11

Flow Cytometry in the Clinical Flow Cytometry in the Clinical LaboratoryLaboratory

Patricia Aoun, M.D., M. P. H.Patricia Aoun, M.D., M. P. H.Jean Bailey, MT-ASCPJean Bailey, MT-ASCPKellie Neth, MT-ASCPKellie Neth, MT-ASCP

The Nebraska Medical CenterThe Nebraska Medical Center

22

DefinitionDefinition

Flow CytometryFlow Cytometry is the measurement of the is the measurement of the cellular properties of cells/particles as they cellular properties of cells/particles as they

move in a fluid past a stationary set of detectors.move in a fluid past a stationary set of detectors.

33

Basics of Flow CytometryBasics of Flow Cytometry

Fluidics

Optics

Electronics

•Cells in suspension flow in a single -file through an illuminated volume . . .

where they scatter light and emit fluorescence that is collected and filtered . . .

then converted to digital values that are stored on a computer.

44

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometer Schematic

Laser

1

2

3

4

Flow cell

Purdue University Cytometry Laboratories

FluidicsFluidics

55

FluidicsFluidicsFluidicsFluidics

Cells Cells in suspension in a sample fluidin suspension in a sample fluid flow flow

single file through an illuminated flow cellsingle file through an illuminated flow cell

In most instruments, accomplished by injecting In most instruments, accomplished by injecting the sample fluid into a the sample fluid into a sheath fluid sheath fluid as it passes as it passes through a small (50-300 µm) orificethrough a small (50-300 µm) orifice

66

FluidicsFluidics

Sample FluidSample Fluid:: An isotonic fluid in which the cells An isotonic fluid in which the cells are suspended and in which they are injected are suspended and in which they are injected into the sheath fluid streaminto the sheath fluid stream

Sheath FluidSheath Fluid:: A different isotonic fluid that is A different isotonic fluid that is forced under pressure through a conical nozzle forced under pressure through a conical nozzle assembly designed to produce laminar flowassembly designed to produce laminar flow

77

Fluidics - Flow ChamberFluidics - Flow ChamberFluidics - Flow ChamberFluidics - Flow Chamber

The flow cellThe flow celldefines the axis and dimensions of sheath and defines the axis and dimensions of sheath and

sample flowsample flowdefines the point of optimal hydrodynamic focusingdefines the point of optimal hydrodynamic focusing

88

Flow Cell

Injector Tip

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheath fluid

Purdue University Cytometry Laboratories

99

Fluidics Fluidics

Laminar Flow:Laminar Flow:

If the flow is set up If the flow is set up correctly, the sheath fluid correctly, the sheath fluid and the sample fluid will and the sample fluid will not mix, and laminar flow not mix, and laminar flow will result.will result.

Hydrodynamic Focusing:Hydrodynamic Focusing: Focusing effect of Focusing effect of laminar flow which laminar flow which places the sample places the sample stream in the center of stream in the center of the sheath streamthe sheath stream

1010

FluidicsFluidicsFluidicsFluidics

V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.

Notice also how the position of the inner ink stream is influenced by the position of the ink source.

1111

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometry Optics

Laser

1

2

3

4

Flow cell

Purdue University Cytometry Laboratories

OpticsOptics

1212

Optics (Light source)Optics (Light source)Optics (Light source)Optics (Light source)

The light source used must be The light source used must be

focused on the same point where focused on the same point where

the cells have been focused the cells have been focused

(The Interrogation Point)(The Interrogation Point)

1313

Flow Cell

Injector Tip

FluorescenceFluorescencesignalssignals

Interrogation pointInterrogation point

Sheath fluid

Purdue University Cytometry Laboratories

1414

Optics (Light Source)Optics (Light Source)

Two types of light Two types of light sources sources are used are used

LasersLasers

Arc-lampsArc-lamps

1515

Optics - Light SourceOptics - Light SourceOptics - Light SourceOptics - Light Source

LasersLaserscan provide a single wavelength of can provide a single wavelength of coherent lightcoherent light

(a laser line)(a laser line)can provide from milliwatts to watts of lightcan provide from milliwatts to watts of lightcan be inexpensive, air-cooled units or can be inexpensive, air-cooled units or

expensive, water-cooled unitsexpensive, water-cooled units

1616

Optics - Light SourceOptics - Light SourceOptics - Light SourceOptics - Light Source

Arc-lampsArc-lampsprovide provide mixturemixture of wavelengths that must be of wavelengths that must be filteredfiltered

to select desired wavelengthsto select desired wavelengthsprovide provide incoherentincoherent light lightprovide milliwatts of lightprovide milliwatts of light inexpensive, air-cooled unitsinexpensive, air-cooled units

1717

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometer Schematic

Laser

1

2

3

4

Flow cell

Purdue University Cytometry Laboratories

Optical Channels

1818

Optics - Optical ChannelsOptics - Optical ChannelsOptics - Optical ChannelsOptics - Optical Channels

