Evaluation of an automated method of percent reactive antibody determination

A fluorescence-based automated method of percent reactive antibody (PRA) analysis is described. This method utilizes the conventional antibody-mediated, C'-dependent lymphocyte microcytotoxicity assay to detect alloantibodies, but replaces the eosin-based method for detection of cell death with a fluorescence-based method. To identify viable cells, lymphocytes were pretreated with carboxy fluorescein diacetate (CFDA), which fluoresces green, to identify viable cells. To identify dead cells after the reaction with antibody and C', they were treated with propidium iodide (PI), which fluoresces red. Pretreatment of lymphocytes with CFDA did not affect their ability or interact with alloantibodies in the microcytotoxicity assays. When visually analyzed, detection of cell death by fluorescence was as sensitive as detection by eosin exclusion. However electronic detection of fluorescence was slightly more sensitive than visual detection. Automation of the fluorescent method required a calculation that converts electronic data to an ASHI score for cell death. One such method is described and evaluated. Both the automated and the conventional methods of analysis were used to obtain PRA values for various sera. There was good correlation between the PRA values obtained with the automated method versus the conventional method. Further, there was good correlation for PRA-derived alloantibody specificities obtained with the automated method versus the conventional method. These data demonstrate that automated fluorescence-based PRA analysis is an effective and practical alternative to conventional PRA analysis.