SEARCH

SEARCH BY CITATION

Keywords:

  • Creutzfeldt-Jakob disease;
  • blood;
  • plasma products

Summary

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

There have been four highly probable instances of variant Creutzfeldt-Jakob disease (vCJD) transmission by non-leucocyte depleted red cell concentrates and it is now clear that the infectious agent is transmissible by blood components. To date there in no reported evidence that the infectious agent has been transmitted by fractionated plasma products, e.g. factor VIII concentrate. This review outlines current and potential risk management strategies including donor deferral criteria, the potential for donor screening, blood component processing and prion reduction filters, plasma product manufacture and the difficulties in identification and notification of those considered ‘at risk of vCJD for public health purposes’.

This review offers an update on our recent assessment and management of the risk of transmission of variant Creutzfeldt-Jakob disease (vCJD) by blood components and plasma products (Ludlam & Turner, 2005). As that review surveyed perceptions on the nature of the prion agent, the spectrum of prion diseases in animals and man, and the range of animal studies relating to pathogenicity and infectivity (much of which still represents the current level of knowledge), these topics are not reviewed again here, other than where significant new relevant studies have been published. This current review focuses on the state of the art in relation to the safety of blood components and plasma products, which has also been reviewed elsewhere (Farrugia et al, 2005; Dolan, 2006; Ironside, 2006 and Clarke et al, 2007).

To date, a total of 203 probable, or definite, cases of vCJD have been reported worldwide, of which 166 have arisen in the UK, 23 in France, four in Eire and Spain, three in the USA, and one in each of Holland, Portugal, Italy, Saudi Arabia, Japan and Canada (http://www.cjd.ed.ac.uk/vcjdworld.htm). Of these, two of the Irish and US cases and those in Canada and Japan are thought to have been infected in the UK. The third US case is thought to have been infected in Saudi Arabia. The other cases are thought to have been infected in their countries of origin either through exported UK meat products or exported animals or animal foodstuffs. The UK outbreak of vCJD appears to have reached a peak around the year 2000 and has waned such that in 2007 there were only five new cases, though the frequency of new cases continues to increase in France and Spain. All clinically affected individuals thus far have been methionine homozygous at codon 129 of the prion protein gene (PRNP). Mathematical projections based on the current incidence of vCJD suggest a maximum likelihood estimate of 70 further cases (95% confidence interval 10–190) (Clarke & Ghani, 2005). This could prove to be an underestimate, however, if individuals of other codon 129 genotypes are also capable of being infected and/or secondary transmissions occur from asymptomatic individuals.

Two observations give pause for thought. The first is that the median age of onset of clinical disease (26 years) has not altered over the past 10 years as one might expect if a cohort of individuals were exposed to infection during a specific window of time. The best fit mathematical model suggests an age-related exposure/susceptibility during the teenage years. The second is the data from a retrospective study of tonsils and appendices (Hilton et al, 2004) in which 3/12 500 samples showed evidence of abnormal prion accumulation, giving a maximum likelihood estimate of 3000 future cases. The discrepancy between this estimate and that based on current clinical incidence is best explained by the proposition that around 93% of infected individuals may experience long-term pre- or sub-clinical infection (Clarke & Ghani, 2005). This is consistent with experimental animal studies and clinical studies in patients with iatrogenic CJD and kuru, which suggest that individuals who are heterozygous or valine homozygous at codon 129 have a longer incubation period and a lower incidence of development of clinical disease than those who are codon 129 methionine homozygous. These observations give rise to concern however that a significant cohort of individuals, maybe as many as 1/4000 of the general population in the UK, may have sub-clinical vCJD infection and be at risk of transmitting the disease through blood and tissue products or surgical and medical instrumentation, despite being asymptomatic themselves.

As there is no currently accepted blood test that reliably identify vCJD infected individuals (see below), further studies have been carried out to try to refine the estimate of the prevalence of sub-clinical disease. The National Anonymised Tonsil Archive aims to test 100 000 tonsil samples. Currently, there have been no confirmed positive samples out of 45 000 tested (http://www.hpa.org.uk/infections/topics_az/cjd/tonsil_archive.htm). However there are reservations around the interpretation of these data, given that the sensitivity of the assays in detecting subclinical vCJD is uncertain, the frequency of involvement of the tonsil as a site of preclinical infection is unknown, and a large proportion of the study population are too young to have been exposed to dietary bovine spongiform encephalopathy (BSE). The Spongiform Encephalopathy Advisory Committee (SEAC) has therefore not felt it appropriate to amend the current prevalence estimates within the UK at present (http://www.seac.gov.uk).

Infectivity in the peripheral blood

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

Infectivity remains undetectable in the peripheral blood of patients with vCJD despite the fact that clinical transmission has clearly occurred. This apparent contradiction is probably explained by the presence of a species barrier between man and mouse and the limited volumes of blood that can be inoculated into test animals.

Studies in hamsters infected with the 263K strain of scrapie showed similar results to those in the Fukuoka-1 GSS strain in mice (Brown et al, 1998; Ludlam & Turner, 2005), with a point estimate of 1–10 infectious doses (ID)/ml of whole blood of which around 40% was associated with the leucocytes and most of the remainder in the plasma (Gregori et al, 2004). Further studies in this model suggest that the majority of cell-associated infectivity is only loosely bound and can be washed off and therefore that the plasma form of infectivity probably predominates. Further studies in mice suggest that the level of infectivity is similar in vCJD-infected animals (Cervenakova et al, 2003a). Studies in sheep naturally infected with scrapie, or experimentally infected with BSE, suggest a transmission frequency of up to 50% from blood taken during the preclinical or clinical phase of disease and transfused into recipients from a scrapie-free flock (Hunter et al, 2002). BSE has also been transmitted through buffy coat to the primate Microcebus (Bons et al, 2002).

Variant CJD transmission by blood transfusion

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

Within the UK, the Transfusion Medicine Epidemiology Review (TMER) has proved an effective system for collating evidence of possible transmission of vCJD by blood components (Hewitt et al, 2006). The UK CJD Surveillance Unit in Edinburgh shares information about new cases of vCJD with the Blood Transfusion Services, which search their databases to ascertain whether these patients have been blood donors in the past. In this event attempts are made to identify the fate of the blood components (http://www.cjd.ed.ac.uk/TMER) and trace, notify and monitor living recipients. The ‘reverse’ arm of the TMER study attempts to identify which individuals who develop vCJD have received blood transfusions and to identify the donors.

