Edsel Allan G.

Salonga, RMT, CBO

Cytogenetics Laboratory
Institute of Human Genetics, UP-Manila
May 29, 2018
Overview
• Different Types of Cabinets
• Basic Principle
• Selection and Installation of BSC
• How to use a BSC
• Biosafety Cabinet Certification
 Types of Cabinets
• Fume Hood

• Laminar Flow Cabinet (LFC)

• Biohazard Safety Cabinet (BSC)

 Fume Hoods
• Removes toxic chemical
(ducting sys./ductless)
• No HEPA filter -> not for
biohazard agents
 Laminar Flow Cabinets
• Product protection (no
personnel protection)
• Not for biohazard
agents or chemical
fumes
 Biosafety Cabinets
• Class I BSC: Personnel and Environment Protection
• Class II & III BSC: Personnel, Product and Environment Protection
• HEPA filters (not for chemical vapors)
 BIOLOGICAL SAFETY CABINETS
• BSCs provide effective primary containment
for work with infectious material or toxins
when they are properly maintained and used
in conjunction with good laboratory
techniques
 Basic Principle
• Personnel protection is provided through a
continuous stream of inward air, known as
inflow, which helps prevent aerosols from
escaping through the front opening.
• The exhaust air, which is exhausted into the
surrounding containment zone or directly to
the outside atmosphere, is HEPA-filtered to
protect the environment.
 HEPA & ULPA Filter
HEPA: High Efficiency Particulate Air
ULPA: Ultra Low Penetration Air

Important definitions:
- HEPA: 99.99% - at 0.3 microns
- ULPA: 99.999% - at 0.12 microns

Note: The “classical” definition of

HEPA filter is 99.97% at 0.3
microns, but nowadays all BSC and
LF in US use 99.99% at 0.3 m
Particle Size Comparison
 HEPA/ULPA Capability
• Removes a broad range of airborne
contaminants:
– Fine dust
– Smoke
– Bacteria (typical size: 500 to 0.3 micron)
– Soot
– Pollen
– Radioactive particles
– Impurity ion -> can affect Integrated Circuit speed
 Class I BSC
• 100% Exhaust
• Inflow velocity 75 fpm minimum
• BSL 1 –3 Usage
• Personnel protection only
• CDC/NIH recommends a glove-
port panel for use with small
amounts of radionuclides when
exhausted
• Typical uses today: Toxic powder
weighing, necropsy
• Maybe thimble/air gap or hard
connected to a exhaust system
when proper precautions are
taken
 Class II BSC
• Negative-pressure ventilated cabinet
• Provides HEPA-filtered, recirculated airflow within
the cabinet
• Exhaust air is HEPA-filtered
• Provides personnel and product protection
• Types of Class II BSCs
– Class II A: HEPA filtered air is discharged into the room
– Class II B: HEPA filtered air is discharged out of the
room
 Class II Type A1
• 30% Exhaust, 70% Re-circulate
• Negative pressure plenum
(Changed 2007)
• Inflow velocity 75 fpm minimum
• BSL 1 –3 Usage
• Personnel and Product protection
• Minute amounts of non-volatile
toxic chemicals and radionuclides
if canopy/thimble exhausted
• Typical uses today: Bacterial,
Viral, Fungal, Parasitic
 Class II Type A2
• 30% Exhaust, 70% Re-circulate
• Negative pressure plenum
• Inflow velocity 100 fpm minimum
• BSL 1 –3 Usage
• Personnel and Product protection
• Minute amounts of volatile toxic
chemicals and radionuclides if
canopy/thimble exhausted
• Typical uses today: Bacterial,
Viral, Fungal, Parasitic, Arbor-
viruses
 Class II Type B1
• 70% Exhaust, 30% Re-circulate
• Negative pressure plenum
• Inflow velocity 100 fpm minimum
• BSL 1 –3 Usage
• Personnel and Product protection
• Minute amounts of volatile toxic
chemicals and radionuclides
• Must be hard connected with typical
exhaust requirement being 300-500
CFM at 1.0” w.g.
• Must have interlocked internal blower
with audible and visual alarm for
exhaust failure
• Typical uses today: Bacterial, Viral,
Fungal, Parasitic, Arbor-viruses
 Class II Type B2
• 100% Exhaust
• Negative pressure plenum
• Inflow velocity 100 fpm minimum
• BSL 1 –3 Usage
• Personnel and Product protection
• Small amounts of volatile toxic
chemicals and radionuclides
• Must be hard connected with typical
exhaust requirement being 700-1,200
CFM at 2.0” w.g.
• Must have interlocked internal
blower with audible and visual alarm
for exhaust failure
• Typical uses today: Bacterial, Viral,
Fungal, Parasitic, Arbor-viruses, Prion,
Cytotoxics
International Standards for Class II
• US Standard ANSI/NSF49
• European Standard EN12469
• Japanese Industrial Standard JIS K3800
• South African Standard SABS VC 8041:2001
• British Standard BS5726*
• German Standard DIN12950 Teil 10*
• French Standard NF X44-201:1984*

*now obsolete. Replaced with the harmonized EN12469

 Class III BSC
• 100% Exhaust Glove Box
• Negative Pressure at 0.5” w.g.
minimum
• Double HEPA Filter Exhaust
• BSL 4
• Personnel and Product Protection
• Small amounts of volatile toxic
chemicals and radionuclides
• Must be hard connected with
typical exhaust requirement being
50-100 CFM at 0.5 w.g.
• Must have negative pressure alarm
for cabinet or exhaust failure
• Typical uses today: Toxic Powders,
BSL 4 Agents
Selection of a Safety Cabinet through
Risk Assessment
Airflow Visualization
Poorly-located BSCs
Well-located BSCs
 Installation of BSCs
• Locating BSCs away from
areas where airflow
patterns may be
disrupted (e.g., room air
supply and exhaust
grilles, doors, open
windows, high traffic
areas, and large pieces of
equipment that generate
heat) will help protect the
fragile air curtain at the
front of the cabinet.
 Recommendations are to be considered
with respect to the installation of BSCs
• Consideration should be
given to the use of bag-
in/bag-out (or another
procedure for the safe
removal of filters) HEPA
filters in situations where
effective in situ
decontamination is not
feasible or possible.
• This allows for subsequent
decontamination and
disposal off-site.
 Recommendation… Cont
• Adequate clearance
should be provided
between the exhaust
outlet on top of the BSC
and any overhead
obstructions.
 Recommendation… Cont

• Adequate clearance should be provided on each side

of the BSC to allow access.
 Recommendation… Cont
• BSCs should not be
located directly opposite
seated work stations,
other BSCs, or chemical
fume hoods.
• A reasonably safe
distance, as determined
by a local risk assessment
(LRA), should be
maintained to avoid
operator collision.
 Recommendation… Cont
• The thimble should be
removable or designed
to allow proper
certification of the BSC
(e.g., isolation damper
to seal off the cabinet
for decontamination,
access port to allow
scan testing of the HEPA
filter).
 Recommendation… Cont
• Hard-ducted BSCs should have
exhaust blowers located at the
terminal end of the ductwork.
• Exhaust flow failure(s) should signal
an alarm to the user and activate an
interlock system to prevent the
cabinet blower from operating
whenever the exhaust flow is
insufficient (e.g., flow/electrical
control) to prevent pressurization of
the cabinet.
• Backdraft protection (i.e., damper) in
the ductwork may be necessary to
prevent reversal of airflow through
the HEPA filter in the cabinet.
 Recommendation… Cont
• Supporting BSCs with
emergency power will
help to maintain
containment during
emergency situations.
 Proper Use
• Standard operating procedures (SOPs) to be
followed by facility personnel is strongly
recommended to encourage the proper and
consistent use of a BSC by personnel to
prevent exposures and the release of
pathogens and toxins.
 Start-Up Considerations
• Check that the sash is at
the appropriate height.
Adjust stool height so
that the user’s
underarms are level
with the bottom of the
sash.
 Start-Up Considerations
• Check the pressure
gauges to verify that
readings are within the
acceptable range.
 Start-Up Considerations
• If present, test the airflow alarm and ensure it is
switched to the "on" position.
 Start-Up Considerations
• Confirm inward airflow
by holding a tissue at
the middle of the edge
of the sash to establish
that it is drawn in.
 Start-Up Considerations
• Disinfect the interior
surfaces with a
disinfectant effective
against the infectious
material and toxins used
in the laboratory, allowing
an appropriate contact
time.
• If a corrosive disinfectant
is used, the surface
should be rinsed with
water after disinfection.
 Start-Up Considerations
• Assemble all materials
required for manipulation
and load into the BSC.
• Care should be taken not
to overcrowd or block the
front or rear grilles to
prevent the appropriate
airflow patterns from
being compromised.
 Start-Up Considerations
• When there is
significant potential for
splatter or splashes to
occur during
manipulations of
infectious material or
toxins, the work area
should be lined with a
plastic-backed
absorbent pad.
 Start-Up Considerations
• Place aerosol
generating
equipment (e.g.,
vortex mixer,
sonicator)
towards the back
of the BSC,
without blocking
the rear grille.
 Start-Up Considerations
• After loading material in
the BSC, allow sufficient
time for the air to purge
and the airflow to
stabilize before
initiating work.
• This will be specified in
the manufacturer's
instructions, and is
generally 3-5 minutes.
 Working in the BSC
• Perform operations as
far to the rear of the
work area as
reasonable.
• Ensure that elbows and
arms do not rest on the
grille or work surface.
 Working in the BSC
• Avoid excessive movement
of hands and arms through
the front opening. Such
movements disrupt the air
curtain at the front of the
BSC, which can allow
contaminants to enter or
escape the BSC.
• Arms should enter and exit
the BSC slowly and
perpendicular to the front
opening.
 Working in the BSC
• Keep a bottle of an
appropriate disinfectant
in the BSC while work is
performed to avoid
having to move hands
outside of the BSC.
 Working in the BSC
• Segregate non-
contaminated ("clean")
items from
contaminated ("dirty")
items. Work should
always flow from
"clean" to "dirty" areas.
 Working in the BSC
• Material should be
discarded in a waste
container located
towards the rear of the
cabinet workspace. Do
not discard materials in
containers outside of
the cabinet.
 Working in the BSC
• Decontaminate the
surface of all objects in
the BSC in the event of a
spill.
• The work area, including
the inside surface of the
window, should be
decontaminated while
the BSC remains in
operation.
 Working in the BSC
• Natural gas and propane should
not be used in a BSC; sustained
open flames (e.g., Bunsen burner)
in BSCs are prohibited. On-
demand open flames (e.g., touch-
plate microburners) are to be
avoided as they create turbulence
in the BSC, disrupt airflow
patterns, and can damage the
HEPA filter (CBS Matrix 4.6).
• Non-flame alternatives (e.g.,
microincinerator, or sterile
disposable inoculation loops)
should be used whenever
possible.
 Working in the BSC
• Equipment creating air
movement (e.g.,
vacuum pumps,
centrifuges) may affect
the integrity of the
airflow and should not
be used within the BSC.
 Working in the BSC
• Windows that open
should be kept closed
when the BSC is in use.
