Blood is thicker...

Viren would repeat his double gold performance four years later in Montreal at the 1976 Games and forever etch his name in Olympic history.

Along with his victories, however, came questions. Questions about practices, such as blood doping, that began to be whispered about in 1972, but were replaced two decades later by more technologically advanced forms of performance enhancement.

Treatments with substances such as erythropoietin (EPO), Hemopure, HES, and other hemoglobin/blood plasma boosting or blood manipulating products have created questions about the validity of performances in endurance sports. They've caused governing bodies and scientists to attempt to develop methods to detect their use, and they've promoted a fear among endurance athletes that pharmacology is playing an increasingly greater role in their sports. For while there are drug testing protocols for other performance-enhancing substances, such as stimulants and anabolic steroids, the controls in place to police use of EPO were introduced just prior to the Sydney Olympics.

Controversy has surrounded the development of tests for rHuEPO, what tests to use, and the effectiveness of the tests currently being used. There have been debates between scientists who believe the tests are valid and other scientists and lawyers who claim the test results will not withstand a legal challenge. There have been arguments among sports administrators over which tests to use. There have been arguments over whether or not any of the tests to detect rHuEPO use should be used right now.

At the World Swimming championships in Japan, FINA, the sport's governing body accepted the advice of its medical committee not to test for rHuEPO. The IAAF opted to use a combined blood and urine test at its World Championships in Canada. The cross country ski federation used a blood test during their World Championships, but for "research purposes only." Athletes did not flunk the tests, they merely had adverse results. A new urine test was introduced for detecting HES, a banned plasma expander, and several Finnish skiers were caught and punished, but nobody who had an adverse blood test was sanctioned, nor did the ski federation test the urine for rHuEPO.

At the Sydney Olympics, there were nine adverse findings on the blood test portion of the combined blood/urine test. None of the urine tests were positive for rHuEPO, and no athletes were sanctioned. This spring UCI, the cycling governing body, announced that it would use the urine only rHuEPO test to sanction athletes. Two athletes have had positive tests, one, Danish cyclist Bo Hamburger challenged the result and it was overturned. Just prior to the IAAF World Championships, the results of the A sample analysis of the urine test for rHuEPO for Russian distance runner Olga Yegorova had produced a positive result.

The French hailed it as the first time an athletics participant would be sanctioned for use of rHuEPO. After weeks of conflicting official pronouncements, charges and countercharges, threats, gossip, media proclamations of a doping scandal, Yegorova was allowed to compete in Edmonton, and she won the 5K gold medal. Yegorova has been branded a cheat by some and a martyr by others. She has become the lightening rod that has generated increased scrutiny of the drug testing system, and a movement led by Paula Radcliffe to petition the IAAF for systemic changes in that system.

In an emotionally charged atmosphere, such as this, it is often hard to separate fact from fiction.

That is what will be attempted here-a review of the facts pertaining to the development of testing for rHuEPO. What is known. What must still be done. Genetically engineered EPO-recombinant human EPO(rHuEPO)-has been around in one form or another since 1984. Jim Stray-Gunderson, an American physician then working for the US cross country ski team, warned in 1988 that rHuEPO, while new to the market, was already being used by endurance athletes. As a research scientist now working in Norway, Stray-Gunderson has attempted to perfect a blood test for use by sports' governing bodies. Robin Parisotto led a team of researchers at the Australian Institute for Sport in successfully developing the blood testing method approved by the IOC prior to Sydney.

Bjorn Ekblom, Bo Berglund and other Swedish researchers at the University Hospital in Uppsala, Sweden discovered in the early 1990s that a urine test could be used to differentiate between endogenous(produced by the body) EPO and rHuEPO. The research paper on the method, known as electrophoresis, was published in 1995. It concluded that this test, which can differentiate between the different molecular charges given off by human and recombinant EPO, was promising for the development of a urine test that could prove use of synthetic EPO by athletes. Further research using the method was completed last year by researchers at the French laboratory in Paris, and the urine test they perfected was approved by the IOC as the second half of a combined blood and urine test that was used to test athletes competing at the Sydney Olympics.

Sydney was not the first attempt to control the use of EPO. Cross country skiers were subjected to blood tests starting in 1989; cyclists were given similar "health screening" tests by 1997. The results did not prove athletes were using EPO. The blood tests were used to determine the hematocrit(the percentage of hemoglobin-the protein that transports oxygen-- compared to plasma in the blood) of the athletes. The test used on the skiers measured hemoglobin concentration in the blood and set limits of 16.5 grams/deciliter of hemoglobin for women; 18.5 g/dl for men. UCI, the governing body for cycling, set a limit of 50% for men/47% for women on the hematocrit as a "health risk."

From 1972 until 1989, researchers had also looked for ways to detect blood doping. Various methods were investigated, but none were used prior to the time that the introduction of rHuEPO into the marketplace made blood doping a less attractive option for attempting to boost performance. Blood doping involves taking out a person's blood, separating the red cells from the plasma, storing the red cells under specific conditions, and later reinfusing them into the blood. It is not something easily accomplished. It requires the help of trained medical personnel and the use of the appropriate storage facilities for the blood. Often times the procedure would not be done properly and the athlete would get no benefit or become ill from the process. The risk of being caught was also greater because more people were involved.

The documented cases of blood doping all involved instances when someone involved in the procedure confessed to the violation. Thus, the introduction of EPO into the mix in 1989 was welcome by both those attempting to cheat and those attempting to catch them. The cheaters because it provided a more dependable, less risky form of blood boosting, and the "police" because they now had a substance to detect. The troubling byproduct, however, is the fact that athletes today could still go back to blood doping as a method of enhancing performance as there are still no testing methods that can detect it.

There are those who would argue that the methods presently being used to detect the use of rHuEPO are not effective either. To understand this issue, it's necessary for a short lesson in physiology. The body is an amazingly sophisticated and complex organism. Through a variety of different mechanisms, control over all human functions are maintained. One of these functions is oxygen transport. Red blood cells play an important role in oxygen transport. EPO is a glycoprotein that stimulates the body to produce red blood cells.

The production of EPO is not a fully understood mechanism, but what is known is that the levels of EPO in the body are regulated by a feedback loop from the brain to the kidneys to the bone marrow that appears to be governed by the body's ability to measure and regulate the amount of oxygen in the blood. If there is too little oxygen in the blood, a condition known as hypoxia, the brain senses this and sends a message to the kidneys. A signal is sent from there to the bone marrow to produce more EPO, which triggers the production of more hemoglobin-rich red blood cells.

So, if one lives at altitude, where less oxygen is available, the body is stimulated to compensate by producing more EPO to increase the oxygen transport in the blood. The short term hypoxia experienced in hard training might also be a stimulus for the body to temporarily increase EPO production. These facts form some of the basis for training, and training and living at altitude. One piece of this picture that is not fully understood is how the body turns on and off this mechanism. Recently, scientists at the University of Toronto, proposed the theory that the kidney serves as a "regulator" of sorts for the hematocrit.

"By regulating red cell mass through erythropoietin and plasma volume through excretion of salt and water, the kidney sets the hematocrit at a normal value of 45%," writes Dr. Sandra Donnelly of the University of Toronto. "This is not a random number, but a value that maximizes oxygen delivery to peripheral tissues. The ability of the kidney to coordinate these two volumes to generate a hematocrit of 45% establishes it as the logical site for erythropoietin production."

Donnelly and colleagues suggest that a portion of the kidney known as the critmeter is the control mechanism in this regulation. Because the exact mechanism for regulating the hematocrit is not known, it's also unclear what effects there are of excess EPO in the body. Some people have a genetic condition that causes them to have an unnaturally high hematocrit, over 50%. Other than the fact that these individuals have a shorter life span and are more susceptible to life threatening conditions, such as a stroke or thrombosis, not much is known about the effects of a chronically high hematocrit.

The training effect of an increased plasma volume of fluid allows endurance athletes to have more circulating red blood cells, but what happens to an athlete who has been given an artificially high level of red cells via the injection of rHuEPO? Is that athlete more prone to life threatening conditions? Or do their bodies adapt by increasing fluid retention and increasing plasma volume? These questions haven't been answered and they form the basis for the theories of the health risks taken by athletes who inject rHuEPO. This risk is compounded, scientists say, by the fact that one cannot measure immediately the production of red cells because there is a lag between the time EPO is given and when the red cells are produced.

So an athlete who is injected with rHuEPO won't produce new red cells immediately. It can take days for the increased EPO to result in new red cells. Those red cells can last in the body for 120 days. Thus, if an athlete takes several injections of EPO in a short period of time, he or she might not realize until it's too late that the red cell concentration has gone out of control and threatens to turn the blood into sludge or overtax a kidney or other organ struggling to deal with an artificially induced imbalance.

But the question asked by most athletes and sports administrators is what does all this mean for the detection of those attempting to use rHuEPO to get an unfair advantage? Does it mean that there are too many variables for a blood test to be a reliable indicator of rHuEPO use? How can the blood or the urine test determine whether or not an athlete took rHuEPO as opposed to being an athlete who is merely adapting to hard training or training at altitude?

The tests currently being used were developed by separate groups, each working to perfect a stand-alone test that could be used to detect athletes using rHuEPO. The group in Australia looked at blood. Researchers at a laboratory in France were looking at similar parameters. The Aussie team developed a model based on this indirect detection method. Instead of attempting to differentiate between EPO produced by the body and rHuEPO, the researchers looked a several physiological markers that respond to an injection of rHuEPO. The key to perfecting their method was determining that rHuEPO administration produced a specific response that was able to be identified by their test and could not be confused with any other physiological response. They have published several papers that detail their findings, and in August of 2000 one of their methods was selected by the IOC as the screening test of a two-step EPO test for Sydney.

The Australian researchers used what they called on ON model, markers that, when measured, could indicate someone was taking rHuEPO near the time the test was administered. They also developed an OFF model that detected rHuEPO for a period of time after the athlete had stopped taking rHuEPO. The ON model derived its name from the fact that its formula detects the use of rHuEPO when an athlete is taking it and is effective in detecting use for several days. The OFF model is so named because it can detect rHuEPO only after the athlete has stopped taking the substance, but it can track use for 
several weeks.

The IOC selected the ON model for use in Sydney, but they kept that information from the athletes by allowing press reports of the ability of the OFF test to detect EPO use for as far off as three weeks after an injection of rHuEPO. This had the desired effect of forcing those who were still going to attempt to use rHuEPO to stop using it for the necessary time prior to arriving in Australia for the Games. IOC officials justified the deception by noting that athletes had been withdrawn from the Games allegedly because of suspected rHuEPO use.

Critics of the methods argued that the EPO testing in Sydney was really not an effective deterrent to rHuEPO use since the increased red blood cells produced by rHuEPO use can last for 120 days, while even the OFF model of rHuEPO blood testing could only catch someone who used it within 21 days.

The tests being used in Sydney, they said, were really only effective as an out-of-competition test. Further, the tests had not been properly validated within the scientific community. They would not stand up to a legal challenge. All of this debate went on behind the scenes in Sydney. None of it really surfaced until the Yegorova affair in Edmonton. Yegorova had been tested at an IAAF meet near Paris on July 6. The athletes were not blood tested and were not told that their urine would be analyzed for rHuEPO. The A sample test was determined to be positive. That result was leaked to L\'Equippe, the French sports daily, and the story was spread over the news wires.

Arne Ljungqvist, the IAAF medical chief, explains what happened in Edmonton.

"As you know, the IOC was put under some pressure, particularly from Australia, to have an EPO test in place for the Sydney Games," said Ljungqvist. "For that purpose a blood test was developed in the Sydney lab, and a urine test in the Paris lab. I was repeatedly consulted by the IOC Medical Director (Dr. Patrick Schamasch) with respect to what should be required for a new test of any kind to be valid. I told him, and many journalists who asked the same question, that the standard way for a new test to be scientifically accepted is that (A) it has been published in an internationally recognized journal using the peer review system, and (B) it has been confirmed (reproduced) by other researchers in a different laboratory and their report likewise published as under A or, at least, accepted for publication in such a journal. Since the waiting time for having articles published in recognized journals is quite long (often more than a year), and a confirmation work in a second lab can not get started until the first lab has concluded their study, I said already in 1999 that I could see no way for a new Epo test to be ready for use in Sydney. Time would simply not allow for it.

"I proved, however, to be unnecessarily pessimistic. The following happened. Instead of waiting for the studies in Sydney and Paris to be properly published and confirmed, a review panel with quite many international experts from various fields was convened by the IOC in Lausanne in the summer of 2000. They accepted to evaluate the tests and declare them scientifically OK or not. They felt that their review would be even more detailed and critical than any peer review for publication in journals. Thus, they took on a great degree of responsibility.

"To make a long story short: the panel did not accept the Sydney so called 'OFF model' (i.e. repeated blood tests for the determination of previous EPO use). They said, however, OK to the 'ON model' (i.e. blood parameters suggesting the ongoing - or very recent - EPO use).

"Why then did the panel accept that part of the Sydney work? The reason was that the blood analysis did not contain any new methodology, only standard procedures used in any routine haematological laboratory. What was new was the interpretation of the data obtained at such a multiparameter analysis, and the panel was satisfied that the ON model interpretation was OK. Since, however, the blood analysis only gives an indirect information on possible rHuEPO use (similar haematological alterations can be produced by e.g. high altitude training) it was felt that the On model could not stand alone. It would require some sort of direct confirmation. And that is where the Paris urine test comes into the picture. That test was accepted because the scientific background to the test had already been properly published by a Swedish research team in the mid 1990s. The Paris modification of the test, however, had appeared in Nature only in the form of an incomplete report (brief report) and not as a full, peer reviewed scientific article. Therefore, it was judged that the Paris test could not stand alone and that a case could be judged positive for rHuEPO only if both the blood and urine analyses showed changes compatible with the use of rHuEPO. And that is where we are today. Further experience has shown that the critical parameters to look for in the blood are hemoglobin concentration, hematocrite and the percentage of reticulocytes ( i.e. immature red blood cells).

"So, to the case of Yegorova. At our (IAAF) Grand Prix meeting in Paris doping controls were conducted in the usual way by the meeting organizers on behalf of IAAF (some 12 urine samples). In addition French authorities took an additional number of samples (5 I have been told) for EPO analysis. This was done by them outside our authority and with reference to the well known French law which allows for French doping controls to be conducted on any athlete competing in France. One could expect the French authorities to consult us (IAAF) before taking such an initiative at our competitions or, at least, inform us beforehand but this was not done. Their initiative, therefore, came as a surprise to us. I take the standpoint, however, that the more testing the better provided that the testing follows the right procedures.

"Not long before Edmonton were we (IAAF) informed that Yegorovas urine sample (A sample) had come out positive for EPO in the Paris laboratory. Since that laboratory is the lab that developed the urine part of the combined EPO test, we took for granted that the necessary procedures had been observed. When we later were informed that only urine (no blood) sample had been taken we understood that this was based on a final validation of the urine test to stand alone, a validation procedure which was announced by the IOC Medical Director (Dr. Schamasch) quite a long time ago and in which the Paris lab was involved. It, therefore, came as a great surprise to me when I met with Schamasch in Edmonton and was informed by him that the validation of the urine test to stand alone was not yet completed! He said that it would probably take some more weeks. This meant that the only EPO test valid at the time was the combination of blood and urine analysis accepted at the expert meeting in Lausanne in the summer of 2000.

"At the time of my meeting with Schamasch in Edmonton we had already gone ahead with the B-analysis, which was reported to have met with technical problems. A re-analysis of the B sample was therefore planned, but when I understood that a urine analysis alone would not stand, and that no blood sample had been taken, I consulted legal experts. They confirmed that from a legal point of view we could not go ahead with the case. We, therefore, told the Paris lab that no re-analysis of the B-sample was needed since we had to drop the case (I was later informed that the lab did, indeed, make a re-analysis but experienced the same technical problem as with the first B-analysis).

"No blood sample was taken on Yegorova in Paris. The B-analysis on her urine was not 'negative' - it could simply not be completed due to technical problems. In such a situation we often make a re-analysis, but this was not done for reasons given above. Therefore, technically a complete B-analysis was never conducted. This means that juridically we had no B-analysis that confirmed the finding in the A sample. This means that it is incorrect to state, which I have seen being made so many times, that "Yegorova has tested positive for Epo". Neither technically, nor juridically has she done that.

"Finally, I should mention that the experience with the incident in Paris has made me look closer into the IOC initiated validation procedure for the urine test to stand alone that Dr. Schamasch has been talking about. It seems rather to be an interlaboratory comparison study with five IOC accredited laboratories involved (Barcelona, Lausanne, Oslo, Paris and Sydney) than a scientific validation of the test as such. My committee in WADA (Health, Medical & Research Committee) will, therefore, make a full review of the "state of art" with respect to Epo analysis. This will be on the agenda at our meeting on the 23 of September at Banbury Center in New York."

What happened in Edmonton was that the Russian officials challenged the Paris test result. Said that Yegorova would come to Edmonton, and that she expected to be cleared to run. Yegorova was suspended by the IAAF based on the A sample result. IAAF officials mistakenly told the media that a blood test had also been done on Yegorova in France. They said Yegorova was suspended pending the results of the B sample analysis. The B sample was done, observed by a Russian scientist, Nikolay Durmanov, who acted as Yegorova's representative. Because of a technical error in the analysis of the B sample, the results of the test could not be interpreted, thus the B test failed to uphold the A test result.

This was announced by the IAAF in Edmonton. Yegorova was reinstated. This lead to more confusion because IAAF officials have acknowledged both publicly and privately that the blood test is really only a screening test. That it alone cannot be used to sanction an athlete, and it's value is that it is quick-the analysis can be done in a matter of hours, not the two to three days it takes to complete the urine test-and cheaper than the urinalysis. It is also an indirect method of determining rHuEPO use and therefore more vulnerable to a legal challenge.

The urine test is considered the more promising of the two methods because it is non-invasive, it doesn't involve drawing blood, and it is a direct detection method.

EPO consists of a protein backbone with carbohydrate groups attached. Both natural and rHuEPO occur in several varieties called isoforms, which differ in the structure of the carbohydrate groups. Some of these isoforms have charge differences, and these differences are the basis of the urine test. The testing process separates the isoforms using isoelectric focusing - a type of electrophoresis--on the gel substrate.

EPO and the two commercially available forms of rHuEPO (alpha and beta, which differ in their carbohydrate content) can be separated using this method into distinct bands, representing each of the different isoforms. Their analysis showed that EPO is distinctly more acidic than either form of rHuEPO The results, when compared with a control urine sample and the pure EPO samples, clearly showed that rHuEPO could be detected.

