Le point sur les techniques de génotypage plaquettaire & le bilan du registre suisse HPA / HLA

1. Le point sur les techniques de génotypage plaquettaire & le bilan du registre suisse HPA / HLA Françoise Boehlen Médecin adjointe, CCService d’angiologie et d’hémostase HUG Swisstransfusion – 6 septembre 2013

2. Presentation • Human platelet antigens and tests • Clinical aspects of platelet alloimmunisation • National HPA / HLA platelet registry

3. Platelet alloantigens Two different categories of clinically relevant platelet alloantigens: • « Common alloantigens » found on platelets and other blood cells or tissues (e.g. antigens of the ABO-system and HLA-class I antigens) • « Platelet-specific antigens » = HPA (Human Platelet Antigen), thought to be present exclusively on platelets

4. GPIV GPVI CD9 GPIIb-IIIa a6b1 a5b1 GPIa-IIa GPIb-IX-V Platelet membrane

5. GPIIb-IIIa Fibrinogenbinding site GPIIIa GPIIb RGDbinding site Ca++ Ca++ Ca++ HPA-4 Ca++ HPA-1 HPA-3 s s HPA-1aa HPA-1ab HPA-1bb COOH COOH

6. Tests • HPA typing • Antiplatelet antibodies detection • External Quality Controls (tests performed in HUG) • NIBSC (National Institute for Biological Standards and Control) • International Platelet Immunology Workshop (ISBT)

7. Platelet typing • Phenotyping methods (serologic typing) • Different methods (immunofluorescence, MAIPA…) • Relatively easy to perform but depend on the availability of appropriate human sera • Genotyping methods • Extraction of genomic DNA from blood leukocytes (or amniocytes) • Amplification by PCR (different techniques: RFLP, SSP, etc.)

8. Example 1a 1b 2a 2b 3a 3b 5a 5b + - + - + - + + Platelet genotype 1aa 2aa 3aa 5ab

9. Antiplatelet antibodies detection • MAIPA (monoclonal antibody-specific immobilisation of platelet antigens) • Gold standard reference technique in platelet immunology • Identification of specific platelet antibodies • GPIIb-IIIa, GPIb-IX, GPIa-IIa, etc. (HLA class I) • Characterisation of platelet alloantibodies by cross-match between • Mother’s serum and father’s platelets • Patient serum and panel of platelets of different genotypes

10. Presentation • Human platelet antigens and tests • Clinical aspects of platelet alloimmunisation • National HPA / HLA platelet registry

11. Alloimmune thrombocytopenia Platelet alloantibodies are responsible for: • NAITNeonatal alloimmune thrombocytopenia • PTPPosttransfusion purpura • PAITPassive alloimmune thrombocytopenia • TAITTransplantation-associated alloimmune thrombocytopenia Platelet alloantibodies may contribute to: • PTRPlatelet transfusion refractoriness

12. Alloantibodies

13. Presentation • Human platelet antigens and tests • Clinical aspects of platelet alloimmunisation • National HPA / HLA platelet registry

14. HPA registry of blood donors • Swiss registry of HLA- and HPA-genotyped platelet donors • Proposed by Swiss Transplant Working Group for Blood and Marrow Transplantation (STABMT) • Goal = 2’000 platelet donors HPA genotyped • Financed by • Swiss Red Cross Humanitarian Foundation • Blood Transfusion Service, Swiss Red Cross

15. Project – Part 1 Platelet Apheresis Centres Selection of regular apheresis and blood donors already HLA-typed and < 50 years old Informed consent sheet  questionnaire (donors data) Blood sample

16. Project – Part 2 Laboratory, Haemostasis Unit, Geneva HPA-genotyping Anti-HPA ab screening in homozygous women with ≥ one pregnancy or 2nd/3rd trimester miscarriage Introduction of results in the registry (website) after quality control

17. Project – Part 3 Platelet Apheresis Centres Documentation of HPA-matched transfusions Update of the list of donors (contraindication) Inclusion of new donors

18. Website www.hpa-hla.org Only for participating centres

19. Registry

20. Example – Mrs Y, 50 year-old • Acute myeloid leukaemia • Platelet transfusion refractoriness with bleeding • Anti-HLA class I & II, and anti-HPA-5a alloantibodies • Patient platelet genotyping: HPA-5bb • Persisting refractoriness including after HLA-matched HPA-5-mismatched products • Myeloablative allogenic HSCT from her HLA-identical brother • Brother platelet genotyping: HPA-5ab • Selection HLA- and HPA-5 compatible platelet donors

21. Example – Registry

22. Registry – Statistics • Beginning of the project in March2003  3113 samples received at the end of August 2013 31 717 2116 249

23. Centres (2013)

24. Registry – Annual statistics Last 12 months (from 01.08.2012 to 31.07.2013) • No documentation of HPA-matched transfusions • How many platelet donors searched/found? • How many products prepared/transfused? • For which reason?

25. Rare platelet genotypes  2116 donors available at the end of August 2013 2.5% 0.8%

26. Registry – Characteristics

27. Summary • Aim of this registry • HPA-compatible platelet in case of • Neonatal alloimmune thrombocytopenia • Platelet transfusion refractoriness • (Posttransfusion purpura) • Many questions to be resolved • Real necessity? • Necessity of a huge registry to propose HLA- and HPA-compatible platelets? • What about detection of alloantibodies in blood donors?

28. Thanks Prof. Philippe de Moerloose Head of Haemostasis Unit Dre Cécile Kaplan & coworkers INTS, Paris Oana Bulla & Yen Lai Technician Claude-Alain Mouthon Website designer Drs Rudolf Schwabe & Guy Levy Drs Grazia Nicoloso de Faveri & Behrouz Mansouri Blood Transfusion Service SRC Transfusion centres & donors