Medical Student lecture

Purdue University Cytometry Laboratories – J. Paul Robinson, Professor

Immunophenotyping and applications of cell analysis in

the hematology laboratory

J.Paul RobinsonProfessor of Immunopharmacology &

Biomedical Engineering

April 5, 2005

This lecture can be found on

http://www.cyto.purdue.edu/class


What can flow cytometry be used for?

•Immunology•Hematology•Pathology•Microbiology•Genetics

•Drug discovery•Toxicity testing•Cell culture studies•Functional studies

Clinical and Research

•Chemical Engineering•Biotechnology•Agronomy•Animal Sciences


CELLULAR ANTIGENS

AdhesionReceptors

Metabolic

cytokines

structureenzymes

Slide courtesy of Jim Bender

T cellsB Cells

Phenotype: …outward physical manifestation…


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


Immunofluorescence staining

specific binding

nonspecific binding

Slide from Dr. Carleton Stewart


Direct staining

• Fluorescent probe attached to antibody

• Specific signal: weak, 3dyes/site

• Nonspecific binding: low

Slide from Dr. Carleton Stewart


Avidin-Biotin method I

biotinylated primary Ab

biotin

avidin

biotinylated dye


CFU-GM

MYELOBLAST

MYELOCYTE META-MYELOCYTE

BAND

PMN

CD16

CD11b

CDw13

CD33

CD34

HLA-Dr

CD38

CD71

MY8

Myelomonocytic Antigen Distribution

Purdue Cytometry Labs

PROGRANULOCYTE


NegativePositive

Decision Tree in Acute Leukemia

HLA-DR

TCD13,33

CD19

TdT

CD10

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.


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


Concepts

Scatter: Size, shape, granularity, polarized scatter (birefringence),

structure

Fluorescence: Intrinsic: Endogenous pyridines and

flavinsExtrinsic: All other fluorescence

profiles

Absorption: Loss of light (blocked)Time: Useful for kinetics, QCCount: Number of cells collected in a histogram


Clinical Analyzers


Cell Sorters (FACS – Fluorescence Activated Cell Sorter)


Optical DesignOptical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample


Hydrodynamic SystemsSample in

Sheath

Sheath in

Laser beam

Piezoelectriccrystal oscillator

FluorescenceSensors

Scatter Sensor

Core

Sheath

Signaldirection

Flow Chamber




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 79Shapiro p 79


Optics for forward scatter

scatterdetector

iris

blocker

Laser beam

Stream in air or a round capillary


Frequency distributionN

umbe

r of

eve

nts

Intensity of parameter (e.g. fluorescence)

histogram


histogram

Intensity of parameter

Num

ber

of e

vent

s


Flow cytometry measurements

L

M

G

SCATTER FLUORESCENCE IMAGE


0 200 400 600 800 1000

0 2

00 4

00 6

00 8

0010

00

Side Scatter Projection

Forw

ard

Sca

tter P

roje

ctio

n

Light Scatter Gating

Forward Scatter Projection

90 Degree Scatter

Neutrophils

Lymphocytes

Monocytes

For

war

d S

catte

r

Human white blood cells


Different size cells

Particle or cell size (log scale)

Num

ber

of e

vent

s

small large

0.1 1 10 100 1000

0.920

90

200

700

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


Light Scatter of white blood cells

• Light scatter can be used to identify populations of cells

x

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


“B” Cells

“T” Cells


3 Parameter Data DisplayIsometric Display


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

• 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.


Normal Cell Cycle

GG00G0 - G1

s G2M

DNA Content2N2N 4N4N

G2M G0

G1

s

0 200 400 600 800 10000

75

150

225

300C

ell

Co

un

t


A typical DNA Histogram

G0-G1

S

G2-M

Fluorescence Intensity

# of

Eve

nts

2n 4n


Flow Cytometry of Apoptotic Cells

PI - Fluorescence

# E

vent

s

Apoptotic cells

Normal G0/G1 cells


Analyzing the DNA Histogram

DNA Content0 50 100 150 200

Num

ber

020

040

060

0


Chromosome Analysis

Most human chromosomes can be separated by flow cytometry


Chromosome Analysis(Bivariate(Bivariate Flow Karyotyping - porcine)Flow Karyotyping - porcine)

chromosome 1chromosome 1

chromosome 2chromosome 2


EthidiumEthidium

PIPI

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488

Spectra of PI and EtBr


log Thiazole Orange.1 1000 100 10 1

Cou

nt0

150

112

75

37

RMI = 0RMI = 0

log Thiazole Orange.1 1000 100 10 1

Cou

nt0

150

112

75

37

RMI = 34RMI = 34

Reticulocyte Analysis


4 colors - simultaneous collection(can go to 17 colors)

Emission wavelength (nm)530 580 630 680 730 780

FITC PE PE-TR

PE-CY5

We separate different subsets by taking bands of light from thelight spectrum and analyzing the intensity of light in that band


