Neonatal Alloimmune Thrombocytopenia Research Articles

Generation of human antibodies targeting human platelet antigen (HPA)-1a.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

Does anti-HPA-1a affect birthweight in fetal and neonatal alloimmune thrombocytopenia?

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) ensues from parental incompatibility for platelet alloantigens with maternal sensitization. HPA-1a/1b incompatibility is the most common cause of FNAIT in Caucasians. Placental villitis and lower birthweight in FNAIT suggest anti-HPA-1a may have effects beyond inducing thrombocytopenia. Does FNAIT secondary to anti-HPA-1a result in smaller newborns and, the corollary, does antenatal management of FNAIT increase birthweight? Birthweights of 270 FNAIT-affected newborns from a randomized clinical trial and a NAITbabies.org survey (135 paired siblings) were compared with those of published controls and treated to untreated FNAIT-affected siblings. Birthweights were converted to percentiles to account for gestational age, sex, and role of birth order in birth weight. Body weights of FNAIT-affected and -unaffected pups in a mouse FNAIT model were analyzed. Untreated siblings in both the clinical trial and NAITbabies.org cohorts were not small, compared with normal controls. However, treated siblings in both cohorts had significantly higher birthweight percentiles compared with their previous untreated affected sibling. After accounting for gestational age, sex, and birth order, increased birthweight percentile in treated compared with the untreated siblings remained significant in both cohorts. FNAIT-affected neonatal mice had lower bodyweights than FNAIT-unaffected pups. Untreated FNAIT-affected newborns were not small; however, treatment of FNAIT-affected pregnancies increased newborn birthweights despite corrections to account for other factors that might have influenced the results. High dose IVIG is believed to "block" FcRn and lower maternal anti-HPA-1a levels, and thus increase birthweights by reducing levels of maternal anti-HPA-1a and reducing placental villitis.

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.

SMC4 Loss of Function Is a New Cause of Inherited Bone Marrow Failure

Inherited bone marrow failure syndromes (IBMFS) are a rare, heterogenous group of syndromes that are characterized by cytopenias, bone marrow hypocellularity, and in many cases an increased risk of transformation to myeloid malignancy. Presentation with severe pancytopenias at birth is reported but occurs very rarely, and more often neonatal pancytopenia is attributed to congenital and acquired infections or other acquired causes. We present the case of a preterm neonate with pancytopenia from birth and bone marrow failure of no known infectious, other acquired, or genetic etiology. Antenatal findings were notable for non-immune hydrops fetalis at 30 weeks of gestation prompting delivery. The initial laboratory evaluation revealed a normocytic, reticulocytopenic, severe anemia; leukopenia with an absolute neutrophil count of 500; and severe thrombocytopenia. Viral testing, including parvovirus, rubella, herpes simplex virus, cytomegalovirus, adenovirus, and enterovirus, was negative. Additional testing, including a chromosomal microarray, mitochondrial genome sequencing, chromosome stress testing, neonatal alloimmune thrombocytopenia panel, and telomere length analysis, was non-diagnostic. Bone marrow biopsy showed a remarkably hypoplastic marrow without evidence of dysplasia or cytogenetic abnormalities. The proband underwent matched unrelated donor hematopoietic stem cell transplant, and died approximately one month later from post-transplantation complications including hepatic veno-occlusive disease, thrombotic microangiopathy, CNS and pulmonary hemorrhages, pseudomonal sepsis, and multi-organ dysfunction. Autopsy was significant for microscopic pancreatic atrophy with lipomatous hypertrophy and osteopenia. The proband and parents underwent clinical exome testing which was non-diagnostic and then consented to research reanalysis of the exome data. Research reanalysis identified the infant was compound heterozygous for predicted deleterious variants in the Structural Maintenance of Chromosomes 4 gene (SMC4), inherited in trans from each unaffected parent (c.2478_2479ins10 splice site variant predicted to alter splicing and c.T591G; p.Y197X). Germline variants in SMC4 have not been reported as a cause of human disease; however, somatic variants are observed in several cancers including in acute myeloid leukemia (AML). SMC4 is a highly conserved protein that forms a heterodimer with Structural Maintenance of Chromosomes 2 (SMC2) in the condensin II protein complex. This complex is important for chromosome condensation, cell division, and DNA repair. Within hematopoietic tissue, SMC4 expression is higher in hematopoietic stem and progenitor cells when compared with mature lineage cells. Given the predicted loss of function from both SMC4 variants, we performed RNAseq on proband and control fibroblasts. This showed a roughly 10-fold decrease in total SMC4 transcript levels (Figure 1A). A significant proportion of the transcript present was comprised of an aberrantly spliced transcript characterized by exon 15 skipping. We therefore hypothesized that loss of SMC4 expression leads to a cell intrinsic defect in hematopoiesis. To test this hypothesis, we used CRISPR-Cas9 gene editing to generate knockout of SMC4 in human cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs) before culturing cells in an all-colony forming unit (CFU) assay. Using two independent small guide RNAs, we were able to achieve approximately 80% knock-down. After 14 days in culture, we observed a striking decrease in CFUs when compared with controls, though the composition of colony types was relatively unchanged (Figure 1B). Using next-generation sequencing, we observed a significant decrease in remaining SMC4 genome edits at the end of the culture period, suggesting a relative disadvantage of SMC4-edited cells for survival and/or proliferation. In summary, we describe a preterm infant with congenital bone marrow failure without an identifiable cause and link this phenotype to biallelic loss-of-function variants in SMC4. This is the first study identifying SMC4 as a novel gene that causes inherited bone marrow failure, and further experiments are underway to validate this finding.

