Enzyme Immunohistochemistry: Review of Technical Aspects and Diagnostic Applications

Enzyme immunohistochemistry: review of technical aspects and diagnostic applications

The era of immunohistochemistry was intro- duced by Coons et al1 in 1941 when antibodies were successfully labeled with a fluorochromatic com- Raymond R. Tubbs, D.O. pound. Shortly thereafter, localization of tissue antigens was successfully accomplished with the use Department of Pathology of fluorochromatic labels.2 Initially a research tool, Khalil Sheibani, M.D. immunofluorescence became an essential diagnostic Sharad D. Deodhar, M.D., technique for the evaluation of many disease states, Ph.D. particulary autoimmune diseases mediated by immune complexes or autoantibody deposition. Department of Immunopathology It soon became clear that certain limitations such William A. Hawk, M.D. as special instrumentation requirements and lack of

Department of Pathology permanency were accorded immunofluorescent procedures. Consequently, immunohistochemical systems were developed that permitted the visual localization of a tissue antigen as a permanent preparation with the potential for visualization of adjacent tissue morphology. The successful conjugation of antibodies with enzymes and unlabeled antibody methods made immunomicroscopy practical. Both enzyme-labeled antibody and unlabeled antibody (antienzyme) methods allowed identification of tissue antigens by formation of permanent color products in histologic sections with excellent morphologic detail. '4 This paper reviews the rationale underlying enzymatic immunomicroscopic procedures, tech- 245

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. 246 Cleveland Clinic Quarterly Vol. 48, No. 1 niques currently available, characteris- enzyme coupled to one molecule of an- tics of currently available chromogens, tibody protein. At a ratio of three to safety for personnel, quality control of four enzyme molecules per protein mol- immunohistochemical systems, and ecule there is loss of antibody binding, clinical diagnostic applications of the enzyme function, and penetration. For procedure. most enzyme-labeled antibody immunomicroscopy tests, a molar ratio of 1 Biochemistry of enzyme immunomi- enzyme molecule per protein molecule croscopy is adequate. This allows high function Many different enzymes are potential of both antibody and enzyme and good antibody labels, including acid phos- penetration (peroxidase + antibody = phatase, ^-glucuronidase, 5'-nucleotid- 40,000 + 160,000 = 200,000 molecular ase, glucose oxidase, and horseradish weight). For enzyme-linked immunoperoxidase.5"7 However, horseradish sorb assay (ELISA) a molar ratio of peroxidase has been the enzyme label between two- and three-enzyme mole- used most frequently since it is readily cules per molecule of antibody may be available and relatively inexpensive; desirable. Currently, numerous com- well-established conjugation methods mercial preparations of high-titer anti- have been developed for conjugation bodies with optimum enzyme-protein with antibody.8 conjugation ratios are available. How- The biochemical reaction that occurs ever, lot-to-lot differences and variations at the histochemical level can be sum- between various companies' antisera ex- marized in the equation below: ist and, therefore, the reactivity and specificity of every commercial reagent H2O2 + H2R Peroxidase must be confirmed. (to be (Chromogen * reduced) donor) Tissue processing

R + 2H20 The choice of type of tissue processing (Oxidized-chromogen is largely dependent upon the individ- donor) ual microscopic system. Extracellular The substrate, hydrogen peroxide, is im- immune complexes and autoantibodies portant in the reaction only in that it may be detected in paraffin-embedded accepts hydrogen from the chromogen. tissue with the use of posttrypsinization Ideally, the molecular change in the techniques. Such preparations do pro- oxidized chromogen results in a reaction vide superior morphology. However, product insoluble in organic solvents frozen tissue is preferable for most stud- which differs in color from its parent ies since 10% to 25% of cases positive compound. The amount of substrate with cryostat frozen section immuno- necessary to make the reaction proceed fluorescence are negative even if de- is very small, with usual useful working waxed paraffin-embedded tissue is pre- concentrations of hydrogen peroxide in treated with trypsin.9'10 most systems ranging from 0.0003% to The effect of tissue fixation is espe- 0.003%. cially important in the evaluation of The proportion of conjugated anti- lymphoproliferative disorders. It has body to enzyme is evaluated by the ratio been shown that fixation using any mor- of enzyme to antibody protein. This is dant solution markedly alters the im- probably best expressed as a molar ratio munoglobulin products associated with implying the number of molecules of non-Hodgkin's and Hodgkin's lym-

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. Summer 1981 Enzyme immunohistochemistry 247 phoma cells and reactive lympho- specimen when placed in fixative. To cytes.11'12 Although paraffin-embedded ensure complete tissue penetration, 1- to tissue sections may be counterstained to 2-mm thick sections should be placed in give excellent cellular detail in immu- abundant volumes of appropriate fixa- nomicroscopic sections,13 spurious im- tive. munostaining of non-Hodgkin's lym- The alleged problems of immuno- phomas not infrequently occurs.11'14 globulin diffusion and spurious staining Furthermore, immunostained cryostat said to occur with cryostat frozen section frozen sections are amenable to counter- immunohistochemistry have not proved staining with hematoxylin and eosin or serious under close scrutiny.11'13 Study other counterstains permitting some of frozen section material yields repro- definition of cellular morphology.15 ducible results and observations con- For paraffin-embedded systems, re- sistent with well-established concepts of ports vary widely as to the superiority monoclonality in non-Hodgkin's lym- 11 12 15,23 of different fixatives.16-22 It has been phomas in most cases. ' ' Further- suggested that 2% formaldehyde is su- more, small amounts of alcohol used in perior to 4% formaldehyde.21 Some in- paraffin embedding markedly alter the vestigators have found cacodylate- immunoglobulin phenotypes of prolif- 11 12 buffered paraformaldehyde superior to erating lymphoid cells. ' Both direct Bouin's solution for cytoplasmic immu- and unlabeled frozen section immuno- noglobulins and Bouin's best to preserve histochemistry readily detect appropri- 23-25 antigenicity of hormones.22 In our ex- ate immunoglobulin phenotypes. perience, the best approach is to evalu- Chromogens ate each immunomicroscopic system in- dependently with regard to optimum Table 1 summarizes data currently fixative solutions. available for chromogens used in im- When submitting tissue for paraffin munoperoxidase methods. Each chrom- embedding, an important consideration ogen offers certain advantages but has is the thickness of the original tissue some disadvantages, and many ques-

Table I. Properties of chromogens

Carcino- Federal genicity regula- Solubility in organic (laboratory tions of Immunohistochemical label solvents Color animals) use

Fluorochromatic ? Fluoroscein isothiocyanate Not applicable Green -

Tetramethyl rhodamine Not applicable Red ? - Enzymatic

Benzidine dihydrochloride - Blue + +

DAB (diaminobenzidine) - Brown-black ?* -

TMB (tetramethyl benzidine) - Blue ?t - HYR (Hanker-Yates reagent, p-phenylenedi- - Black ?t - amine & pyrocatechol)

AEC (aminoethylcarbazole) + Red-brown + -

* One study has reported that diaminobenzide (3,3',4,4'-Tetraminodiphenylether • 4 HC1 or 4,4'-Oxybis-o- phenediamine) did not act as a carcinogen in experimental animals.31 f Commercial sources of these reagents specify that this chromogen is noncarcinogenic but, to our knowledge, studies of carcinogenesis of these compounds sponsored by the federal government have not been done.

