REVIEW ARTICLE |
Correspondence to: Clive R. Taylor, Dept. of Patholgy, University of Southern California School of Medicine, HMR 204A, 2011 Zonal Ave., Los Angeles, CA 90033.
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Summary |
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The antigen retrieval (AR) technique, which is predominantly based on high-temperature heating of tissues, is used as a non-enzymatic pretreatment for immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections. It has been widely applied in pathology and analytical morphology. The existence of a growing body of literature on the AR technique raises a number of interesting issues for the further development of AR. These issues include the use of a "test battery" and the concept of "maximal retrieval" applied to the selection of optimal test protocols for the standardization of AR. (J Histochem Cytochem 45:327-343, 1997)
Key Words: antigen retrieval, immunohistochemistry, microwave, formaldehyde, tissue fixation, antigen-antibody recognition, protein conformation
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Introduction |
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Formaldehyde, originally discovered to be a tissue fixative by Ferdinand Blum in 1893, has been used widely to the present day (
The recent development of IHC has provided a revolutionary approach to histopathology in terms of analytical and functional morphology. However, widespread application of IHC has raised a critical issue concerning traditional formalin fixation because most antigens under investigation are influenced significantly and adversely by formalin fixation (
The development of the hybridoma technique was a major contribution, serving to facilitate the large-scale manufacture of many highly specific monoclonal antibodies (MAbs) (
With the recognition that formalin fixation causes major chemical changes in antigens by unknown mechanisms, several attempts were made to modify the methods of fixation, and a variety of modified fixatives have been proposed for preservation of certain antigens (
The worldwide use of the AR-IHC has, however, raised some questions concerning the methodology of heating, the factors that influence the AR-IHC, the variety of AR solutions used for heating the tissue sections, limitations and possible false positive staining, and the mechanism of AR. The purpose of this review article is to address several critical issues concerning AR-IHC, including the factors that influence the AR-IHC. Proposals for standardization of AR-IHC through the use of a "test battery" with the concept of "maximal retrieval" are also described, together with our hypothesis of the mechanism of AR.
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Recent Developments |
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Methodology
The Effect of Heating.
The fact that high-temperature heating is the most important factor for retrieval of antigens masked by formalin fixation was demonstrated by
Recently, we conducted a test to demonstrate that an optimal result of AR-IHC is correlated with the product of heating temperature (T) x time of AR heating treatment (t); "T x t" means the heating condition. For example, the lower the temperature of heating used for AR, the longer the time required to reach the same intensity of AR-IHC compared with that obtained by higher-temperature heating. Our test was based on AR-IHC of MAb MIB1 in archival paraffin sections, and obtained the same strong intensity by the following heating conditions: 100C for 20 min, 90C for 30 min, 80C for 50 min, and 70C for 10 hr (
The Effect of pH.
Another important factor is the pH of the AR solution (
We conducted a study concerning the influence of pH on AR-IHC and observed three distinct patterns of staining: stable type (which changed slightly with pH); V-form type [which gave the best results at extremes (high or low) of pH], and ascending type (which improved with increasing pH). This study was performed using seven buffer solutions. The pH value achieved proved more valuable than the chemical composition of the buffer solution (
The Effect of Molarity.
The Effect of Metal Ions.
The chemical composition of the AR solution may also play a role in AR-IHC. The use of metal salt as the AR solution was based on earlier studies that used zinc-formalin fixation of tissue for better preservation of antigenicity (
The AR Solution.
Although the chemical component of AR solution may play a role as a possible co-factor in the heating procedure, thus far no single chemical has been identified that is both essential and best for AR. To determine the ideal AR solution, we recommend using a "test battery," because different antigens may require different conditions for retrieval (
Application
Demonstrated Broad Utility in Surgical Pathology.
It has been demonstrated that the AR technique is a simple and effective enhancement method for immunohistochemistry on archival tissue sections. Two major advantages of AR-IHC are (a) reduction of the detection thresholds of immunohistochemical staining (increased sensitivity) for a wide range of antibodies, and (b) retrieval of some antigens, such as Ki-67, MIB1, ER-1D5, bcl-2, androgen receptor, RB, CD5, CD8, CD35, CD38, CD25, CD45R, CD34, CD68, CD44, CD55, VCAM-1, CGA7 (smooth muscle-specific actin), CD19, and thrombospondin (
Demonstrated Utility of AR-IHC in Analytical Morphology.
