New CLSI Document for the Validation of Methods Preformed by Flow Cytometry – Sneak Peek and Update Virginia Litwin, Ph.D. Vice President, Immunology
CLSI H62 – Validation of Methods Performed by Flow Cytometry Sneak Peek Overview Document Writing Committee Process and Timelines Content Highlights 2
H62 Document Writing Committee Leadership • Virginia Litwin, Chair • Teri Oldaker, Vice Chair • Raul Louzao, Secretary • Dave Sterry, CLSI Standards Director Voting Members David Barnett, Jacqueline, Cleary, Tom Denny, Cherie Green, Wolfgang Kern, Natalia Kokorina, Jennifer Stewart, Lili Wang Contributors and Reviewers Elena Afonina, Ahmad Al Samman, Tony Bakke, Fiona Craig, Bruce Davis, Lorella Di Donato, Steve Eck, Nancy Fine, Ben Hedley, Shuguang Huang, Jerry Hussong, Andrea Illingworth, Cassie Jiang, Mike Keeney, Natalia Kokorina, Sarah Maremont, Laura Marszalek, Kathy Muirhead, Andy Rawstron, John Schmitz, Alan Stall, Maryalice Stetler-Stevenson, Horacio Vall, Alessandra Vitaliti-Garami, Paul Wallace, Brent Wood, Yuanxin Xu 3
Document Writing Committee Composition Affiliations Scientific Societies • AAPS • Academia • CAP • Biopharmaceutical • ESCCA • CRO • ICCS • Clinical Laboratories • ISAC • Reagent/Instrument Manufacturers Provenance • Government • Canada - FDA • Germany - NIST • Switzerland • UK • USA 4
Special Reviewers ICCS, Advocacy Committee Thomas Denny, Pranav Dorwal, Jeannine Holden, Jerry Hussong, Wolfgang Kern, Virginia Litwin, Sara Monaghan, Teri Oldaker, Andy Rawstron, Stephanie Toney, Christopher Trindade, Paul Wallace AAPS, Flow Cytometry Action Program Committee Ruth Barnard, Steve Eck, Catherine Fleener, Fiona Germaschewski, Christele Gonneau, Cherie Green, Chris Groves, Michael Hedrick, Shuguang Huang, Shibani Mitra- Kaushik, David Lanham, Virginia Litwin, Thomas McCloskey, Thomas McIntosh, Maxime Moulard, Sam Pine, Kruti Shah, Ulrike Sommer, Soren Sonder, Jennifer Stewart, Yongliang (Steve) Sun, Alessandra Vitaliti, Dave Williams, Sam Witherspoon, Yuanxin Xu, Chelsea Xue 5
Process and Timelines Face-to-face Kick-off (Kansas City, MO) September 2017 Final Draft Approved by Voting Members November 2018 Circulate Draft / Open Comment (60 days), February 19 Prepare responses and finalize comments , June 19 Circulate right to appeal (30 days), July 19 Final Draft, August 19 Final CLSI vote (20 days), September 19 Document publication (Word/InDesign), December 19 6
Impact • Extensive review process • American National Standards Institute (ANSI) compliant • Alignment with International Organization for Standardization (ISO) - CLSI serves as the ANSI-appointed Secretariat for the ISO Technical Committee 212 (ISO/TC 212) • Regulatory agencies often recognize CLSI guidelines
Document Outline Chapter 1 Scope Chapter 2 Quality System Essentials Teri Oldaker Chapter 3 Fit for Purpose / Iterative Approach Pre-Examination Phase Fiona Craig Chapter 4 Instrument Qualification, Setup, and Standardization Cherie Green Chapter 5 Assay Development and Optimization Ben Hedley Chapter 6 Assay Validation Steve Eck Chapter 7 Examination Phase/ Post-Examination Phase Raul Louzao
Chapter 1 Scope
Scope • Recommendations and Practical Instructions - One-stop shopping - Current best practices - Summarize recent white papers and scientific advances • Target Audience - Basic research laboratories (non-regulated) - Clinical (regulated US and ex US) - Drug discovery, development, and manufacturing (regulated and non-regulated) - Reagent, assay, and instrument manufacturers - Regulatory agencies
Out of Scope • Out of Scope - Individual cell type-specific assay development - The validation of flow cytometric assays for soluble analytes - Third-party software and LIS interface validation
Chapter 4 Instrument Qualification, Setup, and Standardization
Chapter 4 Outline 4 Instrument Qualification, Setup, and Standardization 4.1 Installation Qualification and Operational Qualification (IQ, OQ) 4.2 Performance qualification (PQ) 4.2.1 Linearity and Dynamic Range 4.2.2 Electronic Noise 4.2.3 Resolution 4.2.4 Carryover 4.3 Cross-instrument, cross-site standardization 4.3.1 Examples of Cross-standardization 4.4 Compensation: 4.4.1 General factors to consider for calculating compensation: 4.4.2 Types of compensation controls 4.4.3 Compensation and Linearity 4.5 Longitudinal Performance 4.6 Qualification and verification of instrument for intended purpose
Chapter 4--Take Home Message • Instrument qualification is often neglected • The foundation of good data
