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Stemming the Flow of Blood Doping

By Hannah Friesen

Published on August 21, 2012

Each year, world records are broken, lying at the feet of rising athletic stars more than ready to challenge their predecessors. Biomechanics researchers have been investigating different factors that affect an individual's performance, aiming to locate the biological machinery behind success. Over the years, many have been studying how an increase in blood volume and human erythropoietins influences athletic ability. The act of blood doping, or artificially raising the body's natural count of red blood cells (RBC's), improves the amount of oxygen delivered to muscles, allowing for a higher degree of fatigue resistance [1]. This discovery has had a profound impact on the world of competitive sports.

USCience Review

Figure 1: This figure demonstrates the layers present after blood is centrifuged. The hematocrit (HCT) value defines the percentage volume of the red blood cells in the blood sample.

The Rationale behind Blood Doping

An illegal performance enhancing technique, blood doping, or blood building, has become increasingly prevalent in endurance-dependent athletic events, particularly cycling and long distance running. These sports heavily rely on aerobic respiration for energy production since inhaled oxygen is used to metabolize glucose molecules to produce adenosine tri-phosphate, or ATP, the body's primary transporter for chemical energy [3]. Numerous individuals have resorted to blood doping since over 95% of the oxygen in the bloodstream is transported by hemoglobin, a metalloprotein present in all RBC's. Therefore, an increase in the level of hematocrit (HCT), the volume percentage of RBC's in the blood (see Figure 1), would improve the efficiency of oxygen distribution to slow twitch muscles, making the human body more fatigue resistant [4].

Ultimately, athletes wishing to partake in this practice have two different transfusion options: homologous or autologous. In a homologous transfusion, RBC's are harvested from a compatible donor, stored, and transfused into the subject's bloodstream; in an autologous transfusion, RBC's are harvested from the subject, stored, and returned to the subject [5]. The primary goal of these procedures is to extract and administer the blood at optimal times. Autologous transfusions are typically more common in modern athletics, and the extraction phase occurs two to three months prior to any given competition. After extraction, the RBC's are centrifuged out of the blood, and the plasma is returned to the subject to maintain fluid levels in the body [1]. Sealed and placed into bags, the RBC's are stored in freezers at -85°C, a temperature which preserves the cells for virtually indefinite periods of time [6]. Upon the advent of competition, one to several days prior to the event, the RBC's are intravenously returned to the subject [1]. This form of "doping" is favored by many due to its potential to increase the RBC count in the body by up to 20%, as suggested by research [4].

There is another method of increasing the body's RBC count that is able to bypass transfusions altogether: injections of human erythropoietin (EPO). EPO is a hormone naturally produced by the kidneys that performs two main functions: stimulating bone marrow to produce RBC's and triggering the synthesis of hemoglobin [7]. Athletes that increase the concentration of EPO in their bodies pave the way for an increase in both oxygen carrying capacity and energy conversion efficiency [8]. Overall, these injections are, in many modern cases, chosen over transfusions because EPO's are harder to detect, and the technique itself require fewer steps.

National Toxicology Program: U.S. Department of Health and Human Services

Figure 2: Di-(2-ethylhexyl)phthalate (DEHP) is a plasticizer in IV bags that is an indicator of transfusions.

Techniques and Methods for Detection

After the number of competitive athletes using illegal performance enhancing techniques dramatically increased, the International Olympic Committee formed the World Anti-Doping Agency (WADA) in 1999. Blood doping on its own was a late-comer in regards to joining the list of banned substances, which include anabolic steroids and amphetamines; there were numerous cases before blood transfusions were ultimately declared an illicit practice in 1986, and EPO injections in 1990. Consequently, the WADA has been attempting to make up for lost time: in response to the ever-climbing number of transfusions and injections, the WADA has been funding research and laboratories aimed at improving detection techniques [2]. While this effort largely remains a work in progress, there are a few different tests currently in practice.

1. Testing for the Presence of Urinary Plasticizers

A modern test has been developed that aims to detect transfusions of any sort- homologous or autologous. Researchers found that subjects undergoing intravenous transfusions, medical or illicit, are exposed to a specific type of plasticizer found in IV bags: di-(2-ethylhexyl)phthalate (DEHP) (see Figure 2). Results from a 2009 scientific study compared DEHP concentrations in urine samples from three groups: hospitalized patients receiving blood transfusions, athletes, and a control group with no exposure to plasticizers. Comparative analysis of each of the urine samples illustrated that increased levels of urinary DEHP are linked to exposure to the plasticizers present in the IV bags used in transfusions. By this logic, the presence of high DEHP concentrations in urine could indicate practice of illegal blood doping [9]. While promising, this detection method is still new and is not, on its own, enough to charge an athlete with illegal doping, but rather serves as more of a screening process [10].

U.S. National Library of Medicine of the National Institutes of Health

Figure 3: Reticulocytes are immature red blood cells. Low levels of reticulocytes and high levels of mature RBC's can serve as evidence of blood doping.

2. Abnormally High Off Scores and Low Reticulocyte Counts

Another test designed to serve as an indicator for illicit intravenous transfusions is a blood analysis involving comparison of the concentrations of mature and immature RBC's present in an athlete's bloodstream. Under normal circumstances, individuals should possess specific percentages of developed red blood cells as well as undeveloped red blood cells, known as reticulocytes (see Figure 3). A measure known as the "off-score" has been developed by researchers based on a series of equations involving standardized mature and immature concentrations in the human body. A score between 85 and 95 is considered typical; scores over 133 are considered atypical and can suggest the possibility of doping due to increased levels of mature RBC's [11]. Using low levels of reticulocytes and a high off score as evidence for blood doping, the ICU presently excludes athletes with abnormal scores from participating in competitive cycling events [12].

3. Hematological Threshold: Atypically Viscous Blood

A classic method used in many modern tests aims to distinguish blood dopers from other athletes by measuring their HCT. An upper threshold is established and numbers above said threshold can be used as evidence to support a blood doping charge [12]. The standardized limits for HCT are 50% in males and 47% in females [13]. Athletes with scores well above these respective values can be accused of engaging in illicit performance-enhancing techniques [12]. Unfortunately, there are a few instances that will alter the body's HCT values naturally: high altitudes and heavy smoking can potentially increase HCT, and intense training regimens, at the start, can increase HCT temporarily [13]. Therefore, the extraction period for blood samples is highly relevant; both the time and location of collecting samples could be the difference between a false accusation and a rightful claim.

4. Isoelectric Focusing: Distinguishing Recombinant EPO from Natural EPO

Illegal recombinant EPO hormones are distinguishable from naturally endogenous hormones based on the electrical charge of each respective substance [14]. Isoelectric focusing (IEF) separates molecules based on the extent of their charge differences and thus can be used to test athletes for the presence of exogenous or recombinant EPO's. First, various stages of staining reveal the isoelectric band pattern for a given EPO, which serves as a reference point to compare sample results collected from athletes. Portions of blood and urine are then subject to ultrafiltration, undergo IEF, and the fluorescent band patterns are detected through a double-blotting technique. If the patterns match the isoelectric formation of an EPO, it is valid evidence for charging an individual with blood doping. Detection of both first and second generation recombinant EPO has been successful in multiple tests over the years [15]. Even the third generation of illegal EPO's, Continuous Erythropoietin Receptor Activator (CERA), is detectable through IEF screening. However, while both urine and blood are viable samples for this particular test, the drug is much more difficult to detect in urine, a fact that has made it a difficult test to administer on samples collected mid-race [14, 15].

5. Membrane Assisted Isoform Immunoassays: Another form of Detection for EPO's

Due to the fact that IEF testing is, while technically sound, becoming increasingly difficult and expensive to administer given new generations of EPO's, researchers are looking into different ways to distinguish exogenous and endogenous substances in the human body. Potentially more promising than IEF, Membrane Assisted Isoform Immunoassays (MAIIA) may be more sensitive than previous methods. This process is, ultimately, a biological test that calculates the concentrations of specific elements in a given sample (see Figures 4 and 5). Developed by Swedish researchers in early 2012, MAIIA relies on discrepancies in protein carbohydrate structures to distinguish illegal substances from the body's natural hormones. This test is ideal in that it is able to detect differences between illegal recombinant proteins and similar endogenous molecules through cost-effective and efficient mechanisms [13]. However, as it is still an extremely new procedure, the WADA has yet to adopt it.

MAIIA Diagnostics

Figure 4: (Left): A sample is run through a 2D MAIIA chip, which can separate different isoforms of EPO. (Right): MAIIA can be used to uncover recombinant EPO (rhEPO) since rhEPO and normal EPO will have differences in isoform charge.

6. The Athlete Biological Passport (ABP)

The notion of creating biological passports for competitive athletes was first suggested by Mario Cazzola, M.D. in 2000, and there has been extensive research conducted on this project in the 12 years since then [13]. This passport contains results from a comparative analysis between athletes' blood tests over a longitudinal period, allowing an athlete's blood test results to be compared with that individual's previous results rather than with the average values of his/her demographic group [12]. Containing information such as age, sex, genotype, exposure to altitude, as well as past blood testing results, the passport will contain biological markers that current samples can be measured against. When testing for blood doping, the blood collected from a given athlete is first analyzed by ABP software specifically designed to detect discrepancies between the content of the passport and the current sample. The results are then discussed by an expert panel who will ultimately decide if flagged blood profiles have been subject to illicit forms of manipulation. Even though this is one of the only methods capable of testing for autologous blood transfusions, based on the nature of the testing process, there is still room for errors since the first half of the test relies on the software being 100% accurate, and the second half of the test relies on human judgment [13]. Ultimately, while the idea of having a "bank" of stored athletic biological information is a promising step forward for the anti-doping effort, there is still much to be done before the ABP methodology is streamlined and perfected.

Notable Cases in the World of Athletics

As many of the detection tests are still being developed and perfected, there is a significant number of athletes in both the past and the present that have partaken in blood doping without being caught. However, that is not to say there have not been large doping scandals in the history of competitive sports. In May 2006, the Spanish Police launched Operación Puerto, an attack against the doping network orchestrated by Dr. Eufemiano Fuentes, a Spanish sports doctor. This case was thrown into the press when the Spanish Civil Guard found hundreds of packages of blood along with thousands of anabolic steroids at Fuentes' residence. The doping circle ultimately involved more than 200 athletes, one of the largest operations ever discovered [16]. Those involved in this case were tried by WADA; the Union Cycliste Internationale, or International Cycling Union (UCI); and the Italian Olympic Sports Committee [17].

Doping rings aside, individual athletes charged with using performance enhancing techniques are often sent to the Court of Arbitration for Sport (CAS). One particular instance, the case of Alberto Contador, has been in the press since 2010. An appeal court earlier this year finally declared Contador, a three-time Tour de France winner, guilty of doping. His blood tested positive for clenbuterol, a muscle-building and aerobic capacity-increasing drug, and also contained traces of plasticizers, remnants probably from IV usage. Both of these results pointed toward illegal acts of blood doping. However, Contador pleaded innocent by claiming that a cut of Spanish steak was the source of the drug. Seventeen months later, Contador's claims were proven false, and he was stripped of his 2010 Tour de France title in addition to 12 other first place wins. The ultimate verdict was a huge upset, and the cycling and international sports communities did not leave this battle unscathed [18].

Deleterious Side Effects

Regardless of those that have been persecuted in the past, there are still many young athletes willing to resort to drastic measures for a taste of the gold. Nonetheless, the potential for fame and success that blood doping advertises does not overshadow the fact that athletes who partake in this practice are endangering not only putting their reputations and careers but also their bodies. Autologous transfusions, for instance, are extremely double-edged; the boost in RBC count may enhance athletic performance, but there are a number of detrimental and damaging side effects. During heavy aerobic exercise, dehydration is not an uncommon occurrence. Lower fluid levels combined with an increased concentration of RBC's can lead to hyperviscosity syndrome, or an increase in the blood's viscosity; phlebitis, or inflammation of veins; and septicemia, a bacterial infection in the bloodstream [4]. Moreover, blood that is highly viscous or "thick" tends to be more prone to coagulation and forming the dangerous blood clots involved in strokes and heart attacks [19]. As for homologous transfusions, multiple blood-borne diseases can be contracted, such as HIV, hepatitis, or malaria [4]. In addition, there are added risks associated with administering someone else's blood; for example, if the donor's blood is not compatible with the recipient's blood, acute transfusion reactions may occur, such as severe hemolysis, high fevers, and urticaria (hives) in the recipient [20]. As a whole, transfusions for the purpose of increasing RBC levels in healthy individuals are highly discouraged, not only because of the practice's illicit nature but also because of the serious health risks posed by blood doping.

Athletes that opt for EPO injections in place of blood transfusions are subject to similarly adverse side effects. In 2002, a cyclist with high blood concentrations of EPO and Vitamin A was admitted to a hospital due to blood doping complications manifesting themselves in the form of cerebral blood clots [21]. An excess of EPO causes the body to produce large amounts of RBC's and hemoglobin, increasing the HCT to unsafe levels [14]. Like transfusions, EPO injections present the dangerous outcome of overly viscous blood leading to life threatening blood clots.

Controversy Regarding the Legitimacy of Testing Results

The dangers prevalent in blood doping practices are only exemplified by the fact that detection tests are still largely in development. As athletes become privy to new technologies, it has become steadily harder to create a direct and consistent method of testing for blood manipulation. Many of the processes listed above are considered to be "indirect" detection techniques and cannot, by themselves, conclusively charge an individual on counts of blood doping. Thresholds and upper limits for HCT, for instance, in many cases stray from the standardized numbers and are affected by environmental factors [12]. Due to the fact that many tests are unable to control for certain variables, the WADA and UCI are often plagued with inconclusive cases.

Even with the more technically sound tests, such as MAIIA or double-blotting for urinary DEHP, there is an ideal window of time during which urine and blood should be extracted; if samples are collected too late or too early, traces of EPO or plasticizers would be gone from the bloodstream, and an athlete who has partaken in blood doping would appear to be innocent [13]. This limitation suggests that even if more complex and accurate detection methodologies are created, if those tests are not conducted at optimal times, there will still be a large percentage of athletes whose blood doping practices will remain under the radar.

Researchers are currently investing time and money into forming techniques that will provide more conclusive evidence for blood doping cases. One possible test includes measuring hepcidin, a liver-produced hormone that appears to be suppressed by high levels of EPO [13]. However, currently, these breakthroughs have yet to adopted by the WADA due to a lack of results from long term analysis. Persecuting groups in blood doping cases are extremely particular about the evidence they will accept especially because of the heavy implications of a conviction: title stripping, suspensions, and potential fines.

Relevance for the 2012 Olympic Games

Acknowledging that transfusions and EPO injections are, in most cases, extremely difficult to detect, the 2012 Olympic organizers teamed with GlaxoSmithKline in London, a pharmaceutical company that provided researchers, equipment, and laboratories [22]. David Cowan, head of the anti-doping program for the 2012 Olympics, declared earlier in 2012 that these games in particular would be the most dangerous for those willing to resort to performance enhancing drugs [23]. The staff consisted of over 150 scientists whose job was to analyze over 6,250 blood samples. Cowan revealed that one strategy he would enforce is testing for a greater number of illegal substances per sample of urine or blood, thereby maximizing efficiency of detection [22].

New tests were implemented for the purpose of indicating biological traces linked to autologous blood transfusions. Endurance athletes, in order to hide elevated levels of RBC's, often dilute their blood with saline. While this masking method would be successful under normal circumstances, researchers have found that RNA present on the surfaces of stored blood differs from the blood in the body [23]. Cowan and his team aimed to utilize this newfound discovery with the aid of gene chips programmed to detect "old RNA" in blood samples. Moreover, records of blood and urine tests from biological passports were present to corroborate or disprove potential blood doping charges [24].

Ultimately, the 2012 anti-doping effort was aimed at preventing as many false victories derived from performance-enhancing techniques as possible. Cowan and his team strove to restore not only the integrity of the Olympic games, but competitive athletics as a whole.

Works Cited

1. E. K. L. Lerner, B. Wilmoth Lerner. (2007). "Blood Doping". World of Sports Science.

2. British Broadcasting Corporation. (2012). "Types of Drugs". [Online]. Available: http://www.bbc.co.uk/ethics/sport/debate/types_1.shtml.

3. N. Purchon. (1997). "Aerobic Respiration". Gondar Design Biology. [Online]. Available: http://purchon.com/wordpress/biology/?page_id=58.

4. D. Bekham. "Blood Doping: Is It Really Worth It?". Biology 1407 Concepts of Biology II. [Online]. Available: http://old.texarkanacollege.edu/~mstorey/beckham.html.

5. Encyclopedia Britannica. "Homologous Transfusions". [Online]. Available: http://www.britannica.com/EBchecked/topic/1795768/homologous-transfusion.

