Residency · Residency · Medical Genetics Genomics

Fluorescence In Situ Hybridization (FISH) and Its Clinical Applications

Principles of FISH

Fundamental Concept

Fluorescence in situ hybridization (FISH) uses fluorescently labeled DNA probes that hybridize to complementary sequences on chromosomes, enabling the detection of specific chromosomal regions in both metaphase and interphase cells. FISH offers higher resolution than G-banding, typically around 100 to 200 kilobases for locus-specific probes, but it is a targeted rather than genome-wide technique. It can be performed on metaphase spreads, interphase nuclei, tissue sections (including paraffin-embedded specimens), or uncultured cells, making it versatile across many clinical settings.

Technical Workflow

The FISH procedure begins with probe labeling, either by direct fluorochrome conjugation or by indirect labeling using biotin or digoxigenin with subsequent fluorescent detection. Both the probe and target DNA are heat-denatured to single strands, then allowed to hybridize overnight at 37 degrees Celsius. Stringency washes remove nonspecific binding, and the preparation is counterstained with DAPI for chromosome or nuclear visualization. Fluorescent signals are then scored by fluorescence microscopy, either manually or with automated image capture systems.

Probe Types

Centromeric (Alpha-Satellite) Probes

Centromeric probes hybridize to repetitive alpha-satellite sequences at chromosome centromeres. Because these targets are large, the resulting signals are bright and easily scored. Their primary clinical use is rapid aneuploidy detection for chromosomes 13, 18, 21, X, and Y on uncultured amniocytes or chorionic villi. These probes can detect numerical abnormalities in interphase cells without the need for cell culture, but they cannot detect structural rearrangements or mosaicism below approximately 5 to 10%.

Locus-Specific Identifier (LSI) Probes

Locus-specific probes target unique sequences at defined chromosomal loci, typically spanning 100 to 500 kilobases. They detect microdeletions, microduplications, and gene rearrangements. These probes are often used in pairs, with one probe at the locus of interest and one at a control locus. Key clinical examples include the TUPLE1/HIRA probe for the DiGeorge/22q11.2 deletion, the ELN probe at 7q11.23 for Williams syndrome, the SNRPN probe at 15q11.2 for Prader-Willi and Angelman syndromes, and the CSF1R/D5S23 probe at 5p15 for cri-du-chat syndrome.

Whole Chromosome Paint (WCP) Probes

Whole chromosome paints are cocktails of probes spanning an entire chromosome that produce a uniform fluorescent coating. They are ideal for identifying the chromosomal origin of marker chromosomes or for characterizing complex rearrangements, and they can confirm translocations seen on G-banding. However, whole chromosome paints are limited to metaphase cells because interphase signals are too diffuse, and they cannot determine the orientation or precise breakpoint location of rearrangements.

Subtelomeric Probes

Subtelomeric probes target unique sequences near chromosome telomeres. They were historically used to detect cryptic subtelomeric rearrangements in patients with unexplained intellectual disability but have been largely supplanted by chromosomal microarray, which provides higher resolution and genome-wide coverage.

Clinical Applications

Prenatal Rapid Aneuploidy Detection

Interphase FISH on uncultured amniocytes or chorionic villus sampling cells can rapidly screen for aneuploidies of chromosomes 13, 18, 21, X, and Y, with results typically available within 24 to 48 hours compared to 10 to 14 days for a full karyotype. This test is used as an adjunct to, not a replacement for, a full karyotype. Quantitative fluorescent PCR (QF-PCR) is an alternative rapid aneuploidy method.

Microdeletion Syndrome Testing

Targeted FISH remains useful when a specific clinical diagnosis is suspected. Common indications include suspected 22q11.2 deletion syndrome (presenting with conotruncal heart defects, palatal anomalies, and hypocalcemia), suspected Williams syndrome (supravalvular aortic stenosis, elfin facies, hypercalcemia), and suspected Prader-Willi or Angelman syndrome, though methylation testing is the preferred first-tier test for the latter. For most constitutional indications, chromosomal microarray is replacing FISH.

Cancer Cytogenetics

FISH is a mainstay in the diagnosis and monitoring of hematologic malignancies. Key applications include detection of the BCR-ABL1 fusion in chronic myeloid leukemia (the Philadelphia chromosome), PML-RARA fusion in acute promyelocytic leukemia, MYC rearrangement in Burkitt lymphoma, HER2 amplification in breast cancer (which guides trastuzumab therapy), ALK rearrangement in non-small cell lung cancer, and 1p/19q codeletion in oligodendroglioma (both prognostic and predictive). Two main probe strategies are used in this setting: break-apart probes, which place flanking probes on either side of a breakpoint so that signal separation indicates rearrangement, and fusion probes, which place probes on two different chromosomes so that juxtaposed signals indicate a translocation.

Marker Chromosome Characterization

When small supernumerary marker chromosomes are identified on karyotype, FISH with centromeric probes can determine their chromosomal origin. The clinical significance of a marker chromosome depends on its euchromatic content. Examples include cat eye syndrome, caused by a bisatellited marker from chromosome 22, and Pallister-Killian syndrome, caused by isochromosome 12p with tissue-specific mosaicism.

Specialized FISH Techniques

Multicolor FISH (M-FISH) / Spectral Karyotyping (SKY)

These techniques paint all 24 chromosomes simultaneously in different colors, allowing identification of complex rearrangements and marker chromosomes. They are particularly useful in cancer cytogenetics for resolving complex karyotypes, though they cannot detect intrachromosomal rearrangements such as inversions or interstitial deletions.

Fiber FISH

Fiber FISH is performed on stretched chromatin fibers, providing resolution down to approximately 5 to 10 kilobases. It is useful for determining gene order, estimating deletion or duplication size, and mapping breakpoints. However, it remains a research and reference laboratory tool rather than a routine clinical assay.

FISH on Tissue Sections

FISH can be performed on formalin-fixed paraffin-embedded (FFPE) tissue and is the standard for HER2 testing in breast cancer and ALK testing in lung cancer. An important technical consideration is truncation artifact, in which nuclear sectioning can produce false signal loss. Strict scoring criteria are required to account for this phenomenon.

Advantages and Limitations

Advantages over Conventional Karyotyping

FISH can be performed on interphase cells, eliminating the need for cell culture and enabling rapid results. It offers higher resolution than karyotyping (approximately 100 to 200 kilobases versus 5 to 10 megabases), is applicable to formalin-fixed paraffin-embedded tissue sections, and can deliver results within 24 to 48 hours for interphase studies.

Limitations

FISH is inherently a targeted test: it only detects abnormalities at the specific loci tested, so a normal FISH result does not exclude genome-wide imbalances. It cannot detect balanced rearrangements at loci not covered by the probe set, nor can it detect point mutations, methylation abnormalities, or small insertions and deletions. False negatives are possible due to probe design issues or polymorphic signal patterns. For many indications, FISH is being supplanted by microarray for genome-wide CNV detection and by next-generation sequencing approaches.

<image>A composite image showing four FISH signal patterns on interphase nuclei: (1) Normal signal pattern for chromosome 21 (two green signals) and chromosome 13 (two red signals) representing a euploid cell; (2) Trisomy 21 pattern showing three green signals for chromosome 21 and two red signals for chromosome 13; (3) A metaphase spread with a BCR-ABL1 dual-color dual-fusion FISH showing the normal separated red (ABL1 on chromosome 9) and green (BCR on chromosome 22) signals alongside two yellow fusion signals indicating the Philadelphia chromosome t(9;22); (4) A break-apart FISH for ALK gene showing one intact yellow (fused red-green) signal and separated red and green signals indicating ALK rearrangement. Each panel is labeled with the clinical context and probe names.</image>

<image>A diagram comparing four FISH probe types side by side on stylized chromosomes: (A) Centromeric probe shown as a bright signal at the centromere of a chromosome pair with alpha-satellite DNA repeat indicated; (B) Locus-specific probe shown as a small signal at a defined band on the chromosome with a normal two-signal pattern and an adjacent example with one signal missing indicating deletion; (C) Whole chromosome paint shown as uniform fluorescent coating of an entire chromosome pair in green, with a third segment painted green on a different chromosome indicating a translocation; (D) Subtelomeric probes shown as small signals near the tips of chromosome arms in two different colors (green for p-arm, red for q-arm).</image>

<image>A clinical decision flowchart for selecting FISH vs. chromosomal microarray vs. karyotype. Starting with the clinical indication at the top, branches lead to: (1) Suspected specific microdeletion syndrome leads to targeted FISH or microarray; (2) Unexplained intellectual disability/MCA leads to microarray as first-tier; (3) Suspected aneuploidy in pregnancy leads to interphase FISH for rapid screen plus karyotype for definitive; (4) Hematologic malignancy leads to karyotype plus targeted FISH panel; (5) Recurrent pregnancy loss leads to parental karyotypes. Each endpoint notes expected turnaround time and resolution.</image>

Clinical Pearls

Interphase FISH for rapid aneuploidy detection (chromosomes 13, 18, 21, X, and Y) provides results in 24 to 48 hours but should always be confirmed with a full karyotype or microarray. FISH is a targeted test, meaning a normal FISH result only excludes abnormalities at the specific loci tested, not genome-wide imbalances. In cancer diagnostics, break-apart probes detect rearrangements regardless of the partner gene, while fusion probes detect only the specific translocation they are designed for. Chromosomal microarray has replaced FISH as the first-tier test for most constitutional indications such as developmental delay, intellectual disability, and multiple congenital anomalies, but FISH remains essential in cancer cytogenetics and for rapid prenatal aneuploidy screening. When interpreting FISH on tissue sections from FFPE specimens, clinicians should be aware of truncation artifact leading to apparent signal loss; laboratories use strict signal counting criteria, such as scoring a minimum of 20 cells with defined ratio thresholds, to minimize false results.

References

• Wolff DJ et al. Guidance for fluorescence in situ hybridization testing in hematologic disorders. J Mol Diagn. 2007;9(2):134-143.
• Wiktor AE, Van Dyke DL. FISH analysis helps identify low-level mosaicism in Turner syndrome patients. Genet Med. 2004;6:132-135.
• Shaffer LG, Bejjani BA. A cytogeneticist's perspective on genomic microarrays. Hum Reprod Update. 2004;10(3):221-226.
• ACMG Technical Standards for clinical genetics laboratories (FISH). 2010 revision.
• Levy B, Burnside RD. Are all chromosome microarrays the same? What clinicians need to know. Prenat Diagn. 2019;39(3):157-164.

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