Antigen Restoration-The "Hot" Revolution in Immunohistochemistry Jules M. Elias* Grove Educational Technologies, Portland, OR

Abstract formaldehyde. What aldehydes do is to denature tissue mac- High temperature epitope retrieval (HIER) methods re- romolecules, thus making tissue proteins that comprise the

store protein antigens thought to be inaccessible, and they majority of tissue antigens inaccessible to the primary an- have had a dramatically positive effect on immunohisto- tibodies used in IHC (2). chemistry. HIER's extensive use by different investigators has resulted in an array of modifications, most of which History enhance the original method. However, lack of standardiza- In the complete absence of moisture, even a temperature tion of these methods makes it imperative that users of of 10O0C does not suffice to denatllre tissue proteins, but HIER techniques be fully cognizant of the diversity of ap- boiling of small biopsies in physiologic (0.85-0.90%) so- proaches and their shortcomings. (The J Histotechnol dium chloride solution for 2-3 minutes prior to freezing in 24:193, 2001) liquid nitrogen results in rapid fixation. It soon became

apparent that substitution of formaldehyde solutions for sa- Key words: antigen retrieval, denaturation, fixation, heat line had additional immobilizing effect on tissue proteins induced epitope retrieval, low temperature antigen restora- (3). That approach to improving the cytological detail of tion, proteolytic enzyme digestion frozen sections has since been discarded because of the need

to preserve protein antigenicity. If high heat could hasten Introduction the denaturation of proteins by formaldehyde, it was

Immunohistochemistry (IHC) is a merger of 2 different thought that high temperature solutions should be able to disciplines: immunology and histology. IHC is routinely partially reverse these masking effects. Non-aldehyde con- used to determine whether a certain tissue expresses, or taining fixatives, on the other hand, particularly those con- equally important, does not express a particular antigen, and taining mixtures of alcohol and mineral acid, were not to determine the micro-anatomic cellular location of the thought to benefit from exposure to high temperature solu- antigen (1). tions prior to application of the primary antibody as protein

The goal of i~nmunohistochemists is to devise protocols denaturation is not as extensive as with aldehyde based that produce the greatest sensitivity without compromising fixatives. specificity. The quality of primary antibodies has an enor- It is common knowledge that the quality of IHC staining mous effect on specificity, but tissue fixation has the great- of paraffin sections is strongly dependent on real or poten- est impact on sensitivity of IHC methods. Proteins can be tial changes in epitope confirmation induced by aldehyde denatured by diverse means and, in practice, the most com- containing fixatives (4). Biochemical investigations on the mon fixative applied prior to paraffin embedding is form- interactions between formaldehyde and proteins carried out aldehyde. Unfortunately, neutral buffered formaldehyde by Fraenkel-Conrat et a1 in the middle of the last century (NBF) has a negative impact on sensitivity; it can severely showed that aldehyde-induced cross-linkages could be re- chemically mask antigens of interest and cause false nega- versed by heating at high temperatures or by treating with tive IHC staining. For several decades the common belief strong alkalis (5). Nearly a half century later, Shi et a1 was that antigen identification in routine paraffin sections applied these techniques to tissue sections, and demon- was significantly compromised by the masking effect of strated that pretreatment of sections immersed in heavy

metal solutions and heated in a microwave significantly increases the sensitivity of the IHC technique (6). They

' No reprlnts available This article IS part of a chapter In In~m~rnoh~s- their technique the retrieval" but ro~~nrhology A P~nctlcnl App~onch to D~ngrlos~s, 2nd edit~on by Jules M. Elias to be published by ASCP Press In the Wlnter of 2002 No portlon of

since then a number of other heating devices (eg, steamer, the article may be reproduced I" any manner. Copyright Is reserved to Dr. autoclave, hot water bath, Bunsen burner, pressure cooker) Ellas and ASCP Press. E-mall adag10l3@juno.com have been reported to achieve similar effects. The term

The Journal of H~stotechnology I Vol. 24, No. 3 1 September 2001 193

"heat induced epitope retrieval," or HIER, has become a widely accepted descriptive term.

