Clinical Applications of Flow Cytometry

J.Paul RobinsonProfessor of ImmunopharmacologyProfessor of Biomedical EngineeringPurdue UniversitySchool of Veterinary Medicine

Primary areas

• DNA/RNA Analysis• Microbiology• Phenotyping• Cell Function

Purdue University Cancer Center&

Purdue University Cytometry Laboratories

Brief Introduction to Flow Cytometry

• What do these instruments look like?• What does flow cytometry do?• How does it work?• Why is it useful?

Optical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

A Histogram(a frequency distribution graph)

Increase in Fluorescence Intensity

# of

Eve

nts

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

G1

MG2

S G0Quiescent 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

G0-G1

S

G2-M

Fluorescence Intensity

Cel

l Num

ber

A typical DNA Histogram

G0-G1

S

G2-M

Fluorescence Intensity

# of

Eve

nts

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

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

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

log Thiazole Orange.1 1000 100 10 1

Count

0

150

112

75

37

RR11RR22RR33RR44

RMI = 34RMI = 34

Reticulocyte Analysis

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 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)

Flow Cytometry of Apoptotic Cells

PI - Fluorescence

# Ev

ents

Apoptotic cells

Normal G0/G1 cells

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

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 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

Differential Inflammatory Cell Count

Data from Dr. Doug Redelman, Sierra Cytometry

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

Hoechst & PI Fluorescence

Data from Dr. Doug Redelman, Sierra Cytometry

PIHoechst 33342

Boar Sperm

Data from Dr. Doug Redelman, Sierra Cytometry

FL1-Hoechst

FL2-PI

Hoechst/PI

Dead

Human Sperm

Data from Dr. Doug Redelman, Sierra Cytometry

Sybr greenPI

Human Sperm - PI - Sybr-Green I

Sybr-Green

PI

Data from Dr. Doug Redelman, Sierra Cytometry

dead

live

activeinactive

Microbiology

• Detection of unknown organisms• Antibiotic sensitivity testing• Detection of Spores

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

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

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

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

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

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

AdhesionReceptors

Metabolic

cytokines

structureenzymes

courtesy of Jim Bender

T cellsB Cells

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 Ibiotinylated primary Ab

biotin

avidin

biotinylated dye

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

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

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.

Cellular Function

• 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