Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters

Abstract

Numerous diseases, recently reported to associate with elevated microvesicle/ microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidinbiotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic lightscattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease. (Blood. 2011;117(4):e39-e48) IntroductionMembrane vesicles are small subcellular structures surrounded by a phospholipid bilayer. Their release by various cell types is enhanced during activation and apoptosis. 1 They represent heterogeneous structures and can be classified into several groups depending on their size, antigenic features, and mechanism of cellular release. 1 The two best characterized categories include exosomes and microvesicles/microparticles (MPs). Both populations are characterized by the exposure of phosphatidylserine, which allows annexin-V (AX) to bind to these-lipid surfaces. 1 Exosomes are composed of small, 50-to 100-nm-sized structures released on exocytosis of multivesicular bodies. 1 The diameter of MPs, formed by membrane blebbing, is described to be 100 to 1000 nm 2 . However, precise definitions of MPs are still lacking. 1,2 MPs are found in various biologic fluids, including blood plasma, 3 urine, 4 and synovial fluid (SF). 5,6 Numerous flow cytometry (FC) studies using blood plasma have shown correlation of MP counts with human cardiovascular 7 and autoimmune diseases, 8 hematologic disorders, 9 and cancer. 10 Of particular interest, autoimmune diseases were reported to be characterized by elevated levels of MPs. 11 The assessment of exosomes and MPs is complicated by the presence of further known categories of membrane bound subcellular structures, such as apoptotic vesicles, exosome-like vesicles, membrane particles, and ectosomes. 1 There is a substantial size overlap among the aforementioned vesicle categories, and the size distribution of a given vesicle preparation may also be affected by the method used for their isolation. 3,12,13 Recently, attempts have been made to standardize isolation and detection protocols for membrane vesicles. 3,14 Up until now, no systemat...

Changing world of neutrophils

“…In flow cytometry the distinction of the smaller vesicles from the noise can be a hard task. Furthermore, immune complexes were shown to overlap with smaller vesicles [42], thus the vesicular structure should always be verified. Most convenient -and reliable -is the detection of vesicles above 300 nm stained with specific fluorescent ligand.…”

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube

“…2B). MP gate was set on the FSC/SSC plot as described previously [2,12,18] ( Supplementary Fig. 2B and C).…”

“…e l s e v i e r . c o m / l o c a t e / t h r o m r e s flow cytometer [2] and iv) presence of protein aggregates [12] or calcium phosphate microprecipitates [13] that mimic MPs during flow cytometry. Recently, in joint efforts supervised by the International Society on Thrombosis and Haemostasis (ISTH), we and others suggested standard pre-analytical and analytical procedures for the detection of MPs [2,3,10].…”

“…2D). We applied our earlier protocol for staining of MPs in biological fluids [12]. Briefly, 1 μg of anti-CD42a-PerCP antibody (BD Biosciences) was added to 20 μL PFP, and incubated for 30 minutes at RT in dark.…”

Microparticles released by Listeria monocytogenes-infected macrophages are required for dendritic cell-elicited protective immunity

“…Such fluorescent MPs were observed under two-photon fluorescent microscopy (LSM 710 and ConfoCor 3 Microscope Systems; Carl Zeiss, Jena, Germany) or analyzed by flow cytometry, as described previously. 17 Count of MPs Isolated MPs were suspended in 250 ml PBS with 1 ml Polystyrene Latex Beads (LB-30; Sigma). After mixing, the number of MPs was counted by a flow cytometer in accordance with their respective bead sizes.…”