6. C. Huggins. (1985). "Preparation and Usefulness of Frozen Blood". Annual Reviews Medical. (36), pp 499-503.

7. "Erythroprotein (EPO) & The EPO Test". Medicine.Net. [Online]. Available: http://www.medicinenet.com/erythropoietin/article.htm.

8. M. Jenkins. (2005). "Erythroprotein". [Online]. Available: http://www.rice.edu/~jenky/sports/epo.html.

9. N. Monfort, R. Ventura, A. Lotorre, V. Belalcazar, M. Lopez, J. Segura. (2009, Aug 18). "Urinary di-(2-ethylhexyl)phthalate metabolites in athletes as screening measure for illicit blood doping: a comparison study with patients receiving blood transfusion". Transfusion. 50(1).

10. S. Ruibal. (2010, Dec. 5). "Blood Doping: How it Works and How it is Detected". USA Today. [Online]. Available: http://www.usatoday.com/sports/cycling/2010-10-05-blooddopingprimer_N.htm.

11. HealthOracle. "Blood Doping". HealthOracle.Org. [Online]. Available: http://healthoracle.org/downloads/B/Blood%20Doping.pdf.

12. G. D'Onofrio, G. Melioli. (2006, Nov.). "Blood Doping: Present Procedures and Detection Techniques". Expert Review of Endocrinology & Metabolism. 1(6) pp. 793.

13. C. Lundby, P. Robach, B. Saltin. (2012, Feb. 10). "The Evolving Science of Detection in Blood Doping". British Journal of Pharmacology. 165(5), pp 1306-1315.

14. E. R. Eichner. (2007, Apr.). "Blood Doping: Infusions, Erythropoitin, and Artificial Blood". Sports Medicine. 37(4).

15. F. Lasne, L. Martin, J. A. Martin, J. Ceaurriz. (2009, Jun. 1). "Detection of continuous erythropoietin receptor activator in blood and urine in anti-doping control". Haematologica. [Online]. 90(6), pp. 888-890.

16. H. Kroner. (2009, Dec. 11). "Operación Puerto: case closed". Cycling News. [Online]. Available: http://www.cyclingnews.com/news/operacion-puerto-case-closed.

17. S. Stokes. (2012, Jan. 12). "Contador, Basso and Scarponi summoned as witnesses in Puerto trial". Velonation. [Online]. Available: http://www.velonation.com/News/ID/10875/Contador-Basso-and-Scarponi-summoned-as-witnesses-in-Puerto-trial.aspx.

18. I. Austen. (2012, Feb. 6). "2010 Tour de France Winner Found Guilty of Doping". New York Times. [Online]. Available: http://www.nytimes.com/2012/02/07/sports/cycling/alberto-contador-found-guilty-of-doping.html?_r=1.

19. American Heart Association. (1999, Aug. 17). ""Thick" Blood May Increase Stroke Risk". ScienceDaily. [Online]. Available: http://www.sciencedaily.com/releases/1999/08/990817065603.htm.

20. S. G. Sandler. (2012, Jan. 10). "Transfusion Reactions". Medscape. [Online]. Available: http://emedicine.medscape.com/article/206885-overview.

21. American Academy of Neurology. (2002, Feb. 26). "Cyclists' "Blood Doping" Associated With Cerebral Clots". ScienceDaily. [Online]. Available: http://www.sciencedaily.com/releases/2002/02/020226075440.htm.

22. R. Blitz, V. Kortekaas. (2012, Jan. 19). "Science Not Enough to Catch Olympic Drug Cheats". [Online]. Available: http://proquest.umi.com.libproxy.usc.edu/pqdlink?vinst=PROD&fmt=3&startpage=-1&vname=PQD&did=2567486921&scaling=FULL&vtype=PQD&rqt=309&cfc=1&TS=1339500904&clientId=5239.

23. S. Reardon. (2011, Sep. 14). "Cheaters Beware: New Doping Tests Ready for London's 2012 Olympics". Science Insider. [Online]. Available: http://news.sciencemag.org/scienceinsider/2011/09/cheaters-beware-new-doping-tests.html.

24. Sports Network. (2012, July 25). "New tests, new methods make it harder to cheat." FoxNews.com. Available: http://www.foxnews.com/sports/2012/07/25/new-tests-new-methods-make-it-harder-to-cheat/.

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