Catching the drug cheats

By Janette Woodhouse
Monday, 30 July, 2012


Rams testicles gave athletes in ancient Greece a testosterone boost, 1896 Paris-to-Bordeaux cyclists combined heroin and cocaine in a speedball, sprinters at the Berlin Olympics experimented with nitroglycerine in an effort to dilate their coronary arteries … athletes have been experimenting with performance-enhancing drugs and techniques for a long time.

But the scale and the sophistication of doping have escalated dramatically since the 1950s. The advent of human growth hormone (HGH) becoming available and Ciba Pharmaceutical commercialising the oral anabolic steroid methandrostenolone opened a whole new era of athlete doping. In 1964, the Tokyo Olympics became known as the ‘Steroid Olympics’ and it became obvious that a banned substance list and drug testing of athletes were needed if an Olympic Gold Medal was to have any credibility.

Ultimately, this lead to the formation of the World Anti-Doping Agency (WADA) - an international, independent, collaborative worldwide campaign for doping-free sport. WADA monitors the anti-doping efforts at the Olympics and has updated the official list of prohibited drugs each year since 2004. Some, like caffeine, have been removed from the list over time, with more added in nine categories, including anabolic agents, stimulants and growth hormones. Alcohol is prohibited for a few of the more dangerous sports, including archery.

However, the effectiveness of the fight against doping depends on the ability of anti-doping laboratories to reliably identify athletes who are using performance-enhancing drugs or methods.

To this end, WADA has an accreditation process based on ISO/IEC 17025 and the International Standard for Laboratories. To be WADA-accredited, laboratories must undergo a series of rigorous tests to establish their analysis credentials. Assessments focus on the facility, equipment, procedures and staffing during three formal inspections and dummy sample testing. After accreditation, WADA’s External Quality Assessment Scheme evaluates laboratory competency through a continuous assessment of performance and provides laboratories with opportunities to compare their results, with the aim being to enhance harmonisation of test results among accredited laboratories.

There are currently 33 laboratories around the world accredited to conduct human doping control sample analyses. The Australian Sports Drug Testing Laboratory housed with the National Measurement Institute in North Ryde and headed by Dr Catrin Goebel is Australia’s only WADA accredited facility.

In the six months prior to 19 July, WADA-accredited laboratories conducted at least 71,649 tests on samples from potential summer Olympic athletes. As a result, at least 107 athletes received sanctions that made them ineligible to participate in the London Games.

Modern trends in athlete doping

The days of athletes doping with ‘designer drugs’ such as tetrahydrogestrinone (also known as THG or The Clear and the main drug in the Balco scandal) are nearly over because such drugs are now so easy to detect.

The use of medicines that are under development by pharmaceutical companies is also now more difficult because the companies have agreed to provide WADA access to their confidential information about their developmental products that could be abused by athletes. For example, in 2008, Roche Holding worked with Wada to develop tests for its drug CERA, an erythropoietin that boosts red blood cell count and oxygen uptake. Testing following the Beijing Games resulted in six athletes testing positive for CERA.

Athletes are now more likely to dope with drugs which mimic or are naturally produced in the body. The ‘big three’ are erythropoietin (EPO), human growth hormone and testosterone. This requires a whole new level of testing for laboratories as these products are naturally occurring in the body and a simple ‘there’ or ‘not there’ test is inadequate.

A new approach has been developed whereby, rather than looking for actual doping agents, samples are screened for a range of haematological, steroid profile and endocrine markers. Athletes are tested over a period of time and longitudinal profiles of each individual established - the ‘Athlete Biological Passport’ (ABP). Any abnormal variations in the individual’s profile can indicate the use of prohibited substances or methods.

Athlete Biological Passport

The ABP is already proving to be a deterrent - data from the Union Cycliste Internationale 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. Stage-times in the recent Tours de France are slower in recent years than in the years before the ABP came in, suggesting a change in behaviour regarding doping in the elite cyclist population.

The Portuguese long-distance runner, Hélder Ornelas, was the first athlete whose APB was used as sole evidence in support of an anti-doping rule violation.

The International Association of Athletics Federations (IAAF) has been collecting a significant number of blood samples throughout 2012 in the lead-up to the London Games, and 200 more ABP tests will be conducted at the Olympic Village in London. In the lead-up to the London Olympics, nine track-and-field athletes were caught doping, based on their ABPs.

In the ABP, haematological screening blood variables that could be indicative of the use of EPO or related substances are measured. These include:

  • Red blood cell (erythrocyte) count
  • Mean corpuscular volume
  • Haematocrit
  • Haemoglobin
  • Mean corpuscular haemoglobin
  • Mean corpuscular haemoglobin concentration
  • White blood cell (leukocyte) count
  • Platelet (thrombocyte) count
  • Reticulocytes percentage

Abnormal variations observed are referred to scientific and medical experts who will give an opinion as to whether these variations could be the result of doping or not.

The endocrine module could prove to be even more promising in the fight against doping, with key biomarkers creating an athlete’s individual ABP ‘fingerprints’.

The London Olympics

At the London Olympic Games, athletes will undergo the most sophisticated testing program yet devised, with individual tests for substances such as erythropoietin (EPO) and human growth hormone, and combined tests for other banned products.

The pharmaceutical giant GlaxoSmithKline (GSK) has provided a £20 million state-of-the-art laboratory in Harlow, Essex, a short distance from the Olympic Park. Here, King’s College London will operate the facility in coordination with the Olympic Committee and International Paralympic Committee.

The 400 m2 laboratory is fully equipped and can be used to reliably check for more than 240 prohibited substances and give negative results in less than 24 hours. It will be staffed 24 hours a day, seven days a week during the Olympic and Paralympic Games. A team of 150 scientists from all over the world will staff the WADA-accredited facility.

