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Pitfalls in the rEPO test

Special news feature, September 23, 2005

See also: Serious concerns over urinary EPO test

The urinary recombinant erythropoietin detection test

According to the Lausanne Court of Arbitration for Sport (Arbitration CAS 2001/A/343, paragraph 23, page 233), "Erythropoietin (EPO) is a natural human hormone which is produced in the kidney and stimulates the production of red blood corpuscles, which are responsible for transporting oxygen to the muscles. The production of red blood corpuscles can be disturbed or prevented, for example in people with defective kidneys. The consequences are treated by administering EPO obtained by genetic engineering (recombinant EPO = rEPO). EPO and rEPO are practically identical which meant that, until recently, it was not possible to distinguish between the two substances."

The same Arbitration CAS 2001/A/343 indicates in paragraph 24, page 233 that the urinary rEPO detection test "tries to directly detect the presence of rEPO in the urine of the person being tested. This "direct method" combines an isoelectrical focussing with a double immunal blotting. The method is based on the finding that artificially produced rEPO behaves differently in an electrical field than human EPO so the two types of EPO can be distinguished from one another. The test method is also based on a second basic assumption that, as is the case with many steroids, the production of natural hormones is reduced when an artificial hormone is administered endogenously."

The urinary rEPO detection test was developed in 2000 by researchers of the National Doping Detection Laboratory of Châtenay-Malabry, France. Surprisingly, this test was adopted by the authorities responsible for doping control in sports despite its very scarce scientific validation, limited to three international publications:

o Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature 405, 635, 2000;
o Lasne F. Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures. J. Immunol. Methods. 253, 125-131, 2001;
o 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. 311:119-26, 2002.

Therefore, there is a clear and worrying absence of basic research regarding the validity and reliability of the urinary rEPO detection test. This lack of basic research has been acknowledged by independent experts, and also by the directors of accredited laboratories responsible for the application of the test, as will be seen within the present document.

The evaluation of the test assigned by WADA to independent experts

In a meeting of February 8, 2002, in SALT Lake City, the Council of the World Anti-Doping Agency (WADA) decided that an independent review of the EPO urinary test in sport doping control should be conducted by two independent research/laboratory experts. The mandate was given to two internationally well recognized electrophoreticists, namely Dr. Gabriel Peltre (ESPCI, LECA, Paris, France) who is a specialist in gel electrophoresis of proteins and immunoblotting and Prof. Dr. Wolfgang Thormann (Department of Clinical Pharmacology of the University of Bern, Switzerland) who specializes in free solution and theoretical aspects of electrophoresis, including the use of capillary electrophoresis for research and routine purposes in clinical and forensic analysis. Drs. Peltre and Thormann were never involved in the analysis of EPO and can thus be considered as independent reviewers.

The result of this independent evaluation was the following document: G. Peltre y W. Thormann. Evaluation report of the urine EPO test. WADA, March 11, 2003

The limitations of the test according to the independent experts

In pages 6 and 7, Part B of the document, entitled "General conclusions", section 3 indicates that "Improvements should be made at several steps of the method". More precisely, it is said that "Each step of the urine EPO test has to be critically reviewed and the potential improvements listed, tested and validated. According to our expertise we do see room for improvements in the following points:

o 1. assessment of the urine samples prior to analysis
o 2. more selective urine preconcentration step
o 3. controlled solubilization of the urine concentrate
o 4. improved electrophoretic separation
o 5. use of more appropriate blotting membranes and antibodies
o 6. use of more recent luminescence kit
o 7. new approach for the interpretation of the scanned EPO profiles.

In the title of section 5 of the same Part B (page 7), the independent experts indicate that "A temporal EPO profile should be known from each athlete and more basic research about the EPO isoforms should be performed". More specifically, they say that:

o "A survey of the urinary EPO profile upon time should be obtained and registered for each athlete, corresponding to different physical activities including effort and rest. Any abnormal deviation from this basal profile should be investigated and explained.
o More fundamental research about the EPO isoforms should be performed: function, catabolism, half-life, exact pI and age, gender and genetic influences, etc."

Another limitation of the test according to these experts relates to the secrecy and sectarianism displayed by WADA with regards to the urinary rEPO detection test. In page 9, section 6 of the document, it is discussed that "A regular discussion forum should be organized among the IOC/WADA accredited laboratories and this should be open to the scientific and medical community. An international data bank of the existing EPO profiles should document the effects of race, physio-pathology, physical effort etc and monitor the evolution of the different misuses of rhEPO. This "EPO observatory" (or task force!) should consider also the effects of any other drug abuse in association with EPO."

Regarding the uniformity with which the urinary rEPO test is performed in the different accredited laboratories, the independent experts state in Part C, Technical aspects, section 2, pages 10-11 of their report: "Only very few modifications of the original method have been observed, including the total duration of the test varying from 24 to 36 hours, the anti protease treatment modified or omitted, reagents from different sources being used, etc. These modifications were introduced as improvements of the original method. By their accumulation they may lead to a noticeable modification of the overall performance of the test, eventually introducing an uncontrolled drift in the final results. Having exactly the same test worldwide is essential for its credibility."

The lack of uniformity in the interpretation of the results in the different accredited laboratories is also one of the two main fundamental reasons raised by the independent expert Prof. Bogaerts, appointed by the Disciplinary Council of the Flemish Community in Belgium, to state that no solid scientific evidence exist that Belgian triathlete Rutger Beke used rEPO. In an interview with Sporza TV on August 2, 2005, Prof. Bogaerts declared "if people have a pronounced post-exercise proteinuria, one lab interprets the result as a positive test, such as those of Ghent and Cologne, but a another laboratory considers the test as negative. I am certain that they have acted honestly, but the EPO test in itself is not reliable."

It is worth pointing out that none of the recommendations of the independent experts has been put into practice to date, and that the only change that has been applied to the test since March 2003 (date of publication of the Peltre and Thormann report) until today is the addition to the analytical procedure of the so-called "stability test", subsequent to the case of athlete Bernard Lagat, in which the African runner was initially accused of doping with rEPO after the analysis of the A sample, then acquitted when the B sample showed negative results (see references to this case below). Interestingly, this modification has never been validated in the usual way of every scientific method, that is by publication in a renown scientific journal submitted to peer review. It is thus obvious that WADA is a closed organization that does not follow the international rules of scientific publication procedures.

Finally, two very significant elements referring to the Peltre and Thormann report are worth noting:

o 1. Part C, Technical aspects, section 6, page 15: "The development of totally new urinary EPO tests should be encouraged and funded."
o 2. The Peltre and Thormann report was withdrawn from WADA’s website soon after a group of Belgian scientists announced their intention to initiate their own investigations to clarify the origin of the positive rEPO test of Belgian triathlete Rutger Beke, accused of doping after testing positive in a urine test performed immediately after taking part in a triathlon in September 2004.

The limitations of the test according to the accredited laboratory directors

The managers of the accredited laboratory of Barcelona, Professor Jordi Segura and Doctor Toni Pascual clearly expressed the lack of basic research relating to the urinary rEPO detection test. In their interviews of July 2002 with the independent experts Peltre and Thormann (page 23, section 4, paragraph 2), they summarized their general valuation of the rEPO test by stating that "the only direct test available on urine has the merit of existing and is definitely needed", but they insisted on the fact that "much knowledge remains to be obtained on the nature of origin or meaning of the urinary EPO isoforms, from the endogenous as well as the exogenous or recombinant ones. What are the modifications induced by the passage of the rhEPO in the human body?"

