Neurofilament ELISA validation

Department of Neuroinflammation, UCL Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom.
Journal of immunological methods (Impact Factor: 1.82). 10/2009; 352(1-2):23-31. DOI: 10.1016/j.jim.2009.09.014
Source: PubMed


Neurofilament proteins (Nf) are highly specific biomarkers for neuronal death and axonal degeneration. As these markers become more widely used, an inter-laboratory validation study is required to identify assay criteria for high quality performance.
The UmanDiagnostics NF-light (R)enzyme-linked immunoabsorbent assays (ELISA) for the neurofilament light chain (NfL, 68kDa) was used to test the intra-assay and inter-laboratory coefficient of variation (CV) between 35 laboratories worldwide on 15 cerebrospinal fluid (CSF) samples. Critical factors, such as sample transport and storage, analytical delays, reaction temperature and time, the laboratories' accuracy and preparation of standards were documented and used for the statistical analyses.
The intra-laboratory CV averaged 3.3% and the inter-laboratory CV 59%. The results from the test laboratories correlated with those from the reference laboratory (R=0.60, p<0.0001). Correcting for critical factors improved the strength of the correlation. Differences in the accuracy of standard preparation were identified as the most critical factor. Correcting for the error introduced by variation in the protein standards improved the correlation to R=0.98, p<0.0001 with an averaged inter-laboratory CV of 14%. The corrected overall inter-rater agreement was subtantial (0.6) according to Fleiss' multi-rater kappa and Gwet's AC1 statistics.
This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online.

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    • "Most recently published articles that have assessed inter-rater reliability have used Cohen’s Kappa exclusively [19-26], and a recent review of the current methods used for inter-rater reliability does not even mention AC1 [27]. During our research of PubMed (up to February 2013), we found only 2 published articles that mention using Gwet’s AC1 method as part of a study [28,29]. "
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    ABSTRACT: Background Rater agreement is important in clinical research, and Cohen’s Kappa is a widely used method for assessing inter-rater reliability; however, there are well documented statistical problems associated with the measure. In order to assess its utility, we evaluated it against Gwet’s AC1 and compared the results. Methods This study was carried out across 67 patients (56% males) aged 18 to 67, with a mean SD of 44.13 ± 12.68 years. Nine raters (7 psychiatrists, a psychiatry resident and a social worker) participated as interviewers, either for the first or the second interviews, which were held 4 to 6 weeks apart. The interviews were held in order to establish a personality disorder (PD) diagnosis using DSM-IV criteria. Cohen’s Kappa and Gwet’s AC1 were used and the level of agreement between raters was assessed in terms of a simple categorical diagnosis (i.e., the presence or absence of a disorder). Data were also compared with a previous analysis in order to evaluate the effects of trait prevalence. Results Gwet’s AC1 was shown to have higher inter-rater reliability coefficients for all the PD criteria, ranging from .752 to 1.000, whereas Cohen’s Kappa ranged from 0 to 1.00. Cohen’s Kappa values were high and close to the percentage of agreement when the prevalence was high, whereas Gwet’s AC1 values appeared not to change much with a change in prevalence, but remained close to the percentage of agreement. For example a Schizoid sample revealed a mean Cohen’s Kappa of .726 and a Gwet’s AC1of .853 , which fell within the different level of agreement according to criteria developed by Landis and Koch, and Altman and Fleiss. Conclusions Based on the different formulae used to calculate the level of chance-corrected agreement, Gwet’s AC1 was shown to provide a more stable inter-rater reliability coefficient than Cohen’s Kappa. It was also found to be less affected by prevalence and marginal probability than that of Cohen’s Kappa, and therefore should be considered for use with inter-rater reliability analysis.
    BMC Medical Research Methodology 04/2013; 13(1). DOI:10.1186/1471-2288-13-61 · 2.27 Impact Factor
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    • "The comparable results were shown in several studies that used the same NF-H Enzyme-Linked Immunosorbent Assay (ELISA) method [62, 70–72]. However, a poor interlaboratory coefficient of variation in a recent multicenter NF-L ELISA validation study has been shown, mainly due to the lack of preparation of accurate and consistent protein standards [200]. Therefore, a standardization of body fluid sampling and storage [201], as well as the use of the standardized and validated assay procedures [23, 200], are needed. "
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    ABSTRACT: Following emerging evidence that neurodegenerative processes in multiple sclerosis (MS) are present from its early stages, an intensive scientific interest has been directed to biomarkers of neuro-axonal damage in body fluids of MS patients. Recent research has introduced new candidate biomarkers but also elucidated pathogenetic and clinical relevance of the well-known ones. This paper reviews the existing data on blood and cerebrospinal fluid biomarkers of neuroaxonal damage in MS and highlights their relation to clinical parameters, as well as their potential predictive value to estimate future disease course, disability, and treatment response. Strategies for future research in this field are suggested.
    05/2011; 2011(6):767083. DOI:10.1155/2011/767083
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