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Diagnostic Electron Microscopy
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Table of Contents

List of Contributors xvii Preface - Introduction xxi 1 Renal Disease 1 John W. Stirling and Alan Curry 1.1 The Role of Transmission Electron Microscopy (TEM) in Renal Diagnostics 1 1.2 Ultrastructural Evaluation and Interpretation 2 1.3 The Normal Glomerulus 3 1.3.1 The Glomerular Basement Membrane 4 1.4 Ultrastructural Diagnostic Features 5 1.4.1 Deposits: General Features 5 1.4.2 Granular and Amorphous Deposits 6 1.4.3 Organised Deposits: Fibrils and Tubules 7 1.4.4 Nonspecific Fibrils 11 1.4.5 General and Nonspecific Inclusions and Deposits 11 1.4.6 Fibrin 12 1.4.7 Tubuloreticular Bodies (Tubuloreticular Inclusions) 12 1.4.8 The Glomerular Basement Membrane 13 1.4.9 The Mesangial Matrix 14 1.4.10 Cellular Components of the Glomerulus 14 1.4.11 Parietal Epithelium 16 1.5 The Ultrastructural Pathology of the Major Glomerular Diseases 16 1.5.1 Diseases without, or with Only Minor, Structural GBM Changes 16 1.5.2 Diseases with Structural GBM Changes 19 1.5.3 Diseases with Granular Deposits 25 1.5.4 Diseases with Organised Deposits 40 1.5.5 Hereditary Metabolic Storage Disorders 46 References 47 2 Transplant Renal Biopsies 55 John Brealey 2.1 Introduction 55 2.2 The Transplant Renal Biopsy 55 2.3 Indications for Electron Microscopy of Transplant Kidney 56 2.3.1 Transplant Glomerulopathy 56 2.3.2 Recurrent Primary Disease 64 2.3.3 De Novo Glomerular Disease 72 2.3.4 Donor-Related Disease 74 2.3.5 Infection 74 2.3.6 Inconclusive Diagnosis by LM and/or IM 79 2.3.7 Miscellaneous Topics 81 References 84 3 Electron Microscopy in Skeletal Muscle Pathology 89 Elizabeth Curtis and Caroline Sewry 3.1 Introduction 89 3.1.1 The Biopsy Procedure 90 3.1.2 Sampling 90 3.1.3 Tissue Processing 90 3.1.4 Artefacts 91 3.2 Normal Muscle 91 3.3 Pathological Changes 96 3.3.1 Sarcolemma 96 3.3.2 Myofibrils 99 3.3.3 Glycogen 102 3.3.4 Cores 104 3.3.5 Target Fibres 105 3.3.6 Myonuclei 105 3.3.7 Mitochondria 106 3.3.8 Reticular System 108 3.3.9 Vacuoles 109 3.3.10 Capillaries 110 3.3.11 Other Structural Defects 111 References 113 4 The Diagnostic Electron Microscopy of Nerve 117 Rosalind King 4.1 Introduction 117 4.2 Tissue Processing 118 4.2.1 Preparation of Nerve Biopsy Specimens 118 4.3 Normal Nerve Ultrastructure 120 4.3.1 Axons 120 4.3.2 Schwann Cells 120 4.3.3 The Myelin Sheath 120 4.3.4 Node of Ranvier 122 4.3.5 Paranode 123 4.3.6 Juxtaparanode 123 4.3.7 Internode 123 4.3.8 Schmidt-Lanterman Incisures 124 4.3.9 Remak Fibres 124 4.3.10 Fibroblasts 124 4.3.11 Renaut Bodies 125 4.4 Pathological Ultrastructural Features 125 4.4.1 Axonal Degeneration 125 4.4.2 Axonal Regeneration 126 4.4.3 Remak Fibre Abnormalities 128 4.4.4 Polyglucosan Bodies 128 4.4.5 Nonspecific Axonal Inclusions 128 4.4.6 Demyelination and Remyelination 130 4.4.7 Specific Schwann Cell Inclusions 135 4.4.8 Nonspecific Schwann Cell Inclusions 136 4.4.9 Fibroblasts 142 4.4.10 Perineurial Abnormalities 142 4.4.11 Cellular Infiltration 143 4.4.12 Endoneurial Oedema 143 4.4.13 Connective Tissue Abnormalities 143 4.4.14 Endoneurial Blood Vessels 145 4.4.15 Mast Cells 145 4.5 Artefact 145 4.6 Conclusions 147 References 148 5 The Diagnostic Electron Microscopy of Tumours 153 Brian Eyden 5.1 Introduction 153 5.2 Principles and Procedures for Diagnosing Tumours by Electron Microscopy 154 5.2.1 The Objective of Tumour Diagnosis 154 5.2.2 The Intellectual Requirements for Tumour Diagnosis by Electron Microscopy 155 5.2.3 Technical Considerations 156 5.2.4 Identifying Good Preservation 158 5.2.5 Distinguishing Reactive from Neoplastic Cells 162 5.3 Organelles and Groups of Cell Structures Defining Cellular Differentiation 162 5.3.1 Rough Endoplasmic Reticulum 162 5.3.2 Melanosomes 165 5.3.3 Desmosomes 167 5.3.4 Tonofibrils 167 5.3.5 Basal Lamina 169 5.3.6 Glandular Epithelial Differentiation and Cell Processes 171 5.3.7 Neuroendocrine Granules 171 5.3.8 Smooth-Muscle Myofilaments 173 5.3.9 Sarcomeric Myofilaments (Thick-and-Thin Filaments with Z-Disks) 176 References 178 6 Microbial Ultrastructure 181 Alan Curry 6.1 Introduction 181 6.2 Practical Guidance 182 6.3 Viruses 183 6.4 Current Use of EM in Virology 185 6.5 Viruses in Thin Sections of Cells or Tissues 186 6.6 Bacteria 191 6.7 Fungal Organisms 194 6.8 Microsporidia 196 6.9 Parasitic Protozoa 206 6.9.1 Cryptosporidium 207 6.9.2 Isospora belli 211 6.10 Examples of Non-enteric Protozoa 212 6.11 Parasitic Amoebae 213 6.12 Conclusions 214 Acknowledgements 214 References and Additional Reading 214 7 The Contemporary Use of Electron Microscopy in the Diagnosis of Ciliary Disorders and Sperm Centriolar Abnormalities 221 P. Yiallouros, M. Nearchou, A. Hadjisavvas and K. Kyriacou 7.1 Introduction 221 7.2 Ultrastructure of Motile Cilia 224 7.3 Genetics of PCD 226 7.4 Current Diagnostic Modalities 228 7.5 Clinical Features 229 7.6 Procurement and Assessment of Ciliated Specimens 230 7.7 Centriolar Sperm Abnormalities 231 7.8 Discussion 232 Acknowledgements 234 References 234 8 Electron Microscopy as a Useful Tool in the Diagnosis of Lysosomal Storage Diseases 237 Joseph Alroy, Rolf Pfannl and Angelo A. Ucci 8.1 Introduction 237 8.2 Morphological Findings 247 8.3 Conclusion 261 References 262 9 Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) 269 John W. Stirling 9.1 Introduction 269 9.2 Diagnostic Strategies - Comparative Specificity and Sensitivity 271 9.3 Diagnosis by TEM 271 References 274 10 Diagnosis of Platelet Disorders by Electron Microscopy 277 Hilary Christensen and Walter H.A. Kahr 10.1 Introduction 277 10.2 TEM Preparation of Platelets 278 10.3 Whole-Mount EM Preparation of Platelets 280 10.4 EM Preparation of Bone Marrow 281 10.5 Pre-embed Immunogold Labelling of Von Willibrand Factor in Platelets 282 10.6 Ultrastructural Features of Platelets 282 10.7 Normal Platelets 283 10.8 Grey Platelet Syndrome 285 10.9 Arthrogryposis, Renal Dysfunction and Cholestasis Syndrome 285 10.10 Jacobsen Syndrome 285 10.11 Hermansky-Pudlak Syndrome, Chediak-Higashi Syndrome and Other Dense-Granule Deficiencies 287 10.12 Type 2B von Willebrand Disease and Platelet-Type von Willebrand Disease 288 References 290 11 Diagnosis of Congenital Dyserythropoietic Anaemia Types I and II by Transmission Electron Microscopy 293 Yong-xin Ru 11.1 Introduction 293 11.2 Preparation of Bone Marrow and General Observation Protocol 294 11.3 CDA Type I 294 11.3.1 Proerythroblasts and Basophilic Erythroblasts 294 11.3.2 Polychromatic and Orthochromatic Erythroblasts 295 11.3.3 Reticulocytes and Erythrocytes 299 11.4 CDA Type II 299 11.4.1 Erythroblasts 301 11.4.2 Erythrocytes 306 11.5 Summary 306 Acknowledgements 307 References 307 12 Ehlers-Danlos Syndrome 309 Trinh Hermanns-Le, Marie-Annick Reginster, Claudine Pierard-Franchimont and Gerald E. Pierard 12.1 Introduction 309 12.2 Collagen Fibrils 310 12.3 Elastic Fibers 310 12.4 Nonfibrous Stroma and Granulo-Filamentous Deposits 311 12.5 Connective Tissue Disorders 311 12.5.1 Ehlers-Danlos Syndrome 311 12.5.2 Spontaneous Cervical Artery Dissection 317 12.5.3 Recurrent Preterm Premature Rupture of Fetal Membrane Syndrome 319 References 319 13 Electron Microscopy in Occupational and Environmental Lung Disease 323 Victor L. Roggli 13.1 Introduction 323 13.2 Asbestos 324 13.2.1 Preparatory Techniques 324 13.2.2 Analytical Methodology 326 13.2.3 Asbestos-Related Diseases 326 13.2.4 Exposure Categories 330 13.3 Hypersensitivity Pneumonitis and Sarcoidosis 330 13.3.1 Preparatory Techniques and Analytical Methodology 331 13.4 Silicosis 331 13.4.1 Preparatory Techniques and Analytical Methodology 333 13.5 Silicate Pneumoconiosis 333 13.5.1 Talc Pneumoconiosis 333 13.5.2 Kaolin Worker's Pneumoconiosis 334 13.5.3 Mica and Feldspar Pneumoconiosis 334 13.5.4 Mixed Dust Pneumoconiosis 335 13.5.5 Preparatory Techniques and Analytical Methodology 335 13.6 Metal-Induced Diseases 335 13.6.1 Siderosis 336 13.6.2 Aluminosis 336 13.6.3 Hard Metal Lung Disease 336 13.6.4 Berylliosis 337 13.6.5 Preparatory Techniques and Analytical Methodology 337 13.7 Rare-Earth Pneumoconiosis 338 13.8 Miscellaneous Disorders 338 References 339 14 General Tissue Preparation Methods 341 John W. Stirling 14.1 Introduction 341 14.1.1 Specimens Suitable for Diagnostic TEM 341 14.2 Tissue Collection and Dissection 342 14.2.1 Tissue Cut-Up 343 14.3 Tissue Processing 345 14.3.1 Fixatives and Fixation 345 14.3.2 Primary Fixation: Glutaraldehyde 347 14.3.3 Secondary Fixation (Post-fixation): Osmium Tetroxide 347 14.3.4 Fixative Vehicles and Wash Buffers 347 14.3.5 En Bloc Staining with Uranyl Acetate 348 14.3.6 Dehydrant and Transition Fluids 348 14.3.7 Resin Infiltration and Embedding Media 349 14.3.8 Tissue Embedding 352 14.4 Tissue Sectioning 352 14.4.1 Ultramicrotomy 352 14.4.2 Sectioning Technique and Ultramicrotome Setup 355 14.4.3 Common Sectioning Problems and Artefacts 356 14.4.4 Section Staining 362 14.4.5 Section Contamination and Staining Artefacts 363 Protocol 364 Processing Schedules 364 References 379 15 Ultrastructural Pathology Today - Paradigm Change and the Impact of Microwave Technology and Telemicroscopy 383 Josef A. Schroeder 