免疫组织化学染色的定量方法研究

Study on the Quantitative Method of Immunohistochemistry

本文提出阳性单位(PU)新概念,用于定量表达免疫组化阳性反应程度。本文将排除了切片或涂片本底因素后的纯免疫组化反应程度按灰度图像分为0(白)到100(黑)个等级,每一等级定义为1个阳性单位。导出公式如下:a.PU=100(G_α—G_β)÷G_(max);b.PU=100(G_A—G_β)÷(A_(Aα)·G_(max))。式中G_α、G_β分别为待测结α和背景β的平均灰度,G_A为含有α和β的A区域的平均灰度;G_(max)为检测仪器的最大灰度。A_(Aα)为α相在A区域中的面积密度。式a适于测算境界明显的粒子,式b则不论粒子境界如何,对各类样品均适合,包括组织学切片和细胞学涂片。PU值大小与反应强度成正比,PU最大值为100、理论最小值为0。对513个EMA染色的胸腹水腺癌细胞进行了测试,结果表明该法对EMA染色定量判断的灵敏度为94.2％,特异度为100％,准确度为95.6％。讨论了PU值的意义和有关对比染色问题。

A new concept of positive unit(PU) was put forward for the quantitative expression of result of immunohistochemical reaction. Intensity of pure immunohistochemical reaction, which means the factor of background from section or smear had been discharged, was classified from 0 (for white) to 100(for black) based on the gray level of gray scale image. Each grade was defined as a positive unit on the paper. The formulas in the following for PU has been deduced, a. PU=100(G_α- G_β)/G_(max); b. PU=100(G_A-G_β)/A_(Aα)G_(max). In the formulas, G_α, G_β denote separatly the mean gray level of thestudying structure α and back ground β; G_A: mean gray level in an area A which containes α and β; G_(max): the maximum gray level of the instrument. A_(Aα): the area density of α phase in area A. Formula a could been used for the particles which are boundary obviously. Formula b could been used for all kind of sample including histological section and cytological smear despite the particles boundary are obvious or not. The value of PU varied directly with the reaction intensity. 513 cells of adenocarcinoma from hydrothorax and ascitic fluid dyed in EMA were measured. The results showed that the sensitivity is 94.2%, the specificity 100% and the accuracy 95.6%. The significance of PU and the problem about contrast dye were discussed.