Diagnostic Electron Microscopy E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Serie: RMS - Royal Microscopical Society
- Sprache: Englisch
- Veröffentlichungsjahr: 2012
Diagnostic Electron Microscopy Diagnostic Electron Microscopy: A Practical Guide to Interpretation and Technique summarises the current interpretational applications of TEM in diagnostic pathology. This concise and accessible volume provides a working guide to the main, or most useful, applications of the technique including practical topics of concern to laboratory scientists, brief guides to traditional tissue and microbiological preparation techniques, microwave processing, digital imaging and measurement uncertainty. The text features both a screening and interpretational guide for TEM diagnostic applications and current TEM diagnostic tissue preparation methods pertinent to all clinical electron microscope units worldwide. Containing high-quality representative images, this up-to-date text includes detailed information on the most important diagnostic applications of transmission electron microscopy as well as instructions for specific tissues and current basic preparative techniques. The book is relevant to trainee pathologists and practising pathologists who are expected to understand and evaluate/screen tissues by TEM. In addition, technical and scientific staff involved in tissue preparation and diagnostic tissue evaluation/screening by TEM will find this text useful.
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Table of Contents
Series Page
Title Page
Copyright
Acknowledgements and Dedication
List of Contributors
Preface–Introduction
Diagnostic Electron Microscopy
The purpose and use of TEM
The aim and purpose of this book
Chapter 1: Renal Disease
1.1 The Role of Transmission Electron Microscopy (TEM) in Renal Diagnostics
1.2 Ultrastructural Evaluation and Interpretation
1.3 The Normal Glomerulus
1.4 Ultrastructural Diagnostic Features
1.5 The Ultrastructural Pathology of the Major Glomerular Diseases
References
Chapter 2: Transplant Renal Biopsies
2.1 Introduction
2.2 The Transplant Renal Biopsy
2.3 Indications for Electron Microscopy of Transplant Kidney
References
Chapter 3: Electron Microscopy in Skeletal Muscle Pathology
3.1 Introduction
3.2 Normal Muscle
3.3 Pathological Changes
References
Chapter 4: The Diagnostic Electron Microscopy of Nerve
4.1 Introduction
4.2 Tissue Processing
4.3 Normal Nerve Ultrastructure
4.4 Pathological Ultrastructural Features
4.5 Artefact
4.6 Conclusions
References
Chapter 5: The Diagnostic Electron Microscopy of Tumours
5.1 Introduction
5.2 Principles and Procedures for Diagnosing Tumours by Electron Microscopy
5.3 Organelles and Groups of Cell Structures Defining Cellular Differentiation
References
Chapter 6: Microbial Ultrastructure
6.1 Introduction
6.2 Practical Guidance
6.3 Viruses
6.4 Current Use of EM in Virology
6.5 Viruses in Thin Sections of Cells or Tissues
6.6 Bacteria
6.7 Fungal Organisms
6.8 Microsporidia
6.9 Parasitic Protozoa
6.10 Examples of Non-enteric Protozoa
6.11 Parasitic Amoebae
6.12 Conclusions
Acknowledgements
References and Additional Reading
Chapter 7: The Contemporary Use of Electron Microscopy in the Diagnosis of Ciliary Disorders and Sperm Centriolar Abnormalities
7.1 Introduction
7.2 Ultrastructure of Motile Cilia
7.3 Genetics of PCD
7.4 Current Diagnostic Modalities
7.5 Clinical Features
7.6 Procurement and Assessment of Ciliated Specimens
7.7 Centriolar Sperm Abnormalities
7.8 Discussion
Acknowledgements
References
Chapter 8: Electron Microscopy as a Useful Tool in the Diagnosis of Lysosomal Storage Diseases
8.1 Introduction
8.2 Morphological Findings
8.3 Conclusion
References
Chapter 9: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL)
9.1 Introduction
9.2 Diagnostic Strategies—Comparative Specificity and Sensitivity
9.3 Diagnosis by TEM
References
Chapter 10: Diagnosis of Platelet Disorders by Electron Microscopy
10.1 Introduction
10.2 TEM Preparation of Platelets
10.3 Whole-Mount EM Preparation of Platelets
10.4 EM Preparation of Bone Marrow
10.5 Pre-embed Immunogold Labelling of Von Willibrand Factor in Platelets
10.6 Ultrastructural Features of Platelets
10.7 Normal Platelets
10.8 Grey Platelet Syndrome
10.9 Arthrogryposis, Renal Dysfunction and Cholestasis Syndrome
10.10 Jacobsen Syndrome
10.11 Hermansky–Pudlak Syndrome, Chediak–Higashi Syndrome and Other Dense-Granule Deficiencies
10.12 Type 2B von Willebrand Disease and Platelet-Type von Willebrand Disease
References
Chapter 11: Diagnosis of Congenital Dyserythropoietic Anaemia Types I and II by Transmission Electron Microscopy
11.1 Introduction
11.2 Preparation of Bone Marrow and General Observation Protocol
11.3 CDA Type I
11.4 CDA Type II
Acknowledgements
References
Chapter 12: Ehlers–Danlos Syndrome
12.1 Introduction
12.2 Collagen Fibrils
12.3 Elastic Fibers
12.4 Nonfibrous Stroma and Granulo-Filamentous Deposits
12.5 Connective Tissue Disorders
References
Chapter 13: Electron Microscopy in Occupational and Environmental Lung Disease
13.1 Introduction
13.2 Asbestos
13.3 Hypersensitivity Pneumonitis and Sarcoidosis
13.4 Silicosis
13.5 Silicate Pneumoconiosis
13.6 Metal-Induced Diseases
13.7 Rare-Earth Pneumoconiosis
13.8 Miscellaneous Disorders
References
Chapter 14: General Tissue Preparation Methods
14.1 Introduction
14.2 Tissue Collection and Dissection
14.3 Tissue Processing
14.4 Tissue Sectioning
References
Chapter 15: Ultrastructural Pathology Today—Paradigm Change and the Impact of Microwave Technology and Telemicroscopy
15.1 Diagnostic Electron Microscopy and Paradigm Shift in Pathology
15.2 Standardised and Automated Conventional Tissue Processing
15.3 Microwave-Assisted Sample Preparation
15.4 Cyberspace for Telepathology via the Internet
15.5 Conclusions and Future Prospects
Acknowledgements
References
Chapter 16: Electron Microscopy Methods in Virology
16.1 Biological Safety Precautions
16.2 Collection of Specimens
16.3 Preparation of Faeces, Vomitus or Urine Samples
16.4 Viruses in Skin Lesions
16.5 Reagents and Methods
16.6 Coated Grids
16.7 Important Elements in the Negative Staining Procedure
16.8 TEM Examination
16.9 Immunoelectron Microscopy
16.10 Thin Sectioning of Virus-Infected Cells or Tissues
16.11 Virology Quality Assurance (QA) Procedures
Acknowledgements
References
Chapter 17: Digital Imaging for Diagnostic Transmission Electron Microscopy
17.1 Introduction
17.2 Camera History
17.3 The Pixel Dilemma
17.4 Camera Positioning
17.5 Resolution
17.6 Fibre Coupled or Lens Coupled?
17.7 Sensitivity, Noise and Dynamic Range
17.8 CCD Chip Type (Full Frame or Interline)
17.9 Binning and Frame Rate
17.10 Software
17.11 Choosing the Right Camera
References
Chapter 18: Uncertainty of Measurement
18.1 Introduction
18.2 Purpose
18.3 Factors That Influence Quantitative Measurements
18.4 How to Calculate the UM
18.5 Worked Examples
18.6 Conclusion
References
Index
Current and future titles in the Royal Microscopical Society –John Wiley Series
Published
Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-ESEM)
Debbie Stokes
Aberration-Corrected Analytical Electron Microscopy
Edited by Rik Brydson
Diagnostic Electron Microscopy—A Practical Guide to Interpretation and Technique
Edited by John W. Stirling, Alan Curry & Brian Eyden
Forthcoming
Low Voltage Electron Microscopy: Principles and Applications
Edited by David C. Bell & Natasha Erdman
Atlas of Images and Spectra for Electron Microscopists
Edited by Ursel Bangert
Understanding Practical Light Microscopy
Jeremy Sanderson
Focused Ion Beam Instrumentation: Techniques and Applications
Dudley Finch & Alexander Buxbaum
This edition first published 2013
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Library of Congress Cataloging-in-Publication Data
Diagnostic electron microscopy : a practical guide to interpretation and technique / edited by John W. Stirling, Alan Curry, and Brian Eyden.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-119-97399-7 (cloth)
I. Stirling, John W. II. Curry, Alan. III. Eyden, Brian.
[DNLM: 1. Diagnostic Imaging–methods. 2. Microscopy, Electron, Transmission. WN 180]
616.07′54—dc23
2012027835
A catalogue record for this book is available from the British Library.
ISBN: 978-1-119-97399-7
Acknowledgements and Dedication
All three editors wish to thank the many individuals who have helped to make this volume possible. Firstly, they would like to express their appreciation to all the authors for their hard work and generosity in sharing their professional experience, as well as all the ‘behind-the-scenes’ staff and colleagues without whom this book could not have been produced.
John Stirling thanks the staff of the Centre for Ultrastructural Pathology, SA Pathology, Adelaide, for their support and photographic contributions—especially Alvis Jaunzems and Jeffrey Swift—and Dr Sophia Otto of the Department of Surgical Pathology, SA Pathology, for her advice and for proofreading.
Alan Curry acknowledges the contributions to his work of the pathologists, particularly Dr Helen Denley and Dr Lorna McWilliam, and technical staff of the Manchester Royal Infirmary, as well as two inspirational organisations—the Public Health Laboratory Service Electron Microscopy network and the Manchester Electron Microscope Society.
Brian Eyden wishes to thank all of the Pathology Department staff at the Christie NHS Foundation Trust (Manchester), without whose technical and light microscopic input the interpretation of tumour ultrastructure would be compromised, if not, in some instances, impossible.
Secondly, the editors wish to recognise the support and encouragement of their families in this endeavour. John Stirling thanks his partner, Jill, and expresses a special appreciation of his teachers and mentors, particularly Alec Macfarlane who helped him achieve his dream of a career in biology and Andrew Dorey who introduced him to electron microscopy and the wonders of cell ultrastructure. Alan Curry thanks his wife, Collette (particularly for her exceptional computer skills), and Brian Eyden thanks his wife, Freda, for understanding the needs of a writing scientist.
Finally, the editors dedicate this book to diagnostic electron microscopists—wherever they may be—who continue to make uncertain diagnoses more precise as a result of their labours, which, in turn, help clinicians to treat their patients better, the ultimate purpose of our work.
List of Contributors
Preface–Introduction
John W. Stirling, Alan Curry and Brian Eyden
Diagnostic Electron Microscopy
Science progresses as a result of a variety of factors. Critical to progress, however, is the invention and availability of appropriate tools and techniques that can completely transform our ability to investigate and understand the world around us—without such tools our ability to investigate even basic phenomena would be severely restricted. One such ‘transformational’ technology is the electron microscope. Although transmission electron microscopy (TEM) is now taken for granted, its application to the biological and medical sciences in the late 1950s and early 1960s ranks as one of the single most important factors that has impacted on our knowledge in biology and medicine. The resolving power of the transmission electron microscope (∼0.2 nm as compared with the light microscope with a resolution of ∼200 nm) made two important things possible for the first time, these being the visualisation of: (1) cell organelles and cytoplasmic structures at the macromolecular level (both useful indicators of cell differentiation) and; (2) viruses and microorganisms in general. Thus, TEM gave us new fundamental insights into cell structure and function, histogenesis and differentiation, and, following from this, our understanding of disease and disease processes.
TEM was quickly taken up as a diagnostic tool. In the clinical setting, electron microscopy has been used to improve diagnostic precision and confidence in many fields, including renal disease, neuromuscular disease, microbiology (particularly virology), tumour pathology, skin diseases, industrial diseases, haematology, metabolic storage diseases and conditions involving abnormalities of cilia and sperm. A number of encyclopaedic atlases of normal and pathological tissues quickly followed the introduction of electron microscopy and the medical literature contains many articles describing diagnostic applications of TEM in a wide range of conditions and specialist areas. Diagnostic TEM reached a zenith during the 1980s; however, since then, the introduction of new methodologies (particularly molecular techniques and affinity labelling systems) has reduced the need for TEM, particularly in tumour diagnosis. Despite this, TEM continues to play a significant and important role in pathology, and techniques continue to develop and improve. For example, the introduction of microwave processing and digital cameras has transformed tissue processing and screening so that ‘same-day’ reporting is easily achieved.
The purpose and use of TEM
The purpose of TEM is to diagnose disease based on the ultrastructural features of the tissue. These features include:
In general, the use of TEM will be predetermined either as a stand-alone protocol (e.g., CADASIL) or as part of a broad integrated diagnostic strategy (e.g., renal biopsies). However, TEM can also be applied on an ad hoc basis whenever there is a chance it will give an improved diagnosis (and therefore better patient care). The general criteria indicating the use of TEM may be summarised simply as follows:
The aim and purpose of this book
The prime aim and purpose of this book is to summarise the current interpretational applications of TEM in diagnostic pathology. In this respect, we have not attempted to reproduce previous encyclopaedic texts but to provide what we regard as a working guide to the main, or most useful, applications of the technique given the limited space available in a text of this size. In addition, we have also included practical topics of concern to laboratory scientists, including brief guides to traditional tissue and microbiological preparation techniques, microwave processing, digital imaging and measurement uncertainty.
Chapter 1
Renal Disease
John W. Stirling1 and Alan Curry2
1Centre for Ultrastructural Pathology, IMVS—SA Pathology Adelaide Australia
2Health Protection Agency, Clinical Sciences Building Manchester Royal Infirmary, Manchester United Kingdom
1.1 The Role of Transmission Electron Microscopy (TEM) in Renal Diagnostics
The ultrastructural examination of renal biopsies has made a significant contribution to our understanding of renal disease and is fundamental to accurate diagnosis. For overall tissue evaluation, light microscopy (LM), immunolabelling and transmission electron microscopy (TEM) are generally combined as an integrated protocol. LM is used to make an assessment of overall tissue morphology and to identify the major pathological processes present. Immunolabelling (preferably using immunofluorescence or by the immunoperoxidase technique) is used to determine the composition and location of glomerular immune deposits. Local practices vary, but an antibody panel can contain antibodies directed against IgG, IgA, IgM, complement (C3, C1q and sometimes C4), κ and λ light chains and albumin. TEM can play a major role when LM and immunolabelling findings are normal, only mildly atypical or equivocal and difficult to interpret, particularly in respect to conditions where there may be similar LM or immunolabelling findings. Thus, the technique is particularly useful in the setting of familial disease where the structural abnormalities in the glomerular basement membrane (GBM) cannot be resolved by LM (e.g. Alport's syndrome). TEM can also provide critical information not revealed by the other methodologies to identify underlying primary disease and unexpected concomitant disease. Similarly with immunolabelling, the full classification and staging of deposits require ultrastructural analysis. Some transplant biopsies can also benefit from ultrastructural evaluation (see Chapter 2); however, TEM rarely contributes to the diagnosis of tubular, vascular or interstitial disease. Overall, ultrastructural screening is essential; it can change the diagnosis in ∼25% of cases and provides ‘useful’ information in ∼66% of cases (Pearson et al., 1994; Elhefnawy, 2011).
1.2 Ultrastructural Evaluation and Interpretation
Examination of glomeruli (and other areas, if necessary) should be thorough and systematic with all components being evaluated for possibly significant features or changes. During screening, a range of representative images should be taken. These should include low-power images to show overall glomerular morphology, plus a representative selection of higher power images to show the specific and critical diagnostic features. In some instances, it may also be important to show that certain features are, in fact, absent (e.g. deposits) or normal (e.g. foot processes). The principal elements that should be examined are (i) the location, size and morphology of immune-related deposits and other inclusions; (ii) the thickness, overall morphology and texture of the GBM; (iii) the size and morphology of the mesangial matrix and (iv) the number and morphology of the cellular components of the glomerulus (Stirling 2000). Sclerotic glomeruli should be avoided, and only well-preserved functional (or significantly functional) glomeruli should be examined. It is also important to ensure that the glomeruli screened are representative of the LM findings: this means that, ideally, the choice of glomeruli to be screened (from semithin sections) should be done in collaboration with the reporting pathologist. Finally, it should be stressed that screening should be unbiased, although some knowledge of the pathology and immunolabelling results may be useful if the features expected are minor or uncommon. The vascular pole should be avoided during ultrastructural evaluation as it may contain misleading nonpathologic deposits, and likewise Bowman's capsule which has no real diagnostic value, although the presence of crescents can be confirmed.
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