An integrative ‘Omics’ solution to the detectionof recombinant human erythropoietin and blooddopingYannis P Pitsiladis,1,2 Jérôme Durussel,2 Olivier Rabin3

1The Brighton Centre forRegenerative Medicine,University of Brighton,Eastbourne, UK2Institute of Cardiovascular andMedical Sciences, College ofMedical, Veterinary and LifeSciences, University ofGlasgow, Glasgow, UK3World Anti-Doping Agency(WADA), Montreal, Canada

Correspondence toProfessor Yannis P Pitsiladis,The Brighton Centre forRegenerative Medicine,University of Brighton,Eastbourne, UK;Y.Pitsiladis@brighton.ac.uk

Received 4 February 2014Accepted 13 February 2014

To cite: Pitsiladis YP,Durussel J, Rabin O. Br JSports Med Published OnlineFirst: [please include DayMonth Year] doi:10.1136/bjsports-2014-093529

ABSTRACTAdministration of recombinant human erythropoietin(rHumanEPO) improves sporting performance and henceis frequently subject to abuse by athletes, althoughrHumanEPO is prohibited by the WADA. Approaches todetect rHumanEPO doping have improved significantly inrecent years but remain imperfect. A new transcriptomic-based longitudinal screening approach is beingdeveloped that has the potential to improve theanalytical performance of current detection methods. Inparticular, studies are being funded by WADA to identifya ‘molecular signature’ of rHumanEPO doping andpreliminary results are promising. In the first systematicstudy to be conducted, the expression of hundreds ofgenes were found to be altered by rHumanEPO withnumerous gene transcripts being differentially expressedafter the first injection and further transcripts profoundlyupregulated during and subsequently downregulated upto 4 weeks postadministration of the drug; with thesame transcriptomic pattern observed in all participants.The identification of a blood ‘molecular signature’ ofrHumanEPO administration is the strongest evidence todate that gene biomarkers have the potential tosubstantially improve the analytical performance ofcurrent antidoping methods such as the AthleteBiological Passport for rHumanEPO detection. Given theearly promise of transcriptomics, research using an‘omics’-based approach involving genomics,transcriptomics, proteomics and metabolomics should beintensified in order to achieve improved detection ofrHumanEPO and other doping substances and methodsdifficult to detect such a recombinant human growthhormone and blood transfusions.

INTRODUCTIONErythropoietinErythropoietin is a glycoprotein hormone producedprimarily in the kidneys that regulates red blood cellmass by stimulating the survival, proliferation anddifferentiation of erythrocytic progenitors.1 Anaemiais a common complication of chronic kidney diseasecaused principally by a deficiency in the synthesis ofendogenous erythropoietin.2 Recombinant humanerythropoietin (rHumanEPO) was first produced inthe 1980s.3 4 In 1989, a 40-year-old patient withchronic kidney disease-associated anaemia who hadundergone chronic haemodialysis for 7 years, anunsuccessful kidney transplantation and received313 units of red blood cells, was the first patient toreceive rHumanEPO treatment.5 Within 8 weeks ofrHumanEPO therapy, his haematocrit increased from15% to 38%, red blood cell transfusions were nolonger required, he was able to play handball and

resumed his physically demanding work.5 Since thissuccess of almost biblical proportions, many clinicaltrials have demonstrated the efficacy and short-termsafety of rHumanEPO for increasing and maintaininghaemoglobin concentration, for reducing the needfor red blood cell transfusions and improving thequality of life of patients with anaemia.6 These posi-tive effects of rHumanEPO have to be weighedagainst the increased risk of venous thromboembolicevents and potential unknown adverse long-termeffects in patients.6 rHumanEPO is currently widelyused to treat symptomatic anaemia caused by chronickidney disease and anaemia with related symptoms insome patients with cancer receiving chemotherapy, aswell as to increase the amount of blood that can betaken from individuals donating their own bloodbefore surgery.

