Testing for recombinant human erythropoietin in urine: problems associated with current anti-doping testing (original) (raw)
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Sensitivity and specificity of detection methods for erythropoietin doping in cyclists
Drug Testing and Analysis, 2019
Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti‐doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double‐blinded, placebo‐controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin β) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl‐PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl‐PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl‐PAGE and isoe...
Blood, 2006
Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEPO in urine. The adopted test is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and recombinant human Epo. We show here that this widely used test can occasionally lead to the false-positive detection of rhEpo (epoetin-β) in postexercise, protein-rich urine, probably because the adopted monoclonal anti-Epo antibodies are not monospecific.
Erythropoietin and blood doping
British Journal of Sports Medicine, 2006
Objective and method: To outline the direct and indirect approaches in the fight against blood doping in sports, the different strategies that have been used and are currently being used to fight efficiently against blood doping are presented and discussed. Results and conclusions: The paper outlines the different approaches and diagnostic tools that some federations have to identify and target sportspeople demonstrating abnormal blood profiles. Originally blood tests were introduced for medical reasons and for limiting misuse of recombinant human erythropoietin (rHuEPO). In this way it became possible to prevent athletes with haematocrit levels well above normal, and potentially dangerous for their health, competing in sport. Today, with nearly a decade of blood testing experience, sports authorities should be familiar with some of the limitations and specially the ability of blood tests performed prior to competitions to fight efficiently against the misuse of rHuEPO, blood transfusion, and artificial haemoglobin.
Second-generation blood tests to detect erythropoietin abuse by athletes
Haematologica, 2003
We previously developed blood tests that were introduced at the Sydney 2000 Olympic Games to identify athletes injecting recombinant human erythropoietin (rHuEPO). The aim of this study was to re-analyse our existing database to develop models with heightened sensitivity, using wherever possible blood parameters measurable with appropriate standards of analytical performance. The principal database for this study was derived from a double-blind trial in which 57 recreational athletes were administered either rHuEPO or placebo. Standard discriminant analysis was used to derive two ON models (ON-hes and ON-he) and two OFF models (OFF-hr and OFF-hre) sensitive to accelerated and decelerated erythropoiesis respectively, utilising concentrations of hemoglobin (h), erythropoietin (e) and serum transferrin receptor (s), as well as percent reticulocytes (r). The ability of our models to detect rHuEPO administration was assessed by comparing model scores of subjects in the administration tri...
Haematologica
Background and Objectives. The use of recombinant human erythropoietin (r-HuEPO) to enhance athletic performance is prohibited. Existing tests cannot readily differentiate between exogenous and endogenous EPO. Therefore the aim of our study was to investigate possible indirect detection of r-HuEPO use via blood markers of altered erythropoiesis. Design and Methods. Twenty-seven recreational athletes were assigned to three groups prior to a 25 day drug administration phase, with the following protocols: EPO+IM group (n = 10), 50 Ukg-1 r-HuEPO at a frequency of 3wk-1 , 100 mg intramuscular (IM) iron 1wk-1 and a sham iron tablet daily; EPO+OR group (n = 8), 50 U.kg-1 r-HuEPO 3wk-1 , sham iron injection 1wk-1 and 105 mg of oral elemental iron daily; placebo group (n = 9), sham r-HuEPO injections 3wk-1 , sham iron injections 1wk-1 and sham iron tablets daily. Each group was monitored during and for 4 weeks after drug administration. Results. Models incorporating combinations of the variables reticulocyte hematocrit (RetHct), serum EPO, soluble transferrin receptor, hematocrit (Hct) and % macrocytes were analyzed by logistic regression. One model (ON-model) repeatedly identified 94-100% of r-HuEPO group members during the final 2 wk of the r-HuEPO administration phase. One false positive was recorded from a possible 189. Another model (OFF-model) incorporating RetHct, EPO and Hct was applied during the wash-out phase and, during the period of 12-21 days after the last r-HuEPO injection, it repeatedly identified 67-72% of recent users with no false positives. Interpretation and Conclusions. Multiple indirect hematologic and biochemical markers used simultaneously are potentially effective for identifying current or recent users of r-HuEPO.
