Multiplex fluorescence in situ hybridization (M-FISH).

Abstract

Multiplex in situ hybridization (M-FISH) is a 24-color karyotyping technique and is the method of choice for studying complex interchromosomal rearrangements. The process involves three major steps. Firstly, the multiplex labeling of all chromosomes in the genome with finite numbers of spectrally distinct fluorophores in a combinatorial fashion, such that each homologous pair of chromosomes is uniquely labeled. Secondly, the microscopic visualization and digital acquisition of each fluorophore using specific single band-pass filter sets and dedicated M-FISH software. These acquired images are then superimposed enabling individual chromosomes to be classified based on the fluor composition in accordance with the combinatorial labeling scheme of the M-FISH probe cocktail used. The third step involves the detailed analysis of these digitally acquired and processed images to resolve structural and numerical abnormalities.