International Journal of Cell Biology
Volume 2013 (2013), Article ID 839329, 10
pageshttp://dx.doi.org/10.1155/2013/839329
Review Article
Identification of Misfolded Proteins in Body Fluids for the Diagnosis of
Prion Diseases
Francesca Properzi and Maurizio Pocchiari Department of Cell Biology and
Neurosciences, Istituto Superiore di Sanità , Viale Regina Elena 299, 00161 Rome,
Italy
Received 21 May 2013; Revised 10 July 2013; Accepted 11 July 2013
Academic Editor: Alessio Cardinale
Copyright © 2013 Francesca Properzi and Maurizio Pocchiari. This is an open
access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited.
Abstract Transmissible spongiform encephalopathy (TSE) or prion diseases
are fatal rare neurodegenerative disorders affecting man and animals and caused
by a transmissible infectious agent. TSE diseases are characterized by
spongiform brain lesions with neuronal loss and the abnormal deposition in the
CNS, and to less extent in other tissues, of an insoluble and protease resistant
form of the cellular prion protein ( ), named . In man, TSE diseases affect
usually people over 60 years of age with no evident disease-associated risk
factors. In some cases, however, TSE diseases are unequivocally linked to
infectious episodes related to the use of prion-contaminated medicines, medical
devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD).
Clinical signs occur months or years after infection, and during this silent
period , the only reliable marker of infection, is not easily measurable in
blood or other accessible tissues or body fluids causing public health concerns.
To overcome the limit of detection, several highly sensitive assays have been
developed, but attempts to apply these techniques to blood of infected hosts
have been unsuccessful or not yet validated. An update on the latest advances
for the detection of misfolded prion protein in body fluids is provided.
1. Introduction There are several forms of Transmissible Spongiform
Encephalopathy (TSE) diseases or prion diseases affecting humans and different
species of farm and wild animals (i.e., sheep, cattle, and cervids). Some of
them have an apparently spontaneous occurrence (i.e., sporadic and genetic TSEs;
some forms of atypical bovine spongiform encephalopathy or scrapie), while
others are linked to the consumption of prion-contaminated food as in the
variant Creutzfeldt-Jakob disease (CJD) [1], feedstuff in bovine spongiform
encephalopathy (BSE) [2], or medical and surgical devices in iatrogenic CJD [3].
Transmission of variant CJD via blood transfusion and possibly plasma-derived
factor VIII from asymptomatic donors [4] indicates that prion infectivity is
present in blood months or years before clinical onset. Thus, the occurrence of
epidemics in farm animals and episodes of human cases linked to prion infection
pose serious public health issues that are often difficult to solve [5]. An
eloquent example is given by the yet unexplained discrepancy between mortality
(176 death from 1995 to June 2013) [6] and estimated prevalence data (1 in 4,000
to 1 in 10,000 people) of variant CJD in the British population [7]. This
incongruity is causing great concerns because healthy and infected donors who
are not promptly identified might transmit disease by blood transfusion,
surgical instruments, or plasma-derived products.
The only validated surrogate marker of infection is the abnormally
misfolded isoform of the cellular prion protein (PrPC) despite intensive but
disappointing search for the identification of other disease-specific biomarkers
in easily accessible tissues or body fluids [8, 9]. Misfolded PrP (PrPTSE)
accumulates in the CNS and other tissues of infected hosts assuming different
conformations that are related to the strain of prions [10]. PrPTSE is easily
detected by western blot or immunohistochemistry methods after removing the
cellular isoform (PrPC). Most anti-PrP antibodies, in fact, do not distinguish
between PrPTSE and PrPC requiring the removal of the cellular isoform for
achieving disease-specific signals. This is usually realised by pretreating
samples with proteases (usually proteinase K) that partially digest PrPTSE but
completely remove PrPC. The use of proteinase K (PK), however, removes fractions
of poorly aggregated misfolded PrPTSE that is usually present in blood [11] and
likely other body fluids decreasing the chance of detection. Finally, it is
still debated whether PrPTSE is unequivocally associated with prion infectivity
as there are occasions in which PrPTSE is either not associated with infectivity
[12] or absent in infected hosts [13]. Despite these limits, PrPTSE remains the
best available choice for confirming the diagnosis of prion diseases and for the
identification of prion-associated infectivity in tissues and body fluids.
Moreover, the profile that assumes PrPTSE in western blot, reflecting different
pathological conformations, is of great help for making a correct molecular
diagnosis of sporadic CJD and for differentiating sporadic from variant CJD
[14].
In the last 15 years several methods have been developed for increasing the
sensitivity of PrPTSE detection with the aim of finding a reliable assay for an
early diagnosis of prion diseases in easily accessible tissues or body fluids.
An overview of these developments is the objective of this work.
2. Protein-Misfolding Cyclic Amplification (PMCA) In 2001, Saborio and
colleagues [15] developed a novel protocol for the in vitro amplification of the
misfolded prion protein based on the principle that disaggregated PrPTSE
incubated in the presence of a large excess of PrPC produces novel PrPTSE.
Disaggregation of fibrils requires a sonication step, which can be repeated
several times, in a cyclic process, to allow sensitive detection of the
misfolded PrP of the original seed. The protein-misfolding cyclic amplification
(PMCA) was originally developed using hamster brain homogenate and has since
been shown to be an efficient method for the amplification of brain PrPTSE of
other species including mouse, sheep, cattle, bank voles, cervids, and humans
[16–23]. In human samples, the amplification of PrPTSE is strongly influenced by
the correct matching of methionine/valine in the 129 residue of PrP, suggesting
that this polymorphic site of the protein is important for the amplification of
PrP misfolding by the PMCA assay [24–26].
Ten cycles of sonication are sufficient to increase the sensitivity of
standard western blots from 6–12 picograms to 0.3–0.5 picograms of brain PrPTSE
and, with an improved automated protocol which enables a substantial increase in
the number of amplification cycles, up to femtogram levels [27]. PMCA is
therefore a promising platform for prion diagnosis in body fluids (blood, urine,
and CSF) where the level of PrPTSE is estimated in the range of picograms per
mL.
The group led by Soto reported the first successful identification of
PrPTSE in blood (buffy coat) of scrapie affected hamsters with 89% sensitivity
and 100% specificity [27] and positive signals in 50% of samples taken in the
preclinical stage of disease as early as 20 days after intraperitoneal 263K
scrapie injection [28]. The detection of PrPTSE in blood of preclinical
scrapie-infected hamsters is consistent with data on infectivity detection in
blood [8].
Since then, automated PMCA revealed the presence of PrPTSE in plasma
fractions [29], urine [29–31], and cerebral spinal fluid (CSF) [32] of
scrapie-diseased hamsters with sensitivity ranging from 50 (plasma) to 100
percent (CSF) (Table 1). In the CSF samples from scrapie-infected hamsters, PMCA
was performed by using a further improved protocol (rPrP-PMCA) in which PrPC was
replaced by recombinant PrP (recPrP), allowing a sensitivity greater than that
observed with previous PMCA protocols [32].
Table 1: Detection of misfolded PrP in body fluids.
Other than in hamster models, PrPTSE was amplified from blood leukocytes of
both naturally [20, 34] and experimentally scrapie-infected sheep [33] where
PrPTSE bands were detected as early as 90 days postinoculation and correlated
with infectivity titres [33]. On leukocytes of naturally scrapie-infected sheep,
PrPTSE was detected in all tested animals with 100% specificity by using an
enhanced (i.e., addition of poly-A PMCA) protocol [20].
Attempts to detect PrPTSE in blood of other species such as cattle with BSE
and cervids with CWD produced negative or controversial results [34, 37, 58]. In
patients with various forms of prion diseases, the detection of PrPTSE by PMCA
was not attempted (or results were not published) in sample of blood, blood
derivatives, plasma, urine, or CSF despite amplification of PrPTSE was
successfully reported in human brain samples taken from both sporadic and
variant CJDs [16, 24–26].
Finally, PMCA amplification of PrPTSE in samples from body fluids, other
than blood, taken from prion-infected hosts was successfully achieved in a
variety of species and included saliva and urine in sheep with scrapie [36, 36];
saliva, urine, and CSF in cervids with CWD [38, 58]; and CSF and saliva in
cattle with BSE [37]. A list of prion-infected body fluids analysed by PMCA with
the obtained sensitivity and specificity is shown in Table 1.
In conclusion, PMCA has certainly been a breakthrough for detection of
minute amount of PrPTSE that are likely present in body fluids and therefore is
a candidate method for developing sensitive tests for the diagnosis of prion
diseases in animals and humans. Moreover, the amplified product of PMCA retains
the PrPTSE signature of the original seed allowing the molecular diagnosis of
CJD in humans and scrapie in sheep with important public health implications. In
the last 10 years, PMCA has frequently been modified by addition of poly-A [20]
or sulfated dextrans [37], by the use of recombinant PrP instead of brain PrPC
[32], or by coupling with sensitive immunoassays [34] that have on one side
improved the sensitivity of PrPTSE detection but, on the other hand, made the
comparison of data produced by different laboratories difficult. PMCA
coamplifies infectivity together with PrPTSE [59, 60] mimicking the
disease-specific pathogenic event but requiring safety precautions in diagnostic
laboratories. Finally, PrPTSE bands may appear in control preparations after
several PMCA cycles [61]. This finding, whether related to de novo formation of
PrPTSE [20, 60, 61] or cross-contamination of samples [22], raised concern for
the reliability of PMCA in diagnostic applications. This inconvenience, however,
is easily settled by using low PMCA cycles and appropriate technical tips to
avoid possible prion contamination [22].
3. Quacking Induced Conversion (QuIC) A spin-off of the PMCA method was
obtained by substituting sonication with automated tube shaking for the
conversion of recPrP substrates [62]. The novel “quacking induced conversion”
(QuIC) protocol enables the amplification of 1 femtogram of PrPTSE of scrapie
hamster brain homogenate within one day, reducing the complexity and timing of
misfolding amplification. Hamster recPrP promotes the conversion of brain
misfolded proteins of other species such as sheep with scrapie and humans with
sporadic and variant CJDs, regardless of the primary sequence of the PrPTSE seed
[41]. Some spontaneous PK-resistant fragments of less than 12 kD are
occasionally observed in unseeded control samples [32], but they wane out by
reducing the incubation time of the reaction [41].
One of the most significant improvements of misfolding amplification
methods was achieved when western blots were replaced by a real-time fluorescent
colour reaction (real-time QuIC) [40, 42]. This novel read-out system, based on
a fluorescent amyloid-sensitive thioflavin dye (ThT) [63], allowed the
implementation of the whole QuIC procedure to a high-throughput 96-well format.
The real-time QuIC (RT-QuIC) is an efficient quantitative method for the
detection of minute amount of PrPTSE with estimates of the 50% seeding dose
(SD50) of hamster scrapie brain in the same order of magnitude of infectious
doses (LD50) [40].
RT-QuIC protocol has been adapted to the detection of brain PrPTSE of other
species such as CWD-infected deer, scrapie-infected sheep, and sporadic CJD
patients by using species-specific recPrP [40, 42, 64]. Full-length human recPrP
and both truncated and full-length hamster recPrPs are efficient substrates for
the amplification of PrPTSE in sporadic CJD brain irrespective of the 129 codon
phenotypes [43, 64]. A note of disappointment is that the efficacy of variant
CJD brain in seeding RT-QuIC reaction is consistently lower than sporadic CJD
samples [64].
The presence of PrPTSE in the CSF by the QuIC assay was initially revealed
by Atarashi and colleagues [62] in 263K-scrapie infected hamsters and Orrú and
colleagues [41] in scrapie-infected sheep. In 2010, Wilham and colleagues [40]
revealed the presence of PrPTSE in the CSF of 263K scrapie-infected hamsters by
RT-QuIC and estimated a titre of about 10−2 SD50 per µL. CSF samples from
control animals did not revealed any presence of PrPTSE indicating a high
specificity of the assay. These encouraging results on the CSF of
scrapie-infected host promoted further studies in patients with various forms of
prion diseases. A blinded experiment was initially performed on 30 CSF samples
of definite sporadic CJD patients provided by the Australian National CJD
Registry and 155 controls (25 suspected CJD cases and 130 neurological controls)
achieving 87.5% specificity and 100% sensitivity. CJD cases were positive
irrespectively of 129 codon genotypes [42]. Similarly, McGuire and colleagues
[43] screened CSF samples from sporadic CJD patients provided by the National
Creutzfeldt-Jakob Disease Research & Surveillance Unit, Edinburgh, UK,
including all three 129-codon genotype and obtaining 99% specificity and 94%
sensitivity. In the same study, the specificity and sensitivity of the 14-3-3
protein, a surrogate marker currently used for the diagnosis of sporadic CJD,
were 65% and 94%, respectively. An example of the RT-QuIC output in the CSF of a
sporadic CJD patient is given in Figure 1.
Figure 1: RT-QuIC reactions seeded with 15 µL of human CSF samples from one
Italian sporadic CJD patient (12076) and one non-CJD control (13004). 100 fg of
263K prion-infected hamster brain homogenate were used to seed positive control
reactions. Each sample was processed in duplicate. Finally, Sano and colleagues
[44] reported that the RT-QuIC assay on the CSF of patients with genetic prion
diseases has 78% sensitivity in GSS, 100% in FFI, 87% in E200K genetic CJD, and
100% in V203I genetic CJD, suggesting that the RT-QuIC assay for the detection
of PrPTSE in the CSF might become a valid method for improving the diagnosis of
patients with a clinical suspicion of human prion disease.
Besides CSF, the RT-QuIC assay revealed PrPTSE in nasal lavages from
hamsters infected with the transmissible mink encephalopathy (TME) hyper strain
[40] and, by using immunoaffinity beads coupled with the conformational 15B3
anti- PrPTSE antibody (enhanced QuIC), in plasma of scrapie-infected hamsters
[39]. The assay showed 100% sensitivity and specificity and was able to detect a
positive signal long before the appearance of clinical signs of scrapie.
Finally, the application of 15B3-conjugated beads to the QuIC protocol and
the use of a hamster-sheep chimeric recPrP as substrate in the reaction
increased the sensitivity (up to attogram levels) and the speed of detection (28
hrs) of variant CJD brain misfolded proteins spiked into human blood [39].
Despite this good achievement there is still no report on the use of the
enhanced QuIC assay in human blood.
Overall, RT-QuIC methodology is a powerful platform for the detection and
large-scale screening of misfolded PrP in both human and animals. Up to attogram
levels of misfolded PrP can be detected and properly quantified within few hours
by using high-throughput 96-well formats. The high levels of specificity
obtained in a variety of tissues and species by using flexible recombinant
substrates demonstrates the versatility of the novel method. It is of note that
RT-QuIC PK-resistant products are reported to be noninfectious (quoted by [43])
and therefore likely more secure in large-scale screening diagnostic procedures.
The two disadvantages of this assay are the relatively poor performance in
amplifying PrPTSE from variant CJD tissues [64] and the failure to reproduce the
original PrPTSE signature impeding the molecular diagnosis of sporadic CJD and
the distinction between sporadic and variant CJDs.
4. Other Potential Assays for the Detection of Misfolded PrP in Blood 4.1.
Immunocapillary Electrophoresis (ICE) The assay, originally developed by Schmerr
and colleagues [65] and based on a competitive immunoassay with PrP fluorescent
peptides, was soon proven efficient for the detection of PrPTSE in blood of
scrapie-infected sheep and elks with CWD [66]. However, these results were not
confirmed in other laboratories using blood samples from CJD-infected
chimpanzees or sporadic, iatrogenic, genetic, and variant CJD patients [45, 67].
It is therefore unlikely that this assay will be of any use for the diagnosis of
human prion diseases.
4.2. Surface Fluorescence Intensity Distribution Analysis (Surface-FIDA)
This assay consists in the immobilization of single PrP aggregates on a capture
antibody coated surface that are then visualized by the concomitant binding with
two anti-PrP fluorescent antibodies and a double-laser beam scanning system
(surface-FIDA). The method discriminates aggregated PrP forms from monomeric PrP
without the use of the proteinase K (PK) digestion step and therefore recognizes
both PK-resistant and PK-sensitive PrPTSE. Surface-FIDA enabled the counting of
bovine and hamster PrP aggregates in brain homogenates and in bovine
cerebrospinal fluid [47]. PrP aggregates were also blind-detected in blood of
scrapie-infected sheep ( ) with high specificity and sensitivity [46], although
it remains unsettle whether the detection of PrP aggregates correlates with
infectivity. It is of note that spiking of blood plasma with PrPTSE from brain
was unsuccessful suggesting that the properties of PrPTSE from brain are
different from endogenous blood misfolded PrP [46].
4.3. Ligand-Based Immunoassay Terry and colleagues [48] reported the
detection of PrPTSE in 55% of blood mononuclear cells (PBMC) obtained from
scrapie-affected sheep ( ) and 71% of experimentally BSE-affected sheep by a
modified polyanionic ligand assay of the IDEXX HerdCheck methodology [68]. The
assay resulted positive also in a subset of scrapie-infected sheep several
months before the onset of clinical signs suggesting that PrPTSE can be detected
in asymptomatic prion-infected hosts. However, the relatively low sensitivity
observed in prion-infected sheep, the long timings of sample preparations, and
the amount of blood volumes required for the purification of PBMC foretell that
this assay would not be easily applicable to large-scale diagnostic
scopes.
4.4. Solid-State Binding Matrix The assay, based on the affinity that
PrPTSE has for stainless steel particles [69, 70], was adapted for the detection
of misfolded PrP in blood of patients with various forms of CJDs [49]. The
selective absorption of PrPTSE on the metal matrix concentrates misfolded
protein up to the point that the signal can be detected by an ELISA assay.
Because of the selectivity of the metal matrix in binding only misfolded PrP,
there is no need to pretreat samples with PK that likely removes a conspicuous
fraction of PrPTSE in blood. This method was initially tested on human blood
spiked with vCJD brain homogenate where misfolded particles in up to the 10−10
brain dilution were detected. Subsequently, blood of variant and sporadic CJD
patients was analysed on a blinded experiment including samples from patients
with other neurological diseases and controls. Only samples that were reactive
in two separate assays were scored as positive. About two-third of blood samples
from variant CJD but none from sporadic CJD patients and neurological or
nonneurological controls yield positive signals in both assays resulting in 100%
specificity for variant CJD [49].
4.5. EP-vCJD Blood Screening Assay In 2003, Paramithiotis and colleagues
[71] reported the manufacture of an antibody directed against PrP epitopes that
are exposed only upon protein misfolding and therefore specific to PrPTSE. This
conformational anti-PrPTSE antibody was then used for the epitope-protection
(EP) vCJD-screening assay, which was later implemented by Amorfix. The
high-throughput assay achieved 100% sensitivity and specificity on 1,000 blinded
human plasma samples, which included samples that were spiked with variant
CJD-infected and normal brains [50]. In 2009, the specificity of the method was
ascertained on a large-scale screening initiated in France in over 20,000 human
blood samples [51]. Results showed that on the first run 486 samples were
positive (97.6% specificity), 20 of which were then confirmed positive on a
second screening [51]. The repeat-reactive samples were finally considered
negative on a third screening [51]. Subsequently, Amorfix tested three variant
CJD blood samples provided by the National Institute for Biological Standards
and Control (NIBSC, UK) that resulted negative [52]. The sensitivity of the test
was therefore further improved for the detection of 1 : 5,000,000 dilution of
variant CJD-infected brain spiked into blood [52]. However, despite this
enhanced sensitivity, the test was still unable to detect prions in blood of
variant CJD patients, and it was finally concluded that more research is
required before the reevaluation of the assay [53].
4.6. Conformation-Dependent Immunoassay (CDI) In 1998, Safar and colleagues
[10] developed an ELISA-formatted, dissociation-enhanced time-resolved
fluorescence detection system based on specific antibody binding to epitopes
that are accessible in PrPC but that are unmasked only in denatured PrPTSE. This
method does not require PK treatment and is able to recognize both sensitive and
resistant PK misfolded proteins and different PrPTSE conformations. The assay,
improved by incorporating a capture antibody, was able to discriminate PrPTSE
signature in different molecular forms of sporadic CJDs, iatrogenic CJDs and
genetic TSEs [72] and detect up to a 10−5 dilution of PrPTSE from variant CJD
brain used for spiking human normal plasma [54, 73]. However, endogenous PrPTSE
was undetectable in white blood cells of sporadic patients by CDI [55], but we
are not aware of its use in variant CJD blood.
4.7. Misfolded Protein Diagnostic Assay (MPD) This technique is based on a
pyrene-labeled palindromic sequence of prion peptides that converts to β-sheets
in the presence of PrPTSE [56, 74]. This process induces an excimeric signal
from the conjugated pyrenes that propagates to other peptides with the final
goal to amplify the PrPTSE signal. MPD assay detects PrPTSE in brain of 263K
scrapie-infected hamsters during the preclinical and clinical stages of disease
[74] and in small volumes of plasma from prion-infected mice and sheep with
sensitivity up to 1 infectious dose per mL [56]. The same assay discriminated in
blinded small-scale experiments control plasma from that of patients with
sporadic CJD and squirrel monkeys with experimental CJD with 100% specificity
and sensitivity [56].
4.8. Multimer Detection System (MDS) This technique is a modified ELISA
assay that recognizes only multimeric forms of PrPTSE without using any
pretreatment with proteinases, which might remove PrPTSE forms likely present in
body fluids [57]. This assay uses the same principle previously described by Pan
and colleagues [75] and is based on the use of two monoclonal antibodies that
share overlapping epitopes. Monomers (PrPC) are captured by an antibody attached
to the surface of a plate and are not detected by the second antibody due to the
absence of any exposed epitopes. On the other hand, multimers (PrPTSE) are
easily recognized by the second antibody because they expose more copies of the
same epitope. The assay was tested on plasma samples of nine scrapie-infected
and nine control hamsters resulting in 100% specificity and sensitivity [57].
This simple assay, however, requires validation in other laboratories and more
basic work for determining whether the multimeric forms detected by the MDS
assay are related to infectivity.
5. Final Remarks It is unquestionable that in the last 15 years there has
been an outstanding progress in improving the detection of PrPTSE for developing
sensitive and specific diagnostic assays. These sophisticated and highly
sensitive methods successfully detect up to attogram levels of PrPTSE in body
fluids of different species (Table 1). A major breakthrough is the development
of the RT-QuIC technology for the detection of PrPTSE in the CSF of patients
with sporadic [43, 64] and genetic [44] TSEs that as soon as is validated by
other groups will change the diagnostic criteria of human prion diseases.
Endogenous PrPTSE has been identified in blood of scrapie-infected hamster
by PMCA [27, 28] and RT-QuIC [39] assays and of patients with variant CJD by the
solid-state binding matrix assay [49]. Despite these successful observations,
however, there are no published reports on the application of either PMCA or
enhanced RT-QuIC on blood samples of patients with any form of prion diseases
suggesting that both assays still need substantial improvement before their use
in the diagnostic setting. Although the development of the RT-QuIC technology
for the detection of PrPTSE in blood samples is more recent than PMCA, an extra
impediment of the RT-QuIC assay might come from the interference of blood
molecules with the ThT reading. On the other hand, the solid-state binding
matrix assay might be a valid alternative for the development of a blood test
for variant CJD, but the relative low sensitivity (71%) and the finding that
some control samples resulted positive in one of the two runs [49] make the use
of this assay a remote ambition.
