Acquired Glanzmann's thrombasthenia is an uncommon event in association with leukemia. The authors describe a patient with acute lymphoblastic leukemia (ALL) who presented with severe hemorrhagic syndrome, without disseminated intravascular coagulation. The patient's course was complicated by the occurrence of severe hemorrhagic episodes, with a thrombasthenia-like profile, requiring multiple transfusions with packed red cells, platelets, and fresh-frozen plasma. Biological explorations detected anti-GPIIb/IIIa complex antibodies. The patient finally died with refractory disease and persistent bleeding. This case is the first reported of autoantibodies to GPIIb/IIIa in ALL. Such paraneoplastic syndrome is potentially responsible for severe life-threatening hemorrhage.

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1312 Poster Board I-336 Introduction Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3. Patient and methods Genomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling. Results 7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect. Conclusion Seven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction. Disclosures No relevant conflicts of interest to declare.

3690 Fetal/neonatal alloimmune thrombocytopenia (F/NAIT) is the most common cause of severe thrombocytopenia in the fetus and the newborn in maternity wards. To counter the bleeding consequences of severe fetal thrombocytopenia, antenatal therapies have been implemented for subsequent pregnancies with incompatible fetuses. Predictive parameters for fetal severe thrombocytopenia are important for the development of non-invasive strategy and tailored intervention. We report here data concerning 67 HPA-1bb women with 81 managed pregnancies with IVIG. In 51% of the cases, the diagnosis of F/NAIT was made during the first gestation, following an intracranial haemorrhage (ICH) in 8 cases (12%). Severe thrombocytopenia was recorded for 88% of the newborn. Analysis of the index cases did not show any correlation between the severity of the disease and the maternal genetic background (ABO group and HLA-DRB3 allele). Subsequent pregnancies were managed and therapy effectiveness was evaluated. The highest mean newborn plt count was observed for a combination of intravenous immunoglobulin (IVIG) and corticoids (135.109/L; 54 newborn), in comparison with IVIG alone (89.109/L; 27 newborn). No ICH was recorded in these 2 groups. The maternal anti HPA-1a antibody concentration measured before any treatment and before 28 weeks of gestation was predictive of the fetal status: a high antibody concentration (≥28 International Units/mL) was correlated with a severe thrombocytopenia of the fetus (p=0.0016). Follow-up of the antibody concentrations during 34 pregnancies with antenatal management allowed demonstrating for the first time that the areas under curves (AUC) weighted by the weeks of gestation were a predictive parameter of therapy failure. The weighted AUC was significantly higher for women who delivered severely thrombocytopenic newborn than newborn with platelet count above 50.109/L (p=0.0107). To conclude, this large retrospective survey gives new insights on maternal predictive parameters for fetal status and therapy effectiveness allowing non-invasive strategies. Table 1: Maternal anti HPA-1a alloantibody concentrations and fetal platelet counts: statistical analysis. Predictive parameter Platelet count (×109/L) p-value Se (%) Sp (%) PPV (%) NPV (%) <50 ≥50 Number of mothers with antibody concentration (<28wg) <28 IU/mL 3 Fetuses 10 0.0016* 81.2 91.7 92.3 79.9 ≥28 IU/mL 13 2 Number of pregnancies with weighted AUC <24 IU/mL/wg 5 Newborn 18 0.0153* 64.3 82.6 69.2 79.2 ≥24 IU/mL/wg 9 5 * P<0.05. AUC: area under curve; IU: international units; Se: sensitivity, Sp: specificity; PPV: positive-predictive value; NPV: negative-predictive value. Wg: weeks of gestation. Disclosures: No relevant conflicts of interest to declare.

Since the first documented case of neonatal thrombocytopenia (NAIT) due to anti HPA-9bw (Maxa+)1, no clinical data report has appeared in the literature. We have conducted a retrospective analysis of the cases referred to the INTS (Paris) in the recent years. This analysis confirms that anti HPA-9bw (Maxa+) NAIT is a clinically severe syndrome which requires prompt diagnosis, in spite of difficulties, and maternal platelet transfusion therapy. This report concerns 8 families (12 neonates) investigated for neonatal thrombocytopenia (initial case included). The diagnosis was performed by genotyping and identification of the maternal alloantibody. The maternal sera reacted with paternal platelets in the MAIPA technique with anti GPIIb-IIIa monoclonal antibodies. No reaction was observed with a standard panel of typed donors and with their own platelets. In 2 families the diagnosis was ascertained retrospectively and the maternal alloantibody not detectable few months after delivery. Platelet genotyping did not show incompatibilities for the known platelet specific alloantigen systems HPA-1 to 11 and HPA-15 when tested in 7 out of 8 families. In the last one, incompatibility was found for HPA-3 without anti HPA-3b maternal alloantibody. As the father was HPA-9bw heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. In 10/12 cases, the infant was incompatible with the mother and therefore the diagnosis was straightforward. Four index cases were first-born children. Hemorrhage was present in 5/9 neonates, all but two infants had severe thrombocytopenia at birth, platelet counts<20.109/l and intracranial hemorrhage was documented in 1 patient, otherwise sonograms were normal. When bleeding was present maternal platelet transfusion therapy was given, most of the time because of poor response to random platelet transfusion. In 3 families successive pregnancies were followed up. In one case the fetus was compatible , in another case the 2nd child was moderately affected and in the last case maternal antenatal therapy (IvIgG) was given for fetal thrombocytopenia. Since 1999 we screen for the potential implication of rare antigens in suspected cases of NAIT when there is no other incompatibility. This screening has led us to characterize a new platelet antigen2. Anti HPA-9bw NAIT accounts for ~2% of our confirmed NAIT cases (parental antigen incompatibilities and presence of maternal alloantibodies). Difficulties in laboratory diagnosis for HPA-9bw NAIT mainly relies on the identification of the alloantibody and the paternal heterozygous status for the antigen. We have observed that monoclonal antibodies could interfere with the alloantibody binding in the MAIPA technique leading to false negative results. The diagnosis could only be confirmed after infant genotyping, in our series we have found 1 neonate and 1 fetus compatible with their mother excluding this antigen to be implicated in fetal/neonatal thrombocytopenia. In conclusion, laboratory investigation of a suspected NAIT case should be carried out in a well-versed laboratory for optimal testing. Therapy requires a strict collaboration between clinicians and blood bank services. Appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding.