DNA flow cytometry of breast carcinoma after acetic-acid fixation

Aqueous acetic acid was used to fix and store specimens of tissue prior to dissociation into nuclear suspensions for flow cytometric quantitation of DNA. The optimum concentration was 20 volumes of glacial acetic acid in 80 volumes of distilled water. Both neoplastic and benign nuclei were easily released from the fixed tissue blocks by slicing and shaking. Residual undissociated tissue was suitable for microscopic examination to confirm its identity. The nuclei fluoresced brightly after staining with propidium iodide, and yielded histograms similar to those from unfixed samples. Acetic-acid fixation resulted in slightly broader G1 and G0 peaks in the DNA histograms in comparison to unfixed cells, but fluorescent debris was less and correlation between the flow cytometric S-phase fraction (SPF) and in vitro thymidine labelling index (TLI) was better than with unfixed cells. Twenty-one of thirty-nine acetic-acid-fixed breast carcinomas had DNA indices in excess of 1.0 (increased nuclear DNA content in comparison to benign cells), and eighteen had DNA indices of 1.0 (normal or near-normal). The SPF was usually in excess of the TLI, but the two were significantly correlated (r = 0.72, P less than 0.0001). However, a significant correlation of SPF with TLI held only for the group with DNA index greater than 1.0. DNA indices greater than 1.0 were associated with high SPF and TLI, and high SPF and TLI each associated with low content of estrogen receptor.