[Show abstract][Hide abstract] ABSTRACT: With the rapid progress in the development of new clinical biomarkers there is an unmet need of fast and sensitive multiplex analysis methods for disease specific protein monitoring. Immuno-affinity extraction integrated with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis offers a route to rapid and sensitive protein analysis and potentially multiplex biomarker analysis. In this study, the previously reported integrated selective enrichment target (ISET)-MALDI-MS analysis was implemented with ssDNA aptamer functionalized microbeads to address the specific capturing of thrombin in complex samples. The main objective for using an aptamer as the capturing ligand was to avoid the inherently high background components, which are produced during the digestion step following the target extraction when antibodies are used. By applying a thrombin specific aptamer linked to ISET-MALDI-MS detection, a proof of concept of antibody fragment background reduction in the ISET-MALDI-MS readout is presented. Detection sensitivity was significantly increased compared to the corresponding system based on antibody specific binding as the aptamer ligand does not induce any interfering background residues from the antibodies. The limit of detection for thrombin was 10 fmol in buffer using the aptamer/ISET-MALDI-MS configuration as confirmed by MS/MS fragmentation. The aptamer/ISET-MALDI-MS platform also displayed a limit of detection of 10 fmol for thrombin in five different human serum samples (1/10 diluted), demonstrating the applicability of the aptamer/ISET-MALDI-MS analysis in clinical samples.
[Show abstract][Hide abstract] ABSTRACT: Vitamin D, parathyroid hormone (PTH) and calcium in blood are correlated with each other. Previous studies have suggested vitamin D to have anti-proliferative effects on tumor cells, whereas PTH may have carcinogenic effects. A cancer disease may influence calcium levels in blood, but less is known about calcium and its potential effect on cancer risk and survival. The aim of this study was to examine pre-diagnostic levels of vitamin D (25OHD), PTH and calcium in relation to survival after breast cancer.
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that mitochondrial dysfunction and DNA damage are involved in lymphomagenesis. Increased copy number of mitochondrial DNA (mtDNA) as a compensatory mechanism of mitochondrial dysfunction previously has been associated with B-cell lymphomas, in particular chronic lymphocytic leukemia (CLL). However, current evidence is limited and based on a relatively small number of cases. Using a nested case-control study, we extended these findings with a focus on subtype specific analyses. Relative mtDNA copy number was measured in the buffy coat of prospectively collected blood of 469 lymphoma cases and 469 matched controls. The association between mtDNA copy number and the risk of developing lymphoma and histologic subtypes was examined using logistic regression models. We found no overall association between mtDNA and risk of lymphoma. Subtype analyses revealed, significant increased risks of CLL (n=102) with increasing mtDNA copy number (OR=1.34, 1.44 and 1.80 for quartiles 2-4, respectively P-trend=0.001). mtDNA copy number was not associated with follow-up time suggesting that this observation is not strongly influenced by indolent disease status. This study substantially strengthens the evidence that mtDNA copy number is related to risk of CLL and supports the importance of mitochondrial dysfunction as a possible mechanistic pathway in CLL ontogenesis.
