Clomiphene use substantially enhances endogenous testosterone production in males. Consequently, it is included under Section 4.2 of the World Anti-Doping Agency (WADA) Prohibited List and prohibited at all times (in and out of competition). Previous research has highlighted the prolonged excretion and detection windows for clomiphene and its metabolites after cessation of use. In addition to therapeutic use in humans, clomiphene may also be used to increase egg production in laying hens. Accordingly, clomiphene and/or its metabolites may also be detected in human urine after consumption of eggs or meat from treated poultry. To address some of these complexities, WADA has issued targeted guidance in Technical Letter TL26, mandating a minimum reporting level for clomiphene. This short communication evaluates results from clomiphene findings in 325 urine samples over the past 3 years and examines how reporting would be affected by the criteria outlined in TL26.

Steroid esters are a frequently used ergogenic aid among athletes, and their direct detection represents an important approach to anti-doping efforts. However, the presence of esterase enzymes in blood complicates these efforts. Although dried blood spots (DBS) have emerged as a promising solution to the esterase problem, the limited sample volume creates both analytical and practical challenges. The larger collection volume for liquid blood may allow for higher analytical sensitivity, thus offsetting the effects of esterase activity in practical scenarios. First, to evaluate the effects of blood esterases ex vivo, 10 individuals were administered a single dose of either testosterone propionate or testosterone cypionate. Venous blood was collected into serum separator tubes (SST) serum and NaF-containing tubes and stored at varying conditions. Both esters significantly decreased in serum while no ester loss was observed in the NaF-containing plasma. Next, to compare the detectability across matrices stored at conditions meant to mimic real-world scenarios, the individuals administered testosterone propionate collected capillary serum, capillary NaF-containing plasma, and capillary DBS at various times postadministration. Each sample type was stored at different conditions designed to mimic what may be encountered during real-world shipping scenarios. Similar detectability of testosterone propionate was observed between all NaF, DBS, and SST samples stored under cool conditions, whereas detectability was better in NaF and DBS samples under ambient and warm conditions. These results indicate that for samples not shipped in temperature regulated conditions, DBS and NaF-containing plasma are the preferred matrices.

The serum testosterone-to-luteinizing hormone (T/LH) ratio has previously been suggested as a sensitive marker of testosterone use in an anti-doping setting. When measured with an automated immunoassay platform, this ratio represents a quick and cost-effective screening biomarker to further direct isotope ratio mass spectrometry (IRMS) testing efforts in concurrently collected urine samples to confirm testosterone abuse. To evaluate the practicality of implementing the serum T/LH ratio for routine use, testosterone values in 356 serum samples were compared between a "gold-standard" LC-MS/MS method and an automated immunoassay method. Excellent correlation and minimal bias between the two methods were observed, highlighting the validity of the immunoassay method. Next, a testosterone administration study utilizing a transdermal delivery route was conducted to compare the effectiveness of the serum T/LH ratio to the currently used serum testosterone to androstenedione (T/A4) and urinary testosterone to epitestosterone (T/E) ratios. The serum T/LH ratio was more sensitive than the T/A4 ratio and showed similar sensitivity to the urinary T/E ratio with high interindividual variability. Finally, T/LH analysis was conducted on 626 capillary serum samples collected in the field from male Ultimate Fighting Championship athletes. Of the three samples that showed elevated T/LH ratios in this pool, two of the corresponding urine samples were IRMS positive, one of which showed an unremarkable urinary T/E ratio and relatively normal steroid profile. These results indicate serum T/LH ratio monitoring provides a beneficial addition to the anti-doping tool kit and can improve urinary IRMS testing recommendations.

ABSTRACT:This Consensus statement provides an update to the 1996 American College of Sports Medicine Position Stand: The Use of Blood Doping as an Ergogenic Aid. Red blood cell (RBC) mass directly influences exercise performance because RBCs are critical to the transport of oxygen to skeletal muscle, as well as their role in maintaining acid-base status and regulating blood flow. Blood doping is defined as the practice of artificially increasing RBC volume for the purpose of improving exercise performance. Blood doping methods have classically included reinfusion of stored blood as well as administration of pharmaceutical agents such as recombinant human erythropoietin, hypoxia-inducible factor stabilizers or nonpharmaceutical substances, like cobalt or xenon, which stimulate the erythropoietic pathway. It is clear through scientific consensus that other methods to naturally increase hemoglobin, such as hypoxic or hyperoxic exposure, are not considered blood doping. More recently, emerging blood doping threats through gene manipulation have received new attention. In the last 25 yr, the science and technology surrounding oxygen sensing and erythropoiesis, pharmacological intervention, and doping detection has advanced substantially, dictating this updated review. It is the position of the American College of Sports Medicine that any blood doping procedure used with the actual or potential to improve athletic performance is unethical and unfair and exposes the athlete to unwarranted and potentially serious health risks.

