instrument qc and qualification. overview why qc is important lsrii optical configuration lsrii...
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Instrument QC and Qualification
Overview
Why QC is ImportantLSRII Optical ConfigurationLSRII QC
LSRII ValidationLSRII Optimization LSRII Calibration LSRII Standardization
Practice Analysis??
3
Characterizing the Cytometer
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) yes yes• CV (resolution) yes (target) yes (range)• Signal-to-noise ratio NO yes (range)• Linearity Yes (range) yes (range)• Specific fluorescence NO yes (target)
• Automated YES NO
Instrument QC
Validation: optimal voltage range for each detector
Optimization: assay specific optimal voltage for each detector & assay specific target channels
Calibration: set detectors to assay specific target channel values for specimen acquisition
Standardization: Monitor trendlines using assay specific target channels over time
Initial Characterization & After Optical Service
Once Per Assay
Each Experiment &
Troubleshooting
Frequency Purpose
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 Assay-
Specific 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)Median Pk5
= Constant
• Defined as proportionality of output (MFI) to input (Fluorescence/ # of photons)
• 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
0 1000 2000 3000
Ab = X Ab = 2XX Abs2X Abs =
1000 MFI2000 MFI
73,000
365
D
1796
75
B
Linearity: Effect on Compensation
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.
FITC
PE
A
68
80
Detector Median Fluorescence Intensity (MFI)
5921
79
C
• 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
LSRII Qualification
Validation Linearity Resolution (CV) Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization Select Optimal Voltages for Inst Performance with Assay-
Specific 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
16
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
18
• High Br widens negative and dim populations. • High Br value = lower resolution • Low Br value = higher resolution
Why is Br important?
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.
• Residual PI in your sample tube will increase Br, which will reduce sensitivity.
PerCP
0100020003000400050006000700080009000
0 1 2 3 4 5 6PI-free dye (µg)
Br
0
0.01
0.02
0.03
0.04
0.05
Qr
BrQr
23
PMT 1
PMT 2
Qr =
Qr =
Qr# fluorescence molecules
# photoelectrons=
= 0.252 photoelectrons
8 fluorescence molecules
= 0.1251 photoelectron
8 fluorescence molecules
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.
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
Why is Qr important?
•Laser power
•Optical efficiency
•PMT sensitivity (red spectrum)•Poor PMT performance •Dirty flow cell•Dirty or degraded filter
What Factors Affect Qr?
Population resolution for a given fluorescence parameter is decreased by increased spread due to spillover from other fluorochromes.
Spillover Decreases Resolution Sensitivity
Spread from APC Cy-7 background
29
30
31
32
Methods Used for Validation
Which Beads to UseWhat Values to PlotSelection Criteria
Duke LSRII Optical Configuration
34
Green Laser
(532nm)
Duke LSRII
PE
Cy7
PE
Cy5
PE
TR
PE
Cy5.5
PE-
Green780/40
610/20
710/50
660/40
575/25
740LP640LP
600LP
690LP
Violet Laser
(407nm)
Duke LSR II
QDot
705
QDot
605
QDot
545
QDot
585
QDot
655
QDot
565705/70
585/42
660/40
605/40
560/40
670LP595LP
570LP
630LP
557LP
560/40
535LP
Am
Cyan
515/20
505LP
Cas
Blue45
0/50
Blue Laser
(488nm)
Duke LSRII
SSC
Blue
FITC
515/20
505LP
575/25
550LP
PE ミBlue
488LP
Red Laser
(635nm)
Duke LSRII
APC
Alexa
680
710/50
685LP
780/60
740LP
APC
Cy7
660/20
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
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
300 Volts 350 Volts 400 Volts 450 Volts 500 Volts
550 Volts 600 Volts 650 Volts 700 Volts 750 Volts
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
0 102 103 104 105
Green B-A
0
1000
2000
3000
4000
SS
C-A
Criteria for the Selection of Voltage Ranges
Lowest CV possibleLowest Voltage possibleHighest MFI possibleLowest background possible
Example of Optimal PMT Performance & Voltage Selection Criteria (Green B)
GreenB Voltage vs. CV, LIN and Ratio
0
10
20
30
40
50
60
70
300 350 400 450 500 550 600 650 700 750
Voltage
CV
or
LIN
0
50
100
150
200
250
300
350
400
450
500
Rat
io
CV Lin Ratio
Optimal Voltages: 450-650
Selection Criteria:LinearLowest CVHighest S:NLowest Voltage
Linearity = (M2-M1)/M1Ratio = M1/B
Red A (APC-Cy7):Before (30Mar06) & After (10Apr06) Replacing PMT
RedA Voltage vs. CV, LIN and Ratio
0
20
40
60
80
100
120
140
160
180
200
300 350 400 450 500 550 600 650 700 750
VoltageC
V o
r L
IN
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Rat
io
CV Lin Ratio
RedA Voltage vs. CV, LIN and Ratio
0
20
40
60
80
100
120
140
160
180
200
300 350 400 450 500 550 600 650 700 750
Voltage
CV
or
LIN
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Rat
io
CV Lin Ratio
Optimal Voltages: 625-700Optimal Voltages: IndeterminateHigh CV’s; poor S:N
30 March 2006 10 April 2006
Faulty PMT on install…
0 102 103 104 105
Red A-A
0
1000
2000
3000
4000
SS
C-A
650 Volts
0 102 103 104 105
Red C-A
0
1000
2000
3000
4000
SS
C-A
650 Volts
Before After
LSRII Qualification
Validation Linearity Resolution Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization Select Optimal Voltages for Inst Performance with Assay-
Specific 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 celllular 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.
Green A: TNF PE-Cy7460v Baseline & Final
44
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 19 (Green A-A): GeoMean = 5462
(Green B-A): GeoMean = 44.7
(Green C-A): GeoMean = 26.3
(Green D-A): GeoMean = 99.2
(Green E-A): GeoMean = 90.2
(Red A-A): GeoMean = 129
(Red B-A): GeoMean = 5.69
(Red C-A): GeoMean = 15
(Violet A-A): GeoMean = 8.05
(Violet B-A): GeoMean = 47
(Violet C-A): GeoMean = 12.3
(Violet D-A): GeoMean = 21.4
(Violet E-A): GeoMean = 7.82
(Violet F-A): GeoMean = 32.2
(Violet G-A): GeoMean = 9.43
(Violet H-A): GeoMean = 18.8TNFa PE-Cy7 DukeJY_460v baseline.fcs
45
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 14.1 (Green A-A): GeoMean = 617
(Green B-A): GeoMean = 986
(Green C-A): GeoMean = 165
(Green D-A): GeoMean = 3.39
(Green E-A): GeoMean = 0.98
(Red A-A): GeoMean = 295
(Red B-A): GeoMean = 870
(Red C-A): GeoMean = 97.4
(Violet A-A): GeoMean = 1890
(Violet B-A): GeoMean = 206
(Violet C-A): GeoMean = 6.55
