Fluorescence In Situ Hybridization (FISH)

General Description

Fluorescence in situ hybridization (FISH) is a cytogenetic technique that allows for the detection, identification, and localization of specific nucleic acid sequences (DNA or RNA) from a cell or tissue sample.

The technique is based on the use of fluorescent DNA probes and the ability of nucleic acids to hybridize with each other through complementary base pairing. It is a highly sensitive method for identifying clinically significant chromosomal abnormalities and diagnosing the corresponding genetic disorder. Additionally, unlike other conventional cytogenetic techniques, FISH does not require cells to be in the mitotic phase to be performed.

When is it indicated?

The FISH technique is primarily applied in prenatal, preimplantation, and oncological diagnosis:

• Diagnosis of syndromes caused by aneuploidies (alterations in the number of chromosomes), such as Down syndrome, Patau syndrome, or Edwards syndrome.
• Diagnosis of syndromes caused by deletions (loss of a chromosomal segment), such as Prader-Willi syndrome, Williams syndrome, or DiGeorge syndrome.
• Diagnosis of neoplasms caused by translocations (breakage and relocation of a chromosomal segment), such as acute and chronic myeloid leukemia.
• Differential diagnosis of melanoma versus benign lesions.
• Selection of treatment for certain cancers, such as lung or breast cancer: patients may present specific chromosomal abnormalities in tumors that make them respond better to certain treatments.

Therefore, fluorescence in situ hybridization is generally recommended in the following cases:

• During pregnancy, especially if there is a family history of genetic disorders, the parents are carriers of chromosomal alterations, or abnormal results have been obtained in routine ultrasound scans.
• Patients with cancers related to chromosomal abnormalities.
• In vitro fertilization treatments, to identify genetic abnormalities in the embryo before implantation in the mother's uterus.

How is it performed?

To perform the FISH technique, a DNA sample is required. In prenatal studies, this sample is obtained from maternal blood, amniotic fluid, or the placenta (chorionic villi). In cancer patients, DNA is obtained from a tumor tissue sample.

The extracted sample is treated with a fixation agent, such as formaldehyde or ethanol, to preserve the shape of the cells and make them permeable. Then, the DNA must be denatured, meaning the double helix is separated into two single strands.

Once the sample is prepared, a probe is added. This is a specifically designed or selected DNA sequence labeled with fluorescent molecules (fluorophores) and homologous to the DNA sequence of interest. The probe then hybridizes with the target sequence, allowing it to be identified.

There are two methods for visualizing the hybridization results:

• Fluorescence microscopy: The sample is illuminated with high-intensity light that excites the fluorescent molecules in the stain, making them visible under the microscope. This is the standard method.
• Flow cytometry: In this case, individual cells in suspension are identified as they flow through a laser beam. This method is typically used for large cell samples in oncological diagnosis.

In FISH hybridization, different types of probes can be used depending on the procedure’s objective:

• Centromeric probes: Generated from repetitive regions located in the chromosome's centromere. Used to diagnose aneuploidies.
• Locus-specific probes: Hybridize to a specific DNA region. Used to locate specific genes and detect structural alterations.
• Chromosome painting probes: A set of probes that, together, hybridize with an entire chromosome.

More complex FISH techniques provide greater precision. One example is multicolor FISH (M-FISH) or spectral karyotyping (SKY), which allows for the simultaneous observation of all chromosomes, each displayed in a different color, using 24 chromosome-specific probes labeled with different fluorochromes or fluorochrome combinations. A similar procedure is multiband FISH (RxFISH), which generates a multicolored banding pattern for each chromosome. Comparative genomic hybridization (CGH), on the other hand, is used to study tumor cells by employing two probe groups (one derived from tumor DNA and the other from healthy DNA) labeled with two different fluorochromes.

Risks

Invasive procedures to collect prenatal DNA samples carry a minimal risk of miscarriage, bleeding, or infection. The complications arising from extracting DNA from tumor tissue via biopsy vary depending on the specific procedure, the area where it is performed, and the patient’s health condition. Generally, however, these are safe procedures that rarely present severe risks.

What to expect from fluorescence in situ hybridization

To obtain an amniotic fluid sample, a procedure called amniocentesis is performed: using ultrasound imaging as a guide, a needle is inserted through the abdomen into the uterus, where the sample is collected by aspiration. Pain during the puncture and aspiration is common, as are mild vaginal bleeding, slight pain, or a small leakage of amniotic fluid afterward.

The extraction of chorionic villi from the placenta is generally performed by inserting a probe through the vagina to the placenta, where a tissue sample is aspirated, also using ultrasound guidance. Discomfort during the procedure and mild vaginal bleeding afterward are common.

Biopsies can be performed using different techniques depending on each case. The most common options are needle aspiration, surgery, or endoscopy. Anesthesia or sedation is often administered, and discomfort may be experienced in the hours following the procedure.

In all cases, these are outpatient procedures that do not require hospitalization.

Specialties requesting fluorescence in situ hybridization

The FISH technique is performed by genetic specialists and is primarily requested in the fields of oncology, gynecology and obstetrics, and assisted reproduction.