Testosterone, androstenedione, cortisol and cortisone levels in human unstimulated, stimulated and parotid saliva.

BACKGROUND: Recently, measurements of steroids like testosterone, androstenedione, cortisol and cortisone in saliva are more and more applied in diagnostics and scientific studies. This is mainly due to the simple and non-invasive collection of saliva. We aimed to evaluate the optimal way to collect saliva for steroid hormone measurement. METHODS: We investigated in twenty volunteers whether there is a difference between steroid hormone concentrations in unstimulated and stimulated saliva collected while chewing, using cotton and synthetic Salivettes®, citric acid or chewing gum. Furthermore, total unstimulated saliva was compared to parotid gland saliva. Testosterone, androstenedione, cortisol and cortisone were measured using Liquid-Chromatography Tandem Mass Spectrometry (LC-MS/MS). RESULTS: Salivary testosterone, androstenedione and cortisol concentrations were unaffected by stimulation upon mouth and tongue movements, cortisone levels were on average 16% lower. Concentrations of all hormones were lower in parotid gland saliva compared to total unstimulated saliva (on average 51%, 26%, 66% and 49% lower, for testosterone, androstenedione, cortisol and cortisone, respectively). Concentrations of testosterone as well as androstenedione were lower when using synthetic Salivettes® (58% and 41%, respectively) and were higher when using cotton Salivettes® (217% and 46%, respectively). Cortisol levels in saliva were unaffected by using Salivettes®. However, cortisol and testosterone levels were higher in with chewing gum stimulated saliva (16% and 55%, respectively). Cortisone concentrations were lower in all types of stimulations (on average 25%-35%). CONCLUSION: The way saliva is collected should be considered when analysing and interpreting salivary hormone concentrations. We advocate unstimulated saliva collection in simple polypropylene tubes for all steroid measurements.

Nutrients required for phospholipid synthesis are lower in blood and cerebrospinal fluid in mild cognitive impairment and Alzheimer's disease dementia.

INTRODUCTION: Synaptic membrane formation depends on nutrients that fuel metabolic pathways for the synthesis of constituent phospholipids. Consequently, insufficient availability of such nutrients may restrict membrane formation and contribute to synaptic dysfunction in Alzheimer's disease (AD). We assessed whether blood and cerebrospinal fluid (CSF) concentrations of nutrients related to phospholipid synthesis differ among patients with AD, mild cognitive impairment (MCI), and control subjects. METHODS: Concentrations of uridine, choline, folate, homocysteine, and other related metabolites were analyzed in paired blood and CSF samples from subjects selected from the Amsterdam Dementia Cohort with AD (n = 150; age, 66 ± 7 years; 37% female), MCI (n = 148; age, 66 ± 8 years; 37% female), and control subjects (n = 148; age, 59 ± 8 years; 38% female). RESULTS: Age- and gender-adjusted analysis of variance revealed different concentrations of circulating uridine, choline, and folate and CSF uridine, folate, and homocysteine (all P < .05) among the three diagnostic groups. Post hoc pairwise comparison showed that subjects with AD had lower CSF uridine, plasma choline and higher CSF homocysteine concentrations, whereas subjects with MCI had lower plasma and CSF uridine, serum and CSF folate, and higher CSF homocysteine concentrations compared with control subjects (all P < .05), with differences ranging from -11 to +22%. DISCUSSION: AD and MCI patients have lower levels of nutrients involved in phospholipid synthesis. The current observations warrant exploration of the application of nutritional strategies in the early stages of AD.

Comparison of multiplex platforms for cytokine assessments and their potential use for biomarker profiling in multiple sclerosis.

