Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti‐doping application

Abstract

The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADAstipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 μl of blood, but various volumes from 20 to75 μl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 μl of serum/plasma. Different storage temperatures, from À20 C to 37 C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis,

Supra‐physiological doses of anabolic androgenic steroids impact erythropoietin and blood parameters

“…The reason for low/absent uEPO signals in the samples and/or low EPO serum levels can be due to diluted urine, sample manipulation, and EPO doping. However, we did not see any correlation with SG, which was surprising, but in line with our previous finding that the intra‐individual variation (CV %) of uEPO did not depend on SG 35 . Notably, the two samples with highest SG showed an absence of uEPO signal, which is in agreement with Lamon et al who reported that both low and high SG were over‐represented in undetectable samples 36 …”

“…However, we did not see any correlation with SG, which was surprising, but in line with our previous finding that the intra-individual variation (CV %) of uEPO did not depend on SG. 35 Notably, the two samples with highest SG showed an absence of uEPO signal, which is in agreement with Lamon et al who reported that both low and high SG were over-represented in undetectable samples. 36 There are some limitations with the study using self-reported AAS users, including uncontrolled AAS intake in regard to doses, substances, and durations.…”

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“…It was discovered that a blood volume of 60 µL allowed the detection of ERAs at levels comparable to WADA's minimum required performance levels (MRPLs) described for 500 µL of serum or plasma. 37 In another study, CERA was demonstrated to be detected in DBS using a simple enzyme-linked immunosorbent assay with a screening time of just 2 h. 38 Furthermore, a multi-analyte assay was developed and the performance of several non-volumetric (Whatman cards-Treated: Whatman FTA DMPK-A, Whatman FTA DMPK-B;…”

“…A recent critical review by Thevis et al described the advances, potential future perspectives and limitations of blood microsamples in doping controls 157 . Fast and cost‐effective screening methods to detect erythropoietin receptor agonists such as CERA, BRP, NESP and EPO‐Fc using dried blood microsamples were developed and optimised 37,38 . In the study conducted by Heiland et al, different blood sample volumes (20–180 µL) and storage temperatures (−20 to 37°C) were evaluated to determine optimal conditions for detecting prohibited ERAs from DBS.…”

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“…Aiming at expanding ERA analytical approaches to DBSs as test matrix, Heiland et al presented an optimized protocol that specifically addressed storage conditions and sample volume for DBSs using different sampling materials 56 . Upon immunopurification, extracts from DBSs formed from formerly 60 μl of whole blood were found to allow for LODs of 62.5, 62, 31, and 12.6 pg/ml for the continuous EPO receptor activator CERA, EPO‐Fc, the European Pharmacopoeia Biological Reference Preparation (BRP) for EPO, and novel erythropoiesis stimulating protein NESP, respectively.…”