New Insights for Multicolor Panel Design talk panel desig… · The BD Horizon™Tour: New insights for multicolor panel design /9 Stain Index: The Standard Metric of Resolution SD

bdbiosciences.com /1 The BD Horizon™ Tour

Ana Teixeira, PhD

Research Applications Scientist

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

APC-Cy7: US Patent 5,714,386

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New Insights for Multicolor

Panel Design

The BD Horizon™ Tour: New insights for multicolor panel design /2

Elements of Multicolor

Flow Cytometry

Considerations in designing panels:

Fluorochrome Characteristics and Availability

Biology: Cell Type, Antigen Characteristics

Instrument Setup and QC;

Spillover


• Choice of fluorochrome helps understand more about

the biology of the experiment.

• Bright dyes are important when looking at dim antigens.

Fluorochromes Reveal Biology

CD

3BV421

V450 FITCPerCP-Cy5.5

PE PE-CF594 Alexa Fluor 647

Alexa Fluor 700

CD197 (CCR7)


• Eight dyes excited by the violet laser

– Base Polymers: BV421, BV480 and BV510

– Tandems (of BV421): BV605, BV650, BV711, BV750 and BV786

• Bright dyes

• Limited cross laser excitation

• Compatible with surface and intracellular targets

BD Horizon Brilliant™ Violet


BD Horizon Brilliant™ Ultraviolet (BUV)

• Six fluorochromes excited by the 355-nm UV laser

• Base polymer: BUV395

• Tandems: BUV496, BUV563, BUV661, BUV737, BUV805

• Designed for reduced spillover into violet channels

• Brings phenotyping to the UV laser line


• Brighter dyes excited by the blue (488nm) laser

– BB515 is an alternative for FITC, Alexa488

• brighter, less spill into PE

– BB700 is an alternative for PerCP-Cy5.5

• brighter, more compatible with Perm Buffer III

BD Horizon Brilliant™ Blue (BB) Dyes

BB515FITC

PerCP-Cy5.5BB700


Considerations for Intracellular (IC) Panels

Cells were stained with CD4 conjugates of different fluorochromes and then fixed

Untreated

Cytofix/

Cytoperm

Perm Buffer III

Transcription

Factor Buffer

PerCP-Cy5.5 BB515 BV421

The fixation and permeabilization process needed for intracellular

staining can decrease dye fluorescence intensity.


CD4 Resolution Comparison

21161

703

284

125

315

118

333

PE

BV421

BV650

FITC

BV510

BV711

BV605

BV786

Stain Index


Stain Index: The Standard Metric of Resolution

SD

MFI

Negative of Width

Brightness Index Stain

Brightness

Width of negative

• The width of the negative is a function of

– Instrument performance

– Qr, Br, and SDen (single color)

– Spillover (multi-color panels)

• The brightness is a function of the

• Reagent

• Antigen density, fluorochrome used.

• Instrument

• PMT gain setting, laser power


Fluorochrome Resolution Ranking

• Rankings were determined by comparing the resolution of LWB cells stained on several

clones run on a variety of flow cytometers.

• Many factors can influence the relative fluorochrome/reagent performance on a given

instrument, including laser power, PMT voltage, optical filters, antibody clone,

biological sample, and staining methodology.



Flow Cytometry





Spillover

https://www.google.com/url?q=https://archive.4plebs.org/s4s/page/1291/&sa=U&ei=7AYlU8m1JozhoAS60YCQAw&ved=0CI0BEPUBMDA&usg=AFQjCNFjAPSdJnjpXK0QiTLD9SyIIT_-MA



Antigen Tregs

CD4 Naïve T

cells

CD4 Memory T

cells

CD8 Naïve T

cells

CD8 Memory T

cells

CD3 +++ +++ +++ +++ +++

CD4 +++ +++ +++

CD8 +++ +++

CD45RA +++ +++

CD127 ++ +++ +++

CD25 +++

CCR7 ++ ++

Defining the Biology

CD3+

CD4+ CD8+

CD25+

CD127+CD45RA+

CCR7+CD45RA-

CCR7-

CD45RA+

CCR7+CD45RA-

CCR7-

1. Define a population tree based on the goals of the assay.

2. Identify the critical populations.

3. Determine which antigens are co-expressed and at what levels.


• Primary: Well characterized,

easily classified as positive or negative,

typically define broad subsets or lineages

– Examples: CD3, CD4, CD19

Classification of Antigens

Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clin Lab Med. 2007;27:469-485.

Leucocyte antigens can be categorized based upon their

patterns of expression

• Secondary: Well characterized,typically expressed at a higher density, often over a continuum

– Examples: CD27, CD28, CD45RA, CD45RO

CD25

CD4

CD45RA

• Tertiary: Expressed at low levels, variable upon activationunknown, critical

– Examples: CD25, STAT5, FoxP3


Grouping Antigen Density: T Cells

BD LSRFortessa™

BD FACSVerse™

Sample

400

1,000

10,000

4,000

40,000

100,000

No

. P

E M

ole

cu

les

• When evaluating antigen density, it can be useful to group

antigens based on their relative levels of expression.

Average number of molecules on T cells

High

>15,000

Medium

1,000–15,000

Low<1000

100


Antigen/Fluorochrome Combinations

High Low Medium



Flow Cytometry





Spillover






Resolution vs Background

Negative population has

high background;

populations not resolved

“Negative” Dim Bright


low background;

populations well resolved


low background but high rSD (spread);

populations not resolved

Resolution: The degree to which a flow cytometer can distinguish

dimly stained cells from unstained cells.

This can be challenging in a polychromatic scenario.

The ability to resolve populations is a function of both background

and spread of the negative population.


Factors Impacting Resolution


Factors Impacting Resolution


Negative Positive

MFI rSD MFI rSD

No Comp 12 3098

Comp

Fluorescence Spillover Introduces

Background / Spread Into Other Detectors

Fluorochromes spillover into other

detectors; e.g. PerCP-Cy™ 5.5

spills into the PE-Cy7 detector.

This fluorescence spillover

contributes to

• increased background (MFI)

• spread (rSD)

PE-

Cy7

PerCP-Cy5.5

A sample is correctly compensated when, in the

spillover detector (PE-Cy7), the MFI of the positive

population is equivalent to that of the negative

population.

However, the spread (rSD) introduced by the spillover

is not removed by the compensation and reduces the

resolution (SI) of any double positive cells.

rSD

rSD

This “background” is subtracted in

the process called Compensation.

29 291

4 329 289


Spillover: Sources

Similar

Emission

Spectra

(Cross-laser)

BUV737 / BV711

Residual

Base

Fluorescence

BV786 / BV421

Adjacent

Detectors

FITC / PE



Background and Spread

Amount of

SpilloverThe Amount of

the Spread

SOV =

0.86%

FITC

PE

SOV =

9.15%

PE-Cy5

PE

SOV =

26.40%

PE-CF594

PE




Amount of


the Spread

PE-CF594

PE

MFI =

Reagent Brightness

420

35,000

7,000

1,500




Amount of


the Spread

PE-CF594

PE

Antigen

Density( Fluorochrome

Brightness )

Population resolution for a given fluorescence

parameter (for example, PE-CF594) is

decreased by increased spread due to spillover

from other fluorochromes (for example, PE).

• To maximize the resolution of a given double-positive subpopulation:

– Minimize fluorescence spillover into the detector that defines that population


Which Controls are needed?

Controls:

• Panel Design

• CS&T beads (PMT voltage)

• Unstained cells and single stained controls

• FMO (all)

• Daily Setup Controls

• CS&T

• Compensation Controls

• Gating controls (eg, FMO)

• Biological controls (eg, unstimulated samples, healthy

donors)

This will allow you to:

• Obtain consistent setup and compensation

• Gate challenging markers reproducibly

• Make appropriate biological comparisons and conclusions


Four Simple Principles for

Single-Color Compensation Controls

FITC = 17.72

Alexa Fluor® 488 = 14.70

Spillover Values into PEPE Detector

FITC CD4

Compensated using:

FITC SOV Alexa Fluor® 488 SOV

PE

1. The fluorescence spectrum (% spillover) of the compensation control reagent should be identical to the reagent used in the experiment.

a. Critical for tandem reagents when different reagents can have different spillovers.

i. Use tandem-specific compensation controls.

b. Even similar fluorochromes such as FITC and Alexa Fluor® 488 or

APC and Alexa Fluor® 647 have different spillover values.




2. The negative and positive populations should have equivalent

autofluorescence.

a. When using stained cells as compensation controls:

i. Compare CD3+ lymphocytes to CD3- lymphocytes.

ii. Don’t use CD3+ lymphocytes and CD3- monocytes or granulocytes.

iii. Don’t use unstained beads and stained cells to compensate a single

fluorochrome.

= (903-78) / (3,135-95) = 27.1%

Spillover Calculated Using Negative:

Cells

= (903-228) / (3,135-107) = 22.3%Beads

4.8%Difference =

MFI

FITC PE

Positive Cells 3,135 903

Negative Cells 95 78

Negative Bead 107 228




3. The positive population should be as bright as possible.– Ensures values in the spillover channel are above background.

– Provides greater accuracy for calculating low spillovers.• For example: FITC PE-Cy7 (1–2%)

4. Take enough events to get statistically accurate numbers.– Provides greater accuracy for calculating low (but real) spillovers.

– Five thousand events give a 1.7% SD.• BD FACSuite software takes 10,000 events (5,000 positive) for beads and

30,000 for cells.

Primary Detector MFI

Spill

over

Dete

cto

r M

FI

105104103101

CORRECT SOV

102


Primary

Fluor FITC PE

PerCP-

Cy5.5 PE-Cy7 APC

APC-

H7

FITC 68

PE 340

PerCP-Cy5.5 131

PE-Cy7 522

APC 486

APC-H7 116

Resolution Impact MatrixCo-expressed Fluor

Using the Resolution Impact Matrix

• The Resolution Impact Matrix provides a quick visual tool to help assess potential problems with spread when looking at using two fluorochromes for co-expressed markers on a population of cells.

• Adding a PE reagent to a co-

expressed marker will have significant

spread into and a major negative

impact on the resolution of the double-

positive cells in the PerCP-Cy5.5

detector.

The table shows that on a

BD FACSVerse:

• No fluorochromes have a significant

negative impact on the resolution of

FITC+ cells.

• FITC has minimal impact on any other

fluorochrome.SI (Primary)

<20% 20-40%

% loss of SP-SI40-60% 60-80% >80%


How To Minimize the Impact of Fluorescence

Spillover to Maximize Resolution

SD

MFI

Negative ofWidth

Brightness SI Resolution

Understanding the impact of

fluorescence spillover on

spread is the key to good

panel design.

How can we improve the

resolution of this double

positive population?

CD3

CD

56


How To Minimize the Impact of Fluorescence

Spillover to Maximize Resolution

Use a

brighter

fluor for

CD56

Titrate

the CD3

to reduce

the

spread.

Use a fluor

for CD3 with less

spillover into the

CD56 detector.

SD

MFI

Negative ofWidth

Brightness SI Resolution

Understanding the impact of

fluorescence spillover on

spread is the key to good

panel design.

CD3CD3

CD

56

CD

56

CD3

CD

56

CD3

CD

56



Flow Cytometry





Spillover








Building a 6-Color Panel to Identify

Regulatory T-Cell (Treg) Subsets

Experimental Goal:

Identify Treg subsets

Markers Used: CD3, CD4, CD8, CD25,

CD127, CD45RA

Assign antigen expression levels:

CD3+

CD4+CD8+

CD25+

CD127+

CD45RA+ CD45RA-

Gating Strategy

TregsNaïve Tregs

Memory Tregs

CD3

CD4

CD8 - - -

CD45RA - -

CD127

CD25


1st Example- No Rules• Use of available reagents

– Minimal attention to fluorochrome brightness or antigen density

2nd Example- Some Rules• Refined panel with focus on

fluorochrome assignment based on expression of antigens.

3rd Example- Best Practices

• Use of best practices to further optimize the panel to maximize the resolution of Tregs.– Antigen density & co-expression

– Fluorochrome brightness

– Spread due to spillover

Approaches to Panel Design

6-color panel for a 2-laser system

Blue

(488 nm)

FITC/Alexa Fluor®

488/Brilliant™ Blue 515

PE

PerCP-Cy™5.5/PerCP

PE-Cy™7

Red

(640 nm)

APC/Alexa Fluor® 647

APC-H7/APC-Cy7


6 Color T-Cell Panel: 1st Example

6 color panel using

available reagents

FITC CD127

PE CD4

PerCP-Cy5.5 CD3

PE-Cy7 CD8

APC CD45RA

APC-Cy7 CD25

CD25 APC-Cy7C

D1

27

FIT

C

FSC

SSC

SSC

CD4 PE

CD

8 P

E-C

y7

CD25 APC-Cy7

CD3 PerCP-Cy5.5C

D4

5R

A A

PC

Data acquired on a BD FACSCanto™ II flow cytometer.


• Antigens expressed at low density require brighter fluorochromes.

• A highly expressed antigen will usually be detected and resolved

from the negative control with almost any fluorochrome.

Fluorochrome

Matching Antigen Density and

Fluorochrome Brightness

PE

APC

PE-Cy7

FITC

PerCP-Cy5.5

APC-Cy7/APC-H7

CD25

CD127

CD45RA

CD4

CD8

CD3

Specificity

ANTIGEN ASSIGNMENT

Laser


Comparison – 1st vs. 2nd Example

CD4 PE

CD

8 P

E-C

y7

CD25 APC-Cy7

CD

45

RA

AP

C

CD25 APC-Cy7

CD

12

7 F

ITC

CD25 PE

CD

12

7 P

E-C

y7

CD

45

RA

AP

C

CD

8 A

PC

-Cy7

CD4 PerCP-Cy5.5 CD25 PE

Using dimmer fluorochrome for CD4

did not impact resolution of CD4+ cells.

Using brighter fluorochromes for low density CD25

and CD127 markers improved their resolution.

1st

2nd


It remains challenging to gate on Tregs.

Why?

– Because spillover and co-expression

were not taken into consideration.

Troubleshooting Panel Design


Resolution Impact MatrixPrimary

Fluor FITC PEPerCP-Cy5.5 PE-Cy7 APC APC-H7 % loss of SP-SI

FITC 75 <20%

PE 422 20-40%

PerCP-Cy5.5 130 40-60%

PE-Cy7 761 60-80%

APC 635 >80%APC-H7 216 SP-SI (Primary)

The resolution impact matrix

suggests that spread from the

PerCP-Cy5.5 conjugate of the highly

co-expressed CD4 will result in a

significant loss of resolution in the

PE-Cy7 detector (CD127).

Using the Resolution Impact Matrix

to Help in Panel Design

2nd Example

CD

12

7 P

E-C

y7

CD25 PE

FITC CD3

PE CD25

PerCP-Cy5.5 CD4

PE-Cy7 CD127

APC CD45RA

APC-Cy7 CD8

Whenever you have a number of

events significantly below zero that

indicates that there is spread into that

detector.

In this panel something is introducing

spread into the PE-Cy7 detector.

Where is it coming from?


• FMO controls allows us to assess the effect of spillover and spread of a given fluorochrome into other detectors.

Understanding the Effects of Spillover:

FMO Controls

CD25 PE

CD25 PE

FMO PerCP-Cy5.5

Full Panel

CD

12

7 P

E-C

y7

CD

12

7 P

E-C

y7

• In this example, the absence

of PerCP-Cy5.5 significantly

improves CD127 PE-Cy7

resolution.

– To improve CD127 resolution,

combinations of reagents with

less spillover into the CD127

detector are required.

CD

8 A

PC

-Cy7

CD

8 A

PC

-Cy7

CD4 PerCP-Cy5.5

CD4 PerCP-Cy5.5

When evaluating a

multicolor panel all FMO

controls should be run.


Resolution Impact MatrixPrimary

Fluor FITC PEPerCP-Cy5.5 PE-Cy7 APC APC-H7 % loss of SP-SI

FITC 75 <20%

PE 422 20-40%

PerCP-Cy5.5 130 40-60%

PE-Cy7 761 60-80%

APC 635 >80%APC-H7 216 SP-SI (Primary)

• Minimize impact of PerCP-Cy5.5 spillover into the PE-Cy7

channel by assigning PerCP-Cy5.5 to CD8.

Optimizing Panel Design to

Improve Resolution of Tregs

CD8 APC-H7

CD4 PerCP-Cy5.5

CD127 PE-Cy7

CD25 PE

CD3 FITC

CD45RA APC

CD3 FITC

CD4 APC-H7

CD45RA PE-Cy7

CD25 PE

CD8 PerCP-Cy5.5

CD127 Alexa Fluor® 647

• Maximize Treg resolution by spreading CD25 and CD127

fluorochrome assignment across lasers and by choosing

fluorochromes with minimal spillover into each other.

Second Example Third Example


6-Color T-Cell Panel: Third Example

6-color T cell panel

optimizing reagent

selection to minimize

spread due to

fluorescence spillover

FITC CD3

PE CD25

PerCP-Cy5.5 CD8

PE-Cy7 CD45RA

Alexa Fluor® 647 CD127

APC-Cy7 CD4

SSC

CD3 FITC

CD25 PE

CD

8 P

erC

P-C

y5.5

CD4 APC-Cy7

CD

45R

A P

E-C

y7

CD25 PE

Data acquired on a BD FACSCanto II flow cytometerC

D1

27

Ale

xa

Flu

or®

647


Resolving the Treg Subset:

Reviewing the Three Examples

Second Example Third ExampleFirst Example

Panel optimized to

minimize loss of

resolution due to spillover

of co-expressed markers.

Panel optimized to use

bright fluorochromes for

the low expressors.

Resolution of CD127+,

CD25+ cells improved.

The basic rules of

panel design were

not applied.

CD

127 P

E-C

y7

CD25 APC-Cy7

CD

127 F

ITC

CD25 PE

Treg

resolution Suboptimal Improved Maximized

CD25 PE

CD

12

7 A

lex

a F

luo

r® 6

47


Cell Analyzers:

• Enables researchers to study (on a single cell basis):

– Cell size

– Granularity & shape

– Surface and/or intracellular proteins

• Cells are not collected

Cell Sorters:

• Enables researchers to sort (collect) viable cells based on the above criteria

• Sorted cells could be used for downstream applications:

– Functional assessment

– Imaging

– Genomics

– Cell line development

– Cell therapy

– …

BD FACSLyric Flow Cytometer

BD FACSMelody Cell Sorter

Instruments


Sorted cells are functionally capable of cytokine production similar to their freshly isolated counterparts

Unsorted Tresps Sorted Tresps

Unsorted Tregs Sorted Tregs

Comparable IC cytokine response in sorted and

unsorted cells post-stimulation

81.0%

Exp

and

ed

Tre

gsP

BM

Cs

77.5%20.5%

81.6%16.1%

Sorted and expanded Tregs preserve FoxP3 expression and maintain their high purity

TregsCD4+ CD25high/+ CD127low/-

Data acquired on BD FACSLyric Flow Cytometer

Big Features, Small Package

Proven Technology

Easy to Learn & Use

High Performance

Reliable Results

Cost & Time Savings

Operator Safety

A TrustedPartner

© 2018 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 46

• Configurations

– 1-laser (blue), 2-color

– 1-laser (blue), 4-color

– 2-laser (blue, yellow-green), 4-color (2-2)

– 2-laser (blue, red), 6-color (4-2)

– 2-laser (blue, violet), 6-color (3-3)

– 2-laser (blue, yellow-green), 6-color (2-4)

– 3-laser (blue, red, violet), 6-color (2-2-2)

– 3-laser (blue, red, yellow-green), 8-color (2-2-4)

– 3-laser (blue, violet, yellow-green), 8-color (2-2-4)

– 3-laser (blue, red, violet), 9-color (4-2-3)

• Lasers

– 488 nm, blue, 16 mW direct diode laser

– 640 nm, red, 36 mW direct diode laser

– 405 nm, violet, 36 mW direct diode laser

– 561 nm, yellow-green, 40 mW optically pumped semiconductor laser

System specifications


Proven Technology

Easy to Learn & Use

High Performance

Reliable Results

Cost & Time Savings

Operator Safety

A TrustedPartner


• Smart automation minimizes manual steps and saves time

Minimal Expertise Required:Simplified Setup

Compensation is automatically computed from compensation

controls that can be labeled capture beads, cells, BD™ FC

Beads, or a combination of these

Good for up to 60 days!


Proven Technology

Easy to Learn & Use

High Performance

Reliable Results

Cost & Time Savings

Operator Safety

A TrustedPartner


• Fixed alignment flow cell, fast and easy nozzle replacement

• Automated sort setup:

– Fluidic startup removes bubbles and checks fluid levels

– Optimizes droplet formation

– Measures drop delay

– Focuses sort streams

– Steers sort streams into tubes or plates

Minimal Expertise Required:Simplified Setup


Proven Technology

Easy to Learn & Use

High Performance

Reliable Results

Cost & Time Savings

Operator Safety

A TrustedPartner


• Capture protein expression information and sorted location to enable discovery from downstream experiments such as RNAseq

• Index 1 to 100 events per location

Index Sorting Helps Map Protein to Gene Expression

A. Index sort data for a single 96-well plate showing TN, TSCM, TCM , and TEM

cells in their respective gates in the contour plotsB. Expression levels of designated genes across different originating cell types

50

• Follow the rules of panel design to get optimal data:

– Choose fluorochromes based on instrument configuration.

– Classify antigens and pair with fluorochromes based on

expression characteristics.

– Optimize instrument setup and compensation.

• Panel design is an iterative process.

• The only way to determine if a panel design is optimized

is to run an experiment with all relevant controls.

Summary


Tools:

www.bdbiosciences.com/tools

• Detailed fluorochrome information

• Buffer compatibility resource

• Multicolor panel designer

• Spectrum viewer

Thank you

Resources:

[email protected]

[email protected]

877.232.8995, 2, 2

Documents

New Insights for Multicolor Panel Design talk panel desig… · The BD Horizon™Tour: New insights for multicolor panel design /9 Stain Index: The Standard Metric of Resolution SD