Week 2A- Immunophenotyping and cytogenetics Flashcards
what are the 4 main features used to define a disease
morphology
immuniphenotype
genetics
clinical findings
what is immunophenotyping
looking at cells properties and antigens to characterize cells
what can immunophenotyping do
- Can distinguish between normal cells, and neoplastic cells
- Can analyse millions of cells rapidly
- Can look at multiple properties of each cell at once
how can blood cells be distinguished
size - forward scatter
internal complexity - side scatter
what is flow cytometry a measurement of
cells in a flowing medium
how many events, markers and parameters can flow cytometry have
35,000 events / s
up to 10 markers at a time
2 scatter parameters
what is is flow cytometry composed of
fluidics
optics
electronics (detection)
interpretation software
what is the FS, SS and fluorescence split into
defined wavelengths and chanelled by a set of filters and mirrors within the flow cytometry
why is the fluorescent light filtered in flow cytometry
so that each sensor will detect fluorescence only at a specified wavelength
what are the sensors called that detect fluorescence
photomultiplier tubes (PMTs)
what does Long pass filters allow
transmission of photons above a specified wavelength
what do band pass filters allow
transmission of photons that have wavelengths within a narrow range
what do short pass filters allow
transmission of photons below a specified wavelength
what does the electrons system (detection) do
convert photons to electrons, which are then converted from analog to digitized data
what happens to the data in detection
binned and stored in digital data file that can be read and analysed with appropriate software
what does doublet exclusion ensure
no clumps or doublets are analysed
otherwise the clump is interpreted as one event with the combined properties of all cells in the group
what does FACS stand for
Fluorescent activated cell sorting
what does FACS mean
its how flow cytometry can only analyse and sort cells with specific properties using electric charge
why is it essential to choose correct combination of fluorochromes
so they dont overlap and the equipment can actually excite/read them
what is compensation
the removal of spectral overlap of emission spectra between fluorochrome like FITC and PE
what happens if compensation is incorrect
interpreting the data can become extremely difficult or impossible
what is the goal of compensation
to remove the signal from a given fluorochrome from all neighbouring channels where it is also detected
what is gating
refers to the selection of successive subpopulations of cells for analysis in flow cytometry
what are three types of gates
FSC vs SSC
doublet gate to exclude clumps
quadrat parameter plot
how does immunophenotyping help with haemotological malignancies
- distinguishes between lymphoid or myeloid
- distinguishes between benign and malignant
- small diagonistic role
what is CD
cluster of differentiation
what does the + and - mean on markers
presence or absence of a specific antigen from the surface of particular cell population
what does hi or low marks indicate
cellular expression levels
how many defined CD markers are there
<300
what are the two main branches on oncology cytogenetics
FISH
karyotyping
what does FISH stand for
fluorescence in situ hybridisation
what does FISH mean
Microscopic examination of either interphase cells or chromosomes for the presence or absence of a fluorescently tagged probe (DNA sequence)
how does DNA sequence relate to the FISH probe
is complementary to it
what happens if the region of interest is present is FISH
the probe will bind (hybridise) to the target DNA sequence, which can then be visualised using fluorescent microscopy
what happens if the region of interest has been deleted in FISH
the probe will not bind to the target DNA and so will not be visualised.
what is karyotyping
Microscopic examination of the chromosomes – a whole genome screen
what can karyotyping detect
loss or gain of material in any region of any chromosome as well as both balanced and unbalanced structural rearrangements such as translocations and inversions
why is level of detail of karyotyping limited
due to resolution of microscope
what chromosomes does karyotyping need
metaphase chromosomes
what is an ideogram
where staining shows up banding on chromosomes - each is unique
