CSF p-tau/Aß42 ratio and brain FDG-PET may reliably detect MCI "imminent" converters to AD.

PURPOSE: To know whether mild cognitive impairment (MCI) patients will develop Alzheimer's disease (AD) dementia in very short time or remain stable is of crucial importance, also considering new experimental drugs usually tested within very short time frames. Here we combined cerebrospinal fluid (CSF) AD biomarkers and a neurodegeneration marker such as brain FDG-PET to define an objective algorithm, suitable not only to reliably detect MCI converters to AD dementia but also to predict timing of conversion. METHODS: We included 77 consecutive MCI patients with neurological/neuropsychological assessment, brain 18F-FDG-PET and CSF analysis available at diagnosis and a neuropsychological/neurological evaluation every 6 months for a medium- to a long-term follow-up (at least 2 and up to 8 years). Binomial logistic regression models and Kaplan-Meier survival analyses were performed to determine the best biomarker (or combination of biomarkers) in detecting MCI converters to AD dementia and then, among the converters, those who converted in short time frames. RESULTS: Thirty-five out of 77 MCI patients (45%) converted to AD dementia, with an average conversion time since MCI diagnosis of 26.07 months. CSF p-tau/Aß42 was the most accurate predictor of conversion from MCI to AD dementia (82.9% sensitivity; 90% specificity). CSF p-tau/Aß42 and FDG-PET-positive MCIs converted to AD dementia significantly earlier than the CSF-positive-only MCIs (median conversion time, 17.1 vs 31.3 months). CONCLUSIONS: CSF p-tau/Aß42 ratio and brain FDG-PET may predict both occurrence and timing of MCI conversion to full-blown AD dementia. MCI patients with both biomarkers suggestive for AD will likely develop an AD dementia shortly, thus representing the ideal target for any new experimental drug requiring short periods to be tested for.

Cerebrospinal fluid analysis and the determination of oligoclonal bands.

This document presents the guidelines for the cerebrospinal fluid (CSF) analysis and the determination of oligoclonal bands (OCBs) as pivotal tests in neuroinflammatory pathologies of the central nervous system. The guidelines have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on the pathologies in which the CSF analysis is indicated, and, particularly, on those characterized by the presence of OCBs in the intrathecal compartment, indications and limits of CSF analysis and OCB determination, instructions for result interpretation, and agreed laboratory protocols (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Cross-validation of biomarkers for the early differential diagnosis and prognosis of dementia in a clinical setting.

PURPOSE: The aim of this study was to evaluate the supportive role of molecular and structural biomarkers (CSF protein levels, FDG PET and MRI) in the early differential diagnosis of dementia in a large sample of patients with neurodegenerative dementia, and in determining the risk of disease progression in subjects with mild cognitive impairment (MCI). METHODS: We evaluated the supportive role of CSF Aß42, t-Tau, p-Tau levels, conventional brain MRI and visual assessment of FDG PET SPM t-maps in the early diagnosis of dementia and the evaluation of MCI progression. RESULTS: Diagnosis based on molecular biomarkers showed the best fit with the final diagnosis at a long follow-up. FDG PET SPM t-maps had the highest diagnostic accuracy in Alzheimer's disease and in the differential diagnosis of non-Alzheimer's disease dementias. The p-tau/Aß42 ratio was the only CSF biomarker providing a significant classification rate for Alzheimer's disease. An Alzheimer's disease-positive metabolic pattern as shown by FDG PET SPM in MCI was the best predictor of conversion to Alzheimer's disease. CONCLUSION: In this clinical setting, FDG PET SPM t-maps and the p-tau/Aß42 ratio improved clinical diagnostic accuracy, supporting the importance of these biomarkers in the emerging diagnostic criteria for Alzheimer's disease dementia. FDG PET using SPM t-maps had the highest predictive value by identifying hypometabolic patterns in different neurodegenerative dementias and normal brain metabolism in MCI, confirming its additional crucial exclusionary role.

CSF metabolites in the differential diagnosis of Alzheimer's disease from frontal variant of frontotemporal dementia.

The early differentiation between Alzheimer's disease (AD) and frontal variant of frontotemporal dementia (fvFTD) is frequently difficult, albeit critical for the adequate management of patients and their caregivers. In order to assess the accuracy of CSF levels of beta-amyloid 1-42 (Aß), tau (τ) and Thr 181-phosphorilated tau (Pτ) in the early differentiation of AD from fvFTD, we designed a prospective study in which patients have been followed up for at least 2 years. Seventy-two patients with AD and 42 patients with fvFTD showed significantly different CSF levels of Pτ (increased in AD, p = 0.0001), Aß (reduced in AD, p = 0.03), and ratios of Pτ to Aß (p = 0.003). ROC analyses showed that the ratio Pτ/Αß is able to predict diagnosis with an AUC of 0.73 (optimal level being 0.16) corresponding to a sensitivity of 80% and a specificity of 68%. Our findings suggest that CSF metabolites may be the important tools in the early differential diagnosis between AD and fvFTD, albeit to be correlated with clinical, neuropsychological and bio imaging features.

