Funded Projects

The PCC has supported world-class research since 2008, investing more than $35.6 M to drive novel science. Research and grant-making are the foundation of the PCC and are the focus of everyday business activity. PCC-supported research contributes to a movement in addressing doping’s root causes and ultimately decreasing the use of performance-enhancing drugs by all participants in all sports at all levels of play.

Funded PCC projects represent proposals from 23 different countries and three continents. More than 100 principle investigators have been awarded grants to advance anti-doping knowledge through more than 250 unique research projects. Projects which showcase alignment with the PCC research priorities are given funding precedence.

Please contact us if you have questions regarding individual projects listed here.

Fellowships

Funded: 2022

Fellow:
Dr. Jenna Goodrum, Sports Medicine Research and Testing Laboratory (SMRTL)

Mentor:
Dr. Goeff Miller, SMRTL

Research Title: 

Biotin as a masking agent for hCG

Funded: 2022

Fellow:
Dr. Sara Amalie Solheim, Oslo University Hospital and the Norwegian School of Sport Sciences

Mentor:
Dr. Yvette Dehnes, Laboratory and APMU Director, Norwegian Doping Control Laboratory

Research Title:

Further development of blood testing: ABP, deterrence and virtual testing

Funded: 2022

Fellow:
Dr. Huu Hien Huynh, University of Washington

Mentor:
Dr. Andrew Hoofnagle; University of Washington

Research Title:

Quantification of type III procollagen fragments in capillary blood

Funded: 2019

Fellow:
Dr. Federico Ponzetto, University of Turin

Mentor:
Professor Ezio Ghigo; University of Turin

Research Title:

Implementation of a blood steroidal module in the framework of the athlete

Research Summary:

The research project entitled “Implementation of a blood steroidal module in the framework of the Athlete Biological Passport” and carried out at the Department of Medical Sciences of the University of Turin, aimed at providing a robust analytical tool for investigating the variations of Blood Steroid Profile. During the 2 years of the project a novel LC-MS/MS method for the quantification of 27endogenous steroids, including major circulating hormones as well as a wide panel of androgens’ phase II metabolites, was developed and validated according to WADA guidelines. This method was then used for calculating preliminary reference intervals of target analytes, some of which were never reported to date, analyzing serum samples collected from healthy volunteers accessing to the Blood Bank of Turin University Hospital. Furthermore, the potential impact of circadian rhythm and physical exercise on Blood Steroid Profile markers was investigated thanks to the analysis of serum/plasma samples collected during two dedicated clinical studies involving 19 healthy male volunteers and 31professional football players. The outcomes of these studies , beside proving the usefulness of the developed analytical platform for gathering additional information on steroid metabolism, useful for the introduction of blood steroid profiling in anti-doping context, highlighted that serum concentrations of testosterone(T) and dihydrotestosterone (DHT)as well as of androgens phase II metabolites are not significantly perturbated by diurnal variations linked to circadian rhythm and by a moderate physical exercise such as a football training session. These results strongly support the introduction of such markers in the future implementation of Blood Steroid Profile for doping control purposes, remarking the utility of monitoring not only T and DHT but also more concentrated phase II metabolites such as etiocholanolone glucuronide, androsterone glucuronide, epiandrosterone sulphate and androsterone sulphate. During the performed studies it was also observed that the administration of 5α-reductase inhibitors (e.g., Finasteride) could have a strong impact on target markers, stressing the need of dedicated study for better characterizing steroid response to such intake. Finally, with the aim of suggesting an easier sample collection procedure for Blood Steroid Profile, a further analytical method for the measurement of most concentrated steroidal compounds from Volumetric Absorptive Micro-Sampling (VAMS) was developed, validated and applied to assess the medium-term stability of target analytes in blood micro-matrices. The promising results obtained in this proof-of-concept study, underlining the stability of VAMS samples up to 100 days at-80°C,opened the way to the use of such technology for simplifying blood sample collection, storage and transportation in the future studies that are needed to fully characterize the perturbations of endogenous and exogenous potential confounding factors on Blood Steroid Profile. Although the advances obtained thank to the present projects, there is still lack of population studies for the definition of reference intervals in elite athletes’ populations as well as for the assessment of long-term intra-individual variation of most promising serum markers of EAAS doping. These topics could represent the challenge for future research project in the field of steroid analysis in anti-doping context.

