Update of Laboratory Diagnostic Methods in Clinical Immunology
World Small Animal Veterinary Association World Congress Proceedings, 2006
Miroslav Toman1, MVDr, CSc; Martin Faldyna2, MVDr, PhD
1 Professor, Veterinary Research Institute; 2Veterinary Research Institute, Brno, Czech Republic

Immunological tests should be preceded by a thorough evaluation of anamnestic data and clinical examination results, as many symptoms may signalise potential immune-mediated diseases. Also standard laboratory tests, biochemical, haematological, cytological or histological analyses and visualisation (radiography) methods may be of a great value for an immune-mediated disease diagnosis. Accordingly, immunological tests should be only performed in the case of an adequate indication, with respect to their costs and cogency. Diagnostic criteria have been established for various immune-mediated diseases, with laboratory analysis playing major or minor roles, according to their diagnostic value.

Immunological laboratory diagnostic methods can be classified from several aspects:

I. Based on a group of diseases that facilitate diagnosis

 Immunological profile tests for the detection of immunodeficiency

 Hypersensitivity tests

 Autoimmunity tests

II. Based on availability of the methods

 Methods performed in a surgery

 Methods included in haematological or biochemical analysis, and histological or visualisation methods that provide valuable information for immunological diagnosis

 A group of basic methods conducted in a specialised immunological laboratory

 Advanced immunological methods above all, used in clinical research

Whereas laboratory diagnosis of allergic and autoimmune diseases is based on serological examination and the tests are usually available as commercially produced kits, technically demanding methods are necessary for the detection of immunodeficiency or immunosuppression disorders.

Basic Laboratory Examinations

Leukocyte count and differential leukocyte count, i.e., standard haematological parameters should be included in basic laboratory examinations, commonly available in any small animal practice. Cytology and histology of various samples obtained from biopsies are also of great value for immunological diagnosis. Preliminary methods selected for humoural immunity testing involve the assessment of total immunoglobulins using a simple precipitation method or serum electrophoresis. Among inflammatory parameters, the C-reactive protein test is available as a commercial kit in dogs however it is not commonly used, due to its cost versus diagnostic value.

Allergy Tests

Practical veterinary surgeons have available in their consulting rooms the hypersensitivity skin tests for allergy diagnosis, including tests for the detection of a particular allergen. These tests produce quite reliable results especially in canine atopy. Allergens are commercially available and tests are usually used in practice for the detection of hypersensitivity type I. Nevertheless, hypersensitivity type IV detection is also relevant.

Nowadays, the diagnostic value of intradermal skin tests is comparable with that of serological detection of specific IgE antibodies against respective allergens using commercially available ELISA kits.

Detection of Autoantibodies

Detection of autoantibodies is an important diagnostic tool for diagnosis of autoimmune diseases. Despite the fact that their occurrence is not quite specific for a respective disease, it may considerably facilitate the diagnosis. Human immunological laboratories have available a wide range of commercial tests, whereas the offer in veterinary medicine is somewhat limited.

Analysis of circulating immune complexes by their non-specific precipitation in sera with polyethyleneglycol is an auxiliary method for the detection of hypersensitivity type III status. However, elevated concentration of circulating immune complexes is also usually detected during chronic infectious processes and due to this fact, the result of the analysis does not lead to a specific conclusion.

Antinuclear antibodies (ANA) are characteristic for systemic autoimmune diseases, above all: systemic lupus erythematosus (SLE). They are detected by indirect immunofluorescence, in sera. A significant level of antibodies (titre 80-100) and characteristic localisation (granular or homogenous fluorescence of the nucleus) is a precursor for obtaining a positive result in the test. The diagnostic value of the test is relatively high.

Rheumatoid factor (RF) is the antibody against Fc fragment of immunoglobulin, in dogs this is usually against IgG, the RF isotype being IgM, or IgA. Rheumatoid factor detection is performed by various tests: conventional Rose-Waaler test, or most recently by turbidimetry or ELISA methods. The diagnostic value of RF detection that should be characteristic of rheumatoid arthritis is low, because rheumatoid factor is also found in serum during other autoimmune diseases, chronic inflammatory responses and even in the serum of normal (especially older) animals.

Immune-mediated anaemia is characterised by the presence of autoantibodies and/or the C3 component of complement proteins on the surface of erythrocytes from a patient. Direct antiglobulin (Coombs) test is most convenient for their detection, as it reveals when autoantibodies or complement proteins are bound to the surface of a patient's erythrocytes. A positive reaction of agglutination signalises an immune-mediated cause of anaemia, but the primary (idiopathic) autoimmune haemolytic anaemia (AIHA) cannot be distinguished from secondary immune mediated anaemia (IMHA) using this test, which is caused by microbial agents or drugs.

A comparable direct test for the detection of antibodies against thrombocytes in patients with idiopathic thrombocytopenia has also been developed, however it is not commonly available for routine diagnosis.

Tests for the detection of other antibodies, such as antibodies against the acetylcholine receptor, for the diagnosis of myasthenia gravis, or antibodies against thyroglobulin and thyroid peroxidase for diagnosis of autoimmune hypothyroidism, are not commonly available.

Immunohistochemistry techniques are very useful methods for the detection of free or immune complex-bound autoantibodies in biopsy specimens. Those are usually used for the detection of skin autoantibodies, which can distinguish between various types of pemphigus complexes. The use of these methods for the detection of various types of glomerulonephritis, or inflammatory bowel disease, is less common but likewise significant.

A Non-specific Immunological Profile Testing

Laboratory assessment of primary or secondary immunodeficiency is demanding for both methodical background and financial costs and especially tests of cell activities are offered only by specialised laboratories that are usually associated with universities or research institutions. Immunological laboratories have different validated methods available (Table 1); nevertheless, it is always necessary to perform a set of immunological examinations. An isolated finding of a decrease in one of the immunological parameters determined, does not necessarily give evidence of immunodeficiency. Also, if the values of one or more parameters are changed, it is recommended to repeat the examination to confirm persistent immunodeficiency.

Table 1. Methods of immunological profile.




Migration and chemotaxis under agarose, test of synthetic particles or microbes ingestion, chemiluminescence, detection of respiratory burst, microbicidity test

Lymphocyte subsets

Flow cytometry, immunohistochemistry

Lymphocyte activity

Lymphocyte transformation test, mixed lymphocyte reaction


Bioassay, ELISA, PCR

Immunoglobulin levels

Single radial immunodiffusion RID, ELISA


Haemolytic activity, ELISA

CRP, lysozyme and other humoural factors

ELISA, turbidimetry

Cell counts and activity detection follow up the haematological analysis of total and differential leukocyte counts. It occurs in the assessment of functional activity of phagocytic cells and lymphocytes, and sometimes also in lymphocyte subset identification (see below). Phagocytic activity may be studied by means of a number of tests (Table1); however, their diagnostic values vary. The lymphocyte transformation test (LTT) which monitors capability of lymphocyte stimulation, by non-specific mitogens for cell proliferation, is most valuable, however technically demanding. The most common findings in immunosuppression of animals are lymphopenia, together with dysbalance of lymphocyte subsets and a decreased activity of lymphocytes in LTT. These changes were detected in primary immunodeficiencies (e.g., severe combine immunodeficiency disease) and also in German shepherd deep pyoderma, demodicosis, distemper, parvovirosis, in cats in FIV, FeLV and FIP infections, and also in chronic renal failure.

Among the tests of humoural factors, the detection of total concentration of antibody isotypes is crucial. The radial immunodiffusion test is simple and available as a commercial kit. It is mostly used for the assessment of isotypes present in serum in high concentrations (IgG, IgM), whilst the ELISA method is preferred for the detection of isotypes present in serum in low concentrations (IgA, IgE). Reduced concentrations of immunoglobulins indicate humoural immunodeficiency, which may be primary (selective IgA deficiency is most common) or secondary (occurs after some infectious diseases, commonly of viral origin). Decreased levels are sometimes found in newborns with non-sufficient colostrum supply. Elevated levels of immunoglobulins are found in chronic inflammatory processes and infections. Extremely high levels of serum immunoglobulins are detected in myeloma and occasionally in some infectious diseases.

Level or activity of complement proteins are a significant immunological parameter too. It is assessed by the test of haemolysis of sensitised erythrocytes, or immunochemical serological analysis of the respective components, particularly C3.

Advanced Methods in Immunology

Similarly, as in related fields, knowledge in the field of immunology has been enormously extended by application of methods monitoring events in the cell at molecular levels, using the methods of genomics and proteomics. Some of these methods have already been applied to clinical immunology, usually as newly obtained knowledge from clinical and experimental studies. However, due to the fact that they are technically demanding and thus expensive, they are only rarely used for direct diagnosis.

Flow cytometry is the most commonly and recent method used, for the determination of lymphocyte subsets. Lymphocytes appear to be in a uniform population when viewed by microscopy. However, they are divided functionally into a series of subpopulations, which undertake various functions (Table 2). Monoclonal antibodies against phenotypic surface molecules are used for distinguishing between respective subtypes of lymphocytes. Changes in T and B lymphocyte ratios and changes in the ratios of helper (Th) and cytotoxic (Tc) T lymphocytes have been studied intensively. Changes in the ratios of these cells have been found to be indicative of SLE, GSP-associated immunodeficiency or leishmaniosis. More recently, other minority subsets (γδ T cells, NK cells), are studied in dogs in connection with immune diseases. Flow cytometry is also exploited, such as when detecting cytoplasm proteins including cytokines, distinguishing between apoptosis and necrosis and determining cell cycle stages, which is a test used in oncology diagnoses.

Table 2. Lymphocyte subsets in dogs.


Phenotypic molecules

Range in blood

T helper cells (Th)

CD3, CD4, TCRαβ, (CD2, CD5)

30-48 %

T cytotoxic cells (Tc)

CD3, CD8, TCRαβ, (CD2, CD5)

15-25 %

B lymphocytes

CD19, CD21, sIgM


γδ T cells

CD3, CD8±, TCRγδ

1-2 %

double positive T cells

CD3, CD4, CD8

0-1 %

Methods of molecular biology and genomics are even more noteworthy. Detection of gene expressions based on polymerase chain reaction techniques (at present it is above all reverse transcription-PCR, real-time PCR) are used in clinical immunology, above all for the cytokines detection. The most commonly detected cytokines are inflammatory cytokines (IL-1, IL-6, TNFα), regulating cytokines (Th1 cytokines--IL-2, IFNγ, Th2 cytokines--IL-4, IL-5, IL-10 and others) and chemokines. So far, the detection of cytokine in mRNA levels has been used in cells or tissues in dogs with various diseases including immune-mediated.

These methods have been also applied in immunogenetics. Damaged genes that cause primary immunodeficiency may be detected in the case of Severe combined X-linked immunodeficiency disease (SCID) of bassets; C3 deficiency and canine leukocyte adherence deficiency (CLAD). The significance of MHC allotyping is gradually increasing; given its association with a number of immune-mediated diseases, in both animals and humans. The association of a particular type of MHC allotype has been confirmed in thyroidism and diabetes mellitus to date.


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Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Martin Faldyna, MVDr, PhD
Veterinary Research Institute
Brno, Jihomoravský kraj, Czech Republic

Miroslav Toman, Prof. MVDr., CSc.
Veterinary Research Institute
Brno, Jihomoravský kraj, Czech Republic

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