wong cd4 immunophenotyping

7/31/2019 Wong CD4 Immunophenotyping

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Welcome

IQAC at DHVI



CD4Immunophenotyping

for HIV MonitoringFlow Cytometry



3

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.



4

Flow Cytometry

Laser based high speed electronic

cell analyzer

Fluorescent conjugatedmonoclonal antibodies

Analyze surface (and cytoplasmic)

cellular antigens.

Flow rates 500 –

700 cells /second



5

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)



6

Light Scatter Properties



7

Blood Cells



8

Flow Light Scatter Pattern



9

Fluorescence



10

Fluorescence Emission



11

Fluorescence Intensity



12

2-parameter Dot Plot



13

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.



14

Fluidics



15

InjectorTip

Fluorescencesignals

Focused laserbeam

Sheathfluid

Purdue University Cytometry Laboratories

Flow Cell



16

Sample Flow



17

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.



18

Forward Angle Light Scatter

FALS Sensor

Laser




19

90 Degree Light Scatter

FALS Sensor

90LS Sensor

Laser




20

Laser

Fluorescence Detectors

F r e q

Fluorescence

FALS Sensor

Fluorescence detector(PMT3, PMT4 etc.)




21

Optical Filters



22

Optics Scheme



23

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



24

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



25

Electronics

Converts optical signals to proportional

electronic signals (voltage pulses)

Analyzes voltage pulse height, area, or

width

Interfaces with the computer for data

transfer



26

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



27

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



28

DETECTION OF THREE FLUORESCENCEINTENSITY PATTERNS FROM CELL SURFACE

PMT 1

LASER


N

u m b e r o f e v e n

t s

low medium high



29

N u m b e r o f e v e n t

s

PMT 1

LASER


FLUORESCENT SIGNAL PATTERN COLLECTION



30

SINGLE COLOR

HISTOGRAMS



31

Dot Plot



32

FL1

F

L 2

Uncompensated vs. Compensated

S



33

Emissions Spectra



34

Fluorescence Overlap



35

Fluorescence Compensation



36

FITC Compensation



37

Compensation Examples



38

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



39

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



40

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



41

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

A

B

A

B

A

B

++

+

+

--

BIVARIATE QUADRANT HISTOGRAM FOR



42

BIVARIATE QUADRANT HISTOGRAM FORCD3 AND CD4 POSITIVE CELLS

++

+

+

-- +



43

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



44

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



45

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



46

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



47

CD45 BASED HETEROGENEOUS GATINGFOR T-CELL SUBSETS

CD8 FL4

S S

CD45-GatingProtocol

HC, NIH & CDC

GUIDELINES

Bergeron et al. 2002



48

Topics of Discussion

Testing Platforms

Single

Dual Instrumentation

Reagents



49

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.



50

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



51

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



52

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.



53

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)



54

Instrument Maintenance

Daily start-up and shut down

Daily calibration

Monthly and periodic maintenance

Troubleshooting



55

BD FACSCountProcedures

Instrument Start Up

Preparing and Running Controls

Preparing and Running Samples Instrument Cleaning and

Maintenance

Troubleshooting

BD FACSCount Sample



56

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



57

BD FACSCalibur Procedures

Instrument Start up

FACSComp Calibration

Daily Controls Patient Samples

Instrument Cleaning and

Maintenance

Troubleshooting

BD MultiTest Reagent Staining



58

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 /



59

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



60

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



61

Quality Control andQuality Assurance

Controls

Reagents

Instruments

Proficiency Testing Training and Competency

External Quality Assessment

UKNEQAS samples

Patient Reporting and



62

Patient Reporting andData Management

Patient Confidentiality

Reference Ranges

Data List Mode Files Data Storage Devices



63

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)



Conclusion

Thank you for your participation.

wong cd4 immunophenotyping

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