Diagnostic Electron Microscopy

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

“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)

• List of Contributors xviiPreface – Introduction xxi1 Renal Disease 1John W. Stirling and Alan Curry1.1 The Role of Transmission Electron Microscopy (TEM) in Renal Diagnostics 11.2 Ultrastructural Evaluation and Interpretation 21.3 The Normal Glomerulus 31.3.1 The Glomerular Basement Membrane 41.4 Ultrastructural Diagnostic Features 51.4.1 Deposits: General Features 51.4.2 Granular and Amorphous Deposits 61.4.3 Organised Deposits: Fibrils and Tubules 71.4.4 Nonspecific Fibrils 111.4.5 General and Nonspecific Inclusions and Deposits 111.4.6 Fibrin 121.4.7 Tubuloreticular Bodies (Tubuloreticular Inclusions) 121.4.8 The Glomerular Basement Membrane 131.4.9 The Mesangial Matrix 141.4.10 Cellular Components of the Glomerulus 141.4.11 Parietal Epithelium 161.5 The Ultrastructural Pathology of the Major Glomerular Diseases 161.5.1 Diseases without, or with Only Minor, Structural GBM Changes 161.5.2 Diseases with Structural GBM Changes 191.5.3 Diseases with Granular Deposits 251.5.4 Diseases with Organised Deposits 401.5.5 Hereditary Metabolic Storage Disorders 46References 472 Transplant Renal Biopsies 55John Brealey2.1 Introduction 552.2 The Transplant Renal Biopsy 552.3 Indications for Electron Microscopy of Transplant Kidney 562.3.1 Transplant Glomerulopathy 562.3.2 Recurrent Primary Disease 642.3.3 De Novo Glomerular Disease 722.3.4 Donor-Related Disease 742.3.5 Infection 742.3.6 Inconclusive Diagnosis by LM and/or IM 792.3.7 Miscellaneous Topics 81References 843 Electron Microscopy in Skeletal Muscle Pathology 89Elizabeth Curtis and Caroline Sewry3.1 Introduction 893.1.1 The Biopsy Procedure 903.1.2 Sampling 903.1.3 Tissue Processing 903.1.4 Artefacts 913.2 Normal Muscle 913.3 Pathological Changes 963.3.1 Sarcolemma 963.3.2 Myofibrils 993.3.3 Glycogen 1023.3.4 Cores 1043.3.5 Target Fibres 1053.3.6 Myonuclei 1053.3.7 Mitochondria 1063.3.8 Reticular System 1083.3.9 Vacuoles 1093.3.10 Capillaries 1103.3.11 Other Structural Defects 111References 1134 The Diagnostic Electron Microscopy of Nerve 117Rosalind King4.1 Introduction 1174.2 Tissue Processing 1184.2.1 Preparation of Nerve Biopsy Specimens 1184.3 Normal Nerve Ultrastructure 1204.3.1 Axons 1204.3.2 Schwann Cells 1204.3.3 The Myelin Sheath 1204.3.4 Node of Ranvier 1224.3.5 Paranode 1234.3.6 Juxtaparanode 1234.3.7 Internode 1234.3.8 Schmidt–Lanterman Incisures 1244.3.9 Remak Fibres 1244.3.10 Fibroblasts 1244.3.11 Renaut Bodies 1254.4 Pathological Ultrastructural Features 1254.4.1 Axonal Degeneration 1254.4.2 Axonal Regeneration 1264.4.3 Remak Fibre Abnormalities 1284.4.4 Polyglucosan Bodies 1284.4.5 Nonspecific Axonal Inclusions 1284.4.6 Demyelination and Remyelination 1304.4.7 Specific Schwann Cell Inclusions 1354.4.8 Nonspecific Schwann Cell Inclusions 1364.4.9 Fibroblasts 1424.4.10 Perineurial Abnormalities 1424.4.11 Cellular Infiltration 1434.4.12 Endoneurial Oedema 1434.4.13 Connective Tissue Abnormalities 1434.4.14 Endoneurial Blood Vessels 1454.4.15 Mast Cells 1454.5 Artefact 1454.6 Conclusions 147References 1485 The Diagnostic Electron Microscopy of Tumours 153Brian Eyden5.1 Introduction 1535.2 Principles and Procedures for Diagnosing Tumours by Electron Microscopy 1545.2.1 The Objective of Tumour Diagnosis 1545.2.2 The Intellectual Requirements for Tumour Diagnosis by Electron Microscopy 1555.2.3 Technical Considerations 1565.2.4 Identifying Good Preservation 1585.2.5 Distinguishing Reactive from Neoplastic Cells 1625.3 Organelles and Groups of Cell Structures Defining Cellular Differentiation 1625.3.1 Rough Endoplasmic Reticulum 1625.3.2 Melanosomes 1655.3.3 Desmosomes 1675.3.4 Tonofibrils 1675.3.5 Basal Lamina 1695.3.6 Glandular Epithelial Differentiation and Cell Processes 1715.3.7 Neuroendocrine Granules 1715.3.8 Smooth-Muscle Myofilaments 1735.3.9 Sarcomeric Myofilaments (Thick-and-Thin Filaments with Z-Disks) 176References 1786 Microbial Ultrastructure 181Alan Curry6.1 Introduction 1816.2 Practical Guidance 1826.3 Viruses 1836.4 Current Use of EM in Virology 1856.5 Viruses in Thin Sections of Cells or Tissues 1866.6 Bacteria 1916.7 Fungal Organisms 1946.8 Microsporidia 1966.9 Parasitic Protozoa 2066.9.1 Cryptosporidium 2076.9.2 Isospora belli 2116.10 Examples of Non-enteric Protozoa 2126.11 Parasitic Amoebae 2136.12 Conclusions 214Acknowledgements 214References and Additional Reading 2147 The Contemporary Use of Electron Microscopy in the Diagnosis of Ciliary Disorders and Sperm Centriolar Abnormalities 221P. Yiallouros, M. Nearchou, A. Hadjisavvas and K. Kyriacou7.1 Introduction 2217.2 Ultrastructure of Motile Cilia 2247.3 Genetics of PCD 2267.4 Current Diagnostic Modalities 2287.5 Clinical Features 2297.6 Procurement and Assessment of Ciliated Specimens 2307.7 Centriolar Sperm Abnormalities 2317.8 Discussion 232Acknowledgements 234References 2348 Electron Microscopy as a Useful Tool in the Diagnosis of Lysosomal Storage Diseases 237Joseph Alroy, Rolf Pfannl and Angelo A. Ucci8.1 Introduction 2378.2 Morphological Findings 2478.3 Conclusion 261References 2629 Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) 269John W. Stirling9.1 Introduction 2699.2 Diagnostic Strategies – Comparative Specificity and Sensitivity 2719.3 Diagnosis by TEM 271References 27410 Diagnosis of Platelet Disorders by Electron Microscopy 277Hilary Christensen and Walter H.A. Kahr10.1 Introduction 27710.2 TEM Preparation of Platelets 27810.3 Whole-Mount EM Preparation of Platelets 28010.4 EM Preparation of Bone Marrow 28110.5 Pre-embed Immunogold Labelling of Von Willibrand Factor in Platelets 28210.6 Ultrastructural Features of Platelets 28210.7 Normal Platelets 28310.8 Grey Platelet Syndrome 28510.9 Arthrogryposis, Renal Dysfunction and Cholestasis Syndrome 28510.10 Jacobsen Syndrome 28510.11 Hermansky–Pudlak Syndrome, Chediak–Higashi Syndrome and Other Dense-Granule Deficiencies 28710.12 Type 2B von Willebrand Disease and Platelet-Type von Willebrand Disease 288References 29011 Diagnosis of Congenital Dyserythropoietic Anaemia Types I and II by Transmission Electron Microscopy 293Yong-xin Ru11.1 Introduction 29311.2 Preparation of Bone Marrow and General Observation Protocol 29411.3 CDA Type I 29411.3.1 Proerythroblasts and Basophilic Erythroblasts 29411.3.2 Polychromatic and Orthochromatic Erythroblasts 29511.3.3 Reticulocytes and Erythrocytes 29911.4 CDA Type II 29911.4.1 Erythroblasts 30111.4.2 Erythrocytes 30611.5 Summary 306Acknowledgements 307References 30712 Ehlers–Danlos Syndrome 309Trinh Hermanns-Lê, Marie-Annick Reginster, Claudine Piérard-Franchimont and Gérald E. Piérard12.1 Introduction 30912.2 Collagen Fibrils 31012.3 Elastic Fibers 31012.4 Nonfibrous Stroma and Granulo-Filamentous Deposits 31112.5 Connective Tissue Disorders 31112.5.1 Ehlers–Danlos Syndrome 31112.5.2 Spontaneous Cervical Artery Dissection 31712.5.3 Recurrent Preterm Premature Rupture of Fetal Membrane Syndrome 319References 31913 Electron Microscopy in Occupational and Environmental Lung Disease 323Victor L. Roggli13.1 Introduction 32313.2 Asbestos 32413.2.1 Preparatory Techniques 32413.2.2 Analytical Methodology 32613.2.3 Asbestos-Related Diseases 32613.2.4 Exposure Categories 33013.3 Hypersensitivity Pneumonitis and Sarcoidosis 33013.3.1 Preparatory Techniques and Analytical Methodology 33113.4 Silicosis 33113.4.1 Preparatory Techniques and Analytical Methodology 33313.5 Silicate Pneumoconiosis 33313.5.1 Talc Pneumoconiosis 33313.5.2 Kaolin Worker’s Pneumoconiosis 33413.5.3 Mica and Feldspar Pneumoconiosis 33413.5.4 Mixed Dust