Laboratory Methods

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Fluorescence in situ hybridization (FISH)

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

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 tumour 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 labelled 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 (deleted genes) or have been moved (translocated genes). Increased numbers of chromosomes, as seen in certain genetic disorders, can also be diagnosed using FISH technologies (trisomy 21 or Down’s 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 labelled 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’s 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’s 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 the clinical diagnosis of trisomy 21 in this baby. The doctors and genetic counsellors will work with the family to help them understand the results of the test. 
Breast cancer
In Figure 2, FISH is used to assess breast tumour 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 tumours may be 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 will be considered. 
Figure 3 shows FISH used in a particular type of chronic leukaemia, chronic myelogenous leukaemia (CML). The specific probes used in this case detect BCR-ABL, an abnormal gene sequence formed by the exchange (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 may receive benefit from molecular-targeted drugs, such as imatinib. 

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