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Monitoring of biological markers indicative of doping: the athlete biological passport
  1. Martial Saugy1,
  2. Carsten Lundby2,
  3. Neil Robinson1
  1. 1Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, Genève and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Epalinges, Switzerland
  2. 2Center for Integrative Human Physiology, Institute of Physiology, University of Zurich, Zurich, Switzerland
  1. Correspondence to Professor Martial Saugy, Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, Genève and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Ch. Des Croisettes 22, Epalinges 1066, Switzerland; martial.saugy{at}chuv.ch

Abstract

The athlete biological passport (ABP) was recently implemented in anti-doping work and is based on the individual and longitudinal monitoring of haematological or urine markers. These may be influenced by illicit procedures performed by some athletes with the intent to improve exercise performance. Hence the ABP is a valuable tool in the fight against doping. Actually, the passport has been defined as an individual and longitudinal observation of markers. These markers need to belong to the biological cascade influenced by the application of forbidden hormones or more generally, affected by biological manipulations which can improve the performance of the athlete. So far, the haematological and steroid profile modules of the ABP have been implemented in major sport organisations, and a further module is under development. The individual and longitudinal monitoring of some blood and urine markers are of interest, because the intraindividual variability is lower than the corresponding interindividual variability. Among the key prerequisites for the implementation of the ABP is its prospect to resist to the legal and scientific challenges. The ABP should be implemented in the most transparent way and with the necessary independence between planning, interpretation and result management of the passport. To ensure this, the Athlete Passport Management Unit (APMU) was developed and the WADA implemented different technical documents associated to the passport. This was carried out to ensure the correct implementation of a profile which can also stand the challenge of any scientific or legal criticism. This goal can be reached only by following strictly important steps in the chain of production of the results and in the management of the interpretation of the passport. Various technical documents have been then associated to the guidelines which correspond to the requirements for passport operation. The ABP has been completed very recently by the steroid profile module. As for the haematological module, individual and longitudinal monitoring have been applied and the interpretation cascade is also managed by a specific APMU in a similar way as applied in the haematological module. Thus, after exclusion of any possible pathology, specific variation from the individual norms will be then considered as a potential misuse of hormones or other modulators to enhance performance.

  • Doping

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Introduction/overview

For many years, the concept of individual monitoring of biological markers had been studied to detect the potential abuse of doping substances. Manfred Donike, head of the Cologne anti-doping laboratory, worked with his group since the beginning of the 80s on the effect of steroid abuse. In 1989, the Cologne group described the long-term influence of anabolic misuse on the steroid profile.1 The idea of individual and longitudinal follow-up in the field of the fight against doping was then born naturally. The same group made this statement even clearer by observing that for an individual, the homeostasis of biosynthesis and metabolism of endogenous steroids was not disturbed by physical workload2 but of course was influenced by the use of testosterone or other similar substances.

Regarding blood doping also, the idea of individual and longitudinal monitoring was established quite soon at the time when recombinant human erythropoietin (rh-EPO) was introduced in the market. In 1989, blood tests were performed at the World Cross Country ski championships in Lahti, Finland, in order to constitute a database and to show possible abnormal individual variation in blood markers due to rh-EPO doping or to blood transfusion.3

As the abuse of rh-EPO by endurance athletes increased dramatically in the 90s, several proposals of indirect detection by markers were suggested. The percentage of circulating MacroHypo red blood cells (RBCs) as well as the transferrin receptors was proposed.4–7

Then, the percentage of reticulocytes (RETs), was shown to be drastically increased a few days after the beginning of a treatment showing the potential of the red cell line to be used as a diagnostic tool to detect blood doping manipulation in the athlete population.8 All these studies were carried out at the time when blood doping (including rh-EPO and transfusion) was certainly the most severe in endurance sports and disciplines. In 1996 and 1997, two major international sports federations decided to limit blood doping in their population of interest. At that time International Skiing Federation (FIS) and International Cycling Union (UCI) decided to introduce a competition rule based on a population-based upper limit of 18 g/dL haemoglobin (HGB) for FIS and 50% haematocrit (HCT) for UCI. This ‘no-start’ rule was introduced with the double aim to preserve the health of the athlete and also to protect fairness in the competition,9–12 but it became rapidly obvious that a population-based cut-off was not appropriate to maintain the competition fair due to the large interindividual variability distribution in a population of several blood markers.13–15

Large database of blood values was then accessible to the main endurance federations and the concept of individual follow-up slowly entered into the culture. The term of passport was first introduced by Cazzola and later Malcovati who studied the feasibility of the haematological passport for athletes competing in endurance sports.16–18 This group, as other authors previously, made at that time a clear statement that the definition of arbitrary limit in critical haematological markers to evaluate the eligibility to compete was neither a very specific nor a very sensitive strategy. In fact, the adoption of this kind of limit was risky by creating false positive cases (eg, naturally elevated HGB) and many potential false negative cases with athletes using plasma volume expanders.

The athlete biological passport: example of the haematological modules

The haematological passport of an athlete is in fact a statistical representation of the longitudinal follow-up of some of his blood markers. The individual and longitudinal follow-up is of interest when the intraindividual variability of a biological marker is lower than the corresponding interindividual variability.19 ,20 This fact is obvious for most of the blood markers in consideration.21 ,22 Then, from a statistical point of view, the athlete has his own reference values for a biological biomarker.

The indirect detection of blood doping, as described previously, was one of the major tasks of the antidoping community before the 2000 Sydney Summer Olympic Games. Many developments in this field were set up by the Australian Institute of Sport, whose work was mainly dedicated to the detection of rh-EPO for the 2000 Olympics. The basis of these developments was to get a score using several indirect haematological and biochemical markers used simultaneously to identify efficiently current or recent users of rh-EPO.

The first models presented by the group integrated a combination of RETHCT, serum EPO, serum soluble transferrin receptor (sTFR), HCT and percentage of macrocytes. They then distinguished the so-called ON-model, giving a score composed of these markers describing the situation they had during the final 2 weeks of rh-EPO administration phase. The OFF-model integrated RETHCT, EPO and HCT and was applied during the washout phase and, during the period up to 12–21 days after the last rh-EPO injection.23–26 Magnani et al27 in 2001 demonstrated that HCT, RET count, serum sTFR and concentration of β-globin mRNA put together into a new multiparametric formula, could detect rh-EPO abuse in about 60% of the samples examined after low dosage of rh-EPO doping.

Altitude and exercise could of course change the paradigm for multiparametric calculations. The evolution of doping habits among endurance athletes and their spectacular mode of adaptation to respond to the detection policy pushed the scientists to propose new models. Gore et al and Sharpe et al presented the second generation model, which defines better the situation, when lower dosages of rh-EPO are used.28–31

In 2006, Sottas et al32 proposed to use and combine all data contained in a single blood profile of an athlete. The Abnormal Blood Profile Score (ABPS) corresponds to a multiparametric marker which can include up to 12 different blood markers which are affected by the administration of rh-EPO or the use of blood transfusion. More recently, two other statistical tools were suggested to be used for targeting purposes only.33 ,34

Indirect markers used in the haematological passport can be affected by heterogenous and confounding factors.19 These factors will create variability in the multiparametric score which needs to be correctly managed for the proper use of the system. The role played by factors like age, gender, altitude, type of sport and instrumentation has been extensively studied.29 ,30 ,35 ,36 Training at altitude is one of the factors which can produce the highest variability of the model, affecting then the specificity and the sensitivity of any longitudinal tool describing the evolution of haematological markers. It is nowadays generally accepted that an exposure to high altitude during the 2 weeks preceding the test must be taken into account in establishing individual limits19 even if the debate is never ending in regard to the factors influencing the stability of blood markers and then, the specificity of the system.37–39 It has been also argued that seasonal changes of haematological markers, due to training and competitions, should be taken into account.40–45 A greater intravariability factor, if included in the model, will of course be detrimental to the sensitivity of the passport, which seems to be already critical. Recently, this sensitivity to flag abnormal deviations in blood values was evaluated after microdosage of rh-EPO treatment applied to athletes.46 The authors demonstrated that it is possible for athletes to use rh-EPO without eliciting abnormal changes in blood markers currently used by the passport, questioning its sensitivity.

How does the haematological module work?

The different markers of blood doping on which experience has been acquired in recent decades have been implemented into the athlete biological passport (ABP). The concept of the ABP has been discussed and then further elaborated by the WADA. Since the 2006 Torino Winter Olympics, several international federations (IFs) agreed that WADA should harmonise the development and validation of the ABP programme.

As a result, in 2009, WADA published the ABP operating guidelines which are regularly updated.47 This can nowadays be used by anti-doping organisations (ADOs) who want to implement the passport.

In these guidelines, two main objectives are defined in the introduction:

  1. To identify and target athletes for specific analytical testing (eg, EPO urine test, homologous blood transfusion test) by intelligent and timely interpretation of haematological module data.

  2. To pursue possible antidoping rule violations in accordance with the World Anti-Doping Code Article 2.2 (use or attempted use by an athlete of a prohibited substance or a prohibited method).

As described in the WADA document, the aim of the ABP, when implemented in accordance with the appropriate technical documents, is a reliable method for indirectly detecting doping that can resist to legal and scientific challenges.48 ,49 Furthermore, it can also be used to evaluate the prevalence of doping within a population of interest.50

The general scheme of the biological passport is shown in figure 1, where the flowchart of the different steps is summarised.

Figure 1

General scheme of the athlete biological passport (ABP). The anti-doping organisation (ADO) testing authority has a pool of athletes to be tested. Mainly, international sports federations (IFs) and national anti-doping organisations (NADOs) will order for sample collection on athletes from their registered testing pool (RTP). Official Doping and/or Blood Control Officers (DCOs/BCOs) are designated to proceed with the appropriate sample collection. They are in charge then to assure the transport of the samples in the appropriate conditions and time (in less than 36 h) to the accredited laboratory. The analysis results are then immediately introduced in the passport of the athlete, which is constituted of at least 4–6 values per season. The Athlete Passport Management Unit (APMU), linked to a WADA accredited laboratory, is in charge of processing the passport, control its validity and the quality of the data and if presenting abnormality in the sequence of biological data (biological values being significantly outside the individual norms), the full ABP documentation package will be examined by three independent experts who shall provide an unanimous decision to the APMU to release a certificate of ‘Adverse Passport Finding’ (APF) for the ADO result management unit.

Key points for the implementation of the ABP

To resist to legal and scientific challenges, the ABP should be a transparent process with the necessary independence between planning, interpretation and result management of the passport.

A new major actor has been introduced in the system to create a framework of independence: the Athlete Passport Management Unit (APMU). This unit should be the central hub connecting laboratory-generated biological data with active test planning intelligence.51 Ideally, this central hub should be associated with a WADA accredited laboratory, because the employees are independent, trained and used to all steps of the legal procedure engaged in case of doping offences and they must report to the recognised ADO as well as WADA. For the time being, APMUs can either be integrated to WADA accredited laboratories, national anti-doping organisations (NADOs) or IFs.

Moreover, a mathematical model has been designed to identify non-subjectively, unusual longitudinal results of the athlete. This is the adaptive model which calculates the probability of a longitudinal profile of marker values assuming that the athlete has a normal physiological condition.

The adaptive model has been introduced into the ABP software, which has been produced by the Lausanne Laboratory scientists.22 ,36

The APMU will liaise with a panel of experts (as agreed with the ADO) in order to interpret in an independent manner the results of the adaptive model in cases of significant abnormality of the profile. These experts should be knowledgeable in one or more of the fields of clinical haematology, sports medicine or exercise physiology. The ideal (and recommended) administrative sequence of the ABP is the following (adapted from WADA guidelines):

  • Identification of the athlete by the ADO:

    • Identification of the suitable time for collection based on recommendation of the APMU.

    • Sample collection request delivered by the ADO to a sample collection agency or to appropriate doping control personnel.

    • The sample collection authority accesses the whereabouts information of the athlete (localisation).

  • The blood collection officer locates the athlete and collects the biological sample:

    • The sample collection personnel are responsible for the transport of the biological sample to the WADA accredited laboratory.

    • The sample collection personnel are responsible to transcribe the doping control form immediately after the collection into web-based database ADAMS (Anti-Doping Administration and Management System) to provide direct access to the relevant data for the laboratory, APMU and the ADO.

  • The WADA accredited laboratory analyses the sample and reports to ADAMS:

    • Notification is carried out to APMU which updates the ABP and applies the adaptive model using the ABP software.

    • The APMU reviews the updated passport including the results in the adaptive model, and advises the ADO on intelligent testing strategies.

    • When the markers (HGB, and/or OFF-hr Score (OFFs) are beyond the 99th centile of the expected ranges returned by the adaptive model, the APMU shall proceed with the steps of evaluation liaising with the expert panel.

    • The APMU will also regularly provide profiles that do not exceed the 99th centile, in order to provide experts with a more balanced view of the considered athlete population.

Haematological markers and athlete's information for the ABP

The haematological module gathers information on the potential markers for blood doping (eg, rh-EPO doping19 blood transfusion52–56 and gene manipulation).57

In the haematological module, the following markers are considered:

  • HCT

  • HGB

  • RBC: RBC count

  • RET%: the percentage of RETs

  • RET#: RETs count

  • MCV: mean corpuscular volume

  • MCH: mean corpuscular haemoglobin

  • MCHC: mean corpuscular haemoglobin concentration

Some of these markers can be combined to be incorporated into the module such as the OFFs, as described by the Australian group in their presentation of the second generation blood test to detect EPO abuse by athletes.29

The OFFs is calculated by the following formula:

OFFs = HGB−60(RET%)

  • HGB: HGB concentration in g/L

  • RET%: RETs in percentage

Another combination, ABPS, introduced by Sottas et al32 in 2006, is a combination of up to 12 different blood markers such as HCT, HGB, RBC, RET%, MCV, MCHC, serum EPO, serum sTFR, RBCo (optical RBC), RDW (red cell distribution width), RET# (absolute RET count), IRF (immature RET fraction).

The passport will also take into account the individual athlete profile information to provide as precisely as possible the context for a better interpretation of the markers. These additional informations include, but are not restricted to gender, type of sport, whereabouts information for the month before, competition schedule58 use of hypoxic devices or altitude training.59–61

Technical documents associated to the haematological module

To allow a correct implementation of a profile which can resist to any scientific or legal criticism62 ,63 it is mandatory to follow rigorously some steps in the chain of production of the results and in the management of the interpretation of the passport. Four technical documents have been then associated to the guidelines for the haematological module which correspond to the requirements for the passport operation. These documents are linked to the International Standards for Testing51 and International Standards for Laboratories64 but have been put in place exclusively for the implementation of the passport. The four WADA technical documents are shown in table 1.

Table 1

Technical documents associated to the haematological module

The preanalytical procedures, which are associated to the two first documents, are certainly among the most sensitive and critical paths in the overall process.65 With blood testing and the introduction of the haematological module, living material for the first time has been introduced in the fight against doping. This is a major change in comparison to the collection and transport of urine, which is still the most common biological sample used in that field. Many authors studied all these technical and biological aspects and all proposed necessary improvements in the procedures.37 ,38 ,41 ,65

Regarding the blood collection, beside the technical aspects66 ,67 of the blood draw (which must be performed after allowing a timeout period in a sitting position),1 the timing of the sample collection, the exercise68–70 and the exposure of the athlete to altitude (real or simulated)28 ,71 are regarded as very important for the stability of haematological markers. Then, the main points to be confirmed are the following:

  • No training or competition before the last 2 h of the blood test.

  • Did the athlete train, compete or reside at an altitude greater than 1000 m within the previous 2 weeks?

  • Did the athlete use any form of altitude simulation (hypoxic tent, mask, etc) during the previous 2 weeks?

  • Did the athlete receive or donate for any reason blood transfusion the previous 3 months?

For transportation of the sample, the rules are relatively obvious. It must be safe in order to assure the integrity of the sample and assure the chain of custody. It must be rapid, because the blood in the laboratory must be analysed within 36–48 h of sample collection.

It must be cooled (2–12°C) in order to preserve the blood cells in the sample and keep them intact.

The analytical procedure

Many aspects of blood analyses have been debated since the beginning of blood tests in sports. The need for standardisation appeared obviously due to different techniques of flow cytometry applied in cell counting and identification of categories of erythrocytes.10 ,72

As this is a quantitative measurement, it is clear that harmonised reference material must be available.73 The quality control system should be applied in a similar manner in the network of laboratories in order to decrease the variability in these longitudinal measurements.74 Still, some clinical haematologists discuss or are critical to the system in place nowadays.41 ,75 ,76

One of the best solution to standardise the analytical results for all samples included in a passport is to analyse blood samples in an appropriate dedicated network (WADA accredited laboratories) using analysers with comparable technical characteristics—if not identical. The preanalytical procedure including the calibration of the analyser and the chain of custody of the samples in the laboratory must be standardised and well documented.

Each blood sample shall be analysed twice after proper homogenisation. This is not usual in clinical haematology laboratory, but was decided in order to ensure legal validity of the result. This is why the preferable institutions to perform that type of work are the WADA accredited antidoping laboratories. They are accredited for the analyses of biological samples and also used to the legal aspects of the procedure.

Each pair of result for the sample must be examined and must correspond to certain criteria.

Absolute differences between the results of the two analyses shall be equal or less than the following for the analyses to be accepted:

  • 0.1 g/dL for HGB analysis.

  • 0.15 absolute difference for RET% analysis (if both measurements are lower or equal to 1%).

  • 0.25 absolute difference for RET% analysis (if both measurements are higher than 1%).

The data from the second analysis are used to confirm the first analysis data and only the results of the first analysis are reported. The laboratory is requested to report immediately the result into the international ADAMS web database.

Result management: the role of the APMU

The APMU is a key element to make the system work properly and efficiently.49 This unit works on behalf of the ADO. APMU has the responsibility to manage the biological data and such information shall be stored in ADAMS and/or the ABP software which implement the blood data into the adaptive model. The APMU will review all profiles in order to provide targeting recommendations to the ADO, when suitable or to refer to the expert panel if appropriate.

After verifying that the results included in the adaptive model are valid (ie, when all the technical documents have been properly applied for all data), the APMU will evaluate the profiles provided by the adaptive model. The latter predicts for an individual an expected range within which marker values fall assuming a normal physiological condition.

A sample or a longitudinal profile will be considered as atypical if it returns a HGB and/or OFFs value outside the expected individual range as defined by the adaptive model.

The expert review

In case of an atypical result or profile, the APMU is responsible to contact one expert from its expert panel to review the passport properly and suggest further action. Several situations can appear and necessitate actions from the APMU (see table 2).

Table 2

The expert review

Once a pathology has been excluded77 ,78 and if the profile is providing a high suspicion of blood manipulation, the review of the group will follow the same procedure as for the first step of evaluation. The expert panel can request any additional information which is needed for their expertise, including the competition programme and/or training schedule of the particular athlete. The APMU will be the link between the ADO and the experts, in order to keep the entire procedure anonymous and confidential.

A unanimous opinion among the three experts is necessary. If the three experts determine whether abnormal or suspicious passport profiles are the result of a medical condition or doping, then the APMU is responsible to compile all the documentation supporting the so-called ‘Adverse Passport Finding’ (APF).

APMU will thus constitute the ABP documentation package. This shall contain the following information:

  • The age, gender, sport and discipline of the athlete;

  • The biological data and the results obtained by the adaptive model;

  • Information on possible exposure to altitude (whereabouts);

  • The competition information;

  • The documentation of the entire chain of custody (including temperature conditions and transport) of each sample making the passport;

  • The laboratory documentation for each blood sample, including the scattergrams, the chain of custody and the internal/external quality controls;

  • All information collected on the doping control forms from each sample;

  • Any other additional information provided by the ADO;

  • The reports of experts’ opinions.

The APF will be then reported by the APMU to the ADO and to WADA in a similar manner as the antidoping laboratories report an Adverse Analytical Finding after detecting the use of a forbidden substance or a forbidden method.

Then the ADO result management will be in charge to review the case and decide if the APF constitutes an anti-doping rule violation as it is defined in the World Anti-Doping Code.

The steroid module

The steroidal markers

In the 1980s, based on the work of Donike and colleagues, an upper limit of 6.0 for the testosterone/epistestosterone (T/E) ratio was introduced to deter testosterone administration. After exogenous testosterone administration, the clear effect of the latter is an increase in the T/E ratio.79 The T/E has been the first widely used indirect marker of doping with anabolic steroids, with a discrimination principle not based on the distinction between the exogenous substance and its endogenous counterpart, but rather on the effect induced by the intake of the exogenous substance on some selected biological markers. Since then, the T/E ratio has been used as a screening test, with any positive result requiring a subsequent confirmation analysis by GC/C/IRMS. GC/C/IRMS allows measurement of slight differences in 13C/12C ratio of testosterone metabolites. Discrimination between pharmaceutical and natural testosterone is possible because synthetic testosterone is known to display different 13C content than its human counterpart produced by means of cholesterol metabolism.80 ,81

Longitudinal steroid profiling

It was known in the 1990s that participant-based reference ranges are more reliable than population-based reference ranges for androgens2 and that individual T/E values do not deviate from the mean value by more than 30%. It has only been recently that a method was proposed to take into account formally these characteristics.82 Based on empirical Bayesian inferential techniques for longitudinal profiling completely similar to what is used for the haematological markers, the test progressively switches the focus from comparison with a population to the determination of individual values. Interestingly, this test is neither a purely population-based nor a purely subject-based approach, but an intermediate approach that makes the best decision in function of the between-subject and within-subject variance components of the marker and actual individual test results. Using the athlete as his own reference is particularly interesting when the marker presents a low ratio of within-subject to between-subject variations. In a population composed of male Caucasian athletes, this ratio has been estimated to be as low as 0.04 for the T/E.83 Such a low ratio already questions the pertinence of a population-based threshold for the T/E ratio (fixed at 4.0 for a long time by WADA, see TD2004EEAS84).

While the terminology ‘steroid profiling’ is used in the literature to denote a follow-up over time, a steroid profile includes concentration levels of endogenous steroids in urine and their respective ratios. Steroid profiles are employed widely in endocrinology to detect enzyme deficiencies or adrenal problems.85 In antidoping laboratories, the urinary steroid profile usually includes the concentration levels of testosterone, the testosterone's inactive epimer, epistestosterone and four testosterone metabolites, androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (α-diol) and 5β-androstane-3α,17β-diol (β-diol).

Ratios such as T/E, A/Etio, A/T, α-diol/E and α-diol/β-diol are robust and do not change due to circadian rhythm or physiological conditions such as exercise workload for athletes.86 On the other hand, these markers may be altered significantly according to the administered steroid and its application mode.

Heterogeneous factors

Heterogeneous factors refer to the factors specific to an individual that are known to have an influence on a biomarker. For example, sex and age are well-known heterogeneous factors used in the evaluation of a steroid profile. It has long been known for a long time that urinary testosterone glucuronides present a bimodal distribution, this effect being particularly marked between Caucasian and Asian populations.87 It only was recently, however, that it was demonstrated that the significant differences observed in testosterone glucuronide excretion are associated with a deletion mutation in the UDP-glucuronide transferase 2B17 (UGT2B17) gene.88 This discovery has important implications for doping tests. For example, when participants deficient in the UGT2B17 gene (del/del) receive exogenous testosterone, it has been shown that their T/E ratio does not rise significantly, remaining well below current threshold at 4.0.82 This suggests that the knowledge of genetic differences in metabolism and excretion is important in the evaluation of urinary steroid profiles. These studies confirm, again, that unique and non-specific thresholds on markers of steroid doping do not fit for indicating anabolic-androgenic steroids misuse.89

Recently, Van Renterghem et al90 ,91 as well as Boccard et al92 made proposals of new markers for the detection of testosterone in sports using extensive steroid profiling and by using an adaptive model based on Bayesian inference. Apart from T/E ratio, four other steroid ratios (6α-OH-androstenedione/16α-OH-dehydroepiandrostenedione (DHEA), 4-OH-androstenedione/16α-OH-androstenedione, 7α-OH-testosterone/7β-OH-DHEA and dihydrotestosterone (DHT)/5β-androstane-3α,17β-diol) were identified as sensitive urinary markers for testosterone misuse. These selected markers were found suitable for individual referencing within the concept of the ABP. The markers showed improved detection time and discriminative power compared to solely T/E ratio. These markers were supporting the evidence of doping with small oral doses of testosterone. Another subject-based steroid profiling was shown to determine misuse of precursors of testosterone like DHEA or DHT.91

Thus, there is a great potential in using a similar approach as developed for the haematological module to implement the steroid module of the ABP. Like in the first module, there is still need for harmonisation for its reliability. This is not so much in the preanalytical conditions, but rather in the analytical production of results that the laboratories shall pursue to decrease the interlaboratory variability.

Perspectives

The growth hormone module

The haematological module has been in place in some federation since 2008; the steroid module is in place since the beginning of 2014 with the experience accumulated for many years in the analysis of steroid profile in urine in antidoping laboratories. The third part dedicated to the hGH line and called ‘endocrine module’ is certainly the one which still needs to be structured and validated with some emphasis. There is evidence that growth hormone is used by athletes for its anabolic and lipolytic properties. It is often abused in combination with anabolic steroids and insulin, even with insulin-like growth factor-I (IGF-I). The GH-2000 project developed a methodology to detect its abuse using the concentrations of two GH-dependent markers, IGF-I and type 3 procollagen (P-III-nP).93–95 Even if this approach is claimed to be used for a single serum sample collected by the ADO, there is no doubt that a longitudinal sequence of those markers will be more selective to prove a manipulation with human growth hormone.

Nowadays, the only official method which has been officially introduced in the hGH doping detection with some success is the one based on the so-called ‘isoform approach’. It is known that the pituitary gland secretes a variable spectrum of hGH isoforms, whereas recombinant growth hormone (rh-hGH) is the monomeric 22 000 Da isoform only. This isoform becomes predominant after injection of rh-hGH. The isoform test is built on specific immunoassays with preference for one or the other isoform allowing the analysis of the relative abundance of the 22 000 Da isoform. Application of rh-hGH can be proven when the ratio of this isoform relative to the others is increased above a certain threshold. The different detection windows of the so called ‘marker method’ and the ‘isoform method’ makes them complementary and could increase the overall detection window of hGH abuse.96 It is known that both approaches must overcome the interindividual and intraindividual variability, but, as for other hormones, the longitudinal study of markers influenced by the application of rh-hGH can be the right answer to the question of the biological variability. This will then complete the ABP with its endocrine module.

Future of the ABP

If the theoretical aspects of the biological passport have been established and are applied already with the haematological and steroid modules by some IFs and national anti-doping agencies, there are still many factors to be solved in order to implement it in an efficient way for the benefits of the fight against doping. The first implementation into several international sports federations or NADO shows clearly that ‘one size fit all’ cannot be applied here. Depending on the type of sport (individual or team sport) and how it is organised throughout the year, the approach may change. Some sports have many competitions spread over a very long season; some others concentrate on major events for which the athlete prepares the rest of the season.

Some sports are practiced at the international level over the entire planet like soccer or track and field. Some others like cycling are concentrated in a relatively limited geographical area and small population of interest. For these reasons, the strategy in the organisation of this biological profiling will change drastically from a federation to another, mainly due to logistic and financial constraints. The main objective of the implementation of the passport has been created by WADA as a tool to define an antidoping rule violation, but the experience of the last seasons shows that the ABP is also a very good tool for prevention and deterrence. As the number of out of competition tests has generally increased, the athlete will react automatically by being more cautious in his behaviour.

This preventive and deterrent approach could be even improved if an abnormal biological profile prior to an event, can lead to non-participation to a race event. This would certainly bring a lot to the fairness of the competition.

ABP is still in its infancy and thanks to recent development and advances in circulating microRNA analyses,97 ,98 proteomics and especially metabolomics99; the specific fingerprints left by doping could also be included in an individual follow-up and be part of the biological passport.

Role of the experts

  • The role of the expert's panel is to provide scientific evidence for the disciplinary panel to base their verdict on. It is up to the disciplinary panel to decide whether an athlete doped (or not?) and not the scientific experts to give an opinion on guilt.

Current status of the athlete biological passport

  • The steroidal module to fight anabolic steroid abuse is the second module, after the blood module to fight erythropoiesis-stimulating agents and blood transfusions, to be implemented in the athlete biological passport (ABP).

  • Urine and blood data can be implemented in the ABP if the collection, transport, analyses follow rigorous protocols.

  • The evaluation of data included in the ABP by the panel of experts must take into account additional information such as heterogeneous factors which could influence the behaviour of biological variables.

  • The indirect approach of the biological passport has been fully accepted by the Court of Arbitration for Sport (CAS) during previous hearings.

References

Footnotes

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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