Prior to the HIER revolution, proteolytic enzymes were the most popular approach to reversing the masking effects of aldehyde fixation. Although this approach has been largely replaced by HIER methods, some investigators have combined proteolytic digestion with HIER, achieving greater enhancement of signal than with either approach alone (7,8).

Target Amplification Using Proteolytic Enzyme Digestion Methods

In the past, frozen sections were initially used for IHC because routine tissue processing was believed to destroy protein antigens. ~iss'es stored in formalin for days or months were considered unsuitable for IHC analysis. In some instances, the deleterious effects of formalin fixation could be reversed to some extent by prolonged washing of tissues prior to their dehydration and embedding. Prolonged bathing of deparaffinized sections in fresh changes of a 10% sucrose-PBS solution or TRIS buffered solution may re- verse the antigenic masking of some protein antigens (9,10). Helander was able to restore i~nmunoreactivity of tissues or of sections by washing for several weeks in distilled water (DW), but immersion in a high pH solution with elevated temperatures accelerated this process (1 1).

Boosting IHC method sensitivity (the signal) has tradi- tionally been the way to cornpensate for the comprotnised irnrnunoreactivity of tissue antigens caused by fixation in aldehyde-containing fixatives. Because IHC is used to dis- tinguish antigenic differences between cells needed to de- fine the lineage of cell populations, a great deal of research was done in an attempt to increase the sensitivity of IHC to make this method compatible with paraffin sections. The discovery by Bums et al that immi~noglobulins present in plasma cell cytoplasm could be demonstrated in paraffin sections using peroxidase labeled antibodies encouraged others to use routinely fixed and embedded paraffin sections for IHC staining (12). The methods subsequently developed were all focused on improving detection by increasing the signal generated by IHC staining. The direct, indirect, bridge, biotin-avidin, and, the latest, tyramine signal ampli- fication methods were all directed at boosting the signal as manifested by enhancement of the final color reaction prod- uct.

An alternative approach to increase IHC sensitivity was proteolytic digestion to restore lost or compromised target antigenicity. Overfixation of tissue in formalin causes for- mation of excess aldehyde cross-linkage that reduces the penetration of reactants, thus causing false-negative stain- Ing.

In the 1980s, a number of different proteolytic enzymes, including trypsin, pronase, pepsin, and proteinase K were used to predigest routine sections to retrieve antigens masked by forrnaldehyde fixation (13-15). Finley and Pe- trusz showed that predigestion of formalin fixed tissue sec- tions by proteolytic enzymes could expose some antigenic epitopes (16). Proteolytic enzylne digestion of NBF fixed, paraffin embedded tissues raises their sensitivity to that of cold acetone fixed, paraffin embedded tissues. Protease di- gestion is believed to unmask imrnunoreactive sites altered by either fixation or paraffin or plastic embedding media (17-19).

The required duration of enzyme digestion for unmasking is inversely related to the length of fixation. Prolonged en- zyme digestion can result in either a reduction in specific staining or an increase in nonspecific false-positive staining (20). Cattoretti et a1 found that tissues fixed for longer than 24 hr are digested for 8 min sing protease type XIV, 0.05% (w/v) in PBS at 37°C instead of 4 tnin (7). These investi- gators adjusted enzynle treatment schedules by cross- titration of concentration and incubation time on opti~nally fixed specimens and with antibodies known to have a nar- row enzyme concentration range for retrieval.

Some proteolytic enzymes also digest bonds in the native proteins and rnay cause a reduction in intensity of staining or loss of immunoreactivity if the appropriate epitope is cleaved. Staining for S-100 protein, for example, is not ilnproved by trypsinization, and false negative results have been reported (2 1).