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. 248 Cleveland Clinic Quarterly Vol. 48, No. 1 tions regarding the safety of these com- and pyrocatechol) that incorporates the pounds (and fluorochrome markers) re- better qualities of benzidine derivatives main unanswered.26 Benzidine dihydro- and that has no currently identified car- chloride gives a stable blue color reac- cinogenic properties.41 The biochemis- tion product, which has been associated try of this chromogen depends upon the with greater sensitivity than other avail- peroxidation of aromatic alcohols in the able chromogens 27-29 Governmental presence of phenolic compounds.40 regulations have made use of this com- From the results of a recent study 30 pound impractical. comparing nine methods for immuno- The most widely used immunohisto- histochemical chromogen systems it was chemical chromogen has probably been concluded that TMB provided the 3,3-diaminobenzidine dihydrochloride greatest sensitivity and specificity.32 monohydrate (DAB).13 This reagent However, these conclusions have been yields a brown to black color reaction challenged, and Hanker-Yates reagent product, which is not soluble in organic (HYR) has been suggested as a superior solvents and does not crystallize on the immunohistochemical chromogen.42 tissue sections. DAB is not currently reg- Published reports have described vari- ulated to our knowledge and in one study able methodology for HYR procedures, did not demonstrate carcinogenesis in and the differential sensitivity of TMB experimental animals.31 Tetramethyl- and HYR may be attributable to minor benzidine (TMB) has been advocated technical variations. HYR reagent as an alternative chromogen that has works well when a sequence of fresh not been associated with carci- substrate-chromogen solutions are used nogenesis in laboratory animals, but crys- with addition of the substrate just prior tallization on the tissue sections is a to placing the sections into the chrom- problem.28'29'32 ogen solution.41 At present one of the The availability of multiple types of more useful reagents would appear to peroxidative chromogens yielding dif- be HYR since it has no known carcino- 42 ferent color reaction products having genic potential to our knowledge, and different tinctorial properties allows the has been shown to work well in compar- 41 simultaneous visualization of more than ative immunomicroscopic systems. All one antigen in the same tissue section.33" chromogens and fluorescent-labeled 35 These results can be achieved without compounds should be handled as poten- elaborate double incubation steps.35 tially hazardous reagents. Similar double-labeling studies have also been done with the use of a com- Enzyme immunomicroscopic proce- bination of enzyme labels such as glu- dures cose oxidase and horseradish peroxidase.36 Several enzyme immunohistochemi- Aminoethylcarbazole (AEC) has cal procedures are available and these also been advocated as a useful chrom- are diagrammatically summarized in ogen.37,38 However, recent evidence sug- Figures 1-5. Once the enzyme has been gests that carcinogenic potential in lab- localized at the antigen site by any of oratory animals does exist, and this re- these procedures, the techniques for de- agent may be regulated by the govern- velopment of the substrate chromogen ment in the future. reaction product are the same regardless Hanker et al40 have developed a chro- of the immunohistochemical technique mogenic reagent (p-phenylenediamine chosen.

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HPO CONJUGATED GOAT ANTIRABBIT IgG HPO CONJUGATED H PO

Figs. 1-4. Diagrams of four different immunoperoxidase methods to detect IgG in epimembranous immune complexes deposited in membranous glomerulonephritis. Fig. 1, direct technique; Fig. 2, indirect technique; Fig. 3, unlabeled peroxidase-antiperoxidase (PAP) technique; and Fig. 4, protein A modification of PAP technique.

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HPO

PREFORMED AVIDIN (OD) — BIOTINYLATED PEROXIDASE COMPLEX

BIOTINYLATED ( (AFFINITY b• PURIFIEp D GOAT ANTI-RABBIT IgG

RABBIT ANTI-HUMAN GLUCAGON

UCAGON IN ALPHA CELL OF PANCREATIC ISLET

Fig. 5. Diagram of biotin-avidin "ABC" technique to detect glucagon within pancreatic alpha islet cells. Biotinylated affinity purified goat anti-rabbit IgG secondary antibody links the rabbit antiglucagon primary antibody to a preformed complex of avidin and biotinylated horseradish peroxidase.

Enzyme-labeled antibody methods not as high as that for peroxidase-antiperoxidase (PAP) procedure, is ade- Direct method. The direct technique quate for most clinical tissue studies. is the simplest immunomicroscopic pro- The direct technique also works well for cedure (Fig. I). The reagent consists of detection of intracellular and surface a specific antibody conjugated with en- membrane-associated immunoglobulins zyme. This enzyme-antibody conjugate in lymphoproliferative disorders.1'® is overlaid directly on the hydrated tis- Indirect method. The indirect im- sue section. Duration of incubation var- munoperoxidase (IMP) procedure does ies with the individual immunomicros- not differ from its immunofluorescent copic system. After washing of excess (IF) counterpart with respect to the reagent from the tissue surface with an basic technique (Fig. 2). The primary isotonic buffer system, the enzyme-sub- unconjugated antibody binds specifi- strate color reaction product is devel- cally to its antigen in the tissue, and oped with one of the chromogens cur- after washing off excess primary anti- rently available. body from the surface of the tissue, the The direct procedure that uses cryo- peroxidase-labeled secondary antibody stat frozen sections is currently the tech- is applied. Subsequently, the enzyme nique of choice for studying renal tissue. color reaction product is developed. Sensitivity of this procedure, although Since the secondary antibody is labeled

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with the enzyme, a color reaction prod- plex. Similarly, the goat PAP system uct identifies the antigen focus in the consists of a goat primary antibody, a tissue. bridge antibody, e.g., rabbit anti-goat IgG, and a soluble goat PAP complex. Unlabeled antibody methods Protein A modification of PAP Triple antibody bridge method. For method. Protein A from Staphylococcus this method, primary antibody and an- aureus (SPA) has been shown to bind the tiperoxidase antibody are raised in the Fc portion of IgG molecules of several same animal, e.g., rabbit. A bridge an- species.4 This particular reagent can be tibody, e.g., anti-rabbit IgG, is applied used as a conjugate with peroxidase as in sequence after the primary antibody a "labeled secondary antibody" as a and before the addition of the antiper- consequence of its Fc IgG binding.47 oxidase to the tissue surface. This sec- Also, SPA can be substituted for bridge ondary antibody "bridges" the primary antibodies, e.g., goat anti-rabbit IgG or and secondary antibody by virtue of its rabbit anti-goat IgG in the unlabeled specificity for the immunoglobulin class PAP procedure (Fig. 4)i7~49 However, in the primary and tertiary reagents. there are differences in relative avidity Finally, peroxidase is applied to the tis- of SPA for the PAP complexes of differ- sue section and the reaction product ent animal species; for example, rabbit developed. Use of this particular reagent and guinea pig PAP bind more com- has been virtually eliminated by avail- pletely than goat or rat PAP.47"49 ability of the sensitive PAP unlabeled technique, which uses a preformed sol- Biotin/avidin lectin method uble PAP complex. Recent evidence suggests that biotin/ Unlabeled PAP method. The unla- avidin enzyme immunohistochemistry beled PAP procedure is illustrated in compares favorably with established Figure 3. This particular method differs IMP techniques (Fig. 5).50 The recently from the triple antibody method only in developed ABC lectin immunohisto- that the tertiary reagent consists of a chemical system has been found to be 8 soluble complex of peroxidase and an- to 40 times more sensitive than the un- tiperoxidase. Excellent commercial labeled PAP method, yields immuno- sources of PAP are available. This par- stained sections having negligible or no ticular procedure is generally more sen- background staining, and is cost effec- sitive than the other available methods. tive (about 5% of cost of average PAP However, the sensitivity of the labeled procedure).51 The ABC system uses in (indirect) antibody technique with the sequence unconjugated primary anti- use of affinity-purified antibodies is body, biotinylated affinity purified sec- about equal to that of the PAP ondary antibody, and a preformed com- method.44 In some systems, the sensitiv- plex of avidin and biotinylated horse- ity of the PAP procedure approaches radish peroxidase as the tertiary re- that of radioimmunoassay with useful agent. The extraordinary sensitivity and working dilutions of the primary anti- specificity of this method are due to at body approaching 1:100,000.45 Rabbit least three factors: (1) avidin has high PAP systems employ in sequence pri- binding affinity for biotin; (2) the avi- mary rabbit antibody against the tissue din-biotin binding reaction is essentially antigen in question, a bridge antibody irreversible; and (3) unlike the second consisting of goat or swine anti-rabbit antibody of a PAP system (which must IgG, and the soluble rabbit PAP com- be present in excess since one of its two

