Vox Sanguinis (2010) 99 (Suppl.
2), 19–23
Alloimmune
thrombocytopenic purpura
T2-PO05
Alloimmune thrombocytopenia due to anti HPA-1b
Malcata C1, Rangel G2, Alegria A3, Duran JA2, Pinto F3,
Martin MG4, Muñiz-Diaz E4
1
CRSL/IPS, IP, Lisbon, Portugal 2CRSP/IPS, IP, Porto, Portugal
3
MAC, Lisbon, Portugal 4Banc de Sang e Teixits, Barcelona, Spain
Background: Neonatal Alloimmune Thrombocytopenia (NAIT) is
caused by the presence of maternal antibodies against antigens
present in fetal platelets that were inherited from the father,
causing immune destruction of fetal platelets. The platelet anti-
gen most frequently involved in NAIT is HPA1a, followed by
HPA5b, although rarely other antigens like HPA3a and HPA1b
may also be found. A case of NAIT caused by anti HPA1b is
presented.
Methods: Antiplatelet antibodies were studied by the ELISA
technique [PAKPLUS, PAKAUTO (GTI Diagnostics, Waukesha,
WI, USA)), Platelet antibody panel cells of Diamed (Cressier,
Switzerland) on the technique of solid phase (MASPAT (Sanquin,
Amsterdam, Netherlands)] and Monoclonal Antibody Technique
Immobilization of Platelet Antigen (MAIPA). Platelet crossmatch
was performed with solid phase technique (MASPAT).
Results: In the serum and plasma of the mother there were anti-
bodies anti HLA class I and against the glycoprotein IIb/IIIa, with
specificity HPA1b or HPA3b. The use of Platelet antibody panel
cells of Diamed suggested the presence of the antibody anti
HPA1b. The platelet crossmatch between platelets from the father
and serum and plasma from the mother was positive. Genotyping
showed: Mother- HPA 1a1a, 2a2a, 3a3a, 5a5a, 15a15b; Father-
HPA 1a1b, 2a2a, 15a15a, 5a5a, 3b3b; Child- HPA 1a1b, 2a2a,
3a3b, 5a5a, 15a15a, consistent with the existence of an antibody
anti HPA1b or anti HPA3b. The MAIPA technique finally con-
firmed an antiplatelet antibody with specificity for HPA1b.
Conclusion: This case illustrates the need to use multiple labo-
ratory techniques in order to identify the specific antibody
responsible for Neonatal Alloimmune Thrombocytopenia.
T2-PO06
Clinical outcomes of intrauterine platelet transfusions in
fetomaternal alloimmune thrombocytopenia (FMAIT) in
England
Lucas G1, Calvert A1, Green F1, Ranasinghe E2, Roberts D3, Sadani
D4, Green A1
1
NHSBT, Bristol, UK 2NHSBT, Cambridge, UK 3NHSBT, Oxford,
UK 4NHSBT, Manchester, UK
Background: Management of pregnancies at risk of FMAIT
varies according to the previous history and between centres.
There is a trend away from intrauterine platelet transfusions
(IUT) because of the associated risks and towards maternal IVIg
therapy but fetal blood sampling (FBS) continues to be used to
monitor responses to IVIg with IUTs being given to thrombo-
cytopenic fetuses.
Methods: Platelets for IUT were from accredited HPA-1a(-)5b(-)
donors, lacking red cell, HLA and HPA antibodies. Treatment
outcomes were followed by contacting the respective centres.
Results: FBS was performed for 42 pregnancies at risk of severe
FMAIT due to HPA-1a (36), 5b (3), 1a+5b (2), or 3a (1) anti-
bodies over a 2 year period. Weekly IVIg and IUTs were used to
treat 23 pregnancies and in a further three pregnancies steroids
were also used. Five pregnancies did not receive IUTs because
the fetal platelet counts were satisfactory and 11 pregnancies
were treated with IUTs but other treatments were incompletely
documented. A total of 137 IUTs were given (Range: 0–14 per
pregnancy; Mean: 3.7). The mean platelet increment was 429
(range 71–881) for 100 evaluable IUTs. Seven neonates required
further platelet transfusions after birth. There were three deaths
following IUT, two emergency deliveries for bradycardia after
IUT and one intracardiac transfusion following a hepatic vein
bleed. Intracranial haemorrhages were not detected in the
neonates.
