HPA-1a Alloimmunization Research Articles

A Phase 1b PK/PD Study to Demonstrate Antigen Elimination with RLYB212, a Monoclonal Anti-HPA-1a Antibody for FNAIT Prevention.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare bleeding disorder of the fetus/newborn caused by development of maternal alloantibodies against fetal human platelet antigens (HPAs), predominantly HPA-1a. Currently there are no treatments available to prevent maternal alloimmunization to HPAs or FNAIT. This proof-of-concept study (EudraCT Number: 2021-005380-49) was designed to assess the ability of subcutaneous (SC) RLYB212, a monoclonal anti-HPA-1a antibody, to eliminate HPA-1a-positive platelets in an antigen challenge model of a 30 mL fetal-maternal hemorrhage. Subjects were randomized to receive a single SC dose of RLYB212 or placebo on day 1 in a single-blinded manner, followed by transfusion of 10 × 109 HPA-1a-positive platelets on day 8. Four subjects received 0.09 mg SC RLYB212, five received 0.29 mg SC RLYB212, and two received placebo. RLYB212 achieved rapid elimination of HPA-1a-positive platelets in a concentration-dependent manner, with concentrations as low as 3.57 ng/mL meeting the prespecified proof-of-concept criterion of ≥90% reduction in platelet elimination half-life versus placebo. Following HPA-1a-positive platelet transfusion, a rapid decline was observed in the concentration of RLYB212 over a period of 2 to 24 hours, corresponding to the time needed for RLYB212 to bind to ∼10% of HPA-1a on cell surfaces. RLYB212 was well tolerated with no reports of drug-related adverse events. The data from this study are consistent with preclinical efficacy data and support the potential use of RLYB212 as a prophylactic treatment for FNAIT that prevents maternal HPA-1a alloimmunization during at-risk pregnancies.

Identifying Pregnancies at Higher Risk for HPA-1a Alloimmunization and Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT): An International, Prospective, Natural History Study

Background and significance Fetal and neonatal alloimmune thrombocytopenia (FNAIT) results from parental incompatibility of human platelet antigen (HPA) and subsequent maternal sensitization. When this occurs, fetal platelet production is impaired and platelet destruction accelerated. FNAIT is the platelet counterpart of Rhesus disease (RhD); however, FNAIT is rarer, more often occurs in first pregnancies, and alloimmunization may be detected as early as the 2nd trimester. Clinical presentation of FNAIT ranges from no/mild symptoms to severe thrombocytopenia and intracranial hemorrhage, which can take place in the second trimester and cause death or lifelong neurological impairment. Severe FNAIT outcomes impose substantial emotional and financial burden on affected families and healthcare systems. Among pregnant HPA-1a-negative women bearing an HPA-1a-positive fetus at risk of alloimmunization, ~27% carry the HLA-DRB3*01:01 gene variant, increasing sensitization risk ~25 fold and thus leading to much higher FNAIT risk. Screening for FNAIT in pregnancy is not routinely performed, unlike in RhD, resulting in failure to identify at-risk pregnancies prior to birth. To prevent severe FNAIT outcomes, we believe at-risk pregnancies must be identified very early in the second trimester, and prophylactic interventions started immediately. Current management of women with existing alloimmunization due to a prior FNAIT-affected pregnancy involves months of weekly antenatal intravenous immunoglobulin with or without steroid administration. Although fetal platelet counts often improve, this therapy is very burdensome to patients. Most data on FNAIT prevalence and risk are from Caucasian populations; studies in non-Caucasian populations have been too small to be informative. This is the first epidemiological study that seeks to identify HPA-1a-based FNAIT in a racially and ethnically diverse international population of pregnant women. Data to be tracked are: frequency of DRB3*0101 in HPA-1bb women; anti-HPA-1a alloimmunization; pregnancy outcomes; and neonatal thrombocytopenia. These data will provide a contemporary control population preceding future single-arm studies of a novel human anti-HPA-1a monoclonal antibody, RLYB212, in prophylaxis of maternal HPA-1a alloimmunization and FNAIT. The virtual elimination of RhD by screening of all pregnancies and administration of prophylaxis in at-risk cases remains one of the most significant medical advances ever achieved and provides a roadmap for FNAIT. Study design and methods Prospective, non-interventional, natural-history study assessing HPA-1a alloimmunization in pregnant women, conducted across multiple European centers in Norway, Netherlands, UK, and Germany, and in the USA. It will provide insights into anti-HPA-1a-mediated FNAIT risk and frequency among Caucasian and non-Caucasian pregnant women. Up to 30,000 women will be screened at gestational weeks 10-14 to enable early identification and follow-up of those women at higher alloimmunization risk, so that in the future, prophylaxis of the higher-risk women could be initiated. Informed consent will be requested for blood-sample collection at the 10-14 week visit for evaluation of FNAIT risk. Inclusion criteria Pregnant women aged ≥18 years Exclusion criteria Prior history of FNAIT or known HPA-1a alloimmunization Statistical methods Demographic, baseline characteristics and discrete variables will be summarized Primary objective To inform frequency of higher FNAIT risk among pregnant women of diverse racial and ethnic characteristics (all comers). (Figure) Secondary objectives Frequency of HPA-1a maternal alloimmunization at Weeks 10-14 postpartum in pregnant women at higher FNAIT risk and outcomes such as: - Spontaneous abortion: non-deliberate fetal death prior to 19 weeks of gestation - Elective abortion: deliberate termination of pregnancy at any time in gestation - Stillbirth: non-deliberate fetal death after 19 weeks of gestation - Premature birth: live birth prior to 37 completed weeks of gestation - Live births: ≥37 completed weeks of gestation Neonatal and severe neonatal thrombocytopenia: platelet count <150 × 10 9/L and <50 × 10 9/L, respectively, within 72 hours of birth Current status Enrolment for clinical-trial NCT05345561 is ongoing and expected to continue for ~3 years.

