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Laboratory Methods in Immunology

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13 Summary of methods used in immunity testing:
Immune status of an individual (Vladimíra Ďurmanová) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.1 Analysis of innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE
13.2 Analysis of acquired immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Immunological methods are due to their high specificity

and sensitivity widely explored in many fields such as micro-
biology, epidemiology, physiology and pathophysiology, bio-
chemistry, etc. Medicine uses immunological reactions for the
diagnosis, prevention, and treatment of a number of diseases.
The first edition of Laboratory methods in Immunology of-
fers thirteen easy to follow chapters presenting basic groups
of immunological methods with emphasis on their clinical util-
isation. The strength of the text and coverage of key topics re-
flects the needs of medical students studying at our University.
Despite the enormous explosion of new information in im-
munology our textbook follows the outlines of the course Prac-
ticals in Immunology that is complementing the Lectures in
Immunology. Though the textbook is originally designed for
undergraduate students, it is eminently suitable also for grad-
uate student, physicians, biomedical technicians and re-
searchers. Descriptions of methods are written in a concise way
without excessive detail and most of them are reinforced with
clear illustrations. Each chapter contains a short introduction
at the beginning and a Chapter summary at the end, re-empa-
sizing essential concepts for better understanding and learn-
ing. Of course, for those, who develop a deeper interest in the
field, numerous advanced textbooks are available.
The authors would like to acknowledge Prof. MUDr. Milan
Buc, DrSc. for his suggestions and critical comments. Our thanks
also go to Doc. Ing. Sabah Shawkat, PhD. and Jonáš Vavro,
who helped with the preparation of illustrations.

Ivana Shawkatová

9
 Short introduction to immunology

1 Short introduction to immunology

1.1 Immunology

The earliest written mention of immunity can be traced back to antient Greece in 430 B.C.
Thucydides noted that people who had recovered from plague could care about the sick without
contracting the illness a second time. However, it was not until the 19th century before observa-
tions of this phenomenon developed into a scientific theory. The origin of immunology is usually
attributed to an English physician Edward Jenner who discovered in 1796 that cowpox, or vac-
cinia, induced protection against human smallpox. Jenner called this procedure vaccination, and
this term is still used to describe the inoculation of healthy individuals with attenuated strains of
disease-causing agents to provide protection from disease. Particularly important was the work of
Paul Ehrlich, who proposed the side-chain theory to explain the specificity of the antigen-anti-
body reaction, and his contributions to the understanding of humoral immunity were awarded the
Nobel Prize in 1908. Erlich shared this Nobel Prize with the founder of cellular immunology Ilya
Metchnikoff, who discovered the major types and functions of phagocytes.
Nowadays, immunology is an important and advanced branch of biomedical science deal-
ing with the structure and function of the immune system, the immune response, immune-based
disorders as well as immunological methods of analysis. It is closely associated with many other
fields of medical science such as microbiology, neurology and endocrinology.

1.2 Immunity

The Latin word immunis, meaning ”exempt” gave rise the English term immunity, which ref-
fers to all mechanisms used by the organism as protection against foreign agents. The human or-
ganism is often described as being at war. This comes from the fact that the body is constantly
under attack from various invaders. Most of them belong to one of the four broad categories of dis-
ease-causing microorganisms (pathogens): viruses, bacteria, pathogenic fungi, and complex eu-
karyotic organisms collectively termed parasites. Foreign agents include also artificial substances
such as toxins, chemicals, drugs, etc. All of them can cause damage to the human organism and
thus the purpose of the immune system is to act as a defending army. Inadequate defense against
pathogens can occur due to genetic defects, acquired causes or escape strategies developed by
pathogens during their co-evolution with humans.
Simply, immunity is the way in which the organism protects itself from invasion by for-
eign agents and provides defense against their harmful effect. Immunity may be classified into
two major categories: innate and adaptive.

1.2.1 Innate immunity

Innate (also called nonspecific or natural) immunity represents the genetically based de-
fense system which we were born with. It involves barriers that form the so called first line of de-
fence keeping harmful substances from entering the body. Elements of the innate immunity

11
Laboratory Metods in Immunology
include body surfaces such as the skin and the mucous membranes. Effective barriers against in-
vasion of many microorganisms constitute different chemical factors: tears, saliva, sticky mucus
in the respiratoty and gastrointestinal tracks or hydrochloric acid and proteolytic enzymes in the
stomach. If an invader passes these physical and chemical barriers, it is attacked and destroyed by
other internal elements of the immune system, either humoral or cellular. Innate humoral imu-
nity is mainly conferred by the complement system and other soluble substances that are harmfull
to microorganisms (degradative enzymes, acute-phase proteins, interferons). Innate cellular imu-
nity is mediated by phagocytic cells: granulocytes, monocytes and macrophages. Numerous other
components are also features of innate imunity, e.g. pattern-recognition molecules, which can bind
to various microorganisms.
1.2.2 Adaptive immunity
Adaptive (specific, acquired) immunity developed late in evolution and is present only in
vertebrates. It is more specialized than innate imunity and it supplements the protection provided
by innate imunity. Adaptive imunity develops with exposure to various foreign agents (antigens)
and reacts specificly to that antigens only. The initial contact with the antigen (called immuniza-
tion) triggers a chain of events that lead to the activation of the two arms of adaptive immunity
with different sets of participiants. One of these two arms is mediated mainly by B cells and cir-
culating antibodies and it is reffered to as humoral. The other is mediated by several phenotypi-
cally distinct subpopulations of T cells, which either directly kill virus-infected, tumour or foreign
cells (cytotoxic T cells) or synthesize and release various substances called cytokines that affect
other cells and control the entire immune response (helper T cells and regulatory T cells). Hence
this arm of adaptive immunity is termed cellular or cell-mediated.
Some T and B cells become memory cells and provide “immunologic memory”. Memory al-
lows to respond faster and more efficiently when the immune system is exposed to the same anti-
gen repeatedly. In many cases, an adaptive immune response confers lifelong protective immunity
to reinfection with the same pathogen. The third cell type that participates in adaptive immunity
are antigen-presenting cells (APCs) such as dendritic cells, monocytes, macrophages. Their im-
portant function is to process and present the antigen to the T cells.
Innate and adaptive arms of the immune system have devoloped an interrelationship. They
interact through various cytokines and cell adhesion molecules, send each other signals, activate
each other and operate in concert to generate a specific and effective immune response that leads
to the elimination of the invader.
1.3 Structures of the immune system
The immune system is formed by a network of organs, cells and molecules. Though it is
a very complex and highly developed system, its mission is simple: to recognise and destroy for-
eign invaders.
The organs of the immune system are positioned throughout the organism. They are called
lymphoid organs as they are home to lymphocytes, the key cells of the immune system. Primary
lymphoid organs are those, where differentiation, proliferation and maturation of pleuripotent
12
Short introduction to immunology
stem cells into immunocompetent cells take place. Primary lymphoid organs are the thymus and
the bone marrow. Thymus is a bilobed organ located in the mediastinum that is responsible for the
differentiation and maturation of T cell progenitors into T cells. Bone marrow is the ultimate source
of all blood cells and it is responsible for the differentiation and maturation of B cells. Mature T
and B cells exit the primary lymphoid organs and are transported via the bloodstream to the sec-
ondary lymphoid organs.
Secondary lymphoid organs are those where mature lymphocytes are activated to respond
to invading pathogens. They are arranged as a series of “filters” monitoring the contents of the ex-
tracellular fluids, i.e. lymph, tissue fluid and blood. They include spleen, lymph nodes, tonsils,
Peyer's patches, appendix and mucosa-associated lymphoid tissue (MALT). The immunological
function of spleen is to serve as a blood filter as the white pulp regions of the spleen are made of
lymphatic tissue containing macrophages, T lymphocytes, and B lymphocytes that destroy
pathogens. The spleen is not a vital organ; its functions are useful but not essential for life.
Lymph nodes act as filters that capture the antigens brought by the lymph. The lymph nodes
contain two basic parts, the cortex and the medulla.
The outer cortex is populated predominantely with B cells arranged as follicles, which may
develop a germinal center when challenged with an antigen and the deeper cortex mainly con-
tains T cells. Medulla contains large blood vessels, sinuses and medullary cords that contain
plasma cells. Mucosa-associated lymphoid tissues are associated with mucosal surfaces of almost
any organ, but especially those of the digestive, genitourinary, and respiratory tracts, which are
constantly exposed to a wide variety of potentially harmful microorganisms and therefore require
their own system of antigen capture and presentation to lymphocytes.
Apart from lymphocytes (already mentioned above), pluripotent haematopoietic stem cells
give rise to other cell types. Neutrophils, eosinophils, and basophils are collectively known as
granulocytes which circulate in the blood unless recruited to act as effector cells at sites of infec-
tion and inflammation. Macrophages and mast cells complete their differentiation in the tissues
where they act as effector cells in the front line of host defense and initiate inflammation.
Macrophages phagocytose bacteria, and recruit other phagocytic cells as neutrophils, from the
blood. Mast cells are exocytic and they are responsible for the defense against parasites as well
as triggering allergic inflammation; they recruit eosinophils and basophils, which are also exo-
cytic. Dendritic cells enter the tissues as immature phagocytes where they specialize in ingesting
and presenting antigens to the T cells.
As a part of the immune system are also considered antibodies, cytokines, complement pro-
teins, degradative enzymes, acute-phase proteins, different receptors, adhesion molecules, etc.
1.4 Immunological methods
Immunology has always stimulated as well as made use of technology, such as microscopy,
electrophoresis, fluorescence, DNA-based methods, etc. Probably the biggest catalyst of progress
in immunology and in many other biomedical areas was the development of a revolutionary tech-
nique of monoclonal antibody production in 1975 by Köhler and Milstein. Monoclonal antibody
technology is a good example how immunology has influenced not only medicine but also sciences
ranging from molecular biology to agriculture and food industry.
13
Laboratory Metods in Immunology
The specificity of the bond between antibody and antigen is the basis of serological methods
that are an excellent tool for the detection of a variety of substances. In addition, antibody is a key
component of tests used to diagnose infectious diseases. Antibody is used to detect pathogens with
techniques such as latex agglutination, enzyme-linked immunosorbent assay and many others.
Since the 70-ies, immunological laboratory methods have gradually become more progres-
sive and simple to perform. Because of their improved specificity and sensitivity, immunological
techniques have now achieved a major role in modern clinical laboratory science. Many of them
are employed in the laboratory diagnosis of immune system disorders that occur due to an inap-
propriate, excessive, or lacking immune response. Such an altered immune response is the cause
of conditions that include allergies and other hypersensitivity reactions, autoimmune disorders,
immunodeficiencies, posttransfusion and posttransplant reactions. The following chapters of this
textbook contain an overview of the basic immunological methodology.
14
Material collection and processing for immunological examination
2 Material collection and processing
for immunological examination
Collection and material processing depends on the intended immune analyses. To evaluate
immune status of a patient, humoral and cellular immunity screening is usually performed. Each
analysis includes different procedures of material processing.
2.1 Collection and blood processing for humoral immunity screening
To perform humoral immunity screening, serum and plasma are obtained by blood pro-
cessing. Plasma is a pale-yellow fluid that makes up about 55 % of the total blood volume. It con-
tains water with dissolved proteins, clotting factors, hormones, mineral ions and other important
soluble components. Serum is blood plasma without clotting factors. Serum looks clearer than
plasma because of fewer proteins. Soluble immune system components such as antibodies, cy-
tokines, components of the complement system, acute phase proteins and etc. are examined in
serum and plasma.
2.1.1 Serum collection for humoral immunity screening
Serum is obtained from coagulated whole blood by centrifugation (Fig. 2.1). For serum col-
lection, the patient’s blood is taken to a clean sterile tube and left to stay at room temperature for
about 30 min. After the clot has been formed, it may be removed from the sides of the tube with
an applicator stick and then centrifuged at 150g for about 10 min. The resulting fluid top layer (su-
pernatant) is designated serum. If serum looks cloudy, repeated centrifugation is needed. He-
molyzed, icteric or lipemic serum can negatively influence certain tests. Serum should be examined
immediately or aliquoted and stored at -20°C for 2-3 weeks or at -80°C for several months.
Fig. 2.1 Serum and plasma collection
2.1.2 Plasma collection for humoral immunity
screening
Plasma is a pale-yellow fluid that is collected from non-
coagulated whole blood. Thus plasma contains fibrinogen
and other clotting components that are not present in serum.
To obtain plasma, patient’s blood is collected into sterile an-
ticoagulant-treated tubes. There are several types of antico-
agulants, which differ in their mechanism of action. The most
widely used anticoagulants are EDTA (ethylene-diaminete-
traacetic acid), heparin and citrate. EDTA binds a metal, such
as calcium and magnesium that are needed for clotting cas-
cade activation, heparin binds to antithrombin III and in-
15
Laboratory Metods in Immunology Material collection and processing for immunological examination

duces the inactivation of thrombin; citrate also binds calcium. Anticoagulant-treated tubes with dif-
ferently coloured tops (indicating the type of anticoagulant) are commercially available. If we in-
tend to examine plasma or blood cells, EDTA-treated tubes are needed (with lavender tops). Plasma
treated with heparin (green tops) is used for several applications; however, heparin is often con-
taminated with endotoxin, which stimulates white blood cells to release cytokines. There are two
ways for plasma collection. The most common way is based on blood centrifugation; the other
way is based on blood sedimentation. Tubes with non-coagulated blood are centrifuged for 10-15
min at 2,000g at 4°C. Blood cells sedimentate and plasma (the fluid fraction) is removed with
a sterile Pasteur pipette to a fresh sterile tube (Fig. 2.1). For blood plasma processing, it is recom-
mended to perform centrifugation within 1 hour after blood collection. The other, less frequent way
of plasma collection is based on blood sedimentation. During this process the tube with non-co-
agulated blood is left at room temperature for 20 min. in an incline and for other 20 min. in a ver-
tical position (Fig. 2.2). Plasma is stored in the same way as serum. It is important to avoid
freeze-thaw cycles because this is detrimental to many plasma components such cytokines or com-
plement components.
SUMMARY AT A GLANCE
•Collection and material processing depends on the type of immune analysis. To evaluate immune sta-
tus of a patient, humoral and cellular immunity screening is performed.
•To perform humoral immunity screening, serum and plasma are obtained by whole blood processing
(centrifugation).
•Serum is obtained from the coagulated blood after centrifugation; plasma is separated from the non-
coagulated blood and in contrast to serum contains clotting factors. The most widely used anticoag-
ulants are EDTA (ethylene-diaminetetraacetic acid), heparin and citrate. Soluble immune system
components such as antibodies, cytokines, components of the complement system, acute phase pro-
teins and etc. are examined in serum and plasma.

•To perform cellular immunity screening, cells are isolated from non-coagulated blood using separation
solution (polymorphonuclear leukocytes by dextrane, mononuclear leukocytes by Ficoll-Hypaque).

Fig. 2.2 Plasma collection by sedimentation

The tube with non-coagulated blood is left at room temperature for 20 min. in an incline and for other 20 min.
in a vertical position. Upper layer contains plasma.

2.2 Collection and blood processing for cellular immunity screening

For cellular immunity screening, the blood cells are collected. The cells determined for func-
tional analysis (phagocytosis, activity of lymphocytes and NK cells) are isolated from non-coagu-
lated blood. The patient’s whole blood is usually collected into a tube containing heparin (10 i.u. of
heparine/1 ml of blood or green top commercial tubes). Gently mixing to prevent possible blood
clotting is recommended.
Cells for immunogenetic or other DNA-based tests are isolated from non-coagulated whole
blood treated with EDTA (lavender top tubes).

16 17
Laboratory Metods in Immunology
3. 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. The con-
cept of an antigen-antibody reaction resembles a key (antigen), which fits into a lock (antibody).
The bonds that hold the antigen to the antibody combining site are non-covalent, including hy-
drogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Multiple bonds
between the antigen and the antibody ensure that the antibody will tightly bind to its antigen.
Specific antigen-antibody interactions are the bases of serological methods that are extensively
used for the identification or quantitation of antigens, and antibodies.
Classical or simple serological techniques involve direct observation of reactions; they do not
require the participation of accessory factors such as indicators or specialized equipment. Aggluti-
nation and precipitation are the two basic groups of classical serological reactions. Major differ-
ence between them is in the physical form of the antigen. Agglutination is the reaction of antibodies
with large particulate molecules while precipitation occurs due to the interaction of antibodies with
soluble antigens.
3.1. Agglutination
Reaction of an antibody with a particulate antigen results in agglutination (clumping) of the
antigen. The antibody molecules bind multiple particles and join them, creating a large complex
(Fig.3.1). The word agglutination comes from the Latin agglutinare which means “to glue”. Antibodies
of IgG and IgM classes can agglutinate particulate antigens however IgM class, due to its high valence,
is especially efficient. Agglutination reactions enable determination and quantitation of antibodies
using a known antigen. Vice versa, agglutination can be used to identify particulate antigens such as
bacteria, yeasts or blood cells by means of specific antibodies. Clumping reaction occurs quickly and
is easy to perform, which ma
kes agglutination an important
technique in routine diagnosis.
Agglutination occurs op-
timally when antigens and
antibodies react in equiva-
lent proportions. Excess of
an antibody results in forma-
tion of very small complexes
that are not able to clump
and form visible agglutina-
tion. This inhibition of an ag-
glutination reaction is called
the prozone effect.
Fig. 3.1 Agglutination
Antigen particles are cross-linked by antibody molecules.
18
Classical serological methods
3.1.1 Direct agglutination
3.1.1.1 Qualitative agglutination test
Agglutination tests can be performed in a qualitative manner so that they serve to deter-
mine the presence or absence of a particulate antigen using in vitro interaction with the appro-
priate antibody. Vice versa, antibodies can be determined by the means of a defined antigen. The
antibody is mixed with the particulate antigen and a positive test is indicated by the formation of
visible conglomerates. For example, agglutination is used for the determination of blood types or
antibodies to blood group antigens. For blood group typing the term haemagglutination is used.
Blood group typing is based on the presence or absence of inherited antigenic substances on the
surface of red blood cells (RBCs). According to the ABO blood group system, there are four dif-
ferent blood groups: A, B, AB or O. Anti-A and anti-B antibodies (called isohaemagglutinins) ap-
pear in the first years of life and they are of IgM class. In blood group typing red blood cells of the
tested person are mixed with isohaemagglutinins and occurrence of agglutination is directly eval-
uated. On the contrary, when patient´s serum is mixed with RBCs of a known blood type we can
assay for the presence of antibodies to that blood type in the tested serum.
Tab. 3.1 Blood group antigens and isohaemagglutinins
Blood group A
Individuals who belong to the blood group A have A antigens on the surface of their erythrocytes and
anti B-antibodies in blood plasma.
Blood group B
Blood group B individuals have B antigens on the surface of their RBCs and anti-A antibodies in blood
plasma.
Blood group AB
A and B antigens are expressed on the surface of RBCs in individuals belonging to the blood group AB,
however neither anti-A nor anti-B antibodies are detected in their plasma.
Blood group O
Neither A nor B antigens are present on the surface of RBCs in blood group O individuals, however, they
have both, anti-A, and anti-B antibodies in their plasma.
Direct agglutination is widely used also in microbiology as a diagnostic method of identi-
fying antigens specific for certain bacteria. A small amount of bacteria and a defined antiserum are
mixed on a slide or card and allowed to react. After a short period of time the occurrence of ag-
glutination is evaluated. The procedure is called serotyping and it is used e.g. to determine the
members of the Enterobacteriaceae or Streptococcaceae families.
19
Laboratory Metods in Immunology Classical serological methods

3.1.1.2 Semi-quantitative agglutination test
Agglutination tests may be used to measure the level (concentration) of antibodies to par-
ticulate antigens. Serial dilutions of serum containing antibody are prepared in test tubes and then
a fixed amount of antigen (such as bacteria) is added. The maximum dilution that gives aggluti-
nation is determined. The reciprocal of the highest dilution of serum showing visible agglutina-
tion is called the “titer of serum”. For instance, the titer of 160 means that the patient´s serum gives
a positive result (agglutination) at any dilution down to 1/160 (1 part serum to 160 parts of a di-
luter) as shown in Fig. 3.2. The titre is thus a measure of the amount of antibody in a unit volume
of the original serum. Titer evaluation is commonly used to assess bacterial infections as the rise
in titer of an antibody to a particular bacterium indicates an infection with that specific bacterial
type. For instance, tube agglutination is routinely employed for the serological diagnosis of ty-
phoid, brucellosis, tularaemia, typhus fever and other infectious diseases.
Fig. 3.2 Titer determination by direct semi-quantitative tube agglutination
Direct Coombs test is also used for testing newborn babies with Rh-positive blood whose
mothers are Rh-negative. Rh system is the second most important blood group system in humans.
The most significant Rh antigen is the D antigen as it is most likely to provoke an immune re-
sponse. RhD-negative individuals commonly have no anti-RhD antibodies, because these are not
usually produced by sensitization against environmental substances. However, RhD-negative in-
dividuals can produce anti-RhD antibodies (which are of class IgG) following a sensitizing event
like the feto-maternal transfer of blood from a fetus in pregnancy. Occasionally they occur due to
a blood transfusion with RhD-positive RBCs. The test shows whether the mother produces anti-
RhD antibodies and whether these antibodies have been transferred through the placenta to the
fetus. Mother´s anti-RhD antibodies attack the developing RBCs of the fetus and may cause the
haemolytic disease of the newborn.
Fig. 3.3 Direct Coombs test
3.1.2.2 Indirect Coombs test

Indirect Coombs test allows deter-

3.1.2 Coombs test mination of circulating (unbound) anti-
erythrocyte antibodies in serum. It is
Binding of antibodies to erythro- used to screen pregnant women for IgG
cytes not always results in agglutination. class antibodies as well as in transfusion
Agglutination does not occur when anti- cross-matching or detection of antibodies
gen or antibody is in excess or because in the diagnosis of immune-mediated
electrical charges on the red blood cells haemolytic anemias. Indirect Coombs test
prevent the effective cross linking of the is a two-stage test as shown in Fig. 3.4.
cells by relatively “small” antibody mole- Firstly, washed RBCs are incubated with
cules such as IgG molecules. In order to the tested serum. If the serum contains an-
detect the presence of non-agglutinating antibodies, a second antibody directed against the IgG tibodies to antigens on the surface of the
coating the RBCs (called the Coombs reagent) is added. This anti-immunoglobulin enables to RBCs, the antibodies will bind onto the
cross-link the red blood cells what results in agglutination. The Coombs test was first described RBCs. Then, the RBCs are washed and in
in 1945 by the British immunologist R. R. Coombs, after whom it is named. Due to the employment the second stage incubated with antihu-
of anti-immunoglobulins is the Coombs test also refferred to as the antiglobulin test. There are man immunoglobulins (Coombs reagent).
two forms of the Coombs test: direct and indirect. If antibodies have bound to erythrocyte
surface antigens in the first stage, RBCs
3.1.2.1 Direct Coombs test will agglutinate when incubated with the
anti-immunoglobulins in the second stage,
Direct Coombs test is used to detect antibodies that are already attached to the surface of red and the test will be positive.
blood cells. To perform direct Coombs test a blood sample is taken and the RBCs are incubated
with the Coombs reagent. If this incubation leads to visual agglutination of the RBCs, the direct Fig. 3.4 Indirect Coombs test
Coombs test is positive (i.e. antibodies are bound to the surface of RBCs) as shown in Fig. 3.3. When
clumping is not seen the test is negative. Direct Coombs test is used for example to diagnose au-
toimmune haemolytic anaemia, a condition in which antibodies are produced that may destroy the
red blood cells.

20 21
Laboratory Metods in Immunology Classical serological methods

3.1.3 Passive agglutination
Agglutination works only with particulate antigens. Soluble antigens are so highly dis-
persed that an antigen-antibody complex evades detection by the naked eye. To make this reac-
tion readily visible, methods using adsorption of soluble antigens (e.g. viral parts, polysaccharide,
DNA) to a particle were developed. As natural carrier particles can serve RBCs (sheep, horse, and
human) displaying the highest adsorptive capacity. Artificial particles like latex beads or colloidal
charcoal are commonly used as well. Reaction of
such a coated particle with an antibody to the solu-
ble antigen is called passive agglutination and the
test is performed in the same way as the direct ag-
glutination test (Fig. 3.5). For example, in the rubella
antibody test, erythrocytes are coated with rubella
antigen and in the presence of antibody, agglutina-
tion occurs. Commercial test systems are usually per-
formed on cardboard cards or glass slides. Latex tests
are available to detect various microorganisms like
Streptococcus agalactiae, Clostridium difficile toxins, ro-
taviruses, etc. Applications include detection of an-
tibodies to soluble antigens like the rheumatoid
factor (details can be found in Chapter 9.2.2.6), viral
antigens and many others.
used for the determination of antigen amount. For
example in the Kahn’s test for syphilis serial di-
lutions of the antigen (toxin) are added to the
tubes containing a fixed quantity of the antibody
(antitoxin). The titre of the precipitating serum is
the maximum dilution of antigen in which
a clearly expressed precipitin reaction is obtained.
Fig. 3.6 Precipitate formation is affected by the
amount of antigen and antibody
3.3 Immunodiffusion
Immunodiffusion techniques detect anti-
gen-antibody precipitation reactions in a semi-
solid medium, e.g. agar gel, agarose gel or gelatin.
Antigens and antibodies diffuse in the medium until they reach the zone of equivalence. There are
several advantages in allowing precipitation to occur in a semisolid rather than in a liquid medium.
The reaction is visible as a distinct band of precipitation, which is stable and can be stained for
preservation, if necessary. 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.

Fig. 3.5 Passive agglutination

3.3.1 Single radial immunodiffusion (Mancini test)

3.2 Precipitation Radial immunodiffusion test is based on a precipitate ring that is formed as antigen diffuses
into a semisolid medium containing incorporated antibody. The antibody is homogenously spread
Precipitation reactions involve interactions of soluble antigens and antibodies that are cross- in the agar gel and different dilutions of the antigen are applied into holes punched into the gel.
linked so that they form a large macromolecular complex. In fluid, the molecules diffuse until they As the antigen diffuses into the gel, it interacts with the antibody and when the equivalence point
reach the ideal concentration (zone of equivalence). When the antigen concentration is very low is reached a whitish precipitation circle (ring) is formed as illustrated in Fig. 3.7. Incubation (dif-
and the antibody relatively abundant (zone of antibody excess), formation of very small complexes fusion) usually lasts 48-72 hrs. and afterwards the diameters of rings are measured. The diameter
occurs. However, a large complex (lattice) has to be formed in order for the aggregate to be visi- of the ring is proportional to the concentration of antigen since the amount of antibody is con-
ble. The area containing excess antibody is known as the prozone. By high concentrations of anti- stant. By using growing concentrations of a standard antigen a standard line can be generated. By
gen (zone of antigen excess), the lattice size becomes too small to precipitate again. Hence, instead the means of this standard line one can quanti-
of reaching a plateau, the curve comes back down to zero. The area containing excess antigen is tate the amount of an antigen in an unknown
known as the postzone (Fig. 3.6). sample. Thus, radial immunodiffusion is a quan-
The precipitation test may be carried out either as a qualitative or as a quantitative test. titative test. It is commonly used in the clinical
It is very sensitive for detecting antigens and relatively less sensitive for the detection of anti- laboratories for the determination and quantita-
bodies. An example of a qualitative test is the slide test, when a drop, each of the antigen and an- tion of various serum proteins in patient samples
tibody is placed on a slide and mixed by shaking. In case of a positive result, precipitation (immunoglobulins, enzymes, complement com-
appears. The simplest type of precipitation test is the ring test based on layering the antigen so- ponents, acute phase proteins, etc.).
lution over a column of antibody in a narrow tube. A precipitate forms at the junction of the two
Fig. 3.7 Radial immunodiffusion test
liquids. However, ring tests have only few clinical applications now. A quantitative tube test is

22 23
Laboratory Metods in Immunology Classical serological methods

(electrophoresed) according to their charge, size, confor-

Fig. 3.8 Calibration graph
3.3.2 Double immunodiffusion by
Ouchterlony
Double immunodiffusion is based on the
As the amount of antibody in the gel
principle that both, antigen and antibody migrate
is constant, the diameter of the pre-
towards each other in a semisolid medium. Most
cipitin ring is a function of the amount
of antigen applied. often they migrate from separate circular wells cut
in a layer of agar or agarose gel. Antigen and an-
tibody are allowed to diffuse radially from their
wells and when they meet in optimal proportions
they form a visible line of precipitation. Position of the precipitation line depends mainly on con-
centrations of both antigen and antibody. The test can be conducted collaterally with multiple wells
filled with different antigen mixtures and multiple wells with different mixtures of antibodies. Pre-
cipitin lines may indicate identity, partial identity, or non-identity, depending on the resulting pat-
tern. In case that an antigen-antibody complex is identical, a precipitin line forms a coherent dash of
identity with the known reference system. A partial identity pattern is formed when some of the an-
tibodies are identical and others are not (the “spur” represents the components that are unrelated).
A non-identity reaction will occur when the antigen-antibody complexes are different. The result-
ing “X” indicates that two unrelated complexes are present (Fig. 3.9). Previously, the Ouchterlony
double immunodiffusion technique used to be extensively performed to test antisera for the presence
of antibodies specific for a particular antigen and to determine their concentration (e.g. in the diag-
nosis of various bacterial, viral or fungal infections). Nowadays, more progressive techniques like the
ELISA method or Western blotting are being used for this purpose (detailed in Chapter 4.3).
Fig. 3.9 Possible precipitation
patterns occurring in immunodif-
fusion by Ouchterlony
3.4 Immunoelectrophoresis
mation, etc. After electrophoresis, a trough is cut in the gel
and antibodies are poured into the trough. As the anti-
bodies diffuse into the agar, precipitin lines are produced
in the equivalence zone. Each antigen gives an individual
band with the corresponding antibody as illustrated in
Fig. 3.10. Immunoelectrophoresis serves for the qualitative
analysis of complex antigen mixtures based on the num-
ber and arrangement of the occurring lines.
This test is commonly used for the analysis of proteins
in a patient’s serum. Tested serum is placed in the well and
antiserum (antibodies directed to whole serum components)
is applied into the trough. Precipitin lines of the patient’s
serum are compared to normal serum (control) in order to
identify presence of pathological phenomena. By this com-
parison one can determine whether one or more serum com-
ponents are missing or whether there is an overabundance of
some serum components in the patient’s sample.
Fig. 3.10 Immunoelectrophoresis by Grabar and Williams
3.4.2 Countercurrent electrophoresis
Countercurrent immunoelectrophoresis is a rapid and more sensitive variant of the double im-
munodiffusion method in which again the electric current is used to drive the antigen towards the an-
tibody. This test works only if conditions can be found where the antigen and antibody have opposite
charges. In the zone of eqivalence they form a precipitation line as illustrated in Fig. 3.11. Countercur-
rent electrophoresis is a qualitative test used for the determination of antigens in clinical specimen (e.g.
bacterial antigens, acute phase
proteins). The thickness of the
lines gives also partially quan-
titative information. Today, this
technique can be replaced by
the ELISA method (detailed
description in Chapter 4.2.2.1).

