A Simpli ed Method for Measuring the Thickness of Glomerular Basement Membranes

Transcription

1 Ultrastructural Pathology, 27: , 2003 Copyright # Taylor & Francis Inc. ISSN: print/ online DOI: / A Simpli ed Method for Measuring the Thickness of Glomerular Basement Membranes B. Marquez, BSc Department of Electron Microscopy and Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus I. Zouvani, MD Histology Department, Nicosia General Hospital, Nicosia, Cyprus A. Karagrigoriou, PhD Department of Mathematics and Statistics, University of Cyprus, Nicosia, Cyprus E. Anastasiades, MD Measurement of the thickness of glomerular basement membranes is required for the diagnosis of thin membrane nephropathy. Over the years various morphometric methods have been used but some are laborious so there is a need for establishing a simplified method for measuring thickness. In the present study 20 renal biopsies were used to carry out a comparative morphometric analysis between 2 methods. The first method was based on measuring thickness at the maximum number of available points, whereas for the second method, thickness was measured at only 5 points per loop. Since both methods gave mean values that are not statistically different in each patient, the authors recommend that the simplified method be used routinely for diagnosis. Keywords glomerular basement membranes, ultrastructure, morphometry Nephrology Department, Lamaca General Hospital, Lamaca, Cyprus A. Pierides, MD Nephrology Department, Nicosia General Hospital, Nicosia, Cyprus K. Kyriacou, PhD Department of Electron Microscopy and Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus The diagnosis of thin-membrane nephropathy relies on ultrastructural evidence of attenuated, thin glomerular basement membranes (GBMs) [1, 2]. Because in the renal biopsies of some patients, thin membranes may not be detectable by routine electron microscopy, various more advanced electron microscopic morphometric methods have been introduced over the years for estimating GBM thickness [3^13]. Some of these methods are complex and time-consuming and require many measurements at specific anatomical sites [13]. This prevents their wider use in routine clinical and laboratory practice. The aim of the present study was to compare and validate a simplified ultrastructural method for estimating the thickness of GBMs. To achieve this aim, a comparative morphometric study was carried out on Received14 May 2003; accepted 4 August Address correspondence to K. Kyriacou, PhD, Department of Electron Microscopy and Molecular Pathology, The Cyprus Institute of Neurology and Genetics, PO Box 3462, Nicosia, Cyprus. kyriacos@cing.ac.cy renal biopsies from 20 patients. First, a detailed electron microscopic morphometric study was performed in all 20 patients, measuring GBM thickness at all available points, in all glomerular loops, and in all glomeruli present, as previously described [14]. With this method one could distinguish between thin GBMs, having a thickness less than 300 nm, and normal GBMs with a thickness between 300 and 400 nm. This study also revealed the presence of thin GBMs in some patients with idiopathic nephrotic syndrome both with minimal change disease and focal glomerulosclerosis. This finding has rarely been reported previously [15, 16]. Using the detailed method in 2 cases we showed when the number of GBM points was reduced to as low as 5% of the total GBM points measured, the means and SD of each case were not significantly altered. Based on these preliminary results we carried out a more extensive study on all the cases, using a simplified method. The simplified method measurements were taken on the same ultrathin sections at only 5 points per loop per glomerulus in each biopsy. The data show that the simplified method gives values for 409

2 410 B. Marquez et al. the mean thickness of GBMs in each patient that are not statistically different from the previous more laborious and detailed method. Since the simplified method is just as reliable and accurate as the detailed method, we recommend that it be adopted for routine use in the diagnosis of thin-membrane nephropathy. SUBJECTS AND METHODS The study is based on kidney biopsies obtained from 20 patients, 12 male and 8 female, with an age range of 4^69. Twelve of these patients presented with proteinuria, 8 in the nephrotic range, while 6 of these also had microhematuria. The remaining 8 patients presented with hematuria only, 3 with macroscopic hematuria and 5 with microhematuria. Renal Biopsies Renal biopsies were obtained by using a standard percutaneous technique under local anesthesia. The biopsies were examined and sectioned under an ordinary light microscope to select tissue containing glomeruli for light microscopy, immunofluorescence, and electron microscopy. All 20 biopsies included in this study had unremarkable histological findings on light microscopy and negative immunofluorescence. Electron Microscopy=Morphometry Specimens were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, ph 7.2, postfixed for 1 h in 1% osmium tetroxide, dehydrated in a series of graded ethanols, cleared in propylene oxide, and embedded in an Epon=Araldite mixture. Ultrathin sections were stained with uranyl acetate=lead citrate and examined in a JEM-1010 transmission electron microscope. Each resin block from the patient s renal biopsy was serially sectioned to obtain the maximum number of glomeruli. In every glomerulus, each patent peripheral loop was photographed at a magnification of 4,000 on cm Kodak electron microscope film. A grating replica with 2,160 cross lines=mm was used for calibration (Electron Microscope Sciences, Washington, PA). Prints having a final magnification of 12,000, were scanned by using a Hewlett Packard, Scanjet 4C, at a resolution of 150 pixels per inch. The digitized images were displayed in a Trinitron Multiscan 17 sf II monitor and measured using the software NIH Image v.1.61 (NIH Image Web site Two methods were used for estimating the thickness of GBMs in each patient and measurements were made on the same ultrathin sections (Figure 1). In the first method a maximum number of measurements was taken along the GBM at every 1=3 mm actual size, corresponding to a distance of 4 mm on the12,000 working prints (Figure 1a). For the second method, measurements were taken as above but only at 5 points, evenly distributed per loop (Figure 1b). This was done to reduce the workload. The aim was to validate the accuracy of this method as compared to the detailed one. For the statistical analysis, the standard two-sample t test [17] was used to compare the detailed method against the simplified method for the estimation of the thickness of GBMs. The same testing procedure was used for the comparison of the mean values for each patient between the above 2 methods. RESULTS A review of previously published studies involving healthy donors, patients with IgA nephropathy, patients with hematuria and minimal change nephritic syndrome, showing that the adult GBM thickness lies between 300 and 400 nm, is displayed in Table 1a. In contrast, studies in patients with benign hematuria show that the adult GBM width may be significantly thinner, consistent with the thin-membrane syndrome (Table 1b). Based on the above findings and for classifying our own patients, we adopted the normal adult mean GBM thickness to be within 300^400 nm. In the case of children up to the age of 9 years, the range used for normal thickness was between 200 and 300 nm, as suggested by Morita et al. [9]. In each patient the number of glomeruli found ranged between 1 and 9. Up to 21 loops were measured per glomerulus and for the detailed method this amounted to between 42 and 2341 GBM widths being measured per patient. In contrast, for the simplified method, 20^270 GBM point measurements were used per patient. The individual GBM width measurements from all the glomeruli found in each patient s biopsy were pooled to produce a mean GBM thickness and a standard deviation (SD) in each case (Table 2). In addition, for each biopsy, a histogram was plotted of the frequency of GBM points measured against thickness to determine whether the distribution was normal or skewed (Figure 2). This quantitative comparative, ultrastructural morphometric study in these 20 cases resulted in the identification of 3 subsets of patients presenting with (a) normal, (b) thin, and (c) thick, GBMs, as shown in Table 2. Subset a with normal GBM thickness consists of 9 patients, including one child (case 1) age 4 years. The mean GBM thickness of the child was nm. With the detailed method the mean GBM thickness in the 8 adults was nm (range 312^398) (Table 3). The mean GBM thickness with the simplified method was , (p value.24), which is not significantly different. In this subset there was a discrepancy in case 2 since with the simplified method the mean GBM measures nm, and so it should appear in subset c, in the thick GBM group. Subset b consists of 10 patients all with a mean GBM thickness well below the lowest value of the normal range. This thin-membrane group had a mean GBM thickness with the old, detailed method of nm (range 239^290). With the new, simplified method the mean and standard deviations were nm (Table 3). The p value was.11 and the difference was again not significant. The last subset consisted of only

3 Morphometry of GBMs 411 FIG. 1 Electron micrographs of the same glomerular loop showing points at which GBM thickness was measured. (a) Detailed method: GBM thickness was measured every 4 mm (dots). (b) Simplified method: GBM thickness was measured at only 5 points per loop (squares), 12,000.

