News

4th WAADS QA Manager Meeting Seibersdorf, Austria, April 24-25th 2017

On April 24th and 25th, the WADA accredited laboratory of Seibersdorf (Austria) hosted the 4th WAADS QA-meeting. At this occasion, 34 participants from 26 WADA accredited laboratories shared their thoughts on quality assurance in anti-doping analysis.

Mrs Victoria Ivanova from WADA, who is closely involved in drafting the new ISO17025 guideline, gave an excellent overview of the status of the new draft guideline with an emphasis on the changes proposed. Although not operational yet, this new guideline will have a high impact on quality assurance in accredited laboratories.

During the other technical sessions, the elements of the perfect standard operating procedure (SOP) were discussed to reach a consensus and harmonize the contents of these SOPs across the laboratories. Similarly, the first steps towards a consensus document on acceptable validation methodologies were set. This document could serve as a guideline for laboratories.

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ADLQ Annual Symposium will take place on the 2nd and 3rd of May 2017 in Doha

As detection techniques, have become more sophisticated, both in animals and in human, so have doping processes and dopants. Increasingly these sport performance enhancing substances are minimally modified endogenous molecules, perhaps even gene therapy, that escape detection by current methodologies. The use of biomarkers, that potentially target a particular biological process, is amongst the most promising approaches in the fight against doping. The search for such novel markers utilizing big data and the omics platforms, and their validation has proved challenging. The extent to which this approach has been successful and the potential of using biomarkers for detecting doping in sport has been realized is the premise of this year’s symposium. Again, following on from discussions in the previous symposium the aim is to compare and contrast biomarkers of dopants, their nature and detection, in animals as opposed to humans. To facilitate this discourse, experts from both human and animal Anti-Doping arenas, veterinarians and scientists will be brought together.

The ADLQ Annual Symposium will take place on the 2nd and 3rd of May 2017 in Doha.

Abstract submission opens on the 1st February and closes at midnight local time on the 15th March  2017. The meeting is currently being considered for CME accreditation.

This symposium promises to offer new insights into the most recent research findings in the field of biomarkers in doping. We wish all participants a productive and engaging meeting and invite you all to experience Qatari hospitality. We are confident that you will take home significant new scientific information along with fond memories of your time in Doha.

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Sample in Lausanne

Prof. Martial Saugy, former director of WADA accredited Laboratory of Lausanne (LAD) and current Director the of the Center of Research and Expertise in anti-doping sciences (REDs) of the University of Lausanne, disputes allegations of misconduct against the Lausanne laboratory, contained in the McLaren reports.

A formal request to WADA has been made in the letter to attached documentation available to the Public to restore the reputation of the Laboratory of Lausanne as well the one of its former Director, Prof. Martial Saugy.

Further information can be viewed at http://unil.ch/reds/home/news.html?showActu=1484119672992&showFrom=1 or unil.ch/reds

 

 

 

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Dr. Alka Beotra with experts for Open discussion with Pharmaceutical Companies

A two-day International Conference on "Implementation of Latest Guidelines in Human and Horse Doping: Interaction between Testing Authorities and Doping Control Laboratories" was organized by National Dope Testing Laboratory (NDTL), India at India Habitat Centre, New Delhi, India on the 4th & 5th of November, 2016 with a focus on interaction between Testing Authorities and Doping Control Laboratories which included representatives from  World Anti-Doping Agency (WADA), South East Asian Regional Anti-Doping Agency (SEA-RADO), National Anti-Doping Organizations (NADOs) of India, Srilanka, Indonesia, Kazakhstan & Singapore, Indian Pharmacopeia Commission (IPC), All India Institute of Medical Sciences (AIIMS), Horse Racing Clubs, etc. The conference was attended by approximately hundred participants, both national and international, from various countries viz. Canada, India, Indonesia, Italy, Kazakhstan, Singapore and Sri Lanka.

