BOOK
Bancroft's Theory and Practice of Histological Techniques E-Book
Kim S Suvarna | Christopher Layton | John D. Bancroft
(2018)
Additional Information
Book Details
Abstract
For 40 years, Bancroft’s Theory and Practice of Histological Techniques has established itself as the standard reference for histotechnologists and laboratory scientists, as well as histopathologists. With coverage of the full range of histological techniques used in medical laboratories and pathology departments, it provides a strong foundation in all aspects of histological technology – from basic methods of section preparation and staining, to advanced diagnostic techniques such as immunocytochemistry and molecular testing. This revised and updated 8th Edition by Kim S. Suvarna, Christopher Layton, and John D. Bancroft is a one-stop reference for all those involved with histological preparations and applications, from student to highly advanced laboratory professional.
Table of Contents
| Section Title | Page | Action | Price |
|---|---|---|---|
| Front Cover | Cover | ||
| IFC | ES1 | ||
| Bancroft’s THEORY and PRACTICE of HISTOLOGICAL TECHNIQUES \r | i | ||
| Bancroft’s THEORY and PRACTICE of HISTOLOGICAL TECHNIQUES \r | iii | ||
| Copyright\r | iv | ||
| Preface to the eighth edition | v | ||
| Preface to the first edition | vi | ||
| List of contributors | vii | ||
| Acknowledgments | ix | ||
| General acknowledgments | ix | ||
| Acknowledgment to Alan Stevens | ix | ||
| Special acknowledgment | ix | ||
| Contents | xi | ||
| 1 - Pathology laboratory management | 1 | ||
| Introduction | 1 | ||
| Regulation and accreditation | 1 | ||
| Accreditation | 2 | ||
| Quality management | 3 | ||
| Quality control (QC) | 3 | ||
| External quality assurance (EQA) | 3 | ||
| Process improvement | 4 | ||
| Risk management | 4 | ||
| Risk identification | 5 | ||
| Risk analysis and evaluation | 5 | ||
| Risk Assessment Tool | 6 | ||
| Severity and likelihood values | 6 | ||
| Incidents may also be scored 1–5 for likelihood | 6 | ||
| Audit | 6 | ||
| Risk funding | 7 | ||
| Safety | 7 | ||
| Laboratory procedures | 8 | ||
| Departmental organization | 8 | ||
| Workflow | 8 | ||
| Personnel management | 9 | ||
| Staffing the laboratory | 9 | ||
| Premises, equipment and materials | 10 | ||
| Financial management | 10 | ||
| Acknowledgments | 10 | ||
| Further reading | 10 | ||
| 2 - Chemical safety in the laboratory | 12 | ||
| Introduction | 12 | ||
| Classifications of hazardous chemicals | 12 | ||
| Labeling of hazardous chemicals | 12 | ||
| Working safely with hazardous chemicals | 13 | ||
| Safety data sheets (SDS) | 13 | ||
| Section 1: Identification of the substance or mixture and of the supplier | 14 | ||
| Section 2: Hazards identification | 15 | ||
| Section 3: Composition and information on ingredients | 15 | ||
| Section 4: First-aid measures | 16 | ||
| Section 5: Fire-fighting measures | 16 | ||
| Section 6: Accidental release measures | 17 | ||
| Section 7: Handling and storage | 18 | ||
| Section 8: Exposure controls/personal protection | 18 | ||
| Eye protection | 19 | ||
| Skin protection | 19 | ||
| Respiratory protection | 20 | ||
| Ingestion protection | 20 | ||
| Section 9: Physical and chemical properties | 20 | ||
| Section 10: Stability and reactivity | 21 | ||
| Section 11: Toxicological information | 21 | ||
| Section 12: Ecological information | 22 | ||
| Section 13: Disposal considerations | 22 | ||
| Recycling | 22 | ||
| Section 14: Transport information | 22 | ||
| Section 15: Regulatory information | 23 | ||
| Section 16: Other information | 23 | ||
| References | 23 | ||
| 3 - Light microscopy | 25 | ||
| Introduction | 25 | ||
| Light and its properties | 25 | ||
| Retardation and refraction | 26 | ||
| Image formation | 27 | ||
| Image quality | 28 | ||
| The components of a microscope | 29 | ||
| The light source | 29 | ||
| Condensers | 29 | ||
| Object stage | 30 | ||
| Objectives | 30 | ||
| The body tube and eyepiece | 30 | ||
| Using the microscope | 31 | ||
| Magnification | 31 | ||
| Illumination | 31 | ||
| Dark field illumination | 31 | ||
| Phase contrast microscopy | 31 | ||
| Interference microscopy | 33 | ||
| Polarized light microscopy | 33 | ||
| Fluorescence microscopy | 36 | ||
| Transmitted light fluorescence | 36 | ||
| Incident light fluorescence | 37 | ||
| The confocal microscope | 38 | ||
| 4 - Fixation of tissues\r | 40 | ||
| Introduction\r | 40 | ||
| Types of fixation | 41 | ||
| Physical methods of fixation | 42 | ||
| Heat fixation | 42 | ||
| Microwave fixation | 42 | ||
| Freeze-drying and freeze substitution | 42 | ||
| Chemical fixation | 42 | ||
| Coagulant fixatives | 43 | ||
| Dehydrant coagulant fixatives | 43 | ||
| Other types of coagulant fixative | 43 | ||
| Non-coagulant cross-linking fixatives | 43 | ||
| Formaldehyde fixation | 44 | ||
| Reversibility of formaldehyde-macromolecular reactions | 44 | ||
| Glutaraldehyde fixation | 47 | ||
| Osmium tetroxide fixation | 48 | ||
| Cross-linking fixatives for electron microscopy | 48 | ||
| Mercuric chloride fixatives | 48 | ||
| Special fixatives | 49 | ||
| Dichromate and chromic acid fixation | 49 | ||
| Fixatives for DNA, RNA and protein analysis | 49 | ||
| Metallic ions as a fixative supplement | 50 | ||