In their interviews with Peltre and Thormann (pages 23, 24, 25), the managers of the accredited laboratory of Barcelona accepted "to discuss critically the different points that could be potentially improved". Among these, the following could be emphasized:

o "1- Urine preparation and concentration: you are suggesting that an immunoaffinity purification could be a far better alternative to the actual 2-step filtration method. Indeed it will eliminate salts and excess of unrelated (to EPO) urinary proteins which are not compatible with good Iso Electric Focusing (IEF) and reduces the risks of EPO destruction due to potential concentrated proteolytic enzymes. You mentioned the research effort that you are currently doing in that field and which will be put together later in this report.
o 2- IEF step: the use of Immobilized pH Gradients (IPG) should save time and improve the reproducibility and reliability of migration (a critical issue when defending a positive case). As far as it is known, attempts made in this sense have not produced acceptable results so far."

It is thus obvious that already in July 2002 the directors of accredited laboratories were fully aware of the limitations inherent to the urinary rEPO detection test, making especial mention of the problems that could be created by urinary proteins with regards to the validity and reliability of the test.

The importance of protein concentration in the urine sample

The Peltre and Thormann report does not leave a shadow of a doubt regarding the importance of the protein concentration in the urine sample. In section 3, Part C of the report (pages 11-16), these independent experts provide a detailed description of the improvements that should be made on several steps of the method, which have already been listed above. Improvements 1 and 2, which refer to proteins in urine, are particularly relevant:

o 1. Assessment of the urine samples prior to analysis: urine samples should be characterized, prior to analysis, by its total protein content and their state of dilution. An excessive protein load for samples collected after a strenuous physical effort and fraudulent urine dilution can thereby be recognized (…).
o 2. More selective urine preconcentration step: The preconcentration of the urine sample is essential to achieve the current sensitivity of the method. With the 700- to 1000-fold concentration, however, quite a large number of samples do not show a measurable EPO concentration (according to one laboratory, up to 20% using an ELISA for simple recognition of EPO) and can thus not be analysed with the IEF assay. Furthermore, samples unusually rich in proteins, especially those obtained after an important physical effort such as competition, induce some artifacts in the method (poor electrophoretic migration inducing smears, back ground staining, etc). Further removal of EPO unrelated proteins should be investigated, for example by a rapid ion exchange filtration step, eliminating all proteins more basic than the EPO isoforms, reducing sample viscosity and unwanted potential protein-protein interactions. A preenrichment of EPO in the urine concentrate, taking advantage of its affinity to immobilized lectins, has already been studied and tested by the laboratory in Châtenay-Malabry. A similar EPO enrichment is expected by using and immunopurification step, an approach that is currently being investigated by the Oslo and Barcelona laboratories.

In view of the efforts of the different accredited laboratories (particularly those of Châtenay-Malabry in the outskirts of Paris, Oslo and Barcelona) to eliminate proteins not related with EPO present in the urine samples, particularly after intense exercise performed in competition, it is clear that those in charge of the application of the urinary rEPO detection test are fully aware of the fact that there is a lack of specificity problem with the test in urine samples with a high protein content. Despite this, those in charge of the accredited laboratories continue to neglect the assessment of the urinary protein concentration in the urine samples in the preanalytic phase, which is a serious negligence of incalculable consequences for athletes accused of rEPO abuse.

Why do accredited laboratories refuse to measure protein concentration in the athletes’ urine samples?

For decades, it has been a well known and scientifically proven fact that various particularly intense exercise modalities, including triathlon, induce a condition called exercise-induced proteinuria, which results in an unusually large quantity of proteins being filtered to urine.

The following scientific publications, ordered form the most recent to the least recent, bear witness to this condition in several endurance sports:

o 1. Yaguchi et al. The effect of triathlon on urinary excretion of enzymes and proteins. Int. Urol. Nephrol. 30 (2): 107-112, 1998. "(…) There was a significant increase in urinary total protein excretion immediately after triathlon (p<0.01). (...) It appears to be reasonable to presume that glomerular damage may persist in some subjects who do heavy exercise."
o 2. Montelpare et al. Continuous versus intermittent exercise effects on urinary excretion of albumin and total protein. J. Sci. Med. Sport 5 (3): 219-228, 2002. "Several studies have reported post-exercise increases of urinary concentrations of plasma proteins. (...) intermittent exercise had a greater influence than continuous exercise on the total output of urine albumin, and of urine total protein."
o 3. McInnis et al. The effect of exercise intensity on hematuria in healthy male runners. Eur. J. Appl. Physiol. Occup. Physiol. 79 (1): 99-105, 1998. "(…) The 400-m sprint at maximal effort significantly increased both hematuria and proteinuria (P<0.01). (...) it was concluded that exercise-related changes in renal function were associated with weight-bearing exercise intensity rather than non-weight-bearing exercise duration."
o 4. Estivi et al. Urinary protein excretion induced by exercise: effect of a mountain agonistic footrace in healthy subjects. Renal function and mountain footrace. J. Sports Med. Phys. Fitness. 32 (2): 196-200, 1992. "The increase in albuminuria induced by exercise in healthy subjects is variably dependent on an increased glomerular filtration and/or to a decreased tubular reabsorption of albumin, in relation to the intensity of muscular work. (...) Albumin excretion rate increased significantly (...). The increase in albuminuria induced by strenuous exercise of an agonistic mountain footrace is of mixed type (both glomerular and tubular) and is associated to the urinary excretion of cationic proteins."
o 5. Suzuki and Ikawa. The mechanisms of exercise-induced proteinuria – relationship between urinary excretion of proteins and lactate after exhaustive exercise. Nipón Jinzo Gakkai Shi. 33 (4): 357-364, 1991. "These results suggest that exercise-induced organic acids and/or decrease in renal circulatory pH caused by organic acids may alter renal glomerular permeability and inhibit renal tubular reabsorption of low molecular weight proteins (...). The permeability of the glomerular basement membrane (GMB) to macromolecular proteins may be altered by a reduction in the charge barrier of the GMB, which may be caused by over-production of organic acids and a lower pH."
o 6. Clerico et al. Exercise-induced proteinuria in well-trained athletes. Clin. Chem. 36 (3): 562-564, 1990. "(…) Compared with the basal state (overnight collection), exercise significantly increased the excretion rate of albumin (...). The mean exercise-related excretion of alpha 1-microglobulin by the athletes significantly exceeded the overnight value (...). Our study indicates that (a) albuminuria furnishes the greater contribution to the increase in exercise-induced proteinuria; (b) the exercise proteinuria is both glomerular and tubular in origin, and is reversible; (c) the enhanced protein requirement of athletes may in part be due to the recurrent excretion of proteins in the urine after physical effort."
o 7. Gilli et al. Exercise-induced urinary abnormalities in long-distance runners. Int. J. Sports Med. 5 (5): 237-240, 1984. "The post-exercise urine samples from 122 long-distance runners showed evident abnormalities upon microscopic examination in 95% of all subjects. Proteinuria, alone or with microscopic hematuria, was frequently found. (...) It can be concluded that urinary abnormalities are very common after exercise. These abnormalities are most often of a "renal" origin, probably due to a temporary hemodynamic impairment, partially of glomerular but principally of tubular function."

To date, the only study that has investigated the effects of exercise and illness on the validity of the urinary rEPO detection test was financed by WADA itself in the year 2002 and carried out by researchers of the Australian Sports Drug Testing Laboratory (R. Kazlauskas, G. J. Trout, C. Howe, K. Nelson, J. Trotman). The document, entitled "The effects of factors such as exercise and disease on the distribution of urinary erythropoietin isoforms", aimed to "determine the variability of the natural isoform pattern of EPO in a group of subjects and determine the extent to which the isoform distribution can be influenced by external factors such as vigorous exercise and disease."

In addition, the project literally indicates "Whilst some data has been obtained showing that the distribution of urinary EPO isoforms is not significantly affected by external factors such as altitude there has been no large systematic study on factors such as acute, extreme and long term exercise, and disease. It is only a matter of time before the technical aspects of the urinary EPO test are legally challenged and hence it is essential that data be collected and published in advance to establish whether extreme exercise or disease can alter the pattern of isoforms present so that they more closely resemble those found in recombinant EPO. Such data will be essential to support the urinary EPO test if it is ever to be used without a blood sample to confirm doping with recombinant EPO."

