Immunoperoxidase of biopsies (PAP) · JClinPathol 1981;34:859-865 Immunoperoxidasestaining...

J Clin Pathol 1981 ;34:859-865

Immunoperoxidase staining of formalin-fixed,paraffin-embedded, human renal biopsies with acomparison of the peroxidase-antiperoxidase (PAP)and indirect methodsRA SINCLAIR,* J BURNS, MS DUNNILL

From the Department ofPathology, John Radcliffe Hospital, Headington, Oxford

SUMMARY The results of immunoperoxidase staining of 33 routinely processed renal biopsiescorrelated with the results of direct immunofluorescence in 79% of tests performed. Most of thediscrepant results were due to positive immunoperoxidase staining, possibly reflecting greatersensitivity of the method. A comparison of two immunoperoxidase methods showed that the indirectmethod produced less intense staining than the peroxidase-antiperoxidase (PAP) method whenequivalent primary antibody titres were used. Lowering the primary antibody titres for the indirecttechnique resulted in equivalent staining by the two methods. Predigestion by protease VII effectivelyrevealed the antigens under study without causing tissue damage or section loss.

In localisation of tissue antigens, immunoenzymaticexamination of paraffin sections offers severaladvantages over immunofluorescence performed onfresh tissue. These include the use of a microscopewith a conventional light source, the simultaneousvisualisation of tissue antigens and tissue structure,the possibility of retrospective examination, andpermanency of the preparations. A number ofsuccessful applications of immunoenzymaticmethods to fresh frozen renal biopsies'-3 werefollowed by a report from MacIver, Giddings, andMepham,4 who applied the immunoperoxidasemethod to formalin-fixed paraffin-embeddedmaterial. These authors, who utilised the peroxidase-antiperoxidase (PAP) method of Sternberger etal.,5 reported that immunoperoxidase staining oftrypsin-digested paraffin sections provides a reliableand sensitive technique for demonstrating immunedeposits. In the present study we have sought toconfirm the value of the PAP method in paraffin-embedded renal biopsies and have compared itssensitivity with an indirect enzyme-labelled antibodymethod. In routine use the latter technique hasinherent advantages over the PAP method since itinvolves fewer steps.

*Present address: Department of Anatomical Pathology,Alfred Hospital, Commercial Road, Prahran, Victoria 3181,Australia.

Accepted for publication 7 January 1981

Material and methods

Thirty-three renal biopsies (28 needle biopsies and5 wedge biopsies) were examined. These had beenfixed in neutral buffered formol saline for 6-18 hand, after routine processing, had been embedded inparaffin and stored for periods ranging from 2 wk to7 yr. Both the PAP and indirect methods wereperformed on each case, using, with minor modifica-tions, the techniques outlined by Bums.6

PAP METHOD1 Cut thin paraffin sections (microtome setting

2-3 pm), mount on uncoated glass slides, dryovernight at 37°C, dewax in xylene, and hydrate.

2 Digest prewarmed sections at 37°C with 0 05 %protease type VII (Signa Cat No P5255) in phos-phate-buffered saline (PBS), pH 7-3 for 20 min.

3 Terminate digestion in cold running tap waterfor 10 min and transfer to two changes of PBS over10 min.4 Expose sections to normal swine serum, diluted

1/5 with PBS, for 10 min. Drain off excess swineserum before stage 5.

5 Incubate sections with rabbit antihuman sera for30 min. Wash in four changes of PBS over 20 min.

6 Block endogenous peroxidase activity with afresh 0 5% solution of hydrogen peroxide in meth-anol for 10 min then wash in PBS for 10 min.

7 Incubate sections with unlabelled swine anti-859

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Sinclair, Burns, Dunnill

rabbit IgG for 30 min and then wash in four changesof PBS over 20 min.

8 Incubate sections with rabbit PAP solublecomplexes for 30 min and then wash in four changesof PBS over 20 min.

9 Reveal the end-product with a fresh solution of0 05% 3,3'-diaminobenzidine (DAB) in 0-01 %hydrogen peroxide in wash buffer for 5 min. HandleDAB with care because of potential carcinogenicity.

10 Wash in running tap water, counterstain withHarris' haematoxylin, dehydrate, clear and mount inDPX.

INDIRECT METHODProceed as for the PAP method but replace steps 7and 8 by incubation with peroxidase labelled swineantirabbit IgG and then wash in four changes ofPBS over 20 min. Perform all incubations in a moistchamber at room temperature.

ENZYME DIGESTIONThe optimum enzyme digestion time was determinedin preliminary studies by digesting known positivesections for periods ranging from 5 min to 1 h.Staining of serum proteins within glomerularcapillary lumina, which obscured the staining of theantigens under study, regularly occurred with diges-tion times of less than 15 min and staining of immunecomplexes progressively diminished following morethan 25 min digestion. Although no section adhesivewas used, only very occasional sections were lostafter digestion, and histological evidence of tissuedistintegration was not seen.

REAGENTS AND DILUTIONSAll antisera and peroxidase-antiperoxidase com-plexes were obtained from Dakopatts A/S throughMercia Brocades Ltd (UK). Optimal primaryantisera dilutions were determined by titrationagainst a number of known positive sections.Although a positive reaction could often be obtainedat high dilution-for example, 1/6400, lower dilu-tions were selected for use with the aim of detectingeven small amounts of antigen. The dilutions used inthe PAP method were as follows: anti-IgG 1/800;anti-IgM 1/200; anti-IgA 1/400; anti-C3 1/100;anti-C4 1/200; anti-Clq 1/200; anti-fibrinogen1/1600; unconjugated swine antirabbit immuno-globulin 1/20; PAP complexes 1/50. Occasionally,individual cases required increased titres-forexample, anti-IgG 1/1600, to reduce backgroundstaining. In 17 of the biopsies studied the titres ofprimary antibody for the PAP and indirect methodwere identical. This allowed a direct comparison ofthe sensitivity of the two methods. In 22 cases, how-ever, the titres for the indirect method were decreased

fourfold-that is, from 1/800 to 1/200, in an attemptto compensate for an observed relative insensitivityof this technique. Blocking of endogenous peroxi-dase activity was performed after, rather than before,the application of the primary antibodies. This wasdone in order to avoid the possibility that antigenreactivity or specificity might be altered by exposureto methanol and hydrogen peroxide. The dilution ofperoxidase conjugated swine antirabbit IgG used inthe indirect method was 1/30. All dilutions wereperformed in 1% ovalbumin in PBS. Fluorescein-conjugated antisera to IgG, IgM, IgA, complementcomponents C3, C4, and Clq, and fibrinogen, wereobtained from Dakopatts A/S, Hoechst Pharma-ceuticals, and Wellcome Reagents Ltd.Some of the positive immunoperoxidase prepara-

tions were counterstained by the periodic acid-Schiff (PAS) method.

CONTROLSCurrent fluorescein conjugates and all primaryantisera used in the immunoperoxidase methods,with the exception of the anti-Clq sera, showed singleprecipitin lines against human plasma or serum onimmunoelectrophoresis. The animal antisera, PAPcomplexes, and anti-Clq sera showed no reaction.Other controls included the omission of the primaryantibody for the immunoperoxidase methods, theobservation of negative reactions with inappropriateprimary antibodies and the presence of stainingpatterns consistent with the morphological diagnosis.In addition, as recommended by Heyderman,7antibody absorptions with purified antigens (IgG,IgM, IgA) were performed. Elimination of antibodyactivity as shown by negative immunoperoxidasestaining was achieved with each antibody and therewas no cross reactivity with inappropriate antigens.

CASES STUDIEDThe cases examined included: membranous glom-erulonephritis 11; mesangiocapillary glomerulo-nephritis 8; focal proliferative glomerulonephritis 5;diffuse proliferative glomerulonephritis 3; Berger'sdisease 2; crescentic glomerulonephritis 2; and onecase each of microscopic polyarteritis nodosa andgold nephropathy.The results obtained from the immunoperoxidase

preparations were scored on a zero to + + + +scale and then compared with the results of directimmunofluorescence which had been performed onfrozen sections at the time of biopsy.

Results

Both the PAP and indirect methods were effective indemonstrating proteins in glomeruli, arterioles,

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Immunoperoxidase staining offormalin-fixed, paraffin-embedded, human renal biopsies

peritubular capillaries, tubular lumina and tubularcytoplasm. Within glomeruli, characteristic patternsof localisation of immune deposits were readilyrecognised within capillary walls, in the mesangiumand at the periphery of glomerular lobules (Figs.1-5). Localisation of deposits was confused ratherthan clarified by PAS counterstaining of the glomeru-lar basement membrane. Fibrin or fibrinogen wasreadily stained in glomerular tufts and Bowman'sspace. Good results with the quoted enzyme diges-tion time depended on the use of consistently thinparaffin sections. Thicker sections-that is, cut at amicrotome setting of 5 ,um, resulted in markedlyincreased background staining and staining of serumproteins within glomerular capillary lumina. Intra-luminal staining occasionally occurred even withthin sections. Prolonging the digestion time in thissituation diminished, but usually did not completelyremove, the staining.Background staining was kept to a minimum by

enzyme digestion, the use of thin sections, theapplication of normal swine serum and, particularly,by washing of the sections for at least 15 min afterexposure to antibody. Washing for 30 min or moreproduced some loss of staining intensity. Endogen-ous peroxidase activity was never detectable aftermethanol-hydrogen peroxide inhibition.

COMPARISON OF IMMUNOFLUORESCENCE

WITH PAP

Comparison of the immunofluorescence results with

immunoperoxidase staining by the PAP method(Table) showed agreement in 79% of 200 testsperformed. Of the discordant results, the immuno-peroxidase preparations were negative only inrelation to six instances of minor fluorescence posi-tivity. On the other hand there was positive peroxi-dase staining in 37 tests where the immunofluor-escence had been negative, and of these, 15 wereexamples of moderate (+ +) or strong (+ + +)staining reactions. The discrepant results did notinvolve particular antigens or particular histologicaldiagnoses and in these biopsies there was alwayspositive immunofluorescence for one or more anti-gens and electron-dense deposits were present.

There was generally good correlation of theintensity of staining of individual antigens betweenthe immunofluorescence and the two immuno-peroxidase methods. The exception was staining forthe C3 component of complement where in six of 33cases immunoperoxidase staining was inexplicablyweak. This weak staining was not improved byreduction of the enzyme digestion time.

COMPARISON OF PAP WITH INDIRECTMETHODThere was good correlation between the results ofthe PAP method and both the high and low titreindirect methods. With high titres the indirect methodproduced six divergent results in 104 tests and withlower titres there were six divergent results in 119tests. With both high and low titres there were four

Fig. I Mesangiocapillaryglomerulonephritis. Peripherallobular staining of CJq. Indirectmethod x 360.

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Fig. 2 Membranous glomerulonephritis.Finely granular staining of IgG. PAPmethod x 360.

Fig. 3 Detail of Fig. 2. Discrete.A extramembranous deposits of

IgG. PAP method x 900.

negative and two positive results in relation to thePAP method and all of these involved only minimal(±) staining reactions.When reaction intensity rather than positivity was

considered, the indirect method at high titres wasapparently less sensitive than the PAP method. This

was obvious in individual biopsies, and by semi-quantifying individual results-for example, + + =2 points, + + + = 3 points, and comparing thetotals from parallel tests performed on 16 cases, adifference in staining intensity was seen (PAPmethod 132 points, indirect method, 94 points).

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Immunoperoxidase staining offormalin-fixed, paraffin-embedded, human renal biopsies

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Fig. 4 Diffuse proliferativeglomerulonephritis. Diffusegranular staining of C3. PAPmethod x 360.

Fig. 5 IgA nephropathy. Mesangialstaining ofIgA. Indirect method x 360.

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However, when a similar comparison was made Discussionbetween the PAP method and the indirect methodperformed at low titres, there was no loss in staining This study has shown that immunoperoxidaseintensity (PAP method 207 points, indirect method, staining of proteolytically-digested paraffin sections209 points, from parallel tests on 20 biopsies). is at least as reliable and sensitive as direct immuno-

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Comparative results of immunofluorescence (IF) andimmunoperoxidase (IP) staining (PAP method) on 33renal biopsies

Antigen Number Concordant Discordantexamined

IF+ IF- IF+ IF-IP+ IP- lP- IP+

IgG 33 29 4 - -

IgM 29 14 5 2 8IgA 30 10 13 - 7C3 33 33 - - -

C4 27 8 12 2 5Clq 22 8 6 - 8Fibrinogen 26 1 14 2 9Total 200 103 54 6 37

fluorescence on fresh tissue in detecting immunedeposits in renal biopsies. Our results showed anoverall concordance with immunofluorescence of79 %, very similar to that reported by Maclver et al.(81 %).4 However, we found fewer negative and morepositive discrepancies. The positive discrepancieswere fairly numerous and when present they were aconsistent finding by both immunoperoxidasetechniques. Moderate or strong staining reactionswere often involved and the results may reflectgreater sensitivity of immunoperoxidase methodscompared with direct immunofluorescence. How-ever, a definitive statement on relative sensitivities isnot possible because the immunofluorescence andimmunoperoxidase studies were not performed pros-pectively by a single individual.

