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Instrument Standardization: Channeling Clinical Flow Cytometry towards Shangri La Sriram Ramanan1, Christèle Gonneau2, Tony Fazio2, Brahmananda Chitteti3 and Virginia Litwin3*
1Flow Cytometry, Hematology Department, Covance, Singapore; 2 Flow Cytometry, Hematology Department, Covance, Geneva, Switzerland; 3Flow Cytometry, Hematology Department, Covance Inc., Indianapolis, IN, US
Introduction
Current Standardization Process (CS&T Beads)
New Standardization Process (FC Beads)
Results: Current versus New Standardization (Monitoring)
Results: Current versus New Standardization (Inter-Instrument Variability)
As the biopharmaceutical industry focuses on cancer immunotherapy, autoimmunity and chronic viral diseases, the importance of flow cytometry in the drug development process is increasing. The utility of flow cytometry in clinical trials has expanded tremendously over the past several years. The need to generate high-quality data from multiple sites and multiple instruments requires a robust instrument-to-instrument standardization process. In this study, two instrument standardization processes were compared. Using an approach described by Becton Dickinson where hard dyed beads (CS&T) were used for the daily application settings, 22 instruments were cross-standardized globally. Next, Cyto-Cal™ beads (Thermo Scientific Inc.) were used to compare the performance of all cross-standardized instruments. This process was subsequently compared to a new approach in which specific fluorochrome bound beads (FC beads, Becton Dickinson) were used for instrument to instrument standardization and performance monitoring. Our results indicate that standardization using FC beads led to a significant reduction in inter-instrument variability in fluorescence (about 5% inter-instrument CV). In summary, instrument-to-instrument standardization not only leads to closeness and comparability between data obtained across sites and across instruments but also helps in streamlining of the daily work flow process and increases cost efficiencies.
Summary ▶ Fluorescence inter-instrument variability is reduced when instruments are standardized with FC beads ▶ Following the positive outcome of the pilot study presented here, we are currently testing this globally
on our 19 FACSCanto™ II (BD) ▶ Better insight into instrument performance: best choice of instruments globally for specific applications ▶ Better outcome for quantitative assays in which we are reporting fluorescence intensities ▶ The journey to Shangri-La is still protracted as many other aspects of the process are equally important
to achieve good quality global and longitudinal data (standardizing data analysis, SOPs, antibody lot-to-lot evaluation) Presented at CYTO Asia 2017
OVERALL INTER-INSTRUMENT VARIABILITY IS ACCEPTABLE
Inter-instrument variability (%CV) is reduced with new FC beads standardization
530/30 (488)
585/42 (488)
670LP (488)
780/60 (488)
620/20 (633)
780/60 (633)
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0.00 5.00 10.00 15.00 20.00 25.00
Comp beads
Fc beads
Cyto-Cal
Comp beads conjugated with: CD4 FITC, CD27 PE, CD20 PerCP-Cy5.5, CD38 PE-Cy7, CD8 APC, CD3 APC-H7, CD56 V450, CD45 V500
Inter-instrument %CV
Observed inter-instrument variability (%CV) depends on the type of bead used for the monitoring
Higher inter-instrument variability (%CV) is observed when hard-dyed beads are used for the monitoring
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585/42 (488)
670LP (488)
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780/60 (633)
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Comp beads
Fc beads
Cyto-Cal
Inter-instrument %CV
Comp beads conjugated with: CD4 FITC, CD27 PE, CD20 PerCP-Cy5.5, CD38 PE-Cy7, CD8 APC, CD3 APC-H7, CD56 V450, CD45 V500
530/30 (488) 585/42 (488) 670LP (488) 780/60 (488)
Peak
5 M
FI
620/20 (633) 780/60 (633) 450/50 (405) 510/50 (405)
Peak
5 M
FI
Current Standardization Monitoring Process (Cyto-Cal™ Beads)
