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I. Protocol for the histopathological diagnosis of bovine spongiform encephalopathy

I. Protocol for the histopathological diagnosis of bovine spongiform encephalopathy


The fixative, 10 percent formol saline, is prepared by dissolving 8.5 g of sodium chloride in 900 ml of distilled water and mixing with 100 ml of 40 percent formaldehyde. The whole uncut brain is fixed in 10 percent formol saline using 10 to 20 times the volume of brain, i.e. 4 to 8 litres of fixative. Change the fixative after one week and retain for a further week. If necessary, the brain can be transported in a smaller volume of fixative to a diagnostic centre with appropriate packaging to ensure it safe transit (see Barlow, 1983).


The brain should be cut coronally at 3 to 5 mm intervals. Four brain stem blocks are selected for processing. The recommended sites are: the medulla, at the obex; the medulla, through the rostral cerebellar peduncles; the midbrain, at two levels to include the superior colliculus and the red nucleus.


The selected blocks are returned to fresh formal saline for a further week during which time the fixative should be changed three times before processing. Mechanical agitation of the blocks (e.g. with an orbital shaker) enhances penetration of the fixative.


Tissues should be rinsed in 70 percent alcohol before being placed on the processor. The schedule for a carousel-type processor is shown in Box 1.

Box 1. Schedule for a carousel-type processor

70% alcohol 2 x 30 minutes
90% alcohol 2 x 30 minutes
100% alcohol 3 x 1 hour
Chloroform I 1 x 1 hour
Chloroform II 14 hours
Wax I 2 hours
Wax II 1 hour
Wax III 1 hour

i) The time in chloroform should he adjusted to the maximum that the processor will accommodate within the range of 12 to 18 hours.
ii) Reagents should he changed frequently - after three runs would he a guideline.


The staining schedule for haematoxylin and eosin is shown in Box 2.


Bright field, light microscopic examination is carried out according to standard histopathological practice.


The following guidelines for diagnostic criteria should apply:

Positive. The presence of vacuolation affecting grey matter neuropil and neuronal perikarya with a systematic and usually bilaterally symmetrical distribution (see Wells et al., 1987).

Note that neuronal vacuolation in the red nucleus is an incidental finding in cattle brains and its occurrence must be disregarded for the purpose of BSE diagnosis.

Box 2. Staining schedule for haematoxylin and eosin

De-wax sections in xylene 2 x 2 minutes
Absolute alcohol 2 x 1 minute
70% alcohol 30 seconds
Tap-water 2 minutes
Harris' haematoxylin - acidified 8 minutes
Rinse in tap.water 2 minutes
Acid alcohol (0.1% hydrochloric acid) 10 seconds
Wash in tap-water 5 minutes
"Blue" in saturated (1.5%) lithiumcarbonate 30 seconds
Wash in tap-water 10 minutes
Eosin ("yellowish" - 2% in tap water-plus formalin to 0.2%) 4 minutes
Running water 5 minutes
Absolute alcohol 2 x 2 minutes
Xylene 3 x 1 minute
Mount in DPX  

*The time in haematoxylin will need to be adjusted to provide the correct intensity of stain.

i ) Were necessary, use ''Scotts Tap-water Substitute'' instead of tap-water.
ii) Acidified Harris' Haematoxylin: CLIN Tech, 1-2 Faraday; Way, London SEI 8 :STR.
iii) EOSIN. Yellowish C. 1 45380, BDH/MERCK.


i) Inadequate submission of material with poor representation of lesion target sites or severe postmortem change;
ii) vacuolation of the grey matter neuropil and/or neuronal perikarya evident only as a minimal change.

Note that in all cases where the initial examination is inconclusive, furthertissue sampling, processing and examination should be undertaken at the pathologist's discretion.


i) Absence of lesions in a range of sections which adequately represent lesion target sites;
ii) lesions indicating an alternative neuropathological diagnosis with absence of vacuolar changes in BSE lesion target sites.

Note that where clinical signs have strongly suggested BSE, or an alternative diagnosis, further sampling should be undertaken at the pathologist's discretion to reach a diagnosis.

Unresolved. In a few cases, the pathologist may be undecided and/or unable to classify the histopathology, and may wish to have a second opinion before classifying into one of the other three categories.

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