The discovery of erythropoietin (EPO) simplified blood doping in sports, but improved detection methods, for EPO has forced cheating athletes to return to blood transfusion. Autologous blood transfusion with cryopreserved red blood cells (RBCs) is the method of choice, because no valid method exists to accurately detect such event. In endurance sports, it can be estimated that elite athletes improve performance by up to 3% with blood doping, regardless of method. Valid detection methods for autologous blood doping is important to maintain credibility of athletic performances. Recreational male (N = 27) and female (N = 11) athletes served as Transfusion (N = 28) and Control (N = 10) subjects in two different transfusion settings. Hematological variables and physical performance were measured before donation of 450 or 900 mL whole blood, and until four weeks after re-infusion of the cryopreserved RBC fraction. Blood was analyzed for transferrin, iron, Hb, EVF, MCV, MCHC, reticulocytes, leucocytes and EPO. Repeated measures multivariate analysis of variance (MANOVA) and pattern recognition using Principal Component Analysis (PCA) and Orthogonal Projections of Latent Structures (OPLS) discriminant analysis (DA) investigated differences between Control and Transfusion groups over time. Significant increase in performance (15 ± 8%) and VO2max (17 ± 10%) (mean ± SD) could be measured 48 h after RBC re-infusion, and remained increased for up to four weeks in some subjects. In total, 533 blood samples were included in the study (Clean = 220, Transfused = 313). In response to blood transfusion, the largest change in hematological variables occurred 48 h after blood donation, when Control and Transfused groups could be separated with OPLS-DA (R2 = 0.76/Q2 = 0.59). RBC re-infusion resulted in the best model (R2 = 0.40/Q2 = 0.10) at the first sampling point (48 h), predicting one false positive and one false negative. Over all, a 25% and 86% false positives ratio was achieved in two separate trials. In conclusions, autologous re-infusion of RBCs increased VO2max and performance as hypothesized, but hematological profiling by multivariate statistics could not reach the WADA stipulated false positive ratio of <0.001% at any time point investigated. A majority of samples remained within limits of normal individual variation at all times.

OBJECTIVES Autologous blood transfusion (ABT) enhances an athlete's performance, is banned as doping by the World Anti-Doping Agency (WADA). Currently, there is no implemented detection method for ABT. Transfusion of one's 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. Functions of cryo-RBC are maintained for decades, but storage lesions lead to the removal of damaged RBC from circulation days after transfusion, with remaining circulating cryo-RBC displaying normal half-life. METHODS The 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. Aa a control group and for investigation of confounders, 14 elite athletes and 5 recreational subjects were sampled multiple times, also at high altitude. RESULTS Here 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. CONCLUSION Identified 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.