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Background: Increased clusterin levels have been reported in brain, cerebrospinal fluid (CSF), and plasma of Alzheimer's disease (AD) patients. Because changes are also observed in mild cognitive impairment (MCI), a possible relationship between clusterin levels and early neurodegenerative changes in AD was suggested., Objectives: To determine whether clusterin concentrations could 1) serve as a diagnostic marker for AD, 2) predict disease progression in MCI, and 3) correlate with AD-biomarkers., Methods: Clusterin levels in CSF and plasma, as well as AD biomarker levels of Aβ42, Tau, and pTau in CSF and Mini-Mental State Examination scores (MMSE) were determined in 67 controls, 50 MCI, and 107 AD patients. Repeated MMSE was obtained for 44 MCI and 72 AD patients after, on average, 2.7 years., Results: Elevated clusterin concentrations in plasma, but not in CSF, were a risk factor for AD (HR 18.6; 95% CI 2.8-122), and related to cognitive decline in MCI (r =-0.38; p <  0.01). An inverse relation between plasma clusterin levels and cognitive decline was observed in AD patients (r = 0.23; p≤0.05). In CSF, but not in plasma, clusterin levels correlated with Tau and pTau in all groups., Conclusion: Elevated plasma clusterin levels in MCI confer an increased risk for progression to AD, and more rapid cognitive decline. We speculate that clusterin levels in CSF may reflect its involvement in the earliest neurodegenerative processes associated with AD pathology. Whereas neither clusterin levels in CSF nor in plasma had diagnostic value, plasma clusterin levels may serve as a prognostic marker for AD.

Background: Amyloid-β (Aβ)-oligomers are neurotoxic isoforms of Aβ and are a potential diagnostic biomarker for Alzheimer's disease (AD)., Objectives: 1) Analyze the potential of Aβ-oligomer concentrations in cerebrospinal fluid (CSF) to diagnose and predict progression to AD in a large clinical study sample. 2) Monitor Aβ-oligomer concentrations over-time, both in early and advanced stages of AD. 3) Examine the relation between Aβ-oligomer levels in CSF and cognitive functioning., Methods: 24 non-demented, 61 mild cognitive impairment (MCI), and 64 AD patients who underwent lumbar puncture and cognitive testing at baseline and follow-up were selected from the memory clinic based Amsterdam Dementia Cohort. CSF samples were analyzed for standard AD-biomarkers and Aβ-oligomer levels using a validated in-house Aβ-oligomer specific enzyme-linked immunosorbent assay. Aβ-oligomer levels were analyzed as indicators of disease progression (follow-up AD diagnosis) and cognitive decline, respectively., Results: Patient groups did not differ in Aβ-oligomer concentrations at baseline or follow-up. Baseline CSF Aβ-oligomer levels were similar in MCI patients that develop AD as in stable MCI patients. MCI and AD patients showed an annual decrease in Aβ-oligomer levels of 9.4% and 6.8%, respectively. A decrease in Aβ-oligomer levels over time was strongly associated with more severe cognitive decline in AD patients., Conclusion: Despite the limited diagnostic potential of Aβ-oligomer levels in CSF to differentiate between patient groups, and between MCI-AD and MCI-stable patients, changes in CSF Aβ-oligomer levels were related to cognitive decline. Therefore, CSF Aβ-oligomers may aid in the selection of patients with a more aggressive disease course.

The adrenal and gonadal androgens, testosterone, androstenedione and dehydroepiandrosterone (DHEA) play an important role in sexual development as well as in other processes. We developed a method for simultaneous quantitative analysis of serum and plasma testosterone, androstenedione and DHEA levels using Isotope-Dilution Liquid-Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS). Samples underwent liquid-liquid extraction and were analyzed on an Acquity 2D-UPLC-System and a Xevo TQ-S tandem mass spectrometer (Waters). The intra-assay and inter-assay coefficients of variation were <4.0%, <6.3% and <7.0% and <6.0%, <8.1% and <7.7% for testosterone, androstenedione and DHEA, respectively. Inter-assay CVs at the lower limit were 10.6%, 16.9% and 9.0% for testosterone (0.10nmol/L), androstenedione (0.10nmol/L) and DHEA (1.0nmol/L), respectively. Recoveries of spiked analytes were 93-107%. The present testosterone method compared well (y=1.00x-0.04; r=0.998) to a published ID-LC-MS/MS method for testosterone in our lab. The latter method being concordant with a published reference method (Bui et al., 2013). The present method compared well to a published ID-LC-MS/MS method (Kushnir et al., 2010) (y=1.06x-0.06; r=0.996 for testosterone; y=1.04x-0.04; r=0.995 for androstenedione and y=1.03x+0.01; r=0.991 for DHEA). In conclusion, we developed a sensitive and accurate ID-LC-MS/MS method to simultaneously measure serum testosterone, androstenedione and DHEA in serum and plasma., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Primary and secondary progressive forms of multiple sclerosis (PPMS and SPMS) have different pathological characteristics. However, it is unknown whether neurodegenerative mechanisms are shared. We measured cerebrospinal fluid (CSF) levels of neurofilament (Nf) light and heavy isoforms and N-acetylaspartic acid (NAA) in 21 PP, 10 SPMS patients and 15 non-inflammatory neurological disease controls (NINDC). Biomarkers were related to Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Severity Score (MSSS) over a long period of follow-up [median (interquartile range) 9 (5.5-12.5) years] in 19 PPMS and 4 SPMS patients, and to T2 lesion load, T1 lesion load, and brain parenchymal fraction at the time of lumbar puncture. Nf light was higher in PPMS (p < 0.005) and Nf heavy was increased in both SPMS and PPMS (p < 0.05 and p < 0.01) compared to NINDC, but were comparable between the two MS subtypes. Nf heavy was a predictor of the ongoing disability measured by MSSS (R(2) = 0.17, β = 0.413; p < 0.05). Conversely, Nf light was the only predictor of the EDSS annual increase (R(2) = 0.195, β = 0.441; p < 0.05). The frequency of abnormal biomarkers did not differ between the two MS progressive subtypes. Our data suggest that PP and SPMS likely share similar mechanisms of axonal damage. Moreover, Nf heavy can be a biomarker of ongoing axonal damage. Conversely, Nf light can be used as a prognostic marker for accumulating disability suggesting it as a good tool for possible treatment monitoring in the progressive MS forms.

Background: We aimed to identify the most useful definition of the "cerebrospinal fluid Alzheimer profile," based on amyloid-ß1-42 (Aβ42), total tau, and phosphorylated tau (p-tau), for diagnosis and prognosis of Alzheimer's disease (AD)., Methods: We constructed eight Alzheimer profiles with previously published combinations, including regression formulas and simple ratios. We compared their diagnostic accuracy and ability to predict dementia due to AD in 1385 patients from the Amsterdam Dementia Cohort. Results were validated in an independent cohort (n = 1442)., Results: Combinations outperformed individual biomarkers. Based on the sensitivity of the best performing regression formulas, cutoffs were chosen at 0.52 for the tau/Aβ42 ratio and 0.08 for the p-tau/Aβ42 ratio. Ratios performed similar to formulas (sensitivity, 91%-93%; specificity, 81%-84%). The same combinations best predicted cognitive decline in mild cognitive impairment patients. Validation confirmed these results, especially regarding the tau/Aβ42 ratio., Conclusions: A tau/Aβ42 ratio of >0.52 constitutes a robust cerebrospinal fluid Alzheimer profile. We recommend using this ratio to combine biomarkers., (Copyright © 2014. Published by Elsevier Inc.)