The authors wanted "to determine whether exercise can induce a change to more basic isoforms", and to that aim they studied subjects "who underwent a range of exercise regimes ranging from a short duration (10 minute) exercise test to exhaustion, through a full marathon of approximately three hours, to a 100 km cross country run with a typical duration of over 24 hours. In all cases urine samples were collected in order to measure both the concentrations of urinary EPO and the distribution of isoforms." Among the study results, it is stated that:

o "(...) the concentration of EPO in urine is highly variable and for some individuals can vary by more than a factor of four from one collection to the next;
o "(...) it appears that the extreme long duration exercise can produce a small but significant increase in the percent basic isoforms found in the urine. It is not known whether this increase relates to changes in EPO production or changes in EPO excretion."
o "The samples from the subjects who were part of the disease study were all suffering from anaemia resulting from severe kidney disease. It was hoped to determine if the EPO excreted from such subjects was different in isoform distribution possibly due to a greater contribution from the liver. Unfortunately it was not possible to draw any conclusions from this aspect of the project because the current method was found to be unsuitable for such urine samples owing to their high protein content."

It is thus evident that a long duration and intense exercise can induce proteinuria, which would alter in a very significant manner the validity of the test, even making it impossible to apply depending on the urinary protein concentration. Even Dr. Françoise Lasne, one of the main investigators of the research group that created the urinary rEPO detection test admits in a publicity leaflet of Waters Millipore Company that the test is only protected up to a urinary protein level of ± 0,2 g/l. This test validity limit concentration can be increased more than five fold in extreme exercise conditions.

In a communication from Prof. Francis Delbeke (Director of the Ghent accredited laboratory, Belgium) to the Disciplinary Council of the Flemish Community in Belgium, in relation to the positive case of Belgian triathlete Rutger Beke, Prof. Delbeke states that "I have been contacted by Dr. O. Rabin, WADA Scientific Director, by phone on July 12th regarding the EPO test. From this conversation (see enclosed e-mail of July 14th), it was found that there are indications that in exceptional circumstances following short intensive exercise, endogenous EPO bands would shift to the basic part (exogenous EPO). A research program was initiated by WADA in collaboration with Dr. F. Lasne (Châtenay-Malabry) and Dr. M. Saugy (Lausanne). The results of this study will be expected by the end of September. A consequence could be that the criteria to conclude the presence of exogenous EPO, could possibly be adapted."

In view of the body of evidence, why do accredited laboratories continue to not measure urinary protein concentration in the preanalytic phase? The only possible reason is the certainty of those responsible for anti-doping control that the measurement of urinary proteins (which by the way is rapid, simple, reliable and inexpensive) could invalidate for their subsequent analysis for rEPO a great number of urine samples collected immediately after performing intense exercise. In the end, as stated by Prof. Jordi Segura and Dr. Toni Pascual "the only direct test available on urine has the merit of existing and is definitely needed"…

The lack of specificity of the antibody used to bind recombinant EPO

Each antibody is used to identify a precise protein, and in the present case, recombinant EPO. When an antibody has the capacity to identify only and exclusively the protein that is should identify, and no others, it is said that the antibody is specific. However, some antibodies are not specific enough in their function, and they also identify other proteins contained in the sample; this is called cross reactivity.

This is what the independent experts Peltre and Thormann say about the antibodies used in the test to bind EPO: Part C, section 5, page 12. "Use of more appropriate blotting membranes and antibodies. (...) Antibodies to EPO, monoclonals or polyclonals, should be tested for their fine specificity and their potential capacity of distinguishing between endogenous and exogenous EPO (…)."

It is therefore clear that the specificity of the primary and secondary antibodies used by the accredited laboratories has not been evaluated. To this matter, it is important to point out that the primary antibody used in the test, called AE7A5 and produced by the laboratory R & D Systems, has not been designed to be used in anti-doping testing, as shown by a clear indication in the bottle that says "For research use only".