What’s with the boys? Lower birth weight in boys from HPA-1a alloimmunized pregnancies – New insights from a large prospective screening study in Poland

Fetomaternal incompatibility in human platelet antigens (HPAs) can cause maternal alloimmunization, which in turn may lead to thrombocytopenia with or without intracranial hemorrhage (ICH) in the fetus or newborn. Retrospective studies suggest that boys from alloimmunized mothers may have higher risk of ICH and lower birth weight than girls. The objective of this study was to assess how maternal HPA-1a alloimmunization, sex of the neonate and birth weight relates in a large prospective cohort. Through a national screening study in Poland (PREVFNAIT) involving HPA-1 typing of 24,259 pregnant women during 2013–2017, 606 HPA-1a negative pregnant women and their offspring were identified and included. Various multivariate models were used to assess if and how maternal HPA-1a alloimmunization status was associated with birth weight and risk of having a small for gestational age (SGA) neonate, and if and how sex of the neonate mattered. Most immunized pregnancies had male fetuses (69 %). Women carrying a male fetus had increased likelihood of having an SGA newborn if they were HPA-1a alloimmunized compared to non-immunized mothers. Increasing maternal anti-HPA-1a antibody levels were significantly associated with reduced birth weight and SGA risk among male-fetus pregnancies, but not if the fetus was female. In conclusion, anti-HPA-1a antibodies in a male fetus pregnancy is associated with increased risk of SGA and lower birth weight, especially if the antibody level is high. Sex of the fetus may therefore be considered as a new clinical predictor of more severe FNAIT neonatal outcome.

The prevalence of HPA-1a alloimmunization and the potential risk of FNAIT depend on both the DRB3*01:01 allele and associated DR-DQ haplotypes.

Alloimmunization against human platelet antigen (HPA)-1a during pregnancy can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) and severe bleeding in the foetus or newborn and likely depends on several factors. HPA-1a alloimmunization is associated with DRB3*01:01, which is associated with several DR-DQ haplotypes. However, it is not known to what extent these haplotypes contribute to the prevalence of HPA-1a alloimmunization. HPA-1a-alloimmunized women, identified in a prospective study, and random donors were typed for selected DRB3, DRB4, DRB1, DQA1 and DQB1 alleles to determine allele and DR-DQ haplotype frequencies. DRB3*01:01 was carried by 94% HPA-1a-immunized women compared to 27% in the general population. In the first population, the DR3-DQ2 haplotype was overrepresented (P<.003). The prevalence of HPA-1a alloimmunization was estimated to be about twice as frequent with DR3-DQ2 compared to DR13-DQ6, together accounting for about 90% of DRB3*01:01-positive individuals. Further, we examined DQB1*02 and DRB4*01:01 alleles for their reported association with HPA-1a alloimmunization, in the context of DR-DQ haplotypes. Since~80% of DQB1*02 alleles are linked to the DR3-DQ2 haplotype, the association might be coincidental. However, the DQB1*02:02-associated DR7-DQ2 haplotype was also overrepresented in alloimmunized women, suggesting a role for this allele or haplotype in HPA-1a alloimmunization. As DRB4*01:01 is predominantly associated with the DR7-DQ2 haplotype in HPA-1a-alloimmunized individuals, the reported association with FNAIT may be coincidental. Typing for DR-DQ haplotypes revealed important genetic associations with HPA-1a alloimmunization not evident from typing individual alleles, and the presence of different DRB3-associated DR-DQ haplotypes showed different prevalence of HPA-1a alloimmunization.