Background: Clusterin (ApoJ) is an amyloid-associated protein and plays an important role in Alzheimer's disease (AD) pathology. Recent genome-wide association studies have indicated that certain genetic variants increase the risk of developing AD. To determine if the expression of clusterin is different in AD patients, both systemically and locally in the brain, differs between (subgroups of) AD patients and non-AD cases, an assay available that detects clusterin in both plasma and cerebrospinal fluid (CSF) with equal sensitivity would be helpful., Methods: We compared four different commercially available antibodies in their ability to detect recombinant clusterin and immune-purified human clusterin. Specificity was tested on western blot and in ELISA systems, and selection was based on the ability to detect clusterin in CSF and plasma. A sandwich ELISA was developed and validated with monoclonal antibody G7 as capture, and rabbit polyclonal (Alexis) antibodies for detection., Results: Our ELISA measured clusterin concentrations in plasma and CSF with dynamic ranges of 2-70 mg/L and 0.5-40 mg/L, respectively. The assays showed 99.8% recovery in CSF and 97% recovery in plasma. Intra-assay coefficient of variation was 1.4% and inter-assay 8.8%. The assay shows no cross-reactivity with related apolipoproteins. Clusterin quantification is dependent on the type of storage for plasma samples. A single freeze/thaw cycle caused fluctuations of clusterin concentrations in plasma, while clusterin in CSF is stable for up to five cycles., Conclusions: We have successfully developed a clusterin ELISA that reliably measures CSF and plasma clusterin concentrations. In a pilot study, all samples gave results that were well within the dynamic range of the assay, with low variations. Freshly stored plasma samples are crucial for accurate clusterin quantification., (© The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

BRI2 protein binds amyloid precursor protein to halt amyloid-β production and inhibits amyloid-β aggregation via its BRICHOS-domain suggesting a link between BRI2 and Alzheimer's disease (AD). Here, we investigate the possible involvement of BRI2 in human AD pathogenesis. BRI2 containing BRICHOS-domain was increased up to 3-fold in AD hippocampus (p = 0.003, n = 14/group). Immunohistochemistry showed BRI2 deposits associated with amyloid-β plaques in early pathologic stages (Braak-III; Thal-2/3). We observed a decrease in the protein levels of ADAM10 (p = 0.02) and furin (p = 0.066), as well as an increase in SPPL2b (p < 0.0001) in AD hippocampus. Because these enzymes are involved in BRI2 processing, their changes may lead to aberrant processing of BRI2 promoting its deposition and likely affecting BRI2 function. Loss of BRI2 function in AD was supported by the decreased presence of BRI2-amyloid precursor protein complexes in the hippocampus of AD patients compared with control subjects. In conclusion, our data obtained from human samples indicate that in early stages of AD there is an increased deposition of BRI2, which likely leads to impaired BRI2 function thereby influencing AD pathophysiology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Defective clearance of the amyloid-β peptide (Aβ) from the brain is considered a strong promoter in Alzheimer's disease (AD) pathogenesis. Astrocytes and microglia are important mediators of Aβ clearance and Aβ aggregation state and the presence of amyloid associated proteins (AAPs), such as Apolipoproteins E and J (ApoE and ApoJ), may influence Aβ clearance by these cells. Here we set out to investigate whether astrocytes and microglia differ in uptake efficiency of Aβ oligomers (Aβoligo ) and Aβ fibrils (Aβfib ), and whether the Aβ aggregation state and/or presence of AAPs affect Aβ uptake in these cells in vitro. Adult human primary microglia and astrocytes, isolated from short delay post-mortem brain tissue, were exposed to either Aβoligo or Aβfib alone or combined with a panel of certain AAPs whereafter Aβ-positive cells were quantified using flow cytometry. Upon exposure to Aβ combined with ApoE, ApoJ, α1-antichymotrypsin (ACT) and a combination of serum amyloid P and complement C1q (SAP-C1q), a clear reduction in astrocytic but not microglial Aβoligo uptake, was observed. In contrast, Aβfib uptake was strongly reduced in the presence of AAPs in microglia, but not in astrocytes. These data provide the first evidence of distinct roles of microglia and astrocytes in Aβ clearance. More importantly we show that Aβ clearance by glial cells is negatively affected by AAPs like ApoE and ApoJ. Thus, targeting the association of Aβ with AAPs, such as ApoE and ApoJ, could serve as a therapeutic strategy to increase Aβ clearance by glial cells., (Copyright © 2014 Wiley Periodicals, Inc.)

Total testosterone is considered to be decreased during the use of combined oral contraceptives. There is, however, considerable concern about the quality of testosterone assays, especially at low levels. We aimed to confirm testosterone levels measured by direct radioimmunoassay in a recent clinical trial with a state-of-the-art LC-MSMS method. Surplus specimens with known testosterone levels collected during the study (Clinical Trial Registration number ISRCTN06414473) were reanalyzed with an LC-MSMS method. This method was compared to another LC-MSMS method that had shown to concur excellently to a reference method. Follow-up experiments were designed to explain the results. In contrast to our expectation, LC-MSMS measurements did not corroborate the data obtained by radioimmunoassay. Subsequent experiments showed that this could be attributed to a strong dependency of the radioimmunoassay on SHBG. Testosterone results (n = 198) obtained by direct radioimmunoassay showed a negative correlation to SHBG levels (r = -0.676; p<0.001). By contrast, testosterone results obtained by LC-MSMS were not related to SHBG (r = 0.100; NS). In conclusion, our results indicate that total testosterone measurements during oral contraceptive use are unreliable when performed with assays sensitive to the SHBG concentration. The discrepancy with the literature can most likely be explained by the sensitivity of the immunoassay used to SHBG. Given the sharp increase in SHBG during the use of many oral contraceptives, total testosterone may not decrease, whereas its bioavailability, estimated by free testosterone levels, will be diminished. Studies aiming at restoration of testosterone homeostasis during oral contraception need to take this into account., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To study the effect of different government prenatal screening (PNS) policies on the uptake of PNS and prenatal diagnostic testing (PND) over the periods 2001-2003 (PNS on request), 2004-2006 (permission to offer the first-trimester combined test (FCT) to women of advanced maternal age (AMA), with women aged <36 years informed on explicit request) and 2007-2010 (introduction of population screening) and to evaluate whether trends in uptake are related to maternal age. The indication AMA for PND is still warranted, and the costs for FCT are only reimbursed for AMA women., Study Design: Analysis of data on the first- and second-trimester screening program (n=41,600) for Down syndrome (DS) and on PND (n=10,795) performed from 2001 to 2010 in the region North-Holland of the Netherlands. To evaluate the actual participation in PNS and PND in different maternal age groups, estimation of the age distribution of women who underwent a fetal anomaly scan in 2009 (n=14,481) was used as a reference population (participation of 85.2%)., Results: The overall uptake of FCT was 35.2% in 2010. Over the years the number of FCT in all age groups increased significantly (P<0.001). Overall the number of PND decreased significantly; the number of PND for AMA decreased and the number of PND for increased risk at FCT (in women <36 and ≥36 years) increased (P<0.05). Since 2004 significantly more DS cases were detected with FCT in AMA women and fewer with PND for AMA, and since 2007 more DS cases were detected with FCT in women <36 years (P<0.001)., Conclusion: The effect of the national screening program is limited. Significantly more women opt for PNS but the overall uptake remains low, especially in younger women. A significant number of AMA women still opt for PND for AMA. The choice for FCT and PND for AMA seems dependent on background risk. To accomplish a more effective screening policy, reimbursement of the cost of the test should apply to all women and the indication for PND for AMA should be abolished., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Neurofilament (Nf) proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases. The aim of this study was to evaluate the effects of pre-analytical variables on the concentration of neurofilament heavy (NfH) and neurofilament light (NfL) proteins., Methods: For NfH an in-house newly-developed and validated SinglePlex Luminex assay was used; ELISA was used to analyze NfL., Results: For the NfL ELISA assay, the intra- and inter-assay variation was respectively, 1.5% and 16.7%. Analytical performance of the NfH SinglePlex Luminex assay in terms of sensitivity (6.6pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6-1250pg/mL), and inter- and intra-assay variation (<8%) were good. Concentrations of both NfL and NfH appeared not negatively affected by blood contamination, repeated freeze-thaw cycles (up to 4), delayed processing (up to 24hours) and during long-term storage at -20°C, 4°C, and room temperature. A decrease in concentration was observed during storage of both neurofilament proteins up to 21days at 37°C, which was significant by day 5., Conclusions: The newly developed NfH SinglePlex Luminex assay has a good sensitivity and is robust. Moreover, both NfH and NfL are stable under the most prevalent pre-analytical variations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Unlabelled: BACKGROUND; Combined oral contraceptives (COCs) reduce levels of androgen, especially testosterone (T), by inhibiting ovarian and adrenal androgen synthesis and by increasing levels of sex hormone-binding globulin (SHBG). Although this suppressive effect has been investigated by numerous studies over many years, to our knowledge no systematic review concerning this issue had been performed. This systematic review and meta-analysis was performed to evaluate the effect of COCs on concentrations of total T, free T and SHBG in healthy women and to evaluate differences between the various types of COCs (e.g. estrogen dose, type of progestin) and the assays used to assess total T and free T., Methods: A review of the literature was performed using database searches (MEDLINE, EMBASE and the Cochrane Central Register of Clinical Trials) and all publications (from inception date until July 2012) investigating the effect of COCs on androgen levels in healthy women were considered eligible for selection. Three reviewers were involved in study selection, data extraction and critical appraisal. For the meta-analysis, data on total T, free T and SHBG were extracted and combined using random effects analysis. Additional subgroup analyses were performed to evaluate differences between the various types of COCs (e.g. estrogen dose, type of progestin) and the assays used to assess total T or free T., Results: A total of 151 records were identified by systematic review and 42 studies with a total of 1495 healthy young women (age range: 18-40 years) were included in the meta-analysis. All included studies were experimental studies and 21 were non-comparative. Pooling of the results derived from all the included papers showed that total T levels significantly decreased during COC use [mean difference (MD) (95% confidence interval, CI) -0.49 nmol/l (-0.55, -0.42); P < 0.001]. Significantly lower levels of free T were also found [relative change (95% CI) 0.39 (0.35, 0.43); P < 0.001], with a mean decrease of 61%. On the contrary, SHBG concentrations significantly increased during all types of COC use [MD (95% CI) 99.08 nmol/l (86.43, 111.73); P < 0.001]. Subgroup analyses revealed that COCs containing 20-25 µg EE had similar effects on total and free T compared with COCs with 30-35 µg EE. In addition, suppressive effects on T levels were not different when comparing different types of progestins. However, subgroup analyses for the estrogen dose and the progestin type in relation to changes in SHBG levels did show significant differences: COCs containing second generation progestins and/or the lower estrogen doses (20-25 µg EE) were found to have less impact on SHBG concentrations., Conclusions: The current literature review and meta-analysis demonstrates that COCs decrease circulating levels of total T and free T and increase SBHG concentrations. Due to the SHBG increase, free T levels decrease twice as much as total T. The estrogen dose and progestin type of the COC do not influence the decline of total and free T, but both affect SHBG. The clinical implications of suppressed androgen levels during COC use remain to be elucidated.

Introduction: The objective of this study was to evaluate the performance of first-trimester Down syndrome (DS) screening with serum sampling at different weeks of gestation., Material and Methods: We studied 35,431 singleton pregnancies (2005-2011), including 145 DS cases. Screening performance was determined in different maternal age groups with serum sampling between weeks 9 + 0 and 13 + 6., Results: No significant differences were found between the detection rates at different gestational weeks. The false-positive rate (FPR) in week 9 (6%) was comparable to the FPR in week 10 (6.5%; p = 0.214), whereas it was significantly lower compared to weeks 11 (7.2%; p = 0.007), 12 (7.4%; p = 0.003) and 13 (8.5%; p < 0.001). The odds of receiving a false-positive result was significantly increased with serum sampling in week 11 (OR 1.32, 95% CI 1.08-1.63; p = 0.008) for women ≥36 years and from week 12 onwards (OR 1.28, 95% CI 1.01-1.61; p = 0.04) for women <36 years. There were no differences in mean log10 multiple of the median values of pregnancy-associated plasma protein-A, free β-human chorionic gonadotrophin or nuchal translucency between both age groups, nor in mean maternal age between the different gestational weeks in either age group., Discussion: Early serum sampling (<11 weeks) resulted in higher screening performance. The impact of the increase in the FPR was highest in women ≥36 years.

Reference values of PTH depend on vitamin D status of the reference population. This is often not described in package inserts. The aim of the present study was therefore to calculate assay specific PTH reference levels in EDTA plasma for the Architect (Abbott) in relation to 25-hydroxyvitamin D (25OHD) levels. The relation between PTH levels, 25OHD, BMI, age, gender and kidney function was determined in a cohort of older individuals from the Longitudinal Aging Study Amsterdam (LASA, n = 738, age 55-65 years) and in a cohort of healthy individuals from the Netherlands Study of Depression and Anxiety (NESDA, n = 633, 18-65 years). The LASA cohort is a representative sample of the Dutch older population. As expected, PTH reference values were significantly lower in 25OHD sufficient subjects (25OHD>50 nmol/L) than in 25OHD deficient and insufficient subjects. The 97.5th percentile of PTH in 25OHD sufficient subjects was 10 pmol/L (94.3 pg/mL), which was higher than the upper limit stated by the manufacturer (7.2 pmol/L or 68.3 pg/mL). The relation between vitamin D and PTH was independent of age, gender, BMI and kidney function. In conclusion, we have shown that it is important to establish PTH reference values in a local reference population taking 25OHD status into account., (© 2013.)

Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation.

Objective: We assessed whether preclinical Alzheimer disease (AD) based on CSF biomarkers at baseline predicts decline in cognitive functioning as measured by repeated neuropsychological tests for 4 cognitive domains in patients with subjective complaints., Methods: We included 132 patients with subjective complaints from our memory clinic-based Amsterdam Dementia Cohort, who underwent lumbar puncture and had repeated (range 2-7) neuropsychological evaluations. Follow-up was 2 ± 1 years. CSF biomarkers amyloid-β (Aβ42), total tau (Tau), and hyperphosphorylated tau-181 were used to define National Institute on Aging-Alzheimer's Association (NIA-AA) preclinical AD stages. Predictive value of preclinical AD stages as defined by CSF biomarkers, individual biomarkers, and Aβ42/tau ratio was assessed using linear mixed models. Outcome measures were compound z scores for memory, attention, executive functioning, language, and global cognition. Analyses were adjusted for age, sex, and education., Results: Patients were 61 ± 8 years old; 56 (42%) were women. Average baseline Mini-Mental State Examination score was 28.3 ± 1.5. Patients who fulfilled criteria for preclinical AD (stage 1: n = 11 + stage 2: n = 10) showed decline over time in memory (β ± SE -0.41 ± 0.14, p < 0.01), executive functions (-0.21 ± 0.08, p < 0.01), and global cognition (-0.29 ± 0.10, p < 0.01). There were no differences in cognitive decline between NIA-AA preclinical AD stages 1 and 2. In patients with normal CSF biomarkers, we observed memory improvement (0.19 ± 0.07, p < 0.01) and stable performance in all other domains., Conclusions: CSF evidence of preclinical AD in patients with subjective complaints predicted cognitive decline over time, encompassing more than memory alone. Executive functioning and global cognitive functioning also deteriorated. On the other hand, 2-year prognosis for patients without evidence of AD pathophysiology was good.

Background: Dehydroepiandrosterone sulphate (DHEAS) is an important marker of the adrenal gland. Its measurement is required in several adrenal diseases, such as adrenal tumours, adrenal insufficiency and congenital adrenal hyperplasia. Most clinical laboratories measure DHEAS using commercially available immunoassays. The aim of the present study was to investigate the accuracy of currently available DHEAS methods., Methods: Seven commercially available DHEAS assays were compared to Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) by measuring 75 serum samples (concentration range 0.06-20.6 μmol/L measured by ID-LC-MS/MS) with each method. Moreover, recovery and linearity experiments were performed. Data from our present study were compared to DHEAS data of the Dutch, German and British External Quality Assessment Schemes (EQAS's)., Results: Three methods agreed well with ID-LC-MS/MS (R between 0.93 and 0.99 and slopes ranging from 0.92 to 1.07) and showed good recoveries. Four methods showed standardization problems (slopes were 0.84, 1.14, 1.20 and 1.28). Linearity was good in all methods. Intra-assay coefficient of variation was 4.1% using ID-LC-MS/MS and below 5.5% in immunometric methods; one assay had an unacceptably high intra-assay coefficient of variation of 18%. Our data are in agreement with data obtained in three EQAS's., Conclusion: Some of the commercially available DHEAS methods show standardization problems and/or a high imprecision. These problems may potentially have clinically adverse consequences. We advise the manufacturers to improve their assays and laboratory specialists to scrutinize the DHEAS method they employ., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The need to recognize Alzheimer's disease (AD) as early as possible led us to evaluate the predictive value of amyloid β(1-42) (Aβ42), total tau (tau), and phosphorylated tau (ptau) in cerebrospinal fluid (CSF) for clinical progression in patients with subjective complaints., Methods: We recruited nondemented patients with subjective complaints (i.e., criteria for mild cognitive impairment [MCI] not fulfilled) from our memory clinic. We assessed the predictive value of CSF Aβ42, tau, and ptau for clinical progression using Cox proportional hazards models adjusted for age, gender, and baseline findings on the Mini-Mental State Examination (MMSE). Clinical progression was defined as progression to MCI or AD., Results: We included 127 patients with subjective complaints (age 60 ± 10 years, 61 [48%] females, MMSE 29 ± 1). At baseline, Aβ42 and tau were abnormal in 20 patients (both 16%), and ptau in 32 patients (25%). Thirteen patients (10%) progressed to MCI (n = 11) or AD (n = 2). Aβ42 was the strongest predictor of progression to MCI or AD with an adjusted hazard ratio (HR) of 16.0 (3.8-66.4). The adjusted HR associated with tau was 2.8 (0.9-9.2) and with ptau 2.6 (0.8-8.2). Combinations of biomarkers had a lower predictive value than Aβ42 alone., Conclusion: Low Aβ42 was the strongest predictor of clinical progression in patients with subjective complaints. These results are in line with the hypothesis that the cascade of pathologic events starts with deposition of Aβ42, whereas neuronal degeneration and hyperphosphorylation of tau are more downstream events, closer to clinical manifestation of AD., (Copyright © 2013 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

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.

Background: α-Klotho is a protein mainly produced in the kidney. Its circulating form has been suggested to link renal damage and distant tissue pathology. As three assays to measure α-Klotho became commercially available, we performed an evaluation of these commercially available Klotho assays., Methods: We studied within-run variation, between-run variation, matrix effects, linearity, and recovery of added recombinant human Klotho in the α-Klotho assays of IBL (IBL International GmbH, Hamburg, Germany), Cusabio (Cusabio Biotech, Wuhan, China) and USCN (USCN life Science, Inc., Wuhan, China) using both serum and ethylenediaminetetraacetic acid plasma., Results: Within run variation was 4, 13 and 32% for the IBL, Cusabio and USCN assay, respectively. Agreement between serum and EDTA plasma was good in the IBL assay, but poor in the USCN and Cusabio assays however improved after modifications in the Cusabio assay. Standardization and agreement between assays was poor., Conclusions: The commercially available methods for the measurement of α-Klotho differ in quality. Some of the manufacturers should improve their assays in order to produce accurate results so that reliable conclusions can be drawn from studies in which these assays are used.

The objective of this study was to quantitatively assess the relationship between apolipoprotein (APOE) genotype and electroencephalographic oscillatory brain dynamics in Alzheimer's disease (AD) patients and control subjects and its regional distribution. We obtained resting-state electroencephalographs of 320 AD patients and 246 control subjects, categorized into APOE ε4 carriers and noncarriers. Peak frequency and relative power in 4 different frequency bands were calculated. We tested the associations between APOE genotype and relative power in 4 brain regions. Peak frequency was comparable in APOE ε4 carrying and noncarrying control subjects, but lower in APOE ε4 noncarrying AD patients. In control subjects, APOE ε4 carriers had a different regional distribution of alpha power than noncarriers. We found no APOE effect in beta, delta, and theta bands. In AD, APOE ε4 noncarriers had lower alpha and higher delta power than carriers. This difference was most pronounced in the parieto-occipital region. In the theta band, APOE ε4 noncarriers had a different regional distribution of power compared with carriers. In conclusion, the most pronounced effect of genotype was seen in AD patients, and APOE ε4 noncarriers showed slower activity, especially in parieto-occipital regions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to compare screening performance for Down syndrome of the absolute risk (AR) method to the first trimester combined test (FCT) at different maternal ages., Methods: There was a retrospective analysis of 32,448 FCT. AR was defined as final risk divided by maternal age risk., Results: The likelihood of receiving a true prediction was comparable between both methods in all age groups. With the AR method, two extra Down syndrome cases were detected in women <30 years, three cases were missed in women ≥36 years, and the likelihood of receiving a false prediction decreased overall (OR 0.82, CI 0.77-0.87; P < 0.0001), in women aged 36-40 years (0.45, CI 0.41-0.51; P < 0.0001), in women aged 41-45 years (0.18, CI 0.13-0.26; P < 0.0001) and increased in women aged ≤25 years (2.12, CI 1.52-2.96; P < 0.004)., Conclusions: The AR method results in a significant decreased likelihood of receiving a false prediction with a comparable likelihood of receiving a true prediction. Thus, fewer invasive diagnostic tests will be performed. It will take away the misunderstanding about differences in screening performance for women of different ages. This might lead to a higher uptake of first trimester screening resulting in a more efficient screening policy., (© 2013 John Wiley & Sons, Ltd.)

Background: The aim of the study was to assess the screening performance of first trimester maternal serum measurements of A-disintegrin-and-metalloprotease 12-s (ADAM12s) and placental protein 13 (PP13) for preeclampsia (PE), gestational hypertension (GH) and small-for-gestational-age (SGA) fetuses., Methods: In this retrospective case-control study 220 pregnant women were matched for gestational and maternal age at sampling. Results were expressed as multiples of the median (MoM) and compared using Kruskal-Wallis and Mann-Whitney U-test. Screening performance was assessed by receiver operator characteristics (ROC) curves and area under the curve (AUC)., Results: Seventeen cases of PE, 30 cases of GH and eight cases of SGA fetuses were matched with 165 controls. ROC-analysis yielded AUCs for ADAM12s and PP13 of 0.63 and 0.59 for PE, 0.68 and 0.57 for GH and 0.59 and 0.62 for SGA, respectively. Combined ADAM12 and PP13 did not improve the AUC value. When the specificity was set at 80%, corresponding detection rate of ADAM12s was 52% for GH., Conclusions: Combined ADAM12s and PP13 measurements do not predict adverse pregnancy outcome, but decreased first trimester ADAM12s levels are associated with GH.

Objective: In this study we investigated the relationships between cerebrospinal fluid (CSF) biomarkers (tau and amyloid-β1-42 [Aβ1-42]) and cognition or behavior in patients with frontotemporal dementia (the behavioral variant, bvFTD)., Methods: We included 58 patients with bvFTD. All patients underwent a neuropsychological assessment and lumbar puncture. Relationships between CSF biomarkers and cognition or behavior were assessed with linear regression analysis., Results: After correction for age, sex, and education, CSF tau levels were found to be negatively related to the Visual Association Test (standardized β = -0.3, P < .05), whereas CSF Aβ1-42 levels were found to be positively related to the Mini-Mental State Examination (β = 0.3, P < .05), the frontal assessment battery (β = 0.5, P < .05), and digit span backwards test (β = 0.3, P = .01). We did not find relations between CSF biomarkers and behavior (measured by the neuropsychiatric inventory). After excluding all patients with a CSF biomarker profile often seen in Alzheimer's disease (high levels of tau and low levels of Aβ1-42), we still found relations between CSF Aβ1-42 levels and Visual Association Test object naming (β = 0.4, P < .05), as well as between CSF Aβ1-42 levels and the frontal assessment battery (β = 0.5, P < .05, but there was no relation between CSF tau and cognition., Conclusion: Low CSF Aβ1-42 levels are associated with worse general cognitive function and worse executive function in patients with bvFTD. Our results provide circumstantial evidence for a pathophysiological role of Aβ1-42 in bvFTD., (Copyright © 2013 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Background: Multiplex assays such as xMAP have been proposed for the assessment of Alzheimer's disease (AD) biomarkers amyloid β 42 (Aβ42), tau (Tau), and phosphorylated tau (pTau) in cerebrospinal fluid (CSF). Here, we compared the traditional enzyme-linked immunosorbent assay (ELISA) and xMAP with respect to their: (1) absolute biomarker concentration, (2) ability to distinguish AD from nondemented subjects, (3) ability to monitor AD longitudinally, and (4) ability to predict progression from mild cognitive impairment (MCI) to AD., Methods: We selected 68 AD, 62 MCI, and 24 nondemented subjects, performed clinical examinations, and obtained CSF at baseline and 2 years later. Aβ42, Tau, and pTau were measured with both ELISA and xMAP., Results: Biomarker levels differed considerably between the two assays, and the differences were concentration dependent. No differences were observed in ability to distinguish nondemented subjects from AD patients between ELISA (area under curve of 0.84 for Aβ42, 0.79 for Tau, and 0.75 for pTau) and xMAP (area under curve of 0.82 for Aβ42, 0.75 for Tau, and 0.73 for pTau), all P < .05. Increased Aβ42 levels of AD patients at follow-up compared with baseline were detected with ELISA, whereas increased Tau levels for nondemented subjects and MCI patients were only detected with xMAP. The hazard ratios for progression from MCI to AD did not differ between the assays., Conclusion: Both ELISA and multiplex assays can be used to measure AD biomarker levels in CSF to support clinical diagnosis and predict progression from MCI to AD with similar accuracy. Importantly, the assays' output in absolute biomarker concentrations is remarkably different, and this discrepancy cannot be reconciled with simple correction factors., (Copyright © 2013 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Background: We aimed to develop a prediction model based on cerebrospinal fluid (CSF) biomarkers, that would yield a single estimate representing the probability that dementia in a memory clinic patient is due to Alzheimer's disease (AD)., Methods: All patients suspected of dementia in whom the CSF biomarkers had been analyzed were selected from a memory clinic database. Clinical diagnosis was AD (n = 272) or non-AD (n = 289). The prediction model was developed with logistic regression analysis and included CSF amyloid β42, CSF phosphorylated tau181, and sex. Validation was performed on an independent data set from another memory clinic, containing 334 AD and 157 non-AD patients., Results: The prediction model estimated the probability that AD is present as follows: p(AD) = 1/(1 + e (- [-0.3315 + score])), where score is calculated from -1.9486 × ln(amyloid β42) + 2.7915 × ln(phosphorylated tau181) + 0.9178 × sex (male = 0, female = 1). When applied to the validation data set, the discriminative ability of the model was very good (area under the receiver operating characteristic curve: 0.85). The agreement between the probability of AD predicted by the model and the observed frequency of AD diagnoses was very good after taking into account the difference in AD prevalence between the two memory clinics., Conclusions: We developed a prediction model that can accurately predict the probability of AD in a memory clinic population suspected of dementia based on CSF amyloid β42, CSF phosphorylated tau181, and sex., (Copyright © 2013 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

In this longitudinal study we investigated the effect of apolipoprotein E (APOE) genotype on the relation between cognitive decline and cerebrospinal fluid (CSF) F2-isoprostanes, the reference marker for oxidative stress. Twenty non-demented subjects, 58 mild cognitive impairment (MCI) patients, and 63 Alzheimer's disease (AD) patients with measurements of CSF F2-isoprostanes at two time points (with a mean interval of 2.0 ± 1.1 years) and known APOE genotype were included. Mean clinical follow-up time was 3.9 ± 2.4 years. For change in F2-isoprostanes over time and associations with Mini-Mental State Examination scores, age- and gender-adjusted linear mixed models were used. Analyses were done for APOE ε4 carriers and non-carriers separately. In APOE ε4 carriers, annual change in F2-isoprostane levels appeared larger than in APOE ε4 non-carriers (β[SE] 2.5[0.5], p < 0.001 versus 1.8[0.5], p < 0.01). In addition, increase in F2-isoprostanes was associated with further cognitive decline in APOE ε4 carriers (p < 0.05), but not in non-carriers (p = 0.28). Our results reiterate the importance of oxidative stress in neurodegeneration, especially in APOE ε4 carrying patients. Future studies should focus on the possibility of increased vulnerability to oxidative damage in APOE ε4 carriers.

