Prevalence of lymphoreticular prion protein accumulation in UK tissue samples.
ABSTRACT This study aims to provide an estimate of the number of individuals in the UK who may be incubating variant Creutzfeldt-Jakob disease and at risk of causing iatrogenic spread of the disease. Lymphoreticular accumulation of prion protein is a consistent feature of variant Creutzfeldt-Jakob at autopsy and has also been demonstrated in the pre-clinical phase. Immunohistochemical accumulation of prion protein in the lymphoreticular system remains the only technique that has been shown to predict neurological disease reliably in animal prion disorders. In this study, immunohistochemistry was used to demonstrate the presence of prion protein, with monoclonal antibodies KG9 and 3F4, in surgically removed tonsillectomy and appendicectomy specimens. The samples were collected from histopathology departments across the UK and anonymised prior to testing. Samples were tested from 16 703 patients (14 964 appendectomies, 1739 tonsillectomies), approximately 60% of whom were from the age group 20-29 years at operation. Twenty-five per cent of the samples were excluded from the final analyses because they contained inadequate amounts of lymphoid tissue. Three appendicectomy samples showed lymphoreticular accumulation of prion protein, giving an estimated prevalence of 3/12 674 or 237 per million (95% CI 49-692 per million). The pattern of lymphoreticular accumulation in two of these samples was dissimilar from that seen in known cases of variant Creutzfeldt-Jakob disease. Although it is uncertain whether immunohistochemical accumulation of prion protein in the lymphoreticular system is specific for variant Creutzfeldt-Jakob disease, it has not been described in any other disease, including other forms of human prion disease or a range of inflammatory and infective conditions. These findings reinforce the importance of measures taken by the UK Department of Health to reduce the risk of spread of variant Creutzfeldt-Jakob via blood products and surgical instruments, and of the urgency to proceed with large-scale screening of fresh tonsil specimens for the presence of prion protein.
- [show abstract] [hide abstract]
ABSTRACT: New variant Creutzfeldt-Jakob disease (nvCJD) is a novel human transmissible spongiform encephalopathy which was first identified in 1996 in the United Kingdom (UK). Subsequent scientific studies have revealed that the strain of the transmissible agent responsible for nvCJD is identical to that of the bovine spongiform encephalopathy (BSE) agent, and the disease has been considered as 'human BSE'. By 31 December 1999, 52 cases of nvCJD had been reported (49 cases in the UK, two cases in France and one case in the Republic of Ireland). All these individuals were under 53 years of age and all those tested were methionine homozygotes at codon 129 of the prion protein gene. The number of cases of nvCJD likely to occur in the future is impossible to estimate because of multiple uncertainties, in particular the disease incubation period, the degree of exposure to the infective agent and the susceptibility of other genetic subtypes. Continued surveillance of both BSE and CJD is required in the UK and in other countries, to ensure that the scale of this potential epidemic is adequately monitored and that all possible steps are taken to prevent further human exposure to the BSE agent.Revue scientifique et technique (International Office of Epizootics) 05/2000; 19(1):98-120. · 0.69 Impact Factor
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ABSTRACT: Strains of transmissible spongiform encephalopathies are distinguished by differing physicochemical properties of PrPSc, the disease-related isoform of prion protein, which can be maintained on transmission to transgenic mice. 'New variant' Creutzfeldt-Jakob disease (CJD) has strain characteristics distinct from other types of CJD and which resemble those of BSE transmitted to mice, domestic cat and macaque, consistent with BSE being the source of this new disease. Strain characteristics revealed here suggest that the prion protein may itself encode disease phenotype.Nature 11/1996; 383(6602):685-90. · 38.60 Impact Factor
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ABSTRACT: To investigate the extent to which deaths from Creutzfeldt-Jakob disease were misclassified during 1979-96. Structured review of clinical records based on predetermined criteria to determine whether death could have been due to sporadic or variant Creutzfeldt-Jakob disease. 100 health authorities and 275 NHS trusts in England. Subjects: 1485 people who died aged 15-44 years from selected neurological disorders in England during 1979-96. Cause of death. The clinical records of 705 (48%) subjects were successfully traced. Tracing of clinical records was highest in subjects who died during 1990-6. There was sufficient information in the records of 640 (91%) of the 705 subjects to exclude Creutzfeldt-Jakob disease as a cause of death. In 61 (9%) subjects, there was insufficient information to reach any conclusion about the validity of the cause of death recorded on the death certificate. The clinical records of four subjects were examined further by the National Creutzfeldt-Jakob Disease Surveillance Unit; none was thought to have died from Creutzfeldt-Jakob disease. No new cases of sporadic or variant Creutzfeldt-Jakob disease were detected in a sample of deaths most likely to have included misclassified cases. This suggests that the surveillance system is unlikely to have missed a significant number of cases among people aged 15-44 years. Hence, any rapid increase in the number of cases of variant Creutzfeldt-Jakob disease in this age group is likely to be real not artefactual.BMJ 02/2000; 320(7228):145-7. · 14.09 Impact Factor
