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Environmental Health and Safety
Biosafety in Research Laboratories Refresher Training Post-Test
Name:
Title:
Department:
PI/Supervisor:
Work (BLDG/RM):
Email:
*NOTE* By completing this test, you are confirming that you have viewed the entire training presentation.
1. One of the recognized lab acquired infections (LAI) in the presentation noted a thumb
laceration from a scalpel used with a West Nile Virus infected bird. The wound was washed
with soap and water. Which of the following should not be done?
A. Proceed to Occupational Health Clinic or Emergency Department for treatment.
B. Inform your supervisor of the accident.
C. Complete the experiment prior to seeking treatment on the same day.
2. You investigated the accident in the above scenario and recommended a change in personal
protective equipment (PPE) to help prevent the risk of a similar injury in the future. Which
of the following do you recommended?
A. The use of Nitrile coated Kevlar gloves
B. The use of Solid front, fluid barrier gown
C. The use of a N95 mask or other piece of properly fitted aerosol protection
3. Your research, which involves recombinant DNA, is funded by the private Massachusetts
Lions Eye Research Fund. Do you need to comply with the NIH Guidelines?
A. Yes
B. No
4. Which of the following routes of exposure has the potential to lead to a LAI (Laboratory Acquired
Infection)?
A.
B.
C.
D.
E.
F.
Aerosol inhalation
Abrasions
Splash to the eyes
Mouth pipetting
None of the above
All of the above
Biosafety in Research Labs Refresher Training Test
v.2
|2013
5. You are working with baculovirus to express recombinant protein in insect cells. This work
must be registered with the Institutional Biosafety Committee (IBC).
A. True
B. False
6. Human cell lines derived from primary human material are covered by the OSHA
Bloodborne Pathogen Standard while human cell lines purchased from the American Type
Culture collection (ATCC) or Coriell Institute are not.
A. True
B. False
7. Which of the following is not part of an Agent Risk Assessment?
A.
B.
C.
D.
Availability of funding
Infectious dose
Severity of disease
Person-to-person disease transmission
8. Which of the following can disrupt air flow in the biosafety cabinet?
A. Two people working simultaneously and independently
B. Placing items on the front grille
C. A person walking rapidly by the biosafety cabinet (BSC) when someone is working
inside
D. Having an open flame
E. All of the above
9. The LAI (Laboratory Acquired Infection) with Sabia Virus in the presentation may have
occurred when the rotor was opened. What can be done to minimize aerosol exposure related
to centrifugation?
A. Load/unload rotor inside a BSC when the rotor is light enough to move
B. Close rotor and centrifuge upon discovering a spill. Place DO NOT DISTURB sign on
the centrifuge. Initiate clean up in half an hour.
C. Clean up immediately. Someone else is waiting to use the centrifuge.
D. A and B
10. The NIH Office of Biotechnology Activities (OBA) requires notification when an exposure
involves recombinant DNA even if that exposure does not lead to illness. Which does not
need to be reported to OBA?
A. A splash to the eye of a recombinant adenoviral vector
B. Exposure of a clinical laboratory worker to Muchupo virus when a blood tube breaks in
the centrifuge.
2|Page
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Biosafety in Research Labs Refresher Training Test
v.2
|2013
11. An ideal container for bags filled with biowaste prior to autoclaving or vendor disposal would be
one that has the following characteristics:
A.
B.
C.
D.
Hard-sided
A lid that can be closed when not being used
Easily decontaminated
All of the above
12. How should biological materials that need to be transported from the lab to the animal
facility be handled?
A. Transport material in your pocket while wearing a lab coat
B. Carry the material in your hands while wearing gloves
C. Transport the materials in a sealed leak-proof, shatter resistant primary and secondary
container
D. Transport the materials in a primary container stored in an open ice bucket
13. A post-doc asks you to ship a slant of Shigella to a colleague at a different University. You
will need to:
A. Put parafilm on the slant cover, surround it in bubble wrap and put it in a FedEx
envelope.
B. Find out about Federal shipping training requirements. Classes are listed on the TEHS
website.
14. The NIH Guidelines are now applicable to research with synthetic nucleic acids. Which of
the following is NOT TRUE?
A. The Guidelines recognize that appropriate biosafety containment of an agent is necessary
regardless of the technology used the generate the agent.
B. The chemical synthesis process is now regulated.
C. The Guidelines are applicable only after nucleic acids are placed in a biological system
(e.g. cell)
D. Synthetic nucleic acids that cannot replicate or do not contain or origin of replication are
exempt from the Guidelines.
*NOTE* By completing this test, you are confirming that you have viewed the entire training presentation.
Return completed test to Tufts EHS ([email protected]).
If you are unable to send via email, please print out and send interdepartmentally to:
Environmental Health & Safety; Posner 105, 200 Harrison Avenue; Boston
To Be Completed By TEHS Staff:
Completion Date:
3|Page
Expiration Date:
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200 Harrison Avenue, Boston, MA 02111 ι TEL: 617.636.3615 ι FAX: 617.636.2419
Biosafety in Research Labs
Tufts University/Tufts Medical Center
January 2013
Learning Objectives





