A novel approach to the histological diagnosis of pediatric nephrotic syndrome by low vacuum scanning electron microscopy

Abstract

Despite intensive treatment, steroid-resistant nephrotic syndrome (NS) often progresses to endstage renal disease. Therefore, a more accurate and quick histological diagnosis is required to properly treat such patients. The aim of this study was to introduce a novel approach to the histological diagnosis of pediatric NS by low vacuum scanning electron microscopy (LVSEM) and to describe the morphological differences in glomeruli between steroid-sensitive and steroid-resistant NS specimens. The subjects were three patients with steroid-sensitive NS and four patients with steroid-resistant NS. Conventional renal biopsy paraffin sections were stained with platinum-blue (Pt-blue) or periodic acid methenamine silver (PAM) and directly observed under LVSEM at magnifications between ×50 and ×10,000. The Pt-blue-stained sections showed three-dimensional structural alterations in glomerular podocytes and foot processes. PAM-stained sections showed changes in the structure and thickness of the glomerular basement membrane (GBM). Consequently, many round-shaped podocytes and elongated primary foot processes were exclusively recognized in steroid-resistant NS, although irregularities in foot process interdigitation, fusions, effacements, and microvillus transformations were observed in both steroid-sensitive and steroidresistant NS. Irregularities in thickness and the wrinkling of GBMs were clearly detected in steroid-resistant NS. The evaluation by LVSEM is probably useful for the renal histological diagnosis of pediatric NS.Nephrotic syndrome (NS) comprises proteinuria, hypoalbuminemia, hypercholesterolemia, and edema. Pediatric patients with NS are usually treated with corticosteroids (13). Patients who respond to steroids (steroid-sensitive NS) have good clinical outcomes, whereas patients who do not respond to steroids (steroid-resistant NS) are predisposed to the development of end-stage renal disease. Histopathologically, most pediatric patients with steroid-sensitive NS show minor glomerular abnormalities, and patients with steroid-resistant NS generally have focal segmental glomerulosclerosis (FSGS) (25). Under light microscopy (LM), minor glomerular abnormalities are defined as a slight or no increase in the mesangial matrix or cellularity without focal segmental glomerular collapse, scarring, endocapillary proliferation, or adhesions. FSGS shows focal and segmental glomerular collapse, scarring, endocapillary proliferation, or adhesions of glomeruli with minimal abnormalities (21). However, it is not clear whether the glomeruli with no obvious changes in FSGS are different from those in minor glomerular abnormalities. In addition, it is difficult to diagnose FSGS when the NS biopsy section does not include glomeruli with the typical alterations of FSGS described above, although the differential diagnosis between minor glomerular abnormalities and FSGS is very

Low-vacuum scanning electron microscopy may allow early diagnosis of human renal transplant antibody-mediated rejection

“…Endothelial cells could not be examined because they are PAM-negative elements. Meanwhile, we previously developed the Pt-blue staining method to reveal the morphological changes of glomerular cellular components under LVSEM (10,11,15). We hope that further studies will be performed in the future to address tions, and they surmised that these findings denote an "immune complex-mediated glomerular injury" (1).…”

“…It is impossible to utilizing TEM for every case of renal transplant biopsy. We recently reported the usefulness of low vacuum scanning electron microscopy (LVSEM) for the rapid three-dimensional analysis of renal biopsy specimens (11,15,16). LVSEM is comparatively less expensive, and it can facilitate convenient and quick observations because it does not require preliminary processing.…”

Informative three-dimensional survey of cell/tissue architectures in thick paraffin sections by simple low-vacuum scanning electron microscopy

“…For light-microscopic examinations, paraffin wax continues to be the universal embedding medium for histological analysis, immunohistochemistry, and diagnostic histopathology, mainly because it is inexpensive and easily handled for sectioning. Electron microscopy of non-conductive paraffin sections takes advantages of BSE imaging in low-vacuum SEM 10 , 11 . However, the images obtained from such thin sections (5–10 µm in thickness) are basically two-dimensional, and it remains a challenge to reconstruct the missing third dimension by examining many sections of the three-dimensional cell/tissue architectures.…”

Morphological diagnosis of Alport syndrome and thin basement membrane nephropathy by low vacuum scanning electron microscopy

“…Using this technique, some researchers have investigated GBM alterations in a three-dimensional manner (3,5,15,17); however, to our knowledge, the three-dimensional findings of GBMs in AS or TBMN have not yet been reported. We have previously demonstrated the usefulness of low vacuum scanning electron microscopy (LVSEM) to evaluate the histological findings of glomeruli by using conventional renal biopsy paraffin sections for the rapid three-dimensional analysis of glomerular structure (10,14,16). Especially, the GBMs could be visualized through the overlying cellular components without removal, by staining with periodic acid methenamine silver (PAM) and by using the backscattered electron (BSE) mode of LVSEM, owing to the fact that the PAM stain contains silver, a heavy metal, which enhances the BSE signals.…”

“…Regarding the latter issue, the podocytes and endothelial cells were not estimated because they were PAM-negative elements. Fortunately, we have previously developed the Pt-blue staining method to reveal the morphological changes of glomerular cellular components under LVSEM (10,16), and we hope that further studies will be performed in the future to address these issues.…”