Projets de recherche

Code: 09C10PD

In the last years the WADA has founded studies of our groups aiming to develop in vitro test systems for the structure independent identification of anabolic substances. Our test system is a stable transfected yeast transactivation system for the identification of substances with affinity to the androgen receptor. Using this test system we could identify and characterise several designer steroids. In the last funding period we further characterized SC and started with the construction of a new reporter gene system in Schizosaccharomyces pombe. SC was able to detect anabolic steroids and their metabolites with a high specificity and sensitivity in urine of abusers (Zierau et al. 2008). Even selective androgen receptor modulators (SARMs) could be detected with SC. In excretion studies with Methyltestosterone in close cooperation with the doping control lab cologne SC was able to detect 1-Testosterone abuse up to 307 hours (GCMS detection limit was 118 hours). Treatment of the urine (concentration, purification) further increases the sensitivity of SC. Using new reporter gene plasmids we could reduce the duration time of the test from 2 days down to 18 h. In addition SP was successfully generated and is now ready to be further characterized. Reaching these milestones, our future aim is to use the SC to supplement GCMS techniques in routine doping analytics. Therefore we want to develop a standard routine procedure protocol to use the system in routine analysis. We also want to further enhance the sensitivity of the system by validation the newly generated SP system. In addition our SC system will be used to identify new long-term metabolites of anabolic steroids. So SC will in addition further improve the sensitivity of the GCMS detection systems.

Main findings

The classical methodology to detect anabolic steroids or other anabolic substances in anti-doping analytics is MS. However, in particular the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances are sometimes not detected during routine screening by MS, if their exact chemical structure is unknown. Moreover a great number of substances have been developed since the fifties and nowadays many pharmaceutical companies are working on non-steroidal androgen receptor modulators (SARMs) which have a completely different chemical structure and metabolism than classical anabolic steroids. In 2005 and 2006 WADA has funded pilot studies of our groups aiming to develop in vitro test systems for the structure independent identification of anabolic substances.

In these funding periods we have successfully established and validated the use of yeast reporter gene systems (SC) for the detection of substances with the ability to bind to the androgen receptor in urine. Besides that we could demonstrate that SC is able to detect anabolic steroids and SARMS in urine. Samples from athletes abusing anabolic steroids were successfully uncovered using SC. The primary aim of our ongoing (LIVE) project was to use the SC system to supplement MS techniques in routine anti-doping analytics. The system is easy to perform, without high tech equipment, cheap and the results are conclusive. Therefore it is most suitable to be used as a pre-screening system to identify the misuseof anabolic steroids, independent of the chemical structure, especially in training controls. Similar systems are successfully in use to identify anabolic misuse in other fields as for example application for enhancement of meat production in livestock. For this we aimed especially to finish the construction of SP and to validate SP in comparison to SC. The “new” SC and SP yeast construction had been finalized and the characterized, resulting in a backup or complementary system for the already established SC system by the SP. Overall the “new” systems are a bit less sensitive and little higher operating expense make them less efficient compared to the “old” SC system.

The next aim was the use and validation of the SC system in further excretion studies with problematic steroids in comparison to MS. A number of different substances have been analysed and in parts results of this of the project part have already been published. Summarizing this, the obtained results are encouraging, but seem to depend on the substance and their specific metabolism. This information is very helpful to further characterise the advantages but also limitations of SC.

Another aim of this founding period was to validate whether the SC system is capable to detect SARM abusing, which was carried out as an animal experiment. Animal experiments with SARMs were performed and post administration urines analysed with the SC. The results indicate that SC is also able to detect such substances in the urines of excretion studies.

A very important aim was the demonstration of the capability of the SC to identify longterm metabolites. This highlight of our so far conducted experiments was the general test whether the SC is a suitable bio detector to identify stable metabolites in the urine for further structure analysis. The presented results demonstrate that that we succeed in this proof of the principle. We are optimistic that we will be able to succeed in identifying a number of long-term metabolites structures in the future using a larger preparative experimental scale.

Last but not least our aim was to develop a standard operation procedure (SOP) to use SC system in routine anti-doping analysis. In our focused parallel analysis in Cologne and Dresden we have been able to acquire a SOP working in both laboratories with the identical efficiency. The simultaneously analyzed blinded urine samples impressively demonstrated that the results do not vary in quality even if performed in different laboratories. These results confirm the stability as well as the reliability of the SC assay and demonstrate its value as a screening assay in routine anti-doping analysis.

Code: 09D9FB

The project was developed using the observation that “Liposom Forte®”, a pharmaceutical-grade product containing “empty” liposomes, was reportedly used by athletes. Liposom Forte® was found stored together with banned and non-banned drugs during investigations carried out by Italian legal authorities.

Due to the negligible direct effect on the enhancement of performance of “Liposom Forte®” and similar pharmaceutical products containing empty liposomes, we postulate these products could be used as masking agents to make detection of other forbidden drugs more difficult. Liposomes can be used as masking agents by following one or more of the following hypothesized strategies: i) Injection immediately after being mixed with steroids to produce “home-made” slow/sustained release preparations;

ii) Injection as such (“empty”), before an “expected” anti-doping test to promote interaction with steroids/metabolites circulating in the organism and altering the excretion profile;

iii) Direct addition to the sample collected for anti-doping tests to reduce the concentration of “free” (i.e. not bound to liposomes) steroids/metabolites and reducing the efficacy of the laboratory analytical procedures used for detection. We have preliminarily shown that an interaction between liposomes and androgenic anabolic steroids can cause a reduced efficacy of the analytical procedures (normally based on GC/MS analysis of the corresponding TMS-derivatives after enzymatic hydrolysis) followed by anti-doping laboratories. We have also preliminarily demonstrated that direct addition of liposomes to urine samples containing steroids causes a masking effect: the amount of steroid detected in the samples was significantly reduced after liposome addition.

We plan to continue the present research considering the following aspects: i) More thorough evaluation of the binding ability and masking potential of liposomes in vitro.

ii) Assessment of the masking potential of liposomes in vivo.

iii) Characterize the physico-chemical properties of liposomes and improve analytical methods used to detect the presence of liposomes in biological samples

Main Findings

One of the hottest topics in antidoping research is the study of the so called masking agents, i. e. substances or methods capable of “hiding” other forbidden substances, thus reducing the efficacy of the experimental strategies used to detect the abuse of doping agents by the analysis of biological fluids. The class of masking agents was originally limited to substances with diuretic effects, but it has been progressively expanding, now comprising also agents that can interfere with either the pharmacokinetics of other banned substances and/or with the analytical procedures normally applied by the WADA-accredited anti-doping laboratories for the detection and, where required, the quantitative determination of the concentration, in biological fluids, of other banned substances.

To the best of our knowledge, this research project is the first one to specifically consider the possible relevance, as masking agents in sport doping, of liposomes, a class of supramolecular structures constituted by phospholipids extensively studied in the pharmaceutical field for their properties as drug delivery systems. More specifically, we have focused our attention on the potential masking effects of liposomes on anabolic androgenic steroid (AAS), to date the most widely abused class of prohibited substances in sports doping.

The results we have obtained can be summarized as follows: I. Liposomes have been shown to interact with anabolic androgenic steroids, leading to a reduced analytical recovery of both the parent compound and the glucuronide metabolites.

II. The interaction occurs in a relatively wide range of experimental conditions, and it has been verified for several representative pseudo-endogenous anabolic androgenic steroids and for various liposomes, differing in charge, size and chemical composition.

III. The effect is particularly noteworthy whenever a quantitative determination of the target steroid(s) is required, as it is the case of threshold substances (i.e. 19-norandrosterone) and/or of the evaluation of the urinary steroid profile in the framework of longitudinal testing and/or of the Athlete Biological Passport.

