Uses of Flow Cytometry in Virology - Clinical Microbiology Reviews
Clinical Applications of Flow Cytometry
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Transcript of Clinical Applications of Flow Cytometry
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Clinical Applications of Flow Cytometry
J.Paul RobinsonProfessor of ImmunopharmacologyProfessor of Biomedical EngineeringPurdue UniversitySchool of Veterinary Medicine
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Primary areas
• DNA/RNA Analysis• Microbiology• Phenotyping• Cell Function
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Purdue University Cancer Center&
Purdue University Cytometry Laboratories
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Brief Introduction to Flow Cytometry
• What do these instruments look like?• What does flow cytometry do?• How does it work?• Why is it useful?
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Optical Design
PMT 1
PMT 2
PMT 5
PMT 4
DichroicFilters
BandpassFilters
Laser
Flow cell
PMT 3
Scatter
Sensor
Sample
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A Histogram(a frequency distribution graph)
Increase in Fluorescence Intensity
# of
Eve
nts
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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
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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
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The Cell Cycle
G1
MG2
S G0Quiescent cells
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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
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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
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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.
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A DNA histogram
G0-G1
S
G2-M
Fluorescence Intensity
Cel
l Num
ber
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A typical DNA Histogram
G0-G1
S
G2-M
Fluorescence Intensity
# of
Eve
nts
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Multiparameter gating
Human Prostate tumor cell line DU-145
DNA - Hoechst
Cyc
lin -
B1
- FIT
C
P-10
5 C
y5
Endoduplicating population
Mitotic cells
DNA - Hoechst
R1-gate
Data from Dr. James Jacobberger
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0 200 400 600 800 1000
PI Fluorescence
Counts
0
75
150
225
300
DNA Analysis
2N 4N
0 200 400 600 800 1000
PI Fluorescence
Counts
0
75
150
225
300
DNA AnalysisDNA Analysis
Aneuploid peak
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log Thiazole Orange.1 1000 100 10 1
Count
0
150
112
75
37
RMI = 0RMI = 0
log Thiazole Orange.1 1000 100 10 1
Count
0
150
112
75
37
RMI = 34RMI = 34
Reticulocyte Analysis
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log Thiazole Orange.1 1000 100 10 1
Count
0
150
112
75
37
RR11RR22RR33RR44
RMI = 34RMI = 34
Reticulocyte Analysis
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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.
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Detection Methods for Apoptotis
• Phosphatidyl serine, can be detetected by incubating the cells with fluorescein-labeled 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)
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Flow Cytometry of Apoptotic Cells
PI - Fluorescence
# Ev
ents
Apoptotic cells
Normal G0/G1 cells
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Labeling Strand Breaks with dUTP [Fluorescein-deoxyuridine triphosphate (dUTP)]
Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidinRed fluorescence is DNA counter-stained with 20µg/ml PI
PI-Red Fluorescence
Green Fluorescence
Green Fluorescence
Side
Sca
tter
Forward Scatter
Green:apoptotic cells
Red:normal cells
R2: Apoptotic Cells
R1: Normal Cells
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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.
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Nuclear antigens
P-105 -CY5 DNA - Hoechst Cyclin - B1 - FITC (log)
Cyclin - B1 - FITC 90 deg Scatter (log) FALSHuman Prostate tumor cell line DU-145
Data from Dr. James Jacobberger
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Differential Inflammatory Cell Count
Data from Dr. Doug Redelman, Sierra Cytometry
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Simultaneous UV & Visible Light
Hoechst 33342 (UV)
PI -
fluor
esce
nce
Data from Dr. Doug Redelman, Sierra CytometryHoechst
Hoechst binds to all DNA - It is UV excited
PI only binds to DNA where it can gain access to the cell - ie Dead cells
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Hoechst & PI Fluorescence
Data from Dr. Doug Redelman, Sierra Cytometry
PIHoechst 33342
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Boar Sperm
Data from Dr. Doug Redelman, Sierra Cytometry
FL1-Hoechst
FL2-PI
Hoechst/PI
Dead
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Human Sperm
Data from Dr. Doug Redelman, Sierra Cytometry
Sybr greenPI
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Human Sperm - PI - Sybr-Green I
Sybr-Green
PI
Data from Dr. Doug Redelman, Sierra Cytometry
dead
live
activeinactive
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Microbiology
• Detection of unknown organisms• Antibiotic sensitivity testing• Detection of Spores
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Uptake of rhodamine 123 by M.luteus
Data from Dr. Hazel Davey
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.
M.luteus
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Mixed suspensions of bacteria Identification on scatter alone?
Light scatter signature of a mixture of B.subtilis spores (BG) and E.coli cells.
BG doublets
BG spores
E.coli cells
debris
debrisE.coli
BG
doublets ?
log FS log FS
log
SS
Cou
nt
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Light Scatter of Bacterial Spores
Light scatter signals from a mixture of live B.anthracis spores, live B. subtilis spores and gamma irradiated B. anthracis spores.
B.anthracis
B.subtilis
irradiated B.anthracis
SS
FS
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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
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YoYo-1 stained mixture of 70% ethanol fixed E.coli cells and B.subtilis (BG) spores.
mixture
BG E.coli
BG
E.coli
mixture Run on Coulter
XL cytometerSc
atte
r
Fluorescence
Scat
ter
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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
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Phenotyping - Immunophenotyping
• Characterization of white blood cells• Identification of lymphocyte subsets
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CELLULAR ANTIGENS
AdhesionReceptors
Metabolic
cytokines
structureenzymes
courtesy of Jim Bender
T cellsB Cells
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Immunofluorescence staining
specific binding
nonspecific binding
Data from Dr. Carleton Stewart
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Direct staining
• Fluorescent probe attached to antibody
• Specific signal: weak, 3dyes/site
• Nonspecific binding: low
Data from Dr. Carleton Stewart
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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
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Avidin-Biotin method Ibiotinylated primary Ab
biotin
avidin
biotinylated dye
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Three Color Lymphocyte Patterns
CD3
CD4
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD4 -->
CD3
CD4
CD8CD
810 1 10 2 10 3 10 4
CD8 -->
101
102
103
104
CD4 -->
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD8 -->
Data from Dr. Carleton Stewart
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10 1 10 2 10 3 10 4
CD56 -->
101
102
103
104
CD4 -->
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD4 -->
CD3CD3 CD310 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD56 -->
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD8 -->
CD5610 1 10 2 10 3 10 4
CD56 -->
101
102
103
104
CD8 -->
CD56 CD810 1 10 2 10 3 10 4
CD8 -->
101
102
103
104
CD4 -->
FOUR COLOR PATTERN
CD4
CD8
CD56
CD8
CD4
CD4
Data from Dr. Carleton Stewart
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NegativePositive
Decision Tree in Acute Leukemia
HLA-DR
TCD13,33
CD19
TdTCD10
CD20
Mu
B,T
AMLL AML
T-ALL
AML-M3
AUL
?
PRE-BI
PRE-BII
PRE-BIII
PRE-BIVPRE-BV
CD13,33
From Duque et al, Clin.Immunol.News.
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Cellular Function
• Phagocytosis• Killing index of phagocytes• Intracellular cytokines• Calcium flux• Oxidative burst• Membrane potential
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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
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Thank you for your attention
These slides will be available on our website at:www.cyto.purdue.edu/education