Prospects for risk stratification of anti-HPA-1a alloimmunized pregnant women

  • Ulrich J. Sachs
    Correspondence
    Correspondence to: Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Langhansstr. 7, 35392, Giessen, Germany.
    Affiliations
    Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany

    Center for Transfusion Medicine and Hemotherapy, University Hospital Giessen and Marburg, Marburg, Germany

    German Center for Fetomaternal Incompatibility (DZFI), University Hospital Giessen and Marburg, Giessen, Germany
    Search for articles by this author
Published:December 31, 2019DOI:https://doi.org/10.1016/j.transci.2019.102709

      Abstract

      A diagnosis of fetal/neonatal alloimmune thrombocytopenia (FNAIT) is made if a platelet-specific antibody is detected in the mother and the fetus or newborn carries the cognate antigen. Some children will experience very low platelet counts or even intracranial hemorrhage with devastating consequences, whereas others are largely unaffected. At the moment, predictive tools to forecast the severity of FNAIT during pregnancy are not available and over- or under-treatment may put the mother or the fetus at risk. A number of potential modulators of FNAIT severity have been reported. Maternal immune responses differ in respect to the IgG subtype composition, the glycosylation pattern of the antibodies, their fine specificity, and their functional effects on platelets, the trophoblast, and endothelial cells. In addition, antibody levels are variable. The efficacy of IgG transfer and, on the fetal side, gender and inflammatory responses, were also investigated for their potential impact on FNAIT severity. These potential risk modulators are scrutinized for available experimental and clinical evidence. Antibody glycosylation and anti-endothelial activity are hot candidates which, most likely in conjunction with the antibody level, should be explored further as tools to stratify fetal risk.

      Keywords

      1. Introduction

      Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder of the fetus and newborn in which maternal alloantibodies bind to the infant’s platelets and cause their destruction during pregnancy and after birth. In Caucasians, approximately 80 % of FNAIT cases are induced by antibodies against human platelet antigen 1a (HPA-1a) [
      • Mueller-Eckhardt C.
      • Kiefel V.
      • Grubert A.
      • Kroll H.
      • Weisheit M.
      • Schmidt S.
      • et al.
      348 cases of suspected neonatal alloimmune thrombocytopenia.
      ,
      • Kjeldsen-Kragh J.
      • Killie M.
      • Tomter G.
      • Golebiowska E.
      • Randen I.
      • Hauge R.
      • et al.
      A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia.
      ]. Approximately 2000 out of 100,000 pregnancies occur in women which do not carry the HPA-1a antigen; 200 of these women will be immunized [
      • Kamphuis M.M.
      • Paridaans N.
      • Porcelijn L.
      • De Haas M.
      • van der Schoot C.E.
      • Brand A.
      • et al.
      Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review.
      ]. In 60 cases, the resulting fetal or neonatal thrombocytopenia will be severe (less than 50 × 109/L) and of these, intracranial hemorrhage (ICH) will occur in approximately 6 cases [
      • Kamphuis M.M.
      • Paridaans N.
      • Porcelijn L.
      • De Haas M.
      • van der Schoot C.E.
      • Brand A.
      • et al.
      Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review.
      ]. According to data analysed by Tiller and colleagues [
      • Tiller H.
      • Kamphuis M.J.
      • Flodmark O.
      • Papadogiannakis N.
      • David A.L.
      • Sainio S.
      • et al.
      Fetal intracranial haemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry.
      ], most of these intracranial bleeds occur prior to gestational week 28. The prognosis of ICH is very poor, with 33 % mortality until day four; and persistent neurological sequelae in more than 50 % of the surviving children.
      Presence of anti-HPA-1a antibodies in the mother and presence of the HPA-1a antigen on the fetal/neonatal platelets confirms the diagnosis of FNAIT. Unfortunately, we have no predictive tools to forecast the severity of FNAIT in a subsequent child of the same two parents. The platelet count may improve, be worse, or remain unchanged [
      • Tiller H.
      • Husebekk A.
      • Skogen B.
      • Kjeldsen-Kragh J.
      • Kjaer M.
      True risk of fetal/neonatal alloimmune thrombocytopenia in subsequent pregnancies: a prospective observational follow-up study.
      ]; the same is true for bleeding symptoms. For ICH, some authors reported high recurrence rates [
      • Radder C.M.
      • Brand A.
      • Kanhai H.H.
      Will it ever be possible to balance the risk of intracranial haemorrhage in fetal or neonatal alloimmune thrombocytopenia against the risk of treatment strategies to prevent it?.
      ,
      • Bussel J.B.
      • Berkowitch R.L.
      • Hung C.
      • Kolb E.A.
      • Wissert M.
      • Primiani A.
      • et al.
      Intracranial hemorrhage in alloimmune thrombocytopenia: stratified management to prevent recurrence in the subsequent affected fetus.
      ], but it also true that a subsequent sibling may not bleed at all.
      The fact that it is difficult to predict the outcome of a subsequent pregnancy indicates that the presence of anti-HPA-1a antibodies is not enough to explain the extent of thrombocytopenia. Furthermore, in the majority of cases, severe thrombocytopenia does not result in ICH [
      • Sanio S.
      • Javela K.
      • Tuimala J.
      • Koskinen S.
      Usefulness of maternal anti-HPA-1a antibody quantitation in predicting severity of foetomaternal alloimmune thrombocytopenia.
      ,
      • Ghaevert C.
      • Campbell K.
      • Stafford P.
      • Metcalfe P.
      • Casbard A.
      • Smith G.A.
      • et al.
      HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia.
      ]. Apparently, a good predictor for the platelet count may not be suitable to predict the (re-) occurrence of ICH.
      This lack of predictive tools must be considered as one of the most important inhibiting factors for general screening programmes in which anti-HPA-1a immunization is monitored in HPA-1bb pregnant women. Do they all need prophylactic treatment? Or will we soon be able to risk stratify anti-HPA-1a immunized pregnant women in order to allocate appropriate monitoring and treatment? This review will discuss prospects for risk stratification based on current knowledge.

      2. Modulating the severity of FNAIT

      The question which additional factors may influence the natural cause of the disease and, eventually, lead to ICH, has been addressed by several groups over a long period of time. Unfortunately, most studies are restricted to in vitro evidence, have included few patients, and have not been confirmed in follow-up studies. Potential modulating factors discussed in this review are summarized in Fig. 1, and subsumed under the following three headlines: (1) maternal immune response; (2) fetal-maternal interface; and (3) intra-fetal processes.
      Fig. 1
      Fig. 1Attributes of the maternal immune response, the feto-maternal interface and of the fetus that were correlated with the outcome (extent of thrombocytopenia or occurrence of intracranial hemorrhage) in anti-HPA-1a mediated fetal/neonatal alloimmune thrombocytopenia.

      2.1 Maternal immune response

      The effect of the maternal immune response on the natural course of FNAIT is, to our current understanding, restricted to either the amount (quantity) or the physicochemical properties (quality) of anti-HPA-1a. With respect to quality, anti-HPA-1a antibodies can differ by their IgG subtype, glycosylation pattern, specificity, and the biological function they exert on antigen-bearing cells. Besides fetal/neonatal platelets, the trophoblast and fetal endothelial cells have also been identified as target cells.

