Measurement of Systemic Immunity
World Small Animal Veterinary Association World Congress Proceedings, 2009
Michael J. Day, BSc, BVMS(Hons), PhD, DSc, DECVP, FASM, FRCPath, FRCVS
School of Clinical Veterinary Science University of Bristol, Langford, United Kingdom

The major components of the immune system are humoral and cellular immunity. Evaluation of the function of these aspects of canine and feline immunity is widely used in research laboratories--but is less commonly available in a commercial diagnostic setting for application to individual patient animals.

Tests of Antibody Production

The standard means of quantifying serum immunoglobulin [Ig] is via single radial immunodiffusion [SRID]. In this test, an agar gel is cast which incorporates an optimum concentration of antiserum specific for the Ig in question. Wells are cut into the gel and loaded with serum from the test dog and a series of standards of known Ig concentration. The Ig diffuses out of the well into the gel where it is bound by antiserum and forms a zone of precipitation around the well. The higher the concentration of Ig in the sample, the further it will diffuse from the well and the larger the zone of precipitation. Following incubation (24-48 hours) the diameter of the test precipitin ring is measured and used to calculate the Ig concentration in the sample relative to the standard curve constructed with the ring diameters of the standards. SRID may be used to detect IgG, IgM and IgA and has also been used to measure the serum concentration of canine C3 and C4. A more sensitive means of detecting and quantifying Ig is capture ELISA which may be used to determine the concentration of Ig in secretions in addition to serum. More complicated 'competitive-inhibition' capture ELISAs are also described for this purpose.

In addition to determining the overall concentration of immunoglobulin it is sometimes desirable to determine whether an animal is capable of mounting a specific immune response to an immunogen. Conveniently most animals receive vaccinal antigens and tests are relatively widely available for the detection of serum antibody specific for core viral components of vaccines.

Tests of Complement Function

The CH50 Assay tests the function of the classical and terminal pathways of complement. In this test, a range of patient serum dilutions is incubated with a standard suspension of antibody-coated erythrocytes which act as an 'indicator system'. The active complement within the serum sample is 'fixed' by the antigen-antibody indicator and leads to haemolysis via formation of the terminal membrane attack complex. Haemolysis is measured spectrophotometrically--greater amounts of complement will lead to haemolysis in latter tubes of the serum dilution series.

Lymphocyte Phenotyping

The methodology involved in flow cytometric testing for determination of the phenotype of lymphocytes in a blood sample is relatively complex. Optimally, the first stage of the procedure involves the separation of mononuclear cells (lymphocytes and monocytes) from a whole blood sample by density gradient centrifugation.

The cell suspension is then separated into different tubes to test different antibodies and controls. In the most straightforward cytometric analysis, antibodies specific for canine or feline CD4, CD8 or CD21 would be added to separate tubes and would bind selectively to those specific populations. These monoclonal antibodies may be directly conjugated to a fluorochrome (usually FITC), although it is possible to use an unconjugated primary antibody with a secondary fluorochrome-linked reagent (generally an anti-mouse IgG Fab'2 in which the Fc portion is cleaved to prevent non-specific binding to cellular FcRs).

The labelled cell suspension is then drawn up into the flow cytometer, which creates a series of droplets--each containing a single mononuclear cell. The stream of droplets is rapidly fired past a beam of laser light of wavelength appropriate for activating the fluorochrome. Each cell is thus 'interrogated' by the machine to determine if it has been positively labelled by antibody. Each positively labelled cell is recorded by the cytometer as an 'event', and a total of 10,000 cells are counted. The % positive cells is calculated and may be related back to an absolute number of positive cells in the original blood sample (obtained from the differential leukocyte count). The ratio of CD4:CD8 cells can thus be determined.

At present, this testing is most often performed for canine samples but the procedure is equally applicable to feline blood. In normal dogs, the CD4:CD8 is approximately 2 to 2.5:1, but this has been reported to change in dogs with SLE (5.2:1) or deep pyoderma (1.3:1). In dogs with leishmaniasis, the proportion of B cells in blood increases and there is a reduced proportion of CD4+ T cells--and this type of analysis is now regarded as an important diagnostic procedure in endemic areas. The relative proportion of T cell subsets might also be expected to change in cats with retroviral infection.

