Detection Of Autologous Blood Doping

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSObjectivesC_ST_ABSAutologous blood transfusion (ABT) enhances athletes performance, is banned as doping by the World Anti-Doping Agency (WADA). Currently, there is no implemented detection method for ABT. Transfusion of ones own, long-term cryopreserved red blood cells (cryo-RBC) immediately increases circulating RBC count, hemoglobin mass, blood volume and oxygen carrying capacity, resulting in enhanced physical performance. Functional viablity of cryo-RBC are maintained for decades, but storage lesions lead to removal of damaged RBC from circulation days after transfusion, with remaining circulating cryo-RBC displaying normal half-life. MethodsThe cytosolic RBC peptidome from 22 human subjects (12 men and 10 women) was analyzed by UHPLC-MS/MS before and after ABT with cryo-RBC. As a control group and for investigation of confounders, 14 elite athletes and 5 recreational subjects were sampled multiple times, also at high altitude. ResultsHere we report alteration in the cytosolic peptidome of circulating RBC weeks after ABT, discriminating doped from non-doped human subjects. A valid discriminating multivariate model (OPLS-DA) based on <200 peptides was accomplished (R2/Q2 = 0.88/0.59, P CV-ANOVA < 0.0001, ROC AUC = 0.97). Models did not show bias for sex, high altitude or elite endurance training and racing. ConclusionIdentified peptides with low intra- and inter-individual variation, and high multivariate model weight and probability scores, create a direct method for the detection of autologous blood doping.