Impact of autologous blood transfusions on surface marker and microRNA profiles of urinary extracellular vesicles

<b>Aim:</b> Autologous blood transfusions (ABT), especially those involving stored erythrocyte concentrates (ECs), are known to be misused as performance enhancers in recreational and competitive athletes. EC storage not only increases the release of extracellular vesicles (EVs), but also significantly alters the microRNA profiles. Since re-transfused EVs also appear in urine, this study was designed to evaluate whether the urinary EV-associated microRNA load could serve as a valuable indicator in the challenging detection of ABT. <b>Methods:</b> Thirty healthy, recreationally active males were included and equally divided into three groups. The control group did not donate or receive any blood components. Group 1 donated about 500 mL of whole blood once, which was subsequently processed into ECs. These were stored for six weeks and then re-infused into the respective donor. Group 2 donated about 500 mL of whole blood twice, at an interval of two weeks. The obtained ECs were stored for six or four weeks, respectively, until parallel re-infusion. In all groups, urine samples were collected over three consecutive weeks before whole-blood donation to establish each individual's baseline, as well as before re-transfusion and several hours and days afterward. Urine samples were processed and analyzed for general urinary health and creatinine levels. Urinary EVs were further isolated by immunoaffinity and characterized using transmission electron microscopy, fluorescence nanoparticle tracking analysis, and western blotting, 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. <b>Results:</b> Urinary EVs presented with typical morphology of small EVs (< 200 nm) and an overall concentration of 8.79 ± 7.00 × 10¹⁰ particles/g U_Crea (urinary creatinine). Significant increases in urinary EV concentrations were detected up to three days after ABT. Apart from Alix, Syntenin, and tumor susceptibility gene 101 (TSG101), surface markers CD63, CD9, CD133/1, CD24, CD326, CD81, and CD31 were also shown to be highly abundant on urinary EVs. Impurities or contaminations were not detected. Cluster analysis based on surface markers showed a clear separation between the control and ABT group. Furthermore, microRNA profiling revealed 13 microRNAs differently regulated upon ABT with miR-155-5p, miR-320b, and miR-6869-5p being the most abundant. <b>Conclusion:</b> This proof-of-concept study suggests an impact of ABT on the urinary EV-microRNA cargo and yields comprehensive findings into surface markers of urinary EVs. While the adoption of urinary EV-associated microRNAs in routine doping tests requires further exploration, these data serve as a valuable basis for a variety of subsequent investigations.