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Abstract: SA-PO1033

New Method of Immunophenotyping of Urinary Cells by Multicolour Flow Cytometry: A Pilot Study

Session Information

Category: Pathology and Lab Medicine

  • 1800 Pathology and Lab Medicine


  • Rychlík, Ivan, Univerzita Karlova 3 lekarska fakulta, Praha, Czechia
  • Habara, Peter, Univerzita Karlova 3 lekarska fakulta, Praha, Czechia
  • Krátká, Karolína, Univerzita Karlova 3 lekarska fakulta, Praha, Czechia
  • Havrda, Martin, Univerzita Karlova 3 lekarska fakulta, Praha, Czechia
  • Lazanska, Renata, Univerzita Karlova 3 lekarska fakulta, Praha, Czechia

Group or Team Name

  • Nephrology Unit.

Podocyturia,non-invasive option,was used to diagnose glomerulopathies(GN),namely fibrosis and glomerulosclerosis,previously detected only by renal histology(RB). Urinary sediment analysis after labelling with antibodies against podocalyxin and podocyte-specific transcription factor WT-1 was proven as clinically useful.Determination of mRNA was used,too, but hardly usable due to high instability.


Patients(pts) who underwent RB for nephrotic syndrome, regardless of its etiology,were examined before RB. First morning urine sample was collected.Urinary cell concentrates were prepared by ultrafiltration using strainers. Conventional staining protocol was used with panel of antibodies: anti-podocalyxin-PE,anti-CD86-FITC,anti-CD87-BV510,anti-CD24-Pe-Cy7,anti-CD10-PerCp,anti-CD133-APC,anti CD45-APC-Cy7. Heathy volunteers and isotype controls were used. The Hoechst33342 DNA dye was applied to identify cells.Gating strategy:only population of cells binding the DNA dye Hoechst33342 was selected to exclude non-nuclear elements,then exclusion of white blood cells,and then immunophenotypically specific rare cell populations could be observed in scatter plots.


13 pts with histology: DKD 5x, obesity-related glomerulopathy 2x, IgA nephropathy 5x, ANCA associated vasculitis 1x and 5 healthy controls were examined.
Population of PCX+/CD10+/CD133+/CD24+ cells was detected in varying degrees of abundance, as well as a population of PCX-/CD10+/CD133+/CD24+ cells. The former population predominated mainly in patients where the histological findings were dominated by degenerative changes (interstitial fibrosis and glomerulosclerosis). In contrast, the latter population was more prevalent in active inflammatory changes, most notably in AAV and IgAN with the presence of fibroepithelial crescents. None of the above cell populations were present in healthy controls, both isotype controls.


The method significantly reduces the autofluorescence background of urine samples to remove noise that prevented multi-color analysis of urinary cells on flow cytometer and thus capture different rare populations of urine glomerular cells,which no published approach to urine flow cytometry has yet allowed. The method is inexpensive and offers the potential for non-invasive differential diagnosis and monitoring of therapy.
Supported by COOPERATIO 34.