Journal of Pathology
J Pathol 2004; 203: 733–739
Published online 21 May 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1580
Prevalence of lymphoreticular prion protein
accumulation in UK tissue samples
David A Hilton,1* Azra C Ghani,2Lisa Conyers,1Philip Edwards,1Linda McCardle,3Diane Ritchie,3
Mark Penney,1Doha Hegazy1and James W Ironside3
1Department of Histopathology, Derriford Hospital, Plymouth, UK
2Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College, London, UK
3National CJD Surveillance Unit, University of Edinburgh, Edinburgh, UK
Dr David A Hilton, Department
of Histopathology, Derriford
Hospital, Plymouth, PL6
Received: 19 February 2004
Revised: 15 March 2004
Accepted: 22 March 2004
This study aims to provide an estimate of the number of individuals in the UK who may
be incubating variant Creutzfeldt-Jakob disease and at risk of causing iatrogenic spread
of the disease. Lymphoreticular accumulation of prion protein is a consistent feature of
variant Creutzfeldt-Jakob at autopsy and has also been demonstrated in the pre-clinical
phase. Immunohistochemical accumulation of prion protein in the lymphoreticular system
remains the only technique that has been shown to predict neurological disease reliably
in animal prion disorders. In this study, immunohistochemistry was used to demonstrate
the presence of prion protein, with monoclonal antibodies KG9 and 3F4, in surgically
removed tonsillectomy and appendicectomy specimens. The samples were collected from
histopathology departments across the UK and anonymised prior to testing. Samples were
tested from 16703 patients (14964 appendectomies, 1739 tonsillectomies), approximately
60% of whom were from the age group 20–29 years at operation. Twenty-five per cent of the
samples were excluded from the final analyses because they contained inadequate amounts
of lymphoid tissue. Three appendicectomy samples showed lymphoreticular accumulation
of prion protein, giving an estimated prevalence of 3/12674 or 237 per million (95%
CI 49–692 per million). The pattern of lymphoreticular accumulation in two of these
samples was dissimilar from that seen in known cases of variant Creutzfeldt-Jakob disease.
Although it is uncertain whether immunohistochemical accumulation of prion protein in
the lymphoreticular system is specific for variant Creutzfeldt-Jakob disease, it has not been
described in any other disease, including other forms of human prion disease or a range of
inflammatory and infective conditions. These findings reinforce the importance of measures
taken by the UK Department of Health to reduce the risk of spread of variant Creutzfeldt-
Jakob via blood products and surgical instruments, and of the urgency to proceed with
large-scale screening of fresh tonsil specimens for the presence of prion protein.
Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John
Wiley & Sons, Ltd.
Keywords: Creutzfeldt-Jakob disease (CJD); prion; screening; immunohistochemistry
Variant Creutzfeldt-Jakob disease (vCJD) was first
recognized as a new and distinctive disease in the UK
in 1996 . Subsequent transmission studies and strain
typing have shown that the transmissible agent causing
vCJD exhibits identical characteristics to the bovine
spongiform encephalopathy (BSE) agent [2–4] and
there is no evidence that vCJD occurred prior to 1995
[5,6]. These data indicate that vCJD is a new disease,
almost certainly caused by exposure to the BSE agent.
This conclusion has led to concern about a possible
human epidemic of vCJD, particularly as it is likely
that over 400000 infected cattle entered the human
food chain in the UK prior to the introduction of the
specified bovine offal ban in November 1989 and as
the ban was not fully effective for several years, a large
number of infected cattle also entered the food chain
in the early 1990s . There have been a number of
attempts to predict future numbers of vCJD cases using
mathematical models and extrapolating from vCJD
cases seen to date [8–13]. Recent estimates based on
the pattern of clinical cases suggest that the epidemic
of vCJD will be relatively small, with an upper 95%
confidence interval of 540 future cases . However,
remaining uncertainties, including the possibility that
other genetic loci affect susceptibility , make
the distribution and timing of any human epidemic
unclear. Furthermore, such models are unable to
estimate the prevalence of asymptomatic infection and
hence provide any estimate of the potential number
of future infections and cases that could arise from
secondary (human-to-human) transmission of vCJD.
