Projets de recherche

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.

Code: 08B11CR

The detection of doping with recombinant peptide and protein hormones (e.g. erythropoietin – Epo, human growth hormone - hGH) is one of the most challenging analytical problems in doping control. The WADA-accredited method for the detection of doping with recombinant human erythropoietins (rhEpo) is based on isoelectric focusing (IEF). Our laboratory and the anti-doping control laboratory of Montreal developed – independently of each other – an SDS-PAGE method which serves as an additional confirmation tool for the worldwide practiced Epo-IEF method. The advantages and additional benefits of this method were presented at various scientific meetings in 2007 and 2008 (e.g. the capability to distinguish between Epo-biosimilars and effort urines, no interference by active urines). By applying this strategy we were able to clearly and multiply demonstrate the abuse of Dynepo by athletes – which is difficult to uncover with the IEF-method alone.Howewer, both the SDS-PAGE method and the Epo-IEF method use urine as sample matrix. Unfortunately, one of the latest generation Epo-pharmaceuticals (MIRCERA, a PEGylated Epoetin beta) is hardly excreted in urine due to its prolonged serum half-life and molecular mass (ca. 60 kDa). An ELISA test will be offered by the manufacturer for quantifying MIRCERA in blood. The consequences will be that in the future THREE methods will have to be performed in order to unambiguously detect the misuse of recombinant eyrthropoietins and analogs, i.e. the Epo-IEF method, the SDS-PAGE method for additional evidence (e.g. Dynepo, effort urines, biosimilars), and the serum/plasma ELISA for detecting MIRCERA abuse. Two different matrices will have to be used: blood and urine. The aim of this project is to develop a method which is capable of detecting doping with all forms recombinant erythropoietins in blood and in a single experiment.

Main findings

Recombinant erythropoietins perform with different sensitivity on SDS-PAGE after Western blotting. While the sensitivity of the majority of epoetins (e.g. epoetins alfa, beta, delta, omega; darbepoetin alfa) is similar on SDS-PAGE, the sensitivity of MIRCERA (PEGylated epoetin beta) is drastically decreased. Redesigning SDS-PAGE by exchanging the SDS for SARCOSYL in the sample and running buffers specifically enhanced the sensitivity for MIRCERA. SARCOSYL, a methyl glycine-based anionic surfactant with slightly higher CMC but much lower aggregation number than SDS, is not capable of solubilizing PEGs under PAGE-conditions - regardless of their polymerization degree (PEGs 1500 to 35000 were tested). Instead, SARCOSYL is only binding to the protein-part of MIRCERA leading to a sharp band on SAR-PAGE. SDS, on the other hand, is binding to both the PEG- and protein-chains of MIRCERA, which leads to band broadening on SDS-PAGE. As a result, the monoclonal anti-EPO antibody (clone AE7A5) is no longer binding to the fully - i.e. PEG- and protein-chain -solubilized MIRCERA-molecules, but only to those molecules which contain only SDS bound to the protein-chain. Naturally, these molecules are located on top of the band, since their charge density is reduced and their migration behaviour decreased. Because these molecules resemble only a small fraction of the MIRCERA-molecules originally loaded on the gel, a decrease in sensitivity is observed. SARCOSYL, on the other hand, leads to a sharp MIRCERA-band, since no solubilization of PEG-chains occurs. Consequently, the antibody is able to bind to all MIRCERA-molecules and no loss in sensitivity is observed after Western blotting. Besides, SARCOSYL-PAGE detects non-PEGylated epoetins with the same sensitivity and resolution as SDS-PAGE. The applicability of SAR-PAGE for detecting MIRCERA, recombinant epoetins, and endogenous EPO in blood and with high sensitivity could be demonstrated by performing single dose excretion studies. Besides, SAR-PAGE is not restricted to electrophoretic separations using the BisTris buffer system -e.g. MOPS-chloride boundary- but is fully compatible with other discontinuous buffer systems, namely the standard Laemmli (glycine-chloride boundary) [1], Neville (borate-sulfate boundary) [2], and Allen-Moore (e.g. borate-citrate boundary) [3] stacking systems – also indicating that the net-charge of the SARCOSYL-protein (i.e. erythropoietin, MIRCERA) micelles is stable within the pH-range of ca 7-10. The developed method is suitable for blood and urine, is not prone to „active“ and „effort urines“, is highly sensitive (down to femtogram-level, i.e. ca 10 amol) and with enhanced sensitivity compared to the traditional SDS-PAGE method for MIRCERA. The criteria of positivity (qualitative criteria, relative mobility values) are simpler since only one band instead of a series of isoforms and their distribution has to be evaluated. Of special importance is the fact that only one matrix and only one method are necessary for the detection of doping with all forms of recombinant erythropoietins (one matrix – one method approach) – instead of currently 4 methods and two matrices. Also, the sensitivity of SAR-PAGE for MIRCERA is higher than the sensitivity of IEF-PAGE (this was independently shown by the anti-doping control laboratory in Lausanne) – which is especially important for the screening. Hence, we also recommend the usage of SARPAGE as a screening procedure, because otherwise cases of low dosed MIRCERA would be missed by the IEF-PAGE method (false negatives). And finally, the required sample volume for SAR-PAGE is very low: 200 μL of serum are sufficient for the detection of shEPO and all forms of recombinant EPO.

Dr. Elsa Clara CORBELLA, Universidad Nacional de Córdoba, ARGENTINA

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.