Funded: 2018

Fellow:
Dr. Scott Lacombe, Dell Pediatric Research Institute, University of Texas at Austin.

Mentor:
Dr. Herbert Tobias; Dell Pediatric Institute, University of Texas at Austin
Dr. Tom Brenna; Dell Pediatric Institute, University of Texas at Austin

Research Title:

Validation of ARC Catalytic Combustion Reactors for Fast GCC-IRMS and
GC×GCC-IRMS

Funded: 2018

Fellow:
Dr. Jacob Bejder, University of Copenhagen

Mentor:
Dr. Nikolai Nordsborg, University of Copenhagen

Research Title:

Improving detection of erythropoiesis stimulating agents and glucocorticoids

Funded: 2018

Fellow:
Dr. Danielle Moncrieffe, King’s College London Drug Control Centre

Mentor:
Prof. David Cowan; King’s College London Drug Control Centre

Research Title:

Improving sample preparation for the quantification of low level proteins in blood

Funded: 2018

Fellow:
Dr. Laura Lewis; Australian Catholic University

Mentor:
Dr. Daniel Eichner, SMRTL

Research Title:

Influence of Relative Energy Deficiency in Sport (RED-S) on the Athlete Biological Passport

Funded: 2016

Fellow:
Dr. Liying Jiang; King’s College London Drug Control Centre

Mentor:
Dr. David Cowan; King’s College London Drug Control Centre

Research Title:

Strategy for cost reduction in doping test using UPLC-ESPI-TOFMS for urine and DESI-QTOFMS for DBS

Funded: 2014

Fellow:
Dr. Geoff Miller; Sports Medicine Research and Testing Laboratory (SMRTL)

Mentor:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)

Research Title:

Assessing hydration status through evaluation of albumin osmolality and lactate for the ABP

Micro-Grants

Funded: 2016

Principal Investigator:
Dr. Lena Ekstrom; Karolinska Instituet

Funded: 2016

Principal Investigator:
Prof. Dr. Maria Kristina Parr; Freie Universitat Berlin

Funded: 2016

Principal Investigator:
Dr. Jack Henion; Q2 Solutions

Funded: 2018

Principal Investigator:
Dr. James Hopker, University of Kent

Research Summary:

This PCC Micro-grant was used to fund a Research Assistant to work on a 6-month project in order to establish the efficacy of performance monitoring in the identification of doping in sports. We used a retrospective analysis of existing performance results to determine how performance evolves over an athlete’s career by applying various statistical and machine learning methods. We initially aimed to extend our previous work involving the use of a Bayesian Latent Factor Regression model to construct performance trajectories of male shot put athlete’s careers. We hypothesized that these modelled trajectories would enable us to identify differences between the careers of doped and presumed clean athletes. Unfortunately, our model proved not to be sensitive enough to detect the variability in athlete performance in order to detect small instances of performance enhancement from doping, especially across different seasons. We therefore considered an alternative Bayesian methodology called change-point analysis. The change-point approach sought to standardize an athlete’s performance across the course of their career taking into consideration a predicted career trajectory derived from presumed clean athletes in the same dataset. This change-point analysis was able to identify patterns of performance trajectory changes common across doped athletes in the dataset that are not evident in the majority of presumed clean athletes. However, we were also able to identify “clean” athletes within the dataset who had similar performance trajectories to previously sanctioned athletes. The performance trajectories we observed in the male shot put athletes were also persistent across other disciplines of track and field athletes we studied (women’s 800m,female shot put, and men’s 100m). These results suggest there is a need to conduct further work on the use of the Bayesian change-point analysis in order to refine modelling of rate and change in rate of performance. Moreover, there is a need to test the model’s ability to predict doping status in athletes, given their performance trajectory. Alongside the statistical approaches, we also investigated whether unsupervised machine learning was able to identify patterns of performance evolution indicative of doped and clean athletes. Unfortunately, this proved to be unsuccessful and was not able identify common patterns of performance trajectories for doped and presumed clean athletes. Further work is therefore required in order to refine and optimize our machine learning approaches involving clustering and neural network algorithms.

