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METHODS AND TECHNOLOGIES

Fluorescence in situ hybridization (FISH)

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Fluorescence in situ hybridization (FISH) is a molecular technique where fluorescence-labeled DNA probes hybridize directly onto chromosomal preparations. This technique employs various types of probes: centromeric probes that target the centromeres of specific chromosomes, painting probes that stain entire chromosomes, and locus-specific probes designed to mark particular DNA segments.

Applications in hematological samples

FISH offers significant benefits for hematological samples, particularly because it enables rapid and reliable detection of disease-related regions without requiring cell culture. Even in interphase cell nuclei, FISH can identify genomic aberrations with a higher sensitivity than traditional banding techniques, which often underestimate their prevalence. The sensitivity of FISH allows detection of one aberrant cell among 1,000 normal cells. This makes it invaluable for identifying translocations, rearrangements, deletions, and genetic gains using specialized split or break-apart probes and locus-specific probes, even when tumor cell chromosomes are of poor quality or during disease progression.

Applications in tumor diagnostics

FISH can also be performed on formalin-fixed, paraffin-embedded (FFPE) tumor sections. For instance, it is used to detect oncogene amplifications that lead to increased gene activity. An example is the HER2/neu gene, which is amplified in approximately 20–30% of breast cancer cases and results in overexpression of the HER2/neu protein. These patients benefit from targeted therapies with HER2/neu antibodies (like Herceptin).

Prenatal diagnostics with FISH

A specialized application is the FISH rapid test in prenatal diagnostics. FISH is used to examine uncultured amniotic cells for numerical abnormalities in chromosomes 13, 18, 21, X, and Y, providing results within 24 hours.

Subtelomere and structural analysis

FISH is also used in subtelomere diagnostics, where subtelomere-specific probes evaluate the integrity of all subtelomeric regions. Chromosome-specific painting probes are commonly used to identify interchromosomal structural changes such as translocations and insertions. These probes selectively stain the euchromatic regions of individual chromosomes.

Advanced Techniques: M-FISH

An advanced extension of the chromosomal painting technique is multicolor or multiplex FISH (M-FISH). This technique uses combinations of five fluorescent dyes to individually stain all 24 human chromosomes. M-FISH is particularly useful for characterizing marker chromosomes and analyzing complex rearrangements. In molecular cytogenetic tumor diagnostics, it provides additional insights into complex karyotypes.

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