Flow Cytometry Tutorials: All About Compensation

Transcript

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Flow Cytometry Tutorials: All About Compensation

00:04

Objectives:

00:06

By the end of this tutorial, you should be able to understand: 

00:09

- What is compensation?

00:12

- Why we need to do compensation?

00:15

- How to do compensation?

00:18

- Manual compensation correction

00:21

- Some compensation tips

00:26

What is Compensation?

00:29

-It's the process of correcting spectral overlaps between channels

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Spectral overlap

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Why do we need to do compensation? 

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In Flow Cytometry, target-specific signal is detected  in the form of Fluorescence

00:47

Some common sources of Fluorescence for Flow include:

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Fluorochromes (aka Fluorophores)conjugated to antibodies

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Fluorescent proteinsused as reportersof gene expression

01:07

Fluorescent dyessuch as DNA-bindingdyes. eg.7AAD

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-Any given Fluorescent molecule exhibits characteristic excitation   and emission properties.

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For example:

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FITC and PE can be excited using 488nm (Blue laser)

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The emission maxima of FITC is ~520nm

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and the emission maxima of PE is ~575nm

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Notice how FITC emission peak overlaps with the PE emission peak? 

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This FITC overlap contributes false positive signal in PE channel

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Therefore, compensation is needed to correct spectral overlap b/w channels 

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How to do Compensation? 

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-Using proper compensation controls, and optimal instrument   settings one can generate an accurate compensation matrix 

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-Modern (digital) Flow Cytometers come with acquisition software    that allows Auto-compensation

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-However, how good an auto-compensation wizard works depends   on how good the compensation controls are

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So what "proper" controls are needed?

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There are mainly two types of controls needed for compensation:

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An Unstained control(cells only)

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&

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Single Stained Controls(one for each color)

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-Unstained control takes into account intrinsic autofluorescence of a given cell-type

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Therefore, highly recommended to match  unstained control to that of experimental cell type

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-For compensation calculation itself, internal negative(negative peak of the single stains) can be used

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But, unstained control is still required for voltage setup

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-When experimental cells cannot be used as unstainedcontrols (eg. when dealing with very few cells),compensation beads may be used.

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Several commercial beads are available

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Single Stained Controls

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-Single stained controls are needed one for eachfluorochrome in the panel

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-Must be the same fluorochrome as in the samples

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eg. If you have FITC, only FITC can be used butnot GFP or AF488 (even when they fall in same channel)

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-Preferably the same antibody, but can be switched  with another antibody (if marker expression is low)

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substitute antibody must target the same cellsas experimental cells 

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-Compensation beads may be used if short on cells   or marker expression is low.

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-Both beads and cells can also be combined

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eg. when using viability dye, you can use cells for dye, and beads for Fluors  

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Last but not the least, single stained controls must be at least as bright as samples

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How to set the optimal PMT voltages?

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-An optimal voltage is where:

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-The positive signal is detected above background

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-The positive peak is not off scale (because of too high voltage)

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-The resolution of the positive and/or dim population is not lost   (because of too low voltage)

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How to calculate compensation?

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-Following are the 3 requirements to calculate compensation

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1. Once set, the optimal voltages need to be the same across channels

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Unstained tube and all the single color tubes need to be recorded at thesesame voltages

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2. Same gated population needs to be used for compensation calculation 

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For example:

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If you are analyzing lymphocytes,then same lymphocyte gate needs to beapplied to all the comp' controls

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In FACSDiva software, you can do thisby right-clicking on the gate and then selecting "Apply to all..." option  

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3.

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Positive gate on each single color control needs to be adjusted toencompass true positive

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This gate can be different for different single color controls

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Once these requirements are met, you can calculate compensations

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In FACSDiva, you can do this by selectingExperiment>Compensation Setup>Calculate Compensations

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How to do manual compensation correction? 

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Often times, auto-calculated compensation values are not accurate

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This is more true when single color controls lack a distinct positive peak

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Manual compensation correction can be done by :

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- Statistically, by comparing MFIs

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while adjusting compensation matrix created by auto-comp wizard

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Manual correction by comparing Median Fluorescence Intensities (MFIs):

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* This is the most accurate method 

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Let's assume you have FITC & PE Fluors

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FITC-A

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PE-A

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The four possible populations would be...

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DoubleNegative

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FITC+

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DoublePositive

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PE+

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In a well compensated sample:

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The PE MFI value of FITC+ populationaligns closely with the double negative

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PE MFI=9

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PE MFI=8

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The FITC MFI value for the PE+ populationaligns closely with the double negative

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FITC MFI=10

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FITC MFI=11

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When FITC is UNDERcompensated:

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 PE MFI for FITC+ population looks higher(as FITC signal bleeds into PE channel)

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FITC+ UNDERcompensatedPE MFI= 153

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When FITC is OVERcompensated:

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 PE MFI for FITC+ population looks lowerthan the double negative population

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FITC+ OVERcompensatedPE MFI=  in negative values

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Same compensation rules apply for thePE+ population (with only difference is -we compare FITC-MFIs for PE+ population)

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Summary 

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-Compensation is a process of correcting spectral overlaps between channels

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-Modern (digital) Flow Cytometers provide auto-compensation wizards 

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-Auto-compensation wizard works only as good as compensation controls 

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-Proper controls & optimal voltage settings result in an accurate comp' matrix

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-Auto-comps can be corrected manually (if needed) by looking at MFI values

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-Manual correction involves comparing MFIs of single positive populations  with double negative population within a sample

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-3 words to remember: UNDERcompensated, OVERcompensated, and  well-compensated

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Some compensation tips... 

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In FACSDiva software, compensation matrix can be adjusted underCytometer Settings option

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To show MFIs in FACSDiva software, right click on the plot showingparameters that need to be compensated, and select "Create Statistics"

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Here is an example statistics view showing MFIs for FITC and PE plot:

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Compensated Sample

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UNDERCompensated for FITC

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Notice how the PE-MFIs are higher for FITC+ (Q4) vs double negative (Q3)

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When doing manual compensation correction, always make sure touse Biexponential display to show events below zero

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On regular Log Scale

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With Biexponential display

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Without Biexponential display, noticehow the events below zeroare hidden - making it tricky to knowwhere the populations are

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In FACSDiva software, Biexponential display can be turned onin the inspector window.

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What looks uncompensated is NOT always uncompensated...

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Sometimes it could be just biology rather than compensation 

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For example: "Steric hindrance" causes populations to look UNDERcomp'd

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Notice how the populationlooks undercompensated?

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This phenomenon occurs when twoantibodies compete to occupy same space

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Anti-TCRαβ (clone WT31)binds to a conformational epitope formed by the TCR and epsilon chain.

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CD3 antibody binds to theepsilon chain of theTCR complex

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Further Reading...

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- Compensation by Dr. Mario Roderer: http://www.drmr.com/compensation/

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- Technical bulletin on Compensation by BD:https://www.bdbiosciences.com/documents/Compensation_Multicolor_TechBulletin.pdf

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-Compensation guidelines by Excyte:https://expert.cheekyscientist.com/guidelines-for-setting-compensation-controls-in-flow-cytometry-experiments

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CreditsConceptualized and Created ByJavid MohammedBackground Music www.bensound.com

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