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Antidoping testing: friend or foe?
Testing for recombinant human erythropoietin: The bouquet of compounds
Problems associated with current anti doping testing: is quality assessment a reliable solution?
Comments on paper by Lundby and colleagues
A negative test cannot exclude doping
To the Editor:
New strategies in the fight against doping are necessary
Expanding the analysis reporting will lead to more efficient testing
To the Editor:
To the Editor:
To the Editor:
Final Word - Response to Commentaries
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Giuseppe Lippi, Associate Professor of Clinical Biochemistry Sez. Chimica Clinica, Università di Verona, Osp. Policlinico, 37134 - Verona, Italy
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ulippi{at}tin.it Giuseppe Lippi
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Ideally, antidoping testing is designed to ensure fair competition and prevent athletes’ harm from unfair practices. While the former issue is questionable, since fairness in sports is mostly a chimera, the latter is foremost and theoretically viable (1-3). The recent study of Lundy et al. proved what everybody in the field of laboratory and sports medicine has supposed for years: a poor agreement in test results for detecting recombinant human erythropoietin (rHuEpo) comparing two World Anti Doping Agency (WADA) accredited laboratories (4). Laboratory tests are not always foolproof. For various reasons, false-positive and false-negative results can occur for any test in laboratory diagnostics (3). Traditionally, both sensitivity and specificity determine the diagnostic efficiency of a certain test in the clinical practice. However, the extension of this concept to the antidoping context is challenging. Basically, antidoping tests are not intended for diagnosing disorders or monitoring therapies. Therefore, sensitivity is only important to identify cheaters (rarely disorders), but specificity is foremost to prevent unfair sanctioning of clean athletes. Several lines of evidence attest that the current antidoping strategy, based on repression rather than education or harm control, has returned negligible results, if none, in terms of preventing athletes from doping and modifying this upward trend. Probably, it has only contributed to modify the pattern of drugs consumption (5). The report of Lundby et al. must hence be regarded as an additional mainstay in the urgent process of reviewing the current antidoping policy based on analytically questionable tests. References 1. Kayser B, Mauron A, Miah A. Current anti-doping policy: a critical appraisal. BMC Med Ethics 2007;8:2. 2. Lippi G, Banfi G, Franchini M, Guidi GC. New strategies for doping control. J Sports Sci 2008;26:441-5. 3. Lippi G, Franchini M, Guidi GC. Doping in competition or doping in sport? Br Med Bull 2008;86:95-107. 4. Lundby C, Achman-Andersen NJ, Thomsen NJ, Norgaard AM, Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). 5. Lippi G, Franchini M, Guidi GC. Haematological testing and antidoping policies. Int J Sports Med 2005;26:508-9. |
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Wolfgang E Jelkmann, Institute of Physiology University of Luebeck, Luebeck, Germany
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jelkmann{at}physio.uni-luebeck.de Wolfgang E Jelkmann
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Sir, Lundby and coworkers (4) show poor agreement in the results obtained in two WADA accredited laboratories applying isoelectric focusing (IEF) for detection of recombinant human erythropoietin (rhEpo) in urines from male volunteers treated with Epoetin beta. This rhEpo and its analogue, Epoetin alfa, are Epo transfected Chinese hamster ovary (CHO) cell-derived drugs clinically used for almost 20 years. Since process patents for the originator rhEpo have expired in the EU and elsewhere, biosimilar follow- on biologics have entered the market. Recently, two novel CHO cell-derived Epoetin biosimilars have been approved in the EU, which are marketed as Epoetin alfa respectively Epoetin zeta (3). Moreover, several companies outside the US and EU are producing so-called Epoetin alfa. While the amino acid sequence of all Epoetins is identical, the structure of their glycans differs depending on the production procedures. The glycans determine the behaviour on IEF. Some products exhibit more acidic and others more basic isoforms (1). Furthermore, Epoetin delta is available, which is homologously expressed in human cells, thus lacking N- glycolylneuraminic acid similar to endogenous Epo (3). No method is established yet to detect pegylated Epoetin beta in urine. As noted earlier, searching rhEpo in urine is “tilting at windmills” (2). References 1. Combe C, Tredree RL, and Schellekens H. Biosimilar epoetins: an analysis based on recently implemented European medicines evaluation agency guidelines on comparability of biopharmaceutical proteins. Pharmacotherapy 25: 954-962, 2005. 2. Jelkmann W. Novel erythropoietic agents: a threat to sportsmanship. Medicina Sportiva 11: 32-42, 2007. 3. Jelkmann W: Developments in the therapeutic use of erythropoiesis stimulating agents. Brit J Haematol 141: 287-297, 2008. 4. Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM, and Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). Doi:10.1152/japplphysiol.90529.2008. |
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Gian Luca Salvagno, Research Professor of Clinical Biochemistry Sez. Chimica Clinica, Università di Verona, Gian Cesare Guidi
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gianluca.salvagno{at}univr.it Gian Luca Salvagno, et al.
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In the recent article of Lundy et at. (1), a rather poor agreement of results was demonstrated between two World-Antidoping Agency (WADA) accredited laboratories. This is not really surprising, in that laboratory test results have not always been, nor they are, as reliable as they should be due to several incidental variables, which variably affect the pre-analytical, analytical and post-analytical phases (2). Laboratory professionals have struggled for decades with this issue, in an attempt to make test results reliable and, especially, comparable between different laboratories. One of the best solution to standardize or harmonize (when standardization is unreachable) test results is to establish a broad concept of quality control for the whole testing process (3). Basically, quality Control (QC) refers to procedures for monitoring the work processes, detecting problems, and making corrections prior to delivery of products or services. Statistical process control, or statistical quality control, is the major procedure for monitoring the analytical performance of laboratory methods. Quality Assessment (QA) refers to the broader monitoring of other dimensions or characteristics of quality, such as patient preparation, specimen acquisition and handling. Then, proficiency testing provides an external or outside measure of analytical performance (3,4). Regardless of inherent limits of the current technique to detect recombinant human erythropoietin in urine, the implementation of rigorous external quality assessment schemes (EQAS), based on urine samples shipped to the all the accredited WADA laboratories worldwide, would be probably helpful to increase consistency in results between different facilities. References 1. Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM, Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). doi:10.1152/japplphysiol.90529.2008 2. Lippi G, Fostini R, Guidi GC. Quality improvement in laboratory medicine: extra-analytical issues. Clin Lab Med 2008;28:285-294. 3. Westgard JO, Burnett RW, Bowers GN. Quality management science in clinical chemistry: A dynamic framework for continuous improvement. Clin Chem 1990;36:1712-1716. 4 Westgard JO, Barry PL. Beyond quality assurance: Committing to quality improvement. Lab Med 1989;20:241-247. |
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C. Robert Valeri, MD Naval Blood Research Lab, Boston, MA, Gina Ragno
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navblood{at}nbrl.org C. Robert Valeri, MD, et al.