Eighteen patients with vCJD have, or had previously, been blood donors, from whom a total of 66 recipients have been identified, 26 of whom are still alive. Of those who have died, four cases of transmission of vCJD prions have been identified (see below). Many of these patients however will have died of their underlying conditions within 5 years of the implicated transfusion and will not have had time to show clinical evidence of vCJD if infected.

The first symptomatic case of vCJD disease associated with blood transfusion was identified in December 2003. This individual developed vCJD 6·5 years after transfusion of red cells donated by an individual who developed symptoms of vCJD 3·5 years after donation (Llewelyn et al, 2004).

A second case of transmission was identified a few months later in a recipient of red cells from a donor who developed symptoms of vCJD 18 months after donation. This patient died from causes unrelated to vCJD 5 years after transfusion. Postmortem investigations found abnormal prion protein accumulation in the spleen and a cervical lymph node, but not in the brain, and no pathological features of vCJD were found (Peden et al, 2004).

A third patient developed symptoms of vCJD 6 years and died 8·7 years after receiving a transfusion of red blood cells from a donor who developed vCJD about 20 months after this blood was donated (Health Protection Agency 2006).

The fourth case of transmission developed symptoms of vCJD 8·5 years after receiving a transfusion of red blood cells from a donor who developed vCJD about 17 months after this blood was donated. The donor to this patient also donated the vCJD-implicated blood transfused to the third patient (Editorial Team, 2007).

All four patients received transfusions of non-leucodepleted red blood cells between 1996 and 1999. Since October 1999, leucocytes have been removed from all blood used for transfusion in the UK.

These data therefore demonstrate clearly that non-leucodepleted red cells from asymptomatic individuals incubating vCJD can transmit the infection by blood transfusion to other individuals and that the risk of them doing so is relatively high.

Donor deferral criteria

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

There has been little substantive change in blood donor criteria since our previous review (Ludlam & Turner, 2005). Whilst other countries continue to defer those who have spent more than a specified cumulative period of time in the UK, within the UK only those considered by the CJD Incidents Panel to be ‘at risk of vCJD for public health purposes’ on account of exposure to implicated surgical instruments, blood components or plasma products, and those who themselves have received blood components, are deferred (http://www.hpa.org.uk/infections/topics_az/cjd). There is considerable complexity relating to the introduction of similar donor deferral criteria in the context of cell, tissue and organ donation. Broadly, whilst all forms of donation are excluded for patients with CJD or those considered potentially infected, donation of haematopoietic stem cells and solid organs is permitted from those considered ‘at risk for public health purposes’ and those previously transfused, subject to a risk assessment that weighs the risk of vCJD transmission against the potentially life-saving nature of an otherwise suitable transplant. Donation of other tissues is based on the same donor deferral criteria as blood. Donor deferral criteria remain, however, blunt risk management tools with potential deleterious effects on blood, tissue and organ supply.

Importation of blood components

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

Since our last report (Ludlam & Turner, 2005) the use of imported methylene-blue treated fresh frozen plasma (FFP) has been extended to all patients under the age of 16 years and to high users. Solvent detergent-treated FFP is recommended for patients undergoing plasma exchange for thrombotic thrombocytopenic purpura on the grounds that there is some evidence to suggest that methylene-blue treated FFP has a deleterious impact on outcome in this patient group (Alvarez-Larran et al, 2004). Consideration continues to be given around the possibility of importing FFP and cryoprecipitate for additional groups of patients. Importation of platelets is likely to be impractical given the short shelf-life of these products. However, it may be possible to import red cell concentrates for some groups of patients, for example for children up to 16 years of age. Consideration also has to be given to cost, quality and regulatory requirements and countervailing risks of transmission of other infectious diseases or of component shortages.

Advances in the development of a screening test

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

As previously noted (Ludlam & Turner, 2005), neither nucleic acid transmission nor immunological responses have been clearly identified in association with transmission of prion diseases, rendering standard molecular and serological screening assays unfeasible. Surrogate markers, such as 14-3-3, S100 and erythroid differentiation-related factor, have thus far proved insufficiently sensitive and specific to be of clinical value. Considerable progress has however been made in the development of assays for the abnormal conformer of prion protein, PrPTSE.

Normal prion protein (PrPC) is a widely expressed 35 kDa 230 amino acid glycosyl-phosphatidylinositol anchored membrane glycoprotein with two N-linked glycosylation sites and a secondary structure that includes three alpha helices and a single beta-pleated sheet. During the development of prion diseases there is a change in secondary and tertiary structure with a substantial increase in the proportion of beta-pleated sheet which leads to a change in the physico-chemical characteristics of the molecule, rendering it relatively resistant to breakdown by endogenous proteases and leading to deposition of amyloid-like plaques in affected tissues. Because PrPC and PrPTSE have the same primary structure and post-translational modifications, both forms tend to be recognised by most conventional monoclonal antibodies. PrPTSE-based assays, therefore, have to utilise alternative ways of distinguishing the normal from the abnormal conformational form. PrPTSE was originally defined by its resistance to digestion by proteinase K (PK). However this is a relative phenomenon; PrPTSE can be digested by higher concentrations and longer exposure to proteolytic enzymes and, in addition, there is recent evidence for proteolysis-sensitive forms of PrPTSE (Safar et al, 2005).

Five general approaches have been developed for the detection of PrPTSE

Differential proteinase-K digestion.  Immunohistochemistry distinguishes PrPTSE from PrPC by disrupting the latter through the use of proteolytic enzymes or chaotropic agents, coupled with the use of standard anti-PrP monoclonal antibodies and in situ visualisation for detection. This technique enables the demonstration of PrPTSE deposition in the central nervous system and, in the case of vCJD, in follicular dendritic cells in lymphoid tissue of clinically affected patients. Western blot relies on the extraction of PrPTSE from blood or tissues, proteolytic digestion and electrophoresis with visualisation by anti-PrP monoclonal antibodies. Proteolysis leads to complete digestion of PrPC, but removes only the membrane-distal part of PrPTSE, leading to three bands on gel electrophoresis which corresponds to the three different glycosylation states. The migration rates of these bands vary between different strains of prion disease, allowing clarification of strain type (Collinge et al, 1996). Partially selective precipitation of PrPTSE from large sample volumes by sodium phosphotungstic acid coupled with enhanced detection of bound antibody by chemiluminescence have been used to enhance the sensitivity of Western blot and demonstrate PrPTSE in a variety of peripheral tissues in both sporadic and vCJD (Wadsworth et al, 2001). The World Health Organisation Working Group on International Reference Materials for the Diagnosis and Study of Transmissible Spongiform Encephalopathies oversaw a collaborative study in which a number of different laboratories used their versions of the immunoblot (Minor et al, 2004). Generally it was concluded that, at present, immunoblot is not sufficiently sensitive to detect PrPTSE in the peripheral blood of animals or humans with clinical prion disease.

Immunocapillary electrophoresis was amongst the first methods that claimed to be able to detect PrPTSE in the peripheral blood. The test material is treated with proteinase and subject to a competitive antibody inhibition assay using a labelled peptide (as the competitor) and a monoclonal antibody that recognises both PrPTSE and the peptide (Schmerr et al, 1999; Yang et al, 2005). The technique has however proved difficult to reproduce in other laboratories and failed to discriminate between infected and uninfected blood samples in a blinded study (Cervenakova et al, 2003b).

Epitope unmasking/masking.  More success has been achieved with the conformation-dependent immunoassay (CDI), which is predicated on the observation that some PrP epitopes are masked within the PrPTSE aggregate. An increase in signal intensity produced by a labelled monoclonal antibody by a sample denatured using guanidine hydrochloride when compared with the native (un-denatured) sample denotes the presence of PrPTSE (PrPC gives the same signal intensity under both conditions). The sensitivity of the technique is increased through the use of highly sensitive dissociation-enhanced lanthanide fluorescence immunoassay for antibody detection and, in some versions of the assay, the use of PK to reduce background signal (Safar et al, 1998, 2002). CDI appears to achieve greater sensitivity than immunoblot (Bellon et al, 2003) and, in the format including PK, may approximate the sensitivity of infectivity assays (Bruce et al, 2001). In the absence of PK it appears able to detect PK-sensitive forms of PrPTSE, though it remains unclear as to whether these are infectious or not (Bellon et al, 2003).

The epitope-protection assay developed by Amorfix uses a chemical modification process which alters epitopes on normal PrP but not those buried within PrPTSE aggregates. The latter are then disaggregated and the conserved epitopes detected using immunodetection methods (http://www.amorfix.com).

PeopleBio have developed an approach where a single antibody is used for both capture and detection steps leading to the blocking of available epitopes by the capture of PrPC but not PrPTSE.

PrPTSE- specific monoclonal antibodies.  Several antibodies have now been developed that appear to be specific for conformation-dependent epitopes present in PrPTSE but not PrPC (Korth et al, 1997; Paramithiotis et al, 2003; Curin Serbec et al, 2004; Zou et al, 2004). On these, the antibody 15B3, described by Korth et al (1997) and manufactured by Prionics, is the best characterised and has proved capable of detecting infectivity in the peripheral blood of scrapie-infected sheep and BSE-infected cattle in the absence of PK digestion (http://www.fda.gov/ohrms/dockets/AC/06/slides/2006-4240S1_9.ppt). Three other antibodies (Paramithiotis et al, 2003; Curin Serbec et al, 2004; Zou et al, 2004) also appear specific to PrPTSE but have not yet been translated to routine assay format.

PrPTSE– specific ligands.  A variety of other ligands have been shown to bind selectively to the abnormally conformed molecule. Plasminogen has been proposed as a means of selective binding PrPTSE, but as it can also bind to a variety of other proteins it is therefore unlikely to be sufficiently specific for assay development (Fischer et al, 2000).

Polyanionic compounds are known to selectively bind PrPTSE and this property has been employed in the Seprion assay (Lane et al, 2003), which uses coated magnetic beads to capture the molecule. The assay is not dependent on PK treatment and is not species-specific provided a suitable detection antibody is used. It is licensed for postmortem diagnosis of BSE and Chronic Wasting Disease and is reported to be able to distinguish between infected and uninfected blood in scrapie-infected sheep and a small number of human samples.

The approach developed by BioMerieux involves PK digestion, precipitation and denaturation followed by reticulation by streptomycin, chemical capture by calyx-6-arene and detection of the macromolecular aggregates by labelled monoclonal antibody (http://www.fda.gov/ohrms/dockets/AC/06/slides/2006-4240S1_9.ppt). Detection of PrPTSE in a small number of plasma samples from scrapie-infected sheep, BSE-infected cattle and CJD-infected humans has been reported.

Adlyfe have developed a third approach utilising a synthetic peptide based on the region of the PrP molecule involved in the PrPC–PrPTSE conformational transition. The peptide sequence is coupled to its mirror image as a palindromic molecule fluorescently labelled at each end. When incorporated into PrPTSE the peptide folds into a hairpin with a beta-sheet conformation and the flurophores stack and change their fluorescence wavelength. Further, the folded ligand induces further molecules to adopt the folded conformation and thus amplifies the signal (Grosset et al, 2005). The assay is reported to have discriminated infected from uninfected plasma in natural and experimental scrapie, BSE and CJD.

Chiron have utilised (http://www.fda.gov/ohrms/dockets/AC/06/slides/2006-4240S1_9.ppt) a synthetic PrP polypeptide to capture PrPTSE on magnetic beads with detection by monoclonal antibody in an ELISA format.

Amplification.  Two methods have been used to amplify the detection signal. Screening for intensively fluorescent targets utilises double labelled antibodies, more of which bind to PrPTSE aggregates than to PrPC and giving rise to a stronger fluorescence signal (Bieschke et al, 2000). Immuno-polymerase chain reaction (PCR) also provides a method of amplifying the signal from an antibody or ligand conjugated to a nucleotide sequence utilising the PCR (Barletta et al, 2005).

