Pranab Dey - Basic and Advanced Laboratory Techniques in Histopathology and Cytology-Springer (2023)

Basic and Advanced
Laboratory Techniques
in Histopathology
and Cytology
Pranab Dey
Second Edition
123
Basic and Advanced Laboratory
Techniques in Histopathology
and Cytology
Pranab Dey
Basic and Advanced

Laboratory Techniques
in Histopathology
and Cytology
Second Edition
Pranab Dey
Department of Cytology and Gynecologic Pathology
Post Graduate Institute of Medical Education and Research
Chandigarh, India
ISBN 978-981-19-6615-6 ISBN 978-981-19-6616-3 (eBook)

https://doi.org/10.1007/978-981-19-6616-3
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature
Singapore Pte Ltd. 2018, 2022
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher,
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Dedicated to
Shree Shree Satyananda Giri,
Paramahansa Yogananda,
Rini and Madhumanti
Preface to the Second Edition
The present edition of the book includes the basic principles and techniques
of routine and special laboratory methods in histopathology and cytology. In
addition, the book also covers advanced laboratory techniques such as immu-
nocytochemistry, flow cytometry, liquid-based cytology, polymerase chain
reactions, tissue microarray and molecular technology.
In this second edition of the book, several important recent topics have
been covered with many new chapters such as liquid biopsy, artificial neural
networks, digital pathology and next-generation sequencing.
Each chapter of the book provides the basic principle, the practical meth-
ods of the technique, troubleshooting and clinical applications of the tech-
nique. Each technique is illustrated with coloured line drawing,
microphotographs and tables. Overall, I expect that the book is a helpful
guide for the pathology students, laboratory technicians, research students
and private practitioners.
Chandigarh, India Pranab Dey

2022 June
vii
Acknowledgements
I wish to express my thanks to Dr. Naren Aggarwal and Ms. Jagjeet Kaur
Saini of Springer publication who encouraged me in every stage of this work.
I am also thankful to Ms. Beauty Christobel, the production editor for her
constant support in publishing the second edition of the book.
I am thankful to Dr. Parikshaa Gupta, Dr. Uma Nahar Saikia and Dr.
Suvradeep Mitra who provided me with many essential figures. I also wish to
express my thanks to my technical staff who shared their experiences with me
during writing the second edition of the book.
My wife Rini and daughter Madhumanti were a great source of inspiration
to me. They gave me constant support during writing the second edition of the
book.
Lastly, I wish to express my gratitude to Almighty God for His blessing.
Chandigarh, India Pranab Dey

2022 June
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
1.1.1 Aims of Fixation:�� 3
1.2 Ideal Fixative�� 3
1.3 Tissue Changes in Fixation �� 3
1.3.1 Types of Fixation�� 4
1.3.2 Description of Nature of Fixation �� 4
1.4 Essential Precautions for Fixation in General�� 6
1.5 Mechanism of Fixation �� 6
1.5.1 Dehydration and Coagulation of Protein�� 6
1.5.2 Cross-linking Fixatives �� 6
1.6 Factors Affecting Fixation�� 10
1.7 Commonly Used Fixatives in the Laboratory �� 12
1.7.1 Formaldehyde �� 12
1.8 Preparation of Different Formalin Solution�� 13
1.8.1 Glutaraldehyde�� 13
1.9 Osmium Tetroxide�� 13
1.9.1 Advantages�� 13
1.9.2 Disadvantages �� 13
1.9.3 Methyl and Ethyl Alcohol�� 13
1.9.4 Acetone �� 14
1.9.5 Bouin’s Fixative�� 14
1.10 Mercury Salt-containing Fixatives�� 14
1.10.1 Zenker’s Fluid �� 14
1.10.2 Helly’s Fluid �� 14
1.10.3 B5 Fixatives�� 14
1.10.4 Fixatives of Choice �� 15
1.11 Fixation Artifact�� 16
1.11.1 Formalin Pigment�� 16
1.11.2 Troubleshooting in Fixation is Highlighted�� 18
References�� 18
2
Processing of Tissue in the Histopathology Laboratory�� 19
2.1 Factors that Influence Tissue Processing�� 19
2.2 Dehydration�� 20
xi
xii Contents
2.3 Individual Dehydrating Agent�� 20

2.3.1 Alcohol�� 20
2.3.2 Dehydrating Agents Other than Alcohol�� 21
2.4 Clearing�� 22
2.4.1 Individual Clearing Agent�� 23
2.4.2 Other Clear Agents�� 23
2.5 Infiltration and Embedding �� 23
2.5.1 Different Impregnating Medium�� 24
2.5.2 Vacuum Impregnation Method �� 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 Rapid tissue Processing�� 27
References�� 28
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 �� 31
3.3.1 Double Embedding Method�� 32
3.3.2 Nitrocellulose and Paraffin �� 32
3.3.3 Tissue Orientation and Embedding�� 33
3.4 Tissue marking�� 33
References�� 34
4 Decalcification
of Bony and Hard Tissue for Histopathology
Processing �� 35
4.2 Factors Controlling the Rate of Decalcification�� 36
4.3 The Methods of Decalcification�� 36
4.3.1 Acid Decalcification�� 36
4.3.2 Von Ebner’s Fluid �� 36
4.3.3 Perenyi’s fluid �� 36
4.3.4 Weak Acids �� 37
4.3.5 Trichloroacetic acid�� 37
4.3.6 Chelating Agents�� 37
4.4 Ion Exchange Resin Method�� 38
4.4.2 Electrolysis Method�� 38
4.4.3 Surface Decalcification �� 38
4.5 Endpoint Determination of Decalcification�� 39
4.6 Results of Under Decalcification�� 39
4.7 Results of Over Decalcification�� 40
References�� 40
5 Tissue
Microtomy: Principle and Procedure�� 41
Contents xiii
5.2 Microtome Knife�� 43

5.2.1 Disposable Knife�� 44
5.2.2 Materials Used in Knife�� 44
5.2.3 Angles of Knife�� 44
5.3 Microtome Knife Sharpening �� 45
5.3.1 Manual Method�� 45
5.3.2 Factors Involved in Cutting�� 45
5.4 Sectioning the Paraffin Block �� 45
5.4.1 Steps of Tissue Sectioning�� 46
5.4.2 How to Recover the Dried Tissue?�� 50
Reference �� 50
6
Frozen Section: Principle and Procedure�� 51
6.2 Indications of Frozen Sections�� 51
6.2.1 The Principle of the Frozen Section�� 51
6.2.2 Cryostat Machine Proper�� 51
6.3 Cryostat Sectioning�� 53
6.4 Staining �� 55
6.4.1 H & E Staining�� 55
6.4.2 Toluidine Blue Stain �� 56
6.5 Factors Affecting the Good Quality Section �� 56
References�� 56
7 Staining Principle and General Procedure of Staining
the Tissue�� 57
7.2 Dyes Used for Staining �� 57
7.2.1 Types of Dye �� 58
7.2.2 Types of Dye on the Basis of Chemical
Structures and Chromophore Groups�� 59
7.3 Mechanisms and Theory of Staining�� 60
7.4 Factors Influencing Staining �� 63
7.5 The Nomenclature Used Regarding Dye�� 64
7.5.1 Applications�� 64
7.6 Metachromasia�� 64
7.6.1 Metachromatic Dyes �� 65
7.6.2 Applications of Metachromasia�� 66
7.7 Progressive and Regressive Staining�� 67
7.8 Mordant�� 67
7.8.1 Lake�� 67
7.8.2 Type of Application of Mordant �� 68
7.8.3 Accentuators �� 68
7.9 Staining Procedure�� 69
7.9.1 Preparation of Buffer Solutions�� 69
References�� 70
8
Haematoxylin and Eosin Stain of the Tissue Section�� 71
xiv Contents
8.2 Haematoxylin�� 71
8.3 Bluing�� 72
8.3.1 Scott’s Tap Water�� 72
8.3.2 Preparation of Different Haematoxylins
and Their Properties�� 73
8.3.3 Mayer’s Haematoxylin�� 73
8.3.4 Ehrlich’s Haematoxylin�� 74
8.3.5 Cole’s Haematoxylin�� 74
8.4 Counterstain by Eosin �� 74
8.5 Routine Haematoxylin and Eosin stain�� 74
8.5.1 Requirements�� 74
8.5.2 Steps�� 74
8.5.3 Staining Time of Different Haematoxylin�� 76
8.6 Iron Haematoxylin�� 78
8.6.1 Heidenhain’s Iron Haematoxylin�� 78
8.6.2 Verhoeff’s Iron Haematoxylin�� 79
8.6.3 Tungsten Haematoxylin�� 79
8.7 Clearing the Smear�� 80
8.8 Mounting�� 80
8.8.1 Disadvantage�� 81
8.8.2 Application of Mounting Medium�� 81
8.8.3 Coverslip �� 82
8.8.4 The Resin-coated Plastic Film�� 82
8.8.5 Restaining �� 82
References�� 82
9 Special
Stains for the Carbohydrate, Protein, Lipid,
Nucleic Acid and Pigments�� 83
9.2 Carbohydrates �� 83
9.2.1 Simple Carbohydrates�� 84
9.2.2 Significance of Mucin Demonstration�� 87
9.3 Staining of Different Carbohydrates �� 87
9.3.1 Glycogen �� 87
9.3.2 Periodic Acid Schiff’s (PAS) Stain �� 87
9.3.3 Indications to do PAS stain �� 87
9.3.4 Principle�� 87
9.3.5 Alcian Blue �� 88
9.3.6 Combined PAS-Alcian Blue Staining �� 89
9.4 Result�� 90
9.4.1 Mucicarmine Stain�� 90
9.5 Colloidal Iron�� 91
9.5.1 Colloidal Ion Stalk Solution �� 91
9.5.2 Lipids�� 91
9.6 Fixation �� 92
9.7 Stains�� 92
9.7.1 Oil red O �� 92
9.7.2 Preparation of Oil Red O Stain �� 92
Contents xv
9.8 Sudan Black B�� 93

9.8.1 Solution�� 93
9.8.2 Steps�� 93
9.8.3 Ferric haematoxylin for Phospholipid�� 93
9.9 Nucleic Acid and Proteins�� 93
9.9.1 Nucleic Acids�� 93
9.9.2 Proteins �� 94
9.9.3 Feulgen Stain�� 94
9.9.4 Methyl Green Pyronin Stain �� 94
9.9.5 Pigments�� 95
9.9.6 Hemosiderin Pigment�� 95
9.9.7 Bile Pigment �� 95
9.9.8 Argyrophil Pigments�� 96
9.9.9 Melanin �� 97
9.9.10 Schmorl’s Stain �� 97
9.9.11 Calcium �� 98
9.9.12 Von Kossa Technique�� 98
9.9.13 Formalin Pigment�� 98
9.9.14 Malarial Pigment�� 98
9.9.15 Starch�� 98
References�� 99
10
Connective Tissue Stain: Principle and Procedure �� 101
10.1 Fibrous Part of Connective Tissue�� 101
10.1.1 Reticulin Fibres �� 102
10.1.2 Elastic Fibres�� 102
10.1.3 Basement Membrane�� 102
10.2 Stains�� 103
10.2.1 Masson Trichrome�� 103
10.2.2 Van Gieson Stain �� 105
10.2.3 Reticulin Stain�� 106
10.2.4 Gordon and Sweet’s Method for Reticulin Stain�� 107
10.3 Elastic Fibres�� 108
10.3.1 Verhoeff’s Stain for Collagen�� 108
10.3.2 Final Verhoeff’s solution �� 108
10.3.3 Weigert’s Resorcin-Fuchsin Stain �� 108
10.3.4 Orcein for Elastic Fibres �� 109
10.3.5 Fibrin and Cross Striation of the Muscle�� 109
References�� 111
11 Amyloid Staining�� 113
11.2 Primary Amyloidosis�� 114
11.3 Stains for Amyloid�� 114
11.3.1 Alkaline Congo Red Stain�� 114
11.3.2 Congo Red Stain by Highman�� 115
11.3.3 Thioflavin T Stain�� 115
xvi Contents
12 Stains
for the Microbial Organisms �� 117
12.1 Bacteria �� 118
12.1.1 Gram’s Stain�� 118
12.2 Ziehl Neelsen Stain �� 118
12.2.1 Reagents�� 118
12.2.2 Steps of Staining �� 118
12.3 Fite Acid-fast Stain for Leprosy�� 119
12.3.1 Methylene blue�� 119
12.3.2 Carbol-fuchsin�� 119
12.3.3 Sulphuric Acid (5%)�� 119
12.3.4 Xylene in Peanut Oil Solution�� 119
12.4 Fungal Infection�� 120
12.4.1 Grocott’s Methenamine Silver�� 120
12.4.2 Reagents�� 120
12.4.4 Result�� 121
12.5 Spirochaetes�� 121
12.5.1 Warthin and Starry Technique�� 121
12.5.2 Viral Inclusions �� 121
Part II Basic Laboratory Techniques in Cytology Laboratory
13 Cytology
Sample Procurement, Fixation and Processing�� 125
13.2 Sample Collection�� 125
13.2.1 Cervical Cytology �� 125
13.2.2 Collection Proper�� 126
13.3 Respiratory Samples �� 127
13.3.1 Sputum Sample �� 128
13.3.2 Bronchial Brush�� 128
13.3.3 Bronchial Wash �� 128
13.3.4 Bronchoalveolar Lavage (BAL)�� 128
13.3.5 Transbronchial Needle Aspiration�� 128
13.3.6 Gastric Brush�� 128
13.3.7 Gastric Lavage�� 128
13.3.8 Endoscopic Ultrasound-guided (EUS) FNAC�� 128
13.3.9 Effusion Fluid Sample�� 129
13.3.10 CSF and Vitreous Fluid �� 129
13.4 Fixation �� 129
13.4.1 Time of Fixation�� 130
13.4.2 Special Fixatives �� 130
13.5 Processing of Laboratory Samples�� 131
13.5.1 Receiving the Sample�� 131
13.5.2 Glass Slides and Liquid Sample�� 131
13.6 Processing �� 132
13.6.1 Processing of Sputum�� 132
Contents xvii
13.6.2 Processing of Fluid: Urine, Body Fluids, Lavage �� 133

13.6.3 The Basic Principle of Centrifuge�� 133
13.6.4 Millipore Filtration�� 134
13.6.5 Processing of Hemorrhagic Fluid�� 135
13.6.6 Cell Block�� 135
13.6.7 Compact Cell Block Technique�� 136
14
Routine Staining in Cytology Laboratory �� 137
14.1 Papanicolaou’s Stain �� 137
14.1.1 Dyes Used in Papanicolaou’s Staining�� 137
14.1.2 Principle of Basic Steps�� 137
14.1.3 Papanicolaou’s Staining Steps�� 138
14.1.4 Bluing Solution �� 139
14.2 Precautions to Be Taken in Papanicolaou’s Staining�� 139
14.2.1 Staining Solutions �� 139
14.2.2 Coverslip �� 140
14.2.3 Staining Proper�� 140
14.2.4 De-staining and Re-staining the Smear�� 140
14.3 May Grunwald Giemsa Stain�� 140
14.3.1 Steps�� 141
14.3.2 Storage of Slides �� 141
Reference �� 141
15
The Basic Technique of Fine Needle Aspiration Cytology �� 143
15.2 Technique Proper�� 144
15.2.1 Equipment �� 144
15.3 Fine Needle Aspiration Procedure�� 145
15.3.1 Clinical History �� 145
15.3.2 Aspiration�� 146
15.3.3 Smear Preparation �� 146
15.4 Fine Needle Sampling�� 147
15.4.1 Steps�� 147
15.4.2 Limitations�� 147
15.5 FNAC of Deep-Seated Lesions �� 147
15.5.1 Major Indications of Deep Seated Guided
FNAC�� 148
15.5.2 USG Guided FNAC�� 148
15.5.3 Steps�� 149
15.6 Transrectal FNAC of the Prostate �� 149
Part III Advanced Techniques in Histology and Cytology

Laboratories
16
Immunocytochemistry in Histology and Cytology�� 153
16.2 Basic Principles�� 153
xviii Contents
16.3 Basic Immunology�� 153

16.4 Detection System�� 155
16.5 Peroxidase-Anti Peroxidase Method�� 155
16.5.1 Advantage�� 156
16.6 Avidin and Biotin Method�� 156
16.6.2 Disadvantage �� 156
16.7 Avidin and Biotin-Conjugated Procedure �� 156
16.7.2 Disadvantages�� 157
16.8 Biotin-Streptavidin Method�� 157
16.8.2 Alkaline Phosphatase–Anti Alkaline
Phosphatase Method�� 157
16.8.4 Polymer-Based Labelling Method�� 158
16.8.6 Catalyzed Signal Amplification
(Tyramine Signal Amplification)�� 158
16.8.7 Steps�� 158
16.8.8 The Sample of Tissues
for Immunocytochemistry�� 158
16.8.9 Sample Collection �� 159
16.8.10 Precautions to Have a Good Fixation�� 161
16.9 Immunocytochemistry Technique�� 163
16.9.1 Control�� 163
16.9.2 Steps�� 163
16.10 Selection of Primary Antibody�� 165
16.10.1 The Dilution of the Primary Antibody�� 166
16.10.2 Quality Control�� 166
16.10.3 Troubleshooting in Immunocytochemistry �� 166
16.11 Automated Immunostaining Platform�� 167
16.11.1 Types of Automated Immunostaining Platforms�� 167
16.11.2 Reagents Delivery Systems�� 167
16.11.3 Clinical Applications of Immunochemistry�� 168
16.12 Diagnostic Immunocytochemistry�� 168
16.12.1 Mesothelial Markers�� 168
16.12.2 Adenocarcinoma Markers in Effusion Fluid�� 169
16.12.3 Different Epithelial Markers �� 169
16.12.4 Mesenchymal Markers�� 171
16.12.5 Neuroendocrine Markers�� 172
16.12.6 Lymphoid Markers�� 172
16.12.7 Melanoma Markers �� 173
16.12.8 Germ Cell Markers�� 173
16.12.9 Site-specific Antibody in Different Epithelial
Malignancies �� 173
16.12.10 PSA and Androgen Receptor�� 173
Contents xix
16.12.11 Androgen Receptor �� 173

16.12.12 TTF �� 173
16.12.13 Estrogen and Progesterone Receptors
(ER and PR)�� 173
16.13 Immunocytochemistry of Round Cell Tumour �� 173
16.14 Immunocytochemistry for Therapy and Management�� 174
16.14.1 Breast Carcinoma�� 174
16.14.2 Estrogen and Progesterone Receptors�� 174
16.14.3 Her 2/Neu�� 174
16.15 Gastrointestinal Stromal Tumor�� 175
16.15.1 Lung Carcinoma�� 175
17 Flow Cytometry: Basic Principles, Procedure,
and Applications in Pathology�� 179
17.2 Principle of Flow Cytometry�� 179
17.3 The Flow Cytometer Instrument �� 179
17.3.1 Light Emission and Scattering�� 181
17.4 Flow Cytometric Cell Sorting �� 182
17.5 Dye Used�� 182
17.5.1 Fluorochrome Dye for Nucleic Acid�� 182
17.6 Samples for Flow Cytometry�� 182
17.6.1 Cytology Samples �� 182
17.6.2 Single-cell Preparation�� 183
17.6.3 Cellular Fixation�� 183
17.6.4 Permeabilization�� 184
17.6.5 RBC Lysing Solution�� 184
17.6.6 Control�� 184
17.6.7 Sample Processing�� 184
17.6.8 Flow Cytometric Immunophenotyping (FCI) �� 184
17.6.9 Data Aquisition�� 185
17.6.10 Data Display and Interpretation�� 186
17.6.11 Quality Control�� 186
17.7 Targets of Application�� 187
17.8 DNA Content and Ploidy Analysis �� 188
17.8.1 Basic Principle�� 188
17.9 Clinical Application�� 189
17.9.1 DNA Content and Diagnosis�� 189
17.9.2 DNA Content and Prognosis of the Patients �� 189
17.10 Immunophenotyping of Lymphomas�� 189
17.10.1 Diagnosis:�� 190
17.10.2 Sub-classification of Lymphomas:�� 191
17.10.3 Limitations of FCI�� 191
17.10.4 Flow Cytometry Features of Different
Lymphomas �� 191
17.10.5 Diagnosis of Other Lesions by FCI�� 191
xx Contents
17.10.6 Predicting Response to Monoclonal Therapy �� 192

17.10.7 Detection of Minimal Residual Disease�� 192
17.10.8 Assessment of Sub-G1 Fraction of Apoptotic
Cells�� 193
17.10.9 Apoptosis Detection by Annexin V Assay�� 193
18 Digital Pathology�� 195
18.2 What Is Digital Pathology?�� 195
18.2.1 Comparison of Traditional Pathology
and Digital Pathology�� 195
18.2.2 Workflow of Digital Pathology �� 196
18.2.3 Basic Instruments and Software in Digital
Pathology�� 197
18.3 Whole Slide Imaging (WSI) �� 197
18.3.1 Hardware �� 198
18.3.2 Software�� 198
18.3.3 Commercially Available WSI�� 199
18.3.4 Advantages of Digital Slides�� 199
18.3.5 Disadvantages of Digital Slides�� 200
18.3.6 Concordance of Glass Slides and Digital Slides �� 201
18.3.7 Guidelines of Clinical Diagnostic Application
of WSI �� 201
18.3.8 Applications of Digital Pathology�� 201
18.3.9 Limitations and Challenges of Digital Pathology �� 202
19 Automation
in the Laboratory and Liquid-Based Cytology�� 205
19.2 Advantages of Automation in Laboratory�� 205
19.2.1 The Various Stages of Automation�� 206
19.2.2 Tissue Processing�� 206
19.3 Automated Immunostaining Platform (AIP)�� 207
19.3.1 Digitization of Slide�� 207
19.4 Cytology Processing �� 207
19.4.1 Advantages of LBC over Conventional Smear �� 208
19.4.2 Limitations of Liquid-based Cytology�� 208
19.4.3 Collection Procedure of LBC�� 209
19.5 Sample Processing�� 209
19.5.1 ThinPrep (Cytic, UK) �� 209
19.6 Comparison of These Two Techniques �� 211
19.7 Automated Screening Devices in Cytology�� 212
19.7.1 BD FocalPoint GS Imaging System�� 212
19.7.2 BD FocalPoint GS Review Station �� 212
19.7.3 HOLOGIC ThinPrep Imaging System�� 213
19.7.4 Review Scope�� 213
19.7.5 Comparison of Manual and Automated Devices�� 213
19.8 Artificial Neural Network (ANN) in Pathology�� 214
Contents xxi
20 Polymerase Chain Reaction: Principle, Technique

and Applications in Pathology�� 215
20.2 What is PCR and How Does it Work?�� 215
20.3 Steps of PCR �� 215
20.3.1 Essential Ingredients of PCR�� 217
20.4 Procedure Proper�� 217
20.4.1 Basic Precautions�� 217
20.4.2 Equipment �� 217
20.4.3 Addition of the Ingredients in a 50 μL PCR
Tube�� 217
20.4.4 Remember �� 218
20.4.5 Thermal Cycling�� 218
20.4.6 Purification of the Amplified Product �� 218
20.4.7 Troubleshooting�� 218
20.4.8 Enhancing PCR Products Formation�� 220
20.5 Types of PCR�� 220
20.6 Applications of PCR �� 227
21
Fluorescent In Situ Hybridisation Techniques in Pathology:
Principle, Technique and Applications�� 229
21.1.1 Applications of FISH�� 229
21.1.2 The Principles of FISH�� 230
21.2 Steps to do FISH �� 230
21.2.1 Histology and Cytology Specimen�� 230
21.3 Troubleshooting�� 231
21.3.1 Different Types of FISH�� 231
21.3.2 Basic Principles�� 233
21.3.3 CGH Method�� 234
21.3.4 Array-based CGH�� 235
21.4 Different Other Varieties of FISH �� 236
22 Tissue Microarray in Pathology: Principal, Technique
and Applications�� 241
22.2 Tissue Microarray Technique�� 241
22.3 TMA Construction and Generation of Grid�� 242
22.4 Designing the Grid�� 242
22.5 Clinical Applications of TMA�� 245
23 Sanger Sequencing and Next Generation Gene Sequencing:
Basic Principles and Applications in Pathology�� 247
23.1 Sanger Sequencing�� 247
23.1.1 Reagents Needed�� 249
23.1.2 Main Steps�� 249
xxii Contents
23.2 Maxam Gilbert Technique�� 250

23.2.1 Main Steps�� 250
23.3 Next-generation Sequencing �� 251
23.3.1 Second-generation Sequencing �� 251
23.4 Illumina Solexa �� 252
23.5 Ion Semiconductor Sequencing (Ion Torrent)�� 254
23.5.2 Disadvantage �� 254
23.6 Third Generation�� 256
23.6.1 Single-Molecule Real-Time Sequencing�� 256
23.7 Fourth Generation Sequencing �� 257
23.7.1 Nanopore Sequencing �� 257
23.7.2 Other Technologies �� 257
23.7.3 DNA Nanoball Sequencing�� 258
23.7.4 RNA-Seq �� 259
23.7.5 Applications of NGS �� 259
24 Liquid
Biopsy: Basic Principles, Techniques
and Applications�� 263
24.2 Conventional Biopsy Versus Liquid Biopsy?�� 263
24.3 The Components of Liquid Biopsy�� 264
24.4 Enrichment of the Contents of Liquid Biopsy�� 264
24.5 The Molecular Techniques to Do in Liquid Biopsy�� 265
24.6 Clinical Applications�� 265
25 Artificial
Neural Network in Pathology: Basic Principles and
Applications�� 267
25.2 ANN Versus Ordinary Computer�� 267
25.3 Artificial Neural Network Versus Biological Neuron �� 268
25.4 Activation�� 269
25.5 Learning of ANN�� 269
25.5.1 Multilayer Perceptron Architecture�� 270
25.5.2 Steps to Building an ANN�� 270
25.5.3 Main Challenges of ANN�� 274
25.5.4 Application of ANN�� 274
25.5.5 Limitations of ANN�� 274
Contents xxiii
Part IV Microscopy, Quality Control and Laboratory

Organization
26 Compound Light Microscope and Other Different
Microscopes�� 279
26.1 Light�� 279
26.2 Colours�� 280
26.3 Image Generation and Human Vision �� 280
26.3.1 Image Formation by the Light Microscope�� 282
26.4 Optical Components�� 283
26.5 How to Take Care and Handle Your Microscope�� 285
26.6 Other Types of Microscope�� 285
26.6.1 Darkfield Microscope�� 285
26.6.2 Bright-Field Microscope �� 286
26.6.3 Phase-contrast Microscope�� 286
26.6.4 Inverted Microscope�� 286
26.6.5 Dissecting Microscope�� 287
27
Fluorescence Microscope, Confocal Microscope and Other
Advanced Microscopes: Basic Principles and Applications
in Pathology�� 289
27.1 Transmitted Fluorescent Microscope�� 290
27.2 Incident Fluorescent Microscope�� 291
27.2.1 The Dye Used in Fluorescence Microscope�� 291
27.2.2 Applications of Fluorescence Microscope�� 292
27.3 Confocal Microscopy�� 292
27.4 Limitations of CFM�� 294
27.5 Applications of CFM�� 294
27.6 Two-Photon Microscopy�� 295
27.7 4Pi Microscopy �� 297
27.8 Spatially Modulated Illumination Microscopy �� 297
27.8.1 Scanning Probe Microscope�� 297
27.8.2 Laser Capture Microdissection �� 299
27.8.3 Expression Microdissection�� 300
28
Electron Microscopy: Principle, Components, Optics and
Specimen Processing�� 303
28.1 Microscope Column and Electronic Optics�� 306
28.2 Specimen and Electron Interaction �� 306
28.2.1 Backscattered Electrons�� 308
28.2.2 Excited Electrons of the Atom�� 308
28.3 Electron Interaction in the Transmission
Electron Microscope �� 308
xxiv Contents
28.4 Sample Preparation for TEM�� 308

28.4.1 Combined Fixation Technique�� 309
28.4.2 Embedding�� 309
28.4.3 Knives�� 310
28.4.4 Staining of the Sections�� 311
28.4.5 Uranyl Salt�� 312
28.5 Scanning Electron Microscopy �� 312
28.5.1 Operational Principle�� 312
29 Quality
Control and Laboratory Organization�� 315
29.2 Quality Control �� 316
29.2.1 Gold Standard�� 319
29.2.2 Record Keeping�� 319
29.3 Audit �� 320
29.3.1 The Beneficial Points of the Internal Audit�� 320
29.3.2 Stages of Audit�� 320
29.3.3 Components of Audit�� 320
29.4 External Quality Assurance�� 321
29.4.1 Laboratory Accreditations�� 321
29.4.2 Pre-requisite for Laboratory Accreditation �� 321
29.4.3 Process of Accreditatation�� 321
29.4.4 Advantages of Laboratory Accreditations�� 321
29.5 Laboratory Organization �� 321
30 Laboratory
Safety and Laboratory Waste Disposal �� 323
30.1 Laboratory Waste Disposal �� 325
30.1.1 Basic Ways to Waste Management�� 325
30.1.2 Steps of Biomedical Waste Disposal�� 326
Multiple Choice Questions for the Self-Assessment�� 329

About the Author
Pranab Dey is professor in the Department of Cytology and Gynaecologic

Pathology at Post Graduate Institute of Medical Education and Research
(PGIMER), Chandigarh, India. Professor Dey completed M.D. (Pathology)
from PGIMER and FRCPath (Cytopathology) from Royal College of
Pathologists, London, UK. Professor Dey has conducted many research proj-
ects and has pioneered works on DNA flow cytometry, image morphometry,
mono-layered cytology and cytomorphologic findings of various lesions on
cytology smears. Professor Dey has more than 35 years of experience in report-
ing, teaching and research as a consultant. He is a well-published author in
pathology and a member of various societies. He has more than 450 research
paper publications in peer-reviewed journals. He has authored 12 books in
cytology and gynaecologic pathology. Dr. Dey is included in the list of top 2%
scientists in the world.
xxv
Abbreviations
a CGH array-based CGH

Ab Antibody
ACEP 3 aminopropyltriethoxysilane
AFM Atomic force microscope
AIP Automated immunostaining platform
ANN Artificial neural networks
APAAP Alkaline phosphatase–antialkaline phosphatase
APC Allophycocyanin
AR Antigen retrieval
BAL Bronchoalveolar lavage
CEA Carcinoembryonic antigen
CEP Chromosome enumeration probe
cf-DNA cell-free DNA
CFM Confocal microscopy
cf-RNA cell-free RNA
CGH Comparative genomic hybridization
CI Colour index
CK Cytokeratin
CLC Contact-free laser pressure catapulting
CLM Conventional light microscopy
CNN Convolutional neural network
CP Conventional preparation
CT Computerized tomography
CTC Circulating tumour cells
CYM Cyan, yellow and magenta
ddNTP Dideoxy nucleotides
ddPCR Droplet digital PCR
D-FISH Double fusion FISH
DIA Digital image analysis
DL Deep learning
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphates
DP Digital pathology
DPX Dibutylphthalate polystyrene xylene
DS Digital slide
DSRT Desmoplastic small round cell tumour
xxvii
xxviii Abbreviations
EA Eosin Azure
EDTA Ethylenediaminetetraacetic acid
EM Electron microscope
EMA Epithelial membrane antigen
EpCAM Epithelial cell adhesion molecule
ER Estrogen receptors
EUS-FNAC Endoscopic ultrasound-guided FNAC
EV Extracellular vesicles
EVA Ethylene-vinyl acetate
EWS Ewing’s sarcoma
EXO EVs contain exosomes
FCI Flow cytometric immunophenotyping
FCM Flow cytometry
FFPE Formalin-fixed paraffin-embedded section
FISH Fluorescent in situ hybridization
FITC Fluorescein iso-thiocyanate
FNAC Fine needle aspiration cytology
FNS Fine needle sampling
FOV Field of view
FPGS FocalPoint GS Imaging System
FRAP Fluorescence recovery after photobleaching
FSC Forward scattering
GAM Contact-free gravity-assisted microdissection
GFP Green fluorescence protein
GLCM Gray level co-occurrence of matrix
GMS Gomori methenamine silver
H&E Hematoxylin and Eosin
HCG Human chorionic gonadotropin
HIS Hue saturation intensity
HRP Horseradish peroxidase
HSIL High-grade squamous intraepithelial lesions
ICC Immunocytochemistry
IHC Immunohistochemistry
IMS Image management system
IPCR Inverse PCR
LB Liquid biopsy
LBC Liquid-based cytology
LCM Laser capture microdissection
LIS Laboratory information service
LSI Locus-specific identifier probe
M-FISH Multi-coloured FISH
MGG May Grunwald Giemsa
MRD Minimal residual disease
MRI Magnetic resonance image
NB Neuroblastoma
NGS Next-generation sequencing
NHL Non-Hodgkin lymphoma
nM Nano micrometer
Abbreviations xxix
OCT Optimum cutting temperature

OG Orange G
PAP Papanicolaou
PAS Periodic Acid-Schiff
PCR Polymerase chain reaction
PE Phycoerythrin
PerCP Peridinin chlorophyll
PLAP Placental alkaline phosphatase
PMT Photomultiplier tube
PNET Peripheral neuroectodermal tumour
PPi Inorganic pyrophosphate
PR Progesterone receptors
PSA Prostate-specific antigen
PTAH Phosphotungstic acid haematoxylin
QA Quality assurance
QC Quality control
Q-FISH Quantitative FISH
QI Quality improvement
Q-PCR Quantitative PCR
RCF Relative centrifugal force
ReLU Rectified linear unit
RGB Red green blue
RMS Rhabdomyosarcoma
RNA Ribonucleic acid
ROC Receiver operating curve
RT-PCR Reverse transcriptase PCR
SEM Scanning electron microscope
SMRT Single-molecule real-time
SOP Standard operating protocol
SSC Side scattering
SSCP Single-strand conformation polymorphism
ssDNA Single-stranded DNA
STM Scanning tunnelling microscope
TEM Transmission electron microscope
TIP ThinPrep image processor
TMA Tissue microarray
TSA Tyramine signal amplification
TTF-1 Thyroid transcription factor-1
USG Ultrasonography
VS Virtual slides
vWF Von Willebrand factor
WSI whole slide imaging
WSP Whole chromosome probes
WT Wilms’ tumor
WT 1 Wilms’ tumor gene 1
Xmd Expression microdissection
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 1.2 Ideal Fixative
Fixation is the first step of any histological and An ideal fixative should have the following quali-
cytological laboratory technique. It is the process ties [1]:
by which the cells in the tissue are fixed in a chem-
• Prevention of autolysis of the cells or tissue.
ical and physical state, and all the biochemical and
• Prevention of decomposition of the tissue by
proteolytic activities within the cells are prevented
bacteria.
so that the cells or tissues can resist any morpho-
• Maintaining the volume and shape of the cell
logical change or distortion or decomposition after
as far as possible.
subsequent treatment with various reagents. The
• Consistently high-quality staining, mainly
fixation helps to maintain the tissue nearest to its
routine stains such as Hematoxylin and eosin
original state in the living system.
stain and Papanicolaou’s stain.
• Rapid action.
• Cheap.
• Non-toxic.
1.1.1 Aims of Fixation
A large number of fixatives are available in the
The primary purposes of fixation are the
market. Each fixative has its advantages and dis-
following
advantages. It is challenging to find universally
accepted ideal fixatives.
• To preserve the tissue nearest to its living state
• To prevent any change in shape and size of the
tissue at the time of processing
• To prevent any autolysis 1.3 Tissue Changes in Fixation
• To make the tissue firm to hard
• To prevent any bacterial growth in the tissue The following changes may occur in tissue due to
• To make it possible to have a clear stain fixation (Box 1.1):
• To have a better optical quality of the cells 1. Volume changes: Fixatives may change the
volume of the cells. Some fixatives such as
Osmium tetroxide causes cell swelling. The
exact mechanism of the volume change is not
understood correctly. However, the volume
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 3
P. Dey, Basic and Advanced Laboratory Techniques in Histopathology and Cytology,
https://doi.org/10.1007/978-981-19-6616-3_1
4 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives
Table 1.1 Types of fixation and classification of

Box 1.1 Change in Tissue After Fixation fixatives
• Volume changes Types of fixative Classification
–– Shrinkage of the volume by formalin A. Nature of • Immersion fixation
(33%) fixation • Coating fixation
• Vapour fixation
• Hardening of tissue • Perfusion fixation
–– Mild degree hardening may occur • Freeze-dryingng
• Interference of staining • Microwave fixation
–– Inhibits routine stain: Osmium B. Chemical • Aldehyde: Formaldehyde,
properties glutaraldehyde
tetroxide inhibits haematoxylin and
• Oxidising agent: Osmium
eosin staining tetroxide
• Changes of optical density by fixation • Protein denaturing agent: Ethyl
–– Nuclei may look like hyperchromatic alcohol, methyl alcohol
• Cross-linkingng agents:
Carbodiimide
change may be due to (a) altered membrane • Miscellaneous: Picric acid
permeability, (b) inhibition of the enzymes C. Component 1. Simple (only one chemical
present present)
responsible for respiration, and (c) change of (a) Formaldehyde
transport of Na+ ions. Formaldehyde may (b) Ethyl alcohol
cause shrinkage of the volume by 33%. In an (c) Glutaraldehyde
experiment, Bahr et al. noted that tissue (d) Picric acid
(e) Osmium tetroxide
shrinkage is inversely proportional to the con- 2. Compound (more than one
centration of formaldehyde [2]. Similarly, chemical present)
glutaraldehyde also causes significant tissue (a) Bouins fluid
shrinkage. However, when glutaraldehyde (b) Carnoy’s solution
D. Action on 1. Coagulative: Ethyl alcohol,
followed by osmium tetroxide is used as fixa- protein picric acid
tions in epoxy resin, then 70% increases in 2. Non-coagulative:
cell size are noted. Formaldehyde, osmium
2. Hardening of tissue: The fixation changes tetroxide, glutaraldehyde
the consistency of the tissue, and some amount
of hardening occurs due to fixation. 3. Component present
3. Interference of staining: Fixation may cause 4. Action on tissue protein
hindrance to the staining of enzymes.
Formaldehyde inactivates 80% of ribonucle-
ase enzyme [3]. It has been noted that Osmium 1.3.2 Description of Nature
tetroxide inhibits haematoxylin and eosin of Fixation
staining.
4. Changes in optical density by fixation: The 1. Immersion fixation: This is the most familiar
fixation may cause the change in optical den- way of fixation in laboratories. In this tech-
sity of the nuclei, and the nuclei may look nique, the whole specimen is immersed in the
condensed and hyperchromatic [4]. liquid fixative, such as tissue samples are
immersed in 10% neutral buffered formalin or
cytology smear in 95% ethyl alcohol.
2. Coating fixation: This is commonly used in
1.3.1 Types of Fixation the cytology samples. The spray fixative is
used for easy transportation of the slide. The
The fixative can be classified based on the fol- main advantages of spray fixatives are:
lowing criteria (Table 1.1): (a) Fixation of the cells.
1. Nature of fixation (b) To impart a protective covering over the
2. Chemical properties smear and.
1.3 Tissue Changes in Fixation 5
(c) No need to carry liquid fixative in a bottle The freeze-drying technique is applicable
or jar. mainly to study soluble material and tiny
The spraying over the smear should be molecules.
smooth and steady, and the optimum dis- Advantages
tance of 10 to 12 in. should be maintained • Excellent for enzyme study
between the nozzle of the spray and the • No change in proteins
smear. The spray fixative usually consists • No shrinkage of tissue
of alcohol and wax. Therefore, this wax • Preservation of glycogen.
should be removed before the staining 6. Microwave Fixation (Box 1.2)
procedure. Basic principle: Microwave is an elec-
3. Vapour fixation: In this type of fixation, the tromagnetic wave with frequencies between
vapour of chemicals is used to fix either a 300 MHz and 300 GHz, and wavelength
smear or tissue section. The commonly used varies from centimetre to nanometre.
chemicals for vapour fixation are formalde- Scientific and medical microwave ovens
hyde, osmium tetroxide, glutaraldehyde and operate with a frequency of 2.45 GHz and
ethyl alcohol. The vapour converts the soluble 0.915 GHz, respectively. The microwave
material to insoluble material, and these mate- creates the electromagnetic field, and the
rials are retained when the smear comes in dipolar molecules such as water rapidly
contact with the liquid solution. oscillate in this electromagnetic field. This
4. Perfusion fixation: This is mainly used for rapid kinetic motion of these molecules
research purposes. In this technique, the fixa- generates uniform heat. The generated heat
tive solution is infused into the arterial system
of the animal, and the whole animal is fixed. Box 1.2 Microwave Fixation of Tissues
The organ such as the brain or spinal cord can • What it is: electromagnetic wave with
also be fixed by perfusion fixation. frequencies between 300 MHz and
5. Freeze-drying: The tissue is cut into thin sec- 300 GHz
tions and then rapidly frozen at a very low • Mechanism: Microwave creates elec-
temperature. Subsequently, the ice within tromagnetic field and the dipolar mole-
the tissue is removed with the help of a vac- cules rapidly oscillates generating heat
uum chamber at a higher temperature by kinetic motion
(− 30 °C).
Steps Advantages:
• At first, the thin cut tissue section is rapidly • Uniform heat production
frozen at −160 °C by immersing it in liq- • No volume change of tissue
uid coolant. This is known as “quenching”. • Good for electron microscopy after
The commonly used fluids in the quench- osmium tetroxide fixation
ing bath are liquid nitrogen, propane and • Facilitates fixation and other laboratory
isopentane. Alternatively, the tissue section steps
can be frozen by keeping it in close contact • Preservation of the tissue antigen
with chilled metal. Disadvantage:
• In the next step, the ice within the tissue is • Tissue in the formalin for microwave
removed by placing the tissue in the vac- fixation may produce toxic gas and
uum chamber at a higher temperature (−30 overhead hood is required
to −50 °C). The water of the solid tissue is • Heat injury may occur from microwave
removed by sublimation. A suitable drying
agent absorbs the water vapour. Applications:
• In the final step, the tissue is gradually • In routine surgical pathology laboratory
warmed to 4 °C and is finally impregnated • Electron microscopy
with the embedding medium. • Urgent processing of special biopsies.
accelerates the fixation and also other steps • The amount of fixative fluid should be 20
of tissue processing. The essential charac- times more than the tissue volume.
teristics of microwave heat generation are • The tissue with fixative should be in a tightly
the homogeneous increase of temperature screw-capped bottle.
within the tissue, and every part of the tis- • Always check the colour of formalin. It
sue is heated. should be clear, and if there is any change of
Factors controlling the temperature colour, then the solution should be filtered or
rise: The rise of temperature in the microwave mildly heated.
heated media depends mainly on: • Never heat the fixative solution with the inten-
tion of rapid fixation as it may cause tissue
• The dielectric property of the media shrinkage.
• Thermal properties of the material
• Radiation level and
• Orientation and shape of the object. 1.5 Mechanism of Fixation
Advantages: The main benefits of micro-
wave fixation are: The wet fixatives usually work as:
• Rapid processing
• No change in volume of tissue
• Preservation of the tissue antigen and good 1.5.1 Dehydration and Coagulation
for immunohistochemistry of Protein
• It facilitates the staining reaction without
any bad effect Methanol and ethanol are commonly used coagu-
Disadvantages: lative fixatives. These two alcohols remove water
• The tissue immersed in formalin during from the tissue and causing destabilising of the
microwave fixation may generate many hydrogen bonds and disrupting the tertiary struc-
toxic gas. Therefore, an overhead hood is ture of the protein. However, the secondary struc-
required to remove this toxic substance ture of the protein is maintained. Ethanol is a
from the microwave. relatively stronger dehydrating agent than metha-
• Chances of heat injury nol. The ethanol and methanol start work from
Applications: 60% to 80% concentration, respectively. The
• In routine surgical pathology laboratory dehydrating fixative has two disadvantages:
• Electron microscopy after osmium tetrox-
ide fixation 1. Shrinkage of the cells and.
• Urgent processing of biopsy (e.g. kidney 2. Removal of the soluble substances from the
biopsy) tissue.
1.4 Essential Precautions 1.5.2 Cross-linking Fixatives

for Fixation in General
Formaldehyde: Formaldehyde in an aqueous
Certain essential precautions are necessary for solution combines with water to form methylene
proper fixation: hydrate, a methylene glycol:
CH 2 O + H 2 O = OHCH 2 OH
• The tissue should be free from excessive blood
before putting it into a fixative. Formaldehyde + water = Methylene glycol
• Tissue should be thinly cut in 3–5 mm
thickness. This methylene glycol may react with water mol-
ecules and form a polymer known as polyoxy-
1.5 Mechanism of Fixation 7
Fig. 1.1 (a) Schematic

diagram showing the a H
mechanism of formalin OH
H
fixation. Formaldehyde
reacts with the side
chain of the protein and H2O
C O C
forms hydroxymethyl
side group. Later on,
these highly reactive H H OH
substances form
cross-linking, and
methylene bridges are Formaldehyde Methlene glycol
formed. This is a stable
reaction, and simple
washing cannot remove
formalin in this stage. H
(b) Schematic diagram
showing the cross-
linking of protein chain R H C O R CH2OH
by formaldehyde
Bound hydroxymethy group
H
R CH2OH R H R CH2 R H2O
Metyhylene bridge
Cross linking the

b protein
Cross linked
Protein chain protein chain
methylene glycol in a longstanding position. This pounds are highly reactive, and subsequently,
again depolymerised in methylene glycol in a cross-linking occurs by creating a methylene
neutral buffer system. Formaldehyde reacts with bridge. This initial reaction of the hydroxymethyl
various protein side chains and forms a hydroxy- side chain is the primary reaction, and the subse-
methyl side group (Fig. 1.1a, b). These com- quent intermolecular and intramolecular cross-
Fig. 1.2 Schematic

diagram showing the O
mechanism of H H H O
glutaraldehyde fixation.
The aldehyde group of C C C C C
glutaraldehyde reacts H
H H H H
with amino group of the
protein. Rapid and
irreversibly cross- Glutaraldehyde
linking of the protein
takes place
O O
H H H
2
C C C C R -NH2
R1-NH2 C
H H H H H Amino
Amino
group
group
H H H H H
2
R1-N = C C C C C = N-R
H H H
Cross liking with

amino group
linking of the molecules occurs as a slow-growing Glutaraldehyde: It has two aldehyde groups
process. This ultimately produces an insoluble separated by three methylene bridges (Fig. 1.2). The
product. The formalin can be removed from tis- aldehyde group of glutaraldehyde reacts with an
sue by prolonged washing. However, once amino group of the protein, predominantly lysine.
Methylene Bridge is formed in the tissue, the When one aldehyde group reacts with the amino
reaction is stable, and it is challenging to remove group, the other free aldehyde group may help with
formalin from the tissue. Formaldehyde also cross-linking. Glutaraldehyde rapidly and irrevers-
reacts with the nucleic acid by reacting with the ibly cross-links the protein. The penetration of glu-
amino group of nucleotides. taraldehyde is slower than formaldehyde.
1.5 Mechanism of Fixation 9
Fig. 1.3 Schematic

diagram showing the o o
mechanism of osmium
tetroxide fixation. os
Osmium tetroxide reacts
with two unsaturated o o
carbon atoms of the Osmium tetroxide
lipids and cross links
with them
o o o
HC + = HC o
os os
HC HC o
o o
o Hexavalent osmium
HC o +H2O = HCOH + OsO3

os
HC o HCOH
o Osmium trioxide
diol
Osmium tetroxide
o o
HC + os + CH = HC o
os
o CH
HC CH HC o o CH
Unsaturated o o
Unsaturated Cross linking
carbon atom of
carbon atom of
lipid
lipid
Osmium tetroxide: Osmium tetroxide (OsO4) in this reaction. Osmic acid monoester formed in
is mainly used as a fixative in electron micros- this reaction is readily hydrolysed to a diol and
copy. It is used alone or in combination with osmic acid (Fig. 1.3). Osmium tetroxide may
another agent. The compound causes the oxida- react with two unsaturated carbon atoms of the
tion of unsaturated bonds in the biological tissue, lipids and may cross-link (Fig. 1.3).
particularly lipid. It converts the unsaturated fatty Figure 1.4 demonstrates the various mecha-
acid into a stable product known as glycol nisms of fixative.
osmate. The tetravalent Os becomes hexavalent
Fig. 1.4 Schematic Alcohol

Water
diagram showing the
mechanism of different
fixatives. Alcohol works
by removing water
molecules from the
tissue and coagulating
the protein.
Formaldehyde forms Water molecule is
hydroxymethyl side removed: Dehydartion
Alcohol
group and cross-links and coagulation of
with protein. Osmium protein
tetroxide forms glycol
osmate with lipid Formaldehyde
molecules
Hydroxymethyl
Protein
Cross linking with protein

Formaldehyde
Osmium tetroxide
Glycol
osmate
Lipid
Cross linking with lipid
Osmium tetroxide
1.6 Factors Affecting Fixation pH is too low or too high. At a very low pH, the
NH2 group of amino acid is converted to NH3,
The following factors may affect the fixation and the reaction between aldehyde groups of
(Box 1.3): the fixative is reduced. Usually, the buffer solu-
1. Hydrogen ion concentration (pH): Most fixation is added to maintain pH of the fixative. The
tives work better at neutral pH.Good fixation commonly used buffers in the fixatives are
occurs when pH remains 6 to 8, and no morpho- phosphate, bicarbonate, Tris and acetate. The
logical distortion is seen in that pH range. There buffers should be chosen in such a way that they
may be changes in the ultrastructure when the should not react with the fixative.
1.6 Factors Affecting Fixation 11
temperatures (60–65 °C). At higher tempera-

Box 1.3 Factors Affecting Fixation tures, the vibration and movement of the mol-
• pH of the fixative ecules are increased. This increases the
–– Neutral pH is preferable penetration rate of the fixative within the tis-
–– pH 6 to 8 is the best range sue and accelerates the fixation process. In
–– High acidity or alkalinity interferes case of a very high temperature, the antigen
fixation within the tissue may be destroyed. The
• Temperature enzymes are better preserved at lower tem-
–– Room temperature suitable for rou- peratures, and for enzyme histochemistry,
tine work 0–4 °C is suitable.
–– High temperature facilitates fixation 3. Duration of fixation: The depth of penetra-
–– Low temperature (0 °C to 4 °C) suit- tion of fixative is directly proportional to the
able for enzyme histochemistry square root of the time of fixation. The diffus-
• Duration of fixation ibility of different fixatives may also vary.
–– Depth of penetration of fixative is D = k √T
directly proportional to the square D = depth of penetration
root of time of fixation T = Time duration
–– Formalin fixes 1 mm/h k = Coefficient of diffusion of the fixative
–– Small tissue: 6 h in formalin is The penetration rate of formalin solution is
optimum 1 mm/h. The presence of blood may hamper
–– Large tissue: 24 h is the optimum the penetration of the fixative. Therefore it is
time preferable to wash the tissue specimen thor-
–– Prolonged fixation in aldehyde: oughly before putting it in fixative. The tissue
Inhibition of enzymatic activity should be sectioned as 3 to 5 mm. Overall,
• Osmolarity of the fixative solution formalin fixes tissue within 24 h. Prolonged
–– Hypertonic: Cell shrinkage fixation may cause loss of lipid and protein
–– Hypotonic: Cell swelling and a significant reduction of the enzymatic
–– Best: Mild hypertonic (400 to activity of the cell. This may also cause the
450 mOsm) hardening of the tissue.
• Concentration 4. Osmolarity of the fixative solution:
–– Mild lower concentration with neu- Osmolality of the fixative has a considerable
tral pH is preferable effect on fixation. The hypertonic fixative
–– Very low concentration prolongs the solution causes shrinkage of the cell, whereas
time of fixation the hypotonic fixative solution induces swell-
–– Higher concentration causes rapid ing of the cells. Electrolytes (0.9% NaCl) or
fixation with undesirable effect sucrose may be added to the fixative to main-
• Agitation tain the osmolarity of the fixative. Mildly
–– Agitation increases rate of hypertonic fixative (400 to 450 milliosmole)
penetration is preferable for routine use in the laboratory.
–– Rapid agitation: damages delicate 5. Concentration: Very low fixative concentra-
tissue tion prolongs the fixation time, and higher
–– Slow gentle agitation preferable concentration causes rapid fixation. However,
the higher concentration of fixative may cause
tissue hardening, tissue shrinkage and artefac-
2. Temperature: Room temperature is alright tual changes. A mildly lower fixative concen-
for tissue fixation and there is no difference in tration with neutral pH is needed for proper
cell morphology from 0 to 45 °C. However, fixation. The optimal concentration of formal-
the fixation time may be reduced in higher dehyde is 10%.
6. Agitation: Agitation increases the penetration

rate and, therefore, rapidity of fixation. Box 1.4: Formaldehyde Fixative
Optimum agitation is needed as slow agitation • Commercially available as 40% concen-
may not affect fixation, whereas rapid agita- tration (considered as100% formalin)
tion may have detrimental effect on delicate • 10% of this formalin is used to make
tissue. neutral buffered formalin
Mechanism: It reacts with various side

1.7 Commonly Used Fixatives chain of the protein and forms hydroxy-
in the Laboratory methyl side group that subsequently cross
link to form a methylene bridge. Subsequent
1.7.1 Formaldehyde intermolecular and intramolecular cross
linking of the molecules occurs and ulti-
Pure formaldehyde vapour dissolved in the water mately produces an insoluble product.
is available as formaldehyde in 37 to 40% con- Rate of penetration: Formalin pene-
centration. This is also known as formalin and is trates approximately 1 mm/h.
considered 100% formaldehyde. 10% of this for- Volume of formalin: The amount of
malin is used in the laboratory to make neutral formalin should be 20 times the volume of
buffered formalin for routine laboratory fixative tissue.
(Box 1.4). Advantages:
Rate of penetration: Formalin penetrates • High penetration rate
approximately 1 mm/h, and usually, 24 h are • Well preserved cell morphology
needed for fixation of a 1 cm3 tissue. • Cheap
The volume of formalin: For proper fixation, • Stable
the specimen should be sliced 5 mm apart, and
the amount of formalin should be 20 times the Disadvantages
tissue volume. The specimen should be wholly 1. Slow fixation
immersed in formalin and should not be in direct 2. Formalin reaction reversible and it can
contact with the container. There should be for- be removed by washing.
malin soaked clothes in between the container 3. Fails to preserve acid
and the tissue. mucopolysaccharides.
Removal of formalin from the tissue: The 4. Not good for staining of fat and enzymes.
cross-linking of the amino acids and proteins is a 5. Highly vascular tissue may have dark
slow process, so if the tissue is washed for 24 h in brown granules.
the water, then 50% of formalin from the tissue is 6. Exposure to skin may cause dermatitis.
removed. 7. Chronic inhalation may cause bronchitis.
Precaution: Formaldehyde is irritant to the
eye and skin and toxic for inhalation. It is a carci-
nogenic element. Disadvantages
1. Slow fixation
Advantages: 2. Formalin reaction with the tissue is reversible,
• The penetration rate of formalin is high and it can be removed by washing.
• Cell morphology well preserved in formalin 3. Formalin fails to preserve acid
• Cheap mucopolysaccharides.
• Stable 4. Highly vascular tissue may have dark brown
• Easy to make the solution granules (artefact).
• Formalin is an effective fixative for routine 5. Exposure to the skin may cause dermatitis.
laboratory staining of the tissue. 6. Chronic inhalation may cause bronchitis.
1.9 Osmium Tetroxide 13
1.8 Preparation of Different 1.9 Osmium Tetroxide

Formalin Solution
Osmium tetroxide is used for fixation in electron
A. 10% neutral buffered formalin: microscopy. It reacts with unsaturated bonds in
• Formaldehyde, 40% 100.0 ml
the lipid molecules and fixes them. The penetra-
• Distilled water: 900.0 ml
tion of the osmium tetroxide in the tissue is poor,
• Sodium dihydrogen phosphate: 4.0 g
• Disodium hydrogen phosphate: 6.5 g and if it is used alone, a good amount of protein
B. Preparation of 10% formal saline: and carbohydrate may be lost during fixation.
• Formaldehyde, 40% 100.0 ml
• Sodium Chloride 9g
• Distilled water 900.0 ml 1.9.1 Advantages
C. Formal ethanol fixative
• 95% Ethyl alcohol 20 ml 1. This is an excellent fixative for lipid.
• Formaldehyde, 40% 10 ml
2. It preserves cytoplasmic organelles such as
Golgi bodies and mitochondria
3. Does not make the tissue hard
1.8.1 Glutaraldehyde
Glutaraldehyde is used as a fixative for electron 1.9.2 Disadvantages

microscopy because it fixes and preserves the
ultrastructure. The fixation occurs due to the 1. It does not fix the proteins and carbohydrates,
extensive cross-linking of the proteins. The pen- and therefore it should be used in combination
etration power of glutaraldehyde is poor, and with another fixative.
therefore only a small piece of tissue should be 2. Osmium tetroxide may react with the ribose
used for fixation. Glutaraldehyde does not react group and cause DNA clumping. This can be
with lipid or carbohydrates, and therefore it prevented by pre-treatment with potassium
should be used in combination with the other permanganate or post-fixation with uranyl
fixative. acetate.
3. Poor penetration in the tissue.
Advantages
4. Tissue swelling may occur.
1. Better fixation of ultrastructure.
5. Toxic and volatilizes at room temperature
2. Less cell shrinkage.
producing harmful vapour. This vapour is
3. Preservation of protein is better.
toxic to eye and respiratory tract.
4. Good cross-linking with collagen.
6. Expensive.
5. Less irritating.
Disadvantages Laboratory use: It is commercially available in

1. Poor penetration and tissue should be less 0.1 to 1 g sealed vials. An aqueous solution of 4%
than 0.5 mm thick. OsO4 is made. This should be stored in a clean
2. Less stable compound. glass vial away from sunlight. In the laboratory,
3. No lipid fixation. 2–4% OsO4 in a buffer solution of pH 7.2 is used.
4. Glutaraldehyde polymerises above pH 7.5.
5. Costly.
1.9.3 Methyl and Ethyl Alcohol
For electron microscopy:
Glutaraldehyde is used as 2.5% glutaraldehyde in Methyl alcohol (methanol) and ethyl alcohol
100 mM phosphate buffer at pH 7.0. (ethanol) are used as dehydrating agents, and
Glutaraldehyde comes commercially as these two alcohols are primarily utilised as fixa-
25% or 50% solutions in 10 ml. tives for cytology smears. The tissue or smear
containing water should not be put directly in the 2. Formaldehyde stock solution (40%): 5 ml.
higher concentration of alcohol as it may distort 3. Glacial acetic acid: 1 ml.
the cells due to the rapid rush of fluid from the
cell. Therefore, graded alcohol should be used for
dehydration. 1.10 Mercury Salt-containing
Laboratory use: 95% ethyl alcohol for Fixatives
fixation.
Reagent preparation: Among the heavy metals, Mercury is commonly
Absolute alcohol: 950 ml. used as a fixative. This is a rapidly acting fixative.
Water: 50 ml. Mercury is a poisonous substance and should be
Time of fixation: 15–30 min. used carefully. Mercury-containing fixatives may
corrode the metal, so the fixative should be kept
in a glass container.
1.9.4 Acetone
It is mainly used for enzyme study and immuno- 1.10.1 Zenker’s Fluid
cytochemistry. It is a poor fixative for morpho-
It is a good fixative for nuclear chromatin and
logical preparation as it causes significant cell
collagen.
shrinkage. Acetone works by dehydration of
cells. Cold acetone is used at 4 °C for fixation. Preparation
Mercuric chloride: 50 g.
Glacial acetic acid: 50 g.
1.9.5 Bouin’s Fixative Potassium dichromate: 25 g.
Distilled water: 950 ml.
Bouin’s solution contains picric acid. This is an
excellent fixative for glycogen. It reacts with protein
and forms protein picrate. The tissue penetration 1.10.2 Helly’s Fluid
rate of picric acid is high, and it fixes small tissue
biopsies within 3 to 4 h. Bouin’s fixative is not suit- This is an excellent cytoplasmic fixative. It takes
able for DNA quantitative study as it damages the about 12 h for 3 mm tissue to fix.
cell membrane and causes hydrolysis of nuclei acid. Preparation
Advantages • Solution A
1. It is a good fixative for connective tissue and –– Distilled water: 250.
glycogen. –– Potassium dichromate: 6.3 g.
2. Rapid penetration rate. –– Mercuric chloride: 12.5 g.
–– Sodium sulphate: 2.5 g.
Disadvantages • Solution B
1. It produces a yellow stain on the tissue –– 37% Formaldehyde solution
Removal of yellow colour –– Before use, mix 95 ml of Solution A with
1. The tissue should be washed thoroughly in 5 ml of solution B.
70% ethanol.
2. This yellow colour can be removed by dip-
ping the tissue in lithium carbonate in 70% 1.10.3 B5 Fixatives
alcohol.
• Stock A solution
Bouin’s solution preparation –– Mercuric chloride: 12 g.
1. Picric acid solution (1% in distilled water): –– Sodium acetate: 2.5 g.
15 ml. –– Distilled water: 200 ml.
1.10 Mercury Salt-containing Fixatives 15
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 laboratory
• Sodium dihydrogen • Cell morphology mucopolysaccharides staining
phosphate well preserved • dark brown granules in
• Disodium hydrogen • Cheap vascular tissue
phosphate • Stable
Glutaraldehyde • Glutaraldehyde • Better fixation of • Poor penetration in • Best for electron
• Phosphate buffer ultrastructure tissue microscopy
• Less cell • Less stable
shrinkage • No lipid fixation
• Protein • Costly
preservation
better
• Less irritating
Osmium • 2-4% Osmium • Good fixative for • Does not fix the proteins • Good for electron
tetroxide tetroxide in buffer lipid and carbohydrates microscopy
solution • Good for Golgi • Cause clumping of
bodies and DNA.
mitochondria • Toxic and volatilizes at
room temperature
• Costly
Ethyl alcohol • Absolute alcohol • Fast penetration • Inflammable • Good for cytology
• Water • Needs license smear
Bouin’s fixative • Picric acid - • Rapid penetration • Produces yellow stain • Good fixative for
Formaldehyde rate on the tissue connective tissue
• Glacial acetic acid • Very good for and glycogen
trichrome stain
Zenker’s fluid • Mercuric chloride • Rapidly acting • Pigments of dichromate • Organ with very
• Glacial acetic acid • Even penetration and Mercury high vascularity
• Potassium • Mercury is poisonous such as the spleen
dichromate • RBC is poorly preserved
• Distilled water
• Stock B solution. Table 1.3 Choice of fixative based on technique

