A fast screening method for the detection of CERA in dried blood spots

Abstract

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia.However, misuse of CERA for doping in endurance sports has been reported.Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 μg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days postinjection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.

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

“…Although DBSs on a filter paper support were well‐extracted with the MAIIA EPGK, optimization in processing and detection are still possible, particularly for the Tasso‐M20 pebbles, because some variations in the DBS intra‐individual signal intensities may be from unevenness in blood desorption from the polymer, indicating that the 90‐min rotation during extraction/immunopurification is not efficient enough to extract all the EPO from the polymer. Possible ways to improve extraction and sensitivity are prolonging the rotation time, or by sonication 33 or rinsing of the polymer with buffer before incubation with the sepharose gel beads.…”

“…The volumetric/quantitative devices are a good way to standardize the volumes collected and remove haematocrit bias caused by uneven blood distribution on filter paper 45 . Experiments with CERA and VAMS have shown promising results, with DBSs being of particular interest for the detection of this ERA which is not easily filtered in urine 33 . Haematocrit bias does not affect ERA detection, 15 particularly when the entire spot is analysed, but collecting sufficient and precise volumes is important for detection.…”

“…Urine, DBSs from capillary blood (using Tasso-M20 devices), and serum samples (collected in BD vacutainer SST, Becton, Dickson and company, NJ, USA) from five healthy participants (three males, two females; ages [26][27][28][29][30][31][32][33][34][35] were collected to evaluate EPO immunodetection from DBSs compared with other well-established matrices. All participants gave their written informed consent, and the collection was approved by the Swedish Ethical Review Authority (Ethical permit Dnr 2020-04258).…”

“…31 Despite DBSs considered as unfeasible for ERA detection in 2016, 32 recent studies successfully used this matrix for ERA detection: a validated method for hEPO, rEPO, NESP and CERA detection from 25-μl modelled blood DBSs using polyacrylamide gel electrophoresis (PAGE) was described in 2018 15 and a method to successfully detect CERA from patients by using 20-μl modelled blood DBSs and a commercial ELISA kit in 2021. 33 However, in these studies, therapeutic doses of ERAs were investigated, a far cry from the minimum required performance levels (MRPLs) that need to be reached in blood for the four main generations of ERAs recently published by WADA. 34 The aim of this study was to find optimal conditions for sensitive and specific ERA detection from DBSs and to evaluate if WADA's MRPLs could be reached using a single DBS for the four main ERAs (BRP, NESP, CERA and EPO-Fc).…”

Biomarker analysis

“…The potential use of dried blood spots (DBS) for biomarker analysis is presented by Rocca et al 6 and Loria et al 7 for improved detection of recombinant erythropoietins. DBS is a complementary approach to traditional urine and venous blood matrices, which can facilitate simpler sample collection and increase the number of samples available for biomarker analysis due to reduced costs.…”