Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

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.

Tracking immature reticulocyte proteins for improved detection of recombinant human erythropoietin (rhEPO) abuse.

Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.

Effect of head and face insulation on cooling rate during snow burial.

OBJECTIVES: Avalanche victims are subjected to a number of physiological stressors during burial. We simulated avalanche burial to monitor physiological data and determine whether wearing head and face insulation slows cooling rate during snow burial. In addition, we sought to compare 3 different types of temperature measurement methods. METHODS: Nine subjects underwent 2 burials each, 1 with head and face insulation and 1 without. Burials consisted of a 60-minute burial phase followed by a 60-minute rewarming phase. Temperature was measured via 3 methods: esophageal probe, ingestible capsule, and rectal probe. RESULTS: Cooling and rewarming rates were not statistically different between the 2 testing conditions when measured by the 3 measurement methods. All temperature measurement methods correlated significantly. CONCLUSIONS: Head and face insulation did not protect the simulated avalanche victim from faster cooling or rewarming. Because the 3 temperature measurement methods correlated, the ingestible capsule may provide an advantageous noninvasive method for snow burial and future hypothermia studies if interruptions in data transmission can be minimized.

Assessing EPO stability in urine and comparing recombinant EPO detectability in matched urine, venous serum, and capillary serum following a controlled epoetin alfa administration.

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.

Influence of multiple human chorionic gonadotropin administrations on serum and urinary steroid Athlete Biological Passport profiles in males.

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.

EPO and the athlete biological passport: Hematological results from a placebo-controlled, boosting and microdose EPO administration in male recreational athletes.

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.

Growth hormone isoform testing in capillary dried blood spots: Results from single and multiple dose administration studies and large-scale field collections.

A multiphase study was designed to examine the detectability of human growth hormone (GH) use in capillary dried blood spots (DBS). First, 13 subjects self-injected a single, 2-mg dose of somatropin and collected capillary DBS samples for 24 h. Next, nine subjects self-injected 2-mg somatropin, six times over the course of 11 days; DBS were collected intermittently following dosing. Finally, a nondrug, large-scale field study involved DBS collections from an athlete and staff population over 3 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 h of dosing was 86% and 56% sensitive on Kits 1 and 2, respectively. In the multidose study, detection within 12 h was 85% and 69% sensitive on Kits 1 and 2, respectively. No positives were detected outside the 12-h window following a single dose, wherein detection was 5.6% sensitive at 24-h in the multidose study. Combining the 12-h 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 h 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.

Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes.

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.

Evaluation of epiandrosterone as a long-term marker of testosterone use.

Identification and evaluation of long-term markers is crucial in prolonging the detection window for anabolic steroid abuse in sport. Recently, sulfoconjugated epiandrosterone was identified as a potential long-term marker for the abuse of certain endogenous anabolic agents, including testosterone, which continues to be widely used as a performance enhancing agent in sport. To evaluate the applicability of epiandrosterone sulfate as a marker for testosterone use, administration studies were conducted with multiple modes of testosterone administration - transdermal, intramuscular, and subcutaneous. A modified sample preparation method was used to collect both glucuronidated and sulfoconjugated analytes of interest. Carbon isotope ratio measurements from the administration studies are presented here. Epiandrosterone was less effective than the conventionally used target compounds for detection of the low dose application (transdermal gel). With intramuscular administration, epiandrosterone was more diagnostic than with transdermal administration, but it did not prolong the detection window more than the conventional target compounds. With subcutaneous administration, the doses administered to the subjects were varied and the effect on the epiandrosterone values was dependent on the magnitude of the dose administered. Epiandrosterone does not appear to be a useful marker in the detection of low dose testosterone administration. It is responsive to higher dose administration, but it does not provide an extension of the detection window relative to conventional target compounds.

Perceived confidence in the FAST exam before and after an educational intervention in a developing country.

BACKGROUND: Trauma care in developing countries suffers from many limitations related to equipment shortages, disrepair, quality assurance, and lack of training. Health care providers in the three principal hospitals in Cusco, Peru have ultrasound machines, but they do not utilize this for the focused assessment of sonography in trauma (FAST) scan (only one of the three hospitals has a computed tomography scanner). AIMS: The goal of this study was to assess the confidence of physicians in a developing country to conduct a FAST exam after an educational intervention. METHODS: Participants were Peruvian health care workers who attended a 2-day conference on trauma. Participants completed a questionnaire based on a 5-point Likert scale (1 = no confidence, 5 = high confidence) to assess comfort with the FAST scan before and after a FAST teaching workshop, which included hands-on ultrasound training. Thirteen individuals, eight of whom were physicians, completed the training and survey. Results were analyzed using paired t test statistics and are reported as pre- and post-training mean scores (+/- standard error), with p < 0.05 considered statistically significant. RESULTS: Participants rated their confidence in using the FAST exam on a trauma patient with an average score of 3.3 (+/- 0.3) pre-training and 4.5 (+/- 0.2) post-training (p = 0.007). When asked about their comfort level in making clinical decisions based on the FAST scan, pre-training average score was 3.5 (+/- 0.4) and post-training was 4.5 (+/- 0.2), p = 0.016. Participants also answered questions about their comfort with the technical aspects of using the ultrasound machine: ability to choose the correct probe (pre: 3.9, post: 4.6, p = 0.011), choosing the correct probe orientation (pre: 3.9, post: 4.6, p = 0.008), and adjusting the depth and gain (pre: 3.1, post: 4.4, p = 0.001). Finally, participants rated their comfort with the specific views of the FAST scan: ability to find the correct subcostal view (pre: 3.3, post: 4.9, p < 0.001), right upper quadrant view (pre: 3.2, post: 4.6, p < 0.001), left upper quadrant view (pre: 3.2, post: 4.4, p = 0.001), and the pelvic view (pre: 3.2, post: 4.5, p < 0.001). CONCLUSION: After a training session in the use of ultrasound in trauma, health care workers in Cusco, Peru reported increased confidence in their FAST scan ability and in their comfort in using this exam for clinical decision-making. Future research should include objective testing of participants' skill as well as longitudinal follow-up to determine the extent to which the FAST scan has been incorporated into participants' evaluations of trauma patients.