Possible detection of Autologous Blood Transfusion (ABT) based on circulating plasma microRNAs involved in erythroid differentiation and fetal hemoglobin induction

Detection of Autologous Blood Transfusions (ABT) is a key issue in the field of anti-doping for the performance enhancing effects of this prohibited method and the consequent unfair use in sport. Unfortunately, at present no direct detection method of ABT is available. In the last years, after the implementation of the Athlete's Biological Passport, scientists have searched novel parameters, combined biomarkers using mathematical approaches and tested new techniques as 'omics' technologies to detect ABT. The present study was performed to determine whether miRNA associated to erythroid phenotype, fetal haemoglobin production and regulation of gamma-globin genes are modulated following ABT. From 6 healthy subjects 500 ml of blood was withdrawn (T2) and then infused after 35 days (T5). Blood samples for microarray analysis of miRNAs were taken 5 days before (T1) and 10 days after blood withdrawal (T3), at day of infusion (T5), and at 3 days (T6) and 15 days (T8) after infusion. For three subjects the withdrawn blood was stored at -80°C, for three at +4°C before infusion. The global microRNAs profiling was performed for a total of 39 RNA samples (extracted from plasma), using the Agilent Human microRNA microarray v.21.0 (#G4872A). This chip represents 2549 microRNAs, sourced from the Sanger miRBase database (Release 21). Microarray results were analyzed using the GeneSpring GX 13 software (Agilent Technologies). Differentially expressed miRNAs were selected following determination of the fold-change analysis, taking T1 as reference sample. Both up-regulated and down-regulated miRNAs were considered. The first set of microRNAs analyzed was constituted by miRNAs related to erythroid differentiation, HbF production and transcriptional regulation of gamma-globin genes. The results obtained allows to conclude that (a) a person-to-person variability is present when the different ABT subjects were analyzed; (b) miRNAs whose expression was found modified following ABT were miR-766-3p (upregulated), miR-191-3p (upregulated) ad miR-16-5p (down-regulated). These results support the concept that miRNA analysis might be considered for detection of ABT when serum samples in analytical test finalized to detection of doping. Acknowledgements. This work was supported by the World Anti Doping Agency Science grant #14C06FM.