–– 37% formaldehyde solution. Technique Fixative of choice
–– Before use, mix 20 ml stock solution A Routine histopathology 10% neutral buffered
with 2 ml stock B. formalin
Electron microscopy Glutaraldehyde solution or
Table 1.2 highlights the comparison of different osmium tetroxide
fixatives. Immunohistochemistry 10% neutral buffered
formalin, Alcoholic formalin
Immunofluorescence Unfixed cryostat
1.10.4 Fixatives of Choice Enzyme histochemistry Fresh frozen section
The choice of fixatives is very important for spe-

cific purposes. In the case of routine histopathol- The different types of fixative are used accord-
ogy tissue, the best fixative is 10% neutral buffered ing to the tissue have been highlighted in
formalin. Similarly, the fixative of choice for elec- Table 1.4:
tron microscopy and immunocytochemistry is Fixative of choice may be different according
Glutaraldehyde solution and 10% neutral buffered to the chemical compounds to demonstrate
formalin, respectively. Table 1.3 highlights the (Table 1.5).
fixative of choice for different techniques.
Table 1.4 Fixative of choice according to tissue

Tissue Fixative Time Box 1.5 Fixation Artifact
Day to day sample 10% buffered Small tissue: • Formalin pigment: Insoluble brownish
(routine) formalin 6h black granular retractile birefringent
Large tissue: pigment due to reaction of formalin with
12 to 24 h
haemoglobin derivatives
Lymph node B5 solution 18 h
Gastrointestinal 10% buffered 6h • Mercury pigments: Dark brown irregu-
tract formalin lar deposit.
Testis 10% buffered 6h • Fuzzy staining: Due to improper
formalin fixation
Or Bouin’s
• Prolonged fixation: Shrinkage of the
fluid
Bone marrow Bouin’s fluid 3h tissue causes tissue separation and
Spleen Zenker’s fluid 6h empty spaces
Eye 10% buffered 48 h • Dichromate deposit: This deposit may
formalin occur after dichromate fixation if the tis-
Brain Formol saline Week sue is not washed properly
Kidney biopsy Neutral 6h
buffered
formalin
Uterus Neutral 24 h 1.11.1.1 Removing the Pigment
buffered
formalin Picric acid Method
Steps
Table 1.5 Fixative of choice for different substances 1. The sections is immersed in xylene followed
Target substance Fixative of choice by alcohol to bring in water.
Protein 10% buffered formalin 2. Subsequently the section is immersed in 1.8%
Lipid Frozen section or Osmium picric acid in absolute ethyl alcohol for
tetroxide
15 min.
Glycogen Alcohol-based fixative
Mucopolysaccharide Frozen section 3. It is then washed thoroughly.
Enzyme Frozen section 4. Section is re-stained.
DNA and RNA Alcohol-based fixative
Iron Alcohol based fixative Schridde’s Method
1. The sections is immersed in xylene followed
by alcohol to bring in water.
1.11 Fixation Artifact 2. Keep the section for 30 min in 100 ml of 75%
alcohol and 0.5 ml of 25% liquor ammonia.
The following fixation artifact may be encoun- 3. Wash thoroughly.
tered in routine laboratory fixation (Box 1.5):
Verocay”s Method
1. The sections is immersed in xylene followed
1.11.1 Formalin Pigment by alcohol to bring in water.
2. Keep the section for 10 min in 100 ml of 80%
Unbuffered formalin is kept for long time is con- alcohol and 1 ml of aqueous potassium
verted into formic acid that reacts with haemo- hydroxide.
globin derivatives of the blood and produces acid 3. Wash thoroughly.
formaldehyde hematin which is an insoluble
brownish black granular refractile birefringent
pigment (Fig. 1.5 and Box 1.6).
1.11 Fixation Artifact 17
Fig. 1.5 Microphotograph

shows brownish-black
granular formalin pigment.
This is refractile
birefringent pigment.
(Hematoxylin and Eosin
stain ×400)
Box 1.6 Formalin Pigment Fuzzy Staining

• Colour: brownish black Appearance: Here the nuclear and cytoplasmic
• Nature: Granular birefringent refractile details are obscured and the section looks fuzzy
• Position: Extracellular or hazy.
• Mechanism of formation: Formic acid Cause: Improper fixation either due to insuf-
reacts with haemoglobin derivatives of ficient fixative or too little time in fixative.
the blood and produces acid formalde-
hyde hematin. Prolonged Fixation
• How to avoid: Use buffered formalin Prolonged fixation cause shrinkage of the tissue
• How to remove: Treat with 1.8% picric followed by separation. The tissue may show
acid in absolute ethyl alcohol for 15 min. holes or empty spaces within it (Fig. 1.6).
Dichromate Deposit
If the tissue is not properly washed after dichro-
Mercury Pigments mate fixation, then the chromium salt may form.
When tissue is fixed by mercuric chloride, it pro- This chrome salt reacts with alcohol and insolu-
duces a dark brown irregular deposit. ble yellow-brown precipitate may appear.
Location: Throughout the tissue. Removal: By 1% hydrochloric acid in 70%
Removal: The application of iodine converts it alcohol for 30 min.
into mercuric iodide which is removed by sodium
thiosulphate.
Fig. 1.6 Tissue

separation due to
prolonged fixation. The
tissue shows holes and
empty spaces within it.
(Hematoxylin and Eosin
stain ×200)
1.11.2 Troubleshooting in Fixation is

Highlighted in Table 1.6
Table 1.6 Troubleshooting in fixation

Problems Cause Remedies
Nuclear margin is Incomplete fixation • Check the concentration of formalin
indistinct and nuclei are • Keep more time in formalin for fixation
fuzzy with bubbling • Cut thin section for fixation
• Do not put too many cassettes together
Tissue shrinkage with • Poor fixation • Proper fixation time
large artefactual spaces • Prolonged fixation • Check the fixative concentration
• Immediately fix the tissue after biopsy
Insoluble brownish-black Formalin pigment due to acid Use buffered formalin
granular pigment formaldehyde hematin formation by
reaction with blood
Intraepithelial cleft Formalin may evaporate, and calcium Keep formalin in a closely capped bottle
formation carbonate is precipitated
References 3. Jonsson N, Lagerst TS. The effect of formalde-

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):181–20.
tetroxide or formaldehyde and during subsequent
treatment. Exp Cell Res. 1957;12:342–55.
Processing of Tissue
in the Histopathology Laboratory 2
The next step after fixation is the processing of Water molcule

tissue. This is also a crucial step because poor tis- Dehydration is removed
sue processing may significantly affect the sec- from tissue
tion cutting and staining.
Aims of tissue processing: The basic aim of Dehydrating
tissue processing is to provide sufficient rigidity Clearing agent is
replaced by
to the tissue to be cut into thin sections for micro- clearing agent
scopical examination.
Principle of processing: In tissue processing, Tissue is
the water within the tissue is removed, and Impregnation infiltrated with
another medium (usually paraffin wax) is impreg- a supporting
medium
nated in the tissue that provides adequate support
to the tissue. Therefore, the essential steps in tis-
Fig. 2.1 Schematic diagram showing overview of pro-
sue processing (Fig. 2.1): cessing. The basic three steps are dehydrating, clearing
and embedding. Removal of water from the tissue is
1. Dehydration: In this step, water is removed 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
stage, tissue is impregnated with an embedding medium
and therefore, to infiltrate the tissue with wax,
it is necessary to remove water.
2. Clearing: This is needed to clear the dehy- 2.1 Factors that Influence Tissue
drating agent and facilitate the transition of Processing
the dehydration and impregnation stage.
The clearing substance is usually miscible The following factors influence the tissue pro-
to both dehydrating agent and impregnating cessing (Box 2.1):
medium.
3. Infiltration and impregnation: In this stage, 1. Size of the tissue sample: The optimum size
the tissue is infiltrated with a supporting of the tissue is very important for effective
medium suitable for providing adequate rigid- processing. The smaller the size of the tissue,
ity of the tissue to make a thin section. better the infiltration of the embedding
https://doi.org/10.1007/978-981-19-6616-3_2
20 2 Processing of Tissue in the Histopathology Laboratory
medium. The optimum thickness of the tissue

should be kept as 3 to 4 mm only. • Viscosity:
2. Agitation: The tissue gets better contact with –– Higher the viscosity of the medium
the surrounding medium if completely lower the penetration
immersed and gently agitated. The agitation • Negative pressure:
causes continuous removal of the fluid from –– Negative pressure removes trapped
the surface by a fresh medium. This has a air in the tissue
better effect on the action of the fluid on the –– Removal of clearing agent by
tissue. Most of the commercial tissue proces- increasing volatility
sors have the facility of agitation. It is impor-
tant to note that too much rapid agitation may
damage the soft and delicate tissue.
3. Heat: Heat increases the fluid penetration 2.2 Dehydration
rate within the tissue, whereas low tempera-
ture impedes the whole process. The present Every tissue contains some amount of free or
commercial tissue processors have the facil- unbound water molecules. As the commonly
ity to heat the tissue in all processing stages. used supporting medium (paraffin) is not mis-
Overheating may produce hard and brittle cible with water, it is necessary to remove the
tissue. free water molecule from the tissue for the suc-
4. Viscosity: The viscosity of the embedding cessful impregnation of the supporting medium
media also affects the processing. Higher vis- (Box 2.2). The sharp difference in the concen-
cosity of the medium lowers the penetration tration gradient between the tissues and the
rate in the tissue. Heat reduces the viscosity dehydrating fluid may cause a sudden rush of
of the medium and helps in better fluid and damage the delicate tissue. Therefore,
penetration. dehydration should be done gradually from low
5. Vacuum: Application of negative pressure to high concentration of dehydration fluid. The
facilitates tissue processing [1]. The vacuum tissue should be kept in the dehydration fluid for
helps to remove the entrapped air from the optimal time because too much time in the
tissue and thereby enhances the penetration dehydrating fluid may cause the tissue hard and
of fluid within the tissue. The negative pres- brittle. Too little time in dehydration fluid may
sure also increases the volatility of the clear- be insufficient to remove free water molecules.
ing agent and therefore helps to remove the Thin 2–3 mm tissue needs less time in dehydra-
fluid from the tissue. tion fluid than thick 5 mm tissue.
Box 2.1 Influencing Factors of Tissue 2.3 Individual Dehydrating

Processing Agent
• Size of the tissue:
–– Smaller the size, better the 2.3.1 Alcohol
processing
• Agitation: Ethanol: Ethanol or ethyl alcohol is the most
–– Agitation facilitates the contact of popular and commonly used dehydrating agent.
tissue with fresh solution This is a clear and colourless fluid. Ethyl alcohol
• Heat: is a flammable liquid. This is a relatively rapid
–– Increases the better penetration of and efficient dehydrating agent. However, it
fluid needs a licence from the government to purchase
ethyl alcohol for laboratory use.
2.3 Individual Dehydrating Agent 21
As a dehydrating agent, ethyl alcohol is used 2.3.2 Dehydrating Agents Other

in 50%, 70%, 90% and 100% concentrations. For than Alcohol
delicate tissue, the dehydration may be started
with a 30% concentration of ethyl alcohol. In the Dioxane: This is 1,4,diethylene dioxide. Dioxane
routine laboratory, 70%, 90% and 100% alcohol is miscible with both water and molten paraffin
for 2 h each are sufficient for tissue dehydration. wax. This is a rapidly acting dehydrating agent
If tissue is immersed in the ethyl alcohol for a and produces minimal shrinkage. Tissue can be
long time, then the removal of attached water kept in dioxane without any harm. Dioxane liber-
from the carbohydrate and protein molecules ates highly toxic gas, and proper ventilation is
causes hard and brittle tissue. mandatory for its use.
Anhydrous cupric sulphate in the final Ethylene glycol: It is also known as ethylene
container: Anhydrous cupric sulphate (CuSO4) glycol monoethyl ether or cellosolve. It is a
is a white powder that draws water from the colourless and odourless fluid. Cellosolve is a
alcohol and thereby helps in dehydration. About rapidly acting dehydrating, and tissue can be kept
1 cm layer of this powder is kept in the bottom in it for a long duration.
of the container. The cupric sulphate powder
should be covered with two to three layers of
filter paper to prevent any colouring of the tis- Box 2.2 Dehydration
sue. When the CuSO4 becomes hydrated, the • Removes free or unbound water mole-
colour of the powder changes to blue. This cule of the tissue as the supporting
gives a warning signal to change the alcohol medium (paraffin) is not miscible with
and the CuSO4 powder. water
Advantages • Sharp difference of concentration gradi-
ent of the dehydrating fluid may damage
• Increases the life span of alcohol the delicate tissue.
• Better dehydration • Gradual dehydration is necessary
• Good indicator to change alcohol • Too much time in the dehydrating fluid:
the tissue becomes hard and brittle.
Methylated spirit: It is also known as denatured • Routine laboratory: 70%, 90% and
alcohol. Methylated spirit contains 99% ethanol 100% alcohol for 2 h each
and 1% methanol or isopropyl alcohol. • Common dehydrating agents:
Methanol: Methanol is a clear, colourless, –– Ethyl alcohol
volatile and inflammable liquid. It can be used as –– Methylated spirit
a substitute for ethanol, but it is rarely used in the –– Methanol
laboratory because of its volatility and high cost. –– Butyl alcohol
Butyl alcohol: n-Butanol, isobutanol and ter- –– Isopropyl alcohol
tiary butanol are used as dehydrating agents both –– Dehydrating agents other than alco-
in animal tissue and plant histology processing. hol: dioxane, ethylene glycol,
Butyl alcohol is a slowly acting dehydrating acetone
agent and takes a longer time than ethyl alcohol
for dehydration. However, the tissue shrinkage is
less by butyl alcohol.
Isopropyl alcohol: Isopropyl alcohol is avail- Acetone: Acetone is a colourless and inflam-
able as isopropanol (99.8%). This is miscible mable liquid with a pungent ketonic smell. It is
with water and liquid paraffin. It is a relatively miscible with both water and alcohol. Acetone
rapid-acting, nontoxic dehydrating agent causing produces tissue shrinkage, and prolonged use
minimal tissue shrinkage. It is a suitable lipid dis- may cause brittleness of tissue. It is best used in
solving solvent. fatty tissue processing.
Table 2.1 comparison of different dehydrating agents

Dehydrating Box 2.3 Clearing
agents Advantages Disadvantages • Aims of clearing
Ethyl • Rapid and • Needs licence from –– Removal of dehydrating agent (e.g.
alcohol efficient the government
dehydrating • Inflammable alcohol) to facilitate impregnation of
agent • Hard and brittle paraffin wax
tissue if kept for a –– To make the tissue clear and improve
long time the microscopic examination
Methanol • Equally • Volatile
• Ideal clearing agent
effective as • High cost
ethanol –– Low viscosity and high penetration
Isopropyl • Relatively • Not possible to use rate
alcohol rapid action in celloidin –– Low melting point
• Nontoxic technique –– Miscible with both alcohol and mol-
• Minimal tissue
shrinkage ten wax
Dioxane • Rapid action • Highly toxic gas is –– No tissue damage
• No shrinkage generated –– Less toxic
of tissue –– Less inflammable
Ethylene • Rapid • Very expensive –– Cheap
glycol • No graded • Clearing agent is
solution is needed • Selection of appropriate clearing
needed agent: Type of tissue, type of processor,
• Tissue can be processing condition (such as heat, vac-
kept in it for a uum) safety factors, cost
long time
• Volume of clearing agent: 40 times the
Acetone • Rapid action • Quickly evaporates
• Cheaper than • Inflammable volume of the specimen
ethanol • Prolonged use may • Total duration:
• Good for fatty cause shrinkage –– Smaller biopsy: 1 h
tissue and brittleness of
–– Larger tissue: Three changes in
processing tissue
xylene or toluene 60 min each
• End point detection: Tissue becomes
The various dehydrating agents are compared transparent
in Table 2.1. • Prolonged exposure to clearing agent:
The brittle and more friable tissue
• Different clearing agents: xylene, tolu-
2.4 Clearing ene, chloroform, amyl nitrate, cedar-
wood oil, limonene
After removing the free water molecule from
the tissue, the next step of processing is to
remove the dehydrating agent itself because
many dehydrating agents are not miscible with Selection of appropriate clearing agent: This
the impregnating material (paraffin wax). The depends on:
clearing agent should be miscible with both the
dehydrating agent and the embedding medium. • Type of tissue: Large tissue takes more time
The refractive index of the clearing agent is sim- than smaller tissue
ilar to the tissue, and therefore it gives a clear • Type of processor: Manual versus automatic
appearance to the anhydrous tissue (Box 2.3). • Processing condition: Temperature, vacuum
So, the completely transparent tissue indicates • Speed of removal of dehydrating agent
the terminal point of the clearing process. Any • Ease of replacement by molten wax
opacity of tissue signifies incomplete • Safety factors: flammability, toxicity
dehydration. • Cost
2.5 Infiltration and Embedding 23
Table 2.2 Comparison of clearing agents

Properties Xylene Toluene Chloroform Esters
Tissue shrinkage Yes Yes Minimum No
Tissue hardening Yes No No No
Inflammable Yes Yes No Yes
Harmful effect Irritant but less Irritant Dangerous toxic gas liberated in Safe
harmful heating
Cost Cheap Cheap Very expensive High cost
The clearing agent with a low melting point is eas- less toxic and may be used in manual processing.
ily and quickly removed by the molten wax. In con- They do not cause tissue hardening even under
trast, clearing agent with a high melting point takes prolonged exposure.
time to be removed by embedding medium. A Cedarwood oil: This is an expensive rapid
clearing agent with high viscosity has a low pene- clearing agent and is mainly used in clearing
tration rate. Prolonged exposure of the tissue to a dense tissue.
clearing agent may make the tissue brittle and more Limonene: This is a clear liquid. It does not
friable. Therefore, the optimal time for clearing is cause any tissue hardening. However, the removal
necessary. The amount of clearing agent should be of limonene from the tissue by paraffin wax is
40 times the volume of tissue for clearing. difficult.
2.4.1 Individual Clearing Agent 2.5 Infiltration and Embedding
Xylene: This is the most commonly used clearing This is the next step after clearing. The clearing
agent in the laboratory. This is a clear and inflam- agent within the tissue is removed by the process of
mable liquid. The small pieces of tissue are diffusion. The tissue space is now infiltrated with
cleared rapidly by xylene within 30 to 60 min. the embedding media. Usually, molten wax is used
Prolonged exposure to xylene may make the tis- as the embedding medium. After cooling to room
sue hard and brittle. temperature, the molten wax is solidified to provide
Toluene: It has almost similar properties as support for cutting into thin sections (Box 2.4).
that of xylene. However, it does not make the tis- Ideal impregnating medium: An ideal
sue hard even after prolonged exposure, and its impregnating medium should have following
action is slightly slower than xylene. Toluene is qualities:
also flammable and toxic.
Chloroform: It is a highly volatile, non- • Miscible with clearing agent
inflammable, expensive and toxic agent. The • Liquid in higher temperature (30 to 60 °C) and
penetrating power of chloroform is slower than solid at room temperature
xylene. However, it does not cause any tissue • Homogenous and stable
shrinkage and is mainly used in the uterus, mus- • Non-toxic and cheap
cle and other dense tissue. Presently chloroform • Transparent
is rarely used in the laboratory. • Fit for sectioning the tissue
Table 2.2 compares the different clearing
agents commonly used in laboratories: The time duration and the number of changes
required for the impregnation in tissue depend on:
2.4.2 Other Clear Agents 1. Size of tissue: Thicker large tissue takes more
time to impregnate with the embedding
Esters: The different esters are amyl nitrate, medium. It also contains a more clearing
methyl salicylate and methyl benzoate. These are agent to remove.
2. Type of tissue: Hard tissue such as bone and 1. To increase hardness: Addition of stearic
cartilage takes more time for embedding than acid.
soft tissue. 2. Reduction of the melting point: Addition of
3. The type of clearing agent: Certain clearing phenanthrene.
agents are easy to remove than others. Such as 3. Improving adhesiveness with tissue and
xylene and toluene are easy to remove than wax: Addition of 0.5% of ceresin.
cedarwood oil. 4. Dimethyl sulphoxide (DMSO): The addition
4. Type of processing: Vacuum embedding of a small amount of DMSO in paraffin wax
enhances impregnation. reduces the infiltration time of the wax and
removes the residual clearing agent. It produces
a homogenous matrix and better support.
2.5.1 Different Impregnating Medium
2.5.1.1 Paraffin Wax 2.5.2 Vacuum Impregnation

Paraffin wax is a type of hydrocarbon produced as Method
a by-product during refining crude petroleum. This
is the most popular, universally accepted embed- Vacuum impregnation helps to impregnate the
ding medium for tissue processing. This is a non- molten medium in the tissue under decreased
toxic and inexpensive medium. The melting point pressure. This is a rapid method to impregnate
of paraffin wax varies from 39 ° C to 70 ° C. The wax within the tissue. As the time duration of the
wax is sold according to its melting point. Paraffin exposed tissue in the high temperature of the
wax with a low melting point is soft at room tem- molten wax is less in this procedure, so the
perature, whereas paraffin wax with higher melting chances of the hardening of the tissue are less.
point is much harder in consistency. Therefore, it is Moreover, there is total removal of the solvent
necessary to have paraffin wax with a suitable from the tissue, and the reduction of solvent con-
melting point to get a good ribbon of tissue. In this tamination of the wax helps to prolong the life
Indian subcontinent, the paraffin wax with melting span of the wax. The vacuum impregnation
point around 60 ° C is the most suitable for labora- method is applied in the processing of the lung
tory use. Total 3 to 4 h’ time in paraffin wax is suf- and spleen.
ficient for impregnation of tissue by wax.
2.5.1.2 Advantages of Paraffin Wax

Box 2.4 Impregnation of Embedding
• Tissue block can be stored for a long duration Medium
• Non-toxic Aims: To provide support to the tissue.
• Cheap Principle: Clearing agent is removed
• Safe by the process of diffusion and the tissue
space is now infiltrated with the embed-
2.5.1.3 Disadvantages of Paraffin Wax ding media.
Ideal impregnating medium:
• It may cause tissue shrinkage and hardening in
case of prolonged impregnation • Miscible with clearing agent
• Paraffin wax takes a long duration for the • Liquid in higher temperature and solid
impregnation of bone and eye. in room temperature
• Homogenous and stable
2.5.1.4 Additives and Modification • Non-toxic and cheap
of Paraffin Wax • Transparent
To alter the physical characteristics of paraffin • Fit for sectioning the tissue
wax, the following modifications may be done:
2.6 Tissue Processing Methods 25
Time duration and the number of

changes of embedding medium:
• Size of tissue: Large versus small

• Type of tissue: Hard versus soft
• The type of clearing agent: Cedar wood
oil takes longer time
• Type of processing: Vacuum processing
accelerates
Different embedding medium: Paraffin

wax, Dimethyl sulphoxide.
Fig. 2.2 Tissue transfer automatic tissue processor. Here
the whole bucket containing tissue is automatically trans-
ferred to the next fluid station
2.6 Tissue Processing Methods
Tissue processing can be done simply manually

or by an automated processor. Manual tissue pro-
cessing is done only in a small laboratory with a
handful of tissue. Automated tissue processor is
widely used in laboratories.
Automated tissue processors: The basic
principle of tissue processors is to transfer the tis-
sue in different fluids for a specified time in the
desired environment. There are two types of tis-
sue processors:
1. Tissue transfer processor.

2. Fluid transfer processor.
Fig. 2.3 Fluid transfer advanced automatic tissue proces-
1. Tissue transfer processor (Fig. 2.2): In this sor. Here the fluid itself has changed automatically, and
system, the bucket of tissue is transferred the bucket remains static
from one carousel to other after a specified
time. There are several containers with the container containing the tissue. It is again
reagents. Tissue remains in a basket with 30 to filled with the fluid required for the next step.
100 cassettes. The basket containing the tissue In this processor, each step can be customised
is submerged in the specific container for a for vacuum, temperature, and time duration.
particular time and then transferred to the next
container automatically. Gentle agitation is
created by vertical oscillation or rotatory 2.6.1 Advantages
movement of the tissue basket. The time
schedule and transfer of tissue in each con- 1. Vacuum pressure makes the system faster and
tainer are determined by a microprocessor. more efficient.
2. Fluid transfer processor (Fig. 2.3): This is a 2. In this closed system, there is no chance for
completely closed processor. Here the tissue tissue drying.
is kept in the container, and the container is
periodically filled with a particular fluid. After Table 2.3 highlights the comparison of the two
a certain period, the fluid is pumped out from types of tissue processors.
Table 2.3 The comparison of the two types of tissue Alcohol (graded)
processor 70%
50% 90%
Tissue transfer Fluid transfer Alcohol
Factors processor processor Paraffin (100%)
Method Tissue is Reagents are 100%
I Hr I Hr
transferred from pumped within I Hr
one bucket to the the tissue
other bucket cassettes I Hr
I Hr
Rapidity Relatively slow Fast
Dehydration
Embedding
Paraffin
Tissue drying Common problem No chance of 100%
is tissue drying tissue drying
during the transfer
of the tissue I Hr
I Hr
Flexibility to Yes. Flexible No flexibility
use different Paraffin Clearing
reagents 100%
Infiltration of Adequate Better Xylene
reagents infiltration of Xylene Xylene
within the the reagents
tissue I Hr I Hr
Fluid agitation It is done by Fluid agitation
vertical oscillation is done by the I Hr
or rotary tidal action I Hr I Hr
movement of the
tissue basket Fig. 2.4 Schematic diagram showing the time schedule
of overnight processing
2.7 Overall Precautions of Tissue

Processing 90% ethanol: 1 h
Absolute alcohol: 1 h
1. The bulk of the tissue should be optimum for Absolute alcohol: 1 h
adequate penetration of fluid. Absolute alcohol: 1 h
2. The amount of fluid should be adequate, and Xylene/Toluene: 1 h
the fluid level should always be higher than Xylene/Toluene: 1 h
the tissue level. Xylene/Toluene: 1 h
3. The tissue basket and cassettes should be Paraffin wax: 1 h
clean, and any spillage of wax should be Paraffin wax: 1 h
cleaned. Paraffin wax: 1 h
4. The temperature of the infiltrating medium
should be optimum, and it is preferable to
keep the temperature 3 to 4 ° C above the 2.7.2 Manual Tissue Processor
melting point.
5. There should be a proper record of the change It is rarely used in the routine laboratory. The
of fluid, number of tissues processed etc. advantage of manual processing are:
1. A small number of samples can be processed

2.7.1 Time schedule for overnight in a small laboratory.
processing (Fig. 2.4) 2. Careful monitoring in each step is possible.
3. In case of emergency, when the automated tis-
50% ethanol: 1 h sue processor is not working, one can take the
70% ethanol: 1 h help of manual processing.
2.7 Overall Precautions of Tissue Processing 27
4. In the case of manual processing, it is possible sections as conventional tissue processing [3].
to select the reagents of choice with flexibility Both Immunohistochemistry and molecular test-
in time duration. ing can be done on the rapid processing sections.
5. The major disadvantages of manual process-
ing include inconvenience for processing and 2.7.3.1 Advantages
time taken procedure.
• Rapid
• Economical
2.7.3 Rapid tissue Processing • Equally good quality tissue sections
• No night shift for the technologists
Currently, rapid tissue processing has been intro-
duced in many laboratories [2]. In the rapid tissue 2.7.3.2 Limitations
processing system, the entire processing takes
only 2–3 h instead of 8 h duration. Here low • Very costly
voltage microwave technology is used for pro- • Large quantity of cases are needed for effec-
cessing (Fig. 2.5). To get a consistent superior tive use in the laboratory
quality result, the traditional vacuum infiltration • Fatty tissue can be processed rapidly
technology is used. Formalin free and xylene-free
ready to use reagents are used in the rapid pro- Tissue processing for electron microscopy: See
cessing system. Overall, 120 cassettes can be pro- Chap. 26.
cessed in 1 h, so the system is high-speed, Troubleshooting in processing is highlighted
convenient, and economical. Microwave-based in Table 2.4.
rapid tissue processing provides identical tissue
Fig. 2.5 Rapid tissue processor: The machine processes the tissue within 3 h
Table 2.4 Troubleshooting in processing

Problem Cause Remedies
Tissue is soft during embedding 1. Tissue is too thick during 1. Tissue should be properly cut
grossing 2. Change the reagents
2. Reagents are saturated with 3. Change paraffin
water
3. Paraffin is saturated with
clearing agent e.g. Xylene
Tissue is coming out from the Dehydration process is inadequate Change the processing program and
block during sectioning so there is defective paraffin give adequate time for dehydration
infiltration in the tissue
Micro chattering effect around the Excessive dehydration Change the dehydration time. It is
edges of the tissue section better to process the smaller biopsy
tissue separately from the larger tissue
to have proper dehydration
Cracked and folded tissue section Excessive dehydration The block is soaked with a wet gauze
piece before sectioning
Irregular staining of haematoxylin Dehydration process is Change the processing program and
and eosin stain suboptimum give adequate time for dehydration
Brittle tissue after processing Excessive blood in tissue Apply a gauze piece soaked with warm
water on the surface of the block
before sectioning
ing of tissue for histopathology. Med J Armed Forces

References India. 1996;52(3):157–60.
3. Priya AHH, Venkatanarasu VB, Chellaswamy S,
1. Brain EB. Infiltration histological specimens with Jeyaraj M, Francis SF, Rajasekaran S. Evaluation
paraffin wax under vacuum. Basic factors and a new of efficacy of microwave staining over conventional
approach. Br Dent J. 1970;128(2):71–8. staining in replicating tissue architecture: a prospec-
2. Sivadas P, Kumar H, Lakshmanan C, Bhardwaj tive study. J Pharm Bioallied Sci. 2020;12(Suppl
JR. Microwave stimulated fixation and rapid process- 1):S283–8.
Embedding of Tissue
in Histopathology 3
After processing the tissue, the next step is 3.1 Embedding Medium
embedding the tissue to make the block. In the
embedding process, the tissue is surrounded by a 1. Paraffin wax: As described in the previous
molten medium using a mould. Subsequently, chapter, paraffin wax is a solid polycrystalline
this medium is solidified to make a block for cut- hydrocarbon. The paraffin wax is sold in the
ting a thin section of tissue. market with a different melting point. Paraffin
Aims of embedding: The embedding medium wax with melting points ranging from 56 to
has three crucial functions: 62 °C is used in our laboratory. Paraffin wax is
cheaper and easy to use. Little supervision is
1. To give support to the tissue needed to make block by it.
2. To prevent distortion of the tissue during 2. Epoxy resin: Epoxy resin is mainly used in
cutting electron microscopy as it provides better reso-
3. To preserve the tissue for archival use. lution and more excellent tissue details.
3. Acrylic medium: Methacrylate monomer is
The choice of the embedding medium: Various miscible with ethanol. In the presence of a cata-
media are used for embedding, such as paraffin lyst (benzoyl peroxide 2%), methacrylate
wax, epoxy resin, methacrylate, carbowax etc. monomer is polymerised and provides a hard
Paraffin wax is the most commonly used embed- and clear block. Methacrylate monomer is
ding medium. The choice of the embedding available in the market along with hydroqui-
medium depends on the following factors: none which should be removed by treating with
a weak alkali solution followed by thoroughly
1. Type of tissue: The density of the tissue and washing with water. The presence of water may
the embedding medium should be close. lead to small bubbles within the block.
Otherwise, tissue may not be adequately sec- 4. Agar gel: Agar gel helps in the cohesion of
tioned, and tissue will be deformed. friable and fragmented tissue, particularly in
2. Type of microtome. cytology samples and endometrial curetting
3. Type of microscope. and small endoscopic biopsies. It does not
provide good support of the issue for section
The basic technique of embedding is the same cutting. Agar –paraffin wax double embed-
irrespective of the embedding medium. ding is a more suitable technique than agar
alone.
https://doi.org/10.1007/978-981-19-6616-3_3
30 3 Embedding of Tissue in Histopathology
5. Gelatin: It is also used in small friable tissues The two L shaped arms are adjusted to make a
and frozen sections containing friable and convenient size for the block. An adequate
necrotic tissue. The melting point of gelatin is lubricant such as glycerine is applied to the L
35 to 40 °C, and this low melting point makes arms and metal plate for easy removal of the
it unsuitable for embedding. tissue. The molten wax is poured into the space
6. Celloidin medium: Celloidin is nitrocellu- between two L arms, and then the tissue is
lose and was mainly used for embedding hard placed within the bottom of the liquid wax. The
tissue. Nowadays, it is not used in the wax is subsequently cooled, and the block with
laboratory. tissue on one surface is removed for
microtomy.
Stainless still mould: Here, the mould is
3.2 Different Types of Mould made of stainless steel (Fig. 3.2). The base of the
Used for Block mould is flat and well-polished, which helps to
remove liquid paraffin. The mould can be cov-
Leuckhard embedding moulds (Fig. 3.1): ered by a plastic ring.
Leuckhard embedding moulds have two arms. Plastic mould: Here, the mould is made of
One arm of the L is longer than the other arm. chemical-resistant plastic (Fig. 3.3).
Fig. 3.1 L shaped

Leuckhard embedding
moulds
L shaped metal
plate
L shaped metal
plates Joined
together
Tissue
Paraffin wax
3.3 Tissue Embedding Method 31
3.3 Tissue Embedding Method
The tissue embedding method is essentially the

same for all types of embedding media.
The following things are needed for tissue
embedding:
1. Molten paraffin wax.

2. Mould with cover.
3. Metal plate (cold plate) to work.
4. Nowadays, there is a commercially available
Fig. 3.2 Stainless still mould system with different components together
[1]. The tissue tek system is the combination
of (Fig. 3.4):
(a) Dispenser of liquid paraffin at a constant
temperature,
(b) A metal plate to make the tissue block.
(c) Cold plate.
Tissue Tek system I: The steps of this system

are (Fig. 3.5):
• Liquid paraffin is kept at a constant tempera-

ture in the dispenser of the system.
• The tissue is put on the lower surface of the
mould by forceps. The cutting surface should
be faced down.
• Molten paraffin is poured on the metallic
Fig. 3.3 Chemical resistant plastic mould mould.
Wax dispenser
Forceps warmer
Cold plate Tissue warmer

Hot plate
Fig. 3.4 Tissue Tek system consisting of a hot plate, tissue warmer and cold plate
Molten paraffin poured on Tissue embedded in

Tissue trek system Tissue taken out mould paraffin
The mold is covered with Unique number is put in The moulds are put
Tissue is pressed on mould peripheral plastic ring the Plastic ring on the cold plate
Fig. 3.5 Illustrated view of the whole embedding pro- the molten paraffin. The mould is now covered with the
cess. At first, the tissue tek system is put on. The tissue is peripheral plastic ring. A unique number is placed in the
taken out from the processing bucket. The molten paraffin plastic ring, and the mould is now kept on the cold plate to
is poured into the metal mould. The tissue is embedded make it firm
with the help of forceps. The tissue is pressed to keep in
• The mould is covered with a peripheral plastic 3.3.1.1 Method

ring on the upper surface. Agar and Paraffin
• Unique tissue number is put in one corner.
• The whole mould is put on the cold plate. • Take the sample in a filter paper
• The metallic mould is detached from the plastic • Pour molten agar
ring, and the block is used for cutting tissue. • Give time to solidification
• Trim the margin
Tissue Tek system II: The basic steps of tissue • The agar block is now processed
Tek II are similar to those described above. • Embed the agar block in the paraffin wax
Instead of a metallic mould, we use a plastic
mould to hold the liquid paraffin in this system.
3.3.2 Nitrocellulose and Paraffin [2]
3.3.1 Double Embedding Method This technique is suitable for brain tissue.
The double embedding method is a special type 3.3.2.1 Method

of embedding where the tissue is embedded
twice, once by agar or nitrocellulose and the sec- • Fix, wash and dehydrate the tissue
ond by paraffin wax. • Treat the tissue with 0.25. 0.5, 1 and 1.5%
Indications: The double embedding method nitrocellulose in methyl
helps to prevent the loss of small tissues. It is spe- • Transfer the tissue to toluene
cially done in endoscopic biopsies and curettage • Impregnate the tissue with paraffin
samples.
3.4 Tissue marking 33
Fig. 3.6 Illustrated

view of embedding of
the different tissue
sample
Tubular tissue: Endometrial

Skin: all layers
all layers in curetting: Keep
should come
transvers in center
section
Long tissue: keep Intestine: all layer Membrane: Swiss

diagonally should come role
3.3.3 Tissue Orientation 3. Multiple sections of tissue, such as endome-

and Embedding trial curetting, should be placed all in a central
position.
The correct orientation of the tissue is very 4. Linear long tissue should be placed diagonally.
important for proper cutting and microscopic 5. Muscle biopsy should be placed in the longi-
examination. Tissue is usually placed as flat on tudinal and transverse planes.
the central part of the mould. It should be ori- 6. Long, membranous tissue such as an amniotic
ented in such a way so that cutting is easy by the membrane should make a Swiss roll.
knife of the microtome. Some of the tissue needs
special care as described below (Fig. 3.6):
3.4 Tissue marking [3]
1. The tubular tissue (fallopian tube, vas differ-
ence, artery etc.) should be placed in such a The tissue marking by ink is needed for the fol-
manner so that we get a transverse section lowing purposes:
with all the layers.
2. Tissue with epithelial surface should be 1. To identify the resection plane or outer margin
placed vertically and at the right angle to the of the tissue
surface to get all the layers. 2. To help in embedding the tissue
3. Any area of interest to identify, such as the Table 3.1 Troubleshooting in tissue embedding
area of transitional zone in cone biopsy of the Problem Cause Remedies
cervix. Fraction of the Tissues are Give pressure after
The tissue markers should have the follow- embedded embedded on embedding the
tissues is a different tissue in the
ing characteristics and features: cutting level. molten wax in the
mould
• The marker substance should not be dissolved Tiny fragments Tissue is • Clean the
in fixative and tissue processing agents. of tissue are carried over forceps every
seen in the by forceps time after
• The marker should not penetrate the deeper
subsequent embedding
tissue blocks • Deal with only
• It should be recognisable in the stained section one tissue at a
both microscopically and macroscopically. time and so
open only one
• The common tissue markers: The common
cassette at a
tissue markers include time
• India ink: This is the most commonly used Epithelium not Wrong Please mark the
marker in the routine surgical pathology labo- properly seen orientation tissue with ink so
ratory. It takes 15 min to mark the tissue. that the embedding
upper surface can
• Silver nitrate: This is also a good marker. It be identified
produces brown, black colour. Tissue is fallen Air bubbles Properly embed
• Rose Bengal: 1% rose Bengal dye is used for out during are entrapped the tissue in the
surgical margin stain. It stains within 5 min microtomy around the molten wax
tissue
and provides a pink colour.
Application of Ink
• Clean the area and dry the tissue with bloating
paper. Completely dry the tissue
References
• Apply the dye with a cotton swab 1. Mccormick JB. Improved tissue-embedding method
• Allow some time to dry it for paraffin and carbowax, using Tissue-Tek system.
• Put fixer over the dye. Usually, 3% acetic acid Tech Bull Regist Med Technol. 1959;29(1):15–6.
or 50% white vinegar is used as a fixer. 2. Molnar LM. Double embedding with nitrocellulose
and paraffin. Stain Technol. 1974;49(5):311.
• Dry the specimen with a sponge 3. Dimenstein IB. Grossing biopsies: an introduction to
• Process now general principles and techniques. Ann Diagn Pathol.
2009;13(2):106–13.
Troubleshooting in tissue embedding is high-
lighted in Table 3.1.
Decalcification of Bony and Hard
Tissue for Histopathology 4
Processing
4.1 Introduction Box 4.1 Decalcification

Aim: Removal of calcium salt from tissue
The presence of calcium salt in the tissues makes without damaging the morphology of the
them very firm to hard, which may damage the tissue.
knife. Therefore, it is often necessary to remove Calcium containing tissue: (1) bone, (2)
calcium salt from the tissue and make it soft for tooth, (3) pathological calcification in tissue.
cutting in a microtome. The process of removing Requisites for successful
calcium salt from the tissue is known as decalcifi- decalcification:
cation (Box 4.1). • Small tissue
Aim: The basic aims of decalcification are: • Adequate fixation
1. Removal of calcium salt from tissue. • Consistency
2. No damage to tissue morphology. • Adequate volume of decalcifying agent
3. No significant effect in staining. • Suitable choice of the decalcifying agent
• Exact end point detection
Calcium containing tissue: The tissue contain- Factors controlling the rate of
ing a heavy amount of calcium salts are: (1) bone, decalcification:
(2) tooth, (3) pathological tissue such as tubercu- Facilitates decalcification
lous lymph node, dystrophic calcification, certain
tumours such as teratomas etc. • Higher concentration of the decalcify-
Requisites for successful decalcification: ing agent
Following measures are helpful for successful • Higher temperature
decalcification: • Agitation of the solution
• Thin tissue
• Consistency: Exact assessment of the consis- • Low density
tency of the tissue is required for successful Methods of decalcification
decalcification. • Acid decalcification
• Small pieces: The tissue should be cut into 2 • Ion exchange resin
to 6 mm thick sections because thicker tissue • Electrical ionization
may take a longer time to be decalcified. • Chelating solution
• Fixation: Adequate fixation of the tissue is • Surface decalcification
necessary for proper decalcification. End point determination of
• Washing: The fixed tissue should be washed decalcification
thoroughly before decalcification.
https://doi.org/10.1007/978-981-19-6616-3_4
36 4 Decalcification of Bony and Hard Tissue for Histopathology Processing
Weak acids:
• Radiographic examination (a) Formic acid
• Chemical test (b) Trichloroacetic acid
• Physical test
Strong acid: The strong acids are used in 5 to
10% concentration. They are rapid in action.
• Choice of decalcifying agent: Suitable choice However, careful attention is needed to prevent
of the decalcifying agent is required. tissue damage. Neutraliser is also used in order to
• Volume: Optimum volume of the decalcifying avoid any tissue distortion.
agent is a prerequisite for proper decalcification. Aqueous Nitric acid: This is rapid in action.
• Endpoint detection: The endpoint of the It does not impair staining if the endpoint is not
decalcification should be determined correctly. crossed.
Preparation
• Nitric acid: 5 ml
4.2 Factors Controlling the Rate
• Distilled water: 100 ml
of Decalcification
Advantages: (1) Rapid in action, and (2) good
• Concentration: The increased concentration
nuclear stain.
of the decalcifying agent increases the rate of
Precaution: Nitric acid may give yellow
decalcification.
colour to the tissue that can be removed by
• Temperature: Increased temperature fastens
urea.
the decalcification rate.
• Density of bone: Hard bone takes a longer Nitric acid formaldehyde (10%)
time to be decalcified. • Nitric acid: 10 ml
• Agitation: Mild agitation of the decalcifying • Formalin: 10 ml
solution increases the rate. • Distilled water: 80 ml
• Thickness of tissue: Thinner tissue is quickly Advantages
decalcified. • Rapid action, (2) Good nuclear stain, (3) Less
chance of tissue damage and swelling, 4)
Long time washing by water is not needed.
4.3 The Methods
of Decalcification [1]
4.3.2 Von Ebner’s Fluid
1. Acid decalcification.
2. Chelating solution. A saturated solution of Sodium chloride: 175 g.
3. Ion exchange resin. Hydrochloric acid (concentrated): 15 ml.
4. Electrical ionisation. Distilled water: Make it up to 1000 ml.
5. Surface decalcification.
Advantages: (1) Rapid action, (2) Ideal decalci-
fying agent for tooth,
4.3.1 Acid Decalcification Disadvantages: (1) Nuclear staining is not
good.
This is the commonest method of decalcification
in the routine laboratory process. Acid makes the
soluble calcium salt, thereby removing calcium 4.3.3 Perenyi’s fluid
from the tissue.
The strong acids: Nitric acid (10%): 40 ml
(a) Hydrochloric acid Chromic acid (0.5%): 30 ml
(b) Nitric acid Absolute alcohol: 30 ml
4.3 The Methods of Decalcification 37
Advantages: (1) Provides excellent results, (2)

Softens the fibrous tissue, (3) Cellular morphol- Box 4.2 Ethylenediaminetetraacetic Acid
ogy well preserved. (EDTA)
Disadvantages: (1) Slower in action, (2) • Most commonly used
Endpoint detection is difficult. • Chelating agents
• Mode of action: Binds with calcium of
the hydroxy-apatite crystals to form a
4.3.4 Weak Acids non-ionized soluble complex
• Slow and gentle in action
Gooding and Stewart solution
Formic acid 5 ml Advantages
Formalin (40% formaldehyde) 5 ml • Morphological preservation of tissue
Distilled water 90 ml. • Suitable to do various other laboratory
tests
Advantages: (1) Decalcifying agent of choice • Very good for bone marrow trephine
in routine laboratory process, biopsy
Disadvantage: (1) Slow acting and takes
many days for decalcification. Disadvantages
Increased concentration of formic acid may • Very slow process
enhance its capacity. • Maintenance of pH around 7 is
necessary
• Thin tissue is needed
4.3.5 Trichloroacetic acid
10% Formal saline: 95 ml. of EDTA is preserving morphology and main-

Tricloroacitic acid: 5 g. taining the tissue for various other techniques for
Advantages: (1) Good for small biopsies, and (2) research purposes. The action of EDTA is pH-
good nuclear stain. dependent, and it works best in pH 7 to 7.6.
Disadvantages: (1) Not suitable for hard bony
tissue (2) Slower in action and takes 4 to 4.3.6.1 EDTA Solution
5 days. EDTA: 5.5 g
Table 4.1 highlights the advantages and disad- Formalin: 100 ml
vantages of various acid decalcifiers. Distilled water: 900 ml
4.3.6.2 Advantages
4.3.6 Chelating Agents 1. It gives the best morphological preservation
of tissue.
Chelating agents are organic substances that 2. Various other laboratory tests can be done on
adsorb metals. Ethylenediaminetetraacetic the tissue, such as immunohistochemistry,
acid (EDTA): EDTA is the most common chelat- fluorescent in situ hybridisation technique,
ing agent in routine laboratory decalcification etc.
(Box 4.2). It binds with the calcium of the 3. It is perfect for bone marrow trephine biopsy
hydroxy-apatite crystals and forms a non-ionized as glycogen is preserved in the tissue.
soluble complex. The action of EDTA is slow and
gentle, and it may take several weeks to remove 4.3.6.3 Disadvantages
calcium from the tissue. Therefore, EDTA is not 1. Prolonged process.
a suitable decalcification agent for dense bone or 2. Maintenance of pH around 7 is necessary.
urgent removal of calcium. The main advantage 3. Thin tissue is needed.
Table 4.1 Acid decalcifying agents

Chemicals Concentration Advantages Disadvantages
Aqueous Nitric 5% (1) Rapid in action, (2) good nuclear stain It gives the yellow colour to the
acid tissue
Nitric acid 10% (1) Rapid action: 1 to 3 days, (2) less Not a very good nuclear stain
formaldehyde chance of tissue damage and swelling, (3)
long time washing by water is not needed
Hydrochloric 8% (1) Rapid action, (2) ideal decalcifying Nuclear staining is not very
acid agent for tooth, good
Trichloroacetic 5 g in 95 ml (1) Good for small biopsies, (2) good (1) Not suitable for hard bony
acid formal saline nuclear stain tissue, (2) slower in action and
takes 4 to 5 days.
Formic acid 5% Decalcifying agent of choice in routine (1) Slow acting and takes many
laboratory process, days for decalcification.
4.4 Ion Exchange Resin Method current is passed from the tissue to the solution to
the cathode (made of carbon). The Calcium from
In this technique, an ion exchange resin (sulfo- the tissue moves to the cathode plate (Fig. 4.1).
nated polystyrene resin) is used along with an The electrolytic solution contains:
organic acid as decalcifying fluid [2]. The cationic
ion exchange resin replaces the Ca+ ion with H+ in • Distilled water: 820 ml
a weakly acidic solution. The resin is kept in the • Formic acid (88%): 80 ml
lower part of the container about 1 cm in-depth, • Hydrochloric acid: 100 ml
and the specimen is kept over the resin. The speci-
men is submerged under the weak acidic solution. Advantages: This is a very rapid decalcification
method and takes only 5 to 6 h to decalcify the
bone.
4.4.1 Advantages Limitation:
• The heat is generated at the time of decalcifi-
1. It produces faster decalcification. cation due to electric current. The heat can
2. The preservation of morphological details of damage the delicate tissue, and the cellular
the tissue is possible as the weak acid is used. architecture may be destroyed.
Regeneration of resin activity: After • Only one tissue can be processed at one time.
three times use, the resin activity is dimin-
ished significantly. The resin is regenerated by
washing it in 10 N HCl twice. 4.4.3 Surface Decalcification
In the case of surface decalcification, the surface

4.4.2 Electrolysis Method layer of paraffin blocks is inverted in 1% hydro-
chloric acid (HCl) for 1 h. The exposed top
In this process electrolysis of the tissue is done in 30-micron tissue of the paraffin block is decalci-
a solution of hydrochloric acid and formic acid fied. The block should be washed thoroughly
[3]. The tissue is attached to an anode made of before cutting. Only the first few paraffin sec-
platinum (positively charged), and an electric tions are expected to be free from calcium.
4.6 Results of Under Decalcification 39
Fig. 4.1 Schematic

diagram showing the
Electrolysis method of
decalcification of bone Platinum
Carbon
cathode anode
Power unit
Bone
4.5 Endpoint Determination Before using an equal volume of both, the

of Decalcification stock solution is mixed.
Method
The endpoint of decalcification can be detected by: –– Now, for the chemical test, 5 ml of the
1. Radiographic examination decalcifying agent from the container con-
2. Microradiography taining the tissue is withdrawn.
3. Chemical test –– Mix the decalcifying agent with 5 ml of
4. Physical test Ammonium hydroxide and Ammonium
oxalate mixture solution.
1. Radiographic examination: X-ray examina- –– The mixture is kept overnight.
tion of the tissue is the most accurate tech- –– Any precipitation is noted.
nique to detect the endpoint of decalcification. –– The presence of precipitation (calcium
However, this is a costly procedure, and the oxalate) indicates that the decalcifying
pre-decalcification radiograph is also needed agent contains calcium, and decalcification
to assess the extent of decalcification. is not completed.
2. Microradiography: Microradiography is a 4. Physical test: This is a crude test, and it does
high-resolution radiographical technique that not accurately detect the endpoint of decalci-
identifies bone mineral density and fication. The tissue is bent, or a pin is intro-
distribution. The less-dense non-mineral areas duced within the tissue. In case of adequate
are yellowish-grey to black. decalcification, it is expected that the tissue
3. Chemical test: This test is done to assess the will be soft and could be bent easily. The pin
presence of calcium in the decalcifying solu- also should penetrate easily within the tissue.
tion at two successive times. The chemical The major disadvantage of physical tests is
test is applied when a weak acid solution (e.g. tissue damage by making a hole or bending it.
formic acid) is used.
Chemical solution
Stock solution. 4.6 Results of Under
Ammonium hydroxide stock solution. Decalcification
Ammonium hydroxide (28%): 5 ml
Distilled water: 95 ml • There will be incomplete impregnation of
Ammonium oxalate stock solution. paraffin
Ammonium oxalate: 5 ml • It will be difficult to cut the section
Distilled water: 95 ml
• Bony dust may be seen
4.7 Results of Over References

Decalcification
1. Skinner RA, Hickmon SG, Lumpkin CK, et al.
Decalcified bone: twenty years of successful specimen
• Loss of nuclear details management. J Histotech. 1997;20:267–77.
• The damage of the cell membrane 2. Dotti LB, Paparo GP, Clarke BE. The use of ion
• Glycogen of the cell may be lost exchange resin in decalcification of bone. Am J Clin
• The tissue may be swollen Pathol. 1951;21(5):475–9.
3. Cook EF, Toghill HC. The electrolytic decalcification
of bone. J R Microsc Soc. 1952;72(1):70–1.
Tissue Microtomy: Principle
and Procedure 5
5.1 Introduction
After embedding the tissue and preparing the

block, the next step is microtomy. The word
“microtomy” originated from the Greek lan-
guage. “Mikros” means small, and “temnein”
means to cut. So, the word “microtomy” means
to cut the tissue into thin sections. For successful
microscopic examination, it is necessary to have
thin sections of the tissue by microtomy.
Microtomes: It is the main instrument by
which we cut the embedded tissue in the paraffin
block as a thin section. The different types of
microtomes in the traditional histology labora-
tory are:
• Rotary microtome
• Rocking microtome
• Base Sledge microtome
• Sliding microtome
• Cryomicrotome
• Ultramicrotome Fig. 5.1 Semi automated rotary microtome
• Laser microtome
1. Rotary microtome (Fig. 5.1): This is the
most commonly used microtome in the tome can be semi-automated or automated
routine laboratory. The cutting blade is with the adjustment and control of the
kept in a horizontal position, and the movement of the block and the angle of
block containing tissue moves up and the knife.
down with the help of a rotatory handle (a) Advantages:
attached to the microtome. In each 360° • Good quality 2 to 3-micron thin
rotation of the wheel handle, the block section is possible
moves down followed by up, and the tis- • Complete Heavy and stable micro-
sue is cut as a thin ribbon. This microtome automated rotary microtome
https://doi.org/10.1007/978-981-19-6616-3_5
42 5 Tissue Microtomy: Principle and Procedure
reduces health hazards and gives

the best quality section
• Good tissue ribbon production
• Easy to cut various types of tissue:
firm, fragile, small biopsy etc.
(b) Disadvantages
• Expensive
• Unsuitable to cut a large block
• Knife faces up and may be danger-
ous to the technical staff.
2. Rocking microtome: The rocking micro-
tome is also known as the Cambridge rock-
ing microtome. The word “rocking” is used
Fig. 5.2 Cryomicrotome used in frozen section
as there is a rocking action of the micro-
tome, like arm movement. In this type of
microtome, the knife is static, and the block • Difficult to get a thin section.
of tissue moves in a rocking motion (arc- • Large slides are required.
like movement of the block). This is one of 4. Sliding microtome: In this microtome,
the oldest designs of the microtome. The the knife is static, and the block moves
microtome can cut thin sections with rib- horizontally over the knife.
bons and is ideal for serial section. The sec- (a) Advantage:
tions are slightly curved in this microtome. • Large sections can be cut.
(a) Advantage: • Mainly used for celloidin embed-
• Thin section. ded tissue,
• Easy to operate. • Simpler design and easy
• Low-cost instrument and reliable. maintenance.
(b) Disadvantage: • Brain sections can be cut better by
• Tissue is curved, and the micro- this type of microtome.
tome does not provide a flat (b) Disadvantage:
section. • The knife may glide in case of
• As the microtome is lightweight hard tissue and may jump
so vibration may occur • Long knives are difficult to
3. Base Sledge microtome: In the sledge sharpen
microtome, the block is fixed in a static 5. Cryomicrotome: This type of microtome
position within a steel carriage. The knife is used for the cutting tissue for frozen
slides to and fro over the top of the block. samples (Fig. 5.2). The sample is made
This microtome is the best for large tissue hard in liquid nitrogen and then cut by the
samples or hard tissue. The tissue sections microtome in the chamber that contains
are usually thick (more than 10 microns) liquid nitrogen.
in the base sledge microtome. (a) Advantages:
(a) Advantages: • To get a rapid section for rapid
• Hard tissue can be cut, diagnosis.
• Large tissue sample can be cut, • To study nerve biopsy,
• The best microtome for ophthal- • To study enzyme histochemistry.
mology and large neuropathology (b) Disadvantages:
section. • It needs continuous supervision to
(b) Disadvantage: maintain the temperature
5.2 Microtome Knife 43
• Freezing artefacts is often seen

• Very expensive instrument
• Fixed tissue is very difficult to cut
6. Ultramicrotome: Ultramicrotome is used
to cut ultrathin sections for transmission
electron microscopy. Sections are cut
between 40 to 100 nano micron thickness
with the help of a glass knife or diamond
knife. The tissue is at first trimmed to
make a small block of 1 mm × 1 mm size
and then the section is cut by this ultrami-
crotome with the help of an optical micro-
scope. The cut section is allowed to float
on the water held by a boat and then finally
picked up on a metal grid.
7. Laser microtome: In this ultramodern
microtome, the laser beam is used to cut
the biological section without any pro-
cessing or embedding of the material. An
infra-red laser beam with ultra-short pulse
duration is applied and therefore almost
no heat is generated and the tissue is cut
without any thermal effect.
5.2 Microtome Knife
The microtome knife is important to cut uniform

and thin sections of tissue (Box 5.1). These are
made of steel. Various types of knife profiles are Fig. 5.3 Different types of microtomy knives based on
available for different types of microtomes. The shape. Profile C is the most commonly used knife
most commonly used knife profile is Profile C or
wedge profile. The various types of microtome type of knife and often vibrates during
knives include (Fig. 5.3): cutting.
3. Wedge (Profile C): This knife is plain on
1. Plano concave (Profile A): One side of the both sides. This is the most widely used knife
knife is plain and the other side is concave. for routine microtomy and it is compatible
Originally these knives were used for cutting with the different types of the microtome.
celloidin embedded tissue. This is a very This type of knife is also easy to sharpen.
sharp knife and is used for cutting soft tissues. 4. Tool edge (Profile D): The knife resembles a
However, presently these knives are less fre- chisel used in woodworking. Both sides of the
quently used. knife are plain, however, the cutting edge is
2. Bi concave (Profile B): The knife is concave steep. The tool edge knife is mainly used to
on both sides. The knife was used for a rock- cut the hard tissue such as decalcified bone.
ing microtome. The concavity of the knife is The knife is difficult to sharpen and is not rec-
often difficult to identify. This is a less rigid ommended presently.
5.2.1 Disposable Knife The desirable hardness of the knife is between

400 to 900 Vicker hardness scale.
Nowadays disposable blade is used in many labo- The diamond knife: This is a costly knife and
ratories to save time to sharpen. Two types of dis- is used to cut epoxy resin blocks in electron
posable blades are available: microscopy. Special care is needed to sharpen
this knife.
1. Low profile blade: These blades are used to Glass knife: This is used for ultramicrotomy
cut small biopsies or soft large tissue. to cut very hard tissue. The cutting edge of the
2. High profile blade: These are used to cut firm knife is parallel to one surface of the glass.
to relatively hard tissue such as uterus, heart
etc.
5.2.3 Angles of Knife
5.2.1.1 Advantages
The various angles formed by the knife include
1. Easy to replace within seconds (Fig. 5.4):
2. No need to sharpen Rake angle: It is the angle between the upper
3. The overall cost of disposable blade system is bevel of the knife and the perpendicular line
low as there is no need of any knife sharpener drawn from the surface of the block. Increased
or abrasive powder to sharpen the knife. rake angle makes the section cutting easier.
Angle of clearance: It is the angle between
5.2.1.2 Disadvantages the lower bevel of the knife and the surface of the
block. It is usually around 2° to 5°. The angle of
1. They are not very rigid like ordinary knife and clearance is related to the friction between the
therefore vibrations effect may be seen block and the knife. Lower the angle of clear-
ance less will be the compression on the block.
Bevel angle: This is the angle in between the
5.2.2 Materials Used in Knife two planes of the knife. The bevel angle is also
known as the knife-edge angle. This angle varies
Conventional knife: The conventional micro- from 18° to 30°. The knife becomes sharp if the
tome knives are made of very high-quality car- bevel angle is small.
bon or steel material that is usually tempered Cutting edge: This is the straight-line formed
from the edge to inside one-third of the width. by the intersecting of the two planes of the bevel.
Fig. 5.4 Schematic Perpendicular line

diagram of angles of From surface of block
knife
Rake angle
Knife
Cutting angle
Angle of clearance
Upper surface
Block
5.4 Sectioning the Paraffin Block 45
Stropping: It helps to polish the cutting edge

Box 5.1 Microtome Knives of the knife that is already sharped by honing and
Types based on shape: also to remove any “burr” formed during honing.
• Plano concave (Profile A): Strop is made of leather and it should be free
–– Very sharp knife and is used for cut- from any dust or grit.
ting soft tissues Automatic knife sharpener: Presently auto-
• Bi concave (Profile B): matic knife sharpener is available in the market.
–– Used for rocking microtome The knife is placed horizontally on the surface of
–– Vibrates during cutting the rotating plate made of glass or copper coated
• Wedge (Profile C): with abrasive agents.
–– Most commonly used
• Tool edge (Profile D):
–– Used for hard tissue 5.3.2 Factors Involved in Cutting
Types based on disposability 1. Temperature: Lowering the temperature

• Permanent facilitates section cutting.
• Disposable 2. Angle of rake: Higher rake angle helps in the
–– Low profile blade: To cut small smooth flow of ribbons. A lower rake angle is
biopsy used for hard tissue.
–– High profile blade: To cut firm to 3. Consistency of tissue: Soft tissue is cut at a
hard tissue slower rate than hard tissue.
Materials used in knife

Conventional knife: Steel.
Diamond knife: Made of diamond and 5.4 Sectioning the Paraffin Block
used to cut epoxy resin blocks.
The following instruments are essential for sec-
tion cutting:
5.3 Microtome Knife Sharpening
1. Microtome with a blade
Sharpening of the microtome knife is needed to 2. Water bath
get good sections. The following methods help in 3. Paraffin block with embedded tissue to cut
the sharpening of the knife: 4. Ice tray
5. A blunt forceps or camel brush
6. Slide rack with slides
5.3.1 Manual Method
Water bath (floatation chamber): A water bath
Honing: Abrasive grindings are used to sharpen the is used to float the tissue after cutting (Fig. 5.5).
knife. Stone can be used to sharpen the knife. Light The temperature of the water bath is usually con-
oil can be used to lubricate the stone before use. trolled automatically by a thermostat. The tem-
Abrasive such as iron oxide, aluminium oxide perature of water in the water bath should be 8° to
and silicon carbide are usually used as abrasive. 10 °C below the melting point of the embedded
The handle of the knife is held by one hand and paraffin wax and is usually kept at 40 to 50 °C. It
the other hand holds the front end of the knife. is necessary to prevent the formation of any air
The knife is pushed forward and diagonally over bubbles within the water bath. For adequate float-
the slab several times. The same procedure is fol- ing of the tissue, one can add a few drops of alco-
lowed on other surfaces of the knife also. hol or a little amount of detergent. This reduces
–– Glycerol: 100 ml
–– Homogenize the mixture thoroughly and
filter it into gauze pieces. Add a few crys-
tals of thymol to prevent bacterial growth.
• Poly l lysine: This is a good adhesive and does
not produce any background staining. The
slides are coated with poly l lysine diluted
with distilled water (1:10) before use.
• 3 Aminopropyltriethoxysilane (ACEP): The
slides are dipped in the dilute ACEP solution
in acetone (2%) and then dried before use.
• Permanent positively charged slides: Here
the slide is tempered in such a way that the
Fig. 5.5 Water bath used in microtomy. The constant
temperature (usually 40–50 °C) is maintained in the water slide surface is always positively charged. It is
bath. This is usually 5° to 10 ° C lower than the melting easier to lift the tissue section with these
point of the paraffin slides. The positively charged slides are also
excellent for lifting frozen section tissues.
the surface tension of the water and tissue floats • Celloidin: It is a relatively strong adhesive
smoothly. and is specially used for the bone sections.
Blunt forceps and camel hair brush: Blunt Celloidin may take the colour in case of stain-
forceps help to manipulate the floating tissue sec- ing of Periodic acid Schiff’s stain or mucicar-
tion (Fig. 5.6). A Camel hairbrush is used to clean mine stain.
the blade.
Slide rack with clean glass slides: The clean
slides are kept in the slide rack. The slides can be 5.4.1 Steps of Tissue Sectioning
already labelled with a diamond pencil or on the (Fig. 5.7)
frosted side with a lead pencil. Alternatively, this
can be marked after lifting the tissue section. 1. Trimming the tissue: Trimming of the tissue
Adhesive: In the case of routine section and is needed to expose the tissue piece within the
staining no adhesive is required. However, in cer- paraffin wax for cutting. The block is fixed in
tain situations, we use cell adhesive materials the chuck of the microtome and the paraffin is
such as: cut till the tissue is fully exposed. Adequate
(1) In brain sections, (2) decalcified tissue, (3) caution should be taken not to overcut tissue as
using strong alkali at the time of staining. this may produce various artefactual changes.
The most commonly used adhesives include: 2. Cooling the block: After the initial trimming,
the blocks are kept for cooling for 15 to
• Mayer’s egg albumin and Glycerol: This is 20 min. This will help to maintain the same
prepared by mixing consistency of the paraffin and tissue. This
–– White part of egg: 100 ml helps in easy cutting.
Fig. 5.6 Blunt forceps

and brush used in
section cutting
Fig. 5.7 Different steps of section cutting are highlighted brush on the water bath. Then the tissue is picked up by a
here. At first the tissue is trimmed. Then the block is putting a glass slide perpendicularly in front of it and then
cooled in ice. The block is placed in the microtome and when the tissue is touched the slide is withdrawn
the angle of clearance is adjusted to 5 °C. The tissue is vertically
gently cut and the tissue ribbon is placed with the help of
3. Cutting proper: 4. Floating the ribbon: The ribbon of the tissue

–– At first the microtome is set to cut the is floated in the water bath and this makes the
expected thickness. The cellular tissue e.g. tissue flat and removal any wrinkling of the tis-
lymph node is set at 4 μm. The routine sec- sue. If there is any wrinkle in the tissue, a few
tions are set to 6 μm thickness. drops of warm water may be gently poured. It
–– The block is fixed in the chuck of the micro- is advisable not to use any sharp instrument to
tome. The cutting surface of the block unfold the wrinkled tissue. With the help of the
should be parallel to the knife (Fig. 5.8a). If forceps, the individual sections are separated
the block and tissue are not in the correct from each other. As mentioned before, the tem-
position then it should be reset to have a perature of the water bath should be constantly
good quality section (Fig. 5.8b). maintained and it should be 6 to 8° below the
–– Please keep the knife and block dry. melting point of the paraffin wax. In case of
–– The tissue in the block is cut by a gentle, temperature variation in the bath, air bubbles
smooth and slow stroke. The ribbon-like may be formed that may rupture the tissue.
tissue sections are produced by creating 5. Picking up the tissue: The slide is placed verti-
adequate heat and pressure in the block. cally within the water bath in front of the tissue
–– The tip of the ribbon is held by forceps and and when the tissue is touched the slide is with-
the end part of the ribbon is removed from the drawn vertically from the water. The tissue
knife-edge by brush. In case of any difficulty pick-up process must be gentle and smooth. To
to get the flat section, the cutting surface prevent any mix up the water bath should be
should be gently warmed with warm water. cleaned immediately after cutting each block.
Specimen
a holder b
Tissue Specimen
with block holder
Tissue
with block
Knife
Knife bevel
Knife bevel face not
face parallel to parallel to
the tissue the tissue
Knife
Correct position Incorrect position
Fig. 5.8 (a) The schematic figure of the correct position of the tissue and the knife (b) The incorrect alignment of the
knife
6. Drying the section: The slide containing the

picked-up section is kept in the slide rack.
Each slide should be marked with a diamond
pencil. It is a vital process and every attempt
should be taken to prevent mix up. The slides
are now kept in a hot oven to get dry. The tem-
perature of the oven should be slightly more
than the melting point of the paraffin.
7. Storage of the block: Once the section is cut,
the block is covered by molten wax to prevent
the air drying of the tissue.
Various problems may occur in tissue sectioning Fig. 5.9 Tear in the tissue due to uneven cutting edge of
(Figs. 5.9, 5.10, 5.11, 5.12, and 5.13) [1]. These the knife
are enumerated below in Table 5.1:
Fig. 5.10 Large hole in the tissue due to the bad Fig. 5.13 Uneven staining pattern due to poor deparaf-
processing finization of the tissue
Table 5.1 Troubleshooting in tissue sectioning

Faults Source of faults Solution
Ribbons not • Paraffin is • Select low
formed: the hard melting point
tissue is twisted • I ncorrect paraffin
and curled or Clearance • Correct the
sticks to the angle: small knife alignment
knife •D irty knife • Clean the blade
• Too cold or •A djust the
hot room room
temperature temperature
Ribbons are •E dges of the • The block
curved: The block are not holder should
ribbons are not parallel: the be aligned
Fig. 5.11 Multiple air bubbles have ruptured the tissue. straight and are sides of the properly
The air bubbles are produced due to uncontrolled tem- coiled block and the • Trimming is
perature in the water bath. Note the characteristic multiple knife edge is needed
irregular tear of the tissue not in right •S harpen the
angle knife or replace
•S urface of the the blade
block is • Trim and
uneven remove the
•B lade is not excessive
sharp paraffin
• The paraffin is
too much
(continued)
Fig. 5.12 Freezing artifact of the tissue due to immediate

putting the tissue from the refrigerator to formalin. Note
the regular spaces in between the tissues due to melting of
ice crystals
Table 5.1 (continued) Table 5.1 (continued)

Faults Source of faults Solution Faults Source of faults Solution
Ribbons are •B
lock is warm •C ool the block Large holes in • The under • Reprocess
excessively •D
ull blade/ •R eplace the the tissue processed tissue
compressed knife blade or tissue ruptures
and wrinkled •P
araffin is soft sharpen the when comes in
and sticky knife contact with
•K
nife •C ool the block warm water
clearance is and then try to Tissue is • I nadequate • Tissue needs
not optimum cut otherwise shrunken dehydration reprocessing
and is too the paraffin Tissue remains •S uboptimal • Tissue needs
small should be soft at the time fixation reprocessing
•R
otation of the replaced with of trimming
microtome is higher melting At the time of •S
uboptimal • Tissue needs
clumsy point sectioning the infiltration of reprocessing
• I ncrease the tissue is the wax
knife clearance compressed but
•N eeds not the paraffin
consistent and wax
gentle rotation
of the wheel
Thick and thin •K nife or block • Tighten the 5.4.2 How to Recover the Dried
section is loosely knife clamps or Tissue?
(chatter) attached the chucks
•B lunt knife •S harpen the
The tissue may be completely dried and crum-
• The clearance knife or change
angle is very the blade bled due to a faulty processor. In such condition,
small •A djust the the tissue is kept in formol-glycerine mixture for
•P araffin is soft clearance angle 8 h. The exact timing may vary depending on the
and sticks to •C lean the knife
size of the tissue.
the knife and try to
remove the
attached 5.4.2.1 Formol-Glycerol
paraffin Stock solution
Section •S tatic •A pply ionizer
attaches to the electricity is to remove the
block generated in static charge • 2 g sodium citrate
the knife or and /or clean • 10 ml formaldehyde
ribbon the blades, • 90 ml water
• The clearance holder etc. with
angle is alcohol
defective • I ncrease the Working solution
clearance angle
Tear or •D efect in the •H one the knife/ • 90 ml of formol-sodium acetate stock solution
Scratches in the blade: a nick use other parts • 10 ml of glycerol
section or jagged of knife/change
knife-edge the blade
•D irt in the •C lean the blade Treat the tissue in the solution for 8 h and then the
knife •D ecalcify the process the tissue in the tissue processor. The
•S harp particles tissue processing starts from the dehydration stage.
in the tissue • Try to remove
•S harp particle sharp particles
in the paraffin by scalpel
Tissue • Too hot water • Temperature Reference
disintegrates in •F aulty and should be
the water bath incomplete lowered down 1. Peachey LD. Thin sections. I. A study of section thick-
processing •R eprocess the ness and physical distortion produced during microt-
tissue omy. J Biophys Biochem Cytol. 1958;4(3):233–42.
Frozen Section: Principle
and Procedure 6
6.1 Introduction 6.2.1 The Principle of the Frozen

Section
The frozen section is the rapid tissue section by
cooling the tissue with the help of cryostat to give The rapid freezing of the tissue sample converts
an immediate report of the tissue sample. This is the water into ice. The firm ice within the tissue
especially needed in a large hospital to diagnose acts as embedding media to cut the tissue.
the lesion or extent of the lesion at the time of Lowering the temperature makes the tissue
operation. The cryostat is the instrument that has firmer, whereas increasing the temperature makes
the arrangement to freeze the tissue and also cut the tissue softer.
the frozen tissue into the microscopic section.
6.2.2 Cryostat Machine Proper

6.2 Indications of Frozen (Fig. 6.1)
Sections
The temperature range in the machine: The
The frozen section is used mainly for immediate cryostat machine has the usual temperature range
diagnosis of the lesion for management and to from 0 °C to −35 °C. Most of the tissue is sec-
know the extent of the lesion [1–3] (Box 6.1). It tioned properly between −15 °C to −25 °C. The
is also helpful to do enzyme immunochemistry water containing tissues can be sectioned at a
and immunofluorescence study. At times, frozen higher temperature and fat-containing tissue
section tissue is used for the demonstration of fat needs a much lower temperature to cut.
and carbohydrate in the tissue sample. Rotary microtome (Fig. 6.2): Rotary micro-
tome is placed inside the cabinet of the cryostat.
Here the knife is fixed and the tissue is moved
Box 6.1 Indications of frozen section
with the help of a rotary wheel.
• Rapid diagnosis of the lesion for intra-
Tissue shelf (Fig. 6.2): On just one side of the
operative management
microtome there is a tissue shelf to keep the tis-
• To know the extent of the lesion
sue. In this place, the samples are kept for freez-
• To do enzyme immunocytochemistry
ing. Usually, the temperature of the tissue shelf is
• To do immunofluorescent
lower than the overall cabinet temperature.
• To stain lipid and certain carbohydrate
Place to keep the brush and knife holder:
in the tissue
Just in front of the microtome machine, there
https://doi.org/10.1007/978-981-19-6616-3_6
52 6 Frozen Section: Principle and Procedure
Fig. 6.1 Cryostat machine with its parts
remains a small place to keep the brush and knife plate has free space and there is a gap between
holder. the knife and the plate.
Knife or blade: Nowadays low or high profile Alternating to an antiroll plate a cool sable
disposable blades are used. The blade should be hair brush can be used to get unrolled tissue.
properly fixed to the holder to get an even pres- Specimen holder: The specimen holder or chuck is
sure in the whole length. Alternatively, C profile supplied by the manufacturers in different sizes and
steel blade is also used. The angle of the knife is shapes. Usually, these are round metal structures.
kept between 5° to 7°. Embedding medium: This medium is used to
Antiroll plate (Fig. 6.2): Just in front of the hold the tissue over the chuck. Presently opti-
knife there is an antiroll plate that prevents the mum cutting temperature (OCT) compounds are
rolling of the cut tissue. It is usually a glass plate used as embedding medium. The OCT is made of
within a metal frame. The under surface of the water-soluble glycols and resin.
6.3 Cryostat Sectioning 53
Fig. 6.2 Microtome, blade, anti-roll plate and tissue shelves are shown
6.3 Cryostat Sectioning (c) Tissue appearance: The gross appear-

ance of the tissue such as colour, texture,
The process of the cryostat sectioning needs the consistency and any suture to mark the
following steps. anatomical position.
(d) Resection margin: It is very important
1. Grossing and cutting the specimen (Box 6.2): to identify the resection margins of the
The cutting surface of the tissue should be tumour. The resection planes and m argins
smooth. The following steps in grossing of the should be inked thoroughly. The different
tissue are mandatory for accurate reporting: colours of ink can be used for medial and
(a) Identify the tissue sample of the lateral margins identification.
patient and the requisition form: This Cutting the tissue: The tissue
is the first and foremost part of the fro- should be fresh without any fixative.
zen tissue grossing. The tissue should be preferably dry and
(b) Salient clinical information: The essen- it should not be wrapped in a gauze
tial clinical information is very helpful piece. Any suture, staple or sharp hard
as it guides the pathologist to reach a structure should be removed from the
possible differential diagnosis. tissue sample. Now the tissue is cut into
small pieces as it facilitates freezing. 4. Loading the blade: The cutting knife or
Take multiple sections of the tissue to blade is now loaded and the proper alignment
understand the main pathology and to is done.
minimize the error. Use a new sharp 5. Trimming the tissue: The loss of normal or
scalpel blade and first cut the most natural colour to whitish colour indicates that
important area that needs microscopic the tissue is frozen. The frozen tissue in the
examination. It is preferable to use a tissue holder is now placed in the holder of
gentle stroke of the scalpel rather than the microtome. The block is trimmed to
too much pressure. remove the excess OCT and to get the smooth
Cytology of the tissue: At times the tissue surface for sectioning.
imprint of the tissue on the slide provides 6. Sectioning (Fig. 6.3): The tissue is now cut
good morphological details such as lym- gently and is spread over the antiroll plate
phoma of the lymph node. Similarly with the help of a brush. The brush should be
crushing of tissue also provides excellent cooled. The tip of the tissue is guided by the
morphological details such as in the case brush.
of tissue of a brain tumour. 7. Section lifting: The glass slide of normal
2. Tissue embedding in the mould (Fig. 6.3): room temperature is pressed firmly over the
The small piece of the tissue is kept in the tissue section and normally the tissue sticks
centre of the mould and then the OCT is immediately.
poured over it in excess. Then the tissue 8. Fixation: The tissue should be immediately
holder or chuck is firmly placed over the tis- fixed in methanol for 1 min or 95% ethanol
sue with overflown OCT. for a few seconds. Rapid fixation within a
3. Tissue loading in the frozen section cham- few seconds is mandatory. In case of delayed
ber: The tissue is now placed in the frozen fixation, the cells are swollen and the cyto-
section chamber and cold spray can be used plasmic margin may be ruptured giving a
to make the process faster. hazy appearance to the margin of the cells.
a b c
d e f
Fig. 6.3 Cryostat processing: (a) Mould is covered with ing chamber, (e) The brush guides the tip of the tissue, (f)
OCT, (b) tissue is now put on the block, (c) OCT is The tissue section is gently spread over the antiroll plate
flooded over the tissue, (d) Tissue now is put in the cool- and later picked up by touching a glass slide
6.4 Staining 55
Table 6.1 Troubleshooting in frozen section

Problems Cause Solution
Freezing artefact Formation of ice crystal within the tissue. • Freeze the tissue rapidly i.e. Snap
Water containing tissue shows more such freezing
artefact • The tissue specimen should not be in
saline solution before freezing
Uneven tissue embedding The surface of the tissue is uneven and • Make tissue even at the cutting
the vital information may be lost surface before freezing
Block is loosen The chuck may be too cold when the • Take the tissue out and reattach it on
During chucking tissue is placed on it a clean chuck which is not too cold.
Tissue crumpled The tissue in the block is warm or too • Make the block of tissue in the
cold optimum temperature: −15 °C to
−20 °C
Chattering artefact The temperature of the block is too cold • Bring the block in optimum
and tissue becomes hard. The blade will temperature. Pressing the cut surface
cut the tissue thick and thin at regular of the block by gloved finger may
intervals. make the block warmer
Thin stripe in tissue The perpendicular tear in the tissue is due • Replace the blade by a sharper one
to nicks on the blade
Widely striped tissue and This may happen if the tissue is sticking • Clean the blade or replaced it with a
also tearing of the tissue to the blade new one
section tissue. This has been highlighted in

Box 6.2 Grossing for frozen section tissue Table 6.1.
• Identify the tissue sample of the Fixation: Immediate dip in 95% ethyl alcohol
patient for a few seconds to fix the tissue.
• Clinical information: Provides possi-
ble differential diagnosis
• Tissue appearance: Colour, texture, 6.4 Staining
nodule, any suture
• Anatomy of the tissue: Identify the We commonly use haematoxylin and eosin (H&
resection planes and margins E) and toluidine blue stain.
• Colour the resection planes and
margins
• Section cutting: 6.4.1 H & E Staining
–– Use sharp blade
–– First cut the most important area • Rinse the slide in tap water
–– Give gentle pressure and avoid too • Put in haematoxylin for 1 min
much pressure • Rinse in tap water for 5 s
• Cytology preparation: If needed make • Rinse in Scott’s tap water for 5 s for bluing
–– Imprint smear • Dip in eosin for 20 s
–– Scrape smear • Rapidly rinse in tap water
–– Crushed smear • 95% Ethanol for 10 s
• 100% Ethanol for 10 s
• 100% Ethanol for 10 s
Troubleshooting in frozen section: Various • Dip in xylene for 20 s
problems may arise during the cutting of frozen • Mount by DPX
6.4.2 Toluidine Blue Stain Table 6.2 Optimum temperature for frozen section
Optimum
This is a very simple stain and takes only a few Tissue temperature
seconds. The drops of toluidine blue stain are put Brain, liver, spleen −7 °C to −10 °C
on the section and the coverslip is put on the sec- Rectum, uterus, adrenal, muscle, −12 °C to −15 °C
skin,
tion. The slide is now ready to see. The histopa- Heart, lung, intestine, pancreas, −16 °C to −20 °C
thologist feels more comfortable in H & E stain ovary, cervix, prostate
than in this unfamiliar toluidine blue stain. Bone marrow, breast −20 °C to −25 °C
6.5 Factors Affecting the Good –– Collagenous tissue: The firm collagenous
Quality Section tissue is difficult to cut.
–– Necrotic tissue: Soft necrotic tissue may
The common factors responsible for the good create a considerable problem as they may
quality smear include: fall from the slide making hole in the sec-
tion. It is preferable to take only viable tis-
• Temperature: When the temperature falls sue for the frozen section.
water within the tissue becomes frozen and –– Bony hard tissue: Hard tissue like bone or
gives the tissue hard consistency. The optimal cartilage may damage the blade signifi-
temperature of frozen tissue is in between −15 °C cantly. In this situation, a new section can
to −25 °C. Warm tissue remains soft and sec- be processed or a new blade can be used.
tions crumple. On the other hand, the over- • Tissue size: The size of the tissue sample
cooled tissue becomes very hard and brittle and should be small as the larger tissue takes a
produces again bad quality crumpled section. much longer time to freeze.
Moreover, the hard tissue may cause “chatter-
ing” artefact and also thick and thin sections.
Different tissue contains a variable amount of References
fat and water. The consistency of different tissue
varies and therefore the optimum temperature to 1. Hatami H, Mohsenifar Z, Alavi SN. The diagnostic
accuracy of frozen section compared to permanent
cool the tissue varies considerably. Table 6.2 section: a single center study in iran. Iran J Pathol.
shows the optimum temperature of different 2015;10(4):295–9.
organs to have a good frozen section. 2. Ayhan A, Ozler A, Dursun P, Haberal AN. Potential
• Tissue consistency: Other than the optimum role of increasing number of sections in frozen sec-
tion diagnosis of ovarian tumors. J Exp Ther Oncol.
cooling temperature the consistency of tissue 2016;11(4):245–50.
has a significant effect on cutting such as: 3. Chambers KJ, Kraft S, Emerick K. Evaluation of fro-
–– Fatty tissue: It is difficult to cut the fatty tissue zen section margins in high-risk cutaneous squamous
in the frozen section. Fat may smear on the cell carcinomas of the head and neck. Laryngoscope.
2015;125(3):636–9.
knife and may make problems in cutting.
Staining Principle and General
Procedure of Staining the Tissue 7
7.1 Introduction
The tissue section is colourless because the fixed

protein has the same refractive index as that of
glass. We use dyes that have a specific affinity
with the different tissue proteins and colour them
differently. This helps us to understand the mor-
phology of the tissue.
7.2 Dyes Used for Staining
The dye may be natural or synthetic. The natural Fig. 7.1 Benzene ring. Benzene itself is colourless.
dye is extracted from plants and animals. When two H atoms in benzene ring is replaced by two O
Nowadays natural dye is rarely used except for atom then the compound Quinone (C6H4O2) is formed
haematoxylin and carmine. The majority of the which is a chromogen
synthetic dye is petroleum derivatives. All these
synthetic dyes have a central benzene ring
(Fig. 7.1). Benzene has the chemical formula C6H6 650 nm (Fig. 7.2a). The white light contains all
and it is in a ring form which is very flexible. The the seven colours (VIBGYOR: Violet, indigo,
molecule of benzene is colourless, however, if a blue, green, yellow, orange, and red) with a wave-
certain chemical group is inserted into the benzene length between 400 to 650 nm. A chromogenic
ring then it will impart colour. Such as two H dye absorbs the light of a particular wavelength
atoms in the benzene ring if replaced by two O of the white light representing a specific colour
atoms then the compound Quinone (C6H4O2) is and emits the light containing the rest of the
formed which is a chromogen. This grouping in colour. The emitted light produces a particular
the benzene ring that imparts colour is known as a colour known as complementary colour
chromophore and the chemical compound formed (Fig. 7.2a). Therefore, we see a coloured light
by the grouping is known as the chromogen. from the dye. Such as the dye that absorbs the red
How dye produces colour: The visible light light will be visible as green colour to the naked
has a range of wavelengths between 400 to eye.
https://doi.org/10.1007/978-981-19-6616-3_7
58 7 Staining Principle and General Procedure of Staining the Tissue
Fig. 7.2 (a) The

a
wavelength of the
visible colour is ranged
from 400 to 800 nano
microns. A chromogenic
dye absorbs the light of
a particular wavelength
of the white light
representing a specific
colour and emits the
light containing the rest
of the colour. The
emitted light produces a
specific colour which is
known as a
complementary colour. b
(b) Schematic diagram
showing colour
generation by dye. The
chromophore group
absorbs light and
imparts colour to the
stain. The autochrome
group donates more
electrons to the
chromophore group and
helps to absorb light of
longer wavelength in the
visible range
The dye is the most elementary component of 7.2.1 Types of Dye

staining a tissue. In general, the dye has two com-
ponents: Chromophore and auxochrome part The dye can be classified based on electrical
(Fig. 7.2b). charge (Table 7.1):
• Chromophore groups: The chromophore 1. Anionic dye or Acidic dye

group absorbs light and imparts colour to the 2. Cationic dye or basic dye
stain. These groups have many free electrons 3. Neutral dye
that absorb the ultraviolet rays of light which 4. Ligand dye (chelating)
are not in the visible range. 1. Anionic dye or Acidic dye: These dyes
• Auxochrome part: This part of the dye helps carry a negative charge (coloured anions)
to intensify the light. It is an ionising group and migrate towards the anode in an elec-
that also helps to stick the stain with the tissue. trical field. The dye is mostly of low
The auxochrome group augments more free molecular weight and soluble in water.
electrons in the chromophore groups. The Most of the dyes have two or more anionic
increased number of electrons in the system groups, making them soluble in water.
helps absorb light of a longer wavelength in These acidic dyes combine with the basic
the visible range. part of the tissues that carry positive
7.2 Dyes Used for Staining 59
Table 7.1 Types of dye on the basis of electrical charge

Types of dye Charge of the dye Tissue to bind Example
Anionic dye or Negatively charged • Cytoplasmic Eosin
acid dye proteins
• Collagen
Cationic dye or Positively • Nucleic acid Methyl green, ethyl green and Alcian blue.
basic dye charged • Epithelial mucin
Neutral dye Contain both acid and • Both nucleus and Giemsa
basic dye cytoplasm
Ligand or Weak acid so anionic and • Nucleus and • Al ligand with haematoxylin: Harris’
chelating dye negatively charged cytoplasm Hematoxylin, and Mayer’s Hematoxylin
• Fe ligand with hematein: Iron
haematoxylin
charges and those tissues are called as minium (Al) ions or iron (Fe) ions. The
“acidophilic”. metal-ion complex has a surplus charge
Example: Eosin is one of the most that increases the solubility in water and
commonly used anionic (acid) dyes. makes the dye insoluble in alcohol, and
2. Cationic dye or basic dye: Cationic dye therefore dehydration due to ethanol does
carries a positive charge (coloured cation) not occur during staining.
and moves towards the cathode in an elec- Al3+-hematein: It is a type of ligand
trical field. The cationic dyes are mostly dye where the metal aluminium (Al) is
soluble in ethanol. These are basic dyes, combined with hematein. It is used in
and they combine with tissues that carry a Harris’ hematoxylin and Mayer’s hema-
negative charge. These negatively charged toxylin. As Al3+-hematein is insoluble in
tissue combined with basic dye is called ethanol, it can be used along with other
“basophilic” tissue. anionic dyes and both the dyes are retained
Example: The examples of basic dyes even after dehydration with alcohol.
are methyl green, ethyl green and Alcian Iron haematein (Fe2+ − haematin):
blue. Iron haematin is the ligand dye that con-
3. Neutral dye: These are the compound sists of Fe and haematin. The dye is made
dyes that contain both acid and basic dye in by combining iron salt and haematoxylin.
combination. In an aqueous solution the Fe binds with haematoxylin molecule by
acid dye and basic dye exchange electrons oxidizing it to haematin. Iron haematin is
and combine to precipitate in the tissue. used for staining myelin and elastic fibres.
Romanowsky-Giemsa staining is the best
example of the compound dye that under-
goes an electron transfer process. 7.2.2 Types of Dye on the Basis
Romanowsky-Giemsa contains azure B of Chemical Structures
and eosin Y. The azure B dye is the electron and Chromophore Groups
acceptor, and eosin Y is the electron donor.
Example: Giemsa 1. Azo dye: These dyes contain –N=N- chromo-
4. Ligand or chelating dye: Ligand dye is a phore group. The majority of the azo dyes are
complex compound that consists of dye anionic (acidic) dyes. Example: orange G,
and a metal ion. They are also known as Congo red
metallochrome. They are usually weak 2. Thiazine dye: Thiazine dye contains –C-N=C-
acids. Hematein is the oxidised hematoxy- and –C-S=C chromophore group. Example:
lin, and it is used as a combination of alu- Toluidine blue, Methylene blue.
3. Triphenylmethanes: Triphenylmethanes
contain =N- chromophore group. Example:
Methyl violet, light green, Malachite green.
4. Azin dye: This group of dyes contains C-O=C
and C-N=C chromophore.
Example: Celestine blue, Nile blue
sulphate
5. Diphenyl methanes: They contain –NH
chromophore group. Example: Auramine.
6. Xanthene dyes: Example: Eosin, Rose
Bengal, Phloxines
7. Oxazine dyes: They contain C-O=C chromo-
phore group. Example: Cresyl violet,
Celestine blue.
8. Acridine dyes: The dyes of this group is
derived from Acridine. Example: Acridine
orange
9. Anthraquinone dyes: These dyes are derived
from anthraquinone. Example: Carminic acid
Fig. 7.3 Schematic diagram showing the interaction of
the anionic and cationic dye with the oppositely charged
tissue components. The acidic or anionic dye binds with
7.3 Mechanisms and Theory the cytoplasmic protein and collagen, whereas the basic or
cationic dye binds with the nucleic acid of the nucleus
of Staining
The staining is the combination of a coloured basic dye having a positively charged cat-
substance (dye) with the tissue that retains the ionic chromogen binds with the basophilic
dye after washing. The staining is primarily a tissue containing a negative charge.
chemical reaction between the dye and the tissue. The dye is the combination of chromo-
The following chemical reactions are involved gen and auxochrome that are oppositely
between the dye and tissue components (Box 7.1) charged. In the solution, they dissociate
[1, 2]: into oppositely charged chromogen and
auxochrome. Such as basic dye dissoci-
1. Electrostatic bond ates into cationic chromogen (positive)
2. Vander Waals attractions and anionic auxochrome (negative). Now
3. Hydrogen bond the cationic positively charged chromogen
4. Covalent bond of the basic dye combines with negatively
5. Hydrophobic bond charged tissue.
6. Dye aggregations Example: Eosin, the acid dye, stains
1. Electrostatic bond: The electrostatic the cytoplasmic proteins.
bond occurs between two oppositely 2. Vander Waals’ attractions (Fig. 7.4):
charged particles, and coulombic forces Vander Waals’ attraction is a non-coulom-
work between the particles. The oppo- bic force. This is the weakest force due to
sitely charged dye binds with the tissue the intermolecular interactions. The
(Fig. 7.3). Such as, an acid dye containing strength of this force is only 0.4 to 4 kJ/
the anionic (negative charged) chromogen mol compared to 20 kJ/mol in an ionic
binds with the acidophilic tissue contain- bond. When the electrons of an atom con-
ing the positive charge. Similarly, the centrate in one pole of the atom then a
7.3 Mechanisms and Theory of Staining 61
Fig. 7.4 Schematic diagram of Van der Wall force. The induced dipole. In the case of London force, the perma-
positive charge of a permanent dipole interacts with nent dipole induces the adjacent atom as induced dipole
another permanent dipole. In the case of Dipole- induced that further induces a chain of induced dipole and forms a
dipole interaction the permanent dipole interact with the large network of tissue with induced dipole interaction
dipole is formed. This dipole is just like a dipole interaction. This is known as dis-
magnet having two poles. persion or London force.
Dipole-dipole interaction: The posi- Example: Elastin stain by Miller’s
tive charge of a permanent dipole interacts stain, Congo red stain
with other permanent dipoles, and electro- 3. Hydrogen bond: Hydrogen bonding is a
static interaction occurs. weak bond. It is a covalent bond between
Dipole-induced dipole interaction: hydrogen and a strongly electronegative
Similarly, a permanent dipole may induce atom, commonly O, N or F (Fig. 7.5a). It is
an adjacent atom and induces a dipole. a polar covalent bond, and the electrons of
This permanent dipole may interact with the two particles are not shared equally. In
the induced dipole. hydrogen bonding, the electronegative
Dispersion or London force: atom should be small in size because the
Permanent dipole may induce the adjacent smaller atom has a stronger electron affin-
atom as an induced dipole. The induced ity. Hydrogen bonding is weaker in
dipoles further induce a chain of the strength. Water forms hydrogen bonds and
induced dipole. In this way, a large net- so competes with stain-tissue bonds.
work of tissue may undergo induced Therefore, hydrogen bonding in dye-tissue
a b
Fig. 7.5 (a) Schematic diagram showing hydrogen bond formation. (b) Best carmine forming hydrogen bond
Fig. 7.6 Covalent bond

formation
is less likely to occur in an aqueous solu- ecules interact, London force, the disper-
tion. Example: Best’s carmine dye to stain sion type of van der Waals force, interacts.
glycogen (Fig. 7.5b). Therefore, instead of hydrophobic bond-
4. Covalent bond: In the case of the cova- ing, the better terminology is probably
lent bond, the two electrically neutral “hydrophobic interaction” [3].
atoms share electrons to satisfy the outer Example: staining in aqueous solution,
shell’s required number (Fig. 7.6). The metachromatic staining
covalent bond is stronger. 6. Dye aggregations: Dye molecules may
Example: Periodic acid Schiff’s stain- interact, forming dye-dye interaction. The
ing for glycogen and Feulgen reaction. aggregate in the solution then penetrates
5. Hydrophobic bond: This is a misnomer the tissue. The dye-dye aggregate
as there is no such bonding in standard increases when the dye concentration is
chemistry. It is probably a type of van der high, the molecular size of the dye is big-
Waals force. When two hydrophobic mol- ger, and the temperature is low.
7.4 Factors Influencing Staining 63
minimal affinity with basophilic sub-

Box 7.1 Mechanism of Staining stances. The affinity of the dye to the spe-
• Electrostatic bond cific tissue is also influenced by the pH
–– Coulombic force and the presence of the inorganic salt con-
–– The electrostatic bond between two centration of the solvent.
oppositely charged particles: dye and 2. Specimen geometry: Specimen geometry
tissue or topography also influences the staining.
• Vander Waals attractions: Weak non- (a) Thick tissue: If the tissue is thick,
coulombic force then the penetration of dye is difficult,
–– Dipole –dipole interaction and the central part of the tissue takes
–– Dipole- induced dipole interaction a poorer stain
–– Dispersion or London force (b) Surface topography: The surface of
• Hydrogen bond the tissue of the paraffin section is
–– Weak bond more even than the cryostat section,
–– Type of covalent bond so it takes better stain.
• Covalent bond (c) Disturbance of microtopography of
–– The two electrically neutral atoms tissue: The alcohol is a coagulative
share electron with each other fixative that disturbs the topography
• Hydrophobic bond of the cell and tissue. The shattering
–– Misnomer as in standard chemistry effect of alcohol increases the dye
there is no such bonding. penetration rate.
–– Probably a type of van der Waals force (d) Inner geometry of tissue: The biologi-
• Dye aggregations cal geometry of the tissue may also
–– Dye molecules aggregate in solution influence staining, such as bone mar-
and then penetrate into tissue row canaliculi being rapidly stained
by Schmorl’s thionine stain than the
adjacent connective tissue.
3. Target concentration: The concentration
7.4 Factors Influencing Staining of the target tissue affects the staining
intensity as more the amount of the target
Several factors influence staining intensity (Box 7.2): tissue, the more intense will be staining.
4. Rate of reaction: The reaction rate in the
1. Dye affinity to the target tissue specimen target tissue also influences the staining
2. Specimen geometry pattern. In the Feulgen reaction, the short
3. Target concentration reaction time exposes only a few aldehyde
4. Rate of reaction groups producing a weak staining pattern.
5. Rate of stain loss 5. Rate of stain loss: The staining pattern is
1. Dye affinity to the target tissue speci- greatly influenced by the rate of stain loss.
men: The tendency to bind a dye with the At times the stain loss may be intentional,
target tissue is known as dye affinity. The such as differentiation or de-staining. The
acidic dye, such as eosin, binds strongly differentiation often removes the excess
with the acidophilic target, which means stain from the cell and thus helps to dif-
cytoplasmic protein. The acidic dye has ferentiate the organisms.
will contain 9 g pure azure B. So, if the solution

Box 7.2 Factors Influencing Staining of 100 ml needs 10 g of pure azure B then we
Intensity have to add (10/9) 10= 11.11 g of supplied azure
• Dye affinity to the target tissue speci- B to the 100 ml solution.
men: Acidic dye binds with positively Vital stain: The term vital stain means that
charged acidophilic tissue and basic dye the dye can penetrate the living cells and can
binds with negatively charged baso- stain the cells without killing them.
philic tissue. Example: Methylene blue, Nile red.
• Specimen geometry: Supravital stain: If the dye is applied in vitro
–– Thick tissue: Less penetration of dye to the living cells then it is called a supravital
–– Surface topography: More even sur- stain.
face better stain
–– Microtopography of tissue: Alcohol
fixation disturbances microtopography 7.5.1 Applications
–– Inner geometry of tissue
• Target concentration: More the 1. Reticulocyte count: Brilliant cresyl blue is
amount of the target tissue the more used to stain the reticulocyte.
intense will be staining. 2. Immunofluorescence: Supravital stain is often
• Rate of reaction: Short reaction time used in the immunofluorescence technique.
often decreases stain intensity 3. DNA stain: Hoechst 33342, and DAPI are
• Rate of stain loss: Too much differen- vital DNA stains and can be used to quantitate
tiation often removes the stain DNA.
Argyrophil: The affinity of a substance to the silver

salt that is reduced to metallic silver is known as
7.5 The Nomenclature Used argyrophilic. There is a need for a reducing agent in
Regarding Dye the argyrophilic reaction. Neuroendocrine cells of
our body are argyrophilic as they chemically react
Colour index of dye: A specific dye may be with the silver salt and produce brown colour in the
called a different name, or the same name may be presence of an external reducing agent. Grimelius
given to many other dyes. Therefore, the simple silver stain is used to demonstrate the argyrophilic
name of a dye may evoke confusion. To avoid granules of the endocrine cells.
this confusion, the “society of dyers and colour- Argentaffin: The argentaffin substances have
ists” has labelled a specific dye by the unique an affinity for silver salt and reduce them to
code known as colour index or colour index num- metallic silver without any external reducing
ber (CI). The CI number is a compendium of dye. agent. Melanin is argentaffin and it is demon-
With the help of the CI, one can identify the exact strated by Masson Fontana stain without any
dye, such as CI 52000 indicates thionine. external reducing agent.
Total dye content (TDC): TDC indicates the
total amount of dye in the dye material. Many
dyes are sold as a powder and are impure, and 7.6 Metachromasia [4]
may contain 95% of the original dye. The rest
(5%) may be the impurities that contain the iso- The word “meta” means altered and “chroma-
mer or closely related compound of the dye. TDC sia” means colour so “metachromasia” indicates
varies in different batches of the same dye. If the altered colour. Metachromasia is defined as a
TDC of azure B is 90% then 10 g of the azure B staining phenomenon when the tissue is stained
7.6 Metachromasia 65
in a different colour from the original dye (positively charged) dye in an aqueous solution
colour. The metachromatic dye is defined as the reacts with the polyanions of the tissues. The
alteration of the original colour of the dye with- binding of the dye molecule with these polyan-
out any change in the chemical structure of the ions of the tissue neutralize the positively
dye (Box 7.3). charged dye. The non-polar aromatic ring of the
dye binds with the other dye by Van der Wall’s
force. The dye-dye aggregation occurs and
7.6.1 Metachromatic Dyes dimer, tetramer and polymer of the dye mole-
cules are formed. Overall dye-binding becomes
Cationic dye: The majority of the metachromatic stronger due to Van der Wall’s force. The dye
dyes are positively charged cationic or basic dyes absorbs light of a shorter wavelength and the vis-
such as toluidine blue, methylene blue, azure A ible colour of the light emitted from the dye tis-
and B, and methyl violet. Brilliant cresyl blue etc. sue changes. This causes metachromasia or
Anionic dyes: Only a few anionic dyes are altered colour, such as pyronin Y in the tissue
metachromatic such as Biebrich scarlet and bro- giving red to orange colour.
mophenol blue. These anionic dyes are weakly Various types of metachromasia (Fig. 7.8):
metachromatic. In relation to thiazine dye, three types of meta-
Mechanism of metachromasia (Fig. 7.7): chromatic change may be seen [5]:
Glycosaminoglycans of the connective tissue
and epithelial mucins and granules of mast cells • Alpha (orthochromatic): The dye remains in
are negatively charged polyanions. The cationic monomeric form and gives a blue colour
Fig. 7.7 Mechanism of dye aggregation in metachroma- dye aggregates and the absorption of light changes by the
sia. The cationic dye interacts with the polyanionic tissue aggregated dye-tissue complex
and the bound water of the dye molecule is released. The
Box 7.3 Metachromasia

Metachromasia is a staining phenomenon
when the tissue is stained in different
colour from the original dye colour.
Metachromatic dyes
Cationic dye: Toluidine blue, methy-
lene blue, azure A and B, methyl violet.
Brilliant cresyl blue etc.
Anionic dyes: Biebrich scarlet and bro-
mophenol blue.
Mechanism of metachromasia: The
cationic (positively charged) dye in aque-
ous solution is neutralized with the poly-
anions of the tissues. Subsequently the
non-polar aromatic ring of the dye binds
with the other dye by Van der Waal’s
force. The dye absorbs shorter wave-
length of light and the visible colour
Fig. 7.8 The different types of metachromasia are high- changes.
lighted in this schematic diagram
Various types of metachromasia:
• Alpha (orthochromatic): Blue colour
• Beta (di and trimeric form): The dye forms • Beta (di and trimeric form): Purple
a dimeric structure and produces the purple colour
colour • Gamma (polymeric form): Red colour
• Gamma (polymeric form): The dye is in
polymeric form and produces a red colour. Factors enhancing metachromasia
• Higher dye concentration
The colour changes in metachromasia may not be • Low pH
homogenous. At times the anions in the tissue • Decreased temperature
may be widely distributed. In such cases, there • Aqueous solution
may be a purple colouration of tissue due to the
mixture of orthochromatic blue and polychro-
matic red colour.
Factors influencing metachromasia: The 7.6.2 Applications
following factors may influence metachromasia: of Metachromasia
1. Dye concentration: High concentration of dye Metachromatic dye is used in the following
enhances metachromasia. conditions:
2. pH: Low pH increases the metachromatic
effect. • To demonstrate metachromatic granules in the
3. Temperature: Decreased temperature aug- mast cells
ments metachromatic effect. • Demonstration of mucin
4. Aqueous solution: Water enhances Van der • Glycogen
Wall’s force in between the dye molecules • Amyloid material
and increases metachromatic effect. • Sulfatides
7.8 Mordant 67
7.7 Progressive and Regressive in the tissue. The mordant thereby removes the
Staining excess dye from the tissue.
4. One dye is replaced by another less affinity dye
Progressive staining: In the case of progressive
staining the dye is allowed to react with the tissue
until it stains the target structure. In fact, this is a 7.8 Mordant
difficult task to supervise and all other influenc-
ing factors should be controlled such as pH of the Mordant is the multivalent metal ion that com-
dye solution, the thickness of tissue, the concen- bines with certain dyes and helps in the attachment
tration of dye etc. It is always necessary to check of dye with the target tissue (Box 7.4). The metal
the staining at frequent intervals to prevent over- used as mordant is either divalent or trivalent such
staining or to have light staining. as Aluminium, iron, copper etc. The mordant
Regressive staining: Here the tissue is inten- binds with the dye by covalent or coordinate bond-
tionally overstained by dye. The affinity of the ing and this is commonly known as chelation. The
different structures of the tissue with dye is vari- term chelation is derived from the “chela” or large
able and this particular property is exploited to claw of the lobster. The metal ion grips two or
remove the dye from the unwanted part of the tis- more non-metal ions by the coordinate links that
sue. This procedure is also known as differentia- give the image of holding the prey with two claws.
tion. The differentiation is done by using:
1. Acid in basic dye or base in acidic dye such as 7.8.1 Lake

in Papanicolaou’s staining basic dye
Hematoxylin is removed from the cytoplasm The lake is defined as the “chelate compound
by using 1% acid alcohol. formed between the multivalent metal ion and the
2. Oxidising agent: The oxidizing agents are dye”. The word lake came from the lac, an insect
used to oxidize the dye and make it colourless in India from which the mordant was initially
material such as picric acid, potassium per- obtained. In course of time, the word lac is
manganate etc. changed to the lake. In the lake, there are two
3. Mordant: Here the dye-mordant complex at types of bonds: (1) a covalent bond between the
first binds with the tissue. Subsequently excess metal with the hydroxyl oxygen, and (2) a coor-
mordant is used that attracts the attached dye dinate bond with the other oxygen (Fig. 7.9).
Fig. 7.9 In the lake

there are two types of
bonds: (1) a covalent
bond between the metal
with the hydroxyl
oxygen, and (2) a
coordinate bond with the
other oxygen
The mordant-dye combination is basic in

action irrespective of the character of the dye. Box 7.4 Mordant
Mordant is insoluble in most biological fluids • It is the salt and hydroxides of the met-
and therefore the staining is not altered even after als that help in the attachment of dye
subsequent treatment of the tissue. with the target tissue.
How does the mordant act: The mordant is • Makes the dye strong
actually a bridge between the tissue and dye. • The mordant binds with the dye by
Mordant forms a coordinate and covalent bond covalent or co-ordinate bonding known
with the tissue. as “lake”
• Type
–– Pre-mordanting: The tissue is at first
7.8.2 Type of Application treated with mordant followed by
of Mordant dye.
–– Meta-mordanting: Mordant in com-
1. Pre-mordanting: The tissue is at first treated bination with dye is used.
with mordant followed by dye e,g Heidenhain’s –– Post mordanting: The dye material is
iron hematoxylin applied first followed by mordant
2. Meta-mordanting: Mordant in combination
with dye is used e,g Alum hematoxylin Example: Aluminium and hematoxylin
solutions
3. Post mordanting: The dye material is applied
first followed by mordant. It is not used in his-
topathology staining. 7.8.3 Accentuators
7.8.2.1 Example Accentuators are the group of substances that

help to increase the staining intensity of the dye.
1. Hematoxylin itself is a poor dye. However, the Accentuators neither form any dye lake nor do
combination of mordants such as Aluminium they take part in any chemical reaction. A com-
and hematoxylin makes a stronger dye. mon example of an accentuator is using potas-
2. In the case of Papanicolaou’s staining, the mor- sium hydroxide in methylene blue solution.
dant phosphotungstic acid is applied for better Table 7.2 highlights the comparison of mordant
cytoplasmic staining by OG6 and EA 36. and accentuators.
Table 7.2 The comparison of mordant and accentuators

Distinguishing points Mordant Accentuators
Mechanism Mordant makes a bridge between dye Accentuators only accelerates the dye tissue
and tissue. reaction
Chemical reaction It takes part in a chemical reaction by It does not take part in any chemical reaction
forming the lake
Example Alum and hematoxylin. Phenol in carbol fuchsin.
7.9 Staining Procedure 69
7.9 Staining Procedure ries prefer manual staining for small batches of
slides. In that case, the glass troughs are used. It
The proper organization of the staining room is is preferable to use a series of sequential arrange-
mandatory to get a well stained section. The ments of glass troughs for staining. All the
staining room should be well ventilated and well troughs should be well covered to prevent evapo-
illuminated (Box 7.5). ration of the reagents, particularly alcoholic solu-
The workflow of the histology/cytology labora- tions. In addition, the laboratory should have an
tory is fixation, grossing, processing, mounting, ample supply of distilled water.
microtomy and staining. Therefore, the staining The preparation of the staining reagent:
room should also be designed accordingly. The The preparation of staining reagents is one of the
staining bench should be window faced. The bench most important tasks in any laboratory. Adequate
should be cleaned properly with the arrangement cautions should be taken regarding the clean
of fume remover. There should be at least two sup- glassware, using distilled water instead of tap
plies of running tap water with a sink. water, following proper stepwise protocols to
Stains and equipment: The reagents should make the staining solution, and maintaining the
be kept in the rack with proper arrangement and concentration of alcohol in alcoholic solution
label (Box 7.6). The list of the reagents should be (Box 7.7).
in the laboratory catalogue. The glass bottle is the
best container to store the reagents. The use of an
amber colour bottle is preferable for the dye that
Box 7.7 Essential Precautions During
reacts with light. Frequently used reagents can be
Preparation of Staining Solution
kept in a small glass bottle or Coplin jar. A micro-
• Clean glassware
scope is necessary to check the stain. An auto-
• Distilled water instead of tap water
mated strainer can stain large batches of slides
• Alcoholic solution to keep in air tight
containing more than 100 slides. Many laborato-
container
• Stepwise proper protocol to follow
• Fresh solution in case of ammonia
Box 7.5 Staining Room
solution
• Arrange the room according to work flow
• Proper filtration of the staining solution
• Do not congest the staining room
before bacteriological stain
• Clean room and work bench
• Silver containing solution to be kept in
• Well ventilated and well illuminated
dark
• Running water with sink
7.9.1 Preparation of Buffer

Box 7.6 Reagents and Equipment Solutions
• All reagents in the rack in labelled glass
container 7.9.1.1 Molar Solution
• Maintain the readily available catalogue
• Amber coloured bottle for the reagents 1 M= One molar solution means the molecular
that needs protection from light weight of the compound expressed in g dis-
• The glass troughs well covered solved in 1000 ml of water.
• Ample supply of distilled water 0.1 M means one-tenth of the molecular weight
• Light microscope of the compound in gram dissolved in 1000 ml
water
7.9.1.2 Citrate Buffer 7.9.1.4 TRIS-HCl Buffer
Citric acid: Dissolve 2.101 g of citric acid in Stock A: 0.2 M TRIS.

100 ml of distilled water. 2.42 g TRIS (hydroxymethyl aminomethane of
Sodium citrate solution: Dissolve 2.9412 g of molecular weight 121) mix in 100 ml water
sodium citrate in 100 ml of distilled water. Stock B: 0.2 M HCl.
Final preparation: Now mix 46.5 ml of citric acid 1.7 ml HCl (molecular weight 36.46) in 100 ml
solution with 3.5 ml of sodium citrate solu- distilled water
tion. The distilled water is mixed in it to make
up to 100 ml. This will make 0.1 M citrate buf- To get pH 7.2: Mix 25 ml of stock A with 22.1 ml
fer. The pH of the solution is adjusted to 2.5 of stock B and make up to 100 ml by distilled
with the help of pH meter by adding 1 N HCl water.
and 5 N NaOH.
References
7.9.1.3 Phosphate Buffer
1. Horobin RW. Biological staining: mechanisms and
Stock A: 0.2 M of sodium dihydrogen orthophos- theory. Biotech Histochem. 2002;77(1):3–13.
phate: Mix 31.2 g of sodium dihydrogen 2. Prentø P. A contribution to the theory of biologi-
orthophosphate (molecular weight 156) in cal staining based on the principles for structural
organization of biological macromolecules. Biotech
1000 ml water. Histochem. 2001;76(3):137–61.
Stock B: 0.2 m disodium hydrogen orthophos- 3. Dapson RW. Dye-tissue interactions: mechanisms,
phate: Mix 28.3 g disodium hydrogen ortho- quantification and bonding parameters for dyes
phosphate (molecular weight 142) in 1000 ml used in biological staining. Biotech Histochem.
2005;80(2):49–72.
water. 4. D’mello AXP, Sylvester TV, Ramya V, Britto FP,
To get pH 6: Mix 438 ml of stock A with 62 ml of Shetty PK, Jasphin S. Metachromasia and meta-
stock B and make up to 1000 ml by distilled chromatic dyes: a review. Int J Adv Health Sci.
water. 2016;2(10):12–7.
5. Culling CF. Handbook of histopathological and his-
tochemical techniques: including museum techniques.
London: Heinemann; 2013.
Haematoxylin and Eosin Stain
of the Tissue Section 8
8.1 Introduction
Haematoxylin is the most commonly used dye in

the pathology laboratory [1, 2]. In combination
with eosin, this dye is almost indispensable for
routine morphological visualisation of tissue to
every histopathologist. Haematoxylin is a good
nuclear stain, and it stains the nuclei bluish-black.
However, the dye also stains collagenous mate-
rial, minerals, and myelin fibres.
8.2 Haematoxylin
Haematoxylin is extracted from the bark of the

Haematoxylum campechianum tree, which is
mainly seen in the Campeche state of Mexico.
Presently it is also available in West Indies.
Extraction: To get haematoxylin, the freshly Fig. 8.1 Schematic diagram showing the oxidation of
cut tree of Haematoxylum campechianum is haematoxylin and its combination with Aluminium
chopped into small pieces and boiled in water. At
first, the orange-red solution is formed, which
turns into a black solution on cooling. The com- The oxidation of haematoxylin to haematin can
pound haematoxylin is then precipitated with the be done in two ways:
help of urea or ether, and a brownish tan powder
is obtained. This is further purified and is sold 1. Natural: This is done by exposing the haema-
commercially. The compound haematoxylin has toxylin powder to sunlight and air. This is also
almost no staining capability unless a mordant is known as ripening. The oxidation process is
used to enhance its staining capability. slow and takes approximately three months.
Oxidation of haematoxylin (Fig. 8.1): However, the staining life of the dye is longer
Haematoxylin cannot act like a dye. Its oxidation than the natural ripening process
product haematin is a weak purple coloured dye.
https://doi.org/10.1007/978-981-19-6616-3_8
72 8 Haematoxylin and Eosin Stain of the Tissue Section
2. Chemical: This is done by treating the dye 8.3 Bluing

with hydrogen peroxide or sodium iodate, or
mercuric oxide. Sodium iodate is the most Most of the regressive staining of haematoxylin
commonly used oxidising agent. The conver- needs bluing. Removing an excess hydrogen ion
sion of haematoxylin to haematin is instant. from the stain is known as bluing. Here, the
However, the dye has a short useful life span.soluble hemalum is converted to an insoluble

form. Bluing gives the crisp blue colour of the
Dye-mordant complex: Haematin is a weak nuclei. In the process of bluing, the pH of the
anion and cannot combine with nucleic acid in solution is raised to 8.5 (alkaline side). The tissue
the nucleus. When a metallic salt (mordant) is section is treated with an alkaline reagent, and
combined with haematin, then a cationic dye- the acidic reagents are neutralised in the bluing
metal complex is formed that behaves as a strong process. The following methods do bluing:
basic dye and combines with nucleic acid
(Fig. 8.1). The mordant determines the type of • By running tap water for several minutes
tissue affinity of the dye and the colour of the • Treating the section with Scott’s tap water (pH
stain. Commonly aluminium (Al3+), iron (Fe3+), is 8): 2–3 min
molybdenum, tungsten, and lead salts are used as • Ammonium hydroxide (5%): 2–3 min
mordant. • Ammonia vapour: a few seconds
Types of haematoxylin: Haematoxylin can
be classified according to its combination with
different mordant such as: 8.3.1 Scott’s Tap Water
1. Iron haematoxylin Sodium bicarbonate 2g

Magnesium sulphate (anhydrous) 10 g
2. Alum haematoxylin
Water 1L
3. Tungsten haematoxylin
4. Lead haematoxylin Slowly add Magnesium sulphate to water so
5. Molybdenum haematoxylin that it dissolves and heat is dissipated.
6. Only haematoxylin (no mordant attached) Warning: The higher pH of the bluing agent
makes the bluing deeper blue colour quickly.
Table 8.1 highlights different types of haematox- However, be careful the tissue section in high pH
ylins with their mordant and properties. may be shed out from the slide.
Table 8.1 Essential types of haematoxylin

Stain type Mordant used Application Time to stain Comments
Mayer’s Potassium or Popular nuclear 5 to 10 min in progressive
haematoxylin ammonium alum counterstain stain and 10 to 20 min in
case of a regressive stain
Ehrlich’s Potassium alum Nuclear stain, and also 20 to 30 min Natural ripening.
haematoxylin stains mucin, bone and Stain is more
cartilage long-lasting.
Harris’s Ammonium or Good nuclear stain in 5 to 15 min in case of Ripening by
haematoxylin Potassium alum exfoliative cytology regressive stain and 5 to 30 s mercuric oxide
in progressive stain
Gill’s Aluminium Good nuclear stain Regressive stain: 5 to 15 min The stain is stable
haematoxylin sulphate for 1 year
Cole’s potassium alum Good nuclear stain Regressive stain: 30 min Stable for 3
haematoxylin months
8.3 Bluing 73
8.3.2 Preparation of Different 8.3.3 Mayer’s Haematoxylin

Haematoxylins and Their
Properties Mayer’s haematoxylin is used predominantly as a
nuclear counterstain where the cytoplasmic mate-
8.3.2.1 Harris’s Alum Haematoxylin rial is needed to demonstrate, such as in enzyme
This haematoxylin is a regressive stain widely histochemistry, PAS or mucicarmine stain.
used in exfoliative cytology as a nuclear stain. Potassium or ammonium alum is used as mor-
Eosin is used as a cytoplasmic stain. Ammonium dant. Mayer’s haematoxylin is used in progres-
or Potassium alum is used here as mordant. This sive staining, and it is best suited for automated
haematoxylin takes 5 to 15 min in case of regres- staining procedures.
sive staining and 5 to 30 s in progressive
staining. 8.3.3.1 Preparation of the Stain
Haematoxylin 1g
8.3.2.2 Preparation of the Stain Potassium or ammonium alum 50 g
Haematoxylin 5g Sodium iodate 0.2 g
Absolute alcohol 50 ml Citric acid 1g
Ammonium alum 100 g Chloral hydrate 50 g
Distilled water 1000 ml Double distilled water 1000 ml
Mercuric oxide 2.5 g
Glacial acetic acid 40 ml 8.3.3.2 Steps
8.3.2.3 Steps • Dissolve haematoxylin, potassium alum and

sodium iodate in distilled water
• Haematoxylin is dissolved in absolute • Gently heat the solution and stir
alcohol • Cool the solution to room temperature
• Alum in hot water is added • Add chloral hydrate and citric acid to the
• Heat to boil solution
• Mix both the solution • Boil the mixture for 4 to 5 min
• Now slowly add mercuric oxide • Cool the solution
• Heat again till the colour changes to dark • Filter
purple
• Rapidly cool the flask by dipping it in cold The differences between Mayer’s and Harris’
water haematoxylin are highlighted in Table 8.2:
• Add glacial acetic acid to the cold solution
Table 8.2 Differences between Mayer’s and Harris’
8.3.2.4 Cautions haematoxylin
Harris’s Mayer’s
• Addition of glacial acetic acid is optional. It Factors haematoxylin haematoxylin
gives crisp staining but reduces the life span of Solvent Absolute alcohol Distilled
the stain. water
• The life span of the stain decreases within 2 Oxidant Mercuric oxide Sodium
iodate
to 3 months. The formation of precipitate and
A substance Glacial acetic acid Chloral
increased stain time indicates that the stain added to sharpen hydrate
material is deteriorating. Always filter the the stain
sediment before the use and increase stain Stain type Both progressive Progressive
time. and regressive stain stain
8.3.4 Ehrlich’s Haematoxylin • Mix iodine solution and alum

• Boil
It is a strong stain for nuclei and also mucin, bone and • Cool and filter the solution
cartilage. The stain is oxidised naturally by keeping it
in air and sunlight for 2 months. The bottle of stain
can be kept in sunlight near the window. Once the 8.4 Counterstain by Eosin
stain is matured, it can be used in the laboratory.
Eosin is used as a counterstain of cytoplasm. It
8.3.4.1 Preparation of the Stain also stains the connective tissue and matrices. It
stains rosy red to pink colour. This is a xan-
Haematoxylin 2g
thene group of dye with a chemical structure
Absolute alcohol 100 ml
Distilled water 100 ml
“tetrabromofluorescein”. Three types of eosin
Glycerol 100 ml are seen: eosin Y, eosin B and ethyl eosin. Eosin
Glacial acetic acid 10 ml Y is the most widely used eosin among these
Potassium alum 10 to 15 g three types. The term Y indicates yellowish.
Eosin is soluble in both water and alcohol.
8.3.4.2 Steps of Preparation Aqueous solution: 1% eosin in distilled water.
Alcohol solution: 0.5% in 95% ethyl alcohol.
• Dissolve haematoxylin in absolute alcohol Washing the eosin-stained sections with tap water
completely helps in the differentiation of the eosin staining.
• Add the other ingredients
• Finally, add Potassium alum (10 to 15 g) till
8.5 Routine Haematoxylin
the full saturation occurs
and Eosin stain
• Keep the whole flask in sunlight for 2 months
for maturation
8.5.1 Requirements
Note: In case of any emergency to use the dye,
• Haematoxylin solution
one can add sodium iodate 100 mg in this solu-
• Grades of alcohol (100% and 95%)
tion for rapid maturation. In a strict sense, then
• 1% HCl in 70% alcohol
this haematoxylin solution should not be called
• 1% eosin
“Ehrlich’s haematoxylin”.
• Xylene
8.3.5 Cole’s Haematoxylin 8.5.2 Steps (Fig. 8.2)
Cole’s haematoxylin is an alum haematoxylin • Deparaffinization: Keep the sections in

oxidised by iodine in alcohol. This is used as a xylene for 10 min each, and three changes
regressive nuclear stain. • Rehydration:
–– Absolute alcohol: 1 to 2 min
8.3.5.1 Preparation of the stain –– 95% alcohol: 1 to 2 min
Haematoxylin 1.5 g –– 80% alcohol: 1 to 2 min
Saturated aqueous potassium alum 700 ml –– 60% alcohol: 1 to 2 min
Iodine (1%) in absolute ethanol (95%) 50 ml • Rinse in tap water
Distilled water 250 ml • Nuclear stain by Haematoxylin: Harris hae-
matoxylin 15 min (keep according to the type
8.3.5.2 Steps of Preparation of haematoxylin)
• Differentiation: In the case of regressive
• Add haematoxylin to distilled water staining, one to two dips in acid alcohol (1%
• Gently heat and dissolve it completely HCl in 70% alcohol) for differentiation is
8.5 Routine Haematoxylin and Eosin stain 75
Fig. 8.2 Basic steps of haematoxylin and eosin stain are highlighted
n ecessary. Please check by microscope for the

desired staining of the nucleus.
• Rinse in water
• Bluing: Wash by running tap water for 10 to
15 min
• Counterstain by eosin: 1% aqueous eosin Y
for 2 to 3 min
• Dehydration:
–– 95% alcohol: 3 min
–– Absolute alcohol: 3 min
–– Absolute alcohol: 5 min
• Clearing: Xylene 5 min each in two jars
• Mount in DPX
Fig. 8.3 Well stained tissue by haematoxylin and eosin
stain (haematoxylin and eosin stain ×200)
Result (Fig. 8.3):
• Nuclei: Blue to bluish-black.