Funded: 2018

Principal Investigator:
Dr. G. Reverter Fundació, IMIM

Funded: 2018

Principal Investigator:
Dr. Nikolai Nordsborg, University of Copenhagen

Funded: 2018

Principal Investigator:
Dr. Malcolm McLeod, Australian National University

Funded: 2019

Principal Investigator:
Dr. Yvette Dehnes, Norwegian Doping Control Laboratory

Funded: 2020

Principal Investigator:
Dr. Siri Dorum, Norwegian Doping Control Laboratory

Funded: 2020

Principal Investigator:
Dr. Andy Hoofnagle, University of Washington

Funded: 2020

Principal Investigator:
Dr. Dilshadbek Usmanov, Institute of Ion Plasma and Laser Technologies, Uzbekistan Academy of Sciences

Funded: 2020

Principal Investigator:
Dr. Guenter Gmeiner, Seibersdorf Labor GmbH

Funded: 2020

Principal Investigator:
Dr. Francesco Botre, Laboratorio Antidoping FMSI

Funded: 2020

Principal Investigator:
Dr. Janusz Pawliszyn, University of Waterloo

Funded: 2020

Principal Investigator:
Dr. Ole N. Jensen, University of Southern Denmark

Funded: 2020

Principal Investigator:
Dr. Danielle Moncrieffe, King’s College London Drug Control Centre

Funded: 2020

Principal Investigator:
Dr. Geoff Miller, Sports Medicine Research & Testing Laboratory (SMRTL)

Funded: 2020

Principal Investigator:
Dr. James Hopker, University of Kent

Research Summary:

This report provides an overview of the work conducted as part of a Micro Grant aimed at investigating the utility of performance monitoring to be used for anti-doping. For the project we have been working with USADA to explore performance data from male 100m sprinters who are affiliated to USATF. We implemented a Bayesian hierarchical model to analyze athlete career trajectories accounting for the effects of ageing and confounders of 100m sprinting performance (i.e. wind velocity). After building a model of population-wide model we were able to explore how individual performance trajectories differed from the overall average, with those exceeding this level being deemed to display ‘excess performance’. However, as good, or exceptional performance is not necessarily linked to doping, we decided to explore changes in ‘excess performance’ over fixed period of time (1, 2 or 3 years)–termed ‘yearly excess performance’. We identified that shorter periods of time resulted in better model predictions than those of longer duration. To enable us to account for the top performing athletes showing consistent levels of excess performance when compared against the population average, we used an Empirical Distribution Function to explore how performance trajectory is affected by athlete performance level. By dividing our sprinters in to 4 quartiles we were able to adjust our measure of excess performance based upon the performance level of the athlete. Using the probability for an athlete to demonstrate a ‘yearly excess performance’ we subsequently demonstrated that our performance model was able to discriminate between sprinters who have a historical anti-doping rule violation and those who do not (AUC = 0.83).The report also highlights the challenges associated with data quantity and quality which impact on the robustness of its predictive capacity and reduce chances of bias and false positives arising from the analytical process. Finally, we highlight the opportunities for this work to be transferred into anti-doping practice via our collaboration with USADA. However, we also acknowledge that further research is required to explore the transference of our generalized performance model to other sports and disciplines in advance of it becoming incorporated into anti-doping practice within ADOs.

Funded: 2020

Principal Investigator:
Dr. Gustavo Cavalcanti, Laboratorio Brasileiro de Controle de Dopagem (LBCD), Federal University of Rio de Janeiro, Brazil

Funded: 2020

Principal Investigator:
Dr. Dustin Nabhan, United States Olympic and Paralympic Committee

Funded: 2020

Principal Investigator:
Dr. Oscar Pozo, IMIM Hospital del Mar Research Institute – Bioanalysis Research Group

Funded: 2021

Principal Investigator:
Dr. David Handelsman, ANZAC Research Institute

Funded: 2021

Principal Investigator:
Dr. Andy Hoofnagle, University of Washington

Research Summary:

In collaboration with the Collagen Turnover Working Group, the Hoofnagle Laboratory finished development and validation of a novel assay to quantify P-III-CP and type III collagen breakdown products in human plasma, which we hope will be useful for the sensitive detection of growth hormone doping by athletes. The assay uses the approach of trypsin digestion of proteins in serum and peptide immunoaffinity enrichment of surrogate peptides prior to the LC-MS/MS simultaneous quantification of unlabeled, endogenous peptide along with an isotope-labeled analog internal standard peptide. The ratio of the chromatographic peak area of the unlabeled peptide to the labeled peptide, also known as the peak area ratio, is used to quantify the peptides, which is referred back to calculate the protein concentration in the original sample. Our laboratory has developed two monoclonal antibodies to peptides in type III collagen, which we used to develop the assay. The approach to the validation of the assay borrowed from guidelines published by the Clinical and Laboratory Standards Institute (C62 and C64). Experiments included precision, linearity, interferences, limits of quantification, and stability. This work culminated in a paper being accepted to the journal Clinical Chemistry, the most prominent journal in its field.

Funded: 2021

Principal Investigator:
Dr. Nicolas Leuenberger, Swiss Laboratory for Doping Analyses

Research Summary:

Aim: We assessed the feasibility of using hematological parameters (such as hemoglobin and reticulocyte mRNA) in dried blood spot (DBS) samples to test athletes for doping and to improve patient care.

Methods: Hemoglobin and erythropoiesis-related mRNAs were measured in venous blood and DBSs from both healthy athletes and hemodialysis patients.

Results: We accurately measured hemoglobin changes overtime in both venous blood and DBS samples. Combining hemoglobin and mRNA analyses, we detected erythropoietin injection in DBSs more sensitively and with higher efficiency by using the DBS OFF-score than by using the athlete biological passport OFF-score.

Conclusion: DBS-based measurements are practical for calculating hemoglobin levels and athlete biological passport OFF-scores. This approach may help detect blood doping and help predict patient response to EPO.

Funded: 2021

Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry

Research Summary:

A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher-order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the “organ-on-a-chip” principle. In a multi-channel microfluidic cell culture chip, 3-dimensional tissue spheroids, which can constitute artificial and interconnected microscale organs, imitate principles of the human physiology. The objective of this study was to determine if the technology is suitable to adequately predict metabolic profiles of prohibited substances in sport. As model compounds, the frequently misused anabolic steroids, stanozolol and dehydrochloromethyltestosterone (DHCMT) were subjected to human liver spheroids in microfluidic cell culture chips. The metabolite patterns produced and circulating in the chip media were then assessed by LC-HRMS/(MS) at different time-points of up to 14days of incubation at 37°C. The overall profile of observed glucurono-conjugated stanozolol metabolites excellently matched the commonly found urinary pattern of metabolites, including 3’OH-stanozolol-glucuronde and stanozolol-N-glucuronides. Similarly, but to a lower extent, the DHCMT metabolic profile was in agreement with phase-I and phase-II biotransformation products regularly seen in post-administration urine specimens.

Funded: 2021

Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry

Research Summary:

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry(IRMS)-based methods for confirmation. Especially concentration ratios like T over epitestosterone (E)or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17genotypeand/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples.

In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated considering the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone-sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes.

The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T-replacement therapies and excretion study samples were investigated.

Funded: 2021

Principal Investigator:
Dr. Holly Cox, SMRTL

Funded: 2021

Principal Investigator:
Nasser Al Ansari, Anti-Doping Lab Qatar

Funded: 2021

Principal Investigator:
Dr. David Cowan, King’s College London

Funded: 2021

Principal Investigator:
Dr. Guenter Gmeiner, Seibersdorf Labor GmbH

Funded: 2021

Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry

Funded: 2022

Principal Investigator:

Dr. Nicholas Leuenberger, Swiss Laboratory for Doping

Working Groups

Principal Investigator:

Dr. John Higgins; Massachusetts General Hospital
Dr. Nikolai Nordsborg; University of Copenhagen
Dr. Jakob Bejder; University of Copenhagen
Dr. Daniel Eichner, SMRTL
Dr. James Cox, University of Utah
Dr. Merav Socolovsky, U Mass Medical School
Dr. Maziyar Baran Pouyan, University of Pittsburgh Medical School
Dr. John Phillips, University of Utah
Dr. Steve Elliott, PCC Scientific Advisory Board
Dr. Mike Sawka, PCC Scientific Advisory Board

