Projets de recherche

Code: 08E09WS

The aim of blood manipulation is to increase the total hemoglobin mass (tHb-mass), which is directly correlated to maximum aerobic power and hence performance. When using the current doping tests it is not yet possible to detect autologous blood transfusions or the application of all kinds of erythropoiesis boosting stimulants.  
To minimize these illegal practices we recommend monitoring tHb-mass of endurance athletes over time. If the individual profile deviates substantially from that expected, the athlete has to undergo further follow-up testing. Serial measurements of tHb-mass can also be used to demonstrate objectively that an athlete has or had not used blood doping practices.  
Practical experience demonstrates that the recently developed method (optimized CO-rebreathing method) is valid, very reproducible and suitable to measure routinely an athlete’s tHb-mass. The procedure takes about 10 minutes and requires only a few drops of capillary blood. The practicability and significance of the method was evaluated within the last two years (2006-2007) in a multicentre study financially supported by a WADA grant. The individual tHb-mass profiles of >200 elite athletes of different endurance disciplines were screened and the preliminary results indicated good stability of tHb-mass (~2-3% variation).  
Doping with blood or EPO increases tHb-mass by at least 10-15%, whereas our recently obtained data (n>200) provide strong evidence that endurance training at sea level has no or only small effects on tHb-mass. It therefore follows that tHb-mass could be used as a screening parameter for blood manipulations. However, we also observed in individual cases that special influences can affect tHb-mass markedly. Without further systematic investigation, these special circumstances potentially undermine the utility of tHb-mass test to detect blood manipulation. The purpose of this follow-up project, therefore, is to identify physiological and outside influences (possible confounding factors) which could alter the normal stability of tHb-mass in athletes. Special emphasis will be placed the influence on tHb-mass of injury and severe infections, iron deficiency and iron supplementation, the impact of heavy and long-lasting exercise, and on the development of tHb-mass in junior athletes until maturation. Knowledge and quantification of these potentially confounding factors is of high importance to interpret deviations of a normal tHb-mass profile. Additionally, markers of these diseases will be correlated to tHb-mass, which will further increase the specificity of the test. As Germany and Australia provide the advantage of a central sports medical health system, large groups of the above-mentioned athletes can be recruited for the planned measurements. The proposed project will quantify factors that potentially confound the normal stability of tHb-mass athletes; specifically the effects of injury, severe infection, and of intense/long-lasting exercise and maturation. The long-term aim of the research team is to include tHb-mass in the “athlete’s biological passport” and to define subject-based reference ranges with the recently developed probabilistic inference techniques. The results of this ‘confounding factors’ study and its markers are essential for the application of tHb-mass into a “biological passport”. 

Main Findings:

Hemoglobin mass is closely related to aerobic performance and the aim of all kinds of blood manipulation is to increase Hb-mass und thereby the oxygen transport capacity. It is, there-fore, desirable to use Hb-mass as a screening tool for blood manipulation. From anti-doping studies we know, that blood manipulation by erythropoiesis stimulating agents and blood transfusions exceed the normal oscillation of Hb-mass. The effects of confounding factors are, however, not sufficiently investigated and may interfere with interpreting the effects of doping. 
The aim of this project was, therefore, to detect and to judge confounding factors which may explain possible variations in Hb-mass by other reasons than doping. In this study we focused on the effects on maturation, illness and injury, iron deficiency and iron supplementation, training breaks, and altitude effects. Additionally, methodological aspects of the CO-rebreathing method were considered. In total, data sets from 1881 CO-rebreathing tests of 428 elite and recreational athletes were obtained by two research groups from Australia and Germany. 
All those studies which were parallel conducted by both research groups yielded almost identical results and the typical error of the method was between 1.1% and 1.7%, i.e., both facts proved the method to be very robust and reliable. The CO-rebreathing procedure can be performed by the athlete in sitting or supine position, but it is influenced by acute exercise or by preceding intensive bouts of exercise. The half-time of COHb can be remarkably reduced by exercise or by breathing of an O2-enriched gas mixture. 
Illness and injury lasting until 2 weeks do not have a pronounced effect on Hb-mass whereas prolonged health problems leading to >2 week training interruption in rare cases (~1%) may decrease Hb-mass in the same magnitude as a blood donation. Severe iron deficiency and iron supplementation of anemic subjects has pronounced effects on Hb-mass and has to be taken into consideration. 
Normal changes in training volume have almost no effects, while complete training interruptions for more than 2 weeks have to be considered. 
Altitude training markedly increases Hb-mass if the stay lasts longer than 2 weeks and elevated values may exist until 5 weeks after return from altitude. 
Training has no effect in young children until puberty, and the most sensitive erythropoietic phase is between the age of 16 and 21 years. In adults above the age of 21 years no more age-related training effect do exist. Overall, our data show that the impact of the confounding factors on Hb-mass can be quantified and their effects can be included into statistical models, i.e. Bayesian approaches, as they are already used for the athlete’s biological passport (ABP). From the scientific point of view, we, therefore, conclude that the biomarker “Hb-mass” should be included into the ABP.

