Anja Hviid Simonsen

Recommendations to standardize preanalytical confounding factors in Alzheimer's and Parkinson's disease cerebrospinal fluid biomarkers: an update

Early diagnosis of neurodegenerative disorders such as Alzheimers (AD) or Parkinsons disease (PD) is needed to slow down or halt the disease at the earliest stage. Cerebrospinal fluid (CSF) biomarkers can be a good tool for early diagnosis. However, their use in clinical practice is challenging due to the high variability found between centers in the concentrations of both AD CSF biomarkers (Aβ42, total tau and phosphorylated tau) and PD CSF biomarker (α-synuclein). Such a variability has been partially attributed to different preanalytical procedures between laboratories, thus highlighting the need to establish standardized operating procedures. Here, we merge two previous consensus guidelines for preanalytical confounding factors in order to achieve one exhaustive guideline updated with new evidence for Aβ42, total tau and phosphorylated tau, and α-synuclein. The proposed standardized operating procedures are applicable not only to novel CSF biomarkers in AD and PD, but also to biomarkers for other neurodegenerative disorders.

Identification of CSF biomarkers for frontotemporal dementia using SELDI-TOF

This investigation describes the discovery of novel possible cerebrospinal fluid (CSF) biomarkers for frontotemporal dementia (FTD) using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Sixteen clinically diagnosed FTD patients and 12 non-demented controls were included in the study. CSF was collected and analyzed for protein expression by SELDI-TOF MS. The samples were analyzed on four different array surfaces using two different energy-absorbing molecules as matrices. In total each sample was subjected to eight different surface/matrix conditions. About 2000 protein peaks (mass/charge ratios) were detected. Forty-two peaks were differentially expressed in FTD (P < 0.01). After exclusion of peaks with low signal-to-noise ratio and/or poor resolution and peaks representing differentially charged proteins, 10 peaks remained, five of which were increased and five decreased in FTD cases compared to controls. Using partial least square discriminant analysis (PLS-DA), the combination of these biomarkers discriminated FTD from non-demented controls with a sensitivity of 94%, a specificity of 83% and an accuracy of 89%. Five of the peaks were purified further and identified by tandem MS as a fragment of neurosecretory protein VGF, transthyretin, S-cysteinylated transthyretin, truncated cystatin C and a fragment of chromogranin B. With use of these potential biomarkers, FTD can be distinguished from control subjects with high accuracy in this pilot study.

Identification of a novel panel of cerebrospinal fluid biomarkers for Alzheimer's disease

An early and accurate diagnosis of Alzheimers disease (AD) is required to initiate symptomatic treatment with currently approved drugs and will be of even greater importance if disease modifying compounds in development display a clinical effect. Protein profiles of human cerebrospinal fluid samples from AD patients (n=95) and population-based healthy controls (n=72) were analyzed by SELDI-TOF-MS in order to discover and characterize novel candidate biomarker combinations that differentiate AD patients from normal aging in this explorative study. Thirty candidate biomarkers (ROC AUC>0.7) were discovered that could differentiate patients with AD from healthy controls. Protein sequence determination and positive identification of 15 biomarkers revealed potential associations between the identified markers and AD pathogenesis. A multi-marker combination of five peaks could distinguish AD from healthy control individuals with high sensitivity (97%) and specificity (98%). The panel of five markers was tested on a blinded independent data set of 30 AD samples and 28 controls giving 100% sensitivity and 97% specificity. This novel panel of biomarkers could potentially be used to improve the accuracy of diagnosis of AD.

Biomarkers for alzheimer's disease

The development and validation of biomarkers for the latent, prodromal and dementia stages of Alzheimer’s disease (AD) is a pressing issue because of their high prevalence and an emerging set of experimental therapeutics that will soon force decisions regarding risk versus benefit. While genetic risk factors and neuroimaging will certainly have important roles to play, here we have focused on biomarkers assayed in body fluids. There is developing consensus for a central role for cerebrospinal fluid amyloid-β (Aβ)42 and tau species to aid in the diagnosis of AD at different stages; plasma-based assays for Aβ species show some promise, but the picture is much less clear than in the cerebrospinal fluid. Biomarkers of different pathogenic steps thought to contribute to AD will also be important in assessing pharmacologic mechanisms of new therapies. Discovery approaches now underway may develop novel panels of biomarkers for AD. The next 5 years will see standardization of more established approaches, and the combination of different modalities into the most effective means for assessing different stages of AD.

A Novel Panel of Cerebrospinal Fluid Biomarkers for the Differential Diagnosis of Alzheimer’s Disease versus Normal Aging and Frontotemporal Dementia

Background: An early and accurate diagnosis of Alzheimer’s disease (AD) is important in order to initiate symptomatic treatment with currently approved drugs and will be of even greater importance with the advent of disease-modifying compounds. Methods: Protein profiles of human cerebrospinal fluid samples from patients with AD (n = 85), frontotemporal dementia (n = 20), and healthy controls (n = 32) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to verify previously discovered biomarkers. Results: We verified 15 protein biomarkers that were able to differentiate between AD and controls, and 7 of these 15 markers also differentiated AD from FTD. Conclusion: A panel of cerebrospinal fluid protein markers was verified by a proteomics technology which may potentially improve the accuracy of the AD diagnosis.

