Comparison of Methods for Concentration Assessment of Extracellular Vesicles Isolated from Different Biological Fluids

Accurate quantification of extracellular vesicles (EVs) remains a significant challenge in biomedical research. Although various analytical methods have been developed, their reliability is often limited by the presence of non-vesicular nanoparticles and biological contaminants, particularly in biological fluids. Moreover, for some sources of EVs, such as uterine aspirates and gastric juice, quantitative evaluation of EVs has not been investigated. The aim of the study is to perform comparative analysis of three EV quantification methods: total protein content measurement, nanoparticle tracking analysis (NTA), and esterase activity assessment using commercial FluoroCet exosome quantitation kit in EVs isolated from various biological fluids: blood plasma, ascitic fluid, uterine aspirates, gastric juice, and medium conditioned by ovarian and non-small cell lung cancer cells. All three methods demonstrated strong correlation for the EV samples derived from the conditioned medium, supporting their validity for <i>in vitro</i> EV quantification in highly purified samples. In contrast, blood plasma, ascitic fluid, and uterine aspirates exhibited discrepancies between the methods, likely attributable to the presence of non-vesicular nanoparticles. Notably, the EVs from gastric juice demonstrated strong correlation between the protein content and esterase activity, indicating prevalence of the vesicle-associated proteins and, potentially, unique EV composition in this fluid. The findings underscore the necessity for multifactorial approach to EV quantification, taking into account factors such as sample origin and limitations inherent to the specific method employed. These results may serve as a basis for the development of standardized protocols for EV quantification, which is particularly relevant for clinical sample analysis.