practice guidelines for the renal biopsy...the renal biopsy, though a relatively safe medical...

Practice guidelines for the renal biopsy

Patrick D Walker1, Tito Cavallo2, Stephen M Bonsib3, The Ad Hoc Committee on RenalBiopsy Guidelines of the Renal Pathology Society

1Nephropathology Associates, Little Rock, AR, USA; 2Department of Pathology and Laboratory Medicine,University of Cincinnati College of Medicine, Cincinnati OH, USA and 3Department of Pathology andLaboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

Biopsy of the kidney should never be undertaken without careful consideration of the risks vs benefits. Giventhe importance of a correct diagnosis in the treatment and prognosis of renal disease, the pathologicalevaluation should use all available modalities. Native kidney biopsies require examination by light microscopy,immunohistochemistry and electron microscopy. The processing of the renal biopsy is complex and requiresthe support of a fully equipped anatomic pathology laboratory. Technical expertise is required to process thesmall fragments of tissue and to produce sections of highest quality. The correct diagnosis requires a well-trained renal pathologist with thorough knowledge of not only renal pathology but also renal medicine in orderto correlate intricate tissue-derived information with detailed clinical data. In view of the importance andconsequences of the pathologic diagnosis, the Renal Pathology Society appointed an Ad Hoc Committee onPractice Guidelines, to define the essential ingredients necessary to provide quality renal pathology diagnoses.This document incorporates the consensus opinions of the committee and the RPS membership at large.Modern Pathology (2004) 17, 1555–1563, advance online publication, 23 July 2004; doi:10.1038/modpathol.3800239

Keywords: renal biopsy; glomerulonephritis; allograft; transplant rejection

The renal biopsy, though a relatively safe medicalprocedure, should be undertaken only after seriousconsideration of possible morbidity and rare mor-tality that can occur with this invasive technique.1

Owing to the nature of the diseases to be detected,the sample of renal tissue needs to be examined withoptimal methods to allow for a complete evaluation,including light microscopy (LM), immunohisto-chemistry (IHC, includes either immunofluores-cence (IF) or immunoperoxidase (IP)) and electronmicroscopy (EM). The correct diagnosis requires awell-trained renal pathologist with thorough knowl-edge of not only renal pathology but also renalmedicine in order to correlate intricate tissue-derived information with detailed and sometimessubtle clinical data in order to provide the bestpossible clinicopathologic diagnosis or diagnoses.

The Renal Pathology Society formed an Ad HocCommittee on Renal Biopsy Guidelines to developrecommendations regarding the processing andevaluation of renal biopsy specimens. This wasfollowed by an intensive several-year period of

refinement. The guidelines would first be reviewedby the Executive Board of the Renal PathologySociety and subsequently submitted to the member-ship for discussion, debate and suggestions (see thecontributor list in Acknowledgments). The commit-tee then revised the guidelines and the processwould begin anew. The techniques and guidelinesappropriate for adequate evaluation of a renal biopsyand the recommended qualification standards forthose interpreting renal biopsies presented in thefollowing sections are the result of this rigorousprocess.

Renal biopsy fixation and processing

The renal biopsy should be accompanied by ade-quate clinical information to enable proper inter-pretation of findings. Many laboratories provide aspecial clinical information form to ease the record-ing of the overall renal syndrome, symptoms andlaboratory data. Although this does not replacedirect communication with the submitting physi-cian, this basic medical information provides a goodinitial background for overall interpretation of therenal biopsy. The use of a dissecting microscope canbe of assistance in assessing sample adequacy.Another alternative is the use of a standard lightmicroscope. The tissue is placed on a glass slide

Received 29 March 2004; revised 17 June 2004; accepted 24 June2004; published online 23 July 2004

Correspondence: Dr PD Walker, MD, Nephropathology Associ-ates, 10802 Executive Center Drive, Suite 111, Little Rock, AR72211, USA.E-mail: [email protected]

Modern Pathology (2004) 17, 1555–1563& 2004 USCAP, Inc All rights reserved 0893-3952/04 $30.00

www.modernpathology.org

with normal saline and examined with or withouta coverslip producing a wet mount.2 A trainedobserver can recognize fat, skeletal muscle, andother nonrenal tissue. Knowledge of the glomerularcontent of the sample can guide division of tissue forthe various histologic modalities (Figure 1).

