Projets de recherche

Code: ISF17A17OJ 

In this project, we want to detect recombinant EPO in plasma samples by liquid chromatography - mass spectrometry through the analysis of intact glycosylated peptides.

We hypothesize that with the new fragmentation techniques such as EThcD, high energy HCD and laser photodissociation, present in the latest orbitrap MS analyzers, it is feasible to detect EPO in body fluids after thorough purification protocols. We aim thus to develop a purification protocol that after a specific EPO immunocapture from plasma samples, it extracts and fractionates glycopeptides based on their disctinct glycan structures through different LC resins. After comprehensive offline fractionation, then we will quantify these intact glycopeptides through parallel reaction monitoring (PRM) acquisition. We will independently monitor each different type of recombinant EPO through trypsin or AspN proteotypic peptides, characterized by different glycan structures that will elute and fragment distinctively
 

Main Findings:

Electrophoretic methods provide te sensitivity for distinguising recombinant and endogenous EPO in human body fluid samples. However, the detection of EPO in biological samples containing both endogenous and recombinant EPO remains challenging. Mass spectrometry (MS) has emerged as a powerful tool to efficiently provide a direct view of glycoprotein profiles and it is useful for assessing differences in natural and recombinant EPO variants.

In this research project, we demonstrated the potential of native MS analysis at the intact protein level for the determination fo complex glycosylation profiles of EPO. We demonstrated the feasibility of using intact glycopeptide profiles for chracerizing EPO produced in CHO cells and HEK293 cells, respectively. We generated a large list of potential targets for distinguising recombinant and endogenous EPO in human body fluid samples. The selection criteria will include the feasibility of lowering the limit of detection compatible with human sample analysis, and selection of non- overlapping glucoforms for specific and sensitive EPO detection.

We propose an integrative approach combining native MS analysis at the intact EPO protein level with intact EPO-glycopeptide MS analysis to facilitate comprehensive characterization of EPO produced in different cell systems. Integration of data from these two approaches will confirm the feasibility of EPO-glycopeptide characterization, and ultimately facilitate the selection of potential mass spectrometry-based biomarkers for doping control.

Code: 17A07GR 

The World Anti-Doping Agency (WADA) establishes two procedures to detect recombinant human growth hormone abuse (rhGH) in sports. The first, the “hGH Isoforms Test”, is based on the separate measurement of growth hormone (GH) isoforms using immunoassays. The second, the “hGH Biomarkers Test”, involves the analysis of two GH-responsive proteins, IGF-1 and P-III-NP, using either immunoassays and/or mass spectrometry (MS)-based techniques. Although being currently applied in routine analysis in anti-doping laboratories, both present some limitations. Main drawback of the “hGH isoforms Test” is the short window of opportunity whereas for the “hGH biomarkers Test” is their dependence of age and sex parameters and the more rapid elimination of IGF-1 compared to P-III-NP reducing retrospectivity.

Recent studies have described fibronectin (FN) as a long-term biomarker of rhGH abuse. These results have been obtained applying enzyme-linked immunoassays (ELISA) to serum and dried blood spots (DBS). Thus, the inclusion of FN in the evaluation of potential rhGH misuse would improve detection of cheating athletes. However, immunological tests suffer from some limitations that MS-based methodologies could overpass. This project aims to develop and validate a liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) method to measure FN concentrations in serum and DBS. The potential advantages of the MS-based
methodology will be assessed and special focus will be done on pre-analytical effects of storage conditions and the introduction of freeze-thaw cycles. Preliminary results from our laboratory show that FN determination is affected by these pre-analytical effects. The new method will be used to quantify FN in samples from an administration study with subjects treated with rhGH and in a control group of healthy subjects to obtain preliminary reference values and preliminary potential decision limits.

