Prospective diagnostic accuracy evaluation and clinical utilization of a modified assay for platelet-associated immunoglobulin in thrombocytopenic and nonthrombocytopenic dogs

Background: No diagnostic tests reliably distinguish primary immune-mediated thrombocytopenia (pIMT) from other causes of thrombocytopenia. Objectives: The purpose of the study was to evaluate diagnostic sensitivity and specificity using modified direct and indirect platelet-associated immunoglobulin (PAIg) assays and reticulated platelets (RP) by flow cytometry for the classification of thrombocytopenic dogs and differentiating pIMT. Methods: Platelets were isolated from plasma samples of thrombocytopenic dogs and nonthrombocytopenic healthy and ill dogs. For direct PAIg, they were analyzed by flow cytometry after incubation with anti-human amylase fluorescein isothiocyanate (FITC, negative control), anti-canine IgG-FITC, anti-canine IgM-FITC, and anti-human CD61-conjugated fluorochrome (AF647). For indirect PAIg, platelets from normothrombocytic dogs were incubated with thrombocytopenic dog plasma and analyzed similar to direct PAIg. RP percentages were determined based on forward light scatter vs thiazole orange fluorescence. Results: Seventy-five thrombocytopenic dogs, 16 nonthrombocytopenic ill dogs, and 24 healthy dogs were evaluated. Diagnostic sensitivity and specificity utilizing direct IgG was 29.4% and 75.9%, respectively; when combining direct/indirect assays (IgG/IgM), it was 76.5% and 65.5%, respectively, for distinguishing pIMT. For RP, no significant difference between pIMT and sIMT was noted. RP > 8% with positive PAIg had a sensitivity of 94% and specificity of 27.6% for distinguishing pIMT. There was a significant difference in platelet concentration and CD61% staining between control and pIMT. Conclusions: The combined modified assays resulted in fair diagnostic sensitivity and specificity for the diagnosis of pIMT. The modification of the immunoglobulin assays improved diagnostic accuracy; however, a single panel to accurately classify thrombocytopenia remains elusive.