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The Lundby paper (6) and Delanghe and Joyner commentary (7) describe numerous technical issues related to testing blood and urine for the presence of recombinant human erythropoietin (rhEpo). The blood level is affected by plasma volume and the urine level is affected by the renal tubular handling of Epo which is poorly understood. My major concern with this report is that erythropoietin treatment elevates hemoglobin concentration by increasing red cell volume (RCV) and decreasing plasma volume (PV). RCV was measured directly using carbon monoxide. PV was estimated indirectly from the carbon monoxide RCV and the total body hematocrit, using the f-factor of 1.0 to correct the peripheral venous hematocrit value, at baseline, 5, 11 and 13 weeks after Epo treatment. To assert that Epo decreases PV, a direct measurement of PV is needed and it should be done 2 weeks following treatment with Epo to assess the renin-angiotensin-aldosterone activity. Our experience has shown the factor to correct the peripheral venous hematocrit to estimate the total volume hematocrit depends on the method used to measure PV (1). Our data in chronic hypovolemic anemic patients and acute hypovolemic anemic dogs demonstrated that transfusion of viable washed red cells increased RCV, PV and total blood volume (2-5). The information reported that erythropoietin treatment elevated hemoglobin concentration by increasing the RCV and decreasing PV is not supported by our data. A direct measurement of PV is needed to document that erythropoietin decreases PV which will affect the measurement of erythropoietin in blood. REFERENCES 1. Valeri CR, Cooper AG, Pivacek LE. Limitations of measuring blood volume with iodinated I 125 serum albumin. Arch Int Med 132:534-538, 1973. 2. Valeri CR and Altschule MD. Hypovolemic Anemia of Trauma: The Missing Blood Syndrome, Chemical Rubber Company, Boca Raton, Florida, 1981. 3. Valeri CR, Donahue K, Feingold HM, Cassidy GP, Altschule MD. Increase in plasma volume after the transfusion of washed erythrocytes. Surg Gynec Obstet 162:30-36, 1986. 4. Valeri CR. Transfusion medicine and surgical practice. Bull Am Coll Surg 78(9):19-24, 1993. 5. Valeri CR, Crowley JP, Loscalzo. The red cell transfusion trigger: has a sin of commission now become a sin of omission? Transfusion 38:602-610, 1998. 6. Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM, Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). doi:10.1152/japplphysiol.90529.2008 7. Delanghe J and Joyner M. Testing for recombinant human erythropoietin. J Appl Physiol (June 26, 2008). doi:10.1152/japplphysiol.90746.2008 |
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Bengt Kayser, Professor Institute of movement sciences and sports medicine, University of Geneva, Switzerland.
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bengt.kayser{at}unige.ch Bengt Kayser
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Lundby et al. (3) illustrate an Achilles heel of anti-doping policy. The war on doping aims at eradication using a strong repressive system, based, among other strategies, on testing for forbidden substances and metabolites in bodily specimens, mainly urine but also blood. For surprise controls outside competition athletes have to report where they are 365 days/yr to be compelled to provide urine samples, being nude from nipple to knee while a doping officer looks on, making sure the sample is genuine. But assuming that testing protocols accurately expose the use of drugs is scotomizing the fact that testing is condemned to remain one step behind the advances in biomedicine. False negatives and false positives are inherent possibilities with testing technology as clearly shown by Lundby et al (3). This uncertainty of laboratory testing is acceptable in the field of therapeutic medicine but problematic in sport. Athletes can never be declared 'truly' clean, whereas false accusations should be avoided by all means. Marion Jones never tested positive throughout her career. On the other hand, many athletes are treated as cheaters even if there is good reason to doubt their intention to dope (1). Instead of pursuing an increasingly repressive strategy at rising cost and with limited efficacy, there are good reasons to question current anti-doping policies and to plea for a more pragmatic approach aiming at reducing harm to the individual and society at large (2). 1) Babette Pluim. A doping sinner is not always a cheat. Br J Sports Med 2008; 42: 549-550. 2) Kayser B, Smith AC. Globalisation of anti-doping: the reverse side of the medal. BMJ. 2008 Jul 4;337:a584. doi: 10.1136/bmj.a584. 3) Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM & Robach P. Testing for recombinant human erythropoietin in urine: Problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). doi:10.1152/japplphysiol.90529.2008. |
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Francoise Lasne, Head of Biology Analysis department, French agency of fight against doping
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f.lasne{at}afld.fr Francoise Lasne
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As the developer of the test validated for anti-doping control of recombinant erythropoietin (rHuEPO), I have been asked to comment on the article of Lundby et al. “Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing” ^(1) . The French anti-doping laboratory did not participate in this study and the two laboratories involved certainly will give detailed technical explanations about the real conditions and the conclusions of this study. I am fully confident in their ability to set the record straight. However, I would like to address just one question to the authors of this article. The article aims to demonstrate the failure of the test validated for anti-doping control (isoelectric focusing followed by double-blotting ^(2- 4) ) based on the assertion of a discrepancy in the results obtained by the two laboratories. For this, the authors state that both laboratories used the validated method. In fact, this method was used by laboratory A, while laboratory B used another method (SDS electrophoresis), currently under investigation as a possible complementary method. My question is this: What is the value of a scientific procedure that uses the results of method B to prove the failure of method A? 1 Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM, Robach P. <http://www.ncbi.nlm.nih.gov/pubmed/18583375?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum> Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol. 2008 Jun 26. 2 Lasne F, de Ceaurriz J. <http://www.ncbi.nlm.nih.gov/pubmed/10864311?ordinalpos=21&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum> Recombinant erythropoietin in urine. Nature. 2000 Jun 8;405(6787):635. 3 Lasne F. <http://www.ncbi.nlm.nih.gov/pubmed/11384674?ordinalpos=18&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum> Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures. J Immunol Methods. 2001 Jul 1;253(1-2):125-31. 4 Lasne F, Martin L, Crepin N, de Ceaurriz J. <http://www.ncbi.nlm.nih.gov/pubmed/12470670?ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum> Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem. 2002 Dec 15;311(2):119-26. |
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Yorck O. Schumacher Medizinische Universiaetsklinik Freiburg, Torben Pottgiesser
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olaf{at}msm1.ukl.uni-freiburg.de Yorck O. Schumacher, et al.
