Cerebrospinal fluid markers in Creutzfeldt-Jakob disease
Cerebrospinal Fluid Research
Cerebrospinal fluid markers in Creutzfeldt-Jakob disease
Anders Skinningsrud 2
Vidar Stenset 0 1
Astrid S Gundersen 0 1
Tormod Fladby 0 1
0 University of Oslo , Oslo , Norway
1 Department of Neurology, Akershus University Hospital , 1487, Lrenskog , Norway
2 Multidisciplinary Medical Laboratory, Akershus University Hospital , 1478, Lrenskog , Norway
Background: The objective was to assess the utility of total tau protein (tTau), the ratio of (tTau)/ 181 phosphorylated tau protein (P-Tau) and 14-3-3 protein, as diagnostic markers in cerebrospinal fluid (CSF) for Creutzfeldt-Jakob disease (CJD). Methods: CSF samples received from Norwegian hospitals between August 2005 and August 2007 were retrospectively selected from consecutive patients with tTau values > 1200 ng/L (n = 38). The samples from patients clinically diagnosed with CJD (n = 12) were compared to those from patients with other degenerative neurological diseases: Alzheimer's/vascular dementia (AD/VaD, n = 21), other neurological diseases (OND, n = 5). Total Tau, P-Tau, and -Amyloid (A42) were measured with commercial kits. Additionally, 14-3-3 protein was measured semi-quantitatively by immunoblot. Results: The minimum cut-off limits for diagnosis of CJD were chosen from the test results. For tTau the lower limit was fixed at 3000 ng/L, for the tTau/P-Tau ratio it was 60, and for 14-3-3 protein it was 0.75 arbitrary units. For tTau and tTau/P-Tau ratio, all but three CJD patients had levels above the minimum, whereas almost all of the other patients were below. For the 14-3-3 protein, two CJD patients were below the minimum and five were above. Only one of the other patients was higher than the limit. The sensitivities, specificities and diagnostic efficiencies were: tTau 75%, 92%, and 87%; tTau/P-Tau 75%, 96%, and 89%; and 14-3-3 protein 80%, 96%, and 91%. Conclusion: The results suggest that 14-3-3 protein may be the better marker for CJD, tTau/PTau ratio and tTau are also efficient markers, but showed slightly inferior diagnostic properties in this study, with tTau/P-Tau marginally better than tTau.
The prion protein disorder Creutzfeldt-Jakob disease
(CJD) is a human transmissible spongiform
encephalopathy (TSE) that leads to rapid decay of brain tissue.
Human prion diseases are either idiopathic such as
sporadic or spontaneous CJD (sCJD), genetic or familial
(fCJD), acquired such as iatrogenic CJD (iCJD) or variant
CJD (vCJD). Variant CJD is related to bovine spongiform
encephalopathy (BSE), has clinical and pathological
characteristics different from sCJD and has not been described
in Norway. The most common form is sCJD, which
accounts for about 85% of all known cases. Spontaneous
CJD is a rare fatal disorder with rapid progression, an
incidence of approximately 1/million/year  and a mortality
of more than 90% within one year. The etiology of sCJD
is not known, but the pathogenesis is related to
conversion of the normal membrane prion protein PrPc to the
pathological form PrPSc . Immunohistochemical
demonstration of PrPSc provides a definite diagnosis of CJD at
autopsy or by brain biopsy.
In the Norwegian population of about 4.5 million, one
would expect approximately 5 cases of sCJD per year. Due
to the few specific pre-mortal diagnostic signs, it is
difficult to separate sCJD clinically from rapidly progressing
Alzheimer's disease (AD) and other rapidly progressing
neurological diseases. As TSEs can cross species barriers,
there is a public health concern about the ability of TSE to
spread from other species to humans. Classical scrapie in
sheep is endemic in Norway, and an atypical variant, Nor
98, has appeared , but there is no evidence that scrapie
has spread from animals to humans.