In the absence of direct glomerular visualization,a standard protocol for dividing the tissue ob-tained at each ‘pass’ should be used to avoidinadequate glomerular sampling for LM, IF or EM

(Figure 2). There are several acceptable appro-aches; laboratories that perform IHC on fixedembedded tissue have even more options. Thestandard approach is to first procure tissue for EMfrom each core by removing 1mm cubes fromthe ends and placing them in formalin, cooledglutaraldehyde or other fixative suitable for EM.Some clinicians prefer that the pathology laboratoryobtain tissue for EM from the ends of the formalin-fixed tissue.

Figure 1 Renal biopsy specimen as seen with a dissecting microscope. (a) Renal cortex, note the glomeruli, recognized as round red areas(wet preparation � 10). (b) Renal medulla, reddish vasculature is present but no glomeruli seen (wet preparation � 10, photographscontributed by Alexis Harris, MD).

The renal biopsyPD Walker et al

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Modern Pathology (2004) 17, 1555–1563

If the specimen is to be sent to a laboratory thatuses IF, the first core can be cut in half by cross-sectioning and the larger piece placed in formalin oranother fixative suitable for LM; the smaller portionis saved for immunofluorescence evaluation. If asecond core is obtained, the ends should be taken forEM and the specimen again divided almost in halfwith the larger tissue core now kept for IF and thesmaller for LM. Alternatively, if both cores containcortex, one core can used for LM and one core for IF.Tissue from further passes should be divided tobalance whatever was obtained initially.

If tissue is limited, the clinical differentialdiagnosis may drive the division of material, forexample, in cases of rapidly progressive glomerulo-nephritis, an attempt to secure sufficient material forIHC to rule out antiglomerular basement membraneantibodies is important. Although artefacts will be

induced, frozen tissue used for IF can be subse-quently processed for either LM or EM and,depending upon the questions to be addressed,useful information can still be obtained.

The biopsy specimen must be handled gentlywhen removed from the biopsy needle; an 18Gneedle or a thin wooden stick, for example, atoothpick, is ideal. Normal saline can be also usedto wash the sample off the needle. To avoid crushartefact, forceps should not be used. Pulling andstretching the tissue during removal from the needlemust also be avoided. Desiccation artefacts andosmotic injury may result from placing the tissue ondry gauze or gauze soaked with water, respectively.Other sources of artefact include freezing the entiresample, or placing the sample in ice-cold saline. Ifthe biopsy cores are sent to the pathology laboratoryfor division and processing, they may be placed intransport fluid (usually tissue culture medium),physiologic saline-moistened gauze or filter papercarefully folded over the tissue.

The sample should be cut with a fresh scalpel orsingle-edged blade (one that has been cleansed of oiland not been exposed to fixative). The portion for IFis gently picked up with a separate clean tool andplaced in transport solution (some centers use tissueculture medium) remaining untouched by formalinor other fixatives. The remainder is placed promptlyin fixative for LM and EM because good LM and EMmorphology is dependent upon the rapidity of tissuefixation.

If the pathology laboratory uses IP instead of IF,biopsy handling is simplified. The material for IP istaken from the formalin-fixed material also used forLM requiring only that the ends of all pieces besaved for EM.

LM

FixationThe most commonly used fixative for LM isbuffered, 10% aqueous formaldehyde solution (for-malin). Formalin is stable at room temperature,provides acceptable morphology, and allows IHC ormolecular studies to be performed. Some labora-tories prefer alcoholic Bouin’s,3 Duboscq-Brasil4 orZenker’s fixatives that provide better preservation ofcertain morphologic details. However, these fixa-tives limit recovery of material for EM, IP ormolecular studies, and require additional precau-tions and handling. Bouin’s and Duboscq-Brasilfluid contain picric acid that is highly explosivewhen dry and can be a disposal problem. Somelaboratories prefer 4% paraformaldehyde to opti-mize tissue suitability for LM, IHC and EM and forpossible research applications including in situhybridization (ISH) studies. Owing to a decreasein polymerization of critical components, thisfixative provides superior molecular stabilizationfor IHC and ISH. It also provides preservation of

Figure 2 Diagram to illustrate division of renal biopsy cores in theabsence of a dissecting microscope for laboratories using IF. Theends from all cores are taken for EM with the remainder dividedfor LM and IF.


