Verification of Erythropoiesis Stimulating Agents analytical method (sensitivity and specificity) in a second laboratory

Code: DBS20AS4DE

Recent work in the anti-doping field has clearly shown that the future of blood collections is dependent on direct capillary blood collection, moving away from traditional venous draws. In that light, this project will focus only on the detectability of endogenous (blood EPO, or bEPO) and recombinant EPO in samples collected directly with capillary collection devices and will avoid venous blood spotted onto a matrix. Blood samples, both from control volunteers and patients who have been administered rEPO, will be collected using Tasso and OneDraw devices, and finger pricks.

Extraction of blood (and EPO) from the spot will be optimized using in-house protocols. Once extracted, two methods of EPO purification will be attempted. First, a conventional magnetic bead immunopurification method utilizing anti-EPO antibodies to capture the EPO in the dried blood sample will be tested (adapted from Desharnais, 2017). Next, the commercially available MAIIA EPO purification gel kit will also be utilized for efficacy. Once purified, samples will be analyzed via SAR-PAGE and Western Blotting, using the biotinylated monoclonal anti-EPO antibody described in previous Cologne Workshops (Reichel 2018, Dehnes 2020) to provide a higher quality protein signal. Using the methods described above, the sensitivity and specificity of bEPO and rEPO in DBS will be characterized.

Main findings

This project was executed as a follow-up verification from work completed in the Barcelona laboratory in 2018 in which various ESAs were detected in dried blood spots using electrophoretic methods. The purpose of this project was to verify these results in a second laboratory and to answer the following questions:

1. What is the optimal method to extract and purify EPO from a dried capillary blood spot?

2. How does the optimal method perform in samples collected directly from capillary blood, specifically in samples from a drug administration?

Extraction and purification using StemCell ELISA and anti-EPO magnetic bead technology were studied in this project. Although MAIIA technology was also proposed as a purification method, this technology is currently under study in other laboratories and therefore was omitted from this project. Initial and follow-up studies both showed superior extraction and purification using the StemCell ELISA method, and thus this method was deemed more appropriate for ESA analysis in DBS.

Next, a clinical study was designed in which nine male subjects each received a single dose of EPOGEN (epoetin alfa) at a dose of 40 IU/kg s.c. Finger prick (spotted onto DMPK-C cards) and capillary blood from Tasso M20 devices was collected from each patient before their dose and then intermittently up to 72 hours after their dose. All samples were extracted using the StemCell ELISA method and were analyzed by SAR-PAGE and Western Blotting according to laboratory standard operating procedures. On average, rEPO was detectable in both finger prick and Tasso M20 samples up to 48 hours.

In most cases, the rEPO detected in the samples appeared on the gels as a double band, with the recombinant portion migrating on the gel similarly to the Dynepo standard and the endogenous portion migrating further. While this has been seen in urine and blood samples following controlled administrations, it differs from the conventional ‘mixed band’ or ‘smeared band’ commonly seen with rEPO-positive samples.

Finally, while rEPO was detectable in capillary blood samples, due to factors including low concentrations of EPO in the samples and low sample volume, gel quality was an issue throughout the study. In many instances, the EPO content on the gel was low enough such that the contrast between EPO and background provided interpretation issues. It is expected that using larger sample volumes should eliminate some of these concerns.

Overall, results from this study validate the data observed in the Barcelona in 2018, showing that doses of rEPO can be detected in DBS. Prior to implementing into routine doping

control, method improvement is recommended regarding increasing sample volumes (pending MAIIA purification results). Additionally, it is recommended that a technical document be drafted by the EPO Working Group outlining sample preparation proced