Professional Documents
Culture Documents
Guidelines For Safe Work Practices in Human and Animal Clinical Diagnostic Laboratories
Guidelines For Safe Work Practices in Human and Animal Clinical Diagnostic Laboratories
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(5)
3. Fundamental Safety Practices in Diagnostic Laboratories
3.1. Specimen receiving
3.1.1. Leaking containers
3.1.2. Visual contamination of the outside of containers
3.1.3. Loose caps
3.1.4. Operational procedures
3.1.5. Manual removal of sealed caps and specimen aliquoting and pipetting
3.1.6. Pneumatic tube systems
3.2. Personal precautions
3.2.1. Work at the open bench
3.2.2. Personal protective equipment
3.3. Biosafety cabinet
3.4. Disinfection
3.4.1. Good work practices
3.4.2. Bleach solutions
3.5. Waste management
3.5.1. Decontamination of medical waste prior to transport and disposal
3.5.2. Management of Discarded Cultures and Stocks
3.5.3. Autoclave safety
3.6. Dry ice
3.6.1. General information
3.6.2. Disposal of dry ice
3.7. Electrical safety
3.8. Gasses in the laboratory
3.8.1. Compressed gas cylinders
3.9. Liquid gasses (cryogens)
3.9.1. Cold contact burns and freezing
3.9.2. Asphyxiation hazards
3.9.3. Explosion hazards
3.9.4. Cryotube explosions
3.9.5. Embrittlement
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3.9.6. Infectious disease hazards
3.10. Slip, trip and fall hazards
3.10.1. Slips
3.10.2. Trips
3.11. Ultra low temperature freezers
3.12. UV light
3.13. Vacuum devices
3.13.1. Implosion safety
3.13.2. Aerosol generation
3.13.3. Vacuum devise aerosol safety
3.13.4. Disposal of liquid wastes from vacuum-assisted aspiration traps
3.14. Biological hazards
3.14.1. Preventing punctures and cuts
3.14.2. Preventing ingestion of infectious agents
3.14.3. Preventing spills and splashes onto skin and mucus membranes
3.14.4. Prevention of aerosols
3.15. Ultrasonic devices
3.16. Clean vs. dirty areas of the laboratory
3.16.1. Clean areas
3.16.2. Supervisor and laboratory director offices
3.16.3. Dirty areas
3.17. Instrumentation
3.17.1. Water baths and water pans in incubators
3.17.2. Centrifuges and cytocentrifuges
3.17.3. Automated analyzers
3.17.4. Vacuum-assisted aspiration devices
3.17.5. ELISA plate washers in microbiology
3.17.6. Identification instruments, blood culture instruments, PCR instruments
3.18. Rapid testing (kits)
3.19. Unidirectional work flow and spatial separation of work areas
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3.20.
3 FUNDAMENTALS OF LABORATORY SAFETY
There are many safety procedures, guidelines, and principles that apply to all sections of
the diagnostic laboratory and many of those are described here. The recommendations
presented in this section may be repeated in subsequent sections to provide emphasis but will
generally represent a broad view of safety throughout the laboratory. Refer to other documents
for more detail (1,2).
Hospitals, clinical laboratories, state and local health departments, the Centers for
Disease Control and Prevention, and the American Society for Microbiology have established
and/or published guidelines that should be followed when suspected agents of bioterrorism have
been, or could be released in the community. However, routine clinical laboratory testing may
provide the first evidence of an unexpected bioterrorism (BT) event. Routine clinical specimens
may also harbor unusual or exotic infectious agents that are dangerous to amplify in culture.
These agents are often difficult to identify and the routine bench technologist may continue to
work up the culture by passage, repeated staining, nucleic acid testing, neutralization and other
methods. This continued workup places the technologist and others in the laboratory at risk for
infection. Ideally, these specimens should not be processed or tested in the routine laboratory.
These specimens can be removed from the testing stream if the suspect agent is known.
Relationships with the State public health laboratory and subsequently with the Laboratory
Response Network are critical in this effort.
Once the testing process has begun, the bench technologists should have clear and
concise instructions regarding when to seek the assistance of the laboratory supervisor and/or
director.
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3.1 Specimen Receiving and Log-In/Set-Up Station
Microbiology specimens are to be received in uncontaminated containers that are intact
and are consistent with laboratory specimen collection policy.
The use of pneumatic tubes in the transport of specimens is acceptable for most
specimens but may be contraindicated for specimens without sealed caps, such as urine
cups; these should to be delivered by hand (see 3.1.6). Institution should adopt specific
standard operating procedures (SOPs) in the event that irreplaceable specimens are
considered for transportation using these systems.
Ideally, all specimens are to be processed in a biological safety cabinet (BSC) adhering
to safe BSC practices.
Use of a four-foot wide BSC for inoculating plates and preparing smears should be
limited to one employee at a time, wearing appropriate PPE. Six-foot wide BSCs may
accommodate additional testing equipment or materials. Check the manufacturers
recommendations before allowing two employees to work simultaneously in the larger
cabinet.
Minimal PPE for the general set up area is gown and gloves. In microbiology, a mask
is recommended but optional. For the mycobacteriology and virology laboratory where
organism manipulation is conducted, one could wear a fit-tested N-95 respirator or select
other appropriate respiratory protection, based on the risk assessment. An N-95
respirator is normally not required for biocontainment levels up to BSL-2, although it
provides a higher degree of protection than a surgical mask. Safe BSC practices are to
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be adhered to at all times. Mycobacterial, fungal, viral, and molecular specimens may
have specific additional safeguards.
3.1.1. Leaking containers
Specimens should be submitted to the laboratory in transport bags that isolate the
patient requisition from specimens and always limit one patient per bag to prevent
misidentification and cross-contamination.
Broken containers and containers that have spilled their contents are unacceptable for
culture and require notification of the section supervisor. A new specimen should be
requested, the incident documented, and the supervisor notified.
Visually examine containers for leaks upon arrival and before placing on rockers, in
centrifuges, in racks, in closed tube sampling (cap-piercing probe) systems, automated
aliquot stations, automated slide preparation systems, or on conveyor belts.
Track and document all incidents of cracked tubes, loose caps, and leaking containers.
Increases in documented events may indicate the need to clarify or strengthen specimen
acceptance policies, improve specimen collection or transportation practices, or may
identify defective container lot numbers.
Consider all sputum containers as coming from patients with tuberculosis or pneumonia
and handle with care. External contamination because of inappropriate lid closure can
contaminate the gloves of the laboratorian and all contents of the BSC. Change and
discard gloves after cleanup. Document the external contamination for reporting
purposes.
Consider all blood culture bottles as coming from patients potentially infected (e.g. with
HIV or hepatitis) and handle appropriately. If there is any concern about external
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contamination, carefully disinfect the outside of the tubes or bottles prior to inserting
them into the blood culture instruments. Change and discard gloves after cleanup and
decontamination of the immediate area. Document the external contamination for
reporting purposes.
Leaking stool containers could possibly contain Salmonella, Shigella, parasites, or viral
agents. Disinfect the outside of the container before culturing the contents and change
and discard gloves before proceeding. Document the external contamination for
reporting purposes.
Viral specimens with damaged or leaking containers may need to be discarded before
opening. Contact the supervisor and be prepared to notify the submitter, asking for
another specimen.
3.1.2. Visual contamination of outside of container
All specimen containers should be considered contaminated.
Do not rely on visible external contamination to confirm potential source of
contamination.
Wipe off visible contamination using towel or gauze pad moistened with acceptable
decontaminant such as a 1:10 dilution of household bleach or use the established
laboratory disinfectant. Ensure label and bar code are not obscured before advancing
specimen for analysis.
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3.1.3. Loose caps
Always grasp the tube or outside of the specimen container, not the stopper or cap when
picking up tubes/specimen containers to prevent spills and breakage.
Ensure tops are tightly secured on all specimen containers, blood-collection tubes, and
sample tubes before advancing for analysis or storage.
3.1.4. Operational procedures
Specimen placement, specimen flow, and bench operational workflow should be
unidirectional and uniform for all operators to maximize effective use of engineering
controls, i.e. from clean areas to dirty areas.
Appropriate PPE should be determined based upon documented risk and hazard
assessments of all the operations performed at each bench, and should be defined
consistently in the same location in all procedure manuals and clearly posted for each
operation carried out at the bench.
Workstation procedure manuals should include instructions for the organization of all
instruments, materials, and supplies in each area and include any cleaning and
disinfection instructions and the frequency of cleaning and disinfection for all surfaces
and instruments.
Supervisors should routinely inspect for cleanliness of the bench.
Have written procedures for non-laboratory operations, e.g. technical instrument
maintenance, in-house or contracted maintenance, emergency response, housekeeping,
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and construction and utility operations to mitigate exposures associated with assigned
operational tasks.
o Write non-laboratory operation procedures for both bench operators and the non-
laboratory service providers with their input and consultation .
o Document the training and assess the competency of service providers and bench
operators for all written non-laboratory operational bench procedures.
General bacteriology stains
Gram stain
Crystal violet, methylene blue, potassium iodide, and ethanol are all
irritants; crystal violet is also carcinogenic and somewhat toxic; ethanol
is a hepatotoxin.
Eye protection (safety glasses or chemical splash goggles) and
disposable gloves are recommended when staining or preparing stains.
Risks include handling live organisms in concentrated amounts, the
potential for creating aerosols, the potential for skin contamination and
environmental contamination.
Contaminated waste should be placed in a biohazard bag for disposal.
All other stains, including fluorescent conjugates refer to the MSDS sheets
associated with each stain or chemical.
3.1.5. Manual removal of sealed caps and specimen aliquotting and pipetting
Always remove caps behind a bench fixed splash shield or wear additional PPE
appropriate to protect from splashes and aerosols.
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Place a gauze pad over the cap and then slowly pry or push the cap off with an
away-from-body motion. Never reuse a gauze pad since this may contribute to cross-
contamination. Several manufacturers market safety devices to help remove caps
from tubes as well as break open ampoules. (For example, Current Technologies -
Saf De-Cap).
Use automated or semi-automated pipettes and safety transfer devices.
3.1.6 Pneumatic Tube Systems
Establish SOPs for the use and decontamination of the pneumatic tube system (PTS).
Breakage or leakage of specimens when transported using a pneumatic tube system
risks contamination of the transport system itself.
Limitations on use of the PTS should be based on a complete risk/hazard assessment
and should include but not be limited to:
Limitations on specimen size, volume, weight and container types (this is especially
applicable to cytology specimens and certain types of urine containers) sent through
the tube system.
Place all specimens sent through a pneumatic tube system in a sealed zip-lock bag.
Bags should be tested and shown to be leak-proof under the conditions present in
the pneumatic tube system.
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Requisition forms should be protected by a separate pouch or enclosed in a separate
secondary bag to prevent contamination.
A zip-lock bag should only contain specimens from one patient.
Place absorbent wadding between patient bags to help absorb spills and minimize
contamination to the outside of the carrier.
Handle contaminated pneumatic tube carriers in accordance with Standard
Precautions.
Contaminated carriers should be disinfected with bleach solution or other
disinfectant following the protocol recommended by the manufacturer and
approved by the hospitals infection control committee if the system is in use in a
hospital.
Prepare SOPs for both laboratory operators and the non-laboratory
service providers with their input and consultation.
Document training and competency assessment of service providers and
bench operators for pneumatic tube system maintenance and
decontamination procedures.
Wear gloves when opening PTS carriers containing patient specimens.
Decontaminate the outside of tube carriers before sending back to patient care areas.
The hospital should establish a hot-line for immediately reporting problems with
the pneumatic tube system.
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The hospital should establish an emergency PTS shut down plan including roles and
responsibilities and include the implementation of an alternative specimen transport
plan.
Develop a system to track incidents of improperly closed carriers, cracked tubes,
loose caps, and leaking containers.
Increases in documented events may indicate the need to clarify or strengthen
pneumatic tube system use policies, improve specimen collection practices,
identify defective carriers and/or container lot numbers.
Documented training and assessment of competency should include knowledge of
the risks associated with using a pneumatic tube system and the precautions to be
taken to control those risks.
3.2. Personal precautions
If engineering controls are in place to prevent splashes or sprays from occurring, the requirement
for PPE may be modified based on a risk assessment and evidence of the effectiveness of the
engineering control to prevent exposure from splashes or sprays.
3.2.1. Work at the open bench
Since no two workstations are identical, written procedures for each clinical laboratory
workstation should include specific work practices and work practice controls to mitigate
potential exposures.
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A dedicated hand washing sink with hot water should be available in each work area to
wash hands following contamination of hands or gloves with blood or other potentially
infectious materials. Employees should not rely on a sink in a rest room for washing their
hands following work in a technical area. Frequent hand washing is essential. Each
workstation should be supplied with alcohol hand rub to facilitate frequent hand cleaning
and with absorbent work pads to prevent accidental splashes. Safety glasses, splash
shield, surgical mask, and gloves should be available for optional use and when necessary
based on the isolate.
In the general microbiology laboratory, masks and disposable gloves are not required
away from the setup station but may be voluntarily used. If gloves are used, they can
easily become contaminated during routine use therefore gloves should not be washed
and reused. Discard gloves and don a new pair when leaving the workstation.
Splash guards at workstations are recommended when working at the blood culture
bench, when accessing blood bottle contents after Select Agent rule out testing has
produced negative results, or at any station where potential splashing may occur.
Should an accident, splash, or spill occur, regardless of how small, notify nearby
workers and the supervisor.
Sniffing of bacterial cultures growing on artificial media to detect characteristic odors
may or may not increase the risk of laboratory acquired infections but there is little or no
scientific evidence that defines the risk or implicates sniffing as a dangerous activity. It
is prudent, however, to refrain from sniffing plates as a precaution and to reduce any risk
to as low as possible. Plates suspected of containing Neisseria meningitidis, or colonies
shown to be gram-negative coccobacilli should never be sniffed and should be
manipulated within the biosafety cabinet.
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Open flame burners should not be used anywhere in the laboratory. Use disposable
loops and needles or use electric incinerators for metal wire devices.
Disinfectant-containing discard containers and sharps containers should be within easy
reach of the work station.
Computer keyboards located at workstations should have a protective cover that is
easily cleanable and should be disinfected along with the benchtop on a routine basis, but
at least at the end of the work shift..
Blood culture bottles should be tapped behind a safety splash shield or within a
biological safety cabinet. Gram negative coccobacilli from blood culture bottles should
be manipulated as described above and ideally handled within a BSC. Laboratories
without the ability to determine or rule out Brucella or Francisella should consider
directly shipping these isolates to a reference laboratory.
Urine remaining from culture activities can be discarded down the sink drain or into the
sanitary sewer.
Feces and other specimens such as body fluids and respiratory specimens remaining
from culture activities should be discarded with medical waste and autoclaved if
warranted by risk assessment.
Tissue remaining from culture activities of BSL-3 infectious agents should be discarded
into medical waste and autoclaved.
3.2.2 Personal Protective Equipment
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Personal protective equipment includes any of a variety of items including gloves,
laboratory coats, gowns, shoe covers, boots, respirators, face shields, safety
glasses, and goggles that are designed to protect the laboratory worker from
exposure to physical, biological, and chemical hazards. Personal protective
equipment is often used in combination with BSCs and other devices that contain
the agents or materials being handled. In some situations where it is impractical
to work in a biological safety cabinet, personal protective equipment may form
the primary barrier between personnel and hazardous materials(1).
OSHA defines PPE as appropriate if it does not permit blood or other potentially
infectious materials to pass thorough or reach the employees street clothes,
undergarments, skin, eyes, mouth or other mucous membranes under normal conditions
of use.
Sources for PPE standards:
o Lab coats - American Society for Testing and Materials (ASTM).
o Eye and Face Protection - American National Standards Institute (ANSI) Z87.1-
2003 (or earlier ANSI consensus standards) (USA Standard for Occupational and
Educational Eye and Face Protection).
o Hand protection - there is no ANSI standard for gloves, but there are
American Society for Testing and Materials (ASTM) standards (which is now
known as ASTM International) ASTM standards for disposable gloves based
on the specific type of material with which the glove is made. The Food and
Drug Administration (FDA) has indicated that patient examination gloves
used during patient care and vascular access procedures meet its adulteration
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requirements and have a 510(k) medical device registration with this agency.
OSHA recommends that selection be based upon the tasks performed and the
performance and construction characteristics of the glove material. Disposable
gloves must be made available in a variety of sizes to ensure that employees
are able to select the size that best fits their hands. Disposable gloves should
also be available in a variety different materials (e.g. nitrile, chloroprene) in
the event that employees have skin sensitivity issues with either the type of
glove material or the accelerants or other chemicals used in the glove
manufacturing process.
The employee medical history should be evaluated for evidence of the
presence of a latex allergy, if latex gloves are in use in the laboratory.
Using the hazard assessment for a given operation, laboratory management
or an assigned safety officer or safety team should select the most
appropriate glove for the task as well as establish how long it can be worn.
Before purchasing gloves, laboratory management or an assigned safety
officer or safety team should request documentation from the manufacturer
that the gloves meet the appropriate test standard(s) for the hazard(s)
anticipated.
Distributing PPEs to each employee e.g. box of gloves, eye protection, face
shields, and masks helps to ensure access to appropriate PPE.
o Employers are required by OSHA to train employees to know at least the
following:
When PPE is necessary.
What PPE is necessary.
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How to properly put on, take off, adjust and wear the PPE.
The limitations of the PPE.
Proper care, maintenance, useful life and disposal of PPE.
Resource: OSHA Informational Booklet Personal Protective Equipment
OSHA 3151-12R 2003.
Eye and face protection (goggles, mask, face shield or other splatter guard) must be used
whenever a splash or spray event could occur. This includes, but is not limited to opening
containers and pipetting, manipulating, aliquoting, or testing specimens, cultures, biological
agents, or other hazardous materials outside of the biosafety cabinet.
o If eye and face protection becomes contaminated, these devices must either be
decontaminated before reuse or disposed of with other contaminated laboratory
waste.
Neither eyeglasses nor contact lenses are considered to be personal protective equipment.
Laboratory workers who wear contact lenses must use face protection as described above.
Prescription safety glasses with side shields should be worn in the laboratory for those who
need corrected vision. In a chemical splash, contact lenses can intensify eye damage because
the lens will hold the chemical against the eye for a longer period of time.
Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination
of personal clothing. Remove protective clothing before leaving for non-laboratory areas
(e.g., cafeteria, breakroom, administrative offices). Dispose of single use protective clothing
with other contaminated waste or deposit reusable clothing for laundering by the institution.
Do not take laboratory clothing and other personal protective equipment home for
laundering or other uses. The employer must provide laundry service for reusable protective
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laboratory coats, gowns, uniforms or scrubs that are potentially or visibly contaminated with
blood or other potentially infectious materials at no cost to the employee.
3.3 Biosafety Cabinet
The Class II-A1 or II-A2 biological safety cabinet (BSC) is best suited and
recommended for the diagnostic laboratory. See Table 1 for a comparison of BSC uses
(1). An excellent overview and summary of the different classes and types of BSCs is
available in Appendix A of the CDC/NIH publication Biosafety in Microbiology and
Biomedical Laboratories, 5
th
Edition (BMBL-5)
Every diagnostic microbiology laboratory should have one or more BSCs to serve as a
primary means of containment for working safely with infectious organisms. The
College of American Pathologists (CAP) requires a BSC in microbiology in a Phase II
deficiency question for microbiology departments that handle specimens or organisms
considered contagious by airborne routes. There are three basic types of BSCs that are
designated as Class I, Class II, and Class III.
The Class I cabinet is similar to a chemical fume hood and is usually hard-
ducted to the building exhaust system and it protects personnel and the room
environment but is not designed to protect the product inside the cabinet. This
type of cabinet could be used in the general laboratory set-up area as a second
choice of cabinet.
For most diagnostic laboratories where chemicals and toxins will not be
manipulated within the cabinet, the Class II-A2 would be appropriate and easiest
to install without a hard duct to the outside. This cabinet can be used at the
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specimen processing station, in the mycobacteriology laboratory, the mycology
laboratory, virology laboratory, and in chemistry and hematology if needed. Air
can be recirculated back into the room through HEPA filters with little risk if the
cabinet is maintained properly. The A-1 or A-2 BSC in the mycobacteriology
laboratory is also an option with a canopy hood (thimble connection to a building
exhaust duct) and annual certification. Never hard duct the Class A hood to the
building exhaust system since building airflow patterns cannot be matched to the
cabinet. HEPA filters remove 99.97% of particles 0.3m in size which includes
all bacteria, viruses, and spores.
The Class III cabinet is not for routine diagnostic laboratories and is designed
for highly infectious agents and provides maximum protection as would be
needed in a highly complex facility that works with high consequence agents like
Ebola and monkeypox virus.
All BSCs must be certified by trained professionals as per Annex F of ANSI/NSF
Standard No. 49, at least annually and each time the unit is moved. Moving the cabinet
can damage the filter at the glue joint resulting in dangerous leaks, so filter and cabinet
integrity must be tested after each move.
Proper loading of the BSC and proper access by the laboratorian is described in BMBL-
5, but there are some basic rules that are important to remember:
1. Do not sweep your arms into or out of the cabinet. Move arms in and out slowly,
perpendicular to the face opening.
2. Install the BSC in the lab away from walking traffic, room fans, and room doors.
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3. Do not block the front grill where downflow of air is conducted or the rear grill
where air is removed from the cabinet.
4. Let the blowers operate at least 4 minutes before beginning work to allow the cabinet
to purge.
5. At the beginning and end of the day, with the blower running, disinfect all surfaces
with a 1:10 solution of household bleach and remove residual bleach with 70%
alcohol, or use another disinfectant appropriate for the organisms encountered.
