Antigen Retrieval Immunohistochemistry Based Research and Diagnostics E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Serie: Wiley Series in Biomedical Engineering
• Sprache: Englisch

Seitenzahl: 738

Veröffentlichungsjahr: 2011

Beschreibung

The most complete, up-to-date reference on antigen retrieval and immunohistochemistry

An antigen is a substance that prompts the generation of antibodies and can cause an immune response. The antigen retrieval (AR) technique is in wide use across the globe, and is a critical technique used in medical diagnosis of disease, particularly clinical targeted cancer treatment.

Antigen Retrieval Immunohistochemistry Based Research and Diagnostics discusses several scientific approaches to the standardization of quantifiable immunohistochemistry (IHC). Based on the development and application of AR by the editors, this volume summarizes recent achievements in AR-IHC and analyzes numerous cutting-edge issues for future research projects.

Featuring contributions from a worldwide group of leading experts and research scientists in the field, this important work:

• Summarizes the key problems in the four fields of antigen retrieval
• Discusses the advances of AR techniques and their applications
• Provides practical methods and protocols in AR-IHC, such as extraction of nucleic acids and proteins for molecular analysis, cell/tissue sample preparation, and standardization and development of various techniques to meet the future needs of¿clinical and research molecular analysis
• Encourages further research in AR and IHC, particularly how AR methods might be employed for improved test performance and the development of greater reliability and reproducibility of IHC
• Includes an appendix of related laboratory protocols

Antigen Retrieval Immunohistochemistry Based Research and Diagnostics is intended for clinical pathologists, molecular cell biologists, basic research scientists, technicians, and graduate students who undertake tissue/cell morphologic and molecular analysis and wish to use and extend the power of immunohistochemistry. It is also pertinent for most biotechnology companies majoring in development of IHC products.

Wiley Series in Biomedical Engineering and Multi-Disciplinary Integrated Systems / Kai Chang, Series Editor

Leseprobe

Table of Contents

Cover

Table of Contents

Half title page

Series page

Title page

Copyright page

PREFACE

CONTRIBUTORS

Part I: RECENT ADVANCES IN ANTIGEN RETRIEVAL TECHNIQUES AND ITS APPLICATION

CHAPTER 1 STANDARDIZATION OF ANTIGEN RETRIEVAL TECHNIQUES BASED ON THE TEST BATTERY APPROACH

1.1 SEARCHING FOR NOVEL CHEMICAL SOLUTIONS

1.2 ANTIBODY AND DETECTION SYSTEM-DEPENDENT TEST BATTERY

1.3 APPLICATION OF TMA TECHNIQUE FOR TEST BATTERY

1.4 SCIENTIFIC ACCURACY OF IHC RELYING ON OPTIMAL AR PROTOCOL?

1.5 ACCURACY OF AR-IHC AS DEMONSTRATED BY IEM AND OTHERS

1.6 SUMMARY

CHAPTER 2 EXTENDED APPLICATION OF ANTIGEN RETRIEVAL TECHNIQUE IN IMMUNOHISTOCHEMISTRY AND IN SITU HYBRIDIZATION

2.1 BRIEF SUMMARY OF PREVIOUS APPLICATIONS OF AR

2.2 DIAGNOSTIC CYTOPATHOLOGY

2.3 IMMUNOFLUORESCENT STAINING OF FFPE TISSUE SECTIONS

2.4 METHODS OF REDUCING AUTOFLUORESCENCE

2.5 ALDEHYDE-FIXED FROZEN CELL/TISSUE SECTIONS

2.6 OTHER APPLICATIONS

CHAPTER 3 EXTRACTION OF DNA/RNA FROM FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE BASED ON THE ANTIGEN RETRIEVAL PRINCIPLE

3.1 DEVELOPMENT OF SIMPLE AND EFFECTIVE PROTOCOL OF DNA EXTRACTION

3.2 DNA QUALITY EVALUATED BY ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION

3.3 ARTIFACTUAL DNA SEQUENCE ALTERATIONS OF FFPE TISSUE AND RETRIEVAL STRATEGY

3.4 DEVELOPMENT OF HEAT-INDUCED PROTOCOL FOR RNA EXTRACTION FROM FFPE TISSUE

3.5 A DETAILED EXAMPLE OF RNA EXTRACTION FROM FFPE CELLS/TISSUES PERFORMED AT OUR LABORATORY

CONCLUSION

ACKNOWLEDGMENT

Part II: STANDARDIZATION OF IMMUNOHISTOCHEMISTRY

CHAPTER 4 KEY ISSUES AND STRATEGIES OF STANDARDIZATION FOR QUANTIFIABLE IMMUNOHISTOCHEMISTRY

4.1 A TOTAL TEST APPROACH FOR STANDARDIZATION OF IHC

4.2 STANDARDIZATION OF IHC: CURRENT STRATEGIES AND THE LONG RUN

4.3 CUTOFF POINT: HOW TO DEFINE OPTIMAL SCORE?

4.4 STANDARD REFERENCE MATERIAL

4.5 OTHER POTENTIAL APPROACHES FOR QUANTIFIABLE IHC

CHAPTER 5 STANDARDIZATION OF IMMUNOHISTOCHEMISTRY BASED ON ANTIGEN RETRIEVAL TECHNIQUE

5.1 INTERNAL REFERENCE STANDARDS (IRS)

5.2 AN INTERIM APPROACH TO IMPROVED REPRODUCIBILITY BASED ON AR

5.3 HYPOTHESIS

5.4 PRELIMINARY TEST TO CONFIRM PREVIOUS STUDIES

5.5 AN EXPECTED FULL RETRIEVAL RATE AMONG MOST ANTIGENS

CHAPTER 6 STANDARD REFERENCE MATERIAL: CELL LINE DEVELOPMENT AND USE OF REFERENCE CELL LINES AS STANDARDS FOR EXTERNAL QUALITY ASSURANCE OF HER2 IHC AND ISH TESTING

6.1 INTRODUCTION

6.2 HISTORICAL RATIONALE

6.3 DEVELOPMENT AND PREPARATION OF CELL LINES AS STANDARD REFERENCE MATERIAL

6.4 APPLICATIONS AND EDUCATIONAL FEEDBACK VALUE

6.5 CONCLUSION

ACKNOWLEDGMENT

CHAPTER 7 PEPTIDES AS IMMUNOHISTOCHEMISTRY CONTROLS

7.1 INTRODUCTION

7.2 WHY USE PEPTIDES AS IHC CONTROLS?

7.3 IDENTIFYING PEPTIDE EPITOPES

7.4 REPRODUCIBILITY OF PEPTIDE CONTROLS

7.5 STABILITY OF PEPTIDE CONTROLS

7.6 PEPTIDE CONTROLS ARE SENSITIVE INDICATORS OF IHC STAINING PROBLEMS

7.7 PEPTIDE CONTROLS CAN DETECT PROBLEMS WITH ANTIGEN RETRIEVAL

7.8 SUMMARY

ACKNOWLEDGMENTS

CONFLICT OF INTEREST DISCLOSURE?