Development of Autoimmune Diseases in Women with HPA-1a Alloimmunization and Fetal-Neonatal Alloimmune Thrombocytopenia (FNAIT)

Introduction: Fetal-NeonatalAlloimmune Thrombocytopenia (FNAIT) is the result of an incompatibility between maternal and fetal human platelet alloantigens (HPA). Affecting 1 in 1000 births, it is one of the most common causes of thrombocytopenia in the neonate. Prospective studies reveal that only 5-10% of pregnancies with HPA-1a incompatibilities result in maternalalloimmunization. The association of HPA-1a incompatibility andalloimmunization is largely based on an immune response gene: the Human Leukocyte Antigen (HLA) class II DRB3*0101 haplotype. Clinical studies have shown that >90% of women that produce HPA-1a antibodies express HLA-DRB3*0101. The HLA-DRB3*0101 haplotype is found on the HLA-DR3 locus of HLA-A1-B8-DR3 - amultigene haplotype which has been implicated in the development of a number of autoimmune diseases. The presence of HLA-A1-B8-DR3 has been shown to impact susceptibility to, the age of onset, and severity of autoimmune diseases. In this study we seek to investigate whether the HPA-1aalloimmunization that results in FNAIT, which is linked to the autoimmune haplotype, is also associated with an increased incidence of maternal autoimmune diseases following pregnancy.Methods: A retrospective review was conducted to identify patients with HPA-1a cases of FNAIT associated pregnancies. Patients with serologically confirmed anti-HPA-1a FNAIT completed a general health status survey developed by our laboratory in 2013 (n = 51; mean age = 30.6 years). In addition to questions about their health, patients described the course of their pregnancy, complications, and any health problems that followed their pregnancy. A follow-up survey was conducted in 2015 (n = 49, mean age = 33.4 years) with additional questions focusing on the development of autoimmune diseases. The incidence of autoimmune disease in our sample population was compared with that in the general population controlled for age and gender (Centers for Disease Control; Table 1).Results: Analysis revealed that of the 49 patients contacted in 2015, 25 patients (51%) had a confirmed diagnosis of an autoimmune disease. Patients reported Rheumatoid Arthritis (RA) (34.6%), Systemic LupusErythematosus (SLE)(10.2%),Sjogren's Syndrome (SS) (4%), and Type 2 Diabetes Mellitus (T2DM) (2%). When compared with the general population, controlled for age and gender, there was an increased incidence of both RA and SLE in the study population.Conclusions: Our results reveal that patients with HPA-1aalloimmunization have indeed developed autoimmune diseases or symptoms that may suggest an autoimmune disease since initial contact in 2013. Specifically, of the 49 patients that were contacted in 2015 and completed the autoimmune disease questionnaire, 25 had confirmed diagnoses of an autoimmune disease. Given the role of the HLA-DR3*0101 haplotype in HLA-1aalloimmunization and that of HLA-A1-B8-DR3 in the development of autoimmune disease, this data suggests that women who do not encode for HPA-1a (i.e., those that express HPA-1b1b) and develop HPA-1aalloimmunization may be susceptible to the development of autoimmune disease(s) later in life. These findings will be compared with women who do not encode for HPA-1a (i.e., those that express HPA-1b1b) but do not make anti-HPA-1a antibodies.Future studies can be aimed at investigating not only the frequency with which autoimmune diseases occur followingalloimmunization but also if they can be prevented. [Display omitted] DisclosuresBussel:Protalex: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; BiologicTx: Research Funding; Rigel Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Prophylix Pharma: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Physicians Education Resource: Speakers Bureau; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Momenta Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Genzyme: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees; Sysmex: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Platelet antibodies and fetal growth: maternal antibodies against fetal platelet antigen 1a are strongly associated with reduced birthweight in boys

To assess whether maternal HPA 1a alloimmunization is associated with birthweight. A retrospective observational cohort study. The national reference laboratory for clinical platelet immunology at a university hospital. 165 HPA 1a incompatible pregnancies identified from a recent screening study of 100 448 women (124 pregnancies) and the national reference laboratory for clinical platelet immunology (41 pregnancies). A linear mixed model analysis was used to assess whether maternal anti-HPA 1a antibodies were associated with birthweight. A generalized linear model was used to assess maternal anti-HPA 1a antibodies as risk factor for small-for-gestational age neonates. Both models were adjusted for gestational age at time of delivery, maternal age, parity, smoking habits during pregnancy, preeclampsia, diabetes mellitus and fetal sex. MAIN OUTCOME MEASURES. Maternal anti-HPA 1a antibody as risk factor of reduced birthweight and small-for-gestational age neonates. The level of maternal anti-HPA 1a antibodies was significantly associated with birthweight and risk of small-for-gestational age neonates after correcting for confounding variables (p<0.001). However, this association was only significant for boys. When the mother had high levels of anti-HPA 1a antibodies during pregnancy, the adjusted mean birthweight in boys was 530g lower compared with anti-HPA 1a antibody negative pregnancies (p<0.001). A linear relation between maternal anti-HPA 1a antibody levels and reduced birthweight in boys was demonstrated. Reduced birthweight should be considered a possible complication of fetal and neonatal alloimmune thrombocytopenia.

