Yearly intrasubject variability of hematological biomarkers in elite athletes for the Athlete Biological Passport.

Confounding factors including exercise and environments challenge the interpretation of individual Athlete Biological Passports (ABPs). This study aimed to investigate the natural variability of hematological ABP parameters over 1 year in elite athletes compared with healthy control subjects and the validity of a multiparametric model estimating plasma volume (PV) shifts to correct individual ABP thresholds. Blood samples were collected monthly with full blood counts performed by flow cytometry (Sysmex XN analyzers) in 20 elite xc-skiers (ELITE) and 20 moderately trained controls. Individual ABP profiles were generated through Anti-Doping Administration & Management System Training, a standalone version of the ABP's adaptive model developed by the World Anti-Doping Agency. Additionally, eight serum parameters were computed as volume-sensitive biomarkers to run a multiparametric model to estimate PV. Variability in ELITE compared with controls was significantly higher for the Abnormal Blood Profile Scores (P = 0.003). Among 12 Atypical Passport Findings (ATPF) initially reported, six could be removed after correction of PV shifts with the multiparametric modeling. However, several ATPF were additionally generated (n = 19). Our study outlines a larger intraindividual variability in elite athletes, likely explained by more frequent exposure to extrinsic factors altering hematological biomarkers. PV correction for individual ABP thresholds allowed to explain most of the atypical findings while generating multiple new ATPF occurrences in the elite population. Overall, accounting for PV shifts in elite athletes was shown to be paramount in this study outlining the opportunity to consider PV variations with novel approaches when interpreting individual ABP profiles.

Indirect biomarkers of blood doping: A systematic review.

The detection of blood doping represents a current major issue in sports and an ongoing challenge for antidoping research. Initially focusing on direct detection methods to identify a banned substance or its metabolites, the antidoping effort has been progressively complemented by indirect approaches. The longitudinal and individual monitoring of specific biomarkers aims to identify nonphysiological variations that may be related to doping practices. From this perspective, the identification of markers sensitive to erythropoiesis alteration is key in the screening of blood doping. The current Athlete Biological Passport implemented since 2009 is composed of 14 variables (including two primary markers, i.e., hemoglobin concentration and OFF score) for the hematological module to be used for indirect detection of blood doping. Nevertheless, research has continually proposed and investigated new markers sensitive to an alteration of the erythropoietic cascade and specific to blood doping. If multiple early markers have been identified (at the transcriptomic level) or developed directly in a diagnostics' kit (at a proteomic level), other target variables at the end of the erythropoietic process (linked with the red blood cell functions) may strengthen the hematological module in the future. Therefore, this review aims to provide a global systematic overview of the biomarkers considered to date in the indirect investigation of blood doping.

Competitive performance as a discriminator of doping status in elite athletes.

As the aim of any doping regime is to improve sporting performance, it has been suggested that analysis of athlete competitive results might be informative in identifying those at greater risk of doping. This research study aimed to investigate the utility of a statistical performance model to discriminate between athletes who have a previous anti-doping rule violation (ADRV) and those who do not. We analysed performances of male and female 100 and 800 m runners obtained from the World Athletics database using a Bayesian spline model. Measures of unusual improvement in performance were quantified by comparing the yearly change in athlete's performance (delta excess performance) to quantiles of performance in their age-matched peers from the database population. The discriminative ability of these measures was investigated using the area under the ROC curve (AUC) with the 55%, 75% and 90% quantiles of the population performance. The highest AUC values across age were identified for the model with a 75% quantile (AUC = 0.78-0.80). The results of this study demonstrate that delta excess performance was able to discriminate between athletes with and without ADRVs and therefore could be used to assist in the risk stratification of athletes for anti-doping purposes.

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

BACKGROUND: 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. RESULTS: 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. SIGNIFICANCE AND NOVELTY: 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.

Is Recovery Optimized by Using a Cycle Ergometer Between Ski-Mountaineering Sprints?

PURPOSE: To optimize the recovery phase between heats in ski-mountaineering sprint competitions, this study investigated whether an active recovery protocol on an ergocycle could improve subsequent performance compared with a self-selected recovery strategy. METHODS: Thirteen elite ski mountaineers (9 men and 4 women) performed 3 sprints with 2 different recovery conditions (Ergo vs Free) in a randomized order. The Ergo condition involved a 10-minute constant-intensity exercise on an ergocycle performed at 70% of maximum heart rate. For the Free condition, the athlete was asked to self-select modality. At the end of the third sprint, a passive recovery (seated) was prescribed for both protocols. Sprint performance (time) and physiological parameters (lactate concentration [La], heart rate [HR], and rating of perceived exertion [RPE]) were recorded from each sprint and recovery phase. RESULTS: In the Ergo vs Free protocols, sprint times (177 [24] s vs 176 [23] s; P = .63), recovery average HR (70% [2.9%] vs 71% [5.2%] of maximal HR), and RPE (16.7 [1.5] vs 16.8 [1.5]; P = .81) were not significantly different. However, [La] decreased more after Ergo (-2.9 [1.8] mmol·L-1) and Free (-2.8 [1.8] mmol·L-1) conditions compared with passive recovery (-1.1 [1.6] mmol·L-1; P < .05). CONCLUSIONS: The use of an ergocycle between heat sprints in ski mountaineering does not provide additional benefits compared with a recovery strategy freely chosen by the athletes. However, active conditions provide a faster [La] reduction compared with passive recovery and seem to be a more suitable strategy between multiple-heat sprints.

Variability of the urinary and blood steroid profiles in healthy and physically active women with and without oral contraception.

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17ß-diol/5ß-androstane-3α,17ß-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.

Future opportunities for the Athlete Biological Passport.

The Athlete Biological Passport (ABP) was introduced to complement the direct anti-doping approach by indirectly outlining the possible use of prohibited substances or methods in sports. The ABP proved its effectiveness, at least through a deterrent effect, even though the matrices used for longitudinal monitoring (urine and blood) are subject to many intrinsic (e.g., genetic) and extrinsic (e.g., environmental conditions) confounding factors. In that context, new and more specific biomarkers are currently under development to enhance both the sensitivity and the specificity of the ABP. Multiple strategies are presently being explored to improve this longitudinal monitoring, with the development of the current modules, the investigation of new strategies, or the screening of new types of doping. Nevertheless, due to the variability induced by indirect biomarkers, the consideration of confounding factors should continuously support this research. Beyond tremendous advances in analytical sensitivity, machine learning-based approaches seem inevitable to facilitate an expert interpretation of numerous biological profiles and promote anti-doping efforts. This perspective article highlights the current innovations of the Athlete Biological Passport that seem the most promising. Through different research axes, this short manuscript provides an opportunity to bring together approaches that are more widely exploited (e.g., omics strategies) and others in the early stages of investigation (e.g., artificial intelligence) seeking to develop the ABP.

Removal of the influence of plasma volume fluctuations for the athlete biological passport and stability of haematological variables in active women taking oral contraception.

The haematological module of the athlete biological passport (ABP) monitors longitudinal haematological variations that could be indicative of blood manipulation. This study applied a multi-parametric model previously validated in elite cyclists to compare inferred and actual PV variations, whereas the potential influence of the oral contraceptive pill (OCP) cycle on the ABP blood biomarkers and plasma volume (PV) in 14 physically active women taking OCPs was also investigated. Blood and serum samples were collected each week for 8 weeks, and the ABP haematological variables were determined according to the World Anti-Doping Agency guidelines. Transferrin (sTFN), ferritin (FERR), albumin (ALB), calcium (Ca), creatinine (CRE), total protein (TP) and low-density lipoprotein (LDL) were additionally computed as 'volume-sensitive' variables in a multivariate analysis to determine individual estimations of PV variations. Actual PV variations were indirectly measured using a validated carbon monoxide rebreathing method. We hypothesised ABP markers to be stable during a standard OCP cycle and estimated PV variations similar to measured PV variations. Measured PV variations were in good agreement with the predictions and allowed to explain an atypical passport finding (ATPF). The ABP biomarkers, Hbmass and PV were stable over 8 weeks. Significant differences occurred only between Week 7 and Week 1, with lower levels of haemoglobin concentration ([Hb]), haematocrit (HCT) and red blood cell count (RBC)(-4.4%, p < 0.01; -5.1%, p < 0.01; -5.2%, p < 0.01) and higher levels of PV at week 7 (+9%, p = 0.05). We thus concluded that estimating PV variations may help interpret individual ABP haematological profiles in women.

Hematological variables in recreational breath-hold divers: a longitudinal study.

BACKGROUND: The influence of regular breath-hold training on hematological variables is not fully understood. We monitored hematological variables in breath-hold divers (BHDs) and active controls over a year expecting both breath-hold training and seasonal effects. METHODS: In 11 recreational BHDs (36±9 years, 177±8 cm, 72±9 kg) and 12 active controls (22±2 years, 174±8 cm, 70±13 kg) monthly blood samples were analyzed with the hematological module of WADA's athlete biological passport. Hemoglobin mass and plasma volume were measured indirectly by the CO-rebreathing method for the last eight months of the study. Breath-hold training sessions were recorded online. Days without breath-hold training, or the number of hours prior to blood sampling when training was realized within the last 24 hours, were recorded. RESULTS: Hematology did not differ significantly between BHDs and controls over the study time (P>0.05). However, hematological values varied significantly over time for both groups suggesting seasonal effects. Blood sampling 19 hours or more after a breath hold training did not indicate any acute effects of breath holding training. CONCLUSIONS: In comparison with a physically active lifestyle, regular breath-hold training does not induce significant variations over one year for the hematological module of the ABP.

