Using DNA-Typing and Erythrocyte Microparticle Analysis to Detect Blood Doping

Overview

A total of 12 subjects will be recruited for participation in this study. 6 subjects will receive re-infusion of autologous blood, and 6 subjects (anemic patients) will receive a homologous transfusion.

Full Title of Study: “Using DNA-Typing and Erythrocyte Microparticle Analysis to Detect Homologous/Autologous Blood Doping- a Transfusion Study”

Study Type

  • Study Type: Interventional
  • Study Design
    • Allocation: Non-Randomized
    • Intervention Model: Parallel Assignment
    • Primary Purpose: Diagnostic
    • Masking: None (Open Label)
  • Study Primary Completion Date: December 2021

Detailed Description

Homologous blood transfusions (HBT) and autologous blood transfusions (ABT) are abused by athletes to illegally increase their hemoglobin mass and subsequently improve oxygen transport. Anti-Doping labs use flow-cytometry to detect HBT in cheating athletes, but athletes avoid being tested positive by matching their blood for minor blood groups before transfusion. Recent publications suggest that DNA typing by Capillary Electrophoresis or RT-PCR might be an alternative way to detect this kind of doping in athletes. Unfortunately, no data exist on the clearance of DNA after transfusion of one bag of blood using this methodology. For the detection of doping with ABT, there is no direct method available and only the biological passport, a longitudinal collection of hematological parameters can indicate doping. Recently RBC Microparticles (RBC-MPs) have been described as a potential biomarker for autologous transfusion. However, also for this methodology, no data on the clearance time of RBC-MPs are available. Thus, in this World Anti-Doping Agency (WADA) approved and sponsored project. The investigators plan to perform a clinical trial in which six healthy subjects receive an ABT and six healthy subjects or patients a HBT. Blood samples will be collected before and at several time-points after transfusion. For the detection of HBT the samples will be analyzed by the official method (cytometry), and the two genotyping methods (STR and RT-PCR) to compare these different techniques and to see if DNA-typing can replace cytometry. For the ABT the collected samples will be analyzed for RBC-MPs on a cytometer dedicated for Microparticles.

Interventions

  • Biological: Homologous Blood Transfusion
    • Homologous, or allogenic, blood transfusions involves someone collecting and infusing the blood of a compatible donor into him/herself.
  • Biological: Autologous Blood Transfusion
    • Autologous blood transfusion is the collection and re-infusion of the patient’s own blood or blood components.

Arms, Groups and Cohorts

  • Experimental: Healthy Subjects
    • Six healthy subjects will receive an ABT (Autologous Blood Transfusion)
  • Experimental: Anemic Patients
    • Six patients with anemia will receive a HBT (Homologous Blood Transfusion)

Clinical Trial Outcome Measures

Primary Measures

  • Donor DNA (# of loci with triplets or quadruplets):
    • Time Frame: 12 months
    • Clearance Kinetics of donor DNA which is transferred during the transfusion of one bag of homologous blood will be established.
  • Cellular Microparticles (10^3/uL):
    • Time Frame: 12 months
    • Clearance Kinetics of cellular microparticles which are introduced during an autologous blood transfusion and are originating from red blood cells during blood storage will be established.

Participating in This Clinical Trial

Inclusion Criteria

  • both genders, – age 20-50 years and – preferably physically active but no elite athletes subjected to Anti-Doping testing. Exclusion Criteria:

  • vulnerable subjects – not willing to participate – not signing the ICF – patients with end-organ failure

Gender Eligibility: All

Minimum Age: 20 Years

Maximum Age: 50 Years

Are Healthy Volunteers Accepted: Accepts Healthy Volunteers

Investigator Details

  • Lead Sponsor
    • Hamad Medical Corporation
  • Collaborator
    • World Anti-Doping Agency
  • Provider of Information About this Clinical Study
    • Principal Investigator: Dr. Mohamed A Yassin ,MD, Hematology Consultant – Hamad Medical Corporation
  • Overall Official(s)
    • Sven Voss, Study Chair, ADLQ
    • Mohamed Yassin, Principal Investigator, Hamad Medical Corporation
    • Francesco Donati, Principal Investigator, Laboratorio Antidoping FMSI, Rome, Italy
    • Costas Georgakopoulos, Principal Investigator, ADLQ
    • Mohammed Alsayrafi, Principal Investigator, ADLQ
    • Jean-Charles Grivel, Principal Investigator, Sidra Medicine
    • Christophe Raynaud, Principal Investigator, Sidra Medicine
  • Overall Contact(s)
    • Sven C Voss, 0097455481955, svoss@adlqatar.qa

References

Voss SC, Thevis M, Schinkothe T, Schanzer W. Detection of homologous blood transfusion. Int J Sports Med. 2007 Aug;28(8):633-7. doi: 10.1055/s-2007-965076. Epub 2007 Jul 5.

Giraud S, Robinson N, Mangin P, Saugy M. Scientific and forensic standards for homologous blood transfusion anti-doping analyses. Forensic Sci Int. 2008 Jul 18;179(1):23-33. doi: 10.1016/j.forsciint.2008.04.007. Epub 2008 Jun 2.

Krotov G, Nikitina M, Rodchenkov G. Possible cause of lack of positive samples on homologous blood transfusion. Drug Test Anal. 2014 Nov-Dec;6(11-12):1160-2. doi: 10.1002/dta.1736. Epub 2014 Oct 20.

Donati F, Stampella A, de la Torre X, Botre F. Investigation on the application of DNA forensic human identification techniques to detect homologous blood transfusions in doping control. Talanta. 2013 Jun 15;110:28-31. doi: 10.1016/j.talanta.2013.02.042. Epub 2013 Mar 18.

Stampella A, Di Marco S, Pirri D, de la Torre X, Botre F, Donati F. Application of DNA-based forensic analysis for the detection of homologous transfusion of whole blood and of red blood cell concentrates in doping control. Forensic Sci Int. 2016 Aug;265:204-10. doi: 10.1016/j.forsciint.2016.04.021. Epub 2016 Apr 30.

Manokhina I, Rupert JL. A DNA-based method for detecting homologous blood doping. Anal Bioanal Chem. 2013 Dec;405(30):9693-701. doi: 10.1007/s00216-013-7122-8. Epub 2013 Jul 11.

Alizadeh M, Bernard M, Danic B, Dauriac C, Birebent B, Lapart C, Lamy T, Le Prise PY, Beauplet A, Bories D, Semana G, Quelvennec E. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood. 2002 Jun 15;99(12):4618-25. doi: 10.1182/blood.v99.12.4618.

Ni W, Le Guiner C, Moullier P, Snyder RO. Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection. PLoS One. 2012;7(5):e36461. doi: 10.1371/journal.pone.0036461. Epub 2012 May 3.

Almizraq RJ, Seghatchian J, Acker JP. Extracellular vesicles in transfusion-related immunomodulation and the role of blood component manufacturing. Transfus Apher Sci. 2016 Dec;55(3):281-291. doi: 10.1016/j.transci.2016.10.018. Epub 2016 Oct 28.

Straat M, Boing AN, Tuip-De Boer A, Nieuwland R, Juffermans NP. Extracellular Vesicles from Red Blood Cell Products Induce a Strong Pro-Inflammatory Host Response, Dependent on Both Numbers and Storage Duration. Transfus Med Hemother. 2016 Jul;43(4):302-305. doi: 10.1159/000442681. Epub 2015 Dec 16.

van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, Sturk A, van Leeuwen TG, Nieuwland R. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost. 2014 Jul;12(7):1182-92. doi: 10.1111/jth.12602. Epub 2014 Jun 19.

Nielsen MH, Beck-Nielsen H, Andersen MN, Handberg A. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma. J Extracell Vesicles. 2014 Feb 4;3. doi: 10.3402/jev.v3.20795. eCollection 2014.

Rubin O, Crettaz D, Tissot JD, Lion N. Pre-analytical and methodological challenges in red blood cell microparticle proteomics. Talanta. 2010 Jun 30;82(1):1-8. doi: 10.1016/j.talanta.2010.04.025. Epub 2010 Apr 22.

Rank A, Nieuwland R, Crispin A, Grutzner S, Iberer M, Toth B, Pihusch R. Clearance of platelet microparticles in vivo. Platelets. 2011;22(2):111-6. doi: 10.3109/09537104.2010.520373. Epub 2011 Jan 13.

Voss SC, Jaganjac M, Al-Thani AM, Grivel JC, Raynaud CM, Al-Jaber H, Al-Menhali AS, Merenkov ZA, Alsayrafi M, Latiff A, Georgakopoulos C. Analysis of RBC-microparticles in stored whole blood bags – a promising marker to detect blood doping in sports? Drug Test Anal. 2017 Nov;9(11-12):1794-1798. doi: 10.1002/dta.2212. Epub 2017 Jun 20.

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