Red Cell Parameters in Winter Endurance Sports
James Stray-Gundersen, MDM
Norwegian University of Sport and Physical Education
Oslo, Norway
Overview:
Blood doping has been the most significant doping problem in endurance sports over the last 20 years. Blood doping has afforded the greatest performance benefit among doping agents and has been the most difficult to detect. Initially, blood doping took the form of homologous and autologous transfusions; this was followed by the use of r-HuEPO, and most recently by the use of HBOC‚s and other oxygen transport molecules.
The International Ski Federation (FIS) was first to introduce hemoglobin limits to allow participation. The concept was to limit both the degree of the health risk and the degree of performance enhancement provided by blood doping. These efforts were followed by implementing hematocrit limits in the International Cycling Union (UCI) and the International Biathlon Union (IBU). Researchers at the same time have attempted to work on both direct and indirect tests for r-huEPO.
Due to the problem of false positives and false negatives associated with measuring hemoglobin or hematocrit alone, in conjunction with the International Skating Union (ISU) medical committee, we have developed the SAFE (Safe And Fair Events) program for the ISU. The SAFE program is a refinement of measuring hemoglobin or hematocrit concentrations to allow starts and further serves to focus anti-doping efforts where they are most likely to be efficacious.
In 1989, Warren and Cureton came up with a very nice equation that essentially says that for every hundred-gram increase in total hemoglobin mass, there is an increase in maximum oxygen intake of about 600 mL. What this means is that if you have a one gram increase in hemoglobin concentration, the resulting increase in athletic performance, as measured by maximum oxygen intake, will be about 1-4 percent.
At the elite level of sport, contests are won or lost by a fraction of a percent, so performance increases resulting from a small increase in hemoglobin can take an athlete from, for example, 38th place, up to one of the best.
The problem is that in case of the recombinant EPO, or even growth hormone, a paradigm shift is needed in the way we approach doping control. Since the half-life of these drugs is very short, and the half-life of the effect is much longer, there is a large open window where urine tests have almost no ability to detect the EPO. Therefore, traditional direct testing, at this time, cannot work in the second and third weeks after cessation of EPO administration.
With the availability of the ADVIA 120 analyzer, however, it is possible to measure red cells and reticulocytes. The ADVIA 120 is a unique machine, and is the only one that can measure, essentially, the hemoglobin concentration, and, thus, hemoglobin content, of each red cell.
EPO 2000
With the help of the ADVIA 120 technology, in combination with the direct urine test, the EPO 2000 project was developed. It was sponsored by the International Olympic Committee, and is a collaboration between researchers in Australia, China, Canada, France and Norway, with data included from a profiling study of 1100 athletes in thirteen different countries.
The hematological parameters used for this project model were hematocrit reticulocyte, hematocrit, and percent macrocytes ˆ all measured using the ADVIA 120 ˆ in addition to immunoassays for erythropoietin and soluble transferrin receptor.
Basically, what was found in this project was that, using a five-parameter on-model equation and a three-parameter off-model equation to detect when an athlete is on or off EPO, there was a complete separation in the results of athletes receiving EPO and those athletes receiving placebo. This means that we have a very effective change model that can be used in conjunction with a hematological passport to detect the use of EPO.
Later efforts in this project included calculating on-model scores based on a four-week altitude camp, which showed that the mean values for men and women almost never go above the cutoff scores for the regular EPO on-model. In the very few instances where athletes had values that exceeded the cutoff stage in response to altitude, urine tests were able to differentiate between the effects of altitude and those of EPO.
The SAFE Solution
In response to questions concerning the accuracy of hemoglobin and hematocrit limits implemented by sporting organizations, we have come up with the SAFE solution, which represents a significant paradigm shift from standard doping control protocols.
Under the SAFE paradigm, all athletes are screened on a non-competition day, thus allaying athletes‚ concerns about testing interfering with their performance.
To implement this screening we have protocols that take up about ten minutes of an athlete‚s time, and we can process two hundred athletes in a period of about five hours. Further, these protocols are relatively inexpensive, meaning that we are better able to focus anti-doping efforts where they are more likely to be successful, and thereby make better use of funds spent on anti-doping efforts.
The SAFE program is an extension of the EPO 2000 project, and is a refinement of hemoglobin or hematocrit measurement. It forms a basis for an individual athlete‚s hematologic passport.