4 TABLE 1 Mean Values of GBM Thickness from Previous Studies (a) Normal GBM thickness Ref. no. Author Mean GBM SD n=type Age Sex Range 2 Dische et al., ö 11 IgA Not assessed Not assessed Min 356 Max Osawa et al., arithmetic 73 5 N 14 A Non Non 145^437 (mean þ = 2 SD) 4 Jorgensen and Benion, geometric þ m ¼ 8N f ¼ 6N Non Non 226^479 (mean þ 150= 7 130) 5 Osterby, arithmetic N Non f ¼ 90% m 66^554 (mean þ = 7 SD) 6 Steffes et al., 1983 M ¼ 373 F ¼ 326 M ¼ 42 F ¼ 45 m ¼ 59 f ¼ 59 Increase to 40s, decrease thereafter þ ve * M ¼ 247^457 Harmonic Live D F ¼ 236^416 7 Coleman et al., arithmetic 19 7 NH Not assessed Not assessed 356^432 (mean þ = 2 SD) 9 Morita et al., 1988 Arithmetic 35 MCNS þ ve M>F Mean þ = 2 SD 1y¼ y ¼ 60 not significant 1 y ¼ 100^340 3y¼ y ¼ 60 3y¼ 130^380 5y¼ y ¼ 60 5y¼ 160^410 7y¼ y ¼ 65 7y¼ 180^430 9^13 y ¼ 310 9^13 y ¼ 65 9^13 y ¼ 190^ Tiebosch et al., 1989 Arithmetic Not assessed Not assessed * NH 223^ IgA 244^ Others ref 273^ normal 264^

5 11 Dische et al., 1990 M ¼ 418 F ¼ 382 All ¼ 408 Harmonic M ¼ 50.9 F ¼ 44.4 All ¼ 51.5 M ¼ 44 F ¼ 17 All ¼ 61 Dead D Peak to 40s further increase after 60s M>F Mean þ = 1.5 SD M ¼ 342^494 F ¼ 315^448 All ¼ 331^ McLay et al., arithmetic RF due to other causes Not assessed Not assessed * 219^467 (b) Thin GBMs 2 Dische et al., ThinGBM ö ö Min206 Max Coleman et al., arithmetic 23 9 idiopathic hematuria ö ö Mean þ = 2 SD 235^327 8 Abe et al., Tiebosch et al., thin GBM ö ö 153^ microhematuria ö ö * 135^ Dische et al., 1990 M ¼ 293 F ¼ 303 All ¼ 300 Harmonic M ¼ 25 F ¼ 21 All ¼ 23 m ¼ 15 f ¼ 29 All ¼ 44 ö M¼ F Mean þ = SD M ¼ 255^331 F ¼ 272^335 All ¼ 266^ McLay et al., arithmetic 29 9 hematuria ö ö * 167^283 Note.N,healthypatients;A,autopsypatients;D,donors;NH,normalhematurics;MCNS,minimalchangenephroticsyndrome;RF,renalfailure;H,hematuria;P,poteinuria;NS,nephrotic syndrome. 413

6 414 B. Marquez et al. FIG. 2 Frequency histograms showing distribution of measured GBM points. (a) Case 3 displays a near normal distribution pattern. (b) Case 20 displays a distribution exhibiting two peaks, at 150 nm and at 650 nm. This case was diagnosed as Alport syndrome. 1 patient, presenting variable GBM width, due to thinning and thickening, in agreement with the clinical diagnosis of Alport syndrome. The data show that results from the simplified method are not statistically different from the results obtained with the detailed method (Table 3). In addition there was no statistical difference between the values of the mean GBM thickness in each individual patient between the 2 methods. In particular, the p values ranged from.10 to.96 for the normal cases and from.15 to.92 for the thin-membrane cases. The frequency histograms plotted for each patient, as represented by case 3, show a near normal distribution of GBM points in the 19=20 cases (Figure 2a). In only one patient, case 20, the distribution exhibited a double peak and a significant number of thick GBMs, suggesting the simultaneous presence of thinning and thickening in this biopsy (Figure 2b). DISCUSSION Thin membrane nephropathy is not an uncommon pathological entity [11, 18] and its accurate diagnosis is often required. This depends on exact ultrastructural morphometric measurements of the thickness of GBMs. Over the years various methods for estimating GBM thickness have been proposed [6, 13, 14], but many of them are not being used because of their complexity [13, 14]. In a previous report from our center, a detailed morphometric study of GBMs was carried out to establish the in-house normal range of GBM thickness, as also recommended by others [11]. To achieve this and to ensure the highest possible accuracy, a detailed method involving measurements at the maximum number of available GBM points, which ranged from 42 to 2,341, in each biopsy has been described and published [14]. This method, however, proved laborious and time-consuming. The aim of the present work is to introduce a simplified method for measuring GBM thickness that is still reliable and sufficiently accurate to allow its wider use in diagnostic renal pathology. Indeed, this simplified method requiring significantly fewer measurements gave the same results for the mean GBM thickness in each of the 20 patients investigated. The choice to compare 5 GBM points with maximum was largely defined from our previous experience involving 2 of the cases. In these 2 cases, GBM points were randomly selected from the available pool, on a decreasing scale, until a minimum number of points was reached at which the mean GBM values were not affected [14]. This minimum number of measurements

7 Morphometry of GBMs 415 TABLE 2 Classification of Cases Based on Mean Thickness of GBM Detailed method Simpli ed method GBM thickness Case no. Number of glomeruli Number of points Mean and SD (nm) Number of points Mean and SD (nm) Normal Thin Thick Note. Mean is calculated as the average of all measured points. Detailed method: GBM thickness measured every1=3 of a micrometer on every patent loop. Simpli ed method: GBM thickness measured only at 5 evenly distributed points in each patent loop. TABLE 3 Mean Values of GBM Thickness for the Two Subsets Exhibiting Normal and Thin GBMs Detailed method (mean and SD) Simpli ed method (mean and SD) p value Normal GBM (8 adults) nm nm.24 Thin GBM (10 adults) nm nm.11 Note. Case 2 is excluded from the adult normal subset since it is a child. corresponded to about 5 points per loop and this was applied to all 20 cases in the present work. The number of points used for each biopsy in the simplified method varied from 10 to 270, since the criterion used was to measure GBM thickness at only 5 points, per loop, per glomerulus. Nearly all 20 patients investigated were diagnosed just as accurately using the simplified method as with the older and more detailed one. There was only one discrepancy in the diagnosis of case 2, for which the mean GBM with the simplified method measured nm, which just exceeds the normal adult range of 300^400 nm. Furthermore, statistical analysis revealed that there were no significant differences in the mean GBM values in each of the patients studied. Other investigators [7] have previously used frequency distribution histograms to present their GBM thickness data, since this reveals the presence of normal or skewed distributions. To ensure that the mean GBM thickness of each patient was the result of a normal distribution and had not been affected by extreme values, frequency distribution histograms of GBM thickness were plotted of all 20 investigated cases. Indeed, in all but one of our patients a normal distribution curve was obtained. Only one case with the Alport syndrome resulted in a distribution curve exhibiting 2 peaks, indicating the presence of simultaneous thinning and thickening. It was important to perform this part of the study since thinning of GBMs may be focal. Consequently, results from simplified methods employing few measurements may miss this phenomenon. However, as shown by the results of the present study, this simplified method is sufficiently accurate and reliable that it can detect focal GBM alterations. In conclusion, the present data show that the simplified method described, which involves measurements at only 5 points per loop is sufficiently accurate that it can be adopted as the method of choice for measuring the GBM thickness in suspected cases of thin-membrane disease. The wider use of this method will help to determine more accurately the true

