This follows on from the work by Schreuder BEC at Lelystad in Holland in which scrapie was well demonstrated by tonsillar biopsy. Patients were diatnosed by both brain and tonsillar biopsies and the results compared. A stain of the tonsil folicular tissue with anti-PrP ELISA staining histopathology techniques showed the PrPsc to be present in reticulated patterns within the tonsil. It could also be shown that the electrophoretic pattern western blot of the PrPsc could perhaps be identified as nv-CJD by the demonstration of the glycoform.
The diagnosis of Creutzfeldt-Jakob disease (CJD) can only be confirmed by brain biopsy or at necropsy, although a rapidly progressive dementia, myoclonus, other neurological signs, and a characteristic electroencephalogram allows confident ante-mortem diagnosis in typical cases, albeit at a relatively advanced clinical stage. A new clinicopathological type of CJD, new variant CJD (nvCJD) has been reported in the UK and putatively linked on epidemiological grounds with dietary exposure to bovine spongiform encephalopathy (BSE).1
Evidence in support of a causal link between BSE and nvCJD is provided by experimental transmission of BSE to macaques, revealing neuropathological similarity to nvCJD, and by the demonstration that nvCJD is associated with a molecular marker that distinguishes it from other forms of CJD and which resembles that seen in BSE and transmitted BSE in a number of other species.2 To date, 14 nvCJD cases have been neuropathologically confirmed in the UK and one in France.
It is at present unclear how many further cases of nvCJD are likely to appear. In nvCJD, confident diagnosis depends on neuropathology, either from a brain biopsy or at necropsy because the clinical course is atypical and the characteristic electroencephalogram of CJD is absent.1 Furthermore, brain biopsy carries a notable morbidity and may give a falsely negative result if the area of brain sampled is unaffected or lacks the specific pathological features of nvCJD.1 If nvCJD is caused by BSE, and an epidemic ensures, early diagnostic markers will be essential for differential diagnosis as the presenting clinical features (depression and sensory disturbance) are non-specific and many may be concerned that they have developed this condition. Recently, we reported that nvCJD is associated with a specific pattern of protease-resistant prion protein (PrP) on Western blot analysis.2 This marker can already be used to aid differential diagnosis on brain biopsy samples.2 However, since PrP is widely expressed outside the central nervous system, we investigated whether an alternative and more accessible tissue might be biopsied to allow a diagnosis of nvCJD before death and to avoid brain biopsy. PrP is expressed in the lymphoreticular system and prion replication is known to occur in the spleen and other lymphoreticular tissues in experimental rodent scrapie models;3 prion infectivity has also been reported in human lymphoreticular tissues.4
We have studied PrP in tonsillar tissue obtained at necropsy using both immunohistochemistry on periodate-lysine-paraformaldehyde and formalin-fixed tissue, and Western blot analysis of frozen tissue. The patient was a 35-year-old woman who died after a 14-month illness with depression at onset followed by ataxia, hyper-reflexia, memory loss and dementia; a diagnosis of nvCJD was made by neuropathology. Abnormal PrP staining was present within tonsillar germinal centres (figure, top a and b). Western blot analysis revealed the presence of protease resistant PrP (figure, bottom); confirming the diagnosis of prion disease.
Furthermore, the sizes and intensity ratios of the three PrP bands (representing diglycosylated, monoglycosylated, and unglycosylated PrP) were similar to those seen in brain from the same patient (designated a type 4 pattern2) suggesting that it may be possible to make the specific diagnosis of nvCJD by this technique. However, tonsillar tissues from types 13 CJD2 were not available for comparison. Such study of protease-resistant PrP in lymphoreticular tissue may be particularly relevant in iatrogenic CJD after peripheral prion inoculation (for instance after treatment with human cadaveric pituitary-derived growth hormone) where lymphoreticular involvement would be expected.
Figure legend: (a) tonsillar tissue germinal centre in sub-epithelial lymphoid tissue; (b) serial section following hydrolytic autoclaving using anti-PrP monoclonal antibody 3F4 shows strong staining of cells within the germinal centre which has the morphology of follicular dendritic cells. Tissue fixation was with periodate-lysine-paraformaldehyde. A similar staining pattern but with reduced intensity was present in formalin-fixed tissue. No reaction for PrP was seen in two age-matched control cases dying from other neurological illnesses. Bottom: Western blot of tissue homogenates with anti-PrP monoclonal antibody 3F4In experimental murine and sheep scrapie, prion replication occurs initially in spleen and is only detectable in the central nervous system considerably later in the incubation period. Abnormal PrP immunostaining has been reported in tonsils of experimental scrapie-infected sheep long before the occurrence of clinical signs.5 It is possible therefore that Western blot analysis of human tonsil material may allow early clinical, or possibly pre-clinical, diagnosis of CJD and nvCJD although extensive prospective studies of suspected cases will be necessary to assess the clinical usefulness of this investigation.Horizontal lines indicate positions of molecular mass markers (in kilodaltons). Odd numbered lanes are before, and even numbers after, treatment with proteinase K. Lanes 1 and 2: normal human brain; lanes 3 and 4: normal human tonsil; lanes 5 and 6: brain from patient with nvCJD; lanes 7 and 8: tonsil from patient with nvCJD.
