1. Prevalence of Blood Doping in Samples Collected from Elite Track and Field Athletes P.-E. Sottas, N. Robinson, G. Fischetto, G. Dollé, J.M. Alonso, and M. Saugy www.clinchem.org/cgi/content/article/57/5/762 May 2011 © Copyright 2011 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Drug testing in elite sports >World Anti-Doping Agency laboratories statistics:  2008: 1.08% adverse analytical findings  2009: 1.11% adverse analytical findings  Is the prevalence of doping underestimated? > Because false positives must be avoided, anti-doping tests give priority to specificity at the expense of sensitivity > Increasingly sophisticated doping protocols being used to evade detection by drug tests > In addition to problem of undetectable substances, drugs designed specifically to foil drug tests are being produced

3. © Copyright 2009 by the American Association for Clinical Chemistry How to estimate the prevalence of doping?  Questionnaire-based surveys Individual bias in the assessment of a sensitive attribute such as doping > Methods of maintaining confidentiality Randomized response methods for decreasing evasive answer bias > However these methods have never been applied successfully in world elite athletes Athletes may still be reluctant to answer truthfully in an attempt to avoid suspicions directed not only towards themselves but also towards their sport > Any more objective alternative?

4. © Copyright 2009 by the American Association for Clinical Chemistry Method: Use of biomarkers of doping  In the clinics Epidemiological method to characterize a disease has been a cornerstone method of public health research > In epidemiology, biomarkers of disease or biomarkers of exposure are used to provide prevalence measures  Translated to anti-doping Epidemiological method to characterize the abuse of doping substances > Prevalence measures based on biomarkers of doping > Reference cumulative distributions built thanks to data collected in clinical trials

5. © Copyright 2009 by the American Association for Clinical Chemistry Paradigm shift in anti-doping  From drug tests to biomarkers of doping The use of biomarkers of doping recently has been formalized in the so-called Athlete Biological Passport  Biomarkers of blood doping Blood doping refers to any method that aims to increase red cell mass, such as blood transfusion and recombinant EPO (rEPO) > Blood sampling by sport governing bodies Some international sport federations introduced the collection of blood samples and the measurement of red blood cell indices in the 1990's with the aim to limit the abuse of rEPO

6. © Copyright 2009 by the American Association for Clinical Chemistry Biomarkers of blood doping  From a full blood count Seven red blood cell indices are used to form the multiparametric marker of blood doping called the Abnormal Blood Profile Score (ABPS) > ABPS is a universal marker Sensitive to rEPO independently of the administration period > ABPS has proven sensitivity The higher the sensitivity, the lower the number of tests required to provide a precise estimate (low sampling error) > ABPS has good generalization properties Good internal and external validity (low systematic error) > ABPS accounts for known effects of heterogenous factors The marker can be applied to populations stratified according to sex, age and other heterogenous factors (low bias)

7. © Copyright 2009 by the American Association for Clinical Chemistry Blood testing in elite track & field athletes  Full blood counts since 2001  From 2001 to 2009, the International Association of Athletics Federations (IAAF) collected 7289 blood samples from 2737 track and field athletes  Data Collection  Date of test, venue, sport, type of competition (in-, pre-, out-of-competition), instrument technology, date of analysis, sex, birth date and nationality were collected  Exposure to altitude  Altitude of the testing location was identified in 3658 tests; altitude also identified from the athletes’ whereabouts in the three-week pre-competition profile for the 2005 and 2007 World Championships in Athletics (3444 entries)

8. © Copyright 2009 by the American Association for Clinical Chemistry Results Table 1. Descriptive statistics on the blood samples collected from elite athletes between 2001 and 2009. The tested population was highly heterogeneous for many factors (eg 147 nationalities). 79% of samples collected were from endurance athletes running distances equal to or longer than 800 m. These were athletes who could benefit from blood doping to enhance their aerobic metabolism. Out-of-competition tests accounted for approximately a quarter of all the tests (23%).

9. © Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Cumulative distribution functions (CDF) of the biomarker ABPS. Black lines: reference CDFs obtained for a modal population of female athletes; left: assuming no doping; right: assuming doping with microdoses of rEPO. The difference between the left and right reference CDFs represents the discriminative power of the marker ABPS. Other lines: empirical CDF obtained from all tests performed on all female athletes of the modal group (green, 1056 samples), on athletes of country A (red, 67 samples) and on a subgroup that includes athletes from country D (blue,84 samples).

10. © Copyright 2009 by the American Association for Clinical Chemistry Prevalence estimates Table 2. Period prevalence estimates of abnormal blood profiles in elite track and field athletes. n: number of samples from which the estimates were derived. Prevalence M1: minimal estimates without any assumptions on the doping method. Prevalence M2: estimates obtained assuming doping with rEPO microdoses. (): 95% CI estimated by bootstrapping methods, with any negative estimates rounded toward 0%.

11. © Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Graphical representation of the blood module of the Athlete Biological Passport for a female athlete of the modal group. Upper left, hemoglobin (HGB); upper right: stimulation index OFF-score (OFFS); lower left: ABPS; lower right: reticulocyte percentage (RET%). Blue lines: actual test results (8 tests). Red lines: individual limits. Colored bars: percentile in the distribution of expected sequences at which falls the observed sequence. This passport shows variations as expected for a normal physiological condition.

12. © Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Graphical representation of the Athlete Hematological Passport for another female athlete of the modal group (see Figure 2 for details). This passport shows variations and absolute values that are not in accordance with a normal physiological condition. A closer examination was required to determine whether the polycythemia presented on several occasions was due to a medical condition or doping. The increased values were measured before important competitions and most probably implicated a doping behavior.

13. © Copyright 2009 by the American Association for Clinical Chemistry Conclusion  Epidemiological method for doping prevalence When applied at the population level, biomarkers of blood doping can be used to derive prevalence estimates of doping  Prevalence estimates depend on nationality World’s elite athletes are not only heterogenous in physiological and anthropometric factors but also in their doping behavior  The Athlete Biological Passport When applied at the individual level, following the concept of personalized biology, the same biomarkers provide a biological signature that can be used to detect doping

14. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Follow us