The Use of RNA‐based 5’‐Aminolevulinate Synthase 2 Biomarkers in Dried Blood Spots to detect rhEPO Micro Doses and Hypoxic Stimulation

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 e.g. with micro doses of recombinant human erythropoietin (rhEPO) is problematic. For this reason, sensitivity of ABP have to be enhance with the implementation of new biomarkers. Here we demonstrated that RNA-based 5’-Aminolevulinate Synthase 2 (ALAS2) could be a useful biomarker to improve the indirect detection of rhEPO micro dosing. Moreover, satisfying results were obtain also for specificity in order to distinguish rhEPO administrations from a sojourn in hypoxic conditions. Samples from two different clinical studies were used to monitor levels of different RNA markers, linear-circular (LC) and linear (L), of ALAS2 RNA, Carbonate anhydrase 1 (CA1) and Solute Carrier family 4 member 1 (SLC4A1) after rhEPO microdoses administration and under hypoxic condition. Analysis of RNA based biomarkerswere performed using dried blood spots (DBSs), these ones provide many advantages in terms of the samples collection, transport and storage. In order to monitored transcirptomics biomarkers variations, manual extraction of RNA from DBSs were performed follow by a RT-qPCR. Transcriptomics biomarkerswere monitored along 16 days where five micro doses were administrated, ALAS2 markers illustrated an increase up to 300% in comparison with the baseline value and these variations were statistically different from evolution in control group. Additionally, also CA1 variated in a significant way due to micro doses administrations. Moreover, ALAS2 markers variations under hypoxic conditions are not significant when compared with control group, which is the opposite of CA1 or reticulocytes percentage, where significant differences were obtained. Finally, SLC4A1 has no significant variation in both conditions. This study demonstrated that transcriptomic biomarkers, in particular ALAS2 mRNAs, could be used to improve the sensitivity and specificity of the hematological module of the ABP, and are compatible with the use of DBSs for anti-doping analyses. The expression level of SLC4A1 was not increased by micro doses of rhEPO, indicating that it is not a beneficial biomarker to detect micro dosing. CA1 is an interesting candidate, although, unlike ALAS2, its expression level was increased also significantly following exposure to hypoxia.