The incidence of neonatal alloimmune neutropenia due to maternal alloimmunization against human neutrophil alloantigen-3 in the Brazilian population

Abstract

Background: Neonatal alloimmune neutropenia (NAN) is a potentially lethal disorder that results of maternal alloimmunization to fetal human neutrophil antigens (HNAs). The alloantibodies more frequently involved in NAN are directed against the HNA-1 and -2 systems; however anti-HNA-3 has also been associated with cases of NAN. Aims: We investigated the presence of maternal anti-HNA-3 antibodies in HNA-3 allele genotypic maternal-fetal incompatibility cases of neonatal neutropenia. Methods: A cross sectional study included samples from 10,000 unselected neonates born in four obstetric units in Sao Paulo City (SP, Brazil). Neonatal neutropenia was defined as neutrophil count1.5 9 109/l in cord blood. Genotyping studies to detect HNA-3a and HNA-3b alleles were performed by PCR-RFLP technique that amplified the sequence for rs2288904. The amplified product was digested with enzyme Taqa1 specific to nucleotide guanine that codifies the HNA-3a antigen resulting in two bands (171 and 100 pb). Individuals HNA-3a present three bands (271 pb, 171 pb and 100 pb) while HNA-3 subjects present only a 271 pb band corresponding to the whole PCR product. The granulocyte agglutination test (GAT) done in duplicate was used for detecting maternal HNA alloantibodies, while the presence of maternal HLA-I/II antibodies was investigated by ELISA (LAT-One Lambda-). The GAT results were confirmed in the Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt University Greifswald, Germany. Results: Neonatal neutropeina was identified in 88 of 10,000 (0.88%) newborn in the Brazilian population. Genotyping studies of the 88 neutropenic newborns and their mothers (83 mothers with three pairs of twins and one triplet) revealed HNA-3 incompatibility in 13/88(14.8%). In all neutropenic cases the mothers were HNA-3a/ a and neonates typed as HNA-3a/b. In total, HNA-3b alloantibodies were detectable in five out 13(38.5%) maternal sera and no antibody was detected in 6/13(46.1%) mothers. The results of the serological investigations were inconclusive in 2/13 (15.4%) samples. Anti-HLA (Class I+II) antibodies were detected by ELISA in two GAT(+) maternal serum. Summary/Conclusions: We have recently reported that HNA-3 genotypic frequencies in Brazilians (HNA-3a/a = 66.2%; HNA-3a30.2% and HNA-33.6%) are similar to that reported for Europeans and North Americans corresponding to frequencies of 0.80 for the allele HNA-3a and 0.20 for the allele HNA-3b (Haematologica 2011;96 (s2):341-2, abstract0821). The present data indicate that fetal-maternal HNA-3 genotypic incompatibility occurred in 14.8%(13/88) of neutropenic neonates out of a sample population of 10,000 unselected newborns. Nevertheless anti-HNA circulat- ing alloantibodies were confirmed by GAT in five out of 13 mothers. In these cases, just one sample was positive in both laboratories and two samples were positive only in the German laboratory. The discrepancy between the GAT results probably happened because the difference of the HNA-3b expression in the neutrophil donors. Furthermore, excluding the anti-HLA alloantibodies, we could identify three maternal sera with anti-HNA-3b alloantibody in cases of neonatal neutropenia in the Brazilian population.