Added value of cerebrospinal fluid multimarker analysis in diagnosis and progression of dementia.

BACKGROUND AND PURPOSE: Recently, some emerging cerebrospinal fluid (CSF) markers have been proposed as diagnostic tools for Alzheimer disease (AD) that can have an effect on disease progression. We analyze the accuracy of these CSF markers for diagnosis of AD in reference to brain amyloid positron emission tomography (PET). We also investigated whether they help in differentiating AD from other dementias and examined their influence in tracing the progression to dementia. METHODS: Amyloid-ß (Aß) 1-42, total tau (t-tau), phosphorylated tau, Aß40 , Aß38 , beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), neurogranin (ng), phosphorylated neurofilament heavy-chain, and α-synuclein (α-syn) CSF levels were analyzed in 319 subjects, among whom 57 also underwent an amyloid PET scan. We also analyzed longitudinal clinical data from 239 subjects. RESULTS: Emerging CSF markers, especially ng/BACE-1 ratio (area under the curve = 0.77) and their combinations with core AD CSF markers (all AUCs >0.85), showed high accuracy to discriminate amyloid PET positivity. Subjects with AD had higher CSF BACE-1, ng, and α-syn levels than those with non-AD dementia. CSF t-tau/α-syn ratio was higher in subjects with dementia with Lewy bodies than in those with frontotemporal dementia. Most emerging/core AD ratios predicted a faster conversion from mild cognitive impairment (MCI) stage to AD and appeared to be helpful when core AD CSF markers were discordant. In addition, the rate of cognitive decline was associated with all CSF core AD markers, several emerging/core AD two-marker ratios, and CSF ng levels. CONCLUSIONS: These results suggest that emerging biomarkers in conjunction with core AD markers improve diagnosis of AD, are associated with the conversion from MCI into AD, and predict a faster progression of dementia.

The Alzheimer's Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid ß and tau.

The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aß42 and Aß40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high diagnostic performance for AD, including the early phases of the disease. However, between-center differences in pre-analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry. The aim of the group was to develop a simplified and standardized pre-analytical protocol for CSF collection and handling before analysis for routine clinical use, and ultimately to ensure high diagnostic performance and minimize patient misclassification rates. Widespread application of the protocol would help minimize variability in measurements, which would facilitate the implementation of unified cut-off levels across laboratories, and foster the use of CSF biomarkers in AD diagnostics for the benefit of the patients.

Cerebrospinal fluid levels of neurogranin in Parkinsonian disorders.

BACKGROUND: CSF concentration of neurogranin has been suggested as a biomarker for synapse dysfunction. OBJECTIVES: To investigate CSF neurogranin in parkinsonian disorders compared to controls and Alzheimer's disease and the possible correlations between neurogranin and cognitive and motor impairment. METHODS: We included 157 patients with PD, 29 with PD with dementia, 11 with dementia with Lewy bodies, 26 with MSA, 21 with PSP, 6 with corticobasal syndrome, 47 controls, and 124 with Alzheimer's disease. CSF neurogranin was measured using two enzyme-linked immunosorbent assays; from EUROIMMUN and the University of Gothenburg. RESULTS: We found a strong correlation between CSF neurogranin-EI and CSF neurogranin-University of Gothenburg (Rs = 0.890; P < 0.001). Neurogranin was decreased in PD, PD with dementia, MSA, and PSP compared to controls and Alzheimer's disease. Neurogranin did not correlate with motor or cognitive impairment, longitudinal decline, or progression to dementia in PD. CONCLUSIONS: CSF neurogranin is decreased in parkinsonian disorders compared to controls, emphasizing the importance of synaptic dysfunction in these disorders. © 2019 International Parkinson and Movement Disorder Society.

First amyloid ß1-42 certified reference material for re-calibrating commercial immunoassays.

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .

Accurate risk estimation of ß-amyloid positivity to identify prodromal Alzheimer's disease: Cross-validation study of practical algorithms.

