WADA - WADA Technical Document – TD2026DBS

WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 1/9 TD2026DBS – Version 1.0 – 01 January 2026

Dried Blood Spots (DBS) for Doping Control Requirements and Procedures for Analytical Testing and Sample Storage 1.0 Introduction and Scope This Technical Document (TD) has been established to harmonize Dried Blood Spot s (DBS) Testing by providing specific requirements and procedures for DBS Sample Analytical Testing and storage. The term “DBS” refers to a capillary blood sample that is collected by puncture/incision of the skin and then deposited directly onto an absorbent sample support and allowed to dry. DBS Samples may be collected by “spotting” capillary blood directly onto an absorbent sample support, either untreated cellulose or a synthetic polymer, or using a specific device with an integrated microneedle(s)/microlancet(s). This version of the TD DBS specifically covers the requirements for Analytical Testing Procedures which are applied to DBS Samples for the detection of Non-Threshold Substances without a Minimum Reporting Level (MRL) only [1]. 2.0 Analytical Testing of DBS Samples Any aspects of the Analytical Testing of DBS Samples shall be done in accordance with Article 5.0 of the International Standard for Laboratories (ISL) [2] and its related relevant TDs, Technical Letters (TLs) and Laboratory Guidelines (LGs), unless otherwise specified in this TD. 2.1 DBS Collection Devices Requirements The Laboratory shall receive DBS Samples collected by an Anti-Doping Organization (ADO) or Delegated Third Party (DTP) for which the DBS Collection Device meets the following criteria:

Laboratory Guidelines (LGs), unless otherwise specified in this TD. 2.1 DBS Collection Devices Requirements The Laboratory shall receive DBS Samples collected by an Anti-Doping Organization (ADO) or Delegated Third Party (DTP) for which the DBS Collection Device meets the following criteria: a) Has a sample support made of either untreated cellulose or synthetic polymer. b) Collects a minimum (≥) of 15 µL capillary blood in each spot/pebble, with a minimum total of 3 spots/pebbles for the “A” Sample and 1 spot/pebble for the “B” Sample, and [Comment to Article 2.1 b): The use of DBS Collection Devices, which collect low capillary blood volume per spot/pebble, may limit the application of different Analytical Testing Procedures . This applies, in particular , for Analytical Testing Procedures that differ in their Sample preparation/extraction protocols or require the whole spot/pebble to achieve the necessary detection sensitivity. With a minimum three (3) “A” spots/pebbles, the Testing Authority (TA) may choose to request the application of one (1) or two (2) different Initial Testing Procedures (ITPs), while considering that if both procedures produce Presumptive Adverse Analytical Finding(s) (PAAF), only one (1) may be confirmed using the remaining “A” spot/pebble and, if needed, the oneWADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 2/9 TD2026DBS – Version 1.0 – 01 January 2026

available “B” spot/pebble. In those cases, the selection of the PAAF(s) to be confirmed shall be made in consultation with the TA.

Page 2/9 TD2026DBS – Version 1.0 – 01 January 2026

available “B” spot/pebble. In those cases, the selection of the PAAF(s) to be confirmed shall be made in consultation with the TA. Notwithstanding the above, a single spot/pebble may be used for different Analytical Testing Procedure(s) if i) they share the same Sample preparation/extraction protocols, and ii) the Analytical Testing Procedure has been validated to meet Test Method performance criteria in a volume less than a whole spot/pebble. Conversely, if available, the Laboratory may combine two (2) or more spots/pebbles from the same DBS Sample, or from different DBS Samples 1 linked to a single Sample Collection Session (SCS) from the Athlete, to have sufficient volume to perform the required Analytical Testing Procedure(s). Whichever the DBS Collection Device used, the Laboratory’s Analytical Testing Procedure shall meet the expected Test Method performance requirements [i.e., Limit of Detection (LOD) of ITPs or Limit of Identification (LOI) of Confirmation Procedures (CPs), where applicable] by using a single spot/pebble, as determined by the availability of only one (1) “B” spot/pebble.] c) Collects “A” and “B” spots/pebbles that can be sealed separately. [Comment to Article 2.1 c). While the ADOs may select for use any of the DBS Collection Devices that meet the criteria listed in Articles 2.1.a)-c), it is recommended that the Laboratory consults with their customers regarding their plans and capacity for selection and use of DBS Collection Devices before proceeding to the development and validation of the Analytical Testing Procedure(s).] 2.2 Acceptance of DBS Samples for Analysis The Laboratory shall accept for analysis DBS Samples collected by an ADO or DTP, unless the Sample meets any of the following conditions. a) The Laboratory does not have a n Analytical Testing Procedure validated for the DBS Collection

