AccuCount Ultra Rainbow fluoreszierende Partikel

Blood and other body fluids contain cell-derived extracellular vesicles (EVs), which are small pieces of cytoplasm surrounded by a lipid membrane. Electric vehicles are usually classified according to their formation mechanism and their size. EVs, called microparticles or microvesicles, are submicron vesicles shed from the cytoplasmic membrane, while exosomes are 50–100 nm vesicles secreted from cells by exocytosis of multivesicular bodies. In this article, the term EV is used to denote all types of cell-derived vesicles found in plasma.

Originally thought of as cellular debris or waste particles, there is now increasing evidence that EVs are involved in numerous functions. 

In the blood, EVs are involved in physiological processes of coagulation, inflammation, or intercellular communication, while elevated EV levels have been reported in numerous diseases, including cardiovascular disease, cancer, sepsis, and autoimmune diseases. Electric vehicles that release the procoagulant lipid phosphatidylserine (PS) have attracted a lot of interest for several reasons.

  • According to the classical theory of EV formation at the plasma membranes of cells, the exposure of PS molecules on the outer membrane sheet represents an early step in the cell activation process that precedes membrane blebbing and EV shedding. Most studies on plasmatic EVs agree with this theory agree and assume that PS-exposing EVs represent the majority or even all of EVs. In addition, PS and tissue factor-exposing EVs have been associated with thrombotic events associated with various pathologies. 
  • Furthermore, the physiological importance of PS-exposing EVs in hemostasis is underscored by the fact that two bleeding disorders, Scott syndrome and Castaman’s defect, are characterized by impairment in the generation of PS-exposing EVs.
  • However, despite intensive research, current knowledge about electric vehicles is still limited. This is mainly due to the small size of EVs, most of which are smaller than 500 nm‘ and intrinsic limitations of the methods used for their characterization. For the past two decades, flow cytometry (FCM) has been the main method used to characterize electric vehicles.
  • In the classic FCM approach, referred to below as conventional FCM, objects are detected in a two-stage process on the one hand based on their light scattering intensity, which must exceed a threshold value , and on the other hand based on their specific labeling with fluorescent ligands. This approach has demonstrated the existence of EVs of different cellular origins in different body fluids.
  •  However, major issues have arisen regarding the actual size and quantity of EVs detected by FCM. In particular, it is now well known that polymer particles, originally used as size calibrators, are not suitable references for EV sizing due to their different light scattering properties. Recent theoretical studies have reported that 500 nm polymer particles scatter light similar to 800 nm EVs on a standard flow cytometer.
  •  In addition, we recently showed experimentally that only 1% to a few % of horsepower-exposing EVs observed by quantitative electron microscopy (EM) were detected by conventional FCM.

Other methods have been used to detect and quantify EVs, including EM, atomic force microscopy (AFM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), or resistive pulse sensing (RPS). 

These methods offer specific advantages, as discussed recently in . However, FCM remains the method of choice for phenotyping large numbers of samples as required in the biomedical context. It’s worth noting that small object detection is not a new topic in FCM. More than 30 years ago, special flow cytometers were developed that could detect 100 nm viruses based on their light scattering properties. Recently, Marie  et al. leisteten Pionierarbeit bei einer alternativen Strategie, die darin bestand, den Nachweis von Viren anhand eines Fluoreszenzparameters anstelle eines Lichtstreuungsparameters auszulösen. Auf dem Gebiet der Elektrofahrzeuge wurde dieser Ansatz selten angewendet , aber seine Leistungsfähigkeit wurde kürzlich durch den Nachweis von Elektrofahrzeugen in Zellkulturen mit einem High-End-Durchflusszytometer und einer originellen Markierungsstrategie veranschaulicht, in die ein lipophiler Fluorophor eingebaut wurde EV-Membranen .

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Here we present a simple method to detect PS-exposing EVs from plasma samples by fluorescence triggering (FL). Furthermore, we compare results obtained with this method and with a recently introduced quantitative EM approach to count electric vehicles.

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