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morphologic details equivalent to formalin.5,6

Methacarn, a modified Carnoys fixative (methylalcohol 60%, chloroform 30%, glacial acetic acid10%), provides good fixation for LM and EM and, insome studies, has provided excellent recovery ofmRNA,7 though not in others.8

The material processed for LM can serve asreserve material for IHC or EM if either of theseother modalities is found lacking glomeruli. For-malin fixation and paraffin embedding is not animpediment to detailed electron microscopic eva-luation and rapid reprocessing is a relatively simpleprocedure.9–11

Processing and sectioningTo avoid specimen loss during processing, thespecimen for LM should be gently wrapped in lenspaper that has been prewetted with fixative. Neithersponges nor plastic embedding bags should be usedbecause mechanical artefact almost always occurs.Tissues can be processed overnight using a protocolappropriate for small biopsy samples. Alternatively,rapid processing protocols are available, someemploying a microwave fixation step, that can beused for same-day processing.12–14

Sectioning and stainingSerial sections of 2 mm thickness are cut and at leasttwo sections should be placed on each slide. Thereare many acceptable staining protocols; most in-clude staining alternating slides with hematoxylinand eosin stain (H&E) periodic acid–Schiff reaction(PAS), silver methenamine and trichrome stains.

IHC

PreparationIF is best performed on unfixed, frozen sections.Tissues can be transported to the laboratory fresh onsaline-soaked gauze or in Michel’s fixative (Mod-ified Michel’s Tissue Fixative (Wampole Labora-tories, Cranbury, NJ, USA) sometimes referred to as‘Zeus medium’).15 Serial sections are cut at 2–4 mmin a cryostat. IP staining requires no special tissuepreparation in that the same formalin-fixed, paraf-fin-embedded material used for LM is also usedfor IP.

StainingThe antigens that should be routinely examinedinclude: immunoglobulins (primarily IgG, IgM andIgA), complement components (primarily C3, C1q,and C4), fibrin, and kappa and lambda light chains.Additional antibodies may be required in specificcircumstances, for example, amyloid speciation,16,17

collagen IV alpha chains in hereditary nephritis,18,19

IgG subclasses,20,21 virus identification,22–25 lympho-cyte phenotyping in allografts in suspected cases ofPTLD,26,27 C4d in allograft biopsies,28,29 etc. In theabsence of appropriate tissue in the IF sample,

paraffin-embedded material can be examined usingIP techniques (see later).

ControlsIHC controls should include a negative control (noantibody applied) and a positive control (albumincan serve this purpose) for each run. Internalcontrols exist for many of the antigens in routineuse. For example, IgA is generally present in tubularcasts, IgG in protein droplets and C3 in bloodvessels. Each time a new vial of antibody is opened,the correct dilution should be determined usingknown positive slides.

IF vs IP

IP has almost completely replaced IF in routinesurgical pathology and in many renal pathologylaboratories. Well-developed antigen-retrieval pro-cedures and the widespread availability of auto-mated IP stainers provide reproducible results inkidney biopsies. Advantages of IP include correla-tion of the LM and IP since sections come from asingle block, and an expensive IF microscopeattachment is not needed. IP material is also a morepermanent sample allowing repeated examinationwithout fading.

Still, IP has certain drawbacks. Complementantigens require careful, time-consuming antigen-retrieval procedures because of antigen maskingduring processing into paraffin. A false negative canbe a concern. Interpretation of IP slides is alsocomplicated by potentially higher background inrenal tissues and the difficulty of titrating the finalcolor product to avoid over- or understaining. Also,since the IP signal is a reaction product dependenton interaction of substrate and enzyme, diffusionartefacts can create problems in interpretation.