INTRODUCTION: The aim was to create readily available algorithms that estimate the individual risk of ß-amyloid (Aß) positivity. METHODS: The algorithms were tested in BioFINDER (n = 391, subjective cognitive decline or mild cognitive impairment) and validated in Alzheimer's Disease Neuroimaging Initiative (n = 661, subjective cognitive decline or mild cognitive impairment). The examined predictors of Aß status were demographics; cognitive tests; white matter lesions; apolipoprotein E (APOE); and plasma Aß42/Aß40, tau, and neurofilament light. RESULTS: Aß status was accurately estimated in BioFINDER using age, 10-word delayed recall or Mini-Mental State Examination, and APOE (area under the receiver operating characteristics curve = 0.81 [0.77-0.85] to 0.83 [0.79-0.87]). When validated, the models performed almost identical in Alzheimer's Disease Neuroimaging Initiative (area under the receiver operating characteristics curve = 0.80-0.82) and within different age, subjective cognitive decline, and mild cognitive impairment populations. Plasma Aß42/Aß40 improved the models slightly. DISCUSSION: The algorithms are implemented on http://amyloidrisk.com where the individual probability of being Aß positive can be calculated. This is useful in the workup of prodromal Alzheimer's disease and can reduce the number needed to screen in Alzheimer's disease trials.

Comparison of elevated phosphorylated neurofilament heavy chains in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

OBJECTIVE: Phosphorylated neurofilament heavy chain (pNfH) levels are elevated in cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). Instead of CSF, we explored blood as an alternative source to measure pNfH in patients with ALS. METHODS: In this single centre retrospective study, 85 patients with ALS, 215 disease controls (DC) and 31 ALS mimics were included. Individual serum pNfH concentrations were correlated with concentrations in CSF and with several clinical parameters. The performance characteristics of pNfH in CSF and serum of patients with ALS and controls were calculated and compared using receiver operating characteristic (ROC) curves. RESULTS: CSF and serum pNfH concentrations in patients with ALS correlated well (r=0.652, p<0.0001) and were significantly increased compared with DC (p<0.0001) and ALS mimics (p<0.0001). CSF pNfH outperformed serum pNfH in discriminating patients with ALS from DC and ALS mimics (difference between area under the ROC curves: p=0.0001 and p=0.0005; respectively). Serum pNfH correlated inversely with symptom duration (r=-0.315, p=0.0033). CSF and serum pNfH were lower when the disease progression rate was slower (r=0.279, p<0.01 and r=0.289, p<0.01; respectively). Unlike CSF, serum pNfH did not correlate with the burden of clinical and electromyographic motor neuron dysfunction. CONCLUSIONS: CSF and serum pNfH concentrations are elevated in patients with ALS and correlate with the disease progression rate. Moreover, CSF pNfH correlates with the burden of motor neuron dysfunction. Our findings encourage further pursuit of CSF and serum pNfH concentrations in the diagnostic pathway of patients suspected to have ALS.

How to handle adsorption of cerebrospinal fluid amyloid ß (1-42) in laboratory practice? Identifying problematic handlings and resolving the issue by use of the Aß42/Aß40 ratio.

INTRODUCTION: We aimed to investigate factors defining amyloid ß (1-42) (Aß1-42) adsorption during preanalytical workup of cerebrospinal fluid (CSF). METHODS: CSF was transferred to new tubes ≤4 times. Variables tested were different polypropylene tube brands, volumes, CSF Aß1-42 concentrations, incubation times, pipettes, vortex intensities, and other CSF proteins, including hyperphosphorylated tau and Interleukin 1 Receptor Accessory Protein (IL-1RAcP). An enquiry assessed the number of transfers in current practice. RESULTS: In diagnostic practice, the number of transfers varied between 1 and 3. Every tube transfer resulted in 5% loss of Aß1-42 concentration, even 10% in small volumes. Adsorption was observed after 30 seconds and after contact with the pipette tip. Tube brand, vortexing, or continuous tube movement did not influence adsorption. Adsorption for Aß1-40 was similar, resulting in stable Aß1-42/Aß1-40 ratios over multiple tube transfers. DISCUSSION: We confirmed that adsorption of CSF Aß1-42 during preanalytical processing is an important confounder. However, use of the Aß1-42/Aß1-40 ratio overcomes this effect and can therefore contribute to increased diagnostic accuracy.

Oxygen sensing by the prolyl-4-hydroxylase PHD2 within the nuclear compartment and the influence of compartmentalisation on HIF-1 signalling.

Hypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-α subunits and a HIF-ß subunit. Under normoxic conditions the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1α stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1α occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1α and shows less prolyl hydroxylase activity for its target HIF-1α than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1α predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.

Seroprevalence of N-methyl-D-aspartate glutamate receptor (NMDA-R) autoantibodies in aging subjects without neuropsychiatric disorders and in dementia patients.

N-methyl-D-aspartate glutamate receptors (NMDA-R) play a key role in learning and memory. Therefore, they may be involved in the pathophysiology of dementia. NMDA-R autoantibodies directed against the NR1a subunit of the NMDA-R, which were first identified as a specific marker for a severe form of encephalitis, cause a decrease in NMDA-Rs, resulting in cognitive impairment and psychosis. We examined the prevalence of NR1a NMDA-R autoantibodies in the serum and cerebrospinal fluid (CSF) of 24 patients with Alzheimer's disease (AD), 20 patients with subcortical ischemic vascular dementia (SIVD), and 274 volunteers without neuropsychiatric disorder. The latter cases showed an association of seropositivity with age. Notably, the overall seroprevalence was not statistically different between dementia patients and matched controls. Further analysis of the patient samples showed that four patients with AD and three patients with SIVD had positive NMDA-R IgM, IgG, and/or IgA autoantibody titers in serum. These patients suffered from psychosis (with the exception of one case). CSF samples were negative for NMDA-R autoantibodies. We conclude that the seroprevalence of NMDA-R-directed autoantibodies is age-related. It has to be clarified by larger studies whether NMDA-R autoantibodies in peripheral blood may predispose patients with AD and SIVD to susceptibility for psychotic episodes if disturbances of blood-brain-barrier integrity occur.

Endothelial cell-derived nitric oxide enhances aerobic glycolysis in astrocytes via HIF-1α-mediated target gene activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

Nitric oxide induces the expression of the monocarboxylate transporter MCT4 in cultured astrocytes by a cGMP-independent transcriptional activation.

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. © 2011 Wiley-Liss, Inc.

Neurofilament Levels Are Reflecting the Loss of Presynaptic Dopamine Receptors in Movement Disorders.

Aims: Neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) are biomarkers for neuroaxonal damage. We assessed whether NfL and other biomarker levels in the CSF are correlated to the loss of presynaptic dopamine transporters in neurons as detected with dopamine transporter SPECT (DaTscan). Methods: We retrospectively identified 47 patients (17 Alzheimer's dementia, 10 idiopathic Parkinson's disease, 7 Lewy body dementia, 13 progressive supranuclear palsy or corticobasal degeneration) who received a DaTscan and a lumbar puncture. DaTscan imaging was performed according to current guidelines, and z-scores indicating the decrease in uptake were software based calculated for the nucleus caudatus and putamen. The CSF biomarkers progranulin, total-tau, alpha-synuclein, NfL, and pNfH were correlated with the z-scores. Results: DaTscan results in AD patients did not correlate with any biomarker. Subsuming every movement disorder with nigrostriatal neurodegeneration resulted in a strong correlation between putamen/nucleus caudatus and NfL (nucleus caudatus right p < 0.01, putamen right p < 0.05, left p < 0.05) and between pNfH and putamen (right p < 0.05; left p < 0.042). Subdividing in disease cohorts did not reveal significant correlations. Progranulin, alpha-synuclein, and total-tau did not correlate with DaTscan results. Conclusion: We show a strong correlation of NfL and pNfH with pathological changes in presynaptic dopamine transporter density in the putamen concomitant to nigrostriatal degeneration. This correlation might explain the reported correlation of impaired motor functions in PD and NfL as seen before, despite the pathological heterogeneity of these diseases.

