Hema Lec Compiled

MTY 1211 - HEMATOLOGY
Chapter 1 - An Overview of Clinical Laboratory Hematology Lecture - Week 1
OUTLINE
History
Red Blood Cells
Hemoglobin, Hematocrit, and Red Blood Cell Indices
Reticulocytes
White Blood Cells
Platelets
Complete Blood Count
Blood Film Examination
Endothelial Cells
Coagulation
Advanced Hematology Procedures
Additional Hematology Procedures
Hematology Quality Assurance and Quality Control
HISTORY Figure 1.1

Composite of Cells Found in Peripheral Blood of Healthy
● Athanasius Kircher - 1657, described “worms” in the Individuals.
blood.
● Anton van Leeuwenhoek - 1674, gave an account of
RBCs.
● Giulio Bizzozero - late 1800s, described platelets as Hemoglobin, Hematocrit, and RBC Indices
“petites plaques.”
● RBCs - assayed for hemoglobin concentration and
● James Homer Wright - 1920, development of Wright hematocrit.
stain. ● Hemoglobin measurement relies on a weak solution
○ Wright’s Romanowsky-type stain of potassium cyanide and potassium ferricyanide,
- polychromatic, mixture of acidic & basic called Drabkin reagent.
dyes ● Hematocrit
● Morphology - cell appearance (cell color, - also called packed cell volume (PCV)
- it is the ratio of the volume of packed RBCs to the
size, shape, cytoplasmic inclusions, and nuclear
volume of the whole blood.
condensation) - normal ratio approaches 50%
○ Buffy coat - light colored layer between the
RBCs & plasma
- contains WBCs and platelets
RED BLOOD CELLS - excluded from hematocrit determination
○ RBCs - packed cells
● RBCs
● RBC Indices
- anucleate, biconcave, discoid cells filled with
- RBC count, HGB, and HCT are the three
hemoglobin which transports O2 & CO2 numerical results that may use to compute
- appear salmon pink RBC indices (MCV, MCH, MCHC)
- 7 to 8 um in diameter
● Anemia - loss of O2-carrying capacity and is often ○ mean cell volume (MCV) - reflects RBC
reflected in a reduced RBC count or decreased RBC diameter on a Wright-stained blood film;
measurement recorded in femtoliters (fL)
hemoglobin concentration.
● Polycythemia - increased RBC count reflecting ○ mean cell hemoglobin concentration
increased circulating RBC mass, leads to (MCHC) - reflects RBC staining intensity and
hyperviscosity. amount of central pallor; grams per deciliter
● Hemocytometer (g/dL)
- a glass counting chamber
- reported the RBC count in cells per microliter, ○ mean cell hemoglobin (MCH) - expresses
the mass of hemoglobin per cell and
milliliter, or liter
parallels the MCHC; picograms (pg)
. ○ RBC distribution width (RDW) - expresses

● Joseph and Wallace Coulter - invented the first the degree of variation in RBC volume
electronic RBC counter, called Coulter counters.
Go, Manalo, Nepomuceno, and Paguirigan
● RBC morphology - Medical laboratory professionals - Left shift - An increase in bands
routinely use light microscopy at 500X or 1000X also signals bacterial infection.
magnification to visually review ○ Eosinophils (EOs)
- cells with round, bright orange-red
Reticulocytes cytoplasmic granules filled with
proteins involved in immune system
● Polychromatic (polychromatophilic) erythrocytes regulation.
- exceeds 7-8 um, stained slightly blue-gray - Eosinophilia - elevated eosinophil
(Wright-stained blood) count, often signals a response to
- newly released from the bone marrow allergy or parasitic infection.
- indicates the ability of the bone marrow to ○ Basophils (BASOs)
increase RBC production in anemia - cells with dark purple, irregular
● Methylene blue dyes (nucleic acid stains, vital cytoplasmic granules that obscure
stains) the nucleus.
- Used to differentiate and count young RBCs - basophil granules contain
● Vital (supravital) stain histamines and various other
- stain absorbed by live cells proteins.
● Reticulocytes - Basophilia - elevated basophil
- Young RBCs containing ribonucleic acid count, it is rare, and often signals a
(RNA is visualized using vital stains) hematologic disease.
○ Lymphocytes (LYMPHs)
- Comprise a complex system of cells
that provide for host immunity
WHITE BLOOD CELLS - recognize foreign antigens and
mount humoral (antibodies) and
cell-mediated antagonistic
● WBCs or leukocytes - its role is protecting their host
responses.
from infection and injury. - On a Wright-stained blood film,
○ Colorless in an unstained cell suspension. most lymphocytes are nearly round,
○ Transported in the blood from their source, are slightly larger than RBCs, and
usually bone marrow or lymphoid tissue, to have round featureless nuclei and a
their tissue or body cavity destination. thin rim of nongranular cytoplasm
○ Can be counted visually using microscope - Lymphocytosis - increase in the
lymphocyte count, often is
and hemacytometer.
associated with viral infections.
● Medical laboratory professionals who analyze body - Lymphopenia or
fluids such as cerebrospinal fluid or pleural fluid may Lymphocytopenia - abnormally
employ visual WBC counting. low lymphocyte count, often
● Leukopenia - decreased WBC count associated with drug therapy or
● Leukocytosis - increased WBC count immunodeficiency.
● Types of WBCs found in peripheral blood in healthy ○ Monocytes (MONOs)
- Immature macrophage passing
individuals: through the bone marrow, to a
○ Neutrophils (NEUTS, segmented targeted tissue location.
neutrophils, polymorphonuclear neutrophils) - Most abundant cell type in the body
- Phagocytic cells - Role: to identify and phagocytize
- Engulf and destroy microorganisms (engulf and consume) foreign
and foreign materials. particles and assist the
lymphocytes in mounting an
- Segmented - multilobed nuclei
immune response through the
- Cytoplasm of neutrophils contains assembly and presentation of
pink- or lavender-staining antigen epitopes.
granules filled with bactericidal - On a Wright-stained blood film,
substances. monocytes have a slightly larger
- Neutrophilia - increased in diameter than other WBCs,
neutrophils, often signals bacterial blue-gray cytoplasm with fine azure
granules, and a nucleus that is
infection.
usually indented or folded.
- Neutropenia - decrease in - Monocytosis - increase in the
neutrophils, often caused by certain number of monocytes.
medications or viral infection. - Monocytopenia - theoretical term
○ Bands (band neutrophils, BANDs) for decreased monocyte count.
- slightly less mature neutrophils with
a non segmented nucleus in a U or
S shape.
-
rare malignant condition characterized by
● Granulocytes - prominent cytoplasmic granules extremely high platelet counts and
○ Bands, Eosinophils, Neutrophils, Basophils uncontrolled platelet production
(BENB) ● Thrombocytopenia (low platelet count)
● Agranulocytes - no distinct granules in their - common consequence of drug treatment
cytoplasm. - easy bruising
○ Lymphocytes, Monocytes - uncontrolled hemorrhage
● Leukemia - uncontrolled proliferation of a clone of
malignant WBCs
● immunodeficiency. COMPLETE BLOOD COUNT
○ May be chronic (ex. chronic myeloid
leukemia or chronic lymphocytic leukemia) ● complete blood count (CBC)
○ Acute (ex. acute myeloid leukemia or acute
- is performed on automated blood cell analyzers and
lymphoblastic leukemia
includes the RBC, WBC, and platelet measurements
○ Some leukemias are more common in a
- No matter who collects, the medical laboratory
specific age group; chronic lymphocytic
professional is responsible for the integrity of the
leukemia is more prevalent in people older
specimen
than 65 years, whereas acute lymphoblastic
○ appropriate anticoagulant and tube
leukemia is the most common form of
○ free of clots and hemolysis
childhood leukemia.
○ specimen must be of sufficient volume,
○ .Characterization of leukemia using:
because “short draws” result in incorrect
- Wright-stained bone marrow
anticoagulant-to-blood ratios
smears
○ must be tested or prepared for storage within
- Flow cytometric
the appropriate time frame to ensure
immunophenotyping
accurate analysis
- Molecular diagnostic technology
○ must be accurately registered in the work
- Cytogenetics
list, process known as specimen accession
- Cytochemical staining
● When one of the results from the blood cell analyzer
PLATELETS is abnormal, the instrument provides an indication of
this, called a flag. In this case a “reflex” blood film
examination is performed.
● Platelets or thrombocytes
- 2-4 um in diameter, round, oval, anucleate,
slightly granular BLOOD FILM EXAMINATION
- true blood cells
- maintain blood vessel integrity
● Blood film examination
- adhere to the surfaces of damaged blood
- wedge-prep blood film on a glass
vessels (thrombosis, clot formation)
microscope slide
● Hemostasis
- stained with Wright-Giemsa stain
- series of cellular and plasma-based
- performed for comparison with analyzer
mechanisms that seal wounds, repair vessel
counts
walls, and maintain vascular patency
- determines the percentage of the different
(unimpeded blood flow)
white blood cells present (WBC differential)
● Uncontrolled platelet & hemostatic activation is
- determines if there are morphological
responsible for:
abnormalities with RBCs, WBCs, and
- deep vein thrombosis
platelets.
- pulmonary emboli
- acute myocardial infarction
- cerebrovascular accidents
- peripheral artery disease ENDOTHELIAL CELLS
- repeated spontaneous abortions
● Endothelial cells
● Thrombocytosis (elevated platelet counts) - structural and do not flow in the bloodstream
- singal inflammation or trauma but convey - important in maintaining normal blood flow, in
modest intrinsic insignificance tethering platelets during injury, and enabling WBCs
● Essential thrombocythemia to escape from the vessel to tissue when needed.

● Cells of the erythroid series
● Endodermal cells - form the inner surface of the - precursors to RBCs
blood vessel; seldom studied in the hema lab
● Myeloid series cells
- mature to form bands and neutrophils,
eosinophils, and basophils
COAGULATION
● Megakaryocytes
● Coagulation system - produce platelets
- complex sequence of plasma proteins,
enzymes, and enzyme cofactors to produce ● Cytochemical stains
clot formation. - differentiate abnormal myeloid, erythroid,
- another six to eight enzymes control over the and lymphoid cells.
coagulation mechanism - includes: myeloperoxidase, Sudan black B,
- a third system of enzymes and cofactors nonspecific and specific esterase, periodic
digests clots to restore vessel patency acid-Schiff, tartrate-resistant acid
(fibrinolysis). phosphatase, and alkaline phosphatase
● Coagulation tests - replaced by flow cytometry
- platelet count immunophenotyping, molecular diagnostic,
- mean platelet volume (MPV) and cytogenetic techniques
- prothrombin time ● Immunostaining methods
- partial thromboplastin time - detect lineage-specific antigens on the
- thrombin time surface or in the cytoplasm of leukemia and
- fibrinogen assay lymphoma cells
- D-dimer assay
ADVANCED HEMATOLOGY
PROCEDURES ● Clinical flow cytometers (quantitative)
- automated clinical blood cell analyzers that
generate the quantitative parameters of the
● Hematology laboratory also performs: CBC through application of electrical
- bone marrow examinations impedance and laser or light beam
- flow cytometry immunophenotyping interruption
- cytogenetic analysis
- molecular diagnosis assays ● Laser-based flow cytometers (qualitative)
- mechanically simpler but technically more
● Bone marrow aspirates and biopsy specimens demanding
- collected and stained to analyze nucleated
cells that are immature precursors to blood ● Cytogenetics (chromosome analysis)
cells - employed in bone marrow aspirate
- results are compared with CBC results examination to find gross genetic errors
generated from the peripheral blood to - essential to the diagnosis and treatment of
correlate findings and develop diagnoses leukemia
● Wright-stained aspirate smears ● Philadelphia chromosome

- examined for morphological abnormalities, - a reciprocal translocation between
high or low bone marrow cell concentration, chromosomes 9 and 22, that is diagnostic in
and inappropriate cell line distributions chronic myeloid leukemia
● t(15;17)
● Biopsy specimens (enhanced by hematoxylin and - a translocation between chromosomes 15
eosin) and 17 diagnostic in acute promyelocytic
- reveal abnormalities in bone marrow leukemia
architecture indicating leukemia, bone
marrow failure, or one of a host of additional ● Molecular diagnostic techniques
hematologic disorder - fastest growing area of laboratory medicine

● Real-time polymerase chain reaction, microarray

analysis, fluorescence in situ hybridization, and
DNA sequencing systems
- detect various chromosome translocations
and gene mutations that confirm specific
types of leukemia and lymphoma
ADDITIONAL HEMATOLOGY
PROCEDURES
● Glucose-6-phosphate dehydrogenase assay -

detects an inherited RBC enzyme deficiency causing
episodic hemolytic
anemia
● Sickle cell solubility screening assay and its
follow-up tests, hemoglobin electrophoresis and
high-performance liquid chromatography, are
used to detect and diagnose sickle cell anemia and
other inherited qualitative hemoglobin abnormalities
and thalassemias.
● Erythrocyte sedimentation rate - detects
inflammation and roughly estimates its intensity
● Analysis of non blood body fluids is always performed
with a rapid turnaround, bec cells rapidly lose their
integrity in these environments.
HEMATOLOGY QUALITY ASSURANCE

AND QUALITY CONTROL
● Medical laboratory scientists (MLS) employ

particularly complex quality control systems in the
hema lab.
● Moving average- an internal standard methodology
that supports hema lab applications
● MLSs must monitor specimen integrity and test
ordering patterns and ensure the integrity and delivery
of reports
● Hematology laboratory places an enormous
responsibility for accuracy, integrity, judgment, and
timeliness on the MedLab professionals

Chapter 4 - Hematopoiesis Lecture - Week 1
Bone Marrow
OUTLINE
● One of the largest organs in the body
● Located within the cavities of the cortical bones
Hematopoietic Development ● Trabeculae
Mesoblastic Phase - projections of calcified bone
Hepatic Phase
- provides structural support for the developing blood
Medullary (Myeloid) Phase
Adult Hematopoietic Tissue
cells that mature within the sea of interposed mature
Bone Marrow adipocytes
Liver ● Two major components of normal bone marrow:
Spleen ○ Red marrow - active marrow; composed of
Lymph Nodes developing blood cells and their progenitors
Thymus - during infancy and early childhood
Hematopoietic Stem Cells and Cytokines
Stem Cell Theory
○ Yellow marrow - inactive marrow;
Stem Cell Cycle Kinetics
Stem Cell Phenotypic and Functional Characterization
composed primarily of adipocytes, with
Cytokines and Growth Factors undifferentiated mesenchymal
Lineage-Specific Hematopoiesis
Erythropoiesis ● Retrogression - the process of replacing the active
Leukopoiesis marrow by adipocytes (yellow marrow) during
Megakaryopoiesis development
Therapeutic Applications ● Marrow cellularity - the ratio of the red marrow to the
yellow marrow (i.e., the hematopoietic cells to the
HEMATOPOIETIC adipocytes)
DEVELOPMENT ● Stromal cells - endothelial cells, adipocytes (fat
cells), macrophages and lymphocytes, osteoblasts,
osteoclasts, and reticular adventitial cells (fibroblasts)
● Hematopoiesis
● Endothelial cells - regulate the flow of particles
- is the continuous, regulated process of renewal,
entering and leaving hematopoietic spaces in the
proliferation, differentiation, and maturation of all
vascular sinuses
blood cell lines.
● Adipocytes - play a role in regulating the volume of
- formation, development, self-renewal, and directed
the marrow in which active hematopoiesis occurs.
differentiation into all required cell lineages.
They also secrete cytokines or growth factors that
positively stimulate HSC numbers and bone
● Hematopoietic system
homeostasis.
- functional model to study stem cell biology,
● Osteoblasts - bone-forming cells
proliferation, and maturation and their contribution to
● Osteoclasts - bone-resorbing
disease and tissue repair
cells.
● Reticular adventitial cells - form an incomplete layer
of cells on the abluminal surface of the vascular
ADULT HEMATOPOIETIC TISSUE sinuses
● Stromal cells - secrete a semifluid extracellular
matrix that serves to anchor developing hematopoietic
● In adults, hematopoietic tissue is located in the bone
cells in the bone cavity
marrow, lymph nodes, spleen, liver, and thymus.
-play a critical role in the regulation of hematopoietic
● Bone marrow - contains developing erythroid,
stem and progenitor cell survival and differentiation
myeloid, megakaryocytic, and lymphoid cells.
● Extracellular matrix - contains substances such as
● Primary lymphoid tissue - bone marrow and thymus
fibronectin, collagen, laminin, thrombospondin,
and is where T and B lymphocytes are derived.
tenascin, and proteoglycans (such as hyaluronate,
● Secondary lymphoid tissue - lymphoid cells
heparan sulfate, chondroitin sulfate, and dermatan).
respond to foreign antigens; the spleen, lymph nodes,
and mucosa-associated lymphoid tissue.
Red Bone Marrow
● is composed of hematopoietic cells arranged in
extravascular cords
● Extravascular cords - located in spaces between
vascular sinuses and are supported by trabeculae of
spongy bone
● Erythroid precursors or erythroblasts - develop in
small clusters, and the more mature forms are located Liver Pathophysiology
adjacent to the outer surfaces of the vascular sinuses - can maintain hematopoietic stem and progenitor
● Megakaryocytes - located adjacent to the walls of cells to produce various blood cells
the vascular sinuses, which facilitates the release of (extramedullary hematopoiesis) as a response
platelets into the lumen of the sinus. to infectious agents or in pathologic
● Immature myeloid (granulocytic) cells - are located myelofibrosis of the bone marrow
deep within the cords - severe hemolytic anemias increases the
conjugation of bilirubin and the storage of iron
Marrow Circulation - porphyrias result in the accumulation of the
various intermediary porphyrins that damage the
● The nutrient and oxygen requirements of the
hepatocytes, erythrocyte precursors, and other
marrow are fulfilled by the nutrient and
tissues.
periosteal arteries, which enter via the bone
foramina.
● The nutrient artery supplies blood only to the
Spleen
marrow.It coils around the central longitudinal
vein, which passes along the bone canal.
● The arteriole branches that enter the inner lining ● The largest lymphoid in the body.
of the cortical bone (endosteum) form sinusoids ● It is vital but not essential for life and function.
(endosteal beds), which connect to periosteal ● Serves as storage site for Platelets
capillaries that extend from the periosteal artery. ● The spleen has rich blood supply receiving
● The periosteal arteries provide nutrients for the approximately 350mL/min
osseous bone and the marrow The external surface of the spleen is surrounded by a
● Blood exits the marrow via the central layer of Peritoneum.
longitudinal vein, which runs the length of the ● Splenic region
marrow. - White pulp - consist of germinal center
● Hematopoietic cells located in the endosteal bed that contains lymphocytes,
receive their nutrients from the nutrient artery macrophages, dendritic cell
- Marginal zone - zone that surrounds
Hematopoietic Microenvironment the white pulp and form a reticular
meshwork that contains macrophages,
● Hematopoietic inductive microenvironment,
memory B cells and CD4+cells
or niche plays an important role in nurturing and
Red pulp - is composed
protecting HSCs and regulating their
primarily of vascular sinuses separated
quiescence, self-renewal, and differentiation
by cords of reticular cell meshwork
● Stromal cells form an extracellular matrix in the
(cords of Billroth) containing loosely
niche to promote cell adhesion and regulate
connected specialized macrophages.
HSCs through complex signaling networks
-creates a sponge-like matrix that
involving cytokines, adhesion molecules, and
functions as a filter for blood.
maintenance proteins.
● Two methods for removing senescent or
● Key stromal cells thought to support HSCs in
abnormal RBCs from the circulation:
bone marrow niches include osteoblasts,
-Culling, in which the cells are
endothelial cells, mesenchymal stem cells,
phagocytized with subsequent
CXCL12-abundant reticular cells, perivascular
degradation of cell organelles,
stromal cells, glial cells, and macrophages.
- Pitting, in which splenic macrophages
remove inclu-
Liver
sions or damaged surface membrane
from the circulating RBCs.
- consists of two lobes situated beneath the ● Aggregates of T lymphocytes surround the
diaphragm in the abdominal cavity arteries that pass through these germinal
- major site of blood cell production during the centers, forming a region called the
second trimester of fetal development Periarteriolar lymphatic sheath or PALS.
Spleen Pathophysiology
● Hepatocytes ● Splenomegaly - Enlargement of spleen and
- protein synthesis and degradation, often palpable
coagulation factor synthesis, ● Hypersplenism - is an enlargement of the
carbohydrate and lipid metabolism, drug spleen resulting in some degree of
and toxin clearance, iron recycling and pancytopenia. Common cause is congestive
storage, and hemoglobin degradation
splenomegaly, secondary to cirrhosis of the ● In adults, T cell progenitors migrate to the
liver and portal hypertension. thymus from the bone marrow for further
● Two pathway of blood in spleen maturation.
- Slow-transit pathway in which the ● At birth, the thymus is an efficient,
RBCs pass circuitously through the well-developed organ.
macrophage-lined cords before reaching ● It consists of two lobes, each measuring 0.5 to 2
the sinuses. RBCs have a difficult time cm in diameter, and is further divided into
passing through the tiny openings lobules.
created by Interendothelial junctions ● Located in the upper part of the anterior
of the adjacent endothelial cell. This mediastinum at about the level of the great
creates an environment that is vessels of the heart.
acidic,hypoglycemic and hypoxic. ● Cortex
Rapid- transit pathway blood cells - characterized by a blood supply system that is
enter the splenic artery and pass directly unique in that it consists only of capillaries.
to the sinuses in the red pulp and - function seems to be that
continue to the venous system to exit of a “waiting zone” densely populated with
the spleen. progenitor T cells.
- When these progenitor T cells migrate from the
Lymph Node bone marrow and first enter the thymus, they
have no identifiable CD4 and CD8 surface
● These are bean-shaped structures (1-5mm in markers (double negative T cells), and they
diameter) locate to the corticomedullary junction
● Region of lymph node -Under the influence of
- Cortex - outer region chemokines, cytokines, and receptors, these
- Medulla - inner region cells move to the cortex and express both CD4
and CD8 (double positive T cells)
● Follicles with germinal centers are called
secondary follicles Whereas those without are ● Medulla
called primary follicles. - only 15% mature T cells and seems to be a
● Paracortex- a region between the cortex and holding zone for mature T cells until they are
medulla which contains predominantly T Cells needed by the peripheral lymphoid tissues.
and numerous macrophages. ● Gross examination indicates that the size of the
● Three main function of Lymph Nodes thymus is related to age.
- weighs 12 to 15 g at birth,
- They are site of lymphocyte proliferation
- increases to 30 to 40 g at puberty
- They are involved in the initiation of - decreases to 10 to 15 g at later ages.
specific immune responses to foreign
antigen Thymus Pathophysiology
- They filter particulate matter, debris, and
bacteria entering the lymph node. - Nondevelopment of the thymus during gestation
results in the lack of formation of T lymphocytes
Lymph Node Pathophysiology
● By nature, they are vulnerable to the same
organisms that circulate through the tissue . HEMATOPOIETIC STEM CELLS
● Increase number of microorganisms enters the AND CYTOKINES
node, overwhelming the macrophages can
cause Adenitis (infection or inflammation of
lymph node) Stem Cell Theory
● 1961 - Till and McCulloch

Thymus - conducted a series of experiments in which
they irradiated spleens and bone marrow of
mice, creating a state of aplasia.
● Formative intrauterine processes:
- thymus tissue originates from - aplastic mice were given an intravenous
endodermal and mesenchymal tissue injection of marrow cells.
- populated initially by primitive lymphoid - colony-forming units–spleen (CFU-S) -
cells from the yolk sac and the liver colonies that are seen 7 to 8 days late in the

spleens. They are capable of self-renewal - Location: hematopoietic inductive
and production of differentiated progeny microenvironment
- CFU-S represents what we now refer to as - direct cell-to-cell or
committed myeloid progenitors or cellular-extracellular signaling
colony-forming unit–granulocyte, erythrocyte, molecules.
monocyte, and megakaryocyte - Cytokines released include KIT
(CFU-GEMM). ligand, thrombopoietin (TPO), and
● hematopoietic progenitor cells can be divided into two FLT3 ligand. Intrinsic regulation
major types: involves genes such as TAL1.
○ Non Commited or undifferentiated HSCs
○ Committed progenitor cells ○ Intrinsic regulation
● monophyletic theory - all blood cells are derived - involves genes such as TAL1,
from a single progenitor stem cell called a pluripotent which is expressed in cells in the
hematopoietic stem cell hemangioblast, a bipotential
- most widely accepted theory among progenitor cell of mesodermal origin
experimental hematologists. that gives rise to hematopoietic and
● polyphyletic theory - each of the blood cell lineages endothelial lineages; and GATA2,
is derived from its own unique stem cell which is expressed in
● HSCs - capable of self-renewal later-appearing HSCs
- Pluripotent
- Give rise to differentiated progeny ● Changes associated with maturation:
- Able to reconstitute the hematopoietic - Overall decrease in cell volume
system of a lethally irradiated host. - Decrease in the nucleus to cytoplasmic (N to
- Three possible fates: C) ratio
- Self-renewal
- Differentiation ● Changes associated with maturation that occur in
- Apoptosis the cytoplasm:
● Lineage-specific progenitor cells are the common - decrease in basophilia
lymphoid progenitor, which proliferates and - increase in the proportion of cytoplasm
differentiates into T, B, and natural killer lymphocyte - possible appearance of granules in the
and dendritic lineages. cytoplasm.
● common myeloid progenitor, which proliferates and
differentiates into individual granulocytic, erythrocytic, ● Changes associated with maturation that occur in
monocytic, and megakaryocytic lineages. the nucleus:
● Symmetric division - when both daughter cells - Loss of nucleoli
follow the path of differentiation and leave the stem - Decrease in the diameter of the nucleus
cell pool. - Condensation of nuclear chromatin
● Asymmetric division - when one daughter cell - Possible change in the shape of the nucleus
returns to the stem cell pool and the other daughter - Possible loss of the nucleus.
cell follow the path of differentiation or undergo
apoptosis.
● Stochastic model of hematopoiesis - Till and
McCulloch proposed that hematopoiesis is a random Stem Cell Cycle Kinetics
process whereby the HSC randomly commits to
self-renewal or differentiation.
● Instructive model of hematopoiesis - the ● Bone marrow
microenvironment in the bone marrow determines - Capable of producing approx. 2.5 billion
whether the HSC will self-renew or differentiate. erythrocytes
● Multilineage priming model - HSCs receive - 2.5 billion platelets
low-level signals from the hematopoietic inductive - 1 billion granulocytes per kilogram of body
microenvironment to amplify or repress genes weight daily
associated with commitment to multiple lineages. - HSCs exist in the bone marrow in the ratio of
● Cell’s fate: 1 per 1000 nucleated blood cells. They are
○ Extrinsic regulation capable of many mitotic divisions when
- Involves proliferation and stimulated by appropriate cytokines
differentiation signals from
specialized niches.
● Mitotic index Cytokines and Growth Factors
- calculated to establish the percentage of
cells in mitosis in relation to the total number
of cells. ● Hematopoietic growth factors or Cytokines
- 1% to 2% - Group of specific glycoprotein
- Factors affecting: duration of mitosis and the - Regulate the proliferation, differentiation, and
length of the resting state. maturation of hematopoietic precursor cells.
- Increased mitotic index - implies increased ● Cytokines
proliferation (except in the case of - Diverse group of soluble proteins that have
megaloblastic anemia) direct and indirect effects on hematopoietic
cells.
- Include interleukins (ILs), lymphokines,
Stem Cell Phenotypic and Functional monokines, interferons, chemokines, and
Characterization colony-stimulating factors (CSFs)
- responsible for stimulation or inhibition of
● Identification and origin of HSCs can be determined production, differentiation, and trafficking of
by immunophenotypic analysis using flow cytometry. mature blood cells and their precursors
● Earliest identifiable human HSCs capable of initiating - Prevent hematopoietic precursor cells from
long-term cultures are: dying by inhibiting apoptosis; they stimulate
- CD341 them to divide by decreasing the transit time
- CD382 from G0 to G1 of the cell cycle; and they
- HLA-DRlow regulate cell differentiation into the various
- Thy1 low cell lineages.
- Lin2.49
● Expression of CD38 and HLA-DR is associated with a ● Cytokines positive influence on HSCs and
loss of “stemness”. progenitor cells with multilineage potential:
● Acquisition of CD33 and CD38 is seen on committed - Ex. KIT ligand, FLT3 ligand, GM-CSF, IL-1,
myeloid progenitors. IL-3, IL-6, and IL-11)
● Expression of CD10 and CD38 is seen on committed ● Cytokines negative influence on hematopoiesis:
lymphoid progenitors - Transforming growth factor-b, tumor necrosis
● Expression of CD7 is seen on T-lymphoid progenitor factor-a, and the interferons
cells and natural killer cells.
● Expression of CD19 is seen on B-lymphoid
progenitors ● Apoptosis
● Functional characterization of HSCs: - programmed cell death, a normal physiologic
○ vitro techniques using long-term culture process that eliminates unwanted, abnormal,
assays or harmful cells.
- enumeration of colony-forming units - In some disease states apoptosis is “turned
(e.g., CFU-GEMM) on semisolid on,” which results in early cell death.
media, such as methylcellulose. - In other states apoptosis is
Primitive progenitor cells, such as inhibited, which allows uncontrolled
the high proliferative potential proliferation of cells
colony-forming cell
● Colony-Stimulating Factors
○ long-term colony initiating cell - Have a high specificity for their target cells
and are active at low concentrations.
● In vivo functional assays - The primary target of G-CSF is the
- require transplantation of cells into granulocytic cell line
syngeneic - The GM-CSF targets the
- lethally irradiated animals granulocytic-monocytic cell line.
- followed by transference of the engrafted - GM-CSF stimulates the proliferation of
bone marrow cells into a secondary recipient granulocyte and monocyte progenitors, also
● Treatment of hematologic disorders is based on works synergistically with IL-3 to enhance
fundamental understanding of the biologic principles megakaryocyte colony formation
of HSC proliferation and maturation.