The total concentration of tau protein (tTau) in CSF has
been found to separate patients with CJD from those with
AD . As an increased concentration of tTau is regarded
to be a marker for degradation of neurons, the success of
tTau as a marker for CJD depends on whether other
neurological diseases have the same high amount and rate of
neuronal degradation as CJD. A low concentration of the
amyloid precursor protein (APP)-derived 42 amino acid
peptide in CSF (A42), has been correlated with brain
amyloid deposition in AD . Elevation of 181
phosphorylated tau protein (P-Tau) is also related to brain
pathophysiology in AD, possibly as a result of interaction
between amyloid and tau metabolism . As low
CSFA42 and elevated P-tau are more specific for AD, one
would expect the diagnostic specificity for CJD to rise
when the markers are combined. In line with this, the
ratio of tTau/P-tau has been described to separate CJD
from other neurodegenerative diseases without overlap
[7,8]. A separation of sCJD in two clinically different
groups according to P-Tau level and a negative prognostic
value of elevated P-Tau have been described for CJD .
Spontaneous CJD patients with high P-Tau had a shorter
disease duration (i.e. they died earlier), had earlier onset
of akinetic mutism and a higher incidence of typical EEGs.
The occurrence of elevated P-Tau values in CJD will
decrease the value of combining tTau and P-Tau in the
diagnosis of sCJD because the difference in tTau/P-Tau
ratio between the groups would be reduced. The 14-3-3
proteins are evolutionarily conserved proteins present in
the cytoplasm of brain neurons at a concentration of
about 1% of the total protein content. It has been
suggested that 14-3-3 protein as a pre-mortem immunoassay
marker, may obviate the need for a brain biopsy in the
diagnosis of CJD. Although 14-3-3 protein is known to be
We wished to investigate the utility of tTau, tTau/P-Tau
ratio and 14-3-3 protein measurements in CSF as markers
for sCJD. Other diagnostic characteristics for CJD, such as
hyper intense magnetic resonance (MR) signals from the
basal ganglia, sharp wave complexes in EEG and the
examination of the CSF proteins, neuron specific enolase
(NSE) and S100 have been evaluated elsewhere .
Routine laboratory analyses of the three biological variables,
tTau, P-Tau and A42, have been offered at the
Department of Clinical Chemistry, Akershus University Hospital,
since 2003. Routine analysis for 14-3-3 protein has been
performed since November 2007. Neurological,
psychiatric and outpatient departments in Norway have also had
the opportunity to send CSF-samples for analysis.
Patient selection and CSF analysis
CSF samples (n = 691) were received by the Department
of Clinical Chemistry, Akershus University Hospital
between August 2005 and August 2007 from patients
demonstrating symptoms of cognitive decline and
possible neurodegenerative disease. This work was supported
and approved by the Eastern Norway Regional Health
Authority, RHA East, and approved by the Regional
Committee for Medical Research Ethics, which also approved
an exception from collecting informed patient consent.
Total Tau, P-Tau and A42 were analysed in CSF-samples
with commercially available enzyme linked
immunosorbent assay (ELISA) kits from Innogenetics (Gent, Belgium)
adapted to a Tecan Robotic Microplate 150 Processor
(Tecan AG, Switzerland). The analyses were performed
approximately twice a month. The 14-3-3 protein was
analysed by immunoblot with equipment from
Invitrogen Corporation Ltd. (Paisley, U.K.) using an antibody
against the -isoform, anti-14-3-3 gamma, clone CG31-2B
(mouse monoclonal IgG1, Upstate biotechnology, Lake
Placid, NY, USA). The 14-3-3 protein results were assessed
semi-quantitatively using arbitrary units. As standard we
used dilutions of a homogenate from normal brain. For
semi-quantification we performed image analysis using
Fujifilm Multi Gauge software (Fujifilm Corporation,
Tokyo, Japan). Thirty-nine patients studied retrospectively
had tTau values > 1200 ng/L (the highest standard of the
kit). One patient was excluded because no additional
sample was available to analyse tTau in dilution. Total
Tau, P-Tau and A42 were measured at the Akershus
University Hospital between August 2005 and October 2007.
14-3-3 protein was analysed in seven of the 12 CJD
patients and 25 of the 26 AD/VaD/OND patients in
October and November 2007 in Akershus University Hospital.
In one AD patient and in five CJD patients there was no
sample available for analysis. Three of these CJD-patients
had qualitative 14-3-3 protein results from laboratories
outside Norway. Two CJD patients had no 14-3-3 protein
determination. The maximum value for normal clinical
values for tTau was 300 ng/L for patients < 50 years, 450
ng/L for patients 5070 years and 500 ng/L for patients >
70 years . For A42, we have previously used values
above 450 ng/L for normal levels , but after
comparison with the Neurochemistry laboratory at Sahlgrenska
University Hospital, Gothenburg, Sweden (unpublished
data), this has recently been revised to 550 ng/L. The
maximum for normal clinical levels for P-Tau was 80 ng/L
from the Neurochemistry laboratory in Gothenburg.
