Cytokeratin labeling of breast cancer cells extracted from paraffin-embedded tissue for bivariate flow cytometric analysis

Abstract

Flow cytometric analyses of DNA content and proliferative fractions have been found to be important prognostic indicators in a variety of human tumors. However, variability in reported results and interlaboratory differences in single‐parameter DNA measurements have impeded the broader use of this methodology. Multiparameter DNA analysis, especially that which allows the ploidy and cell cycle measurements to be targeted specifically to tumor cells, may improve the quality and reliability of these measurements. Cytokeratin labeling simultaneously with DNA allows the identification of the malignant epithelial cells within tumor samples that have been microdissected for tumor enrichment and, thus, can increase the accuracy of tumor ploidy and cell cycle measurements. There are a limited number of reports of cytokeratin labeling of paraffin‐extracted cells, and results with standard preparation procedures can be highly variable. We have developed an improved technique for cytokeratin labeling of paraffin‐extracted cells that is based on predigestion of tissue with collagenase prior to brief pepsin digestion. This two‐step enzymatic digestion produces a superior cytokeratin vs. DNA bivariate analysis, with increased intensity and greater uniformity of cytokeratin labeling. This method should increase the accuracy of ploidy and proliferation measurements from paraffin‐embedded tissue both for retrospective studies and in clinical settings in which only fixed tissue is available. © 1996 Wiley‐Liss, Inc.

Cervical carcinoma-associated fibroblasts are DNA diploid and do not show evidence for somatic genetic alterations

“…Then, cells were incubated with 200 μl premixed FITC- or RPE-labelled secondary reagents [Goat F(ab2)′ anti-mouse IgG1-FITC and goat F(ab2)′ anti-mouse IgG2b-RPE (Southern Biotechnology Associates, Birmingham, AL), both diluted 1:100 in PBATw.] After 30 min on ice, cells were washed twice with ice-cold PBATw and incubated with 1,000 μl NST DNA staining solution containing 50 μM DAPI (Sigma Aldrich) [22]. Cells were kept at room temperature for 30 min, followed by an overnight storage at 4°C.…”

The significance of bivariate cytokeratin and DNA flow cytometry in paraffin-embedded specimens of non-small cell lung cancer

“…Using this technique of gating cancer cells separately from non-epithelial cells, accurate preliminary studies could also be performed using paraffinembedded tissue. 2,3 The enzymatic dissociation and fluorescent staining of cytokeratin are key points for bivariate cytokeratin and DNA flow cytometry. First, an isolated cell must have enough cytoplasm to stain cytokeratin.…”

“…1 In recent years, an immunofluorescent staining step for the cytoskeletons of cancer cells has been interposed into the preparation for flow cytometry in order to differentiate cancer cells from non-epithelial cells. 2,3 More accurate cancerspecific analysis has been possible with bivariate cytokeratin and DNA flow cytometry.…”

Stratification of the biologic aggressiveness of non-small cell lung cancer using DNA flow cytometry and immunohistochemistry for the retinoblastoma protein

“…However, bivariate cytokeratin and DNA flow cytometry has been shown to decrease this error by distinguishing cancer cells from normal cells [19,20]. Cancer cells with cytokeratin stained can be distinguished from normal cells.…”