Bridging the Gap: High-Resolution RHCE Genotyping by Third-Generation Sequencing Enhances Serological Interpretation...

From January 2022 to June 2023, this study enrolled 124 healthy blood donors. Column agglutination and third-generation sequencing (TGS) were used for RHCE genotyping and RhCE antigen typing. Additional serological screening was performed using conventional tube and column agglutination methods. Sanger sequencing was performed on 22 specimens with abnormal RhCE expression. Among the 124 samples, 102 exhibited normal RhCE expression whereas 22 showed abnormal expression; TGS-predicted phenotypes disagreed with serological results in eight of the 102 normal-expression samples and in seven of the 22 abnormal-expression samples, with discrepancies attributed to reagent limitations and the presence of novel alleles. Indeed, six novel RHCE alleles were identified by TGS and their potential phenotypes inferred based on serological findings, demonstrating that conventional tube and column agglutination methods are effective in determining serological phenotypes, especially in cases of weak RhCE antigen expression, whereas TGS proves valuable for genotyping and elucidating the underlying causes of weakened expression.