Abstract
To develop a FXM for detecting donor specific HLA antibody (DSA) to A, B, C, DR, DQ, DP. DSA-FXM: 0.2X10 6 donor PBMCs were incubated with recipient serum at RT for 30’, washed, and then beads conjugated with anti-HLA class-I (CI) and -II (CII) MoAbs were added. The cells were lysed for 30’ and the complexes of HLA-Ag-Ab were captured by CI or CII beads; and detected by PE-anti IgG. The final washed beads were acquired on a flow cytometer. The PE intensities on CI and CII beads were proportionately correlated with the HLA-DSA levels in the serum. FXM: IgG FXM procedure. LMX-SAB: One Lambda method. Pooled HLA-Ab positive serum (PPS) in various dilutions was tested against different numbers of cells by DSA-FXM, FXM, and LMX-IgG. Results showed that DSA-FXM done with 25,000 cells has comparable sensitivity to FXM. DSA-FXM was able to detect low levels of LMX-DSA <500 MFI. The CI or/and CII DSAs including Cw, DQ, and DP were clearly identified in 75 of 117 samples and 87% of the DSAs were confirmed by LMX-SAB. 8 false positive LMX-DSAs were proven by both FXM and DSA-FXM. Interference by Rituximab and auto-Abs was completely eliminated by DSA-FXM. [Figs. 1 and 2] The DSA-FXM is a novel solid phase XM for selectively detecting HLA-DSA to all HLA loci in recipient serum. It eliminates false positive FXM caused by Non-HLA Ab and is truly donor HLA specific.
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