uu.seUppsala University Publications
Change search
Link to record
Permanent link

Direct link
BETA
Björkesten, Johan
Publications (7 of 7) Show all publications
Björkesten, J. (2019). Dried blood sampling and digital readout to advance molecular diagnostics. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Dried blood sampling and digital readout to advance molecular diagnostics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A drastically increased capacity to measure large sets of molecular features in numerous patient samples in great detail will be required to fulfill the vision of precision medicine and wellness, which may characterize molecular diagnostics in the 21st century. Also sampling procedures need a renaissance to permit continuous sampling at population levels at reasonable cost.

Blood sampling is typically performed via venipuncture to draw several milliliters of blood for plasma isolation. This is inconvenient, time-consuming and costly, as well as hard to standardize. The effect on plasma protein profiles by pre-centrifugation delay was investigated in Paper II, demonstrating time- and temperature-dependent release of proteins from blood cells upon delayed plasma isolation, but almost no protein degradation as analyzed by two 92-plex protein panels (Olink® Proteomics). An alternative sampling method, where blood drops from a finger stick are collected dried on paper, is relatively non-invasive, potentially home-based and cheap. Dried blood spots can also be shipped via regular mail and compactly stored. The effect of drying and long term storage stability of a large set of proteins from dried blood spots was investigated in Paper I using Olink® technology. The main findings were that drying slightly but consistently influenced the recorded levels of blood proteins, and that long-term storage decreased the detected levels of some of the proteins with half-lives of decades.

Some molecular diagnostic investigations require great accuracy to be useful, arguing for digital enumeration of individual molecules. Digital PCR is the gold standard but Paper III presents an alternative approach based on rolling circle amplification of single molecules. Another instance where extreme assay performance is required is for rare mutation detection from liquid biopsies. Paper V presents a new method offering essentially error-free genotyping of individual molecules by majority-vote decisions for counting rare mutant DNA in blood. Yet other diagnostic investigations require very simple assays. Paper IV presents a novel one-step method to detect nucleic acid sequences by combining the power of rolling circle amplification and the specificity of DNA strand displacement in a format simple enough to be used at the point of care.   

Altogether, the thesis spans technologies for advanced molecular diagnostics, from sample collection over assay techniques to an improved readout.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1615
Keywords
molecular diagnostics, dried blood sample, DBS, digital readout, digital enumeration, DNA detection methods, proximity extension assay, protein stability, genotyping, rare mutations, cell free DNA, multiplex protein measurement.
National Category
Medical Biotechnology Biochemistry and Molecular Biology Medical Laboratory and Measurements Technologies
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-396325 (URN)978-91-513-0810-4 (ISBN)
Public defence
2019-12-20, A1:111a, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-28 Created: 2019-11-03 Last updated: 2020-01-13
Siart, B., de Oliveira, F. M., Shen, Q., Björkesten, J., Pekar, T., Steinborn, R., . . . Wallner, B. (2019). Protein measurements in venous plasma, earlobe capillary plasma and in plasma stored on filter paper. Analytical Biochemistry, 566, 146-150
Open this publication in new window or tab >>Protein measurements in venous plasma, earlobe capillary plasma and in plasma stored on filter paper
Show others...
2019 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 566, p. 146-150Article in journal (Refereed) Published
Abstract [en]

In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of rho > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019
Keywords
Cytokines, Earlobe capillary, Dried plasma spots, Extension assay, Inflammation protein biomarkers
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-376729 (URN)10.1016/j.ab.2018.11.016 (DOI)000456354900024 ()30472219 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 316929
Available from: 2019-02-08 Created: 2019-02-08 Last updated: 2019-02-08Bibliographically approved
Landegren, U., Al-Amin, R. A. & Björkesten, J. (2018). A myopic perspective on the future of protein diagnostics. New Biotechnology, 45, 14-18
Open this publication in new window or tab >>A myopic perspective on the future of protein diagnostics
2018 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 45, p. 14-18Article, review/survey (Refereed) Published
Abstract [en]

Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2018
Keywords
Protein leakage markers, Liquid biopsy, Early detection of disease, Tissue-specific proteins, Exocrine secretion, Recombinant protein binders, Dual-recognition assays, Sandwich immune assay, Proximity ligation assay, Proximity extension assay, Dried blood spots, Consecutive patient samples, Wellness testing, Point of care
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-363863 (URN)10.1016/j.nbt.2018.01.002 (DOI)000441913800003 ()29309916 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research EU, European Research Council, 313010
Available from: 2018-11-14 Created: 2018-11-14 Last updated: 2018-11-14Bibliographically approved
Shen, Q., Björkesten, J., Galli, J., Ekman, D., Broberg, J., Nordberg, N., . . . Landegren, U. (2018). Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays. Clinical Chemistry and Laboratory Medicine, 56(4), 582-594
Open this publication in new window or tab >>Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
Show others...
2018 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 56, no 4, p. 582-594Article in journal (Refereed) Published
Abstract [en]

Background: A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles.