BACKGROUND: The levels of pro and anti-inflammatory cytokines can be altered in different autoimmune pathologies, such as multiple sclerosis (MS). It is likely that cytokines in bodily fluids can provide a good reflection of ongoing disease patho-physiology. In this study we aimed to validate multiplex cytokine platforms and evaluate whether these cytokines are differentially expressed in MS. METHODS: Assay validation for simultaneous quantification of IL-1ß, IL-6, IL-8 and TNF-α in serum and CSF were performed using both the Luminex-xMAP (Luminex) and Meso Scale Discovery (MSD) platforms. Next, the relation of the pro-inflammatory cytokine 4-plex with disease progression, symptoms and subtypes was studied in paired serum and CSF of MS patients (n=56), and compared with healthy controls (n=203), with the use of the MSD-platform. RESULTS: The MSD-platform showed overall better assay characteristics such as, sensitivity, recovery and linearity compared to the Luminex for the 4-plex cytokines in CSF and serum. IL-6, IL-8 and TNF-α (p<0.001) levels were significantly increased in MS serum compared to healthy controls. Moreover, serum IL-1ß levels correlated with expanded disability status scale (EDSS) scores (r=-0.34, p<0.05). Additionally, IL-6 and IL-8 CSF levels were both significantly decreased in MS patients compared to non-inflammatory neurological disease controls. Noteworthy, higher IL-8 CSF levels than IL-8 serum levels were observed for MS patients, indicating intrathecal activation of macrophages in MS. CONCLUSION: We have demonstrated that the pro-inflammatory 4-plex kit of the MSD-platform shows better assay characteristics in comparison with Luminex kit for quantification of these cytokines in serum and CSF. Overall, the increased levels of IL-6, IL-8 and TNF-α in serum of MS patients compared to healthy controls, support the use of multiple cytokines for future MS biomarker and disease progression research.

Effect of pubertal suppression and cross-sex hormone therapy on bone turnover markers and bone mineral apparent density (BMAD) in transgender adolescents.

Puberty is highly important for the accumulation of bone mass. Bone turnover and bone mineral density (BMD) can be affected in transgender adolescents when puberty is suppressed by gonadotropin-releasing hormone analogues (GnRHa), followed by treatment with cross-sex hormone therapy (CSHT). We aimed to investigate the effect of GnRHa and CSHT on bone turnover markers (BTMs) and bone mineral apparent density (BMAD) in transgender adolescents. Gender dysphoria was diagnosed based on diagnostic criteria according to the DSM-IV (TR). Thirty four female-to-male persons (transmen) and 22 male-to-female persons (transwomen)were included. Patients were allocated to a young (bone age of <15years in transwomen or <14 in transmen) or old group (bone age of ≥15years in transwomen or ≥14years in transmen). All were treated with GnRHa triptorelin and CSHT was added in incremental doses from the age of 16years. Transmen received testosterone esters (Sustanon, MSD) and transwomen received 17-ß estradiol. P1NP, osteocalcin, ICTP and BMD of lumbar spine (LS) and femoral neck (FN) were measured at three time points. In addition, BMAD and Z-scores were calculated. We found a decrease of P1NP and 1CTP during GnRHa treatment, indicating decreased bone turnover (young transmen 95% CI -74 to -50%, p=0.02, young transwomen 95% CI -73 to -43, p=0.008). The decrease in bone turnover upon GnRHa treatment was accompanied by an unchanged BMAD of FN and LS, whereas BMAD Z-scores of predominantly the LS decreased especially in the young transwomen. Twenty-four months after CSHT the BTMs P1NP and ICTP were even more decreased in all groups except for the old transmen. During CSHT BMAD increased and Z-scores returned towards normal, especially of the LS (young transwomen CI 95% 0.1 to 0.6, p=0.01, old transwomen 95% CI 0.3 to 0.8, p=0.04). To conclude, suppressing puberty by GnRHa leads to a decrease of BTMs in both transwomen and transmen transgender adolescents. The increase of BMAD and BMAD Z-scores predominantly in the LS as a result of treatment with CSHT is accompanied by decreasing BTM concentrations after 24months of CSHT. Therefore, the added value of evaluating BTMs seems to be limited and DXA-scans remain important in follow-up of bone health of transgender adolescents.

Reference values for salivary testosterone in adolescent boys and girls determined using Isotope-Dilution Liquid-Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS).

The measurement of testosterone in saliva is an attractive alternative to serum analysis due to the simple and non-invasive sample collection. In children and adolescents salivary testosterone is mainly measured to investigate whether puberty has started or not. This study aimed to establish reference values for salivary testosterone during puberty in boys and girls. We measured salivary testosterone using ID-LC-MS/MS in a cohort of 131 girls and 123 boys of whom each had salivary testosterone measured at two time points during puberty. Salivary testosterone concentrations start to increase with the start of puberty around eight years and continuously increase up to adult concentrations in the following ten years. Reference values were calculated using the Lambda-Mu-Sigma (LMS)-curve fitting method and provided per year from 8 to 26 years of age in boys and girls. These reference ranges may help clinicians and researchers to interpret salivary testosterone results in both individual patients and study subjects.

Comparison of eight routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione in serum.