● Early-Acting Multilineage Growth Factors - produces a large multi clustered colony that
○ Ogawa - described early-acting growth resembles a cluster of grapes containing
factors (multilineage), intermediate-acting brightly colored hemoglobin.
growth factors (multilineage), and late-acting - contain only a few receptors for EPO
growth factors (lineage restricted - cell cycle activity is not influenced
○ KIT ligand - also known as stem cell factor significantly by the presence of exogenous
(SCF) EPO
- an early-acting growth factor; its - under the influence of IL-3, GM-CSF, TPO,
receptor is the transmembrane and KIT ligand develop into colony-forming
protein unit-erythroid (CFU-E) colonies
- a receptor-type tyrosine-protein
kinase that is expressed on HSCs ● Colony-forming unit-erythroid (CFU-E)
and is downregulated with - many EPO receptors
differentiation. - absolute requirement for EPO
- Activation of the KIT receptor by - responsive to low levels of EPO and do not
KIT ligand is essential in the early have the proliferative capacity of the BFU-E
stages of hematopoiesis
● Pronormoblasts
○ FLT3 - a receptor-type tyrosine-protein - the earliest visually recognized erythrocyte
kinase precursors in the bone marrow
○ IL-3 - regulates blood cell production by
controlling the production, differentiation, and ● EPO
function of granulocytes and macrophages. - serves as a differentiation factor that causes
the CFU-E to differentiate into
● Interleukins - a group of scientists began calling pronormoblasts
some of the cytokines interleukins, numbering them in - a lineage-specific glycoprotein produced in
the order in which they were identified (e.g., IL-1, the renal peritubular interstitial cells
IL-2). - small amounts are produced by the liver
○ Characteristics shared by interleukins - production is activated by the stimulus of
include the following: oxygen availability in the kidney
1. They are proteins that exhibit - effects are exerted by binding to
multiple biologic activities, such as transmembrane receptors expressed by
the regulation of autoimmune and erythroid progenitors and precursors.
inflammatory reactions and - serves to recruit CFU-E from the more
hematopoiesis. primitive BFU-E compartment
2. They have synergistic interactions - prevents apoptosis of erythroid progenitors
with other cytokines. - induces hemoglobin synthesis
3. They are part of interacting systems
with amplification potential.
4. They are effective at very low Leukopoiesis
concentrations.
- divided into myelopoiesis and lymphopoiesis
● factors of CFU-GEMM that differentiates into

LINEAGE-SPECIFIC HEMATOPOIESIS
neutrophils. monocytes, eosinophils, and basophils:
- GM-CSF
Erythropoiesis - G-CSF
- macrophage colony-stimulating factor (M-CSF)
- process for maintaining adequate numbers of RBCs - IL-3
in the peripheral blood - IL-5
- occurs in the bone marrow - IL-11
- KIT ligand
● CFU-GEMM
- Gives rise to the earliest identifiable colony
of BFU-E.
● Burst-forming unit-erythroid (BFU-E)

● GM-CSF - controlling growth and differentiation
- stimulates the proliferation and differentiation - hematopoiesis
of neutrophil and macrophage colonies from - lymphocyte functions (recruitment,
the colony-forming differentiation, and inflammation)
unit–granulocyte-monocyte
● See page 57-58, table 4.2, for overview of selected
● G-CSF and M-CSF cytokines and their major functions and clinical
- stimulate neutrophil differentiation and applications.
monocyte differentiation from the
colony-forming unit–granulocyte and
colony-forming unit–monocyte
● IL-3
- is a multilineage stimulating factor that
stimulates the growth of granulocytes,
monocytes, megakaryocytes, and erythroid
cells.
● Eosinophils differentiation require:

- GM-CSF, IL-5, and IL-3
● Basophil differentiation require:

- unclear, but seems to depend on IL-3 and
KIT ligand
● Lymphoid differentiation is promoted by growth

factors:
- IL-2, IL-7, IL-12, and IL-15 and to some
extent IL-4, IL-10, IL-13, IL-14, and IL-16
Megakaryopoiesis
- earlier influences are GM-CSF, IL-3, IL-6, IL-11, KIT

ligand, and TPO
● TPO and IL-11

- controls the production and release of
platelets
● Liver
- main production site of TPO
THERAPEUTIC APPLICATIONS
● Growth factors
- contributed numerous options in the
treatment of hematologic malignancies and
solid tumors
- used as priming agents to increase the yield
of HSCs during apheresis for transplantation
protocols
● Chemokines (chemotactic cytokines)

- complement cytokine function
- help to regulate the adaptive and innate
immune system
HEMATOLOGY 1
[TRANS] UNIT : ERYTHROCYTE PRODUCTION AND DESTRUCTION
MATURATION PROCESS
OUTLINE
ERYTHROID PROGENITORS
ERYTHROCYTE PRODUCTION AND DESTRUCTION • Erythrocyte precursors develop from two progenitors,
I. NORMOBLASTIC MATURATION burst-forming unit-erythroid (BFU-E) and colony-
1. Terminology forming unit-erythroid (CFU-E), both committed to the
2. Maturation Process erythroid cell line.
3. Criteria Used in Identification of Erythroid • The earliest committed progenitor, BFU-E, gives rise to
Precursors
large colonies because they are capable of multi subunit
4. Maturation Sequence
II. ERYTHOKINETICS colonies (called bursts), whereas CFU-E gives rise to
1. Hypoxia – the Stimulus to Red Blood Cell smaller colonies.
Production • It takes about 1 week for the BFU-E to mature to the CFU-
2. Other Stimuli to Erythropoiesis E and another week for the CFU-E to become a
III. Microenvironment of the Bone Marrow pronormoblast.
IV. Erythrocyte Destruction • CFU-E stage, the cell completes approximately three to
1. Macrophage-Mediated Hemolysis
five divisions before maturing further.
(Extravascular Hemolysis)
2. Mechanical Hemolysis (Fragmentation or • Approximately 18 to 21 days are required to produce a
Intravascular Hemolysis) mature RBC from the BFU-E.
ERYTHROID PRECUSORS
• Normoblastic proliferation
INTRODUCTION o is a process encompassing replication (i.e.,
• Red Blood Cell (RBC) or Erythrocyte division) to increase cell numbers and
o Function: to carry oxygen from the lung to the development from immature to mature cell
tissues, where the oxygen is released. stages.
o Through the attachment of the oxygen to • The earliest morphologically recognizable erythrocyte
hemoglobin, the major cytoplasmic component of precursor, the pronormoblast, is derived via the BFU-E
mature RBCs. and CFU-E from pluripotent hematopoietic stem cells.
o RBCs also plays a role in returning carbon dioxide o Pronormoblast - able to divide, with each
to the lungs and buffering pH of the blood daughter cell maturing to the next stage of
• The mammalian erythrocyte is unique among animal cells- development, the basophilic normoblast.
it does not have a nucleus • Erythrocyte cell line, have three and as many as five
NORMOBLASTIC MATURATION divisions with subsequent nuclear and cytoplasmic
TERMINOLOGY maturation of the daughter cells; from a single
• Red Blood Cells or Erythrocytes pronormoblast, therefore, 8 to 32 mature RBCs usually
o Nucleated RBC precursors, normally restricted to result.
the bone marrow, are called erythroblasts or
normoblasts (developing nucleated RBC CRITERIA USED IN IDENTIFICATION OF ERYTHROID
precursors (i.e., blasts) with normal appearance) PRECURSORS
• Normoblastic terminology
o Commonly used in the United States • Well-stained peripheral blood film or bone marrow
o Descriptive of the appearance of the cells smear is used to identify morphologic classification of
• Rubriblast blood cells.
o Some prefer this terminology because it parallels • Wright or Wright-Giemsa of Romanowsky Stain is
the nomenclature used for granulocyte commonly used in hematology.
development. • In blood cell the stage of maturation is determined by
• Erythroblast examining the nucleus and cytoplasm.
o Used primarily in Europe
The following are also important features in identifying RBCs:
Table 5.1 Three Erythroid Precursor Nomenclature Systems • Nuclear Chromatin Pattern (texture, density, homogeneity)
• Nuclear diameter
• Nucleus-to-cytoplasm (N:C) ratio
• Presence or absence of nucleoli
• Cytoplasmic color
Well-stained peripheral blood film or bone marrow smear is used
GO, KIMBERLY COLE. MEDES, HAIDIE M. REYES, KYLA DIANNE. | FEU MT 2023
1
HEMATOLOGY 1
[TRANS] UNIT : ERYTHROCYTE PRODUCTION AND DESTRUCTION
GENERAL TRENDS THAT AFFECT THE APPEARANCE OF A

MATURING ERYHTROID PRECURSORS
a) Overall diameter of the cell decreases and cytoplasm

changes from blue to salmon pink
b) Diameter of the nucleus decreases more rapidly than does in early stages. The hemoglobin concentration begins to
the diameter of the cell = N:C ratio also decreases. The rise in the basophilic normoblast stage, reaching its peak
color changes from purplish-red to a very dark purple-blue. in reticulocytes and representing most of the protein in
c) Nuclear chromatin pattern becomes coarser, clumped, and more mature cells.
condensed.
d) Nuclear chromatin of Erythroid Precursors is inherently PRONORMOBLAST (RUBRIBLAST)
coarser than that of Myeloid Precursors.
e) As it matures, it becomes coarser and more clumped • Nucleus is round to oval, containing one or two nucleoli.
turning into a raspberry-like appearance. The purple red chromatin is open and contains few, if any,
f) Nucleus becomes condensed, without evident of fine clumps.
parachomatin, nucleus is pyknotic. • Cytoplasm is dark blue because of the concentration of
g) Nucleoli disappear and precedes ultimate cessation of ribosomes and RNA.
protein synthesis. • Division – the pronormoblast undergoes mitosis and gives
rise to two daughter pronormoblasts. There could be
• Basophilia or blueness attracts methylene blue. additional one or more division before maturation into
Cytoplasmic basophilia is connected to the amount of basophilic normoblasts.
ribosomal RNA. During the development of erythroid • Location – the pronormoblast is present only in the bone
precursor where ribosomes and other organelles decline, marrow in healthy states.
the blueness fades. • Cellular Activity - The pronormoblast begins to
accumulate the components necessary for hemoglobin
• Eosinophilia or pinkness attracts eosin. Correlates with production. The proteins and enzymes necessary for iron
the accumulation of hemoglobin as cell matures. uptake and protoporphyrin synthesis are produced. Globin
Activating the protein production in the ribosomes that production begins.
makes up cytoplasmic basophilia, red of the hemoglobin • Length of time in this stage – lasts 24 hours
mixes with the blue. Thus, creating salmon pink color
where ribosomes are gone, and only hemoglobin remains.
BASOPHILIC NORMOBLAST (PRORUBICYTE)
• Nucleus
MATURATION SEQUENCE o the chromatin begins to condense, revealing
STAGES OF ERYHTROID DEVELOPMENT IN ORDER & clumps along the periphery of the nuclear
COMPARISON membrane and a few in the interior. The
parachromatin areas become larger and sharpe
CHANGES IN CELLULAR DIAMETER, RNA SYNTHESIS AND as the chromatin condenses and the N:C ratio
CONTENT, DNA SYNTHESIS AND CONTENT, PROTEIN AND decreases to about 6:1. The chromatin stains
HEMOGLOBIN CONTENT DURING ERYTHROID deep purple-red. Nucleoli may be present early in
DEVELOPMENT the stage but disappear later.
• Cytoplasm
a) Cell diameter shrinks from the pro-normoblast to the o may be a deeper, richer blue than in the
reticulocyte stage. pronormoblast when stained.
b) The rate of RNA synthesis for protein production is at its • Division
peak at the pronormoblast stage and ends in the o undergoes mitosis, giving rise to two daughter
orthochromic normoblast stage. RNA content remains cells. More than one division is possible before
relatively constant, due to the accumulation of RNA, into the daughter cells mature into polychromatic
the orthochromic normoblast stage when it begins to normoblasts.
degrade, being eliminated by the end of the reticulocyte • Location
stage. o present only in the bone marrow in healthy states.
c) The rate of DNA synthesis correlates to those stages of • Cellular activity
development that are able to divide: the pronormoblast, o detectable hemoglobin synthesis occurs, but the
basophilic normoblast, and early polychromatic normoblast many cytoplasmic organelles, including
stages. DNA content of a given cell remains relatively ribosomes and a substantial amount of
constant until the nucleus begins to break up and is messenger ribonucleic acid (mRNA; chiefly for
extruded during the orthochromic normoblast stage. There hemoglobin production), completely mask the
is no DNA (i.e., no nucleus) in reticulocytes. minute amount of hemoglobin pigmentation.
d) The total protein concentration declines slightly during
maturation. Proteins, aside from hemoglobin, predominate
GO, KIMBERLY COLE. MEDES, HAIDIE M. REYES, KYLA DIANNE. | FEU MT 2023
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HEMATOLOGY 1
MTY1211 : ERYTHROCYTE PRODUCTION AND DESTRUCTION
• Length of time in this stage • Length of time in this stage
o lasts slightly more than 24 hours o lasts approximately 30 hours.

Figure 5.5 (A), Basophilic normoblast. (Bone marrow, Wright- Giemsa
stain, 31000.(B), Electron micrograph of a basophilic normoblast (315,575).
Figure 5.6 Polychromatic Normoblasts (Rubricytes).
(A), Polychromatic normoblast. (Bone marrow, Wright-Giemsa stain, X1000.)
(B), Electron micrograph of a polychromatic normoblast (x15,575).
POLYCHROMATIC (POLYCHROMATOPHILIC) NORMOBLAST ORTHOCHROMIC NORMOBLAST (METARUBRICYTE)

(RUBRICYTE)
• Nucleus
o the nucleus is completely condensed (i.e.,
• Nucleus
pyknotic) or nearly so; the N:C ratio is low or
o the chromatin pattern varies during this stage of
approximately 1.2.
development, showing some openness early in
• Cytoplasm
the stage but be- coming condensed by the end.
o the increase in the salmon pink color of the
The condensation of chromatin reduces the
diameter of the nucleus considerably, so the N:C cytoplasm reflects nearly complete hemoglobin
production. The residual ribosomes and RNA
ratio decreases from 4:1 to about 1:1 by the end
react with the basic component of the stain and
of the stage. Notably, no nucleoli are present.
contribute a slightly bluish hue to the cell, but that
• Cytoplasm
fades toward the end of the stage as the RNA and
o the first stage in which the pink color associated
organelles are degraded.
with stained hemoglobin can be seen. The
• Division
stained color reflects the accumulation of
o not capable of division because of the
hemoglobin pigmentation over time and
condensation of the chromatin.
concurrent decreasing amounts of RNA. The
color produced is a mixture of pink and blue, • Location
resulting in a murky gray-blue. o present only in the bone marrow in healthy states.
o Polychromatopholic – many color loving • Cellular activity
• Division o hemoglobin production continues on the
o last stage in which the cell is capable of remaining ribosomes using messenger RNA produced
undergoing mitosis, although likely only early in earlier. the nucleus is ejected from the cell in this stage and
the stage; goes through mitosis, producing it moves to the cell membrane into a pseudopod-like
daughter cells that mature and develop into projection. The loss of vimentin, a protein responsible for
orthochromic normoblasts. holding organelles in proper location in the cytoplasm may
be important in the movement of nucleus to cell periphery.
• Location
o present only in the bone marrow in healthy states. The nucleus-containing projection separates from the cell
by having the membrane seal and pinch off the projection
• Cellular activity
with the nucleus enveloped by cell membrane. Nonmuscle
o hemoglobin synthesis increases, and the
myosin of the membrane is important in this pinching
accumulation begins to be visible as a pinkish
process. Pyrenocyte, an enveloped extruded nucleus is
color in the cytoplasm. Cellular RNA and
then engulfed by bone marrow macrophages; these
organelles are still present, particularly
macrophages recognize phosphatidylserine on the
ribosomes, which contribute a blue color to the
pyrenocyte surface as an eat me flag. Other organelles are
cytoplasm. The progressive condensation of the
extruded and ingested in similar fashion. Often, small frag-
nucleus and disappearance of nucleoli are
ments of nucleus are left behind if the projection is pinched
evidence of progressive decline in transcription of
off before the entire nucleus is enveloped. These
deoxyribonucleic acid (DNA).
fragments are called Howell-Jolly bodies when seen in
peripheral RBCs.
• Length of the time in this stage
GO, K., MEDES, H., REYES, K. D., TRILLES, N., RAMIREZ, JES. | FEU MT 2023 3
HEMATOLOGY 1
o lasts approximately 48 hours suspension before the blood film is made. The
residual ribosomes appear as a mesh of small
blue strands, a reticulum, or, when more fully
digested, merely blue dots. When so stained, the
polychromatic erythrocyte is called a reticulocyte.
• Length of the time in this stage :
o about 3 days, with the first 2 days spent in the
bone marrow and the third spent in the peripheral
blood, although possibly sequestered in the
spleen.
Figure 5.7 Orthochromic Normoblasts (Metarubicytes).

(A), Orthochromic normoblast. (Bone marrow, Wright-Giemsa stain, X1000.)
(B), Electron micrograph of an orthochromic normoblast (320,125).
POLYCHROMATIC (POLYCHROMATOPHILIC) ERYTHROCYTE

OR RETICULOCYTE
• Nucleus
o no nucleus; when a cell loses its nucleus, Figure 5.8 Polychromatic Erythrocytes (Shift Reticulocytes)
regardless of cytoplasmic appearance, it is a (A), Polychromatic erythrocytes (arrows). (Peripheral blood, Wright-Giemsa
stain,X1000)
polychromatic erythrocyte. (B),Scanning electron micrograph of a polychromatic erythrocyte (x5000)
• Cytoplasm
o can be compared with that of the late
orthochromic normoblast in that the predominant
color is that of hemoglobin yet with a bluish tinge
due to some residual ribosomes and RNA. By the
end of the polychromatic erythrocyte stage, the
cell is the same color as a mature RBC, salmon Figure 5.9 Reticulocytes (arrows).
pink. It remains larger than a mature cell, (Peripheral blood, new methylene blue
stain, 31000.)
however. The shape of the cell is not the mature
biconcave disc but is irregular in electron micro-
graphs.
• Division
o cannot divide due to the lack of nucleus
• Location ERYTHROCYTE
o resides in the bone marrow for about 1 to 2 days
and then moves into the peripheral blood for
• Nucleus
about 1 day before reaching maturity. During the
o no nucleus in mature RBCs
first several days after exiting the marrow, the
• Cytoplasm
polychromatic erythrocyte is retained in the
o mature circulating erythrocyte is a biconcave disc
spleen for pitting of inclusions and membrane
measuring 7 to 8 mm in diameter, with a thickness of about
polishing by splenic macrophages, which results
1.5 to 2.5 mm. On a Wright-stained blood film, it appears
in the biconcave discoid mature RBC.
as a salmon-pink stained cell with a central pale area that
• Cellular activity
corresponds to the concavity. The central pallor is about
o completes production of hemoglobin from a small
one-third the diameter of the cell.
amount of residual messenger RNA using the
• Division
remaining ribosomes. Endoribonuclease, digests
o cannot divide
the ribo somes. The acidic components that
attract the basophilic stain decline during this • Cellular activity
stage to the point that the polychromatophilia is o delivers oxygen to tissues, releases it, and returns
only slightly evident in the polychromatic to the lung to be reoxygenated. The interior of the
erythrocytes on a peripheral blood film stained erythrocyte contains mostly hemoglobin, the
with Wright stain. A small amount of residual oxygen carrying component. It has a surface
ribosomal RNA is present and can be visualized area-to-volume ratio and shape that enable
with a vital stain such as new methylene blue, so optimal gas exchange to occur. If the cell was
called because the cells are stained while alive in spherical, it would have hemoglobin at the center
of the cell that would be relatively distant from the
HEMATOLOGY 1
membrane and would not be readily oxygenated o Nondialyzable

and deoxygenated. With the biconcave shape, o Molecular weight of 34 kD.
even hemoglobin molecules that are toward the o Consists of a carbohydrate unit and a terminal
center of the cell are not distant from the sialic acid unit for biological activity.
membrane and are able to exchange oxygen. • ACTION
RBCs must squeeze through small spaces such o Produced at one location (kidney) and acting at a
as the basement membrane of the bone marrow distant location (bone marrow).
venous sinus. Similarly, when a cell enters the red o Initiates an intracellular message to the
pulp of the spleen, it must squeeze between developing erythroid cells; this process is called
epithelial cells to move into the venous outflow. signal transduction.
Deformability is crucial for RBCs to enter and o EPO + Initiators = initiates a cascade of
subsequently remain in the circulation. intracellular which
§ increased cell division and maturation,
• Location and length of time in this stage increased intestinal iron absorption and
o mature RBCs remain active in the circulation for hemoglobin synthesis, and more RBCs
approximately 120 days; aging leads to removal entering the circulation.
by the spleen. o EPO responsive cells vary in their sensitivity.
o EPO receptor is a transmembrane protein
homodimer with extracellular and cytoplasmic
domains.
o EPO + Extracellular Domain of the EPO
Receptor = activates Janus- activated tyrosine
kinase 2 (JAK2) signal transducers that are
associated with the cytoplasmic domains of the
EPO receptor.
o JAK2 then activates downstream signal
transduction pathways (such as the signal
Figure 5.10 Mature Erythrocytes. (A), Mature erythrocytes transduction and activator of transcription 5 or
and one lymphocyte. (Peripheral blood, Wright-Giemsa stain x1000.) STAT5 pathway) that ultimately promotes
(B), Scanning electron micrograph of mature erythrocytes.
transcription of specific genes in the RBC nucleus
ERYTHROKENITICS EPO has three major effects:
• Term that describe the dynamics of RBC production and 1. Allowing early release of reticulocytes
destruction. from the bone marrow,
• Erythron 2. Preventing apoptotic cell death, and
o Name given to the collection of all stages of 3. Reducing the time needed for cells to
erythrocytes throughout the body. mature in the bone marrow.
o An important concept for erythrokenitics.
o Conveys the concept of a unified functional Early Release of Reticulocytes
tissue. o EPO promotes from early release of developing
• Discussion of erythrokinetics begins by looking at erythroid precursors from the bone marrow by two
erythrocytes in the bone marrow and the factors that affect mechanisms.
their numbers, their progressive development, and their o EPO induces changes in the adventitial cell layer
ultimate release into the peripheral blood. of the bone marrow/sinus barrier that increase the
width of the spaces for RBC egress into the sinus
HYPOXIA – THE STIMULUS TO RED BLOOD CELL – that if use alone, it will be is insufficient for the
PRODUCTION cells to leave the marrow.
o RBCs are held in the marrow for adhesive
molecules located on the bone marrow stroma,
• Primary oxygen-sensing system of the body is located in
such as fibronectin.
peritubular fibroblasts of the kidney.
o Downregulating the expression with the use of
• Erythropoietin (EPO) - major stimulatory cytokine for
EPO causes the cells to leave the marrow or
RBCs. earlier than normal.
• Hemorrhage – Increased the production of EPO due to the
disruption of oxygen-carrying capacity of the blood. Shift Reticulocytes - When reticulocytes
normally found in the bone marrow are present in the
ERYTHROPOEITIN peripheral blood
STRUCTURE o Releasing cells from the bone marrow early is a

o Thermostable quick fix, so to speak; it is limited in effectiveness
HEMATOLOGY 1
because the available precursors in the marrow bodies.

