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.

Longitudinal Profiling of Endogenous Steroids in Blood Using the Athlete Biological Passport Approach.

CONTEXT: Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. OBJECTIVE: To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. DESIGN: T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. SETTING: Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). INTERVENTION(S): Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. MAIN OUTCOME MEASURE(S): Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. RESULTS: An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. CONCLUSIONS: Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.

Corrigendum: Hidden figures: Revisiting doping prevalence estimates previously reported for two major international sport events in the context of further empirical evidence and the extant literature.

[This corrects the article DOI: 10.3389/fspor.2022.1017329.].

Hidden figures: Revisiting doping prevalence estimates previously reported for two major international sport events in the context of further empirical evidence and the extant literature.

Background: High levels of admitted doping use (43.6% and 57.1%) were reported for two international sport events in 2011. Because these are frequently referenced in evaluating aspects of anti-doping, having high level of confidence in these estimates is paramount. Objectives: In this study, we present new prevalence estimates from a concurrently administered method, the Single Sample Count (SSC), and critically review the two sets of estimates in the context of other doping prevalence estimates. Methods: The survey featuring the SSC model was completed by 1,203 athletes at the 2011 World Championships in Athletics (WCA) (65.3% of all participating athletes) and 954 athletes at the 2011 Pan-Arab Games (PAG) (28.2% of all participating athletes). At WCA, athletes completed both UQM and SSC surveys in randomised order. At PAG, athletes were randomly allocated to one of the two surveys. Doping was defined as "having knowingly violated anti-doping regulations by using a prohibited substance or method." Results: Estimates with the SSC model for 12-month doping prevalence were 21.2% (95% CI: 9.69-32.7) at WCA and 10.6% (95% CI: 1.76-19.4) at PAG. Estimated herbal, mineral, and/or vitamin supplements use was 8.57% (95% CI: 1.3-16.11) at PAG. Reliability of the estimates were confirmed with re-sampling method (n = 1,000, 80% of the sample). Survey non-compliance (31.90%, 95%CI: 26.28-37.52; p < 0.0001) was detected in the WCA data but occurred to a lesser degree at PAG (9.85%, 95% CI: 4.01-15.69, p = 0.0144 and 11.43%, 95% CI: 5.31-11.55, p = 0.0196, for doping and nutritional supplement use, respectively). A large discrepancy between those previously reported from the UQM and the prevalence rate estimated by the SSC model for the same population is evident. Conclusion: Caution in interpreting these estimates as bona fide prevalence rates is warranted. Critical appraisal of the obtained prevalence rates and triangulation with other sources are recommended over "the higher rate must be closer to the truth" heuristics. Non-compliance appears to be the Achilles heel of the indirect estimation models thus it should be routinely tested for and minimised. Further research into cognitive and behaviour aspects, including motivation for honesty, is needed to improve the ecological validity of the estimated prevalence rates.

Comparison of three different blood matrices for the quantification of endogenous steroids using UHPLC-MS/MS.

Urine is currently the matrix of choice for the detection of exogenous substances but also for the application of the steroidal module of the Athlete Biological Passport (ABP) consisting in a longitudinal monitoring of steroid biomarkers. To fill the limitations related to urine, the longitudinal monitoring of serum steroids concentration in the so-called 'blood steroid profile' has recently been proposed. Although serum samples are collected much less than urine samples, plasma derived from ABP whole blood samples used for the full blood count could be exploited for the quantification of endogenous steroids. Alternatively, dried blood spots (DBS) that are much easier to collect could also serve as matrix for the steroid profile. In this study, we compared the concentration levels of several endogenous steroids measured in three different blood matrices (serum, plasma and DBS) collected from 100 elite athletes participating in the 2019 Doha World Athletics Championships using UHPLC-MS/MS. Plasma and serum samples were collected by venipuncture, whereas DBS were generated from whole blood samples. Although steroids demonstrated a good agreement between the three matrices, a slight but acceptable underestimation (10%-20%) was observed in plasma compared with serum. The difference between DBS and the two other matrices was dependent of the bias between serum and plasma. We also showed that a generic HCT correction for DBS could be a valuable approach for quantitative measurements. This study demonstrates the possibility to use three different matrices for the quantification of endogenous steroids although the slight discrepancies should be considered for longitudinal evaluation.

Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle.

In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers, was investigated for the monitoring of a 28-day T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17ß-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the 'blood steroid profile' represents a powerful complementary approach to the 'urinary module' and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration.

