Cellular microparticles (MPs) are fragments of the plasma membrane that are shed by virtually all cell when subject to a number of stress conditions, including cellular activation and apoptosis (Beaudoin and Grondin 1991; Zwaal and Schroit 1997; Freyssinet et al 1999; Hugel et al 2005). Since the description of “platelet dust” by Wolf (1967), numerous studies have reported the presence of subcellular vesicles in centrifuged plasma. Although long considered to be cellular debris, blood MPs are more recently considered reflective of cellular stimulation, activation, and degeneration/apoptosis (Freyssinet 2003; Morel et al 2005; Boulanger et al 2006). By general consensus, MPs are small in size (≤1.5 μm), expose the anionic phospholipid (PL) phosphatidylserine (PS) on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cell of origin (Jy et al 2004). MPs that arise from the cellular components of blood and the endothelial lining of blood vessels are referred to as blood MPs. Apart from blood MPs, plasma contains smaller membrane vesicles (40–100 nm) termed exosomes and larger vesicles (>1.5 μm) termed apoptotic bodies that are derived from blood and vascular cells (Heijnen et al 1999; Freyssinet 2003). The aim of this article is to review the technical aspects of characterizing the morphological and functional properties of blood MPs with emphasis on both preanalytical and analytical variables.

All cell type’s spontaneously shed blebs from their plasma membranes in an active process that appears to be important for membrane remodeling and antigenic turnover. In resting cells, the negatively charged phospholipids, including PS, are almost exclusively present in the inner leaflet of the cell membrane (Zwaal and Schroit 1997). Among the mechanisms proposed to account for this asymmetry are unidirectional enzymatic movement by an aminophospholipid translocase and/or association between the negatively charged phospholipids and elements in the cytoplasm, including cytoskeletal elements and their accompanying proteins (Bevers et al 1998). MP release is an integral part of the cell membrane remodeling process whereby the normal asymmetric distribution of phospholipids between the two leaflets of the plasma membrane is lost. Calcium entry, activation of calpain and scramblase activity are thought to be important steps in the loss of lipid asymmetry and microparticle formation, but the exact sequence of events is not completely understood (Fox et al 1990; Yano et al 1994; Dachary-Prigent et al 1995, 1997; Pasquet et al 1996, 1998; Bucki et al 1998, 2006; Kunzelmann-Marche et al 2001). The possible pathways leading to membrane budding and MP release have been recently reviewed (Freyssinet 2003; Boulanger et al 2006).

In general, most assays have focused on characterizing the antigenic composition of cellular microparticles using antibody capture-based ELISA (Aupeix et al 1997; Freyssinet et al 1999; Mallat et al 1999) or flow cytometry (Combes et al 1997, 1999). Most investigators have elected to characterize blood MPs using flow cytometry and the technical protocols of some have been reviewed in a recent forum (Jy et al 2004). In addition to analyzing MP antigens, several investigators have assessed the coagulant function of blood microparticles using functional assays (Westerman et al 1984; Mallat et al 1999; Berckmans et al 2001; Joop et al 2001; Shet et al 2003; Aras et al 2004). These assays either measure acceleration of plasmatic coagulation by MP-derived phospholipids or triggering of plasmatic coagulation by MP-associated tissue factor. The PF3 assay, in which exposure of platelet poor plasma to Russell’s viper venom in the presence of calcium causes coagulation, but only in the presence of certain phospholipids (supplied by blood MPs), has also been used as a functional assay (Hardisty and Hutton 1965).

Flow cytometry Flow cytometric analysis of blood MPs appears to be the most favored method to characterize blood MPs (Jy et al 2004). Typically, MPs are identified as particles with a forward angle light scatter (FALS) smaller than an internal standard consisting of 1–1.5 μm sized latex particles (Shet et al 2003). Most investigators do not use a lower size limit but some have arbitrarily chosen 100 nm below which particles that are recognized by the instrument are not considered MPs (Boulanger et al 2006). Others have used platelets as an internal standard and gated particles with a FALS value below that of the smallest platelet (Nieuwland et al 2000). While the identification of MPs on the basis of size using FALS tests the limit of sensitivity of flow cytometry, some have overcome this problem by adjusting the parameters of the instrument to detect fluorescent intensity (Horstman et al 1994). Flow cytometry can also be used to enumerate blood MPs by adding a known number of fluorescent or nonfluorescent latex particles to the sample prior to performing analysis (Combes et al 1997, 1999; Shet et al 2003). The number of MPs present in the sample is derived by dividing the number of MP events counted by the volume of the sample analyzed, ie, (beads counted/beads added) × sample volume, and adjusting for the final dilution of the original blood sample. Alternatively, if the flow cytometry instrument delivers the sample to the optics cell by screw-driven syringe at a known rate, then the sample MP count can theoretically be calculated. This method of enumeration is less desirable since it makes assumptions regarding the instruments fluidics and has unacceptable day-to-day variability (Reverter et al 1996).