6. Do not use open flames inside the cabinet: 1
st
choice disposable loops; 2
nd
choice
electric furnaces;
7. To decontaminate the BSC prior to maintenance, engage a BSC certification
technician to use either formaldehyde gas, hydrogen peroxide vapor, or chlorine
dioxide gas when the BSC is not in use.
8. UV lamps are not required in BSCs and they are not necessary.
9. Sealed rotors or safety cups on high-speed and ultracentrifuges should be opened in
a BSC.
10. Where safety cups or sealed rotors cannot be used, centrifuges should be placed in a
containment device or BSC designed for this purpose.
11. Medical waste generated inside of the BSC is to be collected in bags or sharps
containers which are sealed before their removal and placement in medical waste
containers outside of the BSC.
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Table 1. Comparison of Biosafety Cabinet Characteristics
Applications
BSC
Class
Face Velocity Venting
Nonvolatile Toxic
Chemicals and
Radionuclides
Volatile Toxic
Chemicals and
Radionuclides
I 75
Outside or into the room
through HEPA
Yes
When exhausted
outdoors 1,2
II-A1 75
30% vented back into the room
through HEPA or to outside
through a canopy unit
Yes
(minute amounts)
No
II-A2 100
Similar to II-A1, but has 100
lfpm intake air velocity
Yes
When exhausted
outdoors
(minute amounts)
1,2
II-Bl 100
Exhaust cabinet air must pass
through a dedicated duct to the
outside through a HEPA filter
Yes
Yes
(minute amounts)
1,2
II-B2 100
No recirculation; total exhaust
to the outside through a HEPA
filter
Yes
Yes
(small amounts)
1,2
III N/A
Exhaust air passes through
two HEPA filters in series
and is exhausted to the
outside via a hard connection
Yes
Yes
(small amounts)
1,2
1. Installation may require a special duct to the outside, an in-line charcoal filter, and a spark proof (explosion proof)
motor and other electrical components in the cabinet. Discharge of a Class I or Class II, Type A2 cabinet into a room
should not occur if volatile chemicals are used.
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2. In no instance should the chemical concentration approach the lower explosion limits of the compounds.
In the event that an individual who works at a BSC has an infection that may have
involved material manipulated in the cabinet, such as a tuberculin skin test (TST)
conversion of positive results for a TB Interferon Release Assay in someone working
with M. tuberculosis, an evaluation must be performed that includes:
1. Evaluation and recertification of the BSCs in which the suspect work was performed.
2. Evaluation of procedures to ensure that the individual was using proper technique
working in the BSC and, if needed, re-education of the individual on proper BSC
technique.
3. Evaluation (e.g., TST testing) of others in the laboratory who work at the same BSCs
and, potentially, re-education of these individuals on proper BSC technique.
3.4 Disinfection
3.4.1 Good work practices
Regardless of the method, the purpose of decontamination is to protect the laboratory
worker, the environment, and anyone who enters the laboratory or anyone who handles
laboratory materials that have been carried out of the laboratory. For detailed information
see BMBL-5 Appendix A.
Instructions for disinfecting a laboratory work bench should be contained in each SOP
and should include what PPE to wear, how to clean surfaces, what disinfectant to use, and
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how to dispose of cleaning materials. Contact time is critical and should be a part of the
instructions. The instructions should also be posted in the bench area for easy reference.
Routinely clean environmental surfaces before setting up work areas and again before
leaving work areas.
Clean any item (e.g. timer, pen, telephone, thermometer, etc.) touched with used gloves.
Do not use alcohols or alcohol-based solutions alone to disinfect surface areas because
they evaporate readily, which significantly decreases efficacy. Use disinfectants
recommended for environmental surfaces, such as EPA registered disinfectants effective
against HBV, HIV, and other bloodborne pathogens, or use a 1/10 dilution of household
bleach. For EPA environmental disinfectant product registration information go to:
www.epa.gov/oppad001/chemregindex.htm.
Reserve sterilants and high level disinfectants cleared by FDA for processing reusable
medical devices. FDA has identified manufacturers, active ingredients and contact
conditions for these products. FDA-Cleared Sterilants and High Level Disinfectants list:
http://www.fda.gov/cdrh/ode/germlab.html .
Bench surfaces, stationary racks, clay tiles, rockers, slide staining racks, water/heating
baths and all trays should be cleaned whenever there is a spill. All surfaces should be
cleaned at the end of each shift.
Use of disposable liners may reduce cleaning intervals but does not replace the need to
clean surface areas of equipment. The underlying bench surface should be cleaned
whenever the liner is discarded.
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Disposable flexible polyethylene film-backed non-skid highly absorbent surface liners
are available commercially and help to prevent soak-through of most solutions, including
dyes and corrosive chemicals. Always discard with medical waste after contamination.
Allow dried blood or body fluid at least 20 minutes contact with the laboratory-
specified decontaminating solution to allow permeation and easy removal (1).
Never use a knife or other instrument to scrape dried blood or body fluid from
surface areas as this can generate aerosols.
3.4.2 Bleach Solutions (Sodium Hypochlorite) (3)
Hypochlorite solutions are classified as irritants and corrosives. Bleach solution is
corrosive to stainless steel and thorough rinsing must follow its use in the biosafety
cabinets and stainless steel sinks to remove the residue. Do not autoclave bleach
solutions.
Chlorine solutions should never be mixed or stored with cleaning products containing
ammonia, ammonium chloride, or phosphoric acid. Combining these chemicals could
result in the release of a chlorine gas, which can cause nausea, eye irritation, tearing,
headache, and shortness of breath. These symptoms may last for several hours. If exposed
to an unpleasantly strong odor following the mixing of a chlorine solution with a cleaning
product, leave the room or area immediately until the fumes have cleared completely.
To be an effective disinfectant, working bleach solutions should contain at least 0.5%
but less than 2%. sodium hypochlorite. Hypochlorite concentration in household bleach
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varies by manufacturer. Many (but not all) household bleach solutions contain 5.25%
sodium hypochlorite and a 1:10 dilution will produce a 0.53% hypochlorite solution. Use
of bleach solutions with lower hypochlorite concentrations may not provide the proper
level of disinfection. A 1:10 household bleach solution should be made fresh weekly,
but a fresh daily solution may provide more confidence.
3.5 Waste Management
A clinical laboratory must establish a Waste Management Plan.
As part of an on-site waste management plan, the laboratory management or the
designated safety officer or safety team should:
Establish a waste reduction or minimization program.
Identify and define all categories of waste generated by the laboratory.
For each category of waste generated, determine applicability of federal, state,
and local regulations including how that category of waste will be segregated,
packaged, labeled/color-coded, stored, transported and tracked within the
laboratory, outside the laboratory, and outside the facility to comply with the
applicable regulations.
Segregate all regulated waste to prevent access by the public or clients.
Establish a system for reporting and responding to any issues or problems
regarding medical waste management.
Establish treatment and disposal processes.
Disposal of regulated waste should be by a company meeting state and
local licensure requirements.
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A useful reference is:
CLSI. Clinical Laboratory Waste Management; approved guideline; GP05-A2 ,
Wayne, PA:CLSI 2002.
3.5.1 Decontamination of medical waste prior to transport and disposal
Infectious medical waste is defined as waste that is capable of transmitting disease.
Regulated medical waste is any waste contaminated with significant blood or blood
products in liquid or semi-liquid form and contaminated sharps; and it is considered a
higher level of risk, thus warranting regulatory provisions by state or local authorities.
Clinical laboratories must determine the federal, state, and local laws governing their
organizations regulated medical waste and ensure that the organization is in
compliance with those laws. State and/or local regulations may require:
Permits or registration numbers to generate medical waste.
Development and implementation of a waste management plan.
Specific recordkeeping compliance.
Occupational Health and Safety Administration (OSHA). 29 CFR Part 1910.1030,
Occupational Exposure to Bloodborne Pathogens provides minimal requirements for
labeling and packaging of blood and body fluids when transported or outside a laboratory.
Information may be obtained from your local OSHA office or from the Internet website:
http://www.osha.gov/.
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States Department of Environmental Services (or equivalent) are excellent resources
for assistance in complying with state and local medical waste laws.
To find state laws governing medical waste, visit
http://www.epa.gov/epawaste/nonhaz/industrial/medical/programs.htm.
(choose the state, and then look under the Primary Materials - Cases, Codes and
Regulations. Search the States Administrative Codes or Statutes for
information regarding waste management.) (Note: not all states refer to the
waste as infectious medical waste, some states may refer to the waste as
regulated medical waste, special waste, etc.)
Laboratory management must ensure that employees understand these laws
and ensure regulated medical waste is not mixed with non-medical waste in a
facility.
Employee training and competency assessment should be documented for:
Constructing and properly labeling containers for medical waste that
require assembly prior to their use.
Disposal of medical waste in properly labeled containers.
Use of appropriate supplies - containers, appropriate plastic bags,
labeling etc.
Following all federal, state, and local regulations regarding waste
management: Handling of medical waste, immediate disposal of medical
waste, storage of medical waste, transportation of medical waste which
includes any required Department of Transportation (DOT) labeling (e.g., the
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word Biohazard and the universal biohazard symbol) of transport
containers, and final disposal of medical waste.
3.5.2 Management of Discarded Cultures and Stocks
Consideration of the laboratorys biosafety level is needed when discarding cultures and
stocks of infectious agents.
Discarded cultures and stocks of organisms handled under BLS-3 physical containment
are to be collected and sealed in containers which are closed, leakproof, and posted with
the universal biohazard symbol and the word, Biohazard (i.e. Mycobacterium
tuberculosis). The containers would subsequently need to be autoclaved on site. The
use of other on-site medical waste treatment technologies can be considered if these
technologies sterilize the organisms, if they have been properly validated, and if they
are recognized as medical waste treatment technologies by the appropriate state
environmental regulatory agency.
Decontamination is needed of discarded cultures and stocks of organisms handled at
BSL-2 . If this process is done on-site remote from the microbiology department, then
the discarded cultures and stocks should be placed into durable, leakproof containers
which are secured which are secured when they are moved. Decontamination may be
done by a medical waste treatment contractors facility, if the waste is placed into
medical waste shipping containers and packaged in accordance with applicable
regulatory standards. In order to determine if these activities can be done in a manner
that minimizes possible exposures , a risk assessment should be conducted. The
outcome of this assessment will determine if these wastes can be safely managed off
site or if they should be managed on site.
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3.5.3 Autoclave Safety
Gravity displacement steam sterilizers (autoclaves) are frequently used in microbiology,
including virology laboratories. Autoclaves generate significant heat and pressure and all
users must understand and respect the associated risks.
Personnel who operate the autoclave must be trained to properly package, load, and
label materials to be autoclaved in accordance with the procedures used to validate the
sterilization cycle of the unit. They must also receive training in emergency procedures.
Do not touch the sides or back of older autoclaves ; they have little or no heat shielding
and may cause burns.
Do not stack or store combustible materials (e.g., cardboard, plastic materials, etc.) or
flammable liquids next to the autoclave.
Never autoclave materials that contain toxic agents, containing corrosives (e.g. acids,
bases, phenol, etc.), solvents or volatiles (e.g. ethanol, methanol, acetone, chloroform,
etc.) or radioactive materials.
Place all biomedical waste to be autoclaved in an approved, biohazard-labeled
autoclave bag before autoclaving. Not all red or orange bags are capable of being
autoclaved. Bags selected for use in autoclaving waste must be specifically manufactured
for this purpose. Use only bags represented as being appropriate for use in autoclaves
when autoclaving medical waste.
Place all sharps such as needles, scalpels, pipettes, or broken glass into approved, leak
resistant, labeled, and rigid sharps container before sterilizing.
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When decontaminating a bag of dry goods such as bench paper or paper gowns, place
100 mL of water into the autoclave bag to facilitate steam production within the bag.
Do not overfill bags or the autoclave unit as this may interfere with the sterilization
process due to poor steam circulation.
Close autoclave bags loosely with twist ties or other means that allows steam inside.
Do not place bags directly into the autoclave. Bags should be placed onto stainless steel
or polypropylene trays for autoclaving.
Always allow an autoclave unit to cool down before opening. Stand back and open the
door slowly to allow the excess steam to escape. Open the door and allow the contents to
cool before handling. Always use thick, elbow-length, heat-resistant, liquid-impervious
gloves to remove hot items from the autoclave.
After autoclaving, check the autoclave indicator tape to be sure the bars are black. If
the indicator tape is not activated, resterilize the load.
A biological indicator such as Bacillus stearothermophilus spore strips (or equivalent)
should be used at least weekly to assure that the autoclave is performing properly.
3.6 Dry ice
3.6.1 General information
Note: Under certain circumstances, dry ice can be an explosion hazard! Dry ice is solidified
carbon dioxide (CO
2
) and it is extremely cold (-109F, -79C). Unlike water-ice, dry ice
sublimates (changes directly from solid to gas) as it warms, releasing carbon dioxide gas. Carbon
dioxide vapor is substantially heavier than air and in confined, poorly ventilated spaces it can
displace air, causing asphyxiation.
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Avoid contact with skin and eyes. Dry ice can cause severe frostbite within seconds of direct
contact.
Never place dry ice into glass or sealed containers. Storage in a sealed container can result in a
rupture or explosion of the container from over-pressurization.
Never handle dry ice with bare hands. Always wear insulated gloves and safety glasses. Lab
coats are also recommended. Use tongs to handle blocks of dry ice. Use scoops to move
pelletized dry ice.
Do not put dry ice into the mouth or otherwise ingest it. If dry ice is accidentally ingested, it
can cause severe internal injury. Never put dry ice in beverages to cool them.
When transporting dry ice, place the container in the trunk of the car or truck bed and leave the
car windows open for fresh air circulation. Never leave dry ice in a parked passenger vehicle.
Sublimation of dry ice in a closed passenger vehicle can result in the accumulation of dangerous
concentrations of asphyxiating carbon dioxide vapor. When opening a closed cargo area
containing dry ice, allow the closed space to ventilate for 5 minutes before entering.
Do not place dry ice directly on bench tops, tile, laminated countertops or ceramic sinks. Use an
insulating barrier such as double thickness cardboard or wood. Dry ice can destroy the bonding
agent holding the tile or laminated material in place. Dry ice can also cause the bench tops and
ceramic sinks to crack.
3.6.2 Disposal of dry ice
Allow the dry ice to sublimate or evaporate to the atmosphere in a well-ventilated
area where no build-up of carbon dioxide vapor can occur.
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Do not dispose of dry ice in sewers, sinks or toilets. The extreme cold can fracture
ceramic fixtures, crack PVC piping, and if flushed down the plumbing, the gas
buildup can cause an explosion.
Do not place dry ice in trash cans or similar containers. The extreme cold and
resulting condensation can destroy these receptacles.
3.7 Electrical Safety
Electrical hazards can be divided into two main categories: those that can result in an electrical
shock and those that can cause fires and/or explosions.
Electrical shocks can be avoided by making sure that equipment and electrical cords and
plugs are in good repair, grounded outlets are used, and ground-fault interrupt outlets or
circuit breakers are used near sinks, eyewashes, emergency showers, or other water sources.
Electrical circuits should not be overloaded and the use of multi-outlet power strips should
be discouraged whenever possible. When power strips are necessary, their use should be
approved by the Safety Department or a licensed electrician before being placed into use.
Equipment attached to high voltage or high amperage power sources should be
disconnected from the source or a lockout device should be placed on the breaker box to
prevent circuit activation before maintenance is performed.
Electrical devices could generate sparks and should not be used near flammable or volatile
gasses or liquids.
Never place flammable liquids in a household refrigerator. The spark generated by the
door-activated light switch can ignite fumes trapped in the unit causing an explosion and fire.
Specialized refrigerators must be used when storing chemicals that have explosion potential.
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3.8 Gases in the laboratory
3.8.1 Compressed Gas Cylinders
Compressed CO
2
cylinders are often used to provide gases for CO
2
incubators, and the
risks associated with these incubators are minimal as long as the room is well ventilated.
Gas cylinders represent three major safety hazards:
Gas cylinders are heavy and a falling cylinder can cause injury.
The valve attached to the cylinder is relatively fragile compared to the cylinder
and if broken off, the cylinder can become a dangerous projectile.
Faulty valves or regulators may leak allowing toxic or flammable gasses to
enter the room.
In the electron microscopy laboratory, nitrogen is used to bring vacuum chambers to
atmospheric pressure and critical point driers use CO
2
as a transitional fluid in the drying
process for scanning electron microscopy specimens.
Argon is used in sputter coaters, and some laboratories carry out plasma ashing of
biological specimens which requires oxygen (4).
Many of these potential hazards can be minimized by adopting safe handling practices.
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Cylinders must be securely anchored to the wall with chains or straps to prevent
falling. Cylinders less than 18 inches tall may be secured in approved stands or wall
brackets.
When installing a new cylinder, the protective valve cap should be left in place
until the cylinder is secured. The protective valve cap should be replaced before the
straps or chains are removed from the cylinder.
Special regulators and threading are designed for each gas type. Do not try to force
the threads or use the wrong regulator on a tank.
Regulators are normally supplied with instructions for routine maintenance and
periodic checking to insure safe operation. These instructions should be followed
and checks should be performed.
Cylinders should not be dragged, rolled, or physically carried. Do not pick
cylinders up by the cap. Always use specially designed cylinder carts when moving
cylinders. Cylinders must be secured to the cart and the valve covers must be
attached when moving them.
3.9 Liquid Gases (Cryogens)
Cryogenic liquids are liquefied gases that have a normal boiling point below -150C (-238F).
Liquid nitrogen is used in the microbiology laboratory to freeze and preserve cells and virus
stocks.
The electron microscopy laboratory frequently uses liquid nitrogen and some laboratories also
use liquid helium.
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The principle hazards associated with handling cryogenic fluids include cold contact burns and
freezing, asphyxiation, explosion, and material embrittlement.
3.9.1 Cold Contact Burns and Freezing
Liquid nitrogen is cold (-196C; -320F) and contact with liquid or gas can immediately
cause frostbite. At -268C (-450F), liquid helium is cold enough to solidify atmospheric
air.
Always wear eye protection (face shield over safety goggles). The eyes are extremely
sensitive to freezing and liquid nitrogen or liquid nitrogen vapors can cause eye damage.
Do not allow any unprotected skin to come into contact with uninsulated piping, hoses,
tongs, spargers, or other metal objects because they become extremely cold when
exposed to liquid nitrogen. The skin will stick to the metal and the flesh will tear when
one attempts to withdraw from it.
Long-sleeved shirts or laboratory coats, long trousers (preferably without cuffs which
could trap the liquid), closed shoes (never sandals or open shoes), and insulated
cryogloves, labeled as appropriate for use with cryogenic liquids, should be worn when
filling cryogenic dewars. Do not tuck pant legs into shoes or boots as this could direct
liquid into the foot coverings and trap the cryogenic liquid against the skin.
Loose fitting thermal gloves with elbow-length cuffs should be used when filling
dewars. Gloves should be loose enough to be thrown off quickly if they come into
contact with the liquid.
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Gloved hands should not be placed into liquid nitrogen or into the liquid nitrogen
stream when filling dewars. Gloves are not rated for this type of exposure. Insulated
gloves are designed to provide short-term protection when handling hoses, dispensers,
and incidental contact with the liquid. Use special cryogenic liquid tongs when retrieving
items from liquid nitrogen.
There is a high risk of splattering and jets of liquid nitrogen can be generated when
canes, canisters, and other objects that are at much higher temperatures are placed into
liquid nitrogen. These activities can present a freezing hazard.
Do not insert a hollow tube into the liquid nitrogen because liquefied gas may spurt
from the tube.
3.9.2 Asphyxiation Hazards.
Although nitrogen is nontoxic and inert, it can act as an asphyxiant by displacing the
oxygen in the air to levels below that required to support life. Inhalation of nitrogen in
excessive amounts can cause dizziness, nausea, vomiting, loss of consciousness, and
death without warning.
When liquid cryogens are expelled into the atmosphere at room temperature, they
evaporate and expand to 700 - 800 times their liquid volume. Even small amounts of
liquid can displace large amounts of oxygen gas and decrease the oxygen content of the
atmosphere below a safe level, and the literature illustrates these dangers(3-6).
Do not store dewars or nitrogen containers in a confined space. The venting gas could
displace enough oxygen to become a hazard.
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If enclosed spaces must be used, oxygen monitors should be installed. Personnel should
be trained to leave the area immediately if the alarm sounds. The alarm should be
audible both inside and outside the room to prevent anyone from entering the room.
3.9.3 Explosion Hazards.
Liquid gasses, even those that are considered inert, can be an explosion hazard.
Heat flux into the cryogen is unavoidable regardless of the quality of the insulation
provided. Cryogenic fluids have small latent heats and they will expand 700 to 800 times
as they warm to room temperature. Therefore, even a small heat input can create large
pressure increases within the vessel.
Dewars must be moved carefully. Sloshing liquid into warmer regions of the container
can cause sharp pressure rises.
Do not drop, tip, or roll containers on their sides as this could damage the vessel and/or
cause a sharp increase in internal pressure.
Cryogenic containers are equipped with pressure relief devices designed to control the
internal pressure. Cryogenic containers will periodically vent gasses. This is normal.
Do not plug, remove, or tamper with any pressure relief device.
Vents must be protected against icing and plugging. When all vents are closed, the
expanding gas can cause an explosion. Vents must be maintained open at all times.
Always use special ultra-low temperature containers to hold liquid nitrogen. Never
place liquid nitrogen into domestic thermos flasks because they are not designed to
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withstand the large and rapid temperature changes that occur when liquid nitrogen is
placed in the vessel (4,5).
