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In cases when the temperature data logger accompanies multiple ABP blood Samples , and these ABP blood Samples are analyzed in the same batch by the Laboratory or ABP Laboratory , the temperature data logger shall be stopped before the homogenization of the first ABP blood Sample .
The Laboratory shall proceed with the analysis of all ABP blood Samples associated with the same temperature data logger without delay.
5.2 ABP Blood Sample Analysis The ABP blood Sample shall be analyzed twice.
The Laboratory ’s or ABP Laboratory ’s procedure should minimize the delay between the two analyses .
Absolute differences between the two (2) analyses shall be equal or less than (≤) each of the following criteria in order to accept the results: • 0.1 g/dL for HGB; • 0.15% for RET% if either the first or second measurement is lower or equal to 1.00%; otherw ise 0.25% absolute difference.
The data from the second injection is used to confirm the first injection data.
Therefore, if the absolute differences between the results of the analyses are within the criteria above, then only the first injection data is reported into ADAMS.
If the absolute differences between the results of the two analyses are greater than (>) those defined above, then the ABP blood Sample shall be analyzed twice again in accordance with Article 5.2.
In cases of repeated analysis, the ABP blood Sample shall be mixed prior to re- analysis using the automated mixing feature of the blood analyzer or by appropriate manual inversion.
This reanalysis procedure shall be repeated until the absolute differences between the results of the two (2) most recent analyses are within the criteria specified above.
The requirements for an Initial Testing Procedure (ITP), an “A” Sample Confirmation Procedure (CP) and a “B” Sample CP, as defined in the ISL [1], shall not be applicable to ABP blood Samples analyzed for the purposes of the ABP .
6.0 Reporting 6.1 Temperature Report The Laboratory or ABP Laboratory shall promptly submit into ADAMS the raw temperature profile report recorded by the temperature data logger.
The filename shall consist in the concatenation of the data logger ID with the date of Sample reception by the Laboratory or ABP Laboratory (“YYYY- MM-DD” in local time) separated by an underscore.
For example, for a data logger ID “KG34V10” and a ABP Operating Guidelines – Version 9.0 – July 2023 Page 29/91 date of Sample reception “2015- 03-25”, the Laboratory or ABP Laboratory shall report the temperature profile under the filename “KG34V10_2015- 03-25.txt”.
The Laboratory or ABP Laboratory shall report the temperature profile into ADAMS before the test results of the Sample, when temperature data can be retrieved from the logger.
[Comment: Where the Sample meets the requirements of the ISTI Annex I, Article I.2.7, and is analyzed at the Sample collection site without delay, a temperature data logger is not necessary and the Laboratory shall proceed to reporting the test results of the Sample .
In cases that the Laboratory is unable to upload a suitable temperature profile report from the temperature data logger into ADAMS, the Laboratory shall proceed to upload the test results of the relevant Sample(s).]
6.2 Reporting ABP Blood Sample Test Results The Laboratory or ABP Laboratory should report the ABP blood Sample test results as soon as possible and within three (3) days after Sample reception.
The following shall be reported into ADAMS: • Status (“Submitted” or “Not Analyzed”); • ABP blood Sample code; • Type of test ( Out-of-Competition / In-Competition ); • Sport and discipline; • Date and time of receipt of the ABP blood Sample ; • Date and time of analysis of the ABP blood Sample ; • The name of the Testing Authority ; • The name of the Sample Collection Authority ; • Type of Sample (blood Passport ); • Type of analyzer; • Test results (other variables may be included for quality purposes): Blood Variable Unit(s) Haemoglobin HGB g/dL Hematocrit HCT % Immature Reticulocyte Fraction IRF % Mean Corpuscular Haemoglobin MCH pg Mean Corpuscular Haemoglobin Concentration MCHC g/dL Mean Corpuscular Volume MCV fL OFF-Score - - Platelets PLT 103/µL Red Blood Cell Distribution Width RDW -SD fL Red Blood Cells RBC 106/µL Reticulocytes – in absolute number RET 106/µL Reticulocytes Percentage RET% % White Blood Cells WBC 103/µL ABP Operating Guidelines – Version 9.0 – July 2023 Page 30/91 • Include a comment describing any relevant deviation as part of the ABP blood Sample ’s ADAMS record.
7.0 References [1] The World Anti -Doping Code International Standard for Laboratories (ISL).
[2] WADA Technical Document TD LCOC: Laboratory Internal Chain of Custody .
[Comment: Current versions of WADA ISL and Technical Documents may be found at https://www.wada -ama.org/en/what -we-do/science- medical/laboratories ] ABP Operating Guidelines – Version 9.0 – July 2023 Page 31/91 3.4.