| Compound fixatives | 50 | ||
| Factors affecting the quality of fixation | 50 | ||
| Buffers and pH | 50 | ||
| Duration of fixation and the size of specimens | 51 | ||
| Temperature of fixation | 52 | ||
| Concentration of fixative | 52 | ||
| Osmolality of fixatives and ionic composition | 52 | ||
| Additives | 52 | ||
| Selecting or avoiding specific fixatives | 52 | ||
| Fixation for selected individual tissues | 52 | ||
| Eyes | 52 | ||
| Brain | 55 | ||
| Breast | 55 | ||
| Lungs | 55 | ||
| Lymphoid tissue | 55 | ||
| Testis | 55 | ||
| Muscle biopsies | 55 | ||
| Renal biopsies | 55 | ||
| Useful formulas for fixatives | 56 | ||
| Mercuric fixatives | 56 | ||
| Picric acid fixatives | 57 | ||
| Dehydrant fixatives | 58 | ||
| Dehydrant cross-linking fixatives | 58 | ||
| Alcoholic Bouin’s (Gendre’s solution) | 59 | ||
| Other fixatives | 59 | ||
| Acknowledgment | 60 | ||
| References | 60 | ||
| 5 - The gross room/surgical cut-up including sample handling | 64 | ||
| Introduction | 64 | ||
| Safety first and last | 64 | ||
| Specimen reception | 64 | ||
| Surgical cut-up/specimen dissection/grossing | 65 | ||
| Thinking before dissection | 66 | ||
| Case handling | 66 | ||
| Photography | 68 | ||
| Specimen dissection plans | 69 | ||
| Small samples | 69 | ||
| Core biopsies | 69 | ||
| Skin biopsies | 69 | ||
| Bowel specimens | 70 | ||
| Fat clearance | 70 | ||
| Lung tissues | 71 | ||
| Gynecological samples | 71 | ||
| Breast resections | 71 | ||
| Soft tissue resections | 72 | ||
| Other samples | 72 | ||
| References | 72 | ||
| Websites | 72 | ||
| 6 - Tissue processing | 73 | ||
| Introduction | 73 | ||
| Specimen tracking | 73 | ||
| Factors influencing processing | 73 | ||
| Viscosity | 73 | ||
| Agitation | 74 | ||
| Heat | 74 | ||
| Vacuum & pressure | 74 | ||
| Processing solvent contamination | 74 | ||
| Tissue processing stages | 74 | ||
| Fixation | 74 | ||
| Post-fixation treatment | 74 | ||
| Dehydration | 75 | ||
| Clearing | 75 | ||
| Infiltration | 76 | ||
| Paraffin wax | 76 | ||
| Alternative infiltration media | 76 | ||
| Resin | 76 | ||
| Agar | 76 | ||
| Gelatin | 76 | ||
| Celloidin | 77 | ||
| Embedding (Blocking) | 77 | ||
| Paraffin wax embedding | 77 | ||
| Resin embedding | 77 | ||
| Tissue orientation | 77 | ||
| Tissue processors | 77 | ||
| Microwave processors | 78 | ||
| Advantages of new technology in processing | 78 | ||
| Processor maintenance | 78 | ||
| Important maintenance tips | 79 | ||
| Processing schedules | 79 | ||
| Special considerations | 79 | ||
| Prognostic and predictive markers | 79 | ||
| Restoration of tissue dried in processing | 80 | ||
| Reprocessing of poorly processed paraffin wax-infiltrated specimens | 81 | ||
| Quality control | 81 | ||
| Summary | 81 | ||
| References | 82 | ||
| Useful websites | 82 | ||
| Other useful information | 83 | ||
| 7 - Microtomy for paraffin and frozen sections | 84 | ||
| Introduction | 84 | ||
| Types of microtome | 84 | ||
| Rotary microtome | 84 | ||
| Base sledge microtome | 84 | ||
| Rotary rocking microtome | 84 | ||
| Sliding microtome | 84 | ||
| Ultra-microtome | 84 | ||
| Microtome knives | 85 | ||
| Disposable blades | 85 | ||
| Glass and diamond knives | 85 | ||
| Paraffin section cutting | 85 | ||
| Equipment required | 85 | ||
| Flotation (water) bath | 85 | ||
| Drying oven or hot plate | 85 | ||
| Brush and forceps | 86 | ||
| Slides | 86 | ||
| Section adhesives | 86 | ||
| Poly-L-lysine (PLL) | 86 | ||
| 3-aminopropyltriethoxysilane (APES) | 86 | ||
| Charged or plus slides | 86 | ||
| Practical microtomy | 86 | ||
| Setup of the microtome | 86 | ||
| Sectioning | 87 | ||
| Trimming the tissue blocks | 87 | ||
| Cutting sections | 87 | ||
| Floating out sections | 87 | ||
| Drying sections | 88 | ||
| Cutting hard tissues | 88 | ||
| Surface decalcification | 88 | ||
| Problems and solutions | 88 | ||
| Frozen and related sections | 88 | ||
| Uses of frozen sections | 88 | ||
| Theoretical considerations | 88 | ||
| The cryostat | 90 | ||
| Freezing of fresh unfixed tissue | 90 | ||
| Fixed tissue and the cryostat | 91 | ||
| Cryostat sectioning | 91 | ||
| Cabinet temperature | 91 | ||
| Microtome | 92 | ||
| Cryo-embedding medium | 93 | ||
| Blade or knife | 93 | ||
| Anti-roll plate | 93 | ||
| Sectioning technique | 93 | ||
| Decontamination | 93 | ||
| Rapid biopsy for intraoperative diagnosis | 93 | ||
| Ultracryotomy | 93 | ||
| Freeze drying and freeze substitution | 94 | ||
| Applications and uses of freeze-dried material | 94 | ||
| Frozen section substitution | 94 | ||
| Additional reading | 95 | ||
| 8 - Resin (plastic) embedding for microscopy and tissue analysis | 96 | ||
| Introduction | 96 | ||
| The use of resins | 96 | ||
| Types of resin embedding media and commercial resin ‘kits’ | 97 | ||
| Cryotechniques | 102 | ||
| Super resolution fluorescence microscopy, correlative microscopy and tomography | 102 | ||
| Rapid embedding | 103 | ||
| Sectioning resin-embedded material | 103 | ||
| Acrylic resins | 103 | ||
| Applications and characteristics | 105 | ||
| Tinctorial staining | 105 | ||
| Enzyme histochemistry | 105 | ||
| Immunohistochemistry | 106 | ||
| In situ hybridization | 107 | ||
| Sectioning | 107 | ||
| Staining sections | 108 | ||
| Immunohistochemistry using MMA sections | 108 | ||
