Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs.

Abstract

BACKGROUND: Platelet microparticles (PMPs) are subcellular procoagulant vesicles released upon platelet activation. In people with clinical diseases, alterations in PMP concentrations have been extensively investigated, but few canine studies exist. OBJECTIVES: This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations. METHODS: Microparticles (MP) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Anti-CD61 antibody and Annexin V (AnV) were used to detect platelets and phosphatidylserine, respectively. In 13 healthy dogs, CD61/AnVconcentrations were analyzed with/without a calcium buffer. CD61/AnV, CD61/AnV, and CD61/AnVMP quantification were validated in 10 healthy dogs. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses and detection limits (DLs) were calculated. RESULTS: CD61/AnVconcentrations were higher in calcium buffer; 841,800 MP/&#x3bc;L (526,000-1,666,200) vs without; 474,200 MP/&#x3bc;L (278,800-997,500), P&#xa0;<&#xa0;.05. In WB, PMP were above DLs and demonstrated acceptable (<20%) intra-assay and inter-assay CVs in 9/10 dogs: 1.7% (0.5-8.9) and 9.0% (0.9-11.9), respectively, for CD61/AnVand 2.4% (0.2-8.7) and 7.8% (0.0-12.8), respectively, for CD61/AnV. Acceptable CVs were not seen for the CD61/AnVMP. In PPP, quantifications were challenged by high inter-assay CV, overlapping DLs and hemolysis and lipemia interfered with quantification in 5/10 dogs. CONCLUSIONS: Calcium induced higher in&#xa0;vitro PMP concentrations, likely due to platelet activation. PMP concentrations were reliably quantified in WB, indicating the potential for clinical applications. PPP analyses were unreliable due to high inter-CV and DL overlap, and not obtainable due to hemolysis and lipemia interference.