The first day of the conference i.e. 4th November, 2016 commenced with Special Address delivered by Secretary, Sports & CEO, NDTL Shri Rajiv Yadav. Guests of Honor for the conference were Shri Navin Agarwal, Director General, NADA, Dr. Olivier Rabin, Senior Director Science, WADA, Dr. Alka Beotra, Former Scientific Director, NDTL, and Dr. Shila Jain, Laboratory Director, NDTL.

The First Session of the Conference was on the Second Meeting of WADA & Pharmaceutical Companies. The talk of Dr. Olivier Rabin on “Collaboration between the Biopharmaceutical Industry and Anti-Doping” proved highly beneficial. The session also included open discussions on the Role of Pharmaceutical Industry towards fight against doping in sports, way forward and conclusion with active participation by experts from different fields.

In the second session of the conference, talks were delivered on various important themes like Prohibited Substances and Methods in doping by Dr. Olivier Rabin, accreditation of WADA laboratories by Ms Victoria Ivanova, pseudo endogenous steroids in anti-doping analyses by Dr. Xavier de la Torre Fornell and NADO-RADO collaboration with laboratories by Mr. Gobinathan Nair.

The third session of the conference, conducted on the 5th of November, 2016 included Panel Discussions on reporting and result management in horse dope testing, use of ADAMS for result management, implementation of WADA guidelines from NADO’s perspectives, implementation  of WADA TDSSA (testing of ESAs and GHRHs) and drug administration studies & use of herbs in horse racing.

The Conference concluded with vote of thanks by Dr. Alka Beotra, valuable suggestions by the invited speakers, positive feedback by the participants, and fresh ideas by scientists and professionals for incorporation to make the fight against doping in sports more effective.

Dignitaries on Dias at the Inauguration of First International Conference organised by NDTL
Group Photo of First International Conference on "Implementation of latest guidelines in human and horse doping
NDTL Scientists with pioneers in the field of Anti-Doping
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Image of Mr. Colvert’s A-sample – “no. 7397” IEF test result vs. negative and positive control samples (left) and relative abundances of bands as determined by the densitometric analysis (Gasepo software ) (right) as extracted from the documentation package pp. 23 and 27.

Response to Lab Times biased description of Steven Colvert’s positive test results.

Lab Times Issue 5 October 6th 2016, Front page: WADA’s Testing Procedures – Dubious Doping Detection.

Borderline Analysis Another troubling doping case is questioning WADA’s credibility again.

This article is the second one presenting the views of a group of scientists who are not only challenging the interpretation of an athlete’s EPO test results, but discrediting “WADA’s credibility, again”.

While it may sound seemingly insignificant to refer to “WADA’s credibility”, this one-side vitriolic opus is a charge against skilled, experienced scientists.  The SAR-PAGE and IEF data presented are of excellent quality, the results clear and convincing.  The methods, the interpretation of test results were published in the peer-reviewed scientific literature (more than 40 research articles from anti-doping scientists) and so were the criteria for issuing positive findings that are available on WADA’s website (www.wada-ama.org)[1].  It is worth noting that the four signatories never submitted any data in support to their position.  After all, these techniques are not unique to EPO doping control tests and are common in many molecular biology laboratories.  The anti-bodies and the EPO standards being accessible, nothing prevented the authors to demonstrate their point with simple experiments; they opted instead for voicing unchallenged theoretical objections, in a magazine.

The laboratory in Cologne tested Mr. Colvert’s A- and B-samples five times with the two recognized and widely applied complementary techniques; each time the results were consistent with the presence of a recombinant EPO.  The criteria for reporting an adverse analytical finding were objectively met and the conclusions reached by the laboratory were supported from the independent review made by the experts of a second laboratory located in Austria[2]. The scientists from these two organizations have published on EPO testing, their expertise is recognized.

The tests, as applied for the past 16 years, target the known differences between human (endogenous) and recombinant EPO, the latter being the doping agent.  The first method based on their different isoelectric profiles was published by F. Lasne in Nature[3], a prestigious scientific journal.  Later, the discrimination based on their different apparent weight lead to the development of the SDS- PAGE and finally SAR-PAGE approaches[4].   Both laboratories involved in Mr. Colvert’s case authored these publications. 