What remains elusive is the reproducible detection of endogenous PrPTSE in
blood despite the successful identification of minute amounts of spiked brain
PrPTSE into healthy blood. It becomes more and more evident that the properties
of PrPTSE in brain are different from those in blood and that some components of
blood both inhibit and interfere with PrPTSE detection causing false positive
and negative results and compromising the reproducibility of the assay [46, 51,
76]. A clear example is given by the failure of the EP-vCJD assay that had
excellent and reproducible performances on spiked blood but then completely
failed to identify positive and negative human blood samples [50–53].
These findings pose the question on whether the criteria delineated by the
National Institute for Biological Standards and Control (NIBSC, UK) [77] for
prion diagnostic assay validation in terms of satisfactory sensitivity and
specificity on spiked blood and for the request of variant CJD blood samples are
still relevant for defining the best condition of success of potential prion
test in blood.
We think that the principles for assay validation and accessibility to
variant CJD blood samples should rather focus on reproducible and large scale
blinded studies on blood taken from animal models of prion diseases, such as
scrapie or BSE in sheep followed by a large scale screening of healthy blood
donors to ascertain a sufficient level of specificity.
Finally, our impression is that the research on prion detection in blood
does not really need further sensitive assays but rather requires further work
aiming to the identification of interfering blood components and understanding
prion metabolism in blood.
Acknowledgments The authors thank Anna Ladogana, Anna Poleggi, and Michele
Equestre for kindling provide them data on the detection of PrPTSE in the CSF of
a patient with sporadic CJD by the RT-QuIC assay. Part of this work was
supported by the Joint Program of Neurodegenerative Disease (JPND) research on
“Optimisation, harmonisation and standardisation of CSF RT-QuIC analysis for the
diagnosis of sporadic CJD”.
References
snip...
Letter
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Nature Medicine 11, 982 - 985 (2005) Published online: 28 August 2005 |
doi:10.1038/nm1286
Detection of prions in blood JoaquÃn Castilla1, Paula Saá1,2 & Claudio
Soto1
--------------------------------------------------------------------------------
Abstract
Prion diseases are caused by an unconventional infectious agent termed
prion, composed mainly of the misfolded prion protein (PrPSc)1. The development
of highly sensitive assays for biochemical detection of PrPSc in blood is a top
priority for minimizing the spread of the disease2. Here we show that the
protein misfolding cyclic amplification (PMCA) technology3 can be automated and
optimized for high-efficiency amplification of PrPSc. We show that 140 PMCA
cycles leads to a 6,600-fold increase in sensitivity over standard detection
methods. Two successive rounds of PMCA cycles resulted in a 10 million–fold
increase in sensitivity and a capability to detect as little as 8,000 equivalent
molecules of PrPSc. Notably, serial PMCA enables detection of PrPSc in blood
samples of scrapie-afflicted hamsters with 89% sensitivity and 100% specificity.
These findings represent the first time that PrPSc has been detected
biochemically in blood, offering promise for developing a noninvasive method for
early diagnosis of prion diseases.
also see ;
From: TSS
Subject: Detection of prions in blood
JoaquÃn Castilla1, Paula Saá1, 2 & Claudio Soto1
Date: August 28, 2005 at 3:53 pm PST
Public release date: 28-Aug-2005 Contact: Jim Kelly jpkelly@utmb.edu
409-772-8791 Media Hotline: 409-772-6397 University of Texas Medical Branch at
Galveston
'Mad cow' proteins successfully detected in blood
Biochemical technique expected to yield new, more effective test for
disease-causing prions in cattle and humans
GALVESTON, Texas -- Researchers at the University of Texas Medical Branch
at Galveston (UTMB) have found a way to detect in blood the malformed proteins
that cause "mad cow disease," the first time such "prions" have been detected
biochemically in blood. The discovery, reported in an article scheduled to
appear online in Nature Medicine Aug. 28, is expected to lead to a much more
effective detection method for the infectious proteins responsible for
brain-destroying disorders, such as bovine spongiform encephalopathy (BSE) in
cattle and variant Creutzfeldt-Jakob disease (vCJD) in humans. The blood test
would make it much easier to keep BSE-infected beef out of the human food
supply, ensure that blood transfusions and organ transplants do not transmit
vCJD, and give researchers their first chance to figure out how many people may
be incubating the disease. "The concentration of infectious prion protein in
blood is far too small to be detected by the methods used to detect it in the
brain, but we know it's still enough to spread the disease," said UTMB neurology
professor Claudio Soto, senior author of the Nature Medicine paper. "The key to
our success was developing a technique that would amplify the quantity of this
protein more than 10 million-fold, raising it to a detectable level." Soto and
the paper's other authors, UTMB assistant professor of neurology Joaquin
Castilla and research assistant Paula Saá, applied a method they call protein
misfolding cyclic amplification (PMCA) to blood samples taken from 18
prion-infected hamsters that had developed clinical symptoms of prion disease.
PMCA uses sound waves to vastly accelerate the process that prions use to
convert normal proteins to misshapen infectious forms. Successive rounds of PMCA
led to the discovery of prions in the blood of 16 of the 18 infected hamsters.
No prions were found in blood samples that were taken from 12 healthy control
hamsters and subjected to the same treatment. "Since the original publication of
a paper on our PMCA technology, we've spent four years optimizing and automating
this process to get to this point," Soto said. "The next step, which we're
currently working on, will be detecting prions in the blood of animals before
they develop clinical symptoms and applying the technology to human blood
samples." Tests for infectious prions in cattle and human blood are badly
needed. Because current tests require post-slaughter brain tissue for analysis,
in the United States only cattle already showing clinical symptoms of BSE
(so-called "downer cows") are tested for the disorder. This is true even though
vCJD potentially can be transmitted by animals not yet showing symptoms of the
disease. (Only two cases of BSE have been found in American cows so far.) And
although British BSE cases have been in decline since 1992, scientists believe
the British BSE epidemic of the 1980s could have exposed millions of people in
the UK and Europe to infectious prions. The extent of the vCJD epidemic is yet
unknown. So far the disease has killed around 180 people worldwide, but numbers
could reach thousands or even hundreds of thousands in the coming decades.
Prions have also been shown to be transmissible through blood transfusions and
organ transplants. "Who knows what the real situation is in cattle in the United
States? And with people, we could be sitting on a time bomb, because the
incubation period of this disease in humans can be up to 40 years," Soto said.
"That's why a blood test is so important. We need to know the extent of the
problem, we need to make sure that beef and the human blood supply are safe, and
we need early diagnosis so that when scientists develop a therapy we can
intervene before clinical symptoms appear--by then, it's too late."
### For more information or to schedule an interview request a digital
photo or arrange a taped or live television interview via UTMB's satellite
services, please call the media hotline.
--------------------------------------------------------------------------------
FC5.1.1
Transmission Results in Squirrel Monkeys Inoculated with Human sCJD, vCJD,
and GSS Blood Specimens: the Baxter Study
Brown, P1; Gibson, S2; Williams, L3; Ironside, J4; Will, R4; Kreil, T5;
Abee, C3 1Fondation Alliance BioSecure, France; 2University of South Alabama,
USA; 3University of Texas MD Anderson Cancer Center, USA; 4Western General
Hospital, UK; 5Baxter BioSience, Austria
Background: Rodent and sheep models of Transmissible Spongiform
Encephalopathy (TSE) have documented blood infectivity in both the pre-clinical
and clinical phases of disease. Results in a (presumably more appropriate)
non-human primate model have not been reported.
Objective: To determine if blood components (red cells, white cells,
platelets, and plasma) from various forms of human TSE are infectious.
Methods: Blood components were inoculated intra-cerebrally (0.1 ml) and
intravenously (0.5 ml) into squirrel monkeys from 2 patients with sporadic
Creutzfeldt- Jakob disease (sCJD) and 3 patients with variant Creutzfeldt-Jakob
disease (vCJD). Additional monkeys were inoculated with buffy coat or plasma
samples from chimpanzees infected with either sCJD or
Gerstmann-Sträussler-Scheinker disease (GSS). Animals were monitored for a
period of 5 years, and all dying or sacrificed animals had post-mortem
neuropathological examinations and Western blots to determine the presence or
absence of the misfolded prion protein (PrPTSE).
Results: No transmissions occurred in any of the animals inoculated with
blood components from patients with sporadic or variant CJD. All donor
chimpanzees (sCJD and GSS) became symptomatic within 6 weeks of their
pre-clinical phase plasmapheresis, several months earlier than the expected
onset of illness. One monkey inoculated with purified leukocytes from a
pre-clinical GSS chimpanzee developed disease after 36 months.
Conclusion: No infectivity was found in small volumes of blood components
from 4 patients with sporadic CJD and 3 patients with variant CJD. ***However, a
single transmission from a chimpanzee-passaged strain of GSS shows that
infectivity may be present in leukocytes, and the shock of general anaesthesia
and plasmspheresis appears to have triggered the onset of illness in
pre-clinical donor chimpanzees.
Saturday, September 5, 2009
TSEAC MEETING FEBRUARY 12, 2004 THE BAXTER STUDY GSS
snip...
Saturday, September 5, 2009
TSEAC MEETING FEBRUARY 12, 2004 THE BAXTER STUDY GSS
snip...see full text ;
Monday, May 6, 2013
Warning of mad cow disease threat to blood transfusions
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES ADVISORY
COMMITTEE
MEETING
THURSDAY,
FEBRUARY 12, 2004
This transcript has not been edited
Or corrected, but appears as received
From the commerical transcribing
Service. Accordingly, the Food and
Drug Administration makes no
Representation as to its accuracy.
SNIP...
DR. GAMBETTI: Yes, much more MM. I just wanted to point out that actually
it looks like the differences that are still conspicuous between variant CJD and
sporadic CJD seem to kind of decrease, after a report from Switzerland, in which
I'm sure you know, the scrapie prion protein was found in the spleen and muscle
of about 20 to 30 percent of the cases with sporadic CJD, indicating that there
must be, sometime during the course of the disease or during the entire course
of the disease, some scrapie prion protein in the blood also of sporadic CJD
patients, or at least a portion of sporadic CJD patients.
SNIP...
So in summary for the entire presentation here, from the animal models of
blood-borne infectivity, I think we can say that we've had unequivocal
demonstrations of blood-borne infectivity in rodents, sheep, and possibly now in
monkeys. We've had diverse strains of agent that have been looked at, and this
effect has been seen with familial Creutzfeldt-Jakob disease, the Fukuoka
strain, variant CJD ?- this is Larisa Cervenakova's work ?- BSE, our work, and
scrapie.
We've seen it in natural TSE infections, as well as experimental
infections, and this is the Institute for Animal Health work with the sheep
transfusions.
Next.
SNIP...
Next.
So if we take the presumption, and the FDA has just told us that they will
presume that this was a transfusion transmission, then it fills in most of the
missing gaps in this story. There can be TSE infectivity in human blood. It is
present preclinically, and it is transmissible by transfusion. It may even have
a virulence greater than might be expected from the incubation time in rodents,
based on the incubation time in this particular case.
Next.
The only thing that's inconsistent with this story, and it is a major
inconsistency, is why we haven't seen transfusion transmissions from classical
cases of Creutzfeldt-Jakob disease. That has been discussed by a number of
people already today. I can't really add much to that. Is it that we?re missing
them, our surveillance isn't right? Is there something really truly different?