CHAPTER 8 STANDARD REFERENCE MATERIAL: PROTEIN-EMBEDDING TECHNIQUE AND DESIGN OF BAR CODE

8.1 PROTEIN ABSORPTION METHOD

8.2 DIRECT MIXING PROTEIN INTO MATRIX MEDIA

8.3 COATING PROTEIN ON SURFACE OF BEADS

8.4 A DESIGN OF BAR CODE

ACKNOWLEDGMENTS

CHAPTER 9 THE PROS AND CONS OF AUTOMATION FOR IMMUNOHISTOCHEMISTRY FROM THE PROSPECTIVE OF THE PATHOLOGY LABORATORY

9.1 INTRODUCTION

9.2 DEVELOPMENT OF IHC AS AN ADJUNCT TO PATHOLOGIC DIAGNOSIS

9.3 MANUAL METHODS FOR PERFORMING IHC

9.4 DEVELOPMENT OF AUTOMATION FOR IHC

9.5 OPEN VERSUS CLOSED IHC AUTOMATED STAINING INSTRUMENTS

9.6 PRINCIPLES OF IHC AUTOMATION

9.7 HEAT-INDUCED ANTIGEN RETRIEVAL (HIAR) METHODS: ONLINE VERSUS OFF-LINE

9.8 CONCLUSIONS

ACKNOWLEDGMENT

CHAPTER 10 IMAGE ANALYSIS IN IMMUNOHISTOCHEMISTRY

10.1 IMAGE ACQUISITION

10.2 CAMERA AND OPTICS SELECTION

10.3 IMAGE FORMAT

10.4 IMAGE DISPLAY

10.5 IMAGE ANALYSIS

10.6 MULTIPLE STAINS AND COLOCALIZATION

10.7 SPECIMEN PREPARATION

10.8 STAINING

10.9 IHC STAIN CONTROLS

10.10 CONCLUSIONS

Part III: TISSUE/CELL SAMPLE PREPARATION

CHAPTER 11 TISSUE CELL SAMPLE PREPARATION: LESSONS FROM THE ANTIGEN RETRIEVAL TECHNIQUE

11.1 ANTIGEN RETRIEVAL (AR) DEVELOPMENT BACKGROUND— A CONTINUUM OF PAST, PRESENT, AND FUTURE

11.2 TWO PHILOSOPHICALLY DIFFERENT APPROACHES FOR CELL/TISSUE SAMPLE PREPARATION

CHAPTER 12 MECHANISMS OF ACTION AND PROPER USE OF COMMON FIXATIVES

12.1 INTRODUCTION

12.2 DENATURATION

12.3 PENETRATION

12.4 MANAGING SPECIMEN QUALITY

12.5 FIXATION WITH FORMALDEHYE

12.6 OTHER POPULAR FIXATIVES

CHAPTER 13 CELL SAMPLE PREPARATION FOR CLINICAL CYTOPATHOLOGY: CURRENT STATUS AND FUTURE DEVELOPMENT

13.1 CELL BLOCK TECHNIQUE

13.2 MULTIPLE MARKERS ON CYTOLOGIC SMEARS

13.3 AR AND ICC ON SMEARS

13.4 STANDARDIZATION OF ICC

13.5 CONCLUSIONS

CHAPTER 14 DESIGN OF A TISSUE SURROGATE TO EXAMINE ACCURACY OF PROTEOMIC ANALYSIS

14.1 INTRODUCTION

14.2 STUDIES WITH FFPE CELL BLOCKS AND GEL-EMBEDDED PROTEINS

14.3 STUDIES WITH TISSUE SURROGATES

14.4 EVALUATION OF THE EFFECTS OF HISTOLOGICAL PROCESSING ON TISSUE SURROGATES

14.5 EFFECTS OF DETERGENT AND TEMPERATURE ON RECOVERY EFFICIENCY

14.6 EFFECTS OF OTHER BUFFER FORMULATIONS ON RECOVERY EFFICIENCY

14.7 STUDIES WITH TISSUE SURROGATES FORMED FROM OTHER PROTEINS

14.8 CONCLUSION

Part IV: MOLECULAR MECHANISM OF ANTIGEN RETRIEVAL TECHNIQUE

CHAPTER 15 STUDY OF FORMALIN FIXATION AND HEAT-INDUCED ANTIGEN RETRIEVAL

15.1 INTRODUCTION

15.2 REACTION OF FORMALDEHYDE WITH PROTEINS

15.3 FORMATION OF INTRA- AND INTERMOLECULAR CROSS-LINKS IN FORMALIN-TREATED RNASE A

15.4 EFFECT OF FORMALIN ON THE THERMAL PROPERTIES OF RNASE A

15.5 EFFECT OF FORMALIN ON THE IONIZATION STATE OF RNASE A

15.6 EFFECT OF FORMALIN ON THE SECONDARY AND TERTIARY STRUCTURE OF RNASE A

15.7 RECOVERY OF ENZYMATIC ACTIVITY FROM FORMALIN-TREATED RNASE A

15.8 THE EFFECT OF FORMALIN TREATMENT ON THE IMMUNOREACTIVITY OF RNASE A

15.9 RESTORATION OF IMMUNOREACTIVITY IN FORMALIN-TREATED RNASE A

15.10 EFFECT OF FIXATION ON THE RECOVERY OF RNASE A ACTIVITY FROM TISSUE

15.11 EFFECT OF ETHANOL DEHYDRATION ON THE REVERSAL OF FORMALDEHYDE CROSS-LINKS

15.12 EFFECT OF FIXATION AND ETHANOL DEHYDRATION ON PROTEIN STRUCTURE

15.13 THE ROLE OF ETHANOL DEHYDRATION IN AR

15.14 GENERAL COMMENTS ON THE MECHANISM OF AR

15.15 BIOPHYSICAL METHODS

CHAPTER 16 A LINEAR EPITOPES MODEL OF ANTIGEN RETRIEVAL

16.1 INTRODUCTION

16.2 PEPTIDE ARRAY EXPERIMENTAL MODEL

16.3 SOME PEPTIDES ARE DIRECTLY SUSCEPTIBLE TO FORMALIN FIXATION

16.4 IHC ANTIBODIES BIND TO LINEAR EPITOPES

16.5 ADJACENT PROTEINS ARE IMPORTANT IN UNDERSTANDING ANTIGEN RETRIEVAL

16.6 A MODEL OF ANTIGEN RETRIEVAL REQUIRING LINEAR EPITOPES

16.7 EVALUATION OF THE MODEL

16.8 HETEROGENEITY OF ANTIGEN RETRIEVAL REACTIONS

16.9 SUMMARY

CONFLICT OF INTEREST DISCLOSURE?

ACKNOWLEDGMENT

CHAPTER 17 PH OR IONIC STRENGTH OF ANTIGEN RETRIEVAL SOLUTION: A POTENTIAL ROLE FOR REFOLDING DURING HEAT TREATMENT

17.1 INTRODUCTION

17.2 EFFECTS OF PH

17.3 EFFECT OF IONIC STRENGTH

17.4 MECHANISMS OF HIAR

17.5 CONCLUSION

CHAPTER 18 COMMENTARY: FUTURE DIRECTIONS

18.1 INTRODUCTION

18.2 RECOVERING UNMODIFIED PROTEINS FROM FFPE TISSUE

18.3 QUESTIONS ABOUT THE CHEMISTRY OF FIXATION AND DEMODIFICATION

18.4 NEW TECHNIQUES FOR ASSESSING THE QUANTITY AND FUNCTIONAL STATE OF TISSUE PROTEINS

18.5 CONCLUSIONS

Part V: PROTEOMIC ANALYSIS OF PROTEIN EXTRACTED FROM TISSUE/CELLS

CHAPTER 19 TECHNIQUES OF PROTEIN EXTRACTION FROM FFPE TISSUE/CELLS FOR MASS SPECTROMETRY

19.1 INTRODUCTION

19.2 REACTION OF FORMALDEHYDE FIXATIVES WITH PROTEINS

19.3 HEAT-INDUCED PROTEIN EXTRACTION

19.4 LIQUID TISSUE™ METHOD FOR PROTEIN FROM FFPE TISSUE

19.5 OTHER TISSUE EXTRACTION METHODOLOGIES FOR FFPE TISSUE

19.6 CONCLUSION

CHAPTER 20 APPLICATION OF SHOTGUN PROTEOMICS TO FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUES

20.1 THE PROMISE AND CHALLENGE OF SHOTGUN PROTEOMICS IN ARCHIVAL, FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUES (FFPE)

