Introduction into

Flow Cytometry

September 27, 2014

6th EFIS-EJI South East European

Immunology School – SEEIS 2014

Timisoara, Romania

The Principle of Flow Cytometry

• Resuspendend single cells are moved through a

light source (laser).

• In this process cells emit characteristic light signals

depending on the cell type and the preparation of

cells that are detected by appropriate detectors.

Design of a Flow Cytometer

• Fluidic System

Moves and aligns cells in to the laser focus

• Optical System

Excitation Optics

Detection Optics

• Electronical System

Transfers optical signals into electronical signals, digitalizes the electronical signals for the analysis on a computer

The Fluidic System

• Fluidic Cart

– Sheath Fluid

– Waste

– Cleaning Solution

– Shut Down Solution

• Sample Injection Port

• Cuvette

Overview of the Fluidic System

Sheath Fluid Stream Sample Stream

Waste Aspirator

Flow Cell

Laser Fokus

Waste Tank

Reservoir

Fluid Filter

Bubble filter

“Plenum” (internal reservoir)

Sample Injektion

Tube (SIT)

Sample Tube

Hydrodynamic Focusing

• Sample stream is accelerated due

to the reduction of the cross

section inside the cuvette

resulting in the acceleration and

separation of cells (hydrodynamic

Focusing).

• BD FACSCanto™ II:

– Max. event rate of 10.000 events/sec

– Detection of particles ca. 0,2 – 50 µm.

Sheath Fluid

Sample

Laser beam

SIP (Sample Injection Port) BD FACSCanto II

Sample Injection Tube

(SIT)

Support

Arm

Holder for

Tube Rack

Sample

Tube

Cuvette

Waste Line

Intersection Point

SIT

Rinse Line

Sheath Line

Event Rate

Sample

Red laser

Violet laser Blue laser

Sheath fluid

LOW – low sample pressure

Low sample consumption

~12 µl/min

HIGH – high sample pressure

High sample consumption

~120 µl/min

The Optical System

• Excitation optic

– Laser

– Prism and lenses

• Detection optic

– Filter and mirrors

– Detectors

The Lasers

• Lasers emit light of a single wave length

Often used lasers in flow cytometers: – 488 nm (blue Laser)

– 633 nm (red laser)

– 405 nm (violet laser)

• Various available laser types:

Gas laser Diode laser Solid state laser

Transport & Focusing of Laser Beam

Why do we need a laser?

Excitation of fluorochromes in the cuvette that are bound on or inside a

cell/particle

Blue laser Red laser Cuvette

Glas fiber cable

Optical Filters & Dichroic Mirrors

• Separation of cell signals with filters and mirrors

450 550 650 500 550 600

LP 500 BP550/50

Longpass Bandpass

The Detectors

Amplification of the light signal and transfer of an

optical signal into an electronical signal

• Photodiode

• PMT (Photomultiplier Tube)

Detection Optic — Octagon Signals of the blue laser (4–2, 4–2–2 configurations)

564–606 nm

750–810 nm

483–493 nm

> 670 nm

515–545 nm e.g. FITC

SSC

e.g. PE

e.g. PE-Cy7

e.g. PerCP or

PerCP-Cy5.5

Detection Optic — Trigon Signals of the red and violet laser

Signals of the red laser 4–2, 4–2–2 Configurations

Signals of the violet laser 4–2–2 Configuration

425-475

485-535

650-670 750-810 e.g. APC-H7 e.g. APC

e.g. BD Horizon V450

e.g.

BD Horizon V500

Example: Fluorochrome combination on

Canto II (4–2–2 Configuration)

Laser Fluorochromes Alternatives

405 nm (violet) BV421 BD Horizon V450, PacificBlue

BV510 BD Horizon V500, AmCyan, DAPI

488 nm (blue) FITC GFP, Alexa Fluor® 488

PE PI

PerCP-Cy5.5 PerCP, PE-Cy5.5, 7-AAD, PI

PE-Cy7

633 nm (red) APC Alexa Fluor® 647

APC-H7 APC-Cy7

The Optical System

Violet Laser (405 nm)

Blue Laser (488 nm)

Red Laser (633 nm)

Glas fiber cable

Prisms Fokus lense

Steering disk

Flow cell

564–606 nm

750–810 nm

> 670 nm

SSC

515–545 nm

The Electronic System

• Digital data processing

– Tasks:

• Converts analog signals into digital signals

• Determins Area and Height of each impuls

• Calculates the Width of the signal

• Does the compensation

• Communication (data transfer) between workstation and flow cytometer

The Electronic System

Laser

Laser

Laser

Time V

olta

ge

Time

Vo

lta

ge

Time

Vo

lta

ge

Quantification of pulse

Time

Pulse Area

(Area)

Puls

e H

eig

ht

( H

eig

ht)

Pulse Width

(Width)

0

The Results

39,271 39,271

Event 2

Event 3

0 60 120 90

10 160 65

30 650 160

FCS-Data

185

PE-A

Exportable as FCS File 1900

2,688

3100

3,189

1900

90

185 3100

PE-A

FIT

C-A

1900

90

FIT

C-A

185 3100

39,271 0 60 120 90

10 160 65

30 650 160

PE-A

FIT

C-A

39,271 0 60 120 90

10 65

30 650 160

PE-A

FIT

C-A

39,271 0 60 120 90

10 65

30 650 160

PE-A

1900

90

FIT

C-A

FIT

C-A

39,271 0 60 90

10 65

30 650 160

PE-A PE-A

1900

90

FIT

C-A

FIT

C-A

39,271 0 60 90

65

30 160

PE-A

FIT

C-A

Time

PE-A

FIT

C-A

1900

90

FIT

C-A

PE-A

FIT

C-A

Event 1

PE-A

1900

90

FIT

C-A

PE-A

FIT

C-A

PE FITC SSC FSC

PE-A

1900

90

FIT

C-A

PE-A

FIT

C-A

Flow Cytometry Software

BD FACSCantoTM Software BD FACSDivaTM Software

- Automatic QC Setup

- Automatic analysis of predefined kits

for clinical applications (e.g. BD

Multitest)

- Acquisition and analysis of

individual applications

The Electronic System

Time

Time

Time

Time

Time

Data

Processing

PE

FITC

SS

C

FSC

AP

C

PerCP-Cy5.5

Time

What can you measure?

Algae

Blood

cells

DNA/RNA

Protozoa

Applications used in Flow Cytometry

Which Parameters can be Analyzed?

• Relative Size (Forward Scatter-FSC)

• Relative granularity or internal complexity (Side Scatter-

SSC)

• Relative fluorescence intensity

What can a flow cytometer perform?

• Simultaneous quantification of multiple optical parameters

• With a high flow rate.

The Scatter Parameters: FSC & SSC

• Forward Scatter (FSC) – defracted light

– Proportional to cell surface (cell size)

– Detected along the incident light in forward direction (1-10°)

• Side Scatter (SSC) - reflected and refracted light

– Related to cell granularity and complexity

– Detected in a 90° angle from the laser beam

Forward Scatter Light (488 nm): Cell Surface Area

Light Source (Laser Beam)

Side Scatter Light (488 nm): Cell Complexity/Granularity

Light Source (Laser Beam)

Example:

FSC & SSC of Lysed Whole Blood

Neutrophilic

Granulocytes

Lymphocytes

FSC

0 50 100 150 200 250

Monocytes

*1000

*1000

0

50

10

0

15

0

20

0

250

Debris S

SC

Basophilic

Granulocyte

Lymphocyte

Eosinophilic

Granulocyte Neutrophilic

Granulocyte

Monocyte Thrombocyte

Erythrocyte

What is Fluorescence ?

= 488 nm (blue)

Energy of laser

light (excitation)

Energy of emitted

fluorescence

519 nm (green) Antibody

Step 1: Fluorochrome absorbs the energy of laser light

Step 2: Fluorochrome releases absorbed energy as:

a) Vibration and heat

b) Emission of photons with longer wave length

(= lower energy)

Stoke´s- Shift: Wave length difference between absorbtion and emission

Fluorescein

Isothiocyanate

(FITC)

HO

N

O O

O

HO

C

S

Fluorescence Intensity

Relative Fluorescence Intensity

No.

of events

FITC

100 101 102 103 104

FIT

C

FITC

FIT

C

FITC

FIT

C

FITC

FIT

C

FITC

FIT

C

The emitted fluorescence is proportional to the

amount of bound fluorochromes.

Emission Spectra of common

Fluorochromes

blue

Laser

red

Laser

violet

Laser

The Compensation

Spectral Overlap

FITC PE PerCP-Cy5.5 PE-Cy7

650nm 700nm

PerCP-Cy5.5

670 LP

500nm 600nm

FITC

530/30

Rela

tive

In

ten

sity

Wave length (nm)

550nm

PE

585/42

PE-Cy7

780/60

750nm 800nm

The Principle of Compensation

Correction of spectral overlap

• Depends on:

- Used fluorochromes

- Amp gains of detectors

- But not on: used material

• Sample material:

- Unmarked cells as negative control

- Single marked samples to calculate

compensation

The Correct Compensation Settings

Correct Compensation:

The median of the FITC-positive

population is on the same level than

the median of the unstained

population.

Not compensated Correctly compensated

Missing compensation:

The single FITC-stained population

seems to be double-positive due to the

spectral overlap of FITC into the PE

channel.

Example: Spectral Overlap of FITC

650nm 700nm

PerCP-Cy5.5

670 LP

500nm 600nm

FITC

530/30

Re

lative

In

ten

sity

Wave length (nm)

550nm

PE

585/42

750nm 800nm

Before Compensation

650nm 700nm

PerCP-Cy5.5

670 LP

500nm 600nm

FITC

530/30

Rela

tive

In

ten

sity

Wave length (nm)

550nm

PE

585/42

Increase values

After Compensation

650nm 700nm

PerCP-Cy5.5

670 LP

500nm 600nm

FITC

530/30

Rela

tive

In

ten

sity

Wave length (nm)

550nm

PE

585/42

Increase value to move population down

Influence of the Voltage on Compensation

• FITC PMT Voltage constant (575 V)

• PE PMT Voltage changes from 500 to 600 Volt

Spectral overlap

of FITC into PE

channel varies

from

17% (500 V) to

71% (600 V)

8-Color Panel & the Resulting

Compensation Matrix

CD4 FITC

CD4 PE

CD28 PerCP-Cy5.5

CD45RA PE-Cy7

CD3 Pacific Blue

CD4 AmCyan

CD27 APC

CD8 APC-Cy7

FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8

FITC 100.0 23.5 2.1 0.7 0.0 0.0 0.0 3.0

PE 1.6 100.0 12.3 2.7 0.0 0.0 0.0 0.0

PerCP- Cy5.5

0.2 0.1 100.0 43.0 2.5 5.6 0.0 0.0

PE- Cy7

0.0 0.6 0.1 100.0 0.0 3.6 0.0 0.0

APC 0.1 0.0 0.3 0.2 100.0 2.7 0.0 0.0

APC- Cy7

0.0 0.0 0.1 3.9 19.9 100.0 0.0 0.1

Pacific Blue

0.1 0.0 0.0 0.1 0.0 0.0 100.0 18.1

Am Cyan

38.1 7.0 1.1 0.6 1.5 0.0 17.1 100.0

Single-stained controls:

DET

Auto

matic

Com

pensation

Automatic Compensation

Samples

• Unmarked cells/particles to set the PMTV

• Single stained samples of every used fluorochrome

• Tandem conjugates: single stained samples of each

lot# of the individual tandem conjugates

Why so many Colors?