Liquid nitrogen dewars should be filled slowly to minimize the internal stresses that
occur when any material is cooled. Excessive stress could damage the vessel and cause it
to fail.
Liquid helium is cold enough to solidify atmospheric air. Only helium should be
introduced or allowed to enter the helium volume of a liquid helium dewar. Precautions
should be taken to prevent air from back-diffusing into the helium volume.
Liquid nitrogen and liquid helium have boiling points below that of liquid oxygen and
they are able to condense oxygen from the atmosphere. Repeated replenishment of the
system can cause oxygen to accumulate as an unwanted contaminant. Similar oxygen
enrichment may occur where condensed air accumulates on the exterior of cryogenic
piping. An explosive situation may result if this oxygen-rich liquid is allowed to soak
insulating or other materials which are not compatible with oxygen. In addition, some
oils can form an explosive mixture when combined with liquid oxygen.
3.9.4 Cryotube Explosions
Personal protective equipment should include an ANSI specification impact resistant
face shield, heavy gloves, and a buttoned laboratory coat when removing cryotubes and
ampules from nitrogen tanks.
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Cryotubes and glass ampules used for freezing cells and viruses, may explode without
warning when they are removed from cryogenic storage. It is presumed that these tube
explosions are caused by liquid nitrogen entering the tube through minute cracks. As the
tube thaws, the rapidly expanding gas causes the tube to explode, scattering the contents
of the tube (6).
Whenever possible, ampules should be stored in the gaseous phase of the cryogenic
dewar rather being submerged in the liquid nitrogen. An imperfectly sealed ampule will
pick up less nitrogen in the gaseous phase.
Nitrogen outgassing from an imperfectly sealed vial will sometimes produce a hissing
noise before the vial explodes. The absence of hissing does not mean that the vial is safe.
Cryotubes and ampoules should be placed onto gauze or paper toweling in an
autoclavable heavy-walled container immediately after removal from the nitrogen tank
and the lid of the heavy-walled container should be closed quickly. If an explosion
occurs, the entire vessel should be autoclaved..
3.9.5 Embrittlement
Cryogenic liquids should never be poured down the drain. Laboratory plumbing is one
of many ordinary materials that become brittle at cryogenic temperatures and are easily
fractured.
Wood and other porous materials may trap oxygen at low temperatures and they will
literally explode when subjected to mechanical shock (4),
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3.9.6 Infectious Disease Hazards
Liquid nitrogen can become contaminated when ampules are broken in the dewar and
contaminants can be preserved in the nitrogen (7). These potentially infectious
contaminants can contaminate other vials in the dewar and generate an infectious aerosol
as the liquid nitrogen evaporates.
Plastic cryotubes rated for liquid nitrogen temperatures are recommended for liquid
nitrogen storage because they appear to be sturdier than glass ampules and are less likely
to break in the nitrogen.
3.10 Slip, Trip, and Fall Hazards
Slips, trips, and falls can cause a laboratory worker to drop or spill vessels containing infectious
agents or dangerous chemicals. They can also lead to skin punctures and abrasions that make
laboratory workers more vulnerable to laboratory acquired infections.
Good housekeeping is the most fundamental means for reducing slips, trips and falls. Without
good housekeeping, any other preventive measures such as installation of sophisticated flooring,
specialty footwear or training on techniques of walking and safe falling will never be fully
effective.
3.10.1 Slips
Common causes of laboratory slips include wet or oily surfaces; loose, unanchored rugs or
mats; and flooring or other walking surfaces that do not have some degree of traction in all
areas.
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Water on the floor presents the major slip hazard. Any water on the floor should be cleaned
up promptly to prevent slips and falls.
Paraffin from tissue mounting and cutting can accumulate in tissue processing areas and
can make to floor slick, despite regular cleaning, unless special floor care measures are taken.
Mineral oils, mounting fluids, stainless steel cleaners, and other laboratory chemicals and/or
reagents can create slip hazards if they get on the floor. These items should be cleaned up
with soap and water whenever they are discovered.
Cleaning floors with alcohols should be avoided whenever possible because alcohols will
dissolve the wax on the floor creating areas that have different degrees of traction.
Mats can also present a slip hazard if they are not properly anchored to the floor.
Walking on paper, cardboard, or packaging materials can also present a slip hazard.
3.10.2 Trips
Common causes of tripping include obstructed view; poor lighting; clutter in the walkway;
mats or other items in the walkway, uncovered cables; open drawers or cabinets; and uneven
walking surfaces. Mats and rugs are not recommended in microbiology.
Drawers and cabinets should be closed except when they are being accessed.
Clutter and items that protrude from kneehole spaces can injure workers as they move down
aisles in the laboratory. Keep clutter to a minimum in order to prevent injuries. Make sure
that boxes and other items do not protrude into aisles.
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Do not run electrical, data, or other cords across aisles or other walkways.
The use of ergonomic anti-fatigue mats in other sections of the laboratory should be
assessed by the Safety Officer or management before employing in a specific laboratory area.
Some concerns to be aware of prior to use:
These mats are somewhat thick and the raised surface presents a trip hazard.
They serve as obstructions for carts and chairs and may cause them to tip.
They make it difficult to clean up spills.
They make it difficult for the custodial staff to clean and disinfect the floors.
These mats may also place custodial staff at risk if they pick up or move mats that have
been inadvertently contaminated with chemicals or infectious agents.
Liquids will often wick under the mat, hiding potential contamination problems.
Mats present a trip/fall hazard (see above) that could impede egress from the laboratory
in an emergency.
3.11 Ultra low temperature freezers
Wear thermally resistant gloves and a lab coat when handling items stored at ultralow
temperatures.
Specimens stored at ultralow temperatures are extremely cold (-70 to -85C) and
paradoxically, direct contact with the skin can cause severe burns.
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3.12 Ultraviolet (UV) Light
Short wave UV light has been used for surface disinfection in biological safety cabinets,
in fluorescent microscopes, as a terminal disinfectant in some Type 1 water systems, and
for visualizing nucleic acid bands in ethidium bromide stained gels.
Exposure to short wave UV light has been linked to skin cancers, corneal scarring, and
skin burns. These effects can result from direct or reflected UV light exposure. (8)
UV lights are not required or necessary to assist in decontaminating biological safety
cabinets in laboratories (1). If UV lights must be used for other reasons in biological
safety cabinets, they should be monitored throughout their life by means of intensity
sensors. Calibrated UVC sensors are a reliable and cost effective way to monitor UVC
radiation levels in biological safety cabinets. Lights should be monitored because
germicidal UV lights have an expected life around 9,000 hours.
UV lights should never be used for decontaminating biological safety cabinets.
Organisms in cracks, shadows, and on the underside of equipment are not affected by UV
light treatment. In addition, the radiation and ozone produced by these lights will attack
plastic and rubber items in and around the hood, shortening their lifespan. This exposure
can affect mechanical pipette calibrations and other sensitive equipment functions.
Germicidal UV irradiation for longer than 15 minutes is counterproductive because it
produces no additional germicidal benefit and it accelerates equipment degradation.
The UV lamp should never be on while an operator is working in the cabinet. Not all
protective eyewear will protect laboratory workers from deleterious UV light exposure.
Make sure the protective eyewear is rated for UVC protection.
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UV safety glasses must be worn when performing routine lamp maintenance or when
there is potential for direct or indirect (reflected light) exposure.
Gloves, long sleeved lab coat, and full face shield must be worn when working with UV
view boxes.
Placards stating Caution, Ultraviolet Light, Wear Protective Eyewear must be
displayed in areas where UV light is used.
3.13 Vacuum devices
Vacuum-assisted filtration devices and side-arm suction flasks are used routinely in the general
laboratory, whereas the electron microscopy laboratory uses vacuum-assisted evaporators, freeze-
driers, freeze-fracture, and sputter coater units. Vacuum-assisted devices present implosion
hazards and aerosol generation.
3.13.1 Implosion Safety
Implosions can occur when the pressure differential exceeds the specifications of the
vessel.
Implosions can scatter sharp glass debris in all directions and seriously injure anyone in
the vicinity (4,5).
Implosions will also disperse any infectious agents that are present in the vessel.
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Heavy-walled side arm suction flasks are generally rated to withstand a pressure
differential of one atmosphere (14.7 PSI) and house vacuum systems or vacuum pumps
that provide pressure differentials exceeding that level must be regulated with the use of
an in-line pressure regulator.
Cracks, chips, and scratches in vacuum-flasks and bell jars can weaken the glass and
cause an implosion even when the proper differentials are provided by pressure
regulators.
Care must be taken to prevent damage to bell jars and suction flasks caused by
excessive wear or impacts with hard objects.
Implosion guards made of plastic mesh or plastic boxes have been used with suction
flasks to contain glass pieces if the vessel fails.
3.13.2 Aerosol Generation
Vacuum-assisted aspiration traps consist of one or two suction flasks and plumbed
together in series with an in-line HEPA-filter (e.g., Vacushield Vent Device from Pall
Life Sciences, Port Washington NY or equivalent device) to prevent contamination of the
vacuum pump or house vacuum system (1).
When using a dedicated vacuum pump, many laboratories also include a suction flask
containing coarse Dry-Rite (W.A. Hammond Dririte Co., Ltd, Xenia, OH) or an
equivalent desiccant to remove moisture from the air, thereby protecting the pump.
Aspiration traps are used in virology to remove culture media from tubes, shell vials, and
other vessels prior to re-feeding or other cell manipulations. Aspiration systems are also
used in ELISA plate washers.
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All of these devices generate aerosols by agitating the fluid and placing the fluid surface
under reduced pressure (7). Aerosols can deposit infectious agents on the immediate
surfaces and finer aerosols can be inhaled.
3.13.3 Vacuum-Device Aerosol Safety
Aspiration devices should be used in a Biological Safety Cabinet (BSC) in order to
contain any aerosols.
Operators should wear a disposable laboratory coat and gloves to protect themselves
from infectious droplets.
When the culture aspiration is complete, the BSC blower should be run for 5 minutes to
purge any airborne aerosols and the work surfaces should be decontaminated in the
normal fashion.
The in-line HEPA filters should be replaced every 6 months, when they become wet, or
noticeably blocked.
3.13.4 Disposal of Liquid Wastes from Vacuum-Assisted Aspiration traps
Never pour infectious wastes down the sink.
Decontaminate liquid wastes from aspiration traps with bleach before disposal.
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When using an aspiration trap attached to an individual vacuum pump, laboratories
usually pass the vapors through an activated charcoal trap in order to protect the pump
from chlorine vapor corrosion.
A variety of suction trap configurations are possible and the ultimate configuration will
depend upon workflow and individual laboratory practice. The following procedures
apply to all configurations.
Vacuum flasks should be changed when they are three-quarters full to prevent
overfilling. Some laboratories prefer to mark the maximum fill volume on the flask
and add a sufficient volume of bleach at the beginning of the day to produce a 10%
bleach solution when the aspirated fluids reach the maximum fill mark.
Disinfect the hose by aspirating 10-50 mL of a freshly made bleach solution into the
trap. Lift the hose to allow all the bleach to enter the trap. Wait 20 minutes then
remove the trap from the biological safety cabinet.
Once decontaminated, the fluid is considered non-infectious and may be poured
down the sanitary sewer.
Note for the virology laboratory: Bleach will reduce the phenol red dye in cell
culture media and the solution will go from red to colorless. If this color change does
not occur, the fluid has not been decontaminated and sufficient bleach must be added
to decontaminate the vessel.
3.14 Biological Hazards
3.14.1 Preventing Punctures and Cuts
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Skin punctures and cuts can directly introduce an infectious agent into the body and they can
provide a route whereby a secondary agent can enter.
Preventing Needle Sticks (9)
Clinical laboratories should establish a needlestick and sharps injury prevention
program.
Microtome / cryostat blades used to cut frozen sections are another potential sharp that
must be handled carefully and cut resistant gloves should be worn during disassembly of
the potentially contaminated blade for cleaning and disinfection.
Limit the use of needles and syringes to procedures where there are no alternative
methods. Needle-stick injuries occur most often when needles are returned to their
protective sheathes after use.
Needles should not be resheathed. If resheathing is absolutely required, the procedure
should utilize a needle resheathing device to minimize injury and accidental inoculation.
Do not use needle cutting devices because they can produce infectious aerosols. After
use, place needles and syringes in leak- and puncture-resistant containers appropriately
posted with the word, Biohazard and the universal biohazard symbol for
decontamination and disposal.
Do not bend, shear, recap, or remove needles from disposable syringes, or otherwise
manipulate by hand before disposal.
Breakage
Never pick up broken glass with gloved or bare hands. Use forceps, disposable plastic
scoops, tongs or hemostats to pick up broken glass and dispose of the broken glass into a
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sharps container. Place a broom or hand brush and dustpan in various laboratories or in the
utility closet for picking up non-contaminated glassware.
Do not use broken or chipped glassware. Discard it in the appropriate sharps container
labeled for broken glassware.
Handling broken containers with spilled infectious substances: (1)
Appropriate gloves are to be worn for this procedure (based on risk assessment and
protection needed).
Cover the broken container and spilled infectious substance with a cloth or with
paper towels.
For the routine BSL-2 laboratory, pour a disinfectant or fresh 10% household
bleach over the covered area and leave for a minimum of 20 minutes. According
CDC, it would take 23 minutes to clear the air of airborne M. tuberculosis from a
spill if the room had 12 room air changes per hour at 99% removal efficiency and 35
minutes for this removal with 99.9% efficiency. Given the variability of the number
of room air changes per hour in diagnostic laboratories, the wait time should be
carefully evaluated. (http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5417a1.htm)
The cloth or paper towels and the broken material can be cleared away into
biohazard sharps receptacles. Fragments of glass are to be handled with forceps, not
gloved hands. (Using wadded up tape with forceps facilitates this procedure). Small
HEPA vacuum cleaners are also available for removal of fine glass particulates.
If laboratory forms or other printed or written matter are contaminated, the information
on the forms or written matter are to be copied onto another form and the original is to be
discarded into the biohazard waste container.
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Pasteur Pipettes
Whenever possible, substitute plastic or evaluate the procedure to determine if there is a
newer or better technique.
Both the top and the bottom of a Pasteur pipette can cause puncture wounds.
Before handling glass Pasteur pipettes, examine the top of the pipette to see if it is
broken or cracked. Broken pipettes can produce puncture wounds.
When seating glass Pasteur pipettes into suction lines, hold the pipette at the top and do
not allow your hand to extend below the tip. Improper technique while seating the
pipette can produce puncture wounds if the hand slips or the pipette breaks.
Dispose of used Pasteur pipettes in leak- and puncture-resistant containers.
Other sharp devices
Knives, scissors, and tissue homogenizers are frequently used to dissociate tissue
specimens before testing. These items must be handled carefully in order to prevent cuts
and skin punctures that could injure or inoculate laboratory workers with infectious
materials.
Pointed forceps are often used for fine dissociation and for removing coverslips from
shell vials. These forceps can puncture the unwary user causing injury and/or infection.
Glass slides can break and puncture skin.
Culture tubes, and shell vials can crack or shatter if caps are forced. The resulting
shards can easily penetrate latex gloves and skin.
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The lip of glass vessels may be chipped and these chipped surfaces can cut unwary
laboratory workers. Chipped glassware should be discarded at the earliest opportunity.
Sharps Disposal
Used disposable needles, syringes, scalpels, blades, pipettes, etc. must be carefully
placed into properly labeled leak- and puncture-resistant containers used for disposal.
Locate sharps disposal containers in or near the area where the sharps are used in order
to prevent environmental contamination and injuries associated with accumulating sharps
at the point of generation and from moving sharps from one place to another.
Replace sharps containers that are 2/3 to full. Sharps containers must close securely
for transport to decontamination areas. Injuries can occur when laboratory personnel try
to forcibly close full containers. Once closed, over full containers can pop open, creating
a hazard for other workers.
Place non-disposable sharps into a covered leak-resistant, hard walled container for
transport to a processing area for decontamination, preferably by autoclaving. (1)
Place materials to be decontaminated off-site into a medical waste shipping container
and secure for transport in accordance with applicable state, local and federal regulations
(1).
Place clean, uncontaminated sharps (, e.g., clean broken glassware, chipped clean
pipettes, etc.) into rigid, puncture-resistant containers for disposal in the normal trash
stream. Containers should be taped shut to prevent accidental opening and potential
injuries.
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Never place sharp items directly into the regular trash as they could injure custodial or
other staff members when the trash bags are removed from rigid trash containers.
3.14.2 Preventing Ingestion of Infectious Agents
Refrain from touching eyes, nose, mouth, and lips while in the laboratory.
Do not place pens, pencils, safety glasses, or other laboratory items in the mouth or
against the lips.
Do not store food or beverages for human consumption in the laboratory
Mouth pipetting is prohibited; mechanical pipetting devices must be used.
Eating, drinking, smoking, handling contact lenses, and applying cosmetics is not
permitted in the laboratory.
Wash hands after working with potentially hazardous materials and before leaving the
laboratory. The laboratory must have a sink for handwashing, preferably located near the
laboratory exit.
Gloves must be worn to protect hands from exposure to hazardous materials. In the
molecular biology area, gloves are also used to protect the specimen from nucleases that
are on the skin.
Change gloves when contaminated, integrity has been compromised, or when
otherwise necessary.
Remove gloves and wash hands when work with hazardous materials has been
completed and before leaving the laboratory.
Do not wash or reuse disposable gloves.
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Never touch your face, mouth, eyes, or other mucus membranes when wearing
gloves in the laboratory.
Because gloves worn in the diagnostic laboratory are considered potentially
contaminated, they should be placed into biohazard disposal containers.
Remove gloves when answering the phone or using community equipment like
computers
To prevent contamination of ungloved hands, the laboratory should be designed so that
it can be easily cleaned.
Decontaminate work surfaces with an appropriate disinfectant after completion
of work and after any spill or splash of potentially infectious material.
Bench tops must be impervious to water and resistant to heat, organic solvents,
acids, alkalis, and other chemicals.
Chairs used in laboratory work must be covered with a non-porous material that
is easily cleaned. Uncovered cloth chairs are inappropriate.
Carpets and rugs are not appropriate in laboratories. (1)
Avoid clutter in the laboratory. Boxes and papers are difficult to disinfect and
may mask inadvertent spills or aerosol contamination.
Telephones are an effective vehicle for transferring infectious agents to the face
and mucus membranes.
Never pick up or dial a telephone with gloved hands.
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Disinfect telephones regularly using disinfectants. Note: Alcohols do
not inactivate non-enveloped viruses or destroy DNA.
Use the hands-free or speaker phone features whenever possible to
avoid touching the telephone handset to the face.
Never bring briefcases, purses, backpacks, books, magazines, and other
personal items into the laboratory. These items are difficult to disinfect.
In the laboratory, computer keyboards should be considered dirty and they
should be disinfected regularly. Hands should be washed after using the
keyboard with ungloved hands. Keyboard covers may be used if they are
removed and cleaned daily.
3.14.3 Preventing Spills and Splashes onto Skin and Mucous Membranes
It is the responsibility of all laboratory workers to perform all procedures in a manner
that minimizes the creation of splashes and/or aerosols.
All splashes to the eye must be flushed for a minimum of 15 minutes. If a laboratory
worker wearing contact lenses receives a splash to the eye, the eye must be flushed with
water, the lens should be removed, and the eye should be flushed again. Disposable
contact lenses should be discarded. Re-usable contact lenses should be disinfected before
being returned to the eye. Manufacturer consultation may be warranted.
Specimen containers, culture tubes, shell vials, and other cylindrical vessels used in the
laboratory are easily tipped, they could roll when placed on the bench top, and glass
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vessels can break if dropped. These vessels should be secured in racks whenever
possible to prevent opportunities for breakage.
3.14.4 Prevention of Aerosols and Droplets
Any procedure that imparts energy to a microbial suspension can produce infectious
aerosols. (1,6)
Procedures and equipment frequently associated with aerosol production
include pipetting, mixing with a pipette or a vortex mixer, blenders,
centrifugation, and ultrasonic devices (sonicators) (1, 6, 10) These procedures
and equipment generate respirable particles that remain airborne for protracted
periods.
When inhaled, these tiny particles can be retained in the lungs.
These procedures and equipment also generate larger droplets that can contain
larger quantities of infectious agents. The larger droplets settle out of the air
rapidly, contaminating the gloved hands, work surfaces, and possibly the mucous
membranes of persons performing the procedure.
An evaluation of the release of both respirable particles and droplets from
laboratory operations determined that the respirable particles are relatively small
and do not vary widely. In contrast hand and surface contamination is substantial
and varies widely. (1;11)
The potential risk from exposure to larger sized droplet contamination requires
as much attention in a risk assessment as the respirable component of aerosols.
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Aerosol Generation While Pipetting
As noted, the pipetting action has been shown to generate aerosols, therefore it
is prudent to wear gloves, eye protection, and a lab coat when pipetting and to
perform pipetting operations in a biological safety cabinet.
Serological pipettes
Pipettes and pipetting processes can be a significant source of infectious
aerosols and environmental contamination.
Culture and sample pipetting can be done safely if prudent pipetting practices
are employed.
Expelling the last drops. When the last drop of fluid is forcibly expelled out of
the pipette tip, small and large droplet aerosols are formed that can contaminate
the hands, and the environment (6). To minimize aerosol generation, place the
pipette tip against the inside wall of tubes, flasks, or other vessels and gently
expel the last drops of fluid.
Pipette Mixing. When dispersing cell clumps, virologists frequently draw
fluids into and out of the pipette to homogenize specimens and cell suspensions.
