Laminar Flow and Biological Safety Cabinets
Laminar flow cabinets and biological safety cabinets are widely used in cell culture applications to avoid contamination. The introduction of any foreign material can act as a vehicle for unwanted micro-organisms.
Whilst the use of cabinets is not new, some confusion remains about the various types of cabinets (also known as "hoods") and their varying applications. An introduction to the main cabinet types available in Australia and a review of their main uses follows:
Otherwise known as “clean benches” or “clean workstations”, laminar flow cabinets bathe the work zone in air which is passed through a HEPA (high efficiency particulate air) filter. This air is then (for our purposes), regarded as ‘sterile’.
Laminar flow cabinets are used wherever a super clean environment is required for the handling of various materials (e.g. cultures, specimens, samples and media etc), which are sensitive to the presence of foreign particles. This includes viable matter such as bacteria, fungi and other cell types.
As the air leaving the cabinet is not actively prevented from reaching the operator and the laboratory, a requirement is that the work itself does not present a hazard to the operator, other personnel or the environment in general.
Laminar flow cabinets are available in two styles: Crossflow and Downflow. These terms refer to the plane of the airflow in relation to the work surface.
This type of laminar flow cabinet is perhaps the simplest, offering basic protection for the work only. Air is drawn from the laboratory through a pre-filter and then introduced into the work zone via a HEPA filter located at the rear of the work zone. After a short path of travel (typically about half a meter) across the work zone, the air leaves the cabinet, returning to the laboratory.
Particulate matter released inside the work area, either from the work or by the operator, is ideally swept away before being given an opportunity to contaminate other work. It should however be understood that prevention of cross-contamination is perhaps ensured more by good aseptic technique than by any action of the cabinet itself. The actions of the operator must always complement the operation of the cabinet.
All cabinets relying on filtration for their effective operation are ‘percentage devices’. That is, they can never be totally effective but will reduce the risks of contamination by an extremely high factor. The higher the level of pre-conditioning – i.e. the cleaner the air which the cabinet is drawing from its surroundings – the higher the factor.
Cabinets therefore have the ability to enhance the level of sterility created by the operator, but not to produce it independently of the user and the surroundings.
Air follows the same path as a Crossflow cabinet (via a prefilter and then a HEPA) except that it is configured to flow into and within the work area in a vertical direction. As this air bathes the work zone it divides the ‘breaks’ to leave the cabinet, primarily at the front, but some air is usually encouraged to leave the work zone at the rear of the cabinet. Most cabinets have a slot or a series of perforations running the width of the rear of the work zone for escape of the air.
In some cabinets, rather than allowing all the air to return directly to the laboratory, a portion of air is recirculated to the top housing through these rear (and side) perforations and via one or more return air plenums. This system effectively reduces particulate load to the HEPA’s as this faction of the air is essentially already clean. The result is longer life from the HEPA filter.
Downflow cabinets offer similar product protection to Crossflow in that both achieve a clean work area. There are, however, a number of differences which may mean that one type is preferred to the other for certain purposes.
For a similar overall size of cabinet, Downflow cabinets have a deeper usable work area than Crossflow. Downflow cabinets may be manufactured to give double-sided access, which is useful for certain operations and for demonstrations in teaching situations.
An important difference which is often overlooked concerns the gasketting arrangement for sealing the HEPA filter to the cabinet. In most cabinets, this is achieved using a neoprene gasket on the downstream filter face between the filter frame and the cabinet body. This system can be made more effective by an arrangement (such as a spring-loaded mount) which maintains compression of the gasket, thereby preventing unfiltered air from entering the work zone.
Gaskets generally require some method of exerting a constant pressure to compensate for any movement between the filter and the cabinet. Gaskets are also known to suffer from “loss of memory” which refers to the gasket material’s inability to return to its original thickness when pressure is released. If a spring-loaded mount is not incorporated, air leakage past the gasket and into the work zone may occur. It is essential top quality materials exhibiting good long term memory are used in gasket production. Gaskets tend to suffer a reduction of memory with age. In time then, they may allow filter by-pass, which will contaminate the work area and compromise the integrity of the work.
Crossflow cabinets are, by their very nature, designed to rely on the gasket and its continuing successful compression to prevent this filter by-pass. The fan arrangement of the cabinet causes air pressure not only to the filter face but also to its gasket. Obviously then, if the gasket is leaking for any of the reasons outlined above, contamination may result. Downflow, on the other hand, are similarly fitted with neoprene gaskets (and in better quality units, spring-loaded mounts) but they also feature something not usually available in Crossflow: a negative pressure filter seal.
