SCYM ASCP EVALUATION TEST QUESTIONS AND
ANSWERS SURE A+
✔✔bone marrow maturation - ✔✔During the early maturation process, the immature B
cells that are tolerant of the self-antigens present in the bone marrow gradually release
their hold on the stromal cells of the marrow. Morphologic changes during neutrophil
maturation can be identified by flow cytometry using simultaneous quantitative
assessment of multiple antigens in concordance with the light scattering properties of
the human bone marrow cells.
✔✔CyD3 - ✔✔early TCell Marker
✔✔anti=kappa marker - ✔✔BCells
✔✔Data Analysis in Flow Cytometry - ✔✔Flow cytometry data analysis is built upon the
principle of gating. Gates and regions are placed around populations of cells with
common characteristics, usually forward scatter, side scatter and marker expression, to
investigate and to quantify these populations of interest. Here we will show what the
common flow cytometry graph outputs look like and how in a few simple steps you can
identify different cell populations that have been stained with antibodies conjugated to
fluorophores.
✔✔FSC/SSC - ✔✔The first step in gating is often distinguishing populations of cells
based on their forward and side scatter properties. Forward and side scatter give an
estimation of the size and granularity of the cells respectively, although this can depend
on several factors such as the sample, the wavelength of the laser, the collection angle
and the refractive index of the sample and the sheath fluid. Distinguishing populations of
cells can be relatively straight forward for cell lines where there is only one type of cell,
but it can be more complex for samples where there are multiple cell types.
✔✔Simple Parameter Histograms - ✔✔As we have mentioned gates can be applied to
density plots to exclude populations (e.g. debris) or to positively select populations for
further examination
,✔✔Density Plots - ✔✔These graphs display two measurement parameters, one on the
x-axis and one on the y-axis and the events as a density (or dot) plot. The parameters
can be fluorescence, FCS or SSC depending on what you want to show
✔✔Principles of Gating - ✔✔This simple principle of gating can be applied again and
again to further determine the expression patterns on particular cell types. This is
particularly useful as the number of markers and fluorophores in a single experiment
increases
✔✔Backgating to confirm gating strategies - ✔✔Backgating is a useful method of
identification of cells to confirm a staining pattern or gating method. It allows you to
analyze cells identified in a gate on dot plots with different parameters. This can be
useful if you are unsure of your gates, the expression levels, non-specific binding or the
presence of dead cells and need additional information to identify your cells.
✔✔Instrument Optimization - ✔✔Include protocols useful for determination of laser
power, photoelectron efficiency, testing of filter characteristics, evaluation of signal
synchronization, and laser delay determination.
Central characteristics of the flow cytometer and understanding these values at
installation help ensure that when changes are made, the system is performing as well
as when it was first brought into service.
Whenever the optical pathways are changed, including changing a filter installation,
installation of a new laser, and/or realignment because of a new flow cell. Basically,
whenever an optical pathway is changed. This gives the user a baseline to know how
the instrument is performing and a reference for when there are issues.
✔✔Cytometer Calibration- Sensitivity of PMTs - ✔✔There are two separate protocols
necessary for cytometer calibration:
a) Determining the sensitivity of PMTs
Validation of PMT sensitivity
To complete the first protocol, three bead sets are needed. For this work, you should
use:
Single peak beads - BC Flow-Set Pro Fluorospheres
Multipeak beads - DakoFluorospheres
Unstained beads - DakoFluorospheres
b)PMT linearity, which is measured as difference between MFIs of two adjacent peaks
from multiple-peak beads divided by MFI of lower peak from selected peak pair. Like the
PMT calibration, this value is plotted over the voltage range.
Remember that compensation cannot be correctly calculated if the signal is not in the
linear range of the PMT.
✔✔Cytometer Calibration- ComBeads - ✔✔As a second step, CompBeads were mixed
with a negative control (without any binding capacity) and labeled individually with an
appropriate marker. Finally, the primary detector (FL4 for PC5, FL5 for PC7 and FL1 for
FITC) was set to gain highest fluorescence response
, The next step was to set the secondary channels to a minimal MFI. For this, a wide
range of detector voltages (400-700 V) were tested. The voltage with the lowest MFI
found in PMT linear region was chosen
When calibrating your cytometer, the final step is to measure rainbow single-peak
beads using the same primary/secondary channel settings that were used for your
individual fluorochrome.
The repeated measurements (n=20) serves for determination of the target value range
for CV, which is the highest value found within +- 1 SD or +- 10 % of mean value.
✔✔Implement QC Checkpoints - ✔✔Implementing the proper Quality Assurance (QA)
checkpoints is the only way ensure that your flow cytometer is functioning properly over
time. It's also the only way to determine what the issue is and how to fix it if there are
deviations in these checkpoints.
To implement these QA checkpoints, data from three different bead sets must be
recorded and analyzed. The beads include:
Single peak bead
Multiple peak bead
Unstained bead
The parameters to be monitored or "checked" include:
PMT voltage
CV of the single peak bead
MFI of a defined peak (peak 4)
MFI of the unstained beads
Altogether, this allows for three calculations that can be used to assess the overall
quality of the instrument over time. These calculations are:
Accuracy (voltage setting as a function of time)
Precision (CV as a function of time)
Sensitivity (S-T-B ratio as a function of time)
Once the above data are collected, they must be plotted for analysis. The most common
plot is the Levey-Jennings plot, which shows the daily data, a running average and lines
representing tolerance ranges.
✔✔Aerosol Containment - ✔✔Infectious or potentially infectious materials should not be
sorted unless suitable
containment measures are applied. A droplet containment module should be installed to
reduce the risk of exposure to generated droplets and aerosols.
-Only personnel trained in safety protocols should be permitted to run these flow
cytometry analyzers and cell sorters.
-The efficiency of aerosol control measures on the instruments needs to be tested
periodically.
-Sorters must be engineered with an aerosol evacuation system and this must be on
and operational throughout the sort.
-Operators must have procedures in place to maintain and test the aerosol evacuation
system.
-Records must be maintained to show the aerosol evacuation system is functioning
normally.
ANSWERS SURE A+
✔✔bone marrow maturation - ✔✔During the early maturation process, the immature B
cells that are tolerant of the self-antigens present in the bone marrow gradually release
their hold on the stromal cells of the marrow. Morphologic changes during neutrophil
maturation can be identified by flow cytometry using simultaneous quantitative
assessment of multiple antigens in concordance with the light scattering properties of
the human bone marrow cells.
✔✔CyD3 - ✔✔early TCell Marker
✔✔anti=kappa marker - ✔✔BCells
✔✔Data Analysis in Flow Cytometry - ✔✔Flow cytometry data analysis is built upon the
principle of gating. Gates and regions are placed around populations of cells with
common characteristics, usually forward scatter, side scatter and marker expression, to
investigate and to quantify these populations of interest. Here we will show what the
common flow cytometry graph outputs look like and how in a few simple steps you can
identify different cell populations that have been stained with antibodies conjugated to
fluorophores.
✔✔FSC/SSC - ✔✔The first step in gating is often distinguishing populations of cells
based on their forward and side scatter properties. Forward and side scatter give an
estimation of the size and granularity of the cells respectively, although this can depend
on several factors such as the sample, the wavelength of the laser, the collection angle
and the refractive index of the sample and the sheath fluid. Distinguishing populations of
cells can be relatively straight forward for cell lines where there is only one type of cell,
but it can be more complex for samples where there are multiple cell types.
✔✔Simple Parameter Histograms - ✔✔As we have mentioned gates can be applied to
density plots to exclude populations (e.g. debris) or to positively select populations for
further examination
,✔✔Density Plots - ✔✔These graphs display two measurement parameters, one on the
x-axis and one on the y-axis and the events as a density (or dot) plot. The parameters
can be fluorescence, FCS or SSC depending on what you want to show
✔✔Principles of Gating - ✔✔This simple principle of gating can be applied again and
again to further determine the expression patterns on particular cell types. This is
particularly useful as the number of markers and fluorophores in a single experiment
increases
✔✔Backgating to confirm gating strategies - ✔✔Backgating is a useful method of
identification of cells to confirm a staining pattern or gating method. It allows you to
analyze cells identified in a gate on dot plots with different parameters. This can be
useful if you are unsure of your gates, the expression levels, non-specific binding or the
presence of dead cells and need additional information to identify your cells.