The improved IHC results obtained fro111 the use of pro- teolytic digestion expanded the number of n~onoclonal an- tibodies (MAbs) that could perform in routine paraffin sec- tions. Unfortunately, the cleavage sites of proteolytic enzymes are nonspecific, and the epitopes of interest may themselves be affected. In addition, the need for titration of the incubation time and/or enzylne concentration that is re- quired for different tissue types and fixation tirnes makes standardization of this technique impractical. Because each laboratory had to develop its own protocols, histotechnolo- gists were obliged to perform proteolytic digestion under controlled conditions of concentration and time. Some found that the enzymatic solution sho~ild be pre-warmed by la cement in a thermostatic water bath at 37% to lnaxinlize enzyme effectiveness, and that this temperature should be maintained throughout the entire procedure. Additionally, upon completion of digestion, sections should placed in 4°C PBS for 10 min to stop digestion (15,21-23).

Enhanced sensitivity rnay not always be desirable, how- ever, particularly in cases in which diagnosis and prognosis are based on sparse or absent tissue antigens. The higher CEA content of colonic adenocarcinorna compared with ad- enoma is a useful diagnostic discriminator, and the sparse or absent blood group antigen on turnor cells in transitional cell carcinoma of the bladder has important prognostic i n - plications. In these instances, boosting the signal may oblit- erate ~ ~ s e f u l distinctions.

Increasing Sensitivity by Unmasking Epitopes Although proteolytic digestion continues to have a role in

restoring lost antigenicity, its effectiveness is primarily l i~n- ited to formalin fixed material. Thus, there was a conscien- tious effort by Inany investigators to develop other methods to increase IHC sensitivity. For example, the combination of pronase digestion and the highly sensitive alkaline phospha- tase-anti-alkaline phosphatase (APAAP) method has per- mitted the detection of estrogen receptor (ER) in routinely processed breast biopsies (24). Others realized that enzymes have a limited range of effectiveness in paraffin sections and may cleave only surface protein loops (25). Further- more, most commonly used fixatives cross-link different regions of the protein and may hide but not necessarily abolish epitopes.

These considerations inspired new ways to restore any lost i~n~uunoreactivity due to the masking effect of formal-

Antigen Restoration in IHC 1 Elias

dehyde. The goal was to amplify the number of available binding sites of the target that otherwise would only be partially or totally inaccessible to the primary antibodies used in their detection.

The application of heat formed the basis of all antigen retrieval methods that included treatment of deparaffinized sections with microwaves, exposure to combined action of heat and pressure in a pressure cooker, and combined action of enzyme digestion and microwaves (8,26). HIER methods turned out to be equally as effective in formalin as well as B-5 fixed tissue and EDTA-decalcified bone massow biop- sies (27). HIER was also found to be essential for the de- tection of CD5, a marker used in the classification of low- grade B-cell lymphomas (28).

Target Amplification Using HIER Methods HIER methods are a continuation of attempts to increase

sensitivity of IHC methods by increasing the amount of target antigen available for binding by the primary antibody. It has proved to be superior for a significant percentage of the most commonly used antibodies, both polyclonal and monoclonal, to all previous attempts to restore antigenic epitopes masked by aldehyde containing fixatives. In the early 1990s, 2 independent groups published reports that unleashed the "hot" revolution. Their data demonstrated that high temperatures in an aqueous medium could dra- matically improve the detection of masked antigenic epit- opes, hence all the experiments with different ways of ap- plying moist heat (6,27,28). Pileri et a1 showed that in most instances, higher dilutions of primary antibody or high tem- peratures proved more effective than protease XIV by un- masking otherwise inaccessible epitopes (27). Later, Gillett et a1 placed multiple tissue core samples into boiling 0.01 I ~ M citrate buffer, pH 6.0, in a pressure cooker kept under 15 psi for 2 min to restore immunoreactivity of an array of tissue proteins (29).

Some investigators combined proteolytic digestion with high temperature to boost IHC sensitivity. Elias et a1 used a combination of pronase digestion and microwaves with the alkaline phosphatase enzyme labeled streptavidin technique to boost the detection of MIB-1 in breast tumor paraffin sections. Colnpared to either pretreatment used alone, this combination of antigen retrieval increased sensitivity up- ward to 30%, a significant difference for a prognostic marker (8).

