Basic Histopathology and Cytology Techniques PDF

Basic and Advanced Laboratory Techniques in Histopathology and Cytology Pranab Dey 123 Basic and Advanced Laboratory Techniques in Histopathology and Cytology Pranab Dey Basic and Advanced Laboratory Techniques in Histopathology and Cytology Pranab Dey Department of Cyt...

Basic and Advanced Laboratory Techniques in Histopathology and Cytology Pranab Dey 123 Basic and Advanced Laboratory Techniques in Histopathology and Cytology Pranab Dey Basic and Advanced Laboratory Techniques in Histopathology and Cytology Pranab Dey Department of Cytology and Gynecologic Pathology Post Graduate Institute of Medical Education and Research Chandigarh India ISBN 978-981-10-8251-1    ISBN 978-981-10-8252-8 (eBook) https://doi.org/10.1007/978-981-10-8252-8 Library of Congress Control Number: 2018941817 © Springer Nature Singapore Pte Ltd. 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Printed on acid-free paper This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. part of Springer Nature. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore Dedicated to Shree Shree Satyananda Giri, Rini and Madhumanti Preface Laboratory techniques in histopathology and cytology are the foundation of the diagnostic pathology. It is extremely essential to know all the basic and advanced techniques in laboratory. This book discusses the principles, steps, and troubleshooting areas of all the essential laboratory techniques in both histology and cytology laboratories. It contains multiple illustrations, micro- photographs, tables, and boxes that explain the techniques. In addition to the various advanced techniques, microscopy and quality control in the labora- tory have been discussed. I hope that the book will help all the postgraduate students in pathology, practising pathologists, and laboratory technologists. Chandigarh, India Pranab Dey 2017 September vii Acknowledgements I wish to express my thanks to Dr. Naren Aggarwal and Ms. Jagjeet Kaur Saini of Springer Nature who encouraged me in every stage in this work. I am thankful to Dr. Suvradeep Mitra who gave me his valuable sugges- tions at the time of the preparation of this manuscript. He also provided me many microphotographs. My sincere thanks to Dr. Charan Singh Rayat, Lecturer in Histopathology, who shared his vast experience in laboratory technology with me. I am grateful to my technical staffs who exchanged their views and opinions with me. My wife Rini and daughter Madhumanti constantly encouraged me during the writing of this book. They are my source of inspiration. Lastly I wish to express my gratitude to God because without His blessing nothing can be done. ix Contents Part I Basic Laboratory Techniques in Histopathology Laboratory 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives   3 1.1 Introduction   3 1.2 Aims of Fixation    3 1.3 Ideal Fixative   3 1.4 Tissue Changes in Fixation    3 1.5 Types of Fixation   4 1.5.1 Description of Nature of Fixation    5 1.6 Essential Precautions for Fixation in General   6 1.7 Mechanism of Fixation    6 1.8 Factors Affecting Fixation   8 1.9 Commonly Used Fixatives in the Laboratory  10 1.9.1 Formaldehyde  10 1.9.2 Preparation of Different Formalin Solution 10 1.9.3 Glutaraldehyde 11 1.9.4 Osmium Tetroxide 12 1.9.5 Methyl and Ethyl Alcohol 12 1.9.6 Acetone  12 1.9.7 Bouin’s Fixative 12 1.10 Mercury Salt-Containing Fixatives 13 1.10.1 Zenker’s Fluid  13 1.10.2 Helly’s Fluid  13 1.10.3 B5 Fixatives 13 1.10.4 Fixatives of Choice  13 1.11 Fixation Artefact  15 References 17 2 Processing of Tissue in Histopathology Laboratory  19 2.1 Factors that Influence Tissue Processing 19 2.2 Dehydration 20 2.3 Individual Dehydrating Agent 21 2.3.1 Alcohol 21 2.3.2 Dehydrating Agents Other than Alcohol 21 2.4 Clearing 22 2.4.1 Individual Clearing Agent 22 2.4.2 Other Clear Agents 23 xi xii Contents 2.5 Infiltration and Embedding  24 2.5.1 Different Impregnating Medium 24 2.6 Tissue Processing Methods 25 2.7 Overall Precautions of Tissue Processing  26 2.7.1 Time Schedule for Overnight Processing  26 2.7.2 Manual Tissue Processor 26 2.7.3 Microwave Processing 27 References 27 3 Embedding of Tissue in Histopathology 29 3.1 Embedding Medium 29 3.2 Different Types of Mould Used for Block 30 3.3 Tissue Embedding Method  30 3.4 Tissue Orientation and Embedding 32 3.5 Tissue Marking  32 References 33 4 Decalcification of Bony and Hard Tissue for Histopathology Processing 35 4.1 Introduction 35 4.1.1 Factors Controlling the Rate of Decalcification 36 4.2 The Methods of Decalcification 36 4.3 Chelating Agents 37 4.3.1 Other Procedures of Decalcification  38 4.4 Surface Decalcification  38 4.5 End Point Determination of Decalcification  39 Reference  39 5 Tissue Microtomy: Principle and Procedure 41 5.1 Introduction 41 5.2 Microtomes  41 5.2.1 Microtome Knife 43 5.2.2 Disposable Knife 44 5.2.3 Materials Used in Knife 44 5.2.4 Angles of Knife 44 5.3 Microtome Knife Sharpening  44 5.3.1 Manual Method 44 5.3.2 Factors Involved in Cutting 45 5.4 Sectioning the Paraffin Block  45 5.4.1 Steps of Tissue Sectioning 46 Reference  50 6 Frozen Section: Principle and Procedure 51 6.1 Introduction 51 6.2 Indications of Frozen Sections 51 6.3 The Principle of Frozen Section  51 6.4 Cryostat Sectioning 52 6.5 Staining  54 6.5.1 H&E Staining 54 6.5.2 Toluidine Blue Stain  55 6.6 Factors Affecting the Good-­Quality Section 55 References 55 Contents xiii 7 Staining Principle and General Procedure of Staining of the Tissue 57 7.1 Introduction 57 7.1.1 Dyes Used for Staining  57 7.1.2 Types of Dye  58 7.1.3 Types of Dye Based on Chemical Structures and Chromophore Groups 59 7.2 Mechanisms and Theory of Staining 59 7.3 Factors Influencing Staining  62 7.3.1 Nomenclature Used Regarding Dye  62 7.4 Metachromasia 63 7.4.1 Metachromatic Dyes  63 7.5 Progressive and Regressive Staining 65 7.6 Mordant 65 7.6.1 Accentuators  66 7.7 Staining Procedure 66 7.7.1 Preparation of Buffer Solutions 67 References 67 8 Haematoxylin and Eosin Stain of the Tissue Section 69 8.1 Introduction 69 8.2 Haematoxylin 69 8.3 Bluing 70 8.3.1 Preparation of Different Haematoxylins and Their Properties 71 8.3.2 Mayer’s Haematoxylin 71 8.3.3 Ehrlich’s Haematoxylin 71 8.3.4 Cole’s Haematoxylin 72 8.4 Counterstain by Eosin  72 8.5 Routine Haematoxylin and Eosin Stain 72 8.6 Iron Haematoxylin 73 8.6.1 Heidenhain’s Iron Haematoxylin 75 8.6.2 Verhoeff’s Iron Haematoxylin 76 8.6.3 Tungsten Haematoxylin 76 8.7 Clearing of the Smear  77 8.7.1 Mounting 77 8.7.2 Coverslip  79 References 79 9 Special Stains for the Carbohydrate, Protein, Lipid, Nucleic Acid and Pigments 81 9.1 Introduction 81 9.2 Carbohydrates  81 9.2.1 Simple Carbohydrates 82 9.3 Staining of Different Carbohydrates  84 9.3.1 Glycogen  84 9.3.2 Combined PAS-Alcian Blue Staining  87 9.4 Lipids 88 9.5 Stains 89 xiv Contents 9.5.1 Oil Red O 89 9.5.2 Sudan Black B 90 9.5.3 Ferric Haematoxylin for Phospholipid 91 9.6 Nucleic Acid and Proteins 91 9.6.1 Nucleic Acids 91 9.6.2 Proteins  91 9.6.3 Feulgen Stain 91 9.6.4 Methyl Green-Pyronin Stain  92 9.7 Pigments 92 9.8 Hemosiderin Pigment 92 9.8.1 Prussian Blue Reaction (Perls’ Reaction) for Ferric Iron 92 9.9 Bile Pigment 93 9.9.1 Fouchet’s Stain 93 9.10 Argyrophil Pigments  94 9.10.1 Grimelius Staining 94 9.10.2 Melanin  95 9.10.3 Schmorl’s Stain  95 9.10.4 Calcium  96 9.10.5 Formalin Pigment 97 References 97 10 Connective Tissue Stain: Principle and Procedure  99 10.1 Fibrous Part of Connective Tissue 99 10.1.1 Collagen 99 10.1.2 Reticulin Fibres  100 10.1.3 Elastic Fibres  100 10.1.4 Basement Membrane  100 10.2 Stains 100 10.2.1 Masson Trichrome  100 10.2.2 Van Gieson Stain  102 10.2.3 Reticulin Stain  102 10.2.4 Gordon and Sweet’s Method for Reticulin Stain 103 10.3 Elastic Fibres 105 10.3.1 Verhoeff’s Stain for Collagen 105 10.3.2 Weigert’s Resorcin-Fuchsin Stain  106 10.3.3 Orcein for Elastic Fibres 106 10.3.4 Phosphotungstic Acid Haematoxylin (PTAH)  106 References 108 11 Amyloid Staining 109 11.1 Introduction 109 11.2 Stains for Amyloid 110 11.2.1 Alkaline Congo Red Stain  110 11.2.2 Congo Red Stain by Highman 110 11.2.3 Thioflavine T Stain 111 References 111 Contents xv 12 Stains for the Microbial Organisms  113 12.1 Bacteria  113 12.1.1 Gram’s Stain 113 12.1.2 Ziehl-Neelsen Stain  114 12.1.3 Fite Acid-Fast Stain for Leprosy  115 12.2 Fungal Infection 116 12.2.1 Grocott’s Methenamine Silver 116 12.3 Spirochaetes 117 12.3.1 Warthin and Starry Technique  117 12.4 Viral Inclusions  118 12.4.1 Phloxine-Tartrazine Stain 118 References 118 Part II Basic Laboratory Techniques in Cytology Laboratory 13 Cytology Sample Procurement, Fixation and Processing 121 13.1 Introduction 121 13.2 Sample Collection 121 13.2.1 Cervical Cytology 121 13.2.2 Respiratory Samples 124 13.3 Fixation  125 13.3.1 Special Fixatives 126 13.4 Processing of Laboratory Samples 127 13.4.1 Receiving the Sample 127 13.4.2 Glass Slides and Liquid Sample 127 13.5 Processing  128 13.5.1 Processing of Sputum 128 13.5.2 Processing of Fluid: Urine, Body Fluids and Lavage 128 13.5.3 Millipore Filtration 130 13.5.4 Processing of Haemorrhagic Fluid 130 13.5.5 Cell Block 130 13.5.6 Compact Cell Block Technique  131 References 132 14 Routine Staining in Cytology Laboratory  133 14.1 Papanicolaou’s Stain  133 14.1.1 Dyes Used in Papanicolaou’s Staining 133 14.1.2 Principle of Basic Steps 133 14.1.3 Papanicolaou’s Staining Steps  134 14.1.4 Bluing Solution 136 14.2 Precautions to Be Taken in Papanicolaou’s Staining 136 14.3 May Grunwald Giemsa Stain 137 Reference  138 15 Basic Technique of Fine Needle Aspiration Cytology  139 15.1 Introduction 139 15.2 Technique Proper 140 15.3 Fine Needle Aspiration Procedure 141 xvi Contents 15.4 Fine Needle Sampling 142 15.5 FNAC of Deep-Seated Lesions  143 15.5.1 USG-Guided FNAC 144 15.5.2 CT-Guided FNAC 144 15.5.3 Endoscopic Ultrasound-­Guided FNAC (EUS-FNAC)  144 15.5.4 Complications of Guided FNAC  145 15.6 Transrectal FNAC of the Prostate  145 References 146 Part III Advanced Techniques in Histology and Cytology Laboratories 16 Immunocytochemistry in Histology and Cytology 149 16.1 Introduction 149 16.1.1 Basic Principles  149 16.1.2 Basic Immunology 149 16.2 Detection System 151 16.2.1 Peroxidase-Antiperoxidase Method 151 16.2.2 Avidin and Biotin Method  152 16.2.3 Avidin and Biotin Conjugated Procedure 152 16.2.4 Biotin-Streptavidin Method 153 16.2.5 Alkaline Phosphatase-­Antialkaline Phosphatase Method 153 16.2.6 Polymer-Based Labelling Method 153 16.2.7 Catalysed Signal Amplification (Tyramine Signal Amplification)  154 16.3 The Sample of Tissues for Immunocytochemistry  154 16.3.1 Histopathology 154 16.3.2 Cytology 154 16.3.3 Sample Collection  154 16.3.4 Fixation 157 16.3.5 Antigen Retrieval 157 16.3.6 Microwave Retrieval 157 16.3.7 Pressure Cooker Heating  158 16.3.8 Water Bath Heating  158 16.4 Immunocytochemistry Technique 159 16.4.1 Control 159 16.4.2 Steps 159 16.4.3 Chromogen 161 16.5 Troubleshooting in Immunocytochemistry  161 16.6 Applications 162 16.7 Diagnostic Immunocytochemistry 162 16.7.1 Mesothelial Cells Versus Adenocarcinoma  162 16.7.2 Mesothelial Markers 162 16.7.3 Adenocarcinoma Markers in Effusion Fluid  163 16.8 Different Epithelial Markers  163 16.9 Mesenchymal Markers 163 16.10 Neuroendocrine Markers 165 16.11 Lymphoid Markers 165 Contents xvii 16.12 Melanoma Markers  165 16.13 Germ Cell Markers  165 16.14 Site-Specific Antibody in Different Epithelial Malignancies  166 16.15 Immunocytochemistry of Round Cell Tumor  166 16.16 Immunocytochemistry for Therapy and Management 167 16.16.1 Breast Carcinoma 167 16.16.2 Gastrointestinal Stromal Tumor 168 16.16.3 Lung Carcinoma 168 References 169 17 Flow Cytometry: Basic Principles, Procedure and Applications in Pathology 171 17.1 Introduction 171 17.2 Principle of Flow Cytometry 171 17.3 Dye Used 171 17.3.1 Fluorochrome Dyes for FCM 171 17.3.2 Fluorochrome Dye for Nucleic Acid  172 17.4 Samples for Flow Cytometry 173 17.4.1 Cytology Samples 173 17.4.2 Histology Samples 174 17.4.3 Control 174 17.4.4 Sample Processing 174 17.4.5 Flow Cytometric Immunophenotyping (FCI) 175 17.4.6 Data Acquisition 176 17.5 Targets of Application 176 17.6 DNA Content and Ploidy Analysis  177 17.6.1 Clinical Application 178 17.6.2 Diagnosis  178 17.6.3 Prognosis of the Patients 178 17.7 Immunophenotyping  178 17.7.1 Limitations of FCI  180 17.7.2 Flow Cytometry Features of Different Lymphomas  181 17.7.3 Apoptosis 181 17.7.4 Assessment of Sub-G1 Fraction of Apoptotic Cells 181 17.7.5 Apoptosis Detection by Annexin V Assay 182 References 182 18 Digital Image Analysis and Virtual Microscopy in Pathology 185 18.1 Introduction 185 18.2 Basic Principles of Image Analysis 186 18.3 Details of the Image Analysis Steps 186 18.4 Morphologic Features  189 18.5 The Current Problems of Digital Image Analysis 190 18.6 Virtual Slide and Web-Based Teaching 191 18.6.1 Advantage of Virtual Slides 191 xviii Contents 18.6.2 Disadvantages of Virtual Slides 191 References 192 19 Liquid-Based Cytology and Automated Screening Devices in Cytology Sample 193 19.1 Introduction 193 19.1.1 Advantages of LBC Over Conventional Smear 193 19.1.2 Limitations of Liquid-Based Cytology 194 19.2 Sample Processing 194 19.2.1 ThinPrep (Cytic, UK) 194 19.2.2 SurePath Test  195 19.3 Comparison of These Two Techniques  196 19.4 Automated Screening Devices 196 19.4.1 BD FocalPoint GS Imaging System 197 19.4.2 Hologic ThinPrep Imaging System  197 19.4.3 Comparison of Manual and Automated Devices 198 References 198 20 Polymerase Chain Reaction: Principle, Technique and Applications in Pathology 201 20.1 Introduction 201 20.1.1 What Is PCR and How It Works? 201 20.2 Steps of PCR  201 20.2.1 Essential Ingredients of PCR 202 20.3 Procedure Proper 202 20.3.1 Basic Precautions 202 20.3.2 Equipment 203 20.3.3 Thermal Cycling 203 20.3.4 Troubleshooting 204 20.3.5 Enhancing PCR Products Formation 205 20.4 Types of PCR 205 20.5 Applications of PCR  209 References 210 21 Fluorescent In Situ Hybridization Techniques in Pathology: Principle, Technique and Applications 213 21.1 Introduction 213 21.1.1 The Principles of FISH 214 21.2 Steps to Do FISH 214 21.3 Troubleshooting 215 21.3.1 Different Types of FISH 215 21.3.2 CGH Method  218 21.3.3 Array-Based CGH  219 References 220 22 Tissue Microarray in Pathology: Principal, Technique and Applications 221 22.1 Introduction 221 22.2 Tissue Microarray Technique 221 22.3 TMA Construction and Generation of Grid 221 Contents xix 22.4 Designing the Grid 222 22.5 Limitations of TMA 224 22.6 Clinical Applications of TMA 225 References 225 23 Sanger Sequencing and Next-­Generation Gene Sequencing: Basic Principles and Applications in Pathology 227 23.1 Sanger Sequencing 227 23.2 Next-Generation Sequencing 228 23.2.1 Scope of NGS 230 23.2.2 Limitations 230 23.3 Comparison of Sanger Sequencing and NGS  230 References 231 Part IV Microscopy, Quality Control and Laboratory Organization 24 Compound Light Microscope and Other Different Microscopes 235 24.1 Light 235 24.2 Colours 235 24.3 Image Generation and Human Vision  236 24.4 Anatomical Components of a Light Microscope 238 24.5 Optical Components 239 24.5.1 The Major Aberrations of the Lens  240 24.6 How to Take Care and Handle Your Microscope 241 24.7 Other Types of Microscope 242 24.7.1 Dark-Field Microscope 242 24.7.2 Bright-Field Microscope  242 24.7.3 Phase Contrast Microscope  242 References 243 25 Fluorescence and Confocal Microscope: Basic Principles and Applications in Pathology 245 25.1 Transmitted Fluorescent Microscope 245 25.2 Incident Fluorescent Microscope 246 25.3 