Effects of the standardization of quantitative fluorometry on the automated evaluation of platelet antibodies

An automated fluorescence invertoscope featuring a computer-controlled scanning stage with plate inserts for multiple samples, a photomultiplier tube and software controlling every function of the microscope were used to collect the fluorescence data after analog-to-digital conversion. Principal differences between this method and qualitative platelet tests include the standardization of all test reagents and the quantitative evaluation of possible autofluorescence. The automated data acquisition permitted the separate evaluation of the fluorescence intensities of all materials to ensure low background fluorescence and optimal fluorochrome concentrations. EDTA-anticoagulated platelets of 100 donors were incubated with serum, subsequently with fluorescein- or rhodamine-treated IgG, washed, counted in PBS-glycerol, and mounted on thin glass plates. Each plate contained 5000 cells per test and negative and positive controls.