BACKGROUND: Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC-MS/MS methods is available. We compared eight routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione. METHODS: Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC-MS/MS methods. We performed Passing-Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated. RESULTS: The concentrations ranged from 0.05-1.26 nmol/L, 6.15-24.44 nmol/L and 0.15-4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7-16.0%, 0.9-5.2% and 1.2-9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90-1.25, 0.87-1.24 and 0.94-1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods. CONCLUSION: Although most routine LC-MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved.

Determination of human reference values for serum total 1,25-dihydroxyvitamin D using an extensively validated 2D ID-UPLC-MS/MS method.

BACKGROUND: To assess a patient's vitamin D status the precursor metabolite 25-hydroxyvitamin D can be determined. However, measurement of 1,25-dihydroxyvitamin D is required when disorders of 1a-hydroxylation, extrarenal 1a-hydroxylation, or vitamin D receptor defects are suspected. METHODS: The aim of this study was to determine reference values for 1,25-dihydroxyvitamin D3 and D2 using a 2D ID-UPLC-MS/MS method. RESULTS: The LC-MS/MS method, able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D3 and D2 in human serum, was extensively validated. Intra-assay variations were <5% and 8.5% and <7.5% and 11%, for 1,25-dihydroxyvitamin D3 and D2, respectively, over the whole dynamic range (3.1-376 and 3.1-652pmol/L). Limit of quantitation was 3.4pmol/L for both compounds. Our method correlated well with a published LC-MS/MS method (r=0.87) and with the average 1,25-dihydroxyvitamin D3 results of the vitamin D External Quality Assessment Scheme (DEQAS) determined with LC-MS/MS (r=0.93). Reference ranges, determined in 96 plasma samples of healthy volunteers were 59-159pmol/L and <17pmol/L for respectively 1,25-dihydroxyvitamin D3 and D2. The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D3 concentrations with age. The presence of significantly higher average 1,25-dihydroxyvitamin D3 levels in premenopausal women taking oral contraceptive pills compared to postmenopausal women suggests that this effect is estrogen-related, as estrogens lead to a higher vitamin D binding protein. CONCLUSIONS: The major finding of the present study is a reference interval of 59-159pmol/L for 1,25-dihydroxyvitamin D3 determined with a highly sensitive and precise LC-MS/MS method.

Inaccurate First-Generation Testosterone Assays Are Influenced by Sex Hormone-Binding Globulin Concentrations.

BACKGROUND: The quality of testosterone assays has been a matter of debate for several years. Known limitations of testosterone immunoassays are the cross-reactivity with other steroids and a high variation in the low concentration range. We hypothesized that one of the additional limitations of testosterone immunoassays is an ineffective displacement of testosterone from its binding protein. METHODS: Thirty samples from women not using oral contraceptives (OAC), 30 samples from women using OAC, and 30 samples from pregnant women were used to measure testosterone by an isotope dilution (ID)-LC-MS/MS method and by 6 commercially available testosterone immunoassays (UniCel®, ARCHITECT®, Centaur®, Cobas®, Immulite®, and Liaison®). In addition, sex hormone-binding globulin (SHBG)4 was measured by immunoassay (ARCHITECT). RESULTS: The first-generation immunoassays (UniCel, Centaur, Immulite, and Liaison) showed inaccurate testosterone results in the method comparisons with the ID-LC-MS/MS method (R between 0.61 and 0.86) and for some assays (UniCel and Liaison) also a very poor standardization (slopes of 0.59 and 0.67, respectively). On average, SHBG concentrations were lowest in women not using OAC and highest in pregnant women, and overall ranged from 18.5 to 633 nmol/L. In the first-generation immunoassays, but not in the second-generation immunoassays, we observed an inverse relationship between SHBG concentrations and deviations in testosterone from the ID-LC-MS/MS results. CONCLUSIONS: Widely used first-generation testosterone immunoassays are influenced by SHBG concentrations, which lead to inaccurate results in samples from patients with high or low SHBG concentrations, respectively. Laboratory specialists, clinicians, and researchers should be aware of this limitation in testosterone assays.

Comparison of 7 Published LC-MS/MS Methods for the Simultaneous Measurement of Testosterone, Androstenedione, and Dehydroepiandrosterone in Serum.

BACKGROUND: Recently, LC-MS/MS was stated to be the method of choice to measure sex steroids. Because information on the mutual agreement of LC-MS/MS methods is scarce, we compared 7 published LC-MS/MS methods for the simultaneous measurement of testosterone, androstenedione, and dehydroepiandrosterone (DHEA). METHODS: We used 7 published LC-MS/MS methods to analyze in duplicate 55 random samples from both men and women. We performed Passing-Bablok regression analysis and calculated Pearson correlation coefficients to assess the agreement of the methods investigated with the median concentration measured by all methods, and we calculated the intraassay CV of each method derived from duplicate results and the CVs between the methods. RESULTS: Median concentrations of testosterone were 0.22-1.36 nmol/L for women and 8.27-27.98 nmol/L for men. Androstenedione and DHEA concentrations were 0.05-5.53 and 0.58-18.04 nmol/L, respectively. Intraassay CVs were 2.9%-10%, 1.2%-8.8%, 2.7%-13%, and 4.3%-16% for testosterone in women, testosterone in men, androstenedione, and DHEA. Slopes of the regression lines calculated by Passing-Bablok regression analysis were 0.92-1.08, 0.92-1.08, 0.90-1.13, and 0.91-1.41 for all testosterone values, testosterone in women, androstenedione, and DHEA. Intermethod CVs were 14%, 8%, 30%, and 22% for testosterone in women, testosterone in men, androstenedione, and DHEA. CONCLUSIONS: In general, the LC-MS/MS methods investigated show reasonable agreement. However, some of the assays show differences in standardization, and others show high variation.

Facilitating the Validation of Novel Protein Biomarkers for Dementia: An Optimal Workflow for the Development of Sandwich Immunoassays.

Different neurodegenerative disorders, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), lead to dementia syndromes. Dementia will pose a huge impact on society and thus it is essential to develop novel tools that are able to detect the earliest, most sensitive, discriminative, and dynamic biomarkers for each of the disorders. To date, the most common assays used in large-scale protein biomarker analysis are enzyme-linked immunosorbent assays (ELISA), such as the sandwich immunoassays, which are sensitive, practical, and easily implemented. However, due to the novelty of many candidate biomarkers identified during proteomics screening, such assays or the antibodies that specifically recognize the desired marker are often not available. The development and optimization of a new ELISA should be carried out with considerable caution since a poor planning can be costly, ineffective, time consuming, and it may lead to a misinterpretation of the findings. Previous guidelines described either the overall biomarker development in more general terms (i.e., the process from biomarker discovery to validation) or the specific steps of performing an ELISA procedure. However, a workflow describing and guiding the main issues in the development of a novel ELISA is missing. Here, we describe a specific and detailed workflow to develop and validate new ELISA for a successful and reliable validation of novel dementia biomarkers. The proposed workflow highlights the main issues in the development of an ELISA and covers several critical aspects, including production, screening, and selection of specific antibodies until optimal fine-tuning of the assay. Although these recommendations are designed to analyze novel biomarkers for dementia in cerebrospinal fluid, they are generally applicable for the development of immunoassays for biomarkers in other human body fluids or tissues. This workflow is designed to maximize the quality of the developed ELISA using a time- and cost-efficient strategy. This will facilitate the validation of the dementia biomarker candidates ultimately allowing accurate diagnostic conclusions.

Clusterin Levels in Plasma Predict Cognitive Decline and Progression to Alzheimer's Disease.

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 Aß42, 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.

α-Klotho is unstable in human urine.

α-Klotho is an interesting new biomarker in kidney and cardiovascular disease. As α-Klotho is primarily expressed in renal epithelial tissue, various papers have reported α-Klotho concentrations in urine. As these studies did not address the reliability of urinary α-Klotho measurements and as urine is known to be a complex milieu, we investigated the stability of α-Klotho in both fresh catheter and fresh voided urine. α-Klotho was measured in these fresh urine samples and in the same samples under several other conditions. Storage of fresh catheter urine for 3 h at 37 °C, comparable to storage in the bladder, led to a 82% decrease in α-Klotho concentrations. Compared with fresh voided urine, α-Klotho concentrations decreased on an average of 45 and 82% after storage at -80 °C and -20 °C, respectively. An additional freeze-thaw cycle further decreased α-Klotho concentrations. The addition of a protease inhibitor or 0.1% albumin partly prevented degradation in fresh voided urine. Thus, α-Klotho is highly unstable in urine. Future studies showing results of urinary α-Klotho should mention conservation conditions and prove the reliability of the α-Klotho measurements.