are depleted within several days and still may not
be enough to meet the need in the peripheral • The last stage of degradation produces
blood for more cells. nuclear DNA fragments consisting of
o A more sustained response is required in times of multimers of 180 to 185 base pair segments
increased need for RBCs in the circulation. • Blebbing of the plasma membrane is
observed.
Inhibition of Apoptosis • Apoptotic cell contents remain membrane
o Increasing the number of cells that will be able bound and are ingested by macrophages,
to mature into circulating erythrocyte – which prevents an inflammatory reaction.
second, and probably more important, • The membrane-bound vesicles display so-
mechanism by which EPO increases the number called eat me signals on the membrane
of circulating RBCs surface which promote macrophage
o Happen when decreases the apoptosis, ingestion.
programmed death of RBC progenitors.
Þ Evasion of apoptosis by erythroid progenitors
Þ Apoptosis: programmed cell death and precursors.
• It takes about 18 to 21 days to produce
an RBC from stimulation of the earliest • One effect of EPO is an indirect avoidance of
erythroid progenitor (BFU-E) to release apoptosis by removing an apoptosis
from the bone marrow. induction signal.
• RBCs cannot be stored in the body for • Apoptosis of erythroid precursors and
this sort of eventuality, however, progenitors is a cellular process that depends
because they have a limited life span. on a signal from either the inside or outside
• Body produces more CFU-Es than of the cell.
needed at all times. • When EPO levels are low, cell production
• When there is a steady-state demand for should be at a low rate because hypoxia is
RBCs, the extra progenitors are not present while excess early erythroid
allowed to die. precursors should undergo apoptosis.
• When there is an increased demand for • This occurs when the older FasL-bearing
RBCs, however, the erythroid erythroid precursors, such as polychromatic
progenitors have about an 8- to 10-day normoblasts, cross-link with Fas-marked
head start in the production process. immature erythroid precursors, such as
Þ Process of Apoptosis pronormoblasts and basophilic normoblasts,
• Degradation of chromatin into fragments which are then stimulated to undergo
of varying size that are multiples of 180 apoptosis.
to 185 base pairs long; protein • If the FasL-bearing cells are depleted, as
clustering; and activation of when EPO stimulates early bone marrow
transglutamase. release, the younger Fas-positive precursors
• Contrast to necrosis - cell injury causes are allowed to develop, which increases the
swelling and lysing with release of overall output of RBCs from the marrow.
cytoplasmic contents that stimulate an • Second mechanism for escaping apoptosis
inflammatory response. exists for erythroid progenitors: direct EPO
• Apoptosis is not associated with rescue from apoptosis - major way in which
inflammation. EPO is able to increase RBC production.
• EPO + receptor on the CFU-E = reduce
During the sequential process of production of Fas ligand.
apoptosis, the following morphologic • EPO is able to stimulate production of
changes can be seen: various antiapoptotic molecules, which
allows the cell to survive and mature.
• Condensation of the nucleus, causing • Without EPO, the CFU- E does not survive.
increased basophilic staining of the
chromatin; Reduced Marrow Transit Time
• Nucleolar disintegration; o Another effect of EPO is to increase the rate at
• Shrinkage of cell volume with which the surviving precursors can enter the
concomitant increase in cell density and circulation. This is accomplished by two means:
compaction of cytoplasmic organelles, increased rate of cellular processes and
whereas mitochondria remain normal. decreased cell cycle times.
• Followed by a partition of cytoplasm and o EPO stimulates the synthesis of RNA in erythroid
nucleus into membrane-bound apoptotic precursors and effectively increases the rate of
HEMATOLOGY 1
the developmental process. Among the o The use of EPO is one of the methods of blood doping;
processes that are accelerated is hemoglobin aside from being banned in organized sports events,
production. it increases the RBC count and blood viscosity to
o EPO induces erythroid precursors to secrete dangerously high levels and can lead to fatal arterial
erythroferrone, which acts on hepatocytes to and venous thrombosis.
decrease hepcidin production that allows more
iron to be absorbed from the intestines to support Other Stimuli to Erythropoiesis
the increased hemoglobin synthesis .
o Other process that is accelerated is the cessation • Other factors influence RBC production to a modest
of division. Cell division takes time and would extent.
delay entry of cells into the circulation, so cells • It is well documented that testosterone directly stimulates
enter cell cycle arrest sooner causing the cells erythropoiesis, which partially explains the higher
spend less time maturing in the bone marrow. hemoglobin concentration in men than in women.
o In the circulation, cells are larger because of lost • Also, pituitary and thyroid hormones have been found to
mitotic divisions, and they do not have time before affect the production of EPO and so have indirect effects
entering the circulation to dismantle the protein on erythropoiesis.
production machinery that gives the bluish tinge
to the cytoplasm.
o EPO also can reduce the time it takes for cells to
MICROENVIRONMENT OF THE BONE MARROW
mature in the bone marrow by reducing individual
cell cycle time, specifically the length of time that
cells spend between mitoses. • Hematopoiesis occurs in marrow cords, essentially a
o This effect is only about a 20% reduction, loose arrangement of cells outside a dilated sinus area
however, so that the normal transit time in the between the arterioles that feed the bone and the central
marrow of approximately 6 days from vein that returns blood to efferent veins.
pronormoblast to erythrocyte can be shortened by • ERYTHROID ISLANDS – within the bone marrow where
only about 1 day by this effect. erythropoiesis typically occurs.
o With the decreased cell cycle time and fewer o these islands consist of a central macrophage
mitotic divisions, the time it takes from surrounded by erythroid precursors in various
pronormoblast to reticulocyte can be shortened stages of development.
by about 2 days total. • MACROPHAGES – are known to elaborate cytokines that
o If the reticulocyte leaves the marrow early, are vital to the maturation process of erythroid precursors
another day can be saved, and the typical 6-day and to phagocytize called nuclei.
transit time is reduced to fewer than 4 days under o major cellular anchor for the developing
the influence of increased EPO. normoblasts
• Another role of macrophages in erythropoiesis
o Although movement of cells through the marrow
Measurement of erythropoietin cords is sluggish, developing cells would exit the
marrow prematurely in the outflow were it not for
o Quantitative measurements of EPO are performed on an anchoring system within the marrow that holds
plasma and other body fluids. them there until development is complete.
o EPO can be measured by chemiluminescence. • THREE COMPONENTS TO THE ANCHORING SYSTEM
o Although the reference interval for each laboratory o a stable matrix of accessory and stromal cells to
varies, 10 to 30 U/L is sufficient to maintain steady- which normoblasts can attach
state erythropoiesis in a healthy adult. o bridging (adhesive) molecules for that attachment
o Increased amounts of EPO in the urine are expected o receptors on the normoblast membrane
in most patients with anemia, with the exception of • Several systems of adhesive molecules and normoblast
patients with anemia caused by renal disease. receptors tie the developing normoblasts to the
macrophages. At the same time, normoblasts are
Therapeutic uses of erythropoietin anchored to the extracellular matrix of the bone marrow,
chiefly by fibronectin.
• When it comes time for RBCs to leave the marrow, they
o Recombinant EPO is used as therapy in certain cease production of the receptors for adhesive molecules.
anemias such as those associated with chronic kidney • Without the receptor, cells are free to move from the
disease and chemotherapy. marrow into the venous sinus.
o Used to stimulate RBC production before autologous
• Entering the venous sinus requires the RBC to traverse the
blood donation and after bone marrow transplantation.
barrier created by the adventitial cells on the cord side, the
o Unfortunately, some athletes illicitly use EPO
basement membrane, and the endothelial cells lining the
injections to increase the oxygen-carrying capacity of
sinus.
their blood to enhance endurance and stamina,
especially in long-distance running and cycling.
HEMATOLOGY 1
• Egress through this barrier occurs between adventitial • Senescent changes to leukocyte surface antigen CD47
cells, through holes (fenestrations) in the basement (integrin-associated protein) may also be involved by binding
membrane, and through pores in the endothelial cells thrombospondin-1, which then provides an “eat me” signal to
macrophages
ERYTHROCYTE DESTRUCTION • macrophages are able to recognize senescent cells and
distinguish them from younger cells; thus the older cells are
• Because RBCs lack mitochondria, they rely on glycolysis for
targeted for ingestion and lysis.
production of adenosine triphosphate (ATP). The loss of
glycolytic enzymes is central to this process of cellular aging, • When an RBC lyses within a macrophage, the major compo-
called senescence, which culminates in phagocytosis by nents are catabolized. Iron is removed from the heme. It can
macrophages. This is the major method by which RBCs die be stored in the macrophage as ferritin until transported out.
normally. • The globin of hemoglobin is degraded and returned to the
metabolic amino acid pool. The protoporphyrin component of
MACROPHAGE-MEDIATED HEMOLYSIS
heme is degraded through several intermediaries to bilirubin,
(EXTRAVASCULAR HEMOLYSIS) which is released into the blood and ultimately excreted by the
liver In bile.
• accounts for most normal RBC death
• At any given time, a substantial volume of blood is in the spleen, MECHANICAL HEMOLYSIS (FRAGMENTATION OR
which generates an environment that is inherently stressful on
INTRAVASCULAR HEMOLYSIS)
cells.
o Movement through the red pulp is sluggish. The
available glucose in the surrounding blood is depleted • Although most natural RBC deaths occur in the spleen, a small
quickly as cell flow stagnates, so glycolysis slows. The portion of RBCs rupture intravascularly (within the lumen of
pH is low, which promotes iron oxidation. Maintaining blood vessels). The vascular system can be traumatic to RBCs,
reduced iron is an energy-dependent process, so with turbulence occurring in the chambers of the heart or at
factors that promote iron oxidation cause the RBC to points of bifurcation of vessels. Small breaks in blood vessels
expend more energy and accelerate the catabolism of and resulting clots can also trap and rupture cells.
enzymes. • The intravascular rupture of RBCs from purely mechanical or
• In this hostile environment, aged RBCs succumb to various traumatic stress results in fragmentation and release of the cell
stresses. Their deteriorating glycolytic processes lead to contents into the blood; this is called fragmentation or
reduced ATP production, which is complicated further by intravascular hemolysis.
diminished amounts of available glucose. Membrane systems • When the membrane of the RBC has been breached, regard-
that rely on ATP begin to fail. less of where the cell is located when it happens, the cell con-
• Lack of ATP leads to oxidation of membrane lipids and proteins. tents enter the surrounding blood.
As this system fails, intracellular sodium increases and o Although mechanical lysis is a relatively small
potassium decreases. The effect is that the selective contributor to RBC demise under normal
permeability of the membrane is lost and water enters the cell. circumstances, the body still has a system of
The discoid shape is lost and the cell becomes a sphere. plasma proteins, including haptoglobin and
• RBCs must remain highly flexible to exit the spleen by hemopexin, to salvage the released hemoglobin
squeezing through the so-called splenic sieve formed by the so that its iron is not lost in the urine. Hemolysis
endothelial cells lining the venous sinuses and the basement and the functions of haptoglobin and hemopexin
membrane. Spherical RBCs are rigid and are not able to are discussed in Chapter 20.
squeeze through the narrow spaces; they become trapped
against the endothelial cells and basement membrane. In this
situation, they are readily ingested by macrophages that patrol
along the sinusoidal lining.
• ERYPTOSIS – erythrocyte death as a nonnucleated cell
version of apoptosis
o which is precipitated by oxidative stress, energy
depletion, and other mechanisms that create
membrane signals that stimulate phagocytosis.
• It is highly likely that there is no single signal but rather that
macrophages recognize several.
o Examples of the signals that are being further
investigated include binding of autologous
immunoglobulin G (IgG) to band-3 membrane protein
clusters, exposure of phosphatidylserine on the
exterior (plasma side) of the membrane, and inability
to maintain cation balance.
HEMATOLOGY 1
HEMATOLOGY 1
[TRANS] CHAPTER 7: HEMOGLOBIN METABOLISM
HEME STRUCTURE
OUTLINE Heme – consists of a ring of carbon, hydrogen, and nitrogen atoms
HEMOGLOBIN METABOLISM called protoporphyrin IX, with a central atom of divalent ferrous
I. Hemoglobin Structure iron (Fe2+).
1. Heme Structure • Each of the four heme groups is positioned in a pocket of
2. Globin Structure the polypeptide chain near the surface of the hemoglobin
3. Complete Hemoglobin Molecule molecule.
II. Hemoglobin Biosynthesis • The ferrous iron in each heme molecule reversibly
1. Heme Biosynthesis combines with one oxygen molecule.
2. Globin Biosynthesis
3. Hemoglobin Assembly
o When ferrous ions are oxidized to the ferric state
III. Hemoglobin Ontogeny (Fe3+), they no longer can bind oxygen.
IV. Regulation of Hemoglobin Production Methemoglobin – oxidized hemoglobin.
1. Heme Regulation
2. Globin Regulation
3. Systematic Regulation of Erythropoiesis
V. Hemoglobin Function
1. Oxygen Transport
2. Carbon Dioxide Transport
3. Nitric Oxide Transport
VI. Dyshemoglobins
1. Methemoglobin
2. Sulfhemoglobin
3. Carboxyhemoglobin
VII. Hemoglobin Measurement
Hemoglobin – one of the most studied proteins in the body because

of the ability to easily isolate it from red blood cells (RBCs).
- Main function: transport oxygen from the lungs to the
tissues and transport carbon dioxide from the tissues for GLOBIN STRUCTURE
exhalation. The Four Globin Chains – consists of two identical pair of unlike
- It also contributes to acid-base balance by binding and polypeptide chains, 141 to 146 amino acids each.
releasing hydrogen ions and transports nitric oxide, a • Variations in amino acid sequences gives rise to different
regulator of vascular tone. types of polypeptide chains.
- Comprises approximately 95% of the cytoplasmic content o Each chain is designated by a Greek letter.
of RBCs, which provides protection from denaturation in
the plasma and loss through the kidneys.
- Concentration within RBCs: 34 g/dL
- Molecular weight: 64,000 Daltons
Free (non-RBC) hemoglobin – generated from RBCs through
hemolysis, has a short half-life outside of RBCs.
- When released into plasma, it is rapidly salvaged to
preserve its iron and amino acid components.
o When salvage capacity Is exceeded, it is excreted
by the kidneys.
• Each globin chain is divided into eight (8) helices
HEMOGLOBIN STRUCTURE separated by seven (7) nonhelical segments.
Hemoglobin - First protein whose structure was described using o Helices, designated A to H, contain subgroup
x-ray crystallography. numberings for the sequence of the amino acids
- it is a globular protein consisting of two different pairs of in each helix and are relatively rigid and linear.
polypeptide chains and four heme groups, with one heme o Flexible nonhelical segments: connect the
group imbedded in each of the four polypeptide chains. helices; NA – between N-terminus and A helix, AB
between A and B helices, and so forth, with BC,
CD, DE, EF, FG, GH, and HC between the H helix
and the C-terminus.
ROGIE BAYAUA. BEULAH GO. KRISTLE PRING. REJINOLD SERRANO. KAIZER VANGUARDIA. | FEU MT 2023 1
Predominant Adult Hemoglobin (Hb A) – is composed of two α-
globin chains and two β-globin chains.
• Strong α1-β1 and α2-β2 bonds hold dimers in a stable
form.
• α1-β2 and α2-β1 bonds: important for the stability of
quaternary structure in the oxygenated and deoxygenated
forms. (Figure 7.1)
• A small percentage of Hb A is glycated.
o Glycation is a posttranslational modification
formed by the nonenzymatic binding of various
sugars to globin chain amino groups over the life
span of the RBC.
▪ Hb A1c – most characterized of the
glycated hemoglobins, in which glucose
attaches to the N-terminal valine of the β
chain.
▪ Normally, about 4% to 6% of Hb A
circulates in this form.
▪ In uncontrolled diabetes mellitus, the
amount of A1c is increased proportionally
COMPLETE HEMOGLOBIN MOLECULE to the mean blood glucose level over the
The hemoglobin molecule can be described by: preceding 2 to 3 months.
1. Primary Structure – amino acid sequence of the
polypeptide chains. HEMOGLOBIN BIOSYNTHESIS
2. Secondary Structure – chain arrangements in helices and
HEME BIOSYNTHESIS
nonhelices.
Heme Biosynthesis – occurs in the mitochondria and cytoplasm of
3. Tertiary Structure – arrangement of helices into a pretzel-
bone marrow erythroid precursors, beginning with the
like configuration.
pronormoblast through the circulating polychromatic (aka
polychromatophilic) erythrocyte.
Globin chains – loop to form a cleft pocket for heme.
• Mature Erythrocytes – can no longer make hemoglobin
• Each chain contains a heme group that is suspended
as they lose their ribosomes and mitochondria.
between the E and F helices of the polypeptide chain.
• Heme biosynthesis begins in the mitochondria with the
• The iron atom at the center of the protoporphyrin IX ring
condensation of glycine and succinyl coenzyme A (CoA)
of heme is positioned between two histidine radicals,
catalyzed by aminolevulinate synthase to form
forming a proximal bond with F8 and, through the linked
aminolevulinic acid (ALA).
oxygen, a close association with the distal histidine residue
• In the cytoplasm, aminolevulinic acid dehydratase (aka
in E7.
porphobilinogen synthase) converts ALA to
• Globin chain amino acids in the cleft are hydrophobic,
porphobilinogen (PBG).
whereas amino acids on the outside are hydrophilic, which
• PBG undergoes several transformations in the cytoplasm
makes the molecule water soluble.
from hydroxymethylbilane to coproporphyrinogen III.
• This arrangement helps iron remain in its divalent ferrous
• This pathway then continues in the mitochondria until, in
form regardless of if it is oxygenated or deoxygenated.
the final step of production of heme, Fe2+ combines with
Quaternary Structure – also called tetramer, which describes the
protoporphyrin IX in the presence of ferrochelatase
complete hemoglobin molecule.
(heme synthase) to make heme.
• The complete hemoglobin molecule: spherical, four (4) • Transferrin, a plasma protein, carries iron in the ferric
heme groups attached to four (4) polypeptide chains, and (Fe3+) form to developing erythroid cells.
may carry up to four (4) molecules of oxygen.)
• Transferrin binds to transferrin receptors on erythroid
precursor cell membranes and the receptors and
transferrin (with bound iron) are brought into the cell in
an endosome.
• Iron is transported out of the endosome and into the
mitochondria, where it is reduced to the ferrous state and
is united with protoporphyrin IX to make heme.
• Heme leaves mitochondria and is joined to the globin
chains in the cytoplasm.
GLOBIN BIOSYNTHESIS
•
Six structural genes code for six globin chains.
•
The α- and ζ-globin genes are on the short arm of
chromosome 16.
• The ε-, γ-, β-, and δ-globin gene cluster is on the short
arm of chromosome 11.
ROGIE BAYAUA. BEULAH GO. KRISTLE PRING. REJINOLD SERRANO. KAIZER VANGUARDIA. | FEU MT 2023
• In the human genome, there is one copy of each globin per
chromatid, for a total of two genes per diploid, with the
exception of α and γ.
• There are two copies of the α- and γ-globin genes per
chromatid, for a total of four genes per diploid cell.
• Production of globin chains takes place in erythroid
precursors from the pronormoblast through the circulating
polychromatic erythrocyte, but not in mature erythrocytes.
• Transcription of the globin genes to messenger
ribonucleic acid (mRNA) occurs in the nucleus, and
translation of mRNA to the globin polypeptide chain
occurs on ribosomes in the cytoplasm.
o Although transcription of α-globin genes
produces more mRNA than the β-globin gene,
there is less efficient translation of the α-globin
mRNA.
o Therefore, α and β chains = produced in
approximately equal amounts.
• After translation is complete, chains are released from the
ribosomes in the cytoplasm. HEMOGLOBIN ONTOGENY
• Hemoglobin composition differs with prenatal gestation
HEMOGLOBIN ASSEMBLY time and postnatal age.
• After their release from ribosomes, each globin chain binds • Hemoglobin changes reflect the sequential activation and
to a heme molecule, then forms a heterodimer. inactivation (or switching) of the globin genes, progressing
• The non-α chains have charge difference that determines from the ζ- to the α-globin gene on chromosome 16 and
their affinity to bind to α chains. from the ε- to the γ-, δ -, and β -globin genes on
• The α chain has a positive charge and has the highest chromosome 11.
affinity for a β chain because of its negative charge. • The ζ- and ε-globin chains normally appear only during
• The γ-globin chain has the next highest affinity, followed the first 3 months of embryonic development.
by the δ-globin chain. o These two chains, when paired with the α and γ
• Two heterodimers then combine to form a tetramer which chains, form the embryonic hemoglobins.
then completes the hemoglobin molecule. (Figure 7.6)
• Two α and two β chains form Hb A, the major hemoglobin • During the second and third trimesters of fetal life and at
present from 6 months of age through adulthood. birth, Hb F (α2γ2) is the predominant hemoglobin, with
• Hb A2 – contains two α and two δ chains, and comprises small amounts of Hb A2 (α2δ2) and Hb F.
less than 3.5% of total hemoglobin in adults.
• Production of the δ chain polypeptide is very low due to
owing to a mutation in the promoter region of the δ-globin
gene.
• Hb F – contains two α and two γ chains. In healthy adults,
it comprises 1% - 2% of total hemoglobin, and present only
in small proportion of the RBCs (uneven distribution).
o F or A/F cells: RBCs with Hb F.
• Net charge of hemoglobin tetramer – affected by the
various amino acids that comprise the globin chains.
• Electrophoresis and High-Performance Liquid
Chromatography (HPLC) – used for fractionation,
presumptive identification, and quantification for normal
hemoglobins and hemoglobin variants.
• Molecular genetic testing of globin gene DNA –
provides definitive identification of variant hemoglobins.
REGULATION OF HEMOGLOBIN PRODUCTION

HEME REGULATION
• The key rate-limiting step in heme synthesis is the initial
reaction of glycine and succinyl CoA to form ALA,
catalyzed by ALA synthase. (Figure 7.5)
• Heme inhibits the transcription of the ALA synthase gene,
which leads to a decrease in heme production (a negative
feedback mechanism).
• Heme inhibits other enzymes in the biosynthesis pathway,
including ALA dehydrase and PBG deaminase.
• A negative feedback mechanism by heme or substrate
inhibition by protoporphyrin IX is believed to inhibit the
ferrochelatase enzyme.
• An increased demand for heme induces an increased
synthesis of ALA synthase.
GLOBIN REGULATION
Globin synthesis – highly regulated so that there is a balance
production of globin and heme; this is critical because an excess
of globin chains, protoporphyrin IX, or iron can accumulate and
damage the cell, reducing its life span.
- Also regulated during translation when mRNA coding for
the globin chains associates with ribosomes to produce the
polypeptide.
- Many protein factors are required to control initiation,
elongation, and termination steps of translation.
- Heme – is an important regulator of globin mRNA
translation at the initiation step by promoting activation of
a translation initiation factor and inactivating its
repressor.
o When heme level is low, the repressor
accumulates and inactivates the initiation factor,
thus blocking translation of the globin mRNA.
Globin production – mainly controlled at the transcription level by

a complex interaction of DNA sequences (cis-acting promoters,
enhancers, and silencers) and soluble transcription factors (trans- SYSTEMIC REGULATION OF ERYTHROPOIESIS
acting factors) that bind to DNA or to one another to promote or
• Tissue hypoxia – happens when there is an insufficient
suppress transcription.
quantity of hemoglobin or if the hemoglobin molecule is
• Initiation of transcription of a particular globin gene defective in transporting oxygen.
requires:
• Hypoxia – detected by the peritubular cells of the kidney,
(1) The promoter DNA sequences immediately before
which respond by increasing productions of
the 5’ end or the beginning of the gene.
erythropoietin (EPO).
(2) A key transcription factor called Krüppel-like factor 1
• Erythropoietin (EPO) – increases the number of
(KLF1)
erythrocytes produced and released into the periphery,
(3) A number of other transcription factors (e.g.,
which also accelerates the rate of synthesis of erythrocyte
GATA1, Ikaros, TAL1, p45-NF-E2, and LDB1).
components, including hemoglobin.
(4) An enhancer region of DNAse 1 hypersensitive
nucleic acid sequences located more than 20 • Reference intervals for hemoglobin concentration:
kilobases upstream (before the 5’ start site of the Men: 13.5 – 18.0 g/dL (135-180 g/L)
gene) from the globin gene called locus control Women: 12.0 – 15.0 g/dL (120-150 g/L)
region (LCR). Newborns: 16.5 – 21.5 g/dL (165-215 g/L)
o For example, to activate transcription of the β- o Reference intervals for infants and children vary
globin gene cluster on chromosome 11, the LCR, according to age group.
the promoter for the β-globin gene, and various o Individuals living at high altitudes have slightly
transcription factors join together to from a higher levels of hemoglobin as a compensatory
three-dimensional active chromosome hub mechanism to provide more oxygen to the tissues
(ACH), with KLF1 playing a key role in connecting in the oxygen-thin air.
the complex.
o Because the LCR is located a distance upstream HEMOGLOBIN FUNCTION
from the β-globin gene complex, a loop of DNA
is formed when the LCR and β-globin gene
promoter join together in the chromosome hub. OXYGEN TRANSPORT
The other globin genes in the cluster (ε-, γ-, δ-) • The function of hemoglobin is to readily bind oxygen
are maintained in the inactive state in the DNA molecules in the lung, which requires high oxygen
loop, so only the β-globin gene is transcribed. affinity; to transport oxygen; and to efficiently unload
• Krüppel-like factor 1 (KLF1) – also plays a key regulatory oxygen to the tissues, which requires low oxygen affinity.
role in the switch from γ chain to β chain production (γ-β • During oxygenation, each of the four heme iron atoms in a
switching) that begins in late fetal life and continues hemoglobin molecule can reversibly bind one oxygen
through adulthood. molecule.
o It is an exact match for binding to DNA promoter
• The affinity of hemoglobin for oxygen relates to the partial
sequences of the β-globin gene, whereas the γ-
pressure of oxygen (PO2), often defined in terms of the
globin gene promoter has a slightly different
amount of oxygen needed to saturate 50% of hemoglobin,
sequence; this results in a preferential binding to
called P50 value.
and subsequent activation of transcription of
the β-globin gene.
o KLF1 also regulates the expressions of
repressors of γ-globin gene transcription (e.g.,
BCL11A and MYB).
Hemoglobin-Oxygen Dissociation Curves • The concentration of 2,3-bisphosphoglycerate (2,3-
BPG, formerly 2,3-diphosphoglycerate) also has an
effect on oxygen affinity. In the deoxygenated state, the
hemoglobin tetramer assumes a tense or T conformation
that is stabilized by the binding of 2,3-BPG between the b-
globin chains. The formation of salt bridges between the
phosphates of 2,3-BPG and positively charged groups
on the globin chains further stabilizes the tetramer in the T
conformation. The binding of 2,3-BPG shifts the oxygen
dissociation curve to the right, favoring the release of
oxygen. In addition, a lower pH and higher PCO2 in the
tissues further shifts the curve to the right, favoring the
release of oxygen.
o Cooperation among hemoglobin subunits

contributes to the shape of the curve. Hemoglobin
that is completely deoxygenated has little affinity
for oxygen. However, with each oxygen molecule
that is bound, there is a change in the
conformation of the tetramer that progressively
increases the oxygen affinity of the other heme • Clinical conditions that produce a shift of the oxygen
subunits. dissociation curve to the left include a lowered body
temperature as a result of external causes; multiple
transfusions of stored blood with depleted 2,3-BPG;
• Once one oxygen molecule binds, the remainder of the
alkalosis; and presence of hemoglobin variants with a high
hemoglobin molecule quickly becomes fully oxygenated.
affinity for oxygen. Conditions producing a shift of the
Therefore, with high oxygen tension in the lungs, the
curve to the right include increased body temperature;
affinity of hemoglobin for oxygen is high, and
acidosis; presence of hemoglobin variants with a low
hemoglobin becomes rapidly saturated with oxygen.
affinity for oxygen; and an increased 2,3-BPG
Conversely, with relatively low oxygen tension in the
concentration in response to hypoxic conditions, such as
tissues, the affinity of hemoglobin for oxygen is low,
high altitude, pulmonary insufficiency, congestive heart
and hemoglobin rapidly releases oxygen.
failure, and severe anemia.
o The reference interval for arterial oxygen
saturation is 96% to 100%. If the oxygen CARBON DIOXIDE TRANSPORT
dissociation curve shifts to the left, a patient with
arterial and venous PO2 levels in the reference • A second crucial function of hemoglobin is the transport of
intervals (80 to 100 mm Hg arterial and 30 to 50 carbon dioxide. In venous blood, the carbon dioxide
mm Hg venous) will have a higher percent oxygen diffuses into the RBCs and combines with water to
saturation and a higher affinity for oxygen than a form carbonic acid (H2CO3).
patient for whom the curve is normal. With a shift o This reaction is facilitated by the RBC enzyme
in the curve to the right, a lower oxygen affinity is carbonic anhydrase. Carbonic acid then dissociates
seen. to release 𝐻 + and bicarbonate (HCO3− )
o The 𝐻 + from the second reaction binds
o Shifts of the curve to the left or right also occur if oxygenated hemoglobin (HbO2), and the oxygen is
there are changes in the pH of the blood. In the released from the hemoglobin because of the Bohr
tissues, a lower pH shifts the curve to the right and effect. The oxygen then diffuses out of the cell into the
reduces the affinity of hemoglobin for oxygen, and tissues. As the concentration of the negatively
the hemoglobin more readily releases oxygen. A charged bicarbonate increases, it diffuses across the
shift in the curve because of a change in pH (or RBC membrane into the plasma. Chloride (Cl2), also
hydrogen ion concentration) is termed the Bohr negatively charged, diffuses from the plasma into the
effect. It facilitates the ability of hemoglobin to cell to maintain electroneutrality across the
exchange oxygen and carbon dioxide (CO2). membrane; this is called the chloride shift.
• Approximately 85% of the CO2 produced in the tissues
is transported by hemoglobin as 𝑯+ . In this capacity,
hemoglobin provides a buffering effect by binding and
releasing 𝐻 + . A small percentage of CO2 remains in the
cytoplasm and the remainder binds to the globin chains as
a carbamino group.
• Methemoglobin cannot carry oxygen because oxidized
ferric iron cannot bind it. An increase in methemoglobin
level results in decreased delivery of oxygen to the tissues.
Individuals with methemoglobin levels less than 25% are
generally asymptomatic.
• An increase in methemoglobin, called
methemoglobinemia, can be acquired or hereditary. The
acquired form, also called toxic methemoglobinemia,
occurs in normal individuals after exposure to an
exogenous oxidant, such as nitrites, primaquine,
dapsone, or benzocaine.
• As the oxidant overwhelms the hemoglobin reduction
systems, the level of methemoglobin increases, and the
patient may exhibit cyanosis and symptoms of
hypoxia. In many cases, withdrawal of the offending
oxidant is sufficient for a recovery, but if the level of
methemoglobin increases to 30% or more of total
hemoglobin, intravenous methylene blue is
administered.
• Methylene blue reduces methemoglobin ferric iron to
the ferrous state through NADPH-methemoglobin
reductase and NADPH produced by glucose-6-
phosphate dehydrogenase in the hexose
NITRIC OXIDE TRANSPORT
monophosphate shunt. In life-threatening cases,
exchange transfusion may be required.
• A third function of hemoglobin involves the binding,
• Hereditary causes of methemoglobinemia are rare and
inactivation, and transport of nitric oxide.1,10 Nitric oxide is
include mutations in the gene for NADH-cytochrome
secreted by vascular endothelial cells and causes
b5 reductase 3 (CYB5R3), resulting in a diminished
relaxation of vascular wall smooth muscle and
capacity to reduce methemoglobin, and mutations in the α-
vasodilation. When released, free nitric oxide has a very
, β-, γ-globin gene, resulting in a structurally abnormal
short half-life, but some enters RBCs and can bind to
polypeptide chain that favors the oxidized ferric form of
cysteine in the b chain of hemoglobin, forming S-
iron and prevents its reduction. The methemoglobin
nitrosohemoglobin.
produced by the latter group is called M hemoglobin or
• Some investigators propose that hemoglobin preserves Hb M.
and transports nitric oxide to hypoxic microvascular o Hb M is inherited in an autosomal dominant
areas, which stimulates vasodilation and increases pattern, with methemoglobin comprising 30% to
blood flow (hypoxic vasodilation). In this way, hemoglobin 50% of total hemoglobin.12 There is no effective
may work with other systems in regulating local blood flow treatment for this form of
to microvascular areas by binding and inactivating nitric methemoglobinemia.
oxide (causing vasoconstriction and decreased blood flow) o Cytochrome b5 reductase deficiency is an
when oxygen tension is high and releasing nitric oxide autosomal recessive disorder, and
(causing vasodilation and increased blood flow) when methemoglobin elevations occur in individuals
oxygen tension is low. who are homozygous or compound
heterozygous for a CYB5R3 mutation.
DYSHEMOGLOBINS o Most individuals with Hb M or homozygous
cytochrome b5 reductase deficiency maintain
• Dyshemoglobins (dysfunctional hemoglobins that are methemoglobin levels less than 50%; they
unable to transport oxygen) include methemoglobin, have cyanosis but only mild symptoms of
sulfhemoglobin, and carboxyhemoglobin. hypoxia that do not require treatment.
Dyshemoglobins form and may accumulate to toxic levels, • Methemoglobin is assayed by spectral absorption
after exposure to certain drugs or environmental chemicals analysis instruments such as the CO-oximeter.
or gasses. The offending agent modifies the structure Methemoglobin shows an absorption peak at 630 nm.
of the hemoglobin molecule, preventing it from With high levels of methemoglobin, the blood takes on a
binding oxygen. Most cases of dyshemoglobinemia chocolate brown color and does not revert back to
are acquired; a small fraction of methemoglobinemia normal red color after oxygen exposure.
cases are hereditary.
SULFHEMOGLOBIN
METHEMOGLOBIN • Sulfhemoglobin - is formed by irreversible oxidation of
hemoglobin by drugs (such as sulfanilamides,
• Methemoglobin (MetHb) is formed by the reversible phenacetin, nitrites, and phenylhydrazine) or exposure
oxidation of heme iron to the ferric state (𝐹𝑒 3+ ). to sulfur chemicals in industrial or environmental settings.
• Normally, a small amount of methemoglobin is • It is formed by the addition of a sulfur atom to the
continuously formed by oxidation of iron during normal pyrrole ring of heme and has a greenish pigment.
oxygenation and deoxygenation of hemoglobin. However, Sulfhemoglobin is ineffective for oxygen transport, and
methemoglobin reduction systems, predominantly the patients with elevated levels present with cyanosis.
NADH-cytochrome b5 reductase 3 (NADH- Sulfhemoglobin cannot be converted to normal Hb A;
methemoglobin reductase) pathway, normally limit its it persists for the life of the cell. Treatment consists of
accumulation to only 1% of total hemoglobin.
prevention by avoidance of the offending agent. • Hemoglobin electrophoresis and HPLC are used to
• Sulfhemoglobin has a similar peak to methemoglobin on a separate the different types of hemoglobins such as Hb A,
spectral absorption instrument. The sulfhemoglobin A2, and F.
spectral curve, however, does not shift when cyanide is
added, a feature that distinguishes it from methemoglobin.
CARBOXYHEMOGLOBIN
• Carboxyhemoglobin (COHb) - results from the
combination of carbon monoxide (CO) with heme iron.
o The affinity of carbon monoxide for hemoglobin is
240 times that of oxygen.
o Once one molecule of carbon monoxide binds to
hemoglobin, it shifts the hemoglobin-oxygen
dissociation curve to the left, further increasing its
affinity and severely impairing release of oxygen to the
tissues.
• Carbon monoxide has been termed the silent killer
because it is an odorless and colorless gas, and
victims may quickly become hypoxic.
• Some carboxyhemoglobin is produced endogenously, but
it normally comprises less than 2% of total hemoglobin.
• Exogenous carbon monoxide is derived from the exhaust
of automobiles, tobacco smoke, and from industrial
pollutants, such as coal, gas, and charcoal burning. In
smokers, COHb levels may be as high as 15%. As a
result, smokers may have a higher hematocrit and
polycythemia to compensate for the hypoxia.
o Toxic effects, such as headache, dizziness, and
disorientation, begin to appear at blood levels of 20%
to 30% COHb. Levels of more than 40% of total
hemoglobin may cause coma, seizure,
hypotension, cardiac arrhythmias, pulmonary
edema, and death.
• Carboxyhemoglobin may be detected by spectral
absorption instruments at 540 nm. It gives blood a
cherry red color, which is sometimes imparted to the skin
of victims.
o A diagnosis of carbon monoxide poisoning is made if
the COHb level is greater than 3% in nonsmokers and
greater than 10% in smokers. Treatment involves
removing the carbon monoxide source and
administration of 100% oxygen. Use of hyperbaric
oxygen therapy is controversial; it is primarily used
to prevent neurologic and cognitive impairment after
acute carbon monoxide exposure in patients whose
COHb level exceeds 25%.
HEMOGLOBIN MEASUREMENT
• The cyanmethemoglobin method is the reference

