Fluorescent In Situ Hybridization or FISH Test

Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique used for the detection and localization of chromosomal sequences within tissues or cells.

In this technique, fluorescent probes are used which bind to complementary chromosomal sequences, which can be visualized using a fluorescent microscope.

It helps to visualize and map the genetic material in an individual cell, including specific genes or portions of genes. This makes it an important tool for understanding a variety of chromosomal abnormalities and other genetic mutations.

In classical type of cytogenetic staining, a dye binds to the DNA or protein of a chromosome and allows visualization by light microscopy.

ISH is a technique where instead of a dye; a molecular probe binds to the chromosome. In early studies, the probe was labeled with a radioactive compound usually tritium so that after 5-10 days of exposure to X-ray film, a pattern of silver grains could be detected identifying the chromosomal location of the probe.

This method of autoradiography facilitated gene mapping but had some disadvantages. Only one probe could be used as it required longer time. Moreover it required statistical analysis of data obtained by scattering of silver grains.

So in mid 1980’s a variation of this technique i.e., FISH was developed which was quicker, more specific and allowed the use of multiple probes in a single hybridization procedure.

In this technique, instead of radioactively labeled probes, fluorescent dye is used which is visualized using a fluorescent microscope.

Fluorescent dye, a fluorochrome is a substance which absorbs light of lower wavelength and emits light of longer wavelength.

Principle and Technique of FISH

FISH uses small DNA strands called probes that have a fluorescent label attached to them. The probes are complementary to specific parts of a chromosome. When DNA is heated, the patient’s two DNA strands break apart, or denature, and the probes are able to hybridise to their complementary sequence in the patient’s DNA.  If a small deletion is present in the region complementary to the probe, the probe will not hybridise. If a duplication is present, more of the probe is able to hybridise.  These fluorescent labeled probes are detected using a fluorescent microscope.

Cells are cultured and harvested and slides are made. Like ISH and unlike other hybridization based methodologies like PCR, etc the sampled DNA is detected in the intact cell ie. in situ.

The difference between ISH and FISH is that in FISH, the probes used are tagged with fluorochromes emitting green (fluorescein); or red (rhodamine or texas red) or blue (7-amino 4-methylcoumarin-3 acetic acid or cascade blue fluorescence) colour.

Concurrent hybridization with 2 or more of these fluorochromes allows for simultaneous assessment of 2 or more sequences of interest. A maximum of 3 colours can be visualized on a typical fluorescent microscope. However, superior results are obtained by using a computer assisted system to obtain the fluorescent images.

The most critical element in FISH is selection of a specific probe. The gene must be known and the probe used must be homologous to the region of the gene that is to be detected.

Types of probes

A probe is a single-stranded DNA or RNA fragment used search for a particular gene or other DNA sequence. The probe has a base sequence complementary to the target sequence and will thus attach to it by base pairing.

Locus-specific probes

These probes bind to specific gene-containing parts of the DNA and are used to detect presence or absence of that gene.

Repeat sequence probes

They are isolated from telomere and centromere regions. Centromere probes are used to enumerate chromosomes. Telomere probes are used to detect rearrangements, that is to determine if both telomeres of a chromosome are present and located on correct chromosome or if a deletion or rearrangement has occurred.

Chromosome painting probes

They are a cocktail of unique DNA fragments which bind to the whole chromosome.

Following image provides an example of FISH for deletion.

Samples needed for FISH testing

FISH is most commonly performed on blood samples. Body fluids, bone marrow aspirate and tissue obtained through techniques like FNAC, etc can be used to perform FISH.

For prenatal testing, amniotic fluid obtained by amniocentesis or placental samples obtained by chorionic villus sampling can be used.

It can also be performed on tissue embedded in paraffin blocks.

Important Variations of FISH

Chromosome painting

is an extension of FISH, whereby whole chromosomes can be labeled with a series of fluorescent DNA probes that bind to multiple sites along a particular chromosome.

The number of chromosomes that can be detected simultaneously by chromosome painting is limited by the availability of fluorescent dyes that excite different wavelengths of visible light. Thus, chromosome painting has limited ability to visualize all 46 human chromosomes simultaneously. This hurdle has been overcome by the introduction of spectral karyotyping (SKY).

Spectral karyotyping

Spectral karyotyping uses a combination of five fluorochromes and appropriate computer-generated signals. This enables the entire human genome to be visualized.

