Panel Design: “Multi-Sizing” Your Multi-Color

William Godfrey, Ph.D.Manager, Reagent & Application Development

Beckman Coulter, Miami

CYTO 2010

For more information: www.CoulterFlow.com

“Multi-Sizing” into Your Multi-Color

• Fluor selection• Tandem dyes• Considerations for cocktail design• Krome Orange™ - A new violet fluor

How Many Colors Needed?

CD3-PC5

CD

4-PE

CD45-FITC

Side

Sca

tter

TetraCHROME™ CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 with normal blood

How Many Colors Needed?T-reg cells: CD4+

CD25+

FoxP3+

CD127lo/-

Up to 6-8 colors with addition of CD45 and markers for other leukocyte subpopulations

CD4

Side

Sca

tter

Forward Scatter

Side

Sca

tter

CD127C

D25

CD4

FoxP

3

Labor Efficiency:Fewer Steps

Correlated Data:Defined Populations

High Complexity Multi-Color Flow

Benefits

Single cell interrogation / multiple markers

Expanded utility (Research / Clinical)

Higher throughput / fewer tubes

Minimize sample volume

Challenges

More Colors = Greater Complexity

Complex antibody combinations

Selecting fluorochromes that work together

Requires optimization

Requires greater expertise

Requires validation

Prerequisites and Pitfalls for 8+ Colors

• Specific / avid monoclonal antibodies (clone selection)

• Bright fluorochromes (high extinction coefficients; high quantum yields) with range of Stoke’s shifts

• “Well-behaved” conjugates (stable binding; low spectral overlap, low background)

• Higher-plex flow cytometers with efficient light paths

• BD Canto™ cytometer (8-colors)• BC Cyan™ cytometer (9-colors)• BC GALLIOS™ cytometer (10-colors)

CD56-PEMY31 Clone

CD56-PENKH-1 Clone

Monocyte binding of Cyanine dye

CD3ECD

CD3CD3PC5PC5

CD3CD3PC7PC7

CD56-PE CD56-APC-AF750

Fluorochrome LandscapeIntrinsic Characteristics• Extinction Coefficient• Quantum Yield• Emission Spectral Overlap

Instrument Optics• Filter Selection• PMT Sensitivity• Laser Power

Comparative Intensities of CD8 Conjugates

FITCFITC PEPE ECDECD PC5PC5 PECy5.5PECy5.5 PC7PC7

APCAPC Alexa 700Alexa 700 APCAlexa 700APCAlexa 700 APC-H7APC-H7 APCAlexa 750APCAlexa 750

Pacific BluePacific Blue Pacific OrangePacific Orange

Excitation

Blu

eR

edVi

olet

+Qdot Nanocrystals+Qdot Nanocrystals

( )

( )

Brighter Fluors

Dye Options: 10-Colors

405nm

Pacific BluePacific Blue Pacific OrangePacific Orange

488nm

635nm

FITCFITC PEPE ECDECD PECy7PECy7PECy5.5PECy5.5

APC or AF647APC or AF647 APC-AF700APC-AF700 APCCy7 or APC-AF750APCCy7 or APC-AF750

400 500 600 700 800

Dye Options: Conventional Fluors

405nm

Pacific BluePacific Blue Pacific OrangePacific Orange

488nm

635nm

FITCFITC PEPE

APC or AF647APC or AF647

400 500 600 700 800

R-PE(565/576), Є = 2,000 K

APC: (650/662), Є = 700 K

Stokes Shift

FITC (495/518) Є = 78 K

Spectra of Common Fluorochromes

• Critical Fluorescence Properties• Extinction Coefficient• Quantum Efficiency• Stoke’s Shift

• Consider • Available excitations• Emission filters

Stokes Shift

Stokes Shift

488

488 635

Conjugation ChemistryBrightness: Optimization of F/P molar ratio

• Minimize impact on antibody binding affinity• Maximize fluorescence at saturation dosing

Performance: Influenced by multiple factors• Site of covalent linkage to the antibody

- Fc – minimal impact on binding affinity- F(ab) Region – competition with antigen binding

• Molecular weight (size) of dye molecule • Hyperconjugation

- Fluorescence quenching due to close coupling proximity- Non-specific binding- Dye/Cell aggregation

