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Instrument QC and Qualification. Overview  Why QC is Important  LSRII Optical Configuration  LSRII QC  Validation  Optimization  Calibration  Standardization.

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Presentation on theme: "Instrument QC and Qualification. Overview  Why QC is Important  LSRII Optical Configuration  LSRII QC  Validation  Optimization  Calibration  Standardization."— Presentation transcript:

1 Instrument QC and Qualification

2 Overview  Why QC is Important  LSRII Optical Configuration  LSRII QC  Validation  Optimization  Calibration  Standardization  Practice Analysis

3 3 Characterizing the Cytometer

4 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

5 Two Methods of Instrument Charaterization BD CS&T: Cytometer Setup and Tracking

6 Instrument Performance ParametersCS&TPerfetto Laser (amps vs mW)yesyes CV (resolution)yes (target)yes (range) Signal-to-noise ratioNOyes (range) LinearityYes (range)yes (range) Specific fluorescenceNOyes (target) AutomatedYESNO

7 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 FrequencyPurpose

8 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

9 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

10 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

11 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 Ab = XAb = 2X X Abs 2X Abs = 1000 MFI 2000 MFI

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13 73, D 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 DetectorMedian Fluorescence Intensity (MFI) 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

14 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

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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)

17 Background contributions 17

18 High Br widens negative and dim populations. High Br value = lower resolution Low Br value = higher resolution Why is Br important? Low BrHigh Br

19 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 PI-free dye (µg) Br Qr Br Qr

20 20

21  PMT 1  PMT 2 Qr = Qr # fluorescence molecules # photoelectrons =  = photoelectrons 8 fluorescence molecules = 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.

22 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 QrLow Qr Why is Qr important?

23 Laser power Optical efficiency PMT sensitivity (red spectrum) Poor PMT performance Dirty flow cell Dirty or degraded filter What Factors Affect Qr?

24 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

25 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. Br Qr  ySensitivit relative Instrument performance can have a significant impact on the performance of an assay.

26 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.

27 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

28 Stain Index: Normalized Signal/Background D W unstained W compensated dye 1 Stain Index: metric used by Dave Parks, Stanford – Presented at ISAC 2004 Goal: Normalize the signal to the spread of background where background may be autofluorescence, unstained cells, or compensated cells from another dye dimension. D/W = Relative Brightness (SI)IndexStain  D = difference between positive and negative peak medians W = the spread of the background peak (= 2X rSD negative )

29 29 Electronic Noise (SD EN ): Determining Baseline PMT Voltages Median Fluorescence Intensity CV or SD PMT Voltage CV Standard Deviation PMT Voltage PE: Detailed Performance Plot Dim Bead 500 V For this detector, the SD EN = 18 Fluorescence intensity of dim bead = 10 x SD EN = 180 Determine PMT voltage required to set the dim beads at 180 = 500 volts = baseline voltage As PMT voltage is lowered, CV increases  resolution decreases As PMT voltage is increased CV 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 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% PMT Voltage Offset % Negative in CD4 Gate PMT Voltages: Optimal Gains Can Reduce Classification Errors 550 V 750 V 650 V

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32 Methods Used for Validation  Which Beads to Use  What Values to Plot  Selection Criteria

33 Example LSRII Optical Configuration 33 Green Laser (532nm) Duke LSRII PE Cy7 PE Cy5 PE TR PE Cy5.5 PE- Green 780/40 610/20 710/50 660/40 575/25 740LP 640LP 600LP 690LP Violet Laser (407nm) Duke LSR II QDot 705 QDot 605 QDot 545 QDot 585 QDot 655 QDot /70 585/42 660/40 605/40 560/40 670LP 595LP 570LP 630LP 557LP 560/40 535LP Am Cyan 515/20 505LP Cas Blue 450/50 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 /50 685LP 780/60 740LP APC Cy7 660/20 Duke University Medical Center

34 Beads Used for LSRII Validation 8 Peak Rainbow: v 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: v 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

35 Example of Optimal PMT Performance 300 Volts350 Volts400 Volts450 Volts500 Volts 550 Volts600 Volts650 Volts700 Volts750 Volts

36 Criteria for the Selection of Voltage Ranges  Lowest CV possible  Lowest Voltage possible  Highest MFI possible  Lowest background possible

37 Example of Optimal PMT Performance & Voltage Selection Criteria (Green B) Optimal Voltages: Selection Criteria: Linear Lowest CV Highest S:N Lowest Voltage Linearity = (M2-M1)/M1 Ratio = M1/B

38 Red A (APC-Cy7): Before (30Mar06) & After (10Apr06) Replacing PMT Optimal Voltages: Optimal Voltages: Indeterminate High CV’s; poor S:N 30 March April 2006

39 Faulty PMT on install… 650 Volts BeforeAfter

40 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

41 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

42 Criteria for the Selection Optimal PMT Voltages The primary fluorescence should be the highest in the respective detector relative to all secondary detectors.

43 Green A: TNF  PE-Cy7 460v Baseline & Final 43

44 44 Green B: CD8 PerCP-Cy v Baseline

45 Green B: CD8 PerCP- Cy v Final 45

46 Green C: CD27 PE-Cy5 350v Baseline 46

47 Green C: CD27 PE-Cy5 450v Final 47

48 Green D: CD45RO PE-TR 430v Baseline & Final 48

49 Green E: MIP1b PE 350v Baseline & Final 49

50 Duke LSRII Optimization of Specific Fluorescence Violet Red Green Blue Primary Fluorescence Secondary Fluorescence

51 Neg Comp Beads 51

52 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

53 1x (Target Channel Values) 53

54 Target Channels & Ranges

55 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

56 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

57 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: Median pk6 -Median pk5 /Median pk5 Signal:Noise: Median pk5 /Median Neg 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

58 1x Blue B Trend line 58 Duke University Medical Center

59 Ratio and Voltage Variation Duke University Medical Center

60 LASERs have a limited lifespan…

61 Overview  Why QC is Important  LSRII Optical Configuration  LSRII QC  LSRII Validation  LSRII Optimization  LSRII Calibration  LSRII Standardization  Practice Analysis?? Duke University Medical Center

62 8 peak beads Example: Stain Index, Qr, and Br Across Laboratories Example : FITC Channel LabSIQrBr 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 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 BD Biosciences, San Jose, CA 95131; 2 Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

63 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 2Lab 4Lab 6Lab 7 Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry Assays. Jaimes MC, Stall A, Inokuma M, et al. ISAC 2010

64 64 BD FACSCanto II 1 Assay – 4 Platforms BD LSR II BD LSRFortessa BD FACSAria III CD4 V450 CD8 PE CD3 FITC CD20 APC

65 Thank You!! 65

66 Defining Instrument Performance and Sensitivity: Qr, Br, and SDen

67 Qr: Anti-CD10 PE Example Trans- mission 100% % % % % 14 SI Corrected 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

68 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

69 High Br widens negative and dim populations. High Qr value = lower resolution Low Qr value = higher resolution Why is Br important? Low BrHigh Br

70 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 PI-free dye (µg) Br Qr Br Qr

71 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 , USA.

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

73 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. Contributions to Background Br Factors affecting Br: dirty flow cell, damaged optical component

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