Basics of Flow Cytometry Canto & practical part.pdf

7/30/2019 Basics of Flow Cytometry Canto & practical part.pdf

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Basics of MulticolorFlow Cytometry

June 08, 2012


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The Principle of Flow Cytometry

Resuspendend single cells are moved through alight source (laser).

In this process cells emit characteristic light signalsdepending on the cell type and the preparation ofcells that are detected by appropriate detectors.


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Design of a Flow Cytometer

Fluidic System

Transports and align cells in to the laser focus

Optical System

Excitation Optics

Detection Optics

Electronical System

Transfers optical signals into electronical signals, digitalize the

electronical signals for the analysis on a computer


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The Fluidic System

Fluidic Cart

Sheath Fluid Waste

Cleaning Solution

Shut Down Solution

Sample Injection Port

Cuvette


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Sheath Fluid Stream Sample Stream

Waste Aspirator

Flow Cell

Laser Focus

Waste Tank

Reservoir

Fluid Filter

Bubble filter

Plenum (internalreservoir)

Sample InjektionTube (SIT)

Sample Tube

Overview of the Fluidic System


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Sheath Fluid

Sample

Laser beamSample stream is accelerated due to

the reduction of the cross sectioninside the cuvette resulting in theacceleration and separation of cells(hydrodynamic Focusing).

BD FACSCanto II:Max. event rate of 10.000 events/sec

Detection of particles ca. 0,2 50 m.

Thinning out Cells


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SIP (Sample Injection Port)BD FACSCanto II

Sample Injection Tube

(SIT)

Support

Arm

Holder for

Tube Rack

Sample

Tube


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Cuvette

Waste Line

Intersection Point

SIT

Rinse Line

Sheath Line


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Event Rate

Sample

Red laser

Violet laserBlue laser

Sheath fluid

LOW low sample pressure

Low sample consumption~12 l/min

HIGH high sample pressure

High sample consumption~120 l/min


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The Optical System

Excitation optic

Laser Prism and lenses

Detection optic Filter and mirrors

Detectors


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Gas laser Diode laserSolid state laser

The Lasers

Lasers emit light of a single wave length

Often used lasers in flow cytometers: 488 nm (blue Laser) 633 nm (red laser) 405 nm (violet laser)

Various available laser types:


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Transport & Fokusing of Laser Beam

Why do w e need a laser?

Excitation of fluorochromes in the cuvette that are bound on or inside a

cell/particle

Blue laserRed laser Cuvette

Glas fiber cable


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450 550 650 500 550 600

LP 500LP 500 BP550/50BP550/50

Longpass Bandpass

Optical Filters & Dichroic Mirrors

Separation of cell signals with filters and mirrors


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The Detectors

Amplification of the light signal and transfer of an optical

signal into an electronical signal Photodiode

PMT (Photomultiplier Tube)


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564606 nm

750810 nm

483493 nm

> 670 nm

515545 nme.g. FITC

SSC

e.g. PE

e.g. PE-Cy7

e.g. PerCP orPerCP-Cy5.5

Detection Optic OctagonSignals of the blue laser (42, 422 Configurations)


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Signals of the red laser42, 422 Configurations

Signals of the violet laser422 Configuration

Detection Optic Trigon

425-475

485-535

650-670750-810e.g. APC-H7 e.g. APC

e.g. BD Horizon V450

e.g.BD Horizon V500


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Example: Fluorochrome combination onCanto II (422 Configuration)

Laser Fluorochromes Alternatives

407 nm (violet) BD Horizon V450 PacificBlueBD Horizon V500 AmCyan, DAPI

488 nm (blue) FITC GFP, Alexa Fluor 488

PE PI

PerCP-Cy5.5 PerCP, PE-Cy5.5, 7-AAD, PI

PE-Cy7

633 nm (red) APC Alexa Fluor 647

APC-H7 APC-Cy7


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Violet Laser (405 nm)

Blue Laser (488 nm)

Red Laser (633 nm)

Glas fiber cable

Prisms Focus lense

Steering disk

Flow cell

564606 nm

750810 nm

> 670 nm

SSC

515545 nm

The Optical System


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The Electronic System

Digital data processing

Tasks: converts analog signals into digital signals

determins Area and Height of each pulse

Calculates the Width of the signal

Does the compensation

Communication (data transfer) between workstation and flow cytometer


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Laser

Laser

Laser

Time

Voltage

Time

Voltage

Time

Voltag

e

Quantification of the

pulse

Time

Pulse Area(Area)

PulseHeight

(Height)

Pulse Width(Width)

0



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39,27139,27139,27139,271Event 1Event 2

Event 3

FSC SSC FITC PE

0 60 120 9010 160 65

30 650 160

185

Time

PE-A

Exportable as FCS File19001900

2,6882,688

31003100

3,1893,189

1900

90

185 3100

PE-A

F

ITC-A

1900

90F

ITC-A

185 3100

The Result

39,27139,271Event 1Event 2

Event 3

FSC SSC FITC PE

0 60 120 9010 160 65

30 650 160

Time

PE-A

F

ITC-A

39,27139,271Event 1Event 2

Event 3

FSC SSC FITC PE

0 60 120 9010 160 65

30 650 160

Time

PE-A

F

ITC-A

39,27139,271Event 1Event 2

Event 3

FSC SSC FITC PE

0 60 120 9010 160 65

30 650 160

Time

PE-A

1900

90F

ITC-A

F

ITC-A

39,27139,271Event 1Event 2

Event 3

FSC SSC FITC PE

0 60 120 9010 160 65

30 650 160

Time

PE-A PE-A

1900

90F

ITC-A

F

ITC-A

39,27139,271Event 1Event 2

Event 3

FSC SSC FITC PE

0 60 120 9010 160 65

30 650 160

Time

PE-A

Time FSCTime SSCFSCTime FITCSSCFSCTime PEFITCSSCFSCTime

120

PEFITCSSCFSCTime

60 120

PEFITCSSCFSCTime

0 60 120

PEFITCSSCFSCTime

100 60 120

PEFITCSSCFSCTime

160100 60 120

PEFITCSSCFSCTime

65160100 60 120

PEFITCSSCFSC

65160100 60 120

PEFITCSSCFSC

65160100 60 120

PEFITCSSCFSC

160

65160100 60 120

PEFITCSSCFSC

650 160

65160100 60 120

PEFITCSSCFSC

30 650 160

65160100 60 120

PEFITCSSCFSC

Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

Event 3

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

F

ITC-A

Event 3

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

PE-A

F

ITC-A

Event 3

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

1900

90F

ITC-A

PE-A

F

ITC-A

Event 3

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

PE-A

1900

90F

ITC-A

PE-A

F

ITC-A

Event 3

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

PE-A

1900

90F

ITC-A

PE-A

F

ITC-A

Event 3

Event 2Event 1

30 650 160

65160100 60 120

PEFITCSSCFSC

FCS-Data


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Flow Cytometry Software

BDBD FACSCantoFACSCantoTMTM SoftwareSoftware BDBD FACSDivaFACSDivaTMTM SoftwareSoftware

- Automatic QC Setup

- Automatic analysis of predefined kitsfor clinical applications (e.g. BDMultitest)

- Acquisition and analysis of

individual applications


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Time

Time

Time

Time

Time

DataProcessing

PE

FITC

SSC

FSC

APC

PerCP-Cy5.5

Time



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What can you measure?

Algae

Bloodcells

DNA/RNA

Protozoa


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Applications used in Flow Cytometry


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Which Parameters can be Analyzed?

Relative Size (Forward Scatter-FSC)

Relative granularity or internal complexity (Side Scatter-SSC)

Relative fluorescence intensity

What can a flow cytometer perform?

Simultaneous quantification of multiple optical parameters With a high flow rate.


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The Scatter Parameters: FSC & SSC

Forward Scatter Light (488 nm):Cell Surface Area

Light Source(Laser Beam)

Side Scatter Light (488 nm):Cell Complexity/Granularity

Light Source(Laser Beam)

Forward Scatter (FSC) defracted light

proportional to cell surface (cell size)

detected along the incident light in forward direction (1-10)

Side Scatter (SSC)- reflected and refracted light

Related to cell granularity and complexity

Detected in a 90 angle from the laser beam


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NeutrophilicGranulocytes

Lymphocytes

FSC

0 50 100 150 200 250

Monocytes

*1000

*1000

0

50

100

1

50

200

25

0

Debris

SSC

BasophilicGranulocyte

Lymphocyte

EosinophilicGranulocyteNeutrophilicGranulocyte

MonocyteThrombocyte

Erythrocyte

Example: FSC & SSC of Lysed Whole Blood


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= 488 nm (blue)

Energy of laserlight (excitation)

Energy of emittedfluorescence

519 nm (green)Antibody

Step 1: Fluorochrome absorbs the energy of laser light

Step 2: Fluorochrome releases absorbed energy as:

a) Vibration and heatb) Emission of photons with longer wave length

(= lower energy)

Stokes- Shift: Wave length difference between absorbtion and emission

FluoresceinIsothiocyanate

(FITC)

HO

N

OO

O

HO

C

S

What is Fluorescence ?


