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Proceedings of the 4th National Symposium on Biosafety

Biosafety Issues Related to Xenograft Transplantation Bobby Brown, DVM Animal Resources Branch Centers for Disease Control and Prevention 1600 Clifton Road Atlanta, GA 30333 404-630-1320

I want to emphasize again what Dr. Richmond said - what we do in the Biosafety 3 Level is a continuation of all the things and principles that have been set for the Biosafety 2 Level. Many of you are doing most of these things already. We also hear what we call "2 plus"; it would take some refinement of facilities and procedures to make them ABL-3 Level. The standard practices need to be continued and refined. There are some special practices, though, that have to be implemented in order to make it a full Biosafety Level 3 operation, and I'll review some of these with you.

Besides the biohazard sign, we add "BSL-3" to give added emphasis that there is more at risk. People who have any type of immune-compromised status should not be entering the area, and I think even people under severe stress with certain types of infectious diseases probably should be left out of working in that area for a period of time. One of the big features with the Animal Biosafety Level 3, of course, is autoclaving everything coming out of the room (except for personnel). One slightly different feature is how to handle animal carcasses. They should be autoclaved in order to come out of the facility before they go to the incinerator. Animals should be placed in a metal can that is leak proof and large enough to contain any fluids that may drain out. After the autoclaving procedure is completed, the can should be taken directly to the incinerator and the animal carcass incinerated.

There is additional safety equipment needed to maintain Biosafety Level 3. Personal protective equipment, should alwaysbe worn - the full suit is needed when handling infected animals. We provide all protective equipment at the access point, so everything a person would possibly need for protective equipment is available right there. Procedures with some infectious agents might need additional bio-containment devices such as a respirator. Certainly, in handling necropsies and surgeries you need full protection. Animals might be housed in more containment caging systems to minimize the level of exposure or contamination of the area. Placing cages of larger animals inside a containment device that would minimize contamination of the area also adds a greater amount of protection to the worker.

Some secondary barriers that need to be in the facility include a complete separation from other areas, including defined control access. The ABSL-3 area may have card key access so you can readily know who is going in and out. The card key access maintains a record, so you can very easily limit access to the ABSL3 until personnel have received proper training, immunizations, etc. One of the requirements for ABSL3 is that there are two sets of doors between the hallway and the animal. This provides space for changing of protective clothing, and in more modern facilities, usually this includes at least a shower so that you can shower coming in and out of these facilities as needed. Sealing the penetrations in the wall where pipes or other things come through is important because it makes it much easier then to decontaminate. We depend on gaseous formaldehyde for disinfection at times; it makes it a lot easier to maintain the integrity of the room without allowing fumes to seep out. Sealing penetrations has the added advantage in keeping cockroaches from finding hiding places.

Dr. Richmond mentioned the need to charge the drains with water or disinfectant but in most cases drains are not being used now at ABSL-3, being replaced by mop down wipes up. Animal room doors always should be self-closing, and they must be closed when animal work is being done. Autoclaves should be readily available where hazardous work is done in the ABSL-3, and we prefer to have one near the necropsy table and biosafety cabinet. Air flow should always be inward. Air should be exhausted out away from the building and away from any air intakes. HEPA filtration is not required, but it may be installed. Materials coming out, including caging, are autoclaved. Certainly samples for the lab can be passed out in plastic-wrapped containers that are surface disinfected. When these samples are picked up by outside personnel, they are placed in a hazard transport container, carried to the laboratory, and then the container can be opened inside the biosafety cabinet.

Animal Biosafety Level 4 involves a little different aspect of protection. Most of us have heard about all the work going on with Ebola, Filo virus and Hanta viruses. We tend to concentrate on the suit - that everybody's in a space suit. When you're in the suit working in a laboratory, you are only a few millimeters away from the surrounding hazards.

Animals can still bite or scratch. There should be no sharp objects on the cage that might tear the suit. Leather gloves are certainly important to provide extra protection. Gloves can tear, so it's important that they be checked before the suit is put on and replaced at any sign of defect. Wearing the extra heavy gloves to handle any caging materials is essential.

Suits are connected to air lines, essentially the life line that provides the clean air space for you to work in. Air is pumped in through these hoses located in different areas; as you move from one work station to another, the air hose-suit connection has to be disconnected, then reconnected. However, the connection from the HEPA filter into the suit is never disconnected. These suits are sealable. They have a zipper inside and a double locking seal on the outside.

Another feature of the ABSL-4 facility is the doors, one more reason for providing assurance that what we do is beingdone safely. They are made of thick metal with a rubber "balloon" membrane around them which forms a tight seal around the edges, much as a submarine door does, when the doors are closed and the balloon is inflated. Each of the different compartments of the lab is sealed off from the others, and more importantly, all of the inside is sealed from the outside environment in order to provide the extra protection needed. People come in and out wearing their suit through a chemical shower; it's absolutely essential to decontaminate the suit before returning to the clean space, and that is done in the chemical shower. The shower is automatically controlled - you punch the button and disinfectants are sprayed out through a series of nozzles. The suit is saturated and then rinsed off. Since anything that's automatic can fail, we also have pull handles to provide dumping of chemicals onto the suit to assure disinfection.

Animal waste, other materials from the lab, and everything inside a BSL-4 lab comes out through the autoclave. One of the key things is making sure that everybody who has access to removing materials from this autoclave knows to check the autoclave thoroughly to make sure it has completed all the cycles, reached all the proper temperatures and pressure, and more importantly, run for the proper length of time (which is usually one hour at a minimum).

BSL-4 facilities are complex. The middle laboratory animal space is supported by two additional floors. The top floor maintains all of the air-handling equipment for the entire building. It has HEPA filtration of all the air leaving, and it is duplicated so that there is double protection for backup. On the bottom floor, there are large air compressors that provide the air for the suits. There are two of them, one for backup, and in the event that the systems fail, there are cylinders of compressed air that provide back-up air. An other big feature are large holding/cooking tanks. There are no drains in the floor of the BSL-4 lab. All liquid wastes from the sinks and showers are collected in these large tanks and cooked for a long period of time to insure that all microbes are killed before the liquid is released to the sewage treatment facility.

All these elements combined together are what makes the BSL-4 lab work, providing the safe working environment for our emplyees and the protection to the local community. As with major research institutions that conduct animal research, particularly under containment conditions, we spend a lot of time and effort meeting these safety needs.

Symposium Contents

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Office of Health and Safety, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Mail Stop F05 Atlanta, Georgia 30333, USA Last Modified: 1/2/97
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