FISH

Question 1

Skeleton FISH protocol

Answer 1

Question 2

Two main types of FISH

Answer 2

Question 3

Limits for calling FISH abnormal

Answer 3

Question 4

CDKN2A homozygous deletion

Answer 4

Question 5

Answer 5

Deletion by FISH

Question 6

Interphase FISH

Answer 6

Question 7

Metaphase FISH

Answer 7

Question 8

Probes against ALK 3’ region and ALK 5’ region

Answer 8

Inversion by FISH

Question 9

Reporting a normal FISH result

Question 10

Reporting an abnormal FISH result

Answer 10

Question 11

Where do you stop counting amplifications by FISH?

Answer 11

> 25 is enough

Question 12

FISH steps from glass slide

Answer 12

1. Heat fix
2. Deparaffinize
3. Pretreat (heat, acid, digestive enzyme)
4. Probe (overnight, 37-40 oC)
5. Wash
6. Mount and coverslip
7. Readout

Question 13

Resolution of FISH

Answer 13

~300-500 kbs

Question 14

Bacterial artificial chromosomes

Answer 14

Segment of a chromosome from the human genome project, made to be reproducable in bacteria.

Provide good base structures for FISH probes.

Question 15

Categories of information from looking at FISH

Answer 15

1. Number of signals/cell
2. Location (probe diameter distance units)
3. Intensity

Question 16

Approach to FISH interpretation in lymphomas

Answer 16

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.

Question 17

Interpreting MDM2 in adipose tissue (liposarcoma or atypical lipomatous tumors)

Answer 17

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.

Question 18

When does cold ischemic type become relevant for FISH signal?

Answer 18

Even 1 hour of cold ischemic time prior to formalin fixation and paraffin embedding can reduce the FISH signal.

Question 19

Recommended fixation protocol for FISH

Answer 19

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.

Question 20

Decal and FISH

Answer 20

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.