A significant amount of aerosolization can occur during this process, especially
when the fluid is forcibly expelled from the pipette tip. Aerosols are generated in
a similar manner when pipette mixing culture dilutions. Closed cap vortex
mixing is the preferred method for this type of mixing. If pipette mixing is
required, the pipette tip should be kept below the surface of the fluid and the
entire fluid volume should not be ejected from the pipette. This will reduce
aerosolization and bubble formation.
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Vigorous pipetting. Vigorous pipetting (rapid aspiration of fluid into the
pipette) can generate aerosols within pipettes. Some, but not all of the aerosols
will be trapped by the cotton plug at the proximal end of the pipette. However,
some aerosols can travel through the cotton plug and contaminate the pipetting
device. Some mechanical pipetting devices have HEPA filters that minimize
contamination of the handset. These filters should be replaced regularly and
whenever they become wet. Pipette bulbs should be decontaminated regularly
and whenever they become contaminated.
Mechanical pipetters
Hand-held mechanical pipetting devices are used for EIA testing, molecular
diagnostics, and other activities that require precision.
In molecular diagnostics, pipette contamination is the most frequent cause of
false positive results (12). Pipette contamination can occur by aerosols,
touching the outside of the pipette to a contaminated surface, and by
contaminating the inside of the pipette during the pipetting process.
Expelling the last remaining fluid in the tip will result in droplet splatter and
aerosol formation. These aerosols and droplets can contaminate the other
samples and the environment. Most mechanical pipetting devices have two stops
on the plunger the To Deliver stop and the Expel stop. Pipette volumes are
calibrated for accuracy at the To Deliver stop and there is no need to expel the
final amount of fluid in order to preserve pipetting accuracy.
Pipette tips should be touched to the inside of the well or tube before pressing
the delivery plunger. Never direct the pipetting stream into the middle of the
well as this will cause splashing and contamination.
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Care must be exercised when ejecting used tips into discard containers because
the remaining fluid can splash and splatter widely.
The outside of the pipette barrel can become contaminated through splatter,
aerosols, or by touching the barrel to contaminated objects. Do not extend the
barrel of the pipette into a reagent, sample, or discard container. If normal length
tips cannot reach the fluid in the tube, use extended reach pipette tips to prevent
barrel contamination.
Mechanical pipetters should be disinfected regularly following the
manufacturers instructions or with 10% household bleach followed by 70%
alcohol to remove as much bleach as possible
The use of aerosol-resistant pipette tips can significantly reduce nucleic acid
contamination inside the pipette. Aerosol-resistant tips contain a hydrophobic
microporous filter that is bonded onto the walls of the pipette tip. The
microporous filter traps aerosols before they can contaminate the barrel of the
pipette. These filters can also prevent contamination of the specimen when a
contaminated pipette is inadvertently used (12).
Falling drops
When an accidental falling drop from a pipette tip encounters a hard surface, it
generates aerosols and a series of small droplets, some of which may be large
enough to fall and repeat the process (13). Greater contamination ensues when
drops fall a greater distance onto a hard surface.
Many laboratories use commercial plastic-backed bench paper in BSCs and on
laboratory work benches to contain or absorb this type of contamination.
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When faced with the inevitability of a falling drop, it is best to lower the tip of
the pipette and allow the drop to fall a short distance onto an absorbent towel.
This procedure will minimize the kinetic energy of the drop and its capacity to
splatter.
Liquid films in tubes and other vessels
Thin films sometimes form in the neck of culture tubes, shell vials,
microcentrifuge tubes, specimen vials, and other containers. Breaking or popping
this film produces aerosols and microdroplet splatter that can contain infectious
agents, nucleic acids, or other potential contaminants (6) .
Containers with thin films in the neck should be re-capped and centrifuged
whenever possible to disrupt the film or cause it to merge with the fluid in the
vessel.
If centrifugation is not possible (e.g., culture flasks), gauze or another absorbent
material should be placed over the opening and a pipette should be inserted into
the flask to disrupt the film. Dispose of the pipette and the absorbent material
with other contaminated materials.
Opening microcentrifuge tubes
Microcentrifuge and other plug-topped tubes will often produce aerosols and
splatter when opened.
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Microcentrifuge tubes should be subjected to a quick "pulse"
centrifugation before they are opened in order to minimize the amount of
fluid on the cap.
Microcentrifuge tubes should be opened in a biological safety cabinet
whenever possible.
When opening plug-seal microcentrifuge tubes, cover the top of the
tube with absorbent material (i.e. alcohol moistened gauze) to catch any
splatter that might occur. Dispose of the absorbent material with other
contaminated materials.
Lyophilized Materials, Serum Vials and Ampoules
Opening vials of freeze-dried (lyophilized) material can be hazardous because
these fine dry powders are easily dispersed into the atmosphere when air rushes
into the evacuated vessel (6) . The following procedure may be used to safely
open a serum vial containing lyophilized material.
Move the vial and the suggested diluent (water or medium as
appropriate) to a biological safety cabinet.
Wear gloves and lab coat when opening lyophilized vials.
Use a hemostat to remove the aluminum crimp from the vial. Discard
the crimping material into the sharps container.
Cover the stopper with a moistened gauze pad and carefully lift the
edge of the stopper and allow air to slowly enter the vial. Do not disturb
the contents of the vial.
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Once the vacuum has been released, remove the stopper completely and
place it upside down on absorbent paper.
Add the appropriate amount of diluent to the vial using a sterile pipette.
Replace the stopper and allow the vial contents to hydrate for several
minutes.
Discard the gauze, stopper, and absorbent paper with other
contaminated materials.
Using a pipette, transfer the contents of the vial to an appropriate
container.
Discard the original vial with other contaminated materials.
Using needles to remove infectious agents from serum vials
Needle and syringe methods for removing infectious agents from serum vials
are not recommended because they can contaminate the environment and because
they provide opportunities for needle-stick injuries. Use forceps, not needles, to
remove serum separator tubes that are stuck in centrifuge carriers.
Glass Ampoules
Once opened, glass ampoules can be a cut and puncture hazard.
The use of safety ampoule breakers can prevent injuries by covering the
ampoule during the breaking process.
For ampoules containing infectious materials, cover the score line with gauze
moistened with disinfectant then break as usual using the safety ampoule breaker.
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Place the ampoule breaker into a beaker containing 10% bleach after removing
the ampoule.
3.15 Ultrasonic devices
Ultrasonic devices are principally used to lyse bacteria and viruses, and to clean glassware and
laboratory equipment.
Use the lowest effective power setting to minimize aerosol generation.
Bath sonicators should be covered while in use.
Articles destined for ultrasonic cleaning should be properly decontaminated prior to cleaning to
prevent aerosolization of infectious agents (6).
Organism lysis and homogenization procedures should always be performed in closed
containers.
Bath fluids should be changed frequently to prevent aerosolization of bacterial and fungal
contaminants present in the bath.
3.16 Clean vs dirty areas of the laboratory
In the microbiology laboratory, all of the technical work area of the department should be
considered dirty. The same concepts of demarcation and separation of molecular testing areas
(see below) can be used to establish clean and dirty areas within the other sections of the
diagnostic laboratory.
3.16.1 Clean areas
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Wear different color lab coats in clean vs dirty areas of the laboratory (have them available at
entrance to clean areas) or require no lab coats in clean areas
Decontaminate reusable materials and devices (such as telephone, clocks, computers, tissue
boxes, work books) brought into the clean area unless known to be new and immediately apply
laboratory-designated color coded tape
A visual reminder on small objects such as workbooks, tissue boxes, and pens, can easily
identify items located to a clean area.
Demarcate separation of dirty/clean floor areas with tape (must be tape that will stand up to
floor cleaning) to clearly denote clean/dirty area boundaries.
Develop policy for cleaning and maintaining clean areas
Train all personnel (including service personnel) how to identify and maintain clean areas and to
recognize the significance of the demarcation tape and other means of area identification.
Document training and assess competency in use of and maintaining clean areas
3.16.2 Supervisor and Lab Director Offices
Offices that open into the clinical laboratory represent hybrid areas within the laboratory.
These offices are not typically designed or maintained in a manner that allows for easy or
efficient disinfection.
Keep a supply of hand disinfectant gel in all office and work areas and use the gel frequently
Components of offices that should remain clean but may be overlooked include:
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Laboratory documents reports and records, small equipment, pens, procedure manuals
and other items that have been in the laboratory and (could have been) handled with
gloved hands.
Carpets and chairs that are difficult to disinfect
Books, journals, and other reference materials that can be taken into the lab, taken
home, and/or loaned to others for use outside the laboratory
Personal items (e.g., photographs, awards, briefcases, coats, boots, backpacks, purses,
personal electronic devices, etc.) that are difficult to disinfect and would not be allowed
in the general laboratory.
Food items.
Designating these areas as clean does not necessarily make or keep them uncontaminated,
especially when potentially contaminated items are brought into the office and reference
materials and documents move freely between the office and laboratory. The following
procedures can help to reduce the risk of contamination in laboratory office areas.
Never bring specimens, cultures, proficiency samples and similar items should into
office areas.
Personal protective equipment must be removed before entering the offices and
hands should be washed before entering these areas.
Establish a dedicated and protected clean area for personal items e.g. purses,
briefcases, and similar items.
Disinfect desks and personal workspaces, telephones, and computer keyboards in
office areas regularly.
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Refrain from touching eyes, nose, mouth, and lips while in office areas.
Do not place pens, pencils, eyeglass bows, or other items in the mouth or against
the lips.
Do not apply or permit cosmetics in office areas.
Do not store food in the office.
Wash hands after working in the office and before entering common areas such as
rest rooms, administrative areas, cafeteria, and the library.
Avoid clutter in office areas as much as possible. Boxes and papers and other items
make it difficult to clean and decontaminate.
Laboratory Directors and Supervisors should assess the exposure risks associated
with the use of laboratory documents and reference materials in the dirty areas of the
laboratory and develop use policies to minimize those risks.
3.16.3 Dirty Areas
All areas of the working laboratory, all equipment and keyboards, items, waste, and
surfaces are considered dirty areas.
There are no current standards that describe operating procedures within dirty areas of
the laboratory. Laboratorians must be vigilant in recognizing the potential or risk of
transmitting an etiologic agent by touch.
3.17 Instrumentation
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Whether automated or manual, procedures with the potential for producing specimen aerosols
and droplets (e.g. stopper removal, vortexing, opening or piercing evacuated tubes, automatic
sample dispensers, etc. ) require PPE and engineering controls designed to prevent exposures to
infectious agents
3.17.1 Water baths and Water (humidification) Pans in CO
2
Incubators
FromtheChlorineInstitute
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10. Hematology and phlebotomy laboratory
10.1. Specimen receiving and log-in/set-up station
10.1.1. Leading containers
10.1.2. Visual contamination on outside of container
10.1.3. Loose caps
10.2. Work at the open bench
10.2.1. Operational procedure manuals
10.2.2. Manual removal of sealed caps and specimen aliquoting/pipetting
10.2.3. Unfixed specimens
10.3. Personal precautions
10.3.1. Biosafety cabinet
10.3.2. Personal protective equipment
10.3.3. Disinfection
10.4. Decontamination and disposal of laboratory waste
10.5. Clean vs dirty areas of the laboratory
10.6. Instrumentation
10.6.1. Waterbaths
10.6.2. Centrifuges
10.6.3. Automated hematology/hemostasis analyzers
10.6.4. Flow cytometers
10.6.5. Automated slide strainers
10.6.6. Total or semi-automated hematology test systems
10.7. Rapid testing (kits)
10.8. Molecular testing
10.9. Phlebotomy
10.9.1. Sharps workbook
10.9.2. General recommendations
10.9.3. Clean vs dirty areas in the laboratory
10.9.4. Pneumatic tube systems
10.9.5. Personal precautions
10.9.6. Disinfection of work space
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10.9.7. Disinfection patient room work areas and drawing stations
10.9.8. Equipment decontamination
10.9.9. Documentation of training and competency assessment
10.9.10. State and other regulations
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10.10.
10. Hematology Laboratory ( Including Hemostasis and Flow-Cytometry)
10.1 Specimen Receiving and Log-In/Set-Up Station
10.1.1 Leaking containers
See 3.1.1
10.1.2 Visual contamination on outside of container
See 3.1.2
10.1.3 Loose caps
See 3.1.3
10.2 Work at the open bench
10.2.1 Operational SOPs
See 3.1.4
10.2.2 Manual removal of sealed caps and specimen aliquotting/pipetting
See 3.1.5
10.2.3 Unfixed Specimens
Microscopes:
Disinfect the stage, eyepieces, knobs and any other contaminated parts daily.
Select a disinfectant that will be non-corrosive to the microscope and
appropriate for potential infectious agents.
Slide preparations
Avoid waving slides in the air or use of electric fans at an open bench to air dry
slides.
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Use of an electric fan to air dry slides should be in a BSC or fume hood. Keep
in mind that the use of a fan in the A2 BSC will disrupt airflow and will render
the BSC unsuitable for other laboratory work until the airflow has been
stabilized.
Buffy coat smears
Disposable Wintrobe tubes should be used in place of capillary tubes to avoid
cutting glass tubes and possible exposure to blood and shards of glass.
Hemacytometer
Use extreme caution when using glass hemacytometers and glass coverslips to
avoid punctures from glass shards.
Plastic hemacytometers are commercially available and offer repeatable
and reliable measurements and analysis.
Several hemacytomer designs eliminate the use of coverslips and allow
for exact volume control.
Bleeding Times (CLSI Performance of the Bleeding Time Test -H45A2E)
Wear gloves see 3.2.2 and 8.4.2.
Use appropriate pediatric/adult template safety devices.
Ensure devices are reviewed annually in compliance with OSHA
Bloodborne Pathogens Standard (last amended December 12, 2008
See http://www.osha.gov/SLTC/bloodbornepathogens/index.html for
256
available resources) and
http://www.cdc.gov/sharpssafety/pdf/sharpsworkbook_2008.pdf
Use disposable razors when removal of arm hair from test site is
required.
Dispose of used bleeding time device and razor in sharps container
and filter paper in medical waste container.
Bone marrow aspirates.
Include appropriate hospital infection control policies and procedures for
patient care settings in the laboratory policy and procedure for collection of bone
marrow aspirates when collected in these settings.
Limit what is brought into patient rooms.
Bone marrow safety tray kits are marketed by several manufacturers.
Use of disposable trephine and aspiration needles are recommended.
Collect specimens and prepare squash smears and slides in the laboratory.
If smears must be prepared outside the laboratory, e.g. in patient care settings,
use Standard Precautions; use risk and hazards assessment to determine
what/when additional PPE might be appropriate; use a flat secure surface to
prepare smears and slides; ensure cover/caps on additional specimens are tightly
secured before transport; and ensure slides, squash smears, and additional
specimens are transported in leak proof and breakage-resistant receptacles.
Document training and assess competency.
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Include knowledge of, and adherence to, hospital infection control
policies/procedures in patient settings and the concept of Standard Precautions in
all documented training and competency assessments.
10.3 Personal precautions
See 3.2
10.3.1 Biosafety Cabinet
Class II biosafety cabinet is required for all aerosol-generating processes.
See 3.3
10.3.2 Personal Protective Equipment
See 3.2.1
10.3.3 Disinfection
See 3.4
10.4 Decontamination and disposal of laboratory waste- (regulated medical waste
management) (CLSI Clinical Laboratory Waste Management (GP05A2E)
See 3.5
10.5 Dirty vs. clean areas of the laboratory
See 3.16
10.6 Instrumentation
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Whether automated or manual, procedures with the potential for producing specimen aerosols and
droplets (e.g. stopper removal, vortexing, opening or piercing evacuated tubes, automatic sample
dispensers, etc.) require either PPE or engineering controls designed to prevent exposures to
infectious agents. See 3.17
10.6.1 Waterbaths
See 3.17.1
10.6.2 Centrifuges
See 3.17.2
10.6.3 Automated Hematology/Hemostasis Analyzers
Automated analyzers frequently have added features to help reduce operator exposures
but do not totally eliminate potentials for exposure. A common feature in newer systems
is closed system sampling. See also 3.17.3, 9.1, and 11.6.3
Sample probes that move quickly or deliver fluid rapidly may generate aerosols
and droplets.
Always use instruments according to manufacturer instructions.
Ensure instrument safety shields and containment devices are in place at time of
use.
Limit the amount of hand movement near the sample probe and liquid-level
sensors.
Wear gloves and use gauze pads with impermeable plastic coating on one side
on instruments where the operator is required to wipe sample probes after
sampling.
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Newer instruments have automatic probe wash cycles eliminating this source of
exposure .
Handle sample trays and sample plates with caution and cover them when not
being sampled to prevent spillage.
Fill sample cups and aliquot tubes using mechanical devices and never decant
(pour) them.
Assume that effluents of clinical analyzers are contaminated and disposal
should comply with applicable federal, state, and local environmental
regulations.
Follow manufacturer instructions for routine cleaning and trouble-shooting
specimen spills on or within an instrument, including the appropriate PPE and
type of cleaning solution to be used.
When manufacturer instructions do not include spill containment and clean up
instructions, collaborate with manufacture to develop an SOP that will effectively
protect the operator and maintain and extend the instruments operational life.
10.6.4 Flow cytometers (See 3.17.3)
Occupational exposures in a routine flow cytometry (FCM) laboratory arise either from
sample handling or more specifically from aerosols and droplets generated by the flow
itself. Flow cytometric applications, e.g., phenotypic analysis, calcium flux evaluations,
apoptosis measurements of unfixed cells, when performed using jet-in-air flow
cytometers with extremely high pressure settings can expose operators to potentially
hazardous aerosols.
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FCM biosafety procedures should specifically focus on aerosol containment,
waste management and equipment maintenance. (Clinical Flow Cytometric
Analysis of Neoplastic Hematolymphoid Cells. H43-A2. Wayne, PA. CLSI
2007)
Consider all unfixed materials (peripheral leukocytes, bone marrow, various
body fluids, cultured cells and environmental samples) as biohazardous.
The use of a fixative is not always sufficient to eliminate all types of
biohazards.
Use a BSC or PPE with an equivalent fixed containment device for
sample aspiration.
Disposable gloves and protective clothing should always be worn by flow
cytometer operators.
Only documented-trained and experienced operators (professional consensus is 2
years) should perform potentially biohazardous cell sorting. (See International
Society for Analytical Cytology Biosafety Standard for Sorting of Unfixed Cells,
January 2007).
Cell-sorters are equipped with a nozzle to form a jet of microdroplets: this
step is likely to generate aerosols.
Instrument failures such as clogged sort nozzle or air in the fluidic
system can drastically increase aerosol formation.
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Restrict access to essential personnel during sorting. Post a notice at the
entrance that cell sorting is in process. Personnel wishing to monitor the sort
must wear the same personal protective gear as the operator of the cell sorter.
Newly designed safety attachments for cell sorters have become commercially
available.
Some enclosed fluid system flow cytometers perform cell sorting using a fluid
switching mechanism. These cytometers, in contrast to jet-in-air cell sorters, do
not generate aerosols during cell sorting.
Efficiency of aerosol control measures on sorter instruments should be
tested periodically following manufacturer instructions, particularly when
unfixed human cells and known biohazardous samples are acquired or
sorted.
Simpler bead-based technology for measuring efficiency have been
developed.
Newer flow cytometers have added biosafety features for reduced risk of
operator exposure to instrument-generated sample droplets and aerosols, e.g.,
enclosed flow cells, droplet containment modules, automated samplers.
Ensure the instrument is used in a manner specified by the manufacturer.
Open and close the instrument cover with care.
Keep instrument covers and panels closed and secure while instrument is in
use.
Do not defeat safety interlocks and/or sensors.
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Place sample tubes securely into the sample introduction port, otherwise it
could be blown off once it is pressurized and splash sample onto the operator.
Some sample ports contain a metal sip tube that can pose a risk of damaging
gloves when the tube is not inserted carefully.
For improved splash protection, it is recommended that safety glasses or
chemical splash goggles be worn during acquisition.
Automated sample loading systems reduce sample handling and also perform
sample vortexing in an enclosure which prevents operator exposure to aerosols
and splashes.
Take special care when loading samples onto the instrument tube racks.
For unfixed samples, load instrument tube racks inside a biosafety
cabinet or wear PPE to protect from splashes and aerosols.
Perform daily cleaning of fluidic systems and sampler following
manufacturer instructions.
Collect waste into a waste container that contains fresh concentrated
household bleach in sufficient quantity to achieve a final concentration of
10% when the container is full.
Note - Adding chlorine to a waste container where ammonium chloride
tris buffer is used as a lysing agent may release dangerous chlorine gas
(use a broad-spectrum idophor instead).
10.6.5 Automated slide stainers
Ensure the instrument is used in a manner specified by the manufacturer.
263
Keep instrument covers and panels closed and secure while instrument is
in use.
10.6.6 Total or semi-automated hematology test systems
Hazard and risk assessments should be conducted to identify critical operations that
pose a risk for exposure.
Operational SOPs should include:
Instructions for troubleshooting tube breakage and specimen spills on
conveyor tracts, belts, sorter, aliquot, and cap-piercing probe stations.
Instructions requiring at least daily cleaning and disinfection and cleaning
after tube breakage and/or specimen spills.
If instructions are not provided by the manufacturer then collaborate
with the manufacturer to develop trouble-shooting and cleaning
procedures that will protect the operator and be compatible with and
extend the life of the robotic equipment.
Identification of appropriate PPE to be worn when cleaning and
troubleshooting robotics
10.7 Rapid Testing (kits)
See 3.18
264
Used testing kits should be considered contaminated and disposed of
appropriately in accordance with applicable local and state environmental
regulations.
.
10.8 Molecular Testing
CLSI - Collection, Transport, and Processing of Blood Specimens for Testing
Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; H21-A5,
Wayne, PA: CLSI 2008).
Molecular testing for hematopoietic malignancies
CLSI. Nucleic Acid Amplification Assays for Hematopathology ;(MM05-A,
Wayne, PA; CLSI 2003).
Unidirectional work flow and spatial separation of work areas must be strictly
adhered to in addition to standard laboratory safety guidelines for open bench and
instrument operations.
Reagent preparation is the cleanest area, then specimen preparation area, and finally
product detection area.
Care should be taken to leave transportables e.g. pens, tape, scissors, glove boxes
etc. in each designated area (see 3.19). Color coding each area and using color coded
tape and color coded lab coats helps enforce spatial separations of work areas and
retain transportables in their designated areas.
265
Change laboratory coats and gloves wash hands before entering each area (see
3.19).
266
10.9 Phlebotomy
Evaluate, select, and use engineered sharps injury prevention devices that are acceptable for
clinical care and provide optimal protection against injuries. Evaluation of engineered sharps
injury prevention devices should include their prospective use by employees who use sharps.
Establish a process providing annual evaluation and selection of sharps injury prevention
devices
HTTP://WWW.CDC.GOV/SHARPSSAFETY/PDF/SHARPSWORKBOOK_2008.PDF
10.9.1 Sharps workbook see 3.14
10.9.2 General recommendations
Ensure sharps disposal containers are easily accessible in patient rooms and in patient
drawing areas, never more than full, and included in the annual evaluation, selection,
and use of sharps injury prevention devices.
http://www.cdc.gov/niosh/pdfs/97-111.pdf NIOSH sharps container guide
Ensure all applicable patient-care and infection control polices and procedures are
strictly adhered to in patient drawing areas, with emphasis on:
Wash hands with simple soap and water or antimicrobial solution to protect against
external and internal exposure to blood borne pathogens. Ensure hands are washed under
the following conditions:
o before gloving
o after gloves are removed
o after contact with each patient or patient sample
267
o before leaving the laboratory, drawing station, or patient room
o before eating
o after your hands have touched a possibly-contaminated surface
Establish a process and procedure for specimen transport within and if applicable
outside the facility.
If patient specimens are transported from the drawing station to another area of
the facility ensure they are transported in a secondary container which has a tight
fitting latchable cover and is constructed of material to contain blood spills.
The phlebotomy service procedure manual should include spill response
and spill cleanup instructions for all areas of the hospital where there is
potential for specimen spills ( this includes, elevators, stairwells, etc.).
Ensure courier services employed by the laboratory enforce the laboratory policy
for transportation of specimens and ensure documentation of personnel training and
competency assessment on specimen transport including procedures for spill-
response, clean up, and incident reporting
10.9.3 Dirty vs. clean areas in the phlebotomy area and in drawing station areas
See 3.16
10.9.4 Pneumatic Tube Systems (PTS) see 3.1.6
10.9.5 Personal precautions
See 3.2
10.9.6 Disinfection of work space see 3.4.1
268
Good work practices: Regardless of the method, the purpose of decontamination is to
protect the phlebotomist, the patient and the environment, and anyone who enters a patient
room/drawing station or anyone who handles materials that have been carried into or out of
the patient room/drawing station.
Limit the phlebotomy materials brought into a patient room.
Routinely clean environmental surfaces before setting up the patient-room/drawing station
work area and again before leaving the patient room or after each patient in the drawing
station work area.
Clean any item (e.g. pen, telephone, etc.) touched with used gloves.
Do not use alcohols or alcohol-based solutions to disinfect surface areas because they
evaporate readily, which significantly decreases efficacy; instead use aqueous disinfectants
such as 1/10 dilution of household bleach or the hospital-recommended disinfectant.
Use disinfectants recommended for environmental surfaces, such as 1/10 dilution
of household bleach or other EPA registered disinfectants effective against HBV,
HIV, and other blood borne pathogens. EPA environmental disinfectant product
registration information: www.epa.gov/oppad001/chemregindex.htm.
10.9.7 Disinfecting patient-room work areas and drawing stations (See 3.4.1)
Include instructions in the procedure manual regarding what PPE to wear, how to clean, what
disinfectant to use, and how to dispose of the materials. The instructions should also be posted in
the phlebotomy office for reference (see 3.4).
269
Allow dried blood or body fluid at least 20 minutes contact with the tuberculocidal disinfectant
to allow permeation and easy removal.
Never use a knife or other instrument to scrape dried blood or body fluid from surface areas as
this can generate aerosols
10.9.8 Equipment decontamination:
Examine equipment contaminated with blood or other potentially infectious materials prior
to servicing or shipping and decontaminate as necessary. Contact the manufacturer for
decontamination process.
If decontamination of equipment or portions of such equipment is not feasible, then:
Label the equipment with a biohazard symbol and a second label applied
specifically identifying which portions remain contaminated.
Convey this information to all affected employees, servicing representatives prior to
handling, servicing, or shipping so that appropriate precautions will be taken.
10.9.9 Documentation training and competency assessment in phlebotomy
Assessment should include knowledge of, and adherence to, any applicable hospital infection
control policies/procedures in patient settings and the concept of Standard Precautions.
270
Useful resources for procedures for collecting specimens
CLSI. Blood Collection on Filter Paper for Newborn Screening Programs; LA04-A5,
Wayne, PA: CLSI 2007
CLSI. Collection, Transport, Preparation, and Storage of Specimens for Molecular
Methods; MM13 -A, Wayne, PA: CLSI 2005
CLSI. Procedures for the Collectionof Diagnostic Blood Specimens by Venipunture;
H03-A6, Wayne, PA: CLSI; CLSI 2007
CLSI. Procedures and Devices for the Collection of Diagnostic Capillary Blood
Specimens; H04-A6, Wayne, PA:CLSI 2008
CLSI.Procedures for the Handling and Processing of Blood Specimens;H18-A4, Wayne,
PA: CLSI 2009
Additional useful resources
Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical Laboratory (CLSI
X03RE)
HTTP://WWW.CDC.GOV/SHARPSSAFETY/PDF/SHARPSWORKBOOK_2008.PDF
SHARPS WORKBOOK: This Workbook includes several sections that describe each of the
organizational steps and operational processes. A toolkit of forms and worksheets is included to
help guide program development and implementation. The Workbook also contains:
A comprehensive overview of the literature on the risks and prevention of sharps injuries in
healthcare personnel;
A description of devices with sharps injury prevention features, and factors to consider when
selecting such devices; and
Internet links to websites with relevant information on sharps injury prevention.
271
Transportation of specimens outside the laboratory:
US Department of Transportation publication:
https://hazmatonline.phmsa.dot.gov/services/publication_documents/Transporting%20Infectious%20Subs
tances%20Safely.pdf
272
11. Blood bank
11.1. Transfusion-transmitted diseases
11.2. Blood-borne pathogen rule
11.3. Specimen receiving and log-in/set-up station
11.3.1. leaking containers
11.3.2. visual contamination on outside of container
11.3.3. loose caps
11.4. Work at the open bench
11.4.1. Operational procedure manuals
11.4.2. Unfixed specimens
11.4.3. Biosafety cabinet
11.4.4. Personal protective equipment
11.4.5. disinfection
11.4.6. decontamination and disposal of laboratory waste
11.4.7. Waste management plan
11.4.8. Employee training and competency assessment
11.5. Clean vs dirty areas of the laboratory
11.6. Instrumentation
11.6.1. See 3.18
11.6.2. Refrigerators and freezers
11.6.3. Automated blood bank analyzers
11.6.4. Total or semi-automated test systems
11.7. Test kits and reagent trays
11.8. Donor blood collection, apheresis, and disposition
11.8.1. Processes and procedures
273
11 BLOOD BANK (INCLUDING DONOR COLLECTION AND
COMPONENT PREPARATION)
11.1 Transfusion-Transmitted Diseases
Many infectious agents are transmitted through transfusion of infected blood and includes
hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-
1/2), human T-cell lymphotropic viruses (HTLV-I/II), Cytomegalovirus (CMV),
Parvovirus B19, West Nile Virus (WNV), Dengue virus, trypanosomiasis, malaria, and
variant CJD. The AABB also provides information on transfusion-transmitted diseases at
their website.
11.2 Bloodborne Pathogen Standard
The Bloodborne Pathogen Standard, 29 CFR 1910.1030, is promulgated and enforced
by the Occupational Safety and Health Administration (OSHA) and must be adhered to
in the blood bank laboratory. It is available at:
http://www.osha.gov/SLTC/bloodbornepathogens/index.html
11.3 Specimen Receiving and Log-In/Set-Up Station
11.3.1 Leaking containers
See 3.1.1
11.3.2 Visual contamination on outside of container
See 3.1.2
274
11.3.3 Loose caps
See 3.1.3
11.4 Work at the open bench
Written procedures for blood bank should include specific work practices and work
practice controls to mitigate potential exposures.
11.4.1 Operational SOPs
See 3.1.4
11.4.2 Unfixed Specimens
Microscopes
Disinfect the stage, eyepieces, knobs and any other contaminated parts after
use or according to a specified schedule determined by the laboratory.
Select an appropriate disinfectant that will be non-corrosive to the microscope
(see 3.4.1).
Slide preparations
Replace glass with plastic where possible.
Avoid waving slides in the air or use of electric fans at an open bench to air
dry slides.
If other instruments are used to dry slides, the procedure should have a
risk/hazard assessment performed to measure aerosol risks.
275
Manual cell washing perform a risk/hazard assessment
Saline washing of cell suspensions use of automated cell washers reduces
some but not all of the hazards associated with this procedure.
Dumping saline washes into pour-off containers can generate splashes and
aerosols. Splashing can be minimized by using semi automated pipettes and
keeping liquid levels in pour-off containers below full. Fixed shields or
appropriate water resistant PPE should be worn to protect from splashes.
When shaking small test tubes for resuspending red cell pellets and reading
end-point agglutination, use fixed bench top shields or wear appropriate PPE
noted above to protect from splashes and sprays.
Perform procedures using a plasma extractor or expressor with appropriate face and eye
protection to protect from sprays and possible explosion of the blood/component bags.
Use appropriate face and eye protection when cutting donor segments during either
confirmatory testing or other testing.
Wipe outer surfaces of blood bags and components with a towel moistened with
appropriate disinfectant prior to release for infusion, ensuring that the disinfectant will not
compromise the plastic bag.
If engineering controls are in place to prevent splashes or sprays at blood bank
workbenches then the requirement for PPE may be modified based on assessment and
evidence of the effectiveness of the engineering control to prevent exposure to splashes or
sprays.
276
11.4.3 Biosafety Cabinet
Class II biosafety cabinet (see Section 3.3) is required for all aerosol-generating
processes
11.4.4 Personal Protective Equipment
See 3.2.2
11.4.5 Disinfection
See 3.4
11.4.6 Decontamination and disposal of laboratory waste
See 3.5
11.4.7 Waste management plan See section 3.5
11.5 Clean vs dirty areas of the laboratory
See 3.16
11.6 Instrumentation
11.6.1 See 3.17
11.6.2 Refrigerators and Freezers (also see section 3)
All refrigerators and freezers in blood bank should have an established cleaning and
maintenance protocol that will minimize contamination and extend the life of the equipment and
also maintain the sophisticated cooling systems blood bank refrigerators require to provide
uniform and quick temperature recovery when needed.
Most newer blood bank laboratory refrigerators and freezers are stainless steel and have
painted finishes and removable trays which make cleaning and sanitizing an easier
277
process.
o Manufacturer instructions for use and care of blood bank refrigerators and freezers
usually include recommended PPE and type of disinfectant.
o Collaborate with the manufacturer to establish SOPs for cleaning and
maintenance if instructions are not provided with the equipment.
o Clean up blood spills immediately.
o Clean refrigerator handles and outside doors around handles at the end of each
shift.
o Label the refrigerator with the universal biohazard symbol and the word,
Biohazard
11.6.3 Automated Blood Bank Analyzers
Automated or semi-automated instruments are now available that are adapted either to
donor collection settings or patient transfusion settings. Although these instruments
have the potential to replace much of the open bench testing in blood banks and donor
collection settings, use of manual testing is still being used for some antibody detection
and verification procedures and in smaller laboratories. All of the blood bank automated
analyzers currently approved for use in the U.S. have added features to help reduce
operator exposures but have not totally eliminated potentials for exposure.
Sample probes that move quickly or deliver fluid rapidly may generate aerosols
and droplets.
Ensure instruments are used according to manufacturer instructions.
Ensure instrument safety shields and containment devices are in place at time of use.
Limit the amount of hand movement near sample probes and liquid-level sensors.
278
Wear gloves and use gauze pads with impermeable plastic coating on one side on
instruments where the operator is required to wipe sample probes.
Handle sample trays, sample cards and sample plates with caution and covered when not
being sampled to prevent spillage.
Fill aliquot tubes using mechanical devices and never decant (pour).
Consider effluents of clinical analyzers as contaminated and dispose of in compliance
with state and local regulations.
Follow manufacturer instructions for routine cleaning and trouble-shooting of specimen
spills on or within an instrument, including the appropriate PPE and type of cleaning
solution to be used.
o When manufacturer instructions do not include spill containment and
clean up instructions:
Collaborate with the instrument manufacturer to develop an SOP
that will effectively protect the operator and maintain and
extend the instruments operational life.
11.6.4 Total or semi-automated test systems
See 10.6.6
11.7 Test kits and reagent trays
See 10.7
11.8 Donor Blood Collection, apheresis and disposition
Donor collection and pheresis areas should be considered a patent care setting and all applicable
hospital patient-care and infection control polices/ procedures should be strictly adhered to.
279
11.8.1 Processes and procedures
Establish and maintain processes and procedures to control the quality of infectious disease
testing, and safe disposition and transport of all collected blood and blood products.
Use Standard Precautions.
Use risk and hazard assessments to determine what/when additional PPE might be
appropriate.
Ensure cover/caps on any additional specimens are tightly secured before transport.
Place blood or blood products that are being transported from the collection site to another
location in a secondary container which, in addition to maintaining a specified temperature
range, also has a tight fitting cover and is constructed of material to contain blood spills.
Place single donor units or components issued for transfusion within a secondary
container to contain spills, especially when environmental conditions might cause rupture,
e.g. pneumatic tube systems.
Select and use engineered sharps injury prevention devices that are acceptable for clinical
care and provide optimal protection against injuries (see 3.14.1).
Establish a process to provide annual evaluation of use and selection of sharps injury
prevention devices. (See 3.14.1 CDC Sharps Workbook)
Provide easy access to sharps disposal containers, never fill them more than full, and
include them in the annual evaluation, selection, and use of sharps injury prevention
devices (see 3.14).
Do not store donor blood and components with patient specimens and reagent trays.
280
Document training and assess competency
Include knowledge of, and adherence to, hospital infection control
policies/procedures in patient settings and the concept of Standard Precautions in all
documented training
Outdated blood, blood components, and tissue should be discarded in compliance with
federal, state and local regulations
281
12. Veterinary Diagnostic Laboratories
12.1. Introduction
12.2. Biological Risk Classification and Assessment
12.2.1. Risk Classification
12.2.2. Risk Assessment
12.3. General Biosafety Guidelines
12.3.1. Hand washing
12.3.2. Personal protective equipment
12.3.3. Staff training
12.3.4. Biological Spill Management
12.3.5. Immunization
12.4. Pathology (Necropsy and Surgical Pathology)
12.5. Parasitology
12.6. Mycology
12.7. Virology
12.8. Toxicology
12.9. Hematology/Serology
12.10. Molecular Diagnostics and Rapid Tests
12.11. Storage, Packaging, and Shipping
12.12. Employer and Employee Responsibilities
12.13. Biosafety Education/Training
12.14. Biosafety Quality Improvement
282
12.1. Introduction
The primary purpose of this section is to provide practical guidelines for work practices that
minimize biosafety hazards from veterinary diagnostic specimens. Many of the biosafety practice
guidelines listed in other sections of this document, focused primarily on human clinical
microbiology laboratories, suffice for use in veterinary diagnostic laboratories. Similar to human
clinical microbiology laboratories, the nature of the work performed in veterinary diagnostic
laboratories has inherent risk to laboratory workers. According to the 2008 American Veterinary
Medical Association One Health Initiative task force report (www.avma.org/onehealth) 60% of
infectious diseases in humans are due to multi-host pathogens that move across species lines (5) and
over the last 30 years 75% of the emerging human pathogen diseases (e.g. west nile virus fever,
highly pathogenic avian influenza, Lyme disease) have been zoonotic (transmitted between humans
and animals) (4). Thus, veterinary diagnostic laboratorians too are at risk for laboratory-acquired
infections. All non-human diagnostic specimens are potentially infectious to humans, although the
degree of risk is less so than for handling and examination of human diagnostic specimens.
Potential infectious agents in human diagnostic specimens are by definition human pathogens.
Conversely not all potential infectious agents in animal diagnostic specimens are human pathogens.
The key to managing biosafety risk in veterinary diagnostic laboratories depends not only upon
good general biosafety practices but most importantly on a practical risk assessment of the
unknown diagnostic specimen.
In general, veterinary diagnostic laboratories should utilize BSL-2 practices and facilities for
general veterinary diagnostic work and do practical risk assessment of incoming accessions to
determine whether or not it is warranted to handle the specimens using decreased (BSL-1) or
increased (BSL-3) biosafety practices or facilities. Where biosafety risk and practices differ
between handling of human and animal diagnostic specimens those differences are highlighted in
this section.
12.2. Biological Risk Classification and Assessment
12.2.1. Risk Classification
283
The tables below of risk groups and specific infectious agents are included to assist in risk
assessment. Two lists of risk groups are published to facilitate the assessment of risk from
different microbes and to recommend appropriate safety practices for the handling of those
microbes. The World Organization for Animal Health (OIE) and World Health Organization
(WHO) list four groups of biohazardous agents for humans and animals based upon level of risk
and availability of effected treatment and prevention (1). The Centers for Disease Control and
Prevention/National Institutes of Health (CDC/NIH) guidelines propose four biosafety levels
(BSLs) and recommendations for appropriate containment practices for agents known to cause
laboratory acquired infections (2). The two lists of risk groups are roughly equivalent and neither
makes allowance for persons who are particularly susceptible to infections by pre-existing
conditions such as a compromised immune system or pregnancy. Table 1 lists the two risk group
classifications and Table 2 lists the recommended practices and equipment for handling microbes
within each risk group. In both risk group classification systems increasing risk levels (numbers)
imply increasing occupational risk from exposure to an agent and the need for additional
containment for work with that agent.
Table 1. OIE Risk Groups and CDC/NIH Biosafety level (BSL) Classifications
OIE Risk
Group
Characterization
1 Unlikely to cause disease. Not considered infectious
2 Moderate individual and low community risk. Unlikely to cause serious
disease or be transmitted. Effective treatment and prevention available
3 High individual and community risk. Causes serious infections but not
readily transmitted. Effective treatment and prevention usually available
4 High individual and community risk. Readily transmitted and no effective
treatment or prevention available
CDC/NIH
BSL
Class
1 Well-characterized agents not known to consistently cause disease in
healthy adult humans. Minimal potential hazard to laboratory personnel
and the environment
284
2 Agents of moderate potential hazard to personnel and the environment
3 Indigenous and exotic agents that cause serious or potentially lethal disease
as a result of exposure by the inhalation route
4 Dangerous and exotic agents that pose a high individual risk of aerosol-
transmitted laboratory infections and life-threatening disease
Adapted from OIE Manual for Diagnostic Tests and Vaccines for Terrestrial Animals, 6
th
Edition
2008 Biosafety and Biosecurity in the Veterinary Microbiology Laboratory and Animal
Facilities (1), and CDC/NIH BMBL 5
th
Edition, 2007 (2).
Table 2. CDC/NIH BSL Practices and Equipment
BSL Practices Safety Equipment and Facilities
1 Standard microbiological
practices
None required
2 BSL1 practices
Limited access
Display biohazard signs
Sharps precautions
Staff trained with pathogens
Safety manual available
BSC used for specimen processing and work
producing aerosols or splashes
PPE (coats, gloves, face shields) as needed
Autoclave available
3 BSL2 practices
Controlled access
Collect baseline serum from
personnel
BSL2 equipment/facilities
BSC used for work with all specimens and
cultures
PPE (gowns and masks) as needed
Negative pressure airflow
Self-closing double doors
Exhaust air not re-circulated
4 BSL3 Practices
Clothing change before
entering
Shower on exit
Decontaminate all waste on
exit
BSL3 equipment/facilities
Separate building or facility
BSC and full body, air supplied positive
pressure suite for all procedures
Specialized ventilation and decontamination
system
285
Adapted from CDC BMBL 5
th
Edition, 2007 (2).
Generally work in routine veterinary diagnostic laboratories should assume that clinical specimens
contain Group 2 agents and operate with BSL 2 practices, unless a risk assessment would indicate
otherwise. On occasion veterinary diagnostic laboratories may encounter Group 3 agents and use
BSL3 practices. Only under extraordinary circumstances would veterinary diagnostic specimens
contain Risk Group 4 agents, which will not be included in this document. Some Risk Group 2 and
Group 3 agents commonly encountered in veterinary diagnostic laboratories are listed in Table 3;
the list is not meant to be all inclusive.
