Methodological and Short-Term Diurnal Variation in Surface and Cargo Proteins in Plasma Extracellular Vesicles

Extracellular vesicles (EVs) are known as potential biomarkers for several diseases; nevertheless, the degree of technical and biological variability is not yet adequately characterized. Because pre-analytical factors such as blood collection time and EV subpopulation could confound biomarker studies, we performed a pilot study systematically quantifying methodological and biological variability including EV-Array (surface proteins), and proteome characterization of cargo. Plasma samples from six healthy adults were collected at two time points (morning and afternoon) and plasma was analyzed with EV-Array, and isolated EVs were analyzed using nanoparticle tracking analysis (NTA), and label-free mass spectrometry (LC-MS/MS). Methodological repeatability was high for NTA particle size (3.3% CV) and LC-MS (8.2% CV), and lower for EV-Array surface markers (22.6% CV). Variations between samples were reasonable for NTA-size, EV-Array and LC-MS/MS (5-21%) and substantially lower than between-subject variation. No evidence of systemic morning-afternoon shifts in particle size and concentration or EV cargo was observed, although small effects cannot be excluded. The same was true for most surface markers, but minor but statistically significant reductions in a few specific surface markers occurred in afternoon EV-Array samples. In this pilot we therefore do not observe any major systemic diurnal bias in healthy individuals in samples collected a.m. vs. p.m. Despite the small sample size, this study underscores the importance of accounting for individual variability and methodological standardization when designing EV-based biomarker research.