Comprehensive assessment for serum treatment for single antigen test for detection of HLA antibodies

The single antigen test is widely used in the field of transplantation to determine the specificity of HLA antibodies. It will be beneficial to standardize the procedure of the single antigen test among HLA laboratories. It is not uncommon that single antigen testing on native sera fails to detect antibodies with very high concentrations. It has been shown that cleavage products of activated complement components may mask strongly binding antibodies in single antigen testing. To overcome inhibition by the activated complement products, sera are pretreated with ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), or heat inactivation before single antigen testing. However, no studies have been published to systemically compare the impact of these treatments on single antigen testing. The aim of this study is to understand the different effects these treatments may have on single antigen test results. We found that mean fluorescence intensity (MFI) obtained from sera treated with EDTA and heat inactivation were nearly identical, while DTT treatment was less potent to remove the inhibition. In addition, sera dilution did not further increase MFI of antibodies after EDTA treatment. Our results provide guidance to choose a pretreatment reagent for single antigen testing, and to compare studies obtained from laboratories using different treatments.