20.2 DEVELOPMENT OF SHOTGUN PROTEOMICS IN FFPE TISSUES

20.3 EVALUATION OF LASER CAPTURE MICRODISSECTION OF FFPE TISSUES

20.4 SHOTGUN PROTEOME ANALYSIS OF MICRODISSECTED FORMALIN-FIXED BRAIN TUMOR TISSUE

20.5 EVALUATION OF CONFIDENCE AND REPRODUCIBILITY OF QUANTITATIVE SHOTGUN PROTEOMIC ANALYSES

20.6 EVALUATION OF CONFIDENCE AND REPRODUCIBILITY OF QUANTITATIVE SHOTGUN PROTEOMIC ANALYSES OF FFPE TISSUES

20.7 SHOTGUN PROTEOMICS FOR THE ANALYSIS OF ARCHIVAL FFPE TISSUES

20.8 FUTURE DIRECTIONS FOR SHOTGUN PROTEOMICS APPLIED TO FFPE TISSUES

CHAPTER 21 VISUALIZING PROTEIN MAPS IN TISSUE

21.1 INTRODUCTION

21.2 THE HISTORY OF OUR IMAGING MS SAMPLE PREPARATION TECHNIQUE

21.3 PRACTICAL SAMPLE PREPARATION FOR IMS MEASUREMENT

21.4 PROTEIN MAPPING ON A TISSUE SECTION BY IMS: SCRAPPER KNOCKOUT (KO) ANALYSIS

21.5 CONCLUDING REMARKS

CHAPTER 22 SYMBIOSIS OF IMMUNOHISTOCHEMISTRY AND PROTEOMICS: MARCHING TO A NEW ERA

ACKNOWLEDGMENT

APPENDIX  RELATED LABORATORY PROTOCOLS

THE “TEST BATTERY” APPROACH OF ANTIGEN RETRIEVAL (AR) TECHNIQUE

ANTIGEN RETRIEVAL PROTOCOL WITH USE OF CITRACONIC ANHYDRIDE SOLUTION

DNA/RNA AND PROTEIN EXTRACTION FROM FFPE TISSUE SECTIONS BASED ON AR PRINCIPLE (PROVIDED BY CHENG LIU, HT)

SELECTED PROTOCOLS OF CELL SAMPLE PREPARATION FOR CYTOPATHOLOGY (DATA PROVIDED BY CHIARA SUGRUE, MBA, MS, SCT [ASCP], LONG ISLAND JEWISH MEDICAL CENTER)

BODY FLUID CELL SAMPLE PREPARATION

NON-GYNECOLOGIC SPECIMENS: THINPREP PREPARATION

CYTOCENTRIFUGATION PROCEDURE

GYNECOLOGIC SPECIMENS: THINPREP SLIDE PREPARATION

BRUSHING SPECIMENS PREPARATION

RESULT

FINE NEEDLE ASPIRATION (FNA) PREPARATION

Index

Color Plates

ANTIGEN RETRIEVAL IMMUNOHISTOCHEMISTRY BASED RESEARCH AND DIAGNOSTICS

WILEY SERIES IN BIOMEDICAL ENGINEERING AND MULTI-DISCIPLINARY INTEGRATED SYSTEMS

Kai Chang, Series Editor

Advances in Optical Imaging for Clinical Medicine Nicusor Iftimia, William R. Brugge, and Daniel X. Hammer

Antigen Retrieval Immunohistochemistry Based Research and Diagnostics Shan-Rong Shi and Clive R. Taylor

Copyright © 2010 John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.

Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

Antigen retrieval immunohistochemistry based research and diagnostics / [edited by] Shan-Rong Shi, Clive R. Taylor.

p. cm.—(Wiley series in biomedical engineering and multi-disciplinary integrated systems. ; 1)

Summary: “An antigen is a substance that prompts the generation of antibodies and can cause an immune response. The antigen retrieval (AR) technique is used worldwide and has resulted in a revolution in immunohistochemistry (IHC). Featuring contributors who are distinguished experts and researchers in the field, this book discusses several scientific approaches to the standardization of quantifiable IHC. It summarizes the key problems in the four fields of antigen retrieval and provides practical methods and protocols in AR-IHC. Clinical pathologists, molecular cell biologists, basic research scientists, technicians, and graduate students, will benefit from this fully up-to-date work”—Provided by publisher.

Summary: “This book is based on the development and application of AR by the editors, one of whom is the inventor of AR, together with members of a world-leading research center of AR”—Provided by publisher.

ISBN 978-0-470-62452-4 (hardback)

ISBN 978-1-118-06030-8 (ebk)

 1. Immunohistochemistry. 2. Antigens. I. Shi, Shan-Rong, 1936– II. Taylor, C. R. (Clive Roy)

 QR183.6.A577 2010

 616.07'56–dc22

2010024561

PREFACE

The purpose of this collection of contributions by experts in the field is to set forth current knowledge with respect to antigen retrieval (AR) and immunohistochemistry (IHC). In so doing, we hope to contribute to the ongoing evolution of these methods, and the development of greater reliability and reproducibility of IHC. Effective standardization of AR and IHC would lend improved capabilities to IHC when employed in a “special stain” capacity. In addition, effective standardization would allow the development of IHC methods into tissue-based immunoassays, having true quantitative capabilities, analogous to the ELISA method. In order to attain this latter capability, quantifiable reference standards are required to calibrate the IHC method and assessment of proper tissue preparation. This book deals with all of these complex issues in a manner designed both to inform and to stimulate further research, particularly with respect to how AR methods might be employed for improved test performance.

The two of us (Shan-Rong Shi and Clive Taylor) have worked towards these goals, together for two decades, coming to the problem from different directions, but walking down a common path.

I (Shi) have been asked many times the same question: “What made you think of boiling a slide in a microwave oven before doing immunostaining?” There is no short answer for this question. I would like to share my story of AR to honor those people who touched my life and helped me meet my career goals.

My interest in IHC began in 1981 when I went to Massachusetts Eye and Ear Infirmary (MEEI) and Massachusetts General Hospital in Boston as a research fellow under the guidance of Drs. Harold F. Schuknecht, Max L. Goodman, and Atul K. Bhan. One of my projects was focused on IHC staining using archival formalin-fixed paraffin-embedded (FFPE) tissue sections of nasopharyngeal carcinoma obtained from China. I was deeply impressed by the sharp staining contrast between the cancer cells and the background inflammatory cells highlighted by a series of cytokeratin markers. Without IHC, not a single malignant cell would be identified. Because of the great diagnostic potential of IHC demonstrated by this project, I decided to exploit the application of this technique on thousands of valuable samples of human temporal bone collected by Professor Schuknecht, a world-renowned Otologist at MEEI. Although I tried many different IHC protocols with enzyme digestion for these archival formalin-fixed celloidin-embedded temporal bone sections, only moderate positive results were achieved with one antibody tested. This experience made me realize that the key point for successful IHC on archival formalin-fixed tissue sections was to find a method for the recovery of formalin-masked antigenicity, in the search for an AR approach.

In 1987, I had a research opportunity for a newly developed monoclonal antibody at InTek Laboratories, Inc., in Burlingame, California. This antibody was effective only on frozen sections, and I was asked to try to adapt it to FFPE tissue. At that time, enzyme digestion was the only option of choice, and it was not successful. As a result, I lost my job. I moved to a small room close to San Jose State University (SJSU), and in order to make a living, I started to work at a Chinese supermarket. I was insulted regularly by the sales manager, but these poor working conditions in a way inspired a strong feeling that I have never had before. I spent days and nights searching the literature at the library of SJSU, in order to answer what had become an obsession: “was formalin-masked antigenicity reversible or irreversible?” At that time online searching was not available. I read numerous volumes of the “index” page by page, taking notes line by line. I then looked for the journals one by one. In this way I searched all related literature regarding formalin and proteins starting from the most recent year back to 1940s. Finally, I found key clues to the answer in a series of studies published by Fraenkel-Conrat in the 1940s.1–3 Their studies indicated that cross-linkages between formalin and protein could be disrupted by heating above 100°C or by strong alkaline treatment. However, I did not think of using high-temperature heating of FFPE tissue sections because I believed so strongly that high temperature denatures the protein.

In 1989, after much trying, I obtained a job interview at BioGenex Laboratories, San Ramon. That was a sunny afternoon. I met Dr. Marc E. Key, Director of Research, in his office. As soon as I sat down, he asked me: “What can you do for BioGenex?” I answered: “I intend to develop a new method which enables IHC to be performed on archival FFPE tissues.” He was interested in my answer, and told me: “Many people have tried to find such a way but they all failed. If you could succeed, you would become world-famous.” I was hired. Today, when I look back, I appreciated Marc and Dr. Krishan L. Kalra, President of BioGenex, for giving me the opportunity that made it possible for my dream to come true.