In addition, questions have been raised as to the
Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
734DA Hilton et al
safety of some food products not covered by the
specified bovine offal ban [15,16] and it is not known
if BSE has entered the British sheep flock, factors
which could alter predicted numbers of vCJD cases
. These uncertainties make decisions about health
care planning problematic, particularly measures to
reduce the risk of iatrogenic spread of vCJD. In
order to reduce these uncertainties, some form of
population screening is required. However, the lack
of a conventional immune response and the failure to
date to demonstrate abnormal prion protein (PrP) in
blood in vCJD  have made the development of a
diagnostic blood test difficult. If a blood test becomes
available for symptomatic vCJD, it may be several
years before it is known whether pre-clinical disease
could be reliably detected.
It has been known for some time that lymphoretic-
ular accumulation of PrP occurs early in murine mod-
els of scrapie , even when incubation periods are
long . This lymphoreticular involvement has been
successfully used in the development of a tonsillar
biopsy as a pre-clinical test for scrapie in sheep .
Although widespread lymphoreticular involvement is
not a feature of BSE in cattle , extensive lym-
phoreticular PrP deposition has been found in all cases
of symptomatic vCJD examined to date [22,23] and
in two cases in appendicectomy specimens removed
prior to the onset of symptoms [24,25]. On the basis
of these data, we have screened large numbers of
appendicectomy and tonsillectomy specimens for the
presence of abnormal lymphoreticular PrP deposition.
Although the antibodies used in this study cannot dis-
tinguish PrPcfrom PrPSc, immunohistochemical accu-
mulation of PrP within lymphoid tissue correlates with
the detection of protease-resistant PrP by western blot
analyses in human tissues  and immunohistochem-
istry remains the only technique that has been shown to
predict disease in animals reliably [26,27]. This study
was primarily designed to look for evidence of a large
epidemic, but also to provide information about how
many individuals are at high risk of developing vCJD
and causing iatrogenic spread. Interim results from this
study have been published previously [25,28]. How-
ever, the study has now been completed following the
examination of additional cases.
Materials and methods
Appendicectomy and tonsillectomy samples were
identified by Systematized Nomenclature of Medicine
(SNOMED) searching of the computerized databases
of 63 histopathology departments across the UK. Ini-
tially, samples from the age range 10–50 years were
included. However, following negative findings in the
first 3000 cases , it was decided only to examine
appendix samples from individuals aged 20–29 years,
as this represents the highest risk age group for vCJD.
Tonsil samples included all ages, as fewer samples
were available for examination (most tonsillectomy
samples are discarded rather than sent to histopathol-
ogy departments for diagnosis and archiving). A max-
imum of two tissue blocks was examined for each
case. Only samples removed from 1995 onwards were
included, as these represent a longer time from possi-
ble BSE exposure than earlier samples and therefore a
greater likelihood of PrP being detectable. Tissue sam-
ples were collected into batches of at least 1000 cases
and given a randomly obtained study number prior to
testing, in order to protect the anonymity of positive
individuals. Batches of samples from England were
tested at Plymouth and, from Scotland, at Edinburgh.
The study received approval from the South and
West Multi-centre Research Ethics Committee (MREC
reference 99/6/32) and for each of the centres included,
appropriate local research ethics committee approval.
The ethical approach has been discussed previously
 and in view of the lack of direct patient consent
and uncertainty of the significance of a positive result,
the study design was anonymous.
Four-micrometre sections were cut from tissue blocks
at two levels 100 µm apart. Sections were pretreated
by autoclaving at 121◦C for 10 min, followed by
immersion in 96% formic acid for 5 min and digestion
with proteinase K (10 µg/ml) for 5 min at room
temperature, in order to enhance PrPScdetection and
reduce PrPcdetection. PrP was detected using the well-
characterized and widely used monoclonal antibodies
3F4 (Dako, UK) and KG9 (IAH, TSE Resource
Centre, UK) [22,24] and visualized using the CSA
kit (Dako, UK), which gives superior results in terms
of sensitivity to most other immunohistochemical
detection systems . A section from each case was
stained with haematoxylin and eosin for morphological
assessment. Autopsy tonsil tissues from confirmed
cases of vCJD were used as a positive control for each
group of slides stained by immunohistochemistry for
PrP; negative controls were performed by omitting the
primary antiserum. Thirty cases from each batch of
1000 were exchanged between the study centres and
tested ‘blinded’ to the findings of the other centre, for
quality control and validation of results. In order to
minimize the possibility of human error, the samples
were tested and analysed with each of the antibodies
on separate dates.