Confocal Microscopy 247 25.4 Limitations of CFM 249 25.5 Applications of CFM 249 25.6 Two-Photon Microscopy 250 25.7 4Pi Microscopy  250 25.8 Spatially Modulated Illumination Microscopy  251 References 252 26 Electron Microscopy: Principle, Components, Optics and Specimen Processing 253 26.1 Introduction 253 26.1.1 Essential Components of Electron Microscope 254 26.1.2 Microscope Column and Electronic Optics 256 xx Contents 26.2 Specimen and Electron Interaction  256 26.2.1 Electron Interaction in Transmission Electron Microscope 258 26.3 Sample Preparation for TEM 258 26.3.1 Sample Collection  258 26.3.2 Fixation 258 26.3.3 Dehydration  259 26.3.4 Embedding 259 26.4 Sectioning  260 26.4.1 Staining of the Sections  261 26.5 Scanning Electron Microscopy  262 26.5.1 Operational Principle 262 References 262 27 Quality Control and Laboratory Organization 263 27.1 Introduction 263 27.2 Quality Control  265 27.2.1 Pre-analytic Phase  265 27.2.2 Laboratory Processing  266 27.2.3 Tissue and Sample Processing 266 27.2.4 Analytic Phase  266 27.2.5 Post-analytic Phase 267 27.2.6 Gold Standard 267 27.2.7 Record-Keeping 267 27.3 External Quality Assurance 268 27.4 Laboratory Organization  268 27.4.1 Laboratory Construction, Equipments, etc 268 27.4.2 Laboratory Staffs  268 27.4.3 Organization Set-Up and System Protocol 268 References 269 28 Laboratory Safety and Laboratory Waste Disposal  271 28.1 Laboratory Waste Disposal  273 28.2 Disinfectant Used in for the Contaminants 274 References 275 About the Author Pranab Dey is a professor in the Department of Cytology and Gynecologic Pathology at the Postgraduate Institute of Medical Education and Research, Chandigarh. Professor Dey completed his MBBS at R. G. Kar Medical College and Hospital, Calcutta; M.D. (Pathology) at the Postgraduate Institute of Medical Education and Research, Chandigarh; and his FRCPath (Cytopathology) at the Royal College of Pathologists, London. He has con- ducted several research projects and has pioneered work on DNA flow cytom- etry, image morphometry, mono-layered cytology and cytomorphologic findings in various lesions in cytology smears. He has written numerous pub- lications and is a member of various societies. xxi Abbreviations ACEP 3Aminopropyltriethoxysilane APAAP Alkaline phosphatase–antialkaline phosphatase APC Allophycocyanin Ab Antibody AR Antigen retrieval Acgh Array based CGH CEA Carcinoembryonic antigen CEP Chromosome enumeration probe CI Colour index CGH Comparative genomic hybridization CT Computerized tomography CFM Confocal microscopy CLM Conventional light microscopy CP Conventional preparation CYM Cyan, yellow, and magenta CK Cytokeratin DNA Deoxyribonucleic acid DSRT Desmoplastic small round cell tumor ddNTP Dideoxynucleotides phosphates DIA Digital image analysis EM Electron microscope EUS-FNAC Endoscopic ultrasound guided FNAC EA Eosin Azure EMA Epithelial membrane antigen ER Estrogen receptors EDTA Ethylenediaminetetraacetic acid EWS Ewing’s sarcoma FOV Field of view FNAC Fine needle aspiration cytology FNS Fine needle sampling FCI Flow cytometric immunophenotyping FCM Flow cytometry FITC Fluorescein Iso-thiocyanate FRAP Fluorescence recovery after photobleaching FISH Fluorescent in situ hybridization FPGS FocalPoint GS Imaging System FFPE Formalin fixed paraffin embedded section xxiii xxiv Abbreviations GMS Gomori methenamine silver GLCM Gray level co-occurrence of matrix GFP Green fluorescence protein H&E Hematoxylin and Eosin HRP Horseradish peroxidase HIS Hue saturation intensity HCG Human chorionic gonadotropin ICC Immunocytochemistry IHC Immunohistochemistry Ppi Inorganic pyrophosphate LIS Laboratory information service LBC Liquid based cytology LSI Locus-specific identifier probe MRI Magnetic resonance image MGG May Grunwald Giemsa MRD Minimal residual disease nM Nano micrometer NB Neuroblastoma NGS Next generation sequencing NHL Non-Hodgkin lymphoma OCT Optimum cutting temperature OG Orange G PAP Papanicolaou PerCP Peridinin Chlorophyll PAS Periodic Acid Schiff PNET Peripheral neuroectodermal tumor PTAH Phosphotungstic acid haematoxylin PMT Photomultiplier tube PE Phycoerythrin PLAP Placental alkaline phosphatase PCR Polymerase chain reaction PR Progesterone receptors PSA Prostate specific antigen QA Quality assurance QC Quality control QI Quality improvement RGB Red green blue RCF Relative centrifugal force RMS Rhabdomyosarcoma RNA Ribonucleic acid SEM Scanning electron microscope SSCP Single strand conformation polymorphism ssDNA Single stranded DNA SOP Standard operating protocol TIP ThinPrep image processor TTF-1 Thyroid transcription factor-1 TMA Tissue microarray TEM Transmission electron microscope Abbreviations xxv TSA Tyramine signal amplification USG Ultrasonography VS Virtual slides WT Wilms’ tumor WT 1 Wilms’ tumor gene 1 ZN Ziehl Neelsen  Part I Basic Laboratory Techniques in Histopathology Laboratory  Fixation of Histology Samples: Principles, Methods and Types 1 of Fixatives 1.1 Introduction 1. Prevention of autolysis of the cells or tissue 2. Prevention of decomposition of the tissue by Fixation is the first step of any histological and bacteria cytological laboratory technique. It is the process 3. Maintaining the volume and shape of the cell by which the cells in the tissue are fixed in a chem- as far as possible ical and physical state, and all the biochemical and 4. Consistently high-quality staining particularly proteolytic activities within the cells are prevented routine stain such as haematoxylin and eosin so that the cells or tissues can resist any morpho- stain and Papanicolaou’s stain logical change or distortion or decomposition after 5. Rapid action subsequent treatment with various reagents. The 6. Cheap fixation helps to maintain the tissue nearest to its 7. Non-toxic original state in the living system. Large number of fixatives are available in the market. Each fixative has its own advantages and 1.2 Aims of Fixation disadvantages. In fact it is difficult to find a uni- versally accepted ideal fixatives. The basic aims of fixation are the following: To preserve the tissue nearest to its living state 1.4 Tissue Changes in Fixation To prevent any change in shape and size of the tissue at the time of processing The following changes may occur in tissue due to To prevent any autolysis fixation (Box 1.1): To make the tissue firm to hard To prevent any bacterial growth in the tissue 1. Volume changes: Fixatives may change the To make it possible to have clear stain volume of the cells. Some fixatives such as To have better optical quality of the cells osmium tetroxide cause cell swelling. The exact mechanism of the change in volume is not properly understood. However the vol- 1.3 Ideal Fixative ume change may be due to (a) altered mem- brane permeability, (b) inhibition of the An ideal fixative should have the following quali- enzymes responsible for respiration and (c) ties : change of transport Na+ ions. Formaldehyde © Springer Nature Singapore Pte Ltd. 2018 3 P. Dey, Basic and Advanced Laboratory Techniques in Histopathology and Cytology, https://doi.org/10.1007/978-981-10-8252-8_1 4 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives 2. Hardening of tissue: The fixation changes the Box 1.1 Change in Tissue After Fixation consistency of the tissue, and some amount of Volume changes hardening occurs due to fixation. –– Shrinkage of the volume by formalin 3. Interference of staining: Fixation may cause (33%). hindrance of staining of enzymes. Hardening of tissue Formaldehyde inactivates 80% of ribonucle- –– Mild degree hardening may occur. ase enzyme. It has been noted that Interference of staining osmium tetroxide inhibits haematoxylin and –– Inhibits routine stain: Osmium eosin staining. tetroxide inhibits haematoxylin and 4. Changes of optical density by fixation: The eosin staining. fixation may cause the change of optical den- Changes of optical density by fixation sity of the nuclei, and the nuclei may look like –– Nuclei may look like hyperchromatic. condensed and hyperchromatic. may cause shrinkage of the volume by 33%. 