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http://jhc.sagepub.com/ Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/content/59/1/13 The online version of this article can be found at: DOI: 10.1369/jhc.2010.957191 2011 59: 13J Histochem Cytochem Shan-Rong Shi, Yan Shi and Clive R. Taylor Two Decades Antigen Retrieval Immunohistochemistry : Review and Future Prospects in Research and Diagnosis over Published by: http://www.sagepublications.com On behalf of: Official Journal of The Histochemical Society can be found at:Journal of Histochemistry & CytochemistryAdditional services and information for http://jhc.sagepub.com/cgi/alertsEmail Alerts: http://jhc.sagepub.com/subscriptionsSubscriptions: http://www.sagepub.com/journalsReprints.navReprints: http://www.sagepub.com/journalsPermissions.navPermissions: What is This? - Feb 23, 2011Version of Record >> by guest on September 1, 2012jhc.sagepub.comDownloaded from Journal of Histochemistry & Cytochemistry 59(1) 13 –32 © The Author(s) 2011 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1369/jhc.2010.957191 http://jhc.sagepub.com Just two decades have passed since the first article on anti- gen retrieval (AR) was published in 1991 (Shi et al. 1991). This simple technique of boiling formalin-fixed paraffin- embedded (FFPE) tissue sections in water has played a major role in extending the reach and use of immunohisto- chemistry (IHC) in FFPE tissues (Gown et al. 1993; Taylor and Cote 2005). One notable result is the effective division of all publications with respect to IHC for FFPE tissue sec- tions into two eras: pre-AR and post-AR (Gown 2004; Tay- lor 2001), indicating AR as a milestone (Jagirdar 2008). Since the early 1950s, the Journal of Histochemistry & Cytochemistry has published numerous articles describing new and interesting techniques for morphologic examina- tion of tissues, such as the enzyme-labeled IHC (Nakane and Pierce 1966), avidin-biotin detection system (Hsu et al. 1981), AR (Shi et al. 1991; Shi et al. 1992), tyramide signal amplification (Adams 1992), and so on. All of these valu- able developments have contributed to clinical and basic biomedical research projects worldwide. A number of factors contribute to the major impact that AR has had on diagnostic pathology (Boon and Kok 1995; Dabbs 2008; Jagirdar 2008; Hawes et al. 2010; Taylor et al. 2010). First, for more than one hundred years, FFPE tissues have served as the standard tissue preparation method in sur- gical pathology, providing the basis for most of the criteria for pathological diagnosis in “routine” hematoxylin and eosin– stained FFPE tissue sections. AR critically facilitated the use of FFPE tissues for IHC, retaining the use of existing mor- phologic criteria. Second, as a direct result of this extended capability, the value of archival FFPE tissue blocks, accom- panied by known follow-up data, was enormously enhanced, 57191 JHCXXX10.1369/jhc.2010.957191Shi et al.Journal of Histochemistry & Cytochemistry © The Author(s) 2010 Reprints and permission: sagepub.com/journalsPermissions.nav Received for publication July 27, 2010; accepted October 21, 2010 Corresponding Author: Shan-Rong Shi, Department of Pathology, University of Southern California Keck School of Medicine, 2011 Zonal Avenue, HMR 310A, Los Angeles, CA 90033. E-mail: sshi@usc.edu Antigen Retrieval Immunohistochemistry: Review and Future Prospects in Research and Diagnosis over Two Decades Shan-Rong Shi, Yan Shi, and Clive R. Taylor Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, California (SRS, CRT) and Department of Pathology, New York University, Langone Medical Center (YS). Summary As a review for the 20th anniversary of publishing the antigen retrieval (AR) technique in this journal, the authors intend briefly to summarize developments in AR-immunohistochemistry (IHC)–based research and diagnostics, with particular emphasis on current challenges and future research directions. Over the past 20 years, the efforts of many different investigators have coalesced in extending the AR approach to all areas of anatomic pathology diagnosis and research and further have led to AR-based protein extraction techniques and tissue-based proteomics. As a result, formalin-fixed paraffin- embedded (FFPE) archival tissue collections are now seen as a literal treasure of materials for clinical and translational research to an extent unimaginable just two decades ago. Further research in AR-IHC is likely to focus on tissue proteomics, developing a more efficient protocol for protein extraction from FFPE tissue based on the AR principle, and combining the proteomics approach with AR-IHC to establish a practical, sophisticated platform for identifying and using biomarkers in personalized medicine. (J Histochem Cytochem 59:13–32, 2011) Keywords antigen retrieval (AR), immunohistochemistry (IHC), formalin-fixed, paraffin-embedded (FFPE) tissue, quantitative immuno- histochemistry (QIHC), quantifiable reference standards, protein extraction, proteomics, cytopathology Review by guest on September 1, 2012jhc.sagepub.comDownloaded from 14 Shi et al. providing a valuable resource for translational clinical research and basic research that cannot easily be reproduced. Third, AR is a simple, inexpensive, and effective technique that leads to satisfactory IHC staining results in FFPE tissue for a great number of antibodies tested (Shi et al. 1995; Shi et al. 1997; Taylor and Cote 2005; Yamashita 2007; Shi and Taylor 2010a; Taylor et al. 2010). Finally, and by no means least, there is a growing body of recent literature with respect to use of AR methods for extraction of molecules (DNA, RNA, and proteins) from FFPE tissues that has further extended the utility of archival tissues and promises to allow the combination of proteomics and IHC in a true molecular morphology approach. So effective have these AR-based approaches become that investigators are now actively exploring the possible advantages of FFPE tissues in terms of preservation of both morphology and molecules in cell/tissue samples, in comparison to other methods of sample prepara- tion (Frank et al. 1996; Masuda et al. 1999; Sato et al. 2001; Shi et al. 2002; Shi et al. 2006; Palmer-Toy et al. 2005; Becker et al. 2007; Guo et al. 2007; Jiang et al. 2007; Addis et al. 2009; Fowler CB et al. 2010; Shi and Taylor 2010c). In concept and development, the primary goal of the AR methods was always to meet the needs of clinical practice, specifically to facilitate the performance of IHC on FFPE tissues. There was a considerable prior literature describing the use of IHC on FFPE tissues, with many attempts to improve the quality of results (Taylor and Burns 1974; Taylor and Mason 1974; Huang 1975; Hausen and Dreyer 1982; Taylor 1979; Kitamoto et al. 1987; Abbondanzo et al. 1991). Many of the active pioneers were practicing patholo- gists, acutely aware of the need to enhance the capabilities of IHC on FFPE tissues, so as to retain the key morphologic features that form the basis of diagnostic histopathology (Taylor 1980; Taylor and Kledzik 1981; Pinkus 1982; Colvin et al. 1995; Taylor and Cote 2005; Shi and Taylor 2010f). Although the AR technique is simple in concept and execution, development was difficult, in large part, because the notion of heating tissues to improve “antigenicity” was counterintuitive. As the author (Shi) began to formulate the idea in the 1980s, there were a number of practical and the- oretical issues to be addressed. A key scientific question was whether fixation in formalin modified the structure of antigens in a reversible or irreversible manner. To be more specific, was there any theoretical or prior scientific evi- dence that the effects of formalin fixation on proteins could be reversed, and if reversed, was the structure of protein restored to a sufficient degree for recovery of antigenicity? With these key questions in mind, Shi spent many days and nights in 1988 searching the chemical literature under difficult conditions, prior to the increased efficiency of such searches that is afforded today by the Internet and online databases. The answer was finally found in a series of studies of the chemical reactions between protein and formalin, published in the 1940s (Fraenkel-Conrat et al. 1947; Fraenkel-Conrat and Olcott 1948a, 1948b). These studies indicated that cross-link- ages between formalin and protein could be disrupted by heating above 100C or by strong alkaline treatment. With this knowledge of high temperature heating as a potential retrieval approach, the heat-induced AR technique was developed in 1991 (Shi and Taylor 2010d). Today, 20 years on, the AR technique is widely, almost universally, used in surgical pathology, including veterinary pathology (Ramos-Vara 2005), in all morphology-based sciences, and in pharmacology drug-related research, with thousands of original articles published worldwide, together with more than one dozen review articles, with those by Yamashita and D’Amico being most recent (D’Amico et al. 2009; Yamashita 2007). Two books edited by our group summarize almost all data pertaining to further technical development and application of AR (Shi, Gu, Taylor 2000; Shi and Taylor 2010b). The present review is focused on several critical issues, with an emphasis on current challenges and potentially pro- ductive directions for future study. Five areas of research have been identified within these parameters: (1) the role of AR in improved standardization of IHC, (2) AR as a com- ponent of cell/tissue sample preparation, (3) the mechanism of AR, (4) extraction of nucleic acids and proteins from FFPE tissue sections for genomic and proteomic analysis, and (5) combining proteomics and IHC for quantitative analysis of defined cell populations (molecular morphol- ogy) in research and diagnosis. These five themes are woven throughout the following discussion. Extending the Application of AR-IHC To date, AR has been applied predominantly to archival “paraffin blocks” for IHC in diagnostic surgical pathology as a routine procedure. There are, in addition, many other adaptations of the AR method: for improved IHC staining of plastic-embedded tissue samples both by light and elec- tron microscopy, as a blocking procedure to avoid cross- antigen/antibody reaction during multiple IHC staining procedures, for enhancement of DNA/RNA in situ hybrid- ization in FFPE materials, for in situ end-labeling (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) of apoptotic cells in FFPE tissue sections, and in flow cytometry to achieve stronger positive signals while reducing nonspecific background noise (Shi, Cote, Shi, et al 2000). Application of AR to cytopathology, frozen sections, and immunofluorescence methods will be reviewed in more detail. Major applications of AR technique or its prin- ciples are summarized in Table 1. Cytopathology. Application of IHC (or ICC, signifying immunocytochemistry) in cytopathology has lagged behind use in histopathology, in part because of differences in cell sample preparation, which is quite different from that used by guest on September 1, 2012jhc.sagepub.comDownloaded from Review and Future Prospects in Research and Diagnosis over Two Decades 15 Table 1. Major Applications of Antigen Retrieval Technique and Principle Areas of Application of AR Application of AR Technique and/or Principle Reference IEM AR pretreatment of routine processed Epon-embedded tissue ultra-thin sections after etching the grids by solutionsa to achieve satisfactory positive results or directly heating the grid, followed by some washing procedures, including 50 mM NH 4 Cl and 1% Tween 20 Stirling and Graff 1995; Wilson et al. 1996 ISH High-temperature heating FFPE tissue sections prior to ISH to achieve satisfactory results Sibony et al. 1995; Lan et al. 1996; McMahon and McQuaid 1996 TUNEL Optimal heating time such as 1 min to improve the signal Strater et al. 1995; Lucassen et al. 2000 Multiple IHC staining procedure Adding a microwave heating AR procedure (10 min) between each run of IHC staining procedure to block the cross-reaction by denaturing bound antibody molecules from the previous run Lan et al. 1995 Human temporal bone collections Combining sodium hydroxide–methanol and heating AR treatment provides an effective approach for IHC used in celloidin-embedded temporal bone sections. This method is also used for plastic-embedded tissue sections, including IEM Shi et al. 1992; Shi, Cote, Taylor 2000 Immunofluorescence To enhance intensity and reduce autofluorescence D’Ambra-Cabry et al. 1995 Cytopathology Boiling AR pretreatment for archival Pap smear slides to achieve satisfactory IHC staining for MIB-1 used for fine-tuning diagnoses in cervical cytology. Formalin postfixed air-dried smears Boon et al. 1994; Boon et al. 1995; Boon et al. 2000 Fulciniti et al. 2008; Chivukula and Dabbs 2010 FCM Enzyme digestion followed by heating AR treatment was adopted to achieve enhancement of FCM on FFPE tissue Redkar and Krishan 1999 Floating vibratome section Microwave boiling vibratome section to achieve IHC staining results that further extended the use for whole- mount tissue specimens Evers and Uylings 1994, 2000; Shiurba et al. 1998 En bloc tissue AR heating 4% paraformaldehyde-fixed animal brain or testis tissue blocks to enhance immunoreactivity for most antibodies tested Ino. 2003 Frozen tissue section Aldehyde-fixed frozen tissue section with use of AR treatment to achieve both excellent morphology and IHC staining result Yamashita and Okada 2005a; Shi et al. 2008 DNA extraction from FFPE tissue sections Boiling AR pretreatment prior to DNA extraction to replace enzyme treatment for improved results of DNA extraction Frank et al. 1996; Coombs et al. 1999; Shi et al. 2002; Shi et al. 2004 RNA extraction from FFPE tissue sections Heating treatment prior to RNA extraction to recover fixative-induced modification or to replace enzyme treatment for improved results of RNA extraction Masuda et al. 1999; Shi and Taylor 2010c Protein extraction from FFPE tissue sections Boiling AR pretreatment with AR solution, including 2% SDS and/or other chemicals, to improve efficiency of protein extraction from FFPE tissue sections to replace enzyme digestion. Further development by combining elevated hydrostatic pressure may increase extraction to 80% to 95% of proteins in FFPE tissue sections. Ikeda et al. 1998; Shi et al. 2006; Fowler CB et al. 2007; Fowler CB et al. 2008; Fowler CB et al. 2010 IMS Boiling AR pretreatment is being adopted