Chapter 4 98 DISCUSSION We successfully characterized and phenotyped single uEVs in healthy and KTR urine without prior isolation using IFCM in this study. Our protocol is based on the absolute sizing of EVs with the Mie Theory to realize cross-platform reproducibility.23,29,37 Conversion of SSC signals into particle size has been demonstrated for our instrument to selectively analyze plasma-derived EVs ≤400 nm.23 Here, we expanded the analysis range to investigate uEVs ≤1200 nm because larger uEVs might also be meaningful as a biomarker. Podocytes can release uEVs (≥400 nm) containing abundant RNA and protein markers, serving as an indicator of kidney injury.14,38 In addition to size, EV shape or membrane orientation might also be of interest, but EV signals with IFCM are indicated with only a few pixels. Therefore other uEV morphology information, such as shape, is challenging to be explored. The most significant novelty and improvement is the exclusion of urinary A-F particles from uEVs in minimally processed urine. The presence of autofluorescence/ A-F particles is a natural property of urine.14,39 A-F particles presented similar fluorescent characteristics in all the urine samples (HC or KTR). Those particles can be substantially excluded from uEVs with the same gate and compensation matrix (Supplementary Figure S5), indicating possibly no necessity to adjust the gate or compensation matrix when measuring different urine samples. Following published FCM research,13,14 we also found that A-F particles in all urine samples showed fluorescence with emission wavelengths 505-595 nm and 642-745 nm. The broad emission wavelength range indicates that A-F particles hinder specific fluorescent EV detection using many typical fluorophores, such as Alexa488, fluorescein isothiocyanate (FITC), PE, Alexa647, and APC. This problem might be avoided by using fluorophores with other emission wavelengths, such as near-infrared ones.40 Compared to uEVs, A-F particles might be larger/denser because A-F particles were removed by moderate-speed centrifugation while uEVs were maintained. Large uEVs are co-isolated with A-F particles in the low-centrifugation pellet.14 Luca et al. tried a masking strategy to delimit A-F in particular channels but observed that 30-40 % of“positive uEV” remained after detergent lysis.14 We found that detergent treatment could not entirely remove A-F particles (Figure 3B), suggesting a nonEV structure. We assume A-F particles likely remained in their readouts after their mask strategy. Droste et al. processed urine with a 200 nm filter and did not observe A-F particles using IFCM, but they detected about 104 objects/mL CD63+
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