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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-Lê, Marie-Annick Reginster, Claudine Piérard-Franchimont and Gérald E. Piérard
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
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