This is achieved by surrounding the filter gasket arrangement with negative rather than positive pressure air. With this design, should the gasket form a less than perfect seal between the filter and the cabinet, then clean filtered air will flow from the downstream (clean) side back to the upstream (dirty) side, rather than the reverse. In this way the integrity of the work zone is maintained despite a mechanical failure of the filter seal.
So reliable is this system, that it is included as a mandatory design feature in all Australian biological safety cabinets and cytotoxic drug safety cabinets complying with current Australian Standards. This may be regarded as one reason why, unlike in the US and the UK, Australian safety cabinets are designed to be reliable enough to return their exhaust air to the laboratory or work room rather than required to be exhausted to the outside.
A very important aspect for those involved in cell culture is to consider the implications of the differing planes of airflow. That is, which airflow is least likely to encourage contamination by the operator or cross-contamination within the cabinet. As a general rule, if the operators hands must work primarily up and down (in a vertical plane), then horizontal airflow – Crossflow, may be most suited. If however, the operation tends to require working back and forward in a horizontal plane, then a cabinet offering vertical airflow – Downflow, might be the better choice.
It has been hypothesised that as people naturally prefer not to constantly change focal distance; then most operators will tend to favour working in a vertical plane. This may account for the fact that there are substantially more Crossflow than Downflow in use.
The important part of all this is that operators looking for the best results (no losses due to contamination) will avoid working upstream of the critical area. That is, they will be aware of the relative positioning of the critical area (the work) and the filter face; never carrying out manipulations in the space between the two.
It may be argued that as Downflow cabinets direct less airflow directly toward the operators face, they therefore offer some greater degree of protection against aerosols created when handling potentially dangerous organisms. Whilst there may be some marginal advantage of the Downflow in this area, whenever there is a risk to operators, a biological safety cabinet should be used.
The primary function of a biological safety cabinet (or “biohazard” cabinet) is to act as a containment device, providing a degree of protection for workers and the environment.
There are two types of biological safety cabinets in common use in Australia: Class I for operator and environment protection, and Class II for both operator/environment protection and protection of the work.
Of all the cabinet types, this is the only one which could possibly be described as a “hood” (or perhaps a “biological fume cupboard”). Air is drawn into the cabinet through an access opening, away from the operator, thus providing an air curtain or barrier between the operator and the work. Obviously this pattern of airflow will actively encourage introduced contamination and it is therefore totally unsuitable for cell culture applications.
A Class II Cabinet combines the functions of a Downflow cabinet with those of a Class I cabinet. The result is a cabinet which, correctly adjusted, protects the cultures from invasion by foreign material. At the same time, it prevents the escape of aerosols which may be harmful to the operator, other workers or the environment in general.
This is achieved in the following way: Vertical laminar airflow through a HEPA filter bathes the work area, dividing and passing around the perimeter of the work tray. This provides a biologically clean work area in which to handle cultures etc. In addition, a barrier of air is drawn across the work access opening and down through the barrier grille into the sump area beneath the work tray. This flow reduces the potential risks to personnel from airborne contaminants generated in the work zone. Both air flows combine in the sump prior to recirculation via the return air plenum to the top housing. Exhaust air (to create the barrier flow) is discharged through another HEPA filter, into the laboratory.
Correct adjustment of these two sets of air flows is critical to maintain the design conditions which ensure firstly, that unfiltered (dirty) air drawn through the barrier doesn't’t contaminate the work tray, and secondly, that the laminar airflow doesn't’t sweep generated aerosols out through the work access opening toward the operator. Separate fan/filter arrangements, each with an individual speed controller, allow independent adjustments to maintain correct air flows
To assist in ensuring continuing safe and effective operation of a Class II cabinet, the following points are worth remembering:
- The cabinet should ideally be located in an area isolated from other work and major air movements (doorways, windows, air diffusers etc) and personnel traffic; if possible, in an isolated dedicated room or area.
- Attempt to utilise the middle area of the work tray and, at the very least, avoid using the area of turbulence created by the operators forearms near the barrier grille. Materials which are for use in the operation at hand may be stored in the cabinet, preferably to the sides, but so that there is a space for air movement next to the side walls. Large items should be avoided as they create unacceptable levels of work zone turbulence and increase the potential for cross-contamination. Equipment not required for a particular operation should be removed after disinfection.
- The use of plastic-backed absorbent sheeting or mats is encouraged as they quickly soak up splashed droplets and spillages, assisting containment and making clean ups easier and safer.
- Avoid unnecessary and rapid hand/arm movements, especially in and out of the cabinet across the air barrier. Movements in this region should be careful and deliberate to reduce the chance of inducing dirty air from outside the cabinet. Also, this will allow the laminar flow to ‘shower’ the hands in a fashion which will encourage any released particulate matter to be swept away from the work.
- Laboratory coats worn when using the cabinet should be continuous-fronted and manufactured of low-shedding fabric to reduce the amount of fibrous material released and drawn into the cabinet. Styles which include adjustable or elasticised wrist closures are preferred. The wearing of thin gloves is encouraged as they provide some protection to the operator and help to reduce the likelihood of particle release from the hands.
- Frequent routine cleaning, including areas not immediately accessible (such as below the barrier grille, work tray and in the return air plenum), should be carried out. Apart from the increased likelihood of culture contamination when working in a dirty cabinet, a cabinet which is regularly cleaned is likely to cost less to maintain.
A little known fact is that unlike laminar flow cabinets and Class I cabinets, Class II’s do not include a prefilter system. All particulate matter is therefore collected on the HEPA filters. If not thoroughly and regularly cleaned, dust and fibres which have accumulated in the sump area and in the return air plenum, will eventually be released into the airstream, causing a reduction in effective HEPA filter life, leading to their premature replacement.
Until recently, Class II cabinets were being used for the reconstitution and dispensing of drugs for chemotherapy. These cabinets appeared appropriate as they provide a sterile work area in which to handle the drugs, and in addition, an air barrier containment system which ensures the pharmacist is not exposed to liberated product particles.
However, unlike biologically active materials which contaminate areas inaccessible for manual cleaning (such as the top housing), and are deactivated by fumigation, these cytotoxic materials are not so easily neutralised. They therefore present unique problems when internal access for filter replacement etc becomes necessary. To overcome this, a modified cabinet, one specifically designed for handling these and similar materials, was developed.
A cytotoxic drug safety cabinet is essentially a Class II biological safety cabinet with one very important distinguishing difference. In a Class II cabinet, the work zone airstream in combination with the barrier air, is recirculated via the return air plenum to the top housing, allowing contamination of surfaces, fans and filters.
By comparison, the cytotoxic cabinet features a HEPA filter located beneath the work floor, through which all air passes prior to further movement within the cabinet. In this way, cytotoxic contamination is limited to the upstream face of this lower filter and a special procedure is followed when filter changing becomes necessary. The result is that the rest of the cabinet remains clean and may be accessed without special preparation.
Except for some specialised procedures involving the exposure of cells to agents which are cytotoxic or may present similar hazards in handling, cytotoxic drug safety cabinets have limited application in cell culture.
Laminar flow cabinets, either vertical or horizontal, may be used to enhance sterile conditions required for cell culture procedures. The choice between the two types may be made on the basis of technical differences (in ensuring continuing high air quality), size and configuration of work area, and envisaged techniques to be used. They offer no protection to the operator and environment.
If the procedure presents any risk to operators, a biological safety cabinet Type2 should be employed as it offers protection for both the work, personnel and environment.
Biological safety cabinets Type 1 are not suited for sterile procedures as they provide no means of maintaining the integrity of the culture but offer protection to the operator and environment.
Cytotoxic drug safety cabinets are not often used for cell culture purposes. While they offer the same protections as a type 2 cabinet, the extra costs, of providing control of cytotoxic drug risks, are only worthwhile if you are doing that work.
The UV lamp must never be switched on unless the front covers are in place.
Biological Safety cabinets present an enclosed environment with a forced air supply. Flames and flammable material are, therefore, a particular hazard.
- Avoid flames if at all possible.
- Minimize flammable liquids and solids in the hood when a flame is used (nothing more than 50mls).
- Use a spirit burner if possible.
- Gas flames can only be used if they have a safety gas cut-off, if the flame goes out.
- Bottled gas burners are forbidden in biosafety & laminar flow cabinets.
Biosafety cabinets will be tested annually by a NATA accredited technician. A label showing test status will be attached to the cabinet.
- Testing contractors will have a safety induction before starting the testing program.
- All Biosafety cabinets used with infective agents and toxins will be tested at a time agreed between the contractor and lab supervisor.