Mette Kjær Killie

A prospective study of maternal anti-HPA 1a antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn

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<0.001). A maternal antibody level above 3.0 IU/mL measured in gestational week 22 or 34 had a diagnostic sensitivity and specificity of 93% and 63%, respectively, for predicting the grade of neonatal thrombocytopenia. The women who were negative for HLA DRB3*0101 had significantly lower anti-HPA 1a antibody levels than those who were HLA DRB3*0101 positive (p<0.007). In contrast to primigravida, in whom anti-HPA 1a antibody levels increased during pregnancy, the antibody level decreased in 92 of 147 women who had been pregnant previously (P92 or more of 147 = 0.003). The anti-HPA 1a antibody level regularly increased after delivery. Conclusions Maternal anti-HPA 1a antibody levels in weeks 22 and 34 of pregnancy are good predictors of the degree of thrombocytopenia in the newborn both in the first and subsequent pregnancies. Most mothers became immunized at the time of delivery.

A prominent lack of IgG1 Fc-fucosylation of platelet-alloantibodies in pregnancy

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-β3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a-specific, HLA-DRB3*0101-restricted CD4+ T cells.

T-cell responses have been implicated in the development of HPA-1a-induced neonatal alloimmune thrombocytopenia (NAIT). However, HPA-1a-specific T cells have neither been isolated nor characterized. Here, we aimed to determine whether HPA-1a-specific T cells could be isolated from HPA-1a-immunized women. In the present study, peripheral blood mononuclear cells (PBMCs) from an HPA-1a-alloimmunized woman were cultured for weeks in the presence of HPA-1a peptide, labeled with CFSE, and assayed for antigen-specific proliferation. Individual proliferating cells were isolated by fluorescence-activated cell sorting and expanded in culture. Antigen specificity and HLA restriction were determined by cytokine secretion (enzyme-linked immunospot [ELISPOT]) and proliferation assays. Several CD3(+)CD4(+) T-cell clones were isolated that proliferated and secreted cytokines in response to HPA-1a peptide. Two of these clones have been established in long-term culture in our laboratory. Both of these recognize synthetic as well as naturally processed HPA-1a antigen, and the recognition is restricted by the MHC molecule HLA-DRB3*0101 that is strongly associated with NAIT. These HPA-1a-specific T-cell clones represent unambiguous evidence for the association of T-cell responses with NAIT, and they will serve as unique tools to elucidate the cellular immune response that may result in NAIT.

Maternal human platelet antigen-1a antibody level correlates with the platelet count in the newborns: a retrospective study

BACKGROUND: Maternal plasma and/or serum levels of anti‐HPA‐1a at delivery were compared to neonatal platelet (PLT) counts.

Rapid and reliable genotyping of human platelet antigen (HPA)‐1, ‐2, ‐3, ‐4, and ‐5 a/b and Gov a/b by melting curve analysis

BACKGROUND: The probability for occurrence of neonatal alloimmune thrombocytopenic purpura (NAITP) depends largely on the frequency of each individual phenotype in various populations. In caucasians, antibodies to human platelet antigen (HPA)‐1a are the major cause of neonatal alloimmune thrombocytopenic purpura, whereas in the Japanese population, antibodies to HPA‐4b is most frequently involved in NAITP. Conventional PCR techniques for platelet antigen genotyping rely on sequence‐specific primers (SSPs) and detection by gel electrophoresis, a method which is laborious and time consuming. New PCR technology, measuring the match of a hybridization probe with its target and thereby allowing simultaneous detection of both alleles, provides an efficient tool for genotyping of the HPA systems.

Cost-effectiveness of antenatal screening for neonatal alloimmune thrombocytopenia.

Objectives  To estimate the costs and health consequences of three different screening strategies for neonatal alloimmune thrombocytopenia (NAIT).

Toward a prophylaxis against fetal and neonatal alloimmune thrombocytopenia: induction of antibody‐mediated immune suppression and prevention of severe clinical complications in a murine model

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal antibody–mediated destruction of fetal or neonatal platelets (PLTs). Results from our recent large screening study suggest that the pathophysiology of FNAIT is more similar to hemolytic disease of the fetus and newborn (HDFN) than previously thought. Immunization against HPA‐1a might therefore be preventable by a prophylactic regimen of inducing antibody‐mediated immune suppression (AMIS), which has been documented to be a useful prophylaxis against HDFN. This preclinical proof‐of‐concept study investigated whether passive administration of anti‐β3 integrin could induce AMIS and thereby prevent clinical complications of FNAIT.

Neonatal alloimmune thrombocytopenia in Norway: poor detection rate with nonscreening versus a general screening programme

The implementation of an antenatal screening programme for neonatal alloimmune thrombocytopenia (NAIT) is currently under debate. We evaluated the detection rate for NAIT in a nonscreened population of 661 200 births where NAIT was diagnosed on clinical indication. We did a cross‐sectional comparison with a population of 100 448 human platelet antigen 1a (HPA1a)‐screened pregnancies from three of the five health regions in Norway. In a nonscreening situation, 7.5 cases of NAIT were detected per year compared with 53 cases when screening was applied. The detection rate of NAIT in Norway was therefore 14% of the expected rate.

Spontaneously formed tumorigenic hybrids of Meth A sarcoma cells and macrophages in vivo.

We have recently demonstrated that malignant cells can hybridize with tissue macrophages in vitro, giving rise to tumorigenic hybrids. We now demonstrate that this can occur spontaneously in vivo as a result of fusion between inoculated Meth A sarcoma cells and host cells, presumably macrophages. Thus, from tumor cell suspensions prepared by collagenase perfusion and density centrifugation, hybrid cells could be isolated that were neoplastic but in contrast to Meth A expressed macrophage markers and had phagocytic capacity. Their morphologic features were intermediate between Meth A and macrophages. By taking advantage of a semiallogeneic experimental system by inoculation of Meth A cells from BALB/c (H‐2 Kd) into (BALB.K × BALB/c) F1 (H‐2k/d), hybrid cells from these tumors could be shown to express MHC antigens of both the Meth A and the host haplotypes. Hybrid cells grew faster than Meth A cells in vivo, indicating acquisition of growth‐promoting properties through heterotypic cell fusion.