(Violet D-A): GeoMean = 8.57
(Violet E-A): GeoMean = 3.85
(Violet F-A): GeoMean = 32.2
(Violet G-A): GeoMean = 9.64
(Violet H-A): GeoMean = 18.7CD8 PerCPCy55 DukeJY_450vbasline.fcsGreen B: CD8 PerCP-Cy5.5
450v Baseline
Green B: CD8 PerCP-Cy5.5550v Final46
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 15.1 (Green A-A): GeoMean = 609
(Green B-A): GeoMean = 4343
(Green C-A): GeoMean = 442
(Green D-A): GeoMean = 18
(Green E-A): GeoMean = 10.7
(Red A-A): GeoMean = 549
(Red B-A): GeoMean = 1181
(Red C-A): GeoMean = 114
(Violet A-A): GeoMean = 3418
(Violet B-A): GeoMean = 206
(Violet C-A): GeoMean = 37.2
(Violet D-A): GeoMean = 26.3
(Violet E-A): GeoMean = 28.2
(Violet F-A): GeoMean = 274
(Violet G-A): GeoMean = 192
(Violet H-A): GeoMean = 91.4CD8 PerCPCy55 DukeJY_550v final.fcs
Green C: CD27 PE-Cy5350v Baseline47
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 23.7 (Green A-A): GeoMean = 1497
(Green B-A): GeoMean = 6724
(Green C-A): GeoMean = 2747
(Green D-A): GeoMean = 305
(Green E-A): GeoMean = 284
(Red A-A): GeoMean = 368
(Red B-A): GeoMean = 1324
(Red C-A): GeoMean = 1663
(Violet A-A): GeoMean = 321
(Violet B-A): GeoMean = 267
(Violet C-A): GeoMean = 25.6
(Violet D-A): GeoMean = 53.2
(Violet E-A): GeoMean = 16.8
(Violet F-A): GeoMean = 39.1
(Violet G-A): GeoMean = 11.1
(Violet H-A): GeoMean = 19.4CD27 PE-Cy5 VRC_350v baseline.fcs
Green C: CD27 PE-Cy5450v Final48
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 23.4 (Green A-A): GeoMean = 1507
(Green B-A): GeoMean = 13446
(Green C-A): GeoMean = 17138
(Green D-A): GeoMean = 1379
(Green E-A): GeoMean = 1689
(Red A-A): GeoMean = 709
(Red B-A): GeoMean = 1805
(Red C-A): GeoMean = 1904
(Violet A-A): GeoMean = 563
(Violet B-A): GeoMean = 252
(Violet C-A): GeoMean = 114
(Violet D-A): GeoMean = 129
(Violet E-A): GeoMean = 82.4
(Violet F-A): GeoMean = 306
(Violet G-A): GeoMean = 196
(Violet H-A): GeoMean = 91.5CD27 PE-Cy5 VRC_450v final.fcs
(Blue B-A): GeoMean = 33.7
Green D: CD45RO PE-TR430v Baseline & Final
49
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 33.7 (Green A-A): GeoMean = 614
(Green B-A): GeoMean = 2966
(Green C-A): GeoMean = 4539
(Green D-A): GeoMean = 22260
(Green E-A): GeoMean = 252
(Red A-A): GeoMean = 6.68
(Red B-A): GeoMean = 24.6
(Red C-A): GeoMean = 42.5
(Violet A-A): GeoMean = 139
(Violet B-A): GeoMean = 229
(Violet C-A): GeoMean = 251
(Violet D-A): GeoMean = 50.7
(Violet E-A): GeoMean = 10.9
(Violet F-A): GeoMean = 34.7
(Violet G-A): GeoMean = 9.53
(Violet H-A): GeoMean = 19.1CD45RO PE-TR VRC_430v baeline.fcs
Green E: MIP1b PE350v Baseline & Final50
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 44.3 (Green A-A): GeoMean = 269
(Green B-A): GeoMean = 1604
(Green C-A): GeoMean = 3200
(Green D-A): GeoMean = 22352
(Green E-A): GeoMean = 28006
(Red A-A): GeoMean = 3.41
(Red B-A): GeoMean = 8.53
(Red C-A): GeoMean = 24.4
(Violet A-A): GeoMean = 95.4
(Violet B-A): GeoMean = 222
(Violet C-A): GeoMean = 395
(Violet D-A): GeoMean = 925
(Violet E-A): GeoMean = 244
(Violet F-A): GeoMean = 90.3
(Violet G-A): GeoMean = 10
(Violet H-A): GeoMean = 18.6MIP1b PE DukeJY_450v baseline.fcs
(Blue B-A): GeoMean = 47.2 (Green A-A): GeoMean = 267 (Red A-A): GeoMean = 4.12 (Violet A-A): GeoMean = 175
Duke LSRII Optimization of
Specific Fluorescence Violet
Red
Green
Blue
Primary Fluorescence
SecondaryFluorescence
Neg Comp Beads52
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 15.3 (Green A-A): GeoMean = 9.54
(Green B-A): GeoMean = 51.2
(Green C-A): GeoMean = 72.1
(Green D-A): GeoMean = 250
(Green E-A): GeoMean = 214
(Red A-A): GeoMean = 28.4
(Red B-A): GeoMean = 42.1
(Red C-A): GeoMean = 41.2
(Violet A-A): GeoMean = 10.6
(Violet B-A): GeoMean = 85.3
(Violet C-A): GeoMean = 38.8
(Violet D-A): GeoMean = 30.1
(Violet E-A): GeoMean = 29.5
(Violet F-A): GeoMean = 235
(Violet G-A): GeoMean = 178
(Violet H-A): GeoMean = 90.9Controls_Neg Comp.fcs
LSRII Qualification
Validation Linearity Resolution Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization Select Optimal Voltages for Inst Performance with Assay-
Specific 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
1x(Target Channel Values) 54
0 10 2 10 3 10 4 10 5
Blue B-A0 10 2 10 3 10 4 10 5
Green A-A
0 10 2 10 3 10 4 10 5
Green B-A
0 10 2 10 3 10 4 10 5
Green C-A
0 10 2 10 3 10 4 10 5
Red A-A
0 10 2 10 3 10 4 10 5
Green D-A
0 10 2 10 3 10 4 10 5
Green E-A
0 10 2 10 3 10 4 10 5
Red B-A
0 10 2 10 3 10 4 10 5
Red C-A
0 10 2 10 3 10 4 10 5
Violet A-A
0 10 2 10 3 10 4 10 5
Violet B-A
0 10 2 10 3 10 4 10 5
Violet C-A
0 10 2 10 3 10 4 10 5
Violet D-A
0 10 2 10 3 10 4 10 5
Violet E-A
0 10 2 10 3 10 4 10 5
Violet F-A
0 10 2 10 3 10 4 10 5
Violet G-A
0 10 2 10 3 10 4 10 5
Violet H-A
(Blue B-A): GeoMean = 4587 (Green A-A): GeoMean = 1505
(Green B-A): GeoMean = 11590
(Green C-A): GeoMean = 22057
(Green D-A): GeoMean = 65203
(Green E-A): GeoMean = 30710
(Red A-A): GeoMean = 2172
(Red B-A): GeoMean = 3927
(Red C-A): GeoMean = 3934
(Violet A-A): GeoMean = 712
(Violet B-A): GeoMean = 970
(Violet C-A): GeoMean = 3283
(Violet D-A): GeoMean = 2539
(Violet E-A): GeoMean = 3052
(Violet F-A): GeoMean = 12196
(Violet G-A): GeoMean = 27119
(Violet H-A): GeoMean = 4157Controls_1x.fcs
Target Channels & Ranges
LSRII Qualification
Validation Linearity Resolution Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization Select Optimal Voltages for Inst Performance with Assay-
Specific 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
59
1X Blue B (+/- 5% of Mean)
425
435
445
455
465
475
8/7/
2006
8/14
/200
6
8/21
/200
6
8/28
/200
6
9/4/
2006
9/11
/200
6
9/18
/200
6
9/25
/200
6
10/2
/200
6
10/9
/200
6
10/1
6/20
06
10/2
3/20
06
10/3
0/20
06
11/6
/200
6
11/1
3/20
06
Vo
ltag
e
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
9,000
10,000
Med
ian
1X Voltages
1X Medians
Duke University Medical Center
Ratio and Voltage Variation
0
100
300
500
700
1 2 3 4 5 6 7 8 9 10 11
B515 G610 R660 V585
0
500
1000
1500
2000
2500
3000
1 2 3 4 5 6 7 8 9 1 11
Days
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Ratio CV
Duke University Medical Center
LASERs have a limited lifespan…
Laser Performance
5.68
5.70
5.72
5.74
5.76
5.78
5.80
5.82
5.84
Date
Las
er C
urr
ent
(Am
ps)
14.80
15.00
15.20
15.40
15.60
15.80
16.00
La
se
r P
ow
er
(mW
att
s)
Laser CurrentLaser Power
Overview
Why QC is ImportantLSRII Optical ConfigurationLSRII QC
LSRII Validation LSRII Optimization LSRII Calibration LSRII Standardization
Practice Analysis??