Fig. 3.11 Countercurrent electrophoresis

3.4.1 Immunoelectrophoresis by Grabar and Williams

The precipitin reaction became also the basis of a method proposed by P. Grabar and 3.4.3 Rocket immunoelectrophoresis
K. Williams in 1953. The method is called immunoelectrophoresis and it is a combination of elec-
trophoresis and gel diffusion. At first, a complex mixture of antigens is placed in a well in an agar gel Rocket immunoelectrophoresis is a simple and quick method based on the one-dimensional
and then subjected to electrophoresis via application of electric current. The antigens are separated quantitative immunoelectrophoresis. The technique had been used for determining the levels of

24 25
Laboratory Metods in Immunology
specific proteins (mostly human serum proteins)
before automated methods became available. The
procedure involves comparison of the sample of
unknown concentration with a series of dilutions
of a known concentration of the protein. First, the
samples of proteins (antigens) are loaded side-by-
side in wells along the edge of a gel containing in-
corporated antibody. Afterwards antigens are
electrophoresed into the gel where interaction
with the corresponding antibody occurs. The end
result is a precipitation arc (resembling a “rocket”)
spreading out from the loading well. The height
of the precipitin arc is directly proportional to the
concentration of the antigen (Fig. 3.12).
Fig. 3.12 Rocket electrophoresis
SUMMARY AT A GLANCE
•Serological reaction is an in vitro reaction of an antigen and an antibody. The reaction is strictly spe-
cific; an antigen combines only with its homologous antibody and vice versa.
•Serological methods are based on serological reactions. They are extensively used for the identification
or quantitation of different antigens using known antibodies, or detection of antibodies by the means
of known antigens.
•Agglutination and precipitation are the two basic groups of classical serological reactions. Aggluti-
nation is the reaction of antibodies with large particulate molecules while precipitation occurs due to
the interaction of antibodies with soluble antigens.
•Haemagglutination is used for the determination of blood types or antibodies to blood group anti-
gens. Blood group typing is based on the presence or absence of inherited antigenic substances on the
surface of red blood cells (RBCs).
•Immunodiffusion techniques detect precipitation reactions in a semisolid medium. The most frequent
method is the single radial immunodiffusion test. It is based on detecting precipitate ring formed as
antigen diffuses into a semisolid medium containing incorporated antibody.
26
Progressive serological methods
4. Progressive serological methods
4.1 Progressive serological methods based on precipitation
Precipitates in fluids can be determined by means of methods that are based on the meas-
urement of cloudiness (turbidity) of a solution. Antibody and the antigen are mixed in such con-
centrations that only small precipitates are formed making the solution turbid. Nephelometry and
turbidimetry are the two most commonly used methods due to their speed and ease of use.
Incident light entering a solution containing precipitates is subjected to three reactions: Some
of the light is absorbed, some is transmitted and some will be scattered in various directions. Neph-
elometry is based on the measurement of scattered light, while, in turbidimetry, the intensity of
light transmitted through the sample is measured. The amount of light absorption (or light scat-
ter) is measured and compared to that from known solutions. The quantity of the unknown sam-
ple is then determined from a standard curve. Turbidimetry and nephelometry can be used to
detect either antigen or antibody, however, they are typically performed with antibody as the
reagent and antigen as the unknown. The determined antigens are usually various specific proteins
present in serum or other body fluids, like coagulation factors, complement components, acute
phase proteins, theraputic drugs or immunoglobulins.
Both techniques are widely used in clinical laboratories because they are precise, rapid and
easily automated. However, nephelometry is more sensitive than turbidimetry when analysing
small particles like antigen-antibody complexes.
4.1.1 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. The change in the amount of light absorbed
can be related to the amount of antigen-antibody complexes, thus, the amount of antigen in the
sample can be determined. Measurements are made using a spectrophotometer or an automated
analyzer (turbidimeter).
4.1.2 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 meas-
ures light rays passing directly through the solution). Often
low power lasers are used as light sources. Light passes
through the sample (containing antigen-antibody com-
plexes) in a cuvette using a lens system and a filter; the re-
Fig. 4.1 Nephelometer sultant scattered light is measured using a detector.
measures the intensity Commonly an angle of 90 degrees between the incident
of scattered light
and reflected light is used for detection purposes. The re-
27
Laboratory Metods in Immunology
lationship between antigen concentrations and light scattering approaches linearity. Light scatter
may be directly extrapolated by a computer to give actual concentrations, e.g. in mg/l.
4.2 Immunoassays
All immunoassays work on the same basic principle – reaction of an antigen and an anti-
body, where one of the reactants is labeled by a suitable label. Usually the label is fixed to the an-
tibody molecule, namely to its Fc part, so that the antigen binding function is not affected.
Immunoassays differ in the types of labels used and in the analytical procedure by which the label
is visualised, resp. quantitated. The following types of labels are beeing employed most frequently:
• radioactive labels
• enzymes which induce the breakdown of the substrate with the formation
of stained products
• fluorochromes capable to fluoresce
Immunoassays may be qualitative or quantitative. Quantitative immunoassays are reported in
mass units, along with reference intervals (normal ranges) for the test. An example of a qualitative
immunoassay is the test for pregnancy. This test is based on the detection of human chorionic go-
nadotropin in urine or serum that is either present or not. Quantitative immunoassays are performed
by measuring the intensity of the produced signal. The above mentioned test for pregnancy can be
transformed into a quantitative assay by measuring the concentration of the reaction product.
4.2.1 Radioimmunoassay
Radioimmunoassay (RIA) has been developed as the first of the immunoassay techniques.
It was introduced in 1960 by Berson and Yalow and it has revolutionized research and clinical
practice in many areas. This technique was considered of such importance to science that a Nobel
prize was awarded for its development in 1977.
In RIA, the label is an gamma-radioactive isotope such as 125I, 131I, 3H and 32P. The technique
requires the use of an instrument called a gamma counter, which measures the amount of gamma
radiation given off by the radioactive label. The presence of radioactivity indicates that the inves-
tigated antigen or antibody is present in the sample (qualitative information) and the amount of
radiation is proportional to the amount of the determined component (quantitative information).
The main advantage of RIA is the high sensitivity that can be achieved. Antigen levels that
are as low as few picograms can be routinely measured. For example the detection of allergen-
specific IgE levels reqiures a highly sensitive technique as these antibodies occur in serum in ex-
tremely low levels. The radioallergosorbent test (RAST) used in the diagnosis of allergies meets
these requirements.
4.2.1.1 Radioallergosorbent test (RAST)
RAST test belongs to the group of indirect antiglobulin tests. It is used to detect the causative
allergen for an allergy by determining the presence of the allergen-specific IgE antibody. In RAST
28
Progressive serological methods
test the respective purified allergen is bound to a solid phase such as
a well of a multititer plate. Tested serum is added to the well and in-
cubated with the allergen. If serum contains IgE antibodies specific
for the allergen, they will bind to the well. The well is washed (non-
specific antibodies are removed), and anti-human IgE labeled with
a radioisotope is added. Anti-human labeled IgE attaches to the al-
lergen-antibody complexes. After incubation, the well is washed
again, and its radioactivity is measured (Fig. 4.2). The amount of ra-
dioactivity is directly proportional to the IgE concentration. The RAST
test is often used as a screening test by combining groups of allergens
in a single reaction.
Fig. 4.2 RAST (Radioallergosorbent test)
1. Suscepted allergen (antigen) is fixed to a solid phase.
2. Tested serum is added. If serum contains IgE antibodies specific to the allergen, those will bind.
3. Radiolabeled anti-IgE antibody is added that binds to IgE–allergen complexes.
Unbound anti-IgE antibodies are washed away. The amount of radioactivity is directly
proportional to the concentration of allergen-specific IgE in tested serum.
4.2.1.2 Radioimmunosorbent test (RIST)
Radioimmunosorbent test (RIST) is a competition test performed
in vitro, used to measure the total level of IgE antibodies in serum.
Known amounts of radiolabeled IgE compete with the patient´s unla-
beled IgE to bind to a fixed monoclonal anti-IgE. The reduction in ra-
diolabeled IgE due to the presence of investigated IgE in the patient’s
serum can be determined by comparison with known IgE standards
(explained in Fig. 4.3). From a standard curve, one can determine the
amount of total serum IgE in an unknown sample. A positive test for
total IgE indicates a diagnosis of allergy when allergic symptoms are
present. However, serum IgE levels may be increased also in patients
suffering from parasitic infections, some immunodeficiencies (hyper
IgE syndrome) and some malignant diseases.
Fig. 4.3 RIST (Radioimmunosorbent test)
1. Fixed anti-IgE antibody is first incubated with known amount of radiolabeled IgE.
2. Serum containing unknown amount of tested IgE is added. Tested IgE competes
with radiolabeled IgE for the binding site on fixed anti-IgE.
3. The amount of radioactivity (in counts per minute) is indirectly proportional to the concentration
of IgE in the test sample, thus the amount of the patient's total serum IgE can be determined.
Radioimmunoassays enable to detect and quantify a wide range of proteins in blood, serum,
saliva, and urine. However, the major limitation of these techniques is the generation of radioac-
tive waste. That is why safer alternatives were developed using a nonradioactive signal instead of
29
Laboratory Metods in Immunology
the radioactive one. Fortunately, the sensitivity of nonisotopic labels exeeds nowadays that of RIA.
They are described in the following chapters.
4.2.2 Enzyme immunoassay
Enzyme immunoassay (EIA) was developed as an alternative to radioimmunoassay ten years
later. This method uses as a label an enzyme such as alkaline phosphatase, glucose oxidase and
horseradish peroxidase. The enzyme label reacts with chromogenic substrates in such a way that
a turn from colourless to a colour occurs, which is used as a signal. Results (the intensity of colour
produced in response to the substrate conversion) are quantitated spectrophotometrically. The ac-
tual concentration of the antigen or antibody can be determined by comparison with standards of
known concentrations. The sensitivity of EIA approaches or equals that of RIA, offering more
safety and speed. One of the most widely used EIA techniques is the enzyme linked immunosor-
bent assay (ELISA).
4.2.2.1 Enzyme linked immunosorbent assay (ELISA)
In enzyme immunoassays the enzyme is covalently linked to the Fc portion of an appropri-
ate antibody. This linking process was independently developed by S. Avrameas and G. B. Pierce.
Since it is necessary to remove any unbound antibody or antigen by washing, it is more conven-
ient when antibody or antigen is fixed to a solid phase. A technique to accomplish this principle
was developed by Wide and Porath in 1966 and named ELISA (Enzyme Linked Immunosorbent
Assay). ELISA is a solid-phase assay in which either the antigen or antibody is immobilized onto
a support, mostly the surface of a 96-well polystyrene microtiter plate. By fixing one component
to the solid phase the immune complexes can easily be separated from the other components by
simply washing the plate.
Technical advances led to automated pipetting devices, multichannel pipettes, microtiter
plate washers and plate readers. Later in the 1980s fully automated test systems were manufactured
that are routinely used in medical laboratories. The invention of ELISA generated a whole series
of test formats. The basic variants are direct, indirect and “sandwich” test procedures which are
described in detail in the following chapters.
ELISA is a useful tool for determining specific antibodies in serum to microorganisms that
are difficult to cultivate (e.g. specific antibodies to the hepatitis B virus, HIV or West Nile virus).
ELISA also enables to determine different disease-related proteins including cytokines, hormones,
tumor markers, allergens, pesticides or toxins. Other uses of ELISA include detection of potential
food allergens or rapid screening for certain pharmaceuticals, addictive drugs, etc. Various ELISA
kits used for diagnostic purposes are commercially available. Material provided with the test kits
includes antibody coated microtiter plate with 96 wells, enzyme conjugate reagent, substrate so-
lution, stop solution, wash buffer concentrate, sample diluents, reference standards as well as pos-
itive and negative controls.
The final assay signal is usually measured with a spectrophotometric plate reader. The most
controversial aspect of ELISA is determining the “cut-off point” between a positive and a negative
result. A cut-off point may be determined by comparing the ELISA plate reader value with
30
Progressive serological methods
a known reference standard. Unknowns that generate a signal that is stronger than the known
sample are considered positive and those that generate weaker signal are negative.
4.2.2.2 Direct ELISA
In a direct ELISA test which represents the simplest format of
the method the antigen that has to be determined is first adsorbed
to a plastic plate. An excess of another protein (such as bovine serum
albumin) is added to block all the other binding sites on the plate.
Detection enzyme is linked to an antibody in a separate reaction. By
means of this enzyme-labeled antibody the immobilized antigen is
determined as explained in Fig. 4.4. Excess enzyme-labeled antibody
is washed off, only enzyme-labeled antibody bound to antigen is
left. By adding the chromogenic substrate, the enzyme causes
change of colour. In this way the colour signal is illustrating pres-
ence of the antigen. The more intensive is the final colour, the more
antigen is present in the sample. Enzyme-labeled antibody will not
bind if the respective antigen is not present (negative reaction shown
on the example of antigen B in Fig 4.4).
Fig. 4.4 Direct ELISA
1. Antigen A and antigen B are fixed, each in one well of a plastic plate.
2. Enzyme-labeled anti-A antibody is added to both wells to determine antigen A. Anti-A antibody
binds only to antigen A (positive reaction), not to antigen B (negative reaction).
3. A chromogenic substrate for the enzyme is added to visualize the reaction as substrate
changes colour upon reaction with the enzyme. The intensity of colour is proportional
to the amount of antigen A.
Fig. 4.5 Indirect ELISA
1. Antigen A is fixed in wells of a plastic plate.
2. Tested serum is added to determine the presence of anti-A antibody. If serum contains anti-A
antibody, it will bind to antigen A. Antibodies with other specificities are not able to bind
3. To make the reaction visible, a secondary enzyme-labeled antibody is added that will bind
to the primary antibody already fixed to the antigen. Finally, substrate is added that changes
colour upon reaction with the enzyme. The intensity of colour is proportional to the amount
of anti-A antibody.
4.2.2.3 Indirect ELISA
Indirect ELISA is used to detect antibodies specific to an anti-
gen. The antigen is attached to a solid phase (e.g. a multi-well plate)
and a patient´s diluted serum is applied into wells of the plate. If an-
tibodies specific to the antigen are present in the serum, they will bind to it. The plate is then
washed to remove non-specific antibodies and all other serum components. A specially prepared
“secondary” antibody is then applied to the wells, followed by another wash. This secondary an-
31
Laboratory Metods in Immunology
tibody is an antibody that binds to human IgG and it is chemically coupled with an enzyme. Thus,
the well will contain enzyme in proportion to the amount of secondary antibody bound. As in all
ELISA formats, finally a substrate for the enzyme is applied. Catalysis by the enzyme leads to
a change in colour as shown in Fig. 4.5.
4.2.2.4 Sandwich ELISA
In this assay, a multi-well plate is coated with a primary capture
antibody, which recognizes the target antigen in the test sample and
binds to it. The antigen-antibody complex is then recognized by a sec-
ondary detection antibody that is labeled with an enzyme. By adding
a substrate, the enzyme catalyzes the reaction mixture, yielding a spe-
cific colour. By measuring the optical density of this colour, the amount
of the target antigen can be determined. Again, the intensity of colour
is directly proportional to the amount of the tested antigen (Fig. 4.6).
Fig. 4.6 Sandwich ELISA
1. Capture anti-A antibody is immobilized in the well of a plastic plate.
2. Tested serum is added to determine the presence of antigen A. If antigen A
is present, it will bind to the capture antibody.
3. A secondary enzyme-labeled antibody (detection antibody) is added that
will bind to the antigen A fixed in the capture anti-A antibody.
4. Finally, substrate is added that is converted by the enzyme into a colour
product. The intensity of colour is proportional to the quantity of antigen A.
Fig. 4.7 ELISPOT (Enzyme-linked immunosorbent spot assay)
1. ELISPOT plate is coated with capture antibody that is specific to the secretory
product of interest.
2. Cells whose secretory activity is to be measured are plated and stimulated.
The secreted molecules are captured around the secreting cells during
the entire culture period.
3. Cells are removed, leaving behind their secretory footprint.
4. Enzyme-labeled detection antibody is added.
5. Finally, chromogenic substrate is added that enables coloured spot development.
4.2.2.5 ELISPOT
The enzyme-linked immunosorbent spot assay (ELISPOT)
is based on the sandwich version of ELISA. ELISPOT allows
measuring secretory activity of individual activated or re-
sponding cells. Cellular secretions (like specific antibodies or
cytokines) are captured around the originating cell and man-
ifested as coloured footprints (spots). Each spot that devel-
ops in the assay represents a single reactive cell what allows
32
Progressive serological methods
to identify and enumerate these cells with an extraordinary level of accuracy. Due to its high sen-
sitivity, the ELISPOT has proven particularly useful when studying small populations of active
cells such as those regularly found in specific immune responses. Illustration shows principal steps
of the procedure (Fig. 4.7).
4.2.3 Fluorescent immunosorbent assay (FIA)
Fluorescent immunosorbent assay (FIA), known also as immunofluorescence assay (IFA), is
based on using antibodies chemically coupled with a fluorescent molecule such as phycoerythrin
(PE), fluorescein isothiocyanate (FITC) or rhodamine. These fluorescent indicators are called flu-
orochromes or fluorophores. The sample is illuminated with light of a specific wavelength which
is absorbed by the fluorophores, causing them to emit light of longer wavelengths (that means of
a different colour than the absorbed light). Fluorescence is usually visualised by means of a fluo-
rescence microscope. It can be quantified using a flow cytometer or array scanner. Two basic vari-
ants of FIA are the direct and indirect method.
4.2.3.1 Direct FIA
Similarly as in direct ELISA, also in direct FIA the specific antibody is coupled with the label,
in this instance with a fluorescent molecule. Direct FIA is used to detect the presence and local-
ization of specific antigens by the means of fluorescence emitted by the bound specific antibody
(Fig. 4.8A). Analysis of antigens in fresh, frozen or fixed tissues as well as various bacterial or par-
asitic speciment is possible. The technique is used for tumour typing as well or for the detection
and localization of different sub-cellular antigens, like specific DNA sequences on chromosomes,
etc. Thus, direct FIA is widely used in histology, cytology, pathology or microbiology.
Fig. 4.8 FIA (Fluorescent immunosorbent assay)
A) Direct FIA
Tissue or cell-specific antigen is visualized by
a fluorochrome-labeled specific antibody.
B) Indirect FIA
Secondary detection antibody is used to visualize
the „antigen-specific antibody“ complex.
4.2.3.2 Indirect FIA
Indirect FIA uses two antibodies; the
primary antibody specifically binds the
target antigen, and the secondary antibody marked with a fluorochrome recognises the primary
antibody and binds to it. Indirect immunofluorescence is more sensitive than the direct one since
more than one secondary antibody can attach to the primary leading to amplification of the sig-
nal (Fig. 4.8B). If the sample is positive, first, specific antibodies present in the sample (e.g. serum)
attach to the antigen substrates. In a second step, the attached antibodies are stained with fluo-
rescence-labeled anti-human antibodies and visualized using a fluorescence microscope.
33
Laboratory Metods in Immunology Progressive serological methods

Indirect FIA is the method of choice in the determination of autoantibodies to different cells
or tissues and this is why it is widely used in the diagnosis of autoimmune diseases (detailed in
Chapter 9.2.2.6). Indirect FIA enables also detection of antibodies to various other antigen sub-
strates, like different infectious agents. It is possible to perform simultaneous detection of anti-
bodies against several biochemically different antigens on one single biological substrate.
4.3 Western blot
tibody specific to some cell antigens. After the primary antibody is applied, biotinylated anti-IgG
is added, followed by peroxidase labeled avidin. Visualization is accomplished by adding an ap-
propriate substrate for peroxidase.
Over the time, a variety of different methods have evolved that are using the biotin-
avidin/streptavidin complex. They include immunohistochemical staining, flow cytometry, radio,
enzyme- and fluorescent immunoassays, hybridoma screening, Western blotting, purification of
cell surface antigens, etc.

Western blot, also known as immunoblot, is a widely used technique that detects specific Fig. 4.9
protein antigens in serum or other specimen. It combines electrophoresis with transfer of the sep- Western blot (Immunoblot)
arated proteins onto a membrane (this process is called blotting) and serological detection. Main 1. Mixture of antigens
advantage of Western blotting is that it allows analysis of mixtures of different antigens such as (e.g. serum proteins)
is electrophoretically separated
proteins found in human serum (enzymes, hormones, antibodies, receprots, etc.). Western blot is in a gel.
often used as a follow-up test to confirm the presence of specific antibodies in the diagnosis of in- 2. Separated antigens are blotted from
the gel onto a special membrane.
fectious diseases. Examples of its use include confirmatory hepatitis B and HIV testing (see Chap- 3. Labelled antibody is added to detect
ters 11.2.1 and 11.3.1). a specific protein bound to the
membrane.
To perform a Western blot test, samples containing the proteins are applied to wells along
4. Positive signal (e.g. a colour reaction)
one end of a gel. Electrical current causes the proteins in the samples to move in the gel according occurs in the position
to their charge, size and conformation and thus separating the proteins. As a result, bands are of the corresponding protein.

formed that resemble the steps of a ladder (Fig. 4.9). Separated samples are then transferred (blot-
ted) onto a nylon or nitrocelulose membrane. Labelled antibodies are used to detect the target pro-
teins bound to the membrane. Detection antibodies are labeled usually with an enzyme however
other labels like fluorescent or chemilluminiscent compounds may be used as well. When using
enzyme-labeled detection antibodies, their location is revealed by incubating the membrane with SUMMARY AT A GLANCE
a colourless substrate that the attached enzyme converts to a visible coloured product. Regardless
of the detection method, the signal intensity correlates with the amount of protein and can be vi- •Progressive precipitation techniques are based on mixing antibody and antigen in such concentrations
sually estimated as well as quantitated using appropriate equipment. The presence of analysed that only small precipitates are formed making the solution turbid.
proteins is interpreted by comparison with known negative or positive control samples.
•Nephelometry and turbidimetry are the two most commonly used methods that are based on the
4.4 Biotin-avidin system measurement of turbidity. Nephelometry is based on the measurement of scattered light, while, in tur-
bidimetry, the intensity of light transmitted through the solution is measured.
Avidin is an egg-derived glycoprotein with an extraordinarily high affinity for biotin. The
avidin-biotin complex is the strongest known non-covalent interaction between a protein and a lig- •Serological techniques called immunoassays are based on a reaction of an antigen and an antibody,
and. The bond formation between biotin and avidin is very rapid, and once formed, it is unaf- where one of the reactants is labeled by a suitable label (radioactive isotope, enzyme, fluorochrome).
fected by extreme pH, temperature, organic solvents and other denaturing agents. This is why the Immunoassays may be qualitative or quantitative.
interaction of biotin and avidin is being exploited in a number of methods. Many biotin molecules
can be coupled to a protein, enabling the biotinylated protein to bind more than one molecule of •The most common progressive serological immunoassay is the enzyme-linked immunosorbent assay
avidin. When biotinylation is performed under appropriate conditions, the biological activity of (ELISA) which is used to measure the concentration of antibodies or antigens in solutions. ELISA is
the protein is preserved. rapid and simple to carry out, and a large number of samples can be tested in parallel. It is a widely-
Similar in properties to avidin is streptavidin (from the bacterium Streptomyces avidinii), used method for measuring the concentration of various molecules (e.g., a hormones, enzymes, al-
that can be alternatively used. The biotin-avidin/streptavidin system has proven to be particu- lergens or drugs) in a fluid such as serum or urine.
larly useful in the detection and localization of antigens by employing biotinylated antibodies. For
example such a system can be used for staining a histological section with monoclonal primary an-

34 35
Laboratory Metods in Immunology
5. Evaluation of innate humoral immunity
The human immune system plays a crucial role in defence against infectious microorganisms
and tumours, as well as in removal of old, damaged and foreign cells and material. Immune mech-
anisms can be divided into innate (non-specific) and adaptive (specific). In contrast to adaptive im-
munity, the innate immune mechanisms participate in the first line of defence and provide an
immediate but non-specific response, have only limited diversity and no immunological memory.
In addition to mechanical, chemical and biological barriers of the skin and mucosa, the innate arm
of immunity comprises:
1. Humoral innate immunity components – the complement system, acute phase proteins
and other mediators of inflammation.
2. Cellular innate immunity components – phagocytic cells, NK cells
5.1 The complement system
5.1.1 The role of complement in the immune response
The complement system consists of more than 30 plasma and cell membrane proteins with
important roles in the defence mechanisms of innate immunity. The vast majority of complement
factors are present in an inactive form and get activated by distinct mechanisms. The complement
activation in one of the three pathways, the classical (CP), alternative (AP) or lectin (LP), has pro-
found biological consequences including the direct lysis of target cells (microbes, foreign cells) by
membrane attack complex (MAC), opsonisation and promotion of phagocytosis, induction of
chemotaxis and inflammation, as well as the dissolution of immune complexes and their removal
from circulation. Because of its strong cytolytic and proinflammatory activities, the activation of
the complement system needs to be strictly regulated to prevent damage of own cells and tissues.
This is accomplished by several mechanisms, most importantly by the action of soluble and mem-
brane-associated regulatory factors that inhibit the initiation of complement activation, assembly,
stability and function of convertases, and the assembly and stability of the MAC.
The complement system has been shown to participate in the development of numerous dis-
eases and pathological conditions including the autoimmune disorders, systemic inflammatory
response syndrome (SIRS), sepsis, ischaemia-reperfusion syndrome, graft rejections, neurode-
generative disorders and many more. The most common indications for the laboratory evaluation
of the complement system levels and function are congenital complement deficiencies manifesting
with increased susceptibility to bacterial infections and immune complex autoimmune diseases
(systemic lupus erythematosus, vasculitides), and diseases associated with excessive complement
activation and subsequent complement factors consumption. The former are caused by genetic de-
fects affecting the production or function of particular complement factors, while the latter ap-
pear as a consequence of congenital deficiencies of complement regulators or excessive production
and deposition of immune complexes that subsequently activate the complement system (typi-
cally seen in certain systemic autoimmune diseases), as well as due to the production of autoan-
tibodies that bind and stabilize the C3-convertase C3bBb or inactivate the C1-inhibitor. Finally,
36
Evaluation of innate humoral immunity
decreased complement levels can also result from their decreased synthesis or increased catabo-
lism in certain diseases, including the liver cirrhosis, disseminated intravascular coagulation, ma-
lignancies and some others. In most of these conditions, the evaluation of complement levels and
the integrity and function of complement activation pathways is an important part of the diag-
nostics and monitoring of disease progression and its response to treatment.
5.1.2 Laboratory tests for complement evaluation
5.1.2.1 Specimen collection
Because of the extreme unstableness of complement components, proper collection, prepa-
ration, handling and storage of biological samples are crucial to obtain correct and relevant results
in complement assays. For the quantification of individual complement factors and in vitro com-
plement functional assays, functionally intact serum obtained from whole blood by centrifugation
after clotting must be used. For the evaluation of complement activation products produced in vivo,
plasma obtained from venous blood drawn into the EDTA-containing tubes (at a concentration of
20 mM) should be used instead of the serum. EDTA inhibits the complement activation by chelat-
ing calcium and magnesium, thereby preventing the production of complement activation frag-
ments in vitro after the specimen was collected, what would markedly distort the final results. Other
anticoagulants such as citrate and heparin do not sufficiently block the complement activation and
therefore can not be used for this purpose. EDTA-plasma can also sometimes be used for comple-
ment functional assays, provided that it was mixed with a buffer containing Ca2+ and Mg2+ to en-
able complement activation and specific thrombin inhibitor lepirudin to inhibit coagulation. Serum
and plasma should be obtained from whole blood sample as soon as possible and stored at -70 °C
to avoid degradation of complement factors and activation products with generally short half-life.
Prior to laboratory analysis, the samples should be thawed at 37 °C and then kept on ice.
5.1.2.2 Functional screening tests for total complement activity
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. Absolute
absence, significantly decreased concentration or impaired function of any of individual comple-
ment factors will inevitably affect the whole complement cascade and result in abnormal forma-
tion of terminal complement complex (MAC), which can be confirmed by specific monoclonal
antibodies or by examining its ability to lyse target erythrocytes.
The total haemolytic complement (CH50) assay is a functional laboratory assay for the eval-
uation of classical complement pathway. For its activation, immune complexes formed by IgG rab-
bit antibodies that react with surface antigens on sheep erythrocytes are used. After serial dilutions
of tested serum with Ca2+ and Mg2+-containing buffer are prepared in tubes or wells of a microtiter
plate, a fixed amount of the haemolytic system (erythrocytes with bound antibodies) is added to
each tube/well and incubated at 37 °C for 1 hour. This allows the C1q to bind to the Fc portions
of antibodies and initiate the classical complement pathway. The subsequent formation of the
37
Laboratory Metods in Immunology
MAC, which requires the presence and correct function of all classical pathway and terminal com-
plement factors (C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8 and C9), causes the lysis of red blood
cells and the release of haemoglobin. The colour change of the supernatant is subsequently read
by spectrophotometer (Fig. 5.1). Simultaneously, separate tubes or wells with haemolytic system
and buffer alone to measure spontaneous lysis, and tubes/wells containing haemolytic system
with distilled water for total haemolysis are prepared as controls. The results are calculated by
a software or using von Krogh’s equation and expressed as the reciprocal of the dilution that causes
lysis of exactly 50 % of the red blood cells in the assay (hence CH50 assay). Obtained values are
then compared to reference values for normal human serum. Any quantitative or functional ab-
normalities in CP complement proteins will result in an impaired lysis of the cells, and thus de-
creased amount of released haemoglobin. As a consequence, the serum dilution required for the
lysis of 50 % of RBCs will be lower compared to the reference serum.
Fig. 5.1 Principle of the total haemolytic CH50 assay
The analogous assay for the evaluation of AP activity is the alternative pathway haemolytic
(AH50) assay that uses rabbit or guinea pig erythrocytes capable of directly activating the alterna-
tive pathway. To block the simultaneous activation of the classical pathway, ethyleneglycolte-
traacetic acid (EGTA) is also added to the serum dilutions in order to chelate Ca2+. Like the CH50,
the AH50 is expressed in units that represent the exact amount of serum (dilution) that lyses 50 %
of red blood cells. Any deficiencies in factors B, D, P, C3, C5, C6, C7, C8 and C9 will result in ab-
normal values obtained in AH50 test. Hence, performing both CH50 and AH50 assays in a patient
with suspected complement deficiency can help to localise the complement defect. Low or absent
CH50 values in the presence of normal AH50 results indicate the C1, C2 or C4 abnormality. Con-
trariwise, abnormal AH50 but intact CH50 values localise the defect into the initial factors of the al-
ternative complement pathway (i.e. factor B, D or P). Abnormal values obtained in both CH50 and
38
Evaluation of innate humoral immunity
AH50 assays are typically the consequence of deficiencies of central C3 factor or terminal comple-
ment factors C5–C9. As there is no commercial haemolytic assay for the lectin pathway available yet,
ELISA-based functional assay must be used instead. Complete absence of haemolytic activity usu-
ally indicates the presence of a hereditary complement defect, whereas decreased CH50 or AH50
values point at acquired complement deficiency, e.g. due to complement consumption.
In addition to haemolytic assays performed in tubes or microtiter plates, another simple al-
ternative test performed in the agarose gel and resembling the radial immunodiffusion (RID) assay
has also been developed. The haemolytic system (antibody-coated sheep erythrocytes for CP or
rabbit erythrocytes for AP) is cast in the gel and appropriate amounts of the tested serum along
with positive and negative controls are subsequently added to punched holes in the gel. The serum
complement factors diffuse radially and cause the lysis of
erythrocytes manifesting as a zone of clarification if the com-
plement cascade was successfully activated. The diameter of
the lysis zone depends on the functional activity of the com-
plement system in the serum (Fig. 5.2). Any deficiencies of
complement factors will result in decreased diameter of the
lysis zone or even complete absence of haemolysis.
Fig. 5.2 Haemolytic complement assay in the agarose gel
Fig. 5.3 Solid-phase ELISA assay
for evaluation of classical path-
way of complement activation
Non-haemolytic func-
tional screening tests include
the liposome-based assays
and solid-phase ELISA assays.
The former use enzyme-con-
taining liposomes instead of
sheep red blood cells for the
evaluation of the classical
complement pathway. Its successful activation and the formation of the MAC result in the lysis of
the liposomes and the release of an enzyme that can be read by a chemistry analyser. ELISA as-
says are based upon the use of specific activators bound to the bottom of wells of microtiter plates
and the subsequent detection of deposited complement activation products by monoclonal anti-
bodies. For the evaluation of the classical pathway, IgM antibodies immobilised at the bottom of
wells are used. After the patient's serum was placed into the wells coated with antibodies, the C1q
binds to IgM and initiates the classical pathway of complement activation resulting in the forma-
tion of MAC. Its presence and quantity is then measured by monoclonal antibodies against C9
neoepitope, which only appears when the complete pathway was successfully activated. After the
39
Laboratory Metods in Immunology
wash, the bound anti-C9 antibodies are detected by secondary enzyme-labelled antibodies. In the
final step, a specific substrate is added and converted by the enzyme into a coloured product
(Fig. 5.3). The intensity of colour change is read by an ELISA-reader. For the evaluation of the lectin
pathway, the serum is added into the wells coated with mannan as an activator. The binding of
MBL and subsequent activation of MASP enzymes result in the cleavage of C4 and C2, formation
of C3-convertase and finally the assembly of MAC, which can be detected by anti-C9 monoclonal
antibodies. The alternative pathway is activated by immobilised lipopolysaccharide (LPS) and
then detected by monoclonal antibodies to deposited properdin (factor P) or activated C9. A sim-
ple ELISA method comprising all three assays is currently available.
Functional screening assays are useful as initial tests for the identification of congenital com-
plement deficiencies, and may also help to monitor the course of diseases associated with the con-
sumption of complement factors, such as SLE. The confirmation and precise identification of causal
defect require further assays that measure concentrations and function of individual complement
factors and the production of activation products.
5.1.2.3 Quantitative assays for individual complement components
Any abnormal values obtained in total complement functional assays are the indication for
performing quantitative immunochemical assays. The serum concentrations of soluble comple-
ment factors can be determined by classical immunoprecipitation techniques (radial immunodif-
fusion, nephelometry) or by more sensitive assays including ELISA and Western blot (Chapters 3
and 4). In the routine clinical laboratory praxis, C3, C4 and sometimes factor B are the most fre-
quently measured complement proteins when hereditary or acquired immunodeficiencies are sus-
pected. If necessary, other components can also be evaluated, including the C1-INH, MBL,
properdin and others. Decreased or absent levels of individual complement factors in the presence
of abnormal CH50 and/or AH50 confirm the diagnosis of complement deficiency. On the other
hand, normal values obtained in quantitative assays suggest the presence of a functional defect,
and additional functional tests for individual components are further required to verify this defect.
5.1.2.4 Functional tests for individual complement components
Functional activity of individual complement components should be determined when a ge-
netic defect causing dysfunction or even loss of function of a particular complement factor is sus-
pected. The function of respective complement factors can be examined by using modified CH50
or AH50 assays. The most common way is to prepare an array of test tubes containing appropri-
ate haemolytic system (opsonized sheep RBCs for CP and rabbit RBCs for AP, respectively) and
single complement component-deficient or -depleted human serum. In the final step, tested pa-
tient's serum sample is added to the tubes to determine if it restores the complement activity of
a deficient serum. The lack of haemolysis in particular tube indicates that the tested patient's serum
lacks the same complement protein as the laboratory prepared deficient serum (Fig. 5.4). Alterna-
tively, purified functionally active complement proteins can be added in a step-wise fashion to the
tubes containing haemolytic system and patient's serum to determine which one reconstitutes the
40
Evaluation of innate humoral immunity
complement activity of the re-
spective pathway. The occu-
rence of haemolysis after the
addition of a particular com-
plement components confirms
its deficiency in the patient.
Fig. 5.4 Modified CH50 assay
for detection of individual comple-
ment factor deficiencies
The functional activity of the C1-inhibitor (C1-INH) can be examined by various different as-
says, including the ELISA assay that measures the formation of complexes between biotinylated C1s
and C1-inhibitor that are captured on avidin-coated plate. Another possibility is the radial immun-
odiffusion assay (Chapter 3.3.1) that uses anti-C1r polyclonal antibodies dissolved in the gel. Bind-
ing of the C1-INH to C1r blocks the binding site on C1r for these antibodies. Thus, when patient's
serum is mixed with immune complexes and the complement is activated, C1-INH binds to acti-
vated C1r and blocks the binding site for the anti-C1r antibodies. If the function of C1-INH is im-
paired, it cannot bind to C1r and inhibit its interaction with antibodies. The result will be the
formation of a precipitation ring after the serum is allowed to diffuse radially from a hole in the
agarose gel (Fig. 5.5). The diameter of the precipitation zone is then indirectly proportional to func-
tional activity of the C1-INH – the larger the diameter of the precipitation zone, the lower the ac-
tivity of C1-INH and vice versa. Several assays have also been developed to determine the activity
of alternative pathway regulatory factors H and I, although these are not frequently performed.
Fig. 5.5 Radial immunodiffusion
assay for evaluation of C1-inhibitor
activity
5.1.2.5 Quantitative assays for complement activation products
The quantitation of activation fragments (products) is more sensitive method for the evalu-
ation of pathologically increased in vivo complement activation and consumption than the meas-
urement of native inactive complement factors and total complement activity, because the decrease
of native factors levels and complement activity during the active phase of diseases associated
41
Laboratory Metods in Immunology
with complement consumption (systemic lupus erythematosus, vasculitides, etc.) may sometimes
be masked by their increased production by liver (systemic AID are typically accompanied by in-
flammation and the production of acute phase proteins, including the complement factors!). Ac-
tivation products can be either split fragments after enzymatic cleavage or protein complexes.
They can be measured by the ELISA and microbeads-based multiplex flow cytometric assays as the
markers of classical pathway (C1rs-C1INH, C4a, C4b/c, iC4b, C4d), alternative pathway (Ba, Bb,
C3BbP), central component C3 (C3a, iC3b, C3b/c, C3d) and terminal sequence (C5a, C5b-9) acti-
vation. The assays use specific monoclonal antibodies to the so called neoepitopes: antigenic de-
terminants that are hidden in native complement factors but are exposed after the respective
complement component was activated and underwent a conformational change. For instance, the
detection of the terminal complement complex C5b-9 is performed by using immobilised capture
monoclonal antibody to a neoepitope on poly-C9. The bound C5b-9 is subsequently visualised
with enzyme-labelled anti-C6 monoclonal antibody (Fig. 5.6). As the levels of activation products
also depend on the initial concentrations of the native components, it was suggested that the ratio
between the activation product and the native component (e.g. C3d/C3) is a better indicator of
complement activation than
the activation product alone.
These assays are, however, not
among routine tests and are
only performed by few highly
specialised laboratories.
Fig. 5.6 ELISA assay for the mea-
surement of C5b-C9 complement
activation product
5.1.2.6 Detection of membrane-associated complement receptors and regulatory proteins
Although most complement factors are present in soluble form in plasma and body fluids,
receptors for complement fragments (CR1, CR3, CR4) and some regulatory proteins (DAF, MCP,
protectin, etc.) exist in a membrane-bound form. Their detection can be therefore performed by
flow cytometry using fluorescently-labelled monoclonal antibodies (Chapter 7.2.2.2).
5.1.2.7 Detection of autoantibodies to complement components
Several complement-related diseases are caused by the production of autoantibodies to com-
ponents of the complement system. In patients with membranoproliferative glomerulonephritis
(MPGN) type II, autoantibodies against the alternative pathway C3-convertase C3bBb (C3-
nephritic factor) are typically observed. These antibodies stabilise the convertase what results in
an excessive cleavage of C3, its consumption and the development of secondary C3 deficiency. In
addition to the MPGN, patients also suffer from frequent bacterial infections. The definitive labo-
ratory diagnosis relies upon the detection of C3-nephritic factor by several different assays, in-
42
Evaluation of innate humoral immunity
cluding the ELISA and haemolytic methods. Acquired angioedema (AAE) is a disorder caused by
the production of autoantibodies against the C1-inhibitor. In this case, the binding to the C1-INH
causes its inactivation and resulting insufficient inhibition not only of the classical complement
pathway, but also certain clotting factors, kallikrein and plasmin. The resulting overproduction of
vasoactive mediators causes recurrent episodes of oedema formation identical to hereditary an-
gioedema. The presence of the anti-C1-INH autoantibodies can be verified by ELISA method with
purified C1-INH protein immobilised on the microtiter plate.
5.1.2.8 Genetic analyses of complement components
Congenital complement deficiencies caused by gene mutations are extremely rare; there-
fore, their genetic analyses are mostly performed in few specialised laboratories in situations when
a single complement defect was revealed by functional and quantitative assays. Genetic mutation
analyses offer better understanding of the nature of the complement defect and provide an op-
portunity for screening for potential mutation carriers in the family. The most common way how
to identify gene mutation is the amplification of target DNA region within the candidate gene and
its subsequent sequencing (Chapter 12.3.3.3).
5.2 Acute phase proteins and markers of inflammation
5.2.1 The role of acute phase proteins in the immune response
Acute phase proteins (APPs) are a group of more than 30 soluble plasma proteins produced
by liver hepatocytes and, to a lesser extent, by cells of the immune system. Following the tissue in-
jury induced by pathogens, physical, mechanical and chemical insults as well as other endo- and
exogenous triggers, the hepatic APPs synthesis and their plasma levels may increase significantly
in response to main pro-inflammatory cytokines IL-1β, TNF and IL-6 produced during the acute
phase of inflammatory response. Any infectious or non-infectious process (cancer, autoimmunity,
hypersensitivity, ischaemia, trauma, etc.) developing in the human body may induce a uniform
adaptive inflammatory response with typical local manifestation and systemic changes including
the induction of fever, loss of appetite, lethargy, catabolism and APPs production. Acute phase
proteins subsequently directly participate in immune defence mechanisms by neutralising mi-
crobes, regulating the intensity of immune reactions, preventing excessive tissue damage and par-
ticipating in tissue repair and regeneration (Tab. 5.1).
Certain APPs are normally present in plasma at very low levels but their concentrations may
rise rapidly and significantly (10 – 1,000 times) during the acute phase of inflammation. These so-
called main APPs include C-reactive protein (CRP), pentraxin 3 and serum amyloid A. Other APPs
are synthesized continually as physiological components of plasma and their levels increase slower
ceruloplasmin, haptoglobin, complement proteins, inhibitors of serine proteases, α2-macroglobu-
and to a lesser extent (1.5 to 4-fold) during the inflammatory process. This group of APPs includes
lin and others. The most frequently used marker in the laboratory diagnostics of inflammatory
diseases is the C-reactive protein (CRP). Its physiological levels are very low (below 5 mg/l) but can
increase dramatically during general non-specific inflammatory responses to various stimuli (in-
43
Laboratory Metods in Immunology Evaluation of innate humoral immunity

CRP levels compared to viral and parasitic infections, tumours and autoimmune processes. The
Family Members Function CRP measurement is hence an important diagnostic tool used to monitor the intensity, progress,
Pentraxins C-reactive protein acts as opsonin, activates classical complement activity and treatment response in various inflammatory diseases including infections, malig-
pathway, inhibits neutrophil degranulation, nancies, certain systemic autoimmune diseases (rheumatoid arthritis, vasculitides, etc.), cardio-
participates in the clearance of apoptotic cells
vascular diseases and others. CRP also serves as a helpful tool in the differential diagnostics of
Serum amyloid P has contrasting effects on innate immunity,
participates in the clearance of apoptotic bodies infectious vs. non-infectious inflammatory diseases and as a non-specific marker for the differen-
Pentraxin-3 acts as opsonin, activates classical complement tiation of bacterial infections from those caused by viruses. The CRP-guided approach to man-
pathway and phagocytosis agement of infectious diseases thus allows to rationalise the antibiotic (ATB) therapy and helps to
Metaloproteinases Ceruloplasmin copper transporter, oxidizes iron and enables its reduce the ATB over-prescription and its negative consequences including the development of
binding to transferrin bacterial resistance, risks of toxicity and allergic reactions and excessive waste of resources of pub-
Haptoglobin binds haemoglobin, inhibits chemotaxis, lic health insurance. Recently, the measurement of low CRP levels by high-sensitive methods has
phagocytosis and cidal activity of neutrophils been suggested as a predictive marker for the risk evaluation of cardiovascular diseases.
Haemopexin binds and transports haem In recent years we have witnessed the introduction of new markers into the laboratory diag-
Inhibitors of serine α1-antitrypsin prevents the tissue injury by inhibiting nostics of inflammatory diseases. In particular, procalcitonin and neopterin have gained popular-
proteases neutrophil-derived, complement and ity and proved their usefulness in the discrimination between various aetiological causes of
other serine proteases
α1-antichymotrypsin
inflammation. Procalcitonin (PCT) is a pro-hormone (precursor of the hormone calcitonin) normally
inhibits cathepsin G and other proteases
α1-antiplasmin
and the intestine. Its basal levels are very low (under 0.05 μg/l) but may increase dramatically (1,000
produced by parafollicular cells (C cells) of the thyroid gland and neuroendocrine cells of the lung
inhibitor of plasmin
Immunomodulatory α1-acid glycoprotein binding protein for exo- and endogenous – 10,000-fold) during severe or systemic bacterial and invasive fungal and parasitic infections be-
proteins (AGP, orosomucoid) molecules, down-regulator of neutrophil
activity and complement activation cause of its synthesis by nearly all cell types in the body. In comparison to CRP, the PCT levels begin
α2-macroglobulin binding protein and inactivator of to increase and peak much faster after the infection start (within 8 hours) and also return to normal
numerous molecules and iones values sooner (1 – 1.5 day) when the aetiological agents have been eliminated. PCT levels do not in-
Complement C3, C4, MBL, factor B lysis of microorganisms, chemotaxis, crease significantly in viral or non-infectious inflammatory diseases (autoimmunity, malignancies)
factors and others opsonisation, induction of inflammation and, therefore, PCT serves as useful marker for the prediction and diagnostics of infectious sys-
Coagulation and Fibrinogen, prothrombin, participate in blood clotting or temic inflammatory response syndrome (sepsis) and its differentiation from a non-infectoius SIRS.
fibrinolytic factors plasminogen and others fibrinolysis It also enables guided therapy of severe inflammatory conditions as it helps clinicians to distin-
Plasminogen activator inhibits tissue plasminogen activator and guish between bacterial, mycotic or parasitic infections and viral and non-infectious inflammation,
inhibitor-1 urokinase, and hence fibrinolysis
and provides guidance for initiating and discontinuing antibiotic therapy.
Protein C inhibitor inhibits protein C (an anticoagulant)
Another marker, neopterin, produced by activated monocytes and macrophages, typically in-
Other APPs Serum amyloid A chemotaxin, inhibits the production of creases in viral infections, malignancies and some autoimmune diseases, but not in bacterial in-
oxygen reactive species, influences fections. Its measurement may therefore effectively supplement the CRP and PCT evaluation in the
high-density lipoprotein-cholesterol transport
differential diagnostics of systemic inflammatory conditions.
Hepcidin inhibits iron transport across the gut
mucosa and out of macrophages
Lipopolysaccharide binds LPS and mediates its recognition 5.2.2 Laboratory tests for the evaluation of inflammation
binding protein by CD14 and TLR4/MD2 and acute phase proteins

Tab. 5.1 Most important acute phase proteins and their function 5.2.2.1 Evaluation of white blood cell counts and differential

fections, autoimmune diseases, cancer, trauma, surgery, burns, myocardial infarction, etc.). Under
these conditions, the CRP levels rise after few hours and peak within 24 – 48 hours. As the bio-
logical half-life of the CRP is rather short, its levels also decline rapidly when the aetiological
agents have been eliminated (within 4 – 5 days). The CRP concentrations depend on the intensity
and the nature of the inflammatory process: bacterial infections are often associated with higher
As inflammatory processes are regularly associated with the changes in total leukocyte num-
bers as well as WBC subpopulations, the complete blood cell count (CBC) with differential is an
integral part of the diagnostic algorithm of inflammatory diseases. Due to the increased synthesis
of several growth factors (e.g. colony stimulating factors), increased number of white blood cells
(leukocytosis) is often found in patients with local or systemic inflammation. Changes in the in-

44 45
Laboratory Metods in Immunology
dividual leukocyte subpopulations numbers usually depend on the nature of inflammatory
process and thus may provide useful information on the aetiology of the disease. Neutrophilia
(increased number of neutrophils) is indicative for bacterial infections, but may also appear in
other acute inflammatory conditions (myocardial infarction, burns), while eosinophilia (increased
number of eosinophils) is typically found in patients with parasitic infections and allergic dis-
eases. Increased lymphocyte number (lymphocytosis) is present in acute viral infections, some
protozoal and chronic intracellular bacterial infections (tuberculosis, etc.), but also in leukaemias
and lymphomas. Increased monocyte counts (monocytosis) are indicative for chronic inflamma-
tory diseases including infections, autoimmune diseases (systemic lupus erythematosus, rheuma-
toid arthritis, inflammatory bowel diseases) and haematological malignancies.
5.2.2.2 Evaluation of the erythrocyte sedimentation rate
Evaluation of the erythrocyte sedimentation rate (ESR) by modified FĆhrĺus-Westergren
method is a simple and inexpensive laboratory test for assessing the acute inflammatory response.
ESR is a rate at which red blood cells sediment in a period of one and two hours after the anticoag-
ulated blood was placed in an upright tube or capillary. Under physiological conditions, ESR values
are low (≤ 15 mm/hr in men and ≤ 20 mm/hr in women) due to the presence of a negative charge
on the surface of erythrocytes that hinders their interaction and sedimentation. In the presence of in-
flammation, increased synthesis of fibrinogen (one of the APPs) and its deposition on the red blood
cells causes them to stick to each other and form stacks called “rouleaux”. This eventually leads to
accelerated erythrocyte sedimentation typically observed in inflammatory conditions such as infec-
tions, autoimmune diseases, malignancies and other conditions associated with tissue injury (my-
ocardial infarction, trauma, surgery, burns, etc.). The ESR may be significantly affected by several
factors including the patient’s sex, age, red blood number and morphology, immunoglobulin levels
and technical factors. Therefore, the acute phase proteins (e.g. CRP) are considered to be more reli-
able markers of inflammation, because their values are less prone to these confounding factors.
5.2.2.3 Measurement of acute phase proteins and other markers of inflammation
Measurement of acute-phase proteins is a useful tool in medical clinical pathology, as it allows to
detect the presence of inflammatory process in the body, to monitor its activity, progress and response
to treatment, and often provides useful information on possible aetiology of the disease (infectious vs
C-reactive protein, while other APPs (orosomucoid, α1-antitrypsin, ceruloplasmin, haptoglobin) are
non-infectious, bacterial vs. viral). The most frequently measured soluble inflammatory marker is the
used to a lesser extent. Procalcitonin and neopterin are usually determined in critically ill patients as
a part of a diagnostic algorithm for systemic inflammatory response syndrome (see above).
Laboratory methods for the evaluation of APPs include immunoprecipitation methods in the
gel (single radial immunodiffusion assay) and liquid phase (nephelometry, turbidimetry), as well as
more sensitive methods such as ELISA (Chapters 3 and 4). Because of their low basal levels, pro-
calcitonin and neopterin measurement requires the use of highly sensitive methods including the
enzyme-linked immunosorbent assays, enzyme-linked fluorescent immunoassays, chemiluminis-
cent immunoassays, immunoluminometric assays and immunochromatographic assays.
46
Evaluation of innate humoral immunity
The need to obtain the results as soon as possible forced the development of rapid tests for
the quantitative/semiquantitative evaluation of CRP and procalcitonin. Such assays have found
their application in point-of-care settings and primary care units when a quick determination of
the location of the inflammation and differentiation between acute bacterial and viral infection, or
between infectious and non-infectious inflammation is required for guided therapy. Typical ex-
amples include children suffering from acute respiratory and urinary infections (bacterial vs. viral
upper respiratory tract infection, pneumonia vs. tracheitis/bronchitis, acute pyelonephritis vs.
cystitis, etc.) or critically ill patients with systemic inflammatory response syndrome (bacterial
sepsis vs. viral sepsis or non-infectious SIRS). In general, CRP levels above 40 – 50 mg/l are in-
dicative for bacterial infections and usually warrant the use of antibiotic therapy. One of the sev-
eral assays used for rapid CRP evaluation is the NycoCard CRP Single test (Fig. 5.7). It consists of
ies against the CRP. To measure the CRP levels, 5 μl of the fingerstick capillary blood is drawn
a simple laminated test device containing a porous membrane with bound monoclonal antibod-
liquid. 50 μl of the diluted sample is subsequently pipetted
into the capillary and then diluted in 0.4 ml of the dilution
to the test device and allowed to pass through the mem-
brane. Any CRP in the sample is trapped by membrane-
bound anti-CRP antibodies while the remaining components
of the blood sample are absorbed by the paper layer at the
bottom of the device. In the next step, one drop of a conju-
gate (liquid containing anti-CRP antibodies labelled with
gold particles) is applied to the test device. The binding of
gold-labelled anti-CRP antibodies to the CRP in the mem-
brane leads to the red-brown colour change of the membrane.
In the final step, one drop of
a washing solution is applied
to remove all unbound conju-
gate from the membrane. The
intensity of the colour change
is directly proportional to the
concentration of the CRP in
the sample and is read and
quantified with a reflectome-
ter (NycoCard READER II)
within 5 minutes (Fig. 5.8).
Fig. 5.7 NycoCard CRP Single
test with NycoCard Reader II
Fig. 5.8 Principle of NycoCard
CRP test
47
Laboratory Metods in Immunology
SUMMARY AT A GLANCE
•The innate humoral imunity comprises the complement system, acute phase proteins and various sol-
uble mediators of inflammation.
•The complement activation enables direct lysis of bacteria and supports chemotaxis, opsonisation and
removal of immune complexes. Therefore, deficiencies of individual complement factors may lead to
increased susceptibility to bacterial infections or immune-complex diseases.
•The functional integrity of whole complement activation pathways can be examined by several
haemolytic and non-haemolytic functional assays. Subsequently, quantitative and functional assays
for individual complement components must be performed to identify which complement component
is defect.
•Inflammation is a complex and non-specific immune reaction that develops in response to tissue injury
caused by infectious and non-infectious factors. Several soluble proteins play crucial roles in its devel-
opment and serve as excellent markers of its presence and intensity. The measurement of acute phase
proteins (e.g. C-reactive protein), procalcitonin or neopterin is an important diagnostic tool used to
monitor the intensity, progress and treatment response in various inflammatory conditions including
infections, malignancies, autoimmune and cardiovascular diseases. They are also helpful as non-spe-
cific markers for the differentiation of causative factors.
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Evaluation of innate cellular immunity
6. Evaluation of innate cellular immunity
6.1 Phagocytosis
6.1.1 The role of phagocytosis in the immune response
Phagocytosis is an evolutionary old, non-specific immune mechanism participating in the
first line of defence against invading microbes. It is a process in which specialised cells of the im-
mune system track down, engulf and destroy foreign cells and material as well as own damaged
or dead cells and extracellular matrix, and so participate in the clearance of microbes and the resti-
tution of the damaged tissues. The cells capable of phagocytosis belong to two distinct systems –
the myeloid system and the mononuclear-phagocytic system. The cells of the former system are also
referred to as granulocytes due to the presence of granules with antimicrobial substances in their
cytoplasm, or polymorphonuclears (PMNs) because of the multilubulated shape of their nuclei. The
most abundant cell population of the myeloid system are neutrophils – short-lived cells constitut-
ing about 55 % to 70 % of all white blood cells. Other granulocyte population capable of phagocy-
tosis are eosinophils, although their antimicrobial substances are better suited for extracellular
destruction of large parasites. The mononuclear-phagocytic system includes single cell population
called monocytes (immature cells circulating in the bloodstream) and macrophages present in var-
ious tissues and organs. In contrast to neutrophils, macrophages are longer living cells that partic-
ipate in subacute and chronic phases of inflammation and in addition to phagocytosis, they also
contribute to tissue restoration, wound healing, antigen presentation, cytokine production etc.
The process of phagocytosis can be divided into several phases:
1. Chemotaxis is a directed movement of leukocytes toward the concentration gradient of cer-
tain chemicals, produced in tissues damaged by external insults or as a result of microbial
invasion (formyl peptides of microbes, complement activation products, cytokines and
chemokines, leukotrienes and products of coagulation and fibrinolysis).
2. Opsonisation is a process of coating of microbes by macromolecules called opsonins which
help to neutralise the negative charge on the surface of bacteria and enable their firm adhe-
sion to the surface of phagocytic cells. Opsonins include complement fragments C3b, iC3b and
C4b, immunoglobulins IgG and IgM, mannose-binding lectin, C-reactive protein and others.
3. Following the binding of bacterium, the phagocyte starts to form pseudopods that finally en-
close the germ. Its engulfment (ingestion) results in the formation of a vesicle called the
phagosome. The phagosome subsequently fuses with granules (lysosomes) containing an-
timicrobial enzymes, forming the so called phagolysosome.
4. After the phagolysosmes are formed, various antimicrobial substances contained in the gran-
ules digest foreign material, destroy bacteria or inhibit their growth. These microbicidal
mechanisms include the production of lysosyme, acidic hydrolases, neutral proteases, lacto-
ferrin, bacterial permeability-increasing protein, cathelicidins, defensins and reactive oxygen
and nitrogen species.
5. Indigestible and waste material is finally discharged outside the phagocytic cell.
Because of the crucial role of phagocytosis in innate immune mechanisms, defects of phago-
49
Laboratory Metods in Immunology
cyte production or function often result in impaired protection from bacteria and fungi. Patients
with deficiencies of phagocytosis typically suffer from pyogenic or granulomatous bacterial
(Staphylococcus aureus, Serratia marcescens, Klebsiella sp., Salmonella sp., Escherichia coli, Pseudomonas
aeruginosa, etc.) and fungal (Candida sp., Aspergillus sp.) infections of the respiratory tract, lymph
nodes, skin, mucosa and visceral organs. Primary immunodeficiencies comprise diseases with ab-
normal phagocyte numbers (severe congenital neutropaenia, cyclic neutropaenia etc.) or impaired
ability to migrate, engulf or destroy foreign pathogens (leukocyte adhesion deficiencies, chronic
granulomatous disease, specific granule deficiency, myeloperoxidase deficiency and others). Their
correct diagnosis requires the evaluation of neutrophil and monocyte numbers and subsequent
functional studies on individuals steps and mechanisms of phagocytosis: chemotaxis, ingestion,
microbicidal activity and metabolic activation (respiratory burst).
6.1.2 Laboratory tests for the evaluation of phagocytosis
6.1.2.1 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, whereas the blood smear enables to detect their morphological abnormalities such as
the presence of large granules in neutrophils (Chediak-Higashi syndrome) or their absence (spe-
cific granule deficiency). Neutrophil numbers below 1500/μl are referred to as neutropaenia, num-
bers < 500/μl are the criterium for severe neutropaenia and are associated with a high risk of severe
bacterial infections. Neutropaenia is typically found in primary immunodeficiencies (severe con-
genital neutropaenias, cyclic neutropaenia, Kostmann’s syndrome, Shwachman-Diamond syn-
drome, etc.), but can also occur in malignancies, certain infections (tuberculosis, HIV, EBV, CMV,
hepatitis A/B/C), vitamin B12 and folic acid deficiencies, or as a consequence of an autoimmune
or drug-induced iatrogenic destruction of neutrophils. On the other hand, increased numbers of
neutrophils (neutrophilia) are typical for acute bacterial infections, but may also be found in other
acute inflammatory conditions (myocardial infarction, burns), leukocyte adhesion deficiencies
(LAD) and malignancies (chronic myelogenous leukaemia). Decreased numbers of eosinophils
(eosinopaenia) are usually present in hypercorticoid states (Cushing’s disease, administration of
corticosteroids, stress) and acute infections, whereas increased numbers of eosinophils
(eosinophilia) are indicative for allergic disorders, parasitic infections, certain malignancies and
myeloproliferative disorders (Hodgkin’s lymphoma, hypereosinophilic syndrome, solid tumours),
primary immunodeficiencies (hyper-IgE syndrome, Omenn syndrome, Wiskott-Aldrich syn-
drome) and autoimmune diseases (systemic lupus erythematosus, eosinophilic granulomatosis
with polyangiitis). Decreased monocyte count (monocytopaenia) is usually found in certain
leukaemias (e.g. hairy-cell leukaemia), aplastic anaemia or combined immunodeficiencies, while
increased numbers (monocytosis) may accompany several viral and bacterial infections (tubercu-
losis, brucellosis, listeriosis, etc.), autoimmune diseases and malignancies (chronic monocytic or
myelomonocytic leukaemia).
50
Evaluation of innate cellular immunity
6.1.2.2 Specimen collection and isolation of polymorphonuclears
Phagocytic cell function tests can be performed with either full heparinised blood or iso-
lated polymorphonuclear leukocytes (PMNs). The concentration of the heparin in the collection
tube should not be more than 10 IU per 1 ml of the blood as its excess would inhibit the function
of phagocytes. The use of other anticoagulant EDTA is not recommended as it chelates Ca2+ ions
which are necessary for the phagocytosis. Only fresh blood not older than 2 hours should be used
for functional tests. Alternatively, isolated PMNs can also be employed. In this case, 1.4 ml of 6%
dextran is added to 5 ml of fresh heparinized blood and mixed thoroughly. The mixture is then in-
cubated inclined at an angle of 45° for 20 – 25 minutes at 37 °C and then again for 15 – 20 minutes
in a vertical position. This allows erythrocytes and platelets to settle down at the bottom of the
tube while leukocytes remain suspended in the plasma above. The plasma with WBCs is subse-
quently carefully removed, transferred into a clean tube and centrifuged at 150 g for 10 minutes.
Supernatant is removed and the isolated cells forming the sediment (70 – 75 % of them are PMNs)
are repeatedly washed with Hanks' solution, diluted to a desired concentration and are ready for
use in functional tests (Fig. 6.1). Alternatively, plasma with leukocytes can be carefully layered on
the top of the Ficoll (Lymphoprep) solution and centrifuged. During the density gradient cen-
trifugation (Chapter 7.2.1), PMNs settle down at the bottom of the tube while mononuclear cells
(monocytes, lymphocytes) create a ring on the interface between plasma and Ficoll. PMNs are
then removed, washed and diluted to a desired concentration. Simultaneously, 5 ml of the blood
is also collected in a clean tube and allowed to coagulate. The clot is then removed and the serum
is used along with PMNs for the examination of ingestion and cidal activity.
Fig. 6.1 Isolation of polymorpho-
nuclears with dextran
6.1.2.3 Evaluation of chemotaxis
The successful active migration of phagocytes from the circulation to the infection site de-
pends upon the intact microfilamentous and microtubular system, the production of chemotactic
signals, the ability of phagocytic cells to respond to chemotaxins via their receptors (CXCR, CCR,
C5aR, C3aR, etc.), to adhere to endothelial cells (via selectins and integrins) and increase their
plasma membrane fluidity so that they can crawl between the cells of endothelial lining and migrate
51
Laboratory Metods in Immunology
through the vessel wall and interstitial tissues to the infectious site. 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 pres-
ent in certain diseases (diabetes mellitus, severe malnutrition, infections, malignancies). Although
a wide range of techniques is available to evaluate chemotactic activity of cells, these tests do not
belong to routinely used methods as they are laborious, time consuming, lack uniform standardi-
sation and their results show a high degree of variability. Hence, these assays are only performed
in certain specialized laboratories when a defect of chemotaxis is strongly suspected.
Laboratory methods for the evaluation of chemotactic activity include various chamber assays
and agar-plate assays. The latter use an agar gel cast on a polystyrene plate with three pre-cut holes
(Fig. 6.2). Examined patient’s PMNs are added into the middle hole whereas a rabbit serum activated
with zymozan (for the production of chemotactic stimuli) and a buffer medium are added into the
opposite outer holes. During the incubation at 37 °C the PMNs move radially from the central hole.
Their migration toward the hole with a medium is non-directed (random spontaneous locomotion)
while the migration toward the opposite hole containing activated serum is active and directed by
chemotaxins (chemotaxis). After 1 – 2 hours, the cells are fixed and stained, the length of both
chemotactic and spontaneous migration ways is measured and the chemotactic index is calculated
Defects of the expression of adhesive molecules β2-integrins (CD11a/CD18, CD11b/CD18,
(Fig. 6.2). Its decreased value indicates the presence of a chemotactic defect.
CD11c/CD18) and fucosylated carbohydrate ligand for selectins (sialyl-Lewis X) present in leuko-
cyte adhesion deficiency 1 and 2 (LAD1 and 2) syndromes can be examined by flow cytometry
technique using anti-CD18 and anti-CD15 (anti-sLex) monoclonal antibodies, respectively.
Fig. 6.2 Evaluation of chemotac-
tic activity of polymorphonuclears
6.1.2.4 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. The process of ingestion depends upon
intact opsonisation and adherence of microbes to phagocytes and subsequent formation of
pseudopods that encircle them. Hence, defects in the production of opsonins (complement factors,
immunoglobulins), expression of opsonin receptors (complement receptors, Fc receptors) and de-
formability of neutrophils may lead to impaired ability to ingest microorganism. Defects in the mi-
crobial uptake can appear in individuals with congenital complement and immunoglobulin
52
Evaluation of innate cellular immunity
deficiencies, tuftsin deficiency, leukocyte adhesion deficiency (LAD), actin dysfunction, liver in-
sufficiency and acquired hypogammaglobulinaemia (multiple myeloma, chronic lymphocytic
leukaemia). In addition, phagocytosis defects are often observed in severe infections (sepsis), chronic
systemic diseaes and malignancies or can be caused by drugs (corticosteroids, colchicine, salicilates).
Several tests and their modifications have been developed to evaluate phagocytic activity.
These can be performed with either full heparinised blood or isolated PMNs using various mi-
croorganisms (Candida albicans, Saccharomyces cerevisiae, Staphylococcus aureus, Escherichia coli) or
microspheric hydrophilic particles as a substrate. Frequently used functional method is the eval-
uation of phagocytic activity (PhA) and phagocytic index (PhI) of PMNs with C. albicans. In this test,
a suspension of patient's isolated PMNs is incubated with inactivated candida cells (ratio 1 : 5), au-
tologous serum (for opsonisation) and Hanks’ solution in a thermostat at 37 °C and constantly
shaken to avoid the aggregation of candida cells (Fig. 6.3). After 30 minutes, a smear is prepared
on a microscopic slide and then stained with Wright's stain. The sample is evaluated under the
light microscope and the numbers of phagocytosing PMNs (those with at least one C. albicans in
their cytoplasm) out of 100 – 200 PMNs as well as phagocytosed C. albicans cells are counted. Sub-
sequently, PhA and PhI values are calculated as follows:
N of phagocytosing PMNs N of phagocytosed C. albicans cells
PhA (%) = ––––––––––––––––––––––––– x 100 PhI = ––––––––––––––––––––––––––––
N of all PMNs N of phagocytosing PMNs
Phagocytic activity expresses the proportion of phagocytosing cells out of all PMNs and its
reference values are 77.1 +
– 8.8 %, while the PhI is the average number of microorganisms phago-
cytosed by one polymorphonuclear and its reference values are 3.6 +– 0.8. Gross defects of phago-
cytosis, usually observed in patients with primary phagocytic immunodeficiencies, sepsis and
terminal stages of malignancy, are associated with their decreased values. On rare occasions, the
discrimination between a defect in serum opsonisation (complement, immunoglobulin or MBL
deficiencies) and a cellular defect (e.g. actin dysfunction, lack of receptors) can be done by incu-
bating microorganisms and PMNs with autologous serum (from patient) and homologous serum
(from volunteers with blood
type AB) in two separate tu-
bes. Decreased PhA with au-
tologous serum and normal
PhA with homologous serum
indicates an opsonization de-
fect, whereas impaired phago-
cytosis with both sera hints at
a cellular defect.
Fig. 6.3 Evaluation of phago-
cytic activity and phagocytic index
of polymorphonuclears
53
Laboratory Metods in Immunology
Alternatively, the phagocytic activity may also be examined with full blood instead of iso-
lated PMNs or using microspheric hydrophilic 2-hydroxyethyl methacrylate (HEMA) particles as
a substrate. Their low negative charge limits non-specific adherence to cell surfaces and sponta-
neous self-aggregation. After the incubation of HEMA particles with full blood at 37 °C for 1 hour,
a smear is prepared, fixed, stained with Giemsa-Romanowski stain and evaluated under the mi-
croscope. Cells with 3 and more ingested microparticles are identified as phagocytosing. Subse-
quently, PhA and PhI are calculated.
Finally, the ingestion can also be evaluated by flow cytometric assays (Chapter 7.2.2.2) that
measure neutrophil-associated fluorescence after incubation of cells with microparticles or mi-
croorganisms labelled with pH-sensitive fluorogenic dyes. These dyes are non-fluorescent at neu-
tral pH but turn fluorescent upon ingestion and acidification in the phagolysosomes. This allows
discrimination between ingested particles/microbes and those only adhered to cells. The propor-
tion of phagocytosing PMNs (those producing fluorescence) out of all PMNs and mean fluores-
cence intensity of PMNs with phagocytosed microorganisms/microparticles, respectively, are
examined by flow cytometer and the phagocytic activity and phagocytic index are calculated. Al-
ternatively, conventional fluorescent dyes (e.g. fluorescein isothiocyanate) can also be used to label
bacteria, yeasts or microparticles. To discriminate between internalised and membrane-bound par-
ticles, trypan blue is added to quench surface-bound fluorescence.
6.1.2.5 Evaluation of microbicidal activity
Following their ingestion and the fusion of the phagosome with lysosomes, the internalised
microorganisms are finally destroyed within the phagolysosomes by several microbicidal mech-
anisms. Defects in their synthesis or the formation of phagocytic granules and phagolysosomes re-
sult in the impaired killing and increased survival of ingested microorganisms, and hence
increased susceptibility to infections. Defects of microbicidal activity are typically present in cer-
tain primary immunodeficiencies (Chédiak-Higashi syndrome, chronic granulomatous disease,
specific granule deficiency, myeloperoxidase deficiency, Papillon-LefŹvre syndrome, etc.), but also
often develop in patients with other underlying diseases (severe infections, sepsis, malignancies,
severe malnutrition, etc.). Therefore, the evaluation of microbicidal activity has its firm place in the
diagnostic protocol of numerous disorders associated with the development of reccurent pyogenic
or granulomatous bacterial or fungal infections of skin, mucosa and visceral organs.
Several functional tests for the examination of microbicidal mechanisms have been introduced
in the previous decades. Similarly to other phagocytosis assays, these can be performed with either
full heparinised blood or isolated PMNs using various vital microorganisms (C. albicans, S. aureus,
etc.). One of the most widely used tests is the microscopic evaluation of cidal activity (% of cidia) of
patient's PMNs with C. albicans (Fig. 6.4). Briefly, a suspension of patient’s isolated PMNs is incu-
bated with vital serum-resistant C. albicans strain (ratio 1 : 10), autologous serum (for opsonisation)
and Hanks’ solution at 37 °C for one hour, while continuously shaking. Following the incubation,
the PMNs are lysed with sodium deoxycholate and the released C. albicans are stained with the vital
dye methylene blue. This allows to discriminate between dead and viable candida cells, as the dye
is unable to penetrate viable cells leaving them unstained. On the other hand, dead cells are stained
blue as they are unable to prevent the methylene blue from penetrating their membrane. Subse-
54
Evaluation of innate cellular immunity
quently, a microscopic smear is prepared and evaluated under the light microscope and the num-
ber of dead candida cells out of all cells (usually 400) is counted. Simultaneously, a control reac-
tion with serum-resistant C. albicans, serum for opsonisation and Hanks’ solution, but without
patient's PMNs, is prepared and incubated in a separate control tube. This allows to evaluate a pro-
portion of candida cells that died spontaneously without the active contribution by PMNs. Finally,
the candidacidal activity (% of cidia) is calculated according to the following formula:
N of dead C. albicans in the sample – N of dead C. albicans in the control
% of cidia = –––––––––––––––––––––––––––––––––––––––––––––––––––––––– x 100
N of all C. albicans cells
Cidal activity (% of cidia) expresses the percentage of C. albicans cells killed by the direct ac-
tion of examined PMNs after one hour of incubation. Its values below 25 % indicate a possible
presence of a defect in one or more microbicidal mechanisms.
Fig. 6.4 Evaluation of microbici-
dal activity of polymorphonuclears
Alternatively, the candi-
dacidal activity can also be de-
termined by inoculating the
suspension of C. albicans cells
from sample and control tubes
on the Sabourad agar, instead
of staining the dead cells with
vital dye. Following the inoc-
ulation, the plates with the
agar are incubated at 37 °C for
48 hours and the numbers of
grown colonies from both sample and control tube are counted afterwards. Every grown colony cor-
responds with one viable C. albicans cell. The % of cidia is then calculated as follows:
N of grown colonies in the sample
% of cidia = (1 – ––––––––––––––––––––––––––––– ) x 100
N of grown colonies in the control
The microbicidal activity can also be evaluated from full heparinised blood, or using other vi-
able microorganisms as substrates (Staphylococcus aureus, Escherichia coli). Finally, flow cytometric
assays for the measurement of the microbicidal activity have also been developed in the last decades.
Similarly to chemotaxis tests, also assays for the evaluation of microbicidal activity are labo-
rious, time consuming and show a high degree of variability as various microbial species or strains
used in the testing often differ in their sensitivity/resistance to phagocytosis and microbicidal mech-
anisms. Therefore, the use of these assays in routine laboratory diagnostics is rather limited.
55
Laboratory Metods in Immunology Evaluation of innate cellular immunity