Shortly thereafter, Marc gave me an abstract4, and suggested that I drop zinc sulfate solution on FFPE tissue sections prior to IHC staining for enhancing IHC staining results. After multiple attempts following the reported protocol, I did not observe any improvement. At this most frustrating moment, a microwave oven sitting at the table near my desk caught my attention and reminded me of those long forgotten studies performed by Fraenkel-Conrat. Even though I still doubted their conclusions and worried that high temperature might destroy all the antigens on the tissue sections, I decided to give it a try. I covered the FFPE sections with a few drops of zinc solution and heated them in the microwave oven for a few minutes. Unfortunately this attempt was not successful, because the solution evaporated. I decided to immerse the slides in a Coplin jar containing zinc solution and heated them twice in the oven for five minutes, in order to avoid drying the artifact during the boiling process. To my great surprise, I observed a significantly improved IHC staining signal with a clean background. I could not believe my eyes! I repeated the same experiment several times with similar results. This was “antigen retrieval (AR).”

The President of BioGenex, Dr. Kalra, invited three distinguished experts of IHC, Drs. Clive R. Taylor, Ronald A. DeLellis, and Hector Battifora to evaluate AR. They repeated this heat-induced AR protocol at their labs, and were all impressed by the great effects of this simple method. The first landmark article of AR was quickly accepted by Dr. Paul Anderson, Editor of the Journal of Histochemistry and Cytochemistry and published in 1991.5

At that time I started to work with Dr. Clive R. Taylor, Professor and Chairman of Pathology at the University of Southern California, Keck School of Medicine. Clive is a world renowned pioneer in archival IHC used for pathology since the early 1970s. With his kind help and support, I have been conducting a series of research projects on basic principles, further development, standardization and mechanisms of the AR technique. This work has yielded more than 40 peer reviewed articles and a book. Our AR research has been funded by NIH grant since 2001.

In 2000, we published Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology attempting to summarize major achievements in this interesting field with a wish to stimulate further development of AR-IHC.6 Since then, the AR technique has been accepted not only by pathologists who routinely apply AR-IHC for daily pathologic diagnosis in surgical pathology, but also by all scientists who work with cell/tissue morphology worldwide. Because of the expanded application of AR-IHC, the philosophy embedded in this simple technique has created several approaches for further study. For this second AR-IHC book, we categorize the recent literature concerning the AR technique into five sections: recent advances of AR techniques and their application, standardization of IHC, tissue/cell sample preparation, molecular mechanism of the AR technique, and proteomic analysis of proteins extracted from tissue/cells. Our goal is to summarize current key issues in these five fields, to stimulate future studies. It is our intention to initiate research projects addressing several critical issues such as standardization and quantifiable IHC, a desired topic for targeted cancer treatment as emphasized by the American Society of Clinical Oncology/College of American Pathologists Guideline for human epidermal growth factor receptor 2 testing in breast cancer documented in 2007.

Our plan for editing this book was enhanced by the Histochemical Society Annual Meeting held at the Experimental Biology 2007 Meeting in Washington, DC. Several interesting workshops with respect to tissue fixation for molecular analysis in pathology and cell biology, as well as tissue banking and sample preparation, were presented by world-renowned experts from Europe, the United States and Japan. We greatly appreciated all valuable presentations at these workshops that have been driving us in editing this book.

I (Taylor) find Shan-Rong Shi’s story to be interesting in many ways, not least because during its course the conventional scientific dogma of the day, was overturned, by experimental evidence. When Shan-Rong first spoke to me, in his early days at BioGenex, of the notion of boiling deparaffinized sections in buffer, I assured him that, based on what I know of proteins (which turned out to be remarkably little) the method was unlikely to work. After all if one heats complement to just 56 degrees, it is inactivated. But lurking in the back of my mind there was just enough of my own experience, to temper that initial judgment. Almost two decades earlier, when I had first tried to “stain” immunoglobulins in formalin fixed paraffin embedded tissues, I too had been assured by those senior to me that it would not work. Examination of the literature also supported the view that it was doomed to failure, but with just a few glimmers of hope. Cold alcohol processing of paraffin embedded tissues (Sainte-Marie) did allow demonstration of some antigens by immunofluorescence.

I was then working on my D. Phil thesis in Oxford, under the mentorship of Alistair Robb-Smith, murine models of lymphoma and Hodgkin’s disease. And I had problems. Already after just a year in the pathology department I was disconcerted to find that histopathology was not the definitive discipline that I had imagined, that it was subjective and that senior experienced pathologists could disagree vehemently with the diagnosis of a single slide. Recognition of the individual cells contributing to the development of “reticulum cells sarcomas’ ” in my murine models was even more of a challenge, with differing criteria offered by almost every expert whom I consulted, or every paper that I read. I resolved to try immunologic identification of cells using the specificity of antibodies. Like Shan-Rong, I was inspired by the literature of the 1940s, Albert Coons, and Astrid Fagraeus, and the genesis of the immunofluorescence method. A long story, cut short, by switching from fluorescein to peroxidase labeled antibodies we circumvented the problem of “background” fluorescence in FFPE sections, greatly simplifying the task. With Ian Burns, we obtained our first positive results. The late Dr. David Mason joined me in Oxford shortly thereafter. With his healthy disbelief of most of what was written, we did, what I encouraged Dr. Shi to do 20 years later, we did the experiments, and they worked. This was “immunoperoxidase.”7

In an exhilarating 2-year period we multiplied the world literature in the field, and then watched it grow exponentially. With the distant collaboration of Ludwig Sternberger we improved the “sensitivity.” All that was left then, was to try multitudes of new anti-sera (polyclonal antibodies) and the new monoclonal antibodies that began to pour from labs worldwide. Some of these gave results on FFPE tissue sections, most did not, or at least gave poor or inconsistent results after prolonged tissue manipulations. Thus the world of IHC was ripe for Dr. Shi’s equally unconventional idea, and the time was ripe to perform the experiment. The outcome we all now know. Many antigens can be “retrieved.” I have come to think of AR as “unfixation,” and by the use of AR, IHC has become more straightforward and more widespread.

The very success of AR has, however, added to the problems of performing IHC in a reliable and reproducible manner. Less care is taken, than once it was, with fixation, processing, antibody selection and titration, because with AR the stain “works.” In addition, many different labs perform IHC, treating it much like an H&E stain, without fully controlling the method, all because AR allows that to happen. Then the AR protocol itself has inevitably changed as others have sought to improve upon Shan-Rong’s original formula. The result has been a proliferation of different AR methods, that allow the staining of many antigens, in diverse ways that certainly are not standard, and are difficult to reproduce exactly. While AR unarguably has improved the overall qualitative results of IHC, it has in some ways hindered the development of more quantitative methods that are necessary for “measuring” prognostic or predictive markers. For example ER or HER2 results can be converted from negative to positive, from weak to strong and back again, by different AR protocols. Thus for any particular analyte, where the goal is measurement, AR also must be standardized. This book presents the views of many experts with broad and diverse experience in AR and IHC, about how to consolidate the gains that have been made, and how to extend them for diagnosis and research.

Antigen Retrieval Immunohistochemistry Based Research & Diagnostics is intended for clinical pathologists, molecular cell biologists, basic research scientists, technicians, and graduate students who undertake tissue/cell morphologic and molecular analysis and wish to use and extend the power of immunohistochemistry. It is our hope that the readers will find it informative and useful.

ACKNOWLEDGMENTS

We greatly appreciate those people who have contributed to or are working on the development of the AR technique. We express our sincere appreciation to all contributors for writing excellent chapters for this book. Our appreciation also goes to Dr. Richard J. Cote, for his support and collaboration of research, and to Chen Liu, Lillian Young, Leslie K. Garcia, Carmela Villajin, and William M. Win for their technical assistance. The editors wish to express our deep gratitude for the active support of George J. Telecki, Lucy Hitz, Kellsee Chu, Stephanie Sakson, and the production and sales teams at John Wiley & Sons, and Best-Set Premedia. We also appreciate Lindsey Gendall and Wayne Yuhasz of Artech House, Inc. We are grateful for permission to reproduce illustrations and data of published materials from all publishers appearing in every chapter of this book.