Duke University Medical Center
8 peak beads
0 10 2 10 3 10 4 10 5
0
200
400
600
800
1000
0 10 2 10 3 10 4 10 5
0
200
400
600
800
1000
0 10 2 10 3 10 4 10 5
0
300
600
900
1200
0 10 2 10 3 10 4 10 5
0
200
400
600
800
1000
0 10 2 10 3 10 4 10 5
0
200
400
600
800
0 10 2 10 3 10 4 10 50
200
400
600
# C
ells
Example: Stain Index, Qr, and Br Across LaboratoriesExample : FITC Channel
Lab SI Qr Br
1 77.4 0.015 233
2 134.5 0.028 216
3 55.6 0.015 976
4 80.5 0.01 298
5 66 0.01 613
6 13.7 0.007 2322
7 16.2 0.018 2768
Low SI = Bad Resolution; correlates with low Qr and high Br
High SI = Good Resolution; correlates with high Qr and low Br
Low SI = Bad Resolution; correlates with high Br
0 10 2 10 3 10 4 10 50
300
600
900
1200
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 20101BD Biosciences, San Jose, CA 95131; 2Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Plots gated on lymphocytes
Plots gated on CD3+
cells
Plots gated on CD3+CD8+
cells
Correlation of SI, Qr, and Br with Assay Performance
Lab 2 Lab 4 Lab 6 Lab 7
Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry Assays.Jaimes MC, Stall A, Inokuma M, et al. ISAC 2010
65
BD FACSCanto II
1 Assay – 4 Platforms
BD LSR II
BD LSRFortessaBD FACSAria III
• CD4 V450 • CD8 PE• CD3 FITC• CD20 APC
Thank You!!
66
Defining Instrument Performance and Sensitivity: Qr, Br, and SDen
Qr: Anti-CD10 PE Example
Trans-mission
100% 83
35.5% 55
17.8% 35
7.1% 20
3.5% 14
SICorrected
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=0.087
Qr=0.227
Qr=0.038
Qr=0.014
Qr=0.007
Dim Population
• 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
Spectral overlap on a cell
Cell autofluorescence
Raman scatter
Scatter from the flow cell and ambient light.
Relative Background: Br
• Factors affecting Br: dirty flow cell, damaged optical component
• High Br widens negative and dim populations. • High Qr value = lower resolution • Low Qr value = higher resolution
Why is Br important?
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.
• Residual PI in your sample tube will increase Br, which will reduce sensitivity.
PerCP
0100020003000400050006000700080009000
0 1 2 3 4 5 6PI-free dye (µg)
Br
0
0.01
0.02
0.03
0.04
0.05
Qr
BrQr
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.
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.
•
• 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.
BrQr¥ ySensitivit relative
• Instrument performance can have a significant impact on the performance of an assay.
Resolution vs. Background
NegativePopulation
PositivePopulation
Negative population has low backgroundPopulations well resolvedNegative population has high backgroundPopulations 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.
negative
negativepositive
rSD2
medianmedian
Negative ofWidth
Brightness Index Stain
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
76
Gel (from coupling) found all over the flow cell????