Quantitative analysis of human leucocyte antigen expression during culture of Epstein-Barr virustransformed cell lines using the Dako QIFIKIT

Abstract

Pre-existing donor-specific human leucocyte antigen (HLA) antibodies in renal allograft recipients result in hyperacute and accelerated graft failure. These antibodies can be detected in flow cytometric assay systems using HLA-characterised Epstein-Barr virus (EBV)-transformed B-lymphocyte cell lines. Confident assay performance is predicated by the expression of HLAs on the EBV-transformed B-cell line surface. Surface HLA expression of three EBV-transformed B-cell lines that had previously been used as part of a potential organ recipient serum screening panel at St James’ University Hospital, Leeds, are assessed for changes in the level of HLA expression over the nominal culture duration of eight days using the QIFIKIT (Dako, Denmark), a quantitative flow cytometry kit for assessing cell surface antigens. A comparison of the mean fluorescence intensity (MFI) of the known antigen levels of the beads via a calibration graph permits determination of the antibody binding capacity of the cell lines. Results showed that HLA expression is not consistent throughout the cell culture, with optimal expression occurring during day 2 of culture. Inconsistent HLA expression demonstrated during the cell culture means that no assumption of the level of HLA expression can be made, and that cell lines used as part of a screening panel should have their HLA expression in cell culture determined.