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

Abstract

Objectives: This study aimed to develop and evaluate an in-house flow cytometry-based direct antiglobulin test (DAT) for detecting IgG-bound platelets 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 using 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 cutoff, 105/461 (22.8%) thrombocytopenic cats were classified as DAT positive. DAT-positive cats had significantly lower platelet counts than DAT-negative cats (median 4000/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 castrated 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 castrated 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.