life.
foods that have come into contact with the infected meat. Tox- Up to one-half of pregnant women who become infected can
oplasmosis may also occur when humans ingest oocysts from transmit T gondii across the placenta. The trimester in which
cat feces present in a litter box or in the soil. Third, the tachy- the infection was acquired influences the incidence and sever-
zoites, which are observed during the primary infection, can be ity of congenital toxoplasmosis. The transmission risk during
transmitted transplacentally to the unborn fetus (Fig. 22–2). the first trimester of pregnancy is 10% to 25%, whereas the
The incubation period for T gondii in adults ranges from 10 to transmission risk is 60% to 90% during the third trimester.32,33
23 days following ingestion of undercooked meat and from Toxoplasma infection can result from congenital infection or
5 to 20 days after ingestion of oocysts from cat feces. Following infection after birth.
infection, the organism reproduces asexually and forms tissue Although the diagnosis of toxoplasmosis can be made by
cysts that remain for the life of the host. In the immunocompe- demonstrating the parasite in stained tissue samples or CSF,
tent individual, a majority of initial Toxoplasma infections are diagnosis is usually made through serological means. A com-
asymptomatic or may only present with a mild lymphadenopa- bination of tests needs to be performed to determine whether
thy. Immunity is usually sufficient to contain the latent infec- an individual has been recently infected or had a previous in-
tion. Toxoplasmosis can cause serious symptoms in the brain fection with T gondii. The sensitivity and specificity of different
and other organs in immunocompromised patients, as well as commercial kits vary widely and misinterpretation of the re-
in the developing fetus following congenital infection. sults, particularly in determining the presence and significance
 Toxoplasma gondii life cycle. Sexual replication of T gondii occurs in cats, the definitive host for the parasite. T gondii gametes
are formed after merozoites replicate within enterocytes of the cat gut, a process known as merogony. The gametes fuse to form diploid
oocysts, which are shed in cat feces and undergo meiosis in the environment to produce eight haploid progeny sporozoites. Asexual replica-
tion occurs in intermediate hosts such as rodents. Rapidly replicating forms called tachyzoites disseminate within the host during acute infec-
tion and can differentiate into slow-growing forms called bradyzoites inside tissue cysts. Other intermediate hosts or cats can acquire the
infection by ingesting the tissue cysts, re-initiating the sexual phase of the life cycle. Oocysts can survive in the environment for a long time
and develop into sporulated oocysts, which can be transmitted to intermediate hosts such as farm animals through contaminated food and
water. Humans become infected by ingesting tissue cysts in undercooked meat or sporulated oocysts in contaminated water. (Reprinted by
permission from Macmillan Publishers Ltd: Nature Reviews Microbiology, 10:766–778, copyright 2012.)

of IgM antibodies, may occur. This is concerning because serol- The second sample should demonstrate high levels of IgM and
ogy results can influence decisions regarding continuation or IgG antibodies if the first sample was collected early in the
termination of pregnancies.34 IgM antibodies may persist for infection. If both specimens show the presence of IgM but
up to 18 months after infection with T gondii. Therefore, the absence of IgG, the IgM result should be considered to be a
FDA has recommended that anti-Toxoplasma IgM tests should false positive.
be interpreted with caution and that the sole results of a single When both IgM and IgG antibodies are detected and the
assay should not be used in determining a recent infection.35 patient is pregnant, an IgG avidity test should be performed.
The FDA has published guidelines for the interpretation of Determination of the avidity of the antibody has proven useful
T gondii serology results (Table 22–3). in determining whether the IgG antibodies are from a recent
The greatest value of testing for IgM antibodies is in deter- or a previous infection. The presence of high avidity IgG indi-
mining whether a woman has had a recent infection. If no IgM cates an infection in the past, whereas IgG antibodies with a
antibodies are detected and only IgG is detected, this excludes low avidity suggest a more recent infection. In that low avidity
a recent infection before pregnancy. One of the problems when antibodies may persist for a prolonged period of time, their
testing for Toxoplasma-specific IgM antibodies is that the cur- presence does not necessarily mean that the infection was re-
rent assays lack specificity. If only IgM antibodies are detected, cently acquired. However, the presence of high avidity anti-
additional testing should be performed. A second blood sample bodies indicates an infection for at least 3 to 5 months and
should be obtained from the patient 2 weeks after the first does not pose a risk to the fetus.36-39 The assay for IgG avidity
sample is collected and tested together with the first specimen. utilizes a wash buffer containing urea to differentiate low
 Connections
High Avidity Antibodies
The avidity of an antibody molecule represents the strength by
which its antigen-binding sites can bind to an antigen. During the
course of an immune response, somatic mutations occur in the
immunoglobulin genes in the B cells, causing them to produce
antibodies of higher avidity. This results in tight binding of the an-
tibodies to the antigen, such as a lock and key. The presence of
high avidity antibodies is a sign that the immune response has
been going on for quite some time (see Chapter 5).

avidity from high avidity antibodies. Antibodies with low avid-

ity dissociate from the antigen in the presence of urea. The
avidity is determined by running the assay in duplicate using
buffer with and without urea. The avidity index (AI) is deter-
mined by dividing the T gondii-specific IgG signal (optical den-
sity; O.D.) of the set washed with urea-containing buffer by
Toxoplasma gondii tachyzoites in mouse ascitic fluid the T gondii-specific IgG O.D. of the set washed with non-urea
stained with Giemsa stain. (Courtesy of the CDC/Dr. L.L. Moore, Jr., buffer. AI values lower than 0.20 indicate the presence of low
Public Health Image Library.) avidity antibodies, values between 0.20 and 0.25 suggest in-
termediate avidity, and values greater than 0.25 are obtained
with antibodies of high avidity.40
When using serology to detect congenital toxoplasmosis,
several differences exist. Because of the passive transfer of IgG

Table 22–3 General Guidelines for Interpretation of Toxoplasma Gondii Serology Results
IgG RESULT IgM RESULT REPORT AND INTERPRETATION (EXCEPT INFANTS)
Negative Negative No serological evidence of infection with Toxoplasma.
Negative Equivocal Possible early acute infection or false-positive IgM result. Obtain new specimen for
IgM and IgG testing. If result for the second specimen remains the same, the
patient is probably not infected with Toxoplasma.
Negative Positive Possible acute infection or possible false-positive IgM result. Obtain new specimen
for IgM and IgG testing. If result for the second specimen remains the same, the
IgM reaction is probably a false positive.
Equivocal Negative Indeterminate: obtain a new specimen for testing or retest this specimen for IgG
using a different assay.
Equivocal Equivocal Indeterminate: obtain a new specimen for IgM and IgG testing.
Equivocal Positive Possible acute infection with Toxoplasma. Obtain new specimen for IgM and IgG
testing. If the result for the second specimen remains the same or if the IgG
becomes positive, both specimens should be sent to a reference laboratory with
experience with diagnosing Toxoplasma infection.
Positive Negative Infected with Toxoplasma for 6 months or longer.
Positive Equivocal Infected with Toxoplasma for probably more than 1 year or false-positive IgM
reaction. Obtain a new specimen for IgM testing. If results with the second
specimen remain the same, both specimens should be sent to a reference
laboratory with experience with diagnosing Toxoplasma infection.
Positive Positive Possible recent infection within the past 12 months or false-positive IgM result.
Send the specimen to a reference laboratory with experience with diagnosing
Toxoplasma infection.

Adapted from Centers for Disease Control and Prevention. DPDx—Laboratory identification of parasitic diseases of public health concern: toxoplasmosis.
www.cdc.gov/dpdx/toxoplasmosis/dx.html. Accessed April 20, 2016.
across the placenta, infants whose mothers are chronically in- deadly P falciparum and Plasmodium vivax.42 At the time of this
fected will be born with toxoplasma IgG antibodies of maternal writing, the BinaxNOW® Malaria Test (Alere Inc., Waltham,
origin. Serological diagnosis can be made 5 or 10 days after MA) is the only available RDTS for malaria in the United States.
birth by demonstration of positive Toxoplasma IgM or IgA an- The test detects the histidine-rich protein II (HRPII) specific
tibody titers, respectively, in newborn sera. Detection of anti- to P falciparum (P.f.) and a pan-malarial antigen common to all
Toxoplasma IgA antibodies is more sensitive than detection of four malaria species that can infect humans—P falciparum,
IgM antibodies. It should be noted that commercial assays cur- P vivax (P.v.), Plasmodium ovale (P.o.), and Plasmodium malariae
rently used in the United States have not been cleared by the FDA (P.m.) (Fig. 22–4).
for diagnostic testing of infants. Therefore, samples from neonates
suspected of having congenital toxoplasmosis should be sent to
As with all laboratory testing, it is important to use method-
the Toxoplasma Serology Laboratory, Palo Alto, California, the
ologies that are the most straightforward and cost effective for
site that has the most experience with infant testing.
each test. However, it is also important to consider the speci-
ficity and sensitivity of the method when choosing a procedure.
Rapid antigen detection systems (RDTS) (also called lateral Currently, there is no external proficiency testing program of-
flow assays), are based on immunochromatographic antigen fered in the area of parasitic serology except for diagnosis of
detection and are widely used in many diagnostic laboratories. toxoplasmosis.43 Therefore, it is very difficult to evaluate the
RDTS detect soluble proteins through their ability to bind to quality of commercial assays that are currently available. In the
capture antibodies contained in a nitrocellulose strip. The clin- United States, commercial kit manufacturers must obtain FDA
ical sample is placed on the strip and eluted by adding a few approval before selling their products. The FDA requires only
drops of buffer that contains a labeled antibody. A colored band that a new method be equivalent to a method that has already
representing the antigen–antibody complex can then be seen been approved. Researchers at the CDC have expressed con-
on the membrane. Many of the assays are stable at room tem- cern that, over time, the quality of new test kits may drift in a
perature, easy to perform and interpret, and cost effective. negative direction because a new kit may not be quite as good
Many clinical laboratories have discontinued the use of ELISA as the one used for comparison but may still receive approval.44
assays in favor of immunochromatographic assays. For exam- In addition to the inability to evaluate an assay’s performance
ple, several years ago, ELISA-based assays were the predomi- using proficiency testing, a specific test may not detect a parasitic
nant platform for the detection of Giardia and Cryptosporidium. disease in an individual because of the inability of the assay to
Today, many clinical laboratories are using lateral flow assays. detect all species of the parasite causing the infection. For ex-
One example is the Xpect Giardia/Cryptosporidium assay from ample, some serological tests, such as those for schistosomiasis,
Remel (Lexena, Kansas) (Fig. 22–3). only detect antibodies that are species-specific. Therefore, the
Because of their advantages, many RDTS can be used in
rural regions. Assays have also been developed for the rapid
diagnosis of malaria using this platform.41 The assays can be
used in the field and allow for differentiation between the more

BinaxNOW® Malaria test. The immunochromato-

graphic assay targets the histidine-rich protein II (HRPII) antigen
specific to Plasmodium falciparum (P.f.) and a pan-malarial antigen
Remel Xpect® Giardia/Cryptosporidium im- common to all four malaria species capable of infecting humans—
munochromatographic immunoassay. The test uses sample wicking P falciparum, P vivax (P.v.), P ovale (P.o.), and P malariae (P.m.)—
to capture Giardia and Cryptosporidium antigens on discrete test circulating in human venous and capillary EDTA whole blood.
lines containing antigen-specific antibodies for each organism. (Reprinted by permission BioMed Central Ltd, Malaria Journal 14;10:300,
(Reprinted by permission Thermo Fisher Scientific, Lenexa, Kansas.) copyright 2011.)
same sample can give a negative result in one test and a positive Rochester, Minnesota, and Focus Diagnostics in Cypress,
result in another. Also, the type of specimen submitted from a California) and from the CDC. As of 2014, there are a limited
patient may give conflicting results. In one reported example, number of FDA-approved molecular assays for the diagnosis and
samples of serum, stool, and urine from a patient suspected of identification of gastrointestinal and blood and tissue parasites.
having schistosomiasis were submitted to a commercial labora- Many laboratories have developed in-house assays for the iden-
tory for testing. Although the serum result was antibody negative tification and differentiation of parasites.47 A number of new
and no parasites were found in the stool, the urine examination technologies, including nested multiplex PCR, have the potential
demonstrated eggs of Schistosoma haematobium. An immunoblot for the detection and identification of gastrointestinal parasites.48
was positive for S haematobium and negative for Schistosoma man- Molecular tests are playing an increasingly important role as ad-
soni when the serum specimen was submitted to the CDC for juncts to traditional diagnosis of parasitic infections and, in some
blind retesting. In this example, the tests for schistosomiasis per- cases, may even replace traditional methods as commercial com-
formed by the two laboratories were not equal in detecting the panies develop molecular-based assays and seek FDA approval.
three Schistosoma species that infect humans. Situations such as
these can lead to an inaccurate diagnosis.43
Because of the antigenic complexity of parasites, the antibody
Fungal Immunology
formed may not be detected because the assay uses a different Fungi represent a large heterogeneous group of eukaryotic
antigen from the one to which the patient has formed the anti- organisms that are ubiquitous in the environment. Fungi can
bodies. Also, the sensitivity of an assay may vary, depending on either be considered as parasites, deriving their nutrition from
the stage and type of the patient’s disease. For example, antibod- living matter, or more commonly as saprophytes, living off
ies against Plasmodium species (malaria) rise rapidly in an acute of dead and decaying matter. In the environment, fungi play
infection but decline if the parasite becomes latent or dormant an important role in maintaining the ecosystem with the
in the liver as a hypnozoite. Similarly, because of the potential decomposition of cellulose. The Dictionary of Fungi reported
for cross-reactivity of the antibodies formed in a parasitic infec- 97,330 species of described fungi in the “numbers of fungi”
tion, a positive result should be interpreted with caution. entry.49 New organisms are being added as advances in molec-
Although there are commercially available tests for the sero- ular biology allow for the detection and differentiation of fungi.
logical diagnosis of several parasitic agents, only a few com- Despite the number and diversity of fungi, only about 300 fungal
mercial kits are available in the United States and they are not species are potentially pathogenic to humans and animals. Fewer
used by many clinical laboratories. In the United States, the than 50 of these species are responsible for approximately
majority of tests are performed by the following commercial 90% of all fungal infections.50
laboratories: Focus Technologies, Mayo Medical Laboratories, When a human or animal has an infection or disease caused
Parasitic Disease Consultants, Quest Diagnostics, and Specialty by a fungus, it is referred to as a mycosis (plural: mycoses).
Laboratories.44 Most of the tests performed at the commercial Mycoses are classified into four groups—superficial, cuta-
laboratories and at the CDC are produced and evaluated in- neous, subcutaneous, or systemic—based on the type and
house and use reagents that are not universally standardized. amount of tissue involvement and the host response to the
Because of the differences in the reagents used in the tests, dis- pathogen. Mycotic diseases are of growing importance for a
crepant results obtained by different laboratories are commonly number of reasons:51 (1) Fungi are widely distributed in nature
observed. and are able to maintain themselves in the environment, mak-
ing them difficult to eradicate. (2) Many fungi are able to pre-
Molecular-Based Diagnosis sent with various clinical manifestations, ranging from invasive
disease to localized infection, or allergic manifestations, as is
of Parasitic Disease observed with Aspergillus. (3) Diagnosis is difficult because of
The microscopic detection and diagnosis of parasitic disease, al- the varying clinical presentations. (4) Currently, vaccines are
though once considered to be the gold standard, is now recog- not available. (5) There are only a limited number of antifungal
nized as having limitations in being able to detect agents agents available for treating fungal infections. (6) Most fungal
responsible for parasitic infections. Decreased sensitivity associ- infections are opportunistic in nature and, increasingly, treat-
ated with microscopic methods may be caused by intermittent ment of a primary disease puts individuals at risk for oppor-
shedding of the organism, as is seen with Giardia lamblia, or to tunistic infections.
low levels of the organism being present, as is observed with the Fungi do not possess a large array of virulence factors that
bloodborne parasites such as the causative agents responsible for allow for them to be true pathogens. However, fungal oppor-
malaria or babesiosis. With the development of molecular-based tunistic infections have risen in recent years with the advent of
assays, the diagnosis of parasitic infections using molecular- AIDS and our increasing use of immunosuppressive therapies.
based technology is being used with increasing frequency. Fungal diseases are often the first opportunistic diseases de-
Although microscopy remains the means by which most blood tected in patients with AIDS. One of the most common oppor-
and tissue parasites are detected, rapid real-time polymerase tunistic diseases in AIDS patients is pneumonia caused by
chain reaction (PCR) assays for babesiosis and malaria have Pneumocystis jiroveci (previously Pneumocystis carinii). Although
recently been developed45,46 and are available from commercial originally classified as a parasite, this organism now is desig-
reference laboratories (e.g., Mayo Medical Laboratories in nated a fungus because it has a greater gene sequence homology
with fungi than with parasites.52 Many saprophytic fungi formerly called dermatophytes and can be transmitted through direct
dismissed as cultural contaminants are now reported as oppor- contact with an infected person or animal. The subcutaneous
tunistic pathogens.53 mycoses affect the subcutaneous tissue, where they usually form
deep, ulcerated skin lesions. The causative agents are soil sapro-
phytes and acquisition of the fungal agent is generally caused by
Characteristics of Fungi trauma to the skin, usually the feet or legs. The systemic mycoses
Fungi are eukaryotic cells with nuclei and rigid cell walls com- can involve the deep viscera and spread throughout the body.
posed primarily of chitin, glucans, mannans, and glycoproteins. These infections originate in the lungs and then disseminate to
Fungi can exist in two morphological states. They may exist as other locations. Although considered to be pathogenic to man,
yeast, which are unicellular organisms, or as mycelial fungi, many individuals infected with one of the causative agents are
which are multicellular organisms. Yeast reproduce by budding, asymptomatic. Except for Cryptococcus, the causative agents in
whereas the mycelial fungi reproduce through the production this group are dimorphic in nature. A number of fungi previ-
of spores or conidia. It should be noted that many individuals ously not considered to be pathogenic in humans are responsible
develop allergies to the spores and conidia produced by fungi. for opportunistic infections in the immunosuppressed patient.
Although most fungi are monomorphic (existing in a single Several agents have been long known as causing infections in
form), several fungi can exist as either yeast or mycelial fungi, immunosuppressed individuals, including Candida, Aspergillus,
depending on environmental conditions, and are referred to as and the fungi causing Zygomycosis (Rhizopus species, Mucor
dimorphic fungi. species, Cunninghamella bertholletiae, and Apophysomyces elegan).
Fungi grow more slowly that most bacteria (2 to 4 weeks Table 22–4 lists the most frequent agents associated with the
versus 1 to 2 days) and have relatively simple nutritional re- various classifications. Several fungi are considered to be com-
quirements. Because most fungi are found in the environment, mensalistic in nature, including Candida albicans and Malassezia
they prefer cooler temperatures to grow (25°C to 30°C). furfur; however, these organisms may cause disease or clinical
symptoms in some individuals.

Classification of Mycotic Immune Responses to Fungi

Infections (Mycoses) As is the case with the immune responses to bacterial, viral,
Fungi can produce four types of clinical manifestations. Some and parasitic agents, the immune defenses to fungal agents
individuals exhibit hypersensitivity to certain fungal agents. The range from protective mechanisms that include innate immu-
hypersensitivity reaction is generally because of an allergic reac- nity present early in the infection to specific adaptive mecha-
tion to the spores. Hypersensitivity may occur only when an in- nisms that are induced later. The first line of innate defense
dividual comes in contact with the specific spores he or she is includes skin and the mucous membranes of the respiratory,
allergic to, or it can occur as a chronic condition such as allergic gastrointestinal, and genitourinary tracts that provide physical
bronchopulmonary aspergillosis (ABPA), which develops in barriers that separate the host from the environment. Fungi
response to the fungus Aspergillus (most commonly Aspergillus possess very few factors that allow them to overcome those
fumigatus). Fungi may also be responsible for mycotoxicosis, a physical barriers; because of the nutrients and environmental
poisoning of humans and animals by food products contami- conditions needed for many fungi, those mechanisms have not
nated by fungi that produce toxins from a grain substrate. Major evolved. For example, among the most common fungal infec-
mycotoxins include aflatoxins, deoxynivalenol, fumonisins, zear- tions are those caused by the dermatophytes (Trichophyton
alenone, T-2 toxin, ochratoxin, and certain ergot alkaloids. species, Microsporum species, and Epidermophyton floccosum).
Mushrooms are classified as fungi. Thus, another type of disease These fungi cause infection of the skin, hair, and nails because
that fungi can be responsible for is mycetismus, or mushroom they require dead keratin for growth. Infections caused by
poisoning, when the ingestion of toxin-producing mushrooms these fungi are also sometimes known as “ringworm” or “tinea.”
occurs. Potentially deadly mushrooms include Amanita phal- They do not possess any invasive factors and the symptoms
loides, Amanita verna, Amanita virosa, and certain other species associated with these agents are caused by the inflammatory
that contain neurotoxins. response of the host.
The fourth type of mycotic disease is infection with clinical If the fungi penetrate the physical barriers, there are a variety
manifestations caused by the presence or growth of the fungi on of innate mechanisms for recognizing the organism. Innate im-
or in the host tissue. Grouping the fungi according to their clinical mune cells express various pattern-recognition receptors (PRRs)
manifestation is useful, both from a diagnostic standpoint and to that recognize specific structures and molecules present on bac-
facilitate identification of the agents in the laboratory. The super- teria and fungi. These structures and molecules of the organism,
ficial mycoses are fungal diseases that are restricted to the outer called pathogen-associated molecular patterns (PAMPs), are con-
layers of the skin. These infections are cosmetic in nature and are served among microbial species. PRRs on the innate immune
not life threatening. Cutaneous mycoses are those infections that cells (phagocytes, macrophages, and dendritic cells [DCs]) ini-
involve the keratinized body areas (skin, hair, nails) and rarely tiate the immune response by sensing and recognizing the pres-
invade deeper tissue. Although not life threatening, these infec- ence of PAMPs present on the bacteria, fungi, or viruses.
tions produce itchiness and cracking of the skin. Diseases of hair There are several classes of PRRs that can be either trans-
and nails are termed dermatomycoses. The fungi involved are membrane proteins located on the cell surface or cytoplasmic
 Table 22–4 Mycotic Infections Based on Site of Infection and Level of Tissue Involvement
CLASSIFICATION REPRESENTATIVE AGENT DISEASE
Superficial Malassezia furfur Pityriasis versicolor (skin)
Phaeoanellomyces werneckii Tinea nigra (skin)
Piedraia hortae Black piedra (hair)
Trichosporon species White piedra (hair)
Cutaneous Trichophyton species Tinea (e.g., ringworm, athlete’s foot, jock itch)
Microsporum species
Epidermophyton floccosum
Subcutaneous Sporothrix schenckii Sporotrichosis
Fonsecaea pedrosoi Chromoblastomycosis
Pseudallescheria boydii Eumycotic mycetoma
Systemic Histoplasma capsulatum Histoplasmosis (endemic in Ohio and Mississippi river valleys)
Coccidioides immitis Coccidioidomycosis (endemic in the southwestern United
Paracoccidioides brasiliensis States)
Blastomyces dermatitidis Paracoccidioidomycosis (endemic in Central and South
Penicillium marneffei America, primarily Brazil)
Cryptococcus neoformans Blastomycosis (predominantly a veterinary pathogen in Ohio
and Mississippi river valleys)
Penicilliosis marneffei (predominantly seen in AIDS patients in
Southeast Asia)
Cryptococcosis (causes meningitis in immunosuppressed
patients [e.g., AIDS])
Opportunistic Candida albicans Candidiasis
Aspergillus species Aspergillosis
Rhizopus species Zygomycosis
Commensalistic Candida albicans Urinary tract infections, vaginal yeast infections
Malassezia furfur Tinea versicolor (pityriasis versicolor), dandruff, and seborrheic
dermatitis

proteins contained within the cell. The four different classes chitins (the main component of fungal cell walls). The CLR
include the Toll-like receptors (TLRs) and the C-type lectin transmembrane protein, dectin 1, recognizes β-glucans. Dectin
receptors (CLRs), which are transmembrane proteins, and the 2 recognizes high-mannose structures that are common to many
retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and fungi and binds hyphal forms with higher affinity than yeast
the NOD-like receptors (NLRs), which are cytoplasmic pro- forms.62 Individuals with genetic deficiencies in CLRs are highly
teins (see Chapter 3). The recognition of PAMPs by the PRRs susceptible to fungal infections.63-65
upregulates the inflammatory response.54 Of these, the TLRs There has been a debate as to the roles of cell-mediated im-
play the most important role in defense against pathogenic mi- munity versus humoral immunity in host defense to fungal in-
crobial infection by stimulating production of inflammatory fections. It is now accepted that the cell-mediated immune
cytokines and type I interferons.55,56 In addition to playing an response plays the most important role in the adaptive response
important role in innate responses, TLRs are involved in shap- to fungal infections.66 Once the cells of the innate immune sys-
ing adaptive immunity. tem have been activated, cytokines and chemokines are pro-
To date, 10 different TLRs have been identified in humans duced in response to the infection. Chemokines play a vital role
and 13 TLRs have been discovered in mice.57-60 Each TLR rec- in the recruitment of T cells to the site of the infection.
ognizes specific PAMPs found on viruses, bacteria, fungi, and Chemokines also aid in the formation of the adaptive immune
parasitic-protozoa and helminths. Several fungal components response to fungi, specifically, those mediated by Th1 and Th2
are recognized by TLRs, including phospholipomannans and cells. Th1 cells have been shown in mice to be vitally important
β-glucans, which are recognized by TLR2, and glucuronoxy- to clearance of the organism in cryptococcosis and histoplas-
lomannans, which are recognized by CD14 and TLR4.61 mosis.67-69 Once the T cells have committed themselves in re-
Although TLRs play a role in the immunologic response to sponse to the fungi, they express an effector function through
fungi, CLRs are central for fungal recognition and for the induc- the release of cytokines, primarily IFN-α, TNF-α, and IL-17/22,
tion of the innate and adaptive immune responses to fungi. Fun- contributing to protective immunity to pathogenic fungi.70-72
gal cell walls consist mainly of multiple layers of carbohydrates, Although there is a humoral response to fungal infections,
including mannans (cell wall polysaccharide), β-glucan, and the evidence that antibodies contribute to effective defense
against fungal infections is not conclusive. One study has fungi in bronchial lavage fluid or blood. However, molecular-
shown that humoral immunity can protect against experimen- based diagnostic methods for fungal infections are in their in-
tal fungal infections if certain types of protective antibodies are fancy. In fact, none of the molecular assays are even included
present in sufficient quantities.73 The main functions of anti- in the criteria of the European Organization for Research and
bodies in fungal infections include opsonization, ADCC, pre- Treatment of Cancer/Invasive Fungal Infections Cooperative
vention of adherence, and toxin neutralization.74 However, Group and the National Institute of Allergy and Infectious Dis-
there is little evidence supporting the role of antibodies in host ease Mycoses Study Group (EORTC/MSG) for the definition of
defenses in naturally acquired infections. The absence of an invasive fungal disease.81
association between deficiencies in specific antibodies and sus-
ceptibility to fungal infections in patients with progressive Fungal Pathogens
fungal infections also provides evidence against a protective
role of antibodies in fungal infections.66 The following discussion provides information on five of the
fungal infections for which serological testing is used.
Laboratory Diagnosis
of Fungal Infections Aspergillus is a ubiquitous fungus that is found worldwide in
Because of the increased numbers of individuals who are im- nature (Fig. 22–5). The genus has over 185 species and can
munosuppressed, the incidence of invasive fungal infections be recovered from soil and plant material; its spores (conidia)
associated with significant morbidity and mortality has risen may be recovered from the indoor air environment.82 A fumi-
dramatically in the past several decades.75-79 The clinical diag- gatus is the most commonly isolated species, with Aspergillus
nosis of fungal infections in many instances is difficult. The cli- flavus and Aspergillus niger being the other frequently recovered
nician must consider the patient’s symptoms, history of other species. Less commonly recovered isolates include Aspergillus
past or present infections, medical treatments, the patient’s oc- clavatus, the Aspergillus glaucus group, Aspergillus nidulans, As-
cupation, place of residence, and record of travel in evaluating pergillus oryzae, Aspergillus terreus, and Aspergillus versicolor. In
the likely cause of a fungal infection. Available laboratory meth- humans, Aspergillus is primarily an opportunistic pathogen
ods for diagnosing fungal infections include isolation of the causing a variety of infections. Infections in the immunocom-
organism in the laboratory, histopathological evidence of inva- petent individual include Aspergillus otomycosis, which is a su-
sion, skin testing, and serological detection of antigens or perficial infection of the external auditory canal and auricle
antibodies. The traditional “gold standard” is the recovery of (otitis externa). Most of these infections are caused by A niger.
the organism in the laboratory. However, recovery is often dif- Another infection is caused by growth of Aspergillus in the lung
ficult with uncommon fungi. In addition, once the organism cavity, forming a solid mass of hyphae, which can develop into
is isolated, traditional identification methods may take several a fungus ball referred to as an aspergilloma. Pulmonary as-
weeks because of the slow growth of fungi. Several nonculture pergilloma usually occurs in scarred lung tissue or in a preexisting
laboratory methods, including antibody and antigen detection,
are available for fungal pathogens that are commonly encoun-
tered in the clinical laboratory. These include C albicans,
Aspergillus species, Cryptococcus neoformans, and the dimorphic
fungi, Histoplasma capsulatum and Coccidioides immitis.
Although the humoral response to fungi is not the major
defense against fungal infection, antibodies produced against
the invading organisms may be readily detected and can be
used to demonstrate current or past exposure to the agent.
However, in that many fungal infections are opportunistic,
serological diagnosis many times is of little value because of
the fact that immunosuppressed individuals do not reliably
produce antibodies during acute infections. In addition, in
areas where a fungal agent is endemic to a geographic area,
serological testing for antifungal antibodies is of little value
because most people living in those areas will test positive for
the antibodies.
Recently, molecular assays have been used in the detection
and diagnosis of fungal infections.80 Examples include peptide
nucleic acid fluorescence in situ hybridization (PNA-FISH) for
identification of Candida species in blood cultures, real-time
PCR assays for detection of Aspergillus species and P jirovecii
from bronchial lavage fluid, and multiplex PCR coupled with Aspergillus fumigatus, LPCB stain X 450. (Courtesy of
a bead probe fluid array for detection of numerous species of the CDC, Public Health Image Library.)
lung cavity resulting from a previous infection (e.g., tuberculosis, esophagitis, pneumonia, and septicemia may be observed in the
sarcoidosis).83 Some fungal antigens may elicit allergic responses neutropenic patient because of radiation and chemotherapy or
to Aspergillus, causing ABPA, which results in a long-term use of broad spectrum antibiotics.
allergic response. ABPA is thought to be present in 1% to The diagnosis of Candida infections generally involves the
2% of patients with persistent asthma and in approximately recovery of the causative agent. The organisms grow well in
7% of patients with cystic fibrosis.84,85 routine culture media. Direct examination of the clinical ma-
Invasive forms of Aspergillus most frequently begin in the terial through the use of 10% potassium hydroxide (KOH)
lung, resulting from inhalation of the conidia. The organism preparation may also be used to detect Candida species. The
grows and spreads in the lung tissue. Invasive pulmonary As- serological diagnosis of candidiasis is limited because of col-
pergillosis (IPA) may occur in the neutropenic patient because onization by Candida species of the gastrointestinal tract or
of immunosuppression. Disseminated aspergillosis may occur other body sites that can stimulate antibody production in un-
through hematogenous spread to distant sites or by contiguous infected individuals. In addition, immunocompromised pa-
extension from the lung.86 tients may not mount detectable antibody responses. Current
The diagnosis of aspergillosis generally requires a positive recommendations include the combined detection of mannan
tissue biopsy demonstrating the hyphae or a positive culture and anti-mannan antibodies for the specific identification of
for Aspergillus.83 Nonculture methods may also be used to di- Candida species in serum samples.97 These assays can be pos-
agnose invasive aspergillosis. Serological diagnosis is of limited itive 6 days before blood cultures, on average. They also show
utility because the immunosuppressed patient will fail to a very high negative predictive value (greater than 85%).98
mount an antibody response, even with invasive disease.87 The
detection of galactomannan in serum by EIA has increased the
ability to diagnose invasive aspergillosis.88 This assay is offered The genus Cryptococcus includes 19 species of encapsulated
at larger or reference laboratories. Another assay to detect in- yeasts. C neoformans is a major causative agent of human disease
vasive fungal infections, including aspergillosis, is measuring that went from being a rare human pathogen to a significant
β-D-glucan (BDG) in serum. BDG is a component of the cell opportunistic pathogen as the population of immunocompro-
wall of most fungi.89 The assay is approved and included in mised patients increased. C neoformans is a saprobe in nature,
the latest EORTC/MSG diagnostic criteria for the clinical diag- obtaining its nutrition from dead or decaying organic matter.100
nosis of invasive fungal infection.81 Scientists have developed The organism lives in certain trees and rotting wood and has
molecular diagnostics, including PCR, for Aspergillus, which frequently been isolated from soil contaminated by guano from
appear to be more sensitive than other methods, including birds (e.g., pigeons).101,102 C neoformans enters the host mainly
galactomannan,90-93 and show promise in improving the diag- through the lungs and has a predilection for invading the CNS
nosis of invasive aspergillosis.94 of the susceptible host. Although pulmonary infections are
common, Cryptococcal meningoencephalitis represents the pri-
mary life-threatening infection caused by C neoformans. The
Candida ssp. are yeast (unicellular fungi) that may exist as com- pre-AIDS era had an overall incidence of 0.8 cases per million
mensalistic organisms in the human host. They are commonly
found on skin, the gastrointestinal (GI) tract, and in the female
genital tract. Of the more than 150 species of Candida, only a
small percentage is regarded as frequently pathogenic for hu- Connections
mans. C albicans is the leading cause of human infections. Other Predictive Value
members include Candida guilliermondii, Candida krusei, Candida
The predictive value of a laboratory test is the likelihood that the
parapsilosis, Candida tropicalis, Candida pseudotropicalis, Candida
results it produces will lead to an accurate diagnosis. The pre-
lusitaniae, Candida dubliniensis, and Candida glabrata. Following dictive value depends on the sensitivity and specificity of the
the introduction of antibiotics, Candida infections rose dramat- method, as well as the prevalence of the disease in the popula-
ically in the United States. In recent decades, Candida species tion undergoing testing (see Chapter 9). A negative predictive
have been the fourth most common organisms recovered from value is the likelihood that a negative test result is truly negative.
the blood of hospitalized patients in the United States.95 The Thus, an 85% negative predictive value means that there is an
incidence of candidemia-related hospitalizations has risen by 85% chance that the patient does not have the disease in ques-
52% between 2000 to 2005.96 Increased use of immunosup- tion. In general, negative predictive values are higher in popula-
pressive therapies, the use of advanced life support therapies, tions with a low disease prevalence.
and the incidence of HIV-1 infection have all contributed to the Over the years, tests for various serum antigens have been
increased incidence of Candida infections. Infections in the im- used to detect invasive Candida infections, including latex ag-
glutination (LA), counterimmunoelectrophoresis, or ELISA-based
munocompetent host include diaper rash, vaginitis and urinary
assays; however, these have not proven to be useful. The use of
tract infections in the female, and thrush (oral candidiasis) molecular-based assays shows promise. Direct PCR of blood
caused by the use of broad spectrum antibiotics. Individuals samples is a sensitive and specific method for the diagnosis
with thrush for no obvious reason should be evaluated for of invasive candidiasis and may be used for early diagnosis of
AIDS. The introduction of potent antiretroviral therapy has re- specific Candida species.99
duced the incidence of thrush in AIDS patients. Candida
persons per year. In 1992, during the peak of the AIDS epi- easy to perform and interpret and has high specificity, it shows
demic, the rate in several large U.S. cities reached almost five poor sensitivity (50% to 80%).110 Detection of cryptococcal
cases of cryptococcosis per 100,000 persons per year. With the polysaccharide antigen in serum and CSF can be performed
use of antiviral agents for the treatment of AIDS in developed by LA and enzyme immunoassays (EIA). Both types of tests are
countries, these numbers have declined.103,104 more than 90% sensitive and specific.111 Although the LA test
The most important feature contributing to the organism’s is a rapid and reliable serological method for the diagnosis of
virulence and pathogenicity is its capsule. The capsule con- cryptococcosis, false-positive LA test results can occur. False-
sists of polysaccharide containing an unbranched chain of positive results are thought to be caused by rheumatoid factor
alpha-1,3-linked mannose units substituted with xylosyl and or other interference factors.112-116 Pretreating the specimen
β-glucuronyl groups. The capsule has many effects on the with heat and pronase, or 2-Mercaptoethanol, destroys these
host. These include acting as a barrier to phagocytosis, de- factors and reduces the incidence of false-positive test results.110
pleting complement, producing antibody unresponsiveness, Recently, a lateral flow immunochromatographic assay
dysregulating cytokine secretion, interfering with antigen (LFA) (Immy, Inc., Norman, OK) was approved by the U.S.
presentation, and enhancing HIV replication.105 Food and Drug Administration for detection of cryptococcal
The two most common sites for infection with this encap- antigen in CSF and serum (Fig. 22–7). The assay allows for
sulated yeast are the lungs and CNS, with the respiratory tract the detection of cryptococcal antigen in fewer than 15 minutes.
being the most common portal of entry. The majority of cryp- The assay has been shown to have a sensitivity of 100% and a
tococcal infections are asymptomatic, as suggested by the fact specificity of 99.8% with serum samples,117 as well as a sensi-
that serological and hypersensitivity testing indicate a higher tivity of 99.3% and a specificity of 99.1% with CSF.118
incidence of infections than the actual incidence of cryptococ-
cosis.106,107 The majority of cryptococcal infections are diag-
nosed in individuals with compromised immune systems.
Respiratory symptoms range from asymptomatic colonization
of the airway to life-threatening pneumonia with evidence of
an acute respiratory distress syndrome.108 Individuals with
CNS involvement generally present with subacute meningitis
or meningoencephalitis. Symptoms include headache, fever,
lethargy, and memory loss over several weeks. Some patients
may present with an acute onset of meningitis.109
The laboratory diagnosis of Cryptococcus infections may be
done using direct microscopic examination of the clinical ma-
terial, culturing for the organism, or performing serological
tests. Microscopic examination of the organism can be per-
formed by mixing the biological fluid (usually CSF) with India A
ink. The encapsulated yeast displaces the India ink, creating a
halo around the yeast cell (Fig. 22–6). Although the test is

IMMY CrAg® LFA (cryptococcal antigen lateral flow

assay). (A) Kit components. The assay is a sandwich immunochro-
matographic dipstick assay for the qualitative and semiquantitative
detection of cryptococcal antigen from CSF and serum. (B) Test re-
sults: The dipstick on the left shows one line for the control; the test
India ink prep showing Cryptococcus neoformans in sample is negative. The dipstick on the right shows two lines, indi-
CSF. The organism’s large polysaccharide capsule displaces the India cating a valid control result and the presence of cryptococcal anti-
ink, allowing for visualization of the yeast. (Courtesy of the CDC/Dr. gen in the test sample. (Reprinted by permission Immuno-Mycologics,
Leanor Haley, Public Health Image Library.) Inc. [IMMY], Norman, Oklahoma.)
 Measurement of cryptococcal antigen levels has been shown Connections
to have some clinical use. Serial polysaccharide antigen titers
can be performed on serum or CSF. Initial high titers (1:1,024 “-emia” and “-uria”
or higher) indicate high levels of the yeast in the host and poor The suffix “–emia” indicates the presence of a substance in the
host immunity. These individuals have a greater chance of blood, whereas the suffix “–uria” indicates the presence of a sub-
therapeutic failure. As is the case in many infectious diseases, stance in the urine. Thus, “antigenemia” means that a particular
increasing antigen titers are usually seen with worsening of the antigen (in this case, Histoplasma antigen) can be found in the
disease, whereas declining titers are usually associated with blood, whereas “antigenuria” means that detectable antigen is
clinical improvement. Cryptococcal antigen may be detected present in the urine.
even months after successful therapy, so cryptococcal antigen
tests should not be used to indicate cure.
test becomes positive 2 to 4 weeks after infection and the majority
of infected people are asymptomatic. Repeat testing will usually
H capsulatum is a common cause of infection in the Ohio and give positive results for the rest of a person’s life. Thus, in areas
Mississippi river valleys. The organism is a dimorphic fungus, where the fungus is endemic, the skin test is of limited utility.
growing as a yeast at 35°C to 37°C and as a mycelial fungus Laboratory diagnosis may be made by detecting the polysac-
at environmental temperatures. The organism is found and charide antigen in serum or urine by ELISA. Urinary antigen
grows in soil under favorable conditions. The presence of bird tests have been shown to have a high sensitivity and specificity
and bat guano increases the likelihood of the organism being for detecting Histoplasma infections.121 Histoplasma antigen was
present in the soil. Whereas birds are not infected with the detected in the urine in 95% of AIDS patients with disseminated
fungus, bats carry the fungus in their gastrointestinal tracts disease and in the serum of 86% of these patients.122 Ninety-
and shed it.119 Histoplasmosis is acquired by inhalation two percent of patients without AIDS who have disseminated
of mycelial fragments and microconidia. A majority of the histoplasmosis demonstrate antigenuria, but only about 50%
infections (more than 90%) are self-limiting and asympto- have antigenemia.123,124 Molecular assays appear to have promise
matic. The organism can cause a potentially lethal infection in the diagnosis of histoplasmosis; however, at this point, no mo-
in patients with preexisting conditions. The organism causes lecular assays for routine use are commercially available.
opportunistic infection in patients with weakened immune sys-
tems (e.g., people with HIV infection). Of those that are symp-
tomatic, pulmonary histoplasmosis, either acute or chronic, is Similar to H capsulatum, C immitis is also a dimorphic fungus.
the most frequent clinical presentation. In a primary infection, For many years, coccidioidomycosis was thought to be a rare
the organism disseminates to organs rich in mononuclear and nearly always fatal infection. In 1929, a medical student at
phagocytes. Disseminated disease in the immunocompromised Stanford University was accidentally exposed to the organism
patient is 100% fatal if untreated. Survival rates of the acute and experienced only a mild respiratory infection. His survival
episode exceed 80% when treated. resulted in a reassessment of the coccidioidal infections. Coc-
The diagnosis of histoplasmosis can be made by culture of cidioidomycosis was initially recognized as a common respira-
the organism. The organism is generally recovered within 3 weeks tory condition in the San Joaquin Valley of California (valley
of culture, with 90% of samples exhibiting growth within fever).125 Although originally thought to be only found in the
7 days. The ability to recover the organism is affected by the dry, arid regions of the southwestern United States, coccid-
number of specimens submitted for culture, the type of speci- ioidomycosis has become more prevalent throughout both
men submitted (e.g., respiratory secretions, blood, or CSF), and nonendemic and endemic regions of the world.126 The increased
the severity of the infection.120 Complement fixation (CF) and incidence of coccidioidomycosis can be mostly attributed to
precipitation have been the most common tests used for detec- changes in demography. Populations at risk of exposure are
tion of Histoplasma antibodies in the clinical laboratory. For greatly expanded. Regions in which C immitis is endemic have
CF antibodies, a titer of 1:8 to yeast or mycelial antigen is con- major metropolitan centers, such as Phoenix, Arizona. These
sidered positive and a titer of 1:32 may indicate histoplasmosis. areas were previously sparsely populated. Along with increased
Precipitin band testing looks for the presence of H and M anti- population growth in the southwestern United States, there has
bodies in the serum of individuals. The H and M antigens are been increased tourism leading to movement of people into and
glycoproteins released by mycelial and yeast phase cultures. out of endemic areas, increasing the numbers of people who are
H antibodies are detected in fewer than 10% of patients but, exposed to the organism and acquire coccidioidal infections.127
when present, signify active infection. Antibodies to the M anti- In a vast majority of the cases, infections are the result of in-
gen are detected in up to 80% of individuals who have been haling arthroconidia. A single arthroconidium can be enough
exposed to the fungus. These antibodies may be found in indi- to produce a naturally acquired respiratory infection.128 Up to
viduals who have previously been exposed to the organism or two-thirds of cases caused by C immitis are either inapparent or
who have active disease; thus, it is not useful in discriminating so mild as to not prompt medical evaluation.129 In those indi-
previous from current infection. Skin testing, similar to the viduals who do become symptomatic, the clinical presentation
Mantoux tuberculin skin test, may also determine if an individ- is similar to a community-acquired pneumonia. Coccidioides
ual has been exposed to the organism. The H capsulatum skin was estimated to be responsible for approximately one-third
of all cases of community-acquired pneumonia in southern
Arizona.130 SUMMARY
Once inhaled, there is an incubation period of 1 to 3 weeks
before the development of respiratory symptoms. Thus, travel • Parasites are microorganisms that survive by living off of
history is important if the person has traveled to an endemic other organisms. The three major types of parasites are
area. Once coccidioidomycosis is suspected, diagnosis may be protozoa, helminths, and ectoparasites.
established by identifying the fungus in, or recovering C immitis • Parasitic infections can result in eradication of the organ-
from, a clinical specimen or by detecting anticoccidioidal an- ism by the host’s immune system, death of the host be-
tibodies in serum, cerebrospinal fluid, or other body fluid. The cause of an ineffective immune response, or, in most cases,
most frequent way in which coccidioidomycosis is diagnosed establishment of persistent infection because of the host’s
is through serology. Although the organism can be cultured, inability to completely eliminate the organism.
obtaining a sputum specimen in individuals is not often pos- • Innate defenses (e.g., phagocytosis and cytokine release)
sible because of the lack of sputum production in infected in- and specific humoral and cell-mediated adaptive re-
dividuals. In addition, fungal culturing in an outpatient setting sponses can be demonstrated against parasites. IgE anti-
is not available. The use and limitations of skin testing for Coc- bodies and eosinophils can destroy some parasites by
cidioides infection are similar to those for Histoplasma. Once ADCC and many patients with parasitic infections have
positive, the skin test generally remains positive for the rest of increased levels of IgE and eosinophil numbers in the
one’s life; therefore, it is of limited utility in individuals living blood.
in endemic areas. • Because of their complex, multistage life cycles, the im-
Complement fixation, immunodiffusion (ID), and LA have mune responses to the agents responsible for parasitic dis-
been the most commonly used serological methods for detect- eases are often ineffective.
ing infection. One of the original methods for detecting infec- • Parasites have also developed a number of strategies to
tion was the tube precipitin test (TPT). The detection of IgM avoid the immune system, including antigenic conceal-
antibodies by this test has been used to demonstrate infection. ment, antigenic variation, antigenic shedding, antigenic
The test involves overnight incubation of the patient’s serum mimicry, immunologic subversion, and immunologic
with coccidioidal antigen. Formation of a precipitin button at diversion.
the bottom of a test tube demonstrates the presence of IgM an- • Fungi are eukaryotic cells with nuclei and rigid cell walls.
tibodies. A polysaccharide from the fungal cell wall is used as They can exist in two morphological states: unicellular
the antigen in the test. Tube precipitin antibodies are detected yeasts or multicellular mycelia fungi.
in 90% of patients within the first 3 weeks of symptoms.131 • Fungal infections are known as mycoses. They can pro-
This test is often referred to as the “IgM test.” duce hypersensitivity, mycotoxicosis (a poisoning caused
The other original test involved detection of coccidioidal by fungal toxins), mycetismus (mushroom poisoning),
antibodies using a CF assay. When mixed with a coccidioidal skin infections, or systemic infections that disseminate
antigen, an immune complex is formed with the patient’s an- from the lungs or skin to other sites of the body.
tibodies, resulting in depletion of complement. When the com- • Innate defenses including physical barriers such as the
plement is depleted, antibody-coated red blood cells (RBCs) skin and attachment of immune cells to pattern-recognition
added to the mixture fail to lyse. This test is often referred to receptors are an important first line of defense against fun-
as the “IgG test” because IgG is the immunoglobulin class usu- gal infections. Cell-mediated immunity is the most impor-
ally involved in the formation of this type of immune complex. tant adaptive immune response to fungal infections.
CF antibodies are detected later and for longer periods than • The diagnosis of many parasitic and fungal infections relies
tube precipitin-type antibodies. Immunodiffusion assays for on traditional laboratory techniques either by the direct
detecting both IgG and IgM antibodies are available and have observation of the parasite in clinical material (feces, duo-
served as replacements for the earlier TPT and CF tests. denal fluid, small intestine biopsy specimen, or blood) or
EIAs for coccidioidal antibodies are commercially available. by culture and growth of the fungal agent responsible for
The EIA assays allow for the specific detection of IgM or IgG the infection.
antibodies. A positive EIA result is a highly sensitive indicator • Serological testing for parasitic and fungal diseases is less
for coccidioidal infection.132 Although the EIA tests for IgM routine than serology for other infectious diseases because
and IgG are more sensitive than immunodiffusion tests, false test availability is limited and assays exhibit variable per-
positives have been reported.133 At present, it is recommended formance with respect to sensitivity and specificity. Many
that positive EIA results, particularly positive IgM results, of these tests are only offered by reference laboratories.
should be confirmed with immunodiffusion tube precipitin or • Serology results should be used in conjunction with the
complement-fixing test results. Similar to H capsulatum, there patient’s symptoms, history, and other clinical findings to
are no commercially available molecular assays for the detec- make a diagnosis of a fungal or parasitic infection.
tion of Coccidioides infection.
• Toxoplasmosis is an example of a parasitic infection for involve detection of fungal antibody or antigen, depend-
which serological testing is useful. Detection of IgG, IgM, ing on the organism.
and IgA antibodies to T gondii is helpful in determining • To date, there are only a few commercial molecular tests
whether the infection has been acquired recently or in the that are available for the diagnosis of parasitic and invasive
past and whether an infant has acquired a congenital in- fungal infections, but advances in molecular technology will
fection with the organism. likely result in new diagnostic procedures in the future.
• Fungal infections for which serological tests are useful in-
clude aspergillosis, candidiasis, cryptococcosis, histoplas-
mosis, and coccidioidomycosis. Serological testing can

Study Guide: Escape Mechanisms of Parasites from Protective Host Responses

ESCAPE MECHANISM NATURE OF RESPONSE EXAMPLE(S)
Antigenic concealment Intracellular survival within macrophages Leishmania donovani
Antigenic variation Random mutation Plasmodium species
Genetic recombination Plasmodium falciparum, Trypanosoma cruzi
Gene switching Trypanosoma gambiense, Trypanosoma
Multistage parasitic life cycle rhodesiense
Leishmania species
Antigenic shedding Shedding of surface antigens or components Entamoeba histolytica
Antigenic mimicry Incorporation of host “self” antigens into Schistosoma species
parasite surface
Immunologic subversion Immunosuppression Schistosoma mansoni
Immunologic diversion Polyclonal B-cell activation Plasmodium species

CASE STUDIES
1. An otherwise healthy infant developed a seizure 5 days 2. A 60-year-old male with a medical history of chronic ob-
following birth. The mother and baby returned to the structive pulmonary disease (COPD), diabetes mellitus,
hospital for evaluation. Upon examination of the infant, and hepatitis C was seen in the emergency department.
the physician found that the baby demonstrated chorio- The patient admitted to using intravenous drugs in the
retinitis. Prescreening of the mother during the third past and has been receiving inhaled steroid therapy for his
trimester of pregnancy did not demonstrate the presence COPD. He complained of nausea and severe headaches,
of antibodies against T gondii. At that time, she was advised which interfered with his ability to carry out his normal
to refrain from cleaning the litter boxes of the family’s two activities. The patient also felt unbalanced and weak when
cats. Upon questioning, the mother stated that she had standing. Head computed tomography (CT) and magnetic
been cleaning the litter boxes when other family members resonance imaging (MRI) revealed abnormalities in the
failed to do so. The physician suspected that the child may cerebellum of the patient’s brain. Cryptococcal meningitis
have congenital toxoplasmosis. Serological testing of the was suspected. A lumbar puncture was performed and a
mother and the fetus gave the following results: CSF sample was sent for laboratory testing to confirm the
suspected diagnosis.
Anti- Anti- Questions
T gondii IgM T gondii IgG a. What clinical presentations of the patient point to a
Mother 1:256 1:512 diagnosis of cryptococcosis?
Baby Not done 1:256
b. What laboratory tests should be performed to confirm
the patient’s diagnosis?

Questions
a. Evaluate the baby’s status related to T gondii infection.
b. What additional testing should be performed to con-
firm the baby’s status?
REVIEW QUESTIONS
1. Compared with a host’s response to the mumps virus, 7. In congenital toxoplasmosis, which class of antibodies
overcoming a parasitic infection is more difficult for is the most sensitive in detecting infection?
the host because of which of the following characteris- a. IgA
tics of parasites? b. IgG
a. Large size c. IgM
b. Complex antigenic structures d. IgE
c. Elaborate life cycle
d. All of the above 8. The most significant defense against fungal infections is
a. cellular immunity.
2. Which of the following is indicative of a recent b. humoral immunity.
infection with Toxoplasma gondii? c. phagocytosis.
a. Anti-Toxoplasma IgM d. complement activation.
b. Anti-Toxoplasma IgE
c. High avidity anti-Toxoplasma IgG 9. Clinical manifestations of fungal-related illness
d. Low avidity anti-Toxoplasma IgG include
a. hypersensitivity caused by fungal spores.
3. Parasites are able to evade host defenses by which of b. poisoning caused by ingestion of mycotoxins.
the following means? c. growth of fungi in or on tissue.
a. Production of antigens similar to host antigens d. all of the above.
b. Changing surface antigens
c. Sequestering themselves within host cells 10. Which of the following assay formats are increasingly
d. All of the above being adopted by clinical laboratories for serological de-
tection of fungal infections because of their ease of use?
4. The chronic nature of parasitic infections is caused by a. ELISA assays
the host’s b. Lateral flow assays
a. inability to eliminate the infective agent. c. Radial immunodiffusion assays
b. type I hypersensitivity response to the infection. d. Indirect immunofluorescence assays
c. ability to form a granuloma around the
parasite. 11. The presence of anti-H antibodies indicates which of
d. tendency to form circulating immune the following?
complexes. a. A previous infection with Coccidioides immitis
b. A previous exposure to Histoplasma capsulatum
5. The presence of both IgM and IgG antibody in toxo- c. An active infection with Cryptococcus neoformans
plasmosis infections suggests that the infection d. An active infection with Histoplasma capsulatum
a. occurred more than 2 years ago.
b. occurred more recently than 18 months ago. 12. A limiting factor in reliably being able to detect anti-
c. is chronic. fungal antibodies in an acute infection is
d. has resolved itself. a. the lack of humoral response to fungal agents
caused by immunosuppression.
6. Which of the following is indicative of a parasitic b. current assays lack specificity.
infection? c. antibodies are not normally formed against most
a. Increased IgA levels fungi.
b. Increased IgE levels d. antibodies tend to remain at low titer as a mycosis
c. Increased IgG levels develops.
d. Increased IgM levels
13. False positives may be observed in latex agglutination 15. Which of the following serological tests detects the
tests for the capsular antigen of Cryptococcus neofor- polysaccharide capsule antigen in serum and CSF of
mans because of patients with suspected infection with Cryptococcus
a. the use of serum instead of CSF. neoformans?
b. the presence of rheumatoid factor in the a. Complement fixation (CF)
specimen. b. India ink test
c. cross-reactivity with other fungal antigens. c. Latex agglutination (LA)
d. the low specificity of the assay. d. Hemagglutination test

14. A 27-year-old man from Ohio, diagnosed with AIDS, 16. Which of the following is a nondimorphic fungus
developed chest pains. After a short period of time he that is found in concentrated bird droppings and
also developed severe headaches with dizziness. In his can readily cause meningitis in immunocompromised
free time, his hobby was exploring caves (a spelunker). individuals?
His physician ordered a sputum culture and spinal tap a. Coccidioides immitis
and both were positive for a yeastlike fungus. These b. Candida albicans
findings are most consistent with infection by c. Cryptococcus neoformans
a. Candida albicans. d. Histoplasma capsulatum
b. Coccidioides immitis.
c. Cryptococcus neoformans.
d. Histoplasma capsulatum.
 Serology and Molecular
Detection of Viral
Infections

After finishing this chapter, you should be able to: IMMUNE DEFENSES AGAINST VIRAL
INFECTIONS
1. Describe the immune defenses that are important in protecting
humans from viral infections. VIRAL ESCAPE MECHANISMS
2. Discuss mechanisms by which viruses can escape host defenses. LABORATORY TESTING FOR VIRAL
INFECTIONS
3. Correlate the presence of viral IgM and IgG antibodies with their clin-
ical significance in detecting current infections, congenital infections, HEPATITIS VIRUSES
or immunity to infections. Hepatitis A
4. Discuss the role of molecular tests in diagnosing and monitoring Hepatitis E
patients with viral infections. Hepatitis B
5. Differentiate between the different hepatitis viruses and their modes Hepatitis D
of transmission.
Hepatitis C
6. Correlate the various serological markers of hepatitis with their
HERPES VIRUS INFECTIONS
diagnostic significance.
Epstein-Barr Virus
7. Explain the laboratory methods that are most commonly used to
screen for, confirm, or monitor hepatitis virus infections. Cytomegalovirus
8. Associate the following viruses with the specific diseases they cause: Varicella-Zoster Virus
Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster OTHER VIRAL INFECTIONS
virus (VZV), rubella virus, rubeola virus, mumps virus, and the Rubella
human T-cell lymphotropic virus type I.
Rubeola
9. Discuss the laboratory methods used to diagnose and monitor
Mumps
infections with the preceding viruses.
Human T-Cell Lymphotropic Viruses
10. Correlate the heterophile antibody and EBV-specific antibodies with
their clinical significance and describe the laboratory methods used to SUMMARY
test for these antibodies. CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Anti-HBe Hepatitis B surface antigen Hepatitis E virus (HEV) Mumps virus
Anti-HBs (HBsAg) Heterophile antibodies Parenteral
Cytomegalovirus (CMV) Hepatitis B virus (HBV) Human T-cell lymphotropic Rubella virus
Epstein-Barr virus (EBV) Hepatitis Be antigen virus type I (HTLV-I) Rubeola virus
(HBeAg) Human T-cell lymphotropic
Hepatitis Varicella-zoster virus (VZV )
Hepatitis C virus (HCV) virus type II (HTLV-II)
Hepatitis A virus (HAV)
Hepatitis D virus (HDV) IgM anti-HBc

Viruses are submicroscopic pathogens whose size is measured 8. Release

in nanometers. Their basic structure consists of a core of DNA 1. Attachment
or RNA packaged into a protein coat or capsid. In some viruses,
the capsid is surrounded by an outer envelope of glycolipids and
proteins derived from the host cell membrane (Fig. 23–1). It is
remarkable that these tiny particles are capable of causing severe,
and sometimes lethal, disease in humans, ranging from child- 2. Penetration
hood infections to inflammatory diseases with a predilection for 6. Assembly
a specific organ, disseminated disease in immunocompromised 3. Uncoating
7. Budding
patients, cancer, and congenital abnormalities.
Viruses are obligate intracellular pathogens that rely on the
host cell for their replication and survival. They infect their host mRNA
cells by attaching to specific receptors on the cell surface; pen- 5. Translation
etrating the host cell membrane; and releasing their nucleic Viral
acid, which then directs the host cell’s machinery to produce genome
more viral nucleic acid and proteins. These components assem-
ble to form intact viruses that are released by lysis of the cell or 4. Transcription
by budding off the cell’s surface (Fig. 23–2). Replication can
occur quickly in cytolytic viruses that produce acute infections, Basic steps of a virus life cycle. (1) Attachment of the
or slowly in viruses that result in chronic infections. The free virus to a receptor on the host cell surface. (2) Penetration, or entry
virions that are generated can then infect neighboring host cells of the virus into the host cell through endocytosis or other mecha-
and begin new replication cycles that promote dissemination nisms. (3) Uncoating, or degradation of the viral capsid and subse-
of the infection. Thus, viruses can be present in the host as both quent release of viral nucleic acid. With some viruses, the nucleic
freely circulating particles and intracellular particles. This chap- acid integrates into the host cell genome. (4) Transcription to pro-
ter briefly addresses the immunologic mechanisms required to duce additional viral nucleic acid. (5) Translation of viral nucleic acid
to produce viral proteins. (6) Assembly of the viral components to
attack the virus in its different states. Successful defense against
produce intact virions. (7) Budding off the host cell membrane or
viral infections requires a coordinated effort among innate, hu- host cell lysis results in (8) release of viral progeny. Modifications of
moral, and cell-mediated immune responses (Fig. 23–3). The these steps can occur with different viruses.
remainder of the chapter discusses some of the most important

Envelope protein viral infections detected by serology and molecular methods.

These include the hepatitis viruses, herpes viruses, measles,
Envelope mumps, rubella, and human T-cell lymphotropic viruses. Lab-
oratory tests for the human immunodeficiency virus (HIV) are
Tegument discussed separately in Chapter 24.

Capsid
Immune Defenses Against
Viral DNA or RNA Viral Infections
Innate immunity provides the first line of protection against
viral pathogens. Viruses first encounter naturally occurring
barriers in the body such as the skin and mucous membranes.
If they are able to invade these barriers, other innate defenses
are activated when cells of the innate immune system recognize
Basic structure of a virus. pathogen-associated molecular patterns (PAMPs) on the surface
 Epithelium

B
G
B A

ADCC
C
H
NK

MHC-unR

D MHC-R
Tc
E

B
F

Innate defenses provide the initial barrier to viral infection. Infected cells release interferons α and β, which (A) inhibit viral
replication in surrounding cells and (B) stimulate natural killer (NK) cells. Both NK and cytotoxic T (Tc) cells destroy virus-infected host cells
(C, D), resulting in the release of free virions (E). Virus-specific B cells recognize these free virions (F), as well as virions that have penetrated the
epithelium (G), leading to the production of antibodies (H) that bind free virions and mediate virus neutralization, opsonization, complement
activation, and ADCC.

of or within virus-infected host cells. Two important nonspe- activated.1-3 Virus-specific antibodies are produced by B cells and
cific defenses against viruses involve type I interferons and plasma cells and can attack free virus particles in several ways.
natural killer (NK) cells.1-3 Virus-infected cells are stimulated Antibodies play a key role in preventing the spread of a viral in-
to produce IFN-α and IFN-β following recognition of viral fection through neutralization. This process involves the produc-
RNA by toll-like receptors (TLR). Interferons inhibit viral repli- tion of antibodies that are specific for a component of the virus
cation by inducing the transcription of several genes that code that binds to a receptor on the host cell membrane. When these
for proteins with antiviral activity—for example, a ribonuclease neutralizing antibodies bind to the virus, they prevent it from at-
enzyme that degrades viral RNA. IFN-α and IFN-β also en- taching to and penetrating the host cell. Secretory IgA antibodies
hance the activity of NK cells, which bind to virus-infected play an especially important role in this process because they neu-
cells and release cytotoxic proteins such as perforin and tralize viruses in the mucosal surfaces (e.g., respiratory and di-
granzymes, causing the cells to die and release the viruses. gestive tracts), which often serve as entryways for the pathogens.
These cell-free virions are now accessible to antibody molecules. Meanwhile, IgM and IgG antibodies can bind to viruses in the
When innate defenses are insufficient in preventing viral bloodstream and inhibit dissemination of the infection. In addi-
infection, specific humoral and cell-mediated defenses are tion, IgG antibodies promote phagocytosis of viruses through
their opsonizing activity and promote destruction of viruses
through antibody-dependent cell-mediated cytotoxicity (ADCC).
Connections IgG and IgM antibodies also activate complement, which can me-
Interferons diate opsonization via C3b or lyse enveloped viruses by inducing
formation of the membrane attack complex. IgM antibodies may
Interferons “interfere” with the ability of viruses to replicate by
stimulating infected host cells to produce proteins that degrade
also inactivate viral particles by agglutinating them.
viral nucleic acid and proteins (see Chapter 6). Interferons exert Although antibodies can attack viruses in many different
their effects not only on the original infected cell, but also on ways, they cannot reach viruses that have already penetrated
neighboring uninfected host cells. host cells. Elimination of intracellular viruses requires the ac-
tion of cell-mediated immunity. Type 1 helper (Th1) cells and
cytotoxic T lymphocytes (CTL) play a key role in this mecha- their nucleic acid into the genome of the infected host cells.
nism of defense. Th1 cells produce interferon-γ, which induces In this situation, the virus is only stimulated to replicate again
an antiviral state within the virus-infected cells, and IL-2, if the host is exposed to other infectious agents or if the host’s
which assists in the development of effector CTLs. In this immune defenses decline. Latent viruses can remain silent
process, CD8+ CTL become programmed to expand in number within host cells for years because they are hidden from the
and attack the virus-infected cells.4 To recognize the virus- immune system, although reactivation can occur later in life.
infected host cell, the T-cell receptor (TCR) on the CTL must By using these evasion mechanisms, viruses have established
bind to a viral antigen complexed with class I major histocom- themselves as successful human pathogens that can cause a
patibility complex (MHC) on the surface of the infected cell range of mild to life-threatening diseases. Rapid, reliable labo-
(see Fig. 23–3). CD8 is a co-receptor in this interaction. Inter- ratory detection of these pathogens is essential for early patient
action of costimulatory molecules, such as B7 and CD28, diagnosis and treatment. Laboratory identification also leads to
provides secondary signals necessary for the CTL response. prompt implementation of measures to prevent further spread
These molecular interactions stimulate the granules in the of the virus to other members of the population.
CTL to release a pore-forming protein called perforin, which
produces pores in the membrane of the infected host cell, and
proteases called granzymes, which enter the pores. These Laboratory Testing
enzymes activate apoptosis in the host cell, interrupting the for Viral Infections
viral-replication cycle and resulting in release of assembled
infectious virions. The free virions can then be bound by anti- As our knowledge of viruses has increased, so has the develop-
bodies. The CTL response is powerful and involves a series of ment of laboratory assays to detect viral infections. Serological
cell divisions that can produce up to 50,000 times the original and molecular tests can be easily and rapidly performed by
number of cells in a period of 1 to 3 weeks.4 the clinical laboratory. Therefore, they play an essential role in
helping physicians establish a presumptive diagnosis so that
treatment can be initiated promptly. Serological tests are also
Viral Escape Mechanisms important in monitoring the course of infection, detecting past
infections, and assessing immune status, whereas molecular
Viruses can escape the host’s defense mechanisms in several tests have enhanced our ability to detect active infection and
ways.1-3 First, viruses are rapidly dividing agents that undergo are essential in guiding antiviral therapy.
frequent genetic mutations. These mutations result in the pro- In general, the presence of virus-specific IgM antibodies in
duction of new viral antigens, which are not recognized by the patient serum indicates a current or recent viral infection, whereas
initial immune response to the virus. For example, continual IgG antibodies to a virus signify either a current or past infection
antigenic variation in the influenza virus results in the emer- and, in many cases, immunity. Virus-specific IgM antibody in the
gence of novel infectious strains that require development of newborn’s serum indicates a congenital infection because IgM is
new vaccines every year to protect the population. Antigenic actively made during fetal life. In contrast, IgG antibodies in the
variation is also seen in other viruses, including rhinoviruses, infant’s serum are mainly maternal antibodies that have crossed
which cause the common cold, and HIV, which causes AIDS. the placenta. Current infections in the adult or newborn may also
Second, some viruses can escape the action of components be detected by immunoassays for viral antigens in serum or other
of the innate immune system such as interferons, complement clinical samples or by the presence of viral nucleic acids that can
proteins, or the lysosomal enzymes in phagocytic cells. For ex- be detected by molecular methods.
ample, the hepatitis C virus can block interferon-mediated
degradation of viral RNA and herpes simplex viruses (HSV) pro-
duce a protein that binds to the complement component, C3b, Hepatitis Viruses
resulting in inhibition of the complement pathways.
Third, viruses can evade the host’s defense by suppressing Hepatitis is a general term that means inflammation of the liver.
the adaptive immune system. Some viruses, such as the cy- It can be caused by several viruses and by noninfectious agents,
tomegalovirus (CMV) and HIV, do this by reducing the expres- including ionizing radiation, chemicals, and autoimmune
sion of class I MHC molecules on the surface of virus-infected processes. The primary hepatitis viruses affect mainly the liver.
cells, making them less likely to be recognized by CTLs. Other Other viruses, such as CMV, EBV, and HSV, can also produce
viruses, such as rubeola, can cause decreased expression of class liver inflammation, but it is secondary to other disease processes.
II MHC molecules, resulting in reduced Th cell activity. Some This section will focus on the primary hepatitis viruses. The
viruses can alter the function of certain cells of the immune system hepatitis A virus (HAV) and the hepatitis E virus (HEV) are
after directly infecting them. For example, the Epstein-Barr virus transmitted primarily by the fecal–oral route, whereas the
(EBV) causes polyclonal activation in B lymphocytes, whereas hepatitis B virus (HBV), the hepatitis D virus (HDV), and
HIV suppresses the function of CD4 Th cells. EBV can also inhibit the hepatitis C virus (HCV) are transmitted mainly by the
immune responses by producing a protein that can suppress Th1 parenteral route (i.e., through contact with blood and other body
cells because of its similarity to interleukin-10 (IL-10). fluids). All of the hepatitis viruses may produce similar clinical
Finally, some viruses, such as CMV, varicella-zoster virus manifestations. The early, or acute, stages of hepatitis are charac-
(VZV), and HIV, can remain in a latent state by integrating terized by general flu-like symptoms and mild to moderate pain
in the right upper quadrant (RUQ) of the abdomen.5,6 Progres- more definitively. The specific laboratory tests used to detect
sion of the disease leads to liver enlargement (hepatomegaly) and each type of hepatitis are listed in Table 23–1.
tenderness, jaundice, dark urine, and light feces.
Initial laboratory findings typically include elevations in
bilirubin and in the liver enzymes, most notably alanine
Hepatitis A
aminotransferase (ALT).5,6 These findings are nonspecific in- HAV is a nonenveloped, single-stranded ribonucleic acid (RNA)
dicators of liver inflammation and must be followed by specific virus that belongs to the Hepatovirus genus of the Picornaviridae
serological or molecular tests to identify the cause of hepatitis family.6,7 Two major genotypes of the virus are associated with

Table 23–1 The Hepatitis Viruses and Their Associated Serological and Molecular Markers
PROGRESSION SEROLOGICAL
HEPATITIS TYPE AND TO CHRONIC AND MOLECULAR CLINICAL
VIRUS FAMILY TRANSMISSION STATE COMPLICATIONS MARKERS SIGNIFICANCE
Hepatitis A RNA Fecal-oral No Low risk of • IgM • Acute
(HAV) Picornaviridae Blood fulminant anti-HAV hepatitis A
transfusion liver disease • Total • Immunity to
(rare) anti-HAV hepatitis A
• HAV RNA • Detection
of HAV in
clinical,
food, or
water
samples
Hepatitis B DNA Parenteral, Yes 10% to 90% of • HBsAg • Active
(HBV) Hepadnaviridae sexual, cases may hepatitis B
perinatal develop chronic infection
hepatitis
(depending
on age), with
increased risk
for liver cirrhosis
and hepatocellu-
lar carcinoma
• HBeAg • Active
hepatitis B
with high
degree of
infectivity
• IgM • Current or
anti-HBc recent acute
hepatitis B
• Total • Current
anti-HBc or past
hepatitis B
• Anti-HBe • Recovery
from
hepatitis B
• Anti-HBs • Immunity to
hepatitis B
• HBV DNA • Acute, atypi-
cal, or occult
hepatitis B;
viral load
may be
used to
monitor ef-
fectiveness
of therapy
Table 23–1 The Hepatitis Viruses and Their Associated Serological and Molecular Markers—cont’d
PROGRESSION SEROLOGICAL
HEPATITIS TYPE AND TO CHRONIC AND MOLECULAR CLINICAL
VIRUS FAMILY TRANSMISSION STATE COMPLICATIONS MARKERS SIGNIFICANCE
Hepatitis C RNA Parenteral, Yes Eighty-five percent • Anti-HCV • Current
(HCV) Flaviviridae sexual, develop chronic or past
perinatal infection, with hepatitis C
increased risk infection
of cirrhosis,
hepatocellular
carcinoma, or
autoimmune
manifestations
• HCV RNA • Current
hepatitis C
infection;
viral load
may be
used to
monitor ef-
fectiveness
of therapy;
also used
to deter-
mine HCV
genotype
Hepatitis D RNA Mostly Yes Increased risk • IgM- • Acute or
(HDV) Genus parenteral, of developing anti-HDV chronic
Deltavirus but also fulminant hepati- hepatitis D
sexual, tis, cirrhosis, or
perinatal; hepatocellular
HBV infection carcinoma
required
• IgG- • Recovery
anti-HDV from
hepatitis D
or chronic
hepatitis D
• HDV RNA • Active HDV
infection;
viral load
may be
used to
monitor ef-
fectiveness
of therapy
Hepatitis E RNA Fecal-oral Yes, in Fulminant liver • IgM • Current
(HEV) Hepeviridae Blood immunocom- failure in anti-HEV hepatitis E
transfusion promised pregnant infection
individuals women
• IgG • Current
anti-HEV or past
hepatitis E
infection
• HEV RNA • Current
hepatitis E
infection
human disease and both can be detected by the same serolog- Connections
ical assays (see the text that follows). Hepatitis A is a common
infection responsible for an estimated 1.4 million cases of hep- Reverse Transcriptase Polymerase
atitis worldwide.8 HAV is transmitted primarily by the fecal– Chain Reaction (RT-PCR)
oral route, close person-to-person contact, or ingestion of In RT-PCR, viral RNA is treated with the enzyme reverse transcrip-
contaminated food or water.8,9 Conditions of poor personal tase to generate a complementary DNA (cDNA) sequence. The
hygiene, poor sanitation, and overcrowding facilitate transmis- cDNA is then amplified by the polymerase chain reaction to gen-
sion. Rarely, transmission through transfusion of contaminated erate millions of copies that can be detected in the laboratory
blood has been reported and may occur during a short period (see Chapter 12).
within the acute stage of infection when a high number of viral
particles can be found in the source blood.9
Following an average incubation period of 28 days, the virus been exposed to the virus, prophylactic administration of the
produces symptoms of acute hepatitis in the majority of infected hepatitis A vaccine or injections of immune globulin are rec-
adults; however, most infections in children are asympto- ommended.8,10 The vaccine is the preferred treatment for per-
matic.8,9 The infection does not progress to a chronic state and sons aged 1 to 40 years, but intramuscular injection of immune
is usually self-limiting, with symptoms typically resolving globulin, a sterile preparation of pooled human plasma that
within 2 months. Treatment is mainly supportive, involving bed contains antibodies to HAV, can be used to prevent infection
rest, nutritional support, and medication for fever, nausea, and in individuals of any age. To be effective, these treatments must
diarrhea. Massive hepatic necrosis resulting in fulminant hepa- be administered within 2 weeks of exposure.8
titis and death is rare and occurs mainly in those patients with
underlying liver disease or advanced age.8,10
HAV antigens are shed in the feces of infected individuals
Hepatitis E
during the incubation period and the early acute stage of in- HEV is a nonenveloped, single-stranded RNA virus that be-
fection, but they usually decline to low levels shortly after longs to the genus Hepevirus, in the family Hepeviridae.7,14 HEV
symptoms appear and are not a clinically useful indicator of is a major cause of hepatitis worldwide: the World Health
disease.9 Therefore, serological tests for antibody are critical in Organization (WHO) estimates that annually, the virus causes
establishing diagnosis of the infection. Hepatitis A antibodies 20 million infections, over 3 million cases of acute hepatitis,
are most commonly detected by automated, chemiluminescent and over 56,000 deaths.15 Similar to the HAV, HEV is transmitted
microparticle immunoassays. Acute hepatitis A is routinely primarily by the fecal–oral route; however, person-to-person
diagnosed in symptomatic patients by demonstrating the pres- transmission is uncommon. The four genotypes of the virus
ence of IgM antibodies to HAV.5,6,8,9 IgM anti-HAV is detectable differ in terms of their epidemiology and source of infection.
at the onset of clinical symptoms and declines to undetectable Genotypes 1 and 2 are associated primarily with consumption
levels within 6 months in the majority of infected individu- of fecally-contaminated drinking water in developing regions
als.5,6,9 Because false-positive results can occur, the test should of the world with poor sanitation, including parts of Africa,
be reserved for symptomatic individuals.8,9 Tests for total HAV Asia, the Middle East, and Mexico.14,16,17 Outbreaks commonly
antibodies also detect IgM, but predominantly detect IgG, occur in times of natural disasters such as flooding and earth-
which persists for life. Thus, a positive total anti-HAV test result quakes and can affect thousands of individuals. Genotypes 3
in combination with a negative IgM anti-HAV indicates that and 4 have been increasingly recognized in developed parts of
the patient has developed immunity to the virus, either the world, including Europe, North America, and Japan. HEV3
through natural infection or vaccination. Negative total anti- and HEV4 are zoonotic infections in which pigs are the pri-
HAV tests can be used to identify nonimmune individuals who mary host.14,16 These infections are thought to be transmitted
may have been exposed to the virus.8 mainly by consumption of infected pork and possibly by direct
Although IgM anti-HAV is the primary marker to detect acute contact with infected animals or fecally-contaminated water.14
hepatitis A, false-negative results can occur during the early HEV has also been detected in the blood supply in a number of
phase of the infection.11 Molecular methods to detect HAV RNA countries and can be transmitted through blood transfusions.14,16
have been shown to be more sensitive in this situation.11 The Although HEV infections are often silent, all genotypes are
most common format of these methods is the reverse transcrip- capable of causing an acute hepatitis with symptoms that are
tase polymerase chain reaction (RT-PCR).11,12 Molecular meth- indistinguishable from other types of hepatitis. Following an
ods can also be used to test samples of food or water suspected incubation period of 2 to 6 weeks, HEV infection in most
of transmitting the virus.5,12 Multiplex real-time PCR (RT-PCR) people causes a self-limiting illness with recovery occurring
methods that can simultaneously detect more than one type of by 4 to 6 weeks.14,16 However, the infection can have severe
hepatitis virus in clinical samples have also been developed.11,13 consequences. Some patients may experience extrahepatic
A vaccine consisting of formalin-killed HAV was licensed in symptoms, including neurological syndromes, renal injury,
the mid-1990s to prevent hepatitis A. Vaccination has resulted pancreatitis, and hematologic abnormalities.14 Pregnant women
in a significant decrease in the number of HAV infections in infected with HEV1 or HEV2 have a mortality rate of 20%
the United States and other countries throughout the world.7,9 to 25% because of obstetric complications or development of
To prevent infection in unimmunized individuals who have fulminant hepatitis, which is associated with rapidly progressing
liver disease and failure.14,16 The reason for this is unclear, but has thus been associated with sexual contact, blood transfusions,
it may be caused by the hormonal and immunologic changes sharing of needles and syringes by intravenous drug users, tat-
associated with pregnancy. HEV3 can result in chronic infection tooing, and occupational needlestick injury. Inapparent trans-
in immunocompromised individuals.16 Chronic infection can mission of HBV may occur through close personal contact of
progress to liver fibrosis, cirrhosis, and liver failure, which may broken skin or mucous membranes with the virus. Transmission
require liver transplantation.18 of HBV may also occur via the perinatal route, from infected
Measures to prevent the infection include provision of clean mother to infant, most likely during delivery.
drinking water, improvement of sanitation conditions in devel- Several measures have been introduced to prevent HBV
oping countries, and in the case of HEV3, avoidance of eating infection, including screening of blood donors, treating plasma-
undercooked meat, especially pork.14,15 A vaccine to prevent derived products to inactivate HBV, implementing infection-
HEV1 infection has been licensed for use in the People’s Repub- control measures, and, most importantly, immunizing with a
lic of China.19 The vaccine consists of viruslike particles that hepatitis B vaccine.24,25 The current vaccines, consisting of
have been genetically modified to express a gene that codes for recombinant Hepatitis B surface antigen (HBsAg) produced
a key HEV protein. from genetically engineered yeast or mammalian cells, are some
Because HEV is not easily cultured, diagnosis relies on serol- of the most widely used vaccines throughout the world. Im-
ogy to detect antibodies to the virus and molecular methods munization has been highly successful, resulting in a significant
to detect HEV nucleic acid. Antibodies to HEV are typically decline in the incidence of acute hepatitis B in the United States
identified by sensitive enzyme immunoassays (EIAs) that use since routine immunization was implemented in 1991.24,26
recombinant and synthetic HEV antigens. Rapid immunochro- Increasingly widespread use of the vaccine will likely continue
matographic assays have also been developed. Antibody tests to reduce the incidence of new HBV infections worldwide. The
for HEV can detect all four genotypes of the virus because there vaccine can also be administered to individuals thought to be
is only one viral serotype.14,16 Acute infection is indicated by the exposed to the virus, along with HBIG (hepatitis B immune
presence of IgM anti-HEV, which is detectable at clinical onset, globulin), a preparation derived from donor plasma with high
remains elevated for about 8 weeks, and becomes undetectable concentrations of antibodies to HBV that provides temporary
in most patients by 32 weeks.14 HEV-specific IgG antibodies protection.24
appear soon after IgM, reach peak levels about 4 weeks after Despite the preventative measures that have been imple-
symptoms develop, and persist for several years.14,17 Immunoas- mented, a substantial number of HBV infections continue to
says for IgG anti-HEV may be performed to detect patients in occur as previously discussed. Infection with HBV results in
the later stages of infection, determine past exposure, and iden- an incubation period of 30 to 180 days, followed by a clinical
tify seroprevalence of the infection in a population. course that varies in different age groups.6,21,25 Over 90% of
Immunocompromised persons often yield negative antibody newborns with perinatal HBV infection remain asymptomatic,
test results; molecular testing for HEV RNA is recommended in whereas typical symptoms of acute hepatitis are observed in
these patients.14,18 HEV nucleic acid can be performed by real- about 10% of children aged 1 to 5 years and in approximately
time PCR or a loop-mediated isothermal amplification assay one-third of adolescents and adults. Symptoms may last sev-
(LAMP), which is suitable for resource-limited settings because eral weeks to several months and are usually managed through
it is faster and does not require expensive equipment.14 These bed rest and other supportive treatment. Most HBV-infected
assays can be performed on blood or stool samples. HEV RNA adults recover within 6 months and develop immunity to
can be detected just before clinical symptoms. It becomes un- the virus, but about 1% develop fulminant liver disease with
detectable in the blood about 3 weeks after symptom onset; hepatic necrosis. This highly fatal condition is treated with
in the stool, it becomes undetectable at about 5 weeks.14,16 intensive life support, antiviral drugs, and, in some patients,
Therefore, a negative result for HEV RNA does not exclude the liver transplantation.
possibility of a recent infection. Development of chronic HBV infection, in which the virus
persists in the body for 6 months or more, occurs in about 90%
of infected infants, 30% of young children, and 10% of infected
Hepatitis B adults. Chronic infection is also more likely to develop in per-
Hepatitis B is a major cause of morbidity and mortality sons who are immunosuppressed and those who have HIV.21,25
throughout the world. The WHO estimates that HBV has in- Chronic infection with the virus results in inflammation and
fected 2 billion people worldwide, causing 240 million chronic damage to the liver and places the patient at increased risk of
infections and 780,000 deaths each year because of liver dis- developing cirrhosis or hepatocellular carcinoma.27 Patients
ease.20,21 The virus is highly endemic in the Far East, parts of with chronic infection can be treated with antiviral drugs to
the Middle East, sub-Saharan Africa, and the Amazon areas. In reduce liver inflammation and the risk of developing liver com-
the United States, which is considered a low-prevalence area, plications.26,28 Therapies consist of nucleoside analogues that
HBV is responsible for approximately 1.2 million chronic in- inhibit the polymerase enzyme needed for viral replication and
fections and 1,800 deaths annually.22 interferon alpha, which enhances the immune response against
HBV is transmitted through the parenteral route by intimate the virus.
contact with HBV-contaminated blood or other body fluids, most The virus responsible for hepatitis B, HBV, is a DNA virus
notably semen, vaginal secretions, and saliva.6,23–25 Transmission belonging to the Hepadnaviridae family.6,23,27 Eight genotypes,
designated A through H, have been identified based on nu- monitoring the course of infection and progression to chronic
cleotide sequence differences in their genomes. The genotypes disease, and screening of donor blood.
vary in their geographic distribution, pathogenicity, and re- The HBeAg appears shortly after HBsAg and disappears
sponse to treatment, but can be identified by the same sero- shortly before HBsAg in recovering patients. It may be elevated
logical assays. The intact HBV virion is a 42 nm sphere during chronic infection. This marker is present during periods
consisting of a nucleocapsid core surrounded by an outer en- of active replication of the virus and indicates a high degree of
velope of lipoprotein. The core of the virus contains circular, infectivity. The HBeAg is not detectable in serum because the
partially double-stranded DNA; a DNA-dependent DNA poly- viral envelope masks it.
merase enzyme; and two proteins, the hepatitis B core antigen As the host develops an immune response to the virus,
and the hepatitis Be antigen (HBeAg). A protein called the antibodies appear. First to appear is IgM antibody to the
hepatitis B surface antigen (HBsAg) is found in the outer core antigen, or IgM anti-HBc. This antibody indicates current
envelope of the virus. HBsAg is produced in excess and is or recent acute infection. It typically appears 1 to 2 weeks
found in noninfectious spherical and tubular particles that lack after HBsAg during acute infection and persists in high titers
viral DNA and circulate freely in the blood. for 4 to 6 months and then gradually declines. IgM anti-HBc
These antigens, and antibodies to them, serve as serological is useful in detecting infection in cases in which HBsAg is
markers for hepatitis B and have been used in differential undetectable—for example, just before the appearance of
diagnosis of HBV infection, monitoring the course of infection antibodies to the antigen (commonly referred to as the “core
in patients, assessing immunity to the virus, and screening window” period), in neonatal infections, and in cases of fulmi-
blood products for infectivity.5,6,23,29,30 The levels of these nant hepatitis. Therefore, it is used in addition to HBsAg for
markers vary with the amount of viral replication and the the screening of donor blood. IgG antibodies to the core anti-
host’s immune response. They are useful in establishing gen are produced before IgM anti-HBc disappears and then
the initial diagnosis of hepatitis B and monitoring the course persist for the individual’s lifetime. They are the predominant
of infection. Serological markers for hepatitis B are listed in antibodies detected in the test for total anti-HBc and can be
Table 23–1 and are described in the text that follows. Typical used to indicate a past HBV infection.
patterns of the markers during acute and chronic hepatitis B The appearance of antibodies to the HBe antigen, or anti-HBe,
are shown in Figures 23–4 and 23–5. The HBsAg is the first occurs shortly after the disappearance of HBeAg and indicates
marker to appear, becoming detectable 2 to 10 weeks after that the patient is recovering from HBV infection.
exposure to HBV. Its levels peak during the acute stages of Antibodies to HBsAg, or anti-HBs, also appear during the
infection, then gradually decline as the patient develops recovery period of acute hepatitis B, a few weeks after HBsAg
antibodies to the antigen and recovers. Serum HBsAg usually disappears. These antibodies persist for years and provide pro-
becomes undetectable by 4 to 6 months after the onset of tective immunity. Anti-HBs are also produced after immunization
symptoms in patients with acute hepatitis B. In patients with with the hepatitis B vaccine. Protective titers of the antibody
chronic HBV infection, HBsAg remains elevated for 6 months in the serum are considered to be 10 mIU/mL or higher.24,25
or more. Therefore, HBsAg is an indicator of active infection Anti-HBs is not produced during chronic HBV infection, in
and is an important marker in detecting initial infection, which immunity fails to develop.

Incubation Acute Infection Early recovery Full recovery

8–13 weeks 2 weeks–3 months 3–6 months !6 mo-to-years

Symptoms Total anti-HBc

Anti-HBs

HBsAg Anti-HBe
Concentration

HBeAg IgM
Anti-HBc

Time
Typical serological markers in acute hepatitis B. Solid lines represent viral antigen concentrations, whereas dashed lines
indicate antibody concentrations. Each antigen shares the same color with its associated antibody.
 Incubation Chronic infection
2 weeks–3 months 3–6 months
8–13 weeks !6 mo-to-years

Symptoms Liver damage

Total anti-HBc
HBsAg
Concentration

HBeAg
IgM
Anti-HBc

Time
Typical serological markers in chronic hepatitis B.

Serological markers for hepatitis B are most commonly DNA (bDNA) signal amplification.23,29,30 HBV DNA can be
detected by commercial immunoassays. These are available detected in the serum about 21 days before HBsAg and may
in a variety of formats, such as EIA and chemiluminescent be a useful adjunct in detecting early acute HBV infection in
immunoassay (CLIA). They are typically automated to ease certain situations such as the screening of blood donors, as-
batch testing in the clinical laboratory and have excellent sen- sessing cases of occupational exposure, and evaluating patients
sitivity and specificity.5,23 An example of an immunoassay for with equivocal HBsAg test results.23,30 HBV DNA testing is
detecting HBsAg is shown in Figure 23–6. Although these also used to evaluate the effectiveness of antiviral therapy in
methods are highly sensitive and specific, false-positive and patients with chronic hepatitis B. Successful treatment is
false-negative results can occur. Any initial positive results indicated by a 1log10 reduction in HBV DNA levels by 6 months,
should be verified by repeated testing of the same specimen whereas persistently elevated HBV DNA levels indicate possi-
in duplicate, followed by confirmation with an additional ble drug resistance and a need to change therapy.23 Molecular
assay, such as an HBsAg neutralization test or a molecular test testing is also used to diagnose atypical cases of hepatitis B
that detects HBV DNA. originating from mutations in the HBV genome that cause
Several molecular methods have been developed to detect HBsAg tests to be negative.23,30 Molecular methods to detect
HBV DNA in serum or plasma and are mostly based on target HBV genotypes and HBV mutations associated with antiviral
amplification by traditional or real-time PCR or branched drug resistance have also been developed.23,29 These tests will

" "

Magnet

A B C D E F
Detection of the HBs antigen by chemiluminescence microparticle immunoassay. Patient serum or plasma containing HBsAg
(A) is mixed with magnetic microparticles coated with anti-HBs (B) and acridinium-labeled anti-HBs conjugate (C). During incubation complexes
form, with the antigen sandwiched in between the antibodies (D). Application of a magnetic field holds the microparticles and bound reagents
in the tube while unbound materials are washed away and chemiluminescent reagents are added (E). The magnitude of light produced is
measured in a luminometer (F) and is proportional to the concentration of HBsAg in the sample.
likely be used more widely in the future to determine optimal used to confirm a positive HDV antibody screen.32 Molecular
patient therapy. testing for serum HDV RNA is performed by sensitive, real-
time RT-PCR assays.5,35 These assays also provide quantitative
results that can be used to monitor the response of patients to
Hepatitis D antiviral therapy.
Hepatitis D, also known as delta hepatitis, is a parenterally
transmitted infection that can occur only in the presence of
hepatitis B. HDV is a defective virus that requires the help of
Hepatitis C
HBV for its replication and expression. The only member Hepatitis C is a major public health problem, with an estimated
within the Deltavirus genus, HDV consists of a circular RNA 80 million people infected worldwide.36 It is the most common
genome and a single structural protein called hepatitis delta anti- bloodborne infection in the United States, affecting about 1.3%
gen within its core, surrounded by a viral envelope that is of of the population.36,37 Hepatitis C is also the most frequent
HBV origin and contains the HBsAg.23,31 The eight known cause of chronic liver infection and the leading indicator for
HDV genotypes vary in their geographical distribution, path- liver transplantation in the United States.38
ogenicity, and response to treatment. More than 15 million HCV, the virus that causes hepatitis C, is responsible for
people around the world are believed to be infected with HDV, most of the infections previously classified as “nonA–nonB” be-
which is highly prevalent in Mediterranean Europe, the Middle fore the discovery of the virus in 1989.6 It is an enveloped,
East, the Amazon basin, central Africa, and parts of Asia.32,33 single-stranded, positive-sense RNA virus belonging to the
The number of new infections appears to be increasing in family Flaviviridae and the genus Hepacivirus.6,39,40 Scientists
certain parts of the world. have discovered seven different genotypes of the virus, desig-
Similar to HBV, HDV is transmitted sexually in semen or nated 1 through 7, and numerous subtypes for each, indicated
vaginal secretions; through blood by intravenous drug use, by lowercase letters.6,39,40,41 The genotypes differ in their geo-
needlestick injuries, or transfusions; or perinatally from mother graphic distribution, pathogenicity, and response to antiviral
to infant. Infection with the virus can occur in one of two ways: treatment. Genotype 1, the most common, is responsible for
HDV can be transmitted simultaneously as a co-infection with 46% of hepatitis C infections worldwide and over 70% of HCV
HBV or HDV can be contracted as a superinfection of individuals infections in the United State.36,39 Genotypes 1, 2, and 3 are
who are already chronic HBV carriers. Clinically, most patients predominant in North America, Europe, and Japan; genotypes
with co-infections experience an acute, self-limited hepatitis in 3 and 6 are found throughout south and southeast Asia; and
which both viruses are cleared within a few months.5,32,34 Some genotypes 4, 5, and 7 are most common in parts of Africa.39,41
patients may experience more severe symptoms of acute hepa- The variability of HCV, along with its ability to undergo rapid
titis than those infected with HBV alone, but only about 2% of mutations within its hosts, has created difficulty in developing
cases progress to a chronic state. In contrast, more than 70% of an effective vaccine.
patients with superinfections develop chronic liver disease with Hepatitis C is transmitted mainly by exposure to contami-
an accelerated progression to cirrhosis and liver failure.5,31,32,34 nated blood, with intravenous drug use being the main source
Combinations of interferon alpha and antiviral drugs can be of infection.6,42 Blood transfusion was also a major vehicle of
administered to patients with chronic or severe hepatitis D in an transmission before routine screening of blood donors for HCV
attempt to eradicate the virus.32,33 antibody was implemented in 1992, but transmission by this
Testing for hepatitis D should be performed in all patients who means is rare today. Organ transplantation before 1992 was
are HBsAg positive and involves detection of HDV antibodies and also a route of transmission. Other risk factors for acquiring
HDV RNA.32 Antibodies are detected by immunoassays employ- hepatitis C include occupational exposures to contaminated
ing the hepatitis D antigen.5,23,34 The presence of IgG anti-HDV blood, long-term hemodialysis, and unregulated body piercing
antibodies indicates exposure to the virus and can signify an or tattooing in environments such as correctional facilities
acute, chronic, or past hepatitis D infection. Although IgM anti- where contaminated needles are likely to be used.38,42 Sexual
HDV is produced during acute hepatitis D infections, its appear- transmission of HCV is thought to be less common but is
ance may be delayed, it may persist for only a short period of higher in those who have had multiple sex partners or a history
time, and it may be missed.5,32 IgM antibodies to HDV can also of sexually transmitted diseases.38,42 Perinatal transmission has
persist during chronic infection.32 Serology testing for hepatitis been estimated to occur at a rate of about 6%.43
B can be used to help distinguish HBV and HDV co-infections HCV has an average incubation period of 7 weeks (range is
from HBV and HDV superinfections, which, as previously dis- 2 to 30 weeks). The majority of infections are asymptomatic,
cussed, have different clinical outcomes. In addition to being with symptoms of acute hepatitis occurring in only about 20%
positive for HDV antibodies, patients with co-infections are pos- of cases.6,42,43 Asymptomatic infection is problematic because
itive for IgM anti-HBc, whereas patients with superinfections are chronic infection develops in about 70% of infected persons and
positive for IgG anti-HBc.33 up to half of these individuals develop cirrhosis.6,38,43 Cirrhosis
Detection of hepatitis D has been aided tremendously by occurs slowly over a 25- to 30-year period, causing damage to
the development of molecular methods to detect HDV RNA, a the liver and posing an increased risk of developing hepatocel-
marker of active viral replication that is present in all types of lular carcinoma. Patients with chronic HCV infection may also
active hepatitis D infections.5 HDV RNA testing is routinely develop extrahepatic manifestations, including rheumatological
conditions; glomerulonephritis, vasculitis, or other autoimmune Molecular assays for HCV RNA can be classified as qual-
manifestations; neuropathy; ophthalmological symptoms; and itative or quantitative. Qualitative tests distinguish between
dermatological symptoms.38,43 Early detection would help pre- the presence or absence of HCV RNA in a clinical sample.
vent these complications, but HCV is often missed in its early These tests are used to confirm infection in HCV-antibody-
stages because of the asymptomatic nature of the infection in positive patients (as previously mentioned), detect infection
most individuals. in antibody-negative patients who are suspected of having
Clearance of the infection may occur spontaneously or may HCV, screen blood and organ donors for HCV, and detect
require treatment with antiviral drugs. Until recently, the standard perinatal infections in babies born to HCV-positive moth-
treatment involved a combination of pegylated interferon-α (PEG ers.51 Qualitative RT-PCR and transcription-mediated ampli-
IFN-α) and ribavirin. Although this treatment has been successful fication (TMA) methods are commercially available.39,51,52
in 80% of persons infected with genotypes 2 or 3, it has been These tests can detect as low as 5 International Units (IU) of
effective in only half of those with genotype 1 and is associated HCV RNA per mL of serum (for TMA) or 50 IU/mL HCV
with numerous side effects.41,44 Increased understanding of the RNA (for RT-PCR) and become positive within 1 to 3 weeks
biology of HCV has led to the development of direct-acting an- after infection.39,51 They are generally positive at the onset
tiviral drugs (DAAs) and host-targeted agents (HTAs) that inhibit of symptoms, but, in some patients, can transiently decrease
specific steps of the viral replication cycle.42-44 Combination ther- to undetectable levels during the acute phase of the infection.39
apies employing these agents are being evaluated at a rapid pace Quantitative tests are performed by RT-PCR, real-time PCR,
and are revolutionizing the way hepatitis C is being treated. or bDNA amplification.39,51,52 Commercial tests can detect a
The laboratory plays an essential role in screening for wide range of HCV concentrations, from about 10 IU/mL to
hepatitis C, monitoring patients known to have HCV infection, 10 million IU/mL.39 They are used to monitor the amount of
and guiding therapy. Between 1998 and 1999, the CDC issued HCV RNA, or “viral load,” carried by patients before, during,
recommendations that screening for HCV infection be con- and after antiviral therapy in chronically infected individuals.
ducted in high-risk individuals, including those who received The ultimate goal of such therapy is to achieve a sustained
blood or blood products.45’46 In 2012, the CDC extended these virological response (SVR) in which the patient continuously
recommendations to include a one-time screening of all persons tests negative for HCV RNA 12 or 24 weeks after therapy is
in the United States who were born between 1945 and 1965, completed.42,51 The initial viral load level has also been used
regardless of risk factors.47 In 2013, the U.S. Preventative Task as a prognostic tool because those with a low initial viral load
Force endorsed this recommendation.48 The rationale behind are most likely to achieve an SVR.51
the latest recommendation was that about 75% of individuals Genotyping, to determine the exact genotype and subtype
living with HCV infection in the United States were born during of the virus responsible for the infection, should be performed
this time period but are asymptomatic. Identification of these on all HCV-infected patients before antiviral therapy.39,52 It is
persons could lead to closer monitoring for disease progression important to identify the patient’s HCV genotype in order to
and earlier administration of effective antiviral treatment. determine the most effective treatment because HCV genotypes
Screening and diagnosis of hepatitis C begins with serological vary in their response to different antiviral drugs. For example,
testing for HCV antibodies. Anti-HCV IgG is most commonly as previously mentioned, PEG IFN-α/ribavirin treatment is
detected by sensitive EIAs or CLIAs that use recombinant and more effective in patients with genotypes 2 or 3 than in patients
synthetic antigens developed from the conserved domains of the with genotype 1. Genotyping is also useful in epidemiological
capsid core protein (C) and the nonstructural proteins, NS3, studies to determine the source of HCV infection in specific
NS4, and NS5.5,39,40 Alternatively, a rapid immunoblot assay can populations.52
be used for point-of-care testing.49 Antibodies become detectable Genotyping can be performed by PCR amplification and
8 to 10 weeks after HCV exposure and can remain positive for sequencing of the target gene, PCR followed by identification
a lifetime.39 Thus, a reactive result can indicate the presence of the target gene with genotype-specific probes, or real-time
of a current HCV infection or a past HCV infection that has PCR.39,40 PCR/sequencing is the reference method because it
resolved.37 In addition, despite the excellent specificity of these provides precise information regarding the genomic variability
methods, false-positive results may occur because of cross- of the virus in patients during the course of the disease. How-
reactivity in persons with other viral infections or autoimmune ever, sequencing is primarily performed in research laborato-
disorders.5,37 Therefore, any positive results from an anti-HCV ries because of the specialized equipment and analysis software
screening test should be confirmed to distinguish between the required, whereas clinical laboratories typically use real-time
various interpretations of these results. Current CDC guidelines PCR methods or PCR/probe hybridization.39,40
recommend the use of nucleic acid testing (NAT) for HCV RNA
for confirmation. If HCV RNA is detected, a current HCV infec- Herpes Virus Infections
tion is indicated. In contrast, if the NAT is nonreactive, this
suggests a past HCV infection or false-positive antibody test The herpes viruses are large, complex DNA viruses that are
result.37 To distinguish between a true-positive and false-positive surrounded by a protein capsid, an amorphous tegument,
result, HCV antibody testing can be repeated using a different and an outer envelope.53 These viruses are all capable of
assay from the initial test because a biological false-positive result establishing a latent infection with lifelong persistence in the
is unlikely to occur in two different methods.37,50 host. The Herpesviridae family includes eight viruses that can
cause disease in humans: the herpes simplex viruses (HSV-1 two groups based on their location within the cells: EA-D,
and HSV-2); VZV; EBV; CMV; and the human herpes viruses which has a diffuse distribution in the nucleus and cytoplasm,
HHV-6, HHV-7, and HHV-8, the latter of which has been and EA-R, which is restricted to the cytoplasm only. The late
associated with Kaposi sarcoma. This section presents the antigens of EBV are those that appear during the period of
clinical manifestations and laboratory diagnosis of some of the lytic cycle following viral DNA synthesis. They include the
these viruses. viral capsid antigens (VCAs) in the protein capsid and the
membrane antigens in the viral envelope. Antigens appearing
during the latent phase include the EBV nuclear antigen
Epstein-Barr Virus (EBNA) proteins, EBNA-1, EBNA-2, EBNA-3 (or -3a), EBNA-4
The EBV causes a wide spectrum of diseases, including in- (or -3b), EBNA-5 (or -LP), and EBNA-6 (or -3c), and the latent
fectious mononucleosis, lymphoproliferative disorders, and membrane proteins (LMPs), LMP-1, LMP-2A, and LMP-2B
several malignancies.54,55 EBV infections most commonly (Table 23–2).
result from intimate contact with salivary secretions from The clinical manifestations of EBV vary with the host’s age
an infected individual. Although transmission of the virus and immune status. Infections in infants and young children are
can occur by other means, including blood transfusions, generally asymptomatic or mild, whereas primary infections in
bone marrow and solid organ transplants, sexual contact, healthy adolescents or adults commonly result in infectious
and perinatal exposure, these routes appear to be much less mononucleosis infectious mononucleosis (IM).54,55,59,60 More
frequent.54,56 than half of patients with IM present with three classic symp-
In developing nations of the world and lower socioeconomic toms: fever, lymphadenopathy, and sore throat. Symptoms usu-
groups living under poor sanitation, EBV infections usually ally last for 2 to 4 weeks, but fatigue, myalgias, and need for
occur during early childhood, whereas in industrialized nations sleep can persist for months. Treatment is mainly directed at
with higher hygiene standards, infections are typically delayed alleviating symptoms.60 Although the associated symptoms are
until adolescence or adulthood. However, by adulthood, more essential in diagnosing IM, they can also be caused by many
than 95% of individuals have been infected, as evidenced by other infectious agents, so laboratory testing plays an important
the presence of EBV antibodies in their serum.55,57 role in differentiating IM from other infections.
Initial infection with EBV is believed to occur in the Characteristic laboratory findings in patients with IM
oropharynx, where the virus primarily infects epithelial cells include an absolute lymphocytosis of greater than 50% of
and B lymphocytes.54,55,58 EBV binds to β1 integrins on the the total leukocytes and at least 20% atypical lymphocytes
surface of the epithelial cells, which take up the virus by en- (Fig. 23–7).55,60 The atypical lymphocytes are predominantly
docytosis. Inside the oropharyngeal epithelial cells, EBV enters activated cytotoxic T cells that are responding to the viral
a lytic cycle, characterized by viral replication, lysis of host infection.56,58 Serological findings include the presence of a
cells, and release of infectious virions, until the acute infection heterophile antibody and antibodies to certain EBV antigens.
is resolved. The virions spread to adjacent structures, including By definition, heterophile antibodies are antibodies that
the salivary glands and tonsils. There, EBV infects B lympho- are capable of reacting with similar antigens from two or
cytes, which spread the virus throughout the lymphoreticular more unrelated species. The heterophile antibodies associ-
system. EBV enters the B cells by binding to surface CD21, ated with IM are IgM antibodies produced as a result of poly-
which is also the receptor for the C3d component of comple- clonal B-cell activation and are capable of reacting with horse
ment. The virus-infected B cells become polyclonally activated, red blood cells (RBCs), sheep RBCs, and bovine RBCs. These
proliferating and secreting a number of antibodies, including antibodies are produced by 40% of patients with IM during
EBV-specific antibodies; heterophile antibodies; and autoanti- the first week of clinical illness and by 80% to 90% of patients
bodies such as cold agglutinins, rheumatoid factor, and anti- by the third week.54 They disappear in most patients by
nuclear antibodies.54,55,59 In healthy individuals, this process 3 months after the onset of symptoms but can be detected
is kept in check by the immune response of NK cells and spe-
cific CTLs. However, EBV can persist in the body indefinitely
in a small percentage of memory B cells, where it establishes a Table 23–2 Epstein-Barr Virus Antigens
latent infection.58 In the latent state, EBV nucleic acid exists as
EARLY ACUTE
episomal DNA outside of the chromosomes; in these cases, PHASE LATE PHASE LATENT PHASE
active viral replication does not occur. Periodic reactivation
EA-R (early VCA (viral cap- EBNA (EBV nuclear
results in re-entry of the virus into the lytic cycle, with viral
antigen sid antigen) antigens): EBNA-1,
shedding into the saliva and genital secretions, even in healthy,
restricted) MA (membrane EBNA-2, EBNA-3
asymptomatic individuals.54,55,59 EA-D (early antigen) (3a), EBNA-4 (3b),
Several antigens have been identified in EBV-infected cells antigen EBNA-5 (LP),
that are associated with different phases of the viral infection. diffuse) EBNA-6 (3c)
Antibodies to these antigens have become an important diag-
Latent membrane
nostic tool.55,57,59 Antigens produced during the initial stages
proteins (LMP-1,
of viral replication in the lytic cycle are known as the early
LMP-2A, LMP-2B)
antigens (EAs). These antigens can be further classified into
 serial dilutions of the patient’s serum with sheep RBCs in the
Paul–Bunnell test (see the Lab Exercise on DavisPlus). Today,
these methods have been replaced by more sensitive, rapid
agglutination tests or immunochromatographic assays using
purified bovine RBC extract as the antigen. Although screening
tests for the heterophile antibody are ideal for point-of-care
testing, they are not as sensitive or specific as tests for antibod-
ies to EBV, the direct cause of IM.54 Negative heterophile anti-
body results occur in about 10% of adult patients with IM and
up to 50% of children younger than 4 years old.55 False-positive
results, although uncommon, can occur in patients with lym-
phoma, viral hepatitis, malaria, and autoimmune disease, or can
be caused by errors in result interpretation.54,55
Testing for EBV-specific antibodies can be performed to aid
Atypical lymphocytes from a patient with infectious in the diagnosis of IM, especially in patients with a negative
mononucleosis. Note the variation in size, nuclear:cytoplasmic ratio, heterophile antibody screen, or to determine if individuals have
and chromatin coarseness. (From Harmening D. Clinical Hematology had a past exposure to EBV.54,55,60 These antibodies can be
and Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F. A. Davis; detected by indirect immunofluorescence assays (IFA) using
2009.) EBV-infected cells, enzyme-linked immunosorbent assay (ELISA)
or CLIA using recombinant or synthetic EBV proteins, or flow
cytometric microbead immunoassays.57,61,62 Although all of
in some patients for 1 to 2 years.54,55 Because the heterophile these methods have a high level of sensitivity (95% to 99%),
antibody is present in most patients during the acute phase IFA tests have a higher level of specificity and are considered
of illness, testing for this antibody has been typically performed the “gold standard” of EBV serology methods. However, many
to screen for IM in patients who present with symptoms of the laboratories prefer ELISA or CLIA tests because they are less
disease. time consuming and easier to interpret.57,62
For many years, the heterophile antibody of IM was de- IgM antibody to the VCA is the most useful marker for acute
tected by a rapid slide agglutination method called the IM because it usually appears at the onset of clinical symptoms
“Monospot.” In this test, serum premixed with guinea pig and disappears by 3 months.54,62 IgG anti-VCA is also present
kidney antigen was still capable of agglutinating horse RBCs, at the onset of IM but persists for life and can thus indicate a
whereas serum premixed with beef erythrocyte antigen could past infection. Antibodies to EA-D are also seen during acute
not agglutinate horse RBCs because the heterophile antibody IM, whereas anti-EBNA appears during convalescence.54,55,58
was absorbed during the first step. The test was used to dis- Thus, acute primary infection is typically indicated by the pres-
tinguish the heterophile antibody of IM from heterophile anti- ence of IgM anti-VCA and anti-EA-D, as well as the absence of
bodies produced in other diseases, which had different anti-EBNA. A summary of serological responses during acute,
reactivity. The antibody could then be titered by incubating convalescent, and post-IM is shown in Table 23–3.

Table 23–3 Serological Responses of Patients With Epstein-Barr Virus-Associated Diseases

ANTI-VCA ANTI-EA
Condition IgM IgG IgA EA-D EA-R ANTI-EBNA HETEROPHILE ANTIBODY (IgM)
Uninfected – – – – – – –
Acute IM + ++ + – –
Convalescent IM – + – – +
Past infection IM – + – – – + –
Chronic active infection IM – +++ + ++ –
Post-transplant lymphoproliferative disease – ++ + + –
Burkitt’s lymphoma – +++ – ++ + –
Nasopharyngeal carcinoma – +++ + ++ + –

EA-D = early antigen- diffuse; EA-R = early antigen- restricted; EBNA = EBV nuclear antigen; IM = infectious mononucleosis; VCA = viral capsid antigen.
Adapted from Straus SE, et al. Epstein-Barr virus infections: biology, pathogenesis, and management. Ann Intern Med. 1993;118:45, with permission.
 Some individuals develop chronic active EBV infection, with The clinical consequences of CMV infection are much more
severe, often life-threatening IM-associated symptoms that per- serious in the immunocompromised host, most notably organ-
sist or recur for more than 6 months after the acute illness.56,58 transplant recipients and patients with HIV/AIDS. CMV is the
In addition, EBV can sometimes integrate its DNA into the most important infectious agent associated with organ transplan-
genome of the cells it infects and transform them into cancer cells. tation, with infections resulting from reactivation of CMV in the
As a result, EBV has been associated with several malignancies, recipient or transmission of CMV from the donor organ. CMV
both hematologic (e.g., Burkitt’s lymphoma and Hodgkin disease) infection of a previously unexposed recipient is associated with
and nonhematologic (e.g., nasopharyngeal carcinoma and gastric increased risk for allograft failure or graft-versus-host disease
carcinoma).54-56 EBV can also cause lymphoproliferative disorders (GVHD), and poses a high risk for a variety of syndromes, such
in immunocompromised patients, including central nervous as fever and leukopenia, hepatitis, pneumonia, gastrointestinal
system (CNS) lymphomas in patients with AIDS, X-linked lym- complications, CNS dysfunction, and retinitis.64,65,68 Although
phoproliferative disease in males with a rare genetic mutation, combination antiretroviral therapy has reduced the incidence of
and post-transplant lymphoproliferative disorders (PTLD) in CMV-related illness in patients with HIV infection, CMV remains
patients who have received hematopoietic stem cell or solid a major opportunistic pathogen in patients with low CD4 T-cell
organ transplants.55,56,63 These disorders result from the in- counts.
ability of immunosuppressed patients to control primary EBV Various measures can be undertaken to reduce the risk for
infection, leading to massive polyclonal expansion of the CMV transmission and treat CMV infection in the immunocom-
EBV-infected B cells and life-threatening illness with a high rate promised host.64,68 Serological testing can be performed to iden-
of mortality. tify CMV-positive donors so that transplantation of their organs
EBV-associated malignancies can be diagnosed with the help into CMV-negative recipients can be avoided. If a CMV infection
of serology tests for EBV antibodies and molecular methods to has been established in a transplant patient, immunosuppressive
detect EBV DNA in blood and tissue samples.57,58 Typical pat- treatment should be reduced to the lowest dose possible. In
terns of EBV antibodies seen in some of these disorders are addition, a variety of antiviral drugs are currently used to treat
shown in Table 23–2. Molecular tests may be more reliable CMV infection and may, in some instances, be given prophylac-
than serology in immunocompromised patients who may not tically (i.e., before organ transplantation).64,68 Researchers are
demonstrate a good humoral response. Quantitative real-time also investigating a vaccine design that involves the production
PCR is useful in monitoring viral load in transplant patients; a of specific CMV antigens using genetic technologies.69,70
high or steadily increasing EBV viral load indicates the need to CMV is also the most common cause of congenital infec-
decrease immunosuppressive treatment and administer antivi- tions, occurring in 0.3% to 2.3% of all neonates.71,72 Trans-
ral therapy.58 Detection of EBERs (EBV-encoded RNA tran- mission of the virus may occur through the placenta, by
scripts) by in situ hybridization is the method of choice for passage of the infant through an infected birth canal, or by
detecting EBV in tumor tissue.57,58 postnatal contact with breast milk or other maternal secretions.
About 10% to 15% of infants with congenital CMV infection
are symptomatic at birth.72,73 Mothers who acquire primary
Cytomegalovirus CMV infection during their pregnancy have a significantly
CMV is a ubiquitous virus with worldwide distribution. The higher risk of giving birth to a symptomatic or severely affected
prevalence of CMV ranges from 40% to 100%, depending on infant than do women in whom CMV was reactivated during
the population, and increases with age; however, crowded living pregnancy. Symptomatic infants present with a multitude of
conditions and poor personal hygiene facilitate spread earlier symptoms that reflect platelet dysfunction and CNS involve-
in life.64,65 Transmission of the virus can occur in a variety of ment. Ten percent of infants who are asymptomatic at birth
ways. CMV is spread through close, prolonged contact with progressively develop sensorineural hearing loss.73,74
infectious body secretions; intimate sexual contact; blood trans- Several laboratory methods have been developed to detect
fusions; solid organ transplants; and perinatal exposure from CMV infection; the tests recommended for use depend on the
infected mother to infant. The virus has been isolated in many clinical situation.65 Assays for direct detection of the virus,
body fluids, including saliva, urine, stool, vaginal and cervical such as viral culture, identification of CMV antigens, and molec-
secretions, semen, breast milk, and blood.64,65 ular tests for CMV DNA, are necessary to detect a current
Primary, or initial, infections in healthy individuals are usually CMV infection in individuals who are immunocompromised
asymptomatic. However, some people experience a self-limiting, or in neonates suspected of being congenitally infected with
heterophile antibody-negative IM-like illness with fever, myal- CMV. Serology is most beneficial in determining a past exposure
gias, and fatigue.64,65 A small number of immunocompetent to the virus, for example, in pregnant women or in patients in
individuals who have other underlying disorders may develop need of a transplant.
severe CMV disease, which most commonly involves the gas- Isolation of the virus in culture is the traditional method of
trointestinal tract, CNS, and hematologic abnormalities.66 An direct viral detection. In this method, human fibroblast cell lines
immune response against CMV is stimulated, but the virus are inoculated with CMV-infected specimens, most commonly
persists in a latent state in monocytes, dendritic cells, myeloid urine, respiratory secretions, or anticoagulated whole blood.65
progenitor cells, and peripheral blood leukocytes. It may be Presence of the virus is indicated by characteristic cytopathic
reactivated at a later time in the individual’s life.67 effects (CPE) that produce enlarged, rounded, refractile cells.
Although conventional culture provides definitive results when have been exposed to the infection in the past, or if they are
positive, it is limited because CPE do not appear until a few days susceptible to primary infection. In the latter case, the women
to several weeks after inoculation, depending on the viral titer. could be educated on measures to reduce their chances of
Implementation of the rapid centrifugation-enhanced (shell exposure while pregnant.77
vial) method has reduced the time of detection to within 24 hours Although a single positive CMV IgG result indicates past ex-
after inoculation.65 In this assay, infected cells are grown on cov- posure to the virus, conversion from a negative antibody result
erslips in shell vials and incubated with fluorescent-labeled to a positive antibody result over time indicates a recent CMV
monoclonal antibodies to CMV antigens produced early in the infection. However, serial assays for CMV IgG are not routinely
replication cycle. Fluorescent staining will appear in the nuclei performed. Assays for IgM CMV antibodies have been devel-
of positive cells. oped but are limited in value because of the potential for false-
A widely used method for direct identification of CMV has negative results in newborns and immunocompromised
been the CMV antigenemia assay, which uses immunocytochem- patients and for false-positive results caused by other infections
ical or immunofluorescent staining to detect the CMV lower or the presence of rheumatoid factor.65,74,77 In addition, IgM
matrix protein pp65 in infected leukocytes from peripheral antibodies may not necessarily indicate primary CMV infection
blood or cerebral spinal fluid.65,75 Following lysis of erythrocytes because they can also be produced as a result of CMV reacti-
in the sample, the leukocytes are fixed onto a microscope slide, vation and may persist for up to 18 months.65,74,78 Serological
permeabilized, and stained with labeled monoclonal anti-pp65. methods that distinguish CMV antibody avidity appear to be
Fluorescence appears in the nuclei of the infected cells, which more useful in distinguishing a past exposure from a current
can be counted to give quantitative results. The test can be com- primary infection.65,74,77 Low-avidity IgG antibodies indicate
pleted in 2 to 4 hours, allowing for more rapid diagnosis and a recent infection, whereas high-avidity IgG antibodies reflect
treatment of CMV infection in organ transplant patients and a past exposure because the avidity of the antibody increases
individuals infected with HIV. during the course of the immune response. The presence of
Although the antigenemia assay and shell vial culture meth- both IgM and low-avidity IgG antibodies can help identify
ods are sensitive, specific, and rapid, they are labor-intensive pregnant women who have contracted a primary CMV infection.
and require personnel with expertise in performing and inter- Because of the limitations of serology testing, direct methods of
preting these tests. For these reasons, they are progressively detecting CMV infection are essential.
being replaced with molecular methods that detect CMV DNA
or mRNA.65,76 Real-time PCR is the most widely used molec- Varicella-Zoster Virus
ular method because it is sensitive, simple to perform, and
can provide quantitative results. PCR amplification of CMV VZV is the cause of two distinct diseases: varicella, more com-
DNA has been extremely useful for detecting CMV infections monly known as chickenpox, and herpes zoster, also known
in HIV-infected hosts and establishing the diagnosis of CMV as shingles. The virus is transmitted primarily by inhalation
infection in transplant recipients.65,76 PCR also provides a more of infected respiratory secretions or aerosols from skin lesions
sensitive alternative to culture in diagnosing congenital CMV associated with the infection.79-81 Transplacental transmission
infections. Identification of CMV or CMV DNA in amniotic to the fetus may also occur.
fluid after the 20th week of gestation is considered the gold Primary infection with VZV results in varicella, a highly con-
standard for confirmation of fetal infection. Neonatal infection tagious illness characterized by a blisterlike rash with intense
is established by detecting CMV or CMV DNA in the urine of itching and fever.79-82 Historically, the majority of varicella
the infant during the first 10 days of life.65,76 Quantitative PCR, cases have occurred during childhood. In a typical infection,
which detects the CMV copy number in the peripheral blood, vesicular lesions first appear on the face and trunk, and then
is used to monitor the effectiveness of antiviral treatment in spread to other areas of the body (Fig. 23–8). The illness is
immunocompromised hosts and to identify patients at risk for usually mild and self-limiting in healthy children; however,
developing disseminated CMV disease.76 In addition, increasing in some cases it may produce complications, the most common
CMV DNA levels over time can be helpful in distinguishing an of which are secondary bacterial skin infections caused by
active infection from asymptomatic or latent infections.65,76 scratching of the lesions. CNS involvement may occur in some
Although serology tests for CMV have been commercially cases, but does not usually require hospitalization.81 Primary
available for many years, their clinical utility is limited. The
serology methods performed most commonly are semi- or
fully automated EIAs that employ microtiter plates or mi- Connections
croparticle systems.65,67 Assays for CMV IgG are most useful Rheumatoid Factor
in documenting a past CMV infection and determining if an
Recall that rheumatoid factor (RF) is an antibody (usually
individual is at risk for future infection. For example, screen- of the IgM class) that is directed against the Fc portion of
ing of blood and organ donors for CMV IgG is performed to IgG. RF can cause a false-positive result in some IgM assays
identify those donors who are CMV-positive so that the risk because it binds to IgG antibodies in the patient serum that
of post-transfusion/post-transplant primary CMV infection in are directed against the viral antigen bound to the solid phase
sero-negative recipients can be reduced. In addition, screen- (see Chapter 15).
ing of pregnant women for CMV IgG can determine if they
 individuals. A significant number of patients with herpes zoster
develop complications, the most common being postherpetic
neuralgia, characterized by debilitating pain that persists for
weeks, months, or even years after resolution of the infec-
tion.79,84,85 Life-threatening complications such as herpes oph-
thalmicus that lead to blindness, pneumonia, and visceral
involvement are more common in immunosuppressed persons.
Implementation of a vaccine consisting of a strain of live,
attenuated varicella virus in 1995 has resulted in a significant
decline in the incidence of chickenpox and its associated
complications in the United States.79,86 In 2005, a vaccine was
licensed for use in healthy children that combines the varicella
vaccine with that for measles, mumps, and rubella. In addition,
a single-agent VZV vaccine was licensed in 2006 for prevention
of herpes zoster in persons aged 60 or older, presumably by
boosting T-cell immunity to the virus.85,87 Because these vac-
cines all contain a live agent, they are not recommended for use
in immunocompromised persons. A VZV subunit vaccine is
being studied and may offer a viable alternative in the future.
Diagnosis of varicella and herpes zoster is usually based on
identifying the characteristic vesicular lesions associated with
the infection.83,84 Laboratory testing is most important in the
diagnosis of atypical cases, such as those in which the rash is
absent or delayed, and in immunocompromised patients
with disseminated disease.83,88 Definitive diagnosis is based on
Vesicular lesions characteristic of chickenpox. These
identifying VSV or one of its products in skin lesions, vesicular
blisterlike lesions have a pus-filled center. (Courtesy of the Centers for fluids, or tissue. Older methods of identification involved cell
Disease Control and Prevention, Public Health Image Library.) culture and microscopy, but these have significant disadvan-
tages. Culture of the virus and observation of characteristic
CPE can be performed in a number of cell lines but is time
infections in adults, neonates, or pregnant women tend to be consuming (4 days to 2 weeks) and may not yield productive
more severe, with a larger number of lesions and a greater chance results if clinical specimens do not contain sufficient amounts
of developing other complications such as pneumonia. Varicella of the infectious virus.84,89 Microscopic detection of multinu-
infection in pregnant women may also cause premature labor cleated giant cells called Tzanck cells in stained smears made
or congenital malformations if the infection is acquired during from material from the vesicles allowed for rapid identification
the first trimester of pregnancy or may cause severe neonatal of the virus, but this procedure could not distinguish between
infection if transmission of the virus occurs around the time of VZV and HSV.84,89 Direct immunofluorescence staining of
delivery.79-82 Infections in immunocompromised patients are scrapings from vesicular lesions with monoclonal antibodies
likely to result in disseminated disease, with extensive skin rash, directed against VZV antigens provides a rapid, but more
neurological conditions (e.g., encephalitis), and other compli- sensitive and specific means of detecting the virus.84,89 Today,
cations, including pneumonia, hepatitis, and nephritis.79-82 real-time PCR for VZV DNA is the laboratory method of choice
During the course of primary infection, VZV is thought to for diagnosing varicella zoster infection because it is highly
travel from the skin lesions and the blood to sensory neurons, accurate, sensitive, and rapid.83,84 Quantitative real-time PCR
where it deposits its DNA and establishes a lifelong latent state is also useful in monitoring the response of immunocompro-
in the dorsal root, autonomic, and cranial ganglia.82-84 The mised patients to antiviral drugs. PCR can be performed on a
host’s T-cell–mediated immune response is believed to keep variety of samples, including vesicular fluid or scabs, skin swabs,
the virus under control during this time.84 throat swabs, cerebrospinal fluid, blood, saliva, and tissues
Reactivation of VZV, with active viral replication, occurs in from biopsies or autopsies.83,84
15% to 30% of persons with a history of varicella infection.85 Serology testing is of limited use in detecting current infec-
The number of cases increases with age or development of tions because accurate detection requires demonstration of a
an immunocompromised condition, probably as a result of four-fold rise in antibody titer between acute and convalescent
decreased cell-mediated immunity. During reactivation, the samples, a process that takes 2 to 4 weeks to perform.81,83,89 In
virus moves down the sensory nerve to the dermatome sup- addition, testing for VZV IgM is not performed routinely for
plied by that nerve, resulting in eruption of a painful vesicular several reasons: IgM antibodies to VZV may not be detectable
rash known as herpes zoster herpes zoster, or shingles, shingles until the convalescent stage of illness, they cannot distinguish
in the affected area.79,82,85 The rash may persist for weeks to between primary and reactivated infection, and they may not be
months and is more severe in immunocompromised and elderly free of IgG antibodies when serum is processed for testing.89,90
 Serology is most useful in determining if immunity to and glaucoma; cardiac abnormalities; mental retardation; and
VZV is present in certain individuals, such as health-care motor disabilities. In mild cases, symptoms may not be recog-
workers, pregnant women, and patients about to undergo nized until months to years after birth.
organ transplantation.84 Therefore, most serology tests detect Scientists developed a vaccine consisting of live, attenu-
total VZV antibody, which consists primarily of IgG. Several ated rubella virus with the primary goal of preventing infec-
methods have been developed for this purpose.83,84 The tion of pregnant women by reducing dissemination of the
most sensitive and reliable method of detecting VZV anti- virus in the population as a whole.93,95 The vaccine is part of
body is a fluorescent test called fluorescent antibody to mem- the routine immunization schedule in infants and children
brane antigen (FAMA) that detects antibody to the envelope and is usually given in combination with vaccines for measles
glycoproteins of the virus.83,89 Although FAMA is considered and mumps (measles/mumps/rubella [MMR] vaccine) and
to be the reference method for VZV antibody, it requires live, sometimes with varicella (MMRV). Following licensure of the
virus-infected cells and is not suitable for large-scale routine vaccine in 1969, the number of rubella infections and cases
testing. The most commonly used method to detect VZV of CRS in the United States has dropped dramatically with
antibodies in the clinical laboratory is the ELISA because only limited outbreaks occurring, mostly among unvacci-
it is automated, provides objective results, and does not nated young immigrants to this country. However, rubella
require viral culture.83,84 Although older ELISA methods and CRS are still important health problems in parts of the
that employ a whole antigen extract are less sensitive than world where routine immunization against the virus is not
FAMA, a newer ELISA that detects antibody to a highly established.94,95
purified VZV envelope glycoprotein has been shown to have Laboratory testing is helpful in confirming suspected cases
a high level of sensitivity.84,91 Despite this improvement, of German measles because its symptoms may mimic those of
false-positive results can occur because the method can detect other viral infections. It is essential in the diagnosis of CRS and
low levels of antibodies that do not confer long-term protec- in the determination of immune status in other individuals.
tion to varicella.83,84 Laboratory diagnosis of rubella infection can be accomplished
through culture of the virus, demonstration of viral RNA, or
Other Viral Infections detection of virus-specific antibodies. Rubella virus can be
grown in a variety of cultures inoculated with throat swabs,
nasopharyngeal secretions, or other clinical specimens and can
Rubella be detected from almost all infected infants at the time of
The rubella virus is a single-stranded, enveloped RNA virus of birth.92 However, viral growth is slow and may not produce
the genus Rubivirus, belonging to the family Togaviridae.92-94 It characteristic CPE upon primary isolation, requiring at least
is transmitted through respiratory droplets or through transpla- two successive subpassages.92 In the absence of CPE, viral
cental infection of the fetus during pregnancy. nucleic acid can be identified by RT-PCR or viral proteins can
This virus is the cause of the typically benign, self-limited be detected by IFA or EIA.92 Because culture is time consuming
disease that is also known as German measles. Before wide- and labor intensive, it is increasingly being replaced by molec-
spread use of the rubella vaccine, this was mainly a disease of ular methods that are more practical to perform in the clinical
young children. However, today it occurs most often in young, laboratory and provide more timely results.97 The most widely
unvaccinated adults.95 Following an incubation period of 12 used molecular method is RT-PCR. RT-PCR is a highly sensitive
to 23 days, the virus replicates in the upper respiratory tract and specific aid in prenatal or postnatal diagnosis and can be
and cervical lymph nodes, then travels to the bloodstream. It used to detect rubella RNA in a variety of clinical samples,
produces a characteristic erythematous, maculopapular rash, including chorionic villi, placenta, amniotic fluid, fetal blood,
which appears first on the face, then spreads to the trunk lens tissue, products of conception, pharyngeal swabs, spinal
and extremities, and usually resolves in 3 to 5 days.93,94 In fluid, or brain tissue.78,98
adolescents and adults, this is usually preceded by a pro- Serology tests are the most common means of confirming
drome of low-grade fever, malaise, swollen glands, and upper a rubella diagnosis because they are rapid, cost effective, and
respiratory infection lasting 1 to 5 days. However, up to 50% practical in clinical laboratory settings.93 Several methods
of rubella infections are asymptomatic.93,94 The infection have been developed to detect rubella antibodies, including
usually resolves without complications and no specific treat- hemagglutination inhibition (HI), latex agglutination, and
ment is available. A significant number of infected adult immunoassays.92 Although HI was once the standard tech-
women experience arthralgias and arthritis, but chronic nique for measuring rubella antibodies, the most commonly
arthritis is rare.93,94 used method today is the ELISA because of its sensitivity,
Rubella infection during pregnancy may have severe con- specificity, ease of performance, and adaptability to automa-
sequences, including miscarriage, stillbirth, or congenital tion.92,93 More specific solid-phase capture ELISAs can be
rubella syndrome (CRS).93,94,96 The likelihood of severe con- used to detect IgM rubella antibodies. Automated chemilu-
sequences increases when infection occurs earlier in the preg- minescence assays and a multiplex bead immunoassay that
nancy, especially during the first trimester. Infants born with can simultaneously detect measles, mumps, rubella, and
CRS may present with a number of abnormalities, the most varicella are also available and demonstrate comparable
common of which are deafness; eye defects, including cataracts performance with ELISAs.99,100
 Primary rubella infection is indicated either by the pres- against a bright red background and persist for several days.
ence of rubella-specific IgM antibodies or by a four-fold or The typical rash of measles appears about 14 days after expo-
greater rise in rubella-specific IgG antibody titers between sure to the virus and is characterized by an erythematous,
acute- and convalescent samples collected at least 10 to 14 days maculopapular eruption that begins on the hairline, then
apart.93,94,97 The timing of serum collection is important spreads to the face and neck, and gradually moves down the
because IgM antibodies to rubella do not appear in many body to the trunk, arms, hands, legs, and feet (Fig. 23–9). The
patients until about 5 days after the onset of the rash, whereas rash usually lasts 5 to 6 days.
IgG antibodies may not be detectable until 8 days after the Measles is a systemic infection that can result in complica-
rash.92,97 Only about 50% of patients are positive for IgM tions, including diarrhea, otitis media, croup, bronchitis, pneu-
antibodies on the day that the rash appears; thus, a false-negative monia, and encephalitis.93,103,104 Rarely, a fatal degenerative
result can occur if the sample is obtained too early. False-positive disease of the CNS, called subacute sclerosing panencephalitis
results can also occur. Although IgM antibodies generally (SSPE), can result from persistent replication of measles virus
decline by 4 to 6 weeks, they may persist in low levels for a in the brain.93,106 Measles infection during pregnancy can result
year or more in some cases.78,92 False-positive rubella IgM in a higher risk of premature labor, spontaneous abortion, or
results have also been observed in individuals with par- low birth weight.93
vovirus infections, heterophile antibodies, or rheumatoid The incidence of measles has been greatly reduced in de-
factor.78,93 It is therefore recommended that positive IgM re- veloped nations of the world since the introduction of a live,
sults, particularly in pregnant women, be confirmed by a more attenuated measles virus vaccine in 1968. A vaccine consisting
specific test, such as an EIA that measures the avidity of rubella of killed rubeola virus was originally licensed in 1963 but
IgG antibodies, to distinguish between recent and past rubella was ultimately ineffective because recipients developed a case
infections.78,101,102 In these assays, low antibody avidity indi- of atypical measles if they were subsequently infected with
cates a recent infection (with a high risk for CRS), whereas high the measles virus.93 The newer vaccine is used in the routine
antibody avidity is seen in past infections, reflecting the normal immunization schedule of infants and children, either in com-
change in avidity during the course of an immune response. bination with rubella and mumps (MMR) or in combination
Laboratory diagnosis of congenital rubella infection begins with rubella, mumps, and varicella (MMRV).93,95 Recom-
with serological evaluation of the mother’s antibodies and mended administration of the vaccine is in two doses, the first
measurement of rubella-specific IgM antibodies in fetal blood, between the ages of 12 and 15 months and the second between
cord blood, or neonatal serum, depending on the age of the ages 4 and 6. Administration of the first dose before the age of
fetus or infant. To enhance the reliability of a CRS diagnosis, 12 months may result in vaccine failure because the presence
any positive IgM results should be confirmed by viral culture, of maternal antibodies can interfere with the infant’s immune
RT-PCR–amplification of rubella nucleic acid, or demonstration response. The vaccine was considered to be so successful that
of persistently high titers of rubella IgG antibodies after 3 to the CDC and WHO declared measles to be eliminated from
6 months of age.78 the United States in the year 2000 and from the Americas in
Serology tests are also used to screen for immunity to 2002.95,104 However, measles continues to be a global concern
rubella in populations such as pregnant women or health-care
workers. IgG antibodies provide immunity and persist for life.
Rubella-specific IgG antibodies are produced as a result of
natural infection or immunization. An antibody level of 10 to
15 IU/mL is considered to be protective.78,92

Rubeola
The rubeola virus is a single-stranded RNA virus belonging
to the genus Morbillivirus in the Paramyxoviridae family.103 It
is a highly contagious infection that is spread by direct contact
with aerosolized droplets from the respiratory secretions of
infected individuals. After initial infection of the epithelial cells
in the upper respiratory tract, rubeola virus is disseminated
through the blood to multiple sites in the body, such as the
skin, lymph nodes, and liver.104
Rubeola virus infection is the cause of the disease commonly
known as measles. Following an incubation period of about
10 to 12 days, the virus produces prodromal symptoms of
fever, cough, coryza (runny nose), and conjunctivitis, which last
2 to 4 days.93,103,105 During the prodromal period, characteristic Characteristic rash of measles appearing on the face
areas known as Koplik spots appear on the mucous membranes of a boy. (Courtesy of the Centers for Disease Control and Prevention,
of the inner cheeks or lips; these appear as gray-to-white lesions Public Health Image Library.)
and most cases in the United States and other industrialized Mumps
nations are brought in by unvaccinated individuals from other
countries.93,95 Measles outbreaks have occurred in recent years The mumps virus, similar to rubeola, is a single-stranded
in the United States because some people in the population RNA virus that belongs to the Paramyxoviridae family (genus
refuse to become vaccinated or have their children vaccinated Rubulavirus). It is transmitted from person to person by infected
on the basis of religious reasons or unfounded fears of vaccine respiratory droplets, saliva, and fomites and replicates initially
associations with disorders such as autism. in the nasopharynx and regional lymph nodes.93,110,111
The diagnosis of measles has typically been based on clinical (Fomites are inanimate objects or substances that can transmit
presentation of the patient. However, the success of the U.S. infectious organisms.) Following an average incubation period
immunization program in reducing the number of measles of 14 to 18 days, the virus spreads from the blood to various
cases has decreased the ability of some physicians to recognize tissues, including the meninges of the brain, salivary glands,
the clinical features of measles.93,104,105 In addition, atypical pre- pancreas, testes, and ovaries, and produces inflammation at
sentations of measles can occur in individuals who received the those sites.93 Inflammation of the parotid glands, or parotitis, is
earlier form of the measles vaccine, who have low antibody the most common clinical manifestation of mumps, occurring
titers, or who are immunocompromised.93,103,105 Laboratory in 30% to 40% of cases (Fig. 23–10).93 The illness typically
tests are therefore of value in ensuring rapid, accurate diagnosis resolves in 7 to 10 days and does not require therapy other than
of sporadic cases; in addition, they are important for epidemio- supportive treatment to alleviate the symptoms.93,110 Mumps
logical surveillance and control of community outbreaks.93,105,107 infection in pregnant women results in increased risk for fetal
Isolation of rubeola virus in conventional cell cultures is death when it occurs in the first trimester of pregnancy, but it
technically difficult and slow and is not generally performed is not associated with congenital abnormalities.95
in the routine diagnosis of measles, but it may be useful in The number of mumps cases in the United States has de-
epidemiological surveillance of measles virus strains.93,107 The clined significantly since the introduction of a live, attenuated
optimal time to recover measles virus from nasopharyngeal mumps virus vaccine in 1967 and its routine use in childhood
aspirates, throat swabs, or blood is from the prodrome period immunization schedules in 1977.93,95 The vaccine is most
of 3 to 4 days after rash onset. The virus may be isolated from commonly combined with the vaccines for rubella and mumps
urine up to 1 week after appearance of the rash.93,106 (MMR) or is used in combination with the rubella, mumps,
Serological testing provides the most practical and reliable and varicella vaccines (MMRV).
means of confirming a measles diagnosis.93,105,107 In conjunc- The diagnosis of mumps is usually made on the basis of
tion with clinical symptoms, a diagnosis of measles is indicated clinical symptoms, especially parotitis, and does not require
by the presence of rubeola-specific IgM antibodies or by a four- laboratory confirmation.93,112 However, laboratory testing is
fold rise in the rubeola-specific IgG antibody titer between very useful in cases in which parotitis is absent or when differ-
serum samples collected soon after the onset of rash and 10 to entiation from other causes of parotitis is required. Culture of
30 days later.93 SSPE is associated with extremely high titers the mumps virus from clinical specimens is considered to be
of rubeola antibodies.103,107 IgM antibodies are preferentially the gold standard for laboratory confirmation of acute infec-
detected by an IgM capture ELISA method, which is highly sen- tion.112,113 Within the first few days of illness, the mumps virus
sitive and has a low incidence of false-positive results.92,93,105 can be isolated from saliva, urine, cerebrospinal fluid, or swabs
IgM antibodies are detectable by 3 to 4 days after appearance
of symptoms and persist for 1 to 2 months.104,107 Samples
collected before 72 hours may yield false-negative results and
repeat testing is recommended in that situation.93
A variety of methods have been developed to detect IgG
rubeola antibodies, but the most commonly used is
ELISA.92,93,107 IgG antibodies become detectable 7 to 10 days
after the onset of symptoms and persist for life.107 Presence of
rubeola-specific IgG antibodies indicates immunity to measles
because of past infection or immunization.105,107 Testing for
IgG antibodies is therefore routinely performed on serum
samples of individuals such as health-care workers to deter-
mine their immune status.
Molecular methods to detect rubeola RNA can be used in
cases in which serological tests are inconclusive or inconsistent
and can be used to genotype the virus in epidemiological
studies.107-109 The preferred molecular technique is RT-PCR, per-
formed by traditional or real-time PCR methodologies. These Parotitis characteristic of mumps. Note the swollen
assays are sensitive, can be performed on a variety of clinical neck region caused by an enlargement of the boy’s salivary glands.
samples or on infected cell cultures, and can detect viral RNA (Courtesy of the Centers for Disease Control and Prevention, Public
within 3 days of rash appearance.107 Health Image Library.)
from the area around the excretory duct of the parotid gland. RNA into DNA. The DNA then becomes integrated into the
The preferred specimens are a buccal swab or saliva from the host cell’s genome as a provirus. The provirus can remain in
buccal cavity collected within 3 to 5 days of symptom onset.114 a latent state within infected cells for a prolonged period
The specimen can then be used to inoculate cell lines such as of time. Upon activation of the host cell, the provirus can
primary monkey kidney cells and Vero cells, which are grown proceed to complete its replication cycle to produce more
in shell vial cultures and stained with fluorescein-labeled virions. However, HTLV-I and HTLV-II exist predominantly
monoclonal antibodies to identify mumps antigens.112,113 in the proviral state and are spread directly to uninfected
However, culture methods require experienced personnel and cells through a viral synapse. Additional copies of the viral
specialized reagents and are being increasingly replaced with nucleic acid are produced when the infected host cells
molecular detection of viral nucleic acid.97,110 replicate.116
Standard and real-time RT-PCR methods have been devel- The human T-cell lymphotropic viruses preferentially infect
oped to detect mumps virus RNA in specimens collected from CD4+ T lymphocytes, but can also infect CD8+ T cells, den-
the buccal cavity, throat, cerebral spinal fluid, or urine of patients dritic cells, and macrophages.116,117 CD8+ CTLs effectively
with a suspected mumps infection.112,113 In many laboratories, control proliferation of virus-infected cells in most individuals.
RT-PCR is recommended as the primary diagnostic test for However, inflammatory cytokines released during this immune
mumps because it is more sensitive than serology.114 As with response may contribute to the pathogenesis of HTLV-associated
culture, buccal swabs collected early in the illness provide the diseases. In addition, researchers have reported that HTLV-I
best results and false-negative results are frequent in clinical infection of CD4+ T cells can increase production of proin-
samples collected after 1 week of symptom onset.110 Genotyping flammatory cytokines, impair production of Th1 cytokines
may be performed to track transmission of the virus during necessary for cell-mediated immunity, and induce differentia-
mumps outbreaks.113,114 tion of T regulatory (Treg) cells. The differentiated Treg cells
When indicated, serological testing provides a simple can facilitate viral persistence by suppressing the host’s immune
means of confirming a mumps diagnosis, but it has some im- response to the virus.116 HTLV-I also transforms CD4+ T lym-
portant limitations. ELISA is the most commonly used method phocytes into malignant cells in a small percentage of individ-
to detect mumps antibodies because it is sensitive, specific, uals through mechanisms mediated by the Tax protein, which
cost effective, and readily performed by the routine clinical result in increased cell proliferation and accumulation of harm-
laboratory.93 Use of solid-phase IgM capture assays reduces ful genetic mutations.116,117
the incidence of false-positive results because of rheumatoid HTLV-I and HTLV-II can be transmitted by three major
factor. Current or recent infection is indicated by the presence routes: bloodborne (mainly through transfusions containing
of mumps-specific IgM antibody in a single serum sample or cellular components or through intravenous drug abuse),
by at least a four-fold rise in specific IgG antibody between sexual contact (most commonly from men to women), and
two specimens collected during the acute and convalescent mother-to-child (mainly through breastfeeding).115,118 HTLV-
phases of illness.93,112 However, acute IgG titers are often high I infection is endemic in southwestern Japan, the Caribbean
and a four-fold increase in titer may not be evident in the islands, South and Central Africa, the Middle East, parts of
convalescent sample.110,113,114 IgM antibodies can be detected South America, and Papua New Guinea. Between 5 million
within 3 to 4 days of illness and can persist for at least 8 to and 20 million people are thought to be infected with HTLV-
12 weeks.112 However, a negative IgM test does not rule out I worldwide.119 Infections in the United States and Europe
mumps because negative results can occur if the serum was have resulted mainly from immigrants from endemic areas.
collected too early or too late. In addition, individuals who HTLV-II infections are highest in various Native Indian
received any doses of the mumps vaccine tend to have lower populations in the Americas, a few Pygmy tribes in Central
or absent IgM antibody.110,112 IgG antibodies become de- Africa, and intravenous drug abusers in North America and
tectable within 7 to 10 days and persist for years.112 However, Europe.115,120
the presence of mumps IgG antibodies does not necessarily HTLV-I is the cause of two diseases: adult T-cell leukemia/
correlate with the presence of neutralizing antibodies, which lymphoma (ATL) and HTLV-associated myelopathy/tropical
would confer immunity to the virus.113,114 spastic paraparesis (HAM/TSP). ATL can be classified into four
different subtypes based on clinical manifestations: acute,
lymphomatous, chronic, and smoldering.118,121 Over half of
Human T-Cell Lymphotropic Viruses patients have the acute type, an aggressive variant with a median
Human T-cell lymphotropic virus type I (HTLV-I) and survival of 6 months.118 All four types of ATL are characterized
human T-cell lymphotropic virus type II (HTLV-II) are by a monoclonal proliferation of mature T cells that express the
closely related retroviruses. Both viruses have three struc- surface markers, CD3, CD4, and CD25. The malignant cells
tural genes: gag, which codes for viral core proteins; pol, have lobulated, “flower-shaped” nuclei that contain proviral
which codes for viral enzymes; and env, which encodes pro- HTLV-I nucleic acid.115,118 The lifetime risk of HTLV-I carriers
teins in the viral envelope; as well as a region called pX, which for developing ATL is 3% to 5% and is highest in those who
encodes several regulatory proteins including Tax.115,116 These acquired the infection perinatally.118,119 The disease typically
viruses have RNA as their nucleic acid and the enzyme, re- appears after a latent period of 20 to 30 years following initial
verse transcriptase, whose function is to transcribe the viral infection.
 Individuals infected with HTLV-I also have a 4% lifetime also be used to monitor the proviral load in patients with
risk of developing a progressive neurological disorder called HTLV-associated diseases during therapy and to demonstrate
HTLV-I-associated myelopathy/tropical spastic paraparesis the presence of the virus in cells from patients who are sus-
(HAM/TSP).118 The risk is highest among those who con- pected of having HTLV-associated disease.115,125
tracted the infection through sexual transmission. The disease
is characterized by slowly progressive weakness and stiffness
of the legs, back pain, and urinary incontinence. HTLV-I
has also been associated with a variety of autoimmune and
SUMMARY
inflammatory disorders, including uveitis (intraocular inflam- • Viruses are obligate intracellular pathogens that can
mation of the eyes), infective dermatitis, myositis (inflamma- produce a wide range of diseases in humans.
tion of the muscles), and arthropathy (inflammation of the • Viruses can exist as either free infectious virions or intra-
joints); however, a causal relationship of the virus with these cellular particles in infected host cells. These different
conditions has not been established.115,118 It is unclear states require a combined effort of innate, humoral, and
what factors influence the development and types of clinical cell-mediated immune responses to successfully defend
manifestations of HTLV-I infection, but differences in viral the host against viral infections.
strains, viral load, mode of transmission, HLA haplotypes, • Innate defenses against viruses include the skin and mucous
and immune responses mounted by the host may all play membranes, interferons to inhibit viral replication, and
a role.118 NK cells, which release cytotoxic proteins that destroy
The association of HTLV-II infection with disease is unclear virus-infected host cells.
and most individuals infected with the virus are asymptomatic. • Antibodies directed against specific viral antigens can pre-
However, there is evidence that HTLV-II may rarely be associ- vent the spread of viral infection by neutralizing a virus
ated with a neurological disease that is similar to HAM/TSP, as and preventing it from binding to host cells, opsonizing a
well as certain hematologic and dermatological diseases, and virus to make it more likely to be phagocytized, activating
increased incidence of infections.115,118,122 complement-mediated mechanisms of destruction, and
Serological testing plays an important role in detecting agglutinating viruses.
HTLV-I and HTLV-II infections because culture of the viruses • Cell-mediated immunity is needed to eliminate intracel-
requires sophisticated techniques that cannot be performed lular viruses. Virus-specific CTL bind to viral antigen com-
in routine clinical laboratories. HTLV antibodies develop 30 to plexed with class I MHC on the surface of infected host
90 days after exposure to the virus and persist for life.123 cells and release cytotoxic proteins that cause the cells to
Tests for HTLV-I and HTLV-II antibodies are used to detect undergo apoptosis.
HTLV infections in individuals and to screen blood donors. • Viruses have evolved in several ways to escape the host’s
The tests most commonly used for screening are ELISA or defenses. These include frequent genetic mutations to
CLIA methods that incorporate recombinant antigens or produce new viral antigens; evading the action of inter-
synthetic peptides from both HTLV-I and HTLV-II.115,117 ferons, complement, or other components of the immune
Particle agglutination tests have also been developed. Any system; or suppressing the immune system. Some viruses
sample producing a reactive result in the initial screen is can establish a latent state by integrating their nucleic acid
retested by the same method and subsequently tested by into the host’s genome.
a confirmatory method to reduce the incidence of false- • Serological tests for viral antibodies can be used to indicate
positive results and to distinguish between HTLV-I and exposure to a viral pathogen. In general, the presence of
HTLV-II infection. IgM indicates a current or recent infection or a congenital
Commercially available Western blot assays are most com- infection, if present in infant serum. The presence of IgG
monly used for confirmation; line immunoassays (LIA) and antibodies indicates previous exposure to a virus or immu-
IFA tests have also been developed.115 Western blot and LIA nity as a result of vaccination.
identify antibodies to specific HTLV antigens. Specimens are • Culture, antigen detection, and molecular methods for
considered positive if a particular band pattern representing viral nucleic acid can be used to directly identify viruses
antibodies to the gag and env proteins of HTLV-I or HTLV-II is in clinical samples. Molecular methods have become in-
obtained. According to criteria published by the WHO, for creasingly important in the diagnosis of viral infections
example, a sample is considered positive for HTLV-I antibodies and can also be used to quantitate viral load to determine
if visible bands are produced for one of the env proteins (either the effectiveness of antiviral therapy.
gp46 or gp62/68) and one of the gag proteins (either p19, • The hepatitis viruses are those whose primary effect is
p24, or p 53).124 A major problem with the Western blot and inflammation of the liver. Hepatitis A and E are transmit-
LIA methods is that indeterminate results can be obtained ted mainly by the fecal–oral route, whereas hepatitis B, C,
when a single band is observed or when multiple bands and D are transmitted primarily by the parenteral route.
that do not meet the criteria for positivity are seen.115,124 Hepatitis B, C, and D can lead to chronic infections. Vaccines
PCR can be performed to detect HTLV-I or HTLV-II DNA in have been developed to prevent hepatitis A, hepatitis B, and
provirus-carrying peripheral blood mononuclear cells to clar- hepatitis E.
ify repeatedly indeterminate results.115,125 PCR methods can
• Serological markers of hepatitis infections consist of virus- syndrome, disseminated infection in organ transplant recip-
specific antibodies and antigens that are commonly de- ients and patients with HIV/AIDS, and congenital abnormal-
tected by automated immunoassays. ities in infants born to infected mothers.
• IgM anti-HAV antibodies indicate current or recent hepa- • CMV infection is best detected by molecular assays for
titis A infection, whereas IgG anti-HAV antibodies are de- CMV DNA, CMV antigenemia assays for pp65 antigen, or
veloped later in the infection and indicate immunity to shell vial culture. Quantitative PCR is useful in determin-
hepatitis A. Likewise, IgM anti-HEV antibodies are present ing the CMV DNA copy number in immunocompromised
during current or recent hepatitis E infection and IgG anti- hosts undergoing antiviral treatment. Serological assays
HEV antibodies indicate later infection and immunity. for CMV antibody are most helpful in documenting a past
• Hepatitis B infection is indicated by the presence of the infection in potential blood and organ donors.
antigen HBsAg; HBeAg indicates high infectivity. IgM an- • Primary infection with varicella virus causes chickenpox
tibodies to hepatitis B core antigen are present in acute (varicella), whereas reactivation of the virus in nerve cells
hepatitis B, whereas IgG anti-HBc is present during past supplying the skin causes shingles (zoster). Diagnosis of
or chronic hepatitis B infection. Antibodies to HBsAg current varicella virus infection is usually based on clinical
(anti-HBs) indicate immunity and can be produced as a findings, but detection of varicella virus DNA by PCR may
result of past hepatitis B infection or immunization with be helpful in some clinical settings. Serological methods,
the hepatitis B vaccine. most commonly ELISA, are used mainly to document
• The presence of anti-HCV indicates exposure to the hep- immunity to varicella virus.
atitis C virus, but cannot distinguish between a current • Immunization programs have greatly reduced the inci-
and a past infection. Molecular tests for HCV RNA are dence of three childhood infections: rubella, rubeola,
used to confirm antibody-positive results; if present, they and mumps. Rubella infection is the cause of German
indicate a current infection. Molecular tests for hepatitis measles but can result in severe congenital abnormalities if
C are also used to quantitate viral load to determine the it occurs during pregnancy. Rubeola viruses cause measles,
effectiveness of antiviral therapy. A third application of a systemic infection that can cause complications in some
molecular testing is genotyping of the virus to guide deci- individuals. Mumps virus is the cause of mumps, whose
sions about therapy. classic feature is swelling of the parotid glands, although
• Hepatitis D occurs as a super- or co-infection with hepatitis other complications may occur.
B and is indicated by antibodies to hepatitis D or molecular • Although the diagnosis of rubella, measles, and mumps is
tests to detect HDV RNA. Patients with co-infections are also usually based on clinical findings, laboratory testing may
positive for IgM-anti-HBc, whereas patients with superin- be helpful in confirmation. Current infections are indi-
fections are positive for IgG-anti-HBc. cated by the presence of IgM antibodies specific for the
• The Epstein-Barr virus (EBV) is the cause of infectious appropriate virus or by a four-fold rise in virus-specific
mononucleosis, Burkitt’s lymphoma, Hodgkin disease, na- IgG antibodies in two separate specimens collected during
sopharyngeal and gastric carcinomas, and lymphoprolif- the acute and convalescent phases of disease. Testing for
erative disorders in immunosuppressed individuals. IgG antibodies is most commonly performed to screen for
• Most patients with infectious mononucleosis produce het- immunity to these viruses. RT-PCR is a useful adjunct to
erophile antibodies, which can react with antigens from serology in detecting viral RNA in patients with inconclu-
bovine, horse, or sheep RBCs. These antibodies are routinely sive serology results, in epidemiological studies, and in
screened for by the “Monospot” test, which is performed by the detection of congenital rubella infections.
rapid immunochromatographic or agglutination methods to • The human T-cell lymphotropic viruses, HTLV-I and HTLV-II,
detect antibodies to bovine or horse erythrocyte antigens. are retroviruses that infect CD4 T lymphocytes. HTLV-I
• ELISA or IFA tests for EBV-specific antibodies are used to is the cause of adult T-cell leukemia and lymphoma,
confirm a diagnosis of infectious mononucleosis, detect HTLV-I-associated myelopathy/tropical spastic paraparesis
heterophile-negative cases of infectious mononucleosis, (HAM/TSP), and other inflammatory disorders. HTLV-II
and to diagnose other EBV-associated diseases. Acute in- may be associated with HAM/TSP-like neurological dis-
fectious mononucleosis is indicated by the presence of IgM ease as well as hematologic and skin disorders, but the
anti-VCA and anti-EA-D, as well as the absence of anti- disease associations of this virus are unclear. ELISA and
EBNA. Molecular tests are useful in detecting EBV DNA in CLIA tests are used routinely to screen blood donors for
immunocompromised patients who may not develop a antibodies to HTLV-I and HTLV-II and to detect exposure
good antibody response and in monitoring viral load in to HTLV in other individuals. Positive results are con-
patients with EBV-related malignancies during therapy. firmed by Western blot or line immunoassays. PCR for
• CMV (cytomegalovirus) infection is asymptomatic in most proviral DNA can be used to clarify indeterminate results
healthy individuals but may cause a mononucleosis-like and monitor viral load in patients undergoing therapy.
Study Guide: Immune Escape Mechanisms Commonly Used by Viruses
VIRAL ESCAPE MECHANISM EXAMPLES
Acquisition of genetic mutations that result in Influenza viruses, rhinoviruses, HIV
new viral antigens
Inhibition of immunologic components HCV blocks actions of interferons; HSV inhibits C3b
Suppression of the immune system CMV and HIV reduce expression of class I MHC on the surface of
virus-infected cells, reducing their recognition by CTLs;
HIV destroys infected CD4 Th cells
Establishment of a latent state CMV, VZV, and HIV integrate their nucleic acid into the host cell genome
CMV = cytomegalovirus; CTLs = cytotoxic T lymphocytes; HCV = hepatitis C virus; HIV = human immunodeficiency virus; HSV = herpes simplex viruses;
CMV = cytomegalovirus: MHC = major histocompatibility complex; VZV = varicella zoster virus.

CASE STUDIES
1. A 25-year-old male had been experiencing flu-like symp- 2. A 5-pound infant was born with microcephaly, purpuric
toms, loss of appetite, nausea, and constipation for 2 weeks. rash, low platelet count, cardiovascular defects, and a
His abdomen was tender and his urine was dark in color. cataract in the left eye. The infant’s mother recalled expe-
Initial testing revealed elevations in his serum alanine riencing flu-like symptoms and a mild skin rash early in
aminotransferase (ALT) and aspartate aminotransferase her pregnancy. She had not sought medical attention at
(AST) levels. the time. The infant’s physician ordered tests to investi-
gate the cause of the newborn’s symptoms.
Questions
a. What laboratory tests should be used to screen this Questions
patient for viral hepatitis? a. What virus is the most likely cause of the infant’s
b. If the patient tested positive for hepatitis B, which symptoms?
tests should be used to monitor his condition? b. What laboratory tests would you suggest the doctor
c. If the patient were to develop chronic hepatitis B, order on the mother to support your suggested
which markers would be present in his serum? diagnosis?
c. What tests should be performed on the infant’s serum
to support this diagnosis?

REVIEW QUESTIONS
1. The role of CTLs in immune responses against 3. A patient who has developed immunity to a viral
viruses is to infection would be expected to have which of the
a. neutralize viral activity. following serology results?
b. promote destruction of viruses by a. IgM+, IgG–
ADCC. b. IgM–, IgG+
c. destroy virus-infected host cells. c. IgM+, IgG+
d. attack free virions. d. IgM–, IgG–

2. Viruses can escape immune defenses by 4. A newborn suspected of having a congenital viral
a. undergoing frequent genetic infection should be tested for virus-specific antibodies
mutations. of which class(es)?
b. suppressing the immune system. a. IgM
c. integrating their nucleic acid into the host b. IgG
genome. c. IgA
d. all of the above. d. All of the above classes
 5. Which of the following hepatitis viruses is transmitted 11. A pregnant woman is exposed to a child with a
by the fecal–oral route? rubella infection. She had no clinical symptoms but
a. Hepatitis B had a rubella titer performed. Her antibody titer was
b. Hepatitis C 1:8. Three weeks later, the test was repeated and her
c. Hepatitis D titer was 1:128. She still had no clinical symptoms.
d. Hepatitis E Was the laboratory finding indicative of rubella
infection?
6. An individual with hepatomegaly, jaundice, and a. No, the titer must be greater than 256 to be
elevated liver enzymes has the following laboratory significant.
results: IgM anti-HAV (negative), HBsAg (positive), b. No, the change in titer is not significant if no
IgM anti-HBc (positive), and anti-HCV (negative). clinical signs are present.
These findings support a diagnosis of c. Yes, a greater than four-fold rise in titer indicates
a. hepatitis A. early infection.
b. acute hepatitis B. d. Yes, but clinical symptoms must also correlate
c. chronic hepatitis B. with laboratory findings.
d. hepatitis C.
12. The cause of shingles is the
7. The serum of an individual who received all doses a. cytomegalovirus.
of the hepatitis B vaccine should contain b. rubella virus.
a. anti-HBs. c. varicella-zoster virus.
b. anti-HBe. d. HTLV-I.
c. anti-HBc.
d. all of the above. 13. The method of choice for detecting VZV infection
in immunocompromised hosts is
8. Quantitative tests for HCV RNA are used to a. serology to detect virus-specific IgM antibodies.
a. screen for hepatitis C. b. serology to detect virus-specific IgG antibodies.
b. determine the HCV genotype. c. viral culture.
c. differentiate acute HCV infection from chronic d. real-time PCR.
HCV infection.
d. monitor hepatitis C patients on antiviral therapy. 14. Which of the following is true regarding laboratory
testing for mumps?
9. In the laboratory, heterophile antibodies are routinely a. RT-PCR is recommended as the primary
detected by their reaction with diagnostic test.
a. B lymphocytes. b. Serology is necessary for confirmation of a suspected
b. bovine erythrocyte antigens. clinical case.
c. sheep erythrocyte antigens. c. IgM tests for mumps are highly specific.
d. Epstein-Barr virus antigens. d. An acute infection must be confirmed by a four-fold
rise in IgG titer.
10. The presence of IgM anti-rubella antibodies in the
serum from an infant born with a rash suggests 15. A positive result on a screening test for HTLV-I antibody
a. a diagnosis of measles. should be
b. a diagnosis of German measles. a. considered highly specific for HTLV-I infection.
c. congenital infection with the rubella virus. b. followed by PCR.
d. passive transfer of maternal antibodies to the c. confirmed by Western blot.
infant’s serum. d. validated by viral culture.
 Laboratory Diagnosis
of HIV Infection

After finishing this chapter, you will be able to: HIV TRANSMISSION
1. Describe the classification system used to identify HIV isolates. CHARACTERISTICS OF HIV
2. Explain the conditions under which transmission of human Composition of the Virus
immunodeficiency virus (HIV) can occur. Structural Genes
3. Describe the structure of the HIV particle, including pertinent Viral Replication
antigens and the genes that encode them. IMMUNOLOGIC MANIFESTATIONS
4. Depict the replication cycle of HIV, beginning with entry of the virus Immune Responses to HIV
into host cells.
Effects of HIV Infection on the
5. Describe the host’s immune responses to HIV and the effects of HIV Immune System
on the immune system.
CLINICAL SYMPTOMS OF HIV
6. Describe the clinical manifestations of HIV infection. INFECTION
7. Explain the CDC classification system for HIV infection. TREATMENT AND PREVENTION
8. Discuss antiretroviral treatments and the impact they have had on LABORATORY TESTING FOR HIV
HIV infection. INFECTION
9. Discuss the 2014 CDC-recommended algorithm for screening for HIV SCREENING AND DIAGNOSIS
infection, as well as its advantages compared with previous algorithms
Testing Algorithms
and its limitations.
Serological Test Principles
10. Discuss the principle of enzyme-linked immunosorbent assay (ELISA)
testing for HIV infection and contrast first-generation, second-generation, Qualitative Nucleic Acid Tests (NATs)
third-generation, and fourth-generation ELISA tests for HIV. DISEASE MONITORING
11. Discuss the underlying principle and clinical uses of rapid tests for HIV. CD4 T-Cell Enumeration
12. Describe the principle of the Western blot test, interpretation of the Quantitative Viral Load Assays
results, and limitations of the test. Drug Resistance Testing
13. Give reasons for false positives and false negatives in HIV antibody TESTING OF INFANTS YOUNGER THAN
testing. 18 MONTHS
14. Discuss the principle and clinical utility of flow cytometric methods SUMMARY
for CD4 T-cell enumeration.
CASE STUDIES
15. Differentiate between reverse-transcriptase polymerase chain reaction
REVIEW QUESTIONS
(RT-PCR), quantitative (real-time) RT-PCR, and branched DNA
(bDNA) testing for HIV nucleic acid.
16. Discuss the clinical utility of HIV viral load testing and drug-resistance
testing.
17. Discuss the protocol for HIV testing of infants and children younger
than 18 months of age.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Acquired immunodeficiency CD4 T cells Highly active antiretroviral p24 antigen
syndrome (AIDS) Combination antiretroviral therapy (HAART) Reverse transcriptase
Amplicon therapy (CART ) Human immunodeficiency Viral load tests
Antiretroviral therapy (ART) ELISAs virus (HIV)
Western blot test
Branched chain DNA Flow cytometry Hybridization

Human immunodeficiency virus (HIV) is the etiologic agent HIV Transmission

of acquired immunodeficiency syndrome, or AIDS, a disease
that has posed one of the greatest medical challenges world- Transmission of HIV occurs through one of three major routes:
wide. According to the World Health Organization (WHO), at intimate sexual contact, contact with blood or other body flu-
the end of 2015 over 36 million people were living with HIV ids, or perinatally (from infected mother to infant).6,9 The ma-
infection, 1.1 million people became newly infected, and 1.5 jority of cases of HIV infection have occurred through sexual
million people died of AIDS.1 Since its discovery, the virus has transmission involving either vaginal or anal intercourse.
claimed the lives of more than 39 million people worldwide. Worldwide, about 85% of cases of HIV infection can be attrib-
Although the majority of infected persons reside in developing uted to heterosexual contact, whereas in the United States the
countries, HIV infection has also created a significant problem largest number of cases has resulted from anal intercourse in
in developed nations. For example, in the United States, over homosexual males.2,6,9 The presence of other sexually trans-
1.1 million cases of AIDS and more than 673,000 AIDS-related mitted diseases such as syphilis, gonorrhea, or genital herpes
deaths have been reported since 1981, when the first cases of increases the likelihood of transmission by disrupting protec-
AIDS were identified.2 tive mucous membranes and activating immunologic cells in
The virus that is responsible for causing AIDS, HIV-1, was the genital areas.6,9,11
identified independently by the laboratories of Luc Montag- The second route of transmission is through parenteral ex-
nier of France and Robert Gallo and Jay Levy of the United posure to infected blood or body fluids, which occurs mainly
States in 1983 and 1984, respectively.3-5 Isolates of HIV-1 through the sharing of contaminated needles by intravenous
have been classified into four groups: group M (the main or drug users. Less frequently, transmission can take place via
major group), group O (the outlier group), and two newer blood transfusions, the use of clotting factors by hemophiliacs,
groups, group N and group P.6 Group M viruses are respon- occupational injuries with needlesticks or other sharp objects,
sible for the majority of HIV-1 infections worldwide. This or mucous membrane contact in health-care workers exposed
group contains nine subtypes or clades, designated A, B, C, to infectious fluids.9,12,13 The virus has also been acquired by
D, F, G, H, J, and K. Subtype C is the most predominant sub- transplantation of infected tissue. Screening of blood and organ
type worldwide, whereas subtype B is the most prevalent sub- donors for HIV has dramatically decreased the incidence of in-
type in the United States, Europe, and Australia. Groups N, O, fection in recipients of blood transfusions, clotting factors, and
and P occur much less frequently and are largely confined to organ transplants. In the United States, it is estimated that one
West Africa.6-8 An increasing number of circulating recombi- transmission occurs per 1 to 2 million blood donations, result-
nant forms (CRFs) exist, which are produced as a result of ing in the release of about 11 infectious units each year.9,14
genetic recombination between two subtypes that have in- Studies by the Centers for Disease Control and Prevention
fected a single individual.9 (CDC) have estimated the average risk of transmission to
A related but genetically distinct virus, HIV-2, was dis- health-care workers to be approximately 0.3% after a percuta-
covered in 1986.10 The majority of HIV-2 infections have neous exposure to HIV-infected blood and about 0.09% after a
occurred in West Africa, although the virus has also been mucous membrane exposure. Body fluids considered to be po-
identified in patients in other parts of the world.7,8 HIV-2 tentially infectious include blood, semen, vaginal secretions,
is transmitted in the same manner as HIV-1 and may also cerebral spinal fluid, synovial fluid, pleural fluid, peritoneal
cause AIDS, but it is less pathogenic and has a lower rate of fluid, pericardial fluid, amniotic fluid, saliva from dental pro-
transmission. Although this chapter discusses the differences cedures, other fluids containing visible blood, and any body
between the two viruses, our focus is on HIV-1 because it fluid that cannot be differentiated. The risk of transmission is
is much more prevalent throughout the world. In this chap- increased in exposures involving a large quantity of blood, hol-
ter, the term HIV is used to refer to HIV-1, and HIV-2 is low-bore needles placed directly into an artery or vein, or deep
so named. tissue injury. The risk of infection is also increased if the source
Accurate diagnosis is essential for early intervention and patient is in the acute or advanced stages of HIV infection, when
halting the spread of HIV. This chapter will discuss character- the amount of virus circulating in the bloodstream is high.9,12,15
istics of the virus, immunologic manifestations of the disease, The third route of transmission is perinatal, from infected
and laboratory techniques used to diagnose and monitor HIV mother to her fetus or infant. Transmission through this route
infection. can occur during pregnancy, by transfer of blood at the time of
delivery, or through breastfeeding.9,11 Perinatal transmission has called reverse transcriptase, which transcribes the viral RNA
been markedly reduced through HIV screening during preg- into DNA, a necessary step in the virus’s life cycle. HIV is a
nancy, administration of antiretroviral drugs to HIV-positive preg- spherical particle, 100 to 120 nm in diameter, which contains
nant women and their newborn babies, and use of infant formula an inner core with two copies of single-stranded RNA sur-
by mothers who are infected with the virus. These measures have rounded by a protein coat or capsid and an outer envelope of
decreased the rate of perinatal transmission to less than 2%, as glycoproteins embedded in a lipid bilayer.9,17,18 The glycopro-
compared with rates of 15% to 35% in untreated mothers.9,16 teins are knoblike structures that are involved in binding the
virus to host cells during the infection. Figure 24–1 shows the
Characteristics of HIV structure of the HIV virion.

Composition of the Virus Structural Genes

HIV belongs to the genus Lentivirinae of the virus family Retro- The genome of HIV includes three main structural genes—gag,
viridae.9,17 It is classified as a retrovirus because it contains ri- env, and pol—and a number of regulatory genes. Figure 24–2
bonucleic acid (RNA) as its nucleic acid and a unique enzyme, shows the relative locations of the major HIV genes and indicates

Docking glycoprotein (gp120)

Transmembrane glycoprotein (gp41)

Integrase

Matrix protein (p17)

Reverse transcriptase
Capsid (p24)
Viral RNA

Structure of HIV virion, Phospholipid envelope

showing some of the major components.
p = protein; gp = glycoprotein; the num-
bers refer to the molecular weights of the
proteins in kilodaltons.

tat*
rev*
nef*
vif* vpu* env
3!LTR
5!LTR pol vpr* Codes for viral
envelope proteins
gag Codes for
viral enzymes
Codes for gp160 precursor
p66
nucleocapsid and
p51
core proteins
p31
p55 precursor p10
gp41
gp120

p6
p9
p17
p24

*Regulatory genes
The HIV-1 genome. The relative locations of the major genes in the HIV-1 genome are indicated, as well as their gene products.
* = regulatory genes
their gene products. The gag gene codes for p55, a precursor Table 24–1 Major HIV Genes and Their Products
protein with a molecular weight of 55 kd, from which four core
PROTEIN
structural proteins are formed: p6, p9, p17, and p24.19,20 All
GENE PRODUCT FUNCTION
four are located in the nucleocapsid of the virus. The capsid that
surrounds the internal nucleic acids contains p24, p6, and p9, gag p17 Inner surface of envelope
whereas p17 lies in a layer between the protein core and the en- p24 Core coat for nucleic acids
p9 Core-binding protein
velope, called the matrix, and is actually embedded in the inter-
p6 Binds to genomic RNA
nal portion of the envelope.19
The env gene codes for the glycoproteins gp160, gp120, and env gp120 Binds to CD4 on T cells
gp41, which are found in the viral envelope. Gp160 is a pre- gp41 Transmembrane protein associated
cursor protein that is cleaved to form gp120 and gp41. Gp120 with gp120
forms the numerous knobs or spikes that protrude from the pol p66 Subunit of reverse transcriptase;
outer envelope, whereas gp41 is a transmembrane glycoprotein degrades original HIV RNA
that spans the inner and outer membrane and attaches to p51 Subunit of reverse transcriptase
gp120. Both gp120 and gp41 are involved in fusion and at- p31 Integrase; mediates integration of
tachment of HIV to receptors on host cells.17,20 HIV DNA into host genome
The third structural gene, pol, codes for enzymes necessary p10 Protease that cleaves gag precursor
for HIV replication.17,20 These include reverse transcriptase tat p14 Activates transcription of HIV
(p51); ribonuclease (RNase H; p66), an enzyme involved in provirus
the degradation of the original HIV RNA; integrase (p31), an
rev p19 Transports viral mRNA to the
enzyme which mediates the integration of viral DNA into the cytoplasm of the host cell
genome of infected host cells; and a protease (p10) that cleaves
precursor proteins into smaller active units used to make the nef p27 Enhances HIV replication
mature virions. These proteins are located in the core of the vpu p16 Viral assembly and budding
virus in association with HIV RNA.
Several other genes in the HIV genome code for products that vpr p15 Integration of HIV DNA into host
genome
have regulatory or accessory functions.9,17,20 These genes include
tat (transactivator), rev (regulator of expression of virion pro- vif p23 Infectivity factor
teins), nef (negative effector), Vpu (viral protein “U”), Vpr (viral
protein “R”), and Vif (viral infectivity factor). Although the prod-
ucts of these genes are not an integral part of the viral structure,
Entry of HIV into the host cells to which it has attached re-
they serve important functions in controlling viral replication
quires an additional binding step, involving co-receptors that
and infectivity. The HIV gene sequence is surrounded by 5' and
promote fusion of the HIV envelope with the plasma cell mem-
3' long terminal repeat (LTR) regions, which also play a role in
brane. These co-receptors belong to a family of proteins known
regulating the expression of the genes. Table 24–1 summarizes
as chemokine receptors, whose main function is to direct white
the major HIV-1 genes, their products, and their functions.
blood cells (WBCs) to sites of inflammation. The chemokine
HIV-2 has gag, env, pol, and regulatory or accessory genes that
receptor, CXCR4, is required for HIV to enter T lymphocytes,
have similar functions to those seen in HIV-1. The homology be-
whereas the chemokine receptor, CCR5, is required for entry
tween the genomes of the two viruses is approximately 50%.9,19
into macrophages. In fact, individuals who have a genetic mu-
The gag and pol regions are most similar, whereas the env region
tation in the CCR5 gene have been found to be resistant to HIV
differs greatly. Thus, the viruses can most easily be distinguished
infection.21,22 Binding of the co-receptors allows for HIV entry
on the basis of antigenic differences in their env proteins.
by inducing a conformational change in the gp41 glycoprotein,
which mediates fusion of the virus to the cell membrane.9,17
Viral Replication After fusion occurs, the viral particle is taken into the cell and
The first step in the reproductive cycle of HIV occurs when the uncoating of the particle exposes the viral genome.9,17,20 Action
virus attaches to a susceptible host cell. This interaction is me- of the enzyme reverse transcriptase produces complementary
diated through the host-cell CD4 antigen, which serves as a re- DNA from the viral RNA. Double-stranded DNA is synthesized
ceptor for the virus by binding the gp120 glycoprotein on the and, with the help of the HIV integrase enzyme, becomes inte-
outer envelope of HIV. T helper (Th) cells are the main target for grated into the host cell’s genome as a provirus (Fig. 24–3). The
HIV infection because they express high numbers of CD4 mol- provirus can remain in a latent state for a long time, during which
ecules on their surface and bind the virus with high affinity.20 viral replication does not occur. Eventually, expression of the viral
Other cells such as macrophages, monocytes, dendritic cells, genes is induced when the infected host cell is activated by bind-
Langerhans cells, and microglial brain cells can also be infected ing to antigen or by exposure to cytokines. Viral DNA within the
with HIV because they have some surface CD4. HIV viruses that cell nucleus is then transcribed into genomic RNA and messenger
preferentially infect T cells are known as T-tropic or X4 strains, RNA (mRNA), which are transported to the cytoplasm. Transla-
whereas those strains that can infect both macrophages and tion of mRNA occurs, with production of viral precursor proteins
T cells are called M-tropic or R5 strains. and assembly of viral particles. The intact virions bud out from
 Release responses are unable to eliminate HIV from the body,8,9 as we
gp120 will discuss in the text that follows.
B lymphocytes are stimulated to produce antibodies to HIV,
which can usually be detected in the host’s serum by 6 weeks
after primary infection.9,18 The first antibodies to be detected
Attachment Assembly are directed against the gp41 transmembrane glycoprotein, fol-
and budding lowed by production of antibodies to the gag proteins such
CXCR4 CD4 as p24, and finally production of antibodies to the env, pol, and
Fusion and Viral enzymes regulatory proteins. The most immunogenic proteins are in the
uncoating and proteins viral envelope and elicit the production of neutralizing anti-
Translation bodies. These antibodies usually appear 2 to 3 months after
Reverse infection and prevent the virus from infecting neighboring
transcriptase
vRNA cells.24 Antibodies to the envelope proteins have also been
cDNA
mRNA
shown to bind to Fc receptors on NK cells and participate in
dsDNA Genomic
ADCC-mediated killing of HIV-infected cells. However, these
Transcription RNA antibodies may also participate in the pathogenesis of HIV
infection by facilitating Fc-receptor mediated endocytosis of
Provirus
Integrase opsonized virus by uninfected cells. Furthermore, dense gly-
cosylation of the env proteins can mask important epitopes,
and the enormous diversity of the virus poses challenges to the
host in producing a protective antibody response.9,24
Replication cycle of HIV in a CD4+ T cell. T-cell–mediated immunity is thought to play an important
role in the immune response to HIV, as it does in other viral in-
fections.9,18,19,24 CD8+ cytotoxic T lymphocytes, also known as
the host cell membrane and acquire their envelope during the cytolytic T cells (CTLs), appear within weeks of HIV infection
process. The precursor proteins are cleaved by the viral protease and are associated with a decline in the amount of HIV in the
enzyme in the mature virus particles. These viruses can proceed blood during acute infection. Antibodies can attach only to viri-
to infect additional host cells. Viral replication occurs to the great- ons circulating freely outside of host cells; in contrast, CTLs can
est extent in antigen-activated Th cells. attack host cells harboring viruses internally. This process in-
Because viral replication occurs very rapidly and the re- volves the binding of CTLs containing HIV-specific antigen re-
verse transcriptase enzyme lacks proofreading activity, genetic ceptors to HIV proteins associated with class I MHC molecules
mutations occur at a high rate, producing distinct isolates on the surface of infected host cells. HIV-specific CTLs are stim-
that exhibit an extraordinary level of antigenic variation. In ulated to develop into mature, activated clones through the ef-
fact, the level of HIV diversity in a single individual is greater fects of cytokines released by activated CD4 Th cells, a process
than the diversity of all the influenza virus isolates through- that is common to immune responses against other viruses (see
out the world in a given year!23 This tremendous genetic di- Chapter 4 for details). After the CTLs bind to HIV-infected host
versity of HIV hinders the ability of the host to mount an cells, cytolytic enzymes are released from their granules and de-
effective immune response. stroy the target cells. Free virions are released from the damaged
cells and can be bound by antibodies. CTLs can also suppress
replication and spreading of HIV by producing cytokines such
Immunologic Manifestations as interferon-γ, which have antiviral activity.
Innate immune defenses may play a role in responding to
Immune Responses to HIV HIV in the early stages of the infection.24,25 In particular, NK
cells become activated during acute HIV infection and can me-
When HIV infects a healthy individual, there is typically an
diate cytolysis of host cells infected with the virus. In addition,
initial burst of viral replication followed by a slowing down of
dendritic cells recognize viral components through pattern-
virus production as the host’s immune response develops and
recognition receptors, resulting in release of proinflammatory
keeps the virus in check.9,18,19,24 This initial viral replication
cytokines that have antiviral effects and can activate other cells
can be detected in the laboratory by the presence of increased
of the immune system. Future studies will likely clarify the role
levels of p24 antigen and viral RNA in the host’s bloodstream
of these responses in early control of HIV infection.
(see discussion in the text that follows). As the virus replicates,
some of the viral proteins produced within host cells form
complexes with class I major histocompatibility complex Effects of HIV Infection
(MHC) antigens and are transported to the cell surface, where
they stimulate lymphocyte responses. HIV-specific CD4+
on the Immune System
Th cells are generated and assist both humoral and cell-mediated HIV has developed several mechanisms by which it can escape
immune responses against the virus. However, the interactions immune responses.19,24,26,27 Although the humoral and cell-
between HIV and the immune system are complex and these mediated immune responses of the host usually reduce the
level of HIV replication, they are generally not sufficient to Altered production of cytokines and chemokines has also been
completely eliminate the virus. CTL and antibody responses seen, including increases in the levels of some cytokines during
to HIV are hindered by the virus’s ability to undergo rapid ge- the early stages of disease, followed by declining levels of IL-2
netic mutations, generating escape mutants with altered anti- and interferon-γ and a shift in the cytokine profile from Th1
gens toward which the host’s initial immune responses are to Th2 as the infection progresses toward development of
ineffective. In addition, HIV can downregulate the production AIDS.19,20,27 Extensive damage to the lymphoid tissues is evi-
of class I MHC molecules on the surface of the host cells it in- dent late in the infection, with loss of germinal centers and fol-
fects, protecting them from CTL recognition. Numerous cells licular dendritic cells resulting in an inability to activate T and
in the body can also harbor HIV as a silent provirus for long B lymphocytes.20 Other immunologic abnormalities, including
periods, including resting CD4 T cells, dendritic cells, cells of defective antigen presentation and oxidative burst by mono-
the monocyte and macrophage lineage, and microglial cells in cytes and macrophages and decreased natural killer (NK) cell
the brain. In this proviral state, HIV is protected from attack activity, have also been observed in AIDS patients.9,19,27,28
by the immune system until cell activation stimulates the virus
to multiply and display its viral antigens. Clinical Symptoms of HIV Infection
The ability of HIV to evade the immune response results in
a persistent infection that can destroy the immune system. Be- HIV causes a chronic infection that is characterized by a pro-
cause the virus’s prime targets are the CD4 Th cells, these cells gressive decline in the immune system. Although the manifes-
are most severely affected; a decrease in this cell population is tations of the disease vary in individual patients, the infection
the hallmark feature of HIV infection.28 The gastrointestinal progresses through a clinical course that begins with primary,
immune system (GALT), which is the largest immune organ in or acute, infection, followed by a period of clinical latency that
the body, is the most profoundly affected.29 Early in the infec- eventually culminates in AIDS.9,24,32 The acute, or early, stage
tion, HIV causes a rapid depletion of CCR5+ CD4+ memory of infection is characterized by a rapid burst of viral replication
T cells in the GALT. Th17 helper cells, which play an important before the development of HIV-specific immune responses. In
role in the homeostasis of the epithelial cells lining the intes- this stage, high levels of circulating virus, or viremia, can be seen
tinal mucosa (enterocytes) as well as in the secretion of antimi- in the blood of infected individuals; as a result, HIV begins to
crobial defensins, are also preferentially affected. This depletion disseminate to the lymphoid organs. There is a reduction in the
results in damage to the intestinal epithelial barrier, with leak- peripheral blood CD4 T-cell count, but this usually returns to
age of microbial products such as lipopolysaccharide (LPS) into slightly decreased or, sometimes, normal levels. As the immune
the plasma, and a general state of immune activation.6,29 system becomes activated, an acute retroviral syndrome may
CD4 Th cells are thought to be killed or rendered nonfunc- develop. This syndrome, which has been noted in 50% to 70% of
tional by HIV through a variety of mechanisms such as loss of patients with primary HIV infection, is characterized by flu-like
plasma membrane integrity because of viral budding, destruc- or infectious mononucleosis-like symptoms.9,19,24,32 Symptoms
tion by HIV-specific CTL, and viral induction of apoptosis.19,27,30 of the primary stage usually appear 3 to 6 weeks after the initial
Infected T cells turn over much more rapidly than they can be infection and resolve within 7 days to a few weeks. Many pa-
replaced, having a half-life of 12 to 36 hours.27 There is only a tients, however, are asymptomatic during this stage.
partial recovery of the T cells lost early in the infection, which is As HIV-specific immune responses develop, they begin to
followed by a progressive decrease in CD4 T cells during the curtail replication of the virus and patients enter a period of
natural course of untreated infections.6 In addition to reducing clinical latency. This stage is characterized by a decrease in
T-cell numbers, HIV also causes abnormalities in Th cell function viremia as the virus is cleared from the circulation and clinical
and impairment of memory Th cell responses.9,19,20,28 symptoms are subtle or absent.9,19,24,32 However, studies have
Because CD4 T cells play a central role in the immune demonstrated that the virus is still present in the plasma, albeit
system by regulating the activities of B and T lymphocytes at lower levels, and more so in the lymphoid tissues. The CD4
(see Chapter 4), destruction of these cells results in decreased T-cell count remains stable for a variable period of time and
effectiveness of both antibody- and cell-mediated immune re- then begins to progressively decline. A small proportion of HIV-
sponses. These effects apply not only to the immune responses infected individuals, termed long-term nonprogressors (LTNP),
directed against HIV, but to the broad range of antigens that are have normal or mildly depressed CD4 T-cell counts and low
encountered by the host. Dysregulated immune responses are viral loads; they remain asymptomatic for more than 10 years
also evident. HIV proteins actually stimulate polyclonal activa- in the absence of antiretroviral therapy.9,33 The factors that in-
tion of B cells, resulting in maturational and functional defects fluence this slower rate of progression are not completely un-
with increased circulating immunoglobulin levels, immune derstood, but appear to be associated with certain HLA types,
complexes, and autoantibodies.9,19,20,31 However, B cells in HIV- non-HLA genes, and prevalence of the R5 strain of HIV.9,24
infected individuals have a reduced ability to mount antibody Untreated individuals will ultimately progress to AIDS, the
responses after exposure to specific antigens, caused by the final stage of HIV infection, which is characterized by profound
decrease in T-cell help.9,19,20 immunosuppression with very low numbers of CD4 T cells, a
Cell-mediated immunity is also affected by the reduction in resurgence of viremia, and life-threatening infections and ma-
Th activity, resulting in a decline in CTL activity and delayed- lignancies. The rate at which individuals progress to the devel-
type hypersensitivity responses to specific antigens.9,19,26,27 opment of AIDS varies, but progression typically occurs in
untreated individuals within a median time of 10 years after Symptoms of AIDS in infants include failure to thrive, per-
the initial infection.9,32 The rate of progression has dramatically sistent oral candidiasis, hepatosplenomegaly, lymphadenopa-
decreased with the use of antiretroviral therapies (see Treatment thy, recurrent diarrhea, or recurrent bacterial infections.35,36
and Prevention in the text that follows). Abnormal neurological findings may be present. The rate by
A list of the opportunistic illnesses considered to be indicative which HIV infection progresses in children varies and may be
of AIDS is found in the Clinical Correlations box. These condi- influenced by factors such as maturity of the immune system
tions appear in immunocompromised individuals but do not at the time of infection, the dose of virus to which the child
usually affect people with a healthy immune system. In addition was exposed, and the route of infection.36
to infections and malignancies, HIV-infected individuals often The CDC first defined AIDS as “a disease, at least moder-
demonstrate neurological symptoms resulting from the ability ately predictive of a defect in cell mediated immunity, occur-
of HIV to infect cells in the brain. In early HIV infection, these ring in a person with no known cause for diminished resistance
symptoms may manifest as forgetfulness, poor concentration, to that disease.”37 The definition has been revised several times
apathy, psychomotor retardation, and withdrawal, whereas pro- over the years as more information has been acquired about
gression to late disease may result in confusion, disorientation, HIV and additional laboratory tests for HIV have been devel-
seizures, dementia, gait disturbances, ataxia, or paraparesis.9,34 oped.38-41 The CDC also published a separate case definition
for HIV infection in children.41,42 The latest definition at the
time of this writing was published in 2014.43 It combines the
Clinical Correlations case definitions for persons of all ages into a single definition
that is intended to be used for surveillance of the disease. The
Opportunistic Illnesses Indicative of AIDS
2014 definition bases a confirmed case of HIV infection on ei-
(Stage 3 HIV Infection)
ther laboratory criteria or clinical evidence, with laboratory re-
Bacterial infections, multiple or recurrent* sults being the preferred criteria. Laboratory criteria consist of
Candidiasis of bronchi, trachea, or lungs
positive test results in multitest algorithms for HIV antibody
Candidiasis of esophagus
or combination HIV antigen/antibody, whereas clinical evi-
Cervical cancer, invasive†
Coccidioidomycosis, disseminated or extrapulmonary dence refers to physician documentation of HIV infection in
Cryptococcosis, extrapulmonary the patient’s medical record (see Laboratory Testing for HIV In-
Cryptosporidiosis, chronic intestinal (longer than 1 month’s fection in the text that follows).
duration) HIV-positive patients are further classified into one of five
Cytomegalovirus disease (other than liver, spleen, or nodes), stages (0, 1, 2, 3, or unknown).43 The stage can change for an
onset older than 1 month of age individual patient in either direction over time. Stage 0 includes
Cytomegalovirus retinitis (with loss of vision) those individuals with early HIV infection who had an initial con-
Encephalopathy attributed to HIV firmed HIV-positive laboratory result followed by a negative or
Herpes simplex: chronic ulcers (longer than 1 month’s duration) indeterminate HIV test result within a 6-month period. These
or bronchitis, pneumonitis, or esophagitis (onset older than
patients can be reclassified in one of the other categories 180 days
1 month of age)
or more after initial diagnosis. Patients are classified as being in
Histoplasmosis, disseminated or extrapulmonary
Isosporiasis, chronic intestinal (longer than 1 month’s duration) stages 1, 2, or 3 based on their peripheral blood CD4 T-cell count
Kaposi sarcoma or percentage; if this information is missing, they are classified in
Lymphoma, Burkitt (or equivalent term) the “unknown” category. The CD4 T-cell parameters used in this
Lymphoma, immunoblastic (or equivalent term) classification system are shown in Table 24–2. Stage 3 is also in-
Lymphoma, primary, of brain dicated if any of the opportunistic illnesses listed in the Clinical
Mycobacterium avium complex or Mycobacterium kansasii, Correlations box are present.
disseminated or extrapulmonary
Mycobacterium tuberculosis of any site, pulmonary†, disseminated,
or extrapulmonary
Mycobacterium, other species or unidentified species, dissemi-
Treatment and Prevention
nated or extrapulmonary
Pneumocystis jirovecii (previously known as “Pneumocystis carinii”)
Treatment of HIV infection involves supportive care of the as-
pneumonia sociated infections and malignancies as well as administration
Pneumonia, recurrent† of antiretroviral therapy (ART) to suppress the virus’s repli-
Progressive multifocal leukoencephalopathy cation. Several classes of antiretroviral drugs have been devel-
Salmonella septicemia, recurrent oped to treat HIV infection: nucleoside analogue reverse
Toxoplasmosis of brain, onset older than 1 month of age transcriptase inhibitors, nonnucleoside reverse transcriptase
Wasting syndrome attributed to HIV inhibitors, protease inhibitors, integrase inhibitors, fusion in-
*Only among children aged younger than 6 years.
hibitors, and entry inhibitors (co-receptor antagonists).1,9,44,45
†Only among adults, adolescents, and children aged 6 years or older.
These drugs block various steps of the HIV replication cycle.
Courtesy of Centers for Disease Control and Prevention . Revised surveillance Their mechanisms of action are summarized in Table 24–3.
case definition for HIV infection - United States, 2014. MMWR 2014;63(3):1-10. New drugs continue to be developed as advances in this area
are made. Updated guidelines on the use of these drugs are
 Table 24–2 CD4 T-Cell Parameters Used in HIV Staging
AGE ON DATE OF CD4+ T-LYMPHOCYTE TEST
YOUNGER THAN 1 YEAR 1 TO 5 YEARS 6 YEARS OR OLDER
STAGE CELLS/μl PERCENT CELLS/μl PERCENT CELLS/μl PERCENT
1 1,500 34 1,000 30 500 26
2 750–1,499 26–33 500–999 22–29 200–499 14–25
3 <750 <26 <500 <22 <200 <14
The stage is based primarily on the CD4+ T-lymphocyte count; the percentage is considered only if the count is missing.
(Adapted from Centers for Disease Control and Prevention. Revised surveillance case definition for HIV infection—United States, 2014. MMWR 2014;63(3):1-10.)