Erythropoietin receptor agonist (ERA) testing for antidoping can be achieved in both urine and blood samples. Recent published work showed the comparability between the two matrices and focused on detectability in microvolumetric capillary serum samples collected using the Tasso+ device. Currently, in the antidoping field, blood samples are required to be shipped under cold, temperature-controlled conditions. However, due to the suggested greater stability of EPO in blood compared to urine, it is believed that blood samples should be viable for ERA analysis if shipped under the ambient, not cold temperature-controlled conditions to which urine samples are subjected. In this collaborative study with the Ultimate Fighting Championship, microvolumetric capillary serum samples were collected in the field and shipped under ambient conditions to the laboratory for ERA analysis. Resulting data showed that endogenous EPO was detectable in 100% of these samples, showing no loss in detectability despite shipping under non-controlled conditions. Further, ERA analyses were conducted in the laboratory on additional in-house collected samples and post-EPO administration samples subjected to various storage and shipping conditions, also showing reliable endogenous and recombinant EPO detectability in all samples except those experiencing extreme temperature (50°C) conditions. Taken together, these data highlight the stability of EPO in blood samples and show that ERA blood samples can be collected in the field and shipped without costly temperature-controlled shipping methods and without a loss in detectability.

The applicability of urinary minimum reporting limits (MRLs) to determine in-competition use of prohibited substances is an evolving topic. Most stimulants are subject to a universal MRL, despite the wide range of commercially available dosages for commonly used stimulants. Further, it is unknown whether the urinary MRL is reflective of a pharmacological dose ingested after the start of the in-competition period. To evaluate whether urinary MRLs can distinguish between in-competition and out-of-competition use, a controlled administration study was performed with three commonly used stimulants-amphetamine, methylphenidate, and modafinil at relatively low but therapeutically relevant dosages. Four to six volunteers were administered a particular drug once per day for five consecutive days. Urine, serum, dried blood spots (DBS), and oral fluid (OF) were collected during the active administration period and for 48 h after cessation of use. For all participants, urinary concentrations for all target analytes exceeded the MRL even 48 h after cessation of use. In serum and DBS, most volunteers showed detectable amounts at 48 h post use. Peak concentrations were variable between target compounds even with similar administered dosages. Further, there was a reproducible difference between serum and DBS concentrations. Interpretation of results from OF measurements was challenging due to the inability to normalize for hydration status and OF viscosity. Analyte concentrations decreased steadily over the washout period but did not correlate across matrices for all target analytes. The study reiterates the challenges associated with determining in-competition use by relying on urinary concentrations.

Δ9-Tetrahydrocannabinol (Δ9-THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8-tetrahydrocannabinol (Δ8-THC), have become available for sale. Δ8-THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8-THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9-THC (Δ9-THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8-THC, which metabolizes to Δ8-THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8-THC. This study aims to estimate the prevalence of Δ8-THC use in the athlete community by monitoring for Δ8-THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9-THC-COOH. One thousand samples with a presumptive Δ9-THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8-THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

The monitoring of endogenous steroids in urine has been an important component of the Athlete Biological Passport (ABP) for the last decade. Recently, the quantitation of endogenous steroids in blood has been incorporated into the ABP to increase sensitivity in circumstances where the excretion of urinary ABP biomarkers is low. Current ABP guidelines mandate the use of venous blood draws for blood steroid sample collections, however, recent efforts have focused on investigating the use of less invasive sample collection methods, such as capillary blood collected from the upper arm. The focus of this study was to compare the analytical results of venous and capillary blood collected weekly from 20 individuals, 10 males and 10 females, over six weeks. The two primary biomarkers of the blood steroid ABP module, testosterone (T) and the testosterone/androstenedione (T/A4) ratio, were compared, as well as luteinizing hormone (LH) and the T/LH ratio in male participants, two biomarkers known to be responsive to T use. All biomarkers showed excellent agreement between venous and capillary blood. Longitudinal stability between sample types within individuals was also comparable for all biomarkers. Finally, storage of simultaneously collected capillary samples at room temperature and frozen conditions was compared with evaluate the potential impact of non-cold chain shipping conditions. Most biomarkers showed excellent agreement between frozen and room temperature storage conditions. These results indicate capillary blood collections represent a promising alternative to venous blood collections for the blood steroid module of the ABP.

The amino-terminal propeptide of type III procollagen (P-III-NP) is used with IGF-I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P-III-NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We aimed to develop a high-throughput assay using immunoaffinity enrichment coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously. To facilitate higher throughput, we transferred the assay from microcentrifuge tubes to a 96-well plate format with partially automated pipetting. The method was linear (Pearson's R ≥ 0.994) over an estimated concentration range of 1.35-13.3 nM, 0.04-2.28 nM, and 0.26-5.1 nM for each surrogate peptide of P-III-NP, collagen degradation products, and the carboxyl-terminal propeptide, respectively. Intra-day and inter-day imprecision were both < 13.6%, and the results of robustness testing were also encouraging. The method was successfully applied to capillary blood samples obtained using Tasso+ microsampling devices. Modest correlation of P-III-NP concentration was observed between our new method and a WADA-approved immunoassay (N = 40, Pearson's R = 0.789) with a significant bias of -87.8%. Our method simultaneously quantifies four peptides belonging to three regions of type III procollagen in human serum. High bias between assays highlights the need for common higher-order calibrators or reference materials to help improve the comparability of results across laboratories.

The instability of erythropoietin receptor agonists (ERAs, i.e., EPO) in urine presents a challenge to their detectability in doping control samples; however, this issue is not often seen in blood (serum) samples. With the anti-doping field beginning to transition into alternative blood collection technologies, it is important to understand recombinant EPO (rEPO) detectability in serum samples collected from one such capillary collection device, the Tasso+ SST. Twelve individuals were administered a single, 40 IU/kg dose of rEPO (epoetin alfa, EPOGEN®). Following administration, matched urine, venous serum, and capillary serum samples were concurrently collected. Urine aliquots were subject to various storage times and temperatures mimicking shipping conditions of doping control urine samples to assess EPO stability, while other urine aliquots, venous serum, and capillary serum aliquots were frozen until analysis to understand rEPO detectability across all three matrices. EPO and rEPO instability was identified in urine collected from 8 of 12 participants, especially in aliquots stored at room temperature and 37°C. In some of these unstable samples, rEPO was still detectable, while in others, no recombinant nor endogenous EPO was detectable and would have resulted in negative sample reports. Analyzing the concurrently collected urine, venous, and capillary serum samples, rEPO detectability was identical across the three matrices. In most cases, rEPO was detectable for at least 168 h post-administration. Noting greater stability in blood compared with urine, it is recommended that anti-doping authorities utilize this novel capillary serum collection technology to improve overall ERA detectability in doping control samples.

A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)-clomiphene citrate and consumption of clomiphene-containing eggs by the urinary pattern of four mono-hydroxylated clomiphene metabolites. However, reanalyses of doping-control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene-containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow-up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long-term metabolite in doping controls. METHODS:(E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer-specific HC elimination profiles could be monitored. RESULTS:The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. CONCLUSION:These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 μg hCG injections over 3 weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione.

19-Norandrosterone (19NA) is the preferred urinary target compound to identify doping with nandrolone or related 19-norsteroids. At concentrations between 2.5 and 15 ng/mL, isotope ratio mass spectrometry (IRMS) is required to establish exogenous origin of urinary 19NA. An absolute difference of 3‰ between urinary 19NA and an endogenous reference compound (ERC) constitutes a finding for exogenous origin of 19NA. Over the last 3 years, 77 samples containing urinary 19NA between 2.5 and 15 ng/mL were analyzed at our laboratory. The measured δ13 C values for 19NA ranged from -29.5‰ to -16.8‰. In comparison, the δ13 C values for the corresponding urinary ERCs ranged from -22.4‰ to -16.2‰. Due to the considerable overlap in values between the target compound and the natural range of urinary ERCs, it can be challenging to distinguish between endogenous and exogenous origins of urinary 19NA. In addition, it is well known that consumption of offal from non-castrated pigs can produce 19NA in urine. To determine whether this could cause a positive IRMS finding under the current IRMS positivity criteria, meat from non-castrated boars fed a mixture of corn and soy was consumed by 13 volunteers. Two volunteers produced 19NA findings above 2.5 ng/mL, and the measured isotope values, while inconsistent with documented 19-norsteroid preparations, did meet IRMS positivity criteria. However, these increases in 19NA urinary concentrations were short-lived due to rapid elimination. Timely follow-up collections may help support a claim for dietary exposure when low urinary concentrations of 19NA with pseudo-endogenous isotope values are observed.

Background Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. Methods Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. Results Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG >= 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. Conclusions Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900

This study (1) determined the association of time since initial vaccine regimen, booster dose receipt, and COVID‐19 history with antibody titer, as well as change in titer levels over a defined period, and (2) determined risk of COVID‐19 associated with low titer levels. This observational study used data from staff participating in the National Football League COVID‐19 Monitoring Program. A cohort of staff consented to antibody‐focused sub‐study, during which detailed longitudinal data were collected. Among all staff in the program who received antibody testing, COVID‐19 incidence following antibody testing was determined. Five hundred eighty‐six sub‐study participants completed initial antibody testing; 80% (469) completed follow‐up testing 50−101 days later. Among 389 individuals who were not boosted at initial testing, the odds of titer < 1000 AU/mL (vs. ≥1000 AU/mL) increased 44% (odds ratio [OR] = 1.44, 95% confidence interval [CI]: 1.18−1.75) for every 30 days since final dose. Among 126 participants boosted before initial testing with no COVID‐19 history, 125 (99%) had a value > 2500 AU/ml; 86 (96%) of 90 tested at follow‐up and did not develop COVID‐19 in the interim remained at that value. One thousand fifty‐seven fully vaccinated (330 [29%] boosted at antibody test) individuals participating in the monitoring program were followed to determine COVID‐19 status. Individuals with titer value < 1000 AU/mL had twice the risk of COVID‐19 as those with >2500 AU/mL (HR = 2.02, 95% CI: 1.28−3.18). Antibody levels decrease postvaccination; boosting increases titer values. While antibody level is not a clear proxy for infection immunity, lower titer values are associated with higher COVID‐19 incidence, suggesting increased protection from boosters.

Objectives To quantify levels of potential exposure to SARS-CoV-2 surrounding a typical professional American football game, with a focus on interactions on-field between teammates and opposing players before, during, and immediately after competition. Methods We examined across-Club consecutive interactions >= 2 minutes within 6 feet [1.8 meters] between athletes on opposing Clubs for all 2020 NFL regular season games (n = 256). Cumulative interaction was measured for a representative subset (n = 119; 46%) of games. Wearable proximity tracking devices (Kinexon) were used to measure distance and duration of interactions; these data were combined with game schedule and Club rosters for analyses. Frequency and per-game mean, median, interquartile range for consecutive interactions >= 2/>= 5 minutes and cumulative interactions >= 5/>= 15 were described overall and stratified by pre-game, in-game, and post-game. Results Of the 1964 distinct player-to-opponent contacts >= 2 minutes in NFL regular season games, the majority (n = 1,699; 87%) were fewer than 5 minutes in consecutive length. Among the mean 7.7 distinct contacts >= 2 minutes with opponents each game (median = 4; IQR = 2, 8), very few were >= 5 consecutive minutes at any point (mean = 1.0; median = 0; IQR = 0, 0). Most (n = 849; 43.2%) distinct contacts were pre-game, 546 (27.8%) were during competition, and 569 (29%) were post-game. In games where cumulative interactions were analyzed, there was an average of 17.1 player/opponent interactions with cumulative exposure >= 5 minutes (median = 12; IQR = 4, 30), almost all of which occurred during competition. Conclusion There is limited and short contact between and among competing players in professional American football. In the setting of infectious disease such as the COVID-19 pandemic, a robust prevention program integrating masking, distancing, hygiene, and ventilation when off-field can be created to minimize on- and off-field exposures, which effectively reduces transmission risk in outdoors and/or well-ventilated stadium settings.

Hematological results in the context of the Athlete Biological Passport (ABP) from a placebo-controlled EPO administration study are provided here. Twelve participants administered eight subcutaneous boosting doses of epoetin alfa (at 40 IU/kg) over the course of 20 days. After a 10-day washout period, the same volunteers administered six microdoses (900 IU), intravenously, over 13 days. A blinded placebo cohort followed the same dosing pattern, administering saline instead of EPO. All participants supplemented with oral iron, daily, throughout the entirety of the study. In the EPO cohort, as expected, significant changes from baseline were identified in IRF, RET#, RET%, RDW, HCT, HGB, and RBC. No meaningful changes were identified in the placebo cohort population. From the ABP perspective, atypical passport findings (ATPF) were identified in 49% of the samples collected during the boosting and initial washout phases, and 24% of the samples during the microdosing and final washout phases. ATPFs from this cohort were flagged as late as Day 70, the final day of the study. Only a single ATPF was identified from all samples collected from the placebo cohort. ABPs from all volunteers in the study are provided as an avenue to visually convey differences in magnitude and timing of the hematological changes caused by EPO on the individual level. These data are expected to provide important content for Athlete Passport Management Units and ABP expert panels alike.

