Prognostic value of simple measurement of chronic damage in renal biopsy specimens
Alexander J. Howie,
Maria Alice S. Ferreira and
Dwomoa Adu
Department of Pathology, University of Birmingham, and Department of Nephrology, Queen Elizabeth Hospital, Birmingham, UK
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Abstract
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Background. A simple method of measurement of chronic damage in renal biopsy specimens would be useful in clinical management, prognosis, comparisons between different centres and trials.
Methods. An interactive image analysis system was used to outline and measure areas of chronic damage in 247 renal biopsy specimens to give an index of chronic damage, expressed as a percentage of cortical cross-sectional area. Prognostic value was analysed by the KaplanMeier method to study time between biopsy and onset of permanent dialysis.
Results. There was no significant bias between measurements by the same observer or different observers. The index of chronic damage ranged from 0 to 90%. Increasing severity of chronic damage was associated with shortened renal survival. Each increase of 10% in the index increased the hazard ratio of risk of permanent dialysis by 1.5 times (95% confidence interval 1.41.7, P<0.001).
Conclusions. A simple measure of chronic damage was a powerful indicator of prognosis. This is likely to be clinically useful in routine practice and trials.
Keywords: chronic damage; morphometry; prognosis
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Introduction
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Progression to chronic renal failure is influenced by the extent of irreversible damage [1]. Such damage has been assessed in many ways since the work of Risdon et al. in 1968 [2], e.g. by Striker et al. [3] and Bohle et al. [4]. Subjective grading systems have usually been used rather than morphometric methods. Grading systems such as the chronicity and activity indices in lupus nephritis are easy to use but may be unreliable [5,6]. Objective measures are more difficult but more reliable [7]. The hypothesis behind this study was that the amount of chronic damage in renal biopsy specimens could be measured by a simple method and used to predict renal survival.
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Subjects and methods
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Renal excretory function and renal survival
Serum creatinine concentration was measured at biopsy. The interval was noted between biopsy and the end point, which was introduction of permanent haemodialysis or peritoneal dialysis, or return to permanent dialysis in patients with a renal allograft. No patient died before onset of permanent dialysis.
Selection of renal biopsy specimens
Specimens were selected retrospectively to allow several years of follow up. Diagnoses were made based on conventional criteria [8], using vasculitic glomerulonephritis as a synonym for focal segmental necrotizing glomerulonephritis, crescentic glomerulonephritis and similar terms [9] (Table 1
). Those with IgA nephropathy, Henoch Schoenlein nephritis, vasculitic glomerulonephritis and minimal change nephropathy were consecutive specimens with those diagnoses. Two patients with anti-glomerular basement membrane antibody disease (Goodpasture's syndrome) were included in the vasculitic group. A few patients with minimal change nephropathy had been treated with cyclosporin and a biopsy was performed to see if the drug had damaged the kidney. The other groups consisted of all other consecutive specimens taken over a period of several weeks. The 21 allograft specimens were taken 1 week to 5 years after transplantation (median 2 weeks). All patients were adults.
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Table 1. Details of 247 renal biopsy specimens on which the index of chronic damage was measured, with correlation between index and serum creatinine concentration at biopsy (Index v cr bx)
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Measurement of amount of chronic damage
Figures 1
and 2
illustrate the method used. One routinely prepared, formalin fixed, paraffin embedded, 2 µm section of each specimen stained by periodic acid-methenamine silver was examined under a microscope with a video camera. The section was selected only on the grounds that it was technically satisfactory and as completely representative as possible of the size of the specimen, including all pieces if there were more than one. Images at magnification x10 were captured with an Aequitas IDA image database and image archive management system (Dynamic Data Links, Cambridge, UK). For analysis, each image was converted from Aequitas IDA to Aequitas IA image analysis software (Dynamic Data Links). The threshold was adjusted to highlight everything in a defined area. Cortex was defined as the part inside the renal capsule and outside the medulla, whose outer limit was defined by the cortical aspect of arcuate vessels and the medullary aspect of the deepest glomeruli. All cortex was outlined using the freehand drawing facility. The size of this area was measured by the system in arbitrary units. Freehand drawing was then used to outline areas of chronic damage. This included glomeruli showing global sclerosis but not segmental sclerosis, areas of interstitial fibrosis, which appeared more solid and deeply stained than normal or oedematous interstitial tissues, and atrophic tubules, defined as tubules smaller than normal, with thickened basement membranes, or tubules larger than normal, with thin epithelium, including those large enough to be considered cysts [10]. Arteries and arterioles were not judged to have chronic damage unless they were completely occluded. The total area outlined was measured. Areas of cortex and chronic damage were summed for each specimen, and the percentage of chronically damaged cortex was calculated to the nearest integer to give the index of chronic damage. To test agreement between measurements of the index, 20 biopsy specimens were measured twice at intervals by one observer, and 20 other specimens measured separately by two observers.

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Fig. 1. An image on a computer of part of a renal biopsy specimen stained by periodic acid-methenamine silver. Figure 2 shows the preparation of this image for morphometric determination of the index of chronic damage.
