Nephrol Dial Transplant (1998) 13: 165-172 Nephrology Dialysis Transplantation Original Article On the influence of sample size on the prognostic accuracy and reproducibility of renal transplant biopsy Huijian J. Wang, Carl M. Kjellstraud', Sandra M. Cockfield and Kim Solez Departments of Pathology and Medicine, Faculty of Medicine, University of Alberta Correspondence and offprint requests to: Dr C. Kjellstrand, VP Medical Affairs Aksys Ltd, Two Marriott Drive, Lincolnshire, IL 60069, USA. than has generally been assumed in order for glomerulosclerosis rates to be reproducible or predictive of outcome. Arteriolar hyalinosis is prognostically important and shows good reproducibility independent of sample size. Interstitial fibrosis/tubular atrophy appear useless as predictors, being of no prognostic importance and lacking reproducibility. Our finding clarifies some of the discrepancies found by different investigators regarding the importance of renal biopsy in predicting prognosis. Preliminary, our data indicate that samples containing fewer than 25 glomeruli are unreliable in determining outcome based on glomerulosclerosis. The importance of our findings which are based only on chronic lesions, with respect to acute changes, is unknown. Key words: sample size; renal biopsy; prognosis; transplant outcome; reproducibility Introduction The introduction of renal biopsy into clinical medicine by Alwall in 1944 [1] has greatly advanced the understanding of renal diseases. While an accurate diagnosis can sometimes be rendered in a biopsy containing only a single glomerulus [2], the overall sample size needed to generate accurate prognostic information is more controversial. While several authors find a fairly good correlation between the biopsy and natural outcome in a variety of renal diseases [3-7], others find it much less useful [8-10], and in some instances the renal biopsy did not provide more information than could be obtained by simple routine clinical investigation [11]. The size of the renal biopsy is obviously of importance as some lesions can be quite localized, particularly those involving the glomeruli [12]. Although there are statements in the literature as to how many glomeruli are needed for a satisfactory biopsy [13-16], this information is mainly personal impression. We found two articles [17,18] that carefully analysed the size needed, either for prognostic information, or for reproducibility of findings in successive biopsies: Kellow © 1998 European Renal Association-European Dialysis and Transplant Association Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 Abstract Introduction. The minimal specimen size necessary for accurate interpretation of a renal biopsy has not been identified. We attempted such a determination by three different analyses of a collection of biopsies performed in renal transplants. Methods. First, we studied the influence of three lesions (glomerulosclerosis, arteriolar hyalinosis, interstitial fibrosis/tubular atrophy) in 199 baseline biopsies, obtained at time of transplantation, on transplant outcome. Secondly, we compared the results from the three lesions in baseline biopsy with those from 114 subsequent core biopsies in the same patients. Thirdly, we compared the two baseline biopsies obtained in 118 paired kidneys in cadaver transplantation where both kidneys were used. Results. For statistically significant prediction of outcome from glomerulosclerosis, we found that a specimen containing at least 25 glomeruli was needed in the baseline biopsy. Arteriolar hyalinosis predicted outcome independent of sample size, but became less important than percentage glomerulosclerosis in predicting outcome if only samples containing more than 25 glomeruli were considered. Interstitial fibrosis/tubular atrophy did not predict the outcome of a kidney, independent of sample size. When comparing baseline with subsequent core biopsies, or with paired baseline biopsies, at least 14 glomeruli were necessary to allow even moderate reproducibility of glomerulosclerosis (Cohen's kappa > 0.25) and to allow statistical significance (P < 0.05). The reproducibility of arteriolar hyalinosis was not dependent on sample size but was reproducible in 80% of paired baseline biopsies, and in 67% of the comparison of the baseline with core biopsy. Both precision and significance was lost if sample numbers were reduced by including only