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Le point sur les techniques de génotypage plaquettaire & le bilan du registre suisse HPA / HLA. Françoise Boehlen Médecin adjointe, CC Service d ’ angiologie et d ’ hémostase HUG. Swisstransfusion – 6 septembre 2013. Presentation. Human platelet antigens and tests

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le point sur les techniques de g notypage plaquettaire le bilan du registre suisse hpa hla

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

presentation
Presentation
  • Human platelet antigens and tests
  • Clinical aspects of platelet alloimmunisation
  • National HPA / HLA platelet registry
platelet alloantigens
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
slide4
GPIV

GPVI

CD9

GPIIb-IIIa

a6b1

a5b1

GPIa-IIa

GPIb-IX-V

Platelet membrane

gpiib iiia
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

tests
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)
platelet typing
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.)
example
Example

1a

1b

2a

2b

3a

3b

5a

5b

+

-

+

-

+

-

+

+

Platelet genotype

1aa 2aa 3aa 5ab

antiplatelet antibodies detection
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
presentation1
Presentation
  • Human platelet antigens and tests
  • Clinical aspects of platelet alloimmunisation
  • National HPA / HLA platelet registry
alloimmune thrombocytopenia
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
presentation2
Presentation
  • Human platelet antigens and tests
  • Clinical aspects of platelet alloimmunisation
  • National HPA / HLA platelet registry
hpa registry of blood donors
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
project part 1
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

project part 2
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

project part 3
Project – Part 3

Platelet Apheresis Centres

Documentation of HPA-matched transfusions

Update of the list of donors (contraindication)

Inclusion of new donors

website
Website

www.hpa-hla.org

Only for participating centres

example mrs y 50 year old
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
registry statistics
Registry – Statistics
  • Beginning of the project in March2003

 3113 samples received at the end of August 2013

31

717

2116

249

registry annual statistics
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?
rare platelet genotypes
Rare platelet genotypes

 2116 donors available at the end of August 2013

2.5%

0.8%

summary
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?
thanks
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

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