Pneumoconiosis 33513.5.5 Preparatory Techniques and Analytical Methodology 33513.6 Metal-Induced Diseases 33513.6.1 Siderosis 33613.6.2 Aluminosis 33613.6.3 Hard Metal Lung Disease 33613.6.4 Berylliosis 33713.6.5 Preparatory Techniques and Analytical Methodology 33713.7 Rare-Earth Pneumoconiosis 33813.8 Miscellaneous Disorders 338References 33914 General Tissue Preparation Methods 341John W. Stirling14.1 Introduction 34114.1.1 Specimens Suitable for Diagnostic TEM 34114.2 Tissue Collection and Dissection 34214.2.1 Tissue Cut-Up 34314.3 Tissue Processing 34514.3.1 Fixatives and Fixation 34514.3.2 Primary Fixation: Glutaraldehyde 34714.3.3 Secondary Fixation (Post-fixation): Osmium Tetroxide 34714.3.4 Fixative Vehicles and Wash Buffers 34714.3.5 En Bloc Staining with Uranyl Acetate 34814.3.6 Dehydrant and Transition Fluids 34814.3.7 Resin Infiltration and Embedding Media 34914.3.8 Tissue Embedding 35214.4 Tissue Sectioning 35214.4.1 Ultramicrotomy 35214.4.2 Sectioning Technique and Ultramicrotome Setup 35514.4.3 Common Sectioning Problems and Artefacts 35614.4.4 Section Staining 36214.4.5 Section Contamination and Staining Artefacts 363Protocol 364Processing Schedules 364References 37915 Ultrastructural Pathology Today – Paradigm Change and the Impact of Microwave Technology and Telemicroscopy 383Josef A. Schroeder15.1 Diagnostic Electron Microscopy and Paradigm Shift in Pathology 38315.2 Standardised and Automated Conventional Tissue Processing 38515.3 Microwave-Assisted Sample Preparation 39015.4 Cyberspace for Telepathology via the Internet 39715.5 Conclusions and Future Prospects 400Acknowledgements 404References 40416 Electron Microscopy Methods in Virology 409Alan Curry16.1 Biological Safety Precautions 40916.2 Collection of Specimens 41016.3 Preparation of Faeces, Vomitus or Urine Samples 41016.4 Viruses in Skin Lesions 41016.5 Reagents and Methods 41116.5.1 Negative Stains 41116.6 Coated Grids 41216.7 Important Elements in the Negative Staining Procedure 412 16.8 TEM Examination 41316.9 Immunoelectron Microscopy 41316.9.1 Immune Clumping 41316.9.2 Solid-Phase Immunoelectron Microscopy 41316.9.3 Immunogold Labelling 41416.9.4 Particle Measurement 41416.10 Thin Sectioning of Virus-Infected Cells or Tissues 41416.11 Virology Quality Assurance (QA) Procedures 41516.11.1 External QA 41516.11.2 Internal QA 415Acknowledgements 415References 41617 Digital Imaging for Diagnostic Transmission Electron Microscopy 419Gary Paul Edwards17.1 Introduction 41917.2 Camera History 41917.3 The Pixel Dilemma 42017.4 Camera Positioning 42117.5 Resolution 42217.6 Fibre Coupled or Lens Coupled? 42317.7 Sensitivity, Noise and Dynamic Range 42417.8 CCD Chip Type (Full Frame or Interline) 42617.9 Binning and Frame Rate 42617.10 Software 42717.11 Choosing the Right Camera 428References 42918 Uncertainty of Measurement 431Pierre Filion18.1 Introduction 43118.2 Purpose 43218.2.1 Diagnostic Value 43218.2.2 Internal Quality Control 43218.2.3 External Quality Control and Accreditation 43218.3 Factors That Influence Quantitative Measurements 43318.3.1 Sources of Variation 43318.3.2 Alteration of the Intrinsic Dimension of the Structure 43418.3.3 Variation Due to the Analytical Equipment and Method 43618.3.4 Variation Due to Selection Bias 43818.3.5 Measurement Using a Digital Camera 43918.4 How to Calculate the UM 44018.4.1 Steps Required to Analyse and Calculate the UM 44018.4.2 Type of Error and Distribution of Measurements 44018.4.3 Calculating the UM 44218.4.4 Precision of Measurement and Biological Significance 44318.4.5 The Electronic Spread Sheet as an Aid to Calculating UM 44318.4.6 Reporting the UM 44418.5 Worked Examples 44418.5.1 Diameter of Fibrils in a Glomerular Deposit 44418.5.2 Thickness of the Glomerular Basement Membrane 44518.6 Conclusion 446References 447Index 449