larger samples. There was no reproducibility in any study of interstitial fibrosis/ tubular atrophy when comparing either baseline with subsequent biopsy, or paired baseline biopsies. Summary. Much larger biopsy samples are necessary H. J. Wang et al. 166 Subjectsandmethods From July 1991 to April 1995, renal transplants (n = 199), complete with baseline wedge biopsy, were performed at University of Alberta Hospital, Canada. The observation period ended on November 1, 1995; hence, all transplants were observed for 6 months or until loss of transplant function (defined as a return to dialysis or death of the patient). We investigated the influence of sample size on outcome in a study of 199 patients, all of whom are included in the analysis. We had follow-up biopsies on 114 of the 199 transplanted kidneys, and these cases were used in a second study of the influence of size on reproducibility, comparing baseline with subsequent core biopsies. In 59 pairs of kidneys we had baseline biopsies on both kidneys, and these 118 kidney biopsies form the basis for a third comparison of reproducibility between the biopsies from the paired kidneys. Patients There were 70 female patients and 129 males. The mean age was 42± 13 years (range 11-69). Of 199 transplants, 165 were first transplants, 29 second, and five third. The mean age of the donors was 37 ± 15 years (range 4-71 years). There were 145 cadavers and 54 living donors. Preparation ofbiopsy Intra-operative baseline biopsies were performed by obtaining a small wedge from the renal cortex during the time between vascular anastomosis and ureteral anastomosis. Follow-up needle biopsies were obtained under ultrasound guidance using a Truecutf (Baxter Healthcare Corp., Deerfield, IL, USA) needle in a biopsy gun. The needle size was #16 and usually three cores were obtained. The renal biopsy specimens for light microscopy were fixed in 10% buffered formalin and then embedded in paraffin. Sections of 3 urn thickness were cut and then stained with haematoxylin and eosin, periodic acid Schiff (PAS), silverstain, and Masson trichrome stain. Scoring ofbiopsies The number of globally sclerosed glomeruli were counted and then compared with the total number of glomeruli and reported as a percentage. We also scored interstitial fibrosis/ tubular atrophy and arteriolar hyaline thickening as binary data (absent/present). According to the Banff schema [19], the presence of interstitial fibrous and tubular atrophy was defined as > 55% of area involved in the process. Multiple sections of each biopsy were always studied. Treatment All patients received triple therapy with prednisone, azathioprine, and cyclosporine. After transplantation, daily cyclosporine levels were determined and the dose regulated according to blood levels, aiming at a concentration of 300 J.lg/l in the first 2 weeks and then slowly decreasing the dose, aiming at a concentration of 100 J.lg/l by the end of the first year. In 49 cases, cyclosporine was not used early because of delayed graft function. Rejection episodes were treated with i.v. solu-medrol, and if resistant, with i.v. OKT-3. Outcome ofrenal transplant In total, 33 kidneys were lost, 11 due to patient death, 14 from rejection, five from technical complications, and three from other causes. In all, 20 kidneys were lost within 6 months. There was no difference in long-term outcome between related and cadaver kidneys. The 4 year cumulative survival of living donors was 90 versus 80% for cadaver donors (P=0.120). Data were therefore pooled for all donors. Statistical methods The data were entered on Statview IV Statistical Program, version 4.5 and SPSS 6.1 for the Macintosh computer. Analyses used were Fisher's exact probability test, linear regression analysis, Cohen's kappa coefficient and Cox proportional hazards analysis. P < 0.05 was regarded to be significant. All data are mean ± SD. The number of glomeruli per biopsy was used to decide the biopsy sample size. Influence of lesion and sample size on outcome of renal transplants This was studied in Cox proportional hazards analysis by successively excluding cases depending on the number of glomeruli in the biopsy. The first analysis included all biopsies, the second only those with more than six glomeruli, and then, more than nine, 14, 24 and 29 glomeruli successively. In this study, the three lesions (percentage glomerulosclerosis, interstitial fibrosis/tubular atrophy, and arteriolar hyaline changes) were co-variates. The dependent outcome variable in this study was transplant function defined as patient being alive and not on dialysis. Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 et al. [17] in 1959 found reproducibility in 76%, comparing results from core biopsies with those obtained at autopsy, but did not specifically look at biopsy size. Corwin et al. [18] deduced the minimal size of biopsy from the mathematical model of binomial distribution. Biopsies of transplanted kidneys offer a unique opportunity to study this problem. If baseline biopsies are obtained, it has been shown that chronic lesions, e.g. glomerulosclerosis, can prognosticate the outcome in such kidneys. Secondly, if subsequent biopsies are obtained in the transplanted kidney within a reasonably short time, one can compare the results from those biopsies with the baseline, and if paired kidneys are used and baseline biopsies are obtained from both kidneys in the pair, one has an opportunity of studying reproducibility between the two biopsies. At our centre, surgical baseline wedge biopsies are regularly performed in transplanted kidneys, and follow-up needle biopsies are frequent. Furthermore, clinical follow-up of these patients is almost complete. We felt this offered a unique opportunity to study the influence of renal biopsy sample size, both on ability to predict the outcome of transplant, and on reproducibility. 167 Reliability of renal biopsy Influence ofsample size on reproducibility offindings ° Results General In the baseline biopsy we identified four lesions: glomerulosclerosis, arteriolar hyalinosis and interstitial fibrosis/tubular atrophy. Since the last two lesions always overlapped, they were analysed together. The number of glomeruli in the baseline wedge biopsy was 17.8 ± 12 (range 1-72). In the follow-up biopsy the number of glomeruli was 1O.4±5.6 (range 1-34) (see Figure 1) . In 60/199 (30%) of the baseline and 29/114 (25%) of the follow-up needle biopsies, there was some glomerulosclerosis. The mean percentage of sclerotic glomeruli was 4±7 (range 0-43) and 4±9 (range 0-58) in baseline and follow-up biopsies respectively. Arteriolar hyalinosis was present in 31% of baseline biopsies and Influence ofsample size on prognostic reliability Percentage glomerulosclerosis When all baseline biopsies were related to outcome in Cox proportional hazards analysis, percentage glomerulosclerosis was of no predictive value [relative risk (RR)=0.998, confidence interval (CI) 0.959-1.038, P=0.915) . However, as the biopsy size was increased by excluding cases with few glomeruli, accuracy increased and reached a maximum when samples including more than 25 glomeruli were used (RR = 1.056, CI 1.010-1.105, P=0.017). These relations are demonstrated in Table I and Figure 2. Thus, in order to predict outcome from glomerular changes, our data Table I, Relative risk (RR), confidence intervals (CI) and significance of percentage glomerulosclerosis in predicting long-term graft function related to sample size, expressed as number of glomeruli in baseline biopsy (Cox proportional hazard analyses) Included biopsies n" P RR CI All cases > 6 glomeruli > 9 glomeruli > 14 glomeruli > 19 glomeruli > 24 glomeruli 197 0.915 0.822 0.699 0.173 0.062 0.017 0.998 1.005 1.008 1.031 1.043 1.056 0.959-1 .038 0.965-1.046 0.967-1.051 0.987-1.078 0.998-1.091 1.010-1.105 172 142 97 69 49 "The larger the sample size, the higher is the relative risk of graftloss, as a function of percentage glomerulosclerosis. Statistical significance (P <0.05j, is first reached only when samples with more than 24 glomeruli are included. 45 30 40 25 35 30 20 25 1: g 15 1: ::l 0 0 0 20 15 10 20 30 40 50 lII1JMBER GI.O~n:R UJ.lIN IIASEJ.lNE BIOPSIF$ MF..AN 16.4 :t 11.1. RANGE 1 • 72. 60 70 80 NUMBER GI.OMERUJ.l IN T RANSrl..Al'(J' Mt:AN IU :t S.6. RANGE 1 • .l4 Fig. 1. Histogram of number of glomeruli per biopsy in baseline and subsequent transplant biopsy. 