NEWSLETTER
ISSUE
Jul to Sep, 2021 Volume 15
BRAIN WAVE
Sterilization in BSL-3/BSL-4 Laboratories
Today the world is battling Covid-19 pandemic and the Pharmaceutical & Research institution across the globe are working hard to conquer the virus and save the community. In such circumstances, microbiological research laboratories play a critical role. These laboratories undertake extensive research and manipulation of the deadly agent to develop an effective solution. However, the agent, being highly transmissible, poses a great risk to the Lab Technicians and nearby communities of contracting the lethal disease.
The history indicates that most of the infections caused while handling microbiological agents such as viruses & bacteria were fatal. The major challenge was that around 80% of the reported infection were through infected aerosol. Therefore, challenges in handling research activity of the lethal pathogens require a distinct approach.
The research laboratories handle various types of microorganisms, and each organism poses a different level of risk. Some pathogens may cause lethal disease, and some may not present any threat to personnel or the environment carrying out the research. Based on the risk severity of the pathogens handled at the laboratory, these research labs are assigned with Bio Safety Level designation as BSL-1, BSL-2, BSL-3 & BSL-4.
BSL-1 implies least hazardous and requires no containment, whereas BSL-4 means most hazardous that requires maximum containment.
As BSL-3/BSL-4 laboratories pose a high risk, it requires maximum containment. Achieving maximum containment requires special working procedures, laboratory design, equipment and medical surveillance that prevent occupational hazards.
The research laboratory uses specialized equipment such as Centrifuges, Bio-safety cabinets, Freezers, Sterilizers etc., for various processes. Therefore, while conceptualizing a BSL-3/BSL-4 facility, the design of these types of equipment requires special attention. One of the most critical components of the BSL-3/BSL-4 facility is Autoclave (Sterilizer). The autoclave for such a facility needs additional features against the standard ones used in other facilities. The essential elements of BSL-3/BSL-4 laboratories are Separation, Containment & Decontamination of potentially hazardous agents. Similarly, a sterilizer designed for the BSL-3/BSL-4 laboratory should facilitate Separation, Containment & Decontamination.
Separation
The BSL-3/BSL-4 laboratories ensure physical separation through access corridors, self-closing doors, and double-door access.
Similarly, the sterilizer/autoclave is typically configured with a double-door design wherein one door is connected to the contained side and the other to the non-contained side. Typically, the material movement is from the contained side to the non-contained side. The contaminated or hazardous waste is loaded from the contained side and unloaded to the non-contained side after decontamination. The two major differentiating factors of biocontainment sterilizer are 1). The door sealing gasket should ensure no leakage, and 2). The locking mechanism should ensure that both doors cannot be opened simultaneously.
The gasket design should be passive design instead of active design. A major advantage of passive over the active design is that it eliminates the utility requirement, thus eliminating any chances of microbes escaping in case of utility failure.
The door operation should be provided with an interlock arrangement that ensures the prevention of simultaneous door opening. Moreover, the interlock arrangement should be electro-mechanical, ensuring that the interlock is active even in a power failure. Additionally, the safety feature should prevent door opening till the ambient pressure is attained in the sterilizer chamber. The control system will only initiate a cycle if both the doors are confirmed closed. In case of an aborted or incomplete cycle, the interlock should prevent the opening of the non-contained side door.
Containment
In BSL-3/BSL-4 laboratories, the containment is ensured by adopting two airflow schemes, i.e., a). The Exhausted air is not recirculated, and b). Negative airflow is maintained in the laboratories.
The autoclave for a BSL-3/BSL-4 facility is configured with a Biological sealing flange, commonly known as Bio-seal, to achieve the containment. The Bio-seal provides positive separation and seal between the contained & non-contained rooms and restricts the passing of hazardous microbes from the hazardous side to the safe side.
Bio-seal is installed at both ends, i.e., contained & non-contained side of a sterilizer. The Bio-seal comprises a stainless-steel plate (formed in a box structure) connected/assembled to the sterilizer chamber at one end and to the partition wall at the other end. Assembly at both ends is sealed to provide leak-free joint and prevent contamination. Bio-seal assembly at the sterilizer end is sealed using silicon gasket, whereas, on partition wall end, it is sealed with silicon caulking.
Testing of Bio-seal assembly is necessary to ensure the integrity of the seal and prevent contamination in the non-contained area. Therefore, testing should be performed by trained and experienced personnel who are familiar with the testing standards. The testing of Bio-seal is carried out using the industry-standard bulk smoke leak test. Smoke generated for this test has a particulate size of not more than 0.2 microns. During the test period, the room should be maintained at a negative pressure with a differential of at least 13.7 Pascal. The seal is considered secure if no visible penetration of smoke through the Bio-seal is observed.
Decontamination
The microbes handled at BSL-3/BSL-4 laboratories pose a high risk to the personnel health and nearby community. So, even the effluent from the autoclave must be filtered/decontaminated before releasing it into the environment.
There are different methods to decontaminate effluent from sterilizer such as
- Using a HEPA filter placed within the chamber. The 0.2-micron HEPA filter retains microbes that are aerosolized in the chamber, and the filter gets sterilized during sterilization of the load within the chamber.
- Using HEPA filter enclosed in the filter housing placed outside the sterilizer chamber. The effluent from the sterilizer passes through a 0.2 microns HEPA filter (located on the top of the sterilizer) before going to the drain. The filter housing is heated with steam during the exposure phase to sterilize the filter (with retained microbes) in the housing.
- Using an external decontamination chamber, also known as a Kill tank. Here the effluent is drained to a separate tank that facilitates decontamination using direct steam. In addition, the kill tank enables collecting effluent from multiple equipment (sterilizer, washer etc.) and decontaminate at a single point.
Apart from the above, there are some other considerations to incorporate in sterilizers for the BSL-3/BSL-4 facility.
- The steam entry should be from the bottom of the chamber and exhaust from the top to ensure no virus settles on the drain.
- All the valves should be at least a diaphragm valve to avoid cavities.
- There shall be bacterial filters wherever necessary.
- To avoid any virus escaping while pre and post chamber evacuation, the vacuum pump is connected to the sterilizer through bacterial filters and a Kill tank.
- There shall not be any threaded fittings on sterilizers.
- All the fittings shall be high rated to avoid even nano leaks from the fittings.
- The chamber should be evacuated before the opening of a non-contained door.
- A vacuum break filter should be provided on the non-contained side.
- The Service area consisting of piping and utility components should be located on the non-contained side to prevent contamination and make the area easier for work.
- The autoclave is used to decontaminate the hazardous waste, so the loading side should be on the contained side and unloading on the non-contained.
- Sterilizers are typically equipped with a single control system and printer. The primary control system and printer should be located on the non-contained side so that service technicians need not require to enter the contained side to attend the job. Moreover, having a printer on the non-contained side allows records to be used and stored in the non-contained area. However, it is highly recommended to have a secondary control system in the contained area that enables the start and abort of the cycle from the same area.
- To ensure optimum performance, frequent performance testing of sterilizers should be a part of regular laboratory procedures.
- Only trained personnel should be given the responsibility of operation and routine maintenance of the sterilizer.
References
Laboratory biosafety manual (Third edition) – World Health Organization Geneva 2004
The 1,2,3’s of Biosafety Levels -Jonathan Y Richmond
