National University of Sciences and Technology (NUST), Islamabad, Pakistan.
Nishtar Institute of Dentistry Multan, Pakistan.
Micron. 2022 Oct;161:103341. doi: 10.1016/j.micron.2022.103341. Epub 2022 Aug 5.
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.
国际细胞外囊泡学会 (ISEV) 将细胞外囊泡 (EV) 定义为“细胞自然释放的、由脂质双层限定的颗粒，不能复制，即不含有功能性核”。EV 的大小（直径）在30-1000nm 之间，峰值约为100nm。流式细胞术 (FCM) 是分析 EV 最常用的技术。然而，由于其复杂和可变的组成、小尺寸以及生理浓度上的显著差异，EV 的准确表征、程序标准化、仪器校准和结果解释/验证受到了阻碍。在这里，讨论了 FCM 用于 EV 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)EV 方面的缺陷，以及提高仪器灵敏度和分辨率的相应策略。根据校准用参考物质、荧光 (FL)、侧向散射 (SSC) 和前向散射 (FSC) 信号收集光学器件以及流体动力学，描述了整个 FCM 系统的缺点和改进。这项研究可能为现代 FCM 系统分析 EV 时所面临的挑战和前景提供一个全面的参考。