[Show abstract][Hide abstract] ABSTRACT: The association between vitamin D status and hepatocellular carcinoma has not been well investigated, despite experimental evidence supporting an important role of vitamin D in liver pathophysiology. Our objective was to investigate the association between pre-diagnostic circulating 25-hydroxyvitamin D [25(OH)D] serum levels and risk of hepatocellular carcinoma in a prospective, nested case-control study among 520,000 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Each case (n = 138) diagnosed between 1992 and 2010 was matched to one control by age, sex, study center, date and time of blood collection, and fasting status. Serum baseline levels of 25(OH)D were measured by liquid chromatography/tandem mass spectrometry. Multivariable incident rate ratios (IRR) of hepatocellular carcinoma associated with continuous (per 10 nmol/L) or categorical levels (tertiles or a priori-defined categories) of pre-diagnostic 25(OH)D. Higher 25(OH)D levels were associated with a 49% reduction in the risk of hepatocellular carcinoma (highest vs. lowest tertile: multivariable IRR = 0.51, 95% confidence interval, 0.26 to 0.99; Ptrend = 0.04; per 10 nmol/L increase: IRR = 0.80, 95% confidence interval, 0.68-0.94). The finding did not vary substantially by time from enrolment to diagnosis, and did not change after adjustment for biomarkers of pre-existing liver damage, nor chronic infection with hepatitis B or C viruses. The findings were not modified by body size or smoking status. Conclusion: In this prospective study on Western European populations, serum levels of 25(OH)D were inversely associated with risk of hepatocellular carcinoma. Given the rising incidence of this cancer in low-risk developed countries and the strong public health interest surrounding the potentially cancer-protective roles of vitamin D, additional studies in different populations are required. (Hepatology 2014;).
[Show abstract][Hide abstract] ABSTRACT: To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40fmol to 1pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.
[Show abstract][Hide abstract] ABSTRACT: Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL(-1), 80pgmL(-1), and 800fgmL(-1) when arraying the PSA antibody, H117 at the concentration 15μgmL(-1), 35μgmL(-1), and 154μgmL(-1), respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL(-1) to 500ngmL(-1). The microarray showed a LOD of 800fgmL(-1) and a dynamic range of 800fgmL(-1) to 80ngmL(-1) in serum spiked samples.
[Show abstract][Hide abstract] ABSTRACT: Low blood levels of vitamin D (25-hydroxy D3, 25OHD3) in women have been associated with an increased risk of several diseases. A large part of the population may have suboptimal 25OHD3 levels but high-risk groups are not well known. The aim of the present study was to identify determinants for serum levels of 25OHD3 in women, i.e. factors such as lifestyle, menopausal status, diet and selected biochemical variables.
The study was based on women from the Malmo Diet and Cancer Study (MDCS), a prospective, population-based cohort study in Malmo, Sweden. In a previous case--control study on breast cancer, 25OHD3 concentrations had been measured in 727 women. In these, quartiles of serum 25OHD3 were compared with regard to age at baseline, BMI (Body Max Index), menopausal status, use of oral contraceptives or menopausal hormone therapy (MHT) , life-style (e.g. smoking and alcohol consumption), socio-demographic factors, season, biochemical variables (i.e. calcium, PTH, albumin, creatinine, and phosphate), and dietary intake of vitamin D and calcium. In order to test differences in mean vitamin D concentrations between different categories of the studied factors, an ANOVA test was used followed by a t-test. The relation between different factors and 25OHD3 was further investigated using multiple linear regression analysis and a logistic regression analysis.
We found a positive association between serum levels of 25OHD3 and age, oral contraceptive use, moderate alcohol consumption, blood collection during summer/ autumn, creatinine, phosphate, calcium, and a high intake of vitamin D. Low vitamin D levels were associated with obesity, being born outside Sweden and high PTH levels.
The present population-based study found a positive association between serum levels of 25OHD3 and to several socio-demographic, life-style and biochemical factors. The study may have implications e. g. for dietary recommendations. However, the analysis is a cross-sectional and it is difficult to suggest Lifestyle changes as cause- effect relationships are difficult to assess.
BMC Women s Health 08/2013; 13(1):33. · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The relation between vitamin D status and lymphoma risk is inconclusive.
We examined the association between prediagnostic plasma 25-hydroxyvitamin D [25(OH)D] and lymphoid cancer risk.
We conducted a study nested within the European Prospective Investigation into Cancer and Nutrition cohort of 1127 lymphoma cases and 1127 matched controls with a mean follow-up time of 7.1 y. Conditional logistic regression was used to estimate multivariable-adjusted incidence rate ratios of lymphoma risk in relation to plasma 25(OH)D. Season-standardized and season-specific 25(OH)D quartiles were used. We also analyzed 25(OH)D as a continuous variable and used predefined cutoffs.
