Fluorescence in situ hybridization (FISH): a user's guide to optimal preparation of cytologic specimens

Fluorescence in situ hybridization (FISH) is a reliable method for tagging centromeric regions of specific chromosomes in interphase nuclei. Not only is FISH useful for chromosome enumeration, but as region-specific chromosome probes are developed, the clinical applications and potentials for use by pathologists are extensive. This technique lends itself particularly to use in cytology preparations because the cells are disaggregated and monolayer preparations yield excellent technical hybridization results. Over a 7-mo period we processed cytologic samples in an attempt to define and outline a method for optimal specimen processing for FISH use in cell suspensions, techniques applicable to all fresh cytology specimens which can also be used for the processing of surgical pathology aspirates and other material. All samples should be promptly processed to ensure specimen viability, and triaged on an individual basis to ensure preparation of moderately cellular monolayered cytospins. Equivalent nuclear probe signals have been obtained with several sample fixation methods: air-drying, 95% ethanol, methanol (Diff-Quik fixative), and Carnoy's solution. No difference was noted in the nuclear probe signals or specimen adhesion on positively charged or noncharged slides. After initial fixation our slides remained at room temperature until FISH was performed, without any adverse effects. A short digestion with proteinase K and subsequent rehybridization yielded positive results on samples that originally yielded poor nuclear probe signals.