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.

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.

New K103 ß3 allele identified in a context of severe neonatal thrombocytopenia.

BACKGROUND: A new ß3 allele was identified in a severe case of neonatal alloimmune thrombocytopenia (<7 × 10(9) /L). STUDY DESIGN AND METHODS: Diagnosis was done by use of monoclonal antibody-specific immobilization of platelet (PLT) antigen for serologic analyses and polymerase chain reaction (PCR)-sequence-specific primers (SSP) and PCR-restriction fragment length polymorphism (RFLP) for genotyping. Direct sequencing of PCR product was done and mutant αIIbß3 expressed in HEK-293 cells. RESULTS: Serologic analysis revealed in the maternal serum an anti-human PLT alloantigen (HPA)-1a alloantibody associated to an anti-α2ß1. Anti-HPA-1a alloimmunization diagnosis was confirmed by genotyping showing maternofetal incompatibility. However, investigation of rare HPA polymorphisms revealed discrepant HPA-16b assignation between PCR-RFLP and PCR-SSP. Sequencing revealed a new c.385C>A mutation in the ß3 coding sequence resulting in a false assignation of the HPA-16b allele by PCR-RFLP. This mutation leads to a Q103K substitution in mature ß3. The K103-ß3 form of the complex was expressed in HEK-293 cells but did not react with the maternal serum. CONCLUSION: We have characterized a new rare allele (frequency < 1%) of ß3 that yields false HPA-16b genotyping in PCR-RFLP. This new case of false typing assignation emphasizes the necessity to use two genotyping techniques in diagnosis. This particularly applies for rare HPA polymorphisms when PLT phenotyping cannot be used.

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.

Fetal-neonatal alloimmune thrombocytopenia and unexpected Glanzmann thrombasthenia carrier: report of two cases.

BACKGROUND: Discrepancy between phenotyping and genotyping during the investigation of maternofetal alloimmunization leads to the identification of defects on genes encoding membrane glycoproteins (GPs) IIb and IIIa. STUDY DESIGN AND METHODS: Human platelet (PLT) alloantigen (HPA)-1 and -3 PLT phenotypes and genotypes were performed by use of, respectively, the monoclonal antibody-specific immobilization of PLT antigens and the polymerase chain reaction (PCR)-sequence-specific priming techniques for up to 2400 families with thrombocytopenic newborn. When a discrepancy was detected, genomic DNA from the mother was amplified for coding sequences of either the GPIIb or GPIIIa genes. The genetic abnormality responsible for the discrepancy was determined by direct sequencing of the PCR products. RESULTS: Two cases of discrepancy were identified among 2400 families tested. In the first case, Mother L, serologically assigned as HPA-1b-homozygous, was genotyped HPA-1a/1b. In the second case, Mother S, serogically defined HPA-3b-homozygous, was genotyped HPA-3a/3b. DNA sequence analysis revealed for Mother L a T(1447)-->C a point mutation within exon 10 of the GPIIIa gene and for Mother S a C(480)-->G point mutation within exon 4 of the GPIIb gene. These mutations reported in Glanzmann thrombasthenia (GT) patients account for nonexpression of the implicated allele on the PLT surface. Thus, the mothers are GT carriers. Alloantibodies, hallmarks of immunization, were not detected in the maternal serum samples. CONCLUSION: Although this situation is rarely encountered, it is important to combine phenotyping and genotyping to avoid false PLT typing assignation and erroneous diagnosis when alloimmunization might occur.

Anti-HPA-9bw (Maxa) fetomaternal alloimmunization, a clinically severe neonatal thrombocytopenia: difficulties in diagnosis and therapy and report on eight families.

BACKGROUND: Fetal or neonatal alloimmune thrombocytopenia (FMAIT) results from a maternal alloimmunization against fetal platelet (PLT) antigens. In Caucasian persons, HPA-1a is the most frequently implicated antigen. During the past few years, FMAIT has been reported associated with rare or private antigens. STUDY DESIGN AND METHODS: Since the first documented case of FMAIT due to anti-HPA-9bw (Max(a)), no additional cases have been reported. Here a retrospective analysis is presented of the cases referred to our laboratories in recent years. The diagnosis was performed by genotyping and identification of the maternal alloantibody by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) technique. RESULTS: Parental genotyping showed HPA-9bw (Max(a)) mismatch as the sole antigenic incompatibility in seven of eight families. Because the father was found to be HPA-9bw (Max(a)) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. The maternal alloantibody was identified in the MAIPA technique. These data strongly suggest, however, that recognition of the HPA-9bw (Max(a)) epitope is not uniform. The neonatal thrombocytopenia was severe in most cases with bleeding. The outcome was good in all the cases but one. CONCLUSION: This analysis confirms that anti-HPA-9bw (Max(a)) FMAIT is not uncommon and was found to be approximately 2 percent of our confirmed FMAIT cases. It is a clinically severe syndrome that requires prompt diagnosis, albeit difficult, and maternal PLT transfusion therapy. Laboratory investigation of a suspected FMAIT case should be carried out in a specialist laboratory well-experienced in optimal testing. Appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding.