This month Sonia Nicholas, Clinical Diagnostics Editor, interviews Christiaan Bartlett, Certifying Analyst at the Drug Control Centre, King’s College London. Learn about how EPO testing is conducted in the lab and about how current techniques, trends and research are shaping this invaluable work.
Christiaan Bartlett, please could you describe your job and role at the Kings Anti-Doping Centre?
I am a Certifying Analyst at the Drug Control Centre, King’s College London - the UK’s only World Anti-Doping Agency accredited laboratory. For the Olympics we are working at the Anti-Doping Science Centre, based in Harlow, a facility provided to us via GSK and LOCOG. My areas of responsibility in the lab are immunoassays on blood and urine samples; testing for erythropoietin (EPO), human growth hormone, the athlete’s biological passport, methoxypolyethylene glycol epoetin beta (CERA), human chorionic gonadotropin, (hCG) and luteinising hormone (LH). We also train staff, both from within our laboratory and from other WADA labs; and perform validation of new methods and instrumentation.
Could you briefly describe the method of EPO testing that you use in the laboratory?
Urine samples undergo immunopurification to selectively capture all forms of EPO, and eliminate the other abundant urinary proteins. The EPO isoforms from this purified extract are separated by iso-electric focusing (IEF) in a pH 2-6 gradient. A Western blotting procedure transfers the EPO from the gel onto a first membrane, which is then probed with a specific anti-human EPO antibody. A second blot transfers this antibody to a second membrane, which is then probed with a sequence of antibody conjugates, ending with the generation of chemiluminescence which is captured by a highly sensitive camera. The original position of the separated EPO isoforms is revealed on the second membrane as a series of discrete bands.
How does the EPO urine test distinguish between recombinant EPO and naturally occurring EPO?
Erythropoietin is a glycoprotein hormone molecule composed of an amino acid backbone with carbohydrate chains attached. Endogenous EPO and recombinant EPO differ slightly in the overall charge of the molecule. This is because recombinant EPO is produced by hamster cells, and the carbohydrates attached during post-translational modification are slightly different in hamster vs human cells, leading to a difference in molecular charge. This subtle difference means the isoform bands of recombinant EPO focus in a different region of the pH gradient during IEF, allowing us to distinguish between a naturally occurring endogenous EPO isoform pattern and a recombinant EPO isoform pattern.
Do you use commercially available kits for EPO testing, or do you use an ‘in-house’ developed method?
We use the technique described above, developed by Dr. Francoise Lasne from the WADA accredited lab in Paris, with minor modifications. The immunopurification sample preparation kits are off-the-shelf items, developed by MAIIA Diagnostics in a project supported by WADA, solely for the purpose of urine purification for this EPO test.
Can the urine EPO test detect pharmacological agents such as continuous erythropoietin receptor activators (CERA) and Hydrol Peroxylases?
CERA is epoetin beta with polyethylene glycol attached, giving it twice the molecular weight of EPO. It can be detected by the urine EPO test as described, but the pegylated EPO molecule is too large to pass through the kidney’s glomerular filter into urine under normal physiological conditions, hence CERA is more effectively detected in plasma or serum.
What are the advantages (and disadvantages?) of direct urine testing for rhEPO over indirect methods?
Both direct and indirect methods of testing for EPO have their pluses and minuses, but used together they offer an effective tool in catching those athletes intent on cheating. WADA statistics show 45 positive findings for erythropoiesis stimulating agents (ESA) (recombinant EPO, darbepoetin, CERA) from direct tests in 2010. Indirect methods such as the ABP can detect the results of blood manipulation irrespective of the type of manipulation used - ESA or transfusion.
What is your view on the adoption of the athlete biological passport (ABP)? How does it complement urine EPO testing?
Firstly there have been a number of successful sanctions brought about purely as a result of the indirect evidence of doping offered by the passport scheme. These have been ratified by the Court of Arbitration for Sport. The number of cases can only increase as more international federations implement the ABP.
Secondly the ABP successfully acts as a deterrent – data from the UCI, who have invested considerable time and effort in the ABP in an attempt to clean up their sport, show a significant decrease in the number of blood samples with extreme values for % reticulocytes since the passport scheme was introduced in 2008. Reticulocyte production can be stimulated (extreme high % retics) after recent EPO use, or suppressed (extreme low % retics) after a blood transfusion. If you compare the times of the stages in recent Tours de France, you will see the times in recent years are slower than in the years before the ABP came in, again suggesting a change in behaviour regarding doping in the elite cyclist population.
Lastly, an individual result from the ABP, without knowing that athlete’s history, can trigger a target urine EPO test if one of more of the blood parameters measured lies outside of the population normal range.
What is the future for EPO testing?
Researchers are developing new techniques to detect EPO analogues that mimic the action of EPO in the body but are structurally different to EPO, so may not be detected by conventional tests. The pharmaceutical industry is liaising closely with WADA to identify new therapeutic drugs with performance enhancing potential, sharing information so that detection methods can be developed concurrently.
Christiaan Bartlett, Certifying Analyst, Drug Control Centre
Christiaan has worked at the Drug Control Centre at King’s College London for 10 years. Previously he worked in similar analytical science jobs in industry.
At the Drug Control Centre he is mostly involved in the analysis of protein hormones, such as erythropoietin and growth hormone (hGH), in blood and urine. In the build up to the London 2012 Olympic and Paralympic Games he will be developing new methods, and transferring existing methods onto different instruments.
Christiaan gives tours of the laboratories to visitors and the media, at organised Open Days ahead of major Games. Early in 2010 the Drug Control Centre became the first World Anti-Doping Agency accredited laboratory in the world to report a positive finding for hGH, and as a result of this Christiaan gave interviews to Sky Sports News and BBC.
Drug Control Centre at King's College London
The Drug Control Centre at King's College London has an international reputation for its work in anti-doping control. Staff at the centre analyse samples collected from athletes competing in major sporting events and training worldwide, and also carry out research into the metabolism and detection of substances in the human body.
The Drug Control Centre will run an independent anti-doping facility for the London 2012 Olympic and Paralympic Games, operating from a satellite laboratory accredited by the World Anti-Doping Agency (WADA). The facility will be based at one of the UK research and development sites of GlaxoSmithKline (GSK). The laboratory is the result of a partnership between GSK and King’s, led by Professor David Cowan, Director of the Drug Control Centre. Thousands of samples will be analysed throughout the Olympic and Paralympic Games and the laboratory will be in operation 24 hours a day.