Fetal alloimmune thrombocytopenia: is less invasive antenatal management safe?

Objectives. The aim of this study was to review recent multicenter data on antenatal management of anti-HPA-1a fetal alloimmune thrombocytopenia and, based on this retrospective study and on recent literature, to evaluate if FBS modified the obstetrical management.Material and methods. This retrospective study in France includes 23 pregnancies in 21 women who had a previous thrombocytopenic infant due to anti HPA-1a alloimmunization. All pregnant women received intravenous immunoglobulin (IVIG) treatment, with or without corticosteroids. Fetal blood sampling (FBS) was performed before any therapy (four cases) or during pregnancy (nine cases).Results. Infants whose mother received treatment had a significantly higher neonatal platelet count than the corresponding sibling (p = 0.003). In eight cases, therapy was started late during pregnancy. In three cases, treatment was discontinued 3 or 4 weeks before birth, and this was associated with a poorer result. No in utero intracranial hemorrhage was recorded in the infants for whom maternal therapy continued to term. Adverse effects were not observed in any case. All babies were delivered by cesarean even when FBS was performed. One emergency cesarean was performed for fetal bradycardia after FBS.Conclusion. This study confirmed that maternal therapy with intravenous immunoglobulin for fetal alloimmune thrombocytopenia gives satisfactory results. It also showed that a less invasive approach, especially a reduction in the number of fetal blood samples, is possible without deleterious consequences. This observation suggests also to start IVIG early during pregnancy and to continue treatment up to delivery.

Association Between Maternal Anti-HPA1a Antibodies and Birth Weight of the Newborn.

Abstract 2405Poster Board II-382 Background:When the mother develops antibodies against fetal platelets, fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur, with intracranial haemorrhage as the most severe complication. The frequency of FNAIT is reported to be 1:1,100 live births. The main immunogenic platelet alloantigen system in the Caucasian population is the human platelet antigen 1 (HPA1). The β3 integrin, housing the HPA1 antigen epitope on platelets, is also found on endothelial cells and invasive trophoblasts. It is therefore conceivable that maternal alloantibodies against the β3 integrin could have a direct effect on development and thereby function of the placenta. The aim of the study was to investigate whether the presence of maternal anti-HPA1a antibodies affects birth weight. Methods:We studied 74 term deliveries (≥ 37 gestational weeks) from HPA1a negative women in Norway during 1995-2008 where anti-HPA1a antibody status during pregnancy and corresponding birth weight were known. Most of the participants were selected from a prospective screening study of HPA1a negative pregnancies. Participants were also recruited from a registry in the National reference laboratory for clinical platelet immunology. The study was approved by the Regional Committee for Medical Research Ethics, North Norway. General data concerning the pregnancy and the neonate were \\.{/MAIN;222}recorded. Specific data regarding maternal HPA1a immunization status included anti-HPA1a antibody levels measured during pregnancy and post partum. A linear mixed model was used to analyze the influence of maternal anti-HPA1a antibody level on the newborn birth weight. The dependent variable in the model was birth weight. Maternal age and parity, anti-HPA1a antibody level around 34 weeks of gestation, gestational age at time of delivery, sex of neonate and Apgar scores after 1 and 5 minutes were included as independent variables. All independent variables were classified as covariates with fixed effects. Since birth weights among siblings are not independent observations, the model treated each woman as one subject. Results:The mean birth weight was 3,050 grams when maternal anti-HPA1a antibodies were detected at any level or time during pregnancy (n= 47, 95% C.I. = 2952-3148). If no maternal anti-HPA1a antibodies were detected during pregnancy, the mean birth weight was 3,622 grams (n= 27, 95% C.I. = 3398-3845).We found a highly significant association between anti-HPA1a antibody levels around gestational week 34 and corresponding birth weight in the mixed model analysis, both when using antibody levels as a dichotomy variable (p = 0.006) and when dividing antibody levels into 3 intervals (no antibody detected, less than 3 IU/ml or more than 3 IU/ml, p = 0.011). Anti-HPA1a antibody level treated as a continuous variable was not significantly associated with birth weight (p = 0.057). Conclusions:We have demonstrated a significant correlation between the presence and level of maternal anti-HPA1a antibodies during pregnancy and neonatal birth weight. Our findings support the hypothesis that HPA1a alloimmunization affects more than just neonatal platelet counts, and should be considered even a placental disorder. Disclosures:No relevant conflicts of interest to declare.

Mapping of Helper Epitopes to HPA-1a in Neonatal Alloimmune Thrombocytopenia with T-Cell Clones

Background: The major cause of severe neonatal allo-immune thrombocytopenia (NAITP) in Caucasians is fetal-maternal incompatibility for the human platelet antigen-1 (HPA-1), which is determined by the dimorphism Leu (HPA-1a) or Pro (HPA-1b) at amino acid position 33 of β3 integrin glycoprotein IIIa. Maternal responsiveness to HPA-1a shows a very strong association with DR52a (DRB3*0101) and 33Leu is thought to create a binding motif for this MHC class II molecule, thereby conferring helper T-cell responsiveness. At present, there are no preventative measures, reliable predictors of severity or screening procedures to identify women at risk of HPA-1a alloimmunization. Since the T-cells that help HPA-1a antibody production represent an attractive target for both screening assays and specific therapy, the aim was to define the epitope(s) they recognize in detail.Methods: From the peripheral blood of three HLA DR52a positive mothers with anti-HPA-1a antibodies and affected babies, we generated a total of six stable long-term CD4+ T-cell clones that respond specifically to the HPA-1a+ glycoprotein sequence. They have enabled us to characterize, for the first time, the fine specificity and restriction of the HPA-1a helper epitope. The core epitope was mapped by testing the responsiveness (proliferation and cytokine production) of the clones to panels of synthetic peptides spanning the HPA-1a 33Leu polymorphism, including sequences of different lengths, with selected single amino acid substitutions, and with the polymorphic residue located at different positions. Restriction was defined using antigen-presenting cells sharing HLA-DR and by flow cytometric analysis of staining with peptide-DR52a tetramers. The results, together with structural analyses, were used to model the interactions between MHC class II, HPA-1a peptide and specific helper T-cell receptor.Results: The 6 Th clones showed clear specificity for their HPA-1a epitope, even when naturally processed from whole platelets; they recognized only GPIIIa peptides (or platelets) with 33Leu and not 33Pro. The results of screening panels of linear peptides with 33Leu at different positions are consistent with a “core” epitope of 25WCSDEALPL33. The clones also specifically bound tetramerized DR52a complexed with a peptide spanning these residues. Together, the results show that 25Trp, 28Asp and 33Leu of HPA-1a are each important for anchoring respectively in pockets 1, 4 and 9 in DR52a, whereas 33Pro of HPA-1b sterically hinders docking in pocket 9. Extra residues 34Gly-35Ser did not affect T-cell recognition, but certain clones preferred N-terminal 24Ala or 23Cys-24Ala extensions. It has been previously reported that T-cells from alloimunized women with anti-HPA-1a recognize a 33Leu peptide cyclized by disulfide bridges (circular-Leu; 26C*SDEALPLGSPRC*38) but not to the circularized 33Pro equivalent. Our T-cell clones also responded moderately to this circular-33Leu peptide, demonstrating that 25Trp anchor at pocket 1 may not be essential when anchoring in pocket 4 and 9 are strong.Conclusions: Characterization of the HPA-1a specific Th clones reveals that they all recognize the core epitope GPIIIa 25Trp-33Leu, with the polymorphic 33Leu selectively anchoring it in the strongly predisposing HLA-DR52a. Although predicted to lie outside the peptide-binding groove of DR52a, extra N-terminal sequences promote optimal recognition by some T-cells, but are no longer required if the sequence is cyclized. Despite differences in TCR gene usage, the clones show remarkable consistency in fine specificity, supporting our previous evidence from polyclonal T-cell responses from women with anti-HPA-1a antibodies. The identification of a single “core” epitope, and the uniformity of restriction by DR52a in alloimmunized women, opens the way to both diagnostic and therapeutic exploitation in NAITP due to anti-HPA-1a.

The alloimmune response to the human platelet antigen‐1a is not related to maternal‐fetal killer immunoglobulinlike receptor/HLA‐Cw combinations

Human platelet antigen (HPA)-1a fetomaternal alloimmune thrombocytopenia, responsible in the most severe cases for fetal or neonatal intracranial hemorrhages leading to death or survival with neurologic sequelae, was shown to be restricted to the human leukocyte antigen (HLA) Class II DRB3*0101-encoded molecule. Whereas more than 90 percent of alloimmunized mothers display the DRB3*0101 allele, the positive predictive value of the presence of DRB3*0101 is only 35 percent. Additional genetic risk factors may exist of which elucidation could improve the undertaking of incompatible pregnancies in at-risk families, encouraging an antenatal screening. Interactions of killer immunoglobulinlike receptors (KIRs) on maternal decidual NK cells with HLA-Cw molecules on fetal trophoblasts were reported as one of the mechanisms involved in the fetomaternal tolerance during pregnancy. Genotyping was performed of 16 KIR genes in HPA-1a-negative/DRB3*0101-positive alloimmunized mothers and in HPA-1a-negative/DRB3*0101-positive nonimmunized mothers as well as HLA-Cw genotyping in thrombocytopenic children and their nonaffected siblings. No particular KIR genes or KIR genotypes were observed in the alloimmunized or nonimmunized mothers. Distribution of HLA-Cw genes in affected infants and nonaffected siblings did not reveal any HLA-Cw specificity associated with triggering or modulation of the HPA-1a alloimmunization. No maternal KIR/fetal HLA-Cw combinations were demonstrated in association with a detrimental or a protective effect on the HPA-1a alloimmunization. Maternal KIR/fetal HLA-Cw gene combinations that are involved in the fetomaternal tolerance do not appear to play a role in the HPA-1a alloimmunization.

Massive fetal hemorrhage and fetomaternal alloimmune thrombocytopenia from human platelet antigen 5b incompatibility: an unusual association

Massive fetal hemorrhage and fetomaternal alloimmune thrombocytopenia from human platelet antigen 5b incompatibility: an unusual association

Targeting the Risks of Specific Antiplatelet Alloimmunization in the Populations of Sub-Saharan Africa, Asia, and Oceania.

As specific antiplatelet alloimmunization directed against Human Platelet Antigens (HPA) during pregnancy or after platelet transfusion is not a rare event, this study aims at identifying such a risk in the context of the diversity caused by the population migrations we see today in our hospitals and particularly in the Sub-Saharan African (SSA), south East Asia and Polynesian populations. Samples were collected from 155 Beninese, 118 Cameroonian, 96 Congolese (Kinshasa), 107 Vietnamese and 81 Polynesian Ma'ohis, all unrelated, healthy blood donors. DNA was extracted by salting out method and the platelet genotype was determined by PCR-RFLP. We did not observe any significant deviation from the Hardy-Weinberg equilibrium. As opposed to Caucasian populations, the risk of anti HPA-1a alloimmunization is extremely low, due to the absence, or at least the low frequencies of HPA-1 b homozygous individuals in these populations (Cameroon 0.8 % and Benin 1.3 %). An important risk could be associated with HPA-2 immunization in the SSA population, as we observed a relatively high frequency of HPA-2b homozygous individuals. Moreover we noted the frequency of HPA-3b homozygous to be between 11 and 24%. Given that, as neonatal alloimmune thrombocytopenia (NAIT) caused by anti HPA-3a or 3b is similar in severity to disease caused by incompatibility of HPA-1a, this risk should not be ignored particularly in the Vietnamese population with 24.3 % of HPA3b homozygous. We noted the absence of HPA-4b allele. Finally, in contrast to the frequency of HPA-6 heterozygous in the Polynesian population (17%), we do not observe HPA-6b homozygous individuals, suggesting a small risk for that antigen to be implicated in alloimmunization. The repartition of HPA-15 alleles is heterogeneous in these populations. In conclusion: HPA-2 alloimmunization in SSA populations should be identified for platelet transfusion refractoriness or NAIT, similarly for HPA-3 especially in the Vietnamese population and to a lesser degree, HPA-5 in the Cameroon and in the Congo.Genotype frequenciesHPA-1HPA-2HPA-3HPA-5HPA-6HPA-15bb...ab ↑bb...ab ↑bb...ab ↑bb↑ ...ab bb↑ ...ab bbViet Nam0.0↑ 09.4 0.054. ↑24.305.6 ↑0.002.8 ↑0↑ 57.0 24.3Polynesia0.0↑ 16.3 0.0↑ 37.5 18.705. ↑0.0↑ 17.1 0↑ 52.4 28.4Benin1.3↑ 44.8 7.142.8 ↑11.134.1 ↑0.80.00 ↑0↑ 41.6 14.1Cameroon0.832.7 ↑7.8↑ 50.8 13.129.8 ↑6.4↑ 0.00 0↑ 41.2 10.9Congo0.040.0 ↑2.549. ↑18.645.2 ↑4.1↑ 0.00 0↑ 43.8 05.5

Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia.

Neonatal alloimmune thrombocytopenia (NATP) caused by fetomaternal mismatch for human platelet (PLT) alloantigens (HPAs) complicates approximately 1 in 1000 to 1 in 2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage, and sometimes death of the fetus or neonate. As a national reference center for NATP investigations, our experience with this entity over a 12-year period was reviewed. The laboratory records of all cases of suspected NATP referred for evaluation from January 1, 1990, to December 31, 2002, were analyzed. The spectrum of PLT alloantibody specificities identified was compared with an earlier reported series of serologically verified NATP cases. HPA-specific alloantibodies were identified in 1162 (31%) of 3743 sera of mothers of infants with clinically suspected NATP. Maternal HPA-1a (PlA1) alloimmunization accounted for the majority (79%) of confirmed NATP cases, with HPA-5b (Bra), HPA-3a (Baka), and HPA-1b (PLA2) alloantibodies accounting for 9, 2, and 4 percent of cases, respectively. In addition, an increase in the number of cases in which multiple HPA-specific alloantibodies were present in maternal sera was observed during the study period. Although, as with the earlier series, maternal HPA-1a alloimmunization was the dominant cause of NATP, the identification of an increasing number of cases due to alternative HPA polymorphisms suggests that investigation for HPA-1 incompatibility alone is no longer sufficient to fully evaluate clinically suspect NATP cases.

On investigation of markers that may influence alloantibody responses to HPA-1a

In > 99% of cases, HPA-1a antibody production during pregnancy is associated with maternal DRB3*0101 positivity. However, only 35% of HPA-1a neg/DRB3*0101 women produce antibodies (Ab). This study attempted to identify additional genetic marker(s) that may better predict anti-HPA-1a production in these women. Seventy-eight DRB3*0101 pos HPA-1a neg, women (40 HPA-1a Ab pos, 38 Ab neg) with HPA-1a pos infants, were typed for HLA-DRB1*, -B3*, -B4*, -B5*. Results were compared with those from 83 DRB3*0101 pos normal donors. SNaPshot™ was used to test for a polymorphism of the TNF-α locus. The frequency of DRB1*15 was significantly lower in Ab pos mothers (1/40) compared to controls (16/83; P = 0·03), but not compared to Ab neg mothers (6/38; P = 0·07). DRB1*12 was found only in Ab neg mothers (5/38) (controls 0/83; P = 0·02). A study of TNF-α genotype (n = 30, Ab pos; Ab neg; controls) found no difference between the mothers' groups, or mothers and controls. Presence of DRB1*15 or DRB1*12 may lower the likelihood of HPA-1a Ab production. Raised frequency of DRB4* was seen in mothers with affected neonates (13/22) (unaffected, Ab pos 3/11; P = 0·02). DRB4* may increase the odds of HPA-1a alloimmunization. Three NAITP cases due to anti-HPA-1a have been reported (2 local, 1 published), involving mothers DRB3*0101 neg, but DRB4* pos. TNF-α genotype may not predict anti-HPA-1a production in these women. Studies with larger groups would establish the value of these markers in defining women at high risk of HPA-1a alloimmunization.

Thrombopoietin levels in cord blood plasma and amniotic fluid in fetuses with alloimmune thrombocytopenia and healthy controls.

To extend our knowledge of the kinetics of fetal thrombopoietin (TPO), we studied TPO levels in cord blood plasma and amniotic fluid collected from 15 fetuses considered to be at risk of fetomaternal alloimmune thrombocytopenia and also from 10 healthy controls at caesarean delivery. In the plasma of all 25 fetuses and newborn infants studied, TPO was detected above the lower limit of detection (7 pg/ml) and correlated inversely with platelet counts (r = -0.53, P = 0.006). At term, TPO detected in amniotic fluid was at significantly lower levels (7 pg/ml; range 0-22 pg/ml) than simultaneously obtained cord plasma TPO (114 pg/ml; range 43-201 pg/ml; P < 0.001). There was no correlation between levels of TPO in amniotic fluid and cord plasma or platelet counts. In the serial samples collected from the five fetuses with HPA-1a alloimmunization before 37 weeks' gestation, the TPO levels in amniotic fluid were significantly higher than at term (P = 0.013): from 22 to 28 weeks' gestation, 42 pg/ml (30-78 pg/ml); from 32 weeks', 24 pg/ml (17-33 pg/ml); at term, 8 pg/ml (4-13 pg/ml), correlating inversely with gestational age (r = -0.81, P = 0.003). Thus, TPO is present in amniotic fluid at levels apparently inversely related to gestational age. Whether these high levels seen early in pregnancy are normal or are associated with the HPA-1 alloimmunization remains to be shown.

The Natural History of Fetomaternal Alloimmunization to the Platelet-Specific Antigen HPA-1a (PlA1, Zwa) as Determined by Antenatal Screening

Immunization against the human platelet antigen (HPA)-1 alloantigen is the most common cause of severe fetal and neonatal thrombocytopenia. Fetal therapy has substantial risks and its indications need better definition. Of 24,417 consecutive pregnant women, 618 (2.5%) were HPA-1a negative of whom 385 entered an observational study. All were HLA-DRB3*0101 genotyped and screened for anti–HPA-1a. Their partners and neonates were HPA-1 genotyped and the latter were assessed by cord blood platelet counts and cerebral ultrasound scans. Anti–HPA-1a was detected in 46 of 387 pregnancies (12.0%; 95% CI 8.7%-15.2%). All but one were HLA-DRB3*0101 positive (odds ratio 140; 95% CI 19-1035;P&lt; .00001). One baby died in utero, and of 26 HPA-1a–positive babies born to women with persistent antenatal antibodies, 9 were severely thrombocytopenic (8 with a count &lt;10 × 109/L, 1 with a large porencephalic cyst), 10 were mildly thrombocytopenic, whereas 7 had normal platelet counts. Severe thrombocytopenia was significantly associated with a third trimester anti–HPA-1a titer ≥ 1:32 (P = .004), but was not observed in babies of women with either transient or postnatal-only antibodies. HPA-1a alloimmunization complicates 1 in 350 unselected pregnancies, resulting in severe thrombocytopenia in 1:1,200. HPA-1a and HLA-DRB3*0101 typing combined with anti–HPA-1a titration allows selection of the majority of pregnancies at risk of severe thrombocytopenia.

The Natural History of Fetomaternal Alloimmunization to the Platelet-Specific Antigen HPA-1a (PlA1, Zwa) as Determined by Antenatal Screening

Immunization against the human platelet antigen (HPA)-1 alloantigen is the most common cause of severe fetal and neonatal thrombocytopenia. Fetal therapy has substantial risks and its indications need better definition. Of 24,417 consecutive pregnant women, 618 (2.5%) were HPA-1a negative of whom 385 entered an observational study. All were HLA-DRB3*0101 genotyped and screened for anti–HPA-1a. Their partners and neonates were HPA-1 genotyped and the latter were assessed by cord blood platelet counts and cerebral ultrasound scans. Anti–HPA-1a was detected in 46 of 387 pregnancies (12.0%; 95% CI 8.7%-15.2%). All but one were HLA-DRB3*0101 positive (odds ratio 140; 95% CI 19-1035;P< .00001). One baby died in utero, and of 26 HPA-1a–positive babies born to women with persistent antenatal antibodies, 9 were severely thrombocytopenic (8 with a count <10 × 109/L, 1 with a large porencephalic cyst), 10 were mildly thrombocytopenic, whereas 7 had normal platelet counts. Severe thrombocytopenia was significantly associated with a third trimester anti–HPA-1a titer ≥ 1:32 (P = .004), but was not observed in babies of women with either transient or postnatal-only antibodies. HPA-1a alloimmunization complicates 1 in 350 unselected pregnancies, resulting in severe thrombocytopenia in 1:1,200. HPA-1a and HLA-DRB3*0101 typing combined with anti–HPA-1a titration allows selection of the majority of pregnancies at risk of severe thrombocytopenia.

Frequency of Immune Thrombocytopenia in Newborns: A Prospective Study

AbstractThrombocytopenia is a common condition in distressed newborns, but little is known about thrombocytopenia in an unselected cohort of neonates. In an attempt to address this issue, a multicenter prospective study was conducted in three obstetrical wards of AP-HP in Paris. We found the frequency of neonatal thrombocytopenia (<150 × 109/L) to approximate 0.9% (48 of 5,632 appropriate samples). An immune mechanism was likely to be the cause of thrombocytopenia in 10 of the 33 cases studied, implying an incidence of 0.3% of immune neonatal thrombocytopenia in the general population. The frequency of alloimmune thrombocytopenia was 1.5/1,000 liveborn neonates, and 1/1,000 when considering anti–HPA-1a allo-immunization. Because thrombocytopenia, whatever its cause, was often silent and delayed, it appears that the only way to detect neonatal thrombocytopenia in time to prevent its potential disastrous complications would be to perform a systematic neonatal blood sampling for platelet count. All cases of ascertained thrombocytopenia should then be screened for an immune mechanism to enable early detection of autoimmune diseases in mothers and careful monitoring of subsequent pregnancies and deliveries, leading to appropriate prevention of potential severe deleterious effects in the offspring.

Human Monoclonal Fab Fragments Recovered from a Combinatorial Library Bind Specifically to the Platelet HPA-1a Alloantigen on Glycoprotein IIb—IIIa

Background and objectives: Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient’s serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. Materials and methods: Human antibody Fab fragments that bind to the platelet alloantigen HPA-la on glycoprotein Ilb-IIIa (GPIIbllla) were generated by using a recombinant phage display system. We established an immunoglobulin Gl, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. Results: Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-1a-specific clones reveals at least 3 distinct VL and 3 V(H) gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. Conclusions: These human HPA-laspecific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-1a alloimmunization.