流式细胞术检测血小板微颗粒的方法学探讨

Detection of Platelet-derived Microparticle by Flow Cytometry

目的应用流式细胞术(FCM)-富含血小板血浆(PRP)法检测血小板微颗粒(PMPs),并进行方法学、影响因素探讨及临床意义的评价。方法应用特异性荧光抗体CD61-FITC标记血小板,采用FCM-PRP法,以0.82μm标准微球进行定位对照,设置机器检测条件,调节机器阈值,设门计数PMPs占CD61阳性颗粒的百分比。以二磷酸腺苷(ADP)及胶原诱导血小板活化后释放的PMPs作为阳性对照。同时检测不同保存时间血小板血浆中PMPs的释放量。结果30例健康对照者静息状态下血小板释放的PMPs为(2.19±0.48)%,血小板活化后释放的PMPs数量显著增加,ADP诱导血小板活化后产生的PMPs为(5.43±3.48)%,胶原诱导血小板活化后释放的PMPs为(3.46±1.14)%,静息状态和激活后PMPs差异有显著性意义(均P<0.05)。标本存放时间越长,PMPs释放量越大。37例血栓性疾病患者血小板释放的PMPs显著高于健康对照者(P<0.05)。结论采用FCM-PRP法检测PMPs操作简单,影响因素较少,适合临床常规检测。

Objective To establish optimization of flow cytometry(FCM) method for quantification of platelet-derived microparticles(PMPs).Methods Platelet rich plasma(PRP) was prepared as sample,and then was stained with a fluorochrome-conjugated monoclonal antibody directed towards platelet surface glycoproteins.0.82 μm latex beads were used to calibrate forward scatter threshold parameter and set up optimization of procedure of flow cytometry and as standard for counting PMPs.PMPs generated in vitro using ADP or collagen were detected as positive controls,and the method systemically evaluated.Results PMPs released from platelets under testing status in 30 healthy donors were(2.19±0.48)%,after activation of platelets with ADP,PMPs were(5.43±3.48) %,and after activation of platelets with collagen,PMPs were(3.46±1.14) %(both P<0.05).The storage time of sample impacted the results of PMPs.In 37 patients with thrombus diseases,the PMPs were evidently increased as compared with healthy donors.Conclusion The method of FCM-PRP to detect PMPs is feasible,simple,has less influencing factor,and it can be used as a routine test.PMPs are a useful parameter for monitoring platelet activation and diagnosing thrombus diseases early.