An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH)

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromo somes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we pres ent several significant optimizations of the M-FISH technology. First, a ne w strategy for labeling the probes is described which allows for easy and f ast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter whee l and a new generation of filter sets is presented that allows fully automa tic imaging of a complete metaphase spread within seconds. Third, to determ ine the characteristic spectral signatures for all different combinations o f fluorochromes, we developed a novel multichannel image analysis method. T he spectral analysis is solely guided by the image information itself and d oes not require any user interaction. A complete analysis of a metaphase sp read can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH imag es as a simple means of controlling the computer-generated chromosome class ification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.