Analysis of individual platelet-derived microparticles, comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection.
Analysis of individual platelet-derived microparticles, comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection.
复制标题
分析单个血小板衍生的微粒，将流式细胞术和毛细管电泳与激光诱导荧光检测进行比较。
DOI：
10.1039/b301035j
复制
发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Arriaga,EdgarA
中科院分区：
文献类型：
--
作者：
Xiong,Guohua;Aras,Omer;Shet,Arun;Key,NigelS;Arriaga,EdgarA
血小板活化过程中囊泡形成的血小板衍生微粒（PMP）似乎在血液凝固和病理疾病状态中发挥作用。流式细胞术是目前表征血小板和 PMP 的金标准。使用这种技术，我们根据大小和磷脂酰丝氨酸 (PS) 的存在来区分血小板和 PMP； PMP 被任意定义为小于一微米，并且能够与荧光标记的膜联蛋白 V 形成稳定的复合物，膜联蛋白 V 是一种与 PS 形成钙依赖性复合物的蛋白质。通过使用针对 CD41a 的荧光标记抗体，进一步确认了 PMP 和血小板的身份，CD41a 是一种在血小板和 PMP 表面发现的糖蛋白。在本报告中，我们还介绍了使用毛细管电泳和柱后激光诱导荧光检测 (CE-LIF) 来分析荧光标记的血小板和 PMP。虽然流式细胞术和 CE-LIF 都可以测量单个荧光事件，但只有 CE-LIF 允许我们计算活化血小板和 PMP 的单个电泳迁移率，然后将其表示为分布。分布之间的比较表明，PMP 的负迁移率较小。活化血小板制剂包含 PMP 的事实部分模糊了数据的解释。虽然流式细胞术测定的 PMP 和血小板数量 ml−1 低于 CE-LIF 测定的相同参数，但流式细胞术的信噪比比 CE-LIF 好 20 倍。这是第一次报道这两种技术之间的直接比较。
Platelet-derived microparticles (PMPs) formed by vesiculation during platelet activation seem to play a role in blood coagulation and in pathological disease states. Flow cytometry is currently the gold standard to characterize platelets and PMPs. Using this technique we distinguished between platelets and PMPs based on size and the presence of phosphatidyl serine (PS); PMPs were arbitrarily defined to be smaller than one micrometer and capable of forming a stable complex with fluorescently-labeled Annexin V, a protein that forms a calcium-dependent complex with PS. Further confirmation of PMP and platelet identity was done by use of fluorescently-labeled antibodies against CD41a, a glycoprotein found on the surface of both platelets and PMPs. In this report we also introduce the use of capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) for the analysis of fluorescently labeled platelets and PMPs. While both flow cytometry and CE-LIF can measure individual fluorescent events, only CE-LIF allowed us to calculate individual electrophoretic mobilities of activated platelets and PMPs that were then represented as distributions. A comparison between distributions suggests that PMPs have less negative mobilities. The fact that activated platelet preparations include PMPs partially obscure the interpretation of the data. While PMP and platelet number ml−1 determined by flow cytometry is lower than the same parameter determined by CE-LIF, signal-to-noise ratio was 20 fold better for flow cytometry than for CE-LIF. This is the first time that a direct comparison between these two techniques is reported.