The samples for tTau, P-Tau and A42 were initially frozen
at -20C. In February 2006 all old samples were
transferred to -80C and all new samples were kept at -80C.
The samples for 14-3-3 protein were stored for up to 2
years at -80C before analysis in batches of six. The ELISA
standards were run in duplicate and the samples were run
singly. The analytical results were read from the
corresponding standard curve for each run. No result exceeded
the highest standard for P-Tau and A42, but for tTau all
the patients in this study exceeded the highest standard
point of 1200 ng/L. These samples were diluted and rerun.
The patients were clinically diagnosed according to
ICD10 (International Classification of Diseases -10). The
criteria were clinical suspicion, MR findings highly
suggestive of CJD and three-phase sharp wave complexes on EEG
also considered to be characteristic of CJD. Twelve
patients had definite or probable CJD (Table 1); 21
patients had Alzheimer's disease (AD), vascular dementia
(VaD), mixed dementia (AD/VaD), frontotemporal
dementia (FTD) or unspecified dementia and five patients
had other neurological diagnoses (OND, Table 2). Seven
of the 12 CJD patients had short disease duration, mean
3.7 months (n = 7, SD = 1.5) and a further two patients
had unspecified very rapid progression. Three patients
had CJD with slower progression, disease duration 15, 22
and >23 months.
Receiver operating characteristic (ROC) curve analysis was
performed for tTau and tTau/PTau ratio with the statistical
package Analyse-it (Analyse-it Software, Ltd. Leeds, U.K.).
ROC-curve analysis was not performed for 14-3-3 protein
because some of the results from other laboratories were
qualitative only. The AD, VaD, AD/VaD and other
dementia patients were treated as one group, AD/VaD. The lower
limit chosen for the diagnosis of CJD was 3000 ng/L for
tTau, for the ratio of tTau to P-Tau it was 60 and for
14-33 protein it was 0.75 arbitrary units. The limits, based on
our test results, were set to obtain the best diagnostic
performance for each marker. It could be argued that the
cutoff for tTau and 14-3-3 protein could have been set
slightly higher. In that case for tTau the sensitivity would
decrease and the specificity would increase, and for
14-33 protein the sensitivity would also decrease and the
specificity would increase to 100%. The sensitivity,
specificity, positive and negative predictive values, and
diagnostic efficiency for the three markers were calculated
1The time from disease recognition to death, 2Three-phase sharp wave complexes, 3Positive for immunochemical examination of pathological prion
protein, PrPSc, 4Values above the normal limit used in clinical practice marked with *, 5Values below the normal limit used in clinical practice marked
with*, 6Generalized dysrythmia, 714-3-3 protein found positive in different laboratories outside Norway because some hospitals sent CSF samples
for 14-3-3 protein analysis to laboratories outside Norway in parallel to sending samples to us. (Three patients both have semi quantitative results
from us and a qualitative, positive result, from a foreign laboratory). 8NC, non-specific changes. 9HSC, highly suggestive changes of CJD, M = missing
Table 2: Characteristics, results and diagnosis for patients with Alzheimer's disease, vascular dementia, mixed dementia and other
1Values above lower limit for CJD marked with*, 2Values above maximum for normal in clinical practice marked with*, 3Values below minimum for
normal used in clinical practice marked with*, M = missing value.
Table 1 presents the characteristics for 12 patients who
were diagnosed with CJD, two definite CJD and 10
probable CJD. Eight had MR findings suggestive of CJD.
Generalized dysrhythmia and sharp wave complexes on EEG
were found in nine patients and generalized dysrhythmia
indicating diffuse cerebral pathology in the remaining
three. Table 2 presents the characteristics for patients with
diagnoses other than CJD. Two patients had VaD or
possible VaD, 11 had AD or possible AD, three had AD/VaD
or possible AD/VaD, one had AD and Wernicke's
encephalopathy, two had possible FTD and two had unspecified
dementia. Five patients had OND.