| Processing schedules | 108 | ||
| Epoxy resins | 110 | ||
| Cutting and staining sections for light microscopy | 111 | ||
| References | 111 | ||
| 9 - Theory of histological staining | 114 | ||
| Introduction\r | 114 | ||
| Why and how staining happens | 114 | ||
| Why are stains taken into the tissues? | 114 | ||
| Reagent-tissue interactions | 114 | ||
| Solvent-solvent interactions | 116 | ||
| Stain-stain interactions | 116 | ||
| A minor anomaly | 116 | ||
| Solubility, an unacknowledged factor | 117 | ||
| Why is stain retained in tissue? | 117 | ||
| Why are stains not taken up into every part of the tissue? | 118 | ||
| Numbers and affinities of binding sites | 118 | ||
| Rate of reagent uptake | 118 | ||
| Rate of reaction | 118 | ||
| Rate of reagent loss | 118 | ||
| Metachromatic staining | 119 | ||
| How is staining influenced by tissue fixation? | 119 | ||
| What are the effects of specimen geometry on staining? | 119 | ||
| Simple geometrical influences | 119 | ||
| Effects of more complex specimen geometry | 120 | ||
| What are the effects of resin embedding on staining? | 120 | ||
| Some dyestuff properties | 121 | ||
| General influences of dye chemistry on staining | 121 | ||
| Effects of dye impurities on staining | 121 | ||
| Dye nomenclature | 122 | ||
| Problem avoidance and troubleshooting | 122 | ||
| Strategies for avoiding problems, minimizing the need for troubleshooting | 123 | ||
| Issues concerning staining procedures | 123 | ||
| Issues concerning staining reagents | 123 | ||
| Cues for recognizing errors - before mistakes can be rectified they must be noticed | 123 | ||
| References | 123 | ||
| Further reading | 125 | ||
| 10 - The hematoxylins and eosin | 126 | ||
| Introduction | 126 | ||
| Eosin | 126 | ||
| Hematoxylin | 127 | ||
| Alum hematoxylins | 127 | ||
| Staining times with alum hematoxylins | 130 | ||
| Disadvantages of alum hematoxylins | 130 | ||
| Routine staining procedures using alum hematoxylins | 131 | ||
| Papanicolaou staining method for cervical cytological preparations | 131 | ||
| Iron hematoxylins | 132 | ||
| Weigert’s hematoxylin | 132 | ||
| Heidenhain’s hematoxylin | 133 | ||
| Verhöeff’s hematoxylin (Verhöeff, 1908) | 134 | ||
| Tungsten hematoxylins | 134 | ||
| Molybdenum hematoxylins | 136 | ||
| Lead hematoxylins | 136 | ||
| Hematoxylin without a mordant | 136 | ||
| Quality control in routine H&E staining | 136 | ||
| Difficult sections | 137 | ||
| References | 137 | ||
| 11 - Automation in the histology department | 139 | ||
| Introduction | 139 | ||
| The drivers for change | 139 | ||
| Barcode technology and automated sample tracking | 140 | ||
| Dissection/grossing | 141 | ||
| Processing | 142 | ||
| Embedding | 143 | ||
| Trimming and microtomy | 144 | ||
| Hematoxylin and eosin | 145 | ||
| Tinctorial staining | 147 | ||
| Immunohistochemistry | 147 | ||
| Molecular techniques | 149 | ||
| Slide digitization | 149 | ||
| Automated block filing/ archiving | 150 | ||
| The future of automation in histology | 150 | ||
| References | 151 | ||
| 12 - Connective and other mesenchymal tissues with their stains | 153 | ||
| Introduction | 153 | ||
| Connective tissue | 153 | ||
| Formed or fibrous intercellular substances | 153 | ||
| Collagen fibers | 154 | ||
| Types of collagen | 154 | ||
| Staining reactions of collagen | 155 | ||
| Reticular fibers | 155 | ||
| Elastic fibers | 155 | ||
| Oxytalan fibers | 156 | ||
| Elaunin fibers | 156 | ||
| Basement membranes | 156 | ||
| Methenamine silver microwave method | 157 | ||
| Connective tissue cells | 158 | ||
| Fibroblasts | 158 | ||
| Fat cells or adipocytes | 158 | ||
| Areolar tissue | 158 | ||
| Adipose tissue | 159 | ||
| ‘Myxoid’ connective tissue | 159 | ||
| Dense connective tissue | 159 | ||
| Cartilage | 159 | ||
| Bone | 160 | ||
| Other mesenchymal tissues | 160 | ||
| Muscular tissue | 160 | ||
| Involuntary smooth muscle | 160 | ||
| Voluntary striated muscle | 160 | ||
| Striated cardiac muscle | 161 | ||
| General structure of muscle | 161 | ||
| Fibrin and fibrinoid | 162 | ||
| Connective tissue stains (Table 12.1) | 162 | ||
| Trichrome stains | 162 | ||
| Factors affecting trichrome staining | 162 | ||
| Tissue permeability and dye molecular size | 162 | ||
| Heat | 163 | ||
| pH | 163 | ||
| Nuclear stains | 163 | ||
| Effects of fixation | 163 | ||
| Role of phosphotungstic and phosphomolybdic acids (PTA and PMA) | 165 | ||
| Practical uses of PMA and PTA | 165 | ||
| Heidenhain’s ‘Azan’ | 166 | ||
| The demonstration of fibrin | 166 | ||
| Demonstration of muscle striations | 167 | ||
| Staining of elastic tissue fibers | 167 | ||
| General notes on the mechanism of elastic staining | 167 | ||
| Orcein methods | 168 | ||
| Weigert’s resorcin-fuchsin method | 168 | ||
| Mechanism of Weigert elastin staining | 169 | ||
| Modifications of the Weigert technique | 169 | ||
| Aldehyde fuchsin | 170 | ||
| The demonstration of reticular fibers | 170 | ||
| Metal impregnation techniques | 170 | ||
| Preparation of silver solutions | 171 | ||
| References | 174 | ||
| Further reading | 174 | ||
| 13 - Carbohydrates | 176 | ||
| Introduction | 176 | ||
| Classification of carbohydrates | 176 | ||
| Monosaccharides | 176 | ||
| Polysaccharides | 176 | ||