✔✔Instrument Optimization - ✔✔Include protocols useful for determination of laser
power, photoelectron efficiency, testing of filter characteristics, evaluation of signal
synchronization, and laser delay determination.
Central characteristics of the flow cytometer and understanding these values at
installation help ensure that when changes are made, the system is performing as well
as when it was first brought into service.
Whenever the optical pathways are changed, including changing a filter installation,
installation of a new laser, and/or realignment because of a new flow cell. Basically,
whenever an optical pathway is changed. This gives the user a baseline to know how
the instrument is performing and a reference for when there are issues.
✔✔Cytometer Calibration- Sensitivity of PMTs - ✔✔There are two separate protocols
necessary for cytometer calibration:
a) Determining the sensitivity of PMTs
Validation of PMT sensitivity
To complete the first protocol, three bead sets are needed. For this work, you should
use:
Single peak beads - BC Flow-Set Pro Fluorospheres
Multipeak beads - DakoFluorospheres
Unstained beads - DakoFluorospheres
b)PMT linearity, which is measured as difference between MFIs of two adjacent peaks
from multiple-peak beads divided by MFI of lower peak from selected peak pair. Like the
PMT calibration, this value is plotted over the voltage range.
Remember that compensation cannot be correctly calculated if the signal is not in the
linear range of the PMT.
✔✔Cytometer Calibration- ComBeads - ✔✔As a second step, CompBeads were mixed
with a negative control (without any binding capacity) and labeled individually with an
appropriate marker. Finally, the primary detector (FL4 for PC5, FL5 for PC7 and FL1 for
FITC) was set to gain highest fluorescence response
, The next step was to set the secondary channels to a minimal MFI. For this, a wide
range of detector voltages (400-700 V) were tested. The voltage with the lowest MFI
found in PMT linear region was chosen
When calibrating your cytometer, the final step is to measure rainbow single-peak
beads using the same primary/secondary channel settings that were used for your
individual fluorochrome.
The repeated measurements (n=20) serves for determination of the target value range
for CV, which is the highest value found within +- 1 SD or +- 10 % of mean value.
✔✔Implement QC Checkpoints - ✔✔Implementing the proper Quality Assurance (QA)
checkpoints is the only way ensure that your flow cytometer is functioning properly over
time. It's also the only way to determine what the issue is and how to fix it if there are
deviations in these checkpoints.
To implement these QA checkpoints, data from three different bead sets must be
recorded and analyzed. The beads include:
Single peak bead
Multiple peak bead
Unstained bead
The parameters to be monitored or "checked" include:
PMT voltage
CV of the single peak bead
MFI of a defined peak (peak 4)
MFI of the unstained beads
Altogether, this allows for three calculations that can be used to assess the overall
quality of the instrument over time. These calculations are:
Accuracy (voltage setting as a function of time)
Precision (CV as a function of time)
Sensitivity (S-T-B ratio as a function of time)
Once the above data are collected, they must be plotted for analysis. The most common
plot is the Levey-Jennings plot, which shows the daily data, a running average and lines
representing tolerance ranges.
✔✔Aerosol Containment - ✔✔Infectious or potentially infectious materials should not be
sorted unless suitable
containment measures are applied. A droplet containment module should be installed to
reduce the risk of exposure to generated droplets and aerosols.
-Only personnel trained in safety protocols should be permitted to run these flow
cytometry analyzers and cell sorters.
-The efficiency of aerosol control measures on the instruments needs to be tested
periodically.
-Sorters must be engineered with an aerosol evacuation system and this must be on
and operational throughout the sort.
-Operators must have procedures in place to maintain and test the aerosol evacuation
system.
-Records must be maintained to show the aerosol evacuation system is functioning
normally.
