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Corresponding author at: University and Regional Laboratories, Region Skåne, Department of Clinical Immunology and Transfusion Medicine, Akutgatan 8 22185 Lund, Sweden.
Foetal and neonatal alloimmune thrombocytopenia (FNAIT) is the platelet counterpart of haemolytic disease of the foetus and newborn. Among Caucasians, around 80 % of FNAIT cases and some of the most severe cases, are caused by alloantibodies against the human platelet antigen 1a (HPA-1a). For around 3 decades it has been known that almost all HPA-1a-immunised women are HLA-DRB3*01:01 positive. The HLA molecule encoded by the HLA-DRA/DRB3*01:01 genes seems to be of crucial importance for initiating the immune response against HPA-1a. The HLA-DRB3*01:01 carrier status is not only important as a risk factor for immunisation, but does also have a significant impact on foetal/neonatal outcome. The possible role of HLA-DRB3*01:01 typing as tool for risk stratification is discussed.
Foetal and neonatal alloimmune thrombocytopenia (FNAIT) is considered as the platelet counterpart to haemolytic disease of the foetus and newborn (HDFN). Although physicians and midwifes have known HDFN as this clinical entity for more than 400 years, FNAIT was not described before 1957 [
]. Similar to HDFN, maternal alloantibodies to paternally inherited antigens can traverse the placenta, which in case of FNAIT can cause foetal thrombocytopenia that may result in intracranial haemorrhage.
Among Caucasians, around 80 % of FNAIT cases and some of the most severe cases, are caused by alloantibodies against the human platelet antigen 1a (HPA-1a), which is harboured on the β3 integrin (GPIIIa). There are two allelic variants of the β3 integrin, defined by a single amino acid substitution: HPA-1a (Leu33) and HPA-1b (Pro33) [
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.
]. Shortly after, is was discovered that the association with HPA-1a-immunisation was even stronger for another HLA-molecule: HLA-DR52, which is in linkage disequilibrium with HLA-B8 and HLA-DR3 [
Alloimmunization against the platelet-specific Zwa antigen, resulting in neonatal alloimmune thrombocytopenia or posttransfusion purpura, is associated with the supertypic DRw52 antigen including DR3 and DRw6.
]. In 1990, Valentin et al. finally showed that nearly all HPA-1a-immunised women are HLA-DR52a positive, demonstrating that only one of the three serologically defined subtypes of the HLA-DR52 molecule is associated with HPA-1a-immunisation [
HLA-DR52 is the product of the HLA-DRB3 gene, which like HLA-DRB4 and HLA-DRB5 are separate loci present in some – but not all – HLA class II DR-DQ haplotypes (Fig. 1a). The DRB3*01:01 allele is present in about 28 % of Caucasians [
Fig. 1(A) HPA-1bb women carying the HLA-DRB3*01:01 allele are at risk of immunization, due to efficient presentation of the HPA-1a peptide derived from the foetal β3 integrin, by the HLA molecule encoded by HLA-DRA/DRB3*01:01. The illustration is made by Swiss-PDB viewer of structure 2Q6W from PDB [
]. (B) Processing and presentation of platelet antigens by an antigen-presenting cell. Efficient T cell activation depends on formation of an immunological synapse, by several TCR-peptide-HLA complexes and co-stimulatory and structurally supportive interactions.
The strong association with an HLA class II molecule suggested that production of HPA-1a antibodies is T cell-dependent. This assumption was substantiated by Maslanka et al. who demonstrated that synthetic peptides encompassing the HPA-1a antigen, but not peptides harbouring the HPA-1b antigen, could re-stimulate peripheral blood mononuclear cells (PBMC) isolated from a woman who had given birth to a child affected by FNAIT [
Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia.
], and demonstrated that the β3-integrin-dervied peptide covering the HPA-1a antigen is anchored by three amino acid residues into pockets P1, P4 and P9 of the peptide-binding cleft of the HLA-DR52a molecule, where the hydrophobic residue Leu33, defining the HPA-1a allelic variant, is critical for docking into the P9 pocket. In line with this, a study showed that an antigen-presenting B cell line expressing the HLA molecule encoded by DRA/DRB3*01:01 could process and present a recombinant β3-integrin with the HPA-1a-antigen [
Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells.
]. Together these findings, supported the role of an underlying T cell response in the in vivo development of anti-HPA-1a antibodies in these HPA-1bb women, expressing the HLA-DRB3*01:01 allele. If this mechanism is crucial, such T cells should be present in the circulation of alloimmunised women. In 2009, this hypothesis was further substantiated by two studies in which HPA-1a-specific, DRB3*01:01-restricted CD4 positive T cells were cloned from alloimmunised women who had given birth to FNAIT-affected neonates [
Evidence for the specificity for platelet HPA-1a alloepitope and the presenting HLA-DR52a of diverse antigen-specific helper T cell clones from alloimmunized mothers.
