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
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
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