Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 85 INTRODUCTION Extracellular vesicles (EVs) are phospholipid bilayers widely released by cells into body fluids, such as blood and urine. Their reported size ranges from 30 nm to 8000 nm, with most EVs <200 nm.1–3 EVs reflect parental cell status via variations in EV concentration, composition, or cargo and are considered minimally invasive biomarkers.1 Urinary extracellular vesicles (uEVs) are ideal biomarkers as urine collection is noninvasive and easily repeated.4 uEVs show meaningful values in diagnosing renal and urinary system diseases,5–7 and illnesses of other systems, such as Parkinson’s disease and liver cirrhosis.8,9 Despite the perspective as a clinical marker, uEV quantification and characterization are hampered because of their small size, urine contaminants, and lack of methods for accurate detection.1,10 Nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), and flow cytometry (FCM) are the most commonly used singleEV-quantification techniques.1 However, NTA and RPS are limited in phenotyping capabilities, struggling to distinguish uEVs from other particles, such as protein aggregates.1,11 Although some modern flow cytometers can detect small EVs (< 100 nm) based on light scattering, most flow cytometers in clinical research labs have a size detection limit of >600 nm.12 Moreover, some particles in urine emit autofluorescence, leading to false-positive signals in FCM, regardless of labeling.13,14 The origin of these autofluorescent (A-F) particles is still unclear and how to distinguish them from uEVs needs more research. The direct measurement of uEV is also hampered by Tamm-Horsfall protein (THP), a highly abundant urinary protein, easily entrapping uEVs.13–16 Due to the limitations of traditional techniques and the complex composition of urine, uEV purification is commonly required before detection.17 However, no (combination of) isolation methods, including ultracentrifugation, ultrafiltration, precipitation, or size exclusion chromatography, can reach 100 % uEV purity and yield due to significant loss of uEVs or co-isolation of other particles.1,17 Some purification procedure likely alters uEV properties.14,18–20 Ultrafiltration can disintegrate large EVs to generate smaller particles, misunderstood as natural EVs.18 Ultracentrifugation might cause EV aggregation and encapsulation of multiple small uEV inside bigger uEV.14,19,20
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