The last blog addressed industry accepted standards for performance testing of Class III Biosafety Cabinet in the US.  This blog focuses on other Internationally accepted standards.  These standards contain some areas of overlap with US standards and in other areas provide supplemental information. 

British/European Standard BE EN 12469:2000/EN 12469:2000, 2000 (1) :

• Class III BSC has a completely enclosed workspace and manometer to show pressure drop (manometer range of -500 Pa to + 500 Pa), (500Pa =2”wg)
• Supply air single HEPA, exhaust air double HEPA filtered.  Each exhaust filter must be able to be independently tested
• Leak tightness: </= to 10% loss of test overpressure of 500Pa in the whole enclosed system after 30 min
• Working pressure: at least 200 Pa (200 Pa=0.08”wg) below the pressure of the lab
• Alarm indictors: audible alarm for pressure loss
• Volumetric airflow rate: Minimum of 0.05 cubic m/s for each cubic meter of cabinet volume when cabinet is at -200 Pa.  Measurement is taken through the inlet filter.
• Mean airflow velocity for worker protection: >/= to 0.7 m/s with one glove removed, (0.7 m/s =138 ft/min)

 Public Health Agency Canada, 2004, (2):

 PHAC Laboratory Biosafety Manual 3^rd Edition 2004: Document Submission Requirements for the Recertification Performance and Verification Testing of Containment Level (CL) 4 Laboratories in Accordance with the Laboratory Biosafety Guidelines, 2004, Public Health Agency of Canada (and where applicable, Containment Standards for Veterinary Facilities, 1996, Canadian Food Inspection Agency)

 Class III BSC to be tested in accordance with:

• BS EN 12469:2000: Biotechnology- Performance criteria for microbiological safety cabinets (2000); British Standards Institute
• Laboratory Safety Monograph: A Supplement to NIH Guidelines for Recombinant DNA Research (1979); National Cancer Institute Office of Research Safety and the Special Committee of Safety and Health Experts.
• Acceptance criteria: measured leakage from any point in the cabinet shall not exceed a leak rate of 10 X 10^-7 cc/sec at 750 Pa (750 Pa=3″ wg).
• Provide the calibration certificates for the equipment used for the verification.

 The final blog in this series will describe ‘best practices’ in field testing (annual on-site recertification) the Class III BSC.  As there is no NSF 49 Standard or other regulation that addresses annual retesting, the next blog will consider all of US and International Standards and provide a recommendation for a comprehensive test procedure based on the various standards. 

 References:

1. Biotechnology- Performance criteria for microbiological safety cabinets; BS EN 12469:2000/EN 12469:2000, 2000, Dandy Booksellers Ltd.
2. Laboratory Biosafety Guidelines, 3^rd Edition, 2004, Public Health Agency, Canada

Class III Biological Safety Cabinets: Importance of Hands-on Training

Practical training is particularly useful for students that plan to work in high containment situations. While many students have access to Class II Biosafety Cabinets, too few are able to get hands-on experience with a Class III Biological Safety Cabinet. Class III BSC’s require a familiarization with a wide range of equipment features and configurations such as: Gloves & Gloveports, Pass-throughs / Airlocks, Rapid Transfer Ports, Autoclaves, Decontamination Systems, etc.

Class III BSC Demo

Germfree maintains a Class III BSC training unit at their headquarters. Germfree also participates in student training programs with other facilities, providing institutions with Class III BSC’s and other bio-containment equipment to give their students hands-on experience and training in proper technique.  For example, Germfree’s Biological Safety Cabinets have been used for training in university settings such as The Emory University Science and Safety Training Program as well as workshops in conference settings such as ABSA’s Annual Biological Safety Conference.   

Cliff Colby Demonstrating a Class III Biosafety Cabinet

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Cliff Colby is VP of Sales at Germfree. He has more than 12 years experience as a Biosafety Instructor. He has also worked extensively on multi-media biosafety training materials. And if you want to discuss applications that require chemical containment, his background as a Chemist will also come in handy.

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 The BioTechnical Institute of Maryland  provides training for students that want to enter the biotech field.  According to the BTI website, the institute ”fills a need for specialty scientific training of entry-level biotechnicians for employment in Maryland’s rapidly expanding biotechnology industry.  The BTI Laboratory Associates Program provides tuition-free training in basic laboratory skills to bright, ambitious, unemployed and under-employed Maryland residents. ”

What type of airflow should be used in a Class III BSC?  Is higher velocity better?

Class II BSC vs. Class III BSC Airflow

Unlike Class II BSC which employs laminar flow to protect personnel, the Class III Biological Safety Cabinet does not have an open sash in the front, hence does not require laminar airflow to provide personnel protection.  The main consideration for laminar air flow in a Class III BSC is for product protection.  Laminar flow could be important if an internal process generates copious amounts of aerosol, when work is conducted with fine powders, or if there is a risk of cross-contamination between different procedures being performed in the cabinet. Laminar air flow has a set mass airflow where clean HEPA filtered air comes from one direction at a given speed to entrain particles and carry them directly to the exhaust HEPA. The velocity can be very low, as low as 30 ft/min. However, note that 30 ft/min may cause extremely fine powders to become aerosolized. 

Generally, Class III Biosafety Cabinets use turbulent air flow designs. In a turbulent airflow design clean HEPA filtered air is continuously supplied to the cabinet where it dilutes the concentration of aerosolized particles by carrying them to the exhaust HEPA. This is a more passive mechanism of particle removal as compared to that of the air current generated when laminar airflow is established. The rate at which the particles are exhausted depends on the supply velocity (which is equal to the exhaust velocity). In reality, work in a Class III BSC is conducted methodically and carefully.  Most activities conducted in a Class III BSC produce minimal aerosols so turbulent airflow is the norm.  Turbulent airflows are easily adjustable and can have lower air velocity than those required to maintain laminar airflow, hence can pose less of a problem when working with fine powders.

Another question that comes up is whether the Biological Safety Cabinet should be operated at high airflow velocity to remove particles more rapidly.  Typically higher velocity airflows are used when working with volatile chemicals, but not with microbiological agents or toxins.  High velocity airflow can inadvertently, and very effectively, cause powders to be disseminated throughout the interior of the BSC.

A decision regarding whether laminar or turbulent airflow is needed, and the velocity of supply air required for operations should be made based on the anticipated work and user needs. Use of laminar air flow in a Class III Biosafety Cabinet will typically increase the volumetric supply and exhaust airflow  as compared to a BSC using turbulent airflow.  Higher velocity airflow will similarly increase volumetric supply and exhaust as compared to maintaining low velocity airflow.  Increased exhaust flow rate from the Class III BSC should be considered during facility and HVAC design if the cabinet is connected to facility supply air and is to be exhausted out of the building.

Three events have come together in the past few years that codified a need for rapidly deployable, mobile and cost effective containment equipment.  First, there was recognition that many regions and countries in the world did not have adequate infrastructure, reliable power, or primary containment to provide a safe environment when working with emerging, re-emerging and dangerous infectious agents. In the US and elsewhere following the 2001 anthrax letters Public Health Labs and First Responders began experiencing an increase in their mission scope to collect, transport and perform analysis on unknown samples that may contain biological or chemical hazards. That mission had increased significantly due to copycat, hoax and criminal activity.  In the same timeframe, advances in biomedical research created a need to move samples, animals and materials from room to room, or into and out of imaging suites and equipment while maintaining containment.  In response to these needs portable Class III Biosafety Cabinets, flexible film isolators and compact, easily deployable hybrids were developed and refined.

Portable Class III Biological Safety Cabinet

SEA benchtop (Portable)

Small, bench top units were developed that provide safe, effective and affordable primary containment (i.e. SEA) enabling flexibility for laboratory use or field deployment. It was originally developed for diagnostic screening of unknown and highly pathogenic samples in facilities, laboratories or field settings that lack reliable secondary containment controls.  The closed system decreases the chance of aerosol escape, resultant accidental exposure and potential laboratory acquired illnesses.   