]. By testing a repertoire of designed 15-mer peptide variants of the original integrin β3-derived peptide (residues 24–39), with various single amino acid substitutions, it was demonstrated that the 14 studied T cell clones were heterogenous in their recognition – meaning that although they all have had differential allowance for substitutions, they could all be activated by the cognate HLA-peptide complex [
]. This was in line with the finding that the T cell receptor sequences (TRCα and TCRβ) did not show any specific pattern in the antigen-binding regions [
Evidence for the specificity for platelet HPA-1a alloepitope and the presenting HLA-DR52a of diverse antigen-specific helper T cell clones from alloimmunized mothers.
Although it was clear from the beginning, that FNAIT is an antibody-mediated disease, the discoveries during the last three decades has strongly indicated that HPA-1a antibodies are T cell-dependent. This by itself, could also open for the possibilities of regulating the antibody response on the T-cell level [
]. All these four studies were based on results from analysing samples from clinical cases where FNAIT was suspected. Data from retrospective studies should always be interpreted cautiously, particularly because of the risk of selection bias. Platelet immunology laboratories will only receive samples for laboratory investigations from the most severely affected cases, as many of the FNAIT cases with only mild thrombocytopenia will not be diagnosed [
]. Hence, prospective studies would provide a more reliable picture of the association between HPA-1a-imunisation and HLA-DRB3*01:01 carier status. Pooled results from three prospective studies that have examined this association are presented in Table 1 [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
]. As evident from this table, the vast majority of HPA-1a-immunised women are HLA-DRB3*01:01 positive (P = 0.00001, Fisher’s exact test), and if HLA-DRB3*01:01 carrier status is used for the prediction of HPA-1a-immunisation the negative predictive value is 0.983.
Table 1Association between maternal HPA-1a antibodies and HLA-DRB3*01:01 carrier status – pooled data from 3 prospective FNAIT studies [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
The numbers of HLA-DRB3*01:01 positive and negative non-immunised women were not reported in the study by Maslanka et al. [20] but were calculated from the positive and negative predictive values that were provided in the paper.
.
HPA-1a antibodies
# of HLA-DRB3*01:01 positive (%)
# of HLA-DRB3*01:01 negative (%)
# of positive
73 (26.9)
8 (1.7)
# of negative
198 (73.1)
462 (98.3)
Total
271
470
The positive and negative predictive values for are 0.269 and 0.983, respectively, if HLA-DRB3*01:01 carrier status is used for the prediction of HPA-1a-immunisation.
P = 0.00001 (Fisher’s exact test).
* The numbers of HLA-DRB3*01:01 positive and negative non-immunised women were not reported in the study by Maslanka et al. [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
]. Hence, these studies alone do not provide sufficient information for calculating the risk of HPA-1a-immunisation for one particular woman. The risk of postpartum immunisation for both HLA-DRB3*01:01 positive and negative women was calculated in a recent study by applying Bayes’ theorem on data collected from various sources [
]. In women who were HPA-1a negative and HLA-DRB3*01:01 positive, the risk and 95 % confidence interval (CI) of HPA-1a-immunisation after delivery of an HPA-1a–positive child was estimated to 12.7 % (95 % CI, 8.6%–16.8%) as compared to 0.5 % (95 % CI, 0.1 % – 0.9 %) in women who were both negative for HPA-1a and HLADRB3*01:01 – corresponding to a 25 times higher risk of alloimmunisation. Hence, HPA-1a negative women who are HLA-DRB3*01:01 positive have a higher risk of becoming immunised as compared to RhD negative women who give birth to a RhD-positive child without administration of prophylactic anti-D immunoglobulin after delivery. This latter group of women have an approximately 7 % risk of becoming RhD immunised [
4. HPA-1a antibody levels in relation to HLA-DRB3*01:01 carrier status
Although the risk of becoming HPA-1a-immunised is 25 times higher for HLA-DRB3*01:01 positive women as opposed to women lacking this HLA molecule, there is a small proportion of women who are negative for both HPA-1a and HLA-DRB3*01:01 who become HPA-1a-immunised. As HPA-1a negative women are either HLA-DRB3*01:01 nullizygous (zero copies of the HLA-DRB3*01:01 allele), hetero- or hemizygous (one copy of the HLA-DRB3*01:01 allele) or homozygous (two copies of the HLA-DRB3*01:01 allele), an obvious question is to what extend the dose (zero, one or two copies) of the HLA-DRB3*01:01 allele influences the level of maternal HPA-1a antibodies and neonatal platelet count.
