Labscreen hla class 1 and class 2 antigen beads

Serum samples from recipients before and after transplantation were collected to detect anti-HLA class I (HLA-A, HLA-B) or HLA class II (HLA-DR or HLA-DQ) antibodies. These were further analyzed with a Luminex Single Antigen Assay using the LABScreen HLA class I and class II antigen beads and HLA Fusion software (One Lambda, West Hills, CA, USA). The presence of DSA was determined by comparing various HLA specificities with the donor HLA type.

The complement-dependent cytotoxicity cross-match was negative before transplantation in all patients.
Serum samples from recipients were screened for the presence of HLA antibodies using the Lifecodes Lifescreen Deluxe (LMX) kit, according to the manufacturer's manual (Immucor Transplant Diagnostics Inc. Stamford, CT, USA). Samples that were considered positive, scores 6 and 8 i.e., 2,135 MFI, for either HLA class I (HLA-A, HLA-B, or HLA-C) or HLA class II (HLA-DR or HLA-DQ) antibodies were further analyzed with a Luminex Single Antigen assay, using LABscreen HLA class I and class II antigen beads (One Lambda, Canoga Park, GA, USA) (32 (link)).
Briefly, 4 μl of LABscreen beads and 20 μl of serum were mixed in a test well, protected from light. Serum samples were incubated for 30 min at room temperature on a rotating platform (150 rpm), followed by repeated washings with 260 μl wash buffer. Afterwards, each sample was incubated for 30 min with a goat anti-human PE conjugated antibody (1:100 wash buffer) at room temperature, protected from light, and subsequently washed 5 times with wash buffer. Samples were measured using a Luminex 100 reader (Luminex 100, Luminex Corporation, ‘s-Hertogenbosch, the Netherlands) and the baseline normalized values were used. LABscreen negative control serum (LS-NC, One Lambda) was used as a negative control.

Pre-transplant serum samples from recipients were screened for the presence of anti-HLA antibodies using the Lifecodes Lifescreen Deluxe (LMX) kit, according to the manufacturer’s manual (Immucor Transplant Diagnostics Inc. Stamford, CT, USA). Thereafter, anti-HLA class I (HLA-A, HLA-B, and/or HLA-C) or HLA class II (HLA-DR and/or HLA-DQ) antibodies were analyzed with a Luminex Single Antigen assay using LABscreen HLA class I and class II antigen beads (One Lambda, Canoga Park, GA, USA), as described in our previous study^{13 (link)}. A cut-off mean fluorescence intensity value of 5000 was used to determine the presence of anti-HLA antibodies. The presence of donor-specific antibodies (DSA) was determined by comparing the measured HLA specificities with donor HLA typing.