      2.1.1 Anti-HPA-1a IgG subtype

      Only IgG antibodies can be transported through the placenta into the fetal circulation. IgG subtypes differ in their transport rate through the placenta (IgG1 > IgG4 > IgG3 > IgG2), but no systematic analysis has been published whether this leads to relevant differences in the composition of maternal versus fetal/neonatal anti-HPA-1a. Von dem Borne et al. were among the first to report on the composition of maternal anti-HPA-1a [
      • AEGKr Vandem Borne
      • van Leeuwen E.F.
      • von Riesz L.E.
      • van Boxtel C.J.
      • Engelfriet C.P.
      Neonatal alloimmune thrombocytopenia: detection and characterization of responsible antibodies by the platelet immunofluorescence test.
      ]. IgG1 was identified in 95 % of sera, and 25 % also contained IgG3. IgG2 was undetectable, and IgG4 was a rare finding. Very few sera contained only IgG3. Later, Proulx et al. investigated the IgG subtype composition of 43 anti-HPA-1a sera [
      • Proulx C.
      • Filion M.
      • Goldman M.
      • Bradley A.
      • Devine D.
      • Decary F.
      • et al.
      Analysis of immunoglobulin class, IgG subclass and titre of HPA-1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia.
      ]. IgG1 was present in all sera, together with IgG3 in 25 % of the samples. IgG2 was not detected, and IgG4 was a rare finding (≈ 5 %). This group did have access to clinical data and could show that the isotype composition did not correlate with the degree of thrombocytopenia in the newborn. This finding was confirmed in a smaller study which included only 9 cases [
      • Kelsch R.
      • Hutt K.
      • Cassens U.
      • Sibrowski W.
      Semiquantative measurement of IgG subclasses and IgM of platelet-specific antibodies in a glycoprotein-specific platelet-antigen capture assay.
      ], in which the presence of IgG3 again did not correlate with the neonatal platelet count or bleeding score; and in a larger trial encompassing 27 patients [
      • Mawas F.
      • Wiener E.
      • Williamson L.M.
      • Rodeck C.H.
      Immunoglobulin G subclasses of anti-human platelet antigen 1a in maternal sera: relation to the severity of neonatal alloimmune thrombocytopenia.
      ]. In the latter study, however, higher amounts of IgG3 were detected in the more severely affected cases. In summary, there is no evidence that the anti-HPA-1a IgG subtype composition affects FNAIT severity. However, whether or not increased amounts of anti-HPA-1a IgG3 modulate the natural course of FNAIT has not been re-evaluated. Notably, IgG3 antibodies, compared with other IgG subclasses, are potent mediators of antibody-dependent cytotoxicity, phagocytosis, and complement activation [
      • Damelang T.
      • Rogerson S.J.
      • Kent S.J.
      • Chung A.W.
      Role of IgG3 in infectious diseases.
      ]. More data are necessary before a potential relevance of IgG3 as a risk factor in FNAIT can be fully excluded.

      2.1.2 Anti-HPA-1a glycosylation

      Fc parts of antibodies carry sugar moieties, including N-glycans attached to Asparagine 297. The complex saccharide core of these N-glycans usually contains fucose, and this “core fucosylation” has critical relevance for the effector functions of the antibody [
      • Shinkawa T.
      • Nakamura K.
      • Yamane N.
      • Shoji-Hosaka E.
      • Kanda Y.
      • Sakurada M.
      • et al.
      The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.
      ]. Absence of fucose makes the antibody more efficient through increased binding to Fc-receptors, mediating processes such as, antibody-dependent cytotoxicity and phagocytosis [
      • Shields R.
      • Lai J.
      • Keck R.
      • O’Connell L.Y.
      • Hong K.
      • Meng Y.G.
      • et al.
      Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular cytotoxicity.
      ]. Back in 2009, Wuhrer et al. identified markedly decreased levels of core fucosylation when they studied affinity-purified IgG1 anti-HPA-1a antibodies from 3 different patients, 2 of which were mothers of FNAIT babies [
      • Wuhrer M.
      • Porcelijn L.
      • Kapur R.
      • Koeleman C.A.M.
      • Deelder A.M.
      • de Haas M.
      • et al.
      Regulated glycoprotein patterns of IgG during alloimmune responses against human platelet antigens.
      ]. Subsequently, Kapur and colleagues collected sera from 48 women sensitized to HPA-1a and analyzed the sugar composition of their anti-HPA-1a IgG1 Fc in comparison to their total IgG1 Fc [
      • Kapur R.
      • Kustiawan I.
      • Vestrheim A.
      • Koeleman C.A.M.
      • Visser R.
      • Einarsdottir H.K.
      • et al.
      A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.
      ]. Core fucosylation was markedly decreased. More importantly, the authors could show a correlation between the anti-HPA-1a IgG1 fucose content and the severity of FNAIT, although data were widely scattered and no clear cut-off could be identified. For example, anti-HPA-1a IgG1 from two out of four ICH cases had normal fucose content. Interestingly, the decrease in core fucosylation appeared to be conserved over time and was also present in antibodies from sera taken many years after pregnancy. Both major observations, a correlation between the core fucosylation and severity of FNAIT as well as a conserved Fc glycosylation pattern for subsequent pregnancies, were more recently confirmed in a larger cohort from Finland which consisted of 166 patients, including 26 longitudinal samples [
      • Sonneveld M.E.
      • Natunen S.
      • Sainio S.
      • Koeleman C.A.M.
      • Holst S.
      • Dekkers G.
      • et al.
      Glycosylation pattern of anti-platelet IgG is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia.
      ]. In a statistical approach (principal component analysis), a 1 % decrease in fucosylation was associated with an 8 % increase in the risk of severe bleeding. Specific information on ICH cases was not available from this paper. It remains also unclear if the data are sufficient to determine a decision value.
      When the same approach was used for samples derived from cases with haemolytic disease of the fetus and newborn (HDFN), the authors identified differences in the Fc glycosylation between different antibody types [
      • Sonneveld M.E.
      • Koelewijn J.
      • de Haas M.
      • Admiraal J.
      • Plomp R.
      • Koeleman C.A.M.
      • et al.
      Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn.
      ]. The fucose content was not decreased throughout, possibly indicating antigen-dependency of the underlying regulatory mechanism. This paper also demonstrates that the “imprinting” event that decides on the sugar composition is not pregnancy as such, since the composition of transfusion-triggered antibodies was identical. These data need to be considered thoroughly. Anti-HPA-1a is not a “unique” alloantibody but rather a mixture of specificities (see 2.1.3), and more specific analysis of anti-HPA-1a antibodies and their sugar moieties might be necessary. Speaking of risk stratification in pregnant women with anti-HPA-1a, however, core fucosylation is a “hot candidate” marker, since it was shown to correlate in two separate cohorts.

