Pradella M, Barbasetti di Prun P, Nemetz L, Bovo C, Visentin M, Baldo F, Burlina A
Clinical Chemistry and Microbiology Laboratory, Civil Hospital, Castelfranco Veneto, Italy.
Acta Histochem. 1995 Apr;97(2):189-94. doi: 10.1016/s0065-1281(11)80098-x.
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.
白细胞碱性磷酸酶（L-ALP）作为白血病标志物广为人知，但最近的结果表明其在多种疾病诊断中具有应用价值。本研究的目的是开发一种通过图像分析测量单个白细胞中碱性磷酸酶活性的定量方法。我们通过连接电视摄像机和计算机图像分析仪的显微镜研究了人多形核白细胞中L-ALP的反应速率。所测的光密度（OD）通过具有已知吸光度的灰色滤光片进行标准化。在设定的孵育时间后，通过终点测量法测量单个细胞的积分光密度（IOD）。L-ALP动力学参数的研究是在反应的线性阶段和底物浓度增加时通过单点测量进行的。细胞IOD在长达10分钟的孵育时间内与孵育时间成比例增加。平均米氏常数（KM，单位为mM）和最大反应速率（Vmax，单位为ΔIOD/分钟）值分别为0.70±0.11和1.76±0.2（平均值±标准误，n = 5）。我们的研究结果与先前在显微光度分析中使用聚乙烯醇方法得到的结果相当。细胞L-ALP活性的图像分析似乎是定量研究的一种有价值的工具。
