05How to Choose Abnormal Cell Population for Gating

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How to choose an abnormal population for gating?

Dr. Neha Singh,Senior Resident,

Department of Pathology, Maulana Azad 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. Neha Singh, 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


Advantages of collecting ungated data

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.


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.



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.



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).



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


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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.


What is Gating?

An important principle of FCM data analysis: To selectively visualize the cells of interest while eliminating results 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


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


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.

Rectilinear

Amorphous


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Gating strategies


Gating strategies in use

Reminder: All gating strategies should be performed during the subsequent analysis of originally ungated LMD files.

Widely accepted gating procedures: Conventional FSC/SSC gating SSC/CD45 gating CD19 Gating for B cells CD3 Gating for T cells CD38 gating for plasma cells


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. Neha Singh, MAMCDr. Neha Singh, MAMC

10/1/2012

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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.


CD45 gating should replace the first gating step - FSC/SSC as this latter procedure does not discriminate well between leukemic blasts, lymphocytes and monocytes.


Advantages of SSC/CD45 gating

1. Discriminates well between leukemic blasts and normal marrow cells

2. Excludes normal cells from the 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.


Characterization of different cell types on a SSC/CD45 dot plot


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Lymphocytes

Brightest CD45 expression

Lowest SSC (because of absence of granularity)


Neutrophils

Lower CD45 expression than lymphocytes and monocytes

Much higher SSC due to presence of granules


Monocytes

Slightly lower CD45 expression as compared to lymphocytes

Higher SSC due to fine cytoplasmic granularity


Myeloid Precursors

Sometimes form two parallel or merging clusters on the SSC/ CD45 dot plot.

More mature elements form cluster towards the right side with medium CD45 intensity

Immature myeloid precursors are cluster towards the left with low CD45 expression.


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Eosinophils

Form a cluster on the right of the myeloid cluster.

Very high SSC

Moderate CD45 intensity


Erythroid Precursors

Fall in the CD45 negative region

Very low side scatter

Seen along with debris


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.


Neutrophils

Monocytes

Lymphocytes

Blasts

Visualization of different cell

populations on the SSC/CD45 dot

plot from a peripheral blood

sample with Acute Leukemia

Erythroidcells & debris


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CD45 gating in Acute Leukemia


CD45 positive blasts in ALL


CD45 negative blasts in ALL


Heterogeneous CD45 expression in blasts


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AML-M3 Hypergranular blasts


AML-M5


Gating for blasts in CML


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)


CD19 Gating for B Cells (CLL)


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


CD3 gating in T-ALL


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Gating for plasma cells IPT properties of plasma cells:

Intense CD38 expression Presence of cytoplasmic light chains (monoclonal cIg ) Concomitant absence of surface light chains Expression of CD138 and CD56 Downregulation of CD45, surface pan-B cell antigens and

HLA-DR expression

Therefore unless cKappa and cLambda are performed, FCM analysis on plasma cells may yield negative/ nonhemopoietic cells like pattern.


CD38 gating for Plasma cells Plasma cells are gated on cells with intense CD38

positivity

The expression of cIg needs to be demonstrated on cell population with strong CD38 positivity


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.


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


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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.


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


What is Live Gating?

Live gating refers to data collection restricted to certain predetermined criteria.

Disadvantage: Results in throwing critical cells away as 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


Thank You


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05How to Choose Abnormal Cell Population for Gating

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