1.5 Types of Fixation In an ­experiment Bahr et al. noted that the shrinkage of tissue is inversely proportional The fixative can be classified on the basis of the to the formaldehyde concentration. following criteria (Table 1.1): Similarly glutaraldehyde also causes signif- icant tissue shrinkage. However when glu- A. Nature of fixation taraldehyde and osmium tetroxide are used B. Chemical properties as fixations in epoxy resin then 70% C. Component present increased of cell size is noted. D. Action on tissue protein Table 1.1 Types of fixation and classification of fixatives Types of fixative Classification A. Nature of fixation   Immersion fixation   Coating fixation   Vapour fixation   Perfusion fixation   Freeze-drying   Microwave fixation B. Chemical properties   Aldehyde: formaldehyde, glutaraldehyde   Oxidising agent: osmium tetroxide   Protein denaturing agent: ethyl alcohol, methyl alcohol   Cross-linking agents: carbodiimide   Miscellaneous: picric acid C. Component present 1. Simple(only one chemical present)   Formaldehyde   Ethyl alcohol   Glutaraldehyde   Picric acid   Osmium tetroxide 2. Compound (more than one chemical present)   Bouin’s fluid   Carnoy’s solution D. Action on protein 1. Coagulative: ethyl alcohol, picric acid 2. Noncoagulative: formaldehyde, osmium tetroxide, glutaraldehyde 1.5 Types of Fixation 5 1.5.1 Description of Nature Steps: of Fixation At first the thin cut tissue section is rapidly fro- 1. Immersion fixation: This is the commonest zen at −160 °C by immersing it into liquid cool- way of fixation in the laboratories. In this ant. This is known as “quenching”. The technique the whole specimen is immersed in commonly used fluids in the quenching bath are the liquid fixative such as tissue samples are liquid nitrogen, propane and isopentane. immersed in 10% neutral buffered formalin or Alternatively the tissue section can be frozen by cytology smear in 95% ethyl alcohol. keeping it in close contact with chilled metal. 2. Coating fixation: This is commonly used in In the next step, the ice within the tissue is the cytology samples. The spray fixative is removed by placing the tissue in the vacuum used for easy transportation of the slide. The chamber in higher temperature (−30 to main advantages of spray fixatives are: −50 °C). The water of the solid tissue is (a) Fixation of the cells removed by sublimation. The water vapour is (b) To impart a protective covering over the absorbed by a suitable drying agent. smear In the final step, the tissue is gradually warmed (c) No need to carry liquid fixative in bottle to 4 °C and is finally impregnated with the or jar embedding medium. The spraying over the smear should be smooth Freeze-drying technique is useful mainly to and steady, and the optimum distance of study the soluble material and very small 10–12 inches should be maintained between the molecules. nozzle of the spray and the smear. The spray fixa- tive usually consists of alcohol and wax. Advantages: Therefore, this wax should be removed before the staining procedure. Excellent for enzyme study No change of proteins 3. Vapour fixation: In this type of fixation, the No shrinkage of tissue vapour of chemical is used to fix either a Preservation of glycogen smear or tissue section. The commonly used chemicals for vapour fixation are formalde- 6. Microwave fixation (Box 1.2) hyde, osmium tetroxide, glutaraldehyde and ethyl alcohol. The vapour converts the soluble Basic principle: Microwave is a type of elec- material to insoluble material, and these mate- tromagnetic wave with frequencies between rials are retained when the smear comes in 300 MHz and 300 GHz, and wavelength varies contact with liquid solution. from centimetre to nanometre. Scientific and 4. Perfusion fixation: This is mainly used in medical microwave ovens operate with a fre- research purpose. In this technique the fixative quency of 2.45 GHz and 0.915 GHz, respectively. solution is infused in the arterial system of the The electromagnetic field is created by the micro- animal, and the whole animal is fixed. The wave, and the dipolar molecules such as water organ such as the brain or spinal cord can also rapidly oscillate in this electromagnetic field. be fixed by perfusion fixation. This rapid kinetic motion of these molecules gen- 5. Freeze-drying: In this technique the tissue is erates uniform heat. The generated heat acceler- cut into thin sections and then rapidly frozen ates the fixation and also other steps of tissue into a very low temperature. Subsequently the processing. The most important characteristic of ice within the tissue is removed with the help microwave heat generation is homogeneous of vacuum chamber in higher temperature increase of temperature within the tissue, and (−30 °C). every part of the tissue is heated. 6 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives Preservation of the tissue antigen and good for Box 1.2 Microwave Fixation of Tissues immunohistochemistry. What it is: Electromagnetic wave with fre- It facilitates the staining reaction without any quencies between 300 MHz and 300 GHz. bad effect. Mechanism: Microwave creates electro- magnetic field, and the dipolar mole- Disadvantages: cules rapidly oscillate generating heat by kinetic motion. The tissue immersed in formalin during microwave fixation may generate large Advantages: amount of toxic gas. Therefore overhead hood is required for the removal of this toxic sub- Uniform heat production stance from the microwave. No volume change of tissue Chances of heat injury Good for electron microscopy after osmium tetroxide fixation Applications: Facilitates fixation and other laboratory steps In routine surgical pathology laboratory Preservation of the tissue antigen Electron microscopy after osmium tetroxide fixation Disadvantages: Urgent processing of biopsy (e.g. kidney biopsy) Tissue in the formalin for microwave fixation may produce toxic gas and 1.6 Essential Precautions overhead hood is required. for Fixation in General Heat injury may occur from microwave. Certain essential precautions are necessary for Applications: proper fixation: In routine surgical pathology laboratory The tissue should be free from excessive blood Electron microscopy before putting it into fixative. Urgent processing of special biopsies Tissue should be thinly cut in 3–5 mm thickness. The amount of fixative fluid should be 20 Factors controlling the temperature rise: The times more than the volume of the tissue. rise of temperature in the microwave heated The tissue with fixative should be in a tightly media depends mainly on: screw-capped bottle. The dielectric property of the media Thermal properties of the material 1.7 Mechanism of Fixation Radiation level Orientation and shape of the object The wet fixatives usually work as: Advantages: The main advantages of micro- Dehydration and coagulation of protein: wave fixation are: Methanol and ethanol are commonly used coagulative fixatives. These two alcohols Rapid processing. remove water from the tissue and causing No change in volume of tissue. destabilization of the hydrogen bonds and 1.7 Mechanism of Fixation 7 thereby disruption of the tertiary structure of methylene bridge. This preliminary reaction of protein. However, the secondary structure of hydroxymethyl side chain is the primary reac- the protein is maintained. Ethanol is relatively tion, and the subsequent intermolecular and intra- stronger dehydrating agent than methanol. molecular