The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analyzing EVs due to it being a high‐throughput, multiparametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardization exists for EV flow cytometry, which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard to interpret, compare, and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardization, utilizing flow cytometer postacquisition analysis software (FCM_PASS). FCM_PASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter‐diameter curve and scatter‐refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardized units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCM_PASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.

中文翻译：

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FCMPASS 软件有助于细胞外囊泡光散射标准化。

细胞外囊泡 (EV) 的研究是一个快速发展的领域，因为它们在包括诊断、预后、治疗诊断和治疗在内的许多临床医学领域具有巨大潜力。流式细胞术是目前最流行的分析 EV 的方法之一，因为它是一种高通量、多参数的技术，在大多数研究实验室中都很容易获得。尽管用途广泛，但很少有商用流式细胞仪专门设计用于检测 EV。许多用于 EV 分析的流式细胞仪都在其检测限下工作，无法检测到大多数 EV。目前，EV 流式细胞仪几乎没有标准化，这是一个问题，因为流式细胞仪在收集来自被询问粒子的散射光或荧光的方式上存在很大差异。这使得已发表的研究难以解释、比较，在某些情况下，甚至无法复制。在这里，我们展示了一种流式细胞仪光散射标准化方法，利用流式细胞仪采集后分析软件 (FCM_通过）。FCM _PASS建立在 Mie 理论的基础上，通过分析已知直径和折射率的珠子或输入收集角度（如果已知），可以逼近流式细胞仪的几何参数。然后，该软件能够创建散射直径曲线和散射折射率曲线，使研究人员能够将散射数据和仪器灵敏度转换为标准化单位。此外，通过正确的控制，光散射数据可以转换为直径分布或折射率分布。因此， FCM _PASS提供了一种免费且符合人体工程学的方法，使用流式细胞仪标准化和进一步扩展 EV 表征。