Code: 08B05PP 

Transfusing red blood cells (RBCs) is the oldest form of blood doping and - as the very recent findings around the Spanish cycling team Liberty Seguros, the German Cycling Team Telecom and further personal confessions of athletes demonstrate – is still or again used widely in top level sports. Autologous blood transfusion – up to now – is not detectable. It requires that the blood is stored for a longer period prior to reinfusion. 
Whereas red blood cells were for a long time regarded as mere O2-transport vehicles, there is evidence from recent studies that erythrocytes also possess enzymes that actively synthesize nitric oxide (NOS) and that alterations of NOS activation (translocation and phosphorylation) may be an indicator for autologous erythrocyte manipulation. Furthermore, adhesion receptors like CD47 antigen are present on RBCs and undergo alterations during blood manipulation. 
RBC NOS and adhesion receptors are detectable with immunohistochemical methods. Both are also be detectable by means of fluorescence activated cell sorting (FACS) after adaptation of the method, guaranteeing analysis of big numbers of blood samples in a short time. 
Our research group has demonstrated that the procedure of blood sampling, storage, and re-infusion in patients with hip-endoprothetic surgery induce an increase in RBC NOS activity in the RBC concentrate as well as in the patient’s blood after re-infusion. We also found RBC NOS activation during cardiopulmonary bypass. Furthermore, a progressive decrease in adhesion receptors in stored RBC concentrates could be demonstrated. Altogether, these data indicate, that an autologous blood transfusion induces a longer lasting activation of RBCs NOS system as well as changes in adhesion receptor expression. This has a high chance to be used for the detection of autologous blood transfusions when misused in sports. The relevant diagnostic approaches 
(immunohistochemistry plus gray-scale analysis and fluorescence activated cell sorting (FACS)) to quantify NOS and adhesion receptors of RBCs, therefore, may provide a new effective strategy in the fight against autologous blood doping. 
We will investigate 24 healthy male and female physically active subjects prior to 500 ml blood donation, as well as prior to and several days after re-infusion of the autologous blood concentrate after storage of 2 to 4 weeks. RBC NOS activity and adhesion receptors will be analysed in venous blood samples by means of immunohistochemistry and FACS. Furthermore, changes in blood total haemoglobin mass (tHb) will be analysed several times from prior to the donation until 2 weeks after re-infusion by means of optimized CO re-breathing method, allowing a quantification of the percentage of the re-infused amount of blood. Aerobic performance (V’O2max) will be tested several times for the analysis of the physiological effects of the procedures.

Main Findings:

Blood donation, but not red-blood cell re-infusion activated red-blood cell nitric oxid synthase (NOS) system at serine 116 and serine 1177 in healthy, moderately trained subjects. Therefore, red-blood cell NOS activation as measured by serine 116 and/or serine 1177 phosphorylation does not seem to be a valuable parameter for the detection of autologous blood doping in sports.  
However, there seems to be a physiological correlation between the red-blood cell NOS phosphorylation at serine 116 and the number of reticulocytes in the blood which remains to be elucidated in further studies. 

