DNA flow cytometry of cells obtained from old paraffin-embedded specimens. A comparison with results of scanning absorption cytometry (a methodological study)

Abstract

Suspensions of single cell nuclei were obtained from 31 different samples of 7-9 years old paraffin-embedded bladder cancer biopsies. The DNA content of the ethidium-bromide-stained nuclei was analyzed by flow cytometry (FCM). The tumour stemline ploidy, as determined by FCM, was compared with that obtained by Feulgen scanning absorption cytometry (SACM) in imprints obtained from the same biopsy before it was fixed. Fifteen tumours that were diploid by FCM were also diploid by SACM. All of the 16 tumours with non-diploid DNA-stemlines by FCM were non-diploid by SACM, though minor differences between the ploidy values were occasionally seen when the results of the two methods were compared. The background activity due to cell debris was considerable and resulted in a mean variation coefficient (CV) of 5.1% for human spleen cells fixed and embedded before preparation for FCM in the same way as the tumour samples. In the tumour samples there was a large and unpredictable variation of the ratio between the DNA values of chicken erythrocytes (internal standard) and diploid cells. In some specimens this ratio would have resulted in incorrect evaluation of the FCM DNA histogram. The following method for evaluation of FCM histograms is therefore proposed: Single peak histograms obtained from paraffin embedded tissue should always be interpreted as representative for a diploid cell population. In FCM histograms from paraffin-embedded tissue with more than one peak, the first peak should be considered as representing diploid cells.