I (Shi) greatly appreciate valuable clinical and research training in Sichuan Medical College (currently Huaxi Medical School of Sichuan University, Chengdu, China), and I also would like to thank those who have helped me during the most difficult time in my life, especially Drs. Iwao Ohtani, Masahiro Fujuta, Andrew C. Wong, Jimmy J. Lin, as well as Susan Price, and Victor Jang. It would have been impossible for me to develop this technique without their kindness.

Shan-Rong Shi, MD

Clive R. Taylor, MD, PhD

REFERENCES

1. Fraenkel-Conrat H, Brandon BA, Olcott HS. The reaction of formaldehyde with proteins. IV. Participation of indole groups. J. Biol. Chem. 1947; 168: 99–118.

2. Fraenkel-Conrat H, Olcott HS. Reaction of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole, or indole groups. J. Biol. Chem. 1948; 174: 827–843.

3. Fraenkel-Conrat H, Olcott HS. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. J. Am. Chem. Soc. 1948; 70: 2673–2684.

4. Abbondanzo SL, Allred DC, Lampkin S, et al. Enhancement of immunoreactivity in paraffin embedded tissues by refixation in zinc sulfate-formalin. Proc. Annual Meeting US and Canadian Acad. Pathol. Boston: March 4–9, 1990. Lab. Invest. 1990; 62: 2A.

5. Shi SR, Key ME, Kalra KL. AR in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 1991; 39: 741–748.

6. Shi S-R, Gu, J, Taylor CR. Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology, Natick, MA: Eaton, 2000.

7. Taylor CR, Cote RJ. Immunomicroscopy. A Diagnostic Tool for the Surgical Pathologist, 3rd Edition. Philadelphia: Elsevier Saunders, 2006.

CONTRIBUTORS

BRIAN M. BALGLEY, Chief Scientific Officer, Bioproximity, LLC, Annandale, VA

JOHN M.S. BARTLETT, Professor of Molecular Pathology, Edinburgh University Cancer Research Centre, Edinburgh, UK

STEVEN A. BOGEN, Medical Director, Clinical Chemistry, Tufts Medical Center, Boston, MA

ROBERT E. CUNNINGHAM, Histologist, Department of Biophysics, Armed Forces Institute of Pathology, Rockville, MD

RICHARD W. DAPSON, Dapson & Dapson, LLC, Richland, MI

DAVID L. EVERS, Armed Forces Institute of Pathology, Rockville, MD, and Veterans Health Administration, Washington, DC

ALTON D. FLOYD, ImagePath Systems, Inc., Edwardsburg, MI

CAROL B. FOWLER, Research Associate and Technical Director, Proteomics Facility, Department of Biophysics, Armed Forces Institute of Pathology, Rockville, MD, and Veterans Health Administration, Washington, DC

JIANG GU, Professor of Pathology, Dean, Shantou University Medical College, Shantou, and Professor, School of Basic Medical Sciences, Peking University, Beijing, China

DAVID G. HICKS, Professor and Director, Surgical Pathology Unit, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY

MERDOL IBRAHIM, Manager, United Kingdom National External Quality Assessment Service Immunocytochemistry & In situ Hybridization, London, UK

BHARAT JASANI, Professor of Oncological Pathology, Head of Pathology, School of Medicine, Cardiff University, Cardiff, Wales, UK

JEFFREY T. MASON, Chairman, Department of Biophysics, Armed Forces Institute of Pathology, Rockville, MD

LORALEE MCMAHON, Supervisor, IHC Laboratory, University of Rochester Medical Center, Rochester, NY

KEITH D. MILLER, Chief Scientific Officer, UCL-Advanced Diagnostics, Cancer Institute, Director of the UK National External Quality Assessment Scheme for Immunocytochemistry & In-situ Hybridisation and, Fellow of the Institute of Biomedical Science, London, UK

MICHAEL M. MORGAN, Department of Histopathology, University Hospital of Wales, Wales, UK

MASAHIRO MUKAI, Research Associate, Department of Frontier Bioscience, Hosei University, Tokyo, Japan

TIMOTHY J. O’LEARY, Deputy Chief Research and Development Officer and Director, Clinical Science R&D Service, Veterans Health Administration, Washington, DC

VICKY REID, R&D Programme Manager, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK

KEVIN A. ROTH, Robert and Ruth Anderson Professor and Chair, Depart­ment of Pathology, University of Alabama at Birmingham, Director of Alabama Neuroscience Blueprint Core Center, and Editor-in-Chief of Journal of Histochemistry and Cytochemistry, Birmingham, AL

PAUL SCORER, Senior Project Leader, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK

MITSUTOSHI SETOU, Professor, Department of Molecular Anatomy, Hamamatsu University School of Medicine, Shizuoka, Japan

SHAN-RONG SHI, Professor of Clinical Pathology, University of Southern California Keck School of Medicine, and Associate Editor of Journal of Histochemistry and Cytochemistry, Los Angeles, CA

YAN SHI, Clinical Assistant Professor, and Attending Cytopathologist, New York University, Langone Medical Center, New York, NY

SESHI R. SOMPURAM, V.P. Research, Medical Discovery Partners LLC c/o Tufts Medical Center, Boston, MA

CHIARA SUGRUE, Director, Clinical Laboratory Operations, Division of Cytopathology and Assistant Professor, Hofstra School of Medicine, North Shore-Long Island Jewish Health System, New Hyde Park, NY

CLIVE R. TAYLOR, Professor of Pathology, University of Southern California Keck School of Medicine and Editor-in-Chief, Applied Immuno­histochemistry and Molecular Morphology, Los Angeles, CA

COLIN TRISTRAM, Innovations Manager, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK

JEREMY WALKER, Senior Research Scientist, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK

PATRICIA G. WASSERMAN, Senior Director, Division of Cytopathology, and Director, Cytopathology Fellowship Program, North Shore—Long Island Jewish Health System, Albert Einstein College of Medicine, New Hyde Park, NY

SHUJI YAMASHITA, Assistant Professor, Electron Microscope Laboratory, Keio University School of Medicine, Tokyo, Japan

Part I: RECENT ADVANCES IN ANTIGEN RETRIEVAL TECHNIQUES AND ITS APPLICATION

CHAPTER 1

STANDARDIZATION OF ANTIGEN RETRIEVAL TECHNIQUES BASED ON THE TEST BATTERY APPROACH

SHAN-RONG SHI and CLIVE R. TAYLOR

Following the development of the antigen retrieval (AR) technique in 1991,1 hundreds of articles have been published worldwide that document its application in immunohistochemistry (IHC) for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. In addition, there are numerous articles that focus on standardization of the AR technique, stimulated by the current demand for a more quantitative method of IHC.2–6 The critical importance of standardization of antigen retrieval immunohistochemistry (AR-IHC) has been emphasized by the American Society of Clinical Oncology and the College of American Pathologists in their Guideline Recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer.7 The problem was, however, recognized and addressed to some degree much earlier. To optimize the results of AR-IHC in formalin paraffin sections, a “test battery” approach was proposed in 1996.8 The basic principle of this approach is based on the fact that two major factors influence the achievement of a satisfactory result of AR-IHC, namely, the heating condition (heating temperature × heating time) and the pH value of the AR solution (in which the FFPE tissue sections are immersed during heating).8–12 In practice, it suffices to test the (new) primary antibody using three different pH values, ranging from low (acidic), moderate (neutral), and high (basic) buffer solutions (or other comparable commercial AR solutions) under three heating temperatures: low (below boiling), moderate (boiling), and high (pressure cooker or autoclave), to establish an optimal AR protocol for tested antibodies (Table 1.1). Subsequently, numerous investigators have demonstrated the advantages of using this simple test battery method. As emphasized by O’Leary,2 the use of a “test battery” provides a rapid way to optimize AR for a particular antibody/antigen pair.

