Preparation of Positive Control Platelets for Detection of Canine Platelet Surface-Associated IgG, IgM and Complement (C3) by Flow Cytometry

Abstract

ABSTRACT. An assay for detection of platelet surface-associated (PSA-) IgG, IgM and/or complement (C3) in dogs was modified by preparation of artificial positive control platelets. Flow cytometry of fluorescein isothiocyanate (FITC)-conjugated anti-dog IgG, anti-dog IgM and anti-dog C3 antibodies was used to detect the PSA proteins. IgM single, IgM/C3 double and IgG/IgM/C3 triple positive platelets were prepared. FITC-conjugated anti-IgG antibody bound strongly only to the triple positive platelets. Binding of FITC-conjugated anti-IgM or anti-C3 antibody to the double and triple positive platelets was specifically blocked by preincubation with the respective non-FITC-conjugated same-origin antibodies. These results confirm that FITC-conjugated antibodies specifically detect PSA proteins and that the control platelets prepared in this study are appropriate positive controls for detection of PSA proteins by flow cytometry.KEY WORDS: canine, immune-mediated thrombocytopenia, platelet surface-associated immunological proteins, positive control.J. Vet. Med. Sci. 72(8): 1063-1066, 2010 Primary immune-mediated thrombocytopenia (IMTP) is a common disease in dogs. It is diagnosed clinically by exclusion of other diseases that may cause thrombocytopenia and response to immunosuppressive therapies including glucocorticoid administration. Direct measurement of PSA immunoglobulin (PSA-IgG and/or PSA-IgM) may be performed as a laboratory test to aid in confirmation of the diagnosis of IMTP [10]. In the direct anti-globulin test for canine primary immune-mediated hemolytic anemia (IMHA), not only IgG-and IgM-but also C3-bound erythrocytes are detected [1]. Therefore, it is possible that C3 may also be detected in primary canine IMTP. Complement may mediate adherence of immune complexes to the surface of platelets and may induce secondary IMTP in dogs because nonprimate platelets are presumed to have the complement factor 3b (C3b) receptor [3,8]. Therefore, detection of PSA-C3 may also be useful in diagnosis of canine IMTP.Methods used to detect PSA-IgG (and sometimes PSAIgM) include radioimmunoassay [11], flow cytometry [5,7,13] and single layer enzyme-linked immunosorbent assay [2,6]. For reliable detection of PSA-IgG, PSA-IgM and PSA-C3, preparations of positive and negative control platelets are essential. Although measurement of PSA protein on patient platelets should be evaluated objectively by comparison with a positive control in every assay, positive control platelets are not commercially available. Negative controls are prepared by using fresh platelets from healthy dogs. Previously, IgG positive control platelets have been prepared by incubating normal canine platelets in sera containing antiplatelet antibody. These anti-sera were obtained from either alloimmunized dogs [6,7], heteroimmunized rabbits [2] or dogs diagnosed with primary IMTP [11]. These sources required special efforts to obtain the sera, and furthermore, IgM positive and C3 positive controls were not used in these reports. In the present study, we des...

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet‐bound antibodies in canine immune thrombocytopenia

“…A previous study described endotoxin treatment of healthy dog platelets to generate positive controls for PSAIg. 28 The endotoxin method, however, introduces biologic variability due to inherent relatively low diagnostic sensitivity vs specificity is also a feature of platelet autoantibody assays in human ITP and could be an additional explanation for the low levels of platelet-bound antibody in our cases. 20 While all the immunoglobulin and antibody reagents used in our study were commercially produced, we used a single lot of each reagent and did not assess lot-to-lot variability.…”

“…A previous study described endotoxin treatment of healthy dog platelets to generate positive controls for PSAIg 28 . The endotoxin method, however, introduces biologic variability due to inherent differences in platelet reactivity, and endotoxin‐treated platelets demonstrate nonspecific reactivity with anti‐dog IgG, IgM, and C3.…”