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

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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.

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Acknowledgments

The study was performed within the recently established Clinical Dementia Center at the University Medical Center Göttingen and was supported by grants from the EU Joint Program–Neurodegenerative Disease Research [JPND-DEMTEST (Biomarker based diagnosis of rapid progressive dementias-optimization of diagnostic protocols, 01ED1201A)]. This study was also partly supported by the Robert Koch Institute through funds from the Federal Ministry of Health (grant no. 1369–341) and by a grant from the European Commission (Protecting the food chain from prions: shaping European priorities through basic and applied research (PRIORITY, No. 222887) Project number: FP7-KBBE-2007-2A). Thanks to Michele Equestre for technical assistance. The Australian National CJD Registry is funded by the Commonwealth Department of Health and S Collins is supported by a NHMRC Practitioner Fellowship (#APP1005816).

Conflict of Interests

On behalf of all authors, the corresponding author states that there are no conflicts of interests.

Author information

Authors and Affiliations

  1. 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, Elisabeth Ebert, Katharina Stoeck, André Karch, Franc Llorens & Inga Zerr

  2. Department of Pathology, The University of Melbourne, Parkville, 3010, Australia

    Steven Collins

  3. Chronic Disease Programme, CIBERNED, and CIEN Foundation-Queen Sofia Foundation, Instituto de Salud Carlos III, Madrid, Spain

    Miguel Calero

  4. Pharmaceutical Sciences, Laboratory of Pharmacology, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece

    Theodor Sklaviadis

  5. Service de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, Assistance Publique - Hôpitaux de Paris, 2, rue A Paré, 75475, Paris cedex 10, France

    Jean-Louis Laplanche

  6. Department of Molecular Pathology and Neuropathology, Medical University of Lodz, 8/10 Czechoslowacka Str., 92-216, Lodz, Poland

    Ewa Golanska

  7. Center for Neuroscience and Cell Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

    Ines Baldeiras

  8. Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki, Japan

    Katsuya Satoh

  9. Neurology Department, Hospital Clinic, IDIBAPS, Barcelona, Spain

    Raquel Sanchez-Valle

  10. Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy

    Anna Ladogana

  11. Department of Multidisiplinary Laboratory Medicine and Medical Biochemistry Division of Diagnostics and Technology Akerhus University Hospital 1478 Lørenskog, 1478, Lørenskog, Norway

    Anders Skinningsrud

  12. The Public Health Agency of Sweden, Unit for highly pathogenic viruses, 171 82, Solna, Sweden

    Anna-Lena Hammarin

  13. Department of Prion Diseases, Slovak Medical University Bratislava, Limbová 14, 833-03, Bratislava, Slovakia

    Eva Mitrova

  14. Ilsong Institute of Life Science, College of Medicine, Hallym University, Anyang, Republic of Korea

    Yong Sun Kim

  15. Western General Hospital, National Creutzfeldt-Jakob Disease Surveillance Unit, Crewe Road, Edinburgh, EH4 2XU, UK

    Alison Green

Authors
  1. Matthias Schmitz
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  2. Elisabeth Ebert
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  3. Katharina Stoeck
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  4. André Karch
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  5. Steven Collins
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  6. Miguel Calero
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  7. Theodor Sklaviadis
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  8. Jean-Louis Laplanche
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  9. Ewa Golanska
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  10. Ines Baldeiras
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  11. Katsuya Satoh
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  12. Raquel Sanchez-Valle
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  13. Anna Ladogana
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  14. Anders Skinningsrud
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  15. Anna-Lena Hammarin
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  16. Eva Mitrova
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  17. Franc Llorens
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  18. Yong Sun Kim
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  19. Alison Green
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  20. Inga Zerr
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Corresponding author

Correspondence to Matthias Schmitz.

Electronic supplementary material

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Supplement 1

Determination of 14-3-3 protein level in CSF from neurodegenerative and non-neurodegenerative diseases. No significant differences in 14-3-3 protein level could be observed. (GIF 13 kb)

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Supplement 2

Comparison of diagnostic accuracy of 14-3-3 protein to tau and S100B protein. The diagnostic accuracy of 14-3-3 protein was analysed in comparison to tau and S100B which showed the lowest accuracy. The combination of different biomarker proteins increased the diagnostic accuracy further. (GIF 121 kb)

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Schmitz, M., Ebert, E., Stoeck, K. et al. Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic. Mol Neurobiol 53, 2189–2199 (2016). https://doi.org/10.1007/s12035-015-9167-5

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