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Instrument QC and Qualification

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

paul-compton
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Instrument QC and Qualification

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  1. Instrument QC and Qualification

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

  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. 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

  7. 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

  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. 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

  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. 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

  12. 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.

  13. 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

  14. cvs

  15. 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)

  16. Background contributions

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

  18. 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.

  19. 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

  20. 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

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

  22. 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.

  23. 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.

  24. 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.

  25. 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

  26. = 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

  27. 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

  28. % 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

  29. Methods Used for Validation • Which Beads to Use • What Values to Plot • Selection Criteria

  30. 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 ﾐ 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

  31. 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

  32. 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

  33. Criteria for the Selection of Voltage Ranges • Lowest CV possible • Lowest Voltage possible • Highest MFI possible • Lowest background possible

  34. 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

  35. 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

  36. Faulty PMT on install… Before After 650 Volts 650 Volts

  37. 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

  38. 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

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

  40. Green A: TNF PE-Cy7460v Baseline & Final

  41. Green B: CD8 PerCP-Cy5.5450v Baseline

  42. Green B: CD8 PerCP-Cy5.5550v Final

  43. Green C: CD27 PE-Cy5350v Baseline

  44. Green C: CD27 PE-Cy5450v Final

  45. Green D: CD45RO PE-TR430v Baseline & Final

  46. Green E: MIP1b PE350v Baseline & Final

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

  48. Neg Comp Beads

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