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CYTO 2010
Special Workshop on Biosafety
Steve Perfetto
Kevin Holmes
Special Workshop on Biosafety
• History of Biosafety in Cytometry
• ISAC Biosafety Committee
– Standards update, survey
• Biosafety Principles
– Risk Assessment, Standard Operating Procedure development
• Hazards in Cell Sorting Lab
– Aerosols and protection
• Preliminary Guidelines for Sorting
– Recommendations
Safety in Flow Cytometry: History Highlights
1981: T4 Bacteriophage method described (BD FACS II)
1985‐1988: Universal Precaution Documents issued
1995: ISAC Biohazard Working Group Formed 1997: ISAC Biosafety Guidelines published
2001: Use of GloGerm beads as substitute for T4 2002: Public Meeting on Safety Issues pertaining to Clinical Application of flow cytometry to human cells
• 2004: GloGerm modification: Anderson air sampler
• 2007: ISAC Biosafety Standard published
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•
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•
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ISAC Biosafety Committee
• Steve Perfetto, Kevin Holmes, Hank Pletcher
and Claude Lambert.
• ISAC Biosafety Standards Update
– Clarify risk assessment and containment procedures
• All biosafety levels including human cells in BSL2
– Partner with ABSA or other biosafety groups
– Task Force to develop NIH‐wide standards for biosafety in cell sorter laboratories
ISAC Biosafety committee cell sorter survey
– Preliminary Results/concerns
• 20% do NOT have aerosol containment
• Sorting infectious agents without PPE
• Sorting human samples with surgical masks or no respiratory protection
• Sorting infectious agents in shared lab (no BSC)
• Sorting infectious agents or human samples w/o containment testing
http://www.zoomerang.com/Survey/WEB22AEMDPQVPH
Biosafety Principles
– Goal to educate
• Flow cytometry
community
• Biosafety community
– Final goal not to inhibit research, but to conduct it in a safe manner
Biosafety Principles
• Containment
– Reduce or eliminate exposure of lab workers and outside environment to potentially hazardous agents
– Examples: Biological Safety Cabinet, centrifuge cups, aerosol management on cell sorter
– Includes procedures, i.e. standard microbiological practices
Biosafety Principles
• Risk Assessment: – Definition: an action or series of actions taken to recognize or identify hazards and to measure the risk or probability that something will happen because of that hazard.
– Severity of the consequences is also taken into account.
• Requires careful judgment
– Consequences if risks are underestimated
– Excessive safeguards
• Additional expense and burden with little safety enhancement
• May result in circumvention because of excess burden
Risk Assessment
1.
2.
3.
4.
Identify Agent hazards
Identify laboratory procedure hazards
Make final determination of Biosafety level
Evaluate proficiencies of staff and integrity of safety equipment
5. Review risk assessment with biosafety
professional
Risk Assessment Step 1: Agent Hazards
• Classification of microbiological agents into Risk Groups
• Ability to infect and cause disease
• Virulence (severity of disease)
• Availability of preventative measures and effective treatments
– WHO and NIH/CDC criteria: Risk Groups 1‐4
– Also known as Hazard Groups (UK)
– BMBL http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm)
– MSDS (Public Health Agency of Canada http://www.phac‐
aspc.gc.ca/msds‐ftss ) Agent Hazards: recombinant and viral vectors
• Agent Hazards
– Genetically modified agent hazards
• Samples with recombinant DNA, viruses, bacteria, yeast, etc
• Viral vectors: lentiviral, adenoviral or retroviral vectors
Risk Assessment Step 2: Procedure Hazards
• Laboratory Procedure hazards
– 5 routes of laboratory transmission:
1.
2.
3.
4.
5.
Parenteral inoculations: syringe needles/sharps
Spills/splashes on skin or mucous membranes
Ingestion through mouth pipetting
Animal bites & scratches
Inhalation exposure to infectious aerosols
• 1‐4 account for <20% of LAI’s
• Aerosols are serious hazard
Risk Assessment Step 3: Determination of Biosafety Level
• Make final determination of biosafety level and select additional precautions as indicated by risk assessment
• Biosafety or containment levels
– NIH/CDC and WHO: BSL1, 2, 3 and 4 (UK: CL1,2,3 and 4). – Risk Groups correlate with but do not equate to biosafety levels.
Risk Assessment Step 3: Determination of Biosafety Level
BSL
Agents
Practices
Barriers &
safety equipment
1
No disease in humans
Standard microbiological
None
2
Human disease; not aerosol transmitted
BSL1 & limited access, signage, medical surveillance
BSC Class I or II, lab coat, gloves
3
Aerosol transmission possible with serious or lethal consequences
BLS2 & controlled access, decontamination of waste,
lab clothes, baseline serum
BSC Class II, protective lab clothing, respiratory protection
4
Dangerous/exotic high risk BSL3 & clothing change, exit life threatening disease, shower, all material aerosol transmission likely decontaminating upon exit
Modified from BMBL, 5th Ed.
BSC Class III or Class II with full‐
body suit
Risk Assessment Step 4: Evaluate staff proficiencies safety equipment
– Proper training and/or certification of personnel is essential – Equipment must be available and functional
• Aerosol management system
• Personal Protective Equipment (PPE)
Risk Assessment Step 5: Review
• Review Risk Assessment with safety professional, subject matter expert and IBC
– Sometimes required by regulatory agency
– Cell sorters: not specifically regulated by NIH SOP (Standard Operating Procedure) Development
• Identify hazards and specify practices to minimize hazards
• Instrument design specific features determines procedures
– How is it designed? What are deficiencies?
– Evaluation of containment vs. evacuation
SOP Development
• Preparation before sort
– Check fluids, empty waste
– Containment testing (see below)
– Verify function of droplet containment system (analyzers)
– Verification of automated decontamination procedures
– Preparation of decontamination reagents
– Spare nozzle availability
SOP Development
• Failure or nozzle clog
– Most dangerous – Automated detection or not
• Turn off stream
– Aerosol evacuation
• Increase evacuation rate • Decontamination
– Automated or manual
– Surface cleaning
Factors Affecting the efficacy of Disinfection and Sterilization
• Number and location of microorganisms
• Innate resistance of microorganism
• Concentration and potency of disinfectant
– Quaternary ammonium compounds: Conc exp=1
– Phenol: Conc exp=6
• Physical and chemical factors
– Temperature and pH
• Organic and inorganic matter
• Duration of exposure
• Biofilms
– Bacteria up to 1000 fold more resistant
CDC Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008 SOP Development
• PPE required
– Gloves (single or double)
– Lab coat
– Safety glasses or face shield
– Masks, Respirators
• N‐95
• PAPR
Hazards in Cell Sorting Labs
• Laboratory Acquired infections
• 50% Research labs; 45% Clinical Diagnostic labs
• 82% unknown source
• Risks identical to other HCW & lab personnel
• Potentially infectious samples
• Reason for CFR 1910.1030 (BB pathogens)
• Other risks
– Lasers, chemicals, etc.
Hazards in Cell Sorting Labs
• No documented case of LAI in cell sorter lab, however…
1. Cell Sorting is a Laboratory Procedure Hazard
•
Aerosol generation
2. “General agreement among biosafety
professionals… that an aerosol generated by procedures and operations…is the probable source of many LAI’s” •
BMBL, 5th Ed.
Hazards in Cell Sorting Labs
3. Infections in laboratory may occur even if not normally transmitted via aerosol in nature
• Higher concentrations of organism
• Aerosol generating procedures
• Example: – 1st documented case of aerosol infection of Scrub Typhus (Orientia Tsutsugamushi) (Infection, 2001, 29:54)
– Usually transmitted via insect (mite, chigger) route
– lab worker disrupted cells on open bench
Aerosols
• Aerosol or bio‐aerosols: definitions
– Aerosol: a suspension of solid or liquid particles in a gas
– Bio‐Aerosol: an aerosol comprising particles of biological origin which may affect living things through infectivity, allergenicity, toxicity, pharmacological or other processes. • Aerodynamic particle size of 0.5 to 100 μm
Aerodynamic Diameter
• For a given particle, the diameter of a sphere having the same aerodynamic properties
– Standardizes for shape and density
– Better predictor of behavior in air than size
Irregular Particle
de = 5.0 μm
pp = 4000kg/m3
χ=1.36
de = 8.6 μm
pp = 1000kg/m3
VTS=2.2
mm/s
Aerodynamic equivalent sphere
VTS=2.2
mm/s
Size, Alveolar Deposition and Infectivity
Alveolar deposition efficiency
(Fraction of total inhaled
AD range associated with Infection
Aerodynamic diameter (μm)
Vincent, J.H., 2005, J. Environ. Monit. 7:1037; Brosseau, L.M., et al. 1994 ASHRAE Transactions 100:368; Fennelly, K.P. , et al. 2004 Am J Respir Crit Care Med 169:604.
What is the Size and Concentration of Aerosols Produced by a Cell Sorter?
• Aerodynamic Particle Sizer
– TSI Model 3314 UV‐APS
– capable of measuring particles between 0.5μm to 20μm (aerodynamic diameter)
– Principle of operation
• Uses time of flight measurements to determine aerodynamic particle size
• Incorporates UV laser for measurement UV‐excitable dye (run as sample)
POSTER: 211/P125
FACS Aria II, Fail Mode, Open Sort Chamber: 70 PSI (UV+)
1000
Concentration (#/cm3)
25
100
20
10
15
12
1
9
0.1
6
0.01
3
0.5
5
Aerodynamic Diameter (μm)
1.5
70, 35 and 20 PSI (3.0cm UV+)
70 psi
Concentration (#/cm3)
1000
35 psi
20 psi
9.8x103/cm3 mean: 2.0 μm
100
1.9x103/cm3 mean: 2.4 μm
10
1
90/cm3 mode: 2.65 μm
0.1
0.01
0.5
5
Range of alveolar deposition and infectivity
Aerodynamic Diameter (μm)
Aerosol Protection in Cell Sorters
• Secondary Barriers
– Room design, access to room
– Remote operation
• Primary Barriers and PPE
– Biological Safety Cabinet
– Flow Cytometer specific Aerosol Protection in Cell Sorters
• Biological Safety Cabinet
–
–
–
–
–
BD (BioProtect II or III BSC)
ICyte Reflection
BD Influx
Coulter Astrios
certified (NSF‐49 or
BS EN12469: 2000)?
• BSC’s “are partial containment devices
– level of protection is relative and dependent upon
• mechanical performance of the biological safety cabinet • good laboratory practices that minimize aerosol generation and interference with the protective inward airflow”*
*Biological Safety Cabinetry, Clin Microbiol Rev (1991) 4: 207‐241
Aerosol Protection: Masks and Respirators
• Filtering Facepiece Respirators
– NIOSH rating: N‐95, N‐99 or N‐100
– Require annual fit testing
• PAPR (Powered Air Purifying Respirators)
– Equivalent to N‐100 rating
• Surgical Masks
– Not Respirators
– Poor filtration
efficiency
Aerosol Protection in Cell Sorters
• Biological Safety Cabinet
• Flow Cytometer specific – Containment vs. Evacuation
• Aerosol containment system (evacuation)
Aerosol Containment System
Example: BD AMO
• Evacuation of aerosols by ‘PlumeSafe
Whisper’ unit by BuffaloFilter
– Equipped with ULPA (Ultra Low Penetrating Air) filter •January 2009, Geoffrey Lyon at Yale Univ. Sch. Of Medicine reported a high failure rate of Buffalo Filters supplied with the AMO units for BD and Cytomation
•Within 2‐3 weeks, Buffalo verified problem and modified production procedures and began shipping new (individually tested) filters to customers
•Any safety component can fail!
Epoxy did not form complete seal on filter housing
Methods of Containment Assessment in Cell Sorters
• Any containment system must be assessed for efficacy
• Two published methods:
– Bacteriophage T4 test
– GloGerm bead test
Vacushield
filter
Flow Meter
Vacuum trap
AeroTech
Methods of Containment Assessment in Cell Sorters: Modification of Aria
• FACSAria : after nozzle obstruction, are aerosols evacuated from sort chamber?
– NO
• Modification of Aria: open communication between sort chamber and collection chamber.
• With AMS on, aerosols are cleared in 15sec
• New methodology to assess containment?
POSTER: 211/P125
15 sec after fail, stream off
Guidelines* for determining biosafety levels and procedures for cell sorting
BSL2
BSL2/3
BSL3
BSL4
Uninfected non‐primate Non‐infectious Human /NHP cells
Infectious but with low risk assessment
Infectious samples with high risk assessment All samples containing known aerosol pathogens
Extremely Dangerous Pathogens
Mycobacterium
tuberculosis
Monkeypox
Ebola
human cells)
Normal human blood
Human cell lines
Influenza A
2nd gen Lentivirus or 3rd
gen in human cells
Containment system
validation
Periodically
Periodically
Prior to each sort
Prior to each sort
Aerosol containment operating
Required
Required
Required
Required
Required1
N/A
N/A
Risk Assessment Condition
Example Sample type or Normal murine cells
3rd gen Lentivirus (non‐
Agents
N‐95 or better respirator Optional
PAPR
Optional
Optional
Required
N/A
Eye Protection
Safety Glasses
Face Shield or wrap around safety glasses
N/A
N/A
Lab Coat
Front Closure
Wrap Around rear closure
Coveralls
Special suit
Separate room
Optional
Required or limited access Required
to room2
*Preliminary guidelines
Required
Guidelines for determining biosafety
levels and procedures for sorting
Risk Assessment Condition
BSL2
BSL2/3
BSL3
BSL4
Uninfected non‐primate Non‐infectious Human /NHP cells
Infectious but with low risk assessment
Infectious samples with high risk assessment All samples containing known aerosol pathogens
Extremely Dangerous Pathogens
Example Sample type or Normal murine cells
3rd gen Lentivirus (non‐
Agents
human cells)
Normal human blood
Mycobacterium
Human cell lines
tuberculosis
Influenza A
Monkeypox
1For human pathogens that are classified as 2nd gen Lentivirus or 3rd
gen in human cellsRisk Group 2 and are not respiratory hazards, but which may pose a risk if Periodically
Periodically
Prior to each sort
Prior to each sort
Containment system
exposed to mucous membranes, masks may Per NIH Institute Biosafety Committee: validation
be substituted for respirator PPE. Examples 1)
all primary human samples are to be Required
Required
Required
Required
Aerosol containment of Agents in this category include 2
sorted under BSL2/3
operating
Enclosure of the cell sorter within a Class II Leishmania
and toxoplasmosis in mice.
