Fluorescence In Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) uses fluorescently labeled DNA probes that hybridize to complementary sequences on chromosomes, allowing visualization of specific genomic regions. FISH bridges the gap between conventional cytogenetics and molecular testing, providing targeted detection of specific aneuploidies, microdeletions, and rearrangements.

• Locus-specific identifier (LSI) probes: Hybridize to a specific chromosomal region. Used to detect microdeletions (e.g., 22q11.2 for DiGeorge syndrome) or microduplications. Typically used with a control probe on the same chromosome.
• Centromeric enumeration probes (CEP): Bind to alpha-satellite repeat sequences at the centromere. Used to quickly count chromosome copies (e.g., CEP X, CEP Y, CEP 18, CEP 13/21 for rapid aneuploidy detection).
• Whole chromosome paint (WCP) probes: A cocktail of probes spanning an entire chromosome, used to identify the origin of marker chromosomes or confirm complex translocations. Only works on metaphase spreads.
• Telomeric/subtelomeric probes: Target chromosome ends. Historically used for cryptic subtelomeric rearrangements in unexplained ID; largely replaced by CMA.
• Breakapart (split-signal) probes: Two differently colored probes flank a breakpoint region. A translocation separates the signals. Best for genes with many or variable fusion partners, so a single probe set detects any partner (e.g., KMT2A/MLL in leukemia, ALK in lymphoma and lung cancer).
• Dual-fusion probes: Two probes from different chromosomes produce fusion signals when a translocation brings them together. Best for genes with a single consistent partner (e.g., BCR-ABL1 in CML, ETV6-RUNX1 in ALL). More specific than breakapart probes because they detect both derivative chromosomes.
• Metaphase FISH: Performed on cultured cells arrested in metaphase. Provides chromosomal context (which chromosome, which arm). Required for WCP probes.
• Interphase FISH: Performed on non-dividing (interphase) nuclei. No culture required, with faster turnaround (24–48 hours). Used for rapid aneuploidy screening and enumeration in oncology.

• Rapid prenatal aneuploidy screening: Interphase FISH on uncultured amniocytes for chromosomes 13, 18, 21, X, and Y, with results in 24–48 hours (vs. 10–14 days for karyotype)
• Microdeletion syndromes: Targeted detection when a specific syndrome is suspected (e.g., Williams syndrome 7q11.23, Prader-Willi/Angelman 15q11-q13)
• Hematologic malignancies: Detect prognostically important translocations and aneuploidies (e.g., BCR-ABL1, PML-RARA, MYC rearrangement)
• Confirm CMA findings: FISH can determine the mechanism (e.g., is a duplication tandem or insertional?) and provide information on parental origin

• FISH: When you need a rapid, targeted answer for a specific known region or rearrangement
• Karyotype: When you need a genome-wide view of chromosome structure, especially for balanced rearrangements
• CMA: First-tier for unexplained DD/ID, ASD, or MCA: genome-wide, high-resolution CNV detection
• FISH is NOT a screening test: It only interrogates the regions you probe for. A normal FISH result does not rule out abnormalities elsewhere.

• Targeted only: Only detects abnormalities at the specific loci being tested; does not provide genome-wide screening
• Cannot detect point mutations or small indels
• WCP probes require metaphase cells: Cannot be used on interphase nuclei
• Cannot determine exact breakpoints: Provides approximate localization, not nucleotide-level resolution
• False negatives possible: If the target region is atypical or the deletion does not involve the probe binding site