IMAGE-ANALYSIS AS A TOOL FOR QUANTITATIVE ENZYME DETERMINATION AT THECELLULAR-LEVEL - APPLICATION FOR MONOCYTIC DIFFERENTIATION OF THE UM-384 CELL-LINE

Image analysis has been used to determined enzyme activity at the cell ular level in individual,smeared cells. The counterstains used to visu alize smeared cells were chosen to avoid overlap with the chromogene. The amount of the reaction product was quantified by computerised scan ning cytophotometry when the conditions of incubation, time and temper ature of the reaction, and substrate concentration varied. Under optim al conditions for time, temperature and substrate concentration, a lin ear relationship was found between enzyme activity determined on smear ed cells and in cell lysate. Using these defined conditions, different iation of UM-384 cells was studied by measuring enzyme activity. After a monocytic differentiation process, induced by sodium butyrate, non- specific esterase cell activity was compared either with differentiati on markers (HLA-DR, plasminogen activator inhibitor type 2 and lysozym e) or with markers of proliferation (DNA content) or functional proper ties (nitroblue tetrazolium reduction and phagocytosis). The results s how that, using image analysis, non-specific esterase seems to be a us eful means for the assessment of monocytic differentiation whereas mye loperoxidase is not. More generally, quantification of enzyme activity at the cellular level using image analysis can be applied to the stud y of the differentiation process and may help in the classification of leukemic cells.