Evaluation of fibronectin 1 in one dried blood spot and in urine after rhGH treatment

To detect doping with growth hormone (GH), GH isoform and biomarkers tests are available. Both methods use population-based decision limits. Future testing in anti-doping is progressing toward individual-based reference ranges, and it is possible that with such an approach the sensitivity to detect GH doping may increase. In addition to monitoring different proteins, the use of miRNAs as future GH biomarkers has been discussed. Here we have longitudinally studied the serum concentrations of IGF-I, P-III-NP and the different GH isoforms in nine healthy men prior to, during and after two weeks' administration with low doses (1 and 4 IU/day) of recGH. Moreover, three putative miRNAs were analyzed. The results show that 80% of the participants were identified as atypical findings using the GH isoform test. However, the participants were only positive 1.5-3 hours directly after an injection. Only one of the participants reached a GH-2000 score indicative of doping when a population-based decision limit was applied. When IGF-I and P-III-NP were longitudinally monitored, 88% of the participants were identified above an individual upper threshold arbitrarily calculated as three standard deviations above the mean values of four baseline samples. The miRNA levels displayed large intra-subject variations that did not change in relation to recGH administration. Our results show that the GH isoform test is very sensitive in detecting low doses of recGH but with a short detection window. Moreover, longitudinally monitoring of IGF-I and P-III-NP may be a promising future approach to detect GH doping.

Section: Hours and 3 Hours) After An Injection This Results Is In Agrmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Longitudinally monitoring of P‐III‐NP, IGF‐I, and GH‐2000 score increases the probability of detecting two weeks’ administration of low‐dose recombinant growth hormone compared to GH‐2000 decision limit and GH isoform test and micro RNA markers

Lehtihet

Bhuiyan

Dalby

et al. 2018

“…DBS sample extraction of actual whole blood or modeled‐blood samples spotted on filter paper was performed as previously described with minor modifications. One whole blood spot was punched out manually and the filter paper circle was transferred to clean tubes.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, alternative matrices for blood collection such as dried blood spots (DBS) are being explored . In this sense, recent studies have demonstrated the feasibility of a single DBS to detect recombinant human growth hormone (rhGH) abuse by WADA required methods as well as the detection of new potential indirect biomarkers for such an rhGH abuse . Traditionally used for newborn screening of metabolic diseases, DBS consists of the deposition onto a specific filter paper of drop (s) of blood collected via finger or heel prick .…”

Section: Introductionmentioning

confidence: 99%

Detection of erythropoiesis‐stimulating agents in a single dried blood spot

Reverter-Branchat

Ventura

Din

et al. 2018

The use of dried blood spots (DBS) for anti-doping purposes would facilitate an increase in the number of blood samples because it eliminates the need for specialized personnel and involves minimal invasiveness, reduced costs, stability, and easy transportation and storage. Here, the electrophoretic methodology established by the World Anti-Doping Agency (WADA) to detect erythropoiesis-stimulating agents (ESAs) has been adapted to evaluate their applicability to DBS. A qualitative procedure to detect recombinant erythropoietin (rEPO), novel erythropoiesis-stimulating protein (NESP), and continuous erythropoietin receptor activator (CERA) in a single DBS was optimized and validated. For rEPO and NESP, confirmation was performed in finger-prick DBS from a pilot study and an administration patients study, respectively. For CERA, detection capabilities were evaluated in DBS prepared with modeled-blood spiked with known concentrations of the protein. Main validation parameters concerning DBS sampling such as stability, hematocrit influence, and blood type (capillary vs. venous) described minor variations. Onsite drying appeared not to be essential before transport. Intra- and inter-day variation range was 2.9%-23.5%. Linearity was maintained (r ≥ 0.9) and ESAs were robustly recovered (CV ≤ 20.2%). The validated method permitted the detection of treated subjects after 48 hours and 17 days of rEPO and NESP administration, respectively. The reproduction of a CERA pharmacokinetics showed good possibilities for the method with a detection window that could reach 16 days after its actual administration. Thus, results provided here reinforce the suitability of DBS blood sampling for the analysis of ESA misuse in sports drug testing.

“…Conversely, the retrospectivity of fibronectin 1 analyzed from DBS as a potential biomarker for detecting hGH misuse was found to be particularly long. In the context of a clinical trial with 10 male volunteers (0.026 mg hGH/kg/day for 3 days), Ferro et al determined fibronectin 1 from DBS by means of immunological methods up to 216 hours post‐administration . DBS (25 μL) were extracted, diluted and assayed, demonstrating a significant increase in fibronectin 1 concentrations from 24 hours to 216 hours, which would substantially expand the window of opportunity for doping controls if confounding factors affecting blood fibronectin 1 concentrations can be excluded in future studies.…”

Section: Peptide Hormones Growth Factors Related Substances and MImentioning

confidence: 99%

Annual banned‐substance review: Analytical approaches in human sports drug testing

Thevis

Kuuranne

Geyer

2017

Several high-profile revelations concerning anti-doping rule violations over the past 12 months have outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti-doping system. At this time, the necessity to enhance, expand, and improve analytical test methods in response to the substances outlined in the World Anti-Doping Agency's (WADA) Prohibited List represents an increasingly crucial task for modern sports drug-testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug-testing assays, drug elimination profiles, alternative test matrices, together with recent advances in instrumental developments. This annual banned-substance review evaluates literature published between October 2016 and September 2017 offering an in-depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2017 Prohibited List.