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. 252 Cleveland Clinic Quarterly Vol. 48, No. 1 potential antibody binding sites must be Nonspecific binding of antisera available to bind the PAP complex), Although methanolic H2O2 or en- biotinylated secondary bridge anti- zyme pretreatment destroys or con- bodies can be used in low concentrations sumes endogenous peroxidases, the non- since the biotin is already linked to the specific absorption of heterologous se- antibody. rum to the tissue occasionally yields a Each of these methods has certain problem in background staining. These advantages and disadvantages. The in- problems can be eliminated by a com- direct labeled and unlabeled antibody bination of prolonged incubation with techniques, while offering increased sen- high dilutions of the primary antibody sitivity, require multiple procedural (>1:1000) and preincubation of rehy- steps and involve additional reagents. drated sections in nonimmune serum.13 Also, in some immunohistochemistry systems, increased sensitivity may be Relative merits of immunomicroscopic gained at the expense of specificity. methods Background staining: the problem of The disadvantages and advantages of endogenous peroxidases and fluorochromatic and enzyme-labeled pseudoperoxidases. techniques have been the subject of ex- Both IF and IMP procedures are as- tensive debate. Since many of the initial sociated with certain predictable arti- IMP studies were done on paraffin-em- facts. Autofluorescence of certain mate- bedded tissues, for some time IF was rials in tissue must be recognized and thought to be a procedure most suited interpreted for individual sections. Pseu- to frozen section material and IMP for doperoxidases such as hemoglobin and fixed paraffin-embedded material. It is naturally occurring endogenous peroxi- now known that either IF or IMP tech- dases in human tissue are similarly a niques are readily applied to fixed or source of misleading background stain- frozen tissue sections. The initial lack of ing in enzyme-labeled preparations. correlation observed between IF and One of two approaches can be used to IMP in studying lymphoproliferative circumvent this problem. First, controls disorders in frozen section material now consisting of tissue not exposed to the appears to be a consequence of the specific primary antibody, but allowed methodology employed or antibody to incubate with the substrate chromo- concentrations.11,12,24 Also, the earlier gen solution, will allow visualization of problems encountered in differentiating these endogenous peroxidases, and com- granularity and linearity in renal biopsy parison can be made with specific im- specimens ' were not observed in an munostained sections. However, a more evaluation of large numbers of kidney acceptable alternative is to destroy or biopsies studied by comparative IF, consume the endogenous peroxidase IMP, and electron microscopy.59-61 either by preincubation of tissue sections Certain advantages are accorded the with methanol H2O2 or by trypsin, pro- IMP procedure as compared with IF. IF 9,10 52 56 tease, or pronase digestion. ' " Pro- preparations fade with repeated exami- teolytic enzyme pretreatment appears to nation and storage, but immunohisto- enhance antigenicity by a mechanism chemical preparations yielding stable that is not Well established. color reaction products do not fade.

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Also, it is not usually possible to visual- It seems that the resistance to change ize well simultaneously the imrauno- from immunofluorescence to immunope- stained antigen and adjacent tissue mor- roxidase techniques for the routine ex- phology in IF preparations. Conversely, amination of renal biopsy material cannot IMP preparations are readily adaptable be explained in scientific terms but de- to a variety of counterstains enabling pends largely on emotional ties to a system the observer to (1) more precisely locate which has been established for a considerable number of years.60 the tissue antigen, and (2) evaluate such additional parameters as inflammatory Quality assurance response to the antigen. An IF micro- Quality control of both IF and IMP scope is necessary for examination of the reagents is an essential and often ne- IF preparations, and photographic doc- glected part of fluorescent and enzyme umentation is necessary. immunohistochemistry. Commercially Previously, valid disadvantages were available antibody should not be as- also accorded IMP procedures. These sumed to be monospecific or of adequate included the potential carcinogenic na- immunoreactivity. The specificity and ture of the chromogens used with IMP, sensitivity of each reagent purchased and the lack of reliable commercial re- should be evaluated when received. agents. Both of these objections are no Each laboratory should have a protocol longer valid, since diaminobenzidine for evaluating all new antibodies enter- (3,3', 4,4'-Tetraaminodiphenylether • 4 ing the laboratory. The antisera should HC1 or 4,4'-oxybis-o-phenylenedia- be dated when received, and evaluated mine) may not be a carcinogen31 and at by Ochterlony immunodiffusion, Im- least one chromogen, HYR, is now munoelectrophoresis, competitive bind- available, which has no currently iden- ing radioimmunoassay or immunohis- tified health hazard and yields excellent tochemistry with the use of preabsorp- results. Several manufacturers currently tion and postabsorption with antigen distribute antibody-enzyme conjugates control tissues that have been well char- of excellent quality. Objections relating acterized with respect to the appropriate to the more complex nature of IMP antigen. Many commercial antisera procedures are no longer valid, since the have package inserts that attest to the direct technique using enzyme conju- reliability of the reagents. However, gates can be used for most studies that there may be significant interlot varia- employ immunohistochemistry, i.e., tion and the reagent immunoreactivity renal diseases and lymphoproliferative may be altered by environmental factors disorders. The additional time required during shipping. Individual techniques to develop the substrate chromogen re- should be performed regularly to assure action product is no longer than the continued competence by technical per- additional time required for photogra- sonnel and adequate performance of re- phy and cataloging of photographic agents. A detailed record should be kept slides for IF. of all quality assurance tests and docu- It is ideal to have the capability of mentation of corrective actions taken. doing both IF and IMP procedures. Even when excellent standardization This allows the pathologist versatility in and characterization of antibody have the selection of the appropriate proce- been completed, rigorous in-run con- dure. trols are necessary for valid interpreta-

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tion of results. Several types of controls stitution of nonimmune serum such as that may be used are summarized in normal goat serum or normal swine se- Figures 6-8. The simplest control is omis- rum for the primary antibody. This sion of the primary antibody and sub- technique serves as a control for recog-

CONTROL-UNLABELED TECHNIQUE NON-IMMUNE SERUM SUBSTITUTION BLOCKING CONTROL DIRECT TECHNIQUE

PAP COMPLEX

RABBIT ANTIPEROXIDASE HPO CONJUGATED RABBIT ANTI-IgG

RABBIT ANTI-IgG -

UNLABELED TECHNIQUE ABSORBED ANTIBODY CONTROL

GOAT ANTIRABBIT IgG

RABBIT ANTI-hCG—-/J

Figs. 6-8. Diagrams of three types of controls for immunohistochemistry. Fig. 6. Preincubation of tissue section with unconjugated antibody followed by addition of labeled antibody does not result in staining, since tissue antigen binding sites are occupied by the first reagent. Fig. 7. Substitution of nonimmune normal heterologous serum for the primary antibody is applicable to any immunohistochemical procedure, detects nonspecific binding of secondary or tertiary reagents, and profiles endogenous peroxidase staining. Fig. 8. In vitro preabsorption of primary antibody with antigen in question, followed by addition of filtered or centrifuged antiserum-antigen mixture to the section, is also applicable to any immunohistochemical procedure and is the best available negative control. This type of control detects lack of primary antibody specificity, as well as nonspecific binding of secondary or tertiary reagents and endogenous peroxidase activity.

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nition of binding of secondary and ter- board titrations with varying combina- tiary antibodies, to the tissue and for tions of antibody dilutions can be used endogenous peroxidase in indirect and on control tissue sections to evaluate the PAP methods, but cannot adequately optimal dilutions of each reagent. assess monospecificity of the primary antibody. The direct IMP procedure Diagnostic applications can be controlled by preincubation with Immunohistochemical procedures unlabeled antibody, preferably from the have contributed greatly to the under- same antisera lots from which the con- standing of normal physiology and jugate was prepared. This type of con- functional organization of many animal trol effectively blocks the labeled con- and human biologic systems. This paper jugate from reaching the antigen and will be restricted to reviewing the clini- although satisfactory for the direct pro- cal diagnostic applications of IMP cedure, cannot be applied to indirect or methods (Figs. 9-16). unlabeled modifications of the technique. The most reliable control for all Renal immunohistochemistry IMP methods is an absorbed antibody The value of immunomicroscopy in control, in which the primary antibody delineating deposition of immunoglob- is preincubated with exogenous antigen, ulin and complement components or thus binding all the available antibody- autoantibody in glomeruli of patients reacting sites. When the supernatant with various autoimmune diseases is from the centrifuged mixture of bound well established. Once a tool of aca- antibody-antigen is applied to the tissue demic interest, immunomicroscopy is section, antibody is not available for the now an essential diagnostic method that reaction and immunostaining does not must be applied to every renal biopsy occur. specimen. Patterns of IF have been Preabsorption may be necessary to shown to be highly reproducible and 62 remove nonspecific reactants or reac- predictive histopathologically. tions with related antigens in the tissue. Comparative studies of IMP and IF Not uncommonly, secondary anti- of glomerulonephritis were initially fa- bodies, e.g., goat anti-rabbit IgG, or rab- vorable.8 Subsequently, studies of kid- bit anti-goat IgG, will cross react with ney biopsy specimens with the use of the human immunoglobulins. In such indirect technique and enzyme conjugates stances, absorption with purified human were associated with unacceptable back- gamma globulin followed by centrifu- ground staining, and in some cases a gation of the antibody is necessary to distinction between linear and granular assure specificity of the secondary anti- color reaction product was difficult.57,58 body. For every procedure done on a Both IF and direct IMP procedures were day-to-day basis and for each tissue an- compared to the unlabeled IMP tech- alyzed, an in-run control should be in- nique, and the specificity and sensitivity cluded for adequate verification of pos- of the unlabeled PAP procedure were itive or negative results. Use of affinity demonstrated to be comparable to those purified antibodies may help insure of IF.58 However, the length of the pro- specificity. cedure and the expense of additional Finally, when a new commercial an- reagents make use of the PAP procedure tibody is purchased or antibody is made for frozen renal tissue a poor choice. available from other sources, checker- In the past few years, improved tech-

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••KMKMi ' V I f . r

v *

Fig. 9. Photomicrograph, IgA nephropathy, demonstrating confluent granular deposits of IgA in mesangial areas, (IMPAS X 160). niques of enzyme conjugation have re- plexes in paraffin-embedded tissue.63-65 sulted in better commercial antibodies. Similar results can be obtained with Excellent reagents can be obtained from IMP with the use of enzyme-digested several commercial sources for identifi- sections.9'6''66 Maclver et al9 have sug- cation of immunoglobulins and comple- gested that inconsistent detection of ment components. Three recent large complement in previous studies may be series have described IMP results that a function of over trypsinization of tissue compare favorably with IF performed sections. These investigators demon- on the same cases.59-61 Immunostained strated clear separation of granular and frozen sections can be counterstained linear staining patterns, precise locali- with periodic acid Schiff to locate more zation of deposits within glomeruli using precisely sites of antigen deposition (Fig. posttrypsinization IMP, and a concord- 9)™ ance rate with IF of 81%. Optimum Paraffin-embedded renal tissue, conditions for detection of complement whether fixed in formalin or precipita- were shown to be 0.05% trypsin for 40 tive fixatives under the best of condi- minutes, a concentration also sufficient tions, may not be adequate for demon- to detect immunoglobulin heavy chains stration of extracellular immune com- and Clq. plexes or autoantibodies with standard For the present time, fresh tissue immunohistochemistry. Since trypsin should still be used as the tissue of choice and pronase have been shown to en- for renal immunomicroscopy. With the hance detection of tissue antigens, re- availability of excellent commercial cent attempts have been made to use antibodies and well-established tech- enzyme pretreatment for IF or IMP de- niques, the direct IMP procedure is tection of extracellular immune com- probably the technique of choice. It is

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Fig. 10. Photomicrograph, B-cell immunoblastic sarcoma arising in plasmacytoid lymphocytic lymphoma, lymph node. The neoplastic cells (brown-black cytoplasm) are immunostained for kappa light chains. A serial section was negative for lambda light chains. Direct immunoperoxidase technique, counterstained with hematoxylin and eosin, (X 400). Fig. 11. Photomicrograph, beta cell pancreatic apudoma. The neoplasm and adjacent normal islets contain immunoreactive insulin. Unlabeled PAP technique was used employing aminoethylcarbozole as the chromogen, (X 64). not clear at this time whether trypsin- forms of glomerular disease, since in the ized deparaffinized paraffin-embedded hands of some investigators, enzyme tissue will be acceptable as an immu- pretreatment yields variable tissue nomicroscopic preparation for most digestion and inconsistent immuno-

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Fig. 12. Photomicrograph, double label study using the unlabeled PAP technique demonstrating glucacon (black) and insulin (red) within the same pancreatic islet, (X 160). Fig. 13. Photomicrograph, bladder biopsy. Superficial transitional cell carcinoma at right extending over the residual nonneoplastic urothelium does not express blood group A antigen. Endothelial cells, erythro- cytes, nonneoplastic urothelium express blood group A antigen (brown-black). Indirect immunoperoxidase technique, counterstained with hematoxylin and eosin, (X 160).

staining patterns due to poorly under- Lymphoproliferative diseases stood mechanisms that may include formation of antibody or protein moities of Immunohistochemistry has contrib- different antigenicity.44 uted greatly to our understanding of the

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. Summer 1981 Enzyme immunohistochemistry 259 organization of the immune system and contributed both to understanding and to architectural and functional altera- confusion regarding this group of enti- tions in its various components in a ties. Thus, while documenting the pres- variety of disease states. The technology ence of monoclonal cytoplasmic immu- of immunohistochemistry has evolved noglobulins in many non-Hodgkin's parallel with increased knowledge about lymphomas,14'67"72 these techniques as lymphoid neoplasia and has contributed applied to paraffin-embedded tissues significantly to the understanding of the have yielded polyclonal staining of B- nature of proliferating cells of malignant cell lymphomas in some reports.14,68 lymphoma. Immunohistochemistry of Such observations are not in agreement malignant lymphomas will eventually with the clonal premise upon which play a role similar to that of immuno- most cancer immunology is based.23'73"70 microscopy in evaluating renal disease. Studies emphasizing the immuno- Paradoxically, these techniques have logic basis for classification of lympho-

HYPER1MMUNIZED MICE

SPLEEN I EMOVED

MOUSE PL ASM AS Y TOM A ESTABLISHED CELL CULTURE

IMMUNOBLASTS IN CELL SUSPENSION SUSPENSION OF CULTURED MOUSE FROM SPLEEN OF HYPERIMMUNIZEO MICE MYELOMA CELLS

POLYETHYLENE GLYCOL SELECTIVE CULTURE MEDIA MOST CELLS DIE 4 A FEW FUSED CELLS SURVIVE

FUSED CELL SUBCULTURES

ESTABLISHED HYBRIDOMA CELL LINES PRODUCING ULTRASPECIFIC MONOCLONAL ANTIBODIES

MONOCLONAL Ab. MONOCLONAL Ac, MONOCLONAL AD,

Fig. 14. Diagram of procedure used to obtain monoclonal hybridoma antibodies. Immunoblasts from hyperimmunized mice are fused with cultured mouse plasmacytoma cells in the presence of polyethylene glycol. Although most of the cells die, a few cells survive which contain the genetic content of both the stimulated immunoblasts and mouse myeloma cells. The fused cells are subcultured and cloned, reinjected into mouse peritoneal cavity, and ultraspecific monoclonal antibody harvested as ascitic fluid.

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Fig. 15. Photomicrograph, reactive lymphoid hyperplasia, lingual tonsil. Red staining interfollicular T helper/inducer lymphocytes are identified using aminoethylcarbazole as the chromogen. Biotin-avidin "ABC" technique using mouse monoclonal hybridoma primary antibody specific for inducer/helper T lymphocytes, with methylene blue counterstain, (X 64). Fig. 16. Photomicrograph, malignant mixed germinal neoplasm of testis. Neoplastic syncytia trophoblasts are immunostaincd for chorionic gonadotropin. Indirect immunoperoxidase technique using mouse monoclonal hybridoma primary antibodies specific for beta subunit of human chorionic gonadotropin, peroxidase conjugated affinity purifed goat anti-mouse IgG, and hematoxylin and eosin counterstained, (X 160).

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. Summer 1981 Enzyme immunohistochemistry 261 mas attempt to draw parallels between sis, data of distinct prognostic signifi- the components of the immune system cance are obtained for subsets of lym- and the morphologic diversity of non- phomas.79 However, immunohisto- Hodgkin's lymphomas. Of the several chemical study of paraffin tissue from different classifications available for patients with multiple myeloma has subtyping of non-Hodgkin's lymphoma, shown phenotypic expression of both only the Lukes-Collins classification,69 kappa and lambda light chains in cells which is currently available is directly from patients with well-characterized dependent upon the identification of T- circulating monoclonal immunoglobu- and B-cell marker expression by the neo- lins.68 Thus, the initial enthusiasm for plasm. However, immunologic data can immunohistochemistry was tempered be added to the Rappaport morphologic by these apparent anomalous staining classification.70 patterns that violated basic concepts re- Initial functional characterization of garding monoclonality of B-cell neopla- lymphomas was done principally by cell sia• . 80 suspension (CS) studies.75 With the use A similar evolution of understanding of CS techniques, classification as to T, of immunohistochemistry as applied to B, or non-T/non-B origin can be ef- non-Hodgkin's lymphomas has been ob- fected in a relatively large number of served.81"104 The spurious immunostain- cases.73,74 In recent years, information ing patterns observed in a significant regarding the reliability of CS studies number of cases evaluated by paraffin- has accumulated. In a number of B-cell embedded immunohistochemistry lymphomas marking monoclonal with raised serious questions about the valid- cryostat frozen section immunohisto- ity of results obtained in this man- chemistry, polyclonality in CS has been ner.11'14,105 Initially, it was suggested that observed.76"78 This apparent discrep- such results were a consequence of di- ancy may be due to several factors, most clonal immunoglobulin production by likely selective loss of tumor cells or the non-Hodgkin's lymphoma,14 an ex- sampling error resulting in contamina- planation that is not in concurrence tion of the suspension with nonneoplas- with the overwhelming body of evidence tic lymphocytes, particularly in nodular for monoclonality in human B-cell lym- lymphomas in which a significant per- phomas.23,106"108 centage of the lymphoid parenchyma When results of frozen section and may be spared by the neoplasm. paraffin-embedded immunohistochem- When the sensitive PAP immunohis- istry are compared, it becomes clear that tochemical technique developed by the negative or spurious immunostain- Sternberger was initially applied to lym- ing patterns associated with paraffin- phoproliferative disorders, the use of im- embedded tissue are probably a conse- munologic markers to characterize non- quence of processing.11'12,24,28 Currently, Hodgkin's lymphomas was viewed as an cryostat frozen section immunohisto- academic curiosity rather than a clini- chemistry (CFSIH) provides the most cally useful tool by many pathologists. sensitive and specific procedure for de- Recent evidence suggests that when tection of monoclonal cell populations, non-Hodgkin's lymphomas are ap- since most non-Hodgkins lymphomas proached from the standpoint of Lukes- demonstrate monoclonal immunostain- Collins classification and interpreted in ing with CFSIH.11,15'23,25 However par- conjunction with surface marker analy- affin-embedded techniques provide su-

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. 262 Cleveland Clinic Quarterly Vol. 48, No. 1 perior morphology in the minority of chemical phenotypes can be identified cases that mark in monoclonal fashion for most large cell lymphomas.127"129 (Fig. 10). Optimally, both frozen and Recognition of neoplastic prolifera- paraffin-embedded tissues should be tions of true tissue macrophages is per- evaluated for each case. CFSIH is also haps the most difficult diagnostic chal- helpful in delineating physiologic do- lenge at present. Much of the current mains of lymphoid subpopulations, de- problem is a consequence of various fining the nature of nonneoplastic morphologic criteria used and the arbi- lymphoid infiltrates, and in detecting trary distinctions that have been estab- malignant cellular populations in his- lished to distinguish between malignant tologically reactive lymph nodes.109"111 histiocytosis and histiocytic lymphoma Immunoglobulin negative non-Hodg- of true tissue macrophage origin. True kin's lymphomas consist of unusual lym- tissue macrophages with maturation phomas of T-lymphocytic and dediffer- will express alpha-naphthyl acetate es- entiated B-lymphocytic origin, true null terase activity in diffuse pattern, and lymphomas of non-T non-B cytogenesis, are associated with the expression of and neoplastic proliferations of true tis- muramidase and alpha-1-antitrypsin, la sue macrophages. More precise charac- antigen, and polyclonal cytoplasmic terization of these unusual lymphoma immunoglobulins.130"137 However, in subtypes can be accomplished by iden- poorly differentiated variants of malig- tifying immunohistochemistry profiles nant histiocytosis, the neoplastic cells of lymphocyte differentiation antigens may not express these proteins.134,135 The and elaborated substances (Table 2). most definitive evidence for tissue mac- The growing availability of hybridoma rophage origin will be the identification monoclonal antibodies specific for lym- of antigens peculiar to monocy te/mac- phocyte subpopulations is increasing the rophage differentiation on neoplastic accuracy with which determinations of cells with the use of monoclonal anti- cytogenesis are made.112 Differentiated bodies. B-lymphocytic lymphomas are charac- Terminal transferase, an enzyme terized by monoclonal surface mem- present in lymphoblasts but absent in brane-associated immunoglobulin (CS myeloblasts, may be helpful in subtyp- or CFSIH), cytoplasmic immunoglobu- ing the acute leukemas.138,139 Both bio- lins as detected by immunohistochem- chemical and IF techniques are cur- istry procedures on paraffin-embedded rently used to identify the enzyme. Re- tissues in some cases, la and la-like an- liable immunohistochemistry methods 11,113 118 tigens, and J piece expression. " for in situ demonstration of terminal Recognition of T-cell lymphomas has transferase in tissue have not been de- been previously based upon cytochemi- veloped to our knowledge. Results of cal expression of acid alpha-naphthyl initial studies of a cross-reacting anti- acetate esterase or acid phosphatase ac- body that preferentially immunostains 119 tivity in neoplastic cells. However, myeloblasts in tissue suggest that this monoclonal hybridoma antibodies mon- marker may also be helpful in subtyping ospecific for subsets of T lymphocytes leukemias.14 are now available and are helpful in the Since the monoclonal nature of B-cell recognition of these unusual lympho- 120 126 non-Hodgkin's lymphomas has been mas. " With the use of monoclonal well established, use of objective meth- antibodies and CFSIH, immunohisto- ods has been advocated to distinguish

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Table 2. Immunohistology of lymphoproliferative disorders

OKT3 OKTS OKT4 Ig JP Ia Pan Sup Ind Reactive hyperplasia Follicles

Light zone PC + + OC - OC

Dark zone PC + + -

Sinus PC, E - +

Paracortex OC, DC - OC • • • Non-Hodgkin's lymphomas

B cell lymphomas MC + V - - -

With Fc receptors E + V - - -

T cell lymphomas - - V + + * +*

With Fc receptors E - V + + * +* Dedifferentiated B cell

Lymphomas - - + - - -

With Fc receptors E - + - - - Hodgkin's disease

Plasma cells PC + - - - -

Reactive tissue macrophages PC - + - - -

Neoplastic cells PC - NWE NWE NWE NWE

Abbreviations: Ig = surface and/or cytoplasmic immunoglobulins (best assessed with cryostat frozen section immunohistochemistry). JP = J piece, OKI = monoclonal antibody specific for la antigen, OKT3. Pan = monoclonal antibody specific for all peripheral blood T lymphocytes, OKT8. Sup = monoclonal antibody specific for suppressor/cytotoxic T lymphocytes, OKT4. IND = monoclonal antibody specific for inducer/helper T lymphocytes, PC = polyclonal (both k and A light chains present), + = present, — = absent, OC = occasional cells, DC = dendritic cells, NWE = not well established, V = variable, MC = monoclonal (only one light chain present, either k or À), Fc = receptors for Fc portion of immunoglobulin), E = surface-associated immunoglobulin which can be eluted with acidic buffer. * T cell malignant lymphoproliferations react with either T Supp. or T inducer/helper monoclonal antibody, depending on differentiation of the neoplasm.

reactive lymphoid hyperplasias from macrophage origin. Immunohistochem- non-Hodgkin's lymphomas by charac- istry studies by Taylor14 and others145" terizing surface immunoglobulin phe- 148 showed that Reed-Sternberg cells notypes. " Since CS may yield spu- contain polyclonal IgG. These observa- rious results in non-Hodgkin's lympho- tions are consistent with a tissue mac- mas, possibly due to contamination with rophage origin, the cytoplasmic immu- nonneoplastic populations or selective noglobulin probably representing en- loss of tumor cells, CSFIH should be gulfed exogenous polyclonal immuno- used to help determine the biologic po- globulin. Tissue culture cell lines de- tential of the lymphoproliferative dis- rived from Hodgkin's disease display orders.141"144 cytochemical and immunologic features The origin of Reed-Sternberg cells in of macrophages, and when transplanted Hodgkin's disease has been the subject into experimental animals produce tu- of extensive debate. For years the pref- mors with morphology resembling 149 erential involvement of the lymph node Hodgkin's disease. Furthermore, CS sinus and interfollicular zone and de- from tissues involved by Hodgkin's dis- fects in cellular immunity were inter- ease contain Reed-Sternberg cells that preted as evidence for T-cell or tissue have polyclonal cytoplasmic IgG, and