Center of Excellence (Lab Equipment) Grants

2022 Grants

Principal Investigator:

Dr. Fong HaLui, National Measurement Institute, Australia

Principal Investigator:

Dr. Fong HaLui, National Measurement Institute, Australia

Principal Investigator:

Dr. Jacob Bedjer, University of Copenhagen

2021 Grants

Principal Investigator:

Dr. Andy Hoofnagle, University of Washington

Funded: 2021

Principal Investigator:
Dr. Geoffrey Miller, SMRTL

Funded: 2021

Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry

Funded: 2021

Principal Investigator:
Dr. Bradley Johnson, Texas Tech University

Funded: 2021

Principal Investigator:
Dr. John Eiler, California Institute of Technology

Funded: 2021

Principal Investigator:
Dr. Nikos Ntoumanis, University of Southern Denmark

2020 Grants

Principal Investigator:

Dr. Mario Thevis, German Sport University Cologne

Principal Investigator:

Dr. Herb Tobias, Dell Pediatric Institute

Principal Investigator:

Dr. Christian Reichel, Seibersdorf Labor GmbH Doping Control Laboratory

Principal Investigator:

Dr. Michael Zimmerman, European Molecular Biology Laboratory

Research Summary:

The synthesis of novel performance-enhancing drugs and their detection by analytical laboratories is an arms race, in which timely detection and identification of novel performance-enhancing compounds are central to efficient anti-doping control. In anti-doping laboratories, chromatography-coupled mass spectrometry-based methods have become the standard technique to detect and quantify prohibited substances in the urine. Specifically, LC-MS has been shown to offer specificity, precision, and limits of quantitation, enabling high-throughput analysis with minimal sample preparation. Several untargeted mass spectrometry-based approaches have been developed to specifically pinpoint novel, unknown androgenic anabolic steroids (AAS). However, extensive liver metabolization of steroids leads to the fact that only a fraction of unchanged AAS in found in urine, adding analytical challenges to detection and quantification. In the present project, we have tested the feasibility of a novel approach to identify unknown AAS through a combination of MS/MS prediction and molecular networking of simulated and experimental data acquired with high-mass resolution UHPLC-qTOF systems. Results have shown that the chemical structures of controlled AAS share high structural similarity with known steroids. Hence, molecular networking (MN) could cluster these structures according to the structural features of their steroidal core, classifying these analytes into six chemical classes. Strikingly, the presence of double bonds in rings A and B, the carbonyl group in position 3 and the side chain in position 17 seem to have a high impact on fragmentation. Using the data gathered from MN, we have extracted the most conserved ions from each chemical class of steroids and have used this information to improve the predicted MS/MS fragmentation patterns of these steroids. Predicted MS/MS data were also treated to remove fragments lower than m/z 50, as well as low normalized intensities. These treatments have improved MS/MS prediction by 16%, significantly increasing the efficiency of the detection. Lastly, we have also compiled a theoretical database of over 1,400 steroids, including their molecular characteristics, predicted metabolic products, and MS/MS spectra (of steroid compounds and their known and predicted metabolites) in three collision energies. This comprehensive dataset will help to better identify and annotate previously undescribed steroids.

Principal Investigator:

Dr. Aviv Amirav, Tel Aviv University

Principal Investigator:

Dr. Jen-Tsan Chi, Duke University

Principal Investigator:

Dr. Christopher Chouinard, Florida Institute of Technology/Clemson University

Research Summary:

The World Anti-Doping Agency (WADA) Prohibited List bans anabolic androgenic steroid use by athletes at all times. The list contains over 60 compounds which are most commonly detected and quantified using chromatography and mass spectrometry-based methods. One of the challenges to detection of current and new substances is the presence of isomers, compounds with identical molecular weight that often cannot be differentiated even by tandem mass spectrometry(MS/MS). However, in addition to those compounds explicitly named by WADA’s Prohibited List, also banned are “other substances with a similar chemical structure or similar biological effect(s).” As such, there is an urgent need to develop new methods capable of identifying previously undetected species, especially in the presence of potential endogenous or exogenous isomers. The project proposed a novel method by which to combine experimental approaches (collision cross section measurements and structurally specific reactions) with computational modeling and machine learning to create a predictive database that can be used with LC-IM-MS methods for identification of new compounds.