Code: 08A02JP

Recombinant glycoproteins expressed in non-human cells and in particular EPO and NESP has shown to contain small amounts of N-glycolyl-neuraminic acid (Neu5Gc), a sialic acid for which humans are devoid of the suitable hydroxylase. The detection of such non-human component of EPO (or other proteins) will constitute an unequivocal evidence of their exogenous origin. As part of previous research projects funded by WADA, we showed the presence of such monosaccharide in rEPO and NESP and later developed a capillary hplc method with fluorescence detection for the determination of small amounts of Neu5Gc (LOD ca. 5 fmol). By using such method in combination with an immunoaffinity purification developed in parallel using microwell plates coated with an anti-EPO antibody we were also able to detect the presence of Neu5Gc in plasma samples of EPO users. This method is already at the cutting-edge of the current technology using fluorescence detection. Furthermore, according to anti-doping regulations, the use of mass spectrometry is preferred, or required whenever possible. With this precedent, the aim of the present project is to move on following the same strategy, taking profit of our long experience in the field, and develop a method with a better sensitivity and able to detect Neu5Gc using mass spectrometry as unequivocal identification. The method will use nanoLC (hplc-chip from Agilent technologies) as the optimum sensitivity set-up and triple quadrupole MS. The instrument to be used has as specifications the detection of a chromatographic peak of 0.8 fmol reserpine (S/N =20) under conventional LC conditions. Using the hplc-chip (nanoLC) conditions this sensitivity should be increased ca. 50-100 times, fully supporting the feasibility of the attempt. The low amounts of Neu5Gc expected in urine of EPO users (ca. ≥ 35 fM) will be detectable in 20 mL of urine or less. Although some publications suggest that Neu5Gc can be incorporated into different glycoproteins through its ingestion as part of the diet, our preliminary results suggest that once a sample is immunopurified, there are no traces of Neu5Gc in negative samples, thus making the method specific for the purified glycoprotein. Although we will apply the method for EPO, using already developed immunopurification strategies, the same approach can be applied to the detection of the same analyte in different recombinant glycoproteins and matrices provided the corresponding specific immunopurification is applied.

Main Findings:

The detection of recombinant erythropoietin and/or its analogues is a major anti-doping problem because of the difficulty to differentiate exogenous from endogenous and the very low concentrations found in biological fluids. Current methods are based on electrophoresis and an anti-body recognition, not selective to the recombinant origin. The development of a ChipLC-MS nano-technique allows the analysis of conventional sample volumes through their pre-concentration in a built-in nano pre-column plus the on-line analytical separation through valve switching. All in a plug and play chip that includes the nanospray needle to be connected with an MS/MS detector. The objective of the current project was the development of an ultra sensitive analytical procedure for the detection of N-glycolyl-neuraminic acid as the unequivocal proof of the exogenous origin of EPO. The ChipLC/MS/MS procedure developed allowed a significant increase of sensitivity with respect to conventional instruments with a final limit of detection of 50 fmol Neu5Gc on column (as the DMB derivative) which, thanks to the large volume injection corresponds to 100 fmol/sample. The method showed to be linear and with acceptable precision and accuracy. However the instability of the nanospray needle made it not applicable for routine analysis. Furthermore, the sensitivity achieved, although significantly increased with respect to other MS procedures, is not amenable to the analysis of real samples yet. Still a limit of detection between 20 and 50 times lower would be needed. It is to be expected that as instruments become more sensitive, a method for the analysis of Neu5Gc on its own or a Neu5Gc containing peptide would be feasible in the coming years. 

Code: 08B15RG

The detection of human growth hormone in body fluids (plasma or urine) is a cumbersome task for several reasons being the low concentrations and the molecular heterogeneity the most important ones. Thus, at present for reasons of sensitivity and specificity immunoglobulins are indispensable in any analytical approach and also for the detection of growth hormone abuse antibodies play an important role. It is known that the administration of growth hormone results in a down regulation of the bodies-own production and that during a certain time frame only the exogenous molecule will be found in circulation. As the pharmaceutical product consists in a single isoform (the endogenous material is composed of several isoforms) this difference is used for anti-doping purposes.  Currently, two strategies are being followed: -1- an approach in which the amount one isoform (22 kDa) is compared all other isoforms and -2- an approach in which the amount of one isoform (22 kDa) is compared to the amount of a second isoform (20 kDa). For the second approach only a few specific antibodies are known and available. In order to have a reliable test for anti-doping purposes at least two specific antibodies, with distinct epitopes, should be available. In the course of the development of antibodies to the 20 kDa isoform fourteen different clones were generated by the research team. One, #7-clone 1B3, has now been fully explored. This antibody has shown superior surface properties with respect to the other known anti-20 kDa mAbs. Now, within the framework of this project the other clones will be produced at larger scale and characterised for their binding properties. The most promising clones will be further developed with the aim of having at least two distinct anti-20 kDa antibodies. The characterisation and comparison of the generated antibodies will be done by means of surface plasmon resonance and employing the entire arsenal anti-GH antibodies, GH isoforms, proteolytic GH fragments, synthetic GH isoforms, growth hormone binding proteins, sandwich assays, etc. This approach has already been applied successfully in the characterisation of the antibodies employed in the differential immunoassay that addresses the ratio between “pit” and “rec” isoforms. 