Main Findings:

Detection of recombinant human growth hormone (rhGH) abuse in sports is one of the major concerns of anti-doping agencies. The discovery of new biomarkers targeting rhGH misuse would help to improve the current methods carried out routinely in anti-doping laboratories. Recently, immunoreactive fibronectin (FN) protein has been described as a potential biomarker of rhGH abuse that could increase the window of opportunity of the already approved biomarkers of rhGH misuse, IGF-1 and P-III-NP.  Traditionally, immunoassays have been the method of choice to measure protein levels in biological samples. However, these antibody-based approaches could present analytical issues, as cross-reactivity, dependence on tertiary/quaternary protein structures or interferences with endogenous antibodies, which could impact final determination. To overcome immunoassay drawbacks, this project aimed to develop a mass spectrometry-based method to quantify FN in blood samples. A panel of four tryptic peptides (out of six initially proposed) located in different domains of FN sequence was selected to be used as surrogates of FN protein for quantification. The digestion process with trypsin was improved in view of the matrix characteristics and protein expected levels (in the range of µg/mL). Once optimized, an LC-MRM-MS method was developed and validated in accordance to the World Anti-Doping Agency in the Code International Standard for Laboratories (ISL) in terms of specificity, carryover, LoD (1.2 - 2.2 fmol/µL), LoQ (range 3 - 6 fmol/μL), linearity (mean r2 > 0.99), precision (within-run 3.0 - 3.9 %; between-run 9.5 - 44.3 %), accuracy (-16.7 - 5.7 %), recovery (mean 87.7 %), matrix effect (mean 14.6 %) and stability (-15.4 - 18.3 %). This procedure was applied to a set of samples from two different studies. The first was an administration study in which 10 subjects were treated for three consecutive days with a moderate dose of rhGH (0.027 mg/kg) and compared with two untreated subjects. The analysis of FN concentration using the mass spectrometry validated method indicated that no differences were present due to the drug treatment, in disagreement with previous results by immunoassay. For the second study, and because FN has been described as a potential biomarker of Dystrophic Muscular Duchenne (DMD), a population of DMD (mostly teenagers) patients was compared with a group of healthy control children. Similarly, no differences in FN levels were detected between both groups. However, when these samples were tested by immunoassay, significant differences were observed between both groups.  

These apparent divergences between results obtained with mass spectrometry and those using immunoassay could be potentially explained by the high structural and functional complexity of FN, which could impact differently depending on the antibodies’ selectivity used in immunoanalytical methods. In summary, our results spotlight the importance of method selection in peptide/protein quantification when intended to be used for bioanalytical discriminating purposes.

Code: 17A31MT 

Trenbolone is a veterinary product marketed as an effective growth promoter in animal husbandry and has never obtained medical approval for humans. Nevertheless, it is presumed to be regularly misused by athletes due to its performance enhancing properties and resulted in various adverse analytical findings in the recent years. By the end of 2016, the issue of trenbolone in sports was particularly emphasized by the McLaren report as one of the anabolic steroids extensively misused in Russia. The cocktail administered to athletes consisted of oxandrolone, methenolone and trenbolone. While the first 2 steroids have been carefully investigated in recent years and long-term metabolites were identified, this research is still pending for trenbolone. Since more than 25 years doping control laboratories search for trenbolone itself and its major urinary metabolite epitrenbolone. Detectability of trenbolone was only improved by using liquid chromatography-mass spectrometry instead of gas chromatography-mass spectrometry.

Aim of this study will be the in-depth investigation of the trenbolone metabolism in humans using a recently established methodology developed especially for metabolite detection in sports drug testing.  After administration of deuterium-labeled trenbolone, all metabolites carrying this label will be identified unambiguously by hydrogen isotope ratio mass spectrometry. Then all analytes of interest will be quantified and identified by means of high-resolution and high-accuracy mass spectrometry. Within this study we will focus on the detection of long-term metabolites prolonging the retrospective detection of trenbolone misuse.

Main Findings: 

Trenbolone is a synthetic anabolic-androgenic steroid, which has been misused for performance enhancement in sports. The detection of trenbolone doping in routine sports drug testing programs is complex as methods utilizing gas chromatography/ mass spectrometry are complicated by unspecific derivatization products and artefacts, and liquid chomatography/ mass spectrometry-based assyas have shown to allow for comparably high limits-of-detection only. The number of previously reported metabolites in human urine is limited, and most analytical methods rely on targeting epitrenbolone, trenbolone glucuronide, and epitrenbolone glucuronide.

