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使用现有流式细胞仪和基于散射的触发对循环血小板微粒进行标准化计数:前向散射还是侧向散射?

Standardized counting of circulating platelet microparticles using currently available flow cytometers and scatter-based triggering: Forward or side scatter?

机构信息

R&T Department, BioCytex, Marseille, France.

Faculté De Pharmacie, VRCM, UMR-S1076, Aix-Marseille Université, INSERM, Marseille, France.

出版信息

Cytometry A. 2016 Feb;89(2):148-58. doi: 10.1002/cyto.a.22685. Epub 2015 May 11.

Abstract

Clinical determination of MP counts using flow cytometry has not been fully accepted yet due to the lack of standardization protocols. In the past 5 years, we have proposed two versions of a method with reproducible PMP counts in plasma samples. Both methods use forward scatter (FSC)-based threshold set with reference beads of appropriate sizes; first using 0.5 µm beads and later with 0.3 µm beads. Both systems provide reproducible PMP counts. However, this technique works only with some of currently used commercial flow cytometers. Instruments with limited resolution or generating heterogeneous FSC signals are excluded. Such performances are incompatible with the required interinstrument standardization. Here we show that (i) flow cytometers with sub-optimal FSC capabilities generally have higher SSC resolution and background rejection capacity, and (ii) that the same biological entities, "dim and bright PMP," both can be counted using alternative strategies, either as previously described, based on FSC measurements, or as presented here, based on SSC detection. The critical element in the standardization protocol is the use of different sizes of reference beads. This study was designed to permit simultaneous access to both FSC- and SSC-optimized platforms. A new range of about 0.17-0.6 µm eq. (µm-equivalents) is proposed for an alternative SSC-based MP gate generating the same PMP counts as those obtained in the previously proposed 0.3-1 µm eq. FSC-based MP gate. The two equivalent standardization options reconcile intrinsically different scattering behaviors between SSC- and FCS--triggered instruments and open the opportunity for multicenter studies in the future.

摘要

由于缺乏标准化方案,使用流式细胞术对 MPs 进行临床计数尚未被完全接受。在过去的 5 年中,我们提出了两种方法,可在血浆样本中得到重现性的 PMP 计数。这两种方法都使用与适当大小的参考微球相对应的前向散射(FSC)阈值设定;第一种方法使用 0.5µm 微球,第二种方法使用 0.3µm 微球。这两种系统都能提供重现性的 PMP 计数。然而,这种技术仅适用于目前使用的一些商业流式细胞仪。排除了分辨率有限或产生不均匀 FSC 信号的仪器。这些性能与所需的仪器间标准化不兼容。在这里,我们表明:(i)具有次优 FSC 性能的流式细胞仪通常具有更高的 SSC 分辨率和背景排斥能力;(ii)相同的生物实体,“暗和亮 PMP”,都可以使用替代策略进行计数,要么如前所述,基于 FSC 测量,要么如本文所述,基于 SSC 检测。标准化方案的关键要素是使用不同大小的参考微球。本研究旨在同时访问 FSC 和 SSC 优化平台。提出了一个约 0.17-0.6µm eq.(µm 等效值)的新范围,用于替代基于 SSC 的 MP 门,该门产生与先前提出的基于 0.3-1µm eq. 的 FSC 门相同的 PMP 计数。这两种等效的标准化选项协调了 SSC-和 FCS-触发仪器之间固有的不同散射行为,并为未来的多中心研究提供了机会。

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