Methods for evaluation of

the immune system

Lecture 6

Burygina Ekaterina Vasil’evna

candidate of medical sciences, assistant professor,
Department of Clinical Mycology, Allergy and Immunology

Бурыгина Екатерина Васильевна

к.м.н., ассистент кафедры клинической микологии,
аллергологии и иммунологии
 Lecture plan

1. General principles of evaluation of patient's immune

system
2. White blood cell (WBC) count
3. Clusters of Differentiation and Immunophenotyping
4. Flow Cytometry
5. Assessment of phagocytosis
6. Antibody Assays
Immunological methods are used
for
infections diseases
immunology and allergy
hematology
oncology
transfusiology
transplantology
rheumatology
forensic medicine
and many more…
Assessing the immune system
Tests for evaluating immunological
functions
 General principles of evaluation of patient's
immune system
• Anamnesis (medical history)
• Clinical examination
• Diagnostic technics (e.g. ultrasound, X-ray, computed tomography (CT),
magnetic resonance imaging (MRI))
• Initial laboratory tests (1st level ) –to identify serious defects of the immune
system
• white blood cell count (leukocytes, monocytes, lymphocytes, neutrophiles,
eosinophiles, basophiles)
• total protein and γ-globulin fractions
• levels of serum IgG, IgM, IgA, IgE
• Advanced tests (2nd level ) - in-depth study of the functional state of the
immune system
• T cells subpopulations;
• proliferative activity of T and B cells on mitogens, antigens, allogeneic cells;
• activity of NK;
• components of complement system
• phagocytosis;
• production and reception of interleukins;
• genes responsible for the expression of immunologically significant molecules
 White blood cell (WBC) count

On hemocytometer On hematology analyzer

Neubaur Chamber

Cell counting can be performed using hemocytometer (cells are manually

counted under a microscope in specially designed chambers) or automated
counters (hematology analyzers) that can distinguish each leukocyte population
by laser light scatter.
 White blood cell (WBC) count
Normal Total leucocytes count
4,5 - 11,0 х 10⁹/L white blood cells

• Adult /child = 5,0 to 10,0 х 10⁹/L

• Child ≤2 years = 6,2 to 17,0 х 10⁹/L
• Newborn = 9,0 to 30,0 х 10⁹/L

WBC count in adult

 Total leukocytes level
Leukopenia Leukocytosis
<3,5х10⁹/L >11,0 х10⁹/L
• Viral or bacterial infection • Bacterial, fungal, or parasitic
• Tuberculosis infection

• Diminished bone marrow function • Inflammatory conditions

• Intoxications
• Autoimmune disorders (Lupus, RA)
• Cancer • Burns

• Leukemia • Corticosteroid use

• Aplastic anemia • Stress, exercise

• Cigarette smoking
• Medication side effects
• Chemotherapy or radiation therapy • Pregnancy
• Leukemia
• Myeloproliferative diseases
• Epileptic seizures
 Neutrophils

• The normal range in adults:

1.5-8.0 х10⁹/L.
The normal range of mature/
segmented neutrophils:
2.5-6.0 cells/mm3.
The normal range of immature
neutrophils: 0-0.5 cells/mm3.
• circulate for 7 to 10 hours in
Mature
the peripheral blood before Metamyelocytes Band forms
(segmented)
migrating into the tissues, 0% 0-5%
47-67%
• life span in the tissues - a few Shift to the left Shift to the right
days (~4 days) ↑immature cells ↑mature cells
 Neutrophils
Neutrophilia Neutropenia
>8.0 х10⁹/L <1.5 х10⁹/L
Causes: • Mild - 1000-1500 cells/mL
 bacterial infection • Moderate - 500-100 cells/mL

 inflammation (after heart attacks • Severe (agranulocytosis) - 500 cells/mL

or burns) Causes:
 increased concentration of cortisol  intake of medicines (chemotherapy)
and adrenaline hormones  infections (AIDS, tuberculosis and
 ingestion of some drugs like hepatitis)
systemic steroids (prednisone)  cancer and related bone marrow
diseases
 malignancy (like leukemia)
 deficiency of vitamin B12 and other
 surgical procedures, including minerals
splenectomy and appendicitis  autoimmune diseases (like Crohn’s
disease, lupus and rheumatoid
arthritis)
 Eosinophiles
150–450 cells/mL (1-5 % )
Eosinophilia Eosinopenia
>450-550 cells/mL < 150 cells/mL
Hypereosinophilia >1500 cells/mL • Acute stress reactions with increased
glucocorticoid and epinephrine
Mild - 500–1500 cells/mL
secretion
• allergic diseases, atopy, asthma, drug, allergy,
• bacterial and viral infections • Acute inflammation

Moderate - 1500–5000 cells/ mL • Cushing's syndrome with

corticosteroid therapy
• Parasitic infection, HES Churg-Strauss
Syndrome, cancers, Sezary’s Syndrome, ABPA,
drug rash with eosinophilia and systemic
symptoms (DRESS)
Severe >5000 cells/ mL
• hypereosinophilic syndrome
• eosinophilic leukemia • half-life of 8-18 hours
• myeloproliferative disorders in the bloodstream
• cancer • in tissues can persist
for at least several
weeks
 Basophiles
15–100 cells/mL (0.5–1% )
Basophilia Basopenia
>100 cells/mL <15 cells/mL
• Certain skin diseases
• Chicken pox
• Acute infection
• Chronic myelogenous leukemia
• Allergy
• Chronic sinusitis
• Irradiation
• Adrenocortical stimulation
• Measles • Graves’ disease
• Myeloproliferative disorders • Irradiation
• Myxedema • Pregnancy
• Postsplenectomy • Shock
• Smallpox • Stress
• Ulcerative colitis lifespan - 10 hours
in the bloodstream
 Monocytes
200–800 cells/ mL (3-11%)
Monocytosis Monocytopenia
> 800 cells/ mL <200 cells/ mL
Causes: Causes:
• Infections: such as brucellosis, • Early corticosteroid therapy
tuberculosis and rickettsia • Hairy cell leukemia
• Myeloproliferative disorders
• Hodgkin's disease
• Gastrointestinal disorders,
including inflammatory bowel
diseases and sprue
• 4-8 hours in blood
• lifespan 10-20 days in
tissues
 Lymphocytes
1.0–8.0 х10⁹/L (20–40% )
Lymphocytosis Lymphopenia
>8000 cells/ mL <1000 cells/mL in adults
Causes: <2500 cells/mL in infants
• Acute infections, including Causes:
pertussis, typhoid, and • Immunodeficiency syndromes,
paratyphoid including congenital (DiGeorge
• Infectious mononucleosis, with syndrome, etc) and acquired (AIDS)
"atypical" lymphocytosis conditions
• Viral infections, including • Corticosteroid therapy
measles, mumps, adenovirus, • Neoplasia, including Hodgkin's
enterovirus, and Coxsackie virus disease, non-Hodgkin's lymphomas,
• Toxoplasmosis and advanced carcinomas
• HTLV I • Radiation therapy
• Chemotherapy
 Lymphocyte subtypes
Th (CD4+)
40±5%
T cells (CD3+)
65±5%
Tc (CD8+)
30±5%
B cells (CD19+)
Total 15%
lymphocytes
100% NK cells
(CD3-CD56+)
15%
NKT cells
(CD3+CD56+)
5%
 CD - antigens
• C – cluster
• D – differentiation
• Antigens – can be detected with
monoclonal antibodies
 CD - diagnostics
• Less the 15% of CD-antigens have diagnostic meaning for
immunology (for immunophenotyping),
BUT are valuable for
• oncology, hematology and dermatology,
• infection diseases.
CD-antigens as markers for
identification (determination of CD4+ and CD8+ T cells, B
cells, NK cells, regulatory T cells, or Treg cells, etc., or minor
populations of lymphocytes with limited diversity, e.g. NKT
cells and γδ T cells)
functional assessment (activation status of those
lymphocytes and other immune cells may be monitored
through expression of activation markers CD69, CD25, CD23,
etc.)
 Flow cytometry

Modern technology for quickly measuring various parameters

of a cell, its organelles and the processes occurring in it.

Flow cytometer (Beckman Coulter, USA)

FC-500

Cytoflex
 Applications of flow cytometry
IN THE CLINIC (mainly immunology and oncohematology)
• Analysis of the subpopulation composition of peripheral blood cells
• Diagnosis of autoimmune diseases, immunodeficiencies, allergic disorders, sepsis
and other pathologies
• Differential diagnosis of oncohematological diseases
• Counting and immunophenotypic characterization of stem cells
• HIV monitoring
• Quality control in blood services
IN SCIENCE (depending on the goals of the research)
• Typing of cell subpopulations using specific markers
• Quantitative determination of cytokines and other analytes in biological media
• Analysis of programmed cell death – apoptosis
• Evaluation of transfection efficiency
• Cell cycle stage analysis
• Cytotoxicity study
• Screening of drug compounds
Samples for flow cytometry

Blood
Bone marrow
Liquor Required
Joint fluid prerequisite:
Pleural fluid Single cell
BAL suspension
Ascites
Tissue cells
Tumor cells
 Isolation of lymphocytes

• Peripheral blood mononuclear cells can be isolated from whole blood by

centrifugation. Diluted anticoagulated blood (left panel) is layered over Ficoii-
Hypaque™ and centrifuged. Red blood cells and polymorphonuclear
leukocytes or granulocytes are denser and travel through the Ficoii-Hypaque™,
while mononuclear cells consisting of lymphocytes together with some
monocytes band over it and can be recovered at the interface (right panel).
 Red blood cell lysis reagents
• Whole blood lysing reagents
Lysis of erythrocytes due to formic acid, followed by its neutralization and fixation
of cells with paraformaldehyde.
• OptiLyse C
Lysis of erythrocytes due to saponin and subsequent fixation of cells with
paraformaldehyde.
• VersaLyse
Enzymatic lysis of red blood cells. The main active ingredient in VersaLyse reagent
is a cyclic amine; in the presence of carbonic anhydrase of erythrocytes, it is
converted into a compound that has high lytic activity against these cells
• IOTest® 3 Lysing Solution
A solution based on ammonium chloride (NH4Cl), which is the active ingredient in
the lysis solution.
• ImmunoPrep®Reagent for automatic lysis of red blood cells for no-clean technology.
Qualitative lysis of red blood cells. No morphological changes in other types of
cells. High level of standardization. Lysis of erythrocytes due to formic acid,
followed by its neutralization and fixation of cells with paraformaldehyde.
Flow Cytometry
• Principles of flow cytometry. (a)
Cells introduced into the sample
injection port are focused within a
stream of sheath fluid and pass one
by one in front of the laser beam.
Forward scattered light is detected
by a photodiode. Side-scattered
light and emitted fluorescence of
various wavelengths is detected by
photomultiplier tubes, after
passage through a series of dichroic
mirrors and light filters. All of the
information obtained from
individual cells is integrated by the
software and can be expressed in a
number of formats, such as that
shown in (b). (b) On the left is a
scatter plot of forward scatter
(abscissa) versus side scatter
(ordinate) of a sample of human
white blood cells. Lymphocytes are
gated and displayed in red. On the
right is a plot of lymphocytes
stained with anti-CD4 (ordinate) or
anti-CD8 (abscissa) antibodies.
 Device parameters

Side Scatter (SS) at 90°

~ Cell structure

Laser
beam
Small-angle light scattering
(SAS) at an angle < 19°
~ Cell sizes

Fluorescence intensity
5 colours ~ Antigen density
FITC/RD1/ECD/PC5/PC7
Fluorochromes for multicolor analysis

Of the 40+ known fluorochromes, no more than 18 are used simultaneously

Chemokine results
 Expression of chemokine
receptors on CD45RA-Th

Th1
CD4+CD45RA-
CCR6-CCR4-CXCR3+

Th2
CD4+CD45RA-
CCR6-CCR4+CXCR3-

Th17/Th22
CD4+CD45RA-
CCR6+CCR4+CXCR3-)
Isolation of target cell populations
Isolation of target cell populations
 41
возраст
17.11.2015 КДО
Хроническая герпетическая инфекция № истории болезни
Данилова Е.Ю.
диагноз врач

формула крови (%) абс(×10*9/л) норма% норма абс(×10*9/л)

лейкоциты 4,8 4-9
лимфоциты 28 1,34 20-39 1,27-3,26
моноциты 6 3-11
базофилы 1 0-1
эозинофилы 3 0-5
с/я нейтрофилы 60 47-72
п/я нейтрофилы 2 1-6
субпопуляционный состав лимфоцитов норма (%) норма абс(×10*9/л)
Т-лимфоциты (CD3+CD19-) 61 0,820 60-80 0,900-2,100
Т-хелперы (CD3+CD4+) 29↓ 0,390↓ 35-50 0,580-1,300
Т-цитотоксические (CD3+CD8+) 30 0,403 19-35 0,370-1,000
В-лимфоциты (СD19+CD3-) 15 0,202 6-18 0,110-0,400
Т-актив.(CD3+CD4+СD25+) 3 0,040 1,8-6,5 0,030-0,130
Естеств.киллеры (CD3-СD56+) 21↑ 0,282 8-17 0,120-0,370
ИРИ 1,0↓ 1,5-2,6
функциональная активность нейтрофилов % норма (%)
НСТ спонтанный 33↑ 11-18
НСТ активированный 58 40 - 60
фагоцитарный индекс 71 66 - 74
коэффициент киллинга 25 25-45
иммуноглобулины норма (г/л)
Ig A 1,98 0,7-4,0
Ig M 2,55 0,4-2,6
Ig G 21,61↑ 7,0-15,0
Ig Е 25-100 Ед/мл
ЦИК до 80 Ед/мл
интерферон норма (пг/мл)
ИФН-α спонтанный 27 0-30
ИФН-α индуцированный 87↓ 100-500
ИФН-γ спонтанный 34 0-50
ИФН-γ индуцированный 289↓ 1000-2000
 Rules for evaluating immunograms
 Complex analysis of the immunogram is more informative than the
evaluation of each indicator separately.
 A complete immunogram analysis can be carried out only in
conjunction with an assessment of the clinical picture in this patient.
 Real information in the immunogram carries strong shifts in indicators;
weak shifts only allow to increase confidence in the correctness of the
conclusion made.
 The analysis of the immunogram in dynamics is always more
informative both in diagnostic and in prognostic relation than once
received immunogram.
 In the overwhelming majority of cases, the analysis of the
immunogram makes it possible to make approximate, and not
unconditional, conclusions of a diagnostic and prognostic nature.
 The paramount practical significance in the immunogram is the ratio of
different populations and subpopulations of immunocompetent cells,
rather than their absolute values.
 Evaluating immunograms

Assessing the human immune system is somewhat

difficult because:
individual variability of immunogram parameters;
age differences - with age, the content of leukocytes
and lymphocytes decreases, the number of neutrophils
and the levels of IgG, IgA increases;
seasonal fluctuations - maximum values of T- and B-
lymphocytes in winter, a decrease in the number and
functional activity of T-lymphocytes - in spring, B-
lymphocytes - in summer;
daily fluctuations - the maximum number of
lymphocytes in midnight, the smallest upon awakening.
 Evaluating immunograms
Immunological examination:
1) confirms the presence of immunodeficiency;
2) identifies the broken link;
3) determines the severity of disorders in the immune system;
4) gives possibility of selecting an immunomodulator;
5) assessment of the prognosis of the effectiveness of immunotherapy.
It must be taken into account that
 full information can be obtained in combination with the clinical picture;
 comprehensive analysis is more informative than individual indicators;
 it is necessary to carry out an immunogram over time, this is more
informative for identifying stable changes for diagnosis and prognostic
criteria;
The absence of changes in the immunogram in the presence of a clinical
picture of inflammation is an atypical reaction and is a bad sign.
Evaluation of phagocytosis
1. Assessing of adhesion
- determination of adhesion molecules
using MAT by flow cytometry and indirect
immunofluorescence reaction. To
determine the functional activity of
adhesion molecules, the ability of
phagocytes to attach to glass, plastic, and
epithelial cell culture is also used.
2. Assessing of chemotaxis
The most common method is to assess
spontaneous migration in 5-channel glass
flat capillaries. They are filled 2/3 with
heparinized blood, sealed, centrifuged and
incubated in racks at an angle of 10 for 6
hours. At the end of incubation, the length
of travel of leukocytes to the ends of the
capillaries is measured in a microscope
with an eyepiece micrometer. The
migration length for each individual is
calculated in 5 channels of each capillary
and expressed as an average of 5
Evaluation of phagocytosis (NBT-test)
Nitroblue tetrazolium (NBT) tests
The essence of the method is that
NBT is initially yellow in color, but
under the influence of superoxide
radicals it is converted into dark blue
formazan, which is deposited in the
form of granules inside the cell.
Blood neutrophils are normally in an
inactivated state, so the number of
neutrophils containing formazan
granules does not exceed 10-15%.
This indicator is called a
spontaneous NBT test.
To assess the functional reserve
capabilities of neutrophils,
incubation of neutrophils with
various stimuli is used activated
NBT- test
 Immunodiagnostics
Immunodiagnostics is a diagnostic methodology that uses an
antigen-antibody reaction as their primary means of
detection.
The concept of using immunology as a diagnostic tool was
introduced in 1960 as a test for serum insulin.
Immunodiagnostic tests use monoclonal antibodies as
reagents whose results are used to aid diagnosis and are
widely used in many scientific disciplines.
The different types of test range from simple manual
methods to fully automated systems with sophisticated
integrated detection.
 Hybridoma and monoclonal
antibodies

Georges César
Köhler Milstein
The Nobel Prize in 1984

The conventional polyclonal antiserum produced in response to a complex antigen

contains a mixture of monoclonal antibodies, each specific for one of the four epitopes
shown on the antigen (inset). In contrast, a monoclonal antibody, which is derived from a
single plasma cell, is specific for one epitope on a complex antigen. The outline of the basic
method for obtaining a monoclonal antibody is illustrated here.
Monoclonal antibodies
 Immunoprecipitation- Based
Techniques
• Precipitation reactions are one of the important reactions that occur
when antigen and antibody come to contact. When a soluble antigen
reacts with its antibody in the presence of NaCl at optimal temperature
and pH, the antigen-antibody complex forms an insoluble precipitate.
Generally, liquid media and gels such as agar, agarose, and
polyacrylamide are used for this kind of reaction.

Immunoprecipitation in solution Immunoprecipitation in in agar gels

 Radioimmunoassay
• Radioimmunoassay (RIA) determination of antigens or antibodies in a sample
with the use of radioisotopes.
• RIA measures the presence of an antigen with very high sensitivity.
• RIA was first described in 1960 for the measurement of endogenous plasma
insulin by Solomon Berson and Rosalyn Yalow.
Immunoturbodimetry
Endpoint determination of IgG
concentration by photometric
measurement of the antigen–
antibody reaction.
A turbid suspension is formed
proportional to the
concentration of the analyte
being studied the amount of
light passing through a
solution decreases depending
on its turbidity.
Measurement is carried out at
a wavelength of 340 nm or 600
nm
 Nephelometry

Light from the LED passes through the

test sample and is scattered by Ag-Ab
complexes in solution. Scattered light is
detected by a photodiode

Benchtop nephelometer for rapid

quantitative determination of the
concentration of specific proteins in
biological fluids.
Enzyme Linked Immuno-Sorbent Assay, or ELISA
Photometer HumaReader HS
 Immunofluorescence
• It was described in 1942 and refined by Coons in 1950, which used
a fluorescence microscope able to read the specific
immunological reaction and cellular slide preparations.
• It is an effective method for visualizing intracellular processes,
structures, and conditions as well.
• Detects surface antigens or antibodies.
• Fluorescent dyes are used for the visualization of Ag-Ab reactions.

Dendritic cell infected with

Listeria monocytogenes
Western blotting
Western blotting uses antibodies to identify protein
bands following gel electrophoresis.
In Western blotting, a protein mixture is (a) treated
with SDS, a strong denaturing detergent, (b) then
separated by electrophoresis in an SDS
polyacrylamide gel (SDS-PAGE), which separates the
components according to their molecular weight;
lower-molecular-weight components migrate farther
than higher-molecular-weight components.
(c) The gel is removed from the apparatus and
applied to a protein binding sheet of nitrocellulose or
polyvinylidene fluoride (PVDF), and the proteins in
the gel are transferred to the sheet by the passage of
an electric current.
(d) Addition of enzyme-linked antibodies detects the
antigen of interest, and (e) the position of the
antibodies is visualized by means of an ELISA reaction
that generates a highly colored insoluble product that
is deposited at the site of the reaction. Alternatively,
a chemiluminescent ELISA can be used to generate
light that is readily detected by exposure of the blot
to a piece of photographic film.
Complement Fixation Test
 Interferon-gamma-based in vitro
assays (IGRA)

In the ELISA method (QuantiFERON®-TB Gold In-Tube Test; Quest Diagnostics, USA), whole blood is
stimulated with M. tuberculosis antigens, and the amount of IFN-γ secreted into the supernatant is
quantified by ELISA. (B) In the ELISPOT method (T-SPOT.TB; Oxford Immunotec, UK), PBMCs are
prepared by density gradient centrifugation (Ficoll method). A defined number of cells is then
stimulated with M. tuberculosis antigens for 24 h on plates coated with anti-IFN-γ antibodies.
Antigen-responsive cells secrete IFN-γ, which binds to these antibodies. After removal of the cells,
antigens are detected by a second labeled anti-IFN-γ antibody. The number of spots on the plate
corresponds to the number of IFN-γ+ cells in the sample.
 Thank you!

See you in February!