Immunohistological characterisation of amyloiddeposits in renal biopsy specimens

R J Fitzmaurice, C Bartley, J McClure, P Ackrill

AbstractThe amyloid deposits in 21 renal biopsyspecimens were subjected to a detailedimmunohistochemical analysis using apanel of antibodies against recognisedconstituents of tissue amyloid. This wasa retrospective study of material origin-ally submitted during the investigationof various renal abnormalities andstudied by a routine protocol includinghistochemistry, electron microscopy,and immunofluorescence. The presenceof an amyloid was confirmed in all 21cases. Seventeen cases contained P com-ponent and either amyloid A (AA) (11cases) or an immunoglobulin light chainassociated amyloid (six cases). Fourcases contained amyloid material withunusual immunohistochemical findings;one case had AA and P-component (PC)in the interstitium, one case had A lightchain and 1-2 microglobulin, one casehad K light chain and Clq, and one case

had A light chains only.It was possible, therefore, to identify

precisely the amyloid constituents andthereby "type" the amyloid by immuno-histochemical means. The availability ofthe antibodies used and their applicationusing these techniques could simplify theconfirmation of clinically suspectedamyloidosis.

Department ofPathological Sciences,Stopford Building,University ofManchester,Manchester, M13 9PTR J FitzmauriceC BartleyJ McClureArtificial Kidney Unit,University Hospital ofSouth ManchesterP AckrillCorrespondence to:Professor J McClure

Accepted for publication4 October 1990

In haematoxylin and eosin stained paraffinwax embedded sections amyloid is recognisedas amorphous hyaline and eosinophilicextracellular material. Its presence is said tobe confirmed by the demonstration of apple-green birefringence on polarisation of sectionsstained by alkaline Congo red (ACR),' or byfinding the characteristic complex at theultrastructural level.2Although each type of amyloid has a par-

ticular chemical composition,3 there are twoprincipal types. One, designated amyloid AA,is a protein of molecular weight 8500, thoughtto be derived from an ae-l- globulin serumprecursor by proteolytic cleavage, and theother is derived from either K or A light chainsof immunoglobulins and as a consequence isassociated with B cell (immunoglobulin-secreting) tumours.Under certain conditions, other proteins

may be identified in amyloid deposits includ-ing prealbumin in heredofamilial and seniledisorders, and calcitonin in medullary carcin-oma of the thyroid.

J Clin Pathol 1991;44:200-204

There are now several published paperswhich confirm the presence of amyloid informalin fixed, paraffin wax embedded tissues(including necropsy material) by immunohis-tological techniques using polyclonal anti-sera.'4 More recently, monoclonal antibodieshave been used.7 Generally, these studies haveused limited panels of antisera, typicallyincluding antibodies to AA, K, and A lightchains, P-component, and prealbumin. Oneexception is a study of senile cerebralamyloidosis which used a panel of 14 anti-sera.8 In a small number of instances amyloidof more than one type occurring withinindividual deposits has been described.9 10

Although the distribution of amyloiddeposits and associated underlying conditionsis extremely varied, the kidney is often affec-ted, leading to impaired renal function.Immunohistological studies on localisedamyloidosis of the urogenital tract, renalamyloid AA complicating hairy cell leuk-aemia, the prevalence of amyloidosis in a

necropsy series, and characterisation of renalamyloid using antisera to AA, P-componentimmunoglobulin, and K and A light chainshave been described in needle biopsyspecimens of kidney.' 1-4 To our knowledge,however, there has not been an attempt todefine individual constituents in renal biopsyspecimens using an extended panel of anti-bodies. We therefore investigated the amyloiddiagnosed in 21 renal biopsy specimens, usinga panel of antibodies, to identify as fully as

possible the constituents of the amyloiddeposits.

