Transcript PMT - University of Nebraska Medical Center

Flow Cytometry in the Clinical
Laboratory
Patricia Aoun, M.D., M. P. H.
Jean Bailey, MT-ASCP
Kellie Neth, MT-ASCP
The Nebraska Medical Center
1
Definition

Flow Cytometry is the measurement of the
cellular properties of cells/particles as they
move in a fluid past a stationary set of detectors.
2
Basics of Flow Cytometry
Fluidics
•Cells in suspension flow in a
single -file through an illuminated
volume . . .
Optics
where they scatter light and emit
fluorescence that is collected and
filtered . . .
Electronics
then converted to digital values that
are stored on a computer.
3
Flow Cytometer Schematic
PMT
4
PMT
Dichroic
Filters
Flow cell
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Fluidics
Purdue University Cytometry Laboratories
4
Fluidics

Cells in suspension in a sample fluid flow
single file through an illuminated flow cell

In most instruments, accomplished by injecting
the sample fluid into a sheath fluid as it passes
through a small (50-300 µm) orifice
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Fluidics
Sample Fluid: An isotonic fluid in which the
cells are suspended and in which they are
injected into the sheath fluid stream
Sheath Fluid: A different isotonic fluid that is
forced under pressure through a conical
nozzle assembly designed to produce
laminar flow
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Fluidics - Flow Chamber

The flow cell
defines the axis and dimensions of sheath and
sample flow
 defines the point of optimal hydrodynamic focusing

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Flow Cell
Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
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Purdue University Cytometry Laboratories
Fluidics
Laminar Flow:
If the flow is set up
correctly, the sheath fluid
and the sample fluid will
not mix, and laminar flow
will result.
Hydrodynamic Focusing:
Focusing effect of
laminar flow which
places the sample
stream in the center of
the sheath stream
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Fluidics
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.
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V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Flow Cytometry Optics
PMT
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Optics
Purdue University Cytometry Laboratories
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Optics (Light source)

The light source used must be
focused on the same point where
the cells have been focused
(The Interrogation Point)
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Flow Cell
Injector
Tip
Sheath
fluid
Fluorescence
signals
Interrogation point
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Purdue University Cytometry Laboratories
Optics (Light Source)
Two
types of light sources
are used
Lasers
Arc-lamps
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Optics - Light Source

Lasers
can provide a single wavelength of coherent light
(a laser line)
 can provide from milliwatts to watts of light
 can be inexpensive, air-cooled units or
expensive, water-cooled units

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Optics - Light Source

Arc-lamps
provide mixture of wavelengths that must be filtered
to select desired wavelengths
 provide incoherent light
 provide milliwatts of light
 inexpensive, air-cooled units

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Flow Cytometer Schematic
PMT
4
Optical Channels
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Purdue University Cytometry Laboratories
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Optics - Optical Channels
An optical channel is a path that light can
follow from the illuminated volume to a detector
 Certain types of optical elements (filters, mirrors)
provide separation of channels and wavelength
selection

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Properties Being Measured

Intrinsic Properties
 Forward light scatter
 Side light scatter

Extrinsic Properties
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Optics - Forward Scatter Channel
The amount of light scattered in the forward
direction (along the same axis that the laser light
is traveling) is detected in the forward scatter
channel
 The intensity of forward scatter is proportional to
the size and shape of cells

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Forward Angle Light Scatter
Laser
FALS Sensor
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Purdue University Cytometry Laboratories
Optics - Side Scatter Channel
The amount of light scattered to the side
(perpendicular to the axis that the laser light is
traveling) is detected in the side scatter or 90o
scatter channel
 The intensity of side scatter is proportional to the
complexity of cells

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90 Degree Light Scatter
Laser
FALS Sensor
90LS Sensor
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Purdue University Cytometry Laboratories
Properties Being Measured

