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 Chan, Hong Kong, Chine

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Code: ISF17B03BS 

Recombinant erythropoietins (EPOs) have long been one of the most frequently abused drugs for increasing blood oxygen capacity. However, numerous compounds with EPO-like activity but completely different chemical structures (either short peptides or even nonpeptidic synthetic molecules) have been developed so far and due to such chemical diversity, detection of their potential abuse is notoriously difficult. To our knowledge, no universal and reliable analytical method is available to confirm the presence of all EPO mimetics in biological sample regardless of their chemical structure. Here, we describe development of a rapid and universal assay for detection of any ligand binding to the erythropoietin receptor (EpoR) which could be used as a low-cost screening test in doping control to identify individuals for further detailed examination. Our innovative platform comprises combination of immunoprecipitation steps and two modified ELISA tests where recombinant filamentous phage particles displaying EpoR are used instead of primary antibodies. As evaluated with spiked artificial urine, such assay can detect the presence of EpoR ligands chemically unrelated to EPO in specimens. Site-specific proteolytic cleavage and isolation of EpoR:ligand complex from phage particles enables analysis of the complex by mass spectrometry and potential identification of previously unknown EpoR ligand. The described platform may also be further developed into the lateral flow immunoassay format similar to common over-the-counter pregnancy test which would greatly increase its applicability out of the laboratory. To emphasize, such concept can also be applied to detection and identification of other growth factor or hormone mimetics by implementation of appropriate combination of receptor, ligands and antibodies. Moreover, based on our platform a multiplex assay capable of simultaneous detection of substances with distinct activity can be developed by utilizing Luminex® technology and appropriate combination of recombinant phage particles displaying different hormone and growth factor receptors. 

Code: 17A28PA 

Detection of the use of the anabolic steroid nandrolone is based primarily upon the identification of the main urinary metabolite, 19-norandrosterone. Laboratories use the sophisticated technique of gas chromatography mass spectrometry to detect 19-norandrosterone in athletes' urine. WADA have recently taken steps to harmonize the analysis of nandrolone, issuing WADA Technical Document TD2016NA in 2016.

Reference materials are a vital tool for validating analytical testing methods and for ongoing laboratory quality assurance. International best practice specifies that CRMs beused as part of routine laboratory quality control. However CRMs for the measurement of steroid metabolites in human urine are not readily available. The project involves certification of the stable carbon isotope delta value of 19-norandrosterone (19-NA) in CRM MX017, currently being prepared for WADA project 15J04JM. Laboratories will reconstitute this material with water, and then analyse it alongside samples of urine taken from athletes for doping control. The testing laboratory can the compare the measured values for the CRM to those certified, providing assurance of the validity of the testing procedure.

Main Findings:

A new freeze-dried human urine NMIA MX017 certified reference material (CRM) has been prepared by NMIA, under the funding rom WADA, to replace the depleted MX005 CRM. Approximately 2400 units had been produced and made available to WADA laboratories since April 2019. This material will assist WADA laboratories to demonstrate comparability of measurements for the Athlete Biological Passport introduced in 2014. The replacement material will continue to support the longitudinal profiling measurements for detection of testosterone abuse and to provide SI-traceable values for the mass fraction and mass concentration of the glucuronides of six steroids specified in the WADA technical document TD2018EAAS: testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5a-androstane-3a,17b-diol (5a-Adiol) and 5b-androstane-3a,17b-diol (5b-Adiol).

In February 2020, the carbon isotope delta value of (δ13CVPDB) 19-norandrosterone (19-NA) has been added to the MX017 certificate. The new property value (δ13CVPDB of 19-NA) is traceable to the VPDB reference via the NMIA MX018 steroid carbon isotopic reference materials normalised by two secondary isotopic reference materials IAEA-CH-6 (sucrose) and IAEA-CH-7 (polyethylene). The reference value, δ13CVPDB -29.82 ± 0.41 , was determined using 36 bottles of MX017 analysed in five batches of experiments. The combined standard uncertainties of the reference value was expanded with a coverage factor of 2.0 to provide a 95% level of confidence.

Analysis was performed on 20 mL of reconstituted urine CRM (MX017) after hydrolysis with β-glucuronidase (E.colie). Free steroids were extracted into hexane and purified by two-dimensional HPLC. In-house quality control samples including water spikes, matrix spikes and solvent standards were included to monitor fractionation due to sample transformation procedures. The purified fractions were dried and reconstituted in cyclohexan/2-propanol (4:1) for anaylis by GC-C-IRMS. The identical treatment priniciples was followed to ensure samples and calibration soltions were combusted and transferred the same way into the IRMS. Each samples was injected twice in a randomised order bracketed by calibration standards. Frequent bracketing of the test samples with calibration solutions allowed any drift in the instrument to be fully captured. An approximate δ-value of the internal working gas was used to calculate all raw δ-values for samples and standards. A multi-point isotopic bracketing calibration approach was adopted to normalise all the measured δ-values of samples using steroid isotopic CRM mixtures NMIA MX018-1 and MX018-3. A linear regression line (R2> 0.99) fitted through the measured and the reference delta values from the eight steroid compounds, in the two calibration mixtures, allowed the measured δ-values of 19-norandrosterone in the MX017 to be normalised to δ13CVPDB the VPDB scale (Figure 1). The multi-point steroid isotopic calibration approach has allowed the property value to be assigned with ower uncertainty than the measurement approach employing calibrated reference gas.

