流式细胞术检测血小板活化的影响因素分析

目的:探讨流式细胞术（FCM）检测血小板活化的影响因素。方法:采集6名志愿者静脉血，枸橼酸钠为抗凝剂, 室温放置不同时间后进行三色免疫荧光标记，FCM检测血小板早期活化标志物纤维蛋白原受体（Fib-R，即PAC-1）和晚期活化标志物P选择素（CD62P）的变化。结果:血小板活化标志物PAC-1和CD62P随着放置时间的延长而表达增加（P< 0.05）。抽血后10 min和30 min PAC-1和CD62P测定值分别相差2.7％和3.5％。且抽血后在不同的活化水平下重复检测多次，结果重复性好，其变异系数（CV）<5%。结论:在室温，枸橼酸钠抗凝条件下，放置时间对血小板活化标志物PAC-1和CD62P的检测结果有较大影响，宜在30 min内对采集血液免疫荧光标记后进行流式细胞仪检测。

Assessment of affecting factors in measuring activating platelets with the method of flow cytometry

AIM:To investigate the affecting factors of detecting platelet activation by flow cytometry (FCM).METHODS:Using decoagulant of natrium citricum, anticoagnlated peripheral venous bloods from 6 healthy donors were labeled with the method of three-colour immunofluorescence assay.Platelet activation markers fibrinogen receptor (Fib-R, PAC-1) and P-selectin (CD62P) were measured.In the same time, the reproducibility of FCM was assessed.RESULTS:The platelet activation markers PAC-1 and CD62P at each time point showed significant difference(P<0.05).The ratio was increased with time extending.The positive ratio of PAC-1 and CD62P immediately measured (within 10 min) was 2.7% and 3.5% less than those at time point of 30 min.The results were measured several times under different activation levels.The coefficient of variation was less than 5%.CONCLUSION:In room temperature and with decoagulant of natrium citricum, if the measurements of PAC-1 and CD62P are finished within 30 min after sampling, good reproducibility should be achieved.