RAPID (ONE-SHOT) STAINING METHOD FOR 2-COLOR MULTIPARAMETRIC DNA FLOWCYTOMETRIC ANALYSIS OF CARCINOMAS USING STAINING FOR CYTOKERATIN AND LEUKOCYTE COMMON ANTIGEN

The authors present an improved method for rapid two-color staining wi th direct conjugated antibodies to cytokeratin and CD45 antigen (leuko cyte common antigen) for whole-cell, ethanol-fixed preparations of hum an carcinomas. This method was quality controlled with the T24 human b ladder tumor cell line and compared in parallel analysis of 24 fresh h uman carcinomas with the original two-color method of multiparametric analysis that had been published in 1989. This rapid method was design ed 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 ac complished in a single (one-shot), abbreviated incubation with monoclo nal antibody diluted in propidium iodide-RNase, thereby eliminating tw o incubations and three wash steps required with the original method. The single rinse is done in the propidium iodide-RNase staining soluti on with resuspension in fresh staining solution before analysis. With the rapid method, the preparation time is reduced by 130 minutes, resu lting in a 60% time savings in batch staining mode compared with the o riginal method. The time reduction and fewer wash steps, which should avoid excessive cell loss and cytoplasmic stripping, may advance the a doption of this two-color method in clinical practice.