High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues
High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues
复制标题
DOI：
10.1002/path.1765
复制
发表时间：
2005-06-01
影响因子：
7.3
通讯作者：
Fleuren, GJ
中科院分区：
文献类型：
--
作者：
Corver, WE;ter Haar, NT;Fleuren, GJ
石蜡包埋组织的DNA倍性测量的准确性受到缺乏分辨率和无法在双峰DNA直方图中明确识别DNA二倍体群体的限制。已经开发了一种多参数DNA流式细胞术方法，该方法能够同时检测来自档案组织块的脱蜡50 μ m切片或2 mm直径穿孔的样品中的肿瘤细胞和基质细胞。该方法将柠檬酸钠缓冲液中的热预处理和随后的胶原酶/分散酶混合物的酶解结合起来。在流式细胞术分析之前，同时对细胞进行角蛋白（FITC）、波形蛋白（R-PE）和DNA（PI）染色。该方法被应用于12个石蜡包埋的宫颈癌和4个结直肠癌。在所有宫颈癌中，观察到不同的角蛋白阳性和波形蛋白阳性细胞群。虽然波形蛋白阳性细胞组分总是产生单峰DNA含量分布，但在9个宫颈癌中观察到角蛋白阳性细胞组分的双峰分布，而1个宫颈癌显示出3个不同的G（0）G（1）群体。GOG峰的变异系数为1.70%~ 4.79%。平均背景、聚集体和碎片值分别为14.7%（波形蛋白阳性分数）和33.8%（角蛋白阳性分数）。流式分选证实，专门波形蛋白阳性细胞部分代表不同的正常基质和浸润细胞，可以作为内部倍性参考，使DNA亚二倍体和DNA超二倍体肿瘤细胞亚群之间的歧视。两个宫颈癌的角蛋白-波形蛋白共表达细胞的肿瘤起源通过流式分选样品的基因分型证实，显示6 p杂合性丢失（洛）。这种改进的方法避免了对新鲜/冷冻肿瘤组织进行高分辨率DNA倍性测量的需要，并且能够分离高度纯化的肿瘤亚群用于随后的基因分型。版权所有（c）2005大不列颠和爱尔兰病理学会。出版社：John Wiley & Sons，Ltd
The accuracy of DNA ploidy measurements of paraffin-embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi-parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 mu m sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R-PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin-embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin-positive and vimentin-positive cell populations were observed. While the exclusive vimentin-positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin-positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G(0)G(1) populations. Coefficients of variation of the GOG, peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin-positive fraction) and 33.8% (keratin-positive fraction). Flow sorting confirmed that the exclusively vimentin-positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo-diploid and DNA hyper-diploid tumour cell subpopulations. The neoplastic origin of the keratin-vimentin co-expressing cells from two cervical carcinomas was confirmed by genotyping of flow-sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high-resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.