A new multimodal paradigm for biomarkers longitudinal monitoring: a clinical application to women steroid profiles in urine and blood.

Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application. Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring. The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping

Bayesian detection of abnormal hematological values to introduce a no-start rule for heterogeneous populations of athletes.

Sports authorities exclude athletes with abnormal levels of blood parameters. Here, the consideration of longitudinal blood profiles together with heterogeneous factors such as ethnicity and age produces a model with enhanced sensitivity to detect blood doping. Sports disciplines with heterogeneous populations now have a general method to introduce the no-start rule

Retesting the anti-doping samples: Best tool for deterrence?

Long term storage of the anti-doping samples and their reanalyses becomes today more and more a trend in the anti-doping community. The procedure has been implemented by the anti-doping authorities for the samples of the Tour de France and for the Olympic Games since Athens 2004 and has been always presented as a good tool to deter doping habits in top level sport. Recently, the World Anti-Doping Code introduced the possibility for anti-doping organizations to store the athletes’ samples up to ten years. The anti-doping authorities may ask to reanalyze the samples at any time during that period of time as a function of the implementation of new methods or instruments in the accredited laboratories allowing the detection of prohibited substances or their metabolites at a much lower concentration or for a larger detection window. The most significant technological advances for the detection of doping substances have been done in the characterization of various long-term metabolites of anabolic androgenic steroids. This allowed for increasing the time of detection by even a factor of four

Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids: international reference populations of professional soccer players

BACKGROUND AND OBJECTIVES: The determination of the carbon isotope ratio in androgen metabolites has been previously shown to be a reliable, direct method to detect testosterone misuse in the context of antidoping testing. Here, the variability in the 13C/12C ratios in urinary steroids in a widely heterogeneous cohort of professional soccer players residing in different countries (Argentina, Italy, Japan, South Africa, Switzerland and Uganda) is examined. METHODS: Carbon isotope ratios of selected androgens in urine specimens were determined using gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). RESULTS: Urinary steroids in Italian and Swiss populations were found to be enriched in 13C relative to other groups, reflecting higher consumption of C3 plants in these two countries. Importantly, detection criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol for each population were found to be well below the established threshold value for positive cases. CONCLUSIONS: The results obtained with the tested diet groups highlight the importance of adapting the criteria if one wishes to increase the sensitivity of exogenous testosterone detection. In addition, confirmatory tests might be rendered more efficient by combining isotope ratio mass spectrometry with refined interpretation criteria for positivity and subject-based profiling of steroids

The Influence of Training Load on Hematological Athlete Biological Passport Variables in Elite Cyclists.

The hematological module of the Athlete Biological Passport (ABP) is used in elite sport for antidoping purposes. Its aim is to better target athletes for testing and to indirectly detect blood doping. The ABP allows to monitor hematological variations in athletes using selected primary blood biomarkers [hemoglobin concentration (Hb) and reticulocyte percentage (Ret%)] with an adaptive Bayesian model to set individual upper and lower limits. If values fall outside the individual limits, an athlete may be further targeted and ultimately sanctioned. Since (Hb) varies with plasma volume (PV) fluctuations, possibly caused by training load changes, we investigated the putative influence of acute and chronic training load changes on the ABP variables. Monthly blood samples were collected over one year in 10 male elite cyclists (25.6 ± 3.4 years, 181 ± 4 cm, 71.3 ± 4.9 kg, 6.7 ± 0.8 W <sup>.</sup> kg <sup>-1</sup> 5-min maximal power output) to calculate individual ABP profiles and monitor hematological variables. Total hemoglobin mass (Hbmass) and PV were additionally measured by carbon monoxide rebreathing. Acute and chronic training loads-respectively 5 and 42 days before sampling-were calculated considering duration and intensity (training stress score, TSS <sup>TM</sup> ). (Hb) averaged 14.2 ± 0.0 (mean ± SD) g <sup>.</sup> dL <sup>-1</sup> (range: 13.3-15.5 g·dl <sup>-1</sup> ) over the study with significant changes over time (P = 0.004). Hbmass was 1030 ± 87 g (range: 842-1116 g) with no significant variations over time (P = 0.118), whereas PV was 4309 ± 350 mL (range: 3,688-4,751 mL) with a time-effect observed over the study time (P = 0.014). Higher acute-but not chronic-training loads were associated with significantly decreased (Hb) (P <0.001). Although individual hematological variations were observed, all ABP variables remained within the individually calculated limits. Our results support that acute training load variations significantly affect (Hb), likely due to short-term PV fluctuations, underlining the importance of considering training load when interpreting individual ABP variations for anti-doping purposes

Worldwide distribution of blood values in elite track and field athletes: Biomarkers of altered erythropoiesis.

