Pathological diagnosis of variant Creutzfeldt-Jakob disease

The neuropathological and biochemical features of the 89 histologically confirmed cases of variant Creutzfeldt-Jakob disease (vCJD) diagnosed up to the end of October 2001 in the UK are reviewed. Histology of the central nervous system, lymphoid tissues and other organs was accompanied by immunocytochemistry and Western blot analysis of the disease-associated form of the prion protein (PrP(RES)). All patients with vCJD were methionine homozygotes at codon 129 of the PrP gene. The pathology of vCJD showed relatively uniform morphological and immunocytochemical characteristics, which were distinct from other forms of CJD. PrP(RES) accumulation was widespread in lymphoid tissues in vCJD, but was not identified in other non-neural tissues. PrP(RES) in vCJD brain tissue showed a uniform glycotype pattern distinct from sporadic CJD. Given the increasingly widespread occurrence of bovine spongiform encephalopathy in Europe and Asia, there is a major need for widespread CJD surveillance. This should be accompanied by a multidisciplinary laboratory approach to the investigation and diagnosis of all forms of CJD, with the need to investigate autopsy tissues from suspected cases by the histological and biochemical techniques described herein.     

Neuropathology of variant Creutzfeldt-Jakob disease

The clinical, neuropathological genetic and biochemical features of variant Creutzfeldt-Jakob disease (vCJD) are compared to the 926 other cases of suspected CJD referred to the National CJD Surveillance Unit laboratory from 1990-2001. Histological studies of the central nervous system, lymphoid tissues and other organs were accompanied by immunocytochemistry for prion protein (PrP); Western blot analysis of PrPRES was performed on frozen brain tissue. The pathology of vCJD showed relatively uniform morphological and immunocytochemical characteristics, with PrP accumulation in lymphoid tissues, but not in other non-neural tissues. PrPRES accumulation in vCJD showed a uniform glycotype pattern distinct from sporadic CJD. All cases of vCJD were methionine homozygotes at codon 129 of the PrP gene. In view of the spread of bovine spongiform encephalopathy in Europe and Japan, continuing surveillance is required for all forms of CJD, with histological and biochemical analysis of suspected cases to allow an accurate laboratory diagnosis.     

Expression of the p75 neurotrophin receptor by striatal cholinergic neurons following global ischemia in rats is associated with neuronal degeneration

A protein capture assay was used to measure 14-3-3 (-isoform) in the cerebrospinal fluid (CSF) of patients with either variant or sporadic Creutzfeldt-Jakob disease (CJD). The results were compared with those obtained using Western blotting. Elevated levels of 14-3-3 were found in 58% of variant CJD (vCJD) patients and 82% of sporadic CJD (spCJD) patients using the protein capture assay. Using a Western blotting technique, the presence of CSF 14-3-3 was detected in 58% of vCJD patients and in 89% of spCJD patients. When the results from the protein capture assay and the Western blot were combined, 14-3-3 was detected in 77% of vCJD patients and in 91% of spCJD patients. These results suggest that although analysis of CSF 14-3-3 is not as useful in vCJD as it is in the sporadic form of the disease, a combination of these two techniques results in increased sensitivity of 14-3-3 for the diagnosis of vCJD.     

Laboratory diagnosis of variant Creutzfeldt-Jakob disease

The neuropathological and biochemical features of 33 cases of variant Creutzfeldt-Jakob disease (vCJD) diagnosed up to the end of 1998 are analysed in relation to the 646 cases of suspected CJD referred to the CJD Surveillance Unit laboratory from 1990 to 1998. Morphological studies of the central nervous system, lymphoid tissues and other organs were accompanied by immunocytochemistry; Western blot analysis of PrPRES was performed on frozen brain tissue. The findings were analysed in relation to clinical and genetic data. The pathology of vCJD showed morphological and immunocytochemical characteristics distinct from other cases of CJD. PrP accumulation was widespread in lymphoid tissues in vCJD, but was not identified in other non-neural tissues. PrPRES accumulation in vCJD brain tissue showed a uniform glycotype pattern distinct from sporadic CJD. All analysed cases of vCJD were methionine homozygotes at codon 129 of the PrP gene. No evidence currently exists to suggest that cases of CJD diagnosed in individuals who are MV or VV at codon 129 of the PrP gene represent 'human bovine spongiform encaphalopathy (BSE)'. Continued surveillance is required to further investigate this possibility, with the need to investigate autopsy tissues from suspected cases by histological and biochemical techniques.     

Detection and localization of PrPSc in the skeletal muscle of patients with variant, iatrogenic, and sporadic forms of Creutzfeldt-Jakob disease

Variant Creutzfeldt-Jakob disease (vCJD) differs from other human prion diseases in that the pathogenic prion protein PrP(Sc) can be detected to a greater extent at extraneuronal sites throughout the body, principally within lymphoid tissues. However, a recent study using a high-sensitivity Western blotting technique revealed low levels of PrP(Sc) in skeletal muscle from a quarter of Swiss patients with sporadic CJD (sCJD). This posed the question of whether PrP(Sc) in muscle could also be detected in vCJD, sCJD, and iatrogenic (iCJD) patients from other populations. Therefore, we have used the same high-sensitivity Western blotting technique, in combination with paraffin-embedded tissue blotting, to screen for PrP(Sc) in muscle tissue specimens taken at autopsy from 49 CJD patients in the United Kingdom. These techniques identified muscle PrP(Sc) in 8 of 17 vCJD, 7 of 26 sCJD, and 2 of 5 iCJD patients. Paraffin-embedded tissue blotting analysis showed PrP(Sc) in skeletal muscle in localized anatomical structures that had the morphological and immunohistochemical characteristics of nerve fibers. The detection of PrP(Sc) in muscle tissue from all forms of CJD indicates the possible presence of infectivity in these tissues, suggesting important implications for assessing the potential risk of iatrogenic spread via contaminated surgical instruments.     

Leucodepletion for transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) have been recognised around the world for many years. Creutzfeldt-Jakob disease (CJD), one of the human forms of TSE, has been studied widely and thus far has not proved a great threat to human health. The emergence of two new TSEs--bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt-Jakob disease (vCJD) in humans in the UK--has caused great concern. BSE has had an economic impact and vCJD is a threat to human health. It has been shown that these two diseases are caused by the same prion agent and are linked. Research indicates that vCJD behaves differently to CJD and there is strong evidence to suggest that vCJD is present in lymphoid tissues and B lymphocytes, which presents a theoretical risk that it may be transmitted by transfusion of blood and blood products. To minimise/prevent this risk, the UK government has decided that plasma should be sourced from abroad and has instructed the National Blood Service to leucodeplete all blood and blood products, at a cost of 70 million pounds per annum, although it is not known if this will remove this risk.     

人PrP特异性多肽抗体的鉴定和应用

目的 为了进一步检测制备的人PrP特异性多肽抗体的免疫反应性和特异性。方法 构建人PrPN端缺失原核表达质粒,表达并纯化N端缺失的PrP蛋白;Western blot检测多肽抗体与纯化的各种人全长、N端缺失以及C端缺失PrP蛋白的反应能力;免疫荧光法检测多肽抗体与真核表达的人PrP蛋白反应能力;提取Creutzfeldt-Jakob病（CJD）病人以及Scrapie感染的仓鼠脑组织,Westen blot检测多肽抗体与致病性PrP－res蛋白的反应能力。结果 所制备的PrP多肽抗体能与原核及真核细胞表达的各种不同长度的PrP蛋白反应,同时还能特异性识别Scrapie感染鼠脑组织中和CJD病人脑组织中的PrP－res蛋白。结论 多肽抗体具备良好的免疫反应性和特异性,可以应用于临床CJD病人的检测。     

Pre-clinical diagnosis of transmissible spongiform encephalopathies using rapid tests.

During the past few years, important progress has been made in the post-mortem diagnosis of transmissible spongiform encephalopathies (TSEs) (scrapie and BSE) due to the development of the so-called "rapid test" based on the immunological detection of the abnormal form of the prion protein (PrPres) in the central nervous system. These methods now allow routine and high throughput testing, opening the door to large-scale epidemiological studies and systematic testing at slaughterhouses, thus preventing the entry of contaminated carcasses into the human food chain. It has been shown that some of these rapid tests allow pre-clinical diagnosis, anticipating by few months the appearance of clinical signs. In sheep and goat, PrPres can also be detected in peripheral lymphoid tissues a long time before the onset of clinical symptoms. As a consequence, the same rapid tests are suitable for pre-clinical diagnosis of scrapie in these species. It is very likely that the same kind of early diagnosis could be obtained for vCJD. The real challenge in the field of TSE diagnosis is the establishment of a vCJD test, conducted either on blood or urine, since these are the only biological fluids easily accessible from infected people. This is a very important issue to avoid iatrogenic transmission of vCJD within the human population. This is also very difficult because the quantities of infectious agents in the blood are certainly 100-1000 times lower than those present in the brain.     

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.     

Characterisation of antisera raised against species-specific peptide sequences from scrapie-associated fibril protein and their application for post-mortem immunodiagnosis of spongiform encephalopathies

Summary Transmissible spongiform encephalopathies (TSE), such as scrapie or Creutzfeldt-Jakob disease (CJD), are fatal neurodegenerative diseases of the central nervous system caused by a yet unidentified virus. They are accompanied by a brain specific amyloidosis, during which a host coded protein irreversibly aggregates to form the scrapie-associated fibrils. The diagnosis of TSE relies on histopathological detection of spongiform lesions, on electron microscopical detection of fibrils, or on the immunological detection of SAF protein, which is the most specific diagnostic marker. In order to improve the diagnosis of TSE, we developed a protocol for rapid tissue fractionation and enrichment of SAF protein which subsequently allows the specific detection of SAF protein by western blotting and immunodetection. Using some new antisera raised against synthetic peptides with sequences specific for the hamster, sheep, cattle and human SAF protein, several samples can be diagnosed for TSE within 24 hours, starting with only 10–100 mg of brain tissue from different species.     

Absence of protease-resistant prion protein in the cerebrospinal fluid of Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD), believed to be caused by a protease-resistant isoform of prion protein (PrP(Sc)), usually manifests itself as a clinically distinctive age-related dementia because of its rapid progression, occasionally accompanied by cerebellar ataxia. Recently, a variant CJD (vCJD) has been described, which has prominent early psychiatric symptoms and an earlier age of death. Although cerebrospinal fluid (CSF) is part of the extracellular fluid of the central nervous system (CNS), the bulk of its proteins are derived from the plasma and there is increasing concern about possible transmission of prion disease by blood. As investigation of CSF has played a significant role in the diagnosis and management of several neurological diseases, it was decided to characterize PrP present in the CSF of CJD individuals. Significant variation was observed in the level of PrP in the CSF from both non-CJD and CJD (including vCJD) patients, and the detected PrP forms are protease-sensitive. Using a conformation-dependent immunoassay, it was further demonstrated that the PrP detected in the CSF from CJD patients was broadly similar in conformation to that found in non-CJD patients. Taken together, the results of this study fail to demonstrate any correlation between the presence of protease-resistant PrP isoform (PrP(Sc)) in the CSF and disease manifestation.     

Failure to Detect the Presence of Prions in the Uterine and Gestational Tissues from a Gravida with Creutzfeldt-Jakob Disease

The vertical transmission of a prion disease from infected mothers to their offspring is believed to be one of the routes for the natural spread of animal prion diseases. Supporting this notion is the observation that prion infectivity occurs in the placenta of infected ewes. Furthermore, the prion protein (PrP), both in its cellular form (PrP^C) and its pathological isoform (PrP^Sc), has been observed at the fetal-maternal interface of scrapie-infected sheep. However, whether these features of prion infectivity also hold true for human prion diseases is currently unknown. To begin to address such an important question, we examined PrP in the uterus as well as gestational tissues, including the placenta and amniotic fluid, in a pregnant woman with sporadic Creutzfeldt-Jakob disease (CJD). Although the proteinase K (PK)-resistant prion protein, PrP27-30, was present in the brain tissues of the mother, the PrP detected in the uterus, placenta, and amniotic fluid was sensitive to PK digestion. Unlike PrP^C in the brain and adjacent cerebrospinal fluid, the predominant PrP species in the reproductive and gestational tissues were N-terminally truncated, similar to urine PrP. Our study did not detect abnormal PrP in the reproductive and gestational tissues in this case of CJD. Nevertheless, examination by a highly sensitive bioassay is ongoing to ascertain possible prion infectivity from CJD in the amniotic fluid.The transmissible prion diseases affecting both humans and animals are characterized by the accumulation of an infectious prion protein particle (PrP^Sc) mainly in the central nervous system (CNS) and occasionally in the peripheral tissues.^1,2,3,4 Animal prion diseases such as scrapie in sheep and goats, chronic wasting disease in deer and elks, and bovine spongiform encephalopathy in cattle are believed to spread naturally by oral transmission, close contact between animals, and maternal transmission. Indeed, Western blot analysis and bioassays have demonstrated that PrP^Sc and prion infectivity are present not only in the CNS, but also in many peripheral tissues including the tonsils, spleen, lymph nodes, nasal mucosa, distal colon, ovaries, uterus, skeletal muscle, placenta, and amniotic fluid of affected animals.²In humans, the transmission of prion diseases has been observed in the acquired form of the disease including kuru, iatrogenic Creutzfeldt-Jakob disease (iCJD), and variant CJD (vCJD).⁵ Kuru is associated with cannibalistic rituals,^6,7 whereas iCJD is caused by prion exposure in the course of medical or surgical procedures, and vCJD has been attributed to the consumption of prion-contaminated meat.^8,9 In addition to CNS, PrP^Sc has also been detected in the tonsils, spleen, lymph node, retina, optic nerve, rectum, adrenal gland, and thymus of vCJD and in the spleen and skeletal muscles of sporadic CJD (sCJD).^3,4 However, it remains unknown whether human prion diseases are vertically transmitted in pregnancy. For instance, none of four offspring born to four gravid women with CJD had reportedly developed the disease when they reached the respective ages of 22, 10, 7, and 3 years.¹⁰ In addition, no reports are available concerning examination of PrP in the uterus and gestational tissues from prion-affected patients.We examined PrP^Sc distribution in CNS, uterus, and gestational tissues from a woman affected with prion disease, who had become pregnant and delivered a baby boy during the time she had the disease. Typical PK-resistant PrP core fragments and neuropathological changes, characteristic of sCJD (with PrP^Sc type 1 carrying a valine/valine polymorphism at codon 129 of PrP gene), were detected in the brain tissues obtained at either biopsy or autopsy. Although PrP was detectable in the uterus, placenta, and amniotic fluid, it was PK-sensitive. Moreover, neither conventional nor enrichment-based Western blot analysis revealed the presence of abnormal PrP species. In contrast to the PrP in the brain and cerebrospinal fluid (CSF), the PrP detected in the uterus and gestational tissues, including placenta and amniotic fluid, was N-terminally truncated, similar to that normally found in the urine. Although the presence of prion infectivity in tissue remains to be determined by highly sensitive bioassay, our current study suggests that abnormal PrP species, including both PK-resistant and PK-sensitive forms, are undetectable in the uterus and gestational tissues in sporadic CJD.     

人S100蛋白的表达及其抗血清的制备和脑脊液中S100含量测定方法的建立

目的：建立定量检测脑脊液（CSF）及血清中S100蛋白的方法，探讨S100蛋白的检测在辅助诊断克－雅病（CJD）中的应用。方法：利用脑cDNA文库，经PCR获得了S100基因并克隆至原核表达载体pGEX-2T上，在大肠埃希菌中表达了谷胱甘肽－S－转移酶（GST）－S100融合蛋白；融合蛋白经亲和纯化后，免疫家兔，制备抗体；抗体经纯化后，用生物素（BNHS）标记，建立了可定量检测S100蛋白的生物素－亲和素系统ELISA方法，并初步用于临床脑脊液的检测中。结果：所表达的GST－S100蛋白相对分子质量约为35000，以其为抗原制备的S100特异性抗血清具有良好的免疫反应性。建立了定量检测脑脊液中S100蛋白的双抗体夹心ELISA方法，对3例“可能性的CJD”患者（14－3－3蛋白阳性）和15例无痴呆症状患者脑脊液进行检测，结果显示，3例CJD患者脑脊液S100含量均超过2．900μg/L,而在无痴呆症状患者组中14例患者脑脊液S100含量都低于0．180μg/L。对正常人和CJD患者血清进行检测，显示S100蛋白含量个体间差异很大。结论：所建立的方法可用于脑脊液中S100蛋白的检测，进一步扩大标本量有助于明确脑脊液中S100蛋白的检测在辅助诊断CJD中的价值。     

Approaches to Scrapie diagnosis by applying immunohistochemistry and rapid tests on central nervous and lymphoreticular systems

Comparative studies evaluating the performance of Transmissible Spongiform Encephalopathies (TSE) rapid tests (validated for Bovine Spongiform Encephalopathy samples) on Scrapie samples have not been reported widely, particularly those dealing with lymphoreticular system tissues to a much lesser extent. The main objective of this study was to compare the ability of two current rapid tests (Western blot and Luminiscence Immunoassay Prionics-Check; WB and LIA, respectively) to detect PrPsc using central nervous system as well as lymphoreticular system samples corresponding to naturally infected animals. Thirty-four Scrapie-affected sheep, 26 with clinical signs of the disease, were included in the study. Tonsil, retropharyngeal lymph node and medulla oblongata were assessed by three tests: immunohistochemistry (confirmatory test), WB and LIA (rapid tests). The conclusion which can be drawn from this study is the fact that all animals involved in the study, including those at a preclinical stage, could be diagnosed regardless of the test used (with immunohistochemistry consistently showing higher sensitivity) only when the analyses of both the central nervous system and the lymphoreticular system were considered. The choice of these tissues for routine diagnosis is, therefore, proposed as a valuable tool to highly reduce the number of undetected positive cases.     