The proposed work first involved accurate measurement of collision cross section (CCS) for more than half of the WADA prohibited anabolic steroids. This data, coupled with chromatographic retention time accurate mass, and MS/MS fragmentation pattern can be used to increase confidence of identification in a targeted analysis. Furthermore, measurement of this group also provided structural trends that might be used for identification of future unknowns. These results were presented in a recent publication (Velosa et al. JASMS, 2022, 33, 54-61). We have also started applying these methods to other classes of WADA prohibited substances including glucocorticoids (Neal et al. JMSACL, 2022, 24, 50-56). Next, several reactions were investigated for their improvements to IM resolution and structural characterization. Initial reactions were based on our previous work involving ozonolysis (Maddox et al. JASMS, 2020, 31, 411-417) and Paternò-Büchi reactions (Maddox et al. JASMS, 2020, 31, 2086-2092), but have now expanded to include reactions with Girard’s Reagent P and 1,1,-carbonyldiimidazole. These results demonstrate improved IM separations and structural identification and are the subject of a recently submitted publication (Velosa et al. 2022, In Revision). Finally, the current experimental results are being used to with computational modeling to develop a machine learning algorithm to be used for prediction of theoretical CCS.

Principal Investigator:

Dr. Jean-Francois Naud, Laboratiore de Controle du Dopage

2019 Grants

Principal Investigator:

Prof. Francesco Botré, Federazione Medico Sportiva Italiana

Principal Investigator:

Dr. Alexandre Marchand, French Anti-Doping Agency

Research Summary:

Growth Hormone (GH) use has been prohibited by the World Anti-Doping Agency (WADA) for many years, however detecting doping with GH has proven to be very difficult. Two methods have however been validated by WADA to identify GH doping in serum samples:

1-the GH isoforms test consists of 2 immuno lumino metric assays (ILMA) that measures the full length22KDa form of recombinant GH (recGH) and the isoforms of GH produced by the pituitary gland (pitGH). The ratio of rec GH/pit GH increases after an injection of recombinant GH. 2-an indirect test that is based on the measurements of two biomarkers, IGF-I and P-III-NP, that increase in circulation following GH administration. IGF-I can be measured by an automated immuno-assay and/or by LC-MS/MS, P-III-NP by another automated immuno-assay and/or a radio-immunoassay.

The recent development of Multiplex array technology combining the detection of multiple targets in a single well of ELISA-plate opens new possibilities to simplify the detection of GH doping. Meso Scale Discovery (MSD) is one of the commercial leaders in this field. Assays can be processed in a few hours using protocols that are similar to those used in ELISA assays, although typically with fewer steps. Our aim was to use MSD technology to produce a customized multiplex assay dedicated to GH detection for doping controls. This array could include GH proteins (recGH and pitGH) , GH biomarkers (IGF-I, P-III-NP) and potential new biomarkers: fibronectin1 (FN1) and apolipoprotein 1 (APOL1) and Vitamin D binding protein (VDBP). The development effort included:

– screening available antibodies and selecting the best antibody pairs for each target (specificity, sensitivity, linearity, reproducibility)

– multiplexing analytes in as few MSD assays as possible.

– a final comparison of analytes concentrations in 50 serum samples provided by the French Anti-doping Laboratory (AFLD), including positive controls (serums resulting from GH administration) tested either with MSD multiplex assay(s) and with WADA approved techniques (IGF-I kit on IDS-Isys from Immuno Diagnostic Systems (IDS), P-III-NP kit on Advia Centaur (Siemens), hGH ILMA kits from CMZ-Assay GmbH for recGh and pitGH) or ELISA kits for the potential new biomarkers FN1 and APOL1.

The project only reached part of its goals as more technical issues than anticipated were identified during the development. A single multiplex to detect at once all 7 analytes was not possible. Despite extensive search for antibodies, no P-III-NP antibody pair tested allowed detection of P-III-NP in serum and VDBP assay was not evaluated, as more financial resources were needed for other analytes. IGF-IAssay need a specific acidic pre-treatment that enabled its multiplexing. However two duplex assays were validated : one for detection of recGH+pitGH (with 2 possibilities for pitGH) and one for detection of FN1+APOL1.