Table 3. Examples of some common zoonotic microorganisms in Risk Groups 2 and 3 that
may be present in the veterinary diagnostic laboratory
Group 2
Viruses: Influenza viruses types A, B, C, Newcastle disease virus, Parapox virus (Orf),
West Nile virus
Bacteria: Alcaligenes spp.; Arizona spp.; Campylobacter spp.; Chlamydophila psittaci
(nonavian); Clostridium tetani; Clostridium botulinum; Corynebacterium spp.;
Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus spp.; Leptospira
spp.; Listeria monocytogenes; Moraxella spp.; Mycobacterium avium;
Pasteurella spp.; Proteus spp.; Pseudomonas spp.; Salmonella spp.;
Staphyococcus spp.; Yersinia enterocolitica; Yersinia pseudotuberculosis
Fungi: Aspergillus fumigatus; Microsporum spp.; Trichophyton spp.; Blastomyces
dermatitidis (tissues); Coccidioides immitis (tissues); Cryptococcus neoformans;
Histoplasma capsulatum (tissues); Sporothrix schenkii
Group 3
Viruses: Rabies virus; Equine encephalomyelitis virus (Eastern, Western, Venezuelan),
Japanese encephalitis virus; Louping ill virus
Bacteria: Bacillus anthracis; Burkholderia mallei; Brucella spp.; Chlamydia psittaci
(avian strains only), Coxiella burnetii, Mycobacterium bovis.
Fungi: Blastomyces dermatitidis spores (cultures only); Coccidioides immitis spores
(cultures only); Histoplasma capsulatum spores (cultures only).
286
Adapted from OIE Manual for Diagnostic Tests and Vaccines for Terrestrial Animals, 6
th
Edition
2008 Biosafety and Biosecurity in the Veterinary Microbiology Laboratory and Animal Facilities
(1)
In addition to zoonotic agents, veterinary diagnostic laboratories must assess for the suspected
presence of high consequence livestock pathogens during risk assessment. High consequence
livestock pathogens are defined by the USDA Animal and Plant Health Inspection Service
(APHIS) Agricultural Select Agent program in accordance with select agent and toxin regulations
published in 2008 in the U.S. Federal Register by the U.S. Department of Health and Human
Services (42 CFS part 73) and by USDA (9 CFS part 121 and 7 CFS part 331). Criteria used to
classify high consequence livestock pathogens included severity of effect on animal products,
virulence and transmissibility of the agent, and availability of effective treatment. Although not
necessarily zoonotic agents, high consequence livestock pathogens can have severe detrimental
economic impact on agricultural animal health and require handling using BSL-3 practices and
facilities to prevent environmental dispersement and contamination.
287
Table 4. High Consequence Livestock Pathogens and Select Agents
Livestock
African horse sickness virus
African swine fever virus
Akabane virus
Avian influenza virus (highly pathogenic)
Bluetongue virus (exotic)
Bovine spongiform encephalopathy
Camel pox virus
Classic swine fever virus
Foot-and-mouth disease virus
Goat pox virus
Japanese encephalitis virus
Lumpy skin disease virus
Menangle virus
Mycoplasma capricolum subspecies
Mycoids small colony (MmmSC) (contagious bovine pleuropneumonia)
Peste des petits ruminants virus
Rinderpest virus
Sheep pox virus
Swine vesicular disease virus
Vesicular stomatitis virus (exotic)-Indiana subtypes VSV-IN2, VSV-IN3
Virulent Newcastle disease virus
USDA/HHS Overlap Agents
Bacillus anthracis
Brucella abortus
Brucella melitensis
Brucella suis
Burkholderia mallei
Burkholderia pseudomallei
Hendra virus
Nipah virus
Rift Valley fever virus
Venezuelan equine encephalitis virus
Adapted from USDA/APHIS Select Agent and Toxin List, 2010
(www.aphis.usda.gov/programs/ag_selectagent/ag_bioterr_toxinlist)
288
12.2.2. Risk Assessment Refer to section 2 of this document for detailed risk assessment
guidelines.
There is no official standard approach, method or one correct way to conduct a risk assessment,
but there are several strategies available, such as using a risk prioritization matrix, conducting a
job hazard analysis, or simply listing the potential scenarios of what could go wrong while
conducting a procedure, task or activity. Risk assessment in veterinary diagnostic laboratories
takes into account the likelihood of various risk group or BSL microorganisms being present
within unknown clinical samples, plus the likelihood that routine processing of the clinical
samples would expose laboratory workers to infectious agents within those clinical samples. A
risk assessment should take into consideration the source of the clinical sample (including host
species and clinical history), the suspected pathogen within a specimen with its inherent risk
group characteristics, the work activity during diagnostic workup of the clinical sample in the
laboratory, and the competencies and experience of the laboratory personnel.
The most critical risk assessments for veterinary diagnostic laboratories are consideration of
host species, the known medical condition and clinical history of the patient, clinical signs of the
patient, and endemic local geographical conditions (many laboratories receive samples from wide
and diverse geographical areas).
Some Risk Group 3 agents are endemic to specific geographic regions or specific species and
clinical syndromes and would warrant increasing BSL practices to appropriate levels. Examples
include tularemia in western gray squirrels in the western USA causing sudden death, anthrax in
cattle in the north central USA causing sudden death, Coxiella burnetii causing ovine abortions,
psittacosis resulting in respiratory or enteric disease in aviary birds. Alternatively, risk
assessment may indicate a reduction of biosafety practices from routine BSL-2 practices. An
example would be animal blood samples submitted for serology analysis. Unlike human blood
samples that can harbor blood borne human pathogens such as HIV or hepatitis virus, animal
serum in general does not contain zoonotic blood borne pathogens and could often, based upon a
risk assessment, be handled using BSL-1 practices.
289
The assessment of clinical history and other data provided on a laboratory accession/submission
form depends upon professional judgment and should be conducted or overseen by a qualified
veterinarian familiar with the zoonotic and select agents listed in Table 3 and Table 4 above and
the diseases caused by those agents. If questions arise during case accessioning and login that
cannot be clarified from the accession paperwork, then the submitting veterinarian should be
contacted by telephone. Knowledge regarding typical clinical signs, host range, basic
epidemiology and geographical distribution of diseases caused by these agents is essential. The
importance of biosafety risk assessment for veterinary diagnostic specimens cannot be over
emphasized. It is the primary method to differentiate handling of diagnostic specimens in
veterinary diagnostic laboratories from handling of specimens in human clinical microbiology
laboratories, where the potential of diagnostic specimens containing human pathogens is much
higher.
12.3. General Biosafety Guidelines
Refer to Section 3 for extensive and detailed biosafety guidelines generally applicable to all
subdiscipline areas within a veterinary diagnostic laboratory. A few topics are highlighted below
for emphasis in veterinary diagnostic laboratories.
The person most at risk of exposure and laboratory-acquired infection is the laboratorian
working to identify a suspect infectious agent within the diagnostic specimen so the choice of
laboratory work practices to prevent personal exposure is one of the most important decisions in
designing a laboratory biosafety plan.
In the laboratory, the routes of exposure are limited and include inhalation of fine-droplet
infectious aerosols by the airborne route, direct contact on skin or mucous membranes or
ingestion of large droplet infectious material, or percutaneous transmission by needles or other
sharps. The National Research Council (NRC) Committee on Hazardous Biological Substances in
the Laboratory in 1989 recommended seven basic prudent biosafety practices to avoid exposure
to infectious agents via the most common routes of laboratory infection.
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The basic practices help prevent exposure of laboratory workers and are outlines in Table 1. The
NRC recommendations were identified as the most important biosafety work practices but should
be supplemented by additional practices, equipment and facility design whenever there is an
increased risk of exposure to a biosafety hazard or the possibility of exposure to a BSL-3 agent.
Table 1 Prudent biosafety practices for workplaces that handle biohazardous agents
Prudent practice / barrier protections Exposure route(s)
blocked
Do not mouth pipette A, I, C
Manipulate infectious fluids carefully to prevent aerosol
production
A, C
Restrict use of sharps (needles, syringes) P, A
Use protective laboratory clothing/equipment C, A, I
Wash hands frequently C, I
Decontaminate work surfaces C, I
Do not put contaminated droplets into mouth, eyes or nose (no
eating, drinking, storing foods, smoking, or applying
cosmetics or contact lenses in the laboratory)
C, I
Based upon Biological Safety: Principles and Practices, 4
th
Edition, DO Fleming and DL Hunt,
2006 (3). A, airborne; C, contact (skin, mucous membrane); I, ingestion; P, percutaneous.
12.3.1. Hand washing
Hand washing is the most important procedure to reduce the duration of exposure to an
infectious agent, and prevent dissemination of the infectious agent. Hand contamination
occurs during manipulation of specimens, contact with work surfaces, telephones and
equipment. Laboratory personnel should wash their hands:
Immediately after removing gloves
After obvious contamination
After completion of work
Before leaving the laboratory
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Before hand contact with nonintact skin, eyes or mucous membranes
12.3.2. Personal Protective Equipment (PPE)
For routine work in veterinary diagnostic and clinical laboratories PPE should be
provided, used and maintained in the laboratory workspace. Laboratory workers should
be trained in the use of PPE for specific tasks, know the limitations of PPE and be trained
in appropriate procedures for maintaining and disposing of PPE. The level of PPE use in
routine veterinary diagnostic laboratory work should be sufficient for the BSL practices
appropriate to the suspected or identified risk and at a minimum should include gloves
and protective clothing.
Gloves protect the wearer from exposure to potentially infectious material, should be
changed frequently, and most generally are thin latex, vinyl or nitrile. Protective clothing
should include fully closable long sleeved coats or gowns that extend below the level of
the workbench. Open toed shoes should not be worn in the laboratory to prevent
accidental spillage on bare skin. Protective clothing should not be worn outside the
laboratory or taken home for cleaning or laundering.
Face and eye protection should be used when splashes or sprays of potentially
infectious material may be generated during laboratory processing, including during
laboratory clean up. Face and eye protection equipment could include splash goggles,
face shields or bench top splash guards.
Respiratory protection devices (respirators) should be used as part of BSL practices
appropriate to the risk assessment to prevent inhalation of potentially infectious aerosols.
The decision to use respirators in the laboratory may come either from the inherent risk
of potential infectious agents in a clinical specimen (e.g. BSL 3 or Risk Group 3 agents)
or from laboratory manipulations necessary for agent identification that may produce
infectious aerosols. The type of respiratory protection depends upon the specific hazard.
If an N95 or higher-rated respirator cannot be used (i.e. because of facial hair, asthma,
etc.) then a Powered Air Purifier Respirator (PAPR) should be worn when respiratory
protection is required. Surgical masks are not considered a form of effective respiratory
protection.
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12.3.3. Staff Training
Biosafety training and education of workers about potential hazards and safe work
practices are essential to creating a safe work environment. The size of the safety training
program will vary with needs and could include:
Standard Precautions (prudent biosafety practices shown in Table 1)
Selection, use and limitations of Personal Protective Equipment
Management of biohazardous waste
Post exposure management, accident reporting, and investigation of incidents
Blood-borne pathogen information
Basic understanding of risk groups and risk assessment
Procedures for biohazardous spills
Biosafety training should be documented (date and content of training), placed in the
employees training record and maintained for 3 years. The effectiveness of laboratory
safety training should be evaluated periodically. Safety assessments could include safety
audits, inspections by outside agencies, review of accident or incident reports, and
observations and suggestions made by employees. Although management provides
resources to address and correct safety deficiencies, the efforts of laboratorians working at
the bench top provide the foundation of a safe work environment.
12.3.4. Biological Spill Management
The management of biological spills in clinical laboratories must account for the specific
infectious agent (if known), the volume of infectious material spilled, and the presence of
aerosols. Aerosols may readily transmit in spills involving BSL 3 or Risk Group 3 agents. Thus,
occupants should evacuate the areas immediately, close doors and not re-enter the area for 30-60
minutes.
When breakage occurs in a centrifuge (which inherently would produce aerosols) the equipment
should remain closed for 30 minutes before decontamination commences. PPE for biological
spills should include puncture resistant gloves, N-95 respirators (BSL-2 or 3) fluid impenetrable
shoe covers, coats or gowns, and facial protection.
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For BSL3 agents, a respirator or HEPA-filtered respirator should be used, or calling a
designated, prepared spill emergency response team may be appropriate. Any broken glass in a
spill area should be removed and discarded without contact with the hands (use broom, forceps,
tongs etc.). A typical biological spill clean procedure involving a possible aerosol should include
the following:
Alert personnel in area and evacuate
Close doors and do not re-enter area for 30-60 minutes (post sign forbidding entry to the
area) based on the number of air exchanges.
Alert laboratory supervisor
Don PPE appropriate for type of spill
Wear gown, gloves, facial protection
Remove and discard broken glass or other objects (without contact with hands
Absorb the spill with absorbent material
Discard contaminated material in a biohazardous waste container
Clean spill site with aqueous detergent
Decontaminate area with appropriate disinfectant
Rinse spill site with water and allow site to dry
Copy contaminated laboratory forms and discard into the biohazard waste container
Place all disposable contaminated clean up material in the biohazard bag and treat as
infectious waste
Wash hands
Prepare a spill/incident report, identify cause of spill and determine remedial action
If a spill occurs in a Biosafety Cabinet do not turn off the cabinet fan. Minor spills in a BSC can
be absorbed with absorbent paper. If infectious material flows into the grille, all items in the
cabinet should be wiped with disinfectant and removed. The drain valve should be closed and
disinfectant poured onto the surface and through the grille into the drain pan, allowed appropriate
contact time then drained, rinsed and dried.
If a specimen tube breaks within the plastic screw-capped canister in a centrifuge:
Turn the motor off
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Remove the canister immediately and place under a biological safety cabinet.
Notify senior person in charge and other colleagues working in the area.
While wearing protective clothing, open the canister under the safety cabinet.
Pour a 1:10 dilution of bleach or a non-corrosive disinfectant into the canister to
decontaminate all surfaces; let the canister soak in bleach or disinfectant solution for 10
minutes. Clean canister thoroughly.
Do not pick up broken glass with gloved hands. Use forceps or cotton held in forceps, or
tongs or hemostats and dispose into a biosafety sharps container.
Discard all non-sharp contaminated materials from canister into a red biohazard bag for
biohazard waste disposal.
Unbroken capped tubes should be swabbed with the same disinfectant then swabbed
again, washed with water and dried.
All materials used during the clean-up must be treated as infectious waste.
NOTE: If the specimen tube breaks in a centrifuge that does not have
individual canisters, but does have a biohazard cover and sealed rotor, follow the
manufacturers instructions for cleaning and decontamination.
12.3.5. Immunization.
A current CDC guideline on immunization of health care personnel against specific
pathogens is provided in Section 14.1.4 and includes hepatitis B, influenza, measles,
mumps, rubella and varicella that are diseases whose agents are not present in animal
diagnostic specimens. In veterinary diagnostic laboratories it is not recommended that
laboratorians be immunized routinely against potential Risk Group 3 or BSL-3
pathogens. The only situation in which immunization should be considered is against
rabies virus when individuals are processing a large number of specimens potentially
containing rabies virus (e.g. routinely processing central nervous tissues from animals
with neurological disease compatible with rabies). Table 5 below lists rabies pre-
exposure prophylaxis guidelines for the United States of America according to a 2008
CDC advisory committee on rabies immunization practices.
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12.4. Pathology (Necropsy and Surgical Pathology)
Refer to Section 5 for detailed biosafety guidelines applicable to necropsy, surgical pathology, and
histology working areas within a veterinary diagnostic laboratory.
12.5. Parasitology
Refer to Section 6 for detailed biosafety guidelines applicable to parasitology working areas
within a veterinary diagnostic laboratory.
12.6. Mycology
Refer to Section 7 for detailed biosafety guidelines applicable to mycology working areas within
a veterinary diagnostic laboratory.
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12.7. Virology
Refer to Section 8 for detailed biosafety guidelines applicable to virology working areas within a
veterinary diagnostic laboratory.
12.8. Chemistry/Toxicology/Drug Testing
Refer to Section 9 for detailed biosafety guidelines applicable to chemistry/toxicology working
areas within a veterinary diagnostic laboratory.
12.9. Hematology/Serology
Refer to Section 10 for detailed biosafety guidelines applicable to hematology and serology
working areas within a veterinary diagnostic laboratory.
12.10. Molecular Diagnostics and Rapid Tests
Guidelines pertaining to biosafety best practices when conducting molecular diagnostic testing
(PCR) or using rapid tests such as ELISA can be specific to the particular testing being conducted
and are discussed in many of the subspecialty sections of the main document (Sections 3, 4, 5, 6,
7, 8 and 10). Section 8 (Virology) under subsection 8.10 provides the most thorough biosafety
guidelines for molecular diagnostic testing.
.
12.11. Storage, Packaging, and Shipping
Refer to Section 13 for detailed biosafety guidelines applicable to functions within a veterinary
diagnostic laboratory regarding storage, packaging and shipping of infectious or diagnostic
specimens.
12.12. Biosafety Education/Training
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Refer to Section 15 for practical guidelines regarding biosafety training within a veterinary
diagnostic laboratory.
12.13. Biosafety Quality Improvement
Refer to Section 16 for guidelines regarding continual improvement of biosafety within a veterinary
diagnostic laboratory.
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References
1. Biosafety and Biosecurity in the Veterinary Microbiology Laboratory and Animal Facilities. In: OIE
Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organization of Animal Health
(OIE). Office International Des Epizooties, Paris, France. 2008.
2. CDC/NIH. Biosafety in Microbiological and Biomedical Laboratories, 5
th
Edition, U.S. Government
Printing Office, Washington, D.C., 2007.
3. Fleming DO and Hunt DL. Biological Safety: Principles and Practices. ASM Press, Washington, D.C.,
2006.
4. Taylor LH, Latham SM, Woolhouse ME. Risk factors for human disease emergence. Philos Trans R
Soc Lond B Biol Sci 2001;356:983-989.
5. Torrey EF, Yolken RH. Beasts of the Earth. Rutgers University Press, New Brunswick, NJ. 2005.
6. Manning SE et al. Human Rabies Prevention --- United States, 2008, Recommendations of the
Advisory Committee on Immunization Practices. Morbidity and Mortality Weekly Report
2008;57(RR03): 1-16, 28.
299
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13. Storing, packaging, and shipping infectious substances
13.1. Storage of infectious substances
13.1.1. Recommendations
13.2. Packing and shipping infectious substances
13.2.1. Governing authorities and regulations
13.2.2. Importance of regulations
13.2.3. Exceptions
13.2.4. Specific regulations
13.2.5. United States postal service
13.3. Classification of infectious substances
13.3.1. Classification
13.3.2. Steps of Classification
13.3.3. Category A infectious substance
13.3.4. Category B infectious substance
13.3.5. Exempt human specimens
13.3.6. Exempt substances
13.3.7. Patient specimens
13.3.8. Genetically modified organisms
13.3.9. Biological products
13.3.10. Infected animal
13.3.11. Medical waste
13.4. Naming category A and Category B substances
13.5. Packing instructions and packing substances
13.5.1. Packing instructions
13.5.2. Marking and labeling outer packages
13.5.3. Specific markings and labels
13.6. Documentation
13.6.1. Shippers declaration
13.6.2. Emergency response telephone number
13.7. Refrigerants
13.8. Training and certification
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13 Storing, Packaging, and Shipping Infectious Substances
13.1 Storage of Infectious Substances
Infectious substances in a clinical microbiology laboratory are encountered as fresh and
processed patient specimens, cultures and subcultures, stored isolates, and serum or plasma.
Invariably, all of these substances must occasionally be stored in some form and for some
length of time and many of these substances will be manipulated, relocated, and otherwise
touched by laboratory workers. Therefore, storage of infectious substances is an extremely
important and integral component of worker safety in clinical microbiology laboratories.
Handle all stored infectious substances using Standard Precautions and aseptic technique.
Organisms responsible for external contamination of the storage vial will remain viable
during storage and can be transmitted by manipulating the vial.
13.1.1 Recommendations
Use primary containers with tight-fitting lids
Store as far as possible from common walkways, laboratory cart, and human traffic,
and reagents
Assure restricted access to the storage site
Assure storage at temperatures appropriate to maintain viability of microorganisms
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Use sturdy racks, buckets, or boxes that will assure the item will remain upright
Storage cabinets or refrigerators should have latching doors.
Use separate refrigerators to store long term cultures archives, subcultures, and
processed patient specimens.
Reference
Denys, G.A. and L. D. Gray. 2010. Biohazards and safety. In Clinical Microbiology Procedures Handbook.
American Society for Microbiology, Washington, DC. In press.
13.2 Packing and Shipping Infectious Substances
Use of reference laboratories for routine and esoteric testing has increased over time.
Some of the reference laboratories may not be able to send a courier to collect the
specimens from the submitting laboratory.. Knowledge of specimen transportation
standards and requirements needed to forward specimens using common air and ground
carriers has progressively become more important for diagnostic laboratories.
The American Society for Microbiology has published and made available on-line
comprehensive guidelines for packing and shipping infectious substances (1,5,7). They
are based on guidelines issued by the International Air Transport Association (IATA) and
the United States Department of Transportation (DOT). The information presented here
is an abridged version of these guidelines and is not intended to be an all-inclusive guide
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to packing and shipping infectious substances. Permits may be needed in addition to
these requirements. The information is based on IATA and DOT regulations current as
of January 1, 2010.
Note: The requirements and regulations governing the transport of infectious substances
change frequently. Shippers are responsible for being aware of these changes, adhering
to current regulations, getting permits in advance of shipping, and interpreting applicable
regulations for themselves and their facilities.
13.2.1 Governing Authorities and Regulations
The most recognized, copied, and used packing and shipping guidelines in the world are
those in the annual IATA publication Dangerous Good Regulations (8).
In the United States, the DOT regulates the commercial transportation of dangerous
goods (e.g., explosives, gases, flammable liquids, infectious substances, and radioactive
materials) by both air and ground carriers (9). The DOT regulations are in substantial
agreement with international standards (9).
For practical purposes, shippers of infectious substances can consider compliance with
IATA requirements to be compliance with DOT regulations.
13.2.2 Importance of Regulations
The purpose of the regulations is to protect the public, emergency responders, laboratory
workers, and personnel in the transportation industry from accidental exposure to the
infectious contents of the packages. An important non-safety-related benefit of
adherence to these regulations and requirements is to minimize the potential for damage
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to the contents of the package during transport and to reduce the exposure of the shipper
to criminal and civil liability associated with the improper shipment of dangerous goods.
13.2.3 Exceptions
The transportation of small quantities of non-Category A infectious substances (usually
specimens being transported for clinical, diagnostic, or other patient care purposes) is
exempt from most DOT regulations if the specimens are transported by courier, i.e.
private or contract carrier in a motor vehicle used exclusively to transport such
substances (9).
Pack and secure non-Category A infectious substances inside the vehicle according to
DOT regulations.
These regulations are usually less stringent and state that the substances need only be in
leak proof containers, sealed securely, and secured within the vehicle during transport.
The usual strict OSHA regulations still apply during this type of transportation of
infectious substances.
13.2.4 Specific Regulations
The safe and legal transport of infectious substances is based on the following mandated
activities:
Training every 2 years.for individuals handling infectious substances for shipment
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Classification and naming of infectious substances.
Selection of correct packaging materials.
Packing shipments correctly.
Placing appropriate markings and labels onto the outer package.
Documenting relevant aspects of each package and its contents.
13.2.5 United States Postal Service
The United States Postal Service publishes its own regulations in the USPS
Domestic Mail Manual (10). The USPS regulations for mailing hazardous
materials generally adhere to DOT regulations but the USPS Domestic Mail
Manual should be consulted for specific needs and requirements.
13.3 Classification of Infectious Substances
13.3.1 Classification
All shipped goods must be classified using a three-step process to define dangerous
goods that are shipped by commercial carriers. Classification allows the shipper to select
the proper IATA packing instructions and directions to use, and provides important
information necessary to complete required documentation (a Shippers Declaration) if
the substance is a Category A infectious substance.
13.3.2 Steps of Classification
The material is classified into one of the nine IATA-specified classes (Class 1 through
Class 9) of dangerous goods. Infectious and toxic substances are Class 6 dangerous
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goods; dry ice is a Class 9 dangerous good. Class 6 and Class 9 substances usually are
the only dangerous goods shipped by laboratorians.
Class 6 substances must be divided into either Division 6.1 (toxic substances)
or Division 6.2 (infectious substances).
Division 6.2 infectious substances must be classified into one of nine IATA-specified
types of infectious substances: Category A infectious substance, Category B infectious
substance, Patient Specimens, Exempt Human or Animal Specimens, Genetically
Modified Organisms, Exempt Substances, Biological Products, Infected Animals, or
Medical Waste (Table 1 and Figure 1).
If the substance is determined to be either a Patient Specimen or an Organism and
is not obviously a Category A or Category B substance but it meets the criteria of or has
characteristics of a Category A or Category B substance, the shipper must classify it as a
Category A or Category B substance. Otherwise, the substance must be classified as an
Exempt Human or Animal Specimen or a Genetically Modified Organism (Class 9),
respectively.
13.3.3 Category A Infectious Substance
A Category A substance is an infectious substance which is transported in a
form that, when exposure to it occurs, is capable of causing permanent disability,
or life-threatening or fatal disease to otherwise healthy humans or animals (9).
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Category A substances are specifically designated and listed by IATA and DOT.
The list of Category A substances is not all-inclusive; the shipper is allowed by
IATA to perform a thorough risk assessment to use their discretion and
professional judgment when deciding if a substance meets Category A criteria.
Category A substances must be assigned the UN number UN2814 or UN2900.
See Figure 1.
Note: Some Category A infectious substances are considered Category A only
if the substance is in culture form, i.e. concentrated; this distinction is clearly
indicated in the specific IATA list.
Some Category A pathogens have been designated as agents of bioterrorism and are
known as Select Agents (2,3,4,6). United States Federal regulations require shippers to
have special registration and permits to possess, use, transfer, and receive Select Agents.
13.3.4 Category B Infectious Substance
A Category B substance is an infectious substance that does not meet the criteria for
inclusion in Category A (9). Category B substances are not in a form generally capable of
causing disability, life-threatening illness, or fatal disease. Category B substances must be
assigned UN number UN3373 (Biological Substance, Category B). Examples of possible
Category B substances are the following:
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Typical clinical, diagnostic, or patient specimens, e.g., blood, biopsies, swab
specimens, excreta, secreta, body fluids, tissues, etc., (a) being shipped for
routine culturing or screening testing for non-Category A infectious
microorganism(s) or (b) suspected of containing a non-Category A
microorganism(s),
Typical clinical laboratory cultures (usually on solid or in liquid media) of routinely
encountered non-Category A microorganisms routinely encountered and manipulated
in clinical microbiology laboratories.
See Figure 1.
13.3.5 Exempt Human (or Animal) Specimens
Exempt human or animal body site specimens are those for which there is
minimal likelihood there are pathogens present (9). Examples of such
specimens include urine or serum to be tested for glucose, cholesterol,
hormone levels, prostate-specific antigen, and analytes used to evaluate heart
and kidney function.
Professional judgment and knowledge of patient medical history may used to
determine if the specimen is an infectious risk or contains pathogens.
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Exempt Human or Animal Specimens have less stringent packaging
requirements than do Category A and Category B substances. IATA requires
outer packages which contain Exempt Human or Animal Specimens to be
clearly labeled as Exempt Human Specimen or Exempt Animal
Specimen. DOT does not require this label on outer packages. See Figure 1.
13.3.6 Exempt Substances
Many substances commonly encountered in clinical laboratories are exempt
from strict infectious substance shipping requirements. The following are
examples of such substances:
substances that do not contain infectious substances or are unlikely to
cause disease in humans and animals;
most environmental samples (food, soil, etc.);
substances that contain neutralized or inactivated microorganisms;
substances to be tested for alcohol or drugs, pregnancy indicators, cancer,
and antibodies;
samples submitted for forensic analysis
dried blood spots and fecal occult blood screen specimens;
blood and blood components collected for the purpose of transfusion or
transplantation;
FDA-approved and FDA-licensed biological products; and
<30 mL of 10% formalin per primary container when the formalin is used
as a preservative.
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See Figure 1.
13.3.7 Patient Specimens
A Patient Specimen is material collected directly from humans or animals
for diagnostic, treatment, prevention, investigational, or research purposes.
Patient specimens that have Category A or Category B criteria should be
classified as Category A or Category B substances.
Patient specimens that have neither Category A nor Category B criteria
should be treated as Exempt Human or Animal Specimens. See Figure 1.
13.3.8 Genetically Modified Organisms
Genetically modified organisms usually meet either Category A or Category
B criteria. If this is not the case, the organism must be classified as a
genetically modified microorganism (Class 9; Miscellaneous Dangerous
Goods) and packed and shipped as such.
13.3.9. Biological Products
Virtually all commercially available biological products are exempt from the
regulations. Examples of biological products include bacterial typing sera,
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vaccines, bacterial antigens, antimicrobial agents, reagents for identifying
bacteria, and reagents used in antimicrobial susceptibility testing.
13.3.10 Infected Animal
A live, intentionally infected animal that is known to contain or reasonably
expected to contain an infectious substance cannot be transported by air unless
the substance cannot be transported by any other means. An exemption from
DOT will be required.
Consultation with individual commercial carriers is advised if either live or
dead infected animals need to be shipped.
13.3.11 Medical Waste
Medical Waste that contains Category A or Category B infectious substances
must be packed and shipped as such and assigned UN2814, UN2900, or
UN3373.
Medical waste that is reasonably believed to have a low probability of
containing infectious substances must be packed and shipped as Medical
Waste, n.o.s. (UN3291).
13.4 Naming Category A and Category B Substances
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After classifying the substance, the shipper must identify (officially name) the Category
A and Category B infectious substances by assigning the substance one of the 3,000+
IATA-specified and internationally recognized UN numbers and proper shipping names
listed in the blue pages section of the IATA Dangerous Goods Regulations.
This list provides 14 informational items for each of the proper shipping names and the
items correspond to the information needed to complete the Shippers Declaration.
Fortunately, only seven of the 3,000 proper shipping names are used by most clinical
microbiology laboratories:
two for Category A substances that affect humans,
two for Category A infectious substances that affect animals,
one for Category B infectious substance,
one for genetically modified organisms, and
one for dry ice.
The blue pages provide proper shipping names, UN numbers, packing instructions,
quantity limits, and other information related to packing and shipping substances.
13.5 Packing Instructions and Packing Substances
13.5.1 Packing Instructions and Directions
IATA packing instructions (PI) describe the minimum standards for the safe
transport of infectious substances.
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The instructions used by clinical laboratories are those that relate to shipping
Category A infectious substances (PI 602), Category B infectious substances
(PI 650), and dry ice (PI 904; PI 954 after 2010.
There are no specifically numbered instructions for specimens classified as
Exempt Human or Animal Specimens; however, IATA provides directions
which must be followed. See Table 2 for a comparison of the details of
packing instructions and directions.
13.5.2 Marking and Labeling Outer Packages
Shippers are responsible for the proper marking and labeling of the outer
shipping container (commonly, a cardboard box).
The markings and labels communicate essential information regarding the
shipper and consignee of the package, nature and weight of the contents of the
package, the potential hazard of the substance, how the substance is packed,
and information to be used in case of an emergency.
13.5.3 Specific Markings and Labels
Shipper and Consignee shippers and consignees name and address.
Responsible Person -- name and telephone number of someone who can answer
general questions about the shipment.
Category A Substance a Class 6 diamond-shaped Infectious Substance label, and
a proper shipping name, UN number, and quantity label .
Category B Substance -- Biological Substance, Category B and UN3373 labels.
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Dry Ice -- Class 9 Miscellaneous Dangerous Goods and weight of dry ice
label.
Package Orientation arrows on opposite sides of packages with >50 mL of a
liquid or frozen liquid.
Exempt Patient Specimens -- Exempt Human Specimen or Exempt Animal
Specimen label. This requirement is specified only by IATA, not by DOT.
Outer Package containing Category A infectious substance a UN label; a
UN inside of a circle, and a series of letters and numbers which indicate the
type of package, class of goods the package is designed to carry,
manufacturing date, authorizing agency, and the manufacturer.
Examples of appropriately labeled packages which contain Category A and B
infectious substances are shown in Figures 2 and 3, respectively.
13.6 Documentation
13.6.1 Shippers Declaration.
A Shippers Declaration is a legal contract between the shipper and carrier, is
required to document the shipment of Category A infectious substances, must be
accurate, and must be legible.
Essentially all of the IATA-specified technical information required to complete
the Nature and Quantity of Dangerous Goods section of the Declaration can be
found in the blue pages of IATA Dangerous Goods Regulations.
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13.6.2 Emergency Response Telephone Number.
DOT, but not IATA, regulations state an emergency response telephone number
must be provided on Shippers Declarations which accompany shipments of
Category A infectious substance.
The number must be monitored at all times by a person (not an answering
machine, message service, pager, etc.) who has knowledge of the hazards of the
material being shipped and emergency response and accident mitigation
information in case a handler contacts the released contents of the package. This
number also can be that of a commercial service which can provide the
appropriate mitigation information. If a the phone number of a commercial
service is used, then the contract number or service agreement number assigned
by the commercial service needs to be entered on the document, as well as the
name of the service provider.
13.6.3 Airbills
Airbills are required to be prepared by IATA carriers to describe air cargo, and
accompany shipments in transit. Some Dangerous Goods shipments, such as
Biological Substances Category B shipments, require the preparation of this
document, but not a Shippers Declaration. Specific preparation instructions are
detailed within each IATA package instruction and in the Documentation
section of the Dangerous Goods Regulations.
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13.7 Refrigerants
Packaging must be leak proof when wet ice is used. Dry ice is a Class 9 dangerous good,
it must be packaged according to PI 904 (PI954 after 2010), and its use requires
completion of a Shippers Declaration if it is used to ship a Category A substance.
Note: Dry ice is an explosion hazard and must never be placed into a tightly sealed
container! Dry ice must be placed outside the secondary container, and the outer
packaging must permit the release of CO
2
!
13.8 Training and Certification
Anyone involved in packing and shipping infectious substances must receive formal
training in this activity; every 2 years by IATA, and every 3 years by DOT. The essential
components of a training program must include the following:
general awareness and familiarity with packing and shipping infectious substances.
importance, nature, and contents of IATA and DOT regulations.
hands-on and/or demonstrations of packaging and packing techniques.
marking and labeling.
documentation of shipments of dangerous goods.
safety training.
pre- and post-training testing.
issuance of a certificate after successful completion of the training.
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Acceptable training materials and methods include manuals, training courses, and workshops,
all of which are commercially available from professional organizations and commercial
suppliers of packaging materials for dangerous goods. A training program or workshop which
includes didactic, hands-on training, and demonstrations can be developed by any hospital,
laboratory, school, institution, or other facility.
IATA and DOT require all aspects of training to be documented. The most important
document used to prove appropriate and timely training is a certificate which is issued after
training is complete.
Employers should keep a record for each employee who is trained.
The record should include employees name, location and date of training, name of
the trainer, course content, documentation of testing, and a copy of the certificate of
training. IATA and DOT certification is valid for 2 and 3 years, respectively.
The DOT, through its Pipeline and Hazardous Materials Safety Administration
(PHMSA), and the Federal Aviation Administration have authority to perform unannounced
inspections of clinical laboratories whose employees pack and ship infectious substances, and
to inspect these facilities for compliance with the training regulations, and to inspect training
records at these facilities. Facilities that do not comply with prescribed regulations are
subject to substantial fines.
318
References
1. American Society for Microbiology. 2009. Sentinel Level Clinical Microbiology
Laboratory Guidelines.
http://www.asm.org/?option=com_content&view=article&id=6342&Itemid=639
2. Centers of Disease Control and Prevention. 2009. Regulation of Select Agents and
Toxins. http://emergency.cdc.gov/cotper/dsat.
3. Centers of Disease Control and Prevention. 2008. CDC Select Agent Program: Ensuring
the Safe and Secure Possession, Use, and Transfer of Select Agents in the U.S.
http://emergency.cdc.gov/cotper/dsat/pdf/DSAT-AAG-080408.pdf
4. Centers of Disease Control and Prevention and the Office of the Inspector General,
United States Department of Health and Human Services. Possession, use, and transfer of
select agents and toxins; final rule (42 CFR Parts 72 et al.). Fed. Regist.2005;70:13294-
13325.
5. Denys, G.A., L.D. Gray, and J.W. Snyder. Cumitech 40, Packing and shipping
diagnostic specimens and infectious substances. Coordinating ed., D.L. Sewell. 2008.
American Society for Microbiology, Washington, DC.
6. Department of Health and Human Services. Possession, use, and transfer of select agents
and toxins. 42CFR Parts 72 and 73. Fed. Regist. 2008;70:613633-61366.
7. Gray, L.D. and J.W. Snyder. Chapter 21. Packing and Shipping Biological Materials. In
Biological Safety Principles and Practices (4
th
edition). 2006. D.O. Fleming and D.L.
Hunt, eds. ASM Press, Washington, DC.
319
8. International Air Transport Association. 2010. Dangerous Goods Regulations, 51
st
edition. International Air Transport Association, Montreal, Canada.
9. United States Department of Transportation, Pipeline and Hazardous Materials
Safety Administration. 2006. Hazardous materials: infectious substances; harmonization
with the United Nations recommendations; final rule (49 CFR Parts 171 et al.). Fed.
Regist. 2006;71:32244-32263.
10. United States Postal Service. 2006. Domestic mail manual.
(http://pe.usps.com/DMMdownload.asp).
320
TABLE 1. Types and classifications of IATA Division 6.2 Infectious Substances
Type of Infectious Substance IATA Classification
Category A Substance Category A
Category B Substance Category B
a
Patient Specimen
meets Category A criteria Category A
meets Category B criteria Category B
does not meet Category A or B criteria Exempt Human or Animal Specimen
Exempt Human or Animal Specimen Exempt Human or Animal
Specimen
Genetically Modified Microorganism
meets Category A criteria Category A
meets Category B criteria Category B
does not meet Category A or B criteria Genetically Modified Organism
Exempt Substance none
Biological Product
b
Infected Animal
b
Medical Waste
b
a
The proper shipping name for Category B substances is Biological Substance, Category B.
b
Substance is not addressed in detail in this document
321
TABLE 2. Comparison of IATA and DOT Packing Requirements for Infectious
Substances
Substance
Packing Requirement
Exempt
Human
Specimens
a
Category B
b
Category A
c
Inner Containers
leakproof primary (1
o
) and secondary (2
o
) containers yes yes yes
pressure-resistant 1
o
or 2
o
container --
d
yes yes
absorbent between 1
o
and 2
o
containers
e
yes yes yes
list of contents between 2
o
and outer package -- yes yes
positively sealed 1
o
container -- no yes
Outer Container
rigid outer packaging -- yes yes
strict manufacturing specifications none
f
few many
name and number of responsible person -- yes
g
yes
markings and labels yes
h
Less
i
more
Quantity Limits for Either Passenger or Cargo Aircraft
322
maximum for each 1
o
container -- 1 L (1 kg) 50 mL (50 g)
total maximum for outer package -- 4 L (4 kg) 50 mL (50 g)
Documentation
Shippers Declaration for Dangerous Goods -- no yes
emergency response telephone number -- no yes
Costs
cost of labor and materials to pack substance least more most
a
packing directions for packing and shipping Exempt Human Specimens
b
packing instructions 650
c
packing instructions 602
d
requirement not specified by IATA or DOT
e
not required for solid substances such as tissue and solid agar media cultures or
slant
f
should be of adequate strength for its intended capacity, mass, and intended use
(IATA quote)
g
may be placed either on the outer package or on the air waybill
h
Only Exempt Human Specimen or Exempt Animal Specimen is required.
I
If an air waybill is used, the Nature and Quantity of Goods box/area must show
UN3373, Biological Substance, Category B and the number of packages.
323
1.1.1 Consignee
2 Shipper
UN3373
Biological
Substance,
Category B
Responsible Person
and Phone Number
Dry Ice
3 kg
FIGURE 2. A completely labeled outer package. The primary container inside the
package contains a Biological Substance, Category B infectious substance and is
packed according to PI 650.
325
326
3.1.1 Consignee
4 Shipper
Responsible Person
& Phone Number
Proper Shipping Name,
UN Number, & Quantity
4G/CLASS 6.2/2007
5.1.1.1.1 CAN/8-2 AIRPACK
U
N
Dry Ice
3 kg
FIGURE 3. A completely labeled outer package. The primary container inside the
package contains a liquid Category A infectious substance and is packed according to PI
602.
327
328
14 Employer and employee involvement and responsibilities
14.1 Responsibilities of employers before an exposure
14.1.1 Exposure control plan
14.1.2 Documentation of Potential Exposures
14.1.3 Emergency response facilities
14.1.4 Immunizations
14.1.5 Education of employees
14.2 Responsibilities of employees before an exposure
14.3 Responsibilities of employers after an exposure
14.3.1Determination of the extent of exposure
14.3.2 Documentation of exposures
14.3.3 Consultation with employee health clinicians
14.3.4 Counseling exposed employees
14.3.5 Exposure to Mycobacterium tuberculosis
14.3.6 Exposure to Neisseria meningitidis
14.3.7 Exposure to blood-borne pathogens
14.4 Responsibilities of employees after an exposure
329
14. Employer and Employee Involvement and Responsibilities
Introduction
The risk of obtaining a laboratory-associated infection (LAI) after physically contacting a
microorganism (an exposure) in the workplace is real, always present, and an integral part of
working in a diagnostic laboratory, and in particular the clinical microbiology laboratory. The
potential for an exposure exists whenever a laboratorian manipulates and transports microorganisms,
processes and stores patient specimens, and operates instruments used in the process. Diagnostic
laboratories can be safe places to work if standard and appropriate safe work practices and procedures
are easily accessible, understood by employees, enforced, and followed. These procedures should be
properly outlined in the Exposure Control Plan and laboratory manuals. Well designed plans with the
full support of the Director and higher management can reduce workers chances of exposures to
microorganisms and can help ensure a culture of safety in diagnostic laboratories; these plans are
composed of essential elements related to preventing an exposure. Another equally important
element of these plans is the employer and employee involvement and responsibilities before and
after an exposure. Appropriate actions taken after an exposure can greatly reduce or even eliminate
the chance that an exposure will result in an LAI.
14.1 Responsibilities of Employers Before an Exposure
14.1.1 Exposure Control Plan
330
Employers need to establish a formal plan outlining work practices and procedures to minimize
the potential for an exposure. This plan should outline steps to take to reduce the risk of
exposure.
Employers should have a formal plan to address actions to be taken after an employee is
exposed. The plan should incorporate at least the elements presented here (or the equivalent) and
be easily accessible to employees.
Both the employer and the employee should embrace the plan and rigorously participate in
protecting themselves, their colleagues, the staff, and patients.
Develop the plan prior to a first exposure with input from the director of the laboratory,
laboratory workers, and from representatives of employee health and infection control or local
site medical services provider to assure the inclusion of appropriate immunization, post exposure
prophylaxis, and infection control measures.
Document annual review of Safer Medical devices and other advances in research technology
that can reduce or eliminate potential risks of exposure.
A Safety Culture - Develop a Safety Culture with appropriate buy-in from all levels
of employees including upper management and front-line workers. Develop a non-
punitive reporting structure for potential occupational exposures and have outlined
provisions for how employees that have self-reported changes in health status will be re-
assigned if required without negatively influencing their job performance or review of job
performance.