2.2 Acceptance of DBS Samples for Analysis The Laboratory shall accept for analysis DBS Samples collected by an ADO or DTP, unless the Sample meets any of the following conditions. a) The Laboratory does not have a n Analytical Testing Procedure validated for the DBS Collection Device received. b) In cases where the Laboratory receives two (2) or more DBS Samples, which are linked to a single SCS from the Athlete according to the Doping Control Form (DCF), and if a single analysis has been requested, the Laboratory shall analyze only one (1) of the DBS Samples collected, unless otherwise instructed by the TA and/or required for the performance of the Analytical Testing Procedure. The second DBS Sample shall be stored frozen to allow future analysis on request (see Article 6.1.c). [Comment to Article 2.2 b): However, if multiple Testing menus have been requested, the Laboratory shall use as many of the collected DBS Samples as needed to perform the applicable Analytical Testing Procedures.] c) In cases where the Laboratory receives DBS Samples with irregularities (see Article 2.2.1).

1 “A” and “B” spots/pebbles from different DBS Samples shall not be combined.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 3/9 TD2026DBS – Version 1.0 – 01 January 2026

2.2.1 DBS Samples with Irregularities The Laboratory shall observe and document conditions that exist at the time of DBS Sample reception or registration 2 that may adversely impact on the integrity of a Sample or the performance of Analytical Testing Procedure(s). For DBS Samples specifically, examples of

2.2.1 DBS Samples with Irregularities The Laboratory shall observe and document conditions that exist at the time of DBS Sample reception or registration 2 that may adversely impact on the integrity of a Sample or the performance of Analytical Testing Procedure(s). For DBS Samples specifically, examples of irregularities to be noted include, but are not limited to: a) Insufficient number of spots/pebbles to perform the required Analytical Testing Procedure(s). b) Non-filled spots/pebbles. c) Low spot volume. d) Double spots, i.e., the Sample does not form a single clear spot but instead appears as two (2) overlapping or separate spots. e) Absence of desiccant in the Sample container. f) Sample not dry. g) Sample adhered to the container. h) Spots not visible for inspection. [Comment to Article 2. 2.1: Unlike for other blood Samples (e.g., ABP blood Samples), the freezing of DBS Samples shall not be considered an irregularity, because it would not impact the later performance of the Analytical Testing Procedures.] In these cases, the Laboratory shall immediately inform the TA on the possible impact of the noted irregularity(-ies) on the requested analysis and shall include the noted irregularity( -ies) in the Sample record. The Laboratory may analyze Samples with irregularities if the irregularity does not impact the Sample’s chain of custody/unique identification or the suitability of the Sample to be analyzed with the requested Testing menu. For those Sample irregularities that affect the Sample’s chain of custody/unique identification or its analytical suitability , the Laboratory shall seek instructions from the TA, in writing, on the performance of Analytical Testing on the Sample.

2 For some DBS Collection Devices, irregularities might be detected only after the opening of the Sample.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by:

2 For some DBS Collection Devices, irregularities might be detected only after the opening of the Sample.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 4/9 TD2026DBS – Version 1.0 – 01 January 2026

3.0 Initial Storage of DBS Samples and Aliquoting for Analysis The initial storage of DBS Samples shall adopt the following requirements to ensure the stability and integrity of the Sample and minimize storage time at room temperatures. a) The Laboratory shall maintain the DBS “A” Sample refrigerated or frozen and protected from light and moisture until analysis. Before aliquoting, the Sample shall be allowed to approach room temperature in an airtight and dry container (e.g., desiccator, plastic box containing desiccant) to avoid condensation. b) The Laboratory shall store the DBS “B” Sample frozen with desiccant after reception and until analysis (if applicable) or until disposal. c) However, if the DBS “A” and “B” Samples are in the same container, the “B” Sample can remain refrigerated and protected from light and moisture until the ITP(s) and the “A” CP, if applicable, have been completed. [Comment 1 to Article 3.0: In all circumstances, the Laboratory shall take appropriate steps to ensure the integrity of the Sample(s).] DBS Sample aliquoting 3 shall follow the general requirements described in the ISL [2], with the following specifications for DBS Samples. a) The Laboratory shall aliquot, i.e., take spots/pebbles, from the DBS Sample container by using clean tools (e.g., hole puncher, tweezers) to avoid contamination.

specifications for DBS Samples. a) The Laboratory shall aliquot, i.e., take spots/pebbles, from the DBS Sample container by using clean tools (e.g., hole puncher, tweezers) to avoid contamination. b) After spots/pebbles have been taken for extraction and analysis, and any ITP(s) and/or CP(s) have been completed, any spots/pebbles remain ing in the “A” Sample shall be stored frozen , unless otherwise specified in a WADA TD, TL or LGs. [Comment 2 to Article 3.0: In DBS automated analysis, spots/pebbles are not physically punched out from the DBS Collection Device. Therefore, the entire DBS Collection Device remains at room temperature until the ITP(s) and CP(s), if applicable, have been completed.]

3 For DBS Samples, Aliquots are intended as separate spots/pebbles removed from the Sample support.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 5/9 TD2026DBS – Version 1.0 – 01 January 2026

4.0 Selection and Validation of Analytical Testing Procedures The selection and validation of Analytical Testing Procedures for DBS analysis shall be in accordance with the ISL [2], as applicable to Non-Threshold Substances without an MRL[1], with the following specifications: a) The Laboratory should validate and implement the Analytical Testing Procedures included in the Minimum DBS Testing Menus (see Annex A of this TD). The Laboratory may implement additional Test Methods or target Analytes for the analysis of particular Non-Threshold Substances without MRL4.

a) The Laboratory should validate and implement the Analytical Testing Procedures included in the Minimum DBS Testing Menus (see Annex A of this TD). The Laboratory may implement additional Test Methods or target Analytes for the analysis of particular Non-Threshold Substances without MRL4. b) When available, DBS samples shall be used as Quality Control (QC) samples (for example, DBS samples obtained prior to and after a controlled drug administration may serve as Negative (NQC) and Positive (PQC) QC samples, respectively). However, if there are no available DBS samples, QC samples can be generated, for example, from whole venous or capillary blood containing EDTA as anticoagulant by spotting them and allowing them to dry on the same DBS Sample Collection Device/absorbent support that will be used for Sample analysis. c) For the preparation of PQC, the Laboratory should avoid spiking the Reference Materials (RMs) directly on the Sample absorbent support; it is preferable to spike/fortify whole venous or capillary blood before spotting it on the adsorbent support. d) All validation parameters (as per the ISL[2]) shall be evaluated with representative samples, collected from different individuals, using the same DBS Sample Collection Device/absorbent support and analytical protocol as the one that will be used for the Samples. Whenever possible, it is recommended to use capillary blood for the evaluation of Selectivity. e) Analytical Testing Procedures validated for a specific Sample matrix ( e.g., urine, plasma) shall be revalidated when used for DBS Samples. A Flexible Scope of ISO/IEC 17025 Accreditation (see ISL [2]) does not apply when changing to another Sample matrix (e.g., from plasma to DBS). f) For Analytical Testing Procedure validated in DBS, any of the following conditions requires at least a partial re-validation:

1. Any significant changes to the DBS Sample Collection Device and/or to the absorbent sample support

(e.g., change from untreated cellulose to synthetic polymer, or change between two synthetic

f) For Analytical Testing Procedure validated in DBS, any of the following conditions requires at least a partial re-validation:

1. Any significant changes to the DBS Sample Collection Device and/or to the absorbent sample support

(e.g., change from untreated cellulose to synthetic polymer, or change between two synthetic polymers) shall trigger the reassessment of Selectivity and LOD of ITPs and/or LOI of CPs.

4 Laboratories are not required to validate the Test Methods on multiple DBS Collection Devices/sample supports. The Laboratory should select the most appropriate DBS Collection Device(s)/sample support(s) taking into consideration geographical availability and Testing needs, in consultation with its customers. See also Comment to Article 2.1 c).WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 6/9 TD2026DBS – Version 1.0 – 01 January 2026

1. Any significant changes to the DBS Sample preparation workflow ( e.g., punching and automation specific steps) shall trigger the reevaluation of carryover. 5.0 Sample Analysis DBS Sample analysis of Non-Threshold Substances without an MRL shall be done in accordance with the relevant provisions of the ISL [2] and applicable TDs (e.g., TD IDCR [3], TD EPO [4]), TLs or LGs, with the following specifications for DBS Sample analysis. 5.1 "A" CP A new “A” Aliquot shall be used for the “A” CP. Therefore, the “A” CP Aliquot shall not be obtained

(punched or cut out) from the same spot/pebble as the one used for the ITP. 5.2 “B” CP

