Thin basement membrane nephropathy cannot be diagnosed reliably in deparaffinized, formalin-fixed tissue

Abstract

In diagnostic renal pathology, electron microscopy is ideally performed on glutaraldehyde-fixed, plastic resin-embedded tissue (EM-G). When no glomeruli are present in the portion of the biopsy fixed in glutaraldehyde, formalin-fixed, paraffin-embedded tissue can be reprocessed for electron microscopy (EM-F). The usefulness of this salvage technique for the diagnosis of thin basement membrane nephropathy (TBMN) has not been studied systematically. Here we compare the glomerular basement membrane (GBM) thickness by EM-G vs EM-F in 21 renal biopsies, including TBMN (eight patients), normals (two patients), minimal change disease (MCD) (six patients) and diabetic nephropathy (DN) (five patients). There was significant reduction of the GBM thickness by EM-F compared with EM-G across all diagnostic categories in all 21 cases. The mean percentage reduction in GBM thickness was 23% for the TBMN cases, 40% for the normal/MCD cases and 34% for the DN cases. Four patients with MCD had a mean GBM thickness by EM-F that fell below the defining threshold for diagnosis of TBMN. For the TBMN cases, the 99th percentile for GBM thickness by EM-F was 194 nm, suggesting that the diagnosis of TBMN by EM-F can be excluded with confidence if the GBM thickness is above 200 nm. No clear criteria could be established to diagnose TBMN by EM-F. Renal pathologists should be aware that reprocessing of paraffin tissue for EM causes artifactual GBM thinning that precludes accurate diagnosis of TBMN.

Pathologic Classification of Diabetic Nephropathy

“…The GBM width is then compared with GBM width from normal subjects, as determined previously by Steffes et al 27 and recently updated by Haas. 16 Ideally, glutaraldehyde-fixed, plastic resin-embedded tissue should be used for EM, keeping in mind that other methods, particularly the reprocessing of paraffin tissue for EM, may cause artifactual GBM thinning as recently reported by Nasr et al 28 If computer-assisted measurements are not available, we recommend doing direct GBM measurements as recently modified by Haas. 16 …”

Morphological assessment of thin basement membrane disease

“…In other cases (not included in this study) when the thinning of the lamina densa affected the loops to alesserextentitalwayscoexistedwithotherglomerulopathies. Differentnumbersofmeasurementsarereported dependingonthenumberofglomeruliwithinthebiopsyspecimen [8,17,24,29,30,31].Thenumber ofmeasurementsinourmaterialwasdeterminedby thenumberofglomeruliwithinthepuncturebiopsy.…”

“…Therefore, only the lamina densa was measured in our material. In a similar fashion, the measurements were limited to the lamina densa in thestudybyNasret al [31],whoalsopointedtothe commonlyobservedartifactsduetotheseparationof podocytes from the basement membrane and poor conservationofthelaminarara.Thefactofbasement membranethinningbeinglimitedtothelaminadensawasalsohighlightedbyDischeet al [5],Vogleret al [32],andBasta-Jovanovicet al [33]. Most authors use computer-assisted methods to assess the membrane thickness.…”

“…Most authors use computer-assisted methods to assess the membrane thickness. Some authors, for exampleDaset al [34],usebothmethodsforbetter reliabilityofresults.Followingtheirexample,wedecidedtousethecomputer-assistedmethodaswellas theso-called"manual"methodasusede.g.byOsawaet al [35],Fosteret al [16]andNasret al [31]. The term "manual method" is adapted from Kanenetsky et al [36].…”

Renal Disease

“…For a diagnosis of TBMD to be made, IgA disease, minimal change, mesangiocapillary and some forms of lupus glomerulonephritis must be excluded as these may also show a degree of membrane thinning (Savige et al, 2003). Note that TBMD cannot be reliably diagnosed using tissue retrieved from wax because of artefactual thinning of the GBM (Nasr et al, 2007).…”