All potential incidents need to be reported regardless of whether the employee thinks an
exposure occurred or not . The incident is evaluated by the employer and occupational
physician to determine if post exposure prophylaxis is needed.
331
Medical practices designated to perform these medical evaluations should be provided with the
current U.S. Public Health Service recommendations for medical evaluations and post exposure
prophylaxis for the infectious agents in use.
Outreach to the medical provider is important; the medical provider needs to be prepared to
evaluate clinical laboratory workers that may have had occupational exposure to a variety of
infectious agents.
14.1.2 Documentation of Potential Exposures
Develop a record or form to accommodate the following information:
date, time, and location of the exposure.
employees name and employee number.
names of other employees related to the incident (witnesses, etc.).
details of the incident or exposure.
brand names and manufacturer of any devices or instruments involved including lot
numbers of medical devices and PPE used at the time of exposure.
relevant health information/status of the exposed employee at the time of the
exposure.
immediate or remedial actions which were taken, including first aid.
actions recommended the exposed employee take immediately or the same day,
(visits to employee health or the emergency department, administration of
chemoprophylaxis, consultations with physicians, etc.).
results of discussions with employee health clinicians.
monitoring and follow-up plans.
space to record results of monitoring and follow-up plans.
332
appropriate signatures (minimum: those of the employee and the employees
immediate supervisor).
14.1.3 Emergency Response Facilities
Employees must receive training on the proper use of all emergency equipment.
First aid kits must be visible and easily accessible within the laboratory. ANSI
specification eye wash stations and safety showers must be readily accessible as per the
requirements of the ANSI standard and any OSHA standards which mandate their
placement. Cleansing skin abrasions, flushing eyes, or showering after exposures to
exposed areas of the body can greatly reduce or even eliminate the chance that an
exposure will result in an LAI.
Employees should be made aware and frequently reminded of the importance and
locations of these facilities. Ensure that procedures are in place to document that all
equipment is functioning properly (e.g. ANSI standards for eyewashes and showers) and
within acceptable expiration dates (e.g. first aid kit).
14.1.4 Immunizations
The CDC has published recommendations for the immunization of healthcare workers
against several diseases for which these workers are at high risk (1).
The CDC strongly recommends for human health concerns all healthcare workers be
immunized against hepatitis B, influenza, measles, mumps, rubella, and varicella.
333
For human health, the CDC also recommends immunization of laboratory employees
who work with or in certain specifically listed high-risk situations which can be
encountered in laboratories, e.g., Neisseria meningitidis isolates from sterile sites,
hepatitis A-infected primates, hepatitis A research areas, specimens which can contain
wild polio virus, rabies-infected animals, and Salmonella typhi.
Immunization of employees, in general, and these special situations, in particular, are
most effectively addressed in institutional employee health or infection control policies.
Employers should make laboratorians aware of the existence and availability of all
vaccines.
Laboratory workers who manipulate N. meningitidis isolates from sterile sites should
make an informed decision about vaccination with quadravalent meningococcal vaccine
(serogroups A, C, Y, and W-135) (2). The vaccine will decrease but not eliminate the
risk for meningococcal disease; the vaccine might be used as an adjunctive measure by
microbiologists in clinical laboratories (2,3).
14.1.5 Education of Employees
Employees must receive initial training and refresher training at least annually and this
training should be documented. Training should encompass all the necessary laboratory
procedures employed in the facility that may reduce the risk of exposure. Training should
include information on engineering controls in place (including safety equipment), proper
procedures used in the manipulation of samples, proper personal protective equipment and
procedures to be followed in the event of an exposure. Employees should be made aware and
frequently reminded that (a) many reasonable and helpful actions taken after an exposure can
greatly reduce or even eliminate the chance that an exposure will result in an LAI, (b) these
actions are detailed in an exposure plan, (c) employee health clinicians and infection control
334
practitioners are available for consultation after an exposure, and (d) immunizations
appropriate for workers in CM laboratories are available. They should also be made aware
of symptoms of infections and follow up procedures. Document that the employee has read
and understood all procedures that have been adopted in support of this formal exposure
management plan.
14.2 Responsibilities of Employees Before an Exposure
Follow Standard Precautions and other established institutional laboratory safety practices at all
times.
Never perform laboratory procedures, manipulate microorganisms, process patient specimens,
or operate microbiology identification instruments if doing so will increase their risk for an LAI.
Know what to do immediately if one suspects they have experienced an exposure, be familiar
with laboratory infection control manuals and procedures and where they are located, know the
location of first aid kits, eye wash stations, and showers and how to use them, and keep the
supervisor informed of their health status (immunocompetency, cuts, abrasions, pregnancy,
breathing problems, etc.) to determine the possibility of an increased LAI risk while performing a
particular job.
It is a general recommendation to report changes in health status, but employees
cannot be compelled to do so. There should be a provision in place on how to
handle this situation.
Know any unique signs and symptoms of any aerosol transmitted infectious agent which the
laboratory isolates and manipulates on an ongoing basis and to report them to supervisors if
experienced.
335
14.3 Responsibilities of Employers After an Exposure
14.3.1 Determination of the Extent of Exposure
The likelihood that an exposure will result in an LAI is directly related to several variables,
including:
concentration of the microorganism to which the employee was exposed.
physical form of the microorganism (broth, colony, lyophilized, aerosol, etc).
innate virulence of the microorganism.
length of time worker was exposed to the microorganism.
proper use of personal protective equipment.
immunocompetency of the exposed worker.
portal of entry (intact, inflamed, abraded, or cut skin; needle stick; mucous
membrane; respiratory route; oral route).
Immediately after any exposure, an employer (together with the exposed employee, if
possible) should examine all of details of the exposure incident, determine the degree to
which these variables (and others if applicable) were involved in the exposure, make a
decision regarding the degree of exposure, and agree to an acceptable course of action to
prevent or reduce the chances of the employees developing an LAI. Communication or
consultation with the employees health care provider may be necessary depending upon the
severity of exposure and could be essential to a successful outcome. Fortunately, most
exposures in laboratories are mitigated by good hand washing. Unfortunately, some
336
exposures are determined to be significant enough to document and to take actions beyond
hand washing.
A root cause analysis should be initiated to determine all facts regarding the exposure, their
root causes, and an action plan to correct identified causes needs to be developed,
implemented, and monitored. After an incident, retraining of personnel may be in order. In
addition, procedure manuals may require review to ensure appropriate safety measures are
listed.
14.3.2 Documentation of Exposures
Document on an exposure incident form all of details of the exposure and all decisions
related to the exposure.
Gather information from other personnel if they witnessed the incident or assisted in the
clean-up.
Give employees access to medical services for consultation.
The form should be signed by the employee and the employees immediate supervisor.
14.3.3 Consultation with Employee Health Clinicians
The employee and the supervisor of an employee who has experienced a potential exposure
should contact the employee health physician or nurse and discuss the exposure. These
clinicians are the persons most likely to provide advice regarding timely chemoprophylaxis
and to able to administer appropriate antimicrobial agents.
14.3.4 Counseling Exposed Employees
Keep the exposed employee well informed during the postexposure period as information
continues to be gathered and documented.
337
Tell the employee all the findings of the exposure investigation, the supervisors opinion
regarding the extent of exposure, what and when actions are expected to happen, who he/she
should see for medical consultation, where additional information can be found, which post
exposure prophylaxis is advised and available.
Counsel the employee to seek medical advice and treatment for any acute illnesses that
occurs after the exposure and during follow-up. The employee also should receive a copy of
the initial and final exposure report forms.
14.3.5 Exposure to Mycobacterium tuberculosis
Approaches to situations in which an employee has likely been exposed to M. tuberculosis
are best addressed in institutional employee health or infection control policies, many of
which are based on CDC guidelines (1).
If an employee is exposed to M. tuberculosis, e.g., possible inhalation of aerosolized M.
tuberculosis, the employer should arrange to have the employee skin- or blood-tested to
determine if an exposure has occurred. The results of this test can be compared to the
employees baseline or previous annual test to detect a conversion.
14.3.6 Exposure to Neisseria meningitidis
Employees who are exposed percutaneously to a N. meningitidis isolate from a sterile site
should receive treatment with an appropriate antibiotic.
Employees who have a mucosal exposure to a N. meningitidis isolate from a sterile site
should also receive antimicrobial chemoprophylaxis.
338
14.3.7 Exposure to Blood-Borne Pathogens
Approaches to employee percutaneous or membrane exposures to blood-borne pathogens,
e.g., human immunodeficiency virus and hepatitis viruses B and C, are most effectively
addressed in institutional employee health or infection control policies, many of which are
based on CDC guidelines These policies should address the availability of post exposure
prophylaxis for exposure to hepatitis B and human immunodeficiency viruses.
14.4 Responsibilities of Employees After an Exposure
Never assume a laboratory accident or exposure is insignificant or unimportant.
Employees must be empowered to report all incidents with the goal of protecting themselves,
their colleagues, and their families without fear of reprisal. Report all exposures to the supervisor
immediately and discuss the exposure to determine what, if any, actions need to be taken.
Actively participate in the documentation of the exposure and provide pertinent information that
will be used in the development of the corrective- action plan
Cooperate fully with approved post exposure processes already in place when an exposure
occurs and follow prudent medical advice.
Follow the directions of the supervisor to the degree they feel is reasonable.
References
1. CDC. Immunization of health-Care workers: recommendations of the Advisory Committee
on Immunization Practices (ACIP) and the Hospital Infection Control Practices Advisory
Committee (HICPAC). MMWR 1997;46(RR18):1-45.
339
2. CDC. Laboratory-acquired meningococcal disease United States, 2000. MMWR
2000;51(7):141-144.
3. CDC. Prevention and control of meningococcal disease. Recommendations of the Advisory
Committee on Immunization Practices. MMWR 2005;54(RR7):1-21.
4. CDC. Guidelines for preventing the transmission of Mycobacterium tuberculosis
in health-care settings, 2005. MMWR 2005;54(RR17):1-141.
5. CDC. Updated U.S. Public Health Service guidelines for the management of occupational
exposure to HIV and recommendations for postexposure prophylaxis. MMWR
2005;54(RR09):1-17.
6. CDC. Updated U.S. Public Health Service guidelines for the management of occupational
exposure to HBV, HCV, and HIV and recommendations for postexposure prophylaxis.
MMWR 2001;50(RR11):1-53.
340
15 Biosafety education
15.1 Biosafety exercises
15.2 Annual checklist of critical safety items and procedures
15.3 Regular safety mini-presentations
15.4 Monitoring compliance with safety procedures
15.5 Job aids
16 Continuous quality improvement
341
15. Biosafety Education
The employer must develop an accurate job description so the employee understands the job
responsibilities. Knowledge, skills, and abilities needed for the job are to be defined. The
incoming employee should be evaluated to see if they meet these criteria. A mentoring plan
should be developed and any training gaps should be filled before the employee is placed in a
position that would put them at risk for exposure. The employee competency should be evaluated
and documented before they are allowed to work independently.
Educational opportunities to reinforce safe behaviors must be ongoing and supported by all
levels of management and staff. In accordance with OSHA requirements, education about the
risks of exposure to infectious agents should begin with a new employees first orientation to the
laboratory or assignment to technical work and should be specific to the tasks they perform.
Training must include an explanation of the use and limitations of methods that will reduce or
prevent exposure to infectious materials. These include engineering controls, work practices, and
personal protective equipment. Annual retraining for these employees must be provided within
one year of their original training and should emphasize information on new engineering controls
and practices. Annual safety training offers a chance to review key biosafety measures that may
be forgotten during every day work pressures.
It should be clear who is responsible for overseeing the safety education of the laboratory
personnel. This responsibility may be delegated to the biosafety officer or other staff member
who has been given additional training themselves through specialized courses or work
experience and their competency to perform the training verified. Because laboratory tests may
be performed outside of a traditional laboratory setting (e.g., doctors office, outpatient clinic,
342
community setting), these recommendations for training and education must be adapted to suit the
employees performing tests and the individual who is overseeing them.
15.1 Biosafety exercises
Live demonstrations are most effective because they allow interaction between the laboratory
worker and the expert, who can ask and answer questions. Demonstrations of common problems or
exposures that laboratory scientists may encounter in their normal work duties should be presented
as a learning tool for individuals or groups.
A laboratory area or work station can be rigged (or pre-positioned) with deliberate problems or
safety errors for interactive education. Whats wrong here and how would you address it? What
would you do if you encountered this problem? For example:
Set up a BSC with excess clutter inside or demonstrate someone going in and out of the
hood excessively.
Practice what to do if you drop a liquid culture of possible Mycobacterium tuberculosis
in the mycobacteriology lab.
Discuss what to do if a syringe is sent to the lab with a needle attached.
How would you handle a broken tube with a specimen in the centrifuge?
Walk through what you would do if you got a splash of a bacterial suspension in your
face/eyes/mouth.
Demonstrate someone ejecting pipette tips where they could bounce up and strike the
laboratory scientist in the face.
How to deal with a leaking specimen in the pneumatic tube?
343
All proper behavior for avoiding or dealing with these demo events should be described in the
standard safety practices and procedures in the laboratory safety manual.
Educational reinforcement through communications and quizzes should be ongoing and random.
Unpredictable challenges may be most effective because they force quick, deliberate thought about
how to respond. A specific scenario could be handed to a randomly selected technologist, while
some laboratories may have the IT capacity to send safety challenges to individuals when they sign
on for the day. Alternatively, it may be as simple as having a question of the week posted in the
laboratory. These approaches would facilitate safety exercises on all shifts.
Safety challenges may be created as competitions between laboratory sections (chemistry vs.
hematology vs. microbiology staff). Challenge each section to have the best safety record or
complete the most educational events over a set time period.
The American Biological Safety Association (ABSA) has links on its website
(www.absa.org/resbslinks.html) to obtain biosafety information from government agencies and
other professional organizations covering rules/regulations/guidelines, biosecurity and bioterrorism,
and technical links and papers, information on their week long biosafety review course that can
provide continuing education for laboratory personnel.
15.2 Annual checklist of critical safety items and procedures
There is no one set of questions for an annual safety checklist. While there are common activities
all personnel may perform, the list should be customized to reflect the actual job duties. Each work
station should be analyzed for the type of biosafety risks associated with it, and the checklist should
344
target each of these risks. Try having each individual draft their own checklist for the duties they
perform and have their list reviewed by their supervisor and safety officer.
15.3 Regular safety mini-presentations
Monthly safety vignettes can be presented during the regularly scheduled laboratory meetings with
personnel. Employees who have had a near-miss or actual incident could share their experience
with the rest of their co-workers in accordance with institutional privacy considerations. Having
this information coming from their co-workers would increase its impact. Creative solutions to
prevent biosafety problems should be elicited and discussed. Alternatively each lab employee could
take a turn at presenting what they perceive as a biosafety hazard and present this at the staff
meeting to keep everyone aware of daily safety issues so easily forgotten in a busy work place.
If meetings are not possible, short one-page written safety puzzles can be shared in the break
room over coffee, or posted on the lockers, or on the lab bulletin board. Try a whats wrong with
this picture? format. For example:
Show a photo of a person working outside of the biosafety cabinet with molds
Show someone working in the biosafety cabinet with objects blocking the vent of the
biosafety hood
Show a person vortexing bacterial suspensions for antimicrobial susceptibility testing
without a lid on the tube.
Post the correct answers with the next safety quiz.
Employee training and competency assessment should be documented for:
Constructing and properly labeling containers for medical waste.
345
Following all federal, state, and local regulations regarding waste management including
handling, disposal, and storage of medical waste.
Transportation of medical waste, which includes any required Department of Transportation
(DOT) labeling (e.g., known infectious substance label) of transport containers.
Transportation of Specimens
Employee training and competency assessment should be documented for:
Use of appropriate supplies - containers, appropriate plastic bags, labeling etc. for
transport of all laboratory specimens both within and outside of the hospital facility
Following all federal, state, and local regulations regarding transport of laboratory
specimens.
Transportation of specimens outside the facility: Useful resource
US Department of Transportation publication:
https://hazmatonline.phmsa.dot.gov/services/publication_documents/Transporting%20Infecti
ous%20Substances%20Safely.pdf
15.4 Monitoring compliance with safety procedures
To reinforce the importance of safety training and education, it should be a part of the annual
performance review. Employees must participate in their annual training, follow policies, and
should be active in the laboratory wide safety quizzes. Those who have a series of incidents during
the evaluation period should be counseled and receive more intense re-training in those areas.
346
It is imperative that employees understand the importance of reporting incidents to Employee
Health and their supervisor or manager. This is important for the employees protection to link an
injury or infection to an incident that may have occurred in the workplace in order to compensate
the employee appropriately and improve practices to protect others. An expedited exposure control
plan should be developed whereby a laboratory employee is streamlined through the employee
health process so that they are more likely to report an incident. The supervisor or manager should
follow up on every employee who has incurred a problem to protect the employee and to make sure
these incidents are monitored, and systematic risks are eliminated. Annual review of engineering
controls should be performed since changes in practices and technology may put the laboratory
staff at new risks. New engineering controls should be reviewed by the safety officer, risk
management, purchasing, and employees who may use needles and other sharps in the course of
their duties as warranted.
Laboratory sections can rotate each laboratory employees as the safety captain of the month to
reinforce good safety behaviors and encourage their fellow co-workers to do comply with safety
policies. An award can be given to the safety captain with the best track record for their month as
determined by least infractions observed or most creative idea to prevent safety incidents.
Reserve punitive measures for egregious or repeat violations and create a positive attitude about
safety, not a negative one with administrative consequences. In this regard, a laboratory could
create a cadre of safety mystery shoppers. Periodically someone (or everyone) in the lab is
assigned to be a spy and report on any unsafe practices that they see occurring in the lab. The
purpose is not to report people, but to recognize situations they see where safe practices are not
being followed.
347
It is not only important to monitor compliance with safety guidance but also to track and show
progress on a chart the number of incidents or near- misses that occurred each month and applaud
each month that decreases are realized.
15.5 Job aids
Signage of what not to do may remind personnel to comply with safety policies. OSHA, CDC, and
other organizations provide job aids that can be downloaded and printed. However personnel soon
become oblivious to the same sign in the same place delivering the same message. These signs
should be rotated with new messages that are designed to keep staff alert to the hazards at the work
stations.
348
16. Continuous Quality Improvement
Continuous quality improvement for biosafety should be integrated with the continuous quality
improvement for the entire laboratory. Following the 12 quality system elements as defined by
the Clinical Laboratory Standards Institute (CLSI), the biosafety considerations for each of the 12
elements are listed and cross-defined in the table.
349
Table 1. Quality System Elements (QSE), Definition, and Related Biosafety Considerations for
the QSEs
QSE # QSE Definition Biosafety Considerations
1 Organization Organizational
structure of
responsibility is
defined
Clear reporting structure is established for
24/7 reporting of safety incidents. Each staff
member knows who to consult for biosafety
reporting at all times. A chain of
responsibility for overall compliance leads
directly to the head of the organizational
unit.
2 Documents and
Records
Process and
procedure
documents are
written and
maintained
All incidents and near misses are
recorded. All safety practices and policies
are written, reviewed and approved and
available on all shifts to all personnel and
management. Document control assures that
the most current policies are in use and
available. Vaccination records and safety
training records are appropriately
completed, maintained and easily
recovered.
3 Facilities and
Safety
The physical
environment and
space is
appropriate for the
Facilities are designed and constructed with
safety controls and minimize the risk of
injury and occupational illness. Safe work
practices are followed at all times.
350
work being done
4 Personnel Laboratory
personnel follow
prescribed policies
and procedures
according to their
job descriptions
Personnel have received appropriate safety
training at orientation, annually, and when
their duties change. Documentation of
biosafety training and continuous education
is maintained in their personnel file and
linked to their annual review. Training
requirements are part of the annual resource
planning process.
5 Equipment Instruments and
equipment used to
carry out
laboratory
functions
All biohazard risks associated with
operating instruments are clearly defined in
standard operating procedures (SOPs).
Personnel are aware of procedures to
decontaminate equipment prior to
maintenance or being decommissioned.
6 Purchasing and
Inventory
Processes and
procedure for
purchasing
necessary supplies
and materials
Appropriate safety supplies (masks, gloves,
gowns, biohazard disposal bags and
containers) and vendors are identified and
documented. Sufficient inventory of safety
supplies is available so that personnel do
not compromise their personal safety or the
safety of others.
7 Process Control Workflow is
defined to meet
customer
expectations and
ensure the quality
of the service
Biohazard risks associated with operational
procedures are clearly defined and
referenced in SOPs. Ensure that regulatory
standards are met and procedures are
mapped out with quality and safety as
priority goals.
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8 Information
Management
Ensure effective
flow of information
to comply with
legal and
regulatory
requirements
Document reporting of incidents and
responses back to the employee.
Appropriate retention of documents to meet
legal requirements.
9 Occurrence
Management
Identify and
analyze
information which
results in
laboratory errors or
other events
Document all non-compliance with
established safety procedures and policies.
All incidents are reported to Employee
Health; all near-misses are recorded so
that systems can be improved. A reporting
mechanism exists to enable reports to be
categorized to type of error so that
corrective actions can be established.
10 Assessments Assess the
effectiveness of the
of the system
Audits of the biosafety risks and policies
should be performed annually to initiate
improved methods and engineering
controls.
11 Customer
Service
Expectations of the
customer are met
or exceeded
The requirements for biosafety are met and
personnel are satisfied with policies, work
practices and engineering controls to protect
them. Biological samples, reagents and
other items shipped to reference laboratories
(customers) are clearly labeled for
biosafety hazards.
12 Process
Improvement
Systemic review of
processes identify
areas for
Systematic review of occurrence reports as
well as risks and interventions informs
management planning for systematic
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improvement improvements.