IV. Specific countermeasures to ideally annul the observed “masking effect” include the strict monitoring and control of all quality parameters of the analytical procedure(s) followed for the analysis of AAS, with special emphasis, in the case of GC-MS based methods, on the efficacy of the derivatization step.

V. A novel analytical procedure has been designed, developed and validated, to detect and quantitate a wide variety of liposome constituents (phospholipids and sphingomyelins) in biological matrices (blood and/or urine) and in pharmaceutical products, with the aim to identify suitable markers for the detection of liposome intake by the analysis of blood and/or urine samples.

The results of the present project unearthed a new and effective class of masking agents, that go beyond those with a direct pharmacodynamic effect, that act as true and effective “doping delivery systems”, altering the pharmacokinetic properties (i.e. transport, distribution and elimination) of doping drugs.

Code: R09C1MA

Our previous research has found compelling evidence that gene expression profiles are significantly altered one week after receiving an autologous blood transfusion. These data were generated using a microarray that screened more than 54,000 different genes. The aim of this project is to identify, from amongst the hundreds of genes that were differentially expressed, the most reliable set of candidate genes for use as a diagnostic to identify autologous transfusion by athletes.

Main Findings

Following on from our initial results which showed compelling evidence that blood transfusion produced large changes in blood gene expression profile, we have demonstrated that these results can be replicated in a separate subject cohort, and using different microarray chips. After reinfusion of three bags of blood, gene expression differences were most evident 7 days post-reinfusion, remained strong at 14 days and persisted through to 28 days. There was an indication that a change does occur following transfusion with one bag of blood, but of much smaller magnitude. In order for gene expression to be useable diagnostic in the context of antidoping:

1. Additional work is required to determine whether other genes show detectable signals in response to lower quantities of blood.

2. Reference genes expressed at a uniform level in different individuals under different circumstances must be identified to enable measurement of candidate genes relative to a standard.

3. Variability of the candidate and reference genes must be established in an elite athlete population under a typical training/environmental scenarios.

Code: 09E21SW

In recent years scientific research has shown that medications such as sildenafil (Viagra) have the ability to enhance athletic performance at very high altitudes. This has prompted members of the anti-doping community to take an increasing interest in this class of medications, even though research has not shown that they improve athletic performance at sea level or moderate altitudes. This class of medications is of particular importance to athletes with spinal cord injury. Many athletes with spinal cord injury use these medications to treat erectile dysfunction of neurologic origin. Therefore, it is important to understand what effects, if any, this class of medications has on athletic performance in athletes with spinal cord injury. We will attempt to answer this question with a prospective, randomized, controlled study of athletic performance in athletes with spinal cord injury on sildenafil versus placebo at both low and moderate altitudes.

Main Findings

Whereas the ingestions of Sildenafil Citrate tends to improve some cardiovascular and respiratory parameters such as systemic pulmonary arterial pressure and percentage of arterial oxygen saturation mainly under hypoxic conditions in able-bodied subjects, there seems to be no indication that this is the case in athletes with a spinal cord injury. In contrast, there seems to be a negative impact on exercise performance, oxygen saturation, heart rate and lactate concentrations at moderate altitude in this population. Further, no ergogenic effect of a Sildenafil Citrate ingestion was found concerning exercise capacity at sea level and moderate altitude compared to placebo. Thus, it can be concluded from our study results that the ingestion of sildenafil does not enhance exercise capacity in athletes with a spinal cord injury and rather seems to have a negative impact on performance in this population when competing at altitudes up to 2200m.

Code: 09B2MT

The misuse of the peptide hormone LH-RH (Gonadorelin, GnRH, Kryptocur) in elite sports has frequently been reported during the last years, especially in the course of legal statements and confessions of athletes. LH-RH can be administered by means of infusion or facile intranasal application, which influence the endogenous production of the luteinizing hormone (LH) and, via the gonadal axis, an increased release of androgens into circulation is induced.