Become familiar with the Regulatory Agencies and
Institutional Committees governing use of Biological
Material.
Understand key elements of Risk Assessment
Familiarity with Biosafety Level 1 and Biosafety
Level 2 practices and procedures.
Recognize biosafety practices and procedures extend
to work in the animal facility.
Recognize when there is the need to wear Personal
Protective Equipment (PPE) and be able to
determine what PPE should be worn.
Learning Objectives






Become familiar with use of Biosafety Cabinet
Recognize the risk of using a centrifuge incorrectly
and requirements when used with infectious agents.
Understand spill response procedures.
Review collection, storage, transport and
inactivation of biological material
Know that shipping research materials requires strict
procedures to protect everyone in the transportation
chain.
Understand your responsibilities when working with
biological material.
1
Training Summary
Biosafety Related




BRL – 3 yrs
OSHA BBP
annually
Shipping – 2 yrs
PI Training on
NIH Guidelines
online
Other Areas –
Some are
Institution
Specific


TU – General
Lab Safety via
LearnCenter
TMC – HIPAA,
BBP for Infection
Control
Key Links


http://www.tufts.edu/central/resear
ch/IBC/ (Vice Provost)
http://publicsafety.tufts.edu/ehs/



Left column
Accident & Incident Reporting Forms
Biological Safety


Biological Safety References
TU/TMC Biosafety Manual
The Goal of Biosafety

Minimize the risk of exposure to
biohazardous agents using:





Engineering Controls
Work Practice Controls
Exposure Response Plans
Personal Protective Equipment (PPE)
Preventing/minimizing occupational
exposure
2
Biohazardous Materials Definition

An agent of
biological origin
that has the
ability to cause
disease in a
human, animal,
plant, or insect.







Recombinant DNA
(i.e. viral vectors)
Viruses
Bacteria
Parasites
Fungi
Prions
Human source
material
 Human cell lines
Laboratory Acquired Infections


Infections from laboratory-related
activities whether symptomatic or
asymptomatic
Four primary routes of exposure

Inhalation of aerosols
Percutaneous inoculation (needle and syringe,

Contact between mucous membranes and

cuts, abrasions and animal bites)
contaminated material (hands or surfaces)

Ingestion (aspiration through a pipette, smoking or
eating or after inhalation)
Laboratory Acquired Infections

Studies on the incidence of LAIs:


Pike and Sulkin
 Surveyed 4,000 labs
 50% response
 4,079 LAI from 1935-1978
Harding and Byers
 1979-2005
 1,141 with 24 deaths
 46% clinical labs
 50% research labs
3
Causes of LAIs