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Although several issues concerning the EPO-urine test remain controversial, the article by Lundby et al.(1) emphasizes the fact that the fight against doping might need a significant modification in the future. Since the first in-competition doping tests in the 1960´s and the introduction of out-of-competition urine tests 20 years later, anti doping strategies have considerably evolved. Subsequent to the development of recombinant doping substances which are difficult if not impossible to detect in urine such as EPO, blood data is used to screen for suspicious athletes. Not an abused substance itself, but its effects on the organism are analysed. Through these measures, anti-doping developed from the purely biochemical detection of a forbidden substances to a more biological approach. The study of Lundby et al. strengthens this direction and suggests new options: Despite the fact that only a limited number of samples was found positive according to the WADA criterions, a larger number was defined as “suspicious”. This important information should be included in the so-called “biological passport”, which aims at a longitudinal monitoring of variables indicative of doping: It is likely that the entity of increased Hb mass, increased Haematocrit (as shown in table 1 of the article) and suspicious EPO urine test would result in a high prospect of doping in the examined athletes in any probabilistic calculation (2). With more innovative doping methods looming, the anti doping fight should develop from its pure biochemical/ biological horizon into a probabilistic framework, considering all available information. 1. Lundby C, Achman-Andersen, NJ, Thomsen JJ, Norgaard AM, Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). 2. Sottas PE, Baume N, Saudan C, Schweizer C, Kamber M, Saugy M. Bayesian detection of abnormal values in longitudinal biomarkers with an application to T/E ratio. Biostatistics 2007; 8: 285-296. |
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Jakob Mørkeberg cand.scient, Bo Belhage and Rasmus Damsgaard
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jakobmoerkeberg{at}hotmail.com Jakob Mørkeberg, et al.
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Lundby et al. (1) show marked differences between results obtained from two WADA- accredited laboratories using isoelectric focusing (IEF) to detect recombinant human erythropoietin (rhEpo) in urine samples. As proved by Laboratory A, there is no doubt that the implemented rhEpo-test (2) can detect rhEPO, but the test also has certain limitations i.e. a short window of detection (3) due to the strict criteria used to deem a test result as an ‘adverse analytical finding’. Today, sport federations that receive test results from the rhEpo-test, are only provided with either one of the following ‘results’: ‘Negative’, ‘adverse analytical finding’ or ‘atypical analytical finding’ from the laboratory. Therefore, highly suspicious sample results are likely to be deemed ‘negative’, with no further reporting to the federation. Large amounts of important analysis data are simply wasted, because it never reaches the right body – the federations. Information, that can be used by federations not only to compare previous data with new on a individual level as is currently being done for various blood variables (4) and anabolic steroids (5) but also used in combination with blood screening results to give a better overall picture of the individual athlete. This will not only increase the sensitivity of testing for rhEpo but also make the targeting of urine analysis much more efficient. Therefore, in the future federations must be provided with a full report of the specific analysis, including images and quantitative data from the analysis. The data is already there – why don’t we use it??? 1. Lundby C, Achman-Andersen, NJ, Thomsen JJ, Norgaard AM, Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing. J Appl Physiol (June 26, 2008). 2. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature. 2000 Jun 8;405(6787):635. 3. Ashenden M, Varlet-Marie E, Lasne E, Audran M. Haemmatologica 2006 Aug;91(8):1143-4. Epub 2006 Jul 25. 4. Sharpe K, Ashenden MJ, Schumacher YO. A third generation approach to detect erythropoietin abuse in athletes. Haematologica 2006: 91: 356- 363. 5. Sottas PE, Baume N, Saudan C, Schweizer C, Kamber M, Saugy M. Bayesian detection of abnormal values in longitudinal biomarkers with an application to T/E ratio. Biostatistics 2007; 8: 285-296. |
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Martial Saugy, Laboratoire Suisse d’Analyse du Dopage, Institut Universitaire de Médecine Légale Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland, Neil Robinson and Séverine Lamon
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Martial.Saugy{at}chuv.ch Martial Saugy, et al.
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Context: Since spring 2006, the laboratory of Lausanne has been collaborating with the group of Lundby et al. Among others, we accepted that, during the operational phase, the principle of double blind study would be preserved. We also accepted to discuss about the results to avoid any misunderstanding. This discussion never happened and we were never informed about any publication release from this study. Laboratory A: By reading the data presented in the publication and by carefully reviewing those we originally sent to the group, we can assume that our laboratory is the so-called laboratory A and will produce our comments in that perspective. In order to be fair in our response to the Editor regarding this publication, we recently asked the first author to confirm in written that it was actually the case and again we did not get any formal answer. Material and methods: Four deficient points at least must be highlighted in the description of the methods - 1. Treatment : When the dosage is described, the type of administration is not. It is however known that subcutaneous and I.V. administration of EPO yield very different pharmacokinetic results (1). As the authors aim to provide a good picture of the detection power of a method to the scientific community, they should have known that it is directly related to the application mode of the substance. No clear description exists concerning the treatment schedule during the maintenance period (“one single injection was given weekly”). This lets to the authors the possibility of playing with the detection power of the laboratories by occulting to the reader the precise information about how far from the last injection time the urine collection took place. 2. Parameters of interest : There is no mention of the type of technology used to perform the hematocrit (Hct) measurement. Not any other blood parameter was apparently measured. As the authors state in their conclusions, the longitudinal monitoring of athletes is one solution for the detection of blood manipulation. From then on, it is really surprising to see that the authors limit themselves to using Hct, which is nowadays claimed to be highly insufficient for providing a good picture of blood doping. No reticulocyte (Ret%) count, no hemoglobin (Hb) concentration, as well as no off-score have been provided to the reader, even if these parameters are currently considered as the state of the art and the minimum requirement for this monitoring. 3. Samples: On the contrary to what is asserted by the authors, several shipments were necessary to receive the biological material in acceptable conditions for an optimal application of the EPO analysis method. Multiple analyses were performed in duplicate, in order to insure undisputable results to be delivered. 4. EPO detection method: There is simply no mention nor description of the method(s) that was (were) used by the laboratories to screen the presence of rh-EPO in the urine samples. Discussion: Because of a lack of common and usual definition on the reporting of the results from the two laboratories to the authors, as well as because of no mention that one of the lab did not use the official method of EPO detection, the argumentation is basically not based on the same rationale through the entire discussion. In our particular case, we analyzed all samples using the official accredited EPO detection method and returned all the results according to the WADA positivity criteria (TD2007EPO), allowing us, in case of necessity, to defend all of them in front of a court . Negative Day 30, Positive Day 35: Every scientist involved in the field perfectly knows how the urinary matrix can be difficult to handle. Even for other doping substances, it has been observed that the detection can strongly depend on the matrix. In this particular case, day 30 sample, which is presented as negative by the authors, was in fact an undetectable sample that was easily explainable by a low urinary EPO concentration (3) as well as by a low specific gravity of the sample. The main investigators were obviously informed about that but did not take it into account. Moreover, the authors claim that this pseudo-negativity is a proof of false positivity for day 35 sample. Indeed, they consider that, as the detection window for rh-EPO has been reported to be of about three days, no positive case should be declared after a longer period of time. However, our results are completely in accordance with Breidbach’s paper(2), who reported that, on the seventh day after the last of nine rh- EPO doses, this last was detected in approximately one-half of the participants to his study. Conclusion: In conclusion, for all the previous mentioned reasons, the published results cannot be credited with any scientific credibility and demonstrate of a great lack of knowledge of the authors regarding the EPO detection method and more generally of the whole anti-doping procedures. This publication has to be considered as a major offense to the scientists working since years to help the sports authorities to efficiently deal with doping in their disciplines. Reference List: 1. Barclay PG, Fischer ER and Harris DC. Interpatient variation in response to subcutaneous versus intravenous low dose erythropoietin. Clin Nephrol 40: 277-280, 1993. 