The present project shall investigate all effects of LH-RH applications by healthy individuals on LH and testosterone in plasma, as well as the steroid profile, LH and LH-RH in urine. The measured parameters shall outline the influence of LH-RH administrations on regularly determined analytes of routine doping controls such as LH and the steroid profile, and further provide information on detection windows for LH-RH in specific doping control procedures.

Main Findings

Luteinizing hormone-releasing hormone (LH-RH) is a natural hypothalamic peptide hormone responsible for the stimulation of luteinizing hormone (LH) release from the pituitary. LH in turn stimulates the testosterone production and release from the gonads, regulating plasma testosterone concentrations. Since LH-RH has been available as therapeutic agent in different formulations (i.e. for intranasal and intravenous application), its abuse in sport cannot be excluded and confessing athletes have indicated the misuse of LH-RH during their career. In order to obtain detailed information on the effects and thus measurable parameters to uncover LH-RH abuse, administration studies with intranasal, intravenous, and combined application protocols were conducted with 10 male volunteers, plasma and urine samples were collected and parameters including plasma testosterone, LH, and FSH, as well as urinary LH, LH-RH, and steroid profiles were determined. Established assays (immunological as well as chromatographic-mass spectrometric approaches) as well as new liquid chromatography-high resolution/high accuracy mass spectrometry methods developed in the course of the project were used. LH-RH in bolus and intermittent drug regimen resulted in significant increases of plasma testosterone and LH concentrations. In urine, steroid profiles demonstrated an impact of the LH-RH administrations; however, the effects were not as pronounced as desirable and characterized mainly by increasing androsterone/testosterone and androsterone/epitestosterone ratios towards the end of the study due to suppression effects on testosterone and epitestosterone. The commonly employed testosterone/epitestosterone ratio was found to be insensitive to an LH-RH intervention, even at high therapeutic dosing. Moreover, urinary LH was not substantially affected. In an intra-individual picture, the increase of urinary LH concentrations following an LH-RH application could be correlated; however, LH levels remained within normal reference ranges. Since the steroid profile and urinary LH concentrations did not provide sufficient information allowing to pick up LH-RH misuse, the option to directly detect the peptide hormone in urine was pursued. By means of solid-phase extraction followed by LC-MS/MS, LH-RH was detected in urine specimens after both intranasal and intravenous drug administrations. The detection window was found to be 12-24 h employing state-of-the-art analytical instrumentation available in most doping control laboratories.

Code: 09E4MK

Beta2-agonists are a type of medication that is commonly used in the treatment of asthma. They can have other actions other than treating asthma that may have the potential to improve exercise performance.

In the past 25 years, there has been a trend for an increase in applications for permission to use 2-agonists from athletes competing in Olympic Games. In fact athletes that use these agents win a disproportionately high number of medals. Previous research has looked at unselected groups, and found no doping benefit from these agents. Recent research has shown that there is a large variety in the genes that affect how individuals respond to these 2-agonists. We will look at variations in the genetic response to these medications. Specifically we will divide athletes into those with a genetically high response to these drugs and those with a lower response. We will then compare their exercise performance following the administration of a 2-agonist. We hypothesize that a subgroup of athletes with certain genetic variations will benefit from 2-agonists while the rest will not. If some athletes are achieving enhanced performance from asthma medication, then the rules surrounding their use in sport will need to be reviewed.

Main Findings

The aims of this project were (1) to determine if the A46G single-nucleotide polymorphism (SNP) and the C79G SNP of the adrenergic β2-receptor gene (ADRB2) and the A663T SNP of the sodium channel gene (SCNN1A) affect time-trial cycling performance after the inhalation of salbutamol in male cyclists with and without exercise-induced bronchoconstriction (EIB); (2) to assess if women experience a greater increase in lung function following the inhalation of β2-agonists compared to men and therefore increase their 10-km cycling time-trial performance; and lastly, (3) to investigate if there is an ergogenic effect to the inhalation of 1600 μg of salbutamol, a supratherapeutic dose, in male cyclists with and without EIB.