Table 24–3 Antiretroviral Drugs for HIV Therapy

ANTIRETROVIRAL DRUG
CLASSIFICATION MECHANISM OF ACTION EXAMPLES
Nucleoside analogue Similar in structure to nucleosides; incorpo- Zidovudine, lamivudine, didanosine, abacavir,
reverse-transcriptase rate into HIV nucleic acid and block tenofovir
inhibitors (NRTI) synthesis of viral RNA
Nonnucleoside Bind to and inactivate reverse transcriptase Nevirapine, delavirdine, efavirenz, etravirine
reverse-transcriptase
inhibitors (NNRTI)
Protease inhibitors Prevent cleavage of precursor proteins Saquinavir, indinavir, ritonavir, nelfinavir,
needed for assembly of HIV virions fosamprenavir, lopinavir
Integrase inhibitors Prevent integration of HIV DNA into the Raltegravir
host genome
Fusion inhibitors Block viral infection by preventing fusion of Enfuvirtide
HIV with target cells
Entry inhibitors Block binding of HIV to the chemokine Maraviroc
(co-receptor antagonists) co-receptor CCR5

available from the U.S. Department of Health and Human Serv- this multidrug treatment.9,44 Before the development of ART,
ices and the WHO.44,46 the median survival time of AIDS patients was only 26 weeks
Studies have shown that treatment with multiple drugs is from the time of diagnosis; currently, HIV-infected patients
more effective in killing the virus and avoiding viral resistance who are treated appropriately with CART can be expected to
than treatment with a single drug. Potent regimens involving live 50 years or longer.9 Because of this success, CART is rec-
a combination of drugs from at least two of the antiretroviral ommended for all HIV-infected persons at the time of diagno-
drug classes are the standard of treatment and are referred to sis.44,46 Antiretroviral drugs have also had a significant impact
as combination antiretroviral therapy (CART) or highly in reducing perinatal transmission of HIV, as discussed previ-
active antiretroviral therapy (HAART).6,9,44,47 Currently pre- ously in this chapter.6,16 In 1994, investigators from the United
ferred treatment protocols use combinations of two nucleo- States and France published the results of a large clinical trial
side reverse-transcriptase inhibitors and a nonnucleoside demonstrating that the antiretroviral drug zidovudine, admin-
reverse-transcriptase inhibitor, a protease inhibitor, or an in- istered to HIV-positive women during pregnancy and labor and
tegrase inhibitor.6,9,44 The goal of this therapy is to reduce to the newborn during the first few weeks of life, reduced
the patient’s HIV viral load to a level that is below the de- transmission of HIV to the infant by two-thirds.49 Subse-
tectable limit of quantitative plasma viral load assays (see quently, CART and avoidance of breastfeeding by HIV-positive
Quantitative Nucleic Acid Tests (NATs) in the text that fol- women have reduced the rate of perinatal transmission to 0.1%
lows).48 This is most likely to be achieved if treatment is to 0.5% in developed countries of the world.44
started early in the course of infection and the patient can Although antiretroviral drugs and CART have significantly
adhere to the treatment as prescribed.32,45 improved morbidity and mortality in HIV-infected patients,
CART has had a dramatic effect on the clinical course of they cannot be considered a cure for AIDS. Although blood
HIV infection, as evidenced by a significant decline in the in- levels of the virus are greatly reduced in patients treated with
cidence of opportunistic infections, a delay in progression to antiretroviral drugs, HIV is still harbored in lymphoid organs
AIDS, and decreased mortality in patients who have received throughout the body and progressively destroys the immune
system.9,19 Research is ongoing to develop additional drugs to CD4 T-cell enumeration. Principles of each of these methods
target proviral HIV within infected cells.50 are discussed in the text that follows along with their applica-
Several other approaches for dealing with HIV have been tions to the detection and monitoring of HIV infection. Al-
directed toward preventing initial infection. Community-based though culturing the virus from patient samples is a definitive
education aimed at high-risk groups such as homosexual males method of demonstrating HIV infection, it is not used in clin-
and intravenous drug users has provided beneficial informa- ical settings because it is laborious, time consuming, costly, and
tion on reducing transmission of the virus. The CDC has potentially hazardous to laboratory personnel.
published guidelines for the use of antiretroviral drugs for pre-
exposure prophylaxis (PrEP) to prevent transmission to indi-
viduals who are HIV-negative but at a high risk of contracting
Screening and Diagnosis
the infection, such as those who inject illicit drugs or are sex-
Serological tests for HIV antibody are used in the initial diag-
ually active with an HIV-infected partner.51 In addition, the
nosis of HIV infection because most individuals develop anti-
CDC and the Occupational Safety and Health Administration
body to the virus within 1 to 2 months after exposure.64,65
(OSHA) have published precautions to prevent transmission
Since 1985, these tests have played a critical role in screening
of HIV and other bloodborne pathogens in health-care work-
the donor blood supply to prevent transmission of the virus
ers.52,53 Prophylactic therapy with antiretroviral drugs is also
through blood transfusions or administration of blood prod-
offered to health-care workers who may have been exposed to
ucts. Serological tests are also used in epidemiology studies to
HIV through percutaneous or mucous membrane contact with
provide health officials with information about the extent of
potentially infected blood or body fluids, in hope that early
the infection in high-risk populations. These groups can then
treatment will prevent infection.54 As a result of these mea-
be targeted for counseling, treatment, and vaccine trials, and
sures, fewer than 60 documented cases of occupational HIV
their medical or social concerns can be addressed.
transmission to health-care workers have been reported to the
Different serological methods have been used to test for HIV.
CDC as of 2010.55
Standard screening methods for HIV antibody have involved
The ultimate means of preventing HIV infection would be
enzyme-linked immunosorbent assay (ELISA) methodology
the development of an effective vaccine. Much research has
(see Chapter 11). In addition, rapid tests have been developed
been directed in this area, but the task has been very difficult
that can detect HIV antibody within minutes, making them an
for many reasons.9,18,56-59 For example, HIV can rapidly mutate
attractive alternative to the ELISA in certain situations. Con-
and escape immune recognition and there is no ideal animal
firmatory tests are performed on samples that test positive on
model in which to study vaccine effects. In addition, an effec-
a screening test, to differentiate true-positive from false-positive
tive vaccine would need to induce mucosal immunity because
results. The Western blot test (see section in the text that fol-
HIV is usually transmitted through mucosal surfaces. An effec-
lows) was the standard confirmatory test for HIV for a number
tive vaccine should also stimulate potent CTL and broad neu-
of years, but it has largely been replaced by newer methods.
tralizing antibody responses that could detect many variants
Serological testing for p24 antigen and nucleic acid testing for
of the virus. If a vaccine that can prevent HIV infection in the
HIV RNA have been incorporated into the initial HIV testing
traditional sense cannot be developed, it is possible that a less-
scheme, providing for earlier and more accurate detection. The
than-perfect vaccine may provide some benefits by prolonging
principles of these methods are discussed in the text that fol-
the disease-free period and lowering the level of viremia, thus
lows, along with their use in HIV testing algorithms.
reducing the risk of transmission to others.18,60

Testing Algorithms
Laboratory Testing for HIV Infection
Over the years, the CDC and the Association of Public Health
The laboratory plays a key role in establishing the initial di- Laboratories have developed algorithms that use a combination
agnosis of HIV infection and in monitoring known patients of laboratory tests performed in sequence to screen for the
for their response to antiretroviral therapy. In addition, the presence of HIV infection and resolve any discrepant results.
U.S. Preventative Task Force recommends routine HIV screen- These algorithms have been revised as improvements in labo-
ing (referred to as “opt-out screening”) for all consenting per- ratory tests have been made. In 1989, for example, the stan-
sons between 15 to 65 years of age, whereas the CDC dard diagnostic algorithm recommended that testing begin
recommends routine HIV testing for all persons 13 to 64 years with a sensitive ELISA for HIV-1 antibody, with positive sam-
old and annual testing for individuals in high-risk groups.61-63 ples being retested and then confirmed with a more specific
At the time of this writing, it is believed that only 20% or Western blot test for HIV-1 antibody (or less frequently, an
less of HIV-infected adults know their status.47 Universal HIV-specific indirect immunofluorescence assay (IFA).66 In
screening will hopefully identify more persons infected with 1992, the algorithm was modified so that initial testing was
HIV so they can begin early treatment with ART and be less performed with an HIV-1/HIV-2 antibody test in cases where
likely to transmit the virus to others. HIV-2 was likely to be present.67 In 2004, it was recommended
Several types of laboratory tests have been used to diagnose that rapid HIV antibody tests could also be used for initial
and monitor HIV infection, including HIV antibody detection, screening and that positive results should be confirmed by an
HIV antigen detection, viral nucleic acid testing (NAT), and HIV-1 Western blot or IFA.68
 In 2014, the CDC recommended that initial screening be and accurate identification of HIV infection. This makes it pos-
performed with a combination immunoassay that detects an- sible to begin appropriate medical care and ART sooner in in-
tibodies to HIV-1 and HIV-2 as well as the HIV-1 p24 antigen.69 fected individuals. The newer testing also helps to prevent
These recommendations apply to adults and children who are additional infections by encouraging prompt initiation of coun-
older than 24 months. There are separate recommendations seling to reduce risky behaviors and earlier notification of sex-
for children younger than 2 years, as maternal antibodies may ual partners of diagnosed individuals.63,69
be present that are likely to confuse the interpretation of the Although the 2014 algorithm is highly sensitive in the de-
test results (see the text that follows). All positive specimens tection of HIV infection, it has some limitations, which will be
should then undergo additional testing with a rapid im- discussed in the next section. The CDC will continue to eval-
munoassay that discriminates between HIV-1 and HIV-2 anti- uate its algorithm as additional tests become available for clin-
bodies. Any samples that are reactive in the initial test and ical use. The principles of the major laboratory tests used in
nonreactive in the second test should then undergo nucleic testing for HIV infection are discussed in the text that follows.
acid testing (NAT) to resolve the discrepancy.
The 2014 algorithm is summarized in Figure 24–4. This Serological Test Principles
testing scheme provides significant advantages over previous
algorithms.63,69 First, it allows for earlier detection of infec-
tions, as the time between exposure and detectable results ELISAs have been the cornerstone of screening procedures
on the HIV-1/2/p24 combo assay is typically between 15 and for HIV because they are easy to perform, can be adapted to
17 days. Secondly, it overcomes the limitations associated with test a large number of samples, and are highly sensitive and
use of the Western blot test. The Western blot is a lengthy pro- specific.9,64,65 They were first used to detect HIV antibody in
cedure that is typically performed only by specialized reference the United States in 1985 in response to the need to screen
laboratories. In addition, Western blot testing is less sensitive donated blood. Several manufacturers have developed com-
than the initial ELISA used for screening; therefore, indetermi- mercial kits that are useful in screening blood products and in
nate results can occur, which can take as long as 3 to 6 months diagnosing and monitoring patients. An updated list of kits ap-
to resolve. The 2014 testing algorithm allows for more rapid proved for use in the United States is published by the FDA.70

HIV-1/2 antigen/antibody combination immunoassay

(") (#)
Negative for HIV-1 and HIV-2
antibodies and p24Ag

HIV-1/2 antibody differentiation immunoassay

HIV-1 (") HIV-1 (#) HIV-1 (") HIV-1 (#)

or indeterminate
HIV-2 (#) HIV-2 (") HIV-2 (")
HIV-2 (#)
HIV-1 antibodies HIV-2 antibodies HIV antibodies
detected detected detected

2014 Laboratory
HIV testing algorithm recom- HIV-1 NAT
mended by the CDC. (Adapted from (") indicates reactive test result
Branson BM, Owen SM, Wesolowski
LG, et al. Laboratory testing for the (#) indicates nonreactive test result
diagnosis of HIV infection: Updated
recommendations. CDC Stacks. Web NAT: nucleic acid test
site. http://stacks.cdc.gov/view/cdc/ HIV-1 NAT(") HIV-1 NAT(#)
Acute HIV-1 Negative
23447. Updated 2014. Accessed
infection for HIV-1
December 18, 2014.)
Over the years, five generations of ELISAs have been devel-
oped, resulting in improved sensitivity and specificity.
The first generation of ELISAs were based on a solid-
phase, indirect-assay system that detected antibodies to only
HIV-1.64,65,69 (See Chapter 11 for general principles of ELISA.)
In these tests, HIV antibodies in patient serum were detected
after binding to a solid support coated with viral lysate antigens
from HIV-1 cultured in human T-cell lines, followed by addi-
tion of an enzyme-labeled anti-human IgG conjugate and sub-
strate. These first-generation assays were prone to false-positive
results caused by reactions with HLA antigens or other com-
ponents from the cells used to culture the virus, and they were
unable to detect antibodies to HIV-2.64,65
The second-generation ELISAs, introduced in the late 1980s,
were indirect binding assays that used highly purified recom-
binant (i.e., genetically engineered) or synthetic antigens from
both HIV-1 and HIV-2, rather than crude cell lysates.64,65,69
These assays demonstrated improved specificity and sensitivity
overall and were able to detect antibodies to both HIV-1 and
HIV-2. However, decreased sensitivity resulted when samples
containing antibodies to certain subtypes of HIV that lacked the
limited antigens used in the assays were tested.
Third-generation assays use the sandwich technique, based
on the ability of antibody to bind with more than one anti-
gen.64,65,69 These assays are available in ELISA and chemilumi-
nescent immunoassay (CLIA) formats. In these tests, antibodies
in patient serum or plasma bind to recombinant HIV-1 and
HIV-2 proteins coated onto a solid phase. After washing, enzyme-
or chemiluminescent-labeled HIV-1 and HIV-2 antigens are
added and bind to the already bound HIV-specific patient an-
tibodies. Substrate (or trigger solution, if CLIA is used) is added
next, and the color development (or release of light with CLIA)
is proportional to the amount of antibody in the sample. This
test format improves sensitivity by simultaneously detecting
HIV antibodies of different immunoglobulin classes, including
IgM. Enhancements in this method have increased sensitivity
further by detecting low affinity antibodies and antibodies to
group O HIV-1 as well as the more common group M. These
enhancements resulted in a diagnostic sensitivity of 100% and
diagnostic specificity of 99.9%.71
The most recent, fourth-generation assays can simultane-
ously detect HIV-1 antibodies, HIV-2 antibodies, and p24 anti-
gen.64,65,69 Previously, separate immunoassays were used to
detect the p24 antigen from the core of the HIV-1 virion as a
marker of early infection.65 Recall that levels of p24 in the cir-
culation are high in the initial weeks of infection during the
early burst of viral replication, providing a marker that can be
detected before the appearance of HIV antibody during the
acute stage of infection.69 The antigen becomes undetectable
as antibody to p24 develops and binds the antigen in immune
complexes; levels rise again during the later stages of infection Principle of fourth-generation ELISA test for HIV. This
when impairment of the immune system allows the virus to test detects the HIV-1 p24 antigen as well as HIV-1 antibodies and
replicate. HIV-2 antibodies.
The basic principle of the fourth-generation HIV-1 antibody/
HIV-2 antibody/p24 antigen combination tests is illustrated in
Figure 24–5. These tests employ a sandwich ELISA or CLIA in
which patient serum is incubated with a solid phase onto which
synthetic or recombinant HIV-1 antigens, HIV-2 antigens, and a the presence of autoreactive antibodies, history of multiple
monoclonal antibody to HIV-1 p24 have been attached. If pregnancies, severe hepatic disease, passive immunoglobulin
antibody to HIV is in the sample, it will bind to the HIV-1 or administration, recent exposure to certain vaccines, and certain
HIV-2 antigens (or both, if both infections are present); if malignancies.65 False positives can also result from specimen mix-
p24 antigen is in the sample, it will bind to the anti-p24 on the up or mislabeling.69,72 In addition, combination immunoassays
solid phase. Following a wash step to remove excess sample, a cannot distinguish between HIV-1 and HIV-2 infection. Any pos-
conjugate containing chemiluminescent- or enzyme-labeled itive results obtained from the initial ELISA or CLIA screen must
anti-p24 and HIV-1/HIV-2 antigens is added. After a second therefore be confirmed by additional testing that can differentiate
incubation and wash step, the appropriate trigger solution or the two viruses.
substrate/stop solution is added and the relative light units
released or optical absorbance is measured. As previously men-
tioned, these combination assays are used in the initial step Although ELISAs are ideal tests for the detection of HIV anti-
of the 2014 laboratory testing algorithm recommended by bodies in clinical laboratories that perform large-volume batch
the CDC.69 testing, they require expensive instrumentation and skilled per-
More recently, fifth-generation assays that provide more di- sonnel with technical expertise and may have a turnaround
agnostic information have been developed. One of these assays time of a few days. To overcome these limitations and to en-
is a bead-based immunoassay that can detect both HIV antibod- courage more patients to be tested, simple, rapid methods to
ies and antigens, similar to the fourth-generation assays, but in screen for HIV antibody have been developed. These tests are
addition can differentiate between HIV-1 and HIV-2 infection. available for use around the world and provide results within
In the future, implementation of assays such as these may change 30 minutes.
the testing algorithm for HIV. Several commercially available rapid tests have been ap-
Although the immunoassays used in HIV testing have a high proved by the FDA.70 These kits detect antibodies to HIV-1
level of sensitivity and specificity, they may sometimes give alone or to both HIV-1 and HIV-2; they can be used on serum,
erroneous results. False-negative results for HIV antibody occur plasma, whole blood samples obtained by venipuncture or fin-
infrequently but may be caused by the collection of the test gerstick, or, for some kits, oral fluid. Although each test has
serum before the patient develops HIV antibodies (i.e., before unique features, all are lateral flow or flow-through immunoas-
seroconversion), administration of immunosuppressive therapy says that produce a colorimetric reaction in the case of a posi-
or replacement transfusion, conditions of defective antibody tive result. The flow-through assays require multiple steps in
synthesis such as hypogammaglobulinemia, or technical errors which the sample and reagents are added to a solid support
attributed to improper handling of kit reagents.65 False- encased in a plastic device, whereas the lateral flow assays in-
negative results may also occur if the patient harbors a genet- volve a one-step procedure in which the patient sample migrates
ically diverse, recombinant strain of HIV, or an HIV-1 group O along the test strip by capillary action. With either procedure,
strain that is tested for by an assay that does not detect anti- the patient’s sample is applied to a test strip or membrane con-
body to the group O virus. The likelihood of false negatives taining HIV antigens. The antigen–antibody complexes bind to
occurring has been reduced by implementation of fourth- an enzyme-labeled conjugate or an antibody-binding (protein A)
generation tests that can identify HIV infection several days colloidal gold conjugate and are detected by a colorimetric
earlier than third-generation tests because they detect p24 reaction that produces a colored line or dot in the case of a
antigen in addition to HIV antibodies.69,72,73 This makes it positive result.64,65,74,75 Interpretation of the results is made
possible for the infection to be diagnosed within 2 to 3 weeks through visual observation of the test device and does not
after exposure.63 However, there are rare situations in which require instrumentation.
a patient can persistently test negative for HIV antibody A primary use for rapid tests has been to screen for HIV in-
despite the presence of HIV RNA.69 fection; these are especially suitable in certain circumstances.65,75
False-positive results may also occur in these assays. These Rapid tests are ideal for use in resource-limited settings around
can result from several factors, including heat inactivation of the world that do not have access to expensive equipment and
serum before testing, repeated freezing and thawing of specimens, highly trained personnel. They are also beneficial in situations
in which fast notification of test results is desired. For example,
rapid results are important in guiding decisions to begin pro-
Clinical Correlations phylactic therapy with antiretroviral drugs following occupa-
tional exposures because this therapy appears to be most
Seroconversion
effective when administered soon after exposure. Other situa-
By definition, seroconversion is the change of a serological test tions in which rapid tests are advantageous include testing
result from antibody negative to antibody positive. This occurs women whose HIV status is unknown during labor and delivery
over time when the immune system is responding to HIV
and testing patients in sexually transmitted disease clinics or
infection. Seroconversion can be detected by comparing the
test results from two samples collected from the patient, the
emergency departments who are unlikely to return for their test
first soon after exposure to the virus and the second a few results.
weeks later. Although rapid tests are highly sensitive and specific, false
positives can occur.75 In addition, a negative test result does
not rule out early acute HIV infection.76 For these reasons, in the positions where antigen-specific HIV antibodies are pres-
rapid testing should be followed by the current recommended ent. Separate HIV-1- and HIV-2-specific Western blot tests
testing algorithm whenever possible.63,69 Rapid tests that detect must be used to test for antibodies to each virus.64,65,78
p24 antigen as well as HIV-1 and HIV-2 antibodies are under In HIV-1 infection, antibodies to the gag proteins p24 and
evaluation for use in HIV screening.76 p55 appear relatively early after exposure to the virus, but tend
Some rapid HIV kits are available through the Internet or to decrease or become undetectable as clinical symptoms of
over the counter for home testing; the FDA has recently ap- AIDS appear.66 Antibodies to the envelope proteins gp41,
proved one such kit for the testing of oral fluid.70 These tests gp120, and gp160 appear slightly later but remain throughout
offer the advantages of convenience, privacy, and anonymity, all disease stages in an HIV-infected individual, making them
and have the potential of encouraging more widespread screen- a more reliable indicator of the presence of HIV.79 Other anti-
ing among high-risk individuals.77 However, false-negative bodies commonly detected by this method are those directed
results can occur because home tests are not as sensitive as con- against pol proteins p51 and p66, whereas antibodies against
ventional testing, and there is concern that some people who the regulatory gene products are usually not detectable by con-
test positive might not seek confirmatory testing and the appro- ventional methods.65,66 The bands produced by the test sample
priate medical care.77 are examined visually for the number and types of antibodies
As we previously discussed, rapid immunoassays that dif- present. Densitometry can also be performed to quantitate the
ferentiate between HIV-1 and HIV-2 have also been recom- intensity of the bands, which would reflect the amount of each
mended by the CDC as confirmatory tests for samples that test antibody produced. Patients can be followed over time to
positive in the HIV-1 antibody/HIV-2 antibody/p24 antigen determine whether there is a change in the antibody pattern.
combo screen.69 These assays have several advantages over the Because Western blot testing is highly dependent on the lab-
previously recommended Western blot test (see the text that oratorian’s technical skill and subjective interpretation, it is
follows). Specifically, they detect HIV antibodies earlier, reduce generally performed only in specialized reference laboratories
the incidence of indeterminate results, have a shorter result that have an adequate proficiency testing program. Positive and
turnaround time, are less costly, and can detect HIV-2 infec- negative control sera must be included in the test run to pro-
tions in addition to those caused by HIV-1.69,72,73 vide quality control. For the test to be valid, the negative con-
trol should produce no bands and the positive control should
be reactive with p17, p24, p31, gp41, p51, p55, p66, and
Recall that because of the possibility of obtaining false-positive gp120/160. A negative test result for the patient sample is re-
results, all positive samples from HIV screening tests must be ported if either no bands are present or if none of the bands
referred for testing with a more specific confirmatory method. present correspond to the molecular weights of any of the
The Western blot test, or immunoblot, for HIV antibodies known viral proteins.65
was introduced in 1984 and was the most common method Criteria for determining a positive test result have been pub-
used for systematic confirmation of positive ELISA results from lished by the Association of State and Territorial Public Health
1985 to 2014. This technique is more technically demanding Laboratory Directors and CDC, the Consortium for Retrovirus
than ELISA but can provide an antibody profile of the patient Serology Standardization, the American Red Cross, and the
sample that reveals the specificities to individual HIV antigens. FDA.65,66,78,79 According to these criteria, a result should be
Several commercial kits are available for this type of testing and reported as positive if at least two of the following three bands
can provide results within a few hours.64,74,78 are present: p24, gp41, and gp120/gp160 (Fig. 24–6).
Western blot kits are prepared commercially as nitrocellu- Specimens that have some of the characteristic bands pres-
lose or nylon strips containing individual HIV proteins that ent but do not meet the criteria for a positive test result are
have been separated by polyacrylamide gel electrophoresis considered to be indeterminate. This result may be produced if
and blotted onto the test membrane. The protein antigens are the test serum is collected in the early phase of seroconversion
derived from HIV virus grown in cell culture. Antigens with or if the serum contains antibodies that cross-react with some
low molecular weight migrate most rapidly and are therefore of the immunoblot antigens, producing false-positive results.
positioned toward the bottom of the test strip, whereas anti- False positives may be caused by antibodies to contaminants
gens of high molecular weight remain toward the top of the from the cells used to culture HIV to prepare the antigens for
membrane. the test; to autoantibodies, including those directed against
The testing laboratory then applies patient serum to the test HLA, nuclear, mitochondrial, or T-cell antigens; or to antibod-
strip. During the incubation period, any HIV antibodies pres- ies produced after vaccinations.64,65
ent in the sample will bind to their corresponding antigens on The use of recombinant antigens instead of viral lysates has
the test membrane. Unbound antibody is then removed by reduced the incidence of false-positive results. If an indetermi-
washing. Next, an anti-human immunoglobulin with an en- nate test result is obtained, it is recommended that the test be
zyme label (i.e., the conjugate) is added directly to the test strip repeated with the same or a fresh specimen; if the test is still
and binds to specific HIV antibodies from the patient sample. indeterminate, testing may be performed with a new specimen
Unbound conjugate is removed by washing, whereas bound obtained 4 to 6 weeks later. If the pattern converts to positive,
conjugate is detected after adding the appropriate substrate, it can be concluded that the first specimen was obtained during
which produces a chromogenic reaction. Colored bands appear the early phase of seroconversion. Failure of an indeterminate
 The tests are used to resolve discrepancies between a positive
gp160 result in the initial antigen–antibody combo assay and the
gp120 follow-up HIV-1/HIV-2 antibody differentiation assay.69 If the
HIV-1 antibody/HIV-2 antibody/p24 antigen test is nonreac-
tive, but the NAT is reactive, then this result is considered ev-
idence for acute HIV-1 infection. If the HIV-1/HIV-2 antibody
p66 test is indeterminate and the NAT is reactive, this suggests that
HIV-1 antibodies were indeed present. In contrast, if the NAT
p55 p55
p51 is nonreactive and the HIV-1/HIV-2 antibody test is reactive
or indeterminate, a false-positive result on the initial HIV
gp41 antigen–antibody combo assay is indicated.
HIV nucleic acid testing is highly sensitive; it is able to de-
tect HIV RNA about 10 to 12 days after infection.20,63 Methods
gp31 approved by the FDA include a qualitative polymerase chain
reaction (PCR)-based assay to screen donors of whole blood,
p24 p24 blood components, or organs, and a transcription-mediated
amplification (TMA) assay for diagnosis.70

p17 Disease Monitoring

Negative Indeterminate Positive Once a diagnosis of HIV infection has been established, it is
Western blot, showing results from a negative essential to monitor patients over time to evaluate the effec-
sample, an indeterminate sample, and a positive sample. tiveness of their antiretroviral therapy. This way, signs of disease
progression can be detected early and guide decisions about
further treatments. Two laboratory markers are routinely used
test pattern to convert to positive after a few weeks strongly to monitor patients with HIV infection for disease progression
suggests that the pattern is caused by a false-positive test rather and guide their treatments: (1) the peripheral blood CD4
than HIV infection.9,65 During this period, HIV nucleic acid T-cell count, which is considered to be the best indicator
testing can be performed to provide more conclusive results of immune function in HIV-infected individuals, and (2) the
(see the text that follows). HIV-1 RNA level, or “viral load,” which reflects patient re-
Because of its relative insensitivity, level of technical diffi- sponses to ART.9,44,80 Each of these laboratory markers will be
culty, and long turnaround time to obtain results, the Western discussed in detail in the text that follows.
blot test is inappropriate for use as an initial screen for HIV in-
fection.65 For these same reasons, as we previously discussed, CD4 T-Cell Enumeration
the CDC has recommended that the Western blot be replaced
with rapid HIV-1/HIV-2 antibody tests as the standard method Destruction of the CD4 T lymphocytes is central to the im-
for confirmation of positive screening results.69 Comparison munopathogenesis of HIV infection and CD4 lymphopenia has
studies have shown that rapid tests are more suitable confir- long been recognized as the hallmark feature of AIDS. Therefore,
matory tests because they can be performed more quickly, de- enumeration of CD4 T cells in the peripheral blood has played
tect infection earlier, reduce the incidence of indeterminate a central role in evaluating the degree of immune suppression
results, are less expensive, and can detect HIV-1 and HIV-2 in- in HIV-infected patients for many years. Normally, the peripheral
fections simultaneously.69,72,73 Although other confirmatory blood CD4 T-cell count ranges from 450 to 1,500 cells/µL
methods have been developed, including IFA, radioimmuno- (average, 1,000 cells/µL).81 In untreated patients, there is a pro-
precipitation assay (RIPA), and line immunoassays, they have gressive decline in the number of CD4 T cells during the course
not been widely used in clinical settings.64,65 of infection (Fig. 24–7). The rate of decline varies among pa-
tients and can be rapid or gradual. As we previously discussed,
the CDC classification system uses CD4 T-cell counts to place
Qualitative Nucleic Acid Tests (NATs) patients into various stages of HIV infection, with those whose
Qualitative nucleic acid tests (NATs) are used to determine counts are below 200/µL being categorized as having stage 3
whether or not a detectable level of HIV nucleic acid is present infection.69
in human plasma. These tests can be used to screen for infec- Another important clinical application of CD4 T-cell counts
tion or make an initial patient diagnosis. They are particularly is to monitor the effectiveness of antiretroviral therapy. Physi-
beneficial in cases where serological results are inconclusive cians use CD4 T-cell values to help determine whether a
such as in very early infection or in the diagnosis of infants, change in ART is necessary and if prophylactic drugs for certain
where presence of maternal antibodies can confuse test results. opportunistic infections should be administered. According to
As previously mentioned, they are an integral part of the guidelines published by the U.S. Department of Health and
2014 CDC recommendations for laboratory testing for HIV. Human Services, a baseline CD4 T-cell measurement should
 1200
Primary infection
1100 Death
Possible acute HIV syndrome
1000 Wide dissemination of virus Opportunistic
Seeding of lymphoid organs diseases
900 1:512
Clinical latency

Plasma viremia titer

800 1:256

CD4 T cells/mm3
700 1:128

600 1:64

500 Constitutional 1:32

symptoms
400 1:16

Typical CD4 T-cell 300 1:8

numbers and plasma viremia during the 200 1:4
natural course of HIV infection. (Adapted
from Pantaleo G, Graziosi C, Fauci AS. The 100 1:2
immunopathogenesis of human immun- 0 0
odeficiency virus infection. N Engl J Med. 0 3 6 9 12 1 2 3 4 5 6 7 8 9 10 11
1993;328:327–335.) Weeks Years

be performed before the initiation of ART, then every 3 to The absolute CD4 T-cell count is then compared with the
6 months for the first 2 years. Thereafter, annual measurements reference range, which is typically from 450 to 1,500 cells/µL
can be performed in patients whose CD4 T-cell counts have peripheral blood.81
consistently remained between 300 and 500 cells/µL.44 More Current technologies employ multicolor monoclonal anti-
frequent testing should be performed if there is a change in the body panels in a single-platform approach, which allows CD4
patient’s clinical status. If there is a decline in the CD4 T-cell T-cell percentages and absolute numbers to be obtained from
count of greater than 25%, the physician may change the one tube using a single instrument, the flow cytometer.81,82
CART.9 Patients with a CD4 T-cell count below 200/µL are This is made possible by counting CD4+ T cells in a precisely
placed on prophylactic therapy for Pneumocystis jiroveci pneu- measured blood volume or by incubating the sample with a
monia, whereas those with a count of less than 50/µL are given known number of commercially available fluorescent mi-
prophylactic treatment for Mycobacterium avium complex.9,44 crobeads, which function as an internal calibrator. The counts
The gold standard for enumerating CD4 T cells is im- can then be determined by specific flow cytometry software,
munophenotyping with data analysis by flow cytometry (see according to the following equation:82
Chapter 13). The CDC has published guidelines to standardize # of Events in the Total # Microspheres
the performance of CD4 T-cell determinations by flow cytom- Bright CD45 Region Added
etry.81-83 Early guidelines referred to a dual-platform technol-
ogy in which both a flow cytometer and a hematology analyzer # of Events in the Volume of Blood
were required to make CD4 T-cell measurements. According Microfluorosphere Region Added
to this protocol, the percentage of CD4 T cells in a sample is As with the dual-platform technology, lymphocytes are selected
determined by dividing the number of lymphocytes positive for analysis (or “gated”) on the basis of their low-side scatter
for the CD4 marker by the total number of lymphocytes and ability to stain brightly with CD45 antibody.
counted by the flow cytometer, according to the following
equation:
Connections
# CD4 Lymphocytes
% CD4 T Cells = 100 Flow Cytometry
Total # Lymphocytes
In flow cytometry, histograms display the patterns of light scatter
The percentage of CD4 T cells obtained for the patient sample and fluorescence emitted by individual cell populations. Four-
is compared with a reference range established by the labora- color immunofluorescence assays are used in which the lym-
tory performing the test. phocytes are differentiated from other cell types in the sample
In the dual-platform approach, absolute numbers of CD4 on the basis of their low-side scatter and their ability to stain
T cells were calculated by multiplying the absolute number of brightly with fluorescent-labeled antibody to the CD45 marker,
lymphocytes (determined by the complete blood cell [CBC] which is present on all WBCs. The side scatter represents the
count and differential from a hematology analyzer) by the per- amount of cell complexity as determined by properties such as
centage of CD4 T cells in the sample, according to the follow- granularity and membrane irregularity. Because lymphocytes are
more round and less granular than other types of WBCs, they
ing equation:
reflect the laser light to a smaller degree and can be character-
Absolute # CD4 T Cells = WBC Count % ized by their low light scatter.
Lymphocytes % CD4 T Cells
 In addition to CD4+ T-cell percentages and absolute num- Studies performed by the Multicenter AIDS Cohort and
bers, the ratio of CD4 T cells to CD8 T cells may be reported other groups have demonstrated that information obtained
to assess the dynamics between the two T-cell populations. In from viral load tests has prognostic value.44,80 These studies
HIV-infected patients, particularly those with AIDS, the large have shown that baseline plasma viral load values obtained
decrease in the number of CD4+ T cells, along with a possible in patients before the start of antiretroviral therapy are an
increase in CD8+ cytotoxic T cells, results in an inverted ratio, important predictor of disease progression because a higher
or a ratio that is less than 1:1. number of HIV RNA copies/mL of plasma are associated with
Although flow cytometry is the accepted gold standard for more rapid development of an AIDS-defining illness or
enumeration of CD4 T cells, it is a costly method that requires AIDS-related death.
the need for highly skilled personnel, a stable electricity supply, Viral load tests are used routinely to monitor the effective-
refrigeration of reagents, and regular instrument mainte- ness of antiretroviral therapy in HIV-infected patients and play
nance.84,85 These conditions are not available in many areas of an essential role in the clinical management of these individuals.
the world. As a result, there has been much interest in devel- Patients who attain a lower number of HIV-RNA copies/mL of
oping simpler, less expensive methods to determine CD4 T-cell plasma are more likely to achieve a longer treatment re-
measurements. This need has led to the manufacture of minia- sponse.44,80 The optimal goal of therapy is to reach undetectable
turized flow cytometers that can be easily operated by smaller levels of HIV RNA (i.e., < 20 to 75 copies/mL, depending on
laboratories.81,86 These instruments are dedicated for CD3/CD4 the assay).44,78,87 Patients who have a persistently elevated viral
measurements and use simple procedures that require a small load should undergo resistance testing to determine if an alter-
volume of reagents. native drug regimen is needed (see Drug Resistance Testing in the
Another major advance has been development of point- text that follows).44,46
of-care devices that can be easily transported to remote The U.S. Department of Health and Human Services rec-
regions.84,86 These devices can use blood obtained by finger- ommends that plasma HIV RNA testing be performed before
stick or venipuncture and contain stable, dried reagents that antiretroviral therapy begins to obtain a baseline value; testing
can withstand hot, humid temperatures. Disposable test cas- should be performed periodically thereafter to determine the
settes are used to capture CD4+ cells with labeled antibodies, effectiveness of the therapy.44 To obtain an accurate assessment
and the results are analyzed by small, portable instruments that of viral load dynamics in a single patient, it is recommended
can be powered by a rechargeable battery. These methods are that the same assay be used for sequential viral load measure-
particularly suitable for resource-limited settings in developing ments because values may differ between different molecular
countries, where the incidence of HIV infection is significant. tests.78 A change in viral load is considered to be significant if
Although the performance of these less complex methods is there is at least a threefold or 0.5 log10 increase or decrease in
still under evaluation, preliminary studies show that there is the number of copies/mL.44,78 A change in the antiretroviral
reasonable agreement with conventional flow cytometry and therapy protocol (or initiation of therapy for individuals who
they have already made an impact in providing wider access have not received ART) is recommended for patients whose
to a laboratory test that is an integral part of patient care for HIV RNA levels and CD4 T-cell counts reach the critical values
HIV-infected individuals.84,85 established by the AIDS Clinical Trial Group or the WHO.44,46

Quantitative Viral Load Assays Viral load assays are based on amplification methods that in-
Quantitative viral load tests measure the amount of circulating crease the number of HIV RNA copies (or their derivatives)
HIV nucleic acid and play an essential role in helping physi- in test samples to detectable levels. Several amplification meth-
cians predict disease progression, monitor patient response to ods have been developed for this purpose, including PCR;
antiretroviral therapy, and guide treatment decisions.44,78 The the branched chain DNA assay (bDNA); and nucleic acid
amount of HIV RNA, or viral load, in a patient’s plasma reflects sequence-based amplification (NASBA), which amplifies HIV
the natural history of HIV infection in that individual.9 HIV RNA. The basic principles of PCR and bDNA are discussed
RNA levels become detectable about 11 days after infection briefly here and are covered in more detail in Chapter 12.
and rise to very high levels shortly thereafter, during the initial NASBA, which is based on the amplification of HIV RNA, is
burst of viral replication. Typically, over a period of a few a technically complex method that is not suitable for clinical
months, the viral load drops as the individual’s immune system laboratory settings.
clears viral particles from the circulation and a stable level of Two kinds of PCR meth-
plasma HIV RNA, known as the “set point,” is achieved (see ods have been developed to detect HIV nucleic acid: the
Fig. 24–7). In untreated individuals, this level can persist for RT-PCR and. more recently, quantitative (real-time) PCR, also
a long time and then rise again later as the immune system de- known as qPCR. A commercial RT-PCR was the first assay to
teriorates and the patient progresses to AIDS. In contrast, suc- be licensed by the FDA for quantitative measurement of circu-
cessful therapy with antiretroviral drugs will result in a drop lating HIV nucleic acid. The basic principle of this test is to
in the viral load to an undetectable level, usually by 8 to amplify a DNA sequence that is complementary to a portion
24 weeks after initiation of ART.9,44 of the HIV RNA genome.78,88 In this assay, HIV RNA is isolated
from patient plasma by lysis of the virions and precipitation processing time to obtain results.76,84 Lack of accessibility to
with alcohol. The RNA is treated with a thermostable DNA adequate testing has resulted in higher rates of failed re-
polymerase enzyme that has both reverse-transcriptase activity sponses to ART and development of viral resistance in these
and the ability to initiate DNA synthesis in the presence of the settings.46,84 As a result, there has been much interest in the
appropriate reagents. The reverse-transcriptase activity of the development of simpler technologies to determine viral load.
enzyme transcribes the HIV RNA into complementary DNA These include point-of-care devices such as closed cartridges
(cDNA). The cDNA is then amplified by standard PCR that can provide semiquantitative analysis with simpler in-
methodology (see Chapter 12). strumentation in a shorter period of time. Many of these tech-
Although the development of standard RT-PCR methods nologies, including loop-mediated and helicase-dependent
was a revolution in molecular testing for HIV infection, they amplification, are based on isothermal reactions in which the
have several disadvantages, including a limited dynamic temperature remains constant.76 In addition, the use of dried
range (i.e., the range of HIV RNA copies per mL that can be blood spots could eliminate the need for venous blood draw.
detected). In addition, they are highly susceptible to cross- Costs could be further reduced by pooling of blood samples;
contamination with extraneous nucleic acid. For these rea- if a pool tests negative, then no further testing would be
sons, these assays have largely been replaced with qPCR needed. Reactive pools could be further subdivided to iden-
assays that can detect and quantify the PCR products as they tify positive individuals.84 Further development and imple-
are being produced (see Chapter 12).78,88 This is accom- mentation of these innovative methods into resource-limited
plished by adding a fluorescent probe that binds to the settings could allow clinicians to better monitor the effective-
amplicon during the reaction. The PCR amplification primers ness of ART in individuals living in these regions and have a
target highly conserved regions of the HIV-1 gag or pol genes. profound impact on combating HIV infection throughout the
An internal control consisting of a different nucleic acid world.
sequence is simultaneously amplified with each sample to
compensate for the effects of inhibition and allow for more
accurate quantitation. qPCR is highly sensitive and can de-
Drug-Resistance Testing
tect a broad range of RNA copies, from 20 copies/mL to As we previously discussed, HIV is a rapidly replicating virus
10 million copies/mL.78 Commercially available assays can that has an intrinsically high rate of mutation. Because of these
also detect all subtypes of groups M, N, O, and recombinant properties, it is possible for drug-resistant subpopulations of
strains of HIV.78 the virus to emerge during the course of antiretroviral therapy.
In contrast to RT-PCR, Drug resistance has been a major reason for the failure of ART
which involves amplification of the HIV target sequence, the in many people. As a result, laboratory tests have been devel-
bDNA method is based on amplifying the detection signal gen- oped to assess drug resistance patterns; these tests have had a
erated in the reaction. This is accomplished by using a solid- major impact on guiding the selection of optimal ARTs for in-
phase sandwich hybridization assay that incorporates multiple dividual patients.
sets of oligonucleotide probes and hybridization steps to create Two types of laboratory methods can be used to test for drug
a series of “branched” molecules.78,88 First, RNA isolated from resistance: genotype resistance assays and phenotype resistance
lysed virions in patient plasma is captured on wells of a mi- assays.44,78,88 Genotype resistance assays are performed more
crotiter plate coated with a number of probes. The captured frequently than phenotype resistance assays because they are
RNA is then hybridized with branched amplifier probes and less expensive, more widely available, and have a shorter turn-
incubated with an enzyme-labeled probe that will bind to the around time.
DNA branches. Finally, a chemiluminescent substrate is added Genotype resistance assays detect mutations in the reverse-
and color change is measured with a luminometer. Quantita- transcriptase and protease genes of HIV. These tests are avail-
tive results are generated from a standard curve. able commercially and can be performed in clinical laboratory
The bDNA test can detect 75 to 500,000 copies of HIV settings. In these tests, RNA is isolated from patient plasma,
RNA/mL of plasma. As compared with real-time PCR, the the desired genes are amplified by RT-PCR, and the products
bDNA has higher reproducibility and higher throughput (rate are analyzed for mutations associated with drug resistance by
of producing results). However, it requires a larger sample vol- automated DNA sequencing.78,88 The nucleotide sequences of
ume, lacks an internal control, and has a lower specificity. This the genes of interest are identified and entered into a database,
method is most conducive for laboratories with high testing where they are compared with the corresponding sequences
volumes.78,88 in wild-type HIV. The results are analyzed by commercially
available software and reported qualitatively as “resistance,”
“possible resistance,” or “no evidence of resistance” for each
Although conventional HIV viral load tests have had a large drug tested. Results can generally be obtained in 1 to 2 weeks.
impact on patient care, they pose many difficulties for Although genotyping tests have many advantages as compared
resource-limited areas of the world, including the need for a with phenotypic methods, they can identify only known mu-
continuous power supply, refrigeration, costly instrumenta- tations and cannot assess the effects of combinations of indi-
tion, skilled personnel to perform the testing, and adequate vidual mutations on drug resistance.78
 Phenotype resistance assays determine the ability of clin- been associated with development of hypersensitivity to the
ical isolates of HIV to grow in the presence of antiretroviral drug. The test is typically performed by PCR amplification of
drugs.44,78,88 In these assays, recombinant viruses are created the allele, followed by hybridization with sequence-specific
by inserting the reverse-transcriptase, protease, integrase, or oligonucleotide probes.91
env gene sequences from HIV RNA in the patient’s plasma
into a laboratory reference strain of HIV and transfecting the
recombinant virus into mammalian cells. Varying concentra- Testing of Infants Younger
tions of antiviral drugs are incubated with the transfected Than 18 Months
cells and the IC50 values, or drug concentrations needed to
suppress the replication of the patient’s viral isolate by 50%, Serological tests are not reliable in detecting HIV infection in
are calculated. These values are then compared with the IC50 children younger than 18 months of age because of placental
values of cells transfected with a reference strain of HIV in passage of IgG antibodies from an infected mother to her
order to determine drug resistance. The major advantage of child. These maternal antibodies persist in the bloodstream
phenotypic assays is that they measure drug susceptibility di- of the infant during the first year of life (or longer in a small
rectly, on the basis of all mutations present in the patient’s proportion of infants) and can confuse the interpretation of
isolate. However, these assays are expensive and have a longer serological results from infant samples.93,94 Thus, a child born
turnaround time than genotypic assays (2 to 3 weeks). In ad- to an HIV-positive mother may test positive for HIV antibody
dition, phenotypic assays involve sophisticated technologies during the first 18 months of life even though the child is not
and are only performed by a few highly specialized reference infected.
laboratories.78 Because of the difficulties with serological testing, HIV in-
Both genotypic and phenotypic assays require that a viral fection in infants is best diagnosed using molecular methods.44
load of at least 500 to 1,000 copies of HIV RNA/mL be pres- A qualitative HIV-1 DNA PCR test is the preferred method for
ent in the test sample and for the resistant virus to constitute this purpose. This test, which detects proviral DNA within the
more than 25% of the total viral population in the patient to infants’ peripheral blood mononuclear cells, has a sensitivity
produce detectable results.78,88 Despite these limitations, of 55% at birth, increasing to greater than 90% at 2 to 4 weeks,
studies have shown that patients undergoing drug-resistance and 100% by 3 to 6 months of age; and a specificity of 99.8%
testing, particularly by genotyping methods, have a better at birth and 100% in infants older than 1 month.44 Alterna-
chance of receiving antiretroviral therapy regimens that are tively, quantitative HIV RNA assays may be used to diagnose
more likely to result in greater reductions in viral load.78,89,90 HIV infection in infants. Although they are less sensitive than
Therefore, the U.S. Department of Health and Human Serv- DNA assays, RNA tests can be used to provide a baseline viral
ices recommends that drug-resistance testing be performed load measurement and are more likely to detect infections with
in individuals before initiating antiretroviral therapy, in pa- strains other than subtype B.44,94 They can also be used as a
tients in whom CART has failed as evidenced by viral load confirmatory test for infants who initially had a positive HIV
values that have not been optimally reduced, and in all DNA test.
HIV-positive pregnant women.44,78 Genotypic testing is rec- It is recommended that nucleic acid tests for HIV be per-
ommended for patients upon entry into medical care for HIV formed in infants with known perinatal exposure at the ages
or after one to two unsuccessful treatment regimens, because of 14 to 21 days, 1 to 2 months, and 4 to 6 months, and pos-
of its lower cost, faster turnaround time, and greater sensi- sibly at birth for infants at high risk for HIV infection.44 A
tivity for detecting wild-type/resistant virus mixtures. The positive test result should be confirmed by repeat testing on
addition of phenotypic testing is recommended when pa- a second specimen because false positives can occur. Two or
tients are thought to harbor HIV mutants with complex drug- more negative test results (the first at greater than 1 month
resistance patterns, especially to protease inhibitors.44 and the second at greater than 4 months) provide evidence
Specialized tests have been developed in addition to the for the absence of HIV infection and may be confirmed by
standard tests. These include genotypic tests for resistance to serological tests at 12 to 18 months of age. The HIV status of
integrase inhibitors, fusion inhibitors, or env mutations; geno- breastfed infants, who are continually exposed to the virus,
typic or phenotypic assays for co-receptor tropism; and typing cannot be determined accurately until breastfeeding is
for the HLA-B*27 allele.78,91 Analysis of the co-receptor tro- stopped.94
pism is used to determine whether a patient is eligible for Increased emphasis on screening pregnant women for HIV
treatment with the drug maraviroc, which inhibits entry of infection should also help in the identification of HIV-positive
HIV strains that use the CCR5 co-receptor to bind to host infants.95 Rapid tests for HIV antibody should be performed
cells. Early tests for CCR5 tropism were phenotypic assays, on women whose HIV status is unknown and on their new-
but a more rapid genotypic assay that sequences the third vari- born infants soon after birth.94 Prompt detection of HIV in-
able loop of the HIV-1 env gene is now available.78,92 Pharma- fection in newborns is important because infected infants
cogenetic screening for HLA-B*5701 is helpful for patients have a better prognosis when CART is started early and can
who are being considered for treatment with the nucleoside benefit from treatment with prophylactic drugs for oppor-
reverse-transcriptase inhibitor abacavir because this allele has tunistic infections.16,44,46
SUMMARY • Treatment with antiretroviral therapy (ART) is recom-
mended for all HIV-infected persons and has resulted in a
• Human immunodeficiency virus type 1 (HIV-1) is respon- significant delay in disease progression, decreased mortal-
sible for the majority of AIDS cases throughout the world. ity, and reduction in perinatal transmission.
A related virus, HIV-2, may also cause AIDS but is gener- • Several classes of antiretroviral drugs have been developed—
ally less pathogenic. nucleoside analogue reverse-transcriptase inhibitors,
• Transmission of HIV occurs by three major routes: nonnucleoside reverse-transcriptase inhibitors, protease
(1) intimate sexual contact, (2) contact with contami- inhibitors, integrase inhibitors, and entry inhibitors. These
nated blood or body fluids, or (3) vertical transmission drugs are most effective when administered in combina-
from infected mother to her fetus or infant. tions known as CART (combination antiretroviral therapy)
• HIV belongs to the retrovirus family, which contains RNA or HAART (highly active antiretroviral therapy).
as the genetic material from which DNA is transcribed. • The algorithm recommended by the CDC and the Asso-
HIV has three main structural genes: gag, which codes for ciation of Public Health Laboratories in 2014 to screen for
the core proteins of the virus such as p24; pol, which codes HIV infection consists of a sequence of laboratory tests.
for the enzymes reverse transcriptase, integrase, and pro- The initial test is a fourth-generation ELISA that simulta-
tease; and env, which encodes the envelope proteins gp120 neously detects HIV-1 antibody, HIV-2 antibody, and
and gp41. p24 antigen. Positive test results must be confirmed by a
• The primary target cells for HIV are CD4 T lymphocytes rapid test that discriminates between HIV-1 antibody and
and macrophages, which possess some surface CD4. The HIV-2 antibody. Any samples that give discrepant results
CD4 molecule acts as a receptor for attachment of the should undergo nucleic acid testing.
virus by binding to the gp120 envelope protein. Follow- • Rapid screening tests for HIV antibodies are typically sen-
ing attachment, entry of the virus into the host cells is sitive, lateral flow assays that can provide results in fewer
mediated by the chemokine co-receptors, CXCR4 and than 30 minutes. These tests are especially suitable for use
CCR5. in certain situations, including resource-limited settings,
• A burst of viral replication occurs after initial infection fol- occupational exposures, labor and delivery, and clinics or
lowed by a period of latency during which viral DNA be- emergency departments where patients are unlikely to
comes integrated into the host genome as a provirus. make a return visit.
• Viral production slows down as the host’s immune re- • The Western blot, which was used for many years to con-
sponse develops and keeps the virus in check. The host firm positive HIV antibody screening test results, detects
produces neutralizing antibodies, which prevent the virus antibody specificities to individual HIV antigens. It is no
from infecting neighboring cells and develops HIV-specific longer recommended for initial screening and diagnosis
CTLs that lyse virus-infected target cells. Innate defenses of HIV because it is labor intensive, relatively insensitive,
are also activated. and has a long result turnaround time.
• Although the immune responses of the host reduce the • HIV-infected patients are routinely monitored using two
level of HIV replication, they are usually not sufficient to laboratory measurements: CD4 T-cell enumeration and
completely eliminate the virus. HIV can escape these re- HIV viral load.
sponses by undergoing rapid genetic mutations that gen- • Peripheral blood CD4 T-cell counts and percentages are an
erate altered antigens, downregulating production of class excellent indicator of immune function and are routinely
I MHC molecules on the surface of the infected target cells, measured by multicolor immunofluorescence staining fol-
and existing in a latent proviral state. lowed by analysis with flow cytometry. These measure-
• The hallmark feature of HIV infection is a decline in the ments are used to stage HIV-infected patients and to
number of CD4 Th cells during the natural course of in- monitor patients undergoing ART. Declining numbers can
fection. This decline results in an immunodeficiency that indicate if there is a need to change antiretroviral therapy
affects both cell-mediated and humoral antibody re- or initiate prophylactic therapy for opportunistic infections.
sponses to a variety of antigens. • Qualitative nucleic acid tests can be used to screen for HIV
• The clinical course of untreated HIV infection begins with infection or make an initial patient diagnosis. Quantitative
an acute phase in which patients may experience flu-like tests, which measure the amount of HIV nucleic acid circu-
symptoms. This is followed by a latent, asymptomatic pe- lating in patient plasma, are known as viral load tests. These
riod that lasts an average of 10 years. The infection cul- tests have had an important impact on the clinical manage-
minates in AIDS, which is characterized by profound ment of HIV-infected patients by allowing physicians to pre-
immunosuppression with life-threatening opportunistic dict disease progression, monitor patient response to
infections and malignancies. antiretroviral therapy, and guide treatment decisions.
• The 2014 CDC case definition of HIV infection is based • Nucleic acid tests are performed by one of three molecular
on laboratory criteria or clinical evidence and classifies methods: reverse-transcriptase polymerase chain reaction
patients into one of five stages based on CD4 T-cell mea- (RT-PCR), a method that converts HIV RNA into cDNA and
surements and the presence of opportunistic illnesses. then amplifies the cDNA generated; qPCR, a quantitative
 real-time RT-PCR method, and the branched chain DNA in newborns makes tests for HIV antibody unreliable until
assay (bDNA), which amplifies a labeled signal bound to a a child is over 18 months old.
test plate. • Nucleic acid testing is recommended for diagnosis of HIV
• Drug-resistance testing can be performed by genotypic as- infection in infants younger than 18 months. The pre-
says that use molecular methods or by phenotypic assays in ferred method is a qualitative PCR that detects HIV provi-
which HIV replication in clinical isolates is assessed in the ral DNA in the infant’s peripheral blood mononuclear
presence of varying concentrations of antiretroviral drugs. cells. Careful monitoring of HIV-infected mothers and
• Diagnosis of HIV in neonates is more complex than testing early testing of infants at risk is recommended to facilitate
in adults. The presence of maternally acquired antibody prompt medical intervention.

CASE STUDIES
1. A young woman recently discovered that her boyfriend 2. A pregnant woman had used intravenous drugs in the
tested HIV-positive. She was concerned that she may have past and recently discovered that she was HIV-positive.
also contracted the infection because she had experienced She was concerned that her baby would also contract HIV
flu-like symptoms 1 month ago. She decided to visit her infection and discussed this with her physician.
physician for a medical evaluation.
Questions
Questions a. How is HIV infection transmitted from mother to
a. What initial laboratory test should be performed on the infant and what measures should be taken to reduce
young woman to determine if she has been exposed the risk of HIV infection to the infant?
to HIV? b. Should testing for HIV antibody be performed to deter-
b. If the woman tests positive in the initial evaluation, mine if the infant is HIV-positive after birth? Explain
what follow-up testing should be performed to confirm your answer.
the results? c. What type of laboratory testing would be best to
c. If the woman’s test results are confirmed to be positive, evaluate the infant for HIV infection after birth?
what tests should be done to monitor her over time?

REVIEW QUESTIONS
1. All of the following describe HIV except 4. Which of the following is typical of the latent stage of
a. it possesses an outer envelope. HIV infection?
b. it contains an inner core with p24 antigen. a. Proviral DNA is attached to cellular DNA.
c. it contains DNA as its nucleic acid. b. Large numbers of viral particles are synthesized.
d. it is a member of the retrovirus family. c. A large amount of viral RNA is synthesized.
d. Viral particles with no envelope are produced.
2. HIV virions bind to host T cells through which
receptors? 5. The decrease in T-cell numbers in HIV-infected
a. CD4 and CD8 individuals is caused by
b. CD4 and the IL-2 receptor a. lysis of host T cells by replicating virus.
c. CD4 and CCR5 b. fusion of the T cells to form syncytia.
d. CD8 and CCR2 c. killing of the T cells by HIV-specific cytotoxic T cells.
d. all of the above.
3. Antibodies to which of the following viral
antigens are usually the first to be detected in 6. The most common means of HIV transmission
HIV infection? worldwide is through
a. gp120 a. blood transfusions.
b. gp160 b. intimate sexual contact.
c. gp41 c. sharing of needles in intravenous drug use.
d. p24 d. transplacental passage of the virus.
 7. The drug zidovudine is an example of a 12. The characteristic laboratory finding in HIV
a. nucleoside analogue reverse-transcriptase inhibitor. infection is
b. nonnucleoside reverse-transcriptase inhibitor. a. decreased numbers of CD4 T cells.
c. protease inhibitor. b. decreased numbers of CD8 T cells.
d. fusion inhibitor. c. decreased numbers of CD20 B cells.
d. decreased immunoglobulins.
8. False-negative test results in a laboratory test for HIV
antibody may occur because of 13. Which of the following tests is currently recommended
a. heat inactivation of the serum before testing. by the CDC to confirm a positive screening test result
b. collection of the test sample before seroconversion. for HIV infection?
c. interference by autoantibodies. a. Rapid test for HIV-1 and HIV-2 antibodies
d. recent exposure to certain vaccines. b. Western blot
c. Molecular testing for HIV RNA
9. Which of the following combinations of bands would d. HIV viral culture
represent a positive Western blot for HIV antibody?
a. p24 and p55 14. Which of the following tests would give the least
b. p24 and p31 reliable results in a 2-month-old infant?
c. gp41 and gp120 a. CD4 T-cell count
d. p31 and p55 b. ELISA for HIV antibody
c. PCR for HIV proviral DNA
10. The fourth-generation ELISA tests for HIV detect d. p24 antigen
a. HIV-1 and HIV-2 antigens.
b. HIV-1 and HIV-2 antibodies. 15. Which of the following measurements are routinely
c. p24 antigen. used to monitor patients with HIV infection who are
d. HIV-1 antibodies, HIV-2 antibodies, and p24 antigen. undergoing antiretroviral therapy?
a. HIV antibody titer
11. The conjugate used in the fourth-generation ELISA b. p24 antigen levels
tests for HIV consists of enzyme-labeled c. CD4 T-cell and CD8 T-cell counts
a. anti-human immunoglobulin. d. CD4 T-cell count and HIV RNA copy number
b. HIV-1- and HIV-2-specific antibodies.
c. HIV-1- and HIV-2-specific antigens.
d. HIV-1- and HIV-2-specific antigens plus antibody
to p24.
 Immunization
and Vaccines

After finishing this chapter, you should be able to: VACCINES

1. Differentiate between active immunity, passive immunity, and adoptive Historical Evolution of Vaccines
immunity. Conventional Vaccines
2. Recognize examples of active immunity, passive immunity, and Factors Influencing Immunogenicity
adoptive immunity. Next Generation Vaccines
3. Discuss the historical evolution of vaccines from the early contribu- Benefits and Adverse Effects
tions of Edward Jenner through modern approaches to producing of Vaccines
next generation vaccines.
PASSIVE IMMUNIZATION
4. Define vaccine, toxoid, attenuation, adjuvant, and recombinant protein
Passive Immunization as Therapy for
vaccine.
Infectious Diseases
5. Describe the composition of live attenuated vaccines, inactivated
Advantages and Limitations of
vaccines, and subunit vaccines, and contrast their advantages and
Passive Immunization
limitations. Provide examples of each type of vaccine.
Immunosuppressive Effects of Passive
6. Explain how factors that influence the immune response to vaccines
Immunization
determine the ways in which vaccines are administered.
Monoclonal Antibodies
7. Recognize examples of adjuvants and explain the mechanisms by
which they enhance the immune response to vaccines. ADOPTIVE IMMUNOTHERAPY
8. Contrast the benefits and adverse effects associated with vaccines. SUMMARY
9. Differentiate between standard human immune serum globulin and CASE STUDIES
specific human immune serum globulin and their clinical applications. REVIEW QUESTIONS
10. Differentiate between monoclonal antibodies, chimeric antibodies,
humanized antibodies, and fully human antibodies in terms of their
structure and nomenclature.
11. Discuss some of the clinical applications of monoclonal antibody
therapy and immunosuppressive therapy with gamma globulins.
12. Provide examples of clinical applications of adoptive immunotherapy
in the areas of cancer treatment and transplantation.

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
 Active immunity Attenuation Immunoprophylaxis Serotype
Adjuvant Cross-reactivity Immunotherapy Toxoids
Adoptive immunity Human immune serum Passive immunity Tumor-infiltrating
Adoptive immunotherapy globulin (HISG) Passive immunotherapy lymphocytes (TILs)
Antitoxin Immunization Recombinant protein vaccine Vaccine

As we discussed in Chapter 1, immunity can be defined as the immunity, an individual’s own immune system is responding
condition of being resistant to disease, most notably to infections. to an antigen. In passive and adoptive immunity, immunity is
The process by which this state of protection is acquired is called provided to an individual through the transfer of antibodies or
immunization. There are three types of immunity that can be cells from another source to provide immediate protection.
acquired through immunization: active, passive, and adoptive. This chapter discusses the mechanisms by which active,
Active immunity results from immunization with a specific passive, and adoptive immunity occur in the context of their
antigen by natural exposure to infection or administration of a advantages and limitations. Clinical examples under each cat-
vaccine. Adaptive, or antigen-specific, immune responses to egory are described so that the student can develop a better
bacteria, viruses, fungi, and parasites can all result in active understanding of how knowledge of the basic principles of
immunity. For example, production of antibodies to a specific the immune system can be translated into therapies that can
strain of Group A streptococci bacteria following a streptococ- benefit humankind. A special focus is placed on the topic of
cal sore throat protects the individual from future infection vaccines because their use has significantly improved the
with that strain of bacteria. Active immunity is also stimulated health of populations throughout the world.
through the administration of vaccines (see sections that fol-
low). For example, after receiving all doses of the measles
vaccine, most people develop immunity to the measles virus.
Vaccines
In active immunity, the individual’s own immune system is
A vaccine is an antigen suspension derived from a pathogen.1
stimulated to mount an adaptive immune response to an anti-
Vaccines are routinely administered to healthy individuals to
gen. The advantage of active immunization over other types of
stimulate an immune response to an infectious disease. Vacci-
immunization is that it results in long-term memory to an anti-
nation therefore is a form of immunoprophylaxis, or the pre-
gen, providing potentially lifelong protection against the harm-
vention of disease through immunization. Vaccines have had a
ful effects of a pathogenic organism.
tremendous impact on public health by significantly reducing
Passive immunity results from the transfer of antibodies
the incidence of illness and death from numerous diseases that
from immunized hosts to a nonimmune individual. This state
had devastating effects on civilization. In addition, experimental
of immunity can occur naturally, from transfer of a mother’s
“vaccines” for cancer have been developed as immunotherapy
antibodies to her fetus or infant, or artificially, through passive
for patients who already have the disease (see Chapter 19). In
immunization of an individual with commercial preparations
this section, we will discuss the historical evolution of vaccines,
of antibodies formed by other hosts to prevent or treat a disease.
the different forms of vaccines available, how they are routinely
The latter application is also known as passive immunother-
administered, factors that affect their efficacy, and vaccine design
apy. Use of pooled human antibodies to protect a person
for the future.
who has an immunodeficiency disease is an example of passive
immunotherapy. The main advantage of passive immunization
is that it provides immediate protection to an individual who Historical Evolution of Vaccines
has not developed immunity to a particular antigen. As was previously mentioned in Chapter 1, the science of im-
Adoptive immunity results from the transfer of cells of the munology was born out of early observations of immunity and
immune system, usually lymphocytes, from an immunized studies involving vaccination. The motivation behind these stud-
host to a nonimmune individual. Adoptive immunotherapy ies was the desire to eliminate the death and suffering caused by
involves the administration of these cells to treat patients with infectious disease. Advances in science and technology during
conditions such as immunodeficiency diseases or cancer. This the 20th century and beyond have led to the creation of safer,
type of therapy can be beneficial if the cells transferred are able more effective vaccines and remarkable success toward that goal.
to successfully establish themselves in the recipient. An exam-
ple of adoptive immunotherapy is the transplantation of
hematopoietic stem cells into leukemia patients who have One of the most feared diseases in ancient times was smallpox,
undergone treatment with high doses of irradiation and a highly fatal illness characterized by a high fever and pustular
chemotherapy to destroy the malignant cell population. rash.2 Those who survived the disease were usually left with
The primary difference between the immunity gained by disfiguring scars or blindness. However, it was noted in Greece
active, passive, and adoptive immunization is that in active as early as 430 BC that people who were fortunate enough to
survive became immune to this “speckled monster”; they were protective effects induced by the vaccination were caused by
then asked to care for others afflicted with the deadly disease.3 the phenomenon of cross-reactivity, or antigenic similarity
These observations led to the procedure of variolation, in which between the viruses that caused cowpox and smallpox. This
fresh material taken from a skin lesion of a person recovering early vaccine for smallpox led to the development of the mod-
from smallpox was subcutaneously injected with a lancet into ern smallpox vaccine, which is still derived from the vaccinia
the arm or leg of a nonimmune person. Recall from Chapter 1 virus that causes cowpox (Fig. 25–2). The vaccine was so
that in an older form of the procedure practiced in ancient successful that smallpox has been eradicated from the world,
China, the material was dried into a powder and inhaled by and the vaccine is no longer routinely administered.3,4
the nonimmune person. The practice of variolation usually Despite the early development of the smallpox vaccine,
resulted in milder disease and recovery and was performed many years passed before scientists understood that microbes
for centuries in Africa, India, and China. The practice was were the underlying cause of infectious diseases and that com-
introduced to Europe in the 18th century. At that time, over ponents of these pathogens could be used to produce protective
400,000 people in Europe died each year from smallpox, so vaccines. Thus, it was not until 80 years later that the next vac-
the procedure became popular very quickly.3 However, vario- cine was developed by Louis Pasteur against chicken cholera.6
lation was not without risks; 1% to 2% of the recipients devel- Pasteur later went on to develop vaccines against anthrax and
oped smallpox and died, whereas others contracted infectious rabies. These vaccines were all based on the principle of atten-
diseases such as syphilis or tuberculosis from the injected uation. Attenuation involves the use of bacteria or viruses that
material.4 have been weakened through exposure to modifying condi-
These observations led to the search for a safer procedure. tions such as chemical treatment, elevated or cold tempera-
Farmers such as Benjamin Jesty observed that milkmaids who tures, or repeated in vitro passage in cell culture (a technique
had a similar but milder disease called cowpox were protected in which some of the cells are periodically transferred to a flask
from contracting the deadlier smallpox. The farmers, therefore, containing fresh nutrient medium). These weakened microor-
used cellular material from the cowpox lesions of milkmaids ganisms do not cause disease in healthy individuals, but are
to variolate others and observed the same protective effects able to stimulate the immune response because they contain
without the danger of contracting smallpox (Fig. 25–1). The many of the same antigens as their pathogenic counterpart.5 It
English physician Edward Jenner brought fame to this proce- is thought that Pasteur discovered the effect of attenuation by
dure when, in 1796, he injected fluid from the cowpox lesions accident during his studies of chicken cholera.7 After returning
of milkmaid Sarah Nelmes into an 8-year-old boy named James from a summer vacation in 1881, he noticed that he had left a
Phipps.4,5 When Jenner subsequently inoculated the boy with culture of the bacteria that cause chicken cholera, now known
smallpox, James did not develop the disease, showing that the as Pasteurella multocida, on his laboratory bench. Instead of dis-
method was a success. Jenner called this procedure “vaccina- posing of the aged culture, he decided to use it to inoculate
tion” after the Latin word vacca, which means “cow.”3 The chickens. The chickens did not develop the disease; further-
more, when Pasteur later inoculated them with a fresh culture

Color etching of a hand and wrist with cowpox

lesions, from Edward Jenner’s “Inquiry Into the Causes and Effects Evidence of smallpox vaccination. Individuals who
of the Variolae Vaccinae.” The etching shows several stages of have received the smallpox vaccine develop a blister, which dries up
cowpox, from early blistering to its later dimpled rupture. (Courtesy and forms a scab within the first 2 weeks. They can be easily identi-
of the National Library of Medicine, National Institutes of Health & fied by the scar that forms at the site of injection once the scab falls
Human Services. Bethesda, MD.) off. (Linda Miller.)
of the bacteria, they proved to be resistant to cholera. This series 1800s, Salomen and Smith showed that a killed suspension
of events exemplified Pasteur’s philosophy for science, that of Vibrio cholerae could provide pigeons with protection against
“success favors the prepared mind.”7 Within the next year, he cholera. A few years later, vaccines against human cholera,
developed an attenuated bacterial vaccine to protect sheep typhoid fever, and plague were developed using whole killed
against anthrax. organisms.9
The rabies vaccine, for which Pasteur is most famous, was
developed in 1885. Pasteur did not know the causative agent
of rabies, but recognized that it affected the central nervous The 20th century witnessed a tremendous expansion in the
system (CNS). He prepared an attenuated vaccine by repeat- development of vaccines as a result of advances in scientific
edly infecting rabbits with material contaminated with rabies, research, techniques, and laboratory technology.7,10 New meth-
recovering the rabbits’ spinal cords, and exposing them to dry ods of attenuating microorganisms by repeated culture passage
air. In 1885, after testing the vaccine on dogs, he was con- in special media resulted in the development of live, attenuated
vinced to administer his vaccine preparation to a 9-year-old vaccines against tuberculosis (TB) and typhoid fever. Produc-
boy, Joseph Meister, who had been severely bitten by a rabid tion of live, attenuated vaccines for yellow fever and influenza
dog.8 The boy received a series of subcutaneous injections of A was made possible in the 1930s, following Goodpasture’s
the material over a period of 10 days and never developed development of techniques that permitted viral growth in
rabies (Fig. 25–3).9 Pasteur received worldwide honors for embryonic eggs.9,11
this treatment and used the proceeds to build the famous Another major advance of the 20th century was the use of
Pasteur Institute in Paris, where vaccine studies and other inactivated bacterial toxins in vaccine preparations. These
biomedical research is conducted today. He was the first person preparations, referred to as toxoids, are made by chemically
to use the word vaccination in reference to all immunization treating bacterially derived toxins known to cause pathogenesis
procedures.9,10 so that they cannot cause harm to the host, but retain their
Pasteur believed that only live, attenuated organisms could ability to stimulate an immune response. Using a formalin
be used for effective immunization and these remain the basis treatment, inactivated toxoid vaccines were developed against
for many vaccines that are used today. However, in the late diphtheria by Glenny in 1923 and by Ramon and Zoeller
against tetanus in 1926.9 Inactivated toxoids still serve as the
basis for the diphtheria and tetanus vaccines in use today.
The second half of the 20th century has been referred to as
the “golden age of vaccine development.”6,9 During this period,
researchers developed revolutionary techniques that enabled
successful growth of viruses in cell culture. These important
advances led to the development of numerous attenuated viral
vaccines from 1950 to 1980, including those targeted against
polio, measles, mumps, rubella, and varicella.6,7,9
An important development in the last part of the 20th century
was the use of purified polysaccharides to treat bacterial infec-
tions.9,10 These vaccine preparations were developed in the 1970s
and 1980s to prevent meninogococcal meningitis, pneumococcal
pneumonia, and Haemophilus influenzae type b (Hib). In the late
1980s, these vaccines were made more effective by increasing the
immunogenicity of the polysaccharides. This was accomplished
by forming glycoconjugates consisting of polysaccharides linked
to a protein that can be recognized by T cells.
The last part of the 20th century also saw the first applica-
tions of genetic technologies to vaccine development.6,9,10 The
first recombinant (i.e., genetically engineered) protein vaccine
was produced in 1986 against hepatitis B. This vaccine consists
of purified hepatitis B surface antigen (HBsAg) made by genet-
ically modified yeast cells that have incorporated the gene for
HBsAg. It replaced an older form of the vaccine that used a
less purified preparation of HBsAg derived from the plasma of
patients who were infected with hepatitis B. Genetic engineering
was also used to develop recombinant vaccines to prevent other
diseases, including pertussis and Lyme disease.
Louis Pasteur observes as a young boy receives an
inoculation for hydrophobia (a symptom of rabies). (Courtesy of the
National Library of Medicine, National Institutes of Health & Human The beginning of the 21st century has seen continued use and
Services. Bethesda, MD.) refinements in the vaccine technologies developed in the
1900s. The first decade of the 21st century witnessed the licen- the pathogenic growth of the bacteria.14 Researchers have also
sure of live, attenuated vaccines for influenza, rotavirus, and developed a live, attenuated bacterial vaccine against typhoid
herpes zoster, as well as multivalent, glycoconjugate vaccines fever, which consists of a mutated strain of Salmonella typhi
for pneumococcus and meningococcus. A vaccine to help pre- packaged into capsules that are ingested orally.15 To produce
vent cervical cancer, based on production of genetically recom- this vaccine, the bacteria were chemically treated to induce
binant antigens from the human papilloma virus (HPV), was genetic mutations that weakened them so they were no longer
also introduced.6,9 pathogenic.
Currently, 27 infectious diseases are preventable by vaccines Attenuated vaccines are more easily prepared against viral
licensed in the United States. However, many diseases still infections than bacterial infections.9 A major viral disease for
present a challenge to the medical profession because they which an attenuated vaccine was developed is polio. Polio is a
cannot be prevented through immunization. Future vaccines serious disease that causes aseptic meningitis and leaves its
will likely be based on attempts to target these diseases through victims with disabling paralysis. In the early 1960s, Albert Sabin
application of advanced genetic technologies and stimulation developed an oral polio vaccine from live, attenuated strains of
of innate defenses as well as humoral antibody production and poliovirus cultured in monkey kidney cells.16 The vaccine
cellular components of the adaptive immune system.6,7,10,12,13 contained the three serotypes of poliovirus that are capable of
Some of the strategies that are likely to be used in the future causing the disease. Each serotype is a form of the virus that
development of vaccines will be discussed later in this chapter. can be distinguished by the presence of specific antigens that
can be identified by serological typing. Sabin’s vaccine was the
Conventional Vaccines main poliovirus vaccine used in the United States from 1963 to
1997, before a potent inactivated polio vaccine was licensed.
As we previously mentioned, vaccines are antigen preparations
Although the oral polio vaccine is no longer available in the
that are administered to prevent infectious diseases. Conven-
United States because of its potential for adverse effects (see
tional vaccines in use today consist of live, attenuated (nonpath-
Benefits and Adverse Effects of Vaccines in the text that follows), it
ogenic) microorganisms, inactivated (killed) microorganisms, or
is used commonly in underdeveloped areas of the world because
antigenic components of microorganisms, known as subunit
it is easier to administer than the attenuated vaccine, which re-
vaccines. The main features of each of these vaccine forms will
quires injections. The vaccines for measles, mumps, rubella,
be discussed in more detail in the sections that follow. Newer
and varicella also consist of live strains of viruses that have been
strategies for vaccine development, which incorporate modern
attenuated through repeated passage in cultured cells. The
genetic technologies and approaches to enhance immune
measles and mumps viral strains are produced in chick embryo
responses, will be introduced in a later section.
cells, whereas the rubella strain is produced in human diploid
cells.17 The vaccines to prevent chickenpox consist of live,
Live, attenuated vaccines have been in routine use since Jenner’s attenuated Oka strains of varicella virus, which were originally
discovery of the smallpox vaccine. As we previously discussed, isolated from a Japanese child with chickenpox and sequentially
this vaccine was based on the concept of cross-reactivity, in propagated in cultures of human or embryonic guinea pig
which material from the cowpox virus was used to develop cells.18 These strains are used to produce an individual varicella
immunity against the antigenically similar, but highly patho- vaccine or a combination vaccine that also contains the vaccines
genic, smallpox virus. However, most pathogens do not have for measles, mumps, and rubella (MMR or MMRV). A more
an immunologically similar, but less pathogenic, counterpart. potent formulation of the attenuated Oka varicella strain is used
A number of laboratory techniques are currently used to to prevent herpes zoster (shingles) in adults.
modify bacteria or viruses so that they lose their pathogenic A live, attenuated vaccine has also been developed to pre-
properties but are still capable of stimulating a good immune vent influenza. The vaccine is administered intranasally. The
response. The techniques used to prepare conventional vaccines main antigens targeted by the vaccine are two surface glyco-
involve culture of the microorganism under conditions that are proteins of the influenza virus called hemagglutinin (H) and
different from those present in the host and unfavorable to its neuraminidase (N); these antigens are also used to classify the
growth.13 As we previously discussed, Pasteur used these prin- viruses on the basis of their serotype. Similar to the inactivated
ciples to develop nonpathogenic strains of the bacteria that cause influenza vaccine discussed later, a new attenuated influenza
chicken cholera from aged cultures and attenuated rabies virus vaccine must be prepared each year because of the high muta-
from dried spinal cords of infected rabbits. Another example of tion rate of the influenza viruses, which results in the synthesis
an attenuated preparation is the vaccine for tuberculosis devel- of new antigens. The vaccine consists of a quadrivalent suspen-
oped by Albert Calmette and Camille Guerin at the Pasteur sion containing the most common circulating antigenic strains
Institute in 1927. The vaccine, referred to as BCG (Bacillus of influenza virus from four common virus types: influenza A
Calmette Guerin), uses an attenuated strain of Mycobacterium bovis (H3N2), influenza A (H1N1), and two influenza B strains.19 The
developed by growing the bacteria on culture media containing viral strains have been cultivated in chicken eggs and attenuated
increasing concentrations of bile. After several years, the bacteria for adaptation to colder temperatures, so that they grow opti-
had adapted to growing in media containing a high bile content mally at 25°C rather than body temperature.9,20
and were therefore suitable for use in the human body. This is The primary advantage of live, attenuated vaccines is that
because the body’s lower bile concentration is not conducive to they are able to replicate at a low level in the host and are
therefore capable of inducing both humoral and cell-mediated culture systems or recombinant hemagglutinin antigens.19
immune responses.5,9 This is especially important for viral These vaccines avoid or minimize the use of eggs, allowing for
infections because cytotoxic T cells are required in order to faster production and eliminating or reducing the likelihood
attack viruses during the intracellular phase of the viral life of hypersensitivity to egg proteins.
cycle. Because of the broad immunity induced by live, atten- The hepatitis A vaccine consists of purified hepatitis A
uated vaccines, they generally induce an effective immune virus (HAV) cultured in human fibroblasts and inactivated by
response after just a single dose.5 formalin-treatment.22 Although this vaccine was initially only
Despite these advantages, live, attenuated vaccines also have given to individuals at high risk for contracting hepatitis A, it
some significant limitations.5 It is important not to administer has been incorporated into routine childhood immunization
vaccines containing live organisms to immunocompromised programs to reduce the incidence of this common infection.
individuals. Although the organisms are attenuated, they may A major advantage of inactivated vaccines is that they can
cause severe, disseminated, and potentially fatal infections in safely be given to immunocompromised people because the
patients with immunodeficiency diseases or patients receiving organisms have been killed and cannot replicate in the host.5,14
immunosuppressive treatments. Live vaccines may also not be However, this property makes it necessary to provide a larger
recommended for use in pregnant women. On rare occasions, amount of antigen in order to stimulate an effective immune
mutations may occur in the vaccine organism, causing it to lose response and may require two or more booster doses admin-
its attenuation and revert to the pathogenic form. This unfor- istered over time to produce protective immunity. Because the
tunately occurred during use of the live, attenuated (Sabin) inactivated organisms do not infect host cells, these vaccines
vaccine for polio, which led to the vaccine’s replacement with predominantly induce a humoral immune response, with little
an inactivated polio vaccine in industrialized countries. Use of or no cell-mediated immunity.5,14
genomic techniques to design live, attenuated strains that lack
genes required for pathogenicity will hopefully prevent such
Subunit vaccines consist of one or more purified components
an occurrence in the future. Another limitation of live, attenu-
of a pathogen that are capable of stimulating an immune re-
ated vaccines is potential interference with replication of the
sponse. The forms of subunit vaccines that are routinely used
organism in infants by maternal antibodies, necessitating a
are toxoids, capsular polysaccharides, purified proteins, and
delay in the dosing schedule (see Factors Influencing Immuno-
recombinant protein antigens.
genicity in the text that follows). Finally, careful handling and
storage of attenuated vaccines is very important because expo- The pathology of some bacterial diseases,
sure to heat and light may destroy the live organisms, causing such as diphtheria and tetanus, is caused by a single exotoxin.
the vaccines to be ineffective. This requirement can pose a Diphtheria is a contagious, life-threatening disease of the upper
major problem in developing countries of the world, where respiratory tract characterized by formation of a thick mem-
refrigeration is not readily available. brane that can cover the back of the pharynx, making it difficult
to breathe. Tetanus is a serious bacterial infection that affects
the nervous system, causing painful, prolonged muscle con-
Inactivated vaccines consist of intact, killed viruses or bacteria. tractions and, sometimes, difficulty breathing.
The microorganisms are killed by heat or chemical treatment so As we previously mentioned, toxoids are bacterial exotoxins
that they are not pathogenic but retain their antigenic properties. that have been chemically inactivated so they cannot cause
Chemicals such as formaldehyde or β-propiolactone are used harm to the host, but retain their ability to stimulate an im-
more frequently than heat treatment because they are less likely mune response. Toxoids are used in vaccines to induce the pro-
to alter the chemical structure of the surface epitopes.14 Exam- duction of antibodies that can bind to exotoxins and neutralize
ples of inactivated vaccines are the intramuscular vaccine for their effects.9,14 The first toxoid vaccine was developed in 1923
polio, the classic influenza vaccine, and the hepatitis A vaccine. against diphtheria and consisted of a formalin-inactivated toxin
In the early 1950s, Dr. Jonas Salk developed the first effective from the causative organism, Corynebacterium diphtheriae. In
inactivated vaccine for polio. The vaccine consisted of the three 1926, scientists developed a toxoid vaccine against tetanus,
disease-related serotypes of polio virus killed by formaldehyde which consisted of a formalin-inactivated toxin from the
treatment. A more potent inactivated polio vaccine with greater causative organism, Clostridium tetani. Toxoids are still used in
antigenic content was developed in 1978.16 This form of the the composition of today’s vaccines for diphtheria and tetanus.
vaccine is used routinely today in developed countries of the Another commonly used toxoid, which consists of inactivated
world because of its effectiveness and safety. toxin from the bacterium Bordetella pertussis, is part of the acel-
Similar to the live, attenuated influenza vaccine, the inacti- lular pertussis vaccine (see Purified Protein Vaccines in the text
vated influenza vaccines contain one influenza A (H3N2) virus that follows). The vaccines for diphtheria, tetanus, and pertus-
strain, one influenza A (H1N1) virus strain, and one or two sis are available singly or in combinations known as DTaP,
influenza B virus strains grown in embryonated hen eggs.19,21 TdaP, DT, or Td, depending on the amounts of diphtheria,
However, these viruses have been killed by treatment with tetanus, and acellular pertussis components present.23
formaldehyde or β-propiolactone and the vaccine is adminis- Another virulence factor possessed
tered by intramuscular injection.9,20 Researchers have also de- by some bacteria is the presence of a hydrophilic polysaccha-
veloped new influenza vaccines that use virus grown in cell ride capsule, which covers the bacterial outer membrane. The
capsule allows these bacteria to resist phagocytosis and other specific protein antigen from a pathogenic microorganism is
immune defenses by masking components of the membrane that isolated and incorporated into the genome of nonpathogenic
might otherwise be targets of the immune response. However, bacteria, yeast, or other cells. The genetically modified cells are
if antibodies to the capsular polysaccharides are present, they cultured in large quantities and produce the desired antigen,
can facilitate clearance of the bacteria by inducing opsonization which can then be purified by conventional biochemical meth-
or complement-mediated lysis.9,14 Therefore, vaccines against ods.14 The first recombinant protein vaccine was developed in
encapsulated bacteria contain purified capsular polysaccharides 1986 for hepatitis B and is widely used today. It is safer than
from specific bacterial strains that stimulate the production of the previous hepatitis B vaccine, which consisted of HBsAg
antibodies. Because the structure of these capsular antigens isolated from pooled plasma of infected patients. The recom-
varies with different bacterial serotypes, these vaccines contain binant hepatitis B vaccine is produced by cloning the gene for
multiple polysaccharide types to ensure an immune response HBsAg in yeast cells, then harvesting and purifying the HBsAg
that provides broad protection.9 protein.28 The protein spontaneously assembles into viruslike
The first polysaccharide vaccine was developed against Strep- particles that are not infectious but induce an effective immune
tococcus pneumoniae, the cause of pneumococcal pneumonia. The response because they are similar in structure to the actual
vaccines in use today contain polysaccharides from 13 or 23 dif- hepatitis B virus (HBV).9
ferent S pneumoniae serotypes.24 Another polysaccharide vaccine Recombinant DNA technology is also the basis for vaccines
has been developed against H influenzae type b (Hib), which was developed against the HPV, which can cause cervical cancer,
a major cause of pneumonia and meningitis in infants and young anal cancer, and other genital cancers. These vaccines contain
children before the vaccine was implemented. The vaccine is recombinant L1 major capsid proteins from 2 to 9 HPV virus
composed of the polyribosylribitol phosphate component of the types (including types 16 and 18), which are highly associated
Hib capsule conjugated to a protein carrier.25 A polysaccharide with anal–genital cancers.29,30 The genes for the L1 proteins
vaccine has also been developed against Neisseria meningitidis, are cloned in yeast or insect cell lines infected with baculovirus,
an important cause of bacterial meningitis, especially in young a type of virus that infects invertebrate cells. The isolated
individuals living in close quarters such as dormitory buildings. proteins then combine into viruslike particles that induce an
The vaccine consists of four purified bacterial capsular polysac- effective immune response. HPV vaccination is recommended
charides (A, C, Y, W-135) from N meningitidis.26 for adolescent girls and boys to confer protection before they
A problem with polysaccharide antigens is that they do not become sexually active. The vaccine is also recommended for
induce a good immune response, especially in infants and the young men who have sex with men and immunocompromised
elderly, two populations that are at high risk for severe conse- individuals, including those infected with HIV.
quences of encapsulated bacterial infections. This is because
polysaccharides are T-independent antigens that stimulate IgM Factors Influencing Immunogenicity
production with no immunoglobulin class-switching or long-
term memory response. This problem has been circumvented There are many factors that affect the quality of the immune
through vaccines composed of glycoconjugates, in which the response to a vaccine antigen. Important factors include the
polysaccharide antigens are linked to a carrier protein such as age of the recipient, the individual’s immune status, and the
tetanus toxoid or diphtheria toxoid. These conjugates are able nature of the vaccine.31 All of these factors are considered by
to induce a more effective immune response by activating immunization experts when deciding how a vaccine should be
T helper (Th) cells, resulting in immunoglobulin class switch- administered to achieve an optimal immune response.
ing, with production of polysaccharide-specific IgG antibodies
and generation of memory cells.9,14 Age is an important factor in determining how a vaccine
Vaccines can also be composed of should be provided to individuals in a population. Recom-
proteins from a pathogen. One such vaccine protects against mendations for the age at which a vaccine should be routinely
pertussis, a serious respiratory disease also known as “whoop- administered are based on age-specific risks for contracting
ing cough” because of the characteristic whooping sound patients the disease and developing associated complications, as well
make while trying to breathe during violent coughing fits. The as age-related ability to respond to the vaccine. In general, it
first vaccine against pertussis was composed of whole killed is recommended that vaccines be administered to the youngest
B pertussis bacteria and was thought to be associated with individuals at risk for the vaccine’s targeted disease, as long as
rare, but serious, neurological effects such as encephalitis or effectiveness and safety of the vaccine have been demonstrated
encephalopathy and convulsions, especially in children with in that age group.31
neurological disorders.27 Today’s pertussis vaccines are less fre- For example, in the United States, vaccination schedules rec-
quently associated with side effects because they are composed ommended by the Centers for Disease Control and Prevention’s
of two to five purified proteins from B pertussis rather than whole Advisory Committee on Immunization Practices (ACIP) are
bacterial cells.23 One of these proteins is a toxoid derived from categorized according to age. Routine immunization against
the pertussis toxin (see Toxoid Vaccines in the previous text). hepatitis B should begin at birth because the hepatitis B virus
Recombinant DNA technol- may have been transferred through the placenta, whereas
ogy has made it possible to develop even more highly purified vaccines for diphtheria, pertussis, tetanus, rotavirus, H influenza
protein vaccines. In these methods, the gene coding for a type b, polio, and streptococcal pneumonia should begin at
2 months of age. Multiple inoculations of these vaccines, admin- A diverse group of molecules can function as vaccine adju-
istered at specific time intervals through the first 18 months of vants, including emulsions, mineral salts, microbial products,
life, are necessary to achieve optimal immunity because the small molecules, microparticles, and liposomes.9 These mole-
young infant’s immune system is immature. Additional doses of cules are thought to activate pathways of the innate immune
some of these vaccines are also recommended during childhood, system, inducing the release of cytokines that promote inflam-
adolescence, or adulthood to maintain high antibody titers. mation and stimulate cells of the adaptive immune system.9
Some vaccines, such as the live, attenuated vaccine for Adjuvants can be classified as antigen delivery systems, which
measles, mumps, and rubella, are not started until 12 to enhance the uptake of antigens by antigen-presenting cells
15 months of age because administration before that age does (APCs) or immunopotentiators, which activate dendritic cells
not result in an effective immune response. The response is less to present antigens to T cells in humoral or cell-mediated
than optimal because passively acquired maternal antibodies immune responses.9 In some vaccine formulations, the two
present in the younger infant’s serum limit replication of the at- types of adjuvants have been combined together.
tenuated viruses. Other vaccines, such as those for meningo- The ultimate purpose for using adjuvants in vaccines is to
coccal meningitis and HPV, are not administered until 11 to increase antibody titers and, for some vaccines, to induce cell-
12 years of age because the risk for contracting these infections mediated immunity as well. Effective adjuvants can potentially
is greater during adolescence. Still other vaccines, such as those reduce the dose of antigen needed in a vaccine, decrease the
for varicella zoster (the cause of shingles) and pneumococcal number of inoculations required, and increase the speed and
pneumonia, are not administered until later adulthood because duration of the immune response.9,33 They are capable of
the natural decline of immune function in older individuals enhancing immunity in both young and elderly persons.
makes them more susceptible to developing these infections. A Despite the number of adjuvants that have been discov-
few vaccines, such as those for typhoid fever or yellow fever, ered, only a few have been licensed, based on their safety
are recommended only for individuals traveling to areas of the and efficacy. The most widely used adjuvant, and the only
world where there is a high incidence of these diseases. one licensed in the United States, is “alum,” which consists
Vaccination schedules are revised annually by the ACIP in of aluminum hydroxide and aluminum phosphate.9 The
consultation with the American Academy of Pediatrics (AAP) adjuvant activity of alum was discovered by Glenny in 1926,
and the American Academy of Family Physicians (AAFP).5,31 in his experimentation with diphtheria toxoid.34 Alum is
The 2016 immunization schedules for children, adolescents, routinely used today in vaccine formulations against HAV,
and adults are shown in Figures 25–4 and 25–5. Up-to-date HBV, HPV, diphtheria, tetanus, meningococcus, pneumococ-
schedules can be accessed from the Centers for Disease Control cal conjugates, tick-borne encephalitis, and anthrax, which
and Prevention (CDC) at www.cdc.gov/vaccines.32 are adsorbed to aluminum salts.9
Other commonly used adjuvants are oil-in-water emulsions,
composed of liquid dispersions of oil droplets stabilized with
The nature of the vaccine is another important factor influenc-
surfactants.9 Oil-in-water emulsions are mixed with vaccine
ing the quality of the immune response. In general, the most
antigens and are believed to stimulate the immune response
immunogenic vaccines consist of live, attenuated organisms that
by inducing release of chemokines and enhancing antigen
are able to replicate in the host; the least immunogenic vaccines
uptake and migration of APCs.9 The first such adjuvant was
consist of purified components (subunits) derived from the
discovered by Freund in the 1930s and is known as Freund’s
pathogen. Vaccine antigens with a low level of immunogenicity
complete adjuvant (FCA). FCA is a powerful adjuvant contain-
require an adjuvant, a substance that is co-administered with
ing killed mycobacteria. It has been used in animal studies, but
a vaccine antigen to produce an enhanced immune response.
is not suitable for use in humans because it produces abscesses
The term adjuvant, coined by Ramon in 1926, is derived from
and scar formation at the site of inoculation. Freund’s incom-
the Latin word adjuvare, which means “to help.”6,33
plete adjuvant (FIA), a water-in-oil emulsion without mycobac-
teria, has been used in some human vaccines because it is less
toxic.33 Two squalene-based water-in-oil emulsions, MF59
Connections and AS03, have been licensed in Europe for use in influenza
Principles of Immunologic Memory vaccines.33 Additional water-in-oil emulsions and other forms
Memory B and T lymphocytes are generated as a result of active
of adjuvants are under investigation.
immunity. These memory cells can be activated quickly if the in-
dividual is exposed to the same antigen at a later time, reducing Next Generation Vaccines
the lag period before antibody production. Antibody titers rise Although conventional forms of vaccines have been highly
quickly and reach higher levels than those produced after the effective in preventing many infections, we still have no vaccines
first exposure to the antigen. Antibody concentrations remain
for many diseases that are major causes of illness and death
high for a long period and provide long-lasting immunity. The
protection provided by the memory response serves as the basis
in the world. These diseases are caused by viruses, bacteria,
for repeated vaccine injections during routine immunization and parasites that have complex mechanisms of pathogenesis.
schedules. It also provides the host with lifelong immunity after They may display variability through genetic mutations or
recovery from a natural infection with a pathogen. multistage life cycles, or have developed other methods to
escape attack by the immune system.35 For example, the ability
Figure 1. Recommended immunization schedule for persons aged 0 through 18 years – United States, 2016.
(FOR THOSE WHO FALL BEHIND OR START LATE, SEE THE CATCH-UP SCHEDULE [FIGURE 2]).
These recommendations must be read with the footnotes that follow. For those who fall behind or start late, provide catch-up vaccination at the earliest opportunity as indicated by the green bars in Figure 1.
To determine minimum intervals between doses, see the catch-up schedule (Figure 2). School entry and adolescent vaccine age groups are shaded.

19–23
Vaccine Birth 1 mo 2 mos 4 mos 6 mos 9 mos 12 mos 15 mos 18 mos 2-3 yrs 4-6 yrs 7-10 yrs 11-12 yrs 13–15 yrs 16–18 yrs
mos

Hepatitis B1 (HepB) 1st dose 2nd dose 3rd dose

Rotavirus2 (RV) RV1 (2-dose See

1st dose 2nd dose
series); RV5 (3-dose series) footnote 2

Diphtheria, tetanus, & acellular

1st dose 2nd dose 3rd dose 4th dose 5th dose
pertussis3 (DTaP: <7 yrs)

Haemophilus influenzae type b4 See 3rd or 4th dose,

1st dose 2nd dose
(Hib) footnote 4 See footnote 4

Pneumococcal conjugate5
(PCV13) 1st dose 2nd dose 3rd dose 4th dose

Inactivated poliovirus6
(IPV: <18 yrs) 1st dose 2nd dose 3rd dose 4th dose

Annual vaccination (LAIV or Annual vaccination (LAIV or IIV)

Influenza7 (IIV; LAIV) Annual vaccination (IIV only) 1 or 2 doses
IIV) 1 or 2 doses 1 dose only

Measles, mumps, rubella8 (MMR) See footnote 8 1st dose 2nd dose

Varicella9 (VAR) 1st dose 2nd dose

Hepatitis A1 0 (HepA) 2-dose series, See footnote 10

Meningococcal1 1 (Hib-MenCY
6 weeks; MenACWY-D 9 mos; See footnote 11 1st dose Booster
MenACWY-CRM 2 mos)

Tetanus, diphtheria, & acellular

(Tdap)
pertussis1 2 (Tdap: 7 yrs)

Human papillomavirus1 3 (2vHPV:

females only; 4vHPV, 9vHPV: (3-dose
males and females) series)

See footnote 11
Meningococcal B1 1

Pneumococcal polysaccharide5
See footnote 5
(PPSV23)

Range of recommended Range of recommended ages Range of recommended ages Range of recommended ages for non-high-risk No recommendation
ages for all children for catch-up immunization for certain high-risk groups groups that may receive vaccine, subject to
individual clinical decision making
This schedule includes recommendations in effect as of January 1, 2016. Any dose not administered at the recommended age should be administered at a subsequent visit, when indicated and
feasible. The use of a combination vaccine generally is preferred over separate injections of its equivalent component vaccines. Vaccination providers should consult the relevant Advisory
Committee on Immunization Practices (ACIP) statement for detailed recommendations, available online at http://www.cdc.gov/vaccines/hcp/acip-recs/index.html. Clinically significant adverse
events that follow vaccination should be reported to the Vaccine Adverse Event Reporting System (VAERS) online (http://www.vaers.hhs.gov) or by telephone (800-822-7967). Suspected cases
of vaccine-preventable diseases should be reported to the state or local health department. Additional information, including precautions and contraindications for vaccination, is available from
CDC online (http://www.cdc.gov/vaccines/recs/vac-admin/contraindications.htm) or by telephone (800-CDC-INFO [800-232-4636]).This schedule is approved by the Advisory Committee on
Immunization Practices (http//www.cdc.gov/vaccines/acip), the American Academy of Pediatrics (http://www.aap.org), the American Academy of Family Physicians (http://www.aafp.org), and
the American College of Obstetricians and Gynecologists (http://www.acog.org).

NOTE: The above recommendations must be read along with the footnotes of this schedule.
Recommended immunization schedule for persons aged 0 through 18 years, 2016. (Source: Centers for Disease Control and Prevention.)
 Recommended Adult Immunization Schedule—United States - 2016
Note: These recommendations must be read with the footnotes that follow
containing number of doses, intervals between doses, and other important information.

Figure 1. Recommended immunization schedule for adults aged 19 years or older, by vaccine and age group1
VACCINE AGE GROUP 19-21 years 22-26 years 27-49 years 50-59 years 60-64 years 65 years

Influenza*,2 1 dose annually

Tetanus, diphtheria, pertussis (Td/Tdap)*,3 Substitute Tdap for Td once, then Td booster every 10 yrs

Varicella*,4 2 doses

Human papillomavirus (HPV) Female*,5 3 doses

Human papillomavirus (HPV) Male*,5 3 doses

Zoster6 1 dose

Measles, mumps, rubella (MMR)*,7 1 or 2 doses depending on indication

Pneumococcal 13-valent conjugate (PCV13)*,8 1 dose

Pneumococcal 23-valent polysaccharide (PPSV23)8 1 or 2 doses depending on indication 1 dose

Hepatitis A*,9 2 or 3 doses depending on vaccine

Hepatitis B*,10 3 doses

Meningococcal 4-valent conjugate (MenACWY) or
1 or more doses depending on indication
polysaccharide (MPSV4)*,11

Meningococcal B (MenB)11 2 or 3 doses depending on vaccine

Haemophilus influenzae type b (Hib)*,12 1 or 3 doses depending on indication

*Covered by the Vaccine Injury Compensation Program Report all clinically significant postvaccination reactions to the Vaccine Adverse Event Reporting System (VAERS). Reporting forms and instructions on filing
Recommended for all persons who a VAERS report are available at www.vaers.hhs.gov or by telephone, 800-822-7967.
meet the age requirement, lack Information on how to file a Vaccine Injury Compensation Program claim is available at www.hrsa.gov/vaccinecompensation or by telephone, 800-338-2382.
documentation of vaccination, or To file a claim for vaccine injury, contact the U.S. Court of Federal Claims, 717 Madison Place, N.W., Washington, D.C. 20005; telephone, 202-357-6400.
lack evidence of past infection;
zoster vaccine is recommended
Additional information about the vaccines in this schedule, extent of available data, and contraindications for vaccination is also available at
regardless of past episode of zoster www.cdc.gov/vaccines or from the CDC-INFO Contact Center at 800-CDC-INFO (800-232-4636) in English and Spanish, 8:00 a.m. - 8:00 p.m. Eastern Time,
Monday–Friday, excluding holidays.
Recommended for persons with a risk
factor (medical, occupational, lifestyle, Use of trade names and commercial sources is for identification only and does not imply endorsement by the U.S. Department of Health and Human Services.
or other indication) The recommendations in this schedule were approved by the Centers for Disease Control and Prevention’s (CDC) Advisory Committee on Immunization
No recommendation Practices (ACIP), the American Academy of Family Physicians (AAFP), the America College of Physicians (ACP), the American College of Obstetricians
and Gynecologists (ACOG) and the American College of Nurse-Midwives (ACNM).

