Transplant centers are increasingly using virtual crossmatching to evaluate recipient and donor compatibility. However, the current state of virtual crossmatching fails to incorporate donor human leukocyte antigen (HLA) expression in the assessment, despite numerous studies that have demonstrated the impact of donor HLA expression on physical crossmatch outcomes. Whole-transcriptome sequencing (RNA-Seq) for HLA enables simultaneous determination of HLA genotyping and relative HLA expression. Ultimately the RNA-Seq needs to be faster to be incorporated into the virtual crossmatching process. However, to demonstrate feasibility, the utility of the MinION sequencer (Oxford Nanopore Technologies, Oxford, UK) was evaluated in combination with RNA-Seq to generate HLA genotypes and to determine HLA class I expression. Although HLA class I expression varied among individuals, the pattern of HLA expression remained relatively consistent (HLA-B > HLA-A = HLA-C). HLA-A and -C had similar expression profiles. The impact of donor HLA expression was evaluated using serum samples containing a single donor-specific antibody (DSA). By making DSA consistent, donor HLA expression variability could be assessed. With consistent DSA mean fluorescence intensity, there was a direct relationship between the donor HLA expression to which the DSA is against and flow cytometric crossmatch median channel shifts.