A quantitative method to measure alkaline phosphatase activities in individual leukocytes by image analysis

Leucocyte alkaline phosphatase (L-ALP) is well known as leukemia marker, but recent results suggest its usefulness for the diagnosis of several diseases. The aim of this study was to develop a quantitative method to measure alkaline phosphatase activities in individual leukocytes by image analysis. We studied the reaction rate of L-ALP in human polymorphonuclear leucocytes by a microscope attached to a TV camera and a computerized image analyzer. The optical density (OD) measured was standardized by grey filters with known absorbance. We measured IOD for individual cells after a set incubation time by end-point measurements. Studies of kinetic parameters of L-ALP were performed by single-point measurements in the linear phase of the reaction and at increasing substrate concentrations. Cellular IOD increased proportionally with incubation time up to 10 min. The mean KM(mM) and Vmax(delta IOD/min) values were 0.70 +/- 0.11 and 1.76 +/- 0.2 (mean +/- SE, n = 5) respectively. Our findings are comparable to previous results using a polyvynil alcohol method in microphotometry analysis. The image analysis of cellular L-ALP activity appears a valuable tool for quantitative studies.