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The most important site of the brain to be examined for the diagnosis of CWD is the parasympathetic vagal nucleus in the dorsal portion of the medulla oblongata at the obex; this area is involved early in infection in both deer and elk. (J40.66.w1, P10.67.w1)

The segment of medulla oblongata containing the obex can be removed via the foramen magnum. (P10.67.w1)

• Approach the head from the under side.

• Cut through the tissues of the "throat latch" area, from the base of one ear to the base of the other ear.
  • Cutting the skin from the underside (inside out) will reduce dulling of the blade
• Continue the cut through to the junction between the skull and the spinal column (the atlanto-occipital joint).
• Cut through the spinal cord. The cut can be continued through the remainder of the neck and the neck discarded.
• On all sides of the brainstem, use a thin knife or scissors to cut the cranial nerves which branch off from the brainstem, the attachment of the cerebellum (cerebellar peduncles) and attachments of the brain stem to the dura mater.
• Advance an appropriate tool (e.g. a modified spoon or spatula, or a grapefruit knife) into the skull alongside the brainstem to a depth of about 2 inches / 5 cm (D128) (as far as possible, to at least the level of the pons) (D130) and rotate so that the brain stem is severed.
• Gently pull backwards (caudally) with the tool, with additional gentle traction if required using forceps on the caudal end of the brainstem.
• Take a sample from the brainstem, centred on the obex, which is identifiable by the "Y-shape" on the dorsal (top) surface of the brainstem.
• Place the obex sample in 10% clean fresh buffered formalin for fixing: the volume of formalin should be ten times the size of the tissue to be fixed.
• Place fresh samples of brainstem for Western blotting into plastic specimen bags. (D129)

(D128, D129, D130)

• For obtaining the brainstem, including the obex of the medulla oblongata, for histopathology and/or immunohistochemical staining.

• Leave in the appropriate volume of formalin for 3-4 days to allow fixing. After this time the sample may be shipped in a reduced volume of formalin, in an appropriate, leak-proof container.
• "Proper labeling, maintaining label readability, and preventing label separation from specimens are as critical as proper specimen selection and preservation." (B36.2.w2)

• The portion of brainstem removed must include the medulla oblongata at the obex if a proper diagnosis is to be made.
• It is important to ensure that personnel collecting samples are properly trained and that laboratory technicians are vigilant in identifying and rejecting inappropriate (inadequate) samples. Samples should not be collected by owners of captive cervids or by the general hunting public. (D110.w3) 

Proper regard to diagnostic procedures, carcass disposal and disinfection should be maintained, and this technique should be read in association with:

• CWD CONTROL: CWD Quarantine and Disinfection (Overview of Techniques)
• CWD CONTROL: CWD of Deer and Elk Culling and Carcass Disposal (Overview of Techniques)
• CWD CONTROL: CWD Diagnosis and Surveillance (Overview of Techniques)

• Sharp knife for cutting tissues of the neck.
• Thin knife or scissors for freeing the brainstem from its attachments within the skull.
• Tool with a bent end for cutting through the brainstem and pulling it out through the foramen magnum.
• Forceps for holding the brainstem.
• Sufficient volume of formalin for fixing.
• Appropriate size container to hold sample and adequate formalin.
• Leak-proof containers for transporting tissues in formalin.
• Appropriate labels for samples.

• Training and practice is advisable to ensure that the correct tissue are sampled and that the samples remain in good condition for testing. (D110.w3)

• Costs of consumables (containers, formalin etc.).
• Costs of personnel time in taking and processing samples.

• Although the agent causing CWD is not known to be infectious to humans, appropriate protective clothing including gloves should be worn to minimise the risk of any human exposure to the CWD agent. 

THE FOLLOWING CLASSIFICATION SHOULD BE CONSIDERED, AND BIOSAFETY LEVEL CHECKED FOR RECENT ALTERATIONS PRIOR TO COMMENCING STUDIES:

• In the USA the following Biosafety level classifications for TSE agents are suggested in the CDC's "Biosafety in Micribiological and Biomedical Laboratories":
  • "Biosafety level classification. Human prions and those propagated in apes and monkeys are manipulated at Biosafety Level 2 or 3, depending on the studies being conducted. BSE prions are likewise manipulated at Biosafety Level 2 or 3, due to the possibility that BSE prions have been transmitted to humans in Great Britain and France. All other animal prions are considered Biosafety Level 2 pathogens. Thus, based on our current understanding of prion biology described above, once human prions are passaged in mice and mouse PrPSc is produced, these prions should be considered Biosafety Level 2 prions, even though the human prions are Biosafety Level 3 under most experimental conditions. An exception to this statement is in the case of mice expressing human or chimeric human/mouse transgenes. These transgenic mice produce human prions when infected with human prions and should be treated as Biosafety Level 2 or 3 in accord with the guidelines described above." (D131)
  • In the UK for laboratory work with the agent of CWD (as well as human TSE agents and the agents of BSE, FSE and TME), the recommended Overall Laboratory Containment Level for experimental work is level 3, with Animal Containment Level 3 for small animal work and level 1 for large animal work, although some derogations from full Containment Level 3 may be allowed (subject to local risk assessment) since transmission is considered most likely to occur percutaneously or by ingestion (to a lesser extent). (B320)