• Cytoplasm: Pinkish.
8.5.3 Staining Time of Different • Progressive staining takes less time than
Haematoxylin regressive staining
• Pre-treatment of tissue: Staining time is lon-
8.5.3.1 Staining time of Haematoxylin ger if the tissue undergoes prolonged fixation
Depends on the Various Factors or pre-treatment with acid
• If the age of the stain is old then it takes a lon-
• The types of Haematoxylins, such as Cole’s ger time to stain
Haematoxylin, take 20 to 45 min to take nuclear
stain whereas, Mayer’s progressive Haematoxylin Troubleshooting in haematoxylin staining has
takes only 10 to 20 min (Fig. 8.4). been mentioned in Table 8.3 (Figs. 8.5, 8.6,
• Intensity of stain: Prolonged staining time and 8.7).
may be needed in heavily used Haematoxylin.
Fig. 8.4 Staining duration of different hematoxylin stain
Table 8.3 Troubleshooting in haematoxylin staining

Problems Possible causes Remedies
Pale stained nuclei 1. Too much differentiation 1. Stain in haematoxylin again.
2. Too less time in haematoxylin. 2. Keep in haematoxylin for a longer
3. Due to excessive decalcification duration
4. Haematoxylin is over oxidised. 3. Not possible to correct
4. Change the haematoxylin solution
Darkly stained nuclei 1. Too short differentiation 1. Decolorize and do optimum
2. Too much time in haematoxylin. differentiation
3. Thick section 2. Decolorize and give appropriate time
in haematoxylin.
3. Recut thin section
The nuclei look reddish-brown 1. Insufficient bluing 1. Re-stain by giving more time in the
2. Hematoxylin is degenerating bluing step
2. Check the oxidation status of
haematoxylin
8.5 Routine Haematoxylin and Eosin stain 77
Table 8.3 (continued)

Pale coloured cytoplasm by 1. Too thin section 1. Recut the section properly
eosin 2. The eosin solution has a pH more 2. This may be due to the dilution of
than 5. eosin by the carryover bluing solution.
3. Too much dehydration of the section Check the pH of the eosin solution and
in alcohol adjust the pH by adding acetic acid.
3. Do not keep the slide in alcohol for
long time
Cytoplasmic staining is very 1. Long duration in eosin solution 1. Keep the section in eosin for shorter
dark 2. Over concentrated eosin solution duration
3. Very quick dehydration in alcohol 2. Make optimally diluted eosin
solution
3. Increase time duration in
dehydration
Bluish black precipitate It may be due to the precipitation of Filter the haematoxylin staining
haematoxylin solution
The staining is irregular and Improper deparaffinisation Keep the slide in xylene for a longer
spotty. time to remove the paraffin.
Dark blue stain at the edge of Due to heating artefact for using No solution
the tissue sections electrocautery
Water bubbles in the sections. Incomplete dehydration Remove the mounting medium and
coverslip. Keep the section in absolute
alcohol for dehydration. Do several
changes and then remount.
Milky section after the xylene Incomplete dehydration Change the alcohol solution. Please
rinses before putting the dehydrate the section properly before
coverslip. putting in xylene
Fig. 8.5 Pale stained nuclei (haematoxylin and eosin

stain ×200)
Fig. 8.6 Too dark

nuclear stain
(haematoxylin and eosin
stain ×200)
8.6.1 Heidenhain’s Iron

Haematoxylin
Heidenhain’s iron haematoxylin stains the tissue

jet black. It stains various cellular constituents
such as mitochondria, chromatin and nucleoli. In
addition, it also stains myelin sheath and striations
of muscle. This gives a regressive stain. Iron alum
is used as an oxidising agent of haematoxylin, and
ferric ammonium sulphate is used as mordant. The
approximate time of staining is 30 min.
Fig. 8.7 Imperfect deparaffinization of the tissue section
(haematoxylin and eosin stain ×200) 8.6.1.1 Application
• The stain is widely used for the demonstration

8.6 Iron Haematoxylin of protozoa. The darker stained nuclear struc-
ture is identified clearly in contrast to the
In this type of haematoxylin, iron salts are used cytoplasm.
as mordants, such as ferric chloride or ferric • Cross striations of the muscle fibre
ammonium sulphate. The different types of iron • Mitochondria
haematoxylin are:
8.6.1.2 Preparation
1. Heidenhain’s iron haematoxylin Solution no 1:
2. Weigert’s iron haematoxylin Haematoxylin: 0.5 g
3. Verhoeff’s haematoxylin Absolute alcohol: 10 ml
4. Loyez haematoxylin Distilled water: 90 ml
8.6 Iron Haematoxylin 79
Step: No 2:
Ferric chloride: 10 g
• Haematoxylin is dissolved completely in Distilled water: 100 ml
absolute alcohol No 3:
• Add distilled water Iodine: 1 g
• Keep the solution for 4 to 6 weeks for Potassium iodide: 2 g
ripening Distilled water: 100 ml
No 4: Working solution:
Solution no 2: Add
Ferric ammonium sulphate: 5 g No 1: 40 ml
Distilled water: 100 ml No 2: 16 ml
Step: Dissolve violet crystal of Ferric ammo- No 3: 16 ml
nium sulphate in distilled water. Mix those solutions in order.
Steps
8.6.1.3 Staining
• Dewax
• Dewax the tissue • Serial grade of alcohol for hydration
• Absolute alcohol • Stain with freshly prepared working haema-
• 95% ethyl alcohol toxylin solution for 10 min
• Keep section in mordant solution (solution no • Rinse in water
2) for 60 min • Differentiation: By 2% ferric chloride
• Rinse in distilled water • Wash with tap water
• Stain by solution 1 (Heidenhain’s haematoxy- • Remove iodine by 95% ethyl alcohol: 5 min
lin 0.5%) for 60 min • Counterstain: 1% eosin for 1 to 2 min
• Rinse in water • Dehydrate
• Differentiate in 5% alum solution • Clean by xylene
• Wash in running water: 5 to 10 min • Mount by DPX
• Dehydrate
• Clean by xylene Result: Elastic fibre takes black stain.
• Mount in DPX
Result: Target tissue takes black stain. 8.6.3 Tungsten Haematoxylin
Tungsten haematoxylin demonstrates nerve tissue

8.6.2 Verhoeff’s Iron Haematoxylin (such as astrocyte), muscle tissue and collagen.
Here phosphotungstic acid is used as mordant.
Verhoeff’s iron haematoxylin is used to stain
elastic tissue. The stain provides very good con- 8.6.3.1 Preparation
trast for microphotography. Ferric chloride is Solution A:
used here as mordant. Strong iodine is used as an Haematin: 0.8 g
oxidising agent. The whole solution should be Distilled water: 1 ml
prepared just before use because the solution Steps: Dissolve 0.8 g haematin in 1 ml dis-
may be over oxidised if kept for more than 1 h. tilled water.
Solution B:
8.6.2.1 Preparation Phosphotungstic acid: 0.9 g
No 1: Distilled water: 9 ml
Haematoxylin: 5 g Steps: Mix both the solution A and B to get the
Alcohol (100%): 100 ml final staining solution.
8.6.3.2 Staining 8.7 Clearing the Smear
• Dewax Removal of alcohol or clearing of the sample is

• Serial grade of alcohol for hydration done by putting the sample in xylene. The chemi-
• Treat with 0.25% potassium permanganate for cal compound of xylene is dimethyl benzene.
oxidation - 5 min The clearing agent should be colourless and its
• Wash in distilled water refractive index should be close to the mounting
• Bleach: By oxalic acid (5%) for 5 min. media and coverslip (Box 8.1).
• Wash in tap water
• Stain: PTAH final solution for 12 to 24 h at
room temperature
Box 8.1 Clearing Agent
• Wash in distilled water
• Quick dehydration by 95% alcohol and abso- • Xylene (dimethyl benzene)
lute alcohol
Basic properties
• Clean by xylene
• Colourless
• Mount by DPX
• Refractive index should be same as the
mounting media and coverslip.
Note: Quick dehydration prevents the removal of
• It gives transparent cytoplasm
red stain by alcohol.
Warning: Toxic.
8.6.3.3 Result
Connective tissue materials such as collagen,
reticulin fibres etc.: Red.
Nuclei, centrioles, striated muscle etc.: Blue.
The different uses of haematoxylin are high- 8.8 Mounting
lighted in Table 8.4.
The primary function of the mounting media is to
Table 8.4 Applications of different haematoxylin stains give a protective cover over the smear and to
for other purposes make a permanent bond between the coverslip
Substances Haematoxylin and the slides (Box 8.2). The mounting medium
Routine stain in histology Harris should have the following properties:
sections and cytology smears haematoxylin
Carbohydrates Mayer’s • The same refractive index of the coverslip and
haematoxylin
glass slide. The refractive index of the mount-
Phospholipid Baker’s acid
haematein ing medium should be 1.52 to 1.54.
technique • Mounting media should be colourless
Fibrin, cross striations of skeletal Tungsten • It should quickly dry and stick to the slide
muscle haematoxylin • It should resist contamination, particularly the
Connective tissue fibres Verhoeff’s iron growth of microbes
haematoxylin
• It should not react with the stain or tissue
Amoeba, microfilaria Iron haematoxylin
Nuclear chromatin Gill’s • It should be miscible with the clearing agent
haematoxylin • A neutral pH to prevent fading of the stain.
Photomicrography Heidenhain’s iron • Low viscosity otherwise there may be air bub-
haematoxylin ble formation when putting the coverslip.
Counterstaining in Ehrlich’s These air bubbles are brownish in colour and
immunohistochemistry and haematoxylin
cytochemistry are known as a cornflake artifacts.
8.8 Mounting 81
Canada balsam is not used nowadays.

Box 8.2 Mounting Medium Synthetic resins: DPX is the most widely
Aim: To give a protective cover over the used synthetic resin with a refractive index of
smear and to make a permanent bond 1.523. It is called DPX as it contains.
between the coverslip and the slides. D = Distyrene.
P = Plasticiser (tricresyl phosphate).
Ideal mounting medium X = Xylene.
• The same refractive index of the cover- DPX is colourless and preserves the stains
slip and glass slide (1.52 to 1.54). very well. It also dries very quickly.
• Colourless DPX should be used liberally over the slide,
• It should quickly dry and stick to the slide and the excess DPX should be wiped off from the
• Resist the growth of microbes coverslip margin.
• No reaction with the stain or tissue
• Miscible with clearing agent
• A neutral pH to prevent fading of the stain 8.8.1 Disadvantage
• Low viscosity
• It frequently retracts from the margins of
Types of mounting medium the coverslip. This can be prevented by add-
• Neutral resins: such as Canada balsam, ing a plasticiser that makes a mesh with
Euparal plastic [3].
• Synthetic resins: DPX
• Aqueous media: Glycerine, polyvinyl Aqueous media: The aqueous media are used
alcohol etc. when the stains are affected by dehydration by
alcohol or clearing by xylene e,g fat stain by
Permanent mounting: DPX Sudan black needs aqueous media.
Temporary mounting: Glycerine- Glycerine glycerol: This is the commonly
glycerol, polyvinyl alcohol. used aqueous media for temporary mounting.
Polyvinyl alcohol: This is an alternative to
glycerine jelly and is often used in immunofluo-
Types of mounting media: There are three types rescence or frozen sections of lipid stains.
of mounting media: Table 8.5 highlights the comparison of differ-
ent mounting media.
• Neutral resins: such as Canada balsam,
Euparal
• Synthetic resins: DPX 8.8.2 Application of Mounting
• Aqueous media: Glycerine, polyvinyl alcohol etc. Medium
Neutral resins: The commonly used neutral • Put 1–2 drops of mounting medium on the
resin is Canada balsam. Its refractive index of it is middle of the tissue section over the slide
1.523. Canada balsam is well soluble in xylene • Select an appropriate coverslip for the section
and is made as: • Rapidly invert the slide over the coverslip
Canada balsam: 60 g • Mounting medium slowly spreads under the
Xylene: 100 ml coverslip
Disadvantages:
8.8.2.1 Cautions
• Yellow staining after some time
• Takes time to dry • Too small amount of mounting medium: Air
• The basic dyes are poorly preserved bubbles may appear.
Table 8.5 The comparison of different mounting media

Mounting Refractive
medium index Common use Advantages Disadvantages
Canada 1523 Permanent mounting Well soluble in • Yellow staining after some time
balsam xylene • Takes time to dry
• The basic dyes are poorly preserved
DPX 1523 Permanent mounting • Preserve the • Retraction of margin of the
standard stains coverslip
• Quickly dry
Glycerine- 1.47 • Temporary mounting • Inexpensive • Unsuitable for prolonged
glycerol • Oil red O and Sudan • Safe preservation, and the coverslip
black stain • Quick to apply margin should be sealed
• Fluorescent stain
Polyvinyl 1.5 • Temporary mounting A safe and good • Unsuitable for prolonged
alcohol • Fat stain alternative to preservation
• Fluorescent stain glycerine glycerol
• Too much amount of mounting medium: It 8.8.5 Restaining

may spread beyond the edges of the coverslip,
and the sample may also float. In case of a faded slide, the re-staining may be
needed. The steps of re-staining include:
8.8.3 Coverslip
• Removal of the coverslip: Keep the slide in
Coverslip gives a protective covering over the xylene for 2 days or keep the slide and xylene
smear and prevents fading of the stain or any fur- at 50 °C for 15 min
ther physical damage. The good coverslip should • Removal of the excess mounting medium:
have the following characteristics: Take out the slide and keep it in a slanting
position followed by gentle immersion a few
• Clear glass of 0.130 to 0.170 mm thickness times in xylene.
• Plane surface and straight margin • Hydration: Hydrate the slide through a few
• Sufficiently wide to cover the smear changes in graded alcohol.
• Decolourisation: It is done by dipping the
The liquid coverslip is equally effective. slide in 1% acid alcohol
Nowadays, many laboratories use automatic • Washing: Wash in water to remove any acid
cover slip machines. alcohol
• Re-staining
8.8.4 The Resin-coated Plastic Film
The resin-coated plastic film is a particular type References

of film that completely avoids using any mount-
ing medium and glass coverslip. 1. Chan JK. The wonderful colours of the hematoxylin-
eosin stain in diagnostic surgical pathology. Int J Surg
Pathol. 2014;22(1):12–32.
8.8.4.1 Advantages 2. King DF, King LA. A brief historical note on stain-
ing by hematoxylin and eosin. Am J Dermatopathol.
• Very fast and automatic coverslipping 1986;8:168.
3. Brown PA. A review of technique used in the prepara-
• Long duration tion, curation, and conservation of microscope slides
• Excellent quality at the natural history museum London. Biol Curator.
• Free of any artefact 1997;10:1–33.
• No error during slide scanning
Special Stains for the
Carbohydrate, Protein, Lipid, 9
Nucleic Acid and Pigments
Table 9.1 Commonly used stain for different substances

9.1 Introduction
Material Stain
Carbohydrate • Periodic Acid Schiff (PAS)
Other than routine haematoxylin and eosin stain- • Alcian blue
ing, various special stains are now essential parts • PAS and Alcian blue
of routine laboratory work. Box 9.1 highlights • Mucicarmine
the overall indications of these special stains in Lipid • Oil red O
• Sudan black
the laboratory. This chapter will discuss the basic • Ferric haematoxylin
principles, applications and techniques of differ- Nucleic acid • Feulgen stain
ent stains. • Acridine orange
• Methyl green pyronin
Hemosiderin pigment • Pearl’s reaction
Box 9.1 Applications of Special Stain Bile pigment • Fouchet’s stain
Melanin • Masson Fontana method
• Demonstration of various cellular prod-
• Schmorl’s stain
ucts for diagnosis
–– Carbohydrates
–– Proteins
–– Lipids 9.2 Carbohydrates
–– Pigments
• Demonstration of extracellular material Carbohydrates are compounds that contain poly-
for the identification of diseases such as hydroxy aldehydes or polyhydroxy ketones
amyloid groups. They are represented by the standard
• Identification of microbial organisms formula.
• Estimation of DNA and RNA content of Cn(H2O)m. The carbohydrates can be classi-
the cell fied depending on the number of subunits as
monosaccharides, oligosaccharides and polysac-
charides. They are also further classified depend-
Table 9.1 shows the commonly used special ing on their binding with protein and lipids
stains in histopathology and cytology laboratories. material.
https://doi.org/10.1007/978-981-19-6616-3_9
84 9 Special Stains for the Carbohydrate, Protein, Lipid, Nucleic Acid and Pigments
Polysaccharide (Fig. 9.1): Polysaccharide

Box 9.2 Classification of Carbohydrate consists of multiple monosaccharides linked by
• Simple carbohydrates covalent bonds.
–– Monosaccharide: e.g. glucose, Example: Glycogen, starch. Glycogen is avail-
galactose able in large quantities in liver followed by skel-
–– Oligosaccharide: e.g. maltose, etal and cardiac muscle.
sucrose Glycoconjugates or Proteoglycans: These
–– Polysaccharide: e.g. glycogen, starch are primarily groups of extensively glycosyl-
• Glycoconjugates (Complex ated proteins. The proteoglycans have a central
carbohydrates) core protein that is covalently linked with
–– Acid mucopolysaccharides: polysaccharides. The carbohydrate part of the
Carboxylated: e.g. hyaluronic acid proteoglycan is known as glycosaminoglycans.
Sulphated: e.g. chondroitin The different types of glycosaminoglycans
sulphate include:
–– Mucin
Neutral mucin: Surface epithelial • Chondroitin sulphate: Present in cartilage,
cells of stomach ligament, bone
Acidic mucin • Dermatan sulphate: Present on skin
Sialomucin: Goblet cells, sali- • Heparan sulphate: Present in the aorta
vary glands • Heparin: Present in granules of mast cell
Sulphomucin: Mucus glands of • Hyaluronic acid: Present in synovial fluid
bronchus
–– Others Mucin: Mucins are glycoproteins of high molec-
1. Glycolipid ular weight and are composed of a polysaccha-
2. Membrane protein ride chain and a protein component.
3. Blood group antigen Mucin = 80% carbohydrate (hexosamine con-
taining carbohydrate) + 20% protein.
Mucin covers the epithelial cells and makes a
physical barrier that protects the cells from any
9.2.1 Simple Carbohydrates external injury [1]. Mucin may be of two types:
Monosaccharides: These are the simplest form 1. Secretory mucin: Secreted in the respiratory
of carbohydrates with the empirical formula tree, gastrointestinal tract and cervical part of
(CH2O)n. They are the building blocks of various the female genital tract.
other carbohydrates. They contain an aldehyde or 2. Membrane-associated mucin: The mucin
ketone group and the varying number of carbon attached to the membrane of cells. Mucin may
atoms (five C atoms = pentose, six C atoms = also be noted in non-epithelial tissues.
hexose etc.). The monosaccharides are water-
soluble and, therefore difficult to demonstrate in The amino acid components of the protein core
the routine histology section. may be variable, and depending on the tandem
Example: Glucose, ribose, fructose etc. repeats of the nucleotide sequence of the amino
Figure 9.1 shows the structure of the glucose acid of the protein component of mucin, it may
molecule. be classified as many distinct functional types of
Oligosaccharides (Fig. 9.1): These are the mucin [2, 3] (Table 9.2). So far, 20 such MUC
polymers of monosaccharides that contain two to genes have been described.
ten monosaccharides units. The MUC genes are tissue-specific and have
Example: Sucrose, lactose, maltose. distinct biophysical and biochemical properties.
9.2 Carbohydrates 85
Fig. 9.1 Structure of monosaccharide, oligosaccharide and polysaccharide
Table 9.2 Tissue specificity of MUC genes

MUC gene Normal locations Tumour and MUC
MUC 1 bronchi, breast, Urinary bladder, The majority of adenocarcinomas are positive for MUC1.
Kidney, stomach, pancreas, gall Adrenocortical and hepatocellular carcinomas are always
bladder, prostate, negative for MUC1.
cervix
MUC 2 The small and large intestine, Colonic carcinomas are positive for MUC2.
salivary gland and endometrium
MUC 3 Salivary gland, gall bladder, Colonic carcinomas, pancreatic carcinoma,
small and large intestine
MUC 4 Bronchus, endocervix, stomach, Increased in pancreatic, breast, lung and ovarian carcinomas
small and large intestine
MUC 5 AC Bronchus, stomach, endocervix, Endocervical adenocarcinoma and gastrointestinal carcinomas
are positive
MUC 6 endocervix, endometrium Gastric carcinomas show highly expressed MUC6. In colonic
Stomach, gall bladder, ileum, carcinoma, MUC 6 is increasingly expressed along with low
MUC 2.
MUC 7 Sublingual and submandibular Markers of urothelial carcinoma and the demonstration of MUC
glands 7 in lymph node identifies micro-metastasis from the bladder
The carbohydrate part of mucin consists of Staining: These mucins are positive for Alcian blue
80% of the molecular weight of mucin. The poly- stain at low pH (2.8) and are negative for PAS stain.
saccharide part of the mucin may be neutral, Strongly sulphated acid mucins: This type
weakly acidic or strongly acidic. of mucin consists of
Neutral mucin: The name indicates that the
polysaccharide chain here is neutral. • Connective tissue mucin: Chondroitin sul-
Locations: Neutral mucin is noted in surface phate, keratan sulphate, heparin sulphate
epithelial cells of the stomach, prostate and • Bronchial glands
Brunner’s gland of the duodenum. • Some fractions of goblet cells of the intestine
Staining: These mucins are positive for
Periodic acid Schiff’s stain and negative for Strongly sulphated mucins are PAS negative and
Alcian blue stain. Alcian blue positive at pH 0.5.
Acidic mucin: Here the polysaccharide chain Weakly sulphated acid mucins: They are epi-
is anionic. thelial mucin. This group consists of:
9.2.1.1 Locations • Colonic goblet cells

1. Sialomucin: It contains sialic acid. Sialic acid • Bronchial glands
is derived from the acetylation of neur-
aminic acid. This is weak acidic mucin. Weakly sulphated acid mucins are Alcian blue
Sialomucin is found in goblet cells, salivary positive at pH one and negative for PAS.
glands etc. Both neutral and acidic mucins are positive for
2. Sulphomucin: It contains a sulphate group, mucicarmine.
and it is stronger acidic. This type of mucin is Figure 9.2 highlights the staining pattern of
seen in the mucus glands of the bronchus. different types of mucins.
Fig. 9.2 The flow diagram shows different stains for mucin. AB: Alcian blue, PAS: Periodic acid Schiff
9.3 Staining of Different Carbohydrates 87
9.2.2 Significance of Mucin • Glycolipid: PAS helps to demonstrate cere-

Demonstration brosides and gangliosides. Glucocerebrosides
and galactocerebrosides are accumulated in
• Mucin secreting adenocarcinomas are positive Gaucher’s and Krabbe’s disease, respectively.
for mucin stain. Whereas the lymphomas or Gangliosides are accumulated in rare lyso-
other poorly differentiated carcinomas do not somal storage diseases.
show intracellular mucin. • Pigments: Certain pigments such as lipofus-
• Intestinal metaplasia of the stomach show sul- cin and pigments of Dubin Jonson syndrome
phomucin, which is prone to the development are demonstrated by PAS stain.
of malignancy. • Plasma cells: Russel bodies of plasma cells
• Pleural mesotheliomas are positive for acid are stained by PAS.
mucin
9.3.4 Principle (Fig. 9.3)

9.3 Staining of Different
Carbohydrates • The hydroxyl group (OH) of the carbohydrate
molecule is oxidised to the aldehyde (CHO)
9.3.1 Glycogen group by periodic acid.
• These aldehyde groups react with Schiff’s
Glycogen, the polysaccharide, is demonstrated reagent to form a magenta coloured compound
by periodic acid Schiff’s (PAS) reaction.
9.3.2 Periodic Acid Schiff’s (PAS)

Stain [4]
PAS stain demonstrates neutral polysaccharides

present in the basement membrane and the secre-
tion of various glands in our body.
9.3.3 Indications to do PAS stain
• To demonstrate polysaccharides: PAS

helps to demonstrate glycogen, cellulose and
starch. It demonstrates glycogen in glycogen
storage disorders. The basement membrane
of the glands, glomeruli etc., can also be
demonstrated by PAS stain. The capsule of
the various fungi contains carbohydrate
material, and the capsule is demonstrated by
PAS such as cryptococci, Histoplasma, blas-
tomycosis etc.
• Glycoprotein: Mucin, particularly neutral
Fig. 9.3 Schematic diagram shows the basic principle of
mucin, is demonstrated by PAS. The stain is PAS stain. Periodic acid converts the hydroxyl (OH)
helpful for staining mucin of endocervical group to the aldehyde group (CHO) that reacts with
glands, intestinal glands, and bronchial glands. Schiff’s reagent to give a magenta colour
9.3.4.1 Components of Solutions 9.3.5 Alcian Blue

Solution 1: Periodic acid (1%)
Periodic acid: 1 g Alcian blue stains acid mucin (in acidic pH 2.5),
Distilled water: 100 ml such as silaomucin and sulphomucin. It stains
Solution 2: Schiff’s reagent mucin of salivary glands, prostate, and large
Basic fuchsin: 1 g intestine. Alcian blue also stains proteoglycans of
Distilled water: 200 ml the cartilaginous material.
Potassium Metabisulphite: 2 g
1 N hydrochloric acid (HCl): 20 ml 9.3.5.1 Indications
Activated charcoal: 2 g • Intestinal metaplastic cells in Barrett’s
oesophagus and stomach biopsy: The intesti-
9.3.4.2 Preparation nal metaplastic cells contain acid mucin, and
• Dissolve basic fuchsin (1 g) in 200 ml of boil- these cells in Barrett’s oesophagus are better
ing distilled water. demonstrated by Alcian blue.
• Cool the solution • Mucinous adenocarcinoma of the ovary
• Add 1 N hydrochloric acid and mix well • Pleural mesothelial cell: The pleural mesothe-
• Add Potassium Metabisulphite (2 g) lial cells contain hyaluronic acid that is Alcian
• Add activated charcoal (2 g) blue positive and sensitive to hyaluronidase
• Keep the solution in the dark enzyme. Adenocarcinoma cells are Alcian blue
positive and resistant to hyaluronidase enzyme.
• Myxoma: Mucin secreting tumours such as
9.3.4.3 Steps myxomas are positive for Alcian blue stain.
1. Deparaffinise • Others: Mucinous material in myxoedema,
2. Pass through graded lower concentration of discoid lupus erythematous lesion etc. are also
alcohol and section/smear to bring in demonstrated by Alcian blue stain.
water.
3. Oxidise with Periodic acid (1%) for 5 to Basic principle: Alcian blue is a group of water-
10 min soluble polyvalent basic dyes. The dye is made of a
4. Clean with water copper-containing phthalocyanine ring with a cop-
5. Schiff’s reagent for 20 to 30 min per atom in its centre. The Phthalocyanine ring is
6. Clean in running tap water for 5 min. also attached with four isothiouronium groups that
7. Counterstain with haematoxylin are positively charged (Fig. 9.4). This positively
8. Wash in tap water for blueing charged Alcian blue dye complex has an attraction
9. Absolute alcohol with anionic sites of the mucin. Copper imparts the
10. Clear in xylene blue colour of the dye-mucin complex.
11. Mounting
9.3.5.2 Solution
9.3.4.4 Result
Glycogen and glycoprotein: Magenta colour. Alcian Blue Solution
Materials that are positive for PAS reac- Alcian blue, 8G 1% aqueous solution: 1 g
tion: Glycogen, starch, mucin, reticulin, base- Acetic acid (3%) solution: 100 ml
ment membrane, capsule of fungi etc.
Testing the Schiff’s reagent: Neutral Red Solution
Add drops of Schiff’s reagent to formalin. Neutral fast red: 1 g
Active Schiff’s reagent will quickly change the Aluminium sulphate: 5 g
colour of formalin to pink. Deionized water: 100 ml
9.3 Staining of Different Carbohydrates 89
Fig. 9.4 Schematic

diagram shows the basic
principle of Alcian blue
stain. Copper containing
Pthalocyanine ring is
attached with four
positively charged
isothiouronium groups
that has an attraction
with anionic sites of the
mucin. Copper imparts
the blue colour of the
dye-mucin complex
Steps to Make Solution

• Dissolve Aluminium sulphate in deionized
water and heat
• Mix neutral fast red in hot water
• Filter
9.3.5.3 Method of Staining

1. Deparaffinise
2. Rehydration of the section/smear by graded
alcohol
3. Rinse in deionised water
4. Keep the smear in Alcian blue for 30 min
5. Rinse in running water: 5 min
6. Counterstain with Neutral fast red: 10 min
7. 95% ethyl alcohol
8. Absolute alcohol
9. Xylene
Fig. 9.5 The combined PAS and Alcian blue stain: PAS-
10. Mount Alcian blue stain at pH 2.5 highlighting the intestinal
metaplasia (stained blue by Alcian blue) in an antral
Result: Acid mucin (sialomucin, sulphomucin), biopsy. The magenta stained mucin is the normal neutral
proteoglycans, and hyaluronic acid will take blue mucin of the antrum (40×) (Courtesy of Dr. Suvradeep
Mitra, Assistant professor, Department of Histopathology,
colour. Post Graduate Institute of Medical Education and
Research, Chandigarh, India)
9.3.6 Combined PAS-Alcian Blue (Fig. 9.5). This is frequently applied in gastroin-
Staining testinal biopsy sections.
Solution: Alcian blue, periodic acid and
Indications: Combined use of Alcian blue and Schiff’s reagent solution can be made as described
PAS in the same section helps to demonstrate before.
both acidic and neutral mucin in the same section
9.3.6.1 Method of staining

1. Deparaffinise
2. Rehydration of the section/smear by graded
alcohol
3. Rinse in deionised water
4. Keep the smear in Alcian blue for 30 min
5. Wash in tap water followed by deionised water
6. Periodic acid (1%) for 5 to 10 min
7. Clean in running tap water for 5 min.
8. Schiff’s reagent for 20 to 30 min Fig. 9.6 Schematic diagram shows basic principle of
mucicarmine stain. The positively charged carmine com-
9. Clean in running tap water for 5 min plex binds with negatively charged anionic acid mucin
10. Counterstain with haematoxylin
11. Wash in tap water for blueing
12. Absolute alcohol Alcohol (50%): 100 ml
13. Clear in xylene Anhydrous aluminium chloride: 0.5 g.
14. Mounting
Preparation
• Add carmine and aluminium hydroxide in
9.4 Result 50% alcohol
• Mix anhydrous aluminium chloride 0.5 g
Glycogen: Magenta colour. • Gently shake
Acid mucin: Blue colour. • Boil the whole solution by keeping the flask in
the hot water bath
9.4.1 Mucicarmine Stain [5] • Cool
• Filter the solution and preserve it at 4 °C. This
9.4.1.1 Indications will be fit for use for 4 to 6 months
• Mucicarmine stain demonstrates both neutral
and acid mucin.
• It stains mucin of intestinal adenocarcinoma Mucicarmine working solution
• The capsule of fungi such as cryptococci is Mucicarmine stock solution: 10 ml
stained by mucicarmine Deionized water: 90 ml
Principles: The active dye molecule in mucicar- Steps

mine stain is Carmine. Carmine is composed of a 1. Deparaffinise
multi-ring molecule of carminic acid bound with 2. Rehydration of the section/smear by graded
an aluminium ion. The carmine complex is posi- alcohol
tively charged, and so it binds with negatively 3. Rinse in water
charged anionic acid mucin (Fig. 9.6). 4. Haematoxylin for 5 to 10 min
Applications: Mucicarmine stain is applied 5. Wash in water
to demonstrate acid mucin in the malignant cells 6. Mucicarmine solution 30 min
of adenocarcinoma. It also stains capsules of 7. Rinse in water
Cryptococcus. 8. 95% ethyl alcohol
9. Absolute alcohol
9.4.1.2 Solution 10. Xylene
11. Mount
Southgate Mucicarmine stock solution
Carmine: 1 g Interpretation: Positive mucicarmine stain
Aluminium hydroxide: 1 g shows dark red colour.
9.5 Colloidal Iron 91
9.5 Colloidal Iron 9.5.1.2 Result

Acid mucin: Blue.
The colloidal iron technique is helpful for the Collagen: Red.
detection of acid mucopolysaccharides. Muscle: Yellow.
Principle: The positively charged ferric ion in
the colloidal ferric oxide solution is attracted by
the negatively charged carboxyl and sulphate 9.5.2 Lipids
groups of the acid mucin. The potassium ferro-
cyanide then demonstrates the ferric ion attached Lipid word originated from the Greek word
to the tissue. “Lipos”, which means fat. Lipids are defined as a
group of naturally available organic fatty sub-
stances soluble in alcohol and insoluble in water.
9.5.1 Colloidal Ion Stalk Solution Lipids are the major components of the cell
membrane and the membranous part of many cel-
A total of 4.4 ml of 29% ferric chloride is mixed lular organelles. The myelin component of the
with 250 ml of double-distilled water and boiled nerve sheath is also made of lipid.
until the colour changes to dark red. Lipids are classified as simple, compound,
and derived lipids (Table 9.3).
• Colloidal ion working solution: Simple lipids: These lipids on hydrolysis pro-
• Sock solution of colloidal iron: 20 ml duce fatty acid and glycerol (Fig. 9.7). Example:
• Deionized water: 15 ml fats, oils and waxes.
• Glacial acetic acid: 5 ml Fats: Esters of fatty acid and glycerol.
–– Acetic acid (12%): 12 ml of glacial acetic Wax: Esters of fatty acid and alcohol (other
acid in 100 ml of deionised water. than glycerol).
–– Potassium ferrocyanide: 5 g in 100 ml Compound lipids: These are composed of
deionised water. fatty acid, alcohol and another group such as sul-
–– HCl 5%. phur, phosphorous, or carbohydrate producing
–– -Potassium ferrocyanide-HCl: 50 ml of sulpholipid, phospholipid, glycolipid etc.
each of the above solutions just before the Derived lipids: The derived lipids are the sub-
use. stances derived from the hydrolysis of simple or
–– -Acid fuchsin1 g in 100 ml water. compound lipids.
–– -Van Gieson working mixture: 5 ml Acid Example: Fatty acids, steroids, cholesterol,
fuchsin (1%) in 95 ml picric acid (saturated) Carotenoids etc.
9.5.1.1 Method
• Deparaffinise the section and bring it in water
by serial changes in graded alcohol Table 9.3 Classification of lipids
• Dip in acetic acid for 1 min Simple lipid Compound lipids Derived lipids
• Keep in colloidal iron for 1 h • Fatty acid • Phospholipid • Steroids
• Rinse in acetic acid 3 to 4 changes and 3 min • Simple – Glycerol based • Terpenes
triglycerides Phosphatidyl choline • Carotenoids
each • Mixed Phoshphatidyl serine
• Keep the section in Potassium ferrocyanide- triglycerides Plasminogen
HCl solution for 20 min • Waxes – Sphingosine based
Sphingomyelin
• Keep in running water for 5 min – Phosphosphingosides
• Rinse in deionised water – Phosphoinositides
• Glycolipid
• Keep in Van Gieson working mixture for – Cerebroside
5 min – Sulphatide
• Dehydrate, clear in xylene and mount – Ganglioside
Fig. 9.7 The diagram shows reaction of fatty acid and glycerol forming triglyceride
9.6 Fixation
Most of the dyes are soluble in lipid more than

their solvent. Therefore, it is preferable to use
frozen section tissue for lipid staining. The best
fixative for lipid is formal calcium (10% formalin
with 2% calcium acetate).
9.7 Stains
Figure 9.8 highlights various stains for lipids.

Fig. 9.8 Various stains for lipid materials in tissue
section
9.7.1 Oil red O [6]

Application: Oil red o is particularly useful to
Indications: Oil red O stain is used for demon- demonstrate lipid in renal cell carcinoma. The
stration of lipid material. The lipid material takes lipoblasts are also oil red o positive.
deep red colour in this stain.
Principle: Oil red o is a diazo dye that is
highly soluble in lipid substances. Oil red o is 9.7.2 Preparation of Oil Red O Stain
dissolved in an alcohol solution, and in fat-
containing tissue, the dye leaves the solution and 9.7.2.1 Stock solution
moves to the tissue fat as it is more dissolved in Oil red O: 0.5 g
fat. As fat is dissolved in an alcoholic solution so Isopropanol: 100 ml
the processed tissue cannot be stained by oil red Dissolve oil red o in the alcohol and keep it
o and a frozen section or fresh tissue is needed for overnight.
this stain. The mounting medium of oil red o also
should not be alcohol-based. Aqueous mounting 9.7.2.2 Working solution
is used in oil red o stain. Oil red O stock solution: 30 ml
9.9 Nucleic Acid and Proteins 93
Distilled water: 20 ml • Counterstain by haematoxylin

After mixing the stock solution with distilled • Rinse in water
water, wait for 10 min and then filter to use. • Mount in glycerine jelly (aqueous media)
The stability of the working solution is only
1 h. Result: Fat stain dark black.
Fixation: Fresh frozen section or air-dried smear.
9.7.2.3 Steps 9.8.3 Ferric haematoxylin

1. Put the slides directly into oil red o solution for Phospholipid [8]
for 20 to 30 min
2. Rinse with running water Fixation: Frozen section tissue.
3. Counterstain with haematoxylin for 30 s
4. Rinse in water 9.8.3.1 Preparation of solution
5. Mounting in glycerine
Solution 1
Result: Oil red o stains lipid, lipoprotein, and tri- Ferric chloride: 2.5 g
glycerides. Lipid takes red colour. Ferus sulphate: 4.5 g
Concentrated hydrochloric acid: 2 ml
9.8 Sudan Black B [7]
Solution 2
Principle: Sudan black B is the lipophilic dye Distilled water: 10 ml
and is insoluble in water. This dye, therefore, is Haematoxylin: 100 mg
dissolved in tissue fat and stains them. The
slightly basic dye Sudan black B combines with 9.8.3.2 Working solution
an acidic component of the lipid. Mix 15 ml of Solution 1 with 5 ml of Solution 2.
Fixation: Fresh frozen section or air-dried The working solution is stable for 1 h only.
smear.
9.8.3.3 Steps
• Stain by the working solution for 7 min
9.8.1 Solution • Wash the section in distilled water
• Differentiate in 0.2% hydrochloric acid: 5 s
Sudan Black B: 1 g • Wash well
Propylene glycol: 100 ml • Dehydrate in acetone
Heat the solution gently up to 100 °C, cool it, and • Clean in xylene
filter the solution. • Mount by DPX
Result: Phospholipid takes blue colour.

9.8.2 Steps
• Fresh frozen section 9.9 Nucleic Acid and Proteins

• Fix in 10% formalin if fresh section
• Wash well in distilled water 9.9.1 Nucleic Acids
• Air dry
• Put in Sudan Black B solution for 1–2 h Nucleic acids are of two types: Deoxyribonucleic
• Rinse in ethyl alcohol (70%): twice for 2 min acid (DNA) and ribonucleic acid (RNA). DNA
• Rinse in distilled water consists of two helical strands made of alternate
sugar and phosphate molecules. Four types of

bases are linked with each sugar molecule.
These bases are purine (Adenine and Guanine)
and pyrimidine (Cytosine and Thymine).
Adenine only joins with Thymine, and Cytosine
only joins with Guanine. This is known as com-
plementary base pairing. There is another
pyrimidine base known as Uracil that is found in
RNA.
The constituents of nuclei acid are:
• Sugar
• Phosphate
• Base: Purine and pyrimidine
Fig. 9.9 Structure of amino acid. It contains an amino
and also a carboxyl group
9.9.2 Proteins
Proteins are made of amino acids. Each amino Schiff’s reagent: Described previously.
acid contains a central carbon atom with an Potassium metabisulfite solution
attached amino group and carboxylic group on 10% Potassium metabisulfite: 5 ml
each side (Fig. 9.9). 1 M Hydrochloric acid: 5 ml
Steps
9.9.3 Feulgen Stain [9]
1. Rehydration of section/smear by graded
This stain is specific for DNA, and it demon- alcohol
strates sugar deoxyribose. This is particularly 2. Rinse in water
helpful for DNA ploidy examination. 3. 1 (M) hydrochloric acid (preheated at 60 °C)
Basic principle: In the presence of an acidic for 60 min
environment (hydrochloric acid treatment), the 4. Keep in Schiff’s reagent for 45 min
purine bases of the DNA molecule are detached 5. Immerse in 0.05 M metabisulfite for 2 min
from the deoxyribose. DNA molecule becomes three times each
apurinic. However, the sugar-phosphate back- 6. Counterstain by 0.01% fast-green
bone of DNA is preserved. The aldehyde group 7. Dehydrate in absolute alcohol
of the sugar molecule is exposed, and this group 8. Xylene
subsequently binds with Schiff’s reagent to 9. Mount
impart colour. This hydrolysis part is the most
vital part of this stain. Result: DNA takes reddish-purple colour.
DNA + Hydrochloric acid Exposed
aldehyde group of deoxyribose
Aldehyde group + Schiff’s reagent 9.9.4 Methyl Green Pyronin Stain
Reddish purple colour [10]
Application: Feulgen stain is particularly
helpful for DNA ploidy examination. Methyl green pyronin stain demonstrates DNA
1 M hydrochloric acid solution and also RNA.
Hydrochloric acid: 8.5 ml Fixation: Formalin fixation.
Distilled water: 91.5 ml Solution
Methyl green pyronin (2%) in distilled water: 9.9.6 Hemosiderin Pigment

9 ml
Pyronin Y (2%) in distilled water: 4 ml Hemosiderin is the breakdown product of
Glycerol: 14 ml haemoglobin.
Acetate buffer (pH 4.8): 23 ml
Add the ingredients and mix well. 9.9.6.1 Prussian Blue Reaction (Pearl’s
Steps Reaction) for Ferric Iron
Principle: Hydrochloric acid unmasks the ferric
• Deparaffinise iron. This ferric iron reacts with potassium fer-
• Graded alcohol rocyanide to form insoluble blue ferric
• Wash in water ferrocyanide.
• Wash in Acetate Buffer Solution
• Methyl green pyronin solution 30 min. Solution A: Potassium Ferro cyanide: 2 g
• Wash with buffer Distilled water: 100 ml
• Rinse in distilled water 2% aqueous solution of Potassium Ferro
• Dehydrate by alcohol cyanide.
• Clean by Xylene Solution B: Hydrochloric acid 2% (2 ml
• Mount Hydrochloric acid and 98 ml distilled water).
Counterstain: Neutral red 1% aqueous
Result solution.
DNA take bluish green Steps
RNA takes red
• Deparaffinise
• Graded alcohol to bring in water
9.9.5 Pigments • Rinse in distilled water: 10 to 15 dips
• Dip in mixture of equal parts of Solution A
Various organic and inorganic pigments are pres- and Solution B: 30 min
ent in our bodies. The pigments may be endoge- • Multiple dips in distilled water
nous or exogenous in origin (Table 9.4). • Counterstaining by neutral red for 15 s
• Rinse in distilled water: 10 to 15 dips
Table 9.4 Different types of pigments • Dehydrate in graded alcohol
• Clean by xylene
Endogenous Exogenous Artefact looking
pigment pigment as pigments • Mount
Haematogenous • Calcium • Formalin
• Haemosiderinin • Copper • Starch Result (Fig. 9.10):
• Haemoglobin • Uric acid • Malaria Hemosiderin: blue.
• Bile pigment • Schistosome
• Porphyrin
Nuclei: Red.
Non-
Haematogenous
• Argyrophil 9.9.7 Bile Pigment
pigments
• Melanin
• Lipofuschin Bile pigments include conjugated and unconju-
• Dubin Johnson gated bilirubin and biliverdin. Bile pigment is
• Chromaffin stained by Fouchet’s stain [11].
Fig. 9.10 Pearl’s reaction in the cytology smear shows Fig. 9.11 Fouchet’s stain: The stain highlights bile cast
dark blue Hemosiderin pigments within renal tubules in a case of bile cast nephropathy
(×200) (Courtesy of Dr. Suvradeep Mitra, Assistant profes-
sor, Department of Histopathology, Post Graduate Institute
of Medical Education and Research, Chandigarh, India)
9.9.7.1 Fouchet’s Stain
Solution
Trichloroacetic acid: 25 g 9.9.8 Argyrophil Pigments
Mix it well. 9.9.8.1 Grimelius Staining [12]
Ferric chloride: 1 g Grimelius stain can demonstrate the argyrophilic
Distilled water: 10 ml substances.
Mix it well.
Now mix 100 ml aqueous trichloroacetic acid 9.9.8.2 Principle
with 10 ml aqueous ferric chloride solution and Argyrophil cells: These cells quickly absorb the
keep the mixture in a dark bottle. silver salt, and the cells need a reducing agent to
Van Gieson stain solution make visible silver precipitation by reduction.
Acid fuchsin: 100 mg Argentaffin cells: These cells also absorb the
Aqueous saturated Picric acid: 100 ml silver salt and reduce them without any external
reducing substances.
Steps 9.9.8.3 Acetate Buffer

Solution 1
• Deparaffinise Acetic acid: 1.2 ml
• Graded alcohol to bring in water Distilled water: 100 ml
• Rinse in distilled water: 10 to 15 dips Solution 2
• Dip in fresh Fouchet’s solution: 10 min Sodium acetate: 2.7 g
• Wash in distilled water for 2 min Distilled water: 100 ml
• Counterstain: Van Gieson stain solution: 2 min Final solution: 9 ml Solution 1with 91 ml solu-
• Dehydrate in graded alcohol tion 2, pH adjusted to 5.6.
• Clean by xylene Reducing Solution
• Mount Sodium sulphite, anhydrous: 5 g
Hydroquinone: 1 g
Result (Fig. 9.11) Distilled water: 100 ml
Bile pigment: Green. 2% Silver Nitrate
Collagen: Red. Silver nitrate: 4 ml
Muscle: Yellow. 0.2 M Acetate buffer, pH 5.6: 10 ml
Distilled water: 86 ml Use the solution within a month.

Stapes Steps
• Deparaffinise • Deparaffinise
• Graded alcohol to bring in water • Graded alcohol to bring in water
• Rinse in distilled water: 10 to 15 dips • Rinse in distilled water: 10 to 15 dips
• Dip the slides in silver nitrate solution (2%) in • Dip the slides in an ammoniacal silver nitrate
a Coplin jar: Overnight in a Coplin jar: Overnight
• Drain out the silver nitrate solution • Wash with distilled water three times
• Keep the slides in reducing solution for 1 to • Aqueous sodium thiosulphate (5%): 2 min
2 min • Wash thoroughly in running tap water: 2 min
• Wash in distilled water • Counterstain: Neutral red (0.5% aqueous):5 min
• Dehydrate in graded alcohol • Wash in distilled water
• Clean by xylene • Dehydrate by alcohol
• Mount • Clean in Xylene
• Mount
Result
Argyrophillic cells: Black. Result
Background: Golden yellow. Melanin, argentaffin granules, lipofuscin: Black.
Nucleus: Red.
9.9.9 Melanin
9.9.10 Schmorl’s Stain [13]
Melanin is the yellowish-brown to black pig-
ment. This pigment is present in hair, the epider- Schmorl’s solution
mis of the skin, the eye and substansia niagra of Fresh solution of Ferric chloride (1% aque-
the brain. Melanin is produced from tyrosine by ous): 30 ml
series of reactions. Fresh solution of Potassium ferricyanide (1%
aqueous): 4 ml
9.9.9.1 Masson Fontana Method Distilled water: 6 ml
Masson Fontana stain demonstrates melanin and Steps
argentaffin granules.
Solution • Deparaffinise
10% Silver nitrate solution: 25.0 ml • Graded alcohol to bring in water
Stock solution • Rinse in distilled water: 10 to 15 dips
• Now keep the section in Schmorl’s solution in
• Take 25 ml Silver nitrate solution in a flask a Coplin jar for 15 min
and add drop by drop by concentrated ammo- • Wash thoroughly in running tap water
nium hydroxide • Counterstain by neutral red (0.5%): 5 min
• Shake gently • Dehydrate by alcohol
• Add the ammonia solution until all the initial • Clean in Xylene
precipitate dissolves. • Mount
• Clear solution will form
Result (Fig. 9.12)
Working solution Melanin, argentaffin granules, lipofuscin: Dark
Stock solution: 12.5 ml blue.
Distilled water: 37.5 ml Nucleus: Red.
Fig. 9.12 Schmorl’s stain: Microphotograph showing the Fig. 9.13 Brownish formalin pigments in the tissue.
hyphae of the dematiaceous fungi (Phaeohyphomycosis); (Haematoxylin and eosin ×200)
Schmorl’s stain highlights the peacock green coloured
pigmented fungi (×1000 oil immersion). (Courtesy of Dr.
• Clean in Xylene
Suvradeep Mitra, Assistant professor, Department of
Histopathology, Post Graduate Institute of Medical • Mount
Education and Research, Chandigarh, India)
Result
Calcium deposits: black.
9.9.11 Calcium Nuclei: red.
Inorganic calcium is an important constituent

of bone and teeth. Calcium can be deposited 9.9.13 Formalin Pigment
abnormally in necrotic tissue or in case of
infraction. Formalin pigment is brownish-black in colour.
Acidic formalin fixation commonly produces for-
malin pigment (Fig. 9.13). The pigment can be
9.9.12 Von Kossa Technique [14, 15] removed by alcoholic picric acid. Using buffered
formalin helps to reduce formalin pigment.
Silver nitrate (5%)
Silver nitrate: 5.0 g
Distilled water: 100.0 ml 9.9.14 Malarial Pigment
Thiosulphate solution
Sodium thiosulphate: 5 g The malarial pigment is similar to formalin pig-
Distilled water: 100 ml ment. This is also brownish-black in colour. Unlike
formalin pigment, malarial pigment is intracellular
• Deparaffinise in location. RBCs are usually loaded with malarial
• Graded alcohol to bring in water pigment. This pigment can be removed by treating
• Rinse in distilled water: 10 to 15 dips the section with alcoholic picric acid.
• Flood the section with Silver nitrate: 1 h in
strong sunlight
• Wash thoroughly in running tap water 9.9.15 Starch
• Sodium thiosulphate solution: 5 min
• Wash thoroughly in distilled water The starch pigment is produced by talcum pow-
• Counterstain by neutral red (0.5%): 5 min der used in the gloves of the surgeons. The pig-
• Dehydrate by alcohol ment is positive for PAS stain.
References 99
References the demonstration of phospholipids. Histochemie.

1973;36(2):149–66.
9. Delamater ED, Mescon H, Barger JD. The chemis-
1. Corfield AP. Mucins: a biologically relevant glycan
try of the Feulgen reaction and related histo- and
barrier in mucosal protection. Biochim Biophys Acta.
cytochemical methods. J Invest Dermatol. 1950
2015;1850(1):236–52.
Feb;14(2):133–52.
2. Lau SK, Weiss LM, Chu PG. Differential expression
10. Unna PG. Eine Modifikation der Pappenheimschen
of MUC1, MUC2, and MUC5AC in carcinomas of
Farbung auf Granoplasma. Monatsh Praktische
various sites: an immunohistochemical study. Am J
Dermatol. 1902;35:76.
Clin Pathol. 2004;122(1):61–9.
11. Hall MJ. A staining reaction for bilirubin in sections
3. Gendler SJ, Spicer AP. Epithelial mucin genes. Annu
of tissue. Am J Clin Pathol. 1960;34:313–6.
Rev Physiol. 1995;57:607–34.
12. Grimelius L. A silver nitrate stain for alpha-2 cells
4. McManus JFA. Histological demonstration of mucin
in human pancreatic islets. Acta Soc Med Ups.
after periodic acid. Nature. 1946;158:202.
1968;73(5-6):243–70.
5. Southgate HW. Notes on preparing Mucicarmine. J
13. Lillie RD. Histopathologic technique and practical
Pathol Bacteriol. 1927;30:729.
histochemistry. 2nd ed. New York, NY: Blakiston;
6. Lillie RD, Ashburn LL. Supersaturated solutions of
1954.
fat stains in dilute isopropanol for demonstration of
14. von Kossa J. Ueber die im Organismus kun-
acute fatty degeneration not shown by Herxheimer’s
stlich erzeugbaren Verkalkungen. Beit Path Anat.
technique. Arch Pathol. 1943;36:432–40.
1901;29:163.
7. Sheehan DC, Hrapchak BB. Theory and practice of
15. Meloan SN, Puchtler H. Chemical mechanisms of
histotechnology. 2nd ed. St. Louis: Mosby; 1980.
staining methods: Von Kossa’s Technique: what
p. 204–5.
von Kossa really wrote and a modified reaction for
8. Elleder M, Lojda Z. Studies in lipid histochemis-
selective demonstration of inorganic phosphates. J
try. XI. New, rapid, simple and selective method for
Histotechol. 1985;8:11–3.
Connective Tissue Stain: Principle
and Procedure 10
Connective tissue is one of the major types of tis- 10.1 Fibrous Part of Connective
sue that connects the different parts of tissue and Tissue
supports the body parts. Mature connective tissue
is classified as: The fibrous part of the connective tissue includes:
1. Connective tissue proper • Collagen

(a) Loose areolar tissue • Reticulin
(b) Dense connective tissue • Elastin
2. Specialized connective tissue • Basement membrane
(a) Adipose tissue
(b) Hematopoietic tissue Collagen: Collagen fibres are derived from fibro-
(c) Bone blasts. Collagen fibre may remain as individual
(d) Cartilage single fibre or in bunches. There are predomi-
nantly five types of collagen fibres:
The connective tissue consists of two major Collagen I: These are 300 nm long and much
elements: thicker fibres. They carry the main bulk of con-
nective tissue. Collagen I fibres are present in
• Cells skin, bone, tendon, and blood vessels.
• Non-cellular substances Collagen II: Collagen II fibres are composed
of thin fibrils arranged as fine meshwork. They
The non-cellular substances can be divided into: are present in hyaline and elastic cartilage and
cornea.
• Fibrous part Collagen III: It is present along with Collagen
• Amorphous ground substances, also known as I. The tensile strength of collagen III is much less
matrix than Collagen I. Collagen III is present in the
basement membrane of various organs.
Major cells in the connective tissue are divided into Collagen IV: They are the fine fibrillar struc-
Fixed cells: (1) Fibroblasts, (2) Adipose cells, ture that is randomly arranged. Collagen IV fibres
(3) Macrophages, (4) Pericytes, and (5) Mast cells are present in the glomerular basement mem-
Transient cells: (1) Plasma cells, (2) brane of the kidney.
Lymphocytes, (3) Neutrophils, (4) Monocytes, Collagen V: This is a fibril forming collagen
(5) Eosinophils, and (6) Basophils and is present in minor quantities in specific tis-
https://doi.org/10.1007/978-981-19-6616-3_10
102 10 Connective Tissue Stain: Principle and Procedure
sue such as placenta and atherosclerotic plaque. 10.1.1 Reticulin Fibres

It is also present in the interstitial tissue of the
kidney and inter-alveolar septum of the lung. Reticulin fibres are the fine branching fibres that
Collagen VI: This is a unique member of the are interwoven closely. They are collagen III
collagen family. Collagen VI is connected with fibre. These are argyrophilic fibres. Reticulin
various components of the extracellular matrix fibres support the parenchymal tissue of the liver,
material. spleen and lymph node.
Table 10.1 highlights the different types of
collagen.
10.1.2 Elastic Fibres
Table 10.1 Comparison of the different types of collagen These are fine fibres. They are present as branch-
ing fibres or sheets. The elastic fibres are made of
Types of Fibrillary
collagen pattern Location Function microfibrils organised in a complex pattern with
I Fibrillar, Skin, Bone, Gives tensile the help of calcium. Microfibrils of the elastic
thicker dentin, tendon strength fibres interact with various proteoglycans that
II Fibrillar, Cartilage Gives tensile help in the integration of the supporting tissue.
thin, strength
Elastic fibres provide the elasticity of the blood
III Fibrillar, Blood vessels, Provides a
thin, lymph nodes, structural
vessels, lungs and skin.
associated lung framework of
with type I lymph node,
collagen and spleen 10.1.3 Basement Membrane
and gives
tensile
strength The basement membrane is the connective tissue
to various element that separates the epithelial and endothe-
connective lial cells from the underlying connective tissue.
tissue
The basement membrane is divided into three
IV Network Basement It makes the
forming membrane framework of layers from cell membrane to away:
lamina densa
to provide 1. Lamina Lucida: This is nearer to the surface
support
cells and is made of various carbohydrate
V Fibrillar Present along Gives tensile
with type I, strength
materials. The lamina lucida contains integ-
placenta, rin, laminin and collagen. This layer may be
interstitial simply an artefact.
tissue of 2. Lamina densa: This is the next zone of the
kidney and
inter-alveolar
basement membrane and consists of predomi-
septum of nantly collagen IV, proteoglycan, laminin and
lung fibronectin.
VI Beaded Connective It possibly 3. Lamina fibroreticularis: This is the fibrous
filaments tissue of helps in the component and merged with the underlying
blood vessels, attachment of
uterus etc. cells and connective tissue elements. They are com-
Present along connective posed of a bunch of microfibrils and collagen
with type II, tissue. fibres.
10.2 Stains 103
10.2 Stains Then, a polyacid of larger molecular size is

used to differentiate the tissue. It removes the
10.2.1 Masson Trichrome [1] stain from the bone and collagen.
Finally, another fibre specific contrast dye
Many different colours of dye are used in Masson (aniline blue) is used with a larger molecular
trichrome stain to differentiate the collagen weight than the previous (acid fuchsin) dye. This
fibres, muscle, fibrin and RBCs. dye is kept for the optimum time to stain the col-
lagen fibres only.
10.2.1.1 Indications and Advantages
• To study the diseases of muscle, particularly 10.2.1.2 Solution
muscular dystrophy
• To study the cardiac pathologies Bouin’s Fixative
• Liver biopsy: To assess the degree of fibrosis Saturated picric acid: 75 ml
in the liver tissue, such as chronic liver dis- Formaldehyde (40%): 25 ml
ease, cirrhosis, etc. Glacial acetic acid: 5.0 ml
• Kidney biopsy: To see the changes in the base-
ment membrane of glomeruli. Weigert’s Haematoxylin
• Tumor: Distinguishing collagen and smooth Solution (a)
muscle in soft tissue tumours Haematoxylin: 1.0 g
• It is a relatively better differentiating stain 95% ethanol: 100 ml
than the van Gieson stain. Solution (b):
29% aqueous ferric chloride: 4.0 ml
Principle (Fig. 10.1a): The staining of a par- Distilled water: 95.0 ml
ticular tissue by a specific dye depends on the Concentrated hydrochloric acid: 1.0 ml
size, molecular weight, ionic character and Working Solution: Mix equal proportion of solu-
network of the molecules of the tissue. tions (a) and (b) and use immediately.
Similarly, the molecular weight and ionic char-
acter of the dye are important. Dye of lower Acid Fuchsin Solution
molecular weight will penetrate the tissue Acid Fuchsin: 0.5 g
more easily and stain all the tissue compo- Glacial acetic acid: 0.5 ml
nents. The dye of medium molecular weight Distilled water: 100 ml
will penetrate only through muscle and colla-
gen (leaving RBC uncoloured). In contrast, the Phosphomolybdic Acid Solution (1%)
higher molecular dye will penetrate only Phosphomolybdic acid: 1 g
through collagen and muscle fibres will not be Distilled water: 100 ml
stained. So the smaller dye molecule will stain Mix Phosphomolybdic acid in distilled water.
the tissue, and when the larger dye molecule This solution is stable for 6 months.
penetrates the tissue, it will replace the smaller
dye from the tissue. Aniline Blue Solution
In Masson trichrome stain, the dyes of differ- Aniline blue: 2.5 g
ent molecular weights are used sequentially. Glacial acetic acid: 2.0 ml
Acid fuchsin dye is used to stain both muscle Distilled water: 100 ml
and collagen tissue together. The staining time is Preparation: Mix Aniline blue in 100 ml of boil-
kept for the optimum duration so that the dye ing distilled water. Mix 2 ml glacial acetic acid.
stains muscle and collagen adequately. Cool the solution and then filter.
Fig. 10.1 (a) Masson trichrome stain: Schematic dia- (Normal liver tissue) (×200). (Courtesy of Dr. Suvradeep
gram showing the principle of Masson trichrome stain. (b) Mitra, Assistant professor, Department of Histopathology,
Masson trichrome stain: The stain highlighting the portal Post Graduate Institute of Medical Education and
tract bluish-green while the hepatocytes were stained red. Research, Chandigarh, India)
The nuclei of the cells took a deeper shade of blue.
10.2 Stains 105
Steps to Stain differentiate fibrosarcoma from

• Deparaffinise leiomyosarcoma
• Graded alcohol to bring in water • Amyloid versus collagen: Van Gieson stain is
• Rinse in distilled water: 10 to 15 dips specific for collagenous material
• Fix the section in Bouin’s fixative: 60 min
• Rinse thoroughly in running tap water: 10 min Principle: The principle of this stain is the same
• Weigert’s iron haematoxylin: 10 min. as described in Masson trichrome stain.
• Bluing: By keeping the section in running tap
water 10.2.2.2 Van Gieson’s Stain Solution
• Acid fuchsin: 10 min Acid Fuchsin 1% (aqueous): 10 ml
• Rinse with acetic acid Picric acid (aqueous saturated): 100 ml
• Treat with a phosphomolybdic acid solution Control: Skin or artery.
for 5 min until the collagen is devoid of the red
colour 10.2.2.3 Steps of Staining
• Drain the solution • Deparaffinise
• Aniline blue: 5 min • Graded alcohol to bring in water
• Wash in distilled water: 10–15 dips • Rinse in distilled water: 10 to 15 dips
• Differentiate: 2% acetic acid: 2 min • Weigert’s iron haematoxylin: 10 min
• Wash in distilled water • Wash in tap water
• Rapid dehydration • Differntiation:1% acid alcohol
• Clear in xylene • Wash in water
• Mount • Van Gieson’s solution: 5 min
• Directly go to 95% alcohol
Result (Fig. 10.1b) • Rapid dehydration
Muscle: Red. • Clear in xylene
Collagen: Blue. • Mount
Nuclei: Black or blue.
Fibrin: Red. 10.2.2.4 Result (Fig. 10.2)
Warning Note
• Careful removal of picric acid by running tap
water is crucial because inadequate removal
of the picric acid may produce resistance of
the muscle fibre to take the proper stain.
• The differentiation step to impart a red colour
to the muscle should be controlled carefully
with the help of a microscope.
10.2.2 Van Gieson Stain [2]
Aim: This stain demonstrates collagen fibres.

