Flow cytometry test to detect immune-related low platelets in dogs

Abstract

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.