More on EPO testing

Lisa Dusseault pointed me to to an interesting in CyclingNews on EPO testing. Apparently, at least four athletes (Rutker Beke, Virginia Verasategui, Iban Rodriguez, and Bernard Lagat) have been accused of taking EPO based on positive tests and then ultimately cleared (or at least acquitted.)

The basic problem appears to be, as I indicated earlier that the Lasne technique (1-dimensional gel electrophoresis) has never been calibrated to any known accuracy rate, and indeed WADA has changed the criteria at least once:

Up until now, the urinary EPO test has been called "qualitative", but is in fact quantitative, as there has to be a minimum percentage (80% ) of basic isoforms for a sample to be classed as positive. The Chatenay-Malabry laboratory in Paris is even more stringent, requiring 85% of the bands to be basic for a positive. It should be noted that 80% is quite high when considering normal urine: someone could take EPO and still pass the test if their basic bands percentage was 79%. Thus, the test can give rise to false negatives as well.

In January 2005, WADA recommended that the 80% basic bands criterion should no longer be used, and that a more qualitative system should be used:

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.

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.

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.

None of these criteria were subject to scientific review, but were unilaterally adopted by WADA, it seems. On September 5, 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 31 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.

Obviously, the first thing you need to do before changing your criteria is to scientifically calibrate them for error rate, but there's no evidence that this has been done here.

Once you get past the methodological problem, there appear to be two technical issues that may or (may not, since we have very little data) cause false positives. These are both discussed in quite a bit of detail in an article by Dr. Inigo Mujika (Conflict of Interest alert: he's a coach for Virginia Berasetegui. However, the issues he's raising look like ones that have concerned me as well.)

The first issue is how well we understand the baseline EPO isoform mix in the control group (people who don't take EPO). This appears to be a particular issue with samples taken immediately post exercise. There are two subissues here. The first is overall high protein concentrations which you're supposed to control for before running the Lasne test but Mujika claims that labs aren't doing. The second is what the mix of isoforms is. Mujika cites a WADA-financed article by Kazlaukas et al. (Australian Sports Drug Testing Lab) that indicates that the isoform mix can be changed by exercise. The case of Rutker Beke is another piece of evidence here.

The second issue here is the specificity of the antibody you use to detect EPO. Mujika raises the question of whether it's really EPO-specific (actually, he raises the question of whether it's rEPO specific as well, but it seems to me that this is a non-issue since the whole point of the electrophoresis is to remove the concern about antibody specificity). If it's binding to other proteins, this makes the concern about baseline protein concentrations in urine even more significant.

The bottom line here is that as a layman, it doesn't appear that WADA has done enough work on the test to be able to use it to punish athletes with any confidence. I realize they're in a tight spot because they think a lot of EPO abuse is happening but they're having trouble proving it, so they wanted to roll a test out ASAP, but that doesn't make the science get done any faster.