Title: IQAC at DHVI
1Welcome
2CD4 Immunophenotyping for HIV Monitoring
3Introduction
- 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.
4Flow Cytometry
- Laser based high speed electronic cell analyzer
- Fluorescent conjugated monoclonal antibodies
- Analyze surface (and cytoplasmic) cellular
antigens. - Flow rates 500 700 cells /second
5Flow 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)
6Light Scatter Properties
7Blood Cells
8Flow Light Scatter Pattern
9Fluorescence
10Fluorescence Emission
11Fluorescence Intensity
122-parameter Dot Plot
13Flow 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.
14Fluidics
15Flow Cell
Injector Tip
Sheath fluid
Fluorescence
signals
Focused laser
beam
Purdue University Cytometry Laboratories
16Sample Flow
17Optics
- 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.
18Forward Angle Light Scatter
Purdue University Cytometry Laboratories
1990 Degree Light Scatter
Purdue University Cytometry Laboratories
20Fluorescence Detectors
Laser
Purdue University Cytometry Laboratories
21Optical Filters
22Optics Scheme
23Flow Layout
Example Channel Layout for Laser-based Flow
Cytometry
PMT
4
PMT
Dichroic
3
Filters
Flow cell
PMT
2
Bandpass
Filters
PMT
1
Laser
original from Purdue University Cytometry
Laboratories modified by R.F. Murphy
24Fluidics 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
25Electronics
- Converts optical signals to proportional
electronic signals (voltage pulses) - Analyzes voltage pulse height, area, or width
- Interfaces with the computer for data transfer
26SIGNAL AND PATTERN GENERATION
VOLTAGE
27FLUORESCENCE INTENSITY PATTERN FROM A CELL
POPULATION
LASER
Number of events
PMT 1
Relative Fluorescence Intensity
28DETECTION OF THREE FLUORESCENCE INTENSITY
PATTERNS FROM CELL SURFACE
LASER
Number of events
PMT 1
Relative Fluorescence Intensity
29FLUORESCENT SIGNAL PATTERN COLLECTION
LASER
Number of events
PMT 1
Relative Fluorescence Intensity
30SINGLE COLOR HISTOGRAMS
31Dot Plot
32Uncompensated vs. Compensated
FL2
FL1
33Emissions Spectra
34Fluorescence Overlap
35Fluorescence Compensation
36FITC Compensation
37Compensation Examples
38FUNDAMENTAL 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
39THREE PART DIFFERENTIAL ANALYSIS OF WHOLE BLOOD
BECTON DICKINSON
40BIVARIATE QUADRANTS FOR T-CELL SUBSET MARKERS
Mandy et al., 2001
41COMPONENTS OF BIVARIATE QUADRANT DISPLAY DUAL
T-CELL MARKERS ACD4 and BCD3
42BIVARIATE QUADRANT HISTOGRAM FOR CD3 AND CD4
POSITIVE CELLS
43TWO COLOR PATTERN
color CD3-CD4- black CD3CD4- blue CD3-CD4 cyan
CD3CD4 green
FL2-CD4
FL1-CD3
44THREE COLOR PATTERN
CD4
CD4
CD3
CD8
CD8
CD3
45FOUR COLOR PATTERN
CD4
CD8
CD56
CD3
CD3
CD3
CD8
CD4
CD4
CD56
CD56
CD8
46FOUR 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
47 CD45 BASED HETEROGENEOUS GATING FOR T-CELL
SUBSETS
CD45-Gating Protocol
HC, NIH CDC GUIDELINES
Bergeron et al. 2002
48Topics of Discussion
- Testing Platforms
- Single
- Dual
- Instrumentation
- Reagents
49Testing 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.
50Instrumentation
- 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
51Flow 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
52Reagents
- There are many manufacturers of monoclonal
antibodies. Select IVD reagents to ensure
quality and reliability. - Cytometer manufacturers are a good source for
flow reagents.
53Multi-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)
54Instrument Maintenance
- Daily start-up and shut down
- Daily calibration
- Monthly and periodic maintenance
- Troubleshooting
55BD FACSCount Procedures
- Instrument Start Up
- Preparing and Running Controls
- Preparing and Running Samples
- Instrument Cleaning and Maintenance
- Troubleshooting
56BD 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
57BD FACSCalibur Procedures
- Instrument Start up
- FACSComp Calibration
- Daily Controls
- Patient Samples
- Instrument Cleaning and Maintenance
- Troubleshooting
58BD 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
59Beckman Coulter Epics / FC500 Procedures
- Instrument Start up
- FlowCheck FlowSet Calibrations
- Fluorescence Compensation
- Daily Controls
- Patient Samples
- Instrument Cleaning and Maintenance
- Troubleshooting
60Beckman 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.
61Quality Control and Quality Assurance
- Controls
- Reagents
- Instruments
- Proficiency Testing
- Training and Competency
- External Quality Assessment
- UKNEQAS samples
62Patient Reporting and Data Management
- Patient Confidentiality
- Reference Ranges
- Data List Mode Files
- Data Storage Devices
63Acknowledgements
- 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)
64Conclusion
- Thank you for your participation.