F/P 1.8

F/P 6.8F/P 5.2F/P 3.5

F/P 8.4

Flow M

FI

0

50

100

150

200

250

0 2 4 6 8 10

CD3 F/P Molar Ratio

Emis

sion

Inte

nsity

FluorimeterFlow Cytometry

25

20

15

5

0

10

CD3-Alexa Fluor 488

F/P 3.4

F/P 13.1F/P 9.1F/P 6.4

F/P 16.3

Flow M

FI

0

100

200

300

400

500

0.00 5.00 10.00 15.00 20.00

CD4 F/P Molar Ratio

Emis

sion

Inte

nsity

FluorimeterFlow Cytometry

25

20

15

5

0

10

CD4-Alexa Fluor 488

Performance Impact: Organic Dye Ratio

Dye Options: Tandem Dyes

405nm

488nm

635nm

ECDECD PECy7PECy7PECy5.5PECy5.5

APC-AF700APC-AF700 APCCy7 or APC-AF750APCCy7 or APC-AF750

400 500 600 700 800

• Definition• Excitation is transferred from donor to acceptor without emission of

a photon. • Donor / acceptor molecules must be in close proximity (10–100 Å). • Absorption spectrum of the acceptor must overlap emission

spectrum of the donor • Donor and acceptor transition dipole orientations must be

approximately parallel.

• Advantages• Expands fluorochrome choices from single laser source• Minimize cost and complexity of instrumentation – up to 8 colors

using 2 lasers• Enhanced fluorescence intensity compared to organic dyes with

equivalent emission

Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002

Fluorescence Resonance Energy Transfer

Wavelength (nm)

Fluo

resc

ence

Inte

nsity 488 nm

PE Texas Red

• Definition• Excitation is transferred from donor to acceptor without emission of

a photon. • Donor / acceptor molecules must be in close proximity (10–100 Å). • Absorption spectrum of the acceptor must overlap emission

spectrum of the donor • Donor and acceptor transition dipole orientations must be

approximately parallel.

• Advantages• Expands fluorochrome choices from single laser source• Minimize cost and complexity of instrumentation – up to 8 colors

using 2 lasers• Enhanced fluorescence intensity compared to organic dyes with

equivalent emission

Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002

Fluorescence Resonance Energy Transfer

• Limitations• Lot-to-lot variation

- Fluorescence sensitivity- Energy transfer efficiency- Non-specific binding to myeloid

populations • Stability: Photostability & Chemical interactions

0

50

100

150

200

250

300

350

400

550 650 750 850

PE-Cy7 ConjugatesMultiple Vendors,

Multiple Conjugates

Patented Tandem Dye ProcessNative State

PhycobiliproteinUnfold Protein

Couple Acceptor Dye

Refold to Native State

Conjugation process delivers optimum fluorescence intensity

HIC Purification

Step

PC7 Pre-HICpurification

PC7 Post-HICpurification

HIC discardedfraction

0

100

200

300

400

500

600

2 3 4 5 6 7

Cy7/PE (A/D) Ratio

Cy7

Flu

ores

cenc

e In

tens

ity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cro

ss-O

ver R

atio

Dye Coupling Step Optimal

Ratio

0

50

100

150

200

250

550 600 650 700 750 800

Lot # 3021-101Lot # 1828-47Lot # 1828-95

Fluo

resc

ence

Inte

nsity

Emission Wavelength (nm)

Finished Tandem Dye

Three Lot Comparison of PC5Process controls variability

Patented Tandem Dye Process

MUST treat different vendor tandems as different fluorochromesfor compensation set-up!

BCI Vendor 2BCI

Vendor 2

Under

Over

Impact on Compensation

1.5% Comp

PECy5.5

0

100

200

300

400

500

600

700

500 600 700 800Wavelength (nm)

Emis

sion

Inte

nsity

488nm excitation633nm excitationAPC

47.5% Comp

PECy5

0

100

200

300

400

500

600

700

500 600 700 800Wavelength (nm)

Emis

sion

Inte

nsity

488nm excitation633nm excitationAPC

Tandem Dye Selection: Dual Laser

Minimized Spectral Overlap = Better Resolution

Time (hours) Exposed to Light (80 Lumens)

% C

hang

e FL

4/FL

5 M

FI R

atio

0

40

80

120

160

200

240

280

0 6 12 18 24Time (hours) Exposed to Light (80 Lumens)

% C

hang

e FL

4/FL

5 M

FI R

atio

0

40

80

120

160

200

240

280

0 6 12 18 24

0 Hours 2 Hours 6 Hours

FL4-FL5 Compensation = 10.5%CD3-APC-AF 750A

PC

FL4-FL5 Compensation = 10.5%CD3-APC-Cy7

APC-Alexa Fluor® 750 Photostability

Enhanced on photo-stability of APC-Alexa Fluor 750 conjugate regardless of paraforaldehyde

Performance Impact: Antibody & Dye

Non-Specific Binding

Conjugate/Dye Aggregation

CD14-PECy5 CD3-PECy5

Binding due to Fc receptor

Binding due to Cy dye binding

CD15-FITC (IgM)

0.25 µg 0.125 µg 0.063 µg 0.031 µg

Monocyte binding of

Cyanine dye

Pre-Formulation Post-Formulation

Company “B”

Company “C”

• Low background fluorescence on negative populations

• Optimal signal to noise • Low affinity binding of cyanine dyes

to monocyte populations eliminated

Proprietary Chemistry / Enhanced Specificity

CD3PE-TxRed

CD3PE-Cy5

CD3PE-Cy7

CD3PE-Cy7

CD3PE-Cy5

CD3PC7

CD3PC5

CD3ECD

Beckman Coulter

Fluor Choice?

• Detection of Surface Antigens• Large selection of conjugates• Limited by detection sensitivity and proximity of co-

expressed antigens• Tandem dyes provide ↑ sensitivity over organic dyes• FITC, Alexa Fluor dyes, violet-excited dyes

- Constitutively expressed antigens- Subset gating

• Detection of Intracellular Antigens• Cytoplasmic antigens

- Phycobiliproteins and organic dyes may be better- Alexa Fluor 488 better than FITC – lower background

• Nuclear antigens- Phycobiliproteins or tandem dyes hindered due to

conjugate size?- Close proximity can lead to FRET between dyes

Instrument Contributions

• Channels available• Sensitivity in channels

Comparative Flurochrome Sensitivity between Platforms

0

100

200

300

400

500

600

700

800

CRS FC500

Sig

nal t

o No

ise

FITCPEECDPECY5PECY5.5PECY7APCAlexa Fluor 700APCA700APCA750Pacific BluePacific Orange

GALLIOS FC500

GALLIOS™ cytometer• 3 lasers• 10 colors

Optimizing the Combination

• Determine fluorochrome/conjugate strategy• Organic dyes to maximize spectral separation for gating reagents• PE & APC used for antigens with continuum of expression• Tandems dyes for mid-density → bright antigens

• Perform titration curves for each conjugate• Determine Signal/Noise Ratio• Choose optimal dose: Saturation, Highest S/N

• Prepare combination, verify performance• Always use controls – approach can vary

- Negative Control- Internal negative population- FMO- Isotype controls

- Positive controls- Each antibody as single color - Known positive control material

• Evaluate performance for major interactions

High density Ag/High density Ag/Dim dyeDim dye

CDxCDx

Low density Ag/Low density Ag/Bright dyeBright dye

CDzCDz

What Can Go Wrong?

• Potential conjugate interactions• Non-specific binding

- Aggregate formation between conjugates- Cyanine & Alexa Fluor dye binding to myeloid populations

• Steric Hindrance- Ligand – receptor binding blocked

due to physical interference

• Fluorescence Quenching- Over conjugation of antibody- Concentration and proximity on the cell

• FRET PotentialCDz

CDx

CD

x

Charge

CDz

CDx

CD

x

Size

Hyper-conjugation Concentration &

Proximity

Dose Optimization: Multi-Step ProcessSingle color titrations:• Optimal S/N• Saturation binding when possible

0.0

5.0

10.0

15.0

20.0

0 0.2 0.4 0.6 0.8 1

Dose (ug/test)

Fluo

resc

ence

nce

S/NPositive MFINegative MFI

0.0

5.0

10.0

15.0

20.0

0 0.2 0.4 0.6 0.8 1

Dose (ug/test)

Fluo

resc

ence

nce

S/NPositive MFINegative MFI

Combination Matrix to finalize dosing• Target optimal S/N dose for each component• Evaluate for potential interactions• Evaluate multiple doses:

Simple matrix or DOE ½, ½½, 1 ½, 2 ½ X1,½1,11,21x

2, ½2,12,22XCDyy

½ x1x2X

CDxx

• Spectral Overlap/Compensation• Loss of low end resolution• Display artifacts

• If bright signal overlaps into PMT containing dimmer signal

• Increased “noise”• Spread of the negative population• Difficulty in accurate determination of

low level positivity

PE ECD PC7

CD45-PC7 CD7-PE CD19-PE

S/N=38 S/N=27

CD45-PC7 CD7-PE CD19-PE

S/N=38 S/N=27

CD45-ECD CD7-PE CD19-PE

S/N=19S/N=22

CD45-ECD CD7-PE CD19-PE

S/N=19S/N=22

CD45-ECD vs CD45-PC7 Overlap into PE

Impact on co-expressed antigens

Spectral Overlap

• APC conjugates of CD3, CD8, and CD45 versus PE-labeled tetramer• FRET from PE to APC results in FL3 signal (PerCP channel)• CD3 & CD8 close to TCR; CD45 antigen spatially separated from TCR

Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002

A2/

CM

V -P

E

+ CD4 PerCP

No CD4 PerCP (PE-APC FRET)

CD3 APC Gated CD8 APC Gated CD45 APC Gated

Effect of Antigen Proximity

Interferants: Washing

Kappa/Lambda Resolution• Requires high sensitivity• Dependant on sample preparation methodology• Pre-wash required to remove plasma immunoglobulins

1x 2x 3x

Beckman Coulter Solastra™ Panels*

Aligned with Bethesda Recommendations* Not available for sale in US

Aligned with Bethesda Recommendations* Not available for sale in US

FITC PE ECD PC5.5 PC7

Kappa Lambda CD19 CD5 CD45

CD20 CD10 CD19 CD38 CD45

CD2 CD56 CD7 CD5 CD45

CD8 CD4 ⎯ CD3 CD45

CD15 CD11b CD16 CD14 CD45

HLADR CD56 CD34 CD117 CD45

CD7 CD13 CD34 CD33 CD45

B-cell Kit

T-cell Kit

Myeloid Kit

Peripheral Blood: Solastra B-cell KitsB-CLL #1: CD45++/CD19+/CD5+/-/ CD20++/Kappabright+

Tube 2 Tube 2

Tube 1 Tube 1

New Violet-Excitable Dye

• GALLIOS™ Configuration: 3 laser 10 color instrument• 405nm laser – 2 colors• 488nm laser – 5 colors• 635nm laser – 3 colors

• Krome Orange™ dyeSecond violet-excitable fluor to pair with Pacific Blue™ dye

See Poster #P346

Krome Orange Spectrum

0%

20%

40%

60%

80%

100%

350 400 450 500 550 600 650 700 750

Wavelength (nm)

Rel

ativ

e In

tens

ity

KOV500Pac OrAmCyan

550/40405 nm

Krome Orange Conjugation

0

5

10

15

20

25

30

0.00 0.50 1.00 1.50 2.00 2.50

µg Conjugate per Test

Med

ian

Fluo

resc

ence

F:P 17.3F:P 15.9F:P 14.0F:P 10.8

F:P 6.1

CD45-Krome Orange Titration Curve

0

5

10

15

20

25

30

0.00 0.50 1.00 1.50 2.00 2.50

µg Conjugate per Test

Med

ian

Fluo

resc

ence

F:P 17.3F:P 15.9F:P 14.0F:P 10.8

F:P 6.1

CD45-Krome Orange Titration Curve

CD14 (RMO52)-Krome Orange

SI = 101.3

Lymphocytes

Monocytes

CD16 (3G8)-Krome Orange

SI = 18.6Lymphocytes

CD20 (B9E9)-Krome Orange

SI = 51.2Lymphocytes

SI = 9.2Lymphocytes

CD19 (J4.119)-Krome Orange

Krome Orange vs Other Violet Fluors

AmCyan(525/50)

Pacific Orange Dye(575/26)

Krome Orange Dye(550/40)(standard FL10 filter on Gallioscytometer)

V500 Dye(525/50)

CD3

SI = 20.6 SI = 20.6

SI = 13.5 SI = 13.5

SI = 36.6SI = 36.6

CD8 CD45

29.65

21.20

16.03

SI = 12.0 SI = 12.0

SI = 48.5 SI = 48.5

SI = 33.2 SI = 33.2

SI = 26.1 SI = 26.1

SI = 43.5 SI = 43.5

SI = 81.7 SI = 81.7

SI = 34.7 SI = 34.7

5.19

8.67

6.75

4.19

2.92

CD4

7.16

23.20

9.92

13.32

3.54

UCHT1

UCHT1

SK71 SK3

S3.5

13B8.2 B9.11

3B5

SK1

RPA-T8

2D1

HI30

J.331

Krome Orange vs Other Violet Fluors

Relative compensation values

Krome Orange: 4-Color Stain

CD8-Fluorescein

CD

4-Pa

cific

Ora

nge

CD19-PE

CD

4-P

acifi

c O

rang

e

CD8-Fluorescein

CD

19-P

E

CD8-Fluorescein

CD

4-Kr

ome

Ora

nge

CD19-PE

CD

4-Kr

ome

Ora

nge

CD8-Fluorescein

CD

19-P

E

CD8-Fluorescein

CD

4-Pa

cific

Ora

nge

CD19-PE

CD

4-P

acifi

c O

rang

e

CD8-Fluorescein

CD

19-P

E

CD8-Fluorescein

CD

4-Kr

ome

Ora

nge

CD19-PE

CD

4-Kr

ome

Ora

nge

CD8-Fluorescein

CD

19-P

E

0.0%1.5%

0.3%1.0%

19.4%1.0%

0.0%1.5%

0.0%1.0%

19.4%1.0%

FL9-%FL10 = 0.5%FL10-%FL9 = 5.9%

FL9-%FL10 = 0.0%FL10-%FL9 = 5.8%

CD8-Pacific Blue

CD

4-Kr

ome

Ora

nge

Gated on CD3+

CD3+

Side

Sca

tter

CD3-APC

Side

Sca

tter

CD8-Pacific Blue

CD

4-Pa

cific

Ora

nge

Gated on CD3+

CD3+

CD3-APC

CD45-Krome Orange

Lymphs

Monos

Grans

CD14-PC5

CD

45-P

acifi

c O

rang

e

CD14-PC5

CD

45-K

rom

e O

rang

e

Monos

CD45-Pacific Orange

Lymphs

Monos

Grans Monos

FL9 - %FL10 FL10 - %FL9Krome Orange™ 1.5 9.1Pacific Orange™ 0.0 9.3

Side

Sca

tter

Side

Sca

tter

Krome Orange: 10-Color Stains

Krome Orange: CD45/Side Scatter

Data courtesy of F. Preijers, Radboud University Nijmegen Medical Center, The Netherlands

CD

45-V

500

CD

45-E

CD

CD

45-K

rom

e O

rang

eC

D45

-Pac

ific

Ora

nge

• Multi-parametric flow analysis provides a powerful tool• Dissection of complex cell populations• Identification of underlying mechanisms and alterations in

disease states• Increased efficiency in laboratory testing

• Optimal design is critical for scientifically valid results• Match fluorochrome choices to the platform capability• Optimize sensitivity by pairing dye intensity with antigen

density• ↑ colors = ↑ complexity• Violet-excited fluors can easily add 2 parameters

• Validate, validate, and validate your application specificity prior to initiating studies

Summary

Acknowledgements www.CoulterFlow.comMiami: Reagent Development

Ravinder GuptaSireesha KaanumalleDavid BloodgoodMeryl ForemanJeffrey Cobb

Marseille: Reagent DevelopmentLaura Nieto GligorovskyFranck GailleEmmanuel GautherotFelix Montero

Detroit: Organic ChemistryHashem Akhavan-TaftiRobert EickholtMark SandisonRhonda Federspiel

CollaborationFrank Preijers

(Nijmegen Medical Center)

Research Tools

Practical Flow CytometryHoward Shapiro

History of Flow Cytometry

Spectrios

Experiment Designer

On-Line Knowledge Base

Bone Yard

Lymphocytic Lymphocytic differentiationdifferentiation

MFI: 14.7 MFI: 44.7

CD8-APC-AF700CD8-AF700

Optimal excitation of Alexa Fluor 700 by APC energy transfer= ½ to ¾ log brighter fluorescence

Tandem Dye Selection: Alexa Fluor® 700

Wavelength (nm)

Fluo

resc

ence

Inte

nsity

635 nm

APC Alexa Fluor 700

CD14 (RMO52)-Krome Orange

SI = 101.3

Lymphocytes

Monocytes

CD20 (B9E9)-Krome Orange

SI = 51.2Lymphocytes

CD16 (3G8)-Krome Orange

SI = 18.6Lymphocytes

SI = 9.2Lymphocytes

CD19 (J4.119)-Krome Orange