Methods: Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 degrees C or 22 degrees C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 degrees C and thawing at 22 degrees C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins.

Results: Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 degrees C or 22 degrees C, respectively. Some increases became noticeable after 8 h delay at 4 degrees C but already after 1 h at 22 degrees C. For samples stored at 4 degrees C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 degrees C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 degrees C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles.

Conclusions: Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH, 2018
Keywords
biobank, protein detection, proteome, proximity extension assay (PEA), sample collection and handling
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-350276 (URN)10.1515/cclm-2017-0648 (DOI)000426657400016 ()29040064 (PubMedID)
Funder
Swedish Research Council, 829-2009-6285EU, European Research Council, 313010, 294409
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2019-11-03Bibliographically approved
Björkesten, J., Enroth, S., Shen, Q., Wik, L., Hougaard, D., Cohen, A., . . . Landegren, U. (2017). Stability of Proteins in Dried Blood Spot Biobanks.. Molecular & Cellular Proteomics, 16(7), 1286-1296
Open this publication in new window or tab >>Stability of Proteins in Dried Blood Spot Biobanks.
Show others...
2017 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 7, p. 1286-1296Article in journal (Refereed) Published
Abstract [en]

An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4&deg;C or -24&deg;C.</p> <p>Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4&deg;C and -24&deg;C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24&deg;C compared to at +4&deg;C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4&deg;C or -24&deg;C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.

Keywords
Absolute quantification, Affinity proteomics, Biobanking, Bioinformatics splicing, Biomarkers, Blood*, DBS, Diagnostic, Dried Blood Spot, Multiplex protein detection, PCR, Plasma or serum analysis, Predictive markers*, Protein Stability, Proximity Extension Assay
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-322568 (URN)10.1074/mcp.RA117.000015 (DOI)000404597500009 ()28501802 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 294409Novo Nordisk
Available from: 2017-05-25 Created: 2017-05-25 Last updated: 2019-11-03Bibliographically approved
Lönn, P., Al-Amin, R. A., Heldin, J., Gallini, R., Björkesten, J., Oelrich, J., . . . Landegren, U.High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs.
Open this publication in new window or tab >>High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Interactions and posttranslational modifications (PTMs) of proteins orchestrate cellular responses to cytokines, drugs or other agents, but it has been difficult to monitor and characterize these dynamic events at high-throughput. Here, we have established a semi-automated system for large-scale in situ proximity ligation assays (isPLA). The protocol combines isPLA in microtiter wells with automated microscopy and computer-based image analysis whereby specific protein phosphorylations and interactions are digitally recorded in cells, along with measurements of morphological features. We demonstrate how this platform can improve analysis of cellular signaling by investigating TGF-b responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells. We depict single cell responses in relation to e.g. local cell crowding and cell cycle progression via measurements of DNA content and nuclear size. Finally, we illustrate the application of the protocol for demonstrating drug effects by screening a library of phosphatase inhibitors. In summary, our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.

National Category
Natural Sciences
Research subject
Biochemistry; Molecular Cellbiology; Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-374248 (URN)
Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
Björkesten, J., Patil, S., Fredolini, C., Lönn, P. & Landegren, U.Multiplex digital enumeration of circular DNA molecules on solid supports.
Open this publication in new window or tab >>Multiplex digital enumeration of circular DNA molecules on solid supports
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Digital PCR is a detection method with unprecedented accuracy for DNA quantification, but with some limitations in the form of complexity of instrumentation and limited multiplexing. Here we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy, to overcome limitations of digital PCR. In this method, sets of small DNA circles, resulting from detection reactions, are captured on a streptavidin-coated surface and subjected to rolling circle amplification (RCA). We found that the addition of 15% polyethylene glycol 4000 to RCA on planar surfaces ensured uniform, easily counted signals, each of which represents an individual reaction product. The DNA circles were immobilized and detected with efficiencies of 50 and 100%, respectively, as determined by droplet digital PCR. We confirmed previous reports about the effect on RCA efficiency by sequence composition and size of the RCA templates at the level of individual amplified molecules, and we developed an efficient one-step de- and re-staining procedure for sequential multiplexing via toehold-triggered DNA strand displacement.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-396324 (URN)
Available from: 2019-11-03 Created: 2019-11-03 Last updated: 2019-11-03
Organisations

Search in DiVA

Show all publications