      2.1.3 Anti-HPA-1a specificity

      The HPA-1a epitope is formed by a single amino acid substitution (Leu33Pro) located on the flexible PSI-domain of the integrin β3 chain [
      • Newman P.J.
      • Derbes R.S.
      • Aster R.H.
      The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/ proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing.
      ]. On platelets, the β3 chain forms heterodimers either with αIIb or with αv, which function as fibrinogen or vitronectin receptors, respectively [
      • Bennett J.S.
      • Berger B.W.
      • Billings P.C.
      The structure and function of platelet integrins.
      ]. In contrast to αIIbβ3, the αvβ3 integrin is present on numerous other cells. It was demonstrated previously that αvβ3 is constitutively expressed on endothelial cells, where it binds anti-HPA-1a antibodies [
      • Leeksma O.C.
      • Giltay J.C.
      • Zandbergen-Spaargaren J.
      • Modderman P.W.
      • van Mourik J.A.
      • von dem Borne A.E.
      The platelet alloantigen Zwa or PlA1 is expressed by cultured endothelial cells.
      ,
      • Giltay J.C.
      • Leeksma O.C.
      • von dem Borne A.E.
      • van Mourik J.A.
      Alloantigenic composition of the endothelial vitronectin receptor.
      ]. By the use of absorption/elution experiments, it was recently confirmed that the anti-HPA-1a immune response is not specifically directed against the point mutation in the β3 chain [
      • Santoso S.
      • Wihadmadyatami H.
      • Bakchoul T.
      • Werth S.
      • Al-Fakhri N.
      • Bein G.
      • et al.
      Antiendothelial αvβ3 antibodies are a major cause of intracranial bleeding in fetal/neonatal alloimmune thrombocytopenia.
      ]. Anti-HPA-1a rather comprises of at least three separable specificities: anti-β3 (which binds all β3-expressing cells), anti-αvβ3 (which binds endothelial cells strongly but platelets only weakly), and anti-αIIbβ3 (which binds platelets, but not endothelial cells). It is intriguing to speculate that these different specificities exert different biological activities (see 2.1.6). It is too early to judge whether anti-HPA-1a specificity is a promising marker for risk stratification per se.

      2.1.4 Functional capacity of anti-HPA-1a on platelets

      Anti-HPA-1a is expected to stick to its epitope on glycoprotein IIb/IIIa (the αIIbbβ3 integrin) on the platelet surface and to initiate the platelet’s phagocytosis. In fact, an early approach of testing the functional capacity of anti-HPA-1a antibodies was based on this concept [
      • Ghaevert C.
      • Campbell K.
      • Stafford P.
      • Metcalfe P.
      • Casbard A.
      • Smith G.A.
      • et al.
      HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia.
      ]. Monocyte chemiluminescence (CL) responses to antibody-coated platelets were measured in accordance with previous approaches used to predict the potency of anti-D in cases of HDFN. In this study, CL values did generally correlate well with antibody levels. Where the results between the antibody level and CL were discrepant, CL was unable to predict the clinical outcome (platelet count and/or ICH). The authors concluded that measuring functional activity does not add any clinical value.
      Antibodies may not only lead to platelet removal by phagocytes, but, by binding to their target antigen on the platelet surface, also interfere with the function of this specific receptor. Anti-HPA-1a binds to glycoprotein IIb/IIIa, the major fibrinogen receptor. In a study comprising 43 sera with anti-HPA-1a, 4/43 sera (9 %) contained blocking antibodies which hindered the binding of transfected cell expressing GP IIb/IIIa to fibrinogen [
      • Kroll H.
      • Penke G.
      • Santoso S.
      Functional heterogeneity of alloantibodies against the human platelet antigen (HPA)-1a.
      ]. These antibodies were capable of inducing thrombopathy. All neonates with blocking antibodies suffered from severe bleeding (ICH, n = 3 and organ bleeding, n = 1), but maternal sera from other neonates with similar clinical manifestations did not contain blocking antibodies.
      Indirect evidence for the occurrence of blocking antibodies stems also from studies performed with human recombinant IgG1 (clone B2G1), generated in a phage library approach from anti-HPA-1a immunized women [
      • Griffin H.M.
      • Ouwehand W.H.
      A human monoclonal antibody specific for the leucine-33 (PlA1, HPA-1a) form of platelet glycoprotein IIIa from a V gene phage display library.
      ]. This antibody interferes with the fibrinogen binding site of β3 [
      • Joutsi-Korhonen L.
      • Preston S.
      • Smethurst P.A.
      • Ijsseldijk M.
      • Schaffner-Rechkinger E.
      • Armour K.L.
      • et al.
      The effect of recombinant IgG antibodies against the leucine-33 form of the platelet b3 integrin (HPA-1a) on platelet function.
      ]. Taken together, inhibitory alloantibodies in FNAIT may contribute to severe bleeding complications including, ICH. They seem to occur only rarely, but should be considered as potential markers for risk stratification.

      2.1.5 Functional capacity of anti-HPA-1a on trophoblast

      Placental dysfunction may affect fetal growth and predispose to critical bleeding. Ducbruc et al. examined 21 placentas from FNAIT affected pregnancies in comparison to 42 age-matched controls [
      • Dubruc E.
      • Lebreton F.
      • Giannoli C.
      • Rabilloud M.
      • Huissoud C.
      • Devouassoux-Shisheboran M.
      • et al.
      Placental histological lesions in fetal and neonatal alloimmune thrombocytopenia: a retrospective cohort study of 21 cases.
      ]. Chronic chorioamnionitis, basal chronic villitis and chronic intervillositis were significantly more frequent in FNAIT cases. Only three cases with ICH were present in the cohort, not allowing statistical evaluation. A couple of years before, Althaus et al. analysed 14 placentas and identified chronic villitis only in the untreated (5/6), but not in the IVIG treated (0/7) group [
      • Althaus J.
      • Weir E.W.
      • Askin F.
      • Kickler T.S.
      • Blakemore K.
      Chronic villitis in untreated neonatal alloimmune thrombocytopenia: an etiology for severe early intrauterine growth restriction and the effect of intravenous immunoglobulin therapy.
      ]. In both studies, only some of the women were immunized against HPA-1a (43 % in both papers), the majority had other HPA specificities. Severe chronic villitis was associated with HPA-3a rather than HPA-1a immuniziation. Most women with anti-HPA-1a had nonspecific pathological changes of their placenta in one paper [
      • Althaus J.
      • Weir E.W.
      • Askin F.
      • Kickler T.S.
      • Blakemore K.
      Chronic villitis in untreated neonatal alloimmune thrombocytopenia: an etiology for severe early intrauterine growth restriction and the effect of intravenous immunoglobulin therapy.
      ], whereas chronic intervillositis was present in most women with HPA-1a antibodies in the other [
      • Dubruc E.
      • Lebreton F.
      • Giannoli C.
      • Rabilloud M.
      • Huissoud C.
      • Devouassoux-Shisheboran M.
      • et al.
      Placental histological lesions in fetal and neonatal alloimmune thrombocytopenia: a retrospective cohort study of 21 cases.
      ]. In vitro, interference of anti-HPA-1a with trophoblast cells has been reported [
      • Eksteen M.
      • Heide G.
      • Tiller H.
      • Zhou Y.
      • Nedberg N.H.
      • Martinez-Zubiaurre I.
      • et al.
      Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model.
      ]. All studies were conducted prior to the identification of the different anti-HPA-1a specificities [
      • Santoso S.
      • Wihadmadyatami H.
      • Bakchoul T.
      • Werth S.
      • Al-Fakhri N.
      • Bein G.
      • et al.
      Antiendothelial αvβ3 antibodies are a major cause of intracranial bleeding in fetal/neonatal alloimmune thrombocytopenia.
      ]. Trophoblast cells express αvβ3. Whether or not the observed pathological changes are related to specific anti-HPA-1a subtypes remains speculative.
      There is some evidence in the literature that disturbed placental function, possibly induced by anti-HPA-1a antibodies, affects the fetus, leading to reduced birth weight [
      • Tiller H.
      • Killie M.K.
      • Husebekk A.
      • Skogen B.
      • Ni H.
      • Kjeldsen-Kragh J.
      • et al.
      Platelet antibodies and fetal growth: maternal antibodies against fetal platelet antigen 1a are strongly associated with reduced birthweight in boys.
      ], which may or may not predispose to bleeding. However, birth weight did not differ between ICH and non-ICH cases in a French study [
      • Delbos F.
      • Bertrand G.
      • Croisille L.
      • Ansart-Pirenne H.
      • Bierling P.
      • Kaplan C.
      Fetal and neonatal alloimmune thrombocytopenia: predictive factors of intracranial hemorrhage.
      ], and small for gestational age (SGA) babies were equally present in both cohorts.
      In summary, there is histopathological evidence for inflammatory changes in the placenta from FNAIT babies but these changes are inconsistent. There is currently also no convincing evidence that antibody-mediated placental changes contribute to the bleeding risk of the fetus or newborn. Inflammatory changes in placental tissues can only be diagnosed after delivery. They are of scientific interest but are unsuitable for risk stratification.

      2.1.6 Functional capacity of anti-HPA-1a on endothelial cells

      The interaction between anti-HPA-1a and endothelial cells affects endothelial integrity and interferes with the spreading capability of these cells [
      • Van Gils J.M.
      • Stutterheim J.
      • van Duijn T.J.
      • Zwaginga J.J.
      • Porcelijn L.
      • de Haas M.
      • et al.
      HPA-1a alloantibodies reduce endothelial cell spreading and monolayer integrity.
      ]. These findings indicate that anti-HPA-1a antibody-mediated vascular damage may be involved in the pathomechanism of FNAIT and contribute to intracranial bleeding. In a subsequent study on 36 sera (18 from cases with ICH (+ICH) and 18 from cases without ICH (-ICH)), a stronger binding of + ICH anti-HPA-1a antibodies to endothelial cell-derived αvβ3 was observed [
      • Santoso S.
      • Wihadmadyatami H.
      • Bakchoul T.
      • Werth S.
      • Al-Fakhri N.
      • Bein G.
      • et al.
      Antiendothelial αvβ3 antibodies are a major cause of intracranial bleeding in fetal/neonatal alloimmune thrombocytopenia.
      ]. By absorption experiments, these authors subsequently identified anti-HPA-1a antibodies of anti-αvβ3 specificity in the + ICH but not in the -ICH cohort. Only the anti-αvβ3 subtype, but not the anti-β3 subtype, induced EC apoptosis (so called “anoikis”) of HPA-1a-positive ECs by caspase-3/7 activation, and reactive oxygen species. In addition, only the anti-αvβ3 subtype, but not the anti-β3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation. They concluded that the composition of the anti-HPA-1a antibody subtype(s) of the mother determines whether ICH occurs.
      The target antigen αvβ3 is only accessible for antibodies when a new vessel is formed; rapid development of premature medullary veins into shower-like numerous vessels associated with extensive angiogenesis has been observed between week 19 and 24 of gestation [
      • Pooh R.K.
      • Kurjak A.
      Fetal brain vascularity visualized by conventional 2D and 3D power doppler technology.
      ]. It is feasible that antibodies against αvβ3 interfere with these critical processes to provoke ICH, but direct biological evidence is currently lacking.
      Analysis of the anti-endothelial activity of anti-HPA-1a of the anti-αvβ3 subtype in maternal serum has a potential in the diagnostic prediction of ICH development and is another “hot candidate” marker, although independent confirmation of this observation is still pending.

      2.1.7 Anti-HPA-1a antibody level

      The predictive potential of quantifying the antibody has been a matter of debate for many years. Recently, a systematic, narrative review was published based on ten retrospective and three prospective studies [
      • Kjaer M.
      • Bertrand G.
      • Bakchoul T.
      • Massey E.
      • Baker J.M.
      • Liebermann L.
      • et al.
      Maternal HPA-1a antibody level and its role in predicting the severity of fetal/neonatal alloimmune thrombocytopenia: a systematic review.
      ]. The authors identified two relevant factors that were required to establish a correlation between the maternal antibody level and the platelet count of the fetus or newborn: (1) quantitation of anti-HPA-1a and fetal/neonatal platelet count need to be performed at the same time point; and (2) the monoclonal antibody immobilization of platelet antigens (MAIPA) assay had to be used to quantify the antibody. Restricting their review to studies which meet these criteria, the authors concluded that a positive predictive value (PPV) of 54 % (95 % confidence interval (CI): 43–63%) and a negative predictive value (NPV) of 95 % (95 % CI: 86–98%) do not allow for the use of maternal antibody levels to make a diagnosis of “severe” FNAIT (defined as a platelet count below 20 × 109/L or 50 × 109/L, depending on the study design). Consequently, they judged antibody levels unsuitable for establishing or guiding a treatment regimen. However, they also concluded that concentration of the antibody might be used to identify low-risk cases, since the NPV was in the range of 88–95 %. Based on very preliminary data, a decision value is proposed if the antibody level is determined by the MAIPA assay (not any other technique) against the WHO standard [
      • Allen D.
      • Rigsby R.
      • Bessos H.
      • Berry J.
      • Wilson D.W.
      • Ouwehand W.
      • et al.
      Collaborative study to establish the first international standard for quantitation of anti-HPA-1a.
      ]. A quantitation performed during the third trimester could be used to guide the mode of delivery and whether HPA-1bb platelets should be on stand-by.
      How about the correlation between antibody levels and ICH? Notably, Kjaer et al. did not address this outcome in their analysis. A closer look at the studies selected by these authors (Table 1) retrieves that ICH was underrepresented in most studies. Only 2/8 studies reported details about a relevant number of ICH cases [
      • Sanio S.
      • Javela K.
      • Tuimala J.
      • Koskinen S.
      Usefulness of maternal anti-HPA-1a antibody quantitation in predicting severity of foetomaternal alloimmune thrombocytopenia.
      ,
      • Ghaevert C.
      • Campbell K.
      • Stafford P.
      • Metcalfe P.
      • Casbard A.
      • Smith G.A.
      • et al.
      HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia.
      ]. Although a positive correlation between the antibody level and the presence of ICH was detectable in one study only, both papers clearly state that antibody levels cannot be used to predict ICH. A broad range of variation precluded the identification of decision values in both studies.
      Table 1Correlation between maternal anti-HPA-1a antibody levels and intracranial hemorrhage of the fetus or newborn.
      StudyICH cases/

      total number of cases
      Antibody level in ICH cases versus in non-ICH casesAuthors’ conclusion
      Williamson et al., 1998 [
      • Williamson L.
      • Hackett G.
      • Rennie J.
      • Palmer C.R.
      • Maciver C.
      • Hadfield R.
      • et al.
      The natural history of fetomaternal alloimmunization to the platelet-specific antigen HPA-1a (PlA1, Zwa) as determined by antenatal screening.
      ]
      1/33not different from non-ICH casesn/a
      Killie et al., 2008