Laboratory Guidelines – Analytical Requirements for the Endocrine Module of the Athlete Biological Passport 1.0 Objective These Laboratory Guidelines have been developed to ensure a harmonized application of Analytical Testing Procedures for the measurement of Markers of human Growth Hormone (hGH) as part of the Endocrine Module of the Athlete Biological Passport (ABP).
The document provides guidance on the pre-analytical details, Sample prepar ation 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 Procedures for the quantification of hGH Markers as part of the Endocrine Module of the ABP, which allows the detection of hGH doping and may also have utility in detecting GH secretagogues and IGF -I abuse in sport 1,2.
These Laboratory Guidelines follow the rules established in the WADA International Standard for Laboratories (ISL) 3 and relevant Technical Documents (TDs) regarding the Analyt ical Testing of blood Samples .
3.0 Introduction to the Analytical Testing Procedures The Analytical Testing Procedures for the Endocrine Module involve the measurement of two (2) Markers of hGH biological activity, namely Insulin -like Growth Factor -I (IGF -I) and N -terminal Pro -peptide of Type III Collagen (P -III-NP), which are naturally present in blood and whose concentrations are increased following hGH administration 4–11.
The measured concentrations of these two (2) Markers are then combined in a discriminant function formulae to calculate a GH -2000 score, which is gender -spec ific and includes an adjustment for age to reflect the age- related decline in hGH and Marker concentrations 4.
In order to generate individual Athlete longitudinal data that are comparable between Laboratories , a specific IGF -I / P-III-NP assay pairing is applied for the measurement of concentrations of IGF -I and P-III-NP in blood (serum) for the purposes of the ABP.
The assays used for the Endocrine Module of the ABP are limited to: • Intact IGF -I quantification by top- down Liquid Chromatography -(tandem) Mass Spectrometry (LC-MSn; n ≥ 1) 12, as detailed in Table 2 below.
• P-III-NP quantification using Siemens ADVIA Centaur P -III-NP chemilu minescence immunoassay (Siemens Healthcare Laboratory Diagnostics, Camberley, UK).
The Siemens ADVIA Centaur P -III-NP assay is an automated, two- site sandwich, chemiluminescent ABP Operating Guidelines – Version 9.0 – July 2023 Page 32/91 immunoassay 13.
The assay uses two (2) monoclonal mouse antibodies: the first antibody is an acridinium ester -labeled anti -P-III-NP antibody.
The second antibody is a biotin- labeled anti-P-III-NP antibody.
The solid phase contains streptavidin- coated paramagnetic particles and during the reaction, the light emitted by the acridinium label is directly proportional to the concentration of P -III-NP in the sample.
The Siemens P -III-NP assay is calibrated by the manufacturer using a standard derived from bovine P -III-NP.
For the purposes of the ABP, an initial quantification of the “A” Sample is performed.
When requested, a confirmatory quantification of the “A” Sample may additionally be performed using the same assay pairing (see, Article 6.2) to confirm the concentrations and to perform identification of IGF- I (as per TD IDCR14).
The concentrations of IGF- I and P -III-NP reported by the Laboratories , as well as the GH -2000 score automatically calculated in ADAMS, are integrated in the Endocrine Module of ADAMS using a similar Bayesian approach to that applied in the Steroidal and Hematological Modules of the ABP 15.
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) 16; [Comment : Previous studies have demonstrated that IGF -I and P -III-NP concentrations remain stable if the Sample is maintained at a refrigerated temperature for up to 5 days 17.]
− 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/r efrigerated blood Samples either in the same Sample collection tubes or as separated serum in new tubes; − The Laboratory shall check 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 delive red 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; i unless the blood matrix components have been separated before shipment to the Laboratory .
ABP Operating Guidelines – Version 9.0 – July 2023 Page 33/91 − 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 3).
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 Laboratory .
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 Two (2) Aliquots 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.
For initial quantificat ion: • the Aliquots may be analyzed immediately after aliquoting; or • the Aliquots shall be stored at approximately 4 °C if analyzed within 24h (within a maximum of five (5) days from Sample collection); or • the Aliquots shall be frozen ( -20°C ) if the analysi s will be conducted more than 24h after aliquoting.
For the confirmatory quantification, two (2) new Aliquots 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 sui table 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 ISL3.]
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 analysis.
If the Laboratory transfers the Aliquot into new vials 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 Samples : Reception If Samples are received frozen, they should remain frozen until analysis as described in this Article 4.2.
If Samples are received refrigerated, they should be processed as soon as possible as per Article 4.1.
ABP Operating Guidelines – Version 9.0 – July 2023 Page 34/91 Aliquoting and analysis Once the Sample “A” is thawed, two (2) Aliquots shall be taken for initial quantification.