Main Findings: 

Human growth hormone (GH) in humans is a family of different molecules that come from a single gene. The composition of the family members fluctuates very little both at the intra- and interindividual level. However, when the GH pharmaceutical is administered the composition, and thus the ratio between the family members, is altered, and this sets the basis of the so-called direct approaches. One is the established rec/pit differential immuno assay approach that is implemented in most WADA accredited laboratories. An alternative approach, based on the specific ratio between 22 and 20 kDa GH is in the final stage of development with a multi-lab validation study. The major difficulty of the latter approach is the fact that finding a pair of antibodies (for screening and confirmation) with a different binding epitope for 20 kDa is very difficult as this variant differs only from the 22 kDa in that the amino acid Phe31 is linked to Asn47 instead of Glu32 (splicing-out of AA32-46). In this study we have evaluated and characterised 14 new anti-20 kDa antibody clones. We have identified 2 (antibody #4 and antibody #7) with excellent surface properties that are highly specific for 20 kDa GH. However, all antibodies that displayed binding to 20 kDa after immobilisation appear to have the same epitope as sandwich type studies reveal no complementary binding. When we compared the two best performers with one other established ultra specific and good surface antibody from a different source we could establish that this antibody, and the two identified here (#4 and #7) do bind different epitopes. As such, the 22 vs 20 kDa approach to address GH doping may be set up in compliance with the requirements for screening and confirmation procedures.

Code: 08A17PT

The detection and quantification of small molecule doping agents has become a relatively simple process with mass spectrometry regarded as the definitive, analytical methodology. The threat from biological agents such as proteins, peptides or genetic manipulation, provides a much greater analytical challenge for most doping control laboratories. As a result of this much research has been expended into the use of endogenous proteins as so called biomarkers of abuse.Until recently the only effective tool for quantitative analysis of large molecule targets has been enzyme linked immunosorbent assays (ELISA) or similar immunoassay based techniques. These assays are very effective for the analysis of large molecules, particularly where sandwich assays, involving the use of two complimentary antibodies, are employed. While it is likely that immunoassays will remain the standard for routine quantitation of single proteins, great strides are being taken in the development of quantitative methods based upon mass spectrometry, a highly sensitive and selective technique widely applied in the fields of pharmaceutical and sports testing. More recently, developments in sample handling techniques, chromatographic stationary phases and triple quadrupole instruments have made the quantification of proteins by mass spectrometry a reality which could be transferred into the majority of doping control laboratories. A major advantage of using LC-MS is that it provides an independent means of detecting the target protein compared to immunoassay and is a potential gold standard for validating these methods. This is reinforced by the ability to monitor several peptide fragments from the same protein to increase the confidence in the analytical result and it is also possible to analyse multiple proteins in a single analytical run. The aim of this project is to develop a generic LC-MS approach to the quantification of a range of growth hormone (GH) biomarkers currently used or proposed for use in sports drug testing. The method will be designed for use in a typically equipped doping control laboratory and will make as little use as possible of specialised equipment. 