2. Breidbach A, Catlin DH, Green GA, Tregub I, Truong H and Gorzek J. Detection of recombinant human erythropoietin in urine by isoelectric focusing. Clin Chem 49: 901-907, 2003. 3. Lamon S, Robinson N, Sottas PE, Henry H, Kamber M, Mangin P and Saugy M. Possible origins of undetectable EPO in urine samples. Clin Chim Acta 385: 61-66, 2007. |
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Venke Skibeli, PhD, Norwegian Medicines Agency Oslo, Norway
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venke.skibeli{at}imbv.uio.no Venke Skibeli, PhD
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The report by Lundby et al. (2008) has deficiencies regarding information about the design and performance of the current study, comparing results of EPO analyses from two WADA accredited laboratories. The obvious weakness of this relatively simple study is the lack of a description of how the samples from the EPO treated subjects were submitted for analysis, and also which procedures that were utilized by the laboratories. Did Lundby et al. follow a specific protocol describing how the contact with the two labs should be handled for the results to be comparable? In Methods, the current paper presents shortly the transport of the samples from Copenhagen to the chosen doping laboratories, and refers only to the official WADA web page concerning the performance of the analyses. However, the section does not contain any information of how similar conditions in the labs were pursued. This confers insecurity to the evaluation of the results. The authors state that the two labs were blinded towards the treatment. What does this blinding imply? Did the authors ensure that the same procedure(s) were applied in both labs; and how were the samples introduced to the labs, as research or ordinary doping samples? Lundby et al. is stating ‘In real life WADA requires’, implying that the samples to be tested in fact were presented as test samples not ordinary ones. A prerequisite for a test model like this is a detailed protocol approved in advance to ensure proper quality assurance of every step of the study. References: Lundby et al., Journal of Applied Physiology, June 26, 2008. |
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Wilhelm Schänzer, Institute of Biochemistry German Sport University Cologne, Am Sportpark Müngersdorf, Hans Geyer
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schaenzer{at}biochem.dshs-koeln.de Wilhelm Schänzer, et al.
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Comment of Laboratory B on the publication by Lundby et al. Dear Sir, In the article “Testing for recombinant human erythropoietin in urine: problems associated with current anti doping testing” as published in the Journal of Applied Physiology in 2008 (doi:10.1152/japplphysiol.90529.2008), Lundby and coworkers reported that the analyses of urine samples of rHuEpo-treated volunteers with the World Anti-Doping Agency (WADA)-approved isoelectric focusing (IEF) test (2) in two different laboratories (laboratory A and B) led two entirely different results. This statement of the study is wrong for the following reasons: 1. The two laboratories have not used the same methods. Laboratory B has reported only results obtained with a newly established sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) method (1) and not with the WADA-approved IEF procedure (2) as employed by laboratory A. 2. The two laboratories used different reporting categories. The study design did not include a scheme about how to report results. Laboratory A used three reporting categories termed “negative”, “suspicious” and “positive”, while laboratory B used only the expression “suspicious”. Lundby and coworkers interpreted the “suspicious” results of laboratory B as “no misuse” of rHuEpo. This is a misinterpretation. All 15 “suspicious” results reported from laboratory “B” fulfilled the screening criteria for the presence of rHuEpo (1). As no confirmation analyses were performed and a new non-WADA approved method was used, the term “positive” was not used. 3. The two laboratories have analysed different numbers of samples In Table 1 of the publication it is mentioned that 52 samples have been provided to the laboratories. Laboratory B has received only 48 samples, and only 48 samples were