method for hemoglobin assay.
o A lysing agent present in the cyanmethemoglobin
reagent frees hemoglobin from RBCs. Free
hemoglobin combines with potassium ferricyanide
contained in the cyanmethemoglobin reagent, which
converts hemoglobin iron from the ferrous to the ferric
state to form methemoglobin.
• Methemoglobin combines with potassium cyanide to form
the stable pigment cyanmethemoglobin. The
cyanmethemoglobin color intensity, which is proportional to
hemoglobin concentration, is measured at 540 nm
spectrophotometrically and compared with a
standard.
• Many instruments now use sodium lauryl sulfate (SLS)
to convert hemoglobin to SLS-methemoglobin. This
method does not generate toxic wastes.
HEMATOLOGY 1
[TRANS] CHAPTER 19: BONE MARROW FAILURE
• Ehrlich (1888) - provided the first case report of aplastic

OUTLINE anemia involving a patient with severe anemia,
BONE MARROW FAILURE neutropenia, and a hypocellular marrow on postmortem
I. Pathophysiology of Bone Marrow Failure
examination.
II. Aplastic Anema • The name “aplastic anemia” was given to the disease by
1. Acquired Aplastic Anemia Vaquez and Aubertin in 1904.
2. Inherited/Congenital Bone Marrow Failure • Characteristic features of aplastic anemia include:
Syndromes o pancytopenia
3. Differential Diagnosis o reticulocytopenia
III. Other Forms of Bone Marrow Failure o bone marrow hypocellularity
1. Pure Red Cell Aplasia
2. Congenital Dyserythropoietic Anemia
o depletion of hematopoietic stem cells
3. Myelopthistic Anemia • Approximately 80% to 85% of aplastic anemia cases are
4. Anemia of Chronic Kidney Disease acquired, whereas 15% to 20% are inherited/congenital.
PATHOPHYSIOLOGY OF BONE MARROW FAILURE Acquired Aplastic Anemia

• Bone marrow failure is the reduction or cessation of blood • Two major categories:
cell production affecting one or more cell lines. o Idiopathic
• Seen in most cases of bone marrow failure, particularly in o Secondary
severe or advanced stages: 1. Idiopathic acquired aplastic anemia
o Pancytopenia - decreased numbers of o No known cause.
circulating red blood cells (RBCs), white blood o Approximately 70% of all aplastic anemia cases
cells (WBCs), and platelets. are idiopathic.
2. Secondary acquired aplastic anemia
Pathophysiology of bone marrow failure includes: o associated with an identified cause.
o 10% to 15% cases
1. Destruction of hematopoietic stem cells as a result of injury
▪ Idiopathic and secondary acquired
by drugs, chemicals, radiation, viruses, or autoimmune
aplastic anemia have similar clinical and
mechanisms
laboratory findings.
2. Premature senescence and apoptosis of hematopoietic
stem cells as a result of genetic mutations
3. Ineffective hematopoiesis caused by stem cell mutations or INCIDENCE
vitamin B12 or folate deficiency
4. Disruption of the bone marrow microenvironment that • North America and Europe: the annual incidence is
supports hematopoiesis approximately 1 in 500,000.
5. Decreased production of hematopoietic growth factors or • Asia and East Asia: the incidence is two to three times
related hormones higher than in North America or Europe, which may be due
6. Loss of normal hematopoietic tissue as a result of to environmental and/or genetic differences.
infiltration of the marrow space with abnormal cells.
• Aplastic anemia can occur at any age, with peak incidence
Clinical consequences of bone marrow failure: at 15 to 25 years and the second highest frequency at older
• Severe pancytopenia can be rapidly fatal if untreated. than 60 years.
Some patients may initially be asymptomatic, and their o There is no gender predisposition.
cytopenia may be detected during a routine blood
examination.
ETIOLOGY
• Thrombocytopenia can result in bleeding and increased
bruising.
• Decreased RBCs and hemoglobin can result in fatigue, • Cause of idiopathic aplastic anemia: unknown.
pallor, and cardiovascular complications. • Secondary aplastic anemia is associated with exposure to
• Sustained neutropenia increases the risk of certain drugs, chemicals, radiation, or infections.
lifethreatening bacterial or fungal infections. o Alternatively, approximately 70% of cases
of secondary aplastic anemia occur as a
result of idiosyncratic reactions to drugs or
APLASTIC ANEMIA chemicals.
• Aplastic anemia - a bone marrow failure syndrome • A deficiency in GST as a result of the GSTT1 null genotype
resulting from damaged or defective stem cells. is overrepresented in Caucasians, Hispanics, and Asians
o Rare but potentially fatal bone marrow failure A with aplastic anemia, with a frequency of 30%, 28%, and
deficiency in GST as a result of 75%, respectively.
o the GSTT1 null genotype is overrepresented in o GST - important for metabolism and
Caucasians, neutralization of chemical toxins, and
o Hispanics, and Asians with aplastic anemia, with deficiencies of this enzyme may increase
a frequency of the risk of aplastic anemia.
o 30%, 28%, and 75%, respectively.syndrome.
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• Primary lesion in acquired aplastic anemia: o Increased T cell production of such cytokines as
o Quantitative deficiency of hematopoietic IFN-g and tumor necrosis factor-a (TNF-a), which
stem cells inhibit hematopoiesis and induce apoptosis
o Qualitative deficiency of hematopoietic o Upregulation of T-bet, a transcription factor that
stem cells binds to the promoter of the IFN-g gene
o Increased TNF-a receptors on CD341 cells
• Stem cells of patients with acquired aplastic anemia have o Improvement in cytopenias after
diminished colony formation in methylcellulose immunosuppressive therapy
cultures.
• The hematopoietic stem and early progenitor cell • Possible autoimmune mechanisms include:
compartment - identified by expression of CD34 surface o mutation of stem cell antigens
antigens. o disruption of immune regulation
o The CD341 cell population in the bone marrow of
patients with acquired aplastic anemia can be • Candidate antigens have been identified from aplastic
10% or lower than that seen in healthy individuals. anemia patient sera, including kinectin, diazepam-
o These CD341 cells have increased expression of binding inhibitor-related protein and moesin.
Fas receptors that mediate apoptosis and o These proteins are expressed in hematopoietic
increased expression of apoptosis-related genes. progenitor cells, but their role in the pathogenesis
• Bone marrow stromal cells - functionally normal in of aplastic anemia requires further investigation.
acquired aplastic anemia.
o They produce normal or even increased • Approximately one-third of patients with acquired aplastic
quantities of growth factors and are able to anemia have shortened telomeres in their peripheral
support the growth of CD341 cells from healthy blood granulocytes compared with age-matched controls.
donors in culture and in vivo after transplantation. • Telomeres - protect the ends of chromosomes from
damage and erosion, and cells with abnormally short
• Individuals with aplastic anemia also have elevated telomeres undergo proliferation arrest and premature
serum levels of: apoptosis.
o erythropoietin • Telomerase - an enzyme complex that repairs and
o thrombopoietin maintains telomeres.
o granulocyte colony-stimulating factor (G-CSF) • Cause for shortened telomeres in the other 90% of
o granulocytemacrophage colony-stimulating factor patients:
(GM-CSF). o stress hematopoiesis or other yet unidentified
mutations.
• FLT3 ligand- a growth factor that stimulates proliferation ▪ In stress hematopoiesis there is an
of stem and progenitor cells. increase in progenitor cell turnover, and
o Serum levels of FLT3 is up to 200 times higher the telomeres become shorter with each
in patients with severe aplastic anemia cell division.
compared with healthy controls. • Approximately 4% of patients with acquired aplastic
o However, despite their elevated levels, growth anemia and shortened telomeres have mutations in the
factors are generally unsuccessful in ShwachmanBodian-Diamond syndrome (SBDS) gene.
correcting the cytopenias found in acquired o The SBDS gene product is involved in ribosome
aplastic anemia. biogenesis, and its relationship to telomere
maintenance is currently unknown.
• The severe depletion of hematopoietic stem and progenitor • Immunosuppressive therapy - not nearly as effective in
cells from the bone marrow may be due to: inherited bone marrow failure compared with acquired
o direct damage to stem cells aplastic anemia.
o immune damage to stem cells • Hematopoietic stem cell transplantation - the only
known curative treatment for DC and SBDS and a
✓ Direct damage to stem and progenitor cells – results treatment option for acquired aplastic anemia, should not
from deoxyribonucleic acid (DNA) injury after exposure to be performed with HLA-matched siblings who test positive
cytotoxic drugs, chemicals, radiation, or viruses. for the same genetic mutation.
✓ Immune damage to stem cells - results from exposure to
drugs, chemicals, viruses, or other agents that cause an
autoimmune cytotoxic T lymphocytic destruction of stem CLINICAL FINDINGS
and progenitor cells.
• Symptoms vary in acquired aplastic anemia, ranging from
asymptomatic to severe.
• Autoimmune pathophysiology - was first suggested in
the 1970s when aplastic anemia patients undergoing • Symptoms:
pretransplant immunosuppressive conditioning had an o insidious-onset anemia
improvement in cell counts. o pallor
• Further evidence supporting an autoimmune o fatigue
o weakness
pathophysiology include:
o Elevated blood and bone marrow cytotoxic • Severe and prolonged anemia:
(CD81) T lymphocytes with an oligoclonal o serious cardiovascular complications including:
expansion of specific T cell clones ▪ tachycardia, hypotension, cardiac
failure, and death.
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• Symptoms of thrombocytopenia are also varied and among granulocytes.
include:
o Petechiae
o Bruising
o Epistaxis
o mucosal bleeding
o menorrhagia
o retinal hemorrhages
o intestinal bleeding
o intracranial hemorrhage.
• Fever and bacterial or fungal infections - unusual at
initial presentation but may occur after prolonged periods
of neutropenia.
• Splenomegaly and hepatomegaly - typically absent.
LABORATORY FINDINGS
• Pancytopenia is typical, although initially only one or two
cell lines may be decreased.
• The absolute neutrophil count is decreased, and the
absolute lymphocyte count may be normal or decreased.
• The hemoglobin is usually less than 10 g/dL, the mean
cell volume (MCV) is increased or normal, and the
percent and absolute reticulocyte counts are decreased.
• Neutrophils, monocytes, and platelets are decreased in
the peripheral blood, and the RBCs are macrocytic or
normocytic.
• Toxic granulation may be observed in the neutrophils, but • Bone marrow aspirate and biopsy specimens have
the RBCs and platelets are usually normal in appearance. prominent fat cells with areas of patchy marrow cellularity.
• Leukemic blasts and other immature blood cells are o Biopsyspecimens are required for accurate
characteristically absent. quantitative assessment of marrow cellularity,
• The serum iron level and percent transferrin saturation and severe hypocellularity is a characteristic
may be increased, which reflects decreased iron use for feature of aplastic anemia (Figure 19.2).
• erythropoiesis. • Erythroid, granulocytic, and megakaryocytic cells are
• Liver function test results may be abnormal in cases of decreased or absent.
hepatitis-associated aplastic anemia. • Dyserythropoiesis may be present, but there is typically
• PNH is characterized by an acquired stem cell mutation no dysplasia of the granulocyte or platelet cell lines.
resulting in lack of the glycosylphosphatidylinositol • Blasts and other abnormal cell infiltrates are
(GPI)- linked proteins CD55 and CD59. characteristically absent.
o The absence of CD55 and CD59 on the surface • Reticulin staining is usually normal.
of the RBCs renders them more susceptible to
complement-mediated cell lysis. • In patients receiving immunosuppressive therapy, the risk
of developing an abnormal karyotype:
• It is important to test for PNH in acquired aplastic anemia o 14% at 5 years
because of the increased risk of hemolytic or thrombotic o 20% at 10 years.
complications. • Monosomy 7 and trisomy 8 - the most common
o Historically, PNH diagnosis depended on the cytogenetic abnormalities.
Ham acid hemolysis test: • Cytogenetic analysis using conventional culture
▪ Patients’ cells were placed in acidified techniques often underestimates the incidence of
serum, and a positive result karyotype abnormalities because of bone marrow
demonstrated lysis of RBCs. hypocellularity and scarcity of cells in metaphase.
▪ However, this test was poorly sensitive, o Alternatively, interphase fluorescence in situ
because complement-mediated hybridization (FISH) using DNA probes for
hemolysis was detected only in the specific chromosome abnormalities may be
presence of large numbers of circulating used.
PNH cells. o In comparison to conventional cytogenetic
▪ The Ham test has been replaced by flow analysis, FISH has greater sensitivity in the
cytometric analysis for proteins linked to detection of chromosome abnormalities and
the GPI anchor; CD55 and CD59 on can also be performed using nondividing cells.
RBCs, and CD24 and CD14 on
granulocytes and monocytes. • Patients with an inherited bone marrow failure syndrome
▪ Flow cytometry for the GPI anchor may be misdiagnosed with acquired aplastic anemia if:
(FLAER assay) - the newest and most o symptoms manifest in late adolescence or
sensitive assay for detecting PNH cells adulthood
3
ROGIE BAYAUA. BEULAH GO. KRISTEL PRING. REJINOLD SERRANO. KAIZER VANGUARDIA. | FEU MT 2023
o the patients lack the typical clinical and physical • Low birth weight and developmental delay are
characteristics of an inherited marrow failure also common.
TREATMENT AND PROGNOSIS • The symptoms associated with pancytopenia usually
become apparent at 5 to 10 years of age, though some
syndrome (e.g., abnormal thumbs, short stature). patients may not present until adulthood.
• Severe acquired aplastic anemia • Individuals with FA also have an increased cancer risk.
o requires immediate attention to prevent serious This includes an increased incidence of leukemia in
complications. childhood and solid tumors (e.g., oral, esophageal,
o If a causative agent is identified, its use should be anogenital, cervical) in adulthood.
discontinued. • In approximately 5% of cases a malignancy is diagnosed
o Blood product replacement should be given before the FA is recognized.
judiciously to avoid alloimmunization.
o Platelets should not be transfused at counts
greater than 10,000/mL, unless the patient is
GENETICS AND PATHOPHYSIOLOGY
bleeding. • Patients with FA typically have biallelic mutations or
• BMT is the treatment of choice for patients with severe deletions in one of at least 21 genes: FANCA, FANCB,
aplastic anemia who are younger than 40 years of age and FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF,
have an HLA-identical sibling. FANCG (XRCC9), FANCI, FANCJ (BRIP1/ BACH1),
o Unfortunately, only 20% to 30% of patients meet FANCL, FANCM, FANCN (PALB2), FANCO (RAD51C),
these criteria. FANCP (SLX4), FANCQ (ERCC4 or XPF), FANCR
• IST, consisting of antithymocyte globulin and (RAD51), FANCS (BRCA1), FANCT (UBE2T), FANCU
cyclosporine, is used for patients older than 40 years of age (XRCC2), and FANCV (MAD2L2, REV7).
and for patients without an HLA identical sibling. • The mode of inheritance is autosomal recessive except for
o Antithymocyte globulin decreases the number FANCB, which is X-linked recessive.
of activated T cells, and cyclosporine inhibits T • Mutations in the FANCA gene occur with the highest
cell function, thereby suppressing the frequency (60%); this is followed by FANCC (14%) and
autoimmune reaction against the stem cells. FANCG (10%), whereas mutations in the other FANC
genes are much less common.
• For patients with severe acquired aplastic anemia who • FA cells may also have accelerated telomere shortening
are not responsive to IST, BMT from a high-resolution and apoptosis, a late S-phase cell cycle delay,
HLA-matched unrelated donor is an option. hypersensitivity to oxidants, and cytokine dysregulation.
• Other supportive therapy includes antibiotic and • The FA pathway consists of a nuclear core complex, a
antifungal prophylaxis in cases of prolonged protein ID complex, and effector proteins.
neutropenia. • The FA proteins A, B, C, E, F, G, L, and M form the nuclear
core complex; proteins D2 and I form the ID complex; and
INHERITED/CONGENITAL BONE MARROW the effector proteins are D1, J, N, O, P, Q, R, S, T, U, and
FAILURE SYNDROMES V.
• The three most common inherited/congenital bone marrow • The core complex facilitates the monoubiquitylation and
failure disorders associated with pancytopenia are activation of the ID complex. The ID complex then localizes
Fanconi anemia, dyskeratosis congenita, and with effector DNA repair proteins at foci of DNA damage to
Shwachman-Bodian-Diamond syndrome. effect DNA repair.
FANCONI ANEMIA LABORATORY FINDINGS

• Fanconi anemia (FA) is a chromosome instability disorder • Laboratory results are similar to those in acquired aplastic
characterized by aplastic anemia, physical abnormalities, anemia, with pancytopenia, reticulocytopenia, and a
and cancer susceptibility. hypocellular bone marrow.
• In 1927 Dr. Guido Fanconi first described this syndrome • Macrocytic RBCs are often the first detected abnormality,
in three brothers with skin pigmentation, short stature, and and thrombocytopenia usually precedes the development
hypogonadism. of the other cytopenias.
• FA is the most common of the inherited bone marrow • Fetal hemoglobin (Hb F) may be strikingly elevated, and a
failure syndromes. fetoprotein is also increased.
• Chromosomal breakage analysis is the diagnostic test
CLINICAL FINDINGS for Fanconi anemia.
• Physical malformations may be present at birth, though • Patients’ peripheral blood lymphocytes are cultured with
hematologic abnormalities may not appear until older the DNA cross-linking agents diepoxybutane (DEB) or
childhood or adulthood. mitomycin C (MMC).
• These anomalies vary considerably, though there is a • Compared with normal lymphocytes, FA cells have a
higher frequency of skeletal abnormalities (thumb greater number of characteristic chromosome breaks and
malformations, radial hypoplasia, microcephaly, hip ring chromosomes, indicating increased fragility.
dislocation, and scoliosis); skin pigmentation • Diagnosis is confirmed by genetic testing for FA gene
(hyperpigmentation, hypopigmentation, café-au-lait mutations or deletions.
lesions); short stature; and abnormalities of the eyes,
kidneys, and genitals. TREATMENT AND PROGNOSIS
4
• More than 90% of FA patients develop bone marrow failure ✓ Collagen vascular disease
by 40 years of age. ✓ Exposure to drugs/chemicals
• One-third of patients develop MDS and/or acute myeloid - Cyclosporine has a higher response
leukemia (AML) by a median age of 14 years, and 25% rate (65% to 87%) corticosteroid (30%
develop solid tumors by a median age of 26 years. to 62%) better suited for long-term
• Squamous cell carcinomas of the head and neck, maintenance if needed.
anogenital region, and skin are the most common solid o Transiernt erythroblastopenia of childhood
tumors, followed by tumors of the liver, brain, and kidney. (TEC) – the acquired form of PRCA in a
• Patients with FA have an increased risk of developing young child.
vulvar cancer, esophageal cancer, AML, and • Congenital Pure Red Cell Aplasia: Diamond-Blackfan
head/neck cancer compared with the general population. Anemia (DBA) – a erythroid hypoplastic disorder of early
• Left untreated, death by 20 years of age secondary to bone infancy with an estimated incidence of 7 to 10 cases per
marrow failure or malignancy is common. million live births. Mutations are identified in 17 genes that
• Patients with mutations in the FANCC gene experience encode ribosome proteins. RPS7, RPS10, RPS17,
bone marrow failure at a particularly young age and have RPS19, RPS24, RPS26, RPS27, RPS28, and RPS29 in
the poorest survival. the 40S subunit and RPL5, RPL11, RPL15, RPL26,
RPL27, RPL31, and RPL35A in the 60S subunit, as well as
• Increased telomere shortening in FA cells is associated encoding GATA 1, a transcription factor important for
with more severe pancytopenia and a higher risk of hematopoiesis.
malignancy. ✓ RPS19 gene occur with highest
• Supportive treatment for cytopenia includes transfusions frequency (25%)
and administration of cytokines (G-CSF and GM-CSF). ✓ RPL11 gene & RP26 are second highest
• The only curative treatment is hematopoietic stem cell frequency (6%)
transplant, preferably from an HLA-identical sibling, - Other known mutations are uncommon
although high- resolution (molecular) HLA-matched • A severe macrocytic anemia with reticulocytopenia
unrelated donors can be used with almost the same is the most common finding in peripheral blood. WBC
success rate. counts are normal or slightly lower, and platelet counts
• Gene therapy has been attempted in clinical trials but has are normal or slightly higher. Because myeloid cells
not been successful. and megakaryocytes have normal cellularity and
erythroid cells have hypoplasia, bone marrow testing
DIFFERENTIAL DIAGNOSIS distinguishes DBA from aplastic anemia's hypocellular
• A distinction must be made between acquired aplastic marrow. The karyotype in DBA is usually normal, and
anemia, inherited/congenital bone marrow failure genetic testing can identify a DBA-related mutation in
syndromes, and other causes of pancytopenia, including about 70% of patients. In most cases, Hb F and
PNH, MDS, megaloblastic anemia, and leukemia. erythrocyte adenosine deaminase are increased;
these findings distinguish DBA from TEC, in which
• Alternative diagnoses include lymphoma, myelofibrosis,
these levels are normal
and mycobacterial infections, which also may present with
pancytopenia.
• Bone marrow evaluation and molecular testing for • Congenital Dyserythropoietic Anemia – are a
chromosome abnormalities and gene mutations can further heterogenous group of a rare disorder characterized
distinguish these diagnoses. by:
✓ Anemia
• Anorexia nervosa also may present with pancytopenia. ✓ Reticulocytopenia
• In these cases the bone marrow is hypocellular and has a ✓ Hypercellular bone marrow (marked
decreased number of fat cells. ineffective erythropoiesis)
✓ Distinctive dysplastic changes in bone marrow
OTHER FORMS OF BONE MARROW FAILURE erythroblasts
o Some types of megaloblastic growth exist,
however it is unrelated to vitamin B12 or folate
• Pure Red Cell Aplasia – it is a rare disorder of insufficiency.
erythropoiesis characterized by a selective and severe o Secondary hemosiderosis – arise from chronic
decrease in erythroid precursors in an otherwise normal intermedullary and extreme medullary hemolysis.
bone marrow. Types of CDA:
➢ CDA I - inherited in an autosomal
• Acquired Pure Red Cell Aplasia – it can occur in child
recessive pattern and is characterized by
or in adult and can acute or chronic.
o Primary PRCA – idiopathic or auto immune- a mild to severe chronic anemia caused
by mutations in the CDAN1 or C15orf41
related
genes. Malformations of fingers/toes,
o Secondary PRCA – may occur in association
brown pigmentation, and neurologic
with an underlying:
defects are mostly found in CDA I.
✓ Thymoma ➢ CDA II – most common subtype and it is
✓ Hematologic malignancy inherited in autosomal recessive pattern.
✓ Solid tumor Also known as HEMPAS (hereditary
✓ Infection erythroblast multinuclearity with positive
✓ Chromic hemolytic anemia acidified serum). The anemia in CDA II is
mild to moderate with hemoglobin
5
ranging from 9 g/dL to 12 g/dl and mean
hemoglobin of 11 g/dL.
➢ CDA III – the least common subtype. This
familial autosomal dominant form is
associated with mutations in the KIF23
gene, which codes for a protein involved
in cytokinesis. The anemia is mild, and
the hemoglobin is usually in the range of
8 to 14 g/dL, with a mean of 12 g/dL.
RBCs are macrocytic, and poikilocytosis
and basophilic stippling are evident.
• Myelophthisic Anemia - due to the infiltration of abnormal

cells into the bone marrow and subsequent destruction and
replacement of normal hematopoietic cells.
o Metastatic solid tumor cells (particularly from lung,
breast, and prostate), fibroblasts, and inflammatory
cells (such as those found in miliary tuberculosis and
fungal infections) have been implicated. Cytopenia
results from the release of substances such as
cytokines and growth factors that suppress
hematopoiesis and destroy stem, progenitor, and
stromal cells.
o With normocytic erythrocytes and
reticulocytopenia, the anemia is mild to moderate.
Teardrop erythrocytes and nucleated RBCs, as well
as immature myeloid cells, are found in peripheral
blood.o
• Anemia of Chronic kidney Disease
o The primary cause of anemia in CKD is
inadequate renal production of erythropoietin
without erythropoietin, the bone marrow lacks
adequate stimulation to produce RBCs.
➢ Another contributor is the chronic
inflammation. Inflammatory cytokines
increase production of hepcidin by the
liver, which decreases the iron available
for erythropoiesis.
➢ Uremia also inhibits erythropoiesis and
increases RBC fragility.
➢ Hemodialysis and frequent blood
draws result in chronic blood loss.
Anemia of CKD is normocytic and
normochromic with reticulocytopenia.
o The Kidney Disease: Improving Global
Outcomes (KDIGO) Clinical Practice Guideline
for Anemia recommended to have a periodic
hemoglobin testing in patients with CKD and
investigation of the anemia.
6
HEMATOLOGY 1
[LEC] CHAPTER 16: Anemias: Red Blood Cell Morphology and Approach to Diagnosis
OUTLINE disease, neurologic symptoms, previous medications,