Immunofluorescence is the choice for manynephropathologists. The use of dark field micro-scopy for IF produces a very high signal-to-noiseratio. Accurate localization of deposits is possible inmost laboratories with good frozen section techni-que, and excellent resolution is achievable withfluorescence microscopes having epifluorescenceattachments. Overall, the choice between IF or IPis based on the experience and technical resourcesof the nephropathologist. Both methods can provideexcellent material and, in fact, they can complementone another in various clinical settings.

EM

The tissue for EM may be fixed in 2–3% glutar-aldehyde or 1–4% paraformaldehyde. Adequatefixation can also be obtained when tissue is fixedin buffered formalin. EM cannot be performedon tissues exposed to mercury-based fixatives(Zenker’s, B-5, etc). The tissue sent in Michel’stransport medium would have poor ultrastructural


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preservation since this is not a fixative solution.Rapid placement of the sample into fixative willprovide the best outcome. Tissue can be reprocessedfrom the paraffin block, or the frozen block if noglomeruli are available in the EM sample. Althoughsevere cellular artefacts may result when frozen andparaffin-embedded tissue are processed for EM, theGBM and immune deposits are usually sufficientlyintact for evaluation.

Toluidine blue-stained, 1 mm thick, so-called‘thick’ sections, are examined to identify appropri-ate structures for thin sectioning and examinationwith the electron microscope. Thick sections arealso useful to supplement the paraffin material (eg alesion of focal segmental sclerosis may only bepresent on the thick section). In general, one or twoglomeruli are examined ultrastructurally. Low-,medium- and high-magnification photographs aretaken to include both capillary loops and mesangialareas. The tubulointerstitium and vessels are alsoexamined and pertinent photographs taken toillustrate any abnormalities in these areas.

Interpretation of the renal biopsy

The evaluation of a kidney biopsy includes exam-ination of multiple serial sections each with severaltissue slices, stained with a variety of stains to beexamined by LM and IHC. ‘Thick sections’ for EMare also reviewed for additional information. Carefulevaluation of glomeruli, tubules, the interstitiumand the vessels is required. The final report shouldprovide a glomerular count with the number show-ing global and/or segmental sclerosis. In certainsituations, other glomerular lesions should becounted (eg number of crescents, subtyped intocellular, fibrocellular and fibrous, etc). A descrip-tion of alterations in tubules, interstitium andvessels should also be included. Each slide repre-sents another level that may reveal additionalfindings, re-emphasizing the importance of meticu-lous examination of all materials. Rapid turnaroundis required to obtain maximum utility from informa-tion derived from the renal biopsy. Two days for LMand IF and 3–5 days for EM should be consideredroutine. Finally, a renal biopsy should never beinterpreted in a clinical vacuum. A nephropatho-logist must have a thorough understanding of renaldisease as well as good communication with thenephrologists caring for the patients. This allows anaccurate correlation of clinical information with theobserved pathologic processes and this combinationwill lead to the correct final diagnosis.

The Native Kidney Biopsy

Native kidney biopsies provide maximum informa-tion when evaluated by LM, IHC and EM.30–32

Whether the use of all three modalities is necessaryfor all biopsies has been debated, and certainly some

nephropathologists use their discretion in selectedcases.33 Still, less than a complete workup should bea rare exception, usually resulting from an inade-quate specimen for one of the studies, LM, IF or EM.Biopsy diagnoses generated without IHC or EM datamay result in an erroneous or incomplete diagnosis(Table 1) because a number of entities can only berecognized by IHC or EM.

If one study is intentionally omitted it is usuallyEM. Recent reviews, however, have demonstratedthe utility of EM in renal biopsy diagnosis andreconfirmed the necessity of routinely performingelectron microscopic evaluation.32,34 Haas34 showedthat the usefulness of EM has not declined since theearlier reports documenting the need for EMpublished in the early 1960s and 1970s. While EMwas absolutely necessary to make a correct diagnosisin 21% of cases, EM evaluation resulted in clinicallyrelevant refinement of or addition to the diagnosisin another 24%. Examples of the latter includefindings of thin glomerular basement mem-branes (GBMs) to account for otherwise unexplainedhematuria in a patient with interstitial nephritis oracute tubular necrosis, tubuloreticular inclusions ina reportedly HIV-negative patient with collapsingglomerulopathy, among others. Although in retro-spect, as many as 50% of cases do not require EM, itis not possible in an individual biopsy to determineif it can be completed without EM until the EMfindings are actually known.