The results for tTau by diagnostic group and lower limits
for CJD are presented in Figure 1A. Eleven patients had
tTau values above cut-off (3000 ng/L) and 27 below. Nine
of the 12 patients with tTau-values above cut-off had CJD,
one had OND (cerebral B-cell lymphoma) and one had
possible FTD. Three patients with CJD of long duration
had values (1343, 1880 and 2350 ng/L) which are below
the chosen lower limit, 3000 ng/L. All but one of the OND
patients and all but one of the AD/VaD patients had
values below 3000 ng/L.
Ratio of tTau to P-Tau
The results for the ratio tTau/P-Tau by diagnostic group
and chosen lower limit for CJD (60) are presented in
Figure 1B. Ten patients had tTau/P-Tau ratios above the limit.
Nine of these had CJD and one had cerebral B-cell
lymphoma. The three patients with CJD of long duration, had
values below cut-off (19, 25 and 26). All but one of the
OND patients and all AD/VaD patients had values below
the limit for CJD.
P-Tau results by diagnostic group and maximum for
normal (80 ng/L) are presented in Figure 2A. Six of the 12
CJD patients had P-Tau above normal. In the
AD/VaDgroup (n = 21) 15 patients (71%) had P-Tau above and six
below normal. The P-Tau results in the AD/VaD patients
were distributed in two groups, range 2792 and 200287
ng/L. One of the OND patients (cerebral infarction) had
P-Tau slightly above normal, 81 ng/L.
Beta amyloid (142)
The results for A42 by diagnostic group and lower normal
limit are presented in Figure 2B. Three of 11 patients
(38%) in the CJD group (one missing value) and 11 of the
21 patients (52%) in the AD/VaD group had A42 values
below normal. The two CDJ patients with the lowest
A42values, 372 and 385, also had elevated P-Tau results, 149
and 211 ng/L.
The lower limit for CJD diagnosis was set at 0.75 arbitrary
units. Figure 3 shows results from the home laboratory.
Samples from 32 patients, seven from the CJD group, all
five OND patients and 20 from the AD/VaD group, were
available for analysis. Five of the seven CJD patients tested
positive. Three of the 14-3-3 positive patients had been
tested before and had positive results from laboratories
outside Norway. Three of the five CJD patients not tested
by us, had been tested before and had positive results.
These were included in the estimations of diagnostic
parameters (Table 3). Ten of the 12 patients with CJD had
been examined for 14.3.3-protein by us, other
laboratories or both. Eight of these were positive. One of the five
patients with OND (cerebral B-cell lymphoma) tested
weakly positive, and all the tested patients in the AD/VaD
group were negative. Of the three CJD patients with long
disease duration, two were negative.
Calculations of sensitivity, specificity, positive predictive
value, negative predictive value and diagnostic efficiency
are presented in Table 3. The ROC curve analysis for the
diagnosis of CJD showed that tTau was not significantly
different from tTau/P-Tau for the diagnosis of CJD (area
under curve 0.897 and 0.918, n.s.).
Although the ROC-curve analysis showed no significant
difference between tTau and tTau/P-Tau, the tTau/P-Tau
ratio did separate results around the cut-off value more
clearly than t-Tau and the specificity and predictive values
of a positive test for the CJD diagnosis were slightly better.
Thus, our findings seem to agree with those of
Riemenschneider et al , who found that tTau/P-Tau ratio was a
better marker for CJD than tTau. Buerger et al  came to
the opposite conclusion, but they measured P-Tau
phosphorylated in the 232- position and not in the
181-position as measured here and by Riemenschneider et al. Our
results suggest that 14-3-3 protein may be a slightly better
marker than tTau and tTau/P-Tau.
In contrast to P-Tau, A42 did not contribute to providing
a more definite diagnosis of CJD. Increased P-Tau was a
more specific measure of AD/VaD than low A42, and
contributed slightly to the diagnostic separation of CJD from
the AD/VaD group. Similar values for A42 in the CJD and
AD/VaD groups suggest that comparable amyloid
pathology was present in both groups. This indicates that some
CJD patients may have acquired CJD in addition to an
increased and possibly AD-related, amyloid pathology.
The fact that the two CJD patients with the lowest A42
values, also had elevated P-Tau is consistent with an
interaction between prion protein and the AD pathological
processes . The P-Tau values separated into two
groups for AD/VaD patients (Figure 2a). The group with
results above 200 ng/L was homogenous clinically
because 11 of the 13 patients had the clinical diagnosis of
AD, one had FTD/or possibly AD and one had AD/VaD.
