Standardization of Assay Procedures for Analysis of the CSF Biomarkers Amyloid β(1-42), Tau, and Phosphorylated Tau in Alzheimer's Disease: Report of an International Workshop

Large variation in assay performance and outcomes of CSF Aβ1-42, total Tau (Tau), and phosphorylated Tau (pTau) (at amino acid 181) levels is observed between laboratories. The aim of this study was to assess the differences in assay procedures between several experienced international laboratories, as potential sources of error. 14 groups performed the Aβ42, Tau, and pTau assays according to the guidelines of the manufacturer. Differences in analytical procedures between the laboratories were monitored. At least 23 items in assay procedures were identified that varied between the laboratories, including procedures for washing, pipetting, incubation, finishing, and sample handing. In general, the inter- and intra-assay variation between the groups was generally below 10% for all three assays. We concluded that 17 international centers that use the same assays for Aβ42, Tau and pTau on a regular basis do not uniformly adhere to the procedures recommended by the manufacturer. For harmonization of intercenter results of these biomarkers standardization of protocols is highly needed

Effect of Raloxifene Treatment on Osteocyte Apoptosis in Postmenopausal Women

Increased osteocyte apoptosis, as the result of estrogen deficiency, could play a role in the decrease of bone mass and bone strength seen in postmenopausal osteoporosis. We investigated whether treatment with raloxifene of postmenopausal women with osteoporosis affects osteocyte apoptosis. Transiliac bone biopsies were obtained from 26 osteoporotic women at baseline and after 2 years of treatment with placebo or raloxifene. Immunohistochemical detection of cleaved caspase-3 was performed on sections from nondecalcified bone biopsies to visualize apoptosis. In the trabecular bone total osteocytes, positively stained osteocytes and empty lacunae were counted and percent positive cells and percent empty lacunae determined. Statistical evaluation was performed by Wilcoxon’s paired t-test and Spearman’s rank correlations. There was no significant difference in percentage positive osteocytes between baseline and follow-up biopsies in both the placebo and the raloxifene groups. The percentage empty lacunae increased significantly in the placebo group (11.20 ± 1.43 vs. 9.00 ± 2.25, P = 0.014) but not in the raloxifene group. At baseline in both groups combined, there was a negative correlation between indices of bone remodeling and the percentage positive osteocytes (bone formation rate/bone volume r = −0.67, P = 0.001). We found no direct evidence for an effect of raloxifene treatment on osteocyte apoptosis, but small effects of raloxifene treatment cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual

Pooled Analysis of Prognostic Impact of Urokinase-Type Plasminogen Activator and Its Inhibitor PAI-1 in 8377 Breast Cancer Patients

Background: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). Methods: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. Results: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). Conclusions: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategie

Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). METHODS: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. RESULTS: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). CONCLUSIONS: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategies

The effect of amyloid associated proteins on the expression of genes involved in amyloid-beta clearance by adult human astrocytes

Astrocytes appear to be important mediators in the clearance of amyloid beta1-42 (A beta), the key component of senile plaques characteristic of Alzheimer's disease (AD). Recently, we found the amyloid associated proteins (AAPs) alpha 1-antichymotrypsin (ACT), apolipoprotein J and E (ApoJ and ApoE) and a mixture of serum amyloid P (SAP) and C1q (SAP-C1q) to modify A beta-uptake by human astrocytes. Here we investigated the effect of oligomeric (A beta oligo) and fibrillar A beta (A beta fib), alone and in combination with a panel of AAPs on the astrocytic expression of genes proposed to be involved in A beta-uptake and degradation. Primary human astrocytes (isolated from non-demented control (n = 4) and AD patient (n = 4) brain specimens) were exposed to either A beta oligo or A beta fib preparations with or without the above mentioned AAPs. Quantitative gene expression analysis of A beta-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 A beta-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 A beta oligo or A beta fib alone in any of the investigated astrocyte cultures. Interestingly NEP expression was increased upon exposure to ApoE in combination with both A beta-preparations, and also SCARB1 expression was induced upon treatment with ApoE in combination with A beta fib in astrocytes from non-demented controls. Further, SAP-C1q increased SCARB1 expression in control astrocytes when combined with A beta oligo. These alterations were not found in astrocytes from AD patients. Thus, we conclude that A beta 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 A beta 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. (C) 2011 Elsevier Inc. All rights reserved

Measurement of dehydroepiandrosterone sulphate (DHEAS): a comparison of Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) and seven currently available immunoassays

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. 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). 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. 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 emplo

Accuracy of 6 Routine 25-Hydroxyvitamin D Assays:Influence of Vitamin D Binding Protein Concentration

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. (C) 2011 American Association for Clinical Chemistr