Recommended adult immunization schedule—United States, 2016. (Courtesy of the Centers for Disease Control and Prevention.)
of the immune system to eliminate HIV is hampered by the responses are needed.35,39 One way of stimulating both arms
virus’ capacity to infect and kill CD4+ T cells, integrate into of the adaptive immune response is to use a “prime-boost”
the host genome, and rapidly mutate (see Chapter 24). Plas- strategy, in which the host is initially “primed” through ad-
modium falciparum, the cause of malaria, has posed a challenge ministration of an antigen-carrying vector that induces T-cell
for vaccine development through its ability to alter its surface responses (see the text that follows), followed by a “boost”
antigens in the different stages of its complex life cycle.36 with a subunit vaccine to stimulate antibody production.12
The vaccine for tuberculosis, BCG, is not optimally effective
because mycobacteria can establish a carrier state and become
As we previously discussed, several vaccines require administra-
reactivated during periods of immune suppression. Other
tion with an adjuvant to induce an optimal immune response.
infections that have posed a global challenge for effective vac-
The need for good adjuvants is becoming increasingly important
cine development include hepatitis C, respiratory syncytial
as new, highly specific vaccine antigens are discovered. To this
virus, Epstein-Barr virus, cytomegalovirus, herpes simplex, rhi-
end, scientists are searching for new, effective adjuvants. There
novirus, leishmaniasis, and dengue fever.35
is also ongoing research as to whether these new substances can
New vaccine designs, adjuvants, and methods of delivery
be used in combination with each other or with traditional ad-
need to be developed to conquer these diseases. The potential
juvants. Novel adjuvants targeting pattern-recognition receptors,
for these to be successful will be aided by technical advances
such as the Toll-like receptors (TLRs), Rig-like receptors (RLRs),
in multiple disciplines, including molecular genetics, structural
NOD-like receptors (NLRs), and C-type lectin receptors (see
biology, bioinformatics, systems biology, nanotechnology, for-
Chapter 3), are being studied because of their ability to stimulate
mulation techniques, and immune response monitoring.35
innate immunity and the release of cytokines that affect the
adaptive immune responses.9,12,40 Examples of such adjuvants
are poly-IC, a synthetic analog of double-stranded RNA that
The first step in producing an effective vaccine is the discovery
activates TLR3; monophosphoryl lipid A (MPL) and its deriva-
of antigens from a pathogen that will elicit an effective immune
tives, which bind to TLR4; bacterial flagellin, which activates
response. This step can now be facilitated by reverse vaccinology.
TLR5; and CpG (cytosine-phosphate-guanine) oligodeoxynu-
In this process, computer analysis is used to screen the entire
cleotides, which bind to TLR9.33,40 Saponin-based adjuvants are
genome of a pathogen to identify genes that code for proteins
also under study. These are plant-derived glycosides that are
that would make good vaccine targets.35,37 The process reverses
combined with antigen in nanoparticles called immunostimu-
the order of conventional methods for antigen detection, in
latory complexes (ISCOMs). They are thought to stimulate
which culture of the organism is followed by isolation of po-
strong antibody and cell-mediated responses by increasing
tential antigens, and finally bioinformatic analysis of the genes
antigen uptake and activation of dendritic cells.40 Various cy-
that code for the antigens. Reverse vaccinology allows for fast
tokines, chemokines, and inactivated bacterial toxins are also
identification of candidate vaccine antigens and facilitates
being studied for their ability to act as immunopotentiators.33
development of vaccines that have been difficult to produce.
In order to induce an optimal immune response, it will be
This new approach has been applied successfully to meningo-
important to combine these immunopotentiators with efficient
coccus and is being studied in additional organisms, including
antigen delivery systems. A number of novel vaccine delivery
group A streptococcus, S pneumoniae, Staphylococcus aureus, and
methods are under investigation.35,38,39,41 These include viral
Chlamydia.35 Vaccine antigen detection is also being facilitated
vectors such as vaccinia virus, adenoviruses, and baculoviruses
by other technologies, including screening of gene libraries for
that have been genetically modified so that they carry the antigen
immunogenic proteins and identification of microbial antigens
of interest, but are incapable of causing disease. Another delivery
by mass spectrometry.35
vehicle being studied consists of noninfectious viruslike particles
Once potential vaccine antigens are identified, it must be
formed from viral proteins that self-assemble at the plasma
determined which antigens will induce the most protective
membrane when recombinant viral vectors are used to infect
immune response. For pathogens that show a high degree of
cultured cells. A third vaccine form under study is delivery of
variability, such as the HIV and influenza viruses, finding anti-
“naked” DNA plasmids, encoding the antigen of interest under
gens that can induce broadly neutralizing antibodies will be
control of a mammalian promoter gene sequence. Naked DNA
desirable.12,35,38 These antibodies are directed against epitopes
can be administered through a needleless gene gun that uses
that are highly conserved among different strains of the microor-
pressurized gas. Synthetic delivery systems, consisting of
ganism. The identification of antigens that can induce broadly
nanoparticles, copolymers, DNA nanostructures, or liposomes
neutralizing antibodies may allow for development of a universal
loaded or coated with the antigens of interest, are also being
vaccine, which could provide long-lasting cross-protection
investigated.39 Entrapment of antigens within microparticles
against multiple strains of a pathogen.38 Advanced technologies
has been shown to protect the antigen from degradation in the
also allow for synthesis of “mosaic” antigens, composed of frag-
environment and increase uptake by APCs such as dendritic cells
ments of natural peptides from a pathogen combined to produce
and macrophages to enhance the immune response.41
novel proteins that are highly immunogenic.35
For those pathogens that have both intracellular and extra-
cellular phases in their life cycles, antigen combinations that Nonparenteral routes of antigen delivery such as oral, in-
induce cell-mediated responses as well as humoral antibody tranasal, aerosol, transcutaneous, intradermal, and rectal are
also being studied.10,41 Licensed oral vaccines are available from the United States and Canada because of immunization.43
already for rotavirus and typhoid fever along with an intranasal The success of vaccination continues in the 21st century as
vaccine for influenza.31 Additional needle-free methods will be immunization programs have expanded to countries through-
particularly attractive for developing areas of the world because out the world, preventing about 2.5 million deaths each year
they reduce the risk of transmitting bloodborne diseases and in children aged 5 and under.43 The mission of the Global
do not require sterile equipment or highly trained personnel. Vaccine Action Plan endorsed by the World Health Assembly
They will also avoid the pain associated with administration is to continue this expansion so that access to immunization
by injection. Research is being conducted with oral vaccines will be universal by the year 2020.46
composed of edible transgenic plants such as bananas or toma- An important feature of immunization is that it not only
toes that express the gene for the vaccine antigen of interest. benefits the individuals receiving the vaccine, but also reduces
Oral vaccines have an additional advantage in that they can the risk of nearby persons, who have not been vaccinated, of
potentially stimulate mucosal immunity as well as humoral contracting the infectious disease. When a sufficient proportion
antibody production and cell-mediated responses.41 However, of individuals in a population have been immunized, unvacci-
these systems need to be refined so that effective immune nated individuals, such as newborns and immunocompro-
responses, rather than immunologic tolerance, is induced. mised patients, are offered some protection because there is
little chance for the disease to spread in the community. This
concept of extending protection to others in the population is
Finally, in order to evaluate the effectiveness of a vaccine, more
known as community immunity or herd immunity and is of great
sophisticated methods will also be needed to assess the immune
importance to public health (Fig. 25–6).47
response. Antibody titers have traditionally been used as indi-
Persons who have altered immunocompetence are at risk
cators of vaccine-induced immunity.42 However, by using tech-
from certain immunizations and require special consideration.
niques such as DNA microarray analysis, multiplexed flow
These individuals have decreased levels of humoral or cell-
cytometry, and intracellular staining, the phenotype and acti-
mediated immunity because of inherited primary immunode-
vation status of individual cells of the immune system can be
ficiency diseases or acquired deficiencies secondary to other
analyzed, potentially generating a signature profile of markers
conditions, such as HIV infection, hematologic malignancies,
that more specifically represents the immune response to a
or treatment with immunosuppressive drugs or radiation (see
vaccine.35,37 These techniques can also be used to monitor cells
Chapter 17). The administration of live vaccines is contraindi-
in the tissues, where interaction with the antigen takes place,
cated in immunodeficient individuals because they are highly
as well as the blood.35 Used in conjunction with advances in
susceptible to contracting infections; therefore they should be
vaccine antigen discovery and improved delivery mechanisms,
immunized with inactivated or subunit vaccines.31 Although
immune monitoring methods will surely accelerate the devel-
the organisms contained in live vaccines are attenuated and
opment of new, effective vaccines against major global diseases
usually will not cause pathology in healthy individuals, they
in the next generation.
have the potential for uncontrolled replication and may cause
disseminated disease in immunodeficient persons. For exam-
Benefits and Adverse Effects of Vaccines ple, the vaccine for smallpox was known to cause a highly fatal
Vaccines have been cited as one of the 20th century’s greatest condition in infants with severe combined immunodeficiency
medical achievements.43 Because of routine immunization, (SCID), involving progressive spread of necrotic lesions from
smallpox has been eradicated worldwide and poliomyelitis has the site of vaccine injection to adjacent areas of the skin, bone,
been eliminated from the Western world. Infectious diseases and internal organs. Administration of the BCG vaccine for
that were once leading causes of illness and death in the be- tuberculosis to infants with SCID or HIV infection has also
ginning of the 20th century, such as diphtheria and measles, resulted in disseminated, life-threatening infections.5 The CDC
have a greatly reduced incidence today, especially in developed publishes specific recommendations for vaccination of persons
nations. A study conducted by the CDC in 2007 found that with altered immunocompetence.31
the overall incidence of diseases for which vaccines had been Vaccines can also produce adverse effects in previously
developed before 1980 decreased by over 92%, and mortality healthy individuals, but fortunately, most of these are not severe.
from these diseases decreased by more than 99%.43,44 For A local inflammatory response at the site of injection is fre-
example, before the development of the DTP vaccine, an quently reported because of stimulation of TLRs by the vaccine
estimated 176,000 cases of diphtheria, 1,300 cases of tetanus, antigen or adjuvant. Systemic inflammatory reactions are also
and 147,000 cases of pertussis were reported annually in the common. These manifest with fever, irritability, nausea, vom-
United States. In 2013, these numbers had decreased to 0, 26, iting, and myalgia 24 to 48 hours after injection of killed
and 28,639 cases, respectively.45 Annual cases of measles in vaccines, or 14 to 21 days after receipt of a live vaccine, and
the United States dropped from over 500,000 before 1963 to generally resolve within 72 hours.48 Other adverse effects of
fewer than 200 cases in 2013, and the number of German vaccines include hypersensitivity reactions and effects related
measles/congenital rubella cases in the United States decreased to the vaccine antigen or its administration.5
from 48,000 before 1969 to 9 cases in 2013. H influenzae type Hypersensitivity reactions to vaccines may be local or sys-
b, which was the leading cause of bacterial meningitis and temic and can be immediate or delayed (see Connections box).
invasive pneumonia before 1985, has been virtually eliminated IgE-mediated, type I hypersensitivity (anaphylactic) is usually
 Not immunized Immunized Not immunized,
but still healthy and healthy sick, and contagious

No one
is immunized

Contagious
disease spreads
through the
population

Some of the
population gets
immunized

Contagious
disease spreads
through some
of the population

Most of the
population gets
immunized

Spread of
contagious
disease is
contained

Community immunity (“herd immunity”). (Adapted from the National Institute of Allergy and Infectious Diseases. http://www
.niaid.nih.gov/topics/pages/communityimmunity.aspx Accessed November 30, 2015.)

triggered by vaccine additives such as gelatin (a stabilizer) or and antibiotics such as neomycin.49 Hypersensitivity develops
neomycin (an antibiotic used to prevent bacterial contamina- infrequently in response to vaccines and human serum prepa-
tion). Anaphylactic reactions are rare, occurring in 0.65 cases rations and is more common when serum from animal sources
per 1 million vaccine doses, on average.48 Development of is used for passive immunization.
an Arthus skin reaction because of local immune complex for- Fortunately, other adverse effects associated with vaccines
mation (type III hypersensitivity) has been reported in indi- are rare. Interested readers can learn about effects associated
viduals who have received a booster shot of the tetanus vaccine with specific vaccines from vaccine information sheets and
and who possess residual antibodies from previous tetanus written inserts that accompany vaccine preparations and from
immunization (see Chapter 14).48 Contact dermatitis, a de- the CDC website on vaccines and immunizations.32 The most
layed (type IV) hypersensitivity reaction that appears 48 hours dramatic example of such an effect was the development of
after vaccination, is considered harmless. It has been reported paralytic poliomyelitis after use of the live, attenuated oral
in some individuals in response to vaccine additives, including (Sabin) vaccine for polio. Occurring at a rate of approxi-
adjuvants such as alum, preservatives such as thimerosol and mately 1 case per 2.7 million doses of the vaccine, this event
2-phenoxyethanol, the toxin-inactivating agent formaldehyde, was caused by reversion of the Sabin polio virus type 3 to a
 Connections in 911 cases of the infection, were reported in the United
States, mostly in people who had not been vaccinated against
Hypersensitivity Reactions the disease or who had unknown vaccination status.53 Pertus-
Hypersensitivity to vaccine components can be in the form of sis presents another challenge, because immunity wanes 5 to
type I, II, or III reactions. Recall the mechanisms of these reactions 10 years after vaccination, requiring multiple boosters at
from Chapter 14. In sum: various ages to maintain adequate protection.54 Failure to
Type I reactions: Individuals produce high levels of IgE antibody, maintain immunity with up-to-date immunization has re-
which binds to mast cells and basophils. Binding of allergen to sulted in frequent outbreaks, with over 48,000 cases of per-
adjacent cell-bound IgE antibodies triggers the granules in tussis reported in the United States in the year 2012.55 High
these cells to release chemical mediators, which rapidly induce vaccine coverage is essential to preventing such outbreaks and
inflammatory reactions and smooth muscle contractions.
maintaining a healthy population.
Type III reactions: Persons develop IgG and IgM antibodies that
bind to vaccine or serum components. Complement binds
to these complexes, activating the classical pathway and re- Passive Immunization
lease of inflammatory mediators. Neutrophils are attracted to
the areas of immune complex deposition, where they release As we previously discussed, passive immunity results from the
lysosomal enzymes that destroy surrounding tissues. transfer of preformed antibodies to an unimmunized host.
Type IV reactions: Some individuals may develop a delayed
Antibodies can be transferred naturally to a mother’s fetus or
response to a vaccine component. This is a cell-mediated
infant in two ways: (1) A pregnant woman’s IgG antibodies pass
reaction in which Th1 cells are stimulated to release cytokines
that attract macrophages and cause inflammation. through the placenta to her unborn fetus or (2) maternal IgA
antibodies in breast milk and colostrum are ingested by the
infant during the nursing process (see Chapter 5). These anti-
bodies provide protection to the newborn against pathogens
neurovirulent strain.4 To avoid this tragic consequence, in- to which the mother has developed immunity, either through
dustrialized countries have stopped using the Sabin vaccine natural infection or through vaccination. IgG antibodies protect
and replaced it with the injectable, killed Salk-type polio the infant during its first few months of life, a time when the
vaccine in their routine immunization schedules.50 Another baby’s own immune system is immature and has not yet en-
example of a potentially serious vaccine consequence was the countered many antigens. IgA antibodies provide mucosal
possible association of a vaccine for Lyme disease with devel- immunity, an important mechanism in attacking pathogens at
opment of chronic arthritis that was resistant to treatment. their portals of entry into the body. Antibodies can also be pas-
This condition developed in individuals with the HLA type sively transferred to a host as a means of immunoprophylaxis
DR-4, possibly because of a cross-reactive autoimmune re- or immunotherapy.
sponse.51 Although the association of the vaccine with arthritis
could not be definitively demonstrated, wide media coverage Passive Immunization as Therapy
generated fear from the public and the vaccine was withdrawn
from the market in 2002, just 3 years after its licensure.
for Infectious Diseases
In 1998, public concern arose from a study conducted in The benefits of passive immunization were first discovered in
England by Dr. Andrew Wakefield, who proposed a linkage the late 1800s by von Behring and Kitasto. The two scientists
between the MMR vaccine and development of a form of first developed an antibody preparation against diphtheria and
autism. Wakefield and his colleagues hypothesized that the tetanus by injecting rabbits with small doses of the toxins re-
vaccine caused intestinal inflammation and damage to the in- sponsible for these diseases. They went on to demonstrate that
testinal barrier, allowing pathogenic proteins to enter into the injection of serum from these rabbits into mice could protect the
bloodstream and cause damage to the brain.52 It was not until mice from infection with virulent forms of the bacteria that
2010 that research studies using valid scientific designs proved caused the two diseases.14,56,57 These historical experiments
Wakefield’s findings to be unsubstantiated; his early papers showed that protective substances (now known to be antibodies)
were then retracted.52 Unfortunately, wide media coverage of could be generated in the blood and passively transfer their
Wakefield’s claims had stirred up fears in the public and many immune properties when injected into nonimmune individuals.
parents refused to get their children vaccinated. Today, human serum preparations are used to provide pas-
Fears such as these, as well as religious or personal objec- sive immunity to individuals who have been exposed to a
tions against immunization, have caused some individuals to pathogen but have not been vaccinated or developed immunity
delay or refuse vaccination for themselves or their children. through natural infection. Two types of preparations are avail-
Anti-vaccine public sentiments can result in lower than optimal able: Standard human immune serum globulin (also known
vaccine coverage, leaving a significant number of individuals as HISG or gamma globulin) and specific human immune
in the population unprotected against serious diseases. This serum globulins. Standard HISG is a sterile preparation of
situation, coupled with importation of diseases by unvacci- concentrated antibodies made from pooled serum of several
nated individuals immigrating into a country, can lead to out- thousands of donors.58 In the United States, only donors who
breaks of diseases that would normally be preventable. For test negative for hepatitis B and HIV are used. The plasma from
example, from 2001 to 2011, 63 outbreaks of measles, resulting these individuals is enriched for immunoglobulins by a cold
ethanol precipitation procedure, known as Cohn’s alcohol frac- to a variety of pathogens until its own immune system can
tionation.59 This is followed by depletion of blood coagulation mature.
factors, removal of IgG aggregates, and several virus inactiva- However, passive immunity is not long-lasting. The length
tion steps to further ensure safety of the preparation.58 HISG of the immunity is limited by the biological half-life of the
consists predominantly of IgG; IgM and IgA are found in in- immunoglobulins (23 days for IgG, the predominant im-
significant quantities because of their lower serum concentra- munoglobulin in human serum). Therefore, patients with
tions and rapid half-lives (see Chapter 5).59 HISG has been immunodeficiency diseases require repeated, periodic injec-
administered for more than 60 years as a prophylactic treat- tions or intravenous administration of HISG to be adequately
ment to prevent infections in immunodeficient patients who protected. In addition, no memory lymphocytes are gener-
are unable to produce sufficient amounts of antibodies (see ated, so an individual will not be protected if exposure to
Chapter 17).58 HISG contains antibodies specific for numerous the same antigen occurs at a later time in life. Another dis-
antigens to provide generalized humoral protection against a advantage of passive immunization is that hypersensitivity
variety of pathogens. reactions, although rare with HISG, can occur frequently
Antigen-specific immune globulins, also known as hyper- after therapy with animal serum. These reactions involve
immune globulins, are prepared from pooled serum of human type I hypersensitivity (anaphylaxis) or type III hypersensi-
donors who have developed immunity against a particular tivity (serum sickness) (see Chapter 14).
pathogen through a recent natural infection or vaccination.
These preparations contain a high concentration of antibody
against the pathogen or its product and are used to treat indi- Immunosuppressive Effects
viduals who have been potentially exposed to the pathogen but of Passive Immunization
have not been immunized. The potency of the preparation is In addition to its protective effects, passive immunization of
ensured by determining the antibody titer through laboratory gamma globulins can also have immunosuppressive effects in
testing. Specific HISGs have been developed for a variety of in- certain situations. For example, in Chapter 14, we discussed
fectious diseases, including hepatitis A, hepatitis B (hepatitis B how the administration of Rhogam can prevent hemolytic dis-
immune globulin; HBIG), varicella zoster, rabies, tetanus, and ease of the newborn. Rhogam inhibits the production of anti-Rh
respiratory syncytial virus.5,59,60 antibodies in an Rh-negative mother toward paternally-derived
Specific immune globulins for some antigens have also been Rh antigens on her fetus. In addition, it has been found that in-
prepared from animal sera, usually horse serum. Examples of travenous immunoglobulin therapy can modulate the proinflam-
these include antitoxins for tetanus, diphtheria, and botulism, matory activities of IgG antibodies in patients with autoimmune
as well as antisera against snake venoms. Antitoxins are anti- diseases. Over 30 years ago it was discovered that intravenous
bodies that specifically bind to epitopes on bacterial toxins. infusion of HISG results in an immediate increase in platelet
They protect against the harmful effects of these toxins by counts and improvement of symptoms in patients with immune
neutralizing their activity. thrombocytopenia (ITP).58 Similar results have been found
in patients with other inflammatory disorders. Intravenous
Advantages and Limitations immunoglobulin therapy is approved by the Food and Drug
Administration (FDA) for treatment of ITP, chronic inflammatory
of Passive Immunization demyelinating polyneuropathy (CIDP), Kawasaki’s disease,
As we previously mentioned in this chapter, the main advantage and Guillain-Barre syndrome. Its effects are also being studied
of passive immunization is that it provides immediate immunity in other chronic inflammatory disorders, including multiple
to the host. This is because the antibodies are already present in sclerosis, systemic vasculitis, rheumatoid arthritis, SLE, autoim-
the serum that is being transferred and the host does not have mune hemolytic anemia, autoimmune skin blistering diseases,
to experience the lag period required for its own immune system graft-vs.-host disease (GVHD), and sepsis.58
to be activated by the antigen (see Chapter 5). This immediate The way in which intravenous immunoglobulins inhibit
protection can be especially beneficial in situations in which the inflammatory response is unclear, but several mechanisms
unimmunized individuals have been exposed to a harmful have been proposed.5,58 The antibodies in the HISG prepara-
antigen; they would develop disease symptoms and possibly die tion contain many antigen specificities and may mediate killing
if they had to wait for an immune response to occur. For exam- of target cells by antibody-dependent cellular cytotoxicity
ple, disease could be avoided in an unvaccinated person who (ADCC), prevent interactions of ligands with cell surface re-
had contact with soil that was potentially contaminated with ceptors, inhibit cytokines, neutralize autoantibodies, or bind
tetanus-causing bacteria or who had an accidental needlestick to activated complement components such as C3a and C5a,
involving blood from a hepatitis B patient. Hepatitis A could blocking their activity. Other immunomodulating effects of
be prevented in customers who dined at a restaurant in which HISG may be mediated through the Fc portion of the im-
a food handler was found to have the infection, and death munoglobulins, including saturation of Fc receptors on cells
could be prevented in people who have been bitten by a poi- of the immune system, limiting the access of immune com-
sonous snake or an animal with rabies. When a mother natu- plexes to Fc receptors on phagocytic cells, enhancement of
rally transfers antibodies to her infant through the placenta or T regulatory (Treg) cell activity, modulation of dendritic cell
breast milk, her child is provided with immediate protection activation, or inhibition of B-cell function.
Monoclonal Antibodies therapy because they caused type I (anaphylactic) or type III
(immune complex) hypersensitivity reactions.
Monoclonal antibodies, as we discussed in Chapter 5, are de- Over the years, the development of recombinant DNA
rived from a single clone of B cells; therefore, they have exquisite technology has allowed us to produce monoclonal antibodies
specificity for a particular epitope of an antigen. This specificity that have an increasingly larger human component, making
is being harnessed in the use of monoclonal antibodies as agents them less likely to trigger an adverse immune reaction. Ini-
of passive immunization, most notably for the treatment of tially, chimeric antibodies were developed, which consisted of
cancer and autoimmune diseases.61,62 Numerous monoclonal a mouse-derived immunoglobulin variable region combined
antibodies have been approved by the FDA for treatment of pa- with a human-derived constant region (Fig. 25–7). Chimeric
tients with hematologic malignancies, solid tumors, autoimmune antibodies can be identified by the suffix “–ximab.” This
disorders, and other miscellaneous conditions.61 Examples of was followed by the production of humanized antibodies,
monoclonal antibodies that have been widely used as therapeutic which contain all human sequences except for the antigen-
agents include rituximab, directed against the CD20 antigen on binding complementarity determining regions; the latter are
B cells, for treatment of non-Hodgkin lymphoma; trastuzumab mouse derived. Humanized antibodies are denoted by the
(Herceptin), directed against Her2/neu, for treatment of certain suffix, “–zumab.” Today, it is possible to produce fully human
breast cancers; and adalimumab (Humira), directed against
tumor necrosis factor-α (TNF-α), used for reduction of inflam-
mation in patients with rheumatoid arthritis. These and other
examples of monoclonal antibodies approved by the FDA for Connections
therapeutic use in patients are listed in Table 25–1.
Monoclonal Antibodies and Mice
As was previously discussed in Chapter 5, monoclonal anti-
bodies were originally developed using mouse B cells to produce Monoclonal antibodies were originally produced from mice, by
hybridomas that secreted antibody to the desired antigen. Mouse injecting the mice with the desired antigen, isolating B cells from
the immunized animals, and fusing them with cultured mouse
monoclonal antibodies can be identified by the suffix, “–omab.”
myeloma cells to produce immortal hybrid cell lines known as
When these monoclonal antibodies were used as therapeutic “hybridomas” (see Chapter 5). Cell culture techniques were used
agents in humans, immune responses were likely to occur to the to isolate the hybridomas that produced the desired antibody.
foreign epitopes on the mouse immunoglobulins, resulting in The monoclonal antibodies purified from these cultures con-
the production of human anti-mouse antibodies (HAMA). HAMA sisted entirely of mouse protein.
significantly limited the usefulness of the monoclonal antibody

Table 25–1 Examples of FDA-Approved Monoclonal Antibodies Used for Immunotherapy

NAME* TYPE SPECIFICITY DISEASE INDICATION(S)
Adalimumab (Humira) Human TNF-α Rheumatoid arthritis, psoriatic arthritis, anky-
losing spondylitis, Crohn’s disease, plaque
psoriasis, juvenile idiopathic arthritis
Alemtuzumab (Campath) Humanized CD52 Chronic lymphocytic leukemia
Basiliximab (Simulect) Chimeric CD25 Kidney transplant rejection
Bevacizumab (Avastin) Humanized Vascular endothelial growth Colorectal cancer, non-small cell lung carci-
factor (VEGF) noma, renal cell carcinomas, glioblastoma
Cetuximab (Erbitux) Chimeric Epidermal growth factor Colorectal cancer, squamous cell carcinoma
receptor (EGFR) of the head and neck
Infliximab (Remicade) Chimeric TNF-α Rheumatoid arthritis, psoriatic arthritis, anky-
losing spondylitis, Crohn’s disease, plaque
psoriasis, ulcerative colitis
Ipilimumab (Yervoy) Human CTLA-4 Melanoma
Omalizumab (Xolair) Humanized IgE Allergic asthma
Rituximab (Rituxan) Chimeric CD20 Non-Hodgkin lymphoma, chronic lymphocytic
leukemia, rheumatoid arthritis, Wegener’s
granulomatosis, microscopic polyangitis
Trastuzumab (Herceptin) Humanized HER2/neu Breast cancer, gastric adenocarcinoma, gas-
troesophageal junction adenocarcinoma
*Brand names are in parentheses.
 Mouse Chimeric to increase the cell-mediated immune response of the recipient.
The cells transferred can be derived from a donor individual
or from the recipients themselves. In the case of autologous
transfers, the cells are removed from the patient, treated in vitro
to become more immunologically reactive, and infused back
into the same patient. The cells transferred in adoptive im-
munotherapy can be naturally derived or genetically engi-
neered to target a particular antigen. Heterogeneous cell types
-omab -ximab can be transferred, although most applications have focused
on the transfer of T-cell–mediated activity.
Human studies involving adoptive cell transfer have led to
Humanized Human
the development of some exciting immunotherapies for cancer
patients and transplant recipients. Cancer immunotherapy
with adoptively transferred cells is based on the observation of
immune responses against tumor cells in animal studies. In the
1960s, researchers discovered that lymph node cells from mice
immunized with chemically or virally induced tumors were
able to protect against growth of the same tumor type when
they were transferred to genetically identical recipients.64,65
-zumab -umab
They found that this immune response was enhanced in the
Monoclonal antibodies used as therapeutic agents
presence of the cytokine IL-2.66 Scientists later began to look
can be made in mice, but humans may react to the foreign protein.
for the ability of lymphoid cells actually found within a tumor
The antigen-binding portion of the molecule can be grafted onto
the constant regions of human antibodies to reduce such reactivity. mass to mount an anti-tumor response. Through their work,
It is also possible to humanize such antibodies so that only variable they identified a heterogeneous population of T cells that can
regions are incorporated into human antibody molecules. demonstrate cytotoxic effects against the tumor. These cells
came to be known as tumor-infiltrating lymphocytes (TILs).67
TILs appear to be ineffective in eliminating the tumor at their
monoclonal antibodies by phage display, a technique in which natural site, possibly because they are not present in adequate
genes coding for a specific antibody molecule are cloned in numbers, they become anergic as a result of chronic activation,
bacteriophages or by transgenic mouse technologies involving or they are surrounded by Treg cells and myeloid-derived
incorporation of the immunoglobulin genes of interest into suppressor cells.66,67 However, Rosenberg and his colleagues
genetically modified mice.61 Fully human antibodies are in- at the National Institutes of Health (NIH) found that they could
dicated by the suffix, “–umab.” These antibodies have a low enrich for and expand the TIL population in vitro by incubat-
potential for immunogenicity and are becoming more widely ing cells from the tumor in the presence of IL-2.
available for therapeutic use.61,63 In 1986, Rosenberg’s group demonstrated protective effects
The development of humanized and fully human antibodies of autologous TILs against metastases in mice. In 1988, the
has especially had an impact in the area of cancer treatment. researchers applied a similar approach to humans. In their
Monoclonal antibodies are able to mediate killing of tumor landmark publication, the scientists showed that TILs isolated
cells by a variety of mechanisms (see Chapter 17).61,63 They from patients with metastatic melanoma and expanded in vitro
can directly kill tumor cells by binding to cell-surface receptors could mediate tumor regression when given back to the same
or other membrane-bound proteins, causing apoptosis or in- patients.68 In this adoptive immunotherapy procedure, which
hibition of signaling and cell proliferation. They can also bind has been improved over the years, tumors are surgically re-
to the tumor cells and facilitate their destruction through moved, dissected into fragments, and incubated in culture in
opsonization and phagocytosis, ADCC, activation of the com- the presence of IL-2 for a few weeks. During the incubation
plement cascade, or activation of T cells. Some monoclonal period, the TILs proliferate and kill the residual tumor cells.
antibodies have been conjugated to drugs or toxins, allowing T cells showing potent anti-tumor activity are isolated from the
other therapeutic agents to be specifically delivered to the mixture and further expanded in the presence of IL-2, anti-
tumor cells. Because of their ability to specifically target a CD3 antibody, and irradiated mononuclear cells derived from
diverse range of molecules, monoclonal antibodies are consid- normal peripheral blood.66 These purified, potent TILs are
ered to be one of the most successful cancer therapies in the then infused into the same patient from which the tumor frag-
last 20 years.63 ments were derived. The expanded TILs migrate back to the
tumor, where they mediate its destruction (see Fig 17-8).
Adoptive Immunotherapy This therapy has been cited as the most effective treatment
for patients with metastatic melanoma.66 In a study conducted
Although passive immunization is aimed at providing the in 2011, adoptive transfer of autologous TILs along with IL-2
humoral component of the immune response, adoptive im- into patients with stage IV melanoma resulted in objective clin-
munotherapy involves transferring cells of the immune system ical response rates in up to 72% of the patients. Complete
tumor regression was seen in 22% of the patients; the majority
of these patients were free of the disease at the time of follow- • Adoptive immunotherapy is achieved by transferring cells
up, which ranged from 57 months to over 8 years.67 The suc- of the immune system to a nonimmune host. The cells can
cess of this treatment is thought to be to the result of the high be derived either from a different (allogeneic) host or they
level of immunogenicity of melanoma tumors and pretreat- can be stimulated in vitro before re-introduction into the
ment of patients with total body irradiation, which is thought same individual (autologous).
to deplete regulatory and suppressor cells within the host that • Active immunity takes time to develop, but provides long-
could inhibit the anti-tumor effects of the TILs.66,67 Adoptive lasting protection to the host because of the production
TIL therapy is also being investigated in patients with other of immunologic memory. Passive immunity provides im-
types of tumors who have exhausted other treatment options. mediate protection, but is short lived because antibody
Efforts are underway to improve techniques to reduce the cost titers decline at a rate that is determined by the biological
of this expensive, personalized treatment so that it can be avail- half-life of the immunoglobulins. Adoptive immunity pro-
able to a larger number of patients.67 Research with genetically vides long-lasting protection if the cells become estab-
engineered T cells expressing high-affinity T-cell receptors for lished in the host.
target tumor antigens is also underway.67 • A traditional vaccine is a microbially derived antigen sus-
Hematopoietic stem-cell transplantation is another area in pension that is administered to a healthy host in order to
which medical practitioners are using adoptive immunotherapy. stimulate an immune response that will prevent the host
These stem cells are routinely used as a treatment for certain from developing an infectious disease. Thus, vaccines are
immunodeficiency disorders (see Chapter 19).69 Autologous or used for immunoprophylaxis.
allogeneic transplantation of hematopoietic stem cells can also • In 1796, Dr. Edward Jenner was the first to publicize the
be used to restore the immune system in patients who have procedure of vaccination by using material from cowpox
been treated with chemotherapy and a high dose of total body skin lesions to immunize against smallpox. In the 1800s,
irradiation. The treatment has been used most commonly in Louis Pasteur used the principle of attenuation, or weak-
patients with multiple myeloma, non-Hodgkin lymphoma, or ened microorganisms, to produce vaccines against chicken
acute leukemias.69 cholera, anthrax, and rabies.
Clinical trials are also underway in other exciting areas • In the 20th century, vaccine development grew exponen-
related to transplant immunology. For example, clinical trials tially as a result of new methods to attenuate microorgan-
involving adoptive transfer of Treg cells to transplant patients isms, production of toxoids, and technologies that allowed
are being conducted in an attempt to prevent graft rejection viruses to be grown in cell culture. In the latter part of the
or GVHD.70 In addition, medical scientists are adoptively trans- 20th century, the first recombinant vaccine, produced by
ferring cytotoxic T cells to transplant recipients to treat oppor- genetic engineering, was developed.
tunistic infections with viruses such as CMV, EBV, and adenovirus • Conventional vaccines may be composed of live, attenu-
and their associated complications, which can be life threatening ated (weakened, nonpathogenic) microorganisms; inacti-
in these immunosuppressed patients.71 vated (killed) microorganisms; or antigenic subunits such
as recombinant antigens or toxoids. Toxoids are bacterial
toxins that have been inactivated so that they are no longer
pathogenic, but are still immunogenic.
• Adjuvants are substances that are often included in vac-
SUMMARY cines in order to increase the immune response to the
target antigens. Examples of adjuvants include alum
• Immunity is the condition of resistance to a disease. Im-
(aluminum hydroxide and aluminum phosphate) and
munization is the process by which immunity is acquired.
oil-in-water emulsions. They are believed to enhance the
There are three types of immunization—active, passive,
immune response by promoting migration and antigen
and adoptive.
uptake by APCs and inducing the release of proinflam-
• Active immunization involves stimulation of an individ-
matory cytokines and chemokines.
ual’s own immune system to mount an adaptive immune
• In the future, scientists will develop next generation
response to an antigen, as a result of either natural infec-
vaccines that will identify vaccine target antigens more
tion or receipt of a vaccine.
efficiently by using genetic technologies. In developing
• Passive immunization involves transfer of premade an-
these vaccines, researchers will attempt to induce broadly
tibodies from an immune host to a nonimmune host.
neutralizing antibodies and cell-mediated responses,
This type of immunity can occur naturally, by transfer
employ novel adjuvants, and investigate the use of non-
of maternal IgG antibodies through the placenta to the
parenteral routes of delivery.
fetus, or by transfer of IgA antibodies from a mother
• The effectiveness of a vaccine is not only influenced by the
to her infant through breast milk. Passive immunity
nature of the vaccine, but also by the age and immune status
can also be transferred artificially through commercial
of the host. To obtain an optimal immune response, vaccines
antibody preparations such as human immune serum
must be administered according to recommended schedules.
globulin.
• Vaccines protect not only the individuals who have re- • Passive immunization of immunoglobulins can also be
ceived the vaccines, but also their contacts, a phenomenon used as immunosuppressive therapy for certain autoim-
known as “community immunity” or “herd immunity.” mune and chronic inflammatory disorders, as well as to
• Vaccines are considered to be one of the greatest achieve- prevent hemolytic disease of the newborn.
ments of medicine. The benefits of vaccines greatly out- • Monoclonal antibodies have specificity for a particular
weigh their risks. Rarely, vaccines can cause side effects antigenic epitope. They are being used as therapeutic
such as hypersensitivity reactions. Live vaccines should agents for a variety of cancers and autoimmune diseases.
not be administered to patients with immunodeficiency These antibodies were originally 100% mouse derived
disorders because they can replicate uncontrollably and because they were produced by hybridoma technology.
cause disseminated disease. Advancement in genetic technologies has allowed scien-
• Passive immunization with preformed antibody prepa- tists to synthesize hybrid antibodies that consist partially
rations is administered in order to provide immediate of mouse protein and partially of human protein. These
humoral immunity to unimmunized persons. antibodies can be characterized as chimeric, humanized,
• Patients with humoral immunodeficiencies are routinely or fully human, depending on the amount of the human
treated with standard human immune serum globulin, component. Administration of these antibodies reduces
prepared from pooled serum of many donors, to provide the chance that the recipients will produce human-
protection against a variety of pathogens. Specific human anti-mouse antibodies (HAMA) and develop hypersen-
immune serum globulins containing antibody against a par- sitivity reactions.
ticular pathogen can be used to treat nonimmune, healthy • Adoptive immunotherapy involves the transfer of cells
individuals who have had contact with the pathogen to pro- of the immune system to provide immunity. Examples
vide immediate protection. include the treatment of melanoma patients with autol-
• Antitoxins are antibodies directed against toxins from ogous, IL-2 activated tumor-infiltrating lymphocytes
pathogenic bacteria. They can be isolated from the serum (TIL) and the administration of hematopoietic stem
of laboratory animals that have been injected with the cells to patients with hematologic malignancies who
toxin and used to prevent diseases such as tetanus, diph- have been treated with high doses of chemotherapy and
theria, and botulism. irradiation.

Study Guide: Major Features of Active Immunity, Passive Immunity, and Adoptive Immunity
MECHANISM EXAMPLES ADVANTAGES LIMITATIONS
Active Activation of humoral and Natural infection with Long-term immuno- Delay in initiation of
Immunity cell-mediated responses pathogen logic memory to the immune response
in an individual’s own Immunization with a vaccine antigen is generated
immune system by
exposure to an antigen
Passive Transfer of antibodies Passage of IgG through the Provides immediate Immunity is tempo-
Immunity from immunized placenta, from pregnant protection to the rary, declining with
host(s) to nonimmune woman to her fetus recipient the half-life of the
individuals Passage of IgA through antibodies
breast milk, from mother Immunologic mem-
to infant ory is not generated
Standard human immune Hypersensitivity can
serum globulin develop, especially
Specific human immune when sera of animal
serum globulin origin are used
Antitoxins
Rhogam
Monoclonal antibodies
Adoptive Transfer of cells of the Adoptive immunotherapy Can transfer cell- Patient’s own im-
Immunity immune system to with activated TILs to mediated immunity mune cells must be
nonimmune individuals cancer patients depleted to increase
Hematopoietic stem cell chance of success-
transplantation ful therapy
Allogeneic cells may
be rejected
Study Guide: Characteristics of Conventional Vaccines
COMPOSITION EXAMPLES ADVANTAGES LIMITATIONS
Attenuated Live pathogens that BCG Induce both humoral Cannot be administered
have been weak- Typhoid fever and cell-mediated to immunocompromised
ened by growth Oral polio (Sabin) immunity individuals
under modified Measles (Rubeola) Effective in inducing Rare potential to mutate
culture conditions Mumps immunity after a to a pathogenic form
German measles single dose Maternal antibodies can
(Rubella) interfere with immune
Chickenpox response to the vaccine
(Varicella) in infants
Shingles (Zoster) Require careful handling
Influenza (nasal and storage
mist)
Rotavirus
Inactivated Killed Intramuscular polio Can safely be given Stimulates humoral
microorganisms (Salk) to immunocompro- immunity but little or no
Influenza (intra- mised individuals cell-mediated immunity
muscular or May require two or
intradermal) more booster doses
Hepatitis A to produce protective
immunity
Subunit One or more purified Toxoids Induces an immune Requires two or more
components of a Purified proteins response to the booster doses to produce
pathogen Polysaccharides pathogenic protective immunity
Recombinant component(s) of Requires an
antigens a microorganism adjuvant to increase
Safer than adminis- immunogenicity
tration of an intact Must be multivalent if a
organism broad immune response
is desired
a. Toxoids Bacterial toxins that Diphtheria See information for Requires two or more
have been chemi- Tetanus Subunit vaccines in booster doses to
cally inactivated so this table produce protective
that they are not immunity
pathogenic Requires an adjuvant to
increase immunogenicity
b. Purified Biochemically puri- Pertussis See information for See information for
Components fied components of (whooping Subunit vaccines in Subunit vaccines in this
a microorganism cough) this table table
Produces fewer side
effects than whole
bacteria
c. Polysaccharides Biochemically puri- Streptococcal See information for See information for
fied polysaccharide pneumonia Subunit vaccines in Subunit vaccines in
from bacterial Haemophilus this table this table
capsule influenzae type b Requires conjugation to a
Neisserial carrier protein to induce
meningitis IgG production and long-
term immunity
d. Recombinant Protein produced by Hepatitis B Highly purified pro- See information for
Antigen genetically modified Human papilloma tein that is safer Subunit vaccines in
nonpathogenic virus (cervical, than administration this table
bacteria, yeast, anal, genital of intact organism Cannot be used to
or other cells cancers) produce antigens other
than proteins
CASE STUDIES
1. A 2-year-old boy has made numerous visits to his doctor 2. Suppose you ate lunch at a popular restaurant a few days
because he has suffered from recurring respiratory and ago. Your local health department puts out a notice that
ear infections. An immunologic workup revealed that several of the customers who dined at the restaurant re-
the child had a low number of B cells and decreased cently have confirmed cases of hepatitis A and that the
immunoglobulin concentrations. Based on these results, source of infection was traced to a supply of green onions
the boy was diagnosed with an antibody immunodefi- that had been used in the salads. Public health officials
ciency disease. advise anyone who has eaten at the restaurant in the last
2 weeks to visit the local county health department to
Questions
receive an injection to prevent the infection.
a. What childhood vaccines could safely be adminis-
tered to this child? What is the composition of these Questions
vaccines? a. What would the injection consist of, and why would
b. What childhood vaccines should not be administered it be the treatment of choice?
to this child? Why? b. If you received this treatment and did not develop
c. How can the child be protected against the diseases hepatitis A, would you be immune to this virus 10 years
for which he is unable to receive vaccination? from now? Why or why not?

REVIEW QUESTIONS
1. Suppose an individual develops antibodies in response 5. The antigenic component of the hepatitis B vaccine
to a streptococcal pharyngitis infection. This is an differs from those of many of the conventional
example of vaccines in that it consists of a
a. active immunity. a. live, attenuated virus.
b. passive immunity. b. inactivated virus.
c. adoptive immunity. c. cryptic antigen.
d. immunoprophylaxis. d. recombinant antigen.

2. Which of the following illustrates passive immunity? 6. Which of the following describes the properties
a. Development of high antibody titers in a of a toxoid?
healthy person after receipt of the hepatitis B a. Both pathogenic and immunogenic
vaccine b. Pathogenic but not immunogenic
b. Recovery of a patient from a hepatitis A c. Not pathogenic but immunogenic
infection d. Neither pathogenic nor immunogenic
c. Passage of IgG antibodies through the placenta
of a pregnant woman to her fetus 7. Suppose a vaccine was available in two forms:
d. Transfer of tumor-infiltrating lymphocytes to a attenuated and inactivated. What is an advantage
cancer patient of the attenuated form?
a. It can be used in immunocompromised patients.
3. Which of the following is not a characteristic b. It induces both humoral and cell-mediated
of passive immunity? immunity.
a. Transfer of antibodies c. There is no interference of the immune response in
b. Occurs naturally or as a result of therapy infants by maternal antibodies.
c. Provision of immediate protection d. It does not require special handling and storage to
d. Development of long-term memory maintain its effectiveness.

4. What was one of the major contributions of Louis 8. What factor(s) influence the effectiveness of a person’s
Pasteur to vaccine development? immune response to a vaccine?
a. Development of the smallpox vaccine a. Age of the recipient
b. Use of attenuated microorganisms in vaccines b. The individual’s immune status
c. Inactivation of bacterial toxins for vaccines c. The nature of the vaccine
d. Discovery of recombinant vaccine antigens d. All of the above
 9. An oral vaccine may be advantageous over an injectable 13. HAMA are
vaccine for a pathogen because it a. mouse-derived antibodies that have been used for
a. reduces the risk of transmitting bloodborne therapy.
pathogens in developing areas of the world. b. monoclonal antibodies with therapeutic benefits.
b. avoids the pain associated with injections. c. human antibodies that are produced against mouse
c. induces mucosal immunity. proteins.
d. all of the above. d. antitoxins that can provide immediate immunity.

