The study of chromosomes in the detection of genetic abnormalities is called cytogenetics.
Fluorescence in situ hybridization (FISH) combines conventional cytogenetics with molecular genetics and can be used in hematology, pathology and constitutional cytogenetics. It is a technique that uses fluorescently labeled DNA sequences (probes) to assess specific regions of interest, such as genes and centromeres, using fluorescence microscopy.
The DNA sequence of the FISH probe is complementary to the region of interest. When using a FISH probe, if the region of interest is present, the probe will bind (hybridize) to the target DNA sequence, which can then be visualized using fluorescence microscopy – indicating the presence of the gene/centromere etc. If the region of interest has been deleted, the probe will not bind to the target DNA and so will not be visualized. A breakapart or a translocation FISH probe can be used to detect whether a gene has been rearranged.
Figure 1. Schematic of the fluorescence in situ hybridization (FISH) process
A FISH experiment is a very simple process that requires a double stranded fluorescently labeled probe to be added to your cell sample (bone marrow, peripheral blood, urine) or FFPE tissue (breast, lung, muscle) or, more specifically, target DNA.
Figure 2. Illustration of a double stranded fluorescently labeled probe added to target DNA
FISH on cell samples: As a first step for FISH on cell samples, the sample will be cultured, harvested and fixed to arrest cell division. It’s also important to note that FISH can be carried out on uncultured material such as blood, bone marrow, amniocytes.
The harvested, fixed-cell pellet is resuspended in Carnoy’s solution or alternative fixative solutions, which is used to apply a drop onto a microscope slide. The prepared sample slide will then follow recommended pre-treatment steps. The fluorescently labeled DNA probe will then be applied onto the microscope slide containing the cell sample.
FISH on tissues: As a first step for FISH on tissue samples, the tissue blocks are sectioned into thin ribbons, mounted onto slides and baked overnight. The tissue slides are then treated with organic solvents to remove the paraffin wax, leaving only the tissue attached to the slide.
The tissue slide undergoes pre-treatment and digestion to create an optimal tissue structure for FISH. Then, a fluorescently labelled DNA probe will be applied onto the microscope slide containing the tissue section.
It is important that sample slides and tissue slides are prepared fresh on the same day as the FISH assay is going to be carried out. OGT also recommends that FISH probes should be kept in the dark as much as possible throughout use as they can degrade when exposed to light.
A coverslip is then laid on top of the probe and then the edges of the coverslip are sealed with a rubber glue or equivalent.
View our tips for FISH sample preparation and tips for FISH probe application
The second step in FISH involves the denaturation of the cell sample/tissue and FISH probe together, which requires heating them at 75°C plus or minus 1°C for two minutes (for cell samples) and five minutes (for tissue) in order to separate the DNA strands and allow the fluorescently labeled probe to access the target DNA.
Figure 3. Illustration of denaturation of the DNA sample and FISH probe
This step is most often performed on an automated hybridization unit such as a ThermoBrite or hotplate. Note it is important to put proper QC measures in place to ensure accurate denature temperatures as some units are notorious for inaccurate digital readouts.
View our tips for FISH denaturation
The third step is hybridization. This is where the sample slide or tissue slide is maintained at 37°C (+/- 1°C) in a humid environment for a defined period of time (typically overnight) to enable the binding of the fluorescent probe to the target DNA. Shorter hybridization times are available for specific probe products. The full details can be found within a specific product’s instructions for use, or IFUs.
Figure 4. Illustration of the hybridization of the FISH probe to the target DNA
As a final part of the hybridization step, you must wash the sample slide twice after the hybridization period is finished. The first wash occurs in 0.4xSSC solution at 72°C for two minutes.
The second wash occurs in 2xSSC, 0.05% Tween-20 at room temperature for 30 seconds.
Both solutions should be at pH 7 and will aid in removing any unbound and non-specifically binding probe.
The post-hybridization washes are generally performed in 0.4xSSC and 2xSSC, 0.05% Tween-20, but these concentrations can vary depending on the product undergoing FISH. The full details can be found within a specific product’s instructions for use, or IFUs.
A fluorescent DNA counterstain such as DAPI is then applied after the washing steps have been completed to aid in visualization of interphase nuclei and metaphase chromosomes under a fluorescence microscope.
View our tips for FISH hybridization and tips for post-hybridization washes
The final step requires you to analyze your cell sample/ tissue with the assistance of a fluorescence microscope equipped with different filters, most commonly DAPI, Texas Red, FITC - which is a green fluorophore, Orange, Gold and Aqua. These can be use individually or in combination (dual or triple filters).
This step requires you to identify the fluorescent probe signals and count different color patterns. Normally, the color pattern identified on normal samples/ tissues from healthy patients that do not exhibit the genetic abnormality the probe was manufactured for, would produce two signals in the color your probe was labelled, seen in every analyzable cell. The color pattern identified on abnormal samples/ tissues from sick patients that exhibit the genetic abnormality the probe was manufactured for, would vary depending on the sickness. Refer to specific product IFUs for expected signal patterns.
Figure 4. Illustration of FISH analysis using a fluorescence microscope
There can be significant variability in terms of numbers of cells analyzed depending on the reason for referral, sample type and a laboratory’s own internal guidelines. For example, within OGT’s hematology FISH protocol it is recommended that one technologist count 100 interphase cells and a second technologist counts a further 100 interphase cells from a different area of the hybridization.
View our tips for FISH analysis
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