Code: ISF17D05FM & ISF17E06RG

In a funded WADA study recently completed (“Novel molecular biomarkers for detection of autologous blood transfusion in sport: fetal hemoglobin and microRNAs”) we tested the hypothesis that novel biomarkers as fetal hemoglobin (HbF) and related microRNAs might show changes of interest in a group of trained healthy volunteers exposed to ABT respect to controls. The first result of this study is the production of a validated WADA-UNIFE-Biobank constituted of around 2000 plasma samples from control and ABT trained subjects. It is established that hypoxic and hyperoxic stimuli and blood manipulation during procedures of withdrawal and reinfusion (distress, ageing, apoptosis/degradation of circulating blood cells, effects of preservative substances, etc.) might induce a predictable and an unpredictable series of changes of miRNAs expression. Therefore, we hypothesized that after significant blood collection and autologous reinfusion, the miRNA network in the athlete’s plasma is changed, allowing to generate integrated molecular profiles permitting to predict ABT. Therefore, the hypothesis is that a miRNA pathway might be much more informative than a single miRNA, even if demonstrated to be associated with ABT in a sub-set of athletes. To this end, a small sample of subjects enrolled for the cited study was recently spontaneously tested in our laboratory for a full genome analysis. Therefore, the aim of this study is the validation of the first release of ABT-miRNA list and the identification of novel miRNAs of putative interest in predicting ABT following global miRNA analyses.

Main Findings

This project was aimed at identifying novel molecular markers useful in the anti-doping field to detect autologous blood transfusion (ABT). The hypothesis was that both the phases of ABT, blood withdrawal and reinfusion, might be accompanied by changes in different parameters to be identified by OMICS approaches in the level of microRNAs (miRNAs), small non-coding RNAs that regulate gene expression. In a previous funded study “Novel molecular biomarkers for detection of autologous blood transfusion in sport: fetal hemoglobin and microRNAs” twenty-four healthy trained male subjects were enrolled and randomized into Transfusion (T) and a Control (C) groups. The T subjects underwent nine seriated blood samples before and after the procedures of withdrawal and reinfusion. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects at 12 days time-point after re-infusion.

In the present study, using samples stored in the biobank, we aimed to validate the first release of ABT-miRNA list at other time-points (i.e. D-25 and D+3) to confirm the interest of this miRNA list in predicting ABT. ABT prediction was also demonstrated after global miRNA analyses, thus identifying novel ABT-informative miRNAs. The levels of selected informative microRNAs in relation to sex was determined and protocols/assays for ABT detection were developed. of specific interest. To this aim, microarray analysis and droplet digital RT-ddPCR were also performed for subjects of the T group and C groups for selected time-points.

The data obtained studying the global miRNome pattern demonstrated clustering of ABT-treated plasma samples at D+3 and D+15. These results support the concept that identification of ABT can be performed in one-step using the developed microarrays procedure for miRNA global analysis. The blood withdrawal was not found to be a “confounding factor”. Secondarily, the data obtained by RT-ddPCR performed in plasma samples from ABT trained subjects at D-25, D+3 and D+15 time points were first aimed at validation the “erythroid-associated miRNA list” (let-7a-5p, miR-126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p and miR-92a-3p) representing ABT associated miRNAs involved in hypoxia, erythroid differentiation and fetal hemoglobin production. This set of 8 erythroid associated miRNAs were informative in detecting ABT in most (5/6) of the ABT-treated subjects at all the time-points considered. We then considered miRNA found specifically dysregulated at D-25, D+3 and D+15 time points according with the miRNA microarray analyses. This additional list was proposed in consideration of the miRNA fold changes, the availability of the assays for miRNA amplification and the necessity to have both up- and down-regulated miRNAs to test as possible ABT-associated biomarkers. A final list of six miRNAs was identified and selected for further experimental activity. While the data obtained for down-regulated miRNAs were not informative (no major differences with the control set), differential expression of miR-766-3p (D-25), miR-636 (D-25), miR-425-3p (D+3), miR-4284 (D+3), miR-3151-3p (D+15) and miR-5787 (D+15) was found. When the two miRNA lists (erythroid and global) were used, only one ABT subject was not identified as abnormal or potentially positive for ABT (score 11/12 = 92%). Despite a high heterogeneity within the male and female groups, minimal sex-biased miRNAs differences were observed. Interestingly several miRNAs found up-regulated at the D-25 time point, which is 10 days after the blood withdrawal, may represent “follow-me” markers to identify athletes at risk of ABT to be tested in the proximity of major agonistic events. The results of the present project allow to validate a list of 14 miRNA useful, by RT-ddPCR based analysis, to detect ABT in the majority (over 90%) of the ABT-treated subjects.

