Instrument QC and
Qualification
Overview
Why QC is Important
LSRII Optical Configuration
LSRII QC




Validation
Optimization
Calibration
Standardization
Practice Analysis
Characterizing the Cytometer
3
Challenges…
• Instrument - optical configuration,
optimization, standardization, and calibration
• Reagent - optimization and standardization
• Sample processing
• Staining protocols
• Data Analysis - compensation & gating
• Operators
• Volume of data (death-by-excel!)
Duke University Medical Center
Two Methods of Instrument Charaterization
BD CS&T: Cytometer Setup and Tracking
Instrument Performance
• Parameters
CS&T
Perfetto
•
•
•
•
•
Laser (amps vs mW)
CV (resolution)
Signal-to-noise ratio
Linearity
Specific fluorescence
yes
yes (target)
NO
Yes (range)
NO
yes
yes (range)
yes (range)
yes (range)
yes (target)
•
Automated
YES
NO
Instrument QC
Frequency
Purpose
Initial Characterization &
After Optical Service
Validation: optimal voltage
range for each detector
Once Per Assay
Optimization: assay
specific optimal voltage
for each detector & assay
specific target channels
Each Experiment
&
Troubleshooting
Calibration: set detectors
to assay specific target
channel values for
specimen acquisition
Standardization: Monitor
trendlines using assay
specific target channels over
time
Key Performance Factors in High-Quality Flow
Cytometry Data
•
Resolution of subpopulations, including dim subpopulations
• Sensitivity
•
Relative measured values of fluorescence
• Linearity and accuracy
•
Reproducibility of results and cytometer performance
• Tracking
•
Comparison of results across time and amongst laboratories
• Standardization
LSRII qualification

Validation
 Linearity
 Resolution (CV)
 Signal-to-Noise Ratio (S:N) - LLOD & LLOQ

Optimization




Select Optimal Voltages for Inst Performance with AssaySpecific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)




Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision





Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trendlines
Linearity
 Definition: Proportionality of output to input
 Method: Access linearity using the ratio of
two pulses over voltage range of the PMT
 Significance: Linearity is important for
compensation
(Median Pk6 - Median Pk5)
= Constant
Median Pk5
Linearity
• Defined as proportionality of output (MFI) to input (Fluorescence/
# of photons)
2X Abs
2000 MFI
=
X Abs
1000 MFI
Ab = X
Ab = 2X
0
1000
2000
3000
• Important for fluorescence compensation
• Compensation of data in the last decade involves subtraction of
large numbers
• Small errors (non-linearity) in one or both large numbers can cause
a large absolute error in the result
Linearity: Effect on Compensation
•
Compensation of data in the last decade involves subtraction of large numbers
•
Errors (non-linearity) in one or both large numbers can cause a large absolute error
in the result
A
Detector
FITC
PE
B
C
D
Median Fluorescence Intensity (MFI)
68
80
1796
75
5921
79
BD CompBeads stained with varying levels of FITC-Ab.
Compensation was set using samples A and C.
This cytometer had a 2% deviation from linearity above 50,000 units.
73,000
365
LSRII Qualification


Validation



Optimization




Linearity
Resolution (CV)
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Select Optimal Voltages for Inst Performance with AssaySpecific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)




Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision





Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines
cvs
15
Instrument Sensitivity: Two Definitions
• Defining sensitivity
1. Threshold: Degree to which a flow cytometer can distinguish
particles dimly stained from a particle-free background. Usually
used to distinguish populations on the basis of Molecules of
Soluble Equivalent Fluorochrome (MESF).
2. Resolution: Degree to which a flow cytometer can distinguish
unstained from dimly stained populations in a mixture.
• How to measure instrument-dependent sensitivity?
•
Resolution sensitivity is a function of three independent
instrument factors:
•
•
•
Br
Qr
Electronic noise (SDen)
Background contributions
17
Why is Br important?
• High Br widens negative and dim populations.
• High Br value = lower resolution
• Low Br value = higher resolution
Low Br
High Br
Br: Optical Background from Propidium Iodide
•
Example: It is common to use propidium iodide (PI) to distinguish live
from dead cells. Propidium iodide was added in increasing amounts to
the buffer containing beads, and Qr and Br were estimated.
PerCP
Br
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Br
Qr
0.05
0.04
0.03
0.02
Qr
0.01
0
0 1 PI-free
2 3dye 4(µg) 5 6
•
Residual PI in your sample tube will increase Br, which will reduce
sensitivity.
20
Relative Q: Qr
•
Qr is photoelectrons per fluorescence unit and indicates how bright a
reagent will appear on the sample when measured in a specific
detector.
Qr
=
# photoelectrons
# fluorescence molecules
PMT 1


Qr =
2 photoelectrons
= 0.25
8 fluorescence
molecules
PMT 2

Qr =
1 photoelectron
8 fluorescence
molecules
= 0.125
Why is Qr important?
A system with a higher Qr has a better resolution than
a system with a lower Qr.
Low Qr value = lower resolution
High Qr value = higher resolution
High Qr
Low Qr
What Factors Affect Qr?
•Laser power
•Optical efficiency
•PMT sensitivity (red spectrum)
•Poor PMT performance
•Dirty flow cell
•Dirty or degraded filter
Spillover Decreases Resolution Sensitivity
Spread from APC Cy-7 background
Population resolution for a given fluorescence parameter
is decreased by increased spread due to spillover from
other fluorochromes.
Summary: Instrument Performance
and Sensitivity
• Qr and Br are independent variables, but both affect sensitivity.
• Increases in Br or decreases in Qr can reduce sensitivity and the
ability to resolve dim populations.
• Sensitivityrelative 
Qr
Br
• On digital instruments, BD FACSDiva™ software v6 and CS&T
provides the capability to track performance data for all of these
metrics, also allowing users to compare performance between
instruments.
Instrument performance can have a significant impact on the performance of an
assay.
•
Resolution vs. Background
Negative
Population
Positive
Population
Negative population has
low background
Populations well resolved
Negative population has
high background
Populations not resolved
Negative population has
low background
high CV (spread)
Populations not resolved
The ability to resolve populations is a function of both
background and spread of the negative population.
Measuring Sensitivity: The Stain Index
• The Stain Index is a measure of reagent performance on
a specific cytometer, a normalized signal over
background metric.
median positive  median negative
Brightness
Stain Index 

Width of Negative
2  rSD negative
Brightness
Width of negative
•
•
The brightness is a function of the assay (antigen density, fluorochrome used).
The width of the negative is a function of:
– Instrument performance (Qr, Br, and SDen) [single-color]
– The assay


(Fluorescence spillover / compensation) [multicolor]
The cell population
Stain Index: Normalized Signal/Background
Stain Index (SI)  D/W = Relative Brightness
70
60
Goal: Normalize the signal
to the spread of background
where background may be
autofluorescence, unstained
cells, or compensated cells
from another dye dimension.
D
50
40
Wunstained
30
20
Wcompensated dye
10
0
5.5
6.5
7.5
Stain Index (SI)  D
1Stain
8.5
W
9.5
10.5
11.5
D = difference between positive and negative peak medians
W = the spread of the background peak (= 2X rSDnegative)
Index: metric used by Dave Parks, Stanford – Presented at ISAC 2004
Electronic Noise (SDEN): Determining Baseline
PMT Voltages
The BD FACSDiva™ 6.0 CS&T module analyzes dim particles, which are
similar to dim cells’ brightness, allowing relevant detector baselines to be
visualized by plotting fluorescence intensity vs (PMT gain, CV, and SD)
PE: Detailed Performance Plot
Dim Bead
• For this detector, the SDEN = 18
• Fluorescence intensity of dim bead
= 10 x SDEN = 180
CV
Standard Deviation
10000
1000
CV or SD
• Determine PMT voltage required to
set the dim beads at 180
= 500 volts = baseline voltage
• As PMT voltage is lowered, CV
increases  resolution decreases
500 V
1000
100
18
• As PMT voltage is increased
CV unchanged  resolution unchanged
10
1
10
100
10000
Median Fluorescence Intensity
180
29
1000
100
100000
PMT Voltage
PMT Voltage
PMT Voltages: Optimal Gains Can Reduce
Classification Errors
% Negative in CD4+ Monocyte Gate
12.0%
CD4 dim monocytes
650 V
CD4 negative
CD4+ lymphocytes
% Negative in CD4 Gate
550 V
10.0%
8.0%
6.0%
4.0%
2.0%
750 V
0.0%
-100
0
100
PMT Voltage Offset
30
31
Methods Used for
Validation
Which Beads to Use
What Values to Plot
Selection Criteria
Example LSRII Optical Configuration
Blue Laser
Duke LSRII
(488nm)
Red Laser
Duke LSRII
(635nm)
FITC
Alexa
680
515/20
710/50
SSC
APC
PE ﾐ
Blue
APC
Blue
Cy7
Green Laser
Duke LSRII
Violet Laser
(532nm)
Duke LSR II
(407nm)
QDot
PE
655
Cy5.5
QDot
PE
TR
710/50
585
660/40
Am
Cyan
535LP
QDot
PE-
QDot
Green
565
545
Cas
Blue
PE
33
Cy5
PE
QDot
Cy7
605
QDot
705
Duke University Medical Center
Beads Used for LSRII Validation
• 8 Peak Rainbow: 300-900v in 50v increments (all
PMTs)
• Non-fluorescent to Very Bright; Broad Spectrum Ex & Em
• 8pks
• Run: Once, then after optical service - “High” flow rate &
LOG scale
• Uses: Validation
• Check Linearity: (MedianPk6-MedianPk5)/MedianPk5
• Check CV: CV Pk5
• Check S:N (LLOD & LLOQ): MedianPk5/MedianBlank
• Unstained Comp Beads: 300-900v in 50v increments
(all PMTs)
• Non-fluorescent
• 1pk
• Run: Once, then after optical service- “High” flow rate &
LOG scale
• Uses: Validation
• Check S:N (LLOD & LLOQ): MedianPk5/MedianBlank
Example of Optimal PMT Performance
3000
3000
3000
3000
3000
2000
2000
1000
2000
1000
0
10
2
3
10
Green B-A
10
4
10
2000
1000
0
0
5
300 Volts
10
2
3
10
Green B-A
10
4
10
2000
1000
0
0
S S C-A
4000
S S C-A
4000
S S C-A
4000
S S C-A
4000
S S C-A
4000
5
0
0
350 Volts
1000
10
2
3
10
Green B-A
10
4
10
5
0
0
400 Volts
10
2
3
10
Green B-A
10
4
10
5
0
450 Volts
3000
3000
3000
3000
1000
2000
1000
0
0
0
10
2
3
10
Green B-A
10
4
550 Volts
10
5
2000
1000
10
2
3
10
Green B-A
10
4
600 Volts
10
5
10
2
3
10
Green B-A
10
4
650 Volts
10
5
4
10
5
1000
0
0
10
2000
1000
0
0
3
10
Green B-A
S S C-A
3000
S S C-A
4000
S S C-A
4000
S S C-A
4000
S S C-A
4000
2000
2
500 Volts
4000
2000
10
0
0
10
2
3
10
Green B-A
10
4
700 Volts
10
5
0
10
2
3
10
Green B-A
10
4
750 Volts
10
5
Criteria for the Selection of Voltage
Ranges
Lowest CV possible
Lowest Voltage possible
Highest MFI possible
Lowest background possible
Example of Optimal PMT Performance &
Voltage Selection Criteria (Green B)
GreenB Voltage vs. CV, LIN and Ratio
70
500
450
60
400
50
350
300
40
250
30
200
150
20
100
10
50
0
0
300
350
400
450
500
550
600
650
700
Voltage
Ratio = M1/B
Linearity = (M2-M1)/M1
CV
Lin
Ratio
Optimal Voltages: 450-650
750
R a tio
C V o r L IN
Selection Criteria:
Linear
Lowest CV
Highest S:N
Lowest Voltage
Red A (APC-Cy7):
Before (30Mar06) & After (10Apr06) Replacing PMT
30 March 2006
10 April 2006
5000
200
5000
180
4500
180
4500
160
4000
160
4000
140
3500
140
3500
120
3000
120
3000
100
2500
100
2500
80
2000
80
2000
60
1500
60
1500
40
1000
40
1000
20
500
20
500
0
0
300
350
400
450
500
550
600
650
700
750
C V o r L IN
200
0
0
300
350
400
450
500
Voltage
CV
Lin
550
600
650
700
750
Voltage
Ratio
Optimal Voltages: Indeterminate
High CV’s; poor S:N
CV
Lin
Ratio
Optimal Voltages: 625-700
R a tio
RedA Voltage vs. CV, LIN and Ratio
R a tio
C V o r L IN
RedA Voltage vs. CV, LIN and Ratio
Faulty PMT on install…
Before
After
4000
3000
3000
S S C-A
S S C-A
4000
2000
2000
1000
1000
0
0
0
10
2
3
10
65010RedVolts
A-A
4
10
5
0
10
2
3
10
Red C-A
10
650 Volts
4
10
5
LSRII Qualification


Validation



Optimization




Linearity
Resolution
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Select Optimal Voltages for Inst Performance with AssaySpecific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)




Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision





Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines
Beads Used for LSRII Optimization
• STAINED Comp Beads: validated range in 50v increments (each
PMT)
•
•
•
•
Assay Specific fluorescence
1pk
Run: Once, then after optical service- “High” flow rate & LOG scale
Uses: Optimization
• Optimize PRIMARY Fluorescence (Primary>Secondary)
• Unstained Comp Beads: optimized voltages (all PMTs)
•
•
•
•
Non-fluorescent
1pk
Run: Once, then after optical service- “High” flow rate & LOG scale
Uses: Validation
• Check S:N (LLOD & LLOQ)
• 1x or Midrange Rainbow: optimized voltages (all PMTs)
• Moderate fluorescence, near cellular expression; Broad Spectrum
Ex & Em
• 1pk
• Run: Once (after optimization) - “High” flow rate & LOG scale
• Uses: Validation
• Establish Target Channels Linearity: medians = assay specific target
channels
Criteria for the Selection Optimal PMT
Voltages
The primary fluorescence should
be the highest in the respective
detector relative to all secondary
detectors.
(Blue B-A): GeoMean = 19
0
10
2
10
3
10
4
(Green A-A): GeoMean = 5462
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 129
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
10
5
0
2
10
10
2
R ed B- A
3
10
4
10
5
0
10
2
10
10
4
3
10
4
10
5
0
10
2
10
4
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
(Violet D-A): GeoMean = 21.4
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 90.2
10
10
Violet B- A
G reenD -A
0
3
(Violet C-A): GeoMean = 12.3
R ed C- A
3
10
(Violet B-A): GeoMean = 47
(Red C-A): GeoMean = 15
G reenC -A
10
2
Violet A- A
(Green D-A): GeoMean = 99.2
0
10
(Red B-A): GeoMean = 5.69
(Green C-A): GeoMean = 26.3
0
3
R ed A- A
(Green B-A): GeoMean = 44.7
0
10
(Violet A-A): GeoMean = 8.05
3
10
4
(Violet E-A): Geo Mea n = 7.82
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 32.2
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 9.43
Green A: TNF PE-Cy7
460v Baseline & Final
0
10
2
10
3
10
4
Violet G- A
TNFa PE-Cy7 DukeJY_460v baseline.fcs
(Violet H-A): GeoMean = 18.8
43
0
10
2
10
Violet H- A
3
10
4
(Blue B-A): GeoMean = 14.1
0
10
2
10
3
10
4
(Green A-A): GeoMean = 617
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 295
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
3
10
4
10
5
0
2
10
10
5
0
10
2
10
2
10
4
3
10
4
10
5
0
10
2
10
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 0.98
10
4
(Violet D-A): GeoMean = 8.57
G reenD -A
0
10
(Violet C-A): GeoMean = 6.55
R ed C- A
3
3
Violet B- A
(Red C-A): GeoMean = 97.4
10
10
(Violet B-A): GeoMean = 206
R ed B- A
G reenC -A
10
2
Violet A- A
(Green D-A): GeoMean = 3.39
0
10
(Red B-A): GeoMean = 870
(Green C-A): GeoMean = 165
0
3
R ed A- A
(Green B-A): GeoMean = 986
0
10
(Violet A-A): GeoMean = 1890
3
10
4
(Violet E-A): Geo Mea n = 3.85
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 32.2
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 9.64
Green B: CD8 PerCP-Cy5.5
450v Baseline
0
10
2
10
3
10
4
Violet G- A
CD8 PerCPCy55 DukeJY_450vbasline.fcs
(Violet H-A): GeoMean = 18.7
44
0
10
2
10
Violet H- A
3
10
4
(Blue B-A): GeoMean = 15.1
0
10
2
10
3
10
4
(Green A-A): GeoMean = 609
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 549
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
10
5
0
2
10
10
2
R ed B- A
3
10
4
10
5
0
10
2
10
10
4
3
10
4
10
5
0
10
2
10
4
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
(Violet D-A): GeoMean = 26.3
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 10.7
10
10
Violet B- A
G reenD -A
0
3
(Violet C-A): GeoMean = 37.2
R ed C- A
3
10
Violet A- A
(Red C-A): GeoMean = 114
G reenC -A
10
2
(Violet B-A): GeoMean = 206
(Green D-A): GeoMean = 18
0
10
(Red B-A): GeoMean = 1181
(Green C-A): GeoMean = 442
0
3
R ed A- A
(Green B-A): GeoMean = 4343
0
10
(Violet A-A): GeoMean = 3418
3
10
4
(Violet E-A): Geo Mea n = 28.2
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 274
0
Green B: CD8 PerCPCy5.5
550v Final
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 192
0
10
2
10
3
10
4
Violet G- A
CD8 PerCPCy55 DukeJY_550v final.fcs
(Violet H-A): GeoMean = 91.4
45
0
10
2
10
Violet H- A
3
10
4
(Blue B-A): GeoMean = 23.7
0
10
2
10
3
10
4
(Green A-A): GeoMean = 1497
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 368
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
3
10
4
10
5
0
2
10
10
5
0
10
2
10
2
10
4
3
10
4
10
5
0
10
2
10
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 284
10
4
(Violet D-A): GeoMean = 53.2
G reenD -A
0
10
(Violet C-A): GeoMean = 25.6
R ed C- A
3
3
Violet B- A
(Red C-A): GeoMean = 1663
10
10
(Violet B-A): GeoMean = 267
R ed B- A
G reenC -A
10
2
Violet A- A
(Green D-A): GeoMean = 305
0
10
(Red B-A): GeoMean = 1324
(Green C-A): GeoMean = 2747
0
3
R ed A- A
(Green B-A): GeoMean = 6724
0
10
(Violet A-A): GeoMean = 321
3
10
4
(Violet E-A): Geo Mea n = 16.8
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 39.1
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 11.1
Green C: CD27 PE-Cy5
350v Baseline
0
10
2
10
3
10
4
Violet G- A
CD27 PE-Cy5 VRC_350v baseline.fcs
(Violet H-A): GeoMean = 19.4
46
0
10
2
10
Violet H- A
3
10
4
(Blue B-A): GeoMean = 23.4
0
10
2
10
3
10
4
(Green A-A): GeoMean = 1507
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 709
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
10
5
0
2
10
10
2
R ed B- A
3
10
4
10
5
0
10
2
10
10
4
3
10
4
10
5
0
10
2
10
4
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
(Violet D-A): GeoMean = 129
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 1689
10
10
Violet B- A
G reenD -A
0
3
(Violet C-A): GeoMean = 114
R ed C- A
3
10
Violet A- A
(Red C-A): GeoMean = 1904
G reenC -A
10
2
(Violet B-A): GeoMean = 252
(Green D-A): GeoMean = 1379
0
10
(Red B-A): GeoMean = 1805
(Green C-A): GeoMean = 17138
0
3
R ed A- A
(Green B-A): GeoMean = 13446
0
10
(Violet A-A): GeoMean = 563
3
10
4
(Violet E-A): Geo Mea n = 82.4
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 306
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 196
Green C: CD27 PE-Cy5
450v Final
0
10
2
10
3
10
4
Violet G- A
CD27 PE-Cy5 VRC_450v final.fcs
(Violet H-A): GeoMean = 91.5
47
0
10
2
10
Violet H- A
3
10
4
(Blue B-A): GeoMean = 33.7
0
10
2
10
3
10
4
(Green A-A): GeoMean = 614
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 6.68
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
10
5
0
2
10
10
2
R ed B- A
3
10
4
10
5
0
10
2
10
10
4
3
10
4
10
5
0
10
2
10
4
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
(Violet D-A): GeoMean = 50.7
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 252
10
10
Violet B- A
G reenD -A
0
3
(Violet C-A): GeoMean = 251
R ed C- A
3
10
(Violet B-A): GeoMean = 229
(Red C-A): GeoMean = 42.5
G reenC -A
10
2
Violet A- A
(Green D-A): GeoMean = 22260
0
10
(Red B-A): GeoMean = 24.6
(Green C-A): GeoMean = 4539
0
3
R ed A- A
(Green B-A): GeoMean = 2966
0
10
(Violet A-A): GeoMean = 139
3
10
4
(Violet E-A): Geo Mea n = 10.9
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 34.7
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 9.53
Green D: CD45RO PE-TR
430v Baseline & Final
0
10
2
10
3
10
4
Violet G- A
CD45RO PE -T R VRC_430v baeline.fcs
(Violet H-A): GeoMean = 19.1
48
0
10
2
10
Violet H- A
3
10
4
(Blue B-A): GeoMean = 44.3
0
10
2
10
3
10
4
(Green A-A): GeoMean = 269
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 3.41
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
10
5
0
2
10
10
2
R ed B- A
3
10
4
10
5
0
10
2
10
10
4
3
10
4
10
5
0
10
2
10
4
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
(Violet D-A): GeoMean = 925
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 28006
10
10
Violet B- A
G reenD -A
0
3
(Violet C-A): GeoMean = 395
R ed C- A
3
10
(Violet B-A): GeoMean = 222
(Red C-A): GeoMean = 24.4
G reenC -A
10
2
Violet A- A
(Green D-A): GeoMean = 22352
0
10
(Red B-A): GeoMean = 8.53
(Green C-A): GeoMean = 3200
0
3
R ed A- A
(Green B-A): GeoMean = 1604
0
10
(Violet A-A): GeoMean = 95.4
3
10
4
(Violet E-A): Geo Mea n = 244
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 90.3
Green E: MIP1b PE
350v Baseline &
Final
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 10
0
10
2
10
3
10
4
Violet G- A
M IP1b PE DukeJY _450v baseline.fcs
(Violet H-A): GeoMean = 18.6
49
0
10
2
10
Violet H- A
3
10
4
Blue
Duke LSRII
Optimization of
Specific
Fluorescence
Primary
Fluorescence
Secondary
Fluorescence
Violet
Red
Green
(Blue B-A): GeoMean = 15.3
0
10
2
10
3
10
4
(Green A-A): GeoMean = 9.54
10
5
0
10
2
10
3
10
4
(Red A-A): GeoMean = 28.4
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
2
10
4
10
5
0
10
2
10
3
10
4
10
5
0
10
2
10
2
10
3
10
4
10
5
0
10
2
10
4
3
10
4
10
5
0
10
2
10
4
10
5
10
3
10
4
10
5
2
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
Violet C- A
(Violet D-A): GeoMean = 30.1
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 214
10
10
(Violet C-A): GeoMean = 38.8
G reenD -A
0
3
Violet B- A
R ed C- A
3
10
Violet A- A
(Red C-A): GeoMean = 41.2
G reenC -A
10
2
(Violet B-A): GeoMean = 85.3
(Green D-A): GeoMean = 250
0
10
R ed B- A
(Green C-A): GeoMean = 72.1
10
10
(Red B-A): GeoMean = 42.1
G reenB -A
0
3
R ed A- A
(Green B-A): GeoMean = 51.2
0
10
(Violet A-A): GeoMean = 10.6
3
10
4
(Violet E-A): Geo Mea n = 29.5
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): Geo Mea n = 235
0
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 178
Neg Comp Beads
0
10
2
10
3
10
4
Violet G- A
Controls_Neg Comp.fcs
(Violet H-A): GeoMean = 90.9
51
0
10
2
10
Violet H- A
3
10
4
LSRII Qualification


Validation



Optimization




Linearity
Resolution
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Select Optimal Voltages for Inst Performance with AssaySpecific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)




Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision





Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines
(Blue B-A): GeoMean = 4587
0
10
2
10
3
10
4
(Green A-A): GeoMean = 1505
10
5
0
10
2
10
3
10
4
(Red A-A): Ge oMe an = 2172
10
5
0
10
2
G reenA -A
B lue B-A
10
2
10
3
10
4
10
5
0
10
2
G reenB -A
10
2
10
10
4
10
5
0
10
3
10
4
3
10
4
10
5
0
2
10
10
5
0
10
2
10
2
10
4
3
10
4
10
5
0
10
2
2
10
10
5
10
3
10
4
10
5
10
3
10
4
10
5
10
5
10
5
10
5
10
5
10
5
(Violet D-A): GeoMean = 2539
10
5
0
10
2
10
3
10
4
Violet D- A
(Green E-A): GeoMean = 30710
10
4
Violet C- A
G reenD -A
0
10
(Violet C-A): GeoMean = 3283
R ed C- A
3
3
Violet B- A
(Red C-A): Ge oMe an = 3934
10
10
(Violet B-A): GeoMean = 970
R ed B- A
G reenC -A
10
2
Violet A- A
(Green D-A): GeoMean = 65203
0
10
(Red B-A): Ge oMe an = 3927
(Green C-A): GeoMean = 22057
0
3
R ed A- A
(Green B-A): GeoMean = 11590
0
10
(Violet A-A): GeoMean = 712
3
10
4
(Violet E-A): GeoMea n = 3052
10
5
0
10
2
G reenE-A
10
3
10
4
Violet E-A
(Violet F-A): GeoMea n = 12196
0
1x
(Target Channel
Values)
10
2
10
3
10
4
Violet F-A
(Violet G-A): GeoMean = 27119
0
10
2
10
3
10
4
Violet G- A
Controls_1x.fcs
(Violet H-A): GeoMean = 4157
53
0
10
2
10
Violet H- A
3
10
4
Target Channels & Ranges
LSRII Qualification


Validation



Optimization




Linearity
Resolution
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Select Optimal Voltages for Inst Performance with AssaySpecific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)




Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision





Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines
Duke University Medical Center
Daily Calibration
• Mid-Range or “1x” Rainbow Beads: TC settings
•
•
•
•
Moderate fluorescence, similar to cells; Broad Spectrum Ex & Em
1pk
Run: Morning & Before each Exp - “High” flow rate & LOG scale
Uses: Calibration & Standardization
•
•
•
•
Set Assay-Specific Target Channels (TCs)
Determine Daily Voltage Settings
Daily QC: P/F (CVs, voltages, trends)
LSRII Performance Verification for Exp Run: P/F (CVs & TCs)
• 8 Peak Rainbow & Unstained Comp Beads: TC Settings
•
•
•
•
Non-fluorescent to Very Bright; Broad Spectrum Ex & Em
8pk and 1pk
Run: Once Daily - “High” flow rate & LOG scale
Uses: Standardization
•
•
•
•
Check Linearity
Check S:N (LLOD & LLOQ)
Check Specificity
Check Resolution
• Ultra Rainbow Beads: Mean ch ~150,000
•
•
•
•
VERY Bright; Broad Spectrum Ex & Em
1pk
Run: Once Daily - “Low” flow rate & linear scale
Uses: Manufacturer Recommended
•
•
•
•
Daily QC: P/F (CV & voltages)
Check/Adjust Area Scaling
Check/Adjust Time Delay
Check/Adjust Window Extension
Duke University Medical Center
Daily Standardization
•
•
•
Mid-Range or “1x” Rainbow Beads:
• Record Daily For each PMT & Scatter
• CV
• HV
• Median
• Plot Monthly For each PMT & Scatter
• CV
• HV vs Median
8 Peak Rainbow & Unstained Comp Beads:
• Record Daily For each PMT & Scatter
• Median
• Neg Comp Beads
• Peak 5
• Peak 6
• Frequency
• Peak 5
• Peak 6
• CV: peak 5
• HV
• Plot Monthly For each PMT & Scatter
• Linearity: Medianpk6-Medianpk5/Medianpk5
• Signal:Noise: Medianpk5/MedianNeg
• Q & B???
Ultra Rainbow Beads:
• Record Daily For each PMT & Scatter
• CV
• HV
• Median
• Plot Monthly For each PMT & Scatter
• CV
• HV vs Median
Duke University Medical Center
1x Blue B Trend line
1 X B lu e B (+/- 5 % o f M e a n )
475
10,000
9,000
465
8,000
6,000
V o ltag e
455
5,000
445
M ed ian
7,000
4,000
3,000
435
2,000
1X V oltages
1X M edians
1,000
11/13/2006
11/6/2006
10/30/2006
10/23/2006
10/16/2006
10/9/2006
10/2/2006
9/25/2006
9/18/2006
9/11/2006
9/4/2006
8/28/2006
8/14/2006
8/21/2006
0
8/7/2006
425
58
Duke University Medical Center
Ratio and Voltage Variation
700
500
300
B515
100
G 610
R660
V585
0
1
2
3
4
5
6
7
8
9
10
11
3000
3 .5
2500
3 .0
2 .5
2000
2 .0
1500
1 .5
1000
1 .0
R at io
500
CV
0 .5
0
0 .0
1
2
3
4
5
6
7
8
9
1
11
D ays
Duke University Medical Center
LASERs have a limited lifespan…
Laser Current
Laser Power
Laser Performance
5.84
16.00
5.82
5.80
15.60
5.78
5.76
15.40
5.74
15.20
5.72
15.00
5.70
5.68
14.80
Date
L a s e r P o w e r ( m W a tt s )
L a ser C u r r en t (A m p s)
15.80
Overview
Why QC is Important
LSRII Optical Configuration
LSRII QC




LSRII Validation
LSRII Optimization
LSRII Calibration
LSRII Standardization
Practice Analysis??
Duke University Medical Center
Example: Stain Index, Qr, and Br
Across Laboratories
Example : FITC Channel
Lab
SI
Qr
Br
8 peak beads
12 00
90 0
1
77.4
0.015
233
60 0
30 0
0
0
2
10
3
10
4
10
5
10
10 00
80 0
2
134.5
0.028
216
High SI = Good Resolution;
correlates with high Qr and low Br
60 0
40 0
20 0
0
0
2
10
3
10
4
10
5
10
10 00
80 0
3
55.6
0.015
976
60 0
40 0
20 0
0
0
2
10
10
3
10
4
10
5
12 00
90 0
4
80.5
0.01
298
60 0
30 0
0
0
2
10
3
10
10
4
5
10
80 0
60 0
5
66
0.01
613
40 0
20 0
0
0
10
2
10
3
10
4
10
5
Low SI = Bad Resolution;
correlates with low Qr and high Br
6
13.7
0.007
2322
# C el l s
60 0
40 0
20 0
0
0
10
2
10
3
10
4
10
5
10 00
80 0
7
16.2
0.018
2768
60 0
40 0
20 0
0
0
10
2
10
3
10
4
10
5
Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry
Assays.
Jaimes MC,1 Stall A,1 Inokuma M,1 Hanley MB,1 Maino S,1 D’Souza MP,2 and Yan M.1 ISAC 2010
1BD Biosciences, San Jose, CA 95131; 2Division of AIDS, National Institute of Allergy and
Low SI = Bad Resolution;
correlates with high Br
Correlation of SI, Qr, and Br with
Assay Performance
Lab 2
Lab 4
Lab 6
Plots gated on
lymphocytes
Plots gated
on CD3+
cells
Plots gated on
CD3+CD8+
cells
Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry
Assays.
Jaimes MC, Stall A, Inokuma M, et al. ISAC 2010
Lab 7
1 Assay – 4 Platforms
BD FACSCanto II
BD FACSAria III
64
CD4 V450
• CD8 PE
• CD3 FITC
BD LSR II • CD20 APC
•
BD LSRFortessa
Thank You!!
65
Defining Instrument Performance
and Sensitivity: Qr, Br, and SDen
Qr: Anti-CD10 PE Example
Dim
Population
The laser and detectors
were attenuated by ND
filters over a 30-fold
range to illustrate the
effects of decreasing
detector sensitivity on
population resolution.
Qr=0.227
Transmission
SI
Corrected
100%
83
Qr=0.087
35.5%
55
Qr=0.038
17.8%
35
Qr=0.014
7.1%
20
Qr=0.007
3.5%
14
Relative Background: Br
• Br is a measure of true optical background in the fluorescence
detector, which helps indicate how easily (dim) signals may be
resolved from unstained cells in that detector.
Unbound antibody
or fluorochrome
Scatter from the flow
cell and ambient light.
Raman scatter
Spectral overlap
on a cell
Cell
autofluorescence
• Factors affecting Br: dirty flow cell, damaged optical component
Why is Br important?
• High Br widens negative and dim populations.
• High Qr value = lower resolution
• Low Qr value = higher resolution
Low Br
High Br
Br: Optical Background from Propidium
Iodide
•
Example: It is common to use propidium iodide (PI) to distinguish live
from dead cells. Propidium iodide was added in increasing amounts to
the buffer containing beads, and Qr and Br were estimated.
PerCP
Br
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Br
Qr
0.05
0.04
0.03
0.02
Qr
0.01
0
0 1 PI-free
2 3dye 4(µg) 5 6
•
Residual PI in your sample tube will increase Br, which will reduce
sensitivity.
Electronic Noise (SDen)
• SDen is the background signal due to electronics:
• Contributed by:
• PMT connections / PMT noise
• Cables too near power sources
• Digital error
• Broadens the distribution of unstained or dim particles
• Increases in electronic noise results in decreased
resolution sensitivity
Most important for channels with low cellular autofluorescence
• APC-Cy™7, PE-Cy™7, PerCP-Cy™5.5
Cy™ is a trademark of Amersham Biosciences Corp. Cy™ dyes are subject to
proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and
are made and sold under license from Amersham Biosciences Corp only for research
and in vitro diagnostic use. Any other use requires a commercial sublicense from
Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855-1327,
USA.
Gel (from coupling) found all over the flow
cell????
Green E
Green D
Green C
Green B
Green A
10000
1000
CV
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
100
10
1
0
200
400
600
voltage
800
1000
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