Main Findings: 

The aim of this project was to develop a single methodology based upon LC-MS capable of detecting multiple biomarkers of the administration of growth hormone (GH). The preferred aim was to do so without recourse to antibodies or similar bio-reagents for which continuation of supply could be an issue. Potential applications of the developed method would be as a reference method for the detection of GH biomarkers or possibly for use as a confirmatory procedure for the same.  The approach to the project was broken down into: • the development LC-MS methods to detect the proteins of interest via proteotypical peptides,
• to obtain stable isotope labelled versions of the targeted peptides,
• to develop a generic extraction protocol capable of isolate all the analytes in a single fraction
• to validate the resultant methodology                                                                                                                                   The markers chosen were based upon those previously identified as providing the most definitive proof of GH abuse PIIINP, IGF-I and its binding proteins including IGFBP-3. In addition a number of additional potential biomarkers (IGFBP-2, IGF-II LRH) were included in the assay development phase. In the case of PIIINP, direct determination of potential prototypic peptides using traditional approaches proved impossible given the low levels of standard material available. Significant effort was expended identifying possible approaches to hydrolysis of the protein. By means of computer simulation (in-silico digestion) the most likely enzyme for cleaving PIIINP to produce sufficiently selective proteolytic peptides for use in an assay was determined. The chosen protease, Glu-C, was utilised but detection of the expected peptide fragments remained elusive. Finally, a custom synthesis of the expected peptide was commissioned and using this standard it proved possible to detect a target peptide for the analysis of PIIINP using LC-MS. While a target peptide is now available, this was accomplished a stage in the experimental work to prevent full development of an assay to be undertaken. Having developed LC-MS methodologies for the detection of proteotypic peptides of IGF-I, IGFBP-3 and related peptides attention was switched to optimisation of extraction methodologies to isolate the proteins from human plasma.  In practice, it was found that isolation of IGF-I was related to the concentration of the IGFBP’s present in the sample unless steps were taken to completely disrupt the binding of these proteins. Optimised conditions for extraction of IGF-I could be obtained by acidification of the sample and extraction on a polymeric phase SPE cartridge. This approach gave only low and variable extraction efficiency for the IGFBP’s and a second methodology optimised for these analytes was developed.  Analysis of samples for IGF-I in both plasma and serum using this approach proved successful. Reasonable results could also be obtained for the binding proteins with acceptable data obtained for calibration lines and QC’s. All indications are that these methods, if applied in the correct manner, would provide the basis for successful analysis of IGF-I and IGFBP-3 in plasma / serum samples.  The developed methodologies for IGF-I, the IGF binding proteins and related biomarkers could be used as the basis of confirmatory methods for these markers in human plasma / serum or as reference methods with which immunoassay based methods can be calibrated or controlled. In order to do so reference matrix with known levels of each marker would prove highly beneficial and aid the introduction of LC-MS methodologies into WADA sports testing laboratories. While it proved impossible to develop a methodology for PIIINP in the time frame available, an appropriate proteotypic peptide has been identified which could form the basis for the development of an LC-MS based assay. Publications are in preparation detailing the methodologies developed and initial investigation of PIIINP and this target peptide.

Code: 08A15MT 

This project comprises a combination of the analysis of different prohibited peptide hormones (for example, but nor limited to: sunthetic insulin analogues,synacthen, gonadorelin, synthetic IGF analogues) in plasma and/or urine samples. Former studies provided information about the expected concentratons thatt were estimates in low fol/mL range, their chromatographic separation and their mass spectrometric characterisation. Thus, an efficient and highly specific sample preparation procedure with nanotechnology based immuniaffinity purification employing coated magnetic beads and appropriate primary antibodies will be developed. The subsequent determination and identification of peptides will be performed by means of liquid chromatography - tandem mass spectrometry. The major goal of the planned project is the simultaneous purification and determination of a variety of structurally and physiologically different bioactive peptide hormones from biological fluids, and te installation of a comprehensive screening tool dedicated to the detection of prohibited compounds with peptide-based structure using mass spectrometric approaches is aimed. The assay shall utilize commercially available reagents and equipment only in order to allow a rapid and facile transfer to other anti-doping laboratories, and detailed instructions and protpcpls shall enable a fast implementation of a complementary and highly specific screening tool in sports drug testing laboratories. 