| Connective tissue glycoconjugates, the proteoglycans | 177 | ||
| Mucins | 179 | ||
| Other glycoproteins | 180 | ||
| Fixation | 180 | ||
| Techniques for the demonstration of carbohydrates | 181 | ||
| The periodic acid-Schiff (PAS) technique | 181 | ||
| Mechanism of the PAS technique | 181 | ||
| Schiff reagent | 183 | ||
| Standard alcian blue technique | 184 | ||
| Low pH alcian blue technique | 185 | ||
| Combined alcian blue-PAS | 185 | ||
| Mucicarmine | 186 | ||
| Colloidal iron | 187 | ||
| High iron diamine | 188 | ||
| Metachromatic methods | 189 | ||
| Lectins | 190 | ||
| Immunohistochemistry | 190 | ||
| Enzymatic digestion techniques | 190 | ||
| Diastase digestion | 190 | ||
| Sialidase (neuraminidase) | 191 | ||
| Hyaluronidase | 192 | ||
| Chemical modification and blocking techniques | 192 | ||
| Methylation | 192 | ||
| Saponification | 193 | ||
| References | 194 | ||
| Further reading | 197 | ||
| 14 - Pigments and minerals\r | 198 | ||
| Introduction | 198 | ||
| Pigments can be classified under the following headings | 198 | ||
| Endogenous pigments | 198 | ||
| Hematogenous | 198 | ||
| Hemosiderins | 198 | ||
| Normal and abnormal iron metabolism | 199 | ||
| Demonstration of hemosiderin and iron | 199 | ||
| Hemoglobin | 201 | ||
| Demonstration of hemoglobin | 201 | ||
| Bile pigments | 202 | ||
| Demonstration of bile pigments and hematoidin | 204 | ||
| Porphyrin pigments | 205 | ||
| Non-hematogenous endogenous pigments | 205 | ||
| Melanins | 205 | ||
| The most common sites where melanin can be found are | 206 | ||
| Demonstration of melanin | 207 | ||
| Reducing methods for melanin | 207 | ||
| Enzyme methods for melanin | 210 | ||
| Solubility and bleaching methods for melanin | 210 | ||
| Formalin-induced fluorescence (FIF) | 212 | ||
| Other methods for melanin | 212 | ||
| Immunohistochemistry | 213 | ||
| Lipofuscins | 214 | ||
| Demonstration of lipofuscins | 215 | ||
| Chromaffin | 216 | ||
| Pseudomelanosis pigment (melanosis coli) | 216 | ||
| Dubin-Johnson pigment | 216 | ||
| Ceroid-type lipofuscins | 216 | ||
| Hamazaki-Weisenberg bodies | 216 | ||
| Endogenous minerals | 217 | ||
| Calcium | 217 | ||
| Copper | 218 | ||
| Uric acid and urates | 220 | ||
| Artifact pigments | 221 | ||
| Formalin pigment | 221 | ||
| Malarial pigment | 221 | ||
| Schistosome pigment | 222 | ||
| Mercury pigment | 222 | ||
| Chromic oxide | 222 | ||
| Starch | 222 | ||
| Exogenous pigments and minerals | 222 | ||
| Tattoo pigment | 223 | ||
| Amalgam tattoo | 223 | ||
| Carbon | 223 | ||
| Tobacco | 224 | ||
| Silica | 224 | ||
| Asbestos | 225 | ||
| Lead | 225 | ||
| Beryllium and aluminum | 226 | ||
| Silver | 227 | ||
| References | 228 | ||
| 15 - Amyloid | 231 | ||
| Introduction | 231 | ||
| History | 231 | ||
| Composition | 232 | ||
| Ultrastructure | 232 | ||
| Classification and nomenclature | 233 | ||
| Pathogenesis | 235 | ||
| Amyloidosis | 236 | ||
| Other diseases in which amyloid occurs | 237 | ||
| Diagnosis | 238 | ||
| Differentiation between different amyloid types | 238 | ||
| Demonstration | 239 | ||
| Congo red | 239 | ||
| Sirius red | 241 | ||
| Metachromatic techniques for amyloid | 242 | ||
| Methyl violet | 242 | ||
| Crystal violet method (Hucker & Conn, 1928) | 242 | ||
| Methyl green (Bancroft, 1963) | 242 | ||
| Polarizing microscopy | 242 | ||
| Acquired fluorescence methods | 244 | ||
| Miscellaneous methods | 244 | ||
| Fibril extraction | 245 | ||
| Immunohistochemistry for amyloid | 245 | ||
| Laser microdissection-proteomics for typing amyloid | 246 | ||
| Evaluation of methods | 247 | ||
| The future | 248 | ||
| Acknowledgments | 249 | ||
| References | 249 | ||
| 16 - Traditional stains and modern techniques for demonstrating microorganisms in histology\r | 254 | ||
| Introduction | 254 | ||
| Size | 255 | ||
| Safety | 255 | ||
| General principles of detection and identification | 255 | ||
| Immunohistochemistry (IHC) | 256 | ||
| Molecular methods | 256 | ||
| Detection and identification of bacteria | 257 | ||
| Use of control sections | 258 | ||
| The Gram stain | 258 | ||
| Techniques for mycobacteria | 260 | ||
| Techniques for other important bacteria | 262 | ||
| Some important bacteria | 265 | ||
| Fungal infections | 266 | ||
| Identification of fungi | 267 | ||
| A selection of the more important fungi and actinomycetes | 268 | ||
| The demonstration of rickettsia | 270 | ||
| The detection and identification of viruses | 271 | ||
| Viral infections | 272 | ||
| Prion disease | 274 | ||
| The demonstration of protozoa and other organisms | 275 | ||
| Protozoa | 276 | ||
| Worms | 276 | ||
| References | 277 | ||
| Website | 279 | ||
| Further reading\r | 279 | ||
| 17 - Bone | 280 | ||
| Introduction | 280 | ||
| Normal bone | 280 | ||
| Bone collagen | 280 | ||
| Bone mineral | 281 | ||
| Bone cells | 281 | ||
| Osteoblasts | 281 | ||
| Osteocytes | 281 | ||
| Osteoclasts | 282 | ||
| Development and growth | 282 | ||
| Intramembranous ossification | 282 | ||
| Endochondral ossification | 282 | ||
| Techniques for analyzing bone | 283 | ||
| Biopsies | 283 | ||
| Amputation specimens | 283 | ||
| Resection/replacement specimens | 284 | ||
| Fixation | 284 | ||
| Sawing | 284 | ||
| Fine-detail specimen radiography | 285 | ||
