The Contribution of Quantitative Techniques Including Morphometry to Renal Diagnosis

The use of quantitative techniques in diagnostic renal pathology has not been utilized fully because of the relative inaccessibility and complexity of using statistically valid stereologic techniques. The introduction of digital image capture and computer-assisted image analysis has opened a new door to the analysis of renal biopsies. The potential of quantitative information to give greater prognostic information or better insight into the pathogenesis of many of these lesions is yet to be completely elucidated. Digital imaging and the advent of virtual microscopy should open new avenues of investigation into the pathogenesis of renal disease using clinically obtained renal biopsy material.     

The contribution of quantitative techniques including morphometry to renal diagnosis

The use of quantitative techniques in diagnostic renal pathology has not been utilized fully because of the relative inaccessibility and complexity of using statistically valid stereologic techniques. The introduction of digital image capture and computer-assisted image analysis has opened a new door to the analysis of renal biopsies. The potential of quantitative information to give greater prognostic information or better insight into the pathogenesis of many of these lesions is yet to be completely elucidated. Digital imaging and the advent of virtual microscopy should open new avenues of investigation into the pathogenesis of renal disease using clinically obtained renal biopsy material.     

Role of Electron Microscopy in Transplant Renal Pathology

The crucial role that electron microscopy plays in diagnostic renal pathology is undisputed. By allowing recognition of findings not identifiable by light microscopic evaluation, electron microscopy has contributed significantly to the understanding of renal diseases and has proven to be of unquestionable value in many diagnostic situations. However, the percentage of cases in which electron microscopic examination adds important information that is either key for establishing or confirming a diagnosis or provides valuable data that influence patient's management remains controversial. This figure depends on the renal biopsy service that is surveyed, but it is reported that on the average ultrastructural evaluation is of value in approximately 30 to 45% of the cases. Correct interpretation of a renal biopsy depends on the ability to correlate light, immunofluorescence, and ultrastructural findings. In contrast, the role of electron microscopy in the examination of renal transplant specimens remains controversial. Many centers do not use routine electron microscopy to examine these specimens and insist that there are only a few specific indications that require ultrastructural evaluation. There is general agreement among renal pathologists that electron microscopy is of importance in the evaluation of renal specimens from patients with proteinuria to distinguish between transplant glomerulopathy, recurrent or de novo glomerulonephritis in order to correctly manage these patients and predict survival of the graft. The other possible indications are much more controversial. This paper summarizes and critically reviews the literature available on this subject and defines recommendations based on the information available at the current time.     

Semiautomatic quantitation of macrophages in human renal biopsy specimens in proteinuric states.

AIMS: To develop and validate a rapid and economical semiautomated approach to the measurement of immunostainable tissue components which is applicable to routine diagnostic practice. To apply this approach to the measurement of macrophages in renal biopsy specimens in nephrotic states, as protein in the renal tubules may induce macrophage infiltration, and the morphology of macrophages in tissue sections does not lend itself to cell counting. METHODS: Macrophages were identified by immunostaining with a pan-macrophage marker, followed by digital image capture and analysis using a macro procedure written for the freeware image analysis program NIH-Image. RESULTS: The method was rapid, robust and accurate to within the limits imposed by sampling error inherent in the use of small needle biopsy specimens. Very few macrophages are found in normal kidney (mean volume fraction (+/- 95% confidence limits) 0.04% (0.02%)) but infiltration of macrophages was detected in minimal change nephropathy (0.29% (0.12%)) and in membranous glomerulonephritis (0.42% (0.11%)). A statistically significant correlation was found between macrophage volume fraction and weight of proteinuria in minimal change nephropathy but not in membranous glomerulonephritis. Correlations were found in both diseases between macrophage volume fraction and serum creatinine at time of biopsy. CONCLUSIONS: The equipment is inexpensive and measurement takes less than one minute per biopsy specimen. The results indicate that macrophage infiltration is part of the pathological process in minimal change nephropathy and membranous glomerulonephritis. The correlation with creatinine at time of biopsy suggests that renal impairment in minimal change nephropathy may result from infiltration by immunologically active cells and not merely from haemodynamic changes in nephrons. However, the correlation is not close, indicating that the relation between macrophage infiltration and disease severity is not a simple one.     