Two further approaches have been developed that result in the amplification of PrPTSE itself. The first of these, protein misfolding cyclic amplification (PMCA) has given rise to considerable excitement. PrPTSE seeded into an excess of PrPC leads to formation of new PrPTSE. That PrPTSE is then fragmented through sonication or shaking and leads to a new round of PrPTSE formation (Kocisko et al, 1994; Saborio et al, 2001). Recurrent cycles therefore of incubation and fragmentation lead to amplification of the original PrPTSE (Castilla et al, 2005). Immunoblot and CDI have been used for detection of PrPTSE and infectivity. Studies show that 140 sonication cycles produced an increase in signal intensity of around 6000-fold, whilst a second ‘nested’ set of 118 cycles with a fresh source of normal PrP led to an approximate 107-fold amplification. The technique has proved capable of discriminating infected from uninfected blood from hamsters experimentally infected with scrapie, however there are recent reports of detection of PrPTSE in uninfected animal brain implying the possibility of low levels of abnormally conformed PrP in ‘normal’ individuals.

A number of cell-based amplification techniques have been described in which the rodent cell lines N2a (Nishida et al, 2000), PK-1 (Klohn et al, 2003), Rov9 (Birkett et al, 2001) and CAD-5 are infectable by natural or experimental strains of scrapie and demonstrate amplification of PrPTSE detected by immunoblot. No cell-based amplification has yet been successfully reported for CJD.

Both these kinds of amplification take several days (PMCA) to weeks (cell-based assays) and would therefore be better positioned as confirmatory rather than screening assays.

Considerations with regard to assay assessment.  Whilst the above is not a comprehensive list of all the assays under development, it does provide a flavour of the range and variety of approaches and their relative strengths and weaknesses. Some of these are now approaching the point at which they may be Council of Europe (CE) marked and marketed as potential clinical assays. There are, therefore, a series of further considerations relating to the potential assessment and utility of prion assays prior to clinical implementation.

The required sensitivity is difficult to gauge because the level, spatial distribution and temporal variation of infectivity in the blood of patients with vCJD or healthy individuals with subclinical infection is unknown. The generalizability of experimental data from mouse and hamster experiments to the human condition cannot be assumed (Castilla et al, 2006). Moreover, the relationship between infectivity and PrPTSE is complex. Although many authorities believe PrPTSE to be causal, there is experimental evidence both of infectivity with very low levels of PrPTSE and of the presence of PrPTSE in the absence of infection in human brain (Yuan et al, 2006). Recent studies have shown that following PK digestion, particles in the size range 300–600 kDa have the highest associated specific infectivity (Silveira et al, 2005), suggesting that a PrPTSE oligomer of 14–28 might represent an infectious dose. The contribution of PK-sensitive PrPTSE to infectivity is uncertain. It is reasonable, therefore, to regard PrPTSE as a marker of infection, provided it is recognised that there is not a simple linear correlation with the level of infectivity or the development of disease. The National Institute of Biological Standards and Controls have established a reference panel of homogenised human CJD-infected brain and spleen spiked into plasma, peripheral blood from natural and experimentally infected animals and a small number of peripheral blood samples from patients with vCJD, to provide independent evaluation of the sensitivity of prion assays (http://www.nibsc.ac.uk).

Whereas the sensitivity of an assay is mostly related to its technical aspects, the specificity is also highly dependent on the population into which it is deployed. Thus an assay which may be highly specific (i.e. a high proportion of true to false positives) in a group of patients with suspect clinical disease, can show very poor specificity in a normal population where the number of false positives may significantly exceed the number of true positives. This point is demonstrated in Fig 1. The UK Blood Services have established a Test Assessment Facility containing samples from 5000 UK and 5000 US blood donors to evaluate the likely positivity rates amongst the general population.

image

Figure 1.  Outcome of screening of a ‘normal’ population of one million donors in whom there is a true positive prevalence of 1/10 000 for subclinical variant Creutzfeldt-Jakob disease using an assay with a sensitivity and specificity of 99%. As can be seen, a very poor specificity results where the number of false positives greatly exceeds the number of true positives.

Download figure to PowerPoint

Finally, there are a number of other considerations in weighing the likely impact of the introduction of a vCJD assay. Such an assay is unlikely to only be used amongst blood donors, it may also be used in patients with suspect CJD or other psychiatric or neurological conditions (to exclude CJD); in those considered ‘at risk for public health purposes’ on account of exposure to implicated surgical instruments, blood components or plasma products; or for population prevalence studies and the ‘worried well’. In the absence of a true confirmatory assay (i.e. the ability to demonstrate infectivity) it will prove difficult to discriminate between false and true positive individuals and, of course, the likelihood of a truly positive individual developing clinical vCJD is unknown. Blood donors would not be able to continue to donate if they tested positive, it is illegal for example to take the donation with the intent of discarding it, even if the donor consents to such a strategy. Test positive individuals will therefore have to be told of this outcome and (presumably) managed as ‘at risk for public health purposes’. Clearly this will cause significant distress and give rise to psychological and social problems for some people, act as a disincentive to blood donation and therefore a negative impact on the blood supply. Moreover, it is likely that previous recipients of blood components from these donors will also have to be traced and contacted (lookback), giving rise to a much larger group of individuals in the population considered ‘at risk of public health purposes’ and requiring specific precautionary measures to be taken in the event of surgery or medical investigation (see below). A comprehensive health and economic evaluation will therefore have to weigh the positive impact of reducing potential secondary transmission of vCJD against these potential negative consequences.

Blood component processing

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

Universal leucodepletion was introduced in the UK in 1999 as a measure to reduce the risk of secondary transmission of vCJD. The experimental data from mice infected with the Fukuoka-1 strain of Gerstmann-Straussler-Scheinker disease (Brown et al, 1998, 1999) suggests that leucodepletion filters have little impact on plasma-borne infectivity. Studies in the 263K hamster model (Gregori et al, 2004) similarly suggest a 40–70% reduction in whole blood infectivity, consistent with the removal of leucocyte-associated infectivity, but not that present in the plasma. Table I illustrates the likely distribution of residual infectivity in a unit of leucodepleted red cell concentrate prepared by bottom and top processing method (with a residual plasma volume of around 10–15 ml). Assuming 10 ID/ml infectivity in whole blood, just over 130 ID would be left in the unit and that up to a 3 log further reduction is required to impact upon the risk of transmission (i.e. achieve <1 ID/unit). Red cell concentrates prepared by the more common top-top methodology contain greater amounts of residual plasma (around 20 ml) and would consequently require a 4-log reduction. The absence of data on the level of infection in human blood means an uncertainty of at least 1-log around these point estimates. It can be said in summary, however, that it is unlikely that current blood component processing will suffice to reduce the risk of transmission in most plausible infectivity scenarios.