In total, 130 competitive female and male athletes, aged between 19 and 45 years were screened (103 males, 27 females). Athletes performed two simulated 10-km time-trials on a cycle ergometer following inhalation of either 400 µg (studies I and II) or 1600 µg (study III) of salbutamol or placebo. Change in forced expiratory volume in 1 second (FEV1) was assessed immediately before and following inhalation. Performance was measured by mean power output over the time-trial duration.

Percent change in FEV1 following the inhalation of salbutamol was significantly increased compared to placebo (p < 0.001) in all three studies, regardless of athletes’ susceptibility to EIB. Despite this increase in lung function following salbutamol use, time-trial performance was not improved. Genetic variation at the ADRB2 and SCNN1A genes did not affect the broncho-dilatory response and time-trial performance to inhaled salbutamol in male and female athletes with and without EIB. Furthermore, there was no difference in the percent change in FEV1 following the inhalation of 400 µg of salbutamol between male and female cyclists. However, there was a decrease in mean power output during the salbutamol time-trial of 3 Watts compared to the placebo time-trial in female athletes, but not in male athletes. This could have been caused by an increased salbutamol dosage-to-weight ratio in women compared to men. Lastly, a supra-therapeutic dose of salbutamol did not affect 10-km time-trial performance in male cyclists, but lead to significant increases in heart rate and minute ventilation, common side-effects of IBAs, in athletes without exercise-induced bronchoconstriction.

In conclusion, despite a significant improvement in lung function following the inhalation of salbutamol, 10-km time trial performance was not improved, regardless of asthma status, genetic variation at the ADRB2 and SCNN1A genes, sex and salbutamol dose.

Code: 09A01WS

The abuse of synthetic hormones, which also occur naturally in the human body is difficult to detect. These compounds are chemically identical. However, the carbon, of which these compounds are made, features two stable isotopes, atoms that exhibit slightly differing masses. The ratios of the stable carbon isotopes 13C and 12C are different in synthetic and natural hormones.

The most frequently abused hormone is testosterone (T), the principal male sex hormone. In fact, not the testosterone itself is analyzed for 13C and 12C, but metabolites that are found in the human urine. There are several of them and they have largely differing concentrations. The most abundant metabolites are androsterone (A) and etiocholanolone (E). Due to their large concentrations they can be analyzed with relative ease. But E and A also have sources other than T. Therefore A and E might exhibit only slightly altered isotope ratios following abuse of T.

Two other compounds, so-called androstanediols, AD and BD, are made virtually exclusively from T. It is also possible, if difficult, to analyze T itself. Interestingly, T as well as AD and BD show much larger variation of the isotope ratios in control subjects than A and E. Therefore the message of corresponding analyses is often not quite clear, although the three compounds certainly yield the most sensitive parameters for T doping.

The project aims to elucidate the biological and analytical factors that cause the scatter of the isotope ratios of T, AD, and BD. We thus want to be able to provide unequivocal results in case of T doping. We also expect a much better sensitivity of the method, so that T doping can also be detected when small amounts have been applied or when the administration has been performed a while ago.

Main Findings

Analysis of the ratio of the two stable carbon isotopes 13C and 12C currently represents the methods choice to detect the illicit administration of synthetic steroids. Synthetic testosterone plays an outstanding role amongst these compounds. Typically, it betrays its origin in that it exhibits significantly reduced 13C/12C ratios as compared to endogenous steroid hormones.

The 13C/12C analytical procedure, however, needs not to be restricted to testosterone necessarily. By contrast, it will be even beneficial to extend the analysis to the main testosterone metabolites 5α-androstane-3α,17β-diol (5αAdiol) and 5β-androstane-3α,17β-diol (5βAdiol).