Brief history of drug detection in sport withparticular reference to blood manipulation anderythropoietinDuring the Olympic Games held at moderate alti-tude (2250 m at sea level) in Mexico City in 1968,altitude-induced blood adaptations such as anincrease in haemoglobin concentration were consid-ered primarily responsible for athletes living at alti-tude wining most of the endurance races and thisphenomenon instigated a new research focus onaltitude training in the field of sports physiology.As an example, the pioneering work of Ekblomet al7 demonstrated that elevation in haemoglobinconcentration increased maximal oxygen uptake( _VO2max) and improved performance. It is notablethat the first accounts of blood manipulation insport as well as the term ‘blood doping’ emergedsoon thereafter in the media.8 There is evidencethat blood transfusions were being used in the1970s and 1980s by athletes such as cyclists, mar-athoners and cross-country skiers.9

Doping in sport seems to adapt rapidly tonew available technology. The appearance ofrHumanEPO, which was shown to successfullyincrease haemoglobin concentration in patients,radically transformed blood doping practices.Following the ‘transfusion era’, rHumanEPOdoping took over in the 1990s, as was highlightedby numerous doping cases during the Tour deFrance cycle race and especially the Festina affair in1998.10 In 2000, Birkeland et al11 demonstratedthat rHumanEPO administration for 4 weeks in 20male athletes increased haematocrit from 43% to51%, _VO2max by 7% as well as time to exhaus-tion by 9%. The practice of red cell transfusionshas made a strong comeback in recent years in

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response to the development of antidoping tests. Based on testi-monies from athletes, it is now known that athletes are usingrHumanEPO in combination with blood transfusions.10

In 1990, erythropoietin was included on the list of prohibitedsubstances by the International Olympic Committee becausemisuse by athletes was suspected, although no approved testexisted.12 There were rumours during this time that athletesused large doses of rHumanEPO to induce very high-blood par-ameter values. For example, Bjarne Riis, the 1996 Tour deFrance winner (who admitted doping in 2007), acquired thenickname of ‘Mr 60%’ which referred to his haematocritlevel.13 Because rHumanEPO was directly undetectable at thetime and as well as to protect the health of athletes, the 50%haematocrit rule was introduced in 1997 by the InternationalCycling Union.14 Briefly, any racing cyclist who had a haemato-crit above 50% (50% for male athletes and 47% for female ath-letes) was declared ineligible and was excluded from the race.Despite this rule, the Festina scandal at the Tour de France in1998 provided the proof of organised and widespread dopingin professional cycling and highlighted the need for the creationof an independent international agency, which would set unifiedstandards for antidoping work and coordinate the efforts ofsports organisations and public authorities. The WADAwas con-sequently established in 1999.

Although the detection of rHumanEPO doping is difficultbecause rHumanEPO is structurally very similar to endogenouserythropoietin, a direct urinary test based on the presence ofdifferent isoforms in isoelectric profiles was developed and pub-lished by Lasne et al.15 This test became the first approvedmethod for the direct detection of rHumanEPO doping. Whilethe effects of rHumanEPO administration can last for weeks,the test has a rather limited detection window, that is, a fewhours up to a few days depending on the dose used, the routeof administration and the individual metabolism.16 17 At thesame time as the development of the direct test, potential indir-ect detection of rHumanEPO using blood markers of alterederythropoiesis was being investigated.8 Notably, following theinitial work of Parisotto et al18 (ie, the so-called first generationblood tests), Gore et al19 developed the second generation ofstatistical models using indirect markers of altered erythropoiesisin order to detect rHumanEPO doping during (ON-model) andafter repeated recent (OFF-model) administration. However,despite the promising laboratory-based results, both direct andindirect detection methods are not immune from false nega-tives17 20 21 or indeed false positives.18 22 For example, it isknown that terrestrial or simulated altitude exposure can influ-ence the indirect indices of rHumanEPO doping.23–25 Despiteconsiderable efficacy, there was clearly a need for these originaldetection methods to be improved.

The Athlete Biological Passport (ABP) was recently intro-duced as a new tool to indirectly detect erythropoiesis-stimulating agents such as rHumanEPO, which can lead to adoping sanction and at the same time as intelligently targetingathletes for additional testing.26 The ABP approach relies onidentifying intraindividual abnormal variability over time inselected haematological parameters (figure 1).14 27 The blooddoping behaviour of athletes has changed since the introductionof the ABP.28 Extreme reticulocyte values (ie, <0.4% or >2%)have dramatically decreased and as such, the implementation ofthe ABP has undeniably been a step forward in the antidopingfield.28 Although the ABP approach can identify abnormalenhanced erythropoiesis regardless of the method used, such asautologous blood transfusion, as shown by Pottgiesser et al29 ina field-like longitudinal blinded setting, the sensitivity of the

ABP has been questioned.30 In order to minimise the risk ofbeing caught via the ABP, it is well known that some athletes arenow using ‘microdoses’ of rHumanEPO which allegedly rangefrom 10 to 40 IU/kg body mass.16 20 31 Microdoses ofrHumanEPO aim (1) to increase haemoglobin mass (Hbmass)while avoiding large fluctuations in the ABP blood markers aswell as minimising the detection window for conventional directmethods and/or (2) to ‘normalise’ the ABP blood markers afterblood manipulations such as autologous blood transfusion.20 31

In addition, variation in ABP haematological parameters due tofactors such as training or hypoxia exposure can influence theinterpretation of the ABP results.32 Adjustments by the athletesto antidoping methods impose a constant need for scientists andantidoping authorities to develop and implement new detectionmethods based on innovative concepts and cutting-edgetechnologies.

‘Omics’-based approaches to detect rHumanEPO dopingThe initial ‘omics’ studiesA genome is defined as the entire collection of genetic informa-tion encoded by a particular organism.33 By semantic associ-ation, the words transcriptome, proteome and metabolome referto the entirety of all transcripts, proteins and metabolitesexpressed by a genome at a specific time. The science related tothese terms was named accordingly genomics, transcriptomics,proteomics and metabolomics and collectively encompass theso-called omics cascade34 (figure 2). Using microarray technol-ogy, a characteristic gene expression signature of a particularstimulus can be revealed by assessing the transcription state ofthe cells of the experimental samples.35 Therefore, if the geneexpression profile following rHumanEPO administration yieldsa specific gene expression signature, this could lead to the devel-opment of new methods with potentially improved discrimin-atory power relative to current detection protocols. Some initialsupport for this hypothesis emerged in a WADA funded studyby Varlet-Marie et al.36 Using the serial analysis of gene expres-sion (SAGE) method, Varlet-Marie et al identified 95 geneswhose differential expression was subsequently tested by quanti-tative real-time PCR in two athletes. These athletes were treatedfirst with high doses of rHumanEPO and then with microdoses.Thirty three marker genes for rHumanEPO administration wereidentified during the high-dose regimen and five remaineddifferentially expressed during the microdose regimen.A transcriptomic-based approach has also been applied withsome success to other doping methods and substances used byathletes. For example, characteristic gene expression profileshave been shown in six patients after autologous blood transfu-sion.37 Collectively, these pilot studies, involving only a limitednumber of patients, provide some confirmatory data to supportthe hypothesis that gene expression profiles may provide a sensi-tive method for the detection of rHumanEPO and blooddoping.

To build on the initial promise and develop new methodswith improved discriminatory power relative to current detec-tion protocols using ‘omics’ technologies, a series of studieshave been funded by WADA as follows:1. A gene-microarray-based approach to the detection of

recombinant human erythropoietin doping in enduranceathletes.

2. Application of a minimally invasive method for RNA sam-pling and the addition of miRNA to the detection of recom-binant human rHumanEPO use by athletes.

