10/1/2012

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How to choose an abnormal
population for gating?
Dr. NehaSingh,
Senior Resident,
Department of Pathology,
MaulanaAzad Medical College, New Delhi.
Introduction
Flow cytometry (FCM) identifies hemopoietic
neoplasms by presence or absence of cellular
antigen expression
Data analysis is based on:
Visual appraisal of patterns formed by cell clusters on dot
plots such as FSC/SSC and SSC/CD45.
GATING: forms the basis of data interpretation
Data saved as List Mode Data (LMD) files
Dr. NehaSingh, MAMC
Collection of ungated data
Microscopic examination:
All elements are examined
Only the abnormal ones may be
stressed upon in the final report.
Flow cytometry:
Samples contain a
heterogeneous cell population
Variability in cell size and
granularity.
LMD files should be collected
ungated
Emphasize upon abnormal
population in report
Dr. NehaSingh, MAMC
Advantages of collecting ungateddata
1. Ensures that all abnormal
cells are collected
2. Especially critical when
the nature of the
abnormal population is
not known
3. The presence of other
cells serves as an internal
positive and negative
controls.
Dr. NehaSingh, MAMC
Hence it is judicious to acquire ungated LMDs prior to
phenotypic analysis of any gated population.
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Terminologies used in FCM
Cytogram:A 2D histogram
in which two cell
parameters are correlated
Dot plot: A representation
of a cytogram in which each
individual cell passing the
instrument is represented
by a dot on a 2D graph.
Dr. NehaSingh, MAMC
Terminologies used in FCM
Density plot:
Similar to a dot plots, but
use of different colors
enables abundant and
less abundant cell
populations to be
identified.
The colors give the graph
a 3D feel.
Dr. NehaSingh, MAMC
Terminologies used in FCM
Contour plot:
A display of a cytogram in
which the density of cells
is defined by contours
(similar to those used on
a cartographic map).
Dr. NehaSingh, MAMC
Terminologies used in FCM
Region:refers to an area drawn on a plot displaying
flow cytometry data
Listmode data (LMD) files: consist of a complete
listing of all events corresponding to all the
parameters collected
Discriminator: A channel setting for a parameter that
ignores events below the setting. Eliminates signals
caused by debris
Dr. NehaSingh, MAMC
10/1/2012
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What is a gate?
A gate is selected by
defining a region on a
cytogram.
Only cells falling within
the gate can pass through
to the next stage of
analysis.
Gates are also used to
select desired populations
for cell sorting.
Dr. NehaSingh, MAMC
What is Gating?
An important principle of FCM data analysis:
To selectively visualize the cells of interest while
eliminatingresults from unwanted particles e.g.
dead cells and debris. This procedure is called
gating.
Gating is an electronic selection of a certain
population of cells for immunophenotypic analysis
Dr. NehaSingh, MAMC
Significance of gating
The purpose of gating is to enrich or highlight the
population being searched for - population of
interest
Allows identification of a subset of cells and
detection of parameters specific only to that subset
The subsequent data analysis and interpretation of
the flow results relies on gating
Dr. NehaSingh, MAMC
Types of Gates
Different types of gates:
Rectilinear
Amorphous
Numeric
Amorphous gates:
Most versatile
Any shape or form
Allow a better and flexible
selection of the
population of interest.
Recti l i near
Amorphous
Dr. NehaSingh, MAMC
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Gatingstrategies
Dr. NehaSingh, MAMC
Gating strategies in use
Reminder: All gating strategies should be
performed during the subsequent analysis of
originally ungatedLMD files.
Widely accepted gating procedures:
Conventional FSC/SSC gating
SSC/CD45 gating
CD19 Gatingfor B cells
CD3 Gatingfor T cells
CD38 gatingfor plasma cells
Dr. NehaSingh, MAMC
FSC/SSC dot plot
Separates cells on the
basis of size and
granularity.
Physical properties of
WBCs allow them to be
distinguished from each
other and from cellular
contaminants.
Dr. NehaSingh, MAMC
Dr. NehaSingh, MAMC
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SSC/CD45 Dot plot
CD45 is selected as the basis for gating procedure.
Found in different amounts in mature and
immature hemopoietic cells
CD45 expression in blasts is lower/ dimmer as
compared to mature lymphocytes and monocytes.
More sensitive than the FSC/SSC approach.
Dr. NehaSingh, MAMC
CD45 gating should replace the
first gating step - FSC/ SSC as this
latter procedure does not
discriminate well between
leukemic blasts, lymphocytes and
monocytes.
Dr. NehaSingh, MAMC
Advantages of SSC/CD45 gating
1. Discriminates well between leukemic blasts and normal
marrow cells
2. Excludes normal cells fromthe phenotypic analysis of
leukemic blasts
3. Identifies blast cell heterogeneity in many cases of
leukemia on the basis of different CD45 display
4. Defines all cell sub-populations in the sample
5. Possible to estimate a BM differential count based on the
distribution of cells on the SSC/CD45 plot, provided the
sample is not hemodiluted.