cross-linking of the molecules occurs The ethanol and methanol start work from as a slow-growing process. This ultimately pro- 60–80% concentration, respectively. The duces an insoluble product. The formalin can be dehydrating fixative has two disadvantages: removed from tissue by prolonged washing. –– Shrinkage of the cells However, once methylene bridge is formed in the –– Removal of the soluble substances from the tissue, the reaction is stable, and it is difficult to tissue remove formalin from the tissue. Formaldehyde Cross-linking fixatives: also reacts with the nucleic acid by reacting with Formaldehyde: Formaldehyde in aqueous the amino group of nucleotides. solution combines with water to form methy- Glutaraldehyde: It has two aldehyde groups lene hydrate, a methylene glycol: that are separated by three methylene bridges (Fig. 1.2). The aldehyde group of glutaraldehyde CH 2 O + H 2 O = OHCH 2 OH reacts with amino group of the protein predomi- nantly lysine. When one aldehyde group reacts In a long-standing position, this methylene with the amino group, the other free aldehyde glycol may further react with water molecules group may help to cross-link. Glutaraldehyde and form a polymer known as polyoxymethylene rapidly and irreversibly cross-links the protein. glycol. This again depolymerized in methylene The penetration of glutaraldehyde is slower than glycol in a neutral buffer system. Formaldehyde formaldehyde. reacts with various side chain of the protein and Osmium tetroxide: Osmium tetroxide (OsO4) is forms hydroxymethyl side group (Fig. 1.1). mainly used as a fixative in electron microscopy. It These compounds are highly reactive and is used alone or as a combination with other agent. ­subsequently cross-linking occurs by forming a O H H H O C C C C C H H OH H H H H H H2O O Glutaraldehyde C C H H OH O H H H O Formaldehyde Methylene glycol R1-NH2 + C C C C C + R2-NH2 H H H H H Amino Amino H group group R–H+ C O R – CH2OH Bound hydroxymethy group H H H H H H R – CH2OH + R – H = R – CH2 – R + H2O R1 N C C C C C N R2 Metyhylene bridge H H H Fig. 1.1 Schematic diagram showing the mechanism of Cross liking with formalin fixation. Formaldehyde reacts with the side amino group chain of the protein and forms hydroxymethyl side group. Later on these highly reactive substances form cross-­ Fig. 1.2 Schematic diagram showing the mechanism of linking and methylene bridges are formed. This is a stable glutaraldehyde fixation. The aldehyde group of glutaral- reaction, and simple washing cannot remove formalin in dehyde reacts with amino group of the protein. Rapid and this stage irreversibly cross-linking of the protein takes place 8 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives Fig. 1.3 Schematic O O diagram showing the mechanism of osmium Os tetroxide fixation. O O Osmium tetroxide reacts with two unsaturated Osmium tetroxide carbon atom of the lipids and cross-links with them O O O HC = HC O + Os Os HC HC O O O O Hexavalent osmium O HC O + H2O = HCOH + OsO3 Os HC O HCOH O Osmium trioxide diol Osmium tetroxide O O HC + + CH HC O O CH Os = Os HC CH HC O O CH Unsaturated O O Unsaturated Cross linking carbon atom of carbon atom of lipid lipid The compound causes oxidation of unsaturated bonds in the biological tissue particularly lipid. It converts the unsaturated fatty acid into a stable product known as glycol osmate. The tetravalent Os becomes hexavalent in this reaction. Osmic acid monoester formed in this reaction is easily hydro- lysed to a diol and osmic acid (Fig. 1.3). Osmium tetroxide may react with two unsaturated carbon atom of the lipids and may cross-link (Fig. 1.3). Figure 1.4 demonstrates the various mecha- nisms of fixative. 1.8 Factors Affecting Fixation The following factors may affect the fixation (Box 1.3): 1. Hydrogen ion concentration (pH): Most of the fixatives work better in neutral pH. In fact, good fixation occurs when pH remains 6–8, Fig. 1.4 Schematic diagram showing the mechanism of dif- and no morphological distortion is seen in that ferent fixatives. Alcohol works by removing water molecules range of pH. There may be changes in the from the tissue and coagulating the protein. Formaldehyde ultrastructure when the pH is too low or too forms hydroxymethyl side group and cross-links with protein. Osmium tetroxide forms glycol osmate with lipid molecules high. In very low pH, the NH2 group of amino 18 Factors Affecting Fixation 9 Box 1.3 Factors Affecting Fixation pH of the fixative –– Neutral pH is preferable. –– pH 6–8 is the best range. –– High acidity or alkalinity interferes fixation. Temperature –– Room temperature suitable for routine work. –– High temperature facilitates fixation. –– Low temperature (0–4 °C) suitable for enzyme histochemistry. Duration of fixation –– Depth of penetration of fixative is directly proportional to the square root of time of fixation. –– Formalin fixes 1 mm/h. –– Small tissue: 6 h in formalin is optimum. –– Large tissue: 24 h is the optimum time, –– Prolonged fixation in aldehyde: inhibition of enzymatic activity, Osmolarity of the fixative solution –– Hypertonic: cell shrinkage –– Hypotonic: cell swelling –– Best: mild hypertonic (400–450 mOsm) Concentration –– Mild lower concentration with neutral pH is preferable. –– Very low concentration prolongs the time of fixation. –– Higher concentration causes rapid fixation with undesirable effect. Agitation –– Agitation increases rate of penetration. –– Rapid agitation: damages delicate tissue. –– Slow gentle agitation preferable. acid is converted to NH3, and the reaction enzymes are better preserved in lower tem- between aldehyde groups of the fixative is perature, and for enzyme histochemistry reduced. Usually buffer solution is added to 0–4 °C is suitable. maintain pH of the fixative. The commonly 3. Duration of fixation: The depth of penetration used buffers in the fixatives are phosphate, of fixative is directly proportional to the bicarbonate, Tris and acetate. The buffers square root of time of fixation. The diffusibil- should be chosen in such a way that they ity of different fixatives may also vary: should not react with the fixative. 2. Temperature: Room temperature is alright for D=k T tissue fixation, and there is no difference of cell morphology from 0 to 45 °C. However, D = depth of penetration the fixation time may be reduced in higher T = Time duration temperature (60–65 °C). At higher tempera- k = Coefficient of diffusion of the fixative ture the vibration and movement of the mole- cules are increased. This increases the rate of The penetration rate of formalin solution is penetration of the fixative within the tissue 1 mm/h. The presence of blood may hamper the and accelerates the process of fixation. In penetration of the fixative. Therefore it is prefer- case of very high temperature, the antigen able to wash the tissue specimen thoroughly within the tissue may be destroyed. The before putting it in fixative. The tissue should be 10 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives sectioned as 3–5 mm. Overall formalin fixes tis- Volume of formalin: For proper fixation the sue within 24 h. Prolonged fixation may cause specimen should be sliced in 5 mm apart, and the loss of lipid and protein and significant reduction amount of formalin should be 20 times the vol- of the enzymatic activity of the cell. This may ume of tissue. The specimen should be com- also cause hardening of tissue. pletely immersed in formalin and should not be in direct contact with the container. There should 4. Osmolarity of the fixative solution: Osmolality be a formalin soaked clothes in between the con- of the fixative has considerable effect on fixa- tainer and the tissue. tion. Hypertonic fixative solution causes Removal of formalin from the tissue: As the cross- shrinkage of the cell, whereas hypotonic fixa- linking of the amino acids and proteins is a slow pro- tive solution induces swelling of the cells. cess, so if the tissue is washed for 24 h in water, then Electrolytes (0.9% NaCl) or sucrose may be 50% of formalin from the tissue is removed. added in the fixative to maintain the osmolar- Precaution: Formaldehyde is irritant to the ity of the fixative. Mildly hypertonic fixative eye and skin and toxic for inhalation. It is a carci- (400–450 mOsm) is preferable for routine use nogenic element. in laboratory. 