Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal...

Area of Science:

• Nephrology
• Quantitative Pathology
• Biomedical Engineering

Background:

• Morphometric estimation of glomerular volume (MGV, IGV) is crucial for understanding kidney disease but is often limited by time and expertise.
• Existing methods for glomerular morphometry, such as the Cavalieri (Cav) method, 2-profile, and Weibel-Gomez (WG) methods, have limitations in accuracy, bias, and precision.
• Focal segmental glomerulosclerosis (FSGS) is a significant kidney disease where glomerular morphometry can provide valuable insights.

Purpose of the Study:

• To evaluate and compare the accuracy, bias, and precision of different methods for estimating mean glomerular volume (MGV) and individual glomerular volume (IGV).
• To assess the impact of varying glomerular sampling numbers on the precision of MGV estimates.
• To introduce and validate a novel 3-profile method for glomerular morphometry and compare it with existing techniques.

Main Methods:

• Morphometric analysis of MGV and IGV was performed on plastic- and paraffin-embedded kidney tissue from control and FSGS mice using the Cavalieri (Cav) method, 2-profile, Weibel-Gomez (WG), and a novel 3-profile method.
• Accuracy, bias, and precision were compared across methods and with different numbers of glomeruli sampled (5, 10, 20).
• Tissue shrinkage artifacts between plastic- and paraffin-embedded samples were quantified.

Main Results:

• An acceptable precision for MGV estimation was achieved with 10-glomerular sampling using the Cav method, while 5-glomerular sampling was less precise.
• The 2- and 3-profile methods showed greater concordance with Cav-based MGV in plastic-embedded tissue compared to WG.
• A consistent underestimation bias was observed with 2- and 3-profile methods for IGV compared to the Cav method, with greater variability in FSGS glomeruli.
• The novel 3-profile method demonstrated incremental benefits over the 2-profile method, showing improved correlation, Lin's concordance, and reduced bias.
• A significant shrinkage artifact (52%) was quantified in paraffin-embedded versus plastic-embedded control tissues, with reduced but variable shrinkage in FSGS glomeruli.

Conclusions:

• The novel 3-profile method offers improved concordance and reduced bias for glomerular volume estimation compared to the 2-profile method.
• Glomerular morphometry, particularly with optimized sampling strategies, provides valuable biological data beyond qualitative histology.
• Understanding tissue processing artifacts, like shrinkage, is critical for accurate morphometric analysis in kidney disease studies.