Tonsil tissue can be easily obtained by biopsy under local anaesthetic in most patients and complications are most unusual. Although many adults will have had childhood tonsillectomy, lymphoreticular tissue may still be obtained from the lingual tonsillar remnants. As a result of the extreme resistance of prions to normal sterilisation procedures, current UK recommendations are that neurosurgical instruments from patients with CJD are destroyed. While infective titres of prions in tonsil may be much lower than in brain, similar precautions to avoid cross contamination and iatrogenic transmission of CJD would seem prudent at present. The development of a disposable tonsil-biopsy kit should be feasible. 1 Will RG, Ironside JW, Zeidler M, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 1996; 347: 92125.
2 Collinge J, Sidle KCL, Meads J, Ironside J, Hill AF. Molecular analysis of prion stain variation and the aetiology of "new variant" CJD. Nature 1996; 383: 68590.
3 Kimberlin RH, Walker CA. Incubation periods in six models of intraperitoneally injected scrapie depend mainly on the dynamics of agent replication within the nervous system and not the lymphoreticular system. J Gen Virol 1988; 69: 295360.
4 Brown P, Gibbs CJJ, Rodgers Johnson P, et al. Human spongiform encephalopathy: the National Institutes of Health series of 300 cases of experimentally transmitted disease. Ann Neurol 1994; 35: 51329.
5 Schreuder BEC, van Keulen LJM, Vromans MEW, Langeveld JPM, Smits MA. Preclinical test for prion diseases. Nature 1996; 381: 563.
Diagnosing Creutzfeldt-Jakob disease (CJD) may be possible by testing tonsil tissue for the prion protein marker, scientists have discovered.
Professor John Collinge and colleagues from the neurogenetics unit at Imperial College School of Medicine in London last year identified a prion protein (PrP) in the brains of people who had died of new variant CJD disease (2 November, p 1100).
The same team now report in the Lancet (1997;349:99-100) that they have found this marker in tonsil tissue taken from a 35 year old woman who died of new variant CJD. The finding, although preliminary, is important as diagnosis of new variant CJD is currently possible only after brain biopsy or at necropsy. People with new variant CJD tend to be younger than those with other types of CJD, and the first symptoms of the disease-mood changes and alterations in sensation-occur in other diseases. To date, 14 cases of new variant CJD have been neuropathologically confirmed in the UK and one in France.
Professor Collinge said: "It is very encouraging that we have been able to detect the marker in tonsil as this tissue can be sampled relatively easily, usually under local anaesthetic."
Since the first reports of new-variant Creutzfeldt-Jakob disease (nvCJD) appeared in The Lancet in April last year, and it was suggested that this disease was caused by eating Bovine Spongiform Encepatopathy (BSE)-infected beef, there has been a pressing need for a means of diagnosing the disease early. At present, definite diagnosis depends on finding characteristic changes in brain tissue either by an operation before death or at necropsy afterwards.
Professor John Collinge and colleagues recently found a prion protein (PrP) characteristic of nvCJD in the brains of people who had died of the disease. The same research group report in this week's Lancet that they have now found this PrP in tonsil tissue from a 35-year-old woman who died of nvCJD. Taking a biopsy (a small sample of tissue) from tonsils is an easy and safe procedure that can be done under local anaesthetic unlike brain biopsy which can be dangerous.
The importance of this discovery is that people suspected of having nvCJD can now have a simple test to prove or disprove the diagnosis. People with nvCJD are younger than those with other types of CJD and the first symptoms of the disease that many of them have, mood changes and alterations in sensation, occur in other diseases. Minds may be put at rest by such a test, but it may also be useful in testing people who have been exposed to possible infection but have no symptoms.
This finding has another implication. UK regulations require surgical instruments used in brain operations on people with CJD to be destroyed, because they cannot be adequately cleaned of infectious PrP particles by sterilisation processes usually used. The researchers note, "The development of a disposable tonsil-biopsy kit should be possible".
Contact: Professor John Collinge, Neurogenetics Unit, Imperial College School of Medicine at St Mary's, London, UK; tel +44 171 706 7094.
The scrapie-associated form of the prion protein (PrPSc) accumulates in the brain and lymphoid tissues of sheep with scrapie. In order to assess whether detecting PrPSc in lymphoid tissue could be used as a diagnostic test for scrapie, we studied the localization and distribution of PrPSc in various lymphoid tissues collected at necropsy from 55 sheep with clinical scrapie. Samples collected from the spleen, palatine tonsil, ileum, and five different lymph nodes were immunohistochemically stained for PrPSc. PrPSc was found to be deposited in a reticular pattern in the center of both primary and secondary lymphoid follicles. In addition, granules of PrPSc were seen in the cytoplasm in macrophages associated with the lymphoid follicles. In 54 (98%) of the 55 scrapie-affected sheep, PrPSc was detected in the spleen, retropharyngeal lymph node, mesenteric lymph node, and the palatine tonsil. However, only in the palatine tonsils was PrPSc present in a consistently high percent! age of the lymphoid follicles. PrP was not detected in any of the lymphoid tissues of 12 sheep that had no neurohistopathological signs of a scrapie infection. We conclude that the tonsils are the best-suited lymphoid tissue to be biopsied for the detection of PrPSc in the diagnosis of clinical scrapie in living sheep.