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Abstract: TH-PO968

Urinary Extracellular Vesicles (uEVs) Have Unique Characteristics as Demonstrated by Imaging and Spectral Cytometry

Session Information

Category: Pathology and Lab Medicine

  • 1501 Pathology and Lab Medicine: Basic

Authors

  • Musante, Luca, University of Virginia, Charlottesville, Virginia, United States
  • La salvia, Sabrina, University of Virginia, Charlottesville, Virginia, United States
  • Lannigan, Joanne, University of Virginia, Charlottesville, Virginia, United States
  • Erdbruegger, Uta, University of Virginia Health System, Charlottesville, Virginia, United States
Background

Urinary extracellular vesicles (uEVs) provide a source of valuable biomarkers for kidney and urogenital diseases. Analysis of uEVs is challenging due to its intrinsic complexity. Imaging flow cytometry (iFC) allows detection of particles that are < 200 nm in size and has a high level of sensitivity for small particle fluorescence. In addition, spectral flow cytometry (sFC), which is based on whole spectrum analysis, can be used to further characterize the findings of the iFC analysis.

Methods

First morning void urine, blood and saliva (internal auto fluorescent control) were centrifuged at relative centrifugation force RCF of 4.600g for 30 minutes. The supernatants was further centrifuged at 20,000g to collect EVs. uEVS were stained with different annexin V conjugates: FITC, PE, PerCPCy5.5, Pacific Blue™, Brilliant Violet 421, Brilliant Violet 510, APC, Alexa Fluor® 647 respectively. Gating strategy was based on the low scatter of the unstained uEVs and the negative control was all fluorescent probes alone in buffer. Stained and unstained EVs from urine, plasma and saliva were evaluated

Results

Acquisition of uEVs alone in iFC showed an interesting auto-fluorescence emission in channel 5 (λex 660 nm; λem 740 nm) for camera 1 and channel 11 (λex 660 nm; λem 740 nm) for camera 2. Auto fluorescence emission in channel 11 was caused by excitation from the violet laser (λex 405 nm) and red laser (λex 642 nm). Auto-fluorescence in Channel 5 was caused by excitation of both blue (λex 488 nm) and yellow laser (λex 561 nm). Spectral analysis of unlabelled uEVS, plasma EVs (pEVs), and saliva EVs (sEVs) showed that this auto-fluorescence was unique and specific for uEVs. Spectrum plots showed a distinct signature

Conclusion

While imaging flow cytometry represents a major advancement in the identification of uEVs, our results suggest that unexpected additional complication of the analysis originated from the auto-fluorescence with a peculiar spectral emission that needs to be taken into account when multicolor antibodies panels are planned. Likewise, choice of AV fluorochrome conjugate should be carefully considered.

Funding

  • Other NIH Support