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改变，包括非肿瘤性和肿瘤性改变，从未表现为非整倍体或多倍体。根据这一结果，我们可以推断，测量可疑变化的细胞的DNA含量，如不典型增生或早期癌症，在组织学上没有明显的非典型性，可能揭示它们的真实性质，无论是良性还是恶性。石蜡切片是病理学家常用的重要资料，要对其进行此类调查存在着严重的障碍。DNA细胞荧光法直接不适用于石蜡切片中DNA的原位测量，由于细胞核重叠，切片中发生非特异性光损失。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的石蜡进行了显微断层扫描…更多的切片，计算超过一定阈值的所有像素值的总强度，并估计细胞的DNA含量。或者用正常细胞荧光法测定切片的中期图像，以确定共聚焦测量的有效性。结果证实，通过这种技术，原位DNA测量是可能的，而且这种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。这项技术的应用清楚地表明，人类癌症最初是一种看似良性的准二倍体肿瘤，生长相当缓慢，通过将其DNA从准二倍体突变为新倍体，逐渐发展成更恶性的肿瘤。该研究对了解人类肿瘤在体内的行为具有重要意义。结合这些研究结果，目前的研究表明，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其他不明确的肿瘤识别和正确理解人类癌症在人体中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