Goals of Instrument & Software System Qualification Establish and maintain a controlled environment that can produce reliable data over a long period of time Ensure integrity and reconstruction of data Support lifecycle of the system by establishing procedures from installation to decommission
Installation Qualification I NSTALLATION P ASS /F AIL D OCUMENTATION N OTES PARAMETER C RITERIA Checklist with Consider space Benchtop and vendor requirements for associated lab requirements, instrument/computer Environment space meet positive footprint and vendor notation of additional clearance fo specifications Pass/Fail and future maintenance initial and date Checklist with Temperature vendor Equipment used to and humidity requirements, perform verification Utilities of lab space positive should be documented meets vendor notation of in report appended to specification Pass/Fail and the checklist initial and date Checklist with vendor Equipment used to Electrical requirements, perform verification requirements Electrical positive should be documented meet vendor notation of in report appended to specifications Pass/Fail and the checklist initial and date Document Include all associated instrument components, if any, Verify all specifications including automated Hardware components (model, serial sample acquisition are installed number, modules, manufacturer uninterrupted power date) supplies, etc.
Operation Qualification O PERATIONAL P ASS /F AIL C RITERIA D OCUMENTATION N OTES PARAMETER Screenshot and/or Perform automated Software report with positive Include automated system functions functionality notation of Pass/Fail, maintenance procedures (startup, QC) initial and date Stress the system to demonstrate that Visual cues can also be used system detects to prompt user to change problems and displays fluids Document warnings appropriate warnings displayed with System alerts Example: Fluidics icons screenshots, initial Example: Attempt to change color when low levels and date acquire data with low are detected. System should fluidics level or have warning and not allow disconnected further acquisition until computer cable fluidics issues are addressed Run calibration beads Checklist with Include any automated QC to verify %CVs, vendor report, Optical detector sensitivity requirements, all testing reagents should be precision and laser power positive notation of documented in a report output meets vendor Pass/Fail and initial attached to the checklist specifications and date There is some overlap in PQ; Acquire triplicates of replicate samples could also Checklist with testing material be used to demonstrate Automated positive notation of (beads or cells) in precision. OQ can be sample successful sample randomly distributed performed using beads acquisition acquisition, Pass/Fail locations in carousel whereas PQ requires and initial and date or plate intended use biological samples.
Performance Qualification Optical alignment Linearity and dynamic range Detection efficiency (Q) Electronic noise (SDen) Background signal (B) Overall resolution of the detection system, which is impacted by efficiency, background, electronic noise Acquisition carryover
Look What’s New! • The National Institute for Standards and Technology (NIST) Fluorescence calibration beads with traceable equivalent o number of reference fluorophores • Enable us to speak the same language Traceable ERF Value Assignment to Commercial Microparticles nt to Commercial Microparticles FC Bead Ex Laser (nm) SRM 1934 FITC 488 Fluorescein PE 488 Fluorescein BB515 488 Fluorescein Six Peak Hard Ex Laser (nm) SRM 1934 Dyed Micro- PerCP 488 Nile Red particles PerCP-Cy5.5 488 Nile Red Intensity 2-6 488 Fluorescein PE-Cy7 488 Nile Red Intensity 2-6 488 Nile Red APC 633 APC APC-R700 633 APC Intensity 2-6 633 APC APC-H7 633 APC Intensity 2-6 405 Coumarin 30 APC-Cy7 633 APC V450 405 Coumarin 30 BV421 405 Coumarin 30 V500-C 405 Coumarin 30 BV510 405 Coumarin 30 BV605 405 Coumarin 30 Traceable ERF Value Assignment to Calibration Beads. Flow Cytometry Quantitation Consortium 81 Federal Register 136 (15 July 2016), pp. 46054-46055 ERF Value Assignment to Cytometer Calibration Beads Submitted by Consortium Members Cytometry Part A ● ● ● 73A: 279-288, 2008; Flow Cytometry Protocols: Third Edition, p53-65, 2011 ● 19 Current Protocols in Cytometry, 75:1.29.1-14, 2016; Flow Cytometry Protocols: Fourth Edition (in press)
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