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3. Metabolomic profiling of recombinant erythropoietinrHumanEPO in Caucasian and east-African endurance-trained athletes.

4. A systems biology biomarker-based approach to the detec-tion of microdose recombinant human erythropoietindoping.

5. A new dimension to the detection of recombinant humanerythropoietin doping using blood gene expression profiles:development of direct antidoping applications.

6. A systems biology biomarkers approach to the differentiationof recombinant human erythropoietin doping from con-founding factors.The overall aim of these studies was to identify a molecular

signature of rHumanEPO doping and hence provide a basis forthe development of novel and robust testing models to detectrHumanEPO doping. Using both phenotypic and molecularmethods, the first study was carried out at sea level in Glasgow(Scotland (SCO), UK) with Caucasian endurance-trained men38

and at moderate altitude (∼2150 m) in Eldoret (Kenya (KEN))with KEN endurance runners39 who abstained from officialsporting competition for the entire duration of the study. Thespecific aims of this first study were:1. To determine and compare the effects of rHumanEPO

administration on blood parameters and exercise perform-ance in endurance-trained men living and training at or near

Figure 1 Examples of the AthleteBiological Passport (ABP) includinghaemoglobin concentration (HGB) andreticulocyte percentage (RET%). Bluelines: actual tests results. Red lines:individual ‘normal’ limits calculated bythe ABP software. Athlete A presentednormal variations. Conversely, athleteB presented abnormal variations, andwas consequently suspected of doping.Adapted from Sottas et al.27

Figure 2 The ‘omics’ cascade. The cascade depicts the flow ofinformation from gene to metabolism.

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sea level with another cohort living and training at moderatealtitude (∼2150 m) in KEN.

2. To establish adequate molecular methods and molecularworkflow to enable valid and optimal assessment of bloodgene expression.

3. To assess the effects of rHumanEPO on blood gene expres-sion profiles in endurance-trained volunteers living andtraining at or near sea level.

4. To replicate and compare the effects of rHumanEPO admin-istration on blood gene expression profiles in another cohortliving and training at moderate altitude (∼2150 m) in KEN.

5. To validate the gene-microarray-based findings using anotherspecific and sensitive quantitative gene expression technology.To achieve these ambitious aims, 39 endurance-trained men,

19 based at sea level (Glasgow, SCO) and 20 based at moderatealtitude (Eldoret, KEN, 2100–2800 m at sea level), receivedrHumanEPO injections for 4 weeks. Blood was obtained2 weeks before, during and 4 weeks after administration(figure 3). Blood, urine and saliva samples at 20 time points pervolunteer (780 blood samples ie, 20 time points×39 partici-pants; figure 3) were collected and specifically processed andstored at −80°C for current and future exploitation. Timepoints at key stages were selected for gene expression analysis(figure 3). Phenotypic results from this first study are beginningto emerge.38 39 Briefly, relative to baseline, running performancewas significantly improved following 4 weeks of rHumanEPOadministration in sea level Caucasian-trained men and remainedsignificantly elevated 4 weeks after administration by approxi-mately 6% and 3%, respectively; these performance effectscoincided with significantly rHumanEPO-induced elevated_VO2max and Hbmass.

38 rHumanEPO administration improved_VO2max and time trial performance to a similar extent in KENand SCO runners despite a higher haematocrit and haemoglobinconcentration in KEN compared with SCO prior torHumanEPO and similar at the end of administration.39 Withregard to the transcriptomic data, preliminary results show thatrelative to baseline, hundreds of genes were differentiallyexpressed during rHumanEPO administration.40 Notably, a fewof these genes were already upregulated after only one singlerHumanEPO injection, while further genes were differentiallyexpressed during rHumanEPO administration and remained dif-ferentially expressed up to 4 weeks after administration com-pared with baseline. In summary, a signature pattern of severalgenes upregulated during rHumanEPO and subsequently down-regulated up to 4 weeks after administration in the SCOcohort.40 More recent transcriptomic data to emerge from thisstudy show replication of the SCO microarray data in the KENcohort.41 An example of the characteristic gene expressionpattern in the sea level Caucasian and Kenyan altitude-basedcohorts is illustrated in figure 4. A subset of target genes wasfurther validated using another quantitative gene expressiontechnology.41 From the preliminary data, it is clear that

significant progress has been made towards successfully fulfillingall five objectives. This research has successfully identified, repli-cated and validated the blood ‘molecular signature’ ofrHumanEPO administration and as such provides the strongestevidence to date that gene biomarkers have the potential toimprove the performance of current antidoping methods suchas the ABP for rHumanEPO detection.

The results from the first study are promising, however, it iswell recognised that some athletes are now microdosing withrHumanEPO to minimise the risk of being caught via currentlyapplied detection methods.16 20 Interestingly, five genesremained differentially expressed even when high and micro-doses were taken into account together in the study byVarlet-Marie et al.36 While these results were generated usingSAGE and from only a few participants, they provide some con-firmatory data to support the idea that gene expression profilesmay provide a more sensitive method to detect microdoses ofrHumanEPO, especially when the whole genome is interro-gated. Therefore, a second study was funded by WADA anddesigned to use the genes significantly altered using a fairly highregimen of rHumanEPO injections discovered in the first studyas primary detection candidates of microdosing withrHumanEPO. For this currently ongoing study, 14 healthyendurance-trained individuals not involved in competition wererequired to participate in a randomised, double-blind, placebo-controlled crossover microdose rHumanEPO regimen necessitat-ing the collection of 364 blood samples (ie, 13 samples×14×2;figure 5). The aim of the rHumanEPO microdose regimen was

Figure 3 Experimental design for theWADA project entitled ‘A gene-microarray-based approach to thedetection of recombinant humanerythropoietin doping in enduranceathletes’. Hbmass, haemoglobin mass;rHuEpo, recombinant humanerythropoietin; VO2max, maximaloxygen uptake.

Figure 4 A typical gene expression pattern for illustrative purposes.EPO, erythropoietin; KEN, Kenya; SCO, Scotland

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to increase Hbmass while deliberately avoiding large fluctuationsin reticulocyte production. Briefly, each participant receivedrHumanEPO injections (NeoRecormon, Roche, Welwyn GardenCity, UK) subcutaneously twice per week for 7 weeks (figure 5).Blood samples were collected in triplicate at baseline and thenonce a week during rHumanEPO administration and for3 weeks after rHumanEPO administration (ie, 13 blood samplesper participant; figure 5). Standardised collection and storageprocedures were applied as per the previous study.

Current confounder studiesGiven the ongoing studies and promising preliminary data, it isof paramount importance to evaluate the effects of major con-founding factors on the discovered ‘molecular signature’ ofrHumanEPO doping, such as the effects of altitude. It is knownthat natural or simulated altitude training is used by athletes forperformance enhancement,42 although the optimal regimen andmagnitude of ergogenic effect of natural altitude training isunclear, while there is a small/no ergogenic effect of simulatedaltitude.43 Only the live high –train low method developed byLevin and Stray-Gundersen44 using natural altitude wouldappear to be effective.43 In addition, it remains uncertain towhat extent these ergogenic effects are due to augmentederythropoiesis.43 Regardless of these conflicting results, altitudeexposure may potentially influence haematological indices ofrHumanEPO doping.23 24 As a result, some athletes claim alti-tude exposure as a source of variation of their individual bloodvariables to mask blood doping practices such as the administra-tion of rHumanEPO. There is therefore a need to develop spe-cific and robust testing models which can differentiate altitudetraining from blood doping combined (or not) with altitudeexposure and preliminary unpublished data using transcrip-tomics are promising (WADA unpublished data). Therefore, theaim of a third study recently funded by WADA is to compareblood gene expression profiles altered by rHumanEPO (fairlyhigh-dose and microdose regimens, data from past and ongoingWADA-funded projects) with altitude exposure in order toprovide a set of candidate genes that can be used to differentiaterHumanEPO from altitude training. For this study, at least 20

endurance athletes will spend at least 4–6 weeks at altitudeabove 2000 m on more than one occasion and serial bloodsamples collected prior, during and post altitude exposure. Theprecise timing of blood sample collection will be determinedfrom pilot altitude studies currently underway. Once again, stan-dardised blood collection and storage procedures will be appliedas per previous studies and in doing so add further importantsamples to the ‘omics’ biobank being created for current andfuture doping test development.

In addition to altitude, another major potential confoundingfactor that could impact on the ‘molecular signature’ ofrHumanEPO doping is the effect(s) of prior exercise. Samplesare often collected at sporting or training venues after intenseexercise.45 It has previously been demonstrated that exercise sig-nificantly affects gene expression profiles of peripheral bloodmononuclear cells and white cells count.46 47 It is essential, there-fore, to define the effects of exercise on blood gene expressionprofiles and particularly on the ‘molecular signature’ ofrHumanEPO doping. In order to provide a set of robust candi-date genes that can be used for the detection of rHumanEPOdoping, blood gene expression assessment will be performedduring and post intense exercise after microdose rHumanEPO orplacebo in the double-blinded trial (ie, WADA study 2, figure 5).In particular, a modified Wingate test consisting of 10 sprints of10 s at baseline (following a familiarisation) and during the weekafter microdose rHumanEPO (figure 5) in order to provide a setof robust candidate genes that can be used for the detection ofrHumanEPO doping but also examine the effects ofrHumanEPO on progressive anaerobic fatigue and repeatedsprint ability. The ability to reproduce maximal performance insubsequent short-duration sprints separated by brief recoveryperiods is a key fitness requirement in most team and racketsports.48 Of note, the International Tennis Federation recentlyadopted the ABP programme, while there is anecdotal evidencein football that doping may be higher than perceived by sportauthorities.49 50 In addition, these results will be used to providethe basis for the development of recommendations on the collec-tion of samples in order to ensure the ‘validity’ of the blood geneexpression results. It is hoped that this approach will help reduce

Figure 5 Experimental design for theWADA project entitled ‘A systemsbiology biomarker-based approach tothe detection of microdoserecombinant human erythropoietindoping’. Hbmass, haemoglobin mass;rHuEpo, recombinant humanerythropoietin.

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the pressure on the athletes who need to wait 2 h after trainingor competition before a blood sample is taken for the ABP.

Necessary steps and potential future applications of an‘omics’-based approach to detect rHumanEPO dopingThe ABP approach relies on identifying intraindividual abnor-mal variability over time of selected haematological para-meters.14 More precisely, the ABP approach uses Bayesiannetworks for the evaluation of the likelihood of doping basedon several variables and/or factors which can then be used asevidence for disciplinary sanction. Bayesian networks are inter-pretable and flexible models for representing the probabilities ofcausal relationships between multiple interacting variables.14 51

In the antidoping field, the Adaptive Model based on theBayesian approach directly estimates the probability of blooddoping based on previous individual test history and heteroge-neous factors known to influence blood parameters. Thisfeature allows the model to remove the variance due to interin-dividual differences and heterogeneous factors such as gender,ethnicity, altitude exposure, age and sport discipline.51 Based onthe promising transcriptomic findings to date, blood gene bio-markers can potentially be integrated into the ABP in order tofurther improve its analytical performance. The idea being tobuild on the current ABP approach by combining currently usedhaematological parameters with new molecular signature data inthe first instance and in doing so attempt to create a uniqueABP model with improved specificity and sensitivity, not amen-able to manipulation.