Code: 17D15XD 

The detection of pseudoendogenous steroids is based on the alterations of the parameters of the steroid profile included in the steroid module of the Athletes’s Biological Passport (ABP). A Bayesian statistical inference approach allows fixing the boundaries for the ABP parameters of a given individual based on the previous data collected. In the case of a suspicious sequence of data or data outside the population based ranges when previous values are not available, a specific confirmatory method based on isotope ratio mass spectrometry (GC-C-IRMS) is mandatorily applied to the suspicious sample to disclose the synthetic from endogenous origin of the steroids detected. The current approach, although scientifically valid, has some limitations: (1) delay in taking appropriate decisions by the results management authorities (RMA) if the first value of the sequence is abnormal since the model needs the collection of additional samples to detect it; (2) ineffective for those athletes not tested with some frequency; (3) unable to detect low dosages with physiological effect.

In addition, new pharmaceutical formulations of synthetic steroids are not easily detectable by GC-C-IRMS if isotopic analyses of additional samples of the athlete are not performed. The aim of this study is to expand the use of IRMS at a screening level for those disciplines at higher risk of pseudoendogenous steroids abuse, simplifying the current confirmatory procedure. This would allow reducing the time of evaluating an abuse of pseudoendogenous steroids including those athletes less frequently tested, detecting lower dosages of administrations and for a longer period of time and finally being able to detect those preparations not currently detectable by the longitudinal evaluation of the IRMS data since these data would be now available.

Code: ISF17R02NN

This project is aimed at identifying markers of rHuEPO misuse and separate these from markers of natural hypoxic exposure by metabolomics. Male (n=20) and female (n=20) non-competing athletes are exposed to 4 weeks sea-level investigation and after a full washout period, participants are exposed to hypobaric hypoxia at 2.320 m for four weeks. Four groups are formed in a blinded randomized and gender balanced fashion: One group (n=20) receives EPO during the sea-level period and placebo during the stay
at altitude. Another group (n=8) receives EPO both at sea-level and altitude. A third group (n=8) receives placebo both at sea-level and altitude and the fourth group (n=4) receives placebo at sea-level and EPO at altitude. The design facilitates identification of markers of EPO misuse at sea-level (n=28) as well as markers of natural altitude exposure (n=28). Each participant’s treatment response is analyzed in relation to a 4 week baseline period. Moreover, the design includes an intra-individual possibility to verify identified targets expected to be unique to rHuEPO misuse, since 8 participants are treated with rHuEPO both at sea-level and altitude. Runners are enrolled due to the good possibility of recruitment and conduction of training at altitude. The project results in a highly valuable biobank for identification of markers sensitive to initiation and termination of rHuEPO injections. Metabolomics discovery is carried out in collaboration with a world-leading research group as well as the experts at University of Cologne, Germany. Importantly, the project is a joint effort between world-leading University research, Anti-doping organizations and WADA laboratories.

Main Findings

With the present study we were able to demonstrate that recombinant human erythropoietin (rhEpo) treatment combined with altitude exposure provide an additive erythropoietic response compared to rhEpo treatment or altitude exposure alone. Accordingly, analysing the results in the Athlete

Biological Passport revealed that the passport is more sensitive to rhEpo treatment at altitude than at sea level, but a concurrent compromised specificity exist with altitude exposure.

We also investigated the potential of immature reticulocyte fraction (IRF) and the ratio between immature reticulocyte and red blood cells (IR/RBC) as novel biomarkers for rhEpo treatment. With this study we demonstrate that a low-dose rhEpo treatment alters IRF and IR/RBC compared with placebo, which also exceeds the fluctuations induced by altitude exposure in the post-treatment period. Similarly, during rhEpo treatment at altitude, both biomarkers exceeded the fluctuations induced by placebo injections at altitude. When individual thresholds were calculated, we were able to identify ~90% of the rhEpo treated participants at sea level with each variable, whereas we were able to identify ~30% and ~65% of the rhEpo treated participants at altitude for IRF and IR/RBC, respectively. Importantly, these sensitivities were obtained with a specificity >99%.

Plasma samples have been subjected to untagerted metabolomics followed and bioinformatic analyses. No biomarkers with sufficient sensitivity and specificity to discriminate between sea-level or altitude exposure could be identified. Likewise, no biomarkers able to discriminate rhEpo and placebo treatment could be identified.

Finally, the present study demonstrated that the novel markers of iron homeostasis, hepcidin and erythroferrone, are more sensitive markers of altered iron homeostasis within one week of changes in iron demands than routine iron markers. When individual thresholds were calculated, 30-40% of the rhEpo treated participants exceeded the thresholds. Our results demonstrate that erythroferrone, and to some extent hepcidin, may hold promise as novel biomarkers for detecting rhEpo misuse at altitude, but intra-individual variability is of concern and additional studies are required

Code: ISF17A12MT

In sports drug testing, the use of alternative matrices such as dried blood/plasma spots (DBS/DPS), oral fluid (OF), hair, and exhaled breath (EB) can be favorable in terms of the duration, intrusiveness and invasiveness of the sampling procedure as well as analyte stability and overall costs for transportation and storage. While DBS already have become an emerging complementary matrix for a broad range of clinical and forensic applications, the utility for doping control purposes was just recently demonstrated. For a variety of low molecular weight analytes such as anabolic agents and stimulants, detection strategies were successfully developed, however, only a few protein-and peptide-based drugs (e.g. Synacthen and IGF-I) were considered so far. Therapeutic proteins have emerged to an important class of new pharmaceuticals and comprise many drug candidates with potential performance-enhancing properties. The misuse of specific protein-/peptide-based drugs in sports is prohibited and both immunological as well as mass spectrometry-based proteomic approaches are currently used for their detection from urine and/or serum samples. Within this study, analytical strategies for the analysis of peptide hormones from DBS specimens will be developed by using different proteomic approaches such as (immuno-)affinity purification, proteolytic digestion, and LC-MS/MS. As extraction, processing and analysis of DBS are automatable, a novel workstation specially designed for a standalone-preparation of DBS samples will be tested and, if successful, employed for routine doping controls. Both “classical” and novel protein and peptide therapeutics will be used as model compounds for method development and characterization: Growth hormone releasing peptides (GHRPs), a myostatin inhibiting therapeutic antibody, and erythropoiesis-stimulating Fc fusion proteins (Sotatercept, ActRIIA-Fc, ACE-011). This study will contribute to the expansion and improvement of available test methods for performance-enhancing proteins and peptides from DBS.

Main Findings

Dried blood spots (DBS) are a relatively new alternative matrix in sports drug testing, which is advantageous with regards to the invasiveness and intrusiveness of the sample collection procedure, the effort and costs for sample transportation and storage, and the analyte stability. Most of the existing doping control DBS assays include low molecular mass analytes (e.g. anabolic agents and stimulants) and only a few methods for peptidic drugs such as Synacthen and IGF-I have been developed so far.

Within this research project, a DBS detection method facilitating the analysis of insulin and its synthetic or animal analogues was established and comprehensively characterized. The successful analysis of these substances at physiologically relevant concentrations was realized after ultrasonication-assisted extraction, immunoaffinity purification, and liquid chromatographic separation followed by high resolution mass spectrometric detection (with or without ion mobility). During method development, major challenges were an efficient purification of the target peptides from the DBS matrix in combination with the low sample volume of 20 µL. Therefore, DBS analysis of insulins at the present stage cannot reach the sensitivity and simplicity of established serum or plasma analysis. Thus, classical plasma/serum or urine analysis is still superior and recommended in case of quantitative analysis. But for the analysis of non-fasting / non-basal insulin levels, this method can provide reliable qualitative results and opens the possibility to simplify the sample collection, transfer, and storage procedures.

Additionally, two complementary LC-HRMS detection methods for the emerging erythropoiesis-stimulating agent Sotatercept (ActRIIA-Fc) from DBS were developed and validated: An initial testing and a confirmation procedure. Both methods comprise an ultrasonication-assisted extraction, affinity enrichment, proteolytic digestion, and HRMS detection by Orbitrap MS. Due to the generic extraction, the multi-analyte initial testing procedure enables the collection of retrospective data and therefore a simultaneous detection of different IgG-based drug analytes. As proof-of-concept, artificial samples fortified with the emerging protein drugs Luspatercept and Bimagrumab as well as authentic post-administration samples containing Bimagrumab were successfully analyzed.

Finally, an automated extraction method for GHRPs from DBS was set up by using the Gerstel MultiPurposeSampler (MPS). Different parameters such as the composition of the extraction buffer, the cartridge used for subsequent online solid-phase extraction (SPE), and the SPE elution buffer were optimized, enabling detection limits at ng/mL levels.

Publications:

Lange T, Walpurgis K, Thomas A, Geyer H, Thevis M. Development of two complementary LC–HRMS methods for analyzing sotatercept in dried blood spots for doping controls. Bioanalysis 2019;11:923–40