Transcript CD4 Immunophenotyping for HIV Monitoring

Welcome
IQAC at DHVI
CD4
Immunophenotyping
for HIV Monitoring
Flow Cytometry
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.
3
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
4
Flow Cytometry

What Can a Flow Cytometer Tell
Us About a Cell?
Its relative size (Forward ScatterFSC)
 Its relative granularity or internal
complexity (Side Scatter-SSC)
 Its relative fluorescence intensity
(FL1,FL2,FL3, and FL4)

5
Light Scatter Properties
6
Blood Cells
7
Flow Light Scatter Pattern
8
Fluorescence
9
Fluorescence Emission
10
Fluorescence Intensity
11
2-parameter Dot Plot
12
Flow Cytometry System

Fluidics


Optics


To introduce and focus cells for analysis.
To generate and collect light signals.
Electronics

To convert optical signals to digital electronic
signals for computer analysis.
13
Fluidics
14
Flow Cell
Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
Purdue University Cytometry Laboratories
15
Sample Flow
16
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.

17
Forward Angle Light Scatter
Laser
FALS Sensor
Purdue University Cytometry Laboratories
18
90 Degree Light Scatter
Laser
FALS Sensor
90LS Sensor
Purdue University Cytometry Laboratories
19
Fluorescence Detectors
Laser
Freq
FALS Sensor
Fluorescence
Fluorescence detector
(PMT3, PMT4 etc.)
Purdue University Cytometry Laboratories
20
Optical Filters
21
Optics Scheme
22
Example Channel Layout for
PMT
Laser-based Flow
Cytometry
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Flow Layout
original from Purdue University Cytometry
Laboratories; modified by R.F. Murphy
23
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
24
Electronics



Converts optical signals to proportional
electronic signals (voltage pulses)
Analyzes voltage pulse height, area, or
width
Interfaces with the computer for data
transfer
25
SIGNAL AND PATTERN GENERATION
LASER
LASER
LASER
PMT 1
PMT 1
VOLTAGE
PMT 1
TIME
26
LASER
PMT 1
Number of events
FLUORESCENCE INTENSITY PATTERN FROM A
CELL POPULATION
low
medium
high
Relative Fluorescence Intensity
27
LASER
PMT 1
Number of events
DETECTION OF THREE FLUORESCENCE
INTENSITY PATTERNS FROM CELL SURFACE
low
medium
high
Relative Fluorescence Intensity
28
LASER
PMT 1
Number of events
FLUORESCENT SIGNAL PATTERN COLLECTION
Relative Fluorescence Intensity
29
SINGLE COLOR
HISTOGRAMS
30
Dot Plot
31
FL2
Uncompensated vs. Compensated
FL1
32
Emissions Spectra
33
Fluorescence Overlap
34
Fluorescence Compensation
35
FITC Compensation
36
Compensation Examples
37
FUNDAMENTAL ASPECT OF COLOR COMPENSATION
HOW TO REMOVE GREEN (G) FROM ORANGE (O)
G
A spectral image
generated by fluorochromes G and O
O
Spectral energy from
fluorochrome G is
subtracted from O
38
THREE PART DIFFERENTIAL ANALYSIS
OF WHOLE BLOOD
Light Scatter
Granulocytes
Lymphocytes
Monocytes
BECTON
DICKINSON
Cell Volume
39
BIVARIATE QUADRANTS
FOR T-CELL SUBSET MARKERS
B
FL1
SSC
A
FSC
+ ++
+
FL2
Mandy et al., 2001
40
++
A
+
A
--
+
B
A
COMPONENTS OF BIVARIATE QUADRANT DISPLAY
DUAL T-CELL MARKERS: A=CD4 and B=CD3
B
B
41
BIVARIATE QUADRANT HISTOGRAM FOR
CD3 AND CD4 POSITIVE CELLS
+
--
++
+
+
42
10
color
10
2
CD3-CD4- black
1
CD3+CD4- blue
10
FL2-CD4
CD4 -->
3
10
4
TWO COLOR PATTERN
CD3-CD4+ cyan
10
1
10
2
CD3 -->
10
3
10
4
CD3+CD4+ green
FL1-CD3
43
2
1
10
CD4 -->
CD4
2
10
1
10
10
10
2
10
3
10
4
10
CD3 -->
1
10
2
10
3
10
4
CD8 -->
CD8
10
3
10
4
CD3
2
10
CD8 -->
CD8
1
1
10
10
CD4 -->
CD4
10
10
3
3
10
10
4
4
THREE COLOR PATTERN
10
1
10
2
10
3
10
4
CD3 -->
CD3
44
10
2
10
3
10
4
10
10
1
CD3 -->
10
2
3
10
10
10
4
1
10
CD3
3
10
4
CD3
4
3
1
10
2
CD4
CD4 -->
2
10
10
2
CD4 -->
CD4
10
10
CD8
10
10
1
1
10
CD8 -->
10
3
3
10
10
10
10
4
CD3
2
CD3 -->
CD3 -->
4
1
2
1
10
1
10
10
10
10
CD4 -->
2
10
CD8 -->
10
10
3
CD4
3
10
CD8
3
10
2
10
1
CD56 -->
CD56
10
4
4
4
FOUR COLOR PATTERN
10
1
10
2
CD56 -->
10
3
CD56
10
4
10
1
10
2
CD56 -->
10
3
CD56
10
4
10
1
10
2
10
3
10
4
CD8 -->
CD8
45
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
46
SS
CD45 BASED HETEROGENEOUS GATING
FOR T-CELL SUBSETS
CD45-Gating
Protocol
HC, NIH & CDC
GUIDELINES
CD8 FL4
Bergeron et al. 2002
47
Topics of Discussion

