Post on 25-Mar-2018
Biosafety in the flow cytometry laboratory
Phil Hogarth Flow Cytometry Manager
PI Vaccine Immunology Team Animal & Plant Health Agency
philip.hogarth@APHA.gsi.gov.uk ISAC Biosafety Commi�ee member
1 Courtesy of K Holmes,NIAID, NIH
Who am I?
PI Research Group Also core facility manager (2008) Doing Flow for 17 years Many more experienced in Flow – Audience But, been working with TB & CL3 14 years – Responsible for Risk Assessment & Biosafety of group – Including core facility & TB mouse house
Co-‐opted ISAC Biosafety Commi�ee 2011
2 Courtesy of K Holmes,NIAID, NIH
Outline
Why Biosafety background Biosafety & cell sor�ng What do you need to do? Risk Assessment Sorters in BSC’s Training/ Containment Tes�ng
3 Courtesy of K Holmes,NIAID, NIH
Why? 2000-‐2014
>60 Laboratory Acquired Infec�ons (www.biosafety.be/CU/LAI/Recent_LAI.html)
Include: Plague (a�enuated!): USA 2009, Fatal
Ebola: Russia 2004 Fatal,2005; Germany 2009,2011 Tularemia: USA 2002, 2009, 2012
TB, E. coli, MRSA, Meningi�s, Brucella, & many more (if you can think of it, someone has caught it – in a lab)
Biosafety must be an integral part of ANY research!
4 Courtesy of K Holmes,NIAID, NIH
Laboratory Acquired Infec�ons
From Safe Sor�ng: Principles and Prac�ces, Kevin a. Holmes, Ph.D., NIAID, NIH
Laboratory Procedure hazards and LAI’s 5 routes of laboratory transmission:
1. Self inocula�ons: syringe needles/sharps 2. Spills/splashes on skin / mucous membranes 3. Mouth pipe�ng 4. Animal bites & scratches 5. Inhala�on exposure to infec�ous aerosols
1-‐4 account for <20% of LAI’s The cause of 82% of LAI’s are unknown, but are presumed to be aerosols
5 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 1
Biological Agents, HSE defini�on: ‘a micro-‐organism, cell culture, or …endoparasite, whether or not GM, which may cause infec�on…or otherwise create a hazard to human health.’
Microbiological agents classified into Risk Groups Ability to infect and cause disease Virulence (severity of disease) Availability of preventa�ve measures and effec�ve treatments
UK -‐ Hazard Groups 1-‐4; WHO, USA, Risk groups 1-‐4 HSE: www.hse.gov.uk/biosafety/biologagents.pdf CDC: www.cdc.gov/od/ohs/bios�y/bmbl5/bmbl5toc.htm WHO: www.who.int/csr/resources/publica�ons/biosafety/Biosafety7.pdf?ua=1
6 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 2 All biological agents must be classified
in 1 of 4 Hazard Groups
Increasing hazard to human health
Hazard Group 1
Hazard Group 2
Hazard Group 3
Hazard Group 4
Lab containment Level 1
Lab containment Level 2
Lab containment Level 3
Lab containment Level 4
7 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 3 So how do we deal with Microbiological Agents? Health and Safety Legisla�on – HSE & COSHH1
1 Control of Substances Hazardous to Health Regula�ons 2002 Source: HSE
Containment & Risk Assessment: central components of legisla�on
8 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 4 Dealing with Microbiological Agents
e.g. Containment Lab AMS, BSC
Hierarchy of Control
e.g. Risk Assessment SOP
Most desirable
Least desirable 9 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 5 Engineering control: Containment Laboratories
10 Courtesy of K Holmes,NIAID, NIH
Engineering Instrument design (sort chamber) Aerosol Management (AMS) Biological Safety Cabinet (BSC)
Biosafety Background 6 Engineering & other controls
Contain at source
PPE Gloves, Labcoat, etc. Respirator (HSE much prefer containment at source)
11 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 7 Administra�ve Controls
Risk Assessment! Standard Opera�ng Procedure – Instrument specific – Step by step, describing:
Setup Opera�on Failure (e.g. Nozzle clog) Decontamina�on Shutdown 12 Courtesy of K Holmes,NIAID, NIH
Biosafety Background 9 Risk Assessment
5 Easy steps (slightly different in UK from ISAC standards)
5. Record, REVIEW & Agree Assessment (with Biosafety unit)
1. Iden�fy the HAZARD
2. Iden�fy the Laboratory PROCEDURES
3. Iden�fy the RISKS
4. Consider appropriate CONTROLS
What is going to Harm you?
is going to harm
Likelihood & Severity of Harm
is going to harm e.g. CL level, BSC, SOP, Training, Competence going to harm
13 Courtesy of K Holmes,NIAID, NIH
Biosafety & Cell Sor�ng (finally!)
OK, I can hear you: What’s this got to do with sor�ng? What’s this got to do with me?
Sorters produce aerosols! it’s how they work (you know that)
14 Courtesy of K Holmes,NIAID, NIH
Aerosol Produc�on by Cell Sorters
Cell sorters produce aerosols – ~50-‐300 µm drops & smaller satellite droplets
Depends upon nozzle diameter, pressure & DDF Captured by collec�on tubes and waste drawer
– ‘…secondary aerosols of various and undefined droplet sizes’ produced during failures (clogs) (ISAC 2007, 2014 Biosafety standards)
Satellite droplets
15 Courtesy of K Holmes,NIAID, NIH
Characteriza�on of aerosols by Cell Sorters: Fail Mode
Kevin Holmes at NIH (NIAID) simulated fail mode on BD FACSAria (block waste collector)
Used TSI UV Aerodynamic Par�cle Sizer to measure aerosol produc�on
Holmes, K.L. Cytometry Part A 2011: 79A, p. 1000-‐1008
16 Courtesy of K Holmes,NIAID, NIH
Characteriza�on of aerosols by Cell Sorters: Fail Mode
0.01
0.1
1
10
100
1000
0.5 5
70 psi 35 psi 20 psi
Aerodynamic Diameter (µµm)
Concentra�on (#/cm3)
9.8x103/cm3 mean: 2.0 µm
1.9x103/cm3 mean: 2.4 µm
90/cm3 mean: 2.65 µm
Range of alveolar deposi�on and infec�vity
From Safe Sor�ng: Principles and Prac�ces, Kevin a. Holmes, Ph.D., NIAID, NIH 17 Courtesy of K Holmes,NIAID, NIH
Aerosols and Cell Sorters: Summary
Sorters can produce high concentra�ons of aerosols – 70psi, 18000/cm3 aerosols can be produced in fail condi�on.
– These aerosols are between 1-‐3µm aerodynamic diameter Higher sheath pressure increases concentra�on and decreases size
Aerosols in this size range, i.e. 1-‐3µµm: – May remain airborne almost indefinitely – More likely to deposit in lung alveoli – Have been shown to be associated with increased infec�vity of some organisms
18 Courtesy of K Holmes,NIAID, NIH
Biosafety in the Cell Sorter Lab What do I need to do?
Follow established biosafety principles Develop an SOP for cell sorter opera�on Use resources: – Biosafety professionals in your Ins�tu�on – Published Standards
SSOORRTTEERR
19 Courtesy of K Holmes,NIAID, NIH
Biosafety Principles 1
Two basic principles of biosafety are Containment and Risk Assessment
1. Containment Reduce or eliminate exposure of lab workers and outside environment to poten�ally hazardous agents
Examples: Biological Safety Cabinet, centrifuge cups, Aerosol Management Systems on cell sorters Includes standard microbiological prac�ces and procedures
20 Courtesy of K Holmes,NIAID, NIH
Biosafety Principles 2
2. Risk Assessment: – ‘an ac�on or series of ac�ons taken to recognize or iden�fy hazards and to measure the risk or probability that something will happen because of that hazard.’
