Techniques in Histopathology and Cytopathology

Techniques in Histopathology and Cytopathology

A Guide for Medical Laboratory Technology Students

Sadhana Vishwakarma MSc PhD (Chemistry)

Professor Department of Engineering Chemistry Technocrats Institute of Technology Bhopal, Madhya Pradesh, India Formerly Head, Department of Medical Laboratory Technology Gramin Polytechnic Nanded, Maharashtra, India

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Website: www.jaypeebrothers.com Website: www.jaypeedigital.com © 2017, Jaypee Brothers Medical Publishers The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not necessarily represent those of editor(s) of the book. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing of the publishers. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However, readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the author(s)/editor(s) assume any liability for any injury and/ or damage to persons or property arising from or related to use of material in this book. This book is sold on the understanding that the publisher is not engaged in providing professional medical services. If such advice or services are required, the services of a competent medical professional should be sought. Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary arrangements at the first opportunity. Inquiries for bulk sales may be solicited at: [email protected] Techniques in Histopathology and Cytopathology First Edition: 2017 ISBN 978-93-5270-109-4 Printed at5HSUR,QGLD/WG

Dedicated to My Father and Mother Late Shri Chhotelal Sharma and Shrimati Tara Sharma

Preface

The decision of writing this book is based on my experience as a classroom teacher and unavailability of the textbook related to histopathological and cytopathological techniques for medical laboratory technology (MLT) students. This book is therefore written primarily for students and trainees of medical laboratory technology. This book covers the full range of basic histological and cytological techniques used in medical laboratories and pathology departments. It provides a thorough grounding in all aspects of histological technology, from basic methods of section preparation and staining to advanced diagnostic techniques like cytology. Each chapter is comprehensively divided into basic theory, procedure, difficulties encountered, and important subjective and objective questions. Recent developments in this field like immunochemistry, automation, microarray, etc. are also incorporated to the extent required. This book provides text in simple language with diagrammatic explanation of procedure, and diagrams and figures that are neat and simple so that students can reproduce them in the examinations without any difficulity. It also provides, sufficient material to the students from examination point of view. It is a suitable resource for both the beginners in histological and cytological area and for the fully qualified laboratory technicians. This book is written in such a manner that it covers all the topics of histological and cytological techniques included in PGMLT, BMLT, DMLT, and CMLT of different boards and universities. Students or teachers are ultimately the right judge of whether I have done justice to this book. Since this is the first edition, the possibility of errors and omissions is difficult to rule out. Suggestions for further improvement of the book are most welcomed and will be gratefully acknowledged. Sadhana Vishwakarma

Acknowledgments

Many people have contributed in different ways and to acknowledge their individual advice and assistance is impossible. However, I owe special thanks to Dr PM Patil, Professor and Reader, Nanded Education Society’s Science College, Nanded, Maharashtra, India, for his advice and valuable suggestions. My thanks are also to the colleagues I worked with during the lifetime of this book. My thanks to Dr JG Pakwanne, Mr OS Darak, and Mr C Khan, who assisted in the preparation of manuscript. With deep gratitude, I acknowledge Dr VS Power, Principal, Gramin Polytechnic, Vishnupuri, Nanded, Maharashtra, India, for his continuous, interest and support. I express my deep appreciation to my husband Chandrakant for his moral support, encouragement and time to time help, my in-laws Shri Tulsiramji Vishwakarma and Shrimati Kesar Vishwakarma, who elevated my ambition, and my children Sanskruti and Aditya, who endured gracefully past painful days I devoted to writing this book. I owe my special thanks to Shri PS Kokas for his advice and moral support. My thanks are due to Shri Jitendar P Vij (Group Chairman) and Mr Ankit Vij (Group President) of M/S Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, especially for Shri Prasun Bhattacharya for taking great efforts in bringing out this book in a nice and attractive manner.

Contents

Chapter 1

Cell and Tissues

1

Cell 1 Cell Division  2 Cell Metabolism  5 Tissues 6 Liquid Connective Tissue  11

Chapter 2

Microscopy 17 History of Optical Microscope  17 Basic Terminology in Microscopy  17 Parts of Microscope  18 Care of Microscope  20 Types of Microscopy  21 Bright Field Microscopy  21 Dark Field Microscopy  22 Phase Contrast Microscopy   24 Fluorescence Microscopy  24 Electron Microscopy  25 Types of Electron Microscope  26 Confocal Scanning Optical Microscopy  28 Deconvolution Microscopy and Image Reconstruction  29 Polarization Light Microscopy  29

