Sensitivity and specificity of detection methods for erythropoietin doping in cyclists

Abstract Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti‐doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double‐blinded, placebo‐controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin β) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl‐PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl‐PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl‐PAGE and isoelectric focusing assays for the detection of erythropoietin abuse were 63.8% and 58.6%, respectively, with a false presumptive finding rate of 4.3% and 6%. None of the false presumptive findings tested positive in the confirmation analysis. Sensitivity was highest between 2 and 6 days after dosing, and dropped rapidly outside this window. Sensitivity of the ABP was 91.3%. Specificity of the urine assays was high; however, the detection window of rHuEPO was narrow, leading to questionable sensitivity. The ABP, integrating longitudinal data, is more sensitive, but there are still subjects that evade detection. Combining these methods might improve performance, but will not resolve all observed shortcomings.

Athletes have used drugs to enhance sports performance throughout history, despite all efforts to ban these doping substances. To detect cheating athletes, the World Anti-Doping Agency (WADA) oversees a global doping control system using blood and urine samples that are collected both in competition and out of competition. If an athlete is tested positive, this will usually lead to a suspension of two or four years, depending on the substance found and the circumstances of the case. It can be up to lifetime ineligibility for recidivists. 1 One substance that is widely abused, especially in endurance sports, is recombinant human erythropoietin (rHuEPO). rHuEPO has shown to positively affect maximal performance, 2,3 and although there is a lack of clear scientific proof whether it also improves actual endurance performance, 4  Although doping tests are intended to discourage athletes from using doping, detect the ones that are violating doping rules, and protect the athletes that play fair, their ability to do so completely depends on the performance of the tests and an intelligent testing program collecting samples within the window of detection. Technical documents describing the urine assays for detecting ESA use are available through WADA 6 and articles on the methods have been published. [7][8][9][10][11] Some information on detection windows for isoelectric focusing (IEF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is available for several rHuEPO variants from open label studies. [12][13][14] However, well-designed blinded studies describing the critical assay characteristics of sensitivity and specificity and the detection window, are not sufficiently (publicly) available. 15 We believe that scientific evidence underpinning internationally and widely applied doping tests should be available in order to understand the value of the tests, protect fair-playing athletes, and collaborate on improving doping detection.
For that reason, we aimed to evaluate the performance of three main methods being used in rHuEPO detection by determining sensitivity and specificity of the sarcosyl-PAGE and IEF assays on urine samples, and of the ABP and its hematological module.

| Study design and participants
For the purpose of this study, blood and urine samples were used that were obtained in a double-blind, randomized, placebo-controlled, parallel, single-center study on rHuEPO, in which 48 healthy male cyclists between 18 and 50 years of age were included. The design of this trial was described elsewhere. 4 In short, the main inclusion criteria were maximum power-to-weight ratio during the maximal exercise test at screening exceeding 4 W/kg, Hb between 8.0 and 9.8 mmol/L (equivalent to 12.8-15.7 g/dL) and Ht below 48% at screening and not being subject to anti-doping regulation or using medication that could potentially interact with the study drugs or study assessments. All participants gave written informed consent prior to any study-related

| Randomization and masking
Randomization to the rHuEPO or the placebo group (1:1) was done by a randomization code generated by an unblinded statistician who was otherwise not involved in the execution of the study. Enrolment of subjects was performed by a blinded study physician.

| Treatment
Epoetin β (NeoRecormon, Roche, Basel, Switzerland), prepared from multidose vials containing a lyophilizate of 50 000 IU epoetin β and 10 mL solvent for solution for injection, or saline (0.9% NaCl) was administered subcutaneously with weekly abdominal injections for 8 weeks. Dosing aimed to reach a target Hb of 10%-15% increase compared to the baseline Hb value, similar to previous studies investigating effects of rHuEPO on performance. 16 The first four injections contained 5000 IU per injection. The dose was modulated by an unblinded, non-study-related physician to 6000 IU, 8000 IU, or 10 000 IU in the final four weeks in case the Hb level was below the target range, doses similar to known practices in professional cycling. 17 When the Hb was in the target range, 2000 IU was administered. If the Ht was equal to or exceeded 52% or if the Hb exceeded the upper limit of the Hb range (increase of 15% compared to baseline), a placebo injection was administered.
All participants also received open-label daily oral doses of 200 mg ferrous fumarate and 50 mg ascorbic acid (both Pharmachemie B.V., Haarlem, Netherlands).

