Flow cytometers have been utilized for the analysis of submicron‐sized particles since the late 1970s. Initially, virus analyses preceded extracellular vesicle (EV), which began in the 1990s. Despite decades of documented use, the lack of standardization in data reporting has resulted in a growing body of literature that cannot be easily interpreted, validated, or reproduced. This has made it difficult for objective assessments of both assays and instruments, in‐turn leading to significant hindrances in scientific progress, specifically in the study of EVs, where the phenotypic analysis of these submicron‐sized vesicles is becoming common‐place in every biomedical field. Methods for fluorescence and light scatter standardization are well established and the reagents to perform these analyses are commercially available. However, fluorescence and light scatter calibration are not widely adopted by the small particle community as methods to standardize flow cytometry (FCM) data. In this proof‐of‐concept study carried out as a resource for use at the CYTO2019 workshop, we demonstrate for the first‐time simultaneous fluorescence and light scatter calibration of small particle data to show the ease and feasibility of this method for standardized FCM data reporting. This data was acquired using standard configuration commercial flow cytometers, with commercially available materials, published methods, and freely available software tools. We show that application of light scatter, fluorescence, and concentration calibration can result in highly concordant data between FCM platforms independent of instrument collection angle, gain/voltage settings, and flow rate; thus, providing a means of cross comparison in standard units. © 2020 International Society for Advancement of Cytometry

中文翻译：

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荧光和光散射校准允许跨不同流式细胞术平台和检测器设置以标准单位比较小颗粒数据。

自 1970 年代后期以来，流式细胞仪已被用于分析亚微米级颗粒。最初，病毒分析先于 1990 年代开始的细胞外囊泡 (EV)。尽管已有数十年的记录使用，但数据报告缺乏标准化导致越来越多的文献无法轻松解释、验证或复制。这使得对化验和仪器的客观评估变得困难，进而导致科学进步的重大障碍，特别是在电动汽车的研究中，对这些亚微米大小的囊泡的表型分析在每个生物医学领域都变得司空见惯。场地。荧光和光散射标准化的方法已经成熟，进行这些分析的试剂是市售的。然而，荧光和光散射校准并未被小颗粒社区广泛采用作为标准化流式细胞术 (FCM) 数据的方法。在作为 CYTO2019 研讨会的资源使用的概念验证研究中，我们首次展示了小颗粒数据的同时荧光和光散射校准，以展示该方法对标准化 FCM 数据的简便性和可行性报告。这些数据是使用标准配置的商业流式细胞仪获得的，具有商业上可用的材料、公开的方法和免费提供的软件工具。我们表明，光散射、荧光和浓度校准的应用可以在 FCM 平台之间产生高度一致的数据，而与仪器收集角度、增益/电压设置和流速无关；因此，提供一种以标准单位进行交叉比较的方法。© 2020 国际细胞术进步协会