Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey

Abstract

Objectives: To carry out a further survey of archived appendix samples to understand better the differences between existing estimates of the prevalence of subclinical infection with prions after the bovine spongiform encephalopathy epizootic and to see whether a broader birth cohort was affected, and to understand better the implications for the management of blood and blood products and for the handling of surgical instruments.

Design: Irreversibly unlinked and anonymised large scale survey of archived appendix samples.

Setting: Archived appendix samples from the pathology departments of 41 UK hospitals participating in the earlier survey, and additional hospitals in regions with lower levels of participation in that survey.

Sample: 32,441 archived appendix samples fixed in formalin and embedded in paraffin and tested for the presence of abnormal prion protein (PrP).

Results: Of the 32,441 appendix samples 16 were positive for abnormal PrP, indicating an overall prevalence of 493 per million population (95% confidence interval 282 to 801 per million). The prevalence in those born in 1941-60 (733 per million, 269 to 1596 per million) did not differ significantly from those born between 1961 and 1985 (412 per million, 198 to 758 per million) and was similar in both sexes and across the three broad geographical areas sampled. Genetic testing of the positive specimens for the genotype at PRNP codon 129 revealed a high proportion that were valine homozygous compared with the frequency in the normal population, and in stark contrast with confirmed clinical cases of vCJD, all of which were methionine homozygous at PRNP codon 129.

Conclusions: This study corroborates previous studies and suggests a high prevalence of infection with abnormal PrP, indicating vCJD carrier status in the population compared with the 177 vCJD cases to date. These findings have important implications for the management of blood and blood products and for the handling of surgical instruments.

Fig 1 Process from block collection to returning blocks to participating hospitals (see supplementary file for full details)

Fig 2 Immunolabelling of positive appendix samples and positive control sections stained during same machine run. First and second column: abnormal prion protein (PrP) in positive appendix sample (A28441). A, B: robust immunolabelling with antibody ICSM35 in several follicles (A). Higher magnification (B) shows characteristic labelling of follicular dendritic cells in one follicle. C, D: immunolabelling of section that served as external positive control in same machine cycle, containing section of cerebellum (C) or weak synaptic or perineuronal net labelling in the frontal cortex (D). F, G: antibody KG9 shows weaker but unequivocal immunolabelling of immediately adjacent section, whereas other antibodies (3F4, K, L) and 12F10, P, Q) do not show any signal. All cerebellar sections show robust immunolabelling (C, H, M, R) and there is less intense labelling of a cortical ribbon (D, I, N, S), characteristic of sporadic Creutzfeldt-Jakob disease with type 3 glycopattern. Antibody 12F10 shows slightly weaker detection of cortical PrP (N). Formic acid treated tonsil biopsy shows intense immunolabelling pattern with ICSM35 and KG9. U-Y: distribution of weak, intermediate, and strong immunolabelling of positive appendix (A32182). Red squares in low power overview (Q) correspond to panel R-U. Appendix shows positive labelling in 12 of 15 follicles. U; section corresponding to TS1 (see table 1) is on top, TS2 is located bottom left, and TS3 bottom right. Scale bar corresponds to 400 µm (first, third, and fourth columns; A, C, D, F, H, I, K, M, N, P, R, S), 100 µm (second column, B, G, L, Q), 6 mm (U), and 200 µm (vCJD tonsil column E, J, O, T and V-Y)

Fig 3 Examples of appendix samples diagnosed as positive, suspect, and non-specific for abnormal prion protein (PrP). A-C: examples of positive samples with methionine homozygous genotype at PRNP codon 129. Intrafollicular distribution and intensity of PrP show some variation, and all follicles show crisp, robust staining of follicular dendritic cells. Inset in C shows area with follicular dendritic cell at high magnification. D-F: three samples with variable positive labelling, illustrating variability in size and intensity of staining. Inset in F shows high magnification of follicular dendritic cell in centre. G-I: positive follicular dendritic cell in cases with valine homozygous genotype at PRNP codon 129, showing similar variability as previous cases. K, M: suspect cases, showing weak labelling of structures that may correspond to follicular dendritic cell, but not confirmed in subsequent immunostains. J, L, N: non-specific cases, with antibody binding to structures in follicle centre that do not correspond to viable follicular dendritic cell. J and N are necrotic follicles where follicular dendritic cell structure has disappeared, and L shows antibody binding in an area of poor morphological preservation. Scale bar 200 µm (50 µm in insets)