Flow-cytometric analysis of the DNA content in paraffin-embedded tissue and in fresh tumour samples obtained from renal cell carcinoma: comparison of DNA ploidy using ethidium bromide and mithramycin.

With the help of the deparaffination technique described by Heldly, it has become possible to conduct large-scale flow-cytometric (FCM) studies, including retrospective DNA analysis of renal cell carcinoma (RCC). The quality of these histograms, however, has to be comparable to the quality of the analyses from fresh tumour tissue. The present study compares the application of FCM on fresh tumour tissue from RCC with paraffin-embedded archive material, thus for the first time, the same combination of stains (ethidium bromide/mithramycin) was used for both materials. From a total of 44 tumours, 125 histograms were obtained from fresh tumour tissue. Aneuploidy was found in 78 (62.4%) of the DNA analyses (coefficient of variation, CV = 5.5%). These 78 aneuploid histograms were compared with 228 sections from the archive material which had been deparaffined and stained. This involved, a deparaffinising period of twelve hours as well the application of pepsin and protease, which was necessary to obtain high-quality histograms (CV = 6.0%). Retrospectively, a total of 58 histograms from 36 cases were available for comparison. Of the archive material, 23/36 tumours were classified as aneuploid; thus new stem cell lines could be detected retrospectively in 63.8% of the cases. The comparison of DNA indices (DI) in the two series produced no significant differences, and the comparison of the mean values for the CV-5.5% for native material histograms and 6.0% for archive material histograms-was not statistically significant. The detritus part in the archive histograms revealed a mean value of 6.3% for native histograms the mean value was 13.4%. These differences were also not significant. These results show that, using the staining combination ethidium bromide/mithramycin, the quality of the histograms obtained with archive material is equal to the quality of the histograms obtained from fresh tumour material. Furthermore, when taking into consideration the clonal heterogeneity of RCC, it is possible to use retrospective analysis to detect aneuploidy with a high degree of certainty and accuracy.