About 6250 samples will be assayed during the Games, which is the highest amount ever at an Olympic and Paralympics event. Most tests will be on urine but at least 10% will be blood-based.

The laboratory will provide results for most of the tests within 24 to 48 hours. For a few tests, such as on endurance-boosting drug EPO, longer time will be required (up to 72 hours) to get the results. The turnaround time of a negative result will be 24 hours.

Urine samples will be split in two - A and B. Part of sample A will be prepared for testing and the rest securely stored. It will be screened for more than 60 prohibitive substances in just one test. The initial result of a test (sample A) will be available in just 12 hours. In case of a suspected positive, the result will be confirmed within the next 12 hours.

The blood and urine samples from athletes will be stored after the Games for eight years to enable retrospective testing on the samples when new tests for more drugs are developed.

The team will employ the latest analytical techniques: ultra high performance liquid chromatography (UHPLC) will ensure fast chromatography with excellent resolution, and triple quadrupole mass spectrometers will provide high sensitivity with a speed of operation to match the UHPLC process.

Gas chromatography and isotope-ratio mass spectrometry, as well as clinical chemistry techniques, will also be used to detect as many as 400 different substances across multiple pharmacological categories.

Australia’s contribution

Four scientists from the NMI’s Sports Drug Testing Laboratory in Sydney joined over 150 scientists from around the world who will be conducting the drug testing program for the London Olympic Games.

Lance Brooker, Jill Simpson, Janelle Grainger and Catrin Goebel were invited to work with King’s College London in the provision of sports drug testing services for the London Olympics. Each member of the team will be working in the field of their expertise: Lance Brooker - isotope ratio mass spectrometry, Jill Simpson - erythropoietin, Janelle Grainger - human growth hormone and Catrin Goebel - liquid chromatography high resolution mass spectrometry.

The Australian Government, through the Australian Sports Anti-doping Authority (ASADA), invested more than $1 million in the Pure Performance program to conduct more than 1000 blood and urine tests across Olympic and Paralympic athletes in the lead-up to London.

What about the horses?

Of the 25 positive drug tests at the Beijing Olympics, six were for horses.

Equine participants in the London Olympics will be tested as are the human athletes. Samples of blood and urine will be tested for prohibited substances, particularly anabolic steroids (synthetic derivatives of the male hormone testosterone), Epogen (a man-made form of the human protein erythropoietin) and corticosteroids.

As in humans, anabolic steroids deliver increased muscle mass and strength to the horses while Epogen stimulates bone marrow to produce more red blood cells and so increases the horse’s stamina.

Corticosteroids are used as anti-inflammatories. They are often injected into an arthritic joint to decrease pain and inflammation, enabling an equine athlete to perform better. Administrations of corticosteroids are not absolutely prohibited in performance horses but the level of corticosteroids in blood or urine is be evaluated to determine if an injection occurred within a prohibited time period.

These are not the only tests on horses: following the 2004 Athens Olympics, Irish rider Cian O’Connor had to return his gold medal after his horse, Waterford Crystal, tested positive for the human anti-psychotics fluphenazine and zuclophenthixol.

The Fédération Equestre Internationale, the governing body of the equestrian sports, has been working at enforcing and testing for drugs as well as finding ways to make the inevitable spills less damaging to the horse and rider.

Human growth hormone

WADA has announced that a new test for the abuse of human growth hormone will be used at the Games, just weeks after it was cleared following a near 13-year development process. It is understood that human growth hormone testing will be particularly sensitive in London.

Currently, HGH can only be detected if it has been used a few days prior to the test; this summer, advancements in biomarker technology mean it will show up even if the abuse took place weeks before the test. The ‘biomarker’ test is claimed to be capable of distinguishing between HGH produced naturally in the body and synthetic HGH.

HGH abuse has been hard to test for and is among the three biggest doping worries for the London Games. The first HGH test at the Olympics was in Athens in 2004. The new HGH test will work alongside the old one in London, giving testers a larger window to find traces of the substance.

EPO testing

Erythropoietin is a naturally occurring hormone that stimulates red blood cell production - more red blood cells means more oxygen carrying capacity, means better endurance for athletes.

The glycoprotein hormone molecule is 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 difference is exploited when the EPO isoforms are separated by iso-electric focusing (IEF) in a pH 2-6 gradient. This allows the scientists to distinguish between a naturally occurring endogenous EPO isoform pattern and a recombinant EPO isoform pattern.

Dr Francoise Lasne from the WADA-accredited lab in Paris developed the EPO testing method which, with slight modifications, will be used during the Games.

Urine samples will be immunopurified to selectively capture all forms of EPO and eliminate other proteins. A Western blotting procedure will transfer 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.

Continuous erythropoietin receptor activators (CERA) can be detected by the urine EPO, but because of its size the molecule does not pass through the kidney’s glomerular filter into urine under normal physiological conditions and so is more effectively detected in plasma or serum.

The Olympic Games are considered a coup by the host country as they reap the rewards of developing the infrastructure needed and then the global exposure generated during the Games. It is also good for science as it funds significant research and employs many talented researchers.

Related Articles

Infrared imaging method measures biomolecules in living cells

Researchers used infrared light to capture clear images of biomolecules inside cells —...

Some Victorian-era books contain toxic dyes

If you come across any brightly coloured, cloth-bound books from the Victorian era, you might...

High-performance SEM drives plant and food research

Plant & Food Research is on a mission to improve the way food is grown, harvested, prepared...


  • All content Copyright © 2024 Westwick-Farrow Pty Ltd