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.

Code: 08C06CM

AIMS/SIGNIFICANCE: -differentiate blood from transfused and non-transfused individuals. - Correlate changes in protein pattern to Hb, physical performance and VO2 max. Finding markers of autologous blood doping is important in order to maintain the fundamental aspect of sports: fair play. BACKGROUND/HYPOTHESIS: Physical performance can be enhaced by blood boosting. Doping using the hormone EPO and homologous blood (non-self) can today be detected while autologous (self) blood transfusion is undetectable. Red blood cells (RBC) can be stored for up to 5 weeks in +4C and for several years in -80C. It is highly unlike that blood can be withdrawn from the body, treated and stored without change in any protein. METHODS: By using proteomic methods, thousands of proteins can be separated and identified. In combination with multivariate statistical methods protein markers to detect autologous blood transfusion can be found. Separation of proteins is done by 2D DIGE and proteon identification done by mass spectrometry. Wr can quantitatively detect changes in protein patterns, thereby separate blood from soped and un-soped individuals. RESULTS: A 10% difference in protein abundance can be detected (95%Cl). Over 2300 proteins protein spots can be separated from 100 uL of RBC. Fresh blood was comparared with blood stored for 5 weeks in -80C. 48 proteins were altered, including enzymes (e.g catalase) stress (E.G hsp 71) and structural (e.g actin) proteins. Ongoing experiments have detected -80 proteins changed by storage in +4 C for 5 weeks. STUDY DESIGN: After blood donation (10 subjects) and storage for 4 week at 4C, RBC will be reinfused. Blood samples will be taken from the subjects sevral times before and after donation and reinfusion. Control samples will be taken from a matched groups. Haemoglobin, Physical performance and VO2 max will be measured on 7 occasions.  

Main Findings: 

The specific aims of this study were: I. Differentiate blood from transfused and non-transfused individuals.
II. Correlate changes in protein pattern to Hb, physical performance and VO2max.       We have investigated the possibilities to use proteomics as a tool to screen the human Red blood cell (RBC) membrane proteome for novel and unique biomarkers useful for development of future diagnostic point-of-care tests. A comparison between fresh and freeze-stored (-80° C) RBC’s were performed using the 2D DIGE technique. From findings in freeze-stored blood, 20 candidate proteins were identified. 
A blood transfusion study was subsequently performed where 10 subjects underwent an autologous blood transfusion (2 x 450 mL donated whole was blood and 2 x 300 mL washed RBC’s re-infused) after 16 week freeze-storage of the RBC’s. Blood samples were drawn at 13 time points for hematological and proteomic analyses and physical performance testing done 9 times. 
Forty eight hours after blood transfusion, Hb increased by 5%, physical performance 
(Running time to exhaustion) was increased by 15% and VO2max by 16%. Only a weak correlation (R2 = 0.33) was seen between Running time and VO2max. 
Blood samples taken from the subjects as well as from the transfusion bags were analyzed by proteomic and standard clinical methods. There is a clear separation of blood taken from a freeze stored bag and fresh venous blood. Different protein profiles between blood taken before and after a transfusion can be visualized. Some of these results were confirmed by Western blot. 
Because no method is today available to directly detect an autologous blood transfusion, we believe that our method under development will provide a solution in a near future, and the current work-plan is to have a prototype (alpha-version) ready for testing within 18-24 months, pending funding and the speed of technical advancements. 

Code: 08C20TF 

AIMS OF THE PROJECT. This goal of this project has been to compare global patterns of gene expression as described in disparate WADA-sponsored studies of the effects of doping agents of methods such as erythropoietin or hypoxia, growth factors such as human growth hormone and IGF-1, steroids and others. To achieve that goal, we have continued to refine the informatics infrastructure and have developed protocols for the application of computational methods for large-scale meta analysis of gene expression data sets from three separate and independent doping studies. We approached the directors of a number of WADA-sponsored studies to obtain data bases that could all be subjected to uniform analytical procedures to identify those presumably few common features that might constitute rigorous markers of exposure to doping manipulation. We received extensive data sets from two other WADA-supported investigators – James Rupert of the University of British Columbia and Dr. Tejvir Khurana of the University of Pennsylvania – and have identified preliminary candidate signatures for further validation and comparison with results of additional data sets to be included in future analyses.   

Main Findings: 

 We have successfully used the WADA Informatics facility to down-load and analyze several large transcriptomic datasets, including the one generated in our own laboratory for the IGF-1 study (Bhasker and Friedmann, 2008), as well as datasets rom the WADA-supported studies of James Rupert at the University of British Columbia and Dr. Tejvir Khurana of the University of Pennsylvania. The purpose of these preliminary studies has been to identify and solve the up-loading difficulties that outside users might encounter. The results of that exercise are presented in detail in the attached figures. Briefly, we have demonstrated that a comparison of studies using disparate methods of creating hypoxic conditions in mice reveal similar patterns of transcriptional dysregulation, despite many major differences in experimental design. These similarities include established categories of biological processes, molecular function and specific gene aberrations (slides 4-6) of Powerpoint summary. Those similarities may constitute the beginnings of a rudimentary molecular “signature” for metabolic and gene expression responses to hypoxia and/or to possibly related manipulations such as artificially augmented blood production in a sport setting (Slide 7). In contrast, a comparison of hypoxia conditions with the expected “negative control” effects of IGF-1 exposure of muscle stem cells reveals fewer transcriptional changes in common with the hypoxic conditions, as expected. We emphasize that these results require extensive validation and corroboration with other related and unrelated data sets from other WADA investigators. That will be the emphasis for future studies with this system.

Code: 08C04MM

Different analytical approaches can be foreseen for direct analysis and for the identification of a characteristic signature pattern following gene doping. The pilot project will evaluate the proof of principle of Affinity Based Biosensors (ABB) integrated with bioinformatics and biomolecular approaches for gene doping detection. Our challenge is to provide a total analytical process for the evaluation of the presence of the gene doping event. The heart of the project is a new multi-screening and real time bioanalytical protocol, based on an affinity sensing platform to be used both in direct and indirect based approaches for gene doping detection. We believe affinity-based biosensors (ABBs), flanking conventional and profiling methodologies, can contribute to gene doping detection as fast, low cost and easy to use instrumental approach. In this context, a flexible platform, consisting of a biochip coupled to a label free technology for simultaneous measurements in short time could represent an innovative approach for selectively detecting gene doping markers (direct approach) or secondary effects induced by gene transfer (indirect approach). The feasibility of this project is assured by the high interdisciplinarity of the partners of the proponent team which will contribute with their specific competences to the definition of a total analytical process using bioanalytical, bioinformatics, biomolecular and immunological competences. The pilot project outcome, will be transferred to the Italian reference anti-doping laboratory (Federazione Italiana Medico Sportiva, Roma), accredited by WADA.