Accidents
Failure to apply SOP
Failure to Identify Risk
 Routine
work/commonly
used agent
 Misidentification
Failure of pre or post
preventive measures
 No immunization
 None or wrong
prophylaxis
 Majority
of
Occupational
Infections
are without
known
exposure
incident
LAI -Recognized Exposure

West Nile Virus

August 2002 and October 2002
 Laceration of thumb from scalpel while
removing brain tissue from a bird
 Needle stick when harvesting brain
tissue from infected mouse
 Wounds washed with soap and water
immediately after exposure incident
 3 days post exposure both individuals
developed signs of WNV
 Both recovered fully within 7 days
Old Title vs

NIH Guidelines
for Research
Involving
Recombinant
DNA Molecules
New Title



NIH Guidelines
for Research
Involving
Recombinant or
Synthetic
Nucleic Acid
Molecules
Implement by
3/3/13
4
Is chemical synthesis covered?
synthesizer
Not until in a …
Oversight of Biohazardous Work

NIH Guidelines for work Recombinant DNA





http://oba.od.nih.gov/oba/rac/Guidelines/NIH_G
uidelines.htm
Responsibilities of TU/TMC, IBC, PI, BSO
Containment levels for work in tissue
culture & animal models
Requires notification of Overt Exposures
to rDNA handled at BSL-2/ABSL-2 or
above
IBC review based on Risk Assessment



III-D: IBC Approval Prior to Initiation of Work
III-E: IBC Notice Simultaneous with initiation
III-F: Exempt from NIH rDNA Guidelines
III-F Registrations

Nothing exempt in Boston



Using nonhazardous plasmids (pBR) to
clone gene of interest into host
(nonpathogenic E.coli)
Often the first step in creating a
transgenic animal
New exemptions for synthetic nucleic
acids

No origin of replication, no integration…
5
Oversight of Biohazardous Work

TU/TMC Institutional Biosafety
Committee
Review and approval of:
 Recombinant DNA
 Human Gene Transfer Clinical Trials (TMC)
 Infectious Agents
 Select Agents and Toxins
 Human Source Material including Human Cell Lines (TU)

Develop Exposure Response Plans for
Biohazardous Agents
 Investigation of accidents/exposures
with agents

IBC Website: http://www.tufts.edu/central/research/IBC/
Registration Form

Before work





Approval may
include NIH
Infectious
Exempt
Deadlines
Committee
decision sent by
IBC office
NIH Guidelines: Definition of rDNA


Molecules
constructed by
joining nucleic
acids
& can replicate in
a living cell
6
NIH Guidelines: synthetic

Nucleic acids
that can base pr
w naturally
occurring
nucleic acids
NIH Definitions Continued

Molecules
resulting from
replication of
rDNA or
synthetic
nucleic acids in
past two slides
Other Changes



AAV serotypes needing review
Transfer of drug resistance traits
Human Gene Transfer w nucleic
acids
7
Guidelines Relevance



Research
performed at or
sponsored by Tufts
due to receipt of
funds
Term & condition
of NIH funding
Noncompliance –
suspension,
limitation etc
Oversight of Biohazardous Work



OSHA Bloodborne Pathogen Std 42 CFR
1910.1030 (www.osha.gov – B)
Compliance required for all work with
Bloodborne Pathogens or OPIM.
BBPs: Capable of causing Human Disease:







HBV
HCV
HIV
Human Source Material / Human Cell Lines
Transmitted by direct contact
Annual training
No charge
Researchers Use

Human cell lines &
materials


1o human material
ATCC:


ATCC Biosafety Level is
specific for DOT
transportation requirements
and not the BSL that the
human cell lines should be
used at.
Key points


screening/testing
viral inactivation
8
ATCC Example


CCL-2 aka HeLa
Products: keratin
Comments: human papillomavirus
18 sequences



cause of cervical cancer
most cases clear w/o med. intervention
Biosafety Level: 2
OSHA BBPs and
Universal Precautions

An Approach to Infection Control

all blood & tissues are guilty




assumed to carry disease
no way to assure complete absence
Body Substance Isolation/Standard
Precautions
Exposure Control Plan

access and updates
Hepatitis Signs/Symptoms


None





B – 30%
C – 80%
Jaundice
Fatigue
Loss of appetite
Yellowness of skin
/ eyes/body fluids
due to deposition
of bile pigment
from excess
bilirubin



hemolytic anemia
gallstones
alcoholic cirrhosis
9
Bloodborne Pathogen - HBV
Hep B Virus and Vaccine

Virus infective
after drying on
surface




Blood
Wound exudates
Due to childhood
vaccination
(1991) ~95%
new infections in
adults
Booster not
recommend w/
normal immune
status


Consider if
suppressed
Occ Exposure

HBIG
Bloodborne Pathogen - HCV

Most common chronic BBP infection in
the US

Seroconversion - infected sharps ~1.8%

Rare with mucous membrane exposure


Survive and transmit for 6 hours but not
longer than 4 days
No vaccine
10
Bloodborne Pathogen - HCV

Long term effects




chronic infection: 75-85%
chronic liver disease: 70%
leading indication for adult liver transplants
Treatment





Expect genotyping
Waiting 12 weeks to begin treatment allows for
more spontaneous resolution than 6 months
Combo therapy w/ pegylated interferon and
ribivarin-30-40% resolve
+ protease inhibitors approved 5/2011 ~75%
12/11 CDC symposium – can we improve
screening with more effective drugs?
Bloodborne Pathogen - HIV

6/5/81 MMWR

5 previously
healthy gay men
pneumoncystis
pneumonia

Within yr

Within 2 yrs


Called AIDS
Agent ID - HIV
Bloodborne Pathogen - HIV

Risk after percutaneous injury 0.3%
mucous membrane exposure 0.09%
PEP (post exposure prophylaxis)
HAART(highly active antiretroviral therapy)
 Suppresses viral replication for decades
 Less infective to others
 Drug therapy 1995
 1997-1998
 Decline in diagnoses and deaths began
leveling in 1999



11
LAI - Known Exposure

Human colonic
adenocarcinoma 1986




Percutaneous injury to
hand from needle used
for the injection of
human cancer cell line
into an animal
19 year old with no
identified immune
deficiencies
Sustained growth of
tumor nodule
http://www.nejm.org/doi
/pdf/10.1056/NEJM19861
2043152314
Exposure - Transplant
Transmission (2002)



Kidney – 50 yr
old F, stroke;
recip – 37 yr old
M, diabetic
Autopsy after
transplant
CDC- 1% organ
suspected of
disease trans
Local Oversight of Biohazardous Material

MA Department of Public Health


Boston Public Health Commission



Biohazardous Waste
Disease Surveillance
BPHC Lab Regulation
Boston Fire Department
Ordinance for Research Labs
12
The Risk Assessment





Agent Review
Key to safe use of
biological material

before work with
biological agent
performing new
technique
New equipment




Combined effort of
PI, Lab employee,
Biosafety Officer,
and IBC



Severity of disease
Transmission
Infectious Dose
Incubation period
Signs and Symptoms of
Disease
Increased susceptibility
PPE to lower exposure
Exposure response
Procedures
Risk Assessment & Synthetics


% of genome from each parent
Fn/purpose of each sequence


Assume same fn as original host?
Synergism between sequences &
transgenes
What’s a Biosafety Level?


Risk Assessment End
Result
A combination of





Laboratory practice
Safety equipment
Facility design
Biosafety Levels 1-4
defined in BMBL 5th Ed
NIH Guidelines Risk
Groups 1-4 defined in
NIH rDNA Guidelines.
13
Biosafety levels

BL1:



Organisms that are not associated with
disease in healthy human adults.
Don’t eat it
BL2:


Organisms associated with human
diseases which are rarely serious and
for which preventive or therapeutic
interventions are often available.
Don’t touch it
Examples
BL1




BL2
yeast two hybrid
pBR & pUC
based plasmids
K12 based E. coli
 nonpathogenic
helper free
adenoassociated
virus





pathogens
 Borrelia
viral vectors
 Retrovirus/len
tivirus
BBP material
NHPrimate
material
Oncogenes
BL1 Lab design
14
BL1- Highlights




Lab designed to be easily cleaned
Each lab has a sink for hand washing
 1. after using rDNA materials
 2. before leaving the lab
Work surfaces decontaminated once a day
and when spills occur
 Usual disinfectant?
No eating, drinking, food storage
BL2: Lab Design
BL2-Highlights

All BL1 Facilities/Practices & more stringent
control of lab access, processes and waste

Doors closed and posted

Autoclave / disinfect waste



Report spills, accidents, potential
exposures.
Minimize the creation of aerosols.
All equipment contained in the room as
much as feasible.
15
Animal Biosafety




Don/doff PPE
Decon of work
area
Transporting &
handling
animals
Work with
sharps
Engineering Controls
Isolate or remove
 In - syringes,
razor blades,
contam. glass
 No – chucks,
pipette wrapper
 ¾ full
 Plastic/mylar
coated capillary
tubes
Optimizing Engineering Controls



“Single most
useful piece of
equipment for
prevention of lab
acquired
infections.” Pike
Annual inspection
Air flow



keep grilles clear
no whooshing
recycle - not for
chems
16
BSC Film Clips
Engineering - The Biosafety Cabinet




Design
Protecting air flow
 Bsc is designed
to be used by
only 1/X
Daily practices
Other poor
practices in
previous slide
Total Exhaust BSC


Primarily for
toxic chemicals
& biologic work
Depends on
building exhaust


Energy savings
problems
HNRCA
17
Engineering - BSC - Organize
Liquid Waste Practices



Chemically
disinfect
10% mercury free
bleach
Less than a week
old



Chlorine
concentration
Wait 20 minutes
Pour down drain
Secondary Containment


Nonporous
Holds entire
contents
18
Centrifugation – Avoid Aerosols

Wipe down outside
of sealed rotor or
safety cup w/
disinfectant before
removing from BSC
Double Barrier


Prevent the
release of
aerosols
Environmental
contamination is
an effective
method of
disease
transmission
Avoid Mechanical Failure


Centrifuges are
designed to
contain the rotor
in event of a
failure
Documented rotor
failures


Personnel injury
Property damage
19
Centrifuges

Rotors





Stress from
centrifugal force
Fatigue –
stretching and
relaxing causes
cracks
 Exp or safe
service dates
Corrosion
User error causes
many malfunctions





Secure rotor lid
Secure rotor to
drive
Improper balancing
Overloading
Buckets can’t swing
freely
Open in BSC
LAI - Without Recognized Exposure

Sabia Virus Purification: Yale 1994







6 x 250 ml screw cap bottles w/ O-Ring, sealed
rotor
Rotor opened – one bottle wet & liquid pooled at
the bottom of rotor
No obvious bottle breakage
PPE – solid front disposable gown, gloves (double
during clean up), and surgical mask
Exposure likely occurred via inhalation of aerosol
when rotor was opened or during decon
PAPR was available but not used
Initially BL3; now BL4
Biological Spills










Assist injured/exposed
Notifications if
necessary
Limit access to area
Don PPE, gather
supplies
Ring spill with
absorbent
Cover with more
absorbent & disinfect
Clean up
Disinfect area again
Dispose of materials
20
Broken Glass in Spills
Spill Kit
•
Basic
•Paper
towels
•Concentrated
disinfectant
•Expanded
•Caution
•Other
tape
items
•Assemble
rather than
purchase
Work Practice Controls
W - reduce exposure risk by changing task
performance
21
Work Practices per Mom & OSHA



Immediately after
removal of gloves
or other PPE
Limited
circumstances ok
for antiseptic hand
cleanser /
towellettes
Most not effective
vs. adenovirus
WP- Medical and Biological Waste



Waste causes /contributes to an increase in
mortality, increase in serious irreversible or
incapacitating reversible illness
Pose substantial potential hazard to human
health or the environment when improperly
treated, stored, transported, disposed of,
or otherwise managed
Examples: Cultures and Stocks of Agents and their
associated Biologicals, Pathological Waste,
Contaminated Animal waste, Sharps, Biotechnology
effluents, Blood and Blood products
Treatment Options

Onsite

Chemical


 liquids


Autoclave
Offsite

Packaged and
Transported R
Med Waste

Approved vendor
TU/TMC
differences
Decontamination
– reduction or
removal so safe
to handle
22
Storage



Closed and flyproof
Secondary
containment for
transport to
autoclave
Bag must be
open for steam
penetration
Autoclave Operations



Each Load/Cycle must be Evaluated
Calibrated Annually by Trained
Individual
Quarterly Qualitative (growth/no
growth) Biological Challenge Testing



Test each Cycle if BSL-3/ABSL-3 Waste
Method Approved by IBC
Record-Keeping Log
Autoclave Explosion
23
Autoclave PPE/Practices




Heat resistant
gloves
Keep your Face
away from the
door
Long Pants
Protective
footwear
Focus on PPE

Protect others



Soiled/used lab
clothing is
autoclaved
before
laundering?
Am J Inf Contol
9/2011
ABC New –
neckties
24
At the request of your colleagues
“Staged” poor fit with alternatives
Exposure Response Plans

Some ERPs available on the IBC
website

Review with all staff prior to work

Key information:






Background information on agent.
Signs and symptoms of disease.
Pre-exposure Health Screening
Actions Before an Exposure Incident
Action immediately after exposure
incident
Medical Evaluation and Follow up
25
Exposure Documentation

First Report of Injury / Supervisors
Accident Report / Employee
Incident Report
 workers
compensation
forms on the EH&S website
 TMC intranet, EHO, BSO
 TU

FUP @ recommended intervals
 Update
OSHA 300 log if later
diagnosed with BBP disease
Post Incident Reporting


Biosafety Officer is notified of any incident,
accident, or exposure involving rDNA
NIH notification



Report




Overt exposure or release into the environment
of any rDNA at BL2 or above
Minor incidents within 30 days
Response made
Prevention of a reoccurrence
IBC Notification
NIH Notification made by Office of the Vice
Provost
Transport of Biohazardous Material



Trained & certified
prior to offering
specs for transport
Includes
 Dry ice
 Infectious Sub.
 Animal & human
 Genetically
modified org
 formalin
IATA vs. DOT
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Select Agents and Toxins (SATs)



Department of Health & Human Services
(DHHS) regulates the possession, use and
transfer of Select Agents and Toxins
SATs considered to have the capability of
being used by a terrorist to negatively
impact human, animal, and plant health
Latest changes involve “tiering”


Effective 12/12 (published 10/5/12)
Overall reduction in # of agents
Select Agent Ripples



Work subject to
SA maybe not
NIH Guidelines
Vendors
requesting proof
you allowed to
possess esp w
synthetics
Ricin 100 mg
Staph Entero 5
mg
Visits

Annually




Active participation – usually scheduled
Scope of work as described in BMR
Best practices
Regulatory changes
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LAI - Without A Recognized Exposure

Tularemia






Boston University 2005
3 Lab employees infected
Believed using Francisella tularensis LVS
at BSL-2
Really wt F. tularensis –a BSL-3 agent
Investigation cited failure of several
safety practices.
Strong argument for confirmatory testing
of high risk agents prior to use at BSL-2
Now for the Quiz!
This quiz is needed to document that you have
completed and understand all sections of this
training.
A passing score of 80% is required.
A Certificate of Training will be sent to you if the
test was passed. Please be sure to keep this
certificate for your records.
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