Fluorescence in situ hybridization (FISH)

Principle

• Fluorescently labelled DNA probes hybridise to complementary sequences in chromosomes or nuclei on a slide, showing the number and position of specific DNA regions.
• Works on metaphase chromosomes and on interphase (non-dividing) nuclei, so cell culture is not always required.

Probe types

• Centromeric (enumeration) probes: count chromosomes to detect aneuploidy.
• Locus-specific probes: detect deletions or amplifications of a gene.
• Whole chromosome paint probes: identify chromosome rearrangements in metaphase.
• Dual-fusion probes (e.g. BCR-ABL1 for t(9;22)) and break-apart probes (e.g. ALK, MYC rearrangements).

Specimen

• Peripheral blood, bone marrow, amniotic fluid, chorionic villi, tumour touch preparations and formalin-fixed paraffin-embedded (FFPE) sections.

Procedure

• Prepare slides; pretreat (protease digestion for tissue sections).
• Apply probe; denature probe and target DNA together (about 72–75 °C).
• Hybridise overnight at about 37 °C in a humid chamber.
• Stringent washes remove unbound probe; counterstain nuclei with DAPI.
• View under a fluorescence microscope with appropriate filters; score signals in a defined number of nuclei (often 50–200).

Results

• Normal cells show two signals per locus-specific probe.
• Fusion signals indicate translocation; split signals indicate break-apart rearrangement; extra or clustered signals indicate gain or amplification.
• HER2 in breast cancer (ASCO/CAP): HER2/CEP17 ratio ≥2.0 with average HER2 copy number ≥4.0 per cell is positive; other combinations need IHC correlation.

Quality control and pitfalls

• Use laboratory-validated cut-offs and control probes; count only intact, non-overlapping nuclei.
• Signal loss from photobleaching, poor fixation or over-digestion; truncation of nuclei in tissue sections.
• Detects only targeted regions; cannot detect point mutations or very small deletions.

Clinical use

• Rapid prenatal aneuploidy (13, 18, 21, X, Y), microdeletion syndromes (22q11.2 DiGeorge, Williams, Prader–Willi/Angelman), CML, APL t(15;17), CLL prognostic panel (del 13q, del 11q, del 17p, trisomy 12), HER2 amplification.