Laboratory Biosafety Manual 4ed - Biological Safety Cabinets

LABORATORY BIOSAFETY MANUAL i
FOURTH EDITION
AND
ASSOCIATED MONOGRAPHS
BIOLOGICAL SAFETY CABINETS

AND OTHER PRIMARY
CONTAINMENT DEVICES
ii BIOLOGICAL SAFETY CABINETS AND OTHER PRIMARY CONTAINMENT DEVICES
LABORATORY BIOSAFETY MANUAL
FOURTH EDITION
AND
ASSOCIATED MONOGRAPHS
BIOLOGICAL SAFETY CABINETS

AND OTHER PRIMARY
CONTAINMENT DEVICES
Biological safety cabinets and other primary containment devices
(Laboratory biosafety manual, fourth edition and associated monographs)
ISBN 978-92-4-001133-5 (electronic version)
ISBN 978-92-4-001134-2 (print version)
© World Health Organization 2020

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Design and layout by Paul Bloxham
iii
Contents
Acknowledgements iv
Glossary of terms vi
Executive summary ix
SECTION 1 Introduction to primary containment devices 1
SECTION 2 Working with primary containment systems 5
2.1 Best practice for working with open-fronted devices5
2.2 Working with enclosed devices: additional considerations6
2.3 Decontamination of safety cabinets and isolators7
SECTION 3 Directional airflow9
3.1 High efficiency particulate air filtration10
3.2 Direct recirculation11
3.3 Hard ducting11
3.4 Anti-blowback valves12
3.5 Thimble ducts13
SECTION 4 Selecting a primary containment device15
4.1 Class I BSCs17
4.2 Class II BSCs19
4.3 Class III BSCs24
4.4 Containment isolators27
4.5 Other local exhaust ventilation types29
References31
Further information34
iv BIOLOGICAL SAFETY CABINETS AND OTHER PRIMARY CONTAINMENT DEVICES
Acknowledgements
Principal coordinator
Dr Kazunobu Kojima, World Health Organization, Switzerland
Scientific contributor
Mr Allan Bennett (Deputy team lead), Public Health England (WHO Collaborating
Centre for Applied Biosafety and Training), United Kingdom of Great Britain and
Northern Ireland
Prof. Stuart Blacksell (Team lead), University of Oxford/Mahidol-Oxford Tropical

Medicine Research Unit, Thailand
Mr David Bressler, Centers for Disease Control and Prevention (WHO Collaborating
Centre for Biosafety and Biosecurity), United States of America
Dr Paul Jensen, Centers for Disease Control and Prevention (WHO Collaborating
Centre for Biosafety and Biosecurity), United States of America
Mr Simon Parks, Public Health England (WHO Collaborating Centre for Applied
Biosafety and Training), United Kingdom of Great Britain and Northern Ireland
Mr John Saunders, Health and Safety Executive, United Kingdom of Great Britain and
Northern Ireland
Mr Joe Tanelli, Public Health Agency of Canada (WHO Collaborating Centre for
Biosafety and Biosecurity), Canada
Project management
Ms Lisa Stevens, World Health Organization, France
Ms Rica Zinsky, World Health Organization, Switzerland
Reviewer
Ms Maren Roush, National Sanitary Foundation, United States of America

ACKNOWLEDGEMENTS v
Technical editing
Ms Fiona Curlet
Financial support
Development and publication of this document have been made possible with
financial support from the Global Partnership Program, Global Affairs Canada, the
Biosecurity Engagement Program, United States Department of State and the Defense
Threat Reduction Agency, US Department of Defense.
vi BIOLOGICAL SAFETY CABINETS AND OTHER PRIMARY CONTAINMENT DEVICES
Glossary of terms
Aerosol: Liquid or solid particles suspended in air and of a size that may allow
inhalation into the lower respiratory tract (usually less than 10 micrometres in
diameter).
Aerosol-generating procedure: Any procedure that intentionally or inadvertently

results in the creation of liquid or solid particles, which become suspended in the air
(aerosols).
Biological agent: A microorganism, biological toxin, protein (prions) or human

endoparasite, either naturally occurring or genetically modified, which may have the
potential to cause infection, allergy, toxicity or otherwise create a hazard to human
health, animals or plants.
Biosafety: Containment principles, technologies and practices that are implemented

to prevent unintentional exposure to biological agents or their inadvertent release.
Calibration: Establishment of the relationship between the measurement provided by

the instrument and the corresponding values of a known standard, allowing correction
to improve accuracy. For example, laboratory equipment such as pipetting devices
may need calibration periodically to ensure proper performance.
Certification: A third-party testimony based on a structured assessment and formal

documentation confirming that a system, person or piece of equipment conforms to
specified requirements, for example, to a certain standard.
Clean: Visually free of soil and below specified levels of analytes.
Consequence (of a laboratory incident): The outcome of an incident (exposure to and/

or release of a biological agent) of varying severity of harm, occurring in the course of
laboratory operations. Consequences may include a laboratory-associated infection,
other illness or physical injury, environmental contamination, or asymptomatic carriage
of a biological agent.
Containment: The combination of physical design parameters and operational

practices that protect personnel, the immediate work environment and the community
from exposure to biological agents. The term "biocontainment" is also used in this
context.
Contamination: The introduction of undesired biological agents into tissues and

specimens or onto surfaces.
Decontamination: Reduction of viable biological agents or other hazardous materials

on a surface or object(s) to a pre-defined level by chemical and/or physical means.
GLOSSARY OF TERMS vii
Disinfectants: Agents capable of eliminating viable biological agents on surfaces or in

liquid waste. These will have varying effectiveness depending on the properties of the
chemical, its concentration, shelf life and contact time with the agent.
Exposure: An event during which an individual comes in contact with, or is in close

proximity to, biological agents with the potential for infection to occur. Routes of
exposure can include inhalation, ingestion, percutaneous injury and absorption and
are usually dependent upon the characteristics of the biological agent. However, some
infection routes are specific to the laboratory environment and are not commonly seen
in the general community.
Fumigation: Use of a poisonous gas or vapour to remove contamination of a biological

agent from a surface, piece of equipment or area.
Good microbiological practice and procedure (GMPP): A basic laboratory code of

practice applicable to all types of laboratory activities with biological agents, including
general behaviours and aseptic techniques that should always be observed in the
laboratory. This code serves to protect laboratory personnel and the community from
infection, prevent contamination of the environment, and provide protection for the
work materials in use.
Hazard: An object or situation that has the potential to cause adverse effects when
an organism, system or (sub)population is exposed to it. In the case of laboratory
biosafety, the hazard is defined as biological agents which have the potential to cause
adverse effects to personnel and/or humans, animals, and the wider community and
environment. A hazard does not become a “risk” until the likelihood and consequences
of that hazard causing harm are taken into account.
Laboratory-associated infection: Any infection acquired or reasonably assumed as a

result of exposure to a biological agent in the course of laboratory-related activities. A
person-to-person transmission following the incident may result in linked secondary
cases. Laboratory-associated infections are also known as laboratory-acquired
infections.
Maximum containment measures: A set of highly detailed and stringent risk control
measures described in the fourth edition of the WHO Laboratory biosafety manual that
are considered necessary during laboratory work where a risk assessment indicates
that the activities to be performed pose very high risks to laboratory personnel, the
wider community and/or the environment, and therefore an extremely high level of
protection must be provided. These are especially needed for certain types of work
with biological agents that may have catastrophic consequences if an exposure or
release were to occur.
Pathogen: A biological agent capable of causing disease in humans, animals or

plants.
viii BIOLOGICAL SAFETY CABINETS AND OTHER PRIMARY CONTAINMENT DEVICES
Primary containment device: A contained workspace designed to provide protection

to its operator, the laboratory environment and/or the work materials for activities
where there is an aerosol hazard. Protection is achieved by segregation of the work
from the main area of the laboratory and/or through the use of controlled, directional
airflow mechanisms. Primary containment devices include biological safety cabinets
(BSCs), isolators, local exhaust ventilators and ventilated working spaces.
Risk: A combination of the likelihood of an incident and the severity of the harm
(consequences) if that incident were to occur.
Risk assessment: A systematic process of gathering information and evaluating the

likelihood and consequences of exposure to or release of workplace hazard(s) and
determining the appropriate risk control measures to reduce the risk to an acceptable
risk.
Sharps: Any device or object that is a puncture or wound hazard because of its
pointed ends or edges. In the laboratory, sharps can include needles, syringes with
attached needles, blades, scalpels or broken glass.
Sterile: The state of having a complete absence of viable biological agents and spores.
Sterilization: A process that kills and/or removes all biological agents including spores.
Validation: Systematic and documented confirmation that the specified requirements

