A Serologic and Molecular Study of the HNA-3 System-Associated Neonatal Neutropenia

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. Therefore, in this study we investigated the presence of maternal anti-HNA-3 antibodies in HNA-3 allele genotypic maternal-fetal incompatibility cases of neonatal neutropenia.

A cross sectional study included samples from 10,000 unselected neonates born in 4 obstetric units in São Paulo City (SP, Brazil). Neonatal neutropenia was defined as neutrophil count £1.5×10⁹/L in cord blood. Genotyping studies to detect HNA-3a and HNA-3b alleles were performed in 88 neutropenic newborns (88/10,000 = 0.88%) and their mothers (83 mothers with 3 pairs of twins and 1 triplet) by PCR-RFLP technique that amplified the sequence for rs2288904. The amplified product was digested with enzyme Taq^α1 specific to nucleotide guanine that codifies the HNA-3a antigen resulting in two bands (171 and 100pb). Individuals HNA-3a/b present 3 bands (271pb, 171pb and 100pb) while HNA-3b/b subjects present only a 271pb 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^®).

We found that 13/88 (14.8%) neutropenic neonates showed HNA-3 genotypic incompatibility with their mothers. In all neutropenic cases the mothers were HNA-3a/a and neonates typed as HNA-3a/b. Using the GAT, anti-HNA alloantibodies were found in 2/13 (15.4%) maternal sera, no antibody was detected in 8/13 (61.5%) mothers, while antibody GAT results were inconclusive in 3/13 (23.1%) samples. Anti-HLA (Class I+II) antibodies were detected by ELISA in one GAT(+) maternal serum. Molecular studies with samples from neonate and mother whose serum contain anti-HNA but not anti-HLA antibodies showed genotypic maternal-fetal incompatibility for HNA-3 and HNA-1c. Using a previously typed donor neutrophil panel we observed that the maternal serum was positive by GAT even against donors negative for the HNA-1c antigen suggesting that the maternal antibody was specific for the HNA-3b alloantigen. Samples from all others 12 neutropenic neonates showed maternal-fetal compatibility for the HNA-1 system.

We have recently reported that HNA-3 genotypic frequencies in Brazilians (HNA-3a/a=66.2%; HNA-3a/b=30.2% and HNA-3b/b=3.6%) 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, abstract 0821). The present data indicated 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 circulating alloantibodies were confirmed by GAT in only 2 out of 13 mothers. Using the GAT we could identify an anti-HNA-3b alloantibody in the maternal serum that did not contain anti-HLA alloantibody.

No relevant conflicts of interest to declare.