TABLE 1.1 Test Battery Suggested for Screening an Optimal Antigen Retrieval Protocol

a One more slide may be used for control without AR treatment. Citrate buffer of pH 6.0 may be used to replace Tris–HCl buffer, pH 7.0–8.0, as the results are the same.

b The temperature of super-high at 120°C may be reached by either autoclaving or microwave heating at a longer time.

c The temperature of mid-high at 90°C may be obtained by either a water bath or a microwave oven monitored with a thermometer.

Reprinted with permission from Shi et al., J. Histochem. Cytochem. 1997; 45: 327–343.

Recent studies have further extended the application of this approach to establish and validate the optimal AR protocol for various antibodies (exemplified in Table 1.2) with different detection systems, employing a multi-tissue microarray (TMA) to achieve a rapid and accurate evaluation.26,27 It has become apparent that significant differences can be found in IHC staining results among various primary antibodies and different detection systems with the use of different AR protocols. For example, Pan et al.27 evaluated the consistency of IHC staining for four antibodies to thyroid transcription factor (TTF)-1, manufactured by Dako, Zymed, Novocastra, and Santa Cruz, employing TMA blocks of 77 hepatocellular carcinomas and 334 nonhepatic epithelial tumors, using two solutions for AR treatment. Significantly different cytoplasmic IHC staining results were observed among different antibodies, as well as different AR solutions (e.g., Dako Target Retrieval Solution vs. ethylenediaminetetraacetic acid [EDTA] buffer at pH 8.0). In another study, Gill et al.21 standardized an AR method for IHC staining using antibody to a neuronal nuclear protein, NeuN, as the outcome measure. They compared three different pH values of AR solutions including low, middle, and high pH, with heating at three temperatures of 95, 100, or 105°C, for 15 or 20 min. They found that heating FFPE tissue sections in an alkaline pH buffer at high temperature gave the best results. The utility of the test battery approach used to establish optimal AR protocols has been demonstrated by abundant literature as summarized in Table 1.2.

TABLE 1.2 Randomly Selected Examples of Test Battery Approach Documented in Abundant Literature

Note: All tissue samples are human source unless otherwise noticed.

Ca., carcinoma; MW, microwave.

The increasing attention directed to the adverse effects of variation in sample preparation upon the quality of IHC staining of FFPE tissues has served to reinforce the importance of determining the optimal AR method for each antibody/detection system/antigen to achieve optimal retrieval and optimal staining of tissues that may have been processed and stored in different and unknown ways (see Chapter 5 for details). Practically, in considering the busy workload in a clinical service laboratory, we recommend a two-step procedure based on the typical design of a test battery (Table 1.1): in the first step, test three AR solutions at different pH values under one heating condition (100°C for 10 min) to find the optimal pH value; in the second step, test optimal heating conditions based on the optimal pH identified in step 1.28 Similarly, Hsi29 recommended using microwave pressure cooker as the standard heating condition for testing two commonly used AR solutions, citrate buffer of pH 6.0 and EDTA solution at pH 8.0, along with protease digestion. With the goal of identifying the optimal AR protocol for a new primary antibody, they used five different concentrations of the antibody, including the manufacturer’s recommended dilution, plus two serial twofold dilutions above and below this concentration. As seen in Table 1.2, many investigators have already accepted the basic principle of test battery, incorporating three levels of pH values and three heating conditions (Table 1.1). However, within this model, different investigators have used different heating methods and different AR methods to achieve optimal results for their individual laboratories. With this broad variety of approaches, clearly, we are a long way from achieving a universal method, even if such is possible.

1.1 SEARCHING FOR NOVEL CHEMICAL SOLUTIONS

Namimatsu et al.30 reported a novel AR solution containing 0.05% citraconic anhydride, pH 7.4, for heating FFPE tissue sections at 98°C for 45 min. They compared the IHC staining results using 62 commonly used antibodies and other conventional AR protocols (0.01 M citrate buffer, pH 6.0 in a pressure cooker; or 0.1 M Tris–HCl buffer containing 5% urea, pH 9.0 microwave heating for 10 min), and found that most antibodies showed stronger intensity with the new method. In particular, some difficult-to-detect antigens such as CD4, cyclin D1, granzyme β, bcl-6, and CD25 gave distinct IHC staining signals only by using the new protocol, leading to a claim that the method might be a candidate for the “universal” approach.

We therefore tested Namimatsu’s protocol and also obtained satisfactory results.31 Among 30 antibodies tested, more than half (53%) showed a stronger intensity of IHC when using the citraconic anhydride for AR, as compared to citric acid buffer, whereas 12 antibodies (43%) gave equivalent results. There was only one antibody (OC-125) that, in our hands, gave a stronger intensity using conventional citric buffer for AR. When using citraconic anhydride for AR, the heating conditions of boiling (100°C) or less than boiling (98°C) temperature yielded identical results for most antibodies tested (90%). However, 3 of 30 antibodies showed lower intensity at 100°C. In addition, some antibodies showed nonspecific background staining at 100°C. In particular, we demonstrated that when using antibody to retinoblastoma protein (pRB), the new protocol had advantages over a previously published low pH protocol,8 including superior morphologic preservation, greater reproducibility, and more intense staining signal.

As a further motivation, there is evidence that establishing the optimal AR protocol will also contribute to standardization of IHC, through “equalizing” variable IHC staining results obtained following different times of formalin fixation. In the light of the studies described above, further studies were conducted as to the utility of the citraconic anhydride method.

First Step: A comparative study of IHC staining for pRB was carried out using paired sections of frozen versus FFPE cell/tissue samples, comparing citraconic anhydride as the AR solution under two different temperatures (98oC vs. 100oC), with solutions of low pH buffer (acetate buffer, pH 1–2) and citrate buffer (pH 6.0). Findings are summarized in Table 1.3. Conventional citrate buffer yielded inconsistent and weaker signals for all specimens, except the cell line T24 (Table 1.3, Fig. 1.1). Stronger intensity was found in pRB-positive cases, while using the citraconic anhydride for AR (Fig. 1.1), although more nonspecific background staining was observed using citraconic anhydride under boiling condition (Fig. 1.1, C vs. D, and R vs. S).

TABLE 1.3 Comparison of pRB-IHC between Frozen and Paraffin Sections Using Four Protocols of AR

Notes: T24 and J82 are cell lines of bladder cancer. Cases 1 to 4 are specimens of human bladder cancer.

a Although peripheral area of the slide showed a percentage of positive staining about 50%, the central area of the slide showed significantly weak positive result.

Reproduced with permission from Shi et al., Biotech. Histochem. 2007; 82: 301–309.

Figure 1.1 Comparison of pRB-IHC staining results for frozen and FFPE tissue sections using four AR protocols. All images are arranged in the same order as given in Table 1.3, indicating all scores indicated in the table. T24 and J82 are two cell lines, Ca #1 and Ca #2 are specimens of human bladder cancer, frozen means frozen cells or tissues fixed in acetone, other terms listed in the top line represent FFPE tissue sections after various AR treatments: Low pH, AR solution at low pH value; CAPC, citraconic anhydride solution with boiling; CA98C, citraconic anhydride solution with heating at 98°C; citrate, conventional boiling heating with citrate acid buffer at pH 6.0. Original magnification × 200.

Reproduced with permission from Shi et al., Biotech. Histochem. 2007; 82: 301–309.

See color insert.

Second Step: For further evaluation, a comparative IHC study was performed using citraconic anhydride and conventional AR protocols with a TMA of 31 cases of bladder cancer. Findings are summarized in Table 1.4. Only 27 cases were available for evaluation due to loss of tissue cores for four cases. Among 27 cases, there were 6, 8, and 13 cases for strong, moderate positive, and negative pRB-IHC, respectively. Identical percentages of pRB-positive nuclei were found in all cases, using either of the two protocols for citraconic anhydride or the low pH solution for AR. Inconsistent and significantly weaker nuclear pRB staining results were found when using citrate buffer of pH 6.0 for AR (Table 1.4; Fig. 1.2).

