Definitive Human Neutrophil Antigen allele assignment using Next Generation Sequencing
HNA genotyping is routinely used as part of neonatal alloimmune neutropenia (NAIN), autoimmune neutropenia and transfusion-related acute lung injury (TRALI) investigations. HNA has been shown to be clinically relevant in haematopoietic stem cell transplantation (HSCT) engraftment and organ transplant rejection. HNA genotyping is currently performed in the laboratory by sanger sequence-based typing (SBT). Throughput of HNA genotyping is restricted by the number of electrophoresis capillaries and the runtime alongside competition with use for other laboratory processes (other SBT and fragment analysis). Other limitations of HNA genotyping by SBT include; a) typing ambiguity due to co-amplification of both sense and anti-sense sequence (cis/trans) in CD16 (HNA-1) assignment and b) complication in CD177 (HNA-2) assignment due to alternative transcription patterns and co-amplification of pseudo genes requiring assignment by phenotyping. An NGS method for HNA genotyping could permit identification of all known polymorphisms. NGS chemistry lends itself to creation of a rapid, robust, high-throughput method with the ability to detect novel polymorphisms. It could offer the potential for routine screening of transplant recipients and donors (e.g. renal transplant waiting list) as well as creation of a HNA typing reference service. HNA genotyping by NGS method could also be aligned with the newly introduced workflow for Human Leukocyte Antigen (HLA) typing, by NGS, allowing better use of available resources.