A new “A” Aliquot shall be used for the “A” CP. Therefore, the “A” CP Aliquot shall not be obtained (punched or cut out) from the same spot/pebble as the one used for the ITP. 5.2 “B” CP For the performance of the “B” CP, a new Aliquot(s) shall be obtained from the designated “B” spot(s)/pebble(s). 6.0 Storage of DBS Samples 6.1 Short-term Storage of DBS Samples a) All DBS Samples retained for storage in the Laboratory shall be stored frozen and protected from light with desiccant. b) DBS Samples shall be stored, at a minimum, for six (6) months after reporting all results (“A” and “B” Samples, if applicable) in ADAMS, unless the Sample has been identified for long-term storage (see Article 6.2) or for secondary use (in which case direct DBS Sample identifiers shall be removed - see ISL [2]). c) Not analyzed DBS Samples (including Samples with irregularities) shall also be stored, at a minimum, for six (6) months after Sample reception, or for the storage period requested by the TA (if longer). 6.2 Long-term Storage of DBS Samples At the direction of the TA or Results Management Authority (RMA, if different) or WADA, any DBS Sample may be placed in long-term storage for up to ten (10) years after the Sample collection date for the purpose of Further Analysis, subject to the conditions set out in the ISL [2]. The ADO requesting the long-term storage shall be responsible for any costs associated with an extended DBS Sample storage beyond the minimum six (6) months established for the short-term storage.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group

Approved by: WADA Executive Committee

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 7/9 TD2026DBS – Version 1.0 – 01 January 2026

7.0 References [1] TD MRPL: Technical Document Minimum Required Performance Levels and Applicable Minimum Reporting Levels for Non-Threshold Substances Analyzed by Chromatographic-Mass Spectrometric Analytical Methods. [2] ISL: The World Anti-Doping Code International Standard for Laboratories. [3] TD IDCR: Technical Document on Minimum Criteria for Chromatographic -Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes. [4] TD EPO Technical Document on Harmonization of Analysis and Reporting of Erythropoietin (EPO) and other EPOReceptor Agonists (ERAs) by Polyacrylamide Gel Electrophoretic (PAGE) Analytical Methods.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 8/9 TD2026DBS – Version 1.0 – 01 January 2026

Annex A – Minimum DBS Testing Menus Table A.1 Multi Class Menu 5 Prohibited Class

MRPL (ng/mL) S1.1 Anabolic Androgenic Steroids (AAS) 10 S1.2 Other Anabolic Agents

- SARMs

2

Table A.1 Multi Class Menu 5 Prohibited Class

MRPL (ng/mL) S1.1 Anabolic Androgenic Steroids (AAS) 10 S1.2 Other Anabolic Agents

- SARMs

2 S2.1.2 HIF Activating Agents 200 S3. Beta-2 Agonists 10 S4.1. Aromatase Inhibitors 10 S4.2. Anti-Estrogenic Substances 10 S4.4. Metabolic Modulators 10 S5. Diuretics And Masking Agents6 10

Table A.2 Steroid Esters Menu Prohibited Class (Specific Examples) MRPL (ng/mL) S1.1 Anabolic Androgenic Steroids (AAS) - Steroids Esters - Boldenone 2 - Nandrolone 2 - Testosterone 1

5 For each class of Prohibited Substances listed in Table A.1, this excludes Threshold Substances (e.g., salbutamol, formoterol) and NonThreshold Substances with MRL (e.g., higenamine, salmeterol, tetroquinol, clomifene, meldonium or those diuretics associated with contaminations – see TL24). 6 Brinzolamide and dorzolamide shall not be included as part of the Analytes in the DBS Multi Class Menu.WADA Technical Document – TD2026DBS

Document number: TD2026DBS Version number: 1.0

Written by:

Reviewed by: WADA Science / DBS Working Group

WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 9/9 TD2026DBS – Version 1.0 – 01 January 2026

Table A.3 Small Peptides Menu

Approved by: WADA Executive Committee Date: 11 September 2025 Effective date: 01 January 2026

Page 9/9 TD2026DBS – Version 1.0 – 01 January 2026

Table A.3 Small Peptides Menu Prohibited Class MRPL (ng/mL) S2.2.1 Testosterone-stimulating Peptides in Males 20 S2.2.3 Growth Hormone (GH) Fragments 20 S2.2.4 Growth Hormone Releasing Factors - GH Secretagogues (GHS)

• GH Releasing Peptides (GHRPs)

20

S2.3. Growth Factors and Growth Factor Modulators

• TB-500

20

S5. Diuretics and Masking Agents

• Desmopressin, Vasopressin

20

Table A.4 ERAs Menu Prohibited Class (Specific Examples) MRPL7 S2.1.1 Erythropoietin Receptor Agonists - rEPO 30 IU/L - dEPO 30 pg/mL - CERA 150 pg/mL - EPO-Fc 150 pg/mL

7 See also TD EPO [4]