Cytogenetics and Molecular

Question 1

Name the stages of the cell cycle

Answer 1

Interphase:
- G1 (growth)
- S (synthesis of DNA / replication; 23 pairs ==> 46 pairs)
- G2 (more growth and DNA checks)
Metaphase/Mitosis
- chromosomes line up along centre and separate forming two daughter cells

Question 2

What are the three stages of preparing a sample for karyotype?

Answer 2

1. Culture
   - minimum 2 cultures for each sample. examine cells from all cultures when analysing.
2. Harvest
   - Colcemid (spindle inhibitor)
   - Hypotonic KCL (lyses RBCs, WBCs swell allowing more space for chromosomes)
   - Fix (acetic acid and ethanol)
   - Drop on slides (must have good spread) and put in oven at 60 degrees overnight
3. Banding
   - Trypsin removes proteins
   - Wash with PBS
   - Leishmann’s stain

Question 3

Causes of karyotype failure

Answer 3

Specimen quality is the main reason

• insufficient sample
• old/degraded sample
• reagent problem
• contamination

Question 4

Advantages of FISH

Answer 4

1. Good for leukaemic cells that aren’t dividing e.g. Myeloma and CLL
2. Can detect cryptic translocations
3. Can analyse gene amplification
4. More cells are analysed…can be used for MRD

Question 5

Name one major disadvantage of FISH

Answer 5

Only abnormalities that are specifically sought will be detected

Question 6

How is plasma cell enrichment performed for FISH?

Answer 6

Magnetic beads coated with anti-CD138 are used to select out the plasma cells.

Question 7

Describe the process of FISH (5 steps)

Answer 7

1. Pre-treat sample with pepsin to remove proteins that might interfere with hybridisation.
2. Heat sample ==> DNA strands separate
3. FISH probes are added when sample has been reduced to a lower temperature (annealing)
4. Wash off unbound probes
5. View under fluorescent light

Question 8

JAK2 V617F

Answer 8

Allele specific PCR

Question 9

JAK2 exon 12

Answer 9

PCR then sequencing

Question 10

CALR

Answer 10

PCR then sequencing

Question 11

MPL

Answer 11

PCR then sequencing

Question 12

BCR-ABL1

Answer 12

RT-PCR and qPCR

Question 13

cKIT D816V

Answer 13

PCR then restriction enzyme

systemic mastocytosis

Question 14

MYD88

Answer 14

Allele specific PCR (2 forward primers WT and MT bind at the same place so amplicon is the same size; must be run on 2 gels)

Question 15

BRAF V600E

Answer 15

CD19+ cells are selected first

PCR then sequencing

Question 16

IGH

Answer 16

Semi-nested PCR

Question 17

TCR

Answer 17

PCR using two sets of primers (alpha/beta and gamma/delta)

Question 18

NPM1, CEBPA

Answer 18

PCR then sequencing

CEBPA requires 3 sets of primers as large gene

Question 19

FLT3-ITD

Answer 19

PCR fragment analysis (one of the primers is labelled with a fluorescent probe)

Question 20

FLT3-TKD

Answer 20

PCR then restriction enzyme

Question 21

inv(16), t(8;21) and t(15;17)

Answer 21

RT-PCR (always nested)

Question 22

FVL, prothrombin 20210, HFE (Cys282Tyr, H63D, S65C)

Answer 22

SNP Taqman Genotyping

Question 23

Factor VIII

Answer 23

Long range PCR for inversions and/or sequencing

Question 24

Factor IX

Answer 24

Sequencing (most patients have different mutations)

Question 25

Alpha thalassaemia

Answer 25

Multiplex PCR

Question 26

Beta thalassaemia

Answer 26

Sequencing

Question 27

Sickle cell

Answer 27

PCR then restriction enzyme