Reproducibility of FCM DNA content from replicate paraffin block samples

Fifty-five paraffin blocks of morphologically and clinically normal colon specimens were divided in half and the two halves treated as separate aliquots for determination of DNA content using propidium iodide staining and FCM analysis. Forty-five of the 55 samples showed a peak channel difference between the two aliquots from the same block of greater than 1 channel. Seven of the 55 specimens showed peak channel difference greater than or equal to 10 channels. Similar results were seen if the mean channel of the G0G1 peak was used in place of the peak channel, with 5 of the 55 specimens showing mean channel differences of greater than or equal to 10 channels. The seven samples with peak channel difference greater than or equal to 10 channels were reprocessed and five of the seven showed reduced peak channel difference on reanalysis. The mean peak channel difference of the 55 samples was 4.3 +/- 10.5 (mean +/- 2 S.D.) using the first analysis. These data suggest that caution should be used in interpreting ploidy of nuclei extracted from paraffin block material if the interpretation is based on comparing two aliquots from the same block, even if they are processed and analyzed in the same batch.