For the first time, blood samples were collected in all athletes participating in a major sporting event of the International Association of Athletics Federations (IAAF) (Athletics World Championships 2011, Daegu, Korea). All variables obtained from blood analyses were incorporated into the individual blood profiles of each athlete for the so-called athlete biological passport (ABP). This unprecedented data collection highlighted differences for a few blood biomarkers commonly measured and reported for the ABP on some group of athletes. Subsequently, blood tests analyses for all athletes were repeated during the following World Championships (2013, Moscow, Russia). Both sets of blood tests were then used to set up the distribution of blood values for track and field athletes considering potential confounding factors such as gender, age, discipline, origin of the athlete (continental classification), and time of blood collection. Implementation of well-defined distribution of blood values will allow to improve the estimation of blood doping prevalence among a specific population of athletes in track and field

The UEFA EURO 2012 anti-doping programme - scientific review

The final tournament of the UEFA European Football Championship is one of the top sporting events in the world, and a high-profile event of this kind requires a well-planned and well-executed anti-doping programme to ensure the integrity of results in the competition. UEFA EURO 2012 presented a unique logistical challenge, with the tournament spread across two countries, both covering a large geographical area. This paper discusses the planning and delivery of both the pre tournament out-of-competition (OOC) testing programme and the in-competition (IC) programme, as well as reviewing the activities of doping control officers (DCOs), the whereabouts programme and assessing the sample collection and transport process. The analytical approach applied is also discussed, along with an overview of the distribution of T/E ratios and blood parameters

Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4°C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month perio

Le dialogue nécessaire entre médecine et antidopage pour l’intégrité du sport et de l’athlète = The necessary dialogue between medicine and anti-doping for the integrity of sport and the athlete

En réponse aux actualités antidopage, cet article traite quelques cas particuliers et s’étend aux perspectives futures de développement de stratégies antidopage efficaces. Après avoir rappelé les principes actuels de la lutte antidopage, il aborde l’utilisation de substances tolérées à un certain seuil et évoque les autorisations à usage thérapeutique (AUT). Les substances autorisées mais pouvant présenter un risque sanitaire pour les athlètes sont discutées avant de conclure sur le développement du passeport biologique de l’athlète comme futur terrain commun pour la lutte antidopage et le suivi médical des sportifs. En conclusion, cette approche souligne le dialogue impératif entre organisations antidopage et médecine du sport afin de défendre de bonnes pratiques à même de préserver la valeur intrinsèque du sport. In the light of recurring anti-doping news, this article discusses some special cases and extends to the future prospects of developing effective anti-doping strategies. After recalling the current principles of the fight against doping, the use of substances tole-rated at a certain threshold, and the therapeutic use exemptions (TUE) are discussed. Authorized substances with a health risk for athletes are discussed before concluding on the development of the athlete's biological passport as a future common ground for anti-doping and medical follow-up of athletes. In conclusion, this approach emphasizes the imperative dialogue between anti-doping organizations and sports medicine in order to defend good practices preserving the intrinsic value of sport

Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration.

The quantification of a large panel of endogenous steroids in serum by LC-MS/MS represents a powerful clinical tool for the screening or diagnosis of diverse endocrine disorders. This approach has also demonstrated excellent sensitivity for the detection of testosterone misuse in the anti-doping field, especially in female athlete population. In both situations, the use of dried blood spots (DBS) could provide a viable alternative to invasive venous blood collection. Here, the evaluation of DBS sampling for the quantification of a panel of endogenous steroids using UHPLC-MS/MS is described. The UHPLC-MS/MS method was validated for quantitative analysis of eleven free and eight conjugated steroids and was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28-days testosterone gel treatment (treatment phase). Results were compared with those obtained from serum matrix. Satisfactory performance was obtained for all compounds in terms of selectivity, linearity, accuracy, precision, combined uncertainty, stability as well as extraction recovery and matrix effects. In control phase, high correlation was observed between DBS and serum concentrations for most compounds. In treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on finger(s) used for gel application. Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping