Fluorescence in Situ Hybridisation - a useful tool in cytology diagnostics and research

Fluorescence in situ hybridisation – a useful tool in cytology diagnostics and research Fluorescence in situ hybridisation (FISH) is a hybrid of cytogenetics and molecular biology. It allows the detection of DNA sequences on metaphases and interphase nuclei and can be used on a wide variety of tissues and cells. It is utilised in aneuploidy detection, translocation- and structural breakpoint analysis, microdeletion detection and gene mapping. It is performed in situ thus making it possible to correlate the localisation of the FISH signal with, for example, conventionally stained cytological material. The double-stranded target DNA and the DNA probe are denatured and rendered single-stranded. Under the following cooling process the probe will bind to the target DNA and can be visualised using a fluorescent (FISH) or chromogenic marker (CISH). Air-dried FNA smears are ideal for FISH as they present whole (interphase) nuclei thus avoiding truncation artefacts and pre-treatment procedures are greatly simplified since protease treatment is unnecessary. There are three main types of FISH probes: Whole chromosome probes, centromeric DNA probes and unique-sequence DNA probes. Unique sequence probes target specific DNA sequences that are not repeated in the genome such as genes. Centromere and locus-specific probes are suitable for interphase chromosomes and may be combined to distinguish true gene amplifications from chromosome polysomies and as such are suitable for cytological smears and biopsies. The number of nuclei necessary for the assessment of chromosome or gene amplification has been estimated to be as low as 20 in a section or smear. Problems encountered in the interpretation of FISH may caused by the type of material, smears with necrotic debris or blood, or the age of the smear, though we have successfully hybridised FNA smears after 10 years of storage at -80C. FISH is validated for use in the routine diagnostic situation, for determination of treatment strategies and is a useful tool in translational research projects.