wong cd4 immunophenotyping
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7/31/2019 Wong CD4 Immunophenotyping
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Welcome
IQAC at DHVI
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CD4Immunophenotyping
for HIV MonitoringFlow Cytometry
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Introduction
Absolute CD4 T-lymphocyte
counts are used to evaluate the
immune status of patients with the
human immunodeficiency virus(HIV).
CD4 antigen is the receptor for
HIV. The absolute number of CD4
T lymphocytes is closely
associated with HIV progression.
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Flow Cytometry
Laser based high speed electronic
cell analyzer
Fluorescent conjugatedmonoclonal antibodies
Analyze surface (and cytoplasmic)
cellular antigens.
Flow rates 500 –
700 cells /second
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Flow Cytometry
What Can a Flow Cytometer Tell
Us About a Cell?
Its relative size (Forward Scatter-
FSC)
Its relative granularity or internal
complexity (Side Scatter-SSC)
Its relative fluorescence intensity(FL1,FL2,FL3, and FL4)
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Light Scatter Properties
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Blood Cells
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Flow Light Scatter Pattern
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Fluorescence
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Fluorescence Emission
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Fluorescence Intensity
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2-parameter Dot Plot
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Flow Cytometry System
Fluidics
To introduce and focus cells for analysis.
Optics
To generate and collect light signals.
Electronics
To convert optical signals to digital electronic
signals for computer analysis.
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Fluidics
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InjectorTip
Fluorescencesignals
Focused laserbeam
Sheathfluid
Purdue University Cytometry Laboratories
Flow Cell
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Sample Flow
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Optics
Excitation optics
Laser(s)
Lenses to shape and focus the laser beam
Collection optics
A collection lens to collect light emitted from
the particle-laser beam interaction
A system of optical mirrors and filters toroute specified wavelengths of the collected
light to designated optical detectors.
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Forward Angle Light Scatter
FALS Sensor
Laser
Purdue University Cytometry Laboratories
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90 Degree Light Scatter
FALS Sensor
90LS Sensor
Laser
Purdue University Cytometry Laboratories
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Laser
Fluorescence Detectors
F r e q
Fluorescence
FALS Sensor
Fluorescence detector(PMT3, PMT4 etc.)
Purdue University Cytometry Laboratories
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Optical Filters
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Optics Scheme
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PMT
PMT
PMT
PMT
DichroicFilters
BandpassFilters
Example Channel Layout forLaser-based Flow
Cytometry
Laser
1
2
3
4
Flow cell
original from Purdue University CytometryLaboratories; modified by R.F. Murphy
Flow Layout
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Fluidics and Optics Review
Created an illumination region with
the excitation optics
Passed the cells precisely through
the illumination region using
hydrodynamic focusing
Routed the generated light signals
to the specific detectors bycollection optics
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Electronics
Converts optical signals to proportional
electronic signals (voltage pulses)
Analyzes voltage pulse height, area, or
width
Interfaces with the computer for data
transfer
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SIGNAL AND PATTERN GENERATION
PMT 1
LASER
PMT 1
LASER
PMT 1
LASER
V O L T
A G E
TIME
FLUORESCENCE INTENSITY PATTERN FROM A
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PMT 1
LASER
FLUORESCENCE INTENSITY PATTERN FROM ACELL POPULATION
N
u m b e r o f e v e n t s
Relative Fluorescence Intensity
low medium high
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DETECTION OF THREE FLUORESCENCEINTENSITY PATTERNS FROM CELL SURFACE
PMT 1
LASER
Relative Fluorescence Intensity
N
u m b e r o f e v e n
t s
low medium high
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N u m b e r o f e v e n t
s
PMT 1
LASER
Relative Fluorescence Intensity
FLUORESCENT SIGNAL PATTERN COLLECTION
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SINGLE COLOR
HISTOGRAMS
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Dot Plot
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FL1
F
L 2
Uncompensated vs. Compensated
S
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Emissions Spectra
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Fluorescence Overlap
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Fluorescence Compensation
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FITC Compensation
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Compensation Examples
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A spectral imagegenerated by fluoro-chromes G and O
Spectral energy fromfluorochrome G issubtracted from O
FUNDAMENTAL ASPECT OF COLOR COMPENSATIONHOW TO REMOVE GREEN (G) FROM ORANGE (O)
G O
THREE PART DIFFERENTIAL ANALYSIS
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THREE PART DIFFERENTIAL ANALYSISOF WHOLE BLOOD
L i g h t S c a t t e r
Cell Volume
Lymphocytes
Granulocytes
Monocytes
BECTON
DICKINSON
BIVARIATE QUADRANTS
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BIVARIATE QUADRANTSFOR T-CELL SUBSET MARKERS
FSC
S S C
A B
FL2
F L 1
+ ++
+Mandy et al., 2001
COMPONENTS OF BIVARIATE QUADRANT DISPLAY
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COMPONENTS OF BIVARIATE QUADRANT DISPLAYDUAL T-CELL MARKERS: A=CD4 and B=CD3
A
B
A
B
A
B
++
+
+
--
BIVARIATE QUADRANT HISTOGRAM FOR
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BIVARIATE QUADRANT HISTOGRAM FORCD3 AND CD4 POSITIVE CELLS
++
+
+
-- +
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TWO COLOR PATTERN
10 1 10 2 10 3 10 4
CD3 -->
1 0
1
1 0
2
1 0
3
1 0
4
C
D 4 - - >
FL1-CD3
F L 2 -
C D 4 color
CD3-CD4- black
CD3+CD4- blue
CD3-CD4+ cyan
CD3+CD4+ green
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THREE COLOR PATTERN
CD3
C D 4
10 1 10 2 10 3 10 4
CD3 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 4 - - >
CD3
C D 4
CD8
C D 8
10 1 10 2 10 3 10 4
CD8 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 4 - - >
10 1 10 2 10 3 10 4
CD3 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 8 - - >
FOUR COLOR PATTERN
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10 1 10 2 10 3 10 4
CD56 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 4 - - >
10 1 10 2 10 3 10 4
CD3 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 4 - - >
CD3CD3 CD3
10 1 10 2 10 3 10 4
CD3 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 5 6 - - >
10 1 10 2 10 3 10 4
CD3 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 8 - - >
CD56
10 1 10 2 10 3 10 4
CD56 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 8
- - >
CD56 CD8
10 1 10 2 10 3 10 4
CD8 -->
1 0
1
1 0
2
1 0
3
1 0
4
C D 4
- - >
C D 4
C D 8
C D 5
6
C D 8
C D 4
C D 4
FOUR COLOR PATTERN
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FOUR COLOR DUAL LASERIMMUNOPHENOTYPING
Antibodies labeled with fluorescein
Antibodies labeled with phycoerythrin (PE)
Antibodies labeled with PE/CY5 or PerCP
Antibodies labeled with APC, CY5 or CY7
CD45 BASED HETEROGENEOUS GATING
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CD45 BASED HETEROGENEOUS GATINGFOR T-CELL SUBSETS
CD8 FL4
S S
CD45-GatingProtocol
HC, NIH & CDC
GUIDELINES
Bergeron et al. 2002
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Topics of Discussion
Testing Platforms
Single
Dual Instrumentation
Reagents
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Testing Platforms
Single platform instrument
Single platform testing can be performed
on flow cytometer using calibration
beads. Cost per test is relatively higher.
Dual platform testing relies on a
Hematology Analyzer.
Hematology cost per test is relativelyinexpensive.
Comparison of both platforms.
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Instrumentation Flow Cytometers
Beckman Coulter
FC500, MCL-XL, Elite, Profile, Point Care
Becton Dickinson
Canto, FACSCalibur, FACSCan, FACSort,
FACSCount Guava Technologies Inc.
