A novel mass spectrometry approach for blood group antigen typing

Abstract

Abstract Introduction Many blood group antigens beyond ABO and RhD are clinically important, especially in certain populations. However, the lack of commercial antisera and the limitations of genotyping make typing challenging. We present a novel method for detecting red cell membrane antigens by leveraging the high analytical performance of mass spectrometry in protein analysis, offering a potential complement to conventional serologic and genotyping techniques. Methods Residual patient samples collected for routine clinical testing were used. Red blood cells were isolated from 300 µL of whole blood using Ficoll gradient, and white ghosts were extracted for enzymatic digestion. Proteolytic digestion was performed using one of the enzymes trypsin, chymotrypsin, pepsin, or Asp-N. Targeted antigen analysis was subsequently performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with parallel reaction monitoring. Genotyping was conducted using either the ID CORE XT or next-generation sequencing (NGS), depending on availability. The results were compared with phenotyping using commercial antisera and genotyping to evaluate concordance. Results Jr(a) negativity was confirmed in two samples that were initially presumed to be Jr(a) negative based on our experimental results, and the findings were consistent with NGS results. In three additional samples, five blood group antigens—Lutheran, Diego, Colton, RhD, and Jr—were successfully typed. All three samples were Lu(b), Kp(b), Di(b), Wr(b), Co(a), Jr(a), and RhD positive, and genotyping using the ID CORE XT showed concordant results. The results of phenotyping with commercial antisera were also consistent. Conclusions Our experimental mass spectrometry-based approach demonstrated concordant results with conventional phenotyping and genotyping. These findings suggest that mass spectrometry can serve as a complementary method for red cell antigen typing, particularly when serologic reagents or genotyping are unavailable.