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Dried blood sampling and digital readout to advance molecular diagnostics
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala universitet.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
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: urn:nbn:se:uu:diva-396325ISBN: 978-91-513-0810-4 (print)OAI: oai:DiVA.org:uu-396325DiVA, id: diva2:1367388
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: 2019-11-28
List of papers
1. Stability of Proteins in Dried Blood Spot Biobanks.
Open this publication in new window or tab >>Stability of Proteins in Dried Blood Spot Biobanks.
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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
2. Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
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
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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
3. 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
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(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
4. Rolling circle amplification reporters – a general tool to simplify molecular detections
Open this publication in new window or tab >>Rolling circle amplification reporters – a general tool to simplify molecular detections
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Methods to detect biomolecules via rolling circle amplification (RCA), for example padlock probes and proximity ligation assay (PLA) tend to be complex and include several reaction steps. Herein, we evaluated a new tool for the toolbox of RCA-based detection methods - RCA Reporters. Briefly, RCA Reporters represent inert DNA structures that, upon contact with a specific nucleic acid sequence, unravel via a highly specific strand displacement process to initiate local enzyme-assisted RCA reactions. The RCA Reporters can be used to directly detect ssDNA or RNA in a sample, or proteins via oligonucleotide-conjugated antibodies. The reagents can also enable faster RCA reactions or extremely selective genotyping of RCA products with repeated copies of a target sequence through a majority-vote mechanism. Further amplification of ongoing RCA reactions via RCA Reporters can allow efficient digital enumeration of single molecules via flow cytometry, with potential for simple and highly accurate molecular counting assays. The intrinsic simplicity of RCA Reporter also renders them attractive for applications at the point of care.

National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331743 (URN)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-11-03
5. Rare Mutation Detection in Blood Plasma Using sRCA Molecule Counting Probes
Open this publication in new window or tab >>Rare Mutation Detection in Blood Plasma Using sRCA Molecule Counting Probes
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Problems in biology and medicine frequently require the ability to observe, evaluate, andcount even extremely rare macromolecules in biological samples. In particular, rare tumorspecific mutations in plasma provide valuable insights in the course of malignant disease andresponses to therapy, but simpler assay techniques are needed. We describe herein a rapidand exquisitely specific means to recognize and magnify detection signals from individualmolecules to easily recorded levels via a process we call super rolling circle amplification(sRCA). We demonstrate the ability of this technique to enumerate tumor-specific sequencevariants in plasma from cancer patients via flow cytometry at very high efficiency, with specificityadequate to detect single nucleotide mutant sequences among 100,000 copies of thenormal sequence in a 3 hr protocol. And the mutation analysis data generated from patientctDNA samples with our sRCA method are in high accordance with the patients’ primary tumorsequencing data.

Keywords
Rolling circle amplification, cfDNA, single molecule, digital counting, rare mutation detection, PoC application
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331737 (URN)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-11-03

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