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Doping Journal 3, 1 (24 February 2006)
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Alexei R Koudinov
The Doping Journal, P.O. Box 1665, Rehovot 76100 Israel
Published online: 24 February, 2006 | Last updated: 22 September, 2007
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Copyright © 2006 by Alexei Koudinov, licensee The Doping Journal
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The study published in the major international scientific journal on blood science and medicine 
suggests International Olympic Committee (IOC) and World Anti-Doping
Agency (WADA) testing for Erythropoietin (Epo) is based on faked
Erythropoietin (Epo) is a glycoprotein hormone that is mainly produced by the kidney. It boosts the production of red blood cells by promoting the proliferation, differentiation and survival of progenitor cells of the erythroid lineage. Recombinant human Epo (rhEpo) is widely used for the treatment of various forms of anaemia. Since rhEpo increases the body's maximum oxygen consumption capacity and endurance by increasing red cell mass, it has also been embraced as an aid in endurance sports. However, this use of Epo was prohibited by IOC and WADA, which has led to the screening of athletes for rhEpo abuse.
Endogenous and recombinant human forms of Epo have different pattern of molecule modification called glycosylation. Such a tiny difference between natural Epo, its' biotechnological and structural analogs (such as Darbepoetin) leads to the molecule electric charge differences that have been exploited to distinguish endogenous and recombinant isoforms by a so called isoelectric focusing technique.
The adopted by WADA urine test for EPO is based on a combination of isoelectric focusing on a gel (a semi-analytical separation of proteins according to the molecules' net electric charge), the transfer of proteins from gel to a special paper and biomolecules detection by a double immunoblotting, not well established complicated and operator-dependent procedure to immunologically detect proteins of interest with an antibody color reaction.
In the Blood journal study research group led by Associate Professor Monique Beullens and Professor Mathieu Bollen (of the Department of Molecular Cell Biology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium) and their colleague Dr. Joris R Delanghe (of the Department of Clinical Chemistry, University Hospital, Gent, Belgium) shows that widely-used by IOC and WADA Epo test can lead to the false-positive detection of rhEpo in post-exercise, protein-rich urine of the endurance sports athletes.
As a result of a disputed case of alleged rhEpo-abuse by an endurance athlete with post-exercise proteinuria, European scientists wondered whether the test for rhEpo can lead to false-positive results, perhaps as a result of cross-reactivity of the Epo-antibodies with unrelated to Epo other proteins of urine.
Straightforward experimental protocol of the reported study leaves little doubt that the major urinary protein that WADA test visualizes with the ani-Epo antibodies is not Epo.
| False-positive detection of recombinant human erythropoietin in urine
following strenuous physical exercise
Blood 107(12): 4711-3 (15 June 2006, ePpub 21 Feb 2006) (Ref. 1).
Beullens M, Delanghe JR, Bollen M.
Department of Molecular Cell Biology, Catholic University of Leuven, Belgium.
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-beta) in postexercise, protein-rich urine, probably because the adopted monoclonal anti-Epo antibodies are not monospecific.
Lasne F. Blood 108(5): 1778-9 (1 Sep 2006) [Ref. 8].
Catlin D, Green G, Sekera M, Scott P, Starcevic B. Blood 108(5): 1778 (1 Sep 2006) [Ref. 9]
Beullens M, Delanghe JR, Bollen M. False-positive detection of rhEpo remains a real concern. Blood 108(5): 1778 (1 Sep 2006) [Ref. 16].
The article by Belgian scientists therefore challenges WADA claim  that "the detection method for EPO is valid and reliable. "Similarly faulty now sounds another WADA statement  that the method for Epo detection "has undergone an extensive scientific validation..." and that "it is a well-established procedure widely accepted by the scientific community, as demonstrated by publication in a number of international scientific journals."
Contrary to WADA claim  the Doping Journal analysis of citation impact of earlier publications on Epo testing in urine [3, 4, 5, 6] indicates IOC/WADA method for Epo testing is not scientifically popular or well-established. An in depth analysis of the articles behind the IOCs' urine test for Epo shows these earlier publications missed critical control experiments [3, 4, 5, 6] and were not designed to exclude non-specific false-positive misidentification of other non-Epo urine components.
Please note: web enhanced references below provide no registration free access to documents
1. Beullens M, Delanghe JR, Bollen M. False-positive detection of recombinant human erythropoietin in urine following strenuous physical exercise. Blood 107, 4711-4713 (2006) [ PubMed ][ Free FullText ][ Back2Text ].
2. Clarification about the EPO Detection Method. World Anti-doping Agency (Last viewed 22 September 2006) [ FullText ][ Back2Text ].
3. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature 405, 635 (2000) [ PubMed ]
4. Catlin DH, Breidbach A, Elliott S, Glaspy J. Comparison of the isoelectric focusing patterns of darbepoetin alfa, recombinant human erythropoietin, and endogenous erythropoietin from human urine. Clin Chem 48, 2057-2059 (2002) [ PubMed ][ Free Full Text ][ Back2Text ].
5. Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem 311, 119-126 (2003) [ PubMed ][ Back2Text ].
6. Breidbach A, Catlin DH, Green GA, Trgub I, Trong H, Gorzek J. Detection of recombinant human erythropoietin in urine by isoelectric focusing. Clin Chem 49, 901-907 (2003) [ PubMed ] [ FullText ][ Back2Text ].
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8. Lasne F. No doubt about the validity of the urine test for detection of recombinant human erythropoietin. Blood 108(5), 1778-1779 [ PubMed ][ Free FullText ][ Back2Text ].
9. Catlin D, Green G, Sekera M, Scott P, Starcevic B. False-positive Epo test concerns unfounded.
Blood 108(5), 1778-1778 (1 Sep 2006) [ PubMed ][ Free FullText ][ Back2Text ].
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14. Anatoly Kucherena. Bez Krovi ne Oboitis' (Анатолий Кучерена. Без крови не обойтись) There is no way. Without blood) National Reviews (2003) (ISBN 5-902207-04-5) pp.253 [ Google search for this publication ][ Back2Text ].
15. Muehlegg vs. International Olympic committee. Case No. CAS 2002/A/374. Arbitral Award, delivered by the Court of Arbitration for Sport [ URL (.PDF) ][ URL (.HTML) ][ Back2Text ].
16. Beullens M, Delanghe JR, Bollen M. False-positive detection of rhEpo remains a real concern. Blood 108(5), 1779-1780 [ Free FullText ][ Back2Text ].
17. Franke WW, Heid H. Pitfalls, errors and risks of false-positive results in urinary EPO drug tests. Clin Chim Acta 373(1-2), 189-90 (Nov 2006, ePpub 20 May 2006) [ PubMed ][ Back2Text ].
18. Krimsky S. Science in the private interest: Has the lure of profits corrupted the virtue of biomedical research. Rowman & Littlefield Publishers, Inc. Oxford, UK (2003)(ISBN 0-7425-1479-X) pp. 247 [ Author Web site ][ Google search for this publication ][ Back2Text ].
19. World Anti-Doping Code version 3.0 (20 February 2003) World Anti-Doping Agency. IOC Web Site [ URL (.PDF) ][ Back2Text ].
20. Rushall B S., Jones M. The anti-drugs-in-sport movement: Causes for concern. International Journal of Sports Science and Coaching 1(1), 1-18 (2006) [ Google search for this publication ][ Back2Text ].
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article should be cited in the following way:
Koudinov AR. WADA, IOC testing for Erythropoietin is faked.
The Doping Journal
Vol. 3, 1 (2006) Available at: http://dopingjournal.org/content/3/1/
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