CML 2 Flashcards
what is CML
stem cell disease w excess myeloid cells
clin molecular lab questions
can disorder be defined? pose serious health prob? is there treatment? is there biomarker to assess? easy to measure? correlation to disease?
what % survive 5 years
67%
visualize xsomes by karyotyping
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
karyotyping for diagnosis pros
gold standard, can ID variants of translocatuon, quantify # cells w translocation
kartyotyping cons
need viable dividing cells, expensive, labor intensive, need analyzing expertise, low sensitivity , need invasive test
sensitivity low to high
karyotyping, fish, pcr, nested pcr
specificity
good for karyotyping, fish, pcr, not nested pcr
FISH
denature dna, add hybridization probes (flor labled), expose to UV light to get signal
what size dna for FISH
400-500 kb … span large areas of genome
FISH for diagnosis pros
quantitative, easy to interpret, sensitive to use PB, works for interphase
FISH cons
expensive, time consuming, need enough cells to analyze
PCR diagnosis pros
sensitive economical, use BM or PB, rapid, doesn’t require dividing cells
how sensitive is PCR
1/10k to 100k
PCR diagnosis cons
need isolate RNA (unstable), may miss some rare breakpts (in exons)
BCR ABL colors
bcr - red, abl - green, fusion - yellow
how PCR works
design fwd/reverse primers. fusion gene gives product
ABL breakpt region
intron bw exon 1, 2
pcr starts w rna or dna
rna
small # of pcr primer sets can amplify what
multiple versions of fusion gene
pcr output is easy to read? must include what
easy to read output. must include internal PCR control to differentiate bw neg & failed result
nested pcr test sensitivity for translocation
1 molecule in 10 000 k (rlly high, not great when detect transcrip at low levels which happen all the time)
specific amplification of target dna involves
using product from 1st pcr round as input for 2nd. 1st set primers (outer). 2nd set primers (inner)
qualitative essay
yes or no. for initial diagnosis.
quantitative
info about amt of biomarker. need after treatment to track relative amt of bcr/abl
QRT-PCR
polymerization , strand displacement, cleavage , polymerization complete
qrt-pcr polymerization involves
fwd, reverse primer. probe w reporter, quencher. cleave reporter so it flors, show # copies pcr
reporter shows up more
at later cycles & as you further dilute it
decr in bcr-abl gene transcript (biomarker) levels show
decr in cytogenetic response, correlating to assay sensitivity
cytogenetic vs molecular assays
molecular assays more sensitive, cytogenetic = less
recurrence of disease after initial response assoc w
extra mutations in orig biomarker (not just fusion, but becomes resistant to imatinib)
why use other drugs after treatment
to decr biomarkers again as tumour still evolving
diagnostic test for CML
detect philadelphia xsome
diagnostic testing evolves as
muchs of resistance becomes clear… 1 mutation but at diff breakpts so it still happens in diff ways
