**2. The Mechanism of Microparticle Formation**

The primary settings for MPs release are cellular activation and death. An increase in the intracellular concentration of calcium ions secreted by cytoplasmic reticulum is a response to multiple factors, including: An increase in the number of free radicals, increased shear force, adenosine diphosphate (ADP) secreted by activated platelets, expression of CD40 ligand (CD40L) on T-cells [26]. In these conditions, activation of calcium concentration-dependent enzymes takes place; these include gelsolin, which facilitates the separation of actin fibers from platelet cytoskeleton [27]; aminophospholipid translocase, which transports aminophospholipids from the outer membrane into the cell interior [28]; floppase, which transports phospholipids from the inner lipid bilayer outwards [29]; calpain, which destroys cytoskeleton actin fibers [30]; and scramblase, which affects transmembrane phospholipid transport [31] (Figure 1). The activated enzymes contribute to the loss of asymmetric distribution of phospholipids in the cell membrane in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) are present mainly in the inner cytoplasmic layer, and phosphatidylcholine (PC) and sphingomyelin (SM) are present in the outer layer of the lipid bilayer. The asymmetry loss process resulting from transferring phosphatidylserine and phosphatidylethanolamine to the outer layer of the cell membrane and simultaneous cytoskeleton destabilization allow the formation and secretion of microparticles [26,32].

**Figure 1.** Microparicle formation following cellular activation and cytoskeletal.

An increase in the intracellular concentration of calcium, as a response to cell activation or apoptotic stimuli, results in the activation of calcium concentration-dependent enzymes: Gelsolin, aminophospholipid translocase, floppase, calpain, and scramblase. During this process, membrane asymmetry is lost, leading to the exposure of phosphatidylserine and phosphatidylethanolamine normally present in the inner leaflet of the membrane bilayer. Destabilization of the cytoskeleton results in cellular contraction and membrane blebbing.

#### **3. Methods of Microparticle Detection**

There are no standardized microparticle testing techniques and each laboratory conducting such testing develops its own MPs detection methods and standards. However, common protocol among researchers is to start collecting MPs from blood with a centrifugation to collect platelet-free plasma to avoid the activation and subsequent release of microparticles from platelets. Microparticle identification is a technical challenge because they are much smaller than cells of origin (diameters of 10–100× less). Microparticles can be isolated from blood, other biological fluids, or from cell cultures. Since MPs are cell-derived structures, a cytometric test is the "gold standard" and is the most widely

used method of microparticle detection on the basis of light scattering as well as binding of marker antibodies to identify the cell of origin [33]. The MPs population to the flow cytometry settings is defined by using size calibration beads. However, MPs smaller than approximately 0.5 μm in diameter are not efficiently resolved by conventional flow cytometers. Available digital flow cytometers do not count all MPs because of their limited forward scatter (FS) sensitivity [34]. Recently, high-sensitivity flow cytometers with significantly improved light scatter detection became available and provide sufficient size resolution for the identification of MPs subtypes [35]. Microparticles generally expose phosphatidylserine and membrane antigens of their parent cells. MPs as cell-derived membrane structures in the cytometry test are commonly determined with annexin V, usually stained with phycoerythrin and antibodies against specific cell antigens, stained with fluorescein isothiocyanate. Labeled annexin V binds mainly to phosphatidylserine, which is typical of all types of microparticles and which is present on the MPs membranous surface and less frequently in membrane permeability disorders; it can also connect to phosphatidylserine located inside microparticles [36]. Annexin V is often used to identify microparticles, but some MPs may not show a tendency to bind to this protein [37]. The use of antibodies against different membranous antigens enables identification of the cellular origin of microparticles.

Another method of microparticle determination is based on ELISA (enzyme-linked immunosorbent assay), which makes use of test plates coated with annexin V or antibodies specific to cell membrane antigens [38]. When total phosphorus or phospholipid activity is determined, ELISA enables quantitative determination of microparticles [39].