We just don't know.
snip...
DR. BROWN: Thank you, Chairman Priola. I feel a little bit like I'm coming
home, maybe for the last time, but it's a nice feeling.
I was asked by David Asher to present the results of the study which
several years ago was undertaken by us with the funding of the Baxter
Pharmaceutical Company, and it has henceforth become known as the Baxter
study.
Before I do that, assuming I have my full complement of 15 minutes and I'm
not down to six and a half, I wanted just to make a comment or two about one or
two of the interesting questions that have been raised in the course of the
morning.
The difficulty of proving that sporadic CJD could be transfusion-linked I
think is probably only going to be solved by exactly the reverse of the
situation that is so compelling as evidence for variant CJD transmission; and
that is, instead of having a young, typical variant CJD donating blood to a
person who is elderly -- when I say "elderly,? that's my age -- you're going to
have to have a classic sporadic elderly patient transmitting blood to an
unusually young patient, and then you'll have the same kind of certainty which
is not totally certain, but you'll have some confidence that that has happened.
And that's not going to be easy to find.
snip...
I was asked by David -- and with the kind permission of Corinne Lasmezas ?
to also give you a summary of her studies, the studies of her group, directed by
her now on what the French are up to with respect to primates.
But the first thing is our own study, and as I mentioned, it's a Baxter
primate study, and those are the major participants. And the goal was twofold,
and here is the first one: to see whether CJD, either sporadic or familial --
actually it turns out to be the familial CJD is incorrect. It really should be
the Fukuoka strain of Gerstmann-Straussler-Scheinker disease. So it's really GSS
instead of familial CJD -- when passaged through chimps into squirrel monkeys
using purified blood components, very pure blood components.
So this addresses the question that was raised just recently about whether
or not red cell infectivity that's been found in rodents is really in the red
cells or is it contaminated.
We prepared these samples with exquisite care, and they are
ultra-ultra-ultra purified. There's virtually no contamination of any of the
components that we looked at ? platelets, red cells, plasma, white cells -- with
any other component.
These are a sort of new set of slides, and what I've tried to do is make
them less complicated and more clear, but I'm afraid I haven't included the
build. So you'll just have to try and follow what I explain with this little red
pointer.
There were three initial patients. Two of them had sporadic CJD. One of
them had Gerstmann-Straussler-Scheinker syndrome. Brain tissue from each
individual patient was inoculated intracerebrally into a pair of chimpanzees.
All right?
From those chimps, either plasma or ultra purified -- in fact, everything
is ultra-purified. I'll just talk about purified plasma, purified white cells --
were inoculated intracerebrally and intravenously to get the maximum amount of
infective load into a pair of squirrel monkeys.
The same thing was done for each of these three sets. This monkey died from
non-CJD causes at 34 months post inoculation.
Let me go back for a second. I didn't point out the fact that these were
not sacrificed at this point. These chimpanzees were apheresed at 27 weeks when
they were still asymptomatic. In this instance, we apheresed them terminally
when they were symptomatic.
And before I forget, I want to mention just a little sidelight of this.
Chimpanzees in our experience -- and I think we may be the only people that have
ever inoculated chimpanzees, and that's no longer a possibility, so this was 20,
30 years ago -- the shortest incubation period of any chimpanzee that we have
ever seen with direct intracerebral inoculation is 13 months.
So we chose 27 weeks, which is about seven months, and incidentally
typically the incubation period is more like 16 or 18 months. The shortest was
13 months. We chose the 27th week, which is about six and a half months,
thinking that this would be about halfway through the incubation period, which
we wanted to check for the presence or absence of infectivity.
But within four weeks after the apheresis, which was conducted under
general anesthesia for three or four hours apiece, every single one of the six
chimpanzees became symptomatic. That is another experiment that I would love to
conclude, perhaps because this is simply not heard of, and it very much smells
like we triggered clinical illness. We didn't trigger the disease, but it
certainly looks like we triggered symptomatic disease at a point that was much
earlier than one would have possibly expected.
Maybe it will never be done because it would probably open the floodgates
of litigation. There's no end of little things that you can find out from CJD
patients after the fact. For example, the neighbor's dog comes over, barks at a
patient, makes him fall down, and three weeks later he gets CJD. So you have a
lawsuit against the neighbor.
I mean, this is not an unheard of matter, but I do think that physical
stress in the form of anesthesia and four hours of whatever goes on with
anesthesia, low blood pressure, sometimes a little hypoxemia looks like it's a
bad thing.
So here we have the 31st week. All of the chimps are symptomatic, and here
what we did was in order to make the most use of the fewest monkeys, which is
always a problem in primate research, we took these same three patients and
these six chimps. Only now we pooled these components; that is to say, we pooled
the plasma from all six chimps. We pooled ultra-purified white cells from all
six chimps because here we wanted to see whether or not we could distinguish a
difference between intracerebral route of infection and intravenous route of
infection.
With respect to platelets and red blood cells, we did not follow that. We
inoculated both intracerebral and intravenously, as we had done earlier because
nobody has any information on whether or not platelets and red cells are
infectious, and so we wanted again to get the maximum.
This is an IV versus IC goal. This one, again, is just getting the maximum
load in to see whether there is, in fact, any infectivity in pure platelets, in
pure red cells.
And of all of the above, the only transmission of disease related to the
inoculation was in a squirrel monkey that received pure leukocytes from the
presymptomatic apheresis. So that goes some way to address the question as to
whether or not it's a matter of contamination. To date the red cells have not
been -- the monkeys that receive red cells have not been observed for more than
a year because that was a later experiment.
So we still can't say about red cells, but we're about four and a half
years down the road now, and we have a single transmission from purified
leukocytes, nothing from plasma and nothing from platelets.
That was the first part of the experiment. The second part was undertaken
with the cooperation of Bob Will and others supplying material to us. These were
a couple of human, sporadic cases of CJD and three variant cases of CJD from
which we obtained buffy coat and plasma separated in a normal way. That is,
these are not purified components.
The two cases of sporadic CJD, the plasma was pooled from both patients.
The buffy coat was pooled from both patients, and then inoculated
intracerebrally and intravenously into three squirrel monkeys each. This is a
non-CJD death five years after inoculation. The other animals are still
alive.
For variant CJD we decided not to pool. It was more important to eliminate
the possibility that there was just a little bit of infectivity in one patient
that would have been diluted to extinction, if you like, by mixing them if it
were to so occur with two patients, for example, who did not have infectivity.
So each one of these was done individually, but the principle was the same:
plasma and buffy coat for each patient was inoculated into either two or three
squirrel monkeys. This is, again, a non-CJD related death.
In addition to that, we inoculated rain as a positive control from the two
sporadic disease cases of human -- from the two human sporadic cases at ten to
the minus one and ten to the minus three dilutions. We have done this many, many
times in the past with other sporadic patients. So we knew what to expect, and
we got exactly what we did expect, namely, after an incubation period not quite
two years, all four monkeys developed disease at this dilution and at the minus
three dilution, not a whole lot of difference between the two.
Now, these are the crucial monkeys because each one of these monkeys every
three to four months was bled and the blood transfused into a new healthy
monkey, but the same monkey all the time. So this monkey, for example, would
have received in the course of 21 months about six different transfusions of
blood from this monkey into this monkey, similarly with this pair, this pair,
and this pair. So you can call these buddies. This is sort of the term that was
used. These monkeys are still alive.
In the same way, the three human variant CJD specimens, brain, were
inoculated into four monkeys, and again, each one of these monkeys has been
repeatedly bled at three to four month intervals and that blood transfused into
a squirrel monkey, the same one each time. Ideally we would love to have taken
bleeding at three months and inoculated a monkey and then let him go, watch him,
and then done the same thing at six months. It would have increased the number
of monkeys eightfold and just unacceptably expensive. So we did the best we
could.
That, again, is a non-CJD death, as is this.
This was of interest mainly to show that the titer of infectivity in brain
from variant CJD is just about the same as it from sporadic. We didn't do a
minus five and a minus seven in sporadic because we have an enormous experience
already with sporadic disease in squirrel monkeys, and we know that this is
exactly what happens. It disappears at about ten to the minus five. So the brain
titer in monkeys receiving human vCJD is identical to the brain titer in monkeys
that have been inoculated with sporadic CJD.
That's the experiment. All of the monkeys in aqua are still alive. They are
approaching a five-year observation period, and I think the termination of this
experiment will now need to be discussed very seriously in view of a probable
six-year incubation period in the U.K. case. The original plan was to terminate
the experiment after five years of observation with the understanding that
ideally you would keep these animals for their entire life span, which is what
we used to do when had unlimited space, money, and facilities. We can't do that
anymore.
It's not cheap, but I think in view of the U.K. case, it will be very
important to think very seriously about allowing at least these buddies and the
buddies from the sporadic CJD to go on for several more years because although
you might think that the U.K. case has made experimental work redundant, in
point of fact, anything that bears on the risk of this disease in humans is
worthwhile knowing, and one of the things we don't know is frequency of
infection. We don't know whether this case in the U.K. is going to be unique and
never happen again or whether all 13 or 14 patients have received blood
components are ultimately going to die. Let's hope not.
The French primate study is primarily directed now by Corinne Lasmezas. As
you know, the late Dominique Dromont was the original, originally initiated this
work, and they have very active primate laboratory in France, and I'm only going
to show two very simple slides to summarize what they did.
The first one is simply to show you the basis of their statement that the
IV route of infection looks to be pretty efficient because we all know that the
intracerebral route of infection is the most efficient, and if you look at this
where they inoculated the same infective load either intracerebrally or
intravenously, the incubation periods were not substantially different, which
suggests but doesn't prove, but doesn't prove that the route of infection is
pretty efficient.
Lower doses of brain material given IV did extend the incubation period and
presumably it's because of the usual dose response phenomenon that you see in
any infectious disease.
With a whopping dose of brain orally, the incubation period was even lower.
Again, just one more example of inefficiency of the route of infection and the
necessity to use more infective material to get transmissions.
And they also have blood inoculated IV which is on test, and the final
slide or at least the penultimate slide shows you what they have on test and the
time of observation, that taken human vCJD and like us inoculated buffy coat,
they've also inoculated whole blood which we did not do.
So to a great extent their studies are complementary to ours and makes it
all worthwhile.
We have about -- oh, I don't know -- a one to two-year lead time on the
French, but they're still getting into pretty good observation periods. Here's
three-plus years.
They have variant CJD adapted to the macaque. That is to say this one was
passaged in macaque monkeys, the cynomolgus, and they did the same thing. Again,
we're talking about a study here in which like ours there are no transmissions.
I mean, we have that one transmission from leukocytes, and that's it.
Here is a BSE adapted to the macaque. Whole blood, and then they chose to
inoculate leukodepleted whole blood, in both instances IV. Here they are out to
five years without a transmission.
And then finally oral dosing of the macaque, which had been infected with
-- which was infected with BSE, but a macaque passaged BSE, whole blood buffy
coat and plasma, all by the IC route, and they're out to three years.
So with the single exception of the leukocyte transmission from our chimp
that was inoculated with a sporadic case of CJD or -- excuse me -- with a GSS,
Gerstmann-Straussler, in neither our study nor the French study, which are not
yet completed have we yet seen a transmission.
And I will just close with a little cartoon that appeared in the Washington
Post that I modified slightly lest you get too wound up with these questions of
the risk from blood. This should be a "corrective."
(Laughter.)
DR. BROWN: Thanks.
Questions?
CHAIRPERSON PRIOLA: Yes. Any questions for Dr. Brown? Dr. Linden.
DR. LINDEN: I just want to make sure I understand your study design
correctly. When you mention the monkeys that have the IV and IC inoculations,
the individual monkeys had both or --
DR. BROWN: Yes, yes, yes. That's exactly right.
DR. LINDEN: So an individual monkey had both of those as opposed to some
monkeys had one and some had the other?
DR. BROWN: Correct, correct. Where IC and IV are put down together was IC
plus IV into a given monkey.
DR. LINDEN: Into a given monkey. Okay.
And the IC inoculations, where were those given?
DR. BROWN: Right parietal cortex, Southern Alabama.
(Laughter.)
DR. BROWN: Oh, it can't be that clear. Yeah, here, Pierluigi.
CHAIRPERSON PRIOLA: Dr. Epstein.
DR. BROWN: Pierluigi always damns me with feint praise. He always says
that's a very interesting study, but. I'm waiting for that, Pierluigi.
I think Jay Epstein --
DR. GAMBETTI: I will say that there's an interesting study and will say,
but I just --
(Laughter.)
DR. GAMBETTI: -- I just point of review. You talk about a point of
information. You say that -- you mention GSS, I guess, and the what, Fukuowa
(phonetic) --
DR. BROWN: Yes, Fukuoka 1.
DR. GAMBETTI: Fukuowa, and is that from the 102, if I remember correctly,
of the --
DR. BROWN: Yes, that is correct.
DR. GAMBETTI: Because that is the only one that also --
DR. BROWN: No, it's not 102. It's 101. It's the standard. It's a classical
GSS. Oh, excuse me. You're right. One, oh, two is classical GSS. It's been so
long since I've done genetics. You're right.
DR. GAMBETTI: Because that is the only one I know, I think, that I can
remember that has both the seven kv fragment that is characteristic of GSS, but
also the PrPsc 2730. So in a sense, it can be stretching a little bit compared
to the sporadic CJD.
DR. BROWN: Yeah, I think that's right. That's why I want to be sure that I
made you aware on the very first slide that that was not accurate, that it truly
was GSS.
There's a GSS strain that has been adapted to mice, and it's a hot strain,
and therefore, it may not be translatable to sporadic disease, correct. All we
can say for sure is that it is a human TSE, and it is not variant. I think
that's about it.
DR. GAMBETTI: I agree, but this is also not perhaps the best --
DR. BROWN: No, it is not the best. We understand --
DR. GAMBETTI: -- of GSS either.
DR. BROWN: Yeah. If we had to do it over again, we'd look around for a --
well, I don't know. We'd probably do it the same way because we have two
sporadics already on test they haven't transmitted, and so you can take your
pick of what you want to pay attention to.
Jay?
DR. EPSTEIN: Yes, Paul. Could you just comment? If I understood you
correctly, when you did the pooled apheresis plasma from the six chimps when
they were symptomatic at 31 weeks, you also put leukocytes into squirrel monkeys
in that case separately IV and IC, but in that instance you have not seen an
infection come down in squirrel monkey, and the question is whether it's
puzzling that you got transmission from the 27-week asymptomatic sampling,
whereas you did not see transmission from the 31-week sampling in symptomatic
animals.
DR. BROWN: Yes, I think there are two or three possible explanations, and I
don't know if any of them are important. The pre-symptomatic animal was almost
symptomatic as it turned out so that we were pretty close to the period at which
symptoms would being, and whether you can, you know, make much money on saying
one was incubation period and the other was symptomatic in this particular case
because both bleedings were so close together. That's one possibility.
The other possibility is we're dealing with a very irregular phenomenon and
you're not surprised at all by surprises, so to speak so that a single animal,
you could see it almost anywhere.
The third is that we, in fact, did just what I suggested we didn't want to
do for the preclinical, namely, by pooling we got under the threshold.
See?
You can again take that for what it's worth. It is a possible explanation,
and again, until we know what the levels of infectivity are and whether by
pooling we get under the threshold of transmission, we simply cannot make
pronouncements.
CHAIRPERSON PRIOLA: Dr. DeArmond.
DR. DeARMOND: Yeah, it was very interesting data, but the --
(Laughter.)
DR. BROWN: I just love it. Go ahead.
DR. DeARMOND: Two comments. The first one was that the GSS cases, as I
remember from reading your publications -- I think Gibbs was involved with them
-- when you transmitted the GSS into animals, into monkeys, perhaps I think it
was chimps, the transmission was more typical of CJD rather than GSS. There were
no amyloid plaques. It was vacuolar degeneration so that you may be transmitting
a peculiar form, as I criticized once in Bali and then you jumped all over me
about.
DR. BROWN: I may do it again.
DR. DeARMOND: Calling me a bigot and some other few things like that.
(Laughter.)
DR. BROWN: Surely not. I wouldn't have said that.
DR. DeARMOND: So there could be something strange about that particular
--
DR. BROWN: Yeah. I think you and Pierluigi are on the same page here. This
may be an unusual strain from a number of points of view.
DR. DeARMOND: The other question though has to do with species barrier
because the data you're showing is kind of very reassuring to us that it's hard
to transmit from blood, but the data from the sheep and from the hamsters and
some of the work, I think, that has been done by others, that it's easy in some
other animals to transmit, hamster to hamster, mouse to mouse.
Could you comment on the --
DR. BROWN: That's exactly why we went to primates. That's exactly it,
because a primate is closer to a human than a mouse is, and that's just common
sense.
And so to try and get a little closer to the human situation and not
totally depend on rodents for transferrable data, that is why you would use a
primate. Otherwise you wouldn't use them. They're too expensive and they cause
grief to animal care study people and protocol makers and the whole thing.
Primate studies are a real pain.
DR. DeARMOND: But right now it's inconclusive and you need more time on
it.
DR. BROWN: I believe that's true. I think if we cut it off at six years you
could still say it was inconclusive, and cutting it off at all will be to some
degree inconclusive, and that's just the way it is.
DR. DeARMOND: So what has to be done? Who do you have to convince, or who
do we all have to convince to keep that going?
DR. BROWN: Thomas?
Without trying to be flip at all, the people that would be the first people
to try to convince would be the funders of the original study. If that fails,
and it might for purely practical reasons of finance, then we will have to look
elsewhere because I really don't want to see those animals sacrificed, not those
eight buddies. Those are crucial animals, and they don't cost a whole lot to
maintain. You can maintain eight -- well, they cost a lot from my point of view,
but 15 to $20,000 a year would keep them going year after year.
CHAIRPERSON PRIOLA: Dr. Johnson.
DR. JOHNSON: Yeah, Paul, I'm intrigued as you are by the shortening of the
incubation period. Have you in all of the other years of handling these animals
when they were transfused, when they were flown out to Louisiana at night -- a
lot of the stressful things have happened to some of these chimps. Have you ever
noticed that before or is this a new observation?
DR. BROWN: Brand new.
MR. JOHNSON: Brand new. Okay.
CHAIRPERSON PRIOLA: Bob, did you want to say something? Dr. Rohwer.
DR. ROHWER: The Frederick fire, wasn't that correlated with a lot of
--
DR. BROWN: Not that I k now of, but you may --
DR. ROHWER: Well, that occurred shortly after I came to NIH, and what I
remember is that there were a whole bunch of conversions that occurred within
the few months following the fire. That was fire that occurred adjacent to the
NINDS facility, but in order to protect it, they moved the monkeys out onto the
tarmac because they weren't sure it wouldn't burn as well.
DR. BROWN: Well, if you're right, then it's not brand new, but I mean, I'm
not sure how we'll ever know because if I call Carlton and ask him, I'm not sure
but what I would trust the answer that he gives me, short of records.
You know, Carlot is a very enthusiastic person, and he might say, "Oh,
yeah, my God, the whole floor died within three days," but I would want to
verify that.
On the other hand, it may be verifiable. There possibly are records that
are still extant.
DR. ROHWER: Actually I thought I heard the story from you.
(Laughter.)
DR. BROWN: You didn't because it's brand new for me. I mean, either that or
I'm on the way
(Laughter.)
CHAIRPERSON PRIOLA: Dr. Bracey.
DR. BRACEY: I was wondering if some of the variability in terms of the
intravenous infection route may be related to intraspecies barriers, that is,
the genetic differences, the way the cells, the white leukocytes are processed,
whether or not microchimerism is established, et cetera.
DR. BROWN: I don't think that processing is at fault, but the question, the
point that you raise is a very good one, and needless to say, we have material
with which we can analyze genetically all of the animals, and should it turn out
that we get, for example, -- I don't know -- a transmission in one variant
monkey and no transmissions in another and a transmission in three sporadic
monkeys, we will at that point genetically analyze every single animal that has
been used in this study, but we wanted to wait until we could see what would be
most useful to analyze.
but the material is there, and if need be, we'll do it.
CHAIRPERSON PRIOLA: Okay. Thank you very much, Dr. Brown.
I think we'll move on to the open public hearing section of the
morning.
snip...
FC5.1.1 Transmission Results in Squirrel Monkeys Inoculated with Human
sCJD, vCJD, and GSS Blood Specimens: the Baxter Study
Brown, P1; Gibson, S2; Williams, L3; Ironside, J4; Will, R4; Kreil, T5;
Abee, C3 1Fondation Alliance BioSecure, France; 2University of South Alabama,
USA; 3University of Texas MD Anderson Cancer Center, USA; 4Western General
Hospital, UK; 5Baxter BioSience, Austria
Background: Rodent and sheep models of Transmissible Spongiform
Encephalopathy (TSE) have documented blood infectivity in both the pre-clinical
and clinical phases of disease. Results in a (presumably more appropriate)
non-human primate model have not been reported. Objective: To determine if blood
components (red cells, white cells, platelets, and plasma) from various forms of
human TSE are infectious. Methods: Blood components were inoculated
intra-cerebrally (0.1 ml) and intravenously (0.5 ml) into squirrel monkeys from
2 patients with sporadic Creutzfeldt- Jakob disease (sCJD) and 3 patients with
variant Creutzfeldt-Jakob disease (vCJD). Additional monkeys were inoculated
with buffy coat or plasma samples from chimpanzees infected with either sCJD or
Gerstmann-Sträussler-Scheinker disease (GSS). Animals were monitored for a
period of 5 years, and all dying or sacrificed animals had post-mortem
neuropathological examinations and Western blots to determine the presence or
absence of the misfolded ‘prion’ protein (PrPTSE). Results: No transmissions
occurred in any of the animals inoculated with blood components from patients
with sporadic or variant CJD. All donor chimpanzees (sCJD and GSS) became
symptomatic within 6 weeks of their pre-clinical phase plasmapheresis, several
months earlier than the expected onset of illness. One monkey inoculated with
purified leukocytes from a pre-clinical GSS chimpanzee developed disease after
36 months. Conclusion: No infectivity was found in small volumes of blood
components from 4 patients with sporadic CJD and 3 patients with variant
CJD.
***However, a single transmission from a chimpanzee-passaged strain of GSS
shows that infectivity may be present in leukocytes, and the ‘shock’ of
general anaesthesia and plasmspheresis appears to have triggered the onset of
illness in pre-clinical donor chimpanzees.