reported. The correct comparison of the results of the two laboratories would have led to the following conclusion: Laboratory B obtained the same results compared to Laboratory A, even with a different method. In general the performance of inter-laboratory studies is very difficult and has to be performed by professional institutions. In the field of doping control this is done by WADA. Regarding the study of Lundby et al., it is questionable, if a group, which is neither experienced nor accredited for inter-laboratory studies, performs such studies with non-certified test material. References: 1. Kohler M, Ayotte C, Desharnais P, Flenker U, Lüdke S, Thevis M, Völker-Schänzer E, Schänzer W. Discrimination of recombinant and endogenous urinary erythropoietin by calculating relative mobility values from SDS gels. Int J Sports Med. 29: 1-6, 2008. 2. 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, 2000. |
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Carsten Lundby, PhD, Copenhagen Muscle Research Centre Copenhagen, Denmark
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lundby{at}idraet.au.dk Carsten Lundby, PhD
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It is appreciated that our study on the subject of the EPO analysis (6) has generated the much needed debate on the present World Anti Doping Agency (WADA) test procedure for detection of recombinant human erythropoietin (rHuEpo). Several interesting points have been made, including the comments of Prof Jelkmann (3) arguing that at present no test is available for biosimilars of Epoetin beta or pegylated Epoetin beta, and the comments by Drs Mørkeberg, Belhage and Damsgaard (7) commenting on the too strict criteria used to judge a test result as an “adverse analytical finding”. As suggested by Drs Schumacher and Pottgieser (8), a consequence hereof could be to consider all available information on erythropoiesis in the evaluation of an athlete. This would require introducing the so called blood passport. Another consequence is for the WADA laboratories to supply NADA and the international sport federations with the actual results of the electrophoresis (i.e. the raw data). So far this information is kept in the laboratories. WADA should implement both these procedures in the very near future. In a recent Nature article (2) on the topic of doping it was among others argued that studies testing the current anti doping procedures are needed. In this regard, it should be noticed that our article is at present the only peer-reviewed study investigating the assay technique used to detect rHuEpo during and after a mimicked doping period. The conclusion from our study is clear even if ignoring the reported analytical results obtained from Lab B (see more below): The results supplied from Lab A clearly demonstrate that it is possible to compete in sporting events with the physiological advantages of rHuEpo doping without much risk of getting caught. In light of the letter to the editor by Lab B it would seem that the newly established SDS-PAGE test procedure is not superior to the presently used WADA test, and should thus likely not be implemented in future anti doping work. In regard to the critique/question of our study by Dr. Lasne (4) we would like to point out that our aim with the study was not to “demonstrate the failure of the test validated for anti doping control”, but rather to test the effectiveness of the current tests. Our motivation for this was based on the recent scientific reports doubting the sensitivity of the test. Among these studies is the publication co- authored by Dr. Lasne (1) where it is demonstrated that the detection window following a single epo injection may be as short as 12-18 hours. As we see it, our study took this finding to the next step, demonstrating that with the present WADA procedures it is indeed possible for an athlete to enhance physical performance by rHuEpo doping without great risk of being tested positive. In regard to comment raised by Dr Saugy from Lab A (Lausanne), that the study was a collaborative investigation, it is important to state that this was never discussed or formally agreed upon. The two laboratories were commissioned to perform the analysis in a strict WADA manner and the results were to be used for scientific purposes. It should also be mentioned that we previously have had rHuEpo urine tests analysed (unpublished data) in Lab A without great detection successes. The aim of this previous collaboration was to determine the time course for rHuEpo detection in urine samples from subjects injected with rHuEpo. The Lausanne laboratory was not satisfied with the analytical result and claimed the rHuEpo had been degraded because the samples may have been thawed during transport. However, as I myself, or P. Robach personally delivered all of the