The group with results below 100 ng/L (n = 8) was
clinically more heterogeneous (Table 2). Our results suggest
that tTau, tTau/P-Tau and possibly 14-3-3 protein may
only be good markers for sCJD of short duration and may
not separate the CJD cases with longer duration from the
other dementias and OND. This fact may be of
considerable importance for the diagnosis of CJD and suggests that
brain biopsy should be used more often in dementia
cases. In addition, the use of autopsy should be
None of the patients in this study were tested for
hereditary forms of CJD or AD, and the clinical information did
not raise any suspicion about hereditary conditions.
Seven patients in the AD/VaD group were <65 years. None
of them had a family history of dementia. Our results
show that in addition to AD and VaD, other rapidly
progressing neurological diseases may have the same tTau
and P-Tau biomarker pattern as CJD. One patient with
cerebral lymphoma had tTau and tTau/P-Tau above the
Data plots of 14-3-3 protein by diagnostic group CJD,
OND and AD/VaD with lower limit for CJD (dashed
line): 0.75 arbitrary units (data from Akershus
University Hospital only).
cut-off values for CJD. Another patient with the same
diagnosis was slightly positive for 14-3-3 protein. In spite
of using both tTau, P-Tau and 14-3-3 protein for the
diagnosis of CJD, our data suggest that there will still be a few
patients with AD/VaD and OND that cannot be
distinguished from CJD using the biomarkers tTau, P-Tau and
14-3-3 protein. Other investigations will usually establish
the diagnosis in these cases by imaging or CSF-cytology.
It can be argued that this study should have been
performed prospectively, i.e. patients clinically suspected to
have CJD should have been enrolled consecutively. This
would have required a more active cooperation between
the clinical centres. We were not able to undertake this
task at that time, but it should ideally be done as a
followup of the present study. Starting with high tTau patients
was, however, a cost-effective way to find cases. The
reason for not analysing 14-3-3 protein at the same time as
the other markers was that the analysis was not set up by
us until October 2007.
tTau tTau/P-Tau 14-3-3 protein
Another criticism that could be raised against our study is
the low frequency of histological verification. The reason
for this is the right to refuse autopsy and the low autopsy
frequency in Norway in general. The importance of
obtaining histological verification should be stressed in
future prospective CJD studies. Considering the low
number of histological verifications, it is possible that
some of the CJD patients were wrongly classified. We
would argue that this is less likely as eight of the 12
patients (two proven histologically) had MR findings
suggestive of CJD. Seven of these also had three-phase sharp
wave complexes. Of the remaining four patients, all had
non-specific MR changes and two had three-phase sharp
wave complexes. The two remaining patients both had
non-specific changes on EEG and the disease duration in
one of them was four months, highly suggestive of CJD.
The other patient had disease duration of 22 months and
although 14-3-3 protein was positive, another type of
degenerative brain disease cannot be totally excluded.
This study also shows a higher prevalence of CJD in
Norway than might be expected. We identified 12 cases during
a period of two years. With a prevalence of one case per
million inhabitants, approximately 10 cases would be
expected, which is quite close to the observed number of
12. However, we do not expect that all the Norwegian
cases in this period were known to us. The prevalence of
CJD may therefore be higher than anticipated. This
indicates a need for closer surveillance of human prion
diseases in Norway.
Total Tau, tTau/P-Tau ratio and 14-3-3 protein are useful,
but not entirely sensitive and specific markers for the
diagnosis of CJD in CSF. The results indicate that the
diagnostic performance of 14-3-3 protein may be slightly better
than tTau/P-Tau, which may be slightly better than tTau
alone. There is clearly a need for a specific test for CJD,
preferably for the misfolded prion protein (PrPSc) itself in
readily obtainable biological samples such as blood and
CSF. It remains to be seen whether a specific test for PrPSc
would be as sensitive and specific as the markers used in
The authors declare that they have no competing interests.
AS designed the study, drafted the manuscript, and
performed the statistical analysis. VS and TF participated in
its design and coordination and helped to draft the
manuscript. ASG performed ELISA tests, set up the
immunoblot for 14-3-3 protein and contributed to the manuscript.
All authors read and approved the final manuscript.
The authors wish to thank Lisbeth Johnsen, Randi Otterstad and Britt
Skrogstad for technical assistance and medical writer Kari Skinningsrud
(Limwric Ltd., Lillestoem, Norway) for critical review of the manuscript.
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