| Urine and blood samples for doping detection
Urine samples were collected for each subject and 50 mL was stored in Falcon tubes (Greiner Bio-One International GmbH, Kremsmuenster, Austria) at −70°C. Samples were collected at approximately 1 hour, 2 days, 4 days, and 7 days after the second dose and at approximately 12 days after the last dose. These samples were taken while subjects had not exercised in the preceding hours. To evaluate potential effects of exercise on the outcome of the test, a sample was taken as soon as the subject could urinate after the maximal exercise at approximately 4 days after the second dose and after a race to the top of Mont Ventoux at approximately 12 days after the last dose. Samples were shipped to the WADA-accredited anti-doping laboratory DoCoLab UGent by courier in two separate batches. The lab was aware these samples were research samples, but was blinded to the administered treatment.
All hematology samples were drawn after participants were seated with their feet on the floor for at least 10 minutes, at the following time points: 1-6 weeks before the first dose; directly before the first, third, fifth and seventh doses; approximately 12 days and 4 weeks after the last dose. In general, this means there were 2 weeks between two samples. Samples were analyzed within 2 hours at the Leiden University Medical Centre (LUMC) on a Sysmex XN9000 analyzer (Sysmex Corporation, Kobe, Japan). Samples taken at the Mont Ventoux race were split into two aliquots, one of which was kept at room temperature for determination of leucocytes and leucocytes differentiation and one was kept between 2 and 8°C for determination of hemoglobin, hematocrit, erythrocytes, reticulocytes, MCV, MCH, MCHC, thrombocytes, immature reticulocyte fraction (IRF), and red blood cell distribution width (RDW-SD). Samples were driven by courier to the LUMC and analysis took place within 36 hours of collection.

| Sarcosyl-PAGE urine assay
The sarcosyl-PAGE urine assay was used as described in the WADA technical document. 6 In short, immunopurification of the sample was performed with antibodies other than the one used for immunoblotting before loading 15 μL of sample on pre-cast polyacrylamide gels (NuPAGE BisTris gels, 10% T, 1.5 mm, as described previously 18  In addition, a negative control sample, a positive control sample containing rHuEPO, and a reference to enable to define apparent molecular mass were run in separate slots. Running buffer used was sodium N-lauroyl sarcosinate, and after the electrophoretic separation double-blotting was performed with the monoclonal mouse antihuman EPO antibody clone AE7A5 and a secondary antibody. The secondary antibody was a goat anti-mouse biotinylated polyclonal antibody IgG (H + L), cross-adsorbed, HRP (Thermo Scientific product no. 31432) and used in combination with a streptavidin horseradish peroxidase complex (Biospa, Milan, Italy) and a substrate (SuperSignal West Femto Maximum Sensitivity; Thermo Scientific, Waltham, MA, USA). The electrophoretic patterns of ESAs were revealed by the use of an amplified chemiluminescent system. Detection of exogenous EPO, in this case epoetin β, was done on the basis of the characteristic band shape ("broad band") and different (higher) apparent molecular mass than endogenous EPO. A sample was termed positive if a single band was visible above the negative control EPO, or if a mixed band both at the endogenous and exogenous location or a diffuse or faint area of the band above the corresponding endogenous band were visible, as per the WADA technical document. 6

| Isoelectric focusing urine assay
The IEF urine assay was used as described in the WADA technical document. 6 In short, immunopurification of the sample was performed with antibodies other than the one used for immunoblotting before loading 20 μL of sample on the gel, with 30 slots for test samples per gel. For purification, Stemcell ELISA was used as described previously, elution with 30 μL 4.4% CHAPS. 19  if there were at least three acceptable, consecutive bands in the basic area, and if the two most intense bands measured by densitometry were in the basic area, as per the WADA technical document. 6 It should be noted that the IEF assay is currently not used in routine practice by the DoCoLab and therefore the associated analyses are exploratory.