FC5.1.2 Interim Transmission Results in Cynomolgus Macaques Inoculated with
BSE and
vCJD Blood Specimens
Lasmezas, C1; Lescoutra, N2; Comoy, E2; Holznagel, E3; Loewer, J3; Motzkus,
D4; Hunsmann, G4; Ingrosso, L5; Bierke, P6; Pocchiari, M5; Ironside, J7; Will,
R7; Deslys, JP2 1Scripps Florida, Infectology, USA; 2CEA, France; 3PEI, Germany;
4DPZ, Germany; 5Istituto Superiore di Sanita, Italy; 6SMI, Sweden; 7CJD
Surveillance Unit, UK
BSE and vCJD transmitted to cynomolgus macaques reproduce many features of
human vCJD, including clinical symptoms, neuropathological hallmarks of vCJD,
PrPres electrophoretical pattern and, most importantly, the wide distribution of
infectivity in peripheral organs. The latter characteristic distinguishes vCJD
from sCJD in both humans and cynomolgus macaques, and prompted us to use this
non-human primate model for further investigations of vCJD and its risk for
human health. The occurrence of four vCJD infections in humans transfused with
blood from patients who later developed vCJD has raised concern about blood
transfusion safety in countries with vCJD. In this collaborative European study,
we investigated the infectivity of blood components and whole blood administered
by intracerebral (ic) and intravenous (iv) routes. Buffy-coat and whole blood
was inoculated by ic and iv route, respectively, from two vCJD patients and from
two clinical vCJD-inoculated macaques. Transfusions were also performed from
whole blood and blood leucodepleted according to hospital practice standards
from two clinical BSE inoculated macaques. Blood infectivity during the
preclinical phase is being examined in orally infected macaques. Whole blood was
collected and transfused from one such animal two years after oral challenge,
whereas buffy-coat and plasma from two animals at 2 and 4.5 years
post-challenge, respectively, have been inoculated by the ic route. This is an
ongoing study in which recipient animals continue to be observed at various
times post-inoculation. So far, we have had one positive transmission in one
animal transfused 65 months earlier with 40 ml of whole blood from a vCJD
macaque (the characteristics of the disease in this animal will be shown in a
separate poster by E. Comoy). This positive transmission reproduces transfusion
transmission of vCJD in humans, with an incubation of 5.5 years compatible with
incubation periods observed in humans.
Prion2013
Oral.05:
Contaminated blood products induce a highly atypical prion disease devoid
of PrPres in primates
Emmanuel Corney,1 Nina Jaffre,1 Jacqueline Mikol,1 Valerie Durand,1
Christelle Jas-Duval,1,2 Sophie Luccantoni-Freire,1 Evelyne Correia,1 Nathalie
Lescoutra-Etcheqaray,3 Nathalie Streichenberqer,4 Stephane Haik,5 Chryslain
Sumian,3 Paul Brown1 and Jean-Philippe Deslys1
1Commissariat a l'Energie Atomique; Institute of Emerging Diseases and
Innovative Therapies (iMETI); Division of Prions and Related Diseases (SEPIA);
Fontenay-aux- Roses, France; 2EFS·Nord de France; Lille, France; 3MacoPharm;
Tourcoing, France; 4Hospices Civils de Lyon; Prion Unit; Neurobiology
Department; Bron, France; 5Inserm; U 975·CNRS; UMR 7225 - Universite Pierre et
Marie Curie; Paris, France
Background, Concerns about the blood-borne risk of prion infection have
been confirmed by the occurrence in the UK of four transfusion-related
infections of vCJD and an apparently silent infection in an hemophiliac patient.
Asymptomatic incubation periods in prion diseases can extend over decades in
humans. We present here unexpected results of experiments evaluating blood
transmission risk in a non-human primate model.
Material and Methods, Cynomolgus macaques were inoculated with brain or
blood specimens from vCJD infected humans or monkeys. Neuropathological and
biochemical findings were obtained using current methods used for human
patients.
Results, Thirteen out of 23 primates exposed to various human or macaque
blood products exhibited a previously undescribed myelopathic syndrome, devoid
of the classical features of prion disease, notably abnormal prion protein
(PrPres) deposition, whereas the 14 corresponding brain-inoculated donor animals
and 1 transfused animal exhibited the classical vCJD pattern. In passage
experiments, plasma transfusion induced the same atypical phenotype after two
years (again, with no detectable PrPres), whereas the intracerebral inoculation
of spinal cord led to a typical prion disease with cerebral spongiosis and
PrPres accumulation in the brain of the primate recipient. Interestingly,
passage experiments in transgenic mice were largely unsuccessful.
In another experiment designed to test the efficacy of antiprion filters,
three recipients of filtered red blood cells suspended in plasma are still
healthy 4.5 y after transfusion whereas the recipients of unfiltered inocula
died after 2.5 y with the atypical neurological profile.
Conclusion. We describe a new fatal neurological myelopathic syndrome in
monkeys exposed to various vCJD/BSE-infected blood components.
Secondary transmission in primates confirms
(I) the transmissibility of this myelopathy, and
(2) its prion origin which could not be diagnosed as such in the first
recipients.
This myelopathy might be compared in some respects to certain forms of
human lower motor neuron disease, including neuromyelitis optica, the flail arm
syndrome of amyotrophic lateral sclerosis (ALS), and the recently described
FOSMN (facial onset sensory and motor neuronopathy) syndrome.
snip...
Oral.12:
Preclinical detection of variant CJD and BSE prions in blood
Caroline Lacroux,1 Jean Yves Douet,1 Emmanuel Corney,2 Hugh Simmons,3
Vincent Beringue,4 Jean Philippe Deslys,2 Didier Vilette1 and Olivier
Andreoletti1 1INRA; Toulouse, France; 2CEA; Fontenay aux roses, France; 3AHVLA;
Weybridge, UK; 4INRA VIM; Jouy en Josas, France
The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a
likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy
(BSE) agent. More recently, secondary vCJD cases were identified in patients
transfused with blood products prepared from apparently healthy donors who later
went on to develop the disease. As there is no validated assay for detection of
vCJD/BSE infected individuals the prevalence of the disease in the population
remains uncertain. In that context the risk of vCJD blood borne transmission is
considered as a serious concern by health authorities.
In this study, appropriate conditions and substrates for highly efficient
and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding
Cyclic Assay (PMCA) were first identified. This showed that whatever the origin
(species) of vCJD/BSE agent, the ovine Q171 PrP substrates provided the best
amplification performances. These results indicate that the homology of PrP
amino-acid sequence between the seed and the substrate is not the crucial
determinant of the vCJD agent in vitro propagation.
The ability of this method to detect endogenous vCJD/BSE agent in the blood
was then defined. In both sheep and primate models of the disease, the assay
enabled the identification of infected individuals in the early preclinical
stage of the incubation period. Finally, blood from two vCJD affected patients
and 135 healthy controls were tested. The assay detected the presence of the
vCJD case within a pool of several dozens of human blood samples. The equivalent
0.05 uL of whole blood from the vCJD affected patient was sufficient for
amplifying PrPres. These results open new possibilities for vCJD screening and
prevention of its iatrogenic transmission.
snip...
AD.06:
Detecting prions in the brain and blood of TSE-infected deer and hamsters
Alan Elder,1 Davin Henderson,1 Anca Selariu,1 Amy Nalls,1 Byron Caughey,2
Richard Bessen,1 Jason Bartz3 and Candace Mathiason1 1Colorado State University;
Fort Collins, CO USA; 2NIH Rocky Mountain Laboratories; Hamilton, MT USA;
3Creighton University; Omaha, NE USA While large quantities of protease
resistant prion protein (PrPres) can be demonstrated by western blot or IHC in
lymphoid biopsies or post-mortem brain tissues harvested from prion-infected
animals, these conventional assays are less reliable as means to detect the
small quantities of prions thought to be present in bodily fluids or associated
with early and asymptomatic phases of TSE disease. The Real Time-Quaking Induced
Conversion (RT-QuIC) assay is capable of detecting prions at concentrations
below the level of sensitivity of conventional assays and provides a real-time
fluorescent readout negating the use of proteases. We have made modifications to
the RT-QuIC assay to utilize it for the detection of PrPres in brain and blood
harvested from various species infected with prions. In this study, we analyzed
CWD-infected deer and CWD/TME-infected hamster whole blood to determine the
effect of: (1) various anticoagulants, (2) freezing and (3) NaPTA precipitation.
Brain tissue and blood collected from naive deer and hamsters served as negative
controls. We were able to demonstrate amplifiable prions in (1) brain and blood
samples harvested from CWD/TME-infected animals, (2) heparinized blood, (3)
frozen vs. fresh blood and (4) NaPTA treated samples. The RT-QuIC assay is able
to detect PrPres in various species of animals and shows promise as an
antemortem diagnostic tool for blood-borne TSEs. http://www.prion2013.ca/tiny_uploads/forms/Scientific-Program.pdf
Sunday, June 9, 2013
TSEAC March 14, 2013: Transmissible Spongiform Encephalopathies Advisory
Committee Meeting Webcast
Tuesday, May 21, 2013
CJD, TSE, PRION, BLOOD Abstracts of the 23rd Regional Congress of the
International Society of Blood Transfusion, Amsterdam, The Netherlands, June
2-5, 2013
Tuesday, November 26, 2013
Transmission of multiple system atrophy prions to transgenic mice
Saturday, May 25, 2013
Brain homogenates from human tauopathies induce tau inclusions in mouse
brain
Tuesday, March 5, 2013
Use of Materials Derived From Cattle in Human Food and Cosmetics; Reopening
of the Comment Period FDA-2004-N-0188-0051 (TSS SUBMISSION)
FDA believes current regulation protects the public from BSE but reopens
comment period due to new studies
Sunday, June 9, 2013
TSEAC March 14, 2013: Transmissible Spongiform Encephalopathies Advisory
Committee Meeting Webcast
Wednesday, June 29, 2011
TSEAC Meeting August 1, 2011 donor deferral Saudi Arabia vCJD risk blood
and blood products
Wednesday, June 29, 2011 TSEAC JUNE 2, 1999 Welcome to the FDA traveling
road show
From: TSS
Subject: TSEAC JUNE 2, 1999 Welcome to the FDA traveling road show
Date: October 15, 2007 at 3:18 pm PST
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES ADVISORY COMMITTEE MEETING
Thursday, June 2, 1999
Wednesday, March 2, 2011
Transmissible Spongiform Encephalopathies Advisory Committee Meeting
Transcript Posted: 3/2/2011 Posted: 3/2/2011
October 28, 2010
Transmissible Spongiform Encephalopathies Advisory Committee Meeting
Transcript Posted: 3/2/2011
Monday, February 7, 2011
FDA’s Currently-Recommended Policies to Reduce the Possible Risk of
Transmission of CJD and vCJD by Blood and Blood Products 2011 ???
October 29, 2010
Transmissible Spongiform Encephalopathies Advisory Committee Meeting
Transcript Posted: 3/2/2011
Monday, October 18, 2010
TSEAC Transmissible Spongiform Encephalopathies Advisory Committee Draft
Agenda and Meeting Materials,
Posted: 10/18/2010
Meeting of the Transmissible Spongiform Encephalopathies Advisory Committee
Center Date Time Location
Tuesday, September 14, 2010
Transmissible Spongiform Encephalopathies Advisory Committee; Notice of
Meeting October 28 and 29, 2010 (COMMENT SUBMISSION)
Saturday, September 5, 2009
TSEAC MEETING FEBRUARY 12, 2004 THE BAXTER STUDY GSS
Sunday, May 10, 2009
Meeting of the Transmissible Spongiform Encephalopathies Committee On June
12, 2009 (Singeltary submission)
TO : william.freas@fda.hhs.gov
May 8, 2009
Greetings again Dr. Freas, TSEAC et al,
I would kindly, once again, wish to comment at this meeting about the
urgent actions that need to be taken asap, to the Meeting of the Transmissible
Spongiform Encephalopathies Committee On June 12, 2009. Due to my disability
from my neck injury, I will not be attending this meeting either, however I hope
for my submission to be read and submitted. ...