are adequate to ensure the intended outcome or results. For example, in order
to prove a material is decontaminated, laboratory personnel must validate the
robustness of the decontamination method by measurement of the remaining
biological agents against the detection limit by chemical, physical or biological
indicators.
ix
Executive summary
Biological safety cabinets (BSCs), isolators and local exhaust ventilators are enclosed,
ventilated working spaces that can be used in laboratories as primary containment
devices. These devices protect the operator, the laboratory environment and/or
the work materials from exposure to infectious aerosols and splashes that may be
generated when manipulating materials containing biological agents. Infectious
aerosol particles may be created during a laboratory activity with liquid, semi-liquid
and/or dried material, particularly if the material contains high concentrations of
biological agents. Laboratory activities, such as streaking agar plates, pipetting
liquid suspensions of infectious agents and homogenizing infectious materials, can
generate infectious aerosols if done by personnel who have not been trained in
good microbiology practices. Primary containment devices, when properly used and
maintained, have been shown to be highly effective in reducing laboratory-associated
infections in practice. This monograph provides information on BSCs and other
primary containment devices, such as isolators and local exhaust ventilation devices, in
order to guide the appropriate selection and use of such devices for individual needs
to help ensure laboratory biosafety. The targeted readership for this monograph
is laboratory personnel working with BSCs or other primary containment devices,
laboratory personnel doing the risk assessment and people involved in planning or
renovating a laboratory, such as the senior management or the laboratory manager.
The information in this monograph on biological safety cabinets and other primary
containment devices is designed to accompany and support the fourth edition
of the WHO Laboratory biosafety manual (core document) and other associated
monographs. The manual and the monographs adopt a risk- and evidence-based
approach to biosafety rather than a prescriptive approach in order to ensure
that laboratory facilities, safety equipment and work practices are locally relevant,
proportionate to needs and sustainable. Emphasis is placed on the importance of a
“safety culture” that incorporates risk assessment, good microbiological practice and
procedure and standard operating procedures, relevant introductory, refresher and
mentoring training of personnel, and prompt reporting of incidents and accidents
followed by appropriate investigation and corrective actions. This new approach
aims to facilitate laboratory design and ways of operating that ensures greater
sustainability while maintaining adequate and appropriate control of biosafety.
The other associated monographs provide detailed information and help implement
systems and strategies on the following specialized topics: risk assessment, laboratory
design and maintenance, personal protective equipment, decontamination and waste
management, biosafety programme management and outbreak preparedness and
resilience.
This monograph describes the different types of BSC and other primary containment
devices and best practice for working with these devices. The technical features of
primary containment devices, such as directional airflow, are explained and methods
for their decontamination are discussed.
x BIOLOGICAL SAFETY CABINETS AND OTHER PRIMARY CONTAINMENT DEVICES
1
INTRODUCTION TO
SECTION
1 PRIMARY CONTAINMENT
DEVICES
Biological safety cabinets (BSCs), isolators and local exhaust ventilators are enclosed,
ventilated working spaces that can be used in laboratories as primary containment
devices. These devices protect the operator, the laboratory environment and/or
the work materials from exposure to infectious aerosols and splashes that may be
generated when manipulating materials containing biological agents. Infectious
aerosol particles may be created during a laboratory activity that imparts energy to
liquid, semi-liquid and/or dried material, particularly if the material contains high
concentrations of biological agents (1–3). Laboratory activities, such as streaking
agar plates, pipetting liquid suspensions of infectious agents and homogenizing
infectious materials, can generate infectious aerosols if done by personnel who have
not been trained in good microbiology practice (3). Processes such as centrifugation
of infectious liquids can also generate infectious aerosols, but these activities are
now normally undertaken in intrinsically contained devices (for example, sealed
centrifuges).
The information in this monograph on biological safety cabinets and other primary
containment devices is designed to accompany and support the fourth edition of
the WHO Laboratory biosafety manual (4) (core document) and other associated
monographs. The manual and the monographs adopt a risk- and evidence-based
approach to biosafety rather than a prescriptive approach in order to ensure
that laboratory facilities, safety equipment and work practices are locally relevant,
proportionate to needs and sustainable.
The other associated monographs provide detailed information and help implement
systems and strategies on the following specialized topics: risk assessment
(5), laboratory design and maintenance (6), personal protective equipment (7),
decontamination and waste management (8), biosafety programme management (9)
and outbreak preparedness and resilience (10).
Primary containment devices, when properly used and maintained, have been shown
to be highly effective in reducing laboratory-associated infections in practice (11). They
protect operators from exposure to infectious aerosols in two main ways. Firstly, they
provide a contained workspace so that activities with a higher risk of generating
aerosols can be segregated from the main area of the laboratory. Secondly, and
perhaps most importantly, these devices use various methods to pass a controlled,
directional airflow across the workspace which draws any aerosols generated into its
path and away from the work area.
2 BIOLOGICAL SAFETY CABINETS AND OTHER PRIMARY CONTAINMENT DEVICES
This directional airflow is created using specialized fans attached to or housed within
the primary containment device. Particles that enter and move with the directional
airflow can then be directed into a filter before being exhausted from the device and/
or the laboratory.
There are various types of primary containment device, each of which uses different
mechanisms to introduce an airflow into the device, treat the circulating air and
exhaust it from the device and/or the laboratory. BSCs are the most commonly
used primary containment devices, and three different classes of BSC exist. These
cabinets differ by the type and level of protection their directional airflow provides for
laboratory personnel (device operators), the environment and/or the work materials
inside and whether external fans and ductwork are required for proper operation of
the primary containment device.
The three classes of BSC are:
n Class I BSCs – open-fronted enclosures that draw an inward airflow across the work
surface through the front opening. The air passes upwards through a high efficiency
particulate air (HEPA) filter before being exhausted. They provide personnel and
environmental protection, but do not offer product protection for materials located in
the work area.
n Class II BSCs – open-fronted enclosures, similar to Class I BSC, in which air enters
the cabinet through the front opening to provide operator protection. The inward
air bypasses the work area by being pulled through the front grille and underneath
the workspace and then travels through a plenum (an enclosed air space) to the
BSC blower, where it is either exhausted through a HEPA filter or directed into the
work area after passing through a separate downflow (supply) HEPA filter. Some
of the now-clean air passes over the work surface as a downward flow of air (that
is recirculation) in order to protect work materials from contaminated room air
(“product protection”) and is then recirculated through front and rear vents. The
surplus air in the plenum is not recirculated but passes through a separate HEPA
filter and is exhausted. The National Sanitary Foundation (NSF) standard defines five
types of Class II cabinets: A1, A2, B1, B2 and C1 (12) (More information on Class II BSC
types is given in subsection 4.2).
n Class III BSCs – a closed, sealed, negative-pressure enclosure to which HEPA-filtered

air is supplied; this air then passes through another HEPA filter on extraction. The
enclosure is sealed to ensure safe gaseous decontamination. Operators access
the working area using integrated gloves or gauntlets. Class III BSCs are normally
fitted with a pass box (often independently ventilated) or dunk tank to facilitate the
movement of potentially contaminated work materials in and out of the cabinet. An
autoclave may also be attached for waste management, mainly in facilities with
a maximum containment cabinet line. A sealable and detachable front window
may be fitted to allow the occasional movement of large equipment into and out
of the cabinet, after fumigation. These devices offer the highest degree of operator
protection as the user is physically separated from the device interior.
SECTION 1 INTRODUCTION TO PRIMARY CONTAINMENT DEVICES 3
Several other types of equipment may be used as primary containment devices, and
provide similar levels of primary containment as BSCs. Although there are many
similarities between these devices and BSCs, some of their features do not comply with
international standards for the construction and operation of BSCs. (It should be noted
that horizontal and vertical outflow cabinets (“clean-air work stations”) are not primary
containment systems and should not be used as such). There are two additional
primary containment devices.
n Isolators – closed boxes, similar to Class III safety cabinets but which do not
comply with any international standards for testing and certification. These may be
manufactured from flexible or rigid materials and can be constructed in a range of
sizes. The work surface is normally accessed by operators using integrated gloves or
half suits and they may have pass boxes or dunk tanks to allow inflow and outflow
of materials. Isolators may also be used to house robotic equipment and, in some
cases, animals. The fact that isolators can be designed to incorporate any process
gives them advantages over standard cabinets.
n Other local exhaust ventilation devices – partially open-fronted enclosures which

perform in a similar way to Class I cabinets. They may give a similar degree of
protection but do not always have terminal HEPA filtration. Local exhaust ventilation
devices are primarily used to protect users undertaking activities with a low
likelihood of producing infectious aerosols. They use only directional airflow and are
sometimes known as a ventilated work station (13). Local exhaust ventilation devices
may meet some, but not all, of the requirements outlined in international standards.
The design, construction and testing of BSCs are governed by national and
international standards, for example those in Australia, China, the European Union,
Japan and the United States of America (12,14–17). In recent years, designs of primary
containment systems that do not comply with such standards have come into use
for several reasons; these include cost, portability and the requirement for bespoke
designs – for example to accommodate large equipment or infected animals, or to
allow rapid deployment to the field and avoid the complex installation requirements of
some BSCs. For this reason, isolators were widely used in diagnostic laboratories in the
response to the West African Ebola outbreak that started in 2013 (18).
This monograph describes the usual practices and procedures for the use of primary
containment devices, including more detailed explanations of the features of each
device type, how they can be most effectively used, and the various testing and
calibration requirements that ensure correct performance. It must be remembered
that these devices can only enhance the safety of personnel if used in conjunction with
the good microbiological practice and procedure (GMPP) described in the Laboratory
biosafety manual (4), section 3 core requirements.
5
WORKING WITH PRIMARY

