1 Electron Microscopy Laboratory, 2 Stereological Research Laboratory, 3 University Institute of Pathology, Aarhus Kommunehospital and Departments of 4 Nephrology and 5 Urology, Skejby Sygehus, Aarhus University Hospital, Aarhus, Denmark
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Abstract |
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Methods. Punch biopsies (3 mm) from six regions in each kidney were obtained from seven explanted renal grafts with varying degrees of clinically diagnosed chronic and acute rejection. One kidney, removed for a minor pelvic tumour, served as reference material. Using point counting on PAS-stained sections, the volume fraction of the interstitial tissue per glomerular cortex VV(interstitium/cortex) was estimated. From each kidney, two of the six biopsies were re-evaluated by the same observer.
Results. VV(interstitium/cortex) varied from 0.25 to 0.78 between the explanted kidneys vs 0.26 in the reference kidney. Variations within the kidneys were low, expressed by standard deviations (SD) of between 0.04 and 0.06, and coefficients of variation (CV) between 0.06 and 0.22. The SD estimated from repeated measurements was 0.04 and CV was 0.07.
Conclusions. Biopsies from one region of the kidney were found to be representative for estimates of interstitial tissue in explanted human kidney grafts, and the degree of reproducibility was high when using point counting, as in the present study.
Keywords: human; interstitium; kidney; representative; reproducibility; transplantation
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Introduction |
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The aim of the study was to investigate the extent of changes in one of the often pathologically affected compartments of the human kidney transplant: the interstitial tissue [7,8]. The amount of interstitial tissue has been shown to correlate with the function of the kidney in transplantation [810] and kidney disease [11]. We wanted to elucidate how well a biopsy from one region of the kidney represented biopsies from other regions. Furthermore, the precision of the estimates of the interstitial volume was evaluated since the importance of reproducibility, in conjunction with representativeness, is obvious for both diagnosis and research.
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Subjects and methods |
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Seven human explanted kidney grafts with preceding clinical dysfunction and different diagnoses of rejection were available for the study. Clinical information is given in Table 1. Three grafts had shown primary non-function, all being the third graft for the patients, i.e. renal allotransplantation 3 (RAT 3) (case numbers 2, 3 and 5). The remaining four were first grafts (RAT 1). Stable function was reached at day 5 (range 437 days), except for patient 5, who did not reach stable function at all. Plasma creatinine was 127 µmol/l (range 73186 µmol/l) at the time of stable function. The grafts had been implanted for a mean period of 3.6 years (range 1 month to 9.3 years). Plasma creatinine was 480 µmol/l (range 323579 µmol/l) before dialysis or explantation.
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A kidney that was removed due to a pelvis tumour (diameter 15 mm) served as a reference organ. The preoperative status for the reference was normal blood pressure, and plasma creatinine was 75 µmol/l. Renography showed equal function of the two kidneys.
The kidneys were divided into halves, with a longitudinal section, and were immersion fixed in 4% buffered formaldehyde for 48 h. From six different locations in each kidney, two 3-mm punch biopsies were taken perpendicular to the renal surface. The biopsies were sampled side by side from the centre of the two poles and from the centre of the middle segment of the anterior and posterior parts of each kidney.
Both biopsies from each location were paraffin embedded pair-wise, sectioned longitudinally at 4 µm and stained with Periodic Acid Schiff. One biopsy from each kidney was scored according to the Banff classification [12]. Banff gradings of acute rejection and chronic allograft nephropathy are shown in Table 1.
One of the two biopsies from each location was chosen randomly and one section from each biopsy was used for stereology. The stereological measurements of the sections were performed with computer-assisted microscopy (C.A.S.T.-Grid®; Olympus Denmark A/S). Live video camera images of the microscope field of vision were transmitted to a computer screen. The microscope was equipped with stepping motors controlling stage movements via the computer software. The system makes it possible to mark an inclusion area at a low magnification and then, at a higher magnification, to systematically sample, with a random start, fields of vision for point counting within the demarcated area.
The volume fraction of the interstitium per glomerular cortex, VV(interstitium/cortex), was estimated using a point counting technique based on the principle that points distributed in an independent way onto a given tissue will hit different tissue compartments according to the relative extent of each compartment type. Counting of the individual sections was performed in a random order without prior knowledge of the specimen's histological grading. After 3 months, two randomly selected sections from each kidney were re-counted.
The glomerular cortex of each section was delineated at a magnification of x80, with a border drawn towards the medulla where the glomeruli were absent and/or where arcuate arteries were present. The computer was then programmed to sample fields of vision, with a specified distance of 700 µm between them. Counting was performed at magnification x400. A 1:4 grid was used, with four points related to counting hits on interstitial tissue (P(interstitium)), and with one point for hits on the reference tissue (P(cortex)), which was defined as the glomerular cortex as described above. The area related to one grid unit was 25x104 µm2, corresponding to a distance of 250 µm between the fine points.
Interstitial tissue was defined as that between glomeruli (including the Bowman's capsule) and outside tubular epithelium and larger vessels (including arterioles). Capillaries, vessel adventitia and tubule basement membranes were defined as interstitial constituents. Tubular basement membrane was included since the transition between the basement membrane and the tubular epithelial cells is clearly distinguished, which makes assessment of each single point easy.
Inflammatory cell accumulations were included in the interstitium. Tubules overlaid by inflammatory cells and with dissolved basement membranes were also defined as interstitium. In the initial count procedure, an average of 80 points (range 16251) on interstitial tissue was counted per section.
For each individual section, calculations were performed according to the equation:
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A ratio-of-means was calculated for each kidney from the total number of points.
For the calculated values of interstitial tissue per cortex in the six biopsies, the standard deviation (SD) and the coefficient of variation (CV) (=SD/mean) were estimated for each kidney, as was the coefficient of error (CE) (=SEM/mean).
The CE for the ratios was estimated from equation (9) in Kroustrup and Gundersen [13] if the CE (P(cortex)) was 0.1. If the CE (P(cortex)) was >0.1, the CE of the slope of the correlation between P(interstitium) and P(cortex) was used.
For the repeated measurements, variance was estimated from the summed squares of differences between measurements. CV and CE were estimated.
For correlation analysis between the amount of interstitial tissue and clinical parameters, the Pearson product moment test was used.
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Results |
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The mean SD for the eight kidneys was 0.05 (range 0.040.06) and the mean CV was 0.13 (range 0.060.22). The CE of the ratios varied from 0.04 to 0.18, with a mean CE of 0.08.
Figure 1 illustrates the results for repeated measurements. The SD and CV estimated from these measurements were 0.04 and 0.07, respectively. The CE was 0.02.
VV(interstitium/cortex) is illustrated as a function of the duration of implantation in Figure 2. The amount of interstitial tissue correlated positively with the increase in implantation time (r=0.80, P=0.03 (two-tailed test)).
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Scores according to the Banff classification are shown in Table 1. Histological diagnoses varied from none to severe acute rejection and from none to severe chronic allograft nephropathy.
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Discussion |
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Studies have been performed regarding reproducibility of histological diagnoses and gradings of these. They have mainly been focused on intra- and inter-observer variability regarding reproducibility of specific diagnoses and gradings on the basis of one biopsy [14]. Since the biopsy represents only a very small fraction of the entire kidney, the question of representativeness of the single kidney biopsy is critical for daily clinical work and for kidney disease research.
To our knowledge only a few studies on the topic of representativeness have been published. With regard to rejection following kidney transplantation, Pardo-Mindan et al. [15] determined the value of a renal biopsy with respect to a histological degree of acute rejection. They showed that in 510 cylinders of renal tissue taken from 17 allograft nephrectomies, an accurate assessment of the histological degree of acute rejection was made in 71.8%.
Wang et al. [16] evaluated the prognostic accuracy and reproducibility of renal transplant biopsies and found that wedge biopsies from the outer cortex obtained at procurement can overestimate the total amount of glomerulosclerosis. In the same study, Wang et al. evaluated interstitial fibrosis/tubular atrophy using a semiquantitative method. The estimates were reported to lack reproducibility when comparing right and left kidney wedge biopsies at baseline from the same donor, and also when comparing baseline wedge biopsies with later core biopsies. Our study is quantitative, which might explain the difference in outcome.
Eikmans et al. [17] performed reverse transcription polymerase chain reaction (RT-PCR) on collagen1(IV) in four biopsies each from three kidneys with focal and segmental glomerulosclerosis, and concluded that a single biopsy is representative for the corresponding kidney.
Interstitial expansion with fibrosis is one of the histological signs in the development of chronic rejection and chronic allograft nephropathy [18].