No statistically significant association between plasma 25(OH)D and overall lymphoid cancer risk was observed. A positive association for B-cell non-Hodgkin lymphoma was noted only in those with a diagnosis made during the first 2 y of follow-up (P-heterogeneity = 0.03), which suggests the possibility of reverse causality. Further analysis restricted to participants with ≥2 y of follow-up time showed a significant association between 25(OH)D and chronic lymphocytic leukemia (CLL) (n = 161): adjusted incidence rate ratios were 0.40 (95% CI: 0.18, 0.90; P-trend = 0.05) and 0.31 (95% CI: 0.13, 0.76; P-trend = 0.03) for the top compared with bottom season-standardized and season-specific quartiles, respectively. Data on dietary vitamin D intake provided further support for the observed association (incidence rate ratio: 0.33; 95% CI = 0.12, 0.89; P-trend = 0.006).
Our findings do not support a protective role of high 25(OH)D concentration in lymphoid cancers overall. However, they suggest that higher concentrations of 25(OH)D are associated with a reduced risk of CLL.
American Journal of Clinical Nutrition 07/2013; · 6.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recommendations and outlines for standardization in biobanking processes are presented by a research team with long-term experiences in clinical studies. These processes have important bearing on the use of samples in developing assays. These measurements are useful to document states of health and disease that are beneficial for academic research, commercial healthcare, drug development industry and government regulating agencies. There is a need for increasing awareness within proteomics and genomics communities regarding the basic concepts of collecting, storing and utilizing clinical samples. Quality control and samples suitability for analysis needs to be documented and validated to ensure data integrity and establish contexts for interpretation of results. Standardized methods in proteomics and genomics are required to be practiced throughout the community allowing datasets to be comparable and shared for analysis. For example, sample processing of thousands of clinical samples, performed in 384 high-density sample tube systems in a fully automated workflow, preserves sample content, is presented showing validation criteria. Large studies will be accompanied by biological, molecular information with corresponding clinical records from patients and healthy donors. These developments position biobanks of human patient samples as an increasingly recognized major asset in disease research, future drug development and within patient care.
The current manuscript is of major relevance to the proteomics and genomics fields, as it outlines the standardization aspects of biobanking and the requirements that are needed to run future clinical studies that will benefit the patients where OMICS science will play a major role. A global view of the field is given where best practice and conventional acceptances are presented along with ongoing large-scale biobanking projects. The authors represent broadly stakeholders that cover the academic, pharma, biotech and healthcare fields with extensive experience and deliveries. This contribution will be a milestone paper to the proteomic and genomics scientists to present data in the future that will have impact to the life science area. This article is part of a Special Issue entitled: Standardization and Quality Control.
Journal of proteomics 07/2013; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prostate specific antigen (PSA) is a well-established tumor marker, which is frequently employed as model biomarker to develop and evaluate emerging quantitative proteomics techniques, partially due to wide access to commercialized immunoassays serving as "gold standard". We designed a multiple reaction monitoring (MRM) assay to detect PSA proteoforms in clinical samples (n=72), utilizing specificity and sensitivity of the method. We report for the first time, a PSA proteoform, coded by SNP-L132I (rs2003783), observed in 9 samples in both heterozygous (n=7) and homozygous (n=2) expression profiles. Other isoforms of PSA, derived from protein databases, were not identified by four unique proteotypic tryptic peptides. We have also utilized our MRM assay for precise quantitative analysis of PSA concentrations in both seminal and blood plasma samples. The analytical performance was evaluated, providing close agreement between each quantitation based on three selected peptides (LSEPAELTDAVK, IVGGWECEK and SVILLGR) and a routinely used commercialized immunoassay. Additionally, we have disclosed that the peptide IVGGWECEK is shared with kallikrein-related peptidase 2 and therefore not unique for PSA. Hence, we propose to use another tryptic sequence (SVILLGR) for accurate MRM-quantification of PSA in clinical samples.