10. When one individual becomes immunized by receiving 14. What is a major characteristic of adoptive
a series of vaccine injections according to schedule, the immunotherapy?
resulting protection extends to that individual’s nearby a. It involves the transfer of cells to deliver immunity.
contacts. This concept is known as b. It involves the transfer of cytokines to deliver
a. immunologic memory. immunity.
b. neighborhood immunity. c. It can only occur in the presence of autologous
c. herd immunity. cells.
d. contagious immunity. d. Its purpose is to increase the humoral immune
response.
11. Which preparation would you recommend for
treatment of a patient with an antibody deficiency? 15. Infusion of TILs into a cancer patient is an
a. Monoclonal antibody example of
b. Specific human immune serum globulin a. active immunity.
c. Standard human immune serum globulin b. adoptive immunity.
d. Animal serum antitoxins c. passive immunity.
d. natural immunity.
12. Immunoglobulins consisting of a mouse-derived
variable region combined with a human-derived
constant region are known as
a. monoclonal antibodies.
b. chimeric antibodies.
c. humanized antibodies.
d. fully human antibodies.
 Glossary
Accelerated rejection: A form of graft rejection that occurs within Allelic exclusion: The selection of an allele on one chromosome
1 to 5 days after second exposure to tissue antigens based on only.
reactivation of B- and T-cell responses. Allergen: An antigen that triggers a type I hypersensitivity response
Accuracy: The ability of a test to actually measure what it claims to (i.e., an allergy).
measure. Allergy immunotherapy (AIT): Therapy involving administration
Acquired immunodeficiency syndrome (AIDS): A disease affect- of increasing doses of an allergen over time with the goal of in-
ing the immune system caused by the human immunodeficiency ducing immune tolerance to the allergen.
virus (HIV). Alloantigen: An antigen that is found in another member of the
Activation unit: The combination of complement components C1, host’s species and that is capable of eliciting an immune response
C4b, and C2b that form the enzyme C3 convertase, whose substrate in the host.
is C3. Allograft: Tissue transferred from an individual of one species into
Active immunity: Immunity resulting from natural exposure to an another individual of the same species.
infectious agent or administration of a vaccine. Allotype: A minor variation in amino acid sequence in a particular
Acute graft-versus-host disease (GVHD): Graft-versus-host disease, class of immunoglobulin molecule that is inherited in Mendelian
which occurs shortly after immunocompetent cells are trans- fashion.
planted into a recipient. It is characterized by skin rashes, diarrhea, Alpha-fetoprotein (AFP): A tumor marker that is commonly ele-
and increased susceptibility to infection. vated in patients with primary hepatocellular carcinoma and
Acute-phase reactants: Normal serum proteins that increase rapidly nonseminomatous testicular cancer.
as a result of infection, injury, or trauma to the tissues. Alpha1-antitrypsin (AAT): An acute-phase protein that acts
Acute rejection (AR): A type of rejection that occurs days to weeks as an inhibitor of proteases released from white blood cells
after transplantation as the result of cellular mechanisms and anti- (WBCs).
body formation. Alternative pathway: A means of activating complement proteins
Acute rheumatic fever: A disease that develops as a sequel to group without an antigen–antibody combination. This pathway is trig-
A streptococcal pharyngitis, characterized by the presence of anti- gered by constituents of microorganisms.
bodies that cross-react with heart tissue. Amplicon: A copy of a select portion of DNA that is obtained by the
Adaptive immunity: A type of resistance that is characterized by polymerase chain reaction (PCR).
specificity for each individual pathogen, or microbial agent, and Amplification: Copying of nucleic acids to increase the amount
the ability to remember a prior exposure, which results in an in- available for testing.
creased response to that pathogen upon repeated exposure. Analyte: The substance being measured in an immunoassay.
Adaptive T regulatory 1 (Tr1) cells: CD4+ T cells induced from Analytic sensitivity: The lowest measureable amount of an analyte.
antigen-activated naïve T cells under the influence of interleukin-10. Analytic specificity: An assay’s ability to generate a negative result
They exert suppressive activities. when the analyte is not present.
Adjuvant: A substance administered with an immunogen that enhances Anaphylatoxin: A small peptide formed during complement acti-
and potentiates the immune response. vation that causes increased vascular permeability, contraction
Adoptive immunity: Immunity resulting from the transfer of cells of smooth muscle, and release of histamine from basophils and
of the immune system (usually lymphocytes) from an immunized mast cells.
host to a nonimmune individual. Anaphylaxis: A life-threatening response to an allergen characterized
Adoptive immunotherapy: Administration of immune cells to treat by the systemic release of histamine.
patients with conditions such as immunodeficiency diseases or Anergy: A state of immune unresponsiveness to a specific antigen.
cancer. Antagonism: When the action of one cytokine counteracts the ac-
Affinity: The initial force of attraction that exists between a Fab site tivity of another cytokine.
on an antibody and one epitope or a determinant site on the corre- Antibodies: Glycoproteins produced by B lymphocytes and plasma
sponding antigen. cells in response to foreign substance exposure. Antibodies are also
Agammaglobulinemias: Immunodeficiency diseases in which anti- known as immunoglobulins.
body levels in the blood are significantly decreased. Antibody array: A multiplex assay that uses antibody-coated beads
Agglutination: The process by which particulate antigens such as to identify target antigens, such as tumor antigens.
cells aggregate to form large complexes when a specific antibody Antibody-dependent cell cytotoxicity (ADCC): The process of
is present. destroying antibody-coated target cells by natural killer cells,
Agglutination inhibition: An agglutination reaction based on com- monocytes, macrophages, and neutrophils, all of which have spe-
petition between antigen-coated particles and soluble patient anti- cific receptors for an antibody.
gens for a limited number of antibody-combining sites. Lack of Antibody–drug conjugates: Antibody that is attached to toxins or
agglutination is a positive test result. radioisotopes to help specifically destroy cancer cells.
Agglutinin: An antibody that causes clumping or agglutination of Anticentromere antibodies: Autoantibodies that bind to proteins
the cells that triggered its formation. in the centromere (the middle region of a chromosome where
Allele: An alternate form of a gene that codes for a slightly different the sister chromatids are joined); associated with the CREST
form of the same product. syndrome.
Anticyclic citrullinated peptide (anti-CCP or ACPA): Autoantibod- ASO titer: A test for the diagnosis of poststreptococcal sequelae,
ies to proteins that contain an atypical amino acid called citrulline based on the neutralization of streptolysin 0 by antistreptolysin 0
(a modified arginine), produced by granulocytes and monocytes. found in patient serum.
This antibody is highly specific for rheumatoid arthritis. Aspergillosis: An opportunistic fungal infection predominantly
Anti-DNase B: An antibody directed against DNase B, which is caused by Aspergillus fumigatus.
secreted by group A streptococci. Ataxia-telangiectasia (AT): An autosomal recessive syndrome that
Antigen: Macromolecule that is capable of eliciting formation of results in a combined defect of both cellular and humoral immu-
immunoglobulins (antibodies) or sensitized cells in an immuno- nity. The defect is in a gene responsible for recombination of
competent host. immunoglobulin superfamily genes.
Antigen-dependent phase: The final phase of B-cell development, Atopy: An inherited tendency to respond to naturally occurring
which occurs when a B cell is stimulated by an antigen and un- allergens; it results in the continual production of IgE.
dergoes transformation to a blast stage, resulting in the formation Attenuation: A process of producing nonpathogenic bacteria or
of memory cells and antibody-secreting plasma cells. viruses for use in vaccines. These organisms have been weakened
Antigen-independent phase: The first phase of B-cell development by treatment with a chemical, exposure to elevated or cold tem-
in the bone marrow, which results in mature B cells that have not peratures, or repeated in vitro passage in cell culture.
yet been exposed to antigen. Autoantibody: An antibody produced against an antigen found on
Antigen presentation: The process by which degraded peptides an individual’s own cells, tissues, or organs.
within cells are transported to the plasma membrane with MHC Autoantigen: An antigen that belongs to the host and is not capable
molecules so T cells can then recognize them. of eliciting an immune response under normal circumstances.
Antigen switching: A protecting mechanism used by parasites that Autocrine: Effect produced by a cell that stimulates the same cell
involves varying synthesis of surface antigens to evade an immune to grow.
response by the host. Autograft: Tissues removed from one area of an individual’s body
Antigenic concealment: Means by which parasites conceal their and reintroduced in another area of the same individual.
antigens from the host by remaining inside of the host’s cells with- Autoimmune disease: A condition in which damage to body organs
out their antigens being displayed. results from the presence of autoantibodies or autoreactive cells.
Antigenic mimicry: A mechanism by which parasites express epi- Autoimmune hemolytic anemia: An autoimmune disorder in which
topes that are similar or identical to host molecules, thus protect- patients form antibodies that destroy their own red blood cells (RBCs).
ing the parasite from being recognized and eliminated by the Autoimmune hepatitis (AIH): Autoimmune disease in which patients
immune system. produce antibodies that damage the liver; formerly known as chronic
Antigenic shedding: A mechanism by which parasites can evade active hepatitis.
the immune system by shedding surface antigens that bind to Autoimmune liver disease: An autoimmune disease that mainly
the host’s antibodies and cells. affects the liver, including autoimmune hepatitis (AIH), primary
Antigenic variation: Result of the process of antigen switching. biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).
Anti-HBe: Antibody to a hepatitis B capsid antigen. Autoimmune thyroid disease (AITD): An autoimmune disease that
Anti-HBs: Antibody to hepatitis B surface antigen. affects the function of the thyroid gland, caused by the formation
Antihistone antibodies: Autoantibodies to histones, which are nu- of antibodies or sensitized cells; includes Hashimoto’s disease and
cleoproteins found in chromatin. Elevated levels are associated Graves disease.
with drug-induced lupus. Automatic sampling: Automatic pipetting of a sample that is pro-
Antineutrophil cytoplasmic antibody (ANCA): Autoantibodies pro- grammed into an instrument for testing of that sample.
duced against proteins in the neutrophil granules; these antibodies Avidity: The strength with which a multivalent antibody binds a
are strongly associated with vascular inflammatory syndromes. multivalent antigen.
Antinuclear antibody (ANA): Antibody produced to different com- Basophil: A type of white blood cell (WBC) found in peripheral
ponents of the nucleus during the course of several autoimmune blood, containing granules that release substances that are involved
diseases. Examples include anti-DNA, antideoxyribonucleopro- in allergic reactions.
tein, and antiribonuclear protein antibodies, all of which occur in Batch analyzer: An instrument that permits analysis of several dif-
systemic lupus erythematosus. ferent samples at the same time.
Antiphospholipid antibodies: A heterogeneous group of auto- Bence Jones proteins: Monoclonal immunoglobulin light chains
antibodies that bind to phospholipids or phospholipid-protein found in the urine of patients with multiple myeloma.
complexes. Benign: Tissue that is not malignant.
Antiretroviral therapy (ART): Therapeutic drugs that suppress the Biohazardous material: Patient specimens that may contain poten-
replication of a retrovirus such as HIV. tially harmful infectious agents.
Anti-RNP antibody: Autoantibodies directed against ribonucleopro- Biomarker profiling: The use of proteomic methods to identify
tein (RNP), which consists of several nonhistone proteins com- unique patterns of proteins that are associated with a disease, such
plexed to a small nuclear RNA called U1-nRNP. These antibodies as a specific type of cancer.
are found in some patients with autoimmune rheumatic diseases. Blowout pipette: A type of pipette in which the last drop of liquid
Antitoxin: Antibody used in passive immunization for the purpose must be forced out using a pipetting bulb or other device to deliver
of neutralizing a bacterial toxin. an accurate volume.
Apoptosis: Programmed cell death. Bone marrow: The largest tissue in the body, located in the long
Arthus reaction: A type III hypersensitivity skin reaction that occurs bones. Its role is the generation of hematopoietic cells.
when an animal has a large amount of circulating antibody and is Borrelia burgdorferi: A spirochete bacterium that is the causative
exposed to the antigen intradermally, resulting in localized depo- agent of Lyme disease.
sition of immune complexes. Borrelia miyamotoi: A spirochete bacterium that causes relapsing fever.
Branched DNA (bDNA): A technique used to detect a small amount Chemiluminescence: The production of light energy by a chemical
of DNA via several hybridization steps that create a branching reaction.
effect with several nucleic acid probes. Chemiluminescent immunoassay: A technique that employs a
Bruton’s tyrosine kinase (Btk) deficiency: An X-linked recessive chemical attached to either an antigen or antibody. Light is emitted
immunodeficiency disease that results in a lack of mature B lym- because of a chemical reaction and indicates an antigen–antibody
phocytes and immunoglobulins of all classes. combination has taken place.
Bystander lysis: A phenomenon that occurs in complement activa- Chemokines: A large family of homologous cytokines that promote
tion when C3b becomes deposited on host cells, making them a migration of white blood cells through chemotaxis.
target for destruction by phagocytic cells. Chemotaxin: A protein or other substance that acts as a chemical
C1 inhibitor (C1-INH): A glycoprotein that acts to dissociate Clr messenger to produce chemotaxis.
and C1s from C1q, thus inhibiting the first active enzyme formed Chemotaxis: The migration of cells in the direction of a chemical
in the classical complement cascade. messenger.
C3 glomerulopathies (C3G): Diseases involving the glomeruli of Chronic granulomatous disease (CGD): An immunodeficiency
the kidneys. inherited in either an X-linked or autosomal recessive fashion that
C4-binding protein (C4BP): A protein in the complement system results in an inability of the neutrophils to produce the reactive
that serves as a cofactor for factor 1 in the inactivation of C4b. forms of oxygen necessary for normal bacterial killing.
Cancer: A disease characterized by the presence of a malignant tumor. Chronic rejection: Rejection of a graft that usually occurs after the
Cancer antigen 125 (CA 125): A glycosylated protein that is used first year and results from progressive fibrosis of blood vessels in
clinically as a marker for ovarian cancer. the grafted tissue.
Cancer vaccines: Vaccines that have been developed for the purpose Class I MHC (HLA) molecules: Proteins coded for by genes at three
of preventing or treating cancer. loci (A, B, C) in the major histocompatibility complex. They are
Candidiasis: An opportunistic fungal infection caused by Candida expressed on all nucleated cells and are important to consider in
albicans and other Candida species. the transplantation of tissues.
Capture assay: An enzyme immunoassay using two antibodies: The Class II MHC (HLA) molecules: Proteins coded for by the DR, DP,
first binds the antigen to a solid phase, and the second contains and DQ loci of the major histocompatibility complex. They are
the enzyme label and acts as an indicator. found on B cells, macrophages, activated T cells, monocytes, den-
Carcinoembryonic antigen (CEA): An oncofetal protein that may dritic cells, and endothelium, and are important to consider in the
be elevated in patients with cancers of the breast, gastrointestinal transplantation of tissues.
tract, pancreas, or lung. Class switching: The production of immunoglobulins other than
Carcinogenesis: The process by which a cell is transformed into a IgM by daughter cells of antigen-exposed B lymphocytes.
malignant tumor. Classical pathway: A means of activating complement that begins
Carcinoma: Malignant tumor derived from the skin or epithelial with antigen–antibody combination.
linings of internal organs or glands. Clinical and Laboratory Standards Institute (CLSI): An organiza-
Cascade induction: When a cytokine secreted by a specific type of tion that develops clinical laboratory testing standards based on
cell activates target cells to produce additional cytokines. input from industry, government, and health-care professionals.
CD4 T cell: Type of lymphocyte that provides help to B cells to ini- Clinical Laboratory Improvement Amendments (CLIA): Federal
tiate antibody production. regulatory standards that apply to all clinical laboratory testing in
CD45: A leukocyte marker present on all white blood cells; used to the United States.
identify WBC populations in flow cytometry analyses. Clonal deletion: The process of elimination of clones of lymphocytes
Celiac disease: An autoimmune disease affecting the small intestine that would be capable of an autoimmune response.
and other organs. Clonal selection theory: A theory postulated to explain the
Cell flow cytometry: An automated system for identifying cells specificity of antibody formation, based on the premise that
based on the scattering of light as cells flow single file through a each lymphocyte is genetically programmed to produce a
laser beam. specific type of antibody and is selected by contact with an
Cell-mediated immunity: A type of immunity in which T cells pro- antigen.
duce cytokines that help to regulate both the innate and adaptive Clusters of differentiation (CD): Antigenic features of leukocytes
immune response. that are identified by groups of monoclonal antibodies expressing
Central tolerance: Destruction of potentially self-reactive T and common or overlapping activity.
B cells as they mature in either the thymus or the bone marrow. Coccidioidomycosis: A fungal disease caused by Coccidioides immitis
Ceruloplasmin: An acute-phase reactant that acts as the principal that is endemic to the southwestern United States and may be
copper-transporting protein in human plasma. characterized by primary pulmonary infection.
Chain of infection: A continuous link between three elements—a Coefficient of variation (CV): The average distance each data point
source, a method of transmission, and a susceptible host. in a normal distribution is from the mean. It is expressed as a
Chain termination sequencing: A modification of the DNA repli- percentage of the mean.
cation process, which utilizes modified nucleotide bases called Cold agglutinins: Antibodies that react below 30°C, typically formed
dideoxynucleotide triphosphates (ddNTP). When these are incor- in response to diseases such as Mycoplasma pneumonia and certain
porated into the growing DNA chain, synthesis stops. viral infections.
Chancre: The initial lesion that develops on the external genitalia in Colony stimulating factor (CSF): A protein in human serum that
syphilis. promotes monocyte differentiation.
Chemical Hygiene Plan: A plan that identifies appropriate work Combination antiretroviral therapy (CART): A combination of
practices, standard operating procedures, and safety considerations antiretroviral drugs used to treat individuals with HIV infection
in regard to the use of chemicals in the laboratory. (see also highly active antiretroviral therapy).
Commensalistic: A relationship between two species of organisms Cytomegalovirus (CMV): A virus in the herpes family that is respon-
in which there is no benefit or harm to either organism. sible for infection, ranging from a mononucleosislike syndrome to
Common variable immunodeficiency (CVI): A heterogeneous a life-threatening illness in immunocompromised patients.
group of immunodeficiency disorders that usually appears in Cytotoxic T cell: T cells that bear the CD8 marker. They kill virus-
patients between the ages of 20 and 30 years. It is characterized infected cells and tumor cells by triggering apoptosis.
by a deficiency of one or more classes of immunoglobulins. Decay-accelerating factor (DAF): A glycoprotein found on peripheral
Competitive immunoassay: An immunoassay in which unlabeled red blood cells (RBCs), endothelial cells, fibroblasts, and epithelial
and labeled antigen compete for a limited number of binding sites cell surfaces that is capable of dissociating C3 convertases formed
on reagent antibody. by both the classical and alternative pathways of complement.
Complement: A series of proteins which are normally present in Delayed hypersensitivity: Type IV or T-cell–mediated hypersensi-
serum and whose overall functions are mediation of inflammation tivity; so named because its manifestations are not seen until
and destruction of foreign cells. 24 to 48 hours after exposure to the inducing antigen.
Complement-dependent cytotoxicity (CDC): Killing of cells that Delta check: A QA procedure that compares a patient’s test results
results from attachment of antibody with activation of complement. with the previous results.
Complement receptor type 1 (CR1): A cell-bound regulator of Denaturation: Treating double-stranded DNA to separate it into
complement activation. It assists in degrading C3b and C4b and single strands.
mediates transport of C3b-coated immune complexes to the liver Dendritic cell: Tissue cells covered with long membranous exten-
and spleen. sions that make them resemble nerve cell dendrites.
Complementary or copy DNA (cDNA): DNA made from RNA Deoxyribonucleic acid (DNA): The nucleic acid whose sugar is
using the enzyme reverse transcriptase. deoxyribose. It is the primary genetic material of all cellular
Conformational epitope: Key antigenic site that results from the fold- organisms and DNA viruses.
ing of one chain or multiple chains, bringing certain amino acids Diapedesis: The process by which cells are capable of moving from
from different segments of a linear sequence or sequences into close the circulating blood to the tissues by squeezing through the wall
proximity with each other so they can be recognized together. of a blood vessel.
Congenital syphilis: The transfer of syphilis from an infected mother DiGeorge anomaly: A congenital defect of the third and fourth
to the fetus during pregnancy. pharyngeal pouches that affects thymic development, leading to a
Conidia: Asexual reproductive structures produced by fungi at the T-cell deficiency.
tip of hyphae; also known as spores. Diluent: One of two entities needed for making up a solution. It is
Constant region: The carboxy-terminal segment of antibody mole- the medium into which the solute is added.
cules (half of immunoglobulin light chains or three-quarters of Direct agglutination: An antigen–antibody reaction that occurs
heavy chains) that consists of a polypeptide sequence found in all when antigens are naturally found on a particle.
chains of that type. Direct allorecognition: Pathway in which recipient T cells recognize
Contact dermatitis: A delayed hypersensitivity reaction caused by intact HLA molecules on donor cells.
T-cell sensitization to low molecular weight compounds, such as Direct antiglobulin test (DAT): A technique to determine in vivo
nickel and rubber, that come in contact with the skin. attachment of antibody or complement to red blood cells
Control mean: The average of all data points. (RBCs), using anti-human globulin to cause a visible agglutina-
C-reactive protein (CRP): A trace constituent of serum that in- tion reaction.
creases rapidly following infection or trauma to the body and acts Direct immunofluorescent assay: A technique to identify a specific
as an opsonin to enhance phagocytosis. antigen using an antibody that has a fluorescent tag attached.
CREST syndrome: A subset of scleroderma named after its five Domain: Region of an antibody molecule that consists of approxi-
major features: calcinosis, Raynaud’s phenomenon, esophageal mately 110 amino acids.
dysmotility, sclerodactyly, and telangiectasia. Double-negative (DN) thymocyte: Stage in the development of
Crossmatch: Incubation of donor lymphocytes with recipient serum T cells when neither CD4 nor CD8 is expressed.
to determine the presence of antibodies, which would indicate Double-positive (DP) thymocyte: Stage in the development of
rejection of a potential transplant. T cells when both CD4 and CD8 antigens are expressed.
Cross-reactivity: A phenomenon that occurs when an antibody Double-stranded DNA (dsDNA) antibodies: Autoantibodies pro-
reacts with an antigen that is structurally similar to the original duced against double-stranded DNA; they are diagnostic for SLE.
antigen that induced antibody production. Dual-parameter dot plot: Grouping of cells based on two different
Cryoglobulins: Immunoglobulins of the IgM class that precipitate characteristics, one of which is plotted on the x-axis and the other
at cold temperatures, causing occlusion of blood vessels in the on the y-axis.
extremities if a patient is exposed to the cold. Ectoparasite: Multi-celled parasitic organism that lives on the skin
Cryptococcosis: A fungal disease caused by Cryptococcus neoformans of the host.
and characterized as a pulmonary infection that may spread to the Electropherogram: Sequencing result from capillary gel electrophore-
central nervous system and the brain. sis that appears as a series of fluorescent peaks.
C-type lectin receptors (CLRs): Plasma membrane receptors found Electrophoresis: The separation of molecules in an electrical field
on white blood cells (WBCs) that bind to mannan and β-glucans based on differences in charge and size.
in fungal cell walls. ELISA: See enzyme-linked immunosorbent assay.
Cyst: Inactive form of a parasite that can transmit infection. Endocrine: Internal secretion of substances such as hormones or
Cytogenetics: The branch of genetics devoted to the study of cytokines directly into the bloodstream that causes systemic
chromosomes. effects.
Cytokine: Chemical messenger produced by stimulated cells that Endogenous pyrogen: A substance produced by the body that
affects the function or activity of other cells. causes fever. Interleukin-1 is an example.
Endotoxin: A component of the cell walls of gram-negative bacteria, Flow cytometry: See cell flow cytometry.
which consists of the portion of lipopolysaccharide (LPS) called Fluorescence in situ hybridization (FISH): A technique used to
lipid A. Endotoxin is a potent stimulator of cytokine release that identify a specific region of DNA in a chromosome through bind-
can lead to septic shock. ing of fluorescent-tagged complementary DNA probes.
End-point method: A radial immunodiffusion technique in which anti- Fluorescence polarization immunoassay (FPIA): An immunoassay
gen is allowed to diffuse out until the point of equivalence is reached. based on the change in polarization of fluorescent light emitted
Enzyme-linked immunosorbent assay (ELISA): An immunoassay from a labeled molecule when it is bound by antibody.
that employs an enzyme label on one of the reactants. Fluorescent antinuclear antibody (FANA) testing: Testing to iden-
Eosinophil: A white blood cell (WBC) that contains reddish-orange tify the presence of antibody to nuclear antigens, using animal cells
granules and is involved in allergic reactions. and a fluorescent-labeled anti-human immunoglobulin.
Epigenetics: The study of modifications in gene expression that are Fluorescent treponemal antibody absorption (FTA-ABS) test:
not caused by changes in the nucleotide sequence of DNA. Fluorescent treponemal antibody absorption test, a confirmatory
Epitope: The key portion of the immunogen against which the test for syphilis, which detects antibodies to Treponema pallidum
immune response is directed; also known as the determinant site. by using anti-human immunoglobulin with a fluorescent label.
Epitope spreading: An expansion of an immune response to un- Fluorochrome: A molecule that absorbs light across a spectrum of
related antigens, which may be involved in the development of wavelengths and emits light of lower energy across a spectrum of
autoimmunity. longer wavelengths.
Epstein-Barr virus (EBV): A DNA virus of the herpesvirus family. Forward-angle light scatter (FSC): Light scattered at an angle of
Erythropoietin (EPO): A colony stimulating factor that increases red less than 90 degrees, which indicates the size of a cell.
blood cell (RBC) production in the bone marrow. Fungi: Organisms made up of eukaryotic cells with rigid walls com-
Examination variable: A variable that includes reagent and test posed of chitin, mannan, and sometimes cellulose.
performance, instrument calibration and maintenance, personnel Gate: A set of filters placed around a population of interest to analyze
requirements, and technical competence. various parameters (extrinsic and intrinsic) of the cells contained
Exotoxin: Potent protein released from living bacteria (mostly within the selected region.
gram-positive) that causes harm to the host by binding to a spe- Gel electrophoresis: Method of separating either proteins or DNA
cific cellular receptor. based on their size and electrical charge. Samples are placed in
External defense system: Structural barriers that prevent most wells on the gel and exposed to an electrical current.
infectious agents from entering the body. Genetic code: A set of 3 nucleotides that code for one amino acid.
External quality assessment (EQA): The testing of unknown Germinal center: The interior of a secondary follicle where blast
samples received from an outside agency. transformation of B cells takes place.
Extractable nuclear antigen (ENA): A member of a family of small Goodpasture’s syndrome: An autoimmune disease characterized by
nuclear proteins associated with uridine-rich RNA that can stim- the presence of an autoantibody to collagen in the glomerular (kid-
ulate the production of autoantibodies; examples are Sm and RNP. ney) or alveolar (lung) basement membranes.
Extrinsic parameter: A parameter that is not an inherent part of Graduated pipette: A pipette that has markings all along its length
the cell. These specific cell surface proteins are analyzed through to allow for varying amounts of liquid to be measured.
attachment of fluorescent antibodies. Graft-versus-host disease (GVHD): A condition that results from
F(ab’)2: Fragment of an immunoglobulin molecule obtained by transplantation of immunocompetent cells into an immunodefi-
pepsin cleavage that consists of two light chains and two heavy- cient host. The transfused cells attack the tissues of the recipient
chain halves held together by disulfide bonding. This piece has within the first 100 days posttransplant.
two antigen-binding sites. Granulocyte colony stimulating factor (G-CSF): A cytokine pro-
Fab fragment: Fragment of an immunoglobulin molecule obtained duced by fibroblasts and epithelial cells that enhances the produc-
by papain cleavage that consists of a light chain and one-half of a tion of neutrophils.
heavy chain held together by disulfide bonding. Granulocyte macrophage colony stimulating factor (GM-CSF): A
Factor H: A control protein in the complement system. It acts as a cytokine produced by T cells and other cell lines that stimulates
cofactor with factor I to break down C3b formed during com- an increased supply of granulocytic cells and macrophages.
plement activation. Granuloma: An organized cluster of inflammatory cells formed in
Factor I: A serine protease that cleaves C3b and C4b formed during some type IV hypersensitivity responses.
complement activation. A different cofactor is required for each of Granulomatosis with polyangiitis (PGA): An autoimmune disease
these reactions. involving inflammation of the small- to medium-sized blood ves-
Fc fragment: Fragment of an immunoglobulin molecule obtained by sels and the production of ANCA.
papain cleavage that consists of the carboxy-terminal halves of two Graves disease: An autoimmune disease characterized by hyperthy-
heavy chains. These two halves are held together by disulfide roidism caused by the presence of antibody to thyroid-stimulating
bonds. This fragment spontaneously crystallizes at 4ºC. hormone receptors. Antigen–antibody combination results in con-
Fibrinogen: An acute-phase reactant that changes to fibrin and forms tinual release of thyroid hormones.
clots in the bloodstream. Group A streptococci: Gram-positive, catalase-negative cocci often
Flanking: Placement on either side of the region of the template DNA found in pairs or chains that are responsible for diseases ranging
to be copied. from pharyngitis to necrotizing fasciitis.
Flocculation: The formation of downy masses of precipitate that Gummas: Localized areas of granulomatous inflammation on bones,
occurs over a narrow range of antigen concentration. skin, and subcutaneous tissue caused by tertiary syphilis.
Flow cytometer: An automated system in which single cells in a fluid Hairy cell leukemia: Chronic leukemia characterized by the forma-
are analyzed in terms of intrinsic light-scattering characteristics as tion of large mononuclear cells with irregular cytoplasmic projec-
well as extrinsic properties. tions found in bone marrow.
Haplotype: A set of genes that are located close together on a chro- mucosal surfaces as a result of a deficiency in the complement
mosome and are usually inherited as a single unit. inhibitor C1NH.
Hapten: A simple chemical group that can bind to antibody once it Heteroantigen: An antigen of a species different from that of the host,
is formed but that cannot stimulate antibody formation unless tied such as other animals, plants, or microorganisms.
to a larger carrier molecule. Heterogeneous enzyme immunoassay: Immunoassay in which
Haptoglobin: An acute-phase reactant that binds irreversibly to free enzyme is used as a label and which requires a separation step to
hemoglobin released by intravascular hemolysis. separate free from bound analyte.
Hashimoto’s thyroiditis: An autoimmune disease that results in Heterophile antibody: Antibody that is capable of reacting with
hypothyroidism caused by the presence of antithyroglobulin and similar antigens from two or more unrelated species; commonly
antimicrosomal antibodies, which progressively destroy the found in patients with infectious mononucleosis.
thyroid gland. Heterophile antigen: An antigen that exists in unrelated plants or
Heavy (H) chain: One of the polypeptide units that makes up an animals but is either identical or closely related, so that antibody
immunoglobulin molecule. Each immunoglobulin monomer con- to one will cross-react with antibody to the other.
sists of two heavy chains paired with two light chains. High-dose hook effect: Limitation of antibody-based assays caused
Heavy-chain diseases: B-cell lymphomas characterized by the pro- by massive amounts of tumor marker antigens present.
duction of monoclonal immunoglobulin heavy chains that are not Highly active antiretroviral therapy (HAART): A multidrug reg-
attached to light chains. imen that is the standard of treatment for HIV infection (see also
Helicobacter pylori: A gram-negative spiral bacterium that is a major combination antiretroviral therapy).
cause of gastric and duodenal ulcers. Hinge region: The flexible portion of the heavy chain of an im-
Helminth: Parasitic worms, including flatworms, tapeworms, and munoglobulin molecule that is located between the first and
roundworms. second constant regions. This allows the molecule to bend to let
Hemagglutination: An antigen–antibody reaction that results in the the two antigen-binding sites operate independently.
clumping of red blood cells (RBCs). Histamine: A vasoactive amine released from mast cells and basophils
Hemagglutination inhibition: A test for detecting antibodies to cer- during an allergic reaction.
tain viruses, based on lack of agglutination as a result of antibody Histoplasmosis: An infection caused by the fungus H capsulatum.
neutralizing the virus. HLA antibody screen: Detection of HLA antigens in candidates and
Hematopoietic growth factors: Cytokines or other factors in the recipients of solid-organ transplants by performance of a cross-
blood that stimulate formation and differentiation of blood cells. match test.
Hemolytic disease of the newborn (HDN): A cytotoxic reaction HLA genotype: Actual alleles, for HLA antigens, that are inherited.
that destroys an infant’s red blood cells (RBCs) because of placental HLA matching: The pairing up of donor and recipient in a transplant
transfer of maternal antibodies to Rh antigens. on the basis of similar HLA antigens.
Hemolytic titration (CH50) assay: An assay that measures complement- HLA phenotype: The expression of HLA genes that actually appear
activating ability by determining the amount of patient serum as proteins on cells.
required to lyse 50% of a standardized concentration of antibody- HLA typing: Laboratory testing used to identify the HLA antigens or
sensitized sheep erythrocytes. genes in a transplant candidate or donor.
Hemolytic uremic syndrome (HUS): A condition characterized by Hodgkin lymphoma (HL): A malignant disease that typically begins
hemolytic anemia, low platelet count, and acute renal failure in one lymph node and is characterized by the presence of Reed-
caused by either a Shiga toxin related to an infection or comple- Sternberg cells, giant multinucleated cells that are usually trans-
ment dysregulation. formed B lymphocytes.
Hepatitis: Inflammation of the liver that can be caused by several Homogeneous enzyme immunoassay: An immunoassay in which
viruses as well as noninfectious factors, such as radiation, exposure no separation step is necessary. It is based on the principle of a
to chemicals, or autoimmune diseases. decrease in enzyme activity when specific antigen– antibody com-
Hepatitis A virus (HAV): An RNA virus that can cause hepatitis; bination occurs.
transmitted by the fecal–oral route, close person-to-person con- Human chorionic gonadotropin (hCG): A hormone that is synthe-
tact, or ingestion of contaminated food or water. sized by trophoblasts and used as a marker for tumors of the
Hepatitis B surface antigen (HBsAg): The surface antigen of hepa- ovaries, testes, and trophoblast cells.
titis B virus, the first marker to appear in hepatitis B infection. Human epithelial growth factor receptor 2 (HER2): A transmem-
Hepatitis B virus (HBV): A DNA virus that can cause hepatitis; it brane receptor that binds human epidermal growth factor; is
is transmitted by a parenteral route, through sexual contact, or overexpressed in a certain type of breast cancer.
acquired perinatally. Human immune serum globulin (HISG): A sterile preparation of
Hepatitis Be antigen (HBeAg): Antigen associated with the capsid concentrated antibodies made from pooled serum of thousands of
of hepatitis B virus. donors; used as a prophylactic treatment in patients with antibody
Hepatitis C virus (HCV): An RNA virus that can cause hepatitis; it immunodeficiencies.
is transmitted sexually or through contaminated blood or needles. Human immunodeficiency virus (HIV): A retrovirus that is the
Hepatitis D virus (HDV): An RNA virus that can cause hepatitis; it etiologic agent of AIDS.
requires the presence of HBV for its replication and expression. Human leukocyte antigen (HLA): Protein coded for by the human
Hepatitis E virus (HEV): An RNA virus that can cause hepatitis; it MHC genes that has essential roles in the immune response and
is transmitted by the fecal–oral route or ingestion of contaminated the rejection of foreign transplants.
food or water. Human T-cell lymphotropic virus type I (HTLV-I): An RNA
Hereditary angioedema (HAE): A disease characterized by swelling retrovirus that causes adult T-cell leukemia or lymphoma and
of the extremities, the skin, the gastrointestinal tract, and other HTLV-associated myelopathy/tropical spastic paraparesis.
Human T-cell lymphotropic virus type II (HTLV-II): An RNA retro- Immunohistochemistry: The use of labeled antibodies to directly
virus that is thought to be associated with neurological disease, detect tumor markers in tissue.
certain hematologic and dermatological diseases, and an increased Immunologic diversion: A mechanism by which parasites enhance
incidence of infections. their survival in the host by inducing the production of proteins
Humoral immunity: Protection from disease resulting from sub- that divert the attention of the immune system.
stances in the serum (e.g., antibodies). Immunologic subversion: A mechanism by which parasites can
Hybridization: Specific binding of two single-stranded DNA segments, avoid the effector mechanisms of the immune response by pro-
as in binding of a probe with a known nucleic acid sequence to an ducing proteins that act as homologues of various components of
unknown piece of DNA. the immune system.
Hybridoma: A cell line resulting from the fusion of a myeloma cell Immunologic tolerance: A state of immune unresponsiveness
and a plasma cell. These can be maintained in tissue culture in- directed against a specific antigen.
definitely and can produce a very specific type of antibody known Immunology: The study of the reactions of a host when foreign
as a monoclonal antibody. substances are introduced into the body.
Hyperacute rejection: Rejection of tissue that occurs within minutes Immunophenotyping: Identifying cells according to their surface
or hours following transplantation, because of antibodies to ABO antigen expression.
and HLA antigens which are already present in the graft recipient. Immunoprophylaxis: The use of immunization to prevent disease.
Hypercytokinemia: Dysregulation of cytokines, producing hyper- Immunosubtraction (immunotyping): A procedure that uses cap-
stimulation of the immune response. illary electrophoresis to identify monoclonal immunoglobulin
Hypersensitivity: A heightened state of immune responsiveness. components.
Hyphae: Filamentous tubular branching structures characteristic of Immunosuppressive agent: An agent used to suppress an antigraft
some fungi. immune response to transplanted tissue.
Idiotype: The variable portion of light and heavy immunoglobulin Immunosurveillance: The mechanisms by which the immune
chains that is unique to a particular immunoglobulin molecule. system patrols the body for the presence of cancerous or pre-
This region constitutes the antigen-binding site. cancerous cells and eliminates them before they become clini-
IgM anti-HBc: Antibody that is the first to appear in hepatitis B in- cally evident.
fection. It is of the IgM class and is directed against core antigen Immunotherapy: Treatment which uses the ability of the immune
on the virus particle. system to destroy tumor cells.
Immature B cell: A phase in the growth of B cells characterized by the Immunotoxins: Antibodies conjugated to toxins to help destroy
appearance of complete IgM antibody molecules on the cell surface. cancer cells.
Immediate hypersensitivity: Reaction to an allergen that occurs in Impetigo: A skin infection caused by bacteria such as Group A
minutes and can be life-threatening. streptococci.
Immune adherence: The ability of phagocytic cells to bind complement- Indigenous microbiota: Symbiotic microorganisms that reside on
coated particles. and colonize the surfaces of an individual, also known as “normal
Immunity: The condition of being resistant to infection. flora.”
Immunization: The process by which immunity is acquired. Indirect allorecognition: Pathway by which the immune system
Immunoblotting: A technique used to identify antibodies to complex recognizes foreign HLA proteins on a donor graft; involves the
antigens. It consists of electrophoresis of the antigen mix followed uptake, processing, and presentation of foreign HLA proteins by
by transfer of the pattern to nitrocellulose paper for reaction with the recipient’s APCs to recipient T cells to produce antibodies and
patient serum. cell-mediated responses against the graft.
Immunochromatography: A rapid technique in which the analyte Indirect immunofluorescent assay: A technique to identify anti-
is applied at one end of a strip and migrates toward the distal end, gen by using two antibodies: one that is specific to the antigen
where the results can be visualized in minutes. and a second that is an anti-human immunoglobulin with a flu-
Immunodeficiencies: Inherited or acquired disorders in which a part orescent tag.
of the body’s immune system is missing or dysfunctional. Infection control: Procedures used to control and monitor infections
Immunoediting: The ability of tumor cells to escape immune sur- occurring within health-care facilities.
veillance through suppression of immunogenicity. Infectivity: An organism’s ability to establish an infection; the pro-
Immunofixation electrophoresis (IFE): A semiquantitative gel pre- portion of individuals exposed to a pathogen through horizontal
cipitation technique in which proteins are first separated by elec- transmission (i.e., person-to-person contact) who will become
trophoresis, then incubated with antibodies to individual proteins infected.
that are added directly to the gel surface; used commonly to identify Inflammasome: A protein oligomer that contains caspase enzymes
monoclonal immunoglobulins. and other proteins associated with apoptosis; may be defective in
Immunofluorescent assay (IFA): Identification of antigens on cells some autoinflammatory disorders.
using an antibody with a fluorescent tag. Inflammation: Cellular and humoral mechanisms involved in the
Immunogen: Any substance that is capable of inducing an immune overall reaction of the body to injury or invasion by an infectious
response. agent.
Immunogenetics: The analysis of gene mutations and polymor- Innate (natural) immunity: The ability of the individual to resist
phisms that affect immune function. infection by means of normally present body functions.
Immunogenicity: The ability of an immunogen to stimulate a host Integrins: Molecules on certain leukocytes that cause adhesion to
response. endothelial cells.
Immunoglobulin (Ig): Glycoproteins in the serum portion of the Interferons (IFN): Cytokines produced by T cells and other cell lines
blood that are considered part of humoral immunity. that inhibit viral synthesis or act as immune regulators.
Interleukins (IL): Cytokines or chemical messengers produced by Lymph node: A secondary lymphoid organ that is located along
leukocytes that affect the inflammatory process through an increase a lymphatic duct and whose purpose is to filter lymphatic
in soluble factors or cells. fluid from the tissues and act as a site for processing of foreign
Internal amplification control: A control used in PCR that is a gene antigen.
target always present at a constant level. Lymphocyte: The key white blood cell (WBC) involved in the adap-
Internal defense system: Defense mechanism inside the body in tive immune response.
which both cells and soluble factors play essential parts. Lymphoma: Cancer of the lymphoid cells that tends to proliferate as
Intrinsic parameter: Light-scattering properties that are a part of the a solid tumor.
cell, such as size and granularity. Macrophage: A white blood cell (WBC) that kills microbes and pre-
Invariant chain: A protein that associates with HLA class II antigens sents antigen to T and B cells.
shortly after they are synthesized to prevent interaction of their Macrophage colony stimulating factor (M-CSF): A cytokine that
binding sites with any endogenous peptides in the endoplasmic induces growth of hematopoietic cells destined to become mono-
reticulum. cytes and macrophages.
Isograft (syngeneic graft): Graft that involves the transfer of tissue Major histocompatibility complex (MHC): The genes that control
between two genetically identical members of the same species. expression of a large group of proteins originally identified on
Isohemagglutinin: Antibody that agglutinates red blood cells (RBCs) leukocytes but now known to be found on all nucleated cells in
of other individuals of the same species. the body. These proteins regulate the immune response and play
Isothermal: An amplification process using a reaction that proceeds a role in graft rejection.
at a single temperature. Malignant: A descriptive term for cancerous tumors that can circu-
Isotype: A unique amino acid sequence that is common to all im- late to other parts of the body and invade nearby organs.
munoglobulin molecules of a given class in a given species. Mannose-binding lectin (MBL): Normally present protein in the
Joining (J) chain: A glycoprotein with a molecular weight of 15,000 blood that binds to mannose on bacterial cells and initiates the
that serves to link immunoglobulin monomers together. These are lectin pathway for complement activation.
found only in IgM and secretory IgA molecules. Mast cell: A tissue cell that plays a role in allergic reactions and also
Joint Commission (JC): An independent body that certifies and ac- functions as an antigen-presenting cell.
credits health-care organizations in the United States, Membrane attack complex (MAC): The combination of comple-
Kappa (κ) chain: One of two types of immunoglobulin light chains ment components C5b, C6, C7, CS, and C9 that becomes inserted
that are present in approximately two-thirds of all immunoglobu- into the target cell membrane, causing lysis.
lin molecules. Membrane cofactor protein (MCP): A protein found on all epithelial
Karyotype analysis: A test used to examine the chromosomes in a and endothelial cells that helps to control complement-mediated
cell sample for numerical or structural abnormalities. lysis by acting as a cofactor for Factor I–mediated cleavage of C3b.
Lambda (λ) chain: One of two types of immunoglobulin light chains Memory cell: Progeny of an antigen-activated B or T cell that is able
that are present in approximately one-third of all immunoglobulin to respond to antigen more quickly than the parent cell.
molecules. Metastasis: Process of malignant cells traveling through the body,
Lancefield group: A means of classifying streptococci on the basis thereby causing new foci of malignancy.
of differences in the cell wall carbohydrate. MHC restriction: The selection of thymocytes that will only interact
Lateral flow immunochromatographic assay (LFA): Immunochro- with the MHC antigens found on host cells.
matographic assay for rapid antigen detection. Microarray: A technology that enables simultaneous analysis of
Lattice: The combination of antibody and multivalent antigen to thousands of genes in a sample by hybridization with a panel of
produce a stable network that results in a visible reaction. molecular probes that are complementary to portions of specific
Law of mass action: A law used to mathematically describe the genes or chromosome regions; the probes are spotted onto sepa-
equilibrium relationship between soluble reactants and insoluble rate locations on a small glass slide or nylon membrane.
products. It can be applied to antigen–antibody relationships. Microbiome: The collection of microorganisms that exists on the
Lean system: A system used in the laboratory that focuses on the body, including bacteria, viruses, yeast, and fungi.
elimination of waste to allow a facility to do more with less and at Micropipette: Mechanical pipettes that deliver volumes in the mi-
the same time increase customer and employee satisfaction. croliter (µL) range; used when very small volumes are needed.
Lectin pathway: A pathway for the activation of complement based Minor histocompatibility antigens (mHAs): Non-HLA proteins
on binding of mannose-binding protein to constituents on bacte- that can induce a weak graft rejection response when introduced
rial cell walls. into an individual possessing a different polymorphic variant.
Leukemia: A progressive malignant disease of blood-forming organs, Mitogen: A substance that stimulates mitosis in all T cells or all
characterized by proliferation of leukocytes and their precursors B cells, regardless of antigen specificity.
in the bone marrow. Mixed lymphocyte reaction (MLR): A means of measuring the
Leukocytes: White blood cells (WBCs). proliferation of responder CD8+ T cells to nonself antigens in a
Leukotrienes (LT): A class of secondary mediators released from potential transplant.
mast cells and basophils during type I hypersensitivity reactions. Molecular mimicry: The similarity between an infectious agent and
Light (L) chain: Small chain in an immunoglobulin molecule that a self-antigen that causes antibody formed in response to the for-
is bound to the larger chain by disulfide bonds. The two types of mer to cross-react with the latter.
light chains are called kappa and lambda. Monoclonal antibody: Very specific antibody derived from a single
Linear epitope: Amino acids following one another on a single chain antibody-producing cell that has been cloned or duplicated.
that act as a key antigenic site. Monoclonal gammopathy: A clone of lymphoid cells that causes
Lyme disease: A disease caused by infection with the spirochete overproduction of a single immunoglobulin component called a
bacteria, Borrelia burgdorferi. paraprotein.
Monoclonal gammopathy of undetermined significance (MGUS): Nucleotide: A nucleotide is a unit of DNA or RNA composed of a
A premalignant plasma cell disorder characterized by the presence phosphorylated ribose or deoxyribose sugar and a nitrogen base.
of monoclonal immunoglobulin in the serum, a plasma bone mar- Occupational Safety and Health Administration (OSHA): Moni-
row count of less than 10%, and absence of clinical features. Some tors and enforces safety regulations for workers.
cases may progress to multiple myeloma over time. Oncofetal antigens: Antigens that are expressed in the developing
Monocyte: The largest white blood cell (WBC) in peripheral blood. fetus and in rapidly dividing tissue, such as that associated with
It migrates to the tissues to become a macrophage. tumors, but that are absent in normal adult tissue.
Multiple myeloma: A malignancy of mature plasma cells that results Oncogene: Gene that encodes a protein capable of inducing cellular
in a monoclonal increase in an immunoglobulin component. The transformation.
most common component increased is IgG. Opsonins: Serum proteins that attach to a foreign substance and
Multiple sclerosis (MS): An autoimmune disease in which the enhance phagocytosis (from the Greek word meaning “to prepare
myelin sheath of axons becomes progressively destroyed by anti- for eating”).
bodies to myelin proteins. Ouchterlony double diffusion: A qualitative gel precipitation
Mumps virus: A single-stranded RNA virus that is the causative agent technique in which both antigen and antibody diffuse out from
of mumps, a disease characterized by swelling of the parotid glands. wells cut in the gel. The pattern obtained indicates whether or
Mutation: A permanent change in the nucleotide sequence within a not antigens are identical.
gene or chromosome. Oxidative burst: An increase in oxygen consumption in phago-
Mutualistic: A relationship between a human host and microbial cytic cells, which generate oxygen radicals used to kill engulfed
species in which both organisms benefit. microorganisms.
Myasthenia gravis (MG): An autoimmune disease characterized by p24 antigen: A structural core antigen that is part of the human im-
progressive muscle weakness caused by formation of antibody to munodeficiency virus (HIV).
acetylcholine receptors. Palindromic sites: Nucleotide sequences that read the same 5′ to
Mycelium: A dense mat formed by some fungi that is made up of 3′ on both strands of the DNA.
intertwined hyphae. Paracrine: Secretions such as cytokines that affect only target cells
Mycoplasma pneumoniae: A small gram-negative bacterium that in close proximity.
lacks a cell wall and is the cause of upper respiratory infections. Paraprotein (M protein): A single immunoglobulin component pro-
Mycoses: Diseases produced by fungi. duced by a malignant clone of lymphoid cells in lymphoprolifer-
Natural killer (NK) cell: A type of lymphocyte that has the ability ative diseases.
to kill target cells without prior exposure to them. Parasite: Microorganism that survives by living off of another
Negative predictive value: The probability that a person with a organism.
negative screening test does not have the disease. Parasitic: Relationship in which an organism causes harm to
Negative selection: The process by which T cells that can respond its host.
to self-antigen are destroyed in the thymus. Parenteral: Mode of transmission other than through the intestinal
Neoplasm: An abnormal cell mass; a tumor. tract, most notably bloodborne transmission.
Nephelometry: A technique for determining the concentration of par- Paroxysmal cold hemoglobinuria: A condition in which patients
ticles in a solution by measuring the light scattered at a particular produce a biphasic autoantibody that binds to RBCs at cold tem-
angle from the incident beam as it passes through the solution. peratures and activates complement at 37°C to produce an inter-
Neutrophil: A white blood cell (WBC) with a multilobed nucleus mittent hemolysis.
and a large number of neutral staining granules. Its main function Paroxysmal nocturnal hemoglobinuria (PNH): A disease charac-
is phagocytosis. terized by complement-mediated hemolysis of erythrocytes result-
Next generation sequencing (NGS): A technique that is able to ing from a deficiency of decay-accelerating factor on the red blood
sequence large numbers of templates simultaneously (massively cells (RBCs).
parallel sequencing), yielding hundreds of thousands of short Particle-counting immunoassay (PACIA): A technique for measuring
sequences in a single run. residual nonagglutinating particles in a specimen using nephelometry
Nick: An amplification technique that starts with breaking one phos- to determine the amount of forward light scatter. Antigen–antibody
phodiester bond on one strand of a double-stranded DNA molecule. combination decreases light scatter so that the amount of patient
Noncompetitive immunoassay: An assay that allows any patient antigen present is indirectly proportional to the amount of light
antigen present to be captured. The amount of label measured is scattered.
directly proportional to the amount of patient antigen present. Passive agglutination: A reaction in which particles coated with
Non-Hodgkin or lymphocytic lymphoma (NHL): A wide range of antigens not normally found on their surfaces clump together
cancers of the lymphoid tissue, of which B-cell lymphomas repre- because of their combination with antibody.
sent the majority. Passive immunity: A type of immunity that results from the transfer
Nontreponemal tests: Serological tests for syphilis that detect anti- of antibodies from immunized hosts to a nonimmune individual.
body to cardiolipin and not specific antitreponemal antibody. Passive immunodiffusion: A precipitation reaction in a gel in which
Nucleic acid sequence: A specific ordering of nucleic acid bases that antigen–antibody combination occurs by means of diffusion.
identifies a particular target. Passive immunotherapy: Passive immunization of an individual
Nucleolus: A prominent structure within the nucleus where tran- with commercial preparations of antibodies formed by other hosts
scription and processing of rRNA and assembly of the ribosomes to prevent or treat a disease.
takes place. Pathogen-associated molecular patterns (PAMPs): Structural
Nucleosome antibodies: Autoantibodies against DNA-histone com- patterns of carbohydrates, nucleic acids, or bacterial peptides on
plexes [also known as nucleosomes or deoxyribonucleoprotein microorganisms that are recognized by pathogen recognition
(DNP)]. receptors (PRRs) on the cells of the innate immune system.
Pathogen recognition receptors (PRRs): Receptors on cells of Preexamination variable: A variable that occurs before the actual
the innate immune system that bind to PAMPs on pathogenic testing of the specimen and includes test requests, patient prepa-
microorganisms. ration, timing, specimen collection, handling, and storage.
Pathogenicity: The inherent capacity of an organism to cause Primary biliary cirrhosis (PBC): An autoimmune disease that involves
disease. progressive destruction of the intrahepatic bile ducts.
Percent panel reactive antibody (%PRA): The proportion of lym- Primary follicle: A cluster of B cells that have not yet been stimulated
phocytes with known HLA phenotypes in a test panel that are by antigen.
killed by antibodies in the serum of a transplant patient. Primary immunodeficiency (PID): Inherited diseases in which part
Periarteriolar lymphoid sheath (PALS): White pulp of splenic tissue, of the immune system is absent or dysfunctional.
which is made up of lymphocytes, macrophages, plasma cells, and Primary lymphoid organs: The organs in which lymphocytes mature:
granulocytes. It surrounds the central arterioles. these are the bone marrow and the thymus.
Peripheral tolerance: Destruction or repression of lymphocytes Primary response: The initial response to a foreign antigen, charac-
in the peripheral lymphoid organs that could respond to terized by a long lag phase, a slow rise in antibody, and consisting
self-antigens. of mostly of IgM.
Personal protective equipment (PPE): Items such as gowns, masks, Primer: Short sequences of DNA, usually 20 to 30 nucleotides long,
gloves, and face shields used to protect the body from infectious used to hybridize specifically to a particular target DNA to help
agents. initiate replication of the DNA.
Phagocytosis: From the Greek word phagein, meaning “cell eating,” Pro-B cell: A stage in B-cell development in which rearrangement
the engulfment of cells or particulate matter by leukocytes, of the genes that code for the heavy-chain region of an antibody
macrophages, or other cells. molecule occur.
Phagolysosome: The structure formed by the fusion of cytoplasmic Probe: A nucleic acid, several hundred to a few thousand bases in
granules and a phagosome during the process of phagocytosis. length, with a known sequence that is used to identify the presence
Phagosome: A vacuole formed within a phagocytic cell as pseudopodia of a complementary DNA or RNA sequence in an unknown sample.
surround a particle during the process of phagocytosis. Proficiency testing: The testing of unknown samples received from
Plasma cell: A differentiated B cell that actively secretes antibody. an outside agency.
Plasma cell dyscrasias: Immunoproliferative diseases characterized Properdin: A protein that stabilized the C3 convertase generated in
by overproduction of a single immunoglobulin component by a the alternative complement pathway.
clone of lymphoid cells. Prostate-specific antigen (PSA): A glycoprotein that is produced
Plasmid: A self-replicating genetic element located in the cytoplasm specifically by epithelial cells in the prostate gland; PSA is a widely
of a bacterium, which has a limited number of genes that can be used marker for prostate cancer.
transferred between bacteria. Proteomics: The field of study that involves identification and quan-
Pleiotropy: Many different actions of a single cytokine. The cytokine tification of the array of proteins present in a sample.
may affect the activities of more than one kind of cell and have Proto-oncogenes: Regulatory genes that promote cell division.
more than one kind of effect on the same cell. Protozoa: A diverse group of single-celled organisms that can live
Polyclonal: Derived from many clones of cells. Polyclonal antibodies and multiply inside of human hosts.
are derived from many clones of B cells or plasma cells, and are Prozone phenomenon: Lack of a visible reaction in antigen–antibody
therefore diverse in terms of their antigen specificity. combination caused by the presence of excess antibody. This may
Polymerase chain reaction (PCR): A means of amplifying tiny result in a false-negative reaction.
quantities of nucleic acid using a heat-stable polymerase enzyme Purine-nucleoside phosphorylase (PNP) deficiency: Lack of the
and a primer that is specific for the DNA sequence desired. enzyme purine nucleoside phosphorylase. The deficiency is inherited
Polymorphism: The presence of two or more different genetic com- as an autosomal recessive trait. Accumulation of a purine metabolite
positions (e.g., HLA genes) among individuals in a population. is toxic to T cells, leading to a defect in cell-mediated immunity.
Positive predictive value: The percentage of all positives in a sero- Quality assessment (QA): The overall process of guaranteeing
logical test that are true positives. quality patient care. It involves the continual monitoring of the
Positive selection: The process of selecting immature T lymphocytes entire test process from test ordering and specimen collection
for survival on the basis of expression of high levels of CD3 and through reporting and interpreting results.
the ability to respond to self-MHC antigens. Quality control (QC): The materials, procedures, and techniques that
Postexamination variable: A process that affects the reporting of monitor the accuracy, precision, and reliability of a laboratory test.
results and correct interpretation of data. Quality indicator: Measurements developed by each laboratory to
Postexposure prophylaxis (PEP): Course of preventative treatment determine if the quality system essentials are being met.
provided after exposure to potentially infectious organisms. Quality management (QM): The overall process of guaranteeing
Poststreptococcal glomerulonephritis: A condition that damages quality patient care.
the glomeruli of the kidney, caused by an initial immune response Quality management system (QMS): A system that incorporates
to a streptococcal infection. the objectives of total quality management and continuous qual-
Postzone phenomenon: Lack of a visible reaction in an antigen– ity improvement to ensure quality results, staff competence, and
antibody reaction, caused by an excess of antigen. efficiency within an organization.
Pre-B cell: The stage of development of a B cell where the heavy- Quality system essentials (QSEs): Methods to meet the requirements
chain part of the antibody molecule is present. of regulatory, accreditation, and standard-setting organizations.
Precipitation: The combination of soluble antigen with soluble Quantitative PCR (qPCR): Accumulation of a PCR product in real
antibody to produce visible insoluble complexes. time during amplification.
Precision: The ability to consistently reproduce the same result upon Radial immunodiffusion (RID): A precipitation technique in which
repeated testing of the same sample. antibody is uniformly distributed in the support gel, and antigen
 is applied to a well cut into the gel. As the antigen diffuses out Safety data sheet (SDS): An SDS contains information on physical and
from the well, an antigen–antibody combination occurs until the chemical characteristics, fire, explosion reactivity, health hazards,
zone of equivalence is reached. primary routes of entry, exposure limits and carcinogenic potential,
Radioimmunoassay (RIA): A technique used to measure small con- precautions for safe handling, spill clean-up, and emergency first aid
centrations of an analyte, using a radioactive label on one of the information.
immunologic reactants. Sandwich immunoassays: Immunoassays based on the ability of
Random access analyzer: An analyzer that can run multiple tests on antibody to bind with more than one antigen.
multiple samples using multiple analytes. Sarcoma: A type of cancer derived from bone or soft tissues such as
Rapid antigen detection system (RDTS): See lateral flow immunochro- fat, muscles, tendons, cartilage, nerves, and blood vessels.
matographic assay. Scarlet fever: An illness with a characteristic rash and fever that is
Rapid plasma reagin (RPR) test: A slide flocculation test for syphilis caused by the erythrogenic toxins released from group A strepto-
that detects antibody to cardiolipin. coccal bacteria.
Rate nephelometry: A technique that measures the rate of light scat- Secondary follicle: A cluster of B cells that are proliferating in
tering after the reagent antibody is added to a sample containing response to a specific antigen.
patient antigen. The rate change is directly related to antigen con- Secondary immunodeficiency: An immunodeficiency that is acquired
centration if the concentration of antibody is kept constant. secondary to other conditions, such as certain infections, malignan-
Reagin: An antibody formed during the course of syphilis that is di- cies, autoimmune disorders, and immunosuppressive therapies.
rected against cardiolipin and not against Treponema pallidum itself. Secondary lymphoid organs: Organs that include the spleen,
Recognition unit: The complement component that consists of the lymph nodes, appendix, tonsils, and other mucosal-associated
C1qrs complex. This must bind to at least two Fc regions to initiate lymphoid tissue where the main contact with foreign antigens
the classical complement cascade. takes place.
Recombinant protein vaccine: A vaccine produced by cloning the Secondary response: A second or memory response to an antigen,
gene coding for the vaccine antigen into the genome of bacteria, characterized by a shortened lag period, a more rapid rise in anti-
yeast, or cultured cells. body, and higher serum levels for a longer period of time.
Redundancy: A phenomenon that occurs when different cytokines Secretory component (SC): A protein with a molecular weight of
have the same effect. 70,000 that is synthesized in epithelial cells and added to IgA to
Reference interval: The range of values found in healthy individuals facilitate transport of IgA to mucosal surfaces.
who do not have the condition detected by the assay. Self-tolerance: The ability of the immune system to accept self-antigens
Reliability: The ability to maintain both precision and accuracy in and not initiate a response against them.
laboratory testing. Sensitivity: The lowest amount of an analyte that can be measured.
Reportable range: The range of values that will generate a positive Sensitization: (1) The combination of antibody with a single anti-
result for the specimens assayed by the test procedure. genic determinant on the surface of a cell without agglutination.
Restriction endonucleases: Enzymes that cleave DNA at specific (2) Induction of an immune response.
recognition sites that are typically 4 to 6 base pairs long. Serial dilution: A method of decreasing the strength of an antibody
Restriction fragment length polymorphisms (RFLPs): Variations solution by using the same dilution factor for each step.
in nucleotides within DNA that change where restriction enzymes Serological pipette: A graduated or measuring pipette that has
cleave the DNA. Where mutations occur, different-size pieces of marks all along its length all the way down to the tip.
DNA are obtained, resulting in an altered electrophoretic pattern. Serology: The study of the noncellular portion of the blood known
Reverse passive agglutination: A reaction in which carrier particles as serum.
coated with antibody clump together because of a combination Serotype: A group of related bacteria or viruses that share specific
with antigen. antigens that can be identified by serological testing.
Reverse transcriptase: An enzyme produced by certain RNA viruses Serum: The liquid portion of the blood minus the clotting factors.
to convert viral RNA into DNA. Serum amyloid A (SAA): An acute-phase protein that acts as a
Rheumatoid arthritis (RA): An autoimmune disease that affects the chemical messenger to activate monocytes and macrophages in
synovial membrane of multiple joints. It is characterized by the order to increase inflammation.
presence of the autoantibodies, anti-CCP and rheumatoid factor. Serum sickness: A type III hypersensitivity reaction that results
Rheumatoid factor (RF): An antibody of the IgM class produced by from the buildup of antibodies to animal serum used in passive
patients with rheumatoid arthritis that is directed against IgG. immunization.
Ribonucleic acid (RNA): The nucleic acid containing the sugar ribose. Severe combined immunodeficiency (SCID): An inherited defi-
It is the primary genetic material of RNA viruses and plays a role in ciency of both cell-mediated and antibody-mediated immunity. It
the transcribing of genetic information in cells. results in death in infancy caused by overwhelming infections.
Rickettsiae: Small gram-negative fastidious bacteria that are obligate Shift: An abrupt change in the mean that may be caused by a mal-
intracellular parasites and are responsible for diseases such as function of the instrument or a new lot number of reagents.
Rocky Mountain spotted fever and typhus. Side (right angle) scatter: Light scattered at 90 degrees in a flow cy-
Rocky Mountain spotted fever (RMSF): A disease caused by infec- tometer that indicates the granularity of a cell.
tion with R rickettsii. Single-parameter histogram: Plot of a chosen parameter or mea-
Rubella virus: An RNA virus that causes German measles and con- surement on the x-axis against the number of events on the y-axis.
genital infection. Six Sigma: A method employed by health-care organizations to reduce
Rubeola virus: A single-stranded RNA virus that causes measles. variables and decrease errors.
S protein: A control protein in the complement cascade that inter- Sm antigen: An extractable nuclear antigen; autoantibodies to Sm
feres with binding of the C5b67 complex to a cell membrane, thus are specific for lupus.
preventing lysis. Solute: One of the two entities needed for making a dilution.
Southern blot: Technique for the identification of specific DNA erythematous rash, and deposition of immune complexes in the
sequences in which DNA is cleaved into fragments by enzymes, kidneys.
separated electrophoretically, denatured, transferred to a nitro- T-dependent antigen: An antigen that requires T-cell help in order
cellulose membrane, and incubated with a labeled probe that is for B cells to respond.
specific for the sequence of interest. T helper (Th) cells: Lymphocytes that express the CD4 antigen.
Specificity: The proportion of people who do not have the disease Their function is to provide help to B cells in recognizing foreign
or condition and who have a negative test. antigen and producing antibody to it.
Spleen: The largest secondary lymphoid organ in the body, located T helper 1 (Th1) cells: T cells that are developed through the expres-
in the upper left quadrant of the abdomen. Its function is to filter sion of IL-12 by dendritic cells, and which are primarily responsible
out old cells and foreign antigens. for cell-mediated immunity.
SS-A/Ro: An extractable nuclear antigen consisting of small, uridine- T helper 2 (Th2) cells: T cells which are developmentally regulated
rich RNA complexed to cellular protein; found in a significant by IL-4, and whose main function is to drive antibody-mediated
percentage of patients with Sjögren’s syndrome. immunity.
SS-B/La: An extractable nuclear antigen consisting of small, uridine- T helper 17 (Th17) cells: A subset of T cells that play an important
rich RNA complexed to cellular protein; found in a significant per- role in host defense against bacterial and fungal infections at
centage of patients with Sjögren’s syndrome. mucosal surfaces. They secrete IL-17 which attracts neutrophils
Standard deviation (SD): A measurement statistic that describes the to the site of infection.
average distance each data point in a normal distribution is from T pallidum particle agglutination (TP-PA) test: A particle aggluti-
the mean. nation test that detects antibodies to Treponema pallidum to aid in
Standard of care: The attention, caution, and prudence that a reason- the diagnosis of syphilis.
able person in the same circumstances would exercise in performing T regulatory (Treg) cell: A subpopulation of T cells that play an
laboratory testing. important role in suppressing the immune response to self-antigens.
Standard precautions: Guidelines describing personnel protection Tertiary syphilis: The last stage of syphilis that appears months to
that should be used for the care of all patients, including hand years after secondary infection. It is characterized by granuloma-
washing, gloves, mask, eye protection, face shield, gown, patient- tous inflammation, cardiovascular disease, and central nervous
care equipment, environmental control, linens, taking care to system involvement.
prevent injuries, and patient placement. Tetrapeptide: The basic four-chain unit common to all immunoglob-
Strand cleavage: Breaking the phosphodiester bonds that connect ulin molecules, consisting of two large heavy chains and two
nucleotides in the DNA chain by using physical or enzymatic smaller light chains.
methods. Threshold cycle: The cycle at which the sample fluorescence reaches
Strand displacement amplification (SDA): A method for amplify- a certain optimal level as determined by an instrument performing
ing DNA by using a DNA primer that is nicked by an endonucle- a polymerase chain reaction (PCR).
ase, allowing for displacement of the amplified strands. Thymocyte: Immature lymphocyte, found in the thymus, that under-
Streptococcus pyogenes: Group A Streptococci; gram-positive goes differentiation to become a mature T cell.
cocci that have a number of clinical manifestations, including Thymus: A small, flat, bilobed organ found in the thorax of humans,
pharyngitis, impetigo, scarlet fever, acute rheumatic fever, and which serves as the site for differentiation of T cells.
post-streptococcal glomerulonephritis. Thyroglobulin (Tg): A large iodinated glycoprotein from which the
Streptolysin O: A protein capable of lysing red blood cells (RBCs) active thyroid hormone triiodothyronine (T3) and its precursor,
and white blood cells (WBCs), which is produced by some groups thyroxine (T4), are synthesized.
of streptococci as they grow. Thyroid peroxidase (TPO): An enzyme that oxidizes iodine ions to
Streptozyme: A serological test for infection with group A streptococci form the iodine atoms that are incorporated into thyroglobulin to
that detects five different antibodies to streptococcal products. facilitate the synthesis of the thyroid hormones, T3 and T4.
Stringency: Conditions that affect the ability of a probe to correctly Thyroid-stimulating hormone (TSH): A hormone produced by the
bind to a specific target DNA sequence. These include tempera- thyroid gland that binds to specific receptors, causing thyroglob-
ture, salt concentration, and concentration of formamide or urea. ulin to be broken down into secretable T3 and T4.
Superantigens: Microbial proteins that can act as potent T-cell mito- Thyroid-stimulating hormone receptor antibody (TRAb): An an-
gens because they bind to both class II MHC molecules and T-cell tibody that is directed against the receptor for thyroid-stimulating
receptors, regardless of antigen specificity. hormone. It is associated with Graves disease and results in over-
Surrogate light chain: Two short polypeptide chains noncovalently stimulation of the thyroid gland.
associated with each other that appear before actual light chains Thyrotoxicosis: A condition caused by overproduction of thyroid
are formed by a developing B cell. hormones, as seen in Graves disease.
Symbiotic: A relationship in which two species live together, often Thyrotropin-releasing hormone (TRH): A hormone secreted by the
maintaining a long-term, but not necessarily a beneficial, interac- hypothalamus that acts on the pituitary gland to induce the release
tion (e.g., a bacterium and a human). of thyroid-stimulating hormone (TSH).
Synergistic: Cytokines whose effects complement and enhance each T-independent antigens: Antigens that are able to elicit antibody
other. formation in the absence of T cells.
Syngeneic graft: The transfer of tissue or organs between genetically Tissue transglutaminase (tTG): An intestinal enzyme that con-
identical individuals such as identical twins. verts the glutamine residues in gliadin to glutamic acid; auto-
Syphilis: A sexually transmitted disease caused by the spirochete antibodies to tTG are commonly produced in patients with
bacterium Treponema pallidum. celiac disease.
Systemic lupus erythematosus (SLE): A chronic inflammatory Titer: A figure that represents the relative strength of an antibody. It
autoimmune disease characterized by the presence of antinuclear is the reciprocal of the highest dilution in which a positive reaction
antibodies. Symptoms may include swelling of the joints, an occurs.
Toll-like receptors (TLRs): Receptors found on human leukocytes Type II hypersensitivity: An immune reaction in which IgG or
and other cell types that recognize microorganisms and aid in their IgM antibodies are produced to cell surface receptors, causing
destruction. damage to the cells, dysfunction of the cells, or overstimulation
Toxoid: A chemically inactivated bacterial toxin used in some vaccines. of the function of the cells; also known as antibody-mediated
Toxoplasma gondii: The protozoal organism that causes toxoplasmo- cytotoxic hypersensitivity.
sis, an infection that can have severe consequences in immuno- Type III hypersensitivity: An immune reaction in which IgG or
compromised individuals and congenitally infected infants. IgM antibodies react with soluble antigens to form small complexes
TP-PA test: A particle agglutination test that detects antibodies to that precipitate in the tissues and activate complement to induce
Treponema pallidum to aid in the diagnosis of syphilis. inflammation; also known as complex-mediated hypersensitivity.
Transcription: The process of generating a messenger RNA strand Type IV hypersensitivity: A cell-mediated response involving the
from DNA. This is used to code for protein. release of cytokines that induce inflammation and tissue damage
Transcription-mediated amplification (TMA): Method of increasing 24 to 72 hours after contact with an antigen.
target DNA through the use of two enzymes, an RNA polymerase Typhus: A group of Rickettsiae that causes endemic and epidemic
and a reverse transcriptase, to make new strands of DNA. typhus, diseases characterized by fever, rash, and a cough.
Transforming growth factor-β (TGF-β): A cytokine that induces an- Urease: An enzyme that breaks down urea to form ammonia
tiproliferative activity in a variety of cell types and downregulation and bicarbonate. Presence of urease is used as an indicator of
of the inflammatory response. Helicobacter pylori.
Transient hypogammaglobulinemia: A condition characterized by Vaccine: An antigen preparation derived from a pathogen that is
low immunoglobulin levels that occurs in infants around 2 to administered to healthy individuals in order to produce immunity
3 months of age. It is believed to be caused by delayed maturation to an infectious disease.
of one or more components of the immune system and usually Variable: Anything that can be changed or altered in laboratory
corrects itself spontaneously. testing.
Translation: The process by which messenger RNA is used to make Variable region: The amino-terminal region of an immunoglobulin
functional proteins. molecule (half of a light chain or quarter of a heavy chain) that
Transporters associated with antigen processing (TAP 1 and TAP 2): has a unique amino acid sequence for each different immunoglob-
Proteins that are responsible for the ATP-dependent transport of ulin molecule. This part is responsible for the specificity of a par-
newly synthesized short peptides from the cytoplasm to the lumen ticular immunoglobulin molecule.
of the endoplasmic reticulum for binding to class I HLA antigens. Variants: Changes in a nucleotide sequence.
Trend: A gradual change in the mean in one direction that may be Varicella-zoster virus (VZV): A herpes virus that is responsible for
caused by a gradual deterioration of reagents or deterioration of chickenpox and zoster, or shingles.
instrument performance. Venereal Disease Research Laboratory (VDRL) test: A flocculation
Treponema pallidum: A spirochete that is the causative agent of syphilis. test for the cardiolipin antibody produced in syphilis patients; an
Treponemal tests: Serological tests for syphilis that detect antibodies example of a nontreponemal test.
directed against Treponema pallidum itself. Viral load tests: Quantitative tests for nucleic acid from viruses such
Tumor: An abnormal cell mass that can either be benign or malignant. as HIV. These tests are used to predict disease progression and to
Tumor-associated antigens (TAA): Antigens that are expressed by monitor the effects of antiretroviral therapy.
normal cells as well as tumor cells. Virulence: A quantitative trait of an organism that refers to the extent
Tumor-infiltrating lymphocyte (TIL): Lymphocytes within a of pathology it can cause when it infects a host.
tumor mass that are able to react with antigens on tumor cells to Virulence factors: Characteristics of a microorganism that can in-
help destroy them. crease its pathogenicity by contributing to its ability to establish
Tumor marker: Biological substances found in increased amounts itself in the host, invade or damage host tissue, or evade the host
in the blood, body fluids, or tissues of patients with a specific type immune response.
of cancer. Volumetric pipette: A pipette that is marked and calibrated to de-
Tumor necrosis factor (TNF): A major mediator of the innate de- liver only one volume of the specified liquid.
fense against gram-negative bacteria. Waldenström macroglobulinemia: An immunoproliferative disease
Tumor-specific antigen (TSA): Antigens that are unique to the caused by a malignancy of lymphocytes that results in production
tumor of an individual patient or shared by a limited number of of` IgM paraproteins.
patients with the same type of tumor. Wegener’s granulomatosis (WG): See granulomatosis with polyangiitis.
Tumor suppressor genes: Genes that inhibit the growth of tumors. Western blot test: A confirmatory test for HIV based on separation
Turbidimetry: A technique for determining the concentration of par- of HIV antigens by electrophoresis followed by transfer or blotting
ticles in a solution based on the change in absorbance, caused by of the antigen pattern to a supporting medium for reaction with
the scattering of light that occurs when an incident beam is passed test serum.
through the solution. Wiskott-Aldrich syndrome (WAS): A rare X-linked recessive
Turnaround time (TAT): The amount of time required between the syndrome characterized by immunodeficiency, eczema, and
point at which a test is ordered by the health-care provider and thrombocytopenia.
the results are reported to the health-care provider. Xenograft: The transfer of tissue from an individual of one species
Type 1 diabetes mellitus (T1D): A chronic autoimmune disease to an individual of another species, such as animal tissue trans-
characterized by insufficient insulin production, caused by pro- planted to a human.
gressive destruction of the beta cells of the pancreas. Yeast: A unicellular form of certain fungi that reproduces asexually by
Type I hypersensitivity: An allergic reaction in which antigen- budding, in which the parent cell divides into two unequal parts.
specific IgE antibody binds to mast cells and basophils, trigger- Zone of equivalence: The point in an antigen–antibody reaction at
ing degranulation and the release of chemical mediators; also which the number of multivalent sites of antigen and antibody are
known as anaphylactic hypersensitivity. approximately equal, resulting in optimal precipitation.
 References
21. Robertson P, Poznansky MC. T-lymphocyte development and mod-
1. Keratin.com. Silkworms and chickens—Louis Pasteur. Immunol- els of thymopoietic reconstitution. Transpl Infect Dis. 2003;5:38–42.
ogy History III. www.keratin.com/am/am003.shtml. Accessed 22. Smith L. Hematopoiesis. In: Rodak BF, Fritsma GA, Keohane EM,
December 3, 2014. eds. Hematology: Clinical Principles and Applications. 4th ed.
2. Greenberg S. History of immunology. In: Paul WE, ed. Funda- Philadelphia, PA: Elsevier Saunders; 2012:66–85.
mental Immunology. 7th ed. Philadelphia, PA: Lippincott Williams 23. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential Immunol-
& Wilkins; 2013:22–46. ogy. 12th ed. Chichester, UK: Wiley-Blackwell; 2011:283–312.
3. Berche P. Louis Pasteur, from crystals of life to vaccination. Clin 24. Czader M. Mature lymphoid neoplasms. In: Rodak BF, Fritsma
Microbiol Infec. 2012;18(suppl 5). GA, Keohane EM, eds. Hematology: Clinical Principles and Applica-
4. Gordon S. Elie Metchnikoff: Father of natural immunity. Eur J tions. 4th ed. Philadelphia, PA: Elsevier Saunders; 2012:558–579.
Immunol. 2008;38:3257–3264.
5. Stiene-Martin A. Leukocyte development, kinetics, and func-
tions. In: Rodak BF, Fritsma GA, Keohane EM, eds. Hematology:
Clinical Principles and Applications. 4th ed. Philadelphia, PA: Elsevier 1. Owen JA, Punt J, Stranford SA, Jones PP. Kuby Immunology.
Saunders; 2012:134–151. 7th ed. New York, NY: WH Freeman and Co.; 2013:261–298.
6. Vajpayee N, Graham SS, Bern S. Basic examination of blood and 2. Berzofsky JA, Berkower IJ. Immunogenicity and antigen structure.
bone marrow. In: McPherson RA, Pincus MR, eds. Henry’s Clinical In: Paul WE, ed. Fundamental Immunology. 7th ed. Philadelphia, PA:
Diagnosis and Management by Laboratory Method. 22nd ed. Wolters Kluwer/Lippincott Williams & Wilkins; 2012:539–582.
Philadelphia, PA: Elsevier Saunders; 2011:509–535. 3. Beadling WV, Cooling L. Immunohematology. In: McPherson RA,
7. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential Immunol- Pincus MR, eds. Henry’s Clinical Diagnosis and Management by Lab-
ogy. 12th ed. Chichester, UK: Wiley-Blackwell; 2011:3–34. oratory Methods. 21st ed. Philadelphia, PA: Elsevier Saunders; 2007:
8. McKenzie SB. The leukocyte. In: Clinical Laboratory Hematology. 618–668.
Upper Saddle River, NJ: Prentice Hall; 2004:85–121. 4. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential Immunol-
9. Bell A, Harmening DM, Hughes VC. Morphology of human blood ogy. 12th ed. Chichester, UK: Wiley-Blackwell; 2011:79–112.
and marrow. In: Clinical Hematology and Fundamentals of Hemostasis. 5. Alving CR, Peachman KK, Rao M, Reed SG. Adjuvants for human
5th ed. Philadelphia, PA: F.A. Davis; 2009:1–44. vaccines. Curr Opin Immunol. 2012;24:310–315.
10. McPherson RA, Massey HD. Overview of the immune system and 6. Mohan T, Verma P, Rao DN. Novel adjuvants and delivery vehicles
immunologic disorders. In: McPherson RA, Pincus MR, eds. for vaccine development: a road ahead. Indian J Med Res. 2013;
Henry’s Clinical Diagnosis and Management by Laboratory Method. 138:779–795.
22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:845–850. 7. Fagoaga OR. Human leukocyte antigen: the major histocompat-
11. Mathur S, Schexneider K, Hutchison RE. Hematopoiesis. In: ibility complex of man. In: McPherson RA, Pincus MR, eds.
McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Man- Henry’s Clinical Diagnosis and Management by Laboratory Methods.
agement by Laboratory Method. 22nd ed. Philadelphia, PA: Elsevier 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:933–953.
Saunders; 2011:536–556. 8. Nepom GT. The major histocompatibility complex. In: Longo DL,
12. Sato K, Fujita S. Dendritic cells—nature and classification. Allergol Fauci A, Kasper D, et al., eds. Harrison’s Principles of Internal Med-
Int. 2007;56:183–191. icine. 18th ed. New York, NY: McGraw Hill; 2011:2685–2694.
13. Haynes BF, Soderberg KA, Fauci AS. Introduction to the immune 9. HLA Nomenclature. hla.alleles.org/nomenclature/stats.html.
system. In: Longo DL, Fauci A, Kasper D, et al., eds. Harrison’s Accessed December 3, 2015.
Principles of Internal Medicine. 18th ed. New York, NY: McGraw 10. Schulze M-S ED, Wucherpfennig KW. The mechanism of HLA-DM
Hill; 2011:2650–2685. induced peptide exchange in the MHC class II antigen presentation
14. Altfeld M, Fadda L, Frieta D, Bhardwaj N. DCs and NK cells: Critical pathway. Curr Opin Immunol. 2012;24:105–111.
effectors in the immune response to HIV-1. Nat Rev Immunol. 2011; 11. Loureiro J, Ploegh HL. Antigen presentation and the ubiquitin-
11:176–186. proteosome system in host-pathogen interactions. Adv Immunol.
15. Engel P, Boumsell L, Balderas R, et al. CD nomenclature 2015: 2006;92:226–306.
human leukocyte differentiation antigen workshops as a driving 12. Li P, Gregg JL, Wang N, et al. Compartmentalization of class II
force in immunology. J Immunol. 2015:195:10:4555–4563. antigen presentation: Contribution of cytoplasmic and endosomal
16. Human cell differentiation molecules. hcdm.org. Accessed processing. Immunol Rev. 2005;207:206–217.
December 3, 2014. 13. Koch J, Tampe R. The macromolecular peptide-loading complex
17. Blom B, Spits H. Development of human lymphoid cells. Annu in MHC class I-dependent antigen presentation. Cell Mol Life Sci.
Rev Immunol. 2006;24:287–320. 2006;63:653–662.
18. Sun JC, Lanier LL. NK cell development, homeostasis and function: 14. Cresswell P, Ackerman AL, Giodini A, et al. Mechanisms of MHC
parallels with CD8+ T cells. Nat Rev Immunol. 2011;11:645–657. class-I restricted antigen processing and cross-presentation. Im-
19. Paust S, von Andrian UH. Natural killer cell memory. Nat munol Rev. 2005;207:147–157.
Immunol. 2011;12:500–508. 15. Van den Hoorn T, Paul P, Jongsma M, Neefjes J. Routes to manip-
20. Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural ulate MHC class II antigen presentation. Curr Opin Immunol. 2011;
killer cells. Nature. 2009;457:557–561. 23:88–95.
16. Scholz C, Tampe R. The intracellular antigen transport machinery 14. Fritzma GA. Thrombosis risk testing. In: Rodak BF, Fritzma GA,
TAP in adaptive immunity and virus escape mechanisms. J Bioenerg Keohane EM, eds. Hematology: Clinical Principles and Applications.
and Biomembr. 2005;37:509–515. Philadelphia, PA: Elsevier Saunders; 2011:668–693.
17. Van Kasteren SI, Overkleeft H, Ovaa H, Neefjes J. Chemical 15. Van Holten TC, Waanders LF, de Groot PG, et al. Circulating
biology of antigen presentation by MHC molecules. Curr Opin biomarkers for predicting cardiovascular disease risk; a systemic
Immunol. 2014;26:21–31. review and comprehensive overview of meta-analyses. PLOS
18. Brown JH, Jardetzky TS, Gorga JC, et al. Three-dimensional struc- One. 2013(April);8(4):e62080.
ture of the human class II histocompatibility antigen HLA-DR1. 16. Cushman M, Arnold AM, Psaty BM, et al. C-reactive protein
Nature. 1993;364:33. and the 10-year incidence of coronary heart disease in older
19. Jardetzky TS, Brown JH, Gorga JC, et al. Crystallographic analysis men and women: the cardiovascular health study. Circulation.
of endogenous peptides associated with HLA-DR1 suggests a 2005;112:25–31.
common, polyproline II-like conformation for bound peptides. 17. Boekholdt SM, Matthijs S, Hack CE, et al. C-reactive protein lev-
Proc Natl Acad Sci USA. 1996;93:734–738. els and coronary artery disease incidence and mortality in appar-
20. Stern LJ, Brown JH, Jardetzky TS, et al. Crystal structure of ently healthy men and women: the epic Norfolk prospective
the human class II MHC protein HLA-DR1 complexed with an population study 1993–2003. Atherosclerosis. 2006;187:415–422.
influenza virus peptide. Nature. 1994;368:215–221. 18. Sarwar N, Thompson AJ, Angelantonio ED. Markers of inflam-
21. Spondylitis Association of America. www.spondylitis.org.about. mation and risk of coronary heart disease. Dis Markers. 2009;
Accessed March 11, 2014. 26:217–225.
22. Bordner AJ. Towards universal structure-based prediction of 19. Koenig W. High-sensitivity C-reactive protein and atherosclerotic
class II MHC epitopes for diverse allotypes. PLOS One. 2010; disease: from improved risk prediction to risk-guided therapy. Int
5(12):e14383. J Cardio. 2013;168:5126–5134.
23. Salimi N, Fleri W, Peters B, Sette A. The immune epitope database: 20. Braga F, Panteghini M. Biologic variability of C-reactive protein:
a historical retrospective of the first decade. Immunology. 2012; is the available information reliable? Clinica Chimica Acta. 2012;
137:117–123. 413:1179–1183.
24. Pascal M, Konstantinou GN, Masiliamani M, et al. In silico predic- 21. Algarra M, Gomes D, Esteves da Silva JCG. Current analytical
tion of Ara h 2 T cell epitopes in peanut-allergic children. Clin Exp strategies for C-reactive protein quantification in blood. Clinica
Allergy. 2012;43:116–127. Chimica Acta. 2013;415:1–9.
25. Profaizer T, Eckles D. HLA alleles and drug hypersensitivity reac- 22. Faty A, Ferre P, Commans S. The acute phase protein serum
tions. Int J Immunogenet. 2012, 39:99–105. amyloid A induces lipolysis and inflammation in human adipocytes
through distinct pathways. PLOS One. 2012 (April);7(4):e34031.
www.plosone.org. Accessed May 4, 2014.
1. Deobagkar-Lele M, Bhaskarla C, Dhanaraju R, et al. Innate immu- 23. Malle E, Sodin-Semrl S, Kovacevic A. Serum amyloid A: an acute-
nity and the 2011 Nobel Prize. Resonance. 2012;17(10):974–995. phase protein involved in tumour pathogenesis. Cell Mol Life Sci.
2. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential Immunol- 2009;66:9–26. doi:10.1007/s00018-008-8321-x.
ogy. 12th ed. Chichester, UK: Wiley-Blackwell; 2011:3–34. 24. Bergin DA, Reeves EP, Meleady P, et al. A-1 antitrypsin regulates
3. Carty M, Bowie AG. Recent insights into the role of Toll-like recep- human neutrophil chemotaxis induced by soluble immune
tors in viral infection. Clin Exp Immunol. 2010;161:397–406. complexes and IL-8. J Clin Invest. 2010 (December);120(12):
4. Liu G, Zhang L, Zhao Y. Modulation of immune responses through 4236–4250.
direct activation of Toll-like receptors to T cells. Clin Exp Immunol. 25. Greene DN, Elliott-Jelf MC, Straseki JA, Grenache DG. Facilitating
2010;160:168–175. the laboratory diagnosis of α1-antitrypsin deficiency. Am J Clin
5. Kumar H, Kawai T, Akira S. Pathogen recognition by the innate Path. 2013;139:184–191.
immune system. Int Rev Immunol. 2011;30:16–34. 26. Stoller JK, Aboussouan LS. α1 antitrypsin deficiency. Lancet. 2005;
6. Jeong E, Lee JY. Intrinsic and extrinsic regulation of innate immune 365:2225–2236.
receptors. Yonsei Med J. 2011;52(3):379–392. 27. Yerbury JJ, Rybehyn MS, Esterbrook-Smith SB, et al. The acute
7. Medzhitov R, Shevach EM, Trinchieri G, Mellor AL. Highlights phase protein haptoglobin is a mammalian extracellular chaper-
of 10 years of immunology. Nat Rev Immunol. 2011;11:693–702. one with an action similar to clusterin. Biochemistry. 2005;44:
8. McPherson RA. Specific proteins. In: McPherson RA, Pincus MR, 10914–10925.
eds. Henry’s Clinical Diagnosis and Management by Laboratory 28. Nantasenamat C, Prachayasittikul V, Bulow L. Molecular modeling
Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011: of the human hemoglobin-haptoglobin complex sheds light on
259–272. the protective mechanisms of haptoglobin. PLOS One. 2013(April);
9. Song C, Hsu K, Yamen E, et al. Serum amyloid A induction of 8(4):e62996.
cytokines in monocytes/macrophages and lymphocytes. Ather- 29. Laudicina RJ, Simonian Y. The leukocyte. In: McKenzie S,
sclerosis. 2007;(2009):374–383. doi:10.1016/j.atherosclerosis Williams L, eds. Clinical Laboratory Hematology. 2nd ed. Boston,
.2009.05.007. MA: Pearson; 2010:Chapter 7:85–121.
10. Grad E, Danenberg HD. C-reactive protein and atherosclerosis: 30. Harmening DM, Lawrence LW, Green R, Schaub CR. Hemolytic
cause or effect? Blood Rev. 2013;27:23–29. anemias. In: Harmening DM, ed. Clinical Hematology and Funda-
11. Pepys MB, Hirschfield GM. C-reactive protein: a critical update. mentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis; 2009:
J Clin Invest. 2003;111:1805–1812. 252–279.
12. Hutchinson WL, Koenig W, Frohlich M, et al. Immunoradiometric 31. Fritzma MG, Fritzma GA. Normal hemostasis and coagulation.
assay of circulating C-reactive protein: age-related values in the In: Rodak BF, Fritzma GA, Keohane EM, eds. Hematology: Clinical
adult general population. Clin Chem. 2000;46:934–938. Principles and Applications. Philadelphia, PA: Elsevier Saunders;
13. Woodhouse S. C-reactive protein: from acute phase reactant to car- 2011:626–646.
diovascular disease risk factor. MLO. 2002(March);34(3):12–20.
32. Mahajan RD, Mishra B, Singla P. Ceruloplasmin—an update. Int 17. Bonilla FA, Oettgen HC. Adaptive immunity. J Allergy Clin Immunol.
J Pharm Sci Rev Res. 2011(July-Aug);9(2):116–119. 2010;125:S33–S40.
33. Das SK, Ray K. Wilson’s disease: an update. Nat Clin Pract Neurol. 18. Pieper K, Grimbacher B, Eibel H. B-cell biology and development.
2006;2:482–493. J Allergy Clin Immunol. 2013;131:959–971.
34. Harmening DM, Marty J, Strauss RG. Cell biology, disorders of 19. De Saint Basile G, Menasche G, Fischer A. Molecular mechanisms
neutrophils, infectious mononucleosis, and related lymphocytosis. of biogenesis and exocytosis of cytotoxic granules. Nat Rev Im-
In: Harmening DM, ed. Clinical Hematology and Fundamentals of munol. 2010;11:568–579.
Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis; 2009:305–330. 20. Chaturvedi A, Davey A, Liu W, et al. B lymphocyte receptors, sig-
35. Segal AW. How neutrophils kill microbes. Annu Rev Immunol. naling mechanisms and activation. In: Paul WE, ed. Fundamental
2005;23:197–223. Immunology. 7th ed. Philadelphia, PA: Wolters, Kluwer/Lippincott,
36. Nauseef WM. Assembly of the phagocyte NADPH oxidase. Williams, and Wilkins; 2013:246–260.
Histochem Cell Biol. 2004;122:277–291. 21. Bell A, Harmening DM, Hughes VC. Morphology of human blood
37. Campbell KS, Hasegawa J. Natural killer cell biology: an update and and marrow cells. In: Harmening DM, ed. Clinical Hematology and
future directions. J Allergy Clin Immunol. 2013;132(3):536–544. Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis
38. Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? Co.; 2009: 1–41.
The example of natural killer cells. Science. 2011;33(6013):44–49. 22. Mahnke YD, Brodie TM, Sallusto F, et al. The who’s who of T-cell
39. Deguine J, Bousso P. Dynamics of NK cell interactions in vivo. differentiation: human memory T-cell subsets. Eur J Immunol.
Immunol Rev. 2013;252:154–159. 2013;43:2797–2809.
40. Di Santo JP. Natural killer cell developmental pathways. A ques- 23. Owen JA, Punt J, Stranford SA, Jones PP. Kuby Immunology. 7th ed.
tion of balance. Ann Rev Immunol. 2006;24:257–286. New York, NY: WH Freeman and Co.; 2013:415–450.
41. Orange JS. Human natural killer cell deficiencies. Curr Opin 24. Ewen CL, Kane KP, Bleackley RC. A quarter century of granzymes.
Allergy Clin Immunol. 2006;6:399–409. Cell Death Differ. 2012;19:28–35.
25. Marini JC, Vora KA. Cell cooperation in the antibody response.
In: Male D, Brostoff J, Roth DB, Roitt IM, eds. Immunology. 8th ed.
1. Zielinski CE, Corti D, Mele F, et al. Dissecting the human immuno- Philadelphia, PA: Elsevier Saunders; 2013:157–170.
logical memory for pathogens. Immunol Rev. 2011;240:40–51. 26. Takimori T, Kaji T, Takahashi Y, et al. Generation of memory
2. Vale AH, Schroeder HW. Clinical consequences of defects in B-cell B cells inside and outside germinal centers. Eur J Immunol.
development. J Allergy Clin Immunol. 2010;125:778–787. 2014;44:1258–1264.
3. Rothenberg E, Champhekar A. T lymphocyte developmental biol- 27. Gandour DM. Applications of flow cytometry to hematopathol-
ogy. In: Paul WE, ed. Fundamental Immunology. Philadelphia, PA: ogy. In: Harmening DM, ed. Clinical Hematology and Fundamen-
Wolters, Kluwer/Lippincott, Williams, and Wilkins; 2013:325–354. tals of Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis; 2009:
4. Takahama Y. Journey through the thymus: stromal guides for 882–910.
T-cell development and selection. Nat Rev/Immunol. 2006;6: 28. Myer L, Dakilewicz K, McIntire J, Bekker L-G. Comparison of point-
127–135. of-care versus laboratory-based CD4 cell enumeration in
5. Lydyard PM, Porakishvilli N. Cells, tissues, and organs of the HIV-positive pregnant women. J Int AIDS Soc. 2013; 16:18649.
immune system. In: Male D, Brostoff J, Roth DB, Roitt IM, eds. 29. Cohen JK, Klausner JD. HIV testing update. MLO. 2011(Nov);
Immunology. 8th ed. Philadelphia, PA: Elsevier Saunders; 2013: 43(11):24–31.
17–50.
6. Blom B, Spits H. Development of human lymphoid cells. Annu
Rev Immunol. 2006;24:287–320. 1. McPherson RA, Massey HD. Laboratory evaluation of immuno-
7. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. globulin function and humoral immunity. In: McPherson RA,
New York, NY: WH Freeman and Co.; 2013:299–328. Pincus MR, eds. Henry’s Clinical Diagnosis and Management by
8. Parham P. The Immune System. 3rd ed. New York, NY: Garland Laboratory Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders;
Science, Taylor and Francis Group LLC; 2009:125–158. 2011:899–912.
9. Owen JA, Punt J, Strandford SA. Kuby Immunology. 7th ed. 2. Edelman GM. The structure and function of antibodies. Sci Am.
New York, NY, WH Freeman and Co.; 2103:357–384. 1970;223:34.
10. Becker C, Stoll S, Bopp T, et al. Regulatory T-cells: present and 3. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. New York,
future hopes. Med Microbiol Immunol. 2006;195:113–124. NY: WH Freeman and Company; 2013:65–103.
11. Jabeen R, Kaplan MH. The symphony of the ninth: the development 4. Porter RR. The structure of antibodies. Sci Am. 1967;217:81.
and function of Th9 cells. Curr Opin Immunol. 2012;24:303–307. 5. Schroeder HW Jr, Wald D, Greenspan NS. Immunoglobulins:
12. Kimura A, Kishimoto T. Th17 cells in inflammation. Int Im- structure and function. In: Paul WE, ed. Fundamental Immunology.
munopharmacol. 2001;11:319–322. 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2013:
13. Owen JA, Punt J, Strandford SA. Kuby Immunology. 7th ed. 129–149.
New York, NY: WH Freeman and Co.; 2013:385–414. 6. Male D, Brostoff J, Roth DB, Roitt IM. Immunology. 8th ed. Philadelphia,
14. Eibel H, Kraus H, Sic H, et al. B cell biology: an overview. Curr PA: Elsevier Saunders; 2013:51–70.
Allergy Asthma Rep. 2014;14:434. 7. Schroeder HW, Cavacini L. Structure and function of immunoglob-
15. Owen JA, Punt J, Strandford SA. Kuby Immunology. 7th ed. ulins. J Allergy Clin Immunol. 2010;125(2S2):S41–S52.
New York, NY: WH Freeman and Co.; 2013:329–356. 8. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. New York,
16. Maddaly R, Pai G, Balaji S, et al. Receptors and signaling mechanisms NY: WH Freeman and Company; 2013:329–356.
for B-lymphocyte activation, proliferation and differentiation— 9. Brandtzaeg P, Johansen F. Mucosal B cells: phenotypic character-
insights from both in vivo and in vitro approaches. FEBS Letters. istics, transcriptional regulation, and homing properties. Immunol
2010;584:4883–4894. Rev. 2005;206:32–63.
10. Monteiro RC. Role of IgA and IgA Fc receptors in inflammation.
J Clin Immunol. 2010;30:1–9. 1. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed.
11. Wines B, Hogarth P. IgA receptors in health and disease. Tissue New York, NY: WH Freeman and Co.; 2013:105–140.
Antigens. 2006;68:103–114. 2. HUGO Gene Nomenclature Committee. www.genenames.org/
12. Woof JM, Kerr MA. The function of immunoglobulin A in immu- Accessed March 10, 2016.
nity. J Path. 2006;208:270–282. 3. Heinrich PC, Behrmann I, Muller-Newen G, et al. Interleukin-6-
13. Woof JM, Mestecky J. Mucosal immunoglobulins. Immunol Rev. type cytokine signaling through the gp130/Jak/STAT pathway.
2005;206:64–82. Biochem J. 1998;334:297–314.
14. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. 4. Lucey DR, Clerici M, Shearer GM. Type 1 and type 2 cytokine
New York, NY: WH Freeman; 2013:415–450. dysregulation in human infectious, neoplastic, and inflammatory
15. Johansson SGO. The history of IgE: from discovery to 2010. Curr diseases. Clin Microbiol Rev. 1996;9:532–562.
Allergy Asthm R. 2011:11(2):173-177. 5. Glickstein LJ, Huber BT. Karoushi—death by overwork in the
16. Nezlin R, Ghetie V. Interactions of immunoglobulins outside the immune system. J Immunol. 1995;155:522–524.
antigen-combining site. Adv Immunol. 2004;82:155–215. 6. D’Elia RV, Harrison K, Oyston PC, et al. Targeting the “cytokine
17. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. storm” for therapeutic benefit. Clin Vaccine Immunol. 2013;
New York, NY: WH Freeman; 2013:225–259. 20(3):319–327.
18. MacKay IR. History of immunology in Australia: events and 7. Lim MS, Elenitoba-Johnson KSJ. The molecular pathology of
identities. Int Med J. 2006; 36:394–398. primary immunodeficiencies. J Mol Diag. 2004;6:59–83.
19. Jerne NK. The natural selection theory of antibody production. 8. Krumm B, Xiang Y, Deng J. Structural biology of the IL-1 super-
Proc Natl Acad Sci USA. 1955;41:849–857. family: key cytokines in the regulation of immune and inflam-
20. Burnet FM. A modification of Jerne’s theory of antibody pro- matory responses. Protein Sci. 2014;23(5):526–538.
duction using the concept of clonal selection. Aust J Sci. 1957; 9. Kanczkowski W, Alexaki V, Tran N, et al. Hypothalamo-pituitary
20:67–69. and immune-dependent adrenal regulation during systemic
21. Dreyer WJ, Bennett JC. The molecular basis of antibody forma- inflammation. Proc Natl Acad Sci. 2013;110(36):14801–14806.
tion: a paradox. Proc Natl Acad Sci USA. 1965;54:864. 10. Braun T, Schett G. Pathways for bone loss in inflammatory
22. Dudley DD, Chaudhuri J, Bassing CH, Alt FW. Mechanism disease. Curr Osteoporos Rep. 2012;10(2):101–108.
and control of V(d)J recombination versus class switch recombi- 11. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor
nation: Similarities and differences. Adv Immunol. 2005;86:43–112. superfamilies: integrating mammalian biology. Cell. 2001;104:
23. Cobb RM, Oestreich KJ, Osipovich OA, Oltz EM. Accessibility 487–501.
control of V(D)J recombination. Adv Immunol. 2006;91:45–110. 12. Schett G, Elewaut D, McInnes IB, et al. How cytokine networks
24. Chaplin DD. Overview of the immune system. J Allergy Clin fuel inflammation: toward a cytokine-based disease taxonomy.
Immunol. 2010;125:S3–S23. Nat Med. 2013;19(7):822–824.
25. Chaudhuri J, Basu U, Zarrin A, et al. Evolution of the immunoglob- 13. Lu XT, Zhao YX, Zhang Y, Jiang F. Psychological stress, vascular
ulin heavy chain class switch recombination mechanism. Adv inflammation, and atherogenesis: potential roles of circulating
Immunol. 2007;94:157–214. cytokines. J Cardiovasc Pharmacol. 2013;62(1):6–12.
26. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. 14. Kim CH. Chemokine-chemokine receptor network in immune
New York, NY: WH Freeman; 2013:653–692. cell trafficking. Curr Drug Targets Imune, Endocrine, Metab Disord.
27. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential 2004;4:343–361.
Immunology. 12th ed. Chichester, UK: Wiley-Blackwell; 2011: 15. Reiche EMV, Bonametti AM, Voltarelli JC, et al. Genetic polymor-
141–187. phisms in the chemokine and chemokine receptors: impact on
28. Buss NAPS, Henderson SJ, McFarlane M, et al. Monoclonal clinical course and therapy of the human immunodeficiency virus
antibody therapeutics: history and future. Curr Opin Pharmacol. type 1 infection (HIV-1). Curr Med Chem. 2007;14:1325–1334.
2012;12:615–622. 16. Travis MA, Sheppard D. TGF-β activation and function in immu-
29. Yamada T. Theraputic monoclonal antibodies. Keio J Med. nity. Annu Rev Immunol. 2014;32:51–82.
2011;60(2):37–46. 17. Van Weyenbergh J, Weitzerbin J, Rouillard D, et al. Treatment
30. Sapra P, Shor B. Monoclonal antibody-based therapies in of multiple sclerosis patients with interferon-beta primes mono-
cancer: advances and challenges. Pharmacol Therapeut. 2013; cyte-derived macrophages for apoptotic cell death. J Leukoc Biol.
138:452–469. 2001;70:745–748.
31. Biological therapies for cancer. www:cancer.gov/cancertopics/ 18. Carlson RJ, Doucette JR, Knox K, Nazarali AJ. Pharmacogenomics
factsheet/Therapy/biological. Accessed August 31, 2014. of interferon-β in multiple sclerosis: what has been accomplished
32. Kristensen LE, Saxne T, Nilsson J, Geborek P. Impact of concomi- and how can we ensure future progress? Cytokine Growth Factor
tant DMARD therapy on adherence to treatment with etanercept Rev. 2014;26(2):249–261.
and infliximab in rheumatoid arthritis. Results from a six-year 19. Melian EB, Plosker GL. Interferon alpha-1: a review of its phar-
observational study in southern Sweden. Arthritis Res Ther. macology and therapeutic efficacy in the treatment of chronic
2006;8(6):R174. hepatitis C. Drugs. 2001;61:1661–1691.
33. Abe T, Takeuchi T, Miyasaka N, et al. A multicenter, double-blind, 20. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential
randomized, placebo controlled trial of infliximab combined with Immunology. 12th ed. West Sussex, UK, Wiley-Blackwell; 2011:
low dose methotrexate in Japanese patients with rheumatoid 226–262.
arthritis. J Rheumatol. 2006;33(1):37–44. 21. Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD4+
34. Gurcan HM, Keskin DB, Stern JNH, et al. A review of the cur- T cells in immunity to viruses. Nat Rev Immunol. 2012;12(2):
rent use of rituximab in autoimmune diseases. Int Pharm. 2009; 136–148.
9:10–15.
22. Szabo SJ, Costa GL, Zhang X, Glimcher LH. A novel transcription 44. Bastarache JA, Koyama T, Wickersham NE, et al. Accuracy and
factor, T-bet, directs Th1 lineage commitment. Cell. 2000;100: reproducibility of a multiplex immunoassay platform: a validation
655–669. study. J Immunol Methods. 2011;367:33–39.
23. Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to 45. Lynch HE, Sanchez AM, D’Souza MP, et al. Development and
interferon γ. Ann Rev Immunol. 1997;15:749–795. implementation of a proficiency testing program for Luminex
24. Vahedi G, Poholek AC, Hand TW, et al. Helper T-cell identity and bead-based cytokine arrays. J Immunol Methods. 2014;409:62–71.
evolution of differential transcriptomes and epigenomes. Immunol 46. Bailey L, Moreno L, Manigold T, et al. A simple whole blood bioas-
Rev. 2013;252(1):24–40. say detects cytokine responses to anti-CD28SA and anti-CD52
25. Sugamura K, Asao H, Kondo M, et al. The interleukin-2 receptor antibodies. J Pharmacol Toxicol. 2013;68(2):231–239.
γ chain: its role in multiple cytokine receptor complexes and
T cell development in XSCID. Annu Rev Immunol. 1996;14:
179–205. 1. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed.
26. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. New York, NY: WH Freeman and Co.; 2013:187–224.
New York, NY: WH Freeman and Co.; 2013:299–328. 2. Flierman R, Daha MR. The clearance of apoptotic cells by
27. Assadullah K, Sterry W, Volk HD. Interleukin-10 therapy—review complement. Immunobiology. 2007;212(4–5):363–370.
of a new approach. Pharmacol Rev. 2003;55:241–269. 3. Lewis MJ, Botto M. Complement deficiencies in humans and
28. Wan YY, Flavell RA. The roles for cytokines in the generation animals: links to autoimmunity. Autoimmunity. 2006(Aug);39(5):
and maintenance of regulatory T cells. Immunol Rev. 2006;212: 367–378.
114–130. 4. Morgan P. Complement. In: Paul W, ed. Fundamental Immunology.
29. Fonenot JD, Rudensky AY. A well-adapted regulatory contrivance: 7th ed. Philadelphia, PA: Lippincott-Raven; 2013:863–890.
regulatory T cell development and the forkhead family transcrip- 5. Mak T, Saunder M. The Immune Response: Basic and Clinical
tion factor Foxp3. Nat Immunol. 2005;6:331–337. Principles. Burlington: Elsevier; 2004:553–581.
30. Basu R, Hatton RD, Weaver CT. The Th17 family: flexibility follows 6. Carroll MC, Fischer MB. Complement and the immune response.
function. Immunol Rev. 2013;252:89–103. Curr Opin Immunol. 1997 (Feb);9(1):64–69.
31. McKenz BS, Kastelein, RA, Cua, DJ. Understanding the 7. Pillemer L, Landy M, Shear MJ. The properdin system and immu-
IL-23-IL-17 immune pathway. Trends Immunol. 2006;27(1):17–23. nity. VII. Alterations in properdin levels and resistance to infection
32. Ghoreschi K, Laurence A, Yang XP, et al. T helper 17 cell hetero- in mice following the administration of tissue polysaccharides.
geneity and pathogenicity in autoimmune disease. Trends Immunol. J Exp Med. 1957 (Jul 1);106(1):99–110.
2011;32(9):395–401. 8. Massey H, McPherson R. Mediators of inflammation: comple-
33. Wilke CM, Bishop K, Fox D, Zou W. Deciphering the role of ment, cytokines and adhesion molecules. In: McPherson R,
Th17 cells in human disease. Trends Immunol. 2011;32(12):603–611. Pincus M, eds. Henry’s Clinical Diagnosis and Management by Lab-
34. Silverpil E, Linden A. IL-17 in human asthma. Expert Rev Respir oratory Methods. 21st ed. Philadelphia, PA: Elsevier Saunders;
Med. 2012;6(2):173–186. 2007:850–875.
35. Parissis J, Filippatos G, Adamopoulos S, et al. Hematopoietic 9. Janssen BJ, Christodoulidou A, McCarthy A, et al. Structure of
colony stimulating factors in cardiovascular and pulmonary remod- C3b reveals conformational changes that underlie complement
eling: promoters or inhibitors. Curr Pharmaceu Design. 2006;12: activity. Nature. 2006(Nov 9);444(7116):213–216.
2689–2699. 10. Walport MJ. Complement. First of two parts. N Engl J Med. 2001
36. Sloand EM, Kim S, Maciejewski JP, et al. Pharmacologic doses of (Apr 5);344(14):1058–1066.
granulocyte colony stimulating factor affect cytokine production 11. Matsushita M, Endo Y, Fujita T. Structural and functional
by lymphocytes in vitro and in vivo. Blood. 2000;95:2269–2274. overview of the lectin complement pathway: its molecular basis
37. Varlet-Mar E, Gaudard A, Audran M, et al. Pharmacokinetics/ and physiological implication. Arch Immunol Ther Exp (Warsz).
pharmacodynamics of recombinant human erythropoietins in 2013 (Aug);61(4):273–283.
doping control. Sports Med. 2003;33:301–315. 12. Medzhitov R, Janeway C, Jr. Innate immunity. N Engl J Med. 2000
38. Siddiqui MA, Scott LJ. Infliximab: a review of its use in Crohn’s (Aug 3);343(5):338–344.
disease and rheumatoid arthritis. Drugs. 2005;65(15):2179–2208. 13. Eisen DP, Minchinton RM. Impact of mannose-binding lectin on
39. Scott LJ. Etanercept: a review of its use in autoimmune inflam- susceptibility to infectious diseases. Clin Infect Dis. 2003 (Dec 1);
matory diseases. Drugs. 2014;74(12):1379–1410. 37(11):1496–1505.
40. Nwe SM, Champlain AH, Gordon KB. Rationale and early clinical 14. Frakking FN, Brouwer N, van Eijkelenburg NK, et al. Low
data on IL-17 blockade in psoriasis. Expert Rev Clin Immunol. mannose-binding lectin (MBL) levels in neonates with pneumonia
2013;9(7):677–682. and sepsis. Clin Exp Immunol. 2007 (Nov);150(2):255–262.
41. Genovese MC, Greenwald M, Chul-Soo C, et al. A phase II random- 15. Heitzeneder S, Seidel M, Forster-Waldl E, Heitger A. Mannan-
ized study of subcutaneous ixekizumab, an anti-interleukin-17 binding lectin deficiency—good news, bad news, doesn’t matter?
monoclonal antibody, in rheumatoid arthritis patients who were Clin Immunol. 2012 (Apr);143(1):22–38.
naïve to biologic agents or had an inadequate response to tumor 16. Mollnes TE, Song WC, Lambris JD. Complement in inflamma-
necrosis factor inhibitors. Arthritis Rheum. 2014;66(7):1693–1704. tory tissue damage and disease. Trends Immunol. 2002 (Feb);
42. Busse WW, Holgate S, Kerwin E, et al. Randomized, double-blind, 23(2):61–64.
placebo-controlled study of brodalumab, a human anti-IL-17 17. Schwaeble WJ, Reid KB. Does properdin crosslink the cellular
receptor monoclonal antibody, in moderate to severe asthma. and the humoral immune response? Immunol Today. 1999 (Jan);
Am J Respir Crit Care Med. 2013;188(11):1294–1302. 20(1):17–21.
43. Li Y, Hua S. Mechanisms of pathogenesis in allergic asthma: role 18. Xu Y, Narayana SV, Volanakis JE. Structural biology of the alterna-
of interleukin-23. Respirology. 2014;19:663–669. tive pathway convertase. Immunol Rev. 2001 (Apr);180:123–135.
19. Arlaud GJ, Barlow PN, Gaboriaud C, et al. Deciphering comple- the three pathways of the complement system. In: Detrick B,
ment mechanisms: the contributions of structural biology. Mol Hamilton R, Folds J, eds. Manual of Molecular and Clinical Lab-
Immunol. 2007 (Sep);44(16):3809–3822. oratory Immunology. 7th ed. Washington, DC: ASM Press; 2006:
20. Pangburn MK, Rawal N. Structure and function of complement 124–127.
C5 convertase enzymes. Biochem Soc Trans. 2002 (Nov);30(Pt 6):
1006–1010.
21. Smith BO, Mallin RL, Krych-Goldberg M, et al. Structure of the 1. Strasinger SK, DiLorenzo MS. Urinalysis and Body Fluids. 6th ed.
C3b binding site of CR1 (CD35), the immune adherence receptor. 2014, Philadelphia, PA: F.A. Davis; 2014.
Cell. 2002 (Mar 22);108(6):769–780. 2. Clinical and Laboratory Standards Institute: Protection of labo-
22. DiScipio RG. Ultrastructures and interactions of complement ratory workers from occupationally acquired infections: approved
factors H and I. J Immunol. 1992 (Oct 15);149(8):2592–2599. guideline, ed 4, CLSI Document M29-A4, 2014. Clinical and
23. Carroll MC. The complement system in regulation of adaptive Laboratory Standards Institute, Wayne, PA.
immunity. Nat Immunol. 2004 (Oct);5(10):981–986. 3. Centers for Disease Control and Prevention. Guideline for hand-
24. Carroll MC. The complement system in B cell regulation. Mol washing hygiene in health-care settings. MMWR. 2002;51(rr16):
Immunol. 2004 (Jun);41(2–3):141–146. 1–48. www.cdc.gov/handhygiene/ Accessed May 8, 2015.
25. Carroll MC, Isenman DE. Regulation of humoral immunity by 4. NIOSH Alert. Preventing allergic reactions to natural rubber latex
complement. Immunity. 2012 (Aug 24);37(2):199–207. in the workplace. DHHS (NIOSH) Publication 97–135. National
26. Laursen NS, Magnani F, Gottfredsen RH, et al. Structure, function Institute for Occupational Safety and Health, Cincinnati, OH, 1997.
and control of complement C5 and its proteolytic fragments. Curr 5. Centers for Disease Control and Prevention. Guidelines for isolation
Mol Med. 2012 (Sep);12(8):1083–1097. precautions: preventing transmission of infectious agents in health-
27. Wen L, Atkinson JP, Giclas PC. Clinical and laboratory evaluation care settings. 2007. www.cdc.gov/hicpac/2007IP/2007isolation
of complement deficiency. J Allergy Clin Immunol. 2004 (Apr); Precautions.html. Accessed April 30, 2015.
113(4):585–593; quiz 594. 6. Occupational Exposure to Bloodborne Pathogens, Final Rule.
28. Sjoholm AG, Jonsson G, Braconier JH, et al. Complement defi- Federal Register. 1991 (Dec 6);56:Federal register number 64004:
ciency and disease: an update. Mol Immunol. 2006 (Jan);43(1-2): Standard number 1910.1030.
78–85. 7. Occupational Safety and Health Administration. Enforcement pro-
29. Mayilyan KR. Complement genetics, deficiencies, and disease cedures for the occupational exposure to bloodborne pathogens
associations. Protein Cell. 2012 (Jul);3(7):487–496. standard. Directive CPL 02–02-069. Washington, DC, 2001.
30. Pickering MC, Botto M, Taylor PR, et al. Systemic lupus erythe- https://www.osha.gov/pls/oshaweb/owadisp.show_document?p_t
matosus, complement deficiency, and apoptosis. Adv Immunol. able=directives&p_id=2570. Accessed May 30, 2015.
2000;76:227–324. 8. CDC, Updated U.S. Public Health Service guidelines for the
31. Skattum L, van Deuren M, van der Poll T, Truedsson L. Comple- management of occupational exposures to HBV, HCV, and HIV
ment deficiency states and associated infections. Mol Immunol. and recommendations for post-exposure prophylaxis. MMWR.
2011 (Aug);48(14):1643–1655. 2001;50(RR11):1–42. www.cdc.gov. Accessed April 15, 2015.
32. Kerr FK, Thomas AR, Wijeyewickrema LC, et al. Elucidation of 9. Clinical Laboratory Standards Institute: protection of laboratory
the substrate specificity of the MASP-2 protease of the lectin com- workers from occupationally acquired infections: approved guide-
plement pathway and identification of the enzyme as a major line. 3rd ed. CLSI Document M29-A3, Clinical and Laboratory
physiological target of the serpin, C1-inhibitor. Mol Immunol. Standards Institute, Wayne, PA, 2005, CLSI.
2008 (Feb);45(3):670–677. 10. International Air Transportation Association Regulations. www.
33. Mollnes TE, Jokiranta TS, Truedsson L, et al. Complement analysis iata.org/whatwedo/cargo/dgr/Documents/infectious-substance-
in the 21st century. Mol Immunol. 2007 (Sep);44(16):3838–3849. classification-DGR56-en.pdf. Accessed December 15, 2015.
34. Hughes J, Nangaku M, Alpers CE, et al. C5b-9 membrane attack 11. U.S. Department of Transportation. Transporting infectious
complex mediates endothelial cell apoptosis in experimental substances safely. https://hazmatonline.phmsa.dot.gov/services/
glomerulonephritis. Am J Physiol Renal Physiol. 2000 (May);278(5): publication_documents/Transporting%20Infectious%20
F747–F757. Substances%20Safely.pdf. Accessed December 8, 2014.
35. Frazer-Abel A, Giclas PC. Update on laboratory tests for the diag- 12. Hazardous materials: infectious substances; harmonizing with
nosis and differentiation of hereditary angioedema and acquired the United Nations recommendations. www.federalregister.gov/
angioedema. Allergy Asthma Proc. 2011 (Sep-Oct);32(suppl 1): articles/2005/05/19/05-9717/hazardous-materials-infectious-
S17–S21. substances-harmonization-with-the-UN recommendations.
36. Salvadori M, Bertoni E. Update on hemolytic uremic syndrome: Accessed December 6, 2015.
diagnostic and therapeutic recommendations. World J Nephrol. 13. Baer DM. Standards for transporting specimens. MLO. 2005;
2013 (Aug 6);2(3):56–76. 37(11):38.
37. Nicolas C, Vuiblet V, Baudouin V, et al. C3 nephritic factor asso- 14. Occupational Safety and Health Administration. Needlestick re-
ciated with C3 glomerulopathy in children. Pediatr Nephrol. 2014 quirements take effect April 18. OSHA, Washington, DC, 2001.
(Jan);29(1):85–94. www.osha gov/needlesticks/needlefaq.html. Accessed June 3, 2015.
38. Giclas PC. Analysis of complement in the clinical laboratory. 15. Occupational Safety and Health Administration. Revision to
In: Detrick B, Hamilton R, Folds J, eds. Manual of Molecular and OSHA’s bloodborne pathogens standard. 2001. www.osha.gov/
Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM SLTC/bloodbornepathogens. Accessed July 18, 2016.
Press; 2006:115–117. 16. Occupational exposure to hazardous chemicals in laboratories,
39. Seelen M, Roos A, Wieslander J, Daha M. An enzyme-linked final rule. Federal Register. 1990(Jan 31);55. Accessed December 5,
immunosorbant assay-based method for functional analysis of 2015.
17. Occupational Safety and Health Administration. Laboratory 9. Tille P. Bailey and Scott’s Diagnostic Microbiology. 13th ed. St. Louis:
Safety Chemical Hygiene Plan. OSHA’s Occupational Exposure to Elsevier Mosby; 2014:142–152.
Hazardous Chemical in Laboratories Standard. (29 CFR 1910. 10. Alper CA, Johnson AM. Immunofixation electrophoresis: a
1450). Jan 22, 2013. technique for the study of protein polymorphism. Vox Sang.
18. Centers for Medicare and Medicaid Services, Department of 1969;17:445.
Health and Human Services: Current Clinical Laboratory Improve- 11. Levinson SS. Urine immunofixation electrophoresis remains
ment Amendments regulations and guidelines. www.cms.gov/ important and is complementary to serum free light chain. Clin
Regulations-and-Guidance/CLIA. Accessed May 1, 2015. Chem Lab Med. 2011;49(11):1801–1804.
19. College of American Pathologists: Commission on Laboratory 12. Csako G. Immunofixation electrophoresis for identification of
Accreditation, Immunology Checklist. College of American proteins and specific antibodies. Methods Mol Biol. 2012;869:
Pathologists, Skokie, IL, 2007. 147–171.
20. Strasinger SK, DiLorenzo MS. The Phlebotomy Textbook. 3rd ed. 13. Lapage G. Dr. HE Durham. Nature. 1945;156:742.
Philadelphia, PA: F.A. Davis; 2011. 14. Blaney KD, Howard PR. Basic and Applied Concepts of Blood
21. Centers for Medicare and Medicaid Services, Department of Banking and Transfusion Practices. 3rd ed. St. Louis: Elsevier
Health and Human Services: Clinical Laboratory Improvement Mosby; 2013:1–27.
Amendments, Brochure #1: Equivalent quality control procedures. 15. Gaikwad UN, Rajurkar M. Diagnostic efficiency of Widal slide
www.cms.gov/Regulations-and-Guidance/Legislation/CLIA/ agglutination test against Widal tube agglutination test in enteric
CLIA_Brochures.html. Accessed May 5, 2015. fever. Int J Med Public Health. 2014;4(3):227–230.
22. Clinical and Laboratory Standards Institute (CLSI): Quality Prac- 16. Tille P. Bailey and Scott’s Diagnostic Microbiology. 13th ed. St. Louis:
tices in Noninstrumented Point of Care Testing: An Instructional Elsevier Mosby; 2014:133–141.
Manual and Resources for Health Care Workers. Approved Guide-
line. CLSI document POCT08-A, Wayne, PA, 2010.
23. Centers for Medicare and Medicaid Services, Department of 1. Kricka LJ, Park JY. Principles of immunochemical techniques. In:
Health and Human Services: Clinical Laboratory Improvement Burtis CA, Bruns DE, eds. Tietz Fundamentals of Clinical Chemistry
Amendments, Brochure #4: Equivalent quality control procedures. and Molecular Diagnostics. 7th ed. St. Louis: Elsevier Saunders;
www.cms.gov/Regulations-and- Guidance/Legislation/CLIA/index. 2015:236–253.
html. Accessed May 6, 2015. 2. Ashihara Y, Kasahara Y, Nakamura RM. Immunoassays and im-
munochemistry. In: McPherson RA, Pincus MR, eds. Henry’s
Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
1. Estridge BH, Reynolds AP. Basic Clinical Laboratory Techniques. Philadelphia, PA: Saunders Elsevier; 2013:851–875.
5th ed. Clifton Park, NY: Thomson Delmar Learning; 2008:85–96. 3. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin
2. Lo SF. Principles of basic techniques and laboratory safety. In: in man. J Clin Invest. 1960;39:1157.
Burtis CA, Bruns DE, eds. Tietz Fundamentals of Clinical Chemistry 4. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed.
and Molecular Diagnostics. 7th ed. St. Louis: Elsevier Saunders; New York, NY: WH Freeman and Co.; 2013:653–692.
2015:107–128. 5. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential
3. Ashwood ER, Bruns DE. Clinical evaluation of methods. In: Immunology. 12th ed. Oxford, Blackwell Publishing, Ltd; 2011:
Burtis CA, Bruns DE, eds. Tietz Fundamentals of Clinical Chemistry 141–187.
and Molecular Diagnostics. 7th ed. St. Louis: Elsevier Saunders; 6. Siemens Healthcare Diagnostics. usa.healthcare.siemens.com/
2015:32–39. drug-testing-diagnostics. Accessed January 11, 2015.
7. Coons AH, Creech HJ, Jones RN. Immunological properties of
an antibody containing a fluorescent group. Proc Soc Expt Biol
1. Male D, Brostoff J, Roth DB, Roitt IM. Immunology. 8th ed. Philadel- Med. 1941;47:200–202.
phia, PA: Elsevier Saunders; 2013:51–70. 8. Kricka LJ, Park JY. Optical techniques. In: Burtis CA, Bruns
2. Ashihara Y, Kasahara Y, Nakamura RM. Immunoassay and DE, eds. Tietz Fundamentals of Clinical Chemistry and Molecular
immunochemistry. In: McPherson RA, Pincus MR, eds. Henry’s Diagnostics. 7th ed. St. Louis, MO: Elsevier Saunders; 2015:
Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. 129–150.
Philadelphia, PA: Elsevier Saunders; 2011:851–876. 9. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology.
3. Kricka LJ, Park JY. Immunochemical techniques. In: Burtis CA, Bruns 7th ed. Philadelphia, PA: Elsevier Saunders; 2013:317–321.
DE, eds. Tietz Fundamentals of Clinical Chemistry and Molecular Diag- 10. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 7th ed.
nostics. 7th ed. St. Louis: Elsevier Saunders; 2013:236–253. Philadelphia, PA: Elsevier Saunders; 2013:381–389.
4. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential Immunol- 11. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 7th ed.
ogy. 12th ed. Oxford, UK: Wiley-Blackwell; 2011:113–140. Philadelphia, PA: Elsevier Saunders; 2013:350–363.
5. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. 12. Tozzoli R, Bonaguri C, Melegari A, et al. Current state of diagnostic
New York, NY: WH Freeman; 2013:653–692. technologies in the autoimmunity laboratory. Clin Chem Lab Med.
6. Kricka LJ, Park JY. Optical techniques. In: Burtis CA, Bruns DE, 2013;51(1):129–138.
eds. Tietz Fundamentals of Clinical Chemistry and Molecular Diag- 13. Menegatti E, Berardi D, Messina M, et al. Lab-on-a-chip: emerging
nostics. 7th ed. St. Louis: Elsevier Saunders; 2013:129–150. analytical platforms for immune-mediated diseases. Autoimmun
7. Mancini G, Carbonara AO, Heremans JF. Immunochemical quanti- Rev. 2013;12:814–820.
tation of antigens by single radial immunodiffusion. Immunochem. 14. Abbott Diagnostics. www.abbottdiagnostics.com/en-us/index:
1965;2:235. html. Accessed January 15, 2015.
8. Fahey JL, McKelvey EM. Quantitative determination of serum 15. Medical Solutions—Siemens Healthineers USA. www.usa.
immunoglobulins in antibody-agar plates. J Immunol. 1965;94:84. healthcare.siemens.com/immunoassay. Accessed January 12, 2015.
 syncytial virus or rhinovirus infection. Pediatr Infect Dis J. 2013;
1. Buckingham L. Molecular Diagnostics: Fundamentals, Methods and 32:1199–1204.
Clinical Applications. 2nd ed. Philadelphia, PA: F.A. Davis; 2012. 21. Ngou J, Magooa MP, Gilham C, et al. HARP study group: com-
2. Kuehn HS, Ouyang W, Lo B, et al. Immune dysregulation in parison of careHPV and hybrid capture 2 assays for detection
human subjects with heterozygous germline mutations in of high-risk human papillomavirus DNA in cervical samples
CTLA4. Science. 2014;346:1623–1627. from HIV-1-infected African women. J Clin Microbiol. 2013;51:
3. Lucas CL, Kuehn HS, Zhao F, et al. Dominant-activating germline 4240–4242.
mutations in the gene encoding the PI(3)K catalytic subunit 22. Clavel C, l Bory JP, Rihet S, et al. Comparative analysis of human
p110_ result in T cell senescence and human immunodeficiency. papillomavirus detection by hybrid capture assay and routine
Nat Immunol. 2014;15:88–97. cytologic screening to detect high-grade cervical lesions. Int J
4. Hornakova T, Chiaretti S, Lemaire MM, et al. ALL-associated Cancer. 1998;75:525–528.
JAK1 mutations confer hypersensitivity to the antiproliferative 23. Luu H, Adler-Storthz K, Dillon LM, et al. Comparing the perfor-
effect of type I interferon. Blood. 2010;115:3287–3295. mance of hybrid capture II and polymerase chain reaction (PCR)
5. International HapMap Project. hapmap.ncbi.nim.nih.gov. Accessed for the identification of cervical dysplasia in the screening and
May 24, 2016. diagnostic settings. Clin Med Insights Oncol. 2013;25:247–255.
6. Anthony Nolan Research Institute. hla.alleles.org. Accessed May 24. Schildhaus H, Deml KF, Schmitz K, et al. Chromogenic in situ
25, 2015. hybridization is a reliable assay for detection of ALK rearrange-
7. Kaixiong Y, Lua J, Mac F, et al. Extensive pathogenicity of mito- ments in adenocarcinomas of the lung. Modern Pathol. 2013;
chondrial heteroplasmy in healthy human individuals. P Natl 26:1468–1477.
Acad Sci. 2014;111:10654–10659. 25. Tanner M, Gancberg D, Di Leo A, et al. Chromogenic in situ
8. Palmieri C, Magi G, Creti R, Baldassarri L. Interspecies mobiliza- hybridization: a practical alternative for fluorescence in situ
tion of an ermT-carrying plasmid of Streptococcus dysgalactiae hybridization to detect HER-2/neu oncogene amplification in
subsp. equisimilis by a coresident ICE of the ICESa2603 family. archival breast cancer samples. Am J Pathol. 2000;157:1467–1472.
J Antimicrob Chemoth. 2013;68:23–26. 26. Bint S, Davies AF, Ogilvie CM. Multicolor banding remains an
9. Southern E. Problem solved: an interview with Sir Edwin South- important adjunct to array CGH and conventional karyotyping.
ern. Interviewed by Jane Gitschier. PLOS Genet. 2013;9:e1003344. Mol Cytogenet. 2013;6:55.
10. Glenn G, Andreou LV. Analysis of DNA by Southern blotting. 27. Mullis K. Target amplification for DNA analysis by the polymerase
Method Enzymol. 2013;529:47–63. chain reaction. Ann Biol Clin. 1990;48:579–582.
11. Jeffreys A, Wilson V, Thein SL. Hypervariable “minisatellite” regions 28. Myers T, Gelfand DH. Reverse transcription and DNA amplifica-
in human DNA. Nature. 1985;314:67–73. tion by a Thermus thermophilus DNA polymerase. Biochemistry.
12. Tang S, Halberg MC, Floyd KC, Wang J. Analysis of common mi- 1991;30:7661–7666.
tochondrial DNA mutations by allele-specific oligonucleotide and 29. Chen Q, Lu P, Jones AV, et al. Detection of JAK2 V617F mutation
Southern blot hybridization. Method Mol Biol. 2012;837:259–279. in chronic myeloproliferative disorders. J Mol Diagn. 2007;9:
13. Pezeshkpoor B, Rost S, Oldenburg J, El-Maarri O. Identification 272–276.
of a third rearrangement at Xq28 that causes severe hemophilia 30. Tonks S, Marsh SG, Bunce M, Bodmer JG. Molecular typing for
A as a result of homologous recombination between inverted HLA class I using ARMS-PCR: further developments following
repeats. Journal of Thromb Haemost. 2012;10:1600–1608. the 12th International Histocompatibility Workshop. Tissue
14. Brown T, Mackey K. Analysis of RNA by Northern blot hybridiza- Antigens. 1999;53:175–183.
tion. Curr Protoc Hum Genet. 2001;30:A.3K.1–A.3K.12. 31. Lee H, Ladd C, Bourke MT, et al. DNA typing in forensic sci-
15. Alvarez K, Kash SF, Lyons-Weiler MA, et al. Reproducibility and ence. I. Theory and background. Am J Foren Med Path. 1994;15:
performance of virtual karyotyping with SNP microarrays for the 269–282.
detection of chromosomal imbalances in formalin-fixed paraffin- 32. Moretti T, Baumstark AL, Defenbaugh DA, et al. Validation of
embedded tissues. Diagn Mol Pathol. 2010;19:127–134. STR typing by capillary electrophoresis. J Forensic Sci. 2001;46:
16. Monzon F, Alvarez K, Gatalica Z, et al. Detection of chromosomal 661–676.
aberrations in renal tumors: a comparative study of conventional 33. Deschoolmeester V, Baay M, Wuyts W, et al. Comparison of three
cytogenetics and virtual karyotyping with single-nucleotide commonly used PCR-based techniques to analyze MSI status in
polymorphism microarrays. Arch Pathology Lab Med. 2009;133: sporadic colorectal cancer. J Clin Lab Anal. 2006;20:52–61.
1917–1922. 34. Wang Y, Li J, Cragun J, et al. Lynch syndrome related endometrial
17. Xiaoyang M, Haiquan Z, Huanying Z, et al. Global analysis of cancer: clinical significance beyond the endometrium. J Hematol
differential expressed genes in ECV304 Endothelial-like cells Oncol. 2013;6:22.
infected with human cytomegalovirus. Afr Health Sci. 2013; 35. Seneca S, Lissens W, Endels K, et al. Reliable and sensitive detection
13:243–251. of fragile X (expanded) alleles in clinical prenatal DNA samples
18. Fattal I, Shental N, Molad Y, et al. EBV antibodies mark SLE and with a fast turnaround time. J Mol Diagn. 2012;14:560–568.
scleroderma patients negative for anti-DNA. Immunology. 2014; 36. Jama M, Millson A, Miller CE, Lyon E. Triplet repeat primed PCR
141(2):276–285. simplifies testing for Huntington disease. J Mol Diagn. 2013;
19. Lachmann N, Todorova K, Schulze H, Schönemann C. Luminex(®) 15(2):255–262.
and its applications for solid organ transplantation, hematopoietic 37. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis:
stem cell transplantation, and transfusion. Transfus Med Hemoth. real-time monitoring of DNA amplification reactions. Biotechnol.
2013;40:182–189. 1993;11:1026–1030.
20. Esposito S, Zampiero A, Terranova L, et al. Pneumococcal bacte- 38. von Wurmb-Schwark N, Higuchi R, Fenech AP, et al. Quantifi-
rial load colonization as a marker of mixed infection in children cation of human mitochondrial DNA in a real time PCR. Forensic
with alveolar community-acquired pneumonia and respiratory Sci Int. 2002;126:34–39.
39. Espy M, Uhl JR, Sloan LM, et al. Real-time PCR in clinical micro- polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment
biology: applications for routine laboratory testing. Clin Microbiol and comparison with patient controls. Orphanet J Rare Dis.
Rev. 2006;19:165–256. 2013;8:1–8.
40. Zhao H, Wilkins K, Damon IK, Li Y. Specific qPCR assays for the 58. Maxam A, Gilbert W. A new method for sequencing DNA. P Natl
detection of orf virus, pseudocowpox virus and bovine papular Acad Sci. 1977;74:560–564.
stomatitis virus. J Virol Methods. 2013;194:229–234. 59. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-
41. Otero-Estévez O, Martínez-Fernández M, Vázquez-Iglesias L, terminating inhibitors. P Natl Acad Sci. 1977;74:5463–5467.
et al. Decreased expression of alpha-L-fucosidase gene FUCA1 in 60. Wirtz C, Sayer D. Data analysis of HLA sequencing using Assign-
human colorectal tumors. Int J Mol Sci. 2013;14:16986–16998. SBT v3.6+ from Conexio. Meth Mol Biol. 2012;882:87–121.
42. Korthals M, Sehnke N, Kronenwett R, et al. Molecular monitoring 61. Santamaria P, Lindstrom AL, Boyce-Jacino MT, et al. HLA class I
of minimal residual disease in the peripheral blood of patients with sequence-based typing. Hum Immunol. 1993;37:39–50.
multiple myeloma. Biol Blood Marrow Tr. 2013;19:1109–1115. 62. Dunn P. Human leucocyte antigen typing: techniques and tech-
43. Gendzekhadze K, Gaidulis L, Senitzer D. Chimerism testing by nology, a critical appraisal. Int J Immunogenet. 2011;38:463–473.
quantitative PCR using Indel markers. Method Mol Biol. 2013; 63. Nyrén P. The history of pyrosequencing. Meth Mol Biol. 2007;
1034:221–237. 373:1–14.
44. Eikmans M, Claas FH. HLA-targeted cell sorting of microchimeric 64. Harrington C, Lin EI, Olson MT, Eshleman JR. Fundamentals of
cells opens the way to phenotypical and functional characteriza- pyrosequencing. Arch Pathol Lab Med. 2013;137:1296–1303.
tion. Chimerism. 2011;2:114–116. 65. The International Genome Sample Resource. www.1000genomes
45. Keslar K, Lin M, Zmijewska AA, et al. Multicenter evaluation of .org. Accessed May 21, 2015.
a standardized protocol for noninvasive gene expression profiling. 66. Gabriel C, Danzer M, Hackl C, et al. Rapid high-throughput
Am J Transplant. 2013;13:1891–1897. human leukocyte antigen typing by massively parallel pyrose-
46. Kwoh D, Davis GR, Whitfield KM, et al. Transcription-based quencing for high-resolution allele identification. Hum Immunol.
amplification system and detection of amplified human immun- 2009;70:960–964.
odeficiency virus type 1 with a bead-based sandwich hybridiza- 67. Lank S, Wiseman RW, Dudley DM, O’Connor DH. A novel single
tion format. P Natl Acad Sci. 1989;86:1173–1777. cDNA amplicon pyrosequencing method for high-throughput, cost-
47. Nelson N, Cheikh AB, Matsuda E, Becker MM. Simultaneous effective sequence-based HLA class I genotyping. Hum Immunol.
detection of multiple nucleic acid targets in a homogeneous 2010;71:1011–1017.
format. Biochemistry. 1996;35:8429–8438.
48. Giachetti C, Linnen JM, Kolk DP, et al. Highly sensitive multiplex
assay for detection of human immunodeficiency virus type 1 and 1. Golightly, MG. Dihydrorhodamine (DHR) flow cytometry test for
hepatitis C virus RNA. J Clinical Microbiol. 2002;40:2408–2419. chronic granulomatous disease (CGD): a simple test for routine
49. McClernon D, Vavro C, St Clair M. Evaluation of a real-time nu- clinical flow cytometry. Int Clin Cytom Soc. 2011 (Winter);2(1).
cleic acid sequence-based amplification assay using molecular 2. Borowitz MJ, Craig FE, Digiuseppe JA, et al. Guidelines for the
beacons for detection of human immunodeficiency virus type 1. diagnosis and monitoring of paroxysmal nocturnal hemoglobin-
J Clinical Microbiol. 2006;44:2280–2282. uria and related disorders by flow cytometry. Cytometry B Clin
50. Mercier-Delarue S, Vray M, Plantier JC, et al. Higher specificity Cytom. 2010(Jul);78(4):211–230.
of NASBA isothermal technology versus real-time PCR for HIV-1 3. Kricka LJ, Park, JY. Optical techniques. In: Burtis CA, Bruns DE,
RNA quantification on dried blood spots. J Clinical Microbiol. eds. Tietz Fundamentals of Clinical Chemistry and Molecular Diag-
2013;51(5):787–798. nostics. 7th ed. St. Louis: Elsevier Saunders; 2015:129–150.
51. Gill P, Ramezani R, Amiri MV, et al. Enzyme-linked immunosor- 4. National Committee for Clinical Laboratory Standards. Clinical
bent assay of nucleic acid sequence-based amplification for applications of flow cytometry quality assurance and immunophe-
molecular detection of M tuberculosis. Biochem Bioph Res Co. notyping of peripheral blood lymphocytes. H42-A. National Com-
2006;347:1151–1157. mittee for Clinical Laboratory Standards, Wayne, PA, 1998.
52. Altunay H, Kosan E, Birinci I, et al. Are isolated anti-HBc blood 5. Van Dongen JJM, Lhermitte L, Bottcher S, et al. EuroFlow anti-
donors in high risk group? The detection of HBV DNA in isolated body panels for standardized n-dimensional flow cytometric
anti-HBc cases with nucleic acid amplification test (NAT) based immunophenotyping of normal, reactive and malignant leuko-
on transcription-mediated amplification (TMA) and HBV dis- cytes. Leukemia. 2012;26:1908–1975.
crimination. Transfus Apher Sci. 2010;43:265–268. 6. Renzi P, Ginns LC. Analysis of T cell subsets in normal adults.
53. Barany F. The ligase chain reaction in a PCR world. PCR Meth Comparison of whole blood lysis technique to Ficoll-Hypaque
Appl. 1991;1:5–16. separation by flow cytometry. Immunol Meth. 1987;98:53–56.
54. Walker G, Linn CP. Detection of Mycobacterium tuberculosis DNA 7. Carter PH, Resto-Ruiz S, Washington GC, et al. Flow cytometric
with thermophilic strand displacement amplification and fluo- analysis of whole blood lysis, three anticoagulants, and five cell
rescence polarization. Clin Chem. 1996;42:1604–1608. preparations. Cytometry. 1992;13:68–74.
55. Spears P, Linn CP, Woodard DL, Walker GT. Simultaneous strand 8. Cortelazzo S, Ponzoni M, Ferreri AJ, Hoelzer D. Lymphoblastic
displacement amplification and fluorescence polarization detection lymphoma. Crit Rev Oncol Hematol. 2011 (Sep);79(3):330–343.
of Chlamydia trachomatis DNA. Biochemistry. 1997;247:130–137. 9. Harris HL, Jaffe ES, Stein H, et al. A revised European-American
56. Wheeler H, Skinner CJ, Khunda A, et al. Molecular testing (strand classification of lymphoid neoplasms: a proposal from the Inter-
displacement assay) for identification of urethral gonorrhoea in national Lymphoma Study Group. Blood. 1994;84:1361–1392.
men: can it replace culture as the gold standard? Int J STD AIDS. 10. Vardiman JW. The World Health Organization classification of
2005;16:430–432. neoplastic diseases of the hematopoietic and lymphoid tissues.
57. van Heumen B, Roelofs HM, Te Morsche RH, et al. Duodenal An overview with emphasis on myeloid neoplasms. Chem Biol
mucosal risk markers in patients with familial adenomatous Interact. 2010 (Mar 19);184(1-2):16–20.
11. Wood BL. Immunophenotyping of leukemia and lymphoma by 4. Luccioli S, Escobar-Gutierrez A, Bellanti JA. Allergic diseases
flow cytometry. In: Detrick B, Hamilton RG, Folds JD, et al., eds. and asthma. In: Bellanti JA, Escobar-Gutierrez A, Tsokos GC, eds.
Manual of Molecular and Clinical Laboratory Immunology. 7th ed. Immunology IV: Clinical Applications in Health and Disease.
Washington, DC: ASM Press; 2006:171–186. Bethesda, MD: I Care Press; 2012:685–765.
12. Bleesing JJH, Fleisher TA. Immunophenotyping. Semin Hematol. 5. Stone KD, Prussin C, Metcalfe DD. IgE, mast cells, basophils, and
2001;38(2):100. eosinophils. J Allergy Clin Immunol. 2010;125:S73–S80.
13. Centers for Disease Control and Prevention. Laboratory Testing 6. Boyce JA. The biology of the mast cell. Allergy Asthma Proc.
for the Diagnosis of Human Immunodeficiency Virus (HIV) In- 2004;25:27–30.
fection. Division of HIV/AIDS Prevention, National Center for 7. MacGalshan D. IgE receptor and signal transduction in mast cells
HIV/AIDS, Viral Hepatitis, and TB Prevention. June, 2014. and basophils. Curr Opin Immunol. 2008;20:717–723.
14. Giorgi JVA, Kesson M, Chou CC. Immunodeficiency and infectious 8. Homberger H. Allergic diseases. In: McPherson RA, Pincus MR,
diseases. In: Rose NL, deMacario CE, Fahey JL, Friedman H, Henry JB, eds. Henry’s Clinical Diagnosis and Management by
Penn GM, eds. Manual of Clinical Laboratory Immunology. 4th ed. Laboratory Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders;
Washington, DC: ASM Press; 1992:174–181. 2011:1021–1033.
15. Srinivasula S, Lempicki RA, Adelsberger JW, et al. Differential 9. Brown JM, Wilson TM, Metcalfe DD. The mast cell and allergic
effects of HIV viral load and CD4 count on proliferation of naïve diseases: role in pathogenesis and implications for therapy. Clin
and memory CD4 and CD8 T lymphocytes. Blood. 2011 (Jul 14); Exp Allergy. 2007;38:4–18.
118(2):262–270. 10. Parham P. IgE-mediated immunity and allergy. In: The Immune
16. Margolick JB, Munoz A, Donnenberg A, et al. Failure of T-cell System. 4th ed. New York, NY: Garland Science; 2015:401–431.
homeostasis preceding AIDS in HIV infection. Nat Med. 1995; 11. Vercilli D. Discovering susceptibility genes for asthma and allergy.
1:674–680. Nat Rev Immunol. 2008;8:169–182.
17. Alexiou GA, Vartholomatos E, Gousssia A, et al. DNA content is 12. 12. Holloway JA, Yang IA, Holgate ST. Genetics of allergic disease.
associated with malignancy of intracranial neoplasms. Clin Neurol J Allergy Clin Immunol. 2010;125:S81-94.
Neurosurg. 2013(Sep);115(9):1784–1787. 13. Granada M, Wilk JB, Tuzova M, et al. A genome-wide association
18. Chen JC, Davis BH, Wood B, Warzynski MJ. Multicenter clinical study of plasma total IgE concentrations in the Framingham heart
experience with flow cytometric method for fetomaternal hem- study. J Allergy Clin Immun. 2012;129(3):840–845.e21.
orrhage detection. Cytometry. 2002;50:285–290. 14. Vercelli D. Remembrance of things past: HLA genes come back
19. Fernandes BJ, vonDadelszen P, Fazal I, et al. Flow cytometric on the allergy stage. J Allergy Clin Immunol. 2012;129:846–847.
assessment of feto-maternal hemorrhage; a comparison with 15. Wlasiuk G, Vercelli D. The farm effect, or: when, what and how
Betke-Kleihauer. Prenat Diagn. 2007;27(7):641–643. a farming environment protects from asthma and allergic disease.
20. Remaley AT, Hortin GL. Protein analysis for diagnostic applica- Curr Opin Allergy CI. 2012;12(5):461–466.
tions. In: Detrick B, Hamilton RG, Folds JD, et al., eds. Manual of 16. Fishbein AB, Fuleihan RL. The hygiene hypothesis revisited: does
Molecular and Clinical Laboratory Immunology. 7th ed. Washington, exposure to infectious agents protect us from allergy? Curr Opin
DC: ASM Press; 2006:7–21. Pediatr. 2012;24(1):98–102.
21. College of American Pathologists. www.cap.org/apps/docs/cap_ 17. American Academy of Allergy, Asthma & Immunology. Allergy
today/surveys/0608_ImmunoSurvey.pdf. Accessed May 26, 2015. statistics. 2015. www.aaaai.org/about-the-aaaai/newsroom/allergy-
22. Sunheimer RL, Threatte G. Analysis: clinical laboratory automa- statistics.aspx. Accessed June 19, 2015.
tion. In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis 18. Asthma and Allergy Foundation of America. Allergy facts and
and Management by Laboratory Methods. 27th ed. Philadelphia, figures. www.aafa.org/display.cfm?id=9&sub=30. Updated April 8,
PA: Saunders Elsevier; 2011:56–63. 2015. Accessed June 19, 2015.
23. Appold K. Checklist for buying a chemistry analyzer. Clin Lab 19. Gawchik DO. Latex allergy. Mt Sinai J Med. 2011;78:759–772.
Prod. 2013 (Nov);12–15. 20. Taylor JS, Erkek E. Latex allergy: diagnosis and management.
24. Turgeon ML. Automated procedures. In: Immunology and Serol- Dermatol Ther. 2004;17(4):289–301.
ogy in Laboratory Medicine. 5th ed. St. Louis, MO: Mosby; 2014: 21. Kuhl K, Hanania NA. Targeting IgE in asthma. Curr Opin Pulm
170–182. Med. 2012;18(1):1–5.
25. Armbruster DA, Overcash DR, Reyes J. Clinical chemistry au- 22. Fried AJ, Oettgen HC. Anti-IgE in the treatment of allergic disor-
tomation in the 21st century-amat victoria curam. Clin Biochem ders in pediatrics. Curr Opin Pediatr. 2010;22(6):758–764.
Rev. 2014;35(3)143–153. 23. Casale TB, Stokes JR. Future forms of immunotherapy. J Allergy
26. Moon TC, Legrys VA. Teaching method validation in the clinical Clin Immunol. 2011;127(1):8–15.
laboratory science curriculum. Clin Lab Sci. 2008;21(1):19–24. 24. Burks AW, Calderon MA, Casale T, et al. Update on allergy
27. Association for Molecular Pathology. Association for Molecular immunotherapy: American Academy of Allergy, Asthma &
Pathology statement: recommendations for in-house develop- Immunology/European Academy of Allergy and Clinical
ment and operation of molecular diagnostic tests. Am J Clin Path. Immunology/PRACTALL consensus report. J Allergy Clin Immunol.
199;111:449–463. 2013;131(5):1288–1296.e3.
25. Stokes JR, Casale TB. Allergic rhinitis and asthma: celebrating
100 years of immunotherapy. Curr Opin Immunol. 2011;23(6):
1. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. New York, 808–813.
NY: W.H. Freeman; 2013:485–516. 26. Volcheck GW. Which diagnostic tests for common allergies?
2. Gould HJ, Sutton BJ, Beavil AJ, et al. The biology of IgE and the Where to start when you face an allergy puzzle. Postgrad Med.
basis of allergic disease. Annu Rev Immunol. 2003;21:579–628. 2001;109(5):71–72.
3. Galli SJ, Tsai M. IgE and mast cells in allergic disease. Nature Med. 27. Carr TF, Saltoun CA. Skin testing in allergy. Allergy Asthma Proc.
2012;18:693–704. 2012;33(suppl 1):S6–S8.
28. Hamilton RG. Immunological methods in the diagnostic allergy of Internal Medicine. 19th ed. New York, NY: McGraw-Hill; 2015.
clinical and research laboratory, In: Detrick B, Hamilton RG, accessmedicine.mhmedical.com/content.aspx?bookid=1130
Folds JD, eds. Manual of Molecular and Clinical Laboratory Im- &Sectionid=79731477. Accessed June 19, 2015.
munology. 7th ed. Washington, DC: ASM Press; 2006:955–963. 48. D’Arena G, Taylor RP, Cascavilla N, Lindorfer MA. Monoclonal
29. Kranke B, Aberer W. Skin testing for IgE-mediated drug allergy. antibodies: new therapeutic agents for autoimmune hemolytic
Immunol Allergy Clin. 2009;29(3):503–516. anemia? Endocr Metab Immune Disord Drug Targets. 2008;8(1):
