Fluorescence in situ hybridization method in isolated single nuclei extracted from paraffin-embedded hepatocellular carcinoma tissues

Abstract

Background: Genetic analysis using the fluorescence in situ hybridization (FISH) method applied to intact tissue sections of formalin-fixed paraffin embedded (FFPE) tissue is well known to be relatively difficult. The frequent technical problems include unsuccessful hybridization as a result of poor probe penetration, excessive probe requirement, excessive background, auto-fluorescence, and overlapping or incomplete nuclei. These problems lead to absence or insufficiency of fluorescent signals, resulting in an inaccurate analysis. Formalin-fixed paraffin embedded tissue can be analyzed either as intact tissue sections or as a suspension of disaggregated, but intact, nuclei. Intact tissue sections have the advantage of preserved tissue architecture and morphology but have the intrinsic disadvantage of poor probe penetration, overlapping or incomplete nuclei and auto-fluorescence, accordingly reducing the accuracy of fluorescent signals evaluation. Objective: To present the effective FISH method applied to isolated of single nuclei and the procedures for isolation of a single nuclei from formalin-fixed paraffin embedded tissues of hepatocellular carcinoma. Material and Method: Ten paraffin-embedded blocks of hepatocellular carcinoma tissues from the department of pathology, Ramathibodi hospital, Thailand were studied. Isolated single nuclei were extracted from 10-□m sections of paraffin-embedded blocks of hepatocellular carcinoma tissue and hybridized with alpha-satellite centromeric DNA enumeration probes for chromosomes X (CEP X, spectrum green) and satellite III for chromosomes Y (CEP Y, spectrum orange). The signal of, at least, 200 interphase nuclei were counted from each specimen. Results: The efficacy of this method has been evaluated in 10 formalin-fixed paraffin embedded tissue of hepatocellular carcinoma. The results showed bright, planar and an easy to score signal. Conclusion: FISH procedure described here is particularly suitable for retrospective studies of genetic aberration applied to formalin-fixed paraffin embedded tissues.