The Athlete Biological Passport: a novel tool for the fight against doping in continuous evolution

Analytical Strategy 2019

ETH - Zürich

Dr. Raul Nicoli

12.11.2019

Swiss Laboratory for Doping Analyses (LAD)

• University Center of Legal Medicine – CHUV – UNIL

• WADA and ISO/IEC 17025 accredited laboratory

• ~25 staff members (chemists, pharmacists, biologists, forensic experts, technicians,administrative staff and students)

• Routine Analyses16’000 samples / year(80% urine – 20% blood)

• Research & Development

WADA-accredited anti-doping laboratories

AMERICA

Havana (CUB)

Los Angeles (USA)

Mexico City (MEX)

Montréal (CAN)

Rio de Janeiro (BRA)

Salt Lake City (USA)

EUROPE

Ankara (TUR)

Athens (GRE)

Barcelona (SPA)

Bucharest (ROM)

Cologne (GER)

Ghent (BEL)

Helsinki (FIN)

Kreischa (GER)

Lausanne (SUI)

Lisbon (POR)

London (UK)

Madrid (SPA)

Oslo (NOR)

Paris (FRA)

Rome (ITA)

Seibersdorf (AUT)

Stockholm (SWE)

Warsaw (POL)

ASIA

Bangkok (THA)

Beijing (CHI)

Doha (QAT)

New Delhi (IND)

Seoul (KOR)

Tokyo (JAP)

OCEANIA

Sydney (AUS)

AFRICA

Bloemfontein (ZAF)

Statistics of 2017

Statistics …. Top sports

2016 Anti-Doping Rule Violations (ARDVs)

Statistics… Top nationalities

2016 Anti-Doping Rule Violations (ARDVs)

The Prohibited List

Performance

enhancer

Use against

ethic in sport

Real or potential

health risk1 2 3

In- /out-of-competition

• S0 Non approved substances

• S1 Anabolic agents

• S2 Peptides, growth factors

• S3 β2-agonists

• S4 Hormones and modulators

• S5 Diuretics and other masking agents

In-competition

• S6 Stimulants

• S7 Narcotics

• S8 Cannabinoids

• S9 Glucocorticosteroids

• P2 β-blockers

Prohibited methods

• M1 Enhancement of oxygen transport

• M2 Chemical and physical manipulation

• M3 Gene doping

URINE BLOOD

• Reception

• A and B container

• Routine test menu

• In or Out-of-competition

• Gender → hCG, LH

• Sport → beta-blockers

• Additional tests

• ESAs

• Peptides (GHRFs)

• IRMS

EDTA

• Reception

• A and B tube, or only A

• Temperature logger for BSS

• Never frozen

• Test menu examples

• Sysmex analysis for ABP

• ESAs → if A and B tube

• HBT

SERUM

• Reception

• A and B tube

• Temperature logger for control

• Centrifugation and gradual freezing

• Test menu examples

• Growth hormone isoforms

• Growth hormone markers → age

• ESAs

Biological matrices and tests

https://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj7k8yR1-PVAhUGbBoKHcu8A8cQjRwIBw&url=https://sanguinarians.com/2016/01/13/additives-in-blood-analysis-and-human-consumption/&psig=AFQjCNEibz3qp3qruKH9et2QTU4cLyk4fA&ust=1503245260705387http://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjli7qk1-PVAhUFVxoKHc8fAdAQjRwIBw&url=http://www.tigermedical.com/Products/Vacutainer-SST-Tubes__BD367988-.aspx&psig=AFQjCNGdq6Xr4Iu5NjEEFCZMseG970Moog&ust=1503245296673764

• Direct methods

- Direct detection/identification/quantification of substances included in

the List

Analytical approaches

• Indirect methods

- Athlete Biological Passport:

- the haematological module (2009)

- the steroidal module (2014)

berlinger.com

Direct methods – screening analyses

OH

H

CH3

O O

OH

OHOH

HOOC

sigmaaldrich.com

• Mass Spectrometers

GC/MS (Agilent; n=4)

GC-MS/MS (Agilent; n=3)

LC-MS/MS (Waters Xevo; n=2)

LC-HRMS(/MS) (Thermo QExactive; n=2)

GC-C-IRMS (Thermo Delta Plus; n=2)

• Immunoanalyzers

ADVIA Centaur (P-III-NP)

IDS-iSYS (IGF-1)

Siemens Dimension EXL (hCG)

Immulite 2000 Xpi (LH)

Berthold-luminometer (GH)

• Hematological analyzers

Sysmex XN-1000; n=2

Direct methods - Instrumentation

http://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiJ3M-OruzQAhXEuxQKHWWpCw8QjRwIBw&url=http://www.news-medical.net/AutoLumat-LB-953-Multi-Tube-Luminometer-from-Berthold&psig=AFQjCNFbFmGhzdHH1JX383IqT7ktNzIs8w&ust=1481553252785100http://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiz_oW4ruzQAhVEPRQKHUGuDxEQjRwIBw&url=http://www.diamonddiagnostics.com/equipment/Imm/Siemens_Advia_Centaur.htm&bvm=bv.141320020,d.d24&psig=AFQjCNHR3Eu4k_ZUz52mQD0gETC1L7ZP_w&ust=1481553341928885

Confirmation analyses – identification criteria

• Direct methods

- Direct detection/identification/quantification of substances included in

the List

Analytical approaches

• Indirect methods

- Athlete Biological Passport:

- the haematological module (2009)

- the steroidal module (2014)

1 : Individual

2 : Longitudinal

3 : Based on an adaptive model

4 : Defines normal individual limits for biological parameters

5 : To identify/target athletes for specific analytical testing (e.g. ESA, HBT)

6 : To pursue possible antidoping rule violations in accordance with the World Anti-Doping Code Article 2.2

Early 2000s

2009

Nowadays

Principles of ABP

14

12

13

15

16

17

18

1 2 3 4 5

HGB

17.216.9

16.5 16.6 16.4

12.4

13.4 13.3 13.513.6

15.3

14.6

15.415.0

1. Reference values are set adapted to the athlete on first point

2. New upper and lower limits are calculated for second point

3. This process continues with the next points

4. The more points you have, the more accurate is the model (min. 4)

5. If a value goes out of the limits, the passport is flagged as atypical

17.3

Atypical !

Individual Limits

Athlete’s values

Principles of ABP

Hematological and steroidal modules of ABP

Artificial oxygen

enhancement (agents

affecting erythropoiesis)

INDIRECT

approaches

Administration of

endogenous steroids

https://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj7k8yR1-PVAhUGbBoKHcu8A8cQjRwIBw&url=https://sanguinarians.com/2016/01/13/additives-in-blood-analysis-and-human-consumption/&psig=AFQjCNEibz3qp3qruKH9et2QTU4cLyk4fA&ust=1503245260705387

Blood Doping

veripalvelu.fi

• Administration of erythropoiesis stimulating agents (ESAs)

• Biosimilars to the endogenous EPO

• Injectable formulations

• Often as microdose

• Blood transfusions

• Homologous; from one subject to another

• Autologous; own blood of one subject

• Agents activating endogenous EPO production

• Activators of hypoxia-inducible factor (HIF); molidustat, roxadustat FG-4592

• Cobalt

• Xenon

Consequences: difference in electric charges and weight

3rd generationC.E.R.A.

1st generationrEPO

hEPO2nd generation

NESP

Direct detection of rEPO : SAR/SDS-PAGE

4 blood markers

7 parameters

EPO/ESA abuse

Blood Transfusion

Biological passport for blood profiling

Artificial oxygen

enhancement (agents

affecting erythropoiesis)

• Instruments adapted from clinical chemistry to count and characterize blood cells for disease detection and monitoring

• Same parameters than targeted in hematological test (e.g. Hb, HCT, Ret)

• Highly automated processes use chemical reagents to dye and shape the cells

• The core element is a flow cell which exposes the cell populations with laser light

Sysmex-Europe.com

Hematology analyzers for blood profiling

Parameters to target in blood for ABP

Parameter Abbreviation

Haemoglobin HGB

Haematocrit HCT

Immature reticulocyte fraction IRF

Mean corpuscular haemoglobin MCH

Mean corpuscular haemoglobin concentration MCHC

Mean corpuscular volume MCV

Red cell distribution width RDW

Red blood cells count RBC

Reticulocytes count RET#

Reticulocytes percentage RET%

Stimulation OFF-hr-score

Combination of 7 markers ABPS score

Off-score =

Example : EPO/ESA abuse

Detection of homologous transfusion

Neil ROBINSONLaboratoire Suisse d’Analyse du Dopage

• Everybody has some specific surface protein on red blood cells =

biological signature

• Surface protein (antigen) can be target with fluroescent dye and

detected by flow cytometry

Autologous blood transfusion

WITHDRAWAL

STORAGE 4° C

RE-INFUSION

• Most challenging doping method to detect• Measurement of plasticizers metabolites in urine with LC-MS and Athlete Biological Passport (ABP) are the current strategies for the detection

• Micro-transfusions, undetectable with ABP is now the trend

Example : blood transfusion

• Direct methods

- Direct detection/identification/quantification of substances included in

the List

Analytical approaches

• Indirect methods

- Athlete Biological Passport:

- the haematological module (2009)

- the steroidal module (2014)

• Examples of administered compounds

• Testosterone

• Dihydrotestosterone (DHT)

• Dehydroepiandrosterone (DHEA)

• 4-androstendione, 5-androstendione, 4-androstendiol, 5-androstendiol

Doping use of endogenous steroids

• Direct methods

- Measurement of e.g. steroid esters in blood (exogenous)

- Measurement of carbon isotope ratio 13C/12C

Testosterone undecanoate• Indirect methods

- Measurement of endogenous steroids concentration in urine samples

- Determination of T/E and steroid profiling Athlete Biological Passport

28

Testosterone metabolism

T/E screening statistics

Urinary T/E-ratio

BPXXXXX Male 25 NADO Football

T/E A/T

5aAdiol/5bAdiolA/Etio5a-diol/E

5 urinary markers

6 parameters

Testosterone (T)

Epitestosterone (E)

Androsterone (A)

Etiocholanolone (Etio)

5α-androstane-3α,17β-diol (5aAdiol)

5β-androstane-3α,17β-diol (5bAdiol)

Biological passport for steroid profiling

Administration of

endogenous steroids

• The analysis is based on gas chromatographic (GC) separation and mass spectrometric (MS and/or MS/MS) detection of the target compouds in urine sample

• Sample preparation procedures highly harmonized within anti-doping laboratories (enzymatic hydrolysis, liquid liquid extraction and derivatisation)

Analytical method for steroid profiling

Real case : T administration

T/E A/T

A/Etio 5aAdiol/5bAdiol

DOPING!

Oral T intake

Transdermal T intake

T/EA/T[T], [A], [Etio], [Diols]

T/E5aAdiol/E[T][E]

Influence of Ethanol

T/E5aAdiol/EA/T

Confounding factors

Haematological Module Steroidal Module

GENETIC

METABOLIC

EX-VIVO

UGT2B17 enzyme deletion

Intake of:

‘’Conazols’’5a-reductase inhibitorsAnabolic steroidsAlcohol

Microbial contamination

[T](lower sensitivity)

A/Etio5aAdiol/5bAdiol

[5aAdiol]

5aAdiol/5bAdiol5aAdiol/E( )

Steroidogenesis inhibition

PHYSIOLOGICAL

PATHOLOGICAL

EXTERNAL

ExerciseAltitudeHaemodilution (change of plasmatic volume)Seasonal Fluctuations

RET%

[HGB]

InjuriesIron Supplement

UlceraGastrointestinal Diseases

[HGB]

( )Variations in [HGB]

HIGH EXPERTISEREQUIRED

Factors that are capable of influencing either the measurement of the ABP markers or their interpretation

• Improvements of Athlete Biological Passports

• Research for new biomarkers to more specific follow-up

• Studies on confounding factors and result interpretation

• Instrumental development

• Implementation of new technologies

• Improvement of current analytical processes

• Forensic approach

• Strategies of DNA-analysis

• Sample integrity

• Gene doping

• Identification of specific markers

Research activities of LAD

• Improvements of Athlete Biological Passport

• Research for new biomarkers to more specific follow-up

• Studies on confounding factors and result interpretation

• Instrumental development

• Implementation of new technologies

• Improvement of current analytical processes

• Forensic approach

• Strategies of DNA-analysis

• Sample integrity

• Gene doping

• Identification of specific markers