Antibody capture assays ELISA-based MP capture assays have been devised and tested to simplify and semi-automate the characterization of blood MPs. These assays have utilized annexin V as a means of capturing phospholipids (Aupeix et al 1997; Mallat et al 1999) or antibodies to specific blood MP membrane antigens, eg, αIIbβ3 (Mallat et al 2000), or antibodies to nonspecific blood MP membrane antigens that are shared by several cell types eg, antibody 1B5 (Aras et al 2004) and have been reviewed elsewhere (Freyssinet et al 1999). Such assays have reduced the challenges of analyzing large sample numbers.

Other assays In addition to the above techniques, the antigenic characteristics and the membrane composition of blood MPs have been studied using electron microscopy (Heijnen et al 1999; Aras et al 2004) (Figure 1 and Figure 2), confocal microscopy (Combes et al 1999), high performance liquid chromatography (Weerheim et al 2002), capillary electrophoresis (Xiong et al 2003), and mass spectrometry (Jin et al 2005; Miguet et al 2006) with varying degrees of success. Assays that measure the pro/anticoagulant functions of blood MPs are somewhat under-utilized perhaps in part due to the technical challenges in developing standardized and reproducible assays.

Figure 1 Ultrastructure of blood MPs from a patient with sickle cell anemia using electron microscopy.

Figure 2 Immunogold labeling of blood MPs. Blood MPs obtained by ultracentrifugation were incubated with rabbit polyclonal anti-tissue factor antibody (a; 6 nm gold), anti-CD144 antibody (b; 12 nm gold), and anti-CD14 antibody (c; 12 nm gold). No labeling was (more ...)

The general consensus is that plasma levels of blood MPs reflect equilibrium between their release and their clearance by monocytes-macrophage system. Since clearance of cells undergoing apoptosis or injury is extremely efficient, the presence of blood MPs is considered an objective in vitro parameter that serves as evidence of deleterious cellular alterations in vivo. However, very little is published about the clearance and fate of blood MPs in any specific disease state leaving one to speculate that their presence reflects either a defect in recognition of these particles or an overwhelming of the clearance mechanism by their sheer number (Willekens et al 2005). Experimental evidence both in vitro and in vivo has demonstrated that blood MPs are capable of influencing diverse biological function (Zwaal and Schroit 1997; Barry and FitzGerald 1999; Horstman and Ahn 1999; Mesri and Altieri 1999; Freyssinet 2003; Hugel et al 2005; Boulanger et al 2006). Blood MPs have been associated with several disease processes and while they seem to be useful markers there is insuffiscient evidence to demonstrate that this association is causal. Clearly, elevated blood MPs in several disease processes result from inflammation, cellular activation and apoptosis. Such MPs, through their diverse biological effects, unequivocally contribute to vascular disease and comorbidity and are therefore likely to be very important in the disease process.

Several studies clearly demonstrate that blood MPs could be mediators of disease in some cases, markers of disease in other cases, and have prognostic potential, but the patho-physiological relevance of blood MPs is still in its infancy. Many of the studies mentioned in this review demonstrate that detection, phenotypic characterization, and quantification of blood MPs are interesting and potentially valuable in patients that are at risk for vascular events. However, conversion of this potential into a reality will necessitate a standardized approach to the detection and characterization of blood MP. In addition, attention to the preanalytical and analytical variables and refinement in the techniques used to assess the cellular origin of blood MPs could help shed light on the mechanisms of MP release, function, and clearance.

The author is grateful to Dr Omer Aras, Marcy Krumwiede, and Dr James G White for their excellent technical assistance and expertise with the electron microscopy studies. The author reports no conflicts of interest in this work.