Laboratory Methodsin Immnunology

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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 Short introduction to immunology
include body surfaces such as the skin and the mucous membranes. Effective barriers against in- stem cells into immunocompetent cells take place. Primary lymphoid organs are the thymus and
vasion of many microorganisms constitute different chemical factors: tears, saliva, sticky mucus the bone marrow. Thymus is a bilobed organ located in the mediastinum that is responsible for the
in the respiratoty and gastrointestinal tracks or hydrochloric acid and proteolytic enzymes in the differentiation and maturation of T cell progenitors into T cells. Bone marrow is the ultimate source
stomach. If an invader passes these physical and chemical barriers, it is attacked and destroyed by of all blood cells and it is responsible for the differentiation and maturation of B cells. Mature T
other internal elements of the immune system, either humoral or cellular. Innate humoral imu- and B cells exit the primary lymphoid organs and are transported via the bloodstream to the sec-
nity is mainly conferred by the complement system and other soluble substances that are harmfull ondary lymphoid organs.
to microorganisms (degradative enzymes, acute-phase proteins, interferons). Innate cellular imu- Secondary lymphoid organs are those where mature lymphocytes are activated to respond
nity is mediated by phagocytic cells: granulocytes, monocytes and macrophages. Numerous other to invading pathogens. They are arranged as a series of “filters” monitoring the contents of the ex-
components are also features of innate imunity, e.g. pattern-recognition molecules, which can bind tracellular fluids, i.e. lymph, tissue fluid and blood. They include spleen, lymph nodes, tonsils,
to various microorganisms. 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
1.2.2 Adaptive immunity 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.
Adaptive (specific, acquired) immunity developed late in evolution and is present only in Lymph nodes act as filters that capture the antigens brought by the lymph. The lymph nodes
vertebrates. It is more specialized than innate imunity and it supplements the protection provided contain two basic parts, the cortex and the medulla.
by innate imunity. Adaptive imunity develops with exposure to various foreign agents (antigens) The outer cortex is populated predominantely with B cells arranged as follicles, which may
and reacts specificly to that antigens only. The initial contact with the antigen (called immuniza- develop a germinal center when challenged with an antigen and the deeper cortex mainly con-
tion) triggers a chain of events that lead to the activation of the two arms of adaptive immunity tains T cells. Medulla contains large blood vessels, sinuses and medullary cords that contain
with different sets of participiants. One of these two arms is mediated mainly by B cells and cir- plasma cells. Mucosa-associated lymphoid tissues are associated with mucosal surfaces of almost
culating antibodies and it is reffered to as humoral. The other is mediated by several phenotypi- any organ, but especially those of the digestive, genitourinary, and respiratory tracts, which are
cally distinct subpopulations of T cells, which either directly kill virus-infected, tumour or foreign constantly exposed to a wide variety of potentially harmful microorganisms and therefore require
cells (cytotoxic T cells) or synthesize and release various substances called cytokines that affect their own system of antigen capture and presentation to lymphocytes.
other cells and control the entire immune response (helper T cells and regulatory T cells). Hence Apart from lymphocytes (already mentioned above), pluripotent haematopoietic stem cells
this arm of adaptive immunity is termed cellular or cell-mediated. give rise to other cell types. Neutrophils, eosinophils, and basophils are collectively known as
Some T and B cells become memory cells and provide “immunologic memory”. Memory al- granulocytes which circulate in the blood unless recruited to act as effector cells at sites of infec-
lows to respond faster and more efficiently when the immune system is exposed to the same anti- tion and inflammation. Macrophages and mast cells complete their differentiation in the tissues
gen repeatedly. In many cases, an adaptive immune response confers lifelong protective immunity where they act as effector cells in the front line of host defense and initiate inflammation.
to reinfection with the same pathogen. The third cell type that participates in adaptive immunity Macrophages phagocytose bacteria, and recruit other phagocytic cells as neutrophils, from the
are antigen-presenting cells (APCs) such as dendritic cells, monocytes, macrophages. Their im- blood. Mast cells are exocytic and they are responsible for the defense against parasites as well
portant function is to process and present the antigen to the T cells. as triggering allergic inflammation; they recruit eosinophils and basophils, which are also exo-
Innate and adaptive arms of the immune system have devoloped an interrelationship. They cytic. Dendritic cells enter the tissues as immature phagocytes where they specialize in ingesting
interact through various cytokines and cell adhesion molecules, send each other signals, activate and presenting antigens to the T cells.
each other and operate in concert to generate a specific and effective immune response that leads As a part of the immune system are also considered antibodies, cytokines, complement pro-
to the elimination of the invader. teins, degradative enzymes, acute-phase proteins, different receptors, adhesion molecules, etc.
1.3 Structures of the immune system 1.4 Immunological methods
The immune system is formed by a network of organs, cells and molecules. Though it is Immunology has always stimulated as well as made use of technology, such as microscopy,
a very complex and highly developed system, its mission is simple: to recognise and destroy for- electrophoresis, fluorescence, DNA-based methods, etc. Probably the biggest catalyst of progress
eign invaders. in immunology and in many other biomedical areas was the development of a revolutionary tech-
The organs of the immune system are positioned throughout the organism. They are called nique of monoclonal antibody production in 1975 by Köhler and Milstein. Monoclonal antibody
lymphoid organs as they are home to lymphocytes, the key cells of the immune system. Primary technology is a good example how immunology has influenced not only medicine but also sciences
lymphoid organs are those, where differentiation, proliferation and maturation of pleuripotent ranging from molecular biology to agriculture and food industry.
12 13
Laboratory Metods in Immunology Material collection and processing for immunological examination
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 2 Material collection and processing
component of tests used to diagnose infectious diseases. Antibody is used to detect pathogens with for immunological examination
techniques such as latex agglutination, enzyme-linked immunosorbent assay and many others.
Since the 70-ies, immunological laboratory methods have gradually become more progres- Collection and material processing depends on the intended immune analyses. To evaluate
sive and simple to perform. Because of their improved specificity and sensitivity, immunological immune status of a patient, humoral and cellular immunity screening is usually performed. Each
techniques have now achieved a major role in modern clinical laboratory science. Many of them analysis includes different procedures of material processing.
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 2.1 Collection and blood processing for humoral immunity screening
of conditions that include allergies and other hypersensitivity reactions, autoimmune disorders,
immunodeficiencies, posttransfusion and posttransplant reactions. The following chapters of this To perform humoral immunity screening, serum and plasma are obtained by blood pro-
textbook contain an overview of the basic immunological methodology. 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-
14 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 SUMMARY AT A GLANCE
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 •Collection and material processing depends on the type of immune analysis. To evaluate immune sta-
ways for plasma collection. The most common way is based on blood centrifugation; the other tus of a patient, humoral and cellular immunity screening is performed.
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 •To perform humoral immunity screening, serum and plasma are obtained by whole blood processing
a sterile Pasteur pipette to a fresh sterile tube (Fig. 2.1). For blood plasma processing, it is recom- (centrifugation).
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- •Serum is obtained from the coagulated blood after centrifugation; plasma is separated from the non-
agulated blood is left at room temperature for 20 min. in an incline and for other 20 min. in a ver- coagulated blood and in contrast to serum contains clotting factors. The most widely used anticoag-
tical position (Fig. 2.2). Plasma is stored in the same way as serum. It is important to avoid ulants are EDTA (ethylene-diaminetetraacetic acid), heparin and citrate. Soluble immune system
freeze-thaw cycles because this is detrimental to many plasma components such cytokines or com- components such as antibodies, cytokines, components of the complement system, acute phase pro-
plement components. 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 Classical serological methods
3.1.1 Direct agglutination

3. Classical serological methods
3.1.1.1 Qualitative agglutination test
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- Agglutination tests can be performed in a qualitative manner so that they serve to deter-
cept of an antigen-antibody reaction resembles a key (antigen), which fits into a lock (antibody). mine the presence or absence of a particulate antigen using in vitro interaction with the appro-
The bonds that hold the antigen to the antibody combining site are non-covalent, including hy- priate antibody. Vice versa, antibodies can be determined by the means of a defined antigen. The
drogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Multiple bonds antibody is mixed with the particulate antigen and a positive test is indicated by the formation of
between the antigen and the antibody ensure that the antibody will tightly bind to its antigen. visible conglomerates. For example, agglutination is used for the determination of blood types or
Specific antigen-antibody interactions are the bases of serological methods that are extensively antibodies to blood group antigens. For blood group typing the term haemagglutination is used.
used for the identification or quantitation of antigens, and antibodies. Blood group typing is based on the presence or absence of inherited antigenic substances on the
Classical or simple serological techniques involve direct observation of reactions; they do not surface of red blood cells (RBCs). According to the ABO blood group system, there are four dif-
require the participation of accessory factors such as indicators or specialized equipment. Aggluti- ferent blood groups: A, B, AB or O. Anti-A and anti-B antibodies (called isohaemagglutinins) ap-
nation and precipitation are the two basic groups of classical serological reactions. Major differ- pear in the first years of life and they are of IgM class. In blood group typing red blood cells of the
ence between them is in the physical form of the antigen. Agglutination is the reaction of antibodies tested person are mixed with isohaemagglutinins and occurrence of agglutination is directly eval-
with large particulate molecules while precipitation occurs due to the interaction of antibodies with uated. On the contrary, when patient´s serum is mixed with RBCs of a known blood type we can
soluble antigens. assay for the presence of antibodies to that blood type in the tested serum.
3.1. Agglutination Tab. 3.1 Blood group antigens and isohaemagglutinins
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 Blood group A
(Fig.3.1). The word agglutination comes from the Latin agglutinare which means “to glue”. Antibodies Individuals who belong to the blood group A have A antigens on the surface of their erythrocytes and
of IgG and IgM classes can agglutinate particulate antigens however IgM class, due to its high valence, anti B-antibodies in blood plasma.
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 Blood group B
bacteria, yeasts or blood cells by means of specific antibodies. Clumping reaction occurs quickly and Blood group B individuals have B antigens on the surface of their RBCs and anti-A antibodies in blood
is easy to perform, which ma plasma.
kes agglutination an important
technique in routine diagnosis. Blood group AB
Agglutination occurs op- A and B antigens are expressed on the surface of RBCs in individuals belonging to the blood group AB,
timally when antigens and however neither anti-A nor anti-B antibodies are detected in their plasma.
antibodies react in equiva-
Blood group O
lent proportions. Excess of
an antibody results in forma- Neither A nor B antigens are present on the surface of RBCs in blood group O individuals, however, they
tion of very small complexes have both, anti-A, and anti-B antibodies in their plasma.
that are not able to clump
and form visible agglutina-
tion. This inhibition of an ag- Direct agglutination is widely used also in microbiology as a diagnostic method of identi-
glutination reaction is called fying antigens specific for certain bacteria. A small amount of bacteria and a defined antiserum are
the prozone effect. mixed on a slide or card and allowed to react. After a short period of time the occurrence of ag-
Fig. 3.1 Agglutination
glutination is evaluated. The procedure is called serotyping and it is used e.g. to determine the
Antigen particles are cross-linked by antibody molecules.
members of the Enterobacteriaceae or Streptococcaceae families.
18 19
Direct Coombs test is also used for testing newborn babies with Rh-positive blood whose
3.1.1.2 Semi-quantitative agglutination test 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-
Agglutination tests may be used to measure the level (concentration) of antibodies to par- sponse. RhD-negative individuals commonly have no anti-RhD antibodies, because these are not
ticulate antigens. Serial dilutions of serum containing antibody are prepared in test tubes and then usually produced by sensitization against environmental substances. However, RhD-negative in-
a fixed amount of antigen (such as bacteria) is added. The maximum dilution that gives aggluti- dividuals can produce anti-RhD antibodies (which are of class IgG) following a sensitizing event
nation is determined. The reciprocal of the highest dilution of serum showing visible agglutina- like the feto-maternal transfer of blood from a fetus in pregnancy. Occasionally they occur due to
tion is called the “titer of serum”. For instance, the titer of 160 means that the patient´s serum gives a blood transfusion with RhD-positive RBCs. The test shows whether the mother produces anti-
a positive result (agglutination) at any dilution down to 1/160 (1 part serum to 160 parts of a di- RhD antibodies and whether these antibodies have been transferred through the placenta to the
luter) as shown in Fig. 3.2. The titre is thus a measure of the amount of antibody in a unit volume fetus. Mother´s anti-RhD antibodies attack the developing RBCs of the fetus and may cause the
of the original serum. Titer evaluation is commonly used to assess bacterial infections as the rise haemolytic disease of the newborn.
in titer of an antibody to a particular bacterium indicates an infection with that specific bacterial
Fig. 3.3 Direct Coombs test
type. For instance, tube agglutination is routinely employed for the serological diagnosis of ty-
phoid, brucellosis, tularaemia, typhus fever and other infectious diseases.
3.1.2.2 Indirect Coombs test
Fig. 3.2 Titer determination by direct semi-quantitative tube agglutination
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
3.1.3 Passive agglutination used for the determination of antigen amount. For
example in the Kahn’s test for syphilis serial di-
Agglutination works only with particulate antigens. Soluble antigens are so highly dis- lutions of the antigen (toxin) are added to the
persed that an antigen-antibody complex evades detection by the naked eye. To make this reac- tubes containing a fixed quantity of the antibody
tion readily visible, methods using adsorption of soluble antigens (e.g. viral parts, polysaccharide, (antitoxin). The titre of the precipitating serum is
DNA) to a particle were developed. As natural carrier particles can serve RBCs (sheep, horse, and the maximum dilution of antigen in which
human) displaying the highest adsorptive capacity. Artificial particles like latex beads or colloidal a clearly expressed precipitin reaction is obtained.
charcoal are commonly used as well. Reaction of
such a coated particle with an antibody to the solu- Fig. 3.6 Precipitate formation is affected by the
ble antigen is called passive agglutination and the amount of antigen and antibody
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 3.3 Immunodiffusion
antigen and in the presence of antibody, agglutina- Immunodiffusion techniques detect anti-
tion occurs. Commercial test systems are usually per- gen-antibody precipitation reactions in a semi-
formed on cardboard cards or glass slides. Latex tests solid medium, e.g. agar gel, agarose gel or gelatin.
are available to detect various microorganisms like Antigens and antibodies diffuse in the medium until they reach the zone of equivalence. There are
Streptococcus agalactiae, Clostridium difficile toxins, ro- several advantages in allowing precipitation to occur in a semisolid rather than in a liquid medium.
taviruses, etc. Applications include detection of an- The reaction is visible as a distinct band of precipitation, which is stable and can be stained for
tibodies to soluble antigens like the rheumatoid preservation, if necessary. The test can be qualitative or semi-quantitative. The immunodiffusion
factor (details can be found in Chapter 9.2.2.6), viral techniques that are most often used in clinical laboratories include single radial immunodiffusion
antigens and many others. 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
(electrophoresed) according to their charge, size, confor-

Fig. 3.8 Calibration graph
mation, etc. After electrophoresis, a trough is cut in the gel
and antibodies are poured into the trough. As the anti-
3.3.2 Double immunodiffusion by bodies diffuse into the agar, precipitin lines are produced
Ouchterlony in the equivalence zone. Each antigen gives an individual
band with the corresponding antibody as illustrated in
Double immunodiffusion is based on the Fig. 3.10. Immunoelectrophoresis serves for the qualitative
As the amount of antibody in the gel
principle that both, antigen and antibody migrate analysis of complex antigen mixtures based on the num-
is constant, the diameter of the pre-
towards each other in a semisolid medium. Most
cipitin ring is a function of the amount ber and arrangement of the occurring lines.
of antigen applied. often they migrate from separate circular wells cut This test is commonly used for the analysis of proteins
in a layer of agar or agarose gel. Antigen and an- in a patient’s serum. Tested serum is placed in the well and
tibody are allowed to diffuse radially from their antiserum (antibodies directed to whole serum components)
wells and when they meet in optimal proportions is applied into the trough. Precipitin lines of the patient’s
they form a visible line of precipitation. Position of the precipitation line depends mainly on con- serum are compared to normal serum (control) in order to
centrations of both antigen and antibody. The test can be conducted collaterally with multiple wells identify presence of pathological phenomena. By this com-
filled with different antigen mixtures and multiple wells with different mixtures of antibodies. Pre- parison one can determine whether one or more serum com-
cipitin lines may indicate identity, partial identity, or non-identity, depending on the resulting pat- ponents are missing or whether there is an overabundance of
tern. In case that an antigen-antibody complex is identical, a precipitin line forms a coherent dash of some serum components in the patient’s sample.
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). Fig. 3.10 Immunoelectrophoresis by Grabar and Williams
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 3.4.2 Countercurrent electrophoresis
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 Countercurrent immunoelectrophoresis is a rapid and more sensitive variant of the double im-
ELISA method or Western blotting are being used for this purpose (detailed in Chapter 4.3). 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
Fig. 3.9 Possible precipitation charges. In the zone of eqivalence they form a precipitation line as illustrated in Fig. 3.11. Countercur-
patterns occurring in immunodif- rent electrophoresis is a qualitative test used for the determination of antigens in clinical specimen (e.g.
fusion by Ouchterlony 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
3.4 Immunoelectrophoresis 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 Progressive serological methods
specific proteins (mostly human serum proteins)

before automated methods became available. The 4. Progressive serological methods
procedure involves comparison of the sample of
unknown concentration with a series of dilutions 4.1 Progressive serological methods based on precipitation
of a known concentration of the protein. First, the
samples of proteins (antigens) are loaded side-by- Precipitates in fluids can be determined by means of methods that are based on the meas-
side in wells along the edge of a gel containing in- urement of cloudiness (turbidity) of a solution. Antibody and the antigen are mixed in such con-
corporated antibody. Afterwards antigens are centrations that only small precipitates are formed making the solution turbid. Nephelometry and
electrophoresed into the gel where interaction turbidimetry are the two most commonly used methods due to their speed and ease of use.
with the corresponding antibody occurs. The end Incident light entering a solution containing precipitates is subjected to three reactions: Some
result is a precipitation arc (resembling a “rocket”) of the light is absorbed, some is transmitted and some will be scattered in various directions. Neph-
spreading out from the loading well. The height elometry is based on the measurement of scattered light, while, in turbidimetry, the intensity of
of the precipitin arc is directly proportional to the light transmitted through the sample is measured. The amount of light absorption (or light scat-
concentration of the antigen (Fig. 3.12). 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
Fig. 3.12 Rocket electrophoresis
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
SUMMARY AT A GLANCE easily automated. However, nephelometry is more sensitive than turbidimetry when analysing
small particles like antigen-antibody complexes.
•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. 4.1.1 Turbidimetry
•Serological methods are based on serological reactions. They are extensively used for the identification Turbidimetry is based on the measurement of the reduction in light intensity of the incident
or quantitation of different antigens using known antibodies, or detection of antibodies by the means light beam after it has passed through the solution. The change in the amount of light absorbed
of known antigens. 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
•Agglutination and precipitation are the two basic groups of classical serological reactions. Aggluti- analyzer (turbidimeter).
nation is the reaction of antibodies with large particulate molecules while precipitation occurs due to
the interaction of antibodies with soluble antigens. 4.1.2 Nephelometry
•Haemagglutination is used for the determination of blood types or antibodies to blood group anti- Nephelometry measures the light that is scattered at
gens. Blood group typing is based on the presence or absence of inherited antigenic substances on the a particular angle from the light beam as it passes through
surface of red blood cells (RBCs). the suspension (in contrast to turbidimetry, which meas-
ures light rays passing directly through the solution). Often
•Immunodiffusion techniques detect precipitation reactions in a semisolid medium. The most frequent low power lasers are used as light sources. Light passes
method is the single radial immunodiffusion test. It is based on detecting precipitate ring formed as through the sample (containing antigen-antibody com-
antigen diffuses into a semisolid medium containing incorporated antibody. 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-
26 27
lationship between antigen concentrations and light scattering approaches linearity. Light scatter test the respective purified allergen is bound to a solid phase such as
may be directly extrapolated by a computer to give actual concentrations, e.g. in mg/l. 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
4.2 Immunoassays for the allergen, they will bind to the well. The well is washed (non-
specific antibodies are removed), and anti-human IgE labeled with
All immunoassays work on the same basic principle – reaction of an antigen and an anti- a radioisotope is added. Anti-human labeled IgE attaches to the al-
body, where one of the reactants is labeled by a suitable label. Usually the label is fixed to the an- lergen-antibody complexes. After incubation, the well is washed
tibody molecule, namely to its Fc part, so that the antigen binding function is not affected. again, and its radioactivity is measured (Fig. 4.2). The amount of ra-
Immunoassays differ in the types of labels used and in the analytical procedure by which the label dioactivity is directly proportional to the IgE concentration. The RAST
is visualised, resp. quantitated. The following types of labels are beeing employed most frequently: test is often used as a screening test by combining groups of allergens
• radioactive labels in a single reaction.
• enzymes which induce the breakdown of the substrate with the formation
of stained products Fig. 4.2 RAST (Radioallergosorbent test)
• fluorochromes capable to fluoresce 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.
Immunoassays may be qualitative or quantitative. Quantitative immunoassays are reported in Unbound anti-IgE antibodies are washed away. The amount of radioactivity is directly
mass units, along with reference intervals (normal ranges) for the test. An example of a qualitative proportional to the concentration of allergen-specific IgE in tested serum.
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 4.2.1.2 Radioimmunosorbent test (RIST)
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. Radioimmunosorbent test (RIST) is a competition test performed
in vitro, used to measure the total level of IgE antibodies in serum.
4.2.1 Radioimmunoassay 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-
Radioimmunoassay (RIA) has been developed as the first of the immunoassay techniques. diolabeled IgE due to the presence of investigated IgE in the patient’s
It was introduced in 1960 by Berson and Yalow and it has revolutionized research and clinical serum can be determined by comparison with known IgE standards
practice in many areas. This technique was considered of such importance to science that a Nobel (explained in Fig. 4.3). From a standard curve, one can determine the
prize was awarded for its development in 1977. amount of total serum IgE in an unknown sample. A positive test for
In RIA, the label is an gamma-radioactive isotope such as 125I, 131I, 3H and 32P. The technique total IgE indicates a diagnosis of allergy when allergic symptoms are
requires the use of an instrument called a gamma counter, which measures the amount of gamma present. However, serum IgE levels may be increased also in patients
radiation given off by the radioactive label. The presence of radioactivity indicates that the inves- suffering from parasitic infections, some immunodeficiencies (hyper
tigated antigen or antibody is present in the sample (qualitative information) and the amount of IgE syndrome) and some malignant diseases.
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 Fig. 4.3 RIST (Radioimmunosorbent test)
are as low as few picograms can be routinely measured. For example the detection of allergen- 1. Fixed anti-IgE antibody is first incubated with known amount of radiolabeled IgE.
specific IgE levels reqiures a highly sensitive technique as these antibodies occur in serum in ex- 2. Serum containing unknown amount of tested IgE is added. Tested IgE competes
with radiolabeled IgE for the binding site on fixed anti-IgE.
tremely low levels. The radioallergosorbent test (RAST) used in the diagnosis of allergies meets 3. The amount of radioactivity (in counts per minute) is indirectly proportional to the concentration
these requirements. of IgE in the test sample, thus the amount of the patient's total serum IgE can be determined.
4.2.1.1 Radioallergosorbent test (RAST)

