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.

Cerebrospinal Fluid Biomarker-Based Diagnosis of Sporadic Creutzfeldt-Jakob Disease: A Validation Study for Previously Established Cutoffs

“…The major limitation of 14-3-3 Western blot analysis is the semiquantitative nature of the detection method, leading to a moderate amount of undefined cases for non-CJD groups, decreasing the specificity of the test. In this regard, 14-3-3 ELISA tests are also available, but these tests are sensitive to false positives in inflammatory diseases or when the CSF is blood contaminated [7]. Interestingly, specificities for tau and the p-tau/tau ratio were not considerably higher, indicating that all 3 biomarkers performed comparatively well in this study population.…”

“…Among them, 14-3-3 and tau proteins, 2 surrogate disease markers considered to reflect the degree of neuronal damage, present increased levels in the CSF of sCJD and are currently used in the diagnostic routine [2,3]. 14-3-3 is part of the diagnostic criteria for sCJD; however, there is a debate about if the diagnostic accuracy of 14-3-3 is lower [4,5], similar to that of the tau protein [6,7], or even higher [8]. Although elevated tau levels can also be detected in other neurological and neurodegenerative conditions [9,10], an optimum cutoff for the diagnosis of sCJD has been defined in the range of 1,200-1,300 pg/mL [6,11,12,13].…”

CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt–Jakob disease suspected cases with inconclusive 14-3-3 result

“…Until now, such assays have not been used in clinical routine setting for various reasons: sub-optimal results, optimal conditions for the test unknown, comparative value not determined. Recently, a commercial ELISA kit for the gamma isoform of 14-3-3 protein has been made available [40] and has been validated in a large population of sCJD and non-CJD patients against the conventional WB method [41]. In our recently published work, we demonstrated, in a smaller cohort of definite sCJD and non-CJD, that this quantitative 14-3-3 ELISA is extremely promising, since it alone correctly classified 93 % of cases [42].…”

Developments in auxiliary examination of Creutzfeldt-Jakob disease

“…In addition, the expression of 14-3-3 protein is also found in many conditions of neuronal injury such as infectious diseases of the CNS, metabolic encephalopathy and in the acute stage of cerebral infarction, reducing the specificity of 14-3-3 protein testing. [20] One study showed that only 17 of 32 confirmed sCJD patients (biopsy-or autopsy-confirmed) had positive results of 14-3-3 protein with the sensitivity of only 53%. [21] And in another analysis of 420 CJD patients confirmed by biopsy, the specificity was only 28%.…”