Journal
MOLECULAR NEUROBIOLOGY
Volume 53, Issue 4, Pages 2189-2199Publisher
SPRINGER
DOI: 10.1007/s12035-015-9167-5
Keywords
Biomarker; Cerebrospinal fluid; Creutzfeldt-Jakob disease; Pre-mortem test; 14-3-3 protein; Standardization
Categories
Funding
- EU Joint Program Neurodegenerative Disease Research JPND-DEMTEST (Biomarker based diagnosis of rapid progressive dementias-optimization of diagnostic protocols) [01ED1201A]
- Robert Koch Institute through funds from the Federal Ministry of Health [1369-341]
- European Commission (Protecting the food chain from prions: shaping European priorities through basic and applied research (PRIORITY) [222887]
- Commonwealth Department of Health
- NHMRC Practitioner Fellowship [APP1005816]
- [FP7-KBBE-2007-2A]
- MRC [MR/J016136/1] Funding Source: UKRI
- Medical Research Council [MR/J016136/1] Funding Source: researchfish
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At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96 % that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/mu L. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3.
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