Main Findings

The present project aimed to develop an innovative analytical approach or delivering sampling and analytical protocol to be applied to gene doping detection, eventually setting up a database. A new multi-screening and real time bioanalytical protocol, based on an affinity sensing platform for gene doping detection was developed using an integrated multidisciplinary approaches based on bioanalytical, bioinformatics, biomolecular and immunological competences. In particular, we developed a bioinformatics supported study for the identification of suitable markers for gene doping tracing. The initial purpose of the Gene Doping Detection Database (GDDDB) is to provide functionality for the design of primers on sequences that can be potentially used as Vectors during gene doping. Since it is currently the most commonly used gene therapy vector, the pilot study GDDDB contains Adenovirus sequences only. The database scheme is designed so that it can be interfaced by a biomart engine (www.biomart.org). Primer design is done using the primer-BLAST web service provides by NCBI (www.ncbi.nlm.nih.gov/tools/primer-blast/) which is a web-based graphical interface to the Primer3 and BLAST algorithms. Here we describe the entity relationship diagram of the database, and the show how it is adjusted to a data warehouse scheme as required for use by the biomart engine. The GDDDB can be accessed and tested via the GDD portal at http:/aragon.cl.cam.ac.uk/GDD/dbportal.html. Furthermore a simple discrete Bayesian analysis is done to calculate the posterior probabilities if gene doping. These results are shown and are given for each probe used in the developed assay. The conditional posterior probabilities are also shown, depending on whether a high or low affinity has been observed from samples. The project also developed an animal model (in vivo approach). The in vivo approach has first used transgenic mice to for the EGFP reporter gene to validate the molecular analysis of the marker in different tissue. Once the applicability of the develop method for the analysis of the selected marker has been proved in this first transgenic model, then a second model system based on the injection of the vector, containing the same reporter gene EGFP, in the tibialis anterior and of the femoral quadriceps muscles was developed. The sampling has been executed at different times after transfection and from different tissues: muscles, liver, spleen, kidney, lung, heart, right quadriceps (site of injection) and left quadriceps. Moreover body fluids (urine, blood, tears) have also been used to evaluate the presence oof viral vector signature. To trace the marker gene dedicated approaches have been developed and applied to these animal models. The EGFP expression has also monitored in different tissue of transgenic mice. In order to unambiguously detect the presence of recombinant vector, a protocol for construct-specific sequences was also developed. For this purpose new primers pairs were designed respectively on 3' promoter and 5' end of the EGFP sequences. Finally different region of the EGFP marker was amplified for tracing the marker in the mice after gene-doping event mimicking. The target sequences were found in all the sampled tissue. Surface plasmon Resonance imaging (SPRi)-based sensing for the detection of the gene-doping event was achieved. In particular Affinity Based Biosensors (ABB) have been developed. Both for DNA target sequence detection (DNA sensing) and antibody detection in human serum (immunosensor) are reported. Immobilization chemistries for molecular robe surface binding, analytical protocol were first optimized using standard solutions and further applied to complex samples (PCR mixture for DNA sensing - direct approach and serum for indirect approach) The analytical platform (biochip) allows simultaneous and real-time detection of sequences belonging to the vector.

Code: 08C19YP 

Living at altitude increases haemoglobin and haematocrit, hence altitude-training is popular among endurance athletes. Since increases in haematocrit can be attained using illicit means like blood-doping or erythropoietin (Epo) use and are potentially hazardous, high haematocrit levels are used to exclude athletes from competition, also without evidence of doping. While sea-level athletes can choose to train at altitude, for others living at altitude has been a way of life for generations (e.g. east-Africans). Recent approaches developed to distinguish the effects of altitude on haematological profiles from those of blood-doping are approximately 20-80% successful. When applying these approaches to the haematological profiles of elite athletes, we found indications of systematic blood-doping or cases of naturally elevated blood markers. There is therefore an urgent need for these methods to be revised to remove any possibility of athletes being incorrectly banned from competition or, conversely, avoiding sanction due to broad definition of legal limits. In this project we will investigate standard red cell indices and contrast these following Epo administration in athletes not involved in competition. Gene-expression profiles will be assessed using the very latest gene-microarray technology. These results will be used to formulate new methods with improved discriminatory power relative to current detection protocols and in doing so eliminate the possibility of naturally elevated blood markers due to athletes living and/or training at altitude and unidentified doping due to inadequate detection. The use of gene-arrays, validated in this context, may provide gene-expression profiles relevant to other illegitimate approaches to improving oxygen carriage that may have been, or will be in the future, devised.