IEF Test Result:

The initial test done on a batch of samples including Mr. Colvert’s, was with the SAR-PAGE: the laboratory determined that the profile of sample no. 7397 was suspicious and they decided to proceed with further confirmatory tests on other aliquots of the A-sample (N. B. the identity of the athlete is unknown to the laboratory).

The first confirmation data presented was from the IEF.  In order to interpret the results, regions must first be delimited from the position of bands generated by reference standards analysed simultaneously: basic for recombinant, endogenous for human, acidic for NESP, as shown by the example provided in WADA Technical Document reproduced in figure 1.

Figure 1: Image extracted from WADA Technical Document showing the definition of basic, endogenous and acidic area from the analysis of standards[5].

Profiles composed uniquely of the recombinant EPO like in figure 1 are not the norm.  Doping regimes have evolved to “micro-dosing” and “bio-similar” recombinant EPOs have appeared on the market1,[6].  Their profiles of isoforms were shown to vary slightly from epoetin α and β.  As a result, athletes’ samples often show mixed profiles, as it is the case here e.g. a combination of endogenous and recombinant bands.  The criteria for concluding to the presence of a recombinant EPO are currently as follows: i) the 2 most intense bands “measured” by densitometry must be located in the basic area; ii) the second most intense band in the basic area has to be at least as intense as the most intense one located in the endogenous area1.

These criteria were definitely met with Mr. Colvert’s A-sample IEF test results, as shown by the images extracted from the documentation package (pp. 23 and 27) provided by the Cologne laboratory (Figure 2).  The two, actually the three most intense bands (61.8 to 100 intensity) are located in the basic /recombinant area and are more intense than band α, the strongest one of the endogenous region (42.4 intensity).

Such a profile is not consistent with endogenous human urinary EPO and therefore, points to a recombinant EPO.  The conclusion that was reached by the laboratory in Cologne was correct.

Figure 2: Image of Mr. Colvert’s A-sample – “no. 7397” IEF test result vs. negative and positive control samples (left) and relative abundances of bands as determined by the densitometric analysis (Gasepo software[7]) (right) as extracted from the documentation package pp. 23 and 27.

SAR-PAGE Test Results:

The second test for the A and B-sample confirmations was the SAR-PAGE.  Under these conditions, recombinant EPO show a “characteristic band shape e.g. broad band”1.  As shown in WADA Technical Document, combined endogenous / recombinant profiles as Mr. Colvert’s, result in a mixed band, “consisting of endogenous EPO and rEPO” – “a diffuse or faint area of the band above the corresponding endogenous band is also indicative for the presence of epoetin-α and-β”.  As also stipulated in the technical document: “The centroid or the boundaries of the width of the band can be used to ascertain that its position and shape differs from the position of endogenous EPO run in parallel”.  The example provided in that regulatory document of a mixed endogenous/recombinant band is reproduced in figure 3.

Figure 3: Excerpt of WADA Technical Document representing the characteristically diffuse mixed endogenous /recombinant band (indicated by red arrow)1.

Each time Mr. Colvert’s samples were analysed, the mixed recombinant and endogenous populations were revealed by the diffuse and faint area above the corresponding signal of endogenous EPO indicating the presence of recombinant EPO as shown in figure 4 (sample no. 7397). 

 

Figure 4: Gasepo densitometric analysis of A- (left) and B- (middle) samples SAR-PAGE confirmation tests (negative human EPO standard from the B-sample test is show for comparison (right)).

Deducing the endogenous to recombinant composition of urinary EPO from the IEF and SAR-PAGE profiles of the Gasepo analyses is wrong, particularly for the latter.  Both recombinant and endogenous bands are mixed, overlapped and not resolved[8].  It is not possible to determine from such results the relative abundance of each specie.  If the laboratory expert was correctly quoted, he made a mistake when he stated that the amount of recombinant was small when compared to the endogenous EPO.

With no hesitation, I support the conclusions of my colleagues from Cologne and Seibersdorf.  These profiles depart significantly from human urinary endogenous EPO.  Both the IEF and SAR-PAGE test results are evidence for the presence of a recombinant EPO in Mr. Colvert’s A- and B-samples. 

