Characterization of Extracellular Vesicles in Plasma of Pregnant Women Using Multicolor Flow Cytometry and Nanoparticle Tracking Analysis

Abstract

previously been studied using flow cytometry based on different syncytiotrophoblast specific antibodies. In most of these studies, indirect antibody labeling was applied, which, compared to direct labeling, is more susceptible to nonspecific binding. Systemic inflammation, which is a characteristic feature of PE, leads to activa tion of cells circulating in maternal blood including leucocytes, endothelium, platelets, and red blood cells (RBC) with increased release of EVs. Therefore, the authors of this study decided to explore EVs of these cells together with STBMs in samples of platelet‐free plasma (PFP) in nonpregnant (Non‐ P) controls, normal pregnant (Norm‐P), and women with late onset PE. Correct identification of the proper phenotype is crucial for any study on quantification and functional characterization of STBMs and EVs since the STBMs in plasma from pregnant women only account for a minor fraction of all EVs and because of considerable cr oss reactivity with blood‐cell derived EVs. This identification was achieved by multicolor flow cytometry using directly conjugated antibodies targeting multiple markers of EVs. This very unique approach reduces the problem of unspecific binding. For the development of this assay, EVs were prepared in vitro from pure populations of leucocytes, endothelium, platelets, and RBC and were examined for suitable antibodies to analyze the corresponding EVs in vivo. Antibody labeling of EVs was not only compared to parent cells, but each marker was tested for cross reactivity with STBMs. STBMs were prepared in vitro from maternal‐side eluate of dual‐ perfused placenta lobes (pSTBM). It has been shown before that this elegant method is superior to other in vitro techniques, such as mechanical dissection of placental tissue or culture of villous explants, in providing functionally intact STBMs. Based on the in vitro data, a panel of markers was defined for in vivo characterization of STBMs simultaneously with EVs of the different intravascular cell types using flow cytometry. EVs including STBMs were prepared by ultracentrifugation of platelet free plasma samples of three groups of women: nonpregnant controls, normal pregnant, and women with late onset PE. After labeling with two separate antibody panels, the EVs wer e subjected to flow cytometry. The first panel with three color labeled antibodies aimed to identify the placental‐derived EVs; the second panel with five color labeled antibodies was shown to be very effective in identifying EVs derived from platelets, RBC, leucocytes, and endothelial cells.