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Fig. 2. The image seen in Figure 1 after freehand drawing to illustrate in principle how the index of chronic damage is calculated. The cortex is outlined to exclude the capsule, and areas of chronic damage are outlined and filled, ready for automatic measurement of areas. In practice, the cortex would be outlined and measured before the areas of chronic damage.
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Statistical analysis
Agreement between measurements was assessed by the Bland and Altman method after log transformation, since differences between measurements were proportional to the mean [11]. This method gives the bias, or mean difference between measurements, and limits of agreement, or two standard deviations either side of the mean, with confidence intervals (CIs) for these. The Spearman rank correlation coefficient was used to investigate the correlation between the index and serum creatinine concentration at biopsy. This non-parametric test was selected because the distribution of variables was not assumed to be normal. To study survival to the end point, serum creatinine concentration at biopsy was divided arbitrarily into four groups: <120 µmol/l, 120249 µmol/l, 250499 µmol/l and
500 µmol/l, and the index of chronic damage was divided arbitrarily into five levels: <10%, 1019%, 2039%, 4059% and
60%. Survival rates were examined by the KaplanMeier product limit method. The equality of survivor function across groups was tested by the log rank test. The Cox proportional hazards model was used to study the contribution of initial serum creatinine concentration and index of chronic damage to the relative risk of end-stage renal failure (ESRF). This was done by performing a Cox regression analysis with censored data to estimate the roles of serum creatinine concentration, categorized so that a change of 1 unit meant a change of 100 µmol/l in serum creatinine concentration, and the initial index of chronic damage, categorized so that a change of 1 unit represented a change of 10% in the index, in the relative risk of reaching the end point (Stata Statistical Software, Release 5.0, College Station, TX, USA).
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Results
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All images of cortex were analysed on every specimen. The median number of images was six (range 115). Analysis of an image took between 2 and 3 min. There was no significant bias between measurements by the same observer (mean 0.97; 95% CI 0.921.02) or measurements by two observers (mean 1.0; CI 0.941.06). The limits of agreement of measurements by the same observer were 0.72 (CI 0.610.81) and 1.21 (CI 1.121.31), and the limits of agreement of measurements by two observers were 0.73 (CI 0.620.83) and 1.27 (CI 1.171.38).
The overall index in 247 specimens ranged from 0 to 90%, but was 100% in some images. For all specimens, there was a weak significant correlation between the index and serum creatinine concentration at biopsy (r=0.43, P<0.001) (Table 1
). There was a strong significant correlation (r
0.76) between the index and serum creatinine concentration in four groups, a weak significant correlation in two groups, and no significant correlation in the remaining six.
Table 1
shows that the overall median index was 37% in those specimens associated with progression to the end point, whereas the overall median index was 3% in both those that had not reached the end point at follow up and those without follow up. By the log rank test, the index was a strong predictor of rate of progression to ESRF (
2=138.9, four degrees of freedom (df), P<0.001; Figure 3
). Only three patients with an index<10% reached the end point within 5 years of biopsy, one with Goodpasture's syndrome, one with vasculitic glomerulonephritis and one with Henoch Schoenlein nephritis. Graphs of renal survival related to the index were similar to those of the whole series in the two largest groups, namely IgA nephropathy (
2=44.9, 4 df, P<0.001) and Henoch Schoenlein nephritis (
2=53.6, 4 df, P<0.001). This was also true of the combination of the other three groups, each with at least two patients who reached the end point, namely vasculitic glomerulonephritis, segmental sclerosing diseases and miscellaneous conditions (
2=19.3, 4 df, P<0.001), and the combination of the other seven groups that individually had either one patient or no patient who reached the end point (
2=19.6, 4 df, P<0.001). For simplicity, Figure 4
illustrates the graph of survival for IgA nephropathy and Henoch Schoenlein combined (
2=85.1, 4 df, P<0.001), and Figure 5
illustrates that for the other groups combined (
2=50.7, 4 df, P<0.001).

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Fig. 3. Renal survival after biopsy related to the index of chronic damage for all specimens (n=200). Upper solid line, index <10%; upper interrupted line, index 1019%; middle solid line, index 2039%; lower interrupted line, index 4059%; lower solid line, index 60%.
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Fig. 4. Renal survival after biopsy related to the index of chronic damage for specimens of IgA nephropathy and Henoch Schoenlein nephritis (n=96). Upper solid line, index <10%; upper interrupted line, index 1019%; middle solid line, index 2039%; lower interrupted line, index 4059%; lower solid line, index 60%.
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Fig. 5. Renal survival after biopsy related to the index of chronic damage for specimens excluding IgA nephropathy and Henoch Schoenlein nephritis (n=104). Upper solid line, index <10%; upper interrupted line, index 1019%; middle solid line, index 2039%; lower interrupted line, index 4059%; lower solid line, index 60%.
