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.

Effect of fixation on brain and lymphoreticular vCJD prions and bioassay of key positive specimens from a retrospective vCJD prevalence study

“…PrP Sc -positive 10% w/v homogenates [48] were diluted to 1% w/v with D-PBS and passed through a 25-gauge syringe needle prior to storage at −70 • C. Formalinfixed, paraffin-embedded (FFPE) tissues that had not been exposed to formic acid were each available as three tissue sections each of approximate dimensions 4 µm × 0.5 cm × 1 cm mounted on glass slides. FFPE tissues for investigation comprised vCJD brain and vCJD spleen and two of the three abnormal PrPpositive appendix specimens (both PRNP codon 129 valine homozygous genotype) identified in the prevalence screen by Hilton et al [41]. Tissue was recovered by sequential treatment, firstly to remove paraffin by immersion in two changes of xylene for 5 min, followed by sequential immersion for 5 min in graded ethanol (100% × 2 and 70% × 1), after which tissue was re-hydrated by immersion in D-PBS for 5 min.…”

“…To date, antemortem tonsil biopsy has shown 100% sensitivity and specificity for diagnosis of clinical vCJD [30,33,34], and the fact that lymphoreticular prion infection is not a feature of iatrogenic CJD [33,36] or kuru [21,38] argues that the distinct pathogenesis of vCJD relates to the effect of prion strain rather than to a peripheral route of infection [21,38,39]. Because lymphoreticular colonization is thought to precede neuroinvasion in vCJD, and indeed has been detected in archived surgical samples removed prior to the development of overt clinical symptoms [40,41], anonymous screening of lymphoreticular tissues removed during routine surgery appears to offer a practical means to estimate the population prevalence of asymptomatic vCJD infection. However, the sensitivity and specificity of such testing during the pre-clinical incubation period or in chronic carriers (who may have a distinct pathogenesis which conceivably might not involve the same degree of lymphoreticular colonization) are unknown.…”

“…To date, three prevalence studies have been reported [41,46,47]. The first study used immunohistochemistry to retrospectively examine 11 247 appendicectomy and 1427 tonsillectomy specimens that had been archived as formalin-fixed tissue blocks [41].…”

“…The first study used immunohistochemistry to retrospectively examine 11 247 appendicectomy and 1427 tonsillectomy specimens that had been archived as formalin-fixed tissue blocks [41]. Positive lymphoreticular accumulation of abnormal PrP was found in three appendixes from 10 278 specimens in a patient birth cohort of , giving a population prevalence in this age group of 292 (95% confidence interval 60-853) per million [41,47]. Subsequent studies have prospectively examined only tonsil as this tissue can be readily obtained from large numbers of routine tonsillectomy procedures.…”

“…Inevitably, the failure to identify an unequivocally positive sample in more recent prospective studies has meant that the positive appendix specimens identified by Hilton et al have been the subject of considerable debate [17][18][19]41]. Central to this is whether the identified immuno-reactivity truly reflects authentic prion infection.…”