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Determination of normal glomerular basement membrane thickness at Department of Pathology and Cytology, Dubrava University Hospital, Zagreb

Electron microscopy (EM), along with light microscopy and immunofluorescent analysis, represents key method in renal biopsy specimen analysis. EM is crucial for diagnosing disorders that directly influence structure of glomerular basement membrane (GBM) such as Alport’s syndrome, thin glomerular basement membrane nephropathy (TBMN) and diabetic nephropathy. Thickness of GBM depends on multiple factors. It is age and sex dependent, but it is also influenced by methodology of tissue preparation and processing. Referential span of normal GBM thickness varies in different centers of proficiency and in multiple research papers. Following previously mentioned, many authors emphasize need for referential span of normal GBM thickness for each laboratory.[1, 2] Orthogonal intercept/mean harmonic thickness measurement s considered the most precise method of GBM thickness measurement. Due to complexity of described method, most frequently is used alternative method of direct measurement of GBM thickness. In this research we have applied modification of the direct measurement / arithmetic mean method to measure GBM thickness in Laboratory for Nephropathology at Dubrava University Hospital, Zagreb. Digital EM photographies with magnification of 4000-8000x were selected from our database of EM pictures of kidney biopsy samples. Pictures were taken on multiple capillaries from 1-3 glomeruli. A total of 30 GBM measurements were taken on 10 randomly selected capillaries to determine an average GBM thickness for each biopsy. Position of measurements in capillary loops were determined similarly as described by Haas.[1] Direct measurements were made using iTEM software (Olympus Soft Imaging Solutions GmbH), with digital zoom of 150-400% to measure the distance between the endothelial and podocyte plasma membranes. Using this method we obtained mean ±SD values for the normal GBM thickness of adult males and females of 340±36 nm and 301±44 nm. These values were based on average GBM thickness for each of 23 males and 22 females, aged from 19 to 84 years, with minimal change nephropathy, acute interstitial nephritis, normal renal parenchyma or acute tubular injury. Exclusion criteria were hematuria and diabetes mellitus. We defined normal ranges for each gender (268-412 nm for males, 213-389 for females), as being within 2 SD of these means. With this research we have standardized method for GBM thickness measurement for Laboratory of Pathology and Cytology at University Hospital Dubrava and defined referential span of normal GBM thickness for our laboratory. Results of our research are crucial for more accurate diagnosis of disorders characterized with structural changes of glomerular basement membrane (GBM). References: 1. M. Haas, Arch Pathol Lab Med, 133 (2009) 224- 232. 2. K. Tryggvason, J. Patrakka, J Am Soc Nephrol, 17 (2006) 813-822.

Electron microscopy; glomerular basement membrane thickness

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