Table I.   Residual infectivity distribution in a unit of leucodepleted red cell concentrate.
Log reduction in infectivityResidual leucocytesResidual plasmaResidual infectivity
  1. The data represents the likely distribution of residual infectivity in a unit of leucodepleted red cell concentrate prepared by a bottom and top processing method (with a residual plasma volume of around 10 ml).

  2. Assuming 10 ID/ml infectivity in whole blood with 40% (i.e.4 ID/ml) being removed by leucodepletion and the remainder residing in the plasma (i.e. for a haematocrit of 0·45 a plasma concentration of approximately 13 D/ml), around 130 ID remains in the unit’s plasma. Hence up to approximately a 3 log further reduction is required to reduce the risk of transmission to <1 ID/unit.

Leucodepletion alone0·2130130·2
1 Log0·21313·2
2 Log0·21·31·5
3 Log0·20·130·33
4 Log0·20·0130·213

Three companies are working on the development of prion reduction filters. One has a CE-marked dock-on filter which is used in series with a leucodepletion filter. Published studies using this filter material show >3 log reduction in infectivity on brain homogenate spikes and to the limit of detection (>1 log) in endogenous infectivity studies (Gregori et al, 2006). Two other companies are working on the development of combined leucodepletion/prion reduction filters. All prion reduction filters will have to undergo independent assessment of clinical safety and efficacy within a series of studies managed by the UK and Irish Blood Services and agreed with SEAC and the Advisory Committee on the Safety of Blood, Tissues and Organs (http://www.advisorybodies.doh.gov.uk/acsbto/index.htm). Part of the problem for both manufacturers and Blood Services is the absence of assays capable of detecting either PrPTSE or infectivity in the peripheral blood of patients with vCJD. Assessment of the efficacy of the technology is therefore based on brain homogenate spikes (where baseline infectivity is sufficient to detect a 3–4 log reduction but the physico-chemical form of the spike is unlikely to be similar to that of plasma based infectivity), and endogenous infectivity studies (where the form of infectivity is likely to be more relevant, but the baseline infectivity is sufficiently low that little more than a 1-log reduction is detectable). There remain, therefore, fundamental questions relating to the clinical relevance of different forms of spike material and general applicability of these kinds of studies to the human situation. The potential for deleterious effects on the red cell concentrate itself are also a matter for concern, both in terms of the possibility of alterations to the rheological or antigenic profile of the red cells and the loss in the volume of the additional filter. The latter would have a particular impact if used in conjunction with bottom and top processing, the combined effect of which may reduce the red cell mass in a concentrate below current standards, necessitating additional transfusions for some individuals.

With regard to platelet concentrates, re-suspension in optimal additive solution rather than plasma would reduce the amount of residual plasma by around 65% to 80–90 ml. This would still contain more than enough infectivity to transmit infection to the recipient under even the most optimistic of the current infectivity assumptions and is likely to be ineffectual. Prion reduction filters are not currently applicable to either platelet concentrates or FFP.

Plasma product manufacturing

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

It is reassuring that to date no recipient of a pooled plasma product has developed vCJD. However in 1997, shortly after the first description of vCJD as a new condition, there was concern that the UK plasma supply might have the potential to transmit the infectious agent and that plasma collected from countries where there were few or no cases of vCJD might pose a lower risk (Ludlam, 1997). Although this view gave rise to controversy, the regulatory authorities moved to a position of allowing, and subsequently mandating that pooled plasma products manufactured in the UK should only be made from plasma imported from parts of the world at low risk of vCJD.

In an attempt to help define the risk of PrPTSE transmission by plasma-derived products, detailed studies have been undertaken to assess how prions are partitioned during the plasma fractionation process, mainly by spiking the starting plasma with ‘exogenous’ prion derived from brain homogenates of experimentally infected animals. The strengths and weaknesses of this approach are similar to those described above in the discussion around the assessment of prion filters. In general there was least clearance of prion in the manufacture of factor VIII, IX and antithrombin concentrates, greater clearance in the preparation of intravenous immunoglobulin, and greatest clearance in the manufacture of albumin (Foster, 1999).

The way in which different countries responded to the risk that plasma products might transmit the infectious agent varied and depended partly on the perceived relative number of donors who might be infectious as well as details of the plasma fractionation techniques used in each country.

In the UK, using data on partitioning of prion infectivity during manufacture of plasma products, along with the animal data on the likely range of infectivity in individuals with sub-clinical infection, a risk assessment was undertaken to quantify the risk of recipients of such products being infected. The CJD Incidents Panel have taken the view that an individual with a >1% additional risk of exposure to an infectious dose of vCJD should be notified and managed as ‘at risk for public health purposes’.

To date a total of 174 ‘implicated’ batches of plasma products have been identified as having been manufactured from a pool of plasma to which an individual contributed who subsequently developed vCJD (Hewitt et al, 2006). For each of these batches a detailed risk assessment was carried out that included the total number of donations included in the pool, the details of the plasma fractionation process used during manufacture and (conservative) estimates of the likely cumulative reduction in infectivity over the manufacturing process. The outcome was expressed as the likely mass of product to which an individual would have had to be exposed to increase their risk of exposure to infection by 1% over background. Although these estimates varied between manufacturers dependent on the details of the manufacturing process, broadly speaking plasma products could be classified into ‘high risk of exposure’ products, such as coagulation factor concentrates, where a single adult dose would suffice to place a patient beyond the 1% additional exposure threshold; ‘medium risk of exposure’ products, such as immunoglobulin, where only patients receiving repeated doses of the implicated batch would be likely to pass the 1% threshold; and ‘low risk of exposure’ products, such as albumin, where unfeasibly large exposure to the implicated batch would be required to move the patient beyond the 1% threshold. Categorisation as ‘at risk for public health purposes’ requires that a patient be notified and that precautions be taken in the use of surgical instruments and other invasive medical interventions (such as endoscopy with biopsy), to reduce the risk of onward transmission to other patients.

Consideration was given to whether to categorise as ‘at risk for public health purposes’ only those who had been exposed to specific implicated batches, or whether to categorise all individuals who had received UK manufactured products between 1980 and 2001 (the dates between which implicated plasma products pools could have used). After much debate it was agreed that for ‘low and medium risk of exposure’ products (immunoglobulins and albumin), the former approach would suffice, whereas for ‘high risk of exposure’ products (coagulation factors), an umbrella approach would be more appropriate because it was uncertain how many batches of plasma might be ‘infectious’ from donors with sub-clinical vCJD and because if additional donors developed vCJD, it would be necessary to inform further groups of recipients, who might have previously been told they had not received ‘implicated’ batches.

Other countries have responded differently to the UK. In France the assessment took into account the local plasma fractionation processes and concluded that the risk posed by ‘implicated’ batches to recipients was very small and it was not appropriate to take any special precautions to prevent further spread by surgical instruments. A similar view was formulated in Germany, though no cases of vCJD have yet arisen in that country (Seitz et al, 2007).

Within the UK advice on prevention of spread of PrPTSE by surgical, medical and dental instruments is given by the Advisory Committee on Dangerous Pathogens and advice on individual clinical incidents by the CJD Clinical Incidents Panel. Instrument contamination was estimated to be of ‘high risk’ where there was contact with tissue from the nervous system and ‘moderate’ risk if there was exposure to lymphoid tissue (http://www.advisorybodies.doh.gov.uk/acdp/index.htm). Surgery involving these tissues should be undertaken with disposable instruments where possible, and if not they should be ‘quarantined’ thereafter and not reused because it would not be possible to ensure adequate decontamination prior to use on the next patient. This guidance has led to major difficulties in the performance of biopsies with gastrointestinal endoscopes because the samples obtained would probably contain lymphoid tissue. The financial implications are significant because the endoscopes cannot be decontaminated and must effectively be discarded. Both upper and lower gastrointestinal endoscopies without biopsy do not result in the instrument being considered as potentially ‘contaminated’ and it can therefore be reused on other patients after standard cleaning procedure. The concern about possible contamination of instruments has also led to an increased use of capsule endoscopies, which give good images but cannot be used to biopsy or treat gut lesions.

Although no individuals with haemophilia have thus far developed vCJD and a retrospective study of autopsy samples from individuals with haemophilia in 1998 showed no evidence of sub-clinical infection, it has been important to try and gather more data (Lee et al, 1998). This has not been easy and depends upon procuring appropriate tissue samples prospectively from individuals undergoing clinically necessary surgery in addition to consent for autopsy. In addition it has been important to try and develop a record of the extent of exposure of individuals to ‘implicated’ batches of concentrate, as well as all recipients of UK clotting factor concentrates over the 22-year period of exposure. This is being co-ordinated by UK Haemophilia Centre Doctors’ Organisation by accumulating the data for subsequent anonymised studies.

Communication with patients and the general public

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

Keeping recipients of blood and blood products informed about the current state of knowledge and in particular informing individuals about their individual risks has proved challenging because of the complexity and uncertainty inherent in our understanding of the field. It has been important for there to be close collaboration between those able to assess the risk of vCJD infection, physicians responsible for clinical services and patient organisations representing those potentially affected. For those who have received blood components from donors who subsequently developed vCJD, the risk of exposure to vCJD is judged to be high and these individuals have been contacted on an individual basis and offered counselling and specialist follow-up. Similarly, blood donors who have donated blood administered to a patient who later developed vCJD have been contacted and are managed as ‘at risk for public health purposes’. In 2004, all patients with haemophilia were sent a letter stating whether or not they had or had not received UK plasma-derived clotting concentrates between 1980 and 2001, irrespective of whether or not they had received UK plasma products, because in an earlier mailing about this topic only those in the ‘at risk’ group were contacted and this left non-recipients of letters not knowing whether they had not been potentially exposed or whether their letter had got lost in the post. All were offered the opportunity for individual counselling. It is this attention to the detail of how patients are informed that is critical in trying to ensure that individuals feel confident in the arrangements.

For patients potentially exposed to other implicated plasma products, the issue of traceability and notification have proved more problematic. Whilst patients with primary immunodeficiency share a similar close long-term relationship with their physicians, those receiving immunoglobulin for other clinical indications or high doses of albumin (for example during plasma exchange), are often discharged following their acute care. The absence of a general system of traceability for plasma products and of searchable clinical notes has made the follow-up of the latter groups of potentially exposed patients highly problematic.

Concluding remarks

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References

Three years after our last review (Ludlam & Turner, 2005), the management of the risk of transmission of vCJD by blood and plasma products remains highly challenging. Whilst the diminishing number of clinical cases is reassuring, there are continuing uncertainties surrounding the prevalence of sub-clinical disease, the level of infectivity in peripheral blood of such individuals, and the overall risk of transmission and development of clinical disease. Much progress has been made in the development of new technologies, such as prion filters and prion assays, but assessment of these is problematic and cost and countervailing risks need to be considered. Accurate and timely communication with the general public and with those who are considered to be at increased risk of exposure remains essential given the continuing complexity and uncertainty of the field.