The CSF p-tau181/Aß42 Ratio Offers a Good Accuracy "In Vivo" in the Differential Diagnosis of Alzheimer's Dementia.

BACKGROUND: The incoming disease-modifying therapies against Alzheimer's disease (AD) require reliable diagnostic markers to correctly enroll patients all over the world. CSF AD biomarkers, namely amyloid-ß 42 (Aß42), total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau181), showed good diagnostic accuracy in detecting AD pathology, but their real usefulness in daily clinical practice is still a matter of debate. Therefore, further validation in complex clinical settings, that is patients with different types of dementia, is needed to uphold their future worldwide adoption. METHODS: We measured CSF AD biomarkers' concentrations in a sample of 526 patients with a clinical diagnosis of dementia (277 with AD and 249 with Other Type of Dementia, OTD). Brain FDG-PET was also considered in a subsample of 54 patients with a mismatch between the clinical diagnosis and the CSF findings. RESULTS: A p-tau181/Aß42 ratio higher than 0.13 showed the best diagnostic performance in differentiating AD from OTD (86% accuracy index, 74% sensitivity, 81% specificity). In cases with a mismatch between clinical diagnosis and CSF findings, brain FDG-PET partially agreed with the p-tau181/Aß42 ratio, thus determining an increase in CSF accuracy. CONCLUSION: The p-tau181/Aß42 ratio alone might reliably detect AD pathology in heterogeneous samples of patients suffering from different types of dementia. It might constitute a simple, cost-effective and reproducible in vivo proxy of AD suitable to be adopted worldwide not only in daily clinical practice but also in future experimental trials, to avoid the enrolment of misdiagnosed AD patients.

Cerebrospinal Fluid Amyloid-ß 42, Total Tau and Phosphorylated Tau are Low in Patients with Normal Pressure Hydrocephalus: Analogies and Differences with Alzheimer's Disease.

Co-existence of Alzheimer's disease (AD) in normal pressure hydrocephalus (NPH) is a frequent finding, thus a common pathophysiological basis between AD and NPH has been postulated. We measured CSF amyloid-ß 42 (Aß42), total tau (t-tau), and phosphorylated tau (p-tau) concentrations in a sample of 294 patients with different types of dementia and 32 subjects without dementia. We then compared scores on neuropsychological tests of NPH patients with pathological and normal CSF Aß42 values. Aß42 levels were significantly lower in NPH than in control patients, with no significant differences between AD and NPH. On the contrary, t-tau and p-tau levels were significantly lower in NPH than in AD, with no differences between NPH and controls. NPH patients with pathological Aß42 levels did not perform worse than NPH patients with normal Aß42 levels in any cognitive domains. Our data seem to support the hypothesis of amyloid accumulation in brains of NPH patients. Nevertheless, amyloid does not seem to play a pathogenetic role in the development of cognitive deficits in NPH.

When diagnostics meets translational research: detection of hemoglobin fractions in cellular lysates from in vitro erythroid cultures by Capillarys 2 Flex Piercing analyzer (Sebia).

Detection of hemoglobin (Hb) variants represents an important issue for diagnosis and adequate treatment of hemoglobinopathies. The Capillarys 2 Flex Piercing analyzer (Capillarys) by Sebia is routinely used in our clinical laboratories to detect Hb variants in peripheral blood (PB). This automated method separates Hb fractions by capillary electrophoresis, giving a spectrophotometric measure of their relative proportion. The scientific research in the field of hemoglobinopathies needs robust procedures to evaluate the efficacy of experimental therapies, as gene therapy. We investigated for the first time the feasibility to use Capillarys on cellular lysates from in vitro erythroid cultures. Because total Hb concentration in erythroid lysates is up to 20-fold lower than in hemolysates from PB, we analyzed diluted blood samples, thanks to the manual mode included in the Capillarys setting. We compared analytical precision, accuracy, sensitivity, and specificity of this procedure to the automatic method, routinely used in diagnostics. For instance, adult Hb intra- and interassay precision were estimated as coefficient of variation 0.2% and 0.3%, respectively. The manual mode is less robust for detection of fractions <3% and the lower level of sensitivity is 2 g/L of total Hb. Specificity of manual and automatic settings was equivalent. We confirmed the performance of the method by analyzing erythroid lysates from thalassemic patients' cultures. Our study demonstrated that the Capillarys 2 Flex Piercing manual method is comparable to the automatic one. The analysis is very robust at low Hb concentrations, as in erythroid cultures from patients affected by hemoglobinopathies, representing a useful tool also in translational research.

Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis.

Background. The presence of CSF oligoclonal bands (OBs) is an independent prognostic factor for multiple sclerosis (MS), but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC) levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS), other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p < 0.001 for all comparisons). KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p < 0.001 and p < 0.001, resp.) and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index). Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients.

Combining Cerebrospinal Fluid Biomarkers and Neuropsychological Assessment: A Simple and Cost-Effective Algorithm to Predict the Progression from Mild Cognitive Impairment to Alzheimer's Disease Dementia.

BACKGROUND: Correctly diagnosing Alzheimer's disease (AD) in prodromal phases would allow the adoption of experimental therapeutic strategies that could selectively interrupt the pathogenetic process before neuronal damage becomes irreversible. Therefore, great efforts have been aimed at finding early reliable disease markers. OBJECTIVE: The aim of this study was to identify a simple, cost effective, and reliable diagnostic algorithm to predict conversion from mild cognitive impairment (MCI) to AD. METHODS: 96 consecutive MCI patients admitted to the Neurology department of San Raffaele Hospital in Milan between January 2009 and January 2015 were included. All patients underwent neuropsychological assessment and lumbar puncture with CSF analysis of amyloid-ß 42 (Aß42), total tau (t-tau), and phosphorylated tau (p-tau) levels. Each patient underwent clinical and neuropsychological follow-up, in order to identify a possible progression from MCI to AD. The mean follow up time was 36.73 months. RESULTS: 37 out of 96 MCI converted to AD during follow up. CSF analysis and neuropsychological assessment reliably detected MCI patients who developed AD. In a subsample of 43 subjects, a Composite Cognitive Score (CCS) was calculated including episodic memory, executive function, and verbal fluency tests. Combining together CSF biomarkers and CCS increased the accuracy of the single predictors, correctly classifying 86% of patients with a specificity of 96% and a Positive Predictive Value of 93%. DISCUSSION: Even if preliminary, our data seem to suggest that CSF analysis and neuropsychological assessment could detect MCI patients who will convert to AD with high confidence. Their relative low cost and availability could make them worldwide essential tools in future clinical trials.

Clinical significance of the number of oligoclonal bands in patients with clinically isolated syndromes.

CSF oligoclonal bands (OCBs) in patients with clinically isolated syndromes (CIS) are a risk factor for clinically definite multiple sclerosis (CDMS). We aimed to address the relevance of the number of OCBs in the prognosis of CIS patients. 219 CIS patients were included in the study, and 42% of them developed the disease during follow-up (median: 5.04 years). Patients with a high number of CSF OCBs (third quartile, 8-12 OCBs) had 2.5-fold increase in CDMS risk, while no further increase in the HR of disease was observed for patients with more than 12 OCBs. The results did not change after adjustment for additional correlates of CDMS development. This association may be due to the epitope-spreading phenomenon and may reflect the stage of the disease at the time of the examination.

Evaluation of the performance of an immunoturbidimetric HbA1c reagent applied to the Siemens ADVIA 2400 automatic analyzer.

OBJECTIVES: A new immunochemical reagent based on latex particles and antibodies against HbA1c (Axis-Shield), using Siemens ADVIA 2400 Instrument was evaluated. DESIGN AND METHODS: Intra-assay and total imprecision, interferences studies (bilirubin ~850 µmol/L, triglycerides ~16.9 mmol/L, total protein ~140 g/L, sodium cyanate ~50 mg/dL, ascorbic acid ~50 mg/dL, urea ~24.99 mmol/L, glucose ~105.46 mmol/L, rheumatoid factor ~600 U/mL), method comparison vs Capillary Electrophoresis (Sebia), Lot to Lot reproducibility, linearity and carry over were conducted on ADVIA 2400 according to CLSI protocols. Additionally, 40 samples were measured by the two methods and also by a NGSP reference lab. RESULTS: CVs % obtained by intra-assay imprecision, on 3 human HbA1c specimens at different concentrations (48, 48-64 and >64 mmol/mol) in 20 replicates, were <4%. CVs % by total imprecision, performed over 20 days with 4 calibrations on 3 human HbA1c samples (48, 48-64 and >64 mmol/mol), resulted <4%. Interferences were studied on two human samples (42-53 and>64 mmol/mol) without obtaining significant biases (<10%). Methods comparison vs Capillary Electrophoresis, performed on 120 samples ranging 23-137 mmol/mol, obtaining r = 0.974 as regression coefficient and a mean bias at decision level (48 mmol/mol) <3%. The results obtained with the NGSP samples have allowed the certification of the new reagent. CONCLUSIONS: The ASD reagent met the needs of clinical laboratories, fulfilling both NGSP and IFCC requirements and it is robust to endogenous interferences.

