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Fluorescence In Situ Hybridisation (FISH)

FISH is a relatively new molecular cytogenetics technology utilising fluorescently labelled DNA probes to detect or confirm gene or chromosome abnormalities that are generally either beyond the resolution of routine cytogenetics or can be applied to samples that are unsuitable for conventional cytogenetics analysis. FISH, PCR based techniques and conventional cytogenetics need to be considered together as all these methodologies can be used to detect numerical and structural cytogenetic aberrations. However, each method has its advantages and disadvantages and one may be superior to another in some circumstances, for example, FISH is rarely suitable for the detection of a specific translocation in a minimal residual disease setting whereas PCR is sensitive for the detection of low level disease.

The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured, a process that separates the complimentary strands within the DNA double helix structure. The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridises with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen through a fluorescent microscope as a coloured spot located in the target area/cell/chromosome and the sample DNA scored for the presence or absence of the signal.

Metaphase FISH

FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine Cytogenetics or identify extra material of unknown origin. It can also help in cases where it is difficult to determine from routine Cytogenetics if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.

Interphase FISH

FISH can be used in interphase cells to determine the chromosome number of one or more chromosomes as well as detect some specific chromosome rearrangements that are characteristic for certain cancers. The primary advantage of interphase FISH is that it can be performed very rapidly if necessary, usually within 24 - 48 hours, because cell growth is not required. This is a major advantage in tumours with a low proliferative level.

Click the picture for a tutorial on the use of FISH techniques in the diagnosis of haematological malignancies (7.5Mb PDF)

HMDS strategy for the detection of cytogentic aberrations

• Conventional cytogenetics presentation samples
  • Acute leukaemias
  • Myelodysplastic syndromes
  • Chronic myeloid leukaemia

• PCR based techniques; presentation and follow-up for MRD
  • Acute leukaemias
  • Chronic myeloid leukaemia

• Interphase FISH presentation
  • B-cell lymphoproliferative disorders
  • Plasma cell disorders

Dual fusion assay showing t(8;14) in Burkitt Lymphoma (C-MYC/IgH/alpha 8 probe set from Abbott Molecular Diagnostics)

Deletion assay showing p53 deletion in B-CLL. Loss of p53 is associated with poor prognosis (p53/alpha satellite 17 probe set from QBIOgene)

Main Advantages of FISH Techniques

• Direct FISH technique is relatively rapid and sensitive
• No cell culture needed
• Result may be easier to interpret than interpretation of complex karyotype
• Can combine FISH with immunostaining ie FICTION technique

Main Disadvantages of FISH Techniques

• Need specialised camera and image capture system
• Limited number of commercial probes available
• FISH is only suitable in cases with moderate to high numbers of abnormal cells. Cell selection technique is needed if malignant cells of interest are present at a low level.
• FISH will only provide information about the probe being tested, other aberrations will not be detected

Overview of FISH Technique

1. Interphase cells are obtained directly from:
   • peripharal blood
   • bone marrow aspirates
   • lymph node or tissue biopsy - dab or imprint from unfixed fresh tissue
   • CSF
   • pleural effusions
   • Formalin fixed paraffin-embedded tissue can also be used, however because of the cross-linking effect of formalin it is essential to use a suitable enzymatic digestion technique otherwise the probe cannot penetrate the tissue.
     • thin sections
     • extracted nuclei from thick sections
2. Fix smears /cytospins / dabs in fresh ice-cold methanol/acetic acid (Carnoy's fixative)
3. Pre-treat in 2x saline sodium citrate (SSC) buffer
4. Dehydrate in ethanol series
5. Apply the probe to the smear under a coverslip and seal with rubber-glue
6. Co-denature the probe and smear using a programmable hot-plate. Follow individual manufacturer protocols for denaturation time and temperature - this may be probe specific
7. Hybridise for 1 to 72 hours (depending on probe)
8. Post hybridisation wash in low salt solution
9. Apply DAPI nuclear counter-stain and seal coverslip with varnish
10. Visualisation using fluorescent microscope

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