6.1.2.6 Evaluation of metabolic activation

The most potent microbicidal mechanism of human PMNs is the production of highly toxic
reactive oxygen species in a process called respiratory burst. Following the ingestion of microbes
and activation of phagocytes, the consumption of oxygen dramatically increases in order to produce
a free radical called superoxide anion (˙O2¯) and, subsequently, other reactive species including hy-
drogen peroxide (H2O2), hydroxyl radicals (OH¯), singlet oxygen (1O2) and other toxic molecules
such as HOCl, OCl¯ and Cl2. Hence, defects of the enzymes participating in this process may lead
to significantly impaired ability to destroy internalised pathogens. Probably the most archetypal ex-
ample of such immunodeficiency is the chronic granulomatous disease (CGD) caused by muta-
tions in genes encoding the subunits of the NADPH-oxidase. Affected individuals usually suffer
from recurrent catalase-positive bacterial and fungal infections including pneumonia, abscesses of
the skin and organs, skin infections or osteomyelitis. The proper diagnosis of this rare disorder re-
quires the examination of the metabolic activation (respiratory burst) of patient's PMNs upon stim-
ulation. Several assays have been developed to enable qualitative and quantitative evaluation of
oxygen radicals production using the light microscopy, spectrophotometry or flow cytometry.
The most widely-used qualitative assay is the nitroblue-tetrazolium (NBT) test based upon
the reduction of ingested colourless NBT by oxygen radicals to an insoluble purple formazan.
Briefly, isolated patient's PMNs in the test tube are stimulated with zymozan or phorbol myrisate
acetate (PMA) and incubated with a soluble NBT salt at 37 °C for 45 minutes, while continuously
shaking. A microscopic smear is then prepared and the proportion (percentage) of cells with for- Fig. 6.5 Evaluation of metabolic activation of polymorphonuclears by NBT (INT) test
mazan in their cytoplasm is evaluated and compared to that in a control tube containing the PMNs
that have not been stimulated with zymozan. The defect in oxygen radical production will mani- droethidine (HE). In addition, flow cytometric assays for the combined evaluation of ingestion
fest with absent or greatly decreased number of formazan-positive PMNs. Quantitative modifi- and oxidative burst have also been developed, using FITC-labelled microorganisms and fluore-
cations of the NBT test are performed in the wells of a microtiter plate using the NBT or its cence-producing ROS indicators. In comparison to the classical NBT test and its modifications,
analogues 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT test) or 3-(4, flow cytometric assays are quicker and easier to perform, more sensitive, more reliable, can be per-
5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT test). After the incubation of formed from small whole blood sample volumes without the need for PMNs separation and are
PMNs with a tetrazolium salt, the produced water-insoluble formazan is extracted from cells using suitable for the detection and discrimination of various forms of CGD. Finally, flow cytometry
a detergent. Following the centrifugation, the sediment is dissolved in a solubilisation solution assay can also be used for the detection of individual subunits of NADPH-oxidase in the PMNs
(acetone, ethanol, etc.) to produce a coloured solution. Its absorbance depends upon the amount using fluorescently-labelled monoclonal antibodies.
of produced formazan (and hence the metabolic activity of examined PMNs) and is measured by Measurement of the chemiluminescent activity is another possibility how to examine meta-
a spectrophotometer. The absorbance value of PMNs stimulated with zymozan is then divided by bolic activation and ROS production by polymorphonuclear leukocytes. Hydrogen peroxide
the value obtained in unstimulated PMNs to calculate the index of metabolic burst (IMB) (Fig. 6.5). (H2O2) produced during the respiratory burst from ˙O2¯ serves as a substrate for a subsequent pro-
Its significantly decreased value points at NADPH-oxidase dysfunction. duction of HOCl, OCl¯ and Cl2 catalysed by myeloperoxidase. The generation of electronically ex-
Other alternatives for the quantitative evaluation of oxygen radical production are the flow cited state and its subsequent return to baseline condition during the myeloperoxidase reaction
cytometric assays using various fluorescent probes as respiratory burst indicators. The commonly leads to the emission of a chemical light. This so called chemiluminescence can be further ampli-
used dihydrorhodamine 123 (DHR) test utilizes freely permeable dihydrorhodamine 123 as a fluo- fied by a substance luminol, which, when activated by H2O2, produces luminescence with a strik-
rescent indicator. Following its cultivation with phorbol myristate acetate-activated PMNs, DHR ing blue glow. Hence, following the incubation of PMNs with a stimulator (phorbol myrisate
123 penetrates into their mitochondria and is subsequently oxidised by hydrogen peroxide to rh acetate, zymozan) and luminol at 37 °C, the chemiluminescence is measured with a chemilumi-
damine 123. This leads to the emission of a bright red fluorescent signal upon excitation by light nometer and compared to that obtained in unstimulated PMNs. As the production of chemilu-
beams, which is then detected and measured in a flow cytometer to determine the proportion of minescence depends on intact function of both NADPH-oxidase and myeloperoxidase, this assay
ROS producing cells and mean intensity of fluorescence. Other fluoresent indicators frequently can be used not only to diagnose chronic granulomatous disease but also for the detection of
used in this type of assays include diacetate form of 2’,7’-dichlorofluorescein (DCFH) and hy- myeloperoxidase deficiency.

56 57
Laboratory Metods in Immunology
6.1.2.7 Evaluation of lysozyme levels and activity
Evaluation of the lysozyme is a laboratory test with a potential utilization in the diagnos-
tics of diseases associated with increased or decreased activity of mononuclear-phagocytic sys-
tem. Lysozyme is a ubiquitous hydrolytic enzyme present in cytoplasmic granules of phagocytes
as well as in blood, saliva, tears and other mucus secretions and tissues. It is produced by neu-
trophils and macrophages and released extracellularly to cleave 1–4-β linkages between N-acetyl-
D-glucosamine and N-acetylmuramic acid in peptidoglycan of Gram-positive and some
Gram-negative bacteria, thereby playing an important role in innate immune mechanisms.
Lysozyme levels often reflect functional activity of the mononuclear-phagocytic system and their
measurement can be helpful in the monitoring of certain diseases. Increased serum lysozyme lev-
els are common in diseases such as tuberculosis, sarcoidosis, Crohn’s disease, myelocytic and
myelomonocytic leukaemias, acute bacterial infections or kidney diseases associated with their
impaired function, while decreased levels can be found in individuals with impaired immunity
(immunosuppressive therapy, radiotherapy). The concentrations and activity of lysozyme can be
measured in a variety of ways:
1. The enzymatic methods are based on the ability of lysozyme to lyse cellular walls of bacteria.
In the diffusion method, 2% agar gel mixed with the suspension of Micrococcus lysodeikticus is
prepared and cast on the plate. The serum sample is then pipetted into the holes punched in
the gel along with standard solutions of the lysozyme with increasing concentrations used for
the calibration. The enzyme then diffuses out of a hole and shows its presence by producing
a ring that is cleared off intact cell walls. After one day, the diameter of the zone of lysis is
measured and the concentration of the lysozyme is read from a calibration curve (Fig. 6.6). In
other modification of the enzymatic assay, the spectrophotometric method, the tested serum sam-
ple and standard solutions are incubated with the suspension of micrococcus in the wells of
a microtiter plate. The lysis of a microbial substrate will manifest with a clarification of sus-
pension, the degree of which can be measured spectrophotometrically.
Fig. 6.6 Enzymatic method for evaluation of
lysozyme activity
2. The serologic (immunometric) methods are
based upon the use of specific antibodies against the lysozyme for its detection and meas-
urement. Included are radial immunodiffusion, rocket immunolectrophoresis and ELISA
(Chapters 3 and 4).
58
Evaluation of innate cellular immunity
6.2 Natural killer cells
6.2.1 The role of natural killer cells in non-specific immune responses
Natural killer (NK) cells are lymphoid cells that share a common lymphoid progenitor with
B and T cells but in contrast to them participate in the mechanisms of innate (non-specific) im-
lar lymphocytes (LGL) due to their size (diameter of 10 – 12 μm) and the presence of numerous
munity. They make up about 5 – 10 % of circulating lymphocytes and are defined as large granu-
azurophilic granules. Similarly to cytotoxic T cells, NK cells play crucial roles in immune responses
to virus-infected, tumour and allogeneic cells. In contrast to CTLs, their response is rapid and does
not require the recognition of viral or tumour antigens as NK cells do not possess antigen-specific
receptors. On contrary, NK cells are unique by their ability to recognise and respond to cellular
stress induced in the presence of a virus, malignancy or cell damage. This is possible due to the
presence of several membrane inhibitory and activating receptors that recognise diminished ex-
pression of self-markers of major histocompatibility complex (MHC) class I and appearance of ab-
normal forms of self surface molecules or stress-induced molecules (MIC, ULBP), respectively.
The activation of the NK cells leads to the release of preformed cytotoxic molecules (granzymes,
perforins) that destroy the target cells. Alternatively, an extrinsic pathway of apoptosis of a target
cell can be induced upon the activation of NK cell and the Fas–FasL interactions. Finally, the tar-
get cell can also be destroyed by the antibody-dependent cell-mediated cytotoxicity (ADCC) re-
action following the recognition of antibody-coated target cells by FcγRIII (CD16) receptors.
NK cell deficiencies can result from an abnormal production of NK cells or their impaired
function. Primary functional NK cell deficiencies are very rare and are usually caused by genetic
defects affecting NK cell receptors for target cell recognition (e.g. CD16), proximal signals for NK
cell activation (e.g. WASP, caspase 8, SAP) or NK cell cytotoxicity (perforin, cathepsin C, etc.). They
typically manifest with increased susceptibility to viral infections, particularly those of the her-
pesvirus and papillomavirus families. Secondary NK cell deficiencies may appear in patients with
malignancies and certain autoimmune diseases or as a consequence of chemotherapy and radio-
therapy. The presence of symptoms and signs suspicious for the NK cell deficiency is usually an
indication for the evaluation of NK cell numbers and activity.
6.2.2 Laboratory tests for the evaluation of NK cell cytotoxic activity
Following the enumeration of NK cell by flow cytometry, assays on their cytotoxic activity
can be performed. As these methods are laborious and suffer from great variability of results, their
utilisation in routine diagnostics is rather limited. Out of numerous available tests, the chromium
51
Cr-release assay is probably the most widely used test (Fig. 6.7). To assess the cytotoxic potential
of NK cells, a human erythrolaeukemic cell line K562 labelled with a radioactive chromium 51Cr
is used as a target. Following their incubation with patient's isolated NK cells at several different
ratios, the target cells are recognised by effector NK cells and subsequently lysed by their cytotoxic
molecules. The lysis of K562 cells results in the release of radioactive chromium into the super-
natant, which radioactivity is measured by a gamma counter and expressed in counts per minute
59
Laboratory Metods in Immunology Evaluation of innate cellular immunity

(cpm). The intensity of radioactivity depends upon the amount of 51Cr released from destroyed tar-
get cells and hence upon the cytotoxic activity of tested NK cells. Simultaneously, two control re-
actions are also performed: one with no effector NK cells to determine spontaneous release of
chromium (background) and the second with a detergent that lysis all target cells to determine
maximal release of 51Cr. The cytotoxic activity is then calculated as follows:
of cytotoxic molecules (perforin, granzymes) in NK cells and the expression of apoptotic markers
(annexin V) on target cells by fluorescently-labelled monoclonal antibodies have also been used in
the evaluation of NK cell function.

cpm probe – cpm background

Cytotoxic activity (%) = ––––––––––––––––––––––––––––––– x 100
SUMMARY AT A GLANCE
cpm maximum – cpm background
•Mechanisms of innate cellular imunity include phagocytosis (performed by polymorphonuclear leuko-
cytes and monocytes/macrophages) and destruction of compromised (virus-infected, tumour or dam-
aged) cells by natural killer cells.
Fig. 6.7 Chromium release
assay for evaluation of natural
•Phagocytosis is a process in which specialised cells of the immune system track down, engulf and de-
killer cell cytotoxic activity
stroy foreign cells and material as well as own damaged or dead cells and extracellular matrix.

•Impaired phagocytosis may result from defects in any of its phases. Therefore, several laboratory meth-
Due to the disadvanta-
ods for the evaluation of phagocyte numbers, chemotaxis, ingestion, cidal activity and respiratory
ges associated with the use of
burst were developed. The most common are the assays for the evaluation of ingestion (phagocytic ac-
radioctive marker, several non-
tivity and index) and microbicidal activity of polymorphonuclears and their ability to produce reactive
radioactive functional assays
oxygen species (NBT test, DHR test and their modifications).
employing fluorescent mark-
ers have been developed. The
•The numbers of NK cells can be determined by flow cytometry, while their activity and functional in-
markers used for the target
tegrity can be examined by several functional tests including the chromium release assay, non-radio-
cell labelling include carboxyfluorescein diacetate (CFDA), 2’, 7’-bis-(2-carboxyethyl)-5-(and-6)-
active assays and flow cytometry-based assays.
carboxyfluorescein (BCECF), rhodamine 123, calcein AM and others. The intensity of the fluores-
cence of the supernatant after the incubation of target cells with NK cells is measured by
fluorometer. Another assay uses the pale yellow 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT) salt to label the K562 cells. The tetrazolium salt penetrates the cell
membrane of target cells and is reduced by an intracellular reduction system of living cells into
a purple coloured formazan. The intensity of the colour change depends on the proportion of vi-
able cells and can be measured by spectrophotometer.
In the recent years, several modifications of flow cytometric assays have been introduced in
the diagnostics of NK cell deficiencies. In these assays, the target cells are labelled with a fluores-
cent dye to discriminate them from effector cells. After the incubation of isolated NK cells with la-
belled target K562 cells, the killed target cells are identified with a membrane impermeable
fluorescent DNA stain, which only penetrates through permeabilised plasma membranes of dead
cells but leaves the viable cells untouched. This allows for a clear discrimination between living and
dead target cells. The primary fluorescent dyes used to stain plasma membranes of target cells in-
clude fluorescein isothiocyanate (FITC), 5-(6)-carboxyfluorescein succinimidyl ester (CFSE), 3,3’-
dioctadecyloxacarbocyanine perchlorate (DiO) and others, whereas propidium iodide (PI) is
commonly used as the secondary fluorescent DNA-binding dye. The fluorescence from primary
and secondary dyes can be easily distinguished by flow cytometer, thereby enabling the determi-
nation of the percentage of dead and viable target cells. Recently, the flow cytometric measurement

60 61
Laboratory Metods in Immunology
7 Evaluation of adaptive immunity
Adaptive immune response, also known as specific immune response, is composed of hu-
moral and cellular components. In contrast to innate immunity, the adaptive immunity is evolu-
tionary younger, it develops during the whole life and possesses immune memory. The main
function of adaptive immunity is to recognize and remember specific antigens, leading to induc-
tion of strong response each time the antigen is entering our body. The principal cells of adaptive
immunity are lymphocytes. Humoral adaptive immunity is assured via antibodies produced by
plasma cells, the latest differentiation stage of B lymphocytes. The cellular adaptive immunity in-
volves subpopulations of T lymphocytes, namely helper, cytotoxic, regulatory and memory T lym-
phocytes. There are quantitative methods for evaluation of adaptive immunity components based
on serum immunoglobulins levels analyses (nephelometry, turbidimetry, ELISA) and counting of
lymphocytes (flow cytometry) in blood samples and qualitative methods based on functional eval-
uation of lymphocytes (blastic transformation assay, skin tests).
7.1 Adaptive humoral immunity components
Adaptive humoral immunity components involve antibodies produced by plasma cells. An-
tibodies also known as immunoglobulins are Y-shaped proteins that recognize and neutralize spe-
cific antigens. There are 5 classes of immunoglobulins, namely IgG, IgM, IgA, IgD and IgE differing
in their biological properties. B cells express a unique B cell receptor (BCR), which binds the spe-
cific antigen leading to their further differentiation to plasma cells. Plasma cells secrete specific an-
tibodies, which have different biologic functions as neutralization of toxins, enhancement of
phagocytosis, complement cascade activation and others.
7.1.1. Analysis of serum immunoglobulins levels
Detection of serum levels of IgG, IgM and IgA is usually performed by automatic analyzers
based on turbidimetry and nephelometry. Nephelometry and turbidimetry belong to serological
methods, which are used for antigen or antibodies detection (see Chapter 4.1). The analyzers are
especially helpful for examination of large amount of samples in huge central laboratories and the
advantage of these machines is that they can examine more than 80 various serum proteins in one
test. For detection of serum IgE and IgD levels, the sensitive enzyme immunoaassay methods (EIA)
are used because of their low serum concentrations. ELISA belongs to EIA methods and it is based
on antigen and antibodies detection in a liquid environment using enzymatic reaction (see Chap-
ter 4.2.2). ELISA and their modifications are typically used for detection of specific antibodies,
i.e. to viruses and bacteria.
7.2 Adaptive cellular immunity components
The principal cells of cellular adaptive immunity are lymphocytes. There are four main pop-
ulations of lymphocytes, namely B lymphocytes, T lymphocytes, NK cells and NKT cells. They
62
Evaluation of adaptive immunity
differ in function as well as in their membrane antigens (CD molecules). B lymphocytes express
CD19 and CD20 and after antigens recognition, they differentiate into plasma cells that secrete
specific antibodies to neutralize them. T lymphocytes are heterogeneous population of cells; we
recognize four subpopulations, namely helper, cytotoxic, regulatory and memory T cells. All T
cells express the same membrane molecule CD3. The main function of T helper cells (Th) is the ac-
tivation of other immune cells. Th cells help B cells to differentiate to plasma cells and by pro-
duction of cytokines they activate cytotoxic T cells and macrophages. Mature Th cells express CD4
molecules, which interact with HLA class II molecules on antigen presenting cells during their
cooperation. The CD4 molecule is also the main receptor for HIV entering into T helper cell. Cy-
totoxic T cells (Tc) express CD8 and are involved in destruction of virally-infected, malignant and
allogeneic cells by cytotoxic mechanism. CD8 interacts with HLA class I molecules present in the
membranes of all nucleated cells. Regulatory T cells (Treg) are involved in suppression of immune
response to self-antigens thereby preventing the autoimmune process. They express CD4, CD25
and Foxp3. Memory T cells develop faster and stronger immune response after the repeated ex-
posure to antigen. They are also called as antigen-experienced T cells. In medicine, we profit from
the ability of re-entering antigens to induce a strong immune response in vaccination praxis. NK
cells (natural killer cells) belong to a separate group of lymphocytes and are characterized by CD16
and CD56 markers. According to their morphology, they belong to large granular lymphocytes
(LGL). They are involved in destruction of virus infected and tumour cells by cytotoxic mecha-
nisms (secretion of granzymes and perforins or ADCC – antibody dependent cell cytotoxicity
mechanism). Finally, NKT cells are a subset of lymphocytes that express receptors of both, T cells
After Stimulation, they secrEt a large amo nt /f cytokines (IL-2, IFN-γ, TNF α IL-4) And Are
anD NK celLs
a,So invOlved in d%struction of Target cells by cytotoxic mechanisms. The method for analysis of
lymphocytes includes their separation by gradient centrifugation followed by flow cytometry. In
immune diagnostic, mainly the analysis of helper and cytotoxic T cells are performed by flow cy-
tometry. The functional evaluation of lymphocytes involves methods such as the lymphocyte trans-
formation assay and skin tests.
7.2.1 Isolation and counting of lymphocytes
Isolation of lymphocytes from the peripheral blood belongs to basic methods used in im-
munological testing. Isolated lymphocytes are mainly required for evaluation of their function in
vitro or for histocompatibility testing. They can also serve for further separation to lymphocytic
subpopulations (i.e. helper or cytotoxic T cells). Method of lymphocytes isolation was first re-
ported by Dr. Arne Bøyum in 1968 and is based on gradient centrifugation using a separation so-
lution (Ficoll-Hypaque, Lymphoprep). During centrifugation, blood cells are separated according
to their density differences into layers. The density of lymphocytes varies between 1,063 to 1,078
g/ml. The peripheral blood mononuclear cells (PBMC) including lymphocytes and monocytes,
and platelets are present on the top of separation solution (with a density 1,077 g/ml), because they
have lower density, in contrast to red blood cells and granulocytes, which have a higher density
and remain at the bottom of the tube (Fig. 7.1). Platelets can be separated apart from the mononu-
clear cells by subsequent washing with Hanks balanced salt solution following centrifugation. Out
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Laboratory Metods in Immunology
of mononuclear cells, lymphocytes account for about 80%, other cells belong to monocytes. The iso-
lation procedure of lymphocytes includes following steps:
1) Mix heparinised blood (use 10 units of heparin per 1 ml of blood) with equal volume of PBS
(phosphate-buffered saline).
2) Slowly layer diluted haparinised blood over Ficoll-Hypaque solution (with a density 1,077
g/ml) in the tube. Use 1 ml of separation solution per 2 ml of blood/PBS mixture (propor-
tion 1:2).
3) Centrifuge the tube 20 min. at 2000 rpm (900 g) at 20°C.
4) Using a pipette, remove PBMC cells seen as white layer over Ficoll-Hypaque solution to an-
other sterile tube.
5) Wash cells with excess of PBS and centrifuge 10 min. at 1300 rpm (400 g), repeat two- times.
6) Re-suspend mononuclear cells in the culture medium RPMI-1640, count cells and determine
viability using trypan-blue staining.
10 ml of freshly isolated blood usually contains 1−2.107mononuclear cells (80 % of them be-
longs to lymphocytes).
Fig. 7.1 Isolation of peripheral blood mononuclear cells
(PBMC) by gradient centrifugation
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 solu-
tion, whereas red cells and granulocytes are sedimented at the bottom of
the tube (modified from www. google. com /highered.mcgraw-hill.com).
7.2.1.1 Counting of lymphocytes in microscope
Cell counting belongs to basic quantitative method both in research and diagnostic labora-
tories. Central diagnostic laboratories usually use automatic analysers based on cell density analy-
sis by a spectrophotometry. The simplest method of cell counting uses a counting chamber and
a microscope. The most frequent used chamber is that of Burker counting chamber (Fig. 7.2). It is
a slide that consists of 2 grids fixed with cover glass. Each grid contains 9 large squares (each meas-
uring 1 x 1 mm, with the depth of chamber of 0.1 mm) with their corners bordered by tripled lines.
Each large square is divided into another 16 small squares, each 0,25 mm long and 0,25 mm wide.
The total volume of each large square is 0,1 mm3 or 10-4 cm3. To determine cell density, cells in 3
large squares should be counted under the microscope and the average number of cells per large
square should be determined. Finally, the cells density per 1 ml in a suspension will be counted
according following formula:
Cells per ml = Average count per square x dilution factor x 10,000 (volume factor based on
total volume calculation of each large square). For better cell counting, the cell suspension has to
be stained with vital dye trypan blue or Turk’s solution.
64
Evaluation of adaptive immunity
Dilute 20 μl of lymphocyte suspension with 380 μl of Turk’s solution (1:20). Nuclei of white
The determination of lymphocyte counts includes the following steps:
1)
blood cells stained by Turk's solution (gentian violet and acetic acid) are blue, whereas red
Mix well with a pipette, take out 12 μl of the suspension and fill both sides of the Burker
blood cells are destroyed by hypotonic environment.
2)
chamber
3) Using the microscope, count cells in 3 large squares bordered by tripled lines, omitting cells
lying on 2nd and 3rd lines
4) Calculate the average number of cells per a large square
5) Calculate the mean number of the other side of the counting chamber
5) Calculate the final concentration of cells using the above mentioned formula
An example: counted lymphocytes = 250, the mean number in 3 large squares = 83,3;
The final count per 1 ml = 83,3x2 (dilution factor) x10 000 (volume factor) = 1,66x106 cells/ml
Fig. 7.2 Bürker chamber
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
(modified from www. google. com/aquaculture.ugent.be).
7.2.2 Isolation and counting of lymphocyte subpopulations
Analysis of lymphocyte populations belongs to the most important methods used in the im-
munological diagnostic. Exact count analysis of T and B lymphocytes is used in the diagnostic of
primary as well as secondary immunodeficiencies (e.g. HIV-progression monitoring). Analysis of
lymphocytes populations also involves the immunophenotyping of leukemias and lymphomas.
Method for isolation and counting of lymphocyte populations is based on using fluores-
cently-labelled monoclonal antibodies (mAbs) which bind to their membrane molecules (desig-
nated as CD antigens). CDs are surface antigenic determinants found only on cells of a certain
lineage and at a particular development stage, e.g. monocytes express CD14, B-lymphocytes CD19
and CD20, all T lymphocytes express CD3, NK cells CD16 and CD56, etc. Population of CD3+ T
lymphocytes contains T helper subpopulation expressing CD4 and T cytotoxic subpopulation ex-
pressing CD8. Fluorescently-labelled cells are examined under fluorescence microscope and flow
cytometry (FACS). The principle of fluorescence is based on excitation of fluorochrome by a UV
light. The emitted light has a longer wavelength and is visible to the human eye. The most used
fluorochromes are FITC (fluorescein-isothiocyanate) with excitation wavelength 490 nm and emis-
sion wavelength 521 nm (emits green fluorescence), TRITC (tetrarhodamine-isothiocyanate) with
excitation wavelength 541 nm and emission wavelength 572 nm (emits red/orange fluorescence)
65
Laboratory Metods in Immunology
and PE (phycoerythrin) with excitation wavelength 565 nm and emission wavelength 573 nm
(emits orange-yellow light).
7.2.2.1 Monoclonal antibodies
Monoclonal antibodies (mAbs) are monospecific, i.e. they recognize only a single antigenic
epitope, having an identical primary structure and effector functions. They are produced by a sin-
gle plasma cell clone and represent only one Ig isotype. mAbs are mainly produced in vitro by
a hybridoma technology based on fusion of myeloma cells with spleen lymphocytes. Spleen cells
are obtained from mice previously immunized with a desired antigen. The cells are mixed with
PEG (polyethelenglycol) and then cultured in a selective HAT medium (hypoxanthine,
aminopterin, thymidine); only the fused cells can grow in this medium. Myeloma cells cannot
grow because they lack HGPRT, an enzyme needed for nucleic acid synthesis. However, the fused
cells can utilize the enzyme because it is supplied by the mice spleen cells (Fig. 7.3). Normal spleen
cells also cannot grow because they have a limited life-span. The fused cells are diluted and cul-
tured individually in microtitre plates; the cell will multiply to form a clone – a hybridoma. Anti-
bodies, which are produced by every hybridoma clone are analysed using ELISA or dot blot
techniques. The most producing and stable clone is selected for further processing. Another
method for production of mAbs includes injection of hybridomas into the peritoneal cavity of
a mouse. The hybridoma will grow in a tumor secreting an ascites fluid rich for mAbs. The anti-
bodies are then purified from the culture medium or ascites fluid by filtration and affinity chro-
matography. According to a production method, many types of mAbs were prepared until now.
Recombinant mAbs are produced by cloning of immunoglobulin genes using viruses (phage dis-
play) or yeast (yeast display), chimeric mAbs are prepared by recombination of mouse and human
genes. Nowadays, the fully humanized mAbs have been prepared by transgenic mice and phage
display technology to avoid the side effects of recombinant and chimeric mAbs. The application
of mAbs is wide including diagnostic as well as disease therapy. In diagnostic they are used for in-
stance in phenotyping of leukemia and lymphomas
by imunohistochemistry, immunofluorescence or
flow cytometry. In the therapy, mAbs are used to
treat cancer, autoimmune and inflammatory dis-
eases, transplant rejections, etc.
Fig. 7.3 Production of monoclonal antibodies
Monoclonal antibodies are produced by the hybridoma technology based on
fusion of myeloma cells with spleen lymphocytes. Spleen cells are obtained
from mice immunized with the desired antigen. The cells are mixed and then
cultured in selective HAT medium (containing hypoxanthine, aminopterin
and thymidine). Only the fused cells (hybridomas) are able to grow in this
medium. The most producing and stable hybridoma clone is selected for an-
tibody production (modified from www.google.com /bio.davidson.edu).
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Evaluation of adaptive immunity
7.2.2.2 Isolation and analysis of lymphocyte subpopulations by flow cytometry
FACS (fluorescence-activated cell sorting) is a flow cytometry technique based on immunofluo-
rescence. This method is widely used in research as well as in medical diagnostics. FACS is used for
fast multiparametric analysis of cells in body fluids or cell mixtures. It enables to detect, analyze and
separate different cell types due to their phenotype (size, granularity, surface molecules) in one step.
This method uses fluorescently labelled monoclonal antibodies specific for cell surface molecules (CD
antigens) and is performed by the means of flow cytometers (Fig. 7.4). The main components of a flow
cytometer are: a flow cell – liquid stream unit that carries the fluid with cells, an optical system that
consists of lamps and lasers, detectors which analyze FSC (forward scatter) and SSC (side scatter) sig-
nals as well as fluorescence, an amplification system (for signal amplification) and finally a computer
for analysis of the signals. The principle of this method relies on hydrodynamic cell focusing. The
first step of this method includes incubation of cell suspension with fluorescently labelled monoclonal
antibodies by a room temperature. Then the prepared cell suspension is inserted into a flow-cell unit
of the flow cytometer that creates a thin stream of liquid. A vibrating mechanism causes that the liq-
uid stream is splitting into drops, thus each drop contains one cell. Each drop (cell) then passes indi-
vidually through the laser beam (usually laser light) following analysis of emitting and scattered lights
by several detectors. There are usually three main types of detectors involving multiparametric cell
analysis: forward scatter (FSC) detector measures light reflection and determines cell size, side scat-
ter (SSC) detector measures a scattered light and determines cell granularity and finally a fluores-
cence detector evaluates fluorescence of used mAb and determines cell type. The signals from each
detector are digitized and passed to a computer for result analysis. The present flow cytometers are
capable of analyzing up to 13 parameters (forward scatter, side scatter, 11 colours of immunofluores-
cence) per cell and up to 100,000 cells per second (multiparametric analysis). Some flow cytometers
can also sort cells into different populations according to their fluorescence. In this case, just before
the stream breaks into droplets, they pass through electrical charging ring that provides a charge to
a desired fluorescently labelled cell in the droplet, e.g. the CD4/FITC labelled cell. The droplets then
fall through an electromagnetic sys-
tem that divides droplets according
to their charge into tubes. The sepa-
rated cells, e.g. CD4/FITC labelled,
cells can be used for further analysis.
Fig. 7.4 Flow cytometer
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
(modified from www.google.com/mpi-bremen.de).
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Laboratory Metods in Immunology
The data obtained by the flow cytometer can be displayed as a count or a graph. Two types
of graphs can be generated: one-dimensional histogram that is plotting one parameter, e.g. num-
ber of fluorescently labelled cells of interest (Fig. 7.5) and two dimensional dot plot that is plotting
two or more parameters, e.g. forward vs. side scatter, single colour vs. side scatter and two-colour
fluorescence data. Combinations from forward and side scatter data are usually used for the blood
cell sorting into lymphocytes, monocytes, and
granulocytes and determination of the differen-
tial blood count (Fig. 7.6). Each dot determines one
cell on a dot plot graph. Finally, the regions on the
dot plots can be separated and individually
analysed. This selection is termed “gating”. Thus
we can create a gate around the lymphocytes to
analyze only this cell population (Fig. 7.7).
Fig. 7.5 One – dimensional histogram
Histogram shows the count of CD8+ cells labelled with PE (dark line)
in comparison to non-labelled cells (grey line),
(modified from www.google. com/scbt.com).
Fig. 7.6 Dot plot graph sorting by cell size
Dot plot of data combinations from forward (FS) and side scatter (SC)
shows blood cell sorting into lymphocytes, monocytes and granulocytes
(modified from www.google.com/acb.sagepub.com).
Fig. 7.7 Dot plot graph sorting the cells by fluorescence
Dot plot graph sorting of T helper CD4 lymphocytes labelled with PE
(lower right quadrant, D4) and T cytotoxic CD8 lymphocytes labelled with
FITC (upper left quadrant, D1). Unlabelled cells are present in the lower
left quadrant (D2) (modified from www.google.com/furukawa.co.jp).
68
Evaluation of adaptive immunity
7.2.2.3 Application of flow cytometry in immunodiagnostic
Flow cytometry is widely used in research as well as in diagnostic. Analysis of lymphocyte
subpopulations in patient’s blood samples is used in the diagnostic of primary and secondary im-
munodeficiencies. In HIV-infected individuals (secondary immunodeficiency of CD4 cells)
CD4/CD8 ratio counting is widely used for monitoring of a disease progression and a response
to treatment. The ratio is decreased in HIV-infected individuals (< 0.5), but increased in patients
with autoimmune diseases (> 2.5). Flow cytometry is also used for immunophenotyping of
leukemias and lymphomas. It is very effective in distinguishing myeloid and lymphoid lineages
in acute leukemias and minimally differentiated leukemias. An example is expression of CD10,
which is common in acute lymphoblastic leukemia (ALL) patients. Clinical applications of flow cy-
tometry in organ transplantation include pre-transplant cross-matching, HLA-antibody screen-
ing and post-transplantation antibody monitoring. In the bone marrow transplantation, the count
analysis of CD34+ hematopoietic stem cells in the peripheral blood or bone marrow graft is very
important and correlates with engraftment success and its recovery. Antigen deficiencies in leuko-
cytes can be also assessed by flow cytometry, e.g. decreased expression of neutrophil adhesion
molecules is a cause of leukocytes adhesion deficiency (LAD1 syndrome). The disorder is charac-
terized by defective neutrophil and monocyte migration to the sites of inflammation following re-
current bacterial infections. Innate immune mechanisms of phagocytosis, such as opsonisation,
adhesion and oxidative burst can be also assessed by flow cytometry. Another application of flow
cytometry includes HLA-typing as a part of autoimmune diseases monitoring. An example is ex-
pression of HLA-B27 in patients suffering from Bechterev disease or HLA-DR4 in patients with
rheumatoid arthritis. Finally, the functional tests including lymphocyte transformation assay and
NK cell cytotoxicity testing can be provided by flow cytometry too.
7.2.2.4 Isolation of lymphocytes subpopulations by magnetic beads
Another way for isolation of lymphocyte subpopulations, except flow cytometry, is using
monoclonal antibodies labelled by magnetic beads. Monoclonal antibodies are binding to antigens
present in the membrane (CD molecules) of an immune cell following isolation of the individual
immune cell populations (T and B lymphocytes and their subpopulations, NK cells, dendritic cells,
etc.) using a magnetic field. To isolate T helper lymphocytes, monoclonal antibodies against CD4
molecule are used. First step of their isolation includes separation of mononuclear cells from the
whole blood by gradient centrifugation (see Chapter 7.2.1). Mononuclear cells are then mixed with
anti CD4 mAb labelled with magnetic beads and incubated 15 min. at 4°C. After incubation, the col-
umn with the labelled suspension of cells is placed into a magnetic device (e.g. MACS – magnetic
cell sorter provided by Miltenyi Biotec). In this step, only the cells labelled with magnetic mono-
clonal antibodies stay in the column, whereas non-labelled cells flow through (Fig. 7.8). Afterwards,
the isolated cell subpopulation is prepared for a further analysis.
The cells can be separated positively or negatively. In the positive selection, cells of interest
are usually labelled by one mAb and stayed in a column, while other cells (not expressing desired
CD molecules) flow through. After the column is removed, the labelled cells are eluted to a new tube
and collected. The positive selection is suitable for the isolation of particular cell type such as T helper
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Laboratory Metods in Immunology
(CD3+CD4+CD8-) or T cytotoxic cells (CD3+CD4-CD8+). In the negative selection, unwanted cells are
labelled by mix of mAbs and attached to the column. In this case cells of interest flow through, they
are collected and prepared for the further analysis. The negative selection can be used for NK cell iso-
lation (CD3-CD16+CD56+) or T lymphocytes isolation (CD3+). In the case of T cells isolation, mix of
mAbs binding to CD14, CD16, CD19, CD20, CD36, CD56, CD66b, and CD123 can be used.
In clinical medicine, the magnetic cell isolation is used in transplantation immunology and
oncology. It is known that positive graft outcome relies on graft matches between a donor and
a recipient. In patients who have undergone the peripheral blood stem cell transplantation, the
graft versus host disease (GvHD) is the most serious complication resulting in the graft rejection.
To reduce a contamination of the blood by donors T lymphocytes (inducing immune reactions =
rejection), the method of magnetic cell sorting is now used. By the positive selection, CD34+ pro-
genitor cells can be obtained and CD3+ T cells by the negative selection (the method is known as T
cell depletion). Using this procedure, a patient receives a pure population of CD34+ progenitor
cells what results in a reduced probability of a graft failure. The most used magnetic sorters are
CliniMacs by Miltenyi Biotec (Germany) and Dynabeads by Dynal Biotech (Norway).
Fig. 7.8 Cell separation by magnetic beads
After incubation of cell suspension with magnetic-labelled monoclonal anti-
bodies (CD4+ labelled), the column with cell suspension is placed into a mag-
netic device (MACS – magnetic cell sorter). Only the cells labelled with
magnetic monoclonal antibodies stay in the column, whereas non-labelled
cells flow through (modified from www.google.com/springerimages.com).
7.2.2.5 Isolation of B and T lymphocytes by other methods
Isolation of B and T lymphocytes by rosettes belongs to an old separation technique. Now
it is occasionally used in the research and was replaced by flow cytometry and magnetic cell sort-
ing. The principle of this method relies on the reaction between surface protein of the lymphocyte
and its ligand on red blood cells (RBC) originating from other species. RBCs bind to the cell and
form a cluster that looks like flower (rosette). In general, the rosette is defined as one that binds
three or more RBCs. For the lymphocyte separation, two types of rosettes can be used. E rosette is
used for T cells isolation and is formed after interaction of T cell CD2 molecule and its partner
LFA3 (CD58) present only on the surface of sheep red blood cells. Red blood cells from other
species cannot be used in this type of rosetting. For B cells isolation, M rosette formation is used.
70
Evaluation of adaptive immunity
In this case, B lymphocyte binds mouse RBCs. To perform rosette technique, lymphocytes isolated
by gradient centrifugation (1.107) are mixed with 1% suspension of sheep RBCs and incubated 30
min. at 4 °C. After centrifugation, the cell sediment is stained and rosettes are counted under a mi-
croscope. In general, the rosettes number determines the lymphocytes number. There are two types
of rosettes according to the rate of formation. The active rosettes are formed in the presence of low
number of RBC (1 Ly : 8 RBCs) within 15 min, whereas the total rosettes are formed in excess of
RBCs (1 : 25–50) within long time period (18 hrs.). It was shown that the active rosettes define pop-
ulation of active T lymphocytes with the major clinical significance as they serve as a sign of indu-
ced cell-mediated immunity.
Isolation of B lymphocytes by cotton is an another old separation technique. The first step of
this method includes separation of mononuclear cells from the whole blood by gradient centrifu-
gation. These cells are subsequently let to pass through a column containing cotton wool. Only B
cells bind to the column (because of the spikes in their membrane), whereas other cells flow through.
Another separation method uses SEPHADEX column with bounded protein A. When mononu-
clear cells pass through the column, only B cells, which express surface Ig receptor will stay in the
column, whereas other cells flow through (protein A binds to Fc-fragment of the receptor).
7.2.3 Lymphocyte transformation test
Lymphocyte transformation test (LTT) or blastic transformation assay belongs to methods
used to evaluate lymphocyte function in vitro. The principle of the test is based on lymphocytes re-
sponse to mitogens that induces non-specific cell proliferation and formation of blasts. Blasts are
cells in the mitogenic stage of their division into two daughter cells. The most used mitogens are:
phytohemagglutinin (PhA) and concanavalin A (ConA), which induce proliferation of T lympho-
cytes, protein A (P.A.), which induces proliferation of B lymphocytes, and pokeweed mitogen
(PwM), which activates both B and T cells. For a detection of a specific sensitization, the mitogen
is added in increasing concentrations (typically a three- or ten-fold stepwise increase). Following
incubation, the degree of cell proliferation is measured radioactively by adding of radiolabelled 3H-
thymidine (tritiated thymidine). Thymidine incorporates into newly synthesized DNA according
thymidine. Samples are measured using β-scintillation counter and the results are expressed in
to their proliferation rate. The higher the cell proliferation, the higher is the amount of incorporated
counts per minute (cpm). To avoid working with a radioactive material, new approaches of cell pro-
liferation analysis can be used such measuring of bromodeoxyuridine (BrDU) by ELISA or by
FACS. Bromodeoxyuridine also belongs to DNA-binding substances. In this case, the anti-BrDU
antibody labelled with peroxidase or fluorochrome is used and its intensity is measured.
The general procedure of LTT includes the following steps:
1) Isolate lymphocytes from peripheral venous heparinised blood by a gradient centrifugation
(e.g. a Ficoll – Hypaque solution). Lymphocytes should be isolated within 24 hrs after the
blood was drown.
Dilute mitogens to concentrations 1 µg, 2 µg and 5 µg/50µl, respectively.
2) Dilute isolated lymphocytes to concentration 1.106/ml.
Add cells and mitogens into wells of microwell plates (1.105cell/well, 50 µl/well) according
3)
4)
to a schedule, control wells contain only medium without mitogens
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Laboratory Metods in Immunology
Add 3H thymidine (40 kBq/20 µl/well) and cultivate for another 6 or 18 hrs
5) Cultivate in a culture medium in the 5% CO2 atmosphere at 37 °C for 3 days
6)
7) Precipitate cells onto a filtrate paper using a precipitation device
8) Dry filters at room temperature, trim off and put into the scintillation vessels, add scintilla-
Measure the radioactivity using a β-scintillation counter.
tion fluid and enclose
9)
The radioactivity is expressed as counts per minute (cpm) and the stimulation index (SI) is cal-
culated according to the following formula:
SI = cpm of mitogen-stimulated samples / cpm of control (non-stimulated) samples.
Values SI over 2.0 are considered as positive, i.e. antigens (mitogens) induced proliferation
of analysed lymphocytes (lymphocytes were sensitized by testing antigen).
LTT is used in the research as well as in diagnostics. In routine medicine, LTT is mostly used
in the diagnosis of allergies (food or drug-induced). In this case, lymphocytes obtained from an in-
vestigated person are exposed to allergens instead mitogens. The most tested drugs are gold salts,
amalgam, HgCl2, Ni, and Be. However, LTT measures only the sensitization of lymphocytes but
not an effector reaction. Thus LTT can be positive due to allergy to the tested substance. However,
clinical symptoms must be always considered to establish the diagnosis. To verify the final diag-
nosis of allergy, other tests should be also performed (as the IgE tests and skin tests).
7.2.4 Evaluation of the lymphocyte function in vitro – ELISPOT
ELISPOT (Enzyme-linked Immunospot Assay) belongs to modern methods used for evaluation
of the lymphocyte function. This method enables to evaluate the ability of B lymphocytes to pro-
duce antigen-specific antibodies or T lymphocytes to produce cytokines. ELISPOT is based on the
sandwich ELISA technique as described also in Chapter 4.2.2.5. To perform the analysis, a special
microplate with bound antibodies (either monoclonal or polyclonal) on their bottom is needed. The
antibodies are chosen according to the specificity of a supposed secreted product. The first step of
the method involves culture of isolated PBMC cells with a specific antigen or mitogen in a hu-
midified 37 °C CO2 incubator for 6 to 24 hrs. During this period, the secreted product binds to the
antibody on the nitrocellulose or PVDF-membrane. Following cell lysis and plate washing the en-
zyme-labelled antibody, which recognizes the specific epitope of the analysed product (e.g. cy-
tokine), is added. After washing, the secreted product is visualized by a substrate solution. The
coloured end product forms a spot on the membrane representing an individual cytokine-pro-
ducing cell. The number and size of spots is evaluated either manually under a microscope or by
using an automated plate reader – ELISPOT reader. The number of spots represents the number
of cytokine-producing cells in the analysed sample. The ELISPOT assay is primary used in im-
munology research; the blastic transformation assay is more widely used in immunodiagnostic.
7.2.5 Evaluation of the lymphocytes function in vivo – immunoskin tests
The lymphocytes function in vivo can be evaluated by immunoskin tests. The tests examine
the delayed type of hypersensitivity reaction; thus the cell-mediated immune response is analysed
(T-cell response to foreign antigens). The testing is based on injection of tested antigens into the
72
Evaluation of adaptive immunity
dermis of the forearm. As tested substances are used killed bacteria, their proteins or allergens
(see Chapter 8.1.1). The mostly used bacterial proteins are tuberculin and candidin. The applica-
tion of tuberculin is used for tuberculosis diagnosis and is known as the Mantoux screening test.
After 48 to 72 hours, the immune response to antigens is evaluated as induration and erythema.
These symptoms are caused by increased activity of macrophages, dendritic cells, and T cells in
the site of antigen injection. The diameter of induration is measured in mm and the reaction is
classified as following: induration between 6 to 10 mm is recorded as “+”, between 11 to 15 mm as
“++”, between 16 to 20 mm as “+++” and induration up to 20 mm is recorded as “++++”. Finally,
the skin test index is counted using following formula: the total number of crosses/the number of
tested antigens. If the index value is higher than 1, the test is positive, i.e. the person developed cell-
mediated immune response to the tested antigen. Otherwise, if the value is lower than 1, the per-
son has a decreased activity of cell mediated immune response and in the case of the Mantoux test
it means that tuberculosis (TB) vaccination of the individual is required. High positivity test value
(number of crosses up to 4) can indicate a latent TB infection; however, to confirm the diagnosis,
other tests have to be performed. One of the most widely used tests is Quantiferon TB-Gold test.
The test evaluates production of IFN-γ by patients T-lymphocytes that were sensibilized by My-
cobacterium tuberculosis.
SUMMARY AT A GLANCE
•Adaptive immune response, also known as specific immune response, is composed of humoral (anti-
bodies) and cellular (T and B lymphocytes) components.
•To evaluate serum immunoglobulins levels (specific humoral imunity), methods like nephelometry, tur-
bidimetry and ELISA are performed.
•For quantitative analysis of lymphocytes (specific cellular imunity), their separation by gradient cen-
trifugation (using Ficoll-Hypaque separation solution) followed by flow cytometry is performed. Flow cy-
tometer 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).
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Laboratory Metods in Immunology Diagnostics of allergies

