Immunophenotyping and
applications of cell analysis in the
hematology laboratory
J.Paul Robinson
Professor of Immunopharmacology &
Biomedical Engineering
April 5, 2005
This lecture can be found on
http://www.cyto.purdue.edu/class
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
What can flow cytometry be used for?
Clinical and Research
•Immunology
•Hematology
•Pathology
•Microbiology
•Genetics
•Drug discovery
•Toxicity testing
•Cell culture studies
•Functional studies
•Chemical Engineering
•Biotechnology
•Agronomy
•Animal Sciences
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Phenotype: …outward physical manifestation…
CELLULAR ANTIGENS
cytokines
structure
enzymes
Adhesion
Metabolic
Receptors
T cells
B Cells
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Slide courtesy of Jim Bender
Cluster Designations (CD)
• These are based on the Immunology Workshop an
international committee that meets in Boston
every few years
• Each antigen that is defined on cells is given a
unique number
• Until a final number is agreed, antigens can be
designated CDw (w=workshop a tentative
designation)
• Here is an example of the possible CDs
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Immunofluorescence staining
specific binding
nonspecific binding
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Slide from Dr. Carleton Stewart
Direct staining
• Fluorescent probe
attached to antibody
• Specific signal:
weak, 3dyes/site
• Nonspecific binding:
low
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Slide from Dr. Carleton Stewart
Avidin-Biotin method I
biotinylated
primary Ab
biotin
avidin
biotinylated dye
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Myelomonocytic Antigen Distribution
CDw13
MY8
CD11b
CFU-GM
CD16
PROGRANULOCYTE
MYELOCYTE
MYELOBLAST
HLA-Dr
CD34
CD33
CD38
CD71
Purdue Cytometry Labs
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
METAMYELOCYTE
BAND
PMN
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
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
From Duque et al, Clin.Immunol.News.
What are the principles in flow
cytometry?
•
•
•
•
•
Light scattered by a laser or arc lamp
Specific fluorescence detection
Hydrodynamically focused stream of particles
Electrostatic particle separation for sorting
Multivariate data analysis capability
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Concepts
Scatter:
Size, shape, granularity, polarized
scatter (birefringence), structure
Fluorescence:
Intrinsic: Endogenous pyridines and flavins
Extrinsic: All other fluorescence profiles
Absorption: Loss of light (blocked)
Time:
Useful for kinetics, QC
Count:
Number of cells collected in a
histogram
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Clinical Analyzers
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Cell Sorters (FACS – Fluorescence Activated Cell Sorter)
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
PMT 2
PMT 1
Laser
Bandpass
Filters
Hydrodynamic Systems
Sample in
Sheath
Piezoelectric
crystal oscillator
Sheath in
Fluorescence
Sensors
Signal
direction
Laser beam
Scatter Sensor
Sheath
Core
Flow Chamber
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Light Scatter
• Materials scatter light at wavelengths at which they do
not absorb
• If we consider the visible spectrum to be 350-850 nm
then small particles (< 1/10 ) scatter rather than absorb
light
• For small particles (molecular up to sub micron) the
Rayleigh scatter intensity at 0o and 180o are about the
same
• For larger particles (i.e. size from 1/4 to tens of
wavelengths) larger amounts of scatter occur in the
forward not the side scatter direction - this is called Mie
Scatter (after Gustav Mie) - thus forward scatter is
related to size (at 1-15 microns)
Shapiro p 79
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Optics for forward scatter
iris
Laser
beam
scatter
detector
blocker
Stream in air or a
round capillary
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Number of events
Frequency distribution
histogram
Intensity of parameter (e.g. fluorescence)
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Number of events
histogram
Intensity of parameter
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Flow cytometry measurements
SCATTER
G
M
L
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
FLUORESCENCE
IMAGE
Light Scatter Gating
1000
600
800
Scatter
Forward
Side Scatter Projection
Forward Scatter Projection
Neutrophils
Forward Scatter Projection
200
400
Monocytes
0
Lymphocytes
0
200
400
600
800
1000
90 Degree Scatter
Human white blood cells
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Different size cells
700
Number of events
200
90
20
0.9
small
0.1
large
1
10
100
1000
Particle or cell size (log scale)
While forward light scatter is not always related to cell size, in
The majority of cases between 1-20 microns, it is a reasonable estimate
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Light Scatter of white blood cells
• Light scatter can be used to identify
populations of cells
x
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
In peripheral blood, the three
main populations of leukocytes
can be distinguished. A “gate” or
“bitmap” can be placed around a
region so that further analysis
can be made on this region. The
cells in the region marked “X”
can be evaluated as a population.