In order to probe for the presence of additional trenbolone metabolites and to re-investigate the metabolism, an elimination study was conducted. One single dose of 10 mg of 5-fold deuterated trenbolone was administered to a healthy male volunteer and urine samples were collected for 30 days. For sample processing, published protocols were combined considering unconjugated, glucuronic acid-, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid-phase extractions, liquid-liquid extractions, metabolite de-conjugation, HPLC fractionation, and derivatization. Analytical methods included gas chromatography/ thermal conversion/ hydrogen isotope ratio mass spectrometry combined with single quadrupole mass spectrometry as well as liquid chromatography/ high accuracy/ high resolution mass spectrometry of the hydrolyzed and non-hydrolyzed samples.

Twenty deuterium-labelled metabolites were identified including glucuronic acid-, sulfo- and potential cysteine-conjugates, and characterized by parallel reaction monitoring experiments yielding corresponding produc ion mass spectra. Main metabolites were attributed to trenbolone-diol and potential trenbolone-diketone derivatives excreted as glucuronic acid and sulfo-conjugated analytes with detection windows of 5 respectively 6 days. Further characterization was conducted with pseudo MS3 experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.

Code: 17A14MT 

Compared to the metabolism of low molecular mass drugs (such as anabolic agents, stimulants etc.), the biotransformation of peptide-based drugs after (subcutaneous) administration is largely unknown. Especially for larger peptides and proteins, dedicated in-vitro models simulating the parenteral administration have been missing. Since mass spectrometric methods commonly rely on detailed information about the active drug and particular its metabolites existing in the circulation, studies providing these data are critical. In the present project it is planned to apply a sophisticated in-vitro model using skin tissue microsomes for prohibited peptides such as synacthen, insulin like growth factor, growth hormone releasing hormone and others. Selected amino acids of the utilized reference peptides will be isotopically labeled, which facilitates and accelerates the identification of formed metabolites by means of diagnostic reporter ions but does not affect the metabolic reactions of the substance of interest. High resolution mass spectrometry enables finally to identify the molecular mass and the amino acid sequence of the formed metabolites, which eventually can serve as target peptides for efficient doping controls using this Isotope-labeled Reporter Ion Detection strategy.

Main Findings: 

Investigations into the metabolism of peptidic drugs stil represents a subtantial challenge in doping controls, and the detection of the intact non-metabolized drug (condidate) is the most commonly employed strategy for most of the established detection assays. In contrast to drugs of low molecular mass (e.g. anabolic androgenic steroids, stimulants, etc.), in vitro experimental apporaches baseed on e.g. liver microsomes do not simulate appropriately the conditions for peptide and protein metabolism, as these compunds are almost exclusively applied by parenteral routes and alternative approaches have been required.

In the present project, the strategy to use skin tissue microsomes for in vitro metabolism studies of prohibited peptide hormones (insulin, synacthen and corticotropin) was shown to be a particularly informative option. Combined with the use of stable isotope-labelled peptides, the identification of resulting metabolites was significantly facilitated due to the formation of diagnostic reporter ions (derived from amino acid-generated immonium ions) still bearing the 13C of 2H isotope label(s) by means of high-resolution/high accuracy mass spectrometry. Applying this stable Isotope-labeled Reporter Ion Screening (IRIS) approach to selected prohibited peptides (insulin, synacthen and cortocotropin) yielded nearly 20 metabolites for these peptides with truncated amino acid chains. Especially for metabolically less stable peptides such as synacthen, the identification of these new metabolites will support prolonging the detection window in doping control samples. In addition, also for metabolically more stable peptides such as insulin and corticotropin, identified metabolites showed diagnostic potential concerning the differentiation between endogenous secretion and subcutaneious administration. Finally, a generically applicable approach with simplified sample preparation protocol was developed by means of mixed-mode cation exchange solif-phase extraction, which facilitates the preparation of blood samples for doping control analysis without the need for immunoaffinity purification.

Code: R16SC02SM

To analyze biological samples using SomaLogic's proprietary SOMAscan™ proteomic assay with the objective of potential discovery of new biomarkers of hGH abuse. The services will be provided in accordance with the terms and conditions set forth in the Standard SOMAscan Services Agreement attached hereto as Exhibit A.