8. Diagnostics of allergies Source of allergen Allergen nomenclature Major allergen

Woody plants Birch – Betula verrucosa Bet v 1
The term allergy is used to identify hypersensitivity reactions caused by the excessive de- Alder – Alnus glutinosa Aln g 1
granulation of mast cells and basophils induced by a specific allergen, against which a hypersen- Hazel – Corylus avellana Cor a 1
sitive individual produces IgE antibodies. The most common allergic disorders are allergic Ash – Fraxinus excelsior Fra e 1
rhinoconjunctivitis, allergic bronchial asthma, and atopic dermatitis caused by the inhaled allergen
Grasses Ryegrass – Lolium perenne Lol p 1
pollens of grasses and birch trees. An increasing number of patients with allergic manifestations
Meadow grass – Poa pratensis Poa p 1
are allergic to weed pollens – ragweed and sagebrush, as well as mites that cause year-around Cat's tail grass – Phleum pratense Phl p 1
symptoms. Increasingly there are also food and drug allergies. Cereal rye – Secale cereale Sec c 1

8.1 Allergy assessment Weeds Wormwood – Artemisia vulgaris Art v 1

Ragweed – Ambrosia artemisiifolia Amb a 1

The basis of allergy assessment is the history and nature of symptoms, especially the analy- Moulds Alternaria alternata Alt a 1
sis of triggering factors such as the season, the impact of external factors, home and working en- Cladosporium herbarum Cla h 2
vironment as well as contact with animals. Aspergillus fumigatus Asp f 1

Mites Dermatophagoides pteronyssinus Der p 1

8.1.1 Skin tests
Dermatophagoides farinae Der f 11

The diagnosis of the early type of allergy and thus also the identification of the causative Cat Felis domesticus Fel d 1
allergen is usually made by skin tests. Skin allergy testing is based on provoking a small, con-
Dog Canis familiaris Can f 1
trolled, allergic response by introducing a microscopic amount of an allergen to the patient’s skin
by various means.
Tab. 8.1 The most commonly used allergens in diagnosis of allergy
8.1.1.1 Prick tests

Prick test involves the injection of allergen extract into the skin (epidermis), resulting in its bind-
ing to the membrane IgE of mast cells, causing their degranulation. The release of histamine and
other vasoactive substances from mast cells results in individuals sensitive to the particular allergen
in erythema and induration with pruritus at the application site within 20 minutes of application.
The choice of tested allergens depends on the geographic area, but also on a patient’s his-
tory. In a patient with suspected allergy, we almost always apply the pollen allergens of birch
trees (birch, alder, hazel), a mixture of grass and cereal pollens, ragweed, mugwort, dust mites,
dog epithelium and cat saliva (Table 8.1). The allergens are applied to the skin of the volar aspect
of the forearm, where the skin to which a drop of allergen is applied is cleansed and dried,
whereupon the skin is pierced by a lance to apply it to the epidermis (about 1 mm deep) where
mast cells are present. In addition to the selected allergens, negative and positive controls are
also applied. As negative controls, a solution which is used to dissolve the allergens is used,
and as a positive control we use histamine, resulting in reddness and wheal in each individual.
The reaction is assessed after 20 minutes. A positive reaction is when the size of the wheal is
3 mm greater compared to the negative control. Based on the results of the skin tests, as well as
the clinical manifestations, the physician reaches a final diagnosis and chooses the appropriate
treatment.
8.1.1.2 Prick to Prick test
The Prick to Prick test is a modification of the standard Prick test used almost exclusive-
ly in the diagnosis of food allergy, where no commercially available test allergen concentrates
are used.
Prick to prick is also performed on the volar forearm, with the needle being first inserted in
the fruit or vegetable, trying to have a minimum quantity of tested food on the needle tip. The
needle is then applied directly to the skin to test for the food allergen injected to the mast cells in
the skin. The skin reaction, wheal and erythema, is assessed also after 20 minutes – the same as in
the prick tests. Prick to prick is limited to foods (fruit and vegetables) with higher water content.
Unlike standardized tests, these tests are less specific.
The most common food allergies are caused by milk, eggs, peanuts, apple, kiwi, etc. Food al-
lergies may occur frequently as oral allergy syndrome (swelling of the tongue and gums), diarrhea,
or rash, anaphylaxis is relatively rare. Food allergy is often masked by the cross-reactivity of al-
lergens. Patients hypersensitive for example to birch pollen allergens may be allergic for example
to kiwi, pear, tomato, celery. Cross-reacting allergens are also found among grasses and food al-
lergens in wheat flour, also in ragweed and melon, etc.

74 75
Laboratory Metods in Immunology
8.1.1.3 Intradermal skin test
Intradermal skin tests are relatively rarely used, almost exclusively for the diagnosis of drug
allergy. Only injectable forms of drugs are used for intracutaneous testing, i.e. liquids not forming
a suspension. Intracutaneous tests are used particularly to test for hypersensitivity to anesthetics
like Lidocaine, Mesocain, or to some antibiotics as Penicillin_G. Hospitalized patients on a strict
diet are most commonly applied with an intracutaneous placebo solution (saline solution) and
5 cm from it the tested solution – in the area between the neck and shoulder. The reaction is as-
sessed after 15 minutes and then 8 hours after being administered. The next day, another drug test
(such as procaine penicillin) may be used on another part of the back.
Drug allergy may be caused by various immunopathological mechanisms, and often it can
be masked by adverse drug reaction or photo-toxicity. Intradermal skin tests are also used in the
assessment of specific immune responses.
8.1.1.4 Epicutaneous “patch” test
These tests are used to demonstrate type IV hypersensitivity, which is clinically important in
allergic contact dermatitis. This hypersensitivity results from sensitization (through the skin, but
also orally) by different chemical compounds that usually cause hypersensitive erythematous to
papulomatous skin changes in 2 to 3 days. The most common is the allergic contact dermatitis
caused in susceptible individuals by compounds of chromium or nickel metal contained, for ex-
ample, in jewelry and bone implants. But increasing numbers of people, especially women, are also
allergic to the components of various perfumes.
Allergens are applied to the skin of the back in commercially prepared patches. Individual
patches contain mixtures of various chemicals, such as metals (potassium-chromate, cobalt-chlo-
ride, nickelsulphate) components of perfumes and cosmetics (Fragrance_mix, Quaternium-15,
Kathon_CG), rubber components (Thiuram_mix, IPPD, Merkapto_mix, Rosin), certain drugs
(neomycin, Benzocaine, Budesonide), formaldehyde, lanolin and others.
These patches are left on the back for two days; thereafter they are peeled off and assessed.
The skin symptoms are assessed after removal of the patch, i.e. on the third day after application.
The skin is assessed for the presence of erythema, papules, vesicles or bullae. Patients who had
a positive patch test are recommended to avoid the substance for the rest of their lives.
8.1.2 Laboratory assessments
The development of new laboratory methods has made the diagnosis of allergies without ad-
accompanied by some non-specific laboratory markers, such as eosinophilia (counts above 0.3 × 109/l)
ditional laboratory assessments something that we cannot even imagine. Allergic disorders are often
and elevation of overall IgE (above 240 µg/l, or 180 kIU/l). These parameters may be increased also
due to parasitary infections, tumors or some immunodeficiencies. Among allergy disorders, the high-
est serum levels of the total IgE may be observed in atopic eczema. In the past, the total IgE assessment
was based on the RIST method (Radioimmunosorbent test), a modified radioimmuno assay (Chapter
4.2.1.1). At present, total IgE levels are diagnosed using methods that are capable of determining the
76
Diagnostics of allergies
levels of specific IgE (sIgE). The term specific IgE antibody in allergology means the antibody
against a particular allergen, e.g. against the most common birch allergen (Bet v 1). We know that
if these antibodies are present in patient´s plasma, the patient is with high probability clinically hy-
persensitive to the tested allergen. In a clinical setting, the sIgE assessment is usually indicated in
the case of sensitization against an allergen that is not included in the skin diagnostic set (prick test),
in food, or professional allergy. In the recent past, the specific IgE was assessed most often using
the RAST method (Radioallergosorbent test), but at present this has been replaced by non-radio mod-
ifications of immunoassays. Currently the most commonly used methods of immunoassay are
those where the allergen is bound to a solid phase (UniCAP, RISA, EUROLINE). UniCAP, or so-
called CAP-FEIA, combines the principles of enzyme and fluorescent immunoassay.
Other soluble molecules are being increasingly assessed in allergology, such as tryptase,
which is a mast cell protease, released in their activation together with histamine. As opposed to
histamine, tryptase is released from mast cells at a much lower rate, i.e. 15–120 minutes from ac-
tivation. For this reason, the assessment of tryptase was used mainly as a laboratory confirmation
of the acute anaphylactic reaction. Tryptase levels should normalise within 12 hours after ana-
phylaxis. The levels of serum tryptase are currently most commonly assessed using CAP-FEIA.
8.1.2.1 Basophil activation test
In the case of discrepancy between the clinical condition of the patient, the skin tests and the
results of specific IgE assessment, it is now possible to perform the so called ‘basophil activation
test’. This assessment represents the capability of sensitized basophils to respond to the allergen.
Basophils are the biggest storers of soluble mediators (histamine, leukotrienes, PAF /platelet ac-
tivating factor/, etc.) in blood circulation. If the basophils, expressing a specific IgE antibody in
their membrane against a particular allergen are exposed to its action, degranulation will result.
We can observe these events observe in an optical microscope profiting from the fact that the gran-
ules possess great affinity to basic stains such as the toluidine blue.
Basophil activation is characterized by their expression of CD63 or CD203c. This is used in
Flow-CAST®, which is the commercial designation of the test used to determine the number of ac-
tivated basophils using flow cytometry. The procedure is based on the incubation of heparinized
blood with allergen. After incubation, a monoclonal antibody anti-CD63 or anti-CD203c is added
to the sample, marking the activated basophils stimulated by added allergen. Expression is assessed
using a flow cytometer to quantify the number of activated basophils (CD63+, CD203c) in propor-
nosis of allergy to certain foods and drugs (muscle relaxants β-lactam antibiotics) or insect venom
tion to their total number. The basophil activation test is currently rarely used, usually in the diag-
in anaphylactic or so far non-sensitized patients. Its great advantage lies in the speed of the test,
where the result, as opposed to a specific IgE test, may be available as soon as in 4 hours.
Test of lymphocyte transformation assay is occasionally used in the diagnostics of drug al-
lergies (Chapter 7.2.3). This is used especially in the case of the equivocal result of intracutaneous
and exposure tests or in older patients. In the lymphocyte transformation assay in sensitized in-
dividuals, drugs to which the individual is sensitive results in the blast transformation of lym-
phocytes. Drugs are diluted for the test according to a specific ratio, and phytohemagglutinin is
used as a positive control.
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Laboratory Metods in Immunology Diagnostics of allergies

8.1.3 Additional assessments

Spirometry is essential for the successful diagnosis of bronchial asthma, as well as for mon- SUMMARY AT A GLANCE
itoring treatment success. This assessment of lung function using the spirometry device is used to
measure lung volume and flow rates. In order to determine the degree of bronchial obstruction •Allergies are hypersensitivity reactions caused by excessive IgE-mediated degranulation of mast cells
used to classify the degree of the disorder, forced vital capacity and basophils induced by specific allergens. The most common allergic disorders are allergic rhinocon-
(VFC) and forced expiratory volume in 1 second (FEV1) are used. juctivitis, allergic bronchial asthma and atopic dermatitis usually induced by inhaled tree, pollen or dust
In addition to the basic spirometric assessment, it is also possible to perform dynamic as- mites. However, food and drug allergies are also prevalent.
sessment such as bronchodilation and bronchoprovocation tests. In the bronchodilation test in pa-
tients with reduced FEV1, a bronchodilator is applied and the subsequent measurement assesses the •The main goal of allergy diagnostics is the identification of causative allergens. Therefore, the proper
reversibility of the bronchial obstruction. A change in excess of 12% confirms reversible bronchial diagnosis of allergies should start with detailed anamnesis focusing on the assessment of the history
obstruction. In the case of the bronchoconstriction test, a bronchoconstrictor (histamine or meta- and nature of symptoms and the analysis of triggering factors.
choline) is applied to a patient with normal FEV1 values. Repeated measurements are used to ob-
serve the change of bronchial obstruction with an increasing concentration of the irritant substance. •Following the anamnesis and physical examination, the definitive confirmation of allergy and identifi-
The assessment is used to determine the presence of bronchial hyperreactivity, which is a provo- cation of causative allergen is usually accomplished with the help of skin tests. The most commonly
cation dose resulting in 20% reduction of FEV1 compared to the initial value (PD20). used type is the prick test, suitable for the diagnostics of allergies to airborne allergens. The diagno-
Imaging methods used in the diagnosis of allergic rhinitis include the X-ray assessment of sis od food and drug allergies often requires the use of other skin test types such as prick to prick test
paranasal sinuses capable of identifying chronic inflammatory changes, and other causes of nasal and intradermal test. The diagnostics of allergic contact dermatitis, mediated by type IV hypersensi-
obstruction and discharge may be excluded. tivity, is typically performed by epicutaneous patch test.
In spite of the above mentioned possibility of the diagnosis of food allergies using the prick
to prick test or sIgE determination, the most sensitive diagnostic methods for the assessment of •The laboratory assessment of allergic disorders includes the evaluation of non-specific markers
food allergies are exposure and elimination tests. These are performed mostly on hospitalized pa- (eosinophil counts, total IgE levels etc.) and several specific methods enabling the identification of
tients. They are based on elimination diet (e.g. rice) and subsequent alternative oral administration causative allergens. These are represented by immunoassays for the measurement of specific IgE lev-
of foods and placebo. Upon the intake of a small sample of suspect food, the subject develops skin, els (older method RAST has been replaced by ELISA, CAP-FEIA etc.), basophil activation tests and lym-
respiratory, gastrointestinal and other symptomatologies. In the case of negative clinical symp- phocyte transformation assay.
toms, additional food is added to the diet the next day. These assessments, however, require great
experience on the part of the physician as it is necessary to eliminate the subjective feelings or sub- •Additional assessments used in allergology include spirometry, bronchodilation and bronchoconstric-
jective bias of the patient. Therefore the standard diagnostic tool remains the so called “DBPCFC” tion tests, exposure and elimination tests, physical tests and various imaging methods.
– double blind, placebo-controlled food challenge, where neither the patient nor the physician
know when the patient was administered a placebo or the tested food. Special diagnostic capsules
should be used in the tests with a precisely defined quantity of the assessed allergen or placebo,
administered repeatedly in pre-defined time intervals. A patient´s health condition is monitored
throughout the testing and for 2 days after the last dose. A positive response includes any of the
following symptoms: urticarial, bronchospasm, angioedema, vomiting, diarrhea, abdominal pain
or anaphylactic shock.
Exposure (provocation) tests may also be used in the diagnosis of food allergies. In rare cases,
the skin allergy may be caused by cold, pressure, heat or exertion. This type of allergic reaction is not
caused by IgE-antibodies however by histamine liberators, e.g. anaphylatoxins of the complement
system, some drugs etc. A suitable physical test is selected on the basis of history, in order to diag-
nose the allergy: cold allergy may be tested in several ways. A patient with a suspected allergy is ex-
posed for a certain time to physical effects, e.g. frozen ice test tubes can be applied to the forearm for
10 minutes. Subsequently, we observe erythematous symptoms or urticaria on a patient’s skin. Heat
hypersensitivity is diagnosed in similar way – using a warm object – warm water or a heater.