Fig. 10.2 Elastic van Gieson (EVG) stain: Elastic van
10.2.2.1 Indications Gieson staining in a case of polyarteritis nodosa. The elas-
• Extent of fibrosis: It helps to assess the quan- tic tissue of the artery (elastic lamina) was stained black
while the fat was stained red (×200). (Courtesy of Dr.
titation of fibrosis
Suvradeep Mitra, Assistant professor, Department of
• Tumor: It helps to differentiate collagenous Histopathology, Post Graduate Institute of Medical
material from the smooth muscle, so useful to Education and Research, Chandigarh, India)
Collagen fibres: Red. • Bone marrow: It is a useful stain to demon-

Nuclei Black. strate marrow fibrosis and is particularly help-
Cytoplasm, muscle, fibrin: Yellow. ful in myelofibrosis.
• Lymph node: Lymph nodal architectural pat-
10.2.2.5 Warning Notes tern is better demonstrated by reticulin stain.
• Water may remove the red colour, so one The loss of lymph nodal architecture is seen in
should directly proceed to 95% ethanol for non-Hodgkin lymphomas.
dehydration • Tumors:
• Alcohol may wipe out the yellow colour, so –– In endometrial stromal sarcoma “weave-
this step should be quick basket” appearance is seen.
• Weigert’s hematoxylin resist decolouration by –– Chicken wire pattern of reticulin is noted in
picric acid, and so it is preferable to the other myxoid liposarcomas.
hematoxylin that gives weak nuclear stain. –– The typical Zellballen pattern in paragan-
glioma is better demonstrated by reticulin
stain.
10.2.3 Reticulin Stain –– Gliomas, sarcomas: Abundant reticulin
fibres
10.2.3.1 Indications –– Ewing’s sarcoma: Absent of reticulin
• Liver biopsy: Reticulin stain helps to demon-
strate early cirrhosis. It is an essential stain in Principle: Reticulin fibres are stained reliably by
liver sections. the silver impregnation method (Fig. 10.3). The
• Kidney: It demonstrates Kimmelstiel-Wilson reticulin fibres contain a carbohydrate compo-
lesion of diabetic glomerulosclerosis. nent. Potassium permanganate is used to oxidise
Fig. 10.3 Schematic diagram shows the mechanism of duces metallic silver that reacts with the aldehyde group
reticulin stain. Potassium permanganate oxidise the car- of the tissue. Gold chloride makes this metallic precipita-
bohydrate component of the reticulin fibres to generate tion permanent. In addition, sodium thiosulphate is used
aldehyde group. In the basic medium the silver salt pro- to remove the excess unreactive silver
10.2 Stains 107
the reticulin fibres, and the aldehyde group is initial precipitate will be formed. Add strong
generated from the carbohydrate component. The ammonia drop by drop to dissolve the black
tissue is treated with silver salt at basic pH. In the precipitate. Now add an equal amount of dis-
basic medium, silver salt produces metallic silver tilled water.
that reacts with the aldehyde group of the tissue. Control: Lymph node or liver tissue.
Subsequently,
sodium thiosulphate is used to remove the 10.2.4.2 Steps to Stain
excess unreactive silver. Tissue is further treated • Deparaffinise
with gold chloride to make this precipitation • Graded alcohol to bring in water
permanent. • Rinse in distilled water: 10 to 15 dips
• Potassium permanganate solution: 5 min
• Wash in tap water
10.2.4 Gordon and Sweet’s Method • Bleach: Oxalic acid 2 min
for Reticulin Stain [3] • Wash in tap water
• Iron alum solution, 10 to 15 min
10.2.4.1 Solution • Wash in distilled water with three changes
• Silver nitrate solution: 2 min
Acidified Potassium Permanganate (1%) • Wash in distilled water with two changes
• Potassium permanganate: 0.5 g • 10% formaldehyde solution: 1 min (till it
• 3% Sulphuric (H2SO4) acid: 2.5 ml becomes grey-black)
• Distilled water: 47.5 ml • Rinse thoroughly in tap water
Freshly prepared solution. • Toning: 0.2% Gold chloride solution for 3 min
• Rinse thoroughly in tap water
Oxalic Acid (2%) • Fix with Sodium thiosulphate (5%): 2 min
Dissolve 2.0 g Oxalic acid in 100 ml distilled • Wash in tap water
water. • Counterstain: Neutral red for 2 min (if you need)
Iron Alum (2%) • Rapid dehydration
Ferric ammonium sulphate 2.0 g. • Clear in xylene
Distilled water 100.0 ml. • Mount
10% Formaldehyde 10.2.4.3 Result (Fig. 10.4)

Formaldehyde: 10.0 ml
Gold Chloride (0.2%)

Dissolve 0.2 g Gold chloride in 100 ml distilled
water.
Sodium Thiosulphate (5%)

Sodium thiosulphate: 5 g
Distilled water: 100.0 ml
Silver Nitrate (10%)

Silver nitrate: 10.0 g
Take 5 ml aqueous silver nitrate solution, Fig. 10.4 Reticulin stain: Reticulin stain in liver high-
and add drop by drop sodium hydroxide. The lighting the normal reticulin pattern of the liver (×400)
Reticulin fibres: Black. Solution 3

Nuclei: Red. Iodine: 1 g
Potassium iodide: 2 g
10.2.4.4 Warning Notes Distilled water: 100 ml
• The tissue section may float as the silver solu-
tion is highly alkaline. So use either a well-
albuminized slide or a cellodinized slide. 10.3.2 Final Verhoeff’s solution
• All these solutions should be freshly made.
• Silver salt solution should be properly pre- Solution 1: 20 ml
pared. Do not mix excess ammonia solution in Solution 2: 8 ml
the silver nitrate. Solution 3: 8 ml
• Use a Coplin jar for the silver nitrate solu- Add 8 ml “solution 2” into 20 ml of solution 1
tion treatment. Time is a critical factor in and then add 8 ml solution 3.
this step of the silver salt reaction. Do not
put the slide in the silver salt solution for 10.3.2.1 Steps of Staining
excess time. • Deparaffinise
• Silver salt solution should be away from • Graded alcohol to bring in water
sunlight. • Rinse in distilled water: 10 to 15 dips
• Use only distilled water in each step • Verhoeff’s iron haematoxylin: 20 min
• Clean glassware should be used. Wash them • Differentiation: By 2% ferric chloride. This is
with distilled water thoroughly, at least five a crucial step. Allow differentiating till the
times. The dust particle may precipitate the fibres take black colour. Check the colour by
silver. microscope.
• Wash in water
• Remove iodine by washing in 95% alcohol
10.3 Elastic Fibres • Counterstain: Van Gieson’s stain: 2 min
• Rapid dehydration
The common stains of Elastic fibres include • Clear in xylene
Verhoeff’s stain, orcein stain and Weigert’s • Mount
resorcin-fuchsin stain.
10.3.2.2 Results
Elastic fibres: Black.
10.3.1 Verhoeff’s Stain for Collagen [4] Nuclei: Black.
Collagen: Red.
Principle: The Hematoxylin dye binds with the Other tissue: Yellow.
elastic tissue by ionic interaction. Ferric salt acts
as an oxidiser and helps in the binding of haema-
toxylin and elastic fibres. 10.3.3 Weigert’s Resorcin-Fuchsin
Control: skin tissue. Stain [5]
10.3.1.1 Solutions Solution

Solution 1 Basic fuchsin: 2 g
Hematoxylin: 5 g Resorcin: 4 g
Absolute alcohol: 100 ml Distilled water: 200 ml
It is preferable to use fresh solution.
Solution 2 • Mix basic fuchsin and resorcin in 200 ml dis-
Ferric chloride: 10 g tilled water and boil. Now add 25 ml of 30%
Distilled water: 100 ml ferric chloride to this boiling solution.
10.3 Elastic Fibres 109
• Boil for another 5 min 10.3.4.2 Result

• Cool Elastic fibres: Brown.
• Filter
• Take the precipitate in a filter paper
• Now, take 200 ml of 95% ethanol in a flask 10.3.5 Fibrin and Cross Striation
and dissolve the precipitate by heating of the Muscle
• Remove the filter paper
• Add 4 ml of concentrated HCl Fibrin is best stained by Phosphotungstic acid
haematoxylin (PTAH).
10.3.3.1 Steps of Staining
• Deparaffinise 10.3.5.1 Phosphotungstic acid
• Graded alcohol to bring in water Haematoxylin (PTAH) [6, 7]
• Rinse in distilled water: 10 to 15 dips Aim: PTAH aims to stain fibrin, and cross stria-
• Put the section in the stain solution for 1 h to tion of the muscle and glial fibres.
3 h at room temperature
• Wash in water Solutions
• Differentiate: By 1% acid alcohol Hematoxylin: 1 g
• Wash in water Phosphotungstic acid: 20 g
• Counterstain: Eosin Distilled water: 1000 ml
• Rapid dehydration
• Clear in xylene • Dissolve haematoxylin and Phosphotungstic
• Mount acid separately in distilled water by gently
heating.
10.3.3.2 Result • Cool
Elastic fibres: Purple. • Combine the two solutions
0.25% potassium permanganate

10.3.4 Orcein for Elastic Fibres Potassium permanganate: 0.25 g
Orcein: 1 g 5% oxalic acid
Hydrochloric acid: 1 ml Oxalic acid: 5 g
70% Alcohol: 100 ml Distilled water: 100 ml
• Dissolve orcein in alcohol and heat the solution, 10.3.5.2 Steps of Staining
• Cool • Deparaffinise
• Filter • Graded alcohol to bring in water
• Add hydrochloric acid • Wash in water
• Oxidation: By 0.25% potassium permanga-
10.3.4.1 Steps of Staining nate for 10 min
• Deparaffinise • Wash in water
• Graded alcohol to bring in water • Bleaching: By 5% oxalic acid for 2 to 5 min
• Wash in water (until the colour disappears)
• Orcein solution: 30 min (in 56 °C) • Wash thoroughly in distilled water
• Differentiate: By 1% acid alcohol • Keep the section in PTAH solution for 12 to
• Wash in water 24 h
• Counterstain: Methylene blue • Dehydration: Rapidly by 95% ethyl alcohol
• Rapid dehydration • Absolute alcohol
• Clear in xylene • Clear
• Mount • Mount
10.3.5.3 Result (Fig. 10.5) Figure 10.6 demonstrates different staining

Striated muscle fibres, fibrin, nuclei, astrocytes: patterns for different types of connective tissue.
blue
• Cytoplasm: brown red

• Collagen and bone: brown pink
Fig. 10.5 Phophotungstic acid hematoxylin

(PTAH) stain: This stain highlighted the fibrin
thrombi as hematoxyphilic blue showing
numerous glomerular fibrin thrombi (×400).
(Courtesy of Dr. Suvradeep Mitra, Assistant
professor, Department of Histopathology, Post
Graduate Institute of Medical Education and
Research, Chandigarh, India)
Fig. 10.6 Staining the colour pattern of different connective tissue stains. (Phophotungstic acid hematoxylin = PTAH,
Haematoxylin and Eosin = H & E)
References 111
References 5. Weigert C. Ueber eine method zur Farbung elast-

ischer Fasern. Zentrablatt Fuer Allgemeine Pathol
Pathologische Anat. 1898;9:289.
1. Garvey W. Modified elastic tissue-Masson trichrome
6. Mallory FB. A contribution to staining methods. J Exp
stain. Stain Technol. 1984;59(4):213–6.
Med. 1900;5:15.
2. Van Gieson I. Laboratory notes of technical methods
7. Puchtler H, Waldrop FS, Meloan SN. On the mecha-
for the nervous system. N Y Med J. 1889;50:57.
nism of Mallory’s phosphotungstic acid-haematoxylin
3. Gordon H, Sweets HH. A simple method for the silver
stain. J Microsc. 1980;119(3):383–90.
impregnation of reticulum. Am J Pathol. 1936;12:545–51.
4. Verhoeff FH. Some new staining methods of wide
applicability. Including a rapid differential stain for
elastic tissue. J Am Med Assoc. 1908;50:876–7.
Amyloid Staining
11
11.1 Introduction
Amyloid is the eosinophilic amorphous extracellular

insoluble misfolded fibrillar protein. This extracel-
lular amyloid protein deposition in various organs
and tissues is known as amyloidosis. Amyloid is not
a single disease. The different diseases of variable
etiopathogenesis may show amyloidosis.
Structure of amyloid (Fig. 11.1): Amyloid pro-
tein is the beta-fibrillar structure which gives the
characteristic staining pattern. Amyloid deposit is
composed of fibrillar amyloid protein, plasma, pro-
teoglycan and extracellular matrix material. On
electron microscopy, the amyloid fibril shows non-
branching filaments that are randomly arranged.
The amyloid fibrils are specifically arranged as
beta-pleated sheet configurations, giving their char-
acteristic tinctorial pattern. Each amyloid fibril is
composed of several protofilaments. Each protofila-
ment is 2–3 nm in width and has a micrometre in Fig. 11.1 The structure of amyloid
length. The protofilament shows a cross β structure,
and the β strands are at the right angle of the long
axis of the amyloid fibril and stacked together by is characterised by amyloid deposition in multi-
the hydrogen- bonding and forming a beta-pleated ple organs, whereas localized amyloidosis shows
sheet. They are folded and arranged in antiparallel only single organ involvement [1–3].
orientation with the adjacent polypeptide chains. Systemic amyloidosis is classified broadly as
The stability of the beta-pleated sheet is a critical (Table 11.1):
factor for the further deposition of the amyloid pro-
tein irrespective of the chemical characters of the 1. Primary amyloidosis
protein. 2. Secondary amyloidosis
Amyloid is classified as systemic amyloidosis 3. Familial amyloidosis
or localised amyloidosis. Systemic amyloidosis 4. Other familial types of amyloidosis
https://doi.org/10.1007/978-981-19-6616-3_11
114 11 Amyloid Staining
Table 11.1 Classification of amyloidosis 11.3 Stains for Amyloid

Biochemical
Type variety 11.3.1 Alkaline Congo Red Stain [4]
Systemic
Primary: Related to plasma cell AL
tumour AA
Principle: Congo red intercalates between the
Secondary: Related to chronic Aβm parallel fibrils of the amyloid protein and forms a
diseases non-polar hydrogen bond.
Haemodialysis related
Heredofamilial 11.3.1.1 Solution
Hereditary polyneuropathy ATTR
Familial Mediterranean fever AA
Localized 1% Sodium Hydroxide
Senile cardiac ATTR Sodium Hydroxide: 1 g
Senile cerebral Aβ Distilled water: 100 ml
Endocrine: Medullary carcinoma of A Cal
the thyroid
Saturated Sodium Chloride in Ethanol
(80%)
80% Ethanol: 100 ml
11.2 Primary Amyloidosis Sodium Chloride: 1 g
AL type amyloidosis (AL): AL amyloidosis is Alkaline Alcohol Sodium Chloride Solution

associated with plasma cell neoplasm, and it is Sodium Hydroxide (1%): 1 ml
the commonest type of amyloidosis. The fibril Sodium chloride ethanol (80%): 100 ml
composition of AL type is a variable region of Freshly made solution should be used.
kappa or lambda light chain of immunoglobulin.
The free light chain is present in the circulation Alkaline Congo Red Stock Solution
and deposited in the various organs. The exact Congo red: 0.5 g
cause of the amyloidogenic potential of the light Alkaline alcohol sodium chloride solution:
chain is not surely known. Possibly, it depends on 300 ml
a certain sequence of amino acids.
Secondary amyloidosis (AA type): The Working Solution of Congo Red
fibrillar composition of secondary amyloidosis is Alkaline Congo red Stock solution: 100 ml
AA protein. This type of amyloidosis is second- 1% Sodium Hydroxide: 1 ml
ary to various inflammatory lesions such as Use only fresh solution
tuberculosis, rheumatoid arthritis, etc. The amy-
loid is formed from serum amyloid A (SAA), an 11.3.1.2 Steps of Staining
acute-phase protein. • Deparaffinise
Familial amyloidosis (ATTR): In this auto- • Graded alcohol to bring in water
somal dominant disorder, the amyloid is formed • Rinse in distilled water: 10 to 15 dips
from the mutant transthyretin (ATTR). • Stain by Mayer’s Hematoxylin
Transthyretin is a transport protein of thyroxine • Blueing by running tap water
and retinol-binding protein. • Wash in distilled water
Localized amyloidosis: In the case of local- • Alkaline alcohol sodium chloride solution:
ized amyloidosis, the amyloid is deposited in 20 min
the isolated organ such as the lung, kidney or • Alkaline Congo red: 20 min
tongue. • Rapid dehydration
References 115
• Clear in xylene 11.3.2.4 Result

• Mount Amyloid: Red colour.
11.3.1.3 Result (Fig. 11.2)

Amyloid: Deep pink. 11.3.3 Thioflavin T Stain [6]
Apple green birefringence in polarised light.
Thioflavin T is a very sensitive technique.
However, it is not a specific stain for amyloid.
11.3.2 Congo Red Stain by Highman [5]
11.3.3.1 Thioflavin T Solution
11.3.2.1 Congo red Solution Thioflavin T: 1 g
50% Ethyl alcohol: 100 ml Distilled water: 100 ml
Congo red: 0.5 g
11.3.3.2 Steps
11.3.2.2 Potassium Hydroxide (0.2%) • Deparaffinise
80% Ethyl alcohol: 100 ml • Graded alcohol to bring in water
Potassium hydroxide: 0.2 g • Rinse in distilled water: 10 to 15 dips
• Alum haematoxylin: 2 min
11.3.2.3 Steps • Wash in water
• Deparaffinise • Thioflavin T Solution: 3 min
• Graded alcohol to bring in water • Rinse in water
• Rinse in distilled water: 10 to 15 dips • Differentiation: 1% acetic acid for 20 min
• Congo red: 5 min • Wash in water
• Differentiation by alcoholic Potassium • dehydrate
hydroxide: a few seconds • Clear in xylene
• Wash in water • Mount in glycerine jelly
• Counterstain: Alum Haematoxylin
• Running tap water for blueing 11.3.3.3 Result
• Wash with distilled water Fluorescence microscope using BG12 exciter fil-
• Rapid dehydration ter and K 530 barrier filter: Bright yellow
• Clear in xylene fluorescence.
• Mount
References
1. Blancas-Mejía LM, Ramirez-Alvarado M. Systemic
amyloidoses. Annu Rev Biochem. 2013;82:745–74.
2. Wechalekar AD, Gillmore JD, Hawkins PN. Systemic
amyloidosis. Lancet. 2016;387(10038):2641–54.
3. Falk RH, Comenzo RL, Skinner M. The systemic
amyloidoses. N Engl J Med. 1997;337(13):898–909.
4. Puchtler H, Sweat F, Levine M. On the binding of
Congo red by amyloid. J Histochem Cytochem.
1962;10:355–64.
5. Highman B. Improved methods for demonstrating
amyloid in paraffin sections. Arch Pathol (Chic).
1946;41:559–62.
6. Vassar PS, Culling CF. Fluorescent stains, with spe-
Fig. 11.2 Polarized microscope shows Apple green bire- cial reference to amyloid and connective tissues. Arch
fringence of amyloid material in Congo red stain Pathol. 1959;68:487–98.
Stains for the Microbial Organisms
12
Microbial organisms or microbes are sub- Routine haematoxylin and eosin stain may not
microscopic infective organisms that may pro- distinctly identify the microbial organism tissue
duce disease in humans. or smear and therefore special stain is needed.
Broadly these microbes can be subdivided Special stain helps to delineate the morphology
into (1) Bacteria, (2) fungi, (3) protozoa, and (4) and characteristic colour of the organisms [1–3]
Helminths and (5) viruses. (Table 12.1). It is advisable to have a culture of
the microbes if possible.
Table 12.1 Special stains for microbial organisms

Organisms Stain Comment
Bacteria
Mycobacterium tuberculosis Ziehl Neelsen(Z-N) stain, Auramine Red bacilli in Z-N stain
rhodamine stain Yellow fluorescence in auramine
rhodamine stain
Mycobacterium Leprae Fite acid-fast stain Red bacilli
Actinomycosis Papanicolaou’s stain, May Grunwald Clustered bacilli with sunray
Giemsa, Gomori methenamine silver appearance
Nocardia MGG, Gram stain, Modified acid-fast stain Thin slender long bacilli
Helicobacter MGG, H & E and Gram stain Flying birds like bacilli in rows
Fungi
Candia Papanicolaou’s stain, May Grunwald Thin slender pseudohyphae and
Giemsa, Gomori methenamine silver, PAS spores
Aspergillus PAP, PAS, H & E and GMS Slender bacilli with acute angle
branching
Mucormycosis PAP, H & E and GMS Broad, wide-angle branching,
non-septate
Cryptococci Mucicarmine, Masson-Fontana silver stain Small 3–5 μm round structure
Histoplasma capsulatum GMS, PAS Small 2–3 μm round structure
Pneumocystis jirovecii GMS
Parasites
Amoeba (E Histolytica) PAS, H & E, Iron Hematoxylin Stain Round shaped small organisms
Giardia lamblia H&E Pear-shaped small organisms
Echinococcosis granulosus H & E, GMS, PAS
H & E Hematoxylin and Eosin, PAP Papanicolaou, GMS Gomori methenamine silver, Z-N Ziehl Neelsen, PAS Periodic
acid Schiff
https://doi.org/10.1007/978-981-19-6616-3_12
118 12 Stains for the Microbial Organisms
12.1 Bacteria • Crystal violet solution: 1 to 2 min

• Lugol’s iodine: 1 min
The common stains of the demonstration of bac- • Differentiation by alcohol: 1 or 2 dip
terial organisms are: • Counterstain by basic fuchsin: 2 min
• Wash with water
• Gram’s stain • Dehydrate with absolute alcohol
• Ziehl Neelsen stain • Clear in xylene
• Mount
12.1.1 Gram’s Stain [4, 5] 12.1.1.3 Result

Gram-negative bacteria: Blue-black
This stain is discovered by Hans Christian Gram Gram-positive bacteria: Pink
in 1884. It helps to identify the Gram-positive
organism.
Principle: Gram-positive bacteria contain a 12.2 Ziehl Neelsen Stain
good amount of peptidoglycan on their cell wall
that helps to retain the dye crystal violet on the Aim: Ziehl Neelsen stain demonstrates acid-fast
cell wall. Now Gram’s iodine is added which tubercular bacilli.
helps to bind the crystal violet on the wall of the Principle: The organisms are stained with
bacteria. This dye-iodine complex does not dif- basic fuchsin dye by heating. They resist deco-
fuse out in presence of acetone. Therefore, Gram- lourization by acid alcohol (1%). The methylene
positive organism shows blue colour even after blue counterstain is used to stain decolourized
acetone treatment. Whereas in Gram-negative non-acid fast bacilli.
organism the dye-iodine complex is diffused out
easily from the bacteria and become colourless.
The counterstain Carbol Fuchsin is applied to 12.2.1 Reagents
give Gram-negative organisms a pink colour.
12.2.1.1 Carbol-fuchsin
12.1.1.1 Reagents Basic fuchsin: 0.5 g
Absolute Ethyl alcohol: 5 ml
Crystal violet solution 5% Phenol (aqueous): 100 ml
Crystal violet: 1.0 g
Absolute alcohol: 20 ml 12.2.1.2 Methylene Blue
Ammonium oxalate (1%): 80 ml Methylene blue: 1.4 g
95% Ethyl alcohol: 100 ml
Lugol’s Iodine
Iodine crystal: 1.0 g 12.2.1.3 Acid Alcohol
Potassium iodide: 2.0 g Hydrochloric acid: 10 ml
Distilled water: 300 ml Alcohol: 1000 ml
Basic Fuchsin
Basic fuchsin: 1.0 g 12.2.2 Steps of Staining
• Deparaffinise
12.1.1.2 Steps of Staining • Pass-through graded lower concentration of
• Deparaffinise alcohol and section/smear to bring in water.
• Pass-through graded lower concentration of • In hot carbol-fuchsin solution for 30 min
alcohol and section/smear to bring in water. • Thoroughly washing in water
12.3 Fite Acid-fast Stain for Leprosy 119
12.3.2 Carbol-fuchsin
Basic fuchsin: 0.5 g

Absolute alcohol: 5 ml
5% Phenol (aqueous): 100 ml
12.3.3 Sulphuric Acid (5%)
Sulphuric acid: 5 ml
25% Ethyl alcohol: 95 ml
Fig. 12.1 Abundant acid-fast bacilli in a case of atypical

mycobacteria. (Ziehl Neelsen stain ×1200) 12.3.4 Xylene in Peanut Oil Solution
• Differentiation: By acid alcohol till the tissue Xylene: 50.0 ml

is pale Peanut Oil: 50.0 ml
• Counterstain: By methylene blue for 15 s This solution can be used for 1 year.
• Wash in water
• Dehydrate with absolute alcohol
• Clear in xylene 12.3.5 Steps of Staining
• Mount
• Deparaffinise: Keep the section in xylene/pea-
12.2.2.1 Result (Fig. 12.1) nut oil solution for 2 min
• Acid-fast bacilli red colour • Blot the excess oil
• Nuclei: Blue colour • Wash in water: 5 min
• Put the slide in carbol-fuchsin solution for
30 min (room temperature)
12.3 Fite Acid-fast Stain • Rinse in water: 5 min
for Leprosy [6] • Decolourise: By 5% sulphuric acid
• Wash in water
Aim: Fite acid-fast stain helps to demonstrate • Counterstain: By methylene blue for 1 or 2
mycobacterium leprosy. dip
Principle: Mycobacterial leprae has an acid- • Wash in water: 5 min
fast character. However, they are weak acid-fast • Blot the section
and therefore a weak concentration of sulphuric • Dry
acid (5%) is used. • Clear in xylene
• Mount
12.3.1 Methylene blue 12.3.5.1 Result

Acid fast bacilli and Mycobacterium leprae
Methylene blue: 1.4 g (Fig. 12.2): Red.
95% Ethyl alcohol: 100 ml Background: Blue.
12.4.2.4 Sodium Thiosulphate

Solution (5%)
Sodium thiosulphate: 1.0 g
12.4.2.5 Sodium Bisulphite (1%)

Sodium metabisulphite: 1.0 g
12.4.2.6 Chromic Acid (2%)

Chromium trioxide: 10.0 g
This solution can be used for 6 months.
Fig. 12.2 Abundant lepra bacilli in a lymph node aspirate
in Fite acid-fast stain for leprosy. (Fite acid fast stain for
leprosy ×1200) 12.4.2.7 Gold Chloride Solution
(0.1%)
Gold chloride: 0.1 g
12.4 Fungal Infection Distilled water: 100.0 ml
12.4.1 Grocott’s Methenamine 12.4.2.8 Stock Solution of Light

Silver [7] Green (0.2%)
Light green: 0.2 g
Aim: Grocott’s methenamine silver stains the Distilled water: 100.0 ml
cell wall of the fungi. It stains the outer wall of Glacial acetic acid: 0.2 ml
the pneumocystis carini organisms.
12.4.2.9 Light Green Working
Solution
12.4.2 Reagents Light green stock solution: 10 ml
12.4.2.1 Stock Solution
of Methenamine Silver
Methenamine (3%): 100 ml 12.4.3 Steps of Staining
Silver nitrate (5%): 5 ml
Add silver nitrate solution slowly drop by • Deparaffinise
drop. The white precipitate will appear. It will • Pass-through graded lower concentration of
dissolve slowly. Filter the solution. Keep the alcohol and section/smear to bring in water
solution in a brown coloured bottle (stable for • Oxidation: 2% Chromic Acid: 30 min
3 months). • Wash in distilled water
• Dip: 1% Sodium bisulphite for 1 min
12.4.2.2 Sodium Borate Solution (5%) • Wash in distilled water
Sodium borate: 5.0 g • Woking Methenamine silver solution: 15 min
Distilled water: 100.0 ml in water bath at a temperature of 60 °C. Tissue
This solution can be used for 3 months. will take brown colour
12.4.2.3 Methenamine Silver Working • Tone: Gold chloride solution 5 s
Solution • Wash in distilled water
Stock solution of Methenamine silver: 50 ml • Sodium thiosulphate solution: 5 s
5% sodium borate: 5 ml • Wash in water
12.5 Spirochaetes 121
Developer Solution
Solution A
Hydroquinone: 300 mg
Buffer solution: 10 ml (as prepared above)
The solutions are stored in 37 ° C.
Solution B
Silver nitrate (2%): 3 ml
Mix 10 ml solution A and 3 ml solution B
(pre-warm to 60 ° C) just before use.
12.5.1.2 Steps
• Deparaffinize and bring the section into the
water
Fig. 12.3 Methenamine silver stain for Pneumocystis
carini infection. (Methenamine silver stain ×1200) • Wash in buffer solution
• Stain in 1% silver nitrate solution at 60 ° C: 1
• Counterstain: Light green working solution h
for 10 s • Keep the section in freshly prepared developer
• Dehydrate by absolute alcohol solution at 60 ° C: 3 to 4 min
• Clear in xylene • Wash in water at 60 ° C
• Mount • Wash in buffer solution
• Dehydrate
• Clear
12.4.4 Result (Fig. 12.3) • Mount
Fungi and pneumocystis: Black. Result: Spirochaetes: Black colour, Background:

Background: Green. Brown to yellow.
12.5 Spirochaetes 12.5.2 Viral Inclusions
12.5.1 Warthin and Starry 12.5.2.1 Phloxine Tartrazine Stain

Technique [8]
Reagents
Fixation: Formalin fixed section. Phloxine: 0.5 g
Calcium chloride: 0.5 g
12.5.1.1 Reagents Distilled water: 100 ml
Tartrazine to saturate in 2-Ethoxy ethanol:
Buffer Solution 100 ml
Sodium acetate: 1.64 g
Acetic acid: 2.5 ml 12.5.2.2 Steps of Staining
Distilled water: 200 ml • Deparaffinise
• Pass-through graded lower concentration of
Silver Solution alcohol and section/smear to bring in water
Silver nitrate: 0.5 g • Nuclear stain: Alum haematoxylin for 10 min
Buffer solution: 50 ml • Differentiates in acid alcohol
• Wash in tap water 2. Kwon-Chung KJ, Hill WB, Bennett JE. New, special
• Stain in phloxine solution: 20 min stain for histopathological diagnosis of cryptococco-
sis. J Clin Microbiol. 1981;13(2):383–7.
• Wash in tap water 3. Madison BM. Application of stains in clinical micro-
• Keep in Tartrazine: 5 to10 min biology. Biotech Histochem. 2001;76(3):119–25.
• Dehydration by absolute alcohol 4. Gram HC. Über die isolierte Färbung der
• Xylene Schizomyceten in Schnitt- und Trockenpräparaten.
Fortschritte der Medizin (in German). 1884;2:185–9.
• Mount 5. Engbaek K, Johansen KS, Jensen ME. A new tech-
nique for Gram staining paraffin-embedded tissue. J
12.5.2.3 Result Clin Pathol. 1979;32(2):187–90.
6. Wade HW. A modification of the Fite formaldehyde
Viral inclusion: Bright red, Background: Yellow. (Fite I) method for staining acid-fast bacilli in paraffin
sections. Stain Technol. 1957;32(6):287–92.
7. Grocott RG. A stain for fungi in tissue sections and
smears using Gomori's methenamine-silver nitrate
References technique. Am J Clin Pathol. 1955;25:975–9.
8. Warthin AS, Chronister AC. A more rapid and
1. Woods GL, Walker DH. Detection of infection or improved method of demonstrating spirochetes in tis-
infectious agents by use of cytologic and histologic sues (Warthin and Starry's cover-glass method). Am J
stains. Clin Microbiol Rev. 1996;9(3):382–404. Syphilis. 1920;4:97–103.
Part II
Basic Laboratory Techniques in
Cytology Laboratory
Cytology Sample Procurement,
Fixation and Processing 13
13.1 Introduction • Gastrointestinal tract: Gastric brush, lavage,

Trans endoscopic FNAC
The routine laboratory technique includes the • Urine: Voided urine, catheterized urine, ure-
following components: teric urine
• Effusion cytology: Effusion fluid
• Specimen collection: Unlike histopathology, • Cerebrospinal fluid (CSF): Clean vial.
the specimen collection is variable in different • Vitreous fluid: Clean vial.
body samples in cytology. • Joint fluid: In anticoagulant.
• Fixation: Different types of fixatives are used
in various cytology samples. (B) Fine needle aspiration cytology: This is
• Processing: The processing of the cytology is usually done directly by the cytologist.
widely different in cytology samples. It largely
depends on the type of sample and available
laboratory facilities. 13.2.1 Cervical Cytology [1, 2]
• Staining: The common stains in the cytology
laboratory are Papanicolaou’s and May The cervical cytology sample is usually col-
Grunwald Giemsa (MGG) stains. lected by a gynaecologist or a trained nurse. The
collection of cervical cytology is important
because the success of a cervical cancer screen-
ing program largely depends on adequate
13.2 Sample Collection sampling.
The following broad types of samples are com-
13.2.1.1 Preparation of the Patient
monly received in cytology laboratory:
• Two weeks after the first day of the last men-
(A) Exfoliative Cytology strual period.
• Cervical cytology: cervical smear, liquid- • No sexual intercourse 2 days prior to
based cytology in the vial sampling
• Respiratory samples: Sputum, bronchial wash, • No use of vaginal cream, jellies or tampons
bronchial brush, bronchoalveolar lavage,
transbronchial needle aspiration cytology, fine 13.2.1.2 Collection Devices
needle aspiration cytology (FNAC) under The ideal collection device should have the fol-
radiological guidance lowing properties:
https://doi.org/10.1007/978-981-19-6616-3_13
126 13 Cytology Sample Procurement, Fixation and Processing
• Able to collect materials from both ectocervi- tion, procuring samples from both ecto and
cal and endocervical regions endocervical material, costlier than all the
• Non-traumatic above devices.
• Non-sticky
• Cheaper
• Disposable 13.2.2 Collection Proper (Box 13.1)
The following collection devices are available: • Keep the patient in dorsolithotomy position
• Clean the vagina with a wet swab.
1. Wooden spatula (Fig. 13.1): Cheaper, easy to • Do not use any lubricant jellies
handle, cells may be attached to wood • Introduce a speculum to visualize the cervix
2. Plastic spatula: Costlier than wood, non-sticking • Inspect both the ectocervix and transforma-
3. Endocervical brush: Easy to collect material, tion zone, ectocervix is smooth pink and
traumatic due to stiff bristle opaque whereas the endocervix is dark pink.
4. Cervix-brush (Fig. 13.2): mainly used for • For the conventional smear
liquid-based cytology (LBC) sample collec-
Fig. 13.2 Cervix brush: This is mainly used in liquid

Fig. 13.1 Wooden spatula: This is a disposable spatula based cytology
13.3 Respiratory Samples 127
Box 13.1 Cervical Smear Collection

Basic precautions
• Avoid vaginal cream, jellies or tampons
2 days prior to the test
• Avoid collection during menstruation
period
• No sexual intercourse within 2 days
Collection devices.
For LBC: Cervex brush, LBC collec-
tion fluid in vial.
Fig. 13.3 Schematic diagram of cervical smear collec-
For conventional: tion. The brush is introduced in the cervical canal the tip
of the brush remains in the endocervical canal and the
• Endocervical broom stick, plastic spat- brooms are touched in the ectocervix. The brush is rotated
ula or wooden spatula and then withdrawn
• Clean glass slides
• Permanent slide marker
–– Immediately fix the slide in 95% ethyl
• Fixative: 95% ethyl alcohol
alcohol
Collection proper. • For LBC preparation
Position of the patient: Dorsolithotomy. –– Use Cervex brush
–– Introduce the central part of the broom into
Preparation
the endocervical canal so that the shorter
• Open the vagina by speculum for proper bristles of the broom touch the ectocervix
visualization of cervix (Fig. 13.3).
• Clean the vagina by wet swab with –– Rotate the broom four to five times in a
water clockwise direction
• Insert the cervex brush or plastic spatula –– Withdraw the broom and put it in the fixa-
within in the vagina tive solution as given by the company.
• Rotate full circle • Label the glass slide or the vial (for liquid-
• LBC: Rinse the brush in the fluid within based cytology)
the vial
• Conventional: Vaginal smear: The cytology sample is col-
–– Spread the material on the glass slide lected from the lateral vaginal wall by using a
–– Immerse the smear immediately in spatula.
95% ethanol for fixation Endometrial aspiration smear: With the
help of a sterile cannula and a syringe the endo-
metrial sample is aspirated.
–– A broom or spatula is used

–– Insert the spatula within the vagina so that 13.3 Respiratory Samples [3, 4]
the tip of the spatula fits with the contour of
the cervix The respiratory samples include: Sputum, bron-
–– Rotate the spatula in a complete round turn chial brush, bronchial brush, bronchoalveolar
–– Withdraw it gently lavage, transbronchial needle aspiration cytol-
–– Spread the sample on the spatula on the ogy, fine needle aspiration cytology (FNAC)
glass slide under radiological guidance.
13.3.1 Sputum Sample • The stylet is withdrawn and aspiration is done

by applying negative suction.
• Collect the morning sputum in a wide- • The needle is withdrawn.
mouthed container • The inside material is ejected by reintroducing
• No fixative is needed the stylet within the needle.
• Multiple smears are made (both air-dried and
alcohol fixed).
13.3.2 Bronchial Brush
Gastrointestinal tract: Gastric brush, lavage,
• Visualized the lesion through the transendoscopic FNAC.
bronchoscope
• Make the smear immediately on the slide
• Prepare both alcohol fixed and air-dried smears 13.3.6 Gastric Brush
• A flexible fiberoptic endoscopy is introduced

13.3.3 Bronchial Wash in the stomach. The endoscope contains spe-
cific channels for biopsy forceps and
• Visualized the lesion through the brushes.
bronchoscope • The brush is covered by a Teflon sheath to pre-
• Introduce 5 ml normal saline through the vent the loss of cytology material.
bronchoscope • The mucosal lesion is visualized and the
• Aspirate the solution brush is withdrawn from the outer Teflon
• Send the fluid immediately to the laboratory sheath.
• The brush is gently rubbed on the lesion.
• Finally, the brush is retracted into the Teflon
13.3.4 Bronchoalveolar Lavage (BAL) sheath and is withdrawn from the endoscope.
• Multiple smears are made by rubbing the
• A fiberoptic bronchoscope is introduced under brush on the glass slide.
local anaesthesia in a selected subsegmental
bronchus
• Normal saline (20 ml) is flushed through the 13.3.7 Gastric Lavage
bronchoscope.
• The lavage solution is recollected by gentle • A flexible fiberoptic endoscopy is introduced
suction. in the stomach.
• The procedure is repeated 4 to 5 times. • Through the endoscope 100 ml of normal
• Overall 40 to 50 ml solution is procured. saline is flushed into the lesion
• BAL fluid is sent to the laboratory • By gentle suction the solution is withdrawn
immediately • It is repeated two to three-time
• Sample is sent to laboratory
13.3.5 Transbronchial Needle

Aspiration 13.3.8 Endoscopic Ultrasound-
guided (EUS) FNAC
• The lesion is visualized by endoscopic
ultrasound. • The lesion is visualized by endoscopic
• A 22- gauze needle with an internal stylet is ultrasound.
inserted through the endoscope to the mass • The FNAC needle is introduced through the
under EUS guidance. channel of the fiberoptic endoscope.
13.4 Fixation 129
• The needle is moved to and fro in the lesion 13.3.10 CSF and Vitreous Fluid
maintaining negative suction.
• Finally, the needle is withdrawn and the • To collect as fresh in a clean container
aspirated material is spread on the glass • Process immediately
slide.
• Endoscopic FNAC is particularly helpful in
submucosal tumours and is complementary to 13.4 Fixation
biopsy.
The fixatives in cytology should have the same
Urine: Voided urine, catheterized urine, ureteric essential properties as described in the histopa-
urine. thology sample in Chap. 1. The common fixa-
Voided urine: Preferable for routine cytology tives in cytology include (Box 13.2):
• Collect second voided urine • Ethyl alcohol (95%): It is the most com-
• Collect urine in a clean container monly used fixative. Ethanol causes dehydra-
• No fixative tion of the cell and mild shrinkage.
• Send the sample in the laboratory immedi- • Methanol (100%): Not cost-effective.
ately for processing • Denatured alcohol: This is unsuitable for
human consumption and so less chance of
13.3.8.1 Bladder Wash misuse.
• Introduce a catheter or cystoscope
• Wash the bladder with 50 to 100 ml of normal Box 13.2 Fixation
saline Ideal fixative
• Withdraw the solution
• Sent the sample immediately to the laboratory • Rapid action
without any preservative • Prevention of cellular distortion
• Good nuclear details
13.3.8.2 Ureteric Urine • Facilitation of staining
• The urine is collected from each ureter by a • Inactivation of microbial organisms
separate catheter. • Fixing the cell on glass slide
Routine fixatives for Papanicolaou and
13.3.8.3 Urinary Brush Hematoxylin stains
• With the help of a ureteric catheter the lesion
is brushed • 95% ethyl alcohol
• The smears are made • 100% Methanol
• Denatured alcohol
• Thin prep system: Methanol based
13.3.9 Effusion Fluid Sample preservative
• SurePath system: Ethanol based
• Collect fresh effusion fluid in a clean preservative
container Time of fixation: 15 to 30 min
• No fixative needed Hemorrhagic fluid: Carnoy’s fixative
• To prevent coagulation 1:9 ratio of ammonium Cell Block: 10% neutral buffered formalin
oxalate: fluid can be used Immunocytochemistry: 95% Ethanol,
• If a long delay in processing: an equal cold acetone etc. (cell block preferable).
amount of 50% ethyl alcohol is mixed with Electron microscopy: Glutaraldehyde
the fluid. solution (2.5%).
• Do not allow the fluid to be frozen.
13.4.1 Time of Fixation 13.4.2 Special Fixatives
• At least 15 to 30 min 13.4.2.1 Hemorrhagic Fluid

• If necessary, one can keep the smear in fixa- Carnoy’s fixative: Acetic acid in the solution
tive for a long duration in a closed bottle or jar. lyses the RBCs. Cell morphology is well pre-
served in Carnoy’s fixative. Main disadvantages
13.4.1.1 Coating Fixatives of this fixative are (1) cell shrinkage, (2) nuclear
Optimal distance of spray fixative is 12 in. away overstaining.
from the smear (Box 13.3). This prevents any dis-
tortion of morphology and damage of the smear. 13.4.2.2 Ingredients of Carnoy’s
Fixative
13.4.1.2 Major Advantages • 95% Ethanol (60 ml)
• Easy to carry • Chloroform (30 ml)
• Wax makes a protective covering over the • Glacial acetic acid (10 ml).
smear
Use only fresh fixative and discard the remaining
13.4.1.3 Precautions solution after use.
• The major ingredients of spray fixative are
alcohol and wax. Therefore, wax should be 13.4.2.3 Fixatives for Liquid-based
removed before staining. Preparation
• Maintenance of optimal distance ThinPrep system: Methanol based preservative.
• Bloody smear should not be fixed by spray SurePath: Ethanol-based preservative.
fixative as this may cause RBC clumping
• Fluid samples should not be fixed by spray 13.4.2.4 Fixatives for Cell Block
10% neutral buffered formalin is the best fixative
for cell block materials.
Fixatives for immunocytochemistry:
Box 13.3 Spray Fixatives • Cellblock section should be used for
Advantages immunocytochemistry
• Easy to carry • Routine fixatives such as 95% ethanol can be
Functions used for immunocytochemistry.
• Protection of smear by wax cover
• Cell fixation 13.4.2.5 Fixatives for Electron
Microscopy
Optimal distance of spray
Glutaraldehyde solution (2.5%).
• Overall 10 in. away from the smear
Use 13.4.2.6 Preservation of Sample Prior
• Cervical smear to Processing
• Process the specimen immediately
Not to use
• If the sample is not possible to process imme-
• Smear of fluid specimen
diately then it can be kept depending on
• Smears with blood
–– Mucus content
Precaution: Wax should be removed by –– Protein content
95% alcohol before staining. –– Sugar content
–– pH of the sample
13.5 Processing of Laboratory Samples 131
Fig. 13.4 Outline

shows the maximum
time duration between
the collection of smear
and processing
Specimens with no sugar or protein, with extreme • Slide should be present in a shock-resistant
pH should always be processed immediately container.
(Fig. 13.4). • Smear should be properly fixed and labelled.
• Paper form should be in a separate bag and
slide should not be wrapped by requisition
13.5 Processing of Laboratory form.
Samples
13.5.2.1 Precautions for Liquid
Processing of a laboratory sample includes the
Samples
following steps:
• Container should be airtight
• Receiving • Properly labelled
• Preparing smear • Plastic container is preferable to the glass
• Staining container
• Mounting and final submission of the slide
13.5.2.2 Unique Identification
13.5.1 Receiving the Sample Number
• Check the unique identification number of the
Requisition form: The sample should always be sample and requisition form at the time of
accompanied with a proper requisition form as receiving and processing
mentioned in Fig. 13.5.
13.5.2.3 Laboratory Bar Code
13.5.2 Glass Slides and Liquid • Each sample should have a unique laboratory
Sample bar code number. This is separate from the
unique identification number.
The following precautions are essential regarding • Stick the bar code number on the container,
the receiving of glass slides: smears and forms.
Fig. 13.5 Requisition

form of the cytology Requisition form
specimen
Name Age Sex

Unique identification of the patient
Date of collection
Site
Procedure of collection
Clinician’s name and contact information
Tests to be done
Clinical history
Chief complaints
Physical findings
Radiological features
Important history: Surgery, chemotherapy, radiotherapy,
exposure of chemicals etc.
13.6 Processing Direct

Sputum
smear
The commonly used processing techniques
include:
Moderate amount
1. Direct smear of fluid (50 to 100 ml)
Effusion
2. Centrifuge
Centrifuge
3. Cytocentrifuge
4. Liquid based preparation Less than 1ml (CSF,
Vitreous fluid etc)
5. Millipore technique
6. Cell block Cytocentrifuge
Figure 13.6 shows overview of processing of dif- Large amount of clear

ferent samples. fluid (100 to 500 ml):
Urine
Milipore filter
13.6.1 Processing of Sputum
(Fig. 13.7) Sample for Cell block
immunocytochemistry
• Pour the sputum sample in a Petri dish kept on

Fig. 13.6 Outline of the processing of different speci-
a black background mens. Sputum is traditionally processed by direct smear-
• Carefully examine for any tissue fragments or ing, a moderate amount of fluid (up to 100 ml) is processed
gray white substance or bloody material by centrifuge whereas a small volume (0.5 ml) of fluid is
• Pick up the tissue fragments by a clean better processed by cytocentrifuge. A large amount of
fluid is processed by the Millipore filtration technique
forceps
• Prepare the smears on the clean glass slide.
13.6 Processing 133
Fig. 13.7 Schematic diagram of processing of sputum

Fig. 13.8 Schematic diagram shows the processing of
13.6.2 Processing of Fluid: Urine, the fluid specimen
Body Fluids, Lavage
Centrifuge: The fluid of a moderate amount (50

to 100 ml) should be processed by centrifugation
e.g. effusion fluid, turbid urine etc. (Fig. 13.8).
• Put the fluid sample in clean airtight centri-
fuged tube
• Rotate the tube at 1500 rounds per minute
(RPM) for 10 min
• Discard the supernatant liquid
• Make multiple smears from the sediments.
Cytocentrifuge: Small amount of clean fluid such
as 0.5 to 1 ml is processed by Cytocentrifuge e.g.,
Fig. 13.9 Cytocentrifuge machine to process small quan-
CSF, ureteric urine, vitreous fluid etc. (Fig. 13.9). tity of sample
• Rotate the sample 1000 rounds per minute for
5 min. drives the particle away from the centre. This
• A thin layer of smear is formed on the glass slide. centrifugal force is counteracted by two other
• Fix the smears in 95% ethanol forces (1) buoyant force which means floating
capacity of the particle and (2) frictional force
that develops due to friction of the particle and
13.6.3 The Basic Principle
liquid media. We can express the forces by this
of Centrifuge
equation.
The rapid circular movement of a particle around
RCF = 1.11× 10−5 × ( round per minute ) × r
2
a central axis generates a centrifugal force that

r ω2 13.6.4 Millipore Filtration

RCF =
g
The Millipore filtration technique is done for pro-
Relative centrifugal force = RCF. cessing large quantities of a clear urine sample.
Rotational radius = r.
Angular velocity in radians per unit time = ω. • Put the moistened millipore filter paper with
Gravitational acceleration = g. normal saline on the sieve.
There are two types of commercially available • Attach the filter cup
cytocentrifuge: • Put the sample in the filter cup
• Put on the suction process.
1. The cytocentrifuge that removes the fluid dur-
• The negative pressure at 100 mm Mercury is
ing the time of sedimentation
created and the fluid is drained into the bot-
2. The cytocentrifuge that retains the fluid.
tle leaving the cell on the filter paper
Cytocentrifugation may cause morphological (Fig. 13.10).
distortion of the cells and careful attention should • Make multiple imprint smears on albumin
be given to this aspect. The optimum rotational coated slides
speed of the machine is the most important factor • Fix the slides immediately in 95%alcohol for
in this respect. 30 min.
Fig. 13.10 Basic

principles of the
Millipore filtration
technique. Here negative
suction is used to draws
the fluid from the upper
container. The fluid
passes through the filter
paper and the cell sticks
on the upper surface of
the filter paper
13.6 Processing 135
13.6.5 Processing of Hemorrhagic 3. Commercially available lysing solution

Fluid Presently ThinPrep and SurePath compa-
nies supply the fixative solution that lyse the
1. Carony’s fixative: This can be used for the RBCs of the sample.
processing of hemorrhagic fluid.
2. Buffy coat preparation:
(a) Centrifuge the hemorrhagic sample at 13.6.6 Cell Block [5]
2000 RPM for 10 min.
(b) Discard the supernatant fluid The cell block technique is mainly used for:
(c) Take the residual suspension in a 2 to
3 in. long capillary tube 1. Immunocytochemistry
(d) Seal the one end of the tube 2. Cytochemistry
(e) Centrifuge the capillary tube 3. Preservation of archival tissue for future use
(f) Break the capillary tube in the buffy coat
region The cell block is made by the following steps
(g) Make fish tail preparation of smear. (Fig. 13.11):
Fig. 13.11 Basic steps

of cell block preparation
is highlighted in this
picture
• Collect the specimen in 10% neutral buffered 5. Agitate the mixture gently to make a cell clot
formalin 6. Transfer the clot into the lens paper
• Keep it 4 h in formalin to fix the cells 7. Wrap the compact clot and then fix it into
• Centrifuge the sample 1500 RPM for 10 min formalin
• Wash the sediment twice in PBS by 8. Process the clot in the tissue processor.
centrifugation
• Add 100 microliter of plasma and 30 μL
thrombin References
• Remove the clot and collect it on filter paper
1. Schumann JL, O’Connor DM, Covell JL, Greening
• Process the clot in the tissue processor as SE. Pap smear collection devices:technical, clinical,
usual diagnostic, and legal considerations associated with
their use. Diagn Cytopathol. 1992;8(5):492–503.
2. Martin-Hirsch P, Jarvis G, Kitchener H, Lilford
R. Collection devices for obtaining cervical cytol-
13.6.7 Compact Cell Block Technique ogy samples. Cochrane Database Syst Rev.
2000;2:CD001036.
Young et al. [6] described compact cell block 3. NCCLS. Nongynecologic Cytologic Specimens:
Collection and Cytopreparatory Techniques; Approved
technique that is in a small area and completely Guideline. NCCLS document GP 23-A[ISBN
free of RBCs. 1-56238-380-9]. NCCLS, West Valley Road, Suite
Steps 14000, Wayne Pennsylvania 19087-1898 USA, 1999.
4. Nongynecologic cytology practice guideline. Acta
Cytol. 2004;48(4):521–46.
1. Centrifuge the material in the fluid
5. Jain D, Mathur SR, Iyer VK. Cell blocks in cytopa-
2. Add CytoRich solution (designed for haemol- thology: a review of preparative methods, utility in
ysis and gentle fixation of the cell) in 1:1 ratio diagnosis and role in ancillary studies. Cytopathology.
to the sediment 2014;25(6):356–71.
6. Yang GC, Wan LW, Papellas J, Waisman J. Compact
3. Keep it for 2 min
cell blocks. Use for body fluids, fine needle aspira-
4. Add 4 drops of plasma and three drops of tions and endometrial brush biopsies. Acta Cytol.
thrombin (5000 IU/ml) 1998;42:703–6.
Routine Staining in Cytology
Laboratory 14
The most commonly used two routinely available 14.1.1 Dyes Used in Papanicolaou’s
stains in the cytology laboratory are Staining
1. Papanicolaou’s stain Hematoxylin: Harris hematoxylin for nuclear

2. May Grunwald Giemsa stain stain.
Orange G: OG-6 for cytoplasmic counter-
stain. It also stains the keratin component of the
14.1 Papanicolaou’s Stain [1] cytoplasm.
Eosin Azure (EA): It is a polychrome stain
Dr. George Papanicolaou, the father of and consists of three dyes: Eosin Y, light green SF
Cytopathology, first introduced this stain. yellowish and Bismarck brown Y.
Papanicolaou’s stain (PAP) stain is the most
important stain in cytology and is used in all cer-
vical smears and non-gynecologic exfoliative 14.1.2 Principle of Basic Steps
smears. This stain has the following excellent (Fig. 14.1)
properties:
1. Rehydration of the smear: With the help of
• Nuclear details seen a subsequent dip in the graded concentration
• Transparent stain of alcohol.
• Demonstrates intracytoplasmic keratin 2. Nuclear staining by Hematoxylin: Harris
hematoxylin is a good rapid nuclear stain.
Progressive method: As mentioned before, in Subsequent differentiation is done to remove
the case of progressive stain the nuclei are opti- excess hematoxylin by acid alcohol.
mally stained and the cytoplasm does not take the 3. Bluing: This is done by treating the smear
dye. with running tap water alternatively weak
Regressive method: In regressive staining, alkaline solution can be used.
the nuclei are intentionally over stained by hema- 4. Cytoplasmic staining by Orange G (OG): As
toxylin dye. Subsequently, the excess stain is OG is alcohol soluble dye so the smear is again
removed by acid alcohol. brought into alcohol and stained with OG.
https://doi.org/10.1007/978-981-19-6616-3_14
138 14 Routine Staining in Cytology Laboratory
Fig. 14.1 Basic

principles of
Papanicolaou’s stain is
highlighted in this
diagram
5. Cytoplasmic staining by EA: The cell cyto-

plasm is stained as blue-green colour by EA.
6. Dehydration: It is done by absolute alcohol.
7. Clearing: Xylene
8. Mounting: By DPX mounting medium.
14.1.3 Papanicolaou’s Staining Steps
1. 70% Ethanol: 1 min

2. 50% Ethanol: 1 min
3. Distilled water: 5 dips
4. Harris hematoxylin: three and half minutes
Fig. 14.2 Papanicolaou’s stain of the routine cervical
5. Distilled water: 5 dips smear (Sure Path preparation of Papanicolaou’s stain
6. 0.25% aqueous solution of hydrochloric ×240)
acid: few dips
7. Water: 1 min 14.1.3.1 Results (Figs. 14.2, 14.3,
8. Lithium carbonate: one and half minute and 14.4)
9. Water: few dips Nuclei: Dark blue.
10. 70% Ethanol: 2 min Cytoplasm: Blue-green.
11. 90% Ethanol: 2 min Keratin: Orange.
12. Orange G: few dips Figures 14.2, 14.3 and 14.4 describe the nor-
13. 95% Ethanol: 2 min mal cervical cytology smear, high grade squa-
14. EA modified: 2 min mous intraepithelial lesion and squamous cell
15. Absolute ethyl alcohol: 2 min carcinoma respectively of the routine cervical
16. Absolute ethyl alcohol: 2 min smear. These are the routine cervical smears of
17. Absolute ethyl alcohol and: 2 min the liquid-
based cytology preparation in our
18. Xylene: 5 min laboratory in the Post Graduate Institute of
19. Xylene: till clear Medical Education and Research, Chandigarh,
20. Mounting in D.P.X India.
14.2 Precautions to Be Taken in Papanicolaou’s Staining 139
• Mix thoroughly by stirring till the color

becomes dark purple
• Cool the flask
• Filter
• Store in dark place
14.1.3.3 EA Solution

Eosin: 2 g
Light green: 0.2 g
Water: 240 ml
Alcohol: 250 ml
Phosphotungstic acid: 1 g
Glacial acetic acid: 10 m
Fig. 14.3 Papanicolaou’s stain of the cervical smear in a
case of high grade squamous intraepithelial lesion. (Sure
Path preparation of Papanicolaou’s stain ×440) 14.1.3.4 Orange G Solution
Orange G (10% aqueous): 50 ml
Ethyl alcohol (95%): 950 ml
Phosphotungstic acid: 0.15 g
14.1.4 Bluing Solution
14.1.4.1 Lithium Carbonate Solution
Stock Solution
Lithium carbonate (LiCO3): 1.5 g
Water: 100 ml
Working Solution
Fig. 14.4 Papanicolaou’s stain of the cervical smear in a Water: 1000 ml
case of squamous cell carcinoma of the cervix (Sure Path Stock solution of lithium carbonate: 30 drops
preparation of Papanicolaou’s stain ×440)
14.2 Precautions to Be Taken

14.1.3.2 Hematoxylin Solution
in Papanicolaou’s Staining
for Papanicolaou’s Stain
Harris Hematoxylin: 5 g
The following precautions should be taken in
Papanicolaou’s staining:
Alum: 100 g
Absolute alcohol: 50 ml
Mercuric oxide: 2.5 g 14.2.1 Staining Solutions
• Dissolve hematoxylin 5 g in 50 ml alcohol • The container should be covered otherwise the

• Mix 100 g alum in water and boil it to evaporation of water or alcohol may change
dissolve the concentration of the dye.
• Now mix hematoxylin and alum in water and • The containers should be washed regularly.
boil • Hematoxylin solution: It is a stable solution.
• Take out the flask from heat However, the addition of small amount of
• Put 2.5 g mercuric oxide in the solution fresh dye solution increases staining quality.
140 14 Routine Staining in Cytology Laboratory
Table 14.1 Troubleshooting areas of the Papanicolaou’s stain