These Aliquots may be stored at approximately 4 °C for a maximum of 24h before analysis.
The remaining “A” serum fraction may be kept in the Sample collection tube or aliquoted into new vial(s) with label(s) ensuring Laboratory Internal Chain of Custody is maintained .
For the confirmatory quantification, two (2) new Aliquots of the “A” Sample shall be analyzed immediately after aliquoting.
5.0 Analytical Testing Procedure Requirements 5.1 Analytical Testing Procedure Validation Requirements Prior to the implementation of the Analytical Testing Procedures for the quantification of IGF-I and P -III-NP in routine Doping Control analysis, the Laboratory shall f ulfil the following requisites: − Validate the Analytical Testing Procedures , including the determination of the assays’ Limit of Quantification (LOQ ), Repeatability (sr), Intermediate Precision (sw), Bias and Measurement Uncertainty (uc); − The Analytical Testing Procedures shall meet the acceptance values for the parameters of IGF- I and P -III-NP assay performance, as specified in Table 1 and Table 2 (as applicable).
Table 1 : Acceptance Criteria for Parameters of Assay Performance for the Endocrine Module Validation Parameters IGF-I P-III-NP Maximum LOQ ≤ 50 ng/mL ≤ 1 ng/mL Maximum Relative Combined Standard Measurement Uncertainty (uc_Max, %) ≤ 20% ≤ 15% 5.2 Analytical Testing Procedure Accreditation Requirements − Demonstrate readiness for assay implementation through method validation data and successful participation in at least one WADA -approved educational External Quality Assessment Scheme (EQAS ) round or inter -Laboratory collaborative study.
In cases of identified deficiencies, proper corrective action(s) shall be documented and implemented; − Obtain ISO/IEC 17025 accreditation for the Analytical Testing Procedures for the quantification of hGH Markers in blood as part of t he Endocrine Module from an Accreditation Body that is a full member of the International Laboratory Accreditation Cooperation (ILAC) and a signatory to the ILAC Mutual Recognition Agreement (ILAC MRA) .
ABP Operating Guidelines – Version 9.0 – July 2023 Page 35/91 5.3 Quality Controls (QCs) and Reagents − QC samples: Laboratories shall implement well -characterized and stable internal QC sample(s), which are not subject to assay lot variations, for the performance of the tests under different assay conditions (different assay lots, different analysts, etc .
).
Following preparation/reception by the Laboratory , all QC material should be aliquoted and stored frozen (preferably at – 80°C for long- term storage) until use.
These QC samples should include: o QC low: Serum obtained from healthy individual(s), which is demonstrated to contain concentrations of IGF -I not greater than (≤) 200 ng/mL and P -III-NP not greater than (≤) 5 ng/mL; o QC high: Serum obtained from hGH administration studies or another appropriate source that has been demonstrated to contain concentrations of IGF- I greater than (≥) 500 ng/mL and P -III-NP greater than (≥) 10 ng/mL.
[Comment: Four (4) separate QC samples may also be used, as long as they contain IGF -I and P -III-NP at the necessary concentrations (e.g., QC IGF-I_low, QC IGF-I_high, QC PIIINP_low and QC PIIINP_high).]
− Reagents: With every new batch of reagents (new lot number), the following evaluation steps should be implemented before including the new batch into routine operations for P -III-NP quantification: o Each of the QC samples shall be determined at least three (3) times whenever a new batch of reagents is obtained.
The number of replicates per determination shall be conducted as stipulat ed by the assay manufacturers.
The QCs may be measured in a single assay or over a range of assays.
If, for any QC, the difference between the mean concentration for the new batch and that for the preceding batch is more than 20%, the new batch shall not be implemented into routine operations and an investigation of the new batch shall be conducted.
o In order to detect small but systematic changes over time, it is recommended that the performance of a new batch of reagents is controlled, for example, through a cumulative sum (CUSUM) chart/table, which is established for each QC based on the difference between the mean(s) of the new batch and the initial value(s).
When using the CUSUM, results should be assessed using customary procedures as detailed at http://itl.nist.gov/div898/handbook/pmc/section3/pmc323.htm 6.0 Analytical Testing Procedure and Reporting of Test Results 6.1 Initial Quantification of the Markers − Two (2) Aliquots taken from the original “A” Sample shall be analyzed once (x1) to quantify intact IGF -I and P -III-NP; ABP Operating Guidelines – Version 9.0 – July 2023 Page 36/91 − QC Sample(s), at low- and high- levels of the Marker s (see Article 5.3), shall be included in each i nitial quantification analytical batch; − The coefficient of variation (CV%) between the duplicate determinations of the IGF -I and P -III-NP concentrations shall not be higher (≤) than the associated uc_Max (see Table 1) .