 

Christiane Ayotte, Ph.D.

President World Association of Anti-Doping Scientists

Professor, Director of the Laboratoire de contrôle du dopage

INRS-Institut Armand-Frappier

christiane.ayotte@iaf.inrs.ca




[1] TD2014EPO. 2014, Harmonization of analysis and reporting of erythropoiesis stimulating agents (ESAs) by electrophoretic techniques. Available at: www.wada-ama.org/en/resources/science-medicine/td2014-epo.

[2] In order to ensure that only positive findings are reported, a mandatory second opinion is sought from scientists listed in WADA Technical Document, following the review of the analytical data.

[3] Lasne F. de Ceaurriz J. Recombinant erythropoietin in urine. Nature. 2000, 405, 635; Lasne F et al. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered hormones. Anal Bioch. 2002, 311:119;

[4] M. Kohler, C. Ayotte, P. Desharnais, et al. Discrimination of recombinant and endogenous urinary erythropoietin by calculating relative mobility values from SDS gels. Int. J. Sports Med. 2007, 29, 1; C. Reichel, F. Abzieher, T. Geisendorfer. SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood.Drug Test. Anal. 2009, 1, 494.; Reichel C. Recent developments in doping testing for erythropoietin. Anal Bioanal Chem. 2011, 401:463.

[5] The borders of the basic and acid areas are defined as shown with standards, endogenous is therefore in between.

[6] Y. Dehnes, A. Shalina, L. Myrvold. Detection of recombinant EPO in blood and urine samples with EPO WGA MAIIA, IEF and SAR-PAGE after microdose injections. Drug Test. Anal. 2013, 5, 861; Martin L., Ashenden M., Bejder J., Hoffmann M., Nordsborg N., Karstoft K., Morkeberg J., Sharpe K., Lasne F. and Marchand A., New insights for identification of doping with recombinant human erythropoietin micro-doses after high hydration, Drug Testing and Analysis, 2016, DOI 10.1002/dta.2004

[7] Bajla I, Holländer I, Gmeiner G, Reichel Ch. Quantitative analysis of images in erythropoietin doping control. Med Biol Eng Comput. 2005 May;43(3):403-9.

[8] The overlap of epoetin α/β and human urinary EPO isoforms is known: for example, Ph. Desharnais, J.F. Naud and C. Ayotte, Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE, Drug Testing and Analysis. 2013. DOI 10.1002/dta.1494

 

 

 

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WAADS Statement

The World Association of Anti-Doping Scientists is aware of the tensions exposed in the media between the International Olympic Committee and the World Anti-Doping Agency. We deplore and view these attempts to undermine the credibility of World Anti-Doping Agency as counter-productive. IOC members accusing WADA of being responsible for the actions of the Russian laboratory is incomprehensible and manifestly unfair.

It is well known that the Agency actively tried to document corruption and to suspend the laboratory before the Sochi Games. These allegations could not be proven at the time, with its now loquacious director denying everything, claiming abusive treatment from WADA.

The widespread collusion and subterfuges deployed were fully exposed by two commissions that were put in place by WADA. For years, the Russian laboratory had been assisting and monitoring doping, helping to evade detection by manipulating samples and results of Russian athletes.

Needless to say, all of us but particularly those WAADS members who provided support to the laboratory operations during the Sochi Games were shocked, felt manipulated and betrayed following the revelations made earlier this year by its former director, Grigory Rodchenkov and subsequently verified by independent investigations.

Therefore, in the current circumstances, we support a strong WADA and we oppose any attempt to subvert its role and leadership. No one but the dopers could benefit from these internal fights and we urge all parties, WADA, Olympic movement, governments, anti-doping authorities, athletes and scientists to regroup and show a united front.

 

Notes for Editors

The World Association of Anti-Doping Scientists was established in 1999.

WAADS represents all WADA accredited laboratories as a scientific organization.

Further information about WAADS and this press release should be addressed to:

Christiane Ayotte (christiane.ayotte@iaf.inrs.ca)

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