Value of electron microscopy in diagnosis of renal disease.

AIMS--To assess the role and value of electron microscopy in the diagnosis of renal disease. METHODS--Retrospective evaluation of 88 renal biopsy specimens received for primary diagnosis by assessment of the contribution of electron microscopy to the final diagnosis in the knowledge of the light microscopy and immunofluorescence findings. RESULTS--Electron microscopy had an important diagnostic role in 75% of cases and was essential or necessary for diagnosis in 25%. In 25% of cases electron microscopy was considered unhelpful in diagnosis. CONCLUSION--Electron microscopy has an integral role in the diagnosis of renal disease, and tissue should be taken for electron microscopy in all cases if possible. In some selected cases once the light microscopy and immunofluorescence findings are known it may be possible to forego electron microscopic examination. Electron microscopy is particularly useful in the differential diagnosis of minimal change disease and the nephrotic syndrome.     

Impact of image size on effectiveness of digital imaging systems

Radiologists detect small diagnostic signals in radiographic film images by altering the distance between the eye and the image, effectively zooming in on a particular detail. Details thus enlarged are more perceptible to the viewer. Considering that conventional film images are nearly life-size, the potential for increasing the detection of small signals in this manner is high. Digital images, however, presented in video format are usually smaller than life-size, sometimes more than 50% smaller. While local enlargements using computer-based imaging systems are extremely useful, the radiologist cannot examine a whole, life-size image. The importance of the latter in the diagnostic process is revealed in detection studies using the same images of a chest phantom with small nodular inclusions, in different size formats. A clear positive correlation exists between overall image size and the detection of signals that are of a diagnostically-relevant size. While it is widely accepted that image fidelity is an important determinant in the clinical acceptability of digital radiography, digital image displays should also be large enough to display life-size images.     

Practical use of electron microscopy for the diagnosis of glomerular disease.

In the past few years many clinically significant new glomerular lesions have been reported from observations made on renal biopsy specimens by electron microscopy. The clinical applicability of electron microscopy is illustrated by examples of specific ultrastructural patterns of glomerular reaction making possible a more accurate diagnosis, prognosis, and prediction of the response to treatment.     

How to improve microscopic images obtained with consumer-type digital cameras

AIM: Digital imaging is useful in conventional photography because it immediately provides images, and the image quality can be improved afterwards by the use of computer programs. The major disadvantages of consumer-type digital cameras mounted on microscopes are (i) unequal illumination through the image, and (ii) a coloured background. A computer program was specifically adapted and refined to improve images obtained with consumer-type digital cameras mounted on microscopes. METHODS AND RESULTS: An approach using a division operation between the specimen image and a background image leads to homogeneous illumination throughout the image, with automatically corrected brightness and white background. The correct colour spectrum is preserved by correction of the histogram. This approach was obtained from the freeware computer program 'Image Arithmetic'. In a test, three different consumer-type digital cameras (Sony, Nikon, Olympus) on different microscopes were used to obtain images of different types of histological specimens (cervical smear, bone marrow biopsy, and colonic biopsy). The computer program dramatically improved the quality of images obtained with all tested cameras. CONCLUSION: Using this approach, even low-cost digital cameras mounted on microscopes produce brilliant images with homogeneous illumination and a white background, the image quality being comparable with expensive cameras especially designed for microscopes.     

Electron microscopy and immunocytochemistry in the assessment of renal biopsy specimens: actual and optimal practice.