– Responsibility of PI together with biosafety professionals and Ins�tu�on Biosafety Commi�ee
Requires careful judgment Adverse consequences if risks are underes�mated Excessive safeguards – Addi�onal expense/procedure with li�le safety enhancement – May result in circumven�on
21 Courtesy of K Holmes,NIAID, NIH
Risk Assessment
5 Easy steps (slightly different in UK from ISAC standards)
5. Record, REVIEW & Agree Assessment (with Biosafety unit)
1. Iden�fy the HAZARD
2. Iden�fy the Laboratory PROCEDURES
3. Iden�fy the RISKS
4. Consider appropriate CONTROLS
22 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 1: Iden�fy Agent Hazards:-‐1.
What is the Hazard Group of the biological agent? Agent may be unknown – Source of sample? – Human, mouse, NHP etc. All Human samples CL2 (minimum)! – Country of origin
Does agent have previous history of LAI’s? – BMBL agent summary statements
Mode of transmission – Aerosol?
Lab Personnel – Risk factors (pregnant, immunocompromised, etc.)
23 Courtesy of K Holmes,NIAID, NIH
“Do we really need to treat the sor�ng of human cell lines like 293T, Hela, etc…as human primary samples?”
Lymphocy�c Choriomeningi�s Outbreak Associated With Nude Mice in a Research Ins�tute. JAMA: 1992: 267, 1349.: – Cell lines contaminated with LCMV
ATCC: “strongly recommend…all human/primate cell lines be handled …as a cell line known to carry HIV or hepa��s virus”
NIH Biosafety Policy for Cell Sorters: ‘Treat human lines as infec�ous ..NIH ‘clean’ cell line was infected with HIV’
All human derived cells CL2 or above!
Risk Assessment Step 1: Iden�fy Agent Hazards:-‐2
24 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 1: Iden�fy Agent Hazards:-‐3
recombinant and viral vectors Gene�cally modified agent hazards
Samples with r.DNA, viruses, bacteria, yeast etc. & Viral vectors: len�virus, adenovirus or retrovirus etc. Not classified in NIH/CDC’s BMBL or WHO Laboratory Safety Manual UK see: www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/
NIH Recombinant DNA Advisory Commi�ee NIH Guidelines For Research Involving Recombinant DNA Molecules:
BSL2 or BSL2+ , dependent upon risk factors
Modified From Safe Sor�ng: Principles and Prac�ces, Kevin a. Holmes, Ph.D., NIAID, NIH 25 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 2: Iden�fy Lab Procedure
What happens to the sample in the Lab? Do you filter? Aerosol risk Are there any sharps? Physical hazards? e.g. Lasers Produc�on of sorter generated aerosols – Cell Sor�ng is a Procedural Hazard – “General agreement among biosafety professionals… aerosol genera�on by procedures and opera�ons…is the probable source of many LAI’s”
26 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 3: Iden�fy Risk
How likely are you to come to harm? Do you have right controls for the hazard? Does an aerosol pose added risk? LAI may occur even if infec�on not via aerosol in nature: higher concentra�ons & aerosol genera�ng procedures
Example: 1st documented case of aerosol infec�on of Scrub Typhus (Orien�a Tsutsugamushi) (Infec�on, 2001, 29:54)
– Usually transmi�ed via insect vector – lab worker disrupted cells on open bench
Decide on the Containment Level required 27 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 3: Determina�on of Containment Level
CL Agents Prac�ces Barriers & safety equipment
1 No disease in humans Standard microbiological None 2 Human disease; not
aerosol transmi�ed CL1 & limited access, signage, medical surveillance
BSC Class I or II, lab coat, gloves
3 Aerosol transmission possible with serious or lethal consequences
CL2 & controlled access, decontamina�on of waste, lab clothes, baseline serum
BSC Class I or II, protec�ve lab clothing, respiratory protec�on
4 Dangerous/exo�c high risk life threatening disease, aerosol transmission likely
CL3 & clothing change, exit shower, all material decontamina�ng upon exit
BSC Class III or Class II with full-‐body suit
Modified from BMBL, 5th Ed. Modified From Safe Sor�ng: Principles and Prac�ces, Kevin a. Holmes, Ph.D., NIAID, NIH
Hazard Groups correlate with but do not = containment level
28 Courtesy of K Holmes,NIAID, NIH
CL2 With Enhanced Precau�ons
Previously referred to as CL2 w/CL3 prac�ces – Not a containment level – CL3 may include prac�ces/facili�es not applicable to CL2 lab, i.e. autoclave in lab, HEPA filtered air, etc.
