[Application of Third-Generation Sequencing in Clinical Detection of Difficult Blood Group Samples]

To explore the value of third-generation sequencing (TGS) in resolving discrepancies between serological findings and Sanger sequencing results in difficult blood group cases.

Samples were collected from 3 patients with difficult blood group typing (including family members of 2 patients) treated at our hospital between January 2023 and May 2025. Blood group identification was performed using serological testing, PCR genotyping, and first-generation (Sanger) sequencing. For cases with inconsistent serological phenotypes and genotypes, further analysis was performed using TGS to identify the underlying causes.

All three cases showed inconsistencies between serological and Sanger sequencing results. Case 1 showed an AB₃ serological phenotype, a genotyping result of AB , and Sanger sequencing identified ABO*A1.02/ABO*B.01. TGS revealed a promoter region mutation (-35_-18del) leading to weakened B antigen expression. Case 2 showed an A_elB serological phenotype, a genotyping result of AB, and Sanger sequencing identified ABO*A1.02/ABO*B3.07, which was inconsistent with the weakened A antigen expression suggested by serology. TGS demonstrated that the c.410C>T mutation was located on the A haplotype rather than the B haplotype, resulting in the A_elB phenotype. Case 3 showed a serological phenotype of RhD--, Sanger sequencing identified RHCE*02/RHCE*02, and TGS detected an RHCE-D(2-9)-CE gene recombination.

TGS overcomes the limitations of first-generation sequencing in blood group genotyping by enabling the detection of non-coding region mutations, accurate haplotype phasing, and identification of gene recombination events.