Flow Cytometry

1
Flow Cytometry
The Basics and Beyond Phyllis Kirchner,
MT(ASCP)SH
2
Flow Cytometry

• Fluidics
• Optics
• Electronics

• Cells in suspension
• file thru an illuminated
• volume where they
• scatter light and emit
• fluorescence that is
• collected, filtered and
• converted into digital
• values that are stored on a
• computer

3
Channel Layout for Laser-based Flow Cytometry
PMT
4
PMT
Dichroic
3
Filters
Flow cell
PMT
2
Bandpass
Side Scatter
Filters
PMT
1
Laser
Forward Scatter
original from Purdue University Cytometry
Laboratories modified by R.F. Murphy
4
The cells go marching one-by-one.
Sample
Sheath fluid
Cross section
Flow cell
Sheath fluid
Sample
Laser
To Detectors
Laser
Waste
Flow cell
Hydrodynamic focusing
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Optical Filters

• Lenses focus the laser light
• width height

Laser
6
Optical Filters
500 520 575 620
675
to detector
to detector
to detector
to detector
to detector
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Photomultiplier Tube (PMT)

• Amplifies signal
• Produces voltage signal proportional to light
  pulses
• Analogue to Digital Converter (ADC) measures
  voltage pulse height and converts to channel
  height

Original signal
8
The Cell
Surface Membrane
Cytoplasm
Granules
Nucleus
Nuclear Membrane
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Forward Light Scatter
Measures the SIZE of the cell
Laser
Forward Detector
Laser
Forward Detector
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Side (Orthogonal) Light Scatter
Measures the COMPLEXITY of the cell
Side Scatter Detector
Laser
Laser
Side Scatter Detector
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Light Scatter Gating
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Gating or bit map is an electronic method of
selecting a population you wish to analyze
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Side Scatter (SSC)
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Forward scatter (FSC)
12
Channel Layout for Laser-based Flow Cytometry
PMT
4
PMT
Dichroic
3
Filters
Flow cell
PMT
2
Bandpass
Side Scatter
Filters
PMT
1
Laser
Forward Scatter
original from Purdue University Cytometry
Laboratories modified by R.F. Murphy
13
Light Scatter Gating
Side Scatter Projection
Forward Scatter
Neutrophils
Scale
1000
200
Forward Scatter Projection
100
50
0 200 400 600
800 1000
40
Monocytes
30
20
15
Lymphocytes
8
200
400
600
800
0
1000
90 Degree Scatter
with permission Purdue University Cytometry
Laboratories
14
Monoclonal Antibodies

• Sensitize mouse to antigen
• Harvest spleen B-cells
• Fuse with myloma cells
• Select hybridoma clone(s) for antibody production
  against antigen
• Maximize antibody production
• Label with fluorescent dye

15
Stokes Shift
excitation emission
The Stokes shift is the difference between the
wavelength a fluorochrome is excited and the
wavelength that it emits light
488 500 600 nm
16
Fluorescence Staining
17
Spectral Overlap
Can be reduced with filters and color compensation
520 575 620
675
18
Color Compensation
Uncompensated vs Compensated
FL1
FL1
FL2
FL2
Electronic means to separate colors into distinct
regions
19
Data Analysis

• Histograms
• Single parameter
• Linear scale
• Log scale
• Scatterplots/ Topigraphical plots
• Multiparameter

20
Ideal log amp
with permission Purdue University Cytometry
Laboratories
21
Log amps dynamic range

• Compare the data plotted on a linear scale
  (above) and a 4 decade log scale (below). The
  date are identical, except for the scale of the x
  axis. Note the data compacted at the lower end of
  the the linear scale are expanded in the log
  scale.

with permission Purdue University Cytometry
Laboratories
22
SINGLE COLOR HISTOGRAMS
number of cells
with permission Purdue University Cytometry
Laboratories
23
TWO COLOR PATTERN
A
B
FL1
FL1
FL2
FL2
with permission Purdue University Cytometry
Laboratories
24
Viability Testing

• Are they dead or alive?
• Trypan blue, Ethidium Bromide, Propidium Iodide
• Staining
• Cytoprep/BM/PB by Wrights stain
• Look at what youre doing markers on

25
ANTIBODIES BIND NON-SPECIFICALLY TO DEAD CELLS
ALL CELLS
VIABLE CELLS
A
B
dead cells
PE-LAMBDA
FL-KAPPA
with permission Purdue University Cytometry
Laboratories
26
Cell Marker Studies

• Membrane antigens
• Cytoplasmic constituents
• Nuclear components
• DNA studies
• Cellular uptake mechanisms

27
Cell Marker Studies

• Peripheral blood
• Immune status, (leukemia/lymphoma)
• Bone Marrow/Lymph nodes/ Tumors/Body fluids
• Leukemia/Lymphoma/Malignancy/DNA ploidy
• Archival material
• DNA ploidy

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T4/T8 Studies

• Evaluation of immune status HIV patients
• Recommended markers multiparameter analysis
• CD45 CD3
• CD4 CD8
• CD19 CD16(56)

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T4/T8 studies

• Assessment of T4/T8 cells esp in HIV patients
• Normal 21
• Evaluation of absolute T4 numbers

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T Cell Malignancies

• Follow normal ontogeny
• Show patterns of clonality
• Increased epitope expression
• Absence of epitope expression

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T Cell Ontogeny
32
Typical T-cell Lymphoma vs Reactive Lymph Node
Purdue University Cytometry Laboratory
33
B Cell Ontogeny
Follow normal ontogeny Show patterns of
clonality Increased epitope expression Absence of
epitope expression
34
Normal lymphs
Leukemic blasts
Purdue University Cytometry Laboratory
35
B cell Lymphoma vs Normal LN
Lymphoma
Normal LN
Purdue University Cytometry Laboratory
36
Normal Cell Cycle
M
G0
G2
DNA Analysis
G1
s
Count
DNA content
s
G2M
0
200
400
600
800
1000
DNA content
This nucleus twice as bright
G1
Original from Purdue University Cytometry
Laboratories modified by PA Kirchner
37
DNA Analysis
0
200
400
600
800
1000
4N
2N
PI Fluorescence
with permission Purdue University Cytometry
Laboratories
38
DNA Analysis
Aneuploid peak
0
200
400
600
800
1000
PI Fluorescence
with permission Purdue University Cytometry
Laboratories
39
Platelets

• Flow used to
• Determine antigen typing of patients platelets
• Determine if patient has antibodies to platelet
  antigens
• Example - Antigen typing
• Negative control anti-HPA-1a
  

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Paroxysmal Nocturnal Hemoglobinuria (PNH) Studies

• CD59
• Membrane Inhibitor of Reactive Lysis (MIRL) -
  complement regulating protein
• CD55
• Decay Accelerating Factor (DAF) complement
  defense protein
• Both absent or decreased in PNH

41

• Flow Cytometry
• CD55 CD59
• Normal
• CD55 CD59
• PNH

CD 55
CD 59
CD 55
CD 59
42
Histocompatability Testing

• Percent Reactive Antibody (PRA) Testing
• Class I and Class II beads coated with HLA
  antigens patient serum
• Incubated with Goat anti-human IgG (FITC)
• Flow cross-matching
• Donor cells patient serum anti-CD3(PerCP) and
  anti-CD19(PE)
• Incubated with Goat anti-human IgG (FITC)

43
PRA
Positive patient
Class I
Class II
Class II
Class I
Negative Control
44
Flow Cross-Match
Negative
CD3 Class I
CD19 Class II
Looking for difference in mean channel fluorescenc
e between negative control and test sample along
with other clinical and laboratory criteria
Positive
CD3 Class I
CD19 Class II
45
Other uses

• Reticulocyte counts
• Fetal-Maternal Hemorrhage
• Karyotyping
• Susceptability testing
• Cell membrane phenomena
• In situ hybribization

46
Sources of Bias in Flow Cytometry

• Specimen collection
• Transportation storage
• Sample preparation
• Staining
• Measurement
• Data analysis
• Reporting