TABLE 1.4 Comparison of pRB-IHC in 27 Cases of FFPE Tissues of Bladder Cancer Using Four Protocols of AR

Notes: CA98°C, heating tissue sections in 0.05% citraconic anhydride at 98°C for 45 min; CAPC, heating tissue sections in 0.05% citraconic anhydride in a plastic pressure cooker using microwave oven for 30 min; Low pH, heating tissue sections in acetic buffer of pH 1–2 for shorter time as described in the text; Citrate, conventional citrate acid buffer 0.01 M at pH 6.0 with same heating condition of a plastic pressure cooker described above.

Reproduced with permission from Shi et al., Biotech. Histochem. 2007; 82: 301–309.

Figure 1.2 Examples of immunostaining intensity from comparison of pRB-IHC in 27 cases of FFPE tissues of bladder cancer (Table 1.4). (A–D) Negative (<10%) showing a few weak positive nuclei (arrows); (E–H) moderate positive (>10%); (I–P) strong positive (>50%). Arrows indicate positive nuclear staining for some lymphocytes or other stromal cells as an internal control. Note the lack of nuclear hematoxylin counterstaining due to low pH AR treatment. The order of cases are indicated in Table 1.4.

Reproduced with permission from Shi et al., Biotech. Histochem. 2007; 82: 301–309.

See color insert.

Third Step: The Western blotting technique, applied to cell extracts, was used to confirm the pRB immunostaining results in two bladder cancer cell lines of T24 and J82, giving quantitative results for pRB in the two cell lines, comparable with that demonstrated by IHC (Fig. 1.3).

Figure 1.3 Western blotting of pRB protein extracted from two fresh cell lines, T24 and J82. The pRB proteins in fresh T24 cell line showed a stronger band than that obtained from J82 cell line. The Western blotting results correlated well with IHC staining intensity (Table 1.3 and Fig. 1.1).

Reproduced with permission from Shi et al., Biotech. Histochem. 2007; 82: 301–309.

Although the novel AR protocol using citraconic anhydride improved the intensity of IHC on FFPE tissue sections for more than half of the antibodies tested, compared to that achieved by other conventional AR protocols, not all antibodies benefitted, which would argue that the citraconic anhydride method does not serve as a truly universal AR protocol. Indeed, many investigators (Table 1.2) have concluded that different antigens may require different “specific” AR protocols. In this respect, the “test battery” is a convenient and cost-effective method for assessing the appropriate AR protocol.2,8 Nevertheless, the present data certainly support inclusion of the citraconic anhydride AR method in such a “test battery.” With respect to the two heating temperatures for citraconic anhydride, the ultimate choice of method for any laboratory may depend on the equipment available.

In a study involving decalcified FFPE rat joint tissue sections and a variety of AR methods, Wilson et al.32 reported successful application of 0.2 M boric acid at pH 7.0 as the AR solution combining a low-temperature incubation (60°C for 17 h). The principal advantage of this AR protocol was that it minimized lifting or loss of decalcified hard tissue sections from charged slides. Their basic approach for establishing an optimal AR protocol was a “test battery” as described above. In a separate series of studies, based upon prior literature,33,34 and with the goal of reducing tissue damage due to boiling during AR, Frost et al.35 compared a microwave boiling AR protocol and a combination AR protocol that included predigestion in 0.1% trypsin in phosphate-buffered saline (PBS) for 15 min, followed by low-temperature heating in a water bath at 80°C for 2 h. Although tissue damage was reduced by using the low-temperature AR protocol, not all antigens could be recovered equally by this method. They concluded that prior to setting up a new IHC stain, it is critical to assess AR protocols, and primary antibody concentrations as well as detection systems, their standard end point was that method giving the strongest IHC staining signal (maximal retrieval level). In addition, Frost and colleagues also emphasized that the IHC results should be correlated with clinical behavior of diseases in order to provide data that are directly useful for treatment. With a similar principle in mind, Umemura et al.36 undertook a comparative study of IHC evaluation of hormone receptor status for 861 breast cancer samples with data from IHC and biochemical methods. They demonstrated that optimizing the AR treatment, primary antibodies, and detection systems significantly affects technical validation of IHC data for hormone receptors. They emphasized that the cutoff point should be set higher to reflect the increasing IHC “scores” achieved by more sensitive IHC method, based on the correlation of biochemistry and IHC, as well as clinical follow-up data.

1.2 ANTIBODY AND DETECTION SYSTEM-DEPENDENT TEST BATTERY

Numerous recent articles have emphasized that the application of test battery for establishing an optimal AR protocol is also dependent on the primary antibody and the subsequent detection system. In other words, if an optimal AR protocol is good for antibody clone “1” to protein “A” employing detection system “B,” it is not necessarily good for antibody clone 2 to protein A, using the same or different detection systems, but a different AR protocol might give acceptable results. In this respect AR, while in some respects “leveling the playing field” so that many antigens may be detected, in some instances does add yet another variable to achieving consistency among different laboratories. For example, Pan et al.27 found variable cytoplasmic IHC staining results of TTF-1 for hepatocellular carcinoma, which depended on different sources of the primary antibody and different AR methods. However, they only tested two conditions of AR. Similarly, Slater and Murphy25 showed great variation in the effectiveness of different AR protocols for IHC staining of an anti-mouse IL-6 antibody (purchased from R&D System, MN, USA) using three AR solutions (pH values of 10.0, 7.0, and 2.0) and four heating conditions (100°C for 10 min, 90°C for 30 min, 80°C for 50 min, 70°C for 1 h). They finally found that there was no staining for IL-6 when using AR solution at pH 10.0 or 7.0 but obtained positive IHC staining at pH 2.0 heated at 80°C for 50 min. Higher temperature heating of 100°C resulted in damage of tissue sections, while lower temperature of 70°C resulted in weak IHC staining.

Again using the test battery principle, Kim et al.37 compared IHC staining results of two monoclonal antibodies to CD4 (clone: YG23 and 1F6) and three monoclonal antibodies to CD8 (clone: YG20, DN17, and 1A5) on archival FFPE tissue sections using eight different AR solutions at pH values ranging from 2 to 10, combining two heating conditions (heating in a microwave oven vs. heating in a pressure cooker). They found that among five monoclonal antibodies tested, only 1F6 (CD4), and 1A5 (CD8) worked on FFPE tissue sections, and that an AR solution of borate at pH 8.0, containing 1 mM EDTA, and 1 mM NaCl yielded the best IHC staining results for CD4 and CD8. Note, however, that according to their data, it is clear that the use of Tris buffer at higher pH (9–10) also provides satisfactory IHC staining intensity for these two antibodies, a finding having extensive support in the published literature.14,16,20,21,38–43 Kim et al.19 also studied seven AR solutions at pH ranging from 2 to 10 for 29 commonly used antibodies and concluded that the optimal AR protocol depends on the particular antibody tested; therefore, the best AR solution should be sought for each antibody, and there is no “universal” approach, nor does AR add reproducibility among laboratories in this context.

Vassallo et al.44 compared two routinely used antibodies of estrogen receptor (ER), 1D5 (Dakopatts [Carpinteria, CA], code E7101) and 6F11 (Newmarker [Fremont, CA], code MS391-S1) by using two AR protocols, citrate buffer at pH 6.0 and Tris-EDTA at pH 8.9. For IHC staining, they adopted three different detection systems, EnVision, EnVision Plus, and labeled streptavidin-biotin (LSAB) peroxidase complex (all three systems purchased from Dakopatts). In their study, antibody 6F11, using the citrate AR protocol with EnVision, yielded a poorer IHC signal than that obtained by using Tris–EDTA solution for AR treatment. Kan et al.45 did a similar comparative study to evaluate the efficacy of different AR protocols, using sodium citrate, citric acid, Tris–HCl, and EDTA buffers of pH 4, 6, and 8, with four different clones of monoclonal antibodies for microtubule-associated protein (MAP)-2-IHC. Staining on FFPE guinea pig brain tissue sections, they found that satisfactory IHC staining was obtained only when MAP-2 antibody clone AP18 was used with the use of AR heating in citric acid buffer of pH 6.0. Gutierrez et al.46 tested the immunoreactivity of 25 monoclonal antibodies to different leucocyte antigens on FFPE tissue sections, with differing fixation conditions. Employing the test battery approach and the biotin-tyramide amplification system, they concluded that all 25 antibodies tested were readily detectable using an appropriate combination of antibody, AR method, and signal amplification system. Again, no method was optimal for all.