Microwave use is not without problems, which include evaporation of buffers, revealing hidden epitopes (eg, nucleolar staining with L26), false positive cytoplaslnic staining with CD5, the small number of slides that can be heated in a single run (larger batches result in inconsisten- cies in staining), and the inevitable alteration in the mor- phology of particular tissues (eg, fatty and hypo-cellular tissues).

HIER: Buffers and pH Once the theory of irreversible alteration of epitopes by

formaldehyde fixation was challenged by the introduction of HIER methods, researchers found that dry microwave treatment is totally ineffective and the technique works only with sections immersed in aqueous, usually ionic, solutions. HIER underwent its own modifications, including use of citrate and other buffers instead of heavy metal solutions,

The Journal of Histotechnology I Vol. 24, No. 3 1 September 2001

alteration of pH, and use of lower, non-boiling tempera- tures. Elias et a1 developed a low temperature antigen res- toration (LTAR) ER method to prevent loss of sections exposed to high temperature HIER methods (30). They have has since determined that LTAR provides the same benefit with hypocellular biopsies (eg, needle biopsies) (unpub- lished report).

Selection of the appropriate fixative to maximize HIER was also considered to be of prime importance to the suc- cess of HIER methods (6,7,3 1). According to Cattoretti et a1 the immunoreactivity of some antigens are selectively un- masked by exposure to hot aqueous solutions of citrate or carbonate ions, whereas other antigens fare better with a protein denaturant such as urea; others benefit from a mix- ture of citrate and us& (7). Aluminum chloride is an effec- tive retrieval agent due to its high ionic strength relative to monovalent metal salt solutions (32). However, elevated temperature is probably the most critical factor as preheat- ing of sections at high temperature using deionized water was found to be adequate.

The use of HIER methods has altered our understanding of the effect of chemical denaturation of antigen epitopes. The loss of antigenicity is not due as much to chemical alteration of antigen epitopes but is more commonly a prob- lem of inaccessibility (33). Within a given tissue, the exact number of intact epitopes present before fixation and the number of intact epitopes remaining after fixation are un- known (34). As demonstrated by the use of HIER methods, the reactive epitopes were always there in the cells, but were just inaccessible (34,35). Investigators determined that the use of EDTA-NaOH solution (pH 8.0) or TRIS buffer (pH 8.0-9.0) for retrieval is an effective way to restore some tissue proteins to greater levels than the Inore widely used citrate buffer (pH 6.0-7.0)(8,34,35). Pileri et a1 found EDTA (pH 8.0) to be optimal for 55 of 61 antibodies; TRIS- HC1 buffer was less efficient than EDTA, but often superior to citrate buffer (27).

All these differences were independent of the heat source, nature of the antibodies (polyclonal vs monoclonal), and nlost importantly, type of fixation (formalin vs B-5). 111

particular, EDTA provided superior results with all nuclear antigens tested, including Ki-67, Bcl-6, p53, and Rbl (27). Interestingly, differences in the risk of false positive p53 i~nmunostaining using the identical clone (DO7) may be due to the difference in retrieval solutions; 10 mM citrate buffer (in DW, pH 6.0) did not induce false positive staining, whereas a commercial unmasking fluid containing 35% urea did cause false positive staining (36,37).

Apparently, both the composition and pH of the buffer are critical factors in the success of HIER methods. Werner et a1 showed that MIB-1 and ER MAbs had dramatic de- creases in staining intensity with buffers at pH values be- tween 3.0 and 6.0. They maintain that for the majority of antibodies, pH 8.0-9.0 is optimal for TRIS-HC1 and pH 6.0-7.0 for citrate buffers (34). In contrast, Morgan et a1 rnaintain that a chelating agent (EDTAIEGTA) at a mildly alkaline pH (8.0-9.0) is suitable for retrieving most antigens in forrnalin fixed tissues (32).

