Morphological analysis of red blood cells detects doping by autologous transfusion in a mouse model

• A mouse model of transfusion provides a relevant platform to identify new biomarkers for anti-doping purposes. • Quantifying storage-induced micro-erythrocytes is a promising biomarker to detect autologous blood transfusion in athletes. Doping by autologous blood transfusion (ABT) of red blood cells (RBCs), especially with low volumes, remains challenging to detect. We hypothesized that mouse models developed for transfusion research could be relevant to screen for innovative biomarkers to support anti-doping efforts. In this context, changes in RBC size (projected surface area [PSA]) and morphology (storage-induced micro-erythrocytes [SMEs]) were tested as potential candidates to identify ABT. C57Bl6 mouse RBCs were stored at 4°C for 13 days (similar to 42 days in humans) and two transfusion scenarios were studied: one representing ∼10% (HIGH) and the other representing ∼6% (LOW) expansion of the recipient’s RBC content. First, biomarkers included in the Athlete Biological Passport (ABP) were investigated. Second, PSA and SME% were quantified by imaging flow cytometry. Hemoglobin concentration and reticulocyte percentage were similar to those in prior human studies, despite accelerated kinetics and distinct baseline values in the mouse model. A decrease in PSA was associated with an increased PSA standard deviation following HIGH, although only PSA differed significantly following LOW. SME% increased in both groups and remained elevated up to 2 days post-transfusion for LOW. Changes in ABP biomarkers in this mouse transfusion model align with those documented in humans, supporting its relevance to screen newly-identified biomarkers for anti-doping purposes. Specifically, the SME% biomarker detected low-volume ABT (i.e. LOW). These findings encourage further investigations into RBC properties after transfusion, which can potentially identify ABT in athletes.