title: High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues.
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 microm 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.
authors.affiliation: Department of Pathology, Leiden University Medical Centre, The Netherlands. w.e.corver@lumc.nl
authors.forename: Willem E
authors.initials: WE
authors.lastname: Corver
authors.forename: Natalja T
authors.initials: NT
authors.lastname: Ter Haar
authors.forename: Enno J
authors.initials: EJ
authors.lastname: Dreef
authors.forename: Noel F C C
authors.initials: NF
authors.lastname: Miranda
authors.forename: Frans A
authors.initials: FA
authors.lastname: Prins
authors.forename: Ekaterina S
authors.initials: ES
authors.lastname: Jordanova
authors.forename: Cees J
authors.initials: CJ
authors.lastname: Cornelisse
authors.forename: Gert Jan
authors.initials: GJ
authors.lastname: Fleuren
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D005557:Formaldehyde; D006358:Hot Temperature; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000276:immunology; D019656:Loss of Heterozygosity; D009363:Neoplasm Proteins / Q000032:analysis / Q000276:immunology; D016612:Paraffin Embedding; D011003:Ploidies; D017154:Stromal Cells / Q000473:pathology*; D016707:Tissue Fixation / Q000379:methods; D002583:Uterine Cervical Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D014746:Vimentin / Q000032:analysis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D014746:Vimentin; D005557:Formaldehyde; D007633:Keratins