Rapid, single-subject genotyping to predict red blood cell antigen expression

Genotyping is useful to predict the expression of those RBC antigens for which antisera are difficult to obtain and to determine the probable phenotype of highly transfused patients, and it can be used to test stored DNA when a blood sample is not available. This study assessed a sequence-specific primer (SSP)-based genotyping system for blood group alleles suitable for the rapid testing of a small number of samples and assessed the use of stored whole blood. Genomic DNA was isolated from fresh and 1- and 2-week-old stored blood from 20 donors with known ABO and Rh phenotypes and was used for ABO, RHD, and RHCE genotyping using SSPs. The amplicons were analyzed using gel electrophoresis and a novel microfluidic on-chip electrophoresis system. Analysis of DNA from fresh and 1- and 2-week-old blood by SSP and gel electrophoresis yielded the correct ABO, RHD, and RHCE type in all samples, but with DNA from 2-week-old stored blood the amplicons were more difficult to visualize. Analysis of the same samples with the SSP on-chip electrophoresis assay correctly typed all samples except for one RHCE typing discrepancy of a fresh sample and one RHCE typing discrepancy of a 2-week-old sample. Analysis of amplicons by on-chip electrophoresis required one tenth the DNA that gel electrophoresis did and could be completed within 30 minutes compared with 2 hours with gel electrophoresis. Amplicons were also more readily visualized with on-chip electrophoresis. Fresh and 1- and 2-week-old samples could be ABO and RH genotyped with SSP. Analysis using on-chip electrophoresis was easier and more rapid than that using gel electrophoresis, but test reliability was slightly more variable.