30. Cox L. Overview of serological-specific IgE antibody testing in 62–68.
children. Curr Allergy Asthm R. 2011;11(6):447–453. 49. Miller LE, Ludke HR, Peacock JE, Tomar RH. Manual of Labora-
31. Hamilton RG. Williams PB. Specific IgE Testing Task Force of tory Immunology. 2nd ed. Philadelphia, PA: Lea & Febiger;
the American Academy of Allergy, Asthma & Immunology. 1991:360–364.
American College of Allergy, Asthma and Immunology. Human 50. McNicholl FP. Clinical syndromes associated with cold agglu-
IgE antibody serology: a primer for the practicing North American tinins. Transfus Sci. 2000;22:125–133.
allergist/immunologist. J Allergy Clin Immunol. 2010;126(1): 51. Bellanti JA, Escobar-Gutierrez A. Mechanisms of immunologic
33–38. injury. In: Bellanti JA, Escobar-Gutierrez A, Tsokos GC, eds. Im-
32. Thermo Scientific. ImmunoCAP lab tests. 2012. www.phadia. munology IV: Clinical applications in health and disease. Bethesda,
com/Products/Allergy-testing-products/ImmunoCAP-Assays/. MD: I Care Press; 2012:661–683.
Accessed June 19, 2015. 52. Bellanti JA, Escobar-Gutierrez A, Joost JJ. Cytokines, chemokines,
33. Makhija M, O’Gorman MR. Common in vitro tests for allergy and and the immune system. In: Bellanti JA, Escobar-Gutierrez A,
immunology. Allergy Asthm Proc. 2012;33(suppl 1):S108–S111. Tsokos GC, eds. Immunology IV: Clinical applications in health and
34. Hamilton RG. Clinical laboratory assessment of immediate-type disease. Bethesda, MD: I Care Press; 2012:287–366.
hypersensitivity. J Allergy Clin Immunol. 2010;125(2 suppl 2): 53. Peiser M, Tralau T, Heidler J, et al. Allergic contact dermatitis:
S284–S296. epidemiology, molecular mechanisms, in vitro methods and
35. Mari A, Alessandri C, Bernardi ML, et al. Microarrayed allergen regulatory aspects. Current knowledge assembled at an inter-
molecules for the diagnosis of allergic diseases. Curr Allergy national workshop at BfR, Germany. Cell Mol Life Sci. 2012;
Asthm R. 2010;10(5):357–364. 69(5):763–781.
36. Grotzke M. The thyroid gland. In: Bishop ML, Fody EP, Schoeff 54. Karlberg AT, Bergstrom MA, Borje A, et al. Allergic contact
LE, eds. Clinical Chemistry. 7th ed. Philadelphia, PA: Lippincott, dermatitis—formation, structural requirements, and reactivity
Williams & Wilkins; 2013:489–501. of skin sensitizers. Chem Res Toxicol. 2008;21(1):53–69.
37. Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group 55. Jacob SE, Steele T. Allergic contact dermatitis: early recognition
antigens and genes—historical origins and guidelines in the new and diagnosis of important allergens. Dermatol Nurs. 2006;18(5):
millennium. Transfusion. 2000;40(4):477–489. 433–439, 446.
38. Storry JR, Olsson ML. Genetic basis of blood group diversity. Br 56. Gladman AC. Toxicodendron dermatitis: poison ivy, oak, and
J Haematol. 2004;126(6):759–771. sumac. Wilderness Environ Med. 2006;17(2):120–128.
39. Dzieczkowski JS, Anderson KC. Transfusion biology and therapy. 57. Jacob SE, Zapolanski T. Systemic contact dermatitis. Dermatitis.
In: Kasper D, Fauci A, Hauser S, et al. eds. Harrison’s Principles of 2008;19(1):9–15.
Internal Medicine. 19th ed. New York, NY: McGraw-Hill; 2015. 58. Lee PW, Elsaie ML, Jacob SE. Allergic contact dermatitis in children:
accessmedicine.mhmedical.com/content.aspx?bookid=1130&Sec common allergens and treatment: a review. Curr Opin Pediatr.
tionid=79732248. Accessed June 19, 2015. 2009;21(4):491–498.
40. Davenport RD, Mintz PD. Transfusion medicine. In: McPherson 59. Akuthota P, Wechsler ME. Hypersensitivity pneumonitis and
RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management by pulmonary infiltrates with eosinophilia. In: Kasper D, Fauci A,
Laboratory Methods. 22nd ed. Philadelphia, PA: Saunders Elsevier; Hauser S, et al., eds. Harrison’s Principles of Internal Medicine.
2011:731–745. 19th ed. New York, NY: McGraw-Hill; 2015. accessmedicine.
41. Torres R, Kenney B, Tormey CA. Diagnosis, treatment, and report- mhmedical.com/content.aspx?bookid=1130&Sectionid=797448
ing of adverse effects of transfusion. Lab Medicine. 2012;43(5): 22. Accessed June 19, 2015.
217–231. 60. Patel AM, Ryu JH, Reed CE. Hypersensitivity pneumonitis: current
42. Cooling L, Downs T. Immunohematology. In: McPherson RA, concepts and future questions. J Allergy Clin Immunol. 2001;108:
Pincus MR, Henry JB, eds. Henry’s Clinical Diagnosis and Manage- 661–670.
ment by Laboratory Methods. 22nd ed. Philadelphia, PA: Elsevier 61. McCormick T, Shearer W. Delayed-type hypersensitivity skin test-
Saunders; 2011:674–730. ing. In: Detrick B, Hamilton RG, Folds JD, eds. Manual of Molecular
43. Blaney KD, Howard PR. Concepts of Immunohematology. 2nd ed. and Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM
St. Louis, MO: Mosby, Elsevier; 2009:284–303. Press; 2006:234–240.
44. Kennedy MS. Hemolytic disease of the newborn (HDFN). In: 62. American Thoracic Society. Targeted tuberculin testing and treat-
Harmening DM, ed. Modern Blood Banking & Transfusion Practices. ment of latent tuberculosis infection. MMWR Recommendations &
6th ed. Philadelphia, PA: F.A. Davis; 2012:427–438. Reports. 2000;49(RR-6):1–51.
45. Murray NA, Roberts IA. Haemolytic disease of the newborn. Arch 63. Jensen PA, Lambert LA, Iademarco MF, Ridzon R, CDC. Guidelines
Dis Child Fetal Neonatal Ed. 2007;92(2):F83–F88. for preventing the transmission of mycobacterium tuberculosis in
46. Harmening DM, Rodberg K, Green EB. Autoimmune hemolytic health-care settings, 2005. MMWR Recommendations & Reports.
anemias. In: Harmening DM, ed. Modern Blood Banking & Trans- 2005;54(RR-17):1–141.
fusion Practices. 6th ed. Philadelphia, PA.: F.A. Davis; 2012: 64. Moesgaard F, Lykkegaard Nielsen M, Norgaard Larsen P, et al.
439–473. Cell-mediated immunity assessed by skin testing (multitest). I.
47. Luzzatto L. Hemolytic anemias and anemia due to acute blood loss. Normal values in healthy Danish adults. Allergy. 1987;42(8):
In: Kasper D, Fauci A, Hauser S, et al., eds. Harrison’s Principles 591–596.
 22. Tsokos GC. Systemic lupus erythematosus. A disease with a
1. Kyttaris V, Tsokos GC. Tolerance, autoimmunity, and autoin- complex pathogenesis. Lancet. 2001;358(suppl):S65.
flammation. In: Bellanti JA, Escobar-Gutierrez A, Tsokos GC, 23. Araujo-Fernandez S, Ahijon-Lana M, Isenberg DA. Drug-induced
eds. Immunology IV: Clinical Applications in Health and Disease. lupus: including anti-tumour necrosis factor and interferon
Bethesda, MD: I Care Press; 2012:767–798. induced. Lupus. 2014;23(6):545–553.
2. American Autoimmune Diseases Related Association. Autoimmune 24. Gergely P, Jr, Isaak A, Szekeres Z, et al. Altered expression of fc
statistics. www.aarda.org/autoimmune-information/autoimmune- gamma and complement receptors on B cells in systemic lupus
statistics/. Updated 2014. Accessed July 29, 2014. erythematosus. Ann NY Acad Sci. 2007;1108:183–192.
3. National Institute of Health. The cost burden of autoimmune 25. Cozzani E, Drosera M, Gasparini G, Parodi A. Serology of lupus
disease. www.diabetesed.net/page/_files/autoimmune-diseases erythematosus: correlation between immunopathological features
.pdf. Updated 2011. Accessed July 29, 2014. and clinical aspects. Autoimmune Dis. 2014;2014:321–359.
4. Abbas AK, Lichtman AH, Pillai S. Immunologic tolerance and 26. Marks SD, Tullus K. Autoantibodies in systemic lupus erythe-
autoimmunity. In: Cellular and Molecular Immunology. 7th ed. matosus. Pediatr Nephrol. 2012;27(10):1855–1868.
Philadelphia, PA: Elsevier Saunders; 2012:319–343. 27. Kimberly RP. Prospects for autoimmune disease: research advances
5. Kyttaris V, Tsokos GC. Tolerance, autoimmunity, and autoin- in systemic lupus erythematosus. JAMA. 2001;285(5):650–652.
flammation. In: Bellanti JA, Escobar-Gutierrez A, Tsokos GC, 28. Nowling TK, Gilkeson GS. Mechanisms of tissue injury in lupus
eds. Immunology IV. Clinical Applications in Health and Disease. nephritis. Arthritis Res Ther. 2011;13(6):250.
Bethesda, MD: iCare Press; 2012:767–798. 29. Isenberg DA, Manson JJ, Ehrenstein MR, Rahman A. Fifty years
6. Selmi C, Leung PS, Sherr DH, et al. Mechanisms of environmental of anti-ds DNA antibodies: are we approaching journey’s end?
influence on human autoimmunity: A National Institute of Envi- Rheumatology. 2007;46(7):1052–1056.
ronmental Health Sciences expert panel workshop. J Autoimmun. 30. Baer AN, Witter FR, Petri M. Lupus and pregnancy. Obstet Gynecol
2012;39(4):272–284. Surv. 2011;66(10):639–653.
7. Wahren-Herlenius M, Dorner T. Immunopathogenic mechanisms 31. Smith PP, Gordon C. Systemic lupus erythematosus: clinical
of systemic autoimmune disease. Lancet. 2013;382(9894):819–831. presentations. Autoimmun Rev. 2010;10(1):43–45.
8. Quintero OL, Amador-Patarroyo MJ, Montoya-Ortiz G, et al. 32. von Muhlen A, Nakamura RM. Clinical and laboratory evaluation
Autoimmune disease and gender: plausible mechanisms for the of systemic rheumatic diseases. In: McPherson RA, Pincus MR,
female predominance of autoimmunity. J Autoimmun. 2012; eds. Henry’s Clinical Diagnosis and Management by Laboratory
38(2–3):J109–J119. Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:
9. Sfriso P, Ghirardello A, Botsios C, et al. Infections and autoimmunity: 973–990.
the multifaceted relationship. J Leukoc Biol. 2010;87(3):385–395. 33. Heinlen LD, McClain MT, Merrill J, et al. Clinical criteria for
10. Cusick MF, Libbey JE, Fujinami RS. Molecular mimicry as a systemic lupus erythematosus precede diagnosis, and associated
mechanism of autoimmune disease. Clin Rev Allergy Immunol. autoantibodies are present before clinical symptoms. Arthrit Rheum.
2012;42(1):102–111. 2007;56(7):2344–2351.
11. Chervonsky AV. Microbiota and autoimmunity. Cold Spring 34. Petri M, Orbai AM, Alarcon GS, et al. Derivation and validation
Harbor Perspectives in Biology. 2013;5(3):a007294. of the systemic lupus international collaborating clinics classifi-
12. Proft T, Fraser JD. Bacterial superantigens. Clin Exp Immunol. cation criteria for systemic lupus erythematosus. Arthrit Rheum.
2003;133(3):299–306. 2012;64(8):2677–2686.
13. National Center for Biotechnology Information (NCBI). Epigenetics 35. Rodriguez-Garcia V, Dias SS, Isenberg D. Recent advances in the
help. Epigenomics scientific background. Electronic publication. treatment of systemic lupus erythematosus. Int J Clin Rheumatol.
www.ncbi.nlm.nih.gov/books/NBK45788/#epi_sci_bkgrd.About_ 2014;9(1):89–100.
Epigenetics. Updated 2011. Accessed July 29, 2014. 36. Bradwell AR, Hughes RG, Karim AR. Immunofluorescent anti-
14. Vadasz Z, Toubi E. Frontier issues in autoimmunity: publications nuclear antibody tests. In: Detrick B, Hamilton RG, Folds JD, eds.
in 2009–2010. IMAJ. 2010;12(12):757–761. Manual of Molecular and Clinical Laboratory Immunology. 7th ed.
15. Lu Q. The critical importance of epigenetics in autoimmunity. Washington, DC: ASM Press; 2006:995–1006.
J Autoimmun. 2013;41:1–5. 37. Al-Zougbi A. Antinuclear antibody. Medscape. Antinuclear anti-
16. Pillai S. Rethinking mechanisms of autoimmune pathogenesis. body. Website. Published August 23, 2012. emedicine.medscape
J Autoimmun. 2013;45:97–103. .com/article/2086616-overview. Updated 2012. Accessed August
17. Rahman A, Isenberg DA. Systemic lupus erythematosus. N Engl 5, 2014.
J Med. 2008;358(9):929–939. 38. Kumar Y, Bhatia A, Minz RW. Antinuclear antibodies and their
18. O’Neill S, Cervera R. Systemic lupus erythematosus. Best Pract detection methods in diagnosis of connective tissue diseases: a
Res Clin Rh. 2010;24(6):841–855. journey revisited. Diagn Pathol. 2009;4:1.
19. Hahn B. Systemic lupus erythematosus. In: Longo DL, Fauci AS, 39. Tran TT, Pisetsky DS. Detection of anti-DNA autoantibodies. In:
Kasper DL, et al., eds. Harrison’s Principles of Internal Medicine. Detrick B, Hamilton RG, Folds JD, eds. Manual of Molecular and
18th ed. New York, NY: McGraw-Hill; 2012. accessmedicine Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM
.mhmedical.com/content.aspx?bookid=331&Sectionid=40727120. Press; 2006:1027–1032.
Accessed July 29, 2014. 40. Reeves WH, Satoh M, Lyons R, et al. Detection of autoantibodies
20. Campbell R, Jr, Cooper GS, Gilkeson GS. Two aspects of the clin- against proteins and ribonucleoproteins by double immunodif-
ical and humanistic burden of systemic lupus erythematosus: fusion and immunoprecipitation. In: Detrick B, Hamilton RG,
mortality risk and quality of life early in the course of disease. Folds JD, eds. Manual of Molecular and Clinical Laboratory Im-
Arthritis Rheum. 2008;59(4):458–464. munology. 7th ed. Washington, DC: ASM Press; 2006:1007–1018.
21. Gualtierotti R, Biggioggero M, Penatti AE, Meroni PL. Updating 41. Meroni PL, Bizzaro N, Cavazzana I, et al. Automated tests of ANA
on the pathogenesis of systemic lupus erythematosus. Autoimmun immunofluorescence as throughput autoantibody detection tech-
Rev. 2010;10(1):3–7. nology: strengths and limitations. BMC Med. 2014;12:38.
42. Zeus Scientific IFA ANA HEp-2 pattern identification guide. Web- 61. Lee AN, Beck CE, Hall M. Rheumatoid factor and anti-CCP
site. www.zeusscientific.com/zeus-ifa-hep-2-atlas/. Updated 2014. autoantibodies in rheumatoid arthritis: a review. Clin Lab Sci. 2008;
Accessed August 5, 2014. 21(1):15–18.
43. Boyes RR. Lab CE. Antinuclear antibody testing: methods and 62. Farid SS, Azizi G, Mirshafiey A. Anti-citrullinated protein anti-
pattern interpretation. Website. https://www.labce.com/mls_mt_ bodies and their clinical utility in rheumatoid arthritis. Int J
mlt_lab_continuing_education.aspx. Updated 2014. Accessed Rheum Dis. 2013;16(4):379–386.
August 5, 2014. 63. Arnett FC, Edworthy SM, Bloch DA. The American Rheumatism
44. Hutchison KW, Wener MH, Gilliland BG, Astion ML. Medical Association 1987 criteria for the classification of rheumatoid
training solutions. Antinuclear antibody online training course. arthritis. Arthrit Rheum. 1988;315–324.
University of Washington, Dept. of Laboratory Medicine. Web- 64. Aletaha D, Neogi T, Silman AJ, et al. 2010 Rheumatoid arthritis
site. medtraining.org/labcontent.aspx. Updated 2013. Accessed classification criteria: an American College of Rheumatology/
August 5, 2014. European League Against Rheumatism collaborative initiative.
45. Ghillani P, Rouquette AM, Desgruelles C, et al. Evaluation of the Arthrit Rheum. 2010;62(9):2569–2581.
LIAISON ANA screen assay for antinuclear antibody testing in 65. Kay J, Upchurch KS. ACR/EULAR 2010 rheumatoid arthritis
autoimmune diseases. Ann NY Acad Sci. 2007;1109:407–413. classification criteria. Rheumatology. 2012;51(suppl 6):5–9.
46. Meroni PL, Schur PH. ANA screening: an old test with new 66. Jung YO, Kim HA. Recent paradigm shifts in the diagnosis and
recommendations. Ann Rheum Dis. 2010;69(8):1420–1422. treatment of rheumatoid arthritis. Korean J Intern Med. 2012;
47. Hanly JG, Su L, Farewell V, Fritzler MJ. Comparison between 27(4):378–387.
multiplex assays for autoantibody detection in systemic lupus 67. Scott DL. Biologics-based therapy for the treatment of rheumatoid
erythematosus. J Immunol Methods. 2010;358(1–2):75–80. arthritis. Clin Pharmacol Ther. 2012;91(1):30–43.
48. Hanly JG, Thompson K, McCurdy G, et al. Measurement of 68. Niewold TB, Harrison MJ, Paget SA. Anti-CCP antibody testing
autoantibodies using multiplex methodology in patients with as a diagnostic and prognostic tool in rheumatoid arthritis. QJM.
systemic lupus erythematosus. J Immunol Methods. 2010;352 2007;100(4):193–201.
(1–2):147–152. 69. Weeda LW, Jr, Coffey SA. Wegener’s granulomatosis. Oral &
49. Aarden LA, de Groot ER, Feltkamp TE. Immunology of DNA. III. Maxillofac Surg Clinics N Am. 2008;20(4):643–649.
Crithidia luciliae, a simple substrate for the determination of anti- 70. Schilder AM. Wegener’s granulomatosis vasculitis and granuloma.
dsDNA with the immunofluorescence technique. Ann NY Acad Autoimmun Rev. 2010;9(7):483–487.
Sci. 1975;254:505–515. 71. Tarabishy AB, Schulte M, Papaliodis GN, Hoffman GS. Wegener’s
50. Schmitz JL. Laboratory testing for antibodies associated with granulomatosis: clinical manifestations, differential diagnosis, and
antiphospholipid antibody syndrome. In: Detrick B, Hamilton management of ocular and systemic disease. Surv Ophthalmol.
RG, Folds JD, eds. Manual of Molecular and Clinical Laboratory 2010;55(5):429–444.
Immunology. 7th ed. Washington, DC: ASM Press; 2006:1046–1052. 72. Cartin-Ceba R, Peikert T, Specks U. Pathogenesis of ANCA-
51. Vlachoyiannopoulos PG, Samarkos M, Sikara M, Tsiligros P. associated vasculitis. Curr Rheumatol Rep. 2012;14(6):481–493.
Antiphospholipid antibodies: laboratory and pathogenetic aspects. 73. Moosig F, Lamprecht P, Gross WL. Wegener’s granulomatosis:
Crit Rev Clin Lab Sci. 2007;44(3):271–338. The current view. Clin Rev Allergy Immunol. 2008;35(1–2):
52. Marlar RA, Fink LM, Miller JL. Laboratory approach to thrombotic 19–21.
risk. In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis 74. Alberici F, Martorana D, Bonatti F, et al. Genetics of ANCA-
and Management by Laboratory Methods. 22nd ed. Philadelphia, PA: associated vasculitides: HLA and beyond. Clin Exp Rheum. 2014;
Elsevier; 2011:823–830. 32(2 suppl 82):S90–S97.
53. Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet. 75. Holle JU, Gross WL. Treatment of ANCA-associated vasculitides
2010;376(9746):1094–1108. (AAV). Autoimmun Rev. 2013;12(4):483–486.
54. Shah A, St. Clair E. Rheumatoid arthritis. In: Longo DL, Fauci 76. Leavitt RY, Fauci AS, Bloch DA, et al. The American College of
AS, Kasper DL, et al., eds. Harrison’s Principles of Internal Medicine. Rheumatology 1990 criteria for the classification of Wegener’s
18th ed. New York, NY: McGraw-Hill; 2012. accessmedicine granulomatosis. Arthrit Rheum. 1990;33:1101–1107.
.mhmedical.com/content.aspx?bookid=331&Sectionid=40727122. 77. Radice A, Bianchi L, Sinico RA. Anti-neutrophil cytoplasmic
Accessed August 8, 2014. autoantibodies: methodological aspects and clinical significance
55. Sanmarti R, Ruiz-Esquide V, Hernandez MV. Rheumatoid arthritis: in systemic vasculitis. Autoimmun Rev. 2013;12(4):487–495.
a clinical overview of new diagnostic and treatment approaches. 78. Csernok E. ANCA testing: the current stage and perspectives.
Curr Top Med Chem. 2013;13(6):698–704. Clin Exp Nephrol. 2013;17(5):615–618.
56. Emery P, McInnes IB, van Vollerhoven R, Kraan MC. Clinical 79. Savige J, Gillis D, Benson E, et al. International consensus state-
identification and treatment of a rapidly progressing disease state ment on testing and reporting of antineutrophil cytoplasmic
in patients with rheumatoid arthritis. Rheumatology. 2008;47: antibodies (ANCA). Am J Clin Pathol. 1999;111(4):507–513.
392–398. 80. Sinico RA, Radice A. Antineutrophil cytoplasmic antibodies
57. Hill J. The what, whys, and wherefores of rheumatoid arthritis. (ANCA) testing: detection methods and clinical application. Clin
Nurs Res Care. 2008;10:123–126. Exp Rheum. 2014;32(2 suppl 82):S112–S117.
58. Klareskog L, Catrina AI, Paget S. Rheumatoid arthritis. Lancet. 81. Hutchison KW, Wener MH, Gilliland BG, Astion ML. Medical
2009;373(9664):659–672. training solutions. ANCA online training course. University of
59. Ingegnoli F, Castelli R, Gualtierotti R. Rheumatoid factors: clinical Washington, Dept. of Laboratory Medicine. Medical Training
applications. Dis Markers. 2013;35(6):727–734. Solutions. Website. medtraining.org/labcontent.aspx. Updated
60. Holers VM. Autoimmunity to citrullinated proteins and the 2013. Accessed September 22, 2014.
initiation of rheumatoid arthritis. Curr Opin Immunol. 2013; 82. Flores-Suarez LF. Antineutrophil cytoplasm autoantibodies: useful-
25(6):728–735. ness in rheumatology. Reumatologia Clinica. 2012;8(6):351–357.
 83. Grotzke M. The thyroid gland. In: Bishop ML, Fody EP, Schoeff 106. Bylund DJ, Nakamura RM. Organ-specific autoimmune diseases.
LE, eds. Clinical Chemistry. 7th ed. Philadelphia, PA: Lippincott In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and
Williams and Wilkins; 2013:489–501. Management by Laboratory Methods. 22nd ed. Philadelphia, PA:
84. Jameson JL, Weetman AP. Disorders of the thyroid gland. In: Elsevier Saunders; 2011:1003–1020.
Longo DL, Fauci AS, Kasper DL, et al., eds. Harrison’s Principles 107. Burek CL, Bigazzi PE, Rose NR. Endocrinopathies. In: Detrick
of Internal Medicine. 18th ed. New York, NY: McGraw-Hill; 2012. B, Hamilton RG, Folds JD, eds. Manual of Molecular and Clinical
accessmedicine.mhmedical.com/content.aspx?bookid=331& Laboratory Immunology. 7th ed. Washington, DC: ASM Press;
Sectionid=40727146. Accessed August 28, 2014. 2006:1062–1077.
85. Jacobson EM, Huber A, Tomer Y. The HLA gene complex in 108. Fasano A, Catassi C. Clinical practice. Celiac disease. N Engl J
thyroid autoimmunity: from epidemiology to etiology. J Autoim- Med. 2012;367(25):2419–2426.
mun. 2008;30(1–2):58–62. 109. Green PH, Cellier C. Celiac disease. N Engl J Med. 2007;357(17):
86. Brown RS. Autoimmune thyroid disease: unlocking a complex 1731–1743.
puzzle. Curr Opin Pediatr. 2009;21(4):523–528. 110. Guandalini S, Assiri A. Celiac disease: a review. JAMA Pediatrics.
87. Jacobson EM, Tomer Y. The genetic basis of thyroid autoimmu- 2014;168(3):272–278.
nity. Thyroid. 2007;17(10):949–961. 111. Lundin KE, Sollid LM. Advances in coeliac disease. Curr Opin
88. Ahmed R, Al-Shaikh S, Akhtar M. Hashimoto thyroiditis: a cen- Gastroenterol. 2014;30(2):154–162.
tury later. Adv Anat Pathol. 2012;19(3):181–186. 112. Ma MX, John M, Forbes GM. Diagnostic dilemmas in celiac
89. Caturegli P, De Remigis A, Rose NR. Hashimoto thyroiditis: clinical disease. Expert Rev Gastroenterol Hepatol. 2013;7(7):643–655.
and diagnostic criteria. Autoimmun Rev. 2014;13(4–5):391–397. 113. Leffler DA, Schuppan D. Update on serologic testing in celiac
90. Menconi F, Marcocci C, Marino M. Diagnosis and classification disease. Am J Gastroenterol. 2010;105(12):2520–2524.
of Graves’ disease. Autoimmun Rev. 2014;13(4–5):398–402. 114. Carbone M, Neuberger JM. Autoimmune liver disease, autoim-
91. Gopinath B, Musselman R, Beard N, et al. Antibodies targeting munity and liver transplantation. J Hepatol. 2014;60(1):210–223.
the calcium binding skeletal muscle protein calsequestrin are 115. Heneghan MA, Yeoman AD, Verma S, Smith AD, Longhi MS.
specific markers of ophthalmopathy and sensitive indicators Autoimmune hepatitis. Lancet. 2013;382(9902):1433–1444.
of ocular myopathy in patients with Graves’ disease. Clin Exp 116. Liberal R, Grant CR, Mieli-Vergani G, Vergani D. Autoimmune
Immunol. 2006;145(1):56–62. hepatitis: a comprehensive review. J Autoimmun. 2013;41:
92. Rocchi R. Critical issues on Graves’ ophthalmopathy. MLO. 126–139.
2006;38(5):10; May 12. 117. Liberal R, Grant CR, Longhi MS, Mieli-Vergani G, Vergani D.
93. McKenna TJ. Graves’ disease. Lancet. 2001;357(9270):1793–1796. Diagnostic criteria of autoimmune hepatitis. Autoimmun Rev.
94. Weetman AP. Graves’ disease. N Engl J Med. 2000;343(17): 2014;13(4–5):435–440.
1236–1248. 118. Nguyen DL, Juran BD, Lazaridis KN. Primary biliary cirrhosis.
95. Sinclair D. Clinical and laboratory aspects of thyroid autoanti- Best Pract Res Clin Ga. 2010;24(5):647–654.
bodies. Ann Clin Biochem. 2006;43(Pt 3):173–183. 119. Mells GF, Kaser A, Karlsen TH. Novel insights into autoimmune
96. Lytton SD, Kahaly GJ. Bioassays for TSH-receptor autoantibod- liver diseases provided by genome-wide association studies.
ies: an update. Autoimmun Rev. 2010;10(2):116–122. J Autoimmun. 2013;46:41–54.
97. Powers AC. Diabetes mellitus. In: Longo DL, Fauci AS, Kasper 120. Bowlus CL, Gershwin ME. The diagnosis of primary biliary
DL, et al., eds. Harrison’s Principles of Internal Medicine. 18th ed. cirrhosis. Autoimmun Rev. 2014;13(4–5):441–444.
New York, NY: McGraw-Hill; 2012. accessmedicine.mhmedical 121. Hirschfield GM. Diagnosis of primary biliary cirrhosis. Best Pract
.com/content.aspx?bookid=331&Sectionid=40727149. Res Clin Ga. 2011;25(6):701–712.
Accessed November 1, 2014. 122. Bogdanos DP, Komorowski L. Disease-specific autoantibodies
98. Imam K. Clinical features, diagnostic criteria and pathogenesis in primary biliary cirrhosis. Clinica Chimica Acta. 2011;412(7–8):
of diabetes mellitus. Adv Exp Med Biol. 2012;771:340–355. 502–512.
99. Canivell S, Gomis R. Diagnosis and classification of autoimmune 123. Hauser SL, Goodin DS. Multiple sclerosis and other demyeli-
diabetes mellitus. Autoimmun Rev. 2014;13(4–5):403–407. nating diseases. In: Longo DL, Fauci AS, Kasper DL, et al., eds.
100. Taplin CE, Barker JM. Autoantibodies in type 1 diabetes. Autoim- Harrison’s Principles of Internal Medicine. 18th ed. New York, NY:
munity. 2008;41(1):11–18. McGraw-Hill; 2012. accessmedicine.mhmedical.com/content
101. Cerna M. Genetics of autoimmune diabetes mellitus. Wien Med .aspx?bookid=331&Sectionid=40727196. Accessed November
Wochenschr. 2008;158(1–2):2–12. 18, 2014.
102. Zhang L, Nakayama M, Eisenbarth GS. Insulin as an autoanti- 124. Milo R, Miller A. Revised diagnostic criteria of multiple sclerosis.
gen in NOD/human diabetes. Curr Opin Immunol. 2008;20(1): Autoimmun Rev. 2014;13(4–5):518–524.
111–118. 125. Compston A, Coles A. Multiple sclerosis. Lancet. 2002;359
103. Wenzlau JM, Hutton JC. Novel diabetes autoantibodies and (9313):1221–1231.
prediction of type 1 diabetes. Curr Diabetes Rep. 2013;13(5): 126. Tavazzi E, Rovaris M, La Mantia L. Drug therapy for multiple
608–615. sclerosis. CMAJ. 2014;186(11):833–840.
104. von Herrath M, Peakman M, Roep B. Progress in immune-based 127. Katzmann JA, Kyle RA. Immunochemical characteristics of
therapies for type 1 diabetes. Clin Exp Immunol. 2013;172(2): immunoglobulins in serum, urine, and cerebrospinal fluid.
186–202. In: Detrick B, Hamilton RG, Folds JD, eds. Manual of Molecular
105. Pancreatic islet transplantation. National Diabetes Information and Clinical Laboratory Immunology. 7th ed. Washington, DC:
Clearinghouse (NDIH) Website. http://www.niddk.nih.gov/ ASM Press; 2006:88–100.
health-information/health-topics/Diabetes/pancreatic-islet- 128. Drachman DB. Myasthenia gravis and other diseases of the
transplantation/Pages/index.aspx. Updated 2014. Accessed neuromuscular junction. In: Longo DL, Fauci AS, Kasper DL,
November 18, 2014. et al., eds. Harrison’s Principles of Internal Medicine. 18th ed.
 New York, NY: McGraw-Hill; 2012. accessmedicine.mhmedical 10. Angaswamy N, Tiriveedhi V, Sarma NJ, et al. Interplay between
.com/content.aspx?bookid=331&Sectionid=40727203. immune responses to HLA and non-HLA self-antigens in allograft
Accessed November 20, 2014. rejection. Hum Immunol. 2013;74:1478–1485.
129. Cavalcante P, Bernasconi P, Mantegazza R. Autoimmune 11. Abbas A, Lichtman AH, Pillai S. Cellular and Molecular Immunol-
mechanisms in myasthenia gravis. Curr Opin Neurol. 2012; ogy. 6th ed. Philadelphia, PA: Saunders; 2007:375–396.
25(5):621–629. 12. D’Orsogna LJ, Roelen DL, Doxiadis II, Claas FH. Alloreactivity
130. Zagoriti Z. Recent advances in genetic predisposition of myas- from human viral specific memory T-cells. Transpl Immunol. 2010;
thenia gravis. Biomed Res Int. 2013;2013:1–12. 23:149–155.
131. Spillane J, Higham E, Kullmann DM. Myasthenia gravis. BMJ. 13. Afzali B, Lechler RI, Hernandez-Fuentes MP. Allorecognition and
2012;345:e8497. the alloresponse: clinical implications. Tissue Antigens. 2007;69:
132. Berrih-Aknin S, Le Panse R. Myasthenia gravis: a comprehensive 545–556.
review of immune dysregulation and etiological mechanisms. 14. Leichtman AB. Primer on Transplantation. Thorofare, NJ: American
J Autoimmun. 2014;52:90–100. Society of Transplant Physicians; 1998; 217–222.
133. Dalakas MC. Novel future therapeutic options in myasthenia 15. Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment
gravis. Autoimmun Rev. 2013;12(9):936–941. of donor-reactive, HLA-specific antibodies in renal transplan-
134. Zisimopoulou P, Brenner T, Trakas N, Tzartos SJ. Serological di- tation: Contraindication vs. risk. Am J Transplant. 2003;3:
agnostics in myasthenia gravis based on novel assays and re- 1488–1500.
cently identified antigens. Autoimmunity Reviews. 2013;12(9): 16. Colvin RB. Antibody-mediated renal allograft rejection: diagnosis
924-930. and pathogenesis. J Am Soc Nephrol. 2007;18:1046–1056.
135. Lahmer T, Heemann U. Anti-glomerular basement membrane 17. Colvin RB. Chronic allograft nephropathy. N Eng J Med. 2003;
antibody disease: A rare autoimmune disorder affecting the kid- 349:2288–2290.
ney and the lung. Autoimmunity Reviews. 2012;12(2):169-173. 18. Costello JP, Mohanakumar T, Nath DS. Mechanisms of chronic
136. Bergs L. Goodpasture syndrome. Crit Care Nurse. 2005;25(5): cardiac allograft rejection. Tex Heart Inst J. 2013;40:395–399.
50-54. 19. Goker H, Haznedaroglu IC, Chao NJ. Acute graft vs. host disease:
137. Hellmark T, Segelmark M. Diagnosis and classification of Good- pathobiology and management. Exp Hematol. 2001;29(3):
pasture’s disease (anti-GBM). J Autoimmun. 2014;48-49: 259–277.
108-112. 20. Copelan EA. Hematopoietic stem-cell transplantation. N Eng
138. Collins AB, Colvin RB. Kidney and lung disease mediated by J Med. 2006;354:1813–1826.
anti-glomerular basement membrane antibodies: Detection by 21. Halloran PF, Lui SL. Primer on Transplantation. Thorofare, NJ:
Western blot analysis. In: Folds JD, Hamilton RG, Folds JD, eds. American Society of Transplant Physicians; 1998: 93–102.
Manual of Molecular and Clinical Laboratory Immunology. 22. Augustine JJ, Bodziak KA, Hricik DE. Use of sirolimus in solid
7th ed. Washington, DC: ASM Press; 2006:1110-1115. organ transplantation. Drugs. 2007;67:369–391.
139. Dammacco F, Battaglia S, Gesualdo L, Racanelli V. Goodpasture’s 23. Buhaescu I, Segall L, Goldsmith D, Covic A. New immunosup-
disease: A report of ten cases and a review of the literature. pressive therapies in renal transplantation: monoclonal antibodies.
Autoimmunity Reviews. 2013;12(11):1101-1108. J Nephrol. 2005;18:529–536.
24. Lee SJ, Klein J, Haagenson M, et al. High-resolution HLA match-
ing contributes to the success of unrelated donor marrow trans-
1. United Network for Organ Sharing. Data. https://www.unos.org/ plantation. Blood. 2007;110:4576–4583.
data. Accessed March 31, 2016. 25. Schmitz JL. Molecular Diagnostics for the Clinical Laboratorian.
2. World Health Organization. Haematopoietic stem cell transplan- 2nd ed. New York, NY: Humana Press; 2005:485–493.
tation. www.who.int/transplantation/hsctx/en/. Accessed July 30, 26. Lucas DP, Paparounis ML, Myers L, et al. Detection of
2015. HLA class I-specific antibodies by the QuikScreen enzyme-
3. Simpson E, Scott D, James E, et al. Minor H antigens: genes and linked immunosorbent assay. Clin Diag Lab Immunol. 1997;4:
peptides. Transpl Immunol. 2002;10:115–123. 252–257.
4. Zwirner NW, Dole K, Stastny P. Differential surface expression of 27. Bray RA, Nickerson PW, Kerman RH, Gebel HM. Evolution of
MICA by endothelial cells, fibroblasts, keratinocytes, and mono- HLA antibody detection: technology emulating biology. Immunol
cytes. Hum Immunol. 1999;60:323–330. Res. 2004;29:41–54.
5. Zou Y, Stastny P, Susal C, et al. Antibodies against MICA anti- 28. Colombo MB, Haworth SE, Poli F, et al. Luminex technology for
gens and kidney-transplant rejection. N Eng J Med. 2007;357: anti-HLA antibody screening: evaluation of performance and of
1293–1300. impact on laboratory routine. Cytometry B Clin Cytom. 2007;72:
6. Thielke J, Kaplan B, Benedetti E. The role of ABO-incompatible 465–471.
living donors in kidney transplantation: State of the art. Semin
Nephrol. 2007;27:408–413.
7. Rarag SS, Fehinger TA, Ruggeri L, et al. Natural killer cell recep- 1. World Health Organization. Cancer. http://www.who.int/
tors: new biology and insights into the graft-versus-leukemia effect. mediacentre/factsheets/fs297/en/. Updated 2015. Accessed May 3,
Blood. 2002;100:1935–1947. 2016.
8. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor 2. American Cancer Society. Cancer facts & figures 2014. http://www
natural killer cell alloreactivity in mismatched hematopoietic trans- .cancer.org/research/cancerfactsstatistics/cancerfactsfigures2014/
plants. Science. 2002;295:2097–2100. index. Updated 2014. Accessed March 7, 2014.
9. Taniguchi M, Rebellato LM, Cai J, et al. Higher risk of kidney graft 3. Mak TW, Saunders ME. Tumor immunology. In: The Immune Re-
failure in the presence of anti-angiotensin II type-1 receptor anti- sponse: Basic and Clinical Principles. Boston, MA: Elsevier/Academic;
bodies. Am J Transplant. 2013;13:2577–2589. 2006:826–871.
 4. Owen JA, Punt J., Stranford SA. Kuby Immunology. 7th ed. 25. Gupta S, Bent S, Kohlwes J. Test characteristics of alpha-fetoprotein
New York, NY: WH Freeman and Co.; 2013:627–651. for detecting hepatocellular carcinoma in patients with hepatitis C.
5. National Cancer Institute. Fact sheet: Cancer staging. http://www A systematic review and critical analysis. Ann Intern Med. 2003;
.cancer.gov/cancertopics/factsheet/detection/staging. Updated 2015. 139(1):46–50.
Accessed May 3, 2016. 26. El-Serag HB, Kanwal F, Davila JA, Kramer J, Richardson P.
6. American Cancer Society. How is breast cancer staged? http:// A new laboratory-based algorithm to predict development of
www.cancer.org/cancer/breastcancer/detailedguide/breast-cancer- hepatocellular carcinoma in patients with hepatitis C and cirrhosis.
staging. Updated 2016. Accessed May 3, 2016. Gastroenterology. 2014;146(5):1249–1255.e1.
7. American Cancer Society. Known and probable human carcinogens. 27. Sturgeon CM, Duffy MJ, Stenman UH, et al. National Academy
http://www.cancer.org/cancer/cancercauses/othercarcinogens/ of Clinical Biochemistry laboratory medicine practice guidelines
generalinformationaboutcarcinogens/known-and-probable- for use of tumor markers in testicular, prostate, colorectal, breast,
human-carcinogens. Updated 2015. Accessed May 3, 2016. and ovarian cancers. Clin Chem. 2008;54(12):e11–e79.
8. Benson JR, Liau SS. Cancer genetics: a primer for surgeons. Surg 28. Wald NJ. Prenatal screening for open neural tube defects and
Clin N Am. 2008;88(4):681–704. Down syndrome: three decades of progress. Prenat Diagn. 2010;
9. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000; 30(7):619–621.
100(1):57–70. 29. Gentry-Maharaj A, Menon U. Screening for ovarian cancer in the
10. Hanahan D, Weinberg RA. Hallmarks of cancer: the next genera- general population. Best Pract Res Clin Ob. 2012;26(2):243–256.
tion. Cell. 2011;144(5):646–674. 30. Medeiros LR, Rosa DD, da Rosa MI, Bozzetti MC. Accuracy of CA
11. Abbas AK, Lichtman AH, Pillai S. Immunity to tumors. In: Cellular 125 in the diagnosis of ovarian tumors: a quantitative systematic
and Molecular Immunology. 7th ed. Philadelphia, PA: Elsevier review. Eur J Obstet Gyn R B. 2009;142(2):99–105.
Saunders; 2012:389–405. 31. Buys SS, Partridge E, Black A, et al. Effect of screening on ovarian
12. van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. cancer mortality: the prostate, lung, colorectal and ovarian
Cancer Research Institute. Cancer immunity. Peptide database. (PLCO) cancer screening randomized controlled trial. JAMA.
http://cancerimmunity.org/peptide/. Updated 2013. Accessed 2011;305(22):2295–2303.
March 7, 2014. 32. Duffy MJ. Carcinoembryonic antigen as a marker for colorectal
13. Prestwich RJ, Errington F, Hatfield P, et al. The immune system— cancer: is it clinically useful? Clin Chem. 2001;47(4):624–630.
is it relevant to cancer development, progression and treatment? 33. Tan E, Gouvas N, Nicholls RJ, et al. Diagnostic precision of car-
Clin Oncol-UK (Royal College of Radiologists). 2008;20(2):101–112. cinoembryonic antigen in the detection of recurrence of colorectal
14. Cancer Research Institute. Cancer immunity. Tumor antigens cancer. Surg Oncol. 2009;18(1):15–24.
resulting from mutations. http://cancerimmunity.org/peptide/ 34. Hayes JH, Barry MJ. Screening for prostate cancer with the
mutations/. Updated 2013. Accessed March 7, 2014. prostate-specific antigen test: a review of current evidence. JAMA.
15. Lee P, Jain S, Bowne WB, Pincus MR, McPherson RA. Diagnosis 2014;311(11):1143–1149.
and management of cancer using serologic and tissue tumor 35. Borza T, Konijeti R, Kibel AS. Early detection, PSA screening, and
markers. In: McPherson RA, Pincus MR, eds. Henry’s Clinical management of overdiagnosis. Hematology—Oncology Clinics of
Diagnosis and Management by Laboratory Methods. 22nd ed. North America. 2013;27(6):1091–1110.
Philadelphia, PA: Elsevier Saunders; 2011:1385–1399. 36. Andriole GL, Crawford ED, Grubb RL, et al. Prostate cancer
16. McCudden CR, Willis MS. Circulating tumor markers: basic con- screening in the randomized prostate, lung, colorectal, and ovarian
cepts and clinical applications. In: Bishop ML, Fody EP, Schoeff LE, cancer screening trial: mortality results after 13 years of follow-
eds. Bishop Clinical Chemistry: Principles, Techniques, and Correlations. up. J Natl Cancer Inst. 2012;104(2):125–132.
7th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2013: 37. Schroder FH, Hugosson J, Roobol MJ, et al. Prostate-cancer
664–679. mortality at 11 years of follow-up. N Engl J Med. 2012;366(11):
17. Sokoll L, Chan D. Tumor markers. In: Clarke W, Dufour D, eds. 981–990.
Contemporary Practice in Clinical Chemistry. Washington, DC: AACC 38. Painter JT, Clayton NP, Herbert RA. Useful immunohistochemi-
Press; 2006. cal markers of tumor differentiation. Toxicol Pathol. 2010;38(1):
18. Pritzker KP. Cancer biomarkers: easier said than done. Clin Chem. 131–141.
2002;48(8):1147–1150. 39. Brennan DJ, O’Connor DP, Rexhepaj E, Ponten F, Gallagher WM.
19. Sturgeon C. Practice guidelines for tumor marker use in the Antibody-based proteomics: fast-tracking molecular diagnostics
clinic. Clin Chem. 2002;48(8):1151–1159. in oncology. Nat Rev Cancer. 2010;10(9):605–617.
20. Loeb S, Catalona WJ. Prostate-specific antigen in clinical practice. 40. Emerson JF, Lai KKY. Endogenous antibody interferences in
Cancer Lett. 2007;249(1):30–39. immunoassays. Lab Medicine. 2013;44(1):69–73.
21. Henry NL, Hayes DF. Cancer biomarkers. Mol Oncol. 2012;6(2): 41. Ostrov BE, Amsterdam D. The interference of monoclonal antibod-
140–146. ies with laboratory diagnosis: clinical and diagnostic implications.
22. Diamandis EP, Hoffman BR, Sturgeon CM. National Academy Immunol Invest. 2013;42(8):673–690.
of Clinical Biochemistry laboratory medicine practice guide- 42. Cole LA, Rinne KM, Shahabi S, Omrani A. False-positive hCG
lines for the use of tumor markers. Clin Chem. 2008;54(11): assay results leading to unnecessary surgery and chemotherapy
1935–1939. and needless occurrences of diabetes and coma. Clin Chem. 1999;
23. Sturgeon CM, Lai LC, Duffy MJ. Serum tumour markers: how to 45(2):313–314.
order and interpret them. BMJ. 2009;339:b3527. 43. Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of
24. Sturgeon CM, Duffy MJ, Hofmann BR, et al. National Academy disease evolution in human cancer. Nat Rev Cancer. 2007;7(6):
of Clinical Biochemistry laboratory medicine practice guidelines 441–453.
for use of tumor markers in liver, bladder, cervical, and gastric 44. Bedeir A, Krasinskas AM. Molecular diagnostics of colorectal
cancers. Clin Chem. 2010;56(6):e1–e48. cancer. Arch Pathol Lab Med. 2011;135(5):578–587.
45. De Abreu FB, Wells WA, Tsongalis GJ. The emerging role of the 69. Gajewski TF. Cancer immunotherapy. Mol Oncol. 2012;6(2):
molecular diagnostics laboratory in breast cancer personalized 242–250.
medicine. Am J Pathol. 2013;183(4):1075–1083. 70. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-T immunother-
46. Pusztai L, Mazouni C, Anderson K, Wu Y, Symmans WF. Molecular apy for castration-resistant prostate cancer. N Engl J Med. 2010;
classification of breast cancer: limitations and potential. Oncologist. 363(5):411–422.
2006;11(8):868–877. 71. van den Boorn JG, Hartmann G. Turning tumors into vaccines:
47. Petrucelli N, Daly MB, Feldman GL. BRCA1 and BRCA2 hereditary co-opting the innate immune system. Immunity. 2013;39(1):27–37.
breast and ovarian cancer. http://www.ncbi.nlm.nih.gov/books/ 72. National Cancer Institute. Fact sheet: Biological therapies for
NBK1247/. Updated 2013. Accessed June 25, 2014. cancer. http://www.cancer.gov/cancertopics/factsheet/Therapy/
48. Food and Drug Administration. Protecting and promoting biological. Updated 2013. Accessed June 5, 2014.
your health. Table of pharmacogenomic biomarkers in drug 73. Lieschke GJ, Burgess AW. Granulocyte colony-stimulating factor
labeling. http://www.fda.gov/drugs/scienceresearch/researchareas/ and granulocyte-macrophage colony-stimulating factor (2).
pharmacogenetics/ucm083378.htm. Updated 2014. Accessed N Engl J Med. 1992;327(2):99–106.
June 25, 2014. 74. Saloustros E, Tryfonidis K, Georgoulias V. Prophylactic and ther-
49. Buckingham L. Molecular diagnostics: Fundamentals, Methods, and apeutic strategies in chemotherapy-induced neutropenia. Expert
Clinical Applications. 2nd ed. Philadelphia, PA: F.A. Davis, Co.; 2012. Opin Pharmacother. 2011;12(6):851–863.
50. Portier BP, Gruver AM, Huba MA, et al. From morphologic to 75. Tonia T, Mettler A, Robert N, et al. Erythropoietin or darbepo-
molecular: established and emerging molecular diagnostics for etin for patients with cancer. Cochrane DB Syst Rev. 2012;12:
breast carcinoma. New Biotechnol. 2012;29(6):665–681. 003407.
51. Cherkis KA, Schroeder BE. Molecular testing as a tool for the man- 76. Wu S, Zhang Y, Xu L, et al. Multicenter, randomized study of
agement of metastatic cancer patients. MLO. 2014;46(3):8–10. genetically modified recombinant human interleukin-11 to
52. National Cancer Institute and National Human Genome Research prevent chemotherapy-induced thrombocytopenia in cancer
Institute. The Cancer genome atlas. http://cancergenome.nih patients receiving chemotherapy. Support Care Cancer. 2012;
.gov/. Accessed May 3, 2016. 20(8):1875–1884.
53. Tan HT, Lee YH, Chung MC. Cancer proteomics. Mass Spectrom 77. Tarhini AA, Gogas H, Kirkwood JM. IFN-alpha in the treatment
Rev. 2012;31(5):583–605. of melanoma. J Immunol. 2012;189(8):3789–3793.
54. Aboud OA, Weiss RH. New opportunities from the cancer 78. Tagawa M. Cytokine therapy for cancer. Curr Pharm Des. 2000;
metabolome. Clin Chem. 2013;59(1):138–146. 6(6):681–699.
55. Dunn GP, Old LJ, Schreiber RD. The immunobiology of cancer 79. Dezfouli S, Hatzinisiriou I, Ralph SJ. Use of cytokines in cancer
immunosurveillance and immunoediting. Immunity. 2004;21(2): vaccines/immunotherapy: recent developments improve survival
137–148. rates for patients with metastatic malignancy. Curr Pharm Des.
56. Chow MT, Moller A, Smyth MJ. Inflammation and immune 2005;11(27):3511–3530.
surveillance in cancer. Semin Cancer Biol. 2012;22(1):23–32. 80. Scott AM, Allison JP, Wolchok JD. Monoclonal antibodies in cancer
57. Burnet FM. The concept of immunological surveillance. Progress therapy. Cancer Immunity. 2012;12:14.
in Experimental Tumor Research. 1970;13:1–27. 81. Bhutani D, Vaishampayan UN. Monoclonal antibodies in oncology
58. Klein G, Sjogren HO, Klein E, Hellstrom KE. Demonstration of therapeutics: present and future indications. Expert Opin Biol Th.
resistance against methylcholanthrene-induced sarcomas in the 2013;13(2):269–282.
primary autochthonous host. Cancer Res. 1960;20:1561–1572. 82. Foltz IN, Karow M, Wasserman SM. Evolution and emergence of
59. Prehn RT, Main JM. Immunity to methylcholanthrene-induced therapeutic monoclonal antibodies: what cardiologists need to
sarcomas. J Nat Cancer Inst. 1957;18:769–778. know. Circulation. 2013;127(22):2222–2230.
60. Schreiber H. Cancer immunology. In: Paul WE, ed. Fundamental 83. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy.
Immunology. 7th ed. Philadelphia, PA: Lippincott Williams & Annu Rev Med. 2013;64:15–29.
Wilkins; 2013:1200–1234. 84. Zolot RS, Basu S, Million RP. Antibody-drug conjugates. Nat Rev
61. Finn OJ. Cancer immunology. N Engl J Med. 2008;358(25): Drug Discov. 2013;12(4):259–260.
2704–2715. 85. Antignani A, Fitzgerald D. Immunotoxins: the role of the toxin.
62. Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Toxins. 2013;5(8):1486–1502.
Invest. 2007;117(5):1137–1146. 86. Mach JP. Introduction to monoclonal antibodies. Cancer Immunity.
63. Adam JK, Odhav B, Bhoola KD. Immune responses in cancer. 2012;12:11.
Pharmacol Ther. 2003;99(1):113–132. 87. Mak TW, Saunders ME. Vaccines and clinical immunization. In:
64. Whiteside TL. The tumor microenvironment and its role in The Immune Response: Basic and Clinical Principles. Amsterdam:
promoting tumor growth. Oncogene. 2008;27(45):5904–5912. Boston: Elsevier/Academic; 2006:696–749.
65. McCarthy EF. The toxins of William B. Coley and the treatment 88. Muul LM, Spiess PJ, Director EP, Rosenberg SA. Identification of
of bone and soft-tissue sarcomas. Iowa Orthopedic J. 2006;26: specific cytolytic immune responses against autologous tumor in
154–158. humans bearing malignant melanoma. J Immunol. 1987;138(3):
66. Kawai K, Miyazaki J, Joraku A, Nishiyama H, Akaza H. Bacillus 989–995.
Calmette-Guerin (BCG) immunotherapy for bladder cancer: 89. Rosenberg SA, Dudley ME. Adoptive cell therapy for the treat-
current understanding and perspectives on engineered BCG ment of patients with metastatic melanoma. Curr Opin Immunol.
vaccine. Cancer Sci. 2013;104(1):22–27. 2009;21(2):233–240.
67. Snook AE, Waldman SA. Advances in cancer immunotherapy. 90. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumor-
Disc Med. 2013;15(81):120–125. infiltrating lymphocytes and interleukin-2 in the immunotherapy
68. Goldman B, DeFrancesco L. The cancer vaccine roller coaster. of patients with metastatic melanoma. A preliminary report.
Nat Biotechnol. 2009;27(2):129–139. N Engl J Med. 1988;319(25):1676–1680.
91. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy 16. Küppers R, Engert A, Hansmann M-L. Hodgkin lymphomoma.
for cancer: harnessing the T cell response. Nat Rev Immunol. J Clin Invest. 2012;122(10):3429–3447.
2012;12(4):269–281. 17. Schwering I, Bräuninger A, Klein U, et al. Loss of the B-lineage–
92. Lee S, Margolin K. Tumor-infiltrating lymphocytes in melanoma. specific gene expression program in Hodgkin and Reed-Sternberg
Curr Oncol Rep. 2012;14(5):468–474. cells of Hodgkin lymphoma. Blood. 2003;101(4):1505–1512.
93. Stauss HJ, Morris EC. Immunotherapy with gene-modified T cells: 18. Karube K, Niino D, Kimura Y, Ohshima K. Classical Hodgkin
limiting side effects provides new challenges. Gene Ther. 2013; lymphoma, lymphocyte depleted type: clinicopathological analy-
20(11):1029–1032. sis and prognostic comparison with other types of classical
94. Ruella M, Kalos M. Adoptive immunotherapy for cancer. Immunol Hodgkin lymphoma. Path Res Prac. 2013;209(4):201–207.
Rev. 2014;257(1):14–38. 19. Jaffett RF. Viruses and Hodgkin’s lymphoma. Ann Oncol. 2002;
95. Kalos M, June CH. Adoptive T cell transfer for cancer immunother- 13(suppl 1):23–29.
apy in the era of synthetic biology. Immunity. 2013;39(1):49–60. 20. Kapatai G, Murray P. Contribution of the Epstein-Barr virus to
the molecular pathogenesis of Hodgkin lymphoma. J Clin Pathol.
2007;60:1342–1349.
1. Potter M. Pathogenetic mechanisms in B-cell non-Hodgkin’s lym- 21. Hjalgrim H, Smedby KE, Rostgaard K, et al. Infectious mononu-
phomas in humans. Cancer Res. 1992;52:5522s. cleosis, childhood social environment, and risk of Hodgkin lym-
2. Falini B, Mason DY. Proteins encoded by genes involved in phoma. Cancer Res. 2007;67(5):2382–2388.
chromosomal alterations in lymphoma and leukemia: clinical 22. Office for National Statistics: cancer statistics registrations: regis-
value of their detection by immunocytochemistry. Blood. 2002; trations of cancer diagnosed in 2007, England. Series MBI no. 38.
99(2):409–426. Surrey, UK. Office of Public Sector Information, 2010.
3. Hari SB, Perera BGK, Panjitkar P, et al. Conformation-selective 23. Cancer Research UK, Non-Hodgkin lymphoma statistics, November
inhibitors reveal differences in the activation and phosphate- 2014. publications.cancerresearchuk.org/downloads/product/CS_
binding loops of the tyrosine kinases ABI. Chem Bio. 2013;8: KF_NHL.pdf. Accessed May 3, 2016.
2734–2743. 24. Shankland KR, Armitage JO, Hancock BW. Non-Hodgkin lym-
4. Lin Y-L, Roux, B. Computational analysis of the binding speci- phoma. Lancet. 2012;380:848–857.
ficity of Gleevec to ABI, c-Kit, Lck, and s-Src tyhrosine kinases. 25. Simard JF, Baecklund F, Chang ET, et al. Lifestyle factors, autoim-
J Am Chem Soc. 2013;135:14741–14753. mune disease and family history in prognosis of non-Hodgkin lym-
5. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the phoma overall and subtypes. Int J Cancer. 2012;132:2659–2666.
classification of the acute leukaemias: French-American-British 26. Niels WCJ, Palumbo A, Johnsen HE, et al. The clinical relevance
Cooperative Group. Br J Haematol. 1976;33:451–458. and management of monoclonal gammopathy of undetermined
6. Bennett JM, Catovsky D, Daniel MT, et al. The French-American- significance and related disorders: recommendations from the
British (FAB) Co-operative Group. Proposals for the classifica- European Myeloma Network. Haematologica. 2014;99(6):984–996.
tion of the myelodysplastic syndromes. Br J Haematol. 1982; 51: 27. Caers J, Vekemans M-C, Bries G, et al. Diagnosis and follow-up
189–199. of monoclonal gammopathies of undetermined significance; in-
7. Harris ML, Jaffe ES, Diebold J, et al. The World Health Organiza- formation for referring physicians. Ann Med. 2013;45:413–422.
tion classification of hematological malignancies. Report of the 28. Bianchi G, Ghobrial IM. Does my patient with a serum mono-
Clinical Advisory Committee meeting, Airline House, Virginia, clonal spike have multiple myeloma? Hematol Oncol Clin N Am.
November 1997. Mod Pathol. 2000;13:193. 2012;26:383–393.
8. Swerdlow SH, Campo E, Harris NL, et al., eds. WHO Classification 29. Kyle RA, Durie BG, Rajkumar SV, et al. Monoclonal gammopathy
of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, of undetermined significance (MGUS) and smoldering (asymp-
France: IARC Press; 2008:10–15, 171–175, 194–195. tomatic) multiple myeloma: IMWG consensus perspectives risk
9. Vardiman JV, Thiele J, Arber DA, et al. The 2008 revision of factors for progression and guidelines for monitoring and man-
the World Health Organization (WHO) classification of myeloid agement. Leukemia. 2010;24:1121–1127.
neoplasms and acute leukemia: rationale and important changes. 30. Rajkumar SV, Kyle RA, Therneau TM, et al. Serum free light chain
Blood. 2009;114:937–951. ratio is an independent risk factor for progression in monoclonal
10. Colby-Graham MF, Chordas C. The childhood leukemias. J Pediatric gammopathy of undetermined significance. Blood. 2005;106(3):
Nursing. 2003;18(2):87–95. 812–817.
11. Kebriaei P, Anastasi J, Larson RA. Acute lymphoblastic leukemia: 31. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA
diagnosis and classification. Best Practice Res Clinic Haematol. Cancer J Clin. 2013;63:11–30.
2001;15(4):597–621. 32. American Cancer Society. Multiple myeloma. http://www.cancer
12. Riley RS, Massey D, Jackson-Cook C, et al. Immunophenotypic .org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-
analysis of acute lymphocytic leukemia. Hematol Oncol Clin N Am. key-statistics. Accessed April 5, 2016.
2002;16:245–299. 33. Zingone A, Kuehl WM. Pathogenesis of monoclonal gammopathy
13. Zenz, T, Mertens, D, Küppers, R, et al. From pathogenesis to of undetermined significance and progression to multiple myeloma.
treatment of chronic lymphocytic leukaemia. Nature Rev Cancer. Sem Hematol. 2011;48(1):4–12.
2010;10:37–50. 34. Kuehl WM, Bergsagel PL. Molecular pathogenesis of multiple
14. Venkataraman G, Aguhar C, Kreitman RJ, et al. Characteristic myeloma and its premalignant precursor. J Clin Invest. 2012;
CD103 and CD123 expression pattern defines hairy cell 122(10):3456–3463.
leukemia. Am J Clin Path. 2011;136:625–630. 35. Chiecchio L, Protheroe RKM, Ibrahim AH, et al. Deletion of chro-
15. Tiacci E, Schiavoni G, Forconi F, et al. Simple genetic diagnosis mosome 13 detected by conventional cytogenetics is a critical
of hairy cell leukemia by sensitive detection of the BRAF-V600E prognostic factor in myeloma. Leukemia. 2006;20:1610–1617.
mutation. Blood. 2012;119(1):192–196.
36. Chang WJ, Santanna-Davilia R, Van Wier SA, et al. Prognostic 59. Merker JD, Valouev A, Gotlib J. Next-generation sequencing in
factors for hyperdiploid-myeloma: effects of chromosome 13 hematologic malignancies: what will be the dividends? Ther
deletions and IgH translocations. Leukemia. 2006;20:807–813. Adv Hematol. 2012;3(6):333–339.
37. Bird JM, Owen RG, S’Da S, et al. Guidelines for the diagnosis and
management of multiple myeloma 2011. Br. J. Hematology. 2011; Immunodeficiency
154:32–75. 1. Al-Herz W, Bousfiha A, Casanova J-L, et al. Primary immunodefi-
38. Rajkumar SV. Multiple myeloma: 2013 update on diagnosis, risk- ciency diseases: an update on classification from the International
stratification, and management. Am J Hematol. 2013;88:226–235. Union of Immunological Societies Expert Committee for Primary
39. Saheen SP, Talwalker SS, Lin, P, et al. Waldenström macroglobu- Immunodeficiency. Frontiers in Immunology. 2014;5(162):1–33.
linemia: a review of the entity and its differential diagnosis. Adv 2. McCusker C, Warrington R. Primary immunodeficiency. Allergy
Anat Pathol. 2012;19(1):11–27. Asthma Clin Immunol. 2011;7(suppl 1):2–8.
40. Gertz MA. Waldenström macroglobulinemia: 2013 update on 3. Kelly BT, Tam JS, Verbsky JW, Routes JM. Screening for severe
diagnosis, risk stratification, and management. Am J Hematol. combined immunodeficiency. Clin Epidemiol. 2013;5:363–369.
2013;88:703–711. 4. Maggina P, Gennary AR. Classification of primary immunodefi-
41. Gertz M. Waldenström macroglobulinemia. Hematol. 2012;17(S1): ciencies: need for a revised approach? J Allergy Clin Immunol.
S112–S116. 2012;131:292–294.
42. Owen RG, Treon SP, Al-Katib A, et al. Clinicopathological definition 5. Bousfiha A, Jeddane L, Ailal F, et al. A phenotypic approach for
of Waldenström’s macroglobulinemia: consensus panel recommen- IUIS PID classification and diagnosis: guidelines for clinicians at
dations from the Second International Workshop on Waldenström’s the bedside. J Clin Immunol. 2013;33:1078–1087.
macroglobulinemia. Semin Oncol. 2003;30:110–115. 6. Stiehm RE. The four most common pediatric immunodeficien-
43. Vital A. Paraproteinemic neuropathies. Bran Pathol. 2001;11: cies. Adv Exp Med Biol. 2007; 601:15–26.
399–407. 7. Fleisher TA. Back to basics: primary immune deficiencies: win-
44. Witzig TE, Wahner-Roedler DL. Heavy chain disease. Curr Treat dow into the immune system. Pediatr Rev. 2006;27(10):363–372.
Option On. 2002;3:247–254. 8. Conley ME. Primary antibody deficiency diseases. In: Detrick B,
45. Wahner-Roedler DL, Kyle RA. Heavy chain diseases. Best Pract Hamilton RG, Folds JD, eds. Manual of Molecular and Clinical Lab-
Res Clin Ha. 2005;18(4):729–746. oratory Immunology. Washington, DC: ASM Press; Washington,
46. Bianchi G, Anderson KC, Harris NL, Sohani AR. The heavy chain DC. 2006:906–913.
diseases: clinical and pathologic features. Oncology. 2014;28(1):45. 9. Fischer A. Human primary immunodeficiency diseases. Immunity.
47. Craig FE, Foon KA. Flow cytometric immunophenotyping for 2007;27:835–845.
hematologic neoplasms. Blood. 2008;11(8):3941–3967. 10. Fischer A. Primary immunodeficiency diseases: an experimental
48. Wood BL, Borowitz MJ. The flow cytometric evaluation of model for molecular medicine. Lancet. 2001;357:1863–1869.
hematopoietic neoplasia. In: McPherson RA, Pincus MR, eds. 11. Anonymous. Primary immunodeficiency diseases. Report of an
Henry’s Clinical Diagnosis and Management by Laboratory Methods. IUIS Scientific Committee. International Union of Immunological
22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:656–673. Societies. Clin Exp Immunol. 1999;118(suppl 1):1–28.
49. McPherson RA, Massey HD. Laboratory evaluation of immunoglob- 12. Puck J. Prenatal diagnosis and genetic analysis of X-linked immun-
ulin function and humoral immunity. In: McPherson RA, Pincus MR, odeficiency disorders. Pediatr Res. 1993;33(suppl):S29.
eds. Henry’s Clinical Diagnosis and Management by Laboratory Meth- 13. Garcia JM, Español T, Gurbindo MªD, et al. Update on the treat-
ods. 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:899–913. ment of primary immunodeficiencies. Allergol Immunopath. 2007;
50. The Binding Site. Immunofixation (IFE) Kit. Product Insert. The 35(5):184–192.
Binding Site Group, Ltd., Birmingham, UK, July 2010. 14. Roifman CM. Approach to the diagnosis of severe combined
51. Korbet SM, Schwartz MM. Disease of the month. Multiple immunodeficiency. In: Detrick B, Hamilton RG, Folds JD, eds. Man-
myeloma. J Am Soc Nephrol. 2006;17:2533–2545. ual of Molecular and Clinical Laboratory Immunology. Washington,
52. Dispenzieri A, Kyle R, Gertz M, et al. International Myeloma Work- DC: ASM Press; 2006:895–900.
ing Group guidelines for serum-free light chain analysis in multiple 15. Puck JPIDJ. Primary immunodeficiency diseases. JAMA. 1997;
myeloma and related disorders. Leukemia. 2009;23:215–224. 278:1835–1841.
53. Viva Products application note. Urine protein concentration with 16. Hilman B, Sorensen RU. Management options: SCIDS with
vivaproducts concentrators. www.vivaproducts.com/downloads/ adenosine deaminase deficiency. Ann Allergy. 1994;72.
urine-protein-concentration-w-concentrators.pdf. Accessed 17. Gennery AR, Cant AJ. Cord blood stem cell transplantation in pri-
July 1, 2015. mary immune deficiencies. Curr Opin Allergy CI. 2007;7:528–534.
54. Jenner E. Serum free light chains in clinical laboratory diagnos- 18. Peacocke M, Siminovitch KA. Wiskott-Aldrich syndrome: new
tics. Clin Chim Acta. 2014;427:15–20. molecular and biochemical insights. J Am Acad Dermatol. 1992;27.
55. Katzmann J, Kyle RA, Benson J, et al. Screening panels for detection 19. Taddei I, Morishima M, Huynh T, Lindsay EA. Genetic factors
of monoclonal gammopathies. Clin Chem. 2009;55:1517–1522. are major determinants of phenotypic variability in a mouse
56. Buckingham L. Molecular Diagnostics. Fundamentals, Methods, and model of the DiGeorge/del22q11 syndromes. PNAS. 2001;98(20):
Clinical Applications. 2nd ed. Philadelphia, PA: F.A. Davis Co.; 11428–11431.
2012:369–418. 20. Noël A-C, Pelluard F, Delezoide A-L, et al. Fetal phenotype associ-
57. Simons A, Sikkema-Raddatz B, deLeeuw N, et al. Genome-wide ated with the 22q11 deletion. Am J Med Genet Part A. 2014;
arrays in routing diagnostics of hematologic malignancies. Hum 9999:1–8.
Mutat. 2012;33(6):941–948. 21. Fischer A. Primary immune deficiency diseases. In: Kasper D,
58. Mullighan CG. Genome sequencing of lymphoid malignancies. Fauci A, Hauser S, et al., eds. Harrison’s Principles of Internal Med-
Blood. 2013;122(24):3899–3907. icine. 19th ed. New York, NY: McGraw-Hill; 2015. accessmedicine
 .mhmedical.com.libproxy1.upstate.edu/content.aspx?bookid= 14. Fischetti VA. Streptococcal M protein. Sci Am. 1991;264:58.
1130&Sectionid=79749659. Accessed July 20, 2015. 15. Larson HS. Streptococcaccae. In: Mahan CR, Manuselis G, eds.
22. Buckley R. Primary immunodeficiency diseases due to defects in Textbook of Diagnostic Microbiology. 2nd ed. Philadelphia, PA: WB
lymphocytes. N Engl J Med. 2000;343:1313–1324. Saunders; 2000:345–371.
23. Holland SM, Neutropenia and neutrophil defects. In: Detrick B, 16. Wessels MR. Streptococcal and enterococcal infections. In:
Hamilton RG, Folds JD, eds. Manual of Molecular and Clinical Lab- Braunwald E, Fauci AS, Kasper EL, et al., eds. Harrison’s Principles
oratory Immunology. Washington, DC: ASM Press. 2006:924–932. of Internal Medicine. 15th ed. New York: McGraw-Hill; 2001:
24. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflam- 901–909.
masomes. Nature Rev Immunol. 2013;13:397–411. 17. Sauda M, Wu W, Conran P, et al. Streptococcal pyrogenic exo-
25. Giclas PC. Hereditary and acquired complement deficiencies. In: toxin B enhances tissue damage initiated by other Streptococcus
Detrick B, Hamilton RG, Folds JD, eds. Manual of Molecular and pyogenes products. J Infect Dis. 2001;184:723–731.
Clinical Laboratory Immunology. Washington, DC: ASM Press; 2006: 18. Spellerberg B, Brandt C. Streptococcus. In: Murray PR, Baron EJ,
914–923 Jorgensen JH, et al., eds. Manual of Clinical Microbiology. 9th ed.
26. CDC. Newborn screening: severe combined immunodeficiency Washington, DC: ASM Press; 2007:412–429.
(SCID). www.cdc.gov/newbornscreening/scid.html. Accessed 19. Moses AE, Goldberg S, Korenman Z, et al. Invasive group A strep-
July 17, 2015. tococcal infections. Israel Emerg Infect Dis. 2002;8:421–426.
27. Rosenzweig D, Fleisher TA. Laboratory evaluation for T cell func- 20. Davies HD, McGeer A, Schwartz B, et al. Invasive group A strep-
tion. J Allergy Clin Immunol. 2013;131(2):622–623e.4. tococcal infections in Ontario, Canada, Ontario Group A Strepto-
28. Mallott J, Kwan A, Church J, et al. Newborn screening for SCID coccal Study Group. N Eng J Med. 2001;335:547–554.
identifies patients with ataxia telangiectasia. J Clin Immunol. 21. Active bacterial core surveillance. Active bacterial core surveil-
2013;33:540–549. lance (ABCs) report, emerging infections program network: group
29. Verbsky JW, Grossman WJ. Cellular and genetic basis of primary A streptococcus 2006. http://www.cdc.gov/abcs/index.html.
immune deficiencies. Pediatr Clin N Am. 2006;53:649–684. Accessed May 4, 2016.
22. Brady HR, Brenner BM. Pathogenesis of glomerular injury. In:
Braunwald E, Fauci AS, Kasper DL, et al., eds. Harrison’s Principles
of Internal Medicine. 15th ed. New York, NY: McGraw-Hill; 2001:
1. Tancrede C. Role of human microflora in health and disease. Eur 1572–1580.
J Clin Microbiol Infect Dis. 1992;11(11):1012–1015. 23. Lang MM, Towers C. Identifying poststreptococcal glomeru-
2. The body’s ecosystem. www.the-scientist.com/?articles.view/ lonephritis. Nurse Pract. 2001;26:34.
articleNo/40600/title/The-Body-s-Ecosystem. Accessed December 12, 24. Sajid M, Kawde A-N, Daud M. Designs, formats and applications
2014. of lateral flow assay: a literature review. Journal of Saudi Chemical
3. Gordon GI, Ley RE, Wilson R, et al. Extending our view of self: the Society. 2014;19(6):689–705. dx.doi.org/10.1016/j.jscs.2014.09.
human gut microbiome initiative (HGMI) [white paper]. National 001. Accessed July 27, 2015.
Human Genome Research Institute. 2005. 25. Stevens DL, Kaplan EL. Streptococcal Infections—Clinical Aspects,
4. Farley TA, Cohen DA, Elkins W. Asymptomatic sexually trans- Microbiology, and Molecular Pathogenesis. New York, NY: Oxford
mitted diseases: the case for screening. Prev Med. 2003;36: University Press; 2000.
502–509. 26. Lewis LS. Medscape. Impetigo work-up. emedicine.medscape.
5. Korenromp EL, Sudaryo MK, de Vlas SJ, et al. What proportion com/article/965254-workup. Accessed July 27, 2015.
of episodes of gonorrhoea and chlamydia becomes symptomatic? 27. Marshall BJ. History of the discovery of Campylobacter pylori. In:
Int. J. STD AIDS. 2002;13:91–101. Blaser MJ. Campylobacter pylori in Gastritis and Peptic Ulcer
6. Que Y, Morielloon P. Staphylococcus aureus (including staphylococ- Disease. New York, NY: Igaku Shoin; 1989:7–23.
cal toxic shock). In: Mandell GL, Bennett JE, Dolin R, eds. Principles 28. Malfertheiner P, Megraud F, O’Moran C, and the European
and Practice of Infectious Diseases. 7th ed. Philadelphia, PA: Churchill Helicobacter pylori Study Group (EHPSG). Current concepts in
Livingstone Elsevier; 2009:2543–2578. the management of Helicobacter pylori infection—the Maastricht
7. Findley BB, McFadden G. Anti-immunology: evasion of the host 2-2000 Consensus Report. Alim Pharmacol Ther. 2002;16:
immune system by bacterial and viral pathogens. Cell. 2006;124: 167–180, 200.
767–782. 29. Hazell SL, Lee A, Brady L, et al. Campylobacter pylori and gastritis:
8. Black JG. Microbiology Principles and Explorations. 6th ed. Hoboken, association with intracellular spaces and adaptation to an environ-
NJ: Wiley; 2005:446–469. ment of mucus as important factors in colonization of the gastric
9. Mak TW, Saunders M. The Immune Response: Basic and Clinical Prin- epithelium. J Infect Dis. 1986;153:658–663.
ciples. Burlington, MA: Elsevier Academic Press; 2006:641–694. 30. Everhart JE, Kruszon-Moran D, Perez-Perez GI, et al.
10. Forbes BA, Sahm DF, Weissfeld A. Bailey and Scott’s Diagnostic Seroprevalence and ethnic differences in Helicobacter pylori
Microbiology. 12th ed. St. Louis: Mosby Elsevier; 2007:265–280. infection among adults in the United States. J Infect Dis.
11. Centers for Disease Control and Prevention. Summary of notifi- 2000;181:1359–1363.
able diseases—March 21, 2008. MMWR. 2008;55(53):1–94. 31. National Institutes of Health Consensus Conference. Helicobacter
12. CDC, biotechnology core branch facility. Introduction to emm pylori in peptic ulcers. JAMA. 1994;272:65–69.
typing: M protein gene (emm) typing Streptococcus pyogenes. 32. Segal ED, Cha J, Lo J, et al. Altered states: involvement of
http://www.cdc.gov/streplab/M-ProteinGene-typing.html. Accessed phosphorylated CagA in the induction of host cellular growth
May 4, 2016. changes by Helicobacter pylori. Proc Natl Acad Sci USA. 1999;
13. Shet A, Kaplan E. Diagnostic methods for group A streptococcal 96:14559–14564.
infections. In: Detrick B, Hamilton RG, Folds JD, eds. Manual of 33. Megraud F. Impact of Helicobacter pylori virulence in the outcome
Molecular and Clinical Laboratory Immunology. 7th ed. Washington, of gastroduodenal disease: lessons from the microbiologist. Digest
DC: ASM Press; 2006:428–433. Dis. 2001;19:99–103.
34. Sokic-Milutinovic T, Wex T, Todorovic V, et al. Anti-CagA and 54. Nisar N, Guleria R, Kumar S, et al. Mycoplasma pneumoniae and
anti-VacA antibodies in Helicobacter pylori-infected patients with its role in asthma. Postgrad Med J. 2007;83:100–104.
and without peptic ulcer disease in Serbia and Montenegro. Scand 55. Schalock PC, Dinulos JGH. Mycoplasma pneumoniae-induced
J Gastroenterol. 2004;3:222–226. Stevens-Johnson syndrome without skin lesions: Fact or fiction?
35. Atherton J, Cao P, Peek RM, et al. Mosaicism in vacuolating cyto- J Am Acad Dermatol. 2005;52:312–315.
toxin alleles of Helicobacter pylori: association of specific vacA 56. McCormack JG. Mycoplasma pneumoniae and the erythema
types with cytotoxin production and peptic ulceration. J Biol multiforme–Stevens-Johnson syndrome. J Infect. 1981;3:32.
Chem. 1995;270:1771–1777. 57. Schubothe H. The cold hemagglutinin disease. Semin Hematol.
36. Suzuki M, Miura S, Suematsu M, et al. Helicobacter pylori-associated 1966;3:27.
ammonia production enhances neutrophil-dependent gastric 58. Fink CG, Sillis M, Read SJ, et al. Neurologic disease associated
mucosal cell injury. Am J Physiol. 1992;263:G719–G725. with Mycoplasma pneumoniae infection: PCR evidence against a
37. Letley DP, Rhead JL, Twells RJ, et al. Determinants of non-toxicity direct invasive mechanism. Clin Mol Pathol. 1995;48:51–54.
in the gastric pathogen Helicobacter pylori. J Biol Chem. 2003; 59. Yang J, Hooper W, Phillips D, et al. Cytokines in Mycoplasma
278:26734–26741. pneumoniae infections. Cytokine Growth Factor Rev. 2004;15:
38. D’Elios MM, Manghetti M, De Carli M, et al. T helper 1 effector 157–168.
cells specific for Helicobacter pylori in the gastric antrum of patients 60. Feizi T, Taylor-Robinson D. Cold agglutinin anti-I and Mycoplasma
with peptic ulcer disease. J Immunol. 1997;158:962–967. pneumoniae. Immunology. 1967;13:405.
39. Wang J, Brooks EG, Bamford KB, et al. Negative selection of 61. Beersma MFC, Dirven K, van Dam AP, et al. Evaluation of 12 com-
T cells by Helicobacter pylori as a model for bacterial strain selec- mercial tests and the complement fixation test for Mycoplasma
tion by immune evasion. J Immunol. 2001;167:926–934. pneumoniae-specific immunoglobulin G (IgG) and IgM antibodies,
40. Allen LA, Schlesinger LS, Kang B. Virulent strains of Helicobacter with PCR used as the “gold standard.” J Clin Microbiol. 2005;
pylori demonstrate delayed phagocytosis and stimulate homo- 43:2277–2285.
typic phagosome fusion in macrophages. J Exp Med. 2000;191: 62. Waites KB, Brown MB, Simecka JW. Mycoplasma: immunologic
115–128. and molecular diagnostic methods. In: Detrick B, Hamilton RG,
41. Mohammadi M, Nedrud J, Redline R, et al. Murine CD4 T-cell Folds JE, eds. Manual of Molecular and Clinical Laboratory Im-
response to Helicobacter infection: TH1 cells enhance gastritis munology. 7th ed. Washington, DC: ASM Press; 2006:510–517.
and TH2 cells reduce bacterial load. Gastroenterology. 1997;113: 63. Baum SG. Mycoplasma pneumoniae and atypical pneumoniae. In:
1848–1857. Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of
42. Graham DY, Lew GM, Malaty HM, et al. Factors influencing the Infectious Diseases. 7th ed. Philadelphia, PA: Churchill Livingstone
eradication of Helicobacter pylori with triple therapy. Gastroenterology. Elsevier; 2009:2486.
1992;102:493–496. 64. Rosenfield RE, Schmidt PJ, Calvo RC, et al. Anti-i, a frequent cold
43. Peek RM, Blaser MJ. Helicobacter pylori and gastrointestinal tract agglutinin in infectious mononucleosis. Vox Sang. 1965;10:631.
adenocarcinomas. Nat Rev Cancer. 2002;2:28–37. 65. Lind K, Spencer ES, Anderson HK. Cold agglutinin production
44. Wotherspoon AC, Ortiz Hidalgo C, Falzon MR, et al. Helicobacter and cytomegalovirus infection. Scand J Infect Dis. 1974;6:109.
pylori-associated gastritis and primary B-cell gastric lymphoma. 66. FilmArray® Respiratory Panel Information Sheet. Biofire Diagnos-
Lancet. 1991;338:1175–1176. tics, Salt Lake City, UT. 2014.
45. Dunn BE, Phadnis SH. Serologic and molecular diagnosis of 67. Eremeeva ME, Dasch GA. Rickettsial (spotted & typhus fevers) &
Helicobacter pylori infection and eradication. In: Detrick B, Hamilton related infections (anaplasmosis & ehrlichiosis). Centers for Disease
RG, Folds JE, eds. Manual of Molecular and Clinical Laboratory Control and Prevention, Atlanta, GA. wwwnc.cdc.gov/travel/
Immunology. 7th ed. Washington, DC: ASM Press; 2006:462–467. yellowbook/2014/chapter-3-infectious-diseases-related-to-
46. Megraud F, Lehours P. Helicobacter pylori detection and antimi- travel/rickettsial-spotted-and-typhus-fevers-and-related-infections-
crobial susceptibility testing. Clin Microbiol Rev. 2007;20(2): anaplasmosis-and-ehrlichiosis. Accessed December 12, 2014.
280–322. 68. Rocky Mountain spotted fever statistics and epidemiology. Centers
47. Megraud F, Fox JG. Helicobacter. In: Murray PR, Baron EJ, Jorgensen for Disease Control and Prevention. Atlanta, GA. www.cdc.gov/
JH, et al., eds. Manual of Clinical Microbiology. 9th ed. Washington, rmsf/stats/. Accessed January 5, 2015.
DC: ASM Press; 2007;947–962. 69. Nettleman MD. Biological warfare and infection control. Infect
48. Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity Control Hosp Epidemiol. 1991;12:368–372.
of mycoplasmas. Microbiol Mol Biol Rev. 1998; 62:1094–1156. 70. Perine PL, Chandler BP, Krause DK, et al. A clinico-epidemiological
49. Shmuel R, David Y, Yehudith N. Molecular biology and path- study of epidemic typhus in Africa. Infect Dis. 1992;14:1149–1158.
ogenicity of mycoplasmas. Microbiol Mol Biol Rev. 1998;62: 71. Walker DH, Bouyer DH. Rickettsia and orientia. In: Murray PR,
1094–1156. Baron EJ, Jorgensen JH, et al., eds. Manual of Clinical Microbiology.
50. Waites KB, Taylor-Robinson DT. Mycoplasma and ureoplasma. 9th ed. Washington, DC: ASM Press; 2007:1036–1045.
In: Murray PR, Baron EJ, Jorgensen JH, et al., eds. Manual of 72. Anderson B, Friedman H, Bendinelli M. Rickettsial Infection and
Clinical Microbiology. 9th ed. Washington, DC: ASM Press; Immunity. New York, NY: Plenum Press; 1997.
2007:1004–1020. 73. Rocky Mountain spotted fever (RMSF)—statistics and epidemi-
51. Waites KB. New concepts of Mycoplasma pneumoniae infections ology. Centers for Disease and Control. www.cdc.gov/rmsf/stats/.
in children. Pediatr Pulmonol. 2003;36:267–278. Accessed December 14, 2014.
52. Foy HM. Infections caused by Mycoplasma pneumoniae and pos- 74. Hechemy KE, Rikihisa Y, Macaluso K, et al. The Rickettsiaceae,
sible carrier state in different populations of patients. Clin Infect Anaplasmataceae, Bartonellaceae, and Coxiellaceae. In: Detrick
Dis. 1993;17(suppl. 1):S37–S46. B, Hamilton RG, Folds JE, eds. Manual of Molecular and Clinical
53. Forbes BA, Sahm DF, Weissfeld A. Bailey and Scott’s Diagnostic Laboratory Immunology. 7th ed. Washington, DC: ASM Press;
Microbiology. 12th ed. St. Louis: Mosby Elsevier; 2007:525–532. 2006:527–539.
75. Walker DH, Ismail N. Emerging and re-emerging rickettsioses: 12. Goldmeier D, Hay P. A review and update on adult syphilis, with
endothelial cell infection and early disease events. Nat Rev Micro- particular reference to its treatment. Int J STD AIDS. 1993;4:70–82.
biol. 2008;6:375–386. 13. Schiff E, Lindberg M. Neurosyphilis. South Med J. 2002;95:
76. Uchiyama T, Kawano H, Kusuhara Y. The major outer membrane 1083–1087.
protein rOmpB of spotted fever group rickettsiae functions in the 14. Lukehart SA. Syphilis. In: Kasper D, Fauci A, Hauser S, et al., eds.
rickettsial adherence to and invasion of Vero cells. Microb Infect. Harrison’s Principles of Internal Medicine. 19th ed. New York, NY:
2006:801–809. McGraw-Hill; 2015. accessmedicine.mhmedical.com.libproxy2.
77. Li H, Walker DH. rOmpA is a critical protein for the adhesion upstate.edu/content.aspx?bookid=1130&Sectionid=79737418.
of Rickettsia rickettsii to host cells. Microb Pathog. 1998;24: Accessed September 3, 2015.
289–298. 15. Symptomatic early neurosyphilis among HIV-positive men who
78. Harrell GT, Aikawa JK. Pathogenesis of circulatory failure in have sex with men—four cities, United States, January 2002–
Rocky Mountain spotted fever. Alteration in the blood volume June 2004. MMWR. 2007;56(25):625–628.
and the thiocyanate space at various stages of the disease. Arch 16. Centers for Disease Control and Prevention. Congenital syphilis:
Intern Med. 1949;83:331–347. United States, 2000. MMWR. 2001;50:573–577.
79. Elghetany TM, Walker DH. Hemostatic changes in Rocky Moun- 17. Centers for Disease Control and Prevention. Congenital syphilis:
tain spotted fever and Mediterranean spotted fever. Am J Clin reported cases and rates per 100,000 live births in infants by year
Pathol. 1999;112:159–168. of birth and race/ethnicity of mother, 2008–2012. www.cdc.gov/
80. Walker DH. Rickettsia rickettsii and other spotted fever group std/stats12/tables/43.htm. Accessed April 17, 2014.
Rickettsiae (Rocky Mountain spotted fever and other spotted 18. Chhabra RS, Brion LP, Castro M, Freundlich L, Glaser JH. Com-
fevers). In: Mandell GL, Bennett JE, Dolin R, eds. Principles and parison of maternal sera, cord blood, and neonatal sera for detect-
Practice of Infectious Diseases. 7th ed. Philadelphia, PA: Churchill ing presumptive congenital syphilis: relationship with maternal
Livingstone Elsevier; 2009:2499–2507. treatment. Pediatrics. 1993;91:88–91.
81. Rocky Mountain Spotted Fever (RMSF)—Symptoms, Diagnosis, 19. Doherty L, Fenton KA, Jones J, et al. Syphilis: Old problem, new
and Treatment. Centers for Disease Control and Prevention. strategy. BMJ. 2002;325:153–165.
Atlanta, GA. 20. Michelow IC, Wendel GD Jr, Norgard MV, et al. Central nervous
system infection in congenital syphilis. N Engl J Med. 2002;
346:1792–1798.
1. The spirochetes. In: Tile PM. Bailey and Scott’s Diagnostic Microbi- 21. Larsen SA, Pope V, Johnson RE, Kennedy RJ, eds. A Manual
ology. 13th ed. St. Louis: Mosby; 2014:535–545. of Tests for Syphilis. Washington, DC: American Public Health
2. Pope V, Ari MD, Schriefer M, Levett PN. Immunological methods Association; 1998.
for the diagnosis of spirochetal diseases. In: Detrick B, Hamilton 22. Pope V, Fears MB, Morrill WE, et al. Comparison of the Serodia
RG, Folds JD. Manual of Clinical Laboratory Immunology. 7th ed. Treponema pallidum particle agglutination, Captia Syphilis-G,
Washington, DC: ASM Press; 2006:477–492. and SpiroTek Reagin II tests with standard test techniques for
3. Centers for Disease Control and Prevention. Sexually transmitted diagnosis of syphilis. J Clin Microbiol. 2000;38:2543–2545.
diseases (STDs): data and statistics. www.cdc.gov/std/stats/. 23. Sena AC, White BL, Sparling PF. Novel treponema pallidum
Accessed April 12, 2016. serologic tests: a paradigm shift in syphilis screening for the
4. Centers for Disease Control and Prevention. Primary and second- 21st century. Clin Infect Dis. 2010;51(6):700–708.
ary syphilis—United States, 2005–2013. MMWR. 2014;63(18): 24. Cole MJ, Perry KR, Parry JV. Comparative evaluation of 15 sero-
402–406. logical assays for the detection of syphilis infection. Eur J Clin
5. Centers for Disease Control and Prevention. Primary and second- Microbiol. 2007;26(10):705–713.
ary syphilis among men who have sex with men—New York City, 25. Gomez E, Jespersen DJ, Harring JA, Binnicker MJ. Evaluation of
2001. MMWR. 2002;51(38):853–856. the Bio-Rad BioPlex 2200 syphilis multiplex flow immunoassay
6. Centers for Disease Control and Prevention. Outbreak of syphilis for the detection of IgM- and IgG-class antitreponemal antibod-
among men who have sex with men—Southern California, 2000. ies. Clin Vaccine Immunol. 2010;17:966.
MMWR. 2001;50(38):117–120. 26. Binnicker MJ, Jespersen DJ, Rollins LO. Treponema-specific tests
7. World Health Organization. Global Incidence and Prevalence of for serodiagnosis of syphilis: comparative evaluation of seven
Selected Curable Sexually Transmitted Infections–2008. Geneva: assays. J Clin Microbiol. 2011;49:1313.
World Health Organization; 2012. www.who.int/reproductive- 27. Centers for Disease Control and Prevention. www.cdc.gov/
health/publications/rtis/stisestimates/en/. Accessed August 19, 2015. std/syphilis/DCL-Syphilis-MMWR-2-10-2011.pdf. Accessed
8. Blanco DR, Miller JN, Lovett MA. Surface antigens of the syphilis May 8, 2014.
spirochete and their potential as virulence determinants. Emerg 28. Pope V. Use of treponemal test to screen for syphilis. Infect Med.
Infect Dis. 1997;3:11–20. 2004;21:399–404.
9. LaSala PR, Smith MB. Spirochete infections. In: McPherson 29. Binnicker MJ. Which algorithm should be used to screen for
RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management by syphilis? Curr Opin Infect Dis. 2012;25(1):79–85.
Laboratory Methods. 22nd ed. Philadelphia, PA: WB Saunders; 30. Centers for Disease Control and Prevention. Discordant results
2011:1129–1144. from reverse sequence syphilis screening: five laboratories, United
10. Orton S, Liu H, Dodd R, Williams A. Prevalence of circulating States, 2006–2010. MMWR. 2011;60(05):133–137.
Treponema pallidum DNA and RNA in blood donors with confirmed- 31. Noordhoek G, Wolters EC, De Jonge MEJ, Van Embden JDA.
positive syphilis tests. Transfusion. 2002;42:94–99. Detection by polymerase chain reaction of Treponema pallidum
11. American Red Cross. Blood donation eligibility criteria. www DNA in cerebrospinal fluid from neurosyphilis patients before
.redcrossblood.org/donating-blood/eligibility-requirements/ and after antibiotic treatment. J Clin Microbiol. 1991;29:1976–1984.
eligibility-criteria-topic. Accessed May 27, 2014.
32. Heymans R, vander Helm JJ, deVries HJC, et al. Clinical value of 53. Johnson B. Lyme disease: serologic assays for antibodies to
Treponema pallidum real time PCR for diagnosis of syphilis. J Clin Borrelia burgdorferi. In: Detrick B, Hamilton RG, Folds JD, Manual
Microbiol. 2010;48(2):497–502. of Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM
33. Gayet-Ageron A, Lautenschlager S, Ninet B, et al. Sensitivity, Press; 2006:493–500.
specificity and likelihood ratios of PCR in the diagnosis of 54. Centers for Disease Control and Prevention. Recommendations
syphilis: a systematic review and meta-analysis. Sex Transmitted for test performance and interpretation from the Second National
Dis. 2013;89(3):251–256. Conference on Serological Diagnosis of Lyme Disease. MMWR.
34. Patel JA, Chonmaitree T. Syphilis screen at delivery: a need for 1995;44:590.
uniform guidelines. Am J Dis Child. 1993;147:256–258. 55. Golightly MG. Laboratory considerations in the diagnosis
35. Sánchez PJ, Wendel GD, Grimprel E, et al. Evaluation of molec- and management of Lyme borreliosis. Am J Clin Path. 1993;
ular methodologies and rabbit infectivity testing for the diagnosis 99:168–174.
of congenital syphilis and neonatal central nervous system 56. McKenna C, Schroeter A, Kierland R, et al. The fluorescent tre-
invasion by Treponema pallidum. J Infect Dis. 1993;167:148–157. ponemal antibody absorbed (FTA-ABS) test beading phenomenon
36. Woznicova V, Votava M, Flasarova M. Clinical specimens for in connective tissue diseases. Mayo Clin Proc. 1973;48:545–548.
PCR detection of syphilis. Epidemiol Mikrobiol Immunol. 2007; 57. Brown SL, Hansen SL, Langone JJ. Role of serology in diagnosis
56:66–71. of Lyme disease. JAMA. 1999;282:62–66.
37. Steere A, Malawista SE, Snydman DR, et al. Lyme arthritis: an 58. Pfister HW, Wilske B, Weber K. Lyme borreliosis: basic science
epidemic of oligoarticular arthritis in children and adults in three and clinical aspects. Lancet. 1994;343:1013–1016.
Connecticut communities. Arthritis Rheum. 1977;20:7–17. 59. Golightly MG, Benach J. Tick-borne diseases. Rev Clin Micro.
38. Burgdorfer W, Barbour AG, Hayes SF, et al. Lyme disease—A tick- 1999;10(1):1–10.
borne spirochetosis? Science. 1982;216:1317. 60. Engstrom SM, Shoop E, Johnson RC. Immunoblot interpretation
39. Centers for Disease Control and Prevention. Summary of notifi- criteria for serodiagnosis of early Lyme disease. J Clin Microbiol.
able diseases—United States, 2013. MMWR. 2013;62(53):1–119. 1995;33:419–427.
40. Schriefer ME. Borrelia. In: Versalovic J, Carroll KC, Funke G, 61. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blot-
et al., eds. Manual of Clinical Microbiology. 10th ed. Washington, DC: ting in the serodiagnosis of Lyme disease. J Infect Dis. 1993;
ASM Press; 2011:924–940. 167:392–400.
41. Steere AC. Lyme disease. N Engl J Med. 2001;345:115–124. 62. Centers for Disease Control and Prevention. Notice to readers:
42. Magnarelli LA, Miller JN, Anderson JF, Riviere GR. Cross-reactivity caution regarding testing for Lyme disease. MMWR. 2005;54:
of nonspecific Treponemal antibodies in serologic tests for Lyme 125–126.
disease. J Clin Micro. 1990;28:1276–1279. 63. Rosa PA, Schwan TG. A specific and sensitive assay for the Lyme
43. Grodzicki RL, Steere AC. Comparison of immunoblotting and disease spirochete Borrelia burgdorferi using the polymerase chain
indirect enzyme-linked immunosorbent assay using different reaction. J Infect Dis. 1989;160:1018–1029.
antigen preparations for diagnosing early Lyme disease. J Infect 64. Centers for Disease Control and Prevention. Lyme disease vacci-
Dis. 1988;157(4):790–797. nation. http://www.cdc.gov/lyme/prev/vaccine.html. Accessed
44. Wormser GP, Dattwyler RJ, Shapiro ED, et al. The clinical assess- May 4, 2016.
ment, treatment, and prevention of Lyme disease, human granu- 65. Lyme Info.Net. Lyme disease vaccine. www.lymeinfo.net/vaccine
locytic anaplasmosis, and babesiosis: clinical practice guidelines .html. Accessed April 17, 2014.
by the Infectious Diseases Society of America. Clin Inf Dis. 66. Branda JA, Rosenberg ES. Borrelia miyamotoi: a lesson in disease
2006;43:1089–1134. discovery. Ann Int Med. 2013 (July);159(1):61–62.
45. Steere AC. Lyme borreliosis. In: Kasper D, Braunwald E, Fauci A, 67. Platonov AE, Karan LS, Kolyasnikova NM, et al. Humans infected
et al., eds. Harrison’s Principles of Internal Medicine. 16th ed. with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg
New York, NY: McGraw-Hill; 2005:995–998. Infect Dis. 2011 (Oct);17(10):1–17.
46. Centers for Disease Control and Prevention. Lyme disease diag- 68. Krause PJ, Narasimhan S, Wormser GP, et al. Human Borrelia
nosis and testing. www.cdc.gov/lyme/diagnosistesting/index.html. miyamotoi infection in the United States. N Engl J Med. 2013;
Accessed April 17, 2014. 368(3):291–293.
47. Smith RP, Schoen RT, Rahn DW, et al. Clinical characteristics 69. Gugliotta JL, Goethert HK, Berandi VP, Telford III SR. Menin-
and treatment outcome of early Lyme disease in patients with goencephalitis from Borrelia miyamotoi in an immunocompro-
microbiologically confirmed erythema migrans. Ann Intern mised patient. N Engl J Med. 2013;368:240–245.
Med. 2002;136:421–428. 70. Barbour AG, Bunikis J, Travinsky B, et al. Niche partitioning of
48. Hercogova J. Review: Lyme borreliosis. Int J Dermatol. 2001; Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector
40:547–550. and mammalian reservoir species. Am J Trop Med Hyg. 2009;
49. Nadelman RB, Nowakowski J, Fish D, et al. Prophylaxis with 81(6):1120–1131.
single-dose doxycycline for the prevention of Lyme disease after
an Ixodes scapularis tick bite. N Engl J Med. 2001;345:79–84.
50. Kalish RA, Kaplan RF, Taylor E, et al. Evaluation of study patients
with Lyme disease, 10–20 year follow-up. J Infect Dis. 2001; 1. State and Local Health Departments, CDC. Update: trends in AIDS
183:453–460. incidence, deaths, and prevalence—United States, 1996. MMWR.
51. Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP. Diag- 1997;46:165–173.
nosis of Lyme borreliosis. Clin Micro Rev. 2005;18:484–509. 2. Falella FJ, Delaney KM, Moorman AC, et al. Declining morbidity
52. Feder HM, Johnson BJ, O’Connell S, et al. A critical appraisal of and mortality among patients with advanced human immunode-
“chronic Lyme disease.” N Engl J Med. 2007;357:1422–1430. ficiency virus infection. N Engl J Med. 1998;338(13):853–860.
 3. Murray CJ, Rosenfeld LC, Lim SS, et al. Global malaria mortality 24. Araujo FG. Diagnosis of parasitic diseases. Mem Inst Oswaldo
between 1980 and 2010: a systematic analysis. Lancet. 2012; Cruz Rio J. Suppl. 1, 1988;83(suppl 1):464–465.
379:413–431. 25. Maddison SE. Serodiagnosis of parasitic diseases. Clin Microbiol
4. Hotez PJ. Forgotten people, forgotten diseases: the neglected Rev. 1991;4(4):457–469.
tropical diseases and their impact on global health and develop- 26. Pappas MG. Recent applications of the Dot-ELISA in immunopar-
ment. Washington, DC: ASM Press; 2013. asitology, Vet Parasitol. 1988;29(2):105–129.
5. World Health Organization. Revised Global Burden of Disease 27. Jones JL, Kruszon-Moran D, Wilson M, et al. Toxoplasma gondii
(GBD) 2002 Estimates: Mortality, incidence, prevalence, YLL, infection in the United States: seroprevalence and risk factors.
YLD and DALYs by sex, cause and region, estimates for 2002 as Am J Epidemiol. 2001;154:357–365.
reported in the World Health Report 2004. 28. Bjerkås I. Neuropathology and host–parasite relationship of acute
6. Chandra RK. Immune responses in parasitic diseases. Part B: experimental toxoplasmosis of the blue fox (Alopex lagopus).
mechanisms. Rev Infect Dis. 1982;4(4):756–762. Vet Pathol. 1990;27(6):381–390.
7. Playfair JHL. Effective and ineffective immune responses to par- 29. Ruskin J, Remington JS. Toxoplasmosis in the compromised host.
asites: evidence from experimental models. Curr Top Microbiol Ann Intern Med. 1976;84(2):193–199.
Immunol. 1978;80:37–64. 30. Wong SY, Remington JS. Biology of Toxoplasma gondii. AIDS.
8. Male D, Brostoff J, Roth D, Roitt I. Immunology. 7th ed. Philadelphia, 1993;7(3):299–316.
PA: Mosby Elsevier; 2006:277–298. 31. Luft BJ, Remington JS. Toxoplasmic encephalitis in AIDS. Clin
9. Dunne DW, Butterworth AE, Fulford AJH, et al. Immunity after Infect Dis. 1992;15(2):211–222.
treatment of human schistosomiasis: association between IgE 32. Remington JS, McLeod R, Thulliez P, Desmonts G. Toxoplasmo-
antibodies to adult worm antigens and resistance to reinfection. sis. In: Remington JS, Klein JO, eds. Infectious Diseases of the
Eur J Immunol. 1992;22:1483–1494. Fetus and Newborn Infant. 5th ed. Philadelphia, PA: Saunders;
10. Hagan P, Blumenthal UJ, Dunn D, Simpson AJ, et al. Human IgE, 2001:205–346.
IgG4 and resistance to reinfection with Schistosoma haematobium. 33. Dunn D, Wallon M, Peyron F, et al. Mother-to-child transmission
Nature. 1991;349:243–245. of toxoplasmosis: risk estimates for clinical counselling. Lancet.
11. Jenkins SJ, Hewitson JP, Jenkins GR, et al. Modulation of the 1999;353(9167):1829–1833.
host’s immune response by schistosome larvae. Parasite Immunol. 34. Montoya JG, Remington JS. Management of Toxoplasma
2005;27:385–393. gondii infection during pregnancy. Clin Infect Dis. 2008;47(4):
12. Collier L, Balows A, Sussman M, eds. Topley and Wilson’s Micro- 554–566.
biology and Microbial Infections. 9th ed. New York, NY: Oxford 35. FDA Public Health Advisory: Limitations of Toxoplasma
University Press;1998:69–70. IgM commercial test kits. www.fda.gov/MedicalDevices/Safety/
13. Garcia LS. Diagnostic Medical Parasitology. 5th ed. Washington, AlertsandNotices/PublicHealthNotifications/ucm062411.htm.
DC: ASM Press; 2007:567–591. Updated March 21, 2013. Accessed January 10, 2014.
14. Bhattacharyya MK, Norris DE, Kumar N. Molecular players of 36. Hedman K, Lappalainen M, Seppala I, Makela O. Recent primary
homologous recombination in protozoan parasites: implications Toxoplasma infection indicated by a low avidity of specific IgG.
for generating antigenic variation. Infect Genet Evol. 2004;4(2): J Infect Dis. 1989;159:736–739.
91–98. 37. Pelloux H, Brun E, Vernet G, et al. Determination of anti-
15. John DT, Petri WA. Markell and Voge’s Medical Parasitology. Toxoplasma gondii immunoglobulin G avidity: adaptation to the
9th ed. Philadelphia, PA: Elsevier Saunders; 2006:112. Vidas system (bioMe rieux). Diagn Microbiol Infect Dis. 1998;
16. Barry JD. The relative significance of mechanisms of antigenic 32:69–73.
variation in African Trypanosomes. Parasitol Today. 1997;13: 38. Lappalainen M, Koskiniemi M, Hiilesmaa V, et al. Outcome of
212–218. children after maternal primary Toxoplasma infection during
17. Sadick MD, Raff HV. Differences in expression and exposure of pregnancy with emphasis on avidity of specific IgG. Pediatr Infect
promastigote and amastigote membrane molecules in Leishmania Dis J. 1995;14:354–356.
tropica. Infect Immun. 1985;47(2):395–400. 39. Liesenfeld O, Montoya JG, Kinney S, et al. Effect of testing for
18. Abu-Shakra M, Buskila D, Yehuda Shoenfeld Y. Molecular mimicry IgG avidity in the diagnosis of Toxoplasma gondii infection in
between host and pathogen: examples from parasites and implica- pregnant women: experience in a US reference laboratory. J Infect
tion. Immunol Lett. 1999;67(2):147–152. Dis. 2001;183(8):1248–1253.
19. Salzet M, Capron A, Stefano GB. Molecular crosstalk in host– 40. Lappalainen M, Koskela P, Koskiniemi M, et al. Toxoplasmosis
parasite relationships: schistosome- and leech–host interactions. acquired during pregnancy: improved serodiagnosis based on
Parasitol Today. 2000;16(12):536–540. avidity of IgG. J Infect Dis. 1993;167(3):691–697.
20. Abu-Shakra M, Shoenfeld Y. Parasitic infection and autoimmu- 41. Moody A. Rapid diagnostic tests for malaria parasites. Clin
nity. Autoimmunity. 1991;9(4):337–344. Microbiol Rev. 2002;15(1):66–78.
21. Zandman-Goddard G, Shoenfeld Y. Parasitic infection and 42. Malaria diagnosis (U.S.)–rapid diagnostic test. www.cdc.gov/
autoimmunity, Lupus. 2009;18:1144–1148. malaria/diagnosis_treatment/rdt.html. Released February 8, 2010.
22. Khouri R, Bafica A, Silva Mda P, et al. IFN-beta impairs super- Accessed January 15, 2014.
oxide-dependent parasite killing in human macrophages: evi- 43. Diagnostic procedures. www.cdc.gov/dpdx/diagnosticProcedures/
dence for a deleterious role of SOD1 in cutaneous leishmaniasis. serum/antibodydetection.html. Accessed January 21, 2014.
J Immunol. 2009;182(4):2525–2531. 44. Wilson M, Schantz P, Nutman T, Tsang VCW. Clinical immunopar-
23. Donati D, Mok B, Chêne A, et al. Increased B cell survival and asitology. In: Rose NR, Hamilton RG, Detrick B, eds. Manual of
preferential activation of the memory compartment by a malaria Clinical Laboratory Immunology. 6th ed. Washington, DC: ASM
polyclonal B cell activator. J Immunol. 2006;177:3035–3044. Press; 2002.
45. Swan H, Sloan L, Muyombwe A, et al. Evaluation of a real-time 68. Huffnagle GB. Role of cytokines in T cell immunity to a pulmonary
polymerase chain reaction assay for the diagnosis of malaria Cryptococcus neoformans infection. Biol Signals. 1996;5:215–222.
in patients from Thailand. Am J Trop Med Hyg. 2005;73(5): 69. Zhou P, Sieve MC, Bennett J, et al. IL-12 prevents mortality in
850–854. mice infected with Histoplasma capsulatum through induction of
46. Persing D, Mathiesen D, Marshall WF, et al. Detection of Babesia IFN-alpha. J Immunol. 1995;155:785–795.
microti by polymerase chain reaction. J Clin Microbiol. 1992; 70. Romani L. Cell mediated immunity to fungi: a reassessment. Med
30(8):2097–2103. Mycol. 2008;46:515–529.
47. Lin MH, Chen TC, Kuo TT, et al. Real-time PCR for quantitative 71. Zelante T, Iannitti R, De Luca A, Romani L. IL-22 in antifungal
detection of Toxoplasma gondii. J Clin Microbiol. 2000;38(11): immunity. Eur J Immunol. 2011;41:270–275.
4121–4125. 72. Vautier S, Sousa Mda G, Brown GD. C-type lectins, fungi and
48. FilmArray gastrointestinal panels. www.biofiredx.com/pdfs/ Th17 responses. Cytokine Growth Factor Rev. 2010;21:405–412.
FilmArray/InfoSheet,%20FilmArray%20GI%20Panel-0234.pdf. 73. Polonelli L, Casadevall A, Han Y, et al. The efficacy of acquired
Accessed January 16, 2014. humoral and cellular immunity in the prevention and therapy of
49. Kirk PM, Cannon PF, Minter DW, Stalpers JA. Dictionary of the experimental fungal infections. Med Mycol. 2000;38(suppl. 1):
Fungi. 10th ed. Wallingford, UK:CABI; 2008. 281–292.
50. Brooks GF. Jawetz, Melnick & Adelberg’s Medical Mycology. 24th ed. 74. Casadevall A, Feldmesser M, Pirofski L. Induced humoral immu-
London: McGraw-Hill Medical; 2007. nity and vaccination against major human fungal pathogens. Curr
51. Garcia ME, Blanco JL. Principales enfermedades fu ngicas que Opin Microbiol. 2002;5:386–391.
afectan a los animales dome sticos. Rev Iberoam Micol. 2000; 75. Pfaller MA, Diekema DJ. Rare and emerging opportunistic
17:S2–S7. fungal pathogens: concern for resistance beyond Candida
52. Stringer J, Beard C, Miller R, Wakefield A. A new name (Pneu- albicans and Aspergillus fumigatus. J Clin Microbiol. 2004;42:
mocystis jiroveci) for pneumocystis from humans. Emerging Infec- 4419–4431.
tious Diseases [serial online]. September 2002;8(9):891–896. 76. Wisplinghoff H, Bischoff T, Tallent SM, et al. Nosocomial blood-
Available from: MEDLINE with Full Text, Ipswich, MA. Accessed stream infections in US hospitals: analysis of 24,179 cases from
May 4, 2016. a prospective nationwide surveillance study. Clin Infect Dis.
53. Rippon JW. Medical Mycology: The Pathogenic Fungi and Pathogenic 2004;39:309–317.
Actinomycetes. 3rd ed. Philadelphia, PA: WE Saunders; 1988. 77. Trick WE, Fridkin SK, Edwards JR, et al. Secular trend of hospi-
54. Takeuchi O, Akira S. Pattern recognition receptors and inflam- tal-acquired candidemia among intensive care unit patients in
mation. Cell. 2010;140(6):805–820. the United States during 1989–1999. Clin Infect Dis. 2002;35:
55. Franchi L, Eigenbrod T, Munoz-Planillo R, Nunez G. The 627–630.
inflammasome: a caspase-1-activation platform that regulates 78. Baddley JW, Stroud TP, Salzman D, Pappas PG. Invasive mold
immune responses and disease pathogenesis. Nat Immunol. infections in allogeneic bone marrow transplant recipients. Clin
2009;10:241–247. Infect Dis. 2001;32:1319–1324.
56. Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians 79. Marr KA, Carter RA, Crippa F, et al. Epidemiology and outcome
of the body. Annu Rev Immunol. 2009;27:229–265. of mould infections in hematopoietic stem cell transplant
57. Janeway CA, Medzhitov R. Innate immune recognition. Annu Rev recipients. Clin Infect Dis. 2002;34:909–917.
Immunol. 2002;20:197–216. 80. Zhang SX. Enhancing molecular approaches for diagnosis of
58. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate fungal infections. Future Microbiol. 2013;8:1599–1611.
immunity. Cell. 2006;124:783–801. 81. De Pauw B, Walsh TJ, Donnelly JP, et al. Revised definitions of
59. Medzhitov R. Recognition of microorganisms and activation of invasive fungal disease from the European Organization for
the immune response. Nature. 2007;449:819–826. Research and Treatment of Cancer/Invasive Fungal Infections
60. Beutler BA. TLRs and innate immunity. Blood. 2009;113: Cooperative Group and the National Institute of Allergy and
1399–1407. Infectious Diseases Mycoses Study Group (EORTC/MSG) Con-
61. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev sensus Group. Clin Infect Dis. 2008;46(12):1813–1821.
Immunol. 2001;1(2):135–145. 82. Summerbell R. Ascomycetes. Aspergillus, fusarium, sporothrix,
62. Shinobu S, Yoichiro I. Dectin-1 and dectin-2 in innate immunity piedraia, and their relatives. In: Howard DH. Pathogenic Fungi in
against fungi. Int Immunol. 2011;23(8):467–472. Humans and Animals. 2nd ed. New York, NY: Marcel Dekker;
63. Ferwerda B, Ferwerda G, Plantinga TS, et al. Human dectin-1 2003:237–498.
deficiency and mucocutaneous fungal infections. N Engl J Med. 83. Zmeili OS, Soubani AO. Pulmonary aspergillosis: a clinical update.
2009;361:1760–1767. QJM. 2007;100(6):317–334.
64. Saijo S, Fujikado N, Furuta T, et al. Dectin-1 is required for host 84. Stevens DA, Moss RB, Kurup VP, et al. Allergic bronchopul-
defense against Pneumocystis carinii but not against Candida monary aspergillosis in cystic fibrosis—state of the art: Cystic
albicans. Nat Immunol. 2007:8:39–46. Fibrosis Foundation Consensus Conference. Clin Infect Dis.
65. Taylor PR, Tsoni SV, Willment JA, et al. Dectin-1 is required 2003;37(suppl 3):S225–S264.
for β-glucan recognition and control of fungal infection. Nat 85. Greenberger PA. Allergic bronchopulmonary aspergillosis. J Allergy
Immunol. 2007;8:31–38. Clin Immunol. 2002;110(5):685–692.
66. Romani L. Immunity to fungal infections. Nat Rev Immunol. 86. Wald A, Leisenring W, van Burik J-A, et al. Epidemiology of As-
2004;4(1):1–23. pergillus infections in a large cohort of patients undergoing bone
67. Allendoerfer R, Deepe GS, Jr. Intrapulmonary response to Histo- marrow transplantation. J Infect Dis. 1997;175(6):1459–1466.
plasma capsulatum in alpha interferon knockout mice. Infect 87. Young RC, Bennett JE. Invasive aspergillosis. Absence of de-
Immun. 1997;65:2564–2569. tectable antibody response. Am Rev Resp Dis. 1971;104:710–716.
 88. Mennink-Kersten MA, Donnelly JP, Verweij PE. Detection of 107. Levitz SM. Overview of host defenses in fungal infections. Clin
circulating galactomannan for the diagnosis and management Infect Dis. 1992;14:S37–S42.
of invasive aspergillosis. Lancet Infect Dis. 2004;4:349–357. 108. Henson DJ, Hill AR. Cryptococcal pneumonia: a fulminate
89. Karageorgopoulos DE, Evridiki, K. Vouloumanou K, et al. presentation. Am J Med. 1984;288(5):221–222.
β-D-glucan assay for the diagnosis of invasive fungal infec- 109. Perfect JR. Cryptococcosis. Infect Dis Clin N Am. 1989;3(1):
tions: a meta-analysis. Clin Infect Dis. 2011;52(6):750–770. 77–102.
90. Hebart H, Loffler J, Meisner C, et al. Early detection of As- 110. Snow RM, Dismukes WE. Cryptococcal meningitis: diagnostic
pergillus infection after allogeneic stem cell transplantation by value of cryptococcal antigen in cerebrospinal fluid. Arch Intern
polymerase chain reaction screening. J Infect Dis. 2000;181: Med. 1975;135(9):1155–1157.
1713–1719. 111. Kauffman CA, Bergman AG, Severance PJ, et al. Detection of
91. Loeffler J, Hebart H, Cox P, et al. Nucleic acid sequence-based cryptococcal antigen. Comparison of two latex agglutination
amplification of Aspergillus RNA in blood samples. J Clin tests. Am J Clin Pathol. 1981;75(1):106–109.
Microbiol. 2001;39:1626–1629. 112. Bennett, JE, Bailey JW. Control for rheumatoid factor in the latex
92. White PL, Linton CJ, Perry MD, et al. The evolution and evalu- test for cryptococcosis. Am J Clin Pathol. 1971;56:360–365.
ation of a whole blood polymerase chain reaction assay for the 113. Dolan CT. Specificity of the latex-cryptococcal antigen test. Am
detection of invasive aspergillosis in hematology patients in a J Clin Pathol. 1972;58:358–364.
routine clinical setting. Clin Infect Dis. 2006;42:479–486. 114. Eng RHK, Person A. Serum cryptococcal antigen determination
93. Costa C, Costa JM, Desterke C, et al. Real-time PCR coupled in the presence of rheumatoid factor. J Clin Microbiol. 1981;
with automated DNA extraction and detection of galactoman- 14:700–702.
nan antigen in serum by enzyme-linked immunosorbent assay 115. Gordon MA, Lapa EW. Elimination of rheumatoid factor in the
for diagnosis of invasive aspergillosis. J Clin Microbiol. 2002;40: latex test for cryptococcosis. Am J Clin Pathol. 1974;61:488–494.
2224–2227. 116. Hay RJ, Mackenzie DWR. False positive latex test for cryptococcal
94. Donnelly JP. Polymerase chain reaction for diagnosing invasive antigen in cerebrospinal fluid. J Clin Pathol. 1982;35:244–245.
aspergillosis: Getting closer but still a ways to go. Clin Infect Dis. 117. Binnicker MJ, Jespersen DJ, Bestrom JE, Rollins LO. Compari-
2006;42:487–489. son of four assays for the detection of cryptococcal antigen. Clin
95. Pappas PG. Invasive candidiasis. Infect Dis Clin N Am. 2006; Vaccine Immunol. 2012;19(12):1988–1990.
20:485–506. 118. Boulware DR, Rolfes MA, Rajasingham R, et al. Multisite
96. Zilberberg MD, Shorr AF, Kollef MH. Secular trends in validation of cryptococcal antigen lateral flow assay and quan-
Candidemia-related hospitalization in the United States, tification by laser thermal contrast. Emerg Infect Dis. 2014;
2000–2005. Infect Control Hosp Epidemiol. 2008;29(10):978–980. 20(1):45–53.
97. Mikulska M, Calandra T, Sanguinetti M, Poulain D, Viscoli C. 119. DiSalvo AF, Ajello L, Palmer JW, et al. Isolation of Histoplasma
The Third European Conference on Infections in Leukemia capsulatum from Arizona bats. Am J Epidemiol. 1969;89(5):
Group. The use of mannan antigen and anti-mannan antibodies 606–614.
in the diagnosis of invasive candidiasis: recommendations from 120. Kauffman CA. Histoplasmosis: a clinical and laboratory update.
the Third European Conference on Infections in Leukemia. Crit Clin Micro Rev. 2007;20(1):115–132.
Care. 2010;14:R222. 121. Wheat LJ, Kohler RB, Tewari RP. Diagnosis of disseminated histo-
98. Cuenca-Estrella M, Verweij PE, Arendrup MC. ESCMID guide- plasmosis by detection of Histoplasma capsulatum antigen in
line for the diagnosis and management of Candida diseases serum and urine specimens. N Engl J Med. 1986;314(2):83–88.
2012: diagnostic procedures. Clin Microbiol Infect. 2012;18 122. Wheat LJ, Kauffman CA. Histoplasmosis. Infect Dis Clin N Am.
(suppl 7):9–18. 2003;17:1–19.
99. Avni T, Leibovici L, Mical PM. PCR diagnosis of invasive can- 123. Wheat, LJ. 2001. Laboratory diagnosis of histoplasmosis: update.
didiasis: systematic review and meta-analysis. J Clin Microbiol. Semin Respir Infect. 2000;16:131–140.
2011;49(2):665. 124. Williams, BM, Fojtasek P, Connolly-Stringfield P, Wheat JL.
100. Levitz SM. The ecology of Cryptococcus neoformans and the Diagnosis of histoplasmosis by antigen detection during an
epidemiology of cryptococcosis. Rev Infect Dis. 1991;13: outbreak in Indianapolis, Ind. Arch Pathol Lab Med. 1994;
1163–1169. 118:1205–1208.
101. Emmons CW. Saprophytic sources of Cryptococcus neoformans 125. Gifford MA. San Joaquin fever. In: Annual Report Kern County
associated with the pigeon. Am J Hyg. 1955;62(3):227–232. Health Department for Fiscal Year July 1, 1935 to June 30,
102. Emmons CW. Isolation of Cryptococcus neoformans from soil. 1936, pp. 22–23.
J Bacteriol. 1951;62(6):685–690. 126. Galgiani JN. Coccidioidomycosis: a regional disease of national
103. van Elden LJ, Walenkamp AM, Lipovsky MM. Declining num- importance: rethinking approaches for control. Ann Intern Med.
ber of patients with cryptococcosis in the Netherlands in the 1999;130:293–300.
era of highly active antiretroviral therapy. AIDS. 2000;14(7): 127. Sunenshine RH, Anderson S, Erhart L, et al. Public health
2787–2788. surveillance for coccidioidomycosis in Arizona. Ann NY Acad
104. McNeil JI, Kan VL. Decline in the incidence of cryptococcosis Sci. 2007;1111:96–102.
among HIV-related patients. J Acquir Immune Defic Syndr Hum 128. Shubitz L, Peng T, Perrill R, et al. Protection of mice against
Retrovirol. 1995;9(2):206–208. Coccidioides immitis intranasal infection by vaccination with re-
105. Casadevall A, Perfect JR. Cryptococcus neoformans. Washington, combinant antigen 2/PRA. Infect Immun. 2002;70:3287–3289.
DC: ASM Press; 1988. 129. Smith CE, Beard RR, Whiting EG, et al. Varieties of coccidioidal
106. Murphy JW. Cryptococcal immunity and immunostimulation. infection in relation to the epidemiology and control of the
Adv Exp Med Biol. 1992;319:225–230. disease. Am J Public Health. 1946;36(12):1394–1402.
130. Valdivia L, Nix D, Wright M, et al. Coccidioidomycosis as a 18. Fujiwara S, Yokokawa Y, Morino K, et al. Chronic hepatitis E: a
common cause of community-acquired pneumonia. Emerg Infect review of the literature. J Viral Hepat. 2014;21(2):78–89.
Dis. 2006;12(6):958–962. 19. Zhu FC, Zhang J, Zhang XF, et al. Efficacy and safety of a recom-
131. Smith CE, Whiting EG, Baker EE, et al. The use of coccidioidin. binant hepatitis E vaccine in healthy adults: a large-scale, ran-
Am Rev Tuberc Pulm Dis. 1948;57(4):330–360. domised, double-blind placebo-controlled, phase 3 trial. Lancet.
132. Wieden MA, Lundergan LL, Blum J, et al. Detection of coccid- 2010;376(9744):895–902.
ioidal antibodies by 33-kDa spherule antigen, Coccidioides EIA, 20. World Health Organization. Hepatitis B fact sheet. www.who
and standard serologic tests in sera from patients evaluated for .int/mediacentre/factsheets/fs204/en/. Updated 2014. Accessed
coccidioidomycosis. J Infect Dis. 1996;173(5):1273–1277. March 5, 2015.
133. Blair JE, Currier JT. Significance of isolated positive IgM sero- 21. Nebbia G, Peppa D, Maini MK. Hepatitis B infection: current
logic results by enzyme immunoassay for coccidioidomycosis. concepts and future challenges. Q J Med. 2012;105:109–113.
Mycopathologia. 2008;166(2):77–82. 22. Centers for Disease Control and Prevention. Viral hepatitis sta-
tistics and surveillance. www.cdc.gov/hepatitis/Statistics/index
.htm. Updated 2014. Accessed March 5, 2015.
1. Abbas AK, Lichtman AH, Pillai S. Immunity to microbes. In: Cel- 23. Horvat RT, Tegtmeier GE. Hepatitis B and D viruses. In: Veralovic
lular and Molecular Immunology. Philadelphia, PA: Elsevier Saunders; J, Carroll KC, Funke G, et al., eds. Manual of Clinical Microbiology.
2015:348–352. Vol 2. 10th ed. Washington, DC: ASM Press; 2011:1659–1676.
2. Mak TW, Saunders ME. In: The Immune Response: Basic and Clin- 24. Mast EE, Margolis HS, Fiore AE, et al. A comprehensive immu-
ical Principles. Burlington, MA: Elsevier Academic Press; 2006: nization strategy to eliminate transmission of hepatitis B virus
664–680. infection in the United States. MMWR. 2005;54(RR16):1–23.
3. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. New York, 25. Shepard CW, Simard EP, Finelli L, et al. Hepatitis B virus infection:
NY: WH Freeman and Co.; 2013:555–557. epidemiology and vaccination. Epidemiol Rev. 2006;28:112–125.
4. Williams MA, Bevan MJ. Effector and memory CTL differentia- 26. Wilkins T, Zimmerman D, Schade RR. Hepatitis B: diagnosis and
tion. Annu Rev Immunol. 2007;25:171–192. treatment. Am Fam Physician. 2010;81(8):965–972.
5. Bendinelli M, Pistello M, Freer G, et al. Viral hepatitis. In: Detrick 27. Ganem D, Prince AM. Hepatitis B virus infection—natural his-
B, Hamilton RG, Folds JD, eds. Manual of Molecular and Clinical tory and clinical consequences. N Eng J Med. 2004;350(11):
Laboratory Immunology. 7th ed. Washington, DC: ASM Press; 1118–1129.
2006:724–745. 28. Dienstag JL. Hepatitis B virus infection. N Eng J Med. 2008;
6. Dienstag JL. Acute viral hepatitis. In: Longo DL, Fauci AS, Kasper 359(14):1486–1500.
DL, et al., eds. Harrison’s Principles of Internal Medicine. 18th ed. 29. Kao J. Diagnosis of hepatitis B virus infection through serological
New York, NY: McGraw-Hill; 2012. accessmedicine.mhmedical and virological markers. Exp Rev Gas Hep. 2008;2(4):553–562.
.com/content.aspx?bookid=331&Sectionid=40727099. Accessed 30. Servoss JC, Friedman LS. Serologic and molecular diagnosis of
March 2, 2015. hepatitis B infection. Infect Dis Clin N Am. 2006;20:47–61.
7. Anderson DA, Counihan NA. Hepatitis A and E viruses. In: 31. Alvarado-Mora MV, Locarnini S, Rizzetto M, Pinho JR. An update
Veralovic J, Carroll KC, Funke G, et al., eds. Manual of Clinical on HDV: virology, pathogenesis and treatment. Antivir Ther (Lond).
Microbiology. Vol 2. 10th ed. Washington, DC: ASM Press; 2011: 2013;18(3 Pt B):541–548.
1423–1436. 32. Hughes SA, Wedemeyer H, Harrison PM. Hepatitis delta virus.
8. Matheny SC, Kingery JE. Hepatitis A. Am Fam Physician. 2012; Lancet. 2011;378(9785):73–85.
86(11):1027–1034. 33. Noureddin M, Gish R. Hepatitis delta: epidemiology, diagnosis
9. Fiore AE, Wasley A, Bell BP. Prevention of hepatitis A through and management 36 years after discovery. Curr Gastroenterol
active or passive immunization. MMWR. 2006;55(RR07):1–23. Rep. 2014;16(1):365.
10. Jeong SH, Lee HS. Hepatitis A: clinical manifestations and man- 34. Farci P. Delta hepatitis: an update. J Hepatol. 2003;39(suppl 1):
agement. Intervirology. 2010;53(1):15–19. S212–S219.
11. Heo NY, Lim YS, An J, et al. Multiplex polymerase chain reaction 35. Le Gal F, Gordien E, Affolabi D, et al. Quantification of hepatitis
test for the diagnosis of acute viral hepatitis A. Clin Mol Hepatol. delta virus RNA in serum by consensus real-time PCR indicates
2012;18(4):397–403. different patterns of virological response to interferon therapy
12. Nainan OV, Xia G, Vaughan G, Margolis HS. Diagnosis of in chronically infected patients. J Clin Microbiol. 2005;43(5):
hepatitis A virus infection: a molecular approach. Clin Microbiol 2363–2369.
Rev. 2006;19(1):63–79. 36. Gower E, Estes C, Blach S, et al. Global epidemiology and geno-
13. Qiu F, Cao J, Su Q, Bi S. Multiplex hydrolysis probe real-time type distribution of the hepatitis C virus infection. J Hepatol.
PCR for simultaneous detection of hepatitis A virus and hepatitis 2014;61(1 suppl):S45–S57.
E virus. Int J Mol Sci. 2014;15:9780–9788. 37. Centers for Disease Control and Prevention. Testing for HCV
14. Kamar N, Dalton HR, Abravanel F, Izopet J. Hepatitis E virus infection: an update of guidance for clinicians and laboratorians.
infection. Clin Microbiol Rev. 2014;27(1):116–138. MMWR. 2013;62(18):362–365.
15. Hepatitis E. World Health Organization. Hepatitis E fact sheet. 38. Duddempudi AT, Bernstein DE. Hepatitis B and C. Clin Geriatr
www.who.int/mediacentre/factsheets/fs280/en/. Updated 2014. Med. 2014;30(1):149–167.
Accessed March 4, 2015. 39. Amjad M, Moudgal V, Faisal M. Laboratory methods for diagnosis
16. Dalton HR, Hunter JG, Bendall RP. Hepatitis E. Curr Opin Infect and management of hepatitis C virus infection. Lab Medicine.
Dis. 2013;26(5):471–478. 2013;44(4):292–299.
17. Perez-Gracia MT, Mateos Lindemann ML, Caridad Montalvo 40. Forman MS, Valsamakis A. Hepatitis C virus. In: Veralovic J,
Villalba M. Hepatitis E: Current status. Rev Med Virol. 2013;23(6): Carroll KC, Funke G, et al., eds. Manual of Clinical Microbiology.
384–398. Vol 2. 10th ed. Washington, DC: ASM Press; 2011:1437–1455.
41. Scheel TK, Rice CM. Understanding the hepatitis C virus life cycle 62. Jenson HB. Epstein-Barr virus. In: Detrick B, Hamilton RG, Folds
paves the way for highly effective therapies. Nat Med. 2013; JD, eds. Manual of Molecular and Clinical Laboratory Immunology.
19(7):837–849. 7th ed. Washington, DC: ASM Press; 2006:637–647.
42. Chan J. Hepatitis C. Disease-A-Month. 2014;60(5):201–212. 63. Gottschalk S, Rooney CM, Heslop HE. Post-transplant lymph-
43. Cheney CP, Chopra S, Graham C. Hepatitis C. Infect Dis Clin N proliferative disorders. Annu Rev Med. 2005;56:29–44.
Am. 2000;14(3):633–665. 64. Hirsch MS. Cytomegalovirus and human herpesvirus types 6, 7,
44. DeLemos AS, Chung RT. Hepatitis C treatment: an incipient ther- and 8. In: Longo DL, Fauci AS, Kasper DL, et al., eds. Harrison’s
apeutic revolution. Trends Mol Med. 2014;20(6):315–321. Principles of Internal Medicine. 18th ed. New York, NY: McGraw-Hill;
45. Centers for Disease Control and Prevention. Recommendations 2012. accessmedicine.mhmedical.com/content.aspx?bookid=331
for prevention and control of hepatitis C virus (HCV) infection &Sectionid=40726938. Accessed March 19, 2015.
and HCV-related chronic disease. MMWR. 1998;47(RR-19):1–40. 65. Hodinka RL. Human cytomegalovirus. In: Veralovic J, Carroll KC,
46. Centers for Disease Control and Prevention. 1999 USPHS/IDSA Funke G, et al., eds. Manual of Clinical Microbiology. Vol 2.
guidelines for the prevention of opportunistic infections in per- 10th ed. Washington, DC: ASM Press; 2011:1558–1574.
sons infected with the human immunodeficiency virus. MMWR. 66. Lancini D, Faddy HM, Flower R, Hogan C. Cytomegalovirus disease
1999;48(RR-10):1–59. in immunocompetent adults. Med J Aust. 2014;201(10):578–580.
47. Smith BD, Morgan RL, Beckett GA, et al. Recommendations for 67. St. George K, Hoji A, Rinaldo CR. Cytomegalovirus. In: Detrick
the identification of chronic hepatitis C virus infection among B, Hamilton RG, Folds JD, eds. Manual of Molecular and Clinical
persons born during 1945–1965. MMWR Recommendations & Laboratory Immunology. 7th ed. Washington, DC: ASM Press;
Reports. 2012;61(RR-4):1–32. 2006:648–657.
48. Moyer VA, U.S. Preventive Services Task Force. Screening for 68. Ariza-Heredia EJ, Nesher L, Chemaly RF. Cytomegalovirus dis-
hepatitis C virus infection in adults: U.S. preventive services task eases after hematopoietic stem cell transplantation: a mini-review.
force recommendation statement. Ann Intern Med. 2013;159(5): Cancer Lett. 2014;342(1):1–8.
349–357. 69. Fu TM, An Z, Wang D. Progress on pursuit of human cy-
49. Parisi MR, Soldini L, Vidoni G, et al. Point-of-care testing for HCV tomegalovirus vaccines for prevention of congenital infection
infection: recent advances and implications for alternative screen- and disease. Vaccine. 2014;32(22):2525–2533.
ing. New Microbiologica. 2014;37(4):449–457. 70. Wang D, Fu TM. Progress on human cytomegalovirus vaccines
50. Vermeersch P, Van Ranst M, Lagrou K. Validation of a strategy for prevention of congenital infection and disease. Curr Opin Virol.
for HCV antibody testing with two enzyme immunoassays in a 2014;6:13–23.
routine clinical laboratory. J Clin Virol. 2008;42(4):394–398. 71. Bale JF. Congenital cytomegalovirus infection. In: Tselis AC,
51. Scott JD, Gretch DR. Molecular diagnostics of hepatitis C infec- Booss J, eds. Handbook of Clinical Neurology. Amsterdam, The
tion. JAMA. 2007;297:724–732. Netherlands: Elsevier BV; 2014:319–326.
52. Ghany MG, Strader DB, Thomas DL, Seeff LB, American Associ- 72. Lazzarotto T, Guerra B, Gabrielli L, et al. Update on the preven-
ation for the Study of Liver Diseases. Diagnosis, management, tion, diagnosis and management of cytomegalovirus infection
and treatment of hepatitis C: an update. Hepatology. 2009;49(4): during pregnancy. Clin Microbiol Infect. 2011;17(9):1285–1293.
1335–1374. 73. Ross SA, Boppana SB. Congenital cytomegalovirus infection: out-
53. Robinson BJ, Pierson SL. Clinical virology. In: Mahon CR, Lehman come and diagnosis. Semin Pediatr Infect Dis. 2005;16(1):44–49.
DC, Manuselis G, eds. Textbook of Diagnostic Microbiology. 4th ed. 74. Adler SP, Marshall B. Cytomegalovirus infections. Pediatr Rev.
Maryland Heights, MO: Elsevier Saunders; 2011:703–740. 2007;28(3):92–100.
54. Cohen JI. Epstein-Barr virus infections, including infectious 75. Landry ML, Ferguson D. 2-hour cytomegalovirus pp65 antigen-
mononucleosis. In: Longo DL, Fauci AS, Kasper DL, et al., eds. emia assay for rapid quantitation of cytomegalovirus in blood
Harrison’s Principles of Internal Medicine. 18th ed. New York, NY: samples. J Clin Microbiol. 2000;38(1):427–428.
McGraw-Hill; 2012. accessmedicine.mhmedical.com.libproxy1 76. Pillet S, Roblin X, Cornillon J, et al. Quantification of cy-
.upstate.edu/content.aspx?bookid=331&Sectionid=40726937. tomegalovirus viral load. Exp Rev Anti Infe. 2014;12(2):193–210.
Accessed March 15, 2015. 77. Walker SP, Palma-Dias R, Wood EM, et al. Cytomegalovirus in
55. Jenson HB. Epstein-Barr virus. Pediatr Rev. 2011;32:375–383. pregnancy: to screen or not to screen. BMC Pregnancy and
56. Williams H, Crawford DH. Epstein-Barr virus: the impact of Childbirth. 2013;13:96–103.
scientific advances on clinical practice. Blood. 2006;107(3): 78. Mendelson E, Aboudy Y, Smetana Z, et al. Laboratory assessment
862–869. and diagnosis of congenital viral infections: rubella, cytomegalovirus
57. Gartner BC. Epstein-Barr virus. In: Versalovic J. Carroll KC, (CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV),
Funke G, et al., eds. Manual of Clinical Microbiology. Vol 2. 10th ed. parvovirus B19 and human immunodeficiency virus (HIV). Reprod
Washington, DC: ASM Press; 2011:1575–1584. Toxicol. 2006;21:350–382.
58. Odumade OA, Hogquist KA, Balfour HH. Progress and problems 79. Marin M, Gurtis D, Chaves SS, et al. Prevention of varicella: rec-
in understanding and managing primary Epstein-Barr virus ommendations of the advisory committee on immunization
infections. Clin Microbiol Rev. 2011;24(1):193–209. practices (ACIP). MMWR. 2007;56(RR-4):1–40.
59. Junker AK. Epstein-Barr virus. Pediatr Rev. 2005;26(3):79–85. 80. Stover BH, Bratcher DF. Varicella-zoster virus: infection, control,
60. Ebell MH. Epstein-Barr virus infectious mononucleosis. Am Fam and prevention. Am J Infect Control. 1998;26(3):369–381.
Physician. 2004;70(7):1279–1290. 81. Whitley RJ. Varicella-zoster virus infections. In: Kasper D, Fauci A,
61. Feng Z, Li Z, Sui B, Xu G, Xia T. Serologic diagnosis of infec- Hauser S, et al., eds. Harrison’s Principles of Internal Medicine.
tious mononucleosis by chemiluminescent immunoassay using 19th ed. New York, NY: McGraw-Hill; 2015. accessmedicine
capsid antigen p18 of Epstein-Barr virus. Clin Chim Acta. 2005; .mhmedical.com/content.aspx?bookid=1130&Sectionid=79738
354:77–82. 275Sectionid=40726936. Accessed April 29, 2015.
82. McCrary ML, Severson J, Trying SK. Varicella zoster virus. J Am 100. Portella G, Galli C. Multicentric evaluation of two chemilumi-
Acad Dermatol. 1999;41:1–14. nescent immunoassays for IgG and IgM antibodies towards
83. Gershon AA, Gershon MD. Pathogenesis and current approaches rubella virus. J Clin Virol. 2010;49:105–110.
to control of varicella-zoster virus infections. Clin Microbiol Rev. 101. Hamkar R, Javilvand S, Mokhtari-Azad T, et al. Assessment of
2013;26(4):728–743. IgM enzyme immunoassay and IgG avidity assay for distinguish-
84. Puchhammer-Stockl E, Aberle S. Varicella-zoster virus. In: Ver- ing between primary and secondary immune response to rubella
salovic J, Carroll KC, Funke G, et al., eds. Manual of Clinical vaccine. J Virol Meth. 2005;130:59–65.
Microbiology. Vol 2. 10th ed. Washington, DC: ASM Press; 102. Mubareka S, Richards H, Gray M, Tipples GA. Evaluation of
2011:1545–1557. commercial rubella immunoglobulin G avidity assays. J Clin
85. Kimberlin DW, Whitley RJ. Varicella-zoster vaccine for the pre- Microbiol. 2007;45(1):231–233.
vention of herpes zoster. N Engl J Med. 2007;356:1338–1343. 103. Rainwater-Lovett K, Moss WJ. Measles (Rubeola). In: Kasper D,
86. Zhou F, Harpaz R, Jumaan AO, et al. Impact of varicella vaccina- Fauci A, Hauser S, et al., eds. Harrison’s Principles of Internal
tion on health care utilization. JAMA. 2005;294(7):797–802. Medicine. 19th ed. New York, NY: McGraw-Hill; 2015. ac-
87. Oxman MN, Levin MJ, Johnson GR, et al. A vaccine to prevent cessmedicine.mhmedical.com/content.aspx?bookid=1130&Sect
herpes zoster and postherpetic neuralgia in older adults. N Engl ionid=79739382. Accessed April 29, 2015.
J Med. 2000;343:222. 104. DiPaola F, Michael A, Mandel ED. A casualty of the immuniza-
88. Bader MS. Herpes zoster: diagnostic, therapeutic, and preventive tion wars: the reemergence of measles. JAAPA. 2012;25(6):
approaches. Postgrad Med. 2013;125(5):78–91. 50–54.
89. Schmid DS, Loparev V. Varicella virus. In: Detrick B, Hamilton RG, 105. Hodinka RL, Moshal KL. Childhood infections. In: Storch GA,
Folds JD, eds. Manual of Molecular and Clinical Laboratory Im- ed. Essentials of Diagnostic Virology. New York, NY: Churchill
munology. 7th ed. Washington, DC: ASM Press; 2006:631–636. Livingstone; 2000:167–186.
90. Breuer J, Harper DR, Kangro HO. Varicella zoster. In: Zuckerman 106. Bellini WJ, Rota JS, Lowe LE, et al. Subacute sclerosing panen-
AJ, Banatvala JE, Pattison JR, eds. Principles and Practice of Clinical cephalitis: more cases of this fatal disease are prevented by
Virology. 4th ed. Chichester, England: John Wiley & Sons; measles immunization than was previously recognized. J Infec
2000:47–77. Dis. 2005;192:1686–1693.
91. Sauerbrei A, Wutzler P. Serological detection of varicella-zoster 107. Leland DS. Measles and mumps. In: Detrick B, Hamilton RG,
virus-specific immunoglobulin G by an enzyme-linked im- Folds JD, eds. Manual of Molecular and Clinical Laboratory Im-
munosorbent assay using glycoprotein antigen. J Clin Microbiol. munology. 7th ed. Washington, DC: ASM Press; 2006:707–711.
2006;44(9):3094–3097. 108. Michel Y, Saloum K, Tournier C, et al. Rapid molecular diagno-
92. Bellini WJ, Icenogle JP. Measles and rubella viruses. In: Veralovic sis of measles virus infection in an epidemic setting. J Med Virol.
J, Carroll KC, Funke G, et al., eds. Manual of Clinical Microbiology. 2013;85:723–730.
Vol 2. Washington, DC: ASM Press; 2011:1372–1387. 109. Hummel KB, Lowe L, Bellini WJ, Rota PA. Development of
93. Centers for Disease Control and Prevention. Epidemiology of quantitative gene-specific real-time RT-PCR assays for detection
vaccine-preventable diseases. The Pinkbook. Website. www.cdc of measles virus in clinical specimens. J Virol Meth. 2006;
.gov/vaccines/pubs/pinkbook/index.html. Published May 2012. 132:166–173.
Updated 2012. Accessed April 3, 2015. 110. Rubin SA, Carbone KM. Mumps. In: Kasper D, Fauci A, Hauser
94. Zimmerman LA, Reef SE. Rubella (German measles). In: Kasper S, et al., eds. Harrison’s Principles of Internal Medicine. 19th ed.
D, Fauci A, Hauser S, et al., eds. Harrison’s Principles of Internal New York, NY: McGraw-Hill; 2015. accessmedicine.mhmedical
Medicine. 19th ed. New York, NY: McGraw-Hill; 2015. ac- .com/content.aspx?bookid=1130&Sectionid=79739481. Accessed
cessmedicine.mhmedical.com/content.aspx?bookid=1130&Sect April 29, 2015.
ionid=79739440. Accessed April 29, 2015. 111. Shanley JD. The resurgence of mumps in young adults and
95. McLean HQ, Fiebelkorn AP, Tempte JL, Wallace GS. Preven- adolescents. Cleveland Clinic J Med. 2007;74(1):42–48.
tion of measles, rubella, congential rubella syndrome, and 112. Leland DS. Parainfluenza and mumps viruses. In: Veralovic J.
mumps, 2013. Summary recommendations of the Advisory Carroll KC, Funke G, et al., eds. Manual of Clinical Microbiology.
Committee on Immunization Practices (ACIP). MMWR. 2013; Vol 2. 10th ed. Washington, DC: ASM Press; 2011:1347–1356.
62(4):1–34. 113. Centers for Disease Control and Prevention. Mumps: ques-
96. DeSantis M, Cavaliere AF, Straface G, et al. Rubella infection in tions and answers about lab testing. www.cdc.gov/mumps/
pregnancy. Reprod Toxicol. 2006;21:390–398. lab/qa-lab-test-infect.html#realtime-pcr. Updated 2012.
97. Tipples G, Hiebert J. Detection of measles, mumps, and rubella Accessed April 29, 2015.
viruses. In: Stephenson JR, Warnes A, eds. Methods in Molecular 114. Public Health Agency of Canada. Archived guidelines for the
Biology. Diagnostic Virology Protocols. Vol 665. New York, NY: prevention and control of mumps outbreaks in Canada. Appen-
Springer Science and Business Media; 2011:183–193. dix 4. Laboratory guidelines for the diagnosis of mumps. www
98. Mace M, Cointe D, Six C, et al. Diagnostic value of reverse tran- .phac-aspc.gc.ca/publicat/ccdr-rmtc/10vol36/36s1/appendix-
scription-PCR of amniotic fluid for prenatal diagnosis of con- annexe-4-eng.php. Updated 2009. Accessed April 29, 2015.
genital rubella infection in pregnant women with confirmed 115. Owen SM, Gessain A, Dezzutti CS, et al. Human T-cell lym-
primary rubella infection. J Clin Microbiol. 2004;42(10): photropic virus types 1 and 2. In: Versalovic J, Carroll KC,
4818–4820. Funke G, et al., eds. Manual of Clinical Microbiology. Vol 2. 10th ed.
99. Binnicker MJ, Jespersen DJ, Rollins LO. Evaluation of the Bio- Washington, DC: ASM Press; 2011:1323–1332.
Rad bioplex measles, mumps, rubella, and varicella-zoster virus 116. Satou Y, Matsuoka M. Virological and immunological mecha-
IgG multiplex bead immunoassay. Clin Vaccine Immunol. 2011: nisms in the pathogenesis of human T-cell leukemia virus type
1524–1526. 1. Rev Med Virol. 2013;23(5):269–280.
117. Verdonck K, Gonzalez E, Van Dooren S, et al. Human 10. Clavel F, Guétard D, Brun-Vézinet, F, et al. Isolation of a new
T-lymphotropic virus 1: recent knowledge about an ancient human retrovirus from West African patients. Science. 1986;
infection. Lancet Infect Dis. 2007;7(4):266–281. 233(4761):343–346.
118. Longo DL, Fauci AS. The human retroviruses. In: Kasper D, Fauci 11. Karim SS, Karim QA, Gouws E, Baxter C. Global epidemiology
A, Hauser S, et al., eds. Harrison’s Principles of Internal Medicine. of HIV-AIDS. Infect Dis Clin N Am. 2007;21:1–17.
19th ed. New York, NY: McGraw-Hill; 2015. accessmedicine 12. Centers for Disease Control and Prevention. Updated U.S. Public
.mhmedical.com/content.aspx?bookid=1130&Sectionid=797387 Health Service guidelines for the management of occupational
42. Accessed May 1, 2015. exposures to HIV and recommendations for postexposure pro-
119. Ishitsuka K, Tamura K. Human T-cell leukaemia virus type I and phylaxis. MMWR. 2005;54(RR09):1–17.
adult T-cell leukaemia-lymphoma. Lancet Oncol. 2014;15(11): 13. Centers for Disease Control and Prevention. Public health service
e517–e526. guidelines for the management of health-care worker exposures
120. Chang YB, Kaidarova Z, Hindes D, et al. Seroprevalence and to HIV and recommendations for postexposure prophylaxis.
demographic determinants of human T-lymphotropic virus type MMWR. 1998;47:211–215.
1 and 2 infections among first-time blood donors—United 14. Dodd RY, Notari EP, Stramer SL. Current prevalence and incidence
States, 2000–2009. J Infect Dis. 2014;209(4):523–531. of infectious disease markers and estimated window period risk
121. Qayyum S, Choi JK. Adult T-cell leukemia/lymphoma. Arch in the American Red Cross blood donor population. Transfusion.
Pathol Lab Med. 2014;138(2):282–286. 2002;42:975–979.
122. Araujo A, Hall WW. Human T-lymphotropic virus type II and 15. Centers for Disease Control and Prevention. Updated U.S. Public
neurological disease. Ann Neurol. 2004;56(1):10–19. Health Service guidelines for the management of occupational
123. Martin-Davila P, Fortun J, Lopez-Velez R, et al. Transmission exposures to HBV, HCV, and HIV and recommendations for
of tropical and geographically restricted infections during postexposure prophylaxis. MMWR. 2001;50(RR11):1–42.
solid-organ transplantation. Clin Microbiol Rev. 2008;21(1): 16. Centers for Disease Control and Prevention. Achievements in pub-
60–96. lic health: Reduction in perinatal transmission of HIV infection—
124. Abrams A, Akahata Y, Jacobson S. The prevalence and signifi- United States, 1985–2005. MMWR. 2006;55(21):592–597.
cance of HTLV-I/II seroindeterminate western blot patterns. 17. Collier L, Oxford J. Human Virology. 3rd ed. New York, NY:
Viruses. 2011;3(8):1320–1331. Oxford University Press; 2006:179–188.
125. Waters A, Oliveira AL, Coughlan S, et al. Multiplex real-time 18. Johnston MI, Fauci AS. An HIV vaccine—evolving concepts.
PCR for the detection and quantitation of HTLV-1 and HTLV-2 N Engl J Med. 2007;356(20):2073–2081.
proviral load: addressing the issue of indeterminate HTLV 19. Mak TW, Saunders ME. The Immune Response: Basic and Clinical
results. J Clin Virol. 2011;52(1):38–44. Principles. Boston, MA: Elsevier Academic Press; 2006:785–823.
20. Owen JA, Punt J, Stranford SA, Jones PP. Kuby Immunology. 7th ed.
2013:593–626.
1. World Health Organization. HIV/AIDS fact sheet. Website. www.who 21. Liu R, Paxton WA, Choe S, et al. Homozygous defect in HIV-1
.int/mediacentre/factsheets/fs360/en/. Updated November 2014. co-receptor accounts for resistance of some multiple-exposed
Accessed December 3, 2014. individuals to HIV-1 infection. Cell. 1996;86:367–377.
2. Centers for Disease Control and Prevention. HIV in the United 22. Samson M, Libert F, Doranz BJ, et al. Resistance to HIV-1 infection
States: At a glance. Website. www.cdc.gov/hiv/statistics/basics/ in Caucasian individuals bearing mutant alleles of the CCR-5
ataglance.html. Updated 2014. Accessed April 25, 2016. chemokine receptor gene. Nature. 1996;382:722–725.
3. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a 23. Coffin J, Swanstrom R. HIV pathogenesis: dynamics and genetics
T-lymphotropic retrovirus from a patient at risk for acquired im- of viral populations and infected cells. Cold Spring Harb Perspect
munodeficiency syndrome (AIDS). Science. 1983;220:868–870. Med. 2013;3:1–16.
4. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and 24. Abbas AK, Lichtman AH, Pillai S. Human immunodeficiency
isolation of cytopathic retroviruses (HTLV-III) from patients with virus and the acquired immunodeficiency syndrome. In: Cellular
AIDS and at risk for AIDS. Science. 1984;224 (4648):500–503. and Molecular Immunology. 8th ed. Philadelphia, PA: Elsevier
5. Levy JA, Hoffman AD, Kramer SM, et al. Isolation of lymphocy- Saunders; 2015:451–461.
topathic retroviruses from San Francisco patients with AIDS. 25. Carrington M, Alter G. Innate control of HIV. Cold Spring Harb
Science. 1984;225 (4664):840–842. Perspect Med. 2012;2:a007070.
6. Maartens G, Celum C, Lewin SR. HIV infection: epidemiology, 26. Collins KL. Resistance of HIV-infected cells to cytotoxic T lympho-
pathogenesis, treatment, and prevention. Lancet. 2014;384: cytes. Microbes Infect. 2004;6:494–500.
258–271. 27. Paranjape RS. Immunopathogenesis of HIV infection. Indian
7. Kandathil AJ, Ramalingam S, Kannangai R, et al. Molecular J Med Res. 2005;121(4):240–255.
epidemiology of HIV. Indian J Med Res. 2005;121:333–344. 28. Lane HC, Fauci AS. Immunologic abnormalities in the acquired
8. Kwon DS, Walker BD. Immunology of human immunodeficiency immunodeficiency syndrome. Annu Rev Immunol. 1985;3:
virus infection. In: Paul WE, ed. Fundamental Immunology. 7th ed. 477–500.
Philadelphia, PA: Wolters Kluwer Health/Lippincott Williams & 29. Lackner AA, Lederman MM, Rodriguez B. HIV pathogenesis: The
Wilkins; 2013:1016–1031. host. Cold Spring Harb Perspect Med. 2012:a007005.
9. Fauci AS, Lane H. Human immunodeficiency virus disease: 30. Swanstrom R, Coffin J. HIV pathogenesis: the virus. Cold Spring
AIDS and related disorders. In: Longo DL, Fauci AS, Kasper DL, Harb Perspect Med. 2012;2:a007443.
et al., eds. Harrison’s Principles of Internal Medicine. 18th ed. 31. Frost SDW, Trkola A, Gunthard HF, Richman DD. Antibody re-
New York, NY: McGraw-Hill; 2012. accessmedicine.mhmedical sponses in primary HIV infection. Curr Opin HIV AIDS. 2008;
.com/content.aspx?bookid=331&Sectionid=40726947. Accessed 3(1):45–51.
December 4, 2014.
32. Zetola NM, Pilcher CD. Diagnosis and management of acute HIV 53. Occupational Safety & Health Administration. Bloodborne
infection. Infect Dis Clin N Am. 2007;21:19–48. pathogens standard 29 CFR 1910.1030. https://www.osha.gov/
33. Rodes B, Toro C, Paxinos E, et al. Differences in disease progres- pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p
sion in a cohort of long-term non-progressors after more than _id=10051. Accessed December 16, 2014.
16 years of HIV-1 infection. AIDS. 2004;18(8):1109–1116. 54. Panlilio AL, Cardo DM, Grohskopf LA, et al. Updated U.S. Public
34. Price RW, Brew B, Sidtis J, et al. The brain in AIDS: central Health Service guidelines for the management of occupational
nervous system HIV-1 infection and AIDS dementia complex. exposures to HIV and recommendations for postexposure pro-
Science. 1988;239(4840):586–592. phylaxis. MMWR. 2005;54(RR09):1–17.
35. European Collaborative Study. Children born to women with HIV 55. Centers for Disease Control and Prevention. Occupational HIV
infection: natural history and risk of transmission. Lancet. 1991; transmission and prevention among health care workers. www.cdc
337:253–260. .gov/hiv/risk/other/occupational.html. Updated 2014. Accessed
36. Peckham C, Gibb D. Mother-to-child transmission of the December 15, 2014.
human immunodeficiency virus (HIV). N Engl J Med. 1995; 56. Haynes BF, McElrath MJ. Progress in HIV-1 vaccine development.
333(5):298–302. Curr Opin HIV AIDS. 2013;8:326–332.
37. Centers for Disease Control and Prevention. Update on acquired 57. Kim D, Elizaga M, Duerr A. HIV vaccine efficacy trials: toward the
immunodeficiency syndrome (AIDS)—United States. MMWR. future of HIV prevention. Infect Dis Clin N Am. 2007;21:201–217.
1982;31:507–514. 58. Sahloff EG. Current issues in the development of a vaccine to pre-
38. Centers for Disease Control and Prevention. Revision of the case vent human immunodeficiency virus. Pharmacotherapy. 2005;
definition of acquired immunodeficiency syndrome for national 25(5):741–747.
reporting—United States. MMWR. 1985;34:373–375. 59. Yuki Y, Nichi T, Kiyono H. Progress towards an AIDS mucosal
39. Centers for Disease Control and Prevention. Revision of the vaccine: an overview. Tuberculosis. 2007;87:S35–S44.
CDC surveillance case definition for acquired immunodefi- 60. Shapiro SZ. Clinical development of candidate HIV vaccines:
ciency syndrome. MMWR. 1987;36(suppl No. 1S):3s–15s. different problems for different vaccines. AIDS Res Hum Retrov.
40. Centers for Disease Control and Prevention. 1993 revised clas- 2014;30(4):325–329.
sification system for HIV infection and expanded surveillance 61. Branson BM, Handsfield HH, Lampe MA, et al. Centers for Dis-
case definition for AIDS among adolescents and adults. MMWR. ease Control and Prevention (CDC). Revised recommendations
1992;41(No. RR-17):1–19. for HIV testing of adults, adolescents, and pregnant women in
41. Centers for Disease Control and Prevention. Revised surveillance health-care settings. MMWR. 2006;55(RR-14):1–17.
case definitions for HIV infection among adults, adolescents, and 62. Moyer VA. U.S. Preventive Services Task Force. Screening for
children aged <18 months and for HIV infection and AIDS HIV: U.S. preventive services task force recommendation state-
among children aged 18 months to <13 years. MMWR. 2008; ment. Ann Intern Med. 2013;159(1):51–60.
57(No. RR-10):1–11. 63. Sherin K, Klekamp BG, Beal J, Martin M. What is new in HIV
42. Centers for Disease Control and Prevention. 1994 revised classifi- infection? Am Fam Med. 2014;89(4):265–272.
cation system for human immunodeficiency virus infection in chil- 64. Constantine NT, Zink H. HIV testing technologies after two
dren less than 13 years of age. MMWR. 1994;43(No. RR-12):1–10. decades of evolution. Indian J Med Res. 2005;121(4):519–538.
43. Centers for Disease Control and Prevention. Revised surveillance 65. Dewar R, Highbarger H, Davey R, Metcalf J. Principles and pro-
case definition for HIV infection—United States, 2014. MMWR. cedures of human immunodeficiency virus serodiagnosis. In:
2014;63(3):1–10. Detrick B, Hamilton RG, Folds JD, eds. Manual of Molecular and
44. Dept. of Health and Human Services. AIDS info. aidsinfo.nih.gov/ Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM
guidelines. Updated 2016. Accessed December 16, 2016. Press; 2006:834–846.
45. Chen LF, Hoy J, Lewin SR. Ten years of highly active antiretroviral 66. Centers for Disease Control and Prevention. Interpretation and
therapy for HIV infection. MJA. 2007;186(3):146–151. use of the Western blot assay for serodiagnosis of human immun-
46. World Health Organization. Clinical guidance across the con- odeficiency virus type I infections. MMWR. 1989;38(S-7):1–7.
tinuum of care: antiretroviral therapy. www.who.int/hiv/pub/ 67. O’Brien TR, George JR, Epstein JS, Holmberg SD, Schochetman G.
guidelines/arv2013/art/arv2013_chapter07_low.pdf?ua=1. Testing for antibodies to human immunodeficiency virus type 2
Updated 2013. Accessed December 16, 2014. in the United States. MMWR. 1992;41(RR-12):1–9.
47. Younai FS. Thirty years of the human immunodeficiency virus 68. Centers for Disease Control and Prevention. Protocols for confir-
epidemic and beyond. Int J Oral Science. 2013;5:191–199. mation of rapid HIV tests. MMWR. 2004;53(10):221–222.
48. Hammer SM, Saag MS, Scheter M, et al. Treatment for adult HIV 69. Branson BM, Owen SM, Wesolowski LG, et al. Laboratory testing
infection. JAMA. 2006;296(7):827–843. for the diagnosis of HIV infection: updated recommendations.
49. Connor EM, Sperling RS, Gelber R, et al. Reduction of maternal– CDC Stacks. Website. stacks.cdc.gov/view/cdc/23447. Updated
infant transmission of human immunodeficiency virus type 1 2014. Accessed December 18, 2014.
with zidovudine treatment. N Engl J Med. 1994;331:1173–1180. 70. FDA. U.S. Food and Drug Administration. Vaccines, blood,
50. Marsden MD, Zack JA. HIV/AIDS eradication. Bioorgan Med and biologics. Complete list of donor screening assays for infec-
Chem. 2013;23:4003–4010. tious agents and HIV diagnostic assays. Website. www.fda.gov/
51. U.S. Public Health Service. Preexposure prophlyaxis for the BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/
prevention of HIV infection in the United States—2014. A clin- LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/
ical practice guideline. CDC Stacks. 2014:1–67. ucm080466.htm. Updated 2014. Accessed December 19, 2014.
52. Centers for Disease Control and Prevention. Recommendations 71. Schappert J, Wians FH, Jr, Schiff E, et al. Multicenter evaluation
for prevention of HIV transmission in health-care settings. of the bayer ADVIA centaur HIV 1/O/2 enhanced (EHIV) assay.
MMWR. 1987;36(suppl no. 2S):1S–17S. Clinica Chimica Acta. 2006;372(1-2):158–166.
72. Nasrullah M, Wesolowski LG, Meyer WA, 3rd, et al. Performance 91. Quest Diagnostics. HIV-1 infection: Laboratory tests for select-
of a fourth-generation HIV screening assay and an alternative HIV ing antiretroviral therapy. Website. www.questdiagnostics.com/
diagnostic testing algorithm. AIDS. 2013;27(5):731–737. testcenter/testguide.action?dc=TG_HIV_Antiretroviral_Therapy.
73. Bentsen C, McLaughlin L, Mitchell E, et al. Performance evalu- Updated 2014. Accessed February 20, 2015.
ation of the Bio-Rad laboratories GS HIV combo ag/ab EIA, 92. Obermeier M, Symons J, Wensing AMJ. HIV population geno-
a 4th generation HIV assay for the simultaneous detection of typic tropism testing and its clinical significance. Curr Opin HIV
HIV p24 antigen and antibodies to HIV-1 (groups M and O) and AIDS. 2012;7(5):470–477.
HIV-2 in human serum or plasma. J Clin Virol. 2011;52(suppl 1): 93. Donovan M, Palumbo P. Diagnosis of HIV: challenges and strate-
S57–S61. gies for HIV prevention and detection among pregnant women
74. Delaney KP, Branson BM, Jafa K, et al. Performance of an oral and their infants. Clin Perinatol. 2010;37(4):751–763.
fluid rapid HIV-1/2 test: experience from four CDC studies. AIDS. 94. Read JS, and the Committee on Pediatric AIDS. Diagnosis of
2006;20:1655–1660. HIV-1 infection in children younger than 18 months in the
75. Greenwald JL, Burstein GR, Pincus J, Branson B. A rapid review of United States. Pediatrics. 2007;120:e1547–e1562.
rapid HIV antibody tests. Curr Infect Dis Reports. 2006;8:125–131. 95. Centers for Disease Control and Prevention. Revised recommen-
76. Owen SM. Testing for acute HIV infection: implications for treat- dations for HIV testing of adults, adolescents, and pregnant
ment as prevention. Curr Opin HIV AIDS. 2012;7(2):125–130. women in health care settings. MMWR. 2006;55(RR-14):1–17.
77. Myers JE, El-Sadr WM, Zerbe A, Branson BM. Rapid HIV self-
testing: long in coming but opportunities beckon. AIDS. 2013;
27(11):1687–1695. 1. Vaccine. Taber’s Medical Dictionary Website. www.tabers.com/
78. Griffith BP, Campbell S, Caliendo AM. Human immunodeficiency tabersonline/view/Tabers-Dictionary/739755/all/vaccine?q=
viruses. In: Versalovic J, Carroll KC, Funke G, et al., eds. Manual vaccine#23. Accessed July 31, 2013.
of Clinical Microbiology. Vol 2. 10th ed. Washington, DC: ASM 2. Centers for Disease Control and Prevention. Smallpox disease
Press; 2011:1302–1322. overview. emergency.cdc.gov/agent/smallpox/overview/disease-facts
79. Consortium for retrovirus serology standardizations. Serologic .asp. Updated February 6, 2007. Accessed November 12, 2013.
diagnosis of human immunodeficiency virus infection by Western 3. Riedel S. Edward Jenner and the history of smallpox and vacci-
blot testing. JAMA. 1988;260:674–679. nation. BUMC Proceedings. 2005;18(1):21–25.
80. Mellors JW, Munoz A, Giorgi JV, et al. Plasma viral load and CD4+ 4. Nossal GJV. Vaccines. In: Paul WE, ed. Fundamental Immunology.
lymphocytes as prognostic markers of HIV-1 infection. Ann Intern 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2008:
Med. 1997;126(12):946–954. 1223–1290.
81. Barnett D, Denny TN. Lymphocyte immunophenotyping in 5. Lattanzi M, Rappuoli R, Stadler K. The use of vaccines and anti-
human immunodeficiency virus infection: For richer, for poorer. body preparations. In: Bellanti JA, Escobar-Gutierrez A, Joost JJ,
In: Carey JL, McCoy JP Jr, Keren DF, eds. Flow Cytometry in eds. Immunology IV: Clinical Applications in Health and Disease.
Clinical Diagnosis. 4th ed. Chicago, IL: American Society for Clin- Bethesda, MD: I Care Press; 2012:891–937.
ical Pathology Press; 2007:259–274. 6. Plotkin SA, Plotkin SL. The development of vaccines: how the
82. Centers for Disease Control and Prevention. Guidelines for per- past led to the future. Nat Rev Microbiol. 2011;9(12):889–893.
forming single-platform absolute CD4+ T cell determinations 7. Plotkin SA. Vaccines: past, present and future. Nat Med. 2005;
with CD45 gating for persons infected with human immunode- 11(4 suppl):S5–11.
ficiency virus. MMWR. 2003;52(RR-2):1–13. 8. Centers for Disease Control and Prevention. Historical perspec-
83. Centers for Disease Control and Prevention. 1997 revised guide- tives: a centennial celebration: Pasteur and the modern era of
lines for performing CD4+ T-cell determinations in persons in- immunization. MMWR. 1985;34(26):389–390.
fected with human immunodeficiency virus. MMWR. 1997; 9. DeGregorio E, D’Oro U, Bertholet S, Rappuoli R. Vaccines. In:
46(RR-2):1–29. Paul WE, ed. Fundamental Immunology. 7th ed. Philadelphia, PA:
84. Rowley CF. Developments in CD4 and viral load monitoring in Lippincott Williams & Wilkins; 2013:1032–1068.
resource-limited settings. Clin Infect Dis. 2014;58(3):407–412. 10. Plotkin SA. Six revolutions in vaccinology. Pediatr Infect Dis J.
85. Wade D, Daneau G, Aboud S, et al. WHO multicenter evaluation 2005;24(1):1–9.
of FACSCount CD4 and pima CD4 T-cell count systems: Instru- 11. Hilleman MR. Vaccines in historic evolution and perspective: a
ment performance and misclassification of HIV-infected patients. narrative of vaccine discoveries. J Hum Virol. 2000;3(2):63–76.
J Acq Immun Def Synd. 2014;66(5):e98–e107. 12. De Gregorio E, Rappuoli R. Vaccines for the future: learning from
86. Boyle DS, Hawkins KR, Steele MS, et al. Emerging technologies human immunology. Microb Biotechnol. 2012;5(2):149–155.
for point-of-care CD4 T-lymphocyte counting. Trends Biotechnol. 13. Makela PH. Vaccines: coming of age after 200 years. FEMS
2012;30(1):45–54. Microbiol Rev. 2000;24(1):9–20.
87. Baum P, Heilek G. Viral load monitoring: shifting paradigms in 14. Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed.
clinical practice. MLO. 2013;45(11):8. New York, NY: WH Freeman and Co.; 2013:574–591.
88. Nolte FS, Caliendo AM. Molecular microbiology. In: Versalovic J, 15. Centers for Disease Control and Prevention. Typhoid immunization
Carroll KC, Funke G, et al., eds. Manual of Clinical Microbiology. recommendations of the Advisory Committee on Immunization
Vol 1. 10th ed. Washington, DC: ASM Press; 2011:27–59. Practices (ACIP). MMWR. 1994;43(RR14):1–7.
89. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance geno- 16. Prevots DR, Burr RK, Sutter RW, Murphy TV. Poliomyelitis pre-
typing in HIV-1 therapy. Lancet. 1999;353:2195–2199. vention in the United States. Updated recommendations of the
90. Hirsch M, Günthard, HF. Schapiro JM, et al. Antiretroviral drug Advisory Committee on Immunization Practices (ACIP). MMWR
resistance testing in adult HIV-1 infection. Recommendations of Recommendations & Reports. 2000;49(RR-5):1–22.
an international AIDS Society—USA panel. JAMA. 2000;28: 17. Centers for Disease Control and Prevention. Measles, mumps, and
2417–2426. rubella—vaccine use and strategies for elimination of measles,
 rubella, and congenital rubella syndrome and control of mumps: recommendations of the Advisory Committee on Immunization
recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 2015;64(11):300–304.
Practices (ACIP). MMWR. 1998;47(RR-8):1–57. 31. Kroger AT, Sumaya CV, Pickering LK, Atkinson WL. General
18. Marin M, Guris D, Chaves SS, et al. Prevention of varicella: recom- recommendations on immunization. Recommendations of the
mendations of the Advisory Committee on Immunization Practices Advisory Committee on Immunization Practices. MMWR. 2011;
(ACIP). MMWR Recommendations & Reports. 2007;56(RR-4):1–40. 60(2):1–61.
19. Grohskopf LA, Shay DK, Shimabukuro TT, et al. Prevention and 32. Centers for Disease Control and Prevention. Immunization
control of seasonal influenza with vaccines recommendations of schedules. www.cdc.gov/vaccines/schedules/index.html. Updated
the Advisory Committee on Immunization Practices—United 2015. Accessed May 18, 2015.
States, 2013–2014. MMWR. 2013:1–43. 33. Glenny A, Pope C, Waddington H, Wallace V. The antigenic value
20. Korsman S. Vaccines. In: Kamps BS, Hoffman C, Preiser W, eds. of toxoid precipitated by potassium-alum. J Path Bacteriol. 1926;
Influenza Report. 2006. www.influenzareport.com/ir/vaccines 29:38–45.
.htm. Accessed December 3, 2013. 34. Dekker CL, Gordon L, Klein J. Dose optimization strategies for
21. Fiore AE, Uyeki TM, Broder K, et al. Prevention and control of vaccines: the role of adjuvants and new technologies. Report of
influenza with vaccines: recommendations of the Advisory the subcommittee on vaccine development and supply. https://
Committee on Immunization Practices (ACIP), 2010. MMWR wayback.archive-it.org/3919/20140414150800/ www.hhs.gov/
Recommendations & Reports. 2010;59(RR-8):1–62. nvpo/nvac/meetings/pastmeetings/2008/dekker_gordan_klein.pdf
22. Fiore AE, Wasley A, Bell BP. Prevention of hepatitis A through 2008:1–22. Accessed May 18, 2015.
active or passive immunization: recommendations of the Advi- 35. Koff WC, Burton DR, Johnson PR, et al. Accelerating next-
sory Committee on Immunization Practices (ACIP). MMWR generation vaccine development for global disease prevention.
Recommendations & Reports. 2006;55(RR-7):1–23. Science. 2013;340(6136):1232910-1–1232910-7. doi: 10.1126/
23. Broder KR, Cortese MM, Iskander JK, et al. Preventing tetanus, science.
diphtheria, and pertussis among adolescents: use of tetanus tox- 36. Kristoff J. Malaria stage-specific vaccine candidates. Curr Pharm
oid, reduced diphtheria toxoid and acellular pertussis vaccines Des. 2007;(13):1989–1999.
recommendations of the Advisory Committee on Immunization 37. Six A, Bellier B, Thomas-Vaslin V, Klatzmann D. Systems biology
Practices (ACIP). MMWR Recommendations & Reports. 2006; in vaccine design. Microb Biotechnol. 2012;5(2):295–304.
55(RR-3):1–34. 38. Lambert LC, Fauci AS. Influenza vaccines for the future. N Engl
24. Nuorti JP, Whitney CG. Prevention of pneumococcal disease J Med. 2010;363(21):2036–2044.
among infants and children—use of 13-valent pneumococcal 39. Rueckert C, Guzman CA. Vaccines: from empirical development
conjugate vaccine and 23-valent pneumococcal polysaccharide to rational design. PLOS Pathog. 2012;8(11):1–7.
vaccine—recommendations of the Advisory Committee on Im- 40. Coffman RL, Sher A, Seder RA. Vaccine adjuvants: putting innate
munization Practices (ACIP). MMWR Recommendations & Reports. immunity to work. Immunity. 2010;33(4):492–503.
2010;59(RR-11):1–18. 41. Azad N, Rojanasakul Y. Vaccine delivery—current trends and
25. Centers for Disease Control and Prevention. Haemophilus b con- future. Curr Drug Del. 2006;3(2):137–146.
jugate vaccines for prevention of haemophilus influenzae type b 42. Plotkin SA. Correlates of protection induced by vaccination. Clin
disease among infants and children two months of age and older. Vac Immunol. 2010;17(7):1055–1065.
Recommendations of the Immunization Practices Advisory 43. Hebert CJ, Hall CM, Odoms LN. Lessons learned and applied: what
Committee (ACIP). MMWR Recommendations & Reports. 1991; the 20th century vaccine experience can teach us about vaccines in
40(RR-1):1–7. the 21st century. Hum Vaccines Immunother. 2012;8(5):560–568.
26. Cohn AC, MacNeil JR, Clark TA, et al. Prevention and control 44. Roush SW, Murphy TV. Historical comparisons of morbidity and
of meningococcal disease: recommendations of the Advisory mortality for vaccine-preventable diseases in the United States.
Committee on Immunization Practices (ACIP). MMWR Recom- JAMA. 2007;298(18):2155–2163.
mendations & Reports. 2013;62(RR-2):1–28. 45. Centers for Disease Control and Prevention. Final 2013 reports
27. Centers for Disease Control and Prevention (CDC). Update: of nationally notifiable infectious diseases. MMWR. 2014; 63(32):
vaccine side effects, adverse reactions, contraindications, and 702–715.
precautions. Recommendations of the Advisory Committee on 46. World Health Organization. Global vaccine action plan 2011–2020.
Immunization Practices (ACIP). MMWR Recommendations & www.who.int/immunization/global_vaccine_action_plan/GVAP_
Reports. 1996;45(RR-12):1–35. doc_2011_2020/en/index.html. Published 2013. Updated 2013
28. Mast EE, Margolis HS, Fiore AE, et al. A comprehensive immu- Accessed December 3, 2013.
nization strategy to eliminate transmission of hepatitis B virus 47. National Institute of Allergy and Infectious Diseases. Community
infection in the United States: Recommendations of the Advisory immunity (“herd” immunity). www.niaid.nih.gov/topics/pages/
Committee on Immunization Practices (ACIP) part 1: immuniza- communityimmunity.aspx. Updated 2010. Accessed November
tion of infants, children, and adolescents. MMWR Recommenda- 15, 2013.
tions & Reports. 2005;54(RR-16):1–31. 48. Siegrist CA. Mechanisms underlying adverse reactions to vaccines.
29. Markowitz LE, Dunne EF, Saraiya M, et al. Centers for Disease J Comp Pathol. 2007;137(suppl 1):S46–50.
Control and Prevention (CDC). Quadrivalent human papillo- 49. Heidary N, Cohen DE. Hypersensitivity reactions to vaccine com-
mavirus vaccine: Recommendations of the Advisory Committee ponents. Dermatitis. 2005;16(3):115–120.
on Immunization Practices (ACIP). MMWR Recommendations & 50. Centers for Disease Control and Prevention. Poliomyelitis pre-
Reports. 2007;56(RR-2):1–24. vention in the United States: updated recommendations of the
30. Centers for Disease Control and Prevention (CDC). Use of 9-valent Advisory Committee on Immunization Practices (ACIP). MMWR.
human papillomavirus (HPV) vaccine: updated HPV vaccination 2000;49(RR05):1–22.
51. Nigrovic LE, Thompson KM. The Lyme vaccine: a cautionary tale. 61. Foltz IN, Karow M, Wasserman SM. Evolution and emergence
Epidemiol Infect. 2007;135(1):1–8. of therapeutic monoclonal antibodies: what cardiologists need to
52. American Academy of Pediatrics. Autism and Andrew Wakefield. know. Circulation. 2013;127(22):2222–2230.
Immunization Website. www2.aap.org/immunization/families/ 62. Yuvienco C, Schwartz S. Monoclonal antibodies in rheumatic
autismwakefield.html. Updated 2013. Accessed November 15, 2013. diseases. Medicine & Health, Rhode Island. 2011;94(11):320–324.
53. Kutty P, Rota J, Bellini W, et al. Measles. In: VPD surveillance 63. Scott AM, Allison JP, Wolchok JD. Monoclonal antibodies in
manual. 6th ed. Publication City: Centers for Disease Control and cancer therapy. Cancer Immun. 2012;12:14.
Prevention; 2013:1–21. www.cdc.gov/vaccines/Pubs/surv-manual/ 64. Klein E, Sjogren HO. Humoral and cellular factors in homograft
chpt07-measles.pdf. and isograft immunity against sarcoma cells. Cancer Res. 1960;
54. Centers for Disease Control and Prevention. Pertussis. Vaccines and 20:452–461.
immunizations. The Pink Book. 12th ed. Website. www.cdc.gov/ 65. Slettenmark B, Klein E. Cytotoxic and neutralization tests
vaccines/pubs/pinkbook/pert.html. Updated May 12. Accessed with serum and lymph node cells of isologous mice with in-
November 19, 2013. duced resistance against gross lymphomas. Cancer Res. 1962;
55. Centers for Disease Control and Prevention. About pertussis 22:947–954.
outbreaks. Pertussis (Whooping Cough). Website. www.cdc.gov/ 66. Lee S, Margolin K. Tumor-infiltrating lymphocytes in melanoma.
pertussis/outbreaks/about.html. Updated 2013. Accessed Curr Oncol Rep. 2012;14(5):468–474.
November 15, 2013. 67. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy
56. Kantha SS. A centennial review: the 1890 tetanus antitoxin paper for cancer: harnessing the T cell response. Nat Rev Immunol.
of von Behring and Kitasato and the related developments. Keio 2012;12(4):269–281.
J Med. 1991;40(1):35–39. 68. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumor-
57. Grundmann K. Emil von Behring: the founder of serum therapy. infiltrating lymphocytes and interleukin-2 in the immunotherapy
Nobel Prizes and Laureates. Website. www.nobelprize.org/nobel_ of patients with metastatic melanoma. A preliminary report.
prizes/medicine/laureates/1901/behring-article.html. Updated N Engl J Med. 1988;319(25):1676–1680.
2001. Accessed November 15, 2013. 69. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J
58. Schwab I, Nimmerjahn F. Intravenous immunoglobulin therapy: Med. 2006;354(17):1813–1826.
how does IgG modulate the immune system? Nat Rev Immunol. 70. Schliesser U, Streitz M, Sawitzki B. Tregs: application for solid-
2013;13(3):176–189. organ transplantation. Curr Opin Organ Tran. 2012;17(1):34–41.
59. Stiehm ER. Standard and special human immune serum globu- 71. Pagliara D, Savoldo B. Cytotoxic T lymphocytes for the treatment
lins as therapeutic agents. Pediatrics. 1979;63(2):301–319. of viral infections and posttransplant lymphoproliferative disor-
60. Keller MA, Stiehm ER. Passive immunity in prevention and treat- ders in transplant recipients. Curr Opin Infect Dis. 2012;25(4):.
ment of infectious diseases. Clin Microbiol Rev. 2000;13(4):602–614. 431–437.
 Answer Key

