Harry Twaalfhoven

Serial CSF sampling in Alzheimer's disease: specific versus non-specific markers

In this longitudinal study we investigated change over time in cerebrospinal fluid (CSF) levels of amyloid-beta 40 and 42 (Aβ40 and Aβ42), total tau (tau), tau phosphorylated at threonine 181 (ptau-181), isoprostane, neurofilaments heavy (NfH) and light (NfL). Twenty-four nondemented subjects, 62 mild cognitive impairment (MCI) and 68 Alzheimers disease (AD) patients underwent 2 lumbar punctures, with minimum interval of 6, and a mean ± SD of 24 ± 13 months. Linear mixed models were used to assess change over time. Amyloid-beta 42, tau, and tau phosphorylated at threonine 181, differentiated between diagnosis groups (p < 0.05), whereas isoprostane, neurofilaments heavy, and NfL did not. In contrast, effects of follow-up time were only found for nonspecific CSF biomarkers: levels of NfL decreased, and levels of isoprostane, amyloid-beta 40, and tau increased over time (p < 0.05). Isoprostane showed the largest increase. In addition, increase in isoprostane was associated with progression of mild cognitive impairment to AD, and with cognitive decline as reflected by change in Mini Mental State Examination (MMSE). Contrary to AD-specific markers, nonspecific CSF biomarkers, most notably isoprostane, showed change over time. These markers could potentially be used to monitor disease progression in AD.

Additional Value of CSF Amyloid-beta(40) Levels in the Differentiation between FTLD and Control Subjects

To determine the additional value of cerebrospinal fluid (CSF)amyloid-beta1-40 (Abeta40) next to amyloid-beta1-42 (beta42), total tau (Tau), and tau phosphorylated at threonine-181 (pTau) to distinguish patients with frontotemporal lobar degeneration (FTLD), Alzheimers disease (AD), and controls, we measured CSF levels of Abeta40, Abeta42, pTau, and Tau in 55 patients with FTLD, 60 with AD, and 40 control subjects. Logistic regression was used to identify biomarkers that best distinguished the groups. Additionally, a decision tree (cost=test method; Matlab 7.7) was used to predict diagnosis selecting the best set of biomarkers with the optimal cut-off. Logistic regression showed that Abeta42 and pTau CSF levels provided optimal distinction between AD and FTLD. A combination of Abeta42, Tau, and Abeta40 optimally discriminated FTLD from controls and AD from controls. The decision tree used Abeta42 (cut-off 578 pg/ml) to identify AD (positive predictive value (PPV) 97%), followed by Tau(cut-off 336 pg/ml) to identify FTLD (PPV 67%), and in the last step,Abeta40 (cut-off 10 ng/ml) was used to differentiate controls (PPV68%). Applying CSF Abeta40 levels in the model, the PPV of diagnosis increased to 75% as opposed to 70% when only Abeta42 and Tau were used. CSF Abeta40 levels added to the conventional CSF biomarkers increases the potential to discriminate subjects with dementia from controls. Our findings favor the implementation of CSF Abeta40 in differential diagnosis between FTLD, AD, and control subjects.

Quantification of amyloid-beta 40 in cerebrospinal fluid

BACKGROUND Truncated forms and full-length forms of the amyloid-beta 40 (Abeta40) are key molecules in the pathogenesis of dementia, and are detectable in CSF. Reliable methods to detect these biomarkers in CSF are of great importance for understanding the disease mechanisms and for diagnostic purposes. METHODS VU-alpha-Abeta40, a monoclonal antibody (mAb) specifically detecting Abeta40, was generated and characterized by solid and fluid phase ELISA, surface plasmon resonance spectroscopy (SPRS), immunoprecipitation (IP), immunohistochemical and Western blot (WB) analysis. In addition, an ELISA with VU-alpha-Abeta40 as catching and 6E10 as detecting mAbs was set up and validated. This ELISA was used to measure Abeta40 in CSF of controls (N=27), patients with Alzheimers disease (AD; N=20), frontotemporal lobe dementia (FTLD; N=14), noninflammatory (N=15) and inflammatory (N=15) neurological conditions. RESULTS VU-alpha-Abeta40 specifically recognizes Abeta40 with high affinity (K(A)=1.3x10(9) M(-1)) and detects Abeta40 in AD brain specimens. The developed sandwich ELISA has a detection limit of 0.21 ng/mL, a mean recovery of 90%, and an intra- and inter-assay CV of 1.4% and 7.3%. FTLD patients had a lower mean level of Abeta40 (8.8 (1.9) ng/mL) than controls (12.0 (1.7) ng/mL); p<0.01). CONCLUSIONS VU-alpha-Abeta40 was successfully implemented in an ELISA which enables us to measure Abeta40 accurately in human CSF. Clinical validation revealed lower levels of Abeta40 in FTLD patients. This finding opens new possibilities for early and differential diagnosis of dementia.