15.1 Diagnostic Electron Microscopy and Paradigm Shift in Pathology 383 15.2 Standardised and Automated Conventional Tissue Processing 385 15.3 Microwave-Assisted Sample Preparation 390 15.4 Cyberspace for Telepathology via the Internet 397 15.5 Conclusions and Future Prospects 400 Acknowledgements 404 References 404 16 Electron Microscopy Methods in Virology 409 Alan Curry 16.1 Biological Safety Precautions 409 16.2 Collection of Specimens 410 16.3 Preparation of Faeces, Vomitus or Urine Samples 410 16.4 Viruses in Skin Lesions 410 16.5 Reagents and Methods 411 16.5.1 Negative Stains 411 16.6 Coated Grids 412 16.7 Important Elements in the Negative Staining Procedure 412 16.8 TEM Examination 413 16.9 Immunoelectron Microscopy 413 16.9.1 Immune Clumping 413 16.9.2 Solid-Phase Immunoelectron Microscopy 413 16.9.3 Immunogold Labelling 414 16.9.4 Particle Measurement 414 16.10 Thin Sectioning of Virus-Infected Cells or Tissues 414 16.11 Virology Quality Assurance (QA) Procedures 415 16.11.1 External QA 415 16.11.2 Internal QA 415 Acknowledgements 415 References 416 17 Digital Imaging for Diagnostic Transmission Electron Microscopy 419 Gary Paul Edwards 17.1 Introduction 419 17.2 Camera History 419 17.3 The Pixel Dilemma 420 17.4 Camera Positioning 421 17.5 Resolution 422 17.6 Fibre Coupled or Lens Coupled? 423 17.7 Sensitivity, Noise and Dynamic Range 424 17.8 CCD Chip Type (Full Frame or Interline) 426 17.9 Binning and Frame Rate 426 17.10 Software 427 17.11 Choosing the Right Camera 428 References 429 18 Uncertainty of Measurement 431 Pierre Filion 18.1 Introduction 431 18.2 Purpose 432 18.2.1 Diagnostic Value 432 18.2.2 Internal Quality Control 432 18.2.3 External Quality Control and Accreditation 432 18.3 Factors That Influence Quantitative Measurements 433 18.3.1 Sources of Variation 433 18.3.2 Alteration of the Intrinsic Dimension of the Structure 434 18.3.3 Variation Due to the Analytical Equipment and Method 436 18.3.4 Variation Due to Selection Bias 438 18.3.5 Measurement Using a Digital Camera 439 18.4 How to Calculate the UM 440 18.4.1 Steps Required to Analyse and Calculate the UM 440 18.4.2 Type of Error and Distribution of Measurements 440 18.4.3 Calculating the UM 442 18.4.4 Precision of Measurement and Biological Significance 443 18.4.5 The Electronic Spread Sheet as an Aid to Calculating UM 443 18.4.6 Reporting the UM 444 18.5 Worked Examples 444 18.5.1 Diameter of Fibrils in a Glomerular Deposit 444 18.5.2 Thickness of the Glomerular Basement Membrane 445 18.6 Conclusion 446 References 447 Index 449

About the Author

John W. Stirling The Centre for Ultrastructural Pathology, Adelaide, Australia Alan Curry Manchester Royal Infirmary, Manchester, UK Brian Eyden Christie NHS Foundation Trust, Manchester, UK

Reviews

Thus, this book is a must-have for allpathology departments, even if they are not equipped with an EMfacility, and it is also a solid proof of the current role ofelectron microscopy in health care. (Microscopy& Microanalysis, 1 August 2013)

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