MethodsBetween 1976 and 1988, 22 patients had renalamyloidosis diagnosed by biopsy. In 21 cases

there was sufficient material available to per-form the additional studies detailed herein.Patient details are given in table 1.The original renal biopsy specimen had

been divided into three portions. One hadbeen formalin fixed and embedded in paraffinwax. Sections (4 pm thick) had been stainedwith haematoxylin and eosin, Congo red,periodic acid Schiff, and Gomori's silverstains. Another had been frozen, and cryostatsections had been stained using a one-stepfluorescein labelled antibody method. Thethird had been post-fixed in glutaraldehyde;thin sections had been stained with uranylacetate and lead citrate and examined with an

AE 1 801 electron microscope.In the additional study serial sections

(4 pm) of formalin fixed, paraffin wax embed-

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Table 1 Diagnostic biopsy report

Case Light ElectronNo Age/Sex Diagnosis microscopy* microscopyt Immunofluorescence

1 66 F Rheumatoid arthritis G, BV, T +2 69 F Nephrotic syndrome G, BV +3 59 F Rheumatoid arthritis G, BV, T, I N/A4 40 M Nephrotic syndrome G, BV, T + IgM, C35 58 F Nephrotic syndrome/

rheumatoid arthritis G, BV IgG, IgA, IgM, C3, fibrin6 57 M Nephrotic syndrome G, PT, I + C3, fibrinogen7 56 F Rheumatoid arthritis G, BV, PT, I + Fibrin8 78 M Nephrotic syndrome G, BV, PT, I + Fibrinogen9 65 F Rheumatoid arthritis/

nephrotic syndrome G, BV, I +10 74 F Chronic renal failure G, BV N/A11 64 F Acute renal failure G +12 50 F Rheumatoid arthritis G, BV N/A13 51 F Hypertrophic G, BV, T, I N/A IgG, IgA, IgM, fibrin, C3, Clq

cardiomyopathy/chronic-renal failure

14 72 M Chronic renal failure BV15 72 M Chronic renal failure G, BV + IgG, IgA, C316 51 F Rheumatoid arthritis BV, I N/A17 51 F Acute renal failure G, BV + IgM, C3, Clq18 69 F Polymyalgia rheumatica G, BV + IgG, IgM, C2, Clq, fibrin19 62 F Diabetes mellitus with

proteinuria G, BV, PT, I + Clq20 53 M Chronic renal failure + IgG, IgA, IgM, C3, Clq21 50 M A chains urine G, BV +

*Positive for Congo red staining.G-glomeruli, BV-blood vessels, T-tubules, I-interstitium, PT-peritubular.t + amyloid material identified ultrastructurally- no fibrillar material seen.

ded tissues were stained with haematoxylinand eosin, alkaline Congo red, and alkalineCongo red with potassium permangante as apretreatment.i" Sections were also immuno-stained with the rabbit anti-human polyclonalantibodies detailed in table 2, using a conven-tional peroxidase-antiperoxidase technique.Negative controls were performed using noprimary antiserum. Positive controls foramyloid A, K, and A light chains were availablein the form of representative blocks fromknown cases of amyloid A and systemicamyloid in K and A light chain multiplemyeloma, respectively. Although enhance-ment of amyloid immunostaining has beendescribed, we did not find this necessary in ourstudy.

All sections were examined by twoobservers independently and the results com-pared and pooled. Disagreements as topositivity and anatomical site were resolved bydiscussion.

ResultsThe results are shown in tables 1 and 3. In all 21cases amyloid was positively identified byCongo red staining and characteristic apple-green birefringence in polarised light. In 17 ofthe cases amyloid deposits could be definitelyidentified as containing either AA amyloid (11cases), or immunoglobulin light chainassociated amyloid (six cases: five A, one K). Thedeposits in these cases also contained P-com-ponent.The unusual cases were as follows: (i) case

18, which contained a small amount ofAA andP-component in the interstitium (the specimenconsisted solely of tubules); (ii) case 19, whichcontained A light chains in glomeruli and also/-2 microglobulin focally within glomeruli;

(iii) case 20, in which weakly positive stainingfor K light chains was shown in occasionalglomeruli and their capsules (Clq was alsoshown in capsules and glomeruli of this case, aunique finding in the series); and (iv) case 21,which contained A light chains only, inglomeruli and blood vessels.

Thyroglobulin, calcitonin, and C3c were notidentified immunohistochemically in any of thesections.

In 14 cases the presence of amyloid materialwas confirmed ultrastructurally. Amyloid wasonly seen electron microscopically in thosecases which could be definitely subclassified onimmunohistological analysis. In a further twocases examined fibrillar material was not iden-tified.

Figures 1 and 3 include the results ofimmunostaining.

DiscussionAlthough amyloid has uniform light micro-scopical and ultrastructural appearances, it isnot a homogeneous chemical entity. Ultra-

Table 2 Antibody dilutions and distributors

Antibody Dilutions Distributors

AA 1/500 Calbiochem (CambridgeBiosciences, Cambridge)

P-Component 1/250B-2 microglobulin 1/500K light chain 1/5000A light chain 1/5000Fibrinogen 1/2500Pre-albumin 1/500 Dako Ltd, High Wycombe,

BucksIgA 1/2500IgM 1/2500IgG 1/2500C3c 1/500Clq 1/500Thyroglobulin 1/5000Calcitonin 1/500

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Table 3 Histochemistry and immunostaining

Alkaline B-2Case Congo P- micro- Pre-No red* KMmO4f component AA globulin A1K Fibrinogin albumin IgA IgG Clq

I G,BV Inhibited G,BV G,BV - - - G,±I,+ T T,I T+ -2 G Persists G, I - - G,BV,I - - - -3 G, I Inhibited G G - - - - - - - -4 G,BV Inhibited G G,BV - - - - - - - -5 G, T Inhibited G, I G,BV - I G,T - - - - -6 G, T Persists - - - - G,T - - - - -7 G,BV Inhibited G,BV G,BV - - - - - - -8 G,BV Inhibited G,BV - - G,BV - - -9 G,BV,T Inhibited G,BV,T G, T BV,T± - - G,±I± - - - -10 G,BV Persists G,BV - - G,BV - - - - -11 G,BV Inhibited - G,BV - - - - - - - -12 BV,T Inhibited - BV,T - - - - - - - -13 G,BV,T Persists G,BV,T - - G,+T,+I - - - -14 - BV, I - G,T - - - - -15 G,BV Inhibited G,BV, I G,BV,I - - - - - - - -16 - - - I+ - - - - - - - -

17 G,BV,T Inhibited G, T G,BV,T - - - G,BV,T - - - -18 - - G G,BV - - - - - - -

19 G Persists I G G, I - - - -20 G,BV Persists G+,I - T BV - - - - G21 G,BV - - - - G,BV - - - - - -

*Sites positive by light microscopy.tKMmO4-Alkaline Congo red with potassium pernanganate pretreatment.+ present focally.

structural observation of amyloid depositsshow a complex, predominantly formed (90%)of irregularly arranged, non-branching fibrils(7 5-10 nm in diameter), which are invariablyassociated with P-component, a pentagonalglycoprotein identical with an a- 1-glycoproteinfound in normal serum. It is the aggregation ofthe amyloid fibrils into a cross B-pleated sheetconfiguration that is responsible for the opticalcharacteristics on polarisation.We included the potassium permanganate

pretreatmentmethod as an aid to confirming thepresence of AA as it is supposed to abolishalkaline Congo red staining when this is causedby AA. There are doubts, however, as to itsspecificity. Taken with the non-specific light

and electron microscopic appearances, thismeans that for full characterisation ofamyloid apanel of antibodies to the various possiblecomponents have to be used.The most extensive series of immunohis-

tological studies have been performed in theneurodegenerative diseases in the centralnervous system, and recently new amyloidcomponents have been identified.'6

In our study, 17 cases contained amyloiddeposits, the immunohistological staining reac-tions of which were unequivocal, and a definitesubtype could therefore be stated. We identifiedP-component in 16 cases, in a distributioncorresponding to amyloid, but we did notidentify P-component in isolation. While faint

Figure IA Case 1:section (viewed inpolarised light) from acase of amyloid A.Amyloid deposits are welllocalised (alkaline Congored).

Figure lB Case 1: Samefield as IA. AlkalineCongo red with potassiumpermanganatepretreatment.Examination in polarisedlight shows loss ofmuch ofthe staining of the amyloidmaterial (alkaline Congored).

