WelcomeIQAC at DHVI

CD4 Immunophenotyping for HIV Monitoring

Flow 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 conjugated monoclonal 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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Injector Tip

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheath fluid

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 to

route 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

Freq

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 for Laser-based Flow Cytometry

Laser

1

2

3

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Flow cell

original from Purdue University Cytometry Laboratories; 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 by collection 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

VO

LT

AG

E

TIME

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PMT 1

LASER

FLUORESCENCE INTENSITY PATTERN FROM A CELL POPULATION

Nu

mb

er o

f ev

ents

Relative Fluorescence Intensity

low medium high

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DETECTION OF THREE FLUORESCENCE INTENSITY PATTERNS FROM CELL SURFACE

PMT 1

LASER

Relative Fluorescence Intensity

Nu

mb

er o

f ev

ents

low medium high

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Nu

mb

er o

f ev

ents

PMT 1

LASER

Relative Fluorescence Intensity

FLUORESCENT SIGNAL PATTERN COLLECTION

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SINGLE COLOR HISTOGRAMS

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Dot Plot

32FL1

FL2

Uncompensated vs. Compensated

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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 image generated by fluoro-chromes G and O

Spectral energy fromfluorochrome G is subtracted from O

FUNDAMENTAL ASPECT OF COLOR COMPENSATIONHOW TO REMOVE GREEN (G) FROM ORANGE (O)

G O

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THREE PART DIFFERENTIAL ANALYSIS OF WHOLE BLOOD

Lig

ht

Sca

tter

Cell Volume

Lymphocytes

Granulocytes

Monocytes

BECTONDICKINSON

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BIVARIATE QUADRANTSFOR T-CELL SUBSET MARKERS

FSC

SS

C

A B

FL2

FL

1 + +++

Mandy et al., 2001

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COMPONENTS OF BIVARIATE QUADRANT DISPLAYDUAL T-CELL MARKERS: A=CD4 and B=CD3

A

BA

B

A

B++

+

+

--

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BIVARIATE QUADRANT HISTOGRAM FOR CD3 AND CD4 POSITIVE CELLS

++

+

+

-- +

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TWO COLOR PATTERN

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 -

->

FL1-CD3

FL2-C

D4 color

CD3-CD4- black

CD3+CD4- blue

CD3-CD4+ cyan

CD3+CD4+green

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THREE COLOR PATTERN

CD3

CD

4

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 --

>

CD3

CD

4

CD8C

D8

10 1 10 2 10 3 10 4

CD8 -->

101

102

103

104

CD

4 --

>

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

8 --

>

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10 1 10 2 10 3 10 4

CD56 -->

101

102

103

104

CD

4 --

>

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 --

>

CD3CD3 CD310 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

56 --

>

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

8 --

>

CD5610 1 10 2 10 3 10 4

CD56 -->

101

102

103

104

CD

8 --

>

CD56 CD810 1 10 2 10 3 10 4

CD8 -->

101

102

103

104

CD

4 --

>C

D4

CD

8

CD

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CD

8

CD

4

CD

4

FOUR COLOR PATTERN

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FOUR COLOR DUAL LASER IMMUNOPHENOTYPING

Antibodies labeled with fluorescein

Antibodies labeled with phycoerythrin (PE)

Antibodies labeled with PE/CY5 or PerCP

Antibodies labeled with APC, CY5 or CY7

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CD45 BASED HETEROGENEOUS GATING FOR T-CELL SUBSETS

CD8 FL4C

D4

FL

2

SS

CD45 FL1

CD45-Gating Protocol

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 relatively

inexpensive. 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, and

Cell 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 Antibody Panels 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 FACSCount Procedures Instrument Start Up Preparing and Running Controls Preparing and Running Samples Instrument Cleaning and

Maintenance Troubleshooting

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BD FACSCount Sample Prep 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

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BD FACSCalibur Procedures Instrument Start up FACSComp Calibration Daily Controls Patient Samples Instrument Cleaning and

Maintenance Troubleshooting

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BD MultiTest Reagent Staining Procedure (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

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Beckman Coulter Epics / FC500 Procedures Instrument Start up FlowCheck & FlowSet Calibrations Fluorescence Compensation Daily Controls Patient Samples Instrument Cleaning and

Maintenance Troubleshooting

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Beckman Coulter Cyto-Stat Reagent 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 processing

run. Acquire samples on flow cytometer.

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Quality Control and Quality Assurance

Controls Reagents Instruments Proficiency Testing Training and Competency External Quality Assessment

UKNEQAS samples

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Patient Reporting and Data 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.