Vitamin D supplementation and testosterone concentrations in male human subjects.

OBJECTIVE: A possible association between serum 25-hydroxyvitamin D and testosterone levels has been reported; however, contradictory results have emerged. DESIGN: To investigate a causal link between vitamin D and testosterone status, we studied the effect of vitamin D supplementation on serum testosterone concentrations in three independent intervention studies including male patients with heart failure (study 1), male nursing home residents (study 2) and male non-Western immigrants in the Netherlands (study 3). METHODS: In study 1, 92 subjects were randomized to either vitamin D (2000 IU cholecalciferol daily) or control. Blood was drawn at baseline, after 3 and 6 weeks. In study 2, 49 vitamin D deficient subjects received either vitamin D (600 IU daily) or placebo. Blood was drawn at baseline, after 8 and 16 weeks. In study 3, 43 vitamin D deficient subjects received either vitamin D (1200 IU daily) or placebo. Blood was drawn at baseline, after 8 and 16 weeks. Serum 25-hydroxyvitamin D levels were measured using LC-MS/MS or radioimmunoassay. Testosterone levels were measured using a 2nd generation immunoassay. RESULTS: Serum 25-hydroxyvitamin D levels significantly increased in all treatment groups (median increase of 27, 30 and 36 nmol/l in studies 1, 2 3, respectively) but not in the control groups. The documented increase in 25-hydroxyvitamin D levels, however, did not affect mean testosterone concentrations at the end of the study (median increase of 0, 0.5 and 0 nmol/l in studies 1, 2 and 3, respectively). CONCLUSIONS: In this post hoc analysis of three small clinical trials of limited duration in men with normal baseline testosterone concentrations, vitamin D supplementation was not associated with an increase in circulating testosterone concentrations.

Simultaneous measurement of testosterone, androstenedione and dehydroepiandrosterone (DHEA) in serum and plasma using Isotope-Dilution 2-Dimension Ultra High Performance Liquid-Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS).

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.

Amyloid-ß oligomers relate to cognitive decline in Alzheimer's disease.

BACKGROUND: Amyloid-ß (Aß)-oligomers are neurotoxic isoforms of Aß and are a potential diagnostic biomarker for Alzheimer's disease (AD). OBJECTIVES: 1) Analyze the potential of Aß-oligomer concentrations in cerebrospinal fluid (CSF) to diagnose and predict progression to AD in a large clinical study sample. 2) Monitor Aß-oligomer concentrations over-time, both in early and advanced stages of AD. 3) Examine the relation between Aß-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 Aß-oligomer levels using a validated in-house Aß-oligomer specific enzyme-linked immunosorbent assay. Aß-oligomer levels were analyzed as indicators of disease progression (follow-up AD diagnosis) and cognitive decline, respectively. RESULTS: Patient groups did not differ in Aß-oligomer concentrations at baseline or follow-up. Baseline CSF Aß-oligomer levels were similar in MCI patients that develop AD as in stable MCI patients. MCI and AD patients showed an annual decrease in Aß-oligomer levels of 9.4% and 6.8%, respectively. A decrease in Aß-oligomer levels over time was strongly associated with more severe cognitive decline in AD patients. CONCLUSION: Despite the limited diagnostic potential of Aß-oligomer levels in CSF to differentiate between patient groups, and between MCI-AD and MCI-stable patients, changes in CSF Aß-oligomer levels were related to cognitive decline. Therefore, CSF Aß-oligomers may aid in the selection of patients with a more aggressive disease course.

N-acetylaspartate and neurofilaments as biomarkers of axonal damage in patients with progressive forms of multiple sclerosis.

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, ß = 0.413; p < 0.05). Conversely, Nf light was the only predictor of the EDSS annual increase (R(2) = 0.195, ß = 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.

First-trimester screening for down syndrome with serum sampling at different gestational ages: the effect on screening performance.

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 ß-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.

The cerebrospinal fluid "Alzheimer profile": easily said, but what does it mean?

BACKGROUND: We aimed to identify the most useful definition of the "cerebrospinal fluid Alzheimer profile," based on amyloid-ß1-42 (Aß42), 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/Aß42 ratio and 0.08 for the p-tau/Aß42 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/Aß42 ratio. CONCLUSIONS: A tau/Aß42 ratio of >0.52 constitutes a robust cerebrospinal fluid Alzheimer profile. We recommend using this ratio to combine biomarkers.