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Abstract: FR-PO942

Analysis of Urinary Extracellular Vesicle Autofluorescence by Imaging Flow Cytometry and Spectral Flow Cytometry

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology


  • 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

Urinary extracellular vesicles (uEVs) provide a source of valuable biomarkers for kidney and urogenital diseases. Analysis of uEVs in imaging flow cytometry is challenging for its intrinsic auto fluorescence emission across the whole electromagnetic spectrum. To date it is not known what the rate of the autofluorescence interference is with respect to the detection of specific uEVs markers


First morning void urine and citrate blood from the same donor were centrifuged at relative centrifugation force RCF of 4,600 g for 30 and 15 minutes respectively. The supernatant was further centrifuge at relative centrifugation force of 20,000g to collect urinary (uEVs) and plasma (pEVs) which were stained with the same commercial clone antibody (3D3) anti podocalyxin (PODXL) conjugated with 3 different fluorescent dyes: Alexa Fluor® 405 (AF405), Alexa Fluor® 488 (AF488) and Alexa Fluor® 647 (AF647). Stained EVs were acquired with both imaging flow cytometry and spectral flow cytometry. Gate strategy was based on the low scatter of the unstained uEVs and the negative control was the fluorescent probe alone in buffer.


Acquisition of uEVs alone showed auto-fluorescence emission in channel 2 (λex488 nm; λem 480-560 nm) camera 1 and channel 11 (λex658 nm; λem 660-740 nm) but not channel 7 (λex405 nm; λem 420-505 nm) for camera 2 for the imaging flow cytometry meanwhile the spectral flow cytometry revealed a spectral fingerprint spanning from the violet to the red emission. Autofluorescence was detected for uEVs but not pEVs. Podocalyxin-AF405 conjugated stained both uEVs and pEVs with a double staining for the autofluorescence and PODXL on the same uEV. While PODXL-AF488 and AF647 stained better pEVs than uEVs as per PODXL-AF405. Same results were obtained for both flow cytometry instruments.


Our results showed an unexpected additional complication of the analysis of uEVs in flow cytometry originated from the auto-fluorescence of the uEVs fraction. In fact, the autofluorescence quenched the emission of PODXL-AF488 and AF647 but not AF405. Moreover, uEVs auto-fluorescence needs to be taken into account especially when simultaneous co-detection of uEVs markers of podocyte origin is planned with particular emphasis on the critical selection of the antibody conjugated fluorescent dye.