Proteolytic digestion, usually by trypsin, is a vitalstep in immunoenzymatic staining of paraffinembedded renal tissue. We have shown that proteaseVII, when used at a dilution of 005 %, effectivelyreveals a variety of antigens. The results wereachieved after a conveniently brief period of pro-teolysis and without the effects of overdigestionwhich have been described with both trypsin4 andprotease VII.8 A recent comparative study9 hasshown protease to be more effective than trypsin inantigen revelation. The relatively high cost ofprotease VII is a disadvantage, particularly if largenumbers of tests are being performed.The relative sensitivities of the PAP and indirect

immunoperoxidase methods have been the subject ofconflicting reports.10-'2 In our hands the PAPmethod was slightly more sensitive than the indirectmethod. The relative insensitivity of the indirectmethod was fully compensated for by a decrease inprimary antibody titres. Since the indirect method isquicker to perform than the PAP method, it may bepreferred for routine immunoperoxidase staining.When choosing an immunohistological method forroutine use, the advantages of the immunoperoxi-dase technique must be weighed against its disad-

vantages,'13 particularly its time-consuming natureand the relatively large number of steps which requirestandardisation for consistent results to be obtained.Simplifying the procedure-for example, eliminatingthe need for proteolytic digestion by the use offixatives other than formol saline,13 14 such asBouin's, Susa's, would make the method moreattractive. At this stage, most pathologists willprobably continue to use immunofluorescence forrenal immunohistology. However, immunoperoxi-dase methods certainly have a role, at least inretrospective studies and for those cases, up to 1 in4,15 where needle biopsy produces insufficientmaterial for fluorescence examination.

We thank Dr Z Abdulaziz for providing the purifiedimmunoglobulins for antibody blocking, Mrs JMitchell for performing the immunoelectrophoreses,Dr DY Mason for advice, and comments on themanuscript, and Miss JC Woods and Mr RSDavison for expert technical assistance.

References

' Davey FR, Busch GJ. Immunohistochemistry of glomeru-lonephritis using horseradish peroxidase and fluorosceinlabeled antibody: a comparison of two technics. Am JClin Pathol 1970 ;53:531-6.

2 Elias JM, Miller F. A comparison of the unlabeled enzymemethod with immunofluorescence for the evaluation ofhuman immunologic renal disease. Am J Clin Pathol1975 ;64:464-71.

3 Tubbs RR, Gephardt G, Valenzuela R, Deodhar S. Anapproach to immunomicroscopy of renal disease withimmunoperoxidase and periodic acid-Schiff counterstain(IMPAS stain). Am J Clin Pathol 1980;73:240-4.

4 Maclver AG, Giddings J, Mepham BL. Demonstration ofextracellular immunoproteins in formalin fixed renalbiopsy specimens. Kidney Int 1979;16:632-6.

5 Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG. Theunlabeled antibody enzyme method of immunohisto-chemistry. J Histochem Cytochem 1970;18:315-33.

Burns J. Immunohistochemical methods and their applica-tion in the routine laboratory. In: Anthony PP, Woolf N,eds. Recent Advances in Histopathology. 10th ed.Edinburgh, London: Churchill Livingstone, 1978:337-50.

7 Heyderman E. Immunoperoxidase technique in histo-pathology: applications, methods, and controls. J ClinPathol 1979;32:971-8.

8 Mepham BL, Frater W, Mitchell BS. The use of proteolyticenzymes to improve immunoglobulin staining by thePAP technique. Histochem J 1979;11 :345-57.

Clausen PP. Immunohistochemical demonstration ofalpha-l-antitrypsin in liver tissue. Acta Pathol MicrobiolScand [A] 1980;88:299-306.

0 Burns J. Background staining and sensitivity of theunlabelled antibody enzyme (PAP) method. Comparisonwith the peroxidase labelled antibody sandwich methodusing formalin-fixed paraffin embedded material.Histochemistry 1975;43:291-4.

Heyderman E, Neville M. A shorter immunoperoxidasetechnique for the demonstration of the carcinoembry-onic antigen and other cell products. J Clin Pathol 1977;

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