A third generation approach to detect erythropoietin abuse in athletes.

BACKGROUND AND OBJECTIVES: Information derived from blood analyses can assist in the detection and/or deterrence of blood doping in sport. We investigated whether comparing an athlete's hematologic values against his or her own historical baseline rather than population-derived thresholds enhanced the ability to detect blood doping. DESIGN AND METHODS: We developed an approach whereby an athlete's true baseline value could be estimated with just one prior blood test. We also estimated a universal value for within-subject variability for key hematologic parameters using the highest value obtained among four separate cohorts of male athletes including 80 elite rowers, 124 endurance-trained or team-sport subjects, 288 professional football players and 630 athletes competing at national or international level. The (individual) baseline and (universal) variability were then incorporated so as to define expected thresholds for subsequent blood tests. The sensitivity of our approach was obtained by analyzing data from 49 recreational athletes administered either recombinant human erythropoietin (n=37) or placebo (n=12). RESULTS: We found that removing within-subject variability by comparing new results against an historical baseline heightened the capacity to detect blood doping. It was possible to delineate the longitudinal changes in either hemoglobin (Hb) or the OFF-hr model score (an algorithm using both Hb and percent reticulocytes) caused by recombinant human erythropoietin treatment from the natural biological fluctuations found in subjects treated with placebo. INTERPRETATION AND CONCLUSIONS: Our objective data supported the intuitive belief that longitudinal monitoring of athletes' blood profiles will help detect blood doping. This information could be used to instigate target-testing of suspicious athletes, or even warrant the exclusion from competition of athletes with aberrant variations in key hematologic values.

Effect of pre-competition and altitude training on blood models used to detect erythropoietin abuse by athletes.

This study reports blood model scores used for detection of recombinant human erythropoietin (rHuEPO) abuse by athletes. Elite female rowers were monitored prior to their World Championships, including a period spent training at moderate altitude. In contrast to previous results, no substantial increase in model scores was found following altitude exposure.

Longitudinal variation of hemoglobin and reticulocytes in elite rowers.

Longitudinal monitoring of athlete's hematologic parameters holds considerable promise as a strategy to detect and thereby deter illicit blood doping. This study documents temporal changes of hemoglobin concentration (Hb) and reticulocyte counts in elite rowers. The 'within subject' variation in rowers was comparable to that of athletes from other sports. Reticulocyte results were dependent on the type of instrument used.

The effect of common hematologic abnormalities on the ability of blood models to detect erythropoietin abuse by athletes.

BACKGROUND AND OBJECTIVES: Algorithms that combine scores from multiple blood parameters are demonstrably effective in highlighting recombinant human erythropoietin (rHuEPO) administration, and have been used to deter rHuEPO use by athletes. These models are sensitive to atypical levels of blood parameters encountered during altered states of red cell production. Because hematologic abnormalities can also result in unusual blood profiles, the aim of this study was to document the incidence and magnitude of such abnormalities in an elite athlete population. DESIGN AND METHODS: We screened blood samples obtained from 413 female and 739 male elite athletes from 12 countries for known hematologic abnormalities, and compared the algorithm scores for these athletes with those of their healthy counterparts. We also established the magnitude of blood parameters required for model scores to exceed cut-offs associated with rHuEPO use. RESULTS: We found that 0.7% of male and 2.4% of female athletes were iron deficient either with our without anemia. An additional 1.4% of males and 1.0% of females had hemoglobinopathies. On average these athletes' model scores were at or below the score of their healthy counterparts. The greatest influence on our models was hemoglobin concentration. Values of other parameters must exceed normal ranges by a substantial margin in order for model scores to approach levels associated with rHuEPO use. INTERPRETATION AND CONCLUSIONS: The hematologic disorders we encountered in elite athletes were not associated with model scores that exceeded the nominal cut-offs that we have previously recommended to delineate rHuEPO use. We did not find any abnormalities among elite endurance athletes that were associated with high model scores.

Effect of altitude on second-generation blood tests to detect erythropoietin abuse by athletes.

