CML 2

Question 1

what is CML

Answer 1

stem cell disease w excess myeloid cells

Question 2

clin molecular lab questions

Answer 2

can disorder be defined? pose serious health prob? is there treatment? is there biomarker to assess? easy to measure? correlation to disease?

Question 3

what % survive 5 years

Answer 3

67%

Question 4

visualize xsomes by karyotyping

Answer 4

culture in growth medium, incubate for 2-3 days, add microtubule inhibitor to stop mitosis in metaphase, move cells to tube & centrifuge to conc in layers, transfer to tube (w fixative), stain to enhance xsomes, ID/photograph xsomes

Question 5

karyotyping for diagnosis pros

Answer 5

gold standard, can ID variants of translocatuon, quantify # cells w translocation

Question 6

kartyotyping cons

Answer 6

need viable dividing cells, expensive, labor intensive, need analyzing expertise, low sensitivity , need invasive test

Question 7

sensitivity low to high

Answer 7

karyotyping, fish, pcr, nested pcr

Question 8

specificity

Answer 8

good for karyotyping, fish, pcr, not nested pcr

Question 9

FISH

Answer 9

denature dna, add hybridization probes (flor labled), expose to UV light to get signal

Question 10

what size dna for FISH

Answer 10

400-500 kb … span large areas of genome

Question 11

FISH for diagnosis pros

Answer 11

quantitative, easy to interpret, sensitive to use PB, works for interphase

Question 12

FISH cons

Answer 12

expensive, time consuming, need enough cells to analyze

Question 13

PCR diagnosis pros

Answer 13

sensitive economical, use BM or PB, rapid, doesn’t require dividing cells

Question 14

how sensitive is PCR

Answer 14

1/10k to 100k

Question 15

PCR diagnosis cons

Answer 15

need isolate RNA (unstable), may miss some rare breakpts (in exons)

Question 16

BCR ABL colors

Answer 16

bcr - red, abl - green, fusion - yellow

Question 17

how PCR works

Answer 17

design fwd/reverse primers. fusion gene gives product

Question 18

ABL breakpt region

Answer 18

intron bw exon 1, 2

Question 19

pcr starts w rna or dna

Answer 19

rna

Question 20

small # of pcr primer sets can amplify what

Answer 20

multiple versions of fusion gene

Question 21

pcr output is easy to read? must include what

Answer 21

easy to read output. must include internal PCR control to differentiate bw neg & failed result

Question 22

nested pcr test sensitivity for translocation

Answer 22

1 molecule in 10 000 k (rlly high, not great when detect transcrip at low levels which happen all the time)

Question 23

specific amplification of target dna involves

Answer 23

using product from 1st pcr round as input for 2nd. 1st set primers (outer). 2nd set primers (inner)

Question 24

qualitative essay

Answer 24

yes or no. for initial diagnosis.

Question 25

quantitative

Answer 25

info about amt of biomarker. need after treatment to track relative amt of bcr/abl

Question 26

QRT-PCR

Answer 26

polymerization , strand displacement, cleavage , polymerization complete

Question 27

qrt-pcr polymerization involves

Answer 27

fwd, reverse primer. probe w reporter, quencher. cleave reporter so it flors, show # copies pcr

Question 28

reporter shows up more

Answer 28

at later cycles & as you further dilute it

Question 29

decr in bcr-abl gene transcript (biomarker) levels show

Answer 29

decr in cytogenetic response, correlating to assay sensitivity

Question 30

cytogenetic vs molecular assays

Answer 30

molecular assays more sensitive, cytogenetic = less

Question 31

recurrence of disease after initial response assoc w

Answer 31

extra mutations in orig biomarker (not just fusion, but becomes resistant to imatinib)

Question 32

why use other drugs after treatment

Answer 32

to decr biomarkers again as tumour still evolving

Question 33

diagnostic test for CML

Answer 33

detect philadelphia xsome

Question 34

diagnostic testing evolves as

Answer 34

muchs of resistance becomes clear… 1 mutation but at diff breakpts so it still happens in diff ways