Methods (molecular, IHC, frozen)
Molecular
FISH overview


Topic Completed: 2 December 2020

Minor changes: 15 December 2020

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PubMed Search: FISH[TI] fluorescence in situ hybridization[TIAB] review[PT] full text[SB]

Ana Caramelo, B.Sc.
António Polónia, M.D., Ph.D.
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Cite this page: Caramelo A, Polónia A. FISH overview. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/molecularpathfishgeneral.html. Accessed April 15th, 2021.
Definition / general
  • Detection of specific nucleic acid sequences (DNA / RNA)
  • Use of a labeled complementary nucleic acid probe
  • Visualization in the cell or tissue (in situ) on a slide by fluorescent microscopy
Essential features
  • Detection of specific nucleic acid sequences (DNA / RNA) (foreign or native)
  • Visualization of signals in the cell or tissue (in situ), by either digital imaging or manual microscopy
  • Detection of numerical chromosome abnormalities (congenital or acquired aneuploidies)
  • Detection of amplification / deletion / chromosomal translocation
Terminology
  • ISH: in situ hybridization
  • FISH: fluorescence in situ hybridization
  • BISH: bright-field in situ hybridization
  • SISH: silver in situ hybridization
  • CISH: chromogenic in situ hybridization
  • DDISH: dual hapten dual probe in situ hybridization
  • PCR: polymerase chain reaction
ICD coding
  • ICD-10: Q90-Q99 - Chromosomal abnormalities, not elsewhere classified
Types of ISH
  • Fluorescence ISH (FISH): direct detection of a fluorescent labeled probe
  • Bright-field ISH (BISH): indirect detection through an enzymatic reaction
    • Silver ISH: detection of silver precipitation (black dots)
    • Chromogenic ISH (CISH): detection of chromogen (e.g. red)
    • Dual hapten dual probe ISH (DDISH): CISH + SISH
  • Hybrid techniques
ISH probes
  • Large chromosome sequences (e.g. large regions, short arm, long arm)
  • Repetitive sequences (e.g. centromeres)
  • Unique sequences (e.g. specific genes, parts of genes)
Specimen types
  • Cells in culture
  • Paraffin block sections
  • Aspirate smears
  • Tissue imprints
  • Liquid based preparations
Applications
  • Interphase cytogenetics (analysis of chromosomes in nondividing cells)
  • Viral infections: detection of viral genome (e.g. EBV, HPV, HIV, fungal species)
  • Gene expression: detection of messenger RNA for various peptides (e.g. immunoglobulin light chains, albumin)
  • Single transcript analysis: detection of point mutations, single nucleotide polymorphisms (SNPs), RNA edited transcripts, tissue specific allele expression, alternative splicing
Cell enrichment methods
Interpretation of ISH signals
  • Presence of signals (e.g. presence of EBER in EBV infection)
  • Gain of signals (e.g. ERBB2 amplification in breast / gastric cancer)
  • Loss of signals (e.g. loss of 1p/19q in oligodendroglioma)
  • Positioning of signals
    • Break apart probe: neighboring signals altered when detected at a distance, resulting in 2 separate color signals (e.g. EWSR1 translocation)
      • Normally, 2 different color signals (red and green) are located nearby, sometimes creating a fused color (yellow); the abnormality exists if the 2 signals are separated
    • Fusion probes: distant signals altered when detected nearby, resulting in overlap or juxtaposition of the 2 different color signals (e.g. EWSR1-FLI1 fusion)
      • Normally, 2 different color signals (red and green) are separated; the abnormality exists if the 2 signals are together, sometimes creating a fused color (yellow)
Advantages versus limitations of fluorescence and bright-field ISH
  • FISH requires fluorescence microscope (higher cost) versus bright-field ISH requires light microscope (lower cost)
  • Higher multiplexing in FISH (> 2 probes) versus low multiplexing in bright-field ISH (≤ 2 probes)
  • Low morphologic correlation in FISH versus high morphologic correlation in bright-field ISH
  • Loss of signals with time in FISH slides (photographic record mandatory) versus permanent staining in bright-field ISH slides
  • Reference: Diagn Pathol 2008;3:41
Molecular / cytogenetics images

Contributed by António Polónia, M.D., Ph.D., Ana Caramelo, B.Sc., Ana Ribeiro, M.D., Catarina Eloy, M.D. and João Vale, M.Sc.
Gastric cancer Gastric cancer

Gastric cancer

Gastric cancer Gastric cancer

Gastric cancer

Breast cancer Breast cancer

Breast cancer


Breast cancer Breast cancer

Breast cancer

Gastric cancer Gastric cancer

Gastric cancer


1p/19q codeletion

1p/19q codeletion

Gastric lymphoma

Gastric lymphoma

Lymph node

Lymph node



Contributed by Leica Microsystems, Biosystems division
Immunoglobulin light chain

Immunoglobulin light chain



Images hosted on other servers:
Trisomy 21

Trisomy 21

UroVysion FISH test

UroVysion FISH test

Single molecule RNA FISH (smFISH)

Single molecule RNA FISH (smFISH)

In situ mutation detection of KRAS mutation

In situ mutation detection of KRAS mutation

Albumin Albumin

Albumin

Sample FISH report
  • Example for HER2 FISH quantification in breast cancer:
    • Number of neoplastic cells analyzed: at least 20 cells in 2 separate areas (at least 10/area)
    • Number of HER2 signals:
    • Average HER2 copy number:
    • Number of CEP17 signal:
    • Average CEP17 copy number:
    • HER2/CEP17 ratio:
    • Genomic heterogeneity: absence / presence (percentage of the total tumor population with amplification)
    • Probe:
    • Guidelines: ASCO / CAP 2018
    • Type of fixative:
    • Time to fixation (cold ischemic time): less than 1 hour
    • Duration of tissue fixation: between 6 and 72 hours
    • Commentary on the preanalytic conditions: the preanalytic conditions are (or are not) according to the recommendations of the ASCO / CAP 2018 guidelines for HER2 in breast cancer.
Board review style question #1

What is the pattern of a break apart rearrangement?

  1. A pair of different color signals together and 2 separate color signals
  2. 2 pairs of different color signals together
  3. 3 different color signals separate
  4. 4 different color signals separate
Board review style answer #1
A. A pair of different color signals together and 2 separate color signals

Comment Here

Reference: FISH overview
Board review style question #2
What is the pattern of a fusion rearrangement?

  1. A pair of different color signals together and 2 separate color signals
  2. 2 pairs of different color signals together
  3. 3 pairs of different color signals together
  4. 4 different color signals separate
Board review style answer #2
A. A pair of different color signals together and 2 separate color signals

Comment Here

Reference: FISH overview
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