Radioimmunoassays enable to detect and quantify a wide range of proteins in blood, serum,
RAST test belongs to the group of indirect antiglobulin tests. It is used to detect the causative saliva, and urine. However, the major limitation of these techniques is the generation of radioac-
allergen for an allergy by determining the presence of the allergen-specific IgE antibody. In RAST tive waste. That is why safer alternatives were developed using a nonradioactive signal instead of
28 29
the radioactive one. Fortunately, the sensitivity of nonisotopic labels exeeds nowadays that of RIA. a known reference standard. Unknowns that generate a signal that is stronger than the known
They are described in the following chapters. sample are considered positive and those that generate weaker signal are negative.
4.2.2 Enzyme immunoassay 4.2.2.2 Direct ELISA
Enzyme immunoassay (EIA) was developed as an alternative to radioimmunoassay ten years In a direct ELISA test which represents the simplest format of
later. This method uses as a label an enzyme such as alkaline phosphatase, glucose oxidase and the method the antigen that has to be determined is first adsorbed
horseradish peroxidase. The enzyme label reacts with chromogenic substrates in such a way that to a plastic plate. An excess of another protein (such as bovine serum
a turn from colourless to a colour occurs, which is used as a signal. Results (the intensity of colour albumin) is added to block all the other binding sites on the plate.
produced in response to the substrate conversion) are quantitated spectrophotometrically. The ac- Detection enzyme is linked to an antibody in a separate reaction. By
tual concentration of the antigen or antibody can be determined by comparison with standards of means of this enzyme-labeled antibody the immobilized antigen is
known concentrations. The sensitivity of EIA approaches or equals that of RIA, offering more determined as explained in Fig. 4.4. Excess enzyme-labeled antibody
safety and speed. One of the most widely used EIA techniques is the enzyme linked immunosor- is washed off, only enzyme-labeled antibody bound to antigen is
bent assay (ELISA). left. By adding the chromogenic substrate, the enzyme causes
change of colour. In this way the colour signal is illustrating pres-
4.2.2.1 Enzyme linked immunosorbent assay (ELISA) ence of the antigen. The more intensive is the final colour, the more
antigen is present in the sample. Enzyme-labeled antibody will not
In enzyme immunoassays the enzyme is covalently linked to the Fc portion of an appropri- bind if the respective antigen is not present (negative reaction shown
ate antibody. This linking process was independently developed by S. Avrameas and G. B. Pierce. on the example of antigen B in Fig 4.4).
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 Fig. 4.4 Direct ELISA
was developed by Wide and Porath in 1966 and named ELISA (Enzyme Linked Immunosorbent 1. Antigen A and antigen B are fixed, each in one well of a plastic plate.
Assay). ELISA is a solid-phase assay in which either the antigen or antibody is immobilized onto 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).
a support, mostly the surface of a 96-well polystyrene microtiter plate. By fixing one component 3. A chromogenic substrate for the enzyme is added to visualize the reaction as substrate
to the solid phase the immune complexes can easily be separated from the other components by changes colour upon reaction with the enzyme. The intensity of colour is proportional
to the amount of antigen A.
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 Fig. 4.5 Indirect ELISA
that are routinely used in medical laboratories. The invention of ELISA generated a whole series 1. Antigen A is fixed in wells of a plastic plate.
of test formats. The basic variants are direct, indirect and “sandwich” test procedures which are 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
described in detail in the following chapters. 3. To make the reaction visible, a secondary enzyme-labeled antibody is added that will bind
ELISA is a useful tool for determining specific antibodies in serum to microorganisms that 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
are difficult to cultivate (e.g. specific antibodies to the hepatitis B virus, HIV or West Nile virus). of anti-A antibody.
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 4.2.2.3 Indirect 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- Indirect ELISA is used to detect antibodies specific to an anti-
lution, stop solution, wash buffer concentrate, sample diluents, reference standards as well as pos- gen. The antigen is attached to a solid phase (e.g. a multi-well plate)
itive and negative controls. and a patient´s diluted serum is applied into wells of the plate. If an-
The final assay signal is usually measured with a spectrophotometric plate reader. The most tibodies specific to the antigen are present in the serum, they will bind to it. The plate is then
controversial aspect of ELISA is determining the “cut-off point” between a positive and a negative washed to remove non-specific antibodies and all other serum components. A specially prepared
result. A cut-off point may be determined by comparing the ELISA plate reader value with “secondary” antibody is then applied to the wells, followed by another wash. This secondary an-
30 31
tibody is an antibody that binds to human IgG and it is chemically coupled with an enzyme. Thus, to identify and enumerate these cells with an extraordinary level of accuracy. Due to its high sen-
the well will contain enzyme in proportion to the amount of secondary antibody bound. As in all sitivity, the ELISPOT has proven particularly useful when studying small populations of active
ELISA formats, finally a substrate for the enzyme is applied. Catalysis by the enzyme leads to cells such as those regularly found in specific immune responses. Illustration shows principal steps
a change in colour as shown in Fig. 4.5. of the procedure (Fig. 4.7).
4.2.2.4 Sandwich ELISA 4.2.3 Fluorescent immunosorbent assay (FIA)
In this assay, a multi-well plate is coated with a primary capture Fluorescent immunosorbent assay (FIA), known also as immunofluorescence assay (IFA), is
antibody, which recognizes the target antigen in the test sample and based on using antibodies chemically coupled with a fluorescent molecule such as phycoerythrin
binds to it. The antigen-antibody complex is then recognized by a sec- (PE), fluorescein isothiocyanate (FITC) or rhodamine. These fluorescent indicators are called flu-
ondary detection antibody that is labeled with an enzyme. By adding orochromes or fluorophores. The sample is illuminated with light of a specific wavelength which
a substrate, the enzyme catalyzes the reaction mixture, yielding a spe- is absorbed by the fluorophores, causing them to emit light of longer wavelengths (that means of
cific colour. By measuring the optical density of this colour, the amount a different colour than the absorbed light). Fluorescence is usually visualised by means of a fluo-
of the target antigen can be determined. Again, the intensity of colour rescence microscope. It can be quantified using a flow cytometer or array scanner. Two basic vari-
is directly proportional to the amount of the tested antigen (Fig. 4.6). ants of FIA are the direct and indirect method.
Fig. 4.6 Sandwich ELISA 4.2.3.1 Direct FIA

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 Similarly as in direct ELISA, also in direct FIA the specific antibody is coupled with the label,
is present, it will bind to the capture antibody.
3. A secondary enzyme-labeled antibody (detection antibody) is added that in this instance with a fluorescent molecule. Direct FIA is used to detect the presence and local-
will bind to the antigen A fixed in the capture anti-A antibody. ization of specific antigens by the means of fluorescence emitted by the bound specific 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. 4.8A). 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. 4.7 ELISPOT (Enzyme-linked immunosorbent spot assay)
1. ELISPOT plate is coated with capture antibody that is specific to the secretory Fig. 4.8 FIA (Fluorescent immunosorbent assay)
product of interest. A) Direct FIA
2. Cells whose secretory activity is to be measured are plated and stimulated. Tissue or cell-specific antigen is visualized by
The secreted molecules are captured around the secreting cells during a fluorochrome-labeled specific antibody.
the entire culture period.
3. Cells are removed, leaving behind their secretory footprint. B) Indirect FIA
4. Enzyme-labeled detection antibody is added. Secondary detection antibody is used to visualize
5. Finally, chromogenic substrate is added that enables coloured spot development. the „antigen-specific antibody“ complex.
4.2.3.2 Indirect FIA

4.2.2.5 ELISPOT
Indirect FIA uses two antibodies; the
The enzyme-linked immunosorbent spot assay (ELISPOT) primary antibody specifically binds the
is based on the sandwich version of ELISA. ELISPOT allows target antigen, and the secondary antibody marked with a fluorochrome recognises the primary
measuring secretory activity of individual activated or re- antibody and binds to it. Indirect immunofluorescence is more sensitive than the direct one since
sponding cells. Cellular secretions (like specific antibodies or more than one secondary antibody can attach to the primary leading to amplification of the sig-
cytokines) are captured around the originating cell and man- nal (Fig. 4.8B). If the sample is positive, first, specific antibodies present in the sample (e.g. serum)
ifested as coloured footprints (spots). Each spot that devel- attach to the antigen substrates. In a second step, the attached antibodies are stained with fluo-
ops in the assay represents a single reactive cell what allows rescence-labeled anti-human antibodies and visualized using a fluorescence microscope.
32 33
Indirect FIA is the method of choice in the determination of autoantibodies to different cells tibody specific to some cell antigens. After the primary antibody is applied, biotinylated anti-IgG
or tissues and this is why it is widely used in the diagnosis of autoimmune diseases (detailed in is added, followed by peroxidase labeled avidin. Visualization is accomplished by adding an ap-
Chapter 9.2.2.6). Indirect FIA enables also detection of antibodies to various other antigen sub- propriate substrate for peroxidase.
strates, like different infectious agents. It is possible to perform simultaneous detection of anti- Over the time, a variety of different methods have evolved that are using the biotin-
bodies against several biochemically different antigens on one single biological substrate. avidin/streptavidin complex. They include immunohistochemical staining, flow cytometry, radio,
enzyme- and fluorescent immunoassays, hybridoma screening, Western blotting, purification of
4.3 Western blot 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 Evaluation of innate humoral immunity
decreased complement levels can also result from their decreased synthesis or increased catabo-
5. Evaluation of innate humoral immunity 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 human immune system plays a crucial role in defence against infectious microorganisms the integrity and function of complement activation pathways is an important part of the diag-
and tumours, as well as in removal of old, damaged and foreign cells and material. Immune mech- nostics and monitoring of disease progression and its response to treatment.
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 5.1.2 Laboratory tests for complement evaluation
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 5.1.2.1 Specimen collection
of immunity comprises:
1. Humoral innate immunity components – the complement system, acute phase proteins Because of the extreme unstableness of complement components, proper collection, prepa-
and other mediators of inflammation. ration, handling and storage of biological samples are crucial to obtain correct and relevant results
2. Cellular innate immunity components – phagocytic cells, NK cells 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
5.1 The complement system 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
5.1.1 The role of complement in the immune response 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-
The complement system consists of more than 30 plasma and cell membrane proteins with ments in vitro after the specimen was collected, what would markedly distort the final results. Other
important roles in the defence mechanisms of innate immunity. The vast majority of complement anticoagulants such as citrate and heparin do not sufficiently block the complement activation and
factors are present in an inactive form and get activated by distinct mechanisms. The complement therefore can not be used for this purpose. EDTA-plasma can also sometimes be used for comple-
activation in one of the three pathways, the classical (CP), alternative (AP) or lectin (LP), has pro- ment functional assays, provided that it was mixed with a buffer containing Ca2+ and Mg2+ to en-
found biological consequences including the direct lysis of target cells (microbes, foreign cells) by able complement activation and specific thrombin inhibitor lepirudin to inhibit coagulation. Serum
membrane attack complex (MAC), opsonisation and promotion of phagocytosis, induction of and plasma should be obtained from whole blood sample as soon as possible and stored at -70 °C
chemotaxis and inflammation, as well as the dissolution of immune complexes and their removal to avoid degradation of complement factors and activation products with generally short half-life.
from circulation. Because of its strong cytolytic and proinflammatory activities, the activation of Prior to laboratory analysis, the samples should be thawed at 37 °C and then kept on ice.
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- 5.1.2.2 Functional screening tests for total complement activity
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. Laboratory tests for the evaluation of functional integrity of the classical, alternative and
The complement system has been shown to participate in the development of numerous dis- lectin pathway include several haemolytic and ELISA-based assays that enable to screen for the
eases and pathological conditions including the autoimmune disorders, systemic inflammatory presence of quantitative and qualitative abnormalities in complement activation cascade. Absolute
response syndrome (SIRS), sepsis, ischaemia-reperfusion syndrome, graft rejections, neurode- absence, significantly decreased concentration or impaired function of any of individual comple-
generative disorders and many more. The most common indications for the laboratory evaluation ment factors will inevitably affect the whole complement cascade and result in abnormal forma-
of the complement system levels and function are congenital complement deficiencies manifesting tion of terminal complement complex (MAC), which can be confirmed by specific monoclonal
with increased susceptibility to bacterial infections and immune complex autoimmune diseases antibodies or by examining its ability to lyse target erythrocytes.
(systemic lupus erythematosus, vasculitides), and diseases associated with excessive complement The total haemolytic complement (CH50) assay is a functional laboratory assay for the eval-
activation and subsequent complement factors consumption. The former are caused by genetic de- uation of classical complement pathway. For its activation, immune complexes formed by IgG rab-
fects affecting the production or function of particular complement factors, while the latter ap- bit antibodies that react with surface antigens on sheep erythrocytes are used. After serial dilutions
pear as a consequence of congenital deficiencies of complement regulators or excessive production of tested serum with Ca2+ and Mg2+-containing buffer are prepared in tubes or wells of a microtiter
and deposition of immune complexes that subsequently activate the complement system (typi- plate, a fixed amount of the haemolytic system (erythrocytes with bound antibodies) is added to
cally seen in certain systemic autoimmune diseases), as well as due to the production of autoan- each tube/well and incubated at 37 °C for 1 hour. This allows the C1q to bind to the Fc portions
tibodies that bind and stabilize the C3-convertase C3bBb or inactivate the C1-inhibitor. Finally, of antibodies and initiate the classical complement pathway. The subsequent formation of the
36 37
MAC, which requires the presence and correct function of all classical pathway and terminal com- AH50 assays are typically the consequence of deficiencies of central C3 factor or terminal comple-
plement factors (C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8 and C9), causes the lysis of red blood ment factors C5–C9. As there is no commercial haemolytic assay for the lectin pathway available yet,
cells and the release of haemoglobin. The colour change of the supernatant is subsequently read ELISA-based functional assay must be used instead. Complete absence of haemolytic activity usu-
by spectrophotometer (Fig. 5.1). Simultaneously, separate tubes or wells with haemolytic system ally indicates the presence of a hereditary complement defect, whereas decreased CH50 or AH50
and buffer alone to measure spontaneous lysis, and tubes/wells containing haemolytic system values point at acquired complement deficiency, e.g. due to complement consumption.
with distilled water for total haemolysis are prepared as controls. The results are calculated by In addition to haemolytic assays performed in tubes or microtiter plates, another simple al-
a software or using von Krogh’s equation and expressed as the reciprocal of the dilution that causes ternative test performed in the agarose gel and resembling the radial immunodiffusion (RID) assay
lysis of exactly 50 % of the red blood cells in the assay (hence CH50 assay). Obtained values are has also been developed. The haemolytic system (antibody-coated sheep erythrocytes for CP or
then compared to reference values for normal human serum. Any quantitative or functional ab- rabbit erythrocytes for AP) is cast in the gel and appropriate amounts of the tested serum along
normalities in CP complement proteins will result in an impaired lysis of the cells, and thus de- with positive and negative controls are subsequently added to punched holes in the gel. The serum
creased amount of released haemoglobin. As a consequence, the serum dilution required for the complement factors diffuse radially and cause the lysis of
lysis of 50 % of RBCs will be lower compared to the reference serum. 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
Fig. 5.1 Principle of the total haemolytic CH50 assay and solid-phase ELISA assays.
The former use enzyme-con-
taining liposomes instead of
The analogous assay for the evaluation of AP activity is the alternative pathway haemolytic sheep red blood cells for the
(AH50) assay that uses rabbit or guinea pig erythrocytes capable of directly activating the alterna- evaluation of the classical
tive pathway. To block the simultaneous activation of the classical pathway, ethyleneglycolte- complement pathway. Its successful activation and the formation of the MAC result in the lysis of
traacetic acid (EGTA) is also added to the serum dilutions in order to chelate Ca2+. Like the CH50, the liposomes and the release of an enzyme that can be read by a chemistry analyser. ELISA as-
the AH50 is expressed in units that represent the exact amount of serum (dilution) that lyses 50 % says are based upon the use of specific activators bound to the bottom of wells of microtiter plates
of red blood cells. Any deficiencies in factors B, D, P, C3, C5, C6, C7, C8 and C9 will result in ab- and the subsequent detection of deposited complement activation products by monoclonal anti-
normal values obtained in AH50 test. Hence, performing both CH50 and AH50 assays in a patient bodies. For the evaluation of the classical pathway, IgM antibodies immobilised at the bottom of
with suspected complement deficiency can help to localise the complement defect. Low or absent wells are used. After the patient's serum was placed into the wells coated with antibodies, the C1q
CH50 values in the presence of normal AH50 results indicate the C1, C2 or C4 abnormality. Con- binds to IgM and initiates the classical pathway of complement activation resulting in the forma-
trariwise, abnormal AH50 but intact CH50 values localise the defect into the initial factors of the al- tion of MAC. Its presence and quantity is then measured by monoclonal antibodies against C9
ternative complement pathway (i.e. factor B, D or P). Abnormal values obtained in both CH50 and neoepitope, which only appears when the complete pathway was successfully activated. After the
38 39
wash, the bound anti-C9 antibodies are detected by secondary enzyme-labelled antibodies. In the complement activity of the re-
final step, a specific substrate is added and converted by the enzyme into a coloured product spective pathway. The occu-
(Fig. 5.3). The intensity of colour change is read by an ELISA-reader. For the evaluation of the lectin rence of haemolysis after the
pathway, the serum is added into the wells coated with mannan as an activator. The binding of addition of a particular com-
MBL and subsequent activation of MASP enzymes result in the cleavage of C4 and C2, formation plement components confirms
of C3-convertase and finally the assembly of MAC, which can be detected by anti-C9 monoclonal its deficiency in the patient.
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- Fig. 5.4 Modified CH50 assay
ple ELISA method comprising all three assays is currently available. for detection of individual comple-
ment factor deficiencies
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- The functional activity of the C1-inhibitor (C1-INH) can be examined by various different as-
sumption of complement factors, such as SLE. The confirmation and precise identification of causal says, including the ELISA assay that measures the formation of complexes between biotinylated C1s
defect require further assays that measure concentrations and function of individual complement and C1-inhibitor that are captured on avidin-coated plate. Another possibility is the radial immun-
factors and the production of activation products. 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
5.1.2.3 Quantitative assays for individual complement components 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-
Any abnormal values obtained in total complement functional assays are the indication for paired, it cannot bind to C1r and inhibit its interaction with antibodies. The result will be the
performing quantitative immunochemical assays. The serum concentrations of soluble comple- formation of a precipitation ring after the serum is allowed to diffuse radially from a hole in the
ment factors can be determined by classical immunoprecipitation techniques (radial immunodif- agarose gel (Fig. 5.5). The diameter of the precipitation zone is then indirectly proportional to func-
fusion, nephelometry) or by more sensitive assays including ELISA and Western blot (Chapters 3 tional activity of the C1-INH – the larger the diameter of the precipitation zone, the lower the ac-
and 4). In the routine clinical laboratory praxis, C3, C4 and sometimes factor B are the most fre- tivity of C1-INH and vice versa. Several assays have also been developed to determine the activity
quently measured complement proteins when hereditary or acquired immunodeficiencies are sus- of alternative pathway regulatory factors H and I, although these are not frequently performed.
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 Fig. 5.5 Radial immunodiffusion
of abnormal CH50 and/or AH50 confirm the diagnosis of complement deficiency. On the other assay for evaluation of C1-inhibitor
hand, normal values obtained in quantitative assays suggest the presence of a functional defect, activity
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- 5.1.2.5 Quantitative assays for complement activation products
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 The quantitation of activation fragments (products) is more sensitive method for the evalu-
lacks the same complement protein as the laboratory prepared deficient serum (Fig. 5.4). Alterna- ation of pathologically increased in vivo complement activation and consumption than the meas-
tively, purified functionally active complement proteins can be added in a step-wise fashion to the urement of native inactive complement factors and total complement activity, because the decrease
tubes containing haemolytic system and patient's serum to determine which one reconstitutes the of native factors levels and complement activity during the active phase of diseases associated
40 41
with complement consumption (systemic lupus erythematosus, vasculitides, etc.) may sometimes cluding the ELISA and haemolytic methods. Acquired angioedema (AAE) is a disorder caused by
be masked by their increased production by liver (systemic AID are typically accompanied by in- the production of autoantibodies against the C1-inhibitor. In this case, the binding to the C1-INH
flammation and the production of acute phase proteins, including the complement factors!). Ac- causes its inactivation and resulting insufficient inhibition not only of the classical complement
tivation products can be either split fragments after enzymatic cleavage or protein complexes. pathway, but also certain clotting factors, kallikrein and plasmin. The resulting overproduction of
They can be measured by the ELISA and microbeads-based multiplex flow cytometric assays as the vasoactive mediators causes recurrent episodes of oedema formation identical to hereditary an-
markers of classical pathway (C1rs-C1INH, C4a, C4b/c, iC4b, C4d), alternative pathway (Ba, Bb, gioedema. The presence of the anti-C1-INH autoantibodies can be verified by ELISA method with
C3BbP), central component C3 (C3a, iC3b, C3b/c, C3d) and terminal sequence (C5a, C5b-9) acti- purified C1-INH protein immobilised on the microtiter plate.
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 5.1.2.8 Genetic analyses of complement components
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 Congenital complement deficiencies caused by gene mutations are extremely rare; there-
monoclonal antibody to a neoepitope on poly-C9. The bound C5b-9 is subsequently visualised fore, their genetic analyses are mostly performed in few specialised laboratories in situations when
with enzyme-labelled anti-C6 monoclonal antibody (Fig. 5.6). As the levels of activation products a single complement defect was revealed by functional and quantitative assays. Genetic mutation
also depend on the initial concentrations of the native components, it was suggested that the ratio analyses offer better understanding of the nature of the complement defect and provide an op-
between the activation product and the native component (e.g. C3d/C3) is a better indicator of portunity for screening for potential mutation carriers in the family. The most common way how
complement activation than to identify gene mutation is the amplification of target DNA region within the candidate gene and
the activation product alone. its subsequent sequencing (Chapter 12.3.3.3).
These assays are, however, not
among routine tests and are 5.2 Acute phase proteins and markers of inflammation
only performed by few highly
specialised laboratories. 5.2.1 The role of acute phase proteins in the immune response
Fig. 5.6 ELISA assay for the mea- Acute phase proteins (APPs) are a group of more than 30 soluble plasma proteins produced
surement of C5b-C9 complement by liver hepatocytes and, to a lesser extent, by cells of the immune system. Following the tissue in-
activation product 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
5.1.2.6 Detection of membrane-associated complement receptors and regulatory proteins 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
Although most complement factors are present in soluble form in plasma and body fluids, adaptive inflammatory response with typical local manifestation and systemic changes including
receptors for complement fragments (CR1, CR3, CR4) and some regulatory proteins (DAF, MCP, the induction of fever, loss of appetite, lethargy, catabolism and APPs production. Acute phase
protectin, etc.) exist in a membrane-bound form. Their detection can be therefore performed by proteins subsequently directly participate in immune defence mechanisms by neutralising mi-
flow cytometry using fluorescently-labelled monoclonal antibodies (Chapter 7.2.2.2). crobes, regulating the intensity of immune reactions, preventing excessive tissue damage and par-
ticipating in tissue repair and regeneration (Tab. 5.1).
5.1.2.7 Detection of autoantibodies to complement components 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-
Several complement-related diseases are caused by the production of autoantibodies to com- called main APPs include C-reactive protein (CRP), pentraxin 3 and serum amyloid A. Other APPs
ponents of the complement system. In patients with membranoproliferative glomerulonephritis are synthesized continually as physiological components of plasma and their levels increase slower
ceruloplasmin, haptoglobin, complement proteins, inhibitors of serine proteases, α2-macroglobu-

