Details of Fluorescent In Situ Hybridization

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Details of Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) methods detect the HER2 gene copy number using two fluorescent-labeled DNA probes. FISH is less subject to the problems of fixation, storage, antigen preservation, and inter-observer variability than IHC analysis. Literature has cited FISH methods capable of 96.5% sensitivity and 100% specificity. However, fluorescent signals are not always clearly defined; clumping of signals occurs, and some cases are undecipherable by FISH. Subjectivity is still introduced with signal enumeration.
Compared to IHC analysis, the FISH method is more time-consuming and technically more challenging. Technical assay performance takes approximately 16 hours for FISH, compared with approximately three hours for IHC analysis. Technicians also need to be specially trained for FISH. A pathologist must be involved during the test performance to select the tumor area for analysis. FISH reagent costs are typically at least three times the costs of IHC analysis.
Additional considerations with FISH include:
  • The context of morphology is obscured, and the analysis suffers from heterogeneous tumors.
  • Special equipment is needed.
  • Permanent slides are usually unavailable for review as signals fade during archival.
  • Despite its excellent specificity, FISH can still be “equivocal.”

HER2/neu gene FISH