Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin‐fixed lymphoid tissues

Abstract

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the Sphase fraction was higher in paraffin-embedded tissues than in unfixed cells. This differ--Cellular DNA content in human lymphoma cells has been analyzed extensively using flow cytometry (FCM). These studies have been performed primarily on cell suspensions obtained from unfixed tissues (3,4,(7)(8)(9)(16)(17)(18) and have provided information on ploidy changes and cell kinetic properties which are valuable in the detection and classification of lymphoid tumors. Recently, flow cytometric analyses of DNA content in cells from formalin-fixed, paraffin-embedded tissues have been successfully performed (2,6,10-12,151. This technique has allowed the retrospective clinical study of patients with a variety of neoplasms, including lymphomas (2,15). In our study, we applied this methodology to both neoplastic and nonneoplastic human lymphoid tissues. We tried to reproduce the analytical conditions used in the first flow cytometric study of DNA content in paraffin-embedded tissue by Hedley et al. (121, who used a mercury arc-based system and DAPI staining. The results were compared to those from unfixed samples from the same tissues. Since all unfixed lymphoma tissues had been previously analyzed on a laser-based ence could be attributed to 4', 6'-diamidino-2 phenylindole dihydrochloride (DAPI), a DNAbinding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of Sfraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.

DNA content in high and intermediate grade non-Hodgkin's lymphoma-prognostic significance and clinicopathological correlations

“…IAN --+---A f-ID ------A ..,.+11. AilOl--C (Bauer et al, 1986;McIntire et al, 1987), but not by others (Camplejohn & Macartney, 1985), and raises the possibility that the larger values of CV may mask minor degrees of aneuploidy. However, a comparative study of flow cytometric DNA analysis using fresh tissue (median CV = 3.1) and paraffin embedded tissue (median CV = 6.1) showed consistent identification of ploidy status in 33/35 tumours.…”

Prognostic implications of cell kinetics, histopathology and pathologic stage in non‐Hodgkin's lymphomas

“…Other cell kinetic variables such as mitotic index (Akerman et af., 1987) or flow cytometric Sphase cell fraction (Braylan et al, 1980, Scarffe andCrowther, 1981;Roos et al, 1985;Christensson et al, 1986;Macartney et al, 1986;Lenner et al, 1987;McIntire et al, 1987;Griffin et al, 1988) have been shown to be indicators of clinical aggressiveness. However, in these studies the survival curves of the two kinetic subgroups become similar after some years, whereas in our study they still appeared markedly different after 6 years.…”

“…Different methods, based on measurement of in vitro [3H]-thymidine (Steel, 1977) or bromodeoxyuridine (Gratzner, 1982) incorporation, on flow cytometric DNA content (Baisch et al, 1982) or on immunocytochemical detection of markers supposed to identify cycling cells (Gerdes et al, 1983), have been used to determine proliferative activity. Previous studies have shown that a high proliferating cell fraction determined by any of the different approaches is likely to be associated with high-grade malignancy histology and with a poor prognosis (Silvestrini et al, 1977;Braylan et al, 1980;Brandt et al, 1981;Costa et al, 1981;Scarf€e and Crowther, 1981;Gerdes et al, 1984Gerdes et al, , 1987Roos et al, 1985;Christensson et al, 1986;Del Bino et al, 1986;Macartney er al., 1986;Akerman et al, 1987;Gerdes et al, 1987;Lenner et al, 1987;McIntire et al, 1987).…”

Nuclear DNA Analysis of Prostate Tissues: Correlation with Stage and Grade of Tumour

“…All previous studies [9,11,12,17] relied on paraffin-embedded tis sues which offer the possibility for retrospective studies relating DNA ploidy to patient survival and prognosis. However, this approach suffers from a number of limita tions: the overall quality of DNA histograms of paraffinembedded tissue is generally inferior to that obtained with fresh tissue [13], and the DNA stability of tissue extracted from paraffin may vary from one specimen to another [ 12]. It was therefore decided to limit the present study to unfixed frozen tissues of which some specimens had been stored at -7 0 °C for up to 4 years.…”

“…Furthermore, in an attempt to overcome the reported difficulties associated with the use of paraffinembedded tissue for flow cytometric DNA determina tions [12,13], the present study was confined to unfixed frozen tissue.…”