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.

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.

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.

Functionality of the Crosswise Model for Assessing Sensitive or Transgressive Behavior: A Systematic Review and Meta-Analysis.

Tools for reliable assessment of socially sensitive or transgressive behavior warrant constant development. Among them, the Crosswise Model (CM) has gained considerable attention. We systematically reviewed and meta-analyzed empirical applications of CM and addressed a gap for quality assessment of indirect estimation models. Guided by the PRISMA protocol, we identified 45 empirical studies from electronic database and reference searches. Thirty of these were comparative validation studies (CVS) comparing CM and direct question (DQ) estimates. Six prevalence studies exclusively used CM. One was a qualitative study. Behavior investigated were substance use and misuse (k = 13), academic misconduct (k = 8), and corruption, tax evasion, and theft (k = 7) among others. Majority of studies (k = 39) applied the "more is better" hypothesis. Thirty-five studies relied on birthday distribution and 22 of these used P = 0.25 for the non-sensitive item. Overall, 11 studies were assessed as high-, 31 as moderate-, and two as low quality (excluding the qualitative study). The effect of non-compliance was assessed in eight studies. From mixed CVS results, the meta-analysis indicates that CM outperforms DQ on the "more is better" validation criterion, and increasingly so with higher behavior sensitivity. However, little difference was observed between DQ and CM estimates for items with DQ prevalence estimate around 50%. Based on empirical evidence available to date, our study provides support for the superiority of CM to DQ in assessing sensitive/transgressive behavior. Despite some limitations, CM is a valuable and promising tool for population level investigation.

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.

Doping Prevalence in Competitive Sport: Evidence Synthesis with "Best Practice" Recommendations and Reporting Guidelines from the WADA Working Group on Doping Prevalence.

BACKGROUND: The prevalence of doping in competitive sport, and the methods for assessing prevalence, remain poorly understood. This reduces the ability of researchers, governments, and sporting organizations to determine the extent of doping behavior and the impacts of anti-doping strategies. OBJECTIVES: The primary aim of this subject-wide systematic review was to collate and synthesize evidence on doping prevalence from published scientific papers. Secondary aims involved reviewing the reporting accuracy and data quality as evidence for doping behavior to (1) develop quality and bias assessment criteria to facilitate future systematic reviews; and (2) establish recommendations for reporting future research on doping behavior in competitive sports to facilitate better meta-analyses of doping behavior. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to identify relevant studies. Articles were included if they contained information on doping prevalence of any kind in competitive sport, regardless of the methodology and without time limit. Through an iterative process, we simultaneously developed a set of assessment criteria; and used these to assess the studies for data quality on doping prevalence, potential bias and reporting. RESULTS: One-hundred and five studies, published between 1975 and 2019,were included. Doping prevalence rates in competitive sport ranged from 0 to 73% for doping behavior with most falling under 5%. To determine prevalence, 89 studies used self-reported survey data (SRP) and 17 used sample analysis data (SAP) to produce evidence for doping prevalence (one study used both SRP and SAP). In total, studies reporting athletes totaled 102,515 participants, (72.8% men and 27.2% women). Studies surveyed athletes in 35 countries with 26 involving athletes in the United States, while 12 studies examined an international population. Studies also surveyed athletes from most international sport federations and major professional sports and examined international, national, and sub-elite level athletes, including youth, masters, amateur, club, and university level athletes. However, inconsistencies in data reporting prevented meta-analysis for sport, gender, region, or competition level. Qualitative syntheses were possible and provided for study type, gender, and geographical region. The quality assessment of prevalence evidence in the studies identified 20 as "High", 60 as "Moderate", and 25 as "Low." Of the 89 studies using SRP, 17 rated as "High", 52 rated as "Moderate", and 20 rated as "Low." Of the 17 studies using SAP, 3 rated as "High", 9 rated as "Moderate", and 5 rated as "Low." Examining ratings by year suggests that both the quality and quantity of the evidence for doping prevalence in published studies are increasing. CONCLUSIONS: Current knowledge about doping prevalence in competitive sport relies upon weak and disparate evidence. To address this, we offer a comprehensive set of assessment criteria for studies examining doping behavior data as evidence for doping prevalence. To facilitate future evidence syntheses and meta-analyses, we also put forward "best practice" recommendations and reporting guidelines that will improve evidence quality.

A novel approach to improve detection of glucocorticoid doping in sport with new guidance for physicians prescribing for athletes.

The systemic effect of glucocorticoids (GCs) following injectable routes of administration presents a potential risk to both improving performance and causing harm to health in athletes. This review evaluates the current GC antidoping regulations defined by the World Anti-Doping Agency and presents a novel approach for defining permitted and prohibited use of glucocorticoids in sport based on the pharmacological potential for performance enhancement (PE) and risk of adverse effects on health. Known performance-enhancing doses of glucocorticoids are expressed in terms of cortisol-equivalent doses and thereby the dose associated with a high potential for PE for any GC and route of administration can be derived. Consequently, revised and substance-specific laboratory reporting values are presented to better distinguish between prohibited and permitted use in sport. In addition, washout periods are presented to enable clinicians to prescribe glucocorticoids safely and to avoid the risk of athletes testing positive for a doping test.

'Clean athlete status' cannot be certified: Calling for caution, evidence and transparency in 'alternative' anti-doping systems.

Athletes, sponsors and sport organisations all have a vested interest in upholding the values of clean sport. Despite the considerable and concerted efforts of the global anti-doping system over two decades, the present system is imperfect. Capitalising upon consequent frustrations of athletes, event organisers and sponsors, alternative anti-doping systems have emerged outside the global regulatory framework. The operating principles of these systems raise several concerns, notably including accountability, legitimacy and fairness to athletes. In this paper, we scrutinise the Clean Protocol™, which is the most comprehensive alternative system, for its shortcomings through detailed analysis of its alleged logical and scientific merits. Specifically, we draw the attention of the anti-doping community - including researchers and practitioners - to the potential pitfalls of using assessment tools beyond the scope for which they have been validated, and implementing new approaches without validation. Further, we argue that whilst protecting clean sport is critically important to all stakeholders, protocols that put athletes in disadvantageous positions and/or pose risks to their professional and personal lives lack legitimacy. We criticise the use of anti-doping data and scientific research out of context, and highlight unintended harms that are likely to arise from the widespread implementation of such protocols in parallel with - or in place of - the existing global anti-doping framework.

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.

Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations.

Aim: Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC-MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. Results: The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20%). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. Conclusion: The UHPLC-MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.

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.

Antidoping: From health tests to the athlete biological passport.

The athlete biological passport (ABP) was implemented by the International Cycling Union (UCI) in 2008. However, this improvement in the fight against doping was preceded with different milestones since 1996. In this paper, a detailed evolution of the ABP from traditional direct (urine) testing for antidoping purposes is presented. A chronological overview of the ABP including earlier non-disclosed information and contemporary documentation are shown and documented. The strategic development from on-site competition blood testing, called "health tests", to the structure of the ABP is explained in this historical overview which provides information to the antidoping community and general public regarding the beginning of blood doping tests.

Does body position before and during blood sampling influence the Athlete Biological Passport variables?

INTRODUCTION: The Athlete's Biological Passport (ABP) is a tool for the indirect detection of blood doping. Guidelines from the World Anti-Doping Agency (WADA) require a 2 hours delay after any physical exercise, and to be seated for 10 minutes before collecting an ABP sample. This study investigated posture-related hematological variations with changes in body position during blood sampling. METHODS: Ten successive venous blood samples from 38 subjects were collected in three situations: immediately after 10-minutes of normalized activity (B1), after 10-minutes seated (B2, typical reference sample in an anti-doping context), after a 50 m walk (B3), after 5 and 10-minutes in a seated position again (B4 and B5), and finally after 5-30 minutes supine (B6-B10). Hemoglobin concentration [Hb] and hematocrit (Hct) were determined by flow cytometry to assess putative posture-related variations. RESULTS: Reticulocytes percentage was unchanged in all conditions, [Hb] and Hct were stable after at least 10-minutes in a seated position. Due to shifts in plasma volume, [Hb] and Hct increased slightly but significantly higher after changing posture for a short walk (+0.1 gr/dL [P = .008] and +0.4% [P = .01] respectively), but readjusted to previous levels after only 5 minutes. Supine position (>10 minutes) induced decreases of [Hb] (-0.2 g/dL in average, P < .01) and Hct (-1.1%, in average, P < .01). CONCLUSION: The observed variations in [Hb] and Hct may have minor clinical significance, while they underline the need to follow strict guidelines for posture before and during blood sampling in an anti-doping context.