Department of Urology, Mayo Clinic, Guggenheim 4-97, 200 1st Street SW, Rochester, MN, 55901, USA.
Department of Biomedical Engineering and Physics, Amsterdam University Medical Center, Amsterdam, The Netherlands.
Nanoscale. 2022 Jul 14;14(27):9781-9795. doi: 10.1039/d2nr01160c.
Extracellular vesicles (EVs) are microscopic particles released naturally in biofluids by all cell types. Since EVs inherits genomic and proteomic patterns from the cell of origin, they are emerging as promising liquid biomarkers for human diseases. Flow cytometry is a popular method that is able to detect, characterize and determine the concentration of EVs with minimal sample preparation. However, the limited awareness of the scientific community to utilize standardization and calibration methods of flow cytometers is an important roadblock for data reproducibility and inter-laboratory comparison. A significant collaborative effort by the Extracellular Vesicle Flow Cytometry Working Group has led to the development of guidelines and best practices for using flow cytometry and reporting data in a way to improve rigor and reproducibility in EV research. At first look, standardization and calibration of flow cytometry for EV detection may seem burdensome and technically challenging for non-academic laboratories with limited technical training and knowledge in EV flow cytometry. In this study, we build on prior research efforts and provide a systematic approach to evaluate the performance of a high sensitivity flow cytometer (herein Apogee A60-Micro Plus) and fine-tune settings to improve detection sensitivity for EVs. We performed calibration of our flow cytometer to generate data with comparable units (nanometers, MESF). Finally, we applied our optimized protocol to measure the concentrations of prostate-derived EVs in healthy individuals and prostate cancer patients. In conclusion, our proof-of-feasibility study can serve as a scientific and technical framework for other groups motivated in using flow cytometry for EV research.
细胞外囊泡 (EVs) 是所有细胞类型在生物体液中自然释放的微小颗粒。由于 EVs 从起源细胞继承了基因组和蛋白质组模式，因此它们作为人类疾病有前途的液体生物标志物而出现。流式细胞术是一种流行的方法，能够在最小化样品制备的情况下检测、表征和确定 EV 的浓度。然而，科学界对利用流式细胞仪的标准化和校准方法的认识有限，是数据重现性和实验室间比较的重要障碍。细胞外囊泡流式细胞术工作组的重要协作努力导致了使用流式细胞术的指南和最佳实践的制定，以及以提高 EV 研究严谨性和重现性的方式报告数据。乍一看，对于非学术实验室来说，EV 检测的流式细胞术的标准化和校准可能看起来繁琐且具有技术挑战性，因为它们在 EV 流式细胞术方面的技术培训和知识有限。在这项研究中，我们基于先前的研究工作，提供了一种系统的方法来评估高灵敏度流式细胞仪（此处为 Apogee A60-Micro Plus）的性能，并调整设置以提高 EV 的检测灵敏度。我们对我们的流式细胞仪进行了校准，以生成具有可比单位（纳米、MESF）的数据。最后，我们应用我们优化的方案来测量健康个体和前列腺癌患者中前列腺衍生 EV 的浓度。总之，我们的可行性研究可以为其他热衷于使用流式细胞术进行 EV 研究的团体提供科学和技术框架。
