Assessing extracellular vesicles in human biofluids using flow-based analyzers

ABSTRACT Extracellular vesicles (EVs) are increasingly being analyzed by flow cytometry. Yet, their miniscule size and low refractive index, causes 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 of detecting 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, we aim to compare the EVs phenotyping performance between the Aurora (Cytek) spectral cell analyzer and the NanoFCM (nFCM) nanoflow analyzer. These two devices were specifically chosen given their lead in becoming gold standards in their respective fields. Immune cell-derived EVs remain poorly characterized despite their clinical potentials. We therefore, used B- and T- cell line-derived EVs and donor-matched human biofluid-derived EVs from serum, urine, and saliva in combination with a panel of established immune markers for this comparative study. A comparative evaluation of both cytometry platforms was performed, discussing their potential and suitability for different applications. We found that nFCM can accurately i) analyze small EVs (40 to 200 nm) matching the size accuracy of electron microscopy; ii) measure concentration of single EV particle per volume; iii) identify underrepresented EV marker subsets; and iv) provide co-localization of EV surface markers. We could also show that human sample biofluids have unique EV marker signatures that could have future clinical relevance.