Delanghe, Joris

Joyner, Michael

English

Ghent University Academic Bibliography

 105:395-396, 2008. First published Jun 26, 2008;  doi:10.1152/japplphysiol.90746.2008 J Appl PhysiolJoris R. Delanghe and Michael J. Joyner  You might find this additional information useful...12 articles, 6 of which you can access free at: This article cites  http://jap.physiology.org/cgi/content/full/105/2/395#BIBL2 other HighWire hosted articles: This article has been cited by    [PDF]  [Full Text], December 1, 2008; 105 (6): 1992-1993. J Appl PhysiolM. Saugy, N. Robinson, S. Lamon, W. Schanzer and H. Geyer Lasne, Y. O. Schumacher, T. Pottgiesser, J. Morkeberg, B. Belhage, R. Damsgaard, V. Skibeli, G. Lippi, W. E. Jelkmann, G. L. Salvagno, G. C. Guidi, C. R. Valeri, G. Ragno, B. Kayser, F. anti-doping testing: is quality assessment a reliable solution?problems associated with current anti-doping testing". Problems associated with current Comments on Lundby et al.'s "testing for recombinant human erythropoietin in urine:   [PDF]  [Full Text], December 1, 2008; 105 (6): 1990-1991. J Appl PhysiolG. Lippi, G. Banfi, J. Morkeberg, B. Belhage, R. Damsgaard and V. Skibeli  erythropoietin".Comments on Delanghe and Joyner's editorial "testing for recombinant humanincluding high-resolution figures, can be found at: Updated information and services  http://jap.physiology.org/cgi/content/full/105/2/395 can be found at: Journal of Applied Physiologyabout Additional material and information  http://www.the-aps.org/publications/japplThis information is current as of March 19, 2009 .    http://www.the-aps.org/.ISSN: 8750-7587, ESSN: 1522-1601. Visit our website at Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society.those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American  publishes original papers that deal with diverse areas of research in applied physiology, especiallyJournal of Applied Physiology on March 19, 2009 jap.physiology.orgDownloaded from Invited EditorialTesting for recombinant human erythropoietinJoris R. Delanghe1 and Michael J. Joyner21Department of Clinical Chemistry, Ghent University Hospital, Ghent, Belgium; 2Department of Anesthesiology, Mayo Clinic,Rochester, MinnesotaERYTHROPOIETIN (Epo) may have effects on exercise capacityand physiological regulation beyond a simple increase in redcell mass and the associated improvement in oxygen transport(4). In the context of a larger study on this topic, Lundby andcolleagues (11) also asked questions about the reliability ofurine testing for recombinant human Epo (rHuEpo). Theystudied eight healthy male subjects during a 4-wk “loading”and 2-wk “boosting” phase of Epo use followed by a 2-wkmaintenance phase. In the parent study they showed that theeffects of Epo on exercise performance were confined to itsimpact on red cell mass and not to other physiological effectsof the hormone. These results were consistent with ideas aboutthe relationship between maximal oxygen uptake and red cellmass or total body hemoglobin that emerged in the 1950s. Thefindings are timely and have implications for public policy relat-ing to the control of doping practices. In this short report a numberof challenges related to urine testing for Epo are highlighted.Testing for recombinant Epo in urine may seem practical atfirst sight but appears to be a very difficult task. The amount ofendogenous Epo in urine is extremely low (5). The physiolog-ical background for testing Epo in urine is complex and thehandling of Epo by the renal tubules is poorly understood (16).Furthermore, exercise-induced renal ischemia and the accom-panying postexercise proteinuria may affect the clearance ofthis 32- to 39-kDa protein and the quality of the urine matrix.The Epo test that has been adopted in World Anti-DopingAgency (WADA)-accredited laboratories is based on isoelec-tric focusing (2, 10). Since the introduction of the test in 2000,Epo-abusing athletes have altered their dosing schemes, prov-ing the initial efficiency of the test. By injecting microdoses ofrHuEpo, the window of detection can be reduced to as little as12–18 h postinjection (8).Several problems (e.g., the lengthy sample preparation,the low sample load capacity, difficulties with interlabora-tory standardization, non-specific binding of the secondaryantibody to urinary proteins, sensitivity issues) were alreadyidentified in a WADA-commissioned report in 2003 (14).The test requires a 700- to 1,000-fold concentration of thespecimen before analysis can be carried out, and the con-centrated urine forms a pellet that is difficult to solubilize.Despite this enormous concentration factor, up to 20% ofthe investigated samples do not show detectable Epo (14).The non-specific interaction of the used monoclonal anti-body with human, bacterial, and yeast proteins is worrisome(4, 6). Epo test results are clearly not always interpretedidentically (4). The use of the software processing has beencriticized (6).The American WADA-accredited laboratory has performedthe direct Epo test on more than 2,600 samples, only nine ofthem were found to be positive (3). The low numbers ofathletes caught by the test are somewhat contradictory to theoverall increase of mean hematocrit values since rHuEpobecame available (12). Additionally, in some high profile legalcases in the United States, athletes who were clearly dopingwith a variety of compounds including Epo “passed” hundredsof individual drug tests.Along these lines, Lundby et al. (11) convincingly demon-strated that the performance of the urinary Epo test is some-what disappointing. Although the judgment process of “real”doping cases differs from the one applied in the present study,the high number of false-negative results imply a risk thatathletes doping with Epo will avoid detection and damage thefundamental goal of fair competition. The earlier reportedflaws of the test help to understand the relatively low efficiencyof the direct Epo test and the current results emphasize the needfor improving Epo testing.The detection window for Epo shows an interindividualvariation because the actual positivity criteria take into consid-eration the endogenous Epo production rate, which variesenormously between individuals. In subjects with a naturallyelevated or stimulated Epo production rate (altitude training,hypoxic tent, etc.), there is a reduced detection window. Thepositivity criteria used by anti-doping laboratories are strict and(in the case of the Epo analog darbepoietin) could be adaptedby only taking into account the position and the specificdistribution of the bands in the most acidic area of the gel andno longer the intensity of the bands. This would rule out thedifferences in interpretation of the test due to different endog-enous Epo levels in individual athletes (8). While the perfor-mance of the existing test can likely be improved by payingmore attention to the preanalytical care after prelevation ofurine specimens (4), there also is concern about protease-treated urine specimens that could mask Epo abuse (9).On the other hand, blood-based indirect Epo tests have abetter physiological basis and offer the advantage that otherkinds of blood doping can also be detected (1, 13, 15). Thisapproach focuses on consistent tracking and establishes upperlimits of normal permitted for competition. The so-called“passport” approach will be further facilitated by the increasedavailability of well-validated mobile hematological analysisequipment. Longitudinal monitoring of blood profiles andcomparing an athlete’s individual hematological values againsthis or her own historical baseline rather than a population-derived threshold further enhances the potency of indirecttesting (1). In the Union Cycliste International (UCI), thegoverning body of cycling, anti-doping program “100% AgainstDoping,” an individual hematological profile is created. How-ever, indirect Epo/blood doping detection methods requireblood sampling, which is a practical disadvantage. Addition-ally, various dietary or saline infusion strategies for the pur-poses of short-term hemodilution are likely being practiced tocircumvent the “passport” approach prior to important compe-Address for reprint requests and other correspondence: J. R. Delanghe,Univ. Hospital Ghent, De