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
•
Cytometry, Part A, Volume 85, Issue 5, pages 434453, 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?
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)
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”
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.”
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.”
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.”
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.”
Key Messages in ISAC 2014
 Introduction



Work with manufacturers
LAIs and aerosols
Pertinent regulations
 Biosafety

“Identify and evaluate agent hazards”
 BSPs

Principles and Cell Sorting
best at this
“Identify laboratory procedure biohazards”
 Cell
sorter operators best at this
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
Key Messages in ISAC 2014
 Biosafety


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


Principles and Cell Sorting
all sorters created equal
Minimize # of operators sorting biohazards
(where applicable)
Hands-on training and proficiency
documentation
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
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…)
Yale Cell Sorting Biosafety
Program
 Identification
on campus



of all high speed cell sorters
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)
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
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
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)
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)
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
Yale Flow Cytometry
Core Facility:
Services:
Cell Sorting and Cell Analysis to 220+
Currently:
PIs and 1000+ users
6 – High Speed Cell Sorters
In the Anlyan Center, George St.
And Amistad Building
14 – Analysis Machines
In the Anlyan Center, Amistad, and
300 George St. Buildings
How much has flow cytometry grown?
PubMed Articles Citing Flow Cytometry:
•
•
•
•
•
1974 – 1
1980 – 119
1990 – 2,288
2003 – 5,890
2013 – 10, 306
How many of you have a flow cytometer
or cell sorter?
What is a safety officer likely to encounter:
“We have a FACS machine”, “It’s a cytometer” or “That’s just a FACS machine”
What do you actually have on your hands?
An Analyzer
Or a Cell Sorter
How does Flow Cytometry Work?
+
=
A problem for EHS and everyone else is that we use the terms
FACS and Flow Cytometry Interchangeably:
BD FACSVantage
- Sorter
BD FACSCalibur
- Analyzer*
BD FACSVerse
- Analyzer
BD FACSJazz
- Sorter
BD FACSAria
- Sorter
BD FACSCanto
- Analyzer
BD LSRII
- Analyzer
-Definitely
not sorter by
Today’s
standards
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
Levels of Safety at Yale for analyzing samples:
1) Fixation – all samples run on analyzers*
2) On a sorter with an AMS – samples that cannot be fixed
3) On a sorter in a hood – samples that are potentially hazardous
4) On a sorter in a hood with respirators – samples that are infected/
hazardous
What are the potential risks and hazards associated
with flow cytometers and cell sorters ?
Flow Cytometers/Analyzers:
Greatest potential hazard or risk associated in running a
cytometer:
The users
1) Train every user
2) Establish clearly stated rules
“But, I’ve been doing flow for years at XXXXX.”
Why are you here? Can anything really go wrong with a sorter?
The Good News:
There are currently no laboratory acquired infections
directly attributed to the process of cell sorting.
(Holmes, Perfetto; Cyto 2010)
The Bad News:
Cell sorting is a procedure that
intentionally creates droplets and aerosols.
80% of laboratory acquired infections are from an
unknown route of exposure, but the majority are
likely transmitted via airborne exposure.
(Sulkin & Pike 1951, Pike 1979)
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?
Framework for RA/RM
Risk Assessment
Risk Management
Pathogen
Practices (good work
practices)
Procedures
Protective equipment
(clothing and equipment)
Personnel
Place (facility design)
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)
Pathogen – Initial Risk Assessment
Principal InvestigatorRisk 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)
Personnel• 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 considered
for BSL-2 or BSL-3 sorting
What do you think this is?
The preferred transportation vessel
for analysis users.
Proper sealable secondary
containers
Proper collection tubes that
are properly sealed
Clean Starting Area
Organize all required items
before starting experiments
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-sort
experiment 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
• 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 complete
a BSL-2 or BSL-3 questionnaire that we will keep on record
• The questionnaires are reviewed in conjunction with
the Environmental Health and Safety Office
• We perform a risk assessment of the proposed sort
• General Risk Assessment Guidelines Used
I. Determine the agent, volume, and concentration
II. Proposed practices/procedures (sorting in this instance)
III. Proposed location and PPE
IV. Training, experience, and health status of the workers
Lo pressure sort
(12 psi, 130-micron nozzle)
Hi pressure sort
(70 psi, 70-micron nozzle)
Stream
Askew
Centered
Stream
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
Testing Aerosol Containment
Position
No AMS/No BSC/No Tube
Holder
No AMS/No BSC/Tube
Holder In
AMS/BSC/No Tube
Holder
A
>150
9
0
B
16
0
0
C
0
0
0
D
0
0
0
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-minute
test
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)
Can we perform BSL-3 sorting in your current
Flow Cytometry dungeons?