Effect of Immunization with Recombinant OspA on Serologic Tests for Lyme Borreliosis

This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection with B. burgdorferi in OspA vaccine recipients.     

Latrogenic Creutzfeldt-Jakob disease with florid plaques

Florid plaques indistinguishable from those found in vCJD were identified at a postmortem examination in the brain of a 58-year-old clinical suspect case of Creutzfeldt-Jakob disease (CJD). Western blotting of brain tissue revealed an unusual prion protein type. Since the patient had received a dura mater graft 20 years prior to death and florid plaques are not only found in new variant CJD, the findings argue in favor of an iatrogenic origin of the disease with the longest incubation time following a dura mater graft reported to date even though he may have been exposed to BSE. The peculiar pathological, clinical and biochemical features may define a new type of human prion disease.     

Effect of immunization with recombinant OspA on serologic tests for Lyme borreliosis

This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection with B. burgdorferi in OspA vaccine recipients.     

Biochemical properties of protease resistant prion proteinPrPsc in natural sheep scrapie

Summary. Prions are infectious agents involved in neurodegenerative diseases, such as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cows and Creutzfeldt-Jakob disease (CJD) in humans. These pathogens are characterized by unusual properties, and, in particular, by their strong resistance to common procedures of disinfection used against conventional microorganisms. A major component of highly infectious fractions is a proteinase K-resistant prion protein PrPsc (PrP-res), the normal host prion protein PrPc being sensitive to PK (PrP-sen). We used a biochemical approach to further characterize PrPsc protein in natural sheep scrapie. Western blot analyses using rabbit antiserum that recognized both normal and pathologic sheep prion proteins, were undertaken to study the biochemical behaviour of PrPsc extracted from brains of sheep naturally infected with scrapie after protease digestion and under denaturing conditions. Increasing concentrations of urea (1 – 7 M) or GdnSCN (0.25 – 3 M) and different pH from 2 to 11 were tested for their effects on protease resistance of PrPsc. Alkaline pH (pH10) and high concentrations of urea (M) and GdnSCN (M) greatly decreased the protease resistance of the prion protein. Identical experiments carried out on three different sheep from the same flock gave similar results. The biochemical behaviour of PrPsc under denaturing conditions and in the presence of proteinase K could thus provide a biochemical means for further characterization of different natural scrapie isolates. Received January 25, 1997 Accepted March 11, 1997     

Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt–Jakob disease

Highly sensitive in vitro screening tests are required to prevent the iatrogenic spread of variant Creutzfeldt–Jakob disease (vCJD). Protein misfolding cyclic amplification (PMCA) is a candidate for such a test, but the sensitivity of this method is insufficient. Polyanions were reported to enhance PMCA efficiency, but their effects on vCJD are unclear. We developed a cell-PMCA of vCJD, wherein cell lysate containing exogenously expressed human PrP was used as substrates, to investigate the effects of various sulfated polysaccharides on amplification efficiency. PrP^res amounts after cell-PMCA were analyzed by western blotting. Heparin, dermatan sulfate, and dextran sulfate (average molecular weight [MW] 1400 kDa) enhanced efficiency, but dextran sulfate (average MW 8 kDa) and a heparin pentasaccharide analog had no effect. Pentosan polysulfate inhibited cell-PMCA reaction. The amplification efficiency of cell-PMCA of vCJD increased to >100-fold per round with heparin. The enhancing effects of heparin on cell-PMCA were seed dependent: it was high for vCJD, low for sporadic Creutzfeldt–Jakob disease, and low to negligible for hamster-adapted scrapie-derived 263 K. In multi-round PMCA, signals were detected at earlier rounds with heparin than without heparin, and PrP^Sc in 10⁻¹⁰ diluted vCJD brain was detected by the sixth round. Heparin-assisted cell-PMCA of vCJD represents a significant step toward detecting very minute amounts of PrP^Sc in the body fluids of asymptomatic vCJD patients.