Aim: The One Lambda LabScreen Single Antigen Bead (SAB) assay is known to have false positive antibody reactivities. Surrogate crossmatching using donor lymphocytes can confirm if an antibody is real but finding a suitable donor is difficult for highly sensitized patients. Having a single HLA class II antigen on a cell would be useful for confirming common false positive antibodies. We cloned DQ2(DQA1*05:01/DQB1*02:01) and DRB4-transduced into T2 cell lines and adapted our flow cytometric crossmatch to test if these cells could replace donor lymphocytes. We also determined the limit of detection on the T2 cells as compared to the mean fluorescent intensities (MFIs) detected using the SAB assay.
Methods: The T2 cell lines were cloned to express either the DQB1*02 (DQA1*05:01/DQB1*02:01) or the DR53 (DRB4*01:03) molecule. True negative (TN), known false positives (KFP), or true positive (TP) sera were confirmed using surrogate donor lymphocyte crossmatches. HLA-expressing T2 cells were pronase-treated and incubated with DTT-treated serum. After washing, cells were stained with goat-antihuman-IgG-APC (Jackson ImmunoResearch Labs) and analyzed on the CytoFLEX flow cytometer. To determine the limit of detection, serial dilutions of TP samples were tested on the DRB4- or DQ2- expressing T2 cells.
Results: There was a > 90% concordance between the donor lymphocyte surrogate XM results and the HLA-expressing T2 cell XMs for the TP, KFP, and TN sera.
Conclusion: HLA-expressing T2 cells can be an alternative for surrogate donor lymphocytes for confirming suspected false antibody reactivity in the SAB assay. The T2 cells only express one class II molecule which is advantageous when investigating false positive antibodies in highly sensitized patients. Rare HLA alleles not represented on the SAB panel can also be expressed.