Advantages

• More colors improve efficiency in:

– time consumption

– use of reagents

– preserving sample material

1 tube with a 6 color labeling can replace up to 15 tubes with a two color

labeling

2 colors BD Simultest™

• CD45/CD14

• IgG1/IgG2a

• CD3/CD4

• CD3/CD8

• CD3/CD19

• CD3/CD16+56

3 colors BD Tritest™

• CD3/CD4/CD45

• CD3/CD8/CD45

• CD3/CD19/CD45

• CD3/CD16+56/CD45

6

4 colors BD Multitest™

• CD3/CD4/CD8/CD45

• CD3/CD19/CD16+56/CD45

6 colors BD Multitest™

• CD3/CD4/CD8/CD19/ CD16+56

/CD45

2 1

Advantage - Multicolor Reagents

4

Why so many Colors?

Advantages

• More colors improve efficiency in:

– time consumption

– use of reagents

– preserving sample material

1 tube with a 6 color labeling can replace up to 15 tubes with a two color

labeling

• Exponental increase of information

More Colors, Higher Resolution =

More Details/Information

The higher the resolution, the more you know about your cells and

small differences are being discovered.

Why so many Colors?

Advantages

• More colors improve efficiency in:

– time consumption

– use of reagents

– preserving sample material

1 tube with a 6 color labeling can replace up to 15 tubes with a two color

labeling

• Exponental increase of information

• Identification of rare/new cells (< 0,05%)

A successful multicolor Application

A successful multicolor application depends on:

A. Sample preparation and quality of sample

B. Careful reagent selection

– New fluorochromes: more colors, more choices that improve

population discrimination in multicolor flow cytometry

C. Proper cytometer performance, setup, and data

collection

D. Proper data analysis

Panel Design Issues

• Some antigens are expressed at high levels, some at low levels, some over a range (continuum).

• Some fluorochromes are bright, others are dim.

• Emission spillover from bright markers can degrade the sensitivity of dim markers being measured in an adjacent detector.

• Some antigen markers are only available with certain fluorophores.

• Tandem dyes may be unstable.

• Complexity of the assay/panel increases the likelihood of errors.

Panel Design Principles I

• Know your cytometer.

– Know what lasers, filters, and how many PMT per laser

Instrument Configuration &

Fluorochrome Combinations

BD Accuri™ C6 BD FACSVerse™

BD FACSCanto™ II

BD FACSVerse™

BD FACSCanto™ II

BD LSR™ Fortessa

BD LSR Fortessa X-20

BD LSR™ Fortessa

BD LSR Fortessa X-20

2 laser

4 colors 2 laser

6 colors

3 laser

8 colors

4 laser

14 colors

5 laser

18 colors

355 nm BUV395

BUV737

BUV395

BUV737

405 nm

BV421 / V450

BV510 / V500 BV421 / V450

BV510 / V500

BV605

BV650

BV711

BV786

BV421 / V450

BV510 / V500

BV605

BV650

BV711

BV786

488 nm

BB515 / FITC / AF488

PE

PerCP / PerCP-Cy5.5

BB515 / FITC / AF488

PE

PerCP / PerCP-Cy5.5

PE-Cy7

BB515 / FITC / AF488

PE

PerCP / PerCP-Cy5.5

PE-Cy7

BB515 / FITC / AF488

PE

PE-CF594

PerCP / PerCP-Cy5.5

PE-Cy7

BB515 / FITC / AF488

PerCP / PerCP-Cy5.5

561 nm

PE

PE-CF594

PE-Cy5

PE-Cy7

640 nm APC / AF647

APC-Cy7 / APC-H7

APC / AF647

APC-Cy7 / APC-H7

APC / AF647

APC-Cy7 / APC-H7 APC / AF647

AF700

APC-H7 / APC-Cy7

APC / AF647

AF700

APC-H7 / APC-Cy7

Panel Design Principles I

• Know your cytometer.

– Know what lasers, filters, and how many PMT per laser

• Availability of certain fluorophores

• Match brighter fluorochromes with lower-expressed

antigens (and vice-versa).

