Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet-rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P-selectin and by determining the percentages of platelet aggregates and microparticles using forward-angle vs side-angle light scatter plots. Results were analyzed by using 2-way ANOVA and the Scheffé F-test. Results: Platelets collected in EDTA had minimal expression of P-selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti-P-selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet-rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P-selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P-selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA-anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.