APOL1 did not show evidence of increase in the serum samples from GH administration and seem nota good biomarker for GH. On the contrary FN1 is more promising and showed higher values in GH positive controls than in the athlete population tested. Additional studies with more samples are needed to see if FN1 could be added to the recognized GH biomarkers.

Two pit GH assays were validated and both worked successfully with the recGH assay as a duplex. The results indicated low rec/pit values for serum from athletes and higher values for positive controls (samples from GH administration study or GH-spiked serum). RecGH+pitGH duplex assays might represent a good alternative to the ILMA from CMZ assay GmbH: working in 96-well microplates with fewer manipulations and with only 50μL serum. The recGH/pitGH ratios were however lower than those obtained with ILMA assays (≈55-70%) due to higher concentrations obtained for pitGH, but the decision limits could be reevaluated after more extensive studies of serum samples from athletes and GH-administration studies.

Principal Investigator:

Dr. Federico Ponzetto, University of Turin

Principal Investigator:

Dr. Robert Roach, University of Colorado-Denver

Principal Investigator:

Dr. Soledad Rubio Bravo, University of Cordoba

Research Summary:

Cubosomic supramolecular solvents (SUPRASs) have been designed and synthesized directly in urine, by spontaneous processes of self-assembly and coacervation of the amphiphile 1,2-hexanediol in the presence of sodium sulfate. These SUPRASs, here synthesized for the first time, consist of square and rounded cubosomes, with a size range of 140−240 nm, that are made up of 1,2-hexanediol, salt, and a high water content (36−61%, w/w).It has been proved that these cubosomic nanostructures are highly efficient to extract multiclass prohibited substances in human sport drug testing owing to their large hydrophilicity and surface area.

The applicability of cubosomic SUPRASs for the development of high throughput matrix-and compound-independent sample treatment platforms has been proved by the extraction of around a hundred of prohibited substances, including highly polar and nonpolar ones, belonging to the 10categories of the WADA list. The method involves the addition of 142 mg of sodium sulfate and 200μL of 1,2-hexanediol to 1 mL of hydrolyzed urine, the vortexing of the mixture for 5 min and centrifugation for 10 min. Then, the SUPRAS extract is directly analyzed by LC-ESI-(Q-IT)MS/MSorLC-ESI-QTOF. Chromatographic separation is carried out in a pentafluorophenyl stationary phase, which has proved the best performance in a comprehensive study on the performance of different retention mechanisms in sport drug testing by liquid chromatography-mass spectrometry.

The SUPRAS-LC-ESI (Q-IT)MS/MS and SUPRAS-LC-ESI-QTOF methods have been validated. Around82−95% of drugs were efficiently extracted (recoveries 70−120%) in urine samples, and 81−92% did not present matrix effects. The capability of extraction and interference removal of cubosomic SUPRAS was proved superior to those of other eleven SUPRASs and conventional organic solvents. The proposed SUPRAS-based sample treatment is transferrable to any WADA-accredited lab sincere agents are commercially available and operations do not require special equipment. This approach is as simple as QuEChERS, but the distinctive features of cubosomes confer them high capability in multiclass determinations.

Principal Investigator:

Dr. Daniel Eichner, Sports Medicine Research And Testing Laboratory

Principal Investigator:

Dr. Cleo Van Diemen, University of Groningen

Research Summary:

After optimization of the library prep similar fractions of EPO were in obtained in Ghent for 1%EPO, 0.1% EPO and 0.01% EPO spike in respectively. In the following experiments, the sensitivity of the test was successfully challenged by including 0.001% EPO samples while reducing the sequencing reads per sample, increasing the throughput per sequencing run (number of samples). Furthermore, the analysis pipeline of the NGS data has been modified and improved from a manual to a semi-automated process. As it is not feasible to count millions of reads manually for each test, the bioinformatics team in Ghent evaluated different strategies to reproduce manual read counting. Since the data analysis needed to mimic the manual read counting, different parameters were adjusted and evaluated to establish maximal sensitivity of the test.

After the successful transfer of the wet lab and bioinformatics methodology, the Ghent laboratory is now fully equipped to implement the following adjustments in the next design of the panel:

1. the addition of probes directed to other potential doping genes and plasmid or virus-derived sequences.