1.3 APPLICATION OF TMA TECHNIQUE FOR TEST BATTERY

Multi-tissue technique has been used for many years in IHC staining to screening numerous samples on one single slide.47–49 Based on these early observations, TMAs were introduced in IHC for rapid study and to economize in the use of expensive reagents.50 The TMA technique has the advantage of collecting hundreds of tissue samples on one single slide and provides the additional advantage of increasing the uniformity of staining across the TMA tissues, by reducing diversity of staining signals that result from separate staining of hundreds slides, perhaps on different days, by different technologists. Recent cooperative studies among multiple research centers, such as the BrainNet Europe Consortium, demonstrated the possibility of using the TMA technique in standardization of AR-IHC to achieve reliable results between different laboratories.51 A multi-tissue “spring-roll” section provided a foundation for standardization of AR-IHC based on giving improved reproducibility and performance of AR-IHC staining results.52 Camp et al.53 validated the availability of TMA using three common antigens (ER, progesterone receptor [PR], and HER2) in FFPE tissue sections of invasive breast carcinoma and demonstrated that many proteins retained antigenicity for longer than 60 years using optimal AR pretreatment. Based on numerous studies, a combination of tissue array with AR technique provides an approach to optimize the use of archival FFPE tissue sections with a variety of fields.54 The advantages are further enhanced by the application of recently developed image analysis software (AQUA) that is designed for quantitative IHC in TMA using an automatic scan.55

1.4 SCIENTIFIC ACCURACY OF IHC RELYING ON OPTIMAL AR PROTOCOL

As described above, an optimal AR protocol established by test battery approach produces the best IHC result, defined as the maximal retrieval level (see Chapter 5). It is worthy to note, although not surprising, that not only is “intensity” of staining affected by the choice of the AR method, but also in some cases the distribution and pattern of staining. For example, Mighell et al.56 demonstrated that fibronectin protein expression pattern, using a polyclonal antibody, was dependent on methods of AR. They used archival FFPE specimens of oral pyogenic granuloma and fibroepithelial polyp, and compared four AR protocols: combinations of enzyme digestion, microwave boiling in citrate buffer, or Tris–HCl buffer at pH 6 or 7.8, and autoclave. They found that after enzyme digestion, there was intense IHC staining in vascular endothelial cells but no staining or minimal staining in connective tissue; in contrast, microwave AR yielded IHC positive staining in connective tissue but no specific vascular staining, while autoclave AR showed positive staining in connective tissue and epithelial nuclei. Comparing these findings with the patterns obtained on frozen tissue sections, there was positive labeling in both vascular endothelial cells and connective tissue. They postulated that different protocols might expose different epitopes. The findings again emphasize the need for optimizing AR for IHC staining in FFPE tissue, while highlighting the concern that AR, when applied without rigorous validation, in fact increases variability observed in IHC staining. Potential causes of these diverse IHC patterns were discussed, including such possibilities as cross-reactivity of the different antibody species within the polyclonal antibody. It is critical to emphasize the fact that variable protein expression patterns may result from different AR protocols, and caution must be taken to avoid misinterpretation. Subsequent published studies obtained somewhat contrasting results.57,58 Yamashita and Okada58 studied the mechanism of heat-induced AR employing SDS-PAGE, Western blotting, and IHC. They adopted the same rabbit polyclonal antibody to fibronectin (F-3648, Sigma [St. Louis, MO]) as used by Mighell et al.56 and found that heating FFPE tissue sections in pH 9.0 buffer solution yielded strong positive fibronectin staining along the basal lamina in the hepatic sinusoid of mouse liver tissue, but no staining when using pH 6.0 buffer. Moreover, they found that boiling FFPE tissue sections in pH 9.0 buffer, followed by heating in pH 6.0 buffer also gave absent or minimal staining. However, boiling the same FFPE slide in pH 9.0 buffer could achieve strong positive staining of fibronectin, suggesting that the pH of AR solution may be an essential factor for proper refolding of epitopes to react with antibodies (see Part IV for details on the study of mechanism of AR).

The generation of artifacts has also been an intermittent concern. Hayashi et al.59 reported a heat-induced artifact for conversion of Amadori products of the Maillard reaction to Nε-(carboxymethyl) lysine that had the potential to affect IHC staining. However, among thousands of articles pertaining to numerous antigen/antibody combinations based on AR-IHC in FFPE tissue sections, “false-positive staining” has not been convincingly demonstrated. Nevertheless, caution must be exercised when evaluating a new antibody using AR-IHC staining procedure for FFPE tissue sections. The following issues should be kept in mind to minimize unexpected or spurious staining results: (1) understanding the specificity of the antigen/antibody under test and the distribution in cells/tissues based on information provided by biochemical research; (2) examination of previous IHC staining reports in fresh cell/tissue samples pertaining to this antibody; (3) staining of negative control FFPE tissue section under identical AR treatment but omitting the primary antibody; (4) critical morphological analysis to confirm that observed patterns of distribution are consistent with other known information relating to pathology, molecular biology, and clinical outcome; and (5) in suspicious cases, further confirmation should be sought by using other methods such as Western blotting to confirm the IHC result as emphasized by Wick and Mills.60

1.5 ACCURACY OF AR-IHC AS DEMONSTRATED BY IEM AND OTHERS

In recent years, with more accurate quantitative methods, numerous immunoelectron microscopic (IEM) studies have validated the application of AR in archival Epon or other plastic material embedded tissues fixed in aldehyde, plus other fixatives such as osmium tetroxide.16,57,61–63 Ramandeep et al.62 designed an interesting study using Escherichia coli DH5α cells as a test model, based on quantitative measurements of immunogold labeling IEM, compared to enzyme-linked immunosorbent assay (ELISA) data, to optimize various tissue processing and IEM procedures including AR. They demonstrated that AR can achieve approximately 90–100% retrieval efficiency for osmium-postfixed material, a very interesting finding because cell/tissue samples postfixed with osmium provide the best preservation of ultrastructural morphology for IEM study. Hann et al.57 carried out a quantitative IEM study based on carefully counting gold labeling particles of collagen IV and fibronectin in the basement membrane underlying the cells of Schlemm’s canal from archival aldehyde-fixed LRWhite-embedded eye tissue and found that duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled, and no artifacts were found after AR. They concluded that heat-induced AR can be used on selected extracellular matrix antigens to achieve positive label that would otherwise be lost due to fixation and storage. The test battery approach has also been evaluated by quantitative IEM using gold labeling techniques.16,17,61 Based on comparison of two polyclonal anti-nestin antibodies, Almqvist et al.64 demonstrated precise localization of nestin in pediatric brain tumors, previously a controversial issue in the IHC literature. To confirm the reproducibility of counting neurons and glia in human brain tissue sections by IHC staining, Lyck et al.26 compared 29 different antibodies with various AR protocols using four buffers (Table 1.2). They reported that it is possible to use IHC staining for reproducible cell counting in brain tissue sections, based on optimal AR protocols, with well-preserved sample materials.

1.6 SUMMARY

Standardization of AR technique should be based on the test battery principle. Achieving the “maximal retrieval level” of IHC staining intensity is a guideline for standardization.Three pH values (acidic, neutral, and basic AR solution), and three heating conditions (under boiling, boiling, and pressure heating) are recommended for the basic “test battery.” However, alternative procedures may be applied according to laboratory facilities and routine protocols as described above. Currently, citrate buffer pH 6.0, Tris–EDTA buffer pH 8–9, and certain AR solutions at lower pH, such as boric acid pH 2–3, or acidic acid buffer pH 2, as well as 0.05% citraconic anhydride pH 7.4, may be used to evaluate the optimal AR protocol.TMAs are valuable in rapid and cost-effective evaluation of new antibodies, in determining optimal AR methods.

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