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Fluorescent in situ Hybridization (FISH)
Fluorescent in situ hybridization, or FISH, detects specific DNA sequences in intact chromosomes. Hybridization refers to the ability of DNA to hybridize, or anneal, or "stick" to any DNA which has a complementary sequence. For instance, let's say there is a sequence on a chromosome like the one beginning "C-A-G-C-" as illustrated below. If this is part of a sequence known to be found only in leukemia cells, they synthesize the complementary oligomer and add it to a spread of intact chromosomes. They also tack a fluorescent molecule onto the oligomer. This oligomer-fluorescent dye is called a "probe".
Then, they allow these probes to anneal ("hybridize") to preparations of chromosomes. After washing the preparations to remove any probe bound to non-target sequences, they view the samples by fluorescent microscopy. If the oligomer-dye is hybridized to a chromosome, it shows up quite readily.
FISH techniques require good samples of cells, since the cells must be cultured and dividing. It also requires an appropriate probe, which may or may not be available for a particular disease. Historically, it has been used as a diagnostic tool to look for known chromosome abnormalities, such as chromosome translocations or aneuploidy. According to Molecular Diagnostics(1), in situ hybridization techniques are severely limited by the lack of established laboratory standards with respect to this technique and by the large number of variables that affect the final outcome. Leading investigators in the field use markedly different protocols, making interpretation of data difficult.
FISH in the detection of MRD
Several groups have used FISH technology to detect MRD in ALL.(15, 16, 17, 18, 25) These groups claim their respective protocols have sensitivity on the order of 10-4. FISH has the potential to be used whenever cytogenetic changes are found in a leukemic clone. As of 1999, there has not been a large-number study published which employs FISH to detect MRD in childhood ALL and correlate the presence of MRD with relapse statistics.
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