Investigation of Extracellular Vesicles in Plasma Samples: A Sensitive Method for Analysis

Extracellular vesicles (EV) are particles enclosed by a lipid bilayer and secreted by various cell types under both normal and pathological conditions. EV secretion plays a key role in maintaining normal physiological functions, while aberrant EV-mediated signaling has been associated with numerous diseases. This study aimed to standardize a protocol for the isolation of EVs from human plasma and their characterization by conventional flow cytometry. Peripheral blood (PB) samples from patients with pro-inflammatory conditions were used due to their higher circulating EV levels, facilitating detection and supporting method standardization. Our results demonstrated the feasibility of EV enrichment using serial centrifugation and their detection using conventional flow cytometer, enhancing the accessibility of EV analysis in laboratories with limited infrastructure. However, this approach has inherent limitations, including reduced sensitivity for particles smaller than 200 nm and limited representation of the full heterogeneity of circulating EVs. Therefore, this method should be considered a practical and accessible strategy for EV detection rather than a comprehensive characterization approach, with potential applicability to different clinical conditions in future studies. The analysis of extracellular vesicles (EV) has been proposed as a potential biomarker strategy for various pathological conditions. However, standardized methodologies are required to ensure reliable detection and quantification. Therefore, the aim of this study was to standardize a flow cytometry protocol for the analysis of EV in FACSCanto II, enabling their detection and quantification in human plasma samples.