FISH

Question 1

What is Fluorescence in situ hybridization (FISH)?

Answer 1

Procedure of hybridizing a fluorescent probe (DNA/RNA sequence) to a sample to detect genetic aberrations

FISH is used for detecting specific genetic changes in various samples.

Question 2

What types of samples can be used for FISH?

Answer 2

• Blood/Bone marrow in an EDTA (purple top) vacutainer
• Unstained Blood/Bone marrow smear
• Lithium heparin and sodium heparin tubes
• Tissues, usually paraffin embedded tissue slides

Only one slide per FISH test is required.

Question 3

What is the first step in the FISH assay procedure?

Answer 3

Specimen fixed to a slide (pre-treatment)

Proper fixation is crucial for successful hybridization.

Question 4

What temperature is used to denature the specimen target DNA and the probe in FISH?

Answer 4

76°

Denaturation is essential for effective hybridization.

Question 5

How long can hybridization take in FISH?

Answer 5

2 to 12 hours at 37°

The duration can affect the quality of hybridization.

Question 6

What is a critical factor for FISH success?

Answer 6

Sample quality

Good hybridization is highly dependent on cellularity.

Question 7

List factors that can interfere with hybridization in FISH.

Answer 7

• Hypo-cellular or hyper-cellular samples
• Making smears and pressing too hard
• Tissue fixation longer than 12 hours in formalin

These factors can lead to poor hybridization outcomes.

Question 8

Define ‘Deletion’ in the context of FISH.

Answer 8

Part of a chromosome is deleted, may or may not involve one or more genes

Deletions can impact gene function and lead to various disorders.

Question 9

What does ‘Amplification’ mean in FISH?

Answer 9

Chromosomal rearrangement events resulting in duplication and multiple copies of a chromosomal region targeting one or more genes

Amplifications can lead to overexpression of oncogenes.

Question 10

What is a ‘Reciprocal translocation’?

Answer 10

Usually involves two or more chromosomes, break occurs in each chromosome and the two chromosomes exchange the broken segments

This can lead to gene fusion and potential oncogenesis.

Question 11

What are ‘Unbalanced translocations’?

Answer 11

Translocation with loss of chromosomal material

Example: t(1;19) in ALL can be unbalanced or balanced.

Question 12

What is the significance of the p-arm and q-arm in chromosomes?

Answer 12

P-arm refers to the short arm and q-arm refers to the long arm of the chromosome

Understanding these arms is important for identifying chromosomal abnormalities.

Question 13

What is the result of the translocation t(9;22)?

Answer 13

Involves the BCR-ABL fusion in CML and ALL

This is a well-known chromosomal aberration associated with specific leukemias.

Question 14

What is the purpose of single copy number probes in FISH?

Answer 14

To detect specific gene deletions, such as TP53 in CLL

These probes are locus specific and provide precise information about genetic alterations.

Question 15

What is the implication of detecting Trisomy 8 in MDS?

Answer 15

Indicates an abnormal increase in the number of chromosomes

Trisomy can be associated with specific hematological disorders.

Question 16

What does the Her2/neu probe signal indicate?

Answer 16

Red probe signal for Her2/neu oncogene = 8 copies; green probe = 2 copies for centromere of chromosome 17

This result indicates mild amplification and is important for treatment selection.

Question 17

True or False: Amplification in FISH only detects deletions.

Answer 17

False

Amplification detects both DNA gain/amplification and deletions.