EXPRESS: Flow cytometry-based direct antiglobulin test for suspected feline immune-mediated thrombocytopenia: Assay development and demographic factors associated with DAT positivity.

OBJECTIVES: This study aimed to develop and evaluate an in-house flow cytometry-based direct antiglobulin test (DAT) for detecting IgG-bound platelet in cats, and to investigate the prevalence of DAT positivity and associated risk factors among thrombocytopenic cats. METHODS: Platelets were identified by CD41/61 expression, and platelet-bound IgG was assessed by flow cytometry. A diagnostic cutoff for the percentage of IgG-positive platelets was established using 100 healthy cats and preliminarily evaluated in 50 cats with severe thrombocytopenia (platelet count <20,000/µL) before being applied to the remaining 411 thrombocytopenic cats (platelet count <200,000/µL) to determine the prevalence of DAT positivity and its associations with platelet count, breed, age, and sex. RESULTS: Healthy cats showed 0-10% IgG-positive platelets, and a cutoff of >10% was established. Using this threshold, 105/461 (22.8%) thrombocytopenic cats were classified as DAT positive. DAT positive cats had significantly lower platelet counts than DAT negative cats (median 4,000/µL; mean 12,562/µL vs 34,000/µL; mean 47,956/µL; P <0.05), and the percentage of IgG-bound platelets was inversely correlated with platelet count (r = -0.38; P = 0.048). No breed association was identified. Cats aged 5 years were more likely to be DAT positive, whereas neutered male cats were less likely to be DAT positive. CONCLUSIONS AND RELEVANCE: Flow cytometry-based DAT is a useful adjunctive diagnostic tool for assessing suspected immune-mediated platelet destruction in thrombocytopenic cats. Younger cats may be at increased risk, whereas neutered male cats may be at lower risk. However, the interpretation of a DAT positive test result should be made alongside assessment of clinical findings and investigations for underlying causes of thrombocytopenia.