Use of an antibody-coupled array and mass spectrometry for simultaneous detection of multiple amyloid beta fragments

dementing conditions. In recent years, several studies have found reduced levels of beta-amyloid protein and increased levels of total tau (T-tau) and phosphorylated tau (phosphotau) protein in the cerebrospinal fluid (CSF) of AD patients, suggesting a promising role of this method in the early diagnosis of disease. Similarly, the expression of tau protein was found to occur also in the oral mucosa epithelium of AD patients. Moreover, an increase of inflammatory mediators such as cytokines and chemokines has been identified in the CSF of these patients. This study investigated the levels of T-tau, phosphotau and beta amyloid proteins and of inflammatory mediators in the CSF and also in saliva of dementia patients, aiming to identify additional biomarkers for early AD diagnosis. Methods: We collected saliva and CSF from 75 individuals, namely 24 patients with probable AD, 18 patients with non-AD dementias and 33 cognitively healthy controls. Dosage of proteins in saliva and CSF was performed by ELISA. Phosphotau, T-tau, beta-amyloid protein, IL-08, MCP, IP10 are the markers that were evaluated in CSF and in saliva were measured T-tau, phosphotau and beta amyloid protein. Comparisons between concentrations of the different markers among the three diagnostic groups were undertaken by ANOVA and Kruskal-Wallis statistical tests. Results: The levels of phosphotau and T-tau proteins in the CSF were significantly increased in AD patients in comparison with healthy controls and non-AD patients. No significant difference in beta amyloid levels emerged between the three groups. Concerning the inflammatory mediators, a significant increase in MCP levels was observed in the non-AD group in comparison with AD and non-demented subjects. IL-08 as IP10 levels were not different between the three groups. No differences between the three groups were indentified with respect to the presence of all markers in saliva. Conclusions: The present study confirms the role of CSF T-tau and phosphotau levels as potential biomarkers in AD diagnosis, although not supporting the potential role of saliva as an alternative biological fluid for determination of these proteins.

Three new potential ovarian cancer biomarkers detected in human urine with equalizer bead technology.

Objective. To examine whether urine can be used to measure specific ovarian cancer proteomic profiles and whether one peak alone or in combination with other peaks or CA125 has the sensitivity and specificity to discriminate between ovarian cancer pelvic mass and benign pelvic mass. Methods. A total of 209 women were admitted for surgery for pelvic mass at the Gynaecological Department at Rigshospitalet, Copenhagen. Of the women, 156 had benign gynaecological tumors, 13 had borderline tumors and 40 had malignant epithelial ovarian cancer. The prospectively and preoperatively collected urine samples were aliquotted and frozen at −80° until the time of analysis. The urine was fractionated using equalizer bead technology and then analyzed with surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry. Biomarkers were purified and identified using combinations of chromatographic techniques and tandem mass spectrometry. Results. Benign and malignant ovarian cancer cases were compared; 21 significantly different peaks (p<0.001) were visualized using Mann–Whitney analysis, ranging in m/z values from 1,500 to 185,000. The three most significant peaks were purified and identified as fibrinogen alpha fragment (m/z = 2570.21), collagen alpha 1 (III) fragment (m/z = 2707.32) and fibrinogen beta NT fragment (m/z = 4425.09). The area under the receiver operator characteristic curve (ROC AUC) value for these three peaks in combination was 0.88, and their ROC AUC value in combination with CA125 was 0.96. Conclusion. This result supports the feasibility of using urine as a clinical diagnostic medium, and the ROC AUC value for the three most significant peaks in combination with or without CA125 demonstrates the enhanced prediction performance of combined marker analysis.

A Disease State Fingerprint for Evaluation of Alzheimer's Disease

Diagnostic processes of Alzheimers disease (AD) are evolving. Knowledge about disease-specific biomarkers is constantly increasing and larger volumes of data are being measured from patients. To gain additional benefits from the collected data, a novel statistical modeling and data visualization system is proposed for supporting clinical diagnosis of AD. The proposed system computes an evidence-based estimate of a patients AD state by comparing his or her heterogeneous neuropsychological, clinical, and biomarker data to previously diagnosed cases. The AD state in this context denotes a patients degree of similarity to previously diagnosed disease population. A summary of patient data and results of the computation are displayed in a succinct Disease State Fingerprint (DSF) visualization. The visualization clearly discloses how patient data contributes to the AD state, facilitating rapid interpretation of the information. To model the AD state from complex and heterogeneous patient data, a statistical Disease State Index (DSI) method underlying the DSF has been developed. Using baseline data from the Alzheimers Disease Neuroimaging Initiative (ADNI), the ability of the DSI to model disease progression from elderly healthy controls to AD and its ability to predict conversion from mild cognitive impairment (MCI) to AD were assessed. It was found that the DSI provides well-behaving AD state estimates, corresponding well with the actual diagnoses. For predicting conversion from MCI to AD, the DSI attains performance similar to state-of-the-art reference classifiers. The results suggest that the DSF establishes an effective decision support and data visualization framework for improving AD diagnostics, allowing clinicians to rapidly analyze large quantities of diverse patient data.

The utility of α-synuclein as biofluid marker in neurodegenerative diseases: a systematic review of the literature

The discovery of α-synuclein (α-syn) as a major component of Lewy bodies, neuropathological hallmark of Parkinsons disease (PD), dementia with Lewy bodies and of glial inclusions in multiple system atrophy initiated the investigation of α-syn as a biomarker in cerebrospinal fluid (CSF). Due to the involvement of the periphery in PD the quantification of α-syn in peripheral fluids such as serum, plasma and saliva has been investigated as well. We review how the development of multiple assays for the quantification of α-syn has yielded novel insights into the variety of α-syn species present in the different fluids; the optimal preanalytical conditions required for robust quantification and the potential clinical value of α-syn as biomarker. We also suggest future approaches to use of CSF α-syn in neurodegenerative diseases.

Cystatin C in cerebrospinal fluid and multiple sclerosis.

A recent study using surface‐enhanced laser desorption/ionization time‐of‐flight analysis of cerebrospinal fluid identified a 12.5kDa truncated isoform of cystatin C (CysC) as a specific biomarker for multiple sclerosis (MS).