Introduction: In our hospital, female-to-male (FtM) transgender adolescents from the age of 16 are treated with two- or four-weekly intra-muscular injections of testosterone-esters. Some patients treated with four-weekly injections have complaints of fatigue and experience mood swings towards the end of the inter-injection period, which calls for an evaluation of the time-course of testosterone levels between injections. Evaluation of salivary testosterone is a practical approach for sequential measurements. Since only ∼2% of total serum testosterone is present in saliva, a sensitive assay is necessary. The objective was to develop an isotope dilution-liquid chromatography-tandem mass spectrometry method (ID-LC-MS/MS) for salivary testosterone measurements and to evaluate the testosterone profiles after testosterone-ester mixture injections in FtM-adolescents., Experimental: FtM treated with 125 mg/2 weeks or with 250 mg/4 weeks depots of testosterone-ester mixture collected saliva at different time intervals. Salivary testosterone was measured by a thoroughly validated ID-LC-MS/MS assay., Results: An ID-LC-MS/MS method for measuring salivary testosterone was developed with adequate accuracy and specificity. The reference range was established at 135-400 pmol/L. Testosterone levels peaked supra-physiologically immediately post-injection, and decreased to levels within the male reference range after nine days in all patients. 250 mg/4 weeks depots resulted in values below the reference range at the end of the 4 weeks., Discussion: The development of an adequate ID-LC-MS/MS method for measuring salivary testosterone allowed us to investigate the testosterone profile in FtM-adolescents after testosterone-esters mixture injections. These injections lead to extreme concentrations which may affect the wellbeing of the patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low concentrations confounds this issue. Therefore, our objective was to assess daily testosterone fluctuation during the menstrual cycle by a thoroughly validated isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method and to evaluate whether an ARCHITECT® 2nd Generation Testosterone fully automated immunoassay is equally suited for this purpose., Methods: Testosterone was measured in serum obtained daily during the menstrual cycle of 25 healthy women, characterized by biochemical and physical examination., Results: Performance of the ID-LC-MS/MS method was concordant with a published reference method (y=1.007x-0.056 nmol/L; r=0.9998). Comparison of the immunoassay to ID-LC-MS/MS yielded y=1.095x+0.104 nmol/L (r=0.9031). Overall, testosterone concentrations were higher mid-cycle, but a peak was not discernible in each individual. Apart from a persistent positive bias, the immunoassay measured the same testosterone profiles as the ID-LC-MS/MS method. The reference interval in women was 0.30-1.69 nmol/L (8.7-48.7 ng/dL) for ID-LC-MS/MS and 0.50-2.00 nmol/L (14.4-57.7 ng/dL) for the immunoassay., Conclusion: The elevation of mid-cycle testosterone concentrations is statistically significant, although not clinically relevant since day-to-day variation is higher and independent of the menstrual cycle. In this light, a single testosterone measurement might not be reflective of the overall testosterone status in an individual. Measurements obtained using the 2nd generation immunoassay gave comparable results across the menstrual cycle., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The intake of the n-3 fatty acids alpha-linolenic acid (ALA), acid (EPA) and docosahexaenoic acid (DHA) has been related to testosterone levels in epidemiological analyses. The aim of this study was to assess whether the n-3 fatty acids affects testosterone levels in post-myocardial infarction (MI) patients, who are at risk of testosterone deficiency. In a double-blind, placebo-controlled trial of low-dose supplementation of n-3 fatty acids, we included 1850 male post-MI patients aged 60-80 years who participated in the Alpha Omega Trial. Patients were randomly allocated to margarines that provided 400 mg/day of EPA-DHA (n = 453), 2 mg/day of ALA (n = 467), EPA-DHA plus ALA (n = 458), or placebo (n = 472). Serum testosterone levels were assessed at baseline and after 41 months using whole day blood samples obtained at the subjects' home or at the hospital. Subjects were on average age of 68.4 (SD 5.3) years old and had baseline mean serum total testosterone of 14.8 (SD 5.6) nmol/L. The four randomized groups did not differ for baseline characteristics. ALA, EPA-DHA, and EPA-DHA plus ALA supplementation did not affect serum total testosterone compared to placebo. Moreover, n-3 fatty acid supplementation did not affect the risk of incident testosterone deficiency (n = 76 with total testosterone <8.0 nmol/L). We conclude that n-3 fatty acids supplementation did not affect serum total testosterone in men who had had a MI., (© 2012 The Authors. International Journal of Andrology © 2012 European Academy of Andrology.)

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid β42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid β42.

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 Alzheimer's 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cerebral microbleeds (MBs) may relate to amyloid in dementia. We selected 26 probable Alzheimer's disease (AD) patients with MBs, 26 age- and sex-matched AD patients without MBs, 11 vascular dementia (VaD) patients, and 22 patients with subjective complaints. We measured amyloid beta 1-42 (Aβ42) and 1-40 (Aβ40) in cerebrospinal fluid (CSF) and plasma, and blood-brain barrier (BBB) function using albumin ratios. CSF Aβ42 was lowest in AD with MBs, whereas Aβ40 was selectively decreased in VaD. In plasma, amyloid-beta was nonsignificantly elevated in VaD compared with controls. Higher albumin ratios in VaD suggested blood-brain barrier dysfunction. A MB pattern suggestive of cerebral amyloid angiopathy (CAA) related to lower CSF Aβ42, while a non-cerebral amyloid angiopathy specific MB distribution related to higher plasma Aβ40. Amyloid-beta is differentially implicated in AD with MBs and VaD. MB distribution related to different amyloid profiles, supporting distinct etiologies. Our results suggest that Aβ42 is retained in cerebrovasculature of AD patients with MBs, while in contrast, VaD patients may possibly drain amyloid., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC., Experimental Design: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc(15lox/+) mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA., Results: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA., Conclusion: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions., (©2012 AACR.)

