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Multicenter Study
. 2018 Mar;55(3):2249-2257.
doi: 10.1007/s12035-017-0479-5. Epub 2017 Mar 21.

Validation of α-Synuclein as a CSF Biomarker for Sporadic Creutzfeldt-Jakob Disease

Franc Llorens  1   2 , Niels Kruse  3 , André Karch  4 , Matthias Schmitz  5   6 , Saima Zafar  5   6 , Nadine Gotzmann  5   6 , Ting Sun  5   6 , Silja Köchy  5   6 , Tobias Knipper  5 , Maria Cramm  5   6 , Ewa Golanska  7 , Beata Sikorska  7 , Pawel P Liberski  7 , Raquel Sánchez-Valle  8 , Andre Fischer  6 , Brit Mollenhauer  3   9   10 , Inga Zerr  5   6
Affiliations
Multicenter Study

Validation of α-Synuclein as a CSF Biomarker for Sporadic Creutzfeldt-Jakob Disease

Franc Llorens et al. Mol Neurobiol. 2018 Mar.

Abstract

The analysis of cerebrospinal fluid (CSF) biomarkers gains importance in the differential diagnosis of prion diseases. However, no single diagnostic tool or combination of them can unequivocally confirm prion disease diagnosis. Electrochemiluminescence (ECL)-based immunoassays have demonstrated to achieve high diagnostic accuracy in a variety of sample types due to their high sensitivity and dynamic range. Quantification of CSF α-synuclein (a-syn) by an in-house ECL-based ELISA assay has been recently reported as an excellent approach for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), the most prevalent form of human prion disease. In the present study, we validated a commercially available ECL-based a-syn ELISA platform as a diagnostic test for correct classification of sCJD cases. CSF a-syn was analysed in 203 sCJD cases with definite diagnosis and in 445 non-CJD cases. We investigated reproducibility and stability of CSF a-syn and made recommendations for its analysis in the sCJD diagnostic workup. A sensitivity of 98% and a specificity of 97% were achieved when using an optimal cut-off of 820 pg/mL a-syn. Moreover, we were able to show a negative correlation between a-syn levels and disease duration suggesting that CSF a-syn may be a good prognostic marker for sCJD patients. The present study validates the use of a-syn as a CSF biomarker of sCJD and establishes the clinical and pre-analytical parameters for its use in differential diagnosis in clinical routine. Additionally, the current test presents some advantages compared to other diagnostic approaches: it is fast, economic, requires minimal amount of CSF and a-syn levels are stable along disease progression.

Keywords: Biomarkers; Cerebrospinal fluid; ELISA; Electrochemiluminescence; Sporadic Creutzfeldt-Jakob; α-Synuclein.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Quantification of CSF a-syn by the human ELISA kit from MSD. a Representative plot for quantification of CSF a-syn in samples from CJD and non-CJD cases. The dynamic range is indicated in blue. Calibrators are denoted as blue spots and unknown samples as red spots. CSF samples from diverse aetiology were in detection range using a 1:8 dilution. b Analytical and technical parameters derived from quantification of CSF a-syn with the human ELISA kit from MSD are shown
Fig. 2
Fig. 2
Diagnostic accuracy of CSF a-syn levels as sCJD biomarker. a CSF a-syn levels in non-sCJD and sCJD. Statistically significant differences were detected between non-CJD and sCJD cases (p < 0.001). b ROC curve for a-syn in the comparative analysis between non-sCJD cases and sCJD cases. Sensitivity and specificity, receiver operating characteristic (ROC) curves and derived area under the curve (AUC) were calculated. Based on Youden Index, an optimal cut-off of 820 pg/mL a-syn and a sensitivity of 98% and specificity of 97% were achieved for the discrimination of sCJD from non-CJD cases
Fig. 3
Fig. 3
Influence of timing and disease duration in CSF a-syn levels. a CSF a-syn levels in serial LPs in sCJD cases at different stages of the disease. Samples were grouped in three categories according to whether they underwent LP in the first (time from disease onset to LP/total duration of the disease <0.33), second (0.33–0.66) or third (>0.66) stage of the disease. Dashed line indicates established cut-off for sCJD. b Association between CSF a-syn levels and disease duration (months) in sCJD patients analysed by a fractional polynomial linear regression approach (p < 0.001). Displayed is the functional form of the association with 95% confidence intervals. The expected duration of disease can be estimated by using the following equation: duration in months = 5.70 + 1.43*((a-syn (pg/mL)/10,000)-0.5–0.99)–0.57*((a-syn (pg/mL)/10,000)–1.02)
Fig. 4
Fig. 4
Effect of short- and long-term storage on CSF a-syn concentration in sCJD patients. CSF a-syn levels were determined as indicated in “Material and Methods”. CSF a-syn concentration is shown relative to the reference sample (time point 0), which was set as 100%. The concentration of CSF a-syn from 8 different sCJD patients (n = 8) was determined a before and after storage at room temperature (0 to 6 days), b storage at 4 °C (0 to 6 days) and c after repeated freezing and thawing cycles (0 to 8 cycles). A significant loss of CSF a-syn level could be observed in samples subjected to 8 freezing-thawing cycles. Error bars represent standard deviations (SD)
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