BACKGROUND AND OBJECTIVES: ON- and OFF-model scores derived from blood parameters sensitive to erythropoiesis have been shown to be a useful tool to identify athletes who are currently injecting erythropoietin to enhance performance or those who have recently stopped doing so. We investigated changes in blood parameters and model scores during and after exposure to terrestrial and simulated altitudes. DESIGN AND METHODS: We retrospectively evaluated changes in hematologic data collected from 19 elite cyclists who lived and trained 2690 m above sea level for 26-31 days, from six elite Kenyan runners who lived 2100 m above sea level but descended to compete at sea level competitions, and from 39 well-trained subjects who resided at sea level but slept at a simulated altitude of 2650-3000 m for 20-23 days of either consecutive or intermittent nightly exposure. RESULTS: Upon ascent to a terrestrial altitude, ON- and OFF-model scores increased immediately, mainly because of an increase in hemoglobin concentration. Scores had not returned fully to baseline three weeks after return to sea level, because of the persistence of the raised hemoglobin concentration for the ON and OFF scores and a fall in reticulocyte percentage for OFF scores. Effects were smaller or negligible for simulated altitude. For Kenyan runners, ON- and OFF-model scores decreased within seven days of descent to sea level. INTERPRETATION AND CONCLUSIONS: Our results reinforce the notion that caution should be exercised when interpreting blood results from athletes who have recently been exposed to either terrestrial or simulated altitude, and appropriate allowance should be made for the effect of altitude on blood model scores.

Standardization of reticulocyte values in an antidoping context.

The lack of standardization of reticulocyte results hinders the ability of sports authorities to recognize the telltale fluctuations over time that are typical for athletes using illegal blood doping to improve their performance. Therefore, the aim of the present study was to devise a tenable approach for antidoping authorities to quantify, instrument bias. We evaluated reticulocyte data derived during a 42-week period from 210 hospital patient blood samples measured in duplicate simultaneously on up to 11 hematology analyzers located in a single laboratory. We found that square root transformation of reticulocyte values enabled quantification of interinstrument bias by using the mean reticulocyte value of a cohort of approximately 54 subjects as a de facto calibration agent. We also demonstrated that measurement precision associated with low reticulocyte values was not inferior to that associated with higher values.

Effects of prolonged low doses of recombinant human erythropoietin during submaximal and maximal exercise.

The aim of this study was to characterise the effect of prolonged low doses of recombinant erythropoietin (r-HuEPO) on the responses to submaximal and maximal exercise. Volunteer recreational athletes ( n=21) were divided into three groups: r-HuEPO+intravenous iron (EPO+IV, n=7), r-HuEPO+oral iron (EPO+OR, n=9) and placebo ( n=5). During the 12 week study, r-HuEPO or saline injections were given three times a week for the first 8 weeks and for the final 4 weeks the subjects were monitored but no injections were administered. The r-HuEPO doses were 50 IU x kg(-1) body mass for 3 weeks and 20 IU x kg(-1) body mass for the next 5 weeks. An exercise test comprising three submaximal intensities and then increments to elicit maximal aerobic power (VO2max ) was conducted during weeks 0, 4, 8 and 12. During week 0, the mean intensity of the submaximal stages was 60%, 72% and 81%. Blood taken at rest was analysed twice a week for haematocrit (Hct). The relative increases in at weeks 4, 8 and 12 were 7.7%, 9.7% and 4.5%, respectively, for the EPO+IV group; 6.0%, 4.7% and 3.1% for the EPO+OR group; and -0.5%, -0.1% and -1.0% for the placebo group, where the improvements at week 12 for the EPO+IV and EPO+OR groups remained significantly above week 0 values. The Hct was significantly elevated by 0.06 and 0.07 units at week 3 in the EPO+IV and EPO+OR groups, respectively, and was stable during the 5 weeks of low-dose r-HuEPO. After 8 weeks of r-HuEPO use, plasma lactate concentration tended to be lower at exercise intensities ranging from 60% to 100%. This study confirmed the ability of low doses of r-HuEPO to maintain Hct and at elevated levels.

