Validation of whole-blood transcriptome signature during microdose recombinant human erythropoietin (rHuEpo) administration.

BACKGROUND: Recombinant human erythropoietin (rHuEpo) can improve human performance and is therefore frequently abused by athletes. As a result, the World Anti-Doping Agency (WADA) introduced the Athlete Biological Passport (ABP) as an indirect method to detect blood doping. Despite this progress, challenges remain to detect blood manipulations such as the use of microdoses of rHuEpo. METHODS: Forty-five whole-blood transcriptional markers of rHuEpo previously derived from a high-dose rHuEpo administration trial were used to assess whether microdoses of rHuEpo could be detected in 14 trained subjects and whether these markers may be confounded by exercise (n = 14 trained subjects) and altitude training (n = 21 elite runners and n = 4 elite rowers, respectively). Differential gene expression analysis was carried out following normalisation and significance declared following application of a 5% false discovery rate (FDR) and a 1.5 fold-change. Adaptive model analysis was also applied to incorporate these markers for the detection of rHuEpo. RESULTS: ALAS2, BCL2L1, DCAF12, EPB42, GMPR, SELENBP1, SLC4A1, TMOD1 and TRIM58 were differentially expressed during and throughout the post phase of microdose rHuEpo administration. The CD247 and TRIM58 genes were significantly up- and down-regulated, respectively, immediately following exercise when compared with the baseline both before and after rHuEpo/placebo. No significant gene expression changes were found 30 min after exercise in either rHuEpo or placebo groups. ALAS2, BCL2L1, DCAF12, SLC4A1, TMOD1 and TRIM58 tended to be significantly expressed in the elite runners ten days after arriving at altitude and one week after returning from altitude (FDR > 0.059, fold-change varying from 1.39 to 1.63). Following application of the adaptive model, 15 genes showed a high sensitivity (≥ 93%) and specificity (≥ 71%), with BCL2L1 and CSDA having the highest sensitivity (93%) and specificity (93%). CONCLUSIONS: Current results provide further evidence that transcriptional biomarkers can strengthen the ABP approach by significantly prolonging the detection window and improving the sensitivity and specificity of blood doping detection. Further studies are required to confirm, and if necessary, integrate the confounding effects of altitude training on blood doping.

The use of biomarkers to describe plasma-, red cell-, and blood volume from a simple blood test.

Plasma volume and red cell mass are key health markers used to monitor numerous disease states, such as heart failure, kidney disease, or sepsis. Nevertheless, there is currently no practically applicable method to easily measure absolute plasma or red cell volumes in a clinical setting. Here, a novel marker for plasma volume and red cell mass was developed through analysis of the observed variability caused by plasma volume shifts in common biochemical measures, selected based on their propensity to present with low variations over time. Once a month for 6 months, serum and whole blood samples were collected from 33 active males. Concurrently, the CO-rebreathing method was applied to determine target levels of hemoglobin mass (HbM) and blood volumes. The variability of 18 common chemistry markers and 27 Full Blood Count variables was investigated and matched to the observed plasma volume variation. After the removal of between-subject variations using a Bayesian model, multivariate analysis identified two sets of 8 and 15 biomarkers explaining 68% and 69% of plasma volume variance, respectively. The final multiparametric model contains a weighting function to allow for isolated abnormalities in single biomarkers. This proof-of-concept investigation describes a novel approach to estimate absolute vascular volumes, with a simple blood test. Despite the physiological instability of critically ill patients, it is hypothesized the model, with its multiparametric approach and weighting function, maintains the capacity to describe vascular volumes. This model has potential to transform volume management in clinical settings. Am. J. Hematol. 92:62-67, 2017. © 2016 Wiley Periodicals, Inc.

Adaptive Bayesian analysis of serum creatinine as a marker for drug-induced renal impairment in an early-phase clinical trial.

BACKGROUND: A concern with using creatinine for the identification of drug-induced renal impairment is that small changes in serum creatinine (SCr) that frequently are perceived as measurement bias or imprecision translate into important changes in the glomerular filtration rate. Important drug-generated changes in creatinine are difficult to detect because they are frequently observed within the reference interval. The design of a crossover drug protocol is an opportunity to use study participants as their own control to identify these small but important changes. METHODS: Twenty individuals participating in a phase I clinical trial were evaluated for SCr changes beyond those expected for biological variation according to individual Z scores derived from an adaptive Bayesian model. After 2 screening tests, participants were administered either drug (n = 11) or placebo (n = 9) during the first dosing interval. A washout period followed, and drug was then administered to the group that initially received placebo, and vice versa (10 visits total per participant). RESULTS: Although all creatinine values fell within the reference interval, 8 participants individually showed increased concentrations (Z scores >2.33). These 8 participants were confirmed at unblinding to have received the drug in the identified dosing period, with 1 exception. CONCLUSIONS: The ability to identify a drug effect on an individual-participant basis in early-phase studies permits drug developers to recognize issues early in development and rapidly engage in risk-benefit analysis. These results suggest that SCr monitoring is able to detect early kidney dysfunction when individual-based reference intervals are used.

The athlete biological passport.

BACKGROUND: In elite sports, the growing availability of doping substances identical to those naturally produced by the human body seriously limits the ability of drug-testing regimes to ensure fairness and protection of health. CONTENT: The Athlete Biological Passport (ABP), the new paradigm in testing based on the personalized monitoring of biomarkers of doping, offers the enormous advantage of being independent of this endless pharmaceutical race. Doping triggers physiological changes that provide physiological enhancements. In the same way that disease-related biomarkers are invaluable tools that assist physicians in the diagnosis of pathology, specifically selected biomarkers can be used to detect doping. SUMMARY: The ABP is a new testing paradigm with immense potential value in the current climate of rapid advancement in biomarker discovery. In addition to its original aim of providing proof of a doping offense, the ABP can also serve as a platform for a Rule of Sport, with the presentation before competition of the ABP to objectively demonstrate that the athlete will participate in a healthy physiological condition that is unaltered by performance-enhancing drugs. Finally, the decision-support system used today for the biological monitoring of world top-level athletes can also be advantageously transferred to other areas of clinical practice to reach the goal of personalized medicine.

Prevalence of blood doping in samples collected from elite track and field athletes.

BACKGROUND: No reliable estimate of the prevalence of doping in elite sports has been published. Since 2001, the international governing body for athletics has implemented a blood-testing program to detect altered hematological profiles in the world's top-level athletes. METHODS: A total of 7289 blood samples were collected from 2737 athletes out of and during international athletic competitions. Data were collected in parallel on each sample, including the age, sex, nationality, and birth date of the athlete; testing date; sport; venue; and instrument technology. Period prevalence of blood-doping in samples was estimated by comparing empirical cumulative distribution functions of the abnormal blood profile score computed for subpopulations with stratified reference cumulative distribution functions. RESULTS: In addition to an expected difference between endurance and nonendurance athletes, we found nationality to be the major factor of heterogeneity. Estimates of the prevalence of blood doping ranged from 1% to 48% for subpopulations of samples and a mean of 14% for the entire study population. Extreme cases of secondary polycythemia highlighted the health risks associated with blood manipulations. CONCLUSIONS: When applied at a population level, in this case the population of samples, hematological data can be used to estimate period prevalence of blood doping in elite sports. We found that the world's top-level athletes are not only heterogeneous in physiological and anthropometric factors but also in their doping behavior, with contrasting attitudes toward doping between countries. When applied at the individual level, the same biomarkers, as formalized in the Athlete Biological Passport paradigm, can be used in analysis of the observed different physiological characteristics and behavioral heterogeneities.

A pilot study on subject-based comprehensive steroid profiling: novel biomarkers to detect testosterone misuse in sports.

CONTEXT: Until now, the testosterone/epitestosterone (T/E) ratio is the main marker for the detection of testosterone (T) misuse in athletes. As this marker can be influenced by a number of confounding factors, additional steroid profile parameters indicating T misuse can provide substantiating evidence of doping with endogenous steroids. The evaluation of a steroid profile is currently based upon population statistics. As large inter-individual variations exist, a paradigm shift towards subject-based references is ongoing in doping analysis. OBJECTIVE: Proposition of new biomarkers for the detection of testosterone in sports using extensive steroid profiling and an adaptive model based upon Bayesian inference. SUBJECTS: Six healthy male volunteers were administered with testosterone undecanoate. Population statistics were performed upon steroid profiles from 2014 male Caucasian athletes participating in official sport competition. DESIGN: An extended search for new biomarkers in a comprehensive steroid profile combined with Bayesian inference techniques as used in the athlete biological passport resulted in a selection of additional biomarkers that may improve detection of testosterone misuse in sports. RESULTS: Apart from T/E, 4 other steroid ratios (6α-OH-androstenedione/16α-OH-dehydroepiandrostenedione, 4-OH-androstenedione/16α-OH-androstenedione, 7α-OH-testosterone/7ß-OH-dehydro-epiandrostenedione and dihydrotestosterone/5ß-androstane-3α,17ß-diol) were identified as sensitive urinary biomarkers for T misuse. These new biomarkers were rated according to relative response, parameter stability, detection time and discriminative power. CONCLUSION: Newly selected biomarkers were found suitable for individual referencing within the concept of the Athlete's Biological Passport. The parameters showed improved detection time and discriminative power compared to the T/E ratio. Such biomarkers can support the evidence of doping with small oral doses of testosterone.

Detection of autologous blood doping with adaptively evaluated biomarkers of doping: a longitudinal blinded study.

BACKGROUND: Since no direct detection method for autologous blood transfusions exists, the most promising attempt is the Athlete Biological Passport (ABP) and its adaptive model that enables a longitudinal monitoring of hematologic measures to identify patterns of blood manipulations. The purpose therefore was to evaluate the performance of this adaptive model for the detection of autologous blood transfusions in a longitudinal blinded setting. STUDY DESIGN AND METHODS: Twenty-one subjects were divided into a doped group (multiple transfusions of 1-2 units of red blood cells, n = 11) and a control group (n = 10). The time course of a cycling season (42 weeks) was simulated including three major competitions (Classics, Grand Tour, World Championships). Up to 10 venous blood samples were ordered per subject by a blinded investigator mimicking the intelligent approach in obtaining hematologic data for the adaptive model (hemoglobin [Hb] concentration, reticulocyte percentage, OFF-score). RESULTS: Retrospective analysis allowed identification of four (probability >99%) or three (probability >99.9%) abnormal samples for Hb and eight (probability >99%) or five (probability >99.9%) abnormal samples for OFF-hr in doped subjects. Four doped subjects (36%) presented an abnormal OFF-hr sequence and three doped subjects (27%) an abnormal Hb sequence; there were no false-positive sequence results. The best possible sensitivity was 82% when a combination of all tests was used. CONCLUSIONS: This investigation provides evidence that the adaptive model allows detection of autologous blood transfusions with a good sensitivity. An intelligent testing approach and the adherence to World Anti-Doping Agency's ABP operating guidelines are nevertheless determinant in the success.

Robust and sensitive peptidomics workflow for plasma based on specific extraction, lipid removal, capillary LC setup and multinozzle ESI emitter.

We present a new workflow for the LC-MS determination of native peptides in plasma at picomolar levels. Collected whole blood was quickly diluted with an ice-cold solution in order to stop protease activity. Diluted plasma samples were extracted by protein denaturation followed by solid-phase-extraction with a polymeric stationary phase that removed most proteins and lipids. Using a specific LC-MS setup with 3 pumps, 240 µL of extracts were injected without drying-reconstitution, a step known to cause peptide losses. After an 18-fold dilution on-line, peptides were trapped on a 1 × 10 mm C8 column, back-flushed and resolved on a 0.3 × 100 mm C18 column. Extract reproducibility, robustness (column clogging), extraction yields, matrix effects, calibration curves and limits of detection were evaluated with plasma extracts and spiked-in standards. The sensitivity and applicability of 3 electrospray sources were evaluated at capillary flow rates (10 µL/min). We show that ionization sources must have a spray angle with the MS orifice when "real" extracts are injected and that a multinozzle emitter can improve very significantly peptide detection. Finally, using our workflow, we have performed a peptidomics study on dried-blood-spots collected over 65 h in a healthy volunteer and discovered 5 fragments (2.9-3.8 KDa) of the protein statherin showing circadian oscillations. This is the first time that statherin is observed in blood where its role clearly deserves further investigations. Our peptidomic protocol shows low picomolar limits of detection and can be readily applied with or without minor modifications for most peptide determinations in various biomatrices.

Fully automated dried blood spot sample handling and extraction for serological testing of SARS-CoV-2 antibodies.

At the beginning of 2020, an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reached pandemic dimensions. Throughout the event, diagnostic tests function as an essential tool for understanding, mitigating, and implement strategies to curb and reduce infections. Here, we present a novel method for the fully automated dried blood spot (DBS) sample handling and extraction for serological testing of human IgG antibodies against SARS-CoV-2 using a commercial enzyme-linked immunosorbent assay (ELISA) testing kit. This proof-of-principle pilot study successfully demonstrates the recovery of antibodies in their intact form from DBS using automated, direct sample elution within 100 µl of extraction buffer. The use of minimally invasive DBS sampling provides an alternative to existing analytical procedures such as sampling by venipuncture or nasal swabs. Due to the ease of DBS collection, no third party need be involved, making at-home sampling possible (e.g., during quarantine).

IEF pattern classification-derived criteria for the identification of epoetin-delta in urine.

Epoetin-delta (Dynepo Shire Pharmaceuticals, Basing stoke, UK) is a synthetic form of erythropoietin (EPO) whose resemblance with endogenous EPO makes it hard to identify using the classical identification criteria. Urine samples collected from six healthy volunteers treated with epoetin-delta injections and from a control population were immuno-purified and analyzed with the usual IEF method. On the basis of the EPO profiles integration, a linear multivariate model was computed for discriminant analysis. For each sample, a pattern classification algorithm returned a bands distribution and intensity score (bands intensity score) saying how representative this sample is of one of the two classes, positive or negative. Effort profiles were also integrated in the model. The method yielded a good sensitivity versus specificity relation and was used to determine the detection window of the molecule following multiple injections. The bands intensity score, which can be generalized to epoetin-alpha and epoetin-beta, is proposed as an alternative criterion and a supplementary evidence for the identification of EPO abuse.

Evaluation of two immunoassays for the measurement of human chorionic gonadotropin in urine for anti-doping purposes.

BACKGROUND: Urinary human chorionic gonadotropin (hCG) concentration is routinely measured in all anti-doping laboratories to exclude the misuse of recombinant or urinary hCG preparations. In this study, extended validation of two commercial immunoassays for hCG measurements in urine was performed. Both tests were initially designed for hCG determination in human serum/plasma. METHODS: Access and Elecsys 1010 are two automated immunoanalysers for central laboratories. The limits of detection and quantification, as well as intra-laboratory and inter-technique correlation, precision, and accuracy, were determined. Stability studies of hCG in urine following freezing and thawing cycles (n = 3) as well as storage conditions at room temperature, 4 degrees C and -20 degrees C, were performed. RESULTS: Statistical evaluation of hCG concentrations in male urine samples (n = 2429) measured with the Elecsys 1010 system enabled us to draw a skewed frequency histogram and establish a far outside value equal to 2.3 IU/L. This decision limit corresponds to the concentration at which a sportsman will be considered positive for hCG. Intra-assay precision for the Access analyser was less than 4.0%, whereas the inter-assay precision was closer to 4.5% (concentrations of the official external controls contained between 5.5 and 195.0 IU/L). Intra and interassay precision for the Elecsys 1010 analyser was slightly better. A good inter-technique correlation was obtained when measuring various urine samples (male and female). No urinary hCG loss was observed after two freeze/thaw cycles. On the other hand, time and inappropriate storage conditions, such as temperatures above 10 degrees C for more than 5 days, can deteriorate urinary hCG. CONCLUSIONS: Both analysers showed acceptable performances and are suitable for screening urine for anti-doping analyses. Each laboratory should validate and establish its own reference values because hCG concentrations measured in urine can be different from one immunoassay to another. The time delay between urine collection and analysis should be reduced as much as possible, and urine samples should be transported in optimal conditions to avoid a loss of hCG immunoreactivity.

The athlete's biological passport and indirect markers of blood doping.

In the fight against doping, disciplinary sanctions have up to now been primarily based on the discovery of an exogenous substance in a biological fluid of the athlete. However, indirect markers of altered erythropoiesis can provide enough evidence to differentiate between natural variations and blood doping. Forensic techniques for the evaluation of the evidence, and more particularly Bayesian networks, allow antidoping authorities to take into account firstly the natural variations of indirect markers - through a mathematical formalism based on probabilities - and secondly the complexity due to the multiplicity of causes and confounding effects - through a distributed and flexible graphical representation. The information stored in an athlete's biological passport may be then sufficient to launch a disciplinary procedure against the athlete. The strength of the passport is that it relies on a statistical approach based on sound empirical testing on large populations and justifiable protocols. Interestingly, its introduction coincides with the paradigm shift that is materializing today in forensic identification science, from archaic assumptions of absolute certainty and perfection to a more defensible empirical and probabilistic foundation.

Blood transfusion in sports.

Blood transfusion is an effective and unmediated means of increasing the number of red blood cells in the circulation in order to enhance athletic performance. Blood transfusion became popular in the 1970s among elite endurance athletes and declined at the end of the 1980s with the introduction of recombinant erythropoietin. The successive implementation in 2001 of a direct test to detect exogenous erythropoietin and in 2004 of a test to detect allogeneic blood transfusion forced cheating athletes to reinfuse fully immunologically compatible blood. The implementation of indirect markers of blood doping stored in an Athlete's Biological Passport provides a powerful means to deter any form of blood transfusion.

A novel mixed living high training low intervention and the hematological module of the athlete biological passport.

Exposure to either natural or simulated hypoxia induces hematological adaptations that may affect the parameters of the Athlete Biological Passport (ABP). The aim of the present study was to examine the effect of a novel, mixed hypoxic dose protocol on the likelihood of producing an atypical ABP finding. Ten well-trained middle-distance runners participated in a "live high, train low and high" (LHTLH) altitude training camp for 14 days. The participants spent ˜6 hr.d-1 at 3000-5400 m during waking hours and ˜10 h.d-1 overnight at 2400-3000 m simulated altitude. Venous blood samples were collected before (B0), and after 1 (D1), 4 (D4), 7 (D7), and 14 (D14) days of hypoxic exposure, and again 14 days post exposure (P14). Samples were analyzed for key parameters of the ABP including reticulocyte percentage (Ret%), hemoglobin concentration ([Hb]), and the OFF-score. The ABP adaptive model was administered at a specificity of 99% to test for atypical findings. We found significant changes in [Hb] and Ret% during the hypoxic intervention. Consequently, this led to ABP threshold deviations at 99% specificity in three participants. Only one of these was flagged as an "atypical passport finding" (ATPF) due to deviation of the OFF-score. When this sample was evaluated by ABP experts it was considered "normal". In conclusion, it is highly unlikely that the present hypoxic exposure protocol would have led to a citation for a doping violation according to WADA guidelines.

Substantial advantage of a combined Bayesian and genotyping approach in testosterone doping tests.

Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG) levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0 after testosterone intake. Future test programs will most likely shift from population based- to individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is dependent on an individual's true negative baseline T/E ratio. The aim was to investigate whether it is possible to increase the sensitivity and specificity of the T/E test by addition of UGT2B17 genotype information in a Bayesian framework. A single intramuscular dose of 500mg testosterone enanthate was given to 55 healthy male volunteers with either two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene. Urinary excretion of TG and the T/E ratio was measured during 15 days. The Bayesian analysis was conducted to calculate the individual T/E cut-off ratio. When adding the genotype information, the program returned lower individual cut-off ratios in all del/del subjects increasing the sensitivity of the test considerably. It will be difficult, if not impossible, to discriminate between a true negative baseline T/E value and a false negative one without knowledge of the UGT2B17 genotype. UGT2B17 genotype information is crucial, both to decide which initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian analysis.

Standardization of reticulocyte counts in the athlete biological passport.

INTRODUCTION: The percentage of circulating reticulocytes (RET%) is a useful marker of blood doping in the context of the Athlete Biological Passport (ABP). The viability of the ABP depends on the comparability of sample data obtained across multiple laboratories for a given athlete. With the recent introduction of a different technology for the measurement of reticulocytes, the goal of this study was to compare currently employed Sysmex XT/XE analyzers to the recently introduced Sysmex XN analyzer. METHODS: RET% differences were searched in two independent data sets, the first consisting of 95 369 RET% values coming from 29 laboratories located in five continents as part of routine testing for the ABP, the second from a targeted study involving 510 samples analyzed on both a Sysmex XT and XN analyzers by two different laboratories. RESULTS: A relatively small but significant bias of 0.27 ([0.22-0.35] 95% CI) for the first data set and 0.19% ([0.16-0.22] 95% CI) for the second data set was observed with Sysmex XN analyzers returning higher values than Sysmex XT/XE analyzers. This bias appears constant over most of the range of RET% measured in elite athletes. CONCLUSION: When RET% values are obtained for the same athlete with different technologies (XT/XE vs XN), an adjustment of RET% emanating from the XT/XE instruments through a decrease of 0.22% within the ABP calculated ranges appears to be sufficient to integrate the results from the two technologies.

Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods.

The combination of growth hormone (GH) and recombinant erythropoietin (rEPO) is thought to be used particularly in endurance sports. Our objective was to reproduce a 2-week administration of rEPO microdoses alone or in combination with GH microdoses (three times a week) on healthy and athletic male subjects and to evaluate if GH had any additional effects compared to EPO treatment alone. The effects of the treatments on hematological parameters and VO2max were studied as well as the detection of GH in serum. While the rEPO microdose regimen was associated with a significant increase in reticulocytes, no clear elevation in hemoglobin concentration (HGB) was observed. Using a correction by plasma volume did not reveal more effects of EPO on HGB. Our results did not show any additional effect when the GH microdoses were co-administered. In addition, no clear increase in VO2max was observed after treatment, with an elevation in only half the subjects in both groups (EPO and EPO+GH). A clear effect of GH on insulin-like growth factor I (IGF-I) was seen but it was lower on procollagen III amino-terminal propeptide (P-III-NP). GH detection using the direct isoform test identified only one subject 24 hours after receiving GH. The GH biomarker test combining IGF-I and P-III-NP was not able to detect the GH administration. However, a longitudinal follow-up of the intraindividual variations showed a significant increase in IGF-I 24 and 48 hours after GH administration in most subjects, while the effect of GH microdoses on P-III-NP was less straightforward.

Mammographic texture synthesis: second-generation clustered lumpy backgrounds using a genetic algorithm.

Synthetic yet realistic images are valuable for many applications in visual sciences and medical imaging. Typically, investigators develop algorithms and adjust their parameters to generate images that are visually similar to real images. In this study, we used a genetic algorithm and an objective, statistical similarity measure to optimize a particular texture generation algorithm, the clustered lumpy backgrounds (CLB) technique, and synthesize images mimicking real mammograms textures. We combined this approach with psychophysical experiments involving the judgment of radiologists, who were asked to qualify the visual realism of the images. Both objective and psychophysical approaches show that the optimized versions are significantly more realistic than the previous CLB model. Anatomical structures are well reproduced, and arbitrary large databases of mammographic texture with visual and statistical realism can be generated. Potential applications include detection experiments, where large amounts of statistically traceable yet realistic images are needed.