Extracellular vesicles (EVs) are submicrometer‐sized biological vesicles released by all cells, and can be found in all body fluids or harvested from cell culture supernatants. It is nowadays widely accepted that EVs can serve as vesicular messengers in various physiological and pathophysiological contexts. Over the last 10–15 years, the EV research community has grown almost exponentially and the field has attracted a lot of attention following numerous studies connecting EVs to therapeutic approaches such as vaccination, antitumor therapy, immunomodulation and drug delivery. However, the exact function or mode of action of EVs in most biological contexts is still poorly understood (1-3). Since the protein composition and cargo of EVs is assumed to resemble the cell releasing them, EVs also have come into focus as potential diagnostic biomarkers.

Historically, different EV types have been described mainly based on the cellular pathway they are thought to have originated from. Exosomes, which are derived from the cell's endocytic system and released when multivesicular bodies fuse with the plasma membrane, are one class of EVs that are formed within the cell; however, a lack of specific markers that can be used to resolve exosomes from other EVs of intracellular origin or from similar sized vesicles that can bud directly from the cell surface has called into question many of the compositional and functional characteristics previously assigned to this particular EV subset. Similarly, microvesicles (or ectosomes), which bud from the cell surface, have historically been considered to be larger and bearing distinct cargo, but it is now realized that as for EVs of intracellular origin, these can be produced by several different mechanisms and can have a wide range of sizes that overlap considerably with the so‐called small EVs that are formed internally. Apoptotic bodies, a poorly characterized and defined class of particles, may also be found in cell culture supernatants, but, as for the other EV types, there are no specific markers that allow these to be unambiguously defined. The increasing appreciation for the heterogeneity of EVs, and obvious inadequacy of the traditional terminology, has led to the realization that single vesicle analysis of molecular cargo is required to effectively classify and identify EV subsets and the features that are responsible for their functional effects.

Due to this complexity and their small diameter, it is challenging to accurately measure individual EVs, and to quantify basic parameters such as diameter, size, and concentration of EVs in a sample of interest. While resistive pulse spectroscopy and nanoparticle tracking analysis can detect single EVs and estimate their size, they cannot effectively measure EV molecular cargo. Flow cytometry is widely used to quantify and distinguish cellular subpopulations in highly heterogeneous samples, but conventional flow cytometry instruments and assay approaches struggle to measure EVs and other submicrometer particles. The amount of scattered light or emitted fluorescence from an EV is orders of magnitudes lower than that from a cell, and most conventional flow cytometers are not sensitive enough to detect EVs smaller than 500 nm in diameter (4).

Recently, new flow cytometers with high sensitivity photodetectors have become available that offer significantly improved performance compared with the older generation of PMT‐based instruments. However, a more sensitive instrument does not necessarily translate into a more sensitive measurement, and deficiencies in assay design, especially with respect to the necessary controls and calibration, have limited the interpretation and reproducibility of single EV flow cytometry measurements. To address those challenges, an international Working Group of researchers active in the International Society of Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and/or International Society for Thrombosis and Haemostasis (ISTH) Vascular Biology Scientific Standardization Committee, have developed a framework for reporting EV flow cytometry methods that complements and extends the existing MIFlowCyt (5) and MISEV (6) guidelines. The resulting MIFlowCyt‐EV framework, published earlier this year in the Journal of Extracellular Vesicles (7), describes the essential controls and calibrations necessary for interpretable and reproducible results. This Working Group is actively developing educational resources to support the EV flow cytometry field (refer to http://evflowcytometry.org for details and future updates).

In addition to such consensus guidelines, it will be essential for the field to develop and qualify standards and reference materials being more suitable for EV research and the dim signals derived from single EVs. While bead standards used in cellular flow cytometry often scatter more light or emit more fluorescence than tens to hundreds of EVs, the field is working on beads and synthetic nanoparticles being more suitable in terms of size and material (8). In this context, a few recent studies have explored the use of biological reference materials by creating EVs or virus‐like particles expressing fluorescent reporter proteins through engineering of respective producer cells (9-11). The idea is that such biological reference materials may have the potential to closely resemble signal intensities and other biophysical parameters such as diameter and density obtained from EVs. Especially within the last few years, the EV flow cytometry field also started to implement fluorescence and more recently scatter calibration to improve accuracy and comparability of reported data.

This special issue on Flow Cytometry of Extracellular Vesicles provides a snapshot of some of the current work in the field. Clearly, the distinguishing feature of flow cytometry is its ability to make quantitative multiparameter immunofluorescence measurements of individual particles that, if applied to EVs, would enable the identification of distinct compositional and functional subpopulations. However, because small EVs bear few antigens, EV immunofluorescence requires careful optimization and calibration of a sort not commonly encountered in the published literature. Tertel and colleagues (page 602‐609) provide a clear example of the optimization and reporting of EV immunofluorescence as applied to an imaging flow cytometer (12). By evaluating the key staining parameters of concentration, temperature, and time, and performing appropriate calibration to report fluorescence in absolute units of MESF (molecules of equivalent soluble fluorochrome), they demonstrate how to produce quantitative and reproducible EV immunofluorescence data.

Light scatter is a particularly challenging problem in flow cytometry of EVs. While light scatter signals are commonly used to trigger detection of cells, the small size and low refractive indices of EVs produce very dim signals that can be hard to discriminate from various sources of background. While fluorescence may provide more specific means to detect EVs (13-15), light scatter can, if interpreted correctly, provide additional information on physical properties such as size and refractive index. While most experienced cytometrists understand that polystyrene or silica beads will scatter much more light than a comparably sized vesicle, owing to their higher refractive indices, it is possible to use measurements of beads, in conjunction with Mie theory‐based light scattering models, to estimate EV diameter from light scatter. Welsh et al. (page 569‐581) present FCM_PASS, a software utility for light scatter calibration of EV flow cytometry data postacquisition based upon Mie theory (16). By using a core‐shell model of vesicles that accounts for the predicted change in refractive index with EV size, as well as consideration of collection angle and illumination wavelength, the software will provide an instrument‐specific calibration that allows estimation of EV diameter from scatter intensity. Because EV refractive index can be different between different vesicle types, the software provides estimates assuming high, medium, and low refractive index values. FCM_PASS also performs fluorescence calibration (using commercially available MESF standards) and writes the calibrated data to a new fcs format file, making this software a one‐stop solution for EV calibration.

In a related publication, Welsh and Tang (page 592‐601) illustrate proper fluorescence and light scatter calibration in the context of characterizing an enveloped virus, which can be considered a special class of EVs (17). In this case, the virus has been engineered to express a green fluorescent protein and thus is endogenously fluorescent. In context of a proof‐of‐concept study carried out as a resource for use at a CYTO2019 workshop, the authors demonstrate its characterization in terms of fluorescence and light scatter calibration and show that calibrated data can be compared between different flow cytometry platforms.

While interpreting light scatter data is a key challenge, so is light scatter measurement. Because light scatter signals from EVs are so dim, background from various sources can overwhelm the system and compromise measurements. However, careful optimization of illumination and light collection can reduce this background and improve signal to noise. While conventional wisdom suggests that side scatter is more appropriate for light scatter measurements of small particles, the angular dependence of light scatter may be useful, especially for larger EVs, and forward scatter measurements may provide additional information. Arkesteijn and colleagues (page 610‐619) report on the optimization of forward angle light scatter (FALS) for measurement of small particles (18). They evaluated the effects of different blocker bars and pinhole diameters on the relative signal to noise. By improving the rejection of the illuminating laser and out of focus light, they report significant reduction in background signals and improvements in sensitivity. The ability to make effective measurements of both forward and side scatter may improve estimates EV size and refractive index using flow cytometry data.

De Rond et al (page 582‐591) also address light scatter and describe an approach to characterize and improve scatter sensitivity in both the forward and orthogonal directions on a modified conventional flow cytometer (19). The authors derived quantitative performance metrics based on rigorous characterization of the background and expressed as a separation index. They evaluated obscuration bars and pinhole diameters, as well as sample stream diameter, and report significant improvements in both forward and side scatter performance, pointing the way for instrument designs that might one day be part of a built‐for‐purpose vesicle flow cytometer.

In summary, single EVs still represent an exciting frontier for flow cytometry, with a clear need for more sensitive instruments, improved standards, and validated assays. The development of guidelines to standardize the reporting of methods and results will enable apple‐to‐apple comparison of results, allowing data‐driven instrument and assay performance in absolute terms. This will be a boon to researchers, who will be able to interpret and reproduce data more readily, and to instrument and reagent manufacturers, who will be able to rationally design new products that will have useful and demonstrable performance improvements. The result will be quantitative measurements of EVs that will let us understand their composition, predict their functions, and use these to diagnose, treat, and prevent human disease.

