Morten Mørk

Preanalytical, analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing

Abstract Background: Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) enable measurement of extracellular vesicles (EVs) in blood plasma but also measure other particles present in plasma. Complete isolation of EVs from similarly sized particles with full EV recovery is currently not possible due to limitations in existing isolation techniques. Aim: This study aimed to evaluate preanalytical, analytical, and biological variation of particle measurements with NTA and TRPS on blood plasma. Methods: Blood from 20 healthy subjects was sampled in the fasting and postprandial state. Platelet free plasma (PFP) was analyzed immediately and after a freeze-thaw cycle. Additionally, the effect of prandial state and a freeze-thaw cycle on EV-enriched particle fractions obtained via size-exclusion chromatography (SEC) was examined. Results: We observed analytical linearity in the range of 1.0–10.0 × 108 particles/mL for NTA and 1.0 × 108–1.8 × 109 particles/mL for TRPS. The analytical variation was generally below 10%. A considerable intra- and inter-individual variation was demonstrated with estimated reference intervals of 1.4 × 1011–1.2 × 1012 particles/mL for NTA and 1.8 × 108–1.6 × 109 particles/mL for TRPS. Food intake and to a lesser extent a freeze-thaw cycle affected particle populations in PFP and, similarly, in EV-enriched fractions. Conclusion: In this study NTA and TRPS enabled acceptably precise concentration and size measurement of submicron particles in PFP. An appreciable intra- and inter-individual biological variation was observed. In studies on particle populations in PFP or EV-enriched fractions, we recommend analysis of fresh, fasting samples.

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles

ABSTRACT Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32–72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8–43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.

Postprandial Increase in Blood Plasma Levels of Tissue Factor–Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

Postprandial increase in blood plasma concentration of tissue factor-bearing extracellular vesicles

Introduction: Besides providing nutrition, breast milk delivers important signals that stimulate the infants developing immune system. It has been postulated that extracellular vesicles (EV) in milk support the instruction and/or development of neonatal immunity. However, little is known about the composition of milk-derived EV, partly due to the difficulty to purify EV from other components in milk. Methods: In this study, an extensive LC-MS/MS proteomic analysis was performed, whereby EV were isolated from breast milk of 7 individual donors using our recently established optimized density gradient-based isolation protocol [1]. High-density, non-floating complexes were included to compare the contents of EV to other macromolecular structures in milk. A comprehensive protein network was composed tracing the possible cellular origins of milk-derived EV and the potential targets in the gut. Results: An average of 579 proteins was identified in EV, compared to 205 proteins in the non-floating fraction. Interestingly, EV associated proteins like ANXA5 and Flotillin were exclusively identified in EV, while CD9, CD63 and CD81 were also present in non-floating protein complexes. Additionally, MHC-II was identified in the EV fraction only, suggesting that antigenic epitopes may be delivered via EV released from antigen-presenting cells. Besides MHC-I, the mammary epithelial cell marker beta-1,4-galactosyltransferase (lactose subunit) was identified in the EV fraction only, demonstrating EV of epithelial origin. Furthermore, several adhesion molecules (ICAM-1, CEACAM-1) were associated to EV which could allow EV binding to gut epithelial cells and gut resident immune cells. Summary/conclusion: In-depth proteomic analysis and compilation of an extensive network of EV proteins involved in immunity demonstrates that milk-derived EV originate from multiple cellular sources and have the ability to target various cell types in the gut.ISEV 2015 is organized by The Local Organizing Committee: Kenneth Witwer (Chair, Baltimore), Shilpa Buch (Omaha), Prasun Datta (Philadelphia), Dolores Di Vizio (Los Angeles), Uta Erdbrügger (Charlottesville), Steven Jay (College Park), Dimitrios Kapogiannis (Baltimore), Leonid Margolis (Bethesda) & Susmita Sahoo (New York) Together with the Executive ISEV Board (2014 – 2016) President: Jan Lötvall Secretary General: Clotilde Théry Treasurer: Fred Hochberg Executive Chair Science / Meetings: Marca Wauben Executive Chair Education: Yong Song Gho Executive Chair Communication: Andrew Hill Members at Large: Peter Quesenberry, Kenneth Witwer, Susmita Sahoo, Dolores Di Vizio, Chris Gardiner, Edit Buzas, Hidetoshi Tahara, Suresh Mathivanan, Igor Kurochkin

Comparison of intracardiac and venous blood levels of microvesicles, tissue factor and procoagulant phospholipids in patients with and without atrial fibrillation.

Reference EPFL-CONF-212539doi:10.1111/jth.12993View record in Web of Science Record created on 2015-09-28, modified on 2017-05-12

Removal of lipoproteins from human plasma by anti-Apolipoprotein B antibody-coated magnetic beads

ISEV 2014 is organized by Local Organizing Committee: Marca Wauben (Utrecht), Raymond Schiffelers (Utrecht), Michiel Pegtel (Amsterdam), Rienk Nieuwland (Amsterdam), Hans van Leeuwen (Rotterdam) & Esther Nolte-’t Hoen (Utrecht) Together with Executive ISEV Board (2012 – 2014) President: Jan Lötvall Secretary General: Clotilde Théry Treasurer: Janusz Rak Members at Large: Yong Song Gho, Dwidjendra Gupta, Andrew Hill, Fred Hochberg, Peter Quesenberry, Lawrence Rajendran, Douglas Taylor, Marca Wauben Adjunct members: Chris Gardiner, Melissa Piper †, Hidetoshi Tahara Diamond Sponsor: Caris Life Sciences