5. Concentration: Very low concentration of fixa- Advantages: tive prolongs the time of fixation, and higher concentration causes rapid fixation. However, The penetration rate of formalin is high. higher concentration of fixative may cause tissue Cell morphology well preserved in formalin. hardening, tissue shrinkage and artefactual Cheap. changes. Mildly lower concentration of fixative Stable. with neutral pH is needed for proper fixation. Easy to make the solution. Optimal concentration of formaldehyde is 10%. Formalin is effective fixation for routine labo- 6. Agitation: Agitation increases the rate of pen- ratory staining of the tissue. etration and therefore rapidity of fixation. Optimum agitation is needed as slow agitation Disadvantages; may have no effect of fixation, whereas rapid agitation may have detrimental effect on deli- 1. Slow fixation. cate tissue. 2. Formalin reaction with the tissue is reversible, and it can be removed by washing. 3. Formalin fails to preserve acid 1.9  ommonly Used Fixatives C mucopolysaccharides. in the Laboratory 4. Highly vascular tissue may have dark-brown granules (artefact) 1.9.1 Formaldehyde 5. Exposure to the skin may cause dermatitis. 6. Chronic inhalation may cause bronchitis. Pure formaldehyde vapour dissolved in the water is available as formaldehyde in 37–40% concentration. This is also known as formalin 1.9.2 Preparation of Different and is considered as 100% formaldehyde. In Formalin Solution laboratory 10% of this formalin is used to make neutral buffered formalin for routine labora- A. 10% neutral buffered formalin: tory fixative (Box 1.4). Formaldehyde, 40%: 100.0 ml Rate of penetration: Formalin penetrates Distilled water: 900.0 ml approximately 1 mm/h and usually 24 h is needed Sodium dihydrogen phosphate: 4.0 g for fixation of a 1 cm3 tissue. Disodium hydrogen phosphate: 6.5 g 1.9 Commonly Used Fixatives in the Laboratory 11 Box 1.4 Formaldehyde Fixative Commercially available as 40% concentration (considered as 100% formalin) Ten percent of this formalin is used to make neutral buffered formalin. Mechanism: It reacts with various side chain of the protein and forms hydroxymethyl side group that subsequently cross-link to form a methylene bridge. Subsequent intermolecu- lar and intramolecular cross-linking of the molecules occurs and ultimately produces an insoluble product. Rate of penetration: Formalin penetrates approximately 1 mm/h. Volume of formalin: The amount of formalin should be 20 times the volume of tissue. Advantages: High penetration rate Well-preserved cell morphology Cheap Stable Disadvantages: 1. Slow fixation. 2. Formalin reaction is reversible, and it can be removed by washing. 3. Fails to preserve acid mucopolysaccharides. 4. Not good for staining of fat and enzymes. 5. Highly vascular tissue may have dark-­brown granules. 6. Exposure to the skin may cause dermatitis. 7. Chronic inhalation may cause bronchitis. with lipid or carbohydrate, and therefore it should B. Preparation of 10% formal saline: be used in combination with the other fixative. Formaldehyde, 40%: 100.0 ml Sodium chloride: 9 g Advantages: Distilled water: 900.0 ml C. Formal ethanol fixative: 1. Better fixation of ultrastructure. Ninety-five percent ethyl alcohol: 20 ml 2. Less cell shrinkage. Formaldehyde, 40%: 10 ml 3. Preservation of protein is better. 4. Good cross-linking with collagen. 5. Less irritating. 1.9.3 Glutaraldehyde Disadvantages; Glutaraldehyde is used as a fixative for electron microscopy because it fixes and preserves the 1. Poor penetration and tissue should be less ultrastructure. The fixation occurs due to the than 0.5 mm thick. extensive cross-linking of the proteins. The pen- 2. Less stable compound. etration power of glutaraldehyde is poor and 3. No lipid fixation. therefore only a small piece of tissue should be 4. Glutaraldehyde polymerizes above pH 7.5. used for fixation. Glutaraldehyde does not react 5. Costly. 12 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives For the purpose of electron microscopy: 1.9.5 Methyl and Ethyl Alcohol Glutaraldehyde is used 2.5% glutaraldehyde in 100 mM phosphate buffer at pH 7.0. Methyl alcohol (methanol) and ethyl alcohol (etha- Glutaraldehyde comes commercially as 25% nol) are used as dehydrating agent, and these two or 50% solutions in 10 ml. alcohols are used mainly as fixatives of cytology smears. The tissue or smear containing water should not be put directly in the higher concentra- 1.9.4 Osmium Tetroxide tion of alcohol as it may cause distortion of the cells due to rapid rush of fluid from the cell. Osmium tetroxide is used for fixation in electron Therefore graded alcohol should be used for microscopy. It reacts with unsaturated bonds in the dehydration. lipid molecules and fixes them. The penetration of Laboratory use: 95% ethyl alcohol for the osmium tetroxide in the tissue is poor, and if it fixation is used alone, then a good amount of protein and carbohydrate may be lost during fixation. Reagent preparation: Advantages: Absolute alcohol: 950 ml Water: 50 ml 1. This is a very good fixative for lipid. Time of fixation: 15–30 min 2. It preserves cytoplasmic organelles such as Golgi bodies and mitochondria, 3. Does not make the tissue hard, 1.9.6 Acetone Disadvantages: It is mainly used for enzyme study and immuno- cytochemistry. It is a poor fixative for morpho- 1. It does not fix the proteins and carbohydrates logical preparation as it causes significant cell and therefore it should be used in combination shrinkage. Acetone works by dehydration of with other fixative. cells. Cold acetone is used at 4 °C for fixation. 2. Osmium tetroxide may react with ribose group and may cause clumping of DNA. This can be prevented by pretreatment with potas- 1.9.7 Bouin’s Fixative sium permanganate or post fixation with ura- nyl acetate. Bouin’s solution contains picric acid. This is an 3. Poor penetration in the tissue. excellent fixative for glycogen. It reacts with pro- 4. Tissue swelling may occur. tein and forms protein picrate. The tissue pene- 5. Toxic and volatizes at room temperature pro- tration rate of picric acid is high, and it fixes ducing harmful vapour. This vapour is toxic to small tissue biopsy within 3–4 h. Bouin’s fixative the eye and respiratory tract. is not suitable for DNA quantitative study as it 6. Expensive. damages the cell membrane and causes hydroly- sis of nuclei acid. Laboratory use: It is commercially available in sealed vial 0.1–1 g. Aqueous solution of 4% Advantages: OsO4 is made. This should be stored in clean glass vial away from sunlight. In laboratory 1. It is a good fixative for connective tissue and 2–4% OsO4 in buffer solution of pH 7.2 is glycogen. used. 2. Rapid penetration rate. 1.10 Mercury Salt-Containing Fixatives 13 Disadvantages: Preparation Solution A 1. It produces yellow stain to the tissue. Distilled water: 250 Removal of yellow colour: Potassium dichromate: 6.3 g Mercuric chloride: 12.5 g 1. The tissue should be washed thoroughly in Sodium sulphate: 2.5 g 70% ethanol. Solution B 2. This yellow colour can be removed by dipping Thirty-seven percent formaldehyde solution the tissue in lithium carbonate in 70% Just before use mix 95 ml of Solution A with 5 ml alcohol. of Solution B. Bouin’s solution preparation: 1.10.3 B5 Fixatives Picric acid solution (1% in distilled water): 15 ml Formaldehyde stock solution (40%): 5 ml Stock A solution Glacial acetic acid: 1 ml Mercuric chloride: 12 g Sodium acetate: 2.5 g Distilled water: 200 ml 1.10 Mercury Salt-Containing Stock B solution Fixatives Thirty-seven percent formaldehyde solution Before use mix 20 ml stock solution A with 2 ml Among the heavy metals, mercury is commonly stock B. used as fixative. This is a rapidly acting fixative. Mercury is a poisonous substance and should be Table 1.2 highlights the comparison of differ- used carefully. Mercury containing fixatives may ent fixatives. corrode the metal so the fixative should be kept in glass container. 