Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

The overall goal of this imaging procedure is to demonstrate the safety procedures required to perform medical imaging of animals in an animal biosafety level 4 facility. Through imaging in the animal biosaftey level 4 environment, disease progression is assessed non-invasively, and biomarkers are identified that aid in potential therapeutic and vaccine evaluations. As imaging suites are separated into the hot and cold sides, infected animals are imaged safely on the hot side, while most of the imaging equipment remain outside, on the cold side, for easy maintenance.

Our purpose is to demonstrate that we can image these animal models of lethal virus disease in a safe manner, and address the limitations of working in such an environment. Demonstrating the animal procedures will be Russ Byrum, the animal facility manager at the integrated research facility. Our imaging technologist, Chris Bartos works with the comparative medicine staff to coordinate the placement of the animal in the scanner, and to acquire the images.

Procedures involving animal subjects have been approved by the Animal Care and Use Committee and the Institutional Biosafety Committee. To prepare the computed tomography scanner, verify that no staff member is in either the hot or cold sides of the scan room, and the subject table is empty. Ensure that an object representing the density of the head and body of a subject, referred to as a phantom, which is made of air, water, and Teflon, is positioned in the appropriate holder on the scanning table, on the hot side.

From the cold side, check that the phantom is properly affixed to the bed. Advance the phantom into the center of the bore. Acquire a scan of the phantom, using laboratory standardized quality control parameters to ensure the scanner is within the manufacturer's specifications.

Notify comparative medicine staff that the computed tomography, or CT scanner, is ready for use. Turn on the downdraft table in the animal procedure room at least ten minutes prior to use. Clean and disinfect surfaces of the downdraft table, using an approved disinfectant, such as 5%dual quaternary ammonium disinfectant solution.

Spray the interior surfaces, and wipe the surfaces dry after 10 minutes of contact time, then spray and wipe the surfaces with 70%ethanol to remove residual disinfectant. Place equipment and supplies needed on the downdraft table, including the anesthesia induction box and the animal handling gloves. Set up the animal holding device on the computed tomography scanner bed, then set up the vital signs monitor.

Check the isoflourane volume in the vaporizer, and add more isoflourane if needed. Visually inspect the anesthesia machine for leaks, and if needed, correct any problem leading to a leak. Weight the scavenging canister that captures the waste anesthetic gas.

Transfer the cage housing the guinea pig from one of the rodent holding rooms adjacent to the imaging suite onto the downdraft table. Verify the animal identification number on the cage card. Open the lid to the cage, and don animal protective leather gloves over the positive pressure suit gloves.

Gently pick up the guinea pig. Place the animal in the anesthesia induction box, and cover the box with the lid. Remove animal handling gloves and clean suit gloves with 5%dual quaternary ammonium disinfectant solution.

Remove the anesthesia induction box containing the animal and the cage from the downdraft table, and place them on a transport cart. Using the cart, take the animal to the computed tomography scanning room. In the CT scanner room, immediately connect the scavenging canister and anesthesia machine to the induction box.

After initiating anesthesia as directed in the text protocol, in the hot side of the CT scanner room, monitor the animal during anesthesia induction for adequate depth of anesthesia by checking that the animal is unresponsive to external stimuli, and checking the muscular tone. Remove the anesthetized guinea pig from the induction box and place it on the holding cushion on a prone position on the imaging bed. For additional maintenance anesthesia, place the nose cone on the guinea pig and turn on the gas to deliver oxygen at one liter per minute.

Deliver isoflourane as indicated in the text protocol. Check for stable respiratory and heart rates. Turn off the anesthesia to the induction box when the guinea pig is fully anesthetized.

Apply ophthalmic ointment in both eyes to protect the corneal epithelium from drying out. Activate the laser system on the cold side of the CT scanner room to position the guinea pig for CT scanning. Use the table in and out buttons to cover the anatomy of interest under the laser crosshairs.

To set the field of view, use the laser button to set the starting point over the anatomy of interest. Contact the comparative medicine staff on the hot side to place a lead shield between themselves and the computed tomography scanner. Acquire a survey scan for slice placement for CT study images according to the manufacturer's protocol.

Prescribe the slice placement for CT study images on survey images. Coordinate with hot side personnel if contrast injection is used. Acquire the study scan according the manufacturer's protocol, then reconstruct the computed tomography images at the scanner console, also according to the manufacturer's protocol.

Send the images to a picture archiving and communication system. When needed, perform further qualitative and quantitative analyses on this data saved to the archiving system. Transfer the guinea pig from the scanner bed to a micro-isolater cage with food in the CT scanner room.

Clean nitrile gloves after handling the animal. Using the cart, transport the cage back to the housing area in the animal procedure room. Monitor the animal until fully recovered from anesthesia before returning the cage to the HEPA ventilated racks.

Disinfect both the downdraft table in the animal procedure room, and the scanner bay area with 5%dual quaternary ammonium solution. Wipe surfaces with a 70%ethanol solution after a 10 minute exposure to dual quaternary ammonium solution. Wipe items such as sensitive electronic equipment, like the monitor and anesthesia machine, that cannot be sprayed directly, with a dual quaternary ammonium saturated cloth, followed by an ethanol saturated cloth.

Strict adherence to all safety procedures and standard operating procedures for animal handling is essential for working safely in an ABSL4 laboratory. Transferring infected animals within the induction box from the animal procedure room to the imaging suite minimizes the risk of contamination of common corridors. Institution of communication systems involving Bluetooth phones with headsets for the imaging staff on the cold side to contact the comparative medicine staff on the hot side, and visual communication decreases the potential for miscommunication between the separated staff.

By following the proper procedures, no laboratory acquired infections or cross-contamination of animal subjects has been recorded while conducting animal biosafety level 4 research at the IRF Frederic. ABSL4 viral pathogens can induce lung related pathologies, and CT is a valuable tool for monitoring disease progression in animal models of infection. CT images are created from the absorption of x-ray radiation into tissues.

In CT, the greater the density of the object imaged, such as bone, the brighter the object appears. Dense organ tissues such as heart and liver tend to be of intermediate intensity, while air-filled tissues, such as healthy lungs, appear black. CT acquires multiple radiographs over a 360 degree view that is reconstructed into a 3 dimensional view of the body.

Once mastered, this technique can be done in approximately 30 minutes if it is performed properly. By following similar procedures, other imaging modalities, such as PET, SPECT, and MRI can be utilized to answer additional questions of disease pathogenesis, and perhaps even imaging of the virus itself. After watching this video, you should have a good understanding of the complexity of the procedure and the resources needed to perform medical imaging in laboratory animals in a animal biosafety level 4 facility.