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

Abstract

Anonymous screening of lymphoreticular tissues removed during routine surgery has been applied to estimate the UK population prevalence of asymptomatic vCJD prion infection. The retrospective study of Hilton et al (J Pathol 2004; 203: 733-739) found accumulation of abnormal prion protein in three formalin-fixed appendix specimens. This led to an estimated UK prevalence of vCJD infection of ∼1 in 4000, which remains the key evidence supporting current risk reduction measures to reduce iatrogenic transmission of vCJD prions in the UK. Confirmatory testing of these positives has been hampered by the inability to perform immunoblotting of formalin-fixed tissue. Animal transmission studies offer the potential for 'gold standard' confirmatory testing but are limited by both transmission barrier effects and known effects of fixation on scrapie prion titre in experimental models. Here we report the effects of fixation on brain and lymphoreticular human vCJD prions and comparative bioassay of two of the three prevalence study formalin-fixed, paraffin-embedded (FFPE) appendix specimens using transgenic mice expressing human prion protein (PrP). While transgenic mice expressing human PrP 129M readily reported vCJD prion infection after inoculation with frozen vCJD brain or appendix, and also FFPE vCJD brain, no infectivity was detected in FFPE vCJD spleen. No prion transmission was observed from either of the FFPE appendix specimens. The absence of detectable infectivity in fixed, known positive vCJD lymphoreticular tissue precludes interpreting negative transmissions from vCJD prevalence study appendix specimens. In this context, the Hilton et al study should continue to inform risk assessment pending the outcome of larger-scale studies on discarded surgical tissues and autopsy samples.

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

“…FVB mice in the experimental groups were euthanized when they developed clinical signs of TSE. C57BL mice were euthanized periodically during the incubation period (6,9,12,15, and 20 weeks post inoculation (wpi)) and at clinical onset (ϳ23 wpi). Negative controls were euthanized at the end of the experiment, together with the last mouse from the experimental group.…”

“…Importantly, PrP TSE has been detected in whole blood of vCJD patients (6 -8). Moreover, a number of healthy people have been found to harbor this agent in lymphoreticular tissues (3,9,10); however, it is not known whether PrP TSE circulates in the blood of these individuals and whether they will develop the disease later in life. Recently, infectivity was reported in the plasma of two out of four patients affected by sporadic CJD (sCJD) (11).…”

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

“…This could have an impact in tissues with lower levels of prion infectivity. For example, a recent study has shown that, while homogenates derived from vCJD appendix could transmit disease to 50% of transgenic mice expressing human PrP C , an FFPE vCJD appendix did not cause an infection (43). We therefore cannot exclude the possibility that low levels of infectivity which might have been present in sCJD heart tissue were inactivated following formalin fixation and paraffin embedding.…”

Accelerated, Spleen-Based Titration of Variant Creutzfeldt-Jakob Disease Infectivity in Transgenic Mice Expressing Human Prion Protein with Sensitivity Comparable to That of Survival Time Bioassay

“…However, the level of sensitivity achieved would be compatible with detection of prionemia (18), at variance with RIII mice (26). Such an assay could complement cell-free (47)(48)(49)(50) or standard (7) biochemical assays, when proving or confirming the presence of bona fide infectivity becomes an issue, due, for example, to inconsistencies among the biochemical tests and/or tissue resampling (51) or to the use of fixation treatments known to alter prion infectivity (11). This will be key to ensuring the accuracy of vCJD prevalence estimates in the United Kingdom (2).…”

“…Accurately quantifying prion infectivity requires inoculating permissive laboratory rodents with endpoint dilutions of the tissue ex-tract of interest and estimating the disease attack rate to establish a 50% infectious dose (ID 50 ) (10). One such bioassay in conventional RIII mice suggested that vCJD spleen contained a very low titer, in apparent contradiction with the prominent accumulation of PrP res in this tissue (7,11) and the higher titers measured in some other prion-infected species (12)(13)(14). Interindividual transmission of vCJD has occurred through blood products from asymptomatic donors (15)(16)(17), suggesting that substantial levels of endogenous vCJD infectivity are present in blood at the preclinical stage of disease.…”