Intrinsic properties

Extrinsic properties
 Cellular components detected by
fluorescently-labeled antibodies specific to the
component
 (ex. antigen site)
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Extrinsic Properties
Cell Wall Component
(antigen site)
Membrane
Receptor
Nuclear protein
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Fluorochromes
Fluorescent dyes used to stain or label
Wavelength
400 nm
500 nm
600 nm
700 nm
RelativeIntensity
Excitation
Emission
Fluorescein (FITC)
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Purdue University Cytometry Laboratories
Fluorescent Dyes
Frequently Used Dyes
Dye
Fluorescein (FITC)
Phycoerythrin (PE)
Red-670 (PE/Cy-5)
Emission
Wavelength Color
525
575
670
Green
Orange
Dark Red
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350
300 nm
457 488 514
400 nm
500 nm
Common Laser Lines
610 632
600 nm
700 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
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Purdue University Cytometry Laboratories
Spectral Overlap
Like the colors in a rainbow, the fluorescent
antibodies we use overlap/blend together.
Need to correct for this spectral overlap in order to
detect distinct cell populations
Can be done by several methods
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ADC (Advanced Digital Compensation)
Color Compensation Grid Coulter FC500
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Optics - Fluorescence Channels
The fluorescence emitted by each fluorochrome
is usually detected in a unique fluorescence
channel
 The specificity of detection is controlled by the
wavelength selectivity of optical filters and
mirrors

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Fluorescence Detectors
Laser
Freq
FALS Sensor
Fluorescence
Fluorescence detector
(PMT3, PMT4 etc.)
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Purdue University Cytometry Laboratories
Flow Cytometry Optics
PMT
4
Filters
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
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Purdue University Cytometry Laboratories
Optics - Filter Properties

Long pass filters transmit wavelengths above a
cut-on wavelength

Short pass filters transmit wavelengths below
a cut-off wavelength

Band pass filters transmit wavelengths in a
narrow range around a specified wavelength

Band width can be specified
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Standard Long Pass Filters
Light Source
520 nm Long Pass Filter
Transmitted Light
>520 nm Light
Standard Short Pass Filters
Light Source
575 nm Short Pass Filter
Transmitted Light
<575 nm Light
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Purdue University Cytometry
Laboratories
Standard Band Pass Filters
630 nm Bandpass Filter
White Light Source
Transmitted Light
620 -640 nm Light
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Purdue University Cytometry Laboratories
Dichroic Filter/Mirror
Filter placed at 45o
Light Source
Transmitted Light
Reflected light
original from Purdue
University Cytometry
Laboratories; modified by37
R.F. Murphy
Flow Cytometry- Detectors
PMT
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Photomultiplier Tubes (PMT’s)
38
original from Purdue University Cytometry Laboratories;
modified by R.F. Murphy
Detectors

Photomultiplier tube (PMT)

The PMT converts the light to a voltage pulse which
rises and falls with the amount of light entering.
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PMTs with Filters on the Coulter FC500 series
Notice the close proximity of the detectors, signal stays stronger
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Electronics

Processing of electrical signals from detectors

Preamplification
 Strengthen
signals so that they can travel from remote
detectors to central electronics

Amplification
 Adjust

signal intensity
Conversion of analog electrical signals to digital
signals
41
Data Acquisition

Digital Signals are collected and stored by a
computer, and can be displayed in a variety of
formats
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Gating
F
A
L
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Side Scatter
1000
Side Scatter Projection
Forward Scatter Projection
Neutrophils
800
1000
100
50
600
200
40
20
Monocytes
400
30
15
Lymphocytes
0
8
200
Forward Scatter
Scale
0
200
400
600
800
1000
90 Degree Scatter
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Purdue University Cytometry Laboratories
A Picture is Worth a Thousand Words
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Clinical Applications of
Flow Cytometry
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General Principles
Cellular antigens: Membrane, cytoplasmic or
nuclear components
 Monoclonal antibodies: Developed against a
specific component
 CD (cluster designation) numbers: Universal system
of numbers for grouping different monoclonal
antibodies against the same antigen

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CD Nomenclature


The system used for leukocyte surface molecules,
as identified by monoclonal antibodies.
Examples:
 CD45 Common Leukocyte Antigen
 CD2, CD3, CD5 T cells
 CD19 and CD20 B cells
 CD34 Stem cell marker
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Indications for Flow Cytometry

Quantitation of lymphocyte subsets in
immunodeficiencies, HIV monitoring

Determination of cell type and stage of
differentiation in acute leukemias, including blast
crisis of CML

Detection of lymphoma cells in blood, bone
marrow, fluids and tissues

Detection of CD34+ stem cells in peripheral blood
and peripheral stem cell collection products
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Flow is not particularly
helpful in…

Chronic phase of CML

Hodgkin’s disease

Low grade myelodysplastic syndromes
(better when able to use antigen mapping
technique)

Screening of peripheral blood in patients who
do not have a sustained lymphocytosis or
abnormal findings on the smear
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