Advancements in detection of performance enhancing drugs in dried blood spots : focusing on erythropoietin

The use of erythropoietin (EPO) for performance enhancing purposes is detected mainly in urine and serum samples in anti-doping laboratories. However, dried blood spots (DBS) have emerged as a potential additional sample for EPO and other various doping substances. The aims of these studies were to develop a method for EPO analysis from DBS (study I) and use this method to sensitively detect endogenous EPO, micro-doses of exogenous EPO, and the EPO c.577del protein VAR-EPO (studies I-III). I also observed the stability of EPO and insulin-like growth factor 1 (IGF-I) in DBS (study I and V), along with investigations into the relationship between EPO and testosterone (study IV).

Sample collection: Capillary blood (for DBS), venous blood (for DBS and serum) and urine were collected from healthy volunteers. The DBS devices used were Capitainer®B 50, Mitra® VAMS, Tasso-M20, and Whatman 903 filter paper cards for EPO and IGF-I detection. Urine and serum were also collected from 30 self-reported anabolic androgenic steroid (AAS) users to examine the presence of EPO in such samples.

Methods: The primary method I used for detecting EPO and VAR-EPO in the collected samples was immunopurification with the EPO Purification Gel Kit, SAR- PAGE, and Western blot. EPO concentrations were measured in serum using a commercial immunoassay kit. IGF-I was analyzed by LC-MS/MS and with the automated immunoassay system IDS-iSYS. Serum AAS was quantified using an immunoassay.

Results: Endogenous EPO was sensitively detected on both polymer and paper blood supports (studies I, II, III), providing stable results (study I), while urinary EPO fluctuated more (study I) and occasionally showed degradation or undetectable bands (studies I and III). The instability of IGF-I in DBS at room temperature (study V) and the slight variations of EPO detection indicate that currently DBS is not suited for longitudinally monitoring these markers. In addition, micro-doses of various recombinant EPOs (rEPO) and VAR-EPO were well-detected in 4 DBS devices from healthy volunteers (studies II, III). When rEPO micro-doses were administered with testosterone to healthy volunteers, as expected, there was no increase in EPO signal intensity in DBS, but in AAS users who were positive for testosterone, serum EPO concentrations were slightly elevated, along with some hematological parameters (study IV).

Conclusion: The detection and knowledge of EPO in DBS has made progress, but DBS detection methods, sample collection, and storage strategies still require further discussion and investigation.

List of scientific papers

I. Heiland CE, Ericsson M, Pohanka A, Ekström L, Marchand A. Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application. Drug Testing and Analysis. 2022;14(8): 1377-1386.
https://doi.org/10.1002/dta.3260

II. Heiland CE, Martin L, Zhou X, Zhang L, Ericsson M, Marchand A. Dried blood spots for erythropoietin analysis: Detection of micro- doses, EPO c.577del variant and comparison with in-competition matching urine samples. Drug Testing and Analysis. 2023;1-5.
https://doi.org/10.1002/dta.3596

III. Heiland CE, Lehtihet M, Börjesson A, Ekström L. Evaluation of a single Eporatio® micro-dose in urine and dried blood spots. Drug Testing and Analysis. 2024;1-4.
https://doi.org/10.1002/dta.3651

IV. Heiland CE, Schickel Y, Lehtihet M, Börjesson A, Ekström L. Supra- physiological doses of anabolic androgenic steroids impact erythropoietin and blood parameters. Drug Testing and Analysis. 2023;15(6):599-604.
https://doi.org/10.1002/dta.3452

V. Heiland CE, Mongongu C, Semence F, Pohanka A, Ericsson M, Marchand A, Ekström L. IGF-I intra-individual variation in serum and DBS using immunoassay and LC-HRMS methods. [Manuscript]