ABSTRACT
The supply of blood, blood products and components in the UK, as elsewhere, is safe, although there is no cause for complacency. Use of blood, blood products and components is not without risk of morbidity and mortality. Transfusion-transmitted infections (TTIs) continue to occur and may severely affect the health and welfare of recipients. As indicated by recent and current inquiries, public interest in these TTIs is huge. The risk of TTI can be mitigated but not abolished. Measures to reduce risk include screening of donors, testing of donations and, where appropriate, treatment of donations. The introduction of newer screening tests might identify some infectious donations but come at a cost, which could exceed a justifiable limit. Thus, the recognition, detection, reporting and investigation of cases of possible TTIs need to be improved. Recipients of blood should understand that, although transfusion in the UK is safe, it is not free of risk and so should be provided with full information so that properly informed consent can be given.
Subject(s)
Blood Donors , Transfusion Reaction , Humans , Prevalence , Blood Transfusion , Transfusion Reaction/prevention & control , United KingdomABSTRACT
Following recognition that blood, blood components, tissues and organs donated by infected donors could transmit infectious prions causing variant Creutzfeldt-Jakob Disease (vCJD), several risk reduction measures were introduced in the UK. The Advisory Committee on the Safety of Blood, Tissues and Organs (SaBTO) established a working group to review the measures in place. Factors considered included: ethical issues around the current provisions and potential changes; operational issues for blood establishments and hospitals; a review from the Advisory Committee on Dangerous Pathogens (ACDP) showing the downward trend in the estimated number of future cases of vCJD; and cost-effectiveness. The working group recommended that the current vCJD risk reduction measures for individuals born after 1995 or with thrombotic thrombocytopenic purpura (TTP) could be withdrawn. After consultation with stakeholders, SaBTO accepted these proposals which allow more equal provision of components, less operational complexity and risk, and more resources to be deployed elsewhere in the NHS. The potential saving on plasma will be £500 m and moving to using pooled platelets in additive solution for all recipients will bring potential savings of £280 m over the next 50 to 60 years. There could be small number of additional clinical cases of vCJD: 1-2 (<1-14; 95% CI) from plasma and 3-4 (<1 to 45; 95% CI) from platelets. Local and national guidelines will still be applied for managing individual conditions. UK Ministers for Health accepted SaBTO's recommendations on 9 Sept 2019 and implementation began immediately. This paper describes the review and rationale leading to these recommendations.
Subject(s)
Blood Component Removal , Creutzfeldt-Jakob Syndrome , Transfusion Reaction , Blood Platelets , Creutzfeldt-Jakob Syndrome/prevention & control , Humans , Risk Reduction BehaviorABSTRACT
BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.
Subject(s)
Biological Assay/methods , Prion Proteins/chemistry , Prion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Animals , Chromatography, High Pressure Liquid , Circular Dichroism , Cricetinae , Prion Proteins/genetics , Prion Proteins/isolation & purification , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , SheepABSTRACT
BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy affecting humans, acquired initially through infection with bovine spongiform encephalopathy (BSE). A small number of vCJD cases have been acquired through the transfusion of blood from asymptomatic donors who subsequently developed vCJD. Filter devices that selectively bind the infectious agent associated with prion disease have been developed for removal of infection from blood. This study independently assessed one such filter, the P-CAPT filter, for efficacy in removing infectivity associated with the BSE agent in sheep blood. The sheep BSE model has previously been used to evaluate the distribution of infectivity in clinically relevant blood components. This is the first study to assess the ability of the P-CAPT filter to remove endogenous infectivity associated with blood components prepared from a large animal model. STUDY DESIGN AND METHODS: Paired units of leukoreduced red blood cells (LR-RBCs) were prepared from donors at the clinical stage of infection and confirmed as having BSE. One cohort of recipients was transfused with LR-RBCs alone, whereas a parallel cohort received LR and P-CAPT-filtered RBCs (LR-RBCs-P-CAPT). RESULTS: Of 14 recipients, two have been confirmed as having BSE. These sheep had received LR-RBCs and LR-RBCs-P-CAPT from the same donor. CONCLUSIONS: The results indicate that, after leukoreduction and P-CAPT filtration, there can still be sufficient residual infectivity in sheep RBCs to transmit infection when transfused into a susceptible recipient.
Subject(s)
Erythrocytes , Hemofiltration/instrumentation , Hemofiltration/methods , Prion Diseases/blood , Prions , Animals , Cattle , Humans , Prion Diseases/prevention & control , Prions/blood , Prions/isolation & purification , SheepABSTRACT
BACKGROUND: The P-Capt prion reduction filter (MacoPharma) removes prion infectivity in model systems. This independent evaluation assesses prion removal from endogenously infected animal blood, using CE-marked P-Capt filters, and replicates the proposed use of the filter within the UK Blood Services. STUDY DESIGN AND METHODS: Two units of blood, generated from 263K scrapie-infected hamsters, were processed using leukoreduction filters (LXT-quadruple, MacoPharma). Approximately 100 mL of the removed plasma was added back to the red blood cells (RBCs) and the blood was filtered through a P-Capt filter. Samples of unfiltered whole blood, the prion filter input (RBCs plus plasma and SAGM [RBCPS]), and prion-filtered leukoreduced blood (PFB) were injected intracranially into hamsters. Clinical symptoms were monitored for 500 ± 1 day, and brains were assessed for spongiosis and prion protein deposit. RESULTS: In Filtration Run 1, none of the 50 challenged animals were diagnosed with scrapie after inoculation with the RBCPS fraction, while two of 190 hamsters injected with PFB were infected. In Filtration Run 2, one of 49 animals injected with RBCPS and two of 193 hamsters injected with PFB were infected. Run 1 reduced the infectious dose (ID) by 1.467 log (>1.187 log and <0.280 log for leukoreduction and prion filtration, respectively). Run 2 reduced prion infectivity by 1.424 log (1.127 and 0.297 log, respectively). Residual infectivity was estimated at 0.212 ± 0.149 IDs/mL (Run 1) and 0.208 ± 0.147 IDs/mL (Run 2). CONCLUSION: Leukoreduction removed the majority of infectivity from 263K scrapie hamster blood. The P-Capt filter removed a proportion of the remaining infectivity, but residual infectivity was observed in two independent processes.
Subject(s)
Blood Safety , Disinfection , Leukapheresis , PrPSc Proteins , Scrapie/prevention & control , Animals , Blood Safety/instrumentation , Blood Safety/methods , Cricetinae , Disease Models, Animal , Disinfection/instrumentation , Disinfection/methods , Leukapheresis/instrumentation , Leukapheresis/methods , Scrapie/bloodABSTRACT
OBJECTIVE: Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt-Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14-3-3, which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real time quaking-induced conversion (RT-QuIC). The objective of this study is to investigate whether CSF RT-QuIC analysis could be used as a diagnostic test in sCJD. METHODS: An exploratory study was undertaken that analyzed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples, 56 were from sCJD patients (30 female, 26 male; aged 31-84 years; mean age, 62.3 ± 13.5 years), and 52 were from control patients (26 female, 26 male; aged 43-84 years; mean age, 67.8 ± 10.4 years). A confirmatory group of 118 patients was subsequently examined that consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male; aged 39-82 years; mean age, 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male; aged 36-87 years; mean age, 63.5 ± 11.6 years). RESULTS: The exploratory study showed that RT-QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study, which showed that CSF RT-QuIC analysis had a sensitivity and specificity of 87% and 100%, respectively. INTERPRETATION: This study shows that CSF RT-QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests.
Subject(s)
14-3-3 Proteins/cerebrospinal fluid , Creutzfeldt-Jakob Syndrome/cerebrospinal fluid , Creutzfeldt-Jakob Syndrome/diagnosis , Prions/cerebrospinal fluid , Adult , Aged , Aged, 80 and over , Animals , Brain/metabolism , Brain/pathology , Computer Systems , Cricetinae , Female , Humans , Male , Middle Aged , Sensitivity and SpecificityABSTRACT
Transplanted tissues have transmitted transmissible spongiform encephalopathies and in the UK there have been more cases of variant Creutzfeldt-Jakob disease (vCJD) than elsewhere in the world. A pilot study was undertaken to look at the feasibility of testing for vCJD in deceased donors using tonsillar tissue. This pilot showed that obtaining consent for removal and testing tonsil tissue was feasible. Donor eligibility for inclusion in the pilot was limited to tissue donors from the National Health Service Blood and Transplant, Tissue Services and to donors shared with the Corneal Transplant Service Eye Banks. Obtaining tonsillar tissue in the immediate post-mortem period was limited by the presence of rigor mortis. Tonsillar tissue was suitable for routine analysis for the presence of prion associated with vCJD in deceased tissue donors. Production and processing of tissue was straightforward and a low assay background was obtained from most samples. Since palatine and lingual tonsil tissue can be obtained in pairs it was possible, in the majority of cases, to set aside an intact sample for confirmatory testing if required. In one instance a sample was reactive by Western blot. However, the pattern of reactivity was not typical for that obtained from vCJD patients. Unfortunately the sample was not of sufficient quality for the confirmatory test to provide a conclusive result.
