Severe neonatal thrombocytopenia due to anti-HPA-4b alloantibodies: Third report described in a Caucasian mother.

BACKGROUND: Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) results from maternal platelet alloimmunization against paternal antigens inherited by the fetus, most often due to the Human Platelet Antigen (HPA)-1 system in Caucasians. We investigated in 2023, a 30-year-old Caucasian woman Gravida 2 Para 1 who gave birth at 35 weeks of gestation to a male (body weight 2210 g) without signs of bleeding. A severe thrombocytopenia (platelet count at 3 G/L) was discovered incidentally a few hours after delivery in the context of the management of a respiratory distress. The newborn recovered after one platelet concentrate transfusion and normalized his platelet count at Day 5. STUDY DESIGN AND METHODS: FNAIT investigation was performed according to guideline recommendations. Platelet genotyping was carried out by multiplex PCR. Maternal serological investigation included Monoclonal Antibody-specific Immobilization of Platelet Antigens method (MAIPA) and Luminex technology. RESULTS: Parental and newborn genotyping pointed out an HPA-4 incompatibility between the mother and the newborn and the father. Serological investigation revealed an anti-HPA-4b alloantibody confirming the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: We described the third case of anti-HPA-4b alloantibody discovered in a Caucasian mother. This case strengthens the need for reference laboratory to genotype a panel of HPA alleles reflecting local genetic population diversity and for crossmatch of maternal serum with fresh paternal platelets in clinical suspected cases of neonatal alloimmune thrombocytopenia.

A new efficient tool for non-invasive diagnosis of fetomaternal platelet antigen incompatibility.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the consequence of platelet destruction by maternal alloantibodies against fetal human platelet antigens (HPA). This may result in intracranial haemorrhages (ICH) or even fetal death. Currently, fetal HPA genotyping is performed using invasive procedures. Here, we carried out a proof-of-concept study for non-invasive prenatal diagnosis of fetal platelet genotyping in four HPA systems (HPA-1, -3, -5 and-15) by droplet digital polymerase chain reaction (ddPCR) using cell-free DNA extracts from the plasma of 47 pregnant women with suspected, or history of, FNAIT. Results showed that 74% (35/47) of pregnant women presented incompatibility in at least one HPA system, and 38% (18/47) of cases presented HPA-1 incompatibility, including nine women with multiple incompatibilities. ICH occurred in one case of profound fetal thrombocytopenia with HPA-15 incompatibility, confirming the need for non-invasive prenatal genotyping in systems other than HPA-1. Fetal HPA genotypes predicted by ddPCR were confirmed in all FNAIT cases after amniocentesis or delivery. Fetal HPA genotyping on maternal plasma based on ddPCR is a fast, safe and reliable non-invasive method. This technique will be useful for the early identification of pregnancies at high risk of FNAIT requiring antenatal management to minimize the risk of fetal/neonatal haemorrhage.

The αIIb p.Leu841Met (Cab3(a+) ) polymorphism results in a new human platelet alloantigen involved in neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15). STUDY DESIGN AND METHODS: A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbß3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbß3 expression and functions. RESULTS: Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbß3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbß3 expression and functions. CONCLUSION: The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed.

Safe fetal platelet genotyping: new developments.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is due to maternal alloimmunization against fetal platelet (PLT) antigens. Antenatal management strategies have been developed to avoid complications such as intracranial hemorrhage. The aim of this study was to set up two reliable, noninvasive fetal genotyping assays to determine the fetal risk in pregnancies in which the father is heterozygous for the offending antigen. This study focused on human PLT antigen (HPA)-1, the most frequently implicated antigen in FNAIT in Caucasians. STUDY DESIGN AND METHODS: Two assays based on cell-free fetal DNA extracted from maternal blood samples and on real-time polymerase chain reaction (QPCR) were developed: an allele-specific QPCR specifically targeting the polymorphic sequence in HPA-1 and the study of the variation in the high-resolution melting curve of amplicons containing the polymorphic region. RESULTS: All results from the 49 samples obtained from 29 pregnant women were consistent with expectations. Six women were compatible with their fetuses (three HPA-1aa women and three HPA-1bb women), 41 HPA-1bb women were incompatible with their fetuses, as were two HPA-1aa women. CONCLUSION: Two fetal PLT genotyping assays on maternal blood samples proved to be reliable as of 15 weeks of gestation, thereby avoiding invasive techniques such as amniocentesis.

HPA-13bw neonatal alloimmune thrombocytopenia and low frequency alloantigens: case report and review of the literature.

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) linked to rare or private antigens is not a rare event. STUDY DESIGN AND METHODS: Such a case discovered during the follow-up of a second child with jaundice with mild thrombocytopenia is reported here. Platelet (PLT) genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. Glycoprotein Ia-specific amplification and sequencing were performed for the polymorphism 807 (exon 7). RESULTS: The mother was found to be HPA-13aaw, and the father HPA-13abw. A maternal alloantibody directed against HPA-13bw has been characterized, leading to the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: This report provides further evidence that NAIT associated with low-frequency antigens is not restricted to single families. Therefore, laboratory investigation of a suspected case should be carried out in a specialist laboratory well experienced in optimal testing to propose appropriate management for the index case and subsequent pregnancies.

New mutation in the platelet beta3-integrin gene: implication for the diagnosis of fetomaternal alloimmunization.

BACKGROUND: Fetal ventriculomegaly is a relatively common finding and fetomaternal alloimmune thrombocytopenia may be one of the causes. STUDY DESIGN AND METHODS: Such a case discovered at 21 weeks of gestation leading to platelet (PLT) immunologic testing is reported here. PLT genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers (SSPs) method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. RESULTS: The mother was found to be HPA-1b homozygous and the father HPA-1a homozygous with PCR-SSP, but the mother was found to be HPA-1 heterozygous by phenotyping. This result was confirmed by PCR-RFLP. Sequencing of the glycoprotein IIIa exon 3 revealed a heterozygous mutation 262T > C, which does not induce an amino acid change. It is localized in the sequence of the antisense primer of the HPA-1 PCR-SSP, inducing the sole amplification of the DNA copy bearing the HPA-1b allele. CONCLUSION: Even if such mutations are a rare event, PLT phenotyping is still of interest to avoid rare false PLT typing assignation, the unknown polymorphism being only discovered by such a combination of techniques.