MICR 304 Introduction to Flow Cytometry

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MICR 304 Introduction to Flow Cytometry
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Overview

• What is flow cytometry?
• Development of flow cytometry
• Components of Flow
• Typical applications
• Flow data

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Flow Cytomtery

• Measurement (cytometry) of single cells in
  suspension that pass by (flow) a laser beam
• Not appropriate for analysis of cell clumps or
  tissues
• Discrete measurements from each cell in the
  sample, providing a distribution rather than an
  average of the measured characteristics in the
  cell sample
• Simultaneous measurement of multiple parameters
• Size (volume)
• Granularity (internal complexity)
• Fluorescence

Light scatter signals
Derived from fluorescent labels
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Basic Outline of a Flow Cytometer
Fluidics
Electronics
Optics
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Commercial History

• First commercial particle analyzer Model A
  Coulter counter (1950)
• First commercial fluorescence analyzer Partec
  (1969)
• First commercial cytometer, the Cytograph the
  Cytofluorograph Kamentsky in 1970
• First commercial cell sorter Becton Dickinson
  FACS-1 (1974, tradename) Hertzenberg
• Epics series 1977-79 by Coulter
• First benchtop analyzers about 1981
• 3 Colors available 1985 and 4 colors in 1986
• First Benchtop Sorters 1992
• First commercial high-speed cell sorter
  Cytomation MoFlo (1994)

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Advantages of Flow Cytometry

• Flexibility of the data acquisition
• Speed of measurement
• Thousands of cells can be analyzed in seconds
• Statistical information immediately available
• Ability to reanalyze with new gates gives us new
  information from old acquisitions

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Measurements in Flow Cytometry

• Light Scatter
• All objects passing through a laser beam in a
  cytometer will scatter light
• Large objects will scatter more light in the
  forward direction than small objects
• Forward Angle Light Scatter (FSC), roughly
  indicates size
• Forward light scatter, FALS , FS, FSC
• Side Scatter near 90 (SSC), structure dependent
  - reflective qualities, or granularity of a
  particle
• SS, SSC, 90 light scatter
• Actual laser spot is obscured and the light at 2
  - 20 off the straight laser line is what is
  measured

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Measurements in Flow Cytometry

• Fluorescence
• Excitation light energy is absorbed by
  fluorescent molecule, and molecule is excited
• As excited molecule returns to unexcited
  ground-state, a specific wavelength is emitted.
• Fluorescence emission is always of a longer
  wavelength (lower energy ) than the excitation
  wavelength.
• The longer the wavelength the lower the energy
• The shorter the wavelength the higher the energy
• e.g.. UV light from sun causes the sunburn not
  the red visible light

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Emission Accomplished!

• Fluorophore Excitation / Absorbance
• Wavelength dependent
• Fluorophore Emission / Fluorescence
• The light given off
• or emitted is at a longer wavelength but lower
  energy

Jablonski diagram illustrating the processes
involved in the creation of an excited electronic
singlet state by optical absorption and
subsequent emission of fluorescence.
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Visible Light Region of the Electromagnetic
Spectrum
Human eye can see 380nm-680nm
Spectrum is often shown this way
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Selected Laser Lines
Dyes
514
488
600 nm
300 nm
500 nm
700 nm
400 nm
PI
Ethidium
PE
FITC
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(FITC)
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Where is Fluorescence in Flow Cytometry Coming
from?

• Intrinsic fluorescence
• Genuine feature of the cell
• autofluorescence
• tryptophan, tyrosine, pigment content,
  hemoglobin,
• green fluorescent protein (GFP) - transfection
  assays
• static
• Extrinsic fluorescence
• Experimentally added to the cell
• Fluorescent probes/dyes - FITC, PE, PI, etc
• Static
• Kinetic

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Common Applications

• Immunophenotyping
• Made possible with the advent of Monoclonal
  antibodies
• Large majority of the uses of flow
• Determination of cell surface antigens and after
  permeabilization for intracellular stains
• Clinically important for disease prognosis and
  diagnosis
• The number of subsets of cells that can be
  recognized is growing yearly.
• DNA quantification
• Intercalating dyes like propidium iodide (red
  fluorescent)
• Functional assays
• Calcium probes, probes for oxidative burst (DHR),
  membranes , phagocytosis assays, and many more

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Monoclonal Antibodies

• Immunization
• Isolation of B-cells
• Fusion with metabolically deficient myeloma cell
• Selection
• Cloning by limited dilution

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Example Lymphocyte Typing
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Following the Sample
From the sample tube
Through the aspiration rod
Through the tubing inside the instrument
Through the flow Cell
Intersecting the laser
Down the stream
Into Waste
or Sort collection tubes
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Following the Cytometer signal path
Stream
lens
Cell
Laser
Trigger signal
Cytometer
Slide Courtesy of Joe Trotter, Director, Flow
Cytometry Facility The Scripps Research Institute
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Histogram
IgM
IgD
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Statistics

• What types of statistics are we interested in??
• Percentages of populations
• How bright those are indicates how MUCH antigen
  is present
• Do those change?
• Is there a reaction to a stimulus?

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Example MICR 304 S2008
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Acknowledgement

• This lecture has been drawn from a Dakocytomation
  training PowerPoint presentation
• Credit to Andrew Beernink (abeernink_at_novasite.com)
  Susan DeMaggio MS BSMT(ASCP)Qcym
  (flocyte_at_cox.net)