All sections were examined by an experienced neu-
ropathologist (DAH at Plymouth and JWI at Edin-
burgh). Cases with fewer than five secondary lymphoid
follicles were excluded from the final analyses because
in the original reported case  and those examined
at autopsy (personal observation JWI), PrP could be
demonstrated in only approximately 20% of follicles.
Sections were recorded as positive if PrP staining was
detected in follicular dendritic cells or tingible body
macrophages in lymphoid follicles.
J Pathol 2004; 203: 733–739
PrP accumulation in UK tissue samples735
Simple summary statistics were calculated in Microsoft
Excel. Exact binomial confidence intervals were cal-
culated for the prevalence estimates. The expected
number of individuals incubating vCJD was calculated
using estimates of the UK population size stratified by
The numbers of cases examined and the age distri-
bution are summarized in Table 1 and Figure 1. The
age distribution of our sample is heavily weighted
towards the high-risk age group (based on cases of
vCJD to date, see Figure 2). The majority of the spec-
imens examined were appendicectomies, reflecting the
availability of samples within histopathology depart-
ments (most tonsillectomy specimens are discarded
after surgery in the UK).
The number of secondary lymphoid follicles varied
considerably between appendicectomy cases, but in
about 25%, fewer than five were present on the first
level and these were therefore excluded from the
figures for analyses. Most of these excluded cases
were severely inflamed, although some showed fibrous
obliteration, and none was considered positive. The
median number of secondary lymphoid follicles in
Table 1. Summary of the samples used in the
Appendicectomy specimens tested
Tonsillectomy specimens tested
Excluded from analysis∗
Total included in analysis
∗Due to inadequate amounts of lymphoid tissue.
†10260 from England and 2414 from Scotland.
proportion of sample
age at removal of tissue
Figure 1. Age distribution of the samples included in the study
age at onset
Figure 2. Age distribution at onset of vCJD cases to end
the remaining appendicectomy cases, which were
included in the study, was 22 at the first level and
most had several additional follicles examined at the
second level. Most tonsil specimens included over 100
secondary lymphoid follicles, although in about 10%
of samples, fewer than five were present.
In the majority of samples, fine granular PrP
immunoreactivity was noted in nerve fibres and the
myenteric ganglia with both antibodies, and in a
few cases, PrP immunoreactivity was also noted
in epithelial cells immediately adjacent to acute
inflammation. In three appendicectomy cases, we
identified PrP immunoreactivity in lymphoid follicles,
which was seen in sections tested at both centres. None
of the tonsillectomy samples was positive.
In the first positive case (previously published ),
immunoreactivity was seen in the sections stained
using KG9 and was limited to one of the six sec-
ondary lymphoid follicles present, with a distribution
suggesting that it was within follicular dendritic cells
(Figure 3A). The pattern of staining, in particular the
coarse granularity (Figure 3B), was very similar to
that seen in the two other cases who subsequently
developed vCJD [24,25]. However, staining was less
evident in sections immunostained with the 3F4 anti-
body. The reason for this discrepancy is not entirely
clear, although we feel that the most likely explana-
tion is sampling error due to the focal nature of the PrP
deposition. This positive case also showed evidence of
acute appendicitis in adjacent tissue, but there was no
morphological evidence of any other disease process
in an adjacent haematoxylin and eosin-stained section.
The second positive sample showed extensive
staining in 31 of 68 secondary lymphoid follicles
(Figure 3C); this was seen with both antibodies,
although it was less intense with 3F4. The staining had
a finer granular pattern and appeared confined to fol-
licular dendritic cells (Figure 3D). The appendix did
not show any acute inflammation. A very occasional
J Pathol 2004; 203: 733–739
736DA Hilton et al
Figure 3. Immunoreactivity with monoclonal anti-PrP antibody KG9 in the three study cases. The first positive case shows
granular staining of follicular dendritic cells in one follicle (A), including numerous coarse granular aggregates (B). The second
positive case shows intense PrP immunoreactivity in two follicles (C), with a predominantly finely granular pattern in follicular
dendritic cells (D). The third case shows a mixture of granular follicular dendritic cell staining and accumulation within the
cytoplasm of macrophages (E)
multinucleate cell was noted in the submucosa of this
case, but not within germinal centres.
