FLOW CYTOMETRY

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FLOW CYTOMETRY

• Dr. MOHAMMED H SAIEMA LDAHR
• KAAU
• FACULTY OF APPLIED MEDICAL SCIENCES
• MEDICAL TECHNOLOGY DEPT.
• 2ND YEAR MT
• INSTROMINTATION
• EXT 21060

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OPTICS (FSC)
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Optics Side Scatter Channel (SSC)

• The amount of light scattered at right angle to
  the incident light beam depends on the internal
  complexity of the particle, this known as wide
  angle or Side Scatter (SSC) , side scatter
  detected at 90, to the laser beam.

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SSC)
Optics (
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OPTICSPROPERTIES OF FSC SSC
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PROPERTIES OF FSC SSC

• As the cell passes through the laser beam, light
  is
• scattered in all directions and that scattered
  in the forward direction is proportional to the
  square of the radius of a sphere, and so to the
  size of the cell or particle.
• The cells may be labeled with fluorochrome-linked
  
• antibodies or stained with fluorescent membrane,
  cytoplasmic or nuclear dye.

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What can a Flow Cytometer (FCM) tell us about a
cell?

• Its relative size (Forward ScatterFSC)
• Its relative granularity or internal complexity
  (Side ScatterSSC)
• Its relative fluorescence intensity (FL1, FL2,
  FL3, FL4)

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Optics - Fluorescence Channels

• Any fluorescent molecule present in or on the
  particle will absorb energy from the laser light
  and release the absorbed energy at longer wave
  length, the emitted light is collected by lenses
  and detectors, emitted fluorescence intensity is
  proportional to the amount of fluorescent
  compound on the particle.

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Optical Design
PMT 5
PMT 4
Sample
PMT 3

Dichroic
Filters
Flow cell
PMT 2
PMT 1
Scatter
Laser
Sensor
Band pass
Filters
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Electronics

• The scattered light from particles passing the
  laser light is converted to digital values that
  stored in the computer for analysis.

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Electronics
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FLOW CYTOMETRY

• Gating.
• Gating is in essence electronic window that sets
  upper and lower limits on the type and amount of
  material that passes through.
• It is used to separate a sub-population from
  heterogeneous population.
• It permits very specific questions to be asked
  about a particular population.

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Fluorescence and Fluorochrome.

• The coupling of monoclonal antibodies with
  fluorescent dyes is necessary for recognition and
  enumeration by flow cytometer.
• Each fluorochrome possesses a distinctive
  spectral pattern of absorption (excitation) and
  emission.
• The property of fluorescence is that, the
  fluorochromes present on the cell absorb the
  laser light and re-emit the light at a lower
  energy and longer wave length.
• The most popular fluorochrome used in
  immuno-fluorescent is fluorescein isothiocyanate
  (FITC), which has an absorption maximum between
  450 and 550 nm.

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Fluorescence and Fluorochrome.

• FITC emits light detected in FL1 on the FCM.
• Another example of an excellent combination is
  Phycoerythrin (PE) as a second fluorochrome since
  it can absorb light at higher wave lengths than
  the FITC.
• PE emits light can be detected in FL2 on the FCM

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APPLICATION

• Applications in Clinical Laboratories
• Immunophenotyping (HIV)
• CD4 absolute counts
• Leukemia and lymphoma immunophenotyping
• Cell cycle and ploidy analysis of tumors
• Reticulocyte enumeration
• Flow cross-matching (organ transplantation)
• Stem cell enumeration
• Residual white blood cell detection
• (QC platelet, red blood cells)

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APPLICATION

• Research Laboratories
• Immune function studies
• Hematopoietic stem cells
• Multi-drug resistance studies (cancer)
• Kinetics studies (cell function) Platelet
  analysis (coronary disease)
• Environmental sample analysis
• Flow and FISH

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APPLICATION

• Immunophenotyping
• Is the termed used in the identification of cells
  by labeling with monoclonal antibodies identified
  as cluster designations (CD)
• CDs is a group of antibodies that all recognize
  the same antigen

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Immunophenotyping

• Is performed by labeling the cells with red,
  green, and / or orange labeled monoclonal
  antibody.
• The cells are then interrogated in the Flow
  Cytometry, and the number, percentage of positive
  cells are recorded.

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CD Markers used for Primary Diagnosis
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CD Markers used for Primary Diagnosis
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TYPES OF MEASUREMENTS

• Intrinsic parameters
• Cell size Granularity.
• Extrinsic parameters
• Surface Antigens
• Single or multiple surface membrane antigens are
  detected with fluorescinated monoclonal
  antibodies examples, CD45, CD4, CD8, etc).

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TYPES OF MEASUREMENTS

• Multi-parametric FCM
• In the multi-parameter approach more than one
  feature of the same cell is measured, such as
  three color surface and cytoplasmic staining in
  conjunction with the cellular DNA content
• leukemia and lymphoma.
• Distinction of myeloid leukemia from lymphatic
  can be precisely determined by the analysis of
  surface and cytoplasmic antigens.

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TYPES OF MEASUREMENTS

• FCM analysis of Platelets
• Glanzmanns Thrombasthenia (GPIIb/IIIa).
• Bernard Soulier Syndrome (GPIb).
• Storage Pool Disease
• Dense Granule SPD.
• Gray Platelets SyndromeL.
• Reticulated Platelets.
• Anti-platelets Antibodies.
• Monitoring treatment with GPIIb/IIIa antagonist

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TYPES OF MEASUREMENTS

• DNA content cell cycle analysis
• Aneuploidy and/or elevated S-phase fraction have
  been shown to be ominous prognostic indicators in
  breast, colon, rectal, prostate, and bladder
  tumours.

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Normal Cell Cycle
M
G0
G2
DNA Analysis
G1
s
Count
s
0
200
400
600
800
1000
DNA content
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APPLICATION
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APPLICATION