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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.

SUBMITTER: Sugasawa T

PROVIDER: S-EPMC9317427 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Both cardiopulmonary bypass (CPB) and red blood cell (RBC) storage are associated with detrimental changes in RBC structure and function that may adversely affect tissue oxygen delivery. We tested the hypothesis that in cardiac surgery patients, RBC deformability and aggregation are minimally affected by CPB with autologous salvaged blood alone but are negatively affected by the addition of stored allogeneic blood.In this prospective cohort study, 32 patients undergoing cardiac surgery with CPB were divided into 3 groups by transfusion status: autologous salvaged RBCs alone (Auto; n = 12), autologous salvaged RBCs + minimal (<5 units) stored allogeneic RBCs (Auto+Allo min; n = 10), and autologous salvaged RBCs + moderate (?5 units) stored allogeneic RBCs (Auto+Allo mod; n = 10). Ektacytometry was used to measure RBC elongation index (deformability) and critical shear stress (aggregation) before, during, and for 3 days after surgery.In the Auto group, RBC elongation index did not change significantly from the preoperative baseline. In the Auto+Allo min group, mean elongation index decreased from 32.31 ± 0.02 (baseline) to 30.47 ± 0.02 (nadir on postoperative day 1) (P = 0.003, representing a 6% change). In the Auto+Allo mod group, mean elongation index decreased from 32.7 ± 0.02 (baseline) to 28.14 ± 0.01 (nadir on postoperative day 1) (P = 0.0001, representing a 14% change). Deformability then dose-dependently recovered toward baseline over the first 3 postoperative days. Changes in aggregation were unrelated to transfusion (no difference among groups). For the 3 groups combined, mean critical shear stress decreased from 359 ± 174 mPa to 170 ± 141 mPa (P = 0.01, representing a 54% change), with the nadir at the end of surgery and returned to baseline by postoperative day 1.In cardiac surgery patients, transfusion with stored allogeneic RBCs, but not autologous salvaged RBCs, is associated with a decrease in RBC cell membrane deformability that is dose-dependent and may persist beyond 3 postoperative days. These findings suggest that autologous salvaged RBCs may be of higher quality than stored RBCs, since the latter are subject to the so-called storage lesions.

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

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