1.10.4 Fixatives of Choice 1.10.1 Zenker’s Fluid The choice of fixatives is very important for specific purposes. In case of routine histopa- It is a good fixative for nuclear chromatin and thology tissue, the best fixative is 10% neutral collagen. buffered formalin. Similarly for electron Preparation microscopy and immunocytochemistry, the Mercuric chloride: 50 g fixative of choice is glutaraldehyde solution Glacial acetic acid: 50 g and 10% neutral buffered formalin, respec- Potassium dichromate: 25 g tively. Table 1.3 highlights the fixative of Distilled water: 950 ml choice for different technique. The different types of fixative are used accord- ing to the tissue and have been highlighted in 1.10.2 Helly’s Fluid Table 1.4. Fixative of choice may be different according This is a good cytoplasmic fixative. It takes to chemical compounds to demonstrate nearly about 12 h for 3 mm tissue to fix. (Table 1.5). 14 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives Table 1.2 Comparison of different fixatives Fixative Ingredients Advantages Disadvantages Applications Buffered – Formaldehyde – High penetration – Slow fixation – Effective for formalin (10%) – Water rate – Fails to preserve acid routine – Sodium – Cell morphology mucopolysaccharides laboratory dihydrogen well preserved – Dark-brown granules staining phosphate – Cheap in vascular tissue – Disodium – Stable hydrogen phosphate Glutaraldehyde – Glutaraldehyde – Better fixation of – Poor penetration in – Best for – Phosphate buffer ultrastructure tissue electron – Less cell – Less stable microscopy shrinkage – No lipid fixation – Protein – Costly preservation better – Less irritating Osmium −2–4% Osmium – Good fixative for – Does not fix the – Good for tetroxide tetroxide in buffer lipid proteins and electron solution – Good for Golgi carbohydrates microscopy bodies and – Cause clumping of mitochondria DNA – Toxic and volatizes at room temperature – Costly Ethyl alcohol – Absolute alcohol – Fast penetration – Inflammable – Good for – Water – Needs licence cytology smear Bouin’s fixative – Picric acid – Rapid – Produces yellow stain – Good fixative – Formaldehyde penetration rate to the tissue for connective – Glacial acetic acid – Very good for tissue and trichrome stain glycogen Zenker’s fluid – Mercuric chloride – Rapidly acting – Pigments of – Organ with – Glacial acetic acid – Even penetration dichromate and very high – Potassium mercury vascularity such dichromate – Mercury is poisonous as the spleen – Distilled water – RBC is poorly preserved Table 1.3 Choice of fixative based on technique Table 1.4 Fixative of choice according to tissue Technique Fixative of choice Tissue Fixative Time Routine histopathology 10% neutral buffered Day-to-day sample 10% buffered Small tissue: formalin (routine) formalin 6h Large tissue: Electron microscopy Glutaraldehyde solution or 12–24 h osmium tetroxide Lymph node B5 solution 18 h Immunohistochemistry 10% neutral buffered formalin, alcoholic formalin Gastrointestinal tract 10% buffered 6h formalin Immunofluorescence Unfixed cryostat Testis 10% buffered 6h Enzyme histochemistry Fresh frozen section formalin Or Bouin’s fluid Bone marrow Bouin’s fluid 3h Spleen Zenker’s fluid 6h Eye 10% buffered 48 h formalin 1.11 Fixation Artefact 15 Table 1.5 Fixative of choice for different substances Target substance Fixative of choice Box 1.6 Formalin Pigment Protein 10% buffered formalin Colour: Brownish black. Lipid Frozen section or osmium Nature: Granular birefringent refractile. tetroxide Position: Extracellular. Glycogen Alcohol-based fixative Mechanism of formation: Formic acid Mucopolysaccharide Frozen section reacts with haemoglobin derivatives of Enzyme Frozen section the blood and produces acid formalde- DNA and RNA Alcohol-based fixative Iron Alcohol-based fixative hyde haematein. How to avoid: Use buffered formalin. How to remove: Treat with 1.8% picric 1.11 Fixation Artefact acid in absolute ethyl alcohol for 15 min. The following fixation artefact may be encoun- tered in routine laboratory fixation (Box 1.5): 2. Subsequently the section is immersed in 1.8% A. Formalin Pigment picric acid in absolute ethyl alcohol for Unbuffered formalin is kept for long time and 15 min. is converted into formic acid that reacts with hae- 3. It is then washed thoroughly. moglobin derivatives of the blood and produces 4. Section is re-stained. acid formaldehyde haematein which is an insolu- ble brownish-black granular retractile birefrin- B. Mercury Pigments: When tissue is fixed by gent pigment (Fig. 1.5) (Box 1.6). mercuric chloride, it produces a dark-brown irregular deposit. Removing the pigment: Steps: Location: Throughout the tissue Removal: Application of iodine converts it 1. The section is immersed in xylene followed into mercuric iodide which is removed by sodium by alcohol to bring in water. thiosulphate. C. Fuzzy Staining Box 1.5 Fixation Artefact Appearance: Here the nuclear and cytoplas- Formalin pigment: Insoluble brownish-­ mic details are obscured and the section looks black granular retractile birefringent fuzzy or hazy. pigment due to reaction of formalin with Cause: Improper fixation either due to insuf- haemoglobin derivatives. ficient fixative or too little time in fixative. Mercury pigments: Dark-brown irregu- lar deposit. D. Prolonged fixation: Prolonged fixation cause Fuzzy staining: Due to improper shrinkage of the tissue followed by separa- fixation. tion. The tissue may show holes or empty Prolonged fixation: Shrinkage of the tis- spaces within it (Fig. 1.6). sue causes tissue separation and empty E. Dichromate deposit: If the tissue is not prop- spaces. erly washed after dichromate fixation, then Dichromate deposit: This deposit may the chromium salt may form. This chrome occur after dichromate fixation if the tis- salt reacts with alcohol, and insoluble yellow-­ sue is not washed properly. brown precipitate may appear. 16 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives Removal: By 1% hydrochloric acid in 70% Troubleshooting in fixation is highlighted in alcohol for 30 min Table 1.6. Fig. 1.5 Microphotograph shows brownish-black granular formalin pigment. This is refractile birefringent pigment (haematoxylin and eosin stain ×400) Fig. 1.6 Tissue separation due to prolonged fixation. The tissue shows holes and empty spaces within it (haematoxylin and eosin stain ×200) References 17 Table 1.6 Troubleshooting in fixation Problems Cause Remedies Nuclear margin is indistinct and Incomplete fixation – Check the concentration of nuclei are fuzzy with bubbling formalin – Keep more time in formalin for fixation – Cut thin section for fixation – Do not put too many cassettes together Tissue shrinkage with large – Poor fixation – Proper fixation time artefactual spaces – Prolonged fixation – Check fixative concentration – Immediately fix the tissue after biopsy Insoluble brownish-black granular Formalin pigment due to acid Use buffered formalin pigment formaldehyde haematein formation by reaction with blood Intraepithelial cleft formation Formalin may evaporate, and Keep formalin in closely capped calcium carbonate is precipitated bottle 3. Jonsson N, Lagerst TS. The effect of formalde- References hyde containing fixatives on ribonuclease activity. Histoehemie. 