Rapid (One-shot) Staining Method for Two-color Multiparametric DNA Flow Cytometric Analysis of Carcinomas Using Staining for Cytokeratin and Leukocyte Common Antigen

Abstract

The authors present an improved method for rapid two-color staining with direct conjugated antibodies to cytokeratin and CD45 antigen (leukocyte common antigen) for whole-cell, ethanol-fixed preparations of human carcinomas. This method was quality controlled with the T24 human bladder tumor cell line and compared in parallel analysis of 24 fresh human carcinomas with the original two-color method of multiparametric analysis that had been published in 1989. This rapid method was designed to achieve comparable staining intensities of both green (phenotype directed monoclonal antibody label) and red (propidium iodide labeled DNA) fluorescence, identical DNA indexes, comparable coefficients of variation, and subjective visual quality of DNA histograms. This is accomplished in a single (one-shot), abbreviated incubation with monoclonal antibody diluted in propidium iodide-RNase, thereby eliminating two incubations and three wash steps required with the original method. The single rinse is done in the propidium iodide-RNase staining solution with resuspension in fresh staining solution before analysis. With the rapid method, the preparation time is reduced by 130 minutes, resulting in a 60% time savings in batch staining mode compared with the original method. The time reduction and fewer wash steps, which should avoid excessive cell loss and cytoplasmic stripping, may advance the adoption of this two-color method in clinical practice.

Prediction of Clonal Chromosome Aberration Frequency in Human Blood Lymphocytes

“…Anti-human cytokeratin antibody (aCK18 Ab, Abcam, Tokyo, Japan) was used as the primary antibody to stain epithelial cells (11). Anti-vimentin antibody (Dako Japan, Inc., Tokyo, Japan) and anti-smooth muscle Actin antibody (aSMA Ab, Abcam, Tokyo) were used to stain mesenchymal or myoepithelial cells.…”

Flow cytometric analysis of S-phase fraction in breast carcinomas using gating on cells containing cytokeratin

“…The characteristics of cytokeratin in normal epithelia of the breast are mostly well preserved during malignant progression and, to some extent, even more pronounced in carcinomas (Osborn et al, 1983;Ferrero et al, 1990;Wetzels et al, 1991). A fluorescein isothiocyanate (FITC)-conjugated secondary antibody, together with a primary monoclonal antibody specific for cytokeratin, used with a suspension of cells with preserved antigenicity, allows flow cytometric sorting of the epithelial cell population (Zarbo et al, 1989;Visscher et al, 1990 1977 and 1990, were included in the study. The patients' median age was 57 years and the median follow-up time was 59 months.…”

Expression and loss of heterozygosity of c‐met proto‐oncogene in primary breast cancer

“…Cell suspensions were fixed in 35% ethanol as previously described by Zarbo et al [20]. DNA was stained by propidium iodide to a final concen-DNA extraction.…”