LAUSR

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with micro-doses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'-aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO micro-dosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO micro-dosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO micro-dosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO micro-dosing and hypoxia, while SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO micro-dosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti-doping analyses.