BSL-4 Containment Layers of Protection

Containment Talk 8.

Containment talk articles discuss risk and safety issues in and around microbiological and biomedical laboratories (BSL-2, 3, and 4). Feel free to contact me via LinkedIn messaging if you have any questions or comments about laboratory safety, biosafety, biocontainment, design & equipment, engineering, etc.

1. Introduction

In previous containment talk articles, I explained the Swiss cheese risk management model and how to use it for better understanding of the risk controls or safety measures and their effectiveness in BSL-2 and BSL-3 laboratories ([1], [2]).

In the latest containment talk article, I discuss BSL-4 laboratory multiple layers of protection. In contrast to biosafety level 2 and 3 laboratories, it is theoretically possible in BSL-4 facilities to completely isolate the hazard from laboratory personnel and the environment through structural and engineering risk controls. If a BSL-4 facility is designed, constructed, operated and maintained according to the state of the art, the human factor and failure (errors) become less dominant.

BSL-4 facility and administrative requirements are described in EU Directives ([3], [4]) and other national or international biosafety manuals, guidelines and standards ([5]-[9]). In terms of definitions that can be used for (re-)verification or (re?)qualification of the facility, such descriptive requirements are only a start for the architect and engineer. During the design phase, the requirements for design, construction, commissioning and testing must be precisely defined and qualification criteria must be established and agreed. Consultation with the regulatory authority is strongly recommended [10].

There are two categories of BSL-4 laboratories: cabinet-style and suit-style ([5]-[9]).

2. Cabinet-Style BSL-4 Facilities

Cabinet-style BSL-4 facilities consist of a single class III biosafety cabinet (BSC) or a series of connected class III BSCs that comply with either NSF-49 [11], EN 12469 [12], or both. Class III BSCs should not be confused with "isolators" and "glove boxes". As a minimum, class III BSC specifications include defined extreme tightness, controlled and monitored negative pressure, and single inlet and double outlet HEPA filters. There is a very high degree of isolation between the biohazard, the worker and the immediate environment, provided the class III BSC, including the gloves, is of safe design and construction, the proper functioning of the cabinet is monitored and maintained, there is a safe method of loading and unloading the cabinet, and the chemical decontamination of the workspace has been validated, and each process can be verified.

Cabinet-style BSL-4 laboratory rooms are normally designed, built and operated as a BSL-3 laboratory as illustrated in Figure 1 ([5], [6], [13], [14]).

Inappropriate transfers into and out of the class III BSC, such as samples, waste, consumables, labware, kits, small equipment, etc., can be a source of exposure to the biohazard, contaminating personnel and the laboratory. To mitigate the transfer risk, a state-of-the-art class III BSC must have dunk tanks, rapid transfer ports, or both ([13], [14]). Another option is to attach a class II BSC to the pass-through box of the class III BSC. In particular, the removal of potentially contaminated items from the class III BSC and subsequent workflows and processing are largely dominated by human performance and behaviour [5].

Ideally, the cabinet’s workspace can be decontaminated with automated chemical decontamination unit (hydrogen peroxide) and it also has an integrated autoclave. Attached incubators, microscopes, ultra-centrifuge, and other equipment as needed per the protocols can be used to enhance work efficiency and safety [5], [14].

When working in the cabinet, isolation of the biological hazard from the personnel is ensured by safe cabinet engineering and sturdy gloves, as shown in Figure 2.

The ergonomic factor for working at a class III BSC is very often not recognized or neglected by users. class III BSCs intended for work at BSL-3 and BSL-4 should be designed in such a way that they do not cause musculoskeletal strain even during prolonged work, e.g. in a sitting or standing position, and that people with different physical conditions can work with them [5], [15]. Another significant problem is that only one size of glove is available per cabinet. Dexterity is impaired [5].

Failure to address these points before class III BSC design and procurement may result in a non-functional, unsafe, and non-ergonomic class III biosafety cabinet.

3. Suit-Style BSL-4 Facilities

Main Risk Controls / Safety Measures

In terms of facilities, a suit-style BSL-4 laboratory is characterised by the availability and reliability of architectural and engineering isolation of the work area with regard to environmental protection, which includes the surrounding laboratory rooms and the community [16]. The workers are isolated from the hazard by a full-body pressurised protective suit [17], [18], [19]:

• Consecutive 4 airlocks
• Extremely leaktight containment boundary
• Sophisticated HVAC-system with HEPA-filtered supply air (single filter) and exhaust air (two filters in series), once through air supply with high exchange rate
• Inward airflow (negative pressure cascade)
• Protection of laboratory worker with full-body pressurised protective suit

?The multiple layers of protection to prevent release via the BSL-4 HVAC system are illustrated in Figure 3 using the Swiss cheese model [1]

Two layers of protection, shown in Figure 4, are major cost drivers in terms of capital expenditure (CAPEX) and operating expenses (OPEX):

(i) single-pass or once-through high air change rate (ACH) air supply and

(ii) inward airflow.