In animal research, portable battery powered Class III BSCs are increasingly being used to move animals from holding rooms to procedure rooms.  The supply is single HEPA filtered, the exhaust is double HEPA filtered as required by the CDC (Download BMBL 5th Ed. pdf).  By use of large RTPs integrated into transporter carts of Class III design, the walls of animal holding rooms, and stationary Class III systems, scientists can safely transport exposed animals from holding rooms to procedure areas equipped with devices such as magnetic resonance imaging (MRI), positron emission tomography (PET), and other non-invasive scanning devices. The systems reduce personnel and environmental exposure and reduce the time the animal must be handled and anesthetized.

 

Class III animal transfer with RTP dock

SEA with passthrough and legs built in

        

 

 

 

 

 

 

 

Transportable Class III BSC

All hazard reciept Transportable Class III Biological Safety Cabinet

Transportable Class III Biological Safety Cabinets are used in Public Health Laboratories for the receipt of unknown hazardous samples associated with chemical or biological terrorism or criminal activities.  Workers use the transport BSCs to move the unknown hazardous sample from the loading dock area or other delivery site used by the First Responder, to the containment lab without risking contamination of non-contained and public areas, as well as the containment lab itself.

Taken together, the advent of the use of transport and mobile Class III BSCs provides a significantly safer way of moving and handling infected animals or unknown samples than any past capability.

 

 

Flexible Film Isolator

Transportable flexible film isolator

The negative-pressure flexible-film isolator is a self-contained primary containment device that provides maximum protection against hazardous biological materials. Isolators can be placed on a counter top or on a mobile cart. The workspace is enclosed in a transparent polyvinylchloride (PVC) film that suspended from a plastic or steel framework. Like Class III BSC, the supply air passes through one HEPA filter and exhaust air passes through two HEPA  filters.

WHO recognizes the double HEPA exhaust obviates the need to duct exhaust air outside the building.  Flexible-film isolators are used frequently and very successfully in animal containment, field work and other instances where it is not feasible to install or maintain conventional BSC.  Hybrids (semi-flexible film isolators) exist where some of the panels are made of a rigid material such as polycarbonate, and typically the front panel is soft PVC.

 

 Deployable Isolators 

Field deployable flexible isolator

The deployable isolator unit is a self-contained negative pressure filtration system that operates on two standard D cell batteries.  Supply air is HEPA filtered. Exhaust air is double HEPA or double HEPA and carbon filtered. All filters are readily available and easily replaceable by First Responders and those involved in field collection and preliminary screening and triage. It is a rapidly deployable, light-weight, disposable system that comes in a compact transport case and sets up much like a dome tent. The isolator is made of durable 15mil polyurethane to withstand field use, repeated assembly/disassembly, and can be assembled and operational within minutes for on-demand use requirements.  Large samples and equipment are introduced through a zipper system similar to those on a BSL-4 suit.  Sampling ports are provided for use with external detectors and analytical equipment.         

The diversity in containment equipment is almost limitless and depends on user requirements and design team innovation and advances in materials. 

 

I was at a meeting recently when someone asked, ‘What are mobile and modular BSL-3 labs and when should they be used?”  It is a good question that comes up often.   Deciding  which platform is the best choice depends on the institute mission, size requirements and in the case of mobile labs, the local road conditions.   For example, if the roads are narrow and turns are very tight, a 12.5 meter long truck lab may not be able to navigate the roads, while a Sprinter van would have no problem.

Mobile Labs: Trucks, Sprinter Vans, Trailers

Mobile labs are those labs which can be moved from place to place easily and often by their own power.  They include platforms built into 12 meter long trucks, 4 meter Sprinter vans, and trailers of various sizes (i.e. 6, 7,12 meters).

Rapid Sample Triage and Screening

They are rapidly deployable and often used in incident investigation (i.e. suspicious materials), military and civil preparedness applications (NBC sampling and analysis at high level events), surge capacity at different locations, and in support of testing during natural disease outbreaks.  They are designed to provide on-the-spot rapid sample triage and screening, and presumptive diagnostic capability to assist in prioritizing samples being sent to national reference labs.  
Mobile labs were initially developed in 1986. It wasn’t until the late 1990’s with the advent of decreased size and ease of portability of analytical equipment and prepackaged reagents, and the availability of igh speed communications technology that these labs became increasingly popular.

Hazmat trailer lab interior

They are rapidly available from time of order to time of delivery, and can be cost effective.  Mobile labs are not however meant to be a substitute for fixed labs, rather they augment the mission and provide a capability not possible with a fixed asset.  The advantages and disadvantages various platforms such as trucks, vans, trailers and containers on flatbeds will be discussed in a later blog.

Truck lab with slide out

Modular labs are constructed using containers that are retrofit and finished at the factory then shipped to a location where they are permanently installed.  The containers are fabricated to be strong and durable enough to withstand handling and stacking during shipping while acting as a protective enclosure to the valuable cargo they contain.  Containers come in a variety of sizes and while one container can be turned into a lab, it is not uncommon in the US and overseas to use multiple containers to build a versatile suite of labs with support and change areas, animal housing and a separate mechanical space.  Connecting 3 containers provides a space that is approximately 7.3 meters wide and 12.2 meters long. 

 

Truck lab interior

Once they are installed onsite they become fixed labs and do not move.  These labs are used for teaching, research, public health and as diagnostic reference labs.  

Modular containment labs have become increasing popular over the past 10-15 years.  They do not require the same process for construction approval as ‘stick-built’ labs and are an increasingly attractive solution to providing additional facility space and new technology capability to existing buildings.

  

 

 

 

Truck lab interior 2

Modular labs are designed and assembled by organizations with expertise in biocontainment, making them a sensible solution to providing turn-key containment capability in countries where resident expertise is not yet available or is nascent and would benefit from partnering with experienced entities. 

  

  

  

 

   

 

Modular Laboratories: Rapidly Available | Easily Shipped

They also are rapidly available from time of order to time of delivery (compared to ‘stick built labs’, the container itself is of low relative cost and can house a lab that is custom designed with ample space, and is easily shipped nationally and internationally.

Modular Container - just delivered

Modular container lab interior rm 1

Modular lab interior- rm 2

Sprinter van lab

Sprinter lab interior

   

The other day I was asked about glove selection for use in a Class III BSC and whether there were options other than butyl rubber.

             Gloves are supplied in a variety of materials from several manufacturers.  These materials include neoprene rubber, butyl rubber, and hypalon and have differing permeability rates with various chemical compounds (including decontamination chemicals). The selection of glove type should be made based on risk assessment that considers chemical permeability, operations (handling of animals or sharps), and the requirement for dexterity.  Start by consulting with a glove permeability chart when use of chemicals is anticipated, and for more specific information, contact the glove vendor for break through rates with the particular glove and chemical you plan to use.  This will help in the selection of the correct glove, as well as the time required to change the gloves before they break through. Strict specifications have been developed for butyl gloves by DOD. Consideration of the operation being performed is essential as the glove types differ significantly in the amount of dexterity needed; with butyl offering less dexterity than hypalon.

             Gloves are available in different thicknesses and hand sizes.  A balance between dexterity and protection should be considered in a risk assessment when specifying glove thickness.  Ordering the correct hand size is important for safety and ability to perform the work when using the gloves.  Gloves that are too large create a ballooning effect making it very difficult to manipulate equipment such as pipetters, small tubes, plates and other common items.  This increases the risk of a spill, contamination of the material and causes worker fatigue.

              Two part arm sleeve and glove systems are available.  This enables the user to select from a variety of gloves and sizes (to include exam gloves, nitrile and other gloves commonly used in microbiological laboratories) which greatly increases dexterity.  However the circular connector between the attachable glove and arm sleeve can be difficult to clean and thoroughly decontaminate. If this system is used, SOPs for thorough decontamination should be in place and personnel should practice decontaminating the glove and ring system (attaches the glove to the sleeve) prior to conducting work with pathogens.

hypalon glove

neoprene glove

Two-part glove system

   Gloves should be carefully inspected before commencing work within a Class III BSC.  While the entire glove and arm length should be inspected, particular attention should be given to the fingertips, the webbing between the fingers, and the connection point to the glove port.  These are areas which are most susceptible to wear, stress and operational damage.  Gloves should be visually inspected periodically for cleanliness during work, and especially if you suspect a breach may have occurred.  One advantage of hypalon gloves is that they are white in color making it easy to spot surface contamination. Gloves should be replaced when any cracks, wear areas, or tears are observed or if break through is suspected. 

             Even though work is conducted wearing gloves (typically an under glove and the outer glove attached to the Class III), personnel should thoroughly wash their hands after working in the Class III and prior to exiting the laboratory.

References:

 1. Glove selection chart

http://www.chem.duke.edu/safety/glove.html

 2. Glove permeability

http://www.himnrbehs.com/himnrbehs/pdf/2002-02-19%20Glove%20Safety%20Booklet.pdf

 3. MIL DTL 43976D (Military Standard for gloves); Dept of Defense, 2003

http://assist.daps.dla.mil/quicksearch/basic_profile.cfm?ident_number=24055

    There are several technologies typically used to transfer samples, materials and waste into and out of the Class III BSC. These include dunk tanks, pass through chambers, double door autoclaves and rapid transfer ports (RTP). 

    Dunk tanks allow for the passage of potentially contaminated materials out of the BSC without breaching containment.  It also allows for live samples to be removed for transfer to another lab or for archive.  The exterior of the primary container is surface decontaminated and packaged in a secondary container which is kept in contact with a chemical decontaminating solution in the dunk tank for a prescribed period of time.  The decontaminant and contact time are specified in laboratory SOPs, as is how frequently the decontaminant should be changed, and how recently it should be prepared prior to conducting work in the BSC.  The liquid must also be checked to ensure it is one inch, or the level specified by the manufacturer above the stainless steel divider that bisects the dunk tank.  Having a liquid level above the divider is needed to maintain containment and separation of unfiltered air from the room and Class III BSC.

       Dunk tanks can be designed with coved or oblong shapes for ease of cleaning, may be slightly graded for ease of draining, and can be designed with ‘cages and polls’ to keep containers and bags submerged, and retrieve the cages from the bottom of the tank for easy removal.  Kynar or Halar coatings should be bonded to the interior surfaces of the stainless steel dunk tank to protect them from the corrosive effects of many decontaminants.  A latch and administrative SOP or a timed interlock ensures both lids of the dunk tank are not opened at once, and that the material has been in appropriate contact with the decontaminant.

Oval slide-out dunk tank

    Pass through chambers allow for clean materials to be passed into the Class III BSC.  Potentially contaminated materials should not exit the BSC via the pass through chamber unless they are double contained with the package surfaces decontaminated.  The interior of the chamber should be surface decontaminated when materials are passed from the Class III to another location as even though the containers are surface decontaminated, there could be trace contaminants on the gloves, which could then contaminate the pass through chamber.  Once the inner door has been opened (between the chamber and the BSC) the chamber should be sprayed and surface decontaminated at the end of the experiment, or prior to opening the outer door that interfaces with the room (i.e. when additional supplies are to be introduced into the pass through chamber to the BSC).

       Pass through chambers doors are gasketted and airtight, and the chamber may be non-ventilated, or ventilated with the exhaust air HEPA filtered prior to release.  Typically the chambers have two doors, one that traverses the lab to the interior of the chamber and the other which connects the interior of the chamber to the interior of the BSC.  There can be doors servicing other areas as well. All doors should be interlocked, with an audible and visual alarm indicating when a door is not fully closed after an appropriate period of time has elapsed to transfer materials. 

Pass through chamber view on Class III BSC exterior

   Double door autoclaves are very useful in passing large items into the Class III BSC and to sterilize bulky waste and material at the end of the experiment.  The 5^th Edition BMBL advocates a double door autoclave be integral with the Class III BSC for BSL-4 cabinet laboratories.  

Double door autoclave integral to Class III BSC

    Rapid Transfer Ports (RTP) provide the user a safe, efficient and time saving method to introduce and remove material from a Class III BSC without potentially contaminating the laboratory.  RTP come in a variety of sizes ranging from small to very large cart mounted versions for animal transport in containment.  They can be made in stainless steel or high durability plastic. 

