Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic

• DOI: 10.1007/s12035-015-9167-5
• Publication Date: 2015-05-06T04:31:29Z
• Authors: Matthias Schmitz; Elisabeth Ebert; Katharina Stoeck; André Karch; Steven Collins; Miguel Calero; Theodor Sklaviadis; Jean-Louis Laplanche; Ewa Golanska; Ines Baldeiras; Katsuya Satoh; Raquel Sanchez-Valle; Anna Ladogana; Anders Skinningsrud; Anna-Lena Hammarin; Eva Mitrova; Franc Llorens; Yong Sun Kim; Alison Green; Inga Zerr
• Keywords: Blotting, Western; Preservation, Biological; diagnosis [Creutzfeldt-Jakob Syndrome]; Enzyme-Linked Immunosorbent Assay; tau Proteins; Sensitivity and Specificity; Creutzfeldt-Jakob Syndrome; 03 medical and health sciences; 0302 clinical medicine; ddc:570; metabolism [Creutzfeldt-Jakob Syndrome]; Humans; Protein Isoforms; metabolism [Protein Isoforms]; metabolism [S100 Proteins]; metabolism [14-3-3 Proteins]; S100 Proteins; Reproducibility of Results; cerebrospinal fluid [Creutzfeldt-Jakob Syndrome]; Reference Standards; metabolism [tau Proteins]; 3. Good health; cerebrospinal fluid [14-3-3 Proteins]; 14-3-3 Proteins; Laboratories; Biomarkers; metabolism [Biomarkers]

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/μL. 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.