Sera of highly sensitized patients (HSP) contain complex human leukocyte antigen (HLA) antibodies, minimizing the chance to identify crossmatch-negative donors. Expression of 3-6 HLA class I antigens on lymphocytes hampers identification of acceptable mismatches (AMs) by conventional screening (C-SCR). The single-antigen-expressing cell line (SAL) concept circumvents this problem. As a proof of principle, 26 sera of sensitized patients were tested by flow cytometry for immunoglobulin G antibodies against 16 HLA-A and -B SALs. Results were compared with C-SCR. Mostly, SAL reactions confirmed presence/absence of HLA antibodies. While C-SCR sometimes failed to provide unambiguous antibody specificity, we defined 24 new HLA antibody specificities with SALs and proposed 33 new AM by non-reactivity with SALs. Thus, the SAL concept is useful for confirmation/identification of AM and will enhance transplantation of HSP.