Age-Markers on the Red Blood Cell Surface and Erythrocyte Microparticles may Constitute a Multi-parametric Strategy for Detection of Autologous Blood Transfusion

• Published in: Sports medicine - open Vol. 9; no. 1; pp. 113 - 13
• Main Authors: Biasini, Giorgia M., Botrè, Francesco, de la Torre, Xavier, Donati, Francesco
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.12.2023; Springer Nature B.V; SpringerOpen
• Subjects: Autologous blood transfusions; Band 3 complex; Biomarkers; Blood; Blood doping; Blood transfusions; Doping in sport; Glycophorin-A; Hematology; Medicine; Medicine & Public Health; Morphology; Original; Original Research Article; Peroxiredoxin-2; Protein expression; Proteins; Sports Medicine; Doping in sport; Autologous blood transfusions; Band 3 complex; Glycophorin-A; Red blood cell microparticles; Peroxiredoxin-2; CD47; Blood doping; Phosphatidylserine
• ISSN: 2198-9761; 2199-1170; 2198-9761
• DOI: 10.1186/s40798-023-00662-9

Background Autologous blood transfusion is one of the illicit strategies, banned by the World Anti-Doping Agency, to increase the levels of hemoglobin, with a consequent improvement in the delivery of oxygen to tissues. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of “storage lesions”, targeting (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2, (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles. Results All of the above markers were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh blood samples, collected for official doping control tests, mixed “ex vivo” to simulate an autotransfusion. Although anonymized before the delivery to the laboratory, it was possible to mix samples belonging to the same subject based on the “athlete biological passport” code. Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to: (i) the reduced concentration, on the erythrocyte membrane, of Band 3 protein (decrease of 19% and of 39% after 20 and 40 days of storage respectively) and of glycophorin A (− 47% and − 63% respectively); (ii) the externalization of phosphatidyl serine (with a five-fold increase after 20 days and a further 2× increase after 40 days); (iii) the reduced concentration of CD47; and (iv) increased levels of erythrocyte microparticles. Conclusions The most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena. Key Points Potential biomarkers of “blood storage” were specifically considered in this study to detect autologous blood transfusions in sport doping. The irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to: (i) the reduction, on the erythrocyte membrane, of Band 3 protein and of glycophorin A; (ii) the externalization of phosphatidyl serine; (iii) the reduced concentration of CD47; and (iv) the increased levels of erythrocyte microparticles. Freshly collected samples showed differences from stored or mixed ones in terms of both protein expression on the RBCs membranes and micro-vesiculation phenomena.