– Determine the antigens and classify expression

– Determine a fluorochrome set and classify brightness

– Match antigens with fluorochromes

Antigen Density

• Level of antigen

expression on a cell

– Can vary due to cell

activation level and

functional differences

– Can be a range

(eg. smeared population)

– Depends on cell type

• Classify antigens as

high, intermediate and

low expressed

Density of Common Human Surface Antigens

www.bdbiosciences.com/documents/Human-Antigen-Density-Chart.pdf

Comparison of Fluorochrome Brightness

Laser very bright bright moderate dim

355 nm BD Horizon™ BUV737 BD Horizon™ BUV395

405 nm

BD Horizon™ BV421 BD Horizon™ BV605 BD Horizon™ BV510 BD Horizon™ V500

BD Horizon™ BV650 BD Horizon™ V450

BD Horizon™ BV711

BD Horizon™ BV786

488 nm

BD Horizon™ PE-CF594 BD Horizon™ BB515 FITC, AlexaFluor®488

PE

PE-Cy7 PerCP, PerCP-Cy5.5

561 nm

PE

PE-Cy7

BD Horizon™ PE-CF594

640 nm APC-R700 APC AlexaFluor® 647 APC-H7, APC-Cy7

Importance of Fluorochrome Choice

V450 BV510 BV421 BV605

CD4

CD

3

• Match reagents with Antigens classified as high, intermediate

and low expressed

• Bright dyes are important when looking at dim antigens

• Choice of fluorochrome helps understand more about the

biology of the experiment

Panel Design Principles II

• Avoid significant spectral overlap between markers

on the same population.

Impact of Spillover Background & Resolution

Population resolution is decreased by increased spread due to spillover

from other fluorochromes.

• Maximal resolution & sensitivity of a given subpopulation

• Minimimal fluorescence spillover into the detector that defines that

population

Spread of the

populations due

to spillover

Spread Resolution

Minimize Impact of Fluorescence

Spillover and Maximize Resolution

Understanding the impact of

fluorescence spillover on spread

is the key to good panel design

Use a fluor for CD3

with less spillover into

the CD56 detector

SD

MFI

Negative of Width

Brightness SI Resolution

CD

56

CD3

CD3

CD

56

How can we improve the

resolution of this double

positive population? Use a brighter

fluor for CD56

CD3

CD

56

1

1

2

2

Strategies to Minimize Spillover Issues

• Multiple antigens co-expressed on the same cell

=> spread them across as many lasers as possible

• Combining fluorochromes with high spillover

=> spread across different cell types

• Use optimal compensation controls

=> Treat the compensation controls the same way as cells (i.e. fixation)

=> Use bright and clear expressed markers

Panel Design Principles II

• Avoid significant spectral overlap between markers

on the same population.

• Prefer red-laser fluorochromes for markers on highly

autofluorescent cells.

• Always check for tandem-dye issues.

Tandem Dyes

• Chemical coupling of small organic dyes (e.g. Cy7)

with a fluorescent protein (e.g. PE)

Cyanine 7 (Cy7) photo-active center

of PE

Tandem Dyes Advantages, Challenges & Solutions

• Advantages

– Increasing number of fluorescent dyes

– Bright fluorochromes with a high stain index

• Challenges

– Degradation

– Lot to lot variances

– Antibody-specific variances

• Solutions

− Protect from light & avoid fixation

− Lot-specific compensation

− Label-specific compensation

• Automated on all digital flow

cytometers from BD Biosciences

Summary - Optimizing an Assay Panel Design Process Workflow

Spillovers of fluorochromes

Ranking of fluorochrome brightness / Match antigen specificities to fluorochrome brightness / resolution (high to low) Spillover

Ranking of antigen density (high to low)

Classify as Primary, Secondary, Tertiary Markers

Co-expression of markers on populations of interest Antigens

What Reagents are available? Fluorochrome

Define the Assay; Purpose / Goal

Cell type; biological function (e.g. cytokines); species

Definition of Sub-populations, CD Markers & “critical” populations Biology

Optical Configuration (laser, filter, PMT)

Gain (PMT voltage) optimization Instrument

Optimize Panel Design using the above information

Maximize resolution of critical populations Optimization