Improving the Detection of Homologous Blood Transfusion in Sport Doping: A Full DNA‐based Genotyping Strategy on Dried Blood Spots

Abstract

Homologous blood transfusions (hBT) are prohibited in sport by the World Anti-Doping Agency (WADA) as they represent one of the illicit strategies that athletes can pursue to improve the oxygen supply to the tissues, with a consequent enhancement of the athletic performance. The current method of detection of hBT is based on the phenotypic mismatch analysis of red blood cells (RBC) surface antigens by flow cytofluorimetry. Despite the excellent sensitivity and specificity of the method, some critical issues have progressively emerged, mostly related ti the relatively high risk of false-negative results. At the same time, the method is not applicable to blood samples collected as “dried blood spots”. To overcome these difficulties, we designed and developed a detection strategy based on DNA analysis. The wide inter-individual variability of DNA genotypes allows to completely eliminate the problem represented by the “false negative” results, improving also the overall sensitivity of the anti-doping tests. Our method is based on a combination of the forensic typing of DNA with the genotyping of SNP genetic polymorphisms. The effectiveness of the proposed approach has been confirmed by the analysis of positive samples obtained by mixing whole blood from two different compatible donors, who shared the same panel of RBC surface antigens, collected on DBS cards. DNA was isolated by a DNA forensic extraction kit based on magnetic beads. Our results suggest that the proposed approach, once validated on samples from real transfused subjects, can complement the current testing strategy based on the mismatch of the RBC surface antigens.