Essentially, what happens under this model is that the athlete comes in on a non-competition day, and 3 mL of blood is drawn in a standard position after they have been seated for five minutes. This five-minute period is used to allow for the stabilization of plasma volume. After the blood sample is taken, it is analyzed on the ADVIA 120.
The first decision in the SAFE paradigm is whether or not an athlete‚s hematocrit level is within the limits for their particular sport. For those who have levels exceeding the limit, we look at the other hematologic parameters of erythropoiesis. If these other parameters show signs of normal erythropoiesis, the athlete is approved for participation in the sporting event.
On the other hand, if the hematocrit level is over the limit and the hematological parameters show signs of accelerated or decelerated erythropoiesis, there is an increased degree of confidence that by preventing this athlete from starting you are doing the right thing. This is not necessarily considered a positive doping test, it is just saying that a significant accelerated or decelerated erythropoietic picture here justifies not allowing the athlete to take part in the sporting event.
It is also worth noting that, for those athletes who have hematocrit levels below the cutoff points, these procedures aid in the establishment of a Œnormal‚ picture from an erythropoietic standpoint. These athletes are, of course, allowed to compete.
When an athlete shows signs of abnormal erythropoiesis, but is allowed to compete, a system of focused investigation comes into play, under which the athlete undergoes more comprehensive testing.
One potential problem with these assessments is that when humans are involved in making decisions about which athletes will compete or which athletes will undergo further doping-control tests, a certain amount of bias is unavoidable. Therefore, as part of the program we have designed a computer macro that takes the export file off the ADVIA 120 and analyzes it. This analysis decides if a given athlete starts or not and whether follow-up testing is indicated or not. This computerized categorization, however, is supervised by a medical official from the sport‚s governing body to ensure correct results.
Using the SAFE paradigm, we have obtained more than 1500 samples from about 600 world-class endurance athletes in ISU, IBU and FIS over the course of the last two winter World Cup seasons. Samples were obtained during World Cup or World Championship competitions in Europe, North America and Japan. Approximately 1100 samples were obtained at sea level, 200 samples at 1400m and 200 at 1750m. The specimens were analyzed on six different ADVIA 120 hematology analyzers, which were carefully calibrated prior to use.
If this testing paradigm had been in place as an official function, the hematological parameter measurements would have resulted in only 1 percent of the athletes being excluded from competition, while 22 percent would have been required to undergo further testing.
Our testing using the ADVIA 120, it should be noted, can indicate accelerated erythropoiesis by detecting high reticulocyte counts, or high percent reticulocyte counts, in addition to the size and hemoglobin concentration of reticulocytes. Decelerated erythropoiesis is detected using essentially the same measurements.
It is worth considering the possibility that through efficient detection methods for EPO, we may push athletes into using other forms of doping. However, with the ADVIA 120 testing procedures we use in the SAFE paradigm, we can also detect indicators of the use of hemoglobin-based oxygen carriers, as well as transfusions.
Overall, what we can do in the case of accelerated erythropoiesis as evidenced by hematological parameters, is go ahead and measure EPO and transferrin receptors and calculate an on-model score. In addition, urine samples are collected and tested for recombinant EPO. If these additional tests are positive, it is fairly certain that an incidence of doping will be detected.
In the case of decelerated erythropoiesis you can have an extremely high degree of certainty that the deceleration is due to stoppage of a banned substance, as opposed to altitude changes or any other normal factor that may affect erythropoiesis. This can be verified through additional blood testing after 5-10 days, which will show either another decelerated model or a normal model. A normal or higher measurement is indicative of a positive change model, which is a very strong indication of the use of doping.
In summary, the anti-doping methods described include the measurement of a series of parameters on the ADVIA 120 analyzer that give an indirect indication of accelerated or decelerated erythropoiesis, the use of hemoglobin-based oxygen carrying molecules, or transfusions. Also in place are additional confirmation tests for doping, some of which are still under development.
With current doping methods, it is theoretically possible for an athlete to use such low doses of prohibited substances that their parameters remain within normal levels. However, the performance benefits and repeated testing over time is likely to detect very substantial changes in their hematological parameters if they cease doping.
Finally, the SAFE paradigm offers an effective strategy for detecting and deterring blood doping. It also reduces inconveniences to the athlete, is welcomed by the athletes because it provides them with accurate data on their own hematological parameters and health, and can be used as the basis for a hematological passport to track doping status and health changes over time.
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