Institute of Experimental Immunology, University of Zurich, Zurich, 8057, Switzerland.
NanoFCM, ltd., D6 Thane Rd, Nottingham, NG90 6BH, UK.
Adv Healthc Mater. 2023 Dec;12(32):e2301706. doi: 10.1002/adhm.202301706. Epub 2023 Oct 25.
Extracellular vesicles (EVs) are increasingly being analyzed by flow cytometry. Yet their minuscule size and low refractive index cause the scatter intensity of most EVs to fall below the detection limit of most flow cytometers. A new class of devices, known as spectral flow analyzers, are becoming standards in cell phenotyping studies, largely due to their unique capacity to detect a vast panel of markers with higher sensitivity for light scatter detection. Another class of devices, known as nano-analyzers, provides high-resolution detection of sub-micron-sized particles. Here, the EV phenotyping performance between the Aurora (Cytek) spectral cell analyzer and the NanoFCM (nFCM) nanoflow analyzer are compared. These two devices are specifically chosen given their lead in becoming gold standards in their respective fields. Immune cell-derived EVs remain poorly characterized despite their clinical potential. Therefore, B- and T-cell line-derived EVs and donor-matched human biofluid-derived EVs from plasma, urine, and saliva are used in combination with a panel of established immune markers for this comparative study. A comparative evaluation of both cytometry platforms is performed, discussing their potential and suitability for different applications. It is found that nFCM can accurately i) analyze small EVs (40-200 nm) matching the size accuracy of electron microscopy; ii) measure the concentration of a single EV particle per volume; iii) identify underrepresented EV marker subsets; and iv) provide co-localization of EV surface markers. It can also be shown that human sample biofluids have unique EV marker signatures that can have future clinical relevance. Finally, nFCM and Aurora have their unique strength, preferred fashion of data acquisition, and visualization to fit different research interests.
细胞外囊泡 (EVs) 越来越多地通过流式细胞术进行分析。然而，它们的微小尺寸和低折射率导致大多数 EV 的散射强度低于大多数流式细胞仪的检测极限。一类新型设备，称为光谱流式分析仪，在细胞表型研究中已成为标准，主要是因为它们具有独特的能力，可以用更高的光散射检测灵敏度来检测大量的标记物。另一类设备，称为纳米分析仪，可提供亚微米大小颗粒的高分辨率检测。在此，比较了 Aurora（Cytek）光谱细胞分析仪和 NanoFCM（nFCM）纳流分析仪之间的 EV 表型性能。选择这两种设备是因为它们在各自领域中成为黄金标准的领先地位。尽管免疫细胞衍生的 EV 具有临床潜力，但它们的特征仍然很差。因此，使用 B 细胞和 T 细胞系衍生的 EV 以及与血浆、尿液和唾液来源的供体匹配的人类生物流体衍生的 EV，结合一组已建立的免疫标记物进行这项比较研究。对这两个细胞仪平台进行了比较评估，讨论了它们在不同应用中的潜力和适用性。结果发现，nFCM 可以准确地：i）分析与电子显微镜的尺寸精度匹配的小 EV（40-200nm）；ii）测量每个体积的单个 EV 颗粒的浓度；iii）识别代表性不足的 EV 标记物亚群；iv）提供 EV 表面标记物的共定位。还可以表明，人类样本生物流体具有独特的 EV 标记特征，将来可能具有临床相关性。最后，nFCM 和 Aurora 具有独特的优势、首选的数据采集和可视化方式，以适应不同的研究兴趣。
