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Abstract Background Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by maternal antibodies against paternal human platelet antigens (HPAs) on fetal platelets. Antibodies against HPA-1a are accountable for the majority of FNAIT cases. We have previously shown that high levels of maternal anti-HPA-1a antibodies are associated with clinically significant reduced birth weight in newborn boys. Chronic inflammatory placental lesions are associated with increased risk of reduced birth weight and have previously been reported in connection with FNAIT pregnancies. The HPA-1a epitope is located on integrin β3 that is associated with integrin αIIb (the fibrinogen receptor) on platelets and megakaryocytes. Integrin β3 is also associated with integrin αV forming the αVβ3 integrin heterodimer, the vitronectin receptor, which is expressed on various cell types, including trophoblast cells. It is therefore thinkable that maternal anti-HPA-1a antibodies present during early pregnancy may affect placenta function through binding to the HPA-1a antigen epitope on invasive throphoblasts. The aim of the study was to examine whether interaction of a human anti-HPA-1a monoclonal antibody (mAb) with HPA-1a on trophoblast cells affect adhesion, migration and invasion of extravillous trophoblast cells. Methods An in vitro model with human anti-HPA-1a mAb, clone 26.4, and the first trimester extravillous trophoblast cell line HTR8/SVneo was employed. The xCELLigence system was utilized to assess the possible effect of anti-HPA-1a mAb on adhesion and migration of HTR8/SVneo cells. Specially designed chambers precoated with Matrigel were used to assess the effect on the invasive capacity of cells. Results We found that human anti-HPA-1a mAb 26.4 partially inhibits adhesion and migratory capacity of HTR8/SVneo cells. Conclusions Our findings suggest that anti-HPA-1a antibodies may affect trophoblast functions crucial for normal placental development. Future studies including primary throphoblast cells and polyclonal anti-HPA-1a antibodies are needed to confirm these results.

Heidi Tiller,1 Anne Husebekk,1 Maria Therese Ahlen,2 Tor B Stuge,1 Bjørn Skogen3 1Immunology Research Group, Faculty of Health Sciences, UiT, The Arctic University of Norway, 2Division of Diagnostic Services, Department of Laboratory Medicine, 3Department of Laboratory Medicine, Norwegian National Unit for Platelet Immunology, University Hospital of North Norway, Tromsø, Norway Abstract: Differences in platelet type between the fetus and the mother can lead to maternal immunization and destruction of the fetal platelets, a condition named fetal and neonatal alloimmune thrombocytopenia (FNAIT). FNAIT is reported to occur in ~1 per 1,000 live born neonates. The major risk is intracranial hemorrhage in the fetus or newborn, which is associated with severe neurological complications or death. Since no countries have yet implemented a screening program to detect pregnancies at risk, the diagnosis is typically established after the birth of a child with symptoms. Reports on broader clinical impact have increased clinical concern and awareness. Along with new treatment options for FNAIT, the debate around antenatal screening to detect pregnancies at risk of FNAIT has been revitalized. Keywords: antibodies, screening, alloimmunization, platelets, newborn, pregnancy

Introduction : Pregnant women negative for human platelet antigen 1 a (HPA-1a) are at risk of alloimmunization with fetal HPA-1a antigen inherited from the father, and their offspring may develop fetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to analyze the frequency of HPA-1a alloimmunization in pregnant Polish women, the feasibility of using maternal platelets for intrauterine transfusions in women subjected to diagnostic fetal blood sampling (FBS) and to discuss potential consequences of alloimmunization. Material and methods : Fifteen thousand two hundred and four pregnant women were typed for HPA-1a; HPA-1a negative were screened for anti-HPA-1a. Alloimmunized women received specialist perinatology care; some of them were subjected to FBS, followed by transfusion of HPA-1a negative platelet concentrates (PC) prepared from maternal blood. Results : Three hundred seventy-three (2.5%) women were HPA-1a negative, and 32 (8.6%) tested positively for anti-HPA-1a. Antibodies were detected in 22 women during pregnancy. Diagnostic FBS followed by PC transfusion was performed in 14 woman, who were platelet donors for their 16 unborn babies. Blood donations were tolerated well by the patients, and also intrauterine platelet transfusions were uneventful. Pharmacotherapy with intravenous immunoglobulins was implemented in 11/22 patients. Conclusions : HPA-1a negative women (ca. 2.5% of all pregnant patients) are at risk of alloimmunization with HPA-1a antigen and developing FNAIT. Alloimmunized women can be donors of platelets for their offspring providing removal of antibodies from PC. Owing to potential complications, special care should be taken if an alloimmunized woman was qualified as a blood or stem cell recipient.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that may cause severe bleeding complications with risk of perinatal death or lifelong disability. The main cause of FNAIT is maternal antibodies against human platelet antigen (HPA)-1a. Both fetomaternal bleeding and transplacental trafficking of fetal cells during pregnancy could be the cause of alloimmunization. Persistence of fetal cells in the mother (fetal microchimerism) and maternal cells in the child (maternal microchimerism) are well-recognized phenomena. Thus, it could be envisaged that fetal exposure to the HPA-1a antigen could tolerize an HPA-1a negative female fetus and prevent production of anti-HPA-1a antibodies later in life if she becomes pregnant with an HPA-1a positive fetus. The objective of the current study was to assess if the risk of producing anti-HPA-1a antibodies and the severity of neonatal thrombocytopenia in HPA-1a negative women with HPA-1a positive mothers (i.e. the mother is HPA-1a/b), was lower than in HPA-1a negative women with HPA-1a negative mothers. HPA-1a negative women with HPA-1a antibodies, identified from a Norwegian screening study (1996-2004), where HPA-1 genotype of their mothers was available, were included in the study. The frequency of HPA-1a positive mothers to HPA-1a immunized daughters were compared to the calculated frequency in the general population. We did not find any difference in the frequency of HPA-1ab among mothers to daughters with HPA-1a antibodies as compared with the general population. Furthermore, acknowledging sample-size limitations, we neither found an association between the mothers' HPA type and their daughters' anti-HPA-1a antibody levels or any difference between the two groups of mothers (HPA-1ab vs HPA-1bb), with respect to frequency of thrombocytopenia in the children of their daughters with HPA-1a antibodies. Hence, there was no indication of tolerance against fetal HPA-1a antigen in HPA-1bb women who had been exposed to HPA-1a antigen during fetal development.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.

Background. Maternal alloantibodies against HPA-1a can cross placenta, opsonize foetal platelets, and induce neonatal alloimmune thrombocytopenia (NAIT). In a study of 100, 448 pregnant women in Norway during 1995–2004, 10.6% of HPA-1a negative women had detectable anti-HPA-1a antibodies. Design and Methods. A possible correlation between the maternal ABO blood group phenotype, or underlying genotype, and severe thrombocytopenia in the newborn was investigated. Results. We observed that immunized women with blood group O had a lower risk of having a child with severe NAIT than women with group A; 20% with blood group O gave birth to children with severe NAIT, compared to 47% among the blood group A mothers (relative risk 0.43; 95% CI 0.25–0.75). Conclusion. The risk of severe neonatal alloimmune thrombocytopenia due to anti-HPA-1a antibodies is correlated to maternal ABO types, and this study indicates that the observation is due to genetic properties on the maternal side.

Background Neonatal alloimmune thrombocytopenia is most commonly due to transplacental passage of maternal anti-HPA 1a antibodies. A prospective study was carried out to evaluate the pattern and quantity of maternal anti-HPA 1a antibodies in order to predict the level of thrombocytopenia in the neonates.Design and Methods A monoclonal antibody immobilization of platelet antigen assay was used to detect antibodies in maternal samples from 1,990 HPA 1bb women. HLA DRB3*0101 typing was performed in all immunized women by sequencing the HLA DRB3 gene when present.Results Primary immunization more often took place in connection with delivery than during the first pregnancy. There was a strong correlation between maternal antibody levels and the platelet counts in the newborn (R2=0.49, p