UNIT TITLE previous episodes of jaundice, and various underlying disease
processes that result in anemia.
I. Definition of Anemia
 Alternatively, individuals with anemia may be asymptomatic.
II. Importance of Patient History and Clinical
Findings  Certain features should be evaluated closely during the
III. Physiologic Adaptations in Patients with physical examination:
Anemia o skin (for petechiae)
IV. Mechanisms of Anemia o eyes (for pallor, jaundice, and hemorrhage)
1. Ineffective and Insufficient Erythropoiesis o mouth (for mucosal bleeding)
2. Blood Loss and Hemolysis o Jaundice is important for the assessment of anemia,
V. Laboratory Diagnosis of Anemia because it may be due to increased RBC destruction,
1. Complete Blood Count with Red Blood Cell
Indices
which suggests a hemolytic component to the anemia.
2. Reticulocyte Count  Moderate anemias (hemoglobin concentration of 7 to 10
3. Peripheral Blood Film Examination g/dL) may cause pallor of conjunctivae and nail beds but may
4. Bone Marrow Examination not produce clinical symptoms if the onset of anemia is slow.
5. Other Laboratory Tests  Severe anemias (hemoglobin concentration of less than 7
VI. Approach to Evaluating Anemias g/dL) usually produce tachycardia, hypotension, and other
1. Approach to Evaluating Anemias symptoms of volume loss, in addition to the symptoms listed
2. Morphologic Classification of Anemia
Based on Mean Cell Volume
earlier.
3. Pathophysiologic Classification of Anemias
and the Reticulocyte Count PHYSIOLOGIC ADAPTATIONS IN PATIENTS WITH
ANEMIA
 In cases of acute blood loss, such as with severe
INTRODUCTION hemorrhage, respond with profound changes in physiologic
 Red blood cells (RBCs) – Function to deliver oxygen to processes to ensure adequate perfusion of vital organs and
lungs. maintenance of homeostasis.
 Hemoglobin – has the remarkable capacity to bind oxygen in  In cases of severe blood loss, such as in trauma, blood
the lungs and then release it appropriately in tissues. volume decreases and hypotension develops, resulting in
 Anemia is derived from the Greek word anaimia, meaning decreased blood supply to the brain and heart.
“without blood.”  In severe anemia, blood is preferentially shunted to organs
o A decrease in hemoglobin concentration or number of that are key to survival, including the brain, muscle, and heart.
RBCs results in decreased oxygen delivery to tissue,  Hypoxia triggers an increase in RBC 2,3-bisphosphoglycerate
resulting in tissue hypoxia. that shifts the oxygen dissociation curve to the right
o Anemia should not be thought of as a disease but (decreased oxygen affinity of hemoglobin) and results in
rather as a manifestation of an underlying condition or increased oxygen delivery to tissues
deficiency  Thus with slowly developing and low-grade anemia, the
body develops physiologic adaptations to increase the
oxygen-carrying capacity of a reduced amount of hemoglobin,
DEFINITION OF ANEMIA
which improves oxygen delivery to tissues.
 A functional definition Anemia is a decrease in the oxygen o With persistent and severe anemia, however, the strain
carrying capacity of the blood. on the heart can ultimately lead to cardiac failure.
o arise if there is insufficient hemoglobin or the hemoglobin
 Reduced oxygen delivery to tissues caused by reduced
has impaired function
hemoglobin concentration elicits an increase in
 Anemia is defined operationally as a reduction in the erythropoietin secretion by the kidneys.
hemoglobin content of blood that can be caused by a o Erythropoietin stimulates the erythroid precursors in the
decrease in the following: bone marrow.
o RBC count, hemoglobin concentration, and hematocrit
below the reference interval for healthy individuals of
MECHANISMS OF ANEMIA
similar age, sex, and race, under similar environmental
 Life span of an RBC in circulation is about 120 days
conditions.
 The reference intervals are derived from large pools of  Adequate RBC production requires several nutritional factors
“healthy” individuals; however, the definition of healthy is such as iron, vitamin B12, and folate.
different for each of these groups.  Globin (polypeptide chain) synthesis must also function
normally.
IMPORTANCE OF PATIENT  In conditions with excessive bleeding or hemolysis, the
bone marrow must increase RBC production to compensate
 History and physical examination are important
for the increased RBC loss.
components in making a clinical diagnosis of anemia.
 Classic symptoms associated with anemia, fatigue and
shortness of breath.
INEFFECTIVE AND INSUFFICIENT ERYTHROPOIESIS
 Starts by obtaining a detailed history, which requires carefully  Erythropoiesis is the term used for marrow erythroid
questioning the patient, particularly with regard to diet, drug proliferative activity.
ingestion, exposure to chemicals, occupation, hobbies, travel,
bleeding history, race or ethnic group, family history of
BISCOCHO. NICER. RAMOS. | FEU MT 2023 1
[TRANS] UNIT 3: NATURAL IMMUNITY AND COMPLEMENT SYSTEM
 Normal erythropoiesis is under the control of the hormone  To detect the presence of anemia, a complete blood count
erythropoietin (produced by the kidney) and other growth (CBC) is performed using an automated blood cell analyzer to
factors and cytokines determine:
 Ineffective erythropoiesis refers to the production of o RBC count
erythroid precursor cells that are defective. o Hemoglobin concentration
o Defective precursors often undergo apoptosis o Hematocrit
(programmed cell death) in the bone marrow. o RBC indices
o Several conditions, such as megaloblastic anemia o White blood cell count
(impaired deoxyribonucleic acid [DNA] synthesis as a o Platelet count
result of vitamin B12 or folate deficiency), thalassemia  RBC indices include:
(deficient globin chain synthesis), and sideroblastic o Mean cell volume (MCV)
anemia (deficient protoporphyrin synthesis), involve o Mean cell hemoglobin (MCH)
ineffective erythropoiesis as a mechanism of anemia o Mean cell hemoglobin concentration (MCHC)
 these anemias, peripheral blood hemoglobin  MCV- the most important of the indices which is a measure of
concentration is low, which triggers an increase in the average RBC volume in femtoliters (fL).
erythropoietin production leading to increased  Automated blood cell analyzers provide:
erythropoietic activity. o Red cell distribution width (RDW)
 many of the defective erythroid precursors undergo o Index of variation of cell volume in an RBC population
destruction in the bone marrow resulting to decrease  Reticulocyte count should be performed for every patient
circulating RBCs. with anemia.
 Insufficient erythropoiesis refers to a decrease in the o Automated analyzers provide accurate measurements of
number of erythroid precursors in the bone marrow, resulting reticulocyte counts.
in decreased RBC production and anemia.  RBC histogram provided by the automated analyzer is an
o factors can lead to the decreased RBC production, RBC volume frequency distribution curve with the relative
including number of cells plotted on the ordinate and RBC volume (fL)
 deficiency of iron (inadequate intake, on the abscissa
malabsorption, excessive loss from chronic  In healthy individuals the distribution is approximately
bleeding); Gaussian.
 deficiency of erythropoietin (renal disease); o Abnormalities include a shift in the curve to the left
 loss of the erythroid precursors as a result of an (smaller cell population or microcytosis) or to the right
autoimmune reaction (aplastic anemia, acquired pure (larger cell population or macrocytosis).
red cell aplasia) o A widening of the curve occurs when a population of
 infection (parvovirus B19). RBCs have different volumes causing a greater variation
 Leukemia cells or with nonhematopoietic cells of RBC volume about the mean. The histogram
(metastatic tumors, granulomas, or fibrosis), the latter complements the peripheral blood film examination in
called myelophthisic anemia with characteristic identifying abnormal RBC populations.
teardrop RBCs.  RDW is the coefficient of variation of RBC volume expressed
as a percentage.
o It indicates the variation in RBC volume within the
BLOOD LOSS AND HEMOLYSIS population measured and an increased RDW correlates
 Anemia- can be developed as a result of acute blood loss or with anisocytosis (variation in RBC diameter) on the
chronic blood loss. peripheral blood film.
 Increased hemolysis results in a shortened RBC life span,  Automated analyzers calculate the RDW by dividing the
which elevate the risk for anemia. standard deviation of the RBC volume by the MCV and then
 Chronic blood loss induces iron deficiency as a cause of multiplying by 100 to convert to a percentage.
anemia.
 With acute blood loss and excessive hemolysis, the bone RETICULOCYTE COUNT
marrow takes a few days to increase production of RBCs.  Serves as an important tool to assess the bone marrow’s
o This response may be inadequate to compensate for a ability to increase RBC production in response to an anemia.
sudden excessive RBC loss as in traumatic hemorrhage  Reticulocytes- young RBCs that lack a nucleus but still
or in conditions with a high rate of hemolysis and contain residual ribonucleic acid (RNA) to complete the
shortened RBC survival. production of hemoglobin.
 Causes of hemolysis include: o Normally they circulate peripherally for only 1 day while
o Intrinsic defects in the RBC membrane completing their development.
o Enzyme systems or hemoglobin o The adult reference interval for the reticulocyte count is
o Extrinsic causes such as: 0.5% to 2.5% expressed as a percentage of the total
 Antibody-mediated processes number of RBCs.
 Mechanical fragmentation o The newborn reference interval is 1.5% to 6.0%, but
 Infection-related destruction these values change to approximately those of an adult
within a few weeks after birth
LABORATORY DIAGNOSIS OF ANEMIA  Absolute reticulocyte count- determined by multiplying the
percent reticulocytes by the RBC count.
COMPLETE BLOOD COUNT WITH RED BLOOD CELL o The reference interval for the absolute reticulocyte count
INDICES is 20 to 115 3 109 /L, based on an adult RBC count within
the reference interval (inside front cover).
o A patient with a severe anemia may seem to be
producing increased numbers of reticulocytes if only the
percentage is considered.
 Production of reticulocytes within the reference interval is o Large or macrocytic RBCs are greater than 8 mm in
inadequate to compensate for an RBC count that is diameter.
approximately one-third of normal.  Certain shape abnormalities of diagnostic value (such as
 Two successive corrections are made to the reticulocyte count sickle cells, spherocytes, schistocytes, and oval macrocytes)
to obtain a better representation of RBC production. and RBC inclusions (such as malarial parasites, basophilic
o To obbtain a corrected reticulocyte count, one corrects for stippling, and Howell-Jolly bodies) can be detected only by
the degree of anemia by multiplying the reticulocyte studying the RBCs on a peripheral blood film.
percentage by the patient’s hematocrit and dividing the  Review of the white blood cells and platelets may help show
result by 45 (the average normal hematocrit). that a more generalized bone marrow problem is leading to
o If the reticulocytes are released prematurely from the the anemia.
bone marrow and remain in the circulation 2 to 3 days o Hypersegmented neutrophils can be seen in vitamin B12
(instead of 1 day), the corrected reticulocyte count must or folate deficiency.
be divided by maturation time to determine the o Blast cells and decreased platelets may be an indication
reticulocyte production index (RPI). of acute leukemia.
 RPI is a better indication of the rate of RBC  Information from the blood film examination always
production than is the corrected reticulocyte count. complements the data from the automated blood cell analyzer.
 State-of-the-art automated blood cell analyzers determine the
fraction of immature reticulocytes among the total circulating BONE MARROW EXAMINATION
reticulocytes, called the immature reticulocyte fraction  Bone marrow aspiration and biopsy may help in
(IRF). establishing the cause of anemia
o IRF- helpful in assessing early bone marrow response  Bone marrow examination is indicated for a patient with
after treatment for anemia. an unexplained anemia associated with or without other
 Analysis of the reticulocyte count plays a crucial role in cytopenias, fever of unknown origin, or suspected
determining whether an anemia is due to an RBC production hematologic neoplasm.
defect or to premature hemolysis and shortened survival  It evaluates hematopoiesis and can determine whether
defect. there is an infiltration of abnormal cells into the bone
o If there is shortened RBC survival, as in the hemolytic marrow.
anemias, the bone marrow tries to compensate by  Important findings in bone marrow that can point to the
increasing RBC production to release more reticulocytes underlying cause of the anemia include abnormal
into the peripheral circulation. cellularity, evidence of ineffective erythropoiesis and
o Although an increased reticulocyte count is a hallmark of megaloblastic changes, lack of iron on iron stains of
the hemolytic anemias, it can also be observed after bone marrow, and the presence of granulomata,
acute blood loss. fibrosis, infectious agents, and tumor cells that may
 Chronic blood loss- does not lead to an appropriate
be inhibiting normal erythropoiesis.
increase in the reticulocyte count, but rather leads to
 Other tests that can assist in the diagnosis of anemia can
iron deficiency and a subsequent low reticulocyte
be performed on a bone marrow specimen as well,
count.
including immunophenotyping of membrane antigens
 An inappropriately low reticulocyte count results from
by flow cytometry, cytogenetic studies, and
decreased production of normal RBCs, as a result of
molecular analysis to detect specific genetic
either insufficient or ineffective erythropoiesis.
mutations and chromosome abnormalities in
Figure No. 1. Formulas for Reticulocyte Counts and Blood Cell
leukemia cells.
Indices
OTHER LABORATORY TESTS
 Other laboratory tests that can assist in establishing the
cause of anemia include routine urinalysis (to detect
hemoglobinuria or an increase in urobilinogen) with a
microscopic examination (to detect hematuria or
hemosiderin) and analysis of stool (to detect occult
blood or intestinal parasites).
 Certain chemistry studies are very useful, such as serum
haptoglobin, lactate dehydrogenase, and
unconjugated bilirubin (to detect excessive hemolysis)
and renal and hepatic function tests.
PERIPHERAL BLOOD FILM EXAMINATION  With more patients having undergone gastric bypass
 An important component in the evaluation of an anemia is surgery for obesity, certain rare deficiencies such as
examination of the peripheral blood film, with particular insufficient copper have become more common as
attention to: another nutritional deficiency that can cause anemia.
o RBC diameter  The anemia may be classified based on reticulocyte
o Shape count, MCV, and peripheral blood film findings.
o Color  Serum vitamin B12 and serum folate assays are
o Inclusions helpful in investigating a macrocytic anemia with a low
 Peripheral blood film verify the results produced by automated reticulocyte count.
analyzers.  Direct antiglobulin test can differentiate autoimmune
 Normal RBCs on a Wright-stained blood film are nearly hemolytic anemia from other hemolytic anemias.
uniform, ranging from 7 to 8 mm in diameter.
o Small or microcytic cells are less than 6 mm in diameter. APPROACH TO EVALUATING ANEMIAS
 The approach to the patient with anemia begins with

taking a complete history and performing a physical o Some normocytic anemia develop as a result of
examination. the premature destruction and shortened
 New –onset fatigue and shortness of breath suggest survival of RBCs (hemolytic anemias)
an acute drop in the hemoglobin concentration. o Characterized by an elevated reticulocyte count.
 A large spleen may be an indication of hereditary o Other normocytic anemias develop as a result of
spherocytosis. a decreased production of RBCs and are
 A stool positive for occult blood may indicate iron characterized by a decreased reticulocyte count
deficiency.  Hemolytic anemias can be further divided into those that
 The first step in the laboratory diagnosis of anemia is result from:
detecting its presence by the accurate measurement of o Intrinsic causes:
the hemoglobin concentration, hematocrit, MCV, and  Membrane defects,
RBC count and comparison of these values with the  Hemoglobinopathies,
reference interval for healthy individuals of the same age,  Enzyme deficiencies
sex, race and environment. o Extrinsic causes:
 A reticulocyte count and peripheral blood film  Immune RBC injury
examination are a paramount importance in evaluating  Nonimmune RBC injury
anemia.  A direct antiglobulin test helps differentiate immune-
mediated RBC destruction from other causes of
MORPHOLOGIC CLASSIFICATION OF ANEMIA BASED hemolysis.
ON MEAN CELL VOLUME MORPHOLOGIC CLASSIFICATION OF ANEMIAS AND
 MCV is an extremely important tool and is key in the THE RETICULOCYTE COUNT
morphologic classification of anemia.  The MCV can further classify the anemia into three
 Microcytic anemias are characterized by an MCV of less subgroups:
than 80 fL with small RBCs (less than 6 um in diameter). o Normocytic anemias,
o These are caused by conditions that result in o Microcytic anemias,
reduced hemoglobin synthesis. o Macrocytic anemias
 Microcytosis is often associated with hypochromia  The excessive RBC loss category includes acute
(increased central pallor in RBCs) and an MCHC of less hemorrhage and the hemolytic anemias with shortened
than 32 g/dL. RBC survival.
 Heme synthesis is diminished in iron deficiency, iron MORPHOLOGIC CLASSIFICATIONAND THE RED
sequestration (chronic inflammatory states), and defective BLOOD CELL DISTRIBUTION WIDTH
protoporphyrin synthesis (sideroblastic anemia, lead
 RDW can help determine the cause of an anemia when
poisoning).
used in conjunction with the MCV.
 Globin chain synthesis is insufficient or defective in
 Each of the MCV categories mentioned previously
thalassemia and in Hb E disease.
(normocytic, microcytic, macrocytic) can also be
 Iron deficiency is the most common cause of microcytic
subclassified by the RDW as homogeneous (normal
anemia
RDW) or heterogeneous (increased or hhigh RDW)
 Macrocytic anemias are characterized by an MCV
 A decreased MCV with an increased RDW is suggestive
greater than 100 fL with large RBCs (greater than 8 um in
of iron deficiency.
diameter).
o This arises from conditions that result in
megaloblastic or nonmegaloblastic red cell
PATHOPHYIOLOGIC CLASSIFICATION OF ANEMIAS
development in bone marrow. AND THE RETICULOCYTE COUNT
o Nonmegaloblastic forms of macrocytic anemias
Figure No. 1. Pathophysiologic Classification of Anemias
are also characterized by large RBCs.
 It is rare for the MCV to be greater than
115 fL in nonmegaloblastic anemias.
o Often seen in patients with chronic liver disease,
alcohol abuse, and bone marrow failure.
 Megaloblastic anemias are caused by conditions that
impair synthesis of DNA, such as vitamin B12 and folate
deficiency or myelodysplasia.
o This is characterized by oval macrocytes and
hypersegmented neutrophils in the peripheral
blood and by megaloblasts or large nucleated
erythroid precursors in the bone marrow.
o The MCV in megaloblastic anemia can be
markedly increased (up to 150 fL), but modest
increases (100 to 115 fL) are most common.
o This are typically related to membrane changes
caused by disruption of the cholesterol-to-
phospholipid ratio.
 Pernicious anemia is one cause of vitamin B12
deficiency, whereas pregnancy with increased
requirements is a leading cause of folate deficiency.
 Normocytic anemias are characterized by an MCV in
the range of 80 to 100 fL.

HEMATOLOGY 1
[TRANS] UNIT 20: INTRODUCTION TO INCREASED DESTRUCTION OF ERYTHROCYTES
 PARAXOSYMAL COLD HEMOGLOBINURIA

OUTLINE o experience hemolysis after exposure to cold
 PARAXOSYMAL NOCTURNAL HEMOGLOBINURIA
INTRODUCTION TO INCREASED DESTRUCTION OF
ERYTHROCYTES o may experience intermittent episodes of
hemolysis
I. CLASSIFICATION o an acquired disorder involving an intrinsic defect
II. HEMOLYSIS
 HEMOLYTIC TRANSFUSION
1. Normal Macrophage-Mediated Hemolysis
and Bilirubin Metabolism o example of a single acute incident
2. Plasma Hemoglobin Salvage During  CHRONIC HEMOLYSIS
Normal Fragmentation Hemolysis o may not be evident if the bone marrow is able to
III. EXCESSIVE MACROPHAGE-MEDIATED compensate
(EXTRAVASCULAR) HEMOLYSIS o it may be punctuated over time with hemolytic
IV. EXCESSIVE FRAGMENTATION crises that cause anemia
(INTRAVASCULAR) HEMOLYSIS
o ex)Glucose-6-phosphate dehydrogenase
V. CLINICAL FEATURES
VI. LABORATORY FINDINGS deficiency (G6PD)
1. Tests of Accelerated Red Blood Cell o RBC life span is chronically shortened
Destruction o bone marrow compensation prevents anemia
2. Tests of Increased Erythropoiesis  DRAMATIC ACUTE HEMOLYTIC EVENT
3. Laboratory Tests to Identify Specific o When the cells are challenged with oxidizing
Hemolytic Processes agents (e.g., antimalarial drugs)
VII. DIFFERENTIAL DIAGNOSIS
 Others result in anemia that is so severe that the bone
marrow cannot generate cells fast enough to
compensate for the anemia
HEMOLYSIS OR HEMOLYTIC DISORDER  THALASSEMIA MAJOR
 increased rate of destruction (lysis) of RBCs, shortening their o Although RBC production is brisk, each cell
life span possesses an inadequate complement of one
 REDUCED NUMBER OF CELLS: type of globin chain
o reduced tissue oxygenation o functional hemoglobin production is
o increased erythropoietin production by the kidney decreased overall
 RETICULOCYTOSIS o Result: oxygen-carrying capacity of blood is
o bone marrow responds by accelerating chronically low
erythrocyte production o CELLS LYSE because excess normal globin
 HEMOLYTIC PROCESS chains precipitate inside erythroid cells
o present without anemia if bone marrow is able to  leads to hemolysis and exacerbates the
compensate; chronically reduced hemoglobin
 by accelerating RBC production production
sufficiently to replace RBCs lost through  INHERITED HEMOLYTIC CONDITIONS
HEMOLYSIS o Ex) thalassemia
 HEALTHY BONE MARROW o passed to offspring by mutant genes from the
o can increase its production of RBCs by four to parents
eight times normal  ACQUIRED HEMOLYTIC DISORDERS
Significant RBC destruction must occur before an anemia o develop in individuals who were previously
develops during hemolysis hematologically normal but acquire an agent or
 HEMOLYTIC ANEMIA condition that lyses RBCs
o when the rate of RBC destruction exceeds the o Ex) malaria
increased rate of RBC production
 INTRINSIC HEMOLYTIC DISORDERS:
CLASSIFICATION o Most are inherited
 When hemolysis is the primary feature, anemias can be o subclassified as:
classified as follows:  membrane defects (abnormalities of
o Acute versus chronic the RBC membrane)
o Inherited versus acquired  enzyme defects (enzymatic pathways)
o Intrinsic versus extrinsic  hemoglobinopathies (hemoglobin
o Intravascular versus extravascular molecule)
o Fragmentation versus macrophage-mediated o If RBCs of the affected patient were to be
 ACUTE VERSUS CHRONIC HEMOLYSIS transfused into a healthy individual, they
o delineates the clinical presentation would still have a shortened life span because
 ACUTE HEMOLYSIS the defect is in the RBC
o If normal RBCs are transfused into a patient,
o has a rapid onset and is isolated (sudden),
the transfused cells have a normal life span
episodic, or paroxysmal (paroxysmal cold
because the transfused cells are normal
hemoglobinuria or paroxysmal nocturnal
hemoglobinuria)
 EXTRINSIC HEMOLYTIC CONDITIONS:  IRON
o Most are acquired o released from heme
o those that arise from outside the RBC o returned to plasma via FERROPORTIN
o substances in plasma o bound to its protein carrier molecule
o conditions affecting the anatomy of the circulatory (TRANSFERRIN)
system o recycled to needy cells thus salvaging virtually all
o immunohemolytic, traumatic, or microangiopathic RBC iron
o may be caused by:  PROTOPORPHYRIN
 infectious agents o catabolized
 chemical agents (drugs and venoms) o products are processed in the liver
 physical agents o move to the intestines- facilitate dietary fat
o patient’s RBCs have a normal life span in the absorption
bloodstream of a healthy individual; normal cells o excreted mostly in the stool
are lysed more rapidly in the patient’s circulation  BILIRUBIN AND UROBILINOGEN
o RBC was normal until it was invaded by an o excretory products derived from the
outside agent protoporphyrin component of heme
 NONINFECTIOUS EXTRINSIC AGENTS that can
damage RBCs:
o antibody against RBC antigens
o prosthetic heart valve
 INTRAVASCULAR HEMOLYSIS
o Occurs by FRAGMENTATION
o takes place most often within the bloodstream
o RBCs can lyse by fragmentation in the spleen and
bone marrow
 MACROPHAGE-MEDIATED HEMOLYSIS
o occurs when RBCs are engulfed by macrophages
o lysed inside the phagocyte by their digestive
enzymes
HEMOLYSIS
NORMAL MACROPHAGE-MEDIATED HEMOLYSIS AND
BILIRUBIN METABOLISM
 Detection of hemolysis depends partly on detection of
RBC breakdown products
 BILIRUBIN- prominent product
 NORMAL BILIRUBIN PROCESS
o described to clarify the relationship between
hemolysis and increased bilirubin levels
 RBC Lifespan: 120 days
o they undergo various metabolic and chemical
changes
 membrane alterations
 loss of deformability
o RETICULOENDOTHELIAL SYSTEM
 macrophages of the mononuclear
phagocyte system
 recognize these changes and
phagocytize the aged erythrocyte
 producing a macrophage-mediated
hemolytic process
 removes approximately 1% of
the RBCs per day
 MACROPHAGES (SPLEEN & LIVER)
 process senescent RBCs
primarily
 INSIDE MACROPHAGES
o Where majority of RBC degradation occurs
 HEMOGLOBIN- hydrolyzed into heme and globin
 the latter is further degraded into amino acids that return
to the amino acid pool
Table No. 1 Classification of Selected Hemolytic Anemias by Primary Cause and Type of Hemolysis
EZEKIEL CRUZ. SAMANTHA CRUZ. IVAN ILAGAN. ROMINA LASCANO. KARYLLE SURIAGA | FEU MT 2023
2 2
Normal Macrophage-Mediated Hemolysis  There, unconjugated bilirubin is joined with one to two
and Bilirubin Metabolism molecules of a sugar acid, glucuronic acid, by uridine
diphosphate (UDP) glucuronosyltransferase family 1
member A1 (UGT1A1) or previously known as
glucuronyl transferase.
 The molecules formed include monoglucuronosyl
bilirubin, with just a single glucuronic acid added, and
bisglucuronosyl bilirubin, previously known as
bilirubin diglucuronide, which has two glucuronic
acid molecules added.
 Addition of glucuronic acids makes the bilirubin
molecules polar and water soluble (conjugated
bilirubin or direct bilirubin).
 Thus, the bilirubin originally release from macrophages
that lacks glucuronic acids is termed unconjugated
bilirubin or indirect bilirubin.
Figure 20.1 Normal Catabolism of Hemoglobin. Macrophages lyse

ingested red blood cells (RBCs) and separate hemoglobin (Hb) into
globin chains and heme components. Amino acids from the globin
chains are reused. Heme is degraded to iron and protoporphyrin. Iron
is returned to the blood to be reused. Protoporphyrin is degraded to
unconjugated bilirubin.
 Unconjugated bilirubin – hydrophobic, and must bind

to albumin to be transported to the liver.
 Bilirubin formed in macrophages and secreted in blood
enters the liver sinusoid via the hepatic artery.
 In the liver sinusoid, bilirubin dissociates from albumin
so that it can be carried into the hepatocyte by organic
anion transporter (OAT) proteins, chiefly OATP1B3.
 Once inside the hepatocyte, bilirubin is bound to
glutathione S-transferase, known as ligandin, for
transport to the endoplasmic reticulum.
3 3
Figure 20.3 Normal Macrophage-Mediated Hemolysis.

