Caveats in the use of archival cell material for DNA ploidy analysis by image cytometry

Monolayer preparation from paraffin-embedded tissue from archival biopsy specimens older than 10 years was shown in this study to be more difficult to deal with than recent biopsies. We have studied the disseminative effect of several enzymes on archival biopsies from laryngeal epithelium, as well as mechanical dissemination. Thirty minutes incubation in protease 0.5% in 37 degrees C without syringing provided the best cell yield. Hydrolysis and Schiff staining conditions were also studied. Cell loss was calculated by stereological methods and found to be high in most cases. Cell loss during various steps in monolayer production was examined and we found no proof of selective cell loss. Large variations in Feulgen-Schiff staining intensities were observed, especially in biopsy specimens older than 10 years. The use of reliable internal (intrinsic) control cells is advocated since interspecimen variations in the diploid integrated optical density (IOD) can be expected. Although DNA measurements on archival biopsy material should be treated with caution, we demonstrate that meticulous studies of the preparation procedures will make it possible to obtain reliable histograms from old, archival biopsies.