BACKGROUND: There is currently no direct method to detect the use of autologous blood transfusions in the context of anti-doping controls. The storage of erythrocytes causes changes in membrane proteins, which could be evidenced by proteomics. The aim of the present study was to identify these modifications on blood samples stored in transfusion bags, at different storage time points.METHODS: Twenty-three hemochromatosis patients undergoing periodic phlebotomy sessions for therapeutic purposes at the San Luigi Gonzaga Hospital in Orbassano, Turin, were enrolled in the study. The blood was stored under standard conditions for up to 21 days at the local Blood Bank. The samples were analyzed at 0, 7, 14, and 21 days after phlebotomy, in order to isolate the erythrocyte membranes. After identifying the most valid erythrocyte membrane extraction protocol and obtaining homogeneous protein samples by manual sonication, we performed Western blot assay (WB), by incubating the membranes with the primary anti beta-actin antibody and then with the anti-band 3 antibody. Two-dimensional electrophoresis was then performed on the samples with the aim of highlighting different protein patterns, that were later identified by mass spectrometry (MALDI-TOF).RESULTS: An increase in actin expression over time was evidenced by WB. Two-dimensional electrophoresis, followed by MALDI-TOF analysis, showed an increase in the number of actin spots and its CRA-b isoform from day 0 to day 21. The analysis of band 3 showed a gradual fragmentation of the protein during storage time.CONCLUSIONS: The results of the present study confirmed the observations of scientific literature demonstrating a modification of erythrocyte proteins during red blood cells (RBC) storage. However, further investigations are needed in order to univocally define these modifications and to make the results applicable in the context of the strategies to fight doping.
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