Durmanov, in his letter to the IAAF, says he was "impressed" with the method used by the French lab. He expressed admiration for the sensitivity of the test, but he also expressed reservations about its validity. He challenged the test by saying that before it could be used to sanction athletes, it first must be properly validated and use recognized reference standards. A Finnish physicist and former race walker, Stig Froberg, and Timo Seppala of Finland's Anti-doping agency, supported the Russian challenge. Froberg pointed out that there was only one paper published in the scientific literature on the urine test, and that paper lacked the relevant data needed to properly validate the test.

Christiane Ayotte, head of the IOC accredited drug testing lab in Montreal, responded to Froberg's comments by saying that the issues he raised were not new. That validation of the test was a continuing process. It has been hampered somewhat, Ayotte notes, by the lack of consistency in the testing rules of different federations. For example, the blood test used in Sydney measured seven parameters in the blood. The one used by the IAAF in Edmonton only measured three. The IAAF requires a blood and urine test, UCI will act on results from a urine test.

"There is no description in the IAAF rules as to what constitutes a positive EPO finding and therefore, in the absence of protocol, and until the day the IOC medical commission finally comes out saying that the urine test alone can stand, jurists will consider that only the combination of blood and urine should be applied," said Ayotee. "Scientifically, I do not agree. If the urine test is considered valid to show the presence of synthetic EPO whether the test had been triggered by a suspicious blood screening or by another reason, I don't see the difference: a positive urine EPO test is the proof of administration.

"That is different with the UCI: a clear written protocol is described for EPO and it is said that the EPO urine test can be done following the blood screening or if requested by the physician for example. Therefore, the UCI is fully supported - legally - to conduct only urine EPO testing. The conclusion: this is again a very good example of a lack of harmonisation. The IOC supporting a combination of the Sydney ON urine test, the WADA and International federations conducting a combination of blood screening -with less parameters than the Sydney model - and urine test, the French government doing only urine testing and the UCI having the health haematocrit levels, the blood and urine or only urine test. All of which are very good if properly supported by written regulations but somewhat confusing to the observers."

Jordi Segura, head of the IOC accredited lab in Barcelona, has been doing some of the continuing work on the urine test in order to fully validate the test. The goal is to be able to use the urine test as an out-of-competition test, where it would be most effective as a deterrent to rHuEPO use.

IAAF and IOC officials acknowledge that they are not ready yet. Meanwhile Parisotto and Stray-Gunderson continue to express their belief that the blood test can also be a valid tool in the attempt to detect rHuEPO use. Parisotto noted that athletes have been able to avoid sanctions from the blood tests that just measure hematocrit, as it is easy to manipulate that variable in the blood, but athletes cannot manipulate five parameters.
Thus, he believes, the blood test for rHuEPO could also be used as a stand-alone method of detection.

Work continues on both projects with WADA standing ready to allocate a significant portion of its research budget for the year for rHuEPO detection. (One project, called Science and Industry Against Blood-Doping(SIAB), is a joint effort by labs in five nations to not only test for rHuEPO, but also to close the loophole and develop effective testing for blood doping, as well as the other products used to manipulate the blood.) USADA, the US version of WADA, also has a research fund that will be spent on testing research and development. Radcliffe and the other athletes that support her position want to see these promised improvements to the testing system sooner, rather than later, however. It's been nearly 30 years since the cloud of doping began drifting over distance running, and the athletes want to see everything possible being done to blow that cloud away.

One gesture that could be made by an athlete has not yet been mentioned is a bit far fetched, perhaps, but worth consideration. Yegorova has been quoted recently as saying that she is not running for the money, that her main goal is an Olympic gold medal in Athens. If she wins her final Golden League series meet in Berlin, however, she would split a share of the "pot of gold" on offer from the IAAF for athletes who win 5 of the 7 races. Would it not be a statement of her commitment to rid the sport of drugs if she were to challenge the IAAF by saying that she would donate her share of the grand prize purse to anti-doping research if the IAAF would match that contribution. While the amount of money this would provide for the research would really be comparatively small, the mere gesture would speak loudly for an athlete's desire that the sport be rid of the black cloud of drug use.

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