      [
      • Killie M.
      • Husebekk A.
      • Kjeldsen-Kragh J.
      • Skogen B.
      A prospective study of maternal anti-HPA 1a antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn.
      ]
      2/56n/an/a
      Bessos et al., 2005 [
      • Bessos H.
      • Turner M.
      • Urbaniak S.
      Is there a relationship between anti-HPA 1a concentration and severity of neonatal alloimmune thrombocytopenia?.
      ]
      0/26n/an/a
      Bertrand et al., 2006 [
      • Bertrand G.
      • Martgeix C.
      • Jallu V.
      • Vitry F.
      • Kaplan C.
      Predicitve value of sequential maternal anti-HPA-1a antibody concentrations for the severity of fetal alloimmune thrombocytopenia.
      ]
      7/43“always associated with high maternal antibody concentration”?
      Killie et al., 2007

      [
      • Killie M.K.
      • Husebekk A.
      • Kaplan C.
      • Taaning E.
      • Skogen B.
      Maternal human platelet antigen-1 antibody level correlates with the platelet count in the newborns: a retrospective study.
      ]
      ?n/an/a
      Ghevaert et al., 2007 [
      • Ghaevert C.
      • Campbell K.
      • Stafford P.
      • Metcalfe P.
      • Casbard A.
      • Smith G.A.
      • et al.
      HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia.
      ]
      “new” cases, 15/97

      “previously known” cases, 3/36
      21.5 IU/ml [IQR, 6.2–94.5] versus no bleeding = 10 IU/ml [IQR 3.3-36]

      ?
      differences not significant, no decision level can be defined

      n/a
      Sainio et al., 2013

      [
      • Sanio S.
      • Javela K.
      • Tuimala J.
      • Koskinen S.
      Usefulness of maternal anti-HPA-1a antibody quantitation in predicting severity of foetomaternal alloimmune thrombocytopenia.
      ]
      first pregnancy, 7/79ICH = 61.5 IU/ml [range, 0.8–244] versus

      no bleeding = 10 IU/ml [range, 0.8–244]
      significant differences, but no decision level can be defined
      subsequent pregnancy, 1/1649 IU/ml versus

      no ICH = range, 0.8–70 IU/ml
      n/a
      Bertrand et al., 2014 [
      • Bertrand G.
      • Petermann R.
      • Kaplan C.
      Prediction of IVIG treatment efficacy in fetal/neonatal alloimmune thrombocytopenia.
      ]
      ?n/an/a
      ? = this information could not be retrieved from the manuscript, n/a = not applicable.
      In summary, although there there is as correlation between maternal antibody levels and neonatal thrombocytopenia, no correlation has been established for ICH, and therefore, it has been difficult to define a decision value that can be used as risk stratification. This discrepancy is most likely due to differences in the functional quality of anti-HPA-1a which includes core fucosylation, fine specificity (anti-β3, anti-αIIbβ3, anti-αvβ3), and functional activity (fibrinogen binding to platelets, apoptosis of endothelial cells). Further studies will also have to consider that all standard material currently used for antibody quantitation contains anti-HPA-1a with anti-β3 specificity only and may therefore be less suitable to predict severity of FNAIT [
      • Bayat B.
      • Traum A.
      • Berghöfer H.
      • Werth S.
      • Zhu J.
      • Bein G.
      • et al.
      Current anti-HPA-1a standard antibodies react with the b3 integrin subunit but not with aIIbb3 and avb3 complexes.
      ].

      2.2 Fetal-maternal interface

      All antibodies must pass the placenta before they may potentially exert any effects on fetal cells. The IgG containing fluid is internalized by syncytiotrophoblastic cells into endosomes, which become acidified. The neonatal Fc receptor (FcRn) is present in these endosomes, and binds IgG molecules at acidic pH. After the intracellular traffic to the fetal side, IgG is released when the pH increases once the endosome has fused with the cell‘s membrane [
      • Simister N.E.
      Placental transport of immunoglobulin G.
      ]. A functionally relevant polymorphisms in the FCRN gene which affects IgG binding has been identified [
      • Sachs U.J.
      • Socher I.
      • Braeunlich C.G.
      • Kroll H.
      • Bein G.
      • Santoso S.
      A variable number of tandem repeats polymorphism influences the transcriptional activity of the neonatal Fc receptor alpha-chain promoter.
      ], but a large study on healthy mother/newborn pairs failed to demonstrate an effect of this polymorphism on fetal IgG concentration [
      • Freiberger T.
      • Ravcukova B.
      • Grodecka L.
      • Kurecova B.
      • Jarkovsky J.
      • Bartonkova D.
      • et al.
      No association of FCRN promoter VNTR polymorphism with the rate of maternal-fetal IgG transfer.
      ]. Accordingly, FcRn is not a relevant factor to be considered any further. The fact that anti-HPA-1a antibodies may interfere with placental development and function was discussed above.

      2.3 Inta-fetal factors

      2.3.1 Gender

      Tiller and coworkers performed an observational cohort study of all recorded cases of ICH caused by FNAIT from the international No IntraCranial Haemorrhage (NOICH) registry [
      • Tiller H.
      • Kamphuis M.J.
      • Flodmark O.
      • Papadogiannakis N.
      • David A.L.
      • Sainio S.
      • et al.
      Fetal intracranial haemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry.
      ]. Of the 43 confirmed ICH cases, 28 ( = 65 %) were male, but antibody concentration, birth weight, platelet counts and APGAR scores were not different between boys and girls, although out of ten SGA cases with FNAIT, nine were boys. ICH was more often lethal when the fetus was male. This observation may indicate that male fetuses are somehow more prone to bleeding than female fetuses. However, the paper does not state how many male versus female fetuses were registered overall. If male fetuses were overrepresented in the registry, it is to be expected that they are also overrepresented in the subgroup of ICH positive cases. In fact, in a paper on FNAIT management and outcome based on the same registry, but published a few years later [
      • Kamphuis M.M.
      • Tiller H.
      • van den Akker E.S.
      • Westgren M.
      • Tiblad E.
      • Oepkes D.
      Fetal and neonatal alloimmune thrombocytopenia: management and outcome of a large international retrospective cohort.
      ], boys were overrepresented in the total cohort (55 % of cases). Still, this does not explain worse outcome after ICH in boys. Another observational study from Norway [
      • Tiller H.
      • Killie M.K.
      • Husebekk A.
      • Skogen B.
      • Ni H.
      • Kjeldsen-Kragh J.
      • et al.
      Platelet antibodies and fetal growth: maternal antibodies against fetal platelet antigen 1a are strongly associated with reduced birthweight in boys.
      ] included 165 pregnancies in 88 women who gave birth to 91 boys (55 %). Again, antibody concentration, birth weight, and platelet counts were equal for boys and girls. In a linear mixed model, the authors identified very high anti-HPA-1a levels as a risk factor for lower birth weight in boys, but not in girls. No data on thrombocytopenia in relation to this model were reported, and no ICH cases were included. In summary, we have some preliminary indication that maternal anti-HPA-1a antibodies may affect male fetuses differently, but no biological conclusive explanation for this observation.