Testing Platforms
 Single
 Dual


Instrumentation
Reagents
48
Testing Platforms


Single platform instrument
Single platform testing can be performed
on flow cytometer using calibration
beads.


Dual platform testing relies on a
Hematology Analyzer.


Cost per test is relatively higher.
Hematology cost per test is relatively
inexpensive.
Comparison of both platforms.
49
Instrumentation

Flow Cytometers

Beckman Coulter


Becton Dickinson




Personal Cell Analyzer System (PCA)
Partec - CyFlow
Accuri Cytometers
Hematology Analyzers


Canto, FACSCalibur, FACSCan, FACSort,
FACSCount
Guava Technologies Inc.


FC500, MCL-XL, Elite, Profile, Point Care
Coulter, Sysmex, Cell Dyn
Pipetters
50
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
51
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.
52
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)
53
Instrument Maintenance




Daily start-up and shut down
Daily calibration
Monthly and periodic maintenance
Troubleshooting
54
BD FACSCount
Procedures





Instrument Start Up
Preparing and Running Controls
Preparing and Running Samples
Instrument Cleaning and
Maintenance
Troubleshooting
55
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
56
BD FACSCalibur
Procedures






Instrument Start up
FACSComp Calibration
Daily Controls
Patient Samples
Instrument Cleaning and
Maintenance
Troubleshooting
57
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
58
Beckman Coulter Epics /
FC500 Procedures







Instrument Start up
FlowCheck & FlowSet Calibrations
Fluorescence Compensation
Daily Controls
Patient Samples
Instrument Cleaning and
Maintenance
Troubleshooting
59
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.
60
Quality Control and
Quality Assurance






Controls
Reagents
Instruments
Proficiency Testing
Training and Competency
External Quality Assessment

UKNEQAS samples
61
Patient Reporting and
Data Management




Patient Confidentiality
Reference Ranges
Data List Mode Files
Data Storage Devices
62
Acknowledgements

Beckman Coulter


Becton Dickinson


Training Material (PPT slides)
Purdue U. Cytometry Laboratories


Reagents & Training Material
Health Canada, Francis Mandy, PhD


Reagents & Training Material
Training Material (PPT slides)
Roswell Park Cancer Institute Laboratory of
Flow Cytometry, Carleton C. Stewart, Ph.D

Training Material (PPT slides)
63
Conclusion
Thank you for your participation.
64