Validation of a clinically applicable flow cytometric assay for the detection of immunoglobulin associated platelets in dogs

Thrombocytopenia is commonly encountered in veterinary practice when evaluating canine patients. It can occur in infectious, neoplastic, inflammatory, toxic, and immune-mediated conditions. Elucidating the underlying cause for thrombocytopenia can therefore represent a challenge to veterinary practitioners. Additionally, determination of whether an immune process could be contributing to a patient's thrombocytopenia is important for refining differentials and enhancing understanding of a particular disease process. A possible candidate test for the development of a clinically applicable assay in dogs is flow cytometry. Therefore, the purpose of this study was to develop a clinically applicable direct and indirect flow cytometric assay for the detection of canine immunoglobulin associated platelets. Direct and indirect flow cytometry was performed in nine healthy beagles and twelve client-owned thrombocytopenic dogs at four time points: fresh and after 24, 48, and 72 h of storage at 4 °C. For healthy dogs, there was no significant difference between fresh and 24 and 48 h samples but there was a significant difference between fresh and 72 h samples. There was no significant difference between fresh and 24, 48, or 72 h samples in the thrombocytopenic dogs. A cut-off value of ≤ 10% antibody binding was defined to differentiate negative and positive classifications and was determined by serial direct flow evaluations in a healthy dog. Based on this cut-off value, healthy and thrombocytopenic dogs were consistently categorized at every time point. The average intra-assay coefficient of variation for the thrombocytopenic dogs was 4.32%. The indirect flow cytometric methods evaluated herein did not provide reliable or repeatable results in healthy or thrombocytopenic dogs. Direct flow cytometry represents a potentially clinically useful test for the detection of immunoglobulin associated platelets in dogs that can be processed and evaluated within a realistic amount of time which would allow for testing in a larger number of patients. Based on the findings from this study using our protocols, indirect flow cytometry was not clinically applicable in dogs.