Effect of Decalcification Agents on Immunoreactivity of Cellular Antigens

J Clin Pathol: first published as 10.1136/jcp.40.8.874 on 1 August 1987. Downloaded from J Clin Pathol 1987;40:874-878

Effect of decalcification agents on immunoreactivity of cellular antigens

N A ATHANASOU, J QUINN, A HERYET, C G WOODS,* J O'D McGEE From the University ofOxford, 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 acid on the immunohistochemical staining of cryostat and&d paraffin embedded sections of tissue from a variety of normal and pathological calcified and uncalcified specimens were studied. Even decalcification in strong acids (HCI, HNO3, 5% trichloracetic acid, HCl-edetic acid did not diminish the reactivity of many useful antigens (including leucocyte common antigen, intermediate filaments, S100 protein and epithelial membrane antigen). Weaker acids (formic acid, acetic acid) and edetic acid decalcified more slowly and generally showed greater preservation of antigenic reactivity with better morphology and staining quality. Trichloracetic acid was also useful as a quick one step fixation and decalcifying agent for both cryostat and routinely processed sections. Knowledge of the preservation of antigenic reactivity in decalcified tissue will be useful in the diagnosis of tumours of uncertain histogenesis and origin which affect calcified tissues. copyright. Immunohistochemical analysis of surgical specimens Material and methods using monoclonal antibodies has assumed an important role in histopathological diagnosis. Surgical specimens included normal appendix, thy- Although frozen material and cryostat sections are roid, skin, breast, prostate, bone, and tonsil, as well as optimal for the preservation of the reactivity of many cases ofcarcinoma (lung, kidney), myeloma, and lym- antigens, routine formalin fixation and paraffin phoma affecting bone. Table I shows the antigenic embedding, often associated with protease digestion determinants sought in each of these tissues. http://jcp.bmj.com/ of tissue sections prior to immunostaining, has been The tissues were either snap frozen in liquid nitro- found to interfere only partially with the localisation gen for cryostat sectioning or fixed in 10% formol of many cellular and tissue antigens useful in tissue saline at room temperature for a minimum of 24 diagnosis.' With calified tissues, the situation is hours (range 24 hours-14 days). Slices of the fixed more complex as fixation is generally followed by a tissues were then routinely processed to paraffin wax. decalcification procedure. Although there are several A parallel fixed slice was also placed in one of several decalifying agents in common use, their effect on the decalcifying agents for 24 hours and three days. The on September 24, 2021 by guest. Protected immunoreactivity of the various cellular and tissue agents studied were 5% aqueous nitric acid (HN03), antigens has been little studied.4 5 This is often of par- 5% aqueous hydrochloric acid (HCl), 10% RD0 ticular importance where panels of monoclonal anti- (Bethlehem, Gotheberg), 5% trichloracetic acid bodies are used in the histopathological diagnosis of (TCA), 10% aqueous formic acid (FA), 10% aqueous tumours of uncertain origin.6 acetic acid (AA), and 10% HCl-edetic acid (ethylene In this study we examined the effect of several diaminetetracetic acid) mixture. Specimens were also decalcifying agents, including strong acids, weak decalcified in edetic acid alone (24 hours and seven acids, and chelating agents on immunohistochemical days), as well as being solely fixed and decalcified in staining for several diagnostically useful cellular and TCA for 24 hours. After washing in running tap tissue antigens in both calcified and uncalcified water for four to six hours the treated specimens were surgical specimens. processed via ethanol (six hours) and xylene (three hours) to paraffin wax. Five micron sections were mounted on Multispot slides (Hendley, Essex) and dried at 60°C for 60 minutes. Before immunostaining, Accepted for publication 19 March 1987 all paraffin sections were dewaxed in xylene and 874 J Clin Pathol: first published as 10.1136/jcp.40.8.874 on 1 August 1987. Downloaded from

Effect ofdecalcification agents on immunoreactivity of cellular antigens 875 Table 1 Antigenic determinants sought in surgical specimens examined Specimen Antigenic determinants sought

Appendix Leucocyte common antigen (LCA), HLA-DR Light and heavy immunoglobulin chains (Ig's) Vimentin, SIOO protein, desmin Factor VIII related protein Cytokeratin intermediate filaments Carcinoembryonic antigen (CEA) Epithelial membrane antigen (EMA) Tonsil Ig's LCA Skin normal, compound naevus Melanoma associated antigen (MAA), cytokeratins, SIOO Thyroid Thyroglobulin (Tgb) Breast EMA, cytokeratins Prostate Prostatic acid phosphatase (PAP) Prostate specific antigen (PSA) Bone marrow osteoarthritic femoral headf LCA, Ig's Lymphoma}affecting bone LCA, Ig's Squamous cell carcinoma lung Adenocarcinoma lung affecting bone EMA, cytokeratins Adenocarcinoma kidney rehydrated through graded alcohols to water. Parallel Table 2 Monoclonal andpolyclonal* antibodies usedfor slices of appendix were also processed to cryostat sec- analysis ofroutinely processed decalcified tissues tions after fixation and decalcification (24 hours) in the various agents, as noted above, to assess if speci- Antibody Antigenic determinant mens could be rapidly fixed and decalcified without destroying antigen reactivity. PD7/26 Leucocyte common antigen'

2BI 1 Leucocyte common antigen' copyright. Table 2 gives details ofmonoclonal antibodies used CR3/43 HLA-DR8 Anti-K in this study. Polyclonal rabbit antibodies against Anti-A Light and heavy immunological chains* human immunoglobulin heavy and light chains (Ig), Anti IgG, IgM E29 Epithelial membrane antigen9 thyroglobulin, and prostate specific antigen (PSA) CEA Carcinoembryonic antigen10 were obtained from Dako (UK) and antiprostatic KLI Cytokeratin intermediate filaments11 acid phosphatase (PAP) from Miles Scientific Ltd. CAM 5-2 Cytokeratin intermediate filaments12 SI-61 S1OO protein13 Immunohistochemistry was performed using an DER. I I Desmin14 http://jcp.bmj.com/ indirect immunoperoxidase or alkaline phosphatase V9 Vimentin1I NKl/C3 Melanoma associated antigen16 antialkaline phosphatase (APAAP) procedure, as Anti-PSA Prostate specific antigen* previously described.'7 Both control and decalcified Anti-PAP Prostatic acid phosphatase* Anti-thyroglobulin Thyroglobulin* sections of fixed tissue were digested by 0-1% trypsin Anti-F VIII Factor VIII related antigen17 (Sigma T: 8128) in 0-1% calcium chloride at pH 7-8 before immunostaining with the following antibodies: *Indicates polyclonal antibodies. CR3/43, anti-light chain Ig, anti-heavy chain Ig, anti- carcinoembryonic antigen, anti-PSA, anti-PAP, anti- on September 24, 2021 by guest. Protected Tgb, and anti-factor VIII related antigen (30 ing quality. Undecalcified controls that were pro- minutes); CAM 5-2 (15 minutes); DER- 11 (20 cessed to cryostat sections of formalin fixed paraffin minutes). The histological quality, degree, and embedded sections were all strongly positive with the specificity of immunohistochemical staining was antibodies tested. scored relative to that of fixed undecalcified control Treatment with the strong acids HCI and HN03 sections. and the proprietary decalcifier RDO for 24 hours and three days preserved immunoreactivity of all antigens Results tested with the exception of HLA-DR and epithelial membrane antigen (EMA). Tissue morphology and Table 3 summarises the effect on the immunostaining quality was adversely affected, particularly reactivity of cellular antigens after treatment with the after prolonged (three day) decalcification, but various decalcifying agents (figs 1 and 2). Prolonged immunostaining was still interpretable. HCI-edetic (three day) decalcification produced no noticeable acid treatment was associated with better mor- differences in terms of antigen reactivity or antibody phology and weak EMA reactivity was retained. TCA specificity- but had an effect on morphology and stain- treatment (both with and without prior formalin J Clin Pathol: first published as 10.1136/jcp.40.8.874 on 1 August 1987. Downloaded from 876 Athanasou, Quinn, Heryet, Woods, McGee Table 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.

*~~~~~~~~~~~~~~~~~~~~~~~~... s=u*t-:. ;, '

4,; A |i ; *e ~~~~~~~~~~SE:",;?*. _-'¢_$ _- * *>a

A ..i .1 4. - copyright. .} ;- 9:~~~~ ~ ~~ ~ ~ ~ '_?iS *i4's2

.4 7S,- @-fj- 4_ a6 ': r - en- http://jcp.bmj.com/ i L:J '; *:'"- A L. I' -~'.!; .. .:. .> K

Jta'i." -Y I, 2 * ''S- -'Y. -a~~~~~~~~~~~~~~~~~~Y

.'444 on September 24, 2021 by guest. Protected

444 jI:.: -. -t )8

.g~~~~~~~~~~~~~~~~~~~~I :: ^, '. 0' I P' l ,l ,,*i t

I :: f-/

Fig 1 Squamous cell carcinoma oflung infiltrating bone: Fig 2 Lymphoma infiltrating bone (right) with tumour cells tumour cells are stainedfor cytokeratin intermediate showing membrane stainingfor leucocyte common antigen filaments (CAM5-2). (Immunoperoxidase, decalcification (PD7/26). (Immunoperoxidase, decalcification TCA 24 HCl-edetic acid 24 hours). hours). J Clin Pathol: first published as 10.1136/jcp.40.8.874 on 1 August 1987. Downloaded from Effect ofdecalcification agents on immunoreactivity of cellular antigens 877 fixation) also showed good staining quality and pre- affected, particularly after prolonged decalcification servation of both morphology and antigenic reac- in strong acids. Interpretation of immuno- tivity. No particular advantage in terms of immunohistochemical staining, however, was still clear, and histochemistry was found in using the proprietary most specimen blocks of bone should be decalcified decalcifier (RDO) tested. It should also be noted that, within 24 hours in the above solutions. HCl-edetic although both anti-leucocyte common antigen anti- acid also provided rapid decalcification with excellent bodies tested, PD7/26 and 2B1 1, reacted with leuco- morphology and good preservation of antigenic reac- cytes after 24 hours in all decalcifying agents, 2B1 1 tivity. did not react with leucocytes after three days of Treatment with TCA was useful as it provided a decalcification in HCI, HNO3, RDO and TCA. rapid one step fixation and decalcification procedure Treatment with weak acids (FA, AA) and edetic for that led to retention of many useful antigenic com- 24 hours and three days showed generally better ponents, as well as excellent morphology and staining staining quality and morphology in tissue sections, quality. TCA decalcification was also suitable for use with preservation of the reactivity of most antigens with cryostat and paraffin sections, allowing a quick tested. Decalcification of the bone specimens affected tissue diagnosis to be obtained. Treatment with weak by tumour, however, was rarely complete after 24 acids and edetic acid undoubtedly resulted in better hours and often still incomplete after three days. morphological detail and staining quality, with To determine if tissues could be rapidly fixed and greater retention of antigenic reactivity. These agents, decalcified without destroying antigen reactivity and however, are not suitable for urgent or larger speci- cell morphology cryostat sections of parallel slices of mens as decalcification of the bone specimens was appendix were cut after fixation and decalcification often not complete within 24 hours. for 24 hours in the agents listed (edetic acid, a slow Recognition of antigenic determinants in the decalcifying agent, excepted). Antigenic reactivity in decalcified specimens by the polyclonal and mono- cryostat sections was similar to that seen in the corre- clonal antibodies studied should aid in the histologi- spondingly decalcified routinely processed paraffin cal diagnosis of tumours of uncertain histogenesis embedded sections. In addition, cryostat sections of and origin that affect calcified tissues.4 Antigenic

TCA fixed and decalcified specimens of bone and determinants for tumours of lymphoid or haemo- copyright. appendix showed a similar pattern of antibody stain- poietic (leucocyte common antigen, light and heavy ing to that seen in material embedded in paraffin and immunoglobulin chain), epithelial (cytokeratins, epi- previously treated with TCA. This indicates that TCA thelial membrane antigen), mesenchymal (vimentin, could be useful as a rapid one step fixation and desmin, factor VIII related antigen), or other (MAA, decalcification agent for both cryostat and routinely S100) differentiation can be distinguished, even in processed sections. In immunoperoxidase stained specimens decalcified in strong acid solutions. In cryostat sections background staining of red cells was addition, antigenic components of tumours likely to http://jcp.bmj.com/ strong with all decalcifying agents, even after block- metastasise to bone such as PSA, PAP-prostate,16 17 ing with methanol-H202, and could potentially cause Tgb-thyroid,l8 Carcinoembryonic antigen large bowel difficulty in interpretation. Background staining of cancer'0 can be recognised in decalcified tissue if the red cells was also seen in immunoperoxidase stained appropriate decalcifying agent is used. Knowledge paraffin sections but was not as pronounced. This of the effects of the various decalcifying agents on effect was avoided by use of the APAAP technique. the immunoreactivity of cellular antigens should rationalise prospective and retrospective immuno- Discussion histochemical analysis of tumours in calcified tissue. on September 24, 2021 by guest. Protected The selection of an appropriate decalcifying agent is This work was supported by a grant from the largely governed by two factors: the amount ofminer- Arthritis and Rheumatism Council. We thank Miss L alised tissue present in the specimen; and the urgency Watts for typing the manuscript and Mr J Markham of the specimen requiring decalcification. Subsequent for photographic assistance. staining and investigative procedures, which may need to be carried out on the decalcified specimen, are References also important, however, and it is this last factor that I Taylor CR. Immunoperoxidase techniques: practical and we examined with respect to immunohistochemistry. theoretical aspects. Arch Pathol Lab Med 1978;102:113-22. Even the most common rapid decalcification pro- 2 Heyderman E. Immunoperoxidase technique in histopathology: cedure in general use-that is, strong acids such as applications, methods and controls. J Clin Pathol 1979;32: HCI, HNO3-did not lessen the reactivity of many 971-8. 3 Curran RC, Gregory J. The unmasking of antigens in paraffin diagnostically useful antigenic determinants. Cyto- sections of tissue by trypsin. Experientia 1977;33:1400-1. logical detail and staining quality were adversely 4 Matthews JB. Influence of decalcification on immuno- J Clin Pathol: first published as 10.1136/jcp.40.8.874 on 1 August 1987. Downloaded from 878 Athanasou, Quinn, Heryet, Woods, McGee histochemical staining of formalin-fixed paraffin embedded antigenic determinant common to the alpha and beta chain of tissue. J Clin Pathol 1982;35:1392-4. S100. Lab Invest 1985;52:232-8. 5 Mukai K, Yoshimura MT, Anzar MT. Effects of decalcification 14 Debus E, Weber K, Osborn M. Monoclonal antibodies to desmin on immunoperoxidase staining. Am J Surg Pathol the muscle specific intermediate filament protein. EMBO J 1986;10:413-9. 1983;2:2305-9. 6 Gatter KC, Abdulaziz Z, Beverley P, etal. Use of monoclonal 15 Altmannsberger M, Osborn M, Schauer A, Weber K. Antibodies antibodies for the histopathological diagnosis of human to different intermediate filament proteins: cell type specific malignancy. J Clin Pathol 1982;35:1253-67. markers on paraffin embedded human tissues. Lab Invest 7 Warnke RA, Gatter KC, Falini B, et al. Diagnosis of human lynm- 1981;45:427-34. phoma with monoclonal antileukocyte antibodies. N Engl J 16 Mackie RM, Campbell I, Turbitt ML. Use of NK1 C3 mono- Med 1983;309.1275-81. clonal antibody in the assessment of benign and malignant 8 Naiem M, Gerdes J, Abudlaziz Z, Nash J, Stein H, Mason DY. melanocytic lesions. J Clin Pathol 1984;37:367-72. Production of monoclonal antibodies for the immuno- 17 Gatter KC, Falini B, Mason DY. The use of monoclonal histological analysis of human lymphoma. In: Knapp W, et al, antibodies in histopathological diagnosis. In: Anthony P, eds. Leukaemia markers. New York: Academic Press, MacSween R, eds. Recent advances in histopathology. 1981:117-25. Edinburgh: Churchill Livingstone, 1984:35-67. 9 Cordell J, Richardson TC, Pulford KAF, et al. Production of two 18 Jobsis AC, De Vries GP, Arholt RRH, Sanders GTB. monoclonal antibodies against human mammary epithelial Demonstration of the prostatic origin of metastases. An membrane antigens and their use in diagnostic immuno- immunohistological method for formalin-fixed embedded chemistry. Br J Cancer 1985;52:347-54. tissue. Cancer 1978;41:1788-93. 10 Heydermann E, Neville AM. A shorter immunoperoxidase 19 Wang MC, Papsidero L, Muryama M, etal. Prostate antigen: a technique for the demonstration of carcinoembryonic antigen new potential marker for prostatic cancer. The Prostate and other cell products. J Clin Pathol 1979;30:138-40. 1981;2:89-96. 11 Viac J, Reano A, Brochier J, etal. Reactivity pattern of a 20 Burt A, Goudie RB. Diagnosis of primary thyroid carcinoma by monoclonal antikeratin antibody (KLI). J Invest Dermatol immunohistological demonstration of thryoglobulin. 1981;81:351-4. Histopathology 1979;3:279-86. 12 Makin CA, Bobrow LG, Bodmer WF. Monoclonal antibody to cyotkeratin for use in routine histopathology. J Clin Pathol 1984;37:975-83. Requests for reprints to: Dr N Athanasou, Nuffield 13 Vanstapel MJ, Peeters B, Cordell J, etal. Production and Department of Pathology, John Radcliffe Hospital, identification of monoclonal antibodies directed against an Headington, Oxford OX3 9DU, England. copyright. http://jcp.bmj.com/ on September 24, 2021 by guest. Protected