Pintelaan 185, B-9000 Gent, Belgium (e-mail: joris.delanghe@ugent.be).J Appl Physiol 105: 395–396, 2008;doi:10.1152/japplphysiol.90746.2008.8750-7587/08 $8.00 Copyright © 2008 the American Physiological Societyhttp://www. jap.org 395 on March 19, 2009 jap.physiology.orgDownloaded from titions (7). Does this mean that a combination of regulartracking tests with random elements along with a marker ofwhole body fluid status is needed?In summary, the data provided by Lundby et al. demonstratethat an improvement in the current Epo test is necessary or thata different strategy to detect Epo use and blood doping shouldbe considered. Blood-based indirect Epo tests offer an inter-esting alternative. The rapidly changing blood doping land-scape will definitely encourage the use of indirect Epo/blooddoping testing with a much broader application.REFERENCES1. Ashenden M, Varlet-Marie E, Lasne F, Audran M. The effects ofmicrodose recombinant human erythropoietin regimens in athletes.Haematologica 91: 1143–1144, 2006.2. Catlin D, Nissen-Lie G, Howe C, Pascual JA, Lasne F, Saugy M.Harmonization of the method for the identification of epoetin alfa and beta(rEPO) and darbepoetin alfa (NESP) by IEF-double blotting and chemi-luminescent detection. WADA Technical Document TD2007EPO. Ac-cessed June 16, 2008. Available at http://www.wada-ama.org/rtecontent/document/td2007epo_en.pdf.3. Catlin D, Green G, Sekera M, Scott P, Starcevic B. False-positive Epotest concerns unfounded. Blood 108: 1778, 2006.4. Delanghe J, Bollen M, Beullens M. Testing for recombinant erythropoi-etin. Am J Hematol 83: 237–241, 2008.5. Emslie KR, Howe C, Trout H. Measurement of urinary erythropoietinlevels in athletes. In: Proceedings of the Manfred Donike Workshop, 17thCologne Workshop on Dope Analysis, Recent Advances in Doping Anal-ysis, Vol. 7, edited Scha¨nzer W, Geyer H, Gotzmann A, Mareck-EngelkeU. Cologne: Sport and Buch Strau, 1999, p. 291–299.6. Franke WW, Heid H. Pitfalls, errors and risks of false-positive results inurinary EPO drug tests. Clin Chim Acta 373: 189–190, 2006.7. Freund BJ, Montain SJ, Young AJ, Sawka MN, DeLuca JP, PandolfKB, Valeri CR. Glycerol hyperhydration: hormonal, renal, and vascularfluid responses. J Appl Physiol 79: 2069–2077, 1995.8. Lamon S, Robinson N, Mangin P, Saugy M. Detection window ofDarbepoetin-alpha following one single subcutaneous injection. Clin ChimActa 379: 145–149, 2007.9. Lamon S, Robinson N, Sottas PE, Henry H, Kamber M, Mangin P,Saugy M. Possible origins of undetectable EPO in urine samples. ClinChim Acta 385: 61–66, 2007.10. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature405: 635, 2000.11. Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM,Robach P. Testing for recombinant human erythropoietin in urine: prob-lems associated with current anti-doping testing. J Appl Physiol;doi:10.1152/japplphysiol.90529.2008.12. Morkeberg J, Saltin B, Belhage B, Damsgaard R. Blood profiles in elitecross-country skiers: a 6-year follow-up. Scand J Med Sci Sports. 2008Feb 17. [Epub ahead of print].13. Parisotto R, Wu M, Ashenden MJ, Emslie KR, Gore CJ, Howe C,Kazlauskas R, Sharpe K, Trout GJ, Xie M. Detection of recombinanthuman erythropoietin abuse in athletes utilizing markers of altered eryth-ropoiesis. Haematologica 86: 128–137, 2001.14. Peltre G, Thormann W. Evaluation Report of the Urine EPO Test. Paris,France, and Bern, Switzerland: Council of the World Anti-Doping Agency(WADA), 2003.15. Sharpe K, Ashenden MJ, Schumacher YO. A third generation approach todetect erythropoietin abuse in athletes. Haematologica 91: 356–363, 2006.16. Skibeli V, Nissen-Lie G, Torjesen P. Sugar profiling proves that humanserum erythropoietin differs from recombinant human erythropoietin.Blood 98: 3626–3634, 2001.Invited Editorial396J Appl Physiol • VOL 105 • AUGUST 2008 • www.jap.org on March 19, 2009 jap.physiology.orgDownloaded from 

Testing for recombinant human erythropoietin

https://biblio.ugent.be/publication/524474/file/526367.pdf

Testing for recombinant human erythropoietin

Abstract

Similar works

Full text

Available Versions

Ghent University Academic Bibliography