An An optical channeloptical channel is a path that light can is a path that light can follow from the illuminated volume to a follow from the illuminated volume to a detectordetector

Certain types of optical elements (filters, Certain types of optical elements (filters, mirrors) provide separation of channels and mirrors) provide separation of channels and wavelength selectionwavelength selection

1919

Properties Being MeasuredProperties Being Measured

Intrinsic PropertiesIntrinsic PropertiesForward light scatterForward light scatterSide light scatterSide light scatter

Extrinsic PropertiesExtrinsic Properties

2020

Optics - Forward Scatter ChannelOptics - Forward Scatter ChannelOptics - Forward Scatter ChannelOptics - Forward Scatter Channel

The amount of light The amount of light scatteredscattered in the in the forwardforward direction (along the same axis that the laser light direction (along the same axis that the laser light is traveling) is detected in the is traveling) is detected in the forward scatter forward scatter channelchannel

The intensity of forward scatter is proportional to The intensity of forward scatter is proportional to the the size and shapesize and shape of cellsof cells

2121

Forward Angle Light Scatter

FALS Sensor

Laser

Purdue University Cytometry Laboratories

2222

Optics - Side Scatter ChannelOptics - Side Scatter ChannelOptics - Side Scatter ChannelOptics - Side Scatter Channel

The amount of light The amount of light scatteredscattered to the to the sideside (perpendicular to the axis that the laser light is (perpendicular to the axis that the laser light is traveling) is detected in the traveling) is detected in the side scatter or 90side scatter or 90oo scatter channelscatter channel

The intensity of side scatter is proportional to The intensity of side scatter is proportional to the the complexitycomplexity of cells of cells

2323

90 Degree Light Scatter

FALS Sensor

90LS Sensor

Laser

Purdue University Cytometry Laboratories

2424

Properties Being MeasuredProperties Being Measured

Intrinsic propertiesIntrinsic properties

Extrinsic propertiesExtrinsic propertiesCellular components detected by Cellular components detected by

fluorescently-labeled antibodies specific to the fluorescently-labeled antibodies specific to the componentcomponent

(ex. antigen site)(ex. antigen site)

2525

Extrinsic PropertiesExtrinsic Properties

Cell Wall Component Cell Wall Component (antigen site)(antigen site)

MembraneMembraneReceptorReceptor

Nuclear protein

2626

FluorochromesFluorescent dyes used to stain or label

Fluorescein (FITC)

400 nm 600 nm 700 nm

Relat

ive In

tensit

yWavelength

500 nm

ExcitationEmission

Purdue University Cytometry Laboratories

2727

Fluorescent DyesFluorescent Dyes

EmissionDye Wavelength Color

Fluorescein (FITC) 525 GreenPhycoerythrin (PE) 575 OrangeRed-670 (PE/Cy-5) 670 Dark Red

Frequently Used Dyes

2828

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines

Purdue University Cytometry Laboratories

2929

Spectral OverlapSpectral Overlap

Like the colors in a rainbow, the fluorescent Like the colors in a rainbow, the fluorescent antibodies we use overlap/blend together.antibodies we use overlap/blend together.

Need to correct for this spectral overlap in order to Need to correct for this spectral overlap in order to detect distinct cell populationsdetect distinct cell populations

Can be done by several methodsCan be done by several methods

3030

ADC (Advanced Digital Compensation)ADC (Advanced Digital Compensation)

Color Compensation Grid Coulter FC500Color Compensation Grid Coulter FC500

3131

Optics - Fluorescence ChannelsOptics - Fluorescence ChannelsOptics - Fluorescence ChannelsOptics - Fluorescence Channels

The fluorescence emitted by each fluorochrome The fluorescence emitted by each fluorochrome is usually detected in a unique is usually detected in a unique fluorescence fluorescence channelchannel

The specificity of detection is controlled by the The specificity of detection is controlled by the wavelength selectivity of optical filters and wavelength selectivity of optical filters and mirrorsmirrors

3232

Laser

Fluorescence Detectors

Fre

q

Fluorescence

FALS Sensor

Fluorescence detector(PMT3, PMT4 etc.)

Purdue University Cytometry Laboratories

3333

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometry Optics

Laser

1

2

3

4

Flow cell

Purdue University Cytometry Laboratories

Filters

3434

Optics - Filter PropertiesOptics - Filter PropertiesOptics - Filter PropertiesOptics - Filter Properties

Long pass filtersLong pass filters transmit wavelengths above transmit wavelengths above a a cut-oncut-on wavelength wavelength

Short pass filtersShort pass filters transmit wavelengths below transmit wavelengths below a a cut-offcut-off wavelength wavelength

Band pass filtersBand pass filters transmit wavelengths in a transmit wavelengths in a narrow range around a specified wavelengthnarrow range around a specified wavelengthBand width Band width can be specifiedcan be specified

3535

Standard Long Pass FiltersStandard Long Pass FiltersStandard Long Pass FiltersStandard Long Pass Filters

Transmitted LightTransmitted LightLight SourceLight Source520 nm Long Pass Filter520 nm Long Pass Filter

