Adverse Effect of Prolonged Formalin Fixation on DNA Histograms in Paraffin-Embedded Tissue

In a retrospective analysis of 174 formalin fixed, paraffin-embedded, hepatocellular carcinoma tissue samples, the correlation between the coefficient of variation (CV) as determined by flow cytometry and the duration of formalin fixation (DFF) was investigated. In addition a potential adverse effect of prolonged formalin fixation on CV and fluorescence intensity in paraffin-embedded liver tissue was also investigated prospectively in samples from four patients in which the DFF varied from three days to four weeks. In the retrospective study, there was a significant linear correlation between the CV and DFF (p<0.001), with the CV increasing as the DFF became prolonged. The proportion of samples in which the CV was less than 10.0 was significantly greater (p<0.01) in samples in which the DFF was less than two weeks. In the prospective study, a decrease in fluorescence intensity was observed as the DFF increased. Following two weeks of formalin fixation the fluorescence intensity decreased to 38.1% of the intensity observed in fresh samples. The mean CV was greater than 10.0 in the samples in which the DFF exceeded two weeks. It is concluded that prolonged formalin fixation has an adverse effect on CV. The DFF for flow cytometric analysis of paraffin-embedded tissue should be no greater than two weeks.