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Kansas State Veterinary Diagnostic Laboratory

Clinical Immunology

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The Clinical Immunology laboratory of KSVDL provides high quality services to practicing veterinarians, the Veterinary Health Center, researchers and industry. This is an AAVLD accredited laboratory.

Tests

If you do not find a specific test listed, please contact Client Services at (785) 532-5650, toll free at 1-866-512-5650 or clientcare@vet.k-state.edu for special testing submission and fees.

Platelet-Surface Associated IgG - Click to Read

Platelet-surface associated IgG is performed in animals with with suspect iImmune mediated thrombocytopenia. This test has been validated in dogs and horses. The PSAIgG assay detects IgG bound to the surface of dog or horse platelets. For dogs, we use a monoclonal antibody to IgG that is labeled with a green fluorescent dye. If the patient has IgG antibody on the surface of their platelets, the fluorescent labeled monoclonal antibody will react to this antibody. We use a flow cytometer with a laser that detectsf fluorescently labeled platelets in the sample. This assay was originally developed by Internal Medicine Specialist (Dr. David Lewis) (Lewis et al., 1995). The advantage of this method over a previously developed ELISA is that this test can reliably assay as few as 5,000 platelets/. Therefore, acquisition of large blood samples is not necessary. The result is expressed as the percentage of platelets coated with IgG. In dogs, IgG is reported to be the primary immunoglobulin class bound to canine platelets. The test replaces the platelet factor 3 (PF3) test. The sensitivity of the PSAIgG is 88%, whereas specificity is 90 – 100%. We use an isotype control antibody that does not react with canine platelets as the negative control antibody for this assay. In equine (horses), we use fluorescent labeled polyclonal antibodies that are specific to equine IgG, IgM, and IgA. A positive result is expressed as the percentage of platelets coated with IgG, IgM or IgA. Please note! A positive test result supports a diagnosis of immune-mediated thrombocytopenia (IMT), but does not differentiate primary IMT from secondary IMT. This test does not detect the difference between autoantibody bound to the cells from antibody that cross reacts with platelet proteins and epitopes present on bacteria, viruses, or tumor cells. Plus, antibodies in the form of immune complexes could be detected by this method. We have found that this assay will give us false positives as the sample ages over time. To get the most accurate results, submit whole blood samples (collected in EDTA) within 24 hours (Vet Clin Path. 2001:30:107-113).

Platelet-surface associated IgG is performed in animals with with suspect iImmune mediated thrombocytopenia. This test has been validated in dogs and horses. The PSAIgG assay detects IgG bound to the surface of dog or horse platelets. For dogs, we use a monoclonal antibody to IgG that is labeled with a green fluorescent dye. If the patient has IgG antibody on the surface of their platelets, the fluorescent labeled monoclonal antibody will react to this antibody. We use a flow cytometer with a laser that detectsf fluorescently labeled platelets in the sample. This assay was originally developed by Internal Medicine Specialist (Dr. David Lewis) (Lewis et al., 1995). The advantage of this method over a previously developed ELISA is that this test can reliably assay as few as 5,000 platelets/. Therefore, acquisition of large blood samples is not necessary. The result is expressed as the percentage of platelets coated with IgG. In dogs, IgG is reported to be the primary immunoglobulin class bound to canine platelets. The test replaces the platelet factor 3 (PF3) test. The sensitivity of the PSAIgG is 88%, whereas specificity is 90 – 100%. We use an isotype control antibody that does not react with canine platelets as the negative control antibody for this assay. In equine (horses), we use fluorescent labeled polyclonal antibodies that are specific to equine IgG, IgM, and IgA. A positive result is expressed as the percentage of platelets coated with IgG, IgM or IgA. Please note! A positive test result supports a diagnosis of immune-mediated thrombocytopenia (IMT), but does not differentiate primary IMT from secondary IMT. This test does not detect the difference between autoantibody bound to the cells from antibody that cross reacts with platelet proteins and epitopes present on bacteria, viruses, or tumor cells. Plus, antibodies in the form of immune complexes could be detected by this method. We have found that this assay will give us false positives as the sample ages over time. To get the most accurate results, submit whole blood samples (collected in EDTA) within 24 hours (Vet Clin Path. 2001:30:107-113). 
View PSAIgG Slideshow PowerPoint Presentation.

Immune mediated thrombocytopenia not associated with a disease or condition is referred to as idiopathic IMT, often called primary IMT. Diagnosis of idiopathic IMT is based on exclusion of other causes of thrombocytopenia. The underlying cause may be auto antibodies to platelet surface glycoproteins, but the antibody specificity to normal platelet epitopes is rarely documented in veterinary medicine. Two reports describe the identification of autoantibodies to the glycoproteins gpIIb/IIIa on canine platelets (Lewis et al., 1996; Scott, 2002). The procedure to identify the specificity of the antibody is very elaborate and is as follows: The autoantibody is eluted from the patient’s platelets by acidification. The elute fraction is then incubated with normal platelets labeled with (I125). If autoantibody is present in the elute, it will bind platelet glycoproteins and form an immunoprecipitate that can be visualized by electrophoresis on a polyacrylamide gel. A band will migrate a certain distance on the polyacrylamide gel corresponding to the molecular weight of the glycoprotein that was immunoprecipitated by the eluted antibody. Although this assay determines the specificity of auto antibodies, it is too sophisticated for clinical laboratories.