The remaining positive case showed staining in
three of 46 secondary lymphoid follicles, which was
similar with both antibodies. Fine granular immunore-
activity was present in cells with the morphology of
follicular dendritic cells, and within the cytoplasm of
cells with abundant eosinophilic cytoplasm, presum-
ably macrophages (Figure 3E). Acute inflammation
was not present.
If lymphoreticular immunoreactivity for PrP is a
reliable marker of pre-clinical infection, the estimated
prevalence of vCJD based on these three positive
samples in 12674 tested will be 237 infections per
J Pathol 2004; 203: 733–739
PrP accumulation in UK tissue samples 737
million population (exact 95% CI 49–692 per million).
If we assume that this estimate relates to those aged
10–30 years (83% of the sample), then this translates
to a best estimate of 3808 individuals (95% CI
785–11128) aged 10–30 years incubating vCJD. If
only the one case with a similar pattern to that seen
in previous cases of vCJD is considered, then the
estimates will be correspondingly lower (prevalence
of 79 infections per million population, 95% CI
2–440). In contrast to these high estimates, clinical
case numbers remain at a much lower level and have
been declining since 2000, with only 18 deaths in
This study provides an estimate of the prevalence
in the UK population of abnormal lymphoreticular
accumulation of PrP. However, our findings need
to be interpreted cautiously, in terms of the clinical
significance of both negative and positive results.
One major limitation of this study in estimating
the prevalence of asymptomatic infection and predict-
ing future numbers of vCJD cases is that it is not
known at what stage during the incubation period PrP
can be detected in lymphoid tissue. In murine models
of scrapie, infectivity can be demonstrated in Peyer’s
patches as early as 1 week after oral inoculation 
and immunohistochemistry can detect PrP in Peyer’s
patches 1 month after intraperitoneal inoculation .
In the tonsils of scrapie-infected sheep, immunohis-
tochemical detection of PrP occurs from 4 months of
age in those homozygous for a susceptibility PrP gene
polymorphism, and by 15 months in heterozygotes,
reliably predicting future neurological disease . A
further study examining tissue from the third eye of
sheep at risk of scrapie found that immunohistochem-
ical detection of PrP in lymphoid follicles predicts
neurological disease with an estimated 87% sensitiv-
ity and 94% specificity . Data are only available
in the pre-clinical phase from three cases of vCJD
; the two appendicectomy samples removed in the
1990s (up to 2 years before symptoms and 4 years
before death) were positive and a third case, removed
in 1987, 10 years before the onset of symptoms, was
negative. This retrospective study has only examined
samples taken from 1995 to 1999, several years after
the peak human exposure to BSE, which is likely to
have occurred between 1988 and 1992, in order to
maximize the chances of identifying positive individ-
uals. Furthermore, we have used a highly sensitive
immunohistochemical technique  and because of
the focal nature of PrP deposition, extensive sampling
of appendix tissue, with a minimum of five (and an
average of more than 20) secondary lymphoid folli-
cles assessed in each case. Using this approach, we
have found that 95% of autopsy appendicectomy sam-
ples from cases of vCJD, with adequate amounts of
lymphoid tissue, test positive . The finding of fine
granular PrP in the myenteric plexus of most sam-
ples (and some epithelial cells adjacent to inflamma-
tion in a few samples) suggests that the proteolytic
digestion used during immunocytochemistry does not
completely remove PrPcand also reflects the high lev-
els of PrPcin autonomic nerves .
Although immunohistochemical accumulation of
PrP in lymphoreticular tissues has not been demon-
strated in any disease other than vCJD [22,33,34], the
significance of the positive samples in this study is not
certain. In one case, the immunohistochemical pattern
of immunoreactivity resembled that seen in appendix
tissue from pre-clinical [24,25] and autopsied cases
of vCJD, but in the other two cases, a more finely
granular pattern of staining was present in relation
to follicular dendritic cells, raising the possibility that
these may be false positives. However, we have been
unable to demonstrate PrP immunoreactivity in a range
of other disorders including other human prion dis-
eases, neoplastic disease, or a range of inflammatory
conditions . Other explanations for our finding of
cases with an unusual pattern of lymphoreticular PrP
immunoreactivity include involvement of other geno-
types (genotype is known to affect the morphological
patterns of PrP deposition in the brain ) or differ-
ing strains of BSE . The anonymous study design
prevents detailed investigation of the positive cases.
