Laboratory Methods

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Fluorescence in situ hybridization (FISH)

This molecular testing method uses fluorescent probes to evaluate genes and/or DNA sequences on chromosomes.

DNA double helix

Humans normally have 23 pairs of chromosomes: 22 pairs of non-sex-determining chromosomes (autosomes) and 1 pair of sex chromosomes (XX for females and XY for males). Chromosomes are made up of DNA, repeating sequences of four bases that form the thousands of genes that direct protein production in the body and determine our physical characteristics. DNA consists of two strands bound together in a double helix structure (like a spiral staircase). Each half of the helix is a complement of the other.

For a FISH test, a sample of a person's cells containing DNA is fixed to a glass slide. Samples can include blood, bone marrow, amniotic fluid, or tumor cells, depending on the clinical indication. The slides with the "target" (person's) DNA are heated to separate the double strands of DNA into single strands. Fluorescent probes are then added to the sample. Fluorescent probes are sections of single-stranded DNA that are complementary to the specific portions of DNA of interest. The probe, which is labeled with a fluorescent dye, attaches to the specific piece of DNA. When the slides are examined using a special microscope, the genes that match the probe can be seen as areas of fluorescence, which will appear as bright spots on a dark background.

This technique can be used to show the presence of extra gene copies (duplicated or amplified genes), and genetic sequences that are missing (gene deletions) or have been moved (translocated genes). Increased numbers of chromosomes, as seen in certain genetic disorders, are also diagnosed using FISH technologies (trisomy 21 or Down syndrome, for example). The targeted area(s) or sequences of DNA are determined by the probes that are used. Multiple targeted areas in the DNA can be assessed at the same time using FISH probes labeled with a number of different fluorescent dyes.

The following photographs show cells that have been evaluated using the FISH methodology. These are just a few examples of the use of FISH technique.

Down syndrome
In Figure 1, FISH testing is applied to cells in amniotic fluid, obtained from a pregnant woman carrying a baby suspected of having Down syndrome (trisomy 21). Three copies of chromosome 21 are observed (red signals). The green signals (two copies) are for chromosome 13; these are for control purposes and show that the test is working properly. FISH supports a clinical diagnosis of trisomy 21. The doctors and genetic counselors will work with the woman to help her understand the results of the test.

Cell positive for trisomy 21 by FISH

Breast cancer
In Figure 2, FISH is used to assess breast tumor cells for the presence of an amplified gene, HER-2/neu (red signals). In approximately 25% of breast cancers, HER-2/neu is amplified. Women with amplified HER-2/neu tumors are treated with a drug (Herceptin) that targets the protein that is the product of the abnormal gene. If a woman is NOT positive for HER-2/neu amplification, she is not likely to receive any therapeutic benefit from Herceptin therapy and other drugs are considered.

Breast tumor cells positive for Her-2/neu

Leukemia
Figure 3 shows FISH used in a particular type of chronic leukemia, chronic myelogenous leukemia (CML). The specific probes used in this case detect BCR-ABL, an abnormal gene sequence formed by the translocation of a portion of chromosome 22 (BCR, a green probe) with a portion of chromosome 9 (ABL1, a red probe). The areas of yellow fluorescence signify the abnormal, fusion gene (joining of red and green probes). Finding the BCR-ABL fusion confirms a diagnosis of CML. BCR-ABL positive patients receive benefit from molecular-targeted drugs, such as imatinib.

Cell positive for BCR-ABL by FISH

Sources

(August 16, 2010) Fluorescence In Situ Hybridization (FISH). National Human Genome Research Institute [On-line information]. Available online at http://www.genome.gov/10000206 through http://www.genome.gov. Accessed March 2011.

(August 16, 2010) Frequently Asked Questions about Genetic Testing. National Human Genome Research Institute [On-line information].Available online at http://www.genome.gov/19516567 through http://www.genome.gov. Accessed March 2011.

(March 6, 2006) Genetics Home Reference. Fluorescent in situ hybridization. Available online at http://ghr.nlm.nih.gov/glossary=fluorescentinsituhybridization through http://ghr.nlm.nih.gov. Accessed March 2011.

(June 29, 2011) Hiller B, Bradtke J, Balz H and Rieder H (2004). CyDAS Online Analysis Site. Available online at http://www.cydas.org/OnlineAnalysis/ through http://www.cydas.org. Accessed July 2011. 

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