Insulin analogues and large bioactive peptides may be used by athletes to enhance performance and are banned by the World Anti-Doping Agency (WADA). In addition to insulin analogues, the large peptides include a structurally diverse set of peptides including analogues of growth hormone releasing hormone (GHRH), insulin-like growth factor-1 (IGF-1), and mechano-growth factor (MGF). Detection of this class of peptides is difficult due to their absorptive losses and presence at very low concentrations in urine. In this report, a high throughput method is described that allows sensitive detection of four classes of large peptides in one assay. Sample extraction is performed by ultrafiltration to concentrate the urine followed by solid phase extraction in a 96-well micro-elution plate. Peptides in the urine samples are detected on a triple quadrupole mass spectrometer coupled to standard flow liquid chromatography. The method was validated and evaluated for limit of detection, limit of identification, specificity, precision, carryover, recovery, matrix interference, and post-extraction stability. The limit of detection for insulin analogues is between 5 and 25 pg/ml and between 5 and 50 pg/ml for the other peptide classes. Specificity was good with no detection of interfering peaks in blank urine samples. Carryover from a high concentration sample was not observed and the post-extraction stability was between 77% and 107%. The method was able to detect insulin analogues in three diabetic urine samples. Increased screening for this class of peptides will improve detection and deterrence.

Luspatercept (Reblozyl®) is a newly approved anti-anemic drug prohibited by the World Anti-Doping Agency. It promotes erythropoiesis by limiting apoptosis of immature erythroblasts and the risk of misuse by athletes for doping is high. Proposed detection methods have been published recently but only evaluated in vitro. The objective of this study was to perform the first administration of luspatercept in healthy volunteers for antidoping purpose and to evaluate the detectability in serum, dried capillary blood spots (DBS, collected using TASSO M20 device), and urine. Indirect detection was also evaluated by analyzing hematological parameters for the Athlete Biological Passport. Four volunteers (two males, two females) received one subtherapeutic dose of luspatercept (0.25 mg/kg) followed 3 weeks after by a second dose. Samples were collected from before administration until 7 weeks after the second dose. After immunopurification, electrophoretic separation SDS-/SAR-/IEF- polyacrylamide gel electrophoresis (PAGE), and immunodetection, luspatercept was detected at high levels in serum until the end of the collection, sign of a very slow elimination and similarly detected unchanged at lower levels in urine from 2 days after the first administration until 7 weeks postadministration. DBS showed also the same long window of detection. Luspatercept effects were however of limited amplitude on hematological markers, and only two subjects presented atypical points outside the physiological limits during the study. The direct detection method was very efficient, and change of electrophoretic method and detection antibody can be used for confirmation of suspicious samples.

A multi-phase study was designed to examine the detectability of human growth hormone (GH) use in capillary dried blood spots (DBS). First, thirteen subjects self-injected a single, 2mg dose of somatropin and collected capillary DBS samples for 24 hours. Next, nine subjects self-injected 2mg somatropin, six times over the course of eleven days; DBS were collected intermittently following dosing. Finally, a non-drug, large-scale field study involved DBS collections from an athlete and staff population over three years. All DBS samples were self-collected using the Tasso M20 device and were analyzed for the presence of GH using the WADA-approved GH isoforms test. Following the single dose, positive detection within 12 hours of dosing was 86% and 56% sensitive on Kits 1 and 2, respectively. In the multi-dose study, detection within 12 hours was 85% and 69% sensitive on Kits 1 and 2, respectively. No positives were detected outside the 12-hour window following a single dose, wherein detection was 5.6% sensitive at 24-hours in the multi-dose study. Combining the 12-hour windows from both studies, 100% of samples had measurable recombinant (REC) and pituitary (PIT) GH concentrations above the assay LoD, 0.041 ng/mL. Finally, 1213 samples were collected in the large-scale field study: 189 showed REC and PIT concentrations above the LoD; none returned positive results. GH is detectable in capillary DBS using the isoforms method for 12-24 hours following use. While detection is short lived, transitioning to a DBS self-collection method can allow more frequent testing and increase deterrence to GH abuse.