细胞外囊泡 (EVs) 是所有细胞释放的亚微米大小的生物囊泡，可以在所有体液中找到或从细胞培养上清液中收集。现在人们普遍认为 EV 可以在各种生理和病理生理环境中充当囊泡信使。在过去的 10-15 年中，EV 研究界几乎呈指数增长，并且在将 EV 与疫苗接种、抗肿瘤治疗、免疫调节和药物递送等治疗方法联系起来的大量研究之后，该领域引起了很多关注。然而，电动汽车在大多数生物学环境中的确切功能或作用方式仍知之甚少（1-3）。由于 EV 的蛋白质组成和货物被认为类似于释放它们的细胞，因此 EV 也已成为潜在的诊断生物标志物的焦点。

从历史上看，不同的 EV 类型主要是根据它们被认为起源的细胞途径来描述的。来自细胞内吞系统并在多泡体与质膜融合时释放的外泌体是在细胞内形成的一类 EV；然而，由于缺乏可用于从细胞内来源的其他 EV 或可直接从细胞表面出芽的类似大小的囊泡中分离外泌体的特定标记物，这对先前分配给该特定 EV 的许多组成和功能特征提出了质疑子集。类似地，从细胞表面萌芽的微泡（或外泌体）在历史上被认为更大并且承载着不同的货物，但现在人们意识到，对于细胞内来源的 EV，这些可以通过几种不同的机制产生，并且可以具有广泛的尺寸范围，与内部形成的所谓的小型电动汽车有很大的重叠。细胞培养上清液中也可能发现凋亡小体是一种特征和定义不佳的颗粒，但对于其他 EV 类型，没有特定的标记可以明确定义它们。对 EV 异质性的日益重视，以及传统术语的明显不足，导致人们认识到需要对分子货物进行单囊泡分析才能有效地分类和识别 EV 子集以及对其功能影响负责的特征。

由于这种复杂性和它们的小直径，准确测量单个 EV 并量化感兴趣样本中 EV 的直径、大小和浓度等基本参数具有挑战性。虽然电阻脉冲光谱和纳米粒子跟踪分析可以检测单个 EV 并估计它们的大小，但它们不能有效地测量 EV 分子货物。流式细胞术广泛用于量化和区分高度异质样本中的细胞亚群，但传统的流式细胞仪仪器和分析方法难以测量 EV 和其他亚微米颗粒。EV 的散射光或发出的荧光量比细胞低几个数量级，大多数传统流式细胞仪的灵敏度不足以检测直径小于 500 nm 的 EV。4）。

最近，具有高灵敏度光电探测器的新型流式细胞仪已经面世，与老一代基于 PMT 的仪器相比，其性能显着提高。然而，更灵敏的仪器并不一定会转化为更灵敏的测量，而且检测设计中的缺陷，特别是在必要的控制和校准方面，限制了单 EV 流式细胞术测量的解释和可重复性。为了应对这些挑战，一个活跃于国际细胞外囊泡学会 (ISEV)、国际细胞计量学促进会 (ISAC) 和/或国际血栓与止血学会 (ISTH) 血管生物学科学标准化委员会的国际研究人员工作组,5 ) 和 MISEV ( 6 ) 指南。由此产生的 MIFlowCyt-EV 框架于今年早些时候发表在细胞外囊泡杂志( 7 ) 上，描述了可解释和可重复的结果所需的基本控制和校准。该工作组正在积极开发教育资源以支持 EV 流式细胞术领域（有关详细信息和未来更新，请参阅 http://evflowcytometry.org）。

除了此类共识指南之外，该领域还必须制定和验证更适合 EV 研究和来自单个 EV 的暗淡信号的标准和参考材料。虽然细胞流式细胞术中使用的珠子标准通常比数十到数百个 EV 散射更多的光或发出更多的荧光，但该领域正在研究在尺寸和材料方面更合适的珠子和合成纳米粒子 ( 8 )。在这种情况下，最近的一些研究探索了生物参考材料的使用，通过改造各自的生产细胞来创建表达荧光报告蛋白的 EV 或病毒样颗粒（9-11）。这个想法是，这种生物参考材料可能与信号强度和其他生物物理参数（如从 EV 获得的直径和密度）非常相似。特别是在过去几年中，EV 流式细胞仪领域也开始实施荧光和最近的散射校准，以提高报告数据的准确性和可比性。