Too dark nuclear • If the smear is kept in Harris • Keep less time in Harris hematoxylin
stain hematoxylin for a long time • Maintain the concentration of acid
• Too less hydrochloric acid • Differentiates properly
concentration • Try to take a better smear
• Too less number of dips in hydrochloric
acid
• Too much bloody smear or high protein
content of the smear
Too pale nuclear • If hematoxylin is diluted • Maintain the proper dilution of
stain • Too less bluing hematoxylin or change the solution
• Too much concentration of hydrochloric • Give time for bluing
acid • Maintain the concentration of acid
• Too much dip in hydrochloric acid • Control this step and give less dips in acid
• Air dried smear prior to fixation • Try to fix the smear properly in future
Improper • Smear is air-dried prior to fixation • Try to fix the smear properly in future
cytoplasmic stain • If the slides remain in alcohol for a too • Maintain the time of fixation in alcohol
long time then the cytoplasmic stain • Maintain pH of EA solution
may be pale. • Control the staining time in hematoxylin
• If the pH of EA is not optimum.
• If the slide remains in hematoxylin for
too long time
• Orange G: It is less stable than hematoxylin • Rapid agitation may cause dislodging of the
and the strength reduces quickly within days. cells
Replace OG whenever the cytoplasmic colour • Stain rack should not hit the bottom of the
appears less crispy. container
• Xylene: It may be contaminated with the
staining dye. Change immediately if it is Table 14.1 highlights the troubleshooting areas of
tinted. the Papanicolaou’s stain.
• Alcohol: Check the concentration of the alco-
hol on regular basis. Discard the first container
of alcohol and replace it with the second and 14.2.4 De-staining and Re-staining
third. the Smear
• The stain solution should be free of stain
deposits and should be filtered daily. • Dip the smear in xylene until the coverslip
drops
• Keep the smear in acid alcohol for 20 min
14.2.2 Coverslip (80 ml of 95% ethanol and 20 ml of 0.5%
hydrochloric acid)
• Smears should not dry before placing the • Wash the smear with running water.
coverslip. • Re-stain
• No air bubbles should be present between the
coverslip and smear.
14.3 May Grunwald Giemsa Stain
14.2.3 Staining Proper May Grunwald Giemsa (MGG) is a Romanowski’s

stain and is routinely used in many laboratories.
• Gentle and slow agitation of the slides within This stain provides excellent cytoplasmic details
the staining solution helps to prevent staining character (Fig. 14.5). This is a metachromatic
deposits and also gives better staining. stain. MGG is a convenient stain for FNAC
Reference 141
Table 14.2 Comparison of Papanicolaou’s stain and

May Grunwald Giemsa stain
Features PAP stain MGG stain
Nuclear detail Excellent and The chromatin
very good stain pattern cannot
for chromatin be studied
stain
Keratin Orange G stains Cannot be
demonstration keratin as bright demonstrated
orange colour
Metachromasia Not a Metachromatic
metachromatic stain
stain
Transparency Transparent Not a
stain transparent stain
Fig. 14.5 May Grunwald Giemsa stained smear in a case Background Not good Good for
pleomorphic adenoma. Note the metachromatic staining mucin or demonstration
of the chondromyxoid substances as deep magenta- necrosis of extracellular
coloured material (May Grunwald Giemsa stain ×440) substance
MGG: May Grunwald Giemsa; PAP stain: Papanicolaou’s
smear. Table 14.2 compares the relative advan- stain
tages and disadvantages of MGG stain over
Papanicolaou’s stain. 14.3.2 Storage of Slides
• All the positive slides should be stored for an

14.3.1 Steps indefinite period.
• The negative slide should be stored for a mini-
1. May Grunwald solution: 5 min mum of 5 years.
2. Running water: 1 min
3. Giemsa stain: 15 min
4. Running water: 1 min
5. Air drying Reference
Clearing and mounting have been discussed in 1. Marshall PN. Papanicolaou staining--a review.
Chap. 8. Microsc Acta. 1983;87(3):233–43.
The Basic Technique of Fine Needle
Aspiration Cytology 15
Table 15.1 Tissue biopsy versus fine needle aspiration

15.1 Introduction cytology
Surgical
Fine needle aspiration cytology (FNAC) is a Features FNAC biopsy
major area of cytology. With the help of FNAC Tissue architecture Lost Preserved
cytologists can procure samples from any part of Lymphatic and capsular Impossible Possible to
the body. It is an easy and safe technique and can invasion to assess assess
be done as a routine office procedure [1]. FNAC In situ carcinoma Difficult to Possible to
diagnose diagnose
does not require any anaesthesia or special pre-
Individual Excellent Not so good
cautions. This is an economical, cost-effective cytomorphology as that of
and highly specific technique. FNAC is compa- FNAC
rable or even better than the frozen section in cer- material
tain situations such as thyroid or breast lesions. Immunocytochemistry Possible Possible
and other tests
Presently many ancillary techniques can be done
on FNAC material and therefore it is possible to FNAC: Fine needle aspiration cytology
provide an exact diagnosis with the help of this
technique (Box 15.1). Tissue biopsy versus FNAC (Table 15.1):
The loss of architecture of the tissue is the
major limitation of this technique. In situ ver-
Box 15.1 FNAC: Advantages sus invasive lesions are often difficult to predict
• Simple on FNAC material. Moreover, capsular and
• Easy to do lymphatic invasion cannot be assessed on the
• Rapid FNAC sample. In fact, a special skill is needed
• Cheap to report FNAC material as it is quite different
• Multiple area of sampling from tissue biopsy [2].
• High sensitivity and specificity Exfoliative cytology versus FNAC: FNAC
• Bypass tissue biopsy sample is also different from the exfoliative
• Ancillary techniques possible on FNAC cytology. FNAC sample shows limited tissue
sample architectural pattern along with well-preserved
cell morphology. The overall cellular characteris-
https://doi.org/10.1007/978-981-19-6616-3_15
144 15 The Basic Technique of Fine Needle Aspiration Cytology
Fig. 15.1 Comparison

of exfoliative cytology
and FNAC
tics are important in the FNAC sample compared properly and also to apply negative suction at
to exfoliative cytology (Fig. 15.1). the time of FNAC. One hand of the operator
Complications: FNAC of superficial masses remains free to hold the swelling.
does not have any significant complications • Syringe: Disposable 10 or 20 ml plastic
except minor hematoma. FNAC of deep-seated syringe.
masses is usually safe. Rarely following compli- • Needles: Ordinary disposable hypodermic
cations may occur: needle of 22 to 25 gauge is needed.
–– Large bore needle: For hard and fibrotic
• Surgical emphysema in lung FNAC
lesions or cysts with the viscous
• Rupture of aneurysmal vessel
material.
• Anaphylaxis in case of hydatid cyst
–– Thin bore needle: For small lymph nodes
• Biliary peritonitis and a bowel perforation
or vascular organs like the thyroid.
• Needle tract seedling of cancer cells (if thick
• Clean glass slides: Labelled clean glass
bore needle is used) [3]
slides.
Contraindications: FNAC technique does not • Spirit swabs: Clean sterile spirit swabs to
have any contraindications. It should not be done clean the area of aspiration.
in the vascular organ in case of coagulation disor- • Suitable fixatives: 95% ethanol for fixation of
ders such as haemophilia or thrombocytopenia. slide
• Additional
–– Few capped vials containing 10% formalin
15.2 Technique Proper solution for cell blocks.
–– Few capped vials containing balanced salt
15.2.1 Equipment (Fig. 15.2) solution for flow cytometry
–– Clean sterile vial for culture (bacterial, fun-
• Pistol handle to hold the syringe: The pistol gal etc.).
has the provision to attach a syringe within it. –– Vials for PCR and other molecular
The pistol handle helps to hold the syringe techniques.
15.3 Fine Needle Aspiration Procedure 145
Fig. 15.2 Essential

equipment to do FNAC
15.3 Fine Needle Aspiration

Procedure (Box 15.2) –– Insert the needle within the mass
–– Make to and fro movement of the
15.3.1 Clinical History needle
–– Apply negative suction
The following information is mandatory before –– Stop the suction
FNAC: –– Withdraw the needle
–– Detach the needle
• Exact site of swelling –– Fill the syringe with air
• Size of the lesion –– Reattach the needle
• Chief complaints with duration –– Eject the material on the glass slide
• History of previous FNAC –– Make adequate number of smears
• Any bleeding disorders –– Apply firm pressure on the site of
FNAC with the help of cotton swab
Box 15.2 Fine Needle Aspiration Procedure
• Clinical information:
–– Location Preparation of the patient: The following mea-
–– Size of the swelling sures may help during the preparation of the
–– Duration of the lesion patient:
–– Major complaints
–– Any history of coagulation disorder • Explain the whole procedure in brief.
• Preparation of the patient: • Take proper consent from the patient, particu-
–– Discuss the technique larly for FNAC of deep-seated lesions and
–– Take written consent orbit.
• Aspiration: • Talk with the patient and give assurance to
–– Clean the area of aspiration by spirit make him/her relax.
–– Hold the swelling in between the two • Clean the area of the site of FNAC with a
fingers to make it immobilized. spirit swab.
15.3.2 Aspiration (Fig. 15.3) • Retract the plunger to get enough air within
the syringe.
• The cytopathologist should personally per- • Reattach the needle
form FNAC [4]. • Eject the material on the slide.
• Take the pistol handle with the attached plas-
tic syringe and needle.
15.3.3 Smear Preparation
• Immobilize the swelling by two fingers of one
of your free hands.
• Push the material on a clean glass slide
• Gently introduce the needle and move the nee-
• Material should be a few cm away from the
dle to and fro in the mass.
end of the slide
• Apply negative suction by withdrawing the
• Needle should be parallel to the slide and little
plunger.
bended
• Lastly, the release of the plunger to stop nega-
• Make a smear by gently pressing a clean glass
tive suction
slide over the lower one to spread the
• Withdraw the needle
material
• Apply firm pressure on the site of FNAC to
• Make 3/4th smears
stop any bleeding
• Keep both air-dried and alcohol fixed smears
a b
c d
Fig. 15.3 FNAC technique: (a) The area of FNAC is along with to and fro movement. (c) The material is
cleaned, (b) The needle attached with the syringe is intro- expelled on the glass slide, (d) The smear is made
duced into the swelling and negative suction is given
15.5 FNAC of Deep-Seated Lesions 147
15.4 Fine Needle Sampling

Box 15.3 Fine Needle Sampling Technique
Fine needle sampling (FNS) is done without Indications
using any syringe (Box 15.3). No artificial nega- • All vascular tissue particularly thyroid
tive suction is applied and the material is aspi- • Small mobile swelling that are difficult
rated with the help of negative capillary pressure to fix
of the atmosphere [5]. Advantages
• Completely painless
• Absence of blood
15.4.1 Steps • Rich in material
Limitations
• Clean the area by sprit swab • Chance of spillage in cyst
• Press the swelling in between the two fingers • Less volume
to make it prominent • Unsuitable for bony and fibrotic lesions
• Introduce a thin bore needle within the
swelling
• Move the needle to and fro in the same direc-
tion and also slowly in different directions 15.5 FNAC of Deep-Seated
• The material comes to the hub of the needle by Lesions
capillary pressure of the atmosphere
• Gently withdraw the needle The basic techniques of FNAC is similar in both
• Fill the syringe with air and attach the needle superficial lesion and deep-seated lesions. However,
to the syringe appropriate radiological guidance is needed to local-
• Expel the aspirated material on the glass slide ize the deep-seated lesion. The common types of
• Make the smears guidance of deep-seated FNAC include:
Advantages: The major advantages of FNS are:
• Ultrasonography (USG) guided FNAC
• To get material without any admixture of • Computerized tomography (CT) guided FNAC
blood. • Endoscopic ultrasound-guided FNAC
• FNS is helpful in a vascular organ like the (EUS-FNAC)
thyroid. • Mammographic guided FNAC
• Small swellings are often difficult to fix and • Fluoroscopy guided FNAC
can be done by FNS. • Magnetic resonance image (MRI) guidance
• Easy to manipulate the needle as it is light and
without any attachment of a syringe. Advantages of guided FNAC: The major advan-
tages of guided FNAC are:
• Easy to sample from the area of particular
15.4.2 Limitations interest (representative part of tissue)
• Avoids any injury to major structures
• The overall material is less in volume and • Avoids aspiration from the necrotic tissue or
therefore difficult to get multiple smears. cystic area
• There is a chance of spillage in the cystic • Possible to aspirate from the tiniest lesion
lesion • Helps to aspirate from the deep seated non
• FNS is not suitable for bony and fibrotic lesions. palpable lesion
15.5.1 Major Indications of Deep Fluoroscopy guided FNAC: This is particularly

Seated Guided FNAC helpful in the case of FNAC of the cortical bony
lesion.
• Diagnosis of the primary lesion Advantage: The easiest method to do.
• Assessment of staging of the malignant lesion Disadvantage:
• To collect material for ancillary studies such
as flow cytometry, microbial culture etc. • High radiation exposure so an adequate shield
is necessary
Ideal radiological imaging modality: The ideal
radiological imaging modality should have fol-
lowing qualities: 15.5.2 USG Guided FNAC
• Free from any radiation hazard This is the most popular and simple technique
• High resolution image generation without any radiation hazards. USG machine is
• Real time visualization of the needle portable and cheap. With the help of USG, the
• Smallest lesion to localize radiologist can monitor the passage of the needle
by real-time monitoring at the time of
Essential information for the guided FNAC: FNAC. Therefore, it helps to insert the needle in
The following information are essential before the exact position within the lesion. USG guided
performing guided FNAC: FNAC can be done from any part of the body.
However, this is widely used in intra-abdominal
• Clinical history organs, small thyroid lesions, and breast lesions
• Simple X-ray, USG or CT scan picture (Fig. 15.4).
• Size of the lesion Principle: In the case of USG guidance
• Routine blood test FNAC, the high-frequency sound wave is used.
• Coagulation parameters
Fig. 15.4 USG guided

FNAC of pancreatic
tumor. (Courtesy by Prof
Anupam Lal, Radiology
Department, PGIMER,
Chandigarh)
15.6 Transrectal FNAC of the Prostate 149
The reflection of the wave from the tissue inter- Application: Small lesions situated near vital
face is recorded and the image is constructed. structures such as mediastinal lesions, or small
Advantages: The major advantages of USG lung lesions.
guided FNAC are: Endoscopic ultrasound-guided FNAC
(EUS-FNAC): Here FNAC is done through
• Economic
endoscope under the guidance of USG [6]. EUS-
• Portable
FNAC can be done from mediastinal, lung,
• Rapid
esophageal, biliary tree and pancreatic lesions.
• Easy to do
• Real-time
• No radiation exposure
15.5.3 Steps
• Angular approach can be done
Disadvantages • Localize the lesion by endoscopic ultrasound

• Significant obscuration due to air or bone may • Insert the needle with the stylet through the
affect the image endoscope to the mass under EUS guidance
• Not very high resolution • Withdraw the stylet and aspirate with the
• Completely operator dependent help of a 20 cc syringe by applying negative
suction.
Procedure: • Do FNAC from multiple sites
• Identify the lesion by USG • Finally withdraw the needle and expel the
• Introduce the needle with stylet material on the slide by reintroducing the sty-
• Withdraw the stylet let within the needle
• Apply suction with the help of syringe
• Withdraw the needle and stylet Others: Magnetic resonance imaging (MRI)
• Spread the material over the slide uses radiofrequency energy and is free from
any radiation hazards. MRI guided FNAC is
CT guided FNAC: Computed tomographic scan still not popular because of the longer time to
(CT scan) is also popular and useful for deep- do, high cost and complicated procedure. In
seated FNAC. addition, mammographic guided FNAC or core
Principle: CT uses an X-ray beam to create needle biopsy is also popular nowadays. Core
multiple tomographic or cross-sectional images of needle biopsy has the added advantage to detect
the body depending on the difference in physical the in situ carcinoma of the breast and therefore
density of the tissue. The computer accumulates it has almost replaced the FNAC of the breast
all the data and produces multiple images in differ- [7].
ent planes. Multiple images of a few mm thickness Complications of guided FNAC: There is no
are generated. Therefore, small and inaccessible significant complication in the guided
lesions can also be reached by CT scan. FNAC. However, occasionally, there may be
bleeding, medical pneumothorax or infection.
Advantages
• High resolution
• Exact localization of the needle possible
15.6 Transrectal FNAC
• Operator independent
of the Prostate
• Deep lesion near vital structure needs CT
guidance
Transrectal FNAC of the prostate is easy to do
Disadvantages and it can be done in outdoor basis. It samples
• Costly from the different areas of the prostate in a single
• Time taken procedure sitting.
• Good radiation exposure Steps (Figs. 15.5 and 15.6):
• Fix the Franzen’s guide in the left index finger

with the help of finger stall.
• Now introduce the Franzen’s guide gently
through the rectum to the area of the lesion of
prostate
• Introduce Franzen’s needle with attached
syringe within the guide
• Do FNAC with the help of Franzen’s needle
and syringe
• Take out the needle and syringe
• Expel the aspirated material on the slide
• Make multiple smear
Fig. 15.5 Franzen’s guide is fixed with the left index

finger References
1. Brown LA, Coghill SB. Cost effectiveness of
a fine needle aspiration clinic. Cytopathology.
1992;3(5):275–80.
2. Orell SR. Pitfalls in fine needle aspiration cytology.
Cytopathology. 2003;14(4):173–82.
3. Tyagi R, Dey P. Needle tract seeding: an avoidable
complication. Diagn Cytopathol. 2014;42(7):636–40.
4. Polacarz SV. Why pathologists should take needle
aspirates. Cytopathology. 1995;6(5):358.
5. Dey P, Ray R. Comparison of fine needle sam-
pling by capillary action and Fine needle aspiration.
Cytopathology. 1993;4:299–303.
6. Jhala NC, Jhala DN, Chhieng DC, Eloubeidi MA,
Eltoum IA. Endoscopic ultrasound-guided fine-needle
aspiration. A cytopathologist's perspective. Am J Clin
Pathol. 2003;120(3):351–67.
Fig. 15.6 Franzen’s guide is introduced in the rectum 7. Mitra S, Dey P. Fine-needle aspiration and core biopsy
and then the Franzen’s needle is inserted into the guide for in the diagnosis of breast lesions: A comparison and
the aspiration review of the literature. Cytojournal. 2016;13:18.
• Keep the patient in his left lateral position

with the lower leg extended
• Palpate the prostate first
Part III
Advanced Techniques in Histology and
Cytology Laboratories
Immunocytochemistry
in Histology and Cytology 16
16.1 Introduction
Immunohistochemistry (IHC) / immunocyto-

chemistry (ICC) is the technique for visual rec-
ognition of antigens present in the tissue with the
help of the corresponding antibody. Coon et al.
first time applied the immunofluorescence tech-
nique on the frozen section by using fluorescence
labelled antibodies [1]. The antibody conjugated
with enzymes acid phosphatase and horseradish
peroxidase was used first time by Nakane et al. in
1967 [2]. IHC technique was successfully intro-
duced in routine formalin-fixed paraffin-
Fig. 16.1 Antigenic determinant site provokes antibody
embedded section (FFPE) by Taylor et al. in 1974 formation
[3]. Subsequently, the development of monoclo-
nal antibodies introduced a new era in immuno-
histochemistry [4]. However, it took another 10 body binding sites have complementary geomet-
to 15 years to have routine use of IHC in a pathol- rical and chemical features (Fig. 16.1). This is
ogy diagnostic laboratory. Presently IHC is an responsible for the antigen-antibody reaction.
essential technique in every pathology This antigen-antibody reaction is further visual-
laboratory. ised by attaching a certain label to the primary or
secondary antibody.
16.2 Basic Principles

16.3 Basic Immunology
The basic principle of immunocytochemistry is
to demonstrate the specific antigen in the cell by Antigen: Any substance capable of producing an
applying the corresponding antibody to have an immunogenic response is called an antigen.
antigen-antibody reaction. The antigen contains There is a specific set of chemical components
an epitope or antigenic determinant site that that evoke an immunogenic response of the anti-
evokes a specific immunologic response to gen, which is known as the epitope or antigenic
develop antibodies. The antigen epitope and anti- determinant site.
https://doi.org/10.1007/978-981-19-6616-3_16
154 16 Immunocytochemistry in Histology and Cytology
Antibody (immunoglobulin): Antibody is arm of Y is the hinge region, which is a flexible

also known as immunoglobulin. The antibody is region of the immunoglobulin.
produced by plasma cells in response to antigenic Hybridoma technique: In this technique,
stimulation. Immunoglobulin has a specific affin- the abundant unlimited amount of pure homog-
ity against the epitope of the antigen. Each immu- enous immunoglobulin is produced. The anti-
noglobulin is composed of a pair of light chains body producing B lymphocyte is fused with a
and a pair of heavy chains polypeptides. The light malignant immortal plasma cell. The resultant
chains of immunoglobulin are of two types κ hybrid cell acquires the capability of unlimited
(kappa) and λ (lambda). There are five types of proliferation and production of specific antibod-
heavy chain: α (alpha), γ (gamma), δ (delta), ε ies. The resultant antibody in this condition is
(epsilon), and μ (mu) and depending on the nature known as “monoclonal antibody”. They are
of heavy chain, immunoglobulin are labelled as named “monoclonal” because they are produced
IgA, IgG, IgD, IgE and IgM respectively. The from a single clone of the cell. Each hybridoma
antibody is a Y shaped structure. The two tips of cell produces only a specific antibody for a par-
the Y are known as the antigen-binding site of the ticular antigen.
immunoglobulin (Fig. 16.2). The base of each Polyclonal antibody: Polyclonal antibody
is generated from the different B lymphocytes
in response to the different epitopes of a single
antigen. As they are generated from the differ-
ent clones of B cells, these antibodies are
known as polyclonal. There is a chance of
batch to batch variation in the case of poly-
clonal antibody.
The differences between the two types of anti-
bodies are highlighted in Table 16.1:
Affinity: Affinity represents the strength of
the binding capacity of the antigenic epitope with
the corresponding site of the antibody. It is actu-
ally the three dimensional fit of the epitope site of
the antigen with antibody.
Avidity: Avidity means the overall functional
strength of binding capacity of antibody and anti-
gen complex. The polyclonal antibody reacts
Fig. 16.2 Schematic diagram of immunoglobulin mole-
with multiple epitope sites of the antigen, and
cule. The antibody is a Y shaped structure and the two tips therefore, the overall strength of the antigen-
of the Y are known as antigen binding site of the antibody complex is strong. The avidity of an
immunoglobulin antibody depends on these factors:
Table 16.1 The differences between monoclonal and polyclonal antibody

Monoclonal antibody Polyclonal antibody
High production cost Low production cost
Specialised training is required to produce No special training is required to produce
Directed to a particular epitope of an antigen Directed to multiple epitopes of an antigen
No variation of the batch to batch May vary from batch to batch.
High specificity Low specificity
Less robust of detection as the antibody is directed to a More robust in detection as the antibodies are directed
single epitope to multiple epitopes
16.5 Peroxidase-Anti Peroxidase Method 155
1. Valency: More the valency of the antibody,

greater is the avidity
2. Affinity: The affinity between the individual
epitope of the antigen and the corresponding
antigen binding site of the antibody
3. Structural arrangement: Three-dimensional
structural arrangement of antigen and
antibody
Antibody specificity: The antibody specific-

ity indicates the precise detection of the specific
epitope of the antigen by the antibody. A particu-
Fig. 16.4 Schematic diagram of indirect immunostain-
lar antigenic determinant site (epitope) can be ing. In this method, a labelled secondary antibody is used
present in more than one antigen, and therefore, a against the primary antibody
single antibody may react with different
antigens.
Sensitivity: This is related to the detection of Advantage:
the relative amount of antigen by the antibody in 1. Rapid and simple method
a particular technique. The highly sensitive tech-
nique detects the low amount of antigen, and the Disadvantage:
relative intensity of the signal of antigen-antibody 1. Different primary antibodies should be
reaction is much strong. labelled differently for the antigen.
2. Low sensitivity
16.4 Detection System Indirect method: In the case of the indirect

conjugated method, the primary antibody is unla-
It is not possible to detect an antigen-antibody belled. The secondary antibody is conjugated and
reaction by light microscope. Therefore, a suit- is directed against the primary antibody (Fig. 16.4).
able detection or visualisation system is neces- A suitable chromogen visualizes the antigen-pri-
sary to demonstrate such reaction. The different mary antibody-secondary antibody complex.
types of detection systems are highlighted below:
Direct method: In the direct method, the pri- Advantages:
mary antibody is directly tagged with an enzyme 1. A single conjugated secondary antibody can
or fluorescence (Fig. 16.3). The antibody should be used against different primary antibodies.
be specific for the particular antigen; otherwise, 2. Higher dilution of primary antibody can be
non-specific staining may occur. used.
3. A large amount of secondary antibodies can be
easily produced against the primary antibody.
4. For negative control, the primary antibody
can be omitted.
16.5 Peroxidase-Anti Peroxidase

Method (Fig. 16.5)
The peroxidase-anti peroxidase reagent is a com-

Fig. 16.3 Schematic diagram of direct immunostaining. plex immune substance. It consists of horseradish
In this method the primary antibody is directly tagged
with an enzyme or fluorescence peroxidase antigen and antibodies against horse-
Fig. 16.6 Schematic diagram of avidin-biotin immunos-

taining. In this method the secondary antibody is tagged
with biotin and avidin conjugated with horseradish per-
oxidase is used. The biotinylated secondary antibody
tightly binds with the peroxidase conjugated avidin
Fig. 16.5 Schematic diagram of peroxidase-

antiperoxidase immunostaining. In this method, a second- 16.6.1 Advantage
ary bridging antibody is used between the primary
antibody and Peroxidase-anti peroxidase complex
1. Rapid and sensitive method
radish peroxidase. Here a secondary bridging

antibody is used between the primary antibody 16.6.2 Disadvantage
and Peroxidase-anti peroxidase complex. The
primary antibody and Peroxidase-anti peroxidase 1. Tissue may contain endogenous biotin, which
reagent are from the same species, whereas the may react with the avidin. Therefore, there is
secondary linking antibody is from a different a chance of a false-positive reaction.
species. This secondary antibody is usually 2. The affinity of the biotin and avidin may vary
highly specific against the primary antibody and widely in different batches. This may signifi-
the antibody present in the Peroxidase-anti per- cantly affect the sensitivity and reproducibil-
oxidase complex. ity of the test.
16.5.1 Advantage 16.7 Avidin and Biotin-

Conjugated Procedure
1. High degree of sensitivity: Peroxidase-anti
peroxidase method is 1000 times more sensi- This is the modification of the above-mentioned
tive than the indirect conjugated method. procedure to further increase the sensitivity of the
test. Here a performed complex of avidin-biotin
and horseradish peroxidase is used (Fig. 16.7).
16.6 Avidin and Biotin Method The presence of multiple molecules of horserad-
ish peroxidase enhances the visualisation reac-
The biotin has a high affinity for avidin. In this tion. The steps are such:
method, the secondary antibody is tagged with
biotin. Now avidin conjugated with horseradish • Primary antibody
peroxidase is used. The biotinylated secondary • Biotinylated secondary antibody
antibody is tightly bound with the peroxidase- • Preformed complex of avidin-biotin and
conjugated avidin (Fig. 16.6). horseradish peroxidase
16.8 Biotin-Streptavidin Method 157
Fig. 16.7 Avidin –biotin conjugated staining. In this

method, a preformed complex of avidin-biotin and horse-
radish peroxidase is used Fig. 16.8 Alkaline phosphatase–anti alkaline phospha-
tase method: In this method, a complex of alkaline phos-
phatase–anti alkaline phosphatase (APAAP) is used. The
16.7.1 Advantages secondary antibody or linking antibody is used to bridge
the primary antibody and APAAP complex
1. Highly sensitive
16.8.2 Alkaline Phosphatase–Anti
Alkaline Phosphatase Method
16.7.2 Disadvantages [5, 6]
1. Endogenous biotin may cause a false positive In the alkaline phosphatase–anti alkaline phos-
reaction phatase method, we use a complex of alkaline
phosphatase–anti alkaline phosphatase (APAAP).
The secondary antibody or linking antibody is
16.8 Biotin-Streptavidin Method used to bridge the primary antibody and APAAP
complex (Fig. 16.8). Similar to the Peroxidase-
In this system, the avidin is replaced by the tetra- anti peroxidase method, here also the primary
meric antibody streptavidin directly conjugated antibody and antibody in the APAAP complex
with the enzyme. The streptavidin molecule a has are both from the same species, whereas the sec-
very high affinity against biotin. The biotin– ondary linking antibody is from a different
streptavidin complexes give better amplification species.
and detection than the avidin-biotin complex.
16.8.3 Advantages
16.8.1 Advantages
1. It is used in cases where the tissue contains a
• Streptavadin does not cross-react with the high quantity of endogenous peroxidase such as
lectin-like substances. in bone marrow, lymph node etc. [5]. It is often
• The enzyme is more stable and can be stored used in dual immunostaining such as APAAP
for a longer duration as this is directly bound and Peroxidase-anti peroxidase staining.
with streptavidin 2. APAAP method provides distinct bright red
• There is no non-specific electrostatic binding colour which is easy to identify compared to
with streptavidin as its isoelectric point is near conventional peroxidase stain.
to neutrality. 3. APAAP is stable for a long duration
3. No background staining as it bypasses biotin

and avidin bindings.
16.8.6 Catalyzed Signal

Amplification (Tyramine
Signal Amplification)
Catalyzed reporter deposition technique is also

known as the tyramine signal amplification
(TSA) method [8]. This technique exploits the
catalytic property of HRP in the presence of
hydrogen peroxide. Biotinylated tyramine is
used in the presence of HRP and hydrogen per-
oxide. The HRP converts the biotinylated tyra-
mine to reactive biotinylated tyramide. This
activated biotinylated tyramide further reacts
with tyrosine in the amino acid of the tissue
and deposits biotin. This biotin is deposited
Fig. 16.9 Polymer-based labelling method: In this tech- only in the antigen-antibody reaction site. This
nique a polymer backbone (dextran) is used and large biotin is visualized by the avidin-biotin tech-
number of enzyme molecules and secondary antibodies
are conjugated within the polymer. The primary antibody nique (Fig. 16.10).
followed by dextran-enzyme- secondary antibody com-
plex are applied to enhance the sensitivity
16.8.7 Steps
16.8.4 Polymer-Based Labelling
Method • Do initial horseradish peroxidase-conjugated
system
In this technique, a polymer backbone is used. • Add biotinylated tyramine solution
This may be dextran, polypeptide or dendrimers • Wash
polymer. The dextran polymer is often used as a • Add horseradish peroxidase-conjugated
polymer backbone. A large number of enzyme streptavidin
molecules (at least 100 peroxidases) and more • Add chromogen (DAB) to visualize the
than 20 secondary antibodies are conjugated reaction
within this dextran polymer (Fig. 16.9). At first
primary antibody is applied, followed by a dextran- The comparison of different visualization tech-
enzyme- secondary antibody complex [7]. niques in immunohistochemistry is highlighted
in Table 16.2.
16.8.5 Advantages
16.8.8 The Sample of Tissues
1. This is a simple and rapid two steps technique for Immunocytochemistry
2. Compared to the avidin-biotin technique, this
is more sensitive because a large number of 16.8.8.1 Histopathology
enzymes take part in a single binding of pri- • Formalin-fixed paraffin-embedded tissue
mary and secondary antibody • Frozen section
Fig. 16.10 Tyramine

signal amplification
(TSA) method: In this
technique, biotinylated
tyramine is used in the
presence of HRP and
hydrogen peroxide. The
HRP converts the
biotinylated tyramine to
reactive biotinylated
tyramide that further
reacts with tyrosine in
the amino acid of the
tissue and deposits
biotin. This biotin is
deposited only in the
antigen-antibody
reaction site. This biotin
is visualized by
avidin-biotin technique
16.8.8.2 Cytology Advantages of cell block:

• Cellblock tissue 1. Multiple sections are available
• Smear from liquid-based cytology 2. The tissue can be further used for archival
• Direct cytology smear: Imprint or FNAC use
smear 3. Minimum background staining.
• Cytospin smear
Liquid-based cytology: Multiple smears can
be made by liquid-based cytology (LBC). The
16.8.9 Sample Collection smears are small and contain a good number of
representative cells.
16.8.9.1 Histopathology
Formalin-fixed paraffin-embedded tissue (FFPE): Advantages
This is the most popular sample for IHC. Presently • Clean background
most of the antibodies work on the FFPE tissue • Concentrated cells in a small area
section. • Multiple smears
Frozen section: The use of the frozen section
for IHC is now discouraged. Direct smear: The smears from the FNAC or
Cellblock: The cell block from the cytology other samples are made directly and fixed imme-
material is preferable in the case of cytology diately. The direct smear gives the poor quality of
samples Any specimen can be used for cell block the immunostaining as there may be severe back-
except the specimen with very low cellularity. ground artifact after immunostaining.
160
Table 16.2 Comparison of different visualization techniques in immunohistochemistry

Name of technique Primary antibody Secondary antibody Visualisation system Advantages Comments
Peroxidase- Primary Ab and Different species HRP antigen and Ab against High degree of sensitivity Unsuitable for bone marrow/
antiperoxidase method antibody against than that of primary HRP complex along with DAB blood smear that contains lot
HRP are of same Ab. This is a linking used as chromogen of endogenous peroxidase
species antibody
Avidin and biotin Primary Ab Biotinylated Peroxidase conjugated avidin Rapid and sensitive Endogenous biotin in the
method Secondary Ab is and a substrate for peroxidase tissue may react with the
tagged with biotin avidin
Avidin and biotin Primary Ab Biotinylated Preformed complex of Highly sensitive False positivity due to
conjugated procedure secondary Ab avidin-biotin and horseradish endogenous biotin
peroxidase
Biotin-Streptavidin Primary Ab Biotinylated Streptavidin is directly • No cross reaction’
method secondary Ab conjugated with enzyme • More stable
Alkaline phosphatase– Primary Ab and Ab Secondary Ab Alkaline phosphatase–anti • Used in tissue containing Dual immunostain with
anti alkaline against Alkaline alkaline phosphatase complex high quantity of endogenous Peroxidase-anti peroxidase
phosphatase method phosphatase are of peroxidase method is possible
same species • Stable
• Bright red colour is easy to
identify
Polymer-based Primary Ab Secondary Ab Polymer backbone conjugating • Simple and rapid two steps
labelling method with a large number of enzyme technique
molecules and more than 20 • more sensitive because a
secondary Abs large number of enzymes take
part in a single binding of
primary and secondary Ab
• No background stain
Tyramine signal Primary Ab Secondary Ab Biotinylated tyramine reacts in • Good sensitivity • Time-consuming
amplification tagged with HRP presence of HRP and hydrogen • Non-specific background
peroxide to generate activated stains may appear
biotinylated tyramide that
reacts with tissue tyramine
Ab: Antibody; HRP: horseradish peroxidase
16 Immunocytochemistry in Histology and Cytology
Advantages 2. Cross-linking of proteins by formalin hinders

• No extra sampling the access of the antibody to the epitope of the
• No wet preparation antigen. Therefore, the antigen retrieval is
often necessary for IHC on formalin-fixed tis-
Disadvantages sue [10].
• Background staining artefact
• Limited antibody panel
16.8.10 Precautions to Have a Good
Cytospin preparations: Multiple wet fixed Fixation
slides can be prepared from cytospin preparation
of CSF, effusion or FNAC samples for the appli- The following precautions are recommended to
cation of a panel of antibodies for ICC. Gross have a good fixation for immunochemistry.
blood mixed or mucus mixed materials are
unsuitable for cytospin. Background artifact may • The sample should be immediately fixed.
be seen in cytospin preparation, but overall cyto- • It is better to use 10% neutral buffered forma-
spin preparation gives good results for ICC. lin for fixation.
• At least 8 h are necessary for good fixation.
Advantages • Same protocol of fixation should be used in
• Multiple smears can be made every sample.
• Panel of antibodies possible
• Overall good result Others: Commercially available fixatives are used
in LBC preparations, and they give equally good
Disadvantages results. Various other fixatives are ethanol, metha-
• Specimen not fit for bloody and mucoid nol and acetone are used in cytology. Alcohol fixa-
materials tive is not suitable for the demonstration of HER-2
• Difficult on scanty cellularity neu oncoprotein, estrogen and progesterone recep-
• Best choice for lymphoid neoplasm tors, and buffered formalin solution is the most
desirable fixative in this context.
16.8.9.2 Fixation
Appropriate fixation of the tissue/cell is manda- 16.8.10.1 Antigen Retrieval
tory to get good IHC. Formalin-fixed tissue often gives inconsistent
Buffered neutral formalin: Buffered neutral results of IHC. It is very difficult to replace for-
formalin (10%) is still now the best fixative for malin as a tissue fixative in real life. Enzymatic
tissue and cytology sample. digestion of the tissue also does not always pro-
vide good results. Therefore, there is a great need
Advantages to have antigen retrieval in FFPE tissue. The term
• Cell morphology well preserved “antigen retrieval” (AR) means the recovery of
• Cheap the antigenicity of the tissue which is unmasked
• Sterilizes the specimen from bacteria at the time of formalin fixation [10]. Fraenkel-
• Prevents diffusion of protein from the cell by Conrat H et al. showed that the reaction between
cross-linking the protein formalin and tissue is to some extent reversible if
heat is applied [11]. Based on this theory, subse-
Disadvantages quently, different studies have shown that the AR
1. Buffered formalin is not suitable for RNA is possible by heating the tissue [12, 13].
based IHC. For RNA stain PAX gene, RCL2 The important factors that control the AR:
or Z7-fixative are suggested [7]. PAX gene
fixative gives excellent staining quality for 1. Heating temperature and time period: There
RNA based IHC [9]. is an inverse correlation between the heating
temperature and the time needed for AR. A test should be adequate in amount to immerse the
battery approach may settle the optimum tem- slides completely
perature and time period for successful • Cover the container with a lid
AR. However, a low temperature (around • Set the microvan for 10 min (750 to 850 W)
90 °C) with prolonged heating period helps in • Remove the container and slides
the perseveration of tissue morphology. • Keep it cool for 20 to 30 min at room
2. pH of the AR solution: The majority of the temperature
antibodies work well if pH is used at around 7.4. • Wash the slides in distilled water
• Keep the slides in Tris-buffered Saline
Microwave Retrieval
This is the most effective and popular method of 16.8.10.4 Warning
AR [14]. It is always better to standardise the • Never allow the slides to dry
technique to get a consistent result. The follow- • Never use metallic container or holder
ing factors may need attention:
16.8.10.5 Pressure Cooker Heating
1. Wattage: The range of wattage of the micro- Pressure cooker heating provides uniform heat-
wave oven can be 750 to 800 W. ing to all the slides.
2. AR buffer solution: Usually, 0.01 M citrate
buffer of pH 6 gives a good result. 16.8.10.6 Requirements
3. Volume of AR buffer solution. • Pressure cooker: 4 to 5 L. Approximately
4. Number of slides in an individual 103 kPa will reach a temperature of near about
container. 120 °C
5. Thickness of tissue section: Thinner tissue • AR solution
section (3 microns) requires less time. • Plastic slide holder
6. Type of antigen: Nuclear antigen takes a lon- • AR solution
ger time for AR. • Silanized Slides
• Tris-buffered Saline
16.8.10.2 Requirements
• Microwave oven
• Plastic container for incubation (microwave 16.8.10.7 Steps
oven resistant) • At first the, remove paraffin and rehydrate the
• Plastic slide holder (microwave oven resistant) tissue section
• AR solution • Arrange the slides in a plastic holder
• Silanized Slides • Put AR solution within the pressure cooker
• Tris-buffered saline • Heat the system so that the AR solution is
• Gloves etc. for personal protection heated near to the boiling point
• Keep the plastic holder with slides within the
16.8.10.3 Steps AR solution
• At first the, remove paraffin and rehydrate the • Seal the lid of the cooker
tissue section • Heat the cooker for 30 s to a few minutes
• Arrange the slides in a microwave oven resis- • Cool the cooker for 20 min
tant plastic holder • Open the lid
• Keep the holder with slides in the plastic con- • Transfer the slides to the Tris-buffered
tainer filled with AR solution. The solution Saline
16.9 Immunocytochemistry Technique 163
16.8.10.8 Precautions epitope of the particular antigen and not with the
• The slides should be completely immersed in other antigen.
AR solution Negative control: For negative control, the
• The slides should not be dry under any primary antibody is omitted. In the absence of
circumstances primary antibodies, no staining reaction should
occur.
16.8.10.9 Water Bath Heating Positive control: The known tissue section
Water bath heating is the conventional procedure with the presence of antigen is used for positive
as a water bath is easily available in most control, such as for desmin stain one can take
laboratories. uterine myometrial tissue as a positive control.
The laboratories that work on cellblock only
16.8.10.10 Requirements should have adequate sections from the cell block
• Water bath. This should be temperature of each positive case. Paraffin-embedded sec-
controlled tions should not be kept as a control for smear
• AR solution preparation.
• Container to keep the AR solution
• Plastic slide holder
• AR solution 16.9.2 Steps
• Silanized Slides
• Tris-buffered Saline The basic steps of immunocytochemistry are
(Fig. 16.11):
16.8.10.11 Steps
• At first the, remove paraffin and rehydrate the 1. Antigen retrieval: Described before.
tissue section 2. Blocking of endogenous enzyme: In the case
• Pour a good amount of AR solution to sub- of the immunoperoxidase enzyme method, it
merge the slides is necessary to block the tissue endogenous
• Warm the container with AR solution in the peroxidase enzymes that are commonly pres-
water bath up to 98 °C ent in RBCs, polymorphs and histiocytes. To
• Now submerge the slide holder with slides block this endogenous peroxidase, the tissue
within the already heated AR solution should be kept in 0.5% to 1% hydrogen per-
• Cover the lid of the container and incubate for oxide in absolute methanol for 10 min
30 min (Table 16.3). Alkaline phosphatase (AP) is
• Take out the container and cool it present in WBC, liver, intestine, bone etc.
• Rinse the slides in distilled water When AP is used as a visualizing enzyme, it is
• Transfer the slides in the Tris-buffered Saline necessary to block this enzyme. Usually, AP
is not survived in FFPE tissue. However, it
may give trouble in the frozen section. 1 mM
16.9 Immunocytochemistry levamisole in 0.5 M HCl solution is capable to
Technique block AP enzyme. Endogenous biotin may
create problems in a biotin-based visualisa-
16.9.1 Control tion techniques. Biotin is present in the liver,
kidney, spleen, etc. The polymer-based
In any IHC staining, it is highly essential to have method can effectively overcome this prob-
proper control because it validates the laboratory lem of endogenous biotin staining.
test. The control slides indicate the specificity of 3. Blocking the background staining: Primary
the test because it is essential to know that the antibodies are highly charged molecules, and
antibody is reacted specifically to the specific they may bind by ionic and hydrophobic
Fig. 16.11 Basic steps

of immunocytochemistry
is shown in this diagram
Table 16.3 Enzymes and blockage

Enzymes Present in tissue Blocking or prevention of reaction
Peroxidase RBCs, polymorphs, histiocytes and hepatocytes 0.5% hydrogen peroxide in absolute methanol
for 10 min
Alkaline WBC, liver, intestine, bone, placenta. However, 1 mM levamisole in 0.5 M HCl solution
phosphatase absent in formalin fixed tissue
Biotin Liver, kidney, spleen Polymer-based technique
interaction with other reciprocally charged one should verify it with a different series of
molecules such as collagen or other antibod- dilutions prior to its laboratory use. Only the
ies present in the serum (Box 16.1). Moreover, dilution with intense clear staining should be
Fc receptors are present over the surface of considered as optimum dilution. In most com-
macrophages, lymphocytes, monocytes and mercially available antibodies, the range of
polymorphs. These Fc receptors may also optimal dilution is already mentioned.
bind with the antibody and produce back- 5. Incubation with labelled secondary anti-
ground staining. Preincubation with 2% nor- body: Secondary antibody is usually labelled
mal serum or 5% bovine serum albumin for 1 and directed against the primary antibody.
h may reduce the unwanted nonspecific bind- 6. Visualization: Appropriate chromogen is
ing. The normal serum protein binds with the applied to visualise the reaction.
charged particles and neutralize them. To 7. Counterstaining: Light counterstain is
block the Fc receptors instead of whole IgG applied to visualize the nuclei and tissue
one can use Fab fragments. architecture.
4. Incubation with primary antibody: Optimal 8. Dehydration and mounting: These are done
dilution of primary antibody is needed to avoid after finishing all the previous steps.
false-negative staining. Too much diluted anti-
body may fail to react with the tissue antigen. Immunohistochemistry protocol: We gener-
Therefore, in the case of any untested antibody, ally follow this protocol in our institute.
16.10 Selection of Primary Antibody 165
16.9.2.1 Chromogen
Box 16.1 Background non-specific For HRP: 3,3′-Diaminobenzidine (DAB) is
immunostaining used.
• Charged primary antibody reacts with Preparation:
oppositely charged molecules such as 10 mg DAB.
collagen 10 microliter hydrogen peroxide.
• Fc receptors over cell surface of certain 10 ml Tris Buffered saline (TBS).
cells bind the antibody For AP: AP substrate solution: Fast red kit is
• Blocking: used.
–– Incubate with 5% bovine serum albu-
min for 30 to 60 min 16.9.2.2 Tris Buffered Saline
–– Block the Fc receptors by using Fab Tris base: 61 g
fragments NaCl: 90 g
• Deparaffinize by three changes in xylene

• Rehydrate the tissue section with graded 16.10 Selection of Primary Antibody
alcohol
• Antigen retrieval: If necessary, by microwave The various factors are essential for the appropri-
treatment ate application of the primary antibody.
• Endogenous peroxidase blocking: Apply
3% hydrogen peroxide in methanol for • Exact purpose of the use of the antibody: This
10 min. (3 ml hydrogen peroxide in 100 ml is the most important factor based on which
methanol) we proceed to use the particular antibody
• Wash thoroughly twice by TBS 3 min each • Sensitivity: If the target antigen is present in a
• Blocking nonspecific sites: Apply 3% bovine scanty amount, then the highly sensitive pri-
serum albumin directly on the smear for mary antibody should be used.
30 min • Specificity: If the immunochemistry is used
• Primary antibody: Apply primary antibody for the diagnosis, then the primary antibody
with proper dilution in TBS with 1% bovine should be highly specific for the antigen, and
serum albumin for 30 min no cross-reaction is allowed.
• Wash thoroughly twice by TBS for 3 min each • Specimen type: The selection of primary anti-
• Secondary antibody: Apply secondary antibody may also depend on the type of speci-
body conjugated with HRP or AP for 30 min mens, such as paraffin-embedded tissue versus
(or biotinylated secondary antibody) flow cytometry or frozen section tissue.
• Wash thoroughly twice by TBS for 3 min • Species to generate antibody: The primary
each antibody and the corresponding secondary
• Chromogen: Apply appropriate chromogen antibody is dependent on the source where
and incubate for 3 to 10 min. Check under the these antibodies are generated.
microscope after 3 min. • The dilution of the primary antibody also
• Rinse in water depends on the different type of primary
• Counterstain: By hematoxylin for 10 s antibodies.
• Dehydrate in alcohol • Positive control: A suitable positive control is
• Clear in xylene mandatory that depends on the type of the pri-
• Mount in DPX mary antibody.
16.10.1 The Dilution of the Primary Analytical: The analytical phase includes the
Antibody following areas:
The primary antibodies are usually available in 1. Primary antibody: The proper selection of pri-
concentrated form. It is necessary to dilute the mary antibody, optimal dilution, and volume of
antibody at the time of use. The more the primary the antibody solution are important factors.
antibody concentration, the greater the risk of 2. Antigen retrieval is also an essential compo-
background staining. The optimal dilution of the nent of the analytical phase.
primary antibody means (a) the maximum intense 3. Validation of antibody: Before the clinical
positivity with the minimum background immu- use, the primary antibody should be validated
nostaining, (b) appropriate incubation time, and by an adequate number of negative and posi-
(c) optimum incubation temperature. tive control.
16.10.2 Quality Control Postanalytical phase: The interpretation of

the immunostaining is the main part of this
Quality control (QC) is an essential component phase.
of immunostaining. It is divided into three phases.
Preanalytical: This phase includes.
16.10.3 Troubleshooting
1. Proper fixation: Appropriate fixation means in Immunocytochemistry
proper fixative with optimal time duration.
2. Slide preparation: The slide should be dried The common problems of IHC have been high-
before the staining. lighted in Table 16.4.
Table 16.4 Troubleshooting in immunocytochemistry

Result Possible cause
No stain
• No stain or very little stain in both Positive and test
slide
• No background stain
Possible causes Remedies
Defective primary or secondary antibody • Expiry date may be over. Use fresh reagents
• Defective storage of the antibodies
Forget to add primary antibody, secondary antibody or • Repeat the procedure
substrate material.
Primary antibody is very much diluted and is almost • Concentrate the primary antibody
absent
Chromogen and substrate improperly used • Use proper chromogen and substrate and repeat the test
Overstain by the counterstain • Give less time for counterstain
Insufficient deparaffinization • Give longer time in xylene and use fresh xylene
Very little or no antigen present in the tissue • Use a highly sensitive IHC technique
Antigenic epitope site is destroyed during paraffin • Use wax of melting point below 60 °C
embedding
No stain in the test section but the positive control is
stained properly
Too much fixation or improper fixation Take precaution on fixation time and a fixative solution
Necrotic or crushed tissue Take a better sample from the viable area
Background staining
Both negative and positive controls are stained
16.11 Automated Immunostaining Platform 167

Result Possible cause
Secondary antibody is cross-reacting with the Apply the normal serum to block the cross-reacting
background substance antigen of the tissue
Section or smear is dried during staining • Take proper precautions to avoid drying the tissue
Chromogen is incompletely dissolved • Dissolve the chromogen properly
Positive control and test samples show background
staining, but negative control is free from
background stain
The primary antibody is over-concentrated • Dilute the primary antibody properly
Only test sample showing background stain
Over fixation and possibly the test sample and controls • Standardize the proper fixation of the test sample
are differently fixed
Table 16.5 The comparison of the open and closed

16.11 Automated Immunostaining systems
Platform Characteristics Open system Closed system
Reagents Own choice of Proprietary
Over the years, there has been significant reagents reagents to use
advancement in technology and manual immuno- Protocols Flexible Fixed
chemistry is now mostly replaced by automated Expenses Cheaper Costly
immunostaining platform (AIP) [ 15]. Skill Expert skill Not much skill as
needed everything is
Advantages: The main benefits of automated
pre-fixed
immunostaining include: Results Relatively less More consistent
consistent
1. Reduced technical staff,
2. Minimal knowledge of the technique is Closed system: In this system, the laboratory
needed, has to use only the proprietary reagents. The
3. There is minimal chance of human error in AIP, staining protocol is also fixed.
4. It gives consistent result. Depending on the loading of the slides, the
5. Reagents supervision is not needed AIP can be divided into (1) Batch loading, and
6. A good number of slides can be stained. (2) Continuous loading.
Limitations: The automated immunostaining
1. Batch loading: In this batch loading system
platform has the following disadvantages: a)
of the AIP, the slides are loaded once and in
Limited flexibility of the staining procedure, (b)
between, the loading of the other slides is not
Too much machine dependency, (c) Costly, (d)
possible.
Regular maintenance is needed.
2. Continuous loading: In this type of loading,
the different sets of slides can be loaded in
16.11.1 Types of Automated between the program and so the technicians
Immunostaining Platforms need not to wait for the completion of the
whole program.
Two types of automated immunostaining plat-
forms (AIP) are available: (a) Open, (b) closed
systems (Table 16.5). 16.11.2 Reagents Delivery Systems
Open system: This is a flexible system. The
retrieval of antigen, dilution of antibody, chemi- The delivery of the reagents to the slides may be
cal reagents, counterstaining etc., is all flexible. of two types (Table 16.6):
Therefore, the individual laboratories can pur- Rotary system: In this system, both the slides
chase the reagents of their own choice. and the reagents are in a horizontal position
Table 16.6 The comparison of the matrix and rotary

system
–– Diagnosis of malignancy in effusion
Rotary sample
Features Matrix system system
–– Sub-classification
Arrangement of the Rows and Horizontal
slide and reagents columns Lung carcinomas
Possibility of High Rare Lymphoma
contamination –– Diagnosis of soft tissue neoplasm
Speed Relatively slow. Fast Small round cell sarcomas
Capacity of slides Large capacity Less Spindle cell tumors
number
Pleomorphic sarcomas
placed in a circular disc. The reagents are directly • Prognosis
poured over the slides. –– Cell proliferation analysis
–– Hormone receptors
Matrix system: Here, the slides are arranged –– Prognostic markers
in rows and columns, and the reagents are added • Therapeutic applications: Receptor
with the help of the robotic arm. studies for specific therapy
In the market, different types of AIPs are avail- • Infective organisms to identify
able. According to the individual priorities, the
laboratory should purchase their own AIP. In mod-
ern days, it is expected that the AIP should be con-
nected with the laboratory information system and
data sharing should be free and uninterrupted. 16.12 Diagnostic
Immunocytochemistry
16.11.3 Clinical Applications 16.12.1 Mesothelial Markers

of Immunochemistry
16.12.1.1 Calretinin
IHC is now an essential component in the labora- • The sensitivity and specificity of calretinin in
tory. It has widespread use in the diagnosis, sub- the identification of mesothelial cells are
classification, and prognosis of the tumors [16]. about 100% and 80% respectively [17]
IHC also helps in the identification of various • Positivity in other cells: It is also present in
infective organisms in our body. the steroid producing cells of the ovary and
Box 16.2 highlights the major applications of testis, renal tubular cells and lipocytes.
IHC
16.12.1.2 HBME-1
• HBME-1 stains intensely around the periph-
Box 16.2 Major application of ery of the mesothelial cells.
immunocytochemistry in histology and • The specificity of HBME-1 is about 80%, and
cytology the majority of the mesothelial cells show
• Diagnosis strong positivity.
–– Detection of unknown primary
–– Classification malignant round cell 16.12.1.3 Wilms’ Tumour Gene 1 (WT-1)
tumors • This is a transcription factor isolated in the
–– Differentiating carcinoma, sarcoma, cells of the kidney.
melanoma etc. • It is expressed in the nucleus of the mesothe-
lial cells.
16.12 Diagnostic Immunocytochemistry 169
Table 16.7 Different markers of mesothelial cells

Marker Predominant use Localisation Positive in other cells
Calretinin • Mesothelial cell identification Cytoplasmic • Renal tubular cells and lipocytes
• Steroid producing cells of the ovary and nuclear
and testis
WT-1 • Mesothelial cell identification Nuclear • Desmoplastic small round cell
• -Serous adenocarcinoma of the tumours
ovary • Endometrial stromal sarcoma
• Neuroblastoma
• Rhabdomyosarcoma
• Sex cord stromal tumour
D2-40 • Mesothelial cell identification Membranous • Germ cell tumours
• Lymphatic tumour and • Synovial sarcoma
cytoplasmic • Kaposi’s sarcoma
• Positivity in other cells: The WT-1 positivity 16.12.2.3 MOC 31

is also seen in desmoplastic small round cell • MOC 31 is positive in, all the cases of adeno-
tumours and ovarian serous carcinoma. carcinoma of the lung and many non-
pulmonary adenocarcinomas.
16.12.1.4 D2-40
• It is a relatively sensitive (85%) and specific 16.12.2.4 Leu M1 (CD 15)
(95%) marker of mesothelial cells. • Leu M1 (CD 15) specifically demonstrates
• Positivity in other cells: The endothelial cells carcinoma cells. The sensitivity of Leu M1 is
of the lymphatics. low (29%).
Different markers of mesothelial cells are
highlighted in Table 16.7.
16.12.3 Different Epithelial Markers
16.12.3.1 Cytokeratin
16.12.2 Adenocarcinoma Markers • Depending on their molecular weights, cyto-
in Effusion Fluid keratin is classified into 20 different types.
The demonstration of specific cytokeratin is
16.12.2.1 BER EP4 immensely helpful in pointing out the origin
• BER EP4 antibody is highly specific and sen- of an unknown metastatic tumour.
sitive to adenocarcinoma. • Cytokeratin 7 and cytokeratin 20 phenotyping
are particularly helpful in finding out the pri-
16.12.2.2 Carcinoembryonic Antigen mary tumour in metastasis. A combination of
(CEA) CK 7 and CK 20 often helps to locate the pri-
• It is usually present in a scanty amount in mary site of the malignancy (Figs. 16.12,
normal epithelial cells and a large amount in 16.13 and Table 16.8 illustrate the expression
gastrointestinal adenocarcinoma and lung of CK and the probable origin of tumours.
adenocarcinoma.
• Specificity of CEA is very high (100%) to Epithelial membrane antigen (EMA): This
differentiate mesothelial cells from complex glycoprotein is developed from milk fat
adenocarcinoma. globules, and is unrelated to CK or intermediate
a b c
d e f
Fig. 16.12 The antibody panel helps to determine the moderate nuclear enlargement and pleomorphism. The
exact site of origin of the metastatic tumour. A 55-year- immunocytochemistry panel on cell block sections shows
old female presented with massive ascites. Cytology CK 7 (c), PAX 8 (e) and WT 1 (f) positivity, CK 20 and
smear (a) shows abundant tight ball-like clusters and the negativity (d). The immunocytochemistry result indicates
cell block section shows (b) sheets of malignant cells with the ovarian origin of the carcinoma
Fig. 16.13 The figure

shows the various
combinations of CK 7
and CK 20 positivity
and the possible origin
of the tumors
16.12 Diagnostic Immunocytochemistry 171
Table 16.8 Expression of CK and probable origin of tumors

Tumor type CK7 CK20 CK5/6
Adenocarcinoma of colon − + −
Adenocarcinoma stomach + Occasional focal positive −
Squamous cell carcinoma of lung − − +
Adenocarcinoma of lung + − −
Infiltrating duct carcinoma of breast + − Positive in basal type of carcinoma
Transitional cell carcinoma + Occasional focal positive −
Ovarian adenocarcinoma (non- mucinous) + − −
Renal cell carcinoma, clear cell − − −
Pancreas and bile duct adenocarcinoma + Occasional focal positive −
− = Negative, + = Positive
filament. EMA is present in the majority of the 16.12.4.2 Smooth Muscle

epithelial cells except for certain tumours such as • Smooth muscle actin: It is present in smooth
hepatocellular carcinoma and also adrenocortical muscle cells and myofibroblasts.
carcinoma. Unfortunately, cells other than epi- • H-Caldesmon: The high molecular weight
thelial origins, such as meningeal cells, mesothe- caldesmon (h-caldesmon) is expressed in
lial cells, certain lymphomas (anaplastic large smooth muscle. It is localised in the
cell lymphoma) and some sarcomas are also pos- cytoplasm.
itive for EMA. • Calponin: It is a calcium binding protein and
Carcinoembryonic antigen (CEA): CEA is is used as a marker of both smooth muscle
best demonstrated in the fetal epithelial cells. cells and myoepithelial cells.
This glycoprotein is abundantly present in gas-
trointestinal and lung adenocarcinomas. Normal 16.12.4.3 Peripheral Nerve Sheath
epithelial cells usually do not show CEA. Markers
S-100 protein: S-100 protein is present in the
nucleus and cytoplasm of glial and Schwann
16.12.4 Mesenchymal Markers cells. It is also seen in melanocytes, Langerhan’s
cells and myoepithelial cells and the neoplasm
• Vimentin: Vimentin is usually expressed in derived from those cells.
mesenchymal cells such as fibroblast, endothe- SOX10: This is a transcription factor and is
lial cells, smooth muscle, etc. However, it has used as a marker of peripheral nerve sheath and
been demonstrated in epithelial neoplasms such melanoma.
as renal cell carcinoma and mesothelial cells.
16.12.4.4 Marker of Vascular Tumours
16.12.4.1 Skeletal Muscle CD31: It is a marker of vascular tumour. CD31 is
• Desmin: Desmin is present in smooth muscle, expressed in platelets, endothelial cells and
skeletal muscle and cardiac muscle. megakaryocytes.
• MyoD1 and myogenin: These are markers of CD 34: CD34 is expressed in vascular endo-
skeletal muscle. thelial cells. However, it is also expressed in
• Myoglobin: It is also a marker of skeletal hematopoietic stem cells and gastrointestinal
muscle. stromal tumours.
Von Willebrand factor (vWF): vWF factor is 16.12.5 Neuroendocrine Markers

produced in the endothelial cells and megakaryo-
cytes. It is a marker of vascular tumours. Chromogranin: Chromogranin is present in the
neurosecretory granules in the neuroendocrine
cells.
16.12.4.5 Marker of Miscellaneous Synaptophysin: Synaptophysin is a marker
Soft Tissue Tumour of neuroendocrine and neuroectodermal tumours.
Transducer-like Enhancer of Split 1 (TLE-1):
TLE-1 is expressed in synovial sarcoma, solitary
fibrous tumour, endometrial stromal sarcoma and 16.12.6 Lymphoid Markers
epithelioid sarcoma.
Table 16.9 highlights the different mesenchy- • The detailed immunophenotyping of lym-
mal markers. phoid markers is very useful for identifying
Table 16.9 The different mesenchymal markers