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actively bind and internalize labeled ex- tory study.155'156 Study of the pituitary ogenous immunoglobulins.150 Although tissue of acromegalic patients has docu- a tissue macrophage origin of Hodgkin's mented the presence of growth hormone disease appears likely, definitive evi- within the neoplastic tissue, an obser- dence could be obtained with immu- vation corroborating radioimmunoas- nohistochemistry staining with the use say results.157 However, Fukaya et al157 of lymphocyte-monocyte differentiation have also demonstrated occasional im- monoclonal antibodies. To our knowl- munoreactive cells positive for prolactin edge, such a study has not been done. and luteinizing hormone in an adenoma removed from a patient with acromeg- Endocrine systems aly, raising questions about the signifi- IMP procedures have contributed cance of identifying other hormones greatly to our understanding of the C- within a particular tumor. Such immu- cell neoplasms and preneoplastic state noreactive cells may represent residual of the thyroid gland. Wolfe et al151 have normal pituitary tissue. Conversely, delineated the distribution of C cells in these observations may suggest that pi- the normal gland and in the thyroid tuitary adenomas are polyclonal neo- gland of patients at risk for hereditary plastic proliferations with secretion of medullary carcinoma. When evaluated one hormone dominating the clinical in this fashion, it has been shown that presentation. Recent cases of pituitary the middle and upper portions of the adenomas with ultrastructural and im- lateral thyroid lobes show marked in- munohistochemical evidence for heter- creases and clustering of calcitonin-con- ogeneous cell populations have been de- taining cells in this disease. The immu- scribed that were eosinophilic in tincta- nohistochemical demonstration of thy- torial differentiation but heterogeneous roglobulin within well-differentiated tu- in their elaboration of growth hormone 158 159 mors of the thyroid gland of both pap- and prolactin. ' It has been sug- illary and follicular types has been gested that at least some of these de- shown to be helpful in confirmation of scribed cases may involve technical thyroidal origin, since tumors of nonthy- problems and may not actually repre- 160 roidal histogenesis examined did not sent a stem-cell neoplasm. Immuno- show thyroglobulin synthesis.152'153 histochemistry may prove to be espe- cially helpful in the confirmation of hor- Immunohistochemistry studies have monal homogeneity in small specimens been helpful in evaluation of both nor- of tissue removed as microadenomas mal and neoplastic pituitary tissue. It from patients with Cushing's syndrome. has been shown that adenomas of the Currently, this confirmation is based adenohypophysis, although they may be upon standard histochemical and clini- tinctorially homogeneous, are immuno- cal correlations.161 Study of pituitary histochemically heterogeneous.154' Im- adenomas occurring in association with munohistochemistry studies are helpful MEN I syndrome has confirmed that in delineating the presence of neoplastic most of the adenomas are of either pro- cells containing the hormone circulating in the patient. Thus, the documentation 162 163 T lactin or growth hormone type. ' In of prolactin in resection tissue from the the rate occurrence of pituitary carci- anterior pituitary of a patient with hy- noma metastatic to extracranial sites, perprolactinemia as assessed by ra- immunohistochemistry techniques may dioimmunoassay is a helpful confirma- offer confirmatory evidence for pituitary

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origin of the neoplasm.164 Of greater and the testis. With these techniques, interest is the immunoreactivity demon- both testosterone and estradiol were strable in chromophobe adenomas of identified in Sertoli-Leydig cells and in the adenohypophysis. These tumors are primitive spindled cells in these tu- usually characterized by multiple hor- mors.169"172 Estradiol was localized in monally positive cells for most of the granulosa cells and in luteinized theca hormones endogenous to the anterior cells, and nonluteinized stromal cells pituitary.165 Immunohistochemistry were negative for steroids.178"181 Immu- techniques may also prove helpful in the nohistochemistry techniques may thus characterization of "hypoplasias" or prove helpful in the subclassification of preadenomatous states.166 Other appli- ovarian tumors based upon the predom- cations of immunohistochemistry in the inant hormone elaborated, and also in study of pituitary disease include the the confirmation of gonadal stromal or- delineation of decreased hormonal syn- igin when such neoplasms occur in an thesis or storage in gonadotrophs in pa- extragonadal location.180'181 167 tients with hemochromatosis. Confir- When carcinoid tumors are evaluated mation of elaboration of adenohypo- with immunohistochemistry techniques, physeal hormones by ovarian teratomas, positive immunoreactivity for multiple oat cell carcinomas, and other extrapi- hormones is usually identified.182 How- tuitary tumors may also be done with ever, somatostatin immunoactivity or- the use of immunohistochemistry tech- dinarily predominates and is usually as- 168-171 niques. sociated with immunostaining for other 182 The use of immunohistochemistry in hormones such as gastrin or calcitonin. evaluation of the endocrine pancreas Most of the tumors studied in this has yielded some interesting results fashion have been clinically silent with (Figs. 11, 12). In experimental animals, respect to hormonal elaboration. the distribution and relative frequency The emergence of immunohistochem- of different immunoreactive cell popu- istry techniques specific for prostatic lations have been documented with acid phosphatase have proved to be very IMP and IF.172 Human pancreatic en- helpful in the confirmation of prostatic docrine tumors are characterized by dis- origin of secondary metastasis.183"187 tinct ultrastructural features that allow Nadji et al188 have also recently shown subclassification of pancreatic apudo- that an antibody can be raised against mas.173 Immunohistochemistry tech- specific tumor antigens of prostatic car- niques have been used to identify the cinoma rather than the elaborated acid distribution of insulin-positive cells in phosphatase product. The antibody hyperinsulinemic hypoglycemia of in- thus derived is specific only for carci- fancy,174 to document the presence of noma of prostate histogenesis. hormones such as glandular kallikrein and cholecystokinin-pancreozymin Oncodevelopmental antigens within islet-cell populations175'176 and to Oncodevelopmental antigens are a profile immunohistochemically the hor- group of substances that are produced monal content of islet-cell tumors177 early in fetal life, but which disappear (Fig. 11). with fetal maturation. These substances Kurman et al178 180 have used immu- may reappear in the bloodstream in nohistochemistry to localize elaborated association with a malignant neoplasm steroid molecules in tumors of the ovary apparently through derepression of the

Downloaded from www.ccjm.org on September 30, 2021. For personal use only. All other uses require permission. 266 Cleveland Clinic Quarterly Vol. 48, No. 1 genes responsible for production of these been suggested as a useful diagnostic markers. It has been shown that the tool in the assessment of lung tumors, sensitive PAP technique is satisfactory since mesotheliomas have been shown to demonstrate the presence of human with immunofluorescence not to express chorionic gonadotropin (HCG) in pla- CEA, whereas bronchogenic carcinomas cental syncytial trophoblast and neo- are associated with CEA elaboration.214 plastic trophoblastic elements.189"191 Ra- Peeripheral serologic measurements of dioimmunoassay of beta subunit HCG CEA may be useful in monitoring re- is helpful in monitoring patients with sponse to therapy in patients with gestational trophoblastic and gonadal breast, stomach, and colorectal can- germinal neoplasia.192"196 Ideally, RIA cer.215-221 A distinction between benign baseline follow-up measurements are colonic mucosa and dysplastic or frankly used in conjunction with immunohisto- carcinomatous changes within the chemical study of initially resected tu- bowel mucosal tissue of patients with mor tissue. This approach permits a ulcerative colitis is theoretically possible precise morphologic and immunohisto- with CEA immunostaining, but staining chemistry characterization of the tumor patterns are inconsistent.222'223 It has for definitive subtyping, and suggests also been shown that IMP techniques which serum markers will be most useful are able to identify certain CEA-positive for therapeutic monitoring.197"200 The cervical carcinomas before ovarian CEA amount of choriocarcinomatous differ- concentration is elevated.224 Van Nagell entiation can be best assessed with im- et al225 have shown the usefulness of munohistochemistry for HCG and the immunohistochemistry identification of degree of endodermal sinus and em- CEA expression by ovarian cystadeno- bryonal differentiation best assessed carcinomas and follow-up serologic with a-fetoprotein immunohistochemis- measurements to assess therapeutic suc- try. These techniques may also be help- cess. The presence of CEA seems to ful in confirming germinal nature of correlate best with mucinous rather neoplasms occurring in an extragonadal than serous differentiation of ovarian location such as the intracranial neoplasms.226-228 201 202 vault. ' However, these markers are An antibody reacting with gp52, a by no means specific for germinal neo- 52,000-dalton glycoprotein of the mouse plasia since they may occur in a variety mammary tumor virus has been shown 203 211 of nongonadal neoplasm. " Al- to immunostain selectively breast carci- though perhaps of limited usefulness in noma cells, not reacting with the normal confirming a germinal origin for a par- breast tissue or other malignancies. ticular neoplasm, monitoring of markers Autoantibodies having specificity for may provide an indication of therapeu- the same virus have been shown to occur 212 in sera of breast cancer patients.230 tic success. Similar results have been observed for Blood group antigens carcinoembryonic antigen (CEA). The The expression of ABO blood group immunohistochemistry method works antigens by different human tissues has well for the detection of CEA in been recognized for many years. Normal tissue.213 However, the elaboration of urothelium, for example, expresses ABO this oncodevelopmental antigen by a antigen in agreement with the pheno- variety of neoplasms makes diagnostic type of the patient's red blood cells. In usefulness limited. CEA expression has

