Longitudinal changes in the phenotypic profile of circulating extracellular vesicles in healthy individuals

Extracellular vesicles (EVs) are emerging as valuable biomarkers in clinical diagnostics. Yet, the natural biological variation in EV concentration and phenotype among healthy individuals and across longitudinal time points remains insufficiently characterized, complicating interpretation and reproducibility in biomarker studies.

To assess longitudinal (day-to-day and week-to-week) intra-individual and inter-individual variation in circulating EVs from healthy donors using complementary analytical platforms.

Blood samples were collected longitudinally from four healthy female donors over 5 consecutive days and across 6 weeks. EVs were analyzed using nanoparticle tracking analysis (NTA), high-resolution flow cytometry (hFCM) targeting 3 surface markers, and the EV Array targeting 23 surface markers. Blood cell counts were also measured to explore correlations with EV profiles.

Significant longitudinal intra- and inter-individual variation was observed in EV concentrations and marker expression. CD9-positive EVs were consistently the most abundant across both antibody-based methods, while CD63^-and CD81-positive EVs showed lower and more variable expression. Long-term variation exceeded short-term variation.

This exploratory study underscores the dynamic nature of EV profiles in healthy individuals and highlights the need for standardized reference ranges and harmonized methodologies. Without accounting for biological and technical variability, EV-based biomarker studies risk misinterpretation and compromised reproducibility.