Chapter 3

Introduction and Importance of Histopathology Importance of Histopathology  32 Decomposition and Putrefaction  32 Various Steps of Histological Techniques  33 Specimen Collection  33 Logging of Specimen  34 Tissue Marking and Orientation  36 Methods of Examination of Tissues  37 Special Instructions on Specimen Collection and Handling   37 Duties and Responsibilities  38

32

xii Techniques in Histopathology and Cytopathology

Chapter 4

Tissue Fixation

40

Functions of fixatives  40 Properties of Fixatives  40 Types of Fixatives  41 Properties and Action of Some Important Fixatives  42 Fixation of Specific Substances  48 Artefacts 47 Preservation and Storage of Tissues  48 Points to be Remember During Fixation of Tissues  48 Special Fixatives for Special Tissue Samples  48

Chapter 5

Decalcification 51 Properties of Decalcifying Agents  51 Technique of Decalcification  51 Tests for Completion of Decalcification  52 Decalcifying Agents  53 Treatment after Decalcification  54 Surface Decalcification  54 Factors Affecting the Rate of Decalcification  55 Tips for Proper Decalcification  55

Chapter 6

Tissue Processing

57

Principle of Tissue Processing  57 Selection and Labeling of Tissues  57 Completion of Fixation before Processing  57 Post-fixation Procedures  57 Dehydration 58 Clearing 59 Impregnation and Infiltration  61 Automatic Tissue Processing  64 Manual Tissue Processing  66 Paraffin Wax Embedding  70 Storage of Paraffin Blocks  71 Celloidin and Low Viscosity Nitrocellulose Embedding  72 Gelatin Embedding  72 Plastic Embedding  73 Resin Embedding  73 Double Embedding  74 Re-embedding 74

Chapter 7

Microtomy and Section Cutting Microtomes 77 Microtome Knives  79 Section Cutting  83 Attaching the Sections to Slides  89

77

Contents

Chapter 8

Frozen Sections and Cryostat

xiii 93

Principle of Frozen Sections  93 Theory of Freezing  93 Cryoprotectants 94 Cryogen selection  94 Freezing Microtome  95 Methods of Cutting Frozen Sections  95

Chapter 9

Staining 100 Principle of Staining  100 Types of Dyes and Stains  101 Classification of Dyes or Stains  102 Types of Staining Processes  103 Staining Procedures  106 Hematoxylin Stains  107 Counter Stains  109 Standard Hematoxylin and Eosin Staining Method for Paraffin Sections  110 Special Staining Methods  110 Staining of Frozen Sections  123 Precautions Taken during Staining  125 Causes of Poor Staining  125

Chapter 10

Mounting of Sections

129

Characteristics of Mountant  129 Types of Mountant  129 Restaining 133 Labeling and Storage of Slides  133 Coverslips 133 Ringing Media  133

Chapter 11

Cytopathology 135 Types of Exfoliation  135 General Cytological Changes in Cells during Malignancy  135 Cytology of Normal Genital Tract  136 Methods of Specimen Collection and Submission  137 Gynecological Sample Collection  138 Non-gynecological sample collection  140 Fine Needle Aspiration Cytology  142 Preservation of cytological specimens  144 Preparation of Smears  145 Fixation and Fixatives  146 Cytological Staining Techniques  148 Special Stains  150 Mounting of Smear of Cell Samples  154

xiv Techniques in Histopathology and Cytopathology

Chapter 12

Museum Techniques

156

Functions of Pathology Museum  156 Safety in the Museum  159

Chapter 13

Safety in Histopathology Laboratory

160

Staff Training  160 Basic Organization and Common Sense  160 Infectious Hazards  160 Radiations Hazard  161 Equipment Hazards  162 Practical Safety in Routine Use  162

Chapter 14

Advances in Histopathology and Cytopathology

165

Microwave Irradiation of Primary Tissue Fixation  165 Ultrasonic Decalcification  165 Microwave-stimulated Tissue Processing  166 Ultrasound-stimulated Tissue Processing  167 Automatic Knife Sharpners  168 Ultramicrotomy 168 Automatic Staining Method  169 Endoscopy 169 Colposcopy 170 Autoradiography 171 Immunohistochemistry 172 Tissue Microarrays  173

Chapter 15

Solution and Reagents

176

Important Definitions  176 Preparation of Standard Solutions  176

Bibliography179 Glossary

181

Index

187

1

cHAPTER

Cell and Tissues

have the same common characteristics. The cell is made up of water, protein, carbohydrates, lipids, and inorganic salts.

CELL It is the basic functional unit of all living systems. It can be described as a mass of protoplasm enclosed within a membrane (plasma/cell membrane). The cells are of different types performing specialized functions, but they

Structure of a Cell The various structures present within the protoplasm are nucleus, cytoplasm, and cell membrane (Fig. 1.1).

Fig. 1.1: Structure of a cell

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