Advancing blood group diagnosis with molecular methods: A focus on clinically challenging cases

Abstract

Traditional serological methods often struggle with the accurate identification of rare or complex blood groups. This study addresses the challenges in diagnosing clinically difficult blood groups by employing genotyping and sequencing techniques to enhance blood supply accuracy. Two patients with challenging blood groups underwent serological testing and molecular diagnostics using fluorescent PCR typing and Sanger sequencing. Patient 1 showed a discrepancy in serological typing, indicating a B subgroup confirmed by a B/O\*1 genotype and a 721C>T mutation in the ABO gene Exon 7, establishing the Bw. 03/O.01.01 genotype. Patient 2, with negative Rh antigen detection and typed as CcDEe, was diagnosed with a Rh-null phenotype due to a homozygous frameshift mutation (c.732delC) in the RHAG (Rh-Associated Glycoprotein) gene Exon 5. Integrating serological screening with genotyping and sequencing technologies is recommended for accurately identifying challenging blood groups. This three-step diagnostic process facilitates precise blood group determination, ensuring accurate blood supply management.

Graphic Abstract

Schematic diagram of the three-step diagnostic workflow for difficult blood group typing.

Note: This workflow combines serological initial screening, PCR genotyping, and Sanger sequencing to achieve rapid typing and genetic confirmation of complex or discrepant forward and reverse typing blood groups. The research methods include sample processing, PCR amplification, and gene sequencing. The results indicate that Patient 1 Patient 1 has a B/O1 genotype, and Patient 2 exhibits an Rh-null phenotype caused by a mutation in the RHAG gene. This workflow can assist in identifying rare blood types and optimizing blood supply plans, thereby enhancing transfusion safety.