FISH Flashcards
Skeleton FISH protocol
Two main types of FISH
Limits for calling FISH abnormal
CDKN2A homozygous deletion
Deletion by FISH
Interphase FISH
Metaphase FISH
Probes against ALK 3’ region and ALK 5’ region
Inversion by FISH
Reporting a normal FISH result
Reporting an abnormal FISH result
Where do you stop counting amplifications by FISH?
> 25 is enough
FISH steps from glass slide
- Heat fix
- Deparaffinize
- Pretreat (heat, acid, digestive enzyme)
- Probe (overnight, 37-40 oC)
- Wash
- Mount and coverslip
- Readout
Resolution of FISH
~300-500 kbs
Bacterial artificial chromosomes
Segment of a chromosome from the human genome project, made to be reproducable in bacteria.
Provide good base structures for FISH probes.
Categories of information from looking at FISH
- Number of signals/cell
- Location (probe diameter distance units)
- Intensity
Approach to FISH interpretation in lymphomas
Lymphomas often have many overlapping cells, making FISH interpretation difficult.
Take your time, find as best of monolayers as you can, check 10-15 fields before deciding if something is truly negative.
Interpreting MDM2 in adipose tissue (liposarcoma or atypical lipomatous tumors)
For MDM2 in adipose tissue, just go down the whole piece of adipose tissue on the bicolor filter.
Don’t mess around. Liposarcoma cells can look pleomorphic, but they can also look like unremarkable adipocytes. However, MDM2 amplification will stand out – when MDM2 is amplified it is usually very amplified to achieve good p53 suppression.
When does cold ischemic type become relevant for FISH signal?
Even 1 hour of cold ischemic time prior to formalin fixation and paraffin embedding can reduce the FISH signal.
Recommended fixation protocol for FISH
FFPE with 6-72 hours of formalin fixation
Any more fixation than this and DNA may be too fragmented for good FISH results.
Fixation at 4C has the best results.
Decal and FISH
Do not go well together – try to avoid doing FISH on decalled tissue.
If you have to use decalled tissue, EDTA decal is greatly preferred.