Linda Josephine Christine van Waalwijk van Doorn

Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer's disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well.

Analytical validation of a biomarker assay is essential before implementation in clinical practice can occur. In this study, we analytically validated the performance of assays detecting soluble amyloid-β precursor protein (sAPP) α and β in CSF in two laboratories according to previously standard operating procedures serving this goal. sAPPα and sAPPβ ELISA assays from two vendors (IBL-international, Meso Scale Diagnostics) were validated. The performance parameters included precision, sensitivity, dilutional linearity, recovery, and parallelism. Inter-laboratory variation, biomarker comparison (sAPPα vs. sAPPβ) and clinical performance was determined in three laboratories using 60 samples of patients with subjective memory complaints, Alzheimer's disease, or frontotemporal dementia. All performance parameters of the assays were similar between labs and within predefined acceptance criteria. The only exceptions were minor out-of-range results for recovery at low concentrations and, despite being within predefined acceptance criteria, non-comparability of the results for evaluation of the dilutional linearity and hook-effect. Based on the inter-laboratory correlation between Lab #1 and Lab #2, the IBL-international assays were more robust (sAPPα: r(2) = 0.92, sAPPβ: r(2) = 0.94) than the Meso Scale Diagnostics (MSD) assay (sAPPα: r(2) = 0.70, sAPPβ: r(2) = 0.80). Specificity of assays was confirmed using assay-specific peptide competitors. Clinical validation showed consistent results across the clinical groups in the different laboratories for all assays. The validated sAPP assays appear to be of sufficient technical quality and perform well. Moreover, the study shows that the newly developed standard operating procedures provide highly useful tools for the validation of new biomarker assays. A recommendation was made for renewed instructions to evaluate the dilutional linearity and hook-effect. We analytically validated the performance of assays detecting soluble amyloid-β precursor protein (sAPP) α and β in CSF according to SOPs in agreement with ISO15189 guidelines. The validated sAPP assays appear to be of sufficient technical quality and perform well. Moreover, this study proofs that the newly developed SOPs, with a minor modification, provide highly useful tools for the validation of new biomarker assays.

BACKGROUND: Before implementation in clinical practice, biomarker assays need to be thoroughly analytically validated. There is currently a strong interest in implementation of the ratio of amyloid-β peptide 1-42 and 1-40 (Aβ42/Aβ40) in clinical routine. Therefore, in this study, we compared the analytical performance of six assays detecting Aβ40 in cerebrospinal fluid (CSF) in six laboratories according to a recently standard operating procedure (SOP) developed for implementation of ELISA assays for clinical routine. METHODS: Aβ40 assays of six vendors were validated in up to three centers per assay according to recently proposed international consensus validation protocols. The performance parameters included sensitivity, precision, dilutional linearity, recovery, and parallelism. Inter-laboratory variation was determined using a set of 20 CSF samples. In addition, test results were used to critically evaluate the SOPs that were used to validate the assays. RESULTS: Most performance parameters of the different Aβ40 assays were similar between labs and within the predefined acceptance criteria. The only exceptions were the out-of-range results of recovery for the majority of experiments and of parallelism by three laboratories. Additionally, experiments to define the dilutional linearity and hook-effect were not executed correctly in part of the centers. The inter-laboratory variation showed acceptable low levels for all assays. Absolute concentrations measured by the assays varied by a factor up to 4.7 for the extremes. CONCLUSION: All validated Aβ40 assays appeared to be of good technical quality and performed generally well according to predefined criteria. A novel version of the validation SOP is developed based on these findings, to further facilitate implementation of novel immunoassays in clinical practice.

BACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid-β 1-42 (Aβ42), total and phosphorylated tau (t-tau, p-tau) are increasingly used to assist in the clinical diagnosis of Alzheimer's disease (AD). However, CSF biomarker levels can be affected by confounding factors. OBJECTIVE: To investigate the association of white matter hyperintensities (WMHs) present in the brain with AD CSF biomarker levels. METHODS: We included CSF biomarker and magnetic resonance imaging (MRI) data of 172 subjects (52 controls, 72 mild cognitive impairment (MCI), and 48 AD patients) from 9 European Memory Clinics. A computer aided detection system for standardized automated segmentation of WMHs was used on MRI scans to determine WMH volumes. Association of WMH volume with AD CSF biomarkers was determined using linear regression analysis. RESULTS: A small, negative association of CSF Aβ42, but not p-tau and t-tau, levels with WMH volume was observed in the AD (r2 = 0.084, p = 0.046), but not the MCI and control groups, which was slightly increased when including the distance of WMHs to the ventricles in the analysis (r2 = 0.105, p = 0.025). Three global patterns of WMH distribution, either with 1) a low, 2) a peak close to the ventricles, or 3) a high, broadly-distributed WMH volume could be observed in brains of subjects in each diagnostic group. CONCLUSION: Despite an association of WMH volume with CSF Aβ42 levels in AD patients, the occurrence of WMHs is not accompanied by excess release of cellular proteins in the CSF, suggesting that WMHs are no major confounder for AD CSF biomarker assessment.

Cerebrospinal fluid (CSF) and MRI biomarkers may support the diagnosis of Alzheimer's disease (AD). At equal amyloid beta (Aβ) or tau production rates, variations in CSF volume may lead to variations in CSF protein concentrations, affecting interpretation of results. We studied if the diagnostic power of AD CSF biomarker concentrations, i.e. Aβ42, total and phosphorylated tau (t-tau, p-tau), can be improved by correction for lateral ventricular volume (VV). Biomarker data and MRI scans from 12 centers were obtained from subjects (total: n=730; AD: n=175, controls: n=157), who underwent both lumbar puncture and MRI scan within 6 months of each other. CSF Aβ42, t-tau, p-tau concentrations were determined using Fujirebio ELISAs. We developed a Matlab-algorithm for standardized automated segmentation analysis of T1 scans in SPM8, determining VV as proxy for CSF volume; VV was also used as ratio with total intracranial volume (TIV). All segmentation outcomes were judged by visual inspection. Height, age, gender, diagnosis according to internationally accepted criteria, scanner type, magnetic field strength, were also recorded. Bivariate correlations were determined between the CSF biomarkers and VV/TIV ratio. Spearman correlation and linear regression analyses were performed to define optimal diagnostic models. The diagnostic power of CSF biomarkers (combinations) was determined by ROC analyses and correction for VV/TIV ratio and covariates. The AD CSF biomarkers negatively correlated weakly to lateral ventricular volume (Aβ42: p<0.0001, r2=0.037; p-tau: p=0.4645, r2=0.0063; t-tau: p=0.3450, r2=0.0059). For differentiation of AD and controls, the area under the curve (AUC) for Aβ42 (0.75), but not for p-tau and t-tau, increased after correction for VV/TIV ratio (0.81). Models constructed using logistic regression showed that the combination of Aβ42 and t-tau improved the diagnostic value (AUC: 0.88) to discriminate AD from controls, compared to the single markers. The diagnostic power was further improved by correcting Aβ42 for VV/TIV ratio (AUC: 0.91). We developed and validated a novel algorithm for automated measurement of VV in T1 images. CSF Aβ42 concentrations decreased with increasing VV; correction for differences in this volume improved the differentiation of AD vs. controls based on CSF Aβ42 alone or in combination with t-tau.