Testing for recombinant erythropoietin
American Journal of Hematology, 2008
Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti‐Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false‐positive detection of rhEpo (epoetin‐β) in post‐exercise, protein‐rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of ...
Recombinant erythropoietin and analogues: a challenge for doping control
Therapeutic drug monitoring, 2004
Erythropoietin (EPO) increases the number of circulating erythrocytes and thus muscle oxygenation. The availability of the recombinant protein (rEPO) has increased the risk of its illegal use in sports, its detection being a difficult challenge. Five different hematopoietic parameters were initially chosen as indirect markers of rEPO abuse: concentration of serum EPO, concentration of serum-soluble transferrin receptors (sTFr), hematocrit, percentage of reticulocytes, and percentage of macrocytes. New models considering only hemoglobin, serum EPO concentration, and percentage of reticulocytes are simpler and seem to be more sensitive when low doses of rEPO are used. A more direct method of urine analysis (isoelectrofocusing, double blotting, and chemiluminescent detection) based on the charge differences between rEPO and endogenous EPO, related to their carbohydrate composition, provides proof of rEPO use. Furthermore, this approach permits the detection of darbepoetin, a direct ana...
Recombinant human erythropoietin in sports: a review
Revista Brasileira de Medicina do Esporte, 2003
REVIEW ARTICLE ENGLISH VERSION sis, as they secrete about 90% of all systemic EPO. The liver makes about 10% of all EPO 1,2. Due to its inherent ability to stimulate red cell production, and thus increase oxygen delivery to the tissues, the use of EPO was banned in sports by the International Olympic Committee (IOC) in 1987 3. Depending on the sports modality, EPO administration to elite athletes may lead to an artificial performance improvement, decreasing, for instance, their time marks. In order to maintain health of the individual and sports ethics, IOC and other sports federations currently consider the use of EPO and its analogue drugs as blood doping cases due to exogenous administration of peptide hormone 4. Availability of this class of compounds in the market dramatically increased in the 80s, with the advent of recombinant DNA technology 5. EPO synthetic form, recombinant human EPO (rHuEPO), started to be marketed in 1988 3 ; since then, it has been successfully used in medical care, improving quality of life of patients in constant need of blood transfusions, or with anemia due to chronic renal failure 2,5. In sports, at the same time, one speculates that rHuEPO was routinely used as an artificial way to produce red cells 6 , due to the extra advantage of its presence in the biologic matrix being hard to be detected by conventional analytic methods, in addition to an effective sports performance gain. Difficulties in detection are due to the fact that EPO is a complex macromolecule, present in low concentrations in biologic fluids, with quite a similar structure to its endogenous form, which made impossible for one to accurately identify its illicit use for a long time 7,8. The purpose of this review is to place the current scientific stage in fighting EPO abuse in sports. After a short summary of endogenous EPO chemical and pharmacological properties and its recombinant forms, report of suspicious cases of EPO abuse in sports will be presented. Then, the evolution of the analytical methodology to identify and differentiate physiological EPO and its synthetic recombinant form, to apply it in doping control in sports, will present. Also new trends in the development of detection technique, and the potential use of new EPO analogue drugs available in the market will be discussed.
Hemorheological and cardiovascular effects of erythropoietin in a rat model of sports doping
2010
Recombinant human erythropoietin (rhEPO) has been therapeutically used for correction of anaemia. However, due to the increase in circulating red blood cells (RBCs) it promotes, thus increasing oxygen delivery to muscles and improving performance in sport, it has been also illegally used as sports doping. Besides the well known increase of hematocrit and blood viscosity; which might cause serious complications for the athletes, other disturbances could occur, whose mechanisms remain to be fully elucidated. This study aimed to ...