Conclusion: IUTs continue to be used in cases of FMAIT refrac-
tory to IVIg therapy. The procedure had a combined morbidity/
mortality of 14.3% in this study.
T2-PO07
Developing in vitro and in vivo model systems to
investigate de novo human anti-HPA-1a antibody for-
mation in neonatal alloimmune thrombocytopenia (NAIT)
Eastlake J, Kumpel B
IBGRL/BITS, NHS Blood and Transplant, Bristol, UK
Background: Placental syncytiotrophoblast microparticles
(STMP), expressing fetal antigens, are shed into maternal blood.
NAIT is due to maternal antibodies to fetal platelet antigens of
paternal origin. Anti-HPA-1a, commonly implicated, targets a
polymorphism on CD61, which is present on STMP that may be
responsible for the primary immune response in NAIT.
Methods: In vitro and in vivo (Hu-PBL-SCID mice) immunisation
of naı̈ve HPA-1b1b HLA-DRB3*0101 leucocytes with HPA-1a+
STMP and/or platelets was performed. STMP were prepared from
fresh term placenta. In vitro: dendritic cells were immunised with
antigen then cultured with Thelper and B cells. Cytokine production
was analysed by Luminex to determine Th1/Th2 responses.
In vivo: leucocytes and antigen were injected i.p. into mice. Cul-
ture supernatants or mouse plasma were screened for human anti-
HPA-1a by MAIPA and/or PIFT and the total IgG concentration
determined by ELISA.
Proliferation assays were performed to assess the effects of antigen
and determine optimal concentrations for immunization.
Results: PBMC proliferation showed a dose response to antigen,
with optimal stimulation at 40-60 lg/mL protein. Cytokine pro-
files during in vitro cultures showed increased levels of IL-6 and
IL-8. IgG levels <730 lg/mL but not platelet specific antibodies
were detected. Weak anti-platelet responses and one anti-HPA-1a
were obtained in mice given STMP but not platelets. Human IgG
>10 lg/mL was present in >90% mice; indicating successful
reconstitution.
Conclusion: Optimizing conditions for de novo anti-HPA-1a
generation is continuing.
19
20 Alloimmune thrombocytopenic purpura
T2-PO08
A new low-frequency polymorphism on platelet
glycoprotein IIIa (sec) is associated with neonatal
alloimmune thrombocytopenia and with impaired GP
IIb/IIIa function
Sachs UJ1, Eva O1, Giptner A1, Bein G1, Peterson JA2, Santoso S1
1
Justus Liebig University, Giessen, Germany 2Blood Research
Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA
Background: The first child of a woman with a history of pre-
vious miscarriages was born on term with a platelet count of
25 · 109/L and petechial haemorrhages. Serological and genetic
work-up revealed a strong alloimmunization against GP IIb/IIIa of
unknown specificity. Additional studies were performed to iden-
tify the suspected new alloantigen (Sec).
Methods: ITGB3 was amplified by PCR and sequenced. ITGB3 and
ITGA2B (GPIIb) cDNA were cloned into mammalian expression
vectors. ITGB3 was modified by site-directed mutagenesis to
generate the Sec mutation. Chinese hamster ovary (CHO) cells
were transfected with ITGA2B and either wild-type or mutant
ITGB3 to express wild-type and mutant (Sec) GP IIb/IIIa, respec-
tively. Transfected CHO cells were analyzed for their immo-
reactivity and functional consequences of the mutation.
Results: Sequence analysis revealed a single nucleotide exchange
in exon 10 of ITGB3 (G331T), changing Lys580Asn in the EGF4
domain. CHO cells expressing Sec, but not wild-type GP IIb/IIIa,
showed binding of Anti-Sec. Adhesion of transfected CHO cells to
fibrinogen showed reduced binding of the Sec variant at low
fibrinogen concentrations. LIBS binding was reduced after addition
of RGDW, as was PAC-1 binding to DTT-activated mutant cells.
Conclusion: NAIT was caused by maternal alloimmunization
against a previously unreported, low-frequency polymorphism on
GPIIIa (G331T = Lys580Asn), termed Sec. This is the first low-
frequency polymorphism on GPIIIa which influences the recep-
tor’s function, most likely because of the mutation’s position
within the EGF4 domain which participates in fibrinogen binding.
T2-PO09
The study of anti-HPA-1a affinity in neonatal
alloimmune thrombocytopenia
Bessos H1, Urbaniak S2, Reid S3, Anani Sarab G2, Moss M2,
Brown P3
1
Scottish National Blood Transfusion Service, Edinburgh, UK
2
Division of Applied Medicine, University of Aberdeen, Aberdeen,
UK 3MRC Human Reproductive Sciences Unit, University of
Edinburgh, Edinburgh, UK
Background: The affect of anti-HPA-1a affinity (based on ka and
kd values) in neonatal alloimmune thrombocytopenia (NAIT) has
yet to be addressed. The aim of our study is to determine the
antibody affinity in up to 25 women with different NAIT out-
comes.
Methods: So far we have studied five women, using anti-HPA-1a
purified by immobilised recombinant-HPA-1a, and IgG fraction
by Melon Gel. The antibodies were assessed by ELISA, gel staining,
and surface plasmon resonance technique (SPRT-Biacore X) using
appropriate controls. The ka and kd of antibodies were determined
using the computer software BIAevaluation 3.2 RC1 – Langmuir
1:1 mathematical model.
 2010 The Author(s)
Journal compilation  2010 International Society of Blood Transfusion, Vox Sanguinis (2010) 99 (Suppl. 2), 19–23
Results: Unlike IgG fraction, purified anti-HPA-1a enabled
affinity determination which varied from 3.4 · 10E-9 down to
2.5 · 10E-7 and appeared to be related to NAIT severity. Moreover,
antibodies of similar affinity showed different association and
dissociation patterns. For example antibody from a mild NAIT case
(serum level 10 IU/mL) with an affinity of 3.2 · 10E-7 had ka and
kd values of 1.3 · 10E4 and 4.2 · 10E-3 respectively, while an
antibody from a normal case of similar affinity of 2.5 · 10E-7
(serum level 57 IU/mL) had ka and kd values of 5.1 · 10E3 and
1.3 · 10E-3 respectively, indicating that the latter antibody asso-
ciated less strongly with its target despite its higher serum level.
Conclusion: Our study shows that purified anti-HPA-1a enables
affinity determination as well as ka and kd analysis. We will
therefore continue using purified anti-HPA-1a in the study of its
affinity in NAIT, recruiting more patients with different disease
outcomes and reporting further updates.
T2-PO10
Allo-immune platelet transfusion refractoriness sur-
monted by allogeneic stem cell transplantation – a case
report
Bonstein L, Stemer G, Dann EJ, Zuckerman T, Rowe JM, Haddad N
Rambam Medical Center, Haifa, Israel
Background: Platelet transfusion refractoriness can reduce
overall survival. Here we describe a case of a 34 years old female
diagnosed with acute myeloid leukemia (AML), who underwent
autologous stem cell transplantation in first complete remission.
Neutropenic phase was complicated with uncontrolled infection
which was supported with four granulocyte transfusions. Later she
developed graft failer with severe platelet refractoriness.
Methods: Indirect platelet immunofluorescence test (PIFT), solid
phase ELISA and panel reactive antibodies (PRA) assays were used
to identify and characterize alloantibodies. PIFT was also em-
ployed for donor crossmatch.
Results: Anti-HLA class I antibodies were identified in the pa-
tient’s serum, with PRA of 99%. Only 2/62 platelet donors had
negative crossmatch. HLA typing of matched donors revealed full
match only at the A locus (partial match at B and mismatch at C).