Marker Predominant use Localization Positive in other cells
Vimentin Soft tissue neoplasm Cytoplasmic • Melanoma
• Lymphoma
• Renal cell carcinoma
Desmin • Rhabdomyosarcoma Cytoplasmic • Desmoplastic small round cell
• Rhabdomyoma tumour
• Leiomyoma and leiomyosarcoma • Alveolar soft part sarcoma
• Dedifferentiated liposarcoma
MyoD1 and • Rhabdomyosarcoma Cytoplasmic • Nephroblastoma
myogenin
Myoglobin • Rhabdomyosarcoma Cytoplasmic • Breast carcinoma
• Rhabdomyoma • Colonic carcinoma
Smooth muscle Leiomyoma and leiomyosarcoma Cytoplasmic • Myofibroblastic tumour
actin • Gastrointestinal stromal tumour
• Endometrial stromal sarcoma
H-Caldesmon Leiomyoma and leiomyosarcoma Cytoplasmic • Gastrointestinal stromal tumour
• Epithelioid mesothelioma
• Glomus tumour
Calponin Leiomyoma and leiomyosarcoma Cytoplasmic • Myofibroblastic tumour
•
S-100 protein • Neurofibroma Cytoplasmic • Melanoma
• Malignant peripheral nerve sheath and nuclear • Lipomatous tumour
tumour • Langerhans cell histiocytosis
SOX10 • Neurofibroma Nuclear • Melanoma
• Malignant peripheral nerve sheath • Clear cell sarcoma
tumour • Granular cell tumour
CD31 Vascular tumour Membranous and • Langerhans cell histiocytosis
cytoplasmic • Ewing’s sarcoma
• Plasmacytoma
CD34 Vascular tumour Membranous and • Gastrointestinal stromal tumour
cytoplasmic • Solitary fibrous tumour
• Dermatofibrosarcoma protuberans
• Granulocytic sarcoma
Von Willebrand Vascular tumour Cytoplasmic No other tumour
factor
TLE-1 Synovial sarcoma Nuclear • Solitary fibrous tumour
• Endometrial stromal sarcoma,
Epithelioid sarcoma
16.13 Immunocytochemistry of Round Cell Tumour 173
Table 16.10 Panel of antibodies for lymphoma 16.12.9 Site-specific Antibody

subtyping
in Different Epithelial
Cells and tumours Markers Malignancies
All lymphoid cells CD 45
B cell CD 19, 20, 23, 10
16.12.10 PSA and Androgen Receptor
T cell CD 3, 5, 4, 8
NK cell Positive: CD3, CD56
Negative: CD19, CD10, CD 23 • Prostate-specific antigen (PSA) is positive in
Hodgkin’s lymphoma CD15, CD 30 normal, hyperplastic, and neoplastic prostate
tissue.
lymphoid cells and sub-classification of • PSA is also positive in breast carcinoma and
lymphomas. salivary gland tumors [19].
• The essential lymphoid markers are high-
lighted in Table 16.10
16.12.11 Androgen Receptor
16.12.7 Melanoma Markers • It is present in the nucleus of the prostate epi-

thelial cells.
HMB45: HMB 45 is a highly specific marker of
melanoma. It also identifies neural crest derived
tumours, occasional carcinomas and immuno- 16.12.12 TTF
blastic lymphomas.
Melan A (Mart-1): This is a melanocyte asso- • Thyroid transcription factor-1 (TTF-1) is pres-
ciated marker. Melan A is also positive in periph- ent in follicular epithelial cells of the thyroid,
eral nerve sheath tumours, angiomyolipoma and bronchial epithelial cells of lung and adeno-
steroid producing cells of the adrenal cortex [18]. carcinomas developed from those cells.
16.12.8 Germ Cell Markers 16.12.13 Estrogen and Progesterone

Receptors (ER and PR)
Placental alkaline phosphatase (PLAP): PLAP
is expressed in normal placenta. It is used to dif- • ER and PR are present in the nucleus of the
ferentiate germ cell tumours and any other undif- breast and endometrial adenocarcinomas.
ferentiated malignancy.
Human chorionic gonadotropin (HCG):
HCG is usually secreted by the syncytiotropho- 16.13 Immunocytochemistry
blast cell of the placenta. The beta subunit of of Round Cell Tumour
HCG is hormone specific.
OCT4: OCT4 is a reliable marker of germ Round cell tumours are often difficult to categorise
cells. It is expressed in seminoma/dysgerminoma, by morphology alone, particularly in cytology
embryonal carcinoma, and gonadoblastoma. material. A panel of antibodies is helpful in differ-
SALL4: SALL4 is a sensitive and specific entiating the different round cell tumors
marker of germ cells. It is positive in seminoma/ (Fig. 16.14). Table 16.11 highlights the basic panel
dysgerminoma, yolk sac tumour, embryonal car- to differentiate the different malignant round cell
cinoma and choriocarcinoma. SALL4 is also tumors.
positive in adenocarcinoma of the colon, oesoph-
agus and cervix.
a b c
d e f
Fig. 16.14 Antibody panel to determine the type of a 45 (b) and CD 99 (c), and positive for NSE (d), chromo-
malignant round cell tumor. A 13-year-old male presented granin (e) and synaptophysin (f). The result of the immu-
with 5 cm diameter right upper abdominal mass near the nocytochemistry panel indicates the diagnosis of
adrenal gland. The cytology smear (a) shows discrete neuroblastoma
monomorphic round cells. The cells are negative for CD
Table 16.11 Basic panel to differentiate the different malignant round cell tumors
Tumor type CD 45 CD99 Myogenin Pancytokeratin Desmin Chromogranin
NB − + − − − +
EWS/PNET − + − − − +
NHL + − − − − −
RMS − − + − + −
DSRCT − − − + + −
WT − − − + + −
NB: Neuroblastoma; EWS: Ewing’s sarcoma; PNET: peripheral neuroectodermal tumor; NHL: Non- Hodgkin
lymphoma; RMS: Rhabdomyosarcoma; DSRCT: Desmoplastic small round cell tumor, WT: Wilms’ tumor
16.14 Immunocytochemistry 16.14.2 Estrogen and Progesterone

for Therapy and Management Receptors
16.14.1 Breast Carcinoma • ER /PR nuclear positivity is related to anti-

estrogen hormone therapy of breast cancer.
Various hormonal receptors along with Her 2/
Neu status of breast carcinoma are mandatory for
the therapy and management of breast carcino- 16.14.3 Her 2/Neu
mas (Fig. 16.15).
• The status of Her 2/Neu over expression in
breast cancer is helpful for anti Her 2/Neu
antibody therapy.
16.15 Gastrointestinal Stromal Tumor 175
a b
c d
Fig. 16.15 The cell block of a case of infiltrating duct carcinoma of breast (a). Estrogen receptor (b), progesterone
receptor (c), and Her 2/neu immunostain (d) were done that showed strong positivity
16.15 Gastrointestinal Stromal Tumor 16.15.1 Lung Carcinoma
CD117 (C-Kit): Gastrointestinal stromal tumour A panel of antibody is helpful to differentiate

with C-Kit mutation (high surface level of CD squamous cell carcinoma from adenocarcinoma
117 expression) is responsive to tyrosine kinase of lung (Fig. 16.16). The cases of lung adenocar-
inhibiting agent (imitanib) therapy [20]. cinoma should be investigated for EGFR muta-
tional changes for specific receptor inhibitory
drugs. Table 16.12 shows the basic panel for the
differentiating adenocarcinoma from squamous
cell carcinoma of lung.
a b
c d
Fig. 16.16 A case of lung carcinoma (a) shows p63 (b), and CK 5/6 positivity (c) and TTF-1 negativity (d). This indi-
cates the diagnosis of squamous cell carcinoma
Table 16.12 Markers of lung carcinoma

Type p40 p63 TTF1 Napsin CK 5/6 CK 7
Squamous cell carcinoma + + − − + −
Adenocarcinoma − − + + − +
+ = positive, − = negative
References 4. McMichael AJ, Pilch JR, Galfre G, Mason DY, Fabre

JW, Milstein C. A human thymocyte antigen defined
by a hybrid myeloma monoclonal antibody. Eur J
1. Coons AH, Creech HJ, Jones RN. Immunological
Immunol. 1979;9:205–10.
properties of an antibody containing a fluorescent
5. Erber WN, McLachlan J. Use of APAAP technique on
group. Exp Biol Med. 1941;47:200–2.
paraffin wax embedded bone marrow trephines. J Clin
2. Nakane PK, Pierce GB. Enzyme-labeled antibodies
Pathol. 1989;42:1201.
for the light and electron microscopic localization of
6. Cordell JL, Falini B, Erber WN, et al.
tissue antigens. J Cell Biol. 1967;33:307–18.
Immunoenzymatic label of monoclonal antibodies
3. Taylor CR, Burns J. The demonstration of plasma
using immune complexes of alkaline phosphatase and
cells and other immunoglobulin-containing cells
monoclonal anti-alkaline phosphatase (APAAP) com-
in formalin- fixed, paraffin-embedded tissues
plexes. J Histochem Cytochem. 1984;32:219.
using peroxidase-labelled antibody. J Clin Pathol.
7. Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli
1974;27:14–20.
M, Ceccarelli C, Pieri F, Fraternali-Orcioni G, Pileri
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SA. The EnVision++ system: a new immunohistochemi- 14. Shi S-R, Key ME, Kalra KL. Antigen retrieval
cal method for diagnostics and research. Critical com- in formalin- fixed, paraffin-embedded tissues: an
parison with the APAAP, ChemMate, CSA, LABC, and enhancement method for immunohistochemical stain-
SABC techniques. J Clin Pathol. 1998;51(7):506–11. ing based on microwave oven heating of tissue sec-
8. Toda Y, Kono K, Abiru H, Kokuryo K, Endo M, tions. J Histochem Cytochem. 1991;39:741.
Yaegashi H, Fukumoto M. Application of tyra- 15. Sharma S, Dey P. Automated immunostaining plat-
mide signal amplification system to immunohisto- form in cytology. J Cytol. 2021;38(2):57–63. https://
chemistry: a potent method to localize antigens that doi.org/10.4103/JOC.JOC_145_20. Epub 2021 May
are not detectable by ordinary method. Pathol Int. 10. PMID: 34321770; PMCID: PMC8280864
1999;49:479–83. 16. Dey P. Immunocytochemistry in diagnostic cytol-
9. Staff S, Kujala P, Karhu R, Rökman A, Ilvesaro J, ogy. New Delhi: Jaypee Brothers Medical Publishers;
Kares S, Isola J. Preservation of nucleic acids and tis- 2021.
sue morphology in paraffin-embedded clinical sam- 17. Yaziji H, Battifora H, Barry TS, et al. Evaluation of
ples: comparison of five molecular fixatives. J Clin 12 antibodies for distinguishing epithelioid meso-
Pathol. 2013;66(9):807–10. thelioma from adenocarcinoma: identification of a
10. Shi SR, Cote RJ, Taylor CR. Antigen retrieval tech- three-antibody immunohistochemical panel with
niques: current perspectives. J Histochem Cytochem. maximal sensitivity and specificity. Mod Pathol.
2001;49(8):931–7. 2006;19:514–23.
11. Fraenkel-Conrat H, Brandon BA, Olcott HS. The reac- 18. Busam KJ, Iversen K, Coplan KA, Old LJ,
tion of formaldehyde with proteins. IV: Participation Stockert E, Chen YT, McGregor D, Jungbluth
of indole groups. J Biol Chem. 1947;168:99. A. Immunoreactivity for A103, and antibody to
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for fine-tuning diagnoses in cervical cytology. In: Shi tumors. Am J Surg Pathol. 1998;22:57–63.
S-R, Gu J, Taylor CR, editors. Antigen retrieval tech- 19. Bodey B, Bodey B Jr, Kaiser HE. Immunocytochemical
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Eaton Publishing; 2000. p. 93–113.
Flow Cytometry: Basic Principles,
Procedure, and Applications 17
in Pathology
17.1 Introduction Box 17.1 Principle of Flow Cytometer

• Single cell suspension
Flow cytometry (FCM) is the method by which the • The specific component of the cell are
various characteristics of individual particles or stained with a fluorescent tagged antibody
cells are studied. FCM provides us with a very • DNA binding dye binds with DNA
quick assessment of cell surface antigens, DNA stoichiometrically
content, and intracellular proteins. The instrument • A leaser beam of particular wave length
is now used not only for research but also for rou- hits the cell
tine clinical activities [1–3]. It is now a well-estab- • The cell absorbs light and emits
lished technique for the diagnosis and classification fluorescence
of lymphoid neoplasms and identification of • The emitted fluorescence is recorded
malignant cells in effusion cytology and other with the help of the photodetectors
body fluids. The modern flow cytometer is now • The electronic signal is converted into
much more user-friendly and smaller in size. digital signals
• The results are then displayed as histo-
gram or two dimensional dot plots.
17.2 Principle of Flow Cytometry
Single dissociated cells in a liquid medium are

essential for flow cytometry. The specific compo- 17.3 The Flow Cytometer
nent of the cell is identified by the antibody tagged Instrument
with a fluorescent dye. Similarly, DNA can also be
stained by a DNA stoichiometric dye. The single The flow cytometer instrument has four main
cells rapidly pass in front of a laser bim and the components [2] (Fig. 17.1):
laser beam of a particular wavelength hits the cell. 1. Fluidics system: The main aim of the fluidics
The individual cells absorb the light and emit light system is to maintain a stable flow of a single
of different wavelengths. The emitted light is column of cells/particles within the sheath
detected by the photomultiplier tube and is con- fluid so that each particle/ cell is hit by the
verted into a digital pulse. The intensity of the digi- laser beam. In the fluidic system, the cells
tal signal is stored in the computer and expressed in flow within the sheath fluid that helps to pre-
a relative scale known as a channel. The result of vent any turbulence of the flowing cells and
the events is expressed as a dot plot or histogram. the cells are illuminated uniformly by the
https://doi.org/10.1007/978-981-19-6616-3_17
180 17 Flow Cytometry: Basic Principles, Procedure, and Applications in Pathology
Fig. 17.1 Schematic diagram of the basic principle of The emitted light is detected by the photomultiplier tube
flow cytometry is highlighted in the picture. The single and is converted into a digital pulse. The result of the
cells are hit by the laser beam of a particular wavelength. events is expressed as a dot plot or histogram
laser beam. The sample fluid passes at a collect the light of a specific wavelength.
higher pressure than the sheath fluid. Dichroic long pass mirror transmits the light
2. Optical system: The main part of the optical of higher wavelength and reflects the light of
system is the laser light source. The laser lower wavelength. Dichroic short pass mirror
generates monochromatic light with a spe- prevents the transmission of the light of higher
cific wavelength. Laser light is highly wavelength and allows only the light of
intense, unidirectional, and sharply focused. shorter wavelength.
There are different sources of laser such as 3. Electronic system: The primary function of
Argon, krypton, helium, etc. The argon laser the electronic component is to receive the
source is commonly used. It provides a light light signal and then convert it into the electric
source of 488 nm wavelength that can be current and finally the voltage is converted
used for many commonly used fluorescent into a digital pulse. The photodetector receives
dyes. Moreover, the argon source of laser has the emitted photon of the light and converts it
a high output, higher gain system, and is less into the voltage. The photomultiplier system
divergent. helps to multiply the voltage. Now the voltage
The optical filter is another component of data is converted into digital data, known as
the optical system. There are different types analogue to digital conversion.
of optical filters in the flow cytometer. The 4. Computer system: The computer records the
combination of the optical filters is used to data generated from each cell/ particle.
17.3 The Flow Cytometer Instrument 181
Fig. 17.2 The diagram

shows the principle of
fluorochrome dye
activation. When a
fluorochrome compound
absorbs a quantum of
light, the electron moves
from the lower orbit to
the higher orbit, and the
compound remains in
excited state. When the
compound comes back
to its ground state, the
electron returns to its
original orbit, and the
compound emits the
quantum of light of a
lower wavelength with a
different colour
What is fluorescence: When a fluorochrome The higher the value of Stoke’s shift, the
compound absorbs a quantum of light, the electron greater the separation of the excitation and
moves from a lower orbit to a higher orbit, and the emission spectrum.
compound remains in an excited state. When the
compound comes back to its ground state, the
electron returns to its original orbit, and the com- 17.3.1 Light Emission
pound emits the quantum of light of a lower wave- and Scattering
length with different colours (Fig. 17.2). The
whole phenomenon is known as fluorescence. As In the case of fluorescence dye tagged with cells,
the excited lightwave and emitted light waves are fluorescence is emitted. The emitted fluoresce of
of different wavelengths and colours so the emit- different wavelengths of different dyes are sepa-
ted light is easily visible and can be recorded. rated and collected with the use of a set of optical
Each fluorochrome dye compound has certain filters.
properties: When light (photon) hits a cell, the light is
scattered. The scattering of light may be forward
• Specific excitation spectrum of light scattering (FSC) or side scattering (SSC).In the
• Specific emission spectrum of light case of FSC, the light is scattered in the same
• The quantum efficiency direction as the laser beam. Whereas, in SSC, the
light is scattered right angle to the axis of the
The difference between the wavelength of laser beam. FSC indicates the cell size and SSC
excitation or absorption spectrum and the indicates the granularity and internal complexity
emission spectrum is known as Stoke’s shift. of the cell (Fig. 17.3).
Table 17.1 Fluorchrome dye used in DNA

Excitation Emission
maximum maximum
Flurochrome (nm) (nm)
Propidium Iodide 305, 540 620
Ethidium bromide 493 620
Hoechst 33342 350 461
Hoechst 33258 352 461
diamidinophenylindole 359 461
(DAPI)
Acridine orange 503 530 (DNA),
640 (RNA)
Fig. 17.3 The light scattering property of the cells. The
forward scatter indicates the cell size, and the side scatter Table 17.2 Fluorochrome dye for conjugating with
indicates the granularity and internal complexities antibody
Excitation Emission
17.4 Flow Cytometric Cell Sorting maximum maximum
Fluorochrome (nm) (nm)
A flow cytometric cell sorting system is used to Fluorescein Iso- 495 519
thiocyanate (FITC)
capture and isolate the cell of interest. These iso-
Phycoerythrin (PE) 496 576
lated cells can be analysed for the further functional
Allophycocyanin 650 660
studies. (APC)
Basic principle: In the flow cytometric cell Rhodamine Red-X 570 590
sorter system, the cells are passed rapidly through Texas Red® 595 613
a narrow orifice and the high-frequency PE-Cy7® 488 566 778
piezoelectric crystal is placed near the orifice to Peridinin Chlorophyll 477 678
(PerCP)
make a stable droplet of the individual cells. At
the time of passing in front of the laser beam, the nm: nano micrometer, 10−9 m
data of the individual cell is generated. The
moment a particular cell fits with the set data, it is Dye for RNA content measurement: RNA
immediately charged by an electric plate. Now content is measured with acridine orange, pyro-
the charged cell is deflected with the help of an nin Y, and oxazine 1.
electrically charged deflecting plate (Fig. 17.1).
17.6 Samples for Flow Cytometry

17.5 Dye Used
17.6.1 Cytology Samples
17.5.1 Fluorochrome Dye
for Nucleic Acid [2] The various types of cytology specimens for
FCM include:
The common dyes in DNA FCM include 1. Fine needle aspiration cytology materials:
Propidium Iodide, Ethidium bromide, Hoechst Lymph node, breast, lung, prostate etc.
33342, and diamidinophenylindole (Table 17.1). 2. Exfoliative samples: Effusion fluid, CSF,
Fluorochrome dye for antibody and pro- bladder wash
tein: Various fluorochrome dyes are used for
conjugating antibodies and protein. The most The special advantages of cytology speci-
commonly used fluorochrome dye is mens (Box 17.2) over the histopathology tissue
fluorescein isothiocyanate (FITC). Table 17.2 include:
highlights the excitation and emission spectrum 1. Cytology samples are easy to process and
of different commonly used fluorescein dyes. require less effort for disaggregation thereby
17.6 Samples for Flow Cytometry 183
Box 17.2 Advantages of Cytology Sample for Box 17.3 Collection Fluid for Flow Cytometry
FCM Citrate buffer
• Easy to process • Sucrose: 85.3 g
• Less effort to disaggregate the cells • Trisodium citrate (Sigma): 11.8 g
• Easy to procure the sample • 50 ml of Dimethyl Sulfoxide in 100 ml
• Possible to study from the multiple of water
areas of the tumor or lymph nodes. • pH keep at 7.6.
• Viable cells so functional studies Phosphate buffered solution
possible • 8 g NaCl
• 0.2 g KCl
• 1.15 g Na2HPO4
single-cell preparation is easier to make for • 0.2 g KH2PO4
FCM. • Dissolve the material in 900 mL dis-
2. It is relatively easy to procure cytology tilled water.
samples. • Keep pH to 7.4 with HCl
3. Multiple areas of the tumour or lymph nodes • Add distilled water to make 1000ml
can be collected for processing.
4. The different functional studies can be done
as the cells are viable. Enzymatic digestion: The enzymes such as
trypsin or papain can be used to break the cell to
Collection: The cytology samples can be col- cell attachment. After a specific period of time
lected in 1 ml of citrate buffer solution or the enzymatic action should be stopped or the
phosphate-buffered solution (PBS) (Box 17.3). cells may be totally digested. Currently, commer-
The sample can be processed immediately or cially prepared cocktail enzymes are available in
maybe kept at −70 °C to process later on. the market that are easy to handle. The enzymatic
method is unsuitable for the analysis of cell sur-
17.6.1.1 Histology Samples face antigen.
Mechanical method: In the mechanical
• Frozen section tissue method the cell sample is rinsed through a thin
• Paraffin-embedded tissue bore needle and finally the cells are filtered by
nylon mesh placed between the needle and
The histopathology sample needs thorough dis- syringe hub. The mechanical method of cell dis-
aggregation for flow cytometry. In fact, paraffin- sociation is easy, cheap and suitable for
embedded tissue does not give a good result on immunophenotyping.
FCM.
17.6.3 Cellular Fixation
17.6.2 Single-cell Preparation
Cellular fixation is not required to process the
Single-cell preparation is the vital step of sample fresh sample. However, cell fixation is needed if
preparation because unless the cells are separated the sample is processed at a later date or intracel-
from each other, no meaningful data can be lular antigen is studied. The sample is fixed after
obtained. The lymphoid cells or the cells in the the cellular dissociation. The cells are fixed with
blood are usually less cohesive. But the epithelial 0.4% formaldehyde for 15 min at 37 °C and then
cells are attached by tight and gap junctions. centrifuged and washed with PBS. For DNA con-
These junctions can be ruptured either by tent analysis, one can fix the cells with 95% ethyl
mechanical or by the enzymatic method. alcohol.
17.6.4 Permeabilization • Keep the cell concentration in the buffer is

kept as at least 2 × 106 cells per ml in 0.4 ml
Permeabilization of the cell membrane is needed PBS (pH 7.4).
for the analysis of intracellular antigen or DNA • Add 500 μl of solution A containing Triton
content. The common permeabilizing agents are X-100, RNAse and propidium iodide fresh
Triton X, Tween 20 and saponin. These agents solution (see below)
permeabilize the cell membrane but retain the • Incubate for 30 min at room temperature in
intracellular antigenic content intact. the dark
• Run in flow cytometer within 2 to 3 h
17.6.5 RBC Lysing Solution

17.6.7.2 Stock Solution of Propidium
The RBC lysing solution helps to lyse the RBCs Iodide (PI)
in the cell sample. The commercially available
RBC lysing solutions usually contain ammonium • Propidium iodide (PI): 1 mg
chloride. • Water: 2 ml
Store in 4 °C in dark
17.6.6 Control Solution A
1. RNAse A: 2 mg
DNA FCM control: Lymphocytes from the 2. Triton X-100: 10 ml: 0. 1 % (v/v)
peripheral blood samples of healthy individuals. 3. Propidium iodide: 0.40 ml of stock solution
Flow cytometric immunophenotyping: The (500 μg/ml)
indications for use of controls are: Make fresh solution
• Any suspicion of artefactual change

• To monitor the autofluorescence and non- 17.6.8 Flow Cytometric
specific binding in the sample Immunophenotyping (FCI)
Negative control: Unlabelled processed samples
may work as a negative control. 17.6.8.1 Direct Stain
With the help of commercially available
• Centrifuge the sample: 3000 rounds per min-
fluorescein-labelled beads, one should do the set-
ute for 3 to 5 min
ting of optics, fluidics and electronics. The spe-
• Discard the supernatant fluid
cific company provides certain instructions and
• Re-suspended in citrate buffer
this should be obeyed strictly.
• Prepare single-cell suspension by repeated
syringing through nylon mesh or by trypsin-
17.6.7 Sample Processing ization [1]
• Keep the cell concentration in the buffer is
17.6.7.1 DNA Flow Cytometry [1] kept as at least 2 × 106 cells per ml.
• Take in 100 μl in PBS (pH 7.4).
• Centrifuge the sample: 3000 rounds per min- • Add 10 μl of labelled antibodies for 25 min in
ute for 3 to 5 min a dark place at room temperature.
• Discard the supernatant fluid • Wash the cells three times in PBS solution by
• Re-suspended in citrate buffer 3000 rounds per minute for 3 to 5 min.
• Prepare single-cell suspension by repeated • Discard supernatant
syringing through nylon mesh or by • Resuspend the cells in 500 μl PBS solution
trypsinization • Run in FCM
17.6 Samples for Flow Cytometry 185
17.6.8.2 Indirect Staining Procedure • Keep for 30 min at room temperature

[2, 3] (Fig. 17.4): • Wash three times in PBS
• Discard the supernatant and re-suspended the
• Collect sample in citrate buffer cells in 500 μl PBS
• Wash the cells in PBS (3000 rounds per min- • Run in FCM
ute for 3 to 5 min)
• Discard the supernatant and re-suspended the
cells in resuspending them in ice-cold PBS, 17.6.9 Data Aquisition [2]
3% BSA, 1% sodium azide.
• Keep 100 μl solution of cells (1.5 × 106 cells/ The sample number and the name of the anti-
ml concentration of cells) into multiple small body should be mentioned clearly on the vial.
aliquots Threshold of fluorescence: The cells should be
• Take 100 μl solution of cells and incubate with in the liquid suspension, and the vial containing
50 μl of primary non-conjugated antibody for cells should always be vortexed before data
30 min at room temperature in dark. acquisition. The fragmented cells or small par-
• Wash the cells in PBS ticles may produce background noise. The mini-
• Add 50 μl of a conjugated secondary antibody mum threshold value of the fluorescence should
Fig. 17.4 Schematic

diagram explains the
steps of indirect
immunostaining
be recorded so that the background noise is Single cells are

eliminated. gated
Live gating: “Gating of the cell” means the
selection of the target cells of interest. The tar-
get cells are selected at the time of data acqui-
100 150 200 250

sition based on (a) size of the cells detected by
(x1,000)
FSC, (b) the antibody marker that the cells
show, such as CD45 markers expressed by
lymphoid cells, or (c) based on positive control
FSC-H
at times.
The number of events to record: At least
100,000 events should be recorded in the case of
50
flow cytometry for immunophenotyping.
However, the number of recorded events may
vary. It largely depends on (a) amount of debris
within the sample and probable frequency of the 50 100 150 200 250
FSC-A
cells of interest within the experimental sample. (x 1,000)
For DNA flow cytometry, only 10,000 events can
be recorded. Fig. 17.5 The single cells are gated by adjusting the for-
ward scatter area and height
17.6.10 Data Display
105
and Interpretation
The basic data of the FCM is recorded in “list

104
mode” or standard file. All the individual events'

CD8PE-A
information, such as FSC, SSC, and fluorescence

values are recorded here. Further, the data is dis-
103
played with the help of software packages sup-

plied by the companies.
Dobublet exclusion: The doublet of the cells
are excluded by adjusting FSC and SSC
0
(Fig. 17.5).
-265
Bivariate histogram: Bivariate histogram is

applied when the two parameters, that means two 0 103 104 105
-1,664 CD4 APC-A
attached fluorochromes on a single cell, are stud-
ied. In this histogram, one parameter is kept on Bivariate histogram
the X-axis and the other in the y axis. The four-
quadrant histogram is convenient to study Fig. 17.6 Bivariate histogram
(Fig. 17.6).
Gating: Manual gating can be done on the 17.6.11 Quality Control
acquired data by selecting the population of cells.
The region of interest is identified by drawing a The appropriate quality control (QC) is an essen-
polygon or rectangle (Fig. 17.7). In the case of tial part of the FCM. The QC has internal quality
sequential gating, the cells and subset of the cell control (IQC) and external quality assurance
population are selected step by step. (EQA).
17.7 Targets of Application 187
Internal quality control: IQC has the follow-

ing components: Box 17.4 Measurement of Cellular Features
• DNA ploidy analysis
1. Specimen integrity: The specimen should be
• Cell cycle analysis
free from any blood clot, haemolysed ele-
• Intracellular and cell surface receptors
ments, or necrotic debris. There should be an
• Cell size
adequate number of cells in the specimen for
• Cell viability and apoptotic cells
analysis.
• Enzyme activity: phosphatase, gluo-
2. Antibody: The appropriate primary antibody
curonidase etc.
selection is essential to identify the target
• Metabolic studies: Oxidative burst,
population. Monoclonal antibody gives better
intracellular pH, intracellular Calcium
result than polyclonal antibodies.
• Cellular protein, lipid, hemoglobin,
3. Fluorochrome: Proper selection of
• RNA content
fluorochrome- conjugated antibodies is also
• Mitochondrial function
needed. The weaker antigen, such as CD19,
• Physical phenomena: Phagocytosis,
should be conjugated with highly sensitive
pinocytosis etc.
fluorochrome. Whereas the stronger antigen
can be conjugated with less sensitive
fluorochrome.
4. Acquisition of a number of events: The acqui-
sition of the optimum number of cells is
100 150 200 250
needed to get a meaningful result.

SSC-A (x 1,000)
5. Instrument quality control: The flow cytometer

is a sophisticated instrument. The laser align-
Gated population
ment, the optical set-up of the photomultiplier of CD45 positive
tube and compensation set-up are needed for cells
the successful use of the instrument.
50
Daily set up: The daily set-up of FCM is manda-

tory to get the best performance. The commer-
cially available beads are used to assess laser
101 102 103 104 105
alignment, the voltage check-up of PMT and the 0
compensation of the emitted fluorescence. CD45 APC-Cy7-A
External quality assurance: For the EQA,
Fig. 17.7 CD45 positive lymphoid cells are gated
the part of the same specimen is sent to the differ-
ent laboratories for FCM. The result of the The most common applications of FCM in
assessment of the population of the cells and clinical laboratories include [2, 4, 5] (Fig. 17.8):
coefficient of variation are compared at the end
• Immunophenotyping of lymphoma and
of EQA.
leukaemia
• DNA ploidy analysis and cell cycle analysis
• Detection of malignancy in body fluid
17.7 Targets of Application
In addition, FCM is also used for histocompati-
FCM can be used to quantitatively measure vari- bility cross matching, HLA-B27 detection, CD 4
ous cellular characteristics (Box 17.4). Many of and CD 8 counting for immunodeficiency,
the targets of application of FCM are reticulocyte enumeration and PNH detection in
research-oriented. immunology and haematology laboratories.
Fig. 17.8 Common

applications of flow
cytometry is highlighted
in this diagram
17.8 DNA Content and Ploidy double channel numbers in the DNA histo-
Analysis gram forming a small tetraploid peak.
• In between these two peaks, the cells have
17.8.1 Basic Principle varying amounts of DNA from 2n to 4n, and
they are in the synthetic (S) phase.
• Any peak other than these two peaks is con-
• DNA specific dye stoichiometrically binds sidered as an aneuploid peak.
with the DNA of the nucleus
The following information is obtained
• Emitted fluorescence from the dye-DNA com-
from DNA FCM
plex is directly proportional to the DNA con-
tent of the nucleus 1. Identification of aneuploid cell population
• The data are displayed as a DNA histogram 2. DNA index
• The majority of the normal cells contain dip- 3. Proliferative cell fraction (% of S-phase)
loid (2n) chromosomes, so they emit a certain
DNA ploidy: The clone of cells containing the
amount of fluorescence represented by a chan-
abnormal amount of DNA is known as aneuploid
nel number. This forms a single peak known
cells. In a DNA histogram, the aneuploid popula-
as the diploid peak in DNA histogram
tion will form a peak somewhere other than the
• The cells in the G2M phase contain the double
diploid peak.
amount of DNA (4n), so they emit the double
The relative DNA content of the aneuploid
amount of fluorescence and are present in
population of cells is calculated by DNA index.
Mean channel number of aneuploid peak

DNA index =
Mean channel number of normal diploid peak
The different types of aneuploidy are mentioned Hypodiploid aneuploidy (Fig. 17.9): Here,
below: the aneuploid cell population forms a peak left to
Hyperdiploid aneuploidy (Fig. 17.9): Here, the diploid peak as they contain less than the 2n
the aneuploid peak lies between diploid and tet- amount of DNA. (DNA index is a lower value
raploid peaks (DNA index is between 1 to 2). than 1).
17.10 Immunophenotyping of Lymphomas 189
Fig. 17.9 Schematic

diagram of different
types of aneuploidy in
DNA histogram
Tetraploid aneuploid (Fig. 17.9): Aneuploid lesion [1, 2]. However, we should keep in mind that
population of cells make a peak on G2M peak. It malignant tumours may often show a diploid cell
is often challenging to distinguish a tetraploid population, and uncommonly benign tumours may
peak from a normal G2M peak. However, more have aneuploidy population of cells Diagnosis of
than 20% cell population in this peak suggests a malignancy only on the basis of DNA ploidy has
tetraplod aneuploidy (DNA index is 2). limited use in the detection of malignant cells in
Hypertetraploid aneuploidy (Fig. 17.9): effusion cytology specimen due to its low sensitiv-
Here, the aneuploid population of cells form a ity [6]. Similarly DNA ploidy estimation of bladder
peak beyond the G2M peak as they have more wash and CSF has relatively poor success [7, 8].
than 4n amount of DNA. peak (DNA index is
greater than 2).
S phase: The S-phase fraction of cells has 2n 17.9.2 DNA Content and Prognosis
to 4n amount of DNA, so they remain between of the Patients
G0G1 and G2M peak. The number of such cells
can be calculated from the DNA histogram. DNA aneuploidy and high S phase are poor prog-
nostic factor in various solid tumors such as bladder,
prostate, ovarian and endometrial carcinomas [9].
17.9 Clinical Application
17.9.1 DNA Content and Diagnosis 17.10 Immunophenotyping

of Lymphomas
DNA FCM provides usual information on the
DNA ploidy and S-phase fraction of the tumour A large number of CDs have been described in
cell population. The presence of an aneuploidy cell different cells of lymphoid origin [10]
population and high S- phase suggests a malignant (Table 17.3). Monoclonal antibodies against
various CDs are now also available for

flow cytometric immunophenotyping (FCI)
5
10
(Figs. 17.10, 17.11, 17.12, and 17.13). The
antibodies are labelled by different fluoro-
chromes of the different emission spectrum.
4
10
Therefore judicious use of the fluorochrome
PE-A
tagged antibodies may help to perform mul-
ticoloured FCI. This has a significant impact
3
10
on the application of FCI as one can study a
large number of CDs in a small volume of
samples. Demonstrating various CD markers
2
10
on the lymphoid cell surface helps to diagnose
and sub-classify lymphomas.
102 103 104 105
Table 17.3 CD markers of lymphoid cells CD19 FITC-A
Cells Markers
Fig. 17.11 Dot plot of CD 19 stain (B cell marker) in
All lymphoid cells CD 45 flow cytometry
T lymphocyte CD 2
CD 3
T helper cell CD4
5
10
T cytotoxic cell CD 8
T regulatory cell CD 4
CD 25
NK cell CD 56
4
10
CD20 PE-A
B lymphocytes CD 19
CD 20
Plasma cell CD 138
3
10
Plasmacytoid dendritic cell CD 304

Stem cell and progenitor cell CD 34
CD 117
CD 271
2
10
2 3 4 5
10 10 10 10
FITC-A
105
Fig. 17.12 Dot plot of CD 20 stain (B cell marker) in

flow cytometry
104
17.10.1 Diagnosis:
PE-A
Q1 Q2
• Most the cases of lymphoma are monoclonal.
103
So light chain restriction of either lambda or

kappa chain indicates lymphoma (of B cell
origin). In FCM, the kappa/ lambda ratio of
102
Q3 Q4
more than 4:1 or more than 1:2 is usually con-
sidered evidence of monoclonality.
102 10
3
104 105 • Clonality demonstration in T cell NHL is dif-
CD45 FITC-A
ficult in FCM. However, the aberrant expres-
sion of CD2, CD5 and CD7 expression may
Fig. 17.10 Dot plot of CD 45 stain in flow cytometry. suggest T NHL [11].
with the FCI data. Laser scanning cytometry

5
10 overcomes this problem.
2. Admixture of other material: Benign reac-
tive components of the lymph node such as
vascular or stromal material are often admixed
4
CD3 PE-A
10
with the cells of interest. Appropriate gating is

needed to eliminate such cells.
3
3. Scanty cells may be missed: It is challenging

10
to identify Reed Sternberg’s cell in the speci-

men of Hodgkin’s lymphoma because of
scanty R-S cells in the specimen.
2
10
4. No light chain restriction: All cases of

B-NHL may not always show light chain
2 3 4 5
10 10 10 10 restriction. This may be due to failed expres-
CD2 FITC-A sion of light chain on the surface of the cell.
5. High cost of FCM instrument: FCM is over-
Fig. 17.13 Dot plot of CD 2 and CD 3 stain (T cell all a costly technique and needs good skill.
marker) in flow cytometry
17.10.4 Flow Cytometry Features

17.10.2 Sub-classification of Different Lymphomas
of Lymphomas:
Table 17.4 highlights the different CD markers of
FCI can be done easily on FNAC material lymphoma cases
because the cells are discrete in cytology mate-
rial. The advantages of FCI over immunocyto-
chemistry are: 17.10.5 Diagnosis of Other Lesions
by FCI
• It is a fast technique to analyze a large popula-
tion of cells Effusions: Epithelial cells are usually absent in
• The various CD markers can be applied to the the effusion fluid. So the demonstration of epi-
small volume of aspirate. thelial cells in effusion fluid may help to identify
• Dual expression of CD markers. the carcinomas. EpCAM, and BER EP4 antibod-
• Additional features such as DNA ploidy, cell ies recognise the epithelial cells. Sahu et al. have
cycle analysis etc., can be done simultane- shown the EpCAM on FCM has high sensitivity
ously along with immunophenotyping. (87%) and specificity (100%) in detecting meta-
• Quantitation of antigen positive cells is static carcinoma in effusion [12].
possible. Bladder washings: DNA ploidy estimation in
Figure 17.14 highlights the comparison of FCM the gated population of cytokeratin positive cell
immunophenotyping and immunocytochemistry. is helpful in detecting malignancy in follow up
cases of urothelial carcinoma of the bladder [13].
CSF: Leptomeninges are often involved in
17.10.3 Limitations of FCI metastatic carcinomas. The CSF sample is usu-
ally of scanty cellularity and admixed with histio-
FCI bears certain limitations that include cytes. The identification of carcinoma cells may
be difficult in both cytology and FCM. It has
1. No morphological correlation: This is the been shown that EpCAM based FCM is highly
most significant disadvantage of FCI. It is not sensitive and specific (100%) for the detection of
possible to correlate morphological findings metastatic carcinoma in CSF [14].
Flow cytometric
immunophenotyping Immunohistochemistry
A-2617-Tube_002
102 103 104 105
CD20-PE-A
102 103 104 105

CS45 FITC-A
• Slow process
• Rapid
• Less objective
• More Objective
• Limited number of CD
• Large number of CD
markers can be used
markers can be used
• Manual quantition of CD
• Quantition of CD positive
positive cell possible
cell possible
• Quantitation of intensity of
• Quantitation of intensity of
antigen positivity not
antigen positivity possible
possible
• Morphology of the cells not
• Morphology of the cells can
possible to see
be seen
• No archival storage
• Archival storage possible
Fig. 17.14 The diagram highlights the differences between flow cytometric immunostaining versus
immunocytochemistry
17.10.6 Predicting Response CD10/TdT or CD19/CD34/CD10/CD13 is used

to Monoclonal Therapy in B cell NHL. Aberrant expression of markers
indicates MRD. In the case of T cell NHL, CD34/
As a therapy for low-grade NHL, Rituximab, a CD3 panel is used [16].
monoclonal antibody, is applied against CD20 on FCM in HIV infection: The quantitation of
B cells. The follow up of these cases needs close CD4 and CD8 count in the blood sample of HIV
monitoring of the measurement of monoclonal infected patients is possible by FCM.This is very
antibody CD 20. This is possible with the help of helpful in monitoring HIV patients.
FCM [15]. Reticulocyte count: FCM analysis of reticu-
locytes is a standard and preferred method.
Auramine O and thiazole orange are FDA proved
17.10.7 Detection of Minimal reagents for reticulocyte analysis using FCM.
Residual Disease
17.10.7.1 Steps [17]
Multicolour FCM by using a panel of antibodies 1. Thiazole orange reagent (1 ml)
against a wide range of CD markers is helpful in 2. Add 5 ml peripheral blood
the rapid detection of minimal residual disease 3. Incubate for 30 min
(MRD). A panel consisting of CD19/CD34/ 4. Vortexing the mixture
5. Run in FCM
Table 17.4 CD expression of different lymphomas

Diagnosis Immunophenotyping
Small lymphocytic Positive for: CD5, CD23, CD19, CD 20
Co-expression of CD 5 and CD 23,
Negative for: CD10
Mantle cell lymphoma Positive for: CD5, CD19 , CD 20
Paraffin section: Cyclin D1, FMC7
Negative for: CD10, CD23
Follicular lymphoma Positive for: Surface Ig, CD10, CD19, CD20 , Bcl2, Bcl6
Negative for: CD5
Lymphoplasmacytic lymphoma Positive: Cytoplasmic immunoglobulin, CO I9. CD20. CD22. CD 79a, CD 38
Negative: CD 5, 23, CD 10
Marginal zone lymphoma Positive: Surface Ig, CD 19,CD 20
Negative: CD5, CD10, CD23
Hairy cell leukemia Positive: CD103, CD11c+, CD25+
Negative: CD10, CD3, CD5,
Plasmacytoma/ multiple myeloma Positive: CD38+, CD138+
Negative: CD10, CD3, CD5,CD23
Precursor B lymphoblastic Positive: Tdt , CD10, CD19, CD20
Negative: Surface Ig
Burkitt’s lymphoma Positive: CD10, CD 19 , 20, surface Ig +,
Negative: CD5, CD23
Ki67index is more than 85%.
Diffuse large B cell lymphoma Positive: Surface Ig +/−, CD10 +/−, CD 19, 20
Negative: CD5 (90%)
Ki 67 index less than 90%.
T lymphoblastic Positive: TdT , CD3, CD7, CD4 +/−, CD8
Peripheral T cell Positive: CD 3, CD7 and CD4/CD8
Anaplastic large cell Positive: CD30 (Ki-1) , EMA, ALK
Negative: CD45 +/−, CD3 +/−
17.10.7.2 Apoptosis [18, 19] 6. Wash the cells in PBS by centrifuging

FCM is one of the critical technology to detect 7. Discard the supernatant fluid
apoptotic cell death. Quantitation of the apoptotic 8. Add 1 ml solution containing Propidium
cells is possible with the help of light scatter(a Iodide, RNAse and Triton X. [RNAse 2 mg,
loss in forward light scatter), plasma membrane Triton X 10 ml and PI 0.40 ml of (500 μg/
changes, and DNA content (a sub diploid peak). ml)]
9. Incubate for 1 h in the dark
10. Vortex and then run in FCM
17.10.8 Assessment of Sub-G1 11. Measure the sub diploid population of cells
Fraction of Apoptotic Cells
1. Cells are washed at 1500 RPM for 5 min in 17.10.9 Apoptosis Detection by

PBS. Annexin V Assay
2. Resuspend the cells in PBS and keep the
concentration as 1.5 × 106 cells/ml 1. Wash the cells in PBS by centrifuging
3. Take the cells in 60 μL of PBS 2. Discard the supernatant fluid
4. Add I ml ice-cold 70% ethyl alcohol drop by 3. Keep the cells and keep the concentration of
drop 1.5 × 106 cells/ml
5. Keep for 1 to 2 h in −20 °C for 4. Resuspend cells in 500 μL of binding buffer.
permeabilisation 5. Add 5 μL of annexin V conjugated with FITC
6. Incubate at room temperature for 5 min in the 11. Dey P. The role of ancillary techniques to diag-
dark. nose and sub-classifiy non hodgkin lymphomas
on fine needle aspiration cytology. Cytopathology.
7. Run in FCM for the detection of anexin 2006;17(5):275–87.
binded cells 12. Sahu S, Gupta P, Susheilia S, Gautam U, Dey
P. Application of multicolour flow cytometry in the
detection of metastatic carcinoma in serous effusions:
Special emphasis in atypical cytology. Cytopathology.
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tion with cytologic grade and clinical relapse. Diagn rametric DNA/cytokeratin flow cytometry in urine
Cytopathol. 2000;22:153–6. voided samples from patients with bladder carcinoma.
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Digital Pathology
18
18.1 Introduction The digital image may be the simple acquisition

of an image by a digital microscope, transferring
In the last few decades, there is a massive devel- the image to the distant site at the time of viewing
opment in computer technology followed by or the whole slide imaging (WSI) of the pathol-
marked advancement in the field of high- ogy slide. The DP was the first time used in tele-
resolution image digitization, image storage, pathology where the image file was directly or
extraction of features and analysis. Virtual indirectly sent to the remote site. The recent
microscopy has also significantly advanced in the introduction of WSI has made a revolutionary
last few years. Previously the pathologists used to change in this field and the WSI file can be imme-
give an opinion on holistic viewing of the image diately studied in the onsite laboratory or remote
of the tissue/cells. Presently there is increasing area.
demand to provide semi-quantitative features
along with the quantitative information of vari-
ous biomarkers on the tissue section or cytology 18.2.1 Comparison of Traditional
smear [1]. Pathology and Digital
Digital image analysis (DIA) provides more Pathology
objective and consistent information about the
images and also helps in the diagnosis, grading, The traditional pathology is based on the obser-
and classification of the disease. It provides vari- vation of the glass slides under the light micro-
ous prognostic information and guidance to ther- scope. It is easy to procure a light microscope in
apy. Nowadays it is essential to have good a laboratory. The handling of the light micro-
knowledge of digital analysis and virtual micros- scope is easy and it does not need any special
copy for the effective improvement of reporting skill. The observation of a light microscope and
tissue specimen [2]. its interpretation to some extent are subjective. In
comparison, DP bypasses the study of the glass
slide. One can directly study the virtual slides on
18.2 What Is Digital Pathology? the computer screen. The DP images are easy to
transport across the globe. Moreover, quantitative
The term “digital pathology” (DP) was used by measurement of various features and application
Weinstein [3] in 1986. It means the data collec- of artificial neural networks (ANN) are easy to do
tion, sharing and decision taken on the basis of on DP. However, the implementation of the DP in
digital images along with clinical information. the laboratory needs a complete setup. The cost
https://doi.org/10.1007/978-981-19-6616-3_18
196 18 Digital Pathology
of DP is hundred times more than traditional 1. Allotment of the specimen identification

pathology. Table 18.1 compares the traditional number (Barcoding: The specimen and the
pathology and digital pathology. slides are allotted with unique barcoding. The
barcoding number helps to identify the speci-
men or slide in any part of processing.
18.2.2 Workflow of Digital Pathology 2. Imaging of the gross specimen: The images of
the gross specimen of the patient are taken
The workflow of DP in any laboratory is [4] and those images are incorporated into the
(Fig. 18.1): laboratory information system (LIS).
3. Processing of the tissue blocks or sample: The
Table 18.1 Comparison of traditional pathology and processing of the histopathology tissue or
digital pathology
samples is not a part of the DP. However,
Traditional many laboratories may prefer to have an auto-
Features pathology Digital Pathology
mated tissue processor, auto-stainer and cover
Availability Easily Limited
available availability slipper to support the DP system.
Price Less costly Thousand times 4. Whole-slide-imaging (WSI): This is one of
costly the vital steps of DP. With the help of the
Technical dealing Easy to Needs initial whole slide scanner, the whole section or
handle training cytology slides are digitized.
Quantitative Limited The software
assessment possibility algorithm is
5. Uploading the WSI file and clinical data: The
available digitized images are then uploaded to the sys-
Artificial neural Limited use Increasingly tem along with the details of clinical and
network or another used radiological data.
expert-based system 6. Interpretation: Now the respective patholo-
Transport Takes time Immediate
gists observe the digitized images and inter-
Storage space Physical Virtual space
pret the case along with full clinical details.
Fig. 18.1 Workflow of digital pathology in a laboratory

18.3 Whole Slide Imaging (WSI) 197
7. The validation and circulation: In this step, blocks and slides are needed. The barcoding
the final interpretation is validated by the con- gives the unique identity of the case.
sultant of the team and it is then uploaded in 2. Whole slide imaging (WSI) system: WSI sys-
the hospital information system. tem is one of the mandatory components of
DP. It helps to digitize the entire slide.
3. Work station to observe the slide: There may
18.2.3 Basic Instruments be multiple substations to see the digitized
and Software in Digital image.
Pathology (Box 18.1) 4. Laboratory information system (LIS): The
software of LIS provides the essential labora-
1. Electronic labelling of the specimen and slide tory data of the patient.
by barcoding system: The barcoded specimen, 5. Additional software for quantitative measure-
ment and artificial neural networks.
Box 18.1 Essential Components of a Digital
Pathology Laboratory
• Electronic labelling of the specimen and 18.3 Whole Slide Imaging (WSI)
slide by barcoding system
• Whole slide imaging (WSI) along with WSI is the main component of the DP. With the
Image management system (IMS) help of WSI, the entire slide is captured as a
• Laboratory information system (LIS) digitized image. Each image of the slide is noth-
• Electronic patient record system ing but multiple pixels of different intensities of
• Radiology imaging system red, green and blue colour. The WSI stores the
• Neural network module: Optional optical density each pixels of each colour
(Fig. 18.2). The pathologist can view any area of
Fig. 18.2 The basic principle of the whole slide imaging system
the slide by any magnification in the digital 2. Microscope with lens and objective:
workstation. Microscope with attached objectives are used.
The components of the WSI: The components Mainly 20× and 40× are used. Occasionally
of the WSI can be divided into )a) Hardware and 2×, and 100× objectives are attached with the
(b) Software [5] (Box 18.2). microscope.
3. Focussing and light adjustment components:
The bright field light source is used to illumi-
18.3.1 Hardware nate the slide.
4. Robotics to load the slide and movement: The
The hardware system is composed of: primary function of the robotics is to take the
slide from the slide holder, place it on the
1. Slide loader: The slide loader loads the slide for stage for scanning, move the slide and then
WSI. Depending on the vendor of WSI, the take it back from the stage. WSI system reads
number of loaded slides varies from 100 to 300. the barcode of the slide and then stores the
digitize images. The scanning time varies
from 30 s to 1 min in 20× objective. However,
Box 18.2 Components of Digital Pathology in 40× or to do Z scanning, the scanning time
Hardware is more than 1 min.
• Slide loader 5. Digital camera for image capturing: The
• Microscope with lens and objective images are captured by CCD or LED camera.
• Focussing and light adjustment
components
• Robotics to load the slide and movement
• Digital camera for image capturing 18.3.2 Software
Software
• Image capturing The software of the WSI has the following
• File format components:
• Image viewing and analysing
• Accessory optional software for image 1. Image capturing: The tissue or smear over the
analysis and artificial neural network slide is scanned and the image is captured.
The scanning may be of two types (Fig. 18.3):
Fig. 18.3 Schematic diagram of different types of scanning

(a) Tile-based scanning: In this type of scan- antigenic expression of the specific cellular
ning, a large number of square images are area.
assembled together into a mosaic pattern. 4. Accessory optional software for image analy-
The tiles of images are stitched together sis and artificial neural network: The WSI sys-
to make a complete image. tem may have many additional software
(b) Line-based scanning: The slide is scanned components for image analysing to do quanti-
longitudinally and the group of long unin- tative measurements and also artificial neural
terrupted strips are stitched together to network (ANN) software for the interpreta-
have the total image. tion such as diagnosis, classification, prog-
(c) The scanning is done mainly in the X and nostic assessment and management of
Y-axis directions. However, Z-axis scan- diseases.
ning is done in a cytology slide and 50 to
150 images in the different planes of the
smear are taken and finally, a single image 18.3.3 Commercially Available WSI
is made. Z-scan imaging takes more time
and more disk space to store. Several WSI machines are available in the market
2. File format: The file format may vary from with different file formats, objectives, scanning
machine to machine. The common file format time periods, and Z-scan facilities. Table 18.2
is TIFF and JPEG. The vendor may use their shows the characteristics of different WSI
proprietary file format that can be used only in machines.
their own system. The interoperable file exten-
sion is needed to read the same image by the
different system. The average image takes 600 18.3.4 Advantages of Digital Slides
to 800 MB. However, the Z scan image takes
2 to 3 GB space to store. The digital slide (DS) has revolutionized the area
3. Image viewing and analysing: Image viewing of the conventional concept of glass slides and
is one of the important components of the microscopy. Unlike conventional microscopy,
software that helps to navigate the image. The DS needs only a computer with internet access.
viewer can pan the image, magnify and also Therefore, there is no need for multiple micro-
focus on the digital image to draw the inter- scopes or multi-headed microscopes for teaching
pretation. In addition, with the help of an and training [6].
annotation tool, the viewer can mark the area The greatest advantage of DS is that simulta-
of interest, measure the area and write text neously many people may have the access to the
over the selected area. The co-registering slides and therefore it can be used in teaching or
facility may help to superimpose the immuno- training in pathology (Box 18.3). The same
histochemistry field over the original haema- image is seen by both the students and teachers
toxylin and eosin-stained slide to assess the and therefore there is better interaction in a
Table 18.2 Comparison of some commercial WSI systems

Features Hamamatsu Leica 3D Histech Philips Ventana
Model Nanozoomer Aperio AT2 Pannoramic UltraFast iScan HT
Slide capacity 320 400 250 300 400
Digital slide format JPG TIFF JPG – TIFF, BIF, JPG
Z stack Yes yes Yes –
Special feature Quality scoring Automated Continuous loading LCD LCD integrated
scanning touchscreen
Scan speed 20×: 30 s 20×: 60 s 20×: 36 s 40×: 60 s 20×: 80 s
40×: 270 s
Table 18.3 Digital slide versus conventional glass slide

Box 18.3 Advantages of Digital Slides Features Digital slide Glass slide
• Multiple users can view the slides Resolution Yes, possible Always possible
• Only a computer and internet access are Viewing Multiple viewers A limited
enough to teach can see at the number of
same time viewers can see
• Same images are seen both by students
by multi-headed
and the teacher so better interaction is microscope
possible Remote Possible by a Long procedure
• No need to have multiple microscopes transportation fraction of
and their maintenance second
Storage and Only virtual Physical space is
• Consistent classical cases can be stored
free of damage space is needed needed
as DS Fading of the No such question In course of time
• More than one slides can be viewed in a slide arises the stain fades
same screen to compare the lesion Retrieval Rapid procedure Slow procedure
• The specific area or the particular object Slide library Virtual library Limited access
can be labelled for attention and possible and of the slide to
images of the the viewer
display same type of
• No fading or damage of digital slides lesion can be
• No need of multiple slides and thereby compared
tissue is not exhausted Cost Very high Cheap
• The cases can be accessed instantly and
there is no need of museum staff
• Distant teaching (tele-education) and Box 18.4 Disadvantages of Digital Slides
even self-learning • Initial high cost of the equipment
• Virtual workshop • Need of huge storage space
• Unfamiliarity of the digital slide to
interpret
teaching session. The initial low power image of
the slide provides the orientation of the tissue.
The annotation on the image also highlights the particularly in the case of the small biopsy
important features of the slide for the better specimen.
understanding of the lesion. Moreover, the DSs The comparison of DS and conventional light
may be accompanied by immunocytochemistry microscopy (CLM) has been highlighted in
or other histochemical stains that help in the Table 18.3.
proper diagnosis and classification of the lesion.
In addition, clinical history, simple radiographs,
computed tomography or magnetic resonance 18.3.5 Disadvantages of Digital
imaging can be added along with the DSs, Slides
Therefore, a complete disease picture may be
available along with DS. One of the major disadvantages of the DS is the
Distant learning is possible with the help of initial cost of the equipmemts [7].
DS. One can even conduct an online workshop (Box 18.4). The slide scanner and the acces-
as the DS can also be available on the internet sories of the whole slide scanning system are
site. Moreover, the good quality DS of the clas- very costly. The next important problem of DS is
sical disease can be used for conducting the the storage of high-resolution data. Huge amount
examination. Unlike conventional glass slides, of disc space is required for the storage of the
DS will never fade. And there is no need for individual slides. Therefore, servers with high-
extra slides. So the tissue will not be exhausted capacity storage capability are needed.
The other probable disadvantage is the 2. The validation should include the prepara-
reduced interaction of the students and the light tions that will be used for the reporting by
microscopic examination of the slides. This is not WSI, such as frozen section, cytology smear
a problem in undergraduate training as very few etc.
students get a chance to handle light microscopes 3. The validation study should represent the
in their future careers. However, for the post- real-world situation as far as possible. If the
graduate training overemphasis on training multiple May Grunwald Giemsa stained
through the DS may be detrimental to the routine slides of fine needle aspiration cytology are
examination of the independent diagnosis of the used for the routine diagnosis then the same
glass slide. set of WSI should be used as DS.
4. Each WSI system includes the total system
such as image scanning, file preparation and
18.3.6 Concordance of Glass Slides image viewing. The entire WSI system
and Digital Slides should be considered for the comparison of
the DS and conventional glass slide.
The primary diagnosis of DS is still a burning 5. It is essential to re-validate the entire WSI
issue. Goacher et al. in a meta-analytic study system if there are any significant changes in
compared WSI and CLM for primary diagnosis any component of the system.
in 38 published studies [8]. The mean diagnostic 6. The validation set to include at least 60 cases
concordance of WSI and CLM was 92.4%. The of each category such as 60 cytology cases,
mean kappa coefficient of the agreement of diag- 60 frozen sections, and 60 histopathology
nosis was 0.74 which is significantly high and cases in each validation set.
indicated good agreement. In a recent study, 7. The same pathologist should interpret the
Azam et al. [9] also did a meta-analysis based on same set of glass slides and digital slides.
24 published studies. They noted 98.3% concor- 8. The DS and glass slides should be interpreted
dance rate between DP and CLM. The major randomly.
areas of discordance were related to the assess- 9. There should be at least 2 weeks gap between
ment of mitosis, nuclear atypia, and grade of dys- the viewing of DS and the glass slide.
plasia. In fact, the various studies consistently 10. The entire material of the glass slide should
mentioned that the concerns in DS are recogniz- be digitized for interpretation.
ing microorganisms, nuclear atypia, mitosis and 11. The entire method, concordance rate and
grading of dysplasia. approval should be recorded and documented
for the future use.
18.3.7 Guidelines of Clinical

Diagnostic Application of WSI 18.3.8 Applications of Digital
Pathology
As the WSI technology is a comparatively newer
technology and most pathologists are not aware Digital pathology has the following applications
of its use as a primary diagnostic tool so it is nec- in the field of pathology (Box 18.5):
essary to have proper validation before its use.
The College of American Pathologist (CAP) has 1. Education: Presently the entire slides can be
published strict guidelines on the implementation scanned and stored in the computer system.
of primary diagnosis by WSI [10]. The recom- The slides can be available on the web page or
mendation of the CAP are: on the parent computer. The classical cases
can be used for teaching purposes [11].
1. It is necessary to have own validation study 2. Online opinion (telepathology): With the
of the laboratory before implementing WSI help of a digital imaging system, it is possible
as a primary diagnostic tool. to send the scanned image of the whole slide
tumours in individual persons. It is often seen

