The International Society for Extracellular Vesicles (ISEV) defines the extracellular vesicle (EV) as "the particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate, i.e. do not contain a functional nucleus”. The size (diameter) of EVs ranges in ~30–1000 nm, with peak population at ~ 100 nm. Flow cytometry (FCM) is the most commonly used technique for analysis of EVs. However, accurate characterization, procedure standardization, instrument calibration and results interpretation/validation of EVs is confounded by their complex and variable composition, small size and substantial differences in physiological concentrations. Here, the challenges to and promises of FCM for characterization of EVs are discussed. Specifically, we systematically reviewed the pitfalls of FCM in the detection of (small) EVs and the corresponding strategies for enhancing the sensitivity and resolution of the instrument. The shortcomings and improvement in the overall FCM system are described in terms of reference material for calibration, the collection optics for fluorescence (FL), side scatter (SSC) and forward scatter (FSC) signals and fluidics. This study may provide a comprehensive reference for a brief overview pertaining to the challenges and promises of a modern FCM system for analysis of EVs.

国际细胞外囊泡协会 (ISEV) 将细胞外囊泡 (EV) 定义为“从细胞中自然释放的由脂质双层分隔且不能复制的颗粒，即不包含功能性细胞核”。大小（直径） EV 的范围在 ~ 30–1000 nm，峰值种群在 ~ 100 nm。流式细胞术 (FCM) 是分析 EV 最常用的技术。然而，准确的表征、程序标准化、EV 的仪器校准和结果解释/验证因其复杂多变的组成、体积小和生理浓度的显着差异而感到困惑。在这里，讨论了 FCM 对电动汽车表征的挑战和前景。具体来说，我们系统地回顾了 FCM 在检测（小型）EV 中的缺陷以及提高仪器灵敏度和分辨率的相应策略。在校准参考材料、荧光 (FL)、侧向散射 (SSC) 和前向散射 (FSC) 信号的收集光学器件和流体学方面描述了整个 FCM 系统的缺点和改进。