Main Findings: 

Bioactive peptides such as insulins, the synthetic adrenocorticotrophic hormone analogue Synacthen, Gonadorelin (LHRH), Growth Hormone Releasing Hormones (GHRHs) and insulin-like growth factors (IGF-1 and derivatives) provide a reasonable potential for the misuse as performance enhancing agents and are prohibited in elite sports according to the list of banned substances established by the World Anti-Doping Agency. Currently, the determination of these analytes is possible by dedicated assays only or methods are even not at hand so far. In the present project, a procedure to determine several prohibited peptides, which are excreted into urine (e.g. Gonadorelin, Human insulin, Humalog (Insulin Lispro), Apidra (Insulin Glulisine), Novolog (Insulin Aspart), Lantus (Insulin Glargine), Porcine Insulin, Synacthen, IGF-1, longR3-IGF-1, Geref and CJC-1295), was developed. The method enables the effective, highly sensitive and specific screening for several different target analytes that are simultaneously purified and analysed by means of immunoaffinity purification, subsequent liquid-chromatographic separation and high resolution / high accuracy mass spectrometric determination at low pg/mL concentrations. Central aspect of the approach is the combination of immunoaffinity purification with mass spectrometry. Employing different specific antibodies coupled to paramagnetic beads, the simultaneous isolation of all targets in one sample preparation procedure was accomplished. In general, the approach is extendable to any banned peptide if adequate antibodies are available. At the present status of the project the above mentioned analytes (11 prohibited peptides, 5 internal standards) are covered  and the method is fully validated under consideration of qualitative result interpretation. 

Code: 08B03RK

Luteinizing hormone (LH) is one of the prohibited gonadotrophins named in the WADA 2008 Prohibited List. LH is a naturally occurring hormone which is secreted by the pituitary gland. In males LH stimulates testosterone production by the testes. Whilst LH has been prohibited in sport for some years, it is only relatively recently that human LH has become available as a commercial pharmaceutical product due to recombinant biotechnology. Thus there is now a pressing need to establish valid reference range(s) for endogenous LH levels in urine to assist in the detection of doping with recombinant LH as well as to improve the standardisation of urine LH measurement as an adjunct to detection of suppressed LH levels that accompany doping by use of exogenous androgens and hCG. Whilst there are several commercially available immunoassays optimised for serum but which can be used to measure LH in urine, the numerical values obtained from the different kits are not in good agreement. There is also the question of whether kits calibrated using natural pituitary-purified LH give accurate results when measuring degradation metabolites of recombinant LH excreted in urine. Thus there is a need to establish comparability of LH measurement amongst the over 30 WADA laboratories who use a variety of techniques for the measurement of LH. This project will measure LH in a range of urines using a variety of commercial assays in order to establish comparability of measurement and establish upper and lower reference ranges for normal subjects. An attempt will also be made to determine if any assays or combination of assays can be used to validly detect recombinant LH as well as distinguishing reliably from endogenous LH. 

Main Findings: 

The primary objective of this project was to determine which method or methods that can be successfully used to measure luteinizing hormone (LH) in urine. The measurement of LH is important for two reasons. The first is that the administration of testosterone and other androgenic hormones leads to a suppression of the secretion of endogenous LH and the second is the recent availability of recombinant LH which may elevate LH values. There are several different methods used by WADA laboratories to measure LH in urine all of which are based on antibody reactions. However it was clear that the different methods can give very different numerical values. Controlled studies using a variety of samples and a number of methods have shown that the Siemens Immulite is the preferred method for measuring LH in urine particularly when the suppression of LH is being used as an aid to detect doping with testosterone. Most other methods are not useful for this purpose because the LH or at least that portion of the LH molecule to which the methods respond is not stable in urine and hence low and apparently suppressed values can and do result from degradation on storage. The values of LH in urine as measured by the Immulite are relatively stable on storage for at least two weeks at room temperature and for months at -200C. 
Studies involving the administration of recombinant LH have shown that the injection of the recommended dose of LH does not lead to any elevation of measured LH in urine. This was not unexpected given the dose and the rate of natural LH production. A short term elevation of urinary LH was observed when thirty two times the recommended dose was injected. A method using SDSPAGE with Western blotting has been developed which can distinguish between pituitary and recombinant LH in both standards and in samples extracted from urine. It is clear from this work that the LH excreted in urine has a different apparent molecular weight to the parent LH.  
As a result of this project it is now feasible to use LH suppression as an adjunct procedure with T/E measurement to detect testosterone doping and to detect and confirm the use of large doses of recombinant LH. 