| Area selection for embedding | 285 | ||
| Decalcification | 286 | ||
| Decalcifying agents | 286 | ||
| Acid decalcifiers | 287 | ||
| Strong inorganic acids, e.g. nitric, hydrochloric | 287 | ||
| Weak organic acids, e.g. formic, acetic, picric | 287 | ||
| Chelating agents | 288 | ||
| Proprietary decalcifiers | 288 | ||
| Factors influencing the rate of decalcification | 288 | ||
| Concentration of decalcifying agent | 288 | ||
| Temperature | 289 | ||
| Agitation | 289 | ||
| Suspension | 289 | ||
| Completion of decalcification | 290 | ||
| Decalcification endpoint test | 290 | ||
| Treatment following decalcification | 291 | ||
| Processing decalcified bone | 292 | ||
| Microtomy of bone | 292 | ||
| Microtomes and knives | 292 | ||
| Microtome sectioning of bone | 293 | ||
| Flattening and adhesion | 293 | ||
| Frozen sections of bone | 294 | ||
| Troubleshooting | 295 | ||
| Surface decalcification | 295 | ||
| Poor fixation | 295 | ||
| Poor processing | 296 | ||
| Adhesive tape methods | 296 | ||
| Staining methods for decalcified bone sections | 296 | ||
| Hematoxylin and eosin (H&E) | 297 | ||
| Collagen stains | 299 | ||
| Cartilage and acid mucopolysaccharides | 299 | ||
| Bone canaliculi | 300 | ||
| Immunohistochemistry (IHC) | 301 | ||
| Preparation of mineralized bone | 301 | ||
| Morphometry of bone | 301 | ||
| Microcomputed tomography (microCT) | 302 | ||
| Acknowledgments | 303 | ||
| References | 303 | ||
| Further reading | 305 | ||
| 18 - Neuropathology and muscle biopsy techniques | 306 | ||
| Introduction | 306 | ||
| The components of the normal central nervous system | 306 | ||
| Techniques for staining neurons | 308 | ||
| Tinctorial stains for Nissl substance | 308 | ||
| Immunohistochemistry of neurons | 309 | ||
| Techniques for staining axons and neuronal processes | 310 | ||
| Myelin | 311 | ||
| The neuroglia | 313 | ||
| Ependymal cells | 313 | ||
| Astrocytes | 313 | ||
| Oligodendrocytes | 314 | ||
| Microglia | 315 | ||
| Neurodegeneration | 315 | ||
| Stains for detection of the changes of Alzheimer’s disease | 318 | ||
| Neuropathology laboratory specimen handling | 321 | ||
| Brain and spinal cord biopsies and excision specimens | 321 | ||
| Intraoperative diagnosis from smear preparations and frozen sections | 322 | ||
| Central nervous system tissues taken at autopsy | 322 | ||
| Peripheral nerve biopsies | 323 | ||
| Muscle biopsies | 325 | ||
| Enzyme histochemistry | 328 | ||
| Demonstration of myophosphorylase | 331 | ||
| Demonstration of phosphofructokinase | 332 | ||
| Acknowledgments | 332 | ||
| References | 332 | ||
| Further reading\r | 336 | ||
| 19 - Immunohistochemical and immunofluorescent techniques | 337 | ||
| Introduction | 337 | ||
| Immunohistochemistry theory | 339 | ||
| Definitions | 339 | ||
| Immunohistochemistry (IHC) | 339 | ||
| Antigen | 339 | ||
| Antibody | 339 | ||
| Antibody-antigen binding | 339 | ||
| Affinity | 339 | ||
| Avidity | 340 | ||
| Antibody specificity | 340 | ||
| Sensitivity | 340 | ||
| Production of primary reagents | 340 | ||
| Polyclonal antibodies | 340 | ||
| Monoclonal antibodies | 340 | ||
| Labels | 341 | ||
| Enzyme labels | 341 | ||
| Fluorescent labels | 342 | ||
| Radiolabels | 343 | ||
| Immunohistochemical methods | 343 | ||
| Traditional direct technique | 344 | ||
| Two-step indirect technique | 344 | ||
| Polymer chain two-step indirect technique | 344 | ||
| Unlabeled antibody-enzyme complex techniques (PAP and APAAP) and Immunogold silver staining technique (IGSS) | 344 | ||
| (Strept)avidin-biotin techniques | 344 | ||
| Amplification methods | 346 | ||
| Unmasking of antigen sites | 346 | ||
| Proteolytic enzyme digestion | 346 | ||
| Heat-mediated antigen retrieval techniques | 347 | ||
| Microwave antigen retrieval | 347 | ||
| Pressure cooker antigen retrieval | 348 | ||
| Steamer | 349 | ||
| Water bath | 349 | ||
| Advantages of heat pretreatment | 349 | ||
| Pitfalls of heat pretreatment | 349 | ||
| Commercial antigen retrieval solutions | 349 | ||
| Detection of low levels of antigen | 349 | ||
| Enhancement and amplification | 349 | ||
| Multiple labeling techniques | 351 | ||
| Immunohistochemistry in practice | 352 | ||
| Choice of technique | 352 | ||
| Fixation and paraffin wax block immunohisto | 352 | ||
| Frozen sections | 353 | ||
| Cytological preparations | 353 | ||
| Automation | 354 | ||
| Automated incubation methods | 354 | ||
| Blocking endogenous enzymes | 354 | ||
| Blocking background staining | 355 | ||
| Controls | 356 | ||
| Negative control | 356 | ||
| Positive control | 356 | ||
| Absorption control | 356 | ||
| Practical aspects of immunohistochemical staining | 356 | ||
| Dilution of immune serum/antibodies | 357 | ||
| Washes | 357 | ||
| Add the Tris, EDTA, and acid to the distilled water and adjust pH to 10 with 1M hydrochloric acid, then add the Tween | 358 | ||
| Manual incubation methods | 358 | ||
| Method selection | 358 | ||
| Preparative technique | 358 | ||
| Antigen retrieval techniques | 359 | ||
| Proteolytic enzyme methods | 359 | ||
| Heat-mediated antigen retrieval | 360 | ||
| Examples of immunostaining protocols for routine diagnostic antigens | 361 | ||
| Immunohistochemistry for immunoglobulin light chains in formalin-fixed paraffin-wax sections | 361 | ||
