High‐resolution multi‐parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin‐embedded tissues

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Abstract
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 µm 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 G0G1 populations. Coefficients of variation of the G0G1 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 © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.