A pilot study to evaluate the application of a generic protein standard panel for quality control of biomarker detection technologies

<p>Abstract</p> <p>Background</p> <p>Protein biomarker studies are currently hampered by a lack of measurement standards to demonstrate quality, reliability and comparability across multiple assay platforms. This is especially pertinent for immunoassays where multiple formats for detecting target analytes are commonly used.</p> <p>Findings</p> <p>In this pilot study a generic panel of six non-human protein standards (50 - 10^7 pg/mL) of varying abundance was prepared as a quality control (QC) material. Simulated "normal" and "diseased" panels of proteins were prepared in pooled human plasma and incorporated into immunoassays using the Meso Scale Discovery^®(MSD^®) platform to illustrate reliable detection of the component proteins. The protein panel was also evaluated as a spike-in material for a model immunoassay involving detection of ovarian cancer biomarkers within individual human plasma samples. Our selected platform could discriminate between two panels of the proteins exhibiting small differences in abundance. Across distinct experiments, all component proteins exhibited reproducible signal outputs in pooled human plasma. When individual donor samples were used, half the proteins produced signals independent of matrix effects. These proteins may serve as a generic indicator of platform reliability.</p> <p>Each of the remaining proteins exhibit differential signals across the distinct samples, indicative of sample matrix effects, with the three proteins following the same trend. This subset of proteins may be useful for characterising the degree of matrix effects associated with the sample which may impact on the reliability of quantifying target diagnostic biomarkers.</p> <p>Conclusions</p> <p>We have demonstrated the potential utility of this panel of standards to act as a generic QC tool for evaluating the reproducibility of the platform for protein biomarker detection independent of serum matrix effects.</p

Rationalising the role of Keratin 9 as a biomarker for Alzheimer’s disease

Keratin 9 was recently identified as an important component of a biomarker panel which demonstrated a high diagnostic accuracy (87%) for Alzheimer’s disease (AD). Understanding how a protein which is predominantly expressed in palmoplantar epidermis is implicated in AD may shed new light on the mechanisms underlying the disease. Here we use immunoassays to examine blood plasma expression patterns of Keratin 9 and its relationship to other AD-associated proteins. We correlate this with the use of an in silico analysis tool VisANT to elucidate possible pathways through which the involvement of Keratin 9 may take place. We identify possible links with Dickkopf-1, a negative regulator of the wnt pathway, and propose that the abnormal expression of Keratin 9 in AD blood and cerebrospinal fluid may be a result of blood brain barrier dysregulation and disruption of the ubiquitin proteasome system. Our findings suggest that dysregulated Keratin 9 expression is a consequence of AD pathology but, as it interacts with a broad range of proteins, it may have other, as yet uncharacterized, downstream effects which could contribute to AD onset and progression

Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR)

Abstract Introduction A common feature of neoplastic cells is that mutations in SMADs can contribute to the loss of sensitivity to the anti-tumor effects of transforming growth factor-β (TGF-β). However, germline mutation analysis of SMAD3 and SMAD4, the principle substrates of the TGF-β signaling pathway, has not yet been conducted in breast cancer. Thus, it is currently unknown whether germline SMAD3 and SMAD4 mutations are involved in breast cancer predisposition. Methods We performed mutation analysis of the highly conserved mad-homology 2 (MH2) domains for both genes in genomic DNA from 408 non-BRCA1/BRCA2 breast cancer cases and 710 population controls recruited by the Ontario site of the breast cancer family registry (CFR) using denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing. The results were interpreted in several ways. First, we adapted nucleotide diversity analysis to quantitatively assess whether the frequency of alterations differ between the two genes. Next, in silico tools were used to predict variants' effect on domain function and mRNA splicing. Finally, 37 cases or controls harboring alterations were tested for aberrant splicing using reverse-transcription polymerase chain reaction (PCR) and real-time PCR statistical comparison of germline expressions by non-parametric Mann-Whitney test of independent samples. Results We identified 27 variants including 2 novel SMAD4 coding variants c.1350G > A (p.Gln450Gln), and c.1701A > G (p.Ile525Val). There were no inactivating mutations even though c.1350G > A was predicted to affect exonic splicing enhancers. However, several additional findings were of note: 1) nucleotide diversity estimate for SMAD3 but not SMAD4 indicated that coding variants of the MH2 domain were more infrequent than expected; 2) in breast cancer cases SMAD3 was significantly over-expressed relative to controls (P A was associated with elevated germline expression (> 5-fold); 3) separate analysis using tissue expression data showed statistically significant over-expression of SMAD3 and SMAD4 in breast carcinomas. Conclusions This study shows that inactivating germline alterations in SMAD3 and SMAD4 are rare, suggesting a limited role in driving tumorigenesis. Nevertheless, aberrant germline expressions of SMAD3 and SMAD4 may be more common in breast cancer than previously suspected and offer novel insight into their roles in predisposition and/or progression of breast cancer

The Prevalence of Sexually Transmitted Infections in Papua New Guinea: A Systematic Review and Meta-Analysis

Patellofemoral osteoarthritis (PF OA) is more prevalent than previously thought and contributes to patient's suffering from knee OA. Synthesis of prevalence data can provide estimates of the burden of PF OA

Practical detection of a definitive biomarker panel for Alzheimer's disease; comparisons between matched plasma and cerebrospinal fluid

Previous mass spectrometry analysis of cerebrospinal fluid (CSF) has allowed the identification of a panel of molecular markers that are associated with Alzheimer's disease (AD). The panel comprises Amyloid beta, Apolipoprotein E, Fibrinogen alpha chain precursor, Keratin type I cytoskeletal 9, Serum albumin precursor, SPARC-like 1 protein and Tetranectin. Here we report the development and implementation of immunoassays to measure the abundance and diagnostic capacity of these putative biomarkers in matched lumbar CSF and blood plasma samples taken in life from individuals confirmed at post-mortem as suffering from AD (n = 10) and from screened 'cognitively healthy' subjects (n = 18). The inflammatory components of Alzheimer's disease were also investigated. Employment of supervised learning techniques permitted examination of the interrelated expression patterns of the putative biomarkers and identified inflammatory components, resulting in biomarker panels with a diagnostic accuracy of 87.5% and 86.7% for the plasma and CSF datasets respectively. This is extremely important as it offers an ideal high-throughput and relatively inexpensive population screening approach. It appears possible to determine the presence or absence of AD based on our biomarker panel and it seems likely that a cheap and rapid blood test for AD is feasible

Practical detection of a definitive biomarker panel for Alzheimer's disease; comparisons between matched plasma and cerebrospinal fluid

Previous mass spectrometry analysis of cerebrospinal fluid (CSF) has allowed the identification of a panel of molecular markers that are associated with Alzheimer's disease (AD). The panel comprises Amyloid beta, Apolipoprotein E, Fibrinogen alpha chain precursor, Keratin type I cytoskeletal 9, Serum albumin precursor, SPARC-like 1 protein and Tetranectin. Here we report the development and implementation of immunoassays to measure the abundance and diagnostic capacity of these putative biomarkers in matched lumbar CSF and blood plasma samples taken in life from individuals confirmed at post-mortem as suffering from AD (n = 10) and from screened 'cognitively healthy' subjects (n = 18). The inflammatory components of Alzheimer's disease were also investigated. Employment of supervised learning techniques permitted examination of the interrelated expression patterns of the putative biomarkers and identified inflammatory components, resulting in biomarker panels with a diagnostic accuracy of 87.5% and 86.7% for the plasma and CSF datasets respectively. This is extremely important as it offers an ideal high-throughput and relatively inexpensive population screening approach. It appears possible to determine the presence or absence of AD based on our biomarker panel and it seems likely that a cheap and rapid blood test for AD is feasible