NDT Advance Access originally published online on January 9, 2006
Nephrology Dialysis Transplantation 2006 21(5):1157-1161; doi:10.1093/ndt/gfk037
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What you should know about the work-up of a renal biopsy
Departments of 1 Pathology and 2 Internal Medicine IV, University of Erlangen-Nürnberg, Germany
Correspondence and offprint requests to: Professor Dr med. Kerstin Amann, Department of Pathology, University of Erlangen-Nürnberg, Krankenhausstr. 8–10, D-91054 Erlangen, Germany. Email: kerstin.amann{at}patho.imed.uni-erlangen.de
Keywords: renal biopsy; workup; morphology; immunohistology; immunofluorescence; electron microscopy
To obtain relevant clinical information from a renal biopsy is an interdisciplinary task, requiring close cooperation between clinician and pathologist. The better the nephrologist's understanding, the more rewarding the information from the pathologist. Based on our experience, we will discuss some practical points which are not often known or are poorly handled by our clinical partners.
Since these points require close interaction between clinicians and pathologists, some guidelines concerning the procedure and work-up of routine kidney biopsy have been established by the Renal Pathology Society [1].
Standard procedures for renal biopsy handling and processing were established at the European Union Consensus Meeting, which took place on February 25, 2000 in Vienna, Austria; these procedures cover the following aspects [1]:
- Taking the biopsy
- Transferring the material
- Dividing the sample
- Preserving the tissue
- Cutting the biopsy sample
- Staining the biopsy sample
- Reporting the finding
- Establishing the diagnosis
If these points are taken into consideration, there is an 
40–50% chance that the results of the kidney biopsy will have a direct consequence for patient management [2–6].
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What size of the biopsy is minimal and optimal for the pathologist? |
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Top
What size of the...
What clinical information is...The first important question on which the opinions of the nephrological and pathological communities are divided concerns the necessary size of the kidney biopsy. We recommend obtaining two biopsy cylinders with a minimal length of 1 cm and a diameter of at least 1.2 mm. The quality of a renal biopsy depends on the size, i.e. the number of glomeruli: it is generally agreed that
10–15 glomeruli are optimal; very often 6–10 glomeruli are sufficient and in some cases even one glomerulus is enough to make a diagnosis. However, as correctly pointed out by Corwin et al. [7] ‘If the percentage of glomerular involvement in a biopsy is used to determine the severity of a focal glomerular lesion, a small biopsy sample size will lead to considerable misclassification of disease severity. In addition, a small biopsy sample size will make the exclusion of focal disease difficult’. Therefore, the pathologist has to check the size and quality of the biopsy sample under a light microscope before he decides how to process the material. One of the cylinders should be fixed and used for light microscopy and the second one for immunohistology, electron microscopy and additional investigations. |
What clinical information is indispensable to the pathologist? |
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To answer this delicate question, we wish to quote a statement from computer technology: ‘One cannot feed in garbage and get out fruit juice’. The absence of clinical information is a sore point in many partnerships between clinicians and pathologists. In an ideal world, the pathologist obtains information on the clinical history, recent laboratory values in particular urine (proteinuria, haematuria, leukocyturia, cylindruria) and serum [urea, creatinine, cholesterol, total protein, creatine clearance, C3, C4, anti-nuclear antibody (ANA), anti-neutrophil cytoplasmic antibodies (ANCA), anti-glomerular basement membrane (GBM), argininosuccinate lyase (ASL)], presence of diabetes mellitus or hypertension or other systemic diseases, other parameters of interest (if available) and current therapy (if any).
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How should a renal biopsy be handled? |
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This depends very much on whether a local pathologist is available or whether biopsy samples have to be mailed. Potential media are isotonic NaCl for fast local transport of the material—the ideal procedure which permits all different types of further processing by the pathologist: cryopreservation of one part of the material for immunofluorescence, fixation with paraformaldehyde (PFA) or formaldehyde (4%, buffered, pH 7.2–7.4) and, in parallel, fixation of a small part of the biopsy in 3% glutaraldehyde (usually for electron microscopy) (Table 1).
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There are two ways to divide and fix a kidney biopsy sample:
- The biopsy is put into isotonic saline and sent directly to the pathologist who divides the cylinder, if possible, into two or three parts: the larger portion will be used for light microscopy, the smaller portion will be used for immunofluorescence and another small portion (if available) will be used for fixation with glutaraldehyde for examination by electron microscopy (this is optional).
- The biopsy is put directly into a fixative (PFA or formaldehyde) and mailed to the pathologist. The pathologist will then embed the material in paraffin. This permits routine stains to be performed as well as immunohistology by the indirect method (APAAP or ABC) using the same material. A small piece of the formalin-fixed cylinder can also be worked-up for subsequent electron microscopy.
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What you should know about how the pathologist works-up the renal biopsy |
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After embedding in paraffin, it is recommended to cut several serial sections, i.e. 8–16 paraffin sections (2–3 µm thick) per biopsy, which will then be used for the following light microscopic and immunohistochemical staining procedures [8].
Light microscopy (Figure 1A–D)
Routine stains (paraffin sections)
- Haematoxylin and eosin (HE)
- Periodic acid–Schiff's (PAS)
- Fibrous tissue stain (i.e. Sirius red, Trichrom, Ladewig, etc.)
- Silver stain
- Protein stain
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Optional stains
- Kossa stain (calcifications)
- Congo red stain (amyloid)
It is useful to apply these different stains because the information obtained is complementary. For first hand information in routine diagnostic evaluation, the HE stain is appropriate (Figure 1A). It provides a first impression of the composition of the tissue (i.e. renal cortex vs medulla, number of glomeruli, cellular infiltration, etc.). To analyse the glomerulus, the PAS stain is most useful, since it delineates in great detail glomerular cells, mesangial matrix and potential expansion, as well as potential modifications of the composition of the matrix, changes of the GBM, i.e. thickening, irregularities, doubling, rupture and finally fibrinoid necrosis of the glomerular tuft (Figure 1B). A further advantage of the PAS stain is that alterations of the vessels, particularly arterial hyalinosis and fibrinoid necrosis, are easy to detect. Immune deposits can best be visualized by protein stains (Figure 1C) such as the acid fuchsin–Orange G stain (SFOG). Often immunodeposits can already be seen or suspected by light microscopy, but definite information requires specific stains or additional investigations, i.e. immunohistochemistry or electron microscopy.
The main advantage of the silver stain (Figure 1D) is that it permits the detection of changes of the GBM, i.e. so-called reduplication or ‘spikes’ which develop as a result of overshooting glomerular production of basement membrane material encircling immunodeposits. In order to evaluate the extent of fibrosis in the glomerulus or tubular interstitium, fibrous tissue stains such as Trichrom, Sirius red or Ladewig stains are indispensable [9]. For specific questions, additional staining techniques are available, for instance the Congo red stain for visualization of amyloidosis, or the Kossa stain to detect calcification.
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What are immunohistochemistry and immunofluorescence, and what information can be obtained from them? |
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In order to probe the tissue with antibodies, two techniques are available: immunofluorescence (Figure 1E) uses labelled antisera or antibodies (which require native tissue without fixation) and immunohistochemistry (which can be done with formalin-fixed tissue, whilst more aggressive fixatives destroy the epitopes and preclude immunohistochemical investigations) (Figure 1F).
The workhorses for immunofluorescence/immunohistochemistry are antisera or monoclonal antibodies against immunoglobulins (IgA, IgG and IgM) and components of the classical or alternative complement pathway (C1q, C3c and C4) as well as 
- and 
-light chains, albumin and fibrinogen, although for research purposes many others are available. The pathologist should not only report whether the reaction is positive, but should also comment on the pattern of staining, e.g. mesangial vs capillary staining pattern, linear (or pseudolinear) vs granular staining. If possible, he should also describe where the deposits are located, e.g. in a subendothelial, intramembranous or subepithelial position.
For specific questions, antibodies against amyloid subunits (AA or AL amyloid, transthyretin, etc.) or antibodies against viruses (cytomegalovirus, polyomavirus and adenovirus) are available. In renal transplant biopsies, immunostaining for the C4d fragment of the complement pathway has become extremely popular. Although this is still somewhat controversial, it appears to be helpful in the diagnosis of acute humoral rejection [10,11].
