Introduction And Definition

Fluorescence in situ hybridization (FISH) is a high-resolution molecular cytogenetic technique.
It utilizes fluorochrome-labeled DNA probes to target specific genetic sequences.
These probes hybridize to complementary target sequences on metaphase chromosomes or interphase nuclei.
FISH is designed to identify the presence, absence, or rearrangement of specific DNA segments.
It detects subtle chromosomal abnormalities that fall below the resolution of standard cytogenetic studies.

Principles And Methodology

The procedure begins with DNA denaturation.
Double-stranded chromosomal DNA on a cytogenetic slide is heated to form single-stranded DNA.
A fluorescently labeled, single-stranded DNA probe is introduced.
The slide undergoes renaturation or reannealing.
The probe hybridizes specifically to its complementary DNA sequence within the chromosomal genome.
The resulting probe signal is visualized and imaged using fluorescence microscopy.

FISH can be performed on uncultured, non-dividing cells (interphase) or cultured, dividing cells (metaphase).
Interphase FISH provides rapid results within 24 to 48 hours because it skips the cell culture step.
Metaphase FISH allows documentation of the exact chromosomal location of the probe.
Metaphase analysis is highly useful for visualizing complex structural rearrangements.

Probe Type	Target Sequence	Primary Clinical Utility
Centromeric Probes	Repetitive alpha-satellite sequences at the centromere.	Rapid aneuploidy detection (e.g., trisomies 13, 18, 21).
Locus-Specific Probes	Unique, single-copy DNA sequences.	Identification of microdeletion and microduplication syndromes.
Whole-Chromosome Painting	Multiple-copy probes spanning an entire chromosome.	Detection of complex translocations and structural rearrangements.
Break-Apart Probes	Sequences flanking a specific gene locus.	Identification of gene rearrangements, especially in hematological malignancies.

FISH is highly useful for rapid prenatal diagnosis of common fetal aneuploidies.
It targets chromosomes 13, 18, 21, X, and Y using uncultured amniocytes or chorionic villi.
It is also utilized postnatally on blood or buccal cells for preliminary detection of conditions like Patau or Edwards syndrome.

FISH facilitated the clinical characterization of submicroscopic deletion syndromes involving 50 to 200 kilobases of DNA.
It uses the HIRA (TUPLE1) probe to detect 22q11.2 deletions in DiGeorge syndrome.
It uses the elastin (ELN) specific probe to confirm 7q11.23 deletions in Williams syndrome.

Interphase FISH provides rapid sex assignment in newborns presenting with ambiguous genitalia.
It detects hidden Y-chromosome material using SRY or DYZ3 probes in patients with Turner syndrome.

FISH detects acquired somatic translocations in pediatric malignancies.
It is frequently used to identify the t(9;22) Philadelphia chromosome in leukemia.

Feature	Description
Advantages	Rapid turnaround time of 24 to 48 hours.
	Does not require dividing cells or cell culture when using interphase nuclei.
	Offers higher resolution than conventional G-banded karyotyping.
	Highly sensitive for detecting low-level mosaicism (1% to 5%).
Limitations	It is a targeted approach, meaning it only detects abnormalities in the specific regions probed.
	Cannot detect point mutations, small indels, or genome-wide copy number variations.
	It has largely been replaced by chromosomal microarray (CMA) as a first-tier test for unexplained developmental delay.
	Currently reserved mostly for confirming abnormalities detected by CMA or for targeted rapid screening.