Comparative evaluation of glomerular morphometric techniques reveals differential technical artefacts between FSGS and normal glomeruli

Morphometric estimates of mean glomerular volume (MGV) have clinical implications, over and above histologic data. However, MGV estimation is time-consuming, could waste tissue sections and requires expertise limiting its utility in retrospective clinical studies. Methods We evaluated MGV using both plastic and paraffin-embedded tissue from control and FSGS mice (n=10 each) using the gold-standard Disector/Cavalieri technique (Vglom-Cav) and other reported techniques [2- or 3-profile technique, Weibel-Gomez method (W-G)]. Within Vglom-Cav we examined the precision of MGV estimation while using MGVs obtained from 5- or 10-individual glomeruli measurements vs the true mean (20 glomeruli). Results In both FSGS and controls, we identified an acceptable precision of 10-glomerular sampling vs true MGV within Vglom-Cav technique [88 (79-94) % of MGV obtained were within 10% of the true MGV]. The 5-glomerular sampling was less precise [70 (56, 81) % of MGV obtained were within 10% of true MGV]. In plastic based techniques, 2- or 3-profile MGVs showed greater concordance with Vglom-Cav, than W-G MGV. The new 3-profile technique offered incremental benefit to the existing 2-profile method (improved Lin’s concordance in control and FSGS animals). We observed a consistent reduction of Vglom values within control animals (52+/-0.06%) in paraffin-embedded tissue (vs corresponding methods in plastic) demonstrating a clear shrinkage artefact due to tissue processing. FSGS glomeruli showed significantly less and more variable shrinkage artefact likely due to glomerular fibrosis. Conclusion We report the precision of 5- or 10-glomerular sampling for MGV estimation using controls and FSGS animals. We demonstrate and quantify the shrinkage bias in MGV during tissue processing for paraffin-embedding that also differentiated control animals and FSGS. Our findings have implications for experimental studies using glomerular morphometry. ### Competing Interest Statement The authors have declared no competing interest.