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When is electron microscopy necessary? |
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It is certainly not necessary to perform electron microscopy on every kidney biopsy but, because the result of the pathological investigation cannot be predicted, it is wise to preserve the material in such a fashion that ultimately electron microscopy is still possible. Electron microscopy requires particular fixation and handling of the material. It is therefore time-consuming and not universally available. If electron microscopy is envisaged at the time of a biopsy, e.g. for Alport's disease or thin basement disease, a portion of the material should be fixed with glutaraldehyde. Alternatively, formalin-fixed tissue can be used subsequently for electron microscopy.
For some renal diseases, the definite diagnosis requires electron microscopy, such as Alport's disease, thin basement disease, immunotactoid disease, minimal change nephropathy.
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What information is provided by electron microscopy? |
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Electron microscopy permits assessment of the following:
- The presence and degree of cell proliferation (mesangial vs endothelial cell proliferation)
- Changes in cell structure (i.e. podocyte foot process fusion or podocyte vacuolization)
- Necrosis or apoptosis of cells
- Changes of glomerular basement membrane (i.e. thickening, thinning, splicing, irregularities)
- Localization of immunoglobulin deposits (i.e. mesangial, subendothelial or subepithelial)
- In some renal diseases, such as lupus nephritis, specific morphological changes can be detected by electron microscopy, e.g. fingerprints or tubuloreticular structures.
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How should the pathologist report the findings in a renal biopsy? |
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It is useful for communication between the clinician and the pathologist to adhere to a standard format in the report. In our opinion, the final report of a kidney biopsy should include information on:
- The adequacy of the specimen (number of glomeruli and arteries)—(although this will sometimes be painful for the clinician who performed the biopsy)
- A description of the morphological changes in a systematic fashion for each of the compartments of interest (glomeruli, tubules, interstititum, vessels, see Table 2)
- The results of immunofluorescence/immunohistochemical studies.
- The results of the electron microscopy (this is more time-consuming and will usually require a separate report later on)
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It is useful to give two different types of diagnoses: first, a descriptive diagnosis (e.g. mesangioproliferative glomerulonephritis) and then the final diagnosis (including the results of immunofluorescence/immunohistochemical, electron microscopic studies as well as clinical information), e.g. IgA glomerulonephritis.
It is hoped that adherence to such a systematic procedure will improve the results and heighten the impact of pathology on clinical decision making.
Conflict of interest statement. None declared.
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References |
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- Regele H, Mougenot B, Brown P et al. Report from Pathology Consensus Meeting on Renal Biopsy Handling and Processing, Vienna, February 25, 2000 (http://www.kidney-euract.org/Rbpathologyconsensus.htm)
- Thut MP, Uehlinger D, Steiger J, Mihatsch MJ. Renal biopsy: standard procedure of modern nephrology. Ther Umsch 2002; 59: 110–116[Medline]
- Paone DB, Meyer LE. The effect of biopsy on therapy in renal disease. Arch Intern Med 1981; 141: 1039–1041[Abstract]
- Fuiano G, Mazza G, Comi N et al. Current indications for renal biopsy: a questionnaire-based survey. Am J Kidney Dis 2000; 35: 448–457[ISI][Medline]
- Andreucci VE, Fuiano G, Stanziale P, Andreucci M. Role of renal biopsy in the diagnosis and prognosis of acute renal failure. Kidney Int Suppl 1998; 66: S91–S95[Medline]
- Cameron JS. Indications for renal biospy, history of the procedure, and relationship of the findings to further investigation and treatment. In: Solez K, Racusen L, Olsen S, eds. Diagnostic Renal Pathology. Transpath Inc. (http://www.transpath.com/m-media/DRP.htm)
- Corwin HL, Schwartz MM, Lewis EJ. The importance of sample size in the interpretation of the renal biopsy. Am J Nephrol 1988; 8: 85–89[ISI][Medline]
- McCarthy GP, Roberts IS. Diagnosis of acute renal allograft rejection: evaluation of the Banff 97 Guidelines for Slide Preparation. Transplantation 2002; 73: 1518–1521[CrossRef][ISI][Medline]
- Amann K. New parameters in kidney biopsy diagnostic-morphometry. Kidney Blood Press Res 2000; 23: 181–182[Medline]
- Nickeleit V, Zeiler M, Gudat F, Thiel G, Mihatsch MJ. Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol 2002; 13: 242–251
[Abstract/Free Full Text] - Böhmig GA, Exner M, Habicht A et al. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol 2002; 13: 1091–1099
[Abstract/Free Full Text]
Accepted in revised form: 9.12.05
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