Cremer Signe E, Krogh Anne K H, Hedström Matilda E K, Christiansen Liselotte B, Tarnow Inge, Kristensen Annemarie T
Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark.
Savara Pharmaceuticals, Hørsholm, Denmark.
Vet Clin Pathol. 2018 Jun;47(2):186-196. doi: 10.1111/vcp.12605. Epub 2018 Mar 30.
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.
This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations.
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 /AnV concentrations were analyzed with/without a calcium buffer. CD61 /AnV , CD61 /AnV , and CD61 /AnV MP 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.
CD61 /AnV concentrations were higher in calcium buffer; 841,800 MP/μL (526,000-1,666,200) vs without; 474,200 MP/μL (278,800-997,500), P < .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 /AnV and 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 /AnV MP. In PPP, quantifications were challenged by high inter-assay CV, overlapping DLs and hemolysis and lipemia interfered with quantification in 5/10 dogs.
Calcium induced higher in 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.
血小板微粒（PMPs）是血小板激活后释放的亚细胞促凝小泡。在患有临床疾病的人群中，PMP浓度的变化已得到广泛研究，但犬类研究较少。
本研究旨在验证一种用于PMP定量的犬类流式细胞术方案，并评估钙对PMP浓度的影响。
使用流式细胞术对枸橼酸化全血（WB）和少血小板血浆（PPP）中的微粒（MP）进行定量。分别使用抗CD61抗体和膜联蛋白V（AnV）检测血小板和磷脂酰丝氨酸。在13只健康犬中，分析有无钙缓冲液时的CD61 /AnV浓度。在10只健康犬中验证了CD61 /AnV、CD61 /AnV和CD61 /AnV MP的定量。计算重复（批内）和平行（批间）分析的变异系数（CV）以及检测限（DLs）。
有钙缓冲液时CD61 /AnV浓度更高；分别为841,800个MP/μL（526,000 - 1,666,200）和无钙缓冲液时的474,200个MP/μL（278,800 - 997,500），P < 0.05。在WB中，9/10只犬的PMP高于检测限，批内和批间CV均可接受（<20%）：CD61 /AnV分别为1.7%（0.5 - 8.9）和9.0%（0.9 - 11.9），CD61 /AnV分别为2.4%（0.2 - 8.7）和7.8%（0.0 - 12.8）。CD61 /AnV MP未观察到可接受的CV。在PPP中，批间CV高、检测限重叠以及溶血和脂血干扰了5/10只犬的定量。
钙诱导体外PMP浓度升高，可能是由于血小板激活。在WB中可可靠地定量PMP浓度，表明其具有临床应用潜力。由于批间CV高和检测限重叠，PPP分析不可靠，且由于溶血和脂血干扰无法进行。