Code: T08A02MS

The aim of the project is to evaluate the robustness of the method with athlete’s samples regarding some collection scenarios and to compare the results obtained in different transport and storage conditions on serum samples. The final aim of the global project is to obtain information on sampling practices also called collection scenarios based on supporting data.

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: R08B02GG

In 2003 the development of the software platform GASepo was submitted and succeeded for a WADA research grant. This included the participation of various experts of 9 WADA accredited labs for the development of this software. After two years GASepo now provides a reliable and easy-to-use software platform for quantitative interpretation of gel images deriving from IEF analysis as well as from SDS-Page analysis for the detection of recombinant EPO. As indicated in the initial grant application, further development of the software is anticipated due to the increasing demands of the various procedures and data from rEPO detection. This proposal targets to a novel and additional way for background subtraction of gel images and sample lanes with a special focus on low abundance gel images. The current version of the GASepo software offers two different ways of automated background subtraction:

1 – Subtraction of the background surface deriving from the lane boundaries

2 – Subtraction of the background surface deriving from an artificially created lane near the lane of interest. The proposed background subtraction module shall allow for subtraction of the background for each band separately. In addition some minor adjustments regarding the flexibility of report creation and the new demands of SDS-PAGE shall be implemented.

Main Findings

The project aimed at functionality enhancement of the existing GASepo software platform for quantitative interpretation of gel images from isoelectric focusing analysis. The GASepo software Version 1 has been previously developed and, in the meantime, has become an internationally accepted tool among the vast majority of the WADA-accredited anti-doping laboratories. Due to the recent development in the EPO doping analytics as well as due to accumulated experience with GASepo gained over time, it was necessary to update selected functionality features and adapt the software to the new generation of operating systems called Windows 7, both in its 32 bit as well as its 64 bit version.

Major and minor software modifications improved the usability and adapted the software to the recent demands of the WADA Technical Document TD2010EPO on EPO detection. Final product of this project is a renewed software version 2.1 of the GASepo platform. The software was validated against a phantom image with known pixel numbers and intensities. A validation report as well as the phantom image for revalidation is included in the software package.

Code: R08B01RH

Growth hormone (GH) is a naturally occurring hormone produced by the pituitary gland. Although banned under the International Olympic Committee (IOC) and World Anti- Doping Agency (WADA) list of prohibited substances, the detection of GH abuse poses a formidable challenge. The GH-2000 and GH-2004 projects have worked to develop a test to detect GH that is based on the measurement of GH-sensitive markers, which rise in response to the administration of GH. The magnitude and duration of the elevation is dependent on the dose of GH given, gender and the individual marker. Men were more sensitive to the effects of GH than women. IGF-I and P-III-P were the best of these markers and were selected to construct formulae that gave improved discrimination between those taking GH and those taking placebo compared with a single marker.

The results of the GH-2000 and GH-2004 projects have been reviewed by a panel of international experts at IOC, WADA and USADA workshops in Rome (April 1999), Dallas (March 2004), Nashville (May 2005), Austin (June 2006), Chicago (June 2007) and London (April 2008). These meetings have confirmed the method’s scientific validity and independent confirmation of this approach has been provided by the Australian-Japanese consortium and Kreischa Institute. The major impasse in implementing this methodology is the need for two immunoassays to be used for each analyte. Furthermore, the original IGF-I assay used in the GH-2000 study is no longer available. It is therefore necessary to validate two new assays for IGF-I and a further P-III-P assay to overcome this hurdle. The aim of the current study is to do this. This will be achieved by simultaneously measuring IGF-I and P-III-P by two commercial immunoassays for each protein on samples obtained from 500 elite athletes.

Main Findings

This report presents new threshold levels for the GH-2000 detection algorithm estimated for each assay combination using reference dataset of 496 samples from elite athletes and a false positive rate of 1 in 10,000 (99.99% specificity). A total of 4 different assay methodologies were used for this study; two for IGF-I and two for P-III-NP concentrations. In addition to the proposed threshold levels estimates, a sample size uncertainty limit has been added. Although this study was not designed to test the sensitivity of the test, it is noteworthy that we correctly identified Terry Newton as having been doping with GH and also identified another rugby league player with a suspicious finding.