Subject(s)
Creutzfeldt-Jakob Syndrome/diagnosis , Palatine Tonsil/pathology , Tissue Donors , Biopsy , Feasibility Studies , Humans , Informed Consent , Organization and Administration , Pilot Projects , Tissue ExtractsABSTRACT
Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed largely using rodent-passaged sheep scrapie strains. Given the potential RT-QuIC has for Creutzfeldt-Jakob disease (CJD) research and human prion test development, this study characterized the behaviour of a range of CJD brain specimens with hamster and human recPrP in the RT-QuIC assay. The results showed that RT-QuIC is a rapid, sensitive and specific test for the form of abnormal PrP found in the most commonly occurring forms of sporadic CJD. The assay appeared to be largely independent of species-related sequence differences between human and hamster recPrP and of the methionine/valine polymorphism at codon 129 of the human PrP gene. However, with the same conditions and substrate, the assay was less efficient in detecting the abnormal PrP that characterizes variant CJD brain. Comparison of these QuIC results with those previously obtained using PMCA suggested that these two seemingly similar assays differ in important respects.
Subject(s)
Brain/pathology , Creutzfeldt-Jakob Syndrome/diagnosis , Pathology/methods , Prions/analysis , Amyloid/analysis , Animals , Cricetinae , Humans , Protein Conformation , Protein Folding , Time FactorsABSTRACT
OBJECTIVE: To establish with improved accuracy the prevalence of disease related prion protein (PrP(CJD)) in the population of Britain and thereby guide a proportionate public health response to limit the threat of healthcare associated transmission of variant Creutzfeldt-Jakob disease (vCJD). DESIGN: Cross sectional opportunistic survey. Study samples Anonymised tonsil pairs removed at elective tonsillectomy throughout England and Scotland. SETTING: National anonymous tissue archive for England and Scotland. MAIN OUTCOME MEASURE: Presence of PrP(CJD) determined by using two enzyme immunoassays based on different analytical principles, with further investigation by immunohistochemistry or immunoblotting of any samples reactive in either assay. RESULTS: Testing of 63 007 samples was completed by the end of September 2008. Of these, 12 753 were from the birth cohort in which most vCJD cases have arisen (1961-85) and 19 908 were from the 1986-95 cohort that would have been also exposed to bovine spongiform encephalopathy through infected meat or meat products. None of the samples tested was unequivocally reactive in both enzyme immunoassays. Only two samples were reactive in one or other enzyme immunoassay and equivocal in the other, and nine samples were equivocally reactive in both enzyme immunoassays. Two hundred and seventy six samples were initially reactive in one or other enzyme immunoassay; the repeat reactivity rate was 15% or less, depending on the enzyme immunoassay and cut-off definition. None of the samples (including all the 276 initially reactive in enzyme immunoassay) that were investigated by immunohistochemistry or immunoblotting was positive for the presence of PrP(CJD). CONCLUSIONS: The observed prevalence of PrP(CJD) in tonsils from the 1961-95 combined birth cohort was 0/32 661 with a 95% confidence interval of 0 to 113 per million. In the 1961-85 cohort, the prevalence of zero with a 95% confidence interval of 0 to 289 per million was lower than, but still consistent with, a previous survey of appendix tissue that showed a prevalence of 292 per million with a 95% confidence interval of 60 to 853 per million. Continuing to archive and test tonsil specimens, especially in older birth cohorts, and other complementary large scale anonymous tissue surveys, particularly of post-mortem tissues, will further refine the calculated prevalence of PrP(CJD).
Subject(s)
Creutzfeldt-Jakob Syndrome/epidemiology , Palatine Tonsil/virology , PrPSc Proteins/isolation & purification , Creutzfeldt-Jakob Syndrome/virology , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Prevalence , United Kingdom/epidemiologyABSTRACT
Prion diseases are associated with accumulation of strain-dependent biochemically distinct, disease-related isoforms (PrP(Sc)) of host-encoded prion protein (PrP(C)). PrP(Sc) is characterised by increased beta-sheet content, detergent insolubility and protease resistance. Recombinant alpha-PrP adopts a PrP(C)-like conformation, while beta-PrP conformationally resembles PrP(Sc), to these we raised 81 monoclonal antibodies in Prnp(0/0) mice. The N-terminal residues 91-110 are highly immunogenic in beta-PrP-immunised mice and of (17/41) anti-beta-PrP antibodies that could be epitope-mapped, approximately 70%, recognised this segment. In contrast, only 3/40 anti-alpha-PrP antibodies could be mapped and none interacted with this region, instead recognising residues 131-150, 141-160 and 171-190. Native PrP(C) was recognised by both antibody groups, but only anti-beta-PrP antibodies directed to 91-110 residues recognised native PrP(Sc) with high affinity, where in addition, species heterogeneity was also evident. Within the six anti-beta-PrP antibodies studied, they all recognised PK-treated native human and mouse PrP(Sc), four failed to recognise PK-treated native bovine PrP(Sc), one of which also did not recognise native PK-treated ovine PrP(Sc), showing the epitope becomes exposed on unfolding and disaggregation. These results demonstrate strain-dependent variations in chain conformation and packing within the 91-110 region of PrP(Sc).
Subject(s)
Antibodies, Monoclonal , Epitope Mapping , PrPC Proteins/immunology , PrPSc Proteins/immunology , Animals , Cattle , Humans , Mice , Peptide Fragments/chemistry , Peptide Fragments/immunology , PrPC Proteins/chemistry , PrPSc Proteins/chemistry , Protein Conformation , Sheep , Species SpecificityABSTRACT
BACKGROUND: The null phenotype of the Lutheran blood group system, Lu(null) or Lu(a-b-), is characterized by the lack of all Lutheran system antigens. It can arise from three genetic backgrounds: recessive, dominant, or X-linked. Lu(null) of the recessive type appears to result from homozygosity for an inactive LU gene. STUDY DESIGN AND METHODS: Three unrelated recessive Lu(null) individuals were assessed by standard serologic tests. All exons of the LU gene were directly sequenced from amplified genomic DNA. The validity of the observed mutations within the LU gene was confirmed by the use of either restriction enzymes or allele-specific primers. RESULTS: All three individuals had the serologic characteristics of recessive Lu(null). One individual was doubly heterozygous for a nonsense mutation 691C>T in exon 6 (Arg231STOP) and a deletion of LU exons 3 and 4. The other two samples showed homozygous nonsense mutations: one had 711C>A in exon 6 (Cys237STOP) and the other 361C>T in exon 3 (Arg121STOP). CONCLUSIONS: The results revealed four unique genetic backgrounds from three examples of the rare recessive Lu(null) phenotype, each encoding Lutheran glycoproteins with a disrupted structure.
Subject(s)
Cell Adhesion Molecules/genetics , Lutheran Blood-Group System , Mutation , Neoplasm Proteins/genetics , Phenotype , Base Sequence , DNA Mutational Analysis , Female , Genes, Recessive , Humans , Lutheran Blood-Group System/genetics , Male , Serologic TestsABSTRACT
Inherited prion diseases are caused by PRNP coding mutations and display marked phenotypic heterogeneity within families segregating the same pathogenic mutation. A proline-to-leucine substitution at prion protein (PrP) residue 102 (P102L), classically associated with the Gerstmann-Sträussler-Scheinker (GSS) phenotype, also shows marked clinical and pathological heterogeneity, including patients with a Creutzfeldt-Jakob disease (CJD) phenotype. To date, this heterogeneity has been attributed to temporal and spatial variance in the propagation of distinct protease-resistant (PrP(Sc)) isoforms of mutant PrP. Here, using a monoclonal antibody that recognizes wild-type PrP, but not PrP 102L, we reveal a spectrum of involvement of wild-type PrP(Sc) in P102L individuals. PrP(Sc) isoforms derived from wild-type and mutant PrP are distinct both from each other and from those seen in sporadic and acquired CJD. Such differential propagation of disease-related isoforms of wild-type PrP and PrP 102L provides a molecular mechanism for generation of the multiple clinicopathological phenotypes seen in inherited prion disease.
