Transfusion-dependent changes in uEV cargo

Aim: Autologous blood transfusions (ABT), especially of stored erythrocyte concentrates (ECs), are suspected to be misused as performance enhancers. The storage of ECs not only increases the release of extracellular vesicles (EVs), but also significantly alters the microRNA profile. Since re-transfused EVs also appear in urine, urinary EVs and their microRNA load could serve as valuable indicators in the challenging detection of ABT. Methods: Thirty healthy and recreationally active males were included and equally divided into three groups. While the control group did not donate or receive any blood components, Group 1 donated once around 500 ml of whole blood. The respective ECs were stored for six weeks until re-infusion. Group 2 donated twice around 500 ml of whole blood at an interval of two weeks. Obtained ECs were stored for six or four weeks, respectively, until parallel re-infusion. Urine samples were taken in three consecutive weeks before whole blood donation to create an individual's baseline, before re-transfusion and several hours and days after re-transfusion. Urine samples were processed and analyzed for general urine health and creatinine levels. Further, urinary EVs were separated by immunoaffinity and characterized using transmission electron microscopy, fluorescence nanoparticle tracking analysis, western blot analysis as well as an established multiplexed bead-based flow cytometry method followed by RNA isolation and in-depth small RNA profiling using next-generation sequencing and comprehensive data analyses. Results: Separated urinary EVs presented with typical morphology of small EVs (<200 nm) and an overall concentration of 8.79 ± 7.00 x1010 particles/g UCrea. Significant increases in urinary EV concentrations were detected up to three days after ABT. Apart from Alix, Syntenin, and TSG101, also CD63, CD9, CD133/1, CD24, CD326, CD81, and CD31 were shown to be highly abundant surface markers of urinary EVs. Impurities or contaminations were absent. Cluster analysis based on surface markers showed a clear separation between control and ABT group. Furthermore, microRNA profiling revealed 13 microRNA differently regulated in the ABT groups but not the control group with miR-155-5p, miR-320b, and miR-6869-5p being most abundant. Conclusion: This proof-of-concept study substantiated the impact of ABT on the microRNA cargo and surface marker of urinary EVs. While the integration of urinary EVs in routine doping tests requires further exploration, this study exposed an important and comprehensive overview of blood-borne EVs that are reflected into urine. Thus, generated data can serve as valuable basis for a variety of subsequent investigations.