samples directly to personnel belonging to the Lausanne laboratory from our own laboratory (CMRC), we felt this did not explain their analytical failure because when delivered the samples were still frozen and covered in dry ice. To avoid a recurrence of these problems, we chose to repeat our study and send identical samples to two WADA accredited laboratories following the recommended WADA routines. The clarification of the other issues which are unclear to Dr. Saugy (8) are as follows: 1) all injections were performed subcutaneously, 2) haematocrit and haemoglobin mass were included in the paper simply to provide evidence that the rHuEpo injections had had an effect, 3) other blood parameters useful in anti doping work (reticulocytes, calculated off scores etc) are planned to be published in a separate article, 4) The exact time points for rHuEpo injections/urine sampling are given in the Methods section and in part in table 1, 5) Two shipments of urine to Lab A were necessary because the courier company DHL mistakenly send the first shipment to Swaziland (Africa) instead of Switzerland. The second shipment was received by Lab A within 24 hours of shipment, and as acknowledged by the laboratory the samples arrived in good “condition” (i.e. frozen and in dry ice). The above points seem to have little relevance to the present discussions on the sensitivity and validity of the WADA urine EPO test and Dr. Saugy´s conclusion is out of context to his comments. A final point in regard to the results of Lab. A. They explain that the “negative” sample was reported to be “Indet low EPO : means that the EPO content of the sample is too low to integrate the profile”. This statement further strengthens the comments of Mørkeberg and co-workers (7) that the criteria used to deem a test result as an “adverse analytical finding” are subjective. Our results provide further proof that the WADA urine test in its present form is not sufficient to determine rHuEpo doping at times when performance is enhanced. With regard to the critique raised by Laboratory B (and in part by Dr. Lasne) 1 &2 ) that their results were the same as with Lab A: They were specifically asked to analyse our samples according to WADA regulations. This was reconfirmed by email on May 13th 2008 by Laboratory B which stated that the samples we sent to them the previous fall were analyzed by the IEF method published by Lasne et al. (5). Based on this information, the samples reported to us by Lab B being “suspicious” were obviously also treated as “suspicious”. In the abstract of our paper we state “…Laboratory B found no misuse”, referring to the fact that the laboratory did not identify one sample as being “Positive”. Assuming (as reconfirmed by email) our samples were handled according to the agreement then this statement remains correct. When the article was published in June, the Cologne laboratory after some delay claimed that the samples had not been analysed “properly” because the samples originated from a scientific study. This was also the reason only to report samples as “suspicious”. It should be noted that we previously have had urine originating from scientific studies as well as from athletes’ analysed in Lab B for rHuEpo, and on these occasions the laboratory has treated the sample identically and also reported the results according to WADA nomenclature, 3) We shipped 52 exactly matched samples to both laboratories, and have no explanation why only 48 are reported by Lab B. This however can hardly be blamed on us. In conclusion we feel that our results and the debate have provoked clear support for our conclusion which remains unaltered: The present WADA urine test to determine rHuEpo doping is insufficient to effectively catch potential rHuEpo dopers at times when performance is enhanced. Reference List 1. Ashenden MJ, Varlet-Marie E, Lasne F and Audran M. The effects of microdose recombinant human erythropoietin regimes in athletes. Haematologica 91: 1143-1144, 2006. 2. Berry DA. The science of doping. Nature 454: 692-693, 2008. 3. Jelkmann W. Testing for recombinant human erythropoietin: The bouquet of compounds. J Appl Physiol 2008. 4. Lasne F. To the Editor. J Appl Physiol 2008. 5. Lasne F, Martin L, Crepin N and Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem 311: 119-126, 2002. 6. Lundby C, chman-Andersen NJ, Thomsen JJ, Norgaard AM and Robach P. Testing for recombinant human erythropoietin in urine: problems associated with current anti-doping testing. J Appl Physiol 105: 417-419, 2008. 7. Morkeberg J, Belhage B and Damsgaard R. Expanding the analysis reporting will lead to more efficient testing. J Appl Physiol 2008. 8. Schumacher YO and Pottgiesser T. New strategies in the fight against doping are necessary. J Appl Physiol 2008. |
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