Labscreen single antigen kit

A plasma sample was obtained by centrifugation of a peripheral blood tube with ethylenediaminetetraacetic acid (Vacutainer® spry-coated K2EDTA tubes, BD™), collected from all patients, at baseline, 12 and 52 weeks after control or ASC administration. HLA Abs were detected in a Luminex platform using a LabscreenMixed™ kit (One Lambda Inc.® Canoga Park, CA, US) according to manufacturer instructions. All samples with a signal >800 units of median fluorescence intensity (MFI) were considered positive, and donor specificities for HLA Abs were determined using Labscreen Single Antigen™ kit (One Lambda Inc.® Canoga Park, CA, US). All signals were normalized according to Quantiplex™ beads fluorescence and specificities > 20,000 units of standard fluorescent intensity were considered relevant. Qualitatively, we defined the HLA antibody titer as the resulting MFI sum of all the determinant beads of the HLA class I molecules included in the Labscreen Mixed kit. We will refer to pre-existing HLA Abs detected in patients before ASC administration as HLA Abs whereas donor ASC-induced HLA Abs will be referred as DSA.

Serum samples were collected from the patients for screening anti‐HLA antibodies with a LABScreen panel reactive antibody (PRA) Kit (One Lambda). Positive samples were further tested for the specificity of the antibodies against HLA‐I (ie, HLA‐A/B/C) and HLA‐II (ie, HLA‐DR/DQ) antigens using a LABScreen Single Antigen Kit (One Lambda). Fluorescence was measured using a Luminex100 flow analyzer (Luminex), and the data were analyzed using the LABScan 100 software (One Lambda). The median fluorescence intensity (MFI) of the PRA beads’ reactions was obtained from the output file generated by the flow analyzer, adjusted for the background signal using the formula: sample beads −negative control beads. The fluorescence intensity of the negative and positive control beads was <100 and >9000, respectively. If the sample data did not fit these conditions, the serum or plasma was treated with ADSORB OUT beads (One Lambda) to reduce background fluorescence. All samples with fluorescence intensity >500 were tested with single Ag beads to confirm and identify sample specificity. MFI was adjusted for the background signal using the abovementioned formula. The samples were considered: negative, MFI < 500; weakly positive, MFI 500‐2000; positive, MFI 2000‐10 000; and strongly positive, MFI > 10 000.

Patients were tested for the presence of donor-specific antibodies (DSAs) including class I (i.e., HLA-A, -B, -C) and class II (i.e., HLA-DR) HLA antibodies. Immunoglobulin anti-HLA reactivity in the serum was tested with a bead-based screening assay. Briefly, we used the LABScreen Mixed kit (One Lambda, Canoga Park, CA, USA), which simultaneously detects class I and class II antibodies with microbeads coated with purified class I and class II HLA antigens. For HLA antibody-positive samples with a median fluorescent intensity (MFI) >500, DSAs were further tested using a LABScreen Single Antigen Kit (One Lambda). Above a cut-off value of MFI ≥2000 was considered positive. Patients with positive DSA received rituximab before transplantation, and the co-infusion of umbilical cord blood (26 (link)).

In the main cohort we used 524 pre-transplant serum samples and 225 post-transplant (at 1 year) serum samples to evaluate levels of anti-HLA and MICA DSA with the respective LABScreen Single Antigen kits (One Lambda) following the manufacturer’s instructions. The same kits and conditions were used to evaluate anti-HLA and anti-MICA DSA in an independent cohort of 168 patients who had an episode of ABMR at the time of diagnostic biopsy. Antibodies were detected based on the mean fluorescence intensity (MFI) for each bead coated with an HLA or MICA antigen, as normalized to the value measured with the negative control serum using the baseline method. All beads with normalized MFI higher than 500 or 100 were considered positive for HLA and MICA, respectively. The MFI cut-off for positivity of anti-MICA DSA was chosen based on a receiver operating characteristic analysis (Supplementary Fig. 4). The maximum MFI of DSA was defined as the highest ranked donor-specific bead. For the remaining patients, anti-HLA antibody testing was performed using either complement-dependent cytotoxicity, ELISA or Luminex-based tests.