Instrument QC and Qualification

Instrument QC and Qualification

Overview

Why QC is ImportantLSRII Optical ConfigurationLSRII QC

Validation Optimization Calibration 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




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

• 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

20

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

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 resolved

Negative 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

0

10

20

30

40

50

60

70

5.5 6.5 7.5 8.5 9.5 10.5 11.5

Stain Index: Normalized Signal/Background

D

Wunstained

Wcompensated dye

1Stain Index: metric used by Dave Parks, Stanford – Presented at ISAC 2004

Goal: Normalize the signalto the spread of backgroundwhere background may beautofluorescence, unstained cells, or compensated cellsfrom another dye dimension.

D/W = Relative Brightness(SI)Index Stain =

WD(SI)Index S tain D = difference between positive and negative peak medians

W = the spread of the background peak (= 2X rSDnegative)

29

Electronic Noise (SDEN): Determining Baseline PMT Voltages

10

100

1000

10000

1 10 100 1000 10000 100000

Median Fluorescence Intensity

CV

or

SD

100

1000

PM

T V

olt

age

CV

Standard Deviation

PMT Voltage

PE: Detailed Performance PlotDim Bead

500 V

• For this detector, the SDEN = 18

• Fluorescence intensity of dim bead = 10 x SDEN = 180

• Determine PMT voltage required to set the dim beads at 180 = 500 volts = baseline voltage

180

18

• As PMT voltage is lowered, CV increases resolution decreases

• As PMT voltage is increasedCV unchanged resolution unchanged

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)

30

CD4 dim monocytes

CD4+ lymphocytesCD4 negative

% Negative in CD4+ Monocyte Gate

0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

-100 0 100

PMT Voltage Offset

% N

egat

ive

in

CD

4 G

ate

PMT Voltages: Optimal Gains Can Reduce Classification Errors

550 V

750 V

650 V

31

Methods Used for Validation

Which Beads to UseWhat Values to PlotSelection Criteria

Example LSRII Optical Configuration

33

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


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





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.

Green A: TNF PE-Cy7460v Baseline & Final

43

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

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 = 14.1 (Green A-A): GeoMean = 617

(Green B-A): GeoMean = 986

(Green C-A): GeoMean = 165

(Green D-A): GeoMean = 3.39



(Red B-A): GeoMean = 870

(Red C-A): GeoMean = 97.4

(Violet A-A): GeoMean = 1890







(Violet H-A): GeoMean = 18.7CD8 PerCPCy55 DukeJY_450vbasline.fcsGreen B: CD8 PerCP-Cy5.5

450v Baseline

Green B: CD8 PerCP-Cy5.5550v Final45

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




(Green D-A): GeoMean = 18










(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 Baseline46

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





(Green E-A): GeoMean = 284











(Violet H-A): GeoMean = 19.4CD27 PE-Cy5 VRC_350v baseline.fcs

Green C: CD27 PE-Cy5450v Final47

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











(Violet C-A): GeoMean = 114

(Violet D-A): GeoMean = 129




(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

48

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






(Red A-A): GeoMean = 6.68










(Violet H-A): GeoMean = 19.1CD45RO PE-TR VRC_430v baeline.fcs

Green E: MIP1b PE350v Baseline & Final49

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













(Violet E-A): GeoMean = 244



(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 Beads51

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







(Violet B-A): GeoMean = 85.3






(Violet H-A): GeoMean = 90.9Controls_Neg Comp.fcs

LSRII Qualification






1x(Target Channel Values) 53

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












(Violet E-A): GeoMean = 3052



(Violet H-A): GeoMean = 4157Controls_1x.fcs

Target Channels & Ranges

LSRII Qualification







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


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


1x Blue B Trend line

58

1X Blue B (+/- 5% of Mean)

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Ratio and Voltage Variation

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B515 G610 R660 V585

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LASERs have a limited lifespan…

Laser Performance

5.68

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5.76

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5.84

Date

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er C

urr

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ps)

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se

r P

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Laser CurrentLaser Power

Overview

Why QC is ImportantLSRII Optical ConfigurationLSRII QC

LSRII Validation LSRII Optimization LSRII Calibration LSRII Standardization

Practice Analysis??


8 peak beads

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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

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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

64

BD FACSCanto II

1 Assay – 4 Platforms

BD LSR II

BD LSRFortessaBD FACSAria III

• CD4 V450 • CD8 PE• CD3 FITC• CD20 APC

Thank You!!

65

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

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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.

Gel (from coupling) found all over the flow cell????

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CV

Green E Green D

Green C Green B

Green A

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

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Documents

Instrument QC and Qualification