Freida L Carson Phd, HT(ASCP) Department of Pathology (Retired) Baylor University Medical Center Dallas, Texas
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Histotechnology A Self Instructional Text 4th Edition Freida L Carson PhD, HT(ASCP) Department of Pathology (retired) Baylor University Medical Center Dallas, Texas Christa Hladik Cappellano BS, HT(ASCP)cm, QIHC Manager, Workflow Consultant Roche Tissue Diagnostics Roche Diagnostics Corporation Fishers, IN Table of Contents xi Preface 15 Glyoxal 15 Mercuric chloride xi Glossary 16 Osmium tetroxide 16 Phosphate buffered osmium tetroxide Fixation 16 Picric acid Chapter 01 | 17 Potassium dichromate 17 Zinc salts 2 Definition 19 Other fixative ingredients 2 Functions of fixatives 19 Compound or combined fixatives 2 Actions of fixatives 19 B-5 fixative 20 Bouin solution 4 Factors affecting fixation 20 Gendre solution 4 Temperature 4 Size 21 Hollande solution 5 Volume ratio 21 Formaldehyde-glutaraldehyde (4CF-1G) 5 Time 21 Zenker & Helly (Zenker-Formol) solutions 7 Choice of fixative 22 Orth solution 7 Penetration 22 Zamboni solution (buffered picric acid- formaldehyde or PAF) 8 Tissue storage 22 Zinc formalin solutions 8 pH 23 8 Osmolality Aqueous zinc formalin (original formula) 23 Unbuffered aqueous zinc formalin 9 Reactions of the cell with fixatives 23 Alcoholic zinc chloride formalin 9 The nucleus 9 Proteins 23 Nonaqueous fixatives 9 Lipids 23 Acetone 9 Carbohydrates 24 Alcohol 24 Carnoy solution 9 Simple aqueous fixatives or fixative 24 Clarke fluid ingredients 9 Acetic acid 24 Transport solutions 10 Formaldehyde 24 Michel transport medium 12 10% aqueous formalin 25 PBS buffer stock solution (also used in immunohistochemistry) 12 10% formalin saline 25 PBS-10% sucrose solution 12 Calcium formalin 25 Fixatives for electron microscopy 12 Formalin ammonium bromide 25 Advantages of primary osmium tetroxide fixation 13 Acetate formalin 25 Disadvantages of primary osmium tetroxide fixation 13 10% neutralized formalin 25 Advantages of primary aldehyde fixation 13 10% neutral buffered formalin 25 Disadvantages of primary aldehyde fixation 13 Modified Millonig formalin 25 Advantages of primary buffered PAF fixation 13 Phosphate buffered paraformaldehyde 25 Disadvantages of primary buffered PAF fixation 13 Alcoholic formalin 14 Glutaraldehyde 25 Removal of fixation pigments 14 Phosphate buffered glutaraldehyde 26 Lugol Iodine Solution Histotechnology 4e iii ©ASCP 2015 ISBN 978 - 089189-6319 Contents 26 Hallmarks of good fixation 45 Soft mushy tissue 45 Incorrect orientation 26 Troubleshooting fixation problems 46 26 Autolysis Tissue carryover 26 Incomplete fixation 46 Tissue not embedded at the same level 46 Pieces of tissue missing from the block 29 Learning activities 46 Special techniques in processing 29 References 46 Decalcification 47 Acid methods Processing 48 Chelating agents Chapter 02 | 48 End point of decalcification 32 Dehydration 49 Undecalcified bone 32 Alcohols 49 Troubleshooting decalcification 50 Frozen sections 33 Ethyl alcohol (ethanol) 51 33 Methyl alcohol (methanol) Frozen sectioning formalin fixed tissue 51 33 Isopropyl alcohol (isopropanol) Troubleshooting processing tissue for frozen sections 33 Butyl alcohol (butanol) 52 Learning activities 33 Acetone 52 34 Universal solvents References 34 Clearing Instrumentation 34 Xylene Chapter 03 | 34 Toluene 35 Benzene 54 Microscopes 35 Chloroform 54 Light microscope 35 Acetone 55 Polarizing microscope 35 Essential oils 56 Phase-contrast microscope 35 Limonene reagents (xylene substitute) 56 Darkfield microscope 36 Aliphatic hydrocarbons (xylene substitutes) 56 Fluorescence microscope 36 Other clearing agents 57 Electron microscope 36 Infiltration 58 Microtomes 37 Paraffin 58 Rotary microtome 38 Protocol 1 58 Sliding microtome 38 Protocol 2 58 Clinical freezing microtome 38 Quality control of paraffin processing 58 Microtome blades 39 Microwave oven processing 60 Troubleshooting microtomy 39 Water soluble waxes 60 Crooked ribbons 40 Celloidin 61 Block face unevenly sectioned 40 Plastics 61 Holes in the section 40 Glycol methacrylate 62 Failure of ribbon to form 40 Epoxy resins 62 Lifting of the section from the blade as the block is raised 41 Agar & gelatin 62 Washboarding or undulations in the section 41 Troubleshooting processing 64 Chatter, or microscopic vibration, in the section 41 Precipitate in the processor chamber & in the tub- 64 Skipped or varied thickness of sections (thick & thin sections) ing 64 Compressed, wrinkled, or jammed sections 41 Overdehydration 64 Lengthwise scratches or splits in the ribbon 41 Poor processing 65 Fragmented or torn sections 42 Sponge artifact 66 Sections flying and sticking to nearby objects or other parts of the 42 Tissue accidentally desiccated microtome 42 Embedding & specimen orientation 45 Troubleshooting embedding Histotechnology 