Pure red cell aplasia

In keeping with standards set forth by many international sports associations, the International Cycling Union (UCI) recently tested participants in its Elite Track World Championships--held at the ADT Event Center's velodrome in Carson, CA--for blood doping and erythropoietin (EPO) abuse--methods for artificially increasing red blood cell mass. To ensure accurate results, the UCI engaged the Laboratoire Suisse d' Analyse du Dopage on the campus of Advanced Medical Analysis Laboratory.

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The lab used automated hematology analyzers to screen specimens. In looking at the results of commonly used blood-testing parameters (e.g., hematocrit, hemoglobin, and reticulocyte percentages and numbers), the lab searched for elevated levels that would indicate current or recent EPO doping. With analyzers that use flourescent flow cytometry, medical technologists were assured that there would be a higher sensitivity to the reticulated red blood cell counts.

A short history of blood doping

The term "blood doping" was introduced by the media in the 1970s to describe the use of blood transfusion to artificially increase red cell mass. Many athletes turn to blood doping based on the assumption that an increased oxygen-carrying capacity of the red blood cells leads to enhanced athletic performance, despite physiological evidence that oxygen is abundantly available at a mitochondrial level in skeletal muscle during endurance exercise.

Autologous transfusions have been used extensively for this purpose. Living and training at higher altitudes also stimulates the body to produce more circulating red blood cells in response to the lower oxygen concentration found at higher altitudes. Simulated altitude environments can be created at low altitudes in order to have the same effect on the red cell population. The administration of recombinant human erythropoietin (rHuEPO) is another approach.

Since the late 1980s, blood doping has been achieved through the administration of rHuEPO. rHuEPO is a genetically engineered protein, structurally almost identical to the naturally occurring erythropoietin, which is produced by the kidneys to stimulate red cell production. More than 500,000 patients throughout the world are currently receiving rHuEPO for legitimate medical treatment of renal failure and for testing anemia, which is secondary to destruction of renal tissue.

rHuEPO use is officially prohibited, however, by the International Olympic Committee (IOC) and other major sporting organizations. In 1989, the IOC Medical Commission introduced restrictions on the new doping class of peptide hormone analogues and the releasing factors for these hormones. These efforts parallel the ability to manufacture biological peptides and proteins.

Detecting blood doping

The general approach to detecting blood doping is to use indirect methods for screening and more specific methods for firm evidence of exogenous rHuEPO. The indirect methods vary by institution, but they involve using multiple hematological parameters to identify athletes who are actively practicing blood doping at the time of analysis, as well as those who have recently undergone blood doping of one form or another.

Commonly used blood-testing parameters to identify athletes who are blood doping include hemoglobin, hematocrit, absolute and percentage reticulocyte (immature red blood cells) counts, and ratios that can be used to subclassify the results into ON (currently blood doping) and OFF (recently stopped blood doping). Positive results are graded, and appropriate further action is taken; this usually involves more specific testing for detection of exogenous rHuEPO.

Isoelectric focusing techniques allow reliable detection of exogenous EPO in urine. The purified natural EPO in urine has a pattern of about 10 bands of pl 3.77 to 4.70, while patterns in urine from cyclists "treated with recombinant EPO contained more basic bands, reflecting the presence of recombinant isoforms, and sometimes acidic bands as well, depending on the presence of endogenous isoforms. The presence of exogenous hormone was always evident; any individual injected with recombinant EPO showed a striking transformation of their initial EPO urine pattern." (1)

Specific testing methods, although confirmatory, need to evolve constantly. Currently, rHuEPO can be detected in urine, but this might become more challenging as cell-culture-derived products become available. The expense of conducting specific tests is not justifiable as firstline investigation.

"A primary focus of our WADA (World AntiDoping Agency) ISO 17025-accredited antidoping laboratory," says Neil Robinson, PhD, laboratory supervisor based at the Laboratoire Suisse d'Analyse du Dopage in Lausanne, Switzerland, "is to reduce the risk of serious complications associated with blood doping among athletes. With the evidence of illegal doping leading to an athlete's ban from competitive sports, it is absolutely critical that the diagnostic tools meet a scientific standard of technical superiority and accuracy." The laboratory is part of the Legal Medicine department of the University of Lausanne and routinely performs urine and blood antidoping tests. As the laboratory is attached to the main University Hospital of the Canton de Vaud (CHUV, Lausanne), all other testing areas, such as endocrinology, microbiology, parasitology, and other clinical laboratory disciplines, are covered.

Dangers of blood doping

EPO abuse is not without risk to athletes. Some of the potential health risks include systemic hypertension, thromboembolic events (blood clot formation), PRCA (pure red cell aplasia) due to the development of antierythropoietin antibodies, and the effects of iron overload. Paradoxically, publication of the risks posed by abused substances has done little to discourage the practice.

Reference

1. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature. 2000;405:635.

By Ian Giles, MD

Ian Giles, MD, is director of scientific affairs for Sysmex America Inc. in Mundelein, IL. The diagnostic instrument for the UCI testing was a Sysmex Model XE-2100 automated hematology analyzer. The Model XT-2000i, also used for this testing, is smaller and more transportable. Both use fluorescent flow cytometry and advanced cell-counting methods, coupled with polymethine dyes, to offer a complete blood count and fully automated reticulocyte results.

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