] has addressed this question. In this study, there was a significant dose-dependent effect of the HLA-DRB3*01:01 allele on anti-HPA-1a levels (Fig. 2a). Even more interesting, there was a significant, but opposite, dose-dependent effect of the mother’s HLA-DRB3*01:01 allele on the neonatal platelet count (Fig. 2b). However, the post-hoc analysis did not demonstrate a significant difference in neonatal platelet count in children born of women with one versus two doses of HLA-DRB3*01:01. This could probably be explained by the fact that the causal chain of events between the maternal HLA-DRB3*01:01 allele dose and the mother’s anti-HPA1a level is shorter than the chain of events between maternal HLA dose and neonatal platelet count. The neonatal platelet count depends on number of factors, such as A) the rate of transplacental transport of anti-HPA-1a; B) the density of HPA-1a on foetal platelets; C) the glycosylation pattern of the Fc-part of anti-HPA-1a IgG [
]; and D) the extent to which foetal thrombopoiesis can keep up with peripheral destruction of sensitised platelets.
Fig. 2Maternal dose of HLA-DRB3*01:01 in relation to (A) anti-HPA-1a levels (IU/mL) at delivery and (B) neonatal platelet count (×109). The HPA-1a-immunised women who did not have detectable HPA-1a antibodies at delivery had detectable antibodies earlier in pregnancy (data not shown). The box-and-whisker plots depict the 90th, 75th, 50th, 25th and 10th percentile. Kruskal-Wallis test was used to calculate the global p-value. The other p-values were calculated by using the Mann-Whitney U test.
]. This view is also in accordance with a recent systematic review that demonstrated a significant association between maternal HPA1a-levels and foetal/neonatal outcome [
The dose-effect seen, between women who are negative and women who are hemi- heterozygous for HLA-DRB3*01:01 is considered to be due to a qualitative matter as the specific peptide can only be presented efficiently on the antigen presenting cells (APCs) of HLA-DRB3*01:01-positive individuals. The dose effect seen, between hemi/heterozygous and homozygous, could be mediated by higher density of the peptide/HLA complexes on the surface of homozygous APCs, supporting a more productive immunological synapse with a more efficient antigen-specific T cell stimulation and B cells stimulation, resulting in higher antibody production (Fig. 1b). Studies have shown increased levels of HLA-DRB3 transcripts in homozygous cells [
]. In addition, HPA-1a-peptide binding measurements on HLA-defined cells B-LCLs (antigen-presenting cells), showed that the homozygous HLA-DRB3*01:01 cells bound more HPA1a-peptide on their surface than a heterozygous cells line [
]. Although the minimum number of TCR-peptide-HLA interactions required for T cell activation by a productive immunological synapsis between CD4 positive T cells and APCs has been debated, it is likely that the quantity has an impact of the response [
5. Neonatal outcome in relation to HLA-DRB3*01:01 carrier status
The association between foetal/neonatal outcome and maternal HLA-DRB3*01:01 carrier status in HPA-1a-immunised women has been examined in both prospective and retrospective FNAIT studies [
HLA-DRB3*01:01 is a predictor of immunization against human platelet antigen-1a but not of the severity of fetal and neonatal alloimmune thrombocytopenia.
]. However, the latter studies are prone to selection bias because mother/child samples are usually not sent to platelet laboratories for FNAIT examination in cases where the newborn only have mild thrombocytopenia. For this reason, it is not possible to draw conclusions regarding an association between HLA-DRB3*01:01 and neonatal outcome from retrospective studies.
A total of more than 150,000 women were HPA-1a typed in 4 prospective FNAIT studies [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
]. As evident from Table 2, which shows the pooled data from these four studies, all HPA-1a-immunised women who were HLA-DRB3*01:01 negative gave birth to children with platelet counts ≥ 50 × 109/L. Thus, the outcome is significantly less severe in children born of HPA-1a-immunised women who are HLA-DRB3*01:01 negative as opposed to the women who are HLA-DRB3*01:01 positive (P = 0.001, Fisher’s exact test).
Table 2Association between foetal/neonatal outcome and maternal HLA-DRB3*01:01 carrier status in HPA-1a-immunised women – pooled data from 4 prospective FNAIT studies [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
The numbers of HLA-DRB3*01:01 positive and negative non-immunised women were not reported in the study by Maslanka et al. [20] but were calculated from the positive and negative predictive values that were provided in the paper.
.
Neonatal platelet counts
# of HLA-DRB3*01:01 positive
# of HLA-DRB3*01:01 negative
# of children with ≥ 50 × 109/L
125
18
# of children with < 50 × 109/L (# of children with ICH)
66 (3)
0
Total
191
18
P = 0.001 (Fisher’s exact test).
* The numbers of HLA-DRB3*01:01 positive and negative non-immunised women were not reported in the study by Maslanka et al. [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
], it is also possible to examine the association between foetal/neonatal outcome and HLA-DRB3*01:01 carrier status irrespective of maternal HPA-1a-immunisation status. Table 3, which contains the pooled data from these studies, clearly shows that risk of giving birth to a child with severe FNAIT is extremely low (P = 0.00001, Fisher’s exact test).