However, there is also natural variation of the relevant 13C/12C ratios. But few is known concerning the physiological factors that might take effect here. Moreover, the 13C/12C analysis of these steroids is challenging. Mostly because of their comparably low abundances in human urine. Therefore sufficient separation and purification is challenging.

Consequently, an efficient analytical procedure for 13C/12C analysis of 5αAdiol and 5βAdiol had to be developed first. This method now allows for long-term precisions of 0.2‰ to 0.3‰ on the so-called VPDB scale. In turn, this was supposed to facilitate valid investigations of physiological effects.

It turned out that increased urinary concentrations of the androstanediols 5αAdiol and 5βAdiol and of testosterone generally result in increased 13C/12C ratios.

As an integrated parameter, we chose energy availability (EA) as a proxy for physical workload. EA is defined as the amount of energy available for the maintenance of physiological functions taking into account the energy expenditure for exercise. In general, higher EA results in lower 3C/12C ratios of the investigated steroids, but in particular of 5αAdiol. The effect appears less pronounced in males, however.

No immediate diurnal effects could be observed. Similarly, no systematic effects were observed in respect to the female menstrual cycle. However, there seems to be a tendency towards alternating 13C/12C ratios within few days. The latter effect seems to be cleared by oral contraception. Within given limits and analytical precision the 13C/12C ratios of the investigated steroids then seem to reflect a pure random process.

While still varying randomly in time, specifically the 13C/12C ratios of 5αAdiol appeared lowered in females using oral contraceptives.

Code: T08M05MA

The activities presented in this report have been directed toward optimising the alignment of Sysmex blood analysers housed in WADA-accredited laboratories throughout the world. As is standard practice in laboratory haematology, between-laboratory comparability of those instruments currently relies upon participation in an external quality assurance program. Each month all laboratories are sent test samples whose results must be in close agreement with the remainder of the laboratories. However those samples must comprise stabilised blood cells in order to yield a useful shelf life, and those stabilised cells respond differently to the reagents and stains used in the analyser. Subsequently, Sysmex analysers use different modes of analysis depending whether samples are comprised of fresh or stabilised blood cells. As an alternate approach, we investigated whether alignment procedures could be modified so as to utilise fresh blood samples instead of stabilised materials.

Experimental work first focused on the stability of fresh blood samples during storage, with the intention of establishing the maximum delay that could be tolerated between sample collection and analysis. This had the dual benefit of not only informing our subsequent experiments, but also providing empirical support for WADA’s interest in extending the Athlete Biological Passport’s

(ABP) current sample collection window (i.e., 36 hrs maximum between collection and analysis). We found that the two key blood parameters utilised in the ABP, namely haemoglobin concentration and reticulocyte percentage, remained stable for up to five days post-collection provided that the sample is stored at approximately 6-8 oC. Moreover the red blood cells were found to swell in a predictable manner, enabling us to develop a nomogram that permits users to reverse extrapolate back to a stored sample’s initial characteristics provided that interim temperature and duration of storage are known.

The second phase of the study was instrument-based. We sought to align the reticulocyte counts of three analysers as closely as possible to the readings provided by a ‘reference’ instrument. We collaborated closely with a Sysmex technical representative, which permitted us to derive a deep appreciation of instrument nuances and subsequently to develop a robust alignment protocol. We found that our prototype re-calibration protocol, which was based upon tight calibration to target QC values, yielded excellent comparability between instruments. As a result, incorporation of our alignment protocol offers the possibility to optimise the comparability of Sysmex instruments located in WADA-accredited laboratories without the need to share fresh blood samples

Main findings

Our study has shown that a 0.31% bias in reticulocyte counts can exist between two XT-2000i instruments which are both operating within manufacturer’s specifications. We also demonstrated that recalibrating instruments toward the assigned value of control material, rather than lying within a tolerance range, brought reticulocyte counts into close alignment.