Main findings: 

The use of recombinant human erythropoietin (rHuEpo) is prohibited by the World Anti-Doping Agency. An OMICS-based longitudinal screening approach has the potential to improve further the performance of current detection methods such as the Athlete Biological Passport. For this project, we successfully used gene expression profiling in whole blood to identify genes that are differentially regulated following rHuEpo administration in Caucasian trained males and Kenyan endurance runners living at sea-level and moderate altitude (~2150 m), respectively. Relative to baseline, the expression of hundreds of genes were found to be altered by rHuEpo. In particular, 15 transcripts were profoundly up-regulated during the 4 weeks of rHuEpo administration and subsequently down-regulated up to 4 weeks post administration in both groups. Importantly, the same pattern was observed in all subjects. Furthermore, 30 transcripts were already differentially expressed two days after the first injection and are therefore promising candidate genes to detect microdose rHuEpo doping. The functions of the discovered genes were mainly related to either the functional or structural properties of the erythrocyte or to the cell cycle and its regulation. In summary, this research project successfully identified the blood “molecular signature” of rHuEpo administration and provided a set of candidate genes with potential to be robust biomarkers of rHuEpo doping. These preliminary results provide the strongest evidence to date that OMICS technologies such as gene expression have the potential to substantially improve and add a new dimension to the current anti-doping methods such the Athlete Biological Passport for rHuEpo detection.

Code: 08B01RG 

Protein hormones represent an extremely challenging analytical problem in terms of anti-doping control. The main reasons are that most of these proteins are produced by humans and that the concentrations in body fluids, such as blood or urine, are very low. Whereas the second condition puts stringent demands on the analytical instrument in terms of sensitivity the first condition does similar to the scientist’s ingenuity in order to enable differentiation between like and non-like. Thus far, all protocols addressing protein hormone doping are based on immunological techniques only and suffer from the problems like the unknown specificity of the antibodies in the assays, the potential cross-reactivity under different conditions, and the fact that all sample handlings cannot be monitored and only an end-stage reading is provided. Still, regardless the analytical measurement immunoglobulins will be required to address specifically a particular category of proteins in complex mixtures.  
This project aims at the development of a single step purification of all protein hormones from plasma through a multi-antibody platform followed by in-situ solid-phase proteolysis and nano-LC chip mass spectrometric identification and quantification. Three different phases can be distinguished for each protein hormone:  A- the characterisation of antibodies addressing a particular protein (or category) by means of surface Plasmon resonance (SPR). From this study the best immunoglobulin (in terms of surface bound properties, specificity and cross reactivity, thermodynamic parameters of the interaction, compatibility in a mixed antibody setting) will be selected.
B- extrapolation of the SPR results to a LC-compatible immunoaffinity stationary phase. For this purpose monoliths functionalised to use similar immobilisation chemistry as in SPR will be employed. Again, first single antibody monoliths shall be characterised and subsequently multi antibody monoliths will be build to finally address all protein hormones in a single step.
C- Identification and quantification of the IAC purified material. This will be accomplished by -1- eluting the IAC captured material onto a protease-containing stationary phase with a short (min) stop-flow setting to allow proteolysis -2- elution of the generated peptides into a reversed phase column to allow conventional chromatography -3- identification of the peptides from each hormone. Quantification shall be achieved using diagnostic peptides with specific isotopic labelling that will be injected as internal standards in each analysis.
The outcome should be a “all-in-one” single injection system that reduces sample manipulation/loss and addresses all hormones employing the same sample, recovering the remainder of the sample for other analytical procedures.

Main findings: 

The project AntiProDo aimed at the development of a single instrumental set-up that included purification of four protein hormones from plasma through a multi-antibody platform followed by in-situ solid-phase proteolysis and nano-LC chip mass spectrometric identification and quantification. 
Through a meticulous characterisation of the binding characteristics of multiple antibodies, one was selected for each of the four initial target analytes: hGH, EPO, hCG and IGF-I. Subsequently, customised monolith solid supports in silica capillaries were produced and functionalised with the antibodies. The functional behaviour was verified. Simultaneously, the same capillary support was developed to house proteolytic enzymes and the activity, efficiency and durability established. At another front, the target proteins were submitted to mass spectrometric analysis to establish the proteotypic peptides to target in a final setting. Heavy isotope labelled peptides were produced as internal standards and the analytical method based on nano-LC ms designed and validated. Ultimately, all elements were hyphenated to demonstrate the proof of concept for this approach.  
Further optimisation of the individual steps, particularly at the reproducibility in the manufacture of the functionalised capillaries, is required before this approach can be taken further.