| 1. |
- Malm, Johan, et al.
(författare)
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Developments in biobanking workflow standardization providing sample integrity and stability
- 2013
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 95:SI, s. 38-45
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Tidskriftsartikel (refereegranskat)abstract
- Recommendations and outlines for standardization in biobanking processes are presented by a research team with long-term experience 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 proteomic and genomic communities regarding the basic concepts of collecting, storing and utilizing clinical samples. Quality control and sample suitability for analysis need 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 and is presented showing validation criteria. Large studies will be accompanied by biological and 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. Biological significance: The current manuscript is of major relevance to the proteomic and genomic 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 genomic 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 in Proteomics.
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| 2. |
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| 3. |
- Végvári, Ákos, et al.
(författare)
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Bioinformatic strategies for unambiguous identification of prostate specific antigen in clinical samples
- 2011
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 202-210
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Tidskriftsartikel (refereegranskat)abstract
- Prostate specific antigen (PSA), as a widely used clinical biomarker in prostate cancer diagnostics, exists in multiple molecular forms. However, all of these forms might not be recognized in a given sample by the standard immunoassays. Therefore, we have investigated PSA isoforms separated by size using mass spectrometric analyses. The objective of these developments was to identify and specify the various forms of PSA. To optimize successful identification of different PSA forms, we have developed a bioinformatic strategy, consisting of high resolution MALDI-MS PMF and sequencing MS/MS data searches. To improve sequence-based identification, the recently introduced Proteios software environment was employed, allowing the combination of multiple database search engines in an automated manner. We could unambiguously identify PSA in clinical samples by all detectable tryptic peptides, which were found to be common in several isoforms.
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| 4. |
- Végvári, Ákos, et al.
(författare)
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Identification of prostate specific antigen (PSA) isoforms in complex biological samples utilizing complementary platforms
- 2010
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 73:6, s. 1137-1147
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of the prostate-specific antigen (PSA) levels in blood are widely used as diagnostic, predictive and prognostic marker of prostate disease. The selective detection of molecular forms of PSA can contribute clinically to meaningful enhancements of the conventional PSA-test. As it is plausible that an in-depth search for structural variants of PSA gene products may increase our ability to discriminate distinct patho-biological basis and stages of prostate diseases, we have developed a multi-step protocol comprising gel-based methods followed by mass spectrometric identification. Our current aim was to provide a comprehensive identification of PSA variants occurring in seminal fluid. We provide a proof-of-principle for this multiple step analytical approach to identify multiple PSA variants from complex biological samples that revealed distinct molecular characteristics. In addition, sequence-annotated protein bands in SDS–PAGE gels were compared to those detected by Western blots, and by monitoring the enzymatic activity in zymogram gels, using gelatin as a substrate. The high accuracy annotations were obtained by fast turnaround MALDI-Orbitrap analysis from excised and digested gel bands. Multiple PSA forms were identified utilizing a combination of MASCOT and SEQUEST search engines.
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| 5. |
- Ahmad Tajudin, Asilah, et al.
(författare)
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Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.
- 2013
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 13:9, s. 1790-1796
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Tidskriftsartikel (refereegranskat)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%.
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| 6. |
- Antfolk, Maria, et al.
(författare)
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A single inlet two-stage acoustophoresis chip enabling tumor cell enrichment from white blood cells
- 2015
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Ingår i: Lab on a Chip. - 1473-0189. ; 15:9, s. 2102-2109
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Tidskriftsartikel (refereegranskat)abstract
- Metastatic disease is responsible for most cancer deaths, and hematogenous spread through circulating tumor cells (CTC) is a prerequisite for tumor dissemination. CTCs may undergo epithelial–mesenchymal transition where many epithelial cell characteristics are lost. Therefore, CTC isolation systems relying on epithelial cell markers are at risk of losing important subpopulations of cells. Here, a simple acoustophoresis-based cell separation instrument is presented. Cells are uniquely separated while maintained in their initial suspending medium, thus eliminating the need for a secondary cell-free medium to hydrodynamically pre-position them before the separation. When characterizing the system using polystyrene particles, 99.6 ± 0.2% of 7 μm diameter particles were collected through one outlet while 98.8 ± 0.5% of 5 μm particles were recovered through a second outlet. Prostate cancer cells (DU145) spiked into blood were enriched from white blood cells at a sample flow rate of 100 μL min−1 providing 86.5 ± 6.7% recovery of the cancer cells with 1.1 ± 0.2% contamination of white blood cells. By increasing the acoustic intensity a recovery of 94.8 ± 2.8% of cancer cells was achieved with 2.2 ± 0.6% contamination of white blood cells. The single inlet approach makes this instrument insensitive to acoustic impedance mismatch; a phenomenon reported to importantly affect accuracy in multi-laminar flow stream acoustophoresis. It also offers a possibility of concentrating the recovered cells in the chip, as opposed to systems relying on hydrodynamic pre-positioning which commonly dilute the target cells.
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| 7. |
- Antfolk, Maria, et al.
(författare)
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Continuous flow microfluidic separation and processing of rare cells and bioparticles found in blood – A review
- 2017
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Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670. ; 965, s. 9-35
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Forskningsöversikt (refereegranskat)abstract
- Rare cells in blood, such as circulating tumor cells or fetal cells in the maternal circulation, posses a great prognostic or diagnostic value, or for the development of personalized medicine, where the study of rare cells could provide information to more specifically targeted treatments. When conventional cell separation methods, such as flow cytometry or magnetic activated cell sorting, have fallen short other methods are desperately sought for. Microfluidics have been extensively used towards isolating and processing rare cells as it offers possibilities not present in the conventional systems. Furthermore, microfluidic methods offer new possibilities for cell separation as they often rely on non-traditional biomarkers and intrinsic cell properties. This offers the possibility to isolate cell populations that would otherwise not be targeted using conventional methods. Here, we provide an extensive review of the latest advances in continuous flow microfluidic rare cell separation and processing with each cell's specific characteristics and separation challenges as a point of view.
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| 8. |
- Ekström, Simon, et al.
(författare)
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Integrated selective enrichment target - a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases
- 2004
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835. ; 25:21-22, s. 3769-3777
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Tidskriftsartikel (refereegranskat)abstract
- The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples.
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| 9. |
- Finnskog, David, et al.
(författare)
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High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry
- 2006
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:5-6, s. 1093-1103
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Tidskriftsartikel (refereegranskat)abstract
- Speed and accuracy are crucial prerequisites in the application of proteomic methods to clinical medicine. We describe a microfluidic-based nanovial array for rapid proteolytic processing linked to MALDI-TOF MS. This microscale format consumes only minute amounts of sample, and it is compatible with rapid bioanalytical protocols and high-sensitivity readouts. Arrays of vials (300 mu m in diameter and 25 mu m deep), isotropically etched in silicon wafers were electrochemically porosified. Automated picoliter microdispensing was employed for precise fluid handling in the microarray format. Vials were prefilled with trypsin solution, which was allowed to dry. Porosified and nonporosified nanovials were compared for trypsin digestion and subsequent MS identification of three model proteins: lysozyme, alcohol dehydrogenase, and serum albumin at levels of 100 and 20 fmol. In an effort to assess the rapid digestion platform in a context of putative clinical applications, two prostate cancer biomarkers, prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2), were digested at levels of 100 fmol (PSA), 20 fmol (PSA) and 8 fmol (hK2). All biomarker digestions were completed in less than 30 s, with successful MS identification in the porous nanovial setting.
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| 10. |
- Ku, Anson, et al.
(författare)
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Acoustic Enrichment of Extracellular Vesicles from Biological Fluids
- 2018
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 90:13, s. 8011-8019
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 μL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.
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