10 1 10 2 10 3 10 4

CD56 -->

101

102

103

104

CD

4 --

>

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 --

>

CD3CD3 CD310 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

56 --

>

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

8 --

>

CD5610 1 10 2 10 3 10 4

CD56 -->

101

102

103

104

CD

8 --

>

CD56 CD810 1 10 2 10 3 10 4

CD8 -->

101

102

103

104

CD

4 --

>

FOUR COLOR PATTERN

CD

4

CD

8

CD

56 -

NK

CD

8

CD

4

CD

4

Data from Dr. Carleton Stewart

CD56 – NK CellsCD3 – T cellsCD4 – T cells – HelperCD8 – T cells - Cytotoxic

This is a subset of cellsIt is CD3+ CD56+

This is a subset of cellsIt is CD3+ CD4+


Multicolor Analysis

Roederer, et al



Cellular Response:

• Cell deathCell death• Cell ‘suicide’Cell ‘suicide’• Ignore damageIgnore damage• Damage repairDamage repair• Incorrect repairIncorrect repair


Functional AssaysFunctional Assays

•intracellular pH•intracellular calcium•intracellular glutathione•oxidative burst•phagocytosis


Oxidative BurstOxidative Burst

•generation of toxic oxygen speciesby phagocytic cells

•superoxide anion measuredwith hydroethidine

•hydrogen peroxide measured with2’,7’-dichlorofluorescin diacetate(DCFH-DA)


TIME (seconds)

0 2400 1800 1200 600

Log

DC

F.1

100

0 1

00

10

1

Scale

34511538

124

Neutrophil Oxidative Burst

PMA-StimulatedNeutrophils

UnstimulatedNeutrophils


FITC-Labeled Bacteria

Phagocytosis


Cellular Functions

• Cell Viability

• Phagocytosis

• Organelle Function– mitochondria, ER

– endosomes, Golgi

• Oxidative Reactions– Superoxide

– Hydrogen Peroxide

– Nitric Oxide

– Glutathione levels

• Ionic Flux Determinations–Calcium

–Intracellular pH

• Membrane Potential

• Membrane Polarization

• Lipid Peroxidation


Organelle Function

• Mitochondria Rhodamine 123• Endosomes Ceramides• Golgi BODIPY-Ceramide

• Endoplasmic Reticulum DiOC6(3) Carbocyanine


Fluorescent IndicatorsHow 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


HydroethidineHE EB

NCH2CH3

NH2H2N

H Br-NCH2CH3

NH2H2N

+

O2-

Phagocytic Vacuole

SODH2O2

NADPH

NADP

O2

NADPH Oxidase

OH-

O2-

DCFDCF

HE

OO22--

HH22OO22

DCFDCF

Example: Neutrophil Oxidative Burst


DCFH-DA DCFH DCF

COOHH

Cl

O

O-C-CH3

O

CH3-C-O

Cl

O

COOHH

Cl

OHHO

Cl

O

COOHH

Cl

OHO

Cl

O

Fluorescent

Hydrolysis

Oxidation

2’,7’-dichlorofluorescin

2’,7’-dichlorofluorescin diacetate

2’,7’-dichlorofluorescein

Cellular Esterases

H2O2

DCFH-DA

DCFH-DADCFH-DA

DCFHDCFH

DCF

H OH O 2 22 2Lymphocytes

Monocytes

Neutrophils

log FITC Fluorescence.1

1000

100 10

1

0

20

40

60

cou

nts

PMA-stimulated PMNControl

80


Phagosome

O2

O2-

H2O2

NADPH + H+

NADP+

HMP

NADPH

Oxidase

GSSG

GSH

GRGP

H2O2SOD

O2-

H+

H2O

Catalase

H2O + O2

PCB

SOD

PCB(Reduced GSH level)

Stimulant

PKC

PCB

(PMA)

Human Neutrophil

?

?+

O2-

OH.

Lipid Peroxidation

Phospolipase A2 activityLeukotrienes

H2O2


Hydroethidine Superoxide Production

15 minutes 45 minutes


Cell Sorting

• Physically separating cells based on some measurable characteristic

• Placing these cells into containers

488 nm laser

+-

Fluorescence Activated Cell SortingFluorescence Activated Cell Sorting

Charged Plates

Single cells sortedinto test tubes

FALS Sensor

Fluorescence detector

Purdue University Cytometry Laboratories


SMALL BEAD LARGE BEAD

Frequency Histogram

SMALL BEAD LARGE BEAD

Sample inSheath

Sheath in

Laser beam

Stream Charge

+2KV -2KV

Waste

SORT RIGHTSORT LEFT

SORT DECISIONS

Piezoelectriccrystal oscillator

Last attacheddroplet

LEFT RIGHT

Sensors

Sensor


Cell Sorting

Video of the droplet formation in a sort stream from a Cytomation instrument. Source: Purdue CDROM vol 4, 1998

Video2.mpg

+++ ---


Lab2 Groups of 8 students eachHansen Hall, Room B50 (Basement)Meet with Kathy Ragheb and Cheryl HoldmanOne on April 18, 12:30-4:30Other 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

Technology

Medical Student lecture