Cerebrospinal fluid biomarkers can play a pivotal role in the diagnostic work up of primary progressive aphasia.

BACKGROUND: Three variants of primary progressive aphasia (PPA) have been currently characterized: non fluent/agrammatic (nfv-PPA), semantic (sv-PPA), and logopenic variant (lv-PPA). lv-PPA is most commonly associated with Alzheimer's disease (AD), while nfv-PPA and sv-PPA are related to frontotemporal lobar degeneration. OBJECTIVE: We aimed to determine whether cerebrospinal fluid (CSF) amyloid-ß42 (Aß42), total tau protein (t-tau), and phosphorylated tau (p-tau), frequently abnormal in AD, could constitute a useful tool in the PPA diagnostic work up, in order to identify subjects with an underlying AD pathology. METHODS: We measured CSF biomarker levels in a group of twenty-eight patients, fourteen lv-PPA, nine nfv-PPA, and five sv-PPA. RESULTS: Since there were no significant differences in any of the parameters investigated between nfv-PPA and sv-PPA, the two groups were considered as one (nfv/sv-PPA). At diagnosis, lv-PPA were older than nfv/sv-PPA patients (mean values: 70.7 versus 64.6 years, p = 0.02). CSF biomarker mean concentrations were significantly different in lv-PPA versus nfv/sv-PPA patients (p = 0.000): Aß42 350.64 versus 661.64 ng/L; tau 631.21 versus 232.71 ng/L; p-tau 101 versus 38.21 ng/L. According to the recent AD diagnostic criteria, (Cummings et al., 2013) eleven lv-PPA and only one nfv/sv-PPA showed a liquoral pattern typical for AD. Finally lv-PPA had CSF biomarker levels very similar to a sample of 72 AD patients from our Department. CONCLUSIONS: Our data showed that CSF biomarkers can easily and reliably detect those patients with language disorders due to an underlying AD pathology, thus offering the possibility of targeted therapeutic interventions. However, because of the small sample size, such analyses should be reproduced in larger populations of patients to confirm our data.

Diagnostic value of IgG4 Indices in IgG4-related hypertrophic pachymeningitis.

Diagnosis of IgG4-Related Hypertrophic Pachymeningitis (IgG4-HP) relies on meningeal biopsies, because cerebrospinal fluid (CSF) diagnostic biomarkers are lacking. Here, we determined whether IgG4 intrathecal production could distinguish IgG4-HP from other disorders presenting with HP (OHP). In patients with IgG4-HP, the median CSF IgG4 concentration, IgG4 Index and IgG4Loc were significantly higher than in both controls and OHP. CSF IgG4 levels higher than 2.27mg/dL identified 100% of IgG4-HP and 5% of OHP. An IgG4Loc cut-off of 0.47 identified 100% of IgG4-HP and no cases of OHP. Our results support CSF IgG4 quantification and IgG4 Indices as alternatives to meningeal biopsy for the diagnosis of IgG4-HP when this procedure is contraindicated or uninformative.

Multicenter evaluation of hemoglobin A1c assay on capillary electrophoresis.

BACKGROUND: Hemoglobin A1c (HbA1c) measurement is currently used for the routine monitoring of long-term glycemic status, thus playing a fundamental role in the management of this disease. Since this marker has recently been recommended as an additional tool for diagnosing diabetes, it's of the utmost importance to ensure that the precision and accuracy of HbA1c methods are satisfactory. METHODS: We assessed the analytical performances of the Capillarys 2 Flex Piercing® analyzer and compared the results obtained with those from two other widely used HPLC instruments. Furthermore, we evaluated the convenience and ergonomics of the system in authentic routine work conditions in three centers. RESULTS: Within-laboratory (n=40) and between-laboratory (n=120) imprecision CV% using four blood samples with different concentrations of HbA1c were <3.4% and <3.1% using IFCC units and <2.1% and <2.0% using NGSP units, respectively. The obtained trueness (<3 mmol/mol, <0.3%) was highly satisfactory, nor was HbA1c measurement compromised by the presence of the commonly present hemoglobinopathies. The comparison made with established methods revealed excellent agreement (r>0.985). CONCLUSIONS: The evaluated method is precise, accurate and robust, with a high throughput. It also allows the identification of the most frequent Hb variants and therefore may be a valid alternative to other methods currently proposed for routine use in clinical laboratories.