RNA‐Based Biomarkers of Iron Metabolism in Dried Blood Spots for Detecting Recombinant Human Erythropoietin in Doping Control

Erythropoietin (EPO) abuse in sports has been a major challenge in doping analysis for decades. Although the Athlete Biological Passport (ABP) serves as an indirect method for EPO detection, it requires continual improvement to increase its sensitivity and reliability. Biomarkers related to iron metabolism and erythropoiesis as complementary parameters to enhance the ABP hematology module have the potential to improve its ability to detect EPO indirectly. In this study, RNA was extracted from dried blood spot (DBS) samples collected over 28 days (days 0, 1, 3, 5, 7, 9, 11, 13, 21, and 28) following the administration of therapeutic doses of recombinant human EPO (Yibiao, 50 IU/kg per dose, administered subcutaneously twice). The expression of four genes, namely, mitoferrin 1 (MFRN1), ferrochelatase (FECH), ferroportin (FPN), and ferritin heavy chain (FTH), was analyzed. The results revealed significant expression changes, with MFRN1 demonstrating peak increases of 2.43-fold, and presented detection windows extending beyond Day 9, providing greater sensitivity and longer detection windows than traditional ABP parameters do. The integration of MFRN1 into the ABP framework has the potential to increase the detection of EPO misuse in athletes.