PSAIgG test can be performed to support a diagnosis of an immune mediated cause for thrombocytopenia. The exact cause may not be determined by this test, and will require the clinician to examine the clinical history, clinical signs, and to do other laboratory tests. Causes of secondary IMT include:

  1. Drug-induced (gold salts or sulfonamides that absorb to platelet membrane and include drug dependent antibodies or expose new epitopes that are foreign)
  2. Infections (induces cross reactive antibodies or exposes new epitopes or induction of immune complexes that adhere to the platelet surface). We have identified dogs with PSAIgG that had concurrent infections with Ehrlichia canis, Ricketsia ricketssi, and Babesia gibsoni.
  3. Neoplasia (induces cross reactive antibodies or exposes new epitopes or induction of immune complexes)
  4. Neonatal alloimmune thrombocytopenia is passively acquired from the dam through ingestion of alloantibodies in the colostrum. The alloantibodies are made to paternal epitopes that the dam lacks. The alloantibodies are generated in the mare after fetal red blood cells carrying the paternal epitope enters the circulation of the mare at parturition and stimulate antibodies.

 

If the PSAIgG test is negative, you should consider the following circumstances:

  1. Antibody may have eluted from the platelets during storage or processing (false negative).
  2. Could be IgM antibodies in the dog (Scott, MA, 2002).
  3. Has the patient been on long term corticosteroids? Corticosteroids typically do not decrease antibody production unless at high doses for a chronic duration (> two weeks). Acutely, corticosteroids will inhibit macrophage function and their ability to remove antibody coated platelets.
  4. Have you considered a nonimmunologic cause (i.e. blood loss, activation or clumping in the tube, and consumption by vasculitis, DIC) for decreased platelet survival? Have you considered abnormal platelet production? Myelosuppression due to bone marrow disease, antibodies to megakaryocytes, antibodies to cytokines, toxicants.

 

Reference List Lewis, D. C., McVey, D. S., Shuman, W. S., and Muller, W. B. (1995). Development and characterization of a flow cytometric assay for detection of platelet-bound immunoglobulin G in dogs. American Journal of Veterinary Research 56, 1555-1558. Lewis, D. C. and Meyers, K. M. (1996). Studies of platelet-bound and serum platelet-bindable immunoglobulins in dogs with idiopathic thrombocytopenic purpura. Experimental Hematology 24, 696-701. Scott, M. K. L. D. J. S. K. (2002). Development of a sensitive immunoradiometric assay for detection of platelet surface-associated immunoglobulins in thrombocytopenic dogs. Am.J.Vet.Res. 63, 124-129.

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RBC- surface associated immunoglobin test for dogs and horses with immune mediated anemia

The erythrocyte surface antibody test detects antibodies on the surface of erythrocytes. For dogs, we use fluorescently- labeled polyclonal antisera to canine IgG, IgM, IgA, and C3 and flow cytometry to quantitate the percentage of antibody coated cells in the sample. For horses, we use fluorescently- labeled polyclonal antisera to equine IgG, IgM and IgA. This test is more sensitive that the Coombs anti-globulin agglutination test, because it is not dependent on agglutination as an endpoint and the false negatives that occur at low end (prozone) or high end (post zone) of the dilution of Coombs reagent. The sensitivity is 100% and the specificity is 87.5% determined by population of dogs with immune mediated hemolytic anemia (J. Vet. Intern. Med. 2000, 14:190-196). The test does not differentiate primary (idiopathic or autoimmune) immune mediated hemolytic anemia (IMHA) from secondary causes for IMHA.

We modified this assay to detect penicillin specific antibodies in equine serum in cases of penicillin induced IMHA.

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Anti-Nuclear Antibody (ANA)

Dilutions of patient serum are tested for antibodies to nuclear substances 1:80 through 1:640. We have noted ANA titers of 1:80 and 1:160 in healthy dogs and in dogs with inflammatory conditions. Positive ANA titers should be considered when the titer is >1:160. Interpretation of a positive ANA as diagnostic of SLE should always accompany specific clinical criteria for SLE because some dogs can have positive ANA titers without disease or with inflammatory conditions (Compendium, 1999:21:135 and 402).

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Rheumatoid Factor Assay (RF)

We use a latex agglutination slide test to detect canine rheumatoid factor in serum (antibody to canine IgG). A definitive diagnosis of canine rheumatoid arthritis should be made by meeting 5/11 criteria established by the American Rheumatoid Association. (Berkow et al, Merck Manual of Diagnosis and Therapy, thirteenth edition, Rahaw, N.J.).

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Immunophenotyping

Lymphocyte phenotyping is requested in cases of immunodeficiency, chronic inflammatory conditions (lymphocytosis), and determining the cell lineage of lymphomas or leukemias. Lymphocyte phenotyping is requested in cases of immunodeficiency, chronic inflammatory conditions (lymphocytosis), and lymphoma or leukemia typing.

Monoclonal antibodies (VMRD, Pullman, WA)Surface Determinants% positive lymphocytes*
HB19A + 1.9/3.2CD5 + IgM+ B92.0 ± 5.8
HB19ACD591.8 ± 2.5
HB61ACD466.2 ± 12.3
HT14ACD816.6 ± 2.8
1.9/3.2IgM+B12.6 ± 3.2
TH14BMHC II (DR)70.7 ± 8.3
H42AMHC II (DP)81.1 ± 5.6

Equine lymphocyte subsets determined by immunophenotyping and flow cytometry
*percentages determined on gated lymphocytes excluding monocytes
(values based on 10 adult horses)

Flow Cytometry PowerPoint Presentation

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