Abstract

Clinical Application of Next Gen Sequencing in Complex Blood Group Investigations

Robert Flower, National Leader Transfusion Safety Research, Australian Red Cross Blood Service

A protocol for analysis of all variants in 33/36 blood group systems by NGS was validated. This protocol proved to be powerful tool in resolution of referred clinical cases including RHD/RHCE anomalies complex antibody mixtures and identification rare/novel alleles. Validation of a protocol for analysis of all variants in 33/36 blood group systems by NGS was with 29 samples with known extended genotype (Illumina TruSight One Panel, data alignment with Human Reference Genome CRCh37). For all samples results were concordant with known extended genotype. This sequencing protocol was applied for a series of referred clinical cases. Six samples required analysis of the homologous RHD and RHCE genes with further resolution using Sanger sequencing. This included one sample that phenotyped as very weak RhD positive and SNP typed as RHD(ex9:del/CE). NGS also indicated a RHD(ex9:del/CE) but did not discriminate between a deletion and a rearrangement with the adjacent RHCE gene. NGS provided phenotype predictions for seven cases referred to assist with resolution of complex antibody mixtures. Analysis of NGS data provided a basis for identification rare/novel alleles in a further six samples and resolved a discrepancy between SNP typing and serology in favour of serology (a rare null Jk allele). In another case the Cromer system was suggested as soluble CD55 inhibited the antibody. NGS identified a homozygous novel nucleotide change c.203G>A, predicting p.S68N. In these referred cases NGS was powerful tool for resolution of complex red cell serology.