| Urine sample evaluation (screening)
Samples were designated as screening negative, screening positive or non-detectable by the DoCoLab in Ghent. Samples were nondetectable if no band was detected in the EPO (endogenous or exogenous) designated region.

| Confirmation analysis
After results of the screening analysis were available, the outcome was analyzed by unblinded Centre for Human Drug Research (CHDR) researchers. The WADA procedure describes that samples returning suspicious results at this screening stage are termed "presumptive adverse analytical findings", and need to undergo confirmation in a confirmation procedure. 20 In the current study, slightly different terminology was used to differentiate between suspicious results from a subject treated with rHuEPO or with a placebo. Therefore, samples from placebo-treated subjects that generated a suspicious result in the screening analysis were termed "false presumptive findings". For the sarcosyl-PAGE assay these were re-analyzed with a confirmatory sarcosyl-PAGE assay. Both the initial and the confirmation methodology were the same. In order to keep this confirmation analysis blinded to the DoCoLab, true positive, true negative, and false negative samples were included as well, and this selection of samples was relabeled by CHDR staff for analysis. Samples were only deemed positive, similar to routine analysis, when they would be forwarded for a mandatory second opinion. The WADA technical document requires 6 that all presumptive adverse analytical findings are evaluated by a recognized expert. After evaluation by the laboratory performing the analysis, samples are therefore forwarded to a WADA-recognized expert.
Currently, these experts are co-authors of the WADA technical document 6 and have extensive scientific knowledge of ESAs, their detection methodology and potential issues in the detection of ESAs. In the framework of this study, samples were not subjected to a second opinion and the interpretation was solely based on the laboratory performing the analysis. No WADA-recognized expert as per the technical document 6 was involved. In case of doubt, samples were deemed negative.

| Osmolality
Osmolality was determined by taking an aliquot from the homogenized, centrifuged samples, using the freezing point method on an Osmometer Auto & Stat, OM-6050 (Arkray, Kyoto, Japan). Osmolality determination was initiated after all analyses had been performed to provide insights into potential effects of osmolality on assay results.

| Athlete biological passport
A method similar to the adaptive model of the ABP was constructed internally using Microsoft Excel 2013 (Redmond, WA, USA), as the official WADA software was not made available for this study. Individual curves of each separate hematological parameter from blood samples were produced by plotting the observed value, and the 99.7% confidence interval (CI) around that value. For the first measurement, the 99.7% CI was based on the population mean (Pop. mean) ± 3xSD of all available data points from the screening and baseline hematological samples of all subjects that completed these visits (Pop. SD); for IRF and RDW-SD only the value at baseline was used as they were not determined at screening. For following data points, with each additional available measurement the 99.7% CI was based more on individual data, and calculated as follows: • second data point: The OFF score (OFFS) was calculated using the following formula: ([hemoglobin concentration in g/dL]) * 10 -(60 * √([Reticulocyte percentage])). Graphs of each separate variable belonging to one subject were combined into one document and sent with a coded (blinded) identifier to the DoCoLab in Ghent for evaluation of a subject being negative, suspicious or positive. Two independent researchers at the DoCoLab, which is designated as an Athlete Passport Management Unit (APMU) by WADA, independently evaluated the data and two sets of results were generated and analyzed, so that reproducibility could also be evaluated.

| Exercise
Two urine samples per subject were collected after exercise: one after a maximal exercise test to exhaustion at 4 days (±1 day) after the second dose administration and one approximately 12 days after the last dose after participants competitively completed an uphill road race on the Mont Ventoux. This race was directly preceded by 110 km cycling.

| Data management
All data were stored in a clinical trial database (Promasys, Omnicomm Inc., Fort Lauderdale, USA) and checked for accuracy and completeness. A blinded data review was performed before code-breaking and analysis according to a standard procedure at our unit.

| STATISTICAL ANALYSIS
Standard assay characteristics were calculated based on the reported results. For determining overall urine assay characteristics, only samples that were designated negative or positive were included in the analysis. When determining sensitivity over time, two analyses were done, one including and one excluding the samples designated nondetectable. For this analysis, samples were binned per day, and only bins with more than one sample were included in the analysis. When determining ABP assay characteristics, suspicious and positive were combined as being an indication of rHuEPO use.