Development of reference ranges in elite athletes for markers of altered erythropoiesis.

BACKGROUND AND OBJECTIVES: Our previous research developed two statistical models that are useful indicators of current (ON-model) or recently discontinued (OFF-model) recombinant human erythropoietin (rHuEPO) use by athletes. The component variables of the ON-model are hematocrit (Hct), reticulocyte hematocrit (RetHct), serum erythropoietin (EPO), percent macrocytes (%Macro), and soluble transferrin receptor (sTfr), whilst the OFF-model uses only the first three variables. Genetics and training modalities of elite athletes may conceivably produce unusual values for blood parameters related to erythropoiesis. The aims of this study were to develop reference ranges in elite athletes for key hematologic parameters as well as ON- and OFF-models scores, and to evaluate the effect of ethnicity, gender, residence at moderate altitude (approximately 2000 m) and within-individual variation on the variables and model scores. DESIGN AND METHODS: Over a period of three weeks, 413 female and 739 male elite athletes from 12 countries visited laboratories to provide three blood samples for analysis of blood parameters sensitive to erythropoiesis. For each parameter and for the ON- and OFF-model scores, we used mixed modeling to establish the range within which we could be 95% certain that the value for a randomly chosen athlete would fall, taking into account various random effects (variation within and between subjects and laboratories) and fixed effects (means for different levels of ethnicity, age, sport, altitude of residency). We performed similar analyses for changes in the ON- and OFF-model scores between the three visits. RESULTS: Most fixed effects were accompanied by clear-cut, small to moderate differences in several parameters. However, residency at moderate altitude was accompanied by a much higher hematocrit than residency nearer sea level, with the mean (and 95% confidence limits) for the difference being 2.3 (0.9 to 3.7) and 1.8 (0.1 to 3.5) units for males and females, respectively. Males at altitude also demonstrated a moderately higher ON-model score. Otherwise the influence of these effects was small for ON-, OFF- and changes in model scores. INTERPRETATION AND CONCLUSIONS: Assessment of an athlete's blood parameters and ON- and OFF-model scores may need adjustment for training modalities and other characteristics of the subject. Changes in model scores (together with monitoring of urine samples for the presence of rHuEPO) provide a promising approach to detection of rHuEPO abuse, because they are less sensitive to subject characteristics and less variable than raw model scores.

Second-generation blood tests to detect erythropoietin abuse by athletes.

BACKGROUND AND OBJECTIVES: We previously developed blood tests that were introduced at the Sydney 2000 Olympic Games to identify athletes injecting recombinant human erythropoietin (rHuEPO). The aim of this study was to re-analyse our existing database to develop models with heightened sensitivity, using wherever possible blood parameters measurable with appropriate standards of analytical performance. DESIGN AND METHODS: The principal database for this study was derived from a double-blind trial in which 57 recreational athletes were administered either rHuEPO or placebo. Standard discriminant analysis was used to derive two ON models (ON-hes and ON-he) and two OFF models (OFF-hr and OFF-hre) sensitive to accelerated and decelerated erythropoiesis respectively, utilising concentrations of hemoglobin (h), erythropoietin (e) and serum transferrin receptor (s), as well as percent reticulocytes (r). The ability of our models to detect rHuEPO administration was assessed by comparing model scores of subjects in the administration trial with the model scores of 1152 elite athletes from 12 countries. RESULTS: The ability of the new models to detect rHuEPO administration was generally higher than that of our previous models, particularly during phases when low doses of rHuEPO were used, and after injections had ceased. INTERPRETATION AND CONCLUSIONS. The increased stability of the new blood parameters facilitates transport of samples to central laboratories, and the heightened sensitivity of the new models makes them better than existing models for federations wishing to screen samples for urine testing and to identify and target suspect athletes for out-of-competition testing. However procedures should be incorporated that respect an elevated model score caused by genetic, health or environmental circumstances.