M-FISH, also referred to as multicolor FISH or multiplex FISH, can be viewed as fluorescent multicolor karyotyping and is mainly used for the detection and classification of interchromosomal aberrations (see Chapter 17). In this form of FISH, probes labeled with a combination of different fluors are hybridized with the chromosomes in a metaphase spread. Currently, 5 different fluoro-chromes are being used (7). The five different fluors give 31 (2n -1) color combinations, enough to uniquely identify the 24 different chromosomes in the human genome. However, lately it has been shown that the resolution of using a five-fluorochrome set is not high enough, and certain small aberrations might be missed (8). To improve resolution, a set of eight fluorochromes, to facilitate labeling each probe with a unique combination of two fluors, has been suggested. However, this would require nine filters (eight for the fluors and one for the DAPI counterstain) and would involve

Fig 11. Software interface of a spot counting or interphase FISH system, showing thumbnails of cells and spots located by the system. (Courtesy of Applied Imaging.)

some manual filter changing, as microscopes currently only accommodate eight-position filter wheels. Therefore, experiments with a seven-fluorochrome set have been performed, with promising results (8).

From a hardware perspective, the requirements of an automated system for M-FISH are similar to the requirements of an automated system for interphase FISH: the system should include the fluorescent epi-illuminating light source and a filter wheel containing the appropriate filters. In addition, the system could include a metaphase finding capability as well as motorized focusing. The software for M-FISH (see Fig. 13) incorporates the following:

• Sophisticated algorithms that analyze the images to determine the fluor combination a chromosome is labeled with and then assign a pseudocolor to each fluor combination. These pseudocolors should be user-changeable to improve visualization of rearranged chromosomes.

• Karyotyping capabilities so that the colored chromosomes can be arranged in a karyotype (see Chapter 17, Fig. 17).

• Individual pseudocolor display of a single chromosome to facilitate visualization of chromosomal aberrations.

High-Resolution Comparative Genomic Hybridization and Microarray CGH

The last forms of FISH discussed here are HR-CGH (high-resolution comparative genomic hybridization), and microarray CGH. Whereas M-FISH is a useful technique for determining inter-

Fig 12. Computer-controlled automated filter wheel. (Courtesy of Applied Imaging.)

chromosomal rearrangements, CGH will give insight in losses or gains of DNA within a chromosome (see Chapter 17). In CGH, the probes are generated from two different sources: one from genetically normal cells and the other from the patient sample. The two different probe sets are labeled with different fluors. These two pools of probes are then hybridized to a slide with normal metaphases. As the name indicates, the two probe sets will compete for hybridization to the corresponding loci. The ratio of the of patient DNA to normal DNA will indicate whether the patient DNA is normal (the ratio is 1 : 1) or whether there is an addition or deletion of DNA in any given region. When there is an addition, the ratio will increase; when there is a deletion, the ratio will decrease.

Until recent, this technique was able to pick up additions and deletions in the order of 10 megabase pairs (Mbp). However, current work has increased the resolution to the order of 3 Mbp (9).

Comparative genomic hybridization requires the use of a high-quality and quantitative FISH imaging system with a dedicated CGH suite. This software suite will perform the following:

• Accurately measure and average the ratio of the two fluors over multiple metaphases. This requires sophisticated algorithms.

• Correct the measurements for unequal chromosome length.

• Plot the ratios along the chromosome length for ease of interpretation, highlighting the areas of statistically significant differences (see Chapter 17, Fig. 15).

With microarray CGH, specific DNA targets are "printed" onto a microscope slide and CGH is performed in situ on the slide (see Chapter 17). A scanner reads the slide and sends the data to a computer for analysis (see Fig. 14).

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