title: Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.
abstract: 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.
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Robert-Koch Str. 40, 37075, Göttingen, Germany. Matthias.Schmitz@med.uni-goettingen.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schmitz
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Ebert
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Stoeck
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Karch
authors.affiliation: Department of Pathology, The University of Melbourne, Parkville, 3010, Australia.
authors.forename: Steven
authors.initials: S
authors.lastname: Collins
authors.affiliation: Chronic Disease Programme, CIBERNED, and CIEN Foundation-Queen Sofia Foundation, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Calero
authors.affiliation: Pharmaceutical Sciences, Laboratory of Pharmacology, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece.
authors.forename: Theodor
authors.initials: T
authors.lastname: Sklaviadis
authors.affiliation: Service de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, Assistance Publique - Hôpitaux de Paris, 2, rue A Paré, 75475, Paris cedex 10, France.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Laplanche
authors.affiliation: Department of Molecular Pathology and Neuropathology, Medical University of Lodz, 8/10 Czechoslowacka Str., 92-216, Lodz, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Golanska
authors.affiliation: Center for Neuroscience and Cell Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Ines
authors.initials: I
authors.lastname: Baldeiras
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Neurology Department, Hospital Clinic, IDIBAPS, Barcelona, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Sanchez-Valle
authors.affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Ladogana
authors.affiliation: Department of Multidisiplinary Laboratory Medicine and Medical Biochemistry Division of Diagnostics and Technology Akerhus University Hospital 1478 Lørenskog, 1478, Lørenskog, Norway.
authors.forename: Anders
authors.initials: A
authors.lastname: Skinningsrud
authors.affiliation: The Public Health Agency of Sweden, Unit for highly pathogenic viruses, 171 82, Solna, Sweden.
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Hammarin
authors.affiliation: Department of Prion Diseases, Slovak Medical University Bratislava, Limbová 14, 833-03, Bratislava, Slovakia.
authors.forename: Eva
authors.initials: E
authors.lastname: Mitrova
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Franc
authors.initials: F
authors.lastname: Llorens
authors.affiliation: Ilsong Institute of Life Science, College of Medicine, Hallym University, Anyang, Republic of Korea.
authors.forename: Yong Sun
authors.initials: YS
authors.lastname: Kim
authors.affiliation: Western General Hospital, National Creutzfeldt-Jakob Disease Surveillance Unit, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Green
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Zerr
mesh_terms: D048948:14-3-3 Proteins / Q000134:cerebrospinal fluid / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D007562:Creutzfeldt-Jakob Syndrome / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007753:Laboratories; D011309:Preservation, Biological; D020033:Protein Isoforms / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D009418:S100 Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D016875:tau Proteins / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D015415:Biomarkers; D020033:Protein Isoforms; D009418:S100 Proteins; D016875:tau Proteins
keywords: 14-3-3 protein; Biomarker; Cerebrospinal fluid; Creutzfeldt-Jakob disease; Pre-mortem test; Standardization