DNA image cytometry on sections as compared with image cytometry on smears and flow cytometry in melanoma

Abstract

DNA measurements of 130 melanomas were carried out by flow cytometry (FCM) and image cytometry (ICM). ICM was applied to cytological preparations of fresh material (cICM) and to sections of formalin-fixed paraffin embedded tissue (sICM). The DNA ploidy, the DNA index of G0/G1 peaks (DI), and the proliferation index (PI) were used to compare all the methods. The following parameters reflecting malignancy were calculated only from ICM histograms: the 5c exceeding rate (5cER) and the malignancy grade (MG). In cases found to be DNA aneuploid by FCM, the PI values (FCM versus cICM) and the DIs (between all methods) showed a high correlation, and the concordance in relation to the DNA ploidy status was 96% (FCM versus cICM) and 94% (FCM versus sICM). However, we ascertained essential differences between FCM and ICM in melanomas classified as DNA diploid by FCM. The concordance in DNA ploidy was only 66% (FCM versus cICM) and 64% (FCM versus sICM). In contrast, cICM and sICM yielded similar results in most cases. With the exception of the near diploid range, ICM is superior to FCM in detecting DNA aneuploidy. In particular, DNA tetraploid stem lines can easily be overlooked by FCM. Therefore, DNA measurements of tumours judged to be DNA diploid by FCM must be verified by ICM. ICM on sections proved to be applicable and yielded reliable results provided that a suitable thickness was used, and the measuring of sectioned and overlapping nuclei was largely avoided by careful focusing in either direction.