      2.3.2 C-reactive protein (CRP)

      CRP is an acute phase protein produced by hepatocytes in response to inflammatory cytokines. A previously unrecognized role for CRP in IgG-triggered phagocytosis was described by Kapur et al.: CRP can bind to phosphorylcholine, which is exposed on the platelet surface after antibody binding [
      • Kapur R.
      • Heitinik-Polle K.M.J.
      • Porcelijn L.
      • Bentlaage A.E.H.
      • Bruin M.C.A.
      • Visser R.
      • et al.
      C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.
      ]. Most likely, crosslinking of αIIbβ3 mediated by anti-HPA-1a antibodies initiates signals which lead to oxidative platelet damage through NADPH oxidase, resulting in phosphorylcholine exposure. CRP, attached to phosphorylcholine, is a ligand for Fc receptors on phagocytes, and phagocytosis of CRP-labeled platelets was enhanced. Mechanistically, it is supposable to speculate that elevated CRP levels trigger platelet removal even if the number of anti-HPA-1a IgG attached to the platelet surface by itself is too low to stimulate phagocytosis. CRP does not cross the placenta [
      • Cicarelli L.M.
      • Perroni A.G.
      • Zugaib M.
      • de Albuquerque P.B.
      • Campa A.
      Maternal and cord blood levels of serum amyloid A, C-reactive protein, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 during and after delivery.
      ], so that fetal CRP is required to stimulate the removal of platelets. In a cohort of 21 FNAIT babies studied by Kapur et al. [
      • Kapur R.
      • Heitinik-Polle K.M.J.
      • Porcelijn L.
      • Bentlaage A.E.H.
      • Bruin M.C.A.
      • Visser R.
      • et al.
      C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.
      ], many had elevated CRP levels, but the authors could not establish if elevated CRP levels are causative for severe bleeding. ICH occurred in cases without elevated levels of CRP, and patients with high levels of CRP did not display clinical symptoms. If confirmed, interfering with inflammation (and thereby reducing CRP levels) could prove beneficial in FNAIT. CRP levels can only be measured after delivery. They are of scientific interest but are unsuitable for risk stratification during pregnancy.

      3. Perspectives

      In an HPA-1a immunized pregnant woman, it is currently not possible to predict the outcome of her pregnancy. Risk stratification is warranted to avoid both over- and under-treatment.
      This review was unable to identify strong predictors of bleeding. Bleeding in FNAIT is a rare event and, most likely, occurs only when multiple factors come together. One requirement for bleeding is a wounded vessel. We do currently not understand how the fetal endothelium becomes damaged or how the well-controlled process of fetal vessel sprouting becomes disturbed in FNAIT, although an interference of anti-HPA-1a sub-specificities (anti-αvβ3) has been reported. Another requirement for bleeding in FNAIT is thrombocytopenia. Although we do currently not understand exactly how (and why) severe thrombocytopenia develops, the antibody level has been identified as a potential factor that shows an association with the platelet count. The same is true for the antibodies’ glycosylation pattern.
      Putting aside concerns with regard to antibody quantitation and considering both, anti-αvβ3 specificity/anti-endothelial activity and core fucosylation of antibodies as “hot candidates”, the ideal low-risk pregnancy would have a low anti-HPA-1a titer, a regular anti-HPA-1a IgG1 Fc core fucosylation, and no detectable anti-αvβ3/anti-endothelial activity. The characteristics of blocking antibodies have not been investigated in enough depth; it is still conceivable that an “ideal low-risk pregnancy” is complicated by bleeding because blocking antibodies may not require any of the three characteristics excluded. Apparently, its absence might need to be confirmed before a “low-risk” pregnancy can remain untreated. Future studies will prove these assumptions as either true or false. All identified factors with the slightest potential effect on risk should ideally be considered in future studies. Measurement of the two most promising “hot” candidate markers is, unfortunately, laborious and technically demanding (Table 2). FNAIT researchers will have to enter a new era of intensified international collaboration to cope with these challenges.
      Table 2Risk stratification in HPA-1a immunized pregnant women.
      FactorEvidence of risk modulationTest feasibilityPotential for risk stratificationConsider in further studies?References
      experimental

      (case series

      >20)
      clinical

      (prospective study)
      yes/noyes/noeasy/advanced/difficultnone/low/intermediate/highyes/no
      Anti-HPA-1a isotypenonoeasylowyes[
      • Proulx C.
      • Filion M.
      • Goldman M.
      • Bradley A.
      • Devine D.
      • Decary F.
      • et al.
      Analysis of immunoglobulin class, IgG subclass and titre of HPA-1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia.
      ,
      • Kelsch R.
      • Hutt K.
      • Cassens U.
      • Sibrowski W.
      Semiquantative measurement of IgG subclasses and IgM of platelet-specific antibodies in a glycoprotein-specific platelet-antigen capture assay.
      ,
      • Mawas F.
      • Wiener E.
      • Williamson L.M.
      • Rodeck C.H.
      Immunoglobulin G subclasses of anti-human platelet antigen 1a in maternal sera: relation to the severity of neonatal alloimmune thrombocytopenia.
      ]
      Anti-HPA-1a core fucosylationyesnodifficultintermediate

      (no decision level)
      yes[
      • Kapur R.
      • Kustiawan I.
      • Vestrheim A.
      • Koeleman C.A.M.
      • Visser R.
      • Einarsdottir H.K.
      • et al.
      A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.
      ,
      • Sonneveld M.E.
      • Natunen S.
      • Sainio S.
      • Koeleman C.A.M.
      • Holst S.
      • Dekkers G.
      • et al.
      Glycosylation pattern of anti-platelet IgG is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia.
      ]
      Functional capacity of anti-HPA-1a:plateletsyesnoadvancedintermediate

      (too few reports)
      yes[
      • Kroll H.
      • Penke G.
      • Santoso S.
      Functional heterogeneity of alloantibodies against the human platelet antigen (HPA)-1a.
      ]
      Functional capacity of anti-HPA-1a:endothelial cellsyesnodifficulthighyes[
      • Santoso S.
      • Wihadmadyatami H.
      • Bakchoul T.
      • Werth S.
      • Al-Fakhri N.
      • Bein G.
      • et al.
      Antiendothelial αvβ3 antibodies are a major cause of intracranial bleeding in fetal/neonatal alloimmune thrombocytopenia.
      ]
      Functional capacity of anti-HPA-1a: placentayesnoeasynone
      Results are not available during pregnancy (only after delivery) and are therefore considered to have no potential for risk stratification in a HPA-1a immunized pregnant woman.
      yes[
      • Dubruc E.
      • Lebreton F.
      • Giannoli C.
      • Rabilloud M.
      • Huissoud C.
      • Devouassoux-Shisheboran M.
      • et al.
      Placental histological lesions in fetal and neonatal alloimmune thrombocytopenia: a retrospective cohort study of 21 cases.
      ,
      • Althaus J.
      • Weir E.W.
      • Askin F.
      • Kickler T.S.
      • Blakemore K.
      Chronic villitis in untreated neonatal alloimmune thrombocytopenia: an etiology for severe early intrauterine growth restriction and the effect of intravenous immunoglobulin therapy.
      ]
      Anti-HPA-1a antibody levelyesyeseasyintermediate

      (NPV:95 %

      CI: 86–98%)
      yes[
      • Kjaer M.
      • Bertrand G.
      • Bakchoul T.
      • Massey E.
      • Baker J.M.
      • Liebermann L.
      • et al.
      Maternal HPA-1a antibody level and its role in predicting the severity of fetal/neonatal alloimmune thrombocytopenia: a systematic review.
      ]
      Placenta function: FcRnnonoeasynoneno[
      • Freiberger T.
      • Ravcukova B.
      • Grodecka L.
      • Kurecova B.
      • Jarkovsky J.
      • Bartonkova D.
      • et al.
      No association of FCRN promoter VNTR polymorphism with the rate of maternal-fetal IgG transfer.
      ]
      Fetal genderyesyeseasylowyes[
      • Tiller H.
      • Kamphuis M.J.
      • Flodmark O.
      • Papadogiannakis N.
      • David A.L.
      • Sainio S.
      • et al.
      Fetal intracranial haemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry.
      ]
      Fetal CRPyesnoeasynone
      Results are not available during pregnancy (only after delivery) and are therefore considered to have no potential for risk stratification in a HPA-1a immunized pregnant woman.
      yes[
      • Kapur R.
      • Heitinik-Polle K.M.J.
      • Porcelijn L.
      • Bentlaage A.E.H.
      • Bruin M.C.A.
      • Visser R.
      • et al.
      C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.
      ]
      a Results are not available during pregnancy (only after delivery) and are therefore considered to have no potential for risk stratification in a HPA-1a immunized pregnant woman.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Declaration of Competing Interest

      None.

      References

        • Mueller-Eckhardt C.
        • Kiefel V.
        • Grubert A.
        • Kroll H.
        • Weisheit M.
        • Schmidt S.
        • et al.
        348 cases of suspected neonatal alloimmune thrombocytopenia.
        Lancet. 1989; 1: 363-366
        • Kjeldsen-Kragh J.
        • Killie M.
        • Tomter G.
        • Golebiowska E.
        • Randen I.
        • Hauge R.
        • et al.
        A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia.
        Blood. 2007; 110: 833-839
        • Kamphuis M.M.
        • Paridaans N.
        • Porcelijn L.
        • De Haas M.
        • van der Schoot C.E.
        • Brand A.
        • et al.
        Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review.
        BJOG. 2010; 117: 1335-1343
        • Tiller H.
        • Kamphuis M.J.
        • Flodmark O.
        • Papadogiannakis N.
        • David A.L.
        • Sainio S.
        • et al.
        Fetal intracranial haemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry.
        BMJ Open. 2013; 3: e002490
        • Tiller H.
        • Husebekk A.
        • Skogen B.
        • Kjeldsen-Kragh J.
        • Kjaer M.
        True risk of fetal/neonatal alloimmune thrombocytopenia in subsequent pregnancies: a prospective observational follow-up study.
        BJOG. 2016; 123: 738-744
        • Radder C.M.
        • Brand A.
        • Kanhai H.H.
        Will it ever be possible to balance the risk of intracranial haemorrhage in fetal or neonatal alloimmune thrombocytopenia against the risk of treatment strategies to prevent it?.
        Vox Sang. 2003; 84: 318-325
        • Bussel J.B.
        • Berkowitch R.L.
        • Hung C.
        • Kolb E.A.
        • Wissert M.
        • Primiani A.
        • et al.
        Intracranial hemorrhage in alloimmune thrombocytopenia: stratified management to prevent recurrence in the subsequent affected fetus.
        Am J Obstet Gynecol. 2010; 203 (e1–14): 135
        • Sanio S.
        • Javela K.
        • Tuimala J.
        • Koskinen S.
        Usefulness of maternal anti-HPA-1a antibody quantitation in predicting severity of foetomaternal alloimmune thrombocytopenia.
        Transfus Med. 2013; 23: 114-120
        • Ghaevert C.
        • Campbell K.
        • Stafford P.
        • Metcalfe P.
        • Casbard A.
        • Smith G.A.
        • et al.
        HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia.
        Transfusion. 2007; 47: 1296-1305
        • AEGKr Vandem Borne
        • van Leeuwen E.F.
        • von Riesz L.E.
        • van Boxtel C.J.
        • Engelfriet C.P.
        Neonatal alloimmune thrombocytopenia: detection and characterization of responsible antibodies by the platelet immunofluorescence test.
        Blood. 1981; 57: 649-656
        • Proulx C.
        • Filion M.
        • Goldman M.
        • Bradley A.
        • Devine D.
        • Decary F.
        • et al.
        Analysis of immunoglobulin class, IgG subclass and titre of HPA-1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia.
        Br J Haematol. 1994; 87: 813-817
        • Kelsch R.
        • Hutt K.
        • Cassens U.
        • Sibrowski W.
        Semiquantative measurement of IgG subclasses and IgM of platelet-specific antibodies in a glycoprotein-specific platelet-antigen capture assay.
        Br J Haematol. 2002; 117: 141-150
        • Mawas F.
        • Wiener E.
        • Williamson L.M.
        • Rodeck C.H.
        Immunoglobulin G subclasses of anti-human platelet antigen 1a in maternal sera: relation to the severity of neonatal alloimmune thrombocytopenia.
        Eur J Haematol. 1997; 59: 287-292
        • Damelang T.
        • Rogerson S.J.
        • Kent S.J.
        • Chung A.W.
        Role of IgG3 in infectious diseases.
        Trends Immunol. 2019; 40: 197-211
        • Shinkawa T.
        • Nakamura K.
        • Yamane N.
        • Shoji-Hosaka E.
        • Kanda Y.
        • Sakurada M.
        • et al.
        The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.
        J Biol Chem. 2003; 278: 3466-3473
        • Shields R.
        • Lai J.
        • Keck R.
        • O’Connell L.Y.
        • Hong K.
        • Meng Y.G.
        • et al.
        Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular cytotoxicity.
        J Biol Chem. 2002; 277: 26733-26740
        • Wuhrer M.
        • Porcelijn L.
        • Kapur R.
        • Koeleman C.A.M.
        • Deelder A.M.
        • de Haas M.
        • et al.
        Regulated glycoprotein patterns of IgG during alloimmune responses against human platelet antigens.
        J Prot Res. 2009; 123: 471-480
        • Kapur R.
        • Kustiawan I.
        • Vestrheim A.
        • Koeleman C.A.M.
        • Visser R.
        • Einarsdottir H.K.
        • et al.
        A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.
        Blood. 2014; 123: 471-480
        • Sonneveld M.E.
        • Natunen S.
        • Sainio S.
        • Koeleman C.A.M.
        • Holst S.
        • Dekkers G.
        • et al.
        Glycosylation pattern of anti-platelet IgG is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia.
        Br J Haematol. 2016; 174: 310-320
        • Sonneveld M.E.
        • Koelewijn J.
        • de Haas M.
        • Admiraal J.
        • Plomp R.
        • Koeleman C.A.M.
        • et al.
        Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn.
        Br J Haematol. 2016; 176: 651-660
        • Newman P.J.
        • Derbes R.S.
        • Aster R.H.
        The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/ proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing.
        J Clin Invest. 1989; 83: 1778-1781
        • Bennett J.S.
        • Berger B.W.
        • Billings P.C.
        The structure and function of platelet integrins.
        J Thromb Haemost. 2009; 7: 200-205
        • Leeksma O.C.
        • Giltay J.C.
        • Zandbergen-Spaargaren J.
        • Modderman P.W.
        • van Mourik J.A.
        • von dem Borne A.E.
        The platelet alloantigen Zwa or PlA1 is expressed by cultured endothelial cells.
        Br J Haematol. 1987; 66: 369-373
        • Giltay J.C.
        • Leeksma O.C.
        • von dem Borne A.E.
        • van Mourik J.A.
        Alloantigenic composition of the endothelial vitronectin receptor.
        Blood. 1988; 72: 230-233
        • Santoso S.
        • Wihadmadyatami H.
        • Bakchoul T.
        • Werth S.
        • Al-Fakhri N.
        • Bein G.
        • et al.
        Antiendothelial αvβ3 antibodies are a major cause of intracranial bleeding in fetal/neonatal alloimmune thrombocytopenia.
        Arterioscler Thromb Vasc Biol. 2016; 36: 1517-1521
        • Kroll H.
        • Penke G.
        • Santoso S.
