benjamin fontes & geoffrey lyon yale university
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Integrating Industry Guidance and Protocol Specific Risk Assessment into the Yale Cell Sorting Biosafety Program. Benjamin Fontes & Geoffrey Lyon Yale University. Central Guidance Document. International Society for the Advancement of Cytometry (ISAC) Cell Sorter Biosafety Standards - PowerPoint PPT PresentationTRANSCRIPT
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Integrating Industry Guidance and Protocol Specific Risk Assessment into the Yale Cell Sorting Biosafety Program
Benjamin Fontes &Geoffrey LyonYale University
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Central Guidance Document• International Society for the Advancement of
Cytometry (ISAC) Cell Sorter Biosafety Standards• Cytometry, Part A, Volume 85, Issue 5, pages 434-
453, May 2014 – Ingrid Schmid, Kevin Holmes, Stephen Perfetto, Charles
(Hank) Pletcher, Philip Hogarth, Simon Monard, Robert Wadley
• Pioneers (Schmid, Perfetto, Holmes)– Pletcher, Lyon, Lopez….
– Your institution’s pioneer?
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2014 ISAC Cell Sorting Biosafety Standards
Updates (augmentation of): Lab Design PPE Equipment specific SOPs Validation of aerosol containment
Bacteriophage Glo-Germ Technetium-99 (UCONN – Wallace et al) Fluorescent beads (Pletcher & Lyon) NIH “next” (Holmes and continuous improvement)
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2014 ISAC Cell Sorter Biosafety Standards Update Author(s) obsession (Schmid, Perfetto,
Holmes…others) Obsessive in personal and researcher
protection Obtaining data & continuous
improvement Find your “obsessive” colleagues (Lyon @
Yale) 2014 Update
“NIH Influence”
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2014 ISAC Cell Sorting Biosafety Standards Industry Guidance Document Gives “NIH” recommended Biocontainment Does not take the place of Risk Assessment!
Page 7, bottom of Table 2, footnote a “This list represents examples of biosafety
level determination for cell sorting of specific agents. The final determination of the biosafety level is dependent upon the risk assessment conducted in collaboration with safety specialists, subject matter experts, and the IBC or equivalent.”
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2014 ISAC Cell Sorting Biosafety Standards Flexibility Based on Risk Assessment
Page 13 – 1st full paragraph PPE for BSL3 “Note that given the combination of
engineering controls, aerosol evacuation system and instrument located within a certified biosafety cabinet, may, dependent upon a risk assessment, allow for alternate combination of PPE. This requires approval by the cell sorting operator/facility director, biosafety professional and the Institution’s biosafety committee.”
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2014 ISAC Cell Sorting Biosafety Standards Flexibility Based on Risk Assessment
Page 13, Last Paragraph before Appendix A: Alternate combinations of engineering
controls, personal protective equipment and biosafety procedures that do not perfectly match the recommended BSLs may be selected. The final risk assessment SOP should be selected based on risk assessment and endorsed by the cell sorting facility manager, biosafety professionals and the IBC.”
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2014 ISAC Cell Sorting Biosafety Standards Flexibility Based on Risk Assessment
Page 12 3.1.1.2 Cell Sorters in biological safety cabinets …Class I BSCs enclosing cell sorters must
be manufactured to meet functional certification criteria for personal protection as defined by the BMBL or EN 12469, although it is recommended that the inward airflow velocity be 100 linear feet per minute or greater; HEPA filters must be tested for leakage annually.”