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Relative Fluorescence Intensity

No.ofevents

FITC

100 101 102 103 104

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC FITC

FITC

FITC

FITC

FITC

FIT

C

FITC

FIT

C FITC

FITC

FITC

FITC

FITC

FIT

C

FITC

FIT

CFITC

FITC

FITC

FITC

FITC

FITCF

ITC

FIT

C

FITC

FITC

FITC

FITC

FITC

FIT

C

FITC

FITC

The emitted fluorescence is proportional to theamount of bound fluorochromes.

Fluorescence Intensity


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blue

Laser

redLaser

v io le tLaser

Emission Spectra of commonFluorochromes


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The Compensation


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Spectral Overlap

FITC PE PerCP-Cy5.5 PE-Cy7

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC

530/30

RelativeInt

ensity

Wave length (nm)

550nm

PE585/42

PE-Cy7780/60

750nm 800nm


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The Principle of Compensation

Correction of spectral overlap

Depends on:

- Used fluorochromes

- Amp gains of detectors

- But not on: used material

Sample material:

- Unmarked cells as negative control

- Single marked samples to calculate

compensation


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Correct Compensation:

The median of the FITC-positivepopulation is on the same levelthan the median of the unstainedpopulation.

Not compensated Correctly compensated

Missing compensation:

The single FITC-stained populationseems to be double-positive due tothe spectral overlap of FITC into thePE channel.

The Correct Compensation Settings


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Example: Spectral Overlap of FITC

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC

530/30

RelativeIntensity

Wave length (nm)

550nm

PE585/42

750nm 800nm


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Prior Compensation

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC530/30

RelativeIntensity

Wave length (nm)

550nm

PE585/42

Increase values


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After Compensation

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC530/30

RelativeIntensity

Wave length (nm)

550nm

PE585/42

Increase value to move population down


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Spectral overlapof FITC into PE cchannel varies

from

17% (500 V) to71% (600 V)

Influence of the Voltage on Compensation

FITC PMT Voltage constant (575 V)

PE PMT Voltage changes from 500 to 600 Volt


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Compensation with bright Markers

Small mistakes in the compensation using dim expressed markers

(A) could lead to huge mistakes when using highly expressed

markers (B, C) in the compensation.


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Calculation of the Compensation

100*FITC

FITCSpilloverFITC

FITC

PE

FITC

PE

Blank

Blank

w/o compensation (FITC, PE & Blank Calibrite Beads)

%03.24100*4.1391.23506

916275776SpilloverFITC

..

8 Color Panel and the Resulting


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8-Color Panel and the ResultingCompensation Matrix

CD4 FITC

CD4 PE

CD28 PerCP-Cy5.5

CD45RA PE-Cy7

CD3 Pacific Blue

CD4 AmCyan

CD27 APC

CD8 APC-Cy7

FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8

FITC 100.0 23.5 2.1 0.7 0.0 0.0 0.0 3.0

PE 1.6 100.0 12.3 2.7 0.0 0.0 0.0 0.0

PerCP-Cy5.5

0.2 0.1 100.0 43.0 2.5 5.6 0.0 0.0

PE-Cy7

0.0 0.6 0.1 100.0 0.0 3.6 0.0 0.0

APC 0.1 0.0 0.3 0.2 100.0 2.7 0.0 0.0

APC-Cy7

0.0 0.0 0.1 3.9 19.9 100.0 0.0 0.1

PacificBlue

0.1 0.0 0.0 0.1 0.0 0.0 100.0 18.1

AmCyan 38.1 7.0 1.1 0.6 1.5 0.0 17.1 100.0

Single-stained controls:

DET

Automatisc

he

Kompensation

Automatis

che

Kompensa

tion


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Automatic Compensation

Samples

Unmarked cells/particles to set the PMTV

Single stained samples of every used fluorochrome

Tandem conjugates: single stained samples of each

lot# of the individual tandem conjugates


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Start of automatic compensationExperiment

Compensation Setup

Create Compensation Controls


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The software automatically createsa new specimen Compensation

Control consisting of single colortubes of selected fluorochromes.



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Adjust PMTV of unmarked

sampleFSC/SSC: population should bevisible and P1 adjusted onpopulation of interest.

Fluorochrome channels:unmarked population visible and onthe left.

ATTENTION: stained samplesshould be on scale. If not, reducePMTV. Peaks should be narrow(small CV).



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Acquisition of single stained samples in each individual tube.The Auto-Interval-Gate function finds positive peak automatically.



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Calculation of automatic compensationExperiment

Compensation Setup

Calculate Compensation...


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Why so many Colors?

Advantages

More colors improve efficiency in: time consumption

use of reagents

preserving sample material

1 tube with a 6 color labeling can repalce up to 15 tubes with a two color

labeling

Exponental increase of information

Identification of rare/new cells (< 0,05%)

Provides internal controls


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Panel Design Goals

Obtain sufficient measurement sensitivity in alldetectors.

Ensure that the bright positive populations can be

accurate compensated and kept within the linearrange of the scale.

Avoid false positives and other artifacts of tandemdye degradation


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Panel Design Issues

Some antigens are expressed at high levels, some atlow levels, some over a range (continuum).

Some fluorochromes are bright, others are dim.

Emission spillover from bright markers can degrade the

sensitivity of dim markers being measured in anadjacent detector.

Some antigen markers are only available with certain

fluorophores.

Tandem dyes may be unstable.

Complexity of the assay/panel increases the likelihoodof error.


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Panel Design Issues

CD45 FITCCD4dim PE

Question: Are the CD45-positive cells also CD4-positive?


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FMO (Fluorescence Minus One) Control

1 2 3

CD3 FITC

PerCP

ThresholdIsotype control

ThresholdFMO-control

Sample FITC PE PerCP APC Name

1 mIgG1 mIgG1 mIgG1 mIgG1 Isotype Control

2 CD3 CD4 -/ mIgG1 CD19 FMO Control

3 CD3 CD4 CD8 CD19 4-color staining


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Panel Design Principles I

Know your cytometer.

Know what lasers, filters, and how many PMT are present

Know from the CS&T report the detector range and efficiency

Availability of certain fluorophores

Match brighter fluorochromes with lower-expressedantigens (and vice-versa).

Determine the antigens and classify expression

Determine a fluorochrome set and classify brightness

Match antigens with fluorochromes


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Brightness of Fluorochromes


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Panel Design Principles II

Avoid significant spectral overlap between markerson the same population.

Spread markers across as many lasers as possible

Avoid spillover of a marker into another detector used with thesame cell population-especially into a detector reading a dimmer

signal

Prefer red-laser fluorochromes for markers on highly

autofluorescent cells.

Always check for tandem-dye issues.


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CD45 FITC

CD4dim PE

CD45 PerCP

CD4dim

PE

Panel Design Principles


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FSCdetector

30 s

30 s

90o

90o

90o


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Practical Part

Introduction to the instrument

Performance Check of Instrument (CS&T) CS&T Module

7-color Set-up

Setting up an experiment

Application Settings

Settings based on target values

Compensation

Acquisition & analysis of a cell sample

LNW (Lyse No Wash)

LW (Lyse Wash)

Performance Check


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Performance Check7 color Set-up Beads

FACS Canto Software:

Calibration of instrument

Compensation

Creates LNW and LW settings (availabl from FACSDiva, too)

Performance Check


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Performance CheckCS&T Beads

Instrument Characterization (Baseline) Daily Quality Control (Performance Check)

Application Settings catalogue update

Calibration Beads

+

Automatic module

Instrument Set-up


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Instrument Set upTarget Values

Set brightest bead population always to the sameposition by changing the PMT-V

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37 611

Instrument Set-up


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Instrument Set upApplication Settings

Set optimal PMT-V for your cellpopulation (2.5x SD of EN)

Changes in the CS&T PMT-V(performance check) leads to theadaption of the followingparatmeters:

FSC & SSC setting

Threshold

Area Scaling Factor

PMT-V

Standardization of instrumentperformance => save asapplication settings

Application Settings do not

include any compensationsettings

LNW vs. LW Settings


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LNW vs. LW Settings(7 color Set-up Beads)

Vortex and incubate in the dark at RT for 20 min

Vortex and incubate in the dark at RT for 10 min

Acquire samples

450 L FACS Lysing Solution 2000 L

5 or 20 L Reagent50 L blood

5 or 20 L Reagent100 L blood

300xg; 5 min.

300 L PBS

Basics of Flow Cytometry Canto & practical part.pdf

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Transcript of Basics of Flow Cytometry Canto & practical part.pdf