Labscreen single antigen bead assay

Pre-transplant sera of all patients were tested prospectively for anti-HLA class I and class II antibodies using multianalyte bead assays performed on the Luminex platform including LABScreen® PRA, LABScreen® Mixed methods for screening; the binding level of donor-specific antibody was determined by the LABScreen® Single Antigen bead assay (One Lambda), Part of Thermo Fisher Scientific (Canoga Park, California, USA) per manufacturer's instructions and results were expressed as mean fluorescence intensity (MFI). Briefly, 5 μl of mixed beads, HLA class I and class II single antigen beads were added to 20 μl of sample serum, and incubated for 30 min at room temperature (RT) in the dark with gentle shaking. After washing with wash buffer three times, 100 μl of goat anti-human IgG secondary antibody conjugated with R-phycoerythrin (PE) was added and the samples were incubated in the dark for 30 min at RT. After washing three times, the samples were read on Luminex-based LABScan™ 100 flow analyzer. Antibody specificity and binding level were analyzed and determined through HLA Visual or HLA Fusion software from the manufacturer.

The participating centers performed surveillance DSA testing prior to transplantation and on post-transplant day 7, 14 and month 1, 3, 6, 9, 12, 18 and 24 (coincident with scheduled surveillance bronchoscopy). Patients underwent additional DSA testing for clinical signs or symptoms of allograft dysfunction. DSA was detected at each center by single antigen bead testing using the LABScreen^® Single Antigen Bead assay (One Lambda, Canoga Park, CA) and designated as either positive or negative. A positive test was defined as a mean fluorescence intensity (MFI) ≥ 1000 on one occasion or an MFI between 500–1000 on two serial occasions. DSA was categorized as preformed (present prior to transplantation) or de novo – not detected prior to transplantation.