AIMS: To determine the variation in practice of British renal histopathologists in the use of immunohistochemistry and electron microscopy in the investigation of renal biopsy specimens; to attempt to identify a consensus on what acceptable practice should be; and to satisfy requests from laboratories which have found immunoperoxidase methods unreliable on renal biopsy specimens, by disseminating methods from laboratories which have had success. METHODS: A questionnaire was sent to all 58 laboratories which participate in the UK National Renal Pathology External Quality Assessment Scheme. RESULTS: A response rate of 88% was achieved. Most laboratories use immunocytochemistry and electron microscopy to investigate most renal biopsy specimens, but a few use these methods only rarely and one, never. There is a widespread wish to switch from immunofluorescence to immunoperoxidase, but this is frustrated by the unreliability of the method. This seems to be mainly because of the need to tailor the time of enzyme pretreatment to each biopsy specimen. CONCLUSIONS: The majority view is that electron microscopy and immunocytochemistry are necessary in the investigation of most native renal biopsy specimens, and the few pathologists who report renal biopsy specimens without these methods risk accusations of negligence. Difficulty in using fixed renal tissues for immunocytochemistry stem largely from variations in the requirement for enzyme pre-digestion. Even where immunoperoxidase methods are usually successful, the occasional use of immunofluorescence in parallel to check that false negatives are not occurring is advocated. In all cases it is wise to keep some frozen cortex in reserve in case there is an unexpected or inconsistent immunoperoxidase result.     

Virtual electron microscopy: a simple implementation creating a new paradigm in ultrastructural examination

Ultrastructural examination is a time-consuming and tiring process, requiring search for diagnostic features on a low-contrast screen in a dim environment. This article describes a method to circumvent these problems through the creation of a virtual ultrathin slide. This can be achieved by automated capturing of hundreds of images at high magnification and stitching them together into a digital image with a resolution of 4 nm/pixel. The pathologist can then navigate the virtual slide at his/her workstation computer. The image shows good contrast and resolution for diagnostic purposes, and most important, the pathologist can precisely note where the specific ultrastructural features are located. The setup required to implement virtual electron microscopy includes a transmission electron microscope equipped with motorized stage and automated digital image capture function, 2 free software components, self-developed software, and a desktop-grade computer. Besides use in daily diagnosis, virtual electron microscopy can open up many new applications such as undergraduate teaching, pathology resident training, external quality assurance program, and expert consultation.     

A digital imaging and communications in medicine (DICOM) print service for chest imaging

Large-scale picture archiving and communication systems (PACS) have not been widely implemented in this or other countries. In almost all radiology departments film remains the medium for diagnostic interpretation and image archive. Chest imaging is the dominant screening examination performed within most imaging departments and as such, is an extremely high-volume, low-margin examination. Digital technologies are being applied to chest imaging to overcome limitations of screen-film receptors (limited latitude) and current film management systems (singleimage copy). Efficient management of images and information is essential to the success of a chest imaging program. In this article we report on a digital imaging and communications in medicine (DICOM)-based centralized printing network for chest imaging. The system components and their operational characteristics are described. Our experience integrating DICOM-compliant equipment supplied by several vendors is described. We conclude that the print model supported by DICOM is adequate for cross-sectional (eg, computed tomography and magnetic resonance) imaging but is too simplistic to be generally applied to projection radiography.     

Electron Microscopy in the Diagnosis of Percutaneous Fine Needle Aspiration Specimens

Five years experience in the application of electron microscopy to fine needle aspiration biopsy specimens is reviewed. In an initial evaluation, 200 consecutive unselected specimens were examined; 89 proved diagnostic and, in a third of these, electron microscopy gave additional information that was often essential to diagnosis. Negative specimens resulted almost entirely from failure to obtain an adequate amount of material. Results were improved by the adoption of a preparation technique involving concentration of the cells in bovine serum albumin and by the inspection of a rapidly stained smear at the time of the aspiration procedure, with a further needle pass for electron microscopy being performed if necessary. Despite the small size of specimens, adequate examination was usually possible and electron microscopy has proved of value in the diagnosis of tumor samples acquired by fine needle aspiration in the same way as has been established with the larger sized specimens obtained by conventional biopsy and surgical resection.     

Electron Microscopy in the Diagnosis of Percutaneous Fine Needle Aspiration Specimens

Five years experience in the application of electron microscopy to fine needle aspiration biopsy specimens is reviewed. In an initial evaluation, 200 consecutive unselected specimens were examined; 89 proved diagnostic and, in a third of these, electron microscopy gave additional information that was often essential to diagnosis. Negative specimens resulted almost entirely from failure to obtain an adequate amount of material. Results were improved by the adoption of a preparation technique involving concentration of the cells in bovine serum albumin and by the inspection of a rapidly stained smear at the time of the aspiration procedure, with a further needle pass for electron microscopy being performed if necessary. Despite the small size of specimens, adequate examination was usually possible and electron microscopy has proved of value in the diagnosis of tumor samples acquired by fine needle aspiration in the same way as has been established with the larger sized specimens obtained by conventional biopsy and surgical resection.     

Enterprise Implementation of Digital Pathology: Feasibility,Challenges, and Opportunities

Digital pathology is becoming technically possible to implement for routine pathology work. At our institution, we have been using digital pathology for second opinion intraoperative consultations for over 10 years. Herein, we describe our experience in converting to a digital pathology platform for primary pathology diagnosis. We implemented an incremental rollout for digital pathology on subspecialty benches, beginning with cases that contained small amounts of tissue (biopsy specimens). We successfully scanned over 40,000 slides through our digital pathology system. Several lessons (both challenges and opportunities) were learned through this implementation. A successful conversion to digital pathology requires pre-imaging adjustments, integrated software and post-imaging evaluations.     

Microwave fixation in diagnostic renal pathology

Microwave-fixed tissues were examined in 10 patients undergoing diagnostic renal biopsy. A small portion of renal tissue was fixed by microwave irradiation and subsequently processed by routine methods for light microscopic, immunofluorescent and electron microscopic studies. The remaining portion of specimen was fixed and processed by conventional methods. In light microscopic examination, the renal architecture and cell morphology were well-preserved. Pathological changes were identical to those seen with formalin-fixed tissue. The pattern, distribution and intensity of positive immunofluorescence in microwave-fixed tissue were similar to those in tissues directly snap-frozen and stained. In electron microscopy, the normal and pathological features were well-demonstrated and not different from those observed in glutaraldehyde-fixed specimens. Specific ultrastructural lesions were clearly demonstrated and, apparently, were not altered by microwave irradiation. Our preliminary data indicate that microwave fixation can be effectively applied in the processing of renal biopsies. As the fixation is rapid, this method may be valuable in circumstances when an urgent diagnosis is required.     

Interstitial myofibroblasts: predictors of progression in membranous nephropathy.

AIMS: To determine the role of interstitial myofibroblasts in the progression of membranous nephropathy; and to assess the predictive value of quantifying myofibroblasts in determining long term renal outcome. METHODS: All cases of membranous nephropathy, diagnosed by renal biopsy at University Hospital of South Manchester between 1984 and 1987, were studied retrospectively. The biopsy specimens (n = 26) were reviewed and analysed morphometrically to measure interstitial volume as a proportion of the total volume of renal cortex, and numbers of interstitial myofibroblasts (cells positive for alpha-smooth muscle actin within the interstitium). Clinical data, with a follow up of seven to eight years, was available for 24 patients, and renal outcome was correlated with pathological changes in the initial diagnostic biopsy specimen. RESULTS: The number of myofibroblasts and interstitial volume were inversely correlated with creatinine clearance at the initial biopsy, and at the end of follow up. Percentage sclerosed glomeruli or stage of glomerular disease, assessed by electron microscopy, did not correlate with renal function at initial biopsy or during follow up. The number of myofibroblasts, but not interstitial volume, correlated with severity of proteinuria at initial biopsy. Of 15 biopsy specimens showing no or mild interstitial fibrosis, four showed a notable increase in the number of interstitial myofibroblasts. All of these patients developed chronic renal failure, compared with three of 11 patients whose specimens showed no or a mild increase in myofibroblast numbers. CONCLUSIONS: Interstitial myofibroblasts play a role in the development of interstitial fibrosis and progressive renal failure in membranous nephropathy. Increased numbers of myofibroblasts in biopsy specimens showing only mild fibrosis may predict subsequent chronic renal failure.     