[Show abstract][Hide abstract] ABSTRACT: There is an ever-increasing awareness and interest within the clinical research field, creating a large demand for blood fraction samples as well as other clinical samples. The translational research area is another field that is demanding for blood samples, used widely in proteomics, genomics, as well as metabolomics. Blood samples are globally the most common biological samples that find its use in a broad variety of applications in life science. We hereby introduce a new reference blood plasma standard (heparin) that is aimed as a global resource for the proteomics community. We have developed these reference plasma standards by defining the Control group as those with C-reactive protein levels <3mg/L and a Disease group with C-reactive protein ranges >30 mg/L. In these references we have used both newborn children 1-2 weeks, as well as youngsters 15-30 years, and middle aged 30-50 years, and elderly patients at the ages of 65+. In total there were 80 patients in each group in the reference plasma pools. We provide data on the developments and characteristics of the reference blood plasma standards, as well as what is used by the team members at the respective laboratories. The standards have been evaluated by pilot sample processing in biobanking operations, and are currently a resource that allows the Proteomic society to perform quantitative proteomic studies. By the use of high quality reference plasma samples, global initiatives, such as the Chromosome Human Proteome Project (C-HPP), will benefit as one scientific program when the entire human proteome is mapped and linked to human diseases. The plasma reference standards are a global resource and can be accessed upon request.
Journal of Proteome Research 05/2013; · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 μl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%.
[Show abstract][Hide abstract] ABSTRACT: A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented ( http://www.c-hpp.org ). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.
Journal of Proteome Research 12/2012; · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyzes several prostate cancer biomarkers simultaneously.
Clinica chimica acta; international journal of clinical chemistry 08/2012; 414C:76-84. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine the risk of prostate cancer in relation to pre-diagnostic serum levels of vitamin D (25OHD(2) and 25OHD(3)), PTH, and calcium.
Nine hundred forty-three incident prostate cancer cases were identified in the Malmö Diet and Cancer Study cohort, and each was matched with one control using incidence density matching with age as the underlying timescale. We also matched for calendar time and age at inclusion. Logistic regression analysis yielded odds ratios with 95 % confidence intervals for different quartiles and deciles. All analyses were repeated stratified for age and body mass index (BMI).
We found a weak trend toward increasing prostate cancer risk with rising vitamin D levels (p-trend across quartiles, 0.048). Dividing the cohort into deciles showed a nonlinear association. Compared to decile one, the prostate cancer risk was highest in deciles seven and eight, which corresponded to vitamin D levels of 91-97 nmol/L (1.68; 1.06-2.68), and 98-106 nmol/L (1.80; 1.13-2.85). In the other deciles, there was no association between prostate cancer risk and vitamin D levels. Albumin-adjusted calcium was positively associated with an increased risk for prostate cancer among men aged 55-65 with a BMI <25 (2.07; 1.08-3.97). No association was observed between pre-diagnostic PTH and subsequent prostate cancer incidence, and the stratified analyses revealed no other convincing relationships.
This study suggests a possible weak positive nonlinear association between vitamin D and the risk of prostate cancer.
Cancer Causes and Control 06/2012; 23(8):1377-85. · 3.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: The aim of this study is a novel automated sample-processing concept for future proteomics and clinical research, performing patient studies from resulting blood fractions in various disease areas. Another aim is biobank storage of small sample volumes, where each sample aliquot can be used for a dedicated clinical analysis and end-point measurement in order to preserve sample integrity and value over time. METHODS: 96 and 384 format sample storage tube systems were utilized for preservation and archiving of clinical patient samples. Automated sample processing and aliquoting were achieved using robotic liquid handling instrumentation, followed by biomarker assay quantitations. Sample workflow was documented and tracked by Nautilus LIMS. RESULTS: Validation by repetitive processing and analysis confirmed the reliability of automated high density 384 format aliquoting. This high density scaling allows for reproducible aliquoting of 70-μL volumes of blood. Plasma with EDTA, Li-heparin, and citrate, as anti-coagulants, fractioned along with the buffy coat (leukocytes) and the erythrocyte fraction. Large scale processing of 11,000 sample aliquots resulted in a 99.8% process fulfillment. CONCLUSION: Our results demonstrate that robust results can be generated from an automated sample processing strategy, isolating plasma, buffy coat, erythrocytes, serum and whole blood, proven by quantitation of 23 common markers used in everyday healthcare around the world. This article is part of a Special Issue entitled: Integrated omics.
Journal of proteomics 05/2012; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormones influence both normal breast cell differentiation and breast cancer cell proliferation and stimulate the angiogenesis of certain cancer forms. Several cross-sectional studies have measured thyroid hormones/autoantibodies in breast cancer ceases vs. controls, but it is difficult to determine the cause-effect direction in these studies. Only three prospective studies have reported on the subject so far. The aim of our study was to investigate prediagnostically measured levels of thyroid hormones, thyrotropin (TSH) and thyroid autoantibodies in relation to subsequent risk of breast cancer. The Malmoe Diet and Cancer study examined 17,035 women between 1991 and 1996. Blood samples were collected at baseline and free triiodothyronine (T3), free thyroxin (T4), TSH and thyroid peroxidase autoantibodies (TPO-Ab) levels were measured in 676 cases and 680 controls. Relative risks with 95% confidence intervals were assessed using a logistic regression analysis adjusted for potential confounders. Free T4 levels were positively associated with a high risk of breast cancer, and the OR for women with free T4 levels above vs. below the median was 1.40 (1.10-1.77). This association was most pronounced in overweight women (1.51:1.07-2.12). Women with high levels of TPO-Ab had a lower risk of breast cancer, but only the analysis of TPO-Ab as a continuous variable reached statistical significance. Free T4 was in our study positively associated with a high risk of breast cancer. This association was most pronounced in overweight/obese women. Women with a high level of TPO-Ab had a relatively low risk of breast cancer.
International Journal of Cancer 02/2012; 131(9):2126-33. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An acoustophoresis-based microfluidic flow-chip is presented as a novel platform to facilitate analysis of proteins and peptides loosely bound to the surface of beads or cells. The chip allows for direct removal of the background surrounding the beads or cells, followed by sequential treatment and collection of a sequence of up to five different buffer conditions. During this treatment, the beads/cells are retained in a single flow by acoustic radiation force. Eluted peptides are collected from the outlets and subsequently purified by miniaturized solid-phase extraction and analyzed with matrix assisted laser desorption mass spectrometry. Fundamental parameters such as the system fluidics and dispersion are presented. The device was successfully applied for wash and sequential elution of peptides bound to the surface of microbeads and human spermatozoa, respectively.
[Show abstract][Hide abstract] ABSTRACT: Prostate specific antigen (PSA) is a widely used and clinically valuable marker for prostate disease. In order to enable the development of new PSA assays and progress the understanding of the biology of PSA we have analyzed PSA in seminal plasma.
PSA in seminal plasma from men attending a fertility clinic and healthy controls was analyzed using SDS-PAGE, Western blotting and mass spectrometry.
Using mass spectrometry, different forms of PSA could be identified in 1-9 bands seen on SDS-PAGE analysis of the respective sample. However, a majority of these molecular forms of PSA were not observed on Western blots. Enzymatic activity of PSA isoforms was demonstrated by sequencing data in zymogram gels. Multivariate analysis of clinical data revealed well-separated patient groups.
We demonstrated that PSA in seminal plasma occurs in several isoforms, yet not all were detectable using an antibody based clinical routine method. The heterogeneity of PSA expression might be of clinical significance, by an improved patient phenotyping.