J Clin Pathol 1987;40:874-878

Effect of decalcification agents on immunoreactivity ofcellular antigensN A ATHANASOU, J QUINN, A HERYET, C G WOODS,* J O'D McGEE

From the University of Oxford, Nuffield Department ofPathology, John Radeliffe Hospital, Oxford, and*Nuffield Orthopaedic Centre, Headington, Oxford

SUMMARY The effects of several strong acids, weak acids a proprietary decalcifier, and edetic acidon the immunohistochemical staining of cryostat and&d paraffin embedded sections of tissuefrom a variety of normal and pathological calcified and uncalcified specimens were studied. Evendecalcification in strong acids (HCI, HNO3, 5% trichloracetic acid, HCl-edetic acid did not diminishthe reactivity ofmany useful antigens (including leucocyte common antigen, intermediate filaments,S100 protein and epithelial membrane antigen). Weaker acids (formic acid, acetic acid) and edeticacid decalcified more slowly and generally showed greater preservation of antigenic reactivity withbetter morphology and staining quality. Trichloracetic acid was also useful as a quick one stepfixation and decalcifying agent for both cryostat and routinely processed sections. Knowledge of thepreservation of antigenic reactivity in decalcified tissue will be useful in the diagnosis of tumours ofuncertain histogenesis and origin which affect calcified tissues.

Immunohistochemical analysis of surgical specimensusing monoclonal antibodies has assumed animportant role in histopathological diagnosis.Although frozen material and cryostat sections areoptimal for the preservation of the reactivity of manyantigens, routine formalin fixation and paraffinembedding, often associated with protease digestionof tissue sections prior to immunostaining, has beenfound to interfere only partially with the localisationof many cellular and tissue antigens useful in tissuediagnosis.' With calified tissues, the situation ismore complex as fixation is generally followed by adecalcification procedure. Although there are severaldecalifying agents in common use, their effect on theimmunoreactivity of the various cellular and tissueantigens has been little studied.4 5 This is often of par-ticular importance where panels of monoclonal anti-bodies are used in the histopathological diagnosis oftumours of uncertain origin.6

In this study we examined the effect of severaldecalcifying agents, including strong acids, weakacids, and chelating agents on immunohistochemicalstaining for several diagnostically useful cellular andtissue antigens in both calcified and uncalcifiedsurgical specimens.

Accepted for publication 19 March 1987

Material and methods

Surgical specimens included normal appendix, thy-roid, skin, breast, prostate, bone, and tonsil, as well ascases ofcarcinoma (lung, kidney), myeloma, and lym-phoma affecting bone. Table I shows the antigenicdeterminants sought in each of these tissues.The tissues were either snap frozen in liquid nitro-

gen for cryostat sectioning or fixed in 10% formolsaline at room temperature for a minimum of 24hours (range 24 hours-14 days). Slices of the fixedtissues were then routinely processed to paraffin wax.A parallel fixed slice was also placed in one of severaldecalcifying agents for 24 hours and three days. Theagents studied were 5% aqueous nitric acid (HN03),5% aqueous hydrochloric acid (HCl), 10% RD0(Bethlehem, Gotheberg), 5% trichloracetic acid(TCA), 10% aqueous formic acid (FA), 10% aqueousacetic acid (AA), and 10% HCl-edetic acid (ethylenediaminetetracetic acid) mixture. Specimens were alsodecalcified in edetic acid alone (24 hours and sevendays), as well as being solely fixed and decalcified inTCA for 24 hours. After washing in running tapwater for four to six hours the treated specimens wereprocessed via ethanol (six hours) and xylene (threehours) to paraffin wax. Five micron sections weremounted on Multispot slides (Hendley, Essex) anddried at 60°C for 60 minutes. Before immunostaining,all paraffin sections were dewaxed in xylene and

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Effect of decalcification agents on immunoreactivity of cellular antigensTable 1 Antigenic determinants sought in surgical specimens examined

Antigenic determinants sought

Appendix

TonsilSkin normal, compound naevusThyroidBreastProstate

Bone marrowosteoarthritic femoral headf

Lymphoma}affecting boneSquamous cell carcinoma lungAdenocarcinoma lung affecting boneAdenocarcinoma kidney

rehydrated through graded alcohols to water. Parallelslices of appendix were also processed to cryostat sec-tions after fixation and decalcification (24 hours) inthe various agents, as noted above, to assess if speci-mens could be rapidly fixed and decalcified withoutdestroying antigen reactivity.

Table 2 gives details of monoclonal antibodies usedin this study. Polyclonal rabbit antibodies againsthuman immunoglobulin heavy and light chains (Ig),thyroglobulin, and prostate specific antigen (PSA)were obtained from Dako (UK) and antiprostaticacid phosphatase (PAP) from Miles Scientific Ltd.Immunohistochemistry was performed using anindirect immunoperoxidase or alkaline phosphataseantialkaline phosphatase (APAAP) procedure, aspreviously described.'7 Both control and decalcifiedsections of fixed tissue were digested by 0-1% trypsin(Sigma T: 8128) in 0-1% calcium chloride at pH 7-8before immunostaining with the following antibodies:CR3/43, anti-light chain Ig, anti-heavy chain Ig, anti-carcinoembryonic antigen, anti-PSA, anti-PAP, anti-Tgb, and anti-factor VIII related antigen (30minutes); CAM 5-2 (15 minutes); DER- 11 (20minutes). The histological quality, degree, andspecificity of immunohistochemical staining wasscored relative to that of fixed undecalcified controlsections.

Results

Table 3 summarises the effect on the immuno-reactivity of cellular antigens after treatment with thevarious decalcifying agents (figs 1 and 2). Prolonged(three day) decalcification produced no noticeabledifferences in terms of antigen reactivity or antibodyspecificity- but had an effect on morphology and stain-

Leucocyte common antigen (LCA), HLA-DRLight and heavy immunoglobulin chains (Ig's)Vimentin, SIOO protein, desminFactor VIII related proteinCytokeratin intermediate filamentsCarcinoembryonic antigen (CEA)Epithelial membrane antigen (EMA)Ig's LCAMelanoma associated antigen (MAA), cytokeratins, SIOOThyroglobulin (Tgb)EMA, cytokeratinsProstatic acid phosphatase (PAP)Prostate specific antigen (PSA)LCA, Ig's

LCA, Ig's

EMA, cytokeratins

Table 2 Monoclonal andpolyclonal* antibodies usedforanalysis ofroutinely processed decalcified tissues

Antibody Antigenic determinant

PD7/26 Leucocyte common antigen'2BI 1 Leucocyte common antigen'CR3/43 HLA-DR8Anti-KAnti-A Light and heavy immunological chains*Anti IgG, IgME29 Epithelial membrane antigen9CEA Carcinoembryonic antigen10KLI Cytokeratin intermediate filaments11CAM 5-2 Cytokeratin intermediate filaments12SI-61 S1OO protein13DER. I I Desmin14V9 Vimentin1INKl/C3 Melanoma associated antigen16Anti-PSA Prostate specific antigen*Anti-PAP Prostatic acid phosphatase*Anti-thyroglobulin Thyroglobulin*Anti-F VIII Factor VIII related antigen17

*Indicates polyclonal antibodies.

ing quality. Undecalcified controls that were pro-cessed to cryostat sections of formalin fixed paraffinembedded sections were all strongly positive with theantibodies tested.

Treatment with the strong acids HCI and HN03and the proprietary decalcifier RDO for 24 hours andthree days preserved immunoreactivity of all antigenstested with the exception of HLA-DR and epithelialmembrane antigen (EMA). Tissue morphology andstaining quality was adversely affected, particularlyafter prolonged (three day) decalcification, butimmunostaining was still interpretable. HCI-edeticacid treatment was associated with better mor-phology and weak EMA reactivity was retained. TCAtreatment (both with and without prior formalin

Specimen

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876 Athanasou, Quinn, Heryet, Woods, McGeeTable 3 Effect ofdecalcifying agents on immunoreactivity oftissue antigens4

Antigenic Undecalcified HCl-determninant control HCl HNO3 RDO edetic acid TCAt AA FA Edetic acid$

LCA ++ ++ ++ ++ ++ ++ ++ ++ ++HLA-DR ++ - - - ++ ++ ++ ++ ++Light Igchain + + + + + + + + + + + +Heavy Igchain + + + + + + + + + + + + + + +Cytokeratins ++ +++ +4 ++ ++ + + +4+ ++ ++CEA ++ ++ ++ +Vimentin ++ + + + ++ ++ ++ ++ ++SlOOprotein ++ + + + ++ + + ++ ++ + +FVIII ++ + + + ++ ++ ++ ++ ++MAA ++ + + + +4 4 + +4 ++ ++PAP ++ + ++ +4 ++ 4+ ++ +4 ++PAA +4 +++4+ + ++ ++ ++ +4 ++Tgb ++ 4+ ++ + +4+ ++ +4 ++ ++Desmin ++ ++ ++ + +4 +4 +4 ++ ++EMA + - - + - ++ ++ ++

*Each tissue was decalcified for 24 hours and 3 days: the results were similar for both time periods.+ + = strong staining; + = weak staining.tWith and without formalin pre-fixation. $Also seven days of edetic acid decalcification.

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Fig 1 Squamous cell carcinoma oflung infiltrating bone: Fig 2 Lymphoma infiltrating bone (right) with tumour cellstumour cells are stainedfor cytokeratin intermediate showing membrane stainingfor leucocyte common antigenfilaments (CAM5-2). (Immunoperoxidase, decalcification (PD7/26). (Immunoperoxidase, decalcification TCA 24HCl-edetic acid 24 hours). hours).

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Effect of decalcification agents on immunoreactivity of cellular antigens 877

fixation) also showed good staining quality and pre-servation of both morphology and antigenic reac-tivity. No particular advantage in terms of immuno-histochemistry was found in using the proprietarydecalcifier (RDO) tested. It should also be noted that,although both anti-leucocyte common antigen anti-bodies tested, PD7/26 and 2B1 1, reacted with leuco-cytes after 24 hours in all decalcifying agents, 2B1 1did not react with leucocytes after three days ofdecalcification in HCI, HNO3, RDO and TCA.

Treatment with weak acids (FA, AA) and edetic for24 hours and three days showed generally betterstaining quality and morphology in tissue sections,with preservation of the reactivity of most antigenstested. Decalcification of the bone specimens affectedby tumour, however, was rarely complete after 24hours and often still incomplete after three days.To determine if tissues could be rapidly fixed and

decalcified without destroying antigen reactivity andcell morphology cryostat sections of parallel slices ofappendix were cut after fixation and decalcificationfor 24 hours in the agents listed (edetic acid, a slowdecalcifying agent, excepted). Antigenic reactivity incryostat sections was similar to that seen in the corre-spondingly decalcified routinely processed paraffinembedded sections. In addition, cryostat sections ofTCA fixed and decalcified specimens of bone andappendix showed a similar pattern of antibody stain-ing to that seen in material embedded in paraffin andpreviously treated with TCA. This indicates that TCAcould be useful as a rapid one step fixation anddecalcification agent for both cryostat and routinelyprocessed sections. In immunoperoxidase stainedcryostat sections background staining of red cells wasstrong with all decalcifying agents, even after block-ing with methanol-H202, and could potentially causedifficulty in interpretation. Background staining ofred cells was also seen in immunoperoxidase stainedparaffin sections but was not as pronounced. Thiseffect was avoided by use of the APAAP technique.

Discussion

The selection of an appropriate decalcifying agent islargely governed by two factors: the amount of miner-alised tissue present in the specimen; and the urgencyof the specimen requiring decalcification. Subsequentstaining and investigative procedures, which mayneed to be carried out on the decalcified specimen, arealso important, however, and it is this last factor thatwe examined with respect to immunohistochemistry.Even the most common rapid decalcification pro-

cedure in general use-that is, strong acids such asHCI, HNO3-did not lessen the reactivity of manydiagnostically useful antigenic determinants. Cyto-logical detail and staining quality were adversely

affected, particularly after prolonged decalcificationin strong acids. Interpretation of immuno-histochemical staining, however, was still clear, andmost specimen blocks of bone should be decalcifiedwithin 24 hours in the above solutions. HCl-edeticacid also provided rapid decalcification with excellentmorphology and good preservation of antigenic reac-tivity.

Treatment with TCA was useful as it provided arapid one step fixation and decalcification procedurethat led to retention of many useful antigenic com-ponents, as well as excellent morphology and stainingquality. TCA decalcification was also suitable for usewith cryostat and paraffin sections, allowing a quicktissue diagnosis to be obtained. Treatment with weakacids and edetic acid undoubtedly resulted in bettermorphological detail and staining quality, withgreater retention of antigenic reactivity. These agents,however, are not suitable for urgent or larger speci-mens as decalcification of the bone specimens wasoften not complete within 24 hours.

Recognition of antigenic determinants in thedecalcified specimens by the polyclonal and mono-clonal antibodies studied should aid in the histologi-cal diagnosis of tumours of uncertain histogenesisand origin that affect calcified tissues.4 Antigenicdeterminants for tumours of lymphoid or haemo-poietic (leucocyte common antigen, light and heavyimmunoglobulin chain), epithelial (cytokeratins, epi-thelial membrane antigen), mesenchymal (vimentin,desmin, factor VIII related antigen), or other (MAA,S100) differentiation can be distinguished, even inspecimens decalcified in strong acid solutions. Inaddition, antigenic components of tumours likely tometastasise to bone such as PSA, PAP-prostate,16 17Tgb-thyroid,l8 Carcinoembryonic antigen large bowelcancer'0 can be recognised in decalcified tissue if theappropriate decalcifying agent is used. Knowledgeof the effects of the various decalcifying agents onthe immunoreactivity of cellular antigens shouldrationalise prospective and retrospective immuno-histochemical analysis of tumours in calcified tissue.

This work was supported by a grant from theArthritis and Rheumatism Council. We thank Miss LWatts for typing the manuscript and Mr J Markhamfor photographic assistance.

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