Histological assessment of pre‐transplant kidney biopsies is reproducible and representative

Snoeijs M G J, Boonstra L A, Buurman W A, Goldschmeding R, van Suylen R J, van Heurn L W E & Peutz‐Kootstra C J
(2010) Histopathology 56, 198–202 Histological assessment of pre‐transplant kidney biopsies is reproducible and representative Aims: Histological examination of pre‐transplant renal biopsy specimens can be used to select grafts from older donors after cardiac death (DCD) with a satisfactory transplant outcome. The aim was to determine whether such biopsy specimens can be reproducibly scored between pathologists and are representative of the whole kidney. Methods and results: In renal biopsy specimens from DCD aged ≥60 years (n = 44), globally sclerosed glomeruli, vascular narrowing, tubular atrophy and interstitial fibrosis were scored by three independent pathologists according to the Pirani scoring system. Interobserver agreement on the sum of scores improved considerably with the introduction of a combined tubulo‐interstitial scoring system (intraclass correlation coefficient increased from 0.38 to 0.64). In small needle biopsy specimens (n = 144) obtained at autopsy, estimates of the proportion of globally sclerosed glomeruli were more precise with increasing sample size. Reasonably precise estimates may be obtained from specimens with at least seven glomeruli. Conclusions: It is feasible to implement pre‐transplant renal biopsy specimen analysis as a selection criterion in clinical practice in order to accept kidneys from marginal donors for transplantation.     

Incidental healed postinfectious glomerulonephritis: a study of 1012 renal biopsy specimens examined by electron microscopy

Glomerulonephritis (GN) characterized by immune complex deposits typical of postinfectious GN but with a paucity or absence of overt clinical symptoms and/or urinary abnormalities may occur after a group A streptococcus infection. The overall incidence of this type of subclinical GN is not known. To address this question, electron microscopy findings in 1012 consecutive renal biopsy specimens (952 native kidney, 60 transplant) examined by a single renal pathologist from August 1999 to April 2002 were retrospectively reviewed for the presence of distinct subepithelial and intramembranous deposits indicative of postinfectious GN. Such deposits were noted in 83 biopsy specimens, including 26 with a primary diagnosis of postinfectious GN (acute, persistent, or latent) and 57 in which these deposits were an incidental finding. In each of the latter 57 cases, some or all of the deposits showed partial or extensive loss of electron density typical of partially or largely resorbed deposits. A diagnosis of incidental postinfectious GN was not made in any biopsy specimen exhibiting another immune complex-related glomerular disease that could possibly account for the deposits, composing 443 of the 1012 biopsy specimens examined. Thirty of the 57 biopsy specimens with incidental postinfectious GN showed mesangial hypercellularity, although this was focal and segmental in all but 3 cases and was not accompanied by the endocapillary hypercellularity typical of acute postinfectious lesions. Immunofluorescence microscopy revealed glomerular deposits of C3 in >90% of these biopsy specimens and IgM deposits in 66%, but only rare IgG, IgA, and Cq deposits. Twenty-three (40%) of these 57 biopsy specimens exhibited diabetic nephropathy, either alone or in combination with another lesion; for perspective, only 128 (13%) of the 1012 biopsy specimens examined showed evidence of diabetic nephropathy. In summary, incidental evidence of resolving or largely healed postinfectious GN was noted in up to 10.5% of renal biopsy specimens (57 of 543, not including specimens with a primary diagnosis of an immune complex-related glomerular disease). The recognition of such lesions is potentially important in the interpretation of certain renal biopsy specimens.