After two severe gastro-intestinal bleeding episodes and no re-
sponse to immunosuppressive therapy, an allogeneic BMT from a
matched sibling donor was performed. Conditioning regimen was
mainly immunosuppressive and included plasma exchange.
Engraftment of platelets was documented at day +25 and re-
mained stable with full donor chimerism. Anti-HLA antibody titer
remained persistently high.
Conclusion: Neutrophil transfusions were most likely to aggra-
vate alloimmunization to HLA class I antigens, although the
transfusion was given in a very immunocompromised situation.
High anti-HLA antibody titer revealed after the use of immuno-
suppressive conditioning proves HLA alloimmunization to be a
very strong and lasting reaction.
T2-PO11
Neonatal alloimmune thrombocytopenia (NAIT)
associated to HPA-9bw (Maxa) alloantibody
Lee K
EFS Ile De France, Creteil, France

Background: We report a case of severe recurrent neonatal
thrombocytopenia. A young caucasian woman gave birth in 2007
and 2009 to two infants, birth weight (BW) 4.070 kg/7.109/L
platelets and BW 3.460 kg/17.109/L platelets, respectively. No
cerebral hemorrhage was noticed. At the moment, investigations
were negative. A retrospective analysis was decided.
Methods: Parents and infants HPA genotyping was performed by
PCR-SSP and/or microarray technology for the HPA -1, -2, -3, -4,
-5, -6, -7, -8, -9, -10, -11, -15 systems. Screening and identifi-
cation of platelet antibody in the maternal and infants sera against
a panel of platelet donors, and cross-matches with paternal
platelets were performed by the PSIIFT and the MAIPA test with
monoclonal antibody (MoAb) to IIb/IIIa (clones P2, Gi5, SZ21,
SZ22, Y2/51), Ia/IIa, and IbIX.
Results: HPA genotyping showed that only HPA-9bw (Maxa) was
mismatch in this family. The mother was found to be HPA-9a/a,
the father and the infants were HPA-9a/bw. The MAIPA showed
negative results with the MoAbs against membrane Gps IaIIa and
IbIX. A positive reaction was found by cross-matching sera from
the mother and the first newborn with paternal platelets by MA-
IPA with anti-IIb/IIIa MoAbs P2, SZ21. A weak positivity was
observed with anti-IIb/IIIa MoAb SZ22.
Conclusion: There are strong arguments for thinking that this
recurrent neonatal thrombocytopenia is due to the HPA-9b
incompatibility. This finding should prompt us to retrospectively
investigate other cases of unexplained neonatal thrombocytopenia
previously referred to our lab.
T2-PO12
Platelet antibodies and fetal growth: maternal HPA-1a
alloimmunization is strongly associated with reduced
birth weight
Tiller H1, Killie MK1, Husebekk A2, Skogen B2, Ni H3,
Kjeldsen-Kragh J4, Øian P5
1
Department of Laboratory Medicine, University Hospital North
Norway, Tromso, Norway 2Division of Immunology, Faculty of
Health Sciences, University of Tromso and Department of
Laboratory Medicine, University Hospital North Norway, Tromso,
Norway 3Department of Laboratory Medicine, Keenan Research
Centre in the Li Ka Shing Knowledge Institute of St. Michael‘s
hospital and Canadian Blood Services and Department of
Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, ON, Canada 4Immunology and Transfusion Medicine,
Oslo University Hospital and Institute of Clinical Medicine,
University of Oslo, Oslo, Norway 5Department of Obstetrics and
Gynaecology, University Hospital of North Norway and
Department of Obstetrics and Gynaecology, Institute of Clinical
Medicine, University of Tromso, Tromso, Norway
Background: The aIIbb3 integrin is a platelet specific glycopro-
tein, but the b chain is also part of the aVb3 integrin on vascular
endothelial cells and invasive trophoblasts. The HPA 1a antigen is
located on the b3 integrin. The aim was to study whether maternal
anti-HPA 1a antibodies were associated with altered birth weight.
Methods: In this observational cohort study, 165 HPA 1a
incompatible pregnancies were identified from a screening study
of 100 448 pregnancies (124 pregnancies) and the National ref-
erence laboratory for clinical platelet immunology (41 pregnan-
cies). A linear mixed model analysis was used to assess whether
maternal anti-HPA 1a antibodies were associated with birth
Journal compilation  2010 International Society of Blood Transfusion, Vox Sanguinis (2010) 99 (Suppl. 2), 19–23
Alloimmune thrombocytopenic purpura 21
weight. A generalized linear model assessed maternal anti-HPA 1a
antibodies as risk factor for small for gestational age (SGA) neo-
nates. Both models adjusted for gestational age, maternal age,
parity, smoking habits, pre-eclampsia, diabetes mellitus and fetal
sex.
Results: The level of maternal anti-HPA 1a antibodies was line-
arly associated with reduced birth weight in boys (P < 0.001).
When the mother had high levels of anti-HPA 1a antibodies
(‡15 IU/mL) during pregnancy, the estimated marginal mean birth
weight in boys was 530 g lower compared with antibody negative
pregnancies (P < 0.001). Maternal HPA 1a alloimmunization was
strongly associated with increased risk of SGA neonates
(P < 0.001).
Conclusion: A linear relationship between maternal anti-HPA1a
antibody levels and reduced birth weight was demonstrated,
indicating that maternal HPA 1a alloimmunization may affect
placental function.
Conflict of Interest Disclosure: Bjørn Skogen, Anne Husebekk,
Mette Kjær Killie and Jens Kjeldsen-Kragh have financial rela-
tionships with Prophylix Pharma AS that might have an interest in
the submitted work. None of the authors had support from
Prophylix Pharma AS for the submitted work.
T2-PO13
Anti HPA-1a maternal alloimmunization and high risk
pregnancies: large retrospective survey, analysis of fetal
risk factors and therapy effectiveness
Bertrand G, Martageix C, Kaplan C
INTS, Paris, France
Background: Alloimmune thrombocytopenia is the most com-
mon cause of severe early onset thrombocytopenia in the neonate.
To prevent the deleterious consequence of severe fetal thrombo-
cytopenia, non invasive therapies have been developed.
Aims: To determine maternal predictive parameters for fetal se-
vere thrombocytopenia and assess the most effective antenatal
management.
Methods: Seventy-five HPA-1bb women included in this study
(239 pregnancies, 159 newborns).
Results: In the index case leading to the diagnosis of alloim-
mune thrombocytopenia 84% of the newborns were severely
affected (<50.109 plts/L) and intracranial haemorrhage (ICH) was
recorded in 12% of the cases. The severity of the affection in this
series was not correlated with the maternal ABO group or the
presence of the HLA DRB3*0101 allele, but with the gynaeco-
logic history of the women. Subsequent pregnancies were
managed as follows: Steroids alone, IVIG alone or combination
of both. No ICH was recorded. Steroids alone were poorly
effective on the neonatal plt count (below 50.109/L; 11 cases).
The highest mean newborn plt count was observed with IVIG
and steroids (133.109/L; 55 cases), in comparison with IVIG alone
(89.109/L; 27 cases). Maternal anti HPA-1a antibody concentra-
tions were followed for 32 pregnancies and found to be pre-
dictive of (1) the fetal status when done early in gestation and
before treatment, (2) the therapy failure (area under curve
weighted by the weeks of gestation).
Conclusion: This large retrospective survey gives new insights on
the predictive value of the maternal antibody titrations and
effectiveness of none invasive therapy.
 2010 The Author(s)
22 Alloimmune thrombocytopenic purpura
T2-PO14
Neonatal alloimmune thrombocytopenia is frequently
caused by low-avidity anti-HPA-1a alloantibodies that
are not detectable by current binding assays
Bakchoul T, Kubiak S, Kruatwurst A, Bein G, Sachs UJ, Santoso S
Justus Liebig University Giessen, Giessen, Germany
Background: Neonatal alloimmune thrombocytopenia (NAIT) is
mostly caused by maternal antibodies against human platelet
antigen 1a (HPA-1a) expressed on glycoprotein (GP) IIb/IIIa.
Accumulated evidence indicated that anti-HPA-1a antibodies are
overlooked by standard methods due to lack of their avidity. Low-
avidity HPA-1a antibodies were shown to be detectable by surface
plasmon resonance (SPR). We sought to investigate the frequency
and in vivo relevance of low-avidity anti-HPA-1a.
Methods: A retrospective cohort consisting of 82 HPA-1bb-
mothers of thrombocytopenic HPA-1ab newborns was analyzed
using standard serological methods. Maternal IgG fractions were
investigated for low avidity antibodies in SPR using purified
GPIIb/IIIa (HPA-1a or -1b). The capability of HPA-1a antibodies of
platelet clearance in vivo was analysed using the NOD/SCID mouse
model of alloimmune thrombocytopenia.
Results: HPA-antibodies were detectable in sera from 68/82
(83%) mothers using standard serological methods. In SPR, IgG
fractions of sera reacting positive in MAIPA showed specific
binding to HPA-1a flow cell [mean: 87 ± 21 resonance units
(RU)]. When MAIPA-negative sera were tested, binding was ob-
served in 7/14 to HPA-1a- (mean: 31 ± 5 RU), but not to HPA-1b-
flow cell (mean: 5 ± 2 RU). In vivo, 5/7 low-avidity antibodies
were capable to clear HPA-1ab platelets but not HPA-1bb platelets
in NOD/SCID mouse. Elimination-kinetic was slower than that
observed by MAIPA-positive antibodies.
Conclusion: Low-avidity HPA-1a antibodies are frequently not
detected by standard serological investigations. These antibodies
harbour the capability of platelet destruction in vivo suggesting
that a significant improvement of the diagnosis and therapy of
NAIT can be achieved by the implementation of new methods such
as SPR.
T2-PO15
Development and validation of non-invasive approach
for fetal HPA-1a genotyping using cell-free fetal DNA
present in maternal plasma
Freixa L1, Nogues N1, Ibanez M1, Farssac E1, Gracia M1, Vinyets I1,
Gonzalez Fraile I2, Muniz-Diaz E1
1
Banc de Sang I Teixits, Barcelona, Spain 2Centro De Hemoterapia
Y Hemodonación De Castilla Y Leon, Valladolid, Spain
Background: Fetal/neonatal alloimmune thrombocytopenia
(FNAIT) is caused by maternal IgG antibodies directed against fetal
platelet antigens. In Caucasians, alloimmunization against the
HPA-1a antigen is the cause of 85% of FNAIT cases. At present,
the management of high-risk alloimmunized HPA-1a neg. women
involves treatment with intravenous gammaglobulin (IVIG) and/or
invasive procedures to assess fetal platelet count. Non-invasive
determination of the fetal HPA-1 genotype would allow us to
identify the pregnancies at risk.
Methods: Automated DNA extraction from plasma samples cor-
responding to HPA-1a negative pregnant women was performed
using the QIAsymphony extractor. The HPA1a/1b polymorphism
 2010 The Author(s)
Journal compilation  2010 International Society of Blood Transfusion, Vox Sanguinis (2010) 99 (Suppl. 2), 19–23
was analyzed by Real time PCR with allele-specific primers. A
total of 38 samples (from 8 to 34 weeks of gestation) corre-
sponding to 24 HPA-1a negative pregnant women have been
tested to date. Plasma samples from HPA-1a negative individuals
have been processed in parallel as controls.
Results: Thirty-one out of the total 38 samples corresponded to
pregnant women carrying HPA-1a positive fetus (results concor-
dant with the confirmed genotype at birth). In all cases, we have
successfully detected the fetal HPA-1a allele (16 samples obtained
<20th week of gestation) and no unspecific amplification has been
observed.
Conclusion: We have developed a reliable and sensitive fetal
HPA-1 genotyping method from the maternal plasma DNA. This
non-invasive prenatal diagnostic test might be a safer alternative
to current invasive methods and could also be applied in FNAIT
prevention programs in the future.