2. to allow detection of sample swapping during processing of samples. With the design optimized now, it will be easy to include probes covering a personal genomic barcode. The NGS lab has experience with a 25 nucleotide signature that is included in capturing experiments to identify patients in routine diagnostic testing. The signature will be included in the new extended design together with the new potential doping genes targets

Principal Investigator:

J.L. Clark

2018 Grants

Principal Investigator:

Dr. David Handelsman, ANZAC Research Institute, University of Sydney

Principal Investigator:

Dr. Mario Thevis, Sport University Cologne

Research Summary:

Immunopurification of doping control samples is a mandatory necessity in EPO analysis during a Confirmation Procedure; moreover, it has become common practice to also immunopurify samples for the Initial Testing Procedure. Typically used materials (e.g. Stemcell purification plate, MAIIA purification kit) rely on anti-EPO antibodies for purification. Also, the detection of EPO after electrophoretic separation and Western blotting is based on a monoclonal anti-EPO antibody, clone AE7A5, directed against a 26amino acid sequence of the N-terminal region of human EPO. While the electrophoretic separation and blot transfer can be efficiency monitored with reference standards and quality control samples, it is presently not possible to monitor the functionality of the entire sample preparation procedure. The reliance on antibodies for both purification and detection has complicated the implementation of an internal standard (ISTD). In this study, customized EPO-polyethylene glycol (PEG)-conjugates were synthesized as potential ISTDs and assessed as to their compatibility with existing sample preparation procedures for urine and blood sample analysis using the most common immunopurification techniques. Moreover, probing for the impact of the ISTD on SAR-PAGE-based EPO analysis concerning potential interference with target analytes was conducted. The presented data demonstrate that a 12 kDa PEG residue attached to human erythropoietin represents a particularly useful construct to serve as internal standard for ERA analysis. The conjugate is applicable to both urine and blood testing using the commonly employed purification techniques, supporting and improving result interpretations especially concerning specimens where the natural abundance of human EPO is low.

Principal Investigator:

Dr. Michael Polet, University of Ghent

Principal Investigator:

Dr. Daniel Eichner, SMRTL

Associated Project:

Exhaled Breath

2017 Grants

Principal Investigator:
Dr. Raul Nicoli; Swiss Anti-Doping Laboratory

Principal Investigator:
Dr. Zach Gagnon; John Hopkins University

Principal Investigator:
Dr. Jentsan Chi; Duke University

2016 Grants

Principal Investigator:
Dr. Fred Schaufele; Xcell Assay

2015 Grants

Principal Investigator:
Dr. Christian Reichel; Seibersdorf Labor GmbH Doping Control Laboratory

Research Summary:

Erythropoietin (EPO) is a hormone, which stimulates the production of red blood cells. Due to its performance enhancing effect, it is prohibited by the World Anti-Doping Agency (WADA). In order to reduce the detection window of EPO-doping, athletes have been applying low doses of recombinant EPO (e.g. < 10 IU/kg body weight, daily or every second day) instead of larger doses twice or more per week (e.g. 30 IU/kg).

Microdoses of Retacrit (epoetin zeta), an EPO biosimilar, were administered intravenously and subcutaneously to human males and females. Urine and serum samples were collected and analysed applying the new biotinylated clone AE7A5 EPO-antibody and a further optimized SAR-PAGE protocol. With the improved protocol, microdosed Retacrit (7.5 IU/kg BW) was detectable for at least 52 hours after intravenous administration. Detection windows were approximately the same for serum and urine and doubled after subcutaneous administration (ca.104 hours). Previous studies applying different electrophoretic techniques and the not further optimized SAR-PAGE protocol revealed considerably shorter detection windows for rhEPO-microdoses. Since the new biotinylated antibody performed significantly more sensitive than the non-biotinylated version, the new protocol will improve the sensitivity and hence detectability of recombinant EPO in doping control.

2014 Grants

Principal Investigator:
Dr. Mike Ashenden; Science and Industry Against Blood doping (SIAB)

Principal Investigator:
Dr. Pauline Rudd; NIBRT

2013 Grants

2012 Grants

2011 Grants

2010 Grants

Principal Investigator:
Dr. Svetlana Appolonova; Moscow Anti-Doping Laboratory

2009 Grants

2008 Grants