4e iv ISBN 978 - 089189-6319 ©ASCP 2015 Contents 66 Cryostat 88 Chemical hazards 67 Troubleshooting cryotomy 90 Carcinogens 67 Poorly adjusted antiroll plate 90 Corrosive substances 67 Incomplete sections 91 Fire & explosive hazards 92 Hazardous chemical spills & storage 67 Tissue processors 92 Chemical storage 67 Conventional processor 92 Hazardous chemical disposal 68 Microwave processor 93 Hazard identification 69 Stainers & coverslippers 95 General safety practices 69 Automatic stainer 95 Employees 70 Microwave staining oven 95 Supervisors 71 Automatic coverslipper 95 Learning activities 71 Miscellaneous equipment 71 Flotation baths 96 References 73 Chromium potassium sulfate coated slides 73 Poly-L-lysine coated slides Laboratory Mathematics & 73 Aminoalkylsilane treated slides 76 Dryers & ovens Solution Preparation 77 Slide & cassette printers Chapter 05 | 78 Circulating water bath 98 Percentage solutions 78 Freezers & refrigerators 98 Problems & examples 78 pH meters 79 Balances & scales 99 Use of the gravimetric factor in solution 79 Embedding center preparation 79 Micrometer pipettes 99 Problems & examples 79 Solvent recycler 99 Hydrates 80 Problems incurred with instruments 100 Normal & molar solutions 80 Equipment malfunction 100 Problems 80 Automated stainers and/or tissue processors 101 The metric system 80 Miscellaneous problems 101 Problems 80 Instrument quality control 80 New instrument verification 101 Temperature conversion 80 Quality control program 101 Problems 84 Learning activities 102 Buffers 84 References 102 General guidelines for solution preparation, use & storage Safety 102 Stability of solutions Chapter 04 | 104 Answers to problems 86 Biological or infectious hazards 104 Learning activities 86 Tuberculosis exposure 87 Cryogenic sprays 104 References 87 HIV, hepatitis C virus (HCV) & HBV 87 Creutzfeldt-Jakob disease (CJD) 87 Handling tissue waste 88 Mechanical hazards 88 Ergonomics Histotechnology 4e v ©ASCP 2015 ISBN 978 - 089189-6319 Contents 122 Red or red-brown nuclei Nuclear & Cytoplasmic 122 Pale cytoplasmic staining Staining 123 Dark cytoplasmic staining Chapter 06 | 124 Eosin not properly differentiated 106 Ultrastructure of the cell 124 Blue-black precipitate on top of sections 106 The nucleus 124 Water & slides turn milky when slides are placed in water following 106 Nuclear membrane alcohol during deparaffinization 106 Nuclear pores 124 Slides are hazy or milky in last xylene before applying cover glass 124 Uneven H&E staining 106 Nucleolus 125 Dark basophilic staining of nuclei & cytoplasm, especially around 107 Chromatin tissue edges 107 The cytoplasm 125 Poor contrast between nucleus & cytoplasm 107 Plasmalemma 126 Nucleic acid stains 108 Mitochondria 126 Feulgen reaction 108 Ribosomes 127 Methyl green-pyronin Y 108 Endoplasmic reticulum 129 Polychromatic stains 108 Golgi apparatus 130 May-Grunwald Giemsa stain 108 Centriole 108 Lysosomes 131 Mounting stained sections 109 Staining mechanisms 131 Resinous media 109 Nuclear staining 131 Aqueous mounting media 109 Cytoplasmic staining 132 Coverslips 132 Troubleshooting mounted stained sections 110 The dyes 132 Water bubbles noted in mounted sections 111 Factors affecting dye binding 132 All areas of section cannot be brought into focus 111 Differentiation 134 Cornflaking artifact seen on mounted sections 112 The nuclear dyes 134 Mounted stained sections are not as crisp as usual when viewed 113 Harris hematoxylin microscopically 114 Mayer hematoxylin 135 Retracted mounting medium 114 Ehrlich hematoxylin 114 Gill hematoxylin 1 135 Learning activities 115 Scott solution 136 References 115 Weigert hematoxylin 115 Hematoxylin substitutes Carbohydrates & Amyloid 116 Celestine blue Chapter 07 | 116 Gallein iron hematoxylin 116 Plasma stains 138 Carbohydrates 116 Eosin solution 138 Group 1: neutral polysaccharides (nonionic homoglycans) 117 Eosin-phloxine B solution 138 Group II: acid mucopolysaccharides (anionic 117 H&E staining heteroglycans) 117 Manual progressive staining method 138 Group III: glycoproteins (mucins, mucoid, 118 Manual regressive staining method mucoprotein, mucosubstances) 118 Automated staining 138 Group IV: glycolipids 119 Note on results of H&E staining 139 Special staining techniques 119 Hints to help achieve good H&E staining 139 PAS reaction 120 Restoring tissue basophilia 139 Test for quality of Schiff reagent 121 Troubleshooting the H&E stain 141 PAS reaction with diastase digestion 121 Incomplete deparaffinization 143 Best carmine 121 Nuclear staining is not crisp 144 Mayer mucicarmine 122 Pale nuclear staining 147 Alcian blue, pH 2.5 122 Dark nuclear staining 149 Alcian blue with hyaluronidase 150 Alcian blue-PAS-hematoxylin Histotechnology 4e vi ISBN 978 - 089189-6319 ©ASCP 2015 Contents 151 Müller-Mowry colloidal iron 190 Nerve cell processes 154 Amyloid 190 Neuroglia 154 Alkaline