| Immunohistochemistry for the assessment of HER2 expression | 362 | ||
| Application of fluorescence in situ hybridization (FISH) for HER2 assessment (Fig. 19.19) | 362 | ||
| Application of chromogenic in situ hybridization (CISH) as an alternative to FISH (Figs. 19.20 and 19.21) | 363 | ||
| Immunohistochemistry on frozen section and non-gynecological cytology smears | 364 | ||
| Immunohistochemistry on renal and skin biopsies | 364 | ||
| Immunofluorescence microscopy | 370 | ||
| Immunohistochemical staining techniques | 371 | ||
| Avidin-biotin techniques | 371 | ||
| Polymer techniques | 371 | ||
| Alkaline phosphatase technique | 372 | ||
| Quality control in immunohistochemistry | 372 | ||
| Introduction | 372 | ||
| Factors affecting stain quality | 373 | ||
| Fixation | 373 | ||
| Processing | 374 | ||
| Reversal of fixation/epitope retrieval | 374 | ||
| Reagent factors | 375 | ||
| Buffers and diluents | 375 | ||
| Antibodies | 375 | ||
| Procedural factors | 376 | ||
| Block and slide storage conditions | 376 | ||
| Monitoring stain quality | 376 | ||
| Validation of antibodies | 377 | ||
| Controls | 378 | ||
| Internal and external positive controls | 378 | ||
| Daily slide review | 379 | ||
| External quality assurance | 380 | ||
| Troubleshooting | 380 | ||
| False negative staining | 380 | ||
| Process failure | 381 | ||
| Positive control selection | 381 | ||
| Incomplete deparaffinization | 382 | ||
| Epitope retrieval | 382 | ||
| Temperature | 382 | ||
| Antibody preparation | 383 | ||
| Chromogen incompatibility | 383 | ||
| False positive staining | 383 | ||
| Poor quality of fixation | 383 | ||
| Technical preparation | 383 | ||
| Epitope retrieval | 385 | ||
| Tissue drying and wetting agents | 386 | ||
| Intrinsic tissue factors | 386 | ||
| Antibody concentration | 387 | ||
| Detection system | 387 | ||
| Chromogen | 388 | ||
| Species cross-reactivity | 388 | ||
| Automation error | 388 | ||
| References | 389 | ||
| Further reading | 394 | ||
| 20 - Molecular pathology | 395 | ||
| Introduction | 395 | ||
| Glossary of terms and abbreviations commonly used in this chapter | 396 | ||
| Accreditation | 396 | ||
| Amplification | 396 | ||
| Anneal | 396 | ||
| Base | 396 | ||
| Base pair (bp) | 396 | ||
| Bioinformatics | 396 | ||
| ‘Black box’ system | 396 | ||
| CE-IVD (Conformité Européene, in vitro diagnostic) | 396 | ||
| Chromosome | 396 | ||
| Codon | 396 | ||
| Companion diagnostic | 396 | ||
| Complementary sequence | 396 | ||
| Deletion | 397 | ||
| Denature | 397 | ||
| Deoxyribonucleic acid (DNA) | 397 | ||
| DNA polymerase | 397 | ||
| Driver mutation | 397 | ||
| Exon | 397 | ||
| Extraction | 397 | ||
| Fluorescence in situ hybridization (FISH) | 397 | ||
| Food and drug administration (FDA) | 397 | ||
| Gene | 397 | ||
| Genome | 397 | ||
| Germline mutation | 397 | ||
| Helicases | 397 | ||
| Home-brew assay | 397 | ||
| Hot-spot | 397 | ||
| Hybridization | 397 | ||
| Immunohistochemistry (IHC) | 397 | ||
| Immunotherapy | 397 | ||
| In situ | 397 | ||
| In situ hybridization (ISH) | 397 | ||
| Insertion | 397 | ||
| ISO standards | 398 | ||
| Kilobase (kb) | 398 | ||
| Macrodissection | 398 | ||
| Melting temperature | 398 | ||
| Messenger RNA (mRNA) | 398 | ||
| Methylation | 398 | ||
| Microsatellite | 398 | ||
| Mismatch repair (MMR) | 398 | ||
| Missense mutation | 398 | ||
| Multiplex testing | 398 | ||
| Mutation | 398 | ||
| Nucleic acid (NA) | 398 | ||
| Nucleotide | 398 | ||
| Oligonucleotide | 398 | ||
| Passenger mutation | 398 | ||
| Plasma | 398 | ||
| Polymerase chain reaction (PCR) | 398 | ||
| Probe | 398 | ||
| Replication | 398 | ||
| Ribonucleic acid (RNA) | 398 | ||
| Sensitivity | 399 | ||
| Single nucleotide variant (SNV) | 399 | ||
| Sporadic mutation | 399 | ||
| Targeted therapy | 399 | ||
| Template | 399 | ||
| Topoisomerases | 399 | ||
| Transcription | 399 | ||
| Transfer RNA (tRNA) | 399 | ||
| Translation | 399 | ||
| Translocation | 399 | ||
| Tyrosine kinase inhibitor (TKI) | 399 | ||
| Validation | 399 | ||
| Wild-type | 399 | ||
| Essentials of molecular pathology: nucleic acid structure and function | 399 | ||
| From what is DNA made? | 399 | ||
| How does DNA replicate? | 400 | ||
| From blueprint to building blocks: transcription and translation | 400 | ||
| Techniques in molecular pathology | 401 | ||
| Practical considerations for the laboratory scientist | 401 | ||
| Practicalities of tissue sample workflow | 401 | ||
| Fundamental factors in pre-analytical workflows | 402 | ||
| Optimizing the use of tissue | 402 | ||
| Processing the sample | 402 | ||
| Specimen preparation and assessment | 402 | ||
| Extraction and/or isolation of target molecules | 402 | ||
| Amplification and measurement | 403 | ||
| Analysis and interpretation | 403 | ||
| Reporting of findings | 403 | ||
| Standardization and accreditation | 403 | ||
| Use of control material | 404 | ||
| Techniques testing for mutations in genes: the polymerase chain reaction (PCR) | 404 | ||
| Stages of PCR based analysis | 404 | ||
| Nucleic acid (NA) extraction | 404 | ||
| Spin column purification and ‘on-membrane’ DNA isolation | 405 | ||
| Magnetic bead isolation | 406 | ||
| Ultrasonication methods | 406 | ||
| PCR analysis methods | 407 | ||
| Sanger sequencing | 407 | ||
| Real-time PCR | 407 | ||
| Pyrosequencing | 408 | ||
| Approaches to PCR testing | 409 | ||
| Uses of PCR | 409 | ||
| EGFR1 in non-small cell lung cancer (NSCLC) | 409 | ||
| BRAF in melanoma | 410 | ||
| KRAS and NRAS in colorectal cancer | 410 | ||
| KIT and platelet derived growth factor alpha (PDGFRA) in gastrointestinal stromal tumors (GISTs) | 410 | ||
| Techniques testing for abnormalities in chromosomes: fluorescence in situ hybridization (FISH) | 410 | ||
| Automation of FISH processing | 411 | ||
| Types of probes available for use in FISH | 412 | ||
| Dual-color/single-fusion probes | 412 | ||
| Extra-signal (ES) probes | 412 | ||
| Dual-color/break-apart probes | 412 | ||
| Dual-color/dual-fusion probes | 412 | ||
| Uses of FISH | 412 | ||
| Limitations of FISH | 412 | ||
| FISH methodologies | 414 | ||
| Alternative techniques for examining chromosome structure | 418 | ||
| Techniques testing for abnormalities in RNA: in situ hybridization (ISH) | 418 | ||
| Uses of ISH | 419 | ||
| Techniques testing for abnormalities of protein expression: immunohistochemistry (IHC) (see also Chapter 19) | 419 | ||
| Techniques testing for surrogate markers of molecular alterations | 420 | ||
| HER2 in breast cancer | 420 | ||
| ALK in non-small cell lung cancer (NSCLC) | 420 | ||
| Future directions | 421 | ||
| Techniques testing for multiple molecular alterations: multiplex testing | 421 | ||
| Next-generation sequencing (NGS): single platform multiplex testing | 421 | ||
| Library preparation | 421 | ||
| Sequencing | 421 | ||
| Bioinformatic interpretation | 422 | ||
| Multi-platform multiplex testing | 422 | ||
| New avenues in molecular techniques: circulating tumor DNA (ctDNA) | 422 | ||
| The challenge: choosing the most appropriate molecular technique | 423 | ||
| The technical challenges of molecular testing | 423 | ||
| Tissue size and DNA available for testing | 423 | ||
| The deleterious effects of formalin fixation | 424 | ||
| Sensitivity of molecular tests | 424 | ||
| Clinical correlation in molecular pathology | 425 | ||
| Molecular pathology reports | 425 | ||
| The relevance of a mutation | 425 | ||
| Molecular pathology in specific tumors | 426 | ||
| Non-small cell lung cancer (NSCLC) | 426 | ||
| EGFR mutations | 426 | ||
| ALK translocations | 426 | ||
| PD-L1 expression | 427 | ||
| Colorectal cancer | 427 | ||
| RAS mutations | 427 | ||
| BRAF mutations | 427 | ||
| Mismatch repair protein (MMR) expression | 428 | ||
| Immunoscore | 428 | ||
| Melanoma | 428 | ||
| BRAF mutations | 428 | ||
| KIT mutations | 428 | ||
| Immunotherapy | 429 | ||
| Breast cancer | 429 | ||
| HER2 amplification | 429 | ||
| Oncotype DX® | 430 | ||
| Gastric and esophageal cancers | 430 | ||
| HER2 amplification | 430 | ||
| Immunotherapy | 430 | ||
| Molecular classification of gastric cancers | 430 | ||
| Brain tumors | 430 | ||
| Molecular pathology across tumor types | 430 | ||
| Predictive markers for non-targeted therapy | 430 | ||
| Perspective | 431 | ||
| Acknowledgment | 431 | ||
| References | 432 | ||
| Useful websites | 433 | ||
| 21 - Transmission electron microscopy\r | 434 | ||
| Introduction | 434 | ||
| Tissue preparation for transmission electron microscopy | 434 | ||
| Specimen handling | 434 | ||
| Fixation | 436 | ||
| Fixative concentration | 436 | ||
| Temperature | 436 | ||
| Duration of fixation | 436 | ||
| Buffers | 436 | ||
| Phosphate buffers | 436 | ||
| Alternative buffers | 437 | ||
| Aldehyde fixatives | 437 | ||
| Glutaraldehyde | 437 | ||
| Formaldehyde | 437 | ||
| Aldehyde combinations | 437 | ||
| Osmium tetroxide | 438 | ||
| Wash buffer and staining | 438 | ||
| Dehydration | 439 | ||
| Embedding | 439 | ||
| Epoxy resins | 439 | ||
| Acrylic resins | 440 | ||
| Tissue processing schedules | 440 | ||
| Procedures for other tissue samples | 441 | ||
| Cultured cells | 441 | ||
| Cell suspensions or particulate matter | 441 | ||
| Material embedded in paraffin wax/cell smears | 441 | ||
| Microwave processing | 442 | ||
| Ultramicrotomy | 442 | ||
| Glass knives | 442 | ||
| Diamond knives | 443 | ||
| Trough fluids | 444 | ||
| Block trimming | 444 | ||
| Semi-thin sections | 444 | ||
| Section collection | 445 | ||
| Support films | 445 | ||
| Ultra-thin sectioning | 445 | ||
| Staining | 448 | ||
| Uranyl salts | 448 | ||
| Lead salts | 448 | ||
| Diagnostic applications | 449 | ||
| The use of TEM for diagnostics | 449 | ||
| Renal disease | 449 | ||
| The location and morphology of immune complex deposits | 449 | ||
| Variations in the thickness and/or texture of the GBM | 450 | ||
| Morphological and numerical changes in the cellular components of the glomerulus | 453 | ||
| Renal transplants | 455 | ||
| Malignant tumors | 457 | ||
| Mesothelioma | 457 | ||
| Langerhans’ histiocytosis (Histiocytosis X) | 460 | ||
| Non-neoplastic diseases | 461 | ||
| Skeletal muscle | 461 | ||
| Epidermolysis bullosa (mechanobullous dermatoses) | 461 | ||
| Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) | 464 | ||
| Amyloid | 465 | ||
| Cornea | 465 | ||
| Cilia | 465 | ||
| Microsporidia | 467 | ||
| Acknowledgments | 469 | ||
| References | 469 | ||
| 22 - Digital pathology | 476 | ||
| Key points\r | 476 | ||
| Introduction | 476 | ||
| Benefits of digital images over physical glass slides | 476 | ||
| Digital images | 476 | ||
| Resolution | 477 | ||
| Pixel depth | 477 | ||
| File size compression | 477 | ||
| Histology as digital images | 477 | ||
| Image acquisition | 479 | ||
| Special cases | 479 | ||
| Large blocks | 479 | ||
| Cytology preparations | 480 | ||
| Fluorescent slides | 480 | ||
| Measures to ensure good quality digital images | 480 | ||
| Accessing and viewing whole slide images | 481 | ||
| Image streaming | 481 | ||
| Client software | 481 | ||
| Hardware installation and image file storage | 482 | ||
| Image storage arrangements | 482 | ||
| Applications | 483 | ||
| Non-diagnostic applications | 483 | ||
| Education & training | 483 | ||
| Research, including image analysis | 483 | ||
| Quality assurance | 483 | ||
| Diagnostic applications | 484 | ||
| Remote intraoperative diagnosis | 484 | ||
| Second opinion | 484 | ||
| Multidisciplinary team (MDT) meetings and clinicopathological conference (CPC) | 485 | ||
| Clinical quantification | 485 | ||
| Whole laboratory digitization | 485 | ||
| The digital pathology workstation | 486 | ||
| Validation and regulatory issues | 486 | ||
| Examples of validation studies | 488 | ||
| Regulatory frameworks and standards | 488 | ||
| File storage | 488 | ||
| Future development | 489 | ||
| Realizing the benefits of a digital workflow | 489 | ||
| Integration with existing laboratory management systems | 489 | ||
| Barcoding of slides | 490 | ||
| Report creation | 490 | ||
| Adoption of paperless requesting and reporting | 491 | ||
| Staff training and reprofiling | 491 | ||
| Pathologist workstations | 491 | ||
| Pathologist training | 491 | ||
| Application of specific validation studies | 491 | ||
| Summary | 491 | ||
| References | 491 | ||
| DiagnosticAppendices\r | 493 | ||
| I - Traditional methods\r | 495 | ||
| Introduction | 495 | ||
| Lipids | 495 | ||
| Classification | 495 | ||
| Fixation and microtomy | 495 | ||
| Fat stains and the Sudan dyes | 496 | ||
| Cholesterol | 496 | ||
| Sphingomyelin | 497 | ||
| Cerebrosides | 497 | ||
| Sulfatides | 497 | ||
| Gangliosides | 498 | ||
| Proteins and nucleic acids | 498 | ||
| Phenyl groups | 498 | ||
| Disulfide linkage | 499 | ||
| Indole groups | 499 | ||
| Nucleic acids | 500 | ||
| Demonstration of nucleic acids | 500 | ||
| Fixation | 500 | ||
| Basophilia | 500 | ||
| Deoxyribonucleic acid (DNA) | 501 | ||
| Feulgen reaction | 501 | ||
| Ribonucleic acid (RNA) | 501 | ||
| Methyl green-pyronin | 501 | ||
| Digestion methods for nucleic acids | 502 | ||
| Enzyme histochemistry | 502 | ||
| Fixation for enzyme histochemistry | 502 | ||
| Smears | 503 | ||
| Enzyme types | 503 | ||
| Oxidoreductases | 503 | ||
| Transferases | 503 | ||
| Hydrolases | 503 | ||
| Diagnostic applications | 503 | ||
| Colonic biopsy in cases of suspected Hirschsprung’s disease | 503 | ||
| References | 504 | ||
| II - Tissue microarray This is an abridged version of this topic in Chapter 6 of the 7th edition of this text\r | 505 | ||
| Introduction | 505 | ||
| Types of tissue microarrays | 505 | ||
| Designing the grid | 506 | ||
| Fixation and processing of tissues and controls (see Chapters 4 and 6Chapter 4Chapter 6) | 506 | ||
| Preparation of the donor block | 506 | ||
| Needle sizes | 506 | ||
| Database for tissue microarray analysis (Shaknovich et al., 2003) | 506 | ||
| Arrayers | 507 | ||
| Preparation of the recipient array block | 507 | ||
| Smoothing and sectioning | 508 | ||
| Microtomy | 508 | ||
| Troubleshooting and tips | 508 | ||
| References | 508 | ||
| III - Applications of immunohistochemistry\r | 509 | ||
| Introduction | 509 | ||
| Classification of neoplasia | 509 | ||
| Anaplastic tumors | 509 | ||
| Technical\rAppendices | 517 | ||
| IV - Measurement units\r | 519 | ||
| Introduction | 519 | ||
| Structure of SI units | 519 | ||
| Base units | 519 | ||
| Derived units | 519 | ||
| Volume | 519 | ||
| Length | 520 | ||
| Mass | 520 | ||
| Temperature conversion | 520 | ||
| References | 520 | ||
| V - Preparation of solutions\r | 521 | ||
| Introduction | 521 | ||
| Volume-to-volume solution | 521 | ||
| Weight-to-volume solutions | 522 | ||
| Molar solutions (M) | 522 | ||
| Normal solutions (N) | 523 | ||
| Preparation of useful solutions | 523 | ||
| VI - Buffer solutions\r | 525 | ||
| Introduction | 525 | ||
| General notes regarding buffer solutions | 525 | ||
| References | 528 | ||
| Further reading | 528 | ||
| VII - Solubility of some common reagents and dyes\r | 529 | ||
| VIII - Mounting media and slide coatings\r | 535 | ||
| Introduction | 535 | ||
| Mountants | 535 | ||
| Combined coverslip and mountant | 535 | ||
| Adhesive slides | 536 | ||
| Silanized (APES) slides | 536 | ||
| Polylysine (PLL) slides | 536 | ||
| Albumen-coated slides | 536 | ||
| References | 536 | ||
| Index | 537 | ||
| A | 537 | ||
| B | 538 | ||
| C | 540 | ||
| D | 541 | ||
| E | 542 | ||
| F | 543 | ||
| G | 544 | ||
| H | 545 | ||
| I | 546 | ||
| J | 548 | ||
| K | 548 | ||
| L | 548 | ||
| M | 549 | ||
| N | 552 | ||
| O | 552 | ||
| P | 552 | ||
| Q | 554 | ||
| R | 554 | ||
| S | 555 | ||
| T | 556 | ||
| U | 557 | ||
| V | 557 | ||
| W | 557 | ||
| X | 557 | ||
| Y | 557 | ||
| Z | 557 | ||
| IBC | ES2 |