Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion

Abstract

The World Anti-Doping Agency (WADA) has prohibited the use of autologous blood transfusion (ABT) as a doping method by athletes. It is difficult to detect this doping method in laboratory tests, and a robust testing method has not yet been established. We conducted an animal experiment and used total RNA sequencing (RNA-Seq) to identify novel RNA markers to detect ABT doping within red blood cells (RBCs) as a pilot study before human trials. This study used whole blood samples from Wistar rats. The whole blood samples were mixed with a citrate–phosphate–dextrose solution with adenine (CPDA) and then stored in a refrigerator at 4 °C for 0 (control), 10, or 20 days. After each storage period, total RNA-Seq and bioinformatics were performed following RNA extraction and the purification of the RBCs. In the results, clear patterns of expression fluctuations were observed depending on the storage period, and it was found that there were large numbers of genes whose expression decreased in the 10- and 20-day periods compared to the control. Moreover, additional bioinformatic analysis identified three significant genes whose expression levels were drastically decreased according to the storage period. These results provide novel insights that may allow future studies to develop a testing method for ABT doping.

Annual banned‐substance review—Analytical approaches in human sports drug testing 2021/2022–

“…Research into complementary methods supporting the detection of ABTs, besides approaches offered by the ABP, were discussed on transcriptome and cellular levels. Employing total RNA sequencing, whole‐blood samples (obtained from rats) were collected and stored in conventional citrate–phosphate–dextrose–adenine (CPDA) solutions for 10 and 20 days and analyzed for significant alterations in gene expression profiles by Sugasawa et al 92 Candidate marker RNAs exhibiting changes more than 10‐fold (compared to baseline) were found with Lyz2 , Ifit3 , and Il1b , all of which are associated with immune system regulations. Both specificity and sensitivity of the approach remain to be shown, that is, how these relate to erythropoiesis and whether these can be reliably monitored in humans that received ABTs, especially at the expectedly low volumes and percentage of transfused blood.…”