If the CV% between duplicate determinations of only one Marker (IGF-I or P-III-NP) exceeds the respective u c_Max, the analysis of only that Marker shall be repeated; − The mean Marker concentration from the duplicate measurement of IGF- I and P -III-NP shall be reported in ADAMS in nanograms per milliliter (ng/mL); [Comment: for the purposes of the Endocrine Module of the ABP, the GH -2000 score does not need to be calculated or reported by the Laboratory since it will be automatically calculated in ADAMS 15].
− If the measured Marker concentration is below the LOQ of the assay, the Laboratory shall report a value of “ -1” for its concentration in ADAMS and the Laboratory shall make a comment in the Test Report on why the Marker could not be quantified (e.g., the measurement of the Marker is not possible due to unusual matrix interferences); − An observation of hemolysis of the Sample should be recorded in the comments section of the Laboratory Test Report in ADAMS.
6.2 Confirmator y Quantification of the Markers If requested by the Testing Authority (TA), Results Management Authority (RMA ) or WADA , the Laboratory shall proceed with the confirmatory quantification of the Markers of the Endocrine Module.
[Comment: An APMU or Passport Custodian (PC), where the PC is not the TA, may request a confirmatory quantification on behalf of the TA or RMA .
In such cases, the APMU or PC shall copy the relevant TA or RMA , as applicable, on all written requests to the Laboratory for confirmatory quantifications.]
When a confirmatory quantification analysis is requested: − Two (2) new Aliquots taken from the original “A” Sample shall be analyzed once (x1) to: o quantify intact IGF -I and P -III-NP; and o identify IGF -I (as per the TD IDCR 14); − At least one QC Sample (see Article 5.3), depending on initial quantification results, shall be included in each confirmatory quantification analytical batch; − The CV (%) between the duplicat e determinations of the IGF- I or P -III-NP concentrations shall not be higher (≤) than the associated uc_Max (see Table 1) .
If the CV% between duplicate determinations of only one Marker (IGF-I or P -III-NP) exceeds the respective uc_Max, the analysis of only that Marker shall be repeated; ABP Operating Guidelines – Version 9.0 – July 2023 Page 37/91 − The mean Marker concentration from the duplicate measurement of IGF- I and P -III-NP shall be reported in ADAMS in nanograms per milliliters (ng/mL); − If the measured Marker concentration is below the LOQ of the assay, the Laboratory shall report a value of “ -1” for its concentration in ADAMS and the Laboratory shall make a comment in the Test Report on why the Marker could not be quantified (e.g., the measurement of the Marker is not possible due to unusual matrix interferences); − An observation of hemolysis of the Sample should be recorded in the comments section of the Laboratory Test Report in ADAMS.
ABP Operating Guidelines – Version 9.0 – July 2023 Page 38/91 Table 2.
Analytical Testing Procedure Validation and Performance Requirements for the initial and confirmatory quantification of IGF- I in blood (serum) Samples by top -down LC -MSn for the Endocrine Module of the ABP.
Method and Instrumentation Top-down (intact IGF -I) Liquid Chromatography comb ined with (Tandem) Mass Spectrometry based on triple quadrupole or HRMS (LC -MSn; n ≥ 1).
Range of the Method Shall cover the ranges of IGF -I concentrations normally found in males and females and demonstrate linearity between 50 –1000 ng/mL , at least.
Limit of Quantification (LOQ ) The LOQ shall not be greater than (≤) 50 ng/mL .
Maximum Relative Combined Standard Measurement Uncertainty uc (%) The estimated uc (%) shall not be greater than (≤) 20%.
Sample IGF-I quantification shall be conducted in duplicate (using two Aliquots of the “A” Sample) using a volume not greater than (≤) 50 μL of serum per replicate.
Internal Standard Stable isotope- labeled IGF -I (e.g., NISTii or ProSpeciii 15N-IGF-I).
Calibration A freshly prepared single point calibrator (SPC) shall be included in each analytical batch.
The Recombinant Human IGF -I calibrator from NIST (SRM 2926iv) should be used to prepare the SPC.
Any other calibration material shall be validated against the NIST SRM 2926 calibrator.
Applicable links ii https://shop.nist.gov/ccrz__ProductDetails?sku=2927&cclcl=en_US iii https://www.prospecbio.com/igf1_n15_human iv https://shop.nist.gov/ccrz__ProductDetails?sku=2926&cclcl=en_US ABP Operating Guidelines – Version 9.0 – July 2023 Page 39/91 7.0 Bibliography 1.
Guha N, Erotokritou- Mulligan I, Bartlett C, et al.