810PS IF_~ Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 In the study of reproducibility of the morphological changes, we studied the influence of the size of biopsy by successively excluding cases as described above, and doing repeated l or Fisher's exact analyses, Cohen 's kappa determinations for association and linear regression analyses. Percentage sclerotic glomeruli was always studied as a continuous variable but we studied interstitial fibrosis and arteriolar hyaline to 3 changes both as continuous variables graded from and as categorical variables, using either four groupings (0, I, 2 and 3), or as binary variables (absent or present). Best accuracy always occurred in the latter two lesions with the binary absence/presence analyses, and only those results will be reported. As new lesions potentially could appear or old ones disappear in subsequent biopsies, we also carried out all analyses comparing the baseline with a subsequent core biopsy in two ways: first we included all biopsies independent of the time between the baseline and subsequent biopsy, then we included biopsies only if performed within 60 days of the baseline biopsy. 37% of follow-up biopsies. There was no relationship between the findings of arteriolar hyalinosis and the number of glomeruli (R=0.075 , P=0.303). Interstitial fibrosis was present in 40 and 35% respectively. H. J. Wang et al. 168 -r------------------.......,,......--... •• •• •• 0.9 •• •• •• C 1 1.08 • ~ V) OV) 0.8 I ~V) ~o • ..:l..:l U· V)~ .~ 0.7 0< ..:l=: ;:H" ~=: ~O S~ 0.6 ..:lZ ~o ~E:: ZU ~ .... 0.5 u~ =:~ ~=: ~~ =:~ O~ ••.p •• •• •• •• •• 1.04 • • 0.4 ~O U ~~ •• •• •• •• 0.3 > .... ~ 0.2 C 0.1 C •• •• •• •• •• •• •• •• •• •• C •• • 1.02 1 0 - t - - ' - - ' - - ' - - ' - - + - - ' - - ' - - ' - - ' - - + - - ' - - ' - - ' - - ' - - - r , r - ' - - ' - - ' - - ' - - + - 0.98 40 0 10 20 30 NUMBER OF GLOMERULI Fig. 2. The probability value for glomerulosclerosis (open squares, hatched line) and relative risk (closed circles, solid line) of subsequent transplant failure plotted versus the number of glomeruli per biopsy included in the Cox proportional hazard analyses. As only biopsies with more glomeruli are included, the P value decreases and the relative risk increases. The data indicate that at least 25 glomeruli are necessary for reliable prediction (P < 0.05). P < 0.05 is reached first when samples containing over 24 glomeruli are included. See text for details. indicate that samples with fewer than 25 glomeruli are unreliable. Comparison ofglomerulosclerosis and arteriolar hyalinosis in predicting outcome Arteriolar hyalinosis In stepwise Cox proportional hazards analysis, arteriolar hyalinosis was more important in predicting outcome, until only samples with more than 25 glomeruli were included, at which point percentage glomerulosclerosis became the better predictor of outcome. Arteriolar hyalinosis was the best predictor of transplant failure when all biopsies were included. In kidneys without hyalinosis the RR = 0.435, CI =0.219-0.863, P=0.017. No improvement in prognostic accuracy was obtained when including only larger samples. This was done by excluding samples with retrospectively fewer than seven, 10, 15, and 20 glomeruli, as for the analyses of the number of glomeruli. Interstitial fibrosis/ tubular atrophy There was no correlation between interstitial fibrosis/ tubular atrophy and long-term outcome, independent of sample size. Statistical significance was not reached in any of the Cox proportional hazards analyses. Sample size and reproducibility ofbaseline to subsequent transplant core biopsies Glomerulosclerosis The reproducibility of glomerulosclerosis analysed by linear regression between baseline and subsequent biopsies is demonstrated in Table 2 and Figure 3. We successively included larger and larger samples by excluding cases with too few glomeruli, and also divided our material to include all biopsies, independent of the length of time between the two biopsies, or to include only those biopsies that were done within 60 days in order to exclude new lesions on the glomeruli. Better correlation was found in the analysis includ- Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 ;::l;::l ..:l0 <z 1.06 PVALUE Reliability ofrenal biopsy 169 N .41 R • 0.46. P • 30 • •:I 2S 20 10 • • • • : 15 0 .003 • • 5 o --~- ...__•••- • .• -. - -.-----1•5 .J,....- -.---!- ..---r---r---,.- o 5 ,---,---.----,.- 10 15 20 .----,--.--,.