Towards a unified protocol for handling of CSF before ß-amyloid measurements.

BACKGROUND: Widespread implementation of Alzheimer's disease biomarkers in routine clinical practice requires the establishment of standard operating procedures for pre-analytical handling of cerebrospinal fluid (CSF). METHODS: Here, CSF collection and storage protocols were optimized for measurements of ß-amyloid (Aß). We investigated the effects of (1) storage temperature, (2) storage time, (3) centrifugation, (4) sample mixing, (5) blood contamination, and (6) collection gradient on CSF levels of Aß. For each study participant, we used fresh CSF directly collected into a protein low binding (LoB) tube that was analyzed within hours after lumbar puncture (LP) as standard of truth. Aß42 and Aß40 were measured in de-identified CSF samples using EUROIMMUN and Mesoscale discovery assays. RESULTS: CSF Aß42 and Aß40 were stable for at least 72 h at room temperature (RT), 1 week at 4 °C, and 2 weeks at - 20 °C and - 80 °C. Centrifugation of non-blood-contaminated CSF or mixing of samples before the analysis did not affect Aß levels. Addition of 0.1-10% blood to CSF that was stored at RT without centrifugation led to a dose- and time-dependent decrease in Aß42 and Aß40, while Aß42/Aß40 did not change. The effects of blood contamination were mitigated by centrifugation and/or storage at 4 °C or - 20 °C. Aß levels did not differ between the first to fourth 5-ml portions of CSF. CONCLUSIONS: CSF can be stored for up to 72 h at RT, 1 week at 4 °C, or at least 2 weeks at either - 20 °C or - 80 °C before Aß measurements. Centrifugation of fresh non-blood-contaminated CSF after LP, or mixing before analysis, is not required. In case of visible blood contamination, centrifugation and storage at 4 °C or - 20 °C is recommended. After discarding the first 2 ml, any portion of up to 20 ml of CSF is suitable for Aß analysis. These findings will be important for the development of a clinical routine protocol for pre-analytical handling of CSF.

Cerebrospinal fluid and plasma biomarker trajectories with increasing amyloid deposition in Alzheimer's disease.

Failures in Alzheimer's disease (AD) drug trials highlight the need to further explore disease mechanisms and alterations of biomarkers during the development of AD. Using cross-sectional data from 377 participants in the BioFINDER study, we examined seven cerebrospinal fluid (CSF) and six plasma biomarkers in relation to ß-amyloid (Aß) PET uptake to understand their evolution during AD. In CSF, Aß42 changed first, closely followed by Aß42/Aß40, phosphorylated-tau (P-tau), and total-tau (T-tau). CSF neurogranin, YKL-40, and neurofilament light increased after the point of Aß PET positivity. The findings were replicated using Aß42, Aß40, P-tau, and T-tau assays from five different manufacturers. Changes were seen approximately simultaneously for CSF and plasma biomarkers. Overall, plasma biomarkers had smaller dynamic ranges, except for CSF and plasma P-tau which were similar. In conclusion, using state-of-the-art biomarkers, we identified the first changes in Aß, closely followed by soluble tau. Only after Aß PET became abnormal, biomarkers of neuroinflammation, synaptic dysfunction, and neurodegeneration were altered. These findings lend in vivo support of the amyloid cascade hypotheses in humans.

Serum neurofilament heavy chains as early marker of motor neuron degeneration.

OBJECTIVE: To determine whether serum phosphorylated neurofilament heavy chain (pNfH) levels are elevated before patients were diagnosed with sporadic or familial ALS, and what the prognostic value of these prediagnostic pNfH levels is. METHODS: pNfH was measured via ELISA in leftovers of serum drawn for routine purposes before the time of diagnosis. These prediagnostic samples were retrieved from the biobank of the University Hospitals Leuven for 95 patients who in follow-up received a diagnosis of ALS. Additionally, 35 patients with mild cognitive impairment (MCI) and 85 healthy controls (HC) were included in this retrospective study. RESULTS: The median disease duration (range) from onset to prediagnostic sampling and from onset to diagnosis was 6.5 (-71.9-36.1) and 9.9 (2.0-40.7) months, respectively. Fifty-eight percent of the prediagnostic samples had serum pNfH levels above the 95th percentile of pNfH levels measured in HC. Serum pNfH levels (median (range)) were elevated up to 18 months before the diagnosis of ALS (91 pg/mL (6-342 pg/mL)) in comparison with HC (30 pg/mL (6-146 pg/mL); P = 0.05), and increased during the prediagnostic stage, which was not observed in patients with MCI. Furthermore, prediagnostic pNfH levels were a univariate predictor of survival in ALS (hazard ratio (95% CI): 2.16 (1.20-3.87); P = 0.01). INTERPRETATION: Our findings demonstrate that serum pNfH is elevated well before the time of diagnosis in mainly sporadic ALS patients. These results encourage to prospectively explore if pNfH has an added value to shorten the diagnostic delay in ALS.

Antibodies against neural antigens in patients with acute stroke: joint results of three independent cohort studies.

BACKGROUND AND PURPOSE: Ischemic stroke (IS) and hemorrhagic stroke (HemS) typically lead to a breakdown of the blood-brain barrier with neural antigen presentation. This presentation could potentially generate destructive auto-immune responses. Pre-existing antineuronal and antiglial antibodies (AA), predominantly NMDA receptor antibodies, have been reported in patients with stroke. This article summarizes three independent prospective studies, the Lübeck cohort (LC), Barcelona cohort (BC), and Heidelberg cohort (HC), exploring the frequency and clinical relevance of AA in patients with acute stroke (AS). METHODS: In all cohorts together, 344 consecutive patients admitted with AS (322 × IS, 22 × HemS) were screened for AA in serum at admission. Clinical outcome parameters as well as a second AA screening were available at 30 days in the LC or at 90 days in the BC. A control group was included in the BC (20 subjects free from neurological disease) and the HC (78 neurological and ophthalmological patients without evidence for stroke). RESULTS: The rate of positivity for AA was similar in control subjects and AS patients (13%, 95% CI [7%, 22%] vs. 13%, 95% CI [10%, 17%]; p = 0.46) with no significant difference between cohorts (LC 25/171, BC 12/75, HC 9/98). No patient had developed new AA after 30 days, whereas 2 out of 60 patients had developed new AA after 90 days. AA positive patients did not exhibit significant differences to AA negative patients in stroke subtype (LC, BC), initial stroke severity (BC, LC, HC), infarct volume (BC), and functional status at admission (BC, LC, HC) and follow-up (BC, LC). CONCLUSIONS: AS does not induce AA to a relevant degree. Pre-existing AA can be found in the serum of stroke patients, but they do not have a significant association with clinical features and outcomes.

Cerebrospinal fluid neurogranin/ß-site APP-cleaving enzyme 1 predicts cognitive decline in preclinical Alzheimer's disease.

INTRODUCTION: The cerebrospinal fluid neurogranin (Ng)/ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) ratio may reflect synaptic affection resulting from reduced beta-amyloid (Aß) clearance. We hypothesize that increased Ng/BACE1 ratio predicts the earliest cognitive decline in Alzheimer's disease. METHODS: We compared Ng/BACE1 levels between cases with subjective cognitive decline (n = 18) and mild cognitive impairment (n = 20) both with amyloid plaques and healthy controls (APOE-ε4+, n = 16; APOE-ε4-, n = 20). We performed regression analyses between cerebrospinal fluid levels, baseline hippocampal and amygdala volumes, and pertinent cognitive measures (memory, attention, Mini Mental State Examination [MMSE]) at baseline and after 2 years. RESULTS: Ng/BACE1 levels were elevated in both subjective cognitive decline and mild cognitive impairment compared to healthy controls. Higher Ng/BACE1 ratio was associated with lower hippocampal and amygdala volumes; lower baseline memory functions, attention, and MMSE; and significant decline in MMSE and memory function at 2-year follow-up. DISCUSSION: High Ng/BACE1 ratio predicts cognitive decline also in preclinical cases with amyloid plaques.

Automation on an Open-Access Platform of Alzheimer's Disease Biomarker Immunoassays.

The lack of (inter-)laboratory standardization has hampered the application of universal cutoff values for Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers and their transfer to general clinical practice. The automation of the AD biomarker immunoassays is suggested to generate more robust results than using manual testing. Open-access platforms will facilitate the integration of automation for novel biomarkers, allowing the introduction of the protein profiling concept. A feasibility study was performed on an automated open-access platform of the commercial immunoassays for the 42-amino-acid isoform of amyloid-ß (Aß1-42), Aß1-40, and total tau in CSF. Automated Aß1-42, Aß1-40, and tau immunoassays were performed within predefined acceptance criteria for bias and imprecision. Similar accuracy was obtained for ready-to-use calibrators as for reconstituted lyophilized kit calibrators. When compared with the addition of a standard curve in each test run, the use of a master calibrator curve, determined before and applied to each batch analysis as the standard curve, yielded an acceptable overall bias of -2.6% and -0.9% for Aß1-42 and Aß1-40, respectively, with an imprecision profile of 6.2% and 8.4%, respectively. Our findings show that transfer of commercial manual immunoassays to fully automated open-access platforms is feasible, as it performs according to universal acceptance criteria.

Optimized Standard Operating Procedures for the Analysis of Cerebrospinal Fluid Aß42 and the Ratios of Aß Isoforms Using Low Protein Binding Tubes.

BACKGROUND: Reduced cerebrospinal fluid (CSF) concentration of amyloid-ß1-42 (Aß1-42) reflects the presence of amyloidopathy in brains of subjects with Alzheimer's disease (AD). OBJECTIVE: To qualify the use of Aß1-42/Aß1-40 for improvement of standard operating procedures (SOP) for measurement of CSF Aß with a focus on CSF collection, storage, and analysis. METHODS: Euroimmun ELISAs for CSF Aß isoforms were used to set up a SOP with respect to recipient properties (low binding, polypropylene), volume of tubes, freeze/thaw cycles, addition of detergents (Triton X-100, Tween-20) in collection or storage tubes or during CSF analysis. Data were analyzed with linear repeated measures and mixed effects models. RESULTS: Optimization of CSF analysis included a pre-wash of recipients (e.g., tubes, 96-well plates) before sample analysis. Using the Aß1-42/Aß1-40 ratio, in contrast to Aß1-42, eliminated effects of tube type, additional freeze/thaw cycles, or effect of CSF volumes for polypropylene storage tubes. 'Low binding' tubes reduced the loss of Aß when aliquoting CSF or in function of additional freeze/thaw cycles. Addition of detergent in CSF collection tubes resulted in an almost complete absence of variation in function of collection procedures, but affected the concentration of Aß isoforms in the immunoassay. CONCLUSION: The ratio of Aß1-42/Aß1-40 is a more robust biomarker than Aß1-42 toward (pre-) analytical interfering factors. Further, 'low binding' recipients and addition of detergent in collection tubes are able to remove effects of SOP-related confounding factors. Integration of the Aß1-42/Aß1-40 ratio and 'low-binding tubes' into guidance criteria may speed up worldwide standardization of CSF biomarker analysis.