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1. Introduction
The neuropathology of the classical human prion diseases,Creutzfeldt-Jakob
dtsease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and kuru, is
characterized by four main features* spongiform change, neuronal loss, ghosts,
and amyloid plaque formation (1-3). These features are shared with prton dis-
easesm ammals; the recognition of these similarities prompted the first attempts
to transmit a human priori disease (kuru) to a primate m 1966 (4), followed by
CJD in 1968 (5) and GSS in 1981 (6). These neuropathological features have
formed the basis of the morphological dtagnosts of human prlon diseases for
many years, although it was recognized that these changes are enormously vari-
able both from case to case and within the central nervous system (CNS) m
individual cases (7). In this respect, it 1s interesting to note that the ortgmal
case reported by Creutzfeldt (8,9) and two of the original cases reported by
Jakob (10-12) do not show any of these characteristic neuropathological fea-
tures; the diagnosis m these cases remains uncertain on review (7,23). How-
ever, at least two of Jakob™s original cases show typical neuropathologtcal
changes and other cases subsequently reported from his laboratory (including
the members of the Backer family) also exhibited classical histological features
(22,14). It is also of interest to note that prton protein (PrP) gene analysis has
recently been performed on one of the Backer family cases,showing a codon
178 Asn mutation with met/val at codon 129 (15). This genotype has been
described m other familial forms of human prion disease (16) (see Chapter 2).

From Methods m Molecular Medrnne Prron Dmeases
Edlted by H Baker and R M Rtdley Humana Press Inc , Totowa, NJ

35
36 lronside
Table 1
Classification of Human Prion Diseases
Creutzfeldt-Jakob disease Sporadic
Famlhal
Iatrogemc
Gerstmann-Straussler-Schemker disease Classical
Variants with neurofibrlllary tangles
Kuru
Fatal famihal insomnia
Atypical prlon dementia


Early clmical and neuropathological reports on human prton diseases suf-
fered from a confusion of nomenclature, in which the significance of the dlag-
nostic feature of spongiform change occasionally was overlooked (14). The
subsequent demonstration that human prton diseases were transmissible rem-
forced the tmportance of spongiform change as a diagnosttc neuropathologtcal
feature, reflected m the use of the term “spongiform encephalopathy” for this
group of disorders (7, I3). Recent substantial advances m the understanding of
the infectious agent, m particular the central role of priori protem in relation to
transmissibthty (Z 7-29) along with increasing knowledge on the pathogenettc
stgmficance of mutations and polymorphisms in the human prion protein gene
(20) have prompted a re-evaluatton of classical neuropathology m this group
of diseases, and a tendency to use the generic term “priori disease” rather than
spongiform encephalopathy (21).
Neuropathological assessmentof the structural changes m the CNS has been
the mainstay in diagnosis of human prton diseasesfor many years (Z,3). A new
range of mvesttgattve techniques, mcludmg PrP gene analysts, PrP mnnunocy-
tochemistry and detection by the Western blot, htstoblot, and immunoblot tech-
niques, priori rod/SAF detection by electronmicroscopy, and transmissibility
to both wild-type and transgemc laboratory animals all now have diagnosttc
applications (17) In the laboratory mvestigatton of human priori diseases a
combined morphological, immunocytochemical, and molecular genetic approach
1sdesirable. However, many casescan be diagnosed on morphologtcal assess-
ment alone, mcludmg the vast maJority of cases of sporadic CJD (1,2).
Histortcally, human priori diseases comprised Creutzfeldt-Jakob disease m
sporadic and familial forms, GSS, a rare inherited disease, and km-u, which
was confined to the Fore tribes m New Guinea. A modified classificatton for
human prton diseases to incorporate recent entitles recognized by clmtcal,
tmmunocytochemtcal, and molecular biological studies in addttton to classical
neuropathology 1sgiven in Table 1. This classificatton will be employed when
discussmg morphologtcal aspects of diagnosis.
37
Diagnosis of Human Pnon Disease

2. Neuropathological Diagnosis
2.1. Autopsy Sampling
In the vast majortty of casesthe neuropathological diagnosis of human prion
diseasesis based on the examination of the fixed brain and spinal cord follow-
ing removal at autopsy. Cortical biopsies are now rarely performed for the
dragnosis of dementia; current guidelines m the United Kingdom require
neurosurgtcal instruments to be destroyed after use on a suspected caseof CJD
(22). Furthermore, the marked variabtltty in the distribution of the cortical
lesions in CJD makes for difficulty in choosmg a biopsy site. since a negative
result could be potentially misleadmg. There is also evidence to suggest that
patients with CJD experience a significant clmical deterioration following brain
biopsy (23)
Autopsy procedures for casesof human prion disease must take mto account
the information known on the transmtssibihty of the agents responsible for
these disorders and the spectaltzed techniques required for decontammation
of the mortuary environment and mortuary equipment followmg autopsy. In
the United Kingdom, the agent responsible for human priori diseases has
recently been reclassified as a Category 3 pathogen (24). Provided adequate
mortuary facilittes exist, a full autopsy may be performed m these cases. How-
ever, when facilities and/or staff are limited, then autopsy may be restrtcted to
the removal of the brain (2.5). This can be accomplished with mimmal con-
tammatton to the immediate environment using tools that can be decontamt-
nated according to recommended standards (24) (see Table 2).
2.1 1. Brain Fixation, Examination, and Sampling
After removal at autopsy, the unfixed brain should be sampled tf fresh fro-
zen tissue is required for molecular biological or transmission studies. Because
of the variability in dlstrrbutlon of the lesions in the CNS, it 1s advisable to
sample at least several corttcal areas, the cerebellum, and bramstem. The brain
should then be fixed m 10% formalm (or equivalent) for a period of 2-3 wk
prior to dissection. In the past, it has been recommended that phenol be added
to the formalm solution to help inactivate the transmissible agent. Phenol IS
now known to be ineffective against the agents responsible for human prion
diseases, and can frequently impair tissue tixatton and morphology, thereby
hindering diagnosis (24). Fixation in formalin will substantially reduce but not
abolish the potential infectivity of CNS tissues in prton diseases, so the fixed
brain should be dissected m a Class 1 safety cabinet to minimize contamina-
tion of the environment (24). Since the greatest risk of infectivity results from
inoculation of CNS tissues, it 1srecommended that dissection of the brain be
performed while wearing gloves that will offer protectton against cuts and
lronside
38

Table 2
Autopsy and Decontamination Procedures for Human Prion Diseases
Autopsy procedures
Access to a “high-rusk” autopsy room is desirable but not essentral
Fully tramed medrcal and technical staff required
Restncted access to autopsy room durmg the procedure
Dtsposable protective clothing to be worn
Open body bag recommended to mmrmrze contamination of the autopsy room
All disposable clothing and drsposable instruments to be incinerated on completron
Nondlsposable mstruments should be autoclaved or decontaminated (see the
followuzg) on completion
Working surfaces to be decontammated with sodium hypochlorrte contammg
20,000 ppm chlorme (see the followuzg) on completion
Decontammahon
Heat-stable equipment and nondrsposable protective clothing
Porous load autoclave
A single cycle 134°C (+4/O) (30 lb psi) 18 mm (holding ttme at temperature)
Six temperature cycles 134°C (+4/O) (30 lb PSI) 3 mm (holding time at temperature)
Nondrsposable nonheat stable equtpment and work surfaces
At least 1 h exposure to sodium hypochlorne contammg 20,000 ppm available
chlorine, wrth repeated wetting
Exposure to 2M sodium hydroxide with repeated wetting
All disposable equipment, temporary bench covermgs, contammated fluids, and
tissues to be Incinerated


inoculation. Ftne chain-mail gloves are avatlable for thts purpose (25); these
have the additional advantage of berng readily autoclaved at the required tem-
perature for decontammation.
Macroscopic exammatron of the brain m human prton drseases wtll usually
yield little in the way of specrtic findings (1,3). Most cases of CJD exhibit
cortical atrophy rn a global drstrrbutlon, whrch 1s often accompanied by cer-
ebellar atrophy, particularly in the superior vermis. Cases of GSS and tatrogemc
(pituitary hormone-associated) CJD may show drsproportlonate atrophy of the
cerebellar hemispheres in addition to the vermrs, often with relative sparing of
the cerebral cortex (2). Stmrlar changes have been described m km-u (26). In
CJD, the brain may exhibit a range of age-associated abnormaltties, which
include meningeal fibrosis and atheroma affecting the circle of Willis. A small
but stgmficant percentage of CJD casesoccur m more elderly mdivtduals, m
whom the brain may exhibit a more severe degree of age-related changes both
on external mspectlon and on mrcroscopy (see Table 3).
On coronal sectioning, the deep gray matter structures (basal ganglia and
thalamus) may also appear atrophied, although this IS also a variable finding
39
Diagnosis of Human Prion Disease
Table 3
Age-Related Changes in the CNS
Macroscopic Memngeal thickening and fibrosis
Atheroma/arteriosclerosis m the circle of Wilhs
Mild cerebral cortical and hippocampal atrophy
Depigmentation of substantia mgra and locus ceruleus
Ventricular dilatation
Enlarged perivascular spaces in deep gray and white matter
Microscopic Variable cerebral cortical and hippocampal neuronal loss
Hippocampal Al3 plaques and neurolibrillary tangles
Occasional Lewy bodies in substantta ntgra and locus ceruleus
Perrvascular lacunes in deep gray matter
Cerebral arteriolosclerosis and calcification