Table 3Association between foetal/neonatal outcome and maternal HLA-DRB3*01:01 carrier status irrespective of HPA-1a-immunisation status – pooled data from 3 prospective FNAIT studies [
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
The numbers of HLA-DRB3*01:01 positive and negative non-immunised women were not reported in the study by Maslanka et al. [20] but were calculated from the positive and negative predictive values that were provided in the paper.
.
Neonatal platelet counts
# of HLA-DRB3*01:01 positive
# of HLA-DRB3*01:01 negative
# of children with ≥ 50 × 109/L
253
470
# of children with < 50 × 109/L
18
0
Total
271
470
Although not reported, it is assumed that none of the HLA-DRB3*01:01 negative non-immunised women in the control groups gave birth to severely thrombocytopenic children.
P = 0.00001 (Fisher’s exact test).
* The numbers of HLA-DRB3*01:01 positive and negative non-immunised women were not reported in the study by Maslanka et al. [
As demonstrated in this review, the risk of becoming HPA-1a-immunised is strongly associated with the mother’s HLA-DRB3*01:01 carrier status. Moreover, the risk of severe foetal/neonatal outcome is extremely low for women who are HLA-DRB3*01:01 negative. Hence, HLA-DRB3*01:01 typing could be used as a clinical tool for risk stratification when counselling an HPA-1a-negative pregnant woman. This could be relevant for a pregnant woman if she has been identified as HPA-1a negative by virtue of being a blood donor, or if her sister previously has had a pregnancy complicated by FNAIT. Due to her sister’s obstetric history, the pregnant woman should be HPA-1a typed to determine if she is at risk of developing HPA-1a antibodies. An HPA-1a-negative pregnant woman should subsequently be HLA-DRB3*01:01 typed, and if she does not carry this HLA allele, no further follow-up during pregnancy seems to be necessary. Even if she should become HPA-1a-immunised during pregnancy, the risk of developing anti-HPA-1a levels in levels high enough to produce significant thrombocytopenia in the foetus/newborn is extremely low. However, it is recommended that the neonatal platelet count is determined just after delivery to be able to administer a platelet transfusion to those very few newborns with low platelet counts.
From 2012–2019 the EU-financed PROFNAIT Consortium worked on the development of a hyperimmune anti-HPA-1a IgG to be used as a prophylaxis against HPA-1a-immunisation in an analogous way to anti-D, that has been used successfully during the last 5 decades for the prevention of RhD-immunisation and HDFN [
]. The intellectual property, research and clinical programs related to the development of hyperimmune anti-HPA-1a IgG was recently acquired by Rallybio IPA, LLC (www.rallybio.com); an American biotech company which is focusing on identifying and accelerating the development of transformative breakthrough therapies for patients with severe and rare disorders, such as FNAIT. As the hyperimmune anti-HPA-1a IgG drug product is manufactured from plasma collected from HPA-1a-immunised women, the supply of plasma for drug production is limited. Hence, it is essential to reserve the prophylactic drug for the HPA-1a-negative women with the highest risk of having a child with severe thrombocytopenia. As the risk of having a severely thrombocytopenic child is extremely low for women who are HLA-DRB3*01:01 negative, these women do not need to be treated with this new prophylactic drug.
In conclusion; the close association between the HLA-DRB3*01:01 carrier status on one side and risk of HPA-1a-immunisation and severe neonatal outcome on the other side, renders HLA-DRB3*01:01 typing as an important tool for risk stratification of HPA-1a-negative pregnant women.
Declaration of Competing Interest
JKK belong to a group of founders and owners of Prophylix AS, a Norwegian biotech company which has produced a hyperimmune anti-HPA-1a IgG (NAITgam) for the prevention of HPA-1a-immunisation and FNAIT. JKK is also a consultant for Rallybio IPA, LLC a US biotech company which will continue the development of NAITgam until licensure.
References
Moulinier J.
Iso-immunisation maternelle anti-plaquettaire et purpura néo-natal. Le système de groupe plaquettaire ‘Duzo’.
in: Proc 6th Congress of the European Society of Hematology, Copenhagen, Kager, Basel1957: 817-820 (236)
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.
Alloimmunization against the platelet-specific Zwa antigen, resulting in neonatal alloimmune thrombocytopenia or posttransfusion purpura, is associated with the supertypic DRw52 antigen including DR3 and DRw6.
Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia.
Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells.
Evidence for the specificity for platelet HPA-1a alloepitope and the presenting HLA-DR52a of diverse antigen-specific helper T cell clones from alloimmunized mothers.
Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.
HLA-DRB3*01:01 is a predictor of immunization against human platelet antigen-1a but not of the severity of fetal and neonatal alloimmune thrombocytopenia.
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