Pro and anti-inflammatory cytokines are involved in disease onset and pathophysiology of multiple sclerosis, Alzheimer's disease and Parkinson's disease. It is likely that panels of multiple cytokines provide a good reflection of disease status and can be used as biological markers in body fluids. Different multi-plex platforms, Luminex-xMAP and Meso Scale Discovery, are able to detect multiple analytes in the same sample at the same time. In this literature based review, we offer an overview of the multi-plex platforms and compare them with the golden standard ELISA in their ability to accurately and sensitively detect cytokines in cerebrospinal fluid (CSF) and blood (serum/plasma). The detectability and levels of cytokines in multiple sclerosis, Alzheimer's disease and Parkinson's disease are promising but also show discrepancies between studies. The current immuno-assays lack sensitivity for detection of various cytokines that have low concentrations of cytokines in CSF and blood, and therefore technical improvements are needed. With such improvements the use of large panels of cytokines as inflammatory profiles may offer additional value in diagnosis, prognosis and therapeutic response in neurodegenerative diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Recent recognition of its broad pathophysiological importance has triggered an increased interest in 25-hydroxyvitamin D [25(OH)D]. By consequence, throughput in 25(OH)D testing has become an issue for clinical laboratories, and several automated assays for measurement of 25(OH)D are now available. The aim of this study was to test the accuracy and robustness of these assays by comparing their results to those of an isotope dilution/online solid-phase extraction liquid chromatography/tandem mass spectrometry (ID-XLC-MS/MS) method. We put specific focus on the influence of vitamin D-binding protein (DBP) by using samples with various concentrations of DBP., Methods: We used 5 automated assays (Architect, Centaur, iSYS, Liaison, and Elecsys), 1 RIA (Diasorin) preceded by extraction, and an ID-XLC-MS/MS method to measure 25(OH)D concentrations in plasma samples of 51 healthy individuals, 52 pregnant women, 50 hemodialysis patients, and 50 intensive care patients. Using ELISA, we also measured DBP concentrations in these samples., Results: Most of the examined 25(OH)D assays showed significant deviations in 25(OH)D concentrations from those of the ID-XLC-MS/MS method. As expected, DBP concentrations were higher in samples of pregnant women and lower in samples of IC patients compared to healthy controls. In 4 of the 5 fully automated 25(OH)D assays, we observed an inverse relationship between DBP concentrations and deviations from the ID-XLC-MS/MS results., Conclusions: 25(OH)D measurements performed with most immunoassays suffer from inaccuracies that are DBP concentration dependent. Therefore, when interpreting results of 25(OH)D measurements, careful consideration of the measurement method is necessary.

Objective: To determine how amyloid β 42 (Aβ42), total tau (t-tau), and phosphorylated tau (p-tau) levels in CSF behave in a large cohort of patients with different types of dementia., Methods: Baseline CSF was collected from 512 patients with Alzheimer disease (AD) and 272 patients with other types of dementia (OD), 135 patients with a psychiatric disorder (PSY), and 275 patients with subjective memory complaints (SMC). Aβ42, t-tau, and p-tau (at amino acid 181) were measured in CSF by ELISA. Autopsy was obtained in a subgroup of 17 patients., Results: A correct classification of patients with AD (92%) and patients with OD (66%) was accomplished when CSF Aβ42 and p-tau were combined. Patients with progressive supranuclear palsy had normal CSF biomarker values in 90%. Patients with Creutzfeldt-Jakob disease demonstrated an extremely high CSF t-tau at a relatively normal CSF p-tau. CSF AD biomarker profile was seen in 47% of patients with dementia with Lewy bodies (DLB), 38% in corticobasal degeneration (CBD), and almost 30% in frontotemporal lobar degeneration (FTLD) and vascular dementia (VaD). PSY and SMC patients had normal CSF biomarkers in 91% and 88%. Older patients are more likely to have a CSF AD profile. Concordance between clinical and neuropathologic diagnosis was 85%. CSF markers reflected neuropathology in 94%., Conclusion: CSF Aβ42, t-tau, and p-tau are useful in differential dementia diagnosis. However, in DLB, FTLD, VaD, and CBD, a substantial group exhibit a CSF AD biomarker profile, which requires more autopsy confirmation in the future.

Background: A new ARCHITECT® alpha fetoprotein (AFP) assay was developed to improve the linearity at the upper end of the calibration curve and to enhance other performance characteristics. In addition, this reformulation eliminated the possibility of falsely depressed samples at high AFP concentrations. The purpose of this study was to evaluate its analytical performance at multiple sites., Methods: The assay configuration, the diluent formulation, and the manufacturing process were redesigned. Analytical performance was evaluated at Abbott Laboratories, Sapporo Medical University, VU University Medical Center, and Johns Hopkins University., Results: The limit of quantitation of the assay was 1.00-1.30 ng/mL. Total precision (%CV) across the assay range varied between 1.41 and 3.52. The assay was linear from 1.19 to 2535 ng/mL, and the range of the assay was expanded from 200 ng/mL to 2000 ng/mL. Comparison of this assay with the on-market ARCHITECT, AxSYM, ADVIA Centaur, DxI, AIA-1800, and E 170 systems yielded regression slopes of 0.91-1.08 and correlation coefficients of =0.99 for serum samples. No falsely depressed results were observed in 174 serum samples with AFP concentrations of 2018-1,196,856 ng/mL and in a spiked sample containing up to 10 mg/mL of purified AFP., Conclusions: The new AFP assay has improved an issue of the on-market ARCHITECT AFP assay and demonstrated excellent assay performance.

Androgen deprivation therapy (ADT) and 5-α-reductase (5AR) inhibition are used in the treatment of men with advanced or metastatic prostate cancer and benign prostatic hyperplasia (BPH), respectively. These drugs exert their effect by lowering androgen levels in the serum and allegedly, the prostate gland. It is, however, unknown whether (increased) intraprostatic androgen levels are associated with the pathogenesis of BPH and with the initiation and progression of prostate cancer. Also, it is unclear whether intraprostatic dihydrotestosterone (DHT) levels correlate with a response to initial hormonal therapy or with patient outcome. These uncertainties have resulted from the finding that serum testosterone levels do not necessarily reflect those in the prostate gland. Intraprostatic DHT levels of men being treated with 5AR inhibition, of those treated with ADT for hormone-naive prostate cancer, and of those with castration-resistant prostate cancer are all altered in an equivalent manner because of hormonal manipulation. Increased knowledge of the mechanisms of the androgenic steroid pathways in prostatic diseases, with a special focus on intraprostatic androgen levels, may lead to treatment that is tailored to the needs of the individual patient, and probably to new therapeutic targets as well., (© 2011 THE AUTHORS. BJU INTERNATIONAL © 2011 BJU INTERNATIONAL.)

Owing to inconsistencies and methodological differences, the present peer-reviewed literature lacks conclusive data on the intraprostatic levels of androgens, in particular dihydrotestosterone (DHT), in untreated benign prostatic hyperplasia (BPH) and prostate cancer. To date, no difference has been shown between DHT concentrations in normal prostatic tissue and BPH, and nor has a difference been shown in DHT concentrations between the histologically distinct regions of the prostate. Recent literature has also failed to show a consistent difference in androgen level between BPH and prostate cancer. The role of intraprostatic DHT in the pathogenesis of BPH and in the initiation and progression of prostate cancer thus remains to be established. Increased knowledge of the mechanisms of the androgenic steroid pathways in prostatic diseases, with a special focus on intraprostatic androgen levels may lead to more optimized and more personalized forms of treatment, and probably new therapeutic targets as well., (© 2011 THE AUTHORS. BJU INTERNATIONAL © 2011 BJU INTERNATIONAL.)

Astrocytes appear to be important mediators in the clearance of amyloid beta1-42 (Aβ), the key component of senile plaques characteristic of Alzheimer's disease (AD). Recently, we found the amyloid associated proteins (AAPs) α1-antichymotrypsin (ACT), apolipoprotein J and E (ApoJ and ApoE) and a mixture of serum amyloid P (SAP) and C1q (SAP-C1q) to modify Aβ-uptake by human astrocytes. Here we investigated the effect of oligomeric (Aβoligo) and fibrillar Aβ (Aβfib), alone and in combination with a panel of AAPs on the astrocytic expression of genes proposed to be involved in Aβ-uptake and degradation. Primary human astrocytes (isolated from non-demented control (n=4) and AD patient (n=4) brain specimens) were exposed to either Aβoligo or Aβfib preparations with or without the above mentioned AAPs. Quantitative gene expression analysis of Aβ-receptors Scavenger receptor B1 (SCARB1), macrophage receptor with collagenous structure (MARCO) and low density lipoprotein receptor related protein-2 (LRP2 or megalin) as well as of Aβ-degrading enzymes neprilysin (NEP), insulin-degrading enzyme (IDE) and metalloproteinase-9 (MMP-9) was performed by real-time PCR. Basal expression of NEP, IDE and SCARB1 was easily detected whereas expression of MARCO, LRP2 and MMP-9 could only be detected upon pre-amplification. Basal expression of NEP, IDE and SCARB1 did not change upon exposure to Aβoligo or Aβfib alone in any of the investigated astrocyte cultures. Interestingly NEP expression was increased upon exposure to ApoE in combination with both Aβ-preparations, and also SCARB1 expression was induced upon treatment with ApoE in combination with Aβfib in astrocytes from non-demented controls. Further, SAP-C1q increased SCARB1 expression in control astrocytes when combined with Aβoligo. These alterations were not found in astrocytes from AD patients. Thus, we conclude that Aβ alone apparently does not affect the astrocytic expression of IDE, NEP or SCARB1. However, NEP and SCARB1 expression is increased in astrocytes from non-demented subjects when exposed to Aβ combined with AAPs like ApoE. These astrocytic gene expression-regulatory mechanisms appear to be defective in AD and thus might contribute to the development and progression of AD pathology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To evaluate the performance of the first-trimester combined test (FCT) in different maternal age groups and to discuss whether adjustments in screening policies should be made., Methods: In this retrospective study data (n = 26 274) from a fetal medicine center on FCT (maternal age, fetal NT, free β-human chorionic gonadotrophin, pregnancy-associated plasma protein-A) were studied., Results: 70.6% of cases was <36 years and 43% of the Down syndrome (DS) cases were detected in this age group. For women <36 years and advanced maternal age (AMA) women (≥36 years) detection rate (DR) and false positive rate (FPR) were 94.5% and 4.1%, and 95.8% and 13.0%, respectively (cut-off 1:200). Lowering the cut-off showed an improved balance in DR and FPR. With increasing maternal age FPR and DR increased and odds of being affected given a positive result (OAPR) decreased., Conclusion: FCT is effective in women <36 and ≥36 years. The balance between FPR and DR is more favourable in women <36 years with comparable OAPR. Although FPR increases with increasing maternal age, performance of FCT in AMA women is more effective than screening based on maternal age alone. Lowering the cut-off to 1:100 in AMA women is suggested to improve screening performance. Routinely offering diagnostic testing to AMA women as a screening policy for the detection of DS seems not reasonable., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: To assess first trimester placental growth factor (PlGF) and pregnancy-associated plasma protein-A (PAPP-A) as screening markers for early-onset pre-eclampsia (PE) and intra-uterine growth restriction (IUGR)., Methods: PlGF concentration was retrospectively measured in first trimester serum specimens of 23 cases of early-onset PE (<34 weeks), 26 cases of IUGR (birth weight < 5th centile) and 5 controls per case. Levels were adjusted for gestational age (GA), ethnicity and smoking to obtain multiples of the expected median (MoM). Logistic regression was used to assess PlGF, PAPP-A and maternal characteristics as potential predictors of early-onset PE and IUGR., Results: PlGF MoM levels were significantly lower in the early-onset PE group (P < 0.0001) compared with controls, but not in the IUGR group. PAPP-A MoM levels were significantly lower in the IUGR group (P < 0.01) compared with controls but not in the early-onset PE group. PlGF significantly improved the ability of systolic blood pressure at the first prenatal visit to predict early-onset PE [achieving a receiver-operating characteristics curve with area under the curve (AUC) of 0.8]. Combining systolic blood pressure at the first prenatal visit and PlGF did not significantly improve the predictive ability compared with PlGF alone (AUC = 0.83)., Conclusion: Serum PlGF is an acceptable marker in first trimester screening for early-onset PE, but a poor marker in screening for IUGR. Screening performance of serum PAPP-A is poor for both early-onset PE and IUGR., (Copyright © 2011 John Wiley & Sons, Ltd.)

Background: A recent study reported that an 18-analyte multiplexed plasma panel of signaling proteins differentiated Alzheimer's disease (AD) from controls. This study measured mRNA expression for nine of these promising bio-markers in 23 AD patients and 23 age- and sex-matched controls., Methods: Total RNA was isolated from PaxGene RNA tubes. Relative mRNA expression levels of CCL5 [RANTES], CSF1, ICAM1, IGFBP6, IL1A, IL3, IL8, PDGFB and TNF were determined by Q-RT-PCR, with GAPDH as housekeeping gene., Results: A panel of five markers (CCL5, CSF1, ICAM1, IL8, TNF) with detectable expression levels in all individuals differed between AD patients and controls (p interaction <0.10). Especially, the relative expression level of CCL5 was lower in AD patients than in controls (p<0.005). Across groups, levels of both CCL5 and TNF were correlated to CSF levels of τ (r=0.39, r=0.32), pτ-181 (r=0.38, r=0.33), and MMSE (r=-0.31, r=-0.33, all p<0.05)., Conclusions: The measured panel, and especially CCL5, could aid in the differentiation of AD from controls.

Objective: To study CSF biomarkers amyloid-beta 1-42 (Aβ42) and total tau (tau) in relation to APOE genotype in their ability to predict progression from mild cognitive impairment (MCI) to Alzheimer's disease (AD)., Methods: In 100 MCI patients CSF Aβ42, tau and APOE genotype were determined. At follow-up of 18 (13-24) months 58 patients remained non-progressive and 42 progressed to AD., Results: Cox proportional hazards models showed an interaction between Aβ42 and APOE genotype (p<0.05). Stratification for APOE revealed HR (95% CI) for abnormal Aβ42 of 8.2 (2.1-31.9) for ε4 non-carriers, 3.9 (0.8-18.5) for heterozygotes and 0.3 (0.0-1.7) for homozygotes. Inversely, stratification for Aβ42 revealed that in patients with normal levels of Aβ42, ε4 homozygotes had a strongly increased risk of progression to AD with HR (95% CI) 20.8 (2.4-182.8). Tau and APOE independently predicted progression to AD., Conclusions: Aβ42 was a stronger predictor of progression to AD in APOE ε4 non-carriers than in carriers. Furthermore, the risk of progression for ε4 homozygotes was very high, also in patients with normal levels of Aβ42., (Copyright © 2009 Elsevier Inc. All rights reserved.)