8 416 B. Marquez et al. incidence and extent of thin-membrane nephropathy in most renal pathology centers, since some cases may go unnoticed because of the reluctance to use complicated methods for measuring GBM thickness accurately. REFERENCES 1. Rogers PW, Kurtzman NA, Bunn SM Jr, White MG. Familial benign essential haematuria. Arch Intern Med. 1973; 131: Dische FE, Weston MJ, Parsons V. Abnormally thin glomerular basement membranes associated with haematuria, proteinuria or renal failure in adults. Am J Nephrol. 1985; 5:103^ Osawa G, Kimmelstiel P, Seiling V. Thickness of glomerular basement membranes. AmJClinPathol.1996; 45:7^ Jorgensen F, Bentzon W. The ultrastructure of the normal human glomerulus: thickness of glomerular basement membrane. Lab Invest. 1968; 18:42^ Osterby R. Morphometric studies of the peripheral glomerular basement membrane in early Juvenile diabetes, I: development of initial basement membrane thickening. Diabetologia. 1972; 8:84^ Steffes MW, Barbosa J, Basgen JM, Sutherland DER, Najarian JS, Mauer SM. Quantitative glomerular morphology of the normal human kidney. Lab Invest. 1983; 49:82^ Coleman M, Haynes WDG, Dimopoulos P, Barratt LJ, Jarvis LR. Glomerular basement membrane abnormalities associated with apparently idiopathic haematuria: ultrastructural morphometric analysis. Hum Pathol. 1986; 17:1022^ Abe S, Amagasaki Y, Iyori S, et al. Thin basement membrane syndrome in adults. J Clin Pathol. 1987; 40:318^ Morita M, White XRHR, Raafat F, Barnes JM, Standring DM. Glomerular basement membrane thickness in children. Pediatr Nephrol. 1988; 2:190^ Tiebosch ATMG, Frederik PM, van Breda Vriesman PJC, et al. Thin-basement-membrane nephropathy in adults with persistent haematuria. N Engl J Med. 1989; 16^ Dische FE, Anderson VE, Keane SJ, Taube D, Bewick M, Parsons V. Incidence of thin membrane nephropathy: morphometric investigation of a population sample. JClinPathol. 1990; 43:457^ McLay ALC, Jackson R, Meyboom F, Boulton Jones JM. Glomerular basement membrane thinning in adults: clinicopathological correlations of a new diagnostic approach. Nephrol Dial Transplant. 1992; 7:191^ Jensen EB, Gundersen HJG, Osterby R. Determination of membrane thickness distribution from orthogonal intercepts. JMicrosc.1978; 115:19^ Marquez B, Stavrou F, Zouvani I, et al. Thin glomerular basement membranes in patients with hematuria and minimal change disease. Ultrastruct Pathol. 1999; 23:149^ Coleman M, Startling JW. Glomerular basement membrane thinning is acquired in minimal change disease. Am J Nephrol. 1991; 11:437^ Danilewicz M, Wagowska-Danilewicz M. Glomerular basement membrane thickness in minimal change disease: the ultrastructural quantitative study. Pol J Pathol. 1998; 49:23^ Jones R, Payne B. Clinical Investigation and Statistics in Laboratory Medicine. London: ACB Venture, Monnens LAH. Thin glomerular basement membrane disease. Kidney Intern. 2001; 60:799^800.