Haematological Module Research Articles

Prohibited Olympic Medalist with PIEZO1 VUS Who Claims Innocence

Competitive athletes are often exposed to extreme physiological loading, resulting in over excessive mechanotransduction during their acute intensive training sessions and competitions. Individual differences in their genetics often affect how they cope with these challenges, as reflected in their high performances. Olympic Medalists are prohibited from providing atypical values in the Hematological Module of the Athlete Biological Passport. Since there was no aphysiological result and the Athlete maintained his innocence, a whole genome sequence analysis was carried out on him and his parents, with the primary focus on the PIEZO ion channels encoding gene. PIEZO1 is known to participate in homeostatic regulation even on a whole-body level, including the regulation of physical performance, circulatory longevity of red blood cells and cell fate determination of mesenchymal stem cells in relation to hydrostatic pressure. However, PIEZO2 was found to be the principal mechanosensory ion channel for proprioception. These regulatory mechanisms play a pivotal role in mechanotransduction and intensive exercise moments. Interestingly, two variances of uncertain significance of PIEZO1 were found that may explain the atypical values of the Athlete. Furthermore, two additional variances in SDC2, the syndcan-2 encoding gene, were identified in trans position that may influence the crosstalk between PIEZO2 and PIEZO1, with more likely relevance to the detected atypical values. After all, based on the found variances of PIEZO1 and syndecan-2, it cannot be ruled out that these VUS variants may have caused or impacted the exhibited outlier findings of the ABP Hematological Module of the Athlete.

Comparison of Microcapillary Blood Sampling Devices for Use in Anti-Doping.

Blood collection is an important facet of anti-doping testing, forming the basis of the hematological module of the athlete biological passport (ABP). Presently, whole blood is collected via venepucture under standardized conditions, in accordance with the World Anti-doping Agency's International Standard for Testing and Investigations (ISTI). Advances in capillary whole blood collection technology now afford the ability to collect microvolumetric capillary whole blood from the upper arm (or other suitable vascular location such as the abdomen) that is "needle-free" and virtually painless. Previous work has demonstrated the suitability and feasibility of capillary whole blood compared with venous using the Tasso+ device. Recently, other microcapillary devices have received FDA approval increasing the options available to the anti-doping community. The present study extends previous work, by collecting microliter capillary whole blood samples via two different FDA approved devices (RedDrop and the Tasso+ device) to compare with venous blood collected simultaneously. Ten participants provided three matched blood samples (2× capillary and 1× venous) in accordance with WADA ABP guidelines for blood sample collection, for a total of 30 samples. Capillary samples from both devices showed excellent laboratory agreement with venous blood for all CBC parameters, with the exception of platelets. Excellent laboratory agreement was also observed between the two microcapillary collection devices. Irrespective of the device, microcapillary blood collection provides a valid alternative to venous blood collection for ABP purposes.

Comparison between standard hematological parameters and blood doping biomarkers in dried blood spots within the athlete population of Swiss Sport Integrity.

The study demonstrated the feasibility of incorporating RNA biomarkers, specifically 5-aminolevulinic acid synthase (ALAS2) and carbonic anhydrase 1 (CA1), to improve the hematological module of the Athlete Biological Passport (ABP) in routine antidoping context. The aim was to investigate the implementation of reticulocyte (RET) related biomarkers, specifically ALAS2 and CA1, using quantitative reverse transcription polymerase chain reaction (RT-qPCR) on dried blood spots (DBS) from elite athletes. Hemoglobin changes over time in DBS samples was measured as well. Combining hemoglobin and messenger RNA (mRNA) analyses allowed to monitor alterations of the established marker, "DBS OFF-score". Ten athletes were selected for sampling by the Swiss national antidoping organization, Swiss Sports Integrity (SSI). Samples were collected, transported and analyzed for ABP following the World Anti-Doping Agency (WADA) procedures and spotted onto Protein Saver DBS cards. Most athletes exhibited stable biomarker levels, except for one individual involved in ski mountaineering, who demonstrated a sustained increase in ALAS2 compared to the individual baseline. This elevation could be due to blood withdrawal or other factors, such as doping with substances outside the targeted test menu. In this study, RNA-biomarkers were successfully analyzed in routine blood samples, and the project demonstrated promising results for the implementation of ALAS2 and CA1 in routine analysis to complement the ABP.

Erythropoiesis related biomarkers to detect autologous blood micro-transfusion in antidoping field

Indirect detection of autologous blood transfusion (ABT) abuse in Anti-Doping world is done by hematological module of athlete biological passport (ABP). Actual markers (reticulocytes (%RET) and hemoglobin (HGB)) of this module are enough sensitive to detect ABT. However, a new way uses by athlete to improve their performance is micro ABT and in this case %RET and HGB have some lack of sensitivity to detect this new way of doping. Previous studies have demonstrated that erythropoiesis-related RNAs, genes 5’-aminolevulinate synthase 2 ( ALAS2) and carbonic anhydrase 1 ( CA1) in capillary dried blood spots (DBS) could be potential biomarkers to increased sensitivity of the ABP in case of micro-ABT. In the present study, we monitored ALAS2 and CA1 expression in DBS after a donation of 450ml of blood and after a micro reinfusion (130ml) of this blood. DBSs samples collected from 48 volunteers’ males and females were analyzed. Participants were divided in two groups (micro ABT and placebo). Before the beginning of the study, randomized volunteers from transfused and non-transfused cohort were monitored 2 or 8 times weekly to get baseline points. 5 samples were collected after the blood withdrawal and 6 samples after the micro-ABT. The purpose was to evaluate the performance of RNAs biomarkers for the detection of micro-ABT in a longitudinal blinded setting. Our results illustrated a clear significant increase of ALAS2 until to 300 % after withdrawal. Regarding CA1 the increase is up to 250%. Looking at micro-ABT ALAS2 and CA1 will decrease under the baseline value. Regarding actual ABP markers, %RET slightly increase after the withdrawal up to 140%. In contrast, micro-ABT seem to have any effect with value that came back to baseline value without decreasing under baseline value. Finally, HGB slightly decrease after withdrawal follow by any increase after micro-ABT. Oppositely, regarding placebo group RNA markers and actual ABP markers stay stable all along the study. Erythropoiesis-related RNAs in capillary DBS such as ALAS2 and CA1 were sensitive biomarkers for blood withdrawal detection followed by micro-ABT. Furthermore, this investigation provide evidence that micro ABT can be detected in a longitudinal blinded setting with RNA biomarkers in DBS. World Anti Doping Agency. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Age-Markers on the Red Blood Cell Surface and Erythrocyte Microparticles may Constitute a Multi-parametric Strategy for Detection of Autologous Blood Transfusion

BackgroundAutologous blood transfusion is one of the illicit strategies, banned by the World Anti-Doping Agency, to increase the levels of hemoglobin, with a consequent improvement in the delivery of oxygen to tissues. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of “storage lesions”, targeting (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2, (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles.ResultsAll of the above markers were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh blood samples, collected for official doping control tests, mixed “ex vivo” to simulate an autotransfusion. Although anonymized before the delivery to the laboratory, it was possible to mix samples belonging to the same subject based on the “athlete biological passport” code. Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to: (i) the reduced concentration, on the erythrocyte membrane, of Band 3 protein (decrease of 19% and of 39% after 20 and 40 days of storage respectively) and of glycophorin A (− 47% and − 63% respectively); (ii) the externalization of phosphatidyl serine (with a five-fold increase after 20 days and a further 2× increase after 40 days); (iii) the reduced concentration of CD47; and (iv) increased levels of erythrocyte microparticles.ConclusionsThe most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena.

Effect of low-volume blood withdrawal on hematological biomarkers

Introduction
 The hematological module of the Athlete Biological Passport (ABP) consists of individual and longitudinal monitoring of hematological parameters biomarkers responsive to erythropoiesis alteration. Based on an adaptive Bayesian model, the ABP seeks to identify non-physiological variations that may correspond to different types of blood doping. In this context, the impact of a standard blood donation (500 ml) was evident on the ABP parameters, hence supporting the robustness of the model. However, there is only scarce evidence of single or multiple withdrawals of smaller volumes of blood, closer to a modern blood doping strategy. This study investigated the impact of low-volume blood withdrawal on hematological variables for anti-doping purposes.
 Methods
 Twelve healthy subjects (7 women and 5 men; 26 ±6 yrs, 170 ±7 cm; 65 ±7 kg) were recruited. After baseline measurement, 140 ml whole blood was withdrawn with subsequent measures of hematological parameters by flow cytometry (Sysmex XN-1000, Sysmex Corporation, Japan) for 3 weeks. Blood volumes (BV) and total hemoglobin mass (Hbmass) were determined by means of a validated CO-rebreathing method. Ferritin (FERR) concentration was additionally measured by chemifluminescence immunoassay technique. Individual profiles were generated using the official ABP adaptive model using the official software platform of the World Anti-Doping Agency (WADA). Mixed model repeated measures analyses were run for each parameter to determine whether changes in the dependent (hematological variables) variables differed over time (fixed factor).
 Results
 43.2 ±8 g of hemoglobin was removed during the 140 ml blood withdrawal. A significant decrease in Hbmass (726 ±185 vs. 709 ±189 g; p = 0.007) and Red Blood Cell Volume (RBCV; 2190 ±521 vs. 2129 ±552 L; p = 0.028) was observed at D + 7 with a return to basal value at D + 14. No significant variations were observed for the primary and secondary variables of the ABP. No Atypical Passport Finding (ATPF) were generated in the individual ABP profiles. Conversely, FERR was significantly downregulated at D + 7 (95.8 ±76 vs. 87.1 ±76 μg/l; p = 0.018).
 Discussion/Conclusion
 While the ABP has demonstrated substantial advantages for traditional doping scenarios, our results challenge the indirect detection of low-volume blood withdrawals in a contemporary blood doping scenario. The impact of low-volume autologous blood transfusion on time trial performance has recently been demonstrated, supporting the need for novel biomarkers with extended detection windows. Hbmass could hence represent an additional marker to screen blood doping while technical issues for the use of CO-rebreathing remain. FERR as a marker of altered iron metabolism was there against shown to be sensitive and possibly suitable for outlining low-volume blood transfusion. The large inter-subject variability underlines the importance of individual longitudinal monitoring to provide sufficient sensitivity in the interpretation of hematological variations. Our study finally confirms that even low-volume blood withdrawals are associated with significant hematological changes while frequent blood samples and additional biomarkers are certainly required to better tackle blood doping strategies using such low-volume transfusions.

Ten years of collecting hematological athlete biological passport samples-perspectives from a National Anti-doping Organization.

The hematological module of the Athlete Biological Passport (ABP) aims to reveal blood doping indirectly by looking at selected biomarkers of doping over time. For Anti-Doping Organizations (ADOs), the ABP is a vital tool in the fight against doping in sports through improved target testing and analysis, investigations, deterrence, and as indirect evidence for use of prohibited methods or substances. The physiological characteristics of sport disciplines is an important risk factor in the overall risk assessment and when implementing the hematological module. Sharing of experiences with implementing the hematological ABP between ADOs is key to further strengthen and extend its use. In this study, we present 10 years of experience with the hematological ABP program from the perspectives of a National ADO with special attention to sport disciplines' physiological characteristics as a potential risk factor for blood doping. Not surprisingly, most samples were collected in sport disciplines where the aerobic capacity is vital for performance. The study highlights strengths in Anti-Doping Norway's testing program but also areas that could be improved. For example, it was shown that samples were collected both in and out of season in a subset of the data material that included three popular sports in Norway (Cross-Country Skiing, Nordic Combined, and Biathlon), however, from the total data material it was clear that athletes were more likely to be tested out of competition and on certain days of the week and times of the day. The use of doping control officers with a flexible time schedule and testing outside an athlete's 60 min time-slot could help with a more even distribution during the week and day, and thus reduce the predictability of testing. In addition to promoting a discussion on testing strategies, the study can be used as a starting point for other ADOs on how to examine their own testing program.

Altitude and Erythropoietin: Comparative Evaluation of Their Impact on Key Parameters of the Athlete Biological Passport: A Review.

The hematological module of the Athlete's Biological Passport (ABP) identifies doping methods and/or substances used to increase the blood's capacity to transport or deliver oxygen to the tissues. Recombinant human erythropoietin (rhEPOs) are doping substances known to boost the production of red blood cells and might have an effect on the blood biomarkers of the ABP. However, hypoxic exposure influences these biomarkers similarly to rhEPOs. This analogous impact complicates the ABP profiles' interpretation by antidoping experts. The present study aimed to collect and identify, through a literature search, the physiological effects on ABP blood biomarkers induced by these external factors. A total of 43 studies were selected for this review. A positive correlation (R2 = 0.605, r = 0.778, p < 0.001) was identified between the hypoxic dose and the increase in hemoglobin concentration (HGB) percentage. In addition, the change in the reticulocyte percentage (RET%) has been identified as one of the most sensitive parameters to rhEPO use. The mean effects of rhEPO on blood parameters were greater than those induced by hypoxic exposure (1.7 times higher for HGB and RET% and 4 times higher for hemoglobin mass). However, rhEPO micro-doses have shown effects that are hardly distinguishable from those identified after hypoxic exposure. The results of the literature search allowed to identify temporal and quantitative evolution of blood parameters in connection with different hypoxic exposure doses, as well as different rhEPOs doses. This might be considered to provide justified and well-documented interpretations of physiological changes in blood parameters of the Athlete Biological Passport.

Feasibility of microvolumetric capillary whole blood collections for usage in Athlete Biological Passport analysis.

The hematological module of the Athlete Biological Passport (ABP) represents an important tool in the pursuit to detect blood doping in athletes. Currently, collecting blood samples for ABP analysis can be cumbersome, invasive, and expensive, involving a venous blood draw performed by a trained phlebotomist followed by cold-chain monitored shipping to the analysis laboratory. Developing innovative methods to collect and transport ABP blood samples while adhering to strict preanalytical and analytical requirements has the potential to greatly increase testing frequency and, consequently, the effectiveness of the ABP program globally. The focus of this study was to compare venous blood collections with capillary blood collections to determine if capillary samples could be used for ABP analysis without sacrificing the analytical integrity required for antidoping testing procedures. In this study, capillary blood was collected using the Tasso+ EDTA device (Tasso, Inc.), a novel microvolumetric device that collects liquid, whole blood from skin capillaries on the upper arm. Excellent laboratory agreement was observed between venous and capillary blood samples for the three main ABP parameters: HGB, RET%, and OFF-Score. Additionally, the stability of capillary samples after storage at 4°C, similar to what would be required during transport, was acceptable for up to 72 h following collection. Finally, we generated individual ABP profiles using the adaptive model for 10 participants and observed excellent agreement between venous and capillary profiles. These results indicate capillary blood collection is a viable alternative to venous blood collections for ABP analysis.

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.

Определение кобальта в образцах плазмы крови методом масс-спектрометрии с индуктивно-связанной плазмой после перорального приема содержащих кобальт биологически активных добавок к пище

Salts of inorganic cobalt (Со) prevent the degradation of the alpha subunit of the hypoxia-inducible factor (HIF), imitating the state of hypoxia in the body and increasing the production of the endogenous hormone erythropoietin (EPO), and are used as doping substances that increase blood oxygen capacity and endurance, which give competitive advantages in sports. Currently, a large number of dietary supplements, including Co-containing ones, are offered on free sale. Their uncontrolled intake can affect not only the professional career of athletes, but also their health, due to the fact that this trace element and its salts are the strongest inorganic poisons and carcinogens. Despite this, their availability on the pharmaceutical market, a noticeable effect of erythropoiesis stimulation and a convenient oral form of administration lead to the need for their detection in modern doping control. The purpose of this research was to develop an approach to differentiate cobalt from vitamin B12, present in the body in its natural state, from the intake of cobalt salts by quantifying and comparing blood levels of vitamin B12 and total cobalt. Methods. The study involved 9 healthy volunteers (women and men) aged 25 to 45 years, leading an active lifestyle. Three of them took 2500 μg/day of cobalamin for 20 days (comparison group), three - dietary supplement containing cobalt asparaginate (100 μg/day in terms of pure cobalt), and the rest - dietary supplements with cobalt sulfate heptahydrate (100 μg/day in terms of pure cobalt) (administration groups) at the same time after meals. Blood samples were taken at baseline and on days 5, 9, 14 and 20. The concentrations of total cobalt in blood plasma samples of volunteers were measured by inductively coupled plasma mass-spectrometry (ICP-MS), the levels of cobalamin were determined on a Cobas 6000 immunochemical analyzer using the Elecsys Vitamin B12 II Assay ELISA kits. Results. It was found that oral intake of of cobalamin at a therapeutic dose significantly exceeding the recommended daily intake (3 μg), there was a regular slight increase in the blood concentration of total cobalt (1.1 times). At the same time intake of dietary supplements containing cobalt in the form of sulfate or asparaginate (about 100 μg per day in terms of pure cobalt) was accompanied by 4-6.7 fold increase in the concentration of total cobalt while unchanged vitamin B12 plasma concentration was observed. The detection of such changes can reliably indicate the use of prohibited salts and, of course, will be in demand for anti-doping control. Conclusion. Long-term monitoring of vitamin B12 and total cobalt levels, similar to hematological module of the Athlete Biological Passport program, will unambiguously detect possible abuse of cobalt salts and can be an additional evidence of the presence of these doping substances to other analytical methods, such as a combination of liquid chromatography and ICP-MS (LC-ICP-MS).

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

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. 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. 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. 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.

The use of RNA-based 5'-aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses.

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'‐aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti‐doping analyses.

The Use of RNA‐based 5’‐Aminolevulinate Synthase 2 Biomarkers in Dried Blood Spots to detect rhEPO Micro Doses and Hypoxic Stimulation

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping e.g. with micro doses of recombinant human erythropoietin (rhEPO) is problematic. For this reason, sensitivity of ABP have to be enhance with the implementation of new biomarkers. Here we demonstrated that RNA-based 5’-Aminolevulinate Synthase 2 (ALAS2) could be a useful biomarker to improve the indirect detection of rhEPO micro dosing. Moreover, satisfying results were obtain also for specificity in order to distinguish rhEPO administrations from a sojourn in hypoxic conditions. Samples from two different clinical studies were used to monitor levels of different RNA markers, linear-circular (LC) and linear (L), of ALAS2 RNA, Carbonate anhydrase 1 (CA1) and Solute Carrier family 4 member 1 (SLC4A1) after rhEPO microdoses administration and under hypoxic condition. Analysis of RNA based biomarkerswere performed using dried blood spots (DBSs), these ones provide many advantages in terms of the samples collection, transport and storage. In order to monitored transcirptomics biomarkers variations, manual extraction of RNA from DBSs were performed follow by a RT-qPCR. Transcriptomics biomarkerswere monitored along 16 days where five micro doses were administrated, ALAS2 markers illustrated an increase up to 300% in comparison with the baseline value and these variations were statistically different from evolution in control group. Additionally, also CA1 variated in a significant way due to micro doses administrations. Moreover, ALAS2 markers variations under hypoxic conditions are not significant when compared with control group, which is the opposite of CA1 or reticulocytes percentage, where significant differences were obtained. Finally, SLC4A1 has no significant variation in both conditions. This study demonstrated that transcriptomic biomarkers, in particular ALAS2 mRNAs, could be used to improve the sensitivity and specificity of the hematological module of the ABP, and are compatible with the use of DBSs for anti-doping analyses. The expression level of SLC4A1 was not increased by micro doses of rhEPO, indicating that it is not a beneficial biomarker to detect micro dosing. CA1 is an interesting candidate, although, unlike ALAS2, its expression level was increased also significantly following exposure to hypoxia.

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.

Support of a laboratory-hosted Athlete Biological Passport Management Unit (APMU) to the anti-doping organisations

AbstractThe athlete biological passport (ABP) is an established means for longitudinal monitoring of selected individual biomarkers of an athlete to obtain indirect but potentially long-term indications of the use of substances or methods prohibited in sport. Along the change from population-based reference values to individual profiling, the ABP aims at triggering follow-up investigations concerning the potential use of endogenous substances with doping potential, which might be difficult either to identify with the existing analytical methods or to interpret based only on the results of a single biological sample. The ABP program has been on-going within the World Anti-Doping Agency (WADA) management since 2009, when the hematological module was officially established to discover blood doping practices, such as administration of erythropoietin (EPO) or application of blood transfusion. Since 2014, the ABP has been complemented by the steroid module, with the aim of targeting the prohibited use of testosterone and other endogenous anabolic androgenic steroids with performance enhancing or masking capability. Although the main objective is to guide and assist the anti-doping organizations in their test distribution plans, the ABP may also be used to proceed with a case to an anti-doping rule violation. Evaluation of biological markers, especially in distinguishing between doping from other confounding factors, requires high level and diversity of expertise, which is coordinated by the athlete biological passport management unit (APMU). Since 2019, the WADA accredited anti-doping laboratories are defined as the host organizations for the APMUs. The benefit of such a structure is to obtain a fully anonymous evaluation process for the passports and an additional level of expertise for the interpretation of analytical results as well as to have a fluent communication line with the analyzing laboratories when further details are needed for the analytical testing and documentation.

A multi-parametric approach to remove the influence of plasma volume on the athlete biological passport during a Union Cycliste Internationale cycling stage race.

Fluctuations in plasma volume (PV) present potential confounders within the concentration-based markers of the haematological athlete biological passport (ABP). Here, a multi-parametric approach involving a simple blood test is applied to the current ABP adaptive model in an attempt to remove the influence of PV expansion, induced by a cycling stage race. Blood samples were obtained from 29 professional cyclists (14 male, 15 female) before, during and after 4-5 consecutive days of racing. Whole blood was analysed in accordance with the World Anti-Doping Agency ABP guidelines for haemoglobin ([Hb]) concentration and platelets. Serum and plasma were analysed for transferrin, albumin, calcium, creatinine, total protein and low-density lipoprotein. PV variation (Z-scores) was estimated using a multi-parametric model (consisting of the biomarkers mentioned earlier) and compared against calculated variations in PV (measured via CO-rebreathing). Significant reductions in [Hb] and the OFF-score were observed in female cyclists after 3 and 4 days of racing, with accompanying increases in PV, which returned to baseline values 4 days post competition. Similarly, a significant increase in PV was observed in male cyclists after 3 and 5 days of racing. When individual estimations of PV variance were applied to the adaptive model, the upper and lower reference predictions for [Hb] and the OFF-score were refined such that all outliers consistent with racing-induced PV changes were removed. The PV model appears capable of reducing the influence of PV on concentration-dependent markers during competition. This is an important step towards the inclusion of the PV correction in the ABP haematological module.

Fluctuations in hematological athlete biological passport biomarkers in relation to the menstrual cycle.

The interpretation of athlete biological passport (ABP) is strengthened by understanding the natural fluctuations in its biological parameters. Here we have assessed the influence of the menstrual cycle on the hematological module of the ABP. Seventeen women with regular menses were included. Blood samples were collected once a week for two consecutive cycles and analyzed for hematological parameters. Menstrual phases were hormonally determined. The intra-individual variation in the hematological parameters was similar between the two cycles. Reticulocyte percentage was significantly lower in the follicle phase (median 0.95%) than in the ovulatory (median 1.10%) and luteal phases (median 1.16%), P = 0.006, whereas no differences were found in hemoglobin concentration, hematocrit, red blood cell count, or red blood cell indices. When the values were entered into the ABP model, findings outside the program-calculated individual thresholds were identified in two participants. One woman showed an atypical low OFF-score in the last sample collected, mainly because of increased reticulocyte percentage. This was likely a response to treated insufficient iron stores. One woman displayed an atypical hemoglobin value at the lower limit 2 weeks after ovulation, which was likely due to fluctuations in plasma volume. In conclusion, the ABP parameters in general are stable throughout the menstrual cycle. Significant differences between the menstrual phases were found in reticulocytes; however, the variation was not related to findings outside the individual thresholds, except in one individual. Moreover, our results highlight the importance of having information about iron supplementation available when evaluating hematological passports.

Changes in blood parameters after intramuscular testosterone ester injections - Implications for anti-doping.

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the 'haematological module' of the athlete biological passport (ABP) used by the World Anti-Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo-controlleddouble-blind design. Urine samples and blood samples were collected twice pre-treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post-injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®-treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope-ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF-Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3-10 days after injection. From an anti-doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow-up testing and substantiating likely misuse of testosterone.

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.