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Laboratory Metods in Immunology
9. Diagnostics of autoimmune diseases
9.1 Introduction
Autoimmune diseases (AID) are a heterogenous group of more than 80 different diseases that
affect approximately 5 % of the population of the Western countries. They are characterised by in-
appropriate humoral or cell-mediated immune reactions against host own components and de-
velop as a consequence of complex interactions between numerous predisposing genes and
environmental factors (microorganisms, drugs, chemicals, etc.). The breakdown of central (reces-
sive) and peripheral (dominant) tolerance and the activation of self-reactive T and B cells eventu-
ally lead to the development of specific immune response to own tissues and organs. Their injury
or dysfunction can result from the binding of autoantibodies to membrane antigens and the sub-
sequent lysis of cells by activated complement system or K cells (type II hypersensitivity reactions),
deposition of autoantibody-antigen immune complexes into the tissues and the induction of in-
flammation (type III HR), direct lysis of cells by activated cytotoxic T cells (type IV HR) or stimu-
lation/blocking of membrane receptors by bound autoantibodies (type V HR).
Autoimmune diseases are generally classified into two groups. The organ-specific autoim-
mune diseases are caused by the production of antibodies or cytotoxic cells to certain organs or tis-
sues, whereas systemic autoimmune diseases result from immune reactions to ubiquitous antigens
(nuclear and cytoplasmic molecules that participate in DNA replication, transcription and trans-
lation) and affect multiple different tissues and organs. The former group comprises diseases of
neuromuscular system (multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, etc.), gas-
trointestinal system (Crohn’s disease, ulcerative colitis, primary biliary cirrhosis, autoimmune
hepatitis, etc.), endocrine system (type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroidi-
tis, Addison's disease, etc.), skin and mucosa (pemphigus vulgaris and foliaceus, psoriasis vul-
garis, dermatitis herpetiformis, vitiligo, etc.), blood cells (autoimmune haemolytic anaemia,
autoimmune trombocytopenic purpura, etc.) and other organs and systems. Systemic AID include
connective tissue diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic
sclerosis, idiopathic inflammatory myopathies, Sjögren’s syndrome and spondyloarthropathies.
9.2 Diagnostic strategy
Autoimmune diseases may affect virtually every tissue and organ in the body and often
manifest with a diverse spectrum of non-specific and specific symptoms and signs. Therefore,
their proper diagnosis and treatment usually require cooperation between specialists from multi-
ple medical disciplines. Similarly to other diseases, the diagnostic procedure starts with detailed
anamnesis and physical examination and continues with an array of laboratory tests.
9.2.1 Anamnesis and physical examination
Anamnesis and physical examination should focus on the detection of symptoms and signs
of inflammation, structural damage and/or functional impairment of organs and tissues caused
80
Diagnostics of autoimmune diseases
by the presence of autoantibodies and self-reactive T cells. As noted above, the clinical manifesta-
tion of autoimmunity is variable and largely depends on which organ or tissue is affected, which
pathological immune mechanism is induced and what is the degree of resulting tissue damage. In
many autoimmune diseases, a latent or oligosymptomatic period with none or unclear and non-
specific symptoms and signs often precedes the onset of typical disease symptomatology, and only
abnormal values in basic laboratory tests may draw attention to the possible presence of AID.
Autoimmune diseases have strong genetic background and sometimes run in families. Pos-
itive family history for AID is therefore suggestive for the presence of the disease in patients with
clinical manifestations of tissue and organ injury. However, the definitive diagnosis of AID can
only be established after appropriate laboratory tests have been performed.
9.2.2 Laboratory diagnostics
The laboratory diagnostics of autoimmune diseases employs a variety of basic and advanced
laboratory tests enabling the evaluation of different parameters of immune responses. The hall-
mark of diagnostics, however, remains the detection of circulating or deposited autoantibodies.
Therefore, the proper diagnosis of AID is mostly established only if both typical symptomatology
and specific autoantibodies are present.
9.2.2.1 Complete blood cell counting and enumeration of lymphocyte subpopulations
The complete blood cell count (CBC) with differential provides useful information on the ab-
solute and relative numbers of blood cell populations. These may be significantly altered in certain
autoimmune diseases. AID caused by the production of autoantibodies against blood cell mem-
brane antigens (autoimmune anaemias, neutropaenias, thrombocytopaenias and lymphopaenias)
are usually accompanied by decreased numbers of respective cells. Chronic inflammatory processes
typical for systemic AID often manifest with various changes in the blood picture, such as nor-
mochromic normocytic anaemia, leukocytosis or leukopaenia etc. Moreover, the reduction of leuko-
cyte numbers can also appear as a consequence of immunosuppressive therapy and blood cell
counting is helpful in monitoring these adverse side-effects.
More specialised tests include enumeration of lymphocyte subpopulations by flow cytometry
(Chapter 7.2.2.2). Increased CD4+ helper T cell counts and an increased CD4+/CD8+ T cell ratio
(formerly known as immunoregulatory index) above normal values (1.0 – 2.0) are frequently found
in numerous autoimmune disorders and reflect the chronic activation of self-reactive CD4+ T cells.
On the other hand, a decrease of CD4+ lymphocyte levels and CD4+/CD8+ ratio below reference
values during the long-lasting immunosuppressive therapy indicates an increased risk for the de-
velopment of secondary immunodeficiency.
9.2.2.2 Biochemical examinations and function tests
Autoimmune processes in particular organs usually result in their functional abnormalities
and various changes of biochemical markers. Autoimmune hepatitis will, for instance, manifest
with increased alanine transaminase (ALT), aspartate transaminase (AST) and bilirubin levels. Au-
81
Laboratory Metods in Immunology
toimmune inflammatory myopathies can present themeselves with increased levels of muscle en-
zymes – creatinine kinase (CK), alanine transaminase (ALT) and aspartate transaminase (AST),
while autoimmune-mediated injury of kidneys may lead to increased urea and creatinine concen-
trations, proteinuria and haematuria and decreased glomerular filtration rate and creatinine clear-
ance. Sjögren’s syndrom is characterised by increased levels of amylases due to the affection of
exocrine glands. Type 1 diabetes mellitus is typically characterised by increased plasma glucose
levels and glycated haemoglobin. Coagulation studies in antiphospholipid syndrome will reveal
prolonged activated partial thromboplastin time (aPTT), dilute thromboplastin time (TDT/DTT) or
prothrombin time.
9.2.2.3 Evaluation of inflammatory markers
The presence of an inflammatory process in certain autoimmune diseases can be reflected by
increased levels of acute phase proteins (APP) synthesized by hepatocytes and some other cells
(monocytes, macrophages, neutrophils etc.) in response to proinflammatory cytokines produced
during the tissue injury and inflammation (Chapter 5.2). The measurement of C-reactive protein
by turbidimetry, nephelometry or ELISA is typically used to monitor the course of certain sys-
temic AID such as rheumatoid arthritis, juvenile idiopathic arthritis and some types of systemic
vasculitides. On the other hand, CRP is not increased in systemic lupus erythematosus, autoim-
mune inflammatory myopathies, systemic sclerosis, Sjögren’s syndrome and organ-specific AID.
Erythrocyte sedimentation rate (ESR) is another useful marker, as almost all systemic AID are ac-
companied by increased FW. Its evaluation can therefore be used to monitor disease activity and
response to the treatment.
9.2.2.4 Evaluation of total immunoglobulin levels
Abnormal levels of immunoglobulins are commonly found in various immune-related dis-
eases, including primary and secondary immunodeficiencies, haematological malignancies, in-
fections and autoimmune disorders. Their evaluation by nephelometry is therefore an important
tool in immunologic evaluation (Chapter 4.1). Increased total IgG levels are commonly found in
systemic autoimmune diseases and autoimmune hepatitis, increased IgA can be detected in
Crohn's disease, ulcerous colitis, ankylosing spondylitis and autoimmune hepatitis, and increased
IgM can be seen in primary biliary cirrhosis and rheumatoid arthritis. On the other hand, de-
creased levels of certain immunoglobulin classes are typically found in primary immunodefi-
ciencies (e.g. selective IgA deficiency), which are, in turn, associated with an increased risk of
developing autoimmune diseases.
9.2.2.5 Evaluation of cryoglobulins
Cryoglobulins are monoclonal IgM (type I), mixture of monoclonal IgM and polyclonal IgG
(type II) or mixture of polyclonal IgM and IgG (type III) that precipitate reversibly in cold tem-
peratures. To assess their presence, venous blood is drawn and placed in the tube without anti-
coagulant. After the coagulation has occurred and the clot has been removed, the serum is kept
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Diagnostics of autoimmune diseases
at 4 °C until the precipitation appears and a cryocrit is measured. The final confirmation of the
presence of cryoglobulins is done by warming up the serum to body temperature, leading to re-
dissolution of the precipitate. The occurrence of cryoglobulins is typically associated with gam-
mopathies (multiple myeloma, Waldenström macroglobulinaemia) and lymphoproliferative
disorders, but may also be found in certain autoimmune diseases such as systemic lupus erythe-
matosus, rheumatoid arthritis and vasculitides.
9.2.2.6 Detection of autoantibodies
As mentioned above, the detection of specific high-affinity autoantibodies against various
cell components is a hallmark of AID diagnostics. The potential targets for autoantibodies include
nucleic acids, histones and other nuclear, nucleolar and perinuclear proteins, mitochondrial pro-
teins, membrane receptors and extracellular proteins (Tab. 9.1). Although not all of them directly
participate in the pathogenesis of a particular disease, they serve as important markers for the pres-
ence, nature and intensity of autoimmune processes in the organism. Therefore, qualitative and/or
quantitative evaluation of autoantibodies in the biological samples from patients with suspected
AID plays an essential role in the diagnostics and classification of autoimmune diseases. In addi-
tion, autoantibodies also have predictive and prognostic value as they usually appear several
months to years before the actual autoimmune disease manifests with first symptoms and signs, and
thus may be helpful in the early diagnostics of future disease in currently healthy individuals.
Lastly, the monitoring of changes in antibody quantities and profiles represents a helpful tool for
assessing the activity and severity of the disease and its response to treatment. On the other hand,
the presence of autoantibodies is not entirely specific for AID, as they sometimes appear in other
non-autoimmune diseases including malignancies and infections. Thus, the definite diagnosis of
AID must be based on co-existence of both appropriate clinical symptomatology and spe- cific au-
toantibodies in the biological sample.
Over the last decades, a broad spectrum of immunoassays for the detection of circulating au-
toantibodies has been introduced in the laboratory diagnostics of AID. The discovery of new au-
toantigens, gradual improvements in antigen purification and recombination techniques and
increasing availability of new and improved tests and technologies have allowed for a qualitative
leap in laboratory diagnostics of autoimmune diseases. The conventional immunochemical methods
for the qualitative or semiquantitative evaluation of autoantibodies such as indirect immunofluo-
rescence (IFI), passive agglutination (PA), double radial immunodiffusion (DRID), counter-im-
munoelectrophoresis (CIE), immunoprecipitation (IP) and immunoblot (IB) have been replaced
to a large extent by immunometric methods that are more reliable and allow quantitative measure-
ment of antibody concentrations. These methods comprise radioimmunoassays (RIA), enzyme
immunoassays (EIA), fluorescent immunoassays (FIA), fluorescent enzyme immunoassays (FEIA)
and chemiluminiscent immunoassays (CLIA).
Indirect immunofluorescence (IFI) is one of the most widely used techniques in the laboratory
diagnostics of AID. The detection of autoantibodies in the serum is performed by its incubation
with antigenic native cell structures present in the specific tissue sections or cultured cell lines. The
bound autoantibodies are subsequently visualised by fluorescently-labelled anti-immunoglobulin
(secondary) antibodies directed against the constant Fc fragment of immunoglobulins IgG, IgM
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Laboratory Metods in Immunology Diagnostics of autoimmune diseases

nucleolar, cytoplasmic, centromeric) depending on the nature of target antigen. The identity of
AUTOANTIBODIES AND THEIR TARGETS AUTOIMMUNE DISEASE autoantibodies should be subsequently confirmed by more specific test such as ELISA. For the de-
tection of anti-dsDNA antibodies, a flagellated protozoa Crithidia luciliae with a dsDNA-contain-
Antinuclear antibodies (ANA) ing organelle (kinetoplast) is commonly used as a substrate. The presence of anti-neutrophil
Double stranded DNA, histones, centromere proteins, Systemic lupus erythematosus, systemic sclerosis, cytoplasmic antibodies (ANCA) is routinely screened using human ethanol- and formalin-fixed
ribonucleoproteins, Smith antigen, enzymes (DNA Sjögren’s syndrome, mixed connective tissue neutrophils. The binding of autoantibodies to components of neutrophil granules will yield typi-
topoisomerase I, tRNA synthetases, RNA polymerases) disease, idiopathic inflammatory myopathies
cal staining patterns associated with certain antigens (Tab. 9.1). Their identity should be subse-
and others (dermatomyositis, polymyositis), primary biliary
quently confirmed by ELISA test. Antibodies to organ-specific and other antigens are typically
cirrhosis, ...
detected with the help of microscopic slides containing specific tissue sections as substrates, mostly
Anti-neutrophil cytoplasmic antibodies (ANCA) from primates and rodents. These include kidneys, liver, adrenal glands, thyroid gland, heart mus-
Proteinase 3 (cytoplasmic ANCA), myeloperoxidase, Granulomatosis with polyangiitis, microscopic cle, oesophagus, stomach, small intestine, striated muscle, etc. For a better characterization of spe-
BPI, elastase, lysozyme, lactoferrin, cathepsin G and polyangiitis, Churg-Strauss syndrome, cystic cific antigens recognised by autoantibodies, ELISA method is usually subsequently performed.
other proteins (perinuclear ANCA, atypical ANCA) fibrosis, inflammatory bowel disease, rheumatoid The main advantage of IFI is the presence of various native antigenic targets on tissue sections
arthritis, primary sclerosing cholangitis,
and cell cultures what gives the IFI method its excellent overall sensitivity. On the other hand, its
drug-induced systemic vasculitis, ...
major disadvantages are manual performance with limited possibility for automation, require-
Antimitochondrial antibodies (AMA) ments for skilled and experienced laboratory personnel, subjective result interpretation, only semi-
Cardiolipin, E2 and E1 subunits of 2-oxo-acid Primary antiphospholipid syndrome, systemic quantitative results, the wide variability of results and low reproducibility. Some of these
dehydrogenases, ... lupus erythematosus, primary biliary cirrhosis limitations can be partially overcome by the use of computer-assisted systems with digital micro-
Autoantibodies against some other well-defined antigens scope camera and automated image analysis and interpretation softwares.
Cyclic citrullinated peptides, immunoglobulin G Rheumatoid arthritis
Gangliosides (GD3, GM1,...) Guillan-Barré syndrome Fig. 9.1 Indirect immunofluores-
Type 4 collagen in the glomerular basement membrane Goodpasture’s syndrome cence for detection of autoanti-
Soluble liver antigen, smooth muscle antigens Autoimmune hepatitis type 1 bodies
Liver/kidney microsomes type 1, liver cytosol type 1 Autoimmune hepatitis type 2
antigens
Tissue transglutaminase, endomysium, reticulin Coeliac disease
Passive agglutinantion
Pancreatic islet cells, 65-kD glutamic acid decarboxylase, Type 1 diabetes mellitus
insulin, tyrosine phosphatase-like protein assay is an older simple met-
Thyroid-stimulating hormone receptor Graves’ disease hod used for the quick detec-
Thyroid peroxidase, thyreoglobulin Hashimoto's thyroiditis tion of rheumatoid factor (RF),
Acetylcholine receptor Myasthenia gravis an IgM autoantibody against
Desmogleins 3 and 1 Pemphigus vulgaris, pemphigus foliaceus the Fc fragment of own IgG
Myelin basic protein, myelin oligodendrocyte glycoprotein Multiple sclerosis
commonly found in patients
21-hydroxylase, 17α-hydroxylase, cytochrome p450 Addison’s disease
Intrinsic factor Atrophic gastritis with pernicious anemia
suffering from rheumatoid
arthritis. The detection of RF is
performed by mixing patient's
Tab. 9.1 Examples of autoantibodies found in autoimmune diseases serum with latex particles
coated with human IgG on
and/or IgA (Fig. 9.1). Currently, IFI is used as a sensitive screening or confirmatory assay for the a cardboard card. In the pres-
detection of various nuclear, cytoplasmic and organ-specific antibodies. For the identification of ence of RF in the sample, its
antinuclear antibodies (ANA), the human laryngeal epithelioma HEp-2 cell line is used as an anti- binding to the IgG will cross-
genic substrate. The target antigens for ANA include various cellular components such as nucleic link the latex particles causing
Fig. 9.2 Passive agglutination for detection of rheumatoid factor
acids, histones, chromatin, nuclear and ribonuclear proteins. The binding of ANA to cell struc- them to clump (Fig. 9.2).
tures will produce various staining patterns (nuclear homogenous, nuclear speckled, perinuclear,

84 85
Laboratory Metods in Immunology Diagnostics of autoimmune diseases

Fig. 9.3 Classical indirect ELISA development. Alternatively, a fluorochrome can be used to label the secondary antibody. The mi-
assay (a) and capture-ELISA assay crosphere-based multiplex flow cytometric immunoassay is an example of a non-planar array that
(b) for detection of autoantibodies uses polystyrene microspheres (beads) labelled internally with different ratios of two fluorescent
dyes (red and infrared fluoride). As each of the two fluorochromes can have any of 10 possible
levels of fluorescence intensity, their combination can create up to 100 distinctly coloured micros-
phere sets, each with a unique spectral signature determined by its fluorochrome mixture. Vari-
Enzyme immunoasays ous antigens for the detection of autoantibodies are conjugated the beads so that each of the 100
(EIA) are other widely used bead sets contains an antigen specific for a unique autoantibody. Serum from a patient is incu-
immunoassays in clinical lab- bated in microtiter plate wells with antigen-coated microspheres and subsequently, anti-im-
oratories. Of the several mod- munoglobulin antibodies labelled with another fluorochrome are added for the detection of bound
ifications, the enzyme-linked autoantibodies. After the incubation, the reaction is read by the flow cytometer/luminometer. The
immunosorbent asssay (ELISA) analyser produces beams of light with two different wavelengths (635 nm and 525 nm) which si-
is the most frequently used multaneously excite both the internal fluorochromes that identify each microsphere particle (and
format. In the classical indi- so the specific antigen) and the fluorochrome dye coupled with secondary antibody bound to the
rect ELISA, specific autoanti- specific autoantibody (Fig. 9.4). As multiplex assays are not limited to the detection of a single au-
bodies in biological samples are detected with the help of purified native, recombinant or synthetic toantibody, they could perspectively be used to detect autoantibody profiles consisting of dozens,
antigens directly attached to the solid phase (Fig. 9.3a). In the newer capture-ELISA and anchor- if not hundreds of different autoantibodies that represent the specific fingerprint of patients with
ELISA formats, the antigens are anchored to the solid phase by either mouse monoclonal anti- autoimmune diseases. This would allow to classify patients into different subcategories with dis-
body directed towards an epitope on the specific antigen, or by different little molecules (Fig. 9.3b). tinct clinical patterns, prognosis and therapeutical approach.
In both cases, this indirect coating strategy assures better exposition of antigenic epitopes to au-
toantibodies, resulting in improved sensitivity. Autoantibodies bound to their specific antigens on
the solid phase are subsequently visualised with the help of secondary antibody labelled with an
enzyme and appropriate substrate (Chapter 4.2.2). In comparison with IFI method, ELISA offers
better reproducibility, objectivity, standardization and higher degree of automation, enables quan-
titative analysis of multiple samples in the same plate and is less laborious. It is used to identify
or confirm the specificity of a wide spectrum of autoantibodies, including antinuclear, anti-
dsDNA, anti-nucleosome, anticentromere, anti-neutrophil cytoplasmic and numerous organ-spe-
cific antibodies.
Another technique with good sensitivity and specificity for the qualitative or semiquantita-
tive detection of autoantibodies is the Western blot (WB). It is based on the recognition of autoanti-
bodies with the help of specific antigens that were previously separated by electrophoresis in
polyacrylamide gel and transferred (blotted) to a nitrocellulose membrane (Chapter 4.3). WB offers
the possibility to detect autoantibodies to known antigens with well-defined molecular weight. It
is commonly used to determine specificities of autoantibodies to extractable nuclear antigens (ENA).
In the recent years, the technological progress and introduction of the new-generation mul-
tiplex assays has revolutionised laboratory diagnostics of AID and enabled simultaneous detection
and measurement of a high number of autoantibodies and other analytes in small sample vol-
umes, with lower amounts of reagents and therefore at proportionally lower costs when compared
to classical methods. These assays include planar microarrays and non-planar suspension arrays.
Planar autoantigen microarrays are miniaturised arrays for the detection of hundreds of autoan-
tibodies that use antigens robotically applied in microspots on glass slides, polystyrene microplates
or nitrocellulose membranes. Following the incubation with sample serum, secondary antibody
Fig. 9.4 Microsphere-based multiplex flow cytometric immunoassay for detection of autoantibodies
conjugated with an enzyme is added. Finally, a specific substrate is added for the colour reaction

