Fig 1: Application of reporter cell line assay in multiplex detection of anti-HLA antibodies in serum samples from allo-sensitized patients. (A) K528-derived fluorescence-barcoded reporter cell lines were transduced to express HLA class I molecules along with human ß2M or class II molecules along with human CD74. (A) The resultant cell lines were pooled, and the surface expression of HLA molecules on individual cell lines were validated with a panel of reference monoclonal antibodies. Example gating strategy is shown in Figure S3 . Demultiplexed reporter cell lines with MFI values above three-fold of the average MFI value of internal control cell lines (K528-FP0000 and K528-FP1111) were scored as positive and the histograms were highlighted in orange. (B) Multiplex detection of anti-HLA antibodies with 10 serum samples from allo-sensitized patients. W6/32 (hIgG1) was used as positive control, and hIgG1? and hIgG1? ( Figure S4 ) as negative controls in staining. Positive readouts that are also positive in clinical HLA SAB readouts were considered as true positive (TP) and indicated with green arrows. Negative readouts that are positive in bead readouts were considered as false negative (FN) and indicated with blue arrows. Positive readouts that were bead not tested (BNT) in SAB assay are indicated with gray arrows. MFI values are listed in Table S1 . (C) Clinical HLA SAB MFI readouts. The color codes are in accordance with those in B), with TPs in both assays highlighted in green, and TPs in SAB assay while FNs in reporter cell assay in blue. HLA alleles are listed in abbreviations as indicated in parentheses in (A). For class II molecule lines, bulk cell line after antibiotic selection were used.