| ROLE OF THE FUNDING SOURCE
This was an investigator initiated study by the foundation CHDR in were not collected due to withdrawal from the study or subjects not being able to perform the particular visit. One additional sample from an rHuEPO treated subject was not analyzed with the IEF assay as it was erroneously not included in the sample shipment at the time of the IEF analysis.  (Table 1).
When comparing samples that were collected before and directly after a maximal exercise test on the same day, sensitivity was 100% at 4 days, 92% and 100% at 5 days, 57% and 0% at 11 days, 33% and 0% at 12 days, and 0% at 13 days since last dosing.

| Sarcosyl-PAGE sensitivity over time
When tested approximately 1-2 hours after the second rHuEPO dosing (and 7 days after the first), 50% of rHuEPO treated subjects tested positive ( Figure 1). The sensitivity increased to approximately 81% when urine was collected 2 days after the last dose, and was 95%-100% between day 3 and 6 after the last dose. After 7 days, sensitivity decreased again to approximately 50% and was 29% at 11 days, 18% at 12 days, and 0% at 13 and 16 days after the last dose. When samples designated as non-detectable are excluded, sensitivity slightly increases, but is very similar. and specificity was 94.0% (with 9 false presumptive findings;

| Isoelectric focusing sensitivity over time
When tested approximately 1-2 hours after the second rHuEPO dosing (and 7 days after the first), 42% of rHuEPO-treated subjects tested positive ( Figure 2). The sensitivity increased to approximately 86% when urine was collected 2 days after the last dose, and was 50%-68% between days 3 and 5 after the last dose. After 6 and 7 days, sensitivity decreased again to approximately 25 and 22% and was only 21% at 11 days and 0% at 12, 13, and 16 days after the last dose.
When samples designated as non-detectable are excluded, sensitivity increases significantly (Figure 2).

| Athlete biological passport
A total of 47 ABPs based on the collected blood samples were rated for rHuEPO use by the two independent APMU staff members.
Results of the ABP performance by Evaluator 1 and Evaluator 2 are depicted in Table 3 and a true negative subject's ABP can be seen in Figure 3.

| DISCUSSION
The present study evaluated the performance of current doping detection methods for epoetin β (NeoRecormon) use by athletes.  The sarcosyl-PAGE urine assay based on 330 samples was shown to be highly sensitive for the applied dose of 5000 IU within a small time range of 2−6 days after dosing. Of all non-rHuEPO samples 4.3% was labeled screening positive, but after the standard confirmation analysis no false positives were found. Findings for IEF showed a slightly lower sensitivity and slightly higher false presumptive finding rate of 6%.
Evaluation of the ABP based on hematology samples had high sensitivity of 91.3% for rHuEPO use. Specificity was also high with 100% and 95.8% for the two evaluators. However, this research brought forward some concerns of all three methods.

| Sarcosyl-PAGE urine assay performance
The sarcosyl-PAGE assay performed well 2−6 days after dosing, but outside this window sensitivity falls rapidly. Given the long-lasting hematological effect of rHuEPO (red blood cell lifespan is around 120 days 21 ), high frequency testing and intelligent testing strategies (eg, targeted testing in combination with the ABP) are necessary to attain a high probability to catch athletes abusing rHuEPO. A second concern with this assay is that a small part of the samples (5%) were non-detectable, especially because this result occurred more often in rHuEPO treated subjects (83% of these samples). Non-detectable results in general have been associated with variety of reasons. Degradation of EPO via neuraminidase activity, with consecutive loss of sialic acid moieties will result in a shift in molecular weight of EPO to lower molecular weight sections. 22 Protease activity might lead to the disappearance of EPO. Of course suppression of endogenous production due to a negative feedback mechanism will also lower endogenous EPO levels, potentially below the limit of detection (LOD) of the method. Finally, urine dilution could disturb the assay. 23    The aim of the screening procedure is to forward all potential positives and not to exclude any false negatives and in the confirmation procedure, this 'philosophy is reversed'; ie, not to report any false positives, as described previously. The sarcosyl-PAGE assay partly does allow for this at reasonable economically viable conditions with less than 5% false presumptive findings forwarded to confirmation. What is remarkable however, is that when the results from the same sample in the screening and confirmation analyses are compared, at least in some cases these are clearly different (Figure 4). Sample preparation errors (eg, sample swapping) cannot definitively be excluded as cause of these discrepancies, but given that false presumptive findings were observed in the screening analysis that was performed according to the standard procedure for sample preparation as for official doping samples, this seems unlikely. Furthermore, although no formal positive Finally, sensitivity of the assay did not seem to be affected by the maximal exercise test. The race however did seem to impact sensitivity, with six subjects that tested positive at days 11 and 12 before the race testing negative after the race. Of these six negative post-race samples, two were non-detectable, potentially due to effects of the exercise and hydration. The other four samples, however, were designated negative, indicating the race potentially affected the ability to detect rHuEPO. These four subjects went from positive to negative in approximately 8 hours, which is remarkable ( Figure 5). One explanation could be that the concentration of erythropoietin in the post-race samples was lower (possibly due to changes in urine concentration during the race), and that the faint signal that was detected pre-race was therefore not visible post-race.

| Isoelectric focusing urine assay performance
Findings for the IEF assay were similar to the sarcosyl-PAGE assay, although overall sensitivity slightly worse for IEF, which is similar to a previous study with micro-doses of rHuEPO. The most striking difference between the two assays was a four-fold higher incidence of samples not being detectable for the IEF (21%). This could be due to the fact that the IEF is not currently being used by the DoCoLab in Ghent and was only implemented for this study, and less routine led to more samples being designated as non-detectable. Additionally, it could be due to the effect of previously described neuraminidase activity on IEF results. The LOD for sarcosyl-PAGE as determined with reference standards was the same as for IEF. Samples were stored frozen immediately following collection until analysis. Samples were defrosted overnight in a fridge and aliquoted immediately before analysis. While for sarcosyl-PAGE, mostly protease activity will lead to undetectable results and neuraminidase activity will lead to a shift (lower molecular weight) of the rHuEPO band, we believe that neuraminidase activity might lead to complete "disappearance" of the EPO bands in IEF and not in sarcosyl-PAGE. This may contribute to the observed difference. Similar to the worrying finding for sarcosyl-PAGE, the majority of the non-detectable samples (75%) in IEF belonged to rHuEPO-treated subjects. This higher incidence in samples from doped athletes in both IEF and sarcosyl-PAGE indicates that, while the fact that no EPO can be detected at all is not a proof of doping, this information might be useful to plan target and/or follow up tests as rHuEPO use might be associated with a non-detectable result.
To rule out problems with the individual assay when such a result occurs, it could be useful to add a positive control to each sample, for example using EPO from a different animal species, as is common for most other doping analyses.
There was no overlap in false presumptive findings between the sarcosyl-PAGE and IEF assays, indicating that such a result is not related to the sample.
In contrast to the sarcosyl-PAGE assay, the maximal exercise test potentially did have an effect on the assay result of the IEF assay.
Sensitivity was higher directly after the test compared to before.
For the race, however, we observed a similar outcome as for the sarcosyl-PAGE assay: three subjects tested positive before the race; after the race two tested negative, and one non-detectable. The impairment in signal for these post-race samples as observed in the sarcosyl-PAGE assay ( Figure 5) was not observed in the IEF assay for these two samples. However, this is solely based on observed signal intensities and not on a quantitative observation.
As no internal standard was used, factors such as recovery differences in sample preparation between different batches/samples might also have played a role. If there indeed was an effect of exercise on the IEF test results, this effect was opposite for the race, compared to the maximal exercise test.

| Athlete biological passport performance
In addition to evaluating the urine assays, we constructed an ABP so we could evaluate all tools used to detect rHuEPO use. This method had a high sensitivity of 91.3%. Given that this data is collected longitudinally and the method is less dependent on the time of sampling in relation to dosing compared to the urine assay, this approach not surprisingly has a better chance of detecting doping athletes. Nevertheless, almost 10% of subjects were not identified as suspicious or positive based on their ABP. See as an example the false negative subject in Figure 3, whose variation in hematological parameters is actually very similar to that of the placebo subject depicted in the left column of panels. This means that some athletes might not be at risk for detection based on the ABP despite using rHuEPO. Moreover, in practice, sensitivity of the ABP might fall due to potential lower doses

| Limitations
There are several limitations to our study. First, although unlikely, it cannot be ruled out that placebo-treated subjects administered rHuEPO outside the proceedings of the study. Given that false presumptive findings were distributed over many subjects, this seems an unlikely explanation for these observations. Furthermore, most subjects received higher doses (8000 or 10 000 IU) at the last dose and the accompanying urine samples taken more than 10 days after dosing, than at the second dose (5000 IU), which could have impacted the urine assay results over time. But even with a higher dose sensitivity was well below 40% for these samples.
Additionally, it is not possible to verify if sample swapping in the preparation or re-labeling of samples occurred, which might be an alternative explanation to the discrepancy observed for several samples between results for screening and confirmation analysis. However, this seems unlikely as the screening assay, which showed false presumptive findings, used the standard sample preparation procedure as for official doping samples. Moreover, such events would then also contribute to overall assay performance in the official WADA procedure. The re-labeling procedure performed here between the screening and confirmation analysis is not part of the official WADA procedure, but was also performed by double-checking by two staff members. Finally, observed characteristics for the assays apply to the materials and methods used here. Deviations, such as applying a 1.0 mm gel in contrast to the 1.5 mm gel used in this study (as based on a previous publication 18 ), might change the assay performance to some extent.
For the ABP, a limitation was that samples were analyzed on a different type of analyzer, although from the same manufacturer, than the Sysmex XT 2000i that is used by anti-doping laboratories. In addition, the ABP algorithm used by WADA was not available to us, and so the current method was an approximation of the official procedure.
Nevertheless, these discrepancies should not have a major impact on the ABP profiles and so the outcome of the review of the ABPs, we feel, can be considered indicative of the first stage of ABP performance at the APMU.
Information about factors that might influence markers in the ABP (eg, altitude training) would be available for the evaluator assessment.
This information was not systematically recorded in the present study, and although no such events were reported by subjects, occurrence could have impacted ABP review. It should also be noted that the relatively high sampling frequency applied in this study, with intervals of as short as two weeks, could lead to the individual reference ranges moving along with the observed values more than with longer intervals and thereby reducing the chance values to fall outside the range.
The selected window is however the smallest acceptable window according to the ABP and so these data should still be correctly interpretable. Finally, in the WADA setting, after assessment of an ABP by an APMU, suspicious ABPs will be sent for review to a WADA hematology expert. If this expert confirms the suspicious finding, they and two other experts will determine, based on all available information and possibly additional requested information, whether the profile is indeed indicative of blood doping. For this study, several WADA hematology experts were approached to participate in assessment of the ABPs of this study, but all of them declined. Therefore, the results reported here can only be considered to resemble the first part of the official system. But as only suspicious results are forwarded to the expert, ABP sensitivity would at best stay at the same level.

| CONCLUSION
The sarcosyl-PAGE urine assay for rHuEPO (specifically epoetin β) did not show false positive results after confirmation analysis, but it does have shortcomings such as a limited detection window. In addition, it is of some concern that in this study we observed a higher rate of non-detectable samples in subjects treated with rHuEPO. Adding a control internal standard to each sample and measuring total erythropoietin levels using an immunoassay could address the latter issue.
The IEF assay in our study was inferior to the sarcosyl-PAGE assay.
The ABP had a much higher sensitivity than the assays using a single urine sample, although it too did not classify all passports correctly.
In summary, we showed that all three methods evaluated in this study have their shortcomings and challenges and that it is critical to continue research to improve existing and develop new doping detection methods.

KEY FINDINGS
World Anti-Doping Agency methods for detection of recombinant human erythropoietin were evaluated in trained cyclists. Detection in urine by sarcosyl-PAGE had a sensitivity of 63.8% and isoelectric focusing of 58.6%, with a peak in sensitivity between 2 and 6 days after dosing, rapidly dropping outside this window. False presumptive finding rates were 4.3% and 6%, respectively, but none of the false presumptive findings tested positive in the confirmation analysis. Sensitivity of the athlete biological passport that integrates longitudinal hematological data was 91.3%.

DECLARATION ON INTEREST
We declare no competing interests.