PERSPECTIVES |
Correspondence to: Clive R. Taylor, Dept. of Pathology, University of Southern California Keck School of Medicine, HMR 204, 2011 Zonal Avenue, Los Angeles, CA 90033. E-mail: taylor@pathfinder.hsc.usc.edu
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Summary |
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Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology.
(J Histochem Cytochem 49:931937, 2001)
Key Words: antigen retrieval (AR), immunohistochemistry, ISH, FISH, nucleic acid, formalin
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Introduction |
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IMMUNOHISTOCHEMISTRY (IHC) has created a wide field for functional (analytical or molecular) morphology, particularly since it has rendered immunoperoxidase methods applicable to routine formalin-fixed, paraffin-embedded tissues based on a series of technical developments. These include increasingly sensitive detection systems and several pretreatments before the immunostaining procedure to recover antigenicity masked by formalin fixation. However, the growing interest of pathologists who attempt further to expand the application of IHC staining on formalin-fixed, paraffin-embedded tissue sections were frustrated by inconsistent results on fixed tissues. More than two decades ago, various alternative fixatives were tried in an attempt to replace formalin in an irreversible chemical reaction of formalinprotein, but they have failed, and it is likely that an ideal fixative will never be found (
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Basic Questions: Definition, Application, and Necessary Knowledge for Beginners |
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In the current literature, the term "antigen retrieval" is predominantly (and originally) defined as a high-temperature heating method to recover the antigenicity of tissue sections that had been masked by formalin fixation. Several terms were used to represent the same AR heating method, resulting in confusion of the results of literature search by Medline (
Studies have also revealed that the AR heating method could achieve satisfactory results in IHC for tissues embedded in plastic embedding media used for immunoelectron microscopy (IEM), celloidin-embedded tissues, or non-embedded tissue slices, as well as in cell smear preparations fixed in non-crosslinking fixatives (
Although most publications have reported satisfactory results of AR-IHC, several issues must be kept in mind when this technique is used.
First, not all antigen structures modified by formalin can be restored using conventional AR protocols. Therefore, a "test battery" can be used to establish an optimal AR protocol for certain antigens under investigation (
For a few proteins, higher temperature (boiling) may induce a negative result of IHC staining. In this case, a lower-temperature heating treatment or a combining retrieval protocol (heat and enzyme digestion or CARD) may provide better results (
Knowledge of the exact localization of a certain protein (antigen) in tissue is critical to interpret not only the accuracy of IHC staining results but also the reliability of AR treatment. Many excellent pioneering studies have given examples of approaches to scientific validation of the exact localization of a protein on the basis of morphology and biochemistry or other correlated fields (
Although the AR technique is a simple method, it is necessary to understand the factors that influence the effectiveness of IHC staining, particularly two major factors, the heating conditions and the pH value of the AR solution. The heating condition represents heating temperature (T) and the time of heating (t), appearing to be a reverse correlation as T x t (i.e., the higher the temperature, the shorter the heating time), to develop an optimal AR protocol after a test battery approach (
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Standardization |
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The demand for standardization of IHC for quantitation is ever more pressing because of the emergence of a new field of translational research that requires quantitation of the differential expression of various prognostic markers for cancer, based on retrospective study of clinical cases with known clinical outcomes (
Simplifying the immunostaining procedure is one of the potential strategies for standardization of IHC, and optimal combination of various amplification approaches to achieve a satisfactory result may be based on the principle of simplification of all techniques used (
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Extraction of DNA/RNA by the Heating Retrieval Method |
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An interesting issue for further development of this heat-induced retrieval approach for macromolecules other than protein was raised by the successful application of the heat retrieval method for enhancement of ISH (
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Can the heat-induced retrieval method be used for sufficient extraction of DNA/RNA from archival paraffin-embedded tissue based on the achievements of ISH and FISH using the heating method? Extraction of DNA from archival formalin-fixed, paraffin-embedded tissue was accomplished as early as 1985 without the use of the heat-induced retrieval technique (
Studies of chemical reactions between formaldehyde and nucleic acids also have demonstrated that several basic reactions are similar to those observed in formalinprotein reactions (
It appears that a subtle merger of the high-temperature heating AR technique for protein antigens with high-temperature heating retrieval of nucleic acids is found in recent literature. Based on the similarity of chemical reactions between proteinformalin and nucleic acidformalin discussed previously, it is reasonable to "borrow" from the heat-induced retrieval technique of protein to improve extraction of nucleic acids to further speed development of this new technique for molecular morphology.
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Mechanism of AR |
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A better understanding of the mechanisms of formalin fixation and antigen retrieval may be helpful in the further development of the retrieval technique for enhancement of IHC and ISH, and for more efficient extraction of proteins and DNA/RNA from archival tissue. Although the exact mechanisms of formalin fixation are still unknown, ARIHC has shed some light on the mechanism of proteinformalin interactions. First of all, the success of AR has shown that the modification of protein structure by formalin is reversible under certain conditions, such as high-temperature heating or strong alkaline treatment. Several hypotheses have been proposed to explain the possible mechanism of ARIHC, including a recent study of calcium-induced modification of protein conformation conducted by
We have suggested a possible mechanism of the AR technique, i.e., the loosening or breaking of the crosslinkages induced by formalin fixation (
The hypothesis of the heat-induced re-naturation is based on an essential principle of immunology, i.e., antigenantibody recognition is dependent on protein structure. 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. A conformational change in a protein caused by formalin fixation may mask the epitope and thus affect the antigenicity of proteins in formalin-treated tissue. The AR method may lead to a re-naturation or at least partial restoration of the protein structure (induced by high-temperature heating or other non-heating procedures), with re-establishment of the three-dimensional protein structure to something approaching its native condition.
The exact chemical reaction involved in this modificationre-naturation mechanism is not clear. The most likely process that would restore part of the native configuration of a formalin-modified antigen is the hydrolysis of crosslinkages and other formalin adducts that result from formalinprotein fixation. In support of this hypothesis, evidence in the literature has shown that extending the period of washing formalin-fixed tissue in water may reverse the loss of immunoreactivity (
In conclusion, the heat-induced AR technique has achieved wide application in pathology as well as other fields of morphology, and has provided a potential approach to standardization of routine IHC for approaching quantification of IHC, as well as more effective extraction of nucleic acids from archival paraffin-embedded tissues. Further studies of this AR technique, including the mechanism of AR, may shed new light in molecular morphology.
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Acknowledgments |
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Supported by the University Pathology Associates of the University of Southern California and by an NIH grant 1 R21 CA91249-01.
We greatly appreciate Dr Baskin's invitation to write this article. We wish to thank Ms Lillian Young for management of our laboratory and Dr Yan Shi and Mr Moung Win for technical assistance with FISH.
Received for publication February 14, 2001; accepted May 9, 2001.
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