Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 151 INTRODUCTION Extracellular vesicles (EVs) are lipid bilayer-delimited membrane particles (30-8000 nm in diameter1) excreted by all cell types, which act as signaling intermediaries during normal homeostasis and during pathologic processes 2-6. EVs carry proteins on their surface and/or a variety of macromolecules as cargo (e.g. DNA, RNA, lipids and proteins 7), which are thought to reflect the status of their cell of origin. Indeed, EVs are regarded as “snapshots” of the status of their cell of origin and are examined to assess the presence of various diseases, e.g., cancer or viral infection 8, 9. As EVs are present in body fluids (e.g. blood1 / saliva10 / urine11, 12), they are considered to be minimally-invasive biomarkers and so-called “liquid biopsies” 2, 13-16. The quantification and characterization of EVs is hampered by their physical characteristics such as their small size, low epitope copy number 17, and the variety of protein markers depending on the cell source 18, 19, all of which contribute to the well-documented EV heterogeneity. The identification of EVs in plasma is further hindered by the molecular complexity of the plasma, which contains multiple elements (e.g., lipoproteins, cell debris and soluble proteins), that interfere with EV analysis 18, 20. Moreover, a lack of robust methods and ambiguities in how data should be interpreted for EV analysis, makes comparison between studies challenging 21, 22. To overcome the selection biases or introduction of artefacts that are introduced by performing commonly used EV isolation methods 18, 20, our group recently developed a standardized Imaging Flow Cytometry (IFCM)-based methodology for the direct measurement of single EVs ≤400 nm in diameter in diluted plasma samples, without prior isolation of EVs 23. By omitting the need for sample isolation, this method has the potential to directly show the status of an individual by measuring distinct EV subsets, which is greatly beneficial for the monitoring of EVs in health and disease 21, 22. Expanding upon the previously reported ability of our methodology to directly detect and discriminate human- and mouse-derived EVs in mixed human/mouse plasma samples 23, we here aimed to assess the ability of our protocol to identify, discriminate and analyze EV subsets within human patient plasma samples. To this end, the setting of clinical organ transplantation offers a unique scenario in which tissue-specific EVs originating from the allograft are released into circulation after transplantation 14, 24, 25. We present an application of our methodology that allows for the direct detection of donor-derived EVs (dd-EVs) in plasma samples from kidney
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