When contemplating possible explanations as to how two of our Test instruments demonstrated absolute biases of 0.24% and 0.31% when counting reticulocytes in fresh blood compared to our Comparative instrument, we conclude that the most likely origin stems from how the separate instruments were calibrated during installation. Standard installation procedures do not require the technician to recover QC reticulocyte values from the Sysmex XT-2000i (other than to verify that in terms of precision the reticulocyte percentage and absolute numbers have a CV < 15%). Instead, calibration materials are used to establish the other channels (e.g., the 16-parameter haemogram plus 5-part white blood cell differential), then the technician merely confirms using ten normal range fresh blood samples that the average reticulocyte value for those ten samples lie within the instrument’s reference interval (i.e., an XT’s typical reference range is approximately 0.64% – 1.65%). This would seem to provide relatively generous tolerances. For example, in the case of our first Test instrument reporting 0.31% low based on the average of ten fresh blood samples, that instrument would still yield a result that would fall within an acceptable range provided that the true average value of those ten samples lay between 0.95% and 1.96% (i.e., 0.64% + 0.31% and 1.65% + 0.31%, respectively). Under that hypothetical circumstance there would have been no basis for the installing technician to have refined the instrument’s set up during installation. Likewise, our data show that the manufacturer’s tolerance for RBC-X sensitivity adjustments mean that an instrument’s fresh blood reticulocyte counts can span a range of 0.47% without failing the manufacturer’s performance specifications. In other words, it seems tenable that tolerances specified by the manufacturer could enable a bias in the order of 0.3%-0.5% to exist in the reticulocyte percentage reported by two properly calibrated XT-2000i instruments.

We have shown that it is possible to remove bias between instruments down to at least one decimal place, in fact in our hands a Test instrument replicated the Comparative instrument’s reticulocyte counts down to two decimal places. We consider either to be zero bias in the context of the Athlete Biological Passport. Our original hypothesis was that because the XT-2000i uses different approaches depending whether stabilised or fresh samples are tested, alignment of reticulocyte counts would necessarily require the comparison of fresh blood results between instruments. However we found that excellent alignment could also be achieved merely by calibrating each instrument to the assigned value of control materials. This possibility has important implications for those WADA-accredited labs testing athlete samples, because an alignment protocol which utilised surrogate samples would avoid having to transport fresh blood to remote laboratories within the imited shelf life associated with this live tissue specimen. However, as proposed by the CLSI’s standard on validation, verification and quality assurance of haematology analysers, when possible fresh blood should be part of an overall QC program (9). A sensible compromise to enhance linkage between QC-derived data and reportable patient results might be to fortify a QC-based approach with localised fresh blood ring studies. For example, regional laboratories within close proximity may elect to optimise alignment by sharing fresh blood samples with their immediate neighbours. Without too much coordination one member could compare with a different regional cohort of laboratories and therefore propagate the confirmation beyond their localised region.

The benefit that improved between-laboratory comparability of reticulocyte counts brings to antidoping efforts is important but deceptively subtle. Currently, there is a two-step process followed before an athlete can be sanctioned via data derived from CBC results. The first step entails a statistical program which flags abnormal blood values that lie beyond the athlete’s individual reference range. These reference ranges are generated with a tolerance for both within- and between-subject components of variation, which far exceed the magnitude of between-laboratory variation. Decreasing these variance components by modest amounts has surprisingly little impact on the tolerance thresholds. Subsequently, because the between-laboratory error component is dwarfed by the within-subject component, reducing the variance has little impact on the statistical process.

