Summary
Background. Extracellular vesicles (EV) are secreted by various types of cells into body fluids and play a crucial role in intracellular communication. They are capable of carrying biologically active molecules and are involved in numerous physiological and pathological processes. Due to their widespread presence in the body and diverse functions, extracellular vesicles are considered promising non-invasive biomarkers. However, research on these vesicles is often hindered by methodological challenges – varying and non-standardized pre-analytical factors and isolation techniques result in significant variability across studies. To address these challenges, a systematic evaluation of extracellular vesicles isolation methods is necessary to determine which factors most significantly influence result variation. Meta-analysis enables the integration of data from multiple studies and provides a statistical assessment of the variables affecting outcomes, thereby contributing to the development of methodological recommendations required for research standardization. This study aims to identify the key factors contributing to variability in extracellular vesicles concentration estimates.
Aim. To evaluate the dependence of extracellular vesicle concentration on experimental factors using a meta-analysis approach.
Methods. The meta-analysis was conducted following PRISMA guideline recommendations. A systematic search for relevant scientific studies was performed across PubMed, Web of Science and Cochrane library databases using targeted keywords. Studies were included in the meta-analysis based on predefined inclusion and exclusion criteria. Random-effects models were applied and visualized using Forest plots. Publication bias was assessed using a Funnel plot and Egger’s test, while sensitivity was evaluated using the leave-one-out method. Statistical analysis was carried out using R and RStudio software.
Results. The analysis revealed a high degree of heterogeneity; no publication bias was detected, and sensitivity analysis confirmed that the results were not overly influenced by individual studies. The calculated overall effect size using a random-effects model was positive – 0.48 with a 95% confidence interval [0.30; 0.65]. Subgroup analysis showed that the most influential factors on extracellular vesicles concentration estimates were the isolation methods, the use of EDTA as a tube additive, and sample storage at –80°C.
Conclusions. The meta-analysis and interpretation of results revealed a consistent overall positive effect across studies, with the isolation method being the most significant factor influencing extracellular vesicle concentration estimates.