Fluorescence - e.g. Monoclonal
Antibodies
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
“T” Cells
“B” Cells
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
3 Parameter Data Display
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Isometric Display
The Cell Cycle
G
M
2
S
G1
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
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
• 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
• 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.
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Normal Cell Cycle
300
G0 - G1G0
G2
Cell Count
225
G0
M
G1
s
150
75
0
G2 M
s
0
200
2N
400
600
DNA Content
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
4N
800
1000
A typical DNA Histogram
4n
2n
G0-G1
G2-M
# of Events
S
Fluorescence Intensity
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Flow Cytometry of Apoptotic
Cells
Apoptotic cells
# Events
Normal G0/G1 cells
PI - Fluorescence
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
400
0
200
Number
600
Analyzing the DNA Histogram
0
50
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
100
DNA Content
150
200
Chromosome Analysis
Most human chromosomes can be separated by flow cytometry
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Chromosome Analysis
(Bivariate Flow Karyotyping - porcine)
chromosome 1
chromosome 2
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Spectra of PI and EtBr
350
300 nm
457 488 514
400 nm
500 nm
610 632
600 nm
700 nm
PI
Ethidium
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
75
RMI = 0
37
75
Count
RMI = 34
0
0
37
Count
112
112
150
150
Reticulocyte Analysis
.1
1
10
100
1000
log Thiazole Orange
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
.1
1
10
100
1000
log Thiazole Orange
4 colors - simultaneous collection
(can go to 17 colors)
FITC
PE
PETR
PE-CY5
530
580 630
680 730 780
Emission wavelength (nm)
We separate different subsets by taking bands of light from the
light spectrum and analyzing the intensity of light in that band
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
FOUR COLOR PATTERN
CD56 – NK Cells
CD3 – T cells
CD4 – T cells – Helper
CD8 – T cells - Cytotoxic
This is a subset of cells
It is CD3+ CD4+
1
10
2
10
3
10
4
4
10
10
2
CD4
CD4 -->
1
10
1
10
10
10
10
2
CD8
10
CD8 -->
2
10
CD56 -->
1
10
CD56 - NK
10
3
3
3
10
10
4
4
This is a subset of cells
It is CD3+ CD56+
10
1
CD3 -->
10
2
10
3
10
4
10
1
10
10
3
10
4
CD3
CD3
1
10
2
10
3
10
4
CD56 -->
CD56
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
2
CD4
CD4 -->
1
10
1
10
10
10
10
3
2
10
CD4 -->
CD4
10
10
10
2
CD8
1
CD8 -->
10
3
3
10
10
4
10
4
4
CD3
2
CD3 -->
CD3 -->
10
1
10
2
CD56 -->
CD56
10
3
10
4
10
1
10
2
10
3
CD8 -->
CD8
Data from Dr. Carleton Stewart
10
4
Multicolor Analysis
Roederer, et al
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Cellular
Response:
•
•
•
•
•
Cell death
Cell ‘suicide’
Ignore damage
Damage repair
Incorrect repair
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Functional Assays
•intracellular pH
•intracellular calcium
•intracellular glutathione
•oxidative burst
•phagocytosis
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Oxidative Burst
•generation of toxic oxygen species
by phagocytic cells
•superoxide anion measured
with hydroethidine
•hydrogen peroxide measured with
2’,7’-dichlorofluorescin diacetate
(DCFH-DA)
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
1000
Neutrophil Oxidative Burst
1
Unstimulated
Neutrophils
.1
345
115
38
12
4
10
Scale
Log DCF
100
PMA-Stimulated
Neutrophils
0
600
1200
1800
TIME (seconds)
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
2400
Phagocytosis
FITC-Labeled Bacteria
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Cellular Functions
• Cell Viability
• Phagocytosis
• Organelle Function
– mitochondria, ER
– endosomes, Golgi
• Oxidative Reactions
–
–
–
–
Superoxide
Hydrogen Peroxide
Nitric Oxide
Glutathione levels
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
• Ionic Flux Determinations
–Calcium
–Intracellular pH
• Membrane Potential
• Membrane Polarization
• Lipid Peroxidation
Organelle Function
•
•
•
•
Mitochondria
Endosomes
Golgi
Endoplasmic Reticulum
Carbocyanine
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Rhodamine 123
Ceramides
BODIPY-Ceramide
DiOC6(3)
Fluorescent Indicators
How the assays work:
• Superoxide: Utilizes hydroethidine the sodium borohydride reduced
derivative of EB
• Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell
where cellular esterases hydrolyze the acetate moieties making a polar
structure which remain in the cell. Oxidants (H2O2) oxidize the DCFH
to fluorescent DCF
• Glutathione: In human samples measured using 40 M
monobromobimane which combines with GSH by means of
glutathione-S-transferase. This reaction occurs within 10 minutes
reaction time.
