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Biochemical markers of insulin- like growth factor -I misuse in athletes: the response of serum IGF- I, procollagen type III amino -terminal propeptide, and the GH -2000 score to the administration of rhIGF- I/rhIGF binding protein- 3 complex.
J Clin Endocrinol Metab.
2014;99(6):2259- 2268. doi:10.1210/jc.2013- 3897 2.
Holt RIG, Sönksen PH.
Growth hormone, IGF- I and insulin and their abuse in sport.
Br J Pharmacol .
2008;154(3):542- 556. doi:10.1038/bjp.2008.99 3.
World Anti -Doping Agency.
International Standard for Laboratories - Version 11.0 .
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Accessed June 8, 2023. https://www.wada -ama.org/en/resources/world- anti-doping-program/international -standard- laboratories -isl#resource- download 4.
Powrie JK, Bassett EE, Rosen T, et al.
Detection of growth hormone abuse in sport.
Growth Hormone & IGF Research.
2007;17(3):220 -226. doi:10.1016/j.ghir.2007.01.011 5.
Holt RIG, Erotokritou -Mulligan I, McHugh C, et al.
The GH -2004 project: the response of IGF1 and type III pro -collagen to the administration of exogenous GH in non -Caucasian amateur athletes.
European journal of endoc rinology .
2010;163(1):45 -54. doi:https://doi.org/10.1530/EJE -09-0978 6.
Erotokritou- Mulligan I, Bassett EE, Kniess A, Sönksen PH, Holt RIG.
Validation of the growth hormone (GH) -dependent marker method of detecting GH abuse in sport through the use of independent data sets.
Growth Hormone & IGF Research.
2007;17(5):416- 423. doi:10.1016/j.ghir.2007.04.013 7.
Longobardi S, Keay N, Ehrnborg C, et al.
Growth Hormone (GH) Effects on Bone and Collagen Turnover in Healthy Adults and Its Potential as a Marker of GH Abuse in Sports: A Double Blind, Placebo- Controlled Study.
J Clin Endocrinol Metab.
2000;85(4):1505 -1512. doi:10.1210/jcem.85.4.6551 8.
Wallace JD, Cuneo RC, Lundberg PA.
Responses of Markers of Bone and Collagen Turnover to Exercise, Growth Hormone (GH) Administration, and GH Withdrawal in Trained Adult Males.
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Wallace JD, Cuneo RC, Baxter R, et al.
Responses of the Growth Hormone (GH) and Insulin- Like Growth Factor Axis to Exercise, GH Administration, and GH Withdrawal in Trained Adult Males: A Potential Test for GH Abuse in Sport.
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Dall R, Longobardi S, Ehrnborg C, et al.
The Effect of Four Weeks of Supraphysiological Growth Hormone Administration on the Insulin- Like Growth Factor Axis in Women and Men.
J Clin Endocri nol Metab.
2000;85(11):4193- 4200. doi:10.1210/jcem.85.11.6964 11.
Nelson AE, Meinhardt U, Hansen JL, et al.
Pharmacodynamics of growth hormone abuse biomarkers and the influence of gender and testosterone: a randomized double- blind placebo-controlled study in young recreational athletes.
J Clin Endocrinol Metab.
2008;93(6):2213 -2222. doi:10.1210/jc.2008- 0402 ABP Operating Guidelines – Version 9.0 – July 2023 Page 40/91 12.
Moncrieffe D, Cox HD, Carletta S, et al.
Inter -Laboratory Agreement of Insulin- like Growth Factor 1 Concentrations Measured Intact by Mass Spectrom etry.
Clinical Chemistry .
2020;66(4):579 -586. doi:10.1093/clinchem/hvaa043 13.
Knudsen CS, Heickendorff L, Nexo E. Measurement of amino terminal propeptide of type III procollagen (PIIINP) employing the ADVIA Centaur platform.
Validation, reference interval and comparison to UniQ RIA.
Clinical Chemistry and Laboratory Medicine (CCLM) .
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Technical Document - Minimum Criteria for Chromatographic -Mass Spectrometric Confirmation of the I dentity of Analytes for Doping Control Purposes.
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World Anti -Doping Agency.
International Standard for Testing and Investigations.
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Drug Test Anal .
2015;7(9):745- 755. doi:10.1002/dta.1772 ABP Operating Guidelines – Version 9.0 – July 2023 Page 41/91 3.5.
Laboratory Guidelines - Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport 1.0 Objective These Laboratory Guidelines have been developed to ensure a harmonized application of the Analytical Testing Procedure for the quantification of endogenous steroid Markers measured in blood (serum) as part of the Steroidal Module of the Athlete Biological Passport (ABP).
The document provides guidance on the pre -analytical details, Sample preparation procedure, the performance of the analyses and the reporting of the test results.
2.0 Scope These Laboratory Guidelines contain requirements for the implementation of the Analytical Testing Procedure for the quantification of endogenous steroid Markers in blood (serum) as part of the Steroidal Module of the ABP to uncover use of endogenous anabolic androgenic steroids (EAAS) administered exogenously.
These Laboratory Guidelines follow the rules established in the WADA International Standard for Laboratories (ISL) 1 and relevant Technical Documents (TDs) regarding the Analytical Testing of blood Samples .
3.0 Introduction to the Analytical Testing Procedure The Analytical Testing Procedure involves the measurement of two (2) Markers , namely Testosterone (T) and Androstenedione ( Androst -4-ene-3,17-dione, A4), which are naturally present in blood, and the calculation of the T/A4 ratio.
