A QUANTITATIVE METHOD TO MEASURE ALKALINE-PHOSPHATASE ACTIVITIES IN INDIVIDUAL LEUKOCYTES BY IMAGE-ANALYSIS

Leucocyte alkaline phosphatase (L-ALP) is well known as leukemia marke r, but recent results suggest its usefulness for the diagnosis of seve ral diseases. The aim of this study was to develop a quantitative meth od to measure alkaline phosphatase activities in individual leukocytes by image analysis. We studied the reaction rate of L-ALP in human pol ymorphonuclear leucocytes by a microscope attached to a TV camera and a computerized image analyzer. The optical density (OD) measured was s tandardized by grey filters with known absorbance. We measured IOD for individual cells after a set incubation time by end-point measurement s. Studies of kinetic parameters of L-ALP were performed by single-poi nt measurements in the linear phase of the reaction and at increasing substrate concentrations. Cellular IOD increased proportionally with i ncubation time up to 10 min. The mean K-M(mM) and V-max(Delta IOD/min) values were 0.70 +/- 0.11 and 1.76 +/- 0.2 (mean +/- SE, n = 5) respe ctively. Our findings are comparable to previous results using a polyv ynil alcohol method in microphotometry analysis. The image analysis of cellular L-ALP activity appears a valuable tool for quantitative stud ies.