- . - --f" 2S 30 35 PERCENT GLOMERULOSCLEROSIS IN BASELINE BIOPSY Fig. 3. Percentage glomerulosclerosis in transplant core biopsies, analysed by linear regression as the dependent variable of the percentage glomerulosclerosis in the baseline biopsy. Only 41 samples without time exclusion and all containing more than nine glomeruli per biopsy are included. Although there is statistical significance, in 16 (39%) of the samples > 10% glomerulosclerosis was found in one of the biopsies, while there was none in the other paired biopsy. ing only biopsies done within 60 days, and shows that precision, demonstrated both by increasing R values and decreasing P values, increased in all samples until only samples with 10 or more glomeruli were included. Beyond this, statistical precision is lost because there are only few cases that include more than 10 glomeruli in both biopsies. The correlation coefficient was only moderate, always less than 0.5, in statistically significant analyses. Arteriolar hyalinosis We analysed arteriolar hyalinosis by both sample size and time between biopsies with Cohen 's kappa and Fisher's exact probability test (Table 3). The best statistical significance was obtained when all samples were included, while agreement increased only moderately when sample size was increased. There was no increase in agreement , and always less statistical significance when a time limit of < 60 days between biopsies was used; there was reproducibility in only two-thirds of the cases; in one-third , a lesion was found in only one of the two biopsies. Interstitialfibrosis/tubular atrophy There was no significant relationship in interstitial fibrosis/tubular atrophy between baseline and subsequent biopsies in any combination of time limit or sample size. Comparison ofbaseline biopsies in paired kidneys Percentage glomerulosclerosis Table 2. Reproducibility of glomerulosclerosis in baseline and subsequent biopsies and its relation to sample size and time between biopsies (linear regression analysis) Cases included All > 6 glomeruli > 9 glomeruli > 14 glomeruli > 19 glomeruli No time limit n Ra p 108 66 41 12 2 0.17 0.33 0.46 0.57 0.073 0.006 0.003 0.053 The results of agreement of glomerulosclerosis in paired baseline biopsies studied by linear regression analysis, are demonstrated in Table 4. There was no < 60 days between biopsies correlation when all biopsies were included (r=0 .217, P=O.l081), however, the precision increased with p n Ra sample size and became statistically significant when samples with > 14 glomeruli were used (r=0.83, 80 0.24 0.034 P<O.OOI). However, only in 16cases did both biopsies 51 0.40 0.003 include over 14 glomeruli, and the results were greatly 31 0.49 0.005 influenced by a single outlier; if this were excluded 10 0.56 0.095 r=0.025, P=0.858 (Figure 4) . 2 "Although the R value increases with an increasing number of glomeruli in the biopsies, this becomes statistically insignificant in samples with over 14 glomeruli as statistical precision is lost due to the small number of samples with large number of glomeruli. Arteriolar hyalinosis Including all 54 paired biopsies with arterioli there was agreement in 80%, (Cohen's kappa=0.55) of the Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 -5 H. J. Wang et at. 170 Table 3. Reproducibilit y of arteriolar hyalinosis in baseline and subsequent biopsies, and its relation to sample size and time between biopsies, analyses by Cohen' s kappa No time limit Cases included All > 6 glomeruli > 9 glomeruli > 14 glomeruli Less than 60 days between biopsies 11 Percentage agreement Kapp a pa 11 Percentage agreement Kappa pa 104 69 46 15 67 68 67 73 0.34 0.33 0.31 0.46 0.002 0.012 0.041 0.080 76 53 31 12 68 66 67 75 0.38 0.29 0.31 0.45 0.003 0.043 0.132 0.076 ' Probability calculated using Fisher' s exact test. Table 4. Reproducibility of glomerul osclerosis between paired baseline biopsies and its relation ship to sample size ( linear regression an alysis) Cases included 11 R P All > 6 glomeru li > 9 glomeruli > 14 glomeruli 56 42 31 16 0.22 0.26 0.34 0.83 0.108 0.091 0.061 < 0.001 Interstitial fib rosis/tubular atrophy Percentage agreement Kappa p All >6 >9 > 14 54 41 31 17 80 78 71 71 0.55 0.53 0.32 0.47 0.0001 0.004 0.185 0.162 I • 60 ... 50 ~ 40 .~ For glomerulosclerosis, there was much bett er agreement between the two base line biopsies (Table 4) than between the baseline and subsequent core biopsy (Table 2). Thus, the R value for more than 14 glomeruli between the two baseline biopsies is 0.83, compared with 0.56 for baseline to core biopsy. On the contrary, there is no great difference in the reproducibility of arteriolar hyalinosis. Cohen's kappa is 0.45 (Table 5) versus 0.47 (Table 3) for baseline/baseline and N .31 R . 0.34. p . 0.061 • 1!l s a s 30 . ~ e. 20 5 f:l 0 '" • Co: 10 0 - 10 ·10 0 10 20 30 40 50 60 PERCENTGlm lEUlOSCLE ROSIS BASEll'IE I Fig. 4. Percentage glomerulosclerosis in paired baseline biop sies, studied by linear regression, and including only biopsies with more than nine glomeruli. There is poor agreement , and the R value decreases and the P value increases greatly , by excluding the single outlier with 44 and 32% sclerosis. Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 11 There was no repr odu cibility in interstitial fibrosis/ tubular at rophy between paired baseli ne biops ies, independent of sample or time. Comparison offindings in the baseline/baseline and baseline/core analyses Table S. Reproducibility of ar teriolar hyalinosis between paired baseline biopsies and its relation to sample size. Analysis calculated using Cohen 's kappa and probability by Fisher's exact test Cases included biopsies (P =O .OOOI) . Both precision and probability decreased as the sample number decreased by excluding samples based on nu mb er of glome rul i ( Ta ble 5). Reliability of renal biopsy baseline/core biopsies respectively, when samples including more than 14 glomeruli are considered. Discussion Wehrmann's patients. There were no correlation's between interstitial fibrosis/tubular atrophy either between baseline and subsequent biopsies, nor in paired baseline biopsies. We believe that this evaluation of the importance of size of the renal biopsy clarifies the many differences that have been found between morphological changes in the kidney, and outcome [3-15]. Our findings agree well with the conclusions of Corwin et al. [18] and Madaio [21]. Based on a mathematical model of binomial distribution, they thought at least 20 glomeruli were necessary for reasonable clinical prediction of focal glomerulosclerosis and of the severity of lupus nephritis. They did not discuss other findings such as hyalinosis or fibrosis. Our findings are quantitatively different from those of Gaber et al. [22] who found samples containing more than 10 glomeruli to be prognostically important. Their analysis is different from ours, in that time to graft loss was not considered and the sample size much smaller. Each of the three analyses performed by us has certain strengths and weaknesses. In our trial to relate outcome to morphological changes there are many other factors that influence the fate of a transplanted kidney, and these clinical factors such as tissue-type, drug toxicity, and acute renal failure, make this analysis weak. In the comparison of baseline to core transplant biopsy, obviously new lesions may appear secondary to insults against the kidney after transplantation or old lesions perhaps disappear. However, as we focused only on chronic changes and also used a 60 day timelimited analysis, we believe that the data here are reliable. It appears to us unlikely that any of these chronic changes should either form or disappear within the 60 day time limit of our analysis [14,15]. The comparison of a superficial wedge biopsy with the deeper core biopsy may be affected by quantitative differences. These may include a higher percentage of sclerosed glomeruli in the superficial wedge biopsy than in the deeper core biopsy obtained with a needle [23]. In fact, we found that glomerulosclerosis was less reproducible in the comparison of baseline wedge biopsy to core needle biopsy than hyalinosis. The easiest study to interpret is when two paired baseline biopsies are compared. However, although the latter is morphologically the most sound, it suffers from the disadvantage of containing the fewest number of biopsies, and therefore statistical instability markedly increased as increasing sample sizes were included with subsequent decrease in the number of biopsies. Perhaps the greatest strength in our three analyses is, that although there may be some quantitative differences, all of them essentially showed the same findings: for judgment of glomerular changes a large number of glomeruli (15-25) are necessary; for prognostication and reliability of arteriolar hyalinosis, almost any size of renal biopsy will suffice; and finally, that interstitial fibrosis/tubular atrophy has little value either for prognosis or reproducibility. The second limitation of our study is that only chronic changes in the kidney were studied. Naturally, Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 In this study, we examined