References

  1. Top of page
  2. Summary
  3. Infectivity in the peripheral blood
  4. Variant CJD transmission by blood transfusion
  5. Donor deferral criteria
  6. Importation of blood components
  7. Advances in the development of a screening test
  8. Blood component processing
  9. Plasma product manufacturing
  10. Communication with patients and the general public
  11. Concluding remarks
  12. References
  • Alvarez-Larran, A., Del Rio, J., Ramirez, C., Albo, C., Pena, F., Campos, A., Cid, J., Muncunill, J., Sastre, J.L., Sanz, C. & Pereira, A. (2004) Methylene blue-photoinactivated plasma vs. fresh-frozen plasma as replacement fluid for plasma exchange in thrombotic thrombocytopenic purpura. Vox Sanguinis, 86, 246251.
  • Barletta, J.M., Edelman, D.C., Highsmith, W.E. & Constantine, N.T. (2005) Detection of ultra-low levels of pathological prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR. Journal of Virological Methods, 127, 154164.
  • Bellon, A., Seyfort-Brandt, W., Lang, H., Baron, H., Groner, A. & Vey, M. (2003) Improved conformation dependent immunoassay: suitability for enhance prion detection with enhanced sensitivity. The Journal of General Virology, 84, 19211925.
  • Bieschke, J., Giese, A., Schulz-Schaeffer, W., Zerr, I., Poser, S., Eigen, M., Eigen, M. & Kretzschmar, H. (2000) Ultrasensitive detection of pathological prion protein aggregates by dual colour scanning for intensely fluorescent targets. Proceedings of the National Academy of Sciences of the United States of America, 97, 54685473.
  • Birkett, C.R., Hennion, R.M., Bembridges, D.A., Clarke, M.C., Chree, A., Bruce, M.E. & Bostock, C.J. (2001) Scrapie strains maintain biological phenotypes on propagation in a cell line in culture. EMBO Journal, 20, 33513358.
  • Bons, N., Lehmann, S., Mestre-Francès, N., Dormont, D. & Brown, P. (2002) Brain and buffy coat transmission of bovine spongiform encephalopathy to the primate Microcebus murinus. Transfusion, 42, 513516.
  • Brown, P., Rohwer, R.G., Dunstan, B.C., MacAuley, C., Gajdusek, D.C. & Drohan, W.N. (1998) The distribution of infectivity in blood components and plasma derivatives in experimental models of transmissible spongiform encephalopathy. Transfusion, 38, 810816.
  • Brown, P., Cervenakova, L., McShane, L.M., Barber, P., Rubenstein, R. & Drohan, W.N. (1999) Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood products do not transmit Creutzfeldt Jakob disease in humans. Transfusion, 39, 11691178.
  • Bruce, M.E., McConnell, I., Will, R.G. & Ironside, J.W. (2001) Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues. Lancet, 358, 208209.
  • Castilla, J., Saa, P., Hetz, C. & Soto, C. (2005) In vitro generation of infectious scrapie prions. Cell, 121, 195206.
  • Castilla, J., Saa, P., Morales, R., Abid, K., Maundrell, K. & Soto, C. (2006) Protein misfolding cyclic amplification for diagnosis and prion propagation studies. Methods in Enzymology, 412, 321.
  • Cervenakova, L., Yakovleva, O., McKenzie, C., Kolchinsky, S., McShane, L., Drohan, W.N. & Brown, P. (2003a) Similar levels of infectivity in the blood of mice infected with human-derived vCJD and GSS strains of transmissible spongiform encephalopathy. Transfusion, 43, 16871694.
  • Cervenakova, L., Brown, P., Soukhartev, S., Yaklovleva, O., Diringer, H., Saenko, E.L. & Drohan, W.N. (2003b) Failure of immunocompetitive capillary electrophoresis assay to detect disease specific prion protein in buffy coat from humans and chimpanzees with Creutzfeldt Jakob disease. Electrophoresis, 24, 853859.
  • Clarke, P. & Ghani, A.C. (2005) Projections of the future course of the primary vCJD epidemic in the UK: inclusion of subclinical infection and the possibility of wider genetic susceptibility. Journal of the Royal Society, Interface, 2, 1931.
  • Clarke, P., Will, R.G. & Ghani, A.C. (2007) Is there the potential for an epidemic of variant Creutzfeldt-Jakob disease via blood transfusion in the UK? Journal of the Royal Society, Interface, 4, 675684.
  • Collinge, J., Sidle, K.C.L., Meads, J., Ironside, J. & Hill, A.F. (1996) Molecular analysis of prion strain variation and the aetiology of new variant CJD. Nature, 383, 685690.
  • Curin Serbec, V., Bresjanac, M., Popovic, M., Pretnar Hartman, K., Galvani, V., Rupreht, R., Cernilec, M., Vranac, T., Hafner, I. & Jerala, R. (2004) Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt Jacob’s disease affected and normal brain tissue. Journal of Biological Chemistry, 279, 36943698.
  • Dolan, G. (2006) Clinical implications of emerging pathogens in haemophilia: the variant Creutzfeldt-Jakob experience. Haemophilia, 12(Suppl. 1), 1620.
  • Editorial Team (2007) Fourth case of transfusion-associated variant-CJD. Euro Surveillance, 12, pii. 3117. Available at http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=3117.
  • Farrugia, A., Ironside, J.W. & Giangrande, P. (2005) Variant Creutzfeld-Jacob disease transmission by plasma products: assessing and communicating risk in an era of scientific uncertainty. Vox Sanguinis, 89, 186192.
  • Fischer, M.B., Roeckl, C., Parizek, P., Schwarz, H.P. & Aguzzi, A. (2000) Binding of disease-associated prion protein to plasminogen. Nature, 408, 479488.
  • Foster, P. (1999) Assessment of the potential plasma fractionation processes to remove causative agents of transmissible spongiform encephalopathy. Transfusion Medicine, 9, 314.
  • Gregori, L., McCombie, N., Palmer, D., Birch, P., Sowemimo-Coker, S.O., Giulivi, A. & Rohwer, R.G. (2004) Effectiveness of leucoreduction for removal; of infectivity of transmissible spongiform encephalopathies from the blood. Lancet, 364, 529531.
  • Gregori, L., Gurgel, P.V., Lathrop, J.T., Edwardson, P., Lambert, B.C., Carbonell, R.G., Burton, S.J., Hammond, D.J. & Rohwer, R.G. (2006) Reduction in infectivity of endogenous transmissible spongiform encephalopathies present in blood by adsorption to selective affinity resins. Lancet, 368, 22262230.
  • Grosset, A., Moskowitz, K., Nelsen, C., Pan, T., Davidson, E. & Orser, C.S. (2005) Rapid presymptomatic detection of PrPsc via conformationally responsive palindromic PrP peptides. Peptides, 26, 21932200.