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Immunohistological characterisation of amyloid deposits in renal biopsy specimens

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staining of P-component was seen in normalsites (glomerular basement membrane and cer-tain vessels), this was of no consequence com-pared with that in the amyloid deposits, wasoutside the amyloid deposits, and was thereforenot specifically recorded.

s3-2 microglobulin was seen in four cases,always associated with other amyloid compon-ents, although it is a well recognised form of

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amyloidosis in patients receiving dialysis'7in whom it may be systemic, or responsiblefor carpal tunnel syndrome, or tumoralamyloidosis of bone and intestinal infarction.Two ofour patients were not receiving dialysis,indicating that /3-2 microglobulin deposition isnot always related to this form of treatment.We identified not only cases of light chain

amyloid but also a case (case 5) of AA inassociation with K light chains in a similardistribution. The staining in this case leads usto believe that this is a combined form ofamyloid. In this case, as would be expected, 2light chains were also present, but these werelocated in the interstitium and were notassociated with the amyloid deposits. Kappalight chain disease has been described as acomplication of multiple myeloma in threecases, but in only one of these, and in thekidney, was there an association betweenamyloid deposition and K light chains.'8 In onlyone of our cases (case 1) did we identifyimmunoglobulins but failed to show K and Alight chains. This may have been due to aninability of the antibodies to discriminate thelight chains from the whole molecule in thesesmall deposits, or possibly due to a maskingeffect. We were also unable to confirm byimmunohistochemistry (table 3) the presenceof some immunoglobulins and Clq that hadbeen initially shown using immunofluorescence(table 2).Some of our cases were difficult to subtype

outright. One of these (case 16) consisted solelyof tubules. In the original examination,however, Congo red positivity had beenpresent in blood vessels and interstitium. Weidentified focally AA only within the inter-stitium and therefore feel that this is probably

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Figure 2 Case 4:immunolocalisation ofamyloid A deposits inglomerulus and smallartery (PAP).

Figure 3A Case 5:amyloid A deposits in twoglomeruli (PAP).

Figure 3B Case 5: Samefield as 4A showingcoexistent presence of Klight chains in the sametwo glomeruli revealed byimmunostaining (PAP)(Nomarski phase).

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Fitzmaurice, Bartley, McClure, Ackrill

of AA type. Cases 14 and 18 showed no Congored material in our study, but definite stainingfor P-component and /-2 microglobulin wasseen in 14 and P-component and AA in 18. As aresult of immunohistochemistry these couldtherefore be classified as /3-2 microglobulin andAA, respectively. Case 19 showed /3-2microglobulin and A light chains in theglomeruli, an unusual combination which wasthought to be a probable A light chain amyloid,and case 20, ,B-2 microglobulin and A lightchains, but in differing sites. Case 8 showed Alight chain amyloid in which alkaline Congored staining had been inhibited by potassiumpermanganate pretreatment.

In one of our cases we identified Clq inglomeruli which also stained for A light chainand (B-2 microglobulin. This is only the thirdimmunohistological report of complementcomponents shown in amyloid.'920

Immunohistological analysis is sensitive andreliable and has been proved feasible on resinembedded sections,2' and synovial fluidsediment,22 but in cases where immunohisto-chemistry is inconclusive, microextractionwith immune diffusion techniques23 or, with dotimmunoassay, immunodiffusion, gel electro-phoresis, and Western blotting,24 may beuseful.We have shown that in most cases, using a

combination of histochemistry and immuno-histochemistry on routinely processed tissues,it is possible not only to identify the presence ofamyloid but also to distinguish individually theconstituents of the deposits. These techniquescould therefore be useful in the assessment ofbiopsy specimens where amyloid has beendiagnosed or would be expected clinically, or,where histologically suspicious deposits haveproved Congo red negative. The possibility ofobtaining this increased sensitivity and theability to identify separately amyloid con-stituents may mean that subclinical disease orlight chain disease could be detected earlier.

We gratefully acknowledge the support ofthe South ManchesterHealth Authority who have generously funded this project.

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