T2-PO16
Fetal/neonatal alloimmune thrombocytopenia (FNAIT):
the importance of an accurate diagnosis for future
pregnancies
Canals C, Ibanez M, Gracia M, Farssac E, Vinyets I, Tarrago M,
Nogues N, Muniz-Diaz E
Banc de Sang I Teixits, Barcelona, Spain
Background: We present the first FNAIT case reported in Spain
due to an anti-HPA-2b allo-immunization.
Case Report: A 35-year old healthy woman gave birth to her first
child in the 40th week by caesarean section, for obstetric indi-
cation. No diathesis was observed in the newborn. The platelet
count was: 38 · 109/L. No infections or other causes of early-
onset thrombocytopenia were present.
Methods: Platelet auto-antibodies in the mother’s serum were
excluded by immunofluorescent tests (IF). No HLA antibodies were
detectable (ELISA Quick Screening). The solid phase assay (PAK12)
test did not reveal alloantibodies against IIb-IIIa glycoprotein
(GP), Ib-IX GP or Ia-IIa GP. The MAIPA assay allowed us to
identify an anti-HPA-2b alloantibody. This finding was consistent
with the platelet genotype (PCR-SSP).
Results: Anti-HPA-3b or HPA-15b antibodies were excluded by
IF and MAIPA assays. The mother was initially typed as HPA 1b1b
by PCR-SSP. Further studies showed that she was a carrier of the
polymorphism 262T>C, which may lead to false HPA-1a negative
results by PCR-SSP, and she was actually 1a1b.
HPA-1 HPA-2 HPA-3 HPA-5 HPA-15
Infant 1a1b 2a2b 3a3b 5a5a 15a15b
Mother 1a1b 2a2a 3a3a 5a5a 15a15a
Father 1a1a 2b2b 3a3b 5a5a 15b15b
Conclusion: Antibodies were only detectable by MAIPA using a
GPIb/IX monoclonal antibody. This fact, together with the poly-
morphism found in the mother, stresses the need to perform the
appropriate laboratory investigations to identify the antibodies
implicated in FNAIT cases. An accurate diagnosis is needed in
order to ensure a good clinical management in subsequent preg-
nancies.

T2-PO17
Detection and isolation of human platelet antigen (HPA)-
1a specific CD4 T cells associated with neonatal alloim-
mune thrombocytopenia (NAIT): development of an
optimalized protocol
Killie IL1, Ahlen MT2, Husebekk A2, Skogen B2, Stuge TB2
1
Division of Immunology, University of Tromsø, Tromsø, Norway
2
Division of Immunology, University of Tromsø, Department of
Laboratory Medicine, University hospital North-Norway, Tromsø,
Norway
Background: NAIT is caused by destruction of fetal platelets by
maternal antibodies reactive to HPA-1a. Helper T cells help acti-
vate B cells to differentiate into antibody-secreting plasma cells.
We have used the CFSE-based proliferation assay to identify and
isolate HPA-1a specific CD4 T cells from HPA-1a immunized
women. Although useful for studies of such responses, not only
HPA-1a specific T cells proliferate in this assay, making the
identification difficult. Thus, the detection and isolation procedure
needs to be optimized to increase the yield of specific T cell clones.
Methods: CFSE-labelled PBMCs from an immunized woman were
stimulated with HPA-1aa platelets for 2 weeks in culture and
analyzed for proliferation by flow cytometry. Proliferating
(CFSElow) single cells were isolated by fluorescence-activated cell
sorting from two populations based on the level of CD4 expres-
sion, and expanded with anti-CD3.
Results: The CD4hi region gave rise to 50 proliferating clones,
while the CD4low region gave rise to 30 clones. Three clones were
HPA-1a specific, determined by ELISPOT assay, all originating
from the CD4low region.
Conclusion: HPA-1a specific CD4 T cells down regulate CD4 in
response to antigen-specific stimulation. This may be used as a
marker to distinguish these cells from proliferating cells with
irrelevant specificities.