  • Health Protection Agency (2006) New case of transfusion-associated variant CJD. CDR Weekly, 16, 23.
  • Hewitt, P.E., Llewlyn, C.A., Mackenzie, J. & Will, R.G. (2006) Creutzfeldt-Jakob disease and blood transfusion: results of the UK Transfusion Medicine Epidemiological Review Study. Vox Sanguinis, 91, 221230.
  • Hilton, D.A., Ghani, A.C., Conyers, L., Edwards, P., McCardle, L., Ritchie, D., Penney, M., Hegazy, D. & Ironside, J.W. (2004) Prevalence of lymphoreticular prion protein accumulation in UK tissue samples. Journal of Pathology, 203, 733739.
  • Hunter, N., Foster, J., Chong, A., McCutcheon, S., Parnham, D., Eaton, S., MacKenzie, C. & Houston, F. (2002) Transmission of prion diseases by blood transfusion. Journal of General Virology, 83, 28972905.
  • Ironside, J.W.(2006) Variant Creutzfeldt-Jakob disease: risk of transmission by blood transfusion and blood therapies. Haemophilia, 12(Suppl. 1), 815.
  • Klohn, P.-C., Stolze, E., Flechsig, E., Enari, M. & Weissmann, C. (2003) A quantitative highly sensitive cell-based infectivity assay for mouse scrapie prions. Proceedings of the National Academy of Sciences of the United States of America, 100, 1166611671.
  • Kocisko, D.A., Come, J.H., Priola, S.A., Chesebro, B., Raymond, G.J., Lansbury, P.T. & Caughey, B. (1994) Cell-free formation of protease resistant prion protein. Nature, 370, 471474.
  • Korth, C., Stierli, B., Streit, P., Moser, M., Schaller, O., Fischer, R., Schulz-Schaeffer, W., Kretzschmar, H., Raeber, A., Braun, U., Ehrensperger, F., Hornemann, S., Glockshuber, R., Riek, R., Billeter, M., Wüthrich, K. & Oesch, B. (1997) Prion (PrPsc)-specific epitope defined by a monoclonal antibody. Nature, 390, 7477.
  • Lane, A., Stanley, C.J., Dealer, S. & Wilson, S.M. (2003) Polymeric ligands with specificity for aggregated prion protein. Clinical Chemistry, 49, 17741775.
  • Lee, C.A., Ironside, J.W., Bell, J.E., Giangrande, P., Esiril, M.M. & McLaughlin, J.E. (1998) Retrospective neuropathological review of prion disease in UK haemophilia patients. Thrombosis and Haemostasis, 80, 909911.
  • Llewelyn, C.A., Hewitt, P.E., Knight, R.S., Amar, K., Cousens, S., Mackenzie, J. & Will, R.G. (2004) Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion. Lancet, 363, 417421.
  • Ludlam, C.A. (1997) New-variant Creutzfeldt-Jakob disease and treatment of haemophilia Executive Committee of the UKHCDO. United Kingdom Haemophilia Centre Directors’ Organisation. Lancet, 350, 1704.
  • Ludlam, C.A. & Turner, M.L. (2005) Managing the risk of transmission of variant Creutzfeldt Jakob disease by blood products. British Journal of Haematology, 132, 1324.
  • Minor, P., Newham, J., Jones, N., Bergeron, C., Gregori, L., Asher, D., Van Engelenburg, F., Stroebel, T., Vey, M., Barnard, G. & Head, M. (2004) Standards for the assay of Creutzfeldt-Jakob disease specimens. Journal of General Virology, 85, 17771784.
  • Nishida, N., Harris, D.A., Vilette, D., Laude, H., Frobert, Y., Grassi, J., Casanova, D., Milhavet, O. & Lehmann, S. (2000) Successful transmission of three mouse adapted scrapie strains to murine neuroblastoma cell lines over expressing wild-type mouse prion protein. Journal of Virology, 74, 320325.
  • Paramithiotis, E., Pinard, M., Lawton, T., LaBoissiere, S., Leathers, V.L., Zou, W.Q., Estey, L.A., Lamontagne, J., Lehto, M.T., Kondejewski, L.H., Francoeur, G.P., Papadopoulos, M., Haghighat, A., Spatz, S.J., Head, M., Will, R.G., Ironside, J., O’Rourke, K., Tonelli, Q., Ledebur, H.C., Chakrabartty, A. & Cashman, N.R. (2003) A prion protein epitope selective for the pathologically misfolded conformation. Nature Medicine, 9, 893899.
  • Peden, A.H., Head, M.W., Ritchie, D.E., Bell, J.E. & Ironside, J.W. (2004) Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet, 364, 527529.
  • Saborio, G.P., Permanne, B. & Soto, C. (2001) Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature, 411, 810813.
  • Safar, J., Wille, H., Itri, U., Groth, D., Serban, H., Torchia, M., Cohen, F.E. & Prusiner, S.B. (1998) Eight prion strains have PrPsc molecules with different conformations. Nature Medicine, 4, 11571165.
  • Safar, J.G., Scott, M., Monaghan, J., Deering, C., Didorenko, S., Vergara, J., Ball, H., Legname, G., Leclerc, E., Solforosi, L., Serban, H., Groth, D., Burton, D.R., Prusiner, S.B. & Wailliamson, R.A. (2002) Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice. Nature Biotechnology, 20, 11471150.
  • Safar, J.G., Geschwind, M.D., Deering, C., Didorenko, S., Sattavat, M., Sanchez, H., Serban, A., Vey, M., Baron, H., Giles, K., Miller, B.L., Dearmond, S.J. & Prusiner, S.B. (2005) Diagnosis of human prion disease. Proceedings of the National Academy of Sciences of the United States of America, 102, 35013506.
  • Schmerr, M.J., Jenny, A.L., Bulgin, M.S., Miller, J.M., Hamir, A.N., Cutlip, R.C. & Goodwin, K.R. (1999) Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy. Journal of Chromatography, A 853, 207214.
  • Seitz, R., Von Auer, F., Blumel, J., Burger, R., Buschmann, A., Dietz, K., Heiden, M., Hitzler, W.E., Klamm, H., Kreil, T., Kretzschmar, H., Nübling, M., Offergeld, R., Pauli, G., Schottstedt, V., Volkers, P. & Zerr, I. (2007) Impact of vCJD on blood supply. Biologicals, 35, 7997.
  • Silveira, J.R., Raymond, G.J., Hughson, A.G., Race, R.E., Sim, V.L., Hayes, S.F. & Caughey, B. (2005) The most infectious prion protein particles. Nature, 437, 257261.
  • Wadsworth, J.D., Joiner, S., Hill, A.F., Campbell, T.A., Desbruslais, M., Luthert, P.J. & Collinge, J. (2001) Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet, 358, 171180.
  • Yang, W.C., Yeung, E.S. & Schmerr, M.J. (2005) Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation. Electrophoresis, 26, 17511759.
  • Yuan, J., Xiao, X., McGeehan, J., Dong, Z., Cali, I., Fujioka, H., Kong, Q., Kneale, G., Gambetti, P. & Zou, W.Q. (2006) Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains. Journal of Biological Chemistry, 281, 3484834858.
  • Zou, W.-Q., Zheng, J., Gray, D.M., Gambetti, P. & Shen, S.G. (2004) Antibody to DNA detects scrapie but not normal prion protein. Proceedings of the National Academy of Sciences of the United States of America, 101, 138011385.