Lab Investigations of Malignancy

Question 1

Blood film diagnosis: Large number of cells with abundant cytoplasm. Nucleus is almost OBSCURED by LARGE GRANULES.

Answer 1

Acute promyelocytic leukaemia (diagnostic)

Question 2

Blood film diagnostic: Blast cells

Answer 2

Acute myeloid leukaemia OR acute lymphoblastic leukaemia (B or T)

Needs further investigation

Question 3

Blood film diagnosis: Single cell with round nucleus and ABUNDANT VILLOUS PROJECTIONS

Answer 3

Typical of CLL (B type)

Hairy cell (CeLL!)

Question 4

How are antigens detected on cell surfaces?

Answer 4

Flow cytometry

Question 5

What can flow cytomtrey tell you about cells?

Answer 5

Different characteristics at the same type

SCATTER (size, and cytoplasm granularity)

FLUORESCENCE (surface and cytoplasm antigens)

Question 6

Flow cytometry antigen expression process (recognise)

Answer 6

Different panels of antibodies attach to fluorochromes used

Designed to assess:

• Cell lineage
• Stage of differentiation

Question 7

What WCCs express CD45? How can it be used? (recognise)

Answer 7

expressed on all WCCs, it is the common leukocyte antigen

Using anti-CD45 and side scatter, you can tell the difference between the lymphoblasts of ALL, myeloblasts of AML, and the mature lymphocytes of CLL

Question 8

Anti-CD45 and side scatter diagnosis: Abnormal cell populations express
CD34
CD33
CD13
HLA-DR
CD117

Answer 8

AML

Question 9

Anti-CD45 and side scatter diagnosis: Abnormal cell populations express
weaker CD45
TDT
CD10
CD19
HLA-DR

Answer 9

B cell ALL

Question 10

How is mitosis arrested in cytogenetics?

Answer 10

Spindle poison (colcemid)

The chromoshomes remain attached to the spindle and are in the middle of the cell

Question 11

How is Giemsa staining used in cytogenetics

Answer 11

Produces specific banding patterns which can be used to produce a karyotype

Question 12

Type of FISH probes (recognise)

Answer 12

Chromosome paints: come from library, are used to determine origins of structural abnormalities

Locus specific probes: Target genes of interest in order to identify rearrangements or mutations

Question 13

FISH diagnosis: t(15:17)(q22;q21) resulting in PML-RARA fusion gene

Answer 13

Acute promyelocytic leukaemia

Can be detected with FISH probe for RARA gene on chr 17

Question 14

Karyotype diagnosis: Abnormalities of chr 5, 11, 15, 17 and COMPLETE LOSS OF CHR 8 and long arm of CHR 5

Answer 14

AML

Question 15

PCR method (recognise)

Answer 15

Primers are short segments of nucleotides complementary to a target sequence of DNA,
flanking either side of the region to be amplified. Primers provide the initiation site for elongation of the new strand.

Taq polymerase is the thermostable or heat-stable enzyme, which extends the primers by the sequential addition of nucleotides, to synthesise a complementary DNA strand from the original
template.

Deoxynucleoside triphosphates, or dNTPs, are the building blocks used by Taq polymerase to
synthesise the new DNA strand

Question 16

Denaturation temp for PCR?

Answer 16

95 degrees

Question 17

Annealing temp for PCR

Answer 17

45-60 degrees

Question 18

How si PCR product analysed?

Answer 18

Gel electrophoresis: Identifying their size after migration though an agarose gel alongside a molecular weight marker

Products then stained with EB (ethidium bromide) and UV light

Question 19

What is real time quantitive PCR? RQ-PCR

Answer 19

Real-time quantitative PCR
(RQ-PCR) can be used to measure the absolute or relative amount of the cDNA or DNA molecule in a
sample and this has important and increasing applications for monitoring response to treatment,
particularly in acute promyelocytic leukaemia and in chronic myeloid leukaemia

Question 20

PCR diagnosis: BCR-ABL1 fusion gene

Answer 20

CML and philidelphia positive ALL

Question 21

Allele PCR diagnosis: JAK2, V617F and MPL

Answer 21

Myeloproliferative neoplasma

Question 22

PCR diagnosis: FLT3 and NPM mutations

Answer 22

Provides infor about prognosis in AML

Question 23

PCR diagnosis: PML-RARA

Answer 23

Acute PROmyelocytic leukaemia

Question 24

WHich drugs can be used to treat CML?

Answer 24

Tyrosine kinase inhibitors eg IMATINIB

Question 25

Poor prognostic factos in CLL?

Answer 25

Expression of CD38 antigen

17q13 deletion is v. bad!