The variation in buffers, pH, temperature, and heat sources as used in HIER methods by different laboratories precludes establishing a single, standardized protocol for performing this pretreatment step. When 15 different labo-

ratories assessed the effect of heating time on HIER for ER using antibody ID5 on paraffin sections of breast cancers, the results suggested that each laboratory must determine its optimal time for HIER and should not rely on published reports or information provided in the technical sheets ac- companying commercial antibodies (38). Nonetheless, cer- tain general principles listed in Appendix 1 apply to all microwave methods, the pretreatment most widely used. See Appendix 2 and 3, respectively, for general procedure for pressure cooker and autoclave techniq~ies. In addition, a test battery as devised by Shi et a1 should be performed for optimal retrieval protocols (Appendix 4) (39).

Mechanism of Action of Microwaves The mechanism of HIER microwave antigen retrieval is

not fully understood, but exposure to heat is central to all HIER methods. Exposure of dipolar molecules such as wa- ter and the polar side chains of proteins to the rapidly al- ternating electromagnetic fields results in instant heat, which increases molecular kinetics and hastens molecular reactions (40). Heat pretreatment significantly reduces the threshold at which a given antigen can be detected. For example, without the use of microwave antigen retrieval, Ki-67 antigen immunoreactivity is confined ti mitotic cells using MIB-1 antibody, a member of the Ki-67 family of proteins. However, the HIER method unmasks Ki-67 irn- munoreactivity at phases of the cell cycle where absolute levels of this protein antigen are too low to be otherwise detected (41). Because Ki-67 is categorized as a prognostic marker, underestimation of the labeling index of a tumor may compromise treatment decisions. Cattoretti and Suur- meijer suggest that the combination of heat and hydrolysis may denature and break the cross-links between adjacent amino acids due to exposure to formalin (42).

Because NBF continues to be the predominant fixative in surgical pathology laboratories, HIER methods have been widely incorporated into IHC staining protocols, particu- larly those that make use of the microwave/citrate buffer method of antigen retrieval.

In addition to heat, the pH of the retrieval solution, the type of retrieval solution, and the role of calcium ions or other divalent metal cations must be considered when de- vising a HIER protocol for a particular target antigen (36,43). Calcium ions are the most abundant ions in tissues, and formaldehyde may form crosslinks between calcium ions and oxygen atoms of both side chains of glutamate or aspartate residues as well as the carboxyl groups of poly- peptide chains (44). The use of B-5 fixative (which contains ~nercuric chloride) for lymph node biopsies is essential to retention of optimal morphology without severely compro- mising lymphocyte membrane markers. Divalent metal ions may complex with proteins during fixation, making chela- tion with EDTAIEGTA in the antigen retrieval solution es- sential to the release of these ions from proteins (44).

Although heating from 70-90°C has no adverse effect on antigenicity of formalin fixed target proteins, the chronic loss of fatty tissue samples (eg, breast) poses a significant limitation on accurate scoring of steroid receptors, onco- genes, and other biomarkers in breast tissue. he Elias et a1 LTAR pretreatment step enabled them to sustain the same level of antigen restoration obtained with conventional HIER methods (30). Koophal et a1 achieved optimal stain-

ing with preservation of tissue and cell morphology by heat- ing overnight at lower temperatures (45).

Summary The goal of HIER pretreatment protocols is to recover

sufficient epitopes to reach the threshold of IHC detectabil- ity. Recent attempts to standardize HIER for MIB-1 may induce others to pursue more uniform approaches to restor- ing lost immunoreactivity of other tissue proteins subjected to aldehyde or precipitating fixatives (46). With some tissue proteins, HIER decreases immunoreactivity, and different combinations of antigen retrieval methods must be used on tissue known to contain sufficient amounts of the desired antigen to determine the best protocol. The type of antigen retrieval used, enzymatiic or HIER, is in some way related to the antigen: there are antibodies that detect antigen exclu- sively on slides pre-treated with only one of these retrieval methods. Optimal results using any unmasking method also depend to a large extent on the length of fixation; best results are achieved using cold (4"C), freshly prepared formaldehyde, and fixation for less than 24 hr.