       The RTP maintains containment as its design features a beta flange lid exterior that mates to an alpha flange door exterior that is attached to the Class III BSC.  The lid and door are rotated and sealed and must form an interlock before door inside the BSC can be opened.  This exposes only the interior of the RTP cylinder to air and materials inside the BSC.  The process is reversed to uncouple the RTP.  The contents can be stored or the RTP can be opened with an allen key inside a Class II BSC where the materials are removed and the interior of the RTP is surface decontaminated for reuse.

Exterior of the sealed RTP being mated to the exterior of the BSC

Decontamination is a broadly used term that describes a number of techniques or strategies for reducing or eliminating the presence of hazardous microorganisms and biological toxins from various surfaces, materials and equipment.   Decontamination is important from a safety perspective as well as preventing cross contamination and maintaining the integrity of the work.

 Critical elements involved in the process include choice of decontaminant, site conditioning, biological indicator choice and monitoring for gas or vapor.  Chemicals frequently used in the decontamination process include: chlorine dioxide gas, 70% ethanol, gaseous paraformaldehyde/formaldehyde, liquid sodium hypochlorite/bleach, and hydrogen peroxide vapor.

When selecting a decontaminating agent a number of factors have to be considered to include the susceptibility of the organism to the decontaminating agent; contact time, temperature and relative humidity required; stability of the decontaminating agent; and the physical characteristics, material compatibility and surface properties of what is being decontaminated.  Decontaminating agents are typically harsh and can damage or leave a residue on gloves, stainless steel, electronics and other materials inside the BSC.

 Site preparation is a requirement regardless of which decontaminating agent is being used.  It entails pre-cleaning the interior of the BSC to remove bioburdens and other compounds that may interfere with decontamination such as protein, organic matter, salts, absorptive materials, and liquids and solid wastes to be autoclaved.  In the case of gaseous decontamination, the Class III BSC and its filter housings must be capable of being sealed to prevent the leakage of gas into inhabited spaces resulting in personnel exposure, and to prevent dilution of the concentration required for microbial killing.  Supply and exhaust vents, drain traps or dunk tanks, and any penetrations must be air tight and sealed. All areas requiring decontamination should be accessible for gas penetration, and a fan(s) may be required to disseminate the gas.

 Different biological indicators may be used for different gas decontaminants, and optimally contain the equivalent of 10⁶ organisms per BI to measure the effectiveness of the process.  Indicators should be distributed throughout the enclosure to be decontaminated with attention to stratification across height and areas that may be more difficult for gas or vapor to reach.  Users of BI’s should follow manufacturer instructions regarding incubation parameters and the type of decontamination systems for which they have been validated.  BI’s are not used when decontamination is by bleach or 70% ethanol wipe down.  In this case, successful decontamination can be confirmed by performing swab sampling throughout the area decontaminated and monitoring those swabs for growth of organisms. 

If BSC are to be decontaminated using gas or vapor, the room should be monitored for leaks of the decontaminant.  Dräger tubes, automated Dräger technology, Calibrated Miran SapphIRe 205B, or other equivalent systems are commonly used for monitoring leakage and the presence of monitor for chlorine, hydrogen peroxide, formaldehyde, and ammonia gases during and after decontamination.  The sensitivity of different technologies and models vary.  It is important that the monitoring system is sensitive and has adequate specificity so it can reliably and reproducibly measure concentrations below the OSHA permissible exposure level (PEL) with 95% confidence.

References:

Favero, M. S. and Bond, W. W. (1991). Chemical disinfection of medical and surgical materials, in Disinfection, Sterilization, and Preservation, 4th ed. (Ed. Block, S.), Lea & Febiger, Philadelphia, PA, 621.

 Environmental Protection Agency (March 2005). Compilation of Available Data on Building Decontamination Alternatives Publication # EPA 600/R05/036.

http://www.epa.gov/NHSRC/pubs/600r05036.pdf

 Czarneski, M.A. (2007). Selecting the Right Chemical Agent for Decontamination of Rooms and Chambers. Applied Biosafety, 12(2), 85-92.

 Lever, M.S., Howells, J.L., Bennett, A.M., Parks, S. and Broster, M.G. (2008). The Microbiological Validation of a New Containment Level 4 Cabinet Line. Applied Biosafety, 13(2), 98-104.