Box 18.5 Applications of Digital Pathology that the same tumour with the same type of
• Primary reporting treatment has a different outcome in two dif-
• Education: The digital slides to use for ferent patients. Digital image analysis can
teaching provide useful information on (a) tumour
• Telepathology: Digital slides to use for morphology, (b) tumour classification, (c)
immediate online opinion tumour grading, (d) tumour stroma reaction,
• Pattern recognition and grading of (e) quantification of biomarkers, and (f)
carcinoma molecular phenotypes. The combined data of
• Quantitation of immunohistochemistry all those features may be helpful to plan for
• Assessment of aggressiveness of a personalised treatment [18].
tumor for personalized medicine
18.3.9 Limitations and Challenges

of Digital Pathology
to the distant center. The experts can examine
the virtual slide and give their opinion. Cost: The WSI system and overall set-up of DP
Thereby the physical transfer of the slides can is very costly in comparison to conventional light
be avoided and rapid opinion may be available microscopy.
in a particular case [12]. Storage capacity: The DS does not need any
3. Primary diagnosis: The primary diagnosis of physical space, but it needs a lot of disc space and
the cases is possible by DP. With the integra- so there is a need for the hard disc with huge
tion of hospital information service and the space for storage.
WSI system, the pathologist can get the clini- WSI system: A very fast, efficient WSI sys-
cal, biochemical and other ancillary data along tem is needed for DP.
with WSI. The slide can be interpreted from Integration and compatibility of the differ-
anywhere and a second opinion can be taken. ent system: Laboratory information service and
4. Pattern recognition and grading of carci- hospital information service should be linked
noma: Image analysis has been applied to with DP.
diagnose and grading of carcinomas. WSI has
been used to grade and differentiate neuro-
blastoma [13] WSI along with artificial neural
networks were able to grade glioma [14].
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Automation in the Laboratory
and Liquid-Based Cytology 19
19.1 Introduction cervical cytology slides are available in the

market. In the histopathology laboratories,
Automation in the laboratory is the recent trend various task-based semi-automated soft-
in a modern laboratory. Automation reduces ware systems are available for counting
human intervention as far as possible. Both mitosis, Ki67 indexing, PDL1 indexing
cytology and histopathology laboratories are etc.
increasingly applying automation. The automa-
tion in the laboratory incorporates the following
areas: 19.2 Advantages of Automation
in Laboratory
1. Receiving and barcoding the specimens.
2. Processing the specimen. In the histology lab- 1. Reduction of manual labour: Automation
oratory, it includes tissue processing microt- reduces the manpower of the skilled technical
omy for section cutting, staining and staff. However, for the initial implementation
coverslipping for the final submission. of the automation, we need a trained
The cytology laboratories also have liquid- technologist.
based cytology (LBC) processor that makes 2. Error: The manual error in the processing is
monolayered cytology. Presently both semi- almost nil.
automated and completely automated LBC 3. Turnaround time: The turnaround time of the
systems are available. Similar to the histopa- overall reporting may not be reduced signifi-
thology laboratory, the cytology laboratory cantly. However, in a busy laboratory, the
may also have an automated stainer, and cover overall turnaround time is expected to be less
slipper. in the case of complete automation in differ-
3. Digitalisation of the slide: Presently many ent steps.
laboratories have the whole slide imaging 4. Consistency of excellency: The consistency of
(WSI) system that scans the slide and makes the overall laboratory quality improves in
the digital image for interpretation. automation.
4. Automated interpretation: We currently 5. Laboratory evaluation: Automation helps to
have task-based automated slide inter- evaluate the quality control system as the
preter. Presently commercially available whole information of the cases is systemati-
semiautomated slide screening systems for cally stored in the laboratory.
https://doi.org/10.1007/978-981-19-6616-3_19
206 19 Automation in the Laboratory and Liquid-Based Cytology
Limitations:
Cost: Automated systems are costly and ulti-
mately in developing countries, it is an extra bur-
den to the health care system.
19.2.1 The Various Stages

of Automation
Receiving and barcoding (Fig. 19.1): The bar-

coding of the specimen, request form, cassette,
paraffin block and slides is the first important
step of the automation. Unique bar-coding helps
to eliminate the error of labelling and tracking the
tissue or slide.
19.2.2 Tissue Processing
Histopathology: The automated tissue processor is

now commercially available. The tissue processor
processes the tissue including dehydration, paraffin
infiltration, and tissue embedding (Fig. 19.2). Most
of the work is done by the machine itself with lim-
Fig. 19.2 Automated SMART tissue processor
ited human intervention. The SMART automation

includes the processing, embedding and to some
extent microtomy for the section cutting. The
SMART automation is supposed to increase pro-
ductivity, reduces the turnaround time and reduce
the overall time of reporting the slide. The SMART
processing technique has the facility for continuous
loading of slides in every 20 min and the overall
processing time is reduced to 2 to 3 h only. Low
power microwave and vacuum technology are used
for tissue processing. There is significantly less
error in SMART processing and the quality of the
slides is better than in conventional processing.
Currently, along with processing, fully automated
Fig. 19.1 Barcoding of the sample and request form embedding is also available. Automated embedding
19.4 Cytology Processing 207
Fig. 19.3 Automated stainer
helps to eliminate human mistakes, reduces any done without any manual intervention. The
chance of tissue loss, and as it dispenses the opti- AIP provides consistent high-quality immu-
mum amount of wax, there is no need for block nostaining. Both open and closed systems of
scraping. It is important to note that all types of tis- AIP are available in the market. The detail of
sue are not suitable for SMART processing. AIP has been highlighted in the previous chap-
Particularly neurological tissue, fatty tissue and ter (Chap. 16).
bones are unsuitable for SMART processing.
Cytopathology: In cytology, different liquid-
based cytology preparation systems are avail- 19.3.1 Digitization of Slide
able (ThinPrep and SurePath systems). These
systems are described in the next section of the The digitization of the slide is done with the
chapter. help of Whole slide imaging (WSI) system.
Microtomy: In histopathology, the semiauto- The various types of WSI are available in the
mated microtomes are available. The automated market. The WSI system may have a variable
microtomes can cut the paraffin-embedded tissue number of slide scanning facility systems (100
according to the desired thickness. to 300) with different scanning speeds and
Staining: Currently automated strainers are Z-scan facilities. Each system has a different
available in the market that can stain a large num- file format for storage and display of digitized
ber of slides (Fig. 19.3). The automated stainer images. A detailed description of WSI is given
provides consistent and high-quality staining. It in Chap. 18.
can perform multiple staining protocols.
Coverslipping: The automated cover slipper
helps to mount the slide without DPX. 19.4 Cytology Processing
The liquid-based cytology (LBC) is an increas-

19.3 Automated Immunostaining ingly popular technique for the preparation of
Platform (AIP) the cervical sample and also other exfoliative
samples such as effusion fluid, sputum, bron-
Currently, the majority of the busy laborato- choalveolar lavage etc. This technique is rela-
ries have introduced AIP [1]. With the help of tively more costly than the conventional
AIP, a large volume of immunostaining can be preparation. However, LBC preparation pro-
vides a clean, monolayered smear in a small

area of the slide. As the smear is free of blood,
mucus and drying artefacts so it is easy to inter-
pret [2, 3].
19.4.1 Advantages of LBC over

Conventional Smear
The major advantages of LBC over conventional

preparation (CP) include:
• Majority of the collected cells are available in
the liquid medium of the collection vial.
Whereas, the major part of the collected cells
are stuck in the spatula of the convention
smear preparation and the cells are thrown in
the waste basket.
• LBC preparation is completely free of any air-
drying artefact.
Fig. 19.4 Smear of conventional and liquid-based cytol-
• There is the almost complete absence of any ogy. Note the small area of liquid-based smear
blood, mucus or necrotic debris in LBC prepa-
ration and the cells are present in a small area
which is easy to screen (Fig. 19.4). Box 19.2 Limitations of liquid based
• Monolayered preparation of the cells is pres- cytology
ent in certain LBC preparation. • High cost
• HPV test is possible from the residual material • Trained cytotechnicains needed
of the LBC sample. • Loss of certain background information
• The monolayered cell preparation may be use- • No define evidence of increased sensi-
ful in the automated detection of malignant tivity of the detection of HSIL cases
cells in the smear.
19.4.2 Limitations of Liquid-based

Box 19.1 Advantages of Liquid Based Cytology
Cytology
• No air drying The major limitation of LBC is the initial cost of
• Representative cells in adequate the machine. Moreover, the collection fluid, spat-
number ula, and processing costs are also significantly
• Small smear area is easy to screen high than CP (Box 19.2). The laboratory staffs also
• Clean background need adequate training for the preparation and
• Monlayered cells screening of the smear. The cells are dispersed
• HPV testing from the neighbouring cells and certain architec-
• Multiple slides can be made tural features in adenocarcinoma may be lost [4].
Moreover, the smear may be free of any tumour
19.5 Sample Processing 209
diathesis and therefore the diagnosis of squamous ThinPrep Pap test and SurePath systems. The
cell carcinoma may be difficult. There is no defi- preparation techniques of these two techniques
nite evidence that LBC reduces the inadequate are different and are described below.
smear rate or it detects more number of high grade
squamous intraepithelial lesions (HSIL) [5].
19.5.1 ThinPrep (Cytic, UK) (Fig. 19.5)
19.4.3 Collection Procedure of LBC The steps of ThinPrep processing are:
• Patient should be in lithotomy position 19.5.1.1 Dispersion and Collection

• Inspect the cervix with the help of a speculum of the Cells on the Filter
• Clean any watery discharge or mucus with wet • Collect the sample in PreservCyt fixative solu-
cotton tion supplied by the company.
• Insert the cervical broom within the cervical • The cylindrical tube with an attached filter on
canal so that shorter bristles touch the the lower surface is submerged within the vial
ectocervix. containing the cells in the PreservCyt solution.
• Rotate the broom several times in the clock- • The cylinder rapidly rotates within the vial
wise and anticlockwise direction and disperses the cells mechanically.
• Remove the broom and rinse it vigorously • Simultaneously negative suction pressure is
within the liquid provided by the company. applied within the cylinder that drains the
• Cap the vial tightly for the further processing fluid of the vial.
• The cells cannot pass through the filter and
stick on the undersurface of the filter of the
cylinder tube. The negative sectional pressure
19.5 Sample Processing
can be adjusted by an inbuilt software to con-
trol the flow of the fluid.
Presently there are two commercially available
liquid-based technologies that are approved by Cell transfer: The trapped cells are now accu-
Food and Drug Administration (FDA), USA: mulated on the surface of the filter attached to the
Fig. 19.5 Schematic

diagram highlighting the
basic principle of the
ThinPrep technique. The
rapid movement of the
filter causes cellular
dispersion.
Simultaneously negative
suction is applied within
the vial of the filter that
helps to remove the fluid
and the cell sticks on the
filter
cylindrical tube. The machine now automatically Cell Enrichment

transfers the cells from the surface of the vial to • Mix the sample with PREPSTAIN® Density
the glass slide. The glass slide is automatically Reagent and centrifuge (Fig. 19.8).
submerged in the fixative solution. • Decant the supernatant elements containing
blood, mucus and necrotic debris by applying
19.5.1.2 SurePath Test (Fig. 19.6) vacuum suction
The overall steps of SurePath test are highlighted • Re-centrifuge the remaining fluid to have a
in Fig. 19.6. cell-rich pellet.
Vortexing (Fig. 19.7): At first with the help of
vortexing the cells are mechanically dispersed.
Fig. 19.6 Schematic

diagram highlighting the
basic principle of the
SurPath technique. The
cells are dispersed by
Vortexing. Cell rich
suspension is made by
buffy coat preparation.
These cells are taken out
and resuspended in the
fluid. Finally, the cells
are settled down by
gravity on the glass slide
19.6 Comparison of These Two Techniques 211
Fig. 19.7 Vortexing causes cell dispersion in the Fig. 19.9 The vials are kept in the settling chamber to
SurePath technique settle the cells by gravity
Fig. 19.8 Centrifugation of the sample with the addition

of density gradient mixture helps to enrich the cells
Resuspension
• Re-vortex the cell-rich pellet
• Resuspend the cells
• Transfer the solution to the PrepStain slide
processor
Cell Sedimentation
• Pour the cell suspension into the PREPSTAIN® Fig. 19.10 Completely automated system to process the
lquid based cytology sample
Settling chamber (Fig. 19.9).
• With the help of gravitational force the cells
are sedimented on the pre-coated slide.
19.6 Comparison of These Two
Totally automated system: Currently totally Techniques
automated SurePath system is available that
can process a large number of samples without The processing techniques of the two above men-
any manual intervention (Fig. 19.10). tioned systems are completely different. The
ThinPrep technique is more automated than the
Table 19.1 Comparison of Pap test and SurePath BD FocalPoint ™ Slide Profiler: The slide pro-
techniques filer scans the cervical smear and analyze the spe-
ThinPrep SurePath cific cytological features. The system finally
Methanol based Ethanol based collection classifies the slide as “no further review” or
collection fluid fluid
“review”.
Completely automated Manual
Cell dispersion is done Cell dispersion is done by
by a circular rotatory manual vortexing • Label the cervical smear slide by the specific
filter submerged within barcode and load it into the BD FocalPoint ™
the vial Slide Profiler.
Cells are concentrated Cells are concentrated by • There are total of 36 trays and each tray con-
on the surface of the density gradient and
tains 8 slides.
vial by negative suction therefore the cells may be
present in more than one • Each slide automatically moves from the tray
layer to the microscopic stage
Blood and mucus may Better cellularity as there is • The device reads the barcode and verifies the
block the pores of the no such problem physical integrity of the slide
filter
• The system then scans the slides both in low
Good monolayered cell Cells are in multiple layers
preparation is possible and high power.
• The slides are ranked according to the degree
of abnormality and the device gives a score to
SurePath technique. Both techniques have certain
each slide depending on the probability of
advantages and limitations. Table 19.1 highlights
abnormality.
the comparison of these two techniques.
• The slide profiler classifies the slide as:
–– “No further review”: The highest probabil-
ity that the smears are normal
19.7 Automated Screening
–– Further review: In these slides, a manual
Devices in Cytology
review is needed to confirm the abnor-
mality. Usually, 75% of such cases con-
There is no fully automated screening device in
tain abnormal cells. The slides that are
the market. We only have semi-automated sys-
reported by cytotechnologists as “within
tems. There are two FDA approved semiauto-
normal limit” also need re-screening by
mated screening devices in the market:
cytologists.
1. BD FocalPoint GS Imaging System
2. HOLOGIC ThinPrep Imaging System
19.7.2 BD FocalPoint GS Review
Station
19.7.1 BD FocalPoint GS Imaging
System [6, 7] This is a review station and the cytotechnologists
followed by the cytologist review all the slides to
FDA has approved this screening device for both detect the abnormality.
primary and re-screening of SurePath and con-
ventional smear. • The slides are placed over the printed
The BD FocalPoint GS Imaging System PAPMAP that indicates the maximum possi-
(FPGS) has two parts: bility of the microscopic field of view (FOV)
containing the abnormal cell.
1. BD FocalPoint ™ Slide Profiler. • Screen the areas of FOV.
2. BD FocalPoint GS Review Station. • Reconfirm the cytotechnologist’s findings
19.7 Automated Screening Devices in Cytology 213
19.7.3 HOLOGIC ThinPrep Imaging Table 19.2 Differences of BD FocalPoint GS Imaging

System versus HOLOGIC ThinPrep Imaging System
System [8, 9]
BD FocalPoint GS Imaging HOLOGIC ThinPrep
System Imaging System
HOLOGIC ThinPrep Imaging System has two
Special slide preparation is It is mandatory to have
parts: not mandatory ThinPrep processing
and stain
1. ThinPrep imaging system and. The cellular features The cellular features
2. ThinPrep review system. mainly nuclear size, shape, along with optical
nucleocytoplasmic ratio density of the nuclei are
etc. are picked up and analyzed
ThinPrep image processor: The ThinPrep analyzed
image processor (TIP) system scans the slide, The slides are ranked The most abnormal 22
detects the abnormal cells and marks the field according to “device score” fields are recorded in
of view for re-screening. The system analyze and labelled as “no review” each slide. No ranks to
and “review” the slides are given
the various cellular features along with the
Cytotechnologists can Cytotechnologist has to
optical density of the nucleus. Therefore the screen only “review: screen every slide again
smears have to be stained in ThinPrep 2000 or labelled slides in those 22 marked
3000 Processor with ThinPrep Stain. This par- areas
ticular processing and stain are needed to
maintain the consistency of the stain. The inte- The difference in operating systems of the two
grated optical density of the nucleus is mea- technologies is highlighted in Table 19.2.
sured like the DNA content of the Feulgen
stained nuclei.
19.7.5 Comparison of Manual
• Place the slides in the cartridge that contains and Automated Devices
25 slides. A total of 10 such cartridges can be
operated at a single time. In a large study, these two automated systems
• The slides within the cartridge are imaged. were compared to the manual screening [10]. It
• The system records 22 FOV of the micro- was noted that automated screening devices are
scopic fields that may contain abnormal 8% less sensitive than manual screening. The
cells. study group raised doubts about the implemen-
• It transforms the information to the review tation of automated screening devices because
scope of the reduced sensitivity and cost effectiveness.
There is no doubt that automated screening
devices increase the productivity of the cyto-
19.7.4 Review Scope technologists as less number of fields are needed
to screen by them. Automation may reduce the
• Place the slide in the review scope. load of the tedious job of the cytotechnologists
• Access the stored location of the 22 FOV by (Table 19.3). However, the increased economic
the review scope burden on the screening program may be one of
• Now the areas are screened in a geographi- the major obstacles to automation. Moreover,
cal manner rather than the rank of the HPV testing along with manual screening
abnormality. may give a more meaningful result. Therefore,
• If any abnormal cells are detected, then screen in future, we may have to give serious thought
the entire slides. before we implement automated screening.
Table 19.3 Comparison of the automated screening ver- in a gynaecology outpatient setting in kuwait. Acta
sus manual screening Cytol. 2002;46:303–10.
3. Moseley RP, Paget S. Liquid-based cytology: is the
Automated
way forward? Cytopathology. 2002;13:71–82.
screening Manual screening
4. Herbert A, Johnson J. Personal view. Is it reality
No chance of Tiring and boaring job or an illusion that liquid-based cytology is better
fatigue than conventional cervical smears? Cytopathology.
Automated system Erratic and subjective approach. 2001;12(6):383–9.
follows consistent Inexperienced worker may miss 5. Davey E, Barratt A, Irwig L, Chan SF, Macaskill P,
logic the abnormal cells Mannes P, Saville AM. Effect of study design and
Machine takes There may be variable time quality on unsatisfactory rates, cytology classifica-
fixed amount of period of screening and tions, and accuracy in liquid-based versus conven-
time technologist takes longer time tional cervical cytology: a systematic review. Lancet.
Very costly device Cheap 2006;367(9505):122–32.
6. Colgan TJ, Bon N, Clipsham S, Gardiner G, Sumner
J, Walley V, McLachlin CM. A validation study
of the FocalPoint GS imaging system for gyne-
19.8 Artificial Neural Network cologic cytology screening. Cancer Cytopathol.
2013;121(4):189–96.
(ANN) in Pathology 7. Passamonti B, Bulletti S, Camilli M, D'Amico MR,
Di Dato E, Gustinucci D, Martinelli N, Malaspina M,
ANN is now widely used in various target areas. Spita N. Evaluation of the FocalPoint GS system per-
The identification of the tumour, grading, mitotic formance in an Italian population-based screening of
cervical abnormalities. Acta Cytol. 2007;51(6):865–71.
count, Ki67 indexing, and PDL1 scoring can be 8. Dawson AE. Can we change the way we
done with the help of ANN. The details of ANN screen?: the ThinPrep Imaging System. Cancer.
in automated interpretation of slides have been 2004;102(6):340–4.
described in detail in Chap. 25. 9. Chivukula M, Saad RS, Elishaev E, White S, Mauser
N, Dabbs DJ. Introduction of the Thin Prep Imaging
System (TIS): experience in a high volume academic
practice. Cytojournal. 2007;4:6.
References 10. Kitchener HC, Blanks R, Cubie H, Desai M, Dunn G,
Legood R, Gray A, Sadique Z, Moss S; MAVARIC
1. Sharma S, Dey P. Automated immunostaining plat- Trial Study Group. MAVARIC - a comparison of
form in cytology. J Cytol. 2021;38(2):57–63. automation-assisted and manual cervical screening:
2. Luthra UK, Chishti M, Dey P, Jolly SV, Abdulla M, a randomised controlled trial. Health Technol Assess.
Das DK, et al. Performance of thin prep smear method 2011;15(3):iii–iv, ix–xi, 1–170.
Polymerase Chain Reaction:
Principle, Technique 20
and Applications in Pathology
20.1 Introduction • The DNA strand is now extended with the

help of DNA polymerase (Taq polymerase).
Polymerase chain reaction (PCR) is one of the This polymerase enzyme incorporates the
most important techniques in molecular pathol- nucleotides in the DNA to make it elongated.
ogy [1, 2]. With the help of PCR, the single or the • The cycle is repeated
pieces of target DNA can be amplified in many
folds. This technique is now an integral part of
every modern laboratory for both diagnosis and 20.3 Steps of PCR
research use.
The basic steps of PCR are described as [1, 2]
(Box 20.1, Fig. 20.1a, b):
20.2 What is PCR and How Does it • Denaturation step: 94 °C: DNA is heated to
Work? 94° c to make it single-stranded. Only 1 to 2 min
are given to this heating process in each cycle.
PCR is just like a photocopy machine and acts • Annealing: 54 °C: The temperature is rapidly
like a “molecular photocopying” and amplifies cooled. At this lowered temperature the primer
the millions of copies of specific target DNA. quickly anneals with the respective site of
The basic principle of PCR are: DNA. With the help of Taq polymerase, the
reaction starts at the primer-DNA template site.
• Double-stranded target DNA is made into • Extension: 72 °C
single-stranded DNA by applying heat. • The complementary nucleotides are attached
• Two oligonucleotide strands or primers are from the 3′ to 5′ end of DNA. There is an expo-
added. The oligonucleotide strand binds nential increment in the number of genes in
with its complementary DNA strand to the each cycle. At least 30 cycles of denaturation-
3′ ends. annealing-extension are done in each PCR.
https://doi.org/10.1007/978-981-19-6616-3_20
216 20 Polymerase Chain Reaction: Principle, Technique and Applications in Pathology
Fig. 20.1 (a) Schematic

diagram shows the basic a
principle of PCR. The
basic steps are
denaturing double-
stranded DNA to
single-stranded DNA,
annealing with the
primer and extending of
DNA strand. (b) PCR
products of negative
control and positive
product demonstrated in
gel electrophoresis and
the red arrow indicates
the positive product.
(Courtesy of Prof Uma
Nahar Saikia,
Department of
Histopathology, Post
Graduate Institute of
Medical Education and
Research, Chandigarh,
India)
b
20.4 Procedure Proper 217
of the new DNA strands. The DNA poly-

Box 20.1 Principal of Polymerase Chain merase enzyme Taq polymerase capture
Reaction these dNTPs in the working solution and
The specific gene is amplified by using a attach them to the terminal part of the primer
pair of DNA primer, heat resistant DNA to make it extended.
polymerase enzyme, and nucleotides. • The target DNA from the sample: The target
DNA is extracted from the sample.
Steps:
• Buffer solution: It provides the optimum
• Denaturation: Heat breaks the double
chemical environment for the reaction to
stranded DNA to single stranded DNA
occur.
• Annealing: Forward and reverse DNA
• Magnesium Chloride (MgCl2): Magnesium
primer bind with the complementary
Chloride works as a cofactor of the Taq poly-
DNA strand in 3′ region of each sepa-
merase enzyme.
rated DNA strand.
• Extension: Heat resistant DNA poly-
merase (Taq polymerase) enzyme grab
the nucleotides and extend the DNA
20.4 Procedure Proper [3, 4]
strand from 3′ to 5′ direction.
20.4.1 Basic Precautions
Repetition of this thermal cycle for 25 to 30
times to increase the DNA product. • Always wear gloves to avoid any
contamination
• Completely thaw all the reagents before PCR
20.3.1 Essential Ingredients of PCR to do
• Keep all the reagents in an ice basket through-
• Primers: The primers are the small pieces of out the time of the PCR experiment
artificially made DNA strands that are actually
the complementary strands of the 3′ end of
each strand of target DNA. Primers usually 20.4.2 Equipment
consist of 20 to 30 nucleotides.
• DNA polymerase (Taq polymerase): Taq • Thermal cycler
polymerase is a type of DNA polymerase • PCR tubes and caps
enzyme that extends the new DNA strand. It • Ethanol-resistant marker
combines at the end of the primer and then • Micropipettes set
sequentially adds new nucleotides to the
DNA strand at the 3′ end complementary to
the target DNA. A high temperature (94 °C) 20.4.3 Addition of the Ingredients
is needed to separate the double stranded in a 50 μL PCR Tube
DNA. Ordinary DNA polymerase breaks
down at this temperature. However, the Taq • Target DNA:1 μLDNA template (1 ng total
polymerase has the unique characteristic to amount)
work efficiently at higher temperatures. This • Forward Primer: 1 μL of 50 μM primer
Taq DNA polymerase is extracted from the (final concentration is 1 μM)
bacteria Thermus aquaticus. This bacteria • Reverse Primer: 1 μL of 50 μM primer (final
lives in hot springs and can survive there. concentration is 1 μM)
• Deoxynucleotide triphosphate (dNTP’s): • dNTPS: 4 μL dNTP mix (2.5 mM dNTP, final
Deoxynucleotide triphosphates (dNTPs) are: concentration is 200 μM)
dATP, dCTP, dGTP, and dTTP. These are the • Taq DNA polymerase: 0.25 μL of 5 U/μL of
raw material or the basic “building blocks” Taq polymerase (total amount 1.25 U)
• Buffer: 5 μL 10× polymerase buffer (com- Mf = Final number of DNA molecule.

monly this buffer is supplied by the M = Initial number of DNA molecule.
manufacturer) N = Number of PCR cycle.
• MgCl2: 5 μL of 25 mM MgCl2
20.4.6 Purification of the Amplified

20.4.4 Remember Product
• Add the template DNA and DNA polymerase The following measures are taken to purify the
just before starting PCR. PCR products from the reaction solution:
• Please put at least one negative control and if
possible one positive control • Agarose gel electrophoresis of the product:
Agarose gel electrophoresis is done from the
portion of the PCR product to verify the valid-
20.4.5 Thermal Cycling ity of the test.
Note the following thing:
• Close the cap of the PCR tubes and then put –– Is any band present in the agarose gel elec-
them in the thermal cycler trophoresis or not?
–– Is there any other bands of different sizes?
20.4.5.1 Standard Steps –– Is there a smear pattern?
• Initial denaturation: At 94 °C for 1 min –– The successful PCR amplification product
• Denaturation: At 94 °C for 30 s shows a single sharp band with the expected
• Annealing: 50–60 °C for 30 s: The tempera- size.
ture may vary depending on the primer used. • Cloning of Products: In this technique fur-
The temperature of the annealing stage should ther PCR is done to confirm the PCR product.
be 3 to 4 °C lower than the melting point (Tm) This is done when the gene is present in a very
of the primers. tiny amount.
• Sequencing of Products: This is done by an
( ) ( )
Tm of the primers = 4 ´ [G + C] + 2 ´ [ A + T] °C
automated sequencer machine to analyse the
• G = Guanine, C = Cytosine, A = Adenine, sequence of DNA formed as a PCR product.
T = Thiamine
• Extension: 72 °C for 1 min/kb of the PCR
product 20.4.7 Troubleshooting
• Final extension: 72 °C for 10 min
• Termination: The reaction is terminated by The various problems in PCR are described here
chilling the mixture to 4 °C (Table 20.1):
Cycling time: The PCR thermal cycle rapidly 1. No amplification of DNA: The possible
heats and cools the PCR reagent mixture. The causes may be:
cycling time depends on (1) the size of the DNA (a) Too small amount of DNA template: If
template and (2) the G-C content of DNA. The there is a very less amount of DNA tem-
number of the thermal cycler is usually set as 25 plate then there may not be adequate
to 30 cycles. If the thermal cycle is increased by amplification. The amount of DNA tem-
more than 35 then too many unwanted DNA plate should be increased in such
products may be produced. conditions.
The product is calculated as: (b) Too stringent reaction condition: If the
M f = M ´ 2N reaction condition is kept very strict then
20.4 Procedure Proper 219
Table 20.1 Troubleshooting in PCR

Problem Cause Solution
No PCR product Too small amount of DNA template Increase the amount of DNA template in the
reaction mixture
Too stringent reaction condition Reduce the stringency
Suboptimal number of the thermal cycle Increase 5 to 10 more cycle
Denaturation temperature is either too high Change the temperature to 1 °C low or high at
or too low a time
The primer annealing temperature is very Lower the temperature 2 °C at a time
high
The reagent is omitted by mistake Do the reaction again
Reagents not in optimum concentration or Make fresh reagents one at a time in the
expired reaction mixture
Primer dimer formation Add Dimethyl sulfoxide
Faulty primer Redesign the primer
G-C rich DNA template Add Dimethyl sulfoxide
Spurious product Too less stringency in PCR Stringent PCR condition to maintain
Too many thermal cycles Maintain optimum thermal
Cycles
Too much DNA template: Reduce the amount of DNA template
Too many thermal cycles Reduce the number of thermal cycles 5 to 10.
Magnesium concentration is very high Lower the concentration
Faulty primer Redesign the primer
Carry over contamination Change the place of PCR
there may not be any amplification of sis shows a small product of less than 100
PCR products base pairs. The addition of DMSO may
(c) Reagent is not added: One or more solve this problem. Alternatively, hot start
reagents may not be added to the reaction thermal cycling may resolve this issue.
mixture. The whole reaction should be (h) Suboptimal number of the thermal
done again by carefully adding the cycle: Suboptimal number of the thermal
reagents. cycle may produce less amount of PCR
(d) Reagents are not in optimum concen- product. In such conditions, increase 5 to
tration or expired: The reagents should 10 more number of the thermal cycle.
be made fresh and one new reagent is (i) Faulty primer: The primer may be faulty
changed at a time to find out which and therefore PCR may not function at
reagent created the problem. all. In such a case, redesign the primer.
(e) Denaturation temperature is either too 2. Non-specific product: Non-specific product
high or too low: In such conditions may be formed in PCR and in this case, mul-
change the temperature to 1 °C low or tiple bands with variable lengths are seen in
high at a time. gel electrophoresis. This may be due to:
(f) Primer annealing temperature is very (a) Too less stringency in PCR: Too less
high: In such case, lower the temperature stringent condition generates unwanted
2 °C at a time. DNA products in PCR.
(g) Primer dimer formation: Two primers (b) Too much DNA template: Too much
may self-anneal or anneal with the other DNA template may produce an undesired
primer. In this case, the gel electrophore- product in PCR.
(c) Too many thermal cycles: In such a 3. Asymmetric PCR: In this technique,
case, reduce the number of thermal cycles unequal concentrations of primers are used.
5 to 10. A great excess of primers is used for the tar-
(d) Magnesium concentration is very high: geted DNA strand that we need to amplify.
Adjust the concentration and make it low. As the reaction proceeds, only the adequate
(e) Faulty primer: Redesign the primer amount of primer in the reaction mixture
(f) Carry over contamination: Change the produces the particular DNA strand in
place of PCR excess. Therefore, ultimately single-stranded
DNA (ssDNA) is formed as PCR product. As
the reaction is slow and goes on arithmeti-
20.4.8 Enhancing PCR Products cally so many more cycles are needed in this
Formation technique. Asymmetric PCR is used for
DNA sequencing and hybridization as only
The following measures help to enhance the PCR one strand is needed in such conditions [6].
products [4]: Disadvantages or limitations:
(a) The ssDNA is vulnerable to damage by
• Addition of non-ionic detergents: Triton many physical and chemical factors and
X-100, Tween 20, or NP-40 help to stabilize a more stable second structure may form
the DNA polymerase enzyme and enhance the (b) Different ssDNA may be formed even in
reaction. However, these agents may lower the the same reaction.
PCR stringency and undesirable DNA prod- (c) It needs more thermal cycles.
ucts may be formed. More than 1% concentra- 4. Hot start PCR [7]: Normally DNA poly-
tion of these detergents may have an inhibitory merase acts at room temperature and even
effect on PCR. in the ice pack. Thereby there always
• The addition of Dimethyl sulfoxide (DMSO) remains the possibility of spurious prod-
in the G-C rich DNA template (1 to 2%): ucts. In the hot start PCR technique, the
The addition of DMSO disrupts the base pair DNA polymerase is unreactive at a lower
and enhances the reaction. temperature and works only at a higher tem-
• Optimized annealing temperature: It is nec- perature. This is done by conjugating an
essary to optimize the annealing temperature inhibitor with the polymerase enzyme and
to increase PCR products. at the higher temperature, the inhibitor is
free from the polymerase enzyme and
allows it to work.
20.5 Types of PCR The various ways to do hot start PCR:
(a) Withheld the key agents until the end
There are different types of PCR methods for of the initial denaturation process
diagnostic purposes. These are: • DNA polymerase enzyme or magne-
sium cofactor
1. Direct PCR: This is the standard PCR tech- (b) Mechanical barriers to the reagents:
nique as has been described in the previous • DNA polymerase is encapsulated and
section. is only released at a higher
2. Reverse transcriptase PCR (RT-PCR): In temperature
the case of RT-PCR, at first cDNA is prepared • The wax barrier is used to separate the
from the RNA of the target sample (Fig. 20.2). key components till the temperature is
This cDNA is then amplified by PCR tech- high
niques [1, 2, 5]. This technique is applied to • Microfluidic devices are used to cre-
study the gene expression of different cells. ate a barrier
20.5 Types of PCR 221
Fig. 20.2 Schematic

diagram explains the
basic principle of
reverse transcriptase
PCR
(c) Modification of DNA Polymerase:

• Antibodies are used to inhibit DNA
polymerase activity at lower
temperatures and it releases the

enzyme at a higher temperature
• DNA polymerase enzyme is chemi-
cally modified so that it works only at
a higher temperature.
• The ligand is used that binds with DNA
polymerase in a temperature-
dependent way
• The amino acid mutation is done in
DNA polymerase enzyme to have
reduced activity in lower temperature
(d) Accessory proteins: The accessory pro-

teins can be used that sequester primer at
a lower temperature. Fig. 20.3 Schematic diagram shows the basic principle
of in situ PCR
The main advantage of hot-start
PCR: To reduce any unwanted PCR
product mulated within the cell so it is possible to
5. In situ PCR: In the case of in situ PCR, the locate the origin of the amplified DNA. The
reaction takes place within a cell on the glass specially designed PCR machine is used to
slide (Fig. 20.3). The PCR product is accu- put the slide within it.
This technique is used to amplify the

nucleic acid in the fixed tissue and cell
instead of in solution [8].
Use:
(a) Detection and location of the virus
within the tissue
(b) Detection and also localization of the
cancer cells.
(c) Demonstration of the genetic mutation
in case of inherited genetic disease
(d) Demonstration of the location of gene
expression within the tissue
Advantages
(a) High specificity
(b) High turnaround time
(c) Low background stain
(d) Avoidance of any radioactive elements
6. Inverse PCR: Inverse PCR (IPCR) amplifies
anonymous DNA sequences. It helps to iden-
tify the flanking DNA sequence of the
genome outside the boundary of the known
target sequence [9]. Fig. 20.4 Schematic diagram explains the basic principle
There are four steps of IPCR (Fig. 20.4) of inverse PCR. At first, DNA is isolated followed by cir-
cularization of double-stranded DNA. With the help of the
(a) DNA isolation: This is the initial basic
endonuclease enzyme, the circular DNA is cut to make
step where genomic DNA is isolated linear DNA fragments with two fragments of known DNA
from the sample and then cut into pieces in two ends. With the help of the known primer, the known
by restriction endonuclease enzymes. DNA sequence is amplified along with the attached
unknown DNA sequence
The DNA is cut in such a way that the
known sequences of DNA are in the
inner region with an unknown sequence Applications of IPCR
on two sides. • Identification of flanking sequence
(b) Circularization of double-stranded • Identification of viral gene insertion
DNA: In this step, the linear DNA mol- within the genome
ecule is circularized under low DNA • Chromosomal rearrangement of
concentration. oncogene
(c) Reopening of the circular DNA: With
the help of the endonuclease enzyme, 7. Single-stranded conformation polymor-
the circular DNA is now cut to make lin- phism (SSCP): The basic principle of SSCP
ear DNA fragments. The known DNA is that the single-stranded DNA has a specific
sequence remains in the two ends of the conformation [10, 11]. Any alteration of the
unknown sequence. single base change due to mutation may lead
(d) Amplification of reverse DNA frag- to a different migration pattern of the single-
ment: Now with the help of the known stranded DNA and therefore in electrophoresis
primer, the known DNA sequence is one can distinguish wild type DNA from
amplified along with the attached mutant DNA. The following steps are done in
unknown DNA sequence. SSCP (Fig. 20.5):
Fig. 20.6 Schematic diagram shows the basic principle

Fig. 20.5 Schematic diagram explains the basic principle of the TaqMan probe using in PCR. The ‘TaqMan’ probe
of single-stranded conformation polymorphism. is attached with a fluorescence reporter dye and a quencher
Alteration of the single base change due to mutation leads dye. In case of intact TaqMan’ probe the reporter dye and
to different migration patterns of the single-stranded DNA the quencher dye remains in close proximity and no fluo-
in electrophoresis rescence is emitted. During PCR the endonuclease breaks
down the ‘TaqMan’ probe and the reporter dye is away
from quencher dye that allows emission of fluorescence
(a) PCR amplification of the target DNA which is directly proportional to the amount of PCR
(b) The double-stranded DNA product is products
denatured
(c) The sample is cooled so that denatured quantitate the initial amount of the target
single-stranded DNA undergoes DNA in the sample.
self-annealing. Mechanisms to quantitate the ampli-
(d) Electrophoresis is done to see the mobil- fied DNA:
ity of the single-stranded DNAs. (a) Hydrolysis of the probe (Fig. 20.6): In
Application: The detection of single base this real-time TaqMan ® assay technique
change mutation and polymorphism in we use a ‘TaqMan’ probe. This is an oli-
essential hypertension, carcinoma, diabetes gonucleotide probe which is attached
etc. with a fluorescence reporter dye at its 5′
8. Real-time PCR: Real-time PCR is also terminal and a quencher dye at the 3′ ter-
known as quantitative PCR (qPCR) as it con- minal end. In case of the proper target
stantly monitors the quantity of the amplified sequence, this probe anneals with one of
DNA in the reaction process [12]. In case of the target sequences of the DNA tem-
qPCR the amplified DNA is fluorescently plate. The ‘TaqMan’ probe is cleaved by
labelled and the emitted fluorescent is Taq polymerase during PCR. When the
directly proportional to the amount of the ‘TaqMan’ probe is intact the reporter
amplified fluorescent dye. Therefore, in each dye and the quencher dye remain in
cycle, the amount of the product can be close proximity and therefore fluores-
directly monitored and it is also possible to cence emitted from the reporter dye is
absorbed by the closely placed quencher

dye. So, no fluorescence was emitted.
During PCR the endonuclease breaks
down the ‘TaqMan’ probe and the
reporter dye is away from the quencher
dye that allows the emission of fluores-
cence. The increased intensity of fluo-
rescence is directly proportional to the
amount of PCR products.
(b) DNA binding dye (Fig. 20.7): In this
technique DNA intercalating agents
SYBR ® Green is used. The SYBR ®
Green dye molecules do not exhibit any
fluorescence in the solution. However,
the dye molecules emit fluorescence
when they are intercalated within the
double stranded DNA that are formed
after the primer extension and polymer-
ization. After each cycle, the emitted
fluorescence from the polymerized DNA Fig. 20.8 Schematic diagram explains the basic principle
is measured to estimate the total amount of the dual hybridization technique to quantitate the PCR
products. In this technique, one probe is attached with a
of the amplified DNA donor fluorophore and the other probe is attached with an
(c) Dual Hybridization (Fig. 20.8): In this acceptor fluorophore. During PCR process the donor and
technique, two hybridization probes are the acceptor fluorophore probes hybridize to the target
applied. One probe is attached with a DNA sequence and comes in close contact. This allows
fluorescence resonance energy transfer
donor fluorophore at the 3′ end and the
other probe carries an acceptor fluoro-
ing stage, the donor and the acceptor
phore at the 5′ terminal. In the denatur-
fluorophore probes hybridize to the tar-
ation step, there is no emission of
get DNA sequence and they are adjusted
fluorescence as any fluorescent emission
in head to tail position so that the donor
by the donor fluorophore is degraded by
fluorophore comes in close contact with
the acceptor fluorophore. In the anneal-
the acceptor fluorophore. This allows
fluorescence resonance energy transfer.
The intensity of the fluorescence is mea-
sured which is directly proportional to
the amount of PCR products.
(d) Molecular beacons (Fig. 20.9): In this
case, the hybridized probe is designed
like a hairpin-like loop. The reporter and
quenching dyes are attached to the two
ends of the loop and their close proxim-
ity of them prevents the emission of fluo-
Fig. 20.7 Schematic diagram shows the basic principle rescence. At the time of annealing of the
of using DNA binding dye to quantitate the PCR products. hybridized probe, the hairpin loop
The free SYBR Green dye molecules do not exhibit any
fluorescence in the solution. In PCR extension, the dye
becomes a straight probe and the reporter
molecules are intercalated within the double-stranded and quenching dyes stay away. This
DNA and emit fluorescence allows the emission of fluorescence. The

of molecular beacon technique to quantitate the PCR Fig. 20.10 Schematic diagram shows the basic principle
products. Here the hybridized probe is designed like a of nested PCR. Here more than two pairs of primers are
hairpin like loop and the reporter and quenching dyes are used for DNA amplification. At first primer is used for the
attached in the two ends of the loop. The close proximity conventional PCR of the sample. In secondary PCR the
of them prevents the emission of any fluorescence. During product of the first PCR is used as the target of the second
annealing of the hybridized probe the hairpin loop set of primers
becomes a straight probe and the reporter and quenching
dyes stay away from that allowing the emission of taining material. The original quantity of tar-
fluorescence
get DNA is assessed with the help of suitable
statistical software (Fig. 20.11).
increased fluorescent intensity is mea-
Steps
sured to quantify the PCR products.
(a) Generation of droplets: Multiple drop-
9. Nested PCR (Fig. 20.10): In the case of
lets are generated from the sample with
nested PCR, more than two pairs of primers
the help of the mixture of oil in water.
are used for DNA amplification. The first
Each droplet is a nano chamber that con-
PCR is a conventional PCR and the primer is
tains the target DNA template or no such
used for the DNA template of the sample. In
template.
secondary PCR the product of the first PCR
(b) Amplification of target DNA by PCR:
is used as the target of the second set of prim-
Now the DNA template within the drop-
ers. The DNA sequence of secondary PCR is
let is amplified by the standard PCR
different and therefore there is no chance of
technique using a TaqMan probe-based
undesired PCR product formation.
assay for Real-time PCR.
10. Droplet digital PCR (ddPCR): Droplet
(c) Droplet assay: After the completion of
digital PCR (ddPCR) is an advanced versa-
the PCR technique, the fluorescence
tile technique that can quantitate nucleic acid
emission of each droplet is assayed. The
with precision [13]. In this technique, 20,
PCR positive droplet will give bright
000 droplets per Pico litre are produced.
fluorescence, whereas, PCR negative
Each droplet contains either 1–3 target DNA
droplet will show only background fluo-
or no DNA. PCR occurs within the droplet
rescence. Now the absolute quantity of
and finally, these droplets are analyzed to
the target DNA is assessed by the help of
find out the positive or negative PCR con-
Poisson statistics.
Fig. 20.11 Schematic diagram shows the basic steps of droplet digital PCR
Advantages of ddPCR: There are several DNA over the attached magnetic bead is
advantages of the ddPCR technique. These amplified by the PCR and the magnetic
include: beads coated with the amplified target DNA
(a) Absolute quantitation: Absolute quan- are detected [14].
titation of the target DNA in the sample Steps (Fig. 20.12):
can be done. (a) DNA isolation: The DNA from the sam-
(b) Simple: Easy to quantitate the target ple is at first isolated.
DNA and no calibration curve is (b) Pre-amplification: The target DNA is
required. amplified with the help of appropriate
(c) Increased accuracy: Strict partition of primers.
the individual droplets help to provide (c) c) Emulsion PCR: The magnetic beads
higher precision. coated with the identical sequence of the
(d) Consumption: Overall consumption of target DNA are used to attach to the
the reagents is less. amplified target DNA. Multiple mag-
(e) Multiplex PCR: Multiplex PCR is pos- netic beads form millions of droplets
sible by applying different probes and containing one target DNA. The PCR
fluorescence dye. products are formed over the magnetic
11. Beads, emulsion, amplification, and beads.
magnetics PCR (BEAMing PCR): (d) Hybridization: Fluorescent probes are
BEAMing PCR is a highly sensitive tech- hybridized with DNA.
nique that is mainly used in liquid biopsy (e) Analyzing the beads: The magnetic
specimens. In this technique, the target beads are analyzed by flow cytometry.
20.6 Applications of PCR 227
Fig. 20.12 Schematic diagram shows the basic steps of beads, emulsion, amplification, and magnetics PCR
Table 20.2 Applications of PCR in basic research and

20.6 Applications of PCR clinical field
Applications of PCR
The various applications of PCR in the clinical Basic research Clinical applications
area are described below (Table 20.2) [15]: • DNA sequencing • Diagnosis of infections
• Bioinformatics • Cancer
• Classification of – Detection of
1. DNA sequencing: PCR is often used in the organisms chromosomal
case of DNA sequencing. • Gene expression abnormalities
2. Diagnosis of infection: PCR is widely used studies – Genetic mutation
• Drug discovery – Detection of minimal
for the diagnosis of viral, bacterial and para- residual disease
sitic infections. As mentioned before Q-PCR • Genetic disease: Intranatal
can quantitate the viral load in the body [16]. detection of inherited
PCR rapidly detects tuberculosis within a few genetic disease e.g. Down
syndrome, Gaucher’s
hours whereas the culture of mycobacteria disease etc.
takes a few weeks to develop. As a sensitive • Forensic pathology:
technique, PCR is able to detect tuberculosis – Paternity detection
in the early latent phase. – Identification of
mutilated body
3. Diagnosis and prognostic information on – Crime site investigation
Cancer: PCR technique is extensively used in • Gene therapy
the field of cancer:
(a) Mutation detection: PCR is applied to
(b) Chromosomal changes: PCR helps to
detect mutation of the oncogenes and
identify the specific chromosomal
tumour suppressor genes such as a muta-
changes such as chromosomal transloca-
tion in p53, c-myc, ras gene etc. [17, 18].
tion, gene rearrangement, loss of hetero- 5. Tse WT, Forget BG. Reverse transcriptase and direct
zygosity etc. [16] . amplification of cellular RNA transcripts by Taq poly-
merase. Gene. 1990;88:293–6.
(c) Monoclonality detection: PCR can 6. Gyllensten UB, Erlich HA. Generation of single
detect B and T cell gene rearrangement stranded DNA by the polymerase chain reaction and
and thereby can prove the monoclonality its application to direct sequencing of the HLA-DQA
in doubtful case of lymphoma [17]. locus. Proc Natl Acad Sci U S A. 1988;85:7652–6.
7. Paul N, Shum J, Le T. Hot start PCR. Methods Mol
(d) Minimal residual disease: In case of fol- Biol. 2010;630:301.
low up of a cancer case, PCR particularly 8. O’Leary JJ, Chetty R, Graham AK, McGee JO ’D. In
Q- PCR can detect and quantitate certain situ PCR: pathologist’s dream or nightmare? J Pathol.
genetic changes to detect any minimal 1996;178:11–20.
9. Jong AY, T'ang A, Liu DP, Huang SH. Inverse
residual disease of a patient [18]. PCR. Genomic DNA cloning. Methods Mol Biol.
4. Genetic diseases: PCR technique is very 2002;192:301–7.
helpful to detect various genetic diseases such 10. Orita M, Iwahana H, Kanazawa H, Hayashi K,
as Down’s syndrome, cystic fibrosis, Sekiya T. Detection of polymorphisms of human
DNA by gel electrophoresis as single-strand confor-
Gaucher’s etc. The main advantage of the mation polymorphisms. Proc Natl Acad Sci USA.
PCR technique is that it can bypass the aggres- 1989;86:2766–70.
sive placental bed biopsy to detect these 11. Dong Y, Zhu H. Single-strand conformational poly-
inherited diseases. The minute amount of fetal morphism analysis: basic principles and routine prac-
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Fluorescent In Situ Hybridisation
Techniques in Pathology: 21
Principle, Technique
and Applications
21.1 Introduction Limitations of FISH: FISH has the following

limitations:
The fluorescent in situ hybridisation technique
(FISH) is also known as interphase cytogenetics. • The FISH technique can be used only in case
In this technique, the double-stranded DNA is at of known chromosomal abnormalities as we
first converted into single-stranded DNA and use only the specific probes.
then subsequently a fluorescent-tagged probe is • It is not suitable for a screening test as only
used to visualize the target DNA part [1–3]. known chromosomal probes are used
Advantages: FISH techniques have many whereas in cell culture or conventional
advantages over conventional cytogenetics tech- technique, we may get a wide range of
niques (Box 21.1). It is possible to do FISH on chromosomal abnormalities.
the paraffin-embedded tissue material and there- • FISH does not give any allele-specific
fore it can be used in archival materials. FISH information.
technique bypasses the tedious cell culture tech-
nique. Most importantly cytogenetics abnormal-
ity of the cells can be demonstrated along with 21.1.1 Applications of FISH
the morphology of the cell and these two can be
correlated. • Gain and loss of chromosomes: FISH is
helpful to detect the total gain or loss of chro-
Box 21.1 Advantages of FISH mosomes such as trisomy 12 in chronic lym-
• No need of cell culture phocytic leukaemia can be detected by FISH
• FISH can be done on paraffin cell block in cytology or histology samples.
material • Chromosomal rearrangements: FISH is help-
• Archival tissue can be used for FISH ful to identify typical chromosomal transloca-
• Morphology of the cell can be seen tion such as t (11; 22) (q24; q12) in Ewing’s/
along with cytogenetic abnormalities primitive neuroectodermal tumour [4].
• High resolution • Gene amplification: Gene amplification such
• The slide can be stored for long time as HER-2 in breast carcinoma can be detected
• Fluorescent tags are safe and simple by FISH.
https://doi.org/10.1007/978-981-19-6616-3_21
230 21 Fluorescent In Situ Hybridisation Techniques in Pathology: Principle, Technique and Applications
• Gene deletion: Gene deletion such as 9p21 • To convert double-stranded DNA into single-
deletion in urothelial cell carcinoma can be stranded DNA.
detected by FISH. • DNA probes tagged with fluorescent dyes are
• Disease monitoring: To assess the progres- also made single-stranded.
sion or regression of disease and also to iden- • The florescent tagged single-stranded DNA
tify the minimal residual disease. probes are allowed to bind with the corre-
sponding single-stranded DNA.
• The hybridized probe-target DNA complexes
21.1.2 The Principles of FISH are visualized by a fluorescent microscope.
The basic principles of FISH are (Fig. 21.1a):

Probes applied in FISH: Three types of probes
are applied for FISH and they include:
• Centromeric probes or chromosome enu-

a meration probes (CEP): CEP is directed
against the alpha or beta satellite repeat
sequence of the centromeric region of the
chromosome. Therefore, it helps to enumerate
the number of chromosomes. The CEP can
detect monosomy or aneuploidy.
• Locus-specific identifier probes (LSI): LSI
probe is used to demonstrate the other specific
region of the chromosome. These probes are
used for the demonstration of various struc-
tural abnormalities such as gene deletion,
translocation etc. LSI also helps in the detec-
tion and localization of genes and the demon-
stration of gene amplification.
• Whole chromosome probes (WSP): The
whole chromosome probes consist of multiple
smaller probes with the complementary DNA
sequence of a different part of a single chro-
b mosome. Multiple WSP can be used to stain a
whole chromosome. Therefore, using differ-
ent fluorochrome for different chromosomes
one can paint each chromosome with a differ-
ent colour. WSPs are used to demonstrate
chromosomal structural abnormalities (trans-
location or deletion).
Fig. 21.1 (a) Schematic diagram shows the basic princi-

ple of FISH technique. At first double-stranded DNA and 21.2 Steps to do FISH [5–7]
also primer DNA are converted into single-stranded
DNA. DNA probes are tagged with fluorescent dyes and
are allowed to bind with the corresponding single-stranded 21.2.1 Histology and Cytology
DNA. (b) FISH image showing one fused signal and one Specimen
break apart signal in 2 tumour cells for EWSR1 fusion in
a case of Ewing sarcoma. (Courtesy by Dr. Parikshaa
Gupta, Associate Professor of Cytology, Post Graduate
1. Fixation: The cytology slides are fixed with
Institute of Medical Education and Research, Chandigarh) 4% paraformaldehyde for 10 min. Histology
21.3 Troubleshooting 231
tissues are already fixed in 10% buffered (a) Add 10 μl of denatured probe solution
formalin. over the slide
2. Deparaffinization of the histology section: (b) Put a coverslip over the smear and close
(a) Cut a 5 μ thick section the margins of the coverslip
(b) Deparaffinize the histology section by (c) Incubate it at 37 ° C for 1 day (24 h).
baking the slide overnight at 56 °C. 6. Post-hybridization:
(c) Keep the slide in xylene for 10 min: two (a) After the incubation, remove the cover-
changes slip gently and rinse the slides in SSC for
(d) Dehydrate by treating in 70% and 100% 5 min twice.
ethyl alcohol twice for 5 min (b) Put the slides in a Coplin jar filled with
(e) Dry the smear on a hot plate pre-warmed SSC at 70 °C.
3. Pre-treatment: 20 μg/ml proteinase K for (c) Keep the slide in a Coplin jar filled with
5–15 min SSC at room temperature.
a. Dehydrate the smear in by dipping in 70%, 7. Visualization (Fig. 21.1b): If the probes are
80% and 95% ethyl alcohol and dry the directly labelled with the fluorochrome dye
smear. then no further procedure is needed. In that
b. Treat the smear with proteinase K solution case, counterstain the slide with 5 μL DAPI/
(20 μg/ml) for 15 min at room temperature. antifade solution. Now visualize the cells with
(Proteinase K solution preparation: add an epifluorescence fluorescence microscope.
32 μl of proteinase K solution (25 mg/ml
proteinase K solution) in 40 ml 2 X SSC,
pH 7.4) 21.3 Troubleshooting
c. Gently wash the slide in deionized water
d. Dehydrate the smear Table 21.1 has described the problems in FISH
Saline sodium citrate (SSC): techniques and their remedies.
Add
Sodium chloride: 175.3 g
Sodium citrate and: 88.2 g 21.3.1 Different Types of FISH
Keep pH 7.2 by adding drops of 10 N solu- 1. Three-dimensional FISH (3D FISH): In this
tion of NaOH. Now add water and make it type of FISH multiple images of the nuclei are
1 L). taken and with the help of suitable software a
4. Denaturation: three-dimensional image is made [8]. 3D
Denaturing of target DNA: Denature DNA of FISH helps to study the topology of the genes
the target cells in the smear by treating the with respect to the chromosomal territory
smear with denaturing solution for 2 min time within the nucleus.
at 72 °C. 2. Living cell cytogenetic (Four-dimension
Denaturing solution: 70% Formamide in 2× FISH): Fluorescent tagged nucleotide can be
SSC (add 10 ml of double-distilled water, incorporated into DNA that may help in the
5 ml of 20× SSC, 25 μl of 250 mM EDTA, and simultaneous visualization of DNA
35 ml of formamide). distribution and genomic organization in the
Denaturing of probe DNA: Add 1 μl of the living cells [9].
labelled probe with 9 μl of 65% formamide 3. Multi-coloured FISH (M- FISH)/Spectral
solution, and 10% dextran sulphate in 2× karyotyping (SKY): In the case of the SKY
SSC. technique, DNA is tagged with different fluo-
Now heat the mixture at 75 ° C for 5–6 min. rochrome dyes (the chromosome-specific
5. Hybridization: painting probes) and all the chromosomes are
stained. The different chromosome, therefore,
Table 21.1 Troubleshooting of FISH technique

Problems Possible cause Remedies
Weak signal • Poorly digested tissue section • Check the pH of the proteinase K solution
or no signal • Check the temperature of the tissue digestion step
• Make fresh proteinase K solution
• Low concentration of the probe • Maintain the optimum concentration of the probe
• Faulty hybridization step • Check the total time of hybridization (at least 14 h)
• Check the incubation temperature
• Loss of DNA • The solution should be DNAse free.
• Faulty microscope • Check the bulb of the microscope
Intense • Non-specific attachment of the probe • Add species-specific excess amount of Cot-1 DNA
background and interspersed repetitive sequences • Wash the slide stringently after the hybridization step
staining of DNA
Variable • Uneven distribution of the • Avoid any air bubbles under the coverslip
staining in hybridization solution
different
areas
Tissue • Over digestion of tissue • Check the digestion time and reduce the time period
morphology • Improper fixation • New sample has to be taken
is poorly
preserved
Speckled • Less stringent solutions • Maintain stringent concentration of the buffer
appearance solution, temperature etc.
takes different colour (Fig. 21.2). There are

three essential steps of M-FISH:
(a) Hybridization: The fluorescent-tagged
whole chromosome probes (WCP) are
hybridized with the metaphase chromo-
some spread. WCPs consist of multiple
probes tagged with spectrally different
fluorochrome in a combinatorial manner.
(b) Visualization and image acquisition: In
this step, the images of the chromosomes
are visualized by the fluorescence micro-
scope with attached filters. Subsequently,
the images are acquired by the digital
camera and appropriate software.
(c) Analysis: Finally, the images are anal-
ysed with the help of specialized software
to find out any structural chromosomal
abnormalities.
Steps of M-FISH [10, 11]
Pre-treatment of the Metaphase
Spread by Trypsin:
(a) Incubate the slide in pepsin at 37 °C for
5 min.
(Pepsin 1: 20,000 in 10 mM HCl) of spectral karyotyping. DNA are tagged with different
(b) Wash the slide in PBS: 5 min fluorochrome dyes and the different chromosome takes
(c) Fix the slide with 1% formaldehyde different colour
(d) Wash again in PBS: 5 min • Normal DNA from the control is also extracted
(e) Dehydrate in serially graded alcohol and labelled by red fluorochrome dye.
(70%, 90% and 100%) for 2 min each • The mixture of both green labelled tumour
Denaturation of Chromosome DNA and red labelled control normal DNA is
(a) Incubate the slide in 50 ml denaturing solu- mixed and allowed to hybridize on the normal
tion within a Coplin jar for 3 min at 72 ° C metaphase chromosome.
(b) Immediately dip the slide in ice-cold eth- • With the help of a digital image analyser the
anol 70%, 90% and 100% each for 2 min. green: red ratio is measured.
(c) Dry the slide • Excess green fluorescence represents chromo-
Denaturation of Probe somal gain or amplification
(a) Centrifuge the M-FISH probes • Excess red fluorescence represents a chromo-
(b) Mix the contents gently and take 10 μl somal loss
probe solution for each slide in an
Eppendorf tube. Advantages of CGH: CGH technique is help-
(c) Incubate the Eppendorf tube at 80 °C for ful in a tiny amount of micro dissected tissue.
5 to 7 min to denature the probe. The technique can be done without any prior
Hybridization knowledge of the chromosomal abnormalities in
(a) Add 10 μl denatured M-FISH probes the tumour DNA.
solution over the denatured chromosome Limitations of CGH: CGH is not helpful if
preparation. there are chromosomal abnormalities without
(b) Put coverslip over the smear and seal the any gain or loss of genetic material. Similarly,
margins of the coverslip with rubber from CGH we do not get any information on
cement. the structural changes of the chromosome.
(c) Keep the slide in a humidified chamber CGH is much less sensitive than PCR and the
for 2 days at 37 °C. result may be changed due to contamination of
(d) Remove the slide from the chamber and normal cells with tumour cells.
take out the coverslip by removing the
rubber cement.
(e) Wash the slides in SSC at 72 °C for 2 min. Box 21.2 Advantages and limitations of CGH
(f) Counterstain with DAPI and place a
Advantages
coverslip
• Tiny micro-dissected tissue can be pro-
(g) The slide is now ready to visualize in the
cessed for CGH
fluorescence microscope
• No need of details of chromosomal
4. Comparative genomic hybridization
abnormalities of tumor tissue
(CGH): CGH provides the global view of the
gain or loss of chromosomes of the tumour Limitations
genome [12]. • Ineffective technique if there are chro-
mosomal abnormalities without any
gain or loss of genetic material
21.3.2 Basic Principles (Fig. 21.3) • No information of any structural
abnormalities
• Tumor DNA is extracted from the sample and • Tedious and prolonged process
labelled with green fluorochrome dye.
Fig. 21.3 Schematic

principle of comparative
genomic hybridization.
Tumor DNA is e
labelled by green
fluorochrome dye and
normal DNA from the
control is labelled by red
fluorochrome dye. They
are allowed to hybridize
on the normal metaphase
chromosome. With the
help of digital image
analyser the green: red
ratio is measured. The
excess green
fluorescence represents
chromosomal gain or
amplification, whereas
excess red fluorescence
represents chromosomal
loss
21.3.3 CGH Method [13] Denature target metaphase smear and hybrid-
ization mixture
Preparation of probe mixture: 6. Fix the metaphase slide by 4% paraformal-
1. Add the following substances to the micro- dehyde for 15 min at 4 °C
centrifuge tube 7. Rinse the slide in PBS
(a) Fluorescein tagged test DNA (10 μl 8. Incubate the slide in proteinase K solution
20 μg/ml) for 5 min at 37 °C.
(b) Texas red labelled reference probe DNA 9. Rinse in PBS for 5 min
(10 μl 20 μg/ml) 10. Denature the metaphase spread smear: Keep
(c) Human Cot-1 DNA (20 μl 1 μg/ml) the slide in preheated Coplin jar in a water
2. Add 1/10th volume of 3 M sodium acetate bath containing denaturing solution at 75 °C
(pH 5.2) and mix them. for 5 min.
3. Add 2.5 volumes of 100% ethyl alcohol (ice 11. Dehydrate the slide with graded alcohol and
cold) and vortex again. dry in the air.
4. Remove the supernatant fluid. 12. Simultaneously denature the DNA samples:
5. Re-suspend the pellet by adding a 10 μl Heat the sample at 75 °C for 5 min.
hybridization mixture. Immediately incubate the sample at 37 °C for
20 min.
Hybridization Analysis
13. Pour 10 μl of the probe mixture into the 21. Analyse the slide under a fluorescent micro-
metaphase smear. Cover the area with a cov- scope with an attached digital camera and
erslip and seal the edges by rubber cement. appropriate software
14. Incubate the slide in a humid chamber at
37 °C for 48 h.
21.3.4 Array-based CGH [14]
Washing
15. Peel the rubber cement The basic principle of array-based CGH (aCGH)
16. Wash the slide twice in SSC for 5 min each is similar to CGH. However, aCGH uses a spe-
17. Wash the slide twice with a pre-warmed cific target DNA sequence instead of a metaphase
hybridization buffer at 45 °C for 10 min. chromosome. Microarray plates with multiple
18. Wash the slide in PBS for 5 min at 37 °C. wells contain genomic bacterial artificial chro-
19. Dehydrate the slide with graded alcohol and mosome or cDNA in the array.
air dry the smear
21.3.4.1 Basic Steps of a-CGH
Counterstain (Fig. 21.4)
20. Pour 8 μl DAPI and apply coverslip over the 1. Tumor DNA is extracted and labelled with a
smear. green fluorochrome dye.
Fig. 21.4 Schematic

principle of array based
comparative genomic
hybridization. Tumor
DNA is labelled with a
green fluorochrome dye
and the control DNA is
labelled with red
fluorochrome dye. The
mixture of them are
hybridized with multiple
specific DNA probe in
the microarray plate and
the hybridization
reaction plate is
analysed
2. Control DNA from a healthy person is cell gel electrophoresis and FISH that identi-
extracted and labelled with a red fluorochrome fies the specific DNA sequence. So at first
dye. single cell gel electrophoresis is done to sep-
3. The mixture of the above two samples are arate the fragmented DNA. Then FISH is
hybridized with multiple specific DNA probