Pre-Analytical, Analytical, and Post-

Analytical of Immunology Test

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 Short Introduction

• Immunology is a study about the structure and function of the immune

Immunology system, the immune response, immune-based disorders as well as
immunological methods of analysis.

• Immunity is the way in which the organism protects itself from invasion
Immunity by foreign agents and provides defense against their harmful effect.
Immunity  innate and adaptive.
 Structure of the Immune System

Primary • thymus
Lymphoid
• bone marrow
Organ

• spleen,
• lymph nodes,
Secondary
• tonsils,
lymphoid • Peyer's patches,
organs • appendix and
• mucosa-associated lymphoid tissue (MALT)
 Material collection and processing for
immunological examination
Collection and blood processing for humoral immunity screening

Collection and blood processing for Cellular immunity screening

To perform cellular immunity screening, cells are isolated from non-coagulated blood using separation
solution (polymorphonuclear leukocytes by dextrane, mononuclear leukocytes by Ficoll-Hypaque)
 Classical serological methods
• Serological reaction is an in vitro reaction of an antigen and an antibody. The
reaction is strictly specific; an antigen combines only with its homologous
antibody and vice versa.
• Agglutination  direct (Qualitative agglutination test and semi-quantitative
agglutination test), coombs test (direct and indirect), and passive agglutination
Classical serological methods
 Classical serological methods
Precipitation
• Precipitation reactions involve interactions of soluble antigens and antibodies that are
cross-linked so that they form a large macromolecular complex

Precipitate formation is affected by the amount of

antigen and antibody
 Classical serological methods
Immunodiffusion
• Immunodiffusion techniques detect antigen-antibody precipitation reactions in a semi-
solid medium, e.g. agar gel, agarose gel or gelatin.
• The test can be qualitative or semi-quantitative.
• The immunodiffusion techniques that are most often used in clinical laboratories include
single radial immunodiffusion and double immunodiffusion.

Possible precipitation patterns occurring in immunodiffusion by

Single/Radial immunodiffusion test Ouchterlony (double)
 Progressive serological methods
• Turbidimetry  Turbidimetry is based on the measurement of the reduction in
light intensity of the incident light beam after it has passed through the
solution.
• Nephelometry  Nephelometry measures the light that is scattered at a
particular angle from the light beam as it passes through the suspension (in
contrast to turbidimetry, which measures light rays passing directly through the
solution).
 Progressive serological methods
Radioimmunosorbent test (RIST)
Radioimmunoassay

Radioallergosorbent test (RAST)

• Direct ELISA
Immunoassays
Enzyme immunoassay • Indirect ELISA
• Enzyme linked immunosorbent assay (ELISA)
• Sandwich ELISA
• ELISPOT

• Direct FIA
Fluorescent immunosorbent assay (FIA)
• Indirect FIA
 Progressive serological methods
Western Blot
• Western blot, also known as immunoblot, is a widely used technique that detects specific
protein antigens in serum or other specimen. It combines electrophoresis with transfer of
the separated proteins onto a membrane (this process is called blotting) and serological
detection
 Evaluation of innate humoral immunity
• Because of the extreme unstableness of complement components, proper collection,
preparation, handling and storage of biological samples are crucial to obtain correct and
relevant results in complement assay.
• Laboratory tests for the evaluation of functional integrity of the classical, alternative and
lectin pathway include several haemolytic and ELISA-based assays that enable to screen
for the
presence of quantitative and qualitative abnormalities in complement activation cascade.
• The total haemolytic complement (CH50) assay is a functional laboratory assay for the
evaluation of classical complement pathway
Principle of the total
haemolytic CH50
assay
 Laboratory tests for complement evaluation

Evaluation of the
Evaluation of white blood
erythrocyte sedimentation
cell counts and differential
rate

Measurement of acute
phase proteins and other
markers of inflammation
 Evaluation of innate cellular immunity
• Specimen collection and isolation of
polymorphonuclears
Laboratory tests • Evaluation of chemotaxis
for the evaluation • Evaluation of blood cell numbers
of phagocytosis • Evaluation of ingestion
• Evaluation of microbicidal activity
• Evaluation of metabolic activation
• Evaluation of lysozyme levels and activity
Laboratory tests for the evaluation of phagocytosis

Evaluation of Blood Cell Numbers

• The complete blood cell counting (CBC) with differential is a basic laboratory
test that provides useful information on circulating blood cell numbers including
the neutrophils, eosinophils and monocytes
• The blood smear enables to detect their morphological abnormalities such as
the presence of large granules in neutrophils (Chediak-Higashi syndrome) or
their absence (specific granule deficiency).
• Neutrophil numbers below 1500/μl are referred to as neutropaenia, numbers <
500/μl are the criterium for severe neutropaenia and are associated with a high
risk of severe bacterial infections
Laboratory tests for the evaluation of phagocytosis
Specimen collection and isolation of polymorphonuclears

Isolation of
polymorphonuclears with
dextran
Laboratory tests for the evaluation of phagocytosis
Evaluation of chemotaxis
• Impaired chemotactic activity is typical for certain rare primary immunodeficiencies
including the leukocyte adhesion deficiency (LAD) syndromes, Chédiak-Higashi
syndrome, hyper-IgE syndrome, lazy leukocyte syndrome,complement deficiencies and
others, but is more commonly caused by drugs (colchicine) or present in certain diseases
(diabetes mellitus, severe malnutrition, infections, malignancies)

Evaluation of chemotactic activity of

polymorphonuclears
Laboratory tests for the evaluation of phagocytosis
Evaluation of ingestion
• Evaluation of the ability of PMNs to take up microorganisms or spheric particles is an im-
portant step in the diagnostics of neutrophil dysfunctions.
Frequently used functional method
is the evaluation of phagocytic
activity (PhA) and phagocytic index
(PhI) of PMNs with C. albicans.

Evaluation of phagocytic activity and phagocytic index

of polymorphonuclears
Laboratory tests for the evaluation of phagocytosis
Evaluation of microbicidal activity
The candidacidal activity (% of cidia) is calculated according to the following formula

Fig. Evaluation of microbicidal

activity of polymorphonuclears

The microbicidal activity can also be evaluated

from full heparinised blood, or using other viable
microorganisms as substrates (Staphylococcus
aureus, Escherichia coli).
Laboratory tests for the evaluation of phagocytosis
Evaluation of metabolic activation
Laboratory tests for the evaluation of phagocytosis

Evaluation of lysozyme levels and activity

• The enzymatic methods  based on the ability of lysozyme to lyse cellular walls of
bacteria
• The serologic (immunometric) methods  based upon the use of specific antibodies
against the lysozyme for its detection and measurement

Laboratory tests for the evaluation of NK cell cytotoxic activity

• The numbers of NK cells can be determined by flow cytometry, while their activity and
functional integrity can be examined by several functional tests including the chromium
release assay, non-radioactive assays and flow cytometry-based assays.
 Evaluation of adaptive immunity

• To evaluate serum immunoglobulins levels (specific humoral imunity), methods like

nephelometry, turbidimetry and ELISA are performed.
• For quantitative analysis of lymphocytes (specific cellular imunity), their separation by
gradient centrifugation (using Ficoll-Hypaque separation solution) followed by flow
cytometry is performed.
• Flow cytometer can sort cells into different population according to their size and
membrane antigens (i.e. CD molecules) using fluorescently - labelled monoclonal
antibodies.
• The functional evaluation of lymphocytes is performed by the lymphocyte (blastic)
transformation test (in vitro) and skin tests (in vivo)
 Evaluation of adaptive immunity
Isolation and counting of lymphocytes
Blood diluted with equal volume of saline is layered over
half of volume of separation solution Ficoll-Hypaque.
After centrifugation at 900g for 20 min., PBMC cells are
present as white layer over Ficoll-Hypaque solution,
whereas red cells and granulocytes are sedimented at
the bottom of the tube
Counting of lymphocytes in microscope
Bürker chamber consists of 2 grids covered by glass.
Each grid contains 9 large squares (each measuring 1 x 1
mm, with the depth of 0.1 mm). Corners are bordered by
tripled lines. Each large square is divided into another 16
small squares, each 0.25 mm long and 0.25 mm wide.
Cells are counted in the large squares omitting cells lying
on the 2nd and 3rd lines
Evaluation of adaptive immunity
Flowcytometer.
The principle of cytometry relies on hydrodynamic
cell focusing. After incubation of cell suspension
with fluorescently labelled monoclonal antibodies,
the cell suspension is inserted into a flow-cell unit
that creates a thin stream of liquid. A vibrating
mechanism makes the liquid stream split into
drops thus each drop contains a single cell. The
drop (cell) then passes individually through the
laser beam. Analysis of emitting and scattered ligh
and fluorescence by several detectors is
performed
 Diagnostics of allergies

• The main goal of allergy diagnostics is the identification of causative allergens.

• The most commonly used type is the prick test

• The proper diagnosis of allergies should start with detailed anamnesis focusing on the
assessment of the history and nature of symptoms and the analysis of triggering factors.

• The diagnosis od food and drug allergies often requires the use of other skin test types
such as prick to prick test and intradermal test.
 Diagnostics of allergies
• The laboratory assessment of allergic disorders
includes the evaluation of non-specific markers
• The diagnostics of allergic (eosinophil counts, total IgE levels etc.) and
contact dermatitis, several specific methods enabling the

mediated by type IV identification of causative allergens. These are

represented by immunoassays for the
hypersensitivity, is typically
measurement of specific IgE levels (older method
performed by epicutaneous
RAST has been replaced by ELISA, CAP-FEIA
patch test.
etc.), basophil activation tests and lym-
phocyte transformation assay
Diagnostics of autoimmune diseases
Laboratory tests for autoimmune diseases include :
• complete blood cell counting,
• enumeration of lymphocyte subpopulations,
• evaluation of inflammatory markers (acute phase proteins, erythrocyte
sedimentation rate),
• total immunoglobulin levels and cryoglobulins,
• detection of specific autoantibodies,
• evaluation of the complement system and circulating immune complexes,
• genetic tests.
Diagnostics of autoimmune diseases

Laboratory tests for detection of specific autoantibodies are the hallmark of

autoimmunity diagnostics. They comprise methods such as
• Direct and indirect immunofluorescence,
• ELISA,
• Western blot,
• passive agglutination or microsphere-based multiplex flow
cytometric immunoassays.
 Diagnostics of autoimmune diseases

Passive agglutination for detection of Classical indirect ELISA assay (a) and
rheumatoid factor capture-ELISA assay (b) for detection of
autoantibodies
Diagnostics of immunodeficiencies
• Basic laboratory screening tests include complete blood cell counting with
differential and evaluation of total immunoglobulin levels and specific
antibody response.

• The innate (non-specific) arm of imunity can be examined with the help of specific
tests for the evaluation of phagocytic and cidal activity and the respiratory burst of
polymorphonuclear phagocytes.

• The evaluation of complement system integrity includes complement activity assays,

assays for measurement of complement protein levels and functional assays for
individual complement factors.
 Diagnostics of immunodeficiencies

• The function of NK cells can be evaluated by chromium release assay.

• The numbers of lymphocyte populations can be determined by flow cytometry,
while their function can be examined in-vitro by lymphocyte proliferation test
(blastic transformation test) and in-vivo by delayed-type hypersensitivity skin
tests
• The disease-causing mutations can be detected with the help of PCR or
sequencing methods.
Diagnostics and monitoring of HIV infection

• The diagnosis of HIV infection is routinely performed by indirect methods (ELISA,

Western blot) for thedetection of specific anti-HIV antibodies

• In case of reactivity in the initial screening ELISA, the test should be repeated in duplicity
and consequently confirmed by Western blot.

• After the diagnosis of HIV is established, patient's immune status and the progression of
infection must be monitored. For this purpose, CD4+ helper T cell counts, viral RNA loads
and viral resistance to antiretrovirotics are repeatedly determined
 Diagnostics and monitoring of HIV infection

ELISA assay for detection of anti-HIV antibodies

Western blot for detection of anti-HIV antibodies

 Laboratory methods used in transplantation immunology

• Worldwide, kidneys are the most commonly transplanted organs. Donor and recipient
matching in kidney transplants reffers to three distinct areas: ABO blood type matching,
HLA type matching, and crossmatching.
• The donor and recipient should share at least six HLA antigens (two HLA-A antigens, two
HLA-B antigens, and two HLA-DR antigens) to decreases the risk of transplant rejection

Crossmatch test
 Summary
Examination of immune status includes innate and acquired immunity testing.

In diagnostics of innate (non-specific) immunity, an analysis of humoral components, (complement, acute

inflammation proteins) and cellular components (number and function of phagocytic cells) is performed.

For analysis of complement components and acute phase protein levels, the turbidimetry and
nephelometry are used.

To assess the function of the complement, total complement activity assay

of the classical (CH50) or alternative pathways is performed (AH50).

In diagnostic of acquired (specific) immune response, an analysis of humoral components like antibodies and
cellular components, like B and T cells is performed
 Summary
To evaluate serum immunoglobulins levels, methods like nephelometry, turbidimetry and ELISA are
performed.

For quantitative analysis of lymphocytes, their separation by gradient centrifugation (using Ficoll-
Hypaque separation solution) followed by flow cytometry is performed.

The functional evaluation of lymphocytes includes the lymphocyte (blastic) transformation test
(in vitro) and skin tests (in vivo)

In the diagnostic of autoimmune diseases (AD), an analysis of autoantibodies, rheumatoid factor,

circulating immunocomplexes and HLA alleles is performed.

To establish the diagnosis of allergy, in vitro laboratory tests that involved IgE level screening (RIST,
RAST, ImmunoCap), and in vivo tests based on skin tests (prick test, patch test) are performed.
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