Analytical Morphology: Theory, Applications and Protocols

298 Pages·1996·11.683 MB·English

by  Jiang Gu (auth.)

Analytical Morphology:
Theory, Applications
and Protocols
Analytical Morphology:
Theory, Applications
and Protocols
Soft Cover Edition, Eaton Publishing Co.
Jiang Gu
Institute of Molecular Morphology
Mount Laurel, NJ, USA
Library of Congress Cataloging-in-Publication Data
[CATALOGING IN PROGRESS]
Copyright© 1997 Eaton Publishing Co.
Softcover reprint of the hardcover 1st edition 1997
All rights reserved. No part of this publication may be reproduced, stored in
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of the copyright owner.
Reproduction or translation of any part of this work beyond that permitted
by Section 107 or 108 of the 1976 United States Copyright Act without the
permission of the copyright owner is unlawful. Requests for permission or
further information should be addressed to the Permissions Department,
Eaton Publishing Co.
ISBN 978-1-4612-8683-7 ISBN 978-1-4612-4166-9 (eBook)
DOI 10.1007/978-1-4612-4166-9
987654321
The front cover shows part of a summary screen from the neural network
assisted cervical screening program PAPNET. This smear represents the first
case of cervical carcinoma in situ diagnosed using the program (seeM. Boon
et al. in this volume).
IV
Analytical Morphology: Theory, Applications and Protocols
CONTENTS
Chapter 1. Antigen Retrieval Technique: A Novel Approach to
Immunohistochemistry on Routinely Processed Tissue Sections
S.-R. Shi, J. Gu, K.L. Kalra, T. Chen, R.J. Cote and C.R. Taylor ........................ 1
Chapter 2. Immunogold-Silver Staining-Autometallography:
Recent Developments and Protocols
G.W. Hacker, G. Danscher and C. Hauser-Kronberger .............................. .41
Chapter 3. Elimination of Background Staining in Immunocyto
chemistry
J. Gu and N. Agrawal ..........................................................5 5
Chapter 4. Principles, Applications and Protocols of Microwave
Technology for Morphological Analysis
A.S.-Y. Leong and F.J. Leong ...................................................6 9
Chapter 5. Microwave Immunohistochemistry: Advances in
Temperature Control
T.-S. Choi, M.M. Whittlesey, S.E. Slap, V.M. Anderson and J. Gu .....................9 1
Chapter 6. Applications of In Situ Hybridization and Immunocyto-
chemistry for Localization and Quantification of Peptide Gene
Expression-A Lesson From Islet Amyloid Polypeptide
H. Mulder, B. Ahren and F. Sund1er ............................................. 115
Chapter 7. Fluorescence In Situ Hybridization
S. Luke, V. Belogolovkin, J.A. Varkey and C.T. Ladou1is ........................... 139
Chapter 8. An Optimized Protocol for In Situ Hybridization Using
PCR-Generated 33P-Labeled Riboprobes
L.H. Lu and N.A. Gillett ...................................................... 175
Chapter 9. Histologic Validation and Diagnostic Consistency of
Neural Network-Assisted Cervical Screening: Comparison with the
Conventional Procedure
M.E. Boon, L.P. Kok, S. Beck and M.R. Kok ..................................... 191
Chapter 10. Neuronal Tract Tracing Methods: Recent Developments
for Light and Electron Microscopy
G.E. Meredith .............................................................. 201
Chapter 11. Theory and Applications of Confocal Microscopy
B. Matsumoto, I. Hale and T. Kramer ........................................... 231
Chapter 12. Standardization and Practical Guidelines of Image
DNA Cytometry in Clinical Oncology
U.G. Falkmer and G.W. Hacker ................................................2 45
Index ..................................................................... 262
Addendum ............................................................... A-1
v
CONTRIBUTORS L.P.Kok
Institute for Theoretical Physics
NeeraAgrawal
Groningen, The Netherlands
Deborah Research Institute, Browns Mills, NJ, USA
Myrthe R. Kok
BoAhren
Leiden Cytology and Pathology Laboratory
Department of Medicine, Malmo Univ. Hospital Leiden, The Netherlands
University of Lund, Lund, Sweden
Theresa Kramer
Virginia M. Anderson
Department of Ophthalmology and Pathology
Department of Pathology
University of Arizona School of Medicine
SUNY Health Science Center at Brooklyn Tucson,AZ
Brooklyn, NY, USA
Charles T. Ladoulis
S.Beck
Department of Pathology
Leiden Cytology and Pathology Laboratory
Maimonides Medical Center, Brooklyn, NY, USA
Leiden, The Netherlands
Anthony S.-Y. Leong
Victoria Belogolovokin
Division of Tissue Pathology
Department of Pathology
Institute of Medical and Veterinary Science
Maimonides Medical Center, Brooklyn, NY, USA Adelaide, South Australia
Mathilde E. Boon
F. Joel Leong
Leiden Cytology and Pathology Laboratory
Division of Tissue Pathology
Leiden, The Netherlands
Institute of Medical and Veterinary Science
Taiying Chen Adelaide, South Australia
BioGenex Laboratories, San Ramon, CA, USA LucyH.Lu
Tak-Shun Choi PE Applied Biosystems, Foster City, CA, USA
Department of Pathology
Sunny Luke
SUNY Health Science Center at Brooklyn Department of Pathology
Brooklyn, NY, USA
Maimonides Medical Center, Brooklyn, NY, USA
Richard J. Cote
Gloria E. Meredith
Department of Pathology
Department of Anatomy
University of Southern California Royal College of Surgeons in Ireland
School of Medicine, Los Angeles, CA, USA
Dublin, Ireland
Richard J. Cote
Brian Matsumoto
Department of Pathology
Neuroscience Research and Department of
University of Southern California Biological Sciences
School of Medicine, Los Angeles, CA, USA University of California, Santa Barbara, CA, USA
Gorm Danscher
Hindrik Mulder
The Steno Institute, Department of Neurobiology Department of Physiology and Neuroscience
University of Aarhus, Aarhus, Denmark Section for Neuroendocrine Cell Biology
Ursula G. Falkmer University of Lund, Lund, Sweden
Department of Oncology and Pathology Shan-Rong Shi
Karolinska Institute and Hospital Department of Pathology
Stockholm, Sweden
University of Southern California
Nancy A. Gillett School of Medicine, Los Angeles, CA, USA
Sierra Biomedical, Sparks, NV, USA
Steven E. Slap
JiangGu Energy Beam Sciences, Agawam, MA, USA
Institute of Molecular Morphology
Frank Sundler
Mount Laurel, NJ, USA
Department of Physiology and Neuroscience
Gerhard W. Hacker Section for Neuroendocrine Cell Biology
Medical Research Coordination Center University of Lund, Lund, Sweden
University of Salzburg, Salzburg, Austria Clive Taylor
Irene Hale Department of Pathology
Neuroscience Research Institute and Department University of Southern California
of Biological Sciences School of Medicine, Los Angeles, CA, USA
University of California, Santa Barbara, CA, USA Jerry A. Varkey
Cornelia Hauser-Kronberger Department of Pathology
Institute of Pathological Anatomy Maimonides Medical Center, Brooklyn, NY, USA
Salzburg General Hospital, Salzburg, Austria Michael M. Whittlesey
Krishan L. Kalra Energy Beam Sciences
BioGenex Laboratories, San Ramon, CA, USA Agawam, MA, USA
vi
Analytical Morphology: Theory, Applications and Protocols
Preface
This volume is a collection of chapters contributed by leading experts in
the emerging field of analytical morphology. Its contents cover a wide range
of techniques for morphologic research and diagnosis and it is intended for
anyone who wants to keep abreast of the rapid development in this field.
Analytical morphology is a contemporary science dealing with the analy
sis of shape, size and color arrangement of cell and tissue components by
means of a variety of analytical maneuvers. It differs from conventional mor
phology and histopathology in that it employs methods beyond routine hema
toxylin and eosin or histochemical staining. To a great extent, the advance
ment of analytical morphology is based on new advances in other disciplines,
such as immunology, molecular biology, laser technology, microwave tech
nology and computer science. Using these new methods, particular cellular
components that would otherwise remain invisible, such as peptides, pro
teins, or nucleotide sequences, are highlighted by visible markers through
chemical, physical, immunological or molecular biological reactions. These
methods include immunocytochemistry, in situ hybridization, in situ poly
merase chain reaction, antigen retrieval, image analysis, and the like.
Analytical morphology is the foundation of contemporary medicine. It
provides concrete and visible evidence for many conceptual deductions of
other life science disciplines. A survey of leading medical and biological
journals reveals that more than half of the scientific and clinical articles re
porting original observations were achieved by morphological means in total
or in part. More than ninety percent of the articles published in recent issues
of two popular pathology journals presented results obtained through ad
vanced morphologic methods, particularly immunocytochemistry and in situ
hybridization. This volume offers an extensive overview and practical guid
ance for these topics.
The chapters originated from material published recently in Cell Vision -
Journal of Analytical Morphology and have been updated and enhanced by
the authors to meet with the purpose of this volume. They are written in a
comprehensive manner and generally comprise a review of a particular topic,
examples of applications, protocols, discussions and comments. The authors
have made significant contributions to the development or modification of
the techniques presented.
I am indebted to the contributing authors for their selfless effort in review
ing these subjects and sharing their personal experiences. I want to thank
those who helped me coordinate this project, in particular David R. Kersten,
Tak-Shun Choi, Neera Agrawal and Gayle Englund. I am grateful to Chris
tine McAndrews, Managing Editor of Cell Vision, for her assistance.
Jiang Gu, MD, PhD
vii
CHAPTER!
Antigen Retrieval Technique: A Novel
Approach to Immunohistochemistry
on Routinely Processed Tissue Sections
Shan-Rong ShP, Jiang Gu2, Krishan L. KalraJ, Taiying Chen3,
Richard J. Cote1 and Clive R. Taylor1
1D epartment of Pathology, University of Southern California School of
Medicine, Los Angeles, CA; 2lnstitute of Molecular Morphology, Mount
Laurel, NJ; 3BioGenex Laboratories, San Ramon, CA, USA
SUMMARY
The antigen retrieval (AR) technique is a non-enzymatic antigen unmasking
method that is utilized prior to immunohistochemical staining of formalin-fixed,
paraffin-, celloidin- or plastic-embedded tissue sections. Two variations exist; the
first, based on high-temperature heating, is applicable to formalin-fixed, paraffin
embedded sections; the second is a non-heating method that involves immersing the
tissue section in an AR solution, such as a NaOH-methanol solution, and is suited to
celloidin-embedded tissues. As formalin is the most common fixative used in mor
phology, the heat-induced AR technique has emerged as a very important approach
to the immunohistochemistry of routinely processed tissue sections. Since AR was de
veloped in 1991, there have been more than two hundred articles from all over the
world demonstrating the value ofA Rfor routine immunohistochemistry. More than
200 antibodies have been tested, yielding excellent immunohistochemical staining
using the AR technique on routine tissue sections in almost all instances. This article
reviews the basic principles of the method and describes the application, develop
ment and standardization of the AR technique as a basis for establishing current
standards ofp ractice and as a foundation for further studies.
INTRODUCTION
Immunohistochemistry (IHC) has a long history, extending over half a
century from 1940, when Coons developed an immunofluorescence tech
nique to detect bacteria (25,193). However, only in the last ten years has the
method found general application in surgical pathology (27,193,197). A se
ries of technical developments of IHC have created an increasingly sensitive
detection system. This system is based primarily on immunoperoxidase tech
niques, specifically the peroxidase anti-peroxidase (PAP), avidin-biotin
peroxidase complex (ABC) and biotin-streptavidin (BSA) methods
1
Antigen Retrieval Technique
(24,29,34,193), stemming from the important contributions of Nakane,
Pierce, Avrameas, Sternberger, Hsu and others (67,144). In Europe, alkaline
phosphatase has found extensive use as an alternative label. The development
of the hybridoma technique accelerated development of IHC through the
manufacture of numerous highly specific monoclonal antibodies, many of
which found early application for IHC staining (24,29,193). Initial success in
cryostat sections was eventually extended to routinely processed paraffin-,
celloidin- or other plastic-embedded tissue sections. Only when the IHC
technique can be used for routinely formalin-fixed, paraffin-embedded tissue
sections, will it finally effect a "brown revolution" in pathology (102).
The critical significance of rendering the IHC technique suitable for rou
tinely formalin-fixed, paraffin-embedded tissue sections (routine paraffin
sections) was emphasized by Taylor and colleagues, who, in 1974, showed
that it was possible to demonstrate at least some antigens in routine paraffin
sections (187). The collective appetites of pathologists worldwide, once
whetted by these initial studies, led to serious attempts to further improve the
ability to perform IHC staining on routine paraffin sections (181,187-193).
Enzymatic digestion was introduced by Huang et al. ( 68) as a pretreatment,
prior to IHC staining, to "unmask" some antigens that had been altered by
formalin fixation. However, the enzymatic digestion method, while widely
applied, did not improve IHC staining of the majority of antigens, as pointed
out by Leong et al. (101). Another drawback of enzymatic digestion is that it
proved difficult to control the optimal digestion conditions for individual tis
sue sections when stained with different antibodies. Difficulties in standard
ization provided a powerful incentive to the development of a new technique,
with the requirements that it should be more powerful, more widely applica
ble and easier to use than enzymatic digestion. In addition, any new method
should enhance immunohistochemical staining of routine paraffin sections in
a reproducible and reliable manner.
The antigen retrieval (AR) technique, based on a series of biochemical
studies by Fraenkel-Conrat and coworkers (39-41), was developed by Shi et
al. in 1991 (154). In contrast to enzymatic digestion, the AR technique is a
simple method that involves heating routine paraffin sections at high temper
ature (e.g., a microwave oven or other heating methods) prior to IHC stain
ing procedures (154). An alternative method that did not utilize heating was
developed for celloidin-embedded tissues ( 155-157). The intensity of IHC
staining was increased dramatically after AR pretreatment as demonstrated
by the original articles (154-157) and by numerous articles published subse
quently. All published reports described excellent AR-IHC staining results,
with the exception of one article (124). Only a minority of antibodies failed
to show improvement following the use of the AR method (48,103). Various
modifications of the AR technique have been described, the majority of
which employ different buffer solutions as the AR solution in place of metal
salt solutions used in the initial report (1,20,28,33,48,59,81,103,120,160,179,
2
S.-R. Shi et al.
194,208,212). Other studies explored different heating methods, with differ
ent temperatures and heating times (81,82,109). The purpose of this review
article is to summarize the development, standardization and application of
AR-IHC, both as the basis for current use and for further studies designed to
optimize the method.
AR TECHNIQUE
Heating AR Technique
Microwave (MW) heating method. In the original standard method
(154), after dewaxing, rehydration and blocking of endogenous peroxidase
with 3% H202, the paraffin sections were rinsed in distilled water and then
placed in plastic Coplin jars. The jars were then filled with AR solution, such
as distilled water, buffer solution, metal salt solution, urea, glycine-HCl pH
3.5, etc., covered with loose-fitting screw caps, and heated in the MW oven
for either 5 or 10 min. The 10-min heating time may be divided into two 5-
min cycles, with an interval of 1 min between cycles to check on the fluid
level in the jars. If necessary, more AR solution may be added after the first 5
min to avoid drying the tissue sections.(Note that in our experience, when
more fluid was needed, it was the result of boiling over the solution and not
evaporation. Replacement was therefore with fresh AR solution, not simply
with distilled water as would be the case if the loss were primarily due to
evaporation). After heating, the Coplin jars were removed from the oven and
allowed to cool for 15 min, rinsed in distilled water twice, and then in phos
phate-buffered saline (PBS) for 5 min, following which the IHC staining pro
cedure was performed. A conventional MW oven (as used in household
kitchens) with power ranging from 600 to 800 W was employed for AR heat
ing. Some authors employ a MW oven with 800-W capacity at medium
power (182). We studied the heating process when using a MW with 720-W
power and found that the boiling point (100 ± 5°C) for the AR solutions in
the Coplin jar was reached in 140-145 s. In order to maintain consistent heat
ing conditions, it is necessary to set the same number of jars at the same loca
tions close to the center of the MW oven. The final temperature and the total
time of heating may be established by using a small pilot study ("test bat
tery") to find the optimal heating condition for each antibody.
The process may be modified extensively and still yield satisfactory re
sults. For example, we studied a modified MW heating protocol as follows:
the AR solution was placed in the Coplin jar in the MW oven for 3 min to boil
the solution, after which the slides were immediately immersed in boiling AR
solution for 15 min, while the jar was moved outside the MW oven in a cool
ing mode. Terada et al. (200) reported a similar approach, preheating the AR
solution (PBS) in a Coplin jar in a MW QVen for 5 min to a boiling point
(1 00°C), then setting the slides in the preheated AR solution and heating by
3