Multiparametric Flow Cytometry Advanced Foundations With Correction

ACS 2016
Polychromatic Roadshow
Wellington : Perth : Brisbane : Sydney : Melbourne
Pratip K. Chattopadhyay, Ph.D.

ImmunoTechnology Section
Vaccine Research Center, NIH
ISAC Scholar
These materials were presented at the 2016 ACS
Polychromatic Flow Cytometry Roadshow.
Please obtain permission if presenting these slides

publicly, and please acknowledge ACS and Pratip
Chattopadhyay.
Questions? Contact Pratip Chattopadhyay

(pchattop@mail.nih.gov)
1: Foundations
Laying Proper Groundwork for PFC
Instrument Setup and Monitoring
Reagents
Instrument Setup, Optimization, and QC
How should your instrument be equipped?
How do you verify that instrument hardware is not defective?
How do you know that instrument is performing optimally?
How do you characterize an instruments performance?
How can you compare instrument performance over time, or

across instruments?

There is little consensus.

across instruments?
Equipment Decisions : Laser Choice
Laser selection depends on your applications and dyes available.

Make a chart like this to help with your decision-making:
Wavelength Compatible Dyes Reagent Sources
UV Brilliant UV, Quantum Dots, Vio BD (4), Invitrogen (8), Miltenyi (1)
Violet Brilliant Violet, Horizon, Pacific, BD (8), BioLegend (3), Coulter (2), Invitrogen (8), Miltenyi (2)
Quantum Dots, Vio
Blue Brilliant Blue, FITC, Alexa 488, BD (3), BD, Coulter, BioLegend, Invitrogen, Miltenyi (2)
Cy55PerCP, PE and Tandems, Vio
Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi
Yellow/Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi
Red APC and Tandems , Alexa BD, Coulter, BioLegend, Invitrogen, Miltenyi
Equipment Decisions : Laser Choice
Laser selection depends on your applications and dyes available.

Wavelength Compatible Dyes Reagent Sources
UV Brilliant UV, Quantum Dots, Vio BD (4), Invitrogen (8), Miltenyi (1)
Violet Brilliant Violet, Horizon, Pacific, BD (8), BioLegend (3), Coulter (2), Invitrogen (8), Miltenyi (2)
Quantum Dots, Vio
Blue Brilliant Blue, FITC, Alexa 488, BD (3), BD, Coulter, BioLegend, Invitrogen, Miltenyi (2)
Cy55PerCP, PE and Tandems, Vio
Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi
Yellow/Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi
Red APC and Tandems , Alexa BD, Coulter, BioLegend, Invitrogen, Miltenyi
Proprietary dyes,
Somewhat limited dye selection for multiplexing,
Catalogue of antibodies somewhat limited.
Keep reagent availability in mind for laser decisions.
UV or Violet Laser? Our System
(15 colors/ 2 lasers)
UV Dye V Dye
Well, if you can, do both. BUV390 BV421
Reagents will become available. UV450 (LD) BV510
This maximizes multiplexing. BUV490 BV570
BUV550 BV605
BUV680 BV650
BUV737 BV711
BUV800 BV750
BV785
Otherwise, if you have to choose one:
UV BUV390 UV450 BUV490 QD54 QD58 QD605 QD655 QD705
5 5 BV605 BV650 BV711
BV570
V BV421 V545 BV570 QD60 QD65 QD705 BV750 QD800
BV510 5 5 BV711 BV785
BV605 BV650
Blue or Green?
Blue (488nm) Alone

FITC and PE, Tandems all excited off of blue.
Disadvantage PE and Tandems excited sub-optimally.
Blue + Green (532nm)

Advantage PE and Tandems much brighter than off blue.
Disadvantage
532nm laser line picked up with broad FITC filter. Easy to fix with
narrow filter, higher power blue, or notch.
Blue + Yellow/Green (561nm)

Advantage PE and Tandem excitement optimal, no FITC problem
Disadvantage 561nm laser line in PE filter, excite APC as well
Equipment Decisions : Example Configs
5-Laser, 21-color 4-Laser, 18/19-color

UV Violet Blue Green/YG Red UV or Violet Blue Green/YG Red
Equipment Decisions : Laser Power
UV Violet Blue
CD4 BUV395
CD4 BV650
CD4 BB515
Laser Power (mW) Laser Power (mW) Laser Power (mW X 102)
Green Red
CD4 APC
Stain cells with
CD4 PE
representative conjugate
for each laser.
Laser Power (mW X 102) Laser Power (mW X 102)

UV Violet Blue
CD4 BUV395
CD4 BV650
CD4 BB515
Green Red
CD4 APC
Run samples at different
CD4 PE
laser powers.
Requires tunable lasers.

UV Violet Blue
CD4 BUV395
CD4 BV650
CD4 BB515
Green Red
CD4 APC
CD4 PE
Calculate Staining Index.

Compare across powers.

Results From Our Experiments
30 100 250
15 45 50 150 125 375

Ultraviolet Violet Blue
0 60 0 200 0 500
mW mW mW
500 500
BTW, brief 1 watt 250 750

250 750
exposure does not fry
cells in sort stream. Green Red
0 1000 0 1000
But my fry eyeballs.
mW mW

across instruments?
Verifying Signals
Goal: To ensure that fluorescence measured properly.
Detector-related Cascade Test for photon detection efficiency.
Filter-related Check that they didnt have a bad day at the factory.
Correct wavelengths? Clean optics? Orientation?
Laser-related Laser power, window extension, laser delay
Reference:
Perfetto, et al. Quality Assurance for Polychromatic Flow Cytometry,
Nature Protocols (2006 and 2012, in press).
Verifying Signals
Reference:
Cascade Test
1. All PMT @ 500 volts.
2. Remove all filters, reserve
last set for test.
1 3. Place test filter at first
PMT.
4. Run 1x beads.
5. Record CV.
6. Move test filters to next
4 3 PMT.
7. Return first PMTs
original filters.
8. Repeat for other PMTs.
9. Calculate statistical
photoelectron estimate =
2 1/n2, where n=CV/100.
This value indicates

detector efficiency.
Cascade Test
Helps evaluate sensitivity of detectors.
Photon detection efficiency (PDE) is usually about 800 + 200.
The higher the PDE, the better.
Values below 500 indicate severe problem with detector.
This is rough way to evaluate detector sensitivity, requires no special

equipment. But there is a much better way if you have the right
equipment, and it can help optimize your cytometer (coming up).
Value of Cascade Test
You can also simply track MFI of signal in cascade test.
We did this on our 30-parameter instrument and found loss of signal

across detector positions:
And it was occurring no matter which

detector we put in each position.
Therefore, not detector intrinsic.

Also ruled out filters.
We suspected it was the lens that

focuses light beam into detector
decagon.
Value of Cascade Test
After adjusting lens, problem remains but is a little better.
Were working with vendor on this. Test gives us actionable data!
Before Lens Adjustment After Lens Adjustment
*grrr. Not same signal cascade.

Verifying Signals
Reference:
Troubleshooting Filter Issues
PMT 1 PMT 2 PMT 3
1x Rainbow Beads
3 All PMT @ 500 volts.
Case A
1 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
Case B
2
2 3 4 5 2 3 4 5 2 3 4 5
A = OK! 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
Case C
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
PMT 1 PMT 2 PMT 3
1x Rainbow Beads
Case A
1 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
x
Case B
2
2 3 4 5 2 3 4 5 2 3 4 5
B = PMT2 Dichroic is 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
a poor transmitter, but

it reflects light
adequately. Or, PMT2 Case C
Bandpass is poor.
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
PMT 1 PMT 2 PMT 3
1x Rainbow Beads
Case A
1 x 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
x
Case B
2
2 3 4 5 2 3 4 5 2 3 4 5
C = PMT1 Dichroic is a 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
poor reflector, or PMT2

Dichroic completely
failed. Case C
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Verifying Signals
Reference:

across instruments?
Instrument Optimization
= Choosing Voltages Wisely
Methods that follow serve a few purposes:
Ensure voltages allow detection within linear range of detector

Optimize detection of dim signals
Provide track-able metrics for QC
Provide a means to compare and standardize
Multi-instrument facilities, multi-center studies (!)
Guide panel development
Approaches to
Optimization/Standardization To Date
Standardize Sample
Preparation and Handling
Approaches to
Important for standardization in

Standardize Sample large multicenter studies, but
Preparation and Handling doesnt address instrument
differences. (i.e., ignore the issue)
Approaches to
Standardize Sample Standardize Negative

Preparation and Handling Autofluorescent Signal
Approaches to

Doesnt ensure PMTs signals are

linear and proportional.
Signal in 1o channel < 2o channel

= compensation confusion
Approaches to

Beads have high CVs
Bead-based Protocols Broad-spectrum dyes = flow dyes
CS&T re-done with each new panel.
Perfetto, Chattopadhyay. Nature Protocols.

Most Recent Practices

Q and B Calculations
Bead-Based Protocols Using an LED Pulser
(2014 - )
Q & B: Parameters Defining Separation
(Successful) Flow cytometry allows clear resolution of cells expressing

a marker from background noise.
Electronic Auto- Spread

Noise Fluor.
Positive Gate
Limit of Detection
Negative Gate
Sources of Background
(Successful) Flow cytometry allows clear resolution of cells expressing

a marker from background noise.
Electronic Auto- Spread

Noise Fluor.
Positive Gate
Limit of Detection
Negative Gate
Sources of Background
Beyond this, resolution depends on quality of detector, brightness of dye,

and staining characteristics of marker.
PMT manufacturers know that there are differences in detector quality,
they generate horrifying graphs to characterize this.
PMTs differ in sensitivity, and it even appears these relate to wavelength.
10%
5%
2.5%
1.25%
PMT Quality
(Efficiency of Photon to Signal)
Electronic Auto- Spread Poor -------------------------- Good
Noise Fluor.
Sources of Background Detection Resolution

(Same Dye Across
Different Quality PMTs)
PMT Quality
(Efficiency of Photon to Signal)
Electronic Auto- Spread Poor -------------------------- Good
Noise Fluor.
Sources of Background Detection Resolution
B Q
Q & B Can Be Measured with LED Pulser
LED pulser plugs into flow cell, and flashes

highly reproducible light at the PMTs.
Since the CVs are lower than beads, you get a

true sense of detectors resolution. Measures
are independent of voltages, and fluidics.
SpheroTech Bead Data LED Data

158 6899
119 5174
Count
Count
79 3450
40 1725
0 0
2 1 2 3 4 5 2 1 2 3 4 5
-10 -10 10 10 10 10 -10-10 10 10 10 10
FITC-A FITC-A
Jim Wood, Wake Forest University and Steve Perfetto, VRC, NIH
Q & B Calculations
Hook Up LED Pulser
Set Starting Pulse

Intensity
Set Reasonable
Voltage
Acquire Data into

FCS File
Measure MFI and

SD, Record in XLS
Repeat Lower
Intensities (Dial)
Output = Q and B = rough because B is voltage dependent,
rough B Value and weve selected only one voltage here.
Why do this?
With reproducible, quantifiable measures of

background and resolution, you can optimize
instrument setup. First, by matching PMTs to
their optimal detection range:
1. Test all PMT at three wavelengths (around

510, around 650, and around 800)
2. Calculate Q and B
B Values on Old VRC Instruments
140
120
100
B-Values
B Value (rSD)
80
60
40
20
0
Aria-B Aria-C LSR-A LSR-B LSR-C LSR-D LSR-E pX50
Instrument
Instrument
Huge spread in B values across detectors and instruments.
This is why standardization of sample processing will never fully make data across
instruments comparable. For new instruments, we tried to lessen this issue.
Q & B Values on Detectors for
New VRC Instruments
780nm
B-Values
Q-Values
New VRC Instruments
Exclude detectors with high
background and low 780nm
resolution.
Then, choose best (lower

right corner) to populate far
red slots.
B-Values
Why far red first?
Because these dyes release

fewest photons and are
hardest to detect: best PMTs.
Repeat at two other

wavelengths with remaining
detectors.
Q-Values
New VRC Instruments
Of the remaining detectors, 510nm
one has poor B, one has poor
Q at this wavelength, even
though they were fine at
800nm.
There are three detectors

B-Values
with super Q/B at this

wavelength.
We now know that

instruments with these
detectors will be super at
detecting V510 dyes.
Q-Values
So Far
Established configuration for new instrument.

Laser choice and power
Checked quality of detectors

Two ways, cascade test and Q/B (LED-pulser)
Verified filters
Matched best performing PMTs to most

challenging detection, matched PMTs to their
most sensitive wavelengths.
Next Step: Choose Voltages
Three methods available
Bead-based
Auto-fluorescence / B ratio
Wavelength-based
Bead-based Method for Voltages
1) Check that filters are in place; set all voltages to 350.
1) Run 8X Rainbow Beads or Simply Cellular Beads.
2) Record data; MFI/CV in each detector.
3) Increase voltage by 50 and repeat step 2.
4) Continue until voltage hits 800.
5) Review data with calibration tool.
6) Record voltage range where tool shows

that data remains linear, and
where separation is greatest.
This is prelimary voltage for each detector.

Auto-fluorescence to B Ratio
Concept: We do not want to measure signals in region of noise.
So, our cell signals must clear background noise (B).
We can pick voltages by measuring B and autofluorescence of cells,

then choosing a voltage where autofluorescence clears background
noise.
Auto-fluorescence to B Ratio
Pulser triggers Make sure Set voltage, Increase voltage,
SSC lasers on collect data collect data
Auto-fluorescence to B
Run cells of Make sure Set voltage, Increase voltage,

interest lasers on collect data collect data
B Values at different Gains
Gains = 300v Gains = 430v Gains = 500v

AF signal (black ) overlayed with B value at same gain
Gains = 300v
Gains = 430v Gains = 500v
8000
4000
1500
6000
3000
# Cells
# Cells
# Cells
1000
4000
2000
2000 500
1000
0 0 0
0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5 2 3 4 5
G560-A: Fixed Unst PBMC 0 10 10 10 10
G560-A: Fixed Unst PBMC G560-A: Fixed Unst PBMC
B Values at different Gains
Gains = 300v Gains = 430v Gains = 500v

AF signal (black ) overlayed with B value at same gain
Gains = 300v
Gains = 430v Gains = 500v
8000
4000
1500
6000
3000
# Cells
# Cells
# Cells
1000
4000
2000
2000 500
1000
0 0 0
0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5 2 3 4 5
G560-A: Fixed Unst PBMC 0 10 10 10 10
G560-A: Fixed Unst PBMC G560-A: Fixed Unst PBMC
Theoretical calculations indicate that ideal voltage is where

AF is 80% higher than B.
Instead of theoretical calculation, one can plot AF/B vs. voltage.
Mid-point is
preliminary voltage for
each detector.
Wavelength-based
It turns out that

when you match
PMTs to their optimal
detection
wavelengths, the
voltages you choose
with any of the above
methods are strongly
correlated to
wavelength.
Wavelength-based
This means that if
you go through the
routine for matching
PMTs to wavelength
of interest, you can
use equations we
generate to set
voltage without using
AF/B or bead-based
methods.
= Optimized system,
easy voltage setting.
Clean-up Voltage Choices
To select best voltage for each detector, use Comp beads (IgK capture beads) to capture
antibodies labeled with the specific fluorochromes used in experiments. Run at 50 volt
increments, within range of voltages determined in first part of routine. Select voltage
for each detector where 1o signal for detector is higher than 2o signals.
Clean-up Voltage Choices
This ensures compensation values are <100%.
Does this matter?
Not really, compensation is unit-less, and values dont have to be

less than 100% for good separation.
But keeping compensation less than 100% by setting voltages

correctly makes troubleshooting easier.

across instruments?
Q/B/Voltages =
Metrics of Instrument Performance
Low B / High Q = Very Sensitive Detector
Pair this with a bright dye (BV421, PE) for SuperHiRes Channel.
If Q or B suddenly goes off, then instrument issue.
If Q/B drastically different across centers, this can explain data

that is not comparable.
Application of Q and B
Q and B are directly, mathematically related to number of
photons released by fluorochromes.
With standards containing known numbers of fluorochrome

molecules (under development),
we can transform relative fluorescence channels to absolute

numbers of fluorochromes,
and then to more accurate measurements of protein

molecules. Instrument comparison is now based on
biologically relevant units.
Studies of receptor biology improved.
Channel Numbers to Receptors
Pulsed LED
(light detection)
Standard Beads
(known # FL molecules)
Calibration known # of
dye molecules per mAb
Steve Perfetto
Q and B to Predict Panel Success
Q and B can be used to predict whether a

staining panel will work, based on a few simple
experiments, rather than complex, step-by-step
panel development. This is in-development, and
can be used to develop very precise automated
tools (rather than guidance software currently
available).
Daily QC
Once the best voltages are established, run 1X beads again, record MFI and CV of
the peak in each channel.
Establish a tolerance range, based on 20+ runs. Calculate the CV for the multiple
runs, and make the target MFI +/- CV of 10% your QC limit.
FITC TRPE APC QD-585

B515 G610 R660 V585
CV = 7.6% CV = 4.9% CV = 7.8% CV = 10.1%
MFI = 4200 MFI = 30,000 MFI = 9,900 MFI = 6,400
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
Standardizing Daily Performance
Daily operation:
1.Core sets up instrument in morning with 1X beads and

unstained comps, ensures values fall within tolerance range.
2.If not, then check instrument and beads first, then adjust
voltage (but be sure still within range that calibration routine
defined.
3.Users check 1X beads before their own runs, to ensure

instrument performance is stable. They do not alter voltages
after that. If a fluorochrome (used in calibration routine) is off-
scale, they need to titrate not adjust voltages!!!
4.The values from daily QC are collected and monitored.

Monitoring Daily Performance
Data from single LSR, APC detector monitoring.

rCV spiked up at d154. Problem with steering mirrors.

Once fixed, back to normal.
When rCV went up, MFI did not.

rCV hurts dim separation (dim peak width) not mean, need to monitor all.
Comp beads got contaminated with APC reagent!

QC System Shows Results of Improvements
12
11 LSR-A
14
10
12
9
rCV G560
10
8
rCV G560
7 8
UCL=6.72
6 6
Avg=5.01
5 4
LCL=3.30
4 2
12 36 60 84 108 132 156
3
Time
2
LSR-A
D12 we installed a better dichroic filter on PE channel. Huge drop in rCV.

Better separation between dim and negative.
QC System Shows Results of Improvements
12
11
600
10
9
550
8 New PE detector required lower
rCV G560
7 voltage and gave lower rCV.

Volt G560
500 6
4
450
3
LSR-E LSR-E
Summary: What Did We Learn
Decide configuration based on dyes available, application, and

benefits/disadvantages of each configuration.
Methods to verify that detectors and filters are working.
Survey of methods for QC
Deeper knowledge of Q and B-based methods.
What these values mean
How to set voltages
How to do daily QC
Thanks
Steve Perfetto
Jim Wood

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ACS 2016

Pratip K. Chattopadhyay, Ph.D.

Please obtain permission if presenting these slides

Questions? Contact Pratip Chattopadhyay

Instrument Setup and Monitoring

How should your instrument be equipped?

How do you verify that instrument hardware is not defective?

How do you know that instrument is performing optimally?

How do you characterize an instruments performance?

How can you compare instrument performance over time, or

How should your instrument be equipped?

How do you verify that instrument hardware is not defective?

How do you know that instrument is performing optimally?

How do you characterize an instruments performance?

How can you compare instrument performance over time, or

Laser selection depends on your applications and dyes available.

Wavelength Compatible Dyes Reagent Sources

Yellow/Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi

Laser selection depends on your applications and dyes available.

Yellow/Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi

Blue (488nm) Alone

Blue + Green (532nm)

Blue + Yellow/Green (561nm)

5-Laser, 21-color 4-Laser, 18/19-color

Laser Power (mW X 102) Laser Power (mW X 102)

Laser Power (mW X 102) Laser Power (mW X 102)

Calculate Staining Index.

Laser Power (mW X 102) Laser Power (mW X 102)

15 45 50 150 125 375

BTW, brief 1 watt 250 750

How should your instrument be equipped?

How do you verify that instrument hardware is not defective?

How do you know that instrument is performing optimally?

How do you characterize an instruments performance?

How can you compare instrument performance over time, or

Goal: To ensure that fluorescence measured properly.

Detector-related Cascade Test for photon detection efficiency.

Correct wavelengths? Clean optics? Orientation?

Laser-related Laser power, window extension, laser delay

Goal: To ensure that fluorescence measured properly.

Detector-related Cascade Test for photon detection efficiency.

Correct wavelengths? Clean optics? Orientation?

Laser-related Laser power, window extension, laser delay

This value indicates

Helps evaluate sensitivity of detectors.

Photon detection efficiency (PDE) is usually about 800 + 200.

The higher the PDE, the better.

Values below 500 indicate severe problem with detector.

This is rough way to evaluate detector sensitivity, requires no special

We did this on our 30-parameter instrument and found loss of signal

And it was occurring no matter which

Therefore, not detector intrinsic.

We suspected it was the lens that

Were working with vendor on this. Test gives us actionable data!

Before Lens Adjustment After Lens Adjustment

*grrr. Not same signal cascade.

Goal: To ensure that fluorescence measured properly.

Detector-related Cascade Test for photon detection efficiency.

Correct wavelengths? Clean optics? Orientation?

Laser-related Laser power, window extension, laser delay

a poor transmitter, but

poor reflector, or PMT2

Goal: To ensure that fluorescence measured properly.