to achieve satisfactory results of IMS in recent years. On the basis of comparing different AR solutions, Gustafsson et al. (2010) summarized that the citrate acid AR method is an important step in being able to fully analyze the proteome for FFPE tissue. Groseclose et al. 2008; Ronci et al. 2008; Gustafsson et al. 2010 AR = antigen retrieval; FFPE = formalin-fixed paraffin-embedded; IEM = immunoelectron microscopy; ISH = in situ hybridization; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; FCM = flow cytometry; IMS = imaging mass spectrometry. aTen percent 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. by guest on September 1, 2012jhc.sagepub.comDownloaded from 16 Shi et al. for FFPE tissues in surgical pathology. In cytopathology, the cell sample is limited to a small amount that allows for only a few “smear” or imprint slides for cytologic evaluation to make a diagnosis. This circumstance alone renders it diffi- cult, if not impossible, to undertake a panel of IHC (or ICC) stains, as is frequently used in histopathology. In a minority of instances, when a larger amount of cell sample is obtained, it is possible to use the cell block technique, which does allow for the cutting of serial sections for a panel of IHC stains. On the basis of these conditions, L. J. Fowler and Lachar (2008) highlighted the challenges that exist in appli- cation of ICC to cytopathology. One of the major issues is lack of proper control samples. Another problem is the fre- quent use of inappropriate antibody concentrations due to a lack of appropriate cell samples for optimal titration studies; by default, dilutions established as suitable for FFPE sections are used, with resultant errors. Indeed, it should be empha- sized that ICC controls for cytology specimens must be made from similarly prepared cell specimens for accurate compari- son. Use of FFPE tissue section as a positive control for a cytology sample is not appropriate and is likely to result in misinterpretation. This problem represents a major practical obstacle because most hospital pathology laboratories lack the resources and expertise to establish appropriate cell line control systems. AR Greatly Improves Sensitivity of ICC for Cytological Smears but Needs Standardization Additional difficulties parallel those encountered in diag- nostic surgical pathology and arise from the fact that proto- cols of cell sample preparation and fixation, used in cytopathology, also are not standardized across laborato- ries. More than a decade ago, Suthipintawong et al. (1996) performed a comparative study for 23 fixation protocols of cell preparations for ICC and reported that fixation in 0.1% formal saline overnight at 27C, followed by 10-min fixa- tion in 100% ethanol, coupled with the use of microwave AR pretreatment, gave the best results. Gong et al. (2004) carefully compared ICC staining results of estrogen recep- tor (ER) between cell smears and corresponding tissue sec- tions using the AR technique for several fixatives, including formaldehyde and Carnoy’s fixative, and demonstrated that the use of the AR technique in cytological smears greatly improved ER immunodetectability in both formalin-fixed and Carnoy’s-Pap smears, raising the final concordance rate with FFPE tissue sections from 31% (formalin fixed) and 29.4% (Carnoy’s smears) without AR to 93% with AR. Although AR techniques increasingly are used in ICC for cytopathology, standardization remains a major problem (Dabbs 2010). Fetsch and Abati (2007) concluded that stan- dardization of AR-ICC staining procedures is imperative for the optimal interpretation of HER2 because of variation in sample size, fixative, and preparation method. They obtained widely different results in a study of 54 FFPE cell block sections of metastatic breast cancer, using three pri- mary antibodies to HER2 with one single heat-induced AR protocol (boiling sections in citric acid buffer of pH 6.0 for 20 min). Therefore, establishing an optimal AR protocol, based on the test battery approach for each primary anti- body tested, is essential instead of using one single AR protocol (Shi and Taylor 2010e). Current Trend: Formalin Postfixed Air-Dried Smears with AR Treatment Give Reliable ICC Results and Better Morphology There is a current trend for a wider use of formalin fixation in cytopathology despite the fact that many of the existing sample preparation methods are fast and give good mor- phology (Suthipintawong et al. 1997; Shidham et al. 2000; Liu J and Farhood 2004; Fulciniti et al. 2008). Fulciniti et al. (2008) reported a study of ICC on fine-needle cytol- ogy samples comparing alcohol-wet-fixed and formalin postfixed air-dried cell smears and concluded that the for- malin postfixed air-dried smears gave reliable ICC results with the use of AR treatment. In addition, the visual evalu- ation