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Serum creatinine concentration at biopsy predicted the development of ESRF less strongly than the index of chronic damage (
2=56.9, 3 df, P<0.001). Several patients with a raised serum creatinine concentration recovered. Using the Cox proportional hazards model, after controlling for initial serum creatinine concentration, each 10% increase in the index resulted in reduced survival with a hazard ratio of 1.5 (CI 1.41.7, P<0.001). With this model, after controlling for the index of chronic damage, each increase of 100 µmol/l in the initial serum creatinine concentration resulted in reduced survival with a hazard ratio of 1.3 (CI 1.21.4, P<0.001).
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Discussion
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This study confirms that a major determinant of prognosis of a kidney that is biopsied is the amount of chronic renal damage at diagnosis [1]. Concepts of chronic renal damage in relation to renal function began to emerge when Risdon and others established the connection between tubular atrophy and renal function [2]. The group of Striker emphasized the importance of interstitial changes such as fibrosis and correlated tubular disease and interstitial disease [3]. The group of Bohle gave evidence to support the role of interstitial fibrosis in prognosis [4], and they and Seron et al. [12] showed the relation between obliteration of intertubular capillaries and renal function. Seron et al. also related the obliteration of intertubular capillaries to the proportion of globally sclerosed glomeruli [12]. Alexopoulos and others correlated the chronicity index in lupus nephritis with the amount of interstitial inflammatory infiltrate [13]. Various aspects of chronic damage have been investigated in these previous studies, but the method described now allows simple assessment of all changes by amalgamation of tubular, interstitial, vascular and glomerular abnormalities.
Other methods of measurement of chronic damage have been described but have disadvantages. Point counting is tedious [1416]. Measurements of the renal content of myofibroblasts [17,18] and collagen [19] require specially prepared immunostained sections, and sometimes require sampling and manual counting. Image analysis methods such as texture analysis [20] and Fibrosis HR [21] require extensive and complicated manipulation of images. These methods, like the collagen method [19], give an extent of interstitial fibrosis of
15% in normal kidneys and of much less than 100% in abnormal kidneys. Cysts and thyroidized tubules cause most of these methods to underestimate the amount of damage.
In contrast, the index of chronic damage described here is measured on routine sections, allows the whole of the cortex in a section to be assessed easily, and is reliable and reproducible. Tests of agreement showed that there was no significant bias and that the limits of agreement were about one quarter above and below the initial measurement. The index was 100% in some images and could have the maximum range from 0 to 100% to allow greater precision than methods with a restricted range [1721]. Cysts and thyroidized tubules are not a problem. The index requires interaction between observer and computer, but much less than in automatic measurement [20,21].
Difficulties are more related to the material than to the method. Only one section was examined, as is usual in morphometric studies of renal biopsy specimens [1421]. The small size of the sample meant that it may not have been representative of the kidney and that an asymmetrical distribution of abnormalities in the kidney may not have been reflected in the sample. Variation within the sample was thought to be of little importance compared with any possible difference between the sample and the kidney. Such a difference probably explains why not every specimen with an index of
40% was associated with progression to ESRF (Figures 3
5
). One problem with any method of measurement of chronic damage is that as atrophic tubules disappear, the kidney shrinks and surviving tubules often show compensatory enlargement [10]. Methods that measure extent of chronic damage as a proportion of cortical cross sectional area, which itself changes as the damage progresses, are likely to underestimate the real extent of the damage.
The index of chronic damage is a powerful predictor of the rate of development of ESRF (Figures 3
5
). This is so even when controlled for serum creatinine concentration at biopsy, which is overall only weakly correlated with the index. Serum creatinine concentration is strongly correlated with the index in groups that have mainly chronic damage to explain renal impairment, such as IgA nephropathy and late non-glomerulonephritic damage. In those with mainly acute damage, tubules and renal impairment may recover and the index is not correlated with serum creatinine concentration. This applies to groups such as patients with vasculitic glomerulonephritis. Progression to ESRF is much more related to the amount of chronic damage at the time of biopsy than to the serum creatinine concentration at biopsy. The index can be used to predict the outcome and likely rate of progression, although the amount of chronic damage at biopsy is not the only factor that influences rate of progression. The underlying disease is also important because, for instance, the only three patients with an index<10% who reached the end point within 5 years of biopsy all had vasculitic illnesses.
The present study was retrospective to allow an adequate length of follow up. Whether treatment had any influence on renal survival independently of the amount of chronic damage would be difficult to determine, but now the amount of chronic damage can be controlled for prospectively in any treatment trial. The index avoids the problems of subjective grading systems [6,7] and most image analysis systems should be able to be used to calculate it. There are potential applications in individual patients, clinical studies and trials.
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Acknowledgments
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We are grateful to Mr T Marshall for statistical advice and to our colleagues in the Department of Nephrology.
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Notes
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Correspondence and offprint requests to: Dr A. J. Howie, Department of Pathology, The Medical School, Birmingham B15 2TT, UK. 
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Received for publication: 11. 5.00
Revision received 9. 1.01.