However, spare paraffin wax sections were available
from the second and third positive cases and have
been used for transmission studies, but these may be
inconclusive if negative, because of the small amount
of tissue available and the difficulty in transmitting
from fixed tissue . Commercially available anti-
PrP antibodies for immunohistochemistry detect both
PrPcand PrPSc, and although two groups have devel-
oped PrPSc-specific antibodies [38,39], they do not
appear to work for immunohistochemistry (JWI, per-
If our positive cases represent pre-clinical cases of
vCJD, then this will be of some concern, as the preva-
lence is much higher than expected from the observed
incidence of clinical cases, either indicating a future
increase in numbers of vCJD cases or a significant
number of individuals with a ‘carrier state’ . In the
latter context, it is of interest to note that inoculation
of the BSE agent into transgenic mice which express
only the human PrP gene with methionine homozy-
gosity at codon 129 has revealed a high incidence
of sub-clinical infection . In vCJD, immunohis-
tochemical accumulation of PrP correlates with the
presence of protease-resistant PrP, as determined by
western blot examination  and infectivity .
Individuals with sufficient PrP accumulation to be
detected by immunohistochemistry may therefore pose
a health risk to others by causing iatrogenic spread
via surgical instruments, blood transfusion or organ
donation. Infectivity is not fully inactivated by auto-
claving  and CJD has been transmitted by re-use
of surgical instruments , although this risk is likely
to be small (http://www.doh.gov.uk/cjd/consultation).
J Pathol 2004; 203: 733–739
738 DA Hilton et al
However, there has been a recent increase in con-
cern about surgical transmission of CJD, following
the demonstration of low levels of PrPScin the skeletal
muscle and spleen of some patients with sporadic CJD
[45,46] and epidemiological studies that have shown
an increased incidence of sporadic CJD following sur-
gical procedures [47,48]. Abnormal PrP has not yet
been demonstrated in the blood of patients with vCJD
, but the most sensitive test for infectivity remains
intra-species inoculation and data from sheep infected
with BSE indicate that blood-borne transmission is
possible . A recent case of vCJD occurring in
an individual 6 years after receiving a blood transfu-
sion from a patient who later developed vCJD suggests
that human blood is also able to transmit the disease
. Our findings therefore reinforce the importance
of recent steps taken by the Department of Health to
reduce these potential risks, which include the leucode-
pletion of all UK-sourced blood and the introduction
of more stringent decontamination procedures for sur-
The incubation period of vCJD is not known and
although numbers of cases are currently in decline,
the possibility of further rises cannot be excluded.
The average incubation period of kuru and iatrogenic
CJD following peripheral inoculation has been esti-
mated to be about 12 years, with some cases of kuru
occurring more than 40 years after the cessation of
cannibalism [44,51], but these diseases did not have
to cross a species barrier. Data from a geographi-
cally associated cluster suggested that they resulted
from exposure to BSE prior to 1986 (http://www.leics-
ha.org.uk/Publics/cjdrep.pdf), indicating an incubation
period for these cases of 10–16 years.
Our study has demonstrated how a better under-
standing of the pathology of vCJD has allowed the
investigation of an important epidemiological ques-
tion about this disease using archival tissue collections.
However, the techniques used in our study have been
limited to immunohistochemistry, because of the use
of formalin-fixed tissue sections, and by the study
design, which prevents return to a positive tissue sam-
ple for further verification. These factors have limited
the interpretation of our findings. However, we believe
that they are of some concern and require urgent fur-
ther investigation by prospective screening of tissue
from tonsillectomies. By analysing fresh tissue, sam-
ples could be tested with a sensitive assay that allows
for automation  and positive findings could be
reliably confirmed by transmission studies. However,
about half of tonsillectomies are performed on children
under 10 years of age, so many individuals undergoing
this procedure will soon have had little or no exposure
to BSE and therefore the window of opportunity for
such a study will diminish over time.
We would like to thank all participating histopathology depart-
ments for their cooperation and the relatives of victims of vCJD
who gave consent for autopsy tissues to be used as positive
control material for this study. We would also like to thank
the following for their technical help with this study: Suzanne
Lowrie, Margaret Le Grice, Mary Nicol, Chris-Anne McKen-
zie, Rosemary Baugh, Jo Ford, and Christl Donnelly. This study
was supported by grants from the UK Department of Health
(1216963 DAH; 1216982 JWI).
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