6. Facilitates the analysis of blasts present in low proportions.
Dr. NehaSingh, MAMC
Characterization of different cell
types on a SSC/CD45 dot plot
Dr. NehaSingh, MAMC
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Lymphocytes
Brightest CD45
expression
Lowest SSC (because of
absence of granularity)
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Neutrophils
Lower CD45 expression
than lymphocytes and
monocytes
Much higher SSC due to
presence of granules
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Monocytes
Slightly lower CD45
expression as
compared to
lymphocytes
Higher SSC due to fine
cytoplasmic granularity
Dr. NehaSingh, MAMC
Myeloid Precursors
Sometimes form two
parallel or mergingclusters
on the SSC/ CD45 dot plot.
More mature elements
form cluster towards the
right side with medium
CD45 intensity
Immaturemyeloid
precursors are cluster
towards the left with low
CD45 expression.
Dr. NehaSingh, MAMC
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Eosinophils
Form a cluster on the
right of the myeloid
cluster.
Very high SSC
Moderate CD45
intensity
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Erythroid Precursors
Fall in the CD45
negative region
Very low side scatter
Seen alongwith debris
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Blasts
Low SSC and lower
CD45 intensity than
lymphocytes and
monocytes.
Sometimes
lymphoblasts are
negative for CD45; seen
as a cluster in the
erythroid region.
Dr. NehaSingh, MAMC
Neutrophi l s
Monocytes
Lymphocyte
s
Bl asts
Visualization of
different cell
populations on
the SSC/CD45 dot
plot froma
peripheral blood
sample with
Acute Leukemia
Erythroi d
cel l s & debri s
Dr. NehaSingh, MAMC
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CD45 gating in Acute
Leukemia
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CD45 positive blasts in ALL
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CD45 negative blasts in ALL
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Heterogeneous CD45 expression in blasts
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10/1/2012
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AML-M3 Hypergranular blasts
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AML-M5
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Gating for blasts in CML
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Other gating strategies
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Gating for B cells for eg. in CLL
Features typical of CLL:
Small cell size; low FSC
Intense CD19 expression
Weak and
heterogeneous CD 20
expression
Downregulation of
CD20:
Variable fluorescence
distribution of CD20
(starting in the negative
region)
Dr. NehaSingh, MAMC
CD19 Gatingfor B Cells (CLL)
Dr. NehaSingh, MAMC
Gating for T cells
ATLL cells overlap with
normal gated
lymphocytes
Difficult to gate on
SSC/CD45 plot
Variable FSC from low
to high
Gated on CD3+cells
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CD3 gating in T-ALL
Dr. NehaSingh, MAMC
10/1/2012
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Gating for plasma cells
IPT properties of plasma cells:
Intense CD38 expression
Presence of cytoplasmiclight chains (monoclonal cIg)
Concomitant absence of surface light chains
Expression of CD138 and CD56
Downregulationof CD45, surface pan-B cell antigens and
HLA-DR expression
Therefore unless cKappaand cLambdaare
performed, FCM analysis on plasma cells may yield
negative/ nonhemopoieticcells like pattern.
Dr. NehaSingh, MAMC
CD38 gating for Plasma cells
Plasma cells are gated on cells with intense CD38
positivity
The expression of cIgneeds to be demonstrated on
cell population with strongCD38 positivity
Dr. NehaSingh, MAMC
Reverse Gating
Sometimes the desired population is not clearly
apparent on light scatter dot plot
How to draw the region in such cases?
A fluorescent-labelled antibody is used to pick out the
cells of interest
A gate is set for positive fluorescence
Light scatter of these cells displayed.
A region can now be drawn on the highlighted light
scatter plot.
Dr. NehaSingh, MAMC
Reverse Gating
Selection a population
of interest by gating it
on the basis of its
antibody expression
Thus highlighting it to
locate its correct
position on the light
scatter dot plot
Dr. NehaSingh, MAMC
10/1/2012
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Gating for exclusion of dead/ nonviable cells
Analysis of dead cells is
done by Propium
Iodide exclusion.
Principle:
High uptake of PI by
dead & dying cells
Dead cells have lower
forward scatter
Higher side scatter than
living cells.
Dr. NehaSingh, MAMC
Alternate technique for Gating dead cells
Gating out dead cells on
the FSC/SSC or
CD45/SSC plots
Seen with debris in the
region of lowest FSC,
and as CD45 negative
cells with low SSC
Less accurate
Dr. NehaSingh, MAMC
What is Live Gating?
Live gatingrefers to data collection restricted to
certain predetermined criteria.
Disadvantage:Results in throwing critical cells
awayas the nature of the abnormal cells is not
known at the time of running the sample
Advantage: used to enrich a small population of
cells; e.g. CD34 +stem cells, potential monoclonal B
cells
Dr. NehaSingh, MAMC
Thank You
Dr. NehaSingh, MAMC