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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., (© 2024 John Wiley & Sons Ltd.)

Total hemoglobin mass (Hb mass ) is routinely assessed in studies by the carbon monoxide (CO) rebreathing. Its clinical application is often hindered due to the consequent rise in carboxyhemoglobin (%HbCO) and the concern of CO toxicity. We tested the reproducibility of the CO rebreathing with a CO dose of 0.5 mL/kg body mass (CO 0.5 ) compared to 1.5 mL/kg (CO 1.5 ) and when shortening the CO rebreathing protocol. Therefore, CO rebreathing was performed 1×/day in eight healthy individuals on four consecutive days. On each day, either CO 0.5 (CO 0.5 -1 and CO 0.5 -2) or CO 1.5 (CO 1.5 -1 and CO 1.5 -2) was administered. Venous blood samples to determine %HbCO and quantify Hb mass were obtained prior to, and at 6 (T 6 ), 8 (T 8 ) and 10 min (T 10 ) of CO rebreathing. This protocol was tested at sea level and at 2320 m to investigate the altitude-related measurement error. At sea level, the mean difference (95% limits of agreement) in Hb mass between CO 0.5 -1 and CO 0.5 -2 was 26 g (-26; 79 g) and between CO 1.5 -1 and CO 1.5 -2, it was 17 g (-18; 52 g) . The respective typical error (TE) corresponded to 2.4% (CO 0.5 ) and 1.5% (CO 1.5 ), while it was 6.5% and 3.0% at 2320 m. With CO 0.5 , shortening the CO rebreathing resulted in a TE for Hb mass of 4.4% (T 8 vs. T 10 ) and 14.1% (T 6 vs T 10 ) and with CO 1.5 , TE was 1.6% and 5.8%. In conclusion, the CO dose and rebreathing time for the CO rebreathing procedure can be decreased at the cost of a measurement error ranging from 1.5-14.1%.