rd
may be sorterd
2) 3 generation lentiviruses
Optional
Required1
N/A
N/A
BSC (NSF‐49 certified) may abrogate the N‐95 or better respirator
under BSL2, if documentation showing need to house the sorter in a separate room Optional
Optional
Required
N/A
PAPR
rd
that they are 3 generation is provided within the BSL2 lab space. PPE (as detailed Safety Glasses
Face Shield or wrap N/A
N/A
Eye Protection
and if they are not primary human cells
above) is still required for the operator.
around safety glasses
nd
should be 3) 2 generation lentiviruses
Front Closure
Wrap Around rear closure Coveralls
Special suit
Lab Coat
sorted under BSL2/3
Separate room
Optional
Required or limited access Required
to room2
Required
Biosafety levels for cell sorting by Agent (examples)
Agent
Recommended Biosafety Level
Hepatitis C
Influenza A (strain PR8)
BSL2/3
BSL2/3
Klebsiella pneumonia
LaCrosse virus
BSL2/3
BSL2/3
LCMV
BSL2/3 or BSL3
Leishmania
Malaria
BSL2/3*
BSL2/3*
Respiratory Syncytial Virus
BSL2/3
Restrictions or Comments
Influenza (seasonal) vaccine required
BSL dependent upon strain; pregnant women excluded from lab during sorting
Toxoplasma gondii
BSL2/3*
pregnant women excluded from lab during
sorting
Vaccinia
Avian influenza
H1N1
BSL2/3
BSL3
BSL3
vaccine required
Influenza (seasonal) vaccine required
H1N1 vaccine required
HIV
BSL2/3 or BSL3
Monkeypox
BSL3
TB, Mycobacterium tuberculosis
BSL3
vaccinia vaccine required, every 3 years
*respirator PPE optional (mucous membrane protection required) for this agent except where the sample also contains human/NHP blood cells or fluids.
Recommendations for Sorting Infectious Samples
• Perform Risk Assesment
– Confer with Biosafety specialists and infectious disease experts where appropriate
– Cell sorting is an Aerosol Procedure Hazard
• Develop SOP
– Instrument‐specific design features
– PPE
• Equip with Aerosol Management System
– Validate Containment‐ GloGerm
• ISAC Biosafety Guidelines
Acknowledgments
• Flow Cytometry
Section, NIAID, NIH:
–
–
–
–
–
–
–
–
–
Mehrnoosh Abshari
Calvin Eigsti
Bishop Hague
Carol Henry
Larry Lantz
Tom Moyer
Michele Racine
David Stephany
Elina Stregevsky
• Steve Perfetto, VRC
• Debbie Wilson, Division of Safety, NIH
• NIH Biosafety task force:
–
–
–
–
–
Phil McCoy
Kathy McKinnon
Jeff Potts
Srinivas Rao
Bill Telford
References
• Schmid, I, Lambert, C, Ambrozak, D, Marti GE, Moss, DM, Perfetto, SP. International Society for Analytical Cytology Biosafety Standard for Sorting of Unfixed Cells.. Cytometry 2007; 71A:414
• Perfetto SP, Ambrozak DR, Koup RA, Roederer M. Measuring containment of viable infectious cell sorting in high‐velocity cell sorters. Cytometry 2003; 52A:122. • Perfetto, SP, Ambrozak, DR, Nguyen, R, Roederer, M, Koup, RA and Holmes, KL. Standard Practice for Cell Sorting in a BSL‐3 Facility. Flow Cytometry Protocols, third edition. Ed. Hawley, TS and Hawley, RG. In press.
• Rutala, WA, Weber, DJ. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008 http://www.cdc.gov/ncidod/dhqp/pdf/guidelines/Disinfection_Nov
_2008.pdf
• Survey Link: http://www.zoomerang.com/Survey/WEB22AEMDPQVPH