Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometry

A flow cytometric assay was developed for correlated measurement of DNA con tent and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, para ffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archiv al or fresh tissue/cell specimens. It is based on (a) proteolytic release o f cell nuclei from deparaffinized and rehydrated 90-mu m thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC -labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA s taining with 4'6-diamidino-2-phenyleindole. The fluorescence was recorded w ith a double-beam flow cytometer equipped with a mercury are lamp and an ar gon ion laser. Cytograms obtained with this assay correlated closely with t hose produced using nonembedded material from the same specimen. Furthermor e, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional eva luation of TUNEL on (corresponding) sections (p<0.001). Since necrotic cell s can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important adva ntage of the assay.