Automated contrast fluorometry. I. Lymphocytotoxicity testing.

Automated contrast fluorometry has been carried out in lymphocytotoxicity testing using a computer-controlled microscope and direct comparison of the data with controls. The method uses heparinized lymphocytes and permits simultaneous evaluation of both fluorescein and ethidium bromide fluorescence by calculating the quotient between the green/red measurements. After comparison of the quotient with that of control samples, the raw data are automatically transformed into cytotoxicity scores. Fast and reliable measurements with minimum background fluorescence can be obtained for up to 24 hours after performance of the assay by controlling complement reactivity and the exposure time of the cells to the fluorescein. The evaluation of 15,204 cytotoxic reactions showed excellent correlation between the automated and visual results of 125 tissue typings using 120 different antibodies and of 17 HLA antibody titers using case-by-case analysis of variance. The values for the automated cytotoxicity readings were slightly higher and showed more intermediate scores than those of the visual readings suggesting a high sensitivity and objectivity of the automated system. In conclusion, this is a methods paper comparing parallel lymphocytotoxicity testing using automated and manual methods. The automated method was found to be more sensitive and faster than the manual method.