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Key Messages in ISAC 2014 Introduction
Work with manufacturers LAIs and aerosols Pertinent regulations
Biosafety Principles and Cell Sorting “Identify and evaluate agent hazards”
BSPs best at this “Identify laboratory procedure
biohazards” Cell sorter operators best at this
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Key Messages in ISAC 2014 Biosafety Principles and Cell Sorting Cell sorter operator serves as PI/Lab Director
Must conduct risk assessment Most knowledgeable of the work in their labs Each “operator” knows “their” instrument
Teach Biosafety and work with operators Learn “Cell Sorting” and work with operators
See procedure from start to finish Transport to/from and all steps in between
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Key Messages in ISAC 2014 Biosafety Principles and Cell Sorting
Have SOPs in place that provide a foundation for selection of controls at each BSL (BSL1, BSL2, BSL2-enhanced, BSL3)
Evaluate sorters selected for biohazard use Not all sorters created equal
Minimize # of operators sorting biohazards (where applicable)
Hands-on training and proficiency documentation
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Key Messages in ISAC 2014 Emergency procedures
Know what a spill is for cell sorters (stream deviation)
Use biosafety “common” sense Evacuate, allow aerosols to settle, be removed
Know contamination zone of the sorter before sorting biohazards Develop decontamination SOPs
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Relationship / Partnership Cell Sorting Operators & Biosafety / EHS
Professionals Biosafety/EHS Share:
Risk Assessment and Risk Management Principles of Biosafety
Cell Sorting Professionals Share: Knowledge of equipment (function,
operation) Process flow (and more…)
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Yale Cell Sorting Biosafety Program Identification of all high speed cell sorters
on campus SciQuest (past, new and future purchases) Core labs Department specific sorters
Equipment questions on biohazard registration forms
Training of EHS professionals (equipment recognition on inspections)
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Yale Cell Sorting Biosafety Program Campus user group meetings Lab inspections Core Facility registration form (where
applicable) Testing & certification of HEPA filtered
control devices (AMS’, Class I and II BSCs) Equipment validation testing Negative pressure verification Minimum PPE requirements for
biohazards
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Yale Cell Sorting Biosafety Program Added Core Facility Manager to Yale
BSL3 Subcommittee Share best practices Approved SOPs used as templates for new
locations (starting point) Demo particle challenge testing and
assessment Assist with authorization for biohazard use
with new sorters
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Yale Cell Sorting Biosafety Program Clear “bands” established (CT biohazard
regulations influence work location) Low Risk (BSL1) Moderate Risk (BSL2)
Primary human materials, other human cells, defective pathogen vectors, non-human primate cells
High Risk (BSL2-enhanced and BSL3) Risk Group 2 and 3 human pathogens Samples known to harbor human pathogens if
used for research (CT State DPH registration required)
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Yale Cell Sorting Biosafety Program
Negative pressure rooms for BSL2 (and higher) AMS at BSL2 (and higher)
Containment aerosol challenge verification Trained, experienced operators (all levels) PPE (lab coat, gloves and full face protection) at
BSL2 Gowns, 2 pair gloves, face shield, sleeves at BSL3 with
respirator consideration based on risk Primary containment enclosure and AMS (BSL2-
enhanced and BSL3)
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Power of Professional and Regional Sharing Journal articles Online posts (extra pulmonary TB) Rockefeller University Cell Sorting
Biosafety Summit (Part 1 and Part 2) 2 Full Days on cell sorting biosafety
hazards Best practices shared Many different approaches Q&A Forum
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Yale Flow Cytometry Core Facility:Services:Cell Sorting and Cell Analysis to 220+PIs and 1000+ users
Currently:
6 – High Speed Cell SortersIn the Anlyan Center, George St.And Amistad Building
14 – Analysis MachinesIn the Anlyan Center, Amistad, and 300 George St. Buildings
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How much has flow cytometry grown?
PubMed Articles Citing Flow Cytometry:
•1974 – 1•1980 – 119•1990 – 2,288•2003 – 5,890•2013 – 10, 306
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How many of you have a flow cytometer or cell sorter?
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“We have a FACS machine”, “It’s a cytometer” or “That’s just a FACS machine”
What do you actually have on your hands?
What is a safety officer likely to encounter:
An Analyzer Or a Cell Sorter
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How does Flow Cytometry Work?
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+=
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BD FACSVantage
BD FACSCalibur
BD FACSVerse
BD FACSJazz
BD FACSAria
BD FACSCanto
BD LSRII
A problem for EHS and everyone else is that we use the termsFACS and Flow Cytometry Interchangeably:
- Sorter
- Analyzer*
- Analyzer
- Sorter
- Sorter
- Analyzer
- Analyzer
-Definitely not sorter by Today’s standards
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Cell Analyzers: Cell Sorters:
Pressures:3-6 psi 20-70+ psi
Aerosol Containment:Usually none Frequently
AMS/Hoods
Samples (at Yale):Always fixed Usually non-fixed
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What are the potential risks and hazards associated with flow cytometers and cell sorters ?
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Flow Cytometers/Analyzers:
Greatest potential hazard or risk associated in running a cytometer:
The users
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1) Train every user 2) Establish clearly stated rules
“But, I’ve been doing flow for years at XXXXX.”
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Why are you here? Can anything really go wrong with a sorter?
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The Bad News:
Cell sorting is a procedure that intentionally creates droplets and aerosols. 80% of laboratory acquired infections are from anunknown route of exposure, but the majority are likely transmitted via airborne exposure. (Sulkin & Pike 1951, Pike 1979)
The Good News:
There are currently no laboratory acquired infectionsdirectly attributed to the process of cell sorting. (Holmes, Perfetto; Cyto 2010)
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The Other Bad News:
Manufacturer trainings place little to no emphasis on bio-safety and the potential risks associated with cell sorting and cell analyzers.
So where do you start?