Figure 20.2 Catabolism of Heme to Bilirubin. In cells containing 1. In a macrophage, hemoglobin is degraded to heme, the iron is released, and
heme oxygenase, iron is removed from heme, and the the protoporphyrin ring is converted to unconjugated bilirubin.
protoporphyrin ring is opened up to form an intermediate, biliverdin.
Biliverdin is converted to unconjugated bilirubin by biliverdin 2. Macrophages release unconjugated bilirubin into the blood, where it binds to
reductase. The unconjugated bilirubin is secreted into the blood and albumin for transport to the liver.
binds to albumin for transport to the liver. When unconjugated 3. Unconjugated bilirubin enters the hepatocyte.
bilirubin enters the hepatocyte, UGT1A1 (uridine diphosphate
glucuronosyltransferase family 1 member A1, formerly glucuronyl 4. The hepatocyte converts unconjugated bilirubin to conjugated bilirubin.
transferase) adds two molecules of glucuronic acid to form
bisglucuronosyl bilirubin, also called conjugated bilirubin. 5. Conjugated bilirubin leaves the liver in the bile and enters the small intestine.
6. Bacteria in the large intestine convert conjugated bilirubin to urobilinogen, most

of which is excreted in the stool.
 In the structure of the liver lobule, small ducts called 7. Some of the water-soluble urobilinogen is reabsorbed in the portal circulation,
canaliculi run from the central vein outward along the and most is recycled through the liver for excretion.
lateral sides between hepatocytes. 8. A small component of the reabsorbed urobilinogen is filtered and excreted in the
 They receive the bile excreted by the hepatocytes and urine.
channel it toward the exterior of the lobule where the
canaliculi join into larger bile ducts that ultimately
collect the bile into the common bile duct.
 Conjugated bilirubin is actively excreted by
hepatocytes into the canaliculi by a unidirectional and
adenosine triphosphate (ATP)-dependent membrane
protein, multi-drug resistant protein 2 (MRP2).
 Once in the bile canaliculi, conjugated bilirubin
continues down to the common bile duct and
eventually into the intestines.
 From there, it assists with the emulsification of fats for
absorption from the diet.
 Under normal conditions, hepatocytes at the exterior

of the lobule receive the highest concentration of
unconjugated bilirubin in the blood entering the sinus.
 Having conjugated the bilirubin, they are not always
able to export all of it into the bile duct.
 Therefore, they export conjugated bilirubin into
sinusoidal blood via a second MRP (MRP3), which is
localized to the sinusoidal side of hepatocytes.
 It can then be absorbed downstream by more
centrilobular hepatocytes via a second OAT protein,
OATP1B1, in the sinusoidal membrane.
 OATP1B1 has a very high affinity for conjugated
bilirubin.
 The absorbed, conjugated bilirubin can then be
exported into the canaliculi by these downstream
hepatocytes.
 Once conjugated bilirubin enters the intestines, it is
oxidized by gut bacteria into various water-soluble
compounds, collectively called urobilinogen.
 Most urobilinogen is oxidized further to stercobilin and
similar compounds that give the brown color to stool.
 This is the usual and ultimate route for excretion of
most red cell-derived protoporphyrin.
4 4
 Some conjugated bilirubin and urobilinogen molecules
are absorbed by osmosis from the intestines into the
blood of the portal circulation.
 The portal circulation (blood vessels that surround the
intestines to absorb nutrients) collects these bile
products and carries them directly to the liver in this
enterohepatic circulation.
 These bilirubin derivatives enter the lobule via the

portal veins and flow into the sinusoidal blood.
 Most of the intestinally absorbed and hepatocyte-
secreted conjugated bilirubin in the sinusoidal blood is
absorbed into hepatocytes by OATP1B1 and recycled
directly into the bile.
 Urobilinogen is similarly recycled into the bile.
 A miniscule amount of direct bilirubin remains in the
blood entering the central vein, along with a larger
amount of urobilinogen.
 These compounds are filtered at the renal
glomerulus and excreted in the urine.
 Although conjugated bilirubin is virtually undetectable
in urine because of efficient recycling in the liver, a
measurable amount of urobilinogen can be expected
normally.
 This is the second, but much smaller, route of
excretion for protoporphyrins. Figure 20.4 Normal Fragmentation Hemolysis.
1. Normally a small number of red blood cells lyse within the circulation, forming
 The yellow color of urine is not caused by the schistocytes and releasing hemoglobin (Hb) into the blood, mostly as a/b dimers.
urobilinogen, which is colorless. It is a result of
urobilin, a stool derivative of urobilinogen that is also 2. The plasma protein haptoglobin (Hpt) binds a hemoglobin dimer in a complex.
water soluble and absorbed via the portal circulation. 3. The hemoglobin-haptoglobin complex binds to CD163 on the surface of macrophages
in various organs.
Plasma Hemoglobin Salvage During Normal 4. The complex is internalized into the macrophage, where the hemoglobin dimer is
Fragmentation Hemolysis released. The hemoglobin dimer is degraded to heme, the iron is released, and the
protoporphyrin ring is converted to unconjugated bilirubin.
 Fragmentation hemolysis is the result of trauma to 5. The haptoglobin is degraded.

the RBC membrane that causes a breach sufficient for 6. The unconjugated bilirubin released into the blood is bound to albumin and processed
the cell contents, chiefly hemoglobin, to spill directly through the liver as in Figure 20.3 (steps 2 to 8).
into plasma.
7. When free hemoglobin is released into the blood with fragmentation, the iron is rapidly
oxidized, forming methemoglobin, and the heme (metheme) molecule dissociates from
 Approximately 10% to 20% of normal RBC the globin.
destruction is via fragmentation, usually secondary
to turbulence and anatomic restrictions in the 8, The plasma protein hemopexin (Hpx) binds free metheme into a complex.
vasculature. 9. The hemopexin-metheme complex binds to CD91 on the surface of hepatocytes.
 Because hemoglobin is filtered through the 10. The complex is internalized into the hepatocyte.
glomerulus, some iron could be lost daily with even
11. The iron is released from the metheme, and the protoporphyrin ring is converted to
normal amounts of fragmentation hemolysis. unconjugated bilirubin, ready for conjugation and further processing, as in Figure 20.3
(steps 4 to 8).
 In addition, free hemoglobin, and especially free
12. Hemopexin is recycled to the blood. Note that metheme can also bind to albumin,
heme, can scavenge nitric oxide and cause iron- forming metheme-albumin (not shown), but this complex is temporary because metheme
induced oxidative damage to cells. is rapidly transferred to hemopexin.
 These effects are magnified in conditions such as

sickle cell disease, hemolytic transfusion  When free in plasma, hemoglobin exists mostly as a/b
reactions, and sepsis. dimers, that rapidly complex to a liver-produced
plasma protein called haptoglobin.
 Thus, several mechanisms exist to salvage  By binding to haptoglobin, hemoglobin avoids filtration
hemoglobin iron and prevent oxidation reactions. at the glomerulus and the iron is saved from urinary
 They are collectively called the haptoglobin- loss.
hemopexin-methemalbumin system.  Haptoglobin binds a hemoglobin dimer in a
conformation that is very similar to the binding of the
complementary dimer in the native hemoglobin
5 5
structure, the conformation of the complex has been
termed a pseudotetramer.  Hemopexin-metheme- binds to CD91 receptor,
 In this complex the hemes are sequestered, as they lipoprotein receptor-related protein (LRP1),
are in intact hemoglobin, so that cells are protected o Mostly on hepatocytes.
from their oxidative properties. o Internalized by endocytosis.
 As the plasma is carried to various tissues, the o Internalized heme can be incorporated within the
complex is taken up by macrophages, principally need proteins in the hepatocyte like cytochromes or
those in the liver, spleen, bone marrow, and lung. broken down to bilirubin to reuse the iron contents.
 In these tissues, macrophages express CD163, the o hemopexin is recycled to blood from the hepatocyte.
haptoglobin scavenger receptor, on their  Metheme-albumin system- A third mechanism of iron
membranes. salvage
 Once the hemoglobin-haptoglobin complex binds to o Albumin acts as a carrier for metheme and other
CD163, the entire complex is internalized into the molecules.
macrophage in a lysosome. o Temporary holding state for metheme, due to high
 Inside the lysosome, iron is salvaged, globin is concentration of albumin content in the plasma. But
catabolized, and protoporphyrin is converted to it should be quickly transferred to hemopexin if
unconjugated bilirubin, just as though an intact RBC available, considering that it will have higher binding
had been ingested by the macrophage. affinity for metheme than albumin.
 Haptoglobin is also degraded within the lysosome. o hemopexin-metheme complex travels to the liver to
 The level of haptoglobin in plasma is typically complete the process.
adequate to salvage only the small amount of plasma  These salvage systems works in order.
hemoglobin generated each day because of normal
fragmentation of RBCs. Haptoglobin binds hemoglobin (up until it is saturated and
 If fragmentation hemolysis is accelerated, depleted) -> hemopexin binds metheme (until saturated) ->
haptoglobin is depleted because the liver’s albumin binds metheme. This system works at the same time
production does not increase in response to the (based on recent studies specifically during accelerated
increased consumption of haptoglobin. hemolysis) where hemopexin acts to prevent heme toxicity at all
times.
 A second mechanism of iron and oxidation prevention o Excess (met) hemoglobin and metheme will be
involves hemopexin. filtered into the urine if haptoglobin-hemopexins are
 The iron in free plasma hemoglobin rapidly becomes overloaded. Kidney is not significantly involved in
oxidized, forming methemoglobin. normal iron salvage, given that released amounts
 The heme molecule (actually metheme or hemin) are only in a negligible range.
readily dissociates from the globin and binds to o Proximal tubular cells can reabsorb iron. Given that
another liver-produced plasma protein, hemopexin. iron staining of tubular cells in urinary sediment
 This binding also saves the iron from urinary loss during periods of excessive hemolysis demonstrates
and prevents oxidant injury to cells and tissues. the presence of hemosiderin.
 The latter role of hemopexin may actually be more o In normal instances small amounts of filtered
critical, as free metheme has high redox activity. transferrin can be salvaged by transferrin receptors
in the apical area of proximal tubular cells. Can be a
 It readily diffuses across cell membranes and into
normal source of iron for metabolic needs
extracellular spaces oxidizing proteins, lipids, and
 Ferroportin- found in the basolateral membrane of
nucleic acids. In so doing, it sensitizes cells to
proinflammatory stimuli. proximal tubular cells
o renal cells can transfer additional salvaged iron back
 Metheme toxicity is a major factor in the lethality of
into the blood.
systemic conditions such as sepsis or hemolytic
o Can manage the amount and “form” of iron present
transfusion reactions.
in normal urinary filtrate.
 This observation has led to experiments using
o Other forms of iron are presented to and processed
hemopexin as therapy.
by the kidney, during excessive fragmentation
hemolysis.
EXCESSIVE MACROPHAGE-MEDIATED
(EXTRAVASCULAR) HEMOLYSIS
 Macrophage mediated hemolysis Can cause
various hemolytic anemias.
o Occurs when more than the normal range of RBCs
are being removed from the circulation daily.
Normally, senescent RBCs displays surface
markers for them to be identified by macrophages
as aged cells which requires removal (seen in
Chapter 5).
6 6
o
Pathologic processes are expressed with the
same markers- for cell recognition or removal. o If cytoplasmic volume is lost with less membrane,
o Anemia develops when- number of affected cells the cell becomes spherocyte (characteristic shape
increases beyond the quantity normally removed change that is associated with macrophage-
each day. Which lead to senescence, where bone mediated hemolysis).
marrow cannot compensate. o spherocyte may enter the circulation, with small
- Example: excessive oxidation o hemoglobin survival rate due to its rigidity and inability to
leads to increased aggregation of Heinz traverse splenic sieve in the times of subsequent
bodies, thus the denatured hemoglobin binds passages in the red pulp. Might get trapped in the
to the interior RBC causes changes in the splenic sinus and get fully ingested by a
membrane exterior. macrophage.
- Ergo, it is detected by macrophages and  Total plasma bilirubin level rises as RBCs are lysed
leads to the premature removal of the cell prematurely- macrophage-mediated hemolytic
from circulation. anemias
- Same process happens when intracellular o It is due to the increase of the unconjugated
parasite is present, complement or fraction (See Figure 1).
immunoglobulins are on the surface of the o A healthy liver can process the increased in load
RBC. of unconjugated bilirubin. Hence, producing more
than the usual amount of conjugated bilirubin that
enters the intestine.
o Formation of urobilinogen forms in the intestines
that would be absorbed by the portal circulation
and excreted by the kidney. Which causes the
detection of increase of urobilinogen in the urine.
Considering that it is absorbed more than direct
bilirubin into the portal circulation and not
reprocessed through the liver as completely as
direct bilirubin.
o Hepatocytes and into the bile. Reprocess bilirubin
that is absorbed in the enterohepatic.
o Increase in unconjugated bilirubin in the plasma,
cannot be detected in urine given that it is bound
to albumin and cannot pass through the
glomerulus.
 Hemopexin- may play an important role in macrophage-

mediated hemolysis.
o It can be toxic to a macrophage if they ingest more
than 1 to 2 erythrocyte, due to the released of heme
 FIGURE 1: Excess Macrophage-Mediated Hemolysis. 1, More than the usual o They posses membrane heme exporter, feline
number of red blood cells are ingested each day by macrophages. 2, An increased leukemia virus subgroup C receptor (FLVCR) that
amount of unconjugated bilirubin is produced, released into the blood, and binds permits them to rid themselves of excess heme.
to albumin. 3, When increased unconjugated bilirubin is presented to the liver, an
increased amount of conjugated bilirubin is made and excreted into the intestine. o Hemopexin- is important to accept the exported free
4, When an increased amount of conjugated bilirubin is present in the intestine, heme during macrophagemediated hemolysis to
an increased amount of urobilinogen is formed and excreted in the stool. 5, prevent heme toxicity to other cells.
Increased urobilinogen in the intestine results in increased urobilinogen
reabsorbed into the blood. 6, Increased urobilinogen in the blood results in EXCESSIVE FRAGMENTATION (INTRAVASCULAR)
increased urobilinogen filtered and excreted in the urine. (Adapted from Brunzel,
N. A. [2013]. Fundamentals of Urine and Body Fluid Analysis. [3rd ed., p. 141, HEMOLYSIS
Figure 7-8]. St. Louis: Elsevier.)  Fragmentation hemolysis- is a minor component of
normal RBC wreckage, it can be a major feature of
 RBC is ingested by a macrophage- causes lyse within the pathologic processes.
phagolysosome and only done by the macrophage solely. o Examples of fragmentation hemolysis: traumatic and
o Contents not detectable within the plasma, contents physical lysis of RBCs caused by prosthetic heart
are degraded within the macrophage. Thus, the valves and the exit of mature intracellular RBC
designation extravascular hemolysis. parasites, such as malaria protozoa, by bursting out of
o Due to defective RBCs display markers such as cells.
senescent RBCs- macrophage-mediated hemolysis o With these scenario the fragmentation destruction of
happens often in the spleen and liver, where markers RBCs can cause profound anemias.
are posed by macrophages.  Excessive fragmentation hemolysis is deduced through the
appearance of RBC contents in the plasma composed
 Occasionally, macrophage ingests a portion- leaving a mostly of hemoglobin and the development of (met)
remainder of the membrane, to reseal. hemoglobinemia.
7 7
 This results to the iron salvage proteins form complexes  FIGURE 2 (B): Excess Fragmentation Hemolysis: The Role of the Liver. 1,
If the amount of hemoglobin released from lysing red blood cells exceeds
with their respective ligands; hemoglobinhaptoglobin, the capacity of haptoglobin, the unbound free hemoglobin is rapidly
metheme-hemopexin, and metheme-albumin. oxidized, forming methemoglobin, and the metheme molecule dissociates
from the globin. 2, Hemopexin binds to metheme, and the complex is
captured by the CD91 receptor on hepatocytes. 3, The complex is
internalized by the hepatocyte, the iron is released from the metheme, and
the protoporphyrin ring is converted to unconjugated bilirubin and
ultimately to conjugated bilirubin to be processed, as in Figure 20.5, steps
3 to 6. 4, Although a small of amount of hemopexin is recycled to the
blood, most is degraded. Metheme can also temporarily bind to albumin,
forming metheme-albumin (not shown), but Figure 20.6, cont’d metheme
is rapidly transferred to hemopexin.
 The complexes are cleared within minutes from plasma

through the completion of with their receptors.
 Amount of free haptoglobin will drop due to increase than
usual amounts of the complex will form and be taken up by
macrophages.
o The endocytosed protein is not recycled to the plasma
and no increase in production will occur, that will lead
to the depletion of haptoglobin in the plasma.
o Free hemopexin can decrease even if it is usually
recycled.
 If there would be an increase level of metheme to be
salvaged, hepatic recycling system can become saturated
o Hence, hemopexin gets degraded within the
hepatocyte and plasma levels declined.
 FIGURE 2 (A): Excess Fragmentation Hemolysis: The Role of Macrophages. 1, o However, the drop in hemopexin is not as vigorous
When an increased number of red blood cells lyse by fragmentation, more than
the usual amount of hemoglobin (Hb) is released into the blood, mostly as a/b
compared to haptoglobin. Given that recycling of
dimers. 2, Haptoglobin (Hpt) binds the increased hemoglobin dimers, forming occurs.
more than usual numbers of complexes. 3, The hemoglobin-haptoglobin  Simultaneously, the appearance of hemoglobin in the
complexes are taken up by macrophages bearing the CD163 receptor in various plasma, is also its appearance in the urine
organs. 4, An increased amount of hemoglobin dimers is released from the
complexes. The hemoglobin is degraded to heme, the iron is released, and the (hemoglobinuria).
protoporphyrin ring is converted to unconjugated bilirubin. The increased amount o If the amount of free hemoglobin and heme exceeds
of unconjugated bilirubin is then transported to the liver and processed as with the salvage capacity of the plasma proteins. There
excess macrophage-mediated hemolysis (Figure 20.5, steps 2 to 6). leukemia
virus subgroup C receptor (FLVCR), that permits them to rid themselves of excess
would be an increase in the iron-containing proteins
heme.21 Hemopexin then is important to accept the exported free heme during that would be absorbed into the proximal tubular cells
macrophagemediated hemolysis to prevent heme toxicity to other cells.21
EXCESSIVE FRAGMENTATION (INTRAVASCULAR) HEMOLYSIS Although
fragmentation hemolysis is a minor component of normal RBC destruction, it can
be a major feature of pathologic processes. Dramatic examples of fragmentation
hemolysis are the traumatic, physical lysis of RBCs caused by prosthetic heart
valves and the exit of mature intracellular RBC parasites, such as malaria
protozoa, by bursting out of cells. In these instances the fragmentation destruction
of RBCs can cause profound anemias. Excessive fragmentation hemolysis is
characterized by the appearance in the plasma of the contents of RBCs, chiefly
hemoglobin, and thus the development of (met)hemoglobinemia. As a result, the
iron salvage proteins form complexes with their respective ligands (Figure 20.6A
and B); hemoglobinhaptoglobin, metheme-hemopexin, and metheme-albumin. 5,
Degradation of haptoglobin is accelerated compared with normal.
8 8
FIGURE 2 (C): Excess Fragmentation Hemolysis: The Role of the Kidney. 1, When FIGURE 2 (D): Fate of Iron Removed from Salvaged Hemoglobin in the Kidney. 1, Iron
excess red blood cells lyse by fragmentation and other systems are saturated, free (Fe) salvaged from absorbed hemoglobin can be transported into the circulation by
(met)hemoglobin enters the urinary filtrate. 2, Cubilin (Cb) on the luminal side of the ferroportin on the basolaminal side of the tubular cell. In the blood it will be bound to
proximal tubular cells binds proteins for reabsorption, including hemoglobin. 3, Cubilin transferrin (Tf) for transport. 2, Iron in excess of what can be transported into the
carries hemoglobin into the proximal tubular cells. 4, The hemoglobin is degraded to circulation is stored as ferritin, and some is converted to hemosiderin. If the tubular cell
heme, the iron is released, and the protoporphyrin ring is converted to unconjugated is sloughed into the filtrate and appears in the urine sediment, the hemosiderin can be
bilirubin as in a macrophage and conjugated as in a hepatocyte. 5, The fate of the detected using the Prussian blue stain. (A–C, Adapted from Brunzel, N. A. [2013].
bilirubin is uncertain, as it may be secreted to the filtrate or reabsorbed into the blood. Fundamentals of Urine and Body Fluid Analysis. [3rd ed., p. 141, Figure 7-8]. St. Louis:
6, When the amount of hemoglobin exceeds the capacity of the Figure 20.6, cont’d Elsevier.)
proximal tubular cells to absorb it from the filtrate, hemoglobinuria occurs.
o When a tubular cell is discarded into the filtrate and
 Glomerular filtrate is low in protein compared with plasma shows in the urine, hemosiderin can be detected
o However, it is not deprived of filtered proteins, this through Prussian blue stain. Using this stain it
includes iron carrying or containing proteins, such as showcase the evidence of excess iron.
transferrin, albumin, and lactoferrin. o Elevated levels of plasma indirect bilirubin and urinary
 Transferrin receptors are located in the apical surface of urobilinogen can be measured, however, it takes time.
proximal tubular cells- to help the iron needs of those cells. Hence, the time used in the detection can also be used
o Proximal tubular cells- contains megalin-cubilin- in the differential diagnosis.
amnionless receptor endocytosis system. Not
specifically for heme/iron-containing proteins. But it acts CLINICAL FEATURES
for nonspecific yet efficient reabsorption of proteins in JAUNDICE
the urinary filtrate. o Refers to the yellow color of the skin and sclera,
 Megalin and cubilin have each been shown to bind whereas icterus describes yellow plasma and tissues.
hemoglobin and myoglobin. An increase in plasma bilirubin and subsequent jaundice
 Additionally, megalin can bind lactoferrin, and cubilin can can occur in other conditions besides hemolysis, such
bind transferrin. as hepatitis or gallstones.
 Nonspecific competitive reabsorption favors the iron-  HEMOLYTIC JAUNDICE or PREHEPATIC JAUNDICE
containing proteins if presented in high concentrations o Jaundice is result of hemolysis which reflects the
during hemolytic episode. predominance of unconjugated bilirubin in plasma.
 Proximal tubular cells- able to catabolize heme via o The lipid solubility of unconjugated bilirubin also leads to
deposition in the brain when hemolysis affects newborns
hemeoxygenase-1,
because the blood-brain barrier is not fully developed.
o Freeing and salvaging iron for export to the plasma by
ferroportin in the basolaminal membrane.  can lead to a type of brain damage called
kernicterus
o Proximal tubular cells also possess UGT1A1 that
conjugates bilirubin.  Refers to the yellow coloring
o MRP2 allow secretion of the conjugated bilirubin into the (icterus) of the brain tissue.
filtrate/urine.  GLUCOSE-6- PHOSPHATE DEHYDROGENASE
o Yet, the presence of OAT proteins in the basolaminal DEFICIENCY-jaundice is periodic
membrane may allow reabsorption into the plasma for  THALASSEMIA-jaundice is chronic
ultimate liver excretion.  GALLSTONES (cholelithiasis)
o Can occur whenever hemolysis is chronic; the
 Proximal tubular cell iron in excess of what can be
constantly increased amount of bilirubin in the bile leads
transported into the circulation gets stored as ferritin, and
to the formation of the stones.
some is converted to hemosiderin.
o When hemolysis is chronic in children, the persistent
compensatory bone marrow hyperplasia can lead to
bone deformities because the bones are still forming.
o For patients in whom an acquired, acute hemolytic
process develops, the associated malaise, aches,
vomiting, and possible fever may cause it to be confused
with an acute infectious process.
LABORATORY FINDINGS
 In patients with the clinical features of hemolytic anemia,
laboratory tests typically show evidence of increased
erythrocyte destruction and the compensatory increase in
the rate of erythropoiesis.
TEST OF ACCELERATED RED BLOOD CELL DESTRUCTION
 BILIRUBIN
o Increase bilirubin may be evident visually in icteric
serum or plasma.
o Bilirubin assays should reveal an increase indirect
fraction resulting in an increase in total bilirubin.
9 9
o If liver function is normal, conjugated bilirubin is formed because the cells have less exposure to plasma
and excreted as urobilinogen in the stool. glucose before early lysis.
o not detected in the urine  Use of this for detection of hemolysis is
 Unconjugated bilirubin is bound to albumin for problematic without baseline values for individuals.
plasma transport and the complex is not filtered by  Now widely used as an indicator of diabetes
the glomerulus. mellitus because the increased rate of glycation
 PLASMA HEMOGLOBIN, URINE HEMOGLOBIN, AND with elevated plasma glucose levels leads to an
URINE HEMOSIDERIN increase in the glycated hemoglobin value.
o Presence of methemoglobin, methemalbumin, and o More exact RBC survival assay uses random labeling
hemopexin-heme imparts a coffee-brown color to of blood with chromium-51 radioisotope.
plasma, strongly suggestive of fragmentation  This is the reference method for RBC survival
hemolysis. studies published by the International Committee
 the color is more often described as root for Standardization in Hematology.
beer- or beer-colored  Sample of blood is collected, mixed with the
o In a properly collected blood specimen: isotope, and returned to the patient.
 normal physiologic fragmentation  Method differs from cohort labeling- RBCs are
hemolysis produces a plasma labeled with radioactive iron or heavy nitrogen as
hemoglobin level of less than 1 mg/dL. they are produced in the bone marrow, thus the
o Typical values during hemolytic processes may be as labeled cells are generally the same age.
low as 15 mg/dL, so an increase in plasma hemoglobin These methods are not often used clinically but are used
may not always be visible. for research, particularly in the search for improved
 COMPLETE BLOOD COUNT methods of determining RBC survival.
Spherocytes can be expected to be seen with  LACTATE DEHYDROGENASE
macrophage-mediated hemolysis, whereas o Often increased in patients with fragmentation
fragmented cells, or schistocytes, are noted with hemolysis because of the release of the enzyme from
fragmentation hemolysis. ruptured RBCs
 HAPTOGLOBIN AND HEMOPEXIN  Other conditions, such as myocardial infarction or
o Immunoturbidimetry - suitable technique for the liver disease, also can cause increases.
determination of haptoglobin o When other results point to fragmentation hemolysis,
substantial decline in the serum haptoglobin one should expect an increase in the level of serum
level indicates fragmentation hemolysis. lactate dehydrogenase and other RBC enzymes and
Haptoglobin measurement - prone to both false- should not be misled into assuming that there is other
positive and false-negative results. organ damage.
 Low values suggest hemolysis but may
be due instead to impaired synthesis of TESTS OF INCREASED ERYTHROPOIESIS
haptoglobin caused by liver disease.  If bone marrow is healthy, hypoxia associated with
o haptoglobin is an acute phase reactant. hemolysis leads to increased erythropoiesis
 a patient with hemolysis may have a o increased circulating reticulocytes
relatively normal haptoglobin level if
o nucleated RBCs
there is also a complicating infection or
inflammation o sociated changes in CBC and bone marrow
o Quantification of hemopexin may also demonstrate a  Chronic hemolytic diseases are evident within 3-6 days
low value, yet it is not often measured, relying instead after acute hemolytic episode
on the more dramatic haptoglobin decline to detect
fragmentation hemolysis. COMPLETE BLOOD COUNT AND RED BLOOD CELL
o Hemopexin assays may ultimately be more valuable for MORPHOLOGY
detection of hemopexin depletion in conditions such as  Increase in polychromatic RBCs (retic) and nucleated
sepsis. RBCs = bone marrow compensation for hemolysis/blood
 CARBON MONOXIDE loss
o Values of 2 to 10 times the normal rate have been  Schistocytes
detected in some patients with hemolytic anemia, but o Expected with excessive fragmentation hemolysis
testing for carbon monoxide production is not typically  Spherocytes
required for clinical diagnosis.
o May be seen with macrophage-mediated processes
 RED BLOOD CELL SURVIVAL
 MCV
o Measuring glycated hemoglobin would show general
evidence of reduced RBC survival. o Increase
 Glycated hemoglobin-increases over the life of a  Extreme compensatory reticulocytosis
cell as it is exposed to plasma glucose. Its level
usually is decreased in chronic hemolytic disease
10 10
 resulting from the larger,
prematurely released “shift”
reticulocytes
o Within reference interval
 Hereditary spherocytosis
 Microsphero-cytes
 Larger shift reticulocytes
o Low or normal
 Schistocytes outnumber reticulocytes
 RDW
o Increase
 Reticulocytosis
 Addition of larger reticulocytes can
be expected to extend the range of
cell volumes
DIFFERENTIAL DIAGNOSIS
RETICULOCYTE COUNT
 Rapid decrease in hemoglobin concentration =
 Detects accelerated erythropoiesis
hemolysis (when hemorrhage and hemodilution is ruled
 Expected to rise during hemolysis or hemorrhage
out)
 Healthy bone marrow o Apparent before reticulocytosis and bilirubinemia
o Increase in: develop
 ARC  Hemolytic jaundice
 Relative RC
o Additional confirmation for hemolytic anemia
 RPI
o Only the indirect fraction of the total serum
 IRF
bilirubin is elevated
o Elevated urobilinogen
BONE MARROW EXAMINATION
 Acute hemolysis
 Not usually necessary to diagnose hemolytic anemia
o Hemoglobinemia
 Reveals erythroid hyperplasia
o Hemoglobinuria
o Peripheral blood reticulocytosis
o Erythroid component increases = overall ratio Figure No. Hemolysis timelines
decreases
 Core biopsy specimen rather than aspirated specimen
o More accurate
LABORATORY TESTS TO IDENTIFY SPECIFIC

