Clinical Applications of Flow Cytometry
J.Paul Robinson
Professor of Immunopharmacology
Professor of Biomedical Engineering
Purdue University
School of Veterinary Medicine
Primary areas
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DNA/RNA Analysis
Microbiology
Phenotyping
Cell Function
Purdue University Cancer Center
&
Purdue University Cytometry Laboratories
Brief Introduction to Flow
Cytometry
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What do these instruments look like?
What does flow cytometry do?
How does it work?
Why is it useful?
Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
PMT 2
PMT 1
Laser
Bandpass
Filters
A Histogram
# of Events
(a frequency distribution graph)
Increase in Fluorescence Intensity
DNA Probes
• DNA in cells can be stained with a
fluorescent dye
• DNA probes like Propidium Iodide are
STOICHIOMETRIC – that means the
number of molecules of probe bound is
equivalent to number of molecules of DNA
• So we can measure how much DNA is in a
cell
DNA/RNA Probes
• Propidium Iodide
• Hoechst
• Cyanine Dyes
–
–
–
–
•
•
•
•
TOTO-1 , YOYO-1, TOTO-3
Thiazole Orange, Thiazole Blue, Thioflavin
PRO dyes
SYTO/SYTOX dyes (Sytox green)
Acridine Orange
Pyronin Y
Styryl Dyes
Mithramycin + EtBr
The Cell Cycle
G
M
2
S
G1
G0
Quiescent cells
Definitions & Terms
• Ploidy
– related to the number of chromosomes in a
cell
• Haploid: Number of chromosomes in a
gamete (germ cell) is called the HAPLOID
number for that particular species
• Diploid: The number of cells in a somatic
cell for a particular species
Definitions & Terms
• Hyperdiploid: greater than the normal 2n
number of chromosomes
• Hypodiploid: Less than the normal 2n
number of chromosomes
• DNA Tetraploidy: Containing double the
number of chromosomes
Definitions & Terms
• DNA Index: The ratio between the mode of
the relative DNA content of the test cells (in
G0/G1phase) to the mode of the relative
DNA content in normal G0/G1 diploid cells
• Coefficient of Variation - CV: The ratio
between the SD of the mode of the G0/G1
cell populations expressed as a percentage.
A DNA histogram
Cell Number
G0-G1
G2-M
S
Fluorescence Intensity
A typical DNA Histogram
G0-G1
G2-M
# of Events
S
Fluorescence Intensity
Multiparameter gating
Endoduplicating population
P-105 Cy5
Cyclin - B1 - FITC
R1-gate
DNA - Hoechst
Mitotic cells
DNA - Hoechst
Human Prostate tumor cell line DU-145
Data from Dr. James Jacobberger
75
150
225
300
DNA Analysis
0
DNA Analysis
400
2N
600
800
1000
300
200
4N
225
PI Fluorescence
150
Aneuploid peak
0
75
Counts
0
0
200
400
600
PI Fluorescence
800
1000
112
112
150
150
Reticulocyte Analysis
Count
RMI = 0
0
0
37
37
75
75
Count
RMI = 34
.1
1
10
100
log Thiazole Orange
1000
.1
1
10
100
1000
log Thiazole Orange
112
150
Reticulocyte Analysis
75
R3
R4
R1
R2
0
37
Count
RMI = 34
.1
1
10
log Thiazole Orange
100
1000
Measurement of Apoptosis
• Apoptosis is programmed cell death where the cell
goes through a highly regulated process of “dying”.
• Characteristics are condensation of the chromatin
material
• Blebbing of nuclear material
• Often accompanied by internucleosomal degradation
of DNA giving rise to distinctive 'ladder' pattern on
DNA gel electrophoresis.
Detection Methods for
Apoptotis
• Phosphatidyl serine, can be detetected by
incubating the cells with fluoresceinlabeled Annexin V
• By staining with the dye, Hoechst 33342
(UV)
• By staining with the dye PI (visible)
• By staining with the dye YOPRO-1
(visible)
Flow Cytometry of Apoptotic
Cells
Apoptotic cells
# Events
Normal G0/G1 cells
PI - Fluorescence
Labeling Strand Breaks with
dUTP [Fluorescein-deoxyuridine triphosphate (dUTP)]
Side Scatter
Green:
apoptotic cells
R2: Apoptotic Cells
Green Fluorescence
R1:
Normal
Cells
Red:
normal cells
PI-Red Fluorescence
Green Fluorescence
Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidin
Red fluorescence is DNA counter-stained with 20µg/ml PI
Nuclear Antigens
• Ki-67 - proliferation related antigen
• Ki-S1 - proliferation related antigen
• Cyclin A: expression begins in late G1/early S
phase and increases as cells traverse S phase,
reaching a maximum in G2. Cyclin A is not
expressed in mitotic cells
• Cyclin B1: accumulates in late S phase but is
maximally expressed in G2 and mitosis.
Nuclear antigens
P-105 -CY5
FALS
DNA - Hoechst
Cyclin - B1 - FITC (log)
90 deg Scatter (log)
Cyclin - B1 - FITC
Human Prostate tumor cell line DU-145
Data from Dr. James Jacobberger
Differential Inflammatory Cell Count
Data from Dr. Doug Redelman, Sierra Cytometry
Simultaneous UV & Visible Light
Hoechst binds to
all DNA - It is UV
excited
PI - fluorescence
PI only binds to
DNA where it can
gain access to the
cell - ie Dead cells
Hoechst 33342 (UV)
Hoechst Data from Dr. Doug Redelman, Sierra Cytometry
Hoechst & PI Fluorescence
Hoechst
33342
PI
Data from Dr. Doug Redelman, Sierra Cytometry
Boar Sperm
Hoechst/PI
FL2-PI
Dead
FL1Hoechst
Data from Dr. Doug Redelman, Sierra Cytometry
Human Sperm
PI
Sybr green
Data from Dr. Doug Redelman, Sierra Cytometry
Human Sperm - PI - Sybr-Green I
PI
SybrGreen
live
inactive
active
dead
Data from Dr. Doug Redelman, Sierra Cytometry
Microbiology
• Detection of unknown organisms
• Antibiotic sensitivity testing
• Detection of Spores
Uptake of rhodamine 123 by M.luteus
M.luteus
Changes in light scattering behaviour and in the ability to accumulate Rhodamine 123 during
resuscitation of a starved cultured of M. luteus. Cells were starved for 2.5 months, incubated
with penicillin G for 10 hours, washed, and resuscitated in weak nutrient broth. Data represent
a culture (A) immediately after the penicillin treatment, and (B) 2 days later.
Data from Dr. Hazel Davey
Mixed suspensions of bacteria
Identification on scatter alone?
Count
log SS
BG
BG doublets
E.coli
debris
doublets ?
BG spores
E.coli cells
debris
log FS
log FS
Light scatter signature of a mixture of B.subtilis
spores (BG) and E.coli cells.
Light Scatter of Bacterial Spores
B.anthracis
SS
B.subtilis
irradiated B.anthracis
FS
Light scatter signals from a mixture of live B.anthracis spores,
live B. subtilis spores and gamma irradiated B. anthracis spores.
Nucleic Acid Content
• Distinguish bacteria from particles of similar size
by their nucleic acid content
• Fluorescent dyes
-must be relatively specific for nucleic acids
-must be fluorescent only when bound to
nucleic acids
Examples
-DAPI
-Hoechst 33342
-cyanine dyes YoYo-1, YoPro-1, ToTo-1
mixture
Scatter
mixture
Scatter
BG
BG
E.coli
E.coli
Fluorescence
YoYo-1 stained mixture of 70% ethanol fixed
E.coli cells and B.subtilis (BG) spores.
Run on
Coulter
XL
cytometer
Microbial Identification Using Antibodies
Enumeration & identification of target organisms in
mixed populations
Examples include:
• Legionella spp. in water cooling towers
• Cryptosporidium & Giardia in water reservoirs
• Listeria monocytogenes in milk
• E.coli O157:H7 in contaminated meat
• Bacillus anthracis & Yersinia pestis biowarfare agents
Phenotyping Immunophenotyping
• Characterization of white blood cells
• Identification of lymphocyte subsets
CELLULAR ANTIGENS
cytokines
structure
enzymes
Adhesion
Metabolic
Receptors
T cells
B Cells
courtesy of Jim Bender
Immunofluorescence staining
specific binding
nonspecific binding
Data from Dr. Carleton Stewart
Direct staining
• Fluorescent probe
attached to antibody
• Specific signal:
weak, 3dyes/site
• Nonspecific binding:
low
Data from Dr. Carleton Stewart
Indirect staining
• Fluorescent probe attached to
a 2nd antibody
• Specific signal: strong, 5-6
2nd Ab/each 1st Ab; therefore
15-18 dyes/site
• Nonspecific binding: high
Data from Dr. Carleton Stewart
Avidin-Biotin method I
biotinylated
primary Ab
biotin
avidin
biotinylated dye
3
10
2
1
10
CD4 -->
CD4
10
2
10
CD3
10
2
10
3
10
CD8
4
10
CD3 -->
1
10
2
10
3
10
4
10 3
10 4
CD8 -->
10 2
1
CD8
CD8 -->
10
10 1
CD4 -->
10
1
10
CD4
3
10
10
4
4
Three Color Lymphocyte Patterns
10
1
10
2
10
3
10
4
CD3 -->
CD3
Data from Dr. Carleton Stewart
10
2
10
3
10
4
10
10
1
CD3 -->
10
2
10
3
10
4
10
1
10
CD3
4
3
10
4
CD3
3
1
10
2
CD56 -->
10
3
CD56
10
4
10
1
10
1
10
10
2
CD4
CD4 -->
2
10
10
2
CD4 -->
CD4
10
10
CD8
1
10
CD8 -->
10
3
3
10
10
10
10
4
CD3
2
CD3 -->
CD3 -->
4
1
2
1
10
1
10
10
10
10
CD4 -->
2
10
CD8 -->
10
10
3
CD4
3
10
CD8
3
10
2
10
1
CD56 -->
CD56
10
4
4
4
FOUR COLOR PATTERN
10
1
10
2
CD56 -->
10
3
CD56
10
4
10
1
10
2
10
3
10
4
CD8 -->
CD8
Data from Dr. Carleton Stewart
PRE-BV
PRE-BIV
Mu
Negative
Positive
PRE-BIII
PRE-BII
CD20
AUL
PRE-BI
CD10
TdT
AMLL
AML
AML-M3
?
CD19
B,T
CD13,33
T-ALL
CD13,33
T
HLA-DR
Decision Tree in Acute Leukemia
From Duque et al, Clin.Immunol.News.
Cellular Function
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Phagocytosis
Killing index of phagocytes
Intracellular cytokines
Calcium flux
Oxidative burst
Membrane potential
Conclusions
• Many current research tools have clinical
application
• Frequently used in clinical trials and clinical
research
• Applications in veterinary medicine require
– Cost reduction
– Antibody specificity
– Increased interest from veterinary researchers
Thank you
for your attention
These slides will be available on our
website at:
www.cyto.purdue.edu/education