To improve the enrichment of specific T cell clones, two main
challenges need to be solved:
1 Reduce background proliferation of unspecific CD4 T cells.
2 Find additional markers specific for activated HPA-1a specific
T cells that can be exploited for isolation.
T2-PO18
Maternal HLA-DR-DQ haplotypes associated with HPA-
1a alloimmunization
Heide G1, Ahlen MT2, Husebekk A2, Killie MK3, Skogen B2,
Stuge TB2
1
Division of Immunology, Institute of Medical Biology, University
of Tromsø, Tromsø, Norway 2Division of Immunology, Institute of
Medical Biology, University of Tromsø; Department of Laboratory
Medicine, University hospital of North Norway, Tromsø, Norway
3
Department of Laboratory Medicine, University hospital of North
Norway, Tromsø, Norway
Background: Fetal and neonatal alloimmune thrombocytopenia
(NAIT) can result in severe complications such as intracranial
hemorrhage in fetus or newborn, due to transfer of platelet-
reactive alloantibodies from the mother to the fetus during
Journal compilation  2010 International Society of Blood Transfusion, Vox Sanguinis (2010) 99 (Suppl. 2), 19–23
Alloimmune thrombocytopenic purpura 23
pregnancy. Fetal-maternal incompatibility in the human platelet
antigen (HPA)-1 system is the most common cause of NAIT, and
HPA-1a alloimmunization is associated with the HLA allelic var-
iant DRB3*0101. However, immunization may also depend on
other factors, as only one third of women that are both HPA-1a-
negative and DRB3*0101-positive become immunized.
DRB3*0101 is associated with several different DR-DQ haplo-
types. We hypothesize that there are specific DR-DQ haplotypes
that are more strongly associated with immunization against
HPA-1a than others.
Methods: HPA-1a-immunized women and random donors were
typed for DRB3, DRB1, DQA1 and DQB1. Their DR-DQ haplotypes
were identified based on the known haplotypes in the Norwegian
population.
Results: Preliminary results from typing of 782 random donors
and 167 immunized women showed that 27% of the general
Norwegian population and 94% of the HPA-1a-immunized
women carried DRB3*0101, and that one haplotype (DRB1*0301-
DQA1*0501-DQB1*0201) was overrepresented among DRB3*
0101-positive immunized women (P < 0.005).
Conclusion: The data in the present study suggests that another,
not yet identified, genetic element within this haplotype can
influence HPA-1a-immunization. Identification and understand-
ing of such novel factors, may allow more accurate prediction of
women at risk of alloimmunization.
T2-PO19
HPA-alloimmunization in suspected Finnish NAIT
patients
Javela K, Koskinen S
Finnish Red Cross, Blood Service, Helsinki, Finland
Background: The HPA-1a antigen is by far the most common
antigen implicated in fetal and neonatal alloimmune thrombo-
cytopenia (NAIT). HPA-5b antigen is also often associated with the
complication.
Methods: Anti-HPA-antibodies were detected with the estab-
lished monoclonal antibody induced immobilization of platelet
antigens (MAIPA), which was also applied to determine HPA-
phenotypes. Nucleic acid testing confirmed the HPA-genotypes.
Clinical data from referrals of 353 consecutive suspected NAIT
cases were collated and combined with the laboratory results.
Results: At least, one incompatibility was observed in 229 (65%)
families (Table). Antibodies were detected in 73 of the 229 patients
(32%). The highest number of bleeding complications (intracere-
bral hemorrhages, ICH and petechiae), and spontaneous abortions
were in the families with anti-HPA-1a immunization. When
present, HPA-5b incompatibility was frequently accompanied
with antiHPA-5b antibodies. HPA-1b incompatibility was rela-
tively common but anti-1b-antibodies were detected rarely.
Platelet medians were lowest in HPA-1a group, as expected.
Conclusion: The incidence of the commonest specificities, anti-
HPA-1a and -5b, and the evidence of ICH were comparable with
previously reported results. The incidence of HPA-1b-antibodies
was quite high as well as the number of ICH cases in rare
incompatibility cases without antibodies.
 2010 The Author(s)