1. a. Although the cholesterol levels were within normal lim-

1. a. Because the swelling has occurred within 2 days, it is its for both HDL and total cholesterol, recent studies indicate
most likely caused by an innate immune response. The adap- that an increase in CRP has been associated with a greater risk
tive immune response takes longer to develop because it de- of a future heart attack. Higher fibrinogen levels are also asso-
pends upon lymphocytes recognizing a specific antigen. ciated with an increased risk for a future cardiovascular event,
Swelling and redness in the tissue is caused by neutrophils although increased fibrinogen is not as great a risk factor as in-
leaving the bloodstream by means of diapedesis in response creased CRP. A rise in both of these acute phase reactants indi-
to the presence of bacteria. b. There may be macrophages, cates an underlying inflammatory process. Such a process is
neutrophils, and dendritic cells present. associated with atherosclerosis, a condition that damages coro-
2. a. The adaptive immune system is characterized by speci- nary blood vessels. Rick’s wife should encourage him to follow
ficity and memory. When exposed to the same foreign substance a healthy diet and to lose weight through exercise.
numerous times, the response is increased each time. Thus, 2. a. CRP is one of the first indicators of a possible infection.
with a serious disease such as tetanus, getting a booster shot of Levels also rise in the case of a malignancy, heart attack, or
a similar but harmless substance will stimulate the immune trauma to the body. If the infection was bacterial, an increase
system each time a booster is given. Restimulating the adaptive in the white blood cell (WBC) count should have been seen.
immune response provides greater protection than the innate This increase would mainly be because of recruitment of neu-
immune system on its own, which could possibly be over- trophils to help fight the invading organism. However, if an in-
whelmed by pathogens such as the bacteria that cause tetanus. fection is caused by a virus, there is typically no increase in the
WBC count. As an acute phase reactant, CRP levels increase
dramatically within 24 hours, long before specific antibody can
1. c 2. d 3. a 4. a 5. d 6. b 7. a be detected. b. An increase in CRP would likely be seen if the
student had infectious mononucleosis, a viral infection. How-
8. d 9. c 10. a 11. b 12. a 13. b 14. c
ever, CRP doesn’t specifically indicate which type of viral in-
15. a 16. b 17. c 18. d 19. c 20. b fection may be present. The symptoms are consistent with the
possibility of mono, but other conditions can’t be ruled out.
Repeating the mono test in a few days will allow enough time
for a detectable level of antibody to form, and the diagnosis
could be confirmed.

1. a. Because every child inherits one haplotype (set of genes)

from the mother and one from the father, 50% of the HLA anti- 1. a 2. c 3. c 4. a 5. d 6. b 7. c
gens would match the mother and 50% would match the father.
8. b 9. d 10. c 11. a 12. d 13. a 14. b
It would never be more than that unless the mother and father
have at least one antigen in common. b. According to the law 15. b 16. d
of independent assortment, there would be a 1:4 chance that the
sister would be an exact match, a 1:2 chance that a sister would
share half of the same alleles, and a 1:4 chance that a sister would
share no alleles, having received the opposite haplotype from
each parent. c. It is possible that a cadaver kidney may actually
1. a. The normal CD19+ cell count indicates that there is not
be a better match, if neither sister is an exact match. The most
a lack of B cells, which are presumably capable of responding
important alleles to match are HLA A, B, and DR. If a cadaver
to antigen and producing antibodies. The low CD4+ T cell
match has more than one allele in common with the recipient
count indicates that there is a decrease in T helper (Th) cells.
at each of these loci, then it would be a closer match.
T helper (Th) cells are necessary for a response to T-dependent
antigens. The decreased CD4+ count means that B cells are not
activated by Th cells and class switching to IgG does not occur.
1. d 2. b 3. a 4. c 5. d 6. a 7. b Memory B cells are not produced either. b. Lack of Th cells
8. a 9. a 10. c 11. b 12. c 13. a 14. b severely limits the B-cell response, resulting in increased
15. d 16. c 17. b. 18. a bacterial infections.
2. a. CD8+ T cells are responsible for the destruction of can- alternative pathway as well. b. Levels of C3 and C4 are nor-
cer cells as well as any intracellular pathogens, such as viruses. mal, indicating that a deficiency of one or more of the mem-
Although antibodies may be produced, they are not very effec- brane attack components is involved. Although a lack of C1q
tive against cancerous cells or virally infected cells. Thus, an or C2 cannot absolutely be ruled out, the fact that the alterna-
important arm of the adaptive immune system would not tive pathway is also affected is an additional indicator that the
be working and an individual would be more susceptible to components common to both pathways, C5 through C9, are
certain kinds of infections, or to cancer. the ones involved. Because defense against encapsulated bac-
teria such as meningococci is reduced if there is a decrease in
C5 through C9, the patient’s symptoms are in accord with this
1. b 2. d 3. a 4. a 5. c 6. b 7. d conclusion. c. In order to confirm the actual deficiency, test-
ing for the individual components C5 through C9 should be
8. c 9. a 10. b 11. b 12. d 13. c 14. d
performed. Because this type of deficiency reduces the overall
15. c 16. b 17. a 18. d functioning of the complement system, patients should receive
prompt therapy when signs of infection are noted.
2. a. Although the abdominal pain and vomiting could be
caused by several infectious agents, the normal white blood
cell count decreases the likelihood of a bacterial infection.
1. a. The presence of IgM only is an indicator of an early acute The accompanying swelling of the hands and legs may be an
infection. IgM is the first antibody to appear, followed by IgG. indicator of a possible inflammatory problem associated with
In a reactivated case of mono, a small amount of IgM might be continuous activation of the complement system. Because
present, but IgG would also be present. Thus, the patient is this has been a recurring problem, the likelihood of an im-
encountering the virus for the first time. b. The memory cells mune problem is increased. Because total serum protein is
triggered by the first exposure to the virus would cause produc- within the normal range, it is unlikely that the deficiency is
tion of IgG in a much shorter time and there would be a greater from lack of antibody production. A decrease of one comple-
increase in IgG compared with the amount of IgM present. ment component would not be apparent on a total protein
2. a. The increase in IgE is an indicator that the cold symp- determination. b. Reduced levels of both C4 and C2 could
toms may actually be caused by an allergy. This is especially be from inheritance of defective genes for both components.
evident in the springtime when pollen levels are high. The child However, the possibility of that is extremely rare. A more
should be tested for specific allergies to determine the cause of plausible explanation is that the deficiency of both C2 and
the symptoms. Treatment with antihistamine and avoidance of C4 is caused by overconsumption rather than a lack of pro-
the allergen will help to relieve the symptoms. b. Chronic res- duction. c. A lack of C1-INH would result in overconsump-
piratory infections may be caused by a decrease or lack of IgA, tion of C4 and C2. As this is the most common deficiency of
but this is not the case here. Normal levels of IgG, IgM, and IgA the complement system, this represents a likely explanation
indicate that this child is not immunocompromised. for the symptoms. This can be verified by testing for this par-
ticular component.

1. a 2. a 3. b 4. d 5. b 6. c 7. a
8. d 9. a 10. a 11. c 12. b 13. a 14. c 1. b 2. a 3. d 4. c 5. c 6. a 7. d
15. d 16. b 17. b 18. c 19. b 20. d 21. a 8. a 9. c 10. a 11. b 12. b 13. b 14. a
22. c 15. c 16. d 17. d 18. b 19. c 20. b

1. a. Gloves should never be removed when working with

1. a. G-CSF. b. IFN-gamma and IL-2. c. IL-4 and IL-10.
patient specimens. When they are, hands should be washed
right away using the correct procedure. Any contamination of
the lab bench should be treated with sodium hypochlorite and
1. b 2. a 3. d 4. d 5. c 6. a 7. d the paper towels should be disposed of in the regulated med-
8. b 9. c 10. d 11. b 12. b 13. a 14. c ical waste container. Because the supervisor’s lab coat was dis-
15. b 16. d posable and became contaminated, it should be discarded in
the regulated medical waste container and replaced with a new
coat. Pipetting should have been done behind a Plexiglass
shield because this would have prevented the spill onto the
lab coat.
1. a. A decreased CH50 indicates a problem with the classical 2. a. Take corrective action. b. Take corrective action.
pathway. The decreased AH50 indicates a problem with the c. Accept. d. Take corrective action.
 with the virus. Therefore, she does not have to be concerned
about possible consequences for the fetus. If there is any
1. c 2. a 3. a 4. c 5. c 6. c 7. b
further question about her immunity, further testing to
8. d 9. a 10. c 11. b 12. d 13. a 14. d determine the class of antibody present could be done. The
15. a 16. a 17. d 18. c 19. b presence of IgG antibody indicates a previous exposure,
20. 2, 1, 2, 3, 2, 2 whereas IgM would indicate a recent infection.

1. c 2. d 3. b 4. d 5. b 6. a 7. b
8. a 9. c 10. d 11. a 12. c 13. a 14. c
1. a. The serological pipette must be emptied completely to ob- 15. b 16. c 17. b 18. d 19. a 20. c
tain the correct volume because it is marked to contain (TC)
rather than to deliver (TD). Therefore, it should have been blown
out to obtain the last bit, or the measurement should have been
from point to point, as in filling the pipette up to the 0.8 mL
mark and then letting it drain to the 0.9 mL mark. b. The
1.9 diluent was not correct. In order to make a 1:40 dilution with 1. a. A negative finding only means that no parasites were ob-
0.1 mL of serum, the calculations are as follows: served for that particular specimen at that particular time. It does
not rule out the possibility that parasites may actually be present.
1/40 = 0.1/X b. Capture enzyme immunoassays that are specific for parasites
X = 4.0 mL (This represents the total volume.) such as Giardia and Cryptosporidium are available. Typically, a
4.0 ! 0.1 = 3.9 mL of diluent to make a 1:40 dilution. solid phase such as microtiter wells is coated with specific anti-
body, and very small amounts of antigen can be detected. If a par-
asite is suspected and the traditional results are negative, this
1. b 2. a 3. c 4. a 5. c 6. d 7. b would be the next step. c. Capture enzyme immunoassays are
8. a 9. b 10. a 11. d 12. c 13. b 14. a very sensitive and are capable of detecting minute amounts of
15. d 16. c 17. d 18. a parasitic antigens that may be present. This is important in testing
a stool culture because large amounts of antigen may not be pres-
ent at any one time. Many organisms, such as Giardia and Cryp-
tosporidium, are extremely small and may not be easily found on
a stained slide preparation. d. In addition to the increased sen-
sitivity, enzyme immunoassays are simple to perform and less
time consuming than traditional tests for parasites. Because in-
1. a. The results indicate normal levels of IgG and IgM, but there strumentation is usually used, the results are more easily inter-
is a decreased level of IgA. This most likely indicates a selective preted with less subjectivity than stained smears.
IgA deficiency, the most common genetic immunodeficiency.
Selective IgA deficiency occurs in approximately 1:1,000 indi-
viduals. b. A decrease in serum IgA most likely indicates a de-
1. c 2. a 3. c 4. b 5. a 6. c 7. b
crease in secretory IgA, the immunoglobulin that is found on
mucosal surfaces. Individuals with a selective IgA deficiency are 8. a 9. b 10. d 11. d 12. b 13. c 14. c
more prone to respiratory tract and gastrointestinal tract infections 15. b 16. b
because IgA represents the first line of defense against pathogens
that invade mucosal surfaces. c. Nephelometry is a more sensi-
tive method for measuring immunoglobulin levels. It is able to
detect small quantities of immunoglobulin present. Results are
obtained faster in comparison to RID; because the process is au- 1. a. Controls would include a reagent blank to test for con-
tomated, it is not subject to human error in reading the results. tamination and a negative control for the mutation (a DNA
Other errors that may occur with RID include overfilling or un- template known not to have the mutation) to demonstrate that
derfilling of wells, nicking of wells, and inaccurate incubation time the restriction enzyme will cut the normal product. A positive
or temperature. Therefore, nephelometry has largely replaced RID control for the mutation (a DNA template known to have the
for the measurement of immunoglobulin levels. mutation) will demonstrate that the restriction enzyme will not
2. a. A positive test on an undiluted patient specimen in- cut the mutant product. An internal control for the restriction
dicates immunity to the virus if the patient was tested im- enzyme activity—ideally, a negative control—should be cut in
mediately after exposure to the disease. Testing 2 days after the same reaction as the test sample to demonstrate that the
exposure would not give enough time for antibody to be enzyme is active in that reaction. b. It is not necessary to see
formed if this is a first exposure to the virus. b. The pres- the 11 bp product. The presence of the 80 bp and 59 bp prod-
ence of antibody indicates that the patient has immunity be- ucts indicate the activity of the enzyme and the 139 bp product
cause of her vaccination and likely will not be re-infected shows the presence of the mutation that prevents the enzyme
from cutting. c. The cutting of the normal control indicates the environment. c. This child should be tested for HIV. That
that the enzyme is active and confirms the interpretation of a would explain the decrease in CD4+ T cells.
positive result (presence of mutation). d. If the internal con-
trol was not digested by the restriction enzyme, then the re-
striction enzyme was not active. Therefore, no interpretation 1. a 2. c 3. b 4. c 5. a 6. b 7. d
can be made about the patient’s DNA.
8. c 9. a 10. d 11. c 12. c 13. c 14. d
2. a. The amplification control should always be present to 15. d 16. b 17. d 18. b 19. b 20. d
demonstrate that the PCR is working and to avoid false nega-
tives. A reagent blank (no template) control is included to de-
tect contamination. In a true negative, the amplification control
would be positive, whereas the test target is negative. The pre-
vious results don’t necessarily predict the current test results
should be positive. Because the amplification control did not 1. a. An increase in eosinophils is typically found in allergic
work, the current results are not interpretable. b. Because the individuals. Interleukins released by stimulated Th1 cells are
amplification control is positive, the PCR is working and the involved in the recruitment of eosinophils from the bone mar-
result is a true negative. The reagent blank (no template) con- row. Although there are other causes of eosinophilia, such as a
trol, not the amplification control, is used to detect contami- parasitic infection, an increased number most often indicates
nation. In a true negative, the amplification control would be an allergic reaction. b. The patient can have a skin prick test
positive, whereas the test target is negative. The previous re- performed to determine which allergens he is sensitized to. The
sults may be reviewed for interpretation of the result, but do patient would know his results immediately because a positive
not predict a positive result in the current sample. c. No. The test would be indicated by formation of wheal-and-flare reac-
test sensitivity goes to 50 copies/mL, meaning that there may tions within 20 minutes at the site(s) of injection. If he is un-
be fewer than 50 copies/mL present that will not be detected able to discontinue any antihistamines he might be taking, or
by the test method. Although the previous results don’t predict if a clear area of skin in his forearm or back could not be found,
a positive result because there is a history of the presence of a a solid-phase immunoassay for allergen-specific IgE could be
virus, a residual low level of viral copies could be present. The performed. c. A solid-phase immunoassay for total IgE could
results should be reported as fewer than 50 copies/mL to indi- be performed to monitor the patient’s response to allergen im-
cate the test’s inability to detect a low-level presence of HIV. munotherapy. If the therapy is successful, the IgE concentra-
tion in the patient’s serum should decrease to a level within the
reference age for patients his age.
1. b 2. a 3. a 4. b 5. a 6. a 7. c 2. a. A positive DAT indicates that the red blood cells (RBCs)
8. d 9. c 10. c 11. b 12. a 13. a 14. d are coated with either antibody or complement components.
The destruction of some RBCs is the reason for the man’s symp-
15. d 16. b 17. d 18. b 19. c 20. b 21. c
toms. b. The most likely cause of the positive DAT is the pres-
ence of an antibody of the IgM class. It might be an anti-I,
triggered by Mycoplasma pneumonia. This is a cold-reacting
antibody. c. A DAT that is only positive with anti-C3d indi-
cates that only complement products are present on the RBCs.
This is a further indication that the antibody is an IgM antibody
1. a. The result may represent an error of specificity given that because it does not remain on the cells at 37°C but does trigger
the newer instrument is getting positive results on specimens complement activation, which can cause the cell destruction.
that were negative by the older method. However, the newer
instrument could be more sensitive than the older one, so these
could actually be positive samples. b. To resolve this discrep- 1. c 2. b 3. d 4. b 5. a 6. b 7. d
ancy, known positive and negative controls should be run. The
8. b 9. c 10. c 11. a 12. b 13. d 14. d
positive controls need to include those at the lower limit of de-
tection, as well as more highly positive samples. This would 15. c
help to determine if the new instrument is actually more sen-
sitive rather than lacking in specificity.
2. a. The flow pattern in A indicates that the majority of lym-
phocytes are B cells because they are CD19+. The population
most affected appears to be CD3+, which are T cells. Pattern B 1. a. In systemic lupus erythematosus, a low titer of rheuma-
indicates that of the CD3+ lymphocytes, the majority are CD8+, toid factor is often present. Conversely, a low titer of antinuclear
or cytotoxic T cells. The CD4+ count is very low. b. T helper antibodies can be associated with rheumatoid arthritis. Thus,
cells are necessary to provide help to B cells so they can re- these two conditions cannot be differentiated on the basis of
spond by making antibody. Thus, the child is unable to make the rapid RF and ANA test results alone. b. The decreased red
IgG in response to potential pathogens she might encounter in blood cell (RBC) count may be because of the presence of a
low-level autoantibody directed against RBCs, often associated
with lupus. c. A fluorescent antinuclear antibody (FANA) test
1. b 2. c 3. d 4. d 5. b 6. b 7. a
is a good screening tool to help distinguish between these two
conditions. A homogeneous pattern or a peripheral pattern 8. d 9. a 10. b 11. b 12. c 13. a 14. c
would be indicative of lupus, whereas a speckled pattern can 15. b
sometimes be found in rheumatoid arthritis or lupus. Therefore,
if a speckled pattern is obtained, more specific testing for ENA
antibodies should be done. The presence of anti-Sm antibody
would be diagnostic for lupus. This is what was found in this
case. Testing for anti-CCP could also be performed, as this 1. a. If no further CA 15-3 is being produced by tumor tissue,
antibody is highly specific for rheumatoid arthritis. levels will decrease at the rate of biological half-life for the mol-
2. a. The low T4 level, enlarged thyroid gland, and presence ecule. Because CA 15-3 levels are not decreasing at this rate,
of antithyroglobulin antibody are all indicators of Hashimoto’s residual tumor is suspected. b. HER-2 overexpression indi-
thyroiditis. b. Antithyroglobulin antibodies progressively de- cates that therapy with the monoclonal antibody Herceptin
stroy thyroglobulin produced by the thyroid. Thyroglobulin is may be successful. c. Because the tumor lacks estrogen and
normally cleaved in the thyroid to produce the secretable hor- progesterone receptors, hormone-suppressing therapy is un-
mones triiodothyronine (T3) and thyroxine (T4). The presence likely to improve prognosis.
of antithyroglobulin antibodies causes enlargement of the thy- 2. a. No other tissues in men are known to produce PSA,
roid because of the immune response, and hypothyroidism re- so another source is extremely unlikely. b. PSA velocity is
sults, characterized by fatigue and weight gain. c. Graves the rate of PSA increase between determinations. Because
disease is also an autoimmune illness that affects the thyroid, PSA increases with age and prostatic enlargement, examining
but it is characterized by hyperthyroidism. In this disease, an- PSA velocity is an attempt to separate benign and malignant
tibodies to thyroid-stimulating hormone receptors are pro- conditions, as velocity is higher in malignancy. Current rec-
duced, sending a signal to the thyroid to constantly produce ommendations for biopsy are for PSA velocities that exceed
T3 and T4; consequently, these hormones are elevated in the 0.35 ng/mL per year. c. Although increased to above the
blood. Symptoms include nervousness, insomnia, restlessness, reference interval, the proportion of free PSA remains within
and weight loss, exactly opposite of the characteristics of the interval associated with benign disease. Furthermore, his
Hashimoto’s thyroiditis. PSA velocity did not exceed 0.35 ng/mL per year, and the
digital rectal exam did not detect any obvious sign of malig-
nancy. Given the man’s age, benign prostatic hypertrophy
1. a 2. d 3. d 4. a 5. d 6. b 7. a is likely, and further PSA testing after a waiting period may
8. c 9. d 10. c 11. a 12. b 13. d 14. a be warranted in lieu of a biopsy. d. Once a man’s life
15. c expectancy is fewer than 10 years, PSA testing is no longer
recommended.

1. d 2. b 3. a 4. c 5. d 6. d 7. a
1. a. The most compatible donor for this patient would be 8. c 9. d 10. d 11. a 12. a 13. b 14. a
Friend 2. Sibling 1 has the B35 antigen for which the patient 15. c.
possesses HLA antibody. Sibling 2 also has the B35 antigen
and is also ABO incompatible. Friend 1 is ABO incompatible.
Friend 2 is ABO identical and does not express the HLA-B35
antigen and is thus the most appropriate donor.
2. a. Maybe, for an unrelated donor, one can’t be sure that 1. a. The patient has evidence of anemia and pneumonia.
they have the same alleles at each locus even if they have the The elevated erythrocytic sedimentation rate (ESR) is a non-
same low resolution type. b. The physician requested high- specific indicator of inflammation or elevated serum proteins.
resolution HLA in order to determine if the donor and recip- Based upon these findings, the physician requested the meas-
ient had the same alleles at each locus. Serological typing urement of serum immunoglobulins. Elevated serum im-
(phenotyping) provides low-resolution results as indicated. munoglobulins can produce an elevated ESR. The extremely
The best outcomes for transplant occur if the recipient and high IgG levels indicate that a monoclonal gammopathy is
donor are HLA allele level matched. High-resolution typing probably present. The patient is most likely suffering from
of the donor was performed. The donor’s B locus typing multiple myeloma. Infiltration of cancerous myeloma cells
indicated he or she had the alleles HLA-A*02:05 and into the bone marrow is likely to be responsible for the
HLA-B*44:03. Thus, they were actually mismatched for two patient’s anemia; despite having pneumonia, the white blood
alleles. Based on this finding, this donor was declined and an cell count is only slightly elevated. The back pain could also
additional search was conducted. be caused by infiltration of myeloma cells into the vertebra.
The age of the patient is appropriate for the diagnosis of mul- and the SPE results indicate that she is immunocompromised
tiple myeloma. b. The diagnosis could be confirmed by per- and producing very little antibody at all. The faint IgG band
forming a bone marrow biopsy because having more than would confirm this.
10% plasma cells in the bone marrow is one of the diagnostic
criteria for multiple myeloma. Radiographs could reveal the
presence of lytic bone lesions responsible for the patient’s back 1. c 2. c 3. d 4. a 5. b 6. a 7. d
pain. In addition, serum protein electrophoresis (SPE) could
8. d 9. a 10. b 11. d 12. a 13. c 14. d
be used to detect a monoclonal band and serum immunofix-
ation electrophoresis (IFE) would be used to identify the sus- 15. b
pected monoclonal IgG and possibly detect Bence Jones
proteins in the patient’s urine. Free light chain assays can be
used to determine the concentration of monoclonal light
chains in the serum and the κ/λ ratio. A serum monoclonal
protein concentration of greater than 3 g/dL and a urinary
monoclonal protein greater than 200 mg/day indicate the 1. a. Poststreptococcal glomerulonephritis. b. Streptococcus
presence of multiple myeloma. pyogenes (Group A streptococci). c. Streptococcal antigen–
2. a. Although hairy cell leukemia (HCL) cells are not generally antibody complexes may deposit in the glomeruli of the kidneys
seen in the bone marrow, the hematologic bone marrow studies or antibody formed against the organisms cross-reacts with anti-
describe malignant cells characteristic of HCL. Splenomegaly is gens in the glomeruli. These immune responses stimulate an
often seen in patients with HCL. b. Malignant HCL cells often inflammatory response that causes damage to the glomeruli,
express CD20 and CD25, the markers found in this patient. In leading to renal impairment and function. The rapid GAS test
addition, CD103 is a sensitive and specific marker for this dis- was negative because the organism is no longer present in the
ease. Although not tested for in this patient, CD123 is also a throat and the patient did not present with pharyngitis. d. A
specific marker for HCL. urinalysis is helpful because microscopic hematuria is typically
present in children with acute poststreptococcal glomeru-
lonephritis. The proteinuria rarely exceeds 3+ by dipstick; how-
1. c 2. c 3. a 4. c 5. d 6. a 7. d ever, massive proteinuria and a nephrotic picture may be
8. b 9. a 10. d 11. b 12. a 13. c 14. d observed in a small percentage of patients. e. The streptozyme
15. a test measures antibodies against five extracellular streptococcal
antigens: anti-streptolysin (ASO), anti-hyaluronidase (AHase),
anti-streptokinase (ASKase), anti-nicotinamide-adenine dinu-
Immunodeficiency Diseases cleotidase (anti-NAD), and anti-DNAse B. The streptozyme test
is positive in 95% of patients with acute poststreptococcal
glomerulonephritis caused by GAS pharyngitis.
1. a. The constant bacterial infections coupled with labo-
2. a. Mycoplasma pneumoniae. b. The patient has only been ill
ratory results indicate an immunodeficiency disease, likely
for several days and has not had time to mount a serological re-
Bruton’s tyrosine kinase deficiency or severe combined im-
sponse to the causative agent. IgG levels suggest that the patient
munodeficiency syndrome (SCID). b. In both conditions,
had a previous exposure to the organism and the level may
an X-linked recessive gene can be inherited, which affects
represent residual immunoglobulin G (IgG) from an earlier
males almost exclusively. c. To differentiate between the
exposure. c. Definitive diagnosis of M pneumoniae requires doc-
two immunodeficiency states, several types of testing are rec-
umented seroconversion by paired specimens obtained 2 to
ommended. Measurement of serum IgA, IgM, and IgG levels
4 weeks apart, measuring both IgM and IgG. A four-fold rise in
should be performed to determine if, in fact, all classes of
IgG levels is considered diagnostic. d. Cold agglutinin titers
antibody are absent. Enumeration of classes of lymphocytes
used for the diagnosis of M pneumoniae infections are not very
should also be determined by flow cytometry. In SCID, both
specific or very sensitive. Testing for cold agglutinins is no longer
T- and B-cell development is affected and both lymphocyte
recommended for the detection of M pneumoniae infections be-
populations would be deficient, whereas in Bruton’s tyrosine
cause the development of cold agglutinins occurs in other con-
kinase deficiency, only B-cell development is affected. Be-
ditions, including some viral infections and collagen vascular
cause the differential indicates that some lymphocytes are
diseases. Although not specific for M pneumoniae infection, a high
present, this would point to Bruton’s tyrosine kinase defi-
cold agglutinin titer in a patient with community-acquired pneu-
ciency. Flow cytometry findings confirming the presence of
monia symptoms (>1:64) is likely to be caused by M pneumoniae.
T cells only validate this diagnosis.
2. a. The patient’s specimen is seen in region 4. Note the faint,
diffuse IgG and light chain bands. No IgA or IgM bands are
visible. Specimen 1 is a normal control. Specimen 2 contains 1. d 2. c 3. b 4. b 5. a 6. a 7. b
a monoclonal IgG kappa protein. Specimen 3 is a concentrated 8. d 9. c 10. c 11. a 12. b 13. c 14. c
24-hour urine specimen that contains albumin. b. Her history 15. b
 IgM antibodies is in determining whether a woman has had a
recent infection. If no IgM antibodies are detected and only
IgG is detected, this excludes a recent infection before preg-
1. a. Almost 25% of individuals with Lyme disease do not ex- nancy. The presence of IgG and IgM in the mother may indicate
hibit the characteristic rash; therefore, its absence does not rule a recent infection. b. Tests for IgA and IgM antibodies are
out the possibility of the disease. b. There are several conditions commonly used for the diagnosis of infection in the newborn.
that can cause false-positive results in EIA testing, including If IgG, IgM, and IgA are detected, a diagnosis of congenital tox-
syphilis, other treponemal diseases, infectious mononucleosis, oplasmosis is established. If IgG antibodies are detected but
and autoimmune diseases such as rheumatoid arthritis. Thus, serological test results for IgM and IgA antibodies are negative,
low levels of antibody might indicate one of these other diseases. follow-up serological testing in suspected cases is indicated.
However, false-negative results in Lyme disease are also possible Maternally transferred antibodies usually decrease and disap-
because of a low level of antibody production. Therefore, an in- pear within 6 to 12 months. Established infection in the
determinate test neither rules out nor confirms the presence of mother can also be indicated by the presence of high avidity
Lyme disease. c. If there is a history of tick bite and patient IgG antibodies.
symptoms are consistent with Lyme disease, then a confirmatory 2. a. The majority of patients with symptomatic cryptococ-
Western blot should be performed. The Western blot is fairly cosis have an identified underlying immunocompromised con-
specific for Lyme disease. If 5 of 10 protein bands specific for dition. These include acquired immunodeficiency syndrome
Borrelia burgdorferi IgG antibodies are positive, this confirms the (AIDS), prolonged treatment with corticosteroids, organ trans-
presence of Lyme disease. plantation, advanced malignancy, diabetes, and sarcoidosis.
2. a. Although it is possible that the mother’s positive RPR test The clinical symptoms and outcome of cryptococcal meningitis
could be a false positive, it is also likely that the mother is in vary, in part because of the related underlying medical condi-
the latent stage of syphilis, with no obvious signs of the disease. tions and the immune status of the host. The most common
Although syphilis is not sexually transmitted during this stage, symptoms are headache, altered mental status, personality
it can be transmitted from a mother to her unborn child. Many changes, confusion, lethargy, and coma. Nausea and vomiting
infants do not exhibit clinical signs of the disease at birth; how- are also common and are caused by increased intracranial pres-
ever, if infected and untreated, a large percentage of babies de- sure. Onset of the disease is often subacute and worsens over
velop later symptoms, including neurological deficits such as several weeks. The patient in this case was immunosuppressed
blindness and mental retardation. b. A positive RPR on cord because of his long-term steroid use and presented with se-
blood could be from transplacental passage of the mother’s vere headaches, gait instability, and weakness upon standing.
IgG antibodies. A titer should be performed on the cord blood b. The simplest diagnostic test is an India ink test on the pa-
and a serum sample obtained from the infant in several weeks. tient’s CSF. Because of the large polysaccharide capsule pro-
If infection is present in the infant, the titer will remain duced by the organism, the India ink is displaced, allowing for
the same or increase. An IgM capture assay could also be per- visualization of the yeast. The serological tests for cryptococcal
formed. The presence of specific anti-treponemal IgM would antigen in serum and CSF are highly sensitive and specific for
indicate that the infant had been exposed to Treponema the diagnosis of invasive disease. New immunochromato-
pallidum because IgM antibodies do not cross the placenta. graphic assays may also be used for the detection of the cap-
c. Because there is a good chance that the infant is at risk for sular antigen in serum and CSF.
congenital syphilis, immediate treatment with penicillin can
prevent any further neurological consequences.
1. d 2. d 3. d 4. a 5. b 6. b 7. a
8. a 9. d 10. b 11. d 12. a 13. b 14. d
1. d 2. c 3. b 4. d 5. b 6. c 7. c 15. c 16. c
8. d 9. b 10. d 11. d 12. b 13. c 14. c
15. a

1. a. The patient’s clinical symptoms and increased liver func-

tion enzymes indicate inflammation of the liver. To determine
1. a. It cannot be determined by the test results available whether this inflammation is the result of viral hepatitis and
whether the baby has congenital toxoplasmosis. The IgG anti- to identify the cause, the following tests should be ordered:
bodies in the newborn may reflect maternal antibodies that (1) IgM anti-HAV to screen for hepatitis A, (2) HBsAg to screen
crossed the placenta. The presence of IgG antibodies in the for hepatitis B, (3) IgM anti-HBc to screen for hepatitis B in the
newborn may reflect either past or recent infection in the core window period when HBsAg is absent, and (4) anti-HCV
mother. IgM antibodies may persist for up to 18 months after to screen for hepatitis C. b. To monitor hepatitis B infection,
infection with T gondii. Thus, the greatest value of testing for testing for HBsAg and HBeAg should be performed periodically
to determine how long the infection is persisting and the rela- 2. a. HIV infection is transmitted from mother to infant
tive infectivity of the patient. Tests for anti-HBe and anti-HBs via three routes: (1) passage through the placenta during
are performed to indicate whether the infection has resolved pregnancy, (2) exposure to maternal blood during the deliv-
and whether immunity has been established, respectively. ery process, or (3) through breast milk. To reduce the risk
c. In chronic hepatitis B, HBsAg persists in the serum for more of transmission, antiretroviral therapy should be adminis-
than 6 months. Total anti-HBc is also present; HBeAg may or tered to the mother during her pregnancy and to the infant
may not be present, depending on the degree of disease pro- after birth. In addition, the mother should be advised not to
gression. Anti-HBe and anti-HBs are usually not present, but breastfeed her baby. b. Testing the infant’s serum for HIV
may have a delayed appearance in those individuals who even- antibody would yield confusing results. This is because the
tually recover. IgG HIV antibodies in the mother’s serum would pass
2. a. Many viruses can produce congenital abnormalities in through the placenta during the pregnancy and be detectable
an infant born to a mother infected during pregnancy. These in the infant’s serum. The result would be a false positive if
include cytomegalovirus, rubella virus, and varicella zoster the infant was not HIV-infected. c. Because of the problems
virus. The infant’s symptoms and mother’s history suggest in- associated with antibody testing, molecular methods are pre-
fection with rubella virus. b. Ideally, the mother would have ferred to make a diagnosis of HIV infection in infants
been tested at the time of her illness during her pregnancy for younger than 18 months of age. The preferred molecular test
rubella antibodies. Demonstration of rubella-specific IgM is a PCR that detects the presence of proviral HIV DNA in
antibody, seroconversion from negative to positive for rubella the infant’s peripheral blood mononuclear cells. Alternately,
antibody, or a four-fold rise in antibody titer would have in- a quantitative HIV RNA test could be performed on the
dicated an active rubella infection. However, because this was infant’s plasma.
not done, the mother could be tested for rubella-specific IgG
antibody, which would indicate if rubella exposure occurred.
Tests that measure the avidity of the IgG antibody can be per- 1. c 2. c 3. d 4. a 5. d 6. b 7. a
formed to distinguish between a recent infection (low avidity) 8. b 9. c 10. d 11. d 12. a 13. a 14. b
and a past infection (high avidity). c. The infant’s serum 15. d
should be tested for rubella-specific IgM antibody, preferably
with an IgM antibody capture enzyme immunoassay. IgM
antibodies, which cannot pass through the placenta, would
have been produced by the fetus as a result of active rubella
infection. IgG antibodies, on the other hand, are derived
1. a. The child could safely receive vaccines that do not con-
mainly from the mother’s serum as a result of passive transfer
tain a live component. These include the vaccines for hepatitis
through the placenta. Viral culture or RT-PCR should be used
B (a recombinant antigen); diphtheria and tetanus (toxoids);
to confirm positive IgM results.
pertussis, Haemophilus influenza b, and pneumococcus (subunit
vaccines); and polio, hepatitis A, and influenza (the prepara-
tions containing inactivated virus). b. The child could not re-
1. c 2. d 3. b 4. a 5. d 6. b 7. a ceive any live, attenuated vaccines because the organisms in
8. d 9. b 10. c 11. c 12. c 13. d 14. a these vaccines, although weakened, could not be controlled by
15. c the child’s immune system and could cause serious, dissemi-
nated infections. Such vaccines include those against the viral
diseases: measles, mumps, rubella, varicella, and rotavirus. The
nasal mist form of the influenza vaccine should also not be ad-
ministered because it contains live, attenuated virus. c. The
1. a. The physician could perform a rapid EIA for HIV anti- child could be protected against these infections by receiving
body. If the test is positive, the physician should order a fourth- regular injections of human serum immunoglobulin (gamma
generation ELISA, which simultaneously detects antibodies globulin), a preparation that has been pooled from the serum
to HIV-1, antibodies to HIV-2, and p24 antigen, to verify the of other individuals, and which contains numerous pre-made
result. b. If the fourth-generation ELISA was positive, a rapid antibodies. In addition, family members and close friends of
test for HIV-1 and HIV-2 antibodies should be performed to the child should make sure that they are up-to-date on their
confirm the results and distinguish between infection by the own immunizations. By preventing development of these dis-
two viruses. c. If it is determined that the woman truly has eases in themselves, they are ensuring that they cannot pass the
HIV infection, her CD4 T-cell counts should be monitored pe- pathogen along to the child. This concept, whereby protection
riodically to assess the effects of the virus on her immune sys- against infectious diseases is extended to others in a population,
tem. In addition, she should be placed on antiretroviral therapy is known as “community immunity” or “herd immunity.”
and the effectiveness of the therapy should be evaluated by pe- 2. a. Hepatitis A-specific human immune serum globulin,
riodically performing viral load assays to monitor the amount consisting of a high concentration of antibody to the hepatitis
of HIV RNA in her plasma. A virus, should be used to prevent the infection in anyone
who has dined at the restaurant recently. Because this prepa- need to be immunized with the hepatitis A vaccine. The vac-
ration is derived from individuals who have previously been cine, consisting of inactivated hepatitis A virus, would stimu-
exposed to hepatitis A, the antibodies are premade and pro- late an immune response against the viral antigens and the
vide immediate protection when they are administered within generation of memory lymphocytes that could quickly be
2 weeks after exposure to the pathogen. b. Although human reactivated in case of a later exposure.
immune serum globulin provides immediate protection, that
protection is only temporary because the antibody titers de-
cline over time, according to the half-life of the immunoglob-
ulin molecules. The half-life for IgG, which comprises the 1. a 2. c 3. d 4. b 5. d 6. c 7. b
majority of the preparation, is 23 days. To achieve long-term 8. d 9. d 10. c 11. c 12. b 13. c 14. a
immunity without actually acquiring the infection, you would 15. b
 Index
NOTE: A page number in regular type indicates where the topic is discussed in the text; an “f” following a page number indicates a figure; a “t” following a
page number indicates a table; a “b” following a page number indicates a box.

AAT. See Alpha1-antitrypsin instrumentation for, 149–150 introduction to, 4

ABO blood group principles of, 145–149, 146f, 147f, monoclonal types of, 72–73, 73f
antigens of, 266, 336 148f, 149f in passive immunization, 469–470, 469b,
discovery of, 20, 146–147 quality control and assurance of, 150 469t, 470f
in transfusion reactions, 222 AHase. See Anti-hyaluronidase in passive immunotherapy, 299–301, 330t
ABPA. See Allergic bronchopulmonary AIDS. See Acquired immunodeficiency disease to M. pneumonia, 362
aspergillosis AIH. See Autoimmune hepatitis for parasitic diseases, 392, 393t
Accelerated rejection, 267 AIT. See Allergy immunotherapy response curve of, 354b
Acetylcholine (ACH), 221, 350 AITD. See Autoimmune thyroid disease sensitization of, 146, 146f
ACPA. See Anticyclic citrullinated peptide ALL. See Acute lymphocytic leukemia of systemic lupus erythematosus, 241t, 242
antibody Allelic exclusion, 48, 70 in vaccines, 469–470, 469b, 469t, 470f
Acquired immunodeficiency disease (AIDS), Allergens, 213. See also Type I hypersensitivity Antibody arrays, 294
58, 397 Allergen-specific IgE testing, 219–221, 220f Antibody-dependent cell cytotoxicity (ADCC), 41,
clinical symptoms of, 438–439, 439b Allergic bronchopulmonary aspergillosis 41f, 221, 410
disease monitoring of, 446–450, 447b, 447f (ABPA), 398 Antibody-drug conjugate, 300–301
treatment of, 439–441, 440t Allergy immunotherapy (AIT), 218–219, 218b Antibody-mediated cytotoxic hypersensitivity. See
Activation phase, in Type I hypersensitivity, Alloantigen, 21 Type II hypersensitivity
215, 216t Allograft, 266, 274b Antibody response curve, 354b
Activation unit, of classical complement pathway, Allorecognition, 266–267, 267f Anticentromere antibodies, 242
94–95, 95f Allotype, of immunoglobulin, 64, 64f Anticyclic citrullinated peptide antibody (ACPA),
Active immunity, 455. See also Vaccines Alpha1-antitrypsin (AAT), 35t, 36–37 246, 247
features of, 472b Alpha-fetoprotein (AFP), 285–288, 286t Anticytokine therapy, 86–87
Active immunotherapy, 298–299 ALPS. See Autoimmune lymphoproliferative Anti-DNase B testing, 358–359
Acute graft-versus-host disease, 268. See also syndrome Antigen-antibody binding, 141–142, 141f, 142f
Graft-versus-host disease Alternative pathway Antigen-dependent phase, of B cells, 52, 53f
Acute lymphocytic leukemia (ALL), 200t, 309, of complement system, 96–97, 97f, 98f Antigenic concealment, 391, 405b
309b, 309f, 317t regulation of, 99, 101f Antigenic mimicry, 392, 405b
Acute-phase reactants, 34–37, 35t Amino acids, 173, 173t, 174f, 174t Antigenic shedding, 391–392, 405b
Acute rejection (AR), 267–268 Amplicon, 179–180, 181f, 449 Antigenic variation, 391, 405b
Acute rheumatic fever, 356 Amplification methods. See also Polymerase chain Antigen-independent phase, of mature B cell, 51
ADA deficiency. See Adenosine deaminase reaction Antigen presentation, 23–24
deficiency PCR in, 178–181, 179f, 180f, 181f Antigen-presenting cell (APC), 83f, 84, 295,
Adaptive immune system. See also B cells; probe amplification in, 183–184, 184f 350–351
Lymphocytes; Natural killer cells; T cells qPCR in, 181, 183f, 338, 354 in adaptive immune response, 53–54
antigen-presenting cells in, 53–54 signal amplification in, 184–185, 185f introduction to, 7, 19
B cell role in, 54–58, 56f, 57f transcription-mediated amplification in, in vaccination, 461
cell types of, 46–59 181–183, 183f Antigens
cytokines of, 83–85, 83f, 88b Analyte, 156 detection of bacterial infections by, 354
introduction to, 7–8, 7f, 8t Anaphylactic hypersensitivity. See Type I of Epstein-Barr virus, 420, 420t
responses to bacterial infection of, 366b hypersensitivity introduction of, 3
responses to tumor cells of, 295–296 Anaphylatoxin, 102 tumor-associated types of, 281–282, 282t
T cell role in, 53–54, 55f Anaphylaxis, 217 tumor-specific types of, 280–281, 282f, 282t,
Adaptive T regulatory cell (Tr1), 85 ANAs. See Antinuclear antibodies 283t
ADCC. See Antibody-dependent cell cytotoxicity ANCA. See Antineutrophil cytoplasmic Antigen shedding, 392
Adenosine deaminase (ADA) deficiency, 328f, antibody Antigen switching, 391
331–332 Anergy, 228, 234 Antihistone antibodies, 241, 241t
Adjuvant, 461 Angioedema, 217–218, 217f Anti-hyaluronidase (AHase), 359
introduction to, 20–21 Antagonism, of cytokines, 78–80, 79f Antimicrobial defense peptides (ADPs), 351
novel types of, 464 Anti-animal antibodies, 292, 292f Antineutrophil cytoplasmic antibody (ANCA),
Adoptive immunotherapy, 301–302, 301f, Antibodies. See also Immunoglobulin 248, 249–250, 249f, 250t
470–471, 472b anti-animal types of, 292, 292f Antinuclear antibodies (ANAs)
ADPs. See Antimicrobial defense peptides antineutrophil cytoplasmic types of, 249–250, detection methods of, 242–245, 243f, 244f, 245f
Affinity, 141, 141f 249f, 250t detection using C. luciliae, 244–245, 245f
AFP. See Alpha-fetoprotein antinuclear types of, 240–242, 241t indirect immunofluorescence of, 242–244,
Agammaglobulinemia, 327, 329–330, 329t deficiencies of, 329–330, 329t 243f, 244f
Age, as factor in vaccination, 460–461, 461b, detection of GAS by, 357–358 microsphere multiplex immunoassay in, 244
462f, 463f detection of H. pylori by, 361 Ouchterlony test for, 245, 245f
Agglutination reactions diversity of, 69–70 types of, 240–242, 241t
comparison of, 152b of dsDNA, 241 Antiphospholipid antibodies, 245
inhibition of, 149, 150f, 152b flow cytometry of, 272, 273f Antiretroviral therapy (ART), 439–440, 440t
Anti-RNP antibody, 241t, 242 Batch analyzers, 203–206, 204t, 205t, 206f Cascade induction, of cytokines, 78–80, 79f
Anti-streptokinase (ASKase), 359 B cells Category B infectious substances, 118, 119f
Anti-streptolysin O (ASO), 357–358, 359 adaptive immune response role of, 54–58, CBC. See Complete blood count
Antitoxins, 468 56f, 57f CCP. See Cyclic citrullinated peptide
APC. See Antigen-presenting cell antigen-independent phase of, 51 CD. See Cluster of differentiation
Apoptosis, 279, 279b cell comparison of, 12b CD4 T cells, 23f, 24–26, 26f, 27f, 28b, 438,
AR. See Acute rejection central tolerance of, 52 440t. See also T helper cells
Array analysis, 176–177, 178f comparison with T cells of, 59b enumeration in HIV monitoring,
ART. See Antiretroviral therapy differentiation of, 50–53, 51f, 53f 446–448, 447f
Arthropod vectors, 362–365, 363–364t, introduction to, 7, 8t CD8 T cell, 23f, 24, 25f, 28b, 58. See also
364f, 365f maturation of, 322b Cytotoxic T cell
Arthus reaction, 225–226, 225f mitogen of, 337 CD16 receptors, 40, 41f
ASKase. See Anti-streptokinase BCR/ABL gene rearrangement, 280–281, CD45 marker, 291
ASO. See Anti-streptolysin O 282f, 282t CDC. See Centers for Disease Control and
Aspergillus species, 400–401, 400f bDNA. See Branched DNA Prevention
Ataxia-telangiectasia (AT), 333t, 334 Behring, Emil von, 4, 467 CDC test. See Complement-dependent
Atopy, 214 Bence Jones, Henry, 64 cytotoxicity test
Attenuated vaccines, 458–459, 473b Bence Jones proteins, 312–314 cDNA. See Complementary DNA
Attenuation, 3 Benign prostatic hyperplasia (BPH), 289 CDR. See Complementarity-determining region
Atypical lymphocytes, 420–421, 421f Bevacizumab (Avastin), 72 CEA. See Carcinoembryonic antigen
Autoantibodies, 234 Bexxar. See Tositumomab Celiac disease, 254–255
Autoantigen, 21 Biohazardous material, 114, 115f Cell flow cytometry. See Flow cytometry
Autocrine action, 78, 78f Biological hazards, 113–118, 113t, 115f, 117b, Cell-mediated immunity (CMI), 351
Autograft, 266, 274b 118b disorder of, 337–338, 337t
Autoimmune diseases Biomarker profiling, 294 introduction to, 7
endogenous factors of, 236–237 Bloodborne pathogens, 117, 117b, 118b mature T cell role in, 48–50, 50f
environmental factors in, 236–237, 238f Blood specimen preparation and measuring, Centers for Disease Control and Prevention
epigenetic modifications in, 237 133–134, 133f, 134f (CDC), 36, 114, 218
etiology of, 234–237, 235f Body substance isolation (BSI), 114 estimation of HIV infections by, 434
genetics of, 235–236 Bone marrow in parasitic serology, 396–397
hormonal influence in, 236 biopsy of, 312–313, 313f, 340 study of vaccination by, 465–467
interacting factors in, 237, 238f introduction to, 8, 9f syphilis screening algorithm of, 377–378, 378f
molecular mimicry in, 236–237 lesions in, 313, 313f Central tolerance, 234–235, 235f
systemic type of, 237–250, 238t Bordet, Jules, 92b Centromere, 22f, 179f, 242–243, 243f
tissue trauma in, 236 Borrelia burgdorferi, 379. See also Lyme disease Cerebrospinal fluid (CSF), 378–379
Autoimmune hemolytic anemia, 223–224 Borrelia miyamotoi, 382–383 Ceruloplasmin, 35t, 37
Autoimmune hepatitis (AIH), 256 Botulism toxin, 350 Cetuximab (Erbitux), 72
Autoimmune liver diseases, 255–257 BPH. See Benign prostatic hyperplasia CGD. See Chronic granulomatous disease
Autoimmune lymphoproliferative syndrome Branched DNA (bDNA), 184–185, 185f, 448, 449 CH50 testing, 105–107, 106f
(ALPS), 334 Bruton’s tyrosine kinase (Btk) deficiency, 328f, Chain of infection, 113–114, 115f
Autoimmune thyroid disease (AITD) 329t, 330 Chain termination sequencing. See Sanger
of Grave’s disease, 250–253, 252f, 253t BSI. See Body substance isolation sequencing
of Hashimoto’s thyroiditis, 250–253, 252f, 253t Butterfly rash, 239, 240f Chancre, of syphilis, 372, 372f
treatment of, 252–253 Bystander lysis, 99 Chediak-Higashi syndrome, 328f, 334
Autoinflammatory disorders, 335–336 Chemical hazards, 118–120, 120f
Automatic sampling, in immunoassay, 203–206, C1 inhibitor (C1-INH), 97–99, 99t Chemical hygiene plan, 119–120
204t, 205t, 206f, 206t C3 glomerulopathies (C3G), 104 Chemiluminescent immunoassay (CLIA),
Avastin. See Bevacizumab C4-binding protein (C4BP), 99, 99t 163–164
Avidity, 141–142, 142f CALT. See Cutaneous-associated lymphoid tissue in HIV screening and detection, 442–444, 443f
index of, 395 Cancer. See also Tumor Chemokines, 46, 82–83, 82t
immunoediting in, 296–297, 297f Chemotaxins, 4, 102
Bacterial cell monitoring patients of, 285, 285f Chemotaxis, 38, 38f
culturing of, 353–354, 353f, 357, 357f, 362 passive immunotherapy in, 299–301, 300t Chickenpox, 411, 423–425, 424f
structure of, 349–350, 349f serum tumor markers in, 285–290, 286–288t Chimeric antigen receptor (CAR), 302
Bacterial infections tumor-specific antigens in, 280–281, 282f, 282t Chronic granulomatous disease (CGD), 328f,
antigen detection of, 354 viruses associated with, 281, 283t 334–335
in autoimmune disease, 236–237 Cancer antigen 125, 286t, 288–289 Chronic inflammatory demyelinating
evasion mechanisms in, 351–353, 352f Candida species, 401 polyneuropathy (CIDP), 468
extracellular virulence factors in, 350–351, CAP. See College of American Pathologists Chronic lymphocytic leukemia (CLL), 200t, 224,
350b, 350f Capillary transfer, in DNA analysis, 176, 176f 309, 317t
human-microbe relations in, 347–348 Capsular antigen, 349–350, 350b Chronic myelogenous leukemia (CML), 280–281,
immune defenses against, 351, 351b Capture assay, 160, 165b 282f, 282t
innate immunity in, 366b CAR. See Chimeric antigen receptor Chronic rejection, 268
laboratory diagnosis of, 353–354, 353f Carcinoembryonic antigen (CEA), 286t, 289 CIDP. See Chronic inflammatory demyelinating
molecular detection of, 354 Carcinoma, 279, 280–281, 283t, 285, 469t. See polyneuropathy
virulence factors in, 348–351, 349f, 350b, 350f also Cancer Class I MHC molecules
Bacteriophage, 72 Cardiopulmonary resuscitation (CPR), 120 comparison of, 28b
Basophil, 5–6, 6f, 13b CART. See Combination antiretroviral therapy introduction to, 22–23, 22f, 23f
 in NK cell action, 40, 41f classical pathway of, 92–96, 94f, 95f, 96f, 98f Cytotoxic T cell (Tc), 48, 54, 55f, 56f. See also
role of, 23–26, 25f activation unit of, 94–95, 95f CD8 T cell
Class II invariant chain peptide (CLIP), assays for, 105–107, 106f, 107t in adaptive immune response, 295–296
25–26, 26f recognition unit of, 93, 94f in viral infection, 409–411, 410f
Class II MHC molecules regulation of, 97–99, 100f
comparison of, 28b deficiencies of, 103–104, 103t, 336 DAF. See Decay-accelerating factor
introduction to, 22f, 23, 23f laboratory assays for, 104–107, 105f, Dark-field microscopy, of syphilis, 373
role of, 23–26, 26f 106f, 107t DAT. See Direct antiglobulin test
Classical pathway, of complement system, 92–96, lectin pathway of, 96, 98f ddNTP. See dideoxyribonucleotide
94f, 95f, 96f, 98f regulation of, 97–99, 100f Decay-accelerating factor (DAF), 99, 99t, 102t
regulation of, 97–99, 100f membrane attack complex of, 95–96, 96f Defensin, 39
Class switching, 70–71 pathways of, 92–97 Delayed hypersensitivity. See Type IV
CLIA. See Chemiluminescent immunoassay; proteins of, 93t hypersensitivity
Clinical Laboratory Improvements receptors of, 100–102, 101–102t, 102f Delta check, 125–126
Amendments regulation of, 97–100, 99t, 100f, Dendritic cell, 7, 13b
Clinical and Laboratory Standards Institute 101–102t, 101f Deoxyribonucleic acid (DNA)
(CLSI), 121, 127 terminal components of, 100, 101–102t in antinuclear antibodies, 240–241, 241t
Clinical applications Complete blood count (CBC), 126b, 240, characteristics of, 169–170, 170f, 171f
of flow cytometry, 197–203, 198–199t, 312, 336 next generation sequencing of, 186–189
200–202t Complex-mediated hypersensitivity. See Type III pyrosequencing of, 186, 188f
of tumor markers, 303b hypersensitivity replication of, 170–172, 172f
Clinical Laboratory Improvements Amendments Compound dilutions, 136, 136f sequencing of, 185–189, 185f, 186f, 187f, 188f
(CLIA), 121, 126–127, 126b, 204–206, Confirmatory tests, for immune dysfunction, Deoxyribonucleotidyl transferase (TdT), 52
206t, 354 337–338, 337t, 338f Department of Transportation (DOT),
CLIP. See Class II invariant chain peptide Congenital defects, of phagocytes, 334–335 117–118, 119f
CLL. See Chronic lymphocytic leukemia Congenital rubella syndrome (CRS), 425 Diapedesis, 4, 37, 38f
Clonal deletion, of T cells, 48 Congenital syphilis, 373, 378 Dideoxyribonucleotide (ddNTP), 185–186, 185f.
Clonal selection hypothesis, 70 Conidia, 398 See also Deoxyribonucleic acid
Clostridium difficile, 350, 350f Constant region, of immunoglobulin, 64, 64f Diffuse large B-cell lymphoma (DLBCL), 311
CLOtest, 360, 360f Contact dermatitis, 227, 227f DiGeorge anomaly, 221, 328f, 333–334, 333t
CLP. See Common lymphoid precursor Coombs reagent, 147, 147f Dilutions, 134–137, 136f
CLR. See C-type lectin receptor Courier-delivered specimen transport, 118 Direct agglutination, 147, 148f, 152b
CLSI. See Clinical and Laboratory Standards Cowpox, 456–457, 456f Direct allorecognition, 266–267, 267f
Institute CPR. See Cardiopulmonary resuscitation Direct antiglobulin test (DAT), 224
CLT. See Cytolytic T cell CR1. See Complement receptor type 1 Direct immunofluorescent assay, 162, 162f, 165b
Cluster of differentiation (CD), 7, 8t, 316 C-reactive protein (CRP), 35–36, 35t, 81, Direct microscopy, of syphilis, 373, 384b
CMI. See Cell-mediated immunity 240, 351 DLBCL. See Diffuse large B-cell lymphoma
CML. See Chronic myelogenous leukemia CREST syndrome, 242, 243 DNA. See Deoxyribonucleic acid
CMP. See Common myeloid precursor Crithidia lucilae, 244–245, 245f DOT. See Department of Transportation
CMV. See Cytomegalovirus Crohn’s disease, 73, 80, 81 Double-negative thymocyte, 47–48, 47f
Coccidioides immitis, 403–404 Crossmatch test, 272 Double-positive thymocyte, 48, 49f
Cold agglutinin, 224, 362 Cross-reactivity, 456 Double-stranded DNA (dsDNA) antibodies,
College of American Pathologists (CAP), 127 CRP. See C-reactive protein 241, 241t
Colony stimulating factor (CSF), 85, 299 CRS. See Congenital rubella syndrome Drosophila fruit fly, 33
Combination antiretroviral therapy (CART), 440 Cryoglobulin, 314–315 Drug-resistance testing, 449–450
Commensalism, 348, 353, 390t, 399t Cryptic antigens, 236 dsDNA antibodies. See Double-stranded DNA
Common lymphoid precursor, 4, 5f Cryptococcosis, 399 antibodies
Common myeloid precursor, 4, 5f Cryptococcus neoformans, 401–403, 402f Dual-parameter dot plot, 196, 196f, 197f, 198f
Common tumor markers, 285–290, 286–288t CSF. See Cerebrospinal fluid; Colony stimulating Dye-terminator sequencing, 185–186, 187f, 188f
Common variable immunodeficiency (CVI), 328f, factor
329t, 330–331 C-type lectin receptor (CLR), 34, 399 Early B-cell factor (EBF), 51
Community immunity, 465, 466f Cutaneous-associated lymphoid tissue (CALT), Ebola virus, 81f
Competitive enzyme immunoassay, 156, 157f, 9, 10 EBV. See Epstein-Barr virus
159, 165b CVI. See Common variable immunodeficiency Ectoparasites, 389
Complementarity-determining region (CDR), Cytogenetics, 293 Edelman, Gerald, 63, 63f
65, 65f Cytokines EDTA. See Ethylene diaminetetraacetic acid
Complementary DNA (cDNA), 180, 414b in adaptive immune response, 83–85, 83f, EGTA. See Ethylene glycol tetraacetic acid
Complementary sequences, of DNA, 88b Ehrlich, Paul, 69–70, 92b
169–170, 171f in innate immune response, 80–83, 82f, EIA. See Enzyme immunoassay
Complement control protein (CCP), 392 82t, 88b Electrical hazards, 120
Complement-dependent cytotoxicity (CDC) test, introduction to, 6, 78–80, 78f, 79f Electropherogram, 186, 188f
269–271, 270f major examples of, 80, 80t Electrophoretic techniques, 144–145, 145f
Complement receptor type 1 (CR1), 99, 99t, in passive immunotherapy, 299 ELISA. See Enzyme-linked immunosorbent assay
101t pleiotropy of, 78 EM. See Erythema migrans
Complement system, 35t, 36 T regulatory cell association with, 84–85 ENAs. See Extractable nuclear antigens
activation of, 102–103 Cytokine storm. See Hypercytokinemia Enbrel. See Etanercept
alternative pathway of, 96–97, 97f, 98f Cytolytic T cell (CLT), 437 Endocrine action, 78–78f
regulation of, 99, 101f Cytomegalovirus (CMV), 237, 411, 422–423 Endocytosis, of virus, 409, 409f
Endogenous factors, in autoimmune diseases, Florida panther, 19f GPI. See Glycosylphosphatidylinositol
236–237 Flow cytometry, 58 Graduated pipettes, 133, 133f, 134f
Endogenous pyrogen, 80 antibody detection by, 272, 273f Grafts
Endoplasmic reticulum (ER), 24–25, 25f, 26f clinical applications of, 197–203, 198–199t, rejection of, 275b
Endotoxin, 350–351 200–202t types of, 274t
End-point method, 144, 150 data acquisition and analysis in, 195–197, 196f, Graft-versus-host disease (GVHD), 264, 268,
Enterotoxigenic E. coli (ETEC), 349 197f, 198f 275b, 332–333, 422, 468
Entrocytes, 438 histogram of, 337, 338f Granulocyte colony stimulating factor (G-CSF),
Envelope protein, 409, 409f in HIV monitoring, 447–448, 447b 85, 86f
Environmental factors, in autoimmune diseases, immunophenotyping by, 316–317, 316t, 317t Granulocyte-macrophage colony stimulating
236–237, 238f instrumentation of, 194–195, 195f, 196f factor (GM-CSF), 85, 86f
Enzyme immunoassay (EIA), 158–161, 161f sample preparation for, 197 Granulomas, 226–227
in detection of hepatitis viruses, 415 Fluorescence in situ hybridization (FISH), Granulomatosis with polyangiitis. See Wegener’s
in fungal infections, 401–402 177–178, 179f, 293, 314, 321–322, 322f granulomatosis
for Lyme disease, 381, 385b Fluorescence polarization immunoassay (FPIA), Granzymes, 40, 41f
Enzyme-linked immunosorbent assay (ELISA), 162–163, 163f, 165b Grave’s disease
87, 105, 159–160, 272, 337, 376, Fluorescent antibody testing, of syphilis, 373 clinical manifestations of, 252, 252f
376f, 392 Fluorescent antinuclear antibody (FANA) testing, etiology of, 250–251, 252f
in antinuclear antibody detection, 244 242–244, 243f, 244f immunopathology of, 252, 252f
in bacterial detection, 354 Fluorescent immunoassay (FIA), 161–163, 162f, laboratory diagnosis of, 253, 253t
in HIV screening and diagnosis, 442–444, 443f 163f treatment of, 252–253
in H pylori detection, 360–361 Fluorescent treponemal antibody absorption Group A Streptococci (GAS)
in rheumatoid arthritis diagnosis, 247 (FTA-ABS) test, 375 classification and structure of, 354–355, 355f
in virus detection, 421, 425, 427–429 Fluorochrome, 194 clinical manifestations of, 355–356, 356f
Eosinophil, 13b, 216, 216t Follicle-stimulating hormone (FSH), 289 detection of antibodies to, 357–358
IgE recruitment of, 69 Food and Drug Administration (FDA), 394, detection of antigens of, 357, 358f
introduction to, 5, 5f 396–397, 468 laboratory diagnosis of, 357–359, 357f, 358f
Epidemic typhus, 363, 364t Forward-angle light scatter (FSC), 194–195, 195f, sequelae of, 356–357
Epigenetic modifications, of self-antigens, 237 196f virulence factors of, 355, 355f
Epitope, 18–19, 19f FPIA. See Fluorescence polarization immunoassay Gummas, of syphilis, 372–373, 372b
Epitope spreading, 237 Fragment antigen binding (Fab), 63 Gut microbiome, 347–348
EPO. See Erythropoietin Fragment crystallizable (Fc), 63 GVHD. See Graft-versus-host disease
Epsilon (ε) chain. See Heavy chain French-American-British (FAB) Cooperative
Epstein-Barr virus (EBV), 237, 283t, 311, 411, Group, 308 HAART. See Highly active antiretroviral therapy
420–422, 420t, 421f, 421t Freund’s complete adjuvant (FCA), 461 HAE. See Hereditary angioedema
EQA. See External quality assessment FSC. See Forward-angle light scatter Hairy cell leukemia (HCL), 200t, 309–310, 310f,
Equivalence, zone of, 142, 142f FSH. See Follicle-stimulating hormone 317t
ER. See Endoplasmic reticulum FTA-ABS test. See Fluorescent treponemal HAMA. See Human anti-mouse antibodies
Erbitux. See Cetuximab antibody absorption test Hand hygiene, 114, 116f
Erythema migrans (EM), 379–380, 380f Fungal immunology Haplotype, 22, 264, 264f
Erythropoietin (EPO), 85, 86f characteristics of fungi in, 398 Haptens, 19–20, 20f
Etanercept (Enbrel), 86 immune responses in, 398–400 Haptoglobin, 35t, 37
ETEC. See Enterotoxigenic E. coli introduction to, 397–398 Hashimoto’s thyroiditis
Ethylene diaminetetraacetic acid (EDTA), 197 laboratory diagnosis in, 400, 401b clinical manifestations of, 251–252
Ethylene glycol tetraacetic acid (EGTA), 105 mycotic infection classification in, 398, 399t etiology of, 250–251, 252f
Examination variables, 123–125, 124f, 125f pathogens in, 400–404, 400f, 402f immunopathology of, 251–252
Exotoxins, 350–351 laboratory diagnosis of, 253, 253t
External defense system, 32–33, 32f, 42b Gamma ( ) globulin, 62, 62f. See also treatment of, 252–253
External quality assessment (EQA), 125 Immunoglobulin HAV. See Hepatitis A virus
Extracellular virulence factors, 350–351, 350b, GAS. See Group A Streptococci HBV. See Hepatitis B virus
350f Gastric carcinoma, 359–361 HCC. See Hepatocellular carcinoma
Extractable nuclear antigens (ENAs), 241 Gating, in flow cytometry, 195–197, 196f, 197f, hCG. See Human chorionic gonadotropin
198f HCL. See Hairy cell leukemia
Fab. See Fragment antigen binding G-CSF. See Granulocyte colony stimulating factor HCV. See Hepatitis C virus
FAB Cooperative Group. See French-American- Gel electrophoresis, 176, 176f, 177f HDN. See Hemolytic disease of newborn
British Cooperative Group Geminal center. See Secondary follicle HDV. See Hepatitis D virus
Factor H, 99, 99t, 101f Genetic code, 173, 173t, 174f Heavy chain, 62–65, 63f, 64f
Factor I, 99, 99t, 101f Genetics diseases of, 315
FANA testing. See Fluorescent antinuclear of autoimmune diseases, 235–236 gene rearrangement in, 70–71, 71f, 322, 323f
antibody testing cancer biomarkers of, 293 Helicobacter pylori, 359–361, 360f
Fc. See Fragment crystallizable chromosomal abnormalities of, 321–322, 321t, Helminths, 389
FCA. See Freund’s complete adjuvant 322b, 322f, 323f Hemagglutination inhibition, 149, 150f, 152b
FDA. See Food and Drug Administration hematologic malignancies effect on, 307–308 Hematologic malignancies
FIA. See Fluorescent immunoassay Giardia lamblia, 396, 396f, 397 cell properties of, 307
Fibrinogen, 35t, 37 Glycosylphosphatidylinositol (GPI), 202–203 classification of, 307
Fire and explosive hazard, 120–121, 121f GM-CSF. See Granulocyte-macrophage colony genetic changes in, 307–308
FISH. See Fluorescence in situ hybridization stimulating factor markers for, 316, 316t
Flocculation, 374 Goodpasture’s syndrome, 259–260 Hematopoietic growth factors, 85–86, 86f, 299
Hematopoietic stem cell (HSC), 4, 5f Human immunodeficiency virus (HIV), 58, 114 IL-2. See Interleukin-2
Hemolytic disease of newborn (HDN), 222–223, characteristics of, 435–437, 435f, 436t, 437f IL-4. See Interleukin-4
223f circulating recombinant forms of, 434 IL-6. See Interleukin-6
Hemolytic titration assay, 105–107, 106f clinical symptoms of, 438–439, 439b, 440t IL-10. See Interleukin-10
Hemolytic uremic syndrome (HUS), 104 disease monitoring of, 446–450, 447b, 447f IM. See Infectious mononucleosis
Hepatitis A virus (HAV), 411–414, 412t drug-resistance testing of, 449–450 Immature B cell, 51, 51f, 52
Hepatitis B virus (HBV), 411, 412t, 415–418, ELISA for, 442–445 Immediate hypersensitivity. See Type I
416f, 417f flow cytometry of, 447–448, 447f hypersensitivity; Type III hypersensitivity
association with cancer of, 283t immunologic manifestations of, 437–438 Immune adherence, 99
Hepatitis C virus (HCV), 411, 413t, 418–419 laboratory testing for, 441 Immune organs
association with cancer of, 283t PCR detection of, 448–449 primary lymphoid organs of, 8–9, 9f
Hepatitis D virus (HDV), 411, 413t, 418 quantitative viral load assay of, 448–449 secondary organs of, 9–11, 10f, 11f
Hepatitis E virus (HEV), 411, 413t, 414–415 rapid tests for, 444–445 Immune responses
Hepatocellular carcinoma (HCC), 283t, 285–288 replication of, 436–437, 437f assessing of new methods in, 465
HER2. See Human epithelial growth factor screening and diagnosis of, 441–446, 442f, against bacterial infections, 351,
receptor 2 443f, 444b, 446f 351b, 366b
Herceptin. See Trastuzumab serological tests for, 442–444, 443f, 444b dysfunction of, 336–340, 337t, 338f, 339f
Herd immunity, 465, 466f structural genes of, 435–436, 435f, 436t in fungal immunology, 398–400
Hereditary angioedema (HAE), 104 testing algorithms for, 441–442, 442f against parasites, 390–391, 390t
Herpes viruses testing of infants for, 450 against tumor cells, 294–296, 295f
association with cancer of, 283t transmission of, 434–435 viral escape mechanisms of, 411, 413b
cytomegalovirus of, 422–423 treatment and prevention of, 439–441, 440t against viral infection, 409–411, 410b, 410f
Epstein-Barr virus in, 420–422, 420t, 421f, virus composition of, 435, 435f Immune thrombocytopenia (ITP), 468
421t western blotting of, 445–446, 446f Immunoassay
introduction to, 419–420 Human leukocyte antigen (HLA), 21–22, 22f, automation of, 203–205, 204t, 205t
serological tests for, 419–425, 421t 236, 264–265, 264f, 265t accuracy of, 206, 206f
varicella-zoster virus in, 411, 423–425, 424f antibody screen of, 272, 273f analytic sensitivity and specificity of,
Heteroantigen, 21 diseases of, 27t 206, 206f
Heterogeneous enzyme immunoassay, 157, genotyping of, 270–271, 271b, 271f reference interval of, 206
159–160 phenotyping of, 269–270, 270f reportable range of, 206
Heterophile, 292, 292f Human papilloma virus (HPV), 283t for circulating tumor markers, 291–293, 292f
antibody of, 420 Human T-cell Lymphotrophic Viruses (HTLV), Immunoblotting, for Lyme disease, 381–382,
antigen of, 21 428–429 381f, 385b
HEV. See Hepatitis E virus Humoral immunity Immunochromatography, 161, 161t, 165b
Hexose monophosphate (HMP), 40f B cell differentiation in, 52, 53f Immunodeficiencies
High-dose hook effect, 291–292, 292f disorders of, 337–338, 337t confirmatory tests of, 337–338, 337t, 338f
Highly active antiretroviral therapy (HAART), 440 theory of, 4 in newborns, 338, 339f
Hinge region, 64–65 HUS. See Hemolytic uremic syndrome of Category 1, 331–332, 333t
Histamine, 215, 216t Hybridization methods, 175–178, 176f, 177f, of Category 2, 332–334, 333t
Histocompatibility systems, 264–266, 264f, 265t 178f, 179f of Category 3, 329-330, 329t
Histocompatibility testing, 269–273, 270f, 271f, Hybridoma, 72, 73f of Category 4, 334
273f Hydrogen peroxide (H2O2), 40f of Category 5, 334–335
Histoplasma capsulatum, 403 Hyperacute rejection, 266, 267 of Category 6, 335
Histoplasmosis, 399 Hypercytokinemia, 80, 81f of Category 7, 335–336
HIV. See Human immunodeficiency virus Hypersensitivity. See Type I hypersensitivity; Type of Category 8, 336
HLA. See Human leukocyte antigen II hypersensitivity; Type III of Category 9, 336
HMP. See Hexose monophosphate hypersensitivity; Type IV hypersensitivity screening tests of, 336–337
H2O2. See Hydrogen peroxide Hypersensitivity pneumonitis, 227 Immunodiffusion techniques, 143–144, 144f,
Hodgkin lymphoma (HL), 310–311, 310f, 317t Hyphae, 400 145f, 146t
Homogeneous enzyme immunoassay, 157, 159f, Immunoediting, 296–297, 297f
160, 165b IATA. See International Air Transport Immunofixation electrophoresis (IFE), 144–145,
Hormonal influence, in autoimmune disease, 236 Association 145f, 146t, 340
Host and parasite interactions, 390–391, IBD. See Inflammatory bowel disease in immunoproliferative diseases, 318–321,
390t, 405b Ibritumomab tiuxetan (Zevalin), 73 318f, 319b, 319f, 320f
bacterial evasion mechanisms of, Idiotype, of immunoglobulin, 64, 64f Immunofluorescence assay (IFA)
351–353, 352f IFA. See Immunofluorescence assay in antinuclear antibody detection, 242–244,
HPV. See Human papilloma virus IFE. See Immunofixation electrophoresis 243f, 244f
HSC. See Hematopoietic stem cell IFR-1. See Interferon regulatory factor 1 for Lyme disease, 381, 385b
HSIG. See Human immune serum globulin IFR-8. See Interferon regulatory factor 8 using C. lucilae, 244–245, 245f
HTLV. See Human T-cell Lymphotrophic Viruses IgA. See Immunoglobulin A Immunogenicity, 17
Human anti-mouse antibodies (HAMA), 469 IgD. See Immunoglobulin D influencing factors of, 460–461, 461b,
Human chorionic gonadotropin (hCG), IgE. See Immunoglobulin E 462f, 463f
287t, 289 IgG. See Immunoglobulin G Immunoglobulin, 41, 74b. See also Antibodies
Human epithelial growth factor receptor 2 IgM. See Immunoglobulin M classes of, 74b
(HER2), 282–283 Ii. See Invariant chain constant region of, 64, 64f
Human Genome Project, 186–189 IIF assay. See Indirect immunofluorescent evaluation of, 317, 339–340
Human immune serum globulin (HSIG), assay fold of, 65, 65f
467–468 IL-1. See Interleukin-1 genes for, 70–72, 71f
 heavy chain of Immunotoxin, 300–301 Isohemagglutinin, 222
diseases of, 315 Impetigo, 355–356, 356f Isolated IgG subclass deficiency, 328f, 329t, 331
gene rearrangements in, 70–71, 71f, IMWG. See International Myeloma Working Isotypes, of immunoglobulin, 64, 64f
322, 323f Group ITP. See Immune thrombocytopenia
structure of, 62–65, 63f, 64f Inactivated vaccines, 459, 473b iTregs. See Induced Tregs
hinge region of, 64–65 Indigenous microbiota, 347–348 IUIS. See International Union of Immunologic
light chains of, 62–64 Indirect agglutination, 147–148, 149f, 152b Societies
tetrapeptide structure of, 62–64, 63f Indirect allorecognition, 267, 267f Ixodes tick, 379, 382
variable region of, 64, 64f Indirect immunofluorescent (IIF) assay, 162,
Immunoglobulin A (IgA), 66t, 67–68, 68f, 74b 162f, 165b Janeway, Charles, 33
in bacterial evasion, 351–353, 352f in antinuclear antibody detection, 242–244, Janus kinase-3 deficiency, 328f
deficiency of, 328f, 329t, 330 243f, 244f Jenner, Edward, 3
Immunoglobulin D (IgD), 52, 66t, 68–69, 74b Induced Tregs (iTregs), 84 Joining chain, 67
Immunoglobulin E (IgE), 66t, 69, 69f, 74b, Infectious mononucleosis (IM), 21, The Joint Commission (TJC), 121, 127
391, 391b 420–421, 421f
testing for, 219–221, 220f Inflammasome, 335–336 Kappa (κ) chain, 64
in type I hypersensitivity, 213b, 214–215, Inflammation, 37, 38f Karyotype analysis, 293
215f, 230b Inflammatory bowel disease (IBD), 85 Killer cell immunoglobulin-like receptors (KIRs),
Immunoglobulin G (IgG), 41, 65–66, 66f, 66t, Infliximab (Remicade), 86 40, 266
67f, 74b Influenza A, 35f, 80, 460
antibody response curve of, 354b INF-α/β. See Interferon-α/β Labeled assays, 156–157, 157f, 158f
in fungal infections, 404 INF- . See Interferon-gamma Laboratory diagnosis
in parasitic serodiagnosis, 394–396, 395t Innate immunity of autoimmune thyroid diseases, 253, 253t
subclass deficiency of, 328f, 329t, 331 acute-phase reactants of, 34–37, 35t of bacterial infections, 353–354, 353f
in type II or type III hypersensitivity, 221–222, bacterial infections in, 366b of celiac disease, 255
225, 230b cytokines in, 80–83, 82f, 82t, 85, 88b of complement system, 104–107, 105f,
Immunoglobulin M (IgM), 66–67, 66f, 66t, 67f, external defense system of, 32–33, 32f, 42b 106f, 107t
74b, 394–396, 395t inflammation in, 37, 38f in fungal immunology, 400, 401b
antibody response curve of, 354b internal defense system of, 33–42, 42b of Goodpasture’s syndrome, 259–260
in fungal infections, 404 natural killer cells in, 39–41, 41f of Grave’s disease, 253, 253t
in type II or type III hypersensitivity, 221–222, pathogen recognition receptors of, 33–34, of Group A streptococci, 357–359, 357f, 358f
225, 230b 34f, 34t of Hashimoto’s thyroiditis, 253, 253t
Immunohistochemistry, of tumors, phagocytosis in, 37–39, 39f, 40f of HIV, 441–446, 442f, 443f, 444b, 446f
290–291, 291b response against tumor cells of, 294–295, 295f of immune dysfunction, 336–340, 337t,
Immunologic diversion, 392, 405b response against viral infection of, 338f, 339f
Immunologic subversion, 392, 405b 409–411, 410f of immunoproliferative diseases
Immunologic tolerance, 234–235, 235f Integrin, 82 by flow cytometry, 316–317, 316t, 317t
Immunopathology Interacting factors, in autoimmune diseases, of genetic and chromosomal abnormalities,
of Goodpasture’s syndrome, 259 237, 238f 321–322, 321t, 322b, 322f, 323f
of Grave’s disease, 252, 252f Interferon-gamma (INF- ), 40, 48–50, by immunofixation electrophoresis,
of Hashimoto’s thyroiditis, 251–252 50f, 84 318–318f, 319b, 319f, 320f
of myasthenia gravis, 258, 258f Interferon regulatory factor 1 (IFR-1), 81 by serum free light chain analysis, 321
of rheumatoid arthritis, 246 Interferon regulatory factor 8 (IFR-8), 51 by serum protein electrophoresis, 318–321,
of systemic lupus erythematosus, Interferons, in viral infection, 410b 318f, 319b, 319f, 320f
239, 239b Interferon-α/β (INF-α/β), 83 of multiple sclerosis, 257–258
of type I diabetes mellitus, 254 Interleukin-1 (IL-1), 35, 36, 80, 80t of myasthenia gravis, 258, 258f
Immunophenotyping, 194 Interleukin-2 (IL-2), 84 quality control in, 123–124, 124f, 125f
by flow cytometry, 316–317, 316t, 317t Interleukin-4 (IL-4), 84 of rheumatoid arthritis, 247–248
Immunoproliferative diseases, laboratory Interleukin-6 (IL-6), 35, 81, 82f of systemic lupus erythematosus, 240
evaluation of Interleukin-10 (IL-10), 84 of T helper cells, 58
by flow cytometry, 316–317, 316t, 317t Internal defense system. of tumors, 290–294, 291b, 292f
of genetic and chromosomal abnormalities, action of NK cells in, 39–41, 41f of type I diabetes mellitus, 254
321–322, 321t, 322b, 322f, 323f acute-phase reactants of, 34–37, 35t of viral infection, 411
by immunofixation electrophoresis, 318–321, inflammation of, 37, 38f of Wegener’s granulomatosis, 248
318f, 319b, 319f, 320f pathogen recognition receptors of, 33–34, 34f, Laboratory hazards, 113–121, 113t
by serum free light chain analysis, 321 34t, 35f Lactobacillus species, 348
by serum protein electrophoresis, phagocytosis in, 37–39, 39f, 40f LAD. See Leukocyte adhesion deficiency
317–318, 318f Internal Laboratory Quality Improvement Form, Lambda (λ) chain, 64
Immunoprophylaxis, 455 122–123, 123b Landsteiner, Karl, 20, 20f
Immunosubtraction, 319, 320f International Air Transport Association (IATA), Late phase, of Type I hypersensitivity,
Immunosuppressive agents, 268–269 117–118, 119f 215–216, 216t
Immunosuppressive effects, 468 International Myeloma Working Group Lateral flow immunochromatography (LFA), 354,
Immunosurveillance, 294 (IMWG), 312 357, 358f. See also Rapid antigen detection
Immunotherapy International Union of Immunologic Societies system
adoptive types of, 470–471 (IUIS), 329 LCR. See Ligase chain reaction
monoclonal antibodies in, 469–470, 469b, Invariant chain (Ii), 24–25, 26f Lean system, 127
469t, 470f In vivo skin test, 219, 219f Lectin pathway, 96–99, 98f, 100f
in tumor treatment, 297–302, 300t, 301f Isograft, 266 Leishmania, 392
Leucine-rich repeat (LRR), 34 MBL. See Mannose binding lectin MS. See Multiple sclerosis
Leukemia, 308–310, 309f, 310f MCP. See Membrane cofactor protein Mtb. See Mycobacterium tuberculosis
cytogenetic characteristics of, 312t, 321 M-CSF. See Macrophage colony stimulating factor Mucosal-associated lymphoid tissue (MALT), 9,
surface markers for, 316–317, 317t Measles. See Rubeola virus 9f, 10, 311
Leukocyte adhesion deficiency (LAD), 328f, 335 Medical radioactive waste disposal, 120 Multiple myeloma, 312–314, 313f, 317t
Leukocytes, 4–6. See also Lymphocytes Melanoma antigen gene (MAGE), 282 Multiple sclerosis (MS), 257–258
surface markers of, 197–203, 198–199t, Membrane attack complex (MAC), 93, 95–96, 96f Mumps virus, 427–428, 427f
200–202t Membrane cofactor protein (MCP), 99, 99t Mutation, of nucleic acid sequence,
Leukotrienes (LT), 215, 216t Membrane inhibitor of reactive lysis (MIRL), 173–175, 174t
Levey-Jennings chart, 124, 124f 100, 102t Mutualism, 348
LFA. See Lateral flow immunochromatography Memory B lymphocytes, 461b Myasthenia gravis (MG), 258–259, 258f
LH. See Luteinizing hormone Memory cells, 10 Mycelial fungi, 398
Ligase chain reaction (LCR), 183–184, 184f in immunologic memory, 461b Mycobacterium tuberculosis (Mtb), 226–227,
Light chain, 62–64, 63f, 64f, 65f Messenger ribonucleic acid (mRNA), 70, 71f, 228, 353, 353f
rearrangements of, 71–72, 71f 172–173, 174f Mycoplasma pneumoniae, 353, 361–362
Lipopolysaccharide layer (LPS), 349–350, 349f Metchnioff, Elie, 4 Mycosis, 397
Live vaccines, 458–459 MG. See Myasthenia gravis Mycotic infections, classification of, 398, 399t
Long-term nonprogressors (LTNP), 438 MGUS. See Monoclonal gammopathy of Myeloid plasmacyoid leukemia, 200t
LPS. See Lipopolysaccharide layer undetermined significance Myeloid precursor, common (CMP), 4, 5f
LRR. See Leucine-rich repeat mHA. See Minor histocompatibility antigen Myeloperoxidase (MPO), 40f
LT. See Leukotrienes MHC. See Major histocompatibility complex Myeloproliferative studies, common markers for,
LTNP. See Long-term nonprogressors MIA. See Microsphere multiplex immunoassay 197–203, 200–202t
Luminescence, 186, 188f Microarrays, in cancer diagnosis, 293–294
Luteinizing hormone (LH), 289 Microbial infections. See Bacterial infections NADP. See Nicotinamide adenine dinucleotide
Lyme disease Microbiocidal defects, 335 phosphate
characteristics of, 379, 379f Microbiome, 347–348 NAT. See Nucleic acid testing
diagnosis of, 380–382, 381f, 385b Micropipettes, 133–134, 135f Natural killer (NK) cells, 83
immune response to, 380 Microscopic visualization, of bacterial cells, action of, 39–41, 41f
PCR screening for, 382, 385b 353, 353f cell comparison of, 12b
stages of, 379–380, 380f Microsphere multiplex immunoassay (MIA), 244 introduction to, 7–8, 8t
treatment of, 382 Minor histocompatibility antigen (mHA), 265 in viral infection, 409–411, 410f
Lymph node, 10, 11f MIRL. See Membrane inhibitor of reactive lysis Negative predictive value, 137
Lymphoblast cells, 309, 309b, 309f Mitogen, 337 Negative selection, of double positive T cells,
Lymphocytes Mixed lymphocyte reaction (MLR), 266–267, 267f 48, 49f
cell comparison of, 12b, 13b Molecular analysis. See also Laboratory diagnosis; Neglected tropical diseases (NTDs), 389
Identification of, 58 Polymerase chain reaction Neisseria gonorrhoeae, 349
introduction to, 7, 7f of bacterial infections, 354, 360–361, Neoplasm, 279
types of, 46–59 362, 365 Nephelometry, 143, 143f, 146t
Lymphoid precursor, common (CLP), 4, 5f of cancer diagnosis, 293–294 Neutrophils, 4, 13b
Lymphoma, 310–312, 310f DNA sequencing in, 185–189, 185f, 186f, Newborns
cytogenetic characteristics of, 312t, 321 187f, 188f hemolytic disease of, 222–223, 223f
surface markers of, 316–317, 317t fluorescence in situ hybridization of, HIV testing of, 450
Lymphoproliferative studies, common markers of, 177–178, 179f vaccination of, 460–461, 462f, 463f
197–203, 200–202t hybridization methods of, 175–178, 176f, 177f, Next generation sequencing (NGS), 186–189,
178f, 179f 294, 322
MAC. See Membrane attack complex PCR in, 178–181, 179f, 180f, 181f NHL. See Non-Hodgkin lymphoma
Macroglobulin. See Immunoglobulin M probe amplification in, 183–184, 184f Nicotinamide adenine dinucleotide phosphate
Macrophage colony stimulating factor (M-CSF), quantitative PCR in, 181, 183f (NADP), 39
85, 86f signal amplification in, 184–185, 185f Nisonoff, Alfred, 63–64
Macrophages, 6, 13b solution hybridization of, 177, 179f NK cells. See Natural killer cells
MAGE. See Melanoma antigen gene strand cleavage methods of, 175, 175f NLPHL. See Nodular lymphocytic-predominant
Major histocompatibility complex (MHC) transcription-mediated amplification in, Hodgkin lymphoma
antigens of, 264–265, 264f, 265t 181–183, 183f NNRTI. See Nucleoside analogue reverse-
Class II molecules of, 22f, 23–26, 23f, 26f, 28b Molecular mimicry, 236–237 transcriptase inhibitor
Class I molecules of, 22–26, 22f, 23f, 25f, 28b, Monitoring patient response, using tumor NOD. See Nucleotide-binding oligomerization
40, 41f markers, 285, 285f domain receptor
Class I-related chain A antigens of, 266 Monoclonal antibody, 72–73, 73f Nodular lymphocytic-predominant Hodgkin
clinical significance of, 26–27, 27t in passive immunization, 469–470, 469b, lymphoma (NLPHL), 310, 317t
introduction to, 17–19 469t, 470f Noncompetitive immunoassay, 156–157, 158f,
polymorphism in, 19f in passive immunotherapy, 299–301, 300t 159–160, 165b
Malaria, 396–397, 396f Monoclonal gammopathy of undetermined Non-Hodgkin lymphoma (NHL), 311–312
MALT. See Mucosal-associated lymphoid tissue significance (MGUS), 307, 312 Nonsteroidal anti-inflammatory drugs
Mannose binding lectin (MBL), 92 Monocytes, 6, 6f, 13b (NSAIDs), 247
Mantoux test, 228, 228f Mouse myeloma cells, 469–470, 469b, Nontreponemal test, 374–375, 374f, 375f, 384b
Mass Action, Law of, 142 469t, 470f NTDs. See Neglected tropical diseases
Mast cell, 6–7, 6f, 13b MPO. See Myeloperoxidase Nuclear Regulatory Commission (NRC), 120
Mature B cell, 51f, 52 M protein, 312–314 Nucleic acid, 169–170, 170f
Mature T cell, 48–50, 50f mRNA. See Messenger RNA probes of, 176, 176f, 177f, 178f
Nucleic acid testing (NAT), 442 PAX5. See Paired box protein 5 Pregnancy hormone. See Human chorionic
Nucleolus, 241t, 242, 243 PBC. See Primary biliary cirrhosis gonadotropin
Nucleoside analogue reverse-transcriptase PCR. See Polymerase chain reaction Primary biliary cirrhosis (PBC), 256–257
inhibitor (NNRTI), 439–441, 440t PD. See Prostaglandin Primary follicles, 9, 10f
Nucleosome antibodies, 241t, 242 PDCA. See Plan-Do-Check-Act Primary immunodeficiency (PID)
Nucleotide-binding oligomerization domain PEP. See Postexposure prophylaxis bone marrow biopsy for, 340
receptor (NOD), 34 Peptidoglycan, 349–350, 349f categories of, 329–336, 329t, 333t
Nucleotides. See Nucleic acid Percent panel reactive antibody, 272 classifications of, 341b
Perforins, 40, 41f clinical effects of, 327–329, 328f
Occupational Safety and Health Administration Periarteriolar lymphoid sheath (PALS), 9, 10f family history in, 340
(OSHA), 114–118, 117b Peripheral tolerance, 235 immunoglobulin evaluation of, 339–340
Oncofetal antigen, 285–288, 286t Personal protective equipment (PPE), 114, 117f newborn screening for, 338, 339f
Oncogene, 307–308 PFGE. See Pulse field gel electrophoresis phenocopy of, 336
Opsonin, 38, 39f, 100–103, 101f, 102f Phagocyte screening tests for, 341b
Organ-specific autoimmune diseases, 250–253, bacterial adhesion of, 351–353, 352f Primary lymphoid organ
251t, 252f, 253t defects of, 337–338, 337t function of, 12b
OSHA. See Occupational Safety and Health Phagocytosis, 4, 37–39, 39f, 40f introduction to, 8–9, 9f
Administration Phagolysosome, 38–39, 39f Primary response, 67, 67f
Ouchterlony double diffusion, 144, 145f, 146t, Phagosome, 38, 39f Primers. See also Amplification methods;
245, 245f Phenocopy of primary immunodeficiencies, 336 Polymerase chain reaction
Oxidative burst, 39, 40f Physical hazards, 121 in PCR, 172, 172f
PID. See Primary immunodeficiency Probe amplification, 183–184, 184f
PACIA. See Particle-counting immunoassay Plan-Do-Check-Act (PDCA), 127 Proficiency testing, 125
PAF. See Platelet-activating factor Plasma cell Progenitor B cell, 50–52, 51f
Paired box protein 5 (PAX5), 51 in B cell differentiation, 52–53, 53f Prostaglandin (PD), 215, 216t
Palindromic site, of nucleic acid sequence, 175 dyscrasias of, 307, 312–315, 313f Prostate-specific antigen (PSA), 289–290
PALS. See Periarteriolar lymphoid sheath introduction to, 10, 11f Protein conformation, 18–19, 18f, 19f
PAMPs. See Pathogen-associated molecular Plasmids, 348–350, 439f Protein synthesis, 173–175, 173t, 174f, 174t
patterns Plasmodium malariae, 396–397, 396f Proteomics, in cancer diagnosis, 294
Paracrine action, 78, 78f Platelet-activating factor (PAF), 215, 216t Proto-oncogene, 280, 307–308, 322f
Paraprotein, 312–314 Pleiotropy, of cytokines, 78 Protozoa, 34, 93, 389–392
Parasitic immunology PNH. See Paroxysmal nocturnal hemoglobinuria Provirus, 436, 437f
immune responses in, 390–391, 390t Poison ivy, 20 Prozone, phenomenon of, 142–143, 374
introduction to, 389–390 Polio vaccine, 459 PRRs. See Pathogen recognition receptors
molecular-based diagnosis in, 397 Polymerase chain reaction (PCR), 87 PSA. See Prostate-specific antigen
parasite survival strategies in, 391–392, 405b detection of heavy-chain rearrangements by, Pulse field gel electrophoresis (PFGE), 355
serodiagnosis in, 392–397, 393t, 395t 322, 323f Purified protein derivative (PPD), 228, 228f
Parasitism, 348 detection of H pylori by, 360–361 Purified protein vaccines, 460, 473b
Paroxysmal cold hemoglobinuria, 224 HIV monitoring by, 448–449 Purine-nucleoside phosphorylase deficiency, 328f,
Paroxysmal nocturnal hemoglobinuria (PNH), 104 HLA genotyping by, 270–271, 270b, 270f, 354 332, 333t
Particle agglutination (PA) test, 375–377, 376f Lyme disease screening by, 382, 385b Pyrosequencing, 186, 188f
Particle-counting immunoassay (PACIA), molecular analysis by, 172, 178–181, 179f,
149–150 180f, 181f QC. See Quality control
Passive agglutination, 147–148, 149f, 152b primers in, 172, 172f QM. See Quality management
Passive immunization of qPCR, 181, 183f, 338, 354 QMS. See Quality management system
advantages and limitations of, 468 of real-time PCR, 354, 360–361, 397, 414, qPCR. See Quantitative PCR
antitoxins in, 468 423, 449 QSES. See Quality system essentials
features of, 472b of reverse transcriptase PCR, 414b Quadrant analysis, of flow cytometry, 195–197,
immunosuppressive effects of, 468 syphilis screening by, 378 196f, 197f, 198f
in infectious disease therapy, 467–468 Polymorphism Qualitative nucleic acid test, 446
monoclonal antibodies in, 469–470, 469b, of HLA proteins, 265, 265t Quality control (QC), in laboratory testing,
469t, 470f in nucleic acid sequences, 174–175 123–124, 124f, 125f
Passive immunodiffusion techniques, Polysaccharide vaccines, 459–460, 473b Quality management (QM), 121–123, 123b, 126b
143–144, 144f Porter, Rodney, 63 regulatory issues of, 126–127, 126b
Passive immunotherapy, 299–301, 300t Positive predictive value, 137 Quality management system (QMS),
monoclonal antibodies in, 299–301, 300t Postexamination variables, 125–126, 126b 127–129, 128t
Pasteur, Louis, 3, 3f, 456–457, 457f Postexposure prophylaxis (PEP), 117, 118b Quality system essentials (QSES), 127, 128t
PA test. See Particle agglutination test Poststreptococcal glomerulonephritis, 356–357 Quantitative PCR (qPCR), 181, 183f, 338, 354.
Pathogen-associated molecular patterns (PAMPs), Postzone, phenomenon of, 142–143, 291 See also Polymerase chain reaction
33, 351, 398–399, 409–410. See also PPD. See Purified protein derivative Quantitative viral load assay, 448–449
Toll-like receptors PPE. See Personal protective equipment
Pathogen recognition receptors (PRRs), 33–34, Precipitation, measurement of, 143, 143f RA. See Rheumatoid arthritis
34f, 34t, 35f, 351, 351b, 398–399. See also Precipitation curve, 142–143, 143f Rabies, 457–458, 457f
Pathogen-associated molecular patterns; Precipitation techniques, 145, 146t Radial immunodiffusion (RID), 105, 105f,
Toll-like receptors Precursor B cell, 51–52, 51f 143–144, 144f, 146t
Patient specimen transport, 117–118 Predominantly antibody deficiencies, Radioactive hazards, 120, 120f
Pattern-recognition receptors. See Pathogen 329–330, 329t Radioactive nucleic acid probe, 176, 176f,
recognition receptors; Toll-like receptors Preexamination variables, 122–123, 123b 177f, 178f
Radioallergosorbent test (RAST), 219–220, 220f SAA. See Serum amyloid A Smallpox, 456–457, 456f
Radioimmunoassay (RIA), 157–158 Safety data sheets (SDS), 119, 120f Sm antigen, 241t, 242
Radioimmunosorbent test (RIST), 220f, 221 Safety hazards, 113–121, 113t SNP. See Single nucleotide polymorphism
Random access analyzers, 203 Salk, Jonas, 459 SOD. See Superoxide dismutase
Rapid antigen detection system (RDTS), Sandwich immunoassay, 160, 165b Solution hybridization, 177, 179f
396, 396f Sanger sequencing, 185–186, 185f, 186f, Southern blot analysis, 176, 176f, 177f
Rapid immunoassay, 160–161, 161f 187f, 188f SP. See Standard precautions
Rapid Plasma Reagin (RPR) test, 374–375, 375f Sarcoma, 279, 283t Spanish flu pandemic, 80
Rapid tests, for HIV antibodies, 444–445 Scarlet fever, 356 SPE. See Serum protein electrophoresis
RAST. See Radioallergosorbent test SCID. See Severe combined immunodeficiency Specificity, in serological testing, 137
Rate nephelometry, 143 disease The Specificity of Serological Reactions (Landsteiner),
Raynaud syndrome, 361 Screening tests 20, 20f
RDTS. See Rapid antigen detection system for immune dysfunction, 336–337, 341b Specimen collection, 122–123, 123b
Reagin, 374 for newborns, 338, 339f Spirochete diseases, 371–384
REAL. See Revised European American for tumor markers, 284 diagnostic tests of, 384b, 385b
Lymphoma SDA. See Strand displacement amplification Spleen, 9, 9f, 10f
Real-time PCR, 354, 360–361, 397, 414, 423, SDS. See Safety data sheets Spotted fever group (SFG), 356f, 363–365,
449. See also Polymerase chain reaction Secondary follicle, 10 363t, 364f
Recognition unit, of classical complement Secondary lymphoid organ S protein, 99t, 100
pathway, 93, 94f function of, 12b SSC. See Side scattering
Recombinant protein vaccine, 457, 473b introduction to, 9–11, 9f, 10f, 11f Standard precautions (SP), 114–117, 116f, 117f
Redundancy, of cytokines, 78 Secondary response, 67, 67f Staphylococcus aureus, 35f, 353, 353f, 464
Reed-Sternberg cells, 310, 310f Secretory component, 68, 68f Staphylococcus epidermidis, 348
Relapsing fever group, 382–383 Selective immunoglobulin A deficiency, 328f, Stevens-Johnson syndrome, 361
Remicade. See Infliximab 329t, 330 Strand cleavage methods, 175, 175f
Replication, of DNA, 170–172, 172f Self-antigen, 266 Strand displacement amplification (SDA), 184, 184f
Restriction endonuclease, 175, 175f Self-tolerance, in autoimmune diseases, Streptococcal pharyngitis, 355–356, 356f
Restriction fragment length polymorphism 234–235, 235f Streptococcal pyroderma, 355–356, 356f
(RFLP), 175, 175f Sensitivity, in serological testing, 137 Streptococcus pneumoniae, 35, 349–350, 460, 464
Retinoic acid-inducible gene-I-like receptor Sensitization, of antigen-antibody reaction, Streptococcus pyogenes, 354–359, 355f, 357f, 358f
(RLR), 33 146, 146f Streptozyme test, 357, 359
Reverse passive agglutination, 148–149, Sensitization phase, in type I hypersensitivity, 215, Stringency, in FISH analysis, 178
149f, 152b 215f Substitution, in DNA replication, 173–175, 174t
Reverse transcriptase, 180, 435 Serial dilution, 136, 136f Subunit vaccines, 459–460, 473b
Reverse transcriptase PCR, 414b Serodiagnosis. See also Serological testing Superantigens, 237
Revised European American Lymphoma (REAL), of bacterial infections, 354 Superoxide dismutase (SOD), 39, 40f
308, 310 of Epstein-Barr-associated diseases, Surrogate light chain, 52
RF. See Rheumatoid factor 421–422, 421t Svedberg unit, 63
RFLP. See Restriction fragment length in parasitic immunology, 392–397, 393t, 395t Symbiosis, 347–348, 390t
polymorphism of Rocky Mountain spotted fever, 365 Synergy, of cytokines, 78–80, 79f
Rh. See Rhogam Serological pipettes, 133, 134f, 135f Syngeneic graft, 266, 274b
Rheumatoid arthritis (RA), 73, 80, 81, 226, 237, Serological testing, 133–137, 133f, 134f, 135f, Syphilis
315, 330 136f. See also Serodiagnosis characteristics of, 371, 371f
clinical manifestations of, 246–247 for herpes viruses, 419–425, 421t diagnosis of, 373–379, 374b, 374f, 375f,
etiology of, 246 of syphilis, 373–374 376f, 384b
immunopathology of, 246 Serotype, in conventional vaccines, 458 disease stages of, 372–373, 372b, 372f
laboratory diagnosis of, 247–248 Serum amyloid A (SAA), 35t, 36 immune response to, 373
treatment of, 247 Serum free light chain analysis (sFLC), 321 PCR testing of, 378
Rheumatoid factor (RF), 246 Serum immunofixation electrophoresis, 318–319, testing algorithms for, 377–378,
Rhogam (Rh), 223, 223f 318f, 319b, 319f 377f, 378f
RIA. See Radioimmunoassay Serum protein electrophoresis (SPE), 62f, 312, transmission of, 371–372
Ribonucleic acid (RNA), 70, 435 317–318, 318f, 339–340 Systemic autoimmune diseases, 237–250, 238t.
characteristics of, 169–170, 170f, 171f Serum sickness, 226 See also Antinuclear antibodies
synthesis of, 172–173, 174f Serum tumor markers, 285–290, 286–288t Systemic lupus erythematosus (SLE), 18, 103,
Rickettsial infections, 362–365, 363–364t, 364f, Severe combined immunodeficiency disease 224, 226, 330, 377
365f (SCID), 58, 328f, 331–332, 333t clinical manifestations of, 239–240, 240f
Rickettsia prowazekii, 363–364, 364t SFG. See Spotted fever group etiology of, 238–239
RID. See Radial immunodiffusion sFLC. See Serum free light chain analysis immunopathology of, 239, 239b
RIST. See Radioimmunosorbent test Sharps hazards, 118, 119f laboratory diagnosis of, 240
Rituximab (Rituxan), 72 Side-chain theory, 69–70 Sm antibody of, 241t, 242
RLR. See Retinoic acid-inducible gene-I-like Side scattering (SSC), 194–195, 196f type III hypersensitivity in, 239, 239b
receptor Signal amplification, 184–185, 185f
RNA. See Ribonucleic acid Simple dilutions, 134–136 T1D. See Type 1 diabetes mellitus
Rocky Mountain spotted fever (RMSF), 363–365, Single nucleotide polymorphism (SNP), 174, 322 TAAs. See Tumor-associated antigens
363t, 364f, 365f Single-parameter histogram, 196, 196f Tachyzoites, 393, 394f, 395f
RPR test. See Rapid Plasma Reagin test Six Sigma, 127–129 Tandem mass spectrometry, 294
Rubella virus, 425–426 SLE. See Systemic lupus erythematosus TAP. See Transport associated with antigen
Rubeola virus, 426–427, 426f Small lymphocytic lymphoma (SLL), 309 processing
TAT. See Turnaround time T helper (Th) cells, 26, 27f, 48–50, 50f, 54, 55f TSAs. See Tumor-specific antigens
Tc. See Cytotoxic T cell laboratory identification of, 58 TSH. See Thyroid-stimulating hormone
T-cell lymphotropic viruses, 428–429 subclasses of, 83, 83f tTG. See Tissue transglutaminase
T-cell receptor (TCR), 47–48, 48f Th1 subclass of, 83–84, 83f Tumor. See also Tumor markers
dysfunction of, 338, 339b Th2 subclass of, 83–84, 83f escape mechanisms of, 296–297, 297f
T-cell receptor excision circle (TREC), 338, Th17 cytokines of, 85 immune defense against, 294–296, 295f
339b, 339f Throat culture plate, 357f immunoediting of, 296–297, 297f
T cells. See also Thymocytes Thymocytes. See also T cells immunohistochemistry of, 290–291, 291b
adaptive immune response role of, 53–54, 55f differentiation of, 46–50 interactions with immune system of, 294
cell comparison of, 12b double-negative type of, 47–48, 47f laboratory detection of, 290–294, 291b, 292f
clonal deletion of, 48 double-positive type of, 48, 49f morphology of, 290
comparison to B cells of, 59b introduction to, 7, 8t suppressor gene of, 280
differentiation of, 46–50, 46f, 47f, Thymus, 8–9, 9f Tumor-associated antigens (TAAs),
48f, 49f Thyroglobulin (Tg), 250 281–282, 282t
introduction to, 7, 8t Thyroid peroxidase (TPO), 250 Tumor escape, 296–297, 297f
leukemia of, 200–201t Thyroid-stimulating hormone (TSH), 221–222, Tumor immunology, 279–280, 279b, 280b
mature stage of, 48–50, 50f 250, 252f, 289 Tumor-infiltrating lymphocytes (TILs), 301–302,
mitogen of, 337 Thyrotropin-releasing hormone (TRH), 250, 252f 301f, 470–471
in Type IV hypersensitivity, 226–228, 230b TILs. See Tumor-infiltrating lymphocytes Tumor markers
TCR. See T-cell receptor Tissue cells, 6–7 benefits and limitations of, 284–285, 284b
T cytotoxic cell, 23f, 24, 25f, 28b, 58 Tissue transglutaminase (tTG), 255 categories of, 283–284, 283t
T-dependent antigen, 54, 55f Tissue trauma, in autoimmune disease, 236 clinical applications of, 303b
response to, 55–57, 56f, 57–58, 57f Titer, 136 diagnosis using, 284
TdT. See Deoxyribonucleotidyl transferase TJC. See The Joint Commission immunoassay for, 291–293, 292f
Testing. See also Clinical manifestations; TLRs. See Toll-like receptors patient response analysis using,
Laboratory diagnosis; Molecular analysis TMA. See Transcription-mediated amplification 285, 285f
of allergen-specific IgE, 219–221, 220f TNF. See Tumor necrosis factor prognosis using, 284–285
of anti-DNase B, 358–359 TNF-α. See Tumor necrosis factor-α screening for, 284
for antinuclear antibodies, 245, 245f TNF-β. See Tumor necrosis factor-β Tumor necrosis factor (TNF), 21, 22f,
of biomarker profiling, 294 The TNM system, 279, 280b 80–81
cerebrospinal fluid in, 378–379 Toll-like receptors (TLRs), 33–34, 34f, 34t, 35f, Tumor necrosis factor-α (TNF-α), 35, 36, 40, 73,
for CH50, 105–107, 106f 237, 351b, 399 79f, 80
CLOtest in, 360, 360f deficiencies of, 335 Tumor necrosis factor-β (TNF-β), 48–50, 50f
for complement-dependent cytotoxicity, Tositumomab (Bexxar), 73 Tumor-specific antigens (TSAs), 280–281, 282f,
269–271, 270f Toxoids, 457, 473b 282t, 283t
for crossmatch, 272 Toxoid vaccines, 459 Tumor suppressor gene, 280
for direct antiglobulin, 224 Toxoplasma gondii, 393, 394f, 395f Turbidimetry, 143, 143f
for drug-resistance, 449–450 Toxoplasmosis, 392–396, 394f, 395f, 395t Turnaround time (TAT), 122
for FANA, 242–244, 243f, 244f TPO. See Thyroid peroxidase Type 1 diabetes mellitus (T1D), 253–254
for FTA-ABS, 375 Tr1. See Adaptive T regulatory cell Type I hypersensitivity
for herpes viruses, 419–425, 421t Transcription, 173, 174f, 180 clinical manifestations of, 217–218, 217f
of histocompatibility, 269–273, 270f, Transcription-mediated amplification (TMA), comparisons of, 230b
271f, 273f 181–183, 183f genetic and environmental influences on,
for HIV Transfer RNA (tRNA), 172–173, 174f 216–217
algorithms for, 441–442, 442f Transforming growth factor-β (TGF-β), 83 immunologic mechanism in, 214–216, 215f,
drug-resistance of, 449–450 Transfusion reactions, 222 216t
in infants, 450 Transient hypogammaglobulinemia, 328f, introduction to, 213–214, 214f
rapid types of, 444–445 329t, 330 testing for, 219–221, 219f, 220f
serological analysis in, 442–444, 443f, 444b Translation, 173–175, 173t, 174f, 174t treatment of, 218–219
for immune dysfunction, 337–338, 337t, 338f Transplant rejection, 267–268 to vaccines, 465–467, 467b
for immunoglobulin E, 219–221, 220f Transport associated with antigen processing Type II hypersensitivity, 221–225, 223f
nontreponemal types of, 374–375, 374f, 375f, (TAP1/TAP2), 24 clinical examples of, 222–224, 223f
384b Transport of specimens, 117–118 comparisons of, 230b
for nucleic acid, 442, 446 Trastuzumab (Herceptin), 72 introduction to, 213–214, 214f, 221–222
for particle agglutination, 375–377, 376f Treatment testing for, 224–225
of skin, in vivo, 219, 219f of celiac disease, 255 Type III hypersensitivity, 225–226, 225f
TP-PA test of, 375–377, 376f of multiple sclerosis, 257 comparisons of, 230b
of treponemal test, 374–377, 374f, 376f, 384b of myasthenia gravis, 258, 258f introduction to, 213–214, 214f
for Type III hypersensitivity, 226 of systemic lupus erythematosus, 240 in systemic lupus erythematosus, 239, 239b
for Type IV hypersensitivity, 227–228, 228f of type I diabetes mellitus, 254 to vaccines, 465–467, 467b
for viruses, 411 TREC. See T-cell receptor excision circle Type IV hypersensitivity, 226–228, 227f, 228f
Testing algorithms T regulatory (Treg) cell, 49–50, 83, 83f comparisons to, 230b
of HIV, 441–442, 442f cytokine association with, 84–85 introduction to, 213–214, 214f
of syphilis, 377–378, 377f, 378f deficiency of, 334 to vaccines, 465–467, 467b
Tetanus, 459 Treponemal test, 374–377, 374f, 376f, 384b Typhus group (TG), 363–364t, 363–365
Tg. See Thyroglobulin Treponema pallidum, 371, 371f. See also Syphilis
TG. See Typhus group particle agglutination test of, 375–377, 376f United Nations (UN), 117–118
TGF-β. See Transforming growth factor-β TRH. See Thyrotropin-releasing hormone Universal precautions (UP), 114
Th. See T helper cells tRNA. See Transfer RNA Urinary tract infection (UTI), 33
Urine immunofixation electrophoresis (UPE), Variable region, of immunoglobulin, 47–48, 48f, Waldenström macroglobulinemia, 314–315, 317t
319–321 64, 64f WAS. See Wiskott-Aldrich syndrome
Urticaria, 217–218, 217f Varicella-zoster virus (VZV), 411, 423–425, 424f Waste disposal, 117–121, 119f
Vascular endothelial growth factor (VEGF), 300 WBC. See White blood cell
Vaccines VDJ complex, 70–71, 71f Wegener’s granulomatosis (WG), 248
to bacterial capsular antigens, 349–350, 350b VDRL. See Venereal Disease Research Laboratory Western blot
benefits and adverse effects of, 465–467, Vectors for HIV screening and detection,
466f, 467b in adjuvant discovery, 464 445–446, 446f
characteristics of, 473b of disease, 115f in HTLV detection, 429
conventional forms of, 458–460 in Rickettsial infections, 362–365, 363–364t, for Lyme disease, 381–382, 381f, 385b
for cowpox, 456–457, 456f 364f, 365f White blood cell (WBC). See also Leukocyte;
delivery routes of, 464–465 of Leishmania, 392 Lymphocytes
factors influencing immunogenicity of, of Lyme disease, 379, 382 comparisons of, 13b
460–461, 461b, 462f, 463f of recombinant DNA, 298, 302 introduction of, 4–6
historical evolution of, 455–458, VEGF. See Vascular endothelial growth factor WHO. See World Health Organization
456f, 457f Velocardiofacial syndrome (VFS), 334 Whooping cough, 460
hypersensitivity in, 465–467, 467b Venereal Disease Research Laboratory (VDRL) Wiskott-Aldrich syndrome (WAS), 221, 328f,
inactivated forms of, 459 test, 374 333, 333t
of measles/mumps/rubella, 425–428 Virulence factors, 348–351, 349f, 350b, 350f World Health Organization (WHO), 220
monoclonal antibodies in, 469–470, 469b, of Group A streptococci, 355, 355f in classification of hematologic malignancies,
469t, 470f of H. pylori, 359 308, 309, 310, 321
nature of, 461 Virus, 80, 81f. See also Human immunodeficiency in estimates of hepatitis, 414, 415
new approaches to design of, 464 virus in estimates of parasitic disease, 389
next generation of, 461–465 escape mechanisms of, 411, 431b Wright, Almroth, 4
novel adjuvants as, 464 factors associated with cancer of, 281, 283t
polysaccharide types of, 459–460 immune defenses against infection of, 409–411, Xenograft, 266, 274b
preventable viruses of, 425–428, 426f, 427f 410b, 410f
purified protein types of, 460 laboratory testing for, 411 Yeast, 398
for rabies, 457–458, 457f life cycle of, 409, 409f, 410f
for smallpox, 456–457, 456f Volumetric pipettes, 133, 133f Zevalin. See Ibritumomab tiuxetan
subunit type of, 459–460 VZV. See Varicella-zoster virus Zone of equivalence, 142, 142f