Main findings

As shown in this study, hGH treatment results in detectable changes of SOMAmer-targeted protein analytes. Across the 10-day study period, some analytes exhibit a quadratic, or squared-type change, which generally track the hGH treatment period. Such parabolic responses that have a maximum or minimum at Day 4, and then return toward baseline levels by day 10, represent analytes that change during the administration of hGH, such as those theorized or observed in the previous WADA/SomaLogic pilot study. However, analytes discovered via a quadratic model in which measurement levels return to baseline within 6 days do not represent detectable differences > 6 days after treatment has stopped. The linear models in Section 4.2 show targets that change across the entire 10 day period, remaining elevated (or depressed) several days post-treatment. Finally, the actual magnitude of change at different timepoints may be important. For this reason, this report includes log2 of fold-change compared to baseline at several key timepoints, as well as test statistics from paired Student’s t-tests. These “key” timepoints include baseline vs. 8 hours (the initial post-treatment follow-up), baseline vs. Day 4 (the final treatment day), and baseline vs. Day 10 (the final study day). The 8-hour analysis shows the highest number of significant analyte changes, but the metabolic targets (e.g. insulin and pancreatic hormone) are suggestive of a potential post-prandial effect. The Day 10 analysis shows the least number of significant changes. The paired analysis results should be combined with the linear mixed-model (quadratic or linear) results to suggest protein targets that have plausible magnitude and directional changes as well as longitudinal trends. To this end, FDR and Bonferroni-adjusted p-values are provided, as well as unadjusted p-values are provided in the external spreadsheet “WADA_SomaLogic_HGH.xlsx”). Appropriate usage of statistically-significant results, combined with specific treatment hypotheses and biological knowledge should be combined holistically and purposefully. This report contains annotated visualizations, as well as the combined, interactive spreadsheet mentioned above, to aid in hypothesis testing so that trade-offs can be made between model type, false discovery, and particular protein targets of interest.

Code: 16C11RV

Intraarticular (IA) and periarticular (PA) uses of glucocorticoids (GC) are allowed in sports. Results obtained by our group show that concentrations of some GC in urines collected after these administration routes are greater than 30 ng/mL during 24-48 hours after administration. Therefore, the use of some GC by IA and PA routes may result in false positive results according to the current WADA rules. Due to the widespread use of GC by these routes in

sports medicine, studies need to be performed to evaluate concentrations in urine of the parent drugs after administration of different GC, and look for criteria to distinguish these allowed uses from forbidden administrations (e.g., intramuscular, IM) when needed. On the other hand, the few published data available show a decrease in plasmatic cortisol concentrations after IA administration of some GC which would suggest a potential systemic effect; no data are available for PA administration.

The objective of the present study will be to perform a thorough study on IA and PA administration of GC. The GC most frequently used in IA and PA therapies will be studied: betamethasone, triamcinolone acetonide and triamcinolone hexacetonide. First, urinary concentrations of GC and their metabolites will be evaluated after IA and PA administrations, and compared with those obtained after IM administration, to define discrimination criteria with adequate sensitivity and selectivity between these administration routes. Second, the potential systemic effect after IA and PA use of GC will be evaluated, by measuring plasmatic concentrations of the parent drugs and cortisol.

The successful outcome of the project will be directly applicable to sports drug testing by improving the discrimination criteria between allowed and forbidden administrations of GC and, therefore, by helping in the evaluation of adverse analytical findings detected in routine doping controls.

Main findings

The objective of the project was to perform a thorough study on intra-articular (IA) and periarticular (PA) administrations of glucocorticoids (GC). Three of the GC most frequently used in IA and PA therapies were evaluated: betamethasone (BET), triamcinolone acetonide (TA) and triamcinolone hexacetonide (THA). IA and PA administrations were studied in patients or athletes subjected to treatments; a total of 54 subjects were included in the IA and PA studies. In addition, 20 healthy volunteers received IM administration of BET or TA for comparison purposes.

Results demonstrated that urinary and plasmatic concentrations obtained after IA and PA administrations are similar to the concentrations reported after IM administration. Likewise, all IA and most of the PA administrations studied result in adrenal suppression and, therefore, systemic effect. Therefore, the study provided most of the scientific evidences to support the current WADA regulations regarding IA and PA administrations of GCs, where all injectable routes of administration of GC are prohibited in sport competitions.

In addition, the data obtained in the project was used to propose the minimum reporting levels for BET and TA used in all WADA accredited antidoping laboratories to declare adverse analytical findings of these compounds. Finally, the data has been also used to define washout periods after IA and PA administrations of BET, TA and THA that are currently used for all physicians around the world prescribing GCs treatments to athletes.