Bohle et al. [11] found that renal interstitial volume and level of serum creatinine were correlated in patients suffering from glomerulonephritis. Early changes in the interstitium as well as measurements of interstitial fibrosis have been shown to predict long-term function in transplanted kidneys [9,10].
Recently, studies have been performed using computerized morphometric measurements of kidney biopsies [19]. Nicholson et al. [9] found an SEM of <10% when using point counting to measure the interstitial volume fraction in needle core transplant biopsies, and an intra-observer error that was also <10%. In another study, Nicholson et al. [19] repeated the computerized measurements of the percentage area fraction of collagen III to evaluate reproducibility. The measurements were repeated 10 times in one section, and another 10 sections were stained and analysed on the same day. A further 10 sections were stained on separate days, but analysed on the same day. The variability was found to be greatest between the three test groups, which they concluded to be due to differences in microscope illumination. This illustrates a technical problem in the use of automated procedures.
With respect to the representativeness of values of interstial volume fractions, Kappel and Olsen [20] obtained 10 specimens from one kidney and found a variation of 5%. To estimate the precision of point counting they also performed a reproducibility study by double measurements on 10 specimens. Deviations were between 4.3% and 11.8%.
We have found no studies published on the representativeness of the distribution of histological changes in the interstitium in kidney transplants.
In the present study, the volume fractions of interstitial tissue in the explants varied from 0.25 to 0.78, which is a wide range in grafts with clinical dysfunction. The amount of interstitial tissue increased with prolonged implantation time.
Independent of the grades of interstitial widening, the distribution within the kidneys was acceptably uniform as estimated using the applied technique. The coefficient of variation increased with a decrease in the amount interstitial tissue, indicating that the counting method became less precise, although it was still acceptable.
The total variance is composed of the biological variance and the measurement (error) variance. Variance component analyses showed that the majority of variance inside the kidneys could be attributed to the measurements. Since the measurement variance was indeed low, we conclude that the degree of representativeness was high.
An acute rejection superimposed upon a chronic rejection in explants might be a problem because of oedema. It could contribute to some of the variation in the fractions of interstitial tissue inside the grafts and also among the grafts.
Ideally, the biopsies should have been performed in vivo to avoid a discontinuation of immunosuppressive treatment before explantation, thereby superimposing acute rejection and theoretically increasing oedema and cell accumulation. This is one of the possible sources of error to the exact quantities of interstitial volume, and it seems likely that the influence on results may be at its greatest in kidneys with low quantities of interstitial tissue. On the other hand, it seems hard to imagine that acute rejection with inflammatory cell accumulation and oedema could be restricted to only one part of the kidney. Pardo-Mindan et al. [15] showed an accurate histological degree of acute rejection in 71.8% of 510 biopsies from 17 allograft nephrectomies. Another source to variation in the interstitial space among cases is the sampling error. As discussed by Sund et al. [8] the sampling error might be a problem when assessing low-grade changes, and especially when using a semi-quantitative method.
The fact that we used a larger biopsy than the typical core biopsies used in the clinical routine might be a limitation of this study. However, since only a fraction of the tissue was selected randomly and used for point counting, it does not necessarily mean that a larger biopsy results in more tissue in the actual counting procedure. However, it might be a problem if the results are extrapolated to studies based on other criteria. We have performed another study on 18G baseline biopsies and found an equivalent degree of reproducibility (data not shown).
The definition of interstitial space was chosen for the most unambiguous identification in any section, i.e. to ensure point counting was distinct and precise. Thus, the tubular basement membranes were included, since the interface between basement membrane and interstitial tissue is sometimes unclear (in oblique sections), whereas that between tubular cells and tubular basement membrane is distinct. Likewise, the adventitia of larger vessels does not have a distinct limit towards interstitium, whereas the external surface of the vascular media is distinct. Inclusion of capillary space is necessary due to problems precisely identifying this parameter in paraffin sections.
The aim of this project was to measure the amount of interstitial tissue in different regions of the kidney to clarify whether it is acceptable to conclude that there is equal distribution of the tissue compartment in the renal cortex. Our results indicate that one biopsy is representative of the whole kidney regarding the distribution of interstitial tissue of explanted grafts with different degrees of acute rejection and chronic allograft nephropathy, using point counting as applied in this study.
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Acknowledgments |
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Notes |
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References |
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