Projets de recherche

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

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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.