(1,3). The white matter of the cerebral hemispheres often appears normal, but
will be reduced m bulk If severe cerebral cortical atrophy is present, these
features will also be accompanied by ventricular dilatation. Other age-related
changes may be noted m sections of the cerebrum and brainstem, including a
loss of pigment in the substantra nigra and locus ceruleus, hippocampal atro-
phy, and enlargement of the perivascular space around small blood vessels
within the thalamus, central white matter, and basal ganglia (see Table 3)
The markedly variable dtstrrbutron of the charactertstrc pathologrcal features
m human prton diseasesnecessitatesextensive and comprehensrve htstological
samplmg. Representative material should be examined from the frontal lobes
(including the enterorhmal cortex), temporal lobes and msula, partetal
parasaggttal and convexity regions, and occiprtal lobes to include the visual
cortex The hrppocampi should be examined, along with representative regions
of the basal ganglia (to include the caudate nucleus, putamen, globus pallidus,
amygdala, and basal nucleus), thalamus, and hypothalamus. Brainstem sec-
trons should mclude the midbrain, pons (including the locus ceruleus), and
medulla. Cerebellar blocks should be taken to include the vermrs and both
hemispheres (comprismg the cortex, white matter, and dentate nucleus). In
most cases of CJD the characteristrc neuropathological changes are present
bilaterally, although not always in a symrnetrrcal fashion (1,2), so rt is neces-
sary to ensure that both cerebral and cerebellar hemispheres are sampled.
The spinal cord m human prion diseasesusually shows no srgnificant external
abnormahty. The dorsal root ganglia, dura mater, and spinal nerve roots usually
appear normal and no consrstent abnormahties are present on the external sur-
face of the spinal cord. On cross-section, the spinal cord also appears unremark-
able in most cases.Blocks for hrstology should be taken from representative regions
of the spinal cord, dorsal root ganglia, spinal nerve roots, and cauda equina.
lronside
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Fig. 1. Spongiformchange in CJD consists of numerous rounded vacuoles within
the neuropil that occur both singly and in confluent groups, distorting the cortical
cytoarchitecture. Hematoxylin and eosin.


Only by performing extensive histological sampling will the full nature and
extent of any histological abnormalities be identified. This is particularly
important for clinicopathological correlation and will also facilitate differen-
tial diagnosis. The most common disorders that can be confused clinically with
CJD include Alzheimer™s disease, Pick™s disease, and diffuse Lewy body dis-
ease (27). Using the appropriate histological techniques, the mentioned sam-
pling protocol will allow a full assessment of these possibilities.
2.2. Histological Investigation
2.2.1. Sporadic CJD
Most neuropathological studies in human prion diseases are performed on
paraffin-embedded tissues. Tissue blocks from the CNS and other organs can
be decontaminated in 96% formic acid for 1 h (28) prior to processing into
paraffin wax. Sections are then cut at 5 pm in thickness and stained for routine
analysis with hematoxylin and eosin. Spongiform change is most easily recog-
nized at this tissue section thickness; the use of thicker (10 pm or more) sec-
tions carries the danger of misinterpretation of other sponge-like changes in
the cerebral cortex (see the following).
In most cases of human prion diseases the histological features are distinc-
tive and will allow a diagnosis to be reached without undue difficulty. In CJD,
the most consistent histological abnormality is spongiform change (29), which
is characterized by a fine vacuole-like appearance in the neuropil, with vacu-
oles varying from 20-200 pm in diameter (Fig. 1). These vacuoles can appear
in any layer of the cerebral cortex and may become confluent, resulting in
large vacuoles that substantially distort the cortical cytoarchitecture. Vacuola-
Diagnosis of Human Prion Disease 41




Fig. 2. Spongiform change in the cerebellum comprises multiple small vacuoles in
the molecular layer that usually do not appear confluent, with relatively little distor-
tion of the tissue architecture. Hematoxylin and eosin.


tion may also be seen within the cytoplasm of larger neurons, particularly in
layers 3 and 5 within the cortex. Cortical involvement is detectable in most
cases of CJD, and is usually accompanied by spongiform change in the basal
ganglia, thalamus, and cerebellar cortex. Cerebellar involvement is present in
most cases, although the severity and distribution of the spongiform change
within the cerebellum is markedly variable (29). Confluent spongiform change
is unusual in the cerebellum, which may, however, exhibit a widespread
microvacuolar change with smaller vacuoles 20-50 pm in diameter in the
molecular layer (Fig. 2).
In long-standing cases, the neuronal loss and spongiform change maybe so
severe as to result in status spongiosus (13), where widespread coarse vacuola-
tion is present throughout the cerebral cortex, resulting in collapse of the corti-
cal cytoarchitecture, leaving an irregular distorted rim of gliotic tissue
containing few remaining neurons (Fig. 3). The basal ganglia and thalamus
also may exhibit severe neuronal loss with gliosis and atrophy, and in the cer-
ebellum there is often an irregular loss of neurons in the granular cell and
Purkinje cell populations, with proliferation of Bergmann and radial glia.
Spongiform change in most brain regions is accompanied by neuronal loss and
gliosis involving both astrocytes and microglia (Figs. 4 and 5). Microglial
hypertrophy and hyperplasia occur in a widespread distribution within the CNS
in CJD (30), but no evidence of a classical inflammatory or immune response
occurs. Microglia are also implicated in the pathogenesis of PrP plaques (see
the following) (31).

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