Comparative genomic hybridization or CGH

It compares the DNA content of differentially labeled normal and tumor cell populations by their co-hybridization to normal metaphase chromosome spreads. If a series of genomic DNA clones aligned on glass slides are compared the technique is called array comparative genomic hybridization. In this manner, tumor-specific alterations in gene copy number can be ascertained.

Applications of FISH

FISH can be used to identify the location of a cloned DNA sequence on metaphase chromosomes. Historically, FISH and other in situ hybridization results played a major role in mapping genes on human chromosomes. This information proved useful for compilation of Human Genome Project (HGP). Since the HGP is now complete, FISH is rarely used now to simply identify the chromosomal location of a human gene.  It can however still be used to map the positions of genes on chromosomes in species for which the genome has not yet been sequenced.

Currently, Fish has widespread applications in diagnostic and research techniques.

Diagnosis of genetic diseases

FISH can be used to detect chromosomal abnormalities like deletions, gene fusions, aneuploidy, translocations, trisomies and monosomies in interface and metaphase nuclei. Down syndrome, Prader-Willi syndrome, Angelman syndrome, etc can be detected using this technique.

The most useful application is the detection of microdeletions too small to be seen using classical cytogenetics.

Absence of the fluorescent signal indicates that a deletion exists. As there is no DNA sequence present on the chromosome that is complementary to the probe, no hybridization occurs. An unaffected person will show 2 signals per cells, one signal for each chromosome. In a person with deletion, there will be a single signal per cell showing one normal and one deleted chromosome.

Prenatal diagnosis of disease

FISH performed on sample obtained using chorionic villus sampling and amniocentesis can determine prenatal diagnosis of various chromosomal disorders.

Role in oncology

Fish has a role in diagnosis, evaluating prognosis as well as remission in cancer. It can also be used for detection of minimal residual disease.

In chronic myeloid leukemia (CML), translocation results in BCR/ABL fusion gene also called Philadelphia chromosome. FISH assay is used as the gold standard for detecting this chromosomal translocation and selecting a targeted therapy.

Detection of MDM2 amplification and expression using FISH technique is used to distinguish dedifferentiated liposarcoma from other high-grade pleomorphic sarcomas. This distinction is crucial since dedifferentiated liposarcoma has a much better prognosis as compared to other high-grade pleomorphic sarcomas

Infertility

FISH performed on sperm cells can be used to detect abnormal somatic or meiotic karyotype. Majority of in vitro fertilization (IVF) pregnancy losses are associated with chromosomal abnormalities in the embryo. Performing genetic diagnosis prior to embryo implantation could prevent the initiation of abnormal pregnancies in IVF patients.

Identify Pathogens

Bacteria or virus from a small sample of patient’s tissue can be detected using FISH. This provides superior results and requires a much shorter time as compared to conventional culture methods.

Track origin of cells after bone marrow transplantation

FISH can be used to detect proportion of recipient to donor cells in patients who have received bone marrow transplantation.

To compare the genomes of two biological species to deduce evolutionary relationships.

Research applications

Fish can be used to identify non-random chromosome rearrangements, amplified genes, translocation molecular break point, characterization of somatic cell hybrids and to study the mechanism of rearrangements.

It can be used as a tool to physically map newly isolated genes of clinical interest. Gene mapping or identification of novel oncogenes or genetic aberrations that contribute towards various diseases can be carried out.

Advantages of FISH technique

• It is quicker
• It is more specific
• It can be performed on cytological specimens in addition to paraffin embedded sections
• Multiple probes can be applied to a single slide.

Disadvantages of FISH technique

• Less sensitive than PCR (polymerase chain reaction) and Southern blotting.
• It does not usually screen all chromosomes for changes. Most FISH probes are specific for one particular deletion or duplication within one band of one chromosome so it will only find what it is looking for.
• Requires a fluorescent microscope and maximum limit of visible colors is three. This has been overcome by spectral karotyping ie by using a combination of five fluorochromes and appropriate computer generated signals.
• It requires a unique hybridization probe for each genetic defect.
• In deletion analysis it reads lack of signal as a positive indication of deletion, but this can result from failure of hybridization. To eliminate this as a source of error, a minimum of 20 cells must be evaluated and all the cells must agree in signal count.
• If mosaicism is suspected, additional cells must be surveyed or a control probe with same or different fluorochrome may be used to ensure hybridisation.