(MPGN) type II, autoantibodies against the alternative pathway C3-convertase C3bBb (C3- and to a lesser extent (1.5 to 4-fold) during the inflammatory process. This group of APPs includes
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 lin and others. The most frequently used marker in the laboratory diagnostics of inflammatory
addition to the MPGN, patients also suffer from frequent bacterial infections. The definitive labo- diseases is the C-reactive protein (CRP). Its physiological levels are very low (below 5 mg/l) but can
ratory diagnosis relies upon the detection of C3-nephritic factor by several different assays, in- increase dramatically during general non-specific inflammatory responses to various stimuli (in-
42 43
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 As inflammatory processes are regularly associated with the changes in total leukocyte num-
these conditions, the CRP levels rise after few hours and peak within 24 – 48 hours. As the bio- bers as well as WBC subpopulations, the complete blood cell count (CBC) with differential is an
logical half-life of the CRP is rather short, its levels also decline rapidly when the aetiological integral part of the diagnostic algorithm of inflammatory diseases. Due to the increased synthesis
agents have been eliminated (within 4 – 5 days). The CRP concentrations depend on the intensity of several growth factors (e.g. colony stimulating factors), increased number of white blood cells
and the nature of the inflammatory process: bacterial infections are often associated with higher (leukocytosis) is often found in patients with local or systemic inflammation. Changes in the in-
44 45
dividual leukocyte subpopulations numbers usually depend on the nature of inflammatory The need to obtain the results as soon as possible forced the development of rapid tests for
process and thus may provide useful information on the aetiology of the disease. Neutrophilia the quantitative/semiquantitative evaluation of CRP and procalcitonin. Such assays have found
(increased number of neutrophils) is indicative for bacterial infections, but may also appear in their application in point-of-care settings and primary care units when a quick determination of
other acute inflammatory conditions (myocardial infarction, burns), while eosinophilia (increased the location of the inflammation and differentiation between acute bacterial and viral infection, or
number of eosinophils) is typically found in patients with parasitic infections and allergic dis- between infectious and non-infectious inflammation is required for guided therapy. Typical ex-
eases. Increased lymphocyte number (lymphocytosis) is present in acute viral infections, some amples include children suffering from acute respiratory and urinary infections (bacterial vs. viral
protozoal and chronic intracellular bacterial infections (tuberculosis, etc.), but also in leukaemias upper respiratory tract infection, pneumonia vs. tracheitis/bronchitis, acute pyelonephritis vs.
and lymphomas. Increased monocyte counts (monocytosis) are indicative for chronic inflamma- cystitis, etc.) or critically ill patients with systemic inflammatory response syndrome (bacterial
tory diseases including infections, autoimmune diseases (systemic lupus erythematosus, rheuma- sepsis vs. viral sepsis or non-infectious SIRS). In general, CRP levels above 40 – 50 mg/l are in-
toid arthritis, inflammatory bowel diseases) and haematological malignancies. 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
5.2.2.2 Evaluation of the erythrocyte sedimentation rate a simple laminated test device containing a porous membrane with bound monoclonal antibod-
liquid. 50 μl of the diluted sample is subsequently pipetted

Evaluation of the erythrocyte sedimentation rate (ESR) by modified FĆhrĺus-Westergren into the capillary and then diluted in 0.4 ml of the dilution
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- to the test device and allowed to pass through the mem-
ulated blood was placed in an upright tube or capillary. Under physiological conditions, ESR values brane. Any CRP in the sample is trapped by membrane-
are low (≤ 15 mm/hr in men and ≤ 20 mm/hr in women) due to the presence of a negative charge bound anti-CRP antibodies while the remaining components
on the surface of erythrocytes that hinders their interaction and sedimentation. In the presence of in- of the blood sample are absorbed by the paper layer at the
flammation, increased synthesis of fibrinogen (one of the APPs) and its deposition on the red blood bottom of the device. In the next step, one drop of a conju-
cells causes them to stick to each other and form stacks called “rouleaux”. This eventually leads to gate (liquid containing anti-CRP antibodies labelled with
accelerated erythrocyte sedimentation typically observed in inflammatory conditions such as infec- gold particles) is applied to the test device. The binding of
tions, autoimmune diseases, malignancies and other conditions associated with tissue injury (my- gold-labelled anti-CRP antibodies to the CRP in the mem-
ocardial infarction, trauma, surgery, burns, etc.). The ESR may be significantly affected by several brane leads to the red-brown colour change of the membrane.
factors including the patient’s sex, age, red blood number and morphology, immunoglobulin levels In the final step, one drop of
and technical factors. Therefore, the acute phase proteins (e.g. CRP) are considered to be more reli- a washing solution is applied
able markers of inflammation, because their values are less prone to these confounding factors. to remove all unbound conju-
gate from the membrane. The
5.2.2.3 Measurement of acute phase proteins and other markers of inflammation intensity of the colour change
is directly proportional to the
Measurement of acute-phase proteins is a useful tool in medical clinical pathology, as it allows to concentration of the CRP in
detect the presence of inflammatory process in the body, to monitor its activity, progress and response the sample and is read and
to treatment, and often provides useful information on possible aetiology of the disease (infectious vs quantified with a reflectome-
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 ter (NycoCard READER II)
within 5 minutes (Fig. 5.8).
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). Fig. 5.7 NycoCard CRP Single
Laboratory methods for the evaluation of APPs include immunoprecipitation methods in the test with NycoCard Reader II
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 Fig. 5.8 Principle of NycoCard
enzyme-linked immunosorbent assays, enzyme-linked fluorescent immunoassays, chemiluminis- CRP test
cent immunoassays, immunoluminometric assays and immunochromatographic assays.
46 47
Laboratory Metods in Immunology Evaluation of innate cellular immunity
6. Evaluation of innate cellular immunity

SUMMARY AT A GLANCE
6.1 Phagocytosis
•The innate humoral imunity comprises the complement system, acute phase proteins and various sol-
uble mediators of inflammation. 6.1.1 The role of phagocytosis in the immune response
•The complement activation enables direct lysis of bacteria and supports chemotaxis, opsonisation and Phagocytosis is an evolutionary old, non-specific immune mechanism participating in the
removal of immune complexes. Therefore, deficiencies of individual complement factors may lead to first line of defence against invading microbes. It is a process in which specialised cells of the im-
increased susceptibility to bacterial infections or immune-complex diseases. 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-
•The functional integrity of whole complement activation pathways can be examined by several tution of the damaged tissues. The cells capable of phagocytosis belong to two distinct systems –
haemolytic and non-haemolytic functional assays. Subsequently, quantitative and functional assays the myeloid system and the mononuclear-phagocytic system. The cells of the former system are also
for individual complement components must be performed to identify which complement component referred to as granulocytes due to the presence of granules with antimicrobial substances in their
is defect. 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-
•Inflammation is a complex and non-specific immune reaction that develops in response to tissue injury ing about 55 % to 70 % of all white blood cells. Other granulocyte population capable of phagocy-
caused by infectious and non-infectious factors. Several soluble proteins play crucial roles in its devel- tosis are eosinophils, although their antimicrobial substances are better suited for extracellular
opment and serve as excellent markers of its presence and intensity. The measurement of acute phase destruction of large parasites. The mononuclear-phagocytic system includes single cell population
proteins (e.g. C-reactive protein), procalcitonin or neopterin is an important diagnostic tool used to called monocytes (immature cells circulating in the bloodstream) and macrophages present in var-
monitor the intensity, progress and treatment response in various inflammatory conditions including ious tissues and organs. In contrast to neutrophils, macrophages are longer living cells that partic-
infections, malignancies, autoimmune and cardiovascular diseases. They are also helpful as non-spe- ipate in subacute and chronic phases of inflammation and in addition to phagocytosis, they also
cific markers for the differentiation of causative factors. 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-
48 49
cyte production or function often result in impaired protection from bacteria and fungi. Patients 6.1.2.2 Specimen collection and isolation of polymorphonuclears
with deficiencies of phagocytosis typically suffer from pyogenic or granulomatous bacterial
(Staphylococcus aureus, Serratia marcescens, Klebsiella sp., Salmonella sp., Escherichia coli, Pseudomonas Phagocytic cell function tests can be performed with either full heparinised blood or iso-
aeruginosa, etc.) and fungal (Candida sp., Aspergillus sp.) infections of the respiratory tract, lymph lated polymorphonuclear leukocytes (PMNs). The concentration of the heparin in the collection
nodes, skin, mucosa and visceral organs. Primary immunodeficiencies comprise diseases with ab- tube should not be more than 10 IU per 1 ml of the blood as its excess would inhibit the function
normal phagocyte numbers (severe congenital neutropaenia, cyclic neutropaenia etc.) or impaired of phagocytes. The use of other anticoagulant EDTA is not recommended as it chelates Ca2+ ions
ability to migrate, engulf or destroy foreign pathogens (leukocyte adhesion deficiencies, chronic which are necessary for the phagocytosis. Only fresh blood not older than 2 hours should be used
granulomatous disease, specific granule deficiency, myeloperoxidase deficiency and others). Their for functional tests. Alternatively, isolated PMNs can also be employed. In this case, 1.4 ml of 6%
correct diagnosis requires the evaluation of neutrophil and monocyte numbers and subsequent dextran is added to 5 ml of fresh heparinized blood and mixed thoroughly. The mixture is then in-
functional studies on individuals steps and mechanisms of phagocytosis: chemotaxis, ingestion, cubated inclined at an angle of 45° for 20 – 25 minutes at 37 °C and then again for 15 – 20 minutes
microbicidal activity and metabolic activation (respiratory burst). 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-
6.1.2 Laboratory tests for the evaluation of phagocytosis 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)
6.1.2.1 Evaluation of blood cell numbers 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 complete blood cell counting (CBC) with differential is a basic laboratory test that provides the top of the Ficoll (Lymphoprep) solution and centrifuged. During the density gradient cen-
useful information on circulating blood cell numbers including the neutrophils, eosinophils and trifugation (Chapter 7.2.1), PMNs settle down at the bottom of the tube while mononuclear cells
monocytes, whereas the blood smear enables to detect their morphological abnormalities such as (monocytes, lymphocytes) create a ring on the interface between plasma and Ficoll. PMNs are
the presence of large granules in neutrophils (Chediak-Higashi syndrome) or their absence (spe- then removed, washed and diluted to a desired concentration. Simultaneously, 5 ml of the blood
cific granule deficiency). Neutrophil numbers below 1500/μl are referred to as neutropaenia, num- is also collected in a clean tube and allowed to coagulate. The clot is then removed and the serum
bers < 500/μl are the criterium for severe neutropaenia and are associated with a high risk of severe is used along with PMNs for the examination of ingestion and cidal activity.
bacterial infections. Neutropaenia is typically found in primary immunodeficiencies (severe con-
genital neutropaenias, cyclic neutropaenia, Kostmann’s syndrome, Shwachman-Diamond syn- Fig. 6.1 Isolation of polymorpho-
drome, etc.), but can also occur in malignancies, certain infections (tuberculosis, HIV, EBV, CMV, nuclears with dextran
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 6.1.2.3 Evaluation of chemotaxis
leukaemias (e.g. hairy-cell leukaemia), aplastic anaemia or combined immunodeficiencies, while
increased numbers (monocytosis) may accompany several viral and bacterial infections (tubercu- The successful active migration of phagocytes from the circulation to the infection site de-
losis, brucellosis, listeriosis, etc.), autoimmune diseases and malignancies (chronic monocytic or pends upon the intact microfilamentous and microtubular system, the production of chemotactic
myelomonocytic leukaemia). 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
50 51
through the vessel wall and interstitial tissues to the infectious site. Impaired chemotactic activity deficiencies, tuftsin deficiency, leukocyte adhesion deficiency (LAD), actin dysfunction, liver in-
is typical for certain rare primary immunodeficiencies including the leukocyte adhesion deficiency sufficiency and acquired hypogammaglobulinaemia (multiple myeloma, chronic lymphocytic
(LAD) syndromes, Chédiak-Higashi syndrome, hyper-IgE syndrome, lazy leukocyte syndrome, leukaemia). In addition, phagocytosis defects are often observed in severe infections (sepsis), chronic
complement deficiencies and others, but is more commonly caused by drugs (colchicine) or pres- systemic diseaes and malignancies or can be caused by drugs (corticosteroids, colchicine, salicilates).
ent in certain diseases (diabetes mellitus, severe malnutrition, infections, malignancies). Although Several tests and their modifications have been developed to evaluate phagocytic activity.
a wide range of techniques is available to evaluate chemotactic activity of cells, these tests do not These can be performed with either full heparinised blood or isolated PMNs using various mi-
belong to routinely used methods as they are laborious, time consuming, lack uniform standardi- croorganisms (Candida albicans, Saccharomyces cerevisiae, Staphylococcus aureus, Escherichia coli) or
sation and their results show a high degree of variability. Hence, these assays are only performed microspheric hydrophilic particles as a substrate. Frequently used functional method is the eval-
in certain specialized laboratories when a defect of chemotaxis is strongly suspected. uation of phagocytic activity (PhA) and phagocytic index (PhI) of PMNs with C. albicans. In this test,
Laboratory methods for the evaluation of chemotactic activity include various chamber assays a suspension of patient's isolated PMNs is incubated with inactivated candida cells (ratio 1 : 5), au-
and agar-plate assays. The latter use an agar gel cast on a polystyrene plate with three pre-cut holes tologous serum (for opsonisation) and Hanks’ solution in a thermostat at 37 °C and constantly
(Fig. 6.2). Examined patient’s PMNs are added into the middle hole whereas a rabbit serum activated shaken to avoid the aggregation of candida cells (Fig. 6.3). After 30 minutes, a smear is prepared
with zymozan (for the production of chemotactic stimuli) and a buffer medium are added into the on a microscopic slide and then stained with Wright's stain. The sample is evaluated under the
opposite outer holes. During the incubation at 37 °C the PMNs move radially from the central hole. light microscope and the numbers of phagocytosing PMNs (those with at least one C. albicans in
Their migration toward the hole with a medium is non-directed (random spontaneous locomotion) their cytoplasm) out of 100 – 200 PMNs as well as phagocytosed C. albicans cells are counted. Sub-
while the migration toward the opposite hole containing activated serum is active and directed by sequently, PhA and PhI values are calculated as follows:
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 N of phagocytosing PMNs N of phagocytosed C. albicans cells
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. PhA (%) = ––––––––––––––––––––––––– x 100 PhI = ––––––––––––––––––––––––––––
N of all PMNs N of phagocytosing PMNs
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 Phagocytic activity expresses the proportion of phagocytosing cells out of all PMNs and its
technique using anti-CD18 and anti-CD15 (anti-sLex) monoclonal antibodies, respectively. 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
Fig. 6.2 Evaluation of chemotac- terminal stages of malignancy, are associated with their decreased values. On rare occasions, the
tic activity of polymorphonuclears 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
6.1.2.4 Evaluation of ingestion indicates an opsonization de-
fect, whereas impaired phago-
Evaluation of the ability of PMNs to take up microorganisms or spheric particles is an im- cytosis with both sera hints at
portant step in the diagnostics of neutrophil dysfunctions. The process of ingestion depends upon a cellular defect.
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, Fig. 6.3 Evaluation of phago-
immunoglobulins), expression of opsonin receptors (complement receptors, Fc receptors) and de- cytic activity and phagocytic index
formability of neutrophils may lead to impaired ability to ingest microorganism. Defects in the mi- of polymorphonuclears
crobial uptake can appear in individuals with congenital complement and immunoglobulin
52 53
Alternatively, the phagocytic activity may also be examined with full blood instead of iso- quently, a microscopic smear is prepared and evaluated under the light microscope and the num-
lated PMNs or using microspheric hydrophilic 2-hydroxyethyl methacrylate (HEMA) particles as ber of dead candida cells out of all cells (usually 400) is counted. Simultaneously, a control reac-
a substrate. Their low negative charge limits non-specific adherence to cell surfaces and sponta- tion with serum-resistant C. albicans, serum for opsonisation and Hanks’ solution, but without
neous self-aggregation. After the incubation of HEMA particles with full blood at 37 °C for 1 hour, patient's PMNs, is prepared and incubated in a separate control tube. This allows to evaluate a pro-
a smear is prepared, fixed, stained with Giemsa-Romanowski stain and evaluated under the mi- portion of candida cells that died spontaneously without the active contribution by PMNs. Finally,
croscope. Cells with 3 and more ingested microparticles are identified as phagocytosing. Subse- the candidacidal activity (% of cidia) is calculated according to the following formula:
quently, PhA and PhI are calculated.
Finally, the ingestion can also be evaluated by flow cytometric assays (Chapter 7.2.2.2) that N of dead C. albicans in the sample – N of dead C. albicans in the control
measure neutrophil-associated fluorescence after incubation of cells with microparticles or mi- % of cidia = –––––––––––––––––––––––––––––––––––––––––––––––––––––––– x 100
croorganisms labelled with pH-sensitive fluorogenic dyes. These dyes are non-fluorescent at neu- N of all C. albicans cells
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- Cidal activity (% of cidia) expresses the percentage of C. albicans cells killed by the direct ac-
tion of phagocytosing PMNs (those producing fluorescence) out of all PMNs and mean fluores- tion of examined PMNs after one hour of incubation. Its values below 25 % indicate a possible
cence intensity of PMNs with phagocytosed microorganisms/microparticles, respectively, are presence of a defect in one or more microbicidal mechanisms.
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 Fig. 6.4 Evaluation of microbici-
bacteria, yeasts or microparticles. To discriminate between internalised and membrane-bound par- dal activity of polymorphonuclears
ticles, trypan blue is added to quench surface-bound fluorescence.
Alternatively, the candi-
6.1.2.5 Evaluation of microbicidal activity dacidal activity can also be de-
termined by inoculating the
Following their ingestion and the fusion of the phagosome with lysosomes, the internalised suspension of C. albicans cells
microorganisms are finally destroyed within the phagolysosomes by several microbicidal mech- from sample and control tubes
anisms. Defects in their synthesis or the formation of phagocytic granules and phagolysosomes re- on the Sabourad agar, instead
sult in the impaired killing and increased survival of ingested microorganisms, and hence of staining the dead cells with
increased susceptibility to infections. Defects of microbicidal activity are typically present in cer- vital dye. Following the inoc-
tain primary immunodeficiencies (Chédiak-Higashi syndrome, chronic granulomatous disease, ulation, the plates with the
specific granule deficiency, myeloperoxidase deficiency, Papillon-LefŹvre syndrome, etc.), but also agar are incubated at 37 °C for
often develop in patients with other underlying diseases (severe infections, sepsis, malignancies, 48 hours and the numbers of
severe malnutrition, etc.). Therefore, the evaluation of microbicidal activity has its firm place in the grown colonies from both sample and control tube are counted afterwards. Every grown colony cor-
diagnostic protocol of numerous disorders associated with the development of reccurent pyogenic responds with one viable C. albicans cell. The % of cidia is then calculated as follows:
or granulomatous bacterial or fungal infections of skin, mucosa and visceral organs.
Several functional tests for the examination of microbicidal mechanisms have been introduced N of grown colonies in the sample
in the previous decades. Similarly to other phagocytosis assays, these can be performed with either % of cidia = (1 – ––––––––––––––––––––––––––––– ) x 100
full heparinised blood or isolated PMNs using various vital microorganisms (C. albicans, S. aureus, N of grown colonies in the control
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- The microbicidal activity can also be evaluated from full heparinised blood, or using other vi-
bated with vital serum-resistant C. albicans strain (ratio 1 : 10), autologous serum (for opsonisation) able microorganisms as substrates (Staphylococcus aureus, Escherichia coli). Finally, flow cytometric
and Hanks’ solution at 37 °C for one hour, while continuously shaking. Following the incubation, assays for the measurement of the microbicidal activity have also been developed in the last decades.
the PMNs are lysed with sodium deoxycholate and the released C. albicans are stained with the vital Similarly to chemotaxis tests, also assays for the evaluation of microbicidal activity are labo-
dye methylene blue. This allows to discriminate between dead and viable candida cells, as the dye rious, time consuming and show a high degree of variability as various microbial species or strains
is unable to penetrate viable cells leaving them unstained. On the other hand, dead cells are stained used in the testing often differ in their sensitivity/resistance to phagocytosis and microbicidal mech-
blue as they are unable to prevent the methylene blue from penetrating their membrane. Subse- anisms. Therefore, the use of these assays in routine laboratory diagnostics is rather limited.
54 55
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
6.1.2.7 Evaluation of lysozyme levels and activity