In the transcription of conversations and communication exchange between the independent experts Peltre and Thormann and the managers of the accredited laboratory of Barcelona, "research efforts to improve the actual urine test" are presented (Page 24, section 1). As effort number 1, they mention:

o "1- Production of specific antibodies to the different EPO isoforms. This difficult research is dependent mainly on the ability of the immunized laboratory animals to differentiate the different EPO isoforms injected to them. For that purpose you are injecting selected glycopeptides to rabbits in order to obtain discriminating polyclonal antibodies. Theoretically, depending on the capacity of the antibodies to differentiate between native-urinary and recombinant EPO, detection could be made by an ELISA or similar test. On the other hand your specific antibodies directed against all EPO isoforms will be used to pre-purify the whole content in EPO of the urine samples and advantageously replace the 1st step of the actual urine test as already mentioned above."

The expert Dr. Hans Heid, whom accompanied African runner Bernard Lagat during the B sample analysis in the accredited laboratory of Cologne in July 2003, states the following in his report (see below) with regards to the lack of specificity of the monoclonal antibody used in the urinary rEPO test (Page 8, section E): "Most importantly, the antibody used does not provide an unequivocal identification of rhEPO and specifically does not discriminate between natural and recombinant erythropoietin! A specific monoclonal antibody exclusively reacting with the recombinant glycoproteins is desirable and should be generated."

A recent study published by Australian researchers casts serious doubt on the utilization of this antibody in the tests for detection of rEPO in sports, and it leaves no place to doubt about the lack of specificity of this antibody with regards to urinary proteins that have nothing to do with EPO (Khan, Grinyer, Truong, Breen, Packer. New Urinary EPO drug testing method using two-dimensional gel electrophoresis. Clin. Chim. Acta 358 (1-2): 119-130, Aug. 2005). Indeed, the authors of the study state literally in the abstract of their investigation "We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO (…)." In simple terms, the antibody that should in theory identify only and exclusively recombinant EPO also identifies endogenous EPO, as well as many other urinary proteins that are totally unrelated to EPO. That is, it gives false positive doping results!

The lack of specificity of this antibody to bind rEPO is the second fundamental argument raised by the independent expert Prof. Bogaerts appointed by the Disciplinary Council of the Flemish Community in Belgium to declare that no solid scientific evidence exists that Belgian triathlete Rutger Beke used rEPO. In an interview with Sporza TV on August 2, 2005, Prof. Bogaerts said "laboratories in Leuven have shown that one of the reagents which is used in the test is not specific enough. This means that the antibody picks up other proteins."

In summary, all experts, both independent and those in charge of accredited laboratories are aware (and were aware in July 2002) that the antibodies utilized to identify rEPO are not the most appropriate, given that they are not specific to rEPO and they cross-react with endogenous EPO and many other urinary proteins, which implies that a high risk of false positive results exists, especially in urine samples with a high protein content, such as many of those collected immediately after strenuous exercise.

The supposedly qualitative nature of the test

Accredited laboratories often insist on describing the urinary rEPO test as a qualitative, not a quantitative test. The difference between the two types of tests is that qualitative tests intend to detect the simple presence of a given substance, whereas tests of a quantitative type intend to measure the concentration of such substance in the sample.

Despite the apparent interest of accredited laboratories in describing the urinary rEPO test as a qualitative test, it is clear, as shown by arbitrations of the Lausanne Court of Arbitration for Sport, that the simple presence of bands indicative of rEPO can not be automatically interpreted as a case of doping, and that it is necessary to observe quantitative-type criteria to reach such conclusion. Indeed, in paragraph 44, page 247 of Arbitration CAS 2001/A/345 it is said that: "(…) it is imperative that a positive doping result be supported by objective criteria which can be verified by third parties." Among the criteria used for identification of a positive doping result, a purely quantitative criterion is mentioned: "more than 80% of the evidence of EPO is in the basic range of the results."

