title: Use of immuno-magnetic beads for direct capture of nanosized microparticles from plasma.
abstract: Increased microparticle tissue factor (TF) activity is not only found in cancer patients, but also in patients with cardiovascular and inflammatory diseases. Methods such as flow cytometry and impedance-based flow cytometry allow the analysis of microparticle subsets but provide no insight on which microparticles carry active TF. Conversely, the microparticle-TF activity itself does not reveal the cellular origin of the microparticles carrying the active TF.For this reason, we developed an immuno-magnetic bead method to capture subsets of microparticles directly from plasma. The method was optimized for capture of platelet-derived microparticles (PMPs) from plasma. Only 100 μl platelet-poor plasma (PPP) was needed in combination with 135 μl (27 μg) of biotinylated antihuman CD41 monoclonal antibody (MoAb) and 200 μl of streptavidin beads to achieve complete separation of PMPs from plasma. As a control, biotinylated mouse IgG1 isotype control MoAb was used instead of the anti-CD41 MoAb. Using biotinylated anti-CD14 MoAb, CD14-positive microparticles were captured from normal plasma spiked with microparticles isolated from the supernatant of lipopolysaccharide-stimulated monocytes (MoMPs). TF activity was found both in the positive (selected) and negative (depleted) fractions indicating that both CD14-positive and negative MoMPs carry active TF. We propose that this method can be used in the future to investigate the source of microparticles carrying active TF in plasma of patients with cancer and other diseases.
authors.affiliation: Department of Clinical Oncology, Leiden University Medical Centre, The Netherlands.
authors.forename: Yuana
authors.initials: Y
authors.lastname: Yuana
authors.forename: Susanne
authors.initials: S
authors.lastname: Osanto
authors.forename: Rogier M
authors.initials: RM
authors.lastname: Bertina
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D019921:Biotinylation; D001792:Blood Platelets / Q000737:chemistry; D055252:Cell-Derived Microparticles / Q000737:chemistry*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D018950:Lipopolysaccharide Receptors / Q000737:chemistry / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009000:Monocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D010949:Plasma / Q000737:chemistry*; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry / Q000276:immunology; D019809:Streptavidin / Q000737:chemistry; D013925:Thromboplastin / Q000032:analysis*
chemical_list: D000911:Antibodies, Monoclonal; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D040201:Platelet Membrane Glycoprotein IIb; D019809:Streptavidin; D013925:Thromboplastin