IQAC at DHVI

1
Welcome

• IQAC at DHVI

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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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Flow Cell
Injector Tip
Sheath fluid
Fluorescence
signals
Focused laser
beam
Purdue University Cytometry Laboratories
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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
Purdue University Cytometry Laboratories
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90 Degree Light Scatter
Purdue University Cytometry Laboratories
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Fluorescence Detectors
Laser
Purdue University Cytometry Laboratories
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Optical Filters
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Optics Scheme
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Flow Layout
Example Channel Layout for Laser-based Flow
Cytometry
PMT
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PMT
Dichroic
3
Filters
Flow cell
PMT
2
Bandpass
Filters
PMT
1
Laser
original from Purdue University Cytometry
Laboratories modified by R.F. Murphy
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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
VOLTAGE
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FLUORESCENCE INTENSITY PATTERN FROM A CELL
POPULATION
LASER
Number of events
PMT 1
Relative Fluorescence Intensity
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DETECTION OF THREE FLUORESCENCE INTENSITY
PATTERNS FROM CELL SURFACE
LASER
Number of events
PMT 1
Relative Fluorescence Intensity
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FLUORESCENT SIGNAL PATTERN COLLECTION
LASER
Number of events
PMT 1
Relative Fluorescence Intensity
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SINGLE COLOR HISTOGRAMS
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Dot Plot
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Uncompensated vs. Compensated
FL2
FL1
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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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FUNDAMENTAL ASPECT OF COLOR COMPENSATION HOW TO
REMOVE GREEN (G) FROM ORANGE (O)
A spectral image generated by fluoro-chromes G
and O
Spectral energy from fluorochrome G is
subtracted from O
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THREE PART DIFFERENTIAL ANALYSIS OF WHOLE BLOOD
BECTON DICKINSON
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BIVARIATE QUADRANTS FOR T-CELL SUBSET MARKERS
Mandy et al., 2001
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COMPONENTS OF BIVARIATE QUADRANT DISPLAY DUAL
T-CELL MARKERS ACD4 and BCD3
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BIVARIATE QUADRANT HISTOGRAM FOR CD3 AND CD4
POSITIVE CELLS
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TWO COLOR PATTERN
color CD3-CD4- black CD3CD4- blue CD3-CD4 cyan
CD3CD4 green
FL2-CD4
FL1-CD3
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THREE COLOR PATTERN
CD4
CD4
CD3
CD8
CD8
CD3
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FOUR COLOR PATTERN
CD4
CD8
CD56
CD3
CD3
CD3
CD8
CD4
CD4
CD56
CD56
CD8
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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
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, CD3/1656)
• 3-color panels (CD3/4/8 CD3/4/45, CD3/8/45,
  CD3/19/45, CD3/1656/45)
• 4-color panels (CD3/4/8/45, CD3/19/1656/45)

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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.