HLA Antibody Screening and Identification

The patients and donors underwent HLA allele typing of at least the A, B, and DRB1 loci routinely. The examination was performed as previously [15 (link)]. In brief, patient plasma/serum was screened for class I and class II HLA antibodies with a LABScreen Mixed Kit (One Lambda, Canoga Park, CA, USA). The samples were incubated with mixed HLA class I- and class II-coated microspheres for 30 min in the dark and then washed before being incubated with anti-human immunoglobulin G-conjugated fluorescein isothiocyanate as described above for the first incubation. Finally, the samples were examined by a Luminex 200 flow analyzer (Luminex, Austin, TX, USA), and the data were analyzed with the HLA Fusion 3.2 software (One Lambda). The MFI of anti-HLA antibodies was obtained using the formula: sample beads − negative control beads. The samples with a MFI > 500 were further tested for the specificity of the antibody (DSA), using a LABScreen Single Antigen Kit (One Lambda).