Lymphocyte Function Testing

This procedure aims to determine whether the lymphocytes in a sample (generally from peripheral blood although teased apart lymphoid tissue can also provide a suitable suspension) are able to respond to stimulation. The simplest means of stimulating these cells is via the use of substances called 'mitogens' which bind in a non antigen-specific fashion directly to glycosylated proteins expressed on the lymphocyte cell membrane to activate the cells. An alternative means of stimulating T cells is to incubate them with antibody specific for CD3, which binds to this signal transduction complex and directly activates the cells--again in a non antigen-specific fashion. The most satisfying (but technically complex) procedure is to attempt to stimulate the lymphocytes in an antigen-specific fashion. This generally entails using an antigen to which the animal has been previously exposed and thus carries immunological memory (typically a vaccinal antigen)--although it is also possible to demonstrate a primary immune response in such a culture system to previously unseen antigen (which will have slower kinetics). This latter methodology requires that the lymphocyte culture be supplemented with a source of autologous antigen presenting cells (APC) or that the mononuclear cell suspension incorporates blood monocytes. These are required to process and present the antigen for recognition via the antigen-specific T cell receptor. In practical terms, the sample required to undertake this testing is heparinized blood (generally 10-20 ml) from the test animal and one or two control animals that are closely age and breed matched for comparative purposes.

There are different approaches to determining whether lymphocyte stimulation has occurred. The most commonly used method involves the addition of radiolabeled (3H) thymidine into the culture for the last 18 hours of incubation. The thymidine is incorporated into the DNA of newly dividing cells, such that at the end of the culture period when the cells are washed free of unbound thymidine, the amount of radiolabel within the cell pellet (as determined by a beta irradiation counter) is proportional to the amount of cell division. The simplest means of expressing this cell division is to calculate the ratio of signal in stimulated versus unstimulated cultures (the stimulation index or SI). Another means of determining outcome is to assay the culture supernatant for proteins that may have been elaborated by the stimulated cells. Classically, the production of antigen-specific immunoglobulin can be detected by ELISA. It is also possible to measure the production of cytokine by activated cells--also by capture ELISA.

Tests of Phagocytic Cell Function

A range of tests of the in vitro function of neutrophils and macrophages has been applied to the dog (less often cats). Neutrophils are readily fractionated from blood by the use of density gradient media as described above and monocytes may be separated from lymphocytes by short-term incubation on glass coverslips (the monocytes will adhere to these whereas lymphocytes will not and will be subsequently washed away). The most important functions of phagocytic cells are their ability to migrate towards a chemotactic stimulus, to phagocytose particles and to destroy the particles which have been phagocytosed. The chemotaxis of phagocytes can be detected in a variety of ways. The cells can be placed into one half of a 'Boyden chamber' which is divided by a filter membrane permeable only to protein molecules.

The chemotactic stimulus (e.g., molecules derived from bacteria or serum complement components) is placed into the second half of the chamber and diffuses to establish a chemotactic gradient. Phagocytic cells that are attracted towards the gradient will become caught in the dividing membrane and may be identified by removing and staining the membrane after culture. The phagocytic ability of these cells can also be readily assessed by incubating the purified cells with a target particle of optimum size for phagocytosis. Classically staphylococci or latex spheres have been used for this purpose, and the phagocytosis is generally enhanced when these particles are appropriately opsonized by pre-incubation with fresh autologous serum as a source of antibody and complement. The relative phagocytic ability of cells from test and control animals can be determined by stopping the reaction and staining cytospin preparations of the culture for counting the numbers of intracytoplasmic versus unphagocytosed particles. Alternatively, the process can be automated by the use of fluorescein-labelled particles that can be detected by subsequent flow cytometric analysis.

The efficiency of phagocyte killing of particles and the activation of the intracellular enzymatic pathways involved can also be measured. Viable bacteria (e.g., staphylococci) are used as targets and preincubated with the phagocytic cells such that an optimum number of these are taken into the cytoplasm of the phagocytes. After a period of incubation (or a series of time points throughout an incubation period) phagocytic cells can be removed from the culture well and lysed by exposure to water. The lysates may then be cultured in order to determine the number of viable organisms remaining at that time point, relative to the total number of staring organisms. Alternatively, the respiratory burst of the neutrophil can be measured after phagocytosis of particulate material by an enzymatic reaction producing a colour change (the nitroblue tetrazolium test) or by emission of light in a chemiluminescence reaction.


References are available upon request.

Speaker Information
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Michael J. Day, BSc, BVMS(Hons), PhD, DSc, DECVP, FASM, FRCPath, FRCVS
School of Clinical Veterinary Science
University of Bristol
Langford, United Kingdom

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