IN reply to ;
snip...see full text ;
Sunday, May 10, 2009
Meeting of the Transmissible Spongiform Encephalopathies Committee On June
12, 2009 (Singeltary submission)
TO : william.freas@fda.hhs.gov
Harvard Risk Assessment of Bovine Spongiform Encephalopathy Update, October
31, 2005
INTRODUCTION
The United States Department of Agriculture’s Food Safety and Inspection
Service (FSIS) held a public meeting on July 25, 2006 in Washington, D.C. to
present findings from the Harvard Risk Assessment of Bovine Spongiform
Encephalopathy Update, October 31, 2005 (report and model located on the FSIS
website:
Comments on technical aspects of the risk assessment were then submitted to
FSIS. Comments were received from Food and Water Watch, Food Animal Concerns
Trust (FACT), Farm Sanctuary, R-CALF USA, Linda A Detwiler, and Terry S.
Singeltary. This document provides itemized replies to the public comments
received on the 2005 updated Harvard BSE risk assessment. Please bear the
following points in mind:
From: Terry S. Singeltary Sr.
To: FREAS@CBER.FDA.GOV
Cc: william.freas@fda.hhs.gov ; rosanna.harvey@fda.hhs.gov
Sent: Friday, December 01, 2006 2:59 PM
Subject: Re: TSE advisory committee for the meeting December 15, 2006 [TSS
SUBMISSION
snip...
ONE FINAL COMMENT PLEASE, (i know this is long Dr. Freas but please bear
with me)
THE USA is in a most unique situation, one of unknown circumstances with
human and animal TSE. THE USA has the most documented TSE in different species
to date, with substrains growing in those species (BSE/BASE in cattle and CWD in
deer and elk, there is evidence here with different strains), and we know that
sheep scrapie has over 20 strains of the typical scrapie with atypical scrapie
documented and also BSE is very likely to have passed to sheep. all of which
have been rendered and fed back to animals for human and animal consumption, a
frightening scenario. WE do not know the outcome, and to play with human life
around the globe with the very likely TSE tainted blood from the USA, in my
opinion is like playing Russian roulette, of long duration, with potential long
and enduring consequences, of which once done, cannot be undone.
These are the facts as i have come to know through daily and extensive
research of TSE over 9 years, since 12/14/97. I do not pretend to have all the
answers, but i do know to continue to believe in the ukbsenvcjd only theory of
transmission to humans of only this one strain from only this one TSE from only
this one part of the globe, will only lead to further failures, and needless
exposure to humans from all strains of TSE, and possibly many more needless
deaths from TSE via a multitude of proven routes and sources via many studies
with primates and rodents and other species. ...
Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518
snip... 48 pages...
Wednesday, October 17, 2007 TSEAC MEETINGS ----- Original Message -----
From: Terry S. Singeltary Sr.
To: FREAS@CBER.FDA.GOV
Cc: william.freas@fda.hhs.gov ; rosanna.harvey@fda.hhs.gov
Sent: Wednesday, November 29, 2006 1:24 PM
Subject: TSE advisory committee for the meeting December 15, 2006
[TSSSUBMISSION]November 29, 2006
Greetings FDA, DHH, Dr. Freas, and Dr. Harvey et al,
a kind and warm Holiday Greetings to you all.i kindly wish to submit the
following to the TSE advisory committee for the meeting December 15, 2006, about
the assessment for potential exposure to vCJD in human
plasma-derivedantihemophilic factor (FVIII) productsmanufactured from U.S.
plasma donors and related communication material ;
i see the media picked up on this as a 'low risk', from what the gov.
agency perceived to be to them;
however, i seem to disagree. from my primitive ciphering, i see it another
way. this is a huge catastrophic risk. 3 in 160 is 1.9%. so call that 2% which
is 1 in 50 or twenty per thousand or 20,000 per million. also, wha tabout the
mixed genotypes/mixed susceptibility?
what about the silent carriers that donated tainted blood?
what about the sporadic CJDs of UNKNOWN strain or phenotype?
this risk assessment is just more BSe to me. just another in a long line of
industry fed crap. i pray that my assessment is the one that is wrong. but it is
THEY who roll the dice with your life. it is THEY who refuse to regulate an
industry that has run amok. just from are call aspect of potentially tainted
blood, and these are just recent recalls ;
PRODUCT
Source Plasma, Recall # B-0054-7CODEUnits: 03MMNC5465, 03MMNC6361,
03MMNC6801, 03MMNC7510, 03MMNC7891,03MMNC8252, 03MMNC8801, 03MMNC9144,
03MMND1122, 03MMND1478, 03MMND1969,03MMND2350, 03MMND2825, 03MMND3211,
03MMND3708, 03MMND4072, 03MMND4588,03MMND4831, 03MMND5320, 03MMND5719,
03MMND6268, 03MMND6683, 03MMND7228,03MMND7656, 03MMND8211, 03MMND8652,
03MMND9195, 03MMND9618, 03MMNE0628,03MMNE0884, 03MMNE1597, 03MMNE1979,
03MMNE2644, 03MMNE3064, 03MMNE3707,03MMNE4122, 03MMNE4750, 03MMNE5080,
03MMNE5876, 03MMNE6218, 03MMNE7189,03MMNE7587, 03MMNE8027, 03MMNE8645,
03MMNE9029, 03MMNE9641, 03MMNE9979,03MMNF0491, 03MMNF0685, 03MMNF0937,
03MMNF1260, 04MMNA0351, 04MMNA0707,04MMNA1241, 04MMNA1650, 04MMNA2291,
04MMNA2646, 04MMNA3340, 04MMNA3719,04MMNA4312, 04MMNA4683, 04MMNA5298,
04MMNA5750, 04MMNA6407, 04MMNA6816,04MMNA7482, 04MMNA7915, 04MMNA8632,
04MMNA9076, 04MMNA9723, 04MMNB0063,04MMNB0696, 04MMNB1100, 04MMNB1845,
04MMNB2285, 04MMNB3035, 04MMNB3485,04MMNB4213, 04MMNB4672, 04MMNB5841,
04MMNB6652, 04MMNB7162, 04MMNB7930,04MMNB8453, 04MMNB9239, 04MMNB9747,
04MMNC0456, 04MMNC0931, 04MMNC1578
RECALLING FIRM/MANUFACTURER
BioLife Plasma Services, L.P., Mankato, MN, by facsimile on June 4, 2004.
Firm initiated recall is complete.
REASON
Blood products, collected from a donor who was at increased risk for new
variant Creutzfeldt-Jakob Disease (nvCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
89 units
DISTRIBUTION
CA and Austria
END OF ENFORCEMENT REPORT FOR October 25, 2006
###
USA FDA MAD COW BLOOD HUMANS RECALL (these are dime a dozen)RECALLS AND
FIELD CORRECTIONS: BIOLOGICS -- CLASS II
______________________________
PRODUCTSource Plasma, Recall # B-1708-6CODEUnits: MI180733, MI180927,
MI181625, MI181780, MI182337, MI182519, MI183140,MI183311, MI183955, MI185006,
MI185278, MI185822, MI186081, MI186855,MI187183, MI187903, MI188273, MI188695,
MI189257, MI189553, MI190136,MI190473, MI191073, MI191395, MI191972, MI192303,
MI193473, MI194343,04MINA0377, 04MINA0801, 05MINA7147, 05MINA7451, 05MINA8094,
05MINA8504,05MINA9548, 05MINA9883, 05MINB0489, 05MINB0875, 05MINB1469,
05MINB1874,05MINB3116, 05MINB7192, 05MINB7529, 05MINB8246, 05MINB8612,
05MINB9236,05MINB9366, 05MINB9475, 05MINB9641, 05MINC0031, 05MINC0237,
05MINC0336,05MINC0894, 05MINC0964, 05MINC1138, 05MINC1619, 05MINC1750,
05MINC1907,05MINC1977, 05MINC2375, 05MINC2774, 05MINC3113, 05MINC3484,
05MINC4277,05MINC4623, 05MINC5623, 05MINC5914, 05MINC7545, 05MINC7870,
05MINC8355,05MINC8689, 05MINC9437, 05MINC9775, 05MIND0067, 05MIND0393,
05MIND0892,05MIND0951, 05MIND1836, 05MIND2183 and 05MIND2962
RECALLING FIRM/MANUFACTURER
BioLife Plasma Services L.P., Muncie, IN, by facsimile on November 22,
2005.
Firm initiated recall is complete.
REASON
Blood products, collected from unsuitable donors based on risk factors for
Creutzfeldt-Jakob Disease (CJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
80 units
DISTRIBUTION CA, NC, and MD
______________________________
PRODUCT
a) Red Blood Cells, Leukocytes Reduced, Recall # B-1714-6;b) Fresh Frozen
Plasma, Recall # B-1715-6;c) Platelets, Recall # B-1716-6CODEa),
b), and c) Unit: 2443732RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by letters dated
November 11, 2003 and December 18, 2003. Firm initiated recall is complete.
REASON
Blood products, collected from a donor who was at increased risk for new
variant Creutzfeldt-Jakob Disease (nvCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
3 units
DISTRIBUTION
TX and WI
END OF ENFORCEMENT REPORT FOR SEPTEMBER 13, 2006
###
PRODUCT
Fresh Frozen Plasma, Recall # B-1751-6
CODE
Unit: 4936623
RECALLING FIRM/MANUFACTURER
Gulf Coast Regional Blood Center, Houston, TX, by facsimile dated September
16, 2005.
Firm initiated recall is complete.
REASON
Blood product, which was collected from an unsuitable donor based on risk
factors for variant Creutzfeldt-Jakob Disease (vCJD), was distributed.
VOLUME OF PRODUCT IN COMMERCE
1 unit
DISTRIBUTION
TX
END OF ENFORCEMENT REPORT FOR SEPTEMBER 6, 2006
###
Mon Aug 7, 2006 10:2471.248.132.189
PRODUCT
a) Red Blood Cells, Recall # B-1587-6;b) Cryoprecipitated AHF, Recall #
B-1588-6;c) Recovered Plasma, Recal # B-1589-6
CODE
a), b) and c)
Unit: 2016719
RECALLING FIRM/MANUFACTURER
Walter Shepeard Community Blood Center, Inc., Augusta, GA, by facsimile on
March 13, 2003.
Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
3 units
DISTRIBUTION
GA and Germany
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1590-6;b) Fresh Frozen
Plasma, Recall # B-1591-6
CODE
a) and b)
Unit: 2443595
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile on
June30, 2004.
Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
TX
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1592-6;b) Fresh Frozen
Plasma, Recall # B-1593-6
CODEa) and b)
Unit: 2545596
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile on
December 14, 2004 and January 3, 2005.
Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
TX
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1550-6;b) Fresh Frozen
Plasma, Recall # B-1551-6
CODEa) and b)
Unit 2395371
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by fax on August
20,2003.
Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
TX
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1552-6;b) Platelets,
Recall # B-1553-6;c) Fresh Frozen Plasma, Recall # B-1554-6
CODE
a), b) and c)
Unit 2438702
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by fax on May
29,2003.
Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at
increasedrisk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
3 units
DISTRIBUTION
TX
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1555-6;b) Fresh Frozen
Plasma, Recall # B-1556-6
CODEa) and b)
Unit 2454970
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by fax on July 23 and
December 11. 2003.
Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
TX
______________________________
PRODUCT
a) Red Blood Cells, Recall # B-1494-6b) Cryoprecipitated AHF, Recall #
B-1495-6
CODEa) and b)
Unit 5013100
RECALLING FIRM/MANUFACTURER
Walter L. Shepeard Community Blood Center, Inc., Augusta, GA, by fax on
May17, 2005. Firm initiated recall is complete.REASONBlood products, which were
collected from a donor who may be at increased risk for variant
Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
GA
______________________________
PRODUCT
Source Plasma, Recall # B-1450-6
CODE
Unit numbers ST0824313 and ST0824764
RECALLING FIRM/MANUFACTURER
Stillwater Plasma Center LLC, Stillwater, OK, by fax on November 21, 2003.
Firm initiated recall is complete.REASON
Blood products, which were collected from a donor whose suitability
pertaining to risk factors for Creutzfeldt-Jakob Disease (vCJD) was not
adequately determined, were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
UK
______________________________
PRODUCT
Plasma Frozen, Recall # B-1422-6;Recovered Plasma, Recall # B-1423-6
CODE
a) Unit 03E42218;
b) Unit 03E38153
RECALLING FIRM/MANUFACTURER
American Red Cross Blood Services, Atlanta, GA, by telephone, e-mail
orletter on February 20 or 21, 2004. Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
GA and Switzerland
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1374-6;
b) Recovered Plasma, Recall # B-1375-6CODEa) and b) unit 2453906
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by fax on October 31
and November 5, 2003. Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
TX and Austria
______________________________
PRODUCT
Source Plasma.
Recall # B-1295-6
CODE
Units: NG0046551, NG0045950
RECALLING FIRM/MANUFACTURERD
CI Biologicals Nacogdoches LLC, Nacogdoches, TX, by telephone and fax
onDecember 20, 2002, Firm initiated recall is complete.
REASON
Blood products, collected from a donor who did not answer the questions on
the new variant Creutzfeldt-Jacob disease (nvCJD) questionnaire appropriately,
were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
KY
______________________________
PRODUCT
Source Plasma. Recall # B-1296-6
CODE
Unit: NG 0044520 RECALLING FIRM/MANUFACTURERDCI Biologicals Nacogdoches
LLC, Nacogdoches, TX, by telephone and fax onDecember 12, 2002. Firm initiated
recall is complete.
REASON
Blood product, collected from a donor who did not answer the questions on
the new variant Creutzfeldt-Jacob disease (nvCJD) questionnaire, was
distributed.
VOLUME OF PRODUCT IN COMMERCE
1 unit
DISTRIBUTION
KY
______________________________
PRODUCT
Source Plasma. Recall # B-1297-6
CODE
Units: NG0042874, NG0043139, NG0043312, NG0043618, NG0043797,
NG0044020,NG0044209, NG0044507, NG0044718, NG0044977, NG0045161, NG0045412,
NG0045555RECALLING FIRM/MANUFACTURERDCI Biologicals Nacogdoches LLC,
Nacogdoches, TX, by telephone and fax onDecember 20, 2002. Firm initiated recall
is complete.
REASON
Blood products, collected from a donor considered to be at increased risk
for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
13 units
DISTRIBUTION
KY
______________________________
PRODUCT
Source Plasma, Recall # B-1298-6
CODE
Units: NG 0046823, NG 0046671, NG 0045205, NG 0044635, NG 0043095,
NG0042525, NG 0042341RECALLING FIRM/MANUFACTURERDCI Biologicals Nacogdoches LLC,
Nacogdoches, TX, by telephone and fax onDecember 20, 2002. Firm initiated recall
is complete.
REASON
Blood products, collected from a donor who answered questions on the
variant Creutzfeldt-Jacob disease (vCJD) questionnaire inappropriately, were
distributed.
VOLUME OF PRODUCT IN COMMERCE
7 units
DISTRIBUTION
KY
______________________________
PRODUCT
Recovered Plasma, Recall # B-1299-6 CODE Unit: 4357117
RECALLING FIRM/MANUFACTURER
Department of the Navy, Naval Medical Center, San Diego, CA, by fax and
letter on September 25, 2003. Firm initiated recall is complete.
REASON
Blood product, collected from a donor considered to be at risk of exposure
to Creutzfeldt-Jacob Disease (CJD), was distributed.
VOLUME OF PRODUCT IN COMMERCE
1 unit
DISTRIBUTION
Germany
END OF ENFORCEMENT REPORT FOR July 12, 2006
###
CJD WATCH MESSAGE BOARD
TSS
FDA mad cow nvCJD 'only' blood recalls 1ST WEEK JULY Fri Jul 7, 2006
09:3770.110.83.160
FDA mad cow nvCJD 'only' blood recalls 1ST WEEK JULY
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1379-6;
b) Platelets, Recall # B-1380-6;
c) Fresh Frozen Plasma, Recall # 1381-6;
d) Recovered Plasma, Recall # B-1382-6
CODE
a) Unit numbers: 2343106, 2377779, and 2403533;
b) and c) Unit numbers: 2377779;
d) Unit numbers: 2343106 and 2403533
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile on
June12, 2003. Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
7 units
DISTRIBUTION TX and Austria
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1467-6;
b) Recovered Plasma, Recall # B-1468-6
CODE
a) and b)
Unit numbers: 2329380
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile on May
8,2003. Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTIONTX and Switzerland
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1479-6;
b) Cryoprecipitated AHF, Recall # B-1480-6;
c) Recovered Plasma, Recall # B-1481-6
CODE
a), b), and c)
Unit numbers: 2383280
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile on
July23 and 29, 2004. Firm initiated recall is complete.
REASONBlood products, which were collected from a donor who may be at
increased risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
3 units
DISTRIBUTION
TX and Switzerland
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1482-6;
b) Fresh Frozen Plasma, Recall # B-1483-6
CODE
a) and b)
Unit number: 2501452
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile
onOctober 5, 2004. Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
2 units
DISTRIBUTION
TX and NY
______________________________
PRODUCT
a) Red Blood Cells Leukocytes Reduced, Recall # B-1484-6;
b) Plasma Cryoprecipitated Reduced, Recall # B-1485-6;
c) Recovered Plasma, Recall # B-1486-6
CODE
a) and c)
Unit number: 2554077;
b) Unit number: 2415708
RECALLING FIRM/MANUFACTURER
South Texas Blood and Tissue Center, San Antonio, TX, by facsimile on
August13, 2004. Firm initiated recall is complete.
REASON
Blood products, which were collected from a donor who may be at increased
risk for variant Creutzfeldt-Jakob Disease (vCJD), were distributed.
VOLUME OF PRODUCT IN COMMERCE
3 units
DISTRIBUTION
TX and Austria
_____________________________________
END OF ENFORCEMENT REPORT FOR July 5, 2006
###
Greetings again Dr. Freas et al at FDA,
WITH new atypical TSE in the bovine, in the sheep, goat, and humans, and
the fact that the new BASE TSE in cattle being very very similar to sporadic
CJD, rather than the nvCJD, the fact that now science showing the TSE agent of
the atypical cattle in Japan showing infectivity other than CNS tissue, the fact
that the latest Texas mad cow and the recent Alabama mad cow both being of the
atypical strain, it would seem prudent to include all human TSE in the blood
ban, in my opinion. with sporadic CJD, you have many strains and or phenotypes,
some of which are 'UNKNOWN', so we do not know how this will transmit, what
tissues are infectious and or if blood transmits. that's the bottomline, however
it has been reported that the BASE is more virulent to humans.With this, and the
fact that sporadic CJD has tripled in the past few years or so, i see itas being
prudent to take serious and immediate action ;
snip...see full text ;
Wednesday, October 17, 2007 TSEAC MEETINGS
----- Original Message -----
From: Terry S. Singeltary Sr.
To: FREAS@CBER.FDA.GOV
Cc: william.freas@fda.hhs.gov ; rosanna.harvey@fda.hhs.gov
Sent: Wednesday, November 29, 2006 1:24 PM
Subject: TSE advisory committee for the meeting December 15, 2006
[TSSSUBMISSION]November 29, 2006
Greetings FDA, DHH, Dr. Freas, and Dr. Harvey et al,
a kind and warm Holiday Greetings to you all.i kindly wish to submit the
following to the TSE advisory committee for the meeting December 15, 2006, about
the assessment for potential exposure to vCJD in human
plasma-derivedantihemophilic factor (FVIII) products manufactured from U.S.
plasma donors and related communication material ;
PDF]Freas, William TSS SUBMISSION
File Format: PDF/Adobe Acrobat -
Page 1. J Freas, William From: Sent: To: Subject: Terry S. Singeltary
Sr. [flounder@wt.net] Monday, January 08,200l 3:03 PM freas ...
Thursday, February 24, 2011
The risk of variant Creutzfeldt-Jakob disease among UK patients with
bleeding disorders, known to have received potentially contaminated plasma
products
Monday, October 14, 2013 Researchers estimate one in 2,000 people in the
UK carry variant CJD proteins http://creutzfeldt-jakob-disease.blogspot.com/2013/10/researchers-estimate-one-in-2000-people.html
Sunday, September 29, 2013
Recalls raise questions on safety practices for donated blood CJD TSE PRION
Wednesday, October 09, 2013
*** WHY THE UKBSEnvCJD ONLY THEORY IS SO POPULAR IN IT'S FALLACY,
£41,078,281 in compensation REVISED
Thursday, October 10, 2013
CJD REPORT 1994 increased risk for consumption of veal and venison and lamb
Friday, August 16, 2013
*** Creutzfeldt-Jakob disease (CJD) biannual update August 2013 U.K.
and
***Contaminated blood products induce a highly atypical prion disease
devoid of PrPres in primates
Monday, May 6, 2013
Warning of mad cow disease threat to blood transfusions
WHAT about the sporadic CJD TSE proteins ?
WE now know that some cases of sporadic CJD are linked to atypical BSE and
atypical Scrapie, so why are not MORE concerned about the sporadic CJD, and all
it’s sub-types $$$
snip...see full text and more here ;
Sunday, August 11, 2013
Creutzfeldt-Jakob Disease CJD cases rising North America updated report
August 2013
Creutzfeldt-Jakob Disease CJD cases rising North America with Canada seeing
an extreme increase of 48% between 2008 and 2010
Sunday, October 13, 2013
CJD TSE Prion Disease Cases in Texas by Year, 2003-2012
Saturday, November 2, 2013
Recommendation of the Swiss Expert Committee for Biosafety on the
classification of activities using prion genes and prion protein January 2013
Saturday, November 16, 2013
Management of neurosurgical instruments and patients exposed to
creutzfeldt-jakob disease 2013 December
Infect Control Hosp Epidemiol.
Thursday, November 28, 2013
Department of Justice Former Suppliers of Beef to National School Lunch
Program Settle Allegations of Improper Practices and Mistreating Cows
Saturday, November 2, 2013
Exploring the risks of a putative transmission of BSE to new species
***Together with previous experiments performed in ovinized and bovinized
transgenic mice and hamsters [8,9] indicating similarities between TME and
L-BSE, the data support the hypothesis that L-BSE could be the origin of the TME
outbreaks in North America and Europe during the mid-1900s.
Saturday, November 2, 2013
APHIS Finalizes Bovine Import Regulations in Line with International Animal
Health Standards while enhancing the spread of BSE TSE prion mad cow type
disease around the Globe
I AGREE WITH MR. BULLARD, it’s all about trade and money, BSE TSE PRION aka
mad cow type disease and sound science there from, was thrown out the window by
the USDA et al that fateful day in December 23, 2003, when the USDA lost it’s
‘gold card’ of supposedly being BSE FREE, (that was and still is a sad joke
though), that’s when mad cow junk science was adopted by the USDA...
see why below...kind regards, terry
Monday, November 4, 2013
*** R-CALF Bullard new BSE rule represents the abrogation of USDA’s
responsibility to protect U.S. consumers and the U.S. cattle herd from the
introduction of foreign animal disease
Wednesday, October 30, 2013
SPECIFIED RISK MATERIAL (SRM) CONTROL VERIFICATION TASK FSIS NOTICE 70-13
10/30/13
U.S.A. 50 STATE BSE MAD COW CONFERENCE CALL Jan. 9, 2001
TSS