Personal Cell Analyzer System (PCA)
Partec - CyFlow
Accuri Cytometers Hematology Analyzers
Coulter, Sysmex, Cell Dyn
Pipetters
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Flow Cytometry Software Beckman Coulter EPICS System II and
Expo 32
Becton Dickinson Simultest, Multitest, andCell Quest (Pro) for BD FACS
Becton Dickinson FACSCount Guava PCA System
Partec FloMax
Accuri CFlow Plus
TreeStar, Inc. FlowJo
Verity Software House - WinList
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Reagents
There are many manufacturers of
monoclonal antibodies. Select IVD
reagents to ensure quality and
reliability. Cytometer manufacturers are a
good source for flow reagents.
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Multi-color AntibodyPanels
2-color panels (Leucogate
CD45/CD14, CD3/4, CD3/8,
CD3/19, CD3/16+56)
3-color panels (CD3/4/8; CD3/4/45,
CD3/8/45, CD3/19/45,
CD3/16+56/45)
4-color panels (CD3/4/8/45,CD3/19/16+56/45)
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Instrument Maintenance
Daily start-up and shut down
Daily calibration
Monthly and periodic maintenance
Troubleshooting
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BD FACSCountProcedures
Instrument Start Up
Preparing and Running Controls
Preparing and Running Samples Instrument Cleaning and
Maintenance
Troubleshooting
BD FACSCount Sample
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BD FACSCount SamplePrep Procedure Material
Whole blood sample collected in anticoagulant.
BD FACSCount Reagents
BD FACSCount Control Beads
BD FACSCount Fixative BD Reverse Pipetter
Procedure
Aliquot 50ul whole blood sample to FACSCount
Reagent tubes
Incubate for 1 hour at RT
Add 50 ul of fixative
Run sample on instrument
BD FACSC lib
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BD FACSCalibur Procedures
Instrument Start up
FACSComp Calibration
Daily Controls Patient Samples
Instrument Cleaning and
Maintenance
Troubleshooting
BD MultiTest Reagent Staining
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BD MultiTest Reagent StainingProcedure(Lyse No Wash) Materials
Whole blood collected in anticoagulant.
MultiTest Reagent Panel
BD FACSLyse Solution
BD Falcon 12 x 75mm test tubes Procedure
Add 50ul of blood sample to 10ul of antibody reagent.
Incubate for 15 minutes at RT
Add 450ul of a working dilution of BD FACSLyse solution. Incubate for 15 minutes at RT
Acquire samples on flow cytometer
B k C lt E i /
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Beckman Coulter Epics /FC500 Procedures
Instrument Start up
FlowCheck & FlowSet Calibrations
Fluorescence Compensation Daily Controls
Patient Samples
Instrument Cleaning andMaintenance
Troubleshooting
Beckman Coulter Cyto-Stat
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Beckman Coulter Cyto StatReagent Staining Procedure(Lyse No Wash)
Materials
Whole Blood collected in anticoagulant.
Coulter Cyto-Stat Reagent Panel
Coulter T-Q Prep
12 x 75mm test tubes
Procedure
Add 100ul of blood sample to 10ul of antibody reagent.
Incubate for 15 minutes at RT
Place tubes in T-Q Prep and start sample processingrun.
Acquire samples on flow cytometer.
Quality Control and
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Quality Control andQuality Assurance
Controls
Reagents
Instruments
Proficiency Testing Training and Competency
External Quality Assessment
UKNEQAS samples
Patient Reporting and
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Patient Reporting andData Management
Patient Confidentiality
Reference Ranges
Data List Mode Files Data Storage Devices
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Acknowledgements
Beckman Coulter Reagents & Training Material
Becton Dickinson
Reagents & Training Material
Health Canada, Francis Mandy, PhD Training Material (PPT slides)
Purdue U. Cytometry Laboratories
Training Material (PPT slides)
Roswell Park Cancer Institute Laboratory of Flow Cytometry, Carleton C. Stewart, Ph.D
Training Material (PPT slides)
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Conclusion
Thank you for your participation.
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