the importance of sample size in renal transplant biopsy in three different ways. The succinct findings are that, to make any prognostic conclusions from glomerulosclerosis, more than 25 glomeruli are necessary, but for arteriolar hyalinosis sample size is not important. Interstitial fibrosis/ tubular atrophy does not seem to carry prognostic information. All three separate analyses of glomerulosclerosis, its predictability on outcome, its reproducibility between baseline and subsequent biopsies, as well as the comparisons of the two baseline biopsies, are in agreement. More than 25 glomeruli are necessary to predict outcome from glomerulosclerosis, and more than 14 to result in a coefficient of determination (R 2 ) of > 0.25 in the comparison of baseline to baseline or to subsequent biopsies. To us, these data indicate that glomerulosclerosis is a localized lesion in the kidney, and that many more glomeruli are necessary for evaluation of lesions in the glomeruli than generally assumed. Arteriolar hyalinosis is different. In all three analyses, the highest precision was reached statistically when all samples were included, and the statistical significance rapidly declined as the number of samples was reduced, subsequent to the increase in sample size. We interpret these findings as showing that arteriolar hyalinosis is a diffuse lesion, and that almost any renal biopsy in which a blood vessel is found is diagnostic for this lesion. There were discordant findings in 10% of the comparison of baseline with baseline and in 20-30% of baseline with subsequent core biopsy. These findings at first appears to be in disagreement with the observation by Bell [20] who found renal arteriolar hyalinosis to be unevenly distributed in autopsies. However, he studied only hypertensive patients, and such patients are often excluded by transplant teams. Thus, the age-related lesions seen by us may be more diffuse, and the agreement between baseline to baseline and baseline to core biopsy, was better. The 50% discrepancy in the findings between paired baseline and core biopsy is compatible with sampling error. It may also be due to the qualitative difference between the more superficial baseline wedge biopsy and the deeper core biopsy or to factors which cause new arteriolar hyaline change (cyclosporine/hypertension) or resolution of lesion of donor organs [14,15]. Interstitial fibrosis/tubular atrophy was of no value in predicting outcome of the kidneys, independent of sample size. This observation is at variance with previous observations, particularly those by Wehrmann et al. [3]. These different findings probably reflect the fact that scarring, on a vascular atherosclerotic basis, as present in our patients, is quite patchy and therefore highly prone to sampling error, while it may be more diffuse in glomeulonephritis, the disease present in 171 H. J. Wang et al. 172 References 1. Alwall N. Aspiration biopsy. Acta Med Scand J Int Med 1952; 143: 430 2. Brewer DA. Renal Biopsy. 2nd edn. Williams and Wilkins Co, Baltimore 1973; 3 3. Wehrmann M, Bohle A, Held H, Schumm G, Kendziorra H, Pressler H. Long-term prognosis of focal sclerosing glomerulonephritis. An analysis of 250 cases with particular regard to tubulointerstitial changes. Clin Nephrol1990; 33: 1I5-122 4. Mbakop A, Chatelenat F. Renal puncture biopsy in the aged subject: a propos of 119 cases. Ann Pathol 1985; 5:101-105 5. Whiting-O'Keefe Q, Henke JE, Shearn MA, Hopper J, Biava CG, Epstein WV. The information content from renal biopsy in systemic lupus erythematosus. Ann Int Med 1982;96: 718-723 6. Waltzer WC, Miller F, Arnold A, Jao S, Anaise D, Rapaport FT. Value of percutaneous core needle biopsy in the differential diagnosis of renal transplant dysfunction. J Urol 1987; 137: 1I17-1I21 7. Richards NT, Darby S, Howie AJ, Adu D, Michael J. Knowledge of renal histology alters patient management in over 40% of cases. Nephrol Dial Transplant 1994; 9: 1255-1259 8. Delaney V, Ling BN, Campbell WG et al. Comparison of fineneedle aspiration biopsy, Doppler ultrasound, and radionuclide scintigraphy in the diagnosis of acute allograft dysfunction in renal transplant recipients: sensitivity, specificity, and cost analysis. Nephron 1993; 63: 263-272 9. Gladman DD, Urowitz MB, Cole E, Ritchie S, Chang CH, Churg J. Kidney biopsy in SLE; 1. A clinical-morphologic evaluation. Q J Med 1989; 73: 1I25-1133 10. Hogan SL, Nachman