Altered erythroid‐related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport

Abstract

BACKGROUND: Autologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport; its detection is a key issue in the field of anti-doping. Among novel markers enabling ABT detection, microRNAs (miRNAs) might be considered a promising analytical tool. STUDY DESIGN AND METHODS:We studied the changes of erythroid-related microRNAs following ABT, to identify novel biomarkers. Fifteen healthy trained males were studied from a population of 24 subjects, enrolled and randomized into a Transfusion (T) and a Control (C) group. Seriated blood samples were obtained in the T group before and after the two ABT procedures (withdrawal, with blood refrigerated or cryopreserved, and reinfusion), and in the C group at the same time points. Traditional hematological parameters were assessed. Samples were tested by microarray analysis of a pre-identified set of erythroid-related miRNAs. RESULTS:Hematological parameters showed moderate changes only in the T group, particularly following blood withdrawal. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects in the completed phase of ABT, independently from blood conservation. Particularly, two or more erythropoiesis-related miRNAs within the shortlist constituted of miR- 126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p, and miR-92a-3p were significantly upregulated in T subjects after reinfusion, with a person-to-person variability but with congruent changes. CONCLUSIONS:This study describes a signature of potential interest for ABT detection in sports, based on the analysis of miRNAs associated with erythroid features.A utologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport, 1 with its detection a key issue in the anti-doping field. Given the lack of a method for its direct detection, 2-5 an approach to the indirect detection of ABT has been considered. The athlete biological passport (ABP) hematological module 6-8 has been adopted by the World Anti-Doping Agency (WADA) to identify and, when necessary, sanction athletes showing abnormal changes in hematological parameters. Novel markers enabling ABT indirect detection have also been proposed, [2][3][4][9][10][11][12][13] including those derived from genomics applied to microRNAs (miRNAs), and are considered a promising analytical tool. [14][15][16][17] In this respect, miRNAs are short non-coding RNA molecules, which act as gene regulators by repressing translation or by inducing the cleavage of target RNA transcripts. [18][19][20][21] Recent evidence suggests that miRNAs (including miRNAs present as extracellular molecules in plasma) can be considered a From the

On the trail of blood doping—microRNA fingerprints to monitor autologous blood transfusions in vivo

“…Based on existing literature and guidelines, reasonable recovery from blood donations within 4–6 weeks, with inter‐individual differences regarding the recovery speed, could have been expected 2,16,52‐56 . Our results, however, indicated an overall insufficient recovery from blood withdrawal after 4 weeks (group 2) and, to a lesser extent, after 6 weeks (group 1) (Table 1), which was comparable to the outcomes reported by Bejder et al 57 As a matter of fact, only some study subjects completely returned to their baseline physiological ranges (Figure S1).…”

“…However, due to a poorly placed baseline measurement, this finding has to be considered carefully 68 . Moreover, although correlations between transfusion volume and associated increase or decrease in hematological variables were observed, 2,16,52‐55 a conclusive connection with aerobic capacity remains to be established 57,65 . Since an increase in miRNA changes with increasing transfusion volume cannot be excluded either and a non‐negligible amount of blood of almost 190 mL is collected during multiple samplings post‐ABT, which might reduce the ABT‐dependent effect size in the present exploratory study, a second standard‐volume blood donation of about 500 mL with subsequent EC re‐transfusion after 28 days of storage was conducted in group 2 to increase the ergogenic aid and achieve a more distinctive magnitude of change.…”

“…So far, there is only a small number of studies on miRNA profiles in connection with ABT: Gasparello et al 16 identified seven miRNAs (miR‐126‐3p, miR‐144‐3p, miR‐191‐3p, miR‐197‐3p, miR‐486‐3p, miR‐486‐5p, and miR‐92a‐3p) that were dysregulated 15 days after autologous re‐transfusion of ECs that had been stored for 35 days. However, the post‐ABT time point for fold chance calculation might have been chosen inappropriately, as it appears more likely that competitive athletes are tested within 48 hours after a competition rather than 15 days later.…”

A Distinctive microRNA (miRNA) Signature in the Blood of Colorectal Cancer (CRC) Patients at Surgery

“…Amplified droplets were analyzed for the fluorescence content using QX200 Droplet Digital PCR system. Data analysis was performed using QuantaSoft software, version 1.7.4 (Bio-Rad, Hercules, CA, USA) [ 69 , 70 , 71 ]. The RT-ddPCR assay was chosen in consideration of its efficiency, the quantification of absolute amounts of analyzed molecules and the possibility to identify changes concerning low numbers of miRNA molecules, as expected using plasma preparation.…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Gasparello et al followed up on the option of targeting the organism's response to blood transfusion at the miRNA level as a potential means to uncover blood transfusion practices in sport . Six volunteers underwent blood transfusions with one unit (450 mL) of either refrigerated or cryopreserved origin, and blood samples were collected on five occasions prior to and on four occasions after blood transfusion.…”