HEMOLYTIC PROCESSES
 Direct antiglobulin test
 Heinz body test
 RBC enzyme studies
 Immunophenotyping
Figure No. Morphologic abnormalities associated with hemolytic anemia
11 11
In chronic hemolysis, persistence of hemoglobinemia,
hemoglobinuria, decreased serum haptoglobin level, indirect
bilirubinemia, and reticulocytosis can be expected, depending on
the mechanism of the hemolysis.
 Hemolytic anemias must be differentiated from other
anemias associated with bilirubinemia, reticulocytosis, or
both
o Anemia with reticulocytosis but no bilirubinemia
 Recovery from hemorrhage not treated
with transfusion or with effective
treatment of deficiencies
 ie., iron deficiency
o Anemia that results from hemorrhage into a body
cavity
 Reticulocytosis during recovery
 Bilirubinemia as a result of catabolism
of the hemoglobin in the hemorrhaged
cells
o Anemias associated with ineffective
erythropoiesis (eg., megaloblastic anemia)
 Bilirubinemia
 Elevated serum lactate dehydrogenase
 Low RC
Figure No. Differential diagnosis of hemolytic anemias vs other causes of indirect bilirubinemia and reticulocytosis
12 12
13
13
HEMATOLOGY 1 [TRANS] UNIT 23: EXTRAVASCULAR HEMOLYSIS
OUTLINE
EXTRINSIC DEFECTS LEADING TO INCREASED
ERYTHROCYTE DESTRUCTION – IMMUNE CAUSES
I. OVERVIEW OF IMMUNE HEMOLYTIC
ANEMIAS
1. Pathophysiology of Immune Hemolysis
2. Laboratory Findings in Immune Hemolytic
Anemia
II. AUTOIMMUNE HEMOLYTIC ANEMIA
1. Warm Autoimmune Hemolytic Anemia
2. Cold Agglutinin Disease
3. Paroxysmal Cold Hemoglobinuria
4. Mixed-Type Autoimmune Hemolytic
Anemia
III. ALLOIMMUNE HEMOLYTIC ANEMIAS
1. Hemolytic Transfusion Reaction
2. Hemolytic Disease of the Fetus and
Newborn
IV. DRUG-INDUCED IMMUNE HEMOLYTIC
ANEMIA
1. Mechanisms of Drug-Induced Immune
Hemolysis
2. Antibody Characteristics
3. Nonimmune Drug-Induced Hemolysis
4. Treatment
OVERVIEW OF IMMUNE HEMOLYTIC ANEMIAS

• Immune hemolytic anemia and nonimmune hemolytic anemia
are two broad categories comprising the extrinsic hemolytic
anemias.
o disorders in which red blood cells (RBCs) are
structurally and functionally normal but a condition
outside of the RBCs causes premature hemolysis.
• Nonimmune extrinsic hemolytic anemias are the result of
physical or mechanical injury to RBCs.
• Immune hemolytic anemias are conditions in which RBC PATHOPHYSIOLOGY OF IMMUNE HEMOLYSIS
survival is shortened as a result of an antibody-mediated • In immune hemolysis an antibody binds to an antigen on the
mechanism. surface of RBCs, which signals premature removal of those
• The antibody may be an autoantibody (directed against a self cells from the circulation through extravascular or intravascular
RBC antigen), an alloantibody (directed against an RBC hemolysis.
antigen of another per- son), or an antibody directed against • Two classes or isotypes of antibodies involved in most
a drug (or its metabolite) taken by the patient. immune hemolytic anemias are immunoglobulin G (IgG) and M
• RBCs with bound antibody or complement are prematurely (IgM).
removed from the circulation o IgG is a monomer in a Y-like structure with two
o extravascularly by macrophages (because of their identical heavy chains (g H chains) and two identical
receptors for complement and the Fc component of light chains (either k or l) connected by disulfide
antibody), bonds.
o intravascularly by complement-mediated hemolysis, o IgM is a pentamer consisting of five monomeric units
or by a combination of both mechanisms. connected by disulfide linkages at the C-termini of
• Anemia develops when the amount of hemolysis exceeds the their heavy chains (m H chains).
ability of bone marrow to replace RBCs that are destroyed. The • The classical complement pathway is an important mediator
degree of anemia varies from asymptomatic and mild to severe of immune hemolysis.
and life threatening. • The first protein, C1, has three components: C1q, C1r, and
• Immune hemolytic anemias may be classified into the following C1s.
groups: o After an antibody binds to its specific antigens on the RBC
o autoimmune hemolytic anemia surface, C1q must bind to two adjacent antibody Fc
o alloimmune hemolytic anemia domains to activate the pathway.
o drug-induced immune hemolytic anemia (Box 23.1). • Theoretically only one IgM molecule is needed for complement
• It is important to determine the cause of an immune hemolytic activation because of its larger pentameric structure with five Fc
anemia so that the appropriate therapy can be administered to domains; however, at least two molecules of monomeric IgG in
the patient. close proximity are required for C1q attachment.
1
ROGIE BAYAUA, BEULAH GO, KRISTLE PRING, REJINOLD SERRANO, KAIZER VANGUARDIA. | FEU MT 2023
• Therefore IgM antibodies are highly effective in activating complement is activated, which overwhelms complement
complement, whereas IgG antibodies are unable to activate the inhibitors.
pathway unless there is a sufficient number of IgG molecules • In these cases, complement activation proceeds from C1 to C9
on the RBC surface. and results in rapid intravascular hemolysis.
• In addition, subclasses IgG1 and IgG3 have high binding • Hemolysis mediated by IgG antibodies occurs with or without
affinity for C1q, whereas subclasses IgG2 and IgG4 have complement and predominantly by extravascular mecha-
minimal ability to bind complement. nisms.
• The binding of C1q to adjacent Fc domains requires calcium • RBCs sensitized with IgG are removed from circulation by
and magnesium ions and activates C1r, which then activates macrophages in the spleen, which have receptors for the Fc
C1s. component of IgG1 and IgG3.
o This activated C1q-C1r-C1s complex is an enzyme that • IgG antibodies are not efficient in activating complement, and
cleaves C4 and then C2, which results in the binding of a intravascular hemolysis by full activation of complement from
small number of C4bC2a complexes to the RBC C1 to C9 is rare (except with anti-P in paroxysmal cold
membrane. hemoglobinuria).
• C4bC2a complex is an active C3 convertase enzyme that • However, if there is a high density of IgG1 or IgG3 bound to
cleaves C3 in plasma; the result is the binding of many C3b antigens on RBCs, some complement is activated and C3b
molecules to C4bC2a on the RBC surface. binds to the membrane.
• The last phase of the classical pathway occurs when • If both IgG and C3b are on the RBC membrane, there is faster
C4bC2aC3b (C5 convertase) converts C5 to C5b, which clearance from the circulation by macrophages in both the
combines with C6, C7, C8, and multiple C9s to form the spleen and the liver.
membrane attack complex (MAC). • Often, IgG-sensitized RBCs are only partially phagocytized by
o The MAC resembles a cylinder that inserts into the lipid macrophages, which results in removal of some membrane.
bilayer of the membrane, forming a pore that allows water Spherocytes are the result of this process, and they are the
and small ions to enter the cell, causing lysis (Figure 23.3). characteristic cell of IgG-mediated hemolysis.
• The spherocytes are eventually removed from circulation by
entrapment in the red pulp of the spleen (splenic cords), where
they are rapidly phagocytized by macrophages.
LABORATORY FINDINGS IN IMMUNE HEMOLYTIC ANEMIA

• Laboratory findings in immune hemolytic anemia are similar to
the findings in other hemolytic anemias and include:
o decreased hemoglobin
o increased reticulocyte count
o increased levels of indirect serum bilirubin and lactate
dehydrogenase
o decreased serum haptoglobin level.
• If hemolysis is predominantly intravascular, or extravascular
hemolysis is severe, the haptoglobin level will be moderately to
severely decreased, plasma hemoglobin will be increased, and
the patient may have hemoglobinuria or even hemosiderinuria
(in cases of chronic hemolysis).
• Hemolysis mediated by IgM antibodies requires complement
and can result in both extravascular and intravascular • Mean cell volume (MCV) may be increased because of
hemolysis. reticulocytosis and RBC agglutination (if present).
• When IgM molecules attach to the RBC surface in relatively low • Leukocytosis and thrombocytosis may occur along with
density, complement activation results in C3b binding to the increased erythroid proliferation in bone marrow.
membrane, but complement inhibitors prevent full activation of • Findings on the peripheral blood film include polychromasia
the pathway to the terminal membrane attack complex. (as a result of reticulocytosis), spherocytes (caused by IgG-
• C3b-sensitized RBCs are destroyed by extravascular mediated membrane damage by macrophages), and
hemolysis, predominantly by macrophages (Kupffer cells) in the occasionally RBC agglutination.
liver, which have C3b receptors. • Nucleated RBCs, fragmented RBCs or schistocytes, and
• RBCs sensitized with only C3d are not prematurely removed erythrophagocytosis (phagocytes engulfing RBCs) may also be
from circulation because macrophages lack a C3d receptor. observed on the peripheral blood film.
• In severe cases of immune hemolysis involving heavy • To determine whether the hemolysis is due to an immune
sensitization of RBCs with IgM antibody, significantly more mechanism, a direct antiglobulin test (DAT) is performed.
2
o The DAT detects in vivo sensitization of the RBC surface • WAIHA may be classified as idiopathic or secondary.
by IgG, C3b, or C3d. o In patients with idiopathic WAIHA, the cause is
o In the DAT procedure, polyspecific antihuman globulin unknown.
(AHG) is added to saline-washed patient RBCs. o Secondary WAIHA may occur in many
• Polyspecific AHG has specificity for the Fc portion of human conditions such as lymphoproliferative diseases
IgG and complement components C3b and C3d; agglutination (chronic lymphocytic leukemia, B-lymphocytic
will occur if a critical number of any of these molecules is lymphomas, and Waldenström
present on the RBC surface. macroglobulinemia), nonlymphoid neoplasms
• If the DAT result is positive with polyspecific AHG, then RBCs (thymoma and cancers of the colon, kidney, lung,
are tested with monospecific anti-IgG and anti-C3b/C3d to and ovary), autoimmune disorders (rheumatoid
identify the type of sensitization. arthritis, scleroderma, polyarteritis nodosa,
• If IgG is detected on RBCs, elution procedures are used to Sjögren syndrome, and systemic lupus
remove the antibody from RBCs for identification. erythematosus), immunodeficiency disorders,
• Specificity of the IgG antibody may be determined by assessing and viral infections.
the reaction of the eluate with screening and panel reagent • The onset of WAIHA is usually insidious, with symptoms of
RBCs (RBCs genotyped for the major RBC antigens) using the anemia (fatigue, dizziness, dyspnea), but some cases can
indirect antiglobulin test (IAT). be acute and life threatening with fever, jaundice,
• Identification of any circulating alloantibodies or autoantibodies splenomegaly, and hepatomegaly, especially in children
by the IAT is also important in the investigation. with WAIHA secondary to viral infections. Massive
• The DAT result may be negative in patients with some immune splenomegaly, lymphadenopathy, fever, petechiae,
hemolytic anemias. ecchymosis, or renal failure in adults suggests an
• In addition, other disorders beside immune hemolytic anemia underlying lymphoproliferative disorder.
can cause a positive DAT. • Although most autoantibodies that cause WAIHA are IgG,
• Therefore diagnosis of immune hemolytic anemia cannot rely rare cases involving IgA autoantibodies as well as
solely on the DAT and must take into account the patient cases with fatal outcomes caused by warm-reacting
history; symptoms; recent medications; previous transfusions; IgM antibodies have been reported. Hemolysis is
coexisting conditions, including pregnancy; and results of predominantly extravascular in WAIHA, and cases of
applicable hematologic, biochemical, and serologic tests. fulminant intravascular hemolysis are rare.
Autoimmune Hemolytic Anemia • Warm autoantibodies are usually panreactive; that is,
they will agglutinate all screening and panel cells, donor
Autoimmune Hemolytic Anemia (AIHA) - is a rare disorder
RBCs, and the patient’s own RBCs, so the specificity of the
characterized by premature RBC destruction and anemia caused by
autoantibody is not apparent. In some cases Rh complex
autoantibodies that bind to the RBC surface with or without
specificity can be demonstrated. Rarely, a specific
complement activation.
autoantibody to an antigen in the Rh blood group system
o AIHA can affect both children and adults, and is identified. Autoantibodies to other antigens (such as LW,
its annual incidence is estimated to be between 1 Jk, K, Di, Ge, Lu, M, N, S, U, Ena, and Wrb) are
and 3 per 100,000 individuals. In children, more occasionally identified.
males are affected, but in adults, more females
• For most patients (approximately 80%) the autoantibody
are affected.6
can be detected in the serum. Because the autoantibody
o Autoantibodies may arise as a result of
is panreactive, it may mask reactions of alloantibodies with
immune system dysregulation and loss of
RBC panel cells.
immune tolerance, exposure to an antigen
• Anemia in WAIHA can be mild or severe, with RBC life
similar to an autoantigen, B-lymphocyte
span sometimes reduced to 5 days or less. Laboratory
neoplasm, or other unknown reason. The type,
findings for serum and urine reflect the predominantly
amount, and duration of antigen exposure and
extravascular hemolysis that occurs in IgG-mediated
genetic and environmental factors may also
immune hemolysis. Polychromasia and spherocytes are
contribute to development of autoantibodies
typical findings on the peripheral blood film. Occasionally
• Severity of the anemia depends on autoantibody
WAIHA is accompanied by immune thrombocytopenic
characteristics (titer, ability to react at 37° C, ability to
purpura and a decreased platelet count, a condition
activate complement, and specificity and affinity for the
known as Evans syndrome, which occurs primarily in
autoantigen), antigen characteristics (density on RBCs,
children.
immunogenicity), and patient factors (age, ability of bone
• In symptomatic but non-life threatening WAIHA, a
marrow to compensate for the hemolysis, function of
glucocorticosteroid such as prednisone is the initial
macrophages, complement proteins and regulators, and
treatment of choice. Approximately 70% to 80% of
underlying conditions).
patients show improvement with prednisone, but only
Autoimmune hemolytic anemias may be divided into four major about 20% to 40% achieve a long-term response. Many
categories based on the characteristics of the autoantibody and the
adult patients need to be on a long-term maintenance
mechanism of hemolysis:
dosage to remain asymptomatic. A serious side effect of
1. Warm autoimmune hemolytic anemia, glucocorticosteroid treatment (initially and long term) is an
2. Cold agglutinin disease, increased risk of osteoporosis and bone fractures.
3. Paroxysmal cold hemoglobinuria o Guidelines from the American College of
4. Mixed-type AIHA Rheumatology indicate that patients on
glucocorticosteroids may benefit from vitamin D,
Warm Autoimmune Hemolytic Anemia calcium, and bisphosphonate therapy to
It is the most commonly encountered autoimmune hemolytic prevent osteoporosis, but each patient’s situation
anemia, comprising up to 70% to 80% of cases. should be evaluated on an individual basis.
• Autoantibodies causing WAIHA react optimally at 37° C, • In patients with chronic WAIHA who are refractive to
and the vast majority of them are IgG. prednisone therapy or require long-term, high-dose
3
prednisone therapy, second-line therapy usually Acrocyanosis is a bluish discoloration of the extremities
includes a choice of either splenectomy or rituximab. (fingers, toes, feet, earlobes, nose) as a result of RBC
Splenectomy achieves a favorable response only in about autoagglutination, which causes local capillary stasis and
50% of patients, both initially and long term. Rituximab decreased oxygen delivery to the affected areas
is a monoclonal anti-CD20 antibody that binds to the
corresponding antigen found on B lymphocytes. • In contrast, patients with acute CAD may have mild to
o However, there are few studies on the long-term severe hemolysis that appears abruptly within 2 to 3
efficacy and safety of rituximab in WAIHA, and as weeks after onset of infectious mononucleosis, other viral
of this publication, rituximab is not FDA- infection, or M. pneumoniae infection, but it resolves
approved for this indication. spontaneously within days to a few weeks.
o Immunosuppressive drugs, such as
cyclophosphamide or azathioprine, are the • A cold agglutinin test determines the titer of the
third-line therapy for refractory WAIHA autoantibody at 4° C. Pathologic cold agglutinins can
because the toxicity and side effects may be reach titers of 1:10,000 to 1:1,000,000 at 4° C. Blood
severe. specimens for cold agglutinin testing must be maintained
o Hematopoietic stem cell transplantation at 37° C after collection to prevent binding of the
(HSCT) has been used in the past for severe, autoantibody to the patient’s own RBCs, which can
refractory, life-threatening autoimmune falsely decrease the antibody titer in the serum.
syndromes, including hemolytic anemia and o Alternatively, a specimen anticoagulated with
Evans syndrome, but is a therapy of last resort. ethylenediaminetetraacetic acid (EDTA) can
be warmed for 15 minutes at 37° C to dissociate
Cold Agglutinin Disease auto absorbed antibody before determining the
Cold agglutinins are autoantibodies of the IgM class that react titer. When a high-titer cold agglutinin is
optimally at 4° C and are commonly found in healthy individuals. present, an EDTA anticoagulated blood
o These non-pathologic cold agglutinins are specimen can show visible agglutinates in the
polyclonal, occur in low titers (less than 1:64 at tube at room temperature or cooler.
4° C), and have no reactivity above 30° C.
• Most pathologic cold agglutinins are monoclonal,
occur at high titers (greater than 1:1000 at 4° C), and are
capable of reacting at temperatures greater than 30° C.
These cold agglutinins can react at body temperature;
therefore, they may induce cold agglutinin disease,
• Cold agglutinins that are able to bind RBC antigens near or
at 37° C (high thermal amplitude) cause more severe
symptoms. CAD is also recognized as a clonal
lymphoproliferative B cell disorder. It comprises
approximately 15% to 20% of the cases of autoimmune
hemolytic anemia.
• In CAD, IgM autoantibodies bind to RBCs when the patient
is exposed to cold temperatures, particularly in peripheral
circulation and vessels of the skin, where temperatures can
drop to 30° C.13 During the brief transit of RBCs through
these colder areas, IgM autoantibodies activate the
classical complement pathway.
o Acute CAD occurs secondary to Mycoplasma • Blood specimens from patients with cold agglutinins must
pneumoniae infection, infectious be warmed to 37° C for at least 15 minutes before
mononucleosis, and other viral infections. complete blood count analysis by automated blood cell
These cold agglutinins are polyclonal IgM, analyzers. RBC agglutination grossly elevates the
with a normal distribution of k and l light mean cell volume, reduces the RBC count, and has
chains. unpredictable effects on other indices.
o Chronic CAD is a rare hemolytic anemia that • When the specimen is warmed to 37° C, antibody
typically occurs in middle-aged and elderly dissociates from RBCs and agglutination usually
individuals, and the autoantibody is usually disappears. If not, a new specimen is collected and
monoclonal IgM with k light chains. Chronic maintained at 37° C for the entire time before testing. To
CAD can be idiopathic, but it is usually avoid agglutination on a peripheral blood film, the slide can
secondary because of an underlying also be warmed to 37° C before the application of blood.
lymphoproliferative neoplasm such as B- Cold agglutinins can also interfere with ABO typing.
lymphocytic lymphoma, Waldenström
macroglobulinemia, or chronic lymphocytic • Acute CAD associated with infections is self-limiting,
leukemia. and cold agglutinin titers are usually less than 1:4000. If
• Clinical manifestations are variable in chronic CAD. Most hemolysis is mild, no treatment is required; however,
patients have a mild anemia with a hemoglobin result patients with severe hemolysis require transfusion and
ranging from 9 to 12 g/dL, but others can develop life- supportive care.
threatening anemia with hemoglobin levels falling to less • In chronic CAD with moderate to severe symptoms,
than 5 g/dL, especially after exposure to cold rituximab produces a partial response in about half of
temperatures. patients because of its targeting and ultimate
• Symptoms include fatigue, weakness, dyspnea, pallor destruction of B lymphocytes containing the CD20
(caused by the anemia), and acrocyanosis.13
4
antigen, but median remission is less than a year.

o Rituximab-fludarabine and rituximab- • The patient control tube is kept at 37° C for both
bendamustine combination therapies have a incubations (a total of 60 minutes). After centrifugation
better response rate, 76% and 71% respectively, serum is examined for hemolysis. A positive test result for
with the later combination having fewer side anti-P is indicated by hemolysis in the patient test
effects and better tolerability. specimen incubated first at 4° C and then at 37° C and no
o Plasmapheresis is used in severe cases but hemolysis in the patient control specimen kept at 37° C. In
provides only temporary benefit. Corticosteroid the control tube, anti-P is not able to bind to antigen at 37°
therapy and immunosuppressive therapy with C, so complement is not activated, and hemolysis does not
cyclophosphamide and chlorambucil are not occur.
effective for most patients. Splenectomy is also • Incubating patient serum with complement and papain-
not effective because C3b-sensitized RBCs in treated compatible group O RBCs increases the
IgM-mediated autoimmune hemolysis are cleared sensitivity of the test in detecting anti-P. Enzyme treatment
primarily by the liver. provides greater exposure of the P antigen on the RBC
surface for antibody binding
• In CAD cases involving an autoantibody with wide thermal • PCH is severe but self-limiting and resolves in several
amplitude, detection of coexisting warm-reactive days to a few weeks, with an excellent prognosis.1,13,18
alloantibodies can be time consuming and difficult. During In most patients, anemia is severe and can be life
transfusion, the patient is kept warm, small amounts of threatening, so transfusion is usually needed until the
blood are given while the patient is observed for symptoms resolve. Because anti-P autoantibody reacts
symptoms of a hemolytic transfusion reaction, and a only at lower temperatures and P antigen-negative blood is
blood warmer is used to minimize in vivo reactivity of very rare, P-positive blood can be transfused.
the cold autoantibody.
Mixed-Type Autoimmune Hemolytic Anemia
Paroxysmal Cold Hemoglobinuria • Mixed-type AIHA occurs very infrequently. In this condition
• Paroxysmal cold hemoglobinuria (PCH) is an acute form the patient simultaneously develops an IgG
of cold-reactive hemolytic anemia. PCH can be autoantibody with optimum reactivity at 37°C (WAIHA)
idiopathic or secondary. Secondary PCH was and a pathologic IgM autoantibody that reacts
associated with late-stage syphilis, but now it is most optimally at 0° C to 10° C but has a thermal amplitude of
seen in young children after a viral respiratory infection. greater than 30° C (CAD).
o PCH is rare in adults. The prevalence of PCH has • Patients with WAIHA and a nonpathogenic cold agglutinin
been reported to be as high as 32% to 40% of (i.e., an agglutinin that does not react at a temperature
children with autoimmune hemolytic anemia, with greater than 20° C) should not be classified as having a
a median age at presentation of 5 years. mixed-type AIHA because the cold agglutinin is not
• Anti-P autoantibody, also called the Donath- clinically significant.
Landsteiner antibody, is a complement-binding IgG • Hemolysis results from a combination of extravascular
hemolysin with specificity for P antigen on RBCs. Anti- and intravascular mechanisms. The disease course
P autoantibody is biphasic in that at cold temperatures it appears to be chronic, with intermittent episodes of severe
binds to the P antigen on RBCs and partially activates anemia. The DAT is positive with IgG only, C3d only, or
complement (C1 to C4), but full complement activation (C3 both IgG and C3d. The warm autoantibody is typically
through C9) and hemolysis occur only upon warming to panreactive with unclear specificity, whereas the cold-
37° C. reacting antibody usually has anti-I specificity. Treatment
o Anti-P autoantibody binds RBC antigen optimally is the same as that described for WAIHA
at 4° C and has a thermal amplitude of less
than 20° C. At warmer temperatures, anti-P
autoantibody dissociates from RBCs; the titer is
usually less than 1:64.
• Children typically present with acute fever, malaise, and
back, leg, and/or abdominal pain 1 to 2 weeks after an
upper respiratory tract infection. Pallor, jaundice, and dark
urine caused by hemoglobinuria are often present.
• Exposure to cold temperatures may precipitate hemolytic
manifestations in some patients, but cold exposure is not
required for the majority of patients to manifest symptoms.
• Reticulocytosis is typical but can be preceded by
reticulocytopenia. The peripheral blood film shows
polychromasia and spherocytes, but schistocytes,
nucleated RBCs, anisocytosis, poikilocytosis, and ALLOIMMUNE HEMOLYTIC ANEMIAS
erythrophagocytosis can also be observed. HEMOLYTIC TRANSFUSION REACTION (HTR)
• The classic Donath-Landsteiner screening test for anti-P is • One of the most severe and potentially life-threatening
done by collecting blood specimens in two tubes complications of blood transfusion caused by immune-
(without anticoagulant), one for the patient test and the mediated destruction of donor cells by an antibody in
other for the patient control. The patient test specimen the recipient.
is incubated first at 4° C for 30 minutes (to allow anti-P • HTRs can have an acute or delayed onset.
binding to the P antigen and partial complement activation • Offending antibody – may be IgM or IgG.
on RBCs) and then at 37° C for 30 minutes (to allow full • Complement – may be partially or fully activated or not
activation of the complement pathway to lysis). activated at all.
5
• Hemolysis – may be intravascular or extravascular. activation.

• Principal signs: inadequate posttransfusion
ACUTE HEMOLYTIC TRANSFUSION REACTION (AHTR) hemoglobin increase, positive DAT results for IgG
• Occur within minutes to hours of initiation of a and/or C3d, morphologic evidence of hemolysis
transfusion. (spherocytes, polychromasia), and an increase in serum
• Most common cause: Accidental transfusion of ABO- indirect bilirubin.
incompatible donor RBCs into a recipient. • Management: monitoring of kidney function, especially
o Example: Transfusion of group A RBCs into a in acutely ill patients.
group O recipient. There is rapid, complement-
mediated intravascular hemolysis and activation HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN
of the coagulation system. • Occurs when an IgG alloantibody produced by the mother
o ABO-incompatible RBC transfusions – are crosses the placenta into the fetal circulation and binds to
usually a result of clerical error and have been fetal RBCs that are positive for the corresponding antigen.
estimated to occur in approx. 1 in 40,000, • IgG-sensitized fetal RBCs are cleared from the circulation
whereas the estimated risk of ABO HTR is by macrophages in the fetal spleen (extravascular
1:80,000. hemolysis), and an anemia gradually develops.
• Severity: Variable and is affected by the infusion rate and o Erythroid hyperplasia – in the fetal bone marrow
volume of blood transfused. o Extramedullary erythropoiesis – in the fetal
• Estimated mortality rate: 2% spleen, liver, kidneys, and adrenal glands.
• AHTRs can occur as a result of incompatibilities involving § As a result, many nucleated RBCs are
other blood group systems, but these are rare. released into the fetal circulation.
§ If anemia is severe in utero, it can lead
• Symptoms of Severe Intravascular Hemolysis in ABO- to generalized edema, ascites, and a
related AHTRs: begins within minutes or hours and may condition called hydrops fetalis (fatal if
include chills, fever, urticaria, tachycardia, nausea and untreated).
vomiting chest and back pain, shock, anaphylaxis, • Anti-Kell antibodies are notable because they also cause
pulmonary edema, congestive heart failure, and anemia by suppressing fetal erythropoiesis.
bleeding as a result of disseminated intravascular
coagulation (DIC). RH HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN
• Transfusion should be immediately terminated on the first • In Rh HDFN, which causes the highest number of fetal
appearance of symptoms. fatalities, an Rh (D)-negative mother has preformed anti-
• Treatment: prevent or correct shock, maintain renal D antibodies (IgG, reactive at 37ºC) from exposure to the
circulation, and control DIC. D antigen either through immunization in a previous
• Immediate investigation of suspected HTR: clerical check pregnancy with a D-positive baby or from previous
for errors, an examination of a posttransfusion blood transfusion of blood products with D-positive RBCs.
specimen for hemolysis, and performance of the DAT • In subsequent pregnancies the anti-D crosses the
on RBCs in a posttransfusion specimen. placenta, and if the fetus is D positive, the anti-D binds to
D antigen sites on the fetal RBCs.
• If an AHTR occurred, hemoglobinemia and • These anti-D-sensitized fetal RBCs are cleared from the
hemoglobinuria are detectable, and the DAT result is circulation by macrophages in the fetal spleen, and anemia
positive. and hyperbilirubinemia develop.
• Hemoglobin and serum haptoglobin levels = decrease; • Amniocentesis is accurate at predicting severe fetal
Serum indirect bilirubin = will not begin to rise until 2 to anemia, but it is an invasive procedure and carries some
3 days after the episode. risk of fetal loss.
• ABO and RH typing, antibody screen, and cross- • If severe fetal anemia and HDFN caused by anti-D are
matching – repeated on recipient and donor specimens to suspected, a percutaneous umbilical fetal blood specimen
identify the blood group incompatibility. can be obtained and tested for the hemoglobin level to
• Coagulation tests (D-dimer, fibrinogen, factors V and VIII, determine the severity of anemia.
and platelet count) – can help reveal and assess the risk of • Ultrasound measurement of fetal cerebral blood flow –
DIC. noninvasive assessment of anemia.
DELAYED HEMOLYTIC TRANSFUSION REACTION (DHTR) Laboratory Findings:

• May occur days to weeks after transfusion as the titer • ABO, Rh typing, and antibody screen – are performed
alloantibodies increases. on the mother when the fetus is between 10 and 16 weeks’
• Often the patient has been alloimmunized by pregnancy or gestation and again at 28 weeks’ gestation.
previous transfusion, but the antibody titer was lower than • Antibody screen during pregnancy detects antibodies
the level of serologic detection at the time of transfusion. other than those caused by ABO incompatibility.
• The second exposure to the antigen results in an increase o If positive for clinically significant antibody, an
in titer (anamnestic response). RBC panel is performed to identify the specificity
• The antibody is usually IgG, is reactive at 37ºC, and may of the antibody.
or may not be able to partially or fully activate complement. o anti-I, anti-P1, anti-Lea, and -Leb – certain
• Antibodies most often implicated in DHTRs are directed antibodies ignored for their corresponding
against antigens in the Duffy and Kidd blood group antigens are poorly developed at birth.
systems. o Mothers with initial positive antibody screens
are retested with an antibody screen every month
• Patient’s antibody binds to transfused RBCs, which leads
until 28 weeks, then every 2 weeks thereafter.
to extravascular hemolysis, with or without complement
o Antibody titers are reported from each specimen
6
• Titration of the antibody does not predict the severity of weakly positive and may be negative.
HDFN, rather it helps determine when to monitor for HDFN • Spherocytes and polychromasia on the peripheral blood
by additional methods such as spectrophotometric analysis film are typical.
of amniotic fluid bilirubin. • HDFN can be cause by other IgG antibodies, particularly
• After the first affected pregnancy, the antibody titer is no antibodies to the K, c, and Fya antigens.
longer useful, and other means of monitoring the fetus • HDFN caused by other blood groups is rare.
are used, such as amniocentesis and ultrasonography. • Antibody screening in the first trimester can assist in
identifying rare antibodies that can cause HDFN.
• Immunized D-negative mother receives antenatal Rh • Adverse clinical outcomes: varying degrees of anemia,
immune globulin (RhIG) at 28 weeks’ gestation and again jaundice, and kernicterus.
within 72 hours of delivery of a D-positive infant to prevent
alloimmunization to the D-antigen.
o One (1) antenatal dose of 200µg RhIG – will
reduce by half the risk of the mother developing
anti-D antibodies and having a child with HDFN in
the next pregnancy.
o Rh-negative women who experience
spontaneous or induced abortion also receive Rh
immune globulin.
• At delivery, newborn testing is performed on umbilical
cord blood.
• Neonates with Rh HDFN have a decreased hemoglobin
level, increased reticulocyte count, and increased level of
serum indirect bilirubin.
• The peripheral blood film shows polychromasia and DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA (DIIHA)
many nucleated RBCs. • Is very rare, with an estimated annual incidence of about
• ABO (only forward typing), Rh typing, and DAT – are 1 per million persons.
also performed. • It is suspected when there is a sudden increase in
• DAT result is positive for IgG, and anti-D can be hemoglobin after administration of a drug, clinical and
demonstrated in an eluate of the infant’s RBCs. biochemical evidence of extravascular or intravascular
hemolysis, and a positive DAT result.
Treatment for the Affected Infant: • More than 125 drugs have been reported to cause DIIHA.
• Intrauterine transfusion – pooled hemolyzed blood is The most common categories: antimicrobial (particularly
removed via amniocentesis from the fetal abdomen and penicillins and cephalosporins), anti-inflammatory, and
replaced with a small amount of fresh RBCs. antineoplastic drugs.
o Can be used to correct fetal anemia and prevent • Most common drugs in the last 10 years: piperacillin,
hydrops fetalis. cefotetan, and ceftriaxone.
• Cordocentesis – also used, whereby fresh RBCs are
injected into the umbilical vein. MECHANISMS OF DRUG-INDUCED IMMUNE HEMOLYSIS
• Survival rates of fetuses receiving transfusions: 85% to • Three (3) generally accepted mechanisms involve an
90%. antibody produced by the patient as a result of exposure
• Risk of premature death: 1% to 3%. to the drug and include drug adsorption, drug-RBC
• After delivery, the neonate may need exchange membrane protein immunogenic complex, and RBC
transfusions and phototherapy to reduce the level of autoantibody induction.
serum indirect bilirubin and prevent kernicterus (bilirubin • A fourth (4th) mechanism, drug-induced
accumulation in the brain). nonimmunologic protein adsorption (NIPA), can result
• Prolonged postnatal anemia – can be the result of a slow in a positive DAT result, but no drug or RBC antibody is
decrease of maternal antibody in the newborn’s circulation. produced by the patient.
o Rare cases of prolonged anemia are documented • Several authors suggested that all drug-induced immune
in infants who received intrauterine hemolysis is explained by a single mechanism known as
transfusions. unifying theory.
o It proposes that a drug interacts with the RBC
HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN membrane and generates multiple immunogenic
CAUSED BY OTHER BLOOD GROUP ANTIBODIES epitopes that can elicit an immune response to
• ABO HDFN – more common than Rh HDFN and may the drug alone, to the drug-RBC membrane
occur during first pregnancy. protein combination, or to an RBC membrane
• Unlike Rh HDFN, it is asymptomatic or produces mild protein alone.
hyperbilirubinemia and anemia. • Diagnosis of DIIHA: antibody screen = positive; RBC
• It is seen in some type A or B infants born to type O eluate = contains an antibody.
mothers who produce IgG anti-A and anti-B, which are
capable of crossing the placenta. 1. Drug adsorption: Patient produces an IgG antibody to a
• It is milder than Rh HDFN likely because A and B antigens drug. When the drug is taken by the patient, the drug binds
are poorly developed on fetal and newborn RBCs, and strongly to the patient’s RBCs. The IgG drug antibody
other cells and tissues express A and B antigens, which binds to the drug attached to the RBCs, usually without
reduces the amount of maternal antibody directed against complement activation. Because the offending antibody is
fetal RBCs. IgG and is strongly attached to the RBCs via the drug,
• The DAT result for newborn with ABO HDFN is only hemolysis is extravascular by splenic macrophages, which
7
remove the antibody- and drug-coated RBCs from the

circulation DRUG-INDEPENDENT ANTIBODIES
2. Drug-RBC membrane protein immunogenic complex: • Drug-independent antibodies are warm-reactive, RBC
A drug binds loosely to an RBC membrane protein to form autoantibodies induced by the drug. Hemolysis is
a drug-RBC protein immunogenic complex or epitope.
extravascular, mediated by macrophages predominantly
The patient produces an IgM and/or IgG antibody that
in the spleen. In the IAT, patient's serum and an eluate of
binds to the complex on the RBCs, and complement is fully
activated, which causes acute intravascular hemolysis. cells generally react at 37° C with all screening and panel
3. RBC autoantibody induction: A drug induces the patient RBCs.
to produce IgG warm-reactive autoantibodies against RBC
self-antigens. These autoantibodies react at 37°C, and NONIMMUNE DRUG-INDUCED HEMOLYSIS
the laboratory findings are indistinguishable from those in
WAIHA. Hemolysis is extravascular and is mediated by • In drug-induced nonimmunologic protein adsorption
macrophages predominantly in the spleen. (NIPA), the patient does not produce an antibody to the
drug or to RBCs. This phenomenon is usually
asymptomatic and results in a positive DAT finding, but
only rarely has hemolysis been reported.
TREATMENT
• Most patients will gradually show improvement within a
few days to several weeks after starting treatment. If
anemia is severe, the patient may require RBC
transfusion or plasma exchange. Regardless of
mechanism, future episodes of DIIHA can be prevented
by avoidance of the drug.
(A), Drug adsorption. A drug binds (adsorbs) tightly to the red blood cell
(RBC) membrane. The patient produces an antidrug immunoglobulin G
(IgG) antibody that binds to the drug. RBCs coated with adsorbed drug
bound with antidrug antibodies are removed from circulation by
macrophages in the spleen (extravascular hemolysis). (B), Drug-RBC
membrane protein immunogenic complex. A drug loosely binds to an
RBC membrane protein forming a drug-RBC protein complex. The patient
produces an IgG and/or IgM (not shown) antibody that binds to the
complex, causing complement activation and acute intravascular
hemolysis. (C), RBC autoantibody induction. A drug induces the patient
to produce IgG warm-reactive autoantibodies against RBC self-antigens.
The IgG autoantibodies bind to the corresponding antigens on the RBC
surface. RBCs coated with IgG autoantibodies are removed from circulation
by macrophages in the spleen (extravascular hemolysis)
ANTIBODY CHARACTERISTICS
DRUG-DEPENDENT ANTIBODIES
• Drug-dependent (most frequent) and drug-independent
antibodies are the two kinds of antibodies implicated in
DIIHA. Some medications can cause both types of
antibodies to be produced at the same time.
1. Antibodies that react only with drug-treated cells. In the
IAT, patient's serum and an eluate of the patient's cells
react only with drug-treated RBCs. Complement is not
usually activated. Examples of drugs that elicit antibodies
in this category are penicillin, ampicillin, and many
cephalosporins.
2. Antibodies that react only in the presence of the drug.
Antibodies activate complement and trigger acute
intravascular hemolysis that may progress to renal failure.
Hemolysis occurs abruptly after short periods of drug
exposure or upon read ministration of the drug. Examples
of drugs that elicit antibodies in this category are
piperacillin and some second and third-generation
cephalosporins.
8
BOARD RECALLS AND BASIC REVIEW OF HEMATOLOGY I
MIDTERM
• Source of error in cyanmethemoglobin method in measuring hgb:

o WBC count that exceeds linearity limits
o Lipemic plasma
o Scratched or dirty hemoglobin
o NOTE: EXCESSIVE ANTICOAGULANT DOES NOT AFFECT
• Cyanmethemoglobin measures all hgb except: SULFHEMOGLOBIN
• Sources of falsely low ESR:
o EDTA tube is clotted
o EDTA tube is 1/3 full
o EDTA tube specimen is 24hrs old
• RETICULOCYTE COUNT: most effective means of assessing rbc generation in response to
anemia
• EMBDEN-MEYERHOF: RBC metabolic pathway that generates ATP via glycolysis,
ANAEROBIC
• PENTOSE PHOSPHATE PATHWAY: AEROBIC rbc pathway, reduced GSH protects RBCs
from hemolysis by oxidative free radicals and reduces oxidized sulfhydryl groups in
hemoglobin
• METHEMOGLOBIN REDUCTASE PATHWAY: Converts ferric ions to ferrous ions to form
functional hemoglobin
• RAPOPORT LEUBERING SHUNT: primarily for the production and accumulation of 2,3-DPG
• POSTERIOIR ILIAC CREST AND STERNUM: Best areas for BM aspiration
• MONOCYTE AND T LYMPHOCYTES: produces cytokines like IL and CSF
• POLYCHROMATOPHILIC NORMOBLAST: last stage of erythropoiesis capable of mitosis
• BASOPHILIC NORMOBLAST: last stage with nucleolus
• POLYCHROMATOPHILIC ERYTHROBLAST: last stage with nucleus
• RUBRICYTE: first stage with pink color of RBC
• PRORUBRICYTE: has deeper of rich blue cytoplasm
• NORMOBLASTIC PROLIFERATION: increase the cell number and the development of
immature cells to mature cells
• ERYTHRONS: Collection of RBC stages
• Senescent Rbs has: CONDENSED NUCLEUS
• MYELOBLAST: most mature granulocyte precursor capable of mitosis----SECONDARY
GRANULES
• PROMYELOCYTE STAGE----AZUROPHILIC GRANULES: primary granules
• INDENTATION OF NUCLEUS: differentiate metamyelocyte(Kidney shape) from other
granulocyte stage
• RBC precursor are found in: Surrounding fat cell in apoptotic island
• DIAPEDESIS: cell movement through blood vessel to tissue site
• CHEMOTAXIS: movement of leukocyte to the site of infection
• APOPTOSIS: program cell death
• T or TENSE FORM: Low oxygen affinity state of hemoglobin
• R or REACTIVE FORM/RELAXED: High oxygen affinity state of hgb
• Greatest portion of operational body iron contained in: HEMOGLOBIN
• HGB H: hgb that has chemical confirmation of Beta4
• Asynchronous development of hematopoetic cells within bone marrow is the result of:
IMPAIRED DNA SYNTHESIS
• SPHEROCYTOSIS: Usually associated with hemolytic anemia
• Hereditary Stomatocytosis is manifested physiology by changes in: MEMBRANE CATION
PERMEABILITY
• HEREDITARY SPHEROCYTOSIS: condition that will have a complete hemolysis at 0.45%
OFT
• SPHEROCYTES: increased OFT
• TARGET CELLS: decreased OFT
• SCHISTOCYTES: associated with increased RBC destruction
• CODOCYTES: seen in patient with Lecithin-cholesterol acyl transferase
• HOWELL JOLLY BODIES: composed of DNA
• HEINZ BODIES: composed denatured hgb
• BASOPHILIC STIPPLINGS: RNA remnants, associated in Lead poisoning
• SIDEROTIC GRANULES/PAPPENHEIMER: Iron
• CABOT RINGS: thready, blue ring shaped, twisted or figure-8
• ALDER-REILLY RESEMBLES TOXIC GRANULES
• ALDER-REILLY ANOMALY: dense azurophilic granulation in all types of leukocytes which is
result from abnormal deposition and storage mucopolysaccharides
• MAYHEGGLIN INCLUSION: associated in Scarlet Fever and occasionally seen in patient
with Burns.
• DOHLE-BODIES: RNA remnants and free ribosomes or ReR, positive with PAS staining
• RBC inclusion mistaken read as Reticulocytes when stained with supravital: HOWELL
JOLLY, PAPPENHEIMER AND HEINZ BODIES
• RBC inclusion in MYELODYSPLASTIC SYNDROME: BASOPHILIC STIPLINGS, HOWELL JOLLY
BODY, DIFFUSE BASOPHILIA
• RBC inclusion in Megaloblastic: HOWELL JOLLY BODIES and also include
HYPERSEGMENTED NEUTROPHIL
• MAYHEGGLIN ANOMALY: there is a pseudo-DOHLE BODIES found, thrombocytopenia and
large platelets
• CHEDIAK-HIGASHI INCLUSION: Large, coarse, irregular lysosomal granules in cytoplas of
granulocytes and monocyte
• Anemia found in Chronic Renal Failure most likely caused by: LOSS OF ERYTHROPOETIN
SYNTHESIS
• MYELOPHTHISIC ANEMIA: Associated with bone marrow infiltration and
hyperproliferation by nonerythroid cells
• THALASSEMIA: Result of quantitative defect in globin chain synthesis
• HODGKIN DISEASE: Presence of giant binucleated reed Sternberg cells with prominent
nucleoli
• DIAGNOSTIC TEST FOR HOFGKIN: Lymph node biopsy
• ROLEAUX FORMATION: Usually observed in Multiple Myeloma patient
• HEMACYTOMETER: glass counting chamber for manual counting
• NEUTROPHILIA(increased neutrophils)- indicates bacterial infection
• EOSINOPHILIA: parasitic infection
• BASOPHILIA: usually allergic reaction
• LYMPHOCYTOSIS: usually viral infection
• ESSENTIAL THROMBOCYTOPENIA: increased platelet count and uncontrolled platelet
production
• THROMBOCYTOPENIA: can result to bleeding and bruises
• SUPRAVITAL STAIN: Stain for iron containing, retics like New Methylene Blue
• ROMANOWSKY LIKE WRIGHT-GIEMSA STAIN: for PBS and it contains eosin and methylene
blue
• PRUSSIAN BLUE: Siderocyte stai
• FIBRINOLYSIS: final stage of coagulation
• MEGAKARYOCTE: largest cell in bone marrow
• MYELOID PROGENITOR can give rise to: Granulocytic, Erythrocytic, Monocytic,
Megakaryocytic
• KIDNEY: detects hypoxia
• SPLEEN: site for platelet sequestration and senescent RBC destruction
• EPO: require very early in hematopoetic differentiation
• ERYHTROPOEITIN: Cytokine stimulatory of eryhtropoesis
• INTRAVASCULAR HEMOLYSIS: destruction of the RBC by the vascular system due to
turbulence
• FANCONI ANEMIA: increased chromosome fragility
• APLASTIC ANEMIA: hypoproliferative, pancytopenia
• POIKILOCYTOSIS: Abnormalities in the horizontal and vertical linkages of the
transmembrane and cytoskeletal RBC membrane proteins, SHAPE CHANGES
• HEMOGLOBIN contain: 4 HEME, 4 GLOBIN
• Aminolevulinate synthase: suppression of this enzyme is a key rate-limiting step in heme
synthesis
• QUALITY CONTRIOL: employed to document assay validity, accuracy, and precision
• RBC parameter: Hgb, Hct, RDW and RBC indices
• EDTA(liquid K3) Ethylenediaminetetraacetate : coagulant of choice for CBC
• HEPARIN: coagulant of choice for OFT
• CITRATE: coagulant of choice for coagulation studies
• 3.2% SODIUM CITRATE: can be used in ESR
• RDW: parameter in rbc size
• MCH: parameter in rbs Hgb per unit volume
• MCH: most reliable automated index for classification of anemia
• MCHC: Hgb concentration of the average rbc
• ANEMIA OF CHRONIC DISORDER: decreased transferrin due to being a negative APR,
decrease transferrin receptors on macrophage
• ANEMIA OF CHRONIC RENAL INSUFFICIENCY: failure of kidney to produce EPO
• LEUKOERYTHROBLASTOSIS: increased immature granulocytes and nRBC
• APLASTIC ANEMIA: at least 2 of the following WBC count lower that 500cells/mm3,
platelet count lower than 20,000/mm and a retic count lower than 1%
• HEREDITARY SIDEROBLASTIC ANEMIA: DIMPORPHIC----can be normocytic, normoblastic
or microcytic, hypochromic----------TARGET CELLS AND BASOPHILIC STIPPLINGS
1 Anemias Caused by Defects in DNA Metabolism
INTRODUCTION • Plays an important role in the metabolism of

amino acids and nucleotides.
• Impaired deoxyribonucleic acid (DNA)
metabolism causes systemic effects by • Deficiency of the vitamin leads to impaired
impairing production of all rapidly dividing cell replication.
cells of the body.
Defect in Megaloblastic Anemia Caused By Deficiency
• Megaloblastic anemia is the hallmark of the in Folate and Vitamin B12
diseases affecting DNA metabolism.
• When either folate or vitamin B12 is deficient,
ETIOLOGY thymidine nucleotide production for DNA
synthesis is impaired.
• The root cause of megaloblastic anemia is
impaired DNA synthesis. • The resulting DNA is nonfunctional, and the
DNA replication process is incomplete. Cell
• The anemia is named for the very large cells of
division is halted, resulting in either cell lysis
the bone marrow that develop a distinctive
or apoptosis of many erythroid progenitors
morphology.
and precursors within the bone marrow.
• Because of a reduction in the number
• Cells that survive continue the abnormal
of cell divisions.
maturation with a fewer number of red blood
• Megaloblastic anemia is one example of a cells (RBCs) released into the circulation.
macrocytic anemia.
• This is called ineffective hematopoiesis.
Physiologic Roles of Vitamin B12 and Folate
• The remaining erythroid precursors are
VITAMIN B12 larger than normally seen.
• An essential nutrient which plays a role in the • Their nuclei are immature-appearing
synthesis of DNA in humans. compared with the cytoplasm.
• Vitamin B12 is a coenzyme in two biochemical • In contrast to the normally dense chromatin of
reactions in humans. comparable normoblasts, the nuclei of
megaloblastic erythroid precursors have an
• One is isomerization of open, finely stippled, reticular pattern.
methylmalonyl coenzyme A (CoA) to
succinyl CoA, which requires vitamin • The slower maturation rate of the nucleus
B12 as a cofactor and is catalyzed by compared with the cytoplasm is called
the enzyme methylmalonyl CoA nuclear-cytoplasmic asynchrony.
mutase.
• Pancytopenia is also evident.
• The second reaction is the transfer of
Other Causes of Megaloblastosis
a methyl group from 5-
methyltetrahydrofolate (5-methyl • Vitamin B12 and folate deficiency are not the
THF) to homocysteine. only causes of megaloblastic erythroid
precursors.
FOLATE
MYELODYSPLASTIC SYNDROME (MDS)
• Folate is the general term used for any form of
the vitamin folic acid. • Cells may have megaloblastoid features.
• The function of folate is to transfer carbon • Macrocytic erythrocytes and their precursors
units in the form of methyl groups from donors characteristically show delayed cytoplasmic
to receptors. and nuclear maturation.
CDA TYPES I AND III
Hematology Lecture
• Nuclear-cytoplasmic asynchrony and • Increased Need

megaloblastic erythroid precursors may be
• During lactation or pregnancy.
seen.
• During infancy or childhood.
FAB M6
• Impaired Absorption
• Another rare condition in which erythroid
precursors have a megaloblastic appearance. • Deficiency of a folate transporter
protein (PCFT).
• In this condition the erythroblasts are
macrocytic, and the immature appearance of • Sprue and Celiac Disease
the nuclear chromatin is similar to the more
open appearance of the chromatin in • Impaired Use of Folate
megaloblasts • Numerous drugs decrease absorption
REVERSE TRANSCRIPTASE INHIBITORS of folic acid or impair folate
metabolism.
• Interfere with DNA production and may also
lead to megaloblastic changes • Antineoplasic (methotrexate),
antibacterial, and antiseizure agents.
Systemic Manifestations of Folate and Vitamin B12
Deficiency • Excessive Loss of Folate
• General symptoms related to anemia (fatigue, • Patients with kidney disease.

weakness, and shortness of breath) are
evident.
• Symptoms related to the alimentary tract may
also be seen.
• Loss of epithelium on the tongue results in a
smooth surface and soreness (glossitis).
• The blood picture of Vit B12 and Folate
deficiency are indistinguishable:
• It takes a few years to develop a
vitamin B12 deficiency.
• Only a few months to develop a folate
deficiency.
Vitamin B12 Deficiency
• Neurologic and neuropsychiatric symptoms
may be seen in vitamin B12 deficiency. • Inadequate Intake
Causes of Vitamin Deficiencies • Strict vegetarians (vegans) who do not

eat meat, eggs, or dairy products.
Folate Deficiency
• Increased Need
• Inadequate Intake
• During pregnancy or lactation.
• Poor diet.
• Impaired Absorption – the absorption of
• Good sources of folate include leafy Vit B12 can be impaired by:
green vegetables, dried beans, liver,
beef, fortified breakfast cereals, and • Failure to separate vitamin B12 from
some fruits, especially oranges food proteins in the stomach
Hematology Lecture
• Failure to separate vitamin B12 from absorption of vitamin B12 because of an

haptocorrin in the intestine intrinsic factor deficiency.
• Lack of intrinsic factor • Autoimmune lymphocyte-mediated
destruction of gastric parietal cells severely
• Malabsorption
reduces the amount of intrinsic factor secreted
• Competition for available vitamin in the stomach.
B12
• Autoantibodies may be detected in the serum:
Lack of Intrinsic Factor
• Anti-IF Antibody
• Lack of intrinsic factor constitutes a significant
• Anti-Parietal Cell Antibody
cause of impaired vitamin B12 absorption.
Inherited Errors of Vitamin B12 Absorption and
• It is most commonly a result of autoimmune
Transport
disease, as in pernicious anemia.
• Imerslund-Gräsbeck syndrome
• Can also result from hereditary intrinsic factor
deficiency. • A rare autosomal recessive condition
caused by mutations in the genes for
• Loss of parietal cells in Helicobacter pylori
either cubilin or amnionless.
infection or after total or partial gastrectomy
• This defect results in decreased
endocytosis of the intrinsic factor-
vitamin B12 complex by ileal
enterocytes.
Parasitic Infection
• Diphyllobothrium latum
• Causes Vitamin B12 deficiency anemia.
• Able to split vitamin B12 from intrinsic
factor, rendering the vitamin
unavailable for host absorption.
LABORATORY DIAGNOSIS
Screening Tests
CBC and Reticulocyte Count
• Decreased RBC count, hemoglobin, and
hematocrit.
• Reticulocytopenia and pancytopenia are
evident.
• MCV = 100-150 fL
• MCH = Increased, MCHC = within the
reference range
Pernicious Anemia
• Peripheral Blood Smear:
• Pernicious anemia is an autoimmune
• Oval Macrocytes
disorder characterized by impaired
Hematology Lecture
• Hypersegmented Neutrophils • or at least 1 six-lobed neutrophil is

noted
• Howell-Jolly Bodies
Lactate Dehydrogenase and Bilirubin
• Poikilocytosis
Levels
• Elevated levels of LDH and Bilirubin
Specific Diagnostic Tests

Bone Marrow Examination
• Megaloblastic maturation is evident.
• Presence of nuclear-cytoplasmic asynchrony.
• Hypercellular bone marrow.
• Reduced M:E ratio – 1:1
WBC Manual Differential

• Hypersegmented neutrophils –
pathognomonic of megaloblastic anemias.
• Rules in reporting:
• when there are at least 5 five-lobed
neutrophils per 100 WBCs
Hematology Lecture
NONMEGALOBLASTIC ANEMIAS
• The macrocytic nonmegaloblastic anemias are
macrocytic anemias in which DNA synthesis is
unimpaired.
• Macrocytes are round.
• MCV – 100-110 fL; rarely reaches 120 fL
• Physiologic Macrocytosis – in newborns.
• Pathologic Macrocytosis - liver disease,
Assays for Folate, Vitamin B12, chronic alcoholism, or bone marrow failure.
Methylmalonic Acid, and Homocysteine
• Serum levels of folate and vitamin B12 can be
tested using immunoassays.
• MMA and Homcysteine – increased levels in
serum.
Antibody Assays
• Antibodies to intrinsic factor and gastric
parietal cells.
Hematology Lecture

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MTY 1211 - HEMATOLOGY

Chapter 1 - An Overview of Clinical Laboratory Hematology Lecture - Week 1

HISTORY Figure 1.1

. ○ RBC distribution width (RDW) - expresses

Go, Manalo, Nepomuceno, and Paguirigan

● Wright-stained aspirate smears ● Philadelphia chromosome

Go, Manalo, Nepomuceno, and Paguirigan

● Real-time polymerase chain reaction, microarray

● Glucose-6-phosphate dehydrogenase assay -

HEMATOLOGY QUALITY ASSURANCE

● Medical laboratory scientists (MLS) employ

Go, Manalo, Nepomuceno, and Paguirigan

● 1961 - Till and McCulloch

Go, Manalo, Nepomuceno, and Paguirigan

Go, Manalo, Nepomuceno, and Paguirigan

● factors of CFU-GEMM that differentiates into

● Burst-forming unit-erythroid (BFU-E)

● Eosinophils differentiation require:

● Basophil differentiation require:

● Lymphoid differentiation is promoted by growth

- earlier influences are GM-CSF, IL-3, IL-6, IL-11, KIT

● TPO and IL-11

● Chemokines (chemotactic cytokines)

Well-stained peripheral blood film or bone marrow smear is used

GENERAL TRENDS THAT AFFECT THE APPEARANCE OF A

a) Overall diameter of the cell decreases and cytoplasm

• Length of time in this stage • Length of time in this stage

o lasts slightly more than 24 hours o lasts approximately 30 hours.

POLYCHROMATIC (POLYCHROMATOPHILIC) NORMOBLAST ORTHOCHROMIC NORMOBLAST (METARUBRICYTE)

Figure 5.7 Orthochromic Normoblasts (Metarubicytes).

POLYCHROMATIC (POLYCHROMATOPHILIC) ERYTHROCYTE

membrane and would not be readily oxygenated o Nondialyzable

STRUCTURE o Releasing cells from the bone marrow early is a

because the available precursors in the marrow bodies.

Hemoglobin – one of the most studied proteins in the body because

REGULATION OF HEMOGLOBIN PRODUCTION

Globin production – mainly controlled at the transcription level by

o Cooperation among hemoglobin subunits

• The cyanmethemoglobin method is the reference

• Ehrlich (1888) - provided the first case report of aplastic

PATHOPHYSIOLOGY OF BONE MARROW FAILURE Acquired Aplastic Anemia

FANCONI ANEMIA LABORATORY FINDINGS

• Myelophthisic Anemia - due to the infiltration of abnormal

OUTLINE disease, neurologic symptoms, previous medications,

BISCOCHO. NICER. RAMOS. | FEU MT 2023 3

BISCOCHO. NICER. RAMOS. | FEU MT 2023 4

 PARAXOSYMAL COLD HEMOGLOBINURIA

Figure 20.1 Normal Catabolism of Hemoglobin. Macrophages lyse

 Unconjugated bilirubin – hydrophobic, and must bind

Figure 20.3 Normal Macrophage-Mediated Hemolysis.

6. Bacteria in the large intestine convert conjugated bilirubin to urobilinogen, most

 Under normal conditions, hepatocytes at the exterior

 These bilirubin derivatives enter the lobule via the

 Fragmentation hemolysis is the result of trauma to 5. The haptoglobin is degraded.

 These effects are magnified in conditions such as

 Hemopexin- may play an important role in macrophage-

 The complexes are cleared within minutes from plasma

LABORATORY TESTS TO IDENTIFY SPECIFIC

Figure No. Morphologic abnormalities associated with hemolytic anemia

OVERVIEW OF IMMUNE HEMOLYTIC ANEMIAS