The erythropoiesis-stimulating agents (ESAs) are still considered as major doping agents and their misuses remain a high-ranking threat in horse racing and equestrian sports. In doping control laboratories, the testing of such substances is usually performed from liquid blood samples, using at least 1 or 2 mL of whole blood or plasma per analysis. This situation results in the collection and storage of significant volumes of blood in order to screen for and, if necessary, confirm the presence of all doping substances to be tested. Furthermore, when blood/plasma is in liquid form, the integrity of both the sample and the substance(s) can be rapidly compromised under prolonged storage and transport conditions.
To overcome these issues, the use of DBS for sample collection, transport, storage and analysis in doping control is under investigation at GIE LCH, in collaboration with two WADA laboratories in the context of equestrian sports. Compared to liquid blood samples, DBS allows fixing only a few tens of microlitres of blood in a compact device suitable for easy (long-term) storage and transport. DBS is also known to ensure sample stability, especially for biomolecules such as proteins and nucleic acids.
In this study, the proof of concept of the use of DBS for equine doping control was performed for both direct (ELISA, Western Blot, LC-MS/MS) and indirect (blood counting and transcriptomic biomarkers) detection of ESAs. Horses were administered rhuEPO (Eprex) or HIF stabiliser (Roxadustat) and whole blood collected with conventional material (lithium heparin tubes, EDTA tubes, PAXgene tubes). DBS were then prepared from the blood tubes to compare detection performances.
The most significant results demonstrated that Eprex triggered erythropoiesis stimulation through an increase of circulating reticulocytes and ALAS2 gene expression. Moreover, ESAs’ detection was almost as effective with DBS as with liquid sample for both Eprex and Roxadustat.