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Framework for RA/RMRisk Assessment Risk Management
Pathogen Practices (good work practices)
Procedures Protective equipment (clothing and equipment)
Personnel Place (facility design)
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Pathogen – Initial Risk Assessment
Principal Investigator-
Risk Group Classification-
• BMBL Biosafety in Microbiological and Biomedical Laboratories (BMBL), Current Edition
(http://www.cdc.gov/biosafety/publications/bmbl5/)
• Public Health Agency of Canada – Pathogen Safety Data Sheets and Risk Assessment
(http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php#l)
• NIH Guidelines – Classification of Bio-hazardous Agents by Risk Groups
(http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIXB.htm)
• ABSA Risk Group Tables for Infectious Agents (http://www.absa.org/riskgroups/index.html)
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Pathogen – Initial Risk Assessment
Principal Investigator-
Risk Group Classification-
• Risk Groups (Gwladys Caspar, former Harvard University BSO)– RG1 “don’t drink it” – Analyzer or Sorter– RG2 “don’t touch it” – Sorter with AMS– RG3 “don’t breathe it” – BSL-3 Sorter– RG4 “don’t do it in MA/CT/Yale”
• BARE – “Block all routes of exposure” (Ben Fontes, Yale EHS)
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• Training and extensive knowledge of the cell sorter and hood• Must have experience operating sorter outside of a BSL-3 setting prior
• Health Screens and Blood storage by Employee Health
• Bio-safety, Blood-Borne Pathogen, and BSL-3 Trainings and refresher courses must be completed regularly
• EHS ensures personnel have a good track record in regard to Safety to be consideredfor BSL-2 or BSL-3 sorting
Personnel-
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What do you think this is?
The preferred transportation vesselfor analysis users.
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Proper sealable secondary containers
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Proper collection tubes that are properly sealed
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Clean Starting Area
Organize all required itemsbefore starting experiments
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Cell Sorting Biosafety –
1)Provide sort operators with the appropriate tools to evaluate their ability to conduct sorts
•Pre-sort discussion with the sort operator and pre-sortexperiment questionnaires BSL-2 and BSL-3 sorts
•Identify the health risk•BMBL Biosafety in Microbiological and Biomedical Laboratories (BMBL), Current Edition (http://www.cdc.gov/biosafety/publications/bmbl5/)•Public Health Agency of Canada – Pathogen Safety Data Sheets and Risk Assessment (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php#l)•NIH Guidelines – Classification of Bio-hazardous Agents by Risk Groups (http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIXB.htm)•ABSA Risk Group Tables for Infectious Agents (http://www.absa.org/riskgroups/index.html)
•Check Principal Investigator’s CT State Bio-hazard Registration Form for work with a particular agent
• Add Sorting Facility to protocol as a location for research • Ensure the researchers conducting the research are on the
protocol
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• Pre-sort discussion with the sort operator and pre-sort experiment questionnaires BSL-2 and BSL-3 sorts
• First questions for us, • “Is your material fixed or non-fixed?”• “Is your material mouse, human, or other?”• “Is your material infectious?”• “Has your material been altered/treated in anyway?”
• Based on the above answers we have the research completea BSL-2 or BSL-3 questionnaire that we will keep on record
• The questionnaires are reviewed in conjunction withthe Environmental Health and Safety Office• We perform a risk assessment of the proposed sort
• General Risk Assessment Guidelines UsedI. Determine the agent, volume, and concentrationII. Proposed practices/procedures (sorting in this instance)III. Proposed location and PPEIV. Training, experience, and health status of the workers
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Lo pressure sort (12 psi, 130-micron nozzle)
Hi pressure sort (70 psi, 70-micron nozzle)
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Stream Askew
CenteredStream
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Glo-Germ Bead Modified Testing-
•Glo-Germ particles are not ideal (tough to keep in suspension, clump, irregular shape)
•Hank Pletcher (Penn.) and Yale Sorting Facility modified Glo-Germ protocol using Polysciences Yellow-Green (YG) Microspheres
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Testing Aerosol Containment
Position No AMS/No BSC/No Tube
HolderNo AMS/No BSC/Tube
Holder InAMS/BSC/No Tube
Holder A >150 9 0 B 16 0 0 C 0 0 0 D 0 0 0
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Any guesses on how many particles can be detected on the sash during this?
~ 4300 YG Beads in 8-minutes
273 YG Beads in one 10-minutetest
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So what is the likely route of potential exposure for the sort operator?
80% of known infections in another study were associated with a “break down” in work practices, or adherence to good microbiological practices and safe technique may have prevented these events. (Phillips 1965)
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Can we perform BSL-3 sorting in your currentFlow Cytometry dungeons?