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Amyloidosis, Microscopy, Electrons, Proteinuria, Nephrotic syndrome, Kidney, What&,rsquo s Known Electron microscopy plays an important role in the diagnosis of various renal diseases such as amyloidosis. The main pathological classifications of renal amyloidosis are based on glomerular involvement and the number of involved main renal components (glomerulus, tubule, vessels, and interstitium). However, a comparison between these classifications has not been made. What&,rsquo s New Light microscopy did not lead to a definite diagnosis in approximately half of the patients and one-third tested negative with Congo red staining. Electron microscopy investigation prevents misdiagnosis. Classification based on glomerulus involvement had a higher correlation with clinical features and is a better predictor of the clinical manifestations. IntroductionAmyloidosis encompasses a group of disorders characterized by deposition of insoluble fibrils, predominantly in the extracellular space of the tissues. 1, Although amyloid fibrils can deposit in any organ,2 the kidney is the most common site in systemic amyloidosis.3 Irrespective of other underlying causes, kidney involvement is the main cause of mortality. 4, Clinical findings of renal involvement are proteinuria, nephrotic syndrome, and renal failure. 5,, 6, Direct toxicity of the amyloid fibrils deposition and, as a result, a decrease in renal function leads to increased mortality. 7, Besides, pathological findings such as interstitial fibrosis and tubular atrophy significantly contribute to morbidity. 8,, 9, The diagnosis of renal amyloidosis is established during routine processing of kidney biopsy, using light microscopy (LM) and electron microscopy (EM) investigations, by detecting amyloid deposits in each of the pertinent histological compartments of the kidney. Staining with Congo red is one of the most widely used procedures to identify amyloid deposits. 10, EM is useful in the diagnosis of amyloidosis, particularly in the early amyloid deposition, by confirming the presence of amyloid fibrils. In some cases, amyloidosis might be undetectable with LM since fibril deposition is minimal, and staining with Congo red could be negative. 9, Due to non-specific clinical symptoms of renal amyloidosis in the early stages of the disease, clinical diagnosis is easily missed, which leads to delayed management and a poor clinical outcome. This can also occur in other diseases that are in the early stages and only detectable by EM investigation. 11, Some studies have suggested a scoring scheme for evaluating renal amyloid deposition. 12,, 13, In this regard, the scoring and grading system proposed by Sen and colleagues seems to be the most comprehensive system. 9, Several studies have been conducted on different clinical and pathological aspects of renal amyloidosis. 3,, 8, However, to the best of our knowledge, EM studies on renal amyloidosis in Iran are scarce and have only covered a short time frame. Thus, the objective of the present study was to survey EM investigations, pathological findings, and clinical features of renal amyloidosis over 16 years. Besides, the relative frequency of diagnosed renal amyloidosis using EM was investigated, and a comparison between the two modified classifications was made.Materials and MethodsIn a cross-sectional study, all the 2,770 kidney biopsies of patients referred to our EM unit during 2001-2016 were investigated. The EM unit at Nemazee Hospital, affiliated to Shiraz University of Medical Sciences (Shiraz, Iran), is one of the oldest and the main medical center in southern Iran. All the patients diagnosed with renal amyloidosis by EM investigation were included in the study. Samples from kidney transplant patients and those that did not contain kidney tissue and glomeruli (due to poor kidney biopsy technique) were excluded. Eventually, the biopsies of 27 patients (0.97% of all kidney biopsies) were used in this study. Written informed consent had been obtained prior to renal biopsies. The study was approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (code, IR.sums.med.rec.1397.208).Light Microscopy and Transmission Electron Microscopy The renal biopsy tissues were cut into sections, and processed for LM and EM investigations. In the case of LM, 3-5 &,mu m serial sections were fixed in formalin, embedded in paraffin, and then stained with hematoxylin and eosin (H&,amp amp E), periodic acid-Schiff (PAS), Jones methenamine silver, Masson trichrome, and Congo red. All of the materials were purchased from Sigma-Aldrich, Germany.Based on the estimated amount of renal cortex involvement, interstitial fibrosis and tubular atrophy were graded on a semiquantitative scale. 14, The tubulointerstitial damage was categorized as mild (&,lt 25%) and moderate to severe (&,ge 25%). In addition, a specimen was cut into small blocks, and routinely processed for EM investigation. A set of 1 &,mu sections was taken and stained with toluidine blue (Sigma-Aldrich, Germany) to detect glomeruli under the light microscope (Olympus BX41, Japan). Then, grids were analyzed under the Transmission Electron Microscopy (TEM Leo 906, Germany). For each case, the data were analyzed using LM and the clinical. Then, the laboratory findings were analyzed using EM to characterize ultrastructural changes of the tissues.Diagnosis of Renal AmyloidosisThe final diagnosis of renal amyloidosis was based on the presence of non-branching randomly oriented fibrils in different parts of the renal cortex specimen that lacked hollow center and measured 8-12 nm in thickness in EM at &,lt &,times 80,000 magnification. In all cases, an LM investigation was performed and visualized as extracellular amorphous eosinophilic material on H&,amp amp E. For the detection of amyloid with Congo red staining, the presence of salmon-colored deposits was assessed under polarized light, which showed an apple-green birefringence.Grades and Classes of Amyloid DepositionAll biopsies were evaluated with EM for amyloid deposition in one of the pertinent histological compartments of the kidney, including the glomerular (G), peritubular (T), perivascular (V), and interstitial (I) areas. For detailed grading of the kidney involvement, we developed a modified version of the scoring system proposed by Sen and Sarcik. 9, Four grades of kidney involvement by amyloid deposition were defined according to a semi-quantitative scale. In this grading system, the extent of amyloid deposits in the glomerular, peritubular, interstitial, and perivascular areas w::as char::acterized as either a negative or positive deposit (table 1). Considering the importance of the renal function of glomerular deposition, and in order to obtain a better impression of it, we developed a modified version of the classification proposed by Sen and Sarcik. 9, This classification is based on the percentage of glomerular involvement (table 1,). In addition, for better analyses, particularly multiple logistic regression, we defined two main grades (A and B) based on the grading system (table 1,). Grade A (non-severe) included grades 1, 2, and 3, and grade B (severe) included grade 4. Similarly, glomerular deposition classes 0 to 4 were classified as class A (non-severe) and class 5 was included in class B (severe) (table 1,). All specimens were appraised and scored by an expert renal pathologist. Then, all the plastic sections were stained with toluidine blue, which included both the medulla and cortex of the kidney. These were re-reviewed to check the reliability of the scoring system.Glomerular deposition classesNumber of casesGrading systemNumber of casesClass AClass 0No amyloid deposition2Grade AGrade1G1Class 1Minimal mesangial (&,lt 10%)2Grade2(G+V) or (G+I)16Class 2Mild mesangial (10-25%)2Grade3G+V+T1Class 3Focal mesangiocapillary (26-50%)2Grade BGrade4G+V+T+I9Class 4Diffuse mesangiocapillary (51-75%)7 Class BClass 5Advanced (&,gt 76%)14G, Glomerular amyloid deposition, T, peritubular amyloid deposition, V, Perivascular amyloid deposition, I, Interstitial amyloid deposition |