In order to evaluate the proof of principle concept outlinedhere, interindividual and intraindividual variability as well asfactors which can affect blood gene expression profiles must beprecisely determined. It has been previously demonstrated thatexternal factors can influence blood profiles. For instance,strenuous endurance exercise can induce changes in some bloodparameters not only by the well-described increase in plasmavolume known as the athlete’s pseudoanaemia52 but also byexercise-induced haemolysis due to metabolic modifications,oxidative stress and repeated foot impact (foot strike).53–55 Theeffects of strenuous long distance exercise such as cycling stageracing and distance running on blood gene expression profilesshould consequently be investigated in elite endurance athletes.As most blood parameter values differ substantially betweenmen and women, the currently used reference ranges in theABP were determined separately for each sex.51 56 A similar dif-ferentiation approach could also be applied to blood geneexpression profiles in order to evaluate gender differences andsimilarities. Injury-related immobility also has the potential toaffect blood gene profiles, as it has been reported that immobil-ity for a period of 4 weeks reduces Hbmass by ∼19% in a com-petitive international female athlete.57 Research to date hasdemonstrated that ethnicity and residence at altitude only min-imally influence the blood gene expression signature ofrHumanEPO doping. However, more research is required tomeasure and generate normal reference ranges and the impactof other ‘confounders’ on blood gene expression profiles toenhance further the discriminant power of the ABP. If these keyfollow-up studies are successful, and an ‘omics’-based solution isadopted in the future, one option would be to adopt on mass(eg, full squad/team), announced testing to obtain baseline datathat could also be used as a comparison throughout the seasonand for subsequent seasons, with target testing set up on anypossible anomalies; such testing could even be initiated atyouth/academy level to build up profiles and reference ranges tobe monitored over time. However, in this case, possible

age-related changes in the reference range of the selectedmolecular signatures of doping would need to be considered. Inaddition to determining the biological variability of ‘omics’ mea-sures, markers and confounders, successful implementation ofan ‘omics’ antidoping solution will require the analytical vari-ability of the finally chosen ‘omics’ technology to be well estab-lished given the importance of analytical and preanalyticalfactors in any antidoping rule violation procedure. The truepotential of ‘omics’ technology and antidoping application (eg,potential to reduce the number of traditional antidoping tests)will require this essential process to be undertaken.

CONCLUSIONSThe current research on the molecular signature ofrHumanEPO doping has, so far, provided some evidence that‘omics’ technologies have the potential to significantlystrengthen the ABP approach and contribute to other traditionalantidoping tests. This approach can be applied for detection ofother doping substances and methods difficult to detect such arecombinant human growth hormone and blood transfusions.There is also the interesting possibility that this approach couldhelp reducing the pressure on the athletes’ antidoping obliga-tions such as the ‘athletes whereabouts’. In order to confirmthat an integrative ‘omics’ approach is a solution to improverHumanEPO detection, it is of paramount importance to pre-cisely determine the normal reference values of the bloodmolecular signature of rHumanEPO doping using different doseregimens as well as to carefully assess the potential effects ofexternal factors on blood gene expression profiles, such as priortraining, altitude including different hypoxic ‘dose’ and proto-cols, sport discipline, level of competition, gender, ethnicity andage. These investigations are necessary before potentially includ-ing the promising blood gene biomarkers in the ABP and/or thedevelopment of a stand alone test to reveal doping or identifysuspicious samples for targeting purposes.

Acknowledgements The authors wish to thank the members of the steeringcommittee of their WADA-funded research for their valuable comments and thenumerous students who assisted data collection in Scotland and Kenya. Thecooperation of all the participants is also greatly appreciated.

Funding The series of studies reviewed here were all supported by research grantsfrom the WADA.

Competing interests None.

Provenance and peer review Commissioned; externally peer reviewed.

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Pitsiladis YP, et al. Br J Sports Med 2014;0:1–7. doi:10.1136/bjsports-2014-093529 7

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 Yannis P Pitsiladis, Jérôme Durussel and Olivier Rabin erythropoietin and blood dopingdetection of recombinant human An integrative 'Omics' solution to the

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