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585/42 (488)
670 LP (488)
780/60 (488)
660/20 (633)
780/60 (633)
450/50 (405)
510/50 (405)
grand mean 55972 36023 43115 6540 31193 11897 65389 21424
SD 5211.21 1612.83 2197.54 377.98 2746.78 652.49 7600.81 2548.40
CV 9.31 4.48 5.10 5.78 8.81 5.48 11.62 11.90
Define a 3-Laser 8-Color 4-2-2 Standard Configuration
Define Standard Configuration
Collect the rsDEN (robust standard deviation of Electronic Noise) from Baseline reports of all instruments. Calculate the Average rsDEN for each detector. This is the virtual composite predicate instrument rsDEN
Compile Baseline reports for predicate values
STEP 1: Fix Minimal PMT Voltage: Acquire (lysed) unstained lysed blood and adjust PMT voltages so that the Lymphocyte fluorescence is well above the noise (target rSDunstained = predicate SDen x 2.5) Record voltages Check PMT voltages by staining blood with a bright marker and by verifying that the population is still within the linear max range STEP 2: Create Fluorescence Target Values: CS&T beads are acquired using PMT voltages established in Step1 The median fluorescence intensity (MFI) values of the bright bead population in each detector become the CS&T beads fluorescence target values
Establish ASTV settings on single instrument
Template containing fluorescence target values was exported in a standardization package Standardization package was imported in each cytometer PMT voltages are adjusted so that CS&T bright beads are reaching the fluorescent target values Voltages are saved as “application settings”
Standardize other instruments with ASTV target
WHY A NEW PROCESS?? • To improve inter-instrument variability while monitoring MFI • CS&T and Cyto-Cal™ beads are hard-dyed beads bound with fluorochrome surrogates, whose
excitation and emission spectra are different from specific fluorochromes Advantages of FC Beads • Beads are covalently bound to the real fluorochromes (Fc Bead FITC, Fc Bead PE and so on.) • Exhibit stable levels of fluorescence over time
Same as above
Define Standard Configuration
Same as above
Compile Baseline reports for predicate values
Fix Minimal PMT Voltage: Same as above Create Fluorescence Target Values: Fc beads are acquired using PMT voltages established in Step1 The median fluorescence intensity (MFI) values of the bright bead population in each detector become the Fc beads fluorescence target values
Establish ASTV settings on single instrument
In progress
Standardize other instruments with ASTV target
* Denotes former Covance employee
Pilot Comparison Experiment
Readout: Median Fluorescence Intensity (MFI) across the 4 instruments
Current standardization: CS&T beads
New standardization: Fc beads
Monitoring Fluorescence (MFI) Monitoring Fluorescence (MFI)
4 instruments (FACS Canto™II (BD))
Cyto-Cal™ (hard dyed)
Fc beads (real
fluorochromes)
CompBeads™ (real
fluorochromes)
Cyto-Cal™ (hard dyed)
Fc beads (real
fluorochromes)
CompBeads™ (real
fluorochromes)
BD CompBeads ™ : Compensation beads
SINGLE INSTRUMENTS ARE VERY STABLE OVER TIME
INTER-INSTRUMENT VARIABILITY CAN STILL BE IMPROVED
530/30 (488) 585/42 (488) 670 LP (488) 780/60 (488) 660/20 (633) 780/60 (633) 450/50 (405) 510/50 (405)
highest mean 63566 37630 45777 7045 36620 13241 72987 24191
lowest mean 47367.00 33666.00 38110.00 5894.00 26046.00 10781.00 41745.00 15529.00
% difference 25.48 10.53 16.75 16.34 28.87 18.58 42.80 35.81
Inter-instrument %CV
Monitoring bead
0.00 5.00 10.00 15.00 20.00 25.00
Fcbead FITC
Fcbead PE
Fcbead PerCP-Cy5.5
Fcbead PerCP
Fcbead PE-Cy7
Fcbead APC
Fcbead APC-H7
Fcbead APC-Cy7
Fcbead V450
Fcbead BV421
Fcbead V500-C
Fcbead BV510
new standardization (FC beads)
current standardization (CS&T)
Data from 2 to 4 independent experiments on 4 instruments
(bars: standard deviation)
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780/60 (633)
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NEW STANDARDIZATION PROCESS
CURRENT STANDARDIZATION PROCESS
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