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Instrument QC and Qualification. Why QC is Important LSRII Optical Configuration LSRII QC Validation Optimization Calibration Standardization Practice Analysis. Overview. Characterizing the Cytometer. Challenges….

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slide2
Why QC is Important

LSRII Optical Configuration

LSRII QC

Validation

Optimization

Calibration

Standardization

Practice Analysis

Overview
challenges
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
Two Methods of Instrument Charaterization

BD CS&T: Cytometer Setup and Tracking

instrument performance

Parameters CS&T Perfetto

Laser (amps vsmW) 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 Performance
instrument qc
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
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

LSRII qualification
linearity
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

linearity1
Linearity
  • 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

2X Abs

2000 MFI

=

X Abs

1000 MFI

Ab = X

Ab = 2X

0

1000

2000

3000

linearity effect on compensation

A

C

B

D

Detector

Median Fluorescence Intensity (MFI)

FITC

68

5921

1796

73,000

PE

80

79

365

75

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

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.

lsrii qualification1

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

LSRII Qualification
instrument sensitivity two definitions
Instrument Sensitivity: Two Definitions
  • Defining sensitivity
    • 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).
    • 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)
slide17

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

PerCP

9000

0.05

8000

0.04

7000

6000

0.03

5000

Br

Br

Qr

4000

Qr

0.02

3000

2000

0.01

1000

0

0

0

1

2

3

4

5

6

PI-free dye (µg)

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.
relative q qr
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.

# photoelectrons

Qr

=

# fluorescence molecules

PMT 1

2 photoelectrons

= 0.25

Qr =

8 fluorescence molecules

PMT 2

1 photoelectron

= 0.125

Qr =

8 fluorescence molecules

slide21

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

slide22

What Factors Affect Qr?

  • Laser power
  • Optical efficiency
  • PMT sensitivity (red spectrum)
  • Poor PMT performance
  • Dirty flow cell
  • Dirty or degraded filter
slide23

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

¥

Sensitivit

y

relative

Br

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.
  • 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

Resolution vs. Background

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
Measuring Sensitivity: The Stain Index
  • The Stain Index is a measure of reagent performance on a specific cytometer, a normalized signal over background metric.

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

=

D/W = Relative Brightness

Stain

Index

(SI)

Stain Index: Normalized Signal/Background

Goal: Normalize the signal

to the spread of background

where background may be

autofluorescence, unstained

cells, or compensated cells

from another dye dimension.

D

Wunstained

Wcompensateddye

D = difference between positive and negative peak medians

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

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

electronic noise sd en determining baseline pmt voltages

CV

Standard Deviation

PMT Voltage

500 V

18

180

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

10000

1000

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

1000

CV or SD

PMT Voltage

  • As PMT voltage is lowered, CV increases  resolution decreases

100

10

100

  • As PMT voltage is increasedCV unchanged  resolution unchanged

1

10

100

1000

10000

100000

Median Fluorescence Intensity

pmt voltages optimal gains can reduce classification errors

% Negative in CD4+ Monocyte Gate

12.0%

550 V

CD4 dim monocytes

10.0%

8.0%

% Negative in CD4 Gate

650 V

6.0%

CD4 negative

CD4+ lymphocytes

4.0%

2.0%

750 V

0.0%

-100

0

100

PMT Voltage Offset

PMT Voltages: Optimal Gains Can Reduce Classification Errors
methods used for validation

Methods Used for Validation

  • Which Beads to Use
  • What Values to Plot
  • Selection Criteria
example lsrii optical configuration

Blue Laser

Duke LSRII

Red Laser

Duke LSRII

(488nm)

(635nm)

FITC

Alexa

680

515/20

710/50

505LP

685LP

488LP

550LP

740LP

660/20

575/25

780/60

SSC

APC

PE

APC

Blue

Blue

Cy7

Green Laser

Duke LSRII

Violet Laser

Duke LSR II

(532nm)

(407nm)

QDot

PE

655

Cy5.5

QDot

PE

585

TR

660/40

710/50

585/42

690LP

610/20

630LP

Am

570LP

600LP

Cyan

QDot

505LP

515/20

560/40

545

535LP

557LP

575/25

560/40

450/50

640LP

595LP

740LP

PE-

670LP

QDot

660/40

605/40

Green

Cas

780/40

565

705/70

Blue

PE

QDot

PE

QDot

Cy5

605

Cy7

705

Example LSRII Optical Configuration

Duke University Medical Center

beads used for lsrii validation
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
Example of Optimal PMT Performance

300 Volts

350 Volts

400 Volts

450 Volts

500 Volts

550 Volts

600 Volts

650 Volts

700 Volts

750 Volts

criteria for the selection of voltage ranges
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
Example of Optimal PMT Performance & Voltage Selection Criteria (Green B)

Selection Criteria:

Linear

Lowest CV

Highest S:N

Lowest Voltage

Ratio = M1/B

Linearity = (M2-M1)/M1

Optimal Voltages: 450-650

red a apc cy7 before 30mar06 after 10apr06 replacing pmt
Red A (APC-Cy7):Before (30Mar06) & After (10Apr06) Replacing PMT

30 March 2006

10 April 2006

Optimal Voltages: 625-700

Optimal Voltages: Indeterminate

High CV’s; poor S:N

faulty pmt on install
Faulty PMT on install…

Before

After

650 Volts

650 Volts

lsrii qualification2

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

LSRII Qualification
beads used for lsrii optimization
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
Criteria for the Selection Optimal PMT Voltages

The primary fluorescence should be the highest in the respective detector relative to all secondary detectors.

slide49

Duke LSRII Optimization of Specific Fluorescence

Blue

Violet

Primary Fluorescence

Red

Green

Secondary

Fluorescence

lsrii qualification3

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

LSRII Qualification
lsrii qualification4

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

LSRII Qualification

Duke University Medical Center

daily calibration
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
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
1x Blue B Trend line

Duke University Medical Center

ratio and voltage variation
Ratio and Voltage Variation

Duke University Medical Center

overview

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: Stain Index, Qr, and Br Across Laboratories

Example : FITC Channel

High SI = Good Resolution; correlates with high Qr and low Br

Low SI = Bad Resolution;

correlates with low Qr and high Br

Low SI = Bad Resolution;

correlates with high Br

8 peak beads

Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry Assays.

Jaimes MC,1 Stall A,1Inokuma M,1 Hanley MB,1Maino S,1 D’Souza MP,2 and Yan M.1ISAC 2010

1BD Biosciences, San Jose, CA 95131; 2Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

correlation of si qr and br with assay performance
Correlation of SI, Qr, and Br with Assay Performance

Lab 2

Lab 4

Lab 6

Lab 7

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

1 assay 4 platforms

BD FACSCanto II

BD LSR II

BD FACSAria III

BD LSRFortessa

  • CD4 V450
  • CD8 PE
  • CD3 FITC
  • CD20 APC
1 Assay – 4 Platforms
qr anti cd10 pe example
Qr: Anti-CD10 PE Example

Dim

Population

SI

Corrected

Trans-

mission

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

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

slide67

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.

Scatter from the flow cell and ambient light.

Unbound antibody or fluorochrome

Raman scatter

Spectral overlap on a cell

Cell autofluorescence

  • Factors affecting Br: dirty flow cell, damaged optical component
slide68

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 iodide1

PerCP

9000

0.05

8000

0.04

7000

6000

0.03

5000

Br

Br

Qr

4000

Qr

0.02

3000

2000

0.01

1000

0

0

0

1

2

3

4

5

6

PI-free dye (µg)

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.
electronic noise sden
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.