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most urothelial neoplasia, the capacity try techniques have been used to iden- to express the ABO blood group antigen tify the causative agent of non-A, non-B is lost as the neoplasm becomes more hepatitis in tissue that may also prove aggressive.231-234 In some superficial useful in IMP studies.246 transitional cell carcinomas of the blad- Antisera specific for certain parasites, der, the natural history of the disease is polyoma viruses, herpes virus, and var- not characterized by aggressive biologic icella/zoster virus have been effective in potential, and thus it would be useful to the retrospective identification of infec- have a means to identify this particular tious agents in tissue with the use of IF group of patients who require less ag- or IMP procedures.247"253' gressive therapeutic measures.232 With the mixed red cell agglutination test, as assessed by passive red blood cell im- Other antigens munoadherence, it is possible to identify Other antigens detected in tissue with neoplasms that retain the ability to ex- immunohistochemistry include the dis- press blood group antigens on the tumor tribution of laminin,254 ligandin,255 and cells. The expressin of ABO antigen on Factor VIII coagulation factor.256 Gli- neoplastic urothelium seems to indicate ofibrillary acidic protein can be used to a less aggressive biologic potential, i.e., characterize glial cell populations, and 231- the tumor does not become invasive. other intracranial mass lesions.267"260 234 Passive red cell immunoadherence However, an inverse relationship be- has also been used to identify secretion tween the degree of anaplasia and the of fetal blood group antigens on polyps intensity of immunostaining with anti- of the distal colon, and the loss of ABO GFA antibody has been observed; thus, isoantigen expression in histologically in poorly differentiated neoplasms, the benign lesions and in mammary carci- technique may have limited use- 235 236 noma. ' ABO antigens in tissue can fulness.258 Neoplastic Paneth cells have also be detected by indirect immuno- been identified with lysozyme antisera histochemistry (Fig. 13). in an unusual variant of gastric carcinoma,261 the presence of actin has been Infectious agents documented in meyloepithelial cells in Immunohistochemistry techniques the breast and other tissues of smooth 262 263 are readily adaptable to the detection of muscle cell origin, ' fetal red cells hepatitis-B surface antigen in fixed and identified in placental intervillous 264 frozen tissue.237"241 These techniques can thrombi, myoglobin in normal and 265 be used to confirm the presence of sur- neoplastic human skeletal muscle, ker- face and core hepatitis-B antigen in he- atin in a variety of normal human tis- 266 267 patic cirrhoses or hepatocellular carci- sues, ' and basement membrane an- nomas, although the relationship be- tigen has been demonstrated in Wilm's 268 tween the presence of the antigen and tumor. the pathogenesis of the disease remains Immunohistochemistry techniques problematic.241-244 Recently, it has been have also been shown to be useful in shown that hepatitis surface antigen has serologic studies to detect antinuclear an affinity for free and bound horserad- factor in serum,269 cell surface antigens ish peroxidase, an observation that man- in CS,270 thymus leukemia antigen dates strict use. of controls in the proce- expression on lymphoid cells,271 and an- dure.245 With IF, immunohistochemis- tilymphocyte antibodies.272

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The future of enzyme continue to grow. Immunohistochemis- immunohistochemistry try may be helpful in the initial immu- The future of diagnostic and investi- nologic characterization of such tumors gational immunohistochemistry will be before noninvasive scanning proce- influenced by several things. The role of dures.280'281 this specialized technique in the diag- Prolactin receptors have been profiled nostic surgical pathology laboratory will with the use of IMP methodol- increase substantially as the techniques 282 283 ogy, ' and the potential exists to de- become more widely accepted and as velop peroxidase-labeled histochemical more systems for application are devel- procedures for the detection of a variety oped. However, as the techniques be- of receptors including estrogen, proges- come more widely used, the necessity for terone, and testosterone. good controls and standardization of Perhaps the most exciting develop- reagents and techniques become even ment in immunohistochemistry relates » » 45,273 more important. to hybridoma technology. Since Kohler The federal government will un- and Milstein's284,285 reports of the suc- doubtedly play some role in the practi- cessful fusion of specific antibody pro- cal daily use of immunohistochemistry ducing cells in culture with tumor cell in clinical laboratories. Classification of lines, the applications of this particular benzidine as a carcinogen and subse- biotechnology have increased greatly.286 quent strict regulations governing its use Hybridomas are produced by the fusion have had considerable bearing on the of a myeloma tumor cell line main- use of this reagent in cytochemistry. As tained in tissue culture with cell lines outlined earlier in this paper, alternative derived from splenic immunoblasts re- chromogens are available. However, moved from hyperimmunized animals many chromogens currently in use in (Fig. 14). Selective tissue cultures yield the United States, other than benzidine cloned hybridoma cell lines that elabo- and fluorescent compounds have not rate monoclonal ultraspecific antibody. been fully investigated with regard to As long as the tissue cell lines can be their neoplastogenic potential. A frame- maintained in culture, production of a work for decision-making by the federal standardized antibody is guaranteed. government regarding potential human The fused cells may be inserted into carcinogens has been recently out- 274 mouse peritoneum where functioning lined. hybridoma plasmacytomas grow, and The immunohistochemistry detection ascitic fluid containing the monoclonal of specific tumor antigens associated antibody can subsequently be har- with one particular type of tumor offers vested. Monclonal antibodies have been great promise. Such antigens have been made with specificity for lymphocyte identified in association with ovarian differentiation antigens (Fig. 15), tu- carcinomas, melanoma, breast as mor specific antigens, «-fetoprotein, evaluated by an antigen cross-reacting CEA, beta subunit of human chorionic with mouse mammary tumor-related gonadotropin (Fig. 16), and many antigens, human cervical squamous cell other substances,276"279 and have con- 278 279 carcinoma, and mesothelioma. " tributed greatly to understanding of The role of external photoscanning lymphocyte maturation and immuno- with radiolabeled antibody tracers will pathology of neoplasia.287"305