Recently, the use of AR-IHC in neuroscience, including anatomy and pathology of the central nervous system, has become an attractive field (
The use of the MW heating AR technique for successful immunodetection of androgen receptor was developed in our laboratory (
Other Applications of AR.
For immunoelectron microscopy, the AR technique has successfully been applied by using the MW heating AR method either after etching the grids with a 10% fresh saturated solution of sodium ethoxide diluted with anhydrous ethanol for 2 min or with a saturated aqueous solution of sodium metaperiodate for 1 hr (
The MW heating method was also used for enhancement of mRNA in situ hybridization (
Recently, the AR technique has also been successfully applied to circumvent the loss of p53 antigenicity in stored paraffin tissue sections (
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Current Issues |
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Issues of Standardization
The standardization of IHC is an important issue that involves three areas: antibodies and reagents, technical procedures, and interpretation and reports (
What is the optimal heating condition and optimal pH value of the AR solution to obtain the maximal retrieval level for AR-IHC with the most commonly used antibodies in pathology? Further studies are necessary to address these questions. The potential effect of the AR technique in standardization of IHC has been indicated by some studies regarding ER-AR-IHC based on comparison between frozen, archival paraffin tissue sections, biochemical assay, and clinical behavior. As an example, a comparison of AR-IHC using antibodies to p53 has been summarized in Table 1. First of all, the necessity of adopting AR-IHC routinely for detection of p53 in archival tissue sections has been demonstrated by all the data shown in Table 1, which demonstrated that the intensity of p53 with the use of AR treatment was much stronger than that obtained by regular immunostaining without AR, the increasing rate ranging from 13% to 42% for PAb 1801 and 34% to 56.4% for DO7. The discrepancies in the increasing rate may be caused by the use of different AR protocols. Our recent experience also indicated the same conclusion, and we have established an optimal protocol for detection of p53 using AR-IHC in diagnostic pathology. Second, the possibility of standardizing the AR protocol was indicated by data reported from three independent research groups (
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"Test Battery"
This concept for the development of optimal protocols for AR-IHC is based on previous studies concerning the two major factors that influence the effect of AR-IHC, i.e., heating condition and the pH value of the AR solution (
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The test battery is a rapid screening approach to develop an optimal protocol for any antibody to be tested. It is an important step in establishing the maximal retrieval level for formalin-masked antigens with a variety of fixation times, to standardize immunostaining results. In our recent study of 14 antibodies using the test battery method, we demonstrated that the strongest intensity of AR-IHC for most antibodies tested was achieved either using low-pH buffer as the AR solution with regular MW heating conditions, or high pH with intense heating conditions (such as autoclave heating or MW heating for a prolonged heating time) (Figure 1) (
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Standardization of AR Technique
Heating Conditions.
Recently, a variety of heating methods have been applied for AR-IHC. These studies have given rise to some discrepancies in the AR immunostaining results from different laboratories. This is particularly important for quantitative immunohistochemical studies such as MIB1, PCNA, ER, PR, p53, and other prognostic markers. From the standpoint of convenience, economy, and practicality, we recommend the use of MW and a pressure cooker as the common heating method for AR-IHC; this achieves the high temperatures necessary for optimal AR. To monitor the accurate temperature, some modifications of equipment may be required, such as an alarm and/or a manometer or thermometer to indicate the exact temperature in the pressure cooker. In this way, the heating conditions can be standardized no matter the kind of MW oven or the conventional heating method used for heating. When Coplin jars are used with the regular MW heating method, measuring the heating time from the boiling point, or applying the calibration method (
pH Value of the AR Solution.