The Renal Transplant Biopsy

Donor biopsyQuestions regarding the suitability of a cadaverickidney for transplantation may arise, particularlywith the increasing use of older donors. Intraopera-tive frozen sections are frequently employed in

Table 1 Renal biopsy diagnosis requires LM, IF and EM

Diagnoses overlooked without IHCLight chain-associated diseasesAL amyloidMonoclonal immunoglobulin deposition diseaseLight chain cast nephropathy

IgA nephropathy/Henoch–Shonlein purpuraIgM nephropathyC1q nephropathyAntiglomerular basement membrane diseaseHumoral (C4d) transplant rejectionFibronectin glomerulopathy

Diagnoses overlooked without EMFibrillary glomerulopathy/immunotactoid glomerulopathyNail–patella syndromeLipoprotein glomerulopathyDense deposit diseaseAlport’s syndromeThin glomerular basement membrane nephropathyCollagenofibrotic glomerulopathy


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these situations. The information extractable from afrozen section is directly linked to the quality of thesection generated. A frozen section of acceptablequality is adequate to assess the percentage ofglobally sclerotic glomeruli and the extent ofarteriosclerosis to identify major glomerular lesionsand significant interstitial inflammation. Attemptsto diagnose glomerulonephritis, quantitate intersti-tial fibrosis or identify mild acute tubular necrosisare frequently beyond the capabilities of the usualfrozen tissue section.

The easiest determination is quantitation ofglomerulosclerosis and this is usually the mainpoint of interest to the surgeon. Randhawa et al35

correlated donor biopsy histologic findings with12-month post-transplant outcome. Using rigorousmultivariate analysis, they demonstrated that witheach increment in global sclerosis (graded 0, 1, 2, 3;0, 1–10, 11–20, 21–30%), the odds ratio for a worseoutcome increased by 2.0 (Po0.03) after correctingfor recipient age. In a more recent study, Escofetet al36 have confirmed the importance of glomerulo-sclerosis as a predictor of outcome. Importantly,Wang et al37 have shown that for a statisticallysignificant prediction of outcome based on glome-rulosclerosis, at least 25 glomeruli are needed inthe baseline biopsy. Although there is also a strongcorrelation between the presence of interstitialfibrosis and a worse outcome,38 this determinationis difficult to impossible on a frozen section of donorkidney. As with all frozen sections, the remainingmaterial should be formalin-fixed and submitted forLM. When time is less critical, rapid processing afterformalin fixation can provide information far super-ior to that available on frozen section.

Transplant biopsies for allograft dysfunctionIn many centers, transplant biopsies are handleddifferently than native kidney biopsies. A minimumof two biopsy cores is indicated since the sensitivityof a single core for rejection is 91%; the addition ofthe second core improves the sensitivity to about99%.39 The same histologic staining protocol isusually followed. Often IF and EM are omittedunless there is clinical suspicion of recurrentdisease. Recent appreciation of the correlationbetween antidonor HLA antibodies,28,38 and C4ddeposition along peritubular capillary endotheliumand resistant rejections mandating more aggressivetherapeutic intervention, has led to the recommen-dation of all or most biopsies for acute dysfunctionbe stained for C4d. Both IF and IP procedures existfor this antigen.

The use of a reproducible classification schemefor rejection that provides significant prognosticinformation is important. The most widely acceptedsystem is the Banff Classification.40,41 This is aworking formulation that continues to evolve asevidenced by the recent addition of antibody-mediated rejection criteria.42

New directions

‘It is hard for the present day nephrologist ornephropathologist to conceive how renal pathologywas understood prior to renal biopsy’43 (quoted inPirani44). The renal biopsy was a significant under-pinning for many of the great advances seen in theunderstanding of renal diseases during the last 50years and continues to play an important role.Accurate diagnosis and classification of renal dis-eases has also led to the development of morespecific and superior therapy. Still, the renal biopsyas evaluated now, cannot always suggest the bestchoice between various available therapeutic op-tions,45 and improvements in its prognostic cap-ability are needed.

