Detection of subarachnoid haemorrhage with spectrophotometry of cerebrospinal fluid - a comparison of two methods.

OBJECTIVES: Spectrophotometric absorption curve analysis of cerebrospinal fluid (CSF) for oxyhaemoglobin and bilirubin is necessary to accurately diagnose subarachnoid haemorrhage (SAH) in patients with typical symptoms but with negative findings on X-ray examinations. In this study, we evaluated the performance of two methods for interpreting absorption curves; one method from the United Kingdom National External Quality Assessment Service (UK-NEQAS) and the other from the national quality assurance programme in Sweden (Equalis). METHODS: Consecutive absorbance curves (n=336) were interpreted with two different methods, and their performance was compared to the diagnosis as stated in the patient records. RESULTS: The UK-NEQAS method displayed equal sensitivity to the Equalis method, but the specificity of the UK-NEQAS method was significantly higher than the Equalis method resulting in fewer false positive results. For UK-NEQAS, a positive predictive value (PPV) of 84.6% and a negative predictive value (NPV) of 99.7% were observed, whereas the Equalis method had a PPV of 27.5% and an NPV of 99.7%. CONCLUSIONS: The semi-automated method based on the guidelines from UK-NEQAS provides an efficient and correct interpretation of absorbance curves with short turn-around times. We propose using this method for the routine interpretation of CSF spectrophotometric curves.

Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid.

OBJECTIVES: The core cerebrospinal fluid (CSF) biomarkers; total tau (tTau), phospho-tau (pTau), amyloid ß 1-42 (Aß 1-42), and the Aß 1-42/Aß 1-40 ratio have transformed Alzheimer's disease (AD) research and are today increasingly used in clinical routine laboratories as diagnostic tools. Fully automated immunoassay instruments with ready-to-use assay kits and calibrators has simplified their analysis and improved reproducibility of measurements. We evaluated the analytical performance of the fully automated immunoassay instrument LUMIPULSE G (Fujirebio) for measurement of the four core AD CSF biomarkers and determined cutpoints for AD diagnosis. METHODS: Comparison of the LUMIPULSE G assays was performed with the established INNOTEST ELISAs (Fujirebio) for hTau Ag, pTau 181, ß-amyloid 1-42, and with V-PLEX Plus Aß Peptide Panel 1 (6E10) (Meso Scale Discovery) for Aß 1-42/Aß 1-40, as well as with a LC-MS reference method for Aß 1-42. Intra- and inter-laboratory reproducibility was evaluated for all assays. Clinical cutpoints for Aß 1-42, tTau, and pTau was determined by analysis of three cohorts of clinically diagnosed patients, comprising 651 CSF samples. For the Aß 1-42/Aß 1-40 ratio, the cutpoint was determined by mixture model analysis of 2,782 CSF samples. RESULTS: The LUMIPULSE G assays showed strong correlation to all other immunoassays (r>0.93 for all assays). The repeatability (intra-laboratory) CVs ranged between 2.0 and 5.6%, with the highest variation observed for ß-amyloid 1-40. The reproducibility (inter-laboratory) CVs ranged between 2.1 and 6.5%, with the highest variation observed for ß-amyloid 1-42. The clinical cutpoints for AD were determined to be 409 ng/L for total tau, 50.2 ng/L for pTau 181, 526 ng/L for ß-amyloid 1-42, and 0.072 for the Aß 1-42/Aß 1-40 ratio. CONCLUSIONS: Our results suggest that the LUMIPULSE G assays for the CSF AD biomarkers are fit for purpose in clinical laboratory practice. Further, they corroborate earlier presented reference limits for the biomarkers.

Dynamics of extracellular matrix proteins in cerebrospinal fluid and serum and their relation to clinical outcome in human traumatic brain injury.

Background Brevican, neurocan, tenascin-C and tenascin-R are extracellular matrix proteins present in brain that show increased expression in experimental animal models of brain injury. However, little is known about the dynamics of these proteins in human body fluids, such as cerebrospinal fluid (CSF) and serum, after traumatic brain injury (TBI). The aims of this study were to investigate if matrix proteins in CSF and serum are associated with functional outcome following traumatic brain injury, if their concentrations change over time and to compare their levels between brain injured patients to controls. Methods In total, 42 traumatic brain injury patients, nine healthy controls and a contrast group consisting of 38 idiopathic normal pressure hydrocephalus patients were included. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the concentrations of proteins. Results Increased concentrations of brevican, tenascin-C and tenascin-R in CSF correlated with unfavourable outcome, with stronger outcome prediction ability compared to other biomarkers of brain tissue injury. CSF brevican, tenascin-R and serum neurocan gradually decreased with time (p = 0.04, p = 0.008, p = 0.005, respectively), while serum tenascin-C (p = 0.01) increased. CSF concentrations of brevican, neurocan and tenascin-R (only in time point 3) after TBI were lower than in the idiopathic normal pressure hydrocephalus group (p < 0.0001, p < 0.0001, and p = 0.0008, respectively). In serum, tenascin-C concentration was higher and neurocan lower compared to healthy controls (p = 0.02 and p = 0.0009). Conclusions These findings indicate that levels of extracellular matrix proteins are associated with clinical outcome following TBI and may act as markers for different pathophysiology than currently used protein biomarkers.

Low-dose γ-secretase inhibition increases secretion of Aß peptides and intracellular oligomeric Aß.

γ-Secretase inhibitors have been considered promising drug candidates against Alzheimer's disease (AD) due to their ability to reduce amyloid-ß (Aß) production. However, clinical trials have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of γ-secretase inhibitors may instead increase Aß production. Using a stem cell-derived human model of cortical neurons and low doses of the γ-secretase inhibitor DAPT, the effects on a variety of Aß peptides were studied using mass spectrometry. One major focus was to develop a novel method for specific detection of oligomeric Aß (oAß), and this was used to study the effects of low-dose γ-secretase inhibitor treatment on intracellular oAß accumulation. Low-dose treatment (2 and 20nM) with DAPT increased the secretion of several Aß peptides, especially Aßx-42. Furthermore, using the novel method for oAß detection, we found that 2nM DAPT treatment of cortical neurons resulted in increased oAß accumulation. Thus, low dose-treatment with DAPT causes both increased production of long, aggregation-prone Aß peptides and accumulation of intracellular Aß oligomers, both believed to contribute to AD pathology.