The main reason why it is necessary to establish objective criteria of a quantitative nature to identify a positive rEPO case is that, as stated in page 291 of Arbitration CAS 2002/A/347 "The bands of EPO and rEPO may partly overlap with each other making it difficult to determine if the urine sample contains endogenous EPO or exogenous rEPO. The image will only be considered rEPO if more than 80% of the bands are in the basic range."

It is necessary to underline the lack of uniformity in this quantitative criterion shown by the different accredited laboratories. In paragraph 38, page 236 of Arbitration CAS 2001/A/343, the court indicates that "(…) the Panel learned with surprise that the IOC-accredited laboratory in Paris has, apparently, sometimes required a level of 85%." Moreover, paragraph 57, page 451 of Arbitration CAS 2002/A/452 indicates that "The minimum percentage used by laboratories to find an rEPO violation ranges from 80% to 86%".

In view of this inability to establish objective quantitative criteria, WADA decided in its Technical Document TD2004EPO to adopt the following criteria to identify positive rEPO cases, indicating that "Several CAS cases have referred to the "80% basic bands" rule in making decisions. Further research and experience has indicated that the identification criteria below are more discriminating than the "80% basic bands" rule and therefore the "80% basic bands" criterion should no longer be used. The following identification criteria define the requisites that the image has to fulfil to consider than an adverse analytical finding corresponding to the presence of rEPO:

o 1.- in the basic area there must be at least 3 acceptable, consecutive bands assigned as 1, 2, 3 or 4 in the corresponding reference preparation.
o 2.- the 2 most intense bands either measured by densitometry or assessed visually in the basic area must be consecutive and the most intense band must be 1, 2 or 3. o 3.- the two most intense bands in the basic area must be more intense than any other band in the endogenous area either measured by densitometry or assessed visually.

These new criteria are not supported by any scientific publication, they have not been scrutinized by the international scientific community, and they have not been endorsed by any arbitration body with decision making capacity in the area of doping control.

On September 5th the President of the Spanish National Anti-Doping Commission (NAC) sent a communication to the Disciplinary Committee of the Spanish Triathlon Federation, in which they were advised that the World Anti-Doping Agency (WADA) phoned the accredited laboratory in Madrid on August 31st to communicate new instructions to modify the evaluation criteria for detection of urinary EPO. The new criteria have not been published by WADA and are therefore not known.

The case of Bernard Lagat

In August 2003 the African middle-distance runner Bernard Lagat was tested positive for rEPO. This result was not confirmed by the analysis of the B sample at the accredited laboratory of Cologne, Germany. On October 8, 2003, the expert that accompanied Lagat during the analysis of the B sample, Dr. Hand Heid of the German Cancer Research Center in Heidelberg published a report in which he describes the procedure followed and draws several conclusions. Among these, the following stand out:

o "C) The test used involves many steps with many pitfalls. Only very experienced and critical "bench biochemists", as those working in the Cologne lab, can responsibly handle the complicated and error-prone procedure. There are several problems with background staining, reproducibility, technical artifacts, losses of proteins during the enrichment steps and losses and changes as result of glycoprotein modifications etc.
o D) The test takes too long and is not suitable for mass screening. It should be simplified and improved markedly, e.g. by using ready-made gels, avoiding the double blotting procedure, minimizing glycoprotein modifications etc.
o E) Most importantly, the antibody used does not provide an unequivocal identification of rhEPO and specifically does not discriminate between natural and recombinant erythropoietin! A specific monoclonal antibody exclusively reacting with the recombinant glycoproteins is desirable and should be generated.
o F) IEF as the only criterion is not sufficient to allow a clear decision, certainly not without a specific antibody. Two-dimensional gel electrophoresis, combined with specific mass spectrometric detection, should also be examined as a "second weapon", notably in cases of "confirmation tests".
o G) There is currently no published epidemiological study available that deals with the various possible influences resulting in modifications of the EPO-molecule(s) (nutritional, ethnical, physiological, altitude etc.).
o H) The recently introduced activity tests for enzymatic activities in certain urine samples places utmost importance to the need of a unified, carefully controlled handling and storing of the samples.
o I) In the same line, the internal report for WADA also asks for necessary improvements and changes of the test. On page 15 of this report it is therefore stated: "The development of totally new urinary EPO tests should be encouraged and funded."

The case of Ironman Lanzarote 2005

Of the ten urine samples collected and analysed for rEPO after Ironman Lanzarote on Saturday, May 21, 2001 two were declared "positive" by the accredited laboratory of Madrid; one was "not detectable" (that is, after the complicated and costly analytical procedure the laboratory was unable to detect EPO in the sample, neither endogenous nor recombinant); three were declared "not qualifiable" (which means that given the lack of clarity of the results the laboratory personnel considered that they could not determine if the results of the samples were positive or negative); and four more "negative". In other words, 20% of the samples analysed were "positive" for rEPO, and at least 30% were dubious, as the samples described as "not qualifiable" could be interpreted. If the "not detectable" sample is included in the same group, the incidence of dubious samples would be 40%.

The great incidence of "positive" and dubious samples after Ironman Lanzarote (reaching up to 60%!) is in flagrant contrast with the incidence of positive rEPO cases known to date. As a matter of fact, according to the Peltre and Thormann report (page 26, section 6, Mail exchange with Dr. Mario Zorzoli, Union Cycliste Internationale, Aigle, Switzerland, July 12, 2002), the incidence of rEPO cases in cycling, sport that is suspected to have a high incidence of rEPO abuse, is as follows: in 2001, of 270 urine tests performed 9 were positive (i.e. 3.3%); until the end of June 2002, of 161 tests performed, only 2 were positive (i.e. 1.2%).

No objective information has been provided on the conditions of transport and storage of the samples from collection in Lanzarote until arrival at the accredited laboratory in Madrid…two days and 17 hours later! The importance of this phase of the protocol has been clearly stated by the various expert reports, especially that of Dr. Hans Heid (page 8, section H): "The recently introduced activity tests for enzymatic activities in certain urine samples places utmost importance to the need of a unified, carefully controlled handling and storing of the samples."

No objective information has been provided on the protein concentration of the urine samples that were declared "positive" by the accredited laboratory of Madrid. Given the conditions of strenuous long duration exercise in hot environment of Ironman Lanzarote, it is more than likely that protein concentration was much higher than the limits of reliability of the test, given the poor specificity of the monoclonal antibody used to identify rEPO and differentiate it from endogenous EPO and several other urinary proteins.

According to the results provided by the accredited laboratory of Madrid, the analysis carried out on the A sample (first analysis and confirmation analysis) and B sample of the two athletes who tested "positive" contained the following percentages of EPO in the basic area:

o Athlete 1: 63.15%; 68.21%; 54.72%
o Athlete 2: 50.40%; 57.77%; 67.13%

On these results, two aspects require particular attention:

o None of the analysed samples is even close to the "80% basic bands" criteria required until January 2005, criterion which by the way is the only one that has been endorsed by the Lausanne Court of Arbitration for Sport; o The variability of the results between samples A and B is 33.2% in the case of Athlete 2 and 13.3% in the case of Athlete 1, which clearly shows the lack of reliability of the test.

Summary

In summary, the evidence provided above prove that:

o The urinary rEPO detection test has serious deficiencies and lacks proper scientific validation, particularly with regards to the effects of strenuous exercise on the urinary EPO isoforms;
o The interpretation of the results obtained on urine samples collected after strenuous exercise is not reliable, when a high protein content is present in the samples;
o The monoclonal antibody AE7A5 is not specific for rEPO and interacts with hEPO and other urinary proteins independent of EPO, particularly those that result from exercise-induced proteinuria;
o Conditions of storage and transport of the urine can also seriously affect the validity and reliability of the test.

See also: Serious concerns over urinary EPO test

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