Novel High-Resolution Three-Dimensional Fluorescent Confocal Imaging Resolves Differences in Urinary Sediment

Background: Although microscopic urinalysis is used as a liquid biopsy to determine kidney disease etiology, it is limited by poor resolution, few stains, and sample instability. Methods: Fixed urine sediment from pediatric patients with kidney disease was fluorescently stained with lectins, membrane markers, and nuclear stains and imaged using high-resolution confocal microscopy. Results: Fluorescent stains followed by high-resolution3D confocal imaging resolved urine sediment structures including of acanthocytes, red blood cells (RBC), white blood cells (WBC), mucus fibers, and casts in higher resolution than standard urine microscopic examination. Widefield fluorescent images (A’’-B’’) and 3D confocal (A’’’-B’’’) renderings of urine sediment stained for wheat germ agglutinin (WGA, green), memGlow (red), and DAPI (blue) resolved structure better than transmission (A’-B’) or Sterheimer-Malbin (A-B). Confocal images of urine sediment in disease showed (C) acanthocyte, (D) WBCs, (E) mucous threads, (F and J) eumorphic RBCs, (G) RBC cast, (H) lipid cast, (I) hyaline cast, and (J) WBC cast. GBM=glomerular basement membrane, GN=glomerulonephritis, s.=syndrome, AKI=acute kidney injury. A-B’’: 60×, widefield, A’’’-B’’’, C-J: confocal 63×, z=5 µm, inset 3× zoom. Scale bar=10 µm. Conclusion: The improved resolution of confocal imaging will provide the foundation for quantification and comparison between disease type and response to therapy when affected patients are followed longitudinally. Enhanced analysis of the urine sediment offers a strategy to increase diagnostic insight beyond the standard microscopic evaluation that has been used relatively unchanged for decades.