Immunology

Platelet-surface associated IgG in Dogs, Cats, and Horses with suspect Immune Mediated Thrombocytopenia.

The PSAIgG assay detects IgG bound to the surface of dog, cat, or horse platelets. For dogs and cats, 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 which has a laser that can detect the amount of fluorescent labeled platelets in the sample. This assay was originally developed by a 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, which is no longer available. The sensitivity of the PSAIgG based on a limited number of cases is 88%, whereas specificity is 90 – 100%. This is in comparison to the variable sensitivity and specificity reported for the PF3 test (30- 80%). We use an isotype control antibody that does not react with canine platelets as the negative control antibody for this assay.
 

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• Alterations in normal canine platelets during storage in EDTA anticoagulated blood. Wilkerson MJ, Shuman W. Vet Clin Pathol. 2001;30(3):107-113.

• Platelet size, platelet surface-associated IgG, and reticulated platelets in dogs with immune-mediated thrombocytopenia. Wilkerson MJ, Shuman W, Swist S, Harkin K, Meinkoth J, Kocan AA. Vet Clin Pathol. 2001;30(3):141-149.
   

In equine (horse), 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 either 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 has a cross reactivity to 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, Vet Clin Path website.).

PSAIgG Slideshow
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More information about immune mediated thrombocytopenia
Immune mediated thrombocytopenia not associated with a detected 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 auto antibodies 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 was eluted from the patient’s platelets by acidification. The elute fraction was incubated with normal platelets labeled with (I¹²⁵). The antibody in the elute will bind to the platelet glycoproteins and form an immunoprecipitate that is then electrophoresed on a polyacrylamide gel. A band will migrate a certain distance on the polyacrylamide gel and the migration will correspond to the molecular weight of the glycoprotein that was immunoprecipitated by the antibody in the elute. Although this assay will determine the specificity of auto antibodies, it is too sophisticated for clinical laboratories.

Therefore, the PSAIgG test is test that 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 can 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.

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 for decreased platelet survival? Blood loss, activation and consumption by vasculitis, DIC, neoplasia, drugs

5. 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.