SECTION
2 CONTAINMENT SYSTEMS
2.1 Best practice for working with open-fronted devices

Open-fronted primary containment systems are Class I and Class II BSCs, and local
exhaust ventilation devices.
n The use and limitations of the device should be explained to all potential operators,
with reference to the relevant standards and literature. Written protocols, or safety
operations manuals, should be issued which should cover movement of material
into and out of the cabinet. Appropriate training in GMPP must also be provided. At
the same time, it should be recognized that primary containment devices may not
protect the operator from exposure caused by poor technique or ignoring protocols
and procedures.
n The device must not be used unless it is working properly. Alarms and indicators that
show the device is operating safely should be checked before each use. Sashes and
sliding front windows on class II BSC, should remain at the correct height when the
cabinet is in use. Glass viewing panels, installed over the front opening, must not be
opened when the device is in use.
n Disruption to the airflow must be avoided by ensuring that minimal equipment and
materials are kept inside the device, and that the air-intake grill at the front (and the
rear) is clear of obstructions (for example, pipettes).
n Bunsen burners or alcohol lamps must not be used inside the device as the heat
produced may distort the airflow and/or damage filters. An electric microincinerator
(bacti-cinerator or similar) is permissible, although disposable sterile transfer loops
are preferred.
n Ultraviolet lights are not recommended as the only method of sterilizing the device. If
they are used, they must be cleaned weekly to remove dust or dirt that may reduce
the germicidal effectiveness of the light. Ultraviolet light intensity should be checked
during device recertification to ensure that light emission is appropriate.
n Work should be carried out in the middle or rear part of the work surface to reduce
airflow obstruction and improve visibility through the glass viewing panel for the
operator.
n Human traffic behind the operator should be kept to a minimum.
n Cabinets should not be sited close to any potentially interfering air movement from
air conditioners or opening and closing doors.
n Operators should not disturb the airflow by repeated removal and reintroduction of
their arms.
n The surface of the biological safety device should be wiped using an appropriate
disinfectant after work is completed and at the end of the day.
n Device fan(s) should be run for some time before beginning work and after
completion of work in the device, according to the manufacturer’s instructions or until
the device indicators show that it is safe to use.
n Paperwork should never be placed inside primary containment devices as it may be

entrained in the airflow and partially block the extract duct, disrupting the airflow
system.
n An uninterruptible power supply should be fitted, in line with the mains electrical
supply, so that there is no interruption to the electrical supply of the device in the
event of an electrical outage. This may also act as an electrical line conditioner to
stabilize the electrical supply if there are issues with voltage stability. An interruptible
power supply cannot be used in devices with an external exhaust fan or blower unit,
such as a Class II B2 device.
2.2 Working with enclosed devices: additional considerations

Enclosed primary containment systems include Class III BSCs and isolators. Users of
these fully contained systems should also consider the following areas, as applicable.
n The condition of gloves or half suits should be checked for damage before and after
use. If damage is found, then gloves/suits should be replaced using safe change
procedures, or specified repair should be undertaken.
n As far as reasonably practical, the use of sharps, scissors or other equipment that
may cut or damage integrated gloves or half suits should be avoided.
n Protocols for safe introduction and removal of materials must be in place and be
complied with.
SECTION 2 WORKING WITH PRIMARY CONTAINMENT SYSTEMS 7
2.3 Decontamination of safety cabinets and isolators
2.3.1 Liquid decontamination
The surface of all items within primary containment devices, including equipment,
should be decontaminated before being removed. The interior surfaces of the device
should be decontaminated before and after each use. The work surfaces and interior
walls should be wiped with a disinfectant validated to inactivate any microorganisms
that might be found inside the device. When a corrosive disinfectant such as bleach
is used, surfaces should then be wiped with sterile water or a 70% alcohol solution
to remove residues that may cause degradation. It should be checked that any
disinfectant is compatible with the construction materials of the containment system.
This is particularly important for isolator systems, which may have a wider range of
materials used in their construction.
2.3.2 Gaseous/vapour decontamination
Based on the risk assessment, primary containment devices may be required to

undergo gaseous/vapour decontamination before being physically moved, before
filter changes, between work activities with different agents, and to allow potentially
contaminated equipment to be removed. Decontamination should be carried out
by a trained individual using a validated method. During decontamination, open-
fronted safety cabinets should have doors fitted to seal the opening, and monitors
should be used to detect any leakage. Alternatively, cabinets can be decontaminated
in sealed bag systems. It should be noted that national regulations may also
stipulate when gaseous/vapour decontamination is required. More information on
gaseous decontamination can be found in Monograph: decontamination and waste
management (8).
9
DIRECTIONAL AIRFLOW
SECTION
3
As previously mentioned, primary containment devices act as risk control measures
by providing a segregated workspace within which to perform higher-risk activities.
Much of their protection is provided by the integration of fan mechanisms to create
directional airflow. This controlled movement of air allows contaminated/potentially
contaminated aerosols to be captured within filters, thus preventing exposure of
laboratory personnel, the surrounding environment and/or the work materials within
the device to such aerosols.
A directional airflow is created using one (or more) specialized fans connected
to, or housed within, the primary containment device which force the air to move
in one direction. Laboratory air entering the device from the outside may become
contaminated by aerosols generated in the workspace as a result of specimen
manipulation and processing. The fans pull this air away from the workspace and
direct it, in most cases, through a special filter able to capture and hold biological
agents until such time as the filter itself can be safely decontaminated. After passing
through the filter, the remaining “clean” air may either be:
n reused: by recirculating it within the primary containment device, or by sending it into

the laboratory, or
n exhausted: by directing the filtered airflow out of the primary containment device, via
an exhaust duct, to an external location (usually the outside environment).
The fan is the part of the system that generates air movement. It does this by generating
a pressure gradient. Between the containment device and the fan the pressure is
negative with respect to the surrounding space. From the fan to the discharge point, the
air pressure is positive with respect to the surrounding space. The pressure differences
would be rapidly lost in the event of an electrical or equipment failure, and, without
mechanical intervention, the air pressure would naturally equilibrate and control of
the directional airflow would be lost. For this reason, primary containment devices
should be fitted with airflow monitoring systems and alarms indicating safe and
unsafe conditions, so that corrective action may be taken in the event of a mechanism
failure. These could include systems that indicate airflow velocity or volumetric flow
(anemometer) and, for full enclosures, the negative pressure (manometer) of the air in
the primary containment device.
There are numerous configurations of fans, filters and exhaust mechanisms and the
correct configuration is essential. The following subsections outlines some of the key
features of directional airflow mechanisms and air exhaust configurations. These
must be understood and correctly used to ensure the safe and effective operation of
primary containment devices.
3.1 High efficiency particulate air filtration

HEPA filters were originally designed to filter airborne radioactive particles but are
now used in a wide variety of applications such as vacuum cleaners, motor vehicles,
aerospace, clean rooms including high technology and pharmaceutical clean rooms,
hospitals and laboratory facilities. HEPA filters are composed of many randomly
oriented fibres that create a fibrous matrix through which air can pass. Particles
travelling with the air may be captured by the fibres, effectively filtering the air. Various
mechanisms are used to capture and filter particulate matter including the following:
n Inertial impaction – as large particles flow through the air towards the fibres, their
size prohibits them from effectively adjusting to the altered airflow around the fibre,
causing them to impact the fibre directly.
n Interception – smaller particles flow less than one-particle diameter away from the
fibres, close enough to touch and adhere to them.
n Diffusion - collision, especially of the smallest particles being filtered, occurs with
other air/gas molecules, altering the path of motion of the particle. This diffusion
of energy between particles impedes and delays their path through the filter
and increases the probability that they will be stopped either by interception or
impaction.
Through the combination of the filtering mechanisms described above, HEPA filters
are capable of capturing small particles that pass through them, including biological
agents. There are various classes of HEPA filter, with different filtering efficiencies of
the material used. The efficiency is often determined according to the criteria outlined
in global standards (19–21). Most HEPA filters have a filtering efficiency of more than
99.97% for particles of 0.3 µm in diameter, the most penetrating particle size (22,23).
Particles with a lower or higher diameter will be removed with a higher efficiency.
It is important to note that HEPA filters are not designed to filter gases or vapours.
If protection is needed against both biological agents and gases/vapours, then a
suitable total exhaust system should be used. If this is not possible, then additional
chemical filters should be used in conjunction with the HEPA filters. Exhaust of
chemicals requires specialist installations and is beyond the scope of this monograph.
SECTION 3 DIRECTIONAL AIRFLOW 11
As with any equipment, it is important that HEPA filters are used, maintained and
replaced when necessary according to manufacturers’ instructions to ensure their
effectiveness. However, it has been shown that HEPA filters can maintain performance
for long periods of time and filter failure is comparatively rare (24).
3.2 Direct recirculation

In some cases, the air leaving the primary containment device is returned directly
to the laboratory after passing through a HEPA filter. This is commonly used as an
affordable and practical solution for many laboratories because of its simplicity and
the absence of complex ducting systems. However, this may be associated with a
higher risk of exposure to workers in the rare event of a failure of an exhaust HEPA
filter. This highlights the need for regular performance monitoring and justification
of selecting this method of exhausting based on a thorough and appropriate risk
assessment. In some circumstances, two HEPA filters may be installed in series to
mitigate the risk. It should also be noted that volatile or toxic chemicals must not be
used in containment devices that recirculate exhaust air to the laboratory.
3.3 Hard ducting

Hard ducting is an exhaust arrangement in which the primary containment device
is firmly connected, without any openings, to the general building exhaust system or
(preferably) to a dedicated laboratory exhaust duct system. The containment device
may also be directly connected to a dedicated, standalone cabinet exhaust system,
which can also be used to maintain laboratory ventilation during operation. Due to the
complex mechanisms involved in directing airflow, certain types of Class II BSCs must
always be hard-ducted, preferably to a dedicated exhaust fan and duct (NSF types
B1 and B2), whereas others may not be designed to connect to a hard-ducted system
(certain NSF A2 devices). For more information on Class II BSC types and airflows, see
subsection 4.2.
To facilitate proper functioning and continuous airflow in hard-ducted primary

containment devices, building exhaust extraction systems must be precisely matched
to the airflow requirements for both the volume and static pressure of the containment
device, as specified by the manufacturer. It should be noted that the level of protection
provided by the containment device is heavily dependent on the quality of the
maintenance of this airflow set-up and the integrity of the duct work. Some countries
may have building standards or regulations that specify the minimum requirements
for duct work. It is important to ensure that any hard ducting that contains potentially
contaminated air is properly sealed to prevent leaks, especially at the time of
installation. However, this is less important for ducts containing air that has already
passed through HEPA filtration, unless gaseous disinfection is to be used.
Certification of hard-ducted containment devices may be more time-consuming than

that with other exhaust configurations because holes may need to be drilled in the
sealed duct work to gain access to HEPA filters for integrity checks or to place devices
to determine that the airflow volume being extracted is appropriate. This must be
taken into account if considering a hard-ducted system. During maintenance of the
containment device, the condition of the associated duct work and terminal fans must
be considered.
Hard-ducted primary containment devices can dispose of the exhaust air in two ways.
n External ducting. Air leaves the containment device and passes into the hard duct
where it is exhausted straight out of the laboratory to the external environment. Note:
this exhausting cannot be done with certain Class II BSCs (NSF type A1 and A2).
n Ducting to a heating, ventilation, and air conditioning (HVAC) system. Air leaves the
containment device through the hard duct which is connected to the exhaust ducts
of a dedicated laboratory HVAC system. This system must not recirculate air to the
building. This allows the exhaust air from both systems to be combined and disposed
of from a single extraction point, which reduces the need for multiple complex
ducting systems for the laboratory.
3.4 Anti-blowback valves

In some circumstances, airflow within the duct may be disrupted because of
unexpected events or forces such as wind turbulence in the external environment. This
turbulence may cause a temporary reduction in directional airflow, or even a reversal,
allowing air to flow backwards through the duct and into the containment device.
The valve involves the introduction

of a flap system which is kept open
at normal air speeds.
Figure 3.1 Anti-blowback valve

SECTION 3 DIRECTIONAL AIRFLOW 13
In order to prevent blowback and reduce the risk of contaminated air re-entering the
laboratory, anti-blowback valves should be installed in the ventilation system. The
valve is essentially a flap system that is kept open at normal air speeds. If air speed
decreases, due to external inward velocity, the flaps will retract automatically, cover
the duct and prevent air from flowing backwards to the containment device and the
laboratory. An example of an anti-blowback valve can be seen in Figure 3.1. However,
different versions of the valves may be used depending on local laboratory conditions
(for example, severe weather) and/or the type of containment devices in use.
3.5 Thimble ducts

A thimble duct, or canopy hood, is a specialized duct fitting designed to link primary
containment devices to an exhaust system. A dedicated terminal exhaust fan, which
may be part of the room HVAC, draws the exhaust air out of the device while at the
same time drawing in room air. This can be done either through vents or gaps in a
fitting connected to the containment device, which gives it a “canopy” shape (Figure
3.2), or through a similar arrangement in the room exhaust system to which the
containment device can be connected. The inwards flow of air into the thimble will
prevent leakage through its openings. The additional extracted air through the thimble
will reduce the room pressure further, improving any pressure cascade. Furthermore,
even when primary containment devices are turned off when not in use, the exhaust
may continue to extract room air to ensure the pressure differential is maintained
and no backflow can occur. Thimble ducts also allow the safe removal of gaseous
disinfectants during ventilation of the cabinet without the need for hard ducting.
Air is drawn from the biosafety

cabinet, and from the room
through vents or gaps in this
representation of a “canopy”
type fitting.
Figure 3.2 Thimble duct/canopy hood

It is important to ensure that the building duct system has sufficient capacity for
both the inward flow of room air and the exhaust of the containment device. The
dedicated exhaust fan of the thimble duct must extract a higher volume of air than the
containment device to prevent any overflow of air into the room.
The thimble duct, or canopy hood, is designed for use with some Class I and Class II
BSCs (all NSF type A1, A2, and their European equivalents; for more information on the
types of Class II BSC, refer to subsection 4.2). However, the thimble should preferably
be removable or carefully designed to allow for proper operational testing of the
primary containment device, such as measurement of exhaust airflow rates from
the device, and/or to allow access to HEPA filters for testing, decontamination and
replacement.
15
SELECTING A PRIMARY
SECTION
4 CONTAINMENT DEVICE
Selection of a primary containment device should be based mainly on the type of
protection required (that is operator protection, environmental protection and/or prod-
uct/work material protection) and the risk that needs to be controlled. The selection
of any primary containment device should therefore be based on the outcomes of a
risk assessment to identify and control the risks posed by the procedures being per-
formed and biological agents being handled. Each primary containment device uses
a different mechanism to create and maintain directional airflow. As introduced in
section 3 (directional airflow), some devices will have specific requirements for air-
flow volumes and exhaust configurations to create the pressure differences needed
to maintain directional airflow, even in the event of device failure. Compatibility of the
primary containment device with specialized exhaust configurations may also become
a selection factor, especially where a negative pressure may need to be maintained in
the laboratory even when the primary containment device is not in operation. Table 4.1
provides a summary of the features of various primary containment devices and their
unique specifications that may affect which device is most appropriate for selection.
Table 4.1 Key considerations in the selection of a primary containment device
CLASS WORKSPACE PROTECTION AIRFLOW EXHAUST KEY

OPENING SET-UP REQUIREMENTS CONSIDERATIONS
Class l Fixed, Operator and Inward direction Exhausted to the Simple airflow
open-fronted environment airflow from the outside (remote design is resistant to
protection front opening, fan) or to the disruption
through a HEPA room through Offers a similar
filter at the top of a HEPA filter level of operator
the cabinet (integral fan) protection as BSC
Class II
No product
protection offered
Table 4.1 Key considerations in the selection of a primary containment device (continued)

Class lla Fixed, sliding or Operator, Directional Exhausted to the Addition of HEPA
hinged open- environment airflow includes room through a filtered downward
fronted and product both an inwards HEPA filter or to airflow offers product
protection flow of air from the environment protection
the front opening through an Different types of
Some types and a downward exhaust canopy Class II BSC exist
using single-pass flow of HEPA- with varying airflow
air are suitable filtered air from set-ups
for protection the top of the
Multiple complex
from chemical cabinet onto the
airflow patterns may
vapours work surface
be highly sensitive to
Laminar airflow disruption
in the working
A plenum space may
area protects
act as a secondary
against cross
safety mechanism
contamination
within the Type A2 BSC is
working area most widely used in
clinical and public
health laboratories
Class lll Completely Enhanced, Single-pass Exhausted to the Complete seal

sealed, access by high-level airflow with outside, through provides highest
glove ports operator and dedicated HEPA- HEPA filters with level of operator
environmental filtered supply a remote fan; protection
protection and exhaust air hard-ducted. If Seal allows
Provides product recirculating air, for gaseous
protection a second HEPA decontamination/
filter is often fumigation
used
Specialized
procedures are
required for the
introduction and/
or removal of work
materials and
equipment
SECTION 4 SELECTING A PRIMARY CONTAINMENT DEVICE 17
Table 4.1: Key considerations in the selection of a primary containment device (continued)

Containment Fixed, sliding or May give Dedicated Exhausted to the Available in various
isolators hinged open- enhanced, high- supply of both outside, through sizes, shapes and
fronted level operator HEPA-filtered 1 or 2 HEPA filters, design specifications
protection inflow and with a remote (bespoke design)
Provides product exhaust air. May fan; hard-ducted Rapid installation for
protection involve double emergency use
HEPA filtration
Able to house large
to allow direct
equipment and/or
recirculation to
animals
the work surface
HEPA = high efficiency particulate air; BSC = biological safety cabinet.

a
For more detailed information on various Class II cabinet types, refer to Table 4.2.
The following subsections describe in more detail the operating mechanisms of the
various primary containment devices, including testing and certification procedures
that may be necessary to create and monitor correct performance.
4.1 Class I BSCs

Class I BSCs are designed to protect the operator and the environment from
infectious aerosols generated within the BSC. They do not provide protection against
contamination of materials on the work surface.
Class I BSCs have a very simple airflow design, which allows them to maintain
performance in most laboratory circumstances. Room air is drawn in through the front
opening at a minimum velocity that meets applicable standards. The air passes over
the work surface and is discharged from the cabinet through an exhaust duct. The
front opening allows the operator’s arms to reach the work surface inside the cabinet
while he or she observes the work surface through a glass window at the front. After
completion of work activities, the window can be fully raised to provide access to the
work surface for cleaning or other purposes.
Figure 4.1 provides a schematic diagram of a Class I BSC. For simplicity, this shows one
option of a directly recirculating Class I cabinet; however other exhaust set-ups can
also be used. The air from the cabinet can be exhausted through a HEPA filter and then
either recirculated to the laboratory or exhausted to the outside through an exhaust
duct. Class 1 cabinets can also be connected through a thimble duct to the laboratory
HVAC that uses a 100% exhaust system (that exhausts all the air to the outside).
After passing through the

HEPA filter, clean air is
exhausted from the cabinet.
HEPA filter
An internal fan draws

contaminated air from the
workspace up into a HEPA filter.
Workspace
Room air is drawn in
through the front opening.
Figure 4.1 Class I biological safety cabinet
Class I BSC performance is easy to monitor using an anemometer to measure the

velocity of the inflow air entering the BSC through the opening. This is done by taking
a series of readings across the plane of the front opening, such as those shown in
Figure 4.2. From these readings, the average inflow velocity can be calculated and
compared to that specified by the manufacturer or in the applicable standard. The
airflow velocity should be constant across the front opening; if significant variance is
noted in a single point, this may indicate a problem with the cabinet or its installation.
According to EN 12469:2000 (16), the airflow velocity should be within the range 0.25 -
0.50 m/s. Additionally, no individual measurement should differ more than 20% of the
value indicated by the manufacturer.
1 2 3 4
Workspace
5 6 7 8
Front opening
Figure 4.2 Anemometer reading for a Class I biological safety cabinet -

the squares 1 to 8 indicate the positions where readings should be taken
The integrity of the inflow can also be confirmed using smoke pencils or smoke
generators which help visualize the airflow and ensure it is directed inward over the
whole area of the front opening. Visualization should be done under normal working
conditions, for example with the operation of independent room HVAC systems. Smoke
pencils/generators can also be used to assess the effect on ventilation of any equipment
used within the cabinet (for example, a centrifuge). HEPA filters installed with Class I
BSCs should be tested at least annually to confirm they perform according to the
manufacturer’s specifications. The correct operation of all alarms and indicators should
also be confirmed, as should the functioning of anti-blowback valves, if installed.
4.2 Class II BSCs

The Class II BSC is designed to provide personnel and environmental protection as well
as protection for work surface materials from potentially contaminated room air. The
airflows inside Class II BSCs are considerably more complex than in other BSC classes
because of the addition of airflows designed to provide product protection. HEPA-
filtered air is driven as a downward airflow from the top of the cabinet onto the work
surface. This is in addition to the inward flow of air at the front opening, which provides
operator protection in a similar way to Class I BSCs. This system often involves partial
recirculation of air within the cabinet; filtered air is divided between an exhaust and
the downward flow mechanism.
Five types of Class II cabinets currently exist and are defined in the United States
NSF standard as NSF types A1, A2, B1, B2 and C1 (12). Each NSF type uses different
mechanisms for the intake, recirculation and exhaust of air to achieve the inward and
downward airflow combination.
The comparable European standard (EN 12469) outlines a single design type, which
is broadly in line with the NSF A2 type of cabinet. Many manufacturers therefore build
cabinets that conform to both EN 12469 and the NSF A2. The important shared design
features specified in the two standards for Class II BSCs is the fail-safe mounting of
filters to prevent leaks across the filter seals, and the use of pressure plenums. NSF/
ANSI 49 – 2016 states that Class II cabinets should be designed to have all biologically
contaminated ducts and plenums under negative pressure or surrounded by negative-
pressure ducts and plenums. In most cases, this plenum is held at a negative pressure
so that if any leakage occurred across the filter seals or from contaminated ducts, the
contaminated air would be drawn back into the cabinet, thus preventing release of
potentially infectious aerosols into the laboratory or to the outside environment. These
designs hold the body of the cabinet at negative pressure, preventing any unfiltered air
escaping through construction joints and seals.
NSF type A1 cabinets are no longer widely used, in part due to the lower inward airflow
requirements and, more importantly, because older models do not meet the negative
pressure design requirements outlined earlier. In such cabinets, the contaminated air
is driven into a positive-pressure plenum that is not firmly bonded or airtight before
it is passed through a HEPA filter for exhaust or recirculation as a downward airflow.
As the plenum may be under direct positive pressure to the laboratory, contaminated
air could escape the containment system through construction joins and seals in
the cabinet body. Furthermore, in such designs, air can escape before reaching the
HEPA filters, leading to containment being breached and air being passed to the
environment and laboratory, or possibly contaminating the workspace. For such
cabinets, the integrity of the seals of the positive pressure sections of the cabinet body
should be routinely tested. Replacement of type A1 cabinets with cabinets that are fully
compliant with current standards should be considered.
NSF type B cabinets use a primarily (in B1 cabinets) or exclusively (in B2 cabinets)
single-pass airflow whereby air removed from the workspace is not mixed and
recirculated as downward airflow. The proportion of air recirculated in NSF type
B1 cabinets varies between models but is typically less than 50% (12). This makes B1
cabinets suitable for use with hazardous chemicals where recirculation of vapours
must be avoided. However, this single-pass airflow, particularly in the case of B2
cabinets, is highly sensitive to changes in the room ventilation rate as well as pressure
differences. Furthermore, an intake with a pre-filter exists at the top of B2 BSCs to
provide the single-pass downward airflow. This pre-filter is prone to drawing in dust
and other particulate matter from the room and may become blocked causing alarm
systems to activate. Opening a door to an anteroom may result in a B2 cabinet failing
to meet the manufacturer’s performance specifications. Because of the single-pass
directional airflow of B1 and B2 cabinets, they are not able to use a thimble duct so
they must be hard-ducted to the outside environment with an integral blower unit, or
be connected to a dedicated laboratory HVAC exhaust system.
NSF type C1 cabinets usually operate in a similar way to a B1 cabinet, with a low air
recirculation rate (typically less than 50%), but they have more flexibility in their exhaust
system, which may be hard-ducted, thimble-connected or directly recirculated.
Table 4.2 gives a comparison of Class II BSCs.
Table 4.2 Characteristics of different Class II biological safety cabinets
CHARACTERISTICS
WORKSPACE MINIMUM RECIRCULATED APPROXIMATE EXHAUST INDICATIONS

OPENING AVERAGE AIR (%) EXHAUST REQUIREMENT FOR THE USE
INFLOW VOLUME OF TOXIC
EXHAUST
VELOCITY (M3/S) b CHEMICALS
AIR (%)
(M/S) a AND RADIO-
NUCLIDES
Class II cabinet type A1
Sliding sash or 0.38 70 0.14 (for a 1.2 m Exhausted to the Work does not
hinged with a 30 cabinet) room through include toxic
fixed opening 0.19 (for a 1.8 m a HEPA filter or chemicals or
cabinet) to the outside radionuclides
through a
thimble duct
Class II cabinet type B1
Sliding sash or 0.51 < 50 0.12 (for a 1.2 m Exhausted to Small amounts
hinged with a > 50 cabinet) the outside with of toxic
fixed opening 0.19 (for a 1.8 m a remote fan; chemicals or
cabinet) hard-ducted radionuclides
Class II cabinet type B2
Sliding sash or 0.51 0 0.28 (for a 1.2 m Exhausted to Small amounts of

hinged with a 100 cabinet) the outside with toxic chemicals or
fixed opening 0.47 (for a 1.8 m a remote fan; radionuclides
cabinet) hard-ducted Not suitable for
dusty environment
Table 4.2 Characteristics of different Class II biological safety cabinets (continued)
CHARACTERISTICS
WORKSPACE MINIMUM RECIRCULATED APPROXIMATE EXHAUST INDICATIONS

OPENING AVERAGE AIR (%) EXHAUST REQUIREMENTS FOR THE USE
INFLOW VOLUME OF TOXIC
EXHAUST
VELOCITY (M3/S) b CHEMICALS
AIR (%)
(M/S) a AND RADIO-
NUCLIDES
Class II cabinet type A2
Sliding sash or 0.51 ≈ 70 0.14 (for a 1.2 m Exhausted to the Work does not
hinged with a ≈ 30 cabinet) room through a include toxic
fixed opening 0.19 (for a 1.8 m HEPA filter or to chemicals or
cabinet) the outside with radionuclides
a remote fan
using a thimble
duct
Class II cabinet type C1
Sliding sash or 0.51 < 50 Suitable with Small amounts

hinged with a > 50 any exhaust of chemical or
fixed opening configuration radionuclides
a
= air speed; b = airflow.
Class II type A2 BSCs, or the European equivalent, are the most widely used Class II
BSCs globally because their use of a negative-pressure plenum on the exterior of the
BSC acts an additional safety feature. The operational principles of the Class II type A2
BSC are shown in Figure 4.3.
An internal fan(s) draws room air through the front opening and the front intake
grill, and the air mixes with air from the work area. This air is passed under the work
area and drawn up into a negative-pressure plenum. About 70% of this air is passed
through a HEPA filter positioned across the entire width of the cabinet working area,
providing a unidirectional downflow of filtered air over the work surface. This clean
downflow of air is split between the front and back of the work surface with some
being drawn through the front intake grills and the rest through the rear intake grills.
Any small aerosol particles generated at the work surface are immediately captured
in this downward airflow and passed through the front or rear grills, thereby providing
the highest level of product protection. The remaining air (about 30%) drawn from
the workspace is passed through a HEPA filter and discharged to the laboratory.
The volume of air for recirculation or exhausting can be controlled using a variable
volume damper which may help vary the volume of airflow to meet performance
specifications independently of the duct pressure.
Approximately 30% of the air from

the negative-pressure plenum
passes upwards through
a HEPA filter, and the clean air
enters the exhaust duct.
The amount of air

HEPA filter
exhausted or
recirculated
may be controlled
using a variable
volume damper. Negative pressure
plenum Fan
Approximately HEPA filter

70% of the
air from the
negative-pressure The internal
plenum passes fan draws
downwards, room air and
through a HEPA contaminated
filter, and the air from the
clean air re-enters workspace
the workspace as up into a
downflow air. negative-
pressure
plenum.
Workspace
Room air is drawn

through a front-
intake grill.
Figure 4.3 Class II type A2 biological safety cabinet
To exhaust air from a BSC II type A2, any of the exhaust configurations described
in section 3 can be used; each has its own advantages. Air from the exhaust could
be recirculated to the room, following HEPA filtration, which has the advantage of
lowering building energy costs because heated and/or cooled air is not being passed
to the outside environment, and is kept within the building. Exhaust air may also be
hard-ducted to a dedicated laboratory HVAC exhaust system or to the outside of the
building, which has the advantage that partial failure of the HEPA filter would not
result in contaminated air being passed back into the room where it could endanger
laboratory personnel. A thimble-duct connection can also be used for exhaust to the
laboratory HVAC system, which allows type A2 cabinets to be turned off when not in
use and negative room pressure to be maintained, thus reducing operating costs.
Because of the complex nature of the airflows in all Class II BSC types, containment
performance is more easily affected by factors such as cabinet positioning, local
airflows and working practices than other types of primary containment system. If not
used correctly, the degree of protection they provide may be greatly reduced (22). The
velocity of the airstream flowing into the front opening can be easily disrupted by air
currents generated by people walking too close to the BSC, open windows, air supply
grills and ducts, and opening and shutting doors. Therefore, BSCs should be situated
far away from human traffic and potentially disturbing air currents. It is particularly
important to follow best working practices for minimizing airflow disruption outlined in
subsection 2.1.
The functional operation and integrity of each Class II BSC must be certified – to
national or international performance standards – both at the time of commissioning
(also known as type testing) and again when installed to ensure that the cabinet
continues to meet performance standards in situ (installation or field testing). In situ
testing must be performed by qualified technicians, according to the manufacturer’s
instructions, and should be repeated regularly (usually at least annually) to ensure
the BSC still functions properly. Evaluation of the effectiveness of cabinet containment
includes testing HEPA filters, mapping downflow velocity, measuring intake airflow
velocity at the front of the cabinet opening, measuring the negative pressure/
ventilation rate, and testing containment, airflow smoke patterns and alarms and
interlocks. Special training, skills and calibrated equipment will be required to
perform these tests. Therefore, these evaluations should be done by a qualified
professional who meets local regulatory requirements. For more details on Class
II testing/certification, at both the design stage and after installation, refer to the
relevant national and/or international standards. Whenever possible, adequate space
should be available behind and on each side of the cabinet to allow easy access
for maintenance. Adequate space above the cabinet may also be required for air
velocity measurements across the exhaust filter and for exhaust filter changes. These
spaces are usually in the range of 30–35 cm but may be greater depending upon the
manufacturer’s recommendations.
4.3 Class III BSCs

The Class III BSC (Figure 4.4) is designed to provide the highest level of protection
to personnel. These cabinets are leak-proof and will be stringently tested to check
leakage rates for the completed system at commissioning and installation. Both
the supply and exhaust air is HEPA-filtered, and the rate of air change within the
cabinet is normally high. Airflow is maintained by a dedicated exhaust system outside
the cabinet, which keeps the inside of the cabinet under negative pressure to the
surrounding laboratory space. Access to the work surface is by means of heavy-duty,
chemically-resistant gauntlets or sleeves with integrated gloves, which are attached to
ports in the cabinet.
Air is exhausted through a

dedicated exhaust system,
either hard-ducted or
using a thimble duct with
canopy hood.
Gas-tight construction
ensures a seal that
facilitates gaseous
decontamination
where necessary.
Air passes through
two HEPA filters in
series.
HEPA filters
Pass box
Inlet HEPA
Glove ports with

gauntlets.
Workspace
Workspace maintained
at negative pressure.
Air is drawn from the room through

an inlet HEPA filter, mixing the air prior to
immediate removal.
The inflow-to-exhaust ratio generally results
in a high air change rate, and maintains a
negative pressure in the workspace.
Figure 4.4 Class IIl biological safety cabinet

The Class III BSC therefore provides complete separation between the material being
handled and the operator, laboratory and surrounding environment. The only potential
for breach of containment is through damage to the integrated gloves or through the
movement of materials in and out of the cabinet.
The Class III BSC may be constructed with a pass box in which work materials
and equipment items can be decontaminated. These boxes may have dedicated
ventilation systems. Cabinets may also be fitted with chemical dunk tanks that
allow sealed materials to be passed to the outside environment. In some maximum
containment laboratories, several Class III BSCs may be joined together to form a
cabinet line with an extended work surface that may be connected to a double-door
autoclave for decontamination of all materials entering or exiting the cabinet.
The integrated gloves should be regularly checked, inspected for damage and
replaced during service if necessary. They should also be replaced on a regular basis.
Procedures for safely changing gloves should be developed and all personnel must be
trained to carry them out. In addition, all HEPA filters should be tested after installation
and then on a regular basis, as indicated by the risk assessment. Alarms and indicators
should also be regularly tested, and the cabinet manometer regularly calibrated. The
rate of air change within the cabinet should be measured and any unequal airflow
velocities determined through an open glove port. The negative pressure and airflow
should all be within the manufacturer’s specifications. Procedures should also be
developed for the removal of specimens and waste from the cabinet. These will
involve the use of different layers of containment and surface decontamination. Before
starting work, the operator should check that the cabinet is working at the correct
negative pressure by checking the anemometer or equivalent device.
Class III cabinets can have a simple construction as they consist of a box with an
attached filter, windows and integrated gloves. If the cabinet is moved or modified in
any way, then recommissioning tests need to be done. Suitable tests to monitor the
leak-proof seal for safe gaseous decontamination should also be considered. The
condition of seals and gaskets should be checked regularly. Local regulations may
require specific risk assessments to be undertaken, which consider failure scenarios
and back-ups such as uninterruptible power supply systems to maintain negative
pressure in the event of loss of power.
A method of supply and exhaust air