本期关于细胞外囊泡流式细胞术的特刊简要介绍了该领域当前的一些工作。显然，流式细胞术的显着特征是它能够对单个粒子进行定量多参数免疫荧光测量，如果应用于 EV，将能够识别不同的组成和功能亚群。然而，由于小型 EV 携带的抗原很少，因此 EV 免疫荧光需要仔细优化和校准，这是一种在已发表的文献中不常见的类型。Tertel 及其同事（第 602-609 页）提供了一个清晰的例子，说明了 EV 免疫荧光应用于成像流式细胞仪的优化和报告（12）。通过评估浓度、温度和时间的关键染色参数，并执行适当的校准以报告绝对单位 MESF（等效可溶性荧光染料的分子）的荧光，他们展示了如何产生定量和可重复的 EV 免疫荧光数据。

光散射是 EV 流式细胞术中一个特别具有挑战性的问题。虽然光散射信号通常用于触发细胞检测，但 EV 的小尺寸和低折射率会产生非常微弱的信号，很难将其与各种背景来源区分开来。虽然荧光可以提供更具体的方法来检测 EV ( 13-15)，如果正确解释，光散射可以提供有关物理特性的附加信息，例如大小和折射率。虽然大多数有经验的细胞计数仪都知道聚苯乙烯或二氧化硅珠比同等大小的囊泡散射更多的光，但由于它们的折射率较高，因此可以使用珠的测量值，结合基于 Mie 理论的光散射模型，来估计来自光散射的 EV 直径。威尔士等。（第 569-581 页）目前 FCM _PASS是一种基于 Mie 理论的用于 EV 流式细胞术数据采集后光散射校准的软件实用程序（16）。通过使用囊泡的核壳模型，该模型解释了折射率随 EV 大小的预测变化，以及收集角度和照明波长的考虑，该软件将提供特定于仪器的校准，允许从散射估计 EV 直径强度。由于 EV 折射率在不同囊泡类型之间可能不同，因此软件提供了假设高、中和低折射率值的估计值。FCM _PASS还执行荧光校准（使用市售 MESF 标准）并将校准数据写入新的 fcs 格式文件，使该软件成为 EV 校准的一站式解决方案。

在相关出版物中，Welsh 和 Tang（第 592-601 页）在表征包膜病毒的背景下说明了适当的荧光和光散射校准，包膜病毒可以被认为是一类特殊的 EV ( 17 )。在这种情况下，病毒已被设计为表达绿色荧光蛋白，因此具有内源性荧光。在作为 CYTO2019 研讨会上使用的资源进行的概念验证研究的背景下，作者展示了其在荧光和光散射校准方面的表征，并表明校准数据可以在不同的流式细胞仪平台之间进行比较。

解释光散射数据是一项关键挑战，光散射测量也是如此。由于来自 EV 的光散射信号非常暗淡，来自各种来源的背景可能会淹没系统并影响测量。然而，仔细优化照明和光收集可以减少这种背景并提高信噪比。虽然传统观点认为侧向散射更适合小颗粒的光散射测量，但光散射的角度依赖性可能有用，尤其是对于较大的 EV，前向散射测量可能提供额外的信息。Arkesteijn 及其同事（第 610-619 页）报告了用于测量小颗粒的前向角光散射 (FALS) 的优化 ( 18）。他们评估了不同阻隔条和针孔直径对相对信噪比的影响。通过改善对照明激光和散焦光的抑制，他们报告了背景信号的显着减少和灵敏度的提高。使用流式细胞术数据对前向和侧向散射进行有效测量的能力可以提高对 EV 大小和折射率的估计。

De Rond 等人（第 582-591 页）还解决了光散射问题，并描述了一种在改进的传统流式细胞仪上表征和提高前向和正交方向散射灵敏度的方法 ( 19 )。作者根据严格的背景表征得出定量性能指标，并表示为分离指数。他们评估了遮光条和针孔直径，以及样品流直径，并报告了前向和侧向散射性能的显着改进，为有朝一日可能成为专用囊泡流式细胞仪一部分的仪器设计指明了道路。

总之，单个 EV 仍然是流式细胞术的一个令人兴奋的前沿领域，显然需要更灵敏的仪器、改进的标准和经过验证的检测。制定标准化方法和结果报告的指南将使结果的苹果与苹果之间的比较成为可能，从而允许数据驱动的仪器和测定性能的绝对值。这对研究人员来说是一个福音，他们将能够更容易地解释和再现数据，以及仪器和试剂制造商，他们将能够合理地设计具有有用和可证明的性能改进的新产品。结果将是电动汽车的定量测量，这将使我们了解它们的组成，预测它们的功能，并使用这些来诊断、治疗和预防人类疾病。