Subject(s)
Mutant Proteins/analysis , Prion Diseases/genetics , Prions/genetics , Adult , Animals , Antibodies, Monoclonal/immunology , Brain/metabolism , Brain/pathology , Enzyme-Linked Immunosorbent Assay/methods , Humans , Mice , Mice, Transgenic , Microscopy, Confocal , Middle Aged , Mutant Proteins/immunology , Mutation , Peptide Hydrolases , Phenotype , Prion Diseases/metabolism , Prion Diseases/pathology , Prion Diseases/transmission , Prions/analysis , Prions/immunology , Protein Isoforms/genetics , Protein Isoforms/immunologyABSTRACT
Prion diseases involve conversion of host-encoded cellular prion protein (PrPC) to a disease-related isoform (PrPSc). Using recombinant human beta-PrP, a panel of monoclonal antibodies was produced that efficiently immunoprecipitated native PrPSc and recognized epitopes between residues 93-105, indicating for the first time that this region is exposed in both human vCJD and mouse RML prions. In contrast, monoclonal antibodies raised to human alpha-PrP were more efficient in immunoprecipitating PrPC than PrPSc, and some of them could also distinguish between different PrP glycoforms. Using these monoclonal antibodies, the physical association of PrP glycoforms was studied in normal brain and in the brains of humans and mice with prion disease. It was shown that while PrPC glycoforms can be selectively immunoprecipitated, the differentially glycosylated molecules of native PrPSc are closely associated and always immunoprecipitate together. Furthermore, the ratio of glycoforms comprising immunoprecipitated native PrPSc from diverse prion strains was similar to those observed on denaturing Western blots. These studies are consistent with the view that the proportion of each glycoform incorporated into PrPSc is probably controlled in a strain-specific manner and that each PrPSc particle contains a mixture of glycoforms.
Subject(s)
Antibodies, Monoclonal/immunology , PrPSc Proteins/immunology , Prion Diseases/immunology , Prions/immunology , Protein Isoforms/immunology , Animals , Creutzfeldt-Jakob Syndrome/immunology , Creutzfeldt-Jakob Syndrome/metabolism , Epitope Mapping , Glycosylation , Humans , Immunohistochemistry , Immunoprecipitation , Mice , PrPC Proteins/immunology , PrPSc Proteins/chemistry , Prion Diseases/metabolism , Recombinant Proteins/immunology , Scrapie/immunology , Scrapie/metabolismABSTRACT
Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , PrPSc Proteins/immunology , PrPSc Proteins/metabolism , Animals , Antibody Specificity , Cell Line , Mice , Mice, Mutant Strains , PrPC Proteins/genetics , Precipitin Tests , Protein Binding/immunologyABSTRACT
Prion diseases are a group of invariably fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle. The infectious agent or prion is largely composed of an abnormal isoform (PrPSc) of a host encoded normal cellular protein (PrPc). The conversion of PrPc to PrPSc is a dynamic process and, for reasons that are not clear, the distribution of spongiform change and PrPSc deposition varies among prion strains. An obvious explanation for this would be that the transformation efficiency in any given brain region depends on favourable interactions between conformations of PrPc and the prion strain being propagated within it. However, identification of specific PrPc conformations has until now been hampered by a lack of suitable panels of antibodies that discriminate PrPc subspecies under native conditions. In this study, we show that monoclonal antibodies raised against recombinant human prion protein folded into alpha or beta conformations exhibit striking heterogeneity in their specificity for truncations and glycoforms of mouse brain PrPc. We then show that some of these PrPc isoforms are expressed differentially in certain mouse brain regions. This suggests that variation in the expression of PrPc conformations in different brain regions may dictate the pattern of PrPSc deposition and vacuolation, characteristic for different prion strains.