However regardless of the statistics, an athlete is not considered to have committed an antidoping rule violation until and unless during the second step an expert review of the haematological data concludes that the most likely cause of the abnormal blood result was doping (as opposed to, for example, a pathology or analytical issue). This expert review shares a common lineage with how clinical haematologists evaluate serial change of reported results in a given patient, inasmuch as both groups are obliged to factor into their considerations an allowance for between-laboratory differences. A subjective allowance of 0.2 – 0.3% is typical of the buffer afforded in the athlete’s favour when blood is tested in different laboratories. Reducing the between-laboratory bias to within 0.1% or lower, as we have shown is possible to accomplish, would effectively mean that experts could interpret all results as if they had been collected on the same instrument. This would reduce the subjective tolerances made for potential between-laboratory bias, and thereby provide additional certainty to their opinions.

Code: T08B01MS

The main objectives of the project were: - Testing the sensitivity of the classical WADA positivity criteria when applied to Dynepo™-enriched samples

- Computing a new decisional tool able to discriminate between negative urine profiles, Dynepo™-enriched urine profiles and effort urine profiles.

- Determining the detection window of Dynepo™ following multiple injections on healthy subjects

Main findings

The main outcome of this project was to demonstrate factually that the current WADA criteria, as defined in the WADA2009TD, were not applicable to Dynepo™ detection in urine. Indeed, a formal application of these criteria on the IEF patterns resulting from 126 Dynepo™-enriched samples demonstrated a sensitivity of 9% on 3 weeks for multiple injections of a total of 22’500 IU of Dynepo™. The 3rd criterion, defining the acceptable ratio between the second most intense basic band and the most intense endogenous band, was mainly responsible for this poor sensitivity. We therefore proposed a linear multivariate model (PLS-DA) computed for discriminant analysis on the basis of 196 detectable urine patterns, half of them resulting from Dynepo™-enriched samples. Following cross-validation, this model, based on 3 latent variables (LV), yielded a score characteristic of each individual IEF pattern. This score indicated how representative a sample was of the positive or the negative class. Bootstrap resampling allowed the definition of a cut-off score and consequently, the identification of atypical samples. Applying this new criterion resulted in a sensitivity of 52% on the same 126 samples, without any loss of specificity. This model eventually evaluated Dynepo™ detection window as close to 48 hours, which is in par with the short half-life of the molecule in the organism, when compared to those of epoetins-α and -β

The main interest of this open model is that it is potentially refined each time a new data is computed. In addition, it could be easily generalized to other epoetins, notably alpha and beta. Pattern classification methods have been previously developed for classical epoetins, but the interpretation of Dynepo™ profiles has never been considered. Considering the fact that the current WADA criteria are manifestly not applicable to Dynepo™ detection, our model has returned a good sensitivity versus specificity ratio. It remains however very dependent on the analytical protocol, as any departure from the described procedure would require a specific validation. Altogether, this suggests that the use of the proposed model could be included as an additional piece of evidence in the procedure of EPO doping detection.

Code: T08A04MS

The aim for the second step is to obtain statistically significant comparison between different delays of transport. The most important point to use for this comparison will be the obtained ratios calculated for both GH detection kits. The aim of the third step is to compare ratios obtained from samples analysed as Asample, and samples analysed as B-sample after a frozen or cold storage of 10 days.

Main findings

• For samples separated immediately after clotting, rhGH detection kit appears to have a slightly better sensitivity when samples are stored frozen compared to cooled.

• For samples arriving after 4 days (96h) and centrifuged upon reception, no differences exist for ratio if samples are analyzed immediately or after 7 days if frozen storage is applied.

• For samples separated immediately after clotting, storage must be made frozen. Under cool conditions, ratios is lower after ten days compared to one day. If frozen, ratios do not present any significant difference when analyzed after one day or ten days.

• If delay between end of collection and centrifugation is below 24 hours, no significant differences of ratio is observed between delays.

• Longer delays (up to 3 days; 72h) leads to lower ratio, but difference is close to analytical variation predicted by rhGH kits supplier’s experts.

• Later, ratios get significantly lower and tends to continuously decrease with time.