mRNA biomarkers sensitive and specific to micro‐dose erythropoietin treatment at sea level and altitude

Abstract Recombinant human erythropoietin (rhEPO) is prohibited by the World Anti‐Doping Agency. rhEPO abuse can be indirectly detected via the athlete biological passport (ABP). However, altitude exposure challenges interpretation of the ABP. This study investigated whether 5′‐aminolevulinate synthase 2 ( ALAS2 ) and carbonic anhydrase 1 ( CA1 ) in capillary dried blood spots (DBSs) are sensitive and specific markers of rhEPO treatment at altitude. ALAS2 and CA1 expression was monitored in DBS collected weekly before, during, and after a 3‐week period at sea level or altitude. Participants were randomly assigned to receive 20 IU kg bw −1 epoetin alpha (rhEPO) or placebo injections every second day for 3 weeks while staying at sea level (rhEPO, n = 25; placebo, n = 9) or altitude (rhEPO, n = 12; placebo, n = 27). ALAS2 and CA1 expression increased up to 300% and 200%, respectively, upon rhEPO treatment at sea‐level and altitude ( P ‐values <0.05). When a blinded investigator interpreted the results, ALAS2 and CA1 expression had a sensitivity of 92%. Altitude did not confound the interpretation. Altitude affected ALAS2 and CA1 expression less than actual ABP markers when compared between sea level and altitude results. An individual athlete passport‐like approach simulation confirmed the biomarker potential of ALAS2 and CA1 . ALAS2 and CA1 were sensitive and specific biomarkers of micro‐dose rhEPO treatment at sea level and altitude. Altitude seemed less a confounding factor for these biomarkers, especially when they are combined. Thus, micro‐dose rhEPO injections can be detected in a longitudinal blinded setting using mRNA biomarkers in DBS.