Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?
Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?
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DOI：
10.1111/j.1538-7836.2008.03200.x
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发表时间：
2009-01-01
影响因子：
10.4
通讯作者：
Dignat-George, F.
中科院分区：
文献类型：
--
作者：
Robert, S.;Poncelet, P.;Dignat-George, F.
背景：血小板微粒（PMPs）已被证明可用于识别具有血管风险的患者。然而，PMP计数，这是目前通过流式细胞术（FCM），需要标准化。目的：目的是（i）使用尺寸校准的荧光珠在常规仪器（Cytomics FC 500）上对PMP计数的FCM设置进行标准化;（ii）确定仪器内和仪器间重现性;（iii）在健康受试者中建立PMP值。研究方法：使用混合物的大小校准的荧光珠（0.5和0.9 μ m）在一个固定的数值比（Megamix），我们门控PMP在一个有限的大小窗口。为了测试仪器内和仪器间的重现性，使用膜联蛋白V和CD 41共表达来计数4个月内相同无血小板血浆（PFP）的冷冻等分试样中的PMP和来自10名健康受试者的PFP中的PMP。结果如下：这种校准珠策略允许对基于FCM的微粒方案进行完全长期控制，并且随时间推移可重复PMP计数[变异系数（CV）< 10%]。使用Megamix作为稳定的模板，最佳设置很容易从一台仪器转移到另一台仪器。使用三种仪器获得相似的PMP计数（CV < 12%）。采用这种标准化FCM方案，在健康受试者（n = 60）中建立了PMP值，其中女性的PMP水平显著高于男性[中位数（第一四分位数至第三四分位数）：1775 mu L-1（1014-3039 mu L-1）vs. 656 mu L-1（407-962 mu L-1）]。结论：本策略为PMP计数标准化提供了一种新的选择，从而为多中心研究开辟了道路。
Background: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. Objectives: The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and interinstrument reproducibility; and (iii) to establish PMP values in healthy subjects. Methods: Using a blend of size-calibrated fluorescent beads (0.5 and 0.9 mu m) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. Results: This calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 mu L-1 (1014-3039 mu L-1) vs. 656 mu L-1 (407-962 mu L-1)]. Conclusions: The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.