Immunohistochemical characterization of novel monoclonal antibodies against the N-terminus of amyloid β-peptide

Abstract Amyloid β-peptide (Aβ) is a key molecule in Alzheimer’s disease (AD). Reliable immunohistochemical (IHC) methods to detect Aβ and Aβ-associated factors (AAF) in brain specimens are needed to determine their role in AD pathophysiology. Formic acid (FA) pre-treatment, which is generally used to enable efficient detection of Aβ with IHC, induces structural modifications within the Aβ, as well as in AAF. Consequently, interpretation of double IHC stainings becomes difficult. Therefore, serial stainings of two newly produced monoclonal antibodies (mAbs) VU-17 and IC16 and two other mAbs (6E10 and 3D6) were performed with four different pre-treatments (no pre-treatment, Tris/EDTA, citrate and FA) and additionally six IHC characteristics were scored: diffuse/compact/classic plaques, arteries with cerebral Aβ angiopathy, dyshoric angiopathy, capillaries with dyshoric angiopathy. Subsequently, these stainings were compared with IHC procedures, which are frequently used in a diagnostic setting, employing mAbs 4G8 and 6F/3D with FA pre-treatment. IHC Aβ patterns obtained with VU-17 and, IC16 and 3D6 without the use of FA pre-treatment were comparable to those obtained with 4G8 and 6F/3D upon FA pre-treatment. Omission of FA pre-treatment gives the advantage to allow double IHC stainings, detecting both Aβ and AAF that otherwise would have been structural modificated upon FA pre-treatment.

Validation of soluble amyloid‐β precursor protein assays as diagnostic CSF biomarkers for neurodegenerative diseases

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, Alzheimers 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: r2 = 0.92, sAPPβ: r2 = 0.94) than the Meso Scale Diagnostics (MSD) assay (sAPPα: r2 = 0.70, sAPPβ: r2 = 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.

Facilitating the Validation of Novel Protein Biomarkers for Dementia: An Optimal Workflow for the Development of Sandwich Immunoassays.

Different neurodegenerative disorders, such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD), lead to dementia syndromes. Dementia will pose a huge impact on society and thus it is essential to develop novel tools that are able to detect the earliest, most sensitive, discriminative, and dynamic biomarkers for each of the disorders. To date, the most common assays used in large-scale protein biomarker analysis are enzyme-linked immunosorbent assays (ELISA), such as the sandwich immunoassays, which are sensitive, practical, and easily implemented. However, due to the novelty of many candidate biomarkers identified during proteomics screening, such assays or the antibodies that specifically recognize the desired marker are often not available. The development and optimization of a new ELISA should be carried out with considerable caution since a poor planning can be costly, ineffective, time consuming, and it may lead to a misinterpretation of the findings. Previous guidelines described either the overall biomarker development in more general terms (i.e., the process from biomarker discovery to validation) or the specific steps of performing an ELISA procedure. However, a workflow describing and guiding the main issues in the development of a novel ELISA is missing. Here, we describe a specific and detailed workflow to develop and validate new ELISA for a successful and reliable validation of novel dementia biomarkers. The proposed workflow highlights the main issues in the development of an ELISA and covers several critical aspects, including production, screening, and selection of specific antibodies until optimal fine-tuning of the assay. Although these recommendations are designed to analyze novel biomarkers for dementia in cerebrospinal fluid, they are generally applicable for the development of immunoassays for biomarkers in other human body fluids or tissues. This workflow is designed to maximize the quality of the developed ELISA using a time- and cost-efficient strategy. This will facilitate the validation of the dementia biomarker candidates ultimately allowing accurate diagnostic conclusions.