6.2 Natural killer cells
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- 6.2.1 The role of natural killer cells in non-specific immune responses
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- Natural killer (NK) cells are lymphoid cells that share a common lymphoid progenitor with
trophils and macrophages and released extracellularly to cleave 1–4-β linkages between N-acetyl- 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
D-glucosamine and N-acetylmuramic acid in peptidoglycan of Gram-positive and some munity. They make up about 5 – 10 % of circulating lymphocytes and are defined as large granu-
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 azurophilic granules. Similarly to cytotoxic T cells, NK cells play crucial roles in immune responses
measurement can be helpful in the monitoring of certain diseases. Increased serum lysozyme lev- to virus-infected, tumour and allogeneic cells. In contrast to CTLs, their response is rapid and does
els are common in diseases such as tuberculosis, sarcoidosis, Crohn’s disease, myelocytic and not require the recognition of viral or tumour antigens as NK cells do not possess antigen-specific
myelomonocytic leukaemias, acute bacterial infections or kidney diseases associated with their receptors. On contrary, NK cells are unique by their ability to recognise and respond to cellular
impaired function, while decreased levels can be found in individuals with impaired immunity stress induced in the presence of a virus, malignancy or cell damage. This is possible due to the
(immunosuppressive therapy, radiotherapy). The concentrations and activity of lysozyme can be presence of several membrane inhibitory and activating receptors that recognise diminished ex-
measured in a variety of ways: pression of self-markers of major histocompatibility complex (MHC) class I and appearance of ab-
1. The enzymatic methods are based on the ability of lysozyme to lyse cellular walls of bacteria. normal forms of self surface molecules or stress-induced molecules (MIC, ULBP), respectively.
In the diffusion method, 2% agar gel mixed with the suspension of Micrococcus lysodeikticus is The activation of the NK cells leads to the release of preformed cytotoxic molecules (granzymes,
prepared and cast on the plate. The serum sample is then pipetted into the holes punched in perforins) that destroy the target cells. Alternatively, an extrinsic pathway of apoptosis of a target
the gel along with standard solutions of the lysozyme with increasing concentrations used for cell can be induced upon the activation of NK cell and the Fas–FasL interactions. Finally, the tar-
the calibration. The enzyme then diffuses out of a hole and shows its presence by producing get cell can also be destroyed by the antibody-dependent cell-mediated cytotoxicity (ADCC) re-
a ring that is cleared off intact cell walls. After one day, the diameter of the zone of lysis is action following the recognition of antibody-coated target cells by FcγRIII (CD16) receptors.
measured and the concentration of the lysozyme is read from a calibration curve (Fig. 6.6). In NK cell deficiencies can result from an abnormal production of NK cells or their impaired
other modification of the enzymatic assay, the spectrophotometric method, the tested serum sam- function. Primary functional NK cell deficiencies are very rare and are usually caused by genetic
ple and standard solutions are incubated with the suspension of micrococcus in the wells of defects affecting NK cell receptors for target cell recognition (e.g. CD16), proximal signals for NK
a microtiter plate. The lysis of a microbial substrate will manifest with a clarification of sus- cell activation (e.g. WASP, caspase 8, SAP) or NK cell cytotoxicity (perforin, cathepsin C, etc.). They
pension, the degree of which can be measured spectrophotometrically. 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
Fig. 6.6 Enzymatic method for evaluation of malignancies and certain autoimmune diseases or as a consequence of chemotherapy and radio-
lysozyme activity 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
2. The serologic (immunometric) methods are is used as a target. Following their incubation with patient's isolated NK cells at several different
based upon the use of specific antibodies against the lysozyme for its detection and meas- ratios, the target cells are recognised by effector NK cells and subsequently lysed by their cytotoxic
urement. Included are radial immunodiffusion, rocket immunolectrophoresis and ELISA molecules. The lysis of K562 cells results in the release of radioactive chromium into the super-
(Chapters 3 and 4). natant, which radioactivity is measured by a gamma counter and expressed in counts per minute
58 59
(cpm). The intensity of radioactivity depends upon the amount of 51Cr released from destroyed tar- of cytotoxic molecules (perforin, granzymes) in NK cells and the expression of apoptotic markers
get cells and hence upon the cytotoxic activity of tested NK cells. Simultaneously, two control re- (annexin V) on target cells by fluorescently-labelled monoclonal antibodies have also been used in
actions are also performed: one with no effector NK cells to determine spontaneous release of the evaluation of NK cell function.
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:
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 Evaluation of adaptive immunity
differ in function as well as in their membrane antigens (CD molecules). B lymphocytes express
7 Evaluation of adaptive immunity 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
Adaptive immune response, also known as specific immune response, is composed of hu- recognize four subpopulations, namely helper, cytotoxic, regulatory and memory T cells. All T
moral and cellular components. In contrast to innate immunity, the adaptive immunity is evolu- cells express the same membrane molecule CD3. The main function of T helper cells (Th) is the ac-
tionary younger, it develops during the whole life and possesses immune memory. The main tivation of other immune cells. Th cells help B cells to differentiate to plasma cells and by pro-
function of adaptive immunity is to recognize and remember specific antigens, leading to induc- duction of cytokines they activate cytotoxic T cells and macrophages. Mature Th cells express CD4
tion of strong response each time the antigen is entering our body. The principal cells of adaptive molecules, which interact with HLA class II molecules on antigen presenting cells during their
immunity are lymphocytes. Humoral adaptive immunity is assured via antibodies produced by cooperation. The CD4 molecule is also the main receptor for HIV entering into T helper cell. Cy-
plasma cells, the latest differentiation stage of B lymphocytes. The cellular adaptive immunity in- totoxic T cells (Tc) express CD8 and are involved in destruction of virally-infected, malignant and
volves subpopulations of T lymphocytes, namely helper, cytotoxic, regulatory and memory T lym- allogeneic cells by cytotoxic mechanism. CD8 interacts with HLA class I molecules present in the
phocytes. There are quantitative methods for evaluation of adaptive immunity components based membranes of all nucleated cells. Regulatory T cells (Treg) are involved in suppression of immune
on serum immunoglobulins levels analyses (nephelometry, turbidimetry, ELISA) and counting of response to self-antigens thereby preventing the autoimmune process. They express CD4, CD25
lymphocytes (flow cytometry) in blood samples and qualitative methods based on functional eval- and Foxp3. Memory T cells develop faster and stronger immune response after the repeated ex-
uation of lymphocytes (blastic transformation assay, skin tests). 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
7.1 Adaptive humoral immunity components 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
Adaptive humoral immunity components involve antibodies produced by plasma cells. An- (LGL). They are involved in destruction of virus infected and tumour cells by cytotoxic mecha-
tibodies also known as immunoglobulins are Y-shaped proteins that recognize and neutralize spe- nisms (secretion of granzymes and perforins or ADCC – antibody dependent cell cytotoxicity
cific antigens. There are 5 classes of immunoglobulins, namely IgG, IgM, IgA, IgD and IgE differing 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
in their biological properties. B cells express a unique B cell receptor (BCR), which binds the spe- anD NK celLs
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 a,So invOlved in d%struction of Target cells by cytotoxic mechanisms. The method for analysis of
phagocytosis, complement cascade activation and others. 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-
7.1.1. Analysis of serum immunoglobulins levels tometry. The functional evaluation of lymphocytes involves methods such as the lymphocyte trans-
formation assay and skin tests.
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 7.2.1 Isolation and counting of lymphocytes
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 Isolation of lymphocytes from the peripheral blood belongs to basic methods used in im-
advantage of these machines is that they can examine more than 80 various serum proteins in one munological testing. Isolated lymphocytes are mainly required for evaluation of their function in
test. For detection of serum IgE and IgD levels, the sensitive enzyme immunoaassay methods (EIA) vitro or for histocompatibility testing. They can also serve for further separation to lymphocytic
are used because of their low serum concentrations. ELISA belongs to EIA methods and it is based subpopulations (i.e. helper or cytotoxic T cells). Method of lymphocytes isolation was first re-
on antigen and antibodies detection in a liquid environment using enzymatic reaction (see Chap- ported by Dr. Arne Bøyum in 1968 and is based on gradient centrifugation using a separation so-
ter 4.2.2). ELISA and their modifications are typically used for detection of specific antibodies, lution (Ficoll-Hypaque, Lymphoprep). During centrifugation, blood cells are separated according
i.e. to viruses and bacteria. 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,
7.2 Adaptive cellular immunity components 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
The principal cells of cellular adaptive immunity are lymphocytes. There are four main pop- and remain at the bottom of the tube (Fig. 7.1). Platelets can be separated apart from the mononu-
ulations of lymphocytes, namely B lymphocytes, T lymphocytes, NK cells and NKT cells. They clear cells by subsequent washing with Hanks balanced salt solution following centrifugation. Out
62 63
Dilute 20 μl of lymphocyte suspension with 380 μl of Turk’s solution (1:20). Nuclei of white
of mononuclear cells, lymphocytes account for about 80%, other cells belong to monocytes. The iso- The determination of lymphocyte counts includes the following steps:
lation procedure of lymphocytes includes following steps: 1)
1) Mix heparinised blood (use 10 units of heparin per 1 ml of blood) with equal volume of PBS 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
(phosphate-buffered saline). blood cells are destroyed by hypotonic environment.
2) Slowly layer diluted haparinised blood over Ficoll-Hypaque solution (with a density 1,077 2)
g/ml) in the tube. Use 1 ml of separation solution per 2 ml of blood/PBS mixture (propor- chamber
tion 1:2). 3) Using the microscope, count cells in 3 large squares bordered by tripled lines, omitting cells
3) Centrifuge the tube 20 min. at 2000 rpm (900 g) at 20°C. lying on 2nd and 3rd lines
4) Using a pipette, remove PBMC cells seen as white layer over Ficoll-Hypaque solution to an- 4) Calculate the average number of cells per a large square
other sterile tube. 5) Calculate the mean number of the other side of the counting chamber
5) Wash cells with excess of PBS and centrifuge 10 min. at 1300 rpm (400 g), repeat two- times. 5) Calculate the final concentration of cells using the above mentioned formula
6) Re-suspend mononuclear cells in the culture medium RPMI-1640, count cells and determine An example: counted lymphocytes = 250, the mean number in 3 large squares = 83,3;
viability using trypan-blue staining. The final count per 1 ml = 83,3x2 (dilution factor) x10 000 (volume factor) = 1,66x106 cells/ml
10 ml of freshly isolated blood usually contains 1−2.107mononuclear cells (80 % of them be-
longs to lymphocytes). Fig. 7.2 Bürker chamber
Bürker chamber consists of 2 grids covered by glass. Each grid
Fig. 7.1 Isolation of peripheral blood mononuclear cells contains 9 large squares (each measuring 1 x 1 mm, with the
depth of 0.1 mm). Corners are bordered by tripled lines. Each
(PBMC) by gradient centrifugation large square is divided into another 16 small squares, each
0.25 mm long and 0.25 mm wide. Cells are counted in the
Blood diluted with equal volume of saline is layered over half of volume
large squares omitting cells lying on the 2nd and 3rd lines
of separation solution Ficoll-Hypaque. After centrifugation at 900g for
(modified from www. google. com/aquaculture.ugent.be).
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).
7.2.2 Isolation and counting of lymphocyte subpopulations

7.2.1.1 Counting of lymphocytes in microscope
Analysis of lymphocyte populations belongs to the most important methods used in the im-
Cell counting belongs to basic quantitative method both in research and diagnostic labora- munological diagnostic. Exact count analysis of T and B lymphocytes is used in the diagnostic of
tories. Central diagnostic laboratories usually use automatic analysers based on cell density analy- primary as well as secondary immunodeficiencies (e.g. HIV-progression monitoring). Analysis of
sis by a spectrophotometry. The simplest method of cell counting uses a counting chamber and lymphocytes populations also involves the immunophenotyping of leukemias and lymphomas.
a microscope. The most frequent used chamber is that of Burker counting chamber (Fig. 7.2). It is Method for isolation and counting of lymphocyte populations is based on using fluores-
a slide that consists of 2 grids fixed with cover glass. Each grid contains 9 large squares (each meas- cently-labelled monoclonal antibodies (mAbs) which bind to their membrane molecules (desig-
uring 1 x 1 mm, with the depth of chamber of 0.1 mm) with their corners bordered by tripled lines. nated as CD antigens). CDs are surface antigenic determinants found only on cells of a certain
Each large square is divided into another 16 small squares, each 0,25 mm long and 0,25 mm wide. lineage and at a particular development stage, e.g. monocytes express CD14, B-lymphocytes CD19
The total volume of each large square is 0,1 mm3 or 10-4 cm3. To determine cell density, cells in 3 and CD20, all T lymphocytes express CD3, NK cells CD16 and CD56, etc. Population of CD3+ T
large squares should be counted under the microscope and the average number of cells per large lymphocytes contains T helper subpopulation expressing CD4 and T cytotoxic subpopulation ex-
square should be determined. Finally, the cells density per 1 ml in a suspension will be counted pressing CD8. Fluorescently-labelled cells are examined under fluorescence microscope and flow
according following formula: cytometry (FACS). The principle of fluorescence is based on excitation of fluorochrome by a UV
Cells per ml = Average count per square x dilution factor x 10,000 (volume factor based on light. The emitted light has a longer wavelength and is visible to the human eye. The most used
total volume calculation of each large square). For better cell counting, the cell suspension has to fluorochromes are FITC (fluorescein-isothiocyanate) with excitation wavelength 490 nm and emis-
be stained with vital dye trypan blue or Turk’s solution. sion wavelength 521 nm (emits green fluorescence), TRITC (tetrarhodamine-isothiocyanate) with
excitation wavelength 541 nm and emission wavelength 572 nm (emits red/orange fluorescence)
64 65
and PE (phycoerythrin) with excitation wavelength 565 nm and emission wavelength 573 nm 7.2.2.2 Isolation and analysis of lymphocyte subpopulations by flow cytometry
(emits orange-yellow light).
FACS (fluorescence-activated cell sorting) is a flow cytometry technique based on immunofluo-
7.2.2.1 Monoclonal antibodies 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
Monoclonal antibodies (mAbs) are monospecific, i.e. they recognize only a single antigenic separate different cell types due to their phenotype (size, granularity, surface molecules) in one step.
epitope, having an identical primary structure and effector functions. They are produced by a sin- This method uses fluorescently labelled monoclonal antibodies specific for cell surface molecules (CD
gle plasma cell clone and represent only one Ig isotype. mAbs are mainly produced in vitro by antigens) and is performed by the means of flow cytometers (Fig. 7.4). The main components of a flow
a hybridoma technology based on fusion of myeloma cells with spleen lymphocytes. Spleen cells cytometer are: a flow cell – liquid stream unit that carries the fluid with cells, an optical system that
are obtained from mice previously immunized with a desired antigen. The cells are mixed with consists of lamps and lasers, detectors which analyze FSC (forward scatter) and SSC (side scatter) sig-
PEG (polyethelenglycol) and then cultured in a selective HAT medium (hypoxanthine, nals as well as fluorescence, an amplification system (for signal amplification) and finally a computer
aminopterin, thymidine); only the fused cells can grow in this medium. Myeloma cells cannot for analysis of the signals. The principle of this method relies on hydrodynamic cell focusing. The
grow because they lack HGPRT, an enzyme needed for nucleic acid synthesis. However, the fused first step of this method includes incubation of cell suspension with fluorescently labelled monoclonal
cells can utilize the enzyme because it is supplied by the mice spleen cells (Fig. 7.3). Normal spleen antibodies by a room temperature. Then the prepared cell suspension is inserted into a flow-cell unit
cells also cannot grow because they have a limited life-span. The fused cells are diluted and cul- of the flow cytometer that creates a thin stream of liquid. A vibrating mechanism causes that the liq-
tured individually in microtitre plates; the cell will multiply to form a clone – a hybridoma. Anti- uid stream is splitting into drops, thus each drop contains one cell. Each drop (cell) then passes indi-
bodies, which are produced by every hybridoma clone are analysed using ELISA or dot blot vidually through the laser beam (usually laser light) following analysis of emitting and scattered lights
techniques. The most producing and stable clone is selected for further processing. Another by several detectors. There are usually three main types of detectors involving multiparametric cell
method for production of mAbs includes injection of hybridomas into the peritoneal cavity of analysis: forward scatter (FSC) detector measures light reflection and determines cell size, side scat-
a mouse. The hybridoma will grow in a tumor secreting an ascites fluid rich for mAbs. The anti- ter (SSC) detector measures a scattered light and determines cell granularity and finally a fluores-
bodies are then purified from the culture medium or ascites fluid by filtration and affinity chro- cence detector evaluates fluorescence of used mAb and determines cell type. The signals from each
matography. According to a production method, many types of mAbs were prepared until now. detector are digitized and passed to a computer for result analysis. The present flow cytometers are
Recombinant mAbs are produced by cloning of immunoglobulin genes using viruses (phage dis- capable of analyzing up to 13 parameters (forward scatter, side scatter, 11 colours of immunofluores-
play) or yeast (yeast display), chimeric mAbs are prepared by recombination of mouse and human cence) per cell and up to 100,000 cells per second (multiparametric analysis). Some flow cytometers
genes. Nowadays, the fully humanized mAbs have been prepared by transgenic mice and phage can also sort cells into different populations according to their fluorescence. In this case, just before
display technology to avoid the side effects of recombinant and chimeric mAbs. The application the stream breaks into droplets, they pass through electrical charging ring that provides a charge to
of mAbs is wide including diagnostic as well as disease therapy. In diagnostic they are used for in- a desired fluorescently labelled cell in the droplet, e.g. the CD4/FITC labelled cell. The droplets then
stance in phenotyping of leukemia and lymphomas fall through an electromagnetic sys-
by imunohistochemistry, immunofluorescence or tem that divides droplets according
flow cytometry. In the therapy, mAbs are used to to their charge into tubes. The sepa-
treat cancer, autoimmune and inflammatory dis- rated cells, e.g. CD4/FITC labelled,
eases, transplant rejections, etc. cells can be used for further analysis.
Fig. 7.3 Production of monoclonal antibodies Fig. 7.4 Flow cytometer

Monoclonal antibodies are produced by the hybridoma technology based on The principle of cytometry relies on hydrodynamic
fusion of myeloma cells with spleen lymphocytes. Spleen cells are obtained cell focusing. After incubation of cell suspension
from mice immunized with the desired antigen. The cells are mixed and then with fluorescently labelled monoclonal antibodies,
cultured in selective HAT medium (containing hypoxanthine, aminopterin the cell suspension is inserted into a flow-cell unit
and thymidine). Only the fused cells (hybridomas) are able to grow in this that creates a thin stream of liquid. A vibrating
medium. The most producing and stable hybridoma clone is selected for an- mechanism makes the liquid stream split into
tibody production (modified from www.google.com /bio.davidson.edu). 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).
66 67
The data obtained by the flow cytometer can be displayed as a count or a graph. Two types 7.2.2.3 Application of flow cytometry in immunodiagnostic
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 Flow cytometry is widely used in research as well as in diagnostic. Analysis of lymphocyte
two or more parameters, e.g. forward vs. side scatter, single colour vs. side scatter and two-colour subpopulations in patient’s blood samples is used in the diagnostic of primary and secondary im-
fluorescence data. Combinations from forward and side scatter data are usually used for the blood munodeficiencies. In HIV-infected individuals (secondary immunodeficiency of CD4 cells)
cell sorting into lymphocytes, monocytes, and CD4/CD8 ratio counting is widely used for monitoring of a disease progression and a response
granulocytes and determination of the differen- to treatment. The ratio is decreased in HIV-infected individuals (< 0.5), but increased in patients
tial blood count (Fig. 7.6). Each dot determines one with autoimmune diseases (> 2.5). Flow cytometry is also used for immunophenotyping of
cell on a dot plot graph. Finally, the regions on the leukemias and lymphomas. It is very effective in distinguishing myeloid and lymphoid lineages
dot plots can be separated and individually in acute leukemias and minimally differentiated leukemias. An example is expression of CD10,
analysed. This selection is termed “gating”. Thus which is common in acute lymphoblastic leukemia (ALL) patients. Clinical applications of flow cy-
we can create a gate around the lymphocytes to tometry in organ transplantation include pre-transplant cross-matching, HLA-antibody screen-
analyze only this cell population (Fig. 7.7). 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
Fig. 7.5 One – dimensional histogram important and correlates with engraftment success and its recovery. Antigen deficiencies in leuko-
Histogram shows the count of CD8+ cells labelled with PE (dark line) cytes can be also assessed by flow cytometry, e.g. decreased expression of neutrophil adhesion
in comparison to non-labelled cells (grey line), molecules is a cause of leukocytes adhesion deficiency (LAD1 syndrome). The disorder is charac-
(modified from www.google. com/scbt.com).
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-
Fig. 7.6 Dot plot graph sorting by cell size pression of HLA-B27 in patients suffering from Bechterev disease or HLA-DR4 in patients with
Dot plot of data combinations from forward (FS) and side scatter (SC) rheumatoid arthritis. Finally, the functional tests including lymphocyte transformation assay and
shows blood cell sorting into lymphocytes, monocytes and granulocytes NK cell cytotoxicity testing can be provided by flow cytometry too.
(modified from www.google.com/acb.sagepub.com).
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
Fig. 7.7 Dot plot graph sorting the cells by fluorescence molecule are used. First step of their isolation includes separation of mononuclear cells from the
Dot plot graph sorting of T helper CD4 lymphocytes labelled with PE whole blood by gradient centrifugation (see Chapter 7.2.1). Mononuclear cells are then mixed with
(lower right quadrant, D4) and T cytotoxic CD8 lymphocytes labelled with anti CD4 mAb labelled with magnetic beads and incubated 15 min. at 4°C. After incubation, the col-
FITC (upper left quadrant, D1). Unlabelled cells are present in the lower
left quadrant (D2) (modified from www.google.com/furukawa.co.jp). 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
68 69
(CD3+CD4+CD8-) or T cytotoxic cells (CD3+CD4-CD8+). In the negative selection, unwanted cells are In this case, B lymphocyte binds mouse RBCs. To perform rosette technique, lymphocytes isolated
labelled by mix of mAbs and attached to the column. In this case cells of interest flow through, they by gradient centrifugation (1.107) are mixed with 1% suspension of sheep RBCs and incubated 30
are collected and prepared for the further analysis. The negative selection can be used for NK cell iso- min. at 4 °C. After centrifugation, the cell sediment is stained and rosettes are counted under a mi-
lation (CD3-CD16+CD56+) or T lymphocytes isolation (CD3+). In the case of T cells isolation, mix of croscope. In general, the rosettes number determines the lymphocytes number. There are two types
mAbs binding to CD14, CD16, CD19, CD20, CD36, CD56, CD66b, and CD123 can be used. of rosettes according to the rate of formation. The active rosettes are formed in the presence of low
In clinical medicine, the magnetic cell isolation is used in transplantation immunology and number of RBC (1 Ly : 8 RBCs) within 15 min, whereas the total rosettes are formed in excess of
oncology. It is known that positive graft outcome relies on graft matches between a donor and RBCs (1 : 25–50) within long time period (18 hrs.). It was shown that the active rosettes define pop-
a recipient. In patients who have undergone the peripheral blood stem cell transplantation, the ulation of active T lymphocytes with the major clinical significance as they serve as a sign of indu-
graft versus host disease (GvHD) is the most serious complication resulting in the graft rejection. ced cell-mediated immunity.
To reduce a contamination of the blood by donors T lymphocytes (inducing immune reactions = Isolation of B lymphocytes by cotton is an another old separation technique. The first step of
rejection), the method of magnetic cell sorting is now used. By the positive selection, CD34+ pro- this method includes separation of mononuclear cells from the whole blood by gradient centrifu-
genitor cells can be obtained and CD3+ T cells by the negative selection (the method is known as T gation. These cells are subsequently let to pass through a column containing cotton wool. Only B
cell depletion). Using this procedure, a patient receives a pure population of CD34+ progenitor cells bind to the column (because of the spikes in their membrane), whereas other cells flow through.
cells what results in a reduced probability of a graft failure. The most used magnetic sorters are Another separation method uses SEPHADEX column with bounded protein A. When mononu-
CliniMacs by Miltenyi Biotec (Germany) and Dynabeads by Dynal Biotech (Norway). 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).
Fig. 7.8 Cell separation by magnetic beads 7.2.3 Lymphocyte transformation test
After incubation of cell suspension with magnetic-labelled monoclonal anti-
bodies (CD4+ labelled), the column with cell suspension is placed into a mag- Lymphocyte transformation test (LTT) or blastic transformation assay belongs to methods
netic device (MACS – magnetic cell sorter). Only the cells labelled with
used to evaluate lymphocyte function in vitro. The principle of the test is based on lymphocytes re-
magnetic monoclonal antibodies stay in the column, whereas non-labelled
cells flow through (modified from www.google.com/springerimages.com). 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-
7.2.2.5 Isolation of B and T lymphocytes by other methods 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
Isolation of B and T lymphocytes by rosettes belongs to an old separation technique. Now antibody labelled with peroxidase or fluorochrome is used and its intensity is measured.
it is occasionally used in the research and was replaced by flow cytometry and magnetic cell sort- The general procedure of LTT includes the following steps:
ing. The principle of this method relies on the reaction between surface protein of the lymphocyte 1) Isolate lymphocytes from peripheral venous heparinised blood by a gradient centrifugation
and its ligand on red blood cells (RBC) originating from other species. RBCs bind to the cell and (e.g. a Ficoll – Hypaque solution). Lymphocytes should be isolated within 24 hrs after the
form a cluster that looks like flower (rosette). In general, the rosette is defined as one that binds blood was drown.
Dilute mitogens to concentrations 1 µg, 2 µg and 5 µg/50µl, respectively.

three or more RBCs. For the lymphocyte separation, two types of rosettes can be used. E rosette is 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
used for T cells isolation and is formed after interaction of T cell CD2 molecule and its partner 3)
LFA3 (CD58) present only on the surface of sheep red blood cells. Red blood cells from other 4)
species cannot be used in this type of rosetting. For B cells isolation, M rosette formation is used. to a schedule, control wells contain only medium without mitogens
70 71
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 dermis of the forearm. As tested substances are used killed bacteria, their proteins or allergens
6) (see Chapter 8.1.1). The mostly used bacterial proteins are tuberculin and candidin. The applica-
7) Precipitate cells onto a filtrate paper using a precipitation device tion of tuberculin is used for tuberculosis diagnosis and is known as the Mantoux screening test.
8) Dry filters at room temperature, trim off and put into the scintillation vessels, add scintilla- After 48 to 72 hours, the immune response to antigens is evaluated as induration and erythema.
Measure the radioactivity using a β-scintillation counter.

tion fluid and enclose These symptoms are caused by increased activity of macrophages, dendritic cells, and T cells in
9) the site of antigen injection. The diameter of induration is measured in mm and the reaction is
The radioactivity is expressed as counts per minute (cpm) and the stimulation index (SI) is cal- classified as following: induration between 6 to 10 mm is recorded as “+”, between 11 to 15 mm as
culated according to the following formula: “++”, between 16 to 20 mm as “+++” and induration up to 20 mm is recorded as “++++”. Finally,
SI = cpm of mitogen-stimulated samples / cpm of control (non-stimulated) samples. the skin test index is counted using following formula: the total number of crosses/the number of
Values SI over 2.0 are considered as positive, i.e. antigens (mitogens) induced proliferation tested antigens. If the index value is higher than 1, the test is positive, i.e. the person developed cell-
of analysed lymphocytes (lymphocytes were sensitized by testing antigen). mediated immune response to the tested antigen. Otherwise, if the value is lower than 1, the per-
LTT is used in the research as well as in diagnostics. In routine medicine, LTT is mostly used son has a decreased activity of cell mediated immune response and in the case of the Mantoux test
in the diagnosis of allergies (food or drug-induced). In this case, lymphocytes obtained from an in- it means that tuberculosis (TB) vaccination of the individual is required. High positivity test value
vestigated person are exposed to allergens instead mitogens. The most tested drugs are gold salts, (number of crosses up to 4) can indicate a latent TB infection; however, to confirm the diagnosis,
amalgam, HgCl2, Ni, and Be. However, LTT measures only the sensitization of lymphocytes but other tests have to be performed. One of the most widely used tests is Quantiferon TB-Gold test.
not an effector reaction. Thus LTT can be positive due to allergy to the tested substance. However, The test evaluates production of IFN-γ by patients T-lymphocytes that were sensibilized by My-
clinical symptoms must be always considered to establish the diagnosis. To verify the final diag- cobacterium tuberculosis.
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

SUMMARY AT A GLANCE
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- •Adaptive immune response, also known as specific immune response, is composed of humoral (anti-
duce antigen-specific antibodies or T lymphocytes to produce cytokines. ELISPOT is based on the bodies) and cellular (T and B lymphocytes) components.
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 •To evaluate serum immunoglobulins levels (specific humoral imunity), methods like nephelometry, tur-
antibodies are chosen according to the specificity of a supposed secreted product. The first step of bidimetry and ELISA are performed.
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 •For quantitative analysis of lymphocytes (specific cellular imunity), their separation by gradient cen-
antibody on the nitrocellulose or PVDF-membrane. Following cell lysis and plate washing the en- trifugation (using Ficoll-Hypaque separation solution) followed by flow cytometry is performed. Flow cy-
zyme-labelled antibody, which recognizes the specific epitope of the analysed product (e.g. cytometer can sort cells into different population according to their size and membrane antigens (i.e. CD
tokine), is added. After washing, the secreted product is visualized by a substrate solution. The molecules) using fluorescently - labelled monoclonal antibodies.
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 •The functional evaluation of lymphocytes is performed by the lymphocyte (blastic) transformation test
using an automated plate reader – ELISPOT reader. The number of spots represents the number (in vitro) and skin tests (in vivo).
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 73
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- 8.1.1.2 Prick to Prick test
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 The Prick to Prick test is a modification of the standard Prick test used almost exclusive-
in erythema and induration with pruritus at the application site within 20 minutes of application. ly in the diagnosis of food allergy, where no commercially available test allergen concentrates
The choice of tested allergens depends on the geographic area, but also on a patient’s his- are used.
tory. In a patient with suspected allergy, we almost always apply the pollen allergens of birch Prick to prick is also performed on the volar forearm, with the needle being first inserted in
trees (birch, alder, hazel), a mixture of grass and cereal pollens, ragweed, mugwort, dust mites, the fruit or vegetable, trying to have a minimum quantity of tested food on the needle tip. The
dog epithelium and cat saliva (Table 8.1). The allergens are applied to the skin of the volar aspect needle is then applied directly to the skin to test for the food allergen injected to the mast cells in
of the forearm, where the skin to which a drop of allergen is applied is cleansed and dried, the skin. The skin reaction, wheal and erythema, is assessed also after 20 minutes – the same as in
whereupon the skin is pierced by a lance to apply it to the epidermis (about 1 mm deep) where the prick tests. Prick to prick is limited to foods (fruit and vegetables) with higher water content.
mast cells are present. In addition to the selected allergens, negative and positive controls are Unlike standardized tests, these tests are less specific.
also applied. As negative controls, a solution which is used to dissolve the allergens is used, The most common food allergies are caused by milk, eggs, peanuts, apple, kiwi, etc. Food al-
and as a positive control we use histamine, resulting in reddness and wheal in each individual. lergies may occur frequently as oral allergy syndrome (swelling of the tongue and gums), diarrhea,
The reaction is assessed after 20 minutes. A positive reaction is when the size of the wheal is or rash, anaphylaxis is relatively rare. Food allergy is often masked by the cross-reactivity of al-
3 mm greater compared to the negative control. Based on the results of the skin tests, as well as lergens. Patients hypersensitive for example to birch pollen allergens may be allergic for example
the clinical manifestations, the physician reaches a final diagnosis and chooses the appropriate to kiwi, pear, tomato, celery. Cross-reacting allergens are also found among grasses and food al-
treatment. lergens in wheat flour, also in ragweed and melon, etc.
74 75
8.1.1.3 Intradermal skin test 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
Intradermal skin tests are relatively rarely used, almost exclusively for the diagnosis of drug if these antibodies are present in patient´s plasma, the patient is with high probability clinically hy-
allergy. Only injectable forms of drugs are used for intracutaneous testing, i.e. liquids not forming persensitive to the tested allergen. In a clinical setting, the sIgE assessment is usually indicated in
a suspension. Intracutaneous tests are used particularly to test for hypersensitivity to anesthetics the case of sensitization against an allergen that is not included in the skin diagnostic set (prick test),
like Lidocaine, Mesocain, or to some antibiotics as Penicillin_G. Hospitalized patients on a strict in food, or professional allergy. In the recent past, the specific IgE was assessed most often using
diet are most commonly applied with an intracutaneous placebo solution (saline solution) and the RAST method (Radioallergosorbent test), but at present this has been replaced by non-radio mod-
5 cm from it the tested solution – in the area between the neck and shoulder. The reaction is as- ifications of immunoassays. Currently the most commonly used methods of immunoassay are
sessed after 15 minutes and then 8 hours after being administered. The next day, another drug test those where the allergen is bound to a solid phase (UniCAP, RISA, EUROLINE). UniCAP, or so-
(such as procaine penicillin) may be used on another part of the back. called CAP-FEIA, combines the principles of enzyme and fluorescent immunoassay.
Drug allergy may be caused by various immunopathological mechanisms, and often it can Other soluble molecules are being increasingly assessed in allergology, such as tryptase,
be masked by adverse drug reaction or photo-toxicity. Intradermal skin tests are also used in the which is a mast cell protease, released in their activation together with histamine. As opposed to
assessment of specific immune responses. 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
8.1.1.4 Epicutaneous “patch” test 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.
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 8.1.2.1 Basophil activation test
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 In the case of discrepancy between the clinical condition of the patient, the skin tests and the
caused in susceptible individuals by compounds of chromium or nickel metal contained, for ex- results of specific IgE assessment, it is now possible to perform the so called ‘basophil activation
ample, in jewelry and bone implants. But increasing numbers of people, especially women, are also test’. This assessment represents the capability of sensitized basophils to respond to the allergen.
allergic to the components of various perfumes. Basophils are the biggest storers of soluble mediators (histamine, leukotrienes, PAF /platelet ac-
Allergens are applied to the skin of the back in commercially prepared patches. Individual tivating factor/, etc.) in blood circulation. If the basophils, expressing a specific IgE antibody in
patches contain mixtures of various chemicals, such as metals (potassium-chromate, cobalt-chlo- their membrane against a particular allergen are exposed to its action, degranulation will result.
ride, nickelsulphate) components of perfumes and cosmetics (Fragrance_mix, Quaternium-15, We can observe these events observe in an optical microscope profiting from the fact that the gran-
Kathon_CG), rubber components (Thiuram_mix, IPPD, Merkapto_mix, Rosin), certain drugs ules possess great affinity to basic stains such as the toluidine blue.
(neomycin, Benzocaine, Budesonide), formaldehyde, lanolin and others. Basophil activation is characterized by their expression of CD63 or CD203c. This is used in
These patches are left on the back for two days; thereafter they are peeled off and assessed. Flow-CAST®, which is the commercial designation of the test used to determine the number of ac-
The skin symptoms are assessed after removal of the patch, i.e. on the third day after application. tivated basophils using flow cytometry. The procedure is based on the incubation of heparinized
The skin is assessed for the presence of erythema, papules, vesicles or bullae. Patients who had blood with allergen. After incubation, a monoclonal antibody anti-CD63 or anti-CD203c is added
a positive patch test are recommended to avoid the substance for the rest of their lives. 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
8.1.2 Laboratory assessments tion to their total number. The basophil activation test is currently rarely used, usually in the diag-
The development of new laboratory methods has made the diagnosis of allergies without ad- in anaphylactic or so far non-sensitized patients. Its great advantage lies in the speed of the test,
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 where the result, as opposed to a specific IgE test, may be available as soon as in 4 hours.
and elevation of overall IgE (above 240 µg/l, or 180 kIU/l). These parameters may be increased also
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
due to parasitary infections, tumors or some immunodeficiencies. Among allergy disorders, the high- and exposure tests or in older patients. In the lymphocyte transformation assay in sensitized in-
est serum levels of the total IgE may be observed in atopic eczema. In the past, the total IgE assessment dividuals, drugs to which the individual is sensitive results in the blast transformation of lym-
was based on the RIST method (Radioimmunosorbent test), a modified radioimmuno assay (Chapter phocytes. Drugs are diluted for the test according to a specific ratio, and phytohemagglutinin is
4.2.1.1). At present, total IgE levels are diagnosed using methods that are capable of determining the used as a positive control.
76 77
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 provocation 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.
78 79
Laboratory Metods in Immunology Diagnostics of autoimmune diseases
by the presence of autoantibodies and self-reactive T cells. As noted above, the clinical manifesta-
9. Diagnostics of autoimmune diseases 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
9.1 Introduction 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
Autoimmune diseases (AID) are a heterogenous group of more than 80 different diseases that abnormal values in basic laboratory tests may draw attention to the possible presence of AID.
affect approximately 5 % of the population of the Western countries. They are characterised by in- Autoimmune diseases have strong genetic background and sometimes run in families. Pos-
appropriate humoral or cell-mediated immune reactions against host own components and de- itive family history for AID is therefore suggestive for the presence of the disease in patients with
velop as a consequence of complex interactions between numerous predisposing genes and clinical manifestations of tissue and organ injury. However, the definitive diagnosis of AID can
environmental factors (microorganisms, drugs, chemicals, etc.). The breakdown of central (reces- only be established after appropriate laboratory tests have been performed.
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 9.2.2 Laboratory diagnostics
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), The laboratory diagnostics of autoimmune diseases employs a variety of basic and advanced
deposition of autoantibody-antigen immune complexes into the tissues and the induction of in- laboratory tests enabling the evaluation of different parameters of immune responses. The hall-
flammation (type III HR), direct lysis of cells by activated cytotoxic T cells (type IV HR) or stimu- mark of diagnostics, however, remains the detection of circulating or deposited autoantibodies.
lation/blocking of membrane receptors by bound autoantibodies (type V HR). Therefore, the proper diagnosis of AID is mostly established only if both typical symptomatology
Autoimmune diseases are generally classified into two groups. The organ-specific autoim- and specific autoantibodies are present.
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 9.2.2.1 Complete blood cell counting and enumeration of lymphocyte subpopulations
(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 The complete blood cell count (CBC) with differential provides useful information on the ab-
neuromuscular system (multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, etc.), gas- solute and relative numbers of blood cell populations. These may be significantly altered in certain
trointestinal system (Crohn’s disease, ulcerative colitis, primary biliary cirrhosis, autoimmune autoimmune diseases. AID caused by the production of autoantibodies against blood cell mem-
hepatitis, etc.), endocrine system (type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroidi- brane antigens (autoimmune anaemias, neutropaenias, thrombocytopaenias and lymphopaenias)
tis, Addison's disease, etc.), skin and mucosa (pemphigus vulgaris and foliaceus, psoriasis vul- are usually accompanied by decreased numbers of respective cells. Chronic inflammatory processes
garis, dermatitis herpetiformis, vitiligo, etc.), blood cells (autoimmune haemolytic anaemia, typical for systemic AID often manifest with various changes in the blood picture, such as nor-
autoimmune trombocytopenic purpura, etc.) and other organs and systems. Systemic AID include mochromic normocytic anaemia, leukocytosis or leukopaenia etc. Moreover, the reduction of leuko-
connective tissue diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic cyte numbers can also appear as a consequence of immunosuppressive therapy and blood cell
sclerosis, idiopathic inflammatory myopathies, Sjögren’s syndrome and spondyloarthropathies. counting is helpful in monitoring these adverse side-effects.
More specialised tests include enumeration of lymphocyte subpopulations by flow cytometry
9.2 Diagnostic strategy (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
Autoimmune diseases may affect virtually every tissue and organ in the body and often in numerous autoimmune disorders and reflect the chronic activation of self-reactive CD4+ T cells.
manifest with a diverse spectrum of non-specific and specific symptoms and signs. Therefore, On the other hand, a decrease of CD4+ lymphocyte levels and CD4+/CD8+ ratio below reference
their proper diagnosis and treatment usually require cooperation between specialists from multi- values during the long-lasting immunosuppressive therapy indicates an increased risk for the de-
ple medical disciplines. Similarly to other diseases, the diagnostic procedure starts with detailed velopment of secondary immunodeficiency.
anamnesis and physical examination and continues with an array of laboratory tests.
9.2.2.2 Biochemical examinations and function tests
9.2.1 Anamnesis and physical examination
Autoimmune processes in particular organs usually result in their functional abnormalities
Anamnesis and physical examination should focus on the detection of symptoms and signs and various changes of biochemical markers. Autoimmune hepatitis will, for instance, manifest
of inflammation, structural damage and/or functional impairment of organs and tissues caused with increased alanine transaminase (ALT), aspartate transaminase (AST) and bilirubin levels. Au-
80 81
toimmune inflammatory myopathies can present themeselves with increased levels of muscle en- at 4 °C until the precipitation appears and a cryocrit is measured. The final confirmation of the
zymes – creatinine kinase (CK), alanine transaminase (ALT) and aspartate transaminase (AST), presence of cryoglobulins is done by warming up the serum to body temperature, leading to re-
while autoimmune-mediated injury of kidneys may lead to increased urea and creatinine concen- dissolution of the precipitate. The occurrence of cryoglobulins is typically associated with gam-
trations, proteinuria and haematuria and decreased glomerular filtration rate and creatinine clear- mopathies (multiple myeloma, Waldenström macroglobulinaemia) and lymphoproliferative
ance. Sjögren’s syndrom is characterised by increased levels of amylases due to the affection of disorders, but may also be found in certain autoimmune diseases such as systemic lupus erythe-
exocrine glands. Type 1 diabetes mellitus is typically characterised by increased plasma glucose matosus, rheumatoid arthritis and vasculitides.
levels and glycated haemoglobin. Coagulation studies in antiphospholipid syndrome will reveal
prolonged activated partial thromboplastin time (aPTT), dilute thromboplastin time (TDT/DTT) or 9.2.2.6 Detection of autoantibodies
prothrombin time.
As mentioned above, the detection of specific high-affinity autoantibodies against various
9.2.2.3 Evaluation of inflammatory markers 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-
The presence of an inflammatory process in certain autoimmune diseases can be reflected by teins, membrane receptors and extracellular proteins (Tab. 9.1). Although not all of them directly
increased levels of acute phase proteins (APP) synthesized by hepatocytes and some other cells participate in the pathogenesis of a particular disease, they serve as important markers for the pres-
(monocytes, macrophages, neutrophils etc.) in response to proinflammatory cytokines produced ence, nature and intensity of autoimmune processes in the organism. Therefore, qualitative and/or
during the tissue injury and inflammation (Chapter 5.2). The measurement of C-reactive protein quantitative evaluation of autoantibodies in the biological samples from patients with suspected
by turbidimetry, nephelometry or ELISA is typically used to monitor the course of certain sys- AID plays an essential role in the diagnostics and classification of autoimmune diseases. In addi-
temic AID such as rheumatoid arthritis, juvenile idiopathic arthritis and some types of systemic tion, autoantibodies also have predictive and prognostic value as they usually appear several
vasculitides. On the other hand, CRP is not increased in systemic lupus erythematosus, autoim- months to years before the actual autoimmune disease manifests with first symptoms and signs, and
mune inflammatory myopathies, systemic sclerosis, Sjögren’s syndrome and organ-specific AID. thus may be helpful in the early diagnostics of future disease in currently healthy individuals.
Erythrocyte sedimentation rate (ESR) is another useful marker, as almost all systemic AID are ac- Lastly, the monitoring of changes in antibody quantities and profiles represents a helpful tool for
companied by increased FW. Its evaluation can therefore be used to monitor disease activity and assessing the activity and severity of the disease and its response to treatment. On the other hand,
response to the treatment. 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
9.2.2.4 Evaluation of total immunoglobulin levels AID must be based on co-existence of both appropriate clinical symptomatology and specific au-
toantibodies in the biological sample.
Abnormal levels of immunoglobulins are commonly found in various immune-related dis- Over the last decades, a broad spectrum of immunoassays for the detection of circulating au-
eases, including primary and secondary immunodeficiencies, haematological malignancies, in- toantibodies has been introduced in the laboratory diagnostics of AID. The discovery of new au-
fections and autoimmune disorders. Their evaluation by nephelometry is therefore an important toantigens, gradual improvements in antigen purification and recombination techniques and
tool in immunologic evaluation (Chapter 4.1). Increased total IgG levels are commonly found in increasing availability of new and improved tests and technologies have allowed for a qualitative
systemic autoimmune diseases and autoimmune hepatitis, increased IgA can be detected in leap in laboratory diagnostics of autoimmune diseases. The conventional immunochemical methods
Crohn's disease, ulcerous colitis, ankylosing spondylitis and autoimmune hepatitis, and increased for the qualitative or semiquantitative evaluation of autoantibodies such as indirect immunofluo-
IgM can be seen in primary biliary cirrhosis and rheumatoid arthritis. On the other hand, de- rescence (IFI), passive agglutination (PA), double radial immunodiffusion (DRID), counter-im-
creased levels of certain immunoglobulin classes are typically found in primary immunodefi- munoelectrophoresis (CIE), immunoprecipitation (IP) and immunoblot (IB) have been replaced
ciencies (e.g. selective IgA deficiency), which are, in turn, associated with an increased risk of to a large extent by immunometric methods that are more reliable and allow quantitative measure-
developing autoimmune diseases. ment of antibody concentrations. These methods comprise radioimmunoassays (RIA), enzyme
immunoassays (EIA), fluorescent immunoassays (FIA), fluorescent enzyme immunoassays (FEIA)
9.2.2.5 Evaluation of cryoglobulins and chemiluminiscent immunoassays (CLIA).
Indirect immunofluorescence (IFI) is one of the most widely used techniques in the laboratory
Cryoglobulins are monoclonal IgM (type I), mixture of monoclonal IgM and polyclonal IgG diagnostics of AID. The detection of autoantibodies in the serum is performed by its incubation
(type II) or mixture of polyclonal IgM and IgG (type III) that precipitate reversibly in cold tem- with antigenic native cell structures present in the specific tissue sections or cultured cell lines. The
peratures. To assess their presence, venous blood is drawn and placed in the tube without anti- bound autoantibodies are subsequently visualised by fluorescently-labelled anti-immunoglobulin
coagulant. After the coagulation has occurred and the clot has been removed, the serum is kept (secondary) antibodies directed against the constant Fc fragment of immunoglobulins IgG, IgM
82 83
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
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
86 87
In addition to detecting circulating autoantibodies, the diagnostic approach in some au- 9.2.2.8 Evaluation of the complement system
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- Alterations in complement proteins levels and activity are commonly observed in various
drome characterised by antibodies to glomerular basal membrane, and autoimmune bullous skin autoimmune diseases, either as their primary cause (primary C2, C3 and C4 deficiencies are as-
diseases characterised by the production of autoantibodies against desmosomal proteins (pem- sociated with impaired clearance of immune complexes and increased risk of developing im-
phigus foliaceus, pemphigus vulgaris), adhesion molecules of the dermal-epidermal junction mune complex-mediated diseases) or as a consequence (autoimmune diseases with immune
(pemphigoid) and epidermal transglutaminase (dermatitis herpetiformis). Tissue-bound anti- complex deposition lead to significant consumption of complement proteins; autoimmune hep-
bodies are detected in kidney biopsy samples and skin excision samples, respectively, by adding atitis is associated with decreased complement synthesis; autoimmune diseases associated with
anti-immunoglobulin antibodies labelled with a fluorochrome. Antibody deposits are subse- chronic inflammation often lead to increased synthesis of APPs, including the complement pro-
quently visualised under the UV microscope (Chapter 4.2.3.1). 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
9.2.2.7 Measurement of circulating immune complexes 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-
Certain autoimmune diseases including the systemic lupus erythematosus, autoimmune vas- ity (Chapter 5.1.2).
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 9.2.2.9 Cytokine measurement
to soluble antigens. Immune complexes subsequently deposit in the tissues (kidneys, joints, vessels)
and induce an inflammation (glomerulonephritis, arthritis, vasculitis). Therefore, the measurement of Various autoimmune diseases are typically associated with increased levels of different cy-
CIC may be helpful in the diagnostics of these inflammatory autoimmune diseases and provides use- tokines such as IL-1, IL-6, TNF, IL-2, IL-4, IL-5, IL-17, IFN-γ and others. Although their measure-
ful clinical information regarding the development, prognosis and follow-up. The amount of circu- ment is usually not routinely performed in commercial laboratories, the evaluation of their levels
lating immune complexes can be determined by polyethylene glycol precipitation assay, where mixing by ELISA or flow cytometry is helpful in certain autoimmune diseases in which anti-cytokine bi-
the serum containing immune complexes with polyethylene glycol will create a turbidity, the inten- ologic treatment (e.g. monoclonal antibodies targeting particular cytokine) is used.
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. 9.2.2.10 Genetic tests
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, The development of the majority of autoimmune diseases is associated with the presence of
anti-human IgG antibody conjugated with an enzyme (e.g. horseradish peroxidase) is added and particular risk HLA alleles/molecules, for instance HLA-DQ2 and -DQ8 in type 1 diabetes melli-
binds to the Fc fragment of human IgG in circulating immune complexes. After another incubation tus and coeliac disease, HLA-DR4 in rheumatoid arthritis, HLA-B27 in ankylosing spondylarthri-
and wash, the substrate is ad- tis, etc. Therefore, HLA-typing permorfed by serologic or genetic methods (Chapter 12.3) is used
ded. The enzyme catalyses its as supplementary test in cases of diagnostic insecurity and differential diagnostics. Certain im-
transformation into coloured munodeficiency syndromes associated with the development of autoimmune diaseases are caused
product and the intensity of by single mutation of particular genes. For instance, autoimmune lymphoproliferative syndrome
colourimetric reaction is read (ALP) is associated with mutations in Fas, Fas ligand, caspase 8 or caspase 10 genes, autoimmune
in a spectrophotometer (Fig. polyendocrinopathy with candidiasis and ectodermal dystrophy syndrome (APECED) with mu-
9.5). Another alternative is the tations in AIRE gene, and immune dysregulation-polyendocrinopathy-enteropathy-X-linked syn-
CIC-anti-C3d ELISA, which uses drome (IPEX) with mutations in FoxP3 gene. Their detection by sequencing is an integral part of
the anti-C3d antibody immo- a diagnostic protocol in these immune dysregulation disorders.
bilised on the microtiter plate
instead of C1q. 9.2.2.11 Other examinations
Histological and immunohistochemical examinations of bioptical samples obtained from