Code: ISF16D11XD

The detection of the exogenous administration of synthetic androgens (the called 'pseudo-endogenous" steroids) having the same chemical structure of the compounds produced endogenously (i.e.  testosterone, 5α-dihydrotestosterone or androstenedione) is primarily based on the alterations of the urinary endogenous steroid profiles. 
A Bayesian approach and adaptive model has been adopted by WADA for the management of the steroid profiles and all the parameters obtained by the Accredited Laboratories will be collected starting 1st January 2014 in a global database integrated in the endocrinological module of the Athletes Biological Passport (ABP), permitting to establish the individual reference ranges of every athlete. Once the ABP detects an atypical profile, an isotope ratio mass spectrometric (IRMS) confirmation must be applied.
The ABP will be effective once a sufficient number of data of a given individual will be collected. In normal conditions, almost two years are needed to collect such information. This will delay in any case the investigations and the time to take the appropriate decisions. Although new specific steroid metabolites have been described, the gap between the real confirmation capacity by IRMS and a suspicious finding is still too large.
IRMS data have demonstrate to be much more stable than the parameters of the ABP and a Bayesian approach similar to the one already in force for the ABP could be applied on the IRMS values. By this combined approach, both the effects on the ABP and the direct detection of pseudoendogenous steroids not produced by the athlete are possible.
The main goal of this project is to define and include in the ABP the more relevant and specific IRMS data of pseudoendogenous steroids metabolites. This should reduce the gap between the suspicion and confirmation capacities of laboratories.

Code: ISF17E11GJ 

Erythropoietin (EPO) is a natural glycoprotein hormone in the body that controls red blood cell production, and there is conclusive evidence that administering exogenous EPO results in significant performance enhancement in endurance sports. Current methods of EPO detection are unable to reliably detect micro-dosing where the drug is given at lower doses but more frequently. Exosomes are small cell derived particles in the blood and urine which contain protein. It is only recently that the importance of exosomes containing miRNAs and other proteins in cell-to-cell communication has become apparent in physiological processes, and the functions are complex and still largely unknown. Exosomes appear to have an intricate and important role in a range of blood processes related to hematopoiesis (creation of new blood cells) which is highly relevant to oxygen carrying capacity via red blood cells and endurance performance. Recent developments in sample preparation will allow us to isolate exosomes from blood and urine, and perform proteomics (simultaneous determination of protein levels across a large number of proteins using LC-MS/MS and bioinformatics) on athletes before and after treatment with EPO at low doses. Due to the intricate involvement of exosomes in red blood cell production, it is anticipated that this project will lead to identification of a number of candidate exosome proteins that will be indicative of administration of micro-dose exogenous EPO.

Code: 16B02JB 

Under this project, Operational Technologies Corporation will develop high affinity and highly specific DNA aptamers to bind the GH-releasing peptides GHRP-6, Ipamorelin and the GHRP-2 main metabolite AA-3 (D-Ala-D-(beta-naphthyl)-Ala-Ala-OH). The targets will be covalently attached to magnetic microbeads (MBs) to select for the highest affinity DNA aptamer candidates in commercially available certified disease-free human serum and urine to ensure highly specific aptamers for their cognate targets in real samples.  Following 8-10 rounds of aptamer selection and PCR amplification, aptamer candidates will be cloned and sequenced.  DNA sequences will be analyzed for partial and full-length consensus sequences.  All candidate aptamers will also be screened by ELISA-like (ELASA) colorimetric microplate assays to rank their relative affinities and specificity for their intended targets as well as related and unrelated targets.  The top candidate aptamers from ELASA screening will also be characterized by Surface Plasmon Resonance (SPR) analyses to determine Ka/Kd values versus their intended targets.

The top aptamer candidates will then be attached to MBs and used to probe spiked human serum and urine samples for the three different GHRP or metabolite targets.  Validation of aptamer-MB pulled down methods will be accomplished by mass spectral analysis at the core proteomics laboratory of the University of Texas Health Sciences Center in San Antonio, TX.  The aptamer-MB pull down protocol will be optimized to include aptamer-MB concentration, capture and elution times and chemical conditions such as pH, ionic strength and the addition of various detergents or other potential additives.  Successful development of aptamer-MB pull-down methods may enhance WADA’s ability to concentrate GHRPs or their metabolites from body fluids and enhance mass spectral GHRP detection capabilities.

Main Findings: 

Although not entirely successful for each of the 3 GHRPs, the basic requirements of this grant have been fulfilled in that several aptamers were developed in a 1:10 diluted human serum or undiluted human urine environment. Most of the 6 lead aptamers were subsequently shown to at least somewhat pull down their cognate target GHRPs in whole human serum or urine with successful detection by ESI-TOF mass spectrometry (MS). In some cases, detection by MS following aptamer-MB pull down and acid-elution failed, but could be corrected by adding 5-fold more aptamer-MBs. In another case, the pull down failure may be due to binding of the G6U-18R to a higher MW (45-60 kD) protein which interfered or competed with binding of the spiked G6 peptide. This same G6U-18R aptamer did, however, detect G6 in urine by MS as it was developed to do. The negative controls lacking aptamers on SAv-MBs demonstrated that the pull down assays were dependent on the specific aptamers tethered to the MBs and that non-specific binding of the GHRPs to the SAv-MBs was essentially non-existent.
It is noteworthy, that with the exception of the 45-60 kD protein pulled down in serum by the two lead anti-G6 aptamer candidates, the Coomassie Blue-stained electrophoresis gels of the pull down acid-eluates are clear. This observation suggests very specific and high affinity binding by the aptamers to their cognate GHRPs because very little else was pulled down on the surface of the aptamer-MBs in serum or urine. Of course, these aptamers were intentionally selected in 10% pooled serum and 100% pooled urine to minimize non-specific binding of the final selected lead aptamers.  It is rather surprising that these 6 lead aptamers bound their cognate GHRP targets with low ng detection limits in buffer by ELASA, but failed to bind in serum and urine matrices by ELASA.  Fortunately, these same aptamers on MBs were mostly able to detect their peptide targets in serum and urine by MS. This suggests that: 1) MS is more sensitive than ELASA and/or 2) active probing of a liquid sample using mobile aptamer-MBs binds more target than a static well surface. Taken together, albeit not perfect, the use of aptamers on MBs for pull down, purification or concentration of target peptides in serum or urine is a promising technique especially when coupled to MS detection which could significantly aid WADA in its search for doping athletes. 

Code: ISF16D07NN

In this 3-year international collaborative project, we will consolidate and expand the existing Athletes Biological Passport (ABP) approach as well as evaluate new strategies for detection of blood manipulation. 
The project consists of two clinical trials where samples are collected from four week doping regimes expected to be currently used by cheating athletes: A) autologous blood transfusion of volumes <150 ml packed red blood cells;
B) frequent intravenous injections of <10 IU per kg bw of recombinant human erythropoietin.          Samples collected from these main trials will be subjected to different analytical approaches, comprising ABP analyses including the abnormal blood profile score (ABPS); evaluation of reticulocyte percentage as a standalone marker; evaluation of iron-homeostatic markers potential for revealing blood manipulation including analyses of the newly discovered hormone erythroferrone; metabolomics and proteomic analyses. 
Thus the project covers a broad range of both well-known and new analytical strategies. Another unique and highly required part of the proposed project, is addressing if gender should be taken into account when interpreting fluctuations of existing and novel markers of blood volume manipulation.

Main Findings: 

With the present study we were able to demonstrate that the inclusion of the iron regulatory hormones, hepcidin and erythroferrone, can aid in the Athlete Biological Passport in the indirect detection of a small volume autologous blood transfusion. Our results demonstrate that erythroferrone, and to some extent hepcidin, may hold promise but intra-individual variability is of concern and additional studies are required. Furthermore, we demonstrate that small, frequent doses of recombinant human erythropoietin (rHuEPO) treatment administered intravenously provides sufficient erythropoietic response to increase aerobic-dominated performance. We investigated the potential of immature reticulocyte fraction (IRF) and the ratio between immature reticulocyte and red blood cells (IR/RBC) as novel biomarkers for rHuEPO treatment. With this study we demonstrate that a low dose rHuEPO treatment alters IRF and IR/RBC compared with placebo, and that such changes can aid in the indirect detection of rHuEPO misuse. When combining the current markers in the ABP with IRF and IR/RBC ~79% of the rHuEPO treated subjects were flagged at least once.