1959;1:251–6. 1. Hopwood D. Fixatives and fixation: a review. 4. Kellenberger E, Johansen R, Maeder M, Bohrmann Histochem J. 1969;1(4):323–60. B, Stauffer E, Villiger W. Artefacts and morpho- 2. Bahr GF, Bloom G, Friberg U. Volume changes of tis- logical changes during chemical fixation. J Microsc. sues in physiological fluids during fixation in osmium 1992;168(Pt 2):181–201. tetroxide or formaldehyde and during subsequent treatment. Expl Cell Res. 1957;12:342–55.  Processing of Tissue in Histopathology Laboratory 2 The next step after fixation is the processing of tissue. This is also a very important step because poor processing of tissue may significantly affect the section cutting and staining. Aims of tissue processing: The basic aim of tissue processing is to provide sufficient rigidity to the tissue so that it can be cut into thin section for microscopic examination. Principle of processing: In tissue processing the water within the tissue is removed, and another medium (usually paraffin wax) is impreg- nated in the tissue that provides the adequate sup- port to the tissue. Therefore the essential steps in tissue processing (Fig. 2.1): Fig. 2.1 Schematic diagram showing overview of pro- cessing. The basic three steps are dehydrating, clearing, 1. Dehydration: In this step water is removed and embedding. Removal of water from the tissue is known as dehydration. This is followed by clearing of the from the tissue. Water is immiscible with wax, dehydrating agents by the process clearing. In the final and therefore to infiltrate the tissue with wax, stage tissue is impregnated with an embedding medium it is necessary to remove water. 2. Clearing: This is needed to clear the dehydrat- ing agent and to facilitate the transition of 2.1  actors that Influence Tissue F dehydration and impregnation stage. The Processing clearing substance is usually miscible to both dehydrating agent and impregnating medium. The following factors influence the tissue pro- 3. Infiltration and impregnation: In this stage, cessing (Box 2.1): the tissue is infiltrated with a supporting medium which is suitable to provide adequate 1. Size of the tissue sample: The optimum size of rigidity of the tissue to make thin section. the tissue is very important for effective © Springer Nature Singapore Pte Ltd. 2018 19 P. Dey, Basic and Advanced Laboratory Techniques in Histopathology and Cytology, https://doi.org/10.1007/978-981-10-8252-8_2 20 2 Processing of Tissue in Histopathology Laboratory 5. Vacuum: Application of negative pressure Box 2.1 Influencing Factors of Tissue facilitates tissue processing. Vacuum helps Processing to remove the entrapped air from the tissue Size of the tissue: and thereby enhances the penetration of fluid –– The smaller the size, the better the within the tissue. Negative pressure also processing. increases the volatility of the clearing agent Agitation: and therefore helps to remove the fluid from –– Agitation facilitates the contact of the tissue. tissue with fresh solution. Heat: –– Increases the better penetration of 2.2 Dehydration fluid. Viscosity: Every tissue contains some amount of free or –– The higher the viscosity of the unbound water molecule. As the commonly used medium, lower the penetration. supporting medium (paraffin) is not miscible Negative pressure: with water, so it is necessary to remove the free –– Negative pressure removes trapped water molecule from the tissue for the successful air in the tissue. impregnation of the supporting medium (Box –– Removal of clearing agent by 2.2). The sharp difference of the concentration increasing volatility. gradient between the tissues and the dehydrating fluid may cause sudden rush of fluid, and this may damage the delicate tissue. Therefore the ­ rocessing. The smaller the size of the tissue, p dehydration should be done gradually from low better is the infiltration of the embedding medium. Optimum thickness of the tissue should be kept as 3–4 mm only. Box 2.2 Dehydration 2. Agitation: The tissue gets better contact with Removes free or unbound water mole- the surrounding medium if it is completely cule of the tissue as the supporting immersed and gently agitated. The agitation medium (paraffin) is not miscible with causes continuous removal of the fluid from water. the surface by fresh medium. This has better Sharp difference of concentration gradi- effect of action of fluid on the tissue. Most of ent of the dehydrating fluid may damage the commercial tissue processors have the the delicate tissue. facility of agitation. It is important to note that Gradual dehydration is necessary. too rapid agitation may damage the soft and Too much time in the dehydrating fluid: delicate tissue. the tissue becomes hard and brittle. 3. Heat: Heat increases the rate of penetration of the Routine laboratory: 70, 90 and 100% fluid within the tissue, whereas low temperature alcohol for 2 h each. impedes the whole process. The present com- Common dehydrating agents: mercial tissue processors have the facility to heat –– Ethyl alcohol the tissue in all stages of processing. Overheating –– Methylated spirit may produce hard and brittle tissue. –– Methanol 4. Viscosity: The viscosity of the embedding –– Butyl alcohol media also affects the processing. Higher vis- –– Isopropyl alcohol cosity of the medium lowers the penetration –– Dehydrating agents other than alco- rate in the tissue. Heat reduces the viscosity of hol: dioxane, ethylene glycol and the medium and helps in better penetration. acetone 2.3 Individual Dehydrating Agent 21 to high concentration of dehydration fluid. The Increases the life span of alcohol tissue should be kept in the dehydration fluid for Better dehydration optimal time because too much time in the dehy- Good indicator to change alcohol drating fluid may cause the tissue hard and brit- tle. Too little time in dehydration fluid may be Methylated Spirit It is also known as denatured insufficient for removal of free water molecule. alcohol. Methylated spirit contains 99% ethanol Thin 2–3 mm tissue needs less time in dehydra- and 1% methanol or isopropyl alcohol. tion fluid than thick 5 mm tissue. Methanol Methanol is a clear, colourless, vola- tile and inflammable liquid. It can be used as a 2.3 Individual Dehydrating substitute of ethanol, but it is rarely used in labo- Agent ratory because of its volatility and high cost. 2.3.1 Alcohol Butyl Alcohol n-Butanol, isobutanol and ter- tiary butanol are used as dehydrating agents both Ethanol Ethanol or ethyl alcohol is the most in animal tissue and plant histology processing. popular and most commonly used dehydrating Butyl alcohol is a slowly acting dehydrating agent. This is a clear and colourless fluid. Ethyl agent and takes longer time than ethyl alcohol for alcohol is flammable liquid. This is a relatively dehydration. However the tissue shrinkage is less rapid and efficient dehydrating agent. However, it by butyl alcohol. needs licence from the government to purchase ethyl alcohol for laboratory use. Isopropyl Alcohol Isopropyl alcohol is avail- able as isopropanol (99.8%). This is miscible As a dehydrating agent ethyl alcohol is used in with water and liquid paraffin. It is a relatively 50, 70, 90 and 100% concentration. For delicate rapid acting, non-toxic dehydrating agent caus- tissue, the dehydration may be started from 30% ing minimal tissue shrinkage. It is a good lipid concentration of ethyl alcohol. In routine labora- dissolving solvent. to