WADA EPO Testing Methodology Remains Sound and Strong Despite Colvert Case Discussion

My father, Dr. Don Catlin, has always been one of the most frank and open experts in the anti-doping industry. That is in part what attracts media to him still today. Yet, this style poses challenges as intents and comments can sometimes be misused. His comments have been used recently to suggest that there are flaws with the EPO drug-testing process in place today. To clarify, the WADA EPO testing methodology remains sound and strong despite the Colvert case discussion.

The case of Steven Colvert has been discussed as a potential example of a case that demonstrates the flaws in the WADA EPO testing methodology, but really it is an example of the complexity of the EPO test and why thorough analysis is needed to establish solid results—which the results in this case appear to be. Confusion can arise when visual analysis is considered alone, or when results are not considered in their entirety or without the benefit of scientific tools. To understand the realities of the Colvert case, one must first gain an understanding of the science involved.

240px-Erythropoietin

Erythropoietin (EPO)

EPO testing today includes the use of three primary methods; IEF, SDS-PAGE and SAR-PAGE. All three methods have been carefully validated and peer reviewed across multiple laboratories and they have been in use for many years. There is an array of research showing the breadth and capability of the methods. We have published papers based on the seminal methods Dr. Francoise Lasne and other colleagues in the doping control industry have created in this complex realm of science. We certainly would not have based our own research on these techniques if we did not believe the methods to be valid and strong.

The EPO testing methodology is outlined in the WADA Technical Document – TD2014EPO. We recommend that those who wish to completely understand the methodology review the document. The harmonized methodology outlined is designed to create consistency in results between laboratories. There is an image on page 11 that is useful in evaluating Colvert’s results.

This complexity of EPO sport drug testing stems from the reality that EPO is a naturally present substance in the human body. This requires methods to be able to distinguish natural EPO from synthetic, or exogenous, forms. The three EPO testing methods evaluate band patterns with variable shading that migrate from a natural EPO pattern when a drug is used.

It is pretty easy to see a positive when therapeutic quantities of a drug are used as there are large migrations in the band patterns. The results are much more difficult to visually determine when an athlete has microdosed, or when an athlete has stopped using in an attempt to clear the drug from the system, as these situations present band migration patterns that can be very subtle and difficult to distinguish visually from negatives.

It is important to realize that EPO testing does not rely on subjective visual analysis of band migration patterns. There is underlying science applied in the data review process to take visual subjectivity out of the equation. Densitometry, defined as the quantitative measurement of optical density in light-sensitive materials, is performed to scientifically evaluate the shading of bands. GASepo—a software solution for quantitative analysis of digital images in EPO doping control, has been developed to present a “method of robust calculation of the cut-off line, band segmentation and classification algorithms.” So, there is sound quantitative science applied beyond visual review of results.

The recent documentary Troubling Science – Steven Colvert Doping Conviction, as well as the October 26, 2016 article that preceded it, did not adequately consider the underlying science in our view. The response from our esteemed colleague Dr. Christiane Ayotte, Laboratory Director at the WADA-accredited laboratory in Montreal, outlined the scientific conclusions made and included references to the densitometry and software applications used to produce the results such as this image.

The Norwegian authors of the 2016 article suggest that Colvert’s SAR-PAGE results are not indicative of EPO drug use based on his lane being “not much different from other lanes.” They discuss the diffuse staining above the blue line, which was used to determine a positive result for Colvert, as a standard staining anomaly that PAGE testing is subject to with different sample conditions. That conclusion discounts the fact that sample conditions are standardized prior to analysis and it also fails to consider that the staining that appeared in Colvert’s lane did not appear for other negatives in the sample group run at the same time under the same conditions.

Having performed the EPO testing methods in our own labs, we are certainly familiar with staining challenges. Indeed, the 2-3 day tests that are performed are highly sensitive and require extremely skilled analysts in order to create bands that are consistently free from what WADA describes as, “spots, smears, areas of excessive background or absent signal in a lane that significantly interfere with the application of the identification criteria.” In such circumstances, the WADA technical document calls for “invalidating the lane.” The SAR-PAGE results that include Colvert’s sample appear to be an excellent model of results that are free of any staining anomalies.

When the documentary was filmed, Don was asked to visually evaluate Colvert’s SAR-PAGE results, with the filmmaker pointing and asking if Colvert’s looked negative. Don ultimately agreed with that assertion, going on to say that he has seen 20 like it and that the lab must not know what it is doing. A rather astounding statement on its own.

When I was shown Colvert’s SAR-PAGE results, I was able to visually determine the positive sample. To me the slight diffuse staining above the blue line is visually different than the other negative samples. Perhaps my 40-year-old eyes are better than Don’s as he approaches the 80-year milestone later this year. This shows that two people with expertise in evaluating EPO testing results can come to different visual conclusions, which reinforces the importance of the underlying science used to properly determine a positive or negative result.

Colvert’s SAR-PAGE results are an example of the subtle migration patterns that make EPO testing complex and difficult to properly evaluate visually. The peaks laid over SAR-PAGE results by the software application help in the results review process and take subjectivity out of the equation as this image shows with Colvert’s sample on the left, a positive control in the middle, and a negative at right.

Furthermore, the IEF results in Colvert’s case are also indicative of the presence of exogenous EPO. This test requires the two densest bands to be above the line and in Colvert’s sample there are actually three above the line making the visual results easy to recognize. So, two separate validated testing methodologies were used to establish Colvert’s results.

It should also be noted that two different laboratories confirmed these results. This is in fact required under the WADA technical document in order to avoid the reporting of false positives that could be subject to intra-laboratory differences. Both laboratories came to the same positive conclusion.

Some paranoid theorists might point to laboratory malfeasance painting pictures of scandalous anti-doping scientists purposefully contaminating samples. That notion is absurd as our colleagues in anti-doping laboratories are among the most ethical scientists we know. The recent Russian doping debacle and the gross ethical transgressions of our old friend, former Russian laboratory director Grigory Rodchenkov, have called into question the ethics of the anti-doping industry as a whole, one of the most unfortunate ramifications of his actions. Yet we would point out that even Grigory considered it anathema to purposefully taint an innocent athlete’s urine and he refused to do so despite direct orders from above.

What does bother us about Mr. Colvert’s case is not the results, but rather the vehement and passionate defense Mr. Colvert has lodged on his own behalf. His words, and his strong statements in defense of clean sport, are certainly convincing. But we have seen such convincing statements before, from both innocent and guilty athletes. Even stars like Alex Rodriguez and Lance Armstrong told convincing tales once. These stories are one of the most difficult elements we confront in anti-doping.

The Colvert case discussion demonstrates the complexity of the EPO test and data review process. Even well-meaning, qualified scientists, including Don, can be critical of it in certain circumstances. Yet at its core, and through the complexity, the WADA EPO testing methodology remains sound and strong.

Don and I would like to extend our apologies for the remarks about the Cologne WADA accredited laboratory, which were not intended to be disparaging. The Cologne laboratory and staff are some of the most capable, ethical and committed partners in the global fight against doping, and we very much respect their undeniable work as leaders in the industry.

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