Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.
Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.
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跨不同流式细胞仪平台的微粒枚举的标准化：多中心协作研讨会的结果。
DOI：
10.1111/jth.13514
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发表时间：
2017-01
期刊：
影响因子：
--
通讯作者：
Lacroix R
中科院分区：
文献类型：
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作者：
Cointe S;Judicone C;Robert S;Mooberry MJ;Poncelet P;Wauben M;Nieuwland R;Key NS;Dignat-George F;Lacroix R
微颗粒是由细胞膜出芽产生的细胞外囊泡，作为新兴生物标志物具有很高的潜力；然而，它们的临床相关性受到方法学枚举问题和缺乏标准化的阻碍。流式细胞术（FCM）仍然是最常用的技术，具有确定单个MPs细胞起源的最佳能力。然而，仪器的表现是不同的，这取决于哪种散射参数（正向散射（FSC）或侧散射（SSC））提供了鉴别亚微米粒子的最佳分辨率。为了克服这一问题，最近提出了一种新的方法，基于两组适用于FSC或SSC优化仪器的选择珠，在不同光学系统的仪器中可重复地枚举血小板衍生的MP计数。目的是在一个国际研讨会上评估这一策略，该研讨会包括44个实验室，共有14种类型的52台细胞仪。采用分辨率能力和背景噪声水平作为标准来鉴定仪器，标准化策略被证明与85%（44/52）的仪器兼容。所有仪器都正确地对两种无血小板血浆样本的PMP水平进行了排序。每个样本的PMP计数的实验室间变异性分别为37%和28%。使用前向或侧向散射光作为相对尺寸参数的仪器之间没有发现差异。尽管仍然存在局限性，但本研究首次证明了基于头部的策略在不同FCM平台上的MP枚举标准化的真正潜力。额外的标准化工作仍然是强制性的，以评估多中心水平的MP临床相关性。
Microparticles are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter, (Forward (FSC) or Side scatter (SSC)), provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) instruments. All instruments correctly ranked the PMP levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side scattered light as the relative sizing parameter. Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MP clinical relevance at a multicenter level.