– so ‘enhanced precau�ons’ are defined, i.e. containment tes�ng before sort, defined disinfec�on, etc.
Modified From Biosafety Standards and Risk Assessment for Sor�ng Unfixed Cells, CYTO 2011, Perfe�o and Holmes 29 Courtesy of K Holmes,NIAID, NIH
Biosafety levels for cell sor�ng by Agent (examples)
Agent Recommended Biosafety Level
Restric�ons or Comments
Hepatitis C BSL2/3 Influenza A (strain PR8) BSL2/3 Influenza (seasonal) vaccine required Klebsiella pneumonia BSL2/3 LaCrosse virus BSL2/3
LCMV BSL2/3 or BSL3 BSL dependent upon strain; pregnant women excluded from lab during sor�ng
Leishmania BSL2/3* Malaria BSL2/3* Respiratory Syncytial Virus BSL2/3
Toxoplasma gondii BSL2/3* pregnant women excluded from lab during sorting
Vaccinia BSL2/3 vaccine required Avian influenza BSL3 Influenza (seasonal) vaccine required H1N1 BSL3 H1N1 vaccine required HIV BSL2/3 or BSL3 Monkeypox BSL3 vaccinia vaccine required, every 3 years TB, Mycobacterium tuberculosis BSL3
*respirator PPE op�onal (mucous membrane protec�on required) for this agent except where the sample also contains human/NHP blood cells or fluids.
30 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 4: Iden�fy Controls
Engineering Containment laboratory (usually CL2 minimum)
Sort chamber design AMS BSC PPE Gloves, Labcoat, etc. Respirator (HSE much prefer containment at source) 31 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 4: Iden�fy Controls
Procedural Develop Standard Opera�ng Procedure – Instrument specific – Step by step, describing:
Setup Sor�ng Sort failure (e.g. Nozzle clog) Decontamina�on (sort chamber, fluidics, BSC) Shutdown
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Risk Assessment Step 4: Iden�fy Controls
Procedural Proper training and/or cer�fica�on of personnel is essen�al Equipment must be available and func�onal (Tested)
33 Courtesy of K Holmes,NIAID, NIH
Risk Assessment Step 5: Review
Review/agree RA with Ins�tute Biosafety professionals Agree final containment level RECORD & review regularly Cell sor�ng not specifically covered by HSE, but do state …’procedures genera�ng aerosols at CL2 or higher MUST be conducted in BSC at appropriate CL’ www.hse.gov.uk/biosafety/biologagents.pdf
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Use Published Resources www.hse.gov.uk/biosafety/biologagents.pdf GMO: www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/ The Approved List of Biological Agents h�p://www.hse.gov.uk/pubns/misc208.pdf
2014 ISAC Cell Sorter Biosafety Standards (Cytometry Part A, 85A: 434-‐453, 2014) www.isac-‐net.org/Resources-‐for-‐Cytometrists/Biosafety.aspx
CYTO U Course: Biosafety www.cytou.peachnewmedia.com/store/provider/provider09.php
CYTO ‘14 Tutorials: www.isac-‐net.org/Educa�on-‐Resources/Educa�on-‐Materials/
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Operator training & SOP Review
Staff proficiency in sorter opera�on & biosafety procedures – Training essen�al for cell sorter lab opera�on – Amount of training �ed to risk assessment The higher the risk, the more training required Inexperienced operator more likely to circumvent safety procedures
Review of SOP at regular intervals Review/prac�ce procedures for nozzle clog
36 Courtesy of K Holmes,NIAID, NIH
Cell Sorters in BSC’s Need for a BSC is dependent upon risk assessment Sorters cannot just be placed in a BSC: must be cer�fied – 2014 Standards: “must be manufactured to meet …CSN EN 12469 (Europe).”
