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
 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 ABOsystem and HLA-class I antigens)
2. « Platelet-specific antigens » = HPA (Human Platelet
Antigen), thought to be present exclusively on platelets
Platelet membrane
GPIV
GPVI
CD9
GPIIb-IIIa
a6b1
a5b1
GPIa-IIa
GPIb-IX-V
GPIIb-IIIa
GPIIIa
GPIIb
Fibrinogen binding site
RGD binding site
Ca++
Ca++
Ca++
HPA-4
Ca++
HPA-1
s
s
HPA-1aa
HPA-1ab
HPA-1bb
COOH
COOH
HPA-3
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
-
Platelet genotype
3a
+
1aa 2aa 3aa 5ab
3b
-
5a
+
5b
+
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 crossmatch 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
PTP
PTR
Antibody
Frequency
anti-HPA-1a
75%
anti-HPA-5b
20%
other
5%
Antibody
Frequency
anti-HPA-1a
85%
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
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
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
Website
www.hpa-hla.org
Only for participating centres
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
717
31
2116
249
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
 2116 donors available at the end of August 2013
aa
ab
bb
HPA-1
1526
537
53
2.5%
HPA-5
1715
383
18
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 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