While the endogenous levels of these Markers are gender -specific, they have been identified as relevant target Analytes to detect T abuse with an increased sensitivity in fem ale Athletes 2,3, as well as the transdermal application of T -related drugs in both genders 4–6.
The quantification of T and A4 concentrations is based on Liquid Chromatography (LC) combined with tandem Mass Spectrometry (LC-MSn; n ≥ 1) .
For the purposes of the ABP, an initial quantification from the “A” Sample is performed.
When requested, a confirmatory quantification of the “A” Sample may additionally be performed (see Article 6.2 ) to confirm the concentrations and to perform identification of the Markers (as per TD IDCR7).
The concentrations of T and A4 in blood reported by the Laboratories are integrated in the Steroidal Module of ADAMS, using a similar Bayesian approach to that applied in the other Steroidal (urine), Hematological and Endocrine Modules of the ABP .
ABP Operating Guidelines – Version 9.0 – July 2023 Page 42/91 4.0 Assay Pre -analytical Procedure − The Laboratory should (usually) receive refrigerated (not frozeni) “A” and “B” blood Samples , which have been collected in blood tubes containing an inert polymeric serum separator gel and a clotting activation factor (for example: BD Vacutainer® SSTTM-II Plus tubes, EU ref 367955; BD Vacutainer® SSTTM-II Plus Advance tubes, EU ref 367954; BD Vacutainer® SSTTM tubes, US ref 367986 ) in accordance with the International Standard for Testing and Investigation (ISTI) 7; − Alternatively, if the clotting and centrifugation of the Sample is performed prior to reception at the Laboratory (for example, at the site of Sample collection), Samples may be received at the Laboratory as frozen/refrigerated blood Samples either in the same Sample collection tubes or as separated serum in new tubes; − The Laboratory shall c heck the status of the Sample(s) (e.g., evidence of hemolysis) and the integrity of the collection tubes ( e.g., evidence of breakage of the separating gel).
The Laboratory shall note any unusual condition of the Sample and record such condition(s) in the Test Report in ADAMS; − Any Samples delivered to the Laboratory in tubes containing an anti -coagulant (for example, ABP blood Samples collected in EDTA tubes), or as separated plasma, shall not be analyzed for Markers of the Endocrine Module; − The Laboratory shall notify and seek advice from the Testing Authority regarding rejection or Analytical Testing of Samples for which irregularities are noted (see ISL 1).
4.1 Samples received as non- separated blood in tubes containing an inert polymeric serum separator gel and a clotting activation factor: Reception Both Samples “A” and “B” shall be centrifuged for 10-15 min at 1300 -1500 g as soon as possible after reception at the La boratory .
The “A” Sample shall be used for the initial and confirmatory (if needed) quantifications (see below).
The “B” Sample shall be step- frozen and stored until use, if needed (see below).
Aliquoting and analysis An Aliquot of the “A” Sample serum shall be taken for initial quantification .
The remaining “A” serum fraction may be kept in the Sample collection tube or aliquoted into new vials with label(s) ensuring that Laboratory Internal Chain of Custody is maintained.
i unless the blood matrix components have been separated before shipment to the Laboratory .
ABP Operating Guidelines – Version 9.0 – July 2023 Page 43/91 For initial quantification: • the Aliquot may be analyzed immediately after aliquoting; or • the Aliquot shall be stored at approximately 4 °C°C if analyzed within 24h (within a maximum of five (5) days from Sample collection); or • the Aliquot shall be frozen (-20°C ) if the analysis will be conducted more than 24h after aliquoting.
For the confirmatory quantification, a new Aliquot of the “A” Sample shall be analyzed immediately after aliquoting.
[Comment: When analyses specific to the ABP are requested for blood (serum) Samples (i.e., Markers of the Endocrine Module or blood steroid Markers as part of the Steroidal Module), only the “A” Sample should be considered for the initial and the confirmatory quantifications of the Markers.
In cases where the “A” Sample is not suitable for the performance of ABP Markers quantification (e.g., there is insufficient Sample volume; the Sample container has not been properly sealed or has been broken; the Sample’s integrity has been compromised in any way; the “A” Sample is missing), a splitting procedure of the “B” Sample could be performed, as detailed in the ISL1.]
Storage [The same storage conditions apply for Samples received in conditions described in section 4.2] Storage for up to three (3) months  at approximately -20 °C.
Storage for more than three (3) months  freeze at approximately - 20 °C and transfer to approximately -70 to - 80 °C.
[Comment: If the separated serum fraction is kept in the Sample collection tube, it shall be step-frozen for storage according to the tube manufacturer’s instructions until analy sis.
If the Laboratory transfers the Aliquot into new vial s for frozen storage, the vials should ensure proper sealing for optimal storage (cryovials with an “O -ring”).
Thawing of Sample(s) for analysis should also be done stepwise.
Samples shall not be thawed under hot water or any other similar process that risks raising the temperature of the Sample above room temperature.
Thawing overnight at 4°C is recommended.]
4.2 Samples received as frozen/refrigerated centrifuged blood/serum Sampl es: Reception If Samples are received frozen, they s hould remain frozen until analysis as described in this Article 4.2 .
If Samples are received refrigerated, they s hould be processed as soon as possible as per Article 4.1.
Aliquoting and analysis Once the Sample “A” is thawed, an Aliquot shall be taken for initial quantification.
This Aliquot may be stored at approximately 4 °C for a maximum of 24h before analysis.