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Laboratory Metods in Immunology
In addition to detecting circulating autoantibodies, the diagnostic approach in some au-
toimmune diseases also incorporates the detection and visualisation of tissue-bound (deposited)
autoantibodies by direct immunofluorescence. This is for example the case in Goodpasture’s syn-
drome characterised by antibodies to glomerular basal membrane, and autoimmune bullous skin
diseases characterised by the production of autoantibodies against desmosomal proteins (pem-
phigus foliaceus, pemphigus vulgaris), adhesion molecules of the dermal-epidermal junction
(pemphigoid) and epidermal transglutaminase (dermatitis herpetiformis). Tissue-bound anti-
bodies are detected in kidney biopsy samples and skin excision samples, respectively, by adding
anti-immunoglobulin antibodies labelled with a fluorochrome. Antibody deposits are subse-
quently visualised under the UV microscope (Chapter 4.2.3.1).
9.2.2.7 Measurement of circulating immune complexes
Certain autoimmune diseases including the systemic lupus erythematosus, autoimmune vas-
culitides, rheumatoid arthritis, Sjögren’s syndrome, ankylosing spondylitis or systemic sclerosis are
associated with the presence of circulating immune complexes (CIC) formed of autoantibodies bound
to soluble antigens. Immune complexes subsequently deposit in the tissues (kidneys, joints, vessels)
and induce an inflammation (glomerulonephritis, arthritis, vasculitis). Therefore, the measurement of
CIC may be helpful in the diagnostics of these inflammatory autoimmune diseases and provides use-
ful clinical information regarding the development, prognosis and follow-up. The amount of circu-
lating immune complexes can be determined by polyethylene glycol precipitation assay, where mixing
the serum containing immune complexes with polyethylene glycol will create a turbidity, the inten-
sity of which can be measured by spectrophotometry (Chapter 4.1). More sensitive and specific
method is the CIC-C1q ELISA for the detection of immune complexes that can bind to C1q component.
Briefly, C1q is adsorbed on a microtiter plate and after the serum sample is added, C1q-fixing circu-
lating immune complexes in the sample bind to the immobilised C1q. Following the washing step,
anti-human IgG antibody conjugated with an enzyme (e.g. horseradish peroxidase) is added and
binds to the Fc fragment of human IgG in circulating immune complexes. After another incubation
and wash, the substrate is ad-
ded. The enzyme catalyses its
transformation into coloured
product and the intensity of
colourimetric reaction is read
in a spectrophotometer (Fig.
9.5). Another alternative is the
CIC-anti-C3d ELISA, which uses
the anti-C3d antibody immo-
bilised on the microtiter plate
instead of C1q.
Fig. 9.5 ELISA assay for detection
of circulating immune complexes
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Diagnostics of autoimmune diseases
9.2.2.8 Evaluation of the complement system
Alterations in complement proteins levels and activity are commonly observed in various
autoimmune diseases, either as their primary cause (primary C2, C3 and C4 deficiencies are as-
sociated with impaired clearance of immune complexes and increased risk of developing im-
mune complex-mediated diseases) or as a consequence (autoimmune diseases with immune
complex deposition lead to significant consumption of complement proteins; autoimmune hep-
atitis is associated with decreased complement synthesis; autoimmune diseases associated with
chronic inflammation often lead to increased synthesis of APPs, including the complement pro-
teins). Therefore, the quantitative measurement of C3, C4, factor B and complement activation
products (C3dg, sC5b-9) by nephelometry or ELISA and the evaluation of classical complement
pathway function by total haemolytic complement activity (CH50) assay are among laboratory
tests performed when certain AID are suspected, and can also serve as markers of disease activ-
ity (Chapter 5.1.2).
9.2.2.9 Cytokine measurement
Various autoimmune diseases are typically associated with increased levels of different cy-
tokines such as IL-1, IL-6, TNF, IL-2, IL-4, IL-5, IL-17, IFN-γ and others. Although their measure-
ment is usually not routinely performed in commercial laboratories, the evaluation of their levels
by ELISA or flow cytometry is helpful in certain autoimmune diseases in which anti-cytokine bi-
ologic treatment (e.g. monoclonal antibodies targeting particular cytokine) is used.
9.2.2.10 Genetic tests
The development of the majority of autoimmune diseases is associated with the presence of
particular risk HLA alleles/molecules, for instance HLA-DQ2 and -DQ8 in type 1 diabetes melli-
tus and coeliac disease, HLA-DR4 in rheumatoid arthritis, HLA-B27 in ankylosing spondylarthri-
tis, etc. Therefore, HLA-typing permorfed by serologic or genetic methods (Chapter 12.3) is used
as supplementary test in cases of diagnostic insecurity and differential diagnostics. Certain im-
munodeficiency syndromes associated with the development of autoimmune diaseases are caused
by single mutation of particular genes. For instance, autoimmune lymphoproliferative syndrome
(ALP) is associated with mutations in Fas, Fas ligand, caspase 8 or caspase 10 genes, autoimmune
polyendocrinopathy with candidiasis and ectodermal dystrophy syndrome (APECED) with mu-
tations in AIRE gene, and immune dysregulation-polyendocrinopathy-enteropathy-X-linked syn-
drome (IPEX) with mutations in FoxP3 gene. Their detection by sequencing is an integral part of
a diagnostic protocol in these immune dysregulation disorders.
9.2.2.11 Other examinations
Histological and immunohistochemical examinations of bioptical samples obtained from
tissues affected by autoimmune process are also important diagnostic tools in certain autoim-
mune diseases, and so are imaging and other methods for the visualisation of organ and tissue
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Laboratory Metods in Immunology
affection. These include medical radiography, ultrasonography, magnetic resonance imaging,
electrocardiography, electromyography, electroencephalography and other examinations.
SUMMARY AT A GLANCE
•Autoimmune diseases are a heterogenous group of disorders characterised by tissue/organ injury or
dysfunction induced by an immune reaction to self-antigens.
•Autoimmune diseases often manifest with a diverse spectrum of symptoms and signs and their proper
diagnostics requires the use of an array of basic and advanced laboratory tests.
•Laboratory tests for autoimmune diseases include complete blood cell counting, enumeration of lym-
phocyte subpopulations, evaluation of inflammatory markers (acute phase proteins, erythrocyte sedi-
mentation rate), total immunoglobulin levels and cryoglobulins, detection of specific autoantibodies,
evaluation of the complement system and circulating immune complexes, and genetic tests.
•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.
•The proper diagnosis of an autoimmune disease is mostly established when both typical symptoma-
tology and specific autoantibodies are present.
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Diagnostics of immunodeficiencies
10. Diagnostics of immunodeficiencies
10.1 Introduction
Human immune system plays a crucial role in defence against infectious microorganisms and
tumours, as well as in removal of old, damaged and foreign cells and material. Quantitative or func-
tional defects in any of its components may result in increased susceptibility to infections, malig-
nancies or autoimmune disorders, often with fatal consequences. These so called immunodeficiency
diseases are classiefied into two main groups according to their aetiopathogenesis. The primary (con-
genital) immunodeficiencies are mostly caused by inherited genetic defects that negatively affect the
development and/or function of one or more components of the immune system, resulting in an in-
creased rate and severity of infections typically manifesting in early infancy and childhood. The sec-
ondary (acquired) immunodeficiencies can develop at any stage during the life as a consequence of
a damage caused to the previously normal immune system by factors such as malnutrition, infections
(e.g. HIV, EBV, CMV, measles and others), chronic diseases (diabetes, hepatic and renal insuffi-
ciency, rheumatological, metabolical and other diseases), malignancies, radiation, surgery, trauma,
treatment with immunosuppressive and chemotherapeutic drugs and some others.
To date, more than 150 primary immunodeficiencies (PIDs) have been described and classified
into several categories according to which component of the immune system is primarily affected:
• Antibody (B cell) deficiencies are caused by defects occuring at some stage of B cell develop-
ment, differentiation or maturation, and include X-linked agammaglobulinaemia (XLA) and
other congenital agammaglobulinaemias, common variable immunodeficiency disorders
(CVIDs), hyper-IgM syndrome, selective IgA deficiency and others.
• Combined B and T cell immunodeficiencies include severe combined immunodeficiency
(SCID) syndromes and several other combined immunodeficiencies with predominant T cell
impairment.
• Other well-defined immunodeficiency syndromes comprise several disorders accompanied by
neurological, vascular, mucocutaneous, haematological or musculoskeletal abnormalities,
such as thymic defects (DiGeorge syndrome, Nezelof syndrome), DNA repair defects (ataxia-
teleangiectasia and others), Wiskott-Aldrich syndrome (WAS), immune-osseous dysplasias,
hyper-IgE syndromes, chronic mucocutaneous candidiasis and some other syndromes.
• Diseases of immune dysregulation represent a group of disorders with a complex phenotype
consisting of various infectious, autoimmune and lymphoproliferative diseases. Included
are Chediak-Higashi syndrome and similar syndromes, familial haemophagocytic lym-
phohistiocytosis (HLH) syndromes, autoimmune lymphoproliferative syndromes (ALPs),
autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy syndrome
(APECED) and immune dysregulation-polyendocrinopathy-enteropathy-X-linked syn-
drome (IPEX).
• Defects of phagocyte numbers and/or function comprise several diseases with abnormal
numbers of phagocytes (severe congenital neutropaenia, cyclic neutropaenia, etc.) or their
impaired ability to migrate, engulf or destroy foreign pathogens (chronic granulomatous
disease, leukocyte adhesion deficiencies, specific granule deficiency and others).
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Laboratory Metods in Immunology
• Defects in innate imunity include rare PIDs caused by defects in pathogen recognition re-
ceptor (PRR) signaling (IRAK-4 deficiency, MyD88 deficiency and others).
• Complement deficiencies are caused by genetic defects of classical, alternative or lectin path-
way complement factors or their regulators.
• Autoinflammatory disorders include diseases such as familial Mediterranean fever or TNF re-
ceptor associated periodic syndrome, characterised by periodic fever and excessive inflam-
matory response, rather than infections.
10.2 Diagnostic strategy
Diagnosis of immunodeficiencies often presents a challenge for clinicians and many ID dis-
eases can therefore go undetected for a longer period of time as they may appear as ordinary in-
fections of upper/lower respiratory or gastrointestinal tract. However, early and correct diagnosis
and treatment of PIDs is essential for preventing further damage to the organism, decreasing the
morbidity and mortality and improving the life expectancy and quality. Diagnostic protocol in-
cludes detailed medical history, physical examination and various laboratory tests for the evalua-
tion of immune system function. If secondary immunodeficiency is suspected, appropriate tests
for the diagnosis of underlaying disorder need to be carefully selected and performed, e.g. HIV
tests, tests for the examination of potential gastrointestinal or urinary protein losses, liver tests, etc.
10.2.1 Clinical presentation and history
Diagnostics of immunodeficiencies requires a stepwise approach and should begin with
evaluation of clinical presentation and medical history, with particular regard to age at onset, type,
location and severity of signs and symptoms. Generally, an immunodeficiency should be sus-
pected in individuals who have recurrent and/or chronic infections of upper and lower respira-
tory tract, gastrointestinal tract or skin, although many patients may eventually develop more
severe bacterial infections (sepsis, meningitis, osteomyelitis, abscesses of internal organs). Other
typical features of immunodeficiencies are infections caused by unusual (opportunistic) microor-
ganisms and complicated infections that don't respond well to conventional treatment or manifest
atypically. Age at which infections began to manifest is of great importance. The most severe forms
of PIDs such as X-linked agammaglobulinaemia or SCID usually manifest early in the infancy as
recurrent infections, chronic diarrhea and failure to thrive. Other PIDs such as common variable
immunodeficiency may not manifest until adulthood. Many immunodeficiency syndromes also
present with autoimmune diseases and malignancies.
Primary immunodeficiencies are often hereditary and, therefore, any record of recurrent,
chronic or severe infections in the family history may hint at the potential presence of PID in the
patient. Medical record of a clinical infection following the vaccination with live attenuated mi-
croorganisms in patient's personal history also supports the diagnosis of PID.
The nature of infections depends on a component of the immune system that is defective. Pa-
tients with antibody immunodeficiencies typically suffer from recurrent infections of sinopul-
monary tract caused by encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae,
92
Diagnostics of immunodeficiencies
etc.), although diarrhea caused by Giardia intestinalis, recurrent enteroviral infections and severe
bacterial infections of skin and other organs may also be present. Patients with predominant de-
fects of specific cellular immunity usually develop infections caused by viruses, fungi and intra-
cellularly replicating bacteria such as mycobacteria or salmonella. Combined defects of B and T cell
mediated immunity usually manifest early in the childhood as failure to thrive, chronic diarrhea
and severe infections caused by a wide range of different extra- and intracellular microorganisms
(viruses, bacteria, fungi, protozoa), often opportunistic (e.g. Pneumocystis jiroveci). Phagocytic de-
fects predispose to pyogenic or granulomatous infections of the respiratory tract, lymph nodes,
skin and viscera caused by Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Ser-
ratia marcescens, Klebsiella sp., Salmonella sp., Escherichia coli, etc.) or fungi (Candida sp., Aspergilus
sp.). Complement deficiencies are associated with an increased susceptibility to neisserial infec-
tions (meningitis, sepsis), recurrent pyogenic infection of respiratory tract and systemic autoim-
mune diseases resembling SLE.
10.2.2 Physical examination
Patients suffering from immunodeficiencies may present with various symptoms and signs
of infections as well as other abnormalities accompanying certain IDs. It should be noted that not
every patient appears ill at the time of examination and clinical signs may sometimes be very sub-
tle or absent. Patients with severe forms of IDs (XLA, SCID) show reduction or absence of lym-
phoid tissues (lymph nodes, adenoid vegetations, tonsils). On the other hand, autoimmune
lymphoproliferative syndromes, chronic granulomatous disease (CGD), common variable im-
munodeficiency and some others manifest with lymphadenopathy and hepatosplenomegaly. Pa-
tients with some IDs present with skin lesions including albinism (Chediak-Higashi syndrome),
petechiae (Wiskott-Aldrich syndrome), eczema, candidiasis, rash, warts, pustulae, abscesses or
alopecia. Patients with DiGeorge syndrome (developmental defect of third and fourth pharyngeal
arches) have facial dysmorphism, conotruncal heart abnormalities, agenesis of the thymus and
parathyroid glands. Facial abnormalities and growth retardation can also be seen in patients with
leukocyte adhesion deficiency 2 syndrome (LAD2), the Nijmegen syndrome and some others. Pa-
tients suffering from ataxia-teleangiectasia present with cerebellar and sensory ataxia and ocular
teleangiectasia. Neurological changes, abnormalities of skeletal development resulting in short-
limbed dwarfism and cartilage-hair hypoplasia are other clinical findings in certain PIDs.
10.2.3 Laboratory tests
10.2.3.1 Basic screening tests
The evaluation of an immune system should begin with basic screening tests. The complete
blood cell count (CBC) with differential provides useful information on the number of blood cell
populations, while 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). The finding of reduced numbers of leukocytes and their subpopulations along
with corresponding clinical presentation is indicative for the presence of immunodeficiency. The
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Laboratory Metods in Immunology
presence of lymphopaenia is typical for certain primary immunodeficiencies affecting T cells
(CIDs, DiGeorge syndrome and others), but may also be of secondary nature (HIV infection, treat-
ment with immunosuppresive and cytostatic drugs, etc.). Reduced numbers of granulocytes (gran-
ulocytopaenia), mostly neutrophils (neutropaenia) are found in PIDs such as severe congenital
neutropaenia, cyclic neutropaenia, reticular dysgenesis, hyper-IgM syndrome and WHIM syn-
drome, but are also present after viral infections, in patients with malignancies or can be caused
by autoimmune or iatrogenic destruction of neutrophils. Decreased numbers of erythrocytes
(anaemia) are present in various different conditions, including some primary immunodeficien-
cies (glucose-6-phosphate dehydrogenase deficiency, paroxysmal nocturnal haemoglobinuria,
etc.), while possible explanations for low platelet count (thrombocytopaenia) are Wiskott-Aldrich
syndrome, some combined immunodeficiencies and CVID. Markedly increased numbers of leuko-
cyte subpopulations are usually present in infections, haematological malignancies, myeloprolif-
erative disorders and also in some immunodeficiencies. Neutrophilia is indicative for bacterial
infections, leukaemias and certain primary immunodeficiencies (leukocyte adhesion deficiencies),
eosinophilia for allergies, parasitic infections, certain myeloproliferative disorders (hypere-
osinophilic syndrome), malignancies and primary immunodeficiencies (hyper-IgE syndrome,
Omenn syndrome, Wiskott-Aldrich syndrome), while lymphocytosis is typically present in viral,
some protozoal and intracellular bacterial infections, leukaemias and lymphomas.
Evaluation of total immunoglobulin levels by nephelometry and ELISA methods is also a part
of initial screen, as abnormal levels of one or more Ig classes are typically found in various B cell
and combined immunodeficiency disorders. The results must be compared with age-matched ref-
erence values. Physiologic hypogammaglobulinaemia, typically occuring in infants aged 3 – 6
months, must be taken into consideration when the values are interpreted. On the other hand, ap-
parently normal IgG levels during first 2 – 3 months of life do not exclude the possibility of severe
antibody deficiency, as the IgGs in the blood of the child are of maternal origin. Concentrations of
IgG, IgM and IgA are usually measured by nephelometry (Chapter 4.1), whereas more sensitive
enzymatic immunoassays are used for IgE due to its low levels in blood (Chapter 4.2.2). Signifi-
cytes are typically found in X-linked agammaglobulinaemia and related µ heavy, Igα or Igβ chain
cantly reduced levels of all immunoglobulin classes with virtually absent circulating B lympho-
deficiencies. Low levels of IgG, IgA and/or IgM isotypes along with impaired specific antibody
response is indicative for common variable immunodeficiency syndromes. Reduced levels of only
one Ig class are typically present in selective Ig immunodeficiencies, such as the most frequent
primary ID – the selective IgA immunodeficiency. Increased levels of immunoglobulins are usu-
ally present in infections and monoclonal gammapaties, but may also be found in certain primary
immunodeficiencies (hyper-IgE syndrome, Omenn syndrome, hyper-IgD syndrome). In hyper-
IgM syndrome, lower concentrations of serum IgG and IgA are usually found along with normal
or increased levels of IgM as a result of a defective immunoglobulin class-switching. In certain
cases, quantitation of IgG subclass levels may be considered, although this test is of limited util-
ity. Defects of Ig production can also be evaluated by measuring specific antibody titers in response
to protein (T-dependent) and polysaccharide (T-independent) antigens. In this case, specific anti-
body response to standardized antigens is assessed before and 3 – 4 weeks after boosting immu-
nization with tetanus or diphteria toxoid vaccine to test for protein antigen responsiveness, and
with pneumococcal or Haemophilus influenzae polysaccharide vaccine to test for carbohydrate anti-
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Diagnostics of immunodeficiencies
gen responsiveness, respectively. An adequate 4-fold rise in the specific antibody titer is indicative
for unimpaired humoral specific imunity. Specific antibody production can also be evaluated by
measuring anti-A/anti-B IgM isohemagglutinin titers.
10.2.3.2 Advanced tests for evaluation of acquired (specific) imunity
If the results of basic screening tests are abnormal and/or the history and clinical presentation
are highly indicative for a immunodeficiency disease, further advanced tests must be performed. Spe-
cialised methods for the evaluation of acquired imunity include enumeration of lymphocyte subpop-
ulations (immunophenotyping) by flow cytometry (Chapter 7.2.2.2). The method is based on the usage
of fluorochrome-labelled monoclonal antibodies specific to various membrane (CD) and intracellu-
lar antigens and enables to evaluate the numbers of total T cells (CD3+), helper T cells (CD3+CD4+),
cytotoxic T cells (CD3+CD8+), regulatory T cells (CD4+CD25+FoxP3+), B cells (CD19+CD20+), NK cells
(CD16+CD56+) or activated T cells (HLA-DR+CD69+). Extremely low or absent B cell counts are typi-
cal for X-linked agammaglobulinaemia and related syndromes with markedly reduced Ig isotypes;
however, decreased or absent B lymphocytes can also be found in some severe combined immunod-
eficiencies (reticular dysgenesis, adenosine deaminase deficiency, RAG1/2 deficiency) and occasion-
ally in common variable immunodeficiency. Low or absent T cell numbers are characteristic for
combined immunodeficiencies, DiGeorge and Wiskott-Aldrich syndromes, ataxia-teleangiectasia and
some other well-defined IDs. Low helper T cell counts and a decreased CD4+/CD8+ T cell ratio are typ-
ically found in common variable immunodeficiency and MHC class II deficiency; however, they are
also associated with conditions like the HIV infection, some bacterial and parasitic infections, physi-
cal trauma, treatment with corticosteroids or stress. Decreased numbers of cytotoxic T cells and an in-
creased CD4+/CD8+ ratio are typical for a rare CD8 deficiency, MHC class I deficiency and numerous
autoimmune disorders. An increased presence of CD4-CD8- double negative cells in the peripheral
blood is typically found in autoimmune lymhoproliferative syndromes caused by defects of media-
tors of apoptosis, while low levels of NK cells are found in certain combined deficiencies (IL-2RG de-
ficiency, JAK3 deficiency) and rare isolated NK cell deficiencies.
Flow cytometry assays are also used to screen for altered expression of specific extracellular or
intracellular proteins (receptors, membrane antigens, signalling molecules, cytotoxic molecules, etc.)
associated with specific immune defects, for example an impaired expression of CD40 or CD40L
on T cells in patients with hyper IgM syndrome, Bruton’s tyrosinkinase Btk in monocytes and
platelets (B cells are usually absent) in patients with X-linked agammaglobulinaemia, FoxP3 tran-
scription factor in regulatory CD4+CD25+ T cells in patients with IPEX syndrome and many more.
Specific cellular immune function is routinely evaluated by delayed-type hypersensitivity skin
tests (Chapter 7.2.5). Testing antigen (candidin, tuberculin, etc.) to which the patient has already
been exposed is injected intradermally on the volar forearm and the reaction is evaluated after 48 –
72 hours. The appearance of erythema and induration larger than 5 mm excludes the diagnosis of T
cell disorder. Alternatively, the function of lymphocytes can also be assessed in-vitro by lymphocyte
transformation test (blastic transformation test) using the mitogens, antigens, irradiated allogeneic
white blood cells or monoclonal antibodies to stimulate the proliferation of T and/or B cells (Chap-
ter 7.2.3). Their response to stimulation is subsequently evaluated by measuring the radioactivity
which intensity depends on the amount of 3H-thymidine incorporated into the cells undergoing
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blastic transformation. Low or absent uptake of radioactive thymidine indicates T cell or combined
immunodeficiency. Enzyme-linked immunosorbent spot assay (ELISpot) is another perspective
method that can be used for evaluation of specific cellular imunity function (Chapters 4.2.2.5 and
7.2.4). Patient's lymphocytes are stimulated by an antigen and synthesised cytokines subsequently
bind to specific monoclonal antibodies coating the bottom of a microtiter plate. Captured cytokines
are then visualised by adding another enzymatically-labelled specific antibody and a substrate, and
resulting coloured reaction (spot) is analyzed under the microscope or by automatised analyzer. Al-
ternatively, cytoplasmic cytokine production can also be evaluated by flow cytometry.
Serologic or flow-cytometric HLA-typing can be used to detect the absence of HLA class I and
II molecules on lymphocytes of patients with MHC class I and II deficiencies (Chapter 12.3). En-
zyme assays for adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activ-
ity in red blood cells are performed to confirm the diagnosis of SCID caused by the defects of ADA
and PNP, respectively.
10.2.3.3 Advanced tests for evaluation of innate (non-specific) imunity
If there is a suspicion for phagocytic, NK cell or complement defects, further tests must be
performed to evaluate the function of innate imunity components. After neutropaenia and mor-
phological abnormalities of neutrophils are ruled out, functional assays for phagocytosis must be
carried out. These tests include the evaluation of chemotaxis (rarely performed), ingestion and
cidal activity (Chapter 6.1.2). The neutrophil oxidative burst (metabolic activation) can be exam-
ined with nitroblue tetrazolium test (NBT) or dihydrorhodamine 123 (DHR) flow cytometric assay
(Chapter 6.1.2.6); oxidative burst and the production of reactive oxygen species are abnormal in pa-
tients with chronic granulomatous disease. Flow cytometry is also used to assess the CD18 (and
sion deficiency syndrome type 1 (LAD1) have typically very low to absent expression of β2-inte-
CD11a, CD11b and CD11c) and CD15 expression on granulocytes. Patients with leukocyte adhe-
cells due to the mutation in the gene encoding the common β2 chain (CD18). CD15 (Sialyl-Lewis
grins CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1, CR3) and CD11c/CD18 (CR4) on white blood
X) expression is absent on neutrophils of patients with LAD2.
If clinical presentations suggest a possible presence of complement deficiency, appropriate
tests should be performed to evaluate the integrity of individual pathways of complement activation
and assess the levels of complement proteins. Primary complement deficiencies are very rare and re-
sult in defective direct killing, opsonisation and phagocytosis of bacteria and ineffective clearance of
immune complexes. The intact function of classical complement pathway is evaluated by total
haemolytic complement activity (CH50) assay, while the activity of alternative pathway is evaluated
in similar way by AH50 assay. Alternatively, non-haemolytic functional assays can also be used
(Chapter 5.1.2.2). As these tests only identify a functional abnormality in respective whole comple-
ment activation pathway and do not provide any information about which particular complement
component is affected, further assays for measurement of complement protein levels (Chapter 5.1.2.3)
and/or functional assays for individual complement factors must be carried out (Chapter 5.1.2.4).
Putative defects in TLR-mediated signaling can be evaluated by measuring IL-6 and TNF-α
production after stimulation of whole blood with TLR agonists, however, this test is used only in
a limited number of laboratories. Natural killer cell cytotoxicity can be evaluated in vitro by
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Diagnostics of immunodeficiencies
chromium release assay (see Chapter 6.2.2) in addition to flow cytometry assays detecting the pres-
ence of perforin, SAP and XIAP proteins.
10.2.3.4 Molecular diagnostics of primary immunodeficiencies
Mutation analyses are an integral part of diagnostic strategy in PIDs and an important tool
for identification of specific disease-causing mutations. They further form a basis for adequate
treatment, estimation of prognosis, development of preventive strategies, genetic counselling and
prenatal diagnostics. Detection of genetic defects (single base pair substitutions, small deletion
and insertions or gross lesions) can be performed at DNA or RNA level by various PCR methods
and DNA sequencing. Identification of a new gene variation must usually be supplemented by an
analysis of mRNA and protein expression or function to evaluate its real disease-causing poten-
tial. In addition to these approaches, several novel diagnostic tools based on the methods of mo-
lecular biology are being currently developed and introduced into the clinical practice, including
those for the detection and quantification of defects of T cell receptor assembly and T cell devel-
opment (DiGeorge syndrome, RAG1/2 deficiencies and others).
SUMMARY AT A GLANCE
•Immunodeficiencies (IDs) are disorders caused by quantitative or qualitative defects in immune system
components, which result in increased susceptibility to infections, malignancies or autoimmune diseases.
•If an immunodeficiency is suspected, its proper diagnostics requires detailed anamnesis, physical exam-
ination and various basic and advanced laboratory tests for the evaluation of immune system function.
•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 evalu-
ation of phagocytic and cidal activity and the respiratory burst of polymorphonuclear phagocytes. The
evaluation of complement system integrity includes complement activity assays, assays for measure-
ment of complement protein levels and functional assays for individual complement factors. The func-
tion 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 de-
layed-type hypersensitivity skin tests.
•The disease-causing mutations can be detected with the help of PCR or sequencing methods.
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11. Diagnostics and monitoring of HIV infection
11.1 Introduction
The first mention of what would eventually become acquired immunodeficiency syndrome
(AIDS) was first reported in 1981 in the United States when several cases of pneumonia caused by
rare opportunistic Pneumocystic jiroveci (carinii) followed by other pathologic conditions were ob-
served in the community of healthy gay men and subsequently among intravenous drug users,
haemophiliacs, blood transfused patients and eventually heterosexual majority. The identification
of the causal agent, the human immunodeficiency virus (HIV), followed in 1983. Since then AIDS
has become a global epidemic responsible for approx. 30 million deaths.
HIV is a member of the Lentivirus genus and the Retroviridae family, currently grouped into
two types: HIV-1 and HIV-2. The more prevalent HIV-1 is classified into several groups (M, N,
O and recently described P), the group M is further subdivided into 11 subtypes (clades) and nu-
merous circulating recombinant forms (CRFs). HIV-2 is mostly confined to West and Central
Africa; it shares only 30 – 40% homology with HIV-1 and is further subdivided into 8 groups
(A-H). HIV-1 is of spherical shape and its core contains two identical copies of single-stranded
RNA bound to protein p7 and enzymes reverse transcriptase, integrase and protease. This complex
is enclosed by a capsid composed of protein p24, which is, in turn, surrounded by a matrix made
of protein p17. This is anchored to the inside of the viral envelope composed of phospholipids and
proteins derived from the membrane of infected cell and viral glycoproteins gp120 and gp41
(Fig. 11.1). HIV infects numerous host cells includ-
ing the CD4+ T cells, monocytes and macrophages,
dendritic cells, eosinophils and microglial cell of
the nervous system. The replication of the virus
within the helper T cells and their destruction by
host immune mechanisms results in their progres-
sive depletion, the collapse of the immune system
and the progression of HIV infection to AIDS.
Fig. 11.1 Schematic structure of the human immuno-
deficiency virus
Following its transmission, HIV first infects the cells at the site of the primary contact (cervix,
vaginal or anorectal mucosa) and is eventually tranferred to the regional lymph nodes. The ex-
tensive viral replication within the infected mucosa and lymphatic tissue eventually results in the
spreading of HIV into the bloodstream and its dissemination. The virus RNA becomes detectable
in the blood within 10 – 12 days after infection start and its levels increase rapidly up to 1 million
copies/ml. Simultaneously, HIV p24 antigen may also be detected in the plasma of infected pa-
tients (in general 12 – 14 days after exposure). At the same time, the number of CD4+ T cells in the
blood temporarily decrease (Fig. 11.2). The high levels of HIV viraemia and antigenaemia are short-
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Diagnostics and monitoring of HIV infection
lived due to the activation of host humoral and cellular immune responses, which start to control
the viral replication, drastically reduce RNA titers to low steady level or below detection level and
cause the rise of CD4+ T cell numbers to levels similar to those present before the infection. These
changes corresponds with the appearance of specific antibodies (first IgM, later IgG class) against
the viral antigens in the plasma (the so called seroconversion), which can be detected by modern
serologic methods approx. 3 – 4 weeks (22 days on average) after the exposure to HIV. The time
period in which the infection is present but the antibodies are not detectable yet is known as sero-
logic “diagnostic window period” (Fig. 11.2). In some circumstances, it may take significantly
longer than few weeks to generate specific HIV antibodies, as the duration of serologic “window
period” is rather variable and depends on several factors, including the way of transmission,
dosage, subtype of HIV, age, health and immune status of infected individuals and others. Dur-
ing the acute HIV infection, most of the infected individuals present influenza-like or mononu-
cleosis-like symptoms and signs lasting between 7 – 10 days. After several weeks from the start of
the infection, HIV positive individuals enter the asymptomatic period characterised by the ab-
sence of any symptoms, low viraemia levels and the presence of HIV-specific antibodies in the
plasma (Fig. 11.2). The continuous replication of the virus in the lymphoid compartments of un-
treated patients leads to slow progressive depletion of CD4+ T cells and finally results in the im-
pairment of the immune system. The drop of CD4+ T cell counts below 200/μl is associated with
repeated increase of viraemia and the development of AIDS, the final stage of HIV infection char-
acterised by the presence of
various opportunistic infec-
tions, tumours, generalized
lymphadenopathy, encepha-
lopathy, reduction of weight
and some other symptoms.
Fig. 11.2 Dynamics of changes
of HIV RNA, p24 antigen, anti-HIV
antibody and CD4+ T cell levels in
the course of HIV infection
11.2 Laboratory methods in HIV diagnostics
First blood tests for HIV infection were introduced in 1985 and since then, their quality has
been continuously improving. The main purposes of HIV testing are identification of infected in-
dividuals and screening of blood supply in order to facilitate effective preventive and therapeuti-
cal measures. According to the UNAIDS recommendations, HIV testing should be voluntary,
informed, available to anyone at request and at no charge, taken either confidentially or anony-
mously and coupled with pre- and post-test counselling. Laboratory tests used for the identifica-
tion of HIV infection can be divided into two main categories: indirect and direct tests.
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11.2.1 Methods of indirect diagnostics
Indirect methods comprise several screening and confirmatory tests based on the detection
of specific antibodies to HIV antigens in various body fluids including the full blood, plasma,
serum, urine and oral fluid.
Fig. 11.3 ELISA assay for detection of anti-HIV antibodies
Enzyme-linked immunosorbent assays (ELISA) are the most commonly used quantitative
screening tests in the diagnostics of HIV infection. The first generation assays were indirect ELISA
tests that included whole-cell lysate HIV antigens obtained from infected cell cultures. Briefly,
the viral lysate was bound to the solid phase at the bottom of a microtiter plate and allowed to
react with added tested serum. If the serum contained specific antibodies against HIV anti-
gens, the antigen-antibody binding had occurred. After the washing step, the bound antibod-
ies were detected by secondary antibodies coupled with an enzyme. After another washing
step, a substrate was added and the enzyme generated its chemical conversion to a colou-
red product (Fig. 11.3a). The intensity of the colour reaction was proportional to antibody con-
centration in the tested sample and was measured by spectrophotometer. These assays were
not sufficiently specific and enabled the detection of specific antibodies 6 – 12 weeks after in-
fection onset. The second generation assays were based on the same indirect approach, but used
HIV recombinant antigens instead of viral lysate. This led to improved specificity and reduced
window period to approx. 5 weeks. The third generation assays first introduced in 1990s employs
a sandwich ELISA format. Here, the recombinant HIV-1/2 protein and peptide antigens are
bound on a solid phase (wells of microtiter plate or microparticles) and allowed to react with
specific anti-HIV antibodies contained in tested patient's serum. In the next step, the antigen-
bound antibodies are detected by addition of the same viral antigens already coupled with
an enzyme molecule. The reaction is subsequently visualised by an appropriate substrate
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Diagnostics and monitoring of HIV infection
(Fig. 11.3b). This approach enables to detect any potential classes of anti-HIV antibodies and
thus ensures higher sensitivity and specificity and allows to shorten the diagnostic window to
about 3 weeks. Recently, the fourth generation assays have been introduced, combining the de-
tection of specific HIV-antibodies with the direct detection of HIV p24 antigen. This has al-
lowed further reduction of the diagnostic window to less than 2 weeks and the identification
of approx. 80 % of all acute HIV infection cases in the pre-seroconversion phase, which would
otherwise have been missed by the third generation ELISA tests. Today’s combined ELISA tests
and similar chemiluminiscence immunoassays (CIA) are highly sensitive (antibodies with even
low titers and avidities are detected), sufficiently specific and enable the detection of HIV-1,
HIV-2 as well as M and O group infections.
Rapid tests are qualitative screening diagnostic assays designed to detect antibodies to HIV
within 30 minutes or less. They may be performed with whole venous blood, fingerstick capillary
blood, plasma, serum, urine and oral fluid samples, have easy procedures and some require little
training to perform and interpret. These tests are mostly based on immunofiltration (flow through)
or immunochromatographic (lateral flow) methods and have sensitivity and specificity similar to
standard ELISA antibody tests. Although they are more expensive than standard ELISAs, they are
useful in situations when the result is needed quickly for profylaxis and prevention, for instance
in emergency and autopsy rooms, small blood banks, before emergency operations, in pregnant
women in labor with unknown HIV status, occupational and non-occupational post-exposure set-
tings (e.g. such as needlestick injuries) or when the person who is being tested is unlikely to return
for a follow-up visit to learn the results. Similarly to classic ELISA screening tests, reactive samples
still require further confirmation with Western blot.
Simple tests are screening assays that can be easily performed without instrumentation and
thus are extremely popular in the developing countries. They are based on agglutination method
where the HIV antigens are bound to particles and allowed to react with antibodies contained in
tested serum to form visible clumping. The technique is called passive haemagglutination when
the red blood cells are used, or latex agglutination when the antigens are attached to latex parti-
cles (Chapter 3.1.3).
The Western blot (WB) is the most commonly used confirmatory assay for HIV diagnostics,
due to its high specificity and ability to discriminate between specific antibodies against individ-
ual antigens. Following the disruption of the HIV virus, individual HIV proteins (antigens) are
separated by electrophoresis according to their molecular weight, transferred onto a nitrocellu-
lose membrane and subsequently used to detect specific antibodies in the sample. In case of their
presence in the sample, anti-HIV antibodies bind to antigens on the membrane and are subse-
quently visualised by secondary enzyme-labelled antibodies (Fig. 11.4). According to the presence
of bands on the paper strip, the results can be read as positive, negative or indeterminate (see the
next chapter). Many commercially available WBs now include antigens from both HIV-1 and HIV-
2. The disadvantages of WB are its high costs, a certain degree of subjectivity when interpreting
the results and frequent occurrence of indeterminate results. A similar assay called the line im-
munoassay (LIA) uses recombinant HIV proteins and/or synthetic peptides that are directly ad-
sorbed onto a nitrocellulose test strip, thereby avoiding difficult-to-control processes such as
electrophoresis and blotting. The use of recombinant and/or synthetic antigens provides highly
reproducible results with high sensitivity and specificity.
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Fig. 11.4 Western blot for detection of anti-HIV antibodies
The indirect immunofluorescence assay (IFA) is another confirmatory assay in which the
tested serum is let to react with HIV-infected cells (usually lymphocytes) and with control unin-
fected cells on a microscope slide. The specific antibodies bound to intracellular HIV are subse-
quently visualised by added secondary antibodies labelled with a fluorochrome as the indicator
system. The results are then analysed microscopically by experienced laboratory staff. Although
IFA has less indeterminate results when compared to WB, its use is limited by the need for ex-
pensive equipment and subjective interpretation of results.
11.2.2 Methods of direct diagnostics
Methods that enable the direct detection of virus and its components in the infected organism,
and thus allow for further reduction of the diagnostic window include viral isolation through viral cul-
ture, detection of HIV antigens and nucleic acid amplification testing for the detection of viral RNA
and DNA. Although these methods are highly specific, they all have certain limitations. Viral culture
tests were the first direct method to detect the HIV virus itself, but are slow, expensive and difficult
to perform. Therefore, other direct methods are used instead. The p24 antigen appears in blood within
2 weeks from infection due to the initial burst of virus replication and becomes detectable by HIV anti-
gen tests based on the ELISA methodology. The antigenaemia lasts approx. 3 to 5 months and then
antigen levels drop below detection limit. With the progression to AIDS, p24 antigen becomes de-
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Diagnostics and monitoring of HIV infection
tectable again (Fig. 11.2). The antigen testing is currently used for blood and blood product screening
and is also valuable for detecting acute HIV infection, diagnosing infection in the neonates and mon-
itoring the antiretroviral therapy. The direct p24 antigen detection is also included in the fourth gen-
eration ELISA screening tests for HIV antibodies (see the previous chapter).
The nucleic acid amplification tests (NAAT) are commercial tests for the identification of ei-
ther viral RNA or proviral DNA. They represent the very first method capable of identifying the
presence of the virus in patients with acute HIV infection. However, due to their high costs and
higher requirements for specialised laboratory equipment and trained personell, NAATs have not
been used as standard screening or confirmatory tests. Amplification and detection of proviral DNA
by conventional or real-time PCR methods enables the detection of sequences of HIV genome
within peripheral blood mononuclear cells infected with quiescent provirus DNA as well as cells
with replicating virus. This approach is currently used for the detection of infection in infants born
to mothers infected with HIV. Qualitative and quantitative detection of viral RNA (viral load testing) by
methods based on target or signal amplification, such as reverse transcriptase PCR (RT-PCR), nu-
cleic acid sequence-based amplification (NASBA), branched-DNA (b-DNA), transcription medi-
ated amplification (TMA), real-time PCR, ligase chain reaction (LCR) and some others, can
supplement for antibody detection methods in situations when acute HIV infection is highly sus-
pected and antibodies are not yet detectable. Measurement of blood plasma HIV RNA concentra-
tions (viral load assay) is also a standard test used to monitor the progress of infection in HIV
positive individuals and to evaluate the efficacy of antiretroviral therapy. RNA detection assays are
also used for screening of blood products. Standard assays have a limit of detection of 400
copies/ml, whereas ultrasensitive assays may detect viral loads as low as 5 – 50 copies/ml.
11.3. Diagnostic algorithms
11.3.1 Identification of new cases of HIV infection
The diagnostic protocol for HIV infection commonly requires the identification of infected
individuals by screening tests and the confirmation of the positivity by confirmatory (supple-
mental) test. In most cases, laboratory tests for the detection of specific HIV antibodies are used.
As the antibodies only become detectable 3 – 4 weeks after the infection with HIV, the correct di-
agnosis of acute infection sometimes requires the use of direct methods. HIV testing should always
be accompanied by appropriate pre- and post-test counselling.
The diagnostic procedure starts with the use of appropriate screening test that possess a high
degree of sensitivity i.e. is designed to detect all individuals positive for HIV antibodies, even if their
titers and avidities are low. Such test should produce only minimum false negative results i.e. only very
few HIV-positive individuals will be missed. Possible causes of false negatives may include acute HIV
infection before the seroconversion, primary or secondary antibody immunodeficiencies or technical
errors during the testing procedure. The most commonly used screening tests are the third or fourth
generation HIV-1/2 ELISA tests, while rapid and simple tests are usually used in certain specific set-
tings such as point-of-care testing and in developing countries. Although all these tests are exquisitely
sensitive, their degree of specificity is slightly limited, resulting in the occasional occurrence of false
positive results. These can result from non-specifically reacting antibodies present in certain diseases
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and conditions, such as other acute viral infections, autoimmune diseases, malignancies or recipients
of trial HIV vaccines. For this reason, every sample producing a reactive result must be tested again
in duplicate using tests with different antigen preparations and/or different test principles, and then
verified with confirmatory assays to differentiate between true and false positive results.
The laboratoty tests used for confirmation (usually WB, alternatively IFA, LIA or NAAT) are
characterised by high degree of specificity and thus produce only few false positive results. If per-
formed and interpreted correctly, the use of several screening and confirmatory tests in tandem
produces accurate and reliable results. The results of the confirmatory HIV-1 Western blot can be
read as negative, positive or indeterminate (Fig. 11.4). While negativity corresponds with the ab-
sence of all bands on the WB paper strip, there are no universal criteria for positivity. According to
WHO recommendations, the positivity requires two Env bands (gp41 and gp 120/160), whereas
the Centers for Disease Control recommend the presence of at least two of p24, gp41 and gp120/160
bands. American Red Cross is even stricter and requires the presence of antibodies to at least one
HIV antigen from each group Gag (p17, p24, p39, p55), Pol (p31, p51, p66) and Env (gp41, gp120,
gp160). Sometimes, confirmatory tests may exhibit indeterminate results (i.e. presence of 1 or more
bands, but not fulfilling the criteria of positivity), which can be in reality either true or false posi-
tives. Truly infected individuals with indeterminate WB results are usually those with early HIV in-
fection undergoing the seroconversion in whom the specific antibodies to individual HIV antigens
are just starting to appear in their blood, or those with advanced AIDS in whom the specific anti-
bodies have disappeared due to the deep depression of host immune system. Other possibility of
indeterminate result in HIV-1 confirmatory WB can be the infection with HIV-2 type. Indeterminate
results in non-infected individuals are usually due to the presence of non-specifically reacting or
cross-reacting antibodies that appear in some acute non-HIV viral infections, autoimmune diseases,
hypergammaglobulinaemia, chronic liver diseases or haematological malignancies, pregnant
women, haemodialysis patients or individuals in HIV vaccine trials. In such situations, the sup-
plemental tests must be repeated within a few weeks or, better, the patient should be additionally
tested with other methods including the HIV-2 WB to detect the infection with less common type
HIV-2 virus, and NAAT to search for the presence of viral nucleic acids. Definitive diagnosis of in-
fection can be established if positivity has been demonstrated in two independent samples. The
patient is then informed about his diagnosis and educated about the nature of the infection, its
prognosis and treatment possibilities, and instructed how to prevent its further transmission.
11.3.2 Diagnostics of primary (acute) HIV infection
Primary (acute) HIV infection refers to the early stages of HIV infection when the antibod-
ies to HIV are not yet detectable. Its correct diagnosis presents a clinical challenge but provides an
opportunity for early treatment and prevention of further HIV transmission, the risk of which is
particularly high in newly infected patients. During this stage of infection, the patients have
markedly increased HIV antigen and RNA levels and majority of them experience non-specific
symptoms of acute HIV disease, including fever, fatigue, headache, pharyngitis, lymphadenopa-
thy, rash, myalgia, arthralgia and some others, which usually persist for 2 – 4 weeks.
Antibody testing of these patients will mostly yield negative results. As viral load levels and
antigenaemia are very high during acute infection and become detectable approximately 1 – 2 weeks
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Diagnostics and monitoring of HIV infection
prior to seroconversion, antibody tests should be performed in conjunction with p24 antigen test
(e.g. using the fourth generation combined assay) and plasma HIV RNA assay when the primary
HIV infection is suspected, e.g. in patients reporting recent risk behavior and presenting symptoms
and signs of the acute HIV syndrome. When high levels of viral RNA are detected in plasma, the
tested person can be preliminary considered to be infected with HIV. Standard antibody tests should
be subsequently performed 3 – 6 weeks later to confirm the presence of specific antibodies.
11.3.3 Diagnostics of HIV infection in infants of HIV-infected mothers
Correct early diagnosis and treatment of HIV infected infants is crucial for reducing their
morbidity and mortality. The laboratory diagnosis of HIV infection in newborns is complicated by
the presence of passively transferred maternal anti-HIV IgG antibodies, which can be detectable
in their blood for up to 18 months. Therefore, the diagnosis of HIV infection in HIV-exposed in-
fants should be established using nucleic acid tests to detect proviral DNA or viral RNA in their
blood. Antibody tests can still be used at 12 to 18 months of age to definitely rule out the infection
in children who were previously negatively tested for the presence of viral nucleic acids.
11.3.4 Monitoring of patients with HIV infection
Once the patient has been diagnosed with HIV infection, several tests should be performed
in regular manner to monitor the clinical progression of the disease, evaluate the immune status
of infected individual and test the viral resistance to antiretroviral therapy. The main goal of HIV
infection management is to keep the levels of viraemia at low levels and to prevent the decline of
CD4+ T lymphocyte numbers below critical levels. For this purpose, the highly active antiretrovi-
ral therapy (HAART) was introduced in 1990s and has quickly proved to be very effective in erad-
icating viruses from blood and expanding the life expectancy of patients. The decision to initiate
treatment with antiretrovirotics is made on the basis of continuous CD4+ T cell and viral load
measurements and the presence of clinical symptoms.
Analysis of CD4+ T lymphocyte counts and CD4+/CD8+ T cell ratio is performed by flow cytom-
etry (Chapter 7.2.2.2) at the time of diagnosis and then regularly every 3 – 4 months to monitor the
immune system status and the progress of infection. CD4+ T cell counts of 350 cells/μl or below are
indicative for initiation of antiretroviral treatment (ART), while cell counts ≤ 200 cells/μl meet the
case definition for AIDS.
Viral load assays include several nucleic acid amplification techniques (RT-PCT, RealTime,
b-DNA, NASBA, etc.) used to quantify the amount of HIV RNA circulating in the blood of an in-
fected individual. In addition to monitoring the progression of HIV infection, the assays are also
used to evaluate the efficacy of ART and detect its potential failure. Viral loads above 100,000 RNA
copies/μl are indicative for initiation of ART.
Drug resistance tests are assays used to detect the development of mutation-based resist-
ance to drugs used for HIV treatment. These frequently occur in patients on ART and are the ar-
gument for the use of alternative ART regimens. Moreover, a substantial percentage of
newly-infected patients already have HIV strain resistant to some of the drugs used for ART. There-
fore, resistance testing should be performed in ART-naive patients after the diagnosis has been
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established and before the initiation of therapy, as well as in patients with treatment failure or in-
sufficient viral suppression during ART. The testing can be performed by genotypic or pheno-
typic assays. Genotypic assays are based on the detection of gene mutations known to be associated
with resistance, using the DNA sequencing, RealTime, microarray or other methods. Briefly, after
HIV RNA is isolated from a specimen and transcribed into cDNA, the gene regions known to har-
bor mutations are amplified by PCR and sequenced. Obtained nucleic acid sequences are then
evaluated by computer software for the presence of mutations. Phenotypic assays enable direct eval-
uation of viral drug susceptibility. They are based on cultivation of a recombinant virus carrying
viral cDNA of HIV strain obtained from the patient in a cell culture with several different drugs.
Subsequently, concentrations of tested antiretroviral drugs required to reduce the growth of the
virus by 50 % are measured and compared to values obtained with drug-sensitive reference strain.
Increase of this value for particular drug compared to reference indicates decreased sensitivity of
patient's HIV to the drug.
Both genotypic and phenotypic assays are also used for co-receptor tropism analyses in order
to determine the dominant type of cellular co-receptor that is used by HIV-infected individual’s
virus to gain access to host cells. This information can be used to decide about potential use of
drugs that target the CCR5 co-receptor. DNA sequencing is also used to determine the group and
subtype of HIV virus in the patient.
SUMMARY AT A GLANCE
•Acquired immunodeficiency syndrome (AIDS) is a secondary immunodeficiency and a terminal stage
of HIV infection.
•The diagnosis of HIV infection is routinely performed by indirect methods (ELISA, Western blot) for the
detection of specific anti-HIV antibodies.
•In case of reactivity in the initial screening ELISA, the test should be repeated in duplicity and conse-
quently confirmed by Western blot.
•In some cases, appropriate direct methods for the detection of viral nucleic acids (PCR) or antigens
(ELISA) must be used. Examples of such situations include acute HIV infection before the seroconver-
sion and diagnosis of HIV infection in newborns of HIV positive mothers.
•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.
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Laboratory methods used in transplantation immunology
12 Laboratory methods used in transplantation
immunology
Transplantation is the transfer of human cells, tissues or organs from a donor to a recipient
with the aim of restoring their functions. Organs or tissues that are transplanted within the same
person’s organism are called autografts (e.g., skin). Transplants that are performed between two
genetically non-identical individuals of the same species are called allografts (or allogeneic grafts).
Allografts can either be obtained from a living donor or a deceased (brain-dead) donor. When
transplantation is performed between different species, like for example placement of primate
hearts into human recipients, it is named xenotransplantation and the grafts are called xenogeneic
(or xenografts). Finally, syngeneic grafts (also reffered to as isografts or syngrafts) are those trans-
ferred between individuals who are genetically identical (e.g., monozygous twins in humans or in-
bred strains in animals).
With respect to location, tissues or organs that are placed in their normal anatomic location are
called orthotopic grafts. However, many transplanted tissues or organs can function in other sites as
well. Grafts that are placed into a site other than their normal one are called heterotopic grafts.
Organs that can be transplanted are the kidneys, heart, liver, lungs, pancreas, intestine, or
thymus. Tissues include bones, tendons, cornea, skin, heart valves, and veins. Worldwide, the kid-
neys are the most commonly transplanted organs, followed closely by the liver and then the heart.
Hematopoietic stem cell transplantation involves the intravenous infusion of autologous or allo-
geneic stem cells to re-establish haematopoietic function in patients whose bone marrow or im-
mune system is defective.
Donor selection is based on multiple factors, however, the most important one is matching of
the donor to a recipient based on HLA typing, called also tissue typing. HLA typing means deter-
mining Human Leukocyte Antigen (HLA) types. HLA complex is the human versions of the MHC
(major histocompatibility complex) that is found in most vertebrates. HLA antigens are cell-surface
glycoproteins of two main groups: Class I molecules (HLA-A, -B, -C) and class II molecules (HLA-
DR, -DQ, -DP). HLA class I antigens are found on the surface of all nucleated cells, while HLA class
II antigens are expressed only on antigen presenting cells as dendritic cells, B-cells and macrophages.
It may be confusing that HLA molecules encoded by certain alleles are reffered to as antigens; how-
ever, this is due their historic discovery and their role in transplantations. HLA antigens serve an im-
portant biologic function in alerting the immune system to the presence of antigens derived from
foreign sources such as bacteria, viruses or malignant cells. HLA molecules must be able to capture
and present antigens of every conceivable type. To do so efficiently, they are extremely polymor-
phic, what means that almost each individual has a unique set of slightly different HLA molecules.
However, this diversity in antigens makes the finding of HLA identical transplant donor difficult.
HLA typing is inevitable since the degree of HLA compatibility between the donor and the recipi-
ent has crucial influence on the outcome of the transplant procedure (Chapter 12.3).
HLA molecules are encoded by a large number of genes located close together on chromo-
some 6. Genes of the HLA complex are categorized into three basic groups: class I, class II, and class
III. There are three main HLA class I genes, known as HLA-A, HLA-B, and HLA-C. The proteins
encoded by these genes are the HLA class I molecules. Six main HLA class II genes exist in hu-
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mans: HLA-DPA1 and DPB1, HLA-DQA1 and DQB1, HLA-DRA and DRB1. HLA class II genes de-
termine two chains of the HLA class II molecules. As explained above, the main function of HLA
molecules is to display foreign peptides to the immune system. The proteins encoded by HLA
class III genes have somewhat different functions; they are involved in inflammation and other im-
mune system activities.
Most HLA genes have hundreds of identified versions called alleles. Most variable (polymor-
phic) are the genes HLA-B and HLA-DRB1. Each known allele is given a particular designation. HLA
genes are passed on by traditional Mendelian inheritance patterns within conserved clusters called
haplotypes as shown in Fig. 12.1. Haplotype is a set of genes (alleles) that is present in each chromo-
some. Each heterozygous individual will have two HLA haplotypes, one in each chromosome (as one
is of paternal and the other of
maternal origin).
Fig. 12.1 Inheritance of HLA
genes
The two paternal haplotypes are designated
as A and B, while C and D represent the two
maternal haplotypes. Since the maternal
and paternal haplotypes can combine in
four different ways, there are four possible
HLA types that can be inherited by the chil-
dren. This means that statistically each child
has a 25% chance of being HLA matched
with any one sibling.
12.1 Haematopoetic stem cell transplantation
Stem cell transplantation offers the only possible curative treatment for different types of
leukemia or lymphoma as well as several non-malignant disorders, such as aplastic anemia or se-
vere immunodeficiencies.
Haematopoetic stem cells originating in the bone marrow have the capacity to develop into
mature circulating blood cells. These pluripotent stem cells used to be obtained from the bone
marrow. Recently they are collected also from peripheral blood or umbilical cord blood. Each of
these cell sources has specific advantages and disadvantages, and each has found particular ap-
plications in the treatment of oncologic or immunologic disorders.
When stem cells are donated from another individual, the transplantation is called allo-
geneic. The most suitable donor of stem cells is a fully HLA matched family member but only
some patients have such a donor. HLA antigens are inherited in dominant Mendelian way, thus
the best chance of finding matches is between siblings. As everybody inherits half of the HLA
antigens from the mother and half from the father, a sibling has a 25% chance of matching her sis-
ter or brother (Fig.12.1).
If a suitable donor is not found within the immediate family a wider family search or unre-
lated donor search is needed. Unrelated bone marrow donor registries and cord blood registries
have been developed to help the cca 70 % of patients needing a stem cell transplant who are un-
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Laboratory methods used in transplantation immunology
able to find a suitably matched donor within their family. Today, over 20 million donors from 48
countries and 47 cord blood banks from 31 countries are listed on Bone Marrow Donors Worldwide
(BMDW), an international database managed by its central office in Leiden, Netherlands.
Some people have a lower chance to find a suitable donor or cord blood unit than others. This
is because some HLA types are more common within the population and some are less common.
In addition, certain HLA molecules are found more often in some racial and ethnic groups than
others, thus a suitable donor is usually of a similar racial or ethnic background.
A stem cell transplant from an HLA mismatched donor can result in a condition when the
recipient’s immune system recognises the transplanted cells as “non-self” and attacks them what
results in the host versus graft reaction (HvG), commonly termed also graft rejection or graft fail-
ure. Likewise, lymphocytes from the donor’s immune system, which are introduced along with the
transplanted stem cells (“graft”) can recognise HLA mismatches and attack the recipient’s organ-
ism (“host”). This condition is called the graft versus host disease (GvHD). GvHD targets mostly
the skin, gut and liver and may become very severe, even fatal. The more compatible the donor-
recipient match is, the less likely the rejection or severe GvHD will occur. To minimize the risk of
GvHD, recipient and donor should be ideally matched at all HLA loci.
12.2 Kidney transplantation
The indication for kidney transplantation is the end-stage renal failure, regardless of the pri-
mary cause. Kidney can be donated by a deceased donor (formerly termed cadaveric) or a living
donor. Donor and recipient matching in kidney transplants reffers to three distinct areas: blood
type matching, HLA type matching, and crossmatching. A well matched kidney is one in which
the blood type between the donor and recipient are compatible, the HLA types are correctly de-
fined and matched and the crossmatch test is negative. Fulfilment of these requirements decreases
the risk of transplant rejection and the need for another transplant. Rejection responses fall into
three general categories – chronic, acute and hyperacute – depending on timing and intensity.
Each type involves particular mechanisms of immune response.
12.2.1 Blood type matching in kidney transplants
ABO blood group antigens are relevant to kidney transplantation as they are expressed not
only on RBCs, but also on other cell types, including the endothelium. The recipient and donor
must have either the same ABO blood type or compatible ones. ABO incompatible transplantation
can cause hyperacute rejection of allograft due to preformed natural antibodies (isohaemagglu-
tinins). The Rhesus blood type is not a factor in donor matching because these antigens are not ex-
pressed on endothelial cells.
Untill recently, kidney transplantation could not be successfully performed unless the donor
and recipient had compatible blood types. Today, a number of centers around the world are per-
forming ABO-incompatible transplants and patients with living donors who do not have a match-
ing blood type can still receive a kidney transplant. This is due to a procedure called plasmapheresis.
Plasmapheresis removes the plasma portion of the blood containing harmful antibodies prior to
the transplant.
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12.3 HLA typing
HLA matching has the greatest clinical impact in both, haematopoetic stem cell and kidney
transplantation results. Donor and recipient should ideally match in at least six HLA antigens
(two HLA-A antigens, two HLA-B antigens, and two HLA-DR antigens), which are the most im-
munogenic ones. The six antigen match was the minimum requirement untill lately. With evolv-
ing clinical practice, especially the provision of safer and more potent immunosuppressive therapy,
the significance of HLA matching has diminished. There is however, a benefit to having a “perfect
match” since the transplanted kidney survives significantly longer.
In heart and lung transplantations practical reasons as ischemic time, availability of donors
and clinical urgency override any matching considerations, although studies have shown it would
be an advantage to match especially in HLA-DR antigens. Corneal grafts are not usually influ-
enced by HLA matching.
In addition to transplantation medicine, HLA typing can be used to help identify individu-
als (forensic testing) or determine if people are related (parentage testing), although now there are
other more specfic DNA-based methods available for these purposes. Moreover, HLA-typing plays
a role in the diagnosis of certain autoimmune diseases. It was observed, that some HLA mole-
cules are associated with specific autoimmune disorders; it means that people with certain HLA
molecules are more likely to develop them. To these diseases belong ankylosing spondylitis, type
I diabetes mellitus or coeliac disease. Relationships have also been documented between certain
HLA and sensitivities to specific drugs.
Two basic approaches in HLA typing are serological and DNA-based typing. According
to these approaches there are two parallel systems of nomenclature that are applied to HLA.
The older system is based on serological determination and antigens are assigned letters and
numbers (e.g. HLA-B27). Later, a newer system based on DNA-typing was developed that al-
lows more precise definition of alleles. In this system, term “HLA” is used in conjunction with
a letter, asterisk and a number (e.g. HLA-DR*15:01) to designate a specific allele at a given HLA
locus.
12.3.1 Serological HLA testing – microlymphocytotoxicity test
Serological (antigen-antibody based) techniques have long been the standard for HLA typ-
ing. These methods rely on the reaction of viable lymphocytes (expressing HLA antigens) with
suitable antisera (anti-HLA antibodies with known specificities). The most common variant is the
complement-mediated microlymphocytotoxicity test in which typing antisera are incubated with
the tested lymphocytes in the presence of complement. The test is performed in microtiter wells
of Terasaki plates and contains several steps:
1. First, viable lymphocytes are obtained from peripheral blood, either by discontinous density
gradient centrifugation or by immunomagnetic beads (Chapter 7.2.2).
2. Isolated lymphocytes are distributed into microtiter wells and a battery of specific anti-HLA
antibodies (antisera) is added to them. Each well contains an antibody directed against
a known HLA antigen.
3. Antibodies and lymphocytes are then incubated at room temperature for 30 minutes. If the
lymphocytes possess the respective HLA antigens, specific antibodies will bind to their surface.
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Laboratory methods used in transplantation immunology
4. Complement is added to each well and incubation continues for another 60 minutes. Dur-
ing this period of time, complement will bind to any cell that is coated with antibody. This
results in lysis of the lymphocytes. If no antibody is coating the lymphocyte, the membrane
remains intact.
5. The assessment of lysed and vital lymphocytes is carried out by a vital staining (using eosin
or trypan blue). Damaged lymphocytes allow the dye to enter the cell, whereas intact lym-
phocytes do not.
6. Results are read using a light or inverse phase contrast microscope. Wells with damaged
cells appear coloured (positive reaction) in contrast to wells with intact cells that appear re-
fractile (negative reaction).
Fig. 12.2 Microlymphocytotoxicity test
12.3.2 Cellular HLA typing
A representative cellular assay is the mixed lym-
phocyte culture (MLC), also called the mixed lymphocyte
reaction (MLR). MLC measures in vitro the cell-medi-
ated response to foreign HLA antigens. Nowadays, MLC
is used only rarely as the test involves the use of radioisotopes, and takes several days to perform.
Today, cellular assays are being replaced by faster and more accurate DNA-based typing methods.
Principle steps of the one-way MLC assay:
1. Recipient lymphocytes are incubated with donor lymphocytes. Donor cells are unresponsive
to antigenic stimuli due to previous treatment with irradiation or mitomycin. These inacti-
vated cells provide foreign alloantigens to the responder recipient cell population. If the
donor cells differ in MHC class II antigens, then the recipient lymphocytes will be stimulated
and start to undergo blastic transformation and proliferate.
2. A radioactive precursor of DNA, [3H]-thymidine, is added to the test system. It incorpo-
rates into the DNA. The uptake of the radioactive thymidine is of course higher in those
cells, which underwent the blastic transformation.
3. The amount of radioactivity is measured by a beta-counter as [3H]-thymidine radiates beta
radioactive rays. The higher the radioactivity, the greater the difference in HLA antigens be-
tween donor and recipient.
12.3.3 Molecular HLA typing
The last two decades are characterised by an exponential growth in the application of mo-
lecular HLA typing techniques. They are DNA-based, what means that HLA types are determined
on the level of alleles (different variants of genes). This approach has significantly increased the
accuracy and specificity of HLA typing and allows more precise HLA matching between donors
and patients. Another advantage is greater specimen flexibility as any nucleated cell or any tissue
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can serve as a source of DNA to be tested. Laboratories are not restricted to use only blood cells
from the patient but they can get the DNA from buccal swabs, frozen blood, dried blood stains,
bone marrow, cultured cells, etc. There are numerous methods and commercial kits available for
simple extraction of good quality DNA.
At this time, almost all HLA testing is done by molecular techniques. Molecular methods are
especially required when performing typing for hematopoietic stem cell transplantation as they are
much more sensitive compared to serologic methods and moreover, they disclose also alleles that
code for molecular products for which no specific antibodies exist, i.e. they are undetectable by
serological techniques.
The majority of molecular HLA typing strategies are based on the polymerase chain reaction
(PCR). The HLA region of the DNA is PCR-amplified providing a large amount of DNA that can
be further analyzed (Fig.12.4). A variety of additional procedures are available to obtain the re-
quired typing resolution. These include involvement of sequence-specific oligonucleotide probes
(SSO or SSOP), sequence specific primers (SSP), and sequence-based typing (SBT).
Fig. 12.3 Polymerase chain reac-
tion (PCR)
12.3.3.1 PCR followed by hybridization with sequence specific oligonucleotides (PCR-SSO)
A single generic primer pair is used for the amplification of the HLA region. The amplified
DNA segment (HLA gene) is immobilized on a membrane. Sequence or allele-specific oligonu-
cleotides (SSOs) are used as probes to detect the presence of the respective DNA segment as it hy-
bridises only to probes with complementary sequences. Probes are labeled, usually by a fluorescent
or enzyme label. Interpretation is usually achieved by entering the final band pattern into a com-
puter programme.
12.3.3.2 PCR using sequence specific primer (PCR-SSP)
PCR-SSP typing is based on using a panel of primer pairs unique to a specific HLA allele or
a group of alleles. Only primers that are completely matched for this DNA segment (specific HLA al-
lele) are able to prime the amplification. Each PCR reaction takes place in a separate tube. Finally, every
112
Laboratory methods used in transplantation immunology
tube should produce either
a specific band or not depend-
ent on whether the respective
allele is present or not. Detec-
tion of amplified PCR segments
is done using an agarose gel
electrophoresis. If a product of
the right size is found, the as-
sumption is that the HLA allele
has been identified.
Fig. 12.4 PCR-SSP
12.3.3.3 Sequence based typing (SBT)
SBT is the only technique, which directly detects the nucleotide sequence of an allele allow-
ing an absolutely exact allele assignment. Sequence based typing involves PCR amplification of the
gene of interest, for example the polymorphic HLA-DRB1 gene, followed by determination of the
base sequence. The sequence is then compared with a database to find comparable sequences and
assign alleles. This method also allows detection of new alleles.
Greatest advantage of SBT is its accuracy. Sequencing requires expensive and sofisticated
equipment, however it seems only a matter of time until it becomes more easy to perform and less
expensive, thus leading to wider use in routine HLA-typing. The newest DNA-based HLA typing
methods include also DNA microarrays (commonly known as DNA chips) based on immobilis-
ing allele-specific sequences to the chip.
12.4 Cross-match
Cross-match is the last laboratory test performed prior to transplantation. It is a sensitive
test which determines whether the recipient produces antibodies to the particular HLA mole-
cules of the potential donor or not. There are several reasons why the patient could be positive
for antibodies to the donor HLA antigens. Most common cause of anti-HLA antibody production
is a previous exposure to foreign HLA molecules through transfusions, transplantations or preg-
nancies. Cross-match is performed by mixing a small amount of the recipient´s serum with a small
amount of potentional donor lymphocytes. Complement is added that causes cell membrane lysis
in case that anti-HLA antibody is present in the serum and has bound to the cells (Fig.12.7). Vital
stain like acridine orange is added to determine cell viability. Percentage of dead versus live cells
is determined by microscopy. In case that increased levels of donor cell death over control values
are observed, the result is positive. A positive crossmatch is a strong indication against trans-
plantation, since it signifies that the recipient has the ability to attack the donor cells, and would
most likely attack the transplanted organ. Researchers develop strategies to remove anti-HLA
antibodies and block their effects to reduce the risk of rejection in positive crossmatch kidney
transplants. A more sensitive and specific crossmatch test has been developed on the basis of
113
Laboratory Metods in Immunology Summary of methods used in immunity testing: Immune status of an individual

