EPO Testing Makes
Olympic Debut with IMMULITE Assay

The question of how pervasive drug use truly is among athletes competing
in sporting events around the world surfaced again with the Summer Olympic Games in Sydney, Australia. Withdrawals of athletes before the games, the insertion of a pledge in the Olympic oath to compete drug free, media coverage of drug testing, and disqualifications of medalists reminded the world of the unfortunate need to test competitors. For some banned substances, no approved test is currently available. Such was true for erythropoietin (EPO)—until this year. With the developement and approval of a new test for synthetic EPO using DPC's IMMULITE® EPO assay, at least this drug is now straightforward to detect. Hopefully, the test has rendered EPO abuse impractical to would-be abusers.

EPO, a peptide hormone secreted by the kidneys in response to a decrease in the partial pressure of blood oxygen, stimulates red blood cell production. An elevation in the number of red blood cells increases oxygen transport to muscle tissue, thus enhancing endurance. Athletes who could be tempted to resort to synthetic EPO administration include those involved in endurance events such as cycling, distance running and swimming.

Synthetic EPO became available in 1987. Since then, it has been implicated in the suspicious deaths of at least 25 world-class cyclists. The International Olympic Committee (IOC) banned its use in 1990, but no validated test for EPO doping would be available for a decade. Rumors of EPO use remained just that. In 1997, the French Union Cycliste Internationale (UCI) began suspending cyclists for a 2-week period on the basis of an elevated hematocrit—a parameter unable in itself to point to exogenous EPO use—but only ostensibly as a protective measure to prevent racing "at risk." Hard evidence of EPO abuse surfaced the next year, when a French border guard discovered EPO in the possession of the trainer of a cycling team favored to win the Tour de France.

Several years ago, Gareau from Canada demonstrated that the level of
soluble transferrin receptor in serum increased significantly following EPO administration. However, this marker was not, in itself, sufficient to prove EPO abuse by an athlete. A breakthrough in the search for a detection method came in 1999, when an EPO administration trial conducted by the Australian Institute of Sport (AIS) and the Australian Sports Drug Testing Laboratory (ASDTL) demonstrated that recom-binant EPO leads to the production of reticulocytes larger and more numerous than those arising from endogenous EPO secretion. Aided by grants from the Australian government and the IOC, the Australians developed a promising blood test.

A concurrent effort by a team of French researchers resulted in a direct test for recombinant EPO in urine.

The Australian and French research groups presented their data to the
IOC Medical Commission and independent scientific experts in July and August of this year. The testing procedure using both blood and urine samples was approved in time for use at the Sydney Olympics. More than 300 precompetition blood/urine tests were to be performed during the 2
weeks prior to the Olympics and throughout the Olympic period. The timing
of the testing would have forced abusers to stop EPO intake too early to obtain its full effects, or risk being discovered.

The program demonstrated a degree of effectivenesss: one country withdrew 40 of its athletes before the beginning of the games in response to failed precompetition EPO testing; subsequently, other athletes also withdrew. No athletes were confirmed positive for EPO administration during the games.

The Sydney 2000 Olympics were the first Summer Games in which blood was collected from athletes for drug testing. The blood samples were tested for use of the recombinant form of erythropoietin (EPO), which boosts athletic performance by stimulating red blood cell production, thereby increasing oxygen transport to muscles.

Beginning in March, scientists from the Australian Sports Drug Testing Laboratory (ASDTL) and the Australian Institute of Sport (AIS), together with collaborators from France, Canada and Norway, undertook a major study to validate a test for detecting the use of recombinant EPO by athletes. This study involved collection and analysis of blood and urine samples from over 1,200 athletes from 13 countries. In addition, more than 130 volunteer, recreational athletes from Australia, Norway and China took part in EPO administration trials to determine the changes in blood and urine parameters following EPO injection. As part of the validation, over 6000 blood samples were analyzed at ASDTL and the AIS.

In early August, an expert scientific panel appointed by the International Olympic Committee (IOC) reviewed the validation data and approved the
EPO drug test.

The EPO test, used for the first time at the Sydney 2000 Olympic Games,
is a combination of a blood test and a urine test. The blood test detects the following changes in blood parameters after EPO administration: serum EPO level, serum soluble transferrin receptor, hematocrit, the percentage of macrocytes (large red blood cells), and the number and size of reticulocytes. Serum EPO levels are measured using the IMMULITE® EPO assay. The urine test was developed in the IOC-accredited laboratory in Paris and uses isoelectric focusing techniques to separate the different glycoforms of EPO in urine. EPO is detected by Western blotting techniques, and the pattern of isoforms in recombinant EPO can be distinguished from the pattern observed with endogenous EPO found in urine. All drug testing of athletes at the Sydney 2000 Olympic Games was carried out by ASDTL, an IOC-accredited laboratory.