高灵敏度流式细胞仪已开发用于单个细胞外囊泡 (EV) 的多参数表征，但性能因仪器和校准方法而异。在这里，我们通过三台高灵敏度流式细胞仪、两台商用仪器 CytoFLEX/CellStream 和一台定制单分子流式细胞仪 (SMFC) 比较了源自人结直肠癌 (DiFi) 细胞的相同（分割）EV 样品的表征。 DiFi EV 使用膜染料 di-8-ANEPPS 和 PE 缀合的抗 EGFR 或抗四跨膜蛋白 (CD9/CD63/CD81) 抗体进行染色，以估计 EV 大小和表面蛋白拷贝数。基于使用交叉校准、硬染色珠进行校准的免疫荧光和囊泡大小的检测限 (LOD)，CytoFLEX 为 ∼10 PE/∼80 nm EV 直径，CellStream 为 ∼10 PE/∼67 nm。对于 SMFC，免疫荧光的 LOD 为 1 PE，尺寸≤ 35 nm。每个系统检测到的 EV 数量（di-8-ANEPPS /PE 颗粒）根据系统的 LOD 差异很大；例如，CellStream/CytoFLEX 分别仅检测到 SMFC 检测到的四跨膜蛋白标记 EV 的 5.7% 和 1.5%，并且使用 Super-CytoFLEX 进行测量和验证，CellStream/CytoFLEX 的中值 EV 直径和抗体拷贝数比 SMFC 大得多。分辨率/单分子 TIRF 显微镜。为了获得代表所有三个平台分析的常见 EV 群体的数据集，我们筛选出了低于 CytoFLEX LOD 的 EV 的 SMFC 和 CellStream 测量值（由珠校准 (10 PE/80 nm) 确定）。使用此过滤数据集的平台间一致性明显优于未过滤数据集，但通过应用使用 SMFC 数据进行阈值分析确定的更高截止值 (21 PE/120 nm)，可以获得结果之间更好的一致性。结果证明了指定 LOD 来定义 EV 群体分析对 EV 流式细胞术研究中仪器间重现性的影响，以及 SMFC 数据阈值分析为其他流式细胞仪提供半定量 LOD 值的效用。© 2024 作者（s）。 《Journal of Extracellular Vesicles》由 Wiley periodicals LLC 代表国际细胞外囊泡学会出版。

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.