mRNA Biomarkers in Dried Blood Spots May Improve Detection of Autologous Blood Micro‐Transfusions Using an Individualized Approach

Autologous blood transfusions (ABTs) are prohibited by the World Anti-Doping Agency (WADA), yet detecting autologous blood micro-transfusions (ABMTs) remains a challenge. Due to smaller transfused volumes, ABMTs cause attenuated biomarker changes, limiting detection sensitivity within the Athlete Biological Passport (ABP). This study assessed whether mRNA expression of 5-aminolevulinic acid synthase (ALAS2) and carbonic anhydrase 1 (CA1), measured from dried blood spots (DBS), could serve as sensitive biomarkers of ABMT. In a randomized, placebo-controlled design, 47 trained individuals (24 ♀; mean VO₂peak 56 ± 7 mL·min^-1·kg^-1) were allocated to an ABMT group (n = 23; ♀ = 12) or placebo group (n = 24; ♀ = 12). The ABMT group donated 450 mL of blood and received a 130 mL packed red blood cell reinfusion 4 weeks later. Blood sampling occurred regularly before and after both donation and reinfusion. ALAS2 and CA1 mRNA expression from DBS, and reticulocyte percentage (RET%) from venous blood, were analyzed. Following blood donation, ALAS2, CA1, and RET% increased by 270%, 200%, and 150%, respectively. However, no consistent group-level changes were observed after ABMT. Individualized analysis identified more outliers for ALAS2 than for CA1, and blinded interpretation of individual mRNA profiles achieved > 95% sensitivity and specificity for detecting ABMT. These findings suggest that ALAS2 mRNA expression, assessed via minimally invasive DBS sampling, is a promising biomarker for identifying ABMT. This approach may enhance current anti-doping strategies by improving sensitivity to small-volume autologous transfusions that evade detection through traditional ABP biomarkers.