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Summary 8. Murphy WM, Deodhar SD, Cawley LP. Use of horseradish peroxidase in identification of Immunohistochemical procedures serum antibodies and immune complexes. have contributed greatly to our under- Clin Chem 1973; 19: 1370-3. standing of disease processes and have 9. Maclver AG, Giddings J, Mepham BL. become a necessary tool in the evalua- Demonstration of extracellular immunopro- tion of many disease states. Initial de- teins in formalin-fixed renal biopsy specimens. Kidney Int 1979; 16: 632-6. tection systems utilized IF markers. En- 10. Huang SN, Minassian H, More JD. Appli- zyme immunohistochemical techniques cation of immunofluorescent staining on par- developed during the past decade have affin sections improved by trypsin digestion. circumvented many of the problems in- Lab Invest 1976; 35: 383-90. herent in IF procedures. 11. Warnke R, Pederson M, Williams C, Levy R. A study of lymphoproliferative diseases This paper outlines the technical comparing immunofluorescence with im- aspects and clinical diagnostic applica- munohistochemistry Am J Clin Pathol 1978; tions of enzyme-labeled immunohisto- 70: 867-75. chemistry. The availability of mono- 12. Warnke R. Alteration of immunoglobulin- bearing lymphoma cells by fixation. J His- clonal antibodies and the adaptation of tochem Cytochem 1979; 27: 1195-6. these reagents to immunohistochemistry 13. Taylor CR. Immunoperoxidase techniques. systems will contribute greatly to further Practical and theoretical aspects. Arch Pa- understanding of disease processes and thol Lab Med 1978; 102: 113-21. will have continued utility in clinical 14. Taylor CR. An immunohistological study of diagnosis. follicular lymphoma, reticulum cell sarcoma, and Hodgkin's disease. Eur J Cancer 1976; 12: 61-75. 15. Tubbs RR, Sheibani K, Weiss RA, Sebek References BA. Frozen section immunohistochemistry of 1. Coons AH, Creech HJ, Jones RN. Immu- malignant lymphomas using the direct im- nological properties of an antibody contain- munoperoxidase procedure (abstract). Lab ing a fluorescent group. Proc Soc Exp Biol Invest 1980; 42: 156. Med 1941; 47: 200-2. 16. Burns J, Hambridge M, Taylor CR. Intra- 2. Coons AH, Kaplan MH. Localization of cellular immunoglobulins: A comparative antigens in tissue cells. II. Improvements in study on three standard tissue processing a method for the detection of antigen by methods using horseradish peroxidase and means of fluorescent antibody. J Exp Med fluorochrome conjugates. J Clin Pathol 1950; 91: 1-30. 1974; 27: 548-57. 3. Nakane PK, Pierce GB, Jr. Enzyme-labeled 17. Arnold W, Kalden JR, VonMayersback H. antibodies: preparation and application for Influence of different histologic preparation the localization of antigens. J Histochem methods on preservation of tissue antigens in Cytochem 1966; 14: 929-31. the immunofluorescent antibody technique. 4. Sternberger LA. Immunocytochemistry. 2nd Ann NY Acad Sci 1975; 254: 27-34. ed. New York: John Wiley & Sons, 1978. 18. Miller HRP. Fixation and tissue preserva- 5. Wisdom GB. Enzyme immunoassay. Clin tion for antibody studies: A review. Histo- Chem 1976; 22: 1243-55. chem J 1972; 4: 305-20. 6. Suffin SC, Muck KB, Young JC, Lewin K, 19. Feltkamp VTM. Preparation of tissues and Porter DD. Improvement of the glucose ox- cells for immunohistochemical processing. idase immunoenzyme technic. Use of a tet- Ann NY Acad Sci 1975; 254: 21-6. razolium whose formazan is stable without 20. Davenport WD, Ball CR. Observation on heavy metal chelation. Am J Clin Pathol the results of specific histochemical tech- 1979; 71: 492-6. niques and empirical staining methods on 7. Boorsma DM, Streefkerk JG. Periodate or several tissue/organ types using a variety of glutaraldehyde for preparing peroxidase fixing fluids. Histopathology 1979; 3: 321-7. conjugates? J Immunol Methods 1979; 30: 21. Taylor CR. Immunohistological observa- 245-55. tions upon the development of reticulum cell

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175. Grube D, Maier V, Raptis S, Schlegel W. GF, Franchi L. Postradiotherapy biopsy of Immunoreactivity of the endocrine pancreas. prostatic adenocarcinoma: A light and im- Evidence for the presence of cholecystokinin- munohistochemical study (abstract). Lab In- pancreozymin within the A-cell. Histochem- vest 1980; 42: 132. istry 1978; 56: 13-35. 188. Nadji M, Tabei SZ, Castro A, Ming Chu T, 176. Ole-MoiYoi O, Pinkus GS, Spragg J, Austen Wang MC, Morales AR. Prostatic specific KF. Identification of human glandular kal- antigen. An immunohistologic marker for likrein in the beta cell of the pancreas. N prostatic neoplasms. Cancer. In Press. Engl .] Med 1979; 300: 1289-94. 189. Yorde DE, Hussa RO, Garancis JC, Pattillo 177. Nieuwenhuijzen Kruseman AC, Knijnen- RA. Immunocytochemical localization of burg G, Erutel de la Riviere G, Bosman FT. human choriogonadotropin in human malig- Morphology and immunohistochemically- nant trophoblast. Model for human chorio- defined endocrine function of pancreatic islet gonadotropin secretion. Lab Invest 1979; 40: cell tumors. Histopathology 1978; 2: 389-99. 391-8. 178. Kurman RJ, Andrade D, Goebelsmann U, 190. Nieumenhuijzen-Kruseman AC, VanLent Taylor CR. An immunohistological study of M, Blom AH, Lauw GP. Choriocarcinoma steroid localization in Sertoli-Leydig tumors in mother and child, identified by immu- of the ovary and testis. Cancer 1978; 42: noenzyme histochemistry. Am J Clin Pathol 1772-83. 1977; 67: 679-83. 179. Kurman RJ, Goebelsmann U, Taylor CR. 191. Sheibani K, Tubbs RR, Velasco ME, Ben- Steroid localization in granulosa theca tu- jamin S. Immunocytochemical identification mors of the ovary. Cancer 1979; 43: 2377- of human chronic gonadotropin (hCG) (ab- 84. stract). Lab Invest 1979; 40: 284A. 180. Taylor CR, Kurman RJ, Warner NE. The 192. Von Eyben FE. Biochemical markers in ad- potential value of immunohistologic tech- vanced testicular tumors. Serum lactate de- niques in the classification of ovarian and hydrogenase, urinary chorionic gonadotro- testicular tumors. Human Pathol 1978; 9: pin and total urinary estrogens. Cancer 1978; 417-27. 41: 648-52. 181. Maurer R, Taylor CR, Schmucki O, Hed- 193. Stepanas AV, Samaan NA, Schultz PN, Ho- inger CE. Extratesticular gonodal stromal loye PY. Endocrine studies in testicular tu- tumor in the pelvis. A case report with im- mor patients with and without gynecomas- munoperoxidase findings. Cancer 1980; 45: tia. A report of 45 cases. Cancer 1978; 41: 985-90. 369-76. 182. Dayal Y, O'Brian DS, Wolfe HJ, Reichlin 194. Schultz H, Sell A, Norgaard-Pedersen, Ar- S. Carcinoid tumors: A comparison of their ends J. Serum alpha-feto-protein and human immunocytochemical hormonal profile with chorionic gonadotropin as markers for the morphologic and histochemical characteris- effect of postoperative radiation therapy tics (abstract). Lab Invest 1980; 42: 111. and/or chemotherapy in testicular cancer. 183. Leav I, Savage P, Rule A, De Lellis RA, Cancer 1978; 42: 2182-6. Merk FB. Immunohistochemical detection 195. Javadpour N, Mclntire KR, Waldmann TA. of human prostatic acid phosphotase (ab- Human chorionic gonadotropin (HCG) and stract). Lab Invest 1979; 40: 267. alpha-fetoprotein (AFP) in sera and tumor 184. Jobsis Ac, DeVries GP, Anholt RR, Sanders cells of patients with testicular seminoma. A GT. Demonstration of the prostatic origin of prospective study. Cancer 1978; 42: 2768- metastases. An immunohistochemical 72. method for formalin-fixed embedded tissue. 196. Javadpour N. Serum and cellular biologic Cancer 1978; 41: 1788-93. tumor markers in patients with urologic can- 185. Nadji M, Tabei SZ, Castro A, Chu TM, cer. Human Pathol 1979; 10: 557-68. Morales AR. Prostatic origin of tumors. An 197. Javadpour N. Significance of elevated serum immunohistochemical study. Am J Clin Pa- alpohafetoprotein (AFP) in seminoma. Can- thol 1980; 73: 735-9. cer 1980; 45: 2166-8. 186. Skinner MS, Seckinger D. Evaluation of 198. Kurman RJ, Scardino PT, Mclntire KR, beta-subunit chorionic gonadotropin as an Waldmann TA, Javadpour N. Cellular lo- aid in diagnosis of trophoblastic disease. Ann calization of alpha-fetoprotein and human Clin Lab Sei 1979; 9: 347-52. chorionic gonadotropin in germ cell tumors 187. Manley PN, Mahan DE, Bruce AW, Kipkie of the testis using an indirect immunoperox-

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