Labscan 100

To perform the HLA-B and MICA typing, about 5 mL of blood was collected by venipuncture in vacuum tubes (Vacutainer, Becton and Dickson, Oxford, UK) containing ethylene diamine tetraacetic acid (EDTA) as anticoagulant. Then, we extracted the genomic DNA by the separation-column method, using the Biopur kit for DNA extraction (Biometrix, Curitiba, Paraná, Brazil), following the manufacturer's protocol. After adjusting the DNA concentration, obtained by the optical-density method, we amplified the DNA using polymerase chain reaction-sequence specific primers (PCR-SSO) combined with Luminex technology. The genomic DNA was amplified using biotinylated sequence-specific primers for HLA-B and MICA in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA), followed by hybridization with complementary probes for DNA, conjugated with microspheres (beads) labeled with different fluorochromes to identify complementary sequences of the amplified DNA, using the LABType kit (One Lambda, Inc., Canoga Park, CA, USA), following the manufacturer's protocol. After hybridization, the results were read using the flow cytometry platform LABScan^™100 (One Lambda, Inc.), followed by analysis using the program HLA Fusion version 2.0 (One Lambda, Inc.). The results showed low-medium resolution.

We included LTx patients who underwent LTx within our center between September 2003 and November 2012, and of whom pretransplantation serum was available. Prior to transplantation, patients were assessed for transplant eligibility via classical cross-match testing. Pretransplant HLA antibodies were measured via the LABScan 100 flow analyzer (One Lambda, CA, USA) or ELISA (LAT, One Lambda), as described previously (7 (link)). All patients were treated with standardized immunosuppressive regime consisting of tacrolimus, basiliximab, prednisolone, and mofetil mycophenolate. Patients depicted as being at risk for CMV or EBV reactivation (defined as a CMV−/EBV− patients receiving a graft from a CMV+/EBV+ donor) were prophylactically treated with valganciclovir up until 6 months after transplantation. Informed consent in accordance with the Declaration of Helsinki was obtained from all the patients, and this study was approved by the medical ethical committee of the University Medical Center Utrecht (METC 06-144). All methods were carried out in accordance with the approved guidelines. Serum samples from 20 healthy controls (HC) who donated blood for research purposes were obtained, processed, and stored at −80°C until further usage.

The presence of HLA antibodies in the pretransplant sera, used for pretransplant crossmatch, was assessed retrospectively in 1 central laboratory as described previously.^{7 (link)} Four thousand one hundred eighty-three (89%) of 4724 of the sera were taken within 3 months pretransplant, 377 (8%) of 4724 were taken 3 to 6 moths pretransplant, and only 164 (3%) of 4724 were taken 6 to 12 months pretransplant. In brief, sera were first tested for the presence of HLA class I and class II antibodies using Lifecodes LifeScreen Deluxe (Immucor Transplant Diagnostics, Stamford, CT). Subsequently, the sera positive for HLA class I and/or class II were analyzed using Lifecodes SAB assay class I and/or II kits (Immucor Transplant Diagnostics) to determine the exact specificity of the HLA antibodies. The LABScan 100 flow analyzer (One Lambda, Canoga Park, CA) was used for data acquisition. Bead positivity assignment, the subject of this study, was evaluated for a range of median fluorescence intensities (MFIs) (median and 5%-trimmed mean), signal-to-background ratio (STBR) cutoffs (Table 1), and combinations thereof. The presence of SAB-DSA was determined by comparing the SAB-HLA-A/B/DR/DQB antibody specificities on serological level with the split level HLA-A/B/DR/DQB typing of the donor.

The samples were typed for MICA and HLA-B alleles using the polymerase chain reaction-specific oligonucleotide sequence (PCR-SSO) method by microbeads array luminex technology (One Lambda, CA). Reactions were read with the LABScan 100 flow cytometry analyzer and assignment of allelic types was performed using the HLA Fusion 2.0 software (One Lambda, CA). Data of MICA genotyping were also used to classify patients and controls based on the presence of valine (val) or methionine (met) amino acids at codon 129 of the alfa-2 domain.