>520 nm Light>520 nm Light

Transmitted LightTransmitted LightLight SourceLight Source575 nm Short Pass Filter575 nm Short Pass Filter

<575 nm Light<575 nm Light

Standard Short Pass FiltersStandard Short Pass FiltersStandard Short Pass FiltersStandard Short Pass Filters

Purdue University Cytometry Laboratories

3636

Standard Band Pass FiltersStandard Band Pass Filters

Transmitted LightWhite Light Source

630 nm Bandpass Filter

620 -640 nm Light

Purdue University Cytometry Laboratories

3737

Dichroic Filter/MirrorDichroic Filter/MirrorDichroic Filter/MirrorDichroic Filter/Mirror

Filter placed at 45o

Reflected light

Transmitted LightLight Source

original from Purdue University Cytometry Laboratories; modified by R.F. Murphy

3838

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometry- Detectors

Laser

1

2

3

4

Flow cell

original from Purdue University Cytometry Laboratories; modified by R.F. Murphy

Photomultiplier Tubes (PMT’s)

3939

DetectorsDetectorsDetectorsDetectors

Photomultiplier tube (PMT)Photomultiplier tube (PMT)The PMT converts the light to a voltage pulse which The PMT converts the light to a voltage pulse which

rises and falls with the amount of light entering.rises and falls with the amount of light entering.

4040

PMTs with Filters on the Coulter FC500 series

Notice the close proximity of the detectors, signal stays stronger

4141

ElectronicsElectronicsElectronicsElectronics

Processing of electrical signals from detectorsProcessing of electrical signals from detectorsPreamplificationPreamplification

Strengthen signals so that they can travel from remote Strengthen signals so that they can travel from remote detectors to central electronicsdetectors to central electronics

AmplificationAmplificationAdjust signal intensityAdjust signal intensity

Conversion of analog electrical signals to digital Conversion of analog electrical signals to digital signalssignals

4242

Data AcquisitionData AcquisitionData AcquisitionData Acquisition

Digital Signals are collected and stored by a Digital Signals are collected and stored by a computer, and can be displayed in a variety of computer, and can be displayed in a variety of formatsformats

4343

Side Scatter

F

A

L

Gating

4444

90 Degree Scatter0 200 400 600 800 1000

For

war

d S

catte

r

0 2

00

40

0 6

00

80

01

00

0

8

15

20

30

40

50

100

200

1000

Lymphocytes

Monocytes

Neutrophils

Side Scatter Projection

Fo

rwa

rd S

ca

tte

r P

roje

ctio

n

Scale

Purdue University Cytometry Laboratories

4545

A Picture is Worth a Thousand Words

4646

Clinical Applications of Clinical Applications of Flow CytometryFlow Cytometry

4747

General PrinciplesGeneral Principles

Cellular antigens:Cellular antigens: Membrane, cytoplasmic or Membrane, cytoplasmic or nuclear componentsnuclear components

Monoclonal antibodies:Monoclonal antibodies: Developed against a Developed against a specific componentspecific component

CD (CD (cluster designationcluster designation) numbers:) numbers: Universal system Universal system of numbers for grouping different monoclonal of numbers for grouping different monoclonal antibodies against the same antigenantibodies against the same antigen

4848

CD NomenclatureCD Nomenclature

The system used for leukocyte surface molecules, The system used for leukocyte surface molecules, as identified by monoclonal antibodies.as identified by monoclonal antibodies.

Examples:Examples:CD45CD45Common Leukocyte AntigenCommon Leukocyte AntigenCD2, CD3, CD5CD2, CD3, CD5 T cellsT cellsCD19CD19and CD20 B cellsand CD20 B cellsCD34 Stem cell markerCD34 Stem cell marker

4949

Indications for Flow CytometryIndications for Flow Cytometry Quantitation of lymphocyte subsets in Quantitation of lymphocyte subsets in

immunodeficiencies, HIV monitoringimmunodeficiencies, HIV monitoring

Determination of cell type and stage of Determination of cell type and stage of differentiation in acute leukemias, including blast differentiation in acute leukemias, including blast crisis of CMLcrisis of CML

Detection of lymphoma cells in blood, bone Detection of lymphoma cells in blood, bone marrow, fluids and tissuesmarrow, fluids and tissues

Detection of CD34+ stem cells in peripheral blood Detection of CD34+ stem cells in peripheral blood and peripheral stem cell collection productsand peripheral stem cell collection products

5050

Flow is not particularly Flow is not particularly helpful in…helpful in…

Chronic phase of CMLChronic phase of CML

Hodgkin’s diseaseHodgkin’s disease

Low grade myelodysplastic syndromesLow grade myelodysplastic syndromes(better when able to use antigen mapping (better when able to use antigen mapping technique)technique)

Screening of peripheral blood in patients who Screening of peripheral blood in patients who do not have a sustained lymphocytosis or do not have a sustained lymphocytosis or abnormal findings on the smearabnormal findings on the smear