must be connected.
Air must be passed through a minimum
of one HEPA filter.
If air is to be recirculated into the laboratory
(thus not requiring complex duct-work), two
HEPA filters must be used.
A method of introduction
and removal of materials
must be made available.
Glove ports with

integrated gloves.
Workspace
The workspace is a totally enclosed

transparent envelope suspended from a frame.
Figure 4.5 Flexible-film isolator
4.4 Containment isolators
4.4.1 Flexible-film containment isolators
The flexible-film isolator (Figure 4.5) is a self-contained primary containment device

that provides a high level of user protection against hazardous biological agents. The
workspace is totally enclosed in a transparent envelope suspended from a framework.
The isolator is maintained at an internal pressure lower than the pressure of the
surrounding space.
As with a Class III BSC, both inlet and outlet air are passed through one or two HEPA
filters. This allows the air to be recirculated to the laboratory rather than having to
discharge exhaust air outside the building. Flexible-film isolators operate at a lower
negative pressure than Class II BSCs but they have been shown to be capable of
achieving high levels of operator protection under a range of working conditions (25).
They give a high level of protection even when certain failures occur such as loss of
pressure (because they are completely sealed) or major leaks (because they have high
inflow velocity) (26).
An advantage of flexible-film isolators over BSCs is that they can be designed for a
specific task(s). Many factors can be taken into consideration in their design, such as
workflows, waste streams and ergonomics, which should allow safe and effective use.
Isolators range in size from small, mobile, one-person designs to large enclosures that
can hold a range of laboratory equipment or animal cages. However, the life-span of
a flexible-film isolator is likely to be shorter than that of a Class III BSC, they are more
susceptible to damage and the flexible-film may need to be replaced on a regular
basis.
Flexible-film isolators have been used for work with high-consequence pathogens
during field work where it has not been feasible or appropriate to install or maintain
conventional BSCs (27). The flexible nature of isolators allows work to be done in many
different and difficult conditions. For containment work, the primary aim is to prevent
positive pressure developing during normal working conditions. If forced supply air is
used, then there must be effective interlocks in the extraction system to prevent positive
pressure developing if extraction is lost. The inside of the isolator is normally accessed
through integrated gloves. As done for Class III BSCs, protocols should be established
for the introduction and removal of materials (for example, reagents, single-use items
such as pipette tips or tubes, specimens, waste). This may involve the use of transfer
ports or surface disinfection. Ventilated pass boxes, dunk tanks or rapid transfer ports
can facilitate the introduction and removal of materials. Effective pressure monitors
and alarms are needed to ensure correct operation. For care of infected animals
within the isolator, back-up systems may be needed to maintain both containment and
allow access for animal care if failures occur.
Isolator systems have mostly been used by the pharmaceutical or nuclear industry.
However, the specifications of these industries are not all directly applicable to
biological containment systems. Other guidance documents for isolators specific to
biological containment have been developed, for example in the United Kingdom (28),
but this is primarily for the use of isolator systems for animal containment. The United
Kingdom guidance contains useful recommendations for both validation and routine
testing of isolator systems. Apart from this, it is common practice to apply methods
similar to those used for testing and validation of Class III cabinets to isolator systems.
Routine tests should be undertaken to ensure the correct operation of alarms,
indicators and back-up systems, if fitted. As no specific standards currently exist for
the certification of isolators, airflows should be measured against the original design
specifications. HEPA filters of isolators should be tested using a suitable method and
shown to be in line with the manufacturer’s specifications.
4.4.2 Rigid containment isolators
Rigid-walled isolator systems are a moveable alternative to the flexible-film model

and can also provide the highest level of containment, using sleeved and half-suit
designs. However, as with Class III BSCs, they need to be operated at much higher
negative pressures than flexible-film isolators to prevent positive pressure developing
during use. This is particularly important with half-suit designs where movement of the
suit can significantly affect the pressure within the containment isolator.
4.4.3 Unventilated isolators
When having to provide a rapid response, deployable safety containment systems

may be needed for use in the field or in temporary laboratories. In such cases, simple
HEPA-filtered isolators have been used successfully for both semi-permanent (18,27)
and mobile pop-up diagnostic facilities. However, the complexity and size of these
systems need to be balanced against the ability to transport and install them in a field
facility (29). While ventilated, HEPA-filtered units offer the highest level of protection
to the operators; the use of unventilated systems can also be considered, based on a
risk assessment. If the aerosol risk is low, then simple film isolator pods or tents may
offer high levels of physical separation from the material being handled, and allow the
use of strong disinfection procedures, whilst minimizing operator exposure to both the
agent and disinfectant. It must be noted that as the effectiveness of the containment
system is reduced, safe working practices must be strengthened.
4.5 Other local exhaust ventilation types
4.5.1 Ventilated work stations
For some operations, a ventilated work station will be adequate to control any aerosols
generated by a procedure. This may be one simple part of a process that has been
identified as the only aerosol risk. These stations can be constructed by connecting
a suitable box, with an open front or loose door, to a HEPA filter attached to a fan
to provide an internal airflow and prevent release of potential pathogens from the
process. Downdraft necropsy tables for postmortems can also be used to capture
aerosols if the exhaust systems contain HEPA filters. However, unless specifically
designed for biological containment work, the performance may not be in line with
BSCs. Since these systems are not covered by biological containment standards,
specific test protocols may need to be developed to ensure correct and satisfactory
operation. Certain guidance documents may offer standardization for some disease-
specific work, such as the use of ventilated work stations for sputum smears for acid
fast bacterial staining in tuberculosis microscopy laboratories (13).
Full containment systems are not always appropriate for animal work, especially
for non-human primates and larger animal species. In such situations, the use of
respiratory protective equipment may be required to protect against any aerosol
exposure. However, the use of directional flow booths and containment systems with
terminal HEPA filter extraction can be used for animal husbandry and to minimize the
possible exposure of operators, thus reducing the amount of aerosol the respiratory
protective equipment may have to filter. Such systems should be designed to provide
good husbandry conditions, but also provide directional airflow away from the
operators to allow safer operation. Again, there are no international standards for these
systems and detailed validation when used in real-world situations is required for each
installation.
There may be cases where dedicated HEPA-filtered ventilation systems are not
available or practical because of the location and available resources. In such
situations, other alternatives may be suitable. For diagnostic work, an alternative
could be a simple system similar to a BSC I design, with air being drawn through an
open-fronted box or cabinet by ducts connected to a remotely located terminal fan.
The exhaust air can then be discharged at height to the atmosphere; this relies on the
exhaust air being highly diluted to minimize environmental contamination. While such
an approach does not prevent release to the environment, it provides high levels of
protection for the laboratory personnel. However, the discharge needs to be carefully
positioned to prevent any aerosols re-entering the laboratory area. In addition, without
the protection of HEPA filters, the ducts and fan should also be considered potentially
contaminated.
4.5.2 Individually ventilated cages
Individually ventilated cages are designed to house small laboratory animals and
come in many forms. Early systems were designed either to protect the animals from
external contamination or to minimize the release of animal allergens. In recent years,
high-containment systems have been developed. Some are designed to operate at
positive pressure, others at negative pressure so care is needed when selecting cages.
To protect the operator, they must be used in conjunction with other types of primary
containment for animal husbandry, normally a dedicated cage change system
(for cleaning or removing an animal) or modified Class I or II BSC, and operational
procedures must be followed. For highly hazardous work, the cage change systems
should be designed to meet the performance criteria of BSCs.
Typically, the cages are held on a manifold rack system which will provide ventilation
to each cage through a dedicated control unit. Depending on the use, both the
supply and extracted air may be HEPA-filtered. For containment work, the system is
balanced to keep the cages at negative pressure while on the rack. The cage design
will depend on the proposed use, but two types are generally used. For lower-hazard
work, the fully enclosed cage has a simple filter integrated in the cage top, allowing
air from the laboratory to be drawn into the cage. This system only provides protection
when the cage is held on the rack. For highly hazardous work, where the animal is
likely to generate infectious aerosols, a fully sealed cage with an integral HEPA filter
can be used; air is supplied and extracted only by the manifold. The cage should
be sealed when removed from the system. This will allow it to be safely transported
to a suitable containment cabinet before opening or handling. With these systems,
the cage, including the external surfaces, can become contaminated, so effective
decontamination steps are required to maintain safety.
31
References
1. Dimmick, RL, Fogl, WF, Chatigny, MA. Potential for accidental microbial aerosol
transmission in the biological laboratory. In: Hellman A, Oxman MN, Pollack R,
editors. Biohazards in Biological Research. Proceedings of a Conference Held at
the Asilomar Conference Center, Pacific Grove, California, January 22–24, 1973. New
York (NY): Cold Spring Harbor Laboratory; 1973:246–66.
2. Bennett A, Parks S. Microbial aerosol generation during laboratory accidents and

subsequent risk assessment. J Appl Microbiol. 2006;100(4):658–63. doi: 10.1111/j.1365-
2672.2005.02798.x
3. Pottage T, Jhutty A, Parks S, Walker J, Bennett A. Quantification of microbial aerosol

generation during standard laboratory procedures. Appl Biosaf. 2014;19(3):124–
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4. Laboratory biosafety manual, fourth edition. Geneva: World Health Organization;

2020 (Laboratory biosafety manual, fourth edition and associated monographs).
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manual, fourth edition and associated monographs).
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(Laboratory biosafety manual, fourth edition and associated monographs).
7. Personal protective equipment. Geneva: World Health Organization; 2020

8. Decontamination and waste management. Geneva: World Health Organization;

2020 (Laboratory biosafety manual, fourth edition and associated monographs).
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10. Outbreak preparedness and resilience. Geneva: World Health Organization; 2020
11. Sayin-Kutlu S, Kutlu M, Ergonul O, Akalin S, Guven T, Demiroglu YZ, et al. Laboratory-
acquired brucellosis in Turkey. J Hosp Infect. 2012;80(4):326–30. doi: 10.1016/j.
jhin.2011.12.020
12. NSF/ANSI 49-2016. Biosafety cabinetry: design, construction, performance, and

field certification. NSF International/American National Standards Institute; 2016.
13. Ventilated workstation manual for AFB smear microscopy. Global Laboratory
Initiative, Centers for Disease Control and Prevention, International Union Against
Tuberculosis and Lung Disease, Association of Public Health Laboratories; 2011
(http://www.stoptb.org/wg/gli/assets/documents/vwm4afbsm.pdf, accessed 15
January 2019).
14. AS 2252.1. Biological safety cabinets – biological safety cabinets (Class I) for
personnel and environment protection. Standards Australia; 2002.
15. CFDA YY-0569. Biological safety cabinets. China Food and Drug Administration;
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16. EN 12469:2000. Biotechnology. Performance criteria for microbiological safety

cabinets. European Standards; 2000.
17. JIS K 3800. Class II biological safety cabinets. Japanese Standards Association; 2009.
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and operation of Ebola diagnostic laboratories led by Public Health England in
Sierra Leone during the West African Ebola outbreak 2013–2015. Clin Microbiol
Infect Dis. 2016;1(4):1–6.
19. BS EN ISO 29463-5:2018. High-efficiency filters and filter media for removing
particles in air. Test method or filter elements. London: British Standards Institute;
2018.
20. DOE-STD-3020-2015. DOE technical standard: specification for HEPA filters used
by DOE contractors. United States Department of Energy; 2015.
21. ISO 29463-1:2017. High efficiency filters and filter media for removing particles from
air – Part 1: classification, performance, testing and marking. Geneva: ISO; 2017.
22. Abraham G, McCabe P. Reliability of ULPA filters in air handling systems. Appl Biosaf.
2007;12(3):184–6. doi: 10.1177/153567600701200308
23. Dubey SC, Murugkar HV, Kaushik RK, Kulkarni DD. The efficiency of HEPA filters
in the air-handling system of a biocontainment laboratory in India. Appl Biosaf.
2009;14(3):121–6. doi: 10.1177/153567600901400302
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biological laboratory. Appl Biosaf. 1998;3(4):134–42. doi: 10.1177/109135059800300405
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program: experiences in Southeast Asia. Appl Biosaf. 2016;21(3):121–7. doi:
10.1177/1535676016661769
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26. Bennett AM, Parks SR, Dennis MJ. Containment testing of negative pressure
isolators used to house laboratory animals infected with BL3 agents. In: Gamble
MR, Millington S, editors. Internationalisation and harmonisation of laboratory
animal care and use issues. Proceedings of the 9th FELASA Symposium, 4–17 June
2004, Nantes, France. London; FELASA; 2004:137–44.
27. Bennett, AM, Parks, SR, Pottage T. Containment and Ebola in an outbreak setting.
Clean Air and Containment Review. 2015;24:24–6.
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laboratory and animal isolators for the containment of biological agents. (http://
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al. Mobile diagnostics in outbreak response, not only for Ebola: a blueprint for a
modular and robust field laboratory. Euro Surveill. 2015;20(44). doi: 10.2807/1560-
7917. ES.2015.20.44.30055
Further information
Biological safety cabinet (BSC) 1: Introduction [Biosafety video series]. Geneva: World
Health Organization; 2019 (https://youtu.be/KHCT9OJqxPo, accessed 6 December
2019).
Biological safety cabinet (BSC) 2: Preparatory steps [Biosafety video series]. Geneva:
World Health Organization; 2019 (https://youtu.be/4DoHJS8JL4U, accessed 6 December
2019).
Biological safety cabinet (BSC) 3: Best practices for safe usage [Biosafety video
series]. Geneva: World Health Organization; 2019 (https://www.youtube.com/
watch?v=18QEJUA9XBs, accessed 6 December 2019).
Biological safety cabinet (BSC) 4: Incident management [Biosafety video series].

Geneva: World Health Organization; 2019 (https://www.youtube.com/watch?v=aS_
TCZTCcsI, accessed 6 December 2019).
37

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LABORATORY BIOSAFETY MANUAL i

BIOLOGICAL SAFETY CABINETS

BIOLOGICAL SAFETY CABINETS

© World Health Organization 2020

SECTION 1 Introduction to primary containment devices 1

SECTION 2 Working with primary containment systems 5

2.1 Best practice for working with open-fronted devices5

2.2 Working with enclosed devices: additional considerations6

2.3 Decontamination of safety cabinets and isolators7

SECTION 3 Directional airflow9

3.1 High efficiency particulate air filtration10

3.2 Direct recirculation11

3.3 Hard ducting11

3.4 Anti-blowback valves12

3.5 Thimble ducts13

SECTION 4 Selecting a primary containment device15

4.1 Class I BSCs17

4.2 Class II BSCs19

4.3 Class III BSCs24

4.4 Containment isolators27

4.5 Other local exhaust ventilation types29

Dr Kazunobu Kojima, World Health Organization, Switzerland

Prof. Stuart Blacksell (Team lead), University of Oxford/Mahidol-Oxford Tropical

Ms Lisa Stevens, World Health Organization, France

Ms Rica Zinsky, World Health Organization, Switzerland

Ms Maren Roush, National Sanitary Foundation, United States of America

Aerosol-generating procedure: Any procedure that intentionally or inadvertently

Biological agent: A microorganism, biological toxin, protein (prions) or human

Biosafety: Containment principles, technologies and practices that are implemented

Calibration: Establishment of the relationship between the measurement provided by

Certification: A third-party testimony based on a structured assessment and formal

Clean: Visually free of soil and below specified levels of analytes.

Consequence (of a laboratory incident): The outcome of an incident (exposure to and/

Containment: The combination of physical design parameters and operational

Contamination: The introduction of undesired biological agents into tissues and

Decontamination: Reduction of viable biological agents or other hazardous materials

Disinfectants: Agents capable of eliminating viable biological agents on surfaces or in

Exposure: An event during which an individual comes in contact with, or is in close

Fumigation: Use of a poisonous gas or vapour to remove contamination of a biological

Good microbiological practice and procedure (GMPP): A basic laboratory code of

Laboratory-associated infection: Any infection acquired or reasonably assumed as a

Pathogen: A biological agent capable of causing disease in humans, animals or

Primary containment device: A contained workspace designed to provide protection

Risk assessment: A systematic process of gathering information and evaluating the

Validation: Systematic and documented confirmation that the specified requirements

The three classes of BSC are:

n Class III BSCs – a closed, sealed, negative-pressure enclosure to which HEPA-filtered

n Other local exhaust ventilation devices – partially open-fronted enclosures which

WORKING WITH PRIMARY

2.1 Best practice for working with open-fronted devices

n Human traffic behind the operator should be kept to a minimum.

n Paperwork should never be placed inside primary containment devices as it may be

2.2 Working with enclosed devices: additional considerations

2.3 Decontamination of safety cabinets and isolators

2.3.1 Liquid decontamination

2.3.2 Gaseous/vapour decontamination

Based on the risk assessment, primary containment devices may be required to

n reused: by recirculating it within the primary containment device, or by sending it into

3.1 High efficiency particulate air filtration

SECTION 1 Introduction to primary containment devices 1

SECTION 2 Working with primary containment systems 5

2.1 Best practice for working with open-fronted devices5

2.2 Working with enclosed devices: additional considerations6

2.3 Decontamination of safety cabinets and isolators7

SECTION 3 Directional airflow9

3.1 High efficiency particulate air filtration10

3.2 Direct recirculation11

3.3 Hard ducting11

3.4 Anti-blowback valves12

3.5 Thimble ducts13

SECTION 4 Selecting a primary containment device15

4.1 Class I BSCs17

4.2 Class II BSCs19

4.3 Class III BSCs24

4.4 Containment isolators27

4.5 Other local exhaust ventilation types29