Natural history of human platelet antigen 1a-alloimmunised pregnancies: a prospective observational cohort study

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare disease that untreated can lead to intracranial haemorrhage or death. The natural history of FNAIT is still unclear; therefore, the benefits of screening cannot be estimated and no routine screening is yet in place. We aimed to assess the incidence of clinically detectable FNAIT among pregnancies in human platelet antigen-1a (HPA-1a)-immunised women. We did a prospective observational cohort study of pregnant women negative for rhesus D (RhD) and rhesus c (Rhc) antigens, without age limits, who underwent routine antenatal screening for red cell antibodies at 27 weeks' gestation and were typed for HPA-1a between March 1, 2017, and May 1, 2020. HPA-1a-negative women were tested for HPA alloantibodies. Health-care professionals were masked to all test results. The main outcome was the proportion of neonates with severe, clinically detectable FNAIT, defined as having an intracranial bleed, organ bleed, or bleeding-related death observed during pregnancy or within the first week of life. Cases of clinically detectable FNAIT not categorised as severe were categorised as mild. This study is registered with ClinicalTrials.gov (NCT04067375). Of 153 106 women typed for HPA-1a, 3722 (2·4%) were negative for HPA-1a. 913 HPA-1a-negative women gave informed consent, underwent HPA-1a antibody screening, and were included in the study. Anti-HPA-1a antibodies were detected in 85 HPA-1a-negative participants, among whom three with HPA-1a-negative fetuses and one with a previous child with FNAIT were excluded. As controls, 820 HPA-1a-negative, non-immunised pregnancies and 2704 randomly selected pregnancies of women negative for RhD and Rhc who were typed HPA-1a positive were included. Of 81 fetuses included, one (1·2%) was diagnosed with severe HPA-1a-mediated intracranial haemorrhage and three (3·7%) had mild FNAIT. Gravidity and parity did not seem to be risk factors for HPA-1a immunisation. 73 (90·1%) of 81 HPA-1a-immunised women were positive for HLA-DRB3*01:01. Our data suggest that, without intervention, the incidence of major clinically detectable bleeding in FNAIT is estimated as 11 (95% CI 0-32) per 10 000 HPA-1a-negative pregnancies. These findings imply that severe bleeding is a rare event that potentially could be prevented by a screening programme. Landsteiner Foundation for Blood Transfusion Research and Sanquin.

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

OC 02.1 Dose-Dependent Elimination of HPA-1a Platelets by Subcutaneous RLYB212, a Monoclonal Antibody to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia

OC 02.1 Dose-Dependent Elimination of HPA-1a Platelets by Subcutaneous RLYB212, a Monoclonal Antibody to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia

O-linked sialic acid residues on platelet membrane glycoprotein IIb mask the human HPA-9b alloepitope

AbstractSialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins (GP). The polymorphisms of human platelet alloantigens (HPA)-3 and HPA-9, located near the C-terminus of the extracellular domain of platelet membrane GPIIb, are adjacent to sialyl-core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase and those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2 dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these 2 O-glycan chains contribute to the presentation of the HPA-9b epitope—perhaps by stabilizing the conformation of the GP in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues but retain the glycan chains to which they are attached.

Fetal and neonatal alloimmune thrombocytopenia in siblings

Fetal and neonatal alloimmune thrombocytopenia in siblings

Pregnant Women at Low Risk of Having a Child with Fetal and Neonatal Alloimmune Thrombocytopenia Do Not Require Treatment with Intravenous Immunoglobulin.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare condition in which maternal alloantibodies to fetal platelets cause fetal thrombocytopenia that may lead to intracranial hemorrhage (ICH). Off-label intravenous immunoglobulin (IVIg) has for 30 years been the standard of care for pregnant women who previously have had a child with FNAIT. The efficacy of this treatment has never been tested in a placebo-controlled clinical trial. Although IVIg treatment may improve the neonatal outcome in women who previously have had a child with FNAIT-associated ICH, the question is whether IVIg is necessary for all immunized pregnant women at risk of having a child with FNAIT. The results from some recent publications suggest that antenatal IVIg treatment is not necessary for women who are (1) HPA-1a-immunized and HLA-DRB3*01:01-negative, (2) HPA-1a-immunized with a previous child with FNAIT but without ICH or (3) HPA-5b-immunized. If IVIg is not used for these categories of pregnant women, the amount of IVIg used in pregnant women with platelet antibodies would be reduced to less than ¼ of today's use. This is important because IVIg is a scarce resource, and the collection of plasma for the treatment of one pregnant woman is not only extremely expensive but also requires tremendous donor efforts.

Diagnosis of Neonatal Alloimmune Thrombocytopenia: Determination of the Specificity of Antiplatelet Alloantibodies in the Maternal Blood Plasma Using a Molecular and Genetic Method

   Background: Thrombocytopenia occurs in 1-5 % of newborns (platelet count < 150 × 109/L). Low platelet count of 50 × 109/L leads to the hemorrhagic syndrome, with one of its causes being neonatal alloimmune thrombocytopenia resulting from incompatibility between the mother and the fetus with human platelet antigens (HPA) inherited from the father and absent in the mother, which leads to the formation of maternal antibodies. Anti-HPA-1a, anti-HPA-5b, anti-HPA-3a, and anti-HPA-3b antibodies are clinically significant as they destroy fetal/neonatal platelets causing severe complications (intracranial hemorrhage in 20 % of cases and prenatal or postnatal death in 10 % of cases). Adequate diagnosis is a key to a successful treatment approach, which largely depends on the thrombocytopenia cause.   Objective: To determine the alloimmune nature of neonatal thrombocytopenia and the specificity of antibodies in the mother’s blood.   Materials and methods: We studied blood samples of parents (21 pairs) of newborns with thrombocytopenia in Saint Petersburg, Russian Federation. We used flow cytometry to determine alloantibodies in the maternal plasma after incubation with paternal platelets and staining with Goat F(ab’)2 Anti-Human IgG-FITC and CD41-PE monoclonal antibodies. Allosensitization index was calculated as the percentage of IgG-positive cells to the number of cells fixing anti-CD41 antibodies. At the value of ≥ 15 %, antiplatelet alloantibodies were considered present in a sample. We used a molecular detection system of the FluoVista analyzer (Inno-Train, Germany) for genetic testing with allele-specific primers. Alleles of genes encoding the expression of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6, HPA-9, and HPA-15 antigens were determined by a real-time polymerase chain reaction using a set of HPA-FluoGene reagents (Inno-Train, Germany). Genomic DNA was isolated using the DNA-sorb-B set (AmpliSens, Russian Federation).   Results: We found that 8 of 21 (38 %) mothers had antibodies against paternal platelets. During genotyping in mother/father pairs, incompatible combinations of platelet antigens were revealed: HPA-1b/HPA-1a in 9 pairs (HPA-1a antigen absent on the maternal platelets and present on the paternal platelets), of which 5 mothers (55%) had antibodies with a probable specificity to anti-HPA-1a; HPA-1a/HPA-1b incompatibility in 4 pairs, with 2 (50 %) mothers having antibodies with an anti-HPA-1b specificity. HPA-3a/HPA-3b incompatibility was observed in 4 pairs, with antibodies (probably anti-HPA3b) in 1 mother (25%). HPA-2a/HPA-2b, HPA-5a/HPA-5b, HPA-15a/HPA-15b, HPA-15b/HPA-15a incompatibilities were detected (1 case each in 21 pairs), with no antibodies found in mothers. The probable specificity of the antibodies was distributed as follows: 62 % for anti-HPA-1a, 25% for anti-HPA-1b, and 13 % for anti-HPA-3a.   Conclusions: We confirmed the immune nature of neonatal thrombocytopenia and determined the probable specificity of maternal alloantibodies in 8 of 21 cases.

Fetal and neonatal alloimmune thrombocytopenia: No evidence of systemic inflammation as a modulator of disease severity. Couldplacental inflammation be key?

In fetal/neonatal alloimmune thrombocytopenia (FNAIT), maternal alloantibodies against paternal human platelet antigens (HPA) cross the placenta and lead to platelet destruction. The extent of thrombocytopenia varies among neonates, and inflammation may constitute an important trigger. A set of stable inflammatory markers was measured in serum samples from neonates with low platelet counts, of which n = 50 were diagnosed with FNAIT due to anti-HPA-1a antibodies and n = 50 were thrombocytopenic without detectable maternal HPA antibodies. Concentrations of C-reactive protein, soluble CD14, procalcitonin, and sFlt-1 did not differ between the two cohorts. There was no correlation between C-reactive protein or soluble CD14 and the platelet count, but a negative correlation between procalcitonin concentrations and the neonatal platelet count in both cohorts. sFlt-1 concentration and the platelet count were correlated in FNAIT cases exclusively. None of the inflammatory markers was statistically different between cases with and without intracranial haemorrhage. We were unable to identify systemic inflammation as a relevant factor for thrombocytopenia in FNAIT. The antiangiogenic enzyme sFlt-1, released by the placenta, did correlate with the platelet count in FNAIT cases. Our findings may give rise to the hypothesis that placental inflammation rather than systemic inflammation modulates disease severity in FNAIT.

Genetic landscape of human platelet antigen variants in the Indian population analysed from 1029 whole genomes

AbstractGenetic variants in human platelet antigens (HPAs) considered allo‐ or auto antigens are associated with various disorders, including neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and post‐transfusion purpura. Although global differences in genotype frequencies were observed, the distributions of HPA variants in the Indian population are largely unknown. This study aims to explore the landscape of HPA variants in India to provide a basis for risk assessment and management of related complications. Population‐specific frequencies of genetic variants associated with the 35 classes of HPAs (HPA‐1 to HPA‐35) were estimated by systematically analysing genomic variations of 1029 healthy Indian individuals as well as from global population genome datasets. Allele frequencies of the most clinically relevant HPA systems in the Indian population were found as follows, HPA‐1a – 0.884, HPA‐1b – 0.117, HPA‐2a – 0.941, HPA‐2b – 0.059, HPA‐3a – 0.653, HPA‐3b – 0.347, HPA‐4a – 0.999, HPA‐4b – 0.0010, HPA‐5a – 0.923, HPA‐5b – 0.077, HPA‐6a – 0.998, HPA‐6b – 0.002, HPA‐15a – 0.582 and HPA‐15b – 0.418. This study provides the first comprehensive analysis of HPA allele and genotype frequencies using large scale representative whole genome sequencing data of the Indian population.

HLA antibodies in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by antibodies against human platelet antigens (HPA). However, in many cases that meet clinical criteria for the condition, maternal sera do not have HPA antibodies. In studies examining whether human leukocyte antigen (HLA) antibodies cause FNAIT, the results are limited and inconclusive. This study sought to examine whether clinically suspected FNAIT cases with absent maternal HPA antibodies had different HLA antibody strength and specificity compared to controls. A retrospective case-control study assessed class I HLA antibody strength and specificity in cases submitted for testing to Versiti, Wisconsin. There were 813 cases that met initial screening criteria, but written consent could only be obtained for 50. After review of medical records and expert panel review, 31 cases with clinical criteria of FNAIT and maternal HLA but not HPA antibodies were included. Each case was matched for maternal age, gestational age at delivery, parity, and race/ethnicity to two controls from unaffected pregnancies that had maternal serum HLA antibodies. FNAIT cases were found to have both significantly higher HLA antibody strength, measured by mean fluorescence index (MFI), and broader HLA antibody specificity at antigen epitope level, compared to matched controls (p < .001). p-values remained significant after controlling for parity and gestational age at delivery. Additional studies are needed to further examine whether the strong HLA antibodies identified in HPA-antibody-negative cases directly cause neonatal thrombocytopenia and whether prenatal treatment may be warranted in select cases to prevent recurrence.

Neonatal Thrombocytopenia: Differing Characteristics of NAIT Versus Non-NAIT.

While neonatal alloimmune thrombocytopenia (NAIT) is the most common cause of severe neonatal thrombocytopenia good clinical predictors are lacking. We analyzed cases of neonatal thrombocytopenia in Schneider Children's Medical Center of Israel to pinpoint qualifiers of NAIT (NAIT+) in comparison to non-NAIT (NAIT-) thrombocytopenia. Patient and maternal data were retrospectively collected on all thrombocytopenic newborns undergoing a workup for NAIT in our tertiary center between 2001 and 2016. Among 26 thrombocytopenic neonates, the mean nadir in NAIT+ patients (25×10 9 /L) was significantly lower than NAIT- patients (64×10 9 /L) ( P <0.001). 61.5% of NAIT+ infants required treatment compared with 23% of non-NAIT ( P =0.015). NAIT+ patients also required more therapeutic modalities than infants with NAIT- thrombocytopenia. Human platelet antigen (HPA)-1a and HPA-5b alloantibodies most frequently caused NAIT. In summary, thrombocytopenia in NAIT+ was significantly more severe compared with NAIT- and more likely to require treatment. In addition, despite the varied ethnic population in Israel, the HPA alloantibodies found in our population were most similar to those common in Western countries. In the absence of rigorous prenatal screening options, we suggest platelet counts below 40 to 50×10 9 /L in a healthy newborn be considered most suggestive for NAIT and warrant urgent NAIT-specific analysis.

Children Newly Diagnosed with Fetal and Neonatal Alloimmune Thrombocytopenia: Neurodevelopmental Outcome at School Age

To evaluate the neurodevelopmental outcome at school age in children newly diagnosed with fetal neonatal alloimmune thrombocytopenia (FNAIT). This observational cohort study included children diagnosed with FNAIT between 2002 and 2014. Children were invited for cognitive and neurological testing. Behavioral questionnaires and school performance results were obtained. A composite outcome of neurodevelopmental impairment (NDI) was used, defined and subdivided into mild-to- moderate and severe NDI. Primary outcome was severe NDI, defined as IQ < 70, cerebral palsy with Gross Motor Functioning Classification Scale (GMFCS) level ≥ III, or severe visual/hearing impairment. Mild-to-moderate NDI was defined as IQ 70-85, minor neurological dysfunction or cerebral palsy GMFCS level ≤ II or mild visual/hearing impairment. In total, 44 children were included at a median age of 12 years (range 6-17 years). Neuroimaging at diagnosis was available in 82% (36/44) of children. High-grade intracranial hemorrhage (ICH) was detected in 14% (5/36). Severe NDI was detected in 7% (3/44); two children had high-grade ICH and one low-grade ICH and perinatal asphyxia. Mild-to- moderate NDI was detected in 25% (11/44); one child had high-grade ICH and eight were without ICH, yet for two children neuroimaging was not performed. Adverse outcome (perinatal death or NDI) was 39% (19/49). Four children (9%) attended special needs education, three of whom had severe NDI and one with mild-to-moderate NDI. Total behavioral problems within the clinical range were reported in 12%, which is comparable with 10% in the general Dutch population. Children who are newly diagnosed with FNAIT are at increased risk for long- term neurodevelopmental problems, even those without ICH.

What to Know about Neonatal Alloimmune Thrombocytopenia (NAIT) ?

What to Know about Neonatal Alloimmune Thrombocytopenia (NAIT) ?

Pregnancy and Child Outcomes Following Fetal Intracranial Hemorrhage

BackgroundThe prenatal and early postnatal outcomes of fetal intracranial hemorrhage (ICH) prenatally diagnosed by fetal magnetic resonance imaging (MRI) have not been well described. MethodsA retrospective study of cases with fetal ICH diagnosed by fetal MRI at Children's National Hospital, Washington, DC, from 2012 to 2020 was conducted. Maternal characteristics, prenatal imaging, pregnancy outcome, and child developmental outcomes were recorded. Abnormal outcomes were categorized as mild for required physical/occupational therapy without other delays, moderate for intermediate multidomain developmental delays, and severe if nonambulatory, nonverbal, or intellectual disability. ResultsFifty-seven cases with fetal ICH were included. The mean (S.D.) maternal age was 31.1 (6.9) years, gestational age at fetal evaluation was 28.1 (5.3) weeks, and gestational age at birth was 38.2 (1.3) weeks. Pregnancy outcomes were 75% (n = 43) live birth, 14% (n = 8) termination of pregnancy, and 11% (n = 6) intrauterine demise (IUD). Live births decreased from 90% to 33% and IUD increased 10% to 22% when comparing unilateral intraventricular hemorrhage with more extensive hemorrhages. Among the 37 live-born infants with clinical follow-up to age 1.8 (1.6) years, neurodevelopmental outcome was normal in 57%, mildly abnormal in 24%, moderately abnormal in 14%, and severely abnormal in 5%. In five cases, an etiology was identified: two had placental pathologies, two had genetic findings (fetal neonatal alloimmune thrombocytopenia and COL4A1 mutation), and one had congenital cytomegalovirus infection. ConclusionPerinatal and early child outcomes following fetal ICH have a wide spectrum of outcomes. Fetal MRI description of ICH location may aid in pregnancy and postnatal outcome prediction.

In vitro analysis of anti-HPA-1a dependent platelet phagocytosis and its inhibition using a new whole blood phagocytosis assay (WHOPPA).

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14+CD16-), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin-semaphoring-integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future.

Neonatal alloimmune thrombocytopenia associated with fetomaternal hemorrhage.

Neonatal alloimmune thrombocytopenia associated with fetomaternal hemorrhage.