High Air Exchange Rates and Inward Airflow

Dilution ventilation systems and high air exchange rates were discussed previously in a containment talk [20]. In essence, the air exchange rate is not the appropriate tool for controlling airborne hazards. Hazards should be controlled at source (e.g., in biosafety cabinets), which is much more efficient and effective than dilution ventilation. Dilution ventilation helps to wash microbial aerosols out of the room, for example after a spill [20].

For BSL-3 and BSL-4 laboratories, an inward airflow must be maintained (Figure 4).

Various guidelines and regulations specify that the inward airflow must be maintained "sustainably", "always", "at all the times", etc. Essentially, it is understood that the HVAC system must provide the inward airflow during normal and special operations such as maintenance, HVAC equipment failure and malfunctions.

For commissioning and certification, the inward airflow through closed doors is verified by the differential pressure across the door using pressure loggers and sampling times of ≤ 5 seconds [10], [21]. Although there is no evidence that cascaded negative pressure prevents cross-contamination of air from higher-risk rooms to lower-risk rooms, it is required by standards and regulations [22]. It is interesting to note that when a BSL-3 or BSL-4 containment door is opened, regardless of whether it is leaky or tight by design, the inward air flow velocity through the door opening is only in the order of 2 (two) centimetres per second!

This low velocity is not relevant to the separation of the two air spaces. It has been known for decades that the turbulence caused by the movement of the door leaf and the movement of people through the door causes significant mixing of the air between the two spaces [23], [24].

BSL-4 Protective Suit

Within the laboratory area, personnel use pressurised suits with their own autonomous breathing air supply. Suit selection and validation of chemical decontamination in the chemical shower airlock must be completed before the facility is commissioned. [17], [18], [19].

The suit is checked for leakage before each use. Adherence to SOPs, such as not bending down and standing up quickly to prevent negative pressure in the suit, or how to deal with punctures and tears in the suit, will reduce the risk of exposure.

The validation, SOPs, and safety checks should be reviewed annually.

Chemical Shower Airlock

When correctly designed, validated and applied, the chemical shower lock is a perfect barrier when the process has been properly tested and validated. From the microbiologists' and virologists' point of view, the containment boundary could therefore only include the chemical shower lock and not the subsequent series of anterooms/body showers, for two reasons:

First, because human risk group 4 pathogens include only viruses (arena viruses, crimean-congo haemorrhagic fever virus, filoviruses, certain tick-borne viruses, hendra viruses) that are highly susceptible to chemical decontamination. Second, if the chemical shower were not reliable, taking off the protective suit would create a potential exposure situation that could not be mitigated by the subsequent body shower (Figures 4 and 5).

However, most BSL-4 regulations and standards consider the suit and body shower room to be part of the containment zone or area. Some regulations even include the personal changing room or outer changing room as part of the containment area.

4. Conclusion

BSL-4 laboratories should be used for work "with hazardous and exotic biological agents that pose a high individual risk of life-threatening disease that can be transmitted via the aerosol route and for which no vaccine or therapy is available" [25]. BSL-4 laboratories therefore provide almost complete isolation of employees and the environment from the biological hazard. Although they are often overengineered and very expensive, it may be better to err on the side of caution when dealing with biological agents in risk group 4.

5. References

[1]????? Basler & Hofmann (2023). The Swiss Cheese Risk Management Model for BSL-2 LaboratoriesCInternet: https://dx.doi.org/10.13140/RG.2.2.20799.89769. Accessed April 2024.

[2]????? Basler & Hofmann (2023). Risk Management for BSL-3 Laboratories Through Multiple Layers of Protection. Internet: https://dx.doi.org/10.13140/RG.2.2.25833.06243. Accessed April 2024.

[3]????? Directive 2009/41/EC of the European Parliament and of the Council on the contained use of genetically modified micro-organisms. Internet: https://eur-lex.europa.eu/eli/dir/2009/41/oj. Accessed February 2024.

[4]????? Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC) consolidated 26. Apr. 2020. Internet: https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX%3A32000L0054. Accessed February 2024.

[5]????? Bressler, D.S.; Hawley, R.J. (2017). Safety considerations in the biosafety level 4 maximum containment laboratory. In Biological Safety: Principles and Practices, 5th Edition. Dawn P. Wooley (Editor), Karen B. Byers (Editor). pp. 695-717. ISBN: 978-1-683-67313-2. Internet: https://www.wiley.com/en-us/Biological+Safety%3A+Principles+and+Practices%2C+5th+Edition-p-9781683673132. Accessed April 2024.

[6]????? PHAC (2022). Canadian Biosafety Standard, 3rd ed. Internet: https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines/third-edition.html. Accessed February 2024.

[7]????? BMBL (2020). Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th Edition. HHS Publication No. (CDC) 300859. Internet. https://www.cdc.gov/labs/BMBL.html. Accessed April 2024.

[8]????? AS/NZS 2243.3:2022. Safety in laboratories Microbiological safety and containment. Internet: https://infostore.saiglobal.com/en-us/standards/as-nzs-2243-3-2022-117305_saig_as_as_3202662/. Accessed February 2024.

[9]????? WHO (2020). Laboratory biosafety manual, fourth edition. Geneva: World Health Organization; (Laboratory biosafety manual, fourth edition and associated monographs). Internet: https://www.who.int/publications/i/item/9789240011311. Accessed February 2024.

[10]?? Basler & Hofmann (2022). Containment Talk: BSL-3 Laboratory Certification: An Introduction and Overview. Internet: https://dx.doi.org/10.13140/RG.2.2.17444.45449. Accessed April 2024.

[11]?? NSF/ANSI 49 Annex I-1 (2020). (Formerly Annex E) details biosafety cabinet selection, installation, use, lifespan and decommissioning. Internet: https://www.nsf.org/knowledge-library/nsf-ansi-49-annex-i-1.? Accessed November 2023.

[12]?? EN 12469:2000. Biotechnology - Performance criteria for microbiological safety cabinets. Internet: https://connect.snv.ch/de/din-en-12469-2000. Accessed April 2024.

[13]?? HSE (2005). Biological agents. The principles, design and operation of Containment Level 4 facilities. Internet: https://levcentral.com/resources/hse-biological-agents-the-principles-design-and-operation-of-containment-level-4-facilities/. Accessed April 2024.

[14]?? Lackemeyer at al. ABSL-4 Aerobiology Biosafety and Technology at the NIH/NIAID Integrated Research Facility at Fort Detrick. Internet: https://www.mdpi.com/1999-4915/6/1/137. Accessed April 2024.

[15]?? Eagleson, D.C. et al. (2017). Primary Barriers: Biological Safety Cabinets, Fume Hoods, and Glove Boxes. In Biological Safety: Principles and Practices, 5th Edition. Dawn P. Wooley (Editor), Karen B. Byers (Editor). pp. 375-398. ISBN: 978-1-683-67313-2. Internet: https://www.wiley.com/en-us/Biological+Safety%3A+Principles+and+Practices%2C+5th+Edition-p-9781683673132. Accessed April 2024.

[16]?? Basler & Hofmann (2024). Understanding ‘Airtight’ and ‘Gastight’ in Relation to the Design and Construction of BSL-3 and BSL-4 Facilities. Internet: https://dx.doi.org/10.13140/RG.2.2.31256.57601. Accessed April 2024.

[17]?? Kümin, D. et al. How to Choose a Suit for a BSL-4 Laboratory—The Approach Taken at SPIEZ LABORATORY. Internet: https://doi.org/10.1177/153567601101600205. Accessed April 2024.

[18]?? Kasloff.? S.B. et al. (2018). Evaluation of Nine Positive Pressure Suits for Use in the Biosafety Level-4 Laboratory. Internet: https://doi.org/10.1177/1535676018793151. Accessed April 2024.

[19]?? Parks, S. et al. Showering BSL-4 Suits to Remove Biological Contamination. Internet: https://doi.org/10.1177/153567601301800402. Accessed April 2024.

[20]?? Basler & Hofmann (2024). Containment Talk: BSL-2 and BSL-3 Laboratory Air Exchange Rates: A Novel Proposal. Internet: https://dx.doi.org/10.13140/RG.2.2.19903.28329. Accessed April 2024.

[21]?? ANSI (2020). ANSI/ASSP Z9.14-2020. Testing and Performance-Verification. Methodologies for Biosafety Level 3 (BSL3) and Animal Biosafety Level 3 (ABSL3) Ventilation Systems. Internet: https://webstore.ansi.org/standards/asse/ansiasspz9142020?gclid=EAIaIQobChMIzuzfkIaE9AIVg_hRCh2MHwYzEAAYASAAEgL9wvD_BwE. Accessed April 2024.

[22]?? ?Kurth A. et a. (2022). Maintaining differential pressure gradients does not increase safety inside modern BSL-4 laboratories. Internet: https://www.frontiersin.org/articles/10.3389/fbioe.2022.953675/full. Accessed April 2024.

[23]?? Sansone E.B. and Keimig S.D. (1987). The influence of door swing and door velocity on the effectiveness of directional airflow. Proceedings of ASHRAE IAQ '87. 1987: 372–381.

[24]?? Hendinger at al. (2016). Influence of the Pressure Difference and Door Swing on Heavy Contaminants Migration between Rooms. Internet: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155159. Accessed April 2024.

[25]?? BMBL (2020). Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th Edition. HHS Publication No. (CDC) 300859. Internet. https://www.cdc.gov/labs/BMBL.html. Accessed April 2024.