In situ DNA cytofluorometry in paraffin sections and its application to malignacy diagnosis.

最近已经清楚地表明，通过测量细胞的DNA含量，可以区分恶性变化和良性变化。胃肠道上皮细胞的良性变化，包括非肿瘤性和肿瘤性改变，从不显示非整倍体或多倍体。根据这一结果，可以推断，在组织学上没有明显异常的异常增生或早期癌症等可疑病变中，通过测定细胞的DNA含量，可以揭示其良性或恶性的真实性质。在病理学家常用的重要物品石蜡切片上进行这种研究，存在严重的障碍。由于石蜡切片中存在细胞核重叠、非特异性光损失等问题，DNA细胞荧光法不适用于石蜡切片中DNA的原位检测。今年，作为前一年项目的延续，我们应用共聚焦显微镜对荧光标记石蜡切片进行了显微层析成像 ...更多信息 部分以计算高于特定阈值的所有像素值的总强度和细胞的估计DNA含量。或者通过对切片中的中期图像应用正常的细胞荧光测定法以确定共聚焦测量的有效性。结果证实，通过该技术原位DNA测量是可能的，并且该技术实际上揭示了恶性细胞克隆（更好地说是亚克隆）的精确分布。这种技术的应用已经清楚地表明，人类癌症开始于明显良性的准二倍体肿瘤，其生长相当缓慢，通过将其DNA从准二倍体突变为整倍体条件，逐渐发展为更恶性的肿瘤。结合这些研究结果，本研究表明，进一步改进共聚焦显微镜和与DNA结合的染料对于正确诊断否则模糊的肿瘤识别和正确理解人类癌症在人体内的整个过程是重要的。少

It has recently been made clear that by measuring DNA contents of cells one can differentiate malignant changes from benign ones. Benign changes including non-neoplasmic and neoplasmic alteration of cells in the gastro-intestinal epithelium never show aneuploidy nor polyploidy. Extrapolating this result, we can infer that measurements of DNA contents of cells in suspicious changes such as dysplasia or incipient cancers showing no overt atypia in histology may reveal their true nature whether benign or malignant.To carry out this kind of investigation on paraffin sections which are common and important goods among pathologists, there have been serious obstacles. DNA cytofluorometry is straight-forward inapplicable to in situ measurement of DNA in paraffin sections, due to overlapping of nuclei, non-specific light-loss occurring in the sections.This year, as a continuation of the former year project, we applied confocal microscopy to get microtomography of fluoresently labelled paraffin … More sections to calculate total intensity of all the pixel values that are above certain threshold and estimated DNA content of a cell. Or by applying normal cytofluorometry to metaphase figures in a section in order to ascertain validity of the confocal measurement.As a result, it is confirmed that by this technique in situ DNA measurements are possible and that this technique actually revealed precise distribution of malignant cell clones (better to say subclones). Application of this technique has made it clear that human cancers start as an apparently benign, quasi-diploid tumor, which grows rather slowly, to progress gradually toward more malignant tumors, by mutating their DNA from quasi-diploid to neuploid conditions. The investigation is proved very important to the understanding of behaviors of the human cancer in vivo.Together with these results of the research, the present investigation suggests that further improvement of the confocal microscope and dyes that bind to DNA would be important to correct diagnosis of otherwise ambiguous tumor-identification and to proper understanding of the entire course of human cancer progression in the human body. Less