Although it is useful to follow the reco~nmendations of others in terms of the optimal unmasking method, the out- come of immunostaining after HIER is often unpredictable. As the permutations for HIER methods are numerous, labo- ratories should experiment with protocols to determine the benefits and disadvantages very carefully. The manual on antigen retrieval methods by Shi and colleagues should be consulted for a comprehensive overview of all aspects of this vital topic (47).

Appendix 1. HIER Basic Procedural Steps Using Microwave Heating

Determine optimal pH of antigen retrieval solution for each antigen and batch slides according to most desir- able pH. In genesal, buffers at pH 8.0-9.0 give the best results.

@ Determine desired temperature based on the type of tissue sections. Fatty tissues will lift off at boiling tem- perature, so lower 90°C temperature is recommended, with adjustment of the microwave time.

@ Place antigen retrieval solution containing slides in the center of the microwave (in the center of a rotary plate to ensure unifor~n heating of slides), and cover with a loose fitting cap. Turn 011 microwave and check temperature of the re- trieval buffer with a temperature probe. Use maximum power setting; a power setting from 7-10 is recommended. Begin timing antigen retrieval time when the buffer begins to boil. Use retrieval time previously deter- mined with sections known to contain the protein of interest. After 5 minutes of n~icrowaving, check buffer level and add fresh heated buffer from an adjacent jar. Be sure to keep slides fully immersed in the buffer before restarting oven.

e Repeat above step as often as needed. Maintain the level of retrieval fluid by adding DW following each cycle. Remove jar from the oven, and cool sections at room temperature for 20 min.

Antigen Restoration in IHC I Elias

@ Rinse several times in 0.5M PBS (pH 7.5). D o not reuse retrieval buffer.

Appendix 2. HIER Basic Procedural Steps Using Pressure Cooker Heating

@ Use approximately one-third capacity of a pressure cooker volume for antigen retrieval fluid.

@ Bring retrieval solution to a boil using an electric hot plate, without sealing the lid.

@ Place metal racks containing rehydrated sections into boiling retrieval solution.

@ Seal pressure cooker and bring to fill1 pressure. Begin t iming when the pressure indicator valve reaches maximum.

@ Determine optimal autoclaving time by using different heating times (1 min, 1.5 min, 2 min).

@ Depressurize and cool pressure cooker under running water before removing the lid.

@ Wash sections in several changes of 0.05M PBS (pH 7.5) to avoid drying.

Appendix 3. HIER Basic Procedural Steps Using an Autoclave (48)

@ Immerse deparaffinized sections in antigen retrieval fluid previously heated to 80°C.

@ Using a stainless steel autoclave equipped with a 1850 W heating filament, autoclave at 120°C for 10 min at 15 psi. Cool in running water for a minimum of 15 min.

@ Transfer sections into 0.05 M PBS at room tempera- ture.

Appendix 4. Recommended Test Battery for Optimal Retrieval Protocols as Devised by Shi et a1 (39)

Ten tissue sections are used, with 1 slide without antigen retrieval reserved for the control. Either TRIS-HCI (ph 7.0- 8.0) or citrate buffer (pH 6.0) may be used.

Table

Temperature"' pH 1.0 pH 6.0 pH 10.0 Very high @ 120°C Slide #I Slide #4 Slide #7 High @ 100°C

for 10 min Slide #2 Slide #5 Slide #8 Mid-high @ 90°C

for 10 ~ n i n Slide #3 Slide #6 Slide #9 :':120°C can be achieved by either microwaving for a prolonged time or by autoclaviny. :':90°C can be reached by a water bath or microwave monitored by a teiiiperature probe.

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Antigen Restoration in IHC 1 Elias