Comparison of multiplex platforms for cytokine assessments and their potential use for biomarker profiling in multiple sclerosis

BACKGROUND The levels of pro and anti‐inflammatory cytokines can be altered in different autoimmune pathologies, such as multiple sclerosis (MS). It is likely that cytokines in bodily fluids can provide a good reflection of ongoing disease patho‐physiology. In this study we aimed to validate multiplex cytokine platforms and evaluate whether these cytokines are differentially expressed in MS. METHODS Assay validation for simultaneous quantification of IL‐1&bgr;, IL‐6, IL‐8 and TNF‐&agr; in serum and CSF were performed using both the Luminex‐xMAP (Luminex) and Meso Scale Discovery (MSD) platforms. Next, the relation of the pro‐inflammatory cytokine 4‐plex with disease progression, symptoms and subtypes was studied in paired serum and CSF of MS patients (n = 56), and compared with healthy controls (n = 203), with the use of the MSD‐platform. RESULTS The MSD‐platform showed overall better assay characteristics such as, sensitivity, recovery and linearity compared to the Luminex for the 4‐plex cytokines in CSF and serum. IL‐6, IL‐8 and TNF‐&agr; (p < 0.001) levels were significantly increased in MS serum compared to healthy controls. Moreover, serum IL‐1&bgr; levels correlated with expanded disability status scale (EDSS) scores (r = −0.34, p < 0.05). Additionally, IL‐6 and IL‐8 CSF levels were both significantly decreased in MS patients compared to non‐inflammatory neurological disease controls. Noteworthy, higher IL‐8 CSF levels than IL‐8 serum levels were observed for MS patients, indicating intrathecal activation of macrophages in MS. CONCLUSION We have demonstrated that the pro‐inflammatory 4‐plex kit of the MSD‐platform shows better assay characteristics in comparison with Luminex kit for quantification of these cytokines in serum and CSF. Overall, the increased levels of IL‐6, IL‐8 and TNF‐&agr; in serum of MS patients compared to healthy controls, support the use of multiple cytokines for future MS biomarker and disease progression research. HIGHLIGHTSAssessment of multiplex immunoassays for IL‐1&bgr;, IL‐6, IL‐8 and TNF‐&agr; quantification.MSD platform overall showed better assay characteristics for IL‐6, IL‐8 and TNF‐&agr;.IL‐1&bgr;, IL‐6, IL‐8 serum levels were higher in MS patients compared to controls.IL‐6 and TNF‐&agr; CSF levels are lower in RRMS compared to NINDC.

Multicenter Analytical Validation of Aβ40 Immunoassays

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.

CSF AMYLOID-β PEPTIDES IN DEMENTIA WITH LEWY BODIES AND ALZHEIMER’S DISEASE

Sample 297 82 215 Age 75.15 75.57 75.00 0.89 Mean (s.d.) (6.95) (6.78) (7.02) % Male 61.28 64.63 60 0.46 % ApoE 49.8% 9.75 65.12 < 0.001 Clinical Diagnostic Category (HC:MCI: AD) 83 : 145 : 69 49 : 29 : 4 34 : 116 : 65 <0.001 Education (years) Mean (s.d.) 15.63 (2.95) 15.68 (2.87) 15.61 (2.99) 0.94 CSF Ab1-42 (pg/mL) Mean (s.d.) 167.65 (53.10) 244.60 (25.17) 138.30 (22.94) <0.001 CSF t-tau (pg/mL) Mean (s.d.) 98.80 (54.52) 58.21 (15.51) 114.28 (56.11) <0.001 CSF P-tau (pg/mL) Mean (s.d.) 35.52 (22.10) 20.14 (7.94) 41.43 (22.94) <0.001 Abbreviations: pg/mL1⁄4 picograms per milliliter, s.d.1⁄4 StandardDeviation,%APOE1⁄4 proportion of APOE ε4 carriers, HC1⁄4Healthy Controls,MCI1⁄4Mild Cognitive Impairment, AD 1⁄4 Alzheimer’s disease dementia.

Standardization of a method for diagnostic biomarker validation for neurodegenerative diseases: App assays as example

acceptable range in 144 out of 146 runs. Average values for each reagent lot are within 10% of the training set mean values. The plot shows longitudinal performance of amyloid-b results for the 2 pools. Conclusions:Using our protocol we were able to maintain acceptable performance of Ab1-42 throughout 17 months and 4 different lots of reagents. References: LM Shaw, H. Vanderstichele, M. Knapik-Czajka, et al. Acta Neuropathol. 2011 May;121(5):597-609.