PH, Wilkman AS, Jennette JC, Falk RJ. Prognostic markers in patients with antineutrophil cytoplasmic autoantibody-associated microscopic polyangitis and glomerulonephritis. J Am Soc Nephrol1996; 7: 23-32 11. Malleson PN. The role of the renal biopsy in childhood onset systemic lupus erythematosus: a viewpoint. Clin Exp Rheumatol 1989; 7: 563-566 12. Fuiano G, Comi N, Magri P et al. Serial morphometric analysis of sclerotic lesions in primary 'focal' segmental glomerulosclerosis. J Am Soc Nephrol1996; 7: 49-44 13. Oberho1zer M, Torhorst J, Perret E, Mihatsch MJ. Minimum sample size of kidney biopsies for semiquantitative and quantitative evaluation. Nephrology 1983; 34: 192-195 14. Morozumi K, Thiel G, Albert FW, Banfi G, Gudet F, Mihatsch MJ. Studies on morphological outcome of cyclosporineassociated arteriolopathy after discontinuation of cyclosporine in renal allografts. Clin NephroI1992; 38: 1-8 15. Solez K, Racusen LC, Marcussen N et al. Morphology of ischemic acute renal failure, normal function, and cyclosporine toxicity in cyclosporine-treated renal allograft recipients. Kidney Int 1993; 43: 1058-1067 16. Pirani CL. Evaluation of kidney biopsy specimens. In: Tisher CC, Brenner BM (eds). Renal Pathology with Clinical and Functional Correlation. Vol 1. J.B.Lippincott Co, Philadelphia; 1989 17. Kellow WF, Cotsonas NJ, Chomet B, Zimmerman MJ. Evaluation of the adequacy of needle-biopsy specimens of the kidney. Arch Int Med 1959; 104: 353-359 18. Corwin HL; Schwartz MM; Lewis EJ. The importance of sample size in the interpretation of the renal biopsy Am J Nephrol1988; 8: 85-89 19. Solez K, Axelsen RA, Benediktsson H et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 1993; 44: 41I-422 20. Bell ET. Renal Diseases. l st edn. Lea and Febiger, Philadelphia, 1946 21. Madaio MP. Renal biopsy. Kidney Int 1990; 38: 529-543 22. Gaber LW, Moore LW, Alloway RR, Amiri H, Ver SR, Gaber AO. Glomerulosclerosis as a determinant of posttransplant function of older donor renal allografts. Transplantation 1995; 60: 334-339 23. Schmidt A, Baker R. Renal biopsy in children: analysis of 61 cases of open wedge biopsy and comparison with percutaneous biopsy. J Uro11976; 116: 79-80 24. Olsen S. Renal histopathology in various forms of acute anuria in man. Kidney Int 1976: 10: S2-S8 25. Houghton DC, Hartnett M, Campbell-Boswell M, Porter G, Bennett W. A light and electro1 microscopic analysis of gentamicin nephrotoxicity in rats. Am J Patho11976: 83: 589-612 26. Solez K, Morel-Maroger L, Sraer J-D. The morphology of 'acute tubular necrosis' in man: analysis of 57 renal biopsies and a comparison with the glycerol model. Medicine (Baltimore) 1979; 58: 362-376 Received for publication: 3.1.97 Accepted in revised form: 5.9.97 Downloaded from http://ndt.oxfordjournals.org/ by guest on October 6, 2014 there was no acute disease found in the baseline biopsies of the transplanted kidneys, therefore, one cannot extrapolate our findings to biopsies of acute ongoing disease in the kidney. It has been shown that a single glomerulus may suffice to make diagnosis [2]. However similar quantitative influences found by us may potentially be present in acute diseases of the kidney. Thus, the correlation between histology and renal function and acute tubular necrosis has always been poor [24-26]. This poor correlation and some of the confusion of the prognostic reliability and the controversy if different lesions of lupus nephritis can change into each other [4,5,9,11], may be partially explained by too small sample size in analyses. Since the earliest countings of the number of glomeruli obtained in a needle biopsy, the total number found, as a mean, is 10 [2]. This has remained unchanged and is the same as we found our in our core biopsies. These 10 glomeruli represent only 0.001% of the 1 x 106 glomeruli in the kidney. A practical conclusion from our findings is that the average size of baseline biopsies should be increased so that better representation is obtained. The size of the biopsy needle and the number of cores that should be obtained in a needle biopsy of the kidney, also needs reconsideration. Based on our observations in transplants and Corwin's general mathematical analyses, we conclude that there must be a minimum of 20-25 glomeruli for reliable prognosis and probably at least 15 for true representation of chronic changes.
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