Tocchetti E V, Flower R L, Lloyd J V
Division of Haematology, Institute of Medical and Veterinary Science, PO Box 14, Rundle Mall, SA 5000, Adelaide, Australia.
Thromb Res. 2001 Jul 1;103(1):47-55. doi: 10.1016/s0049-3848(01)00263-8.
Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytometric methods for detection and quantification of PMPs has not been systemically evaluated. This study reports the optimisation of flow cytometric procedures for the detection of PMPs, the determination of limits of size detection using microbeads, and the characterisation of PMP generation by in vitro activation of platelets using collagen and adenosine 5' diphosphate (ADP). Fluorescent and plain microbeads proved useful for defining the limits of the flow cytometer in detecting PMPs. A systematic calibration of the forward scatter (FS) threshold parameter (size) of the flow cytometer using microbeads allowed for the detection of very small particles (down to 0.1 microm diameter). PMPs generated in vitro using ADP and collagen were reliably detected by flow cytometry using monoclonal antibodies (MAb) directed towards platelet surface membrane glycoproteins (Gp). The PMP events were detected in the FS low (i.e., small size events) and fluorescence (FL) high (i.e., platelet Gp MAb-labelled events) region. PMPs of different size profiles were observed for each of the agonists. Flow cytometry can be used as a tool in the assessment of PMPs. As detection of particles of this type is at the limit of resolution of flow cytometers, careful attention is required with the choice of platelet-specific MAb, isotype control, and optimisation of procedure setup and performance.
血小板微粒（PMPs）的定量检测可能是体内血小板活化检测的一个有用指标。用于检测和定量PMPs的流式细胞术方法的优化尚未得到系统评估。本研究报告了用于检测PMPs的流式细胞术程序的优化、使用微珠确定尺寸检测限以及通过使用胶原蛋白和5'-二磷酸腺苷（ADP）体外激活血小板来表征PMPs的生成。荧光微珠和普通微珠被证明有助于确定流式细胞仪检测PMPs的限度。使用微珠对流式细胞仪的前向散射（FS）阈值参数（尺寸）进行系统校准，能够检测到非常小的颗粒（直径低至0.1微米）。使用针对血小板表面膜糖蛋白（Gp）的单克隆抗体（MAb），通过流式细胞术可靠地检测了使用ADP和胶原蛋白在体外产生的PMPs。PMP事件在FS低（即小尺寸事件）和荧光（FL）高（即血小板Gp MAb标记事件）区域被检测到。对于每种激动剂，观察到了不同尺寸分布的PMPs。流式细胞术可作为评估PMPs的一种工具。由于这类颗粒的检测处于流式细胞仪分辨率的极限，因此在选择血小板特异性MAb、同型对照以及优化程序设置和性能时需要格外小心。