For the AR solution, we emphasize the critical role of the pH value because we have demonstrated that the citrate, phosphate, and acetate buffers may yield a similar intensity of AR-IHC if the pH value is the same (
Reevaluation of the Clinical Interpretation of AR-IHC
To demonstrate the clinical significance of AR-IHC, it is necessary to compare the intensity of AR-IHC with that obtained by routine IHC (without AR) of frozen and paraffin sections and to study the correlations among the three conditions. This type of comparison was, in fact, done in a recent study on Ki-67, MIB1, and PCNA expression on frozen and paraffin sections of breast cancer (
One issue that must be addressed is the threshold of p53 nuclear reactivity which should be considered as prognostically significant in cancers, as pointed out by
In summary, using the test battery may help to establish the optimal protocol for the immunostaining intensity of any antibody at a maximal retrieval level. The development of optimal protocols will make it possible to compare results among different laboratories and may provide a meaninful cut-off point for clinical significance after comparison with standard techniques and clinical follow-up.
Remaining Problems and Artifacts
Is there any false positivity of immunostaining after the AR technique is used for immunostaining? This is a critical issue in interpreting the results of AR-IHC. Thus far, most studies have reported satisfactory results, without false positivity, for a variety of antibodies tested (
Further Development of AR
Heating at Lower Temperature.
At present, one of the crucial limitations of the AR-IHC method is that some antigens, particularly surface markers, are still not stained with the use of AR.
Non-heating Method.
Non-heating methods for retrieving antigens have a long history in IHC. The simplest non-heating method is immersing the formalin-fixed tissue in water for long periods of time (
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Study of the Mechanism of AR |
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AR-IHC has shed some light on our understanding of the mechanism(s) of protein-formalin interactions. First of all, it showed that the modification of protein structure by formalin is reversible under certain conditions, such as high-temperature heating or strong alkaline treatment. We have suggested a possible mechanism of the AR technique, i.e., loosening or breaking of the crosslinkages caused by formalin fixation (
Crosslinking is a special form of chemical modification of protein (
The secondary, tertiary, and quaternary structures of proteins are dependent on the primary structure or the amino acid sequence. In general, if the primary structure of protein is still intact it may be possible to recover its original three-dimensional conformation under an optimal retrieval system. Because crosslinkages are complicated processes that depend on a variety of conditions (e.g., pH, temperature, conditions of tissue and fixation), they may lead to a variety of protein alterations (
Antibodies recognize specific epitopes localized in a particular spatial configuration within the protein molecule. This is particularly true for discontinuous antigenic determinants, which are composed of residues from different parts of the amino acid sequence (
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This "modification-re-modification" hypothesis has been supported by recent studies. Using MW heating followed by trypsin digestion,
Recently,
We conducted a test of AR-IHC in the presence of CaCl2, as summarized in Table 3. We found that (a) The same negative staining of MIB1 could be obtained by using CaCl2 alone as the AR solution; (b) not all antibodies showed a negative result, e.g., the intensity of AR-IHC for keratin remained the same after use of CaCl2 (Figure 3); and (c) re-heating the slides in Tris-HCl buffer, pH 1.5, restored a stronger positive staining for both MIB1 and p53. Although the fact that calcium interferes with AR (at least for some antigens) is consistent with the hypothesis that calcium-induced modification of protein may play a role in the retrieval process, it does not prove that binding of endogenous calcium is responsible for widespread loss of antigenicity in formalin fixation. Another issue that arouses suspicion of the calcium-binding hypothesis is the fact that the same intensity of AR-IHC with MIB1 can be obtained by a variety of AR solutions other than ci-trate buffer or with any calcium chelating agents. In particular, a stronger intensity can be produced by using a low pH or a higher pH of Tris-HCl buffer for AR-IHC of MIB1 (
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Conclusion |
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Although the AR technique is still in the developmental stage, it has been widely used in immunohistochemical studies. Studies on the mechanism of AR-IHC are ongoing, as are attempts to standardize the AR procedure. Widespread application of standardized protocols for AR may lead to valuable retrospective studies in various fields of pathology. Further studies may examine the practical use of AR-IHC in both diagnostic pathology and analytical morphology. Studies on the mechanism of AR-IHC may create a new field in molecular morphology.
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Acknowledgments |
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Supported by the University Pathology Associates of the University of Southern California.
We greatly appreciate Dr Denis G. Baskin's valuable comments and suggestions for this manuscript. We thank Ms Lillian Young and Elisabeth Yee and Mr Moung Win for technical assistance, and Ms Christina Yang for drawing illustrations and composition of the microphotographs.
Received for publication September 9, 1996; accepted September 25, 1996.
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