RECOGNITION AND REDUCTION OF ARTIFACTS FROM AUTOLYSIS IN PARAFFIN-EMBEDDED TISSUE USING DNA NUCLEAR PROTEIN FLOW-CYTOMETRY/

Artifacts from autolysis can be a problem in retrospective flow-cytome tric analyses of DNA content in paraffin-embedded tissues. Autolyzed t issue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffin ized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propi dium iodide (PI) and fluorescein isothiocyanate (FITC) were used to st ain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4-degrees-C for 24-48 h before fixation, ther e was a progressive broadening of the G1 and G2M DNA peaks and a sligh t increase in the average DNA contents of these peaks. Nuclei that sta ined more intensely with PI also stained more intensely with FITC. Sim ilar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The dis tinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolys is, whereas the extended pepsin treatment provides a means for reducin g these artifacts.