• Nitric Oxide: DCFH-DA can indicate for nitric oxide in a similar
manner to H2O2 so care must be used. DAF is a specific probe
available for Nitric Oxide
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Hydroethidine
HE
EB
H2N
NH2
H
N
O2-
H2N
NH2
N + Br
CH2CH3
-
CH2CH3
Phagocytic Vacuole
NADPH Oxidase
NADPH
O2
HE
O2-
NADP
SOD
O 2H2O2
DCF
H2O2
OH-
Example: Neutrophil Oxidative Burst
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
DCF
DCFH-DA
DCFH
DCF
2’,7’-dichlorofluorescin diacetate
O
O
CH3-C-O
O
O-C-CH3
Cl
2’,7’-dichlorofluorescin
Cl
H
COOH
O
HO
Cellular Esterases Cl
OH
Fluorescent
Cl
H
COOH
Hydrolysis
2’,7’-dichlorofluorescein
O
HO
O
H2O2
Cl
Cl
H
Oxidation
DCFH-DA
COOH
Neutrophils
DCFH-DA
80
Monocytes
DCFH H O
2 2
Lymphocytes
DCF
counts
60
PMA-stimulated PMN
Control
40
20
0
.
1
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
1log
100
FITC 10
Fluorescence
1000
Human Neutrophil
Phospolipase A2 activity
Leukotrienes
Lipid Peroxidation
OH.
Phagosome
H2O2
O2-
+
Stimulant
(PMA)
?
H2O2
SOD
O2
O2-
O2-
PKC
?
PCB
Oxidase
GR
NADP+
SOD
GP
GSH
NADPH + H+
Catalase
H2O2
GSSG
NADPH
H2O + O2
+
H
H2O
HMP
PCB
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
PCB
(Reduced GSH level)
Hydroethidine
Superoxide Production
15 minutes
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
45 minutes
Cell Sorting
• Physically separating cells based on some
measurable characteristic
• Placing these cells into containers
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
-
+
Charged Plates
Single cells sorted
into test tubes
Purdue University Cytometry Laboratories
Sample in
Sheath
Stream
Charge
Piezoelectric
crystal oscillator
Sheath in
Sensors
Sensor
Laser beam
SORT DECISIONS
SMALL BEAD LARGE BEAD
Last attached
droplet
SORT LEFT
SORT RIGHT
-2KV
+2KV
RIGHT
LEFT
Waste
SMALL BEAD
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
LARGE BEAD
Frequency Histogram
Cell Sorting
+++
Video of the droplet formation in a sort stream from a Cytomation
instrument. Source: Purdue CDROM vol 4, 1998
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor
--Video2.mpg
Lab
2 Groups of 8 students each
Hansen Hall, Room B50 (Basement)
Meet with Kathy Ragheb and Cheryl Holdman
One on April 18, 12:30-4:30
Other April 25, 12:30-4:30
Work in groups of 2 and you will take blood
• Phenotype your own blood for T cell and B cells
• Blood film and total blood count
• Coulter count to obtain total cell numbers
• Look at T and B cells under fluorescence scope
Purdue University Cytometry Laboratories – J. Paul Robinson, Professor