Similar Supine Heart Rate Variability Changes During 24-h Exposure to Normobaric vs. Hypobaric Hypoxia.

Purpose: This study aimed to investigate the differences between normobaric (NH) and hypobaric hypoxia (HH) on supine heart rate variability (HRV) during a 24-h exposure. We hypothesized a greater decrease in parasympathetic-related parameters in HH than in NH. Methods: A pooling of original data from forty-one healthy lowland trained men was analyzed. They were exposed to altitude either in NH (FIO2 = 15.7 ± 2.0%; PB = 698 ± 25 mmHg) or HH (FIO2 = 20.9%; PB = 534 ± 42 mmHg) in a randomized order. Pulse oximeter oxygen saturation (SpO2), heart rate (HR), and supine HRV were measured during a 7-min rest period three times: before (in normobaric normoxia, NN), after 12 (H12), and 24 h (H24) of either NH or HH exposure. HRV parameters were analyzed for time- and frequency-domains. Results: SpO2 was lower in both hypoxic conditions than in NN and was higher in NH than HH at H24. Subjects showed similarly higher HR during both hypoxic conditions than in NN. No difference in HRV parameters was found between NH and HH at any time. The natural logarithm of root mean square of the successive differences (LnRMSSD) and the high frequency spectral power (HF), which reflect parasympathetic activity, decreased similarly in NH and HH when compared to NN. Conclusion: Despite SpO2 differences, changes in supine HRV parameters during 24-h exposure were similar between NH and HH conditions indicating a similar decrease in parasympathetic activity. Therefore, HRV can be analyzed similarly in NH and HH conditions.

Factors Confounding the Athlete Biological Passport: A Systematic Narrative Review.

BACKGROUND: Through longitudinal, individual and adaptive monitoring of blood biomarkers, the haematological module of the athlete biological passport (ABP) has become a valuable tool in anti-doping efforts. The composition of blood as a vector of oxygen in the human body varies in athletes with the influence of multiple intrinsic (genetic) or extrinsic (training or environmental conditions) factors. In this context, it is fundamental to establish a comprehensive understanding of the various causes that may affect blood variables and thereby alter a fair interpretation of ABP profiles. METHODS: This literature review described the potential factors confounding the ABP to outline influencing factors altering haematological profiles acutely or chronically. RESULTS: Our investigation confirmed that natural variations in ABP variables appear relatively small, likely-at least in part-because of strong human homeostasis. Furthermore, the significant effects on haematological variations of environmental conditions (e.g. exposure to heat or hypoxia) remain debatable. The current ABP paradigm seems rather robust in view of the existing literature that aims to delineate adaptive individual limits. Nevertheless, its objective sensitivity may be further improved. CONCLUSIONS: This narrative review contributes to disentangling the numerous confounding factors of the ABP to gather the available scientific evidence and help interpret individual athlete profiles.

Cases reports: Unintended anti-doping rule violation after dorzolamide use several months prior to a doping control.

The use of specific medicine up to several months before a doping control is not be reported on the doping control form, while the drug could then still be detectable in urine in case of a very slow elimination. It may lead to a positive test result. For example, dorzolamide, a carbonic anhydrase inhibitor for topical ophthalmic application, has a very slow elimination rate via the renal route (half-life > 4 months). This substance can be a source of unintended anti-doping rule violations.

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.kg-1 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, TSSTM). (Hb) averaged 14.2 ± 0.0 (mean ± SD) g.dL-1 (range: 13.3-15.5 g·dl-1) 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.

Repeated Sprint Training in Hypoxia: Case Report of Performance Benefits in a Professional Cyclist.

Repeated sprint training in hypoxia (RSH) has gained unprecedented popularity among the various strategies using hypoxia as an additional stimulus to improve performance. This case study reports the benefits of 150 repeated sprints in normobaric hypoxia over 10 days in a professional cyclist. After 3 weeks of endurance training in November, the cyclist performed five RSH sessions at a simulated altitude of 3,300 m on his own bicycle attached to an indoor trainer in a hypoxic chamber (FiO2 14.1 ± 0.1%, PiO2 94.6 ± 1.4 mm Hg). Each session consisted of four blocks of seven all-out sprints of 6 s interspersed with 14 s active recovery (for a total of 126 s per block). After 12 min of warm-up with a single isolated 6 s reference sprint, the sessions included a first and a second sprinting block with 4 min 54 s active recovery in-between. After 9 min 54 s active recovery including an isolated 6 s reference sprint, a third and a fourth block were performed with 4 min 54 s active recovery in-between, before an active cool-down of 9 min 54 s. The total duration was thus of 50 min per session for a total hypoxic exposure of 250 min exercising. Power output and heart rate were monitored at 1 Hz. Lactate concentration ([La]) and pulse oxygen saturation (SpO2) were measured at the start and end of each block during the first and fifth training session. Basal SpO2 was of 83% during session one and 85.5% during session five. When comparing the first and fifth training session, peak power increased for the best 1 s value (+8%) and the best 5 s average (+10%) to reach 1,041 W and 961 W, respectively. Average power for all blocks (including active recoveries) increased from 334 to 354 W with a similar average heart rate during the sessions (146'.min-1). Peak [La] was increased from 12.3 to 13.8 mmol.l-1. In conclusion, this case report illustrates a 10-days RSH intervention perceived as efficient in a professional cyclist and shown to improve total work (6-s sprints) produced for a similar physiological strain.

Is pain temporary and glory forever? Detection of tramadol using dried blood spot in cycling competitions.

Tramadol is a synthetic opioid drug used in the treatment of chronic and acute pain. An abnormal prevalence of its misuse in elite sport to overcome pain resulting from prolonged physical effort was recently reported. However, besides its antinociceptive effects, tramadol consumption is associated with negative effects such as numbness, confusion, and reduced alertness. This fact prompted the Union Cycliste Internationale to ban the use of tramadol in cycling competitions. Herein, we present the development of a dried blood spot (DBS) sample collection and preparation method followed by a liquid-chromatography mass spectrometry (LC-MS) analysis to rapidly determine the presence of tramadol and its two main metabolites in blood samples. The detection window of each analyte was evaluated and the analysis of performance on various MS platforms (HRMS and MS/MS) was assessed. Tramadol and its two main metabolites were detected up to 12 h after the intake of a single dose of 50 mg of tramadol in positive controls. In professional cycling competitions, 711 DBS samples collected from 361 different riders were analysed using the developed methodology, but all returned negative results (absence of parent and both metabolite compounds). In the context of professional cycling, we illustrate a valid method bringing together the easiness of collection and minimal sample preparation required by DBS, yet affording the performance standards of MS determination. The proposed method to detect tramadol and its metabolites was successfully implemented in cycling races with a probable strong deterrent effect.

Prevalence Estimate of Blood Doping in Elite Track and Field Athletes During Two Major International Events.

In elite sport, the Athlete Biological Passport (ABP) was invented to tackle cheaters by monitoring closely changes in biological parameters, flagging atypical variations. The hematological module of the ABP was indeed adopted in 2011 by World Athletics (WA). This study estimates the prevalence of blood doping based on hematological parameters in a large cohort of track and field athletes measured at two international major events (2011 and 2013 WA World Championships) with a hypothesized decrease in prevalence due to the ABP introduction. A total of 3683 blood samples were collected and analyzed from all participating athletes originating from 209 countries. The estimate of doping prevalence was obtained by using a Bayesian network with seven variables, as well as "blood doping" as a variable mimicking doping with low-doses of recombinant human erythropoietin (rhEPO), to generate reference cumulative distribution functions (CDFs) for the Abnormal Blood Profile Score (ABPS) from the ABP. Our results from robust hematological parameters indicate an estimation of an overall blood doping prevalence of 18% in 2011 and 15% in 2013 (non-significant difference) in average in endurance athletes [95% Confidence Interval (CI) 14-22 and 12-19% for 2011 and 2013, respectively]. A higher prevalence was observed in female athletes (22%, CI 16-28%) than in male athletes (15%, CI 9-20%) in 2011. In conclusion, this study presents the first comparison of blood doping prevalence in elite athletes based on biological measurements from major international events that may help scientists and experts to use the ABP in a more efficient and deterrent way.

The fatigue-induced alteration in postural control is larger in hypobaric than in normobaric hypoxia.

To test the hypothesis that postural control would be more affected by plantar flexors fatigue during acute exposure in hypobaric (HH) than in normobaric (NH) hypoxia or normobaric normoxia (NN). Twelve young male adults performed in a random order three experimental sessions (in HH and NH (FiO2 0.139) at an altitude of 2950 m, and in NN at 500 m) composed of a bipedal postural control with eyes open on a posturographic platform before and after a plantar flexors fatiguing protocol. Center of pressure (CoP) trajectory and stabilogramm diffusion analyses (SDA) parameters were assessed. A two-way repeated measures analysis of variance was used to identify differences by examination of the group and time interaction. Surface of CoP trajectory analysis, increased at POST in HH (p < 0.001) and in NH (p < 0.01) compared to NN. SDA confirmed that PC was more altered in HH than in NH (p < 0.001) and NN (p < 0.05) at POST. The plantar flexor fatigue-induced alteration in postural control increased to a larger extent in HH than in NH or NN, suggesting an alleviating influence of the decreased barometric pressure per se and a mechanical influence of the higher breathing frequency in HH.