Fig. 9.5 ELISA assay for detection
tissues affected by autoimmune process are also important diagnostic tools in certain autoim-
of circulating immune complexes
mune diseases, and so are imaging and other methods for the visualisation of organ and tissue
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Laboratory Metods in Immunology Diagnostics of immunodeficiencies
affection. These include medical radiography, ultrasonography, magnetic resonance imaging,

electrocardiography, electromyography, electroencephalography and other examinations. 10. Diagnostics of immunodeficiencies
10.1 Introduction
Human immune system plays a crucial role in defence against infectious microorganisms and
SUMMARY AT A GLANCE 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-
•Autoimmune diseases are a heterogenous group of disorders characterised by tissue/organ injury or
nancies or autoimmune disorders, often with fatal consequences. These so called immunodeficiency
dysfunction induced by an immune reaction to self-antigens.
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
•Autoimmune diseases often manifest with a diverse spectrum of symptoms and signs and their proper
development and/or function of one or more components of the immune system, resulting in an in-
diagnostics requires the use of an array of basic and advanced laboratory tests.
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
•Laboratory tests for autoimmune diseases include complete blood cell counting, enumeration of lym-
a damage caused to the previously normal immune system by factors such as malnutrition, infections
phocyte subpopulations, evaluation of inflammatory markers (acute phase proteins, erythrocyte sedi-
(e.g. HIV, EBV, CMV, measles and others), chronic diseases (diabetes, hepatic and renal insuffi-
mentation rate), total immunoglobulin levels and cryoglobulins, detection of specific autoantibodies,
ciency, rheumatological, metabolical and other diseases), malignancies, radiation, surgery, trauma,
evaluation of the complement system and circulating immune complexes, and genetic tests.
treatment with immunosuppressive and chemotherapeutic drugs and some others.
To date, more than 150 primary immunodeficiencies (PIDs) have been described and classified
•Laboratory tests for detection of specific autoantibodies are the hallmark of autoimmunity diagnostics.
into several categories according to which component of the immune system is primarily affected:
They comprise methods such as direct and indirect immunofluorescence, ELISA, Western blot, passive
agglutination or microsphere-based multiplex flow cytometric immunoassays. • 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
•The proper diagnosis of an autoimmune disease is mostly established when both typical symptoma-
(CVIDs), hyper-IgM syndrome, selective IgA deficiency and others.
tology and specific autoantibodies are present.
• 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).
90 91
etc.), although diarrhea caused by Giardia intestinalis, recurrent enteroviral infections and severe
• Defects in innate imunity include rare PIDs caused by defects in pathogen recognition re- bacterial infections of skin and other organs may also be present. Patients with predominant de-
ceptor (PRR) signaling (IRAK-4 deficiency, MyD88 deficiency and others). fects of specific cellular immunity usually develop infections caused by viruses, fungi and intra-
• Complement deficiencies are caused by genetic defects of classical, alternative or lectin path- cellularly replicating bacteria such as mycobacteria or salmonella. Combined defects of B and T cell
way complement factors or their regulators. mediated immunity usually manifest early in the childhood as failure to thrive, chronic diarrhea
• Autoinflammatory disorders include diseases such as familial Mediterranean fever or TNF re- and severe infections caused by a wide range of different extra- and intracellular microorganisms
ceptor associated periodic syndrome, characterised by periodic fever and excessive inflam- (viruses, bacteria, fungi, protozoa), often opportunistic (e.g. Pneumocystis jiroveci). Phagocytic de-
matory response, rather than infections. 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-
10.2 Diagnostic strategy 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-
Diagnosis of immunodeficiencies often presents a challenge for clinicians and many ID dis- tions (meningitis, sepsis), recurrent pyogenic infection of respiratory tract and systemic autoim-
eases can therefore go undetected for a longer period of time as they may appear as ordinary in- mune diseases resembling SLE.
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 10.2.2 Physical examination
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- Patients suffering from immunodeficiencies may present with various symptoms and signs
tion of immune system function. If secondary immunodeficiency is suspected, appropriate tests of infections as well as other abnormalities accompanying certain IDs. It should be noted that not
for the diagnosis of underlaying disorder need to be carefully selected and performed, e.g. HIV every patient appears ill at the time of examination and clinical signs may sometimes be very sub-
tests, tests for the examination of potential gastrointestinal or urinary protein losses, liver tests, etc. 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
10.2.1 Clinical presentation and history lymphoproliferative syndromes, chronic granulomatous disease (CGD), common variable im-
munodeficiency and some others manifest with lymphadenopathy and hepatosplenomegaly. Pa-
Diagnostics of immunodeficiencies requires a stepwise approach and should begin with tients with some IDs present with skin lesions including albinism (Chediak-Higashi syndrome),
evaluation of clinical presentation and medical history, with particular regard to age at onset, type, petechiae (Wiskott-Aldrich syndrome), eczema, candidiasis, rash, warts, pustulae, abscesses or
location and severity of signs and symptoms. Generally, an immunodeficiency should be sus- alopecia. Patients with DiGeorge syndrome (developmental defect of third and fourth pharyngeal
pected in individuals who have recurrent and/or chronic infections of upper and lower respira- arches) have facial dysmorphism, conotruncal heart abnormalities, agenesis of the thymus and
tory tract, gastrointestinal tract or skin, although many patients may eventually develop more parathyroid glands. Facial abnormalities and growth retardation can also be seen in patients with
severe bacterial infections (sepsis, meningitis, osteomyelitis, abscesses of internal organs). Other leukocyte adhesion deficiency 2 syndrome (LAD2), the Nijmegen syndrome and some others. Pa-
typical features of immunodeficiencies are infections caused by unusual (opportunistic) microor- tients suffering from ataxia-teleangiectasia present with cerebellar and sensory ataxia and ocular
ganisms and complicated infections that don't respond well to conventional treatment or manifest teleangiectasia. Neurological changes, abnormalities of skeletal development resulting in short-
atypically. Age at which infections began to manifest is of great importance. The most severe forms limbed dwarfism and cartilage-hair hypoplasia are other clinical findings in certain PIDs.
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 10.2.3 Laboratory tests
immunodeficiency may not manifest until adulthood. Many immunodeficiency syndromes also
present with autoimmune diseases and malignancies. 10.2.3.1 Basic screening tests
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 The evaluation of an immune system should begin with basic screening tests. The complete
patient. Medical record of a clinical infection following the vaccination with live attenuated mi- blood cell count (CBC) with differential provides useful information on the number of blood cell
croorganisms in patient's personal history also supports the diagnosis of PID. populations, while the blood smear enables to detect their morphological abnormalities such as the
The nature of infections depends on a component of the immune system that is defective. Pa- presence of large granules in neutrophils (Chediak-Higashi syndrome) or their absence (specific
tients with antibody immunodeficiencies typically suffer from recurrent infections of sinopul- granule deficiency). The finding of reduced numbers of leukocytes and their subpopulations along
monary tract caused by encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae, with corresponding clinical presentation is indicative for the presence of immunodeficiency. The
92 93
presence of lymphopaenia is typical for certain primary immunodeficiencies affecting T cells gen responsiveness, respectively. An adequate 4-fold rise in the specific antibody titer is indicative
(CIDs, DiGeorge syndrome and others), but may also be of secondary nature (HIV infection, treat- for unimpaired humoral specific imunity. Specific antibody production can also be evaluated by
ment with immunosuppresive and cytostatic drugs, etc.). Reduced numbers of granulocytes (gran- measuring anti-A/anti-B IgM isohemagglutinin titers.
ulocytopaenia), mostly neutrophils (neutropaenia) are found in PIDs such as severe congenital
neutropaenia, cyclic neutropaenia, reticular dysgenesis, hyper-IgM syndrome and WHIM syn- 10.2.3.2 Advanced tests for evaluation of acquired (specific) imunity
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 If the results of basic screening tests are abnormal and/or the history and clinical presentation
(anaemia) are present in various different conditions, including some primary immunodeficien- are highly indicative for a immunodeficiency disease, further advanced tests must be performed. Spe-
cies (glucose-6-phosphate dehydrogenase deficiency, paroxysmal nocturnal haemoglobinuria, cialised methods for the evaluation of acquired imunity include enumeration of lymphocyte subpop-
etc.), while possible explanations for low platelet count (thrombocytopaenia) are Wiskott-Aldrich ulations (immunophenotyping) by flow cytometry (Chapter 7.2.2.2). The method is based on the usage
syndrome, some combined immunodeficiencies and CVID. Markedly increased numbers of leuko- of fluorochrome-labelled monoclonal antibodies specific to various membrane (CD) and intracellu-
cyte subpopulations are usually present in infections, haematological malignancies, myeloprolif- lar antigens and enables to evaluate the numbers of total T cells (CD3+), helper T cells (CD3+CD4+),
erative disorders and also in some immunodeficiencies. Neutrophilia is indicative for bacterial cytotoxic T cells (CD3+CD8+), regulatory T cells (CD4+CD25+FoxP3+), B cells (CD19+CD20+), NK cells
infections, leukaemias and certain primary immunodeficiencies (leukocyte adhesion deficiencies), (CD16+CD56+) or activated T cells (HLA-DR+CD69+). Extremely low or absent B cell counts are typi-
eosinophilia for allergies, parasitic infections, certain myeloproliferative disorders (hypere- cal for X-linked agammaglobulinaemia and related syndromes with markedly reduced Ig isotypes;
osinophilic syndrome), malignancies and primary immunodeficiencies (hyper-IgE syndrome, however, decreased or absent B lymphocytes can also be found in some severe combined immunod-
Omenn syndrome, Wiskott-Aldrich syndrome), while lymphocytosis is typically present in viral, eficiencies (reticular dysgenesis, adenosine deaminase deficiency, RAG1/2 deficiency) and occasion-
some protozoal and intracellular bacterial infections, leukaemias and lymphomas. ally in common variable immunodeficiency. Low or absent T cell numbers are characteristic for
Evaluation of total immunoglobulin levels by nephelometry and ELISA methods is also a part combined immunodeficiencies, DiGeorge and Wiskott-Aldrich syndromes, ataxia-teleangiectasia and
of initial screen, as abnormal levels of one or more Ig classes are typically found in various B cell some other well-defined IDs. Low helper T cell counts and a decreased CD4+/CD8+ T cell ratio are typ-
and combined immunodeficiency disorders. The results must be compared with age-matched ref- ically found in common variable immunodeficiency and MHC class II deficiency; however, they are
erence values. Physiologic hypogammaglobulinaemia, typically occuring in infants aged 3 – 6 also associated with conditions like the HIV infection, some bacterial and parasitic infections, physi-
months, must be taken into consideration when the values are interpreted. On the other hand, ap- cal trauma, treatment with corticosteroids or stress. Decreased numbers of cytotoxic T cells and an in-
parently normal IgG levels during first 2 – 3 months of life do not exclude the possibility of severe creased CD4+/CD8+ ratio are typical for a rare CD8 deficiency, MHC class I deficiency and numerous
antibody deficiency, as the IgGs in the blood of the child are of maternal origin. Concentrations of autoimmune disorders. An increased presence of CD4-CD8- double negative cells in the peripheral
IgG, IgM and IgA are usually measured by nephelometry (Chapter 4.1), whereas more sensitive blood is typically found in autoimmune lymhoproliferative syndromes caused by defects of media-
enzymatic immunoassays are used for IgE due to its low levels in blood (Chapter 4.2.2). Signifi- tors of apoptosis, while low levels of NK cells are found in certain combined deficiencies (IL-2RG de-
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- ficiency, JAK3 deficiency) and rare isolated NK cell deficiencies.
Flow cytometry assays are also used to screen for altered expression of specific extracellular or
deficiencies. Low levels of IgG, IgA and/or IgM isotypes along with impaired specific antibody intracellular proteins (receptors, membrane antigens, signalling molecules, cytotoxic molecules, etc.)
response is indicative for common variable immunodeficiency syndromes. Reduced levels of only associated with specific immune defects, for example an impaired expression of CD40 or CD40L
one Ig class are typically present in selective Ig immunodeficiencies, such as the most frequent on T cells in patients with hyper IgM syndrome, Bruton’s tyrosinkinase Btk in monocytes and
primary ID – the selective IgA immunodeficiency. Increased levels of immunoglobulins are usu- platelets (B cells are usually absent) in patients with X-linked agammaglobulinaemia, FoxP3 tran-
ally present in infections and monoclonal gammapaties, but may also be found in certain primary scription factor in regulatory CD4+CD25+ T cells in patients with IPEX syndrome and many more.
immunodeficiencies (hyper-IgE syndrome, Omenn syndrome, hyper-IgD syndrome). In hyper- Specific cellular immune function is routinely evaluated by delayed-type hypersensitivity skin
IgM syndrome, lower concentrations of serum IgG and IgA are usually found along with normal tests (Chapter 7.2.5). Testing antigen (candidin, tuberculin, etc.) to which the patient has already
or increased levels of IgM as a result of a defective immunoglobulin class-switching. In certain been exposed is injected intradermally on the volar forearm and the reaction is evaluated after 48 –
cases, quantitation of IgG subclass levels may be considered, although this test is of limited util- 72 hours. The appearance of erythema and induration larger than 5 mm excludes the diagnosis of T
ity. Defects of Ig production can also be evaluated by measuring specific antibody titers in response cell disorder. Alternatively, the function of lymphocytes can also be assessed in-vitro by lymphocyte
to protein (T-dependent) and polysaccharide (T-independent) antigens. In this case, specific anti- transformation test (blastic transformation test) using the mitogens, antigens, irradiated allogeneic
body response to standardized antigens is assessed before and 3 – 4 weeks after boosting immu- white blood cells or monoclonal antibodies to stimulate the proliferation of T and/or B cells (Chap-
nization with tetanus or diphteria toxoid vaccine to test for protein antigen responsiveness, and ter 7.2.3). Their response to stimulation is subsequently evaluated by measuring the radioactivity
with pneumococcal or Haemophilus influenzae polysaccharide vaccine to test for carbohydrate anti- which intensity depends on the amount of 3H-thymidine incorporated into the cells undergoing
94 95
blastic transformation. Low or absent uptake of radioactive thymidine indicates T cell or combined chromium release assay (see Chapter 6.2.2) in addition to flow cytometry assays detecting the pres-
immunodeficiency. Enzyme-linked immunosorbent spot assay (ELISpot) is another perspective ence of perforin, SAP and XIAP proteins.
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 10.2.3.4 Molecular diagnostics of primary immunodeficiencies
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 Mutation analyses are an integral part of diagnostic strategy in PIDs and an important tool
resulting coloured reaction (spot) is analyzed under the microscope or by automatised analyzer. Al- for identification of specific disease-causing mutations. They further form a basis for adequate
ternatively, cytoplasmic cytokine production can also be evaluated by flow cytometry. treatment, estimation of prognosis, development of preventive strategies, genetic counselling and
Serologic or flow-cytometric HLA-typing can be used to detect the absence of HLA class I and prenatal diagnostics. Detection of genetic defects (single base pair substitutions, small deletion
II molecules on lymphocytes of patients with MHC class I and II deficiencies (Chapter 12.3). En- and insertions or gross lesions) can be performed at DNA or RNA level by various PCR methods
zyme assays for adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activ- and DNA sequencing. Identification of a new gene variation must usually be supplemented by an
ity in red blood cells are performed to confirm the diagnosis of SCID caused by the defects of ADA analysis of mRNA and protein expression or function to evaluate its real disease-causing poten-
and PNP, respectively. 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
10.2.3.3 Advanced tests for evaluation of innate (non-specific) imunity 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).
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 SUMMARY AT A GLANCE
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 •Immunodeficiencies (IDs) are disorders caused by quantitative or qualitative defects in immune system
(Chapter 6.1.2.6); oxidative burst and the production of reactive oxygen species are abnormal in pa- components, which result in increased susceptibility to infections, malignancies or autoimmune diseases.
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- •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.
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
•Basic laboratory screening tests include complete blood cell counting with differential and evaluation
X) expression is absent on neutrophils of patients with LAD2. of total immunoglobulin levels and specific antibody response.
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 •The innate (non-specific) arm of imunity can be examined with the help of specific tests for the evalu-
and assess the levels of complement proteins. Primary complement deficiencies are very rare and re- ation of phagocytic and cidal activity and the respiratory burst of polymorphonuclear phagocytes. The
sult in defective direct killing, opsonisation and phagocytosis of bacteria and ineffective clearance of evaluation of complement system integrity includes complement activity assays, assays for measure-
immune complexes. The intact function of classical complement pathway is evaluated by total ment of complement protein levels and functional assays for individual complement factors. The func-
haemolytic complement activity (CH50) assay, while the activity of alternative pathway is evaluated tion of NK cells can be evaluated by chromium release assay.
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- •The numbers of lymphocyte populations can be determined by flow cytometry, while their function can
ment activation pathway and do not provide any information about which particular complement be examined in-vitro by lymphocyte proliferation test (blastic transformation test) and in-vivo by de-
component is affected, further assays for measurement of complement protein levels (Chapter 5.1.2.3) layed-type hypersensitivity skin tests.
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 •The disease-causing mutations can be detected with the help of PCR or sequencing methods.
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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Laboratory Metods in Immunology Diagnostics and monitoring of HIV infection
lived due to the activation of host humoral and cellular immune responses, which start to control
11. Diagnostics and monitoring of HIV infection 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
11.1 Introduction 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
The first mention of what would eventually become acquired immunodeficiency syndrome serologic methods approx. 3 – 4 weeks (22 days on average) after the exposure to HIV. The time
(AIDS) was first reported in 1981 in the United States when several cases of pneumonia caused by period in which the infection is present but the antibodies are not detectable yet is known as sero-
rare opportunistic Pneumocystic jiroveci (carinii) followed by other pathologic conditions were ob- logic “diagnostic window period” (Fig. 11.2). In some circumstances, it may take significantly
served in the community of healthy gay men and subsequently among intravenous drug users, longer than few weeks to generate specific HIV antibodies, as the duration of serologic “window
haemophiliacs, blood transfused patients and eventually heterosexual majority. The identification period” is rather variable and depends on several factors, including the way of transmission,
of the causal agent, the human immunodeficiency virus (HIV), followed in 1983. Since then AIDS dosage, subtype of HIV, age, health and immune status of infected individuals and others. Dur-
has become a global epidemic responsible for approx. 30 million deaths. ing the acute HIV infection, most of the infected individuals present influenza-like or mononu-
HIV is a member of the Lentivirus genus and the Retroviridae family, currently grouped into cleosis-like symptoms and signs lasting between 7 – 10 days. After several weeks from the start of
two types: HIV-1 and HIV-2. The more prevalent HIV-1 is classified into several groups (M, N, the infection, HIV positive individuals enter the asymptomatic period characterised by the ab-
O and recently described P), the group M is further subdivided into 11 subtypes (clades) and nu- sence of any symptoms, low viraemia levels and the presence of HIV-specific antibodies in the
merous circulating recombinant forms (CRFs). HIV-2 is mostly confined to West and Central plasma (Fig. 11.2). The continuous replication of the virus in the lymphoid compartments of un-
Africa; it shares only 30 – 40% homology with HIV-1 and is further subdivided into 8 groups treated patients leads to slow progressive depletion of CD4+ T cells and finally results in the im-
(A-H). HIV-1 is of spherical shape and its core contains two identical copies of single-stranded pairment of the immune system. The drop of CD4+ T cell counts below 200/μl is associated with
RNA bound to protein p7 and enzymes reverse transcriptase, integrase and protease. This complex repeated increase of viraemia and the development of AIDS, the final stage of HIV infection char-
is enclosed by a capsid composed of protein p24, which is, in turn, surrounded by a matrix made acterised by the presence of
of protein p17. This is anchored to the inside of the viral envelope composed of phospholipids and various opportunistic infec-
proteins derived from the membrane of infected cell and viral glycoproteins gp120 and gp41 tions, tumours, generalized
(Fig. 11.1). HIV infects numerous host cells includ- lymphadenopathy, encepha-
ing the CD4+ T cells, monocytes and macrophages, lopathy, reduction of weight
dendritic cells, eosinophils and microglial cell of and some other symptoms.
the nervous system. The replication of the virus
within the helper T cells and their destruction by Fig. 11.2 Dynamics of changes
host immune mechanisms results in their progres- of HIV RNA, p24 antigen, anti-HIV
sive depletion, the collapse of the immune system antibody and CD4+ T cell levels in
and the progression of HIV infection to AIDS. the course of HIV infection
Fig. 11.1 Schematic structure of the human immuno-

deficiency virus
11.2 Laboratory methods in HIV diagnostics

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- First blood tests for HIV infection were introduced in 1985 and since then, their quality has
tensive viral replication within the infected mucosa and lymphatic tissue eventually results in the been continuously improving. The main purposes of HIV testing are identification of infected in-
spreading of HIV into the bloodstream and its dissemination. The virus RNA becomes detectable dividuals and screening of blood supply in order to facilitate effective preventive and therapeuti-
in the blood within 10 – 12 days after infection start and its levels increase rapidly up to 1 million cal measures. According to the UNAIDS recommendations, HIV testing should be voluntary,
copies/ml. Simultaneously, HIV p24 antigen may also be detected in the plasma of infected pa- informed, available to anyone at request and at no charge, taken either confidentially or anony-
tients (in general 12 – 14 days after exposure). At the same time, the number of CD4+ T cells in the mously and coupled with pre- and post-test counselling. Laboratory tests used for the identifica-
blood temporarily decrease (Fig. 11.2). The high levels of HIV viraemia and antigenaemia are short- tion of HIV infection can be divided into two main categories: indirect and direct tests.
98 99
11.2.1 Methods of indirect diagnostics (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
Indirect methods comprise several screening and confirmatory tests based on the detection about 3 weeks. Recently, the fourth generation assays have been introduced, combining the de-
of specific antibodies to HIV antigens in various body fluids including the full blood, plasma, tection of specific HIV-antibodies with the direct detection of HIV p24 antigen. This has al-
serum, urine and oral fluid. 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
Fig. 11.3 ELISA assay for detection of anti-HIV antibodies 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
Enzyme-linked immunosorbent assays (ELISA) are the most commonly used quantitative where the HIV antigens are bound to particles and allowed to react with antibodies contained in
screening tests in the diagnostics of HIV infection. The first generation assays were indirect ELISA tested serum to form visible clumping. The technique is called passive haemagglutination when
tests that included whole-cell lysate HIV antigens obtained from infected cell cultures. Briefly, the red blood cells are used, or latex agglutination when the antigens are attached to latex parti-
the viral lysate was bound to the solid phase at the bottom of a microtiter plate and allowed to cles (Chapter 3.1.3).
react with added tested serum. If the serum contained specific antibodies against HIV anti- The Western blot (WB) is the most commonly used confirmatory assay for HIV diagnostics,
gens, the antigen-antibody binding had occurred. After the washing step, the bound antibod- due to its high specificity and ability to discriminate between specific antibodies against individ-
ies were detected by secondary antibodies coupled with an enzyme. After another washing ual antigens. Following the disruption of the HIV virus, individual HIV proteins (antigens) are
step, a substrate was added and the enzyme generated its chemical conversion to a colou- separated by electrophoresis according to their molecular weight, transferred onto a nitrocellu-
red product (Fig. 11.3a). The intensity of the colour reaction was proportional to antibody con- lose membrane and subsequently used to detect specific antibodies in the sample. In case of their
centration in the tested sample and was measured by spectrophotometer. These assays were presence in the sample, anti-HIV antibodies bind to antigens on the membrane and are subse-
not sufficiently specific and enabled the detection of specific antibodies 6 – 12 weeks after in- quently visualised by secondary enzyme-labelled antibodies (Fig. 11.4). According to the presence
fection onset. The second generation assays were based on the same indirect approach, but used of bands on the paper strip, the results can be read as positive, negative or indeterminate (see the
HIV recombinant antigens instead of viral lysate. This led to improved specificity and reduced next chapter). Many commercially available WBs now include antigens from both HIV-1 and HIV-
window period to approx. 5 weeks. The third generation assays first introduced in 1990s employs 2. The disadvantages of WB are its high costs, a certain degree of subjectivity when interpreting
a sandwich ELISA format. Here, the recombinant HIV-1/2 protein and peptide antigens are the results and frequent occurrence of indeterminate results. A similar assay called the line im-
bound on a solid phase (wells of microtiter plate or microparticles) and allowed to react with munoassay (LIA) uses recombinant HIV proteins and/or synthetic peptides that are directly ad-
specific anti-HIV antibodies contained in tested patient's serum. In the next step, the antigen- sorbed onto a nitrocellulose test strip, thereby avoiding difficult-to-control processes such as
bound antibodies are detected by addition of the same viral antigens already coupled with electrophoresis and blotting. The use of recombinant and/or synthetic antigens provides highly
an enzyme molecule. The reaction is subsequently visualised by an appropriate substrate reproducible results with high sensitivity and specificity.
100 101
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.
Fig. 11.4 Western blot for detection of anti-HIV antibodies
11.3. Diagnostic algorithms
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- 11.3.1 Identification of new cases of HIV infection
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 The diagnostic protocol for HIV infection commonly requires the identification of infected
system. The results are then analysed microscopically by experienced laboratory staff. Although individuals by screening tests and the confirmation of the positivity by confirmatory (supple-
IFA has less indeterminate results when compared to WB, its use is limited by the need for ex- mental) test. In most cases, laboratory tests for the detection of specific HIV antibodies are used.
pensive equipment and subjective interpretation of results. 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
11.2.2 Methods of direct diagnostics be accompanied by appropriate pre- and post-test counselling.
The diagnostic procedure starts with the use of appropriate screening test that possess a high
Methods that enable the direct detection of virus and its components in the infected organism, degree of sensitivity i.e. is designed to detect all individuals positive for HIV antibodies, even if their
and thus allow for further reduction of the diagnostic window include viral isolation through viral cul- titers and avidities are low. Such test should produce only minimum false negative results i.e. only very
ture, detection of HIV antigens and nucleic acid amplification testing for the detection of viral RNA few HIV-positive individuals will be missed. Possible causes of false negatives may include acute HIV
and DNA. Although these methods are highly specific, they all have certain limitations. Viral culture infection before the seroconversion, primary or secondary antibody immunodeficiencies or technical
tests were the first direct method to detect the HIV virus itself, but are slow, expensive and difficult errors during the testing procedure. The most commonly used screening tests are the third or fourth
to perform. Therefore, other direct methods are used instead. The p24 antigen appears in blood within generation HIV-1/2 ELISA tests, while rapid and simple tests are usually used in certain specific set-
2 weeks from infection due to the initial burst of virus replication and becomes detectable by HIV anti- tings such as point-of-care testing and in developing countries. Although all these tests are exquisitely
gen tests based on the ELISA methodology. The antigenaemia lasts approx. 3 to 5 months and then sensitive, their degree of specificity is slightly limited, resulting in the occasional occurrence of false
antigen levels drop below detection limit. With the progression to AIDS, p24 antigen becomes de- positive results. These can result from non-specifically reacting antibodies present in certain diseases
102 103
and conditions, such as other acute viral infections, autoimmune diseases, malignancies or recipients prior to seroconversion, antibody tests should be performed in conjunction with p24 antigen test
of trial HIV vaccines. For this reason, every sample producing a reactive result must be tested again (e.g. using the fourth generation combined assay) and plasma HIV RNA assay when the primary
in duplicate using tests with different antigen preparations and/or different test principles, and then HIV infection is suspected, e.g. in patients reporting recent risk behavior and presenting symptoms
verified with confirmatory assays to differentiate between true and false positive results. and signs of the acute HIV syndrome. When high levels of viral RNA are detected in plasma, the
The laboratoty tests used for confirmation (usually WB, alternatively IFA, LIA or NAAT) are tested person can be preliminary considered to be infected with HIV. Standard antibody tests should
characterised by high degree of specificity and thus produce only few false positive results. If per- be subsequently performed 3 – 6 weeks later to confirm the presence of specific antibodies.
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 11.3.3 Diagnostics of HIV infection in infants of HIV-infected mothers
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 Correct early diagnosis and treatment of HIV infected infants is crucial for reducing their
WHO recommendations, the positivity requires two Env bands (gp41 and gp 120/160), whereas morbidity and mortality. The laboratory diagnosis of HIV infection in newborns is complicated by
the Centers for Disease Control recommend the presence of at least two of p24, gp41 and gp120/160 the presence of passively transferred maternal anti-HIV IgG antibodies, which can be detectable
bands. American Red Cross is even stricter and requires the presence of antibodies to at least one in their blood for up to 18 months. Therefore, the diagnosis of HIV infection in HIV-exposed in-
HIV antigen from each group Gag (p17, p24, p39, p55), Pol (p31, p51, p66) and Env (gp41, gp120, fants should be established using nucleic acid tests to detect proviral DNA or viral RNA in their
gp160). Sometimes, confirmatory tests may exhibit indeterminate results (i.e. presence of 1 or more blood. Antibody tests can still be used at 12 to 18 months of age to definitely rule out the infection
bands, but not fulfilling the criteria of positivity), which can be in reality either true or false posi- in children who were previously negatively tested for the presence of viral nucleic acids.
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 11.3.4 Monitoring of patients with HIV infection
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 Once the patient has been diagnosed with HIV infection, several tests should be performed
indeterminate result in HIV-1 confirmatory WB can be the infection with HIV-2 type. Indeterminate in regular manner to monitor the clinical progression of the disease, evaluate the immune status
results in non-infected individuals are usually due to the presence of non-specifically reacting or of infected individual and test the viral resistance to antiretroviral therapy. The main goal of HIV
cross-reacting antibodies that appear in some acute non-HIV viral infections, autoimmune diseases, infection management is to keep the levels of viraemia at low levels and to prevent the decline of
hypergammaglobulinaemia, chronic liver diseases or haematological malignancies, pregnant CD4+ T lymphocyte numbers below critical levels. For this purpose, the highly active antiretrovi-
women, haemodialysis patients or individuals in HIV vaccine trials. In such situations, the sup- ral therapy (HAART) was introduced in 1990s and has quickly proved to be very effective in erad-
plemental tests must be repeated within a few weeks or, better, the patient should be additionally icating viruses from blood and expanding the life expectancy of patients. The decision to initiate
tested with other methods including the HIV-2 WB to detect the infection with less common type treatment with antiretrovirotics is made on the basis of continuous CD4+ T cell and viral load
HIV-2 virus, and NAAT to search for the presence of viral nucleic acids. Definitive diagnosis of in- measurements and the presence of clinical symptoms.
fection can be established if positivity has been demonstrated in two independent samples. The Analysis of CD4+ T lymphocyte counts and CD4+/CD8+ T cell ratio is performed by flow cytom-
patient is then informed about his diagnosis and educated about the nature of the infection, its etry (Chapter 7.2.2.2) at the time of diagnosis and then regularly every 3 – 4 months to monitor the
prognosis and treatment possibilities, and instructed how to prevent its further transmission. 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
11.3.2 Diagnostics of primary (acute) HIV infection case definition for AIDS.
Viral load assays include several nucleic acid amplification techniques (RT-PCT, RealTime,
Primary (acute) HIV infection refers to the early stages of HIV infection when the antibod- b-DNA, NASBA, etc.) used to quantify the amount of HIV RNA circulating in the blood of an in-
ies to HIV are not yet detectable. Its correct diagnosis presents a clinical challenge but provides an fected individual. In addition to monitoring the progression of HIV infection, the assays are also
opportunity for early treatment and prevention of further HIV transmission, the risk of which is used to evaluate the efficacy of ART and detect its potential failure. Viral loads above 100,000 RNA
particularly high in newly infected patients. During this stage of infection, the patients have copies/μl are indicative for initiation of ART.
markedly increased HIV antigen and RNA levels and majority of them experience non-specific Drug resistance tests are assays used to detect the development of mutation-based resist-
symptoms of acute HIV disease, including fever, fatigue, headache, pharyngitis, lymphadenopa- ance to drugs used for HIV treatment. These frequently occur in patients on ART and are the ar-
thy, rash, myalgia, arthralgia and some others, which usually persist for 2 – 4 weeks. gument for the use of alternative ART regimens. Moreover, a substantial percentage of
Antibody testing of these patients will mostly yield negative results. As viral load levels and newly-infected patients already have HIV strain resistant to some of the drugs used for ART. There-
antigenaemia are very high during acute infection and become detectable approximately 1 – 2 weeks fore, resistance testing should be performed in ART-naive patients after the diagnosis has been
104 105
Laboratory Metods in Immunology Laboratory methods used in transplantation immunology
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- 12 Laboratory methods used in transplantation
typic assays. Genotypic assays are based on the detection of gene mutations known to be associated immunology
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- Transplantation is the transfer of human cells, tissues or organs from a donor to a recipient
bor mutations are amplified by PCR and sequenced. Obtained nucleic acid sequences are then with the aim of restoring their functions. Organs or tissues that are transplanted within the same
evaluated by computer software for the presence of mutations. Phenotypic assays enable direct eval- person’s organism are called autografts (e.g., skin). Transplants that are performed between two
uation of viral drug susceptibility. They are based on cultivation of a recombinant virus carrying genetically non-identical individuals of the same species are called allografts (or allogeneic grafts).
viral cDNA of HIV strain obtained from the patient in a cell culture with several different drugs. Allografts can either be obtained from a living donor or a deceased (brain-dead) donor. When
Subsequently, concentrations of tested antiretroviral drugs required to reduce the growth of the transplantation is performed between different species, like for example placement of primate
virus by 50 % are measured and compared to values obtained with drug-sensitive reference strain. hearts into human recipients, it is named xenotransplantation and the grafts are called xenogeneic
Increase of this value for particular drug compared to reference indicates decreased sensitivity of (or xenografts). Finally, syngeneic grafts (also reffered to as isografts or syngrafts) are those trans-
patient's HIV to the drug. ferred between individuals who are genetically identical (e.g., monozygous twins in humans or in-
Both genotypic and phenotypic assays are also used for co-receptor tropism analyses in order bred strains in animals).
to determine the dominant type of cellular co-receptor that is used by HIV-infected individual’s With respect to location, tissues or organs that are placed in their normal anatomic location are
virus to gain access to host cells. This information can be used to decide about potential use of called orthotopic grafts. However, many transplanted tissues or organs can function in other sites as
drugs that target the CCR5 co-receptor. DNA sequencing is also used to determine the group and well. Grafts that are placed into a site other than their normal one are called heterotopic grafts.
subtype of HIV virus in the patient. 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.
SUMMARY AT A GLANCE 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-
•Acquired immunodeficiency syndrome (AIDS) is a secondary immunodeficiency and a terminal stage mining Human Leukocyte Antigen (HLA) types. HLA complex is the human versions of the MHC
of HIV infection. (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-
•The diagnosis of HIV infection is routinely performed by indirect methods (ELISA, Western blot) for the DR, -DQ, -DP). HLA class I antigens are found on the surface of all nucleated cells, while HLA class
detection of specific anti-HIV antibodies. 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-
•In case of reactivity in the initial screening ELISA, the test should be repeated in duplicity and conse- ever, this is due their historic discovery and their role in transplantations. HLA antigens serve an im-
quently confirmed by Western blot. 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
•In some cases, appropriate direct methods for the detection of viral nucleic acids (PCR) or antigens and present antigens of every conceivable type. To do so efficiently, they are extremely polymor-
(ELISA) must be used. Examples of such situations include acute HIV infection before the seroconver- phic, what means that almost each individual has a unique set of slightly different HLA molecules.
sion and diagnosis of HIV infection in newborns of HIV positive mothers. 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-
•After the diagnosis of HIV is established, patient's immune status and the progression of infection ent has crucial influence on the outcome of the transplant procedure (Chapter 12.3).
must be monitored. For this purpose, CD4+ helper T cell counts, viral RNA loads and viral resistance HLA molecules are encoded by a large number of genes located close together on chromo-
to antiretrovirotics are repeatedly determined. 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- able to find a suitably matched donor within their family. Today, over 20 million donors from 48
termine two chains of the HLA class II molecules. As explained above, the main function of HLA countries and 47 cord blood banks from 31 countries are listed on Bone Marrow Donors Worldwide
molecules is to display foreign peptides to the immune system. The proteins encoded by HLA (BMDW), an international database managed by its central office in Leiden, Netherlands.
class III genes have somewhat different functions; they are involved in inflammation and other im- Some people have a lower chance to find a suitable donor or cord blood unit than others. This
mune system activities. is because some HLA types are more common within the population and some are less common.
Most HLA genes have hundreds of identified versions called alleles. Most variable (polymor- In addition, certain HLA molecules are found more often in some racial and ethnic groups than
phic) are the genes HLA-B and HLA-DRB1. Each known allele is given a particular designation. HLA others, thus a suitable donor is usually of a similar racial or ethnic background.
genes are passed on by traditional Mendelian inheritance patterns within conserved clusters called A stem cell transplant from an HLA mismatched donor can result in a condition when the
haplotypes as shown in Fig. 12.1. Haplotype is a set of genes (alleles) that is present in each chromo- recipient’s immune system recognises the transplanted cells as “non-self” and attacks them what
some. Each heterozygous individual will have two HLA haplotypes, one in each chromosome (as one results in the host versus graft reaction (HvG), commonly termed also graft rejection or graft fail-
is of paternal and the other of ure. Likewise, lymphocytes from the donor’s immune system, which are introduced along with the
maternal origin). 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
Fig. 12.1 Inheritance of HLA the skin, gut and liver and may become very severe, even fatal. The more compatible the donor-
genes recipient match is, the less likely the rejection or severe GvHD will occur. To minimize the risk of
The two paternal haplotypes are designated GvHD, recipient and donor should be ideally matched at all HLA loci.
as A and B, while C and D represent the two
maternal haplotypes. Since the maternal
and paternal haplotypes can combine in 12.2 Kidney transplantation
four different ways, there are four possible
HLA types that can be inherited by the chil-
The indication for kidney transplantation is the end-stage renal failure, regardless of the pri-
dren. This means that statistically each child
has a 25% chance of being HLA matched mary cause. Kidney can be donated by a deceased donor (formerly termed cadaveric) or a living
with any one sibling. 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-
12.1 Haematopoetic stem cell transplantation 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
Stem cell transplantation offers the only possible curative treatment for different types of three general categories – chronic, acute and hyperacute – depending on timing and intensity.
leukemia or lymphoma as well as several non-malignant disorders, such as aplastic anemia or se- Each type involves particular mechanisms of immune response.
vere immunodeficiencies.
Haematopoetic stem cells originating in the bone marrow have the capacity to develop into 12.2.1 Blood type matching in kidney transplants
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 ABO blood group antigens are relevant to kidney transplantation as they are expressed not
these cell sources has specific advantages and disadvantages, and each has found particular ap- only on RBCs, but also on other cell types, including the endothelium. The recipient and donor
plications in the treatment of oncologic or immunologic disorders. must have either the same ABO blood type or compatible ones. ABO incompatible transplantation
When stem cells are donated from another individual, the transplantation is called allo- can cause hyperacute rejection of allograft due to preformed natural antibodies (isohaemagglu-
geneic. The most suitable donor of stem cells is a fully HLA matched family member but only tinins). The Rhesus blood type is not a factor in donor matching because these antigens are not ex-
some patients have such a donor. HLA antigens are inherited in dominant Mendelian way, thus pressed on endothelial cells.
the best chance of finding matches is between siblings. As everybody inherits half of the HLA Untill recently, kidney transplantation could not be successfully performed unless the donor
antigens from the mother and half from the father, a sibling has a 25% chance of matching her sis- and recipient had compatible blood types. Today, a number of centers around the world are per-
ter or brother (Fig.12.1). forming ABO-incompatible transplants and patients with living donors who do not have a match-
If a suitable donor is not found within the immediate family a wider family search or unre- ing blood type can still receive a kidney transplant. This is due to a procedure called plasmapheresis.
lated donor search is needed. Unrelated bone marrow donor registries and cord blood registries Plasmapheresis removes the plasma portion of the blood containing harmful antibodies prior to
have been developed to help the cca 70 % of patients needing a stem cell transplant who are un- the transplant.
108 109
12.3 HLA typing 4. Complement is added to each well and incubation continues for another 60 minutes. Dur-
HLA matching has the greatest clinical impact in both, haematopoetic stem cell and kidney ing this period of time, complement will bind to any cell that is coated with antibody. This
transplantation results. Donor and recipient should ideally match in at least six HLA antigens results in lysis of the lymphocytes. If no antibody is coating the lymphocyte, the membrane
(two HLA-A antigens, two HLA-B antigens, and two HLA-DR antigens), which are the most im- remains intact.
munogenic ones. The six antigen match was the minimum requirement untill lately. With evolv- 5. The assessment of lysed and vital lymphocytes is carried out by a vital staining (using eosin
ing clinical practice, especially the provision of safer and more potent immunosuppressive therapy, or trypan blue). Damaged lymphocytes allow the dye to enter the cell, whereas intact lym-
the significance of HLA matching has diminished. There is however, a benefit to having a “perfect phocytes do not.
match” since the transplanted kidney survives significantly longer. 6. Results are read using a light or inverse phase contrast microscope. Wells with damaged
In heart and lung transplantations practical reasons as ischemic time, availability of donors cells appear coloured (positive reaction) in contrast to wells with intact cells that appear re-
and clinical urgency override any matching considerations, although studies have shown it would fractile (negative reaction).
be an advantage to match especially in HLA-DR antigens. Corneal grafts are not usually influ-
enced by HLA matching. Fig. 12.2 Microlymphocytotoxicity test
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- 12.3.2 Cellular HLA typing
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 A representative cellular assay is the mixed lym-
I diabetes mellitus or coeliac disease. Relationships have also been documented between certain phocyte culture (MLC), also called the mixed lymphocyte
HLA and sensitivities to specific drugs. reaction (MLR). MLC measures in vitro the cell-medi-
Two basic approaches in HLA typing are serological and DNA-based typing. According ated response to foreign HLA antigens. Nowadays, MLC
to these approaches there are two parallel systems of nomenclature that are applied to HLA. is used only rarely as the test involves the use of radioisotopes, and takes several days to perform.
The older system is based on serological determination and antigens are assigned letters and Today, cellular assays are being replaced by faster and more accurate DNA-based typing methods.
numbers (e.g. HLA-B27). Later, a newer system based on DNA-typing was developed that al- Principle steps of the one-way MLC assay:
lows more precise definition of alleles. In this system, term “HLA” is used in conjunction with 1. Recipient lymphocytes are incubated with donor lymphocytes. Donor cells are unresponsive
a letter, asterisk and a number (e.g. HLA-DR*15:01) to designate a specific allele at a given HLA to antigenic stimuli due to previous treatment with irradiation or mitomycin. These inacti-
locus. 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
12.3.1 Serological HLA testing – microlymphocytotoxicity test and start to undergo blastic transformation and proliferate.
2. A radioactive precursor of DNA, [3H]-thymidine, is added to the test system. It incorpo-
Serological (antigen-antibody based) techniques have long been the standard for HLA typ- rates into the DNA. The uptake of the radioactive thymidine is of course higher in those
ing. These methods rely on the reaction of viable lymphocytes (expressing HLA antigens) with cells, which underwent the blastic transformation.
suitable antisera (anti-HLA antibodies with known specificities). The most common variant is the 3. The amount of radioactivity is measured by a beta-counter as [3H]-thymidine radiates beta
complement-mediated microlymphocytotoxicity test in which typing antisera are incubated with radioactive rays. The higher the radioactivity, the greater the difference in HLA antigens be-
the tested lymphocytes in the presence of complement. The test is performed in microtiter wells tween donor and recipient.
of Terasaki plates and contains several steps:
1. First, viable lymphocytes are obtained from peripheral blood, either by discontinous density 12.3.3 Molecular HLA typing
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 The last two decades are characterised by an exponential growth in the application of mo-
antibodies (antisera) is added to them. Each well contains an antibody directed against lecular HLA typing techniques. They are DNA-based, what means that HLA types are determined
a known HLA antigen. on the level of alleles (different variants of genes). This approach has significantly increased the
3. Antibodies and lymphocytes are then incubated at room temperature for 30 minutes. If the accuracy and specificity of HLA typing and allows more precise HLA matching between donors
lymphocytes possess the respective HLA antigens, specific antibodies will bind to their surface. and patients. Another advantage is greater specimen flexibility as any nucleated cell or any tissue
110 111
can serve as a source of DNA to be tested. Laboratories are not restricted to use only blood cells tube should produce either
from the patient but they can get the DNA from buccal swabs, frozen blood, dried blood stains, a specific band or not depend-
bone marrow, cultured cells, etc. There are numerous methods and commercial kits available for ent on whether the respective
simple extraction of good quality DNA. allele is present or not. Detec-
At this time, almost all HLA testing is done by molecular techniques. Molecular methods are tion of amplified PCR segments
especially required when performing typing for hematopoietic stem cell transplantation as they are is done using an agarose gel
much more sensitive compared to serologic methods and moreover, they disclose also alleles that electrophoresis. If a product of
code for molecular products for which no specific antibodies exist, i.e. they are undetectable by the right size is found, the as-
serological techniques. sumption is that the HLA allele
The majority of molecular HLA typing strategies are based on the polymerase chain reaction has been identified.
(PCR). The HLA region of the DNA is PCR-amplified providing a large amount of DNA that can Fig. 12.4 PCR-SSP
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). 12.3.3.3 Sequence based typing (SBT)
Fig. 12.3 Polymerase chain reac- SBT is the only technique, which directly detects the nucleotide sequence of an allele allow-
tion (PCR) 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

12.3.3.1 PCR followed by hybridization with sequence specific oligonucleotides (PCR-SSO) 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
A single generic primer pair is used for the amplification of the HLA region. The amplified for antibodies to the donor HLA antigens. Most common cause of anti-HLA antibody production
DNA segment (HLA gene) is immobilized on a membrane. Sequence or allele-specific oligonu- is a previous exposure to foreign HLA molecules through transfusions, transplantations or preg-
cleotides (SSOs) are used as probes to detect the presence of the respective DNA segment as it hy- nancies. Cross-match is performed by mixing a small amount of the recipient´s serum with a small
bridises only to probes with complementary sequences. Probes are labeled, usually by a fluorescent amount of potentional donor lymphocytes. Complement is added that causes cell membrane lysis
or enzyme label. Interpretation is usually achieved by entering the final band pattern into a com- in case that anti-HLA antibody is present in the serum and has bound to the cells (Fig.12.7). Vital
puter programme. 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
12.3.3.2 PCR using sequence specific primer (PCR-SSP) 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
PCR-SSP typing is based on using a panel of primer pairs unique to a specific HLA allele or most likely attack the transplanted organ. Researchers develop strategies to remove anti-HLA
a group of alleles. Only primers that are completely matched for this DNA segment (specific HLA al- antibodies and block their effects to reduce the risk of rejection in positive crossmatch kidney
lele) are able to prime the amplification. Each PCR reaction takes place in a separate tube. Finally, every transplants. A more sensitive and specific crossmatch test has been developed on the basis of
112 113
Laboratory Metods in Immunology Summary of methods used in immunity testing: Immune status of an individual
flow cytometry or Luminex

bead assay. These techniques 13 Summary of methods used in immunity testing:
can detect the presence of re- Immune status of an individual
cipient antibodies on the sur-
face of donor lymphocytes Examination of patient’s immune status includes innate and acquired immunity testing.
independent of complement First step of the examination relies on the anamnesis checking, it means the complete case history
binding. However, they are examinations of the patients are reviewed. It involves patient’s previous physical and biochemi-
much more expensive and cal testings. Next step of the patient’s examination involves his own immune status analysis. Both
labour intensive. 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
Fig.12.5 Crossmatch test 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 Innate (non-specific) immunity is activated as first after entering a foreign invander to the host.
aim of restoring their functions. 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-
•Central role in graft rejection play HLA genes that encode for the HLA molecules also reffered to as ponents and acute inflammation proteins (CRP) and cellular components, such number and func-
transplantation antigens, or histocompatibility antigens. The degree of histocompatibility between tion of phagocytic cells, is usually performed. For analysis of complement components, mainly C3
donor and recipient can be determined by serologic or molecular HLA typing and by the MLC test. 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).
•Worldwide, kidneys are the most commonly transplanted organs. Donor and recipient matching in kid- The assay is based on spectrophotometric measurement of the heterologous red cells haemolysis by
ney transplants reffers to three distinct areas: ABO blood type matching, HLA type matching, and presence of the complement system in a patient serum. If no haemolysis occurs (or haemolytic unit
crossmatching. The donor and recipient should share at least six HLA antigens (two HLA-A antigens, CH50 = 0), it means that one complement component in the patient serum is missing. Absence of
two HLA-B antigens, and two HLA-DR antigens) to decreases the risk of transplant rejection. 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-
•Graft-versus host disease (GvHD) can develop as a life-threatening consequence of a stem cell trans- ment components deficiencies are those of C3 and C4 , which are manifested in the patient as re-
plantation. To minimize the risk of GvHD, recipient and donor should be ideally matched in all HLA genes. 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
whereas during bacterial infection the CRP level increases over 50 mg/ml. In such case the pa- 13.2 Analysis of acquired immunity
tient requires antibiotic treatment.
Cellular part of innate immunity involves process of phagocytosis. Number and percent Acquired immune response, also known as specific immune response, developes after an ex-
value of neutrophils is determined from the whole blood using automated blood analyzer. Neu- posure of our body to antigens. It is composed of humoral (antibodies, cytokines) and cellular
trophils, as the main phagocyting cells, account for 60 to 70% out of whole blood cells. The func- (lymphocytes) components. There are quantitative methods for evaluation of acquired immunity
tional analysis of phagocytosis is based on evaluation of chemotaxis (chemotactic index), ingestion components based on serum immunoglobulins levels analyses (ELISA) and counting of lympho-
(phagocytic activity and phagocytic index), cidial activity and metabolic activation (INT test). The cytes (flow cytometry) in blood samples and qualitative methods based on functional evaluation
first step of phagocytosis examination involves isolation of leukocytes, namely polymorphonu- of lymphocytes (blastic transformation assay, immunoskin tests).
clear leukocytes (PMNL), from peripheral heparinised blood using 6% dextrane. PMNL contain Adaptive humoral immunity components involve antibodies produced by plasma cells,
approx. 75% of all isolated leukocytes. Such cells are used in function screening tests. To evaluate which are the latest differentiating state of B lymphocytes. There are five classes of immunoglob-
the chemotaxis of leukocytes, the length that cells migrate in the presence or absence of a chemoat- ulins, namely IgG, IgD, IgM, IgE and IgA. For detection of serum levels of IgG, IgM and IgA, au-
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- 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-
in chemotaxis are usually common in patients with recurrent phlegmonic inflammations of mu- tions, are used for detection of specific antibodies, i.e. to viruses and bacteria in serum samples.
cous tissues and dermis, moreover defects in the number and motility of phagocytes can result in Adaptive cellular immunity components involve lymphocytes. To isolate lymphocytes, the
the development of sepsis. non-coagulated heparin-treated peripheral blood is layered on a separation solution Ficoll-Hypaque
To evaluate ingestion of leukocytes, phagocytic activity and phagocytic index is determined. followed by a gradient centrifugation. PBMC cells including lymphocytes are present as white layer
To do this analysis, patient’s PMNL, serum for opsonisation and inactivated candidas are cultured over a separation solution. Isolated lymphocytes can be used either for count analysis of individ-
by 37°C for 30 mins. Cells smears are stained with the Wright`s dye and analysed under micro- ual lymphocytes subpopulations or for functional evaluation. An exact count analysis of lympho-
(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 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-
das ingested by 1 phagocyte (leukocyte) (reference value 3.6±0.8). Impaired PhA and PhI can be phocytes CD19 and CD20, T lymphocytes CD3 and NK cells express CD16 and CD56. The popula-
observed in patients suffering from recurrent bacterial infections, sepsis, terminal stages of mation of CD3+ T lymphocytes can be subdivided into three basic subpopulations, which express
lignancies or some congenital defects of phagocytosis. different CD molecules: T helper – CD4, T cytotoxic – CD8, and T regulatory – CD4 and CD25. This
Cidal activity evaluates an ability of phagocytes to kill engulfed candida or bacteria. To do method is used in the diagnostic of primary and secondary immunodeficiency (e.g. HIV-progres-
this analysis, patients PMNLs, serum for opsonisation and lived candidas are cultured by 37 °C sion monitoring) as well as in phenotyping of leukemias and lymphomas. In HIV-infected indi-
for 60 mins. After cell lysis, the smears are stained with vital dye and analysed under micro- viduals (secondary immunodeficiency of CD4 cells) CD4/CD8 ratio counting (previously known
scope. Only dead candidas are coloured, the lived candidas are non-stained. Candidacidal ac- as immunoregulation index – IRI) is widely used for monitoring of disease progression and response
tivity (%) is determined as the percentage of killed candidas to the whole number of candidas to treatment. The ratio is decreased in HIV-infected individuals (IRI<0.5), but increased in patients
(reference value 25.4±10.0%). The impaired cidal activity can be found in patients with defects with autoimmune diseases (IRI>2.5). Another way for the isolation of lymphocyte subpopulations,
in microbicidal mechanisms (proteolytic enzymes, production of free radicals, etc.). Finally, we except flow cytometry, involves monoclonal antibodies labelled by magnetic beads; however, it is
can also examine metabolic activity of phagocytes using INT test. The principle of the test is mainly used for research purposes. In clinical medicine, transplantation immunology and oncology
based on the reduction of tetrazolium salts (INT – iodophenyl-nitrophenyl-tetrazolium chlo- mostly utilise this technique. By this procedure, a patient receives a pure population of CD34+ pro-
ride, faint-yellow) to the insoluble formasan (rose to violet) by reactive metabolites of oxygen genitor cells, thus the induction of graft failure can be reduced.
and reductases, which are produced by respiratory burst in phagocytes. To do this analysis, To evaluate patient’s lymphocyte function in vitro, lymphocyte transformation test (LTT) or
patients PMNLs, a serum for opsonisation, INT and zymosan are incubated in wells of a mi- blastic transformation assay is used. The test examines lymphocytes response to mitogens or anti-
crotitre plate. A control sample including all above-mentioned components without zymosan gens (allergens) in vitro by induction of non-specific cell proliferation and formation of blasts. The
is also examined. The absorbance in the analysed and control sample is measured spectropho- degree of cell proliferation is measured radioactively after adding of radiolabelled 3H-thymidine
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 (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
of phagocytes are common in patients with chronic granulomatous disease that is manifested sensitized by testing antigen. The LTT is mostly used in the diagnosis of allergy (food or drug-in-
as recurrent intracellular bacterial and fungal infections of the skin and organs and formation duced). In this case, an exposure of examined person lymphocytes to allergens is analysed; gold
of granulomas. salts, amalgam, HgCl2, Ni and Be are those the most tested. However, LTT measures only the sen-
116 117
sitization of lymphocytes but not an effector reaction. Thus the positive LTT indicates only that the ent to a cross-match positivity grade. Cellular methods of HLA screening involves mostly MLC
person suffers from allergy to tested substances, however to confirm it, clinical symptoms should test (mixed lymphocyte culture test). The test determinates the HLA class II molecules matching
be always considered. For verification of the diagnosis, other tests should be also performed (IgE between a donor and a recipient. Using this assay, lymphocytes from the donor and the recipient
tests, skin tests). are mixed and incubated for 5 days. During this period, lymphocytes from one individual prolif-
To evaluate patient’s lymphocyte function in vivo, immunoskin test is used. The test is based erate in response to foreign HLA antigens of other individual. Lymphocytes are then incubated
on an injection of tested antigens into dermis of the forearm. After 24 to 48 hrs, the immune re- with tritiated thymidine for 6 hours (thymidine incorporates into DNA of proliferating cells), fol-
sponse (induration and erythema) is evaluated. These symptoms are caused by delayed type of hy- lowing radioactivity measuring. Finally the stimulation index (SI) is calculated. If the SI value is
persensitivity reaction (increased activity of macrophages, dendritic cells and T cells). By means over 2, it indicates the identity in HLA- antigens between tested individuals.
of immunoskin tests, we can examine immune response to killed bacteria, their proteins or aller- Methods for HLA-genotyping involve DNA-based methods like PCR-SSP or sequencing. PCR-
gens, e.g. tuberculin is used to establish the diagnosis of tuberculosis and similarly, allergens for SSP method is based on a direct DNA amplification of one specific HLA allele using sequence
the diagnosis of contact allergy. specific primers (SSP). The advantage of this method is the typing of several alleles in one DNA
Screening of autoantibodies plays a key role in establishing the diagnosis of autoimmune sample at once. Another method for HLA-genotyping is sequencing. This method detects exact nu-
diseases (AD). There are many autoantibodies recognizing autoantigens of various organs and cleotide order of an analysed DNA fragment and thus determines the patient′s HLA allele. The
tissues. The most common are antinuclear antibodies (ANA) and rheumatoid factor. Methods for HLA-typing is also used in diagnostic of autoimmune diseases because of associations of some
the autoantibodies screening involve indirect IF assay, ELISA and immunoblotting. Rheumatoid HLA alleles to certain AD, e.g. HLA-B27 in patients suffering from morbus Bechterev, HLA-DR3,
factor (RF) involves autoAb of IgM class to Fc domain of IgG and is mostly present in patient suf- HLA-DR4 or HLA-DQ8 in patients with type I diabetes mellitus or HLA-DR2 in those who devel-
fering from rheumatoid arthritis. RF screening is based on latex agglutination or ELISA method. oped sclerosis multiplex.
To perform latex agglutination, latex particles coated by human IgGs are quickly mixed with a pa- To establish the diagnosis of allergy, in vitro and in vivo laboratory tests are performed. In
tient’s serum in the well of special agglutination plates. The test is positive, when clumping of vitro laboratory tests involved IgE level screening. Total serum IgE level is determined by ra-
latex particles – agglutination occurs. dioimunosorbent test (RIST), which is based on competition of known amounts of radiolabeled IgE
Analysis of circulating immunocomplexes (CIK) levels is performed by mixing a patient′s with the patient's unlabeled IgE to bind to a surface coated with anti-IgE (competitive radioim-
serum with PEG (polyethylenglycol); an incubation for 1 hour follows. During this time, a tur- munoassay). The reduction of radioactivity of control IgE level due to the presence of IgE in the
bidity is forming and its level is measured by nephelometry or turbidimetry. The test is positive patient's serum is determined by comparison with known IgE standards. Values of IgE between
(high concentration of CIK), when the double of a reference value has been measured (reference 100 to 150 IU/ml indicate the presence of allergy. To analyse specific IgE levels, radioallergosorbent
value: 25–60). To detect IK in tissues directly, immunohistochemistry and immunofluorescence is test (RAST, non-competitive radioimmunoassay) is mostly used. The test is based on detection of
performed. Screening of patients with AD also includes determination of Ig level (ELISA), analy- specific serum IgE antibodies to suspected or known allergens. Allergens are bound to carriers
sis of C3 and C4 complement components level (nephelometry and turbidimetry), determination and the patient's serum is added. After incubation radioactive-labelled secondary anti-IgE is
of number of T cells in the peripheral blood (flow cytometry), determination of CD4+/CD8+ ratio added, that binds to the patient′s IgE. After washing, the radioactivity is measured; its amount is
by flow cytometry (IRI is increased over 2 by autoimmunity) and HLA-typing/sequencing. proportional to the serum specific IgE antibodies. RAST test is scored on a scale from 0 to 6, the
The immunodiagnosis also involves methods to determine HLA alleles or antigens (HLA typ- positivity determines up to the score 3.
ing methods). Besides ABO blood group compatibility, HLA matching between donor and recipient Nowadays, other sophisticated methods for in vitro diagnostic test of allergy have been used.
belongs to important requirements reducing a graft rejection, especially for kidney transplantations. The mostly performed is ImmunoCap method based on modification of ELISA. This test is highly
It has been shown that six HLA antigen-match is important for a successful outcome, i.e. donors and sensitive and involves simultaneous measurement of specific IgE antibodies to multiple allergens
recipients should be indentical in HLA-DR, -B, and A alleles (DR>B>A). There are serological (mi- in a single test. Allergens are immobilised to specific carrier consisting of cellulose and agarose
crolymphocytotoxicity assay), cellular (MLC test) and molecular-biological (PCR-SSP, sequencing polymer (CAP). In the assay, IgE in the patient’s serum are bound to the allergens. After a wash-
methods used in HLA typing, PCR-SSP being the most widely used method. Microlymphocytotoxi- ing step, allergens – bound antibodies are detected by enzyme-labeled antibodies. After a sub-
city assay belongs to serological methods of HLA-molecules typing and is based on screening of cell strate addition, the fluorescence is measured. The latest modification of this method known as
membrane HLA-molecules by antisera (include Abs against HLA-molecules). After complement ImmunoCap ISAC uses fluorescence-labelled antibody for detection of allergen bound antibodies.
adding and eosin staining only dead cells (destructed by activation of complement) are red-stained. Test results are measured by means of a laser scanner and evaluated using a software. Results are
The percentage of dead cells is determined under microscope and the result is positive, when more reported in ISAC Standardised Units (ISU).
than 50% of cells are dead. Microlymphocytotoxicity assay is used in immunodiagnostic for cross- Basophil activation test (BAT) is based on the detection of activatory antigens CD63 or
match screening and is based on detection of HLA-Abs in patient’s blood (recipient) against donor CD203c in membranes of basophils by flow cytometry. These CD molecules are being expressed
lymphocytes. A positive cross-match is an absolute contraindication of transplantation independ- in their surface after stimulation only, in our case when whole blood cells or isolated leucocytes
118 119
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. SUMMARY AT A GLANCE
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. •Examination of immune status includes innate and acquired immunity testing. Both, the humoral and
pollens, pet dander, dust mites, etc. By this skin test, purified allergens available in commercial test- cellular immunity screening can be performed depending on the patient’s condition. Firstly, the ab-
ing sets are applied on a cleaned skin of volar forearm or upper back (children) as tiny droplets. solute and differential blood cell count is analysed. According to the changes in blood count we can
They are subsequently pricked into skin’s surface (1 mm deeply) with a special needle or lancet; diagnose the disease and proceed with further immune analysis.
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 •In diagnostics of innate (non-specific) immunity, an analysis of humoral components, (complement,
the diameter is more than 3 mm wide. In patch test, the allergens are mixed with a non-allergic acute inflammation proteins) and cellular components (number and function of phagocytic cells) is
material (base) to a suitable concentration. They are then placed in direct contact with the skin, usu- performed. For analysis of complement components and acute phase protein levels, the turbidimetry
ally on the upper back, within small aluminium discs. Adhesive tape is used to fix them in place. and nephelometry are used. To assess the function of the complement, total complement activity assay
The patches are left in place for 48 hours (diagnosis of type IV hypersensitivity), when a skin re- of the classical (CH50) or alternative pathways is performed (AH50). The functional analysis of phago-
action is evaluated; its intensity can vary from erythema without or with papula or pustule. In cytosis is based on evaluation of chemotaxis (chemotactic index), ingestion (phagocytic activity and
some cases, epithalaxia (separation of epidermis) can be observed. Allergens represent substances phagocytic index), cidial activity and metabolic activation (INT test).
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- •In diagnostic of acquired (specific) immune response, an analysis of humoral components like anti-
centration was not used or the body failed to elicit a response. 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.
120 121
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Laboratory Methods in Immunology

Book · May 2019

Ivana Shawkatová Vladimira Durmanova

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Immunological methods are due to their high specificity

1 Short introduction to immunology

1.2.1 Innate immunity

1.3 Structures of the immune system 1.4 Immunological methods

2.1.1 Serum collection for humoral immunity screening

Fig. 2.1 Serum and plasma collection

2.1.2 Plasma collection for humoral immunity

Plasma is a pale-yellow fluid that is collected from non-

Fig. 2.2 Plasma collection by sedimentation

2.2 Collection and blood processing for cellular immunity screening

3.1.1 Direct agglutination

3.1. Agglutination Tab. 3.1 Blood group antigens and isohaemagglutinins

Reaction of an antibody with a particulate antigen results in agglutination (clumping) of the

Indirect Coombs test allows deter-

Fig. 3.5 Passive agglutination

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

Fig. 3.11 Countercurrent electrophoresis

specific proteins (mostly human serum proteins)

4.2.1.1 Radioallergosorbent test (RAST)

4.2.2 Enzyme immunoassay 4.2.2.2 Direct ELISA

4.2.2.4 Sandwich ELISA 4.2.3 Fluorescent immunosorbent assay (FIA)

Fig. 4.6 Sandwich ELISA 4.2.3.1 Direct FIA

4.2.3.2 Indirect FIA

Fig. 5.2 Haemolytic complement assay in the agarose gel

Fig. 5.3 Solid-phase ELISA assay

5.1.2.4 Functional tests for individual complement components

Functional activity of individual complement components should be determined when a ge-

ceruloplasmin, haptoglobin, complement proteins, inhibitors of serine proteases, α2-macroglobu-

liquid. 50 μl of the diluted sample is subsequently pipetted

6. Evaluation of innate cellular immunity

Defects of the expression of adhesive molecules β2-integrins (CD11a/CD18, CD11b/CD18,

6.1.2.6 Evaluation of metabolic activation

6.1.2.7 Evaluation of lysozyme levels and activity

6.2.2 Laboratory tests for the evaluation of NK cell cytotoxic activity

cpm probe – cpm background

7.2.2 Isolation and counting of lymphocyte subpopulations

Fig. 7.3 Production of monoclonal antibodies Fig. 7.4 Flow cytometer

7.2.2.4 Isolation of lymphocytes subpopulations by magnetic beads

Dilute mitogens to concentrations 1 µg, 2 µg and 5 µg/50µl, respectively.

Measure the radioactivity using a β-scintillation counter.

7.2.4 Evaluation of the lymphocyte function in vitro – ELISPOT

7.2.5 Evaluation of the lymphocytes function in vivo – immunoskin tests

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

8.1 Allergy assessment Weeds Wormwood – Artemisia vulgaris Art v 1

Mites Dermatophagoides pteronyssinus Der p 1

8.1.3 Additional assessments

Histological and immunohistochemical examinations of bioptical samples obtained from

affection. These include medical radiography, ultrasonography, magnetic resonance imaging,

Fig. 11.1 Schematic structure of the human immuno-

11.2 Laboratory methods in HIV diagnostics

Cross-match is the last laboratory test performed prior to transplantation. It is a sensitive