Le point sur les techniques de génotypage

plaquettaire & le bilan du registre suisse

HPA / HLA

Swisstransfusion – 6 septembre 2013

Françoise Boehlen

Médecin adjointe, CC

Service d’angiologie et d’hémostase

HUG

Presentation

Human platelet antigens and tests

Clinical aspects of platelet alloimmunisation

National HPA / HLA platelet registry

Platelet alloantigens

Two different categories of clinically relevant platelet

alloantigens:

1. « Common alloantigens » found on platelets and

other blood cells or tissues (e.g. antigens of the ABO-

system and HLA-class I antigens)

2. « Platelet-specific antigens » = HPA (Human Platelet

Antigen), thought to be present exclusively on platelets

GPIIb-IIIa

GPIV

GPIa-IIa

51

CD9

GPVI

GPIb-IX-V

61

Platelet membrane

Fibrinogen binding site

RGD binding siteGPIIIa GPIIb

COOHCOOH

HPA-3

HPA-4

HPA-1

CaCa++++

CaCa++++

CaCa++++

CaCa++++

s

s

GPIIb-IIIa

HPA-1aaHPA-1abHPA-1bb

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

• 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

1a

1b

2a

2b

3a

3b

5a

5b

+

-

+

-

+

-

+

+

Platelet genotype

1aa 2aa 3aa 5ab

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

Presentation

Human platelet antigens and tests

Clinical aspects of platelet alloimmunisation

National HPA / HLA platelet registry

Alloimmune thrombocytopenia

Platelet alloantibodies are responsible for:

• NAIT Neonatal alloimmune thrombocytopenia

• PTP Posttransfusion purpura

• PAIT Passive alloimmune thrombocytopenia

• TAIT Transplantation-associated alloimmune

thrombocytopenia

Platelet alloantibodies may contribute to:

• PTR Platelet transfusion refractoriness

Alloantibodies

NAIT Antibody Frequency

anti-HPA-1a 75%

anti-HPA-5b 20%

other 5%

PTP Antibody Frequency

anti-HPA-1a 85%

PTR Antibody Frequency

anti-HPA-5b 50%?

anti-HPA-1b ?%

anti-HPA-5a ?%

Presentation

Human platelet antigens and tests

Clinical aspects of platelet alloimmunisation

National HPA / HLA platelet registry

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

Selection of regular apheresis and blood donors

already HLA-typed and < 50 years old

Informed consent sheet questionnaire (donors data)

Blood sample

Project – Part 1

Platelet Apheresis Centres

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

Platelet Apheresis Centres

Documentation of HPA-matched transfusions

Update of the list of donors (contraindication)

Inclusion of new donors

www.hpa-hla.org

Only for participating centres

Website

Registry

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

Example – Registry

Registry – Statistics

Beginning of the project in March 2003

3113 samples received at the end of August 2013

2116

249

71731

Centres (2013)

Centre % of HPA-genotyped donors

Basel Beider 36.1%

Geneve - CTS 24.1%

Lausanne SRTS VD 16.5%

Svizzera Italiana CRS 6.3%

St Gallen SRK 4.4%

Aarau RBSZ 3.3%

Bern - Inselspital 2.9%

Neuchâtel et Jura CRS 2.2%

Luzern Central Schweiz 1.8%

Valais CRS 1.2%

Fribourg CTS 1.2%

Registry – Annual statistics

Last 12 months (from 01.08.2012 to 31.07.2013)

Total number of n

Visit of the registry 146

Search for a donor 248

No documentation of HPA-matched transfusions How many platelet donors searched/found? How many products prepared/transfused? For which reason?

Rare platelet genotypes

aa ab bb

HPA-1 1526 537 53

HPA-5 1715 383 18

2116 donors available at the end of August 2013

2.5%

0.8%

Registry – Characteristics

HPA-genotype Anti-HPA antibodies Frequency

HPA-1bb anti-HPA-1a 0/19 (0%)

HPA-5aa anti-HPA-5b 9/454 (2.0%)

HPA-5bb anti-HPA-5a 1/6 (16.7%)

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

Prof. Philippe de MoerlooseHead of Haemostasis Unit

Dre Cécile Kaplan & coworkersINTS, Paris

Oana Bulla & Yen LaiTechnician

Claude-Alain MouthonWebsite designer

Drs Rudolf Schwabe & Guy Levy

Drs Grazia Nicoloso de Faveri & Behrouz MansouriBlood Transfusion Service SRC

Transfusion centres & donors