New directions include45–51 genomics and proteo-mics of renal disease. This arena is currentlyhandicapped by the need for tissue fixation thatmay limit recovery for RNA and protein analysis. Inaddition, the complex structural organization of thekidney with diseases limited to specific segments ofthe nephron makes interpretation of whole kidneybiopsy proteomic or genomic patterns problematic.The latter makes the genomic or proteomic study ofglomerulonephritis or tubulointerstitial diseasemuch more difficult than the evaluation of, forexample, neoplasms that are far more uniform andoften available in much greater quantity. Laser-capture microdissection provides part of the solu-tion by allowing selection of glomeruli or a specifictubular segment for study.52–54 Still, the minuteamount of available tissue severely limits explora-tion and further technological advance is needed tovalidate clinical utility prior to routine applicationof these exciting techniques.

Conversely, ISH has been successfully appliedto various renal diseases during the last 10–15years.55–59 As the availability of reagents and toolshas expanded, this technique is being applied morewidely. In the future, ISH will provide confirmatoryand specific information for the extensive data beinggenerated by genomic studies.

Conclusions

This document incorporates the consensus opinionsof the Ad Hoc Committee on Practice Guidelines,appointed by the Renal Pathology Society. Asignificant percentage of the membership of theRenal Pathology Society contributed to the manu-script (see Acknowledgements) and the manuscripthas received almost universal support from themembership at large. It is intended to highlight theessential ingredients necessary to provide qualityrenal pathology diagnoses.

Optimum evaluation of a renal biopsy requires atechnically proficient laboratory that can generatewell-fixed and thinly sectioned slides stained with apanel of special stains. The laboratory must be ableto provide complete immunohistochemical analysis


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and ultrastructural examination. Any deviation fromthis comprehensive array of studies risks incorrector incomplete diagnostic information.

There is currently no subspecialty certification bythe American Board of Pathology for renal patho-logists. However, to provide quality diagnoses inthis arena, the pathologist should have receivedmore than the usual brief experience in renalpathology during residency. A renal pathologyfellowship or a concentrated exposure to renalpathology under the auspices of an experiencednephropathologist is highly recommended.

Since every year new entities are identified andnew classification schema are formulated, theprovider of renal pathology diagnoses must keeppace with this rapidly evolving subspecialty. This ischallenging because the majority of renal pathologycontributions to the literature appear in nephrologyrather than pathology journals. Furthermore,although the Renal Pathology Society has excellentsponsored programs at the United States andCanadian Academy of Pathology Meeting and otherPathology meetings worldwide, the most extensiverenal pathology presentations occur at Nephrologymeetings, particularly the American Society ofNephrology Meeting. Thus, significant extra time,effort and expense are required of those desiring toremain current in their knowledge of renal disease.

Renal pathology is an exciting and challengingdiscipline. Patients with renal disease encompass abroad spectrum of medical diseases that are fre-quently complex and multisystem in nature. Therenal biopsy, appropriately processed and inter-preted will yield the correct clinicopathologicdiagnosis leading to the appropriate therapeuticstrategy while, at the same time, providing keyprognostic information.

Acknowledgements

The members of the Renal Pathology Society whocontributed to the development of this articleinclude (in alphabetical order):Arend LJ, Benediktsson H, Bonsib SM, Brisson M-L,Bruijn JA, Cavallo T, Chander PN, Charney DA,Cohen AH, Colvin RB, Crary GS, D’Agati V, Fogo AB,Ferrario F, Gokden N, Haas M, Harris A, Herrera GA,Iskander S, Jennette C, Kashgarian M, King JA,Liapis H, Markowitz G, Meleg-Smith S, Mengel M,Molne J, Moura LA, Nair R, Nasr H, Nast CC, Neff JC,Nickeleit V, Noel L-H, Olsen S, Phillips C, RacusenL, Reinholt FP, Rosen S, Ruiz P, Salinas-Madrigal L,Sarioglu S, Schwartz MM, Sens MA, Seshan SV,Solez K, Strom EH, Truong LD, Walker PD, Young-berg GA, Zhou XJ.

Duality of interest

None declared.

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