Can abrogate requirements for separate room for sorter and PPE (respirators) for occupants of shared lab Does not eliminate need for AMS
37 Courtesy of K Holmes,NIAID, NIH
Why you need an AMS inside a BSC
32/cm3
6.8/cm3 Escaping BSC containment
Data from I-‐Cyt Reflec�on, Poster, J. Lannigan & K. Holmes, ISAC 2011
Condi�ons: BSC: On Sorter in Fail mode No AMS loca�ons as shown: Aerosols measured using UV-‐APS par�cle sizer
657/cm3
It’s about containment at the source!
38 Courtesy of K Holmes,NIAID, NIH
Cell Sorters in BSC’s
Decontamina�on of BSC cri�cal What to use? – Formaldehyde, corrosive, can leave ‘film’, dangerous – H2O2, less corrosive, safer
Needs valida�on against biological agent, H2O2 kills (all) bacteria & virus – spore strips preferred
39
Containment tes�ng: why?
Any system can fail Instrument manufacturers generally make no claims about efficacy of the AMS Visual tests like smoke tests are unreliable for aerosols in this size range
40 Courtesy of K Holmes,NIAID, NIH
Containment tes�ng: When
1. Based upon Risk Assessment, but must be: 2. Following service or any maintenance of sort
chamber and/or AMS. 3. Following ini�al installa�on or reloca�on. 4. Following change of the AMS filter. 5. For BSL3/4 labs: – Prior to every sort if the frequency of sor�ng is once/week or less
– Weekly, if the frequency of sor�ng is mul�ple sorts/week
41 Courtesy of K Holmes,NIAID, NIH
Containment tes�ng: How
GloGerm assay – Published standard Cyclex d assay using beads being validated Other methods in development
42 Courtesy of K Holmes,NIAID, NIH
Food for thought: ISAC Biosafety Commi�ee Survey
Survey sent to ISAC members-‐ April 2010 59 ques�ons: – Infec�ous Sor�ng performed? – Aerosol evacua�on systems? – Containment tes�ng? – Types of samples sorted? – PPE worn (respirators, etc.)?
From Biosafety Standards and Risk Assessment for Sor�ng Unfixed Cells, CYTO 2011, Perfe�o and Holmes 43 Courtesy of K Holmes,NIAID, NIH
Food for thought: Survey Results: No Aerosol Evacua�on
No Containment with poten�ally infec�ous samples: 7 responses – Either NHP, human blood or human cell lines – No aerosol management system or containment tes�ng
– No respiratory protec�on – Of these 7 labs, 4 were in shared laboratory space
ISAC Standards: NHP, Human samples and cell lines are BSL2 with enhanced precau�ons
From Biosafety Standards and Risk Assessment for Sor�ng Unfixed Cells, CYTO 2011, Perfe�o and Holmes 44 Courtesy of K Holmes,NIAID, NIH
Finally
What about Analysis? Fix samples (nearly all assays can be fixed now) Or, treat as sort (loading/removing samples= aerosol) ABOVE ALL
Do Risk Assessment 45
Acknowledgments ISAC Biosafety Commi�ee – Current: Kevin Holmes & Steve Perfe�o (co-‐chairs), Ben Fontes Phil Hogarth, Rich Konz, Simon Monard, Hank Pletcher, Ingrid Schmid, Rob Wadley. Special thanks to Kevin Holmes, NIAID, NIH; for many slides
ISAC Biosafety Commi�ee – Past members NIH Biosafety Task Force London Cytometry Club
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