flow cytometry or Luminex

bead assay. These techniques
can detect the presence of re-
cipient antibodies on the sur-
face of donor lymphocytes
independent of complement
binding. However, they are
much more expensive and
labour intensive.
Fig.12.5 Crossmatch test
13 Summary of methods used in immunity testing:
Immune status of an individual
Examination of patient’s immune status includes innate and acquired immunity testing.
First step of the examination relies on the anamnesis checking, it means the complete case history
examinations of the patients are reviewed. It involves patient’s previous physical and biochemi-
cal testings. Next step of the patient’s examination involves his own immune status analysis. Both
the humoral and cellular immunity screening can be performed depending on patient’s condi-
tions. As first, the patient’s absolute and differential blood cell count is analysed. Today the process
of counting is being automated using blood analysers. Normal blood cell count has following pa-
rameters: red blood cells 4–5.106/μl, white blood cells 4–10.103/μl and platelets 150–300.103/μl. Dif-
ferential white blood cell count includes 50–75% of neutrophils, 3–5% of eosinophils, 0–1% of
basophils, 15–45% of lymphocytes and 3–10% of monocytes. According to the changes in blood
count we can define the certain disease. For example leukocytosis (increase number of white blood
cells) can be sign of bacterial infection, whereas leukopenia (decrease number of white blood cells)
can be caused by recent cold or influenza infection.

SUMMARY AT A GLANCE 13.1 Analysis of innate immunity

•Transplantation is the transfer of human cells, tissues or organs from a donor to a recipient with the
aim of restoring their functions.
•Central role in graft rejection play HLA genes that encode for the HLA molecules also reffered to as
transplantation antigens, or histocompatibility antigens. The degree of histocompatibility between
donor and recipient can be determined by serologic or molecular HLA typing and by the MLC test.
•Worldwide, kidneys are the most commonly transplanted organs. Donor and recipient matching in kid-
ney 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.
•Graft-versus host disease (GvHD) can develop as a life-threatening consequence of a stem cell trans-
plantation. To minimize the risk of GvHD, recipient and donor should be ideally matched in all HLA genes.
Innate (non-specific) immunity is activated as first after entering a foreign invander to the host.
Its main function is to induce defence against extracelullar microorganisms (e.g. Pneumoccocus,
Streptoccocus, Neisseriae). In diagnostic, an analysis of humoral components, such complement com-
ponents and acute inflammation proteins (CRP) and cellular components, such number and func-
tion of phagocytic cells, is usually performed. For analysis of complement components, mainly C3
and C4, 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).
The assay is based on spectrophotometric measurement of the heterologous red cells haemolysis by
presence of the complement system in a patient serum. If no haemolysis occurs (or haemolytic unit
CH50 = 0), it means that one complement component in the patient serum is missing. Absence of
hamolysis in both the CH50 and AH50 assays is a strong indication for deficiency in one of the
complement terminal pathway components (C5 to C9) or C3 protein. The most frequent comple-
ment components deficiencies are those of C3 and C4 , which are manifested in the patient as re-
current bacterial infections. The deficiency of C1 inhibitor is the cause of hereditary angioedema,
an autosomal dominant disorder, which is characterized as swelling of subcutaneous tissues.
Methods for analysis of acute phase inflammatory proteins levels include radial immunod-
ifusion, rocket immunoelectrophoresis, turbidimetry, and nephelometry. The inflammatory pro-
teins are present in plasma in low concentration, but in response to infections, injuries or
malignancies their concentration increases rapidly (more than 100 to 1000-fold). The main acute
phase proteins are CRP (C-reactive protein), pentraxin 3 and serum amyloid A. Today, the quick
CRP test from 1 blood droplet can be performed by a physician using small device based on tur-
bidimetric principle (NycoCard reader). The CRP test is mostly used as a diagnostic tool to indi-
cate bacterial or viral cause of infection. In most viral infection, the CRP level is below 20 mg/ml,

114 115
Laboratory Metods in Immunology
whereas during bacterial infection the CRP level increases over 50 mg/ml. In such case the pa-
tient requires antibiotic treatment.
Cellular part of innate immunity involves process of phagocytosis. Number and percent
value of neutrophils is determined from the whole blood using automated blood analyzer. Neu-
trophils, as the main phagocyting cells, account for 60 to 70% out of whole blood cells. The func-
tional analysis of phagocytosis is based on evaluation of chemotaxis (chemotactic index), ingestion
(phagocytic activity and phagocytic index), cidial activity and metabolic activation (INT test). The
first step of phagocytosis examination involves isolation of leukocytes, namely polymorphonu-
clear leukocytes (PMNL), from peripheral heparinised blood using 6% dextrane. PMNL contain
approx. 75% of all isolated leukocytes. Such cells are used in function screening tests. To evaluate
the chemotaxis of leukocytes, the length that cells migrate in the presence or absence of a chemoat-
tion ways, the chemotactic index is calculated and compared to reference value (2,76 ± 0,83). Defects
tractant (zymosan) is measuring in the gel. From values of chemotactic and spontaneous migra-
in chemotaxis are usually common in patients with recurrent phlegmonic inflammations of mu-
cous tissues and dermis, moreover defects in the number and motility of phagocytes can result in
the development of sepsis.
To evaluate ingestion of leukocytes, phagocytic activity and phagocytic index is determined.
To do this analysis, patient’s PMNL, serum for opsonisation and inactivated candidas are cultured
by 37°C for 30 mins. Cells smears are stained with the Wright`s dye and analysed under micro-
(reference value 77.1 ± 8.8%), whereas phagocytic index (PhI) expresses average number of candi-
scope. Phagocytic activity (PhA) expresses percentage of PMNL that have phagocyted candidas
das ingested by 1 phagocyte (leukocyte) (reference value 3.6±0.8). Impaired PhA and PhI can be
observed in patients suffering from recurrent bacterial infections, sepsis, terminal stages of ma-
lignancies or some congenital defects of phagocytosis.
Cidal activity evaluates an ability of phagocytes to kill engulfed candida or bacteria. To do
this analysis, patients PMNLs, serum for opsonisation and lived candidas are cultured by 37 °C
for 60 mins. After cell lysis, the smears are stained with vital dye and analysed under micro-
scope. Only dead candidas are coloured, the lived candidas are non-stained. Candidacidal ac-
tivity (%) is determined as the percentage of killed candidas to the whole number of candidas
(reference value 25.4±10.0%). The impaired cidal activity can be found in patients with defects
in microbicidal mechanisms (proteolytic enzymes, production of free radicals, etc.). Finally, we
can also examine metabolic activity of phagocytes using INT test. The principle of the test is
based on the reduction of tetrazolium salts (INT – iodophenyl-nitrophenyl-tetrazolium chlo-
ride, faint-yellow) to the insoluble formasan (rose to violet) by reactive metabolites of oxygen
and reductases, which are produced by respiratory burst in phagocytes. To do this analysis,
patients PMNLs, a serum for opsonisation, INT and zymosan are incubated in wells of a mi-
crotitre plate. A control sample including all above-mentioned components without zymosan
is also examined. The absorbance in the analysed and control sample is measured spectropho-
sample/absorbance of control sample, reference value 5.3 ± 1.3). Defects in metabolic activation
tometrically following the calculation of index of respiratory burst (absorbance of analysed
of phagocytes are common in patients with chronic granulomatous disease that is manifested
as recurrent intracellular bacterial and fungal infections of the skin and organs and formation
of granulomas.
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Summary of methods used in immunity testing: Immune status of an individual
13.2 Analysis of acquired immunity
Acquired immune response, also known as specific immune response, developes after an ex-
posure of our body to antigens. It is composed of humoral (antibodies, cytokines) and cellular
(lymphocytes) components. There are quantitative methods for evaluation of acquired immunity
components based on serum immunoglobulins levels analyses (ELISA) and counting of lympho-
cytes (flow cytometry) in blood samples and qualitative methods based on functional evaluation
of lymphocytes (blastic transformation assay, immunoskin tests).
Adaptive humoral immunity components involve antibodies produced by plasma cells,
which are the latest differentiating state of B lymphocytes. There are five classes of immunoglob-
ulins, namely IgG, IgD, IgM, IgE and IgA. For detection of serum levels of IgG, IgM and IgA, au-
tomatic analyzers based on principle of turbidimetry and nephelometry are used. To detect IgD
and IgE levels, a modified ELISA method was developed. In general, ELISA and their modifica-
tions, are used for detection of specific antibodies, i.e. to viruses and bacteria in serum samples.
Adaptive cellular immunity components involve lymphocytes. To isolate lymphocytes, the
non-coagulated heparin-treated peripheral blood is layered on a separation solution Ficoll-Hypaque
followed by a gradient centrifugation. PBMC cells including lymphocytes are present as white layer
over a separation solution. Isolated lymphocytes can be used either for count analysis of individ-
ual lymphocytes subpopulations or for functional evaluation. An exact count analysis of lympho-
cytes subpopulation is based on flow cytometry that uses the fluorescence-labelled monoclonal
antibodies specific for cell-surface molecules (i.e. CD molecules). Monocytes express CD14, B-lym-
phocytes CD19 and CD20, T lymphocytes CD3 and NK cells express CD16 and CD56. The popula-
tion of CD3+ T lymphocytes can be subdivided into three basic subpopulations, which express
different CD molecules: T helper – CD4, T cytotoxic – CD8, and T regulatory – CD4 and CD25. This
method is used in the diagnostic of primary and secondary immunodeficiency (e.g. HIV-progres-
sion monitoring) as well as in phenotyping of leukemias and lymphomas. In HIV-infected indi-
viduals (secondary immunodeficiency of CD4 cells) CD4/CD8 ratio counting (previously known
as immunoregulation index – IRI) is widely used for monitoring of disease progression and response
to treatment. The ratio is decreased in HIV-infected individuals (IRI<0.5), but increased in patients
with autoimmune diseases (IRI>2.5). Another way for the isolation of lymphocyte subpopulations,
except flow cytometry, involves monoclonal antibodies labelled by magnetic beads; however, it is
mainly used for research purposes. In clinical medicine, transplantation immunology and oncology
mostly utilise this technique. By this procedure, a patient receives a pure population of CD34+ pro-
genitor cells, thus the induction of graft failure can be reduced.
To evaluate patient’s lymphocyte function in vitro, lymphocyte transformation test (LTT) or
blastic transformation assay is used. The test examines lymphocytes response to mitogens or anti-
gens (allergens) in vitro by induction of non-specific cell proliferation and formation of blasts. The
degree of cell proliferation is measured radioactively after adding of radiolabelled 3H-thymidine
(tritiated thymidine). According to the values of stimulation index (SI), extent of blastic transfor-
mation is evaluated. SI values over 2.0 are considered as positive, it means that lymphocytes were
sensitized by testing antigen. The LTT is mostly used in the diagnosis of allergy (food or drug-in-
duced). In this case, an exposure of examined person lymphocytes to allergens is analysed; gold
salts, amalgam, HgCl2, Ni and Be are those the most tested. However, LTT measures only the sen-
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sitization of lymphocytes but not an effector reaction. Thus the positive LTT indicates only that the
person suffers from allergy to tested substances, however to confirm it, clinical symptoms should
be always considered. For verification of the diagnosis, other tests should be also performed (IgE
tests, skin tests).
To evaluate patient’s lymphocyte function in vivo, immunoskin test is used. The test is based
on an injection of tested antigens into dermis of the forearm. After 24 to 48 hrs, the immune re-
sponse (induration and erythema) is evaluated. These symptoms are caused by delayed type of hy-
persensitivity reaction (increased activity of macrophages, dendritic cells and T cells). By means
of immunoskin tests, we can examine immune response to killed bacteria, their proteins or aller-
gens, e.g. tuberculin is used to establish the diagnosis of tuberculosis and similarly, allergens for
the diagnosis of contact allergy.
Screening of autoantibodies plays a key role in establishing the diagnosis of autoimmune
diseases (AD). There are many autoantibodies recognizing autoantigens of various organs and
tissues. The most common are antinuclear antibodies (ANA) and rheumatoid factor. Methods for
the autoantibodies screening involve indirect IF assay, ELISA and immunoblotting. Rheumatoid
factor (RF) involves autoAb of IgM class to Fc domain of IgG and is mostly present in patient suf-
fering from rheumatoid arthritis. RF screening is based on latex agglutination or ELISA method.
To perform latex agglutination, latex particles coated by human IgGs are quickly mixed with a pa-
tient’s serum in the well of special agglutination plates. The test is positive, when clumping of
latex particles – agglutination occurs.
Analysis of circulating immunocomplexes (CIK) levels is performed by mixing a patient′s
serum with PEG (polyethylenglycol); an incubation for 1 hour follows. During this time, a tur-
bidity is forming and its level is measured by nephelometry or turbidimetry. The test is positive
(high concentration of CIK), when the double of a reference value has been measured (reference
value: 25–60). To detect IK in tissues directly, immunohistochemistry and immunofluorescence is
performed. Screening of patients with AD also includes determination of Ig level (ELISA), analy-
sis of C3 and C4 complement components level (nephelometry and turbidimetry), determination
of number of T cells in the peripheral blood (flow cytometry), determination of CD4+/CD8+ ratio
by flow cytometry (IRI is increased over 2 by autoimmunity) and HLA-typing/sequencing.
The immunodiagnosis also involves methods to determine HLA alleles or antigens (HLA typ-
ing methods). Besides ABO blood group compatibility, HLA matching between donor and recipient
belongs to important requirements reducing a graft rejection, especially for kidney transplantations.
It has been shown that six HLA antigen-match is important for a successful outcome, i.e. donors and
recipients should be indentical in HLA-DR, -B, and A alleles (DR>B>A). There are serological (mi-
crolymphocytotoxicity assay), cellular (MLC test) and molecular-biological (PCR-SSP, sequencing
methods used in HLA typing, PCR-SSP being the most widely used method. Microlymphocytotoxi-
city assay belongs to serological methods of HLA-molecules typing and is based on screening of cell
membrane HLA-molecules by antisera (include Abs against HLA-molecules). After complement
adding and eosin staining only dead cells (destructed by activation of complement) are red-stained.
The percentage of dead cells is determined under microscope and the result is positive, when more
than 50% of cells are dead. Microlymphocytotoxicity assay is used in immunodiagnostic for cross-
match screening and is based on detection of HLA-Abs in patient’s blood (recipient) against donor
lymphocytes. A positive cross-match is an absolute contraindication of transplantation independ-
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Summary of methods used in immunity testing: Immune status of an individual
ent to a cross-match positivity grade. Cellular methods of HLA screening involves mostly MLC
test (mixed lymphocyte culture test). The test determinates the HLA class II molecules matching
between a donor and a recipient. Using this assay, lymphocytes from the donor and the recipient
are mixed and incubated for 5 days. During this period, lymphocytes from one individual prolif-
erate in response to foreign HLA antigens of other individual. Lymphocytes are then incubated
with tritiated thymidine for 6 hours (thymidine incorporates into DNA of proliferating cells), fol-
lowing radioactivity measuring. Finally the stimulation index (SI) is calculated. If the SI value is
over 2, it indicates the identity in HLA- antigens between tested individuals.
Methods for HLA-genotyping involve DNA-based methods like PCR-SSP or sequencing. PCR-
SSP method is based on a direct DNA amplification of one specific HLA allele using sequence
specific primers (SSP). The advantage of this method is the typing of several alleles in one DNA
sample at once. Another method for HLA-genotyping is sequencing. This method detects exact nu-
cleotide order of an analysed DNA fragment and thus determines the patient′s HLA allele. The
HLA-typing is also used in diagnostic of autoimmune diseases because of associations of some
HLA alleles to certain AD, e.g. HLA-B27 in patients suffering from morbus Bechterev, HLA-DR3,
HLA-DR4 or HLA-DQ8 in patients with type I diabetes mellitus or HLA-DR2 in those who devel-
oped sclerosis multiplex.
To establish the diagnosis of allergy, in vitro and in vivo laboratory tests are performed. In
vitro laboratory tests involved IgE level screening. Total serum IgE level is determined by ra-
dioimunosorbent test (RIST), which is based on competition of known amounts of radiolabeled IgE
with the patient's unlabeled IgE to bind to a surface coated with anti-IgE (competitive radioim-
munoassay). The reduction of radioactivity of control IgE level due to the presence of IgE in the
patient's serum is determined by comparison with known IgE standards. Values of IgE between
100 to 150 IU/ml indicate the presence of allergy. To analyse specific IgE levels, radioallergosorbent
test (RAST, non-competitive radioimmunoassay) is mostly used. The test is based on detection of
specific serum IgE antibodies to suspected or known allergens. Allergens are bound to carriers
and the patient's serum is added. After incubation radioactive-labelled secondary anti-IgE is
added, that binds to the patient′s IgE. After washing, the radioactivity is measured; its amount is
proportional to the serum specific IgE antibodies. RAST test is scored on a scale from 0 to 6, the
positivity determines up to the score 3.
Nowadays, other sophisticated methods for in vitro diagnostic test of allergy have been used.
The mostly performed is ImmunoCap method based on modification of ELISA. This test is highly
sensitive and involves simultaneous measurement of specific IgE antibodies to multiple allergens
in a single test. Allergens are immobilised to specific carrier consisting of cellulose and agarose
polymer (CAP). In the assay, IgE in the patient’s serum are bound to the allergens. After a wash-
ing step, allergens – bound antibodies are detected by enzyme-labeled antibodies. After a sub-
strate addition, the fluorescence is measured. The latest modification of this method known as
ImmunoCap ISAC uses fluorescence-labelled antibody for detection of allergen bound antibodies.
Test results are measured by means of a laser scanner and evaluated using a software. Results are
reported in ISAC Standardised Units (ISU).
Basophil activation test (BAT) is based on the detection of activatory antigens CD63 or
CD203c in membranes of basophils by flow cytometry. These CD molecules are being expressed
in their surface after stimulation only, in our case when whole blood cells or isolated leucocytes
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inhalants, hymenoptera venoms, some food allergens and medicaments (e.g. β-lactam antibiotics).
are exposed to tested allergens. The test is used for diagnosis of allergies induced by latex, some
Other laboratory test to prove the drug hypersensitivity reaction includes LTT test.
In vivo diagnostic tests of allergy involved skin tests. The mostly used are prick test and
patch test. Prick test is used in diagnostics of type I hypersensitivity, i.e. allergies on inhalants, e.g.
pollens, pet dander, dust mites, etc. By this skin test, purified allergens available in commercial test-
ing sets are applied on a cleaned skin of volar forearm or upper back (children) as tiny droplets.
They are subsequently pricked into skin’s surface (1 mm deeply) with a special needle or lancet;
the distance between the pricks should be at least 5cm. 10-20 minutes later, induration and ery-
thema (wheal and flare) can be observed and its diameter is measured. The test is positive when
the diameter is more than 3 mm wide. In patch test, the allergens are mixed with a non-allergic
material (base) to a suitable concentration. They are then placed in direct contact with the skin, usu-
ally on the upper back, within small aluminium discs. Adhesive tape is used to fix them in place.
The patches are left in place for 48 hours (diagnosis of type IV hypersensitivity), when a skin re-
action is evaluated; its intensity can vary from erythema without or with papula or pustule. In
some cases, epithalaxia (separation of epidermis) can be observed. Allergens represent substances
causing contact dermatitis, e.g. nickel, leather, chemicals, fragrances, medications, hair dyes. A neg-
ative test does not mean that the patient is not allergic, simply it reflects that either the right con-
centration was not used or the body failed to elicit a response.
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Summary of methods used in immunity testing: Immune status of an individual
SUMMARY AT A GLANCE
•Examination of immune status includes innate and acquired immunity testing. Both, the humoral and
cellular immunity screening can be performed depending on the patient’s condition. Firstly, the ab-
solute and differential blood cell count is analysed. According to the changes in blood count we can
diagnose the disease and proceed with further immune analysis.
•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). The functional analysis of phago-
cytosis is based on evaluation of chemotaxis (chemotactic index), ingestion (phagocytic activity and
phagocytic index), cidial activity and metabolic activation (INT test).
•In diagnostic of acquired (specific) immune response, an analysis of humoral components like anti-
bodies and cellular components, like B and T cells is performed. 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 lym-
phocyte (blastic) transformation test (in vitro) and skin tests (in vivo).
•In the diagnostic of autoimmune diseases (AD), an analysis of autoantibodies, rheumatoid factor, cir-
culating 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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