        Functional heterogeneity of alloantibodies against the human platelet antigen (HPA)-1a.
        Thromb Haemost. 2005; 94: 1224-1229
        • Griffin H.M.
        • Ouwehand W.H.
        A human monoclonal antibody specific for the leucine-33 (PlA1, HPA-1a) form of platelet glycoprotein IIIa from a V gene phage display library.
        Blood. 1996; 86: 4430-4436
        • Joutsi-Korhonen L.
        • Preston S.
        • Smethurst P.A.
        • Ijsseldijk M.
        • Schaffner-Rechkinger E.
        • Armour K.L.
        • et al.
        The effect of recombinant IgG antibodies against the leucine-33 form of the platelet b3 integrin (HPA-1a) on platelet function.
        Thromb Haemost. 2004; 91: 743-754
        • Dubruc E.
        • Lebreton F.
        • Giannoli C.
        • Rabilloud M.
        • Huissoud C.
        • Devouassoux-Shisheboran M.
        • et al.
        Placental histological lesions in fetal and neonatal alloimmune thrombocytopenia: a retrospective cohort study of 21 cases.
        Placenta. 2016; 48: 104-109
        • Althaus J.
        • Weir E.W.
        • Askin F.
        • Kickler T.S.
        • Blakemore K.
        Chronic villitis in untreated neonatal alloimmune thrombocytopenia: an etiology for severe early intrauterine growth restriction and the effect of intravenous immunoglobulin therapy.
        Am J Obstet Gynecol. 2005; 193: 1100-1104
        • Eksteen M.
        • Heide G.
        • Tiller H.
        • Zhou Y.
        • Nedberg N.H.
        • Martinez-Zubiaurre I.
        • et al.
        Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model.
        Reprod Biol Endocrinol. 2017; 15: 28
        • Tiller H.
        • Killie M.K.
        • Husebekk A.
        • Skogen B.
        • Ni H.
        • Kjeldsen-Kragh J.
        • et al.
        Platelet antibodies and fetal growth: maternal antibodies against fetal platelet antigen 1a are strongly associated with reduced birthweight in boys.
        Acta Obstet Gynecol Scand. 2012; 91: 79-86
        • Delbos F.
        • Bertrand G.
        • Croisille L.
        • Ansart-Pirenne H.
        • Bierling P.
        • Kaplan C.
        Fetal and neonatal alloimmune thrombocytopenia: predictive factors of intracranial hemorrhage.
        Transfusion. 2016; 56: 59-66
        • Van Gils J.M.
        • Stutterheim J.
        • van Duijn T.J.
        • Zwaginga J.J.
        • Porcelijn L.
        • de Haas M.
        • et al.
        HPA-1a alloantibodies reduce endothelial cell spreading and monolayer integrity.
        Mol Immunol. 2009; 46: 406-415
        • Pooh R.K.
        • Kurjak A.
        Fetal brain vascularity visualized by conventional 2D and 3D power doppler technology.
        DSJUOG. 2010; 4: 249-258
        • Kjaer M.
        • Bertrand G.
        • Bakchoul T.
        • Massey E.
        • Baker J.M.
        • Liebermann L.
        • et al.
        Maternal HPA-1a antibody level and its role in predicting the severity of fetal/neonatal alloimmune thrombocytopenia: a systematic review.
        Vox Sang. 2019; 114: 79-94
        • Allen D.
        • Rigsby R.
        • Bessos H.
        • Berry J.
        • Wilson D.W.
        • Ouwehand W.
        • et al.
        Collaborative study to establish the first international standard for quantitation of anti-HPA-1a.
        Vox Sang. 2005; 89: 100-104
        • Williamson L.
        • Hackett G.
        • Rennie J.
        • Palmer C.R.
        • Maciver C.
        • Hadfield R.
        • et al.
        The natural history of fetomaternal alloimmunization to the platelet-specific antigen HPA-1a (PlA1, Zwa) as determined by antenatal screening.
        Blood. 1998; 92: 2280-2287
        • Killie M.
        • Husebekk A.
        • Kjeldsen-Kragh J.
        • Skogen B.
        A prospective study of maternal anti-HPA 1a antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn.
        Haematologica. 2008; 93: 870-877
        • Bessos H.
        • Turner M.
        • Urbaniak S.
        Is there a relationship between anti-HPA 1a concentration and severity of neonatal alloimmune thrombocytopenia?.
        Immunohematology. 2005; 21: 102-108
        • Bertrand G.
        • Martgeix C.
        • Jallu V.
        • Vitry F.
        • Kaplan C.
        Predicitve value of sequential maternal anti-HPA-1a antibody concentrations for the severity of fetal alloimmune thrombocytopenia.
        J Thromb Haemost. 2006; 4: 628-637
        • Killie M.K.
        • Husebekk A.
        • Kaplan C.
        • Taaning E.
        • Skogen B.
        Maternal human platelet antigen-1 antibody level correlates with the platelet count in the newborns: a retrospective study.
        Transfusion. 2007; 47: 55-58
        • Bertrand G.
        • Petermann R.
        • Kaplan C.
        Prediction of IVIG treatment efficacy in fetal/neonatal alloimmune thrombocytopenia.
        Blood. 2014; 124 (letter): 654-655
        • Bayat B.
        • Traum A.
        • Berghöfer H.
        • Werth S.
        • Zhu J.
        • Bein G.
        • et al.
        Current anti-HPA-1a standard antibodies react with the b3 integrin subunit but not with aIIbb3 and avb3 complexes.
        Thromb Haemost. 2019; (e-pub ahead of print)
        • Simister N.E.
        Placental transport of immunoglobulin G.
        Vaccine. 2003; 2: 3365-3369
        • Sachs U.J.
        • Socher I.
        • Braeunlich C.G.
        • Kroll H.
        • Bein G.
        • Santoso S.
        A variable number of tandem repeats polymorphism influences the transcriptional activity of the neonatal Fc receptor alpha-chain promoter.
        Immunology. 2006; 119: 83-89
        • Freiberger T.
        • Ravcukova B.
        • Grodecka L.
        • Kurecova B.
        • Jarkovsky J.
        • Bartonkova D.
        • et al.
        No association of FCRN promoter VNTR polymorphism with the rate of maternal-fetal IgG transfer.
        J Reprod Immunol. 2010; 85: 193-197
        • Kamphuis M.M.
        • Tiller H.
        • van den Akker E.S.
        • Westgren M.
        • Tiblad E.
        • Oepkes D.
        Fetal and neonatal alloimmune thrombocytopenia: management and outcome of a large international retrospective cohort.
        Fetal Diagn Ther. 2017; 41: 251-257
        • Kapur R.
        • Heitinik-Polle K.M.J.
        • Porcelijn L.
        • Bentlaage A.E.H.
        • Bruin M.C.A.
        • Visser R.
        • et al.
        C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.
        Blood. 2015; 125: 1793-1802
        • Cicarelli L.M.
        • Perroni A.G.
        • Zugaib M.
        • de Albuquerque P.B.
        • Campa A.
        Maternal and cord blood levels of serum amyloid A, C-reactive protein, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 during and after delivery.
        Mediat Inflamm. 2005; 2005: 96-100