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Diagnostic Electron Microscopy: A Practical Guide to Interpretation and Technique

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Diagnostic Electron Microscopy: A Practical Guide to Interpretation and Technique

John Stirling (Editor), Alan Curry (Editor), Brian Eyden (Editor)

ISBN: 978-1-119-97399-7 December 2012 492 Pages

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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.

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

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