 Completion of Work in the BSC
• Upon completion of work,
allow sufficient time for
the air in the BSC to purge
(i.e., pass through the
filter) before disrupting
the air curtain by
removing hands or
unloading material from
the BSC.
• The purge time will vary
by model and can be up
to several minutes.
 Completion of Work in the BSC
• Close or cover all
containers.
 Completion of Work in the BSC
• Surface decontaminate
items before removing
them from the BSC.
 Completion of Work in the BSC
• Disinfect the interior surfaces
of the BSC, including sides,
back, lights, and interior of the
glass, with a disinfectant
effective against the
pathogens in use, allowing an
appropriate contact time.
• If a corrosive disinfectant is
used, the surface should be
rinsed with water after
disinfection to avoid corrosion
of the stainless steel surfaces.
 Completion of Work in the BSC
• Routinely remove the
work surface and
disinfect the tray
beneath it.
 Completion of Work in the BSC
• Routinely wipe the
surface of the lights
within the BSC with a
suitable cleaner or
disinfectant (e.g.,
ethanol).
 UV Lamps
• Germicidal UV lamps
are not substitutes for
proper cleaning of BSC
workzone
• May cause
performance
degradation
• May compromise
personnel safety when
proper precautions are
not taken
 Annual Testing
• Recommended
recertification for BSC’s:
– Upon installation at
customer’s site
– On-site annually in
accordance with
international standards
– When the cabinet is
moved
 Field Production Production Type Type
No Test
EN & NSF Per EN Per NSF Per EN Per NSF
1 Inflow velocity √ √ √ √ √
2 Downflow velocity √ √ √ √ √
3 HEPA / ULPA filter leak test √ √ √ √ √
4 Smoke pattern / airflow visualization √ √ √ √ √
5 Site installation assessment (ex: alarm) √ √ √ √ √
6 Light intensity √ √ √ √
7 Noise level √ √ √ √
8 Vibration level √ √ √ √
9 Electrical safety testing to IEC 61010 √ √ √ √
10 Pressure retention / soap bubble √ √ √
11 Microbiological personnel protection √ √
12 Microbiological product protection √ √
13 Microbiological cross-contamination √ √
14 Microbiological performance envelope √
15 Motor / blower performance √
16 Drain spillage trough leakage √
17 Resistance to overturning √
18 Resistance to distortion √
19 Resistance to deflection √
20 Resistance to tipping √
21 Secondary inflow velocity correlation to DIM √
22 Powder coating chemical resistance √
23 Powder coating abrasive resistance √
24 Cabinet design evaluation √
 Cleaning and disinfection:
• Check electrical requirement before use.
• Turn UV lamp at least 30 minutes before use. Be sure
to turn it off during the operation.
• For worktable and sides, use sterile or non-use cloths,
which do not shed particles or fibers.
• To disinfect, use cloths damped with a disinfecting
solution which do not damage or affect the paintwork,
stainless steel or glass.
• A pre-cleanine to disinfecting can also be done with
water and soap.
 1. HEPA filter leak test (LT)
• Purpose:
– This test is conducted to check the HEPA
impulsion/downflow and exhaust filters, the
filter housing, and the mounting frames for
possible leakage.
• Tools:
– Aerosol generator (cool or hot) (DEHS, DOB or
PAO are recommended)
– Aerosol Photometer with extended
logarithmic scale
– Accuracy: particle counter for individual
particles with a dilution range capable of
detecting a penetration of 0.01% or less of
particle exceeding 0.3 µm (HEPA filter of
purity class 100).
• Test conditions
– The sample chamber of the safety cabinet
must be completely empty, widows and doors
of the operating room must be closed (no
draft).
• Methodology:
1. Turn on the photometer and adjust it
for the measurement are in
accordance to the manufacture’s
instruction
2. Introduce aerosol to the airflow
before the filter (entrance of the
BSC)
3. With the probe nozzle approximately
at 2.5 cm from the surface, scan the
downstream side of HEPA filter,
including the perimeter of the filter
passing the photometer probe over
the entire surface.
4. Scan the entire periphery of the filter
and junction between the filter and
the frame a speed not exceed 5cm/s.
• Acceptation criteria:
– The leak (impulsion/downflow or
exhaustion) should be not more than
0.01% of the specific before the filter
(escaping volume of the aerosol).
• Trouble shooting: replace downflow
filter/ replace exhaust filter
 2. Inflow velocity test (IV)
• Purpose: This test is conducted to determine the nominal value of
the inflow velocity (air entered to the BSC)
• Value to be tested: inflow velocity V1 (m/s or ft/min)
• Reference: ISO/IEC 17025:2005 NSF 49 (Annex F) and EU EN 12469
• Test method:
– The inflow velocity (V1) is calculated using the values of the exhaust
volume and of the sample chamber inflow surface.
– If the BSC is connected to technical ventilation, inflow velocity can also
be determined at the work aperture.
• Test Conditions: For all test methods, the sample chamber of the
safety cabinet must be completely empty, windows and doors of
the operating room must be closed (no draft).
• Testing equipment:
– Suitable anemometer measure
the velocity (m/s or ft/min)
• Accuracy of testing equipment
– Accuracy of ± 0.01 m/s or
maximal deviation 3% from the
indicated airflow velocity.
• Calibration of test equipment
– The equipment must be
calibrated in an accredited
laboratory or national
metrological institute in
accordance to the requirement
of ISO/IEC 17025:2005.
• Methodology
– The inflow velocity is measured at several measuring points in the work
aperture. For this purpose, the front window is moved up to height A=7.3 cm.
– The anemometer is installed directly to the inside of the work aperture front
window.
– The measuring points are located at distance C of 3.8 cm immediately at the
inside of the front window lower edge and at distance B of 10 cm to each
other. The lateral distance to the work aperture edges is also 10 cm.
• Methodology
– Install the anemometer to the
following grid point.
– Move the front window to
the measuring position and
operate the air system
blowers at full speed for
approx. 20 min.
– Perform measurement at all
nine measuring points above
the exhaust aperture for a
minimum of one minute for
each measuring point.
• Calculations
– The exhaust velocity can be tabulated in the following table to
calculate the average exhaust velocity (V2)
• Acceptation Criteria
– The nominal value is 0.45 m/s. The average value must
be within ±10% of the nominal value.
– Exhaust airflow volume = 580m3/h
• Troubleshooting
– Raise exhaust bower speed
– Check exhaust blower control voltage
– Replace exhaust filter
– Check installed exhaust system for correct function.
 3. Downflow velocity test
• Purpose of the test
– This test is conducted to check the
nominal value of the downflow velocity
(displacement airflow) in the work area
of the safety cabinet.
• Value to be tested: Downflow
velocity (m/s or ft/min)
• Reference: ISO/IEC 17025:2005 NSF
49 (Annex F) and EU EN 12469
• Test Conditions:
– For all test methods, the sample
chamber of the safety cabinet must be
completely empty, windows and doors
of the operating room must be closed
(no draft).
• Testing equipment:
– Suitable anemometer measure the
velocity (m/s or ft/min)
– The anemometer measuring head must
be secured tightly at the measuring
point to allow accurate measurements.
– The distance between the measuring
points must be correct; the grid must
not be distorted.
• Accuracy of testing equipment
– Accuracy of ± 0.01 m/s or maximal
deviation 3% from the indicated airflow
velocity.
• Calibration of test equipment
– The equipment must be calibrated in an
accredited laboratory or national
metrological institute in accordance to
the requirement of ISO/IEC 17025:2005.
• Methodology:
– Move the door to the
working position.
– Install the measuring head
at 10 cm above the door
edge
– For BSC of 1200 mm, leave
1/8x120 from all sides.
Draw the grid and define
the measuring point as the
following grid.
• Calculating the average value for the
downflow velocity
Average= Sum of measured values/number
of measuring points
• Accepting criteria
– The average value must be within ±10%
of the nominal value (0.36 m/s).
– The difference between measured
values at the individual measuring
points must not exceed 20%.
• Troubleshooting
– Raise downflow blower speed
– Check exhaust blower control
– Replace downflow and or exhaust filters
– Check installed exhaust system for
correct function.
 4. Airflow pattern test
• Purpose of the test- This test is conducted to check the behavior of the
airflows in the sample chamber. Check to see if the displacement airflow
passes along the entire work area, if the vertical passage is correct, and of
air escapes through joints or seals of the housing and of the front window.
• Reference: ISO/IEC 17025:2005 NSF 49 (Annex F) and EU EN 12469
• Test Conditions:
– For all test methods, the sample chamber of the safety cabinet must be
completely empty, windows and doors of the operating room must be closed
(no draft).
– Inflow and downflow should be adjusted to the nominal accepted values.
• Testing equipment:
– Smoke tube or any safe smoke producer.
• Methodology:
– Move the door to the working
position.
– Using a smoke tube, scan the work
area along its centerline from one
end to the other. Hold the smoke
tube so that its tip is at a distance
of approximately =15cm above the
work surface.
• Accepting criteria
– The smoke shows smooth
downward flow with no dead spots
or reflux. No smoke escapes
through the work aperture.
 Site Installation Assessment Test
• Primary
– Alarm Functions
– Blower interlock, Type B1/B2
– Exhaust System Performance
– Canopy connection. Type A2
– Room Influences
• Secondary
– UV
– Light intensity
– Vibration
– Noise
 Summary
• BSCs provide effective primary containment
for work with infectious material or toxins
• This is achieved when the BSCs are:
– Properly installed
– Properly certified or validated and maintained
– Proper training of end-user
– Used in conjunction with good laboratory
techniques