In situ hybridization (ISH) is a technique that uses labeled nucleic acid probes to detect specific DNA or RNA sequences directly in tissue sections or cytology preparations. Unlike extraction-based molecular methods (PCR, sequencing), ISH preserves tissue architecture and shows exactly which cells contain the target sequence.
Principles
ISH uses a probe — a single-stranded DNA or RNA molecule complementary to the target sequence. The probe is labeled with a detectable marker (radioactive isotope, fluorophore, hapten). After denaturation of target DNA (for DNA ISH) or without denaturation (for RNA ISH), the probe hybridizes to the target sequence under controlled temperature and salt conditions. Unbound probe is washed away, and the bound probe is detected by autoradiography, fluorescence microscopy, or enzyme-based chromogenic detection.
Stringency — the specificity of hybridization is controlled by temperature and salt concentration. High stringency (high temperature, low salt) allows only perfectly matched probe-target hybrids to form. Lower stringency permits partial mismatches, which may be desirable for detecting related sequences but increases background.
Fluorescence In Situ Hybridization (FISH)
FISH uses fluorophore-labeled probes detected by fluorescence microscopy. It is the most widely used ISH method in diagnostic pathology. FISH probes include: centromere enumeration probes (CEP) — target chromosome-specific alpha satellite DNA in centromeres, used to detect aneuploidy; locus-specific indicator probes (LSI) — target unique sequences at specific chromosomal loci, used to detect gene amplification (HER2, EGFR) and deletion (1p/19q in oligodendroglioma); break-apart probes — pairs of probes flanking a gene locus; separation indicates translocation (ALK, ROS1, MYC, BCL2, BCL6, MALT1); fusion probes — one probe on each side of a translocation breakpoint; fusion signals indicate the translocation.
HER2 FISH — the gold standard for HER2 status determination in breast and gastric cancer. A ratio of HER2 (17q12) signals to CEP17 signals >2.0 is amplified. FISH is performed when IHC is equivocal (2+ score).
ALK FISH — break-apart FISH for ALK (2p23) rearrangement in lung adenocarcinoma detects patients who respond to ALK inhibitors (crizotinib, alectinib). ALK IHC (D5F3 clone) has largely replaced FISH as the primary screening test, but FISH remains the confirmatory method.
Chromogenic In Situ Hybridization (CISH)
CISH uses hapten-labeled probes detected by an enzyme-based chromogenic reaction (DAB for brown, Fast Red for red). The signal is visible under a brightfield microscope, allowing simultaneous evaluation of morphology and hybridization signal. CISH is cheaper than FISH (no fluorescence microscope required), produces permanent slides, and is more accessible in routine histopathology laboratories.
RNA In Situ Hybridization
RNA ISH detects specific mRNA transcripts in tissue sections. RNAscope (Advanced Cell Diagnostics) is a commercially available RNA ISH technology using a probe design strategy (double Z probes) that eliminates background from non-specific probe binding. Each target mRNA appears as a dot-like signal in the cytoplasm (or nucleus for nascent transcripts). RNAscope is up to 100 times more sensitive than traditional RNA ISH and can detect as few as 1-2 mRNA copies per cell.
Applications of RNA ISH include: detecting viral RNA (EBV EBER — the standard method for identifying EBV-infected cells in Hodgkin lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma); HPV RNA (E6/E7 mRNA detection confirms transcriptionally active HPV infection); gene expression analysis (PD-L1 mRNA, cytokine expression in inflammatory infiltrates); lineage determination (detecting tissue-specific transcripts in poorly differentiated tumors).
Diagnostic Applications
Lymphoma diagnosis — FISH for MYC, BCL2, and BCL6 translocations in diffuse large B-cell lymphoma (double-hit and triple-hit lymphomas). EBER ISH for EBV in Hodgkin lymphoma and post-transplant lymphoproliferative disorder.
Sarcoma diagnosis — FISH for SS18 (synovial sarcoma), FKHR (alveolar rhabdomyosarcoma), EWSR1 (Ewing sarcoma, desmoplastic small round cell tumor), and MDM2 (well-differentiated/dedifferentiated liposarcoma).
Lung cancer — ALK FISH, ROS1 FISH, and MET amplification FISH for targeted therapy selection.
Breast cancer — HER2 FISH for equivocal IHC cases.
Limitations
FISH requires fluorescence microscopy and specialized software for signal enumeration. Probe penetration in thick tissue sections may be incomplete. Signal fading — fluorophores photobleach with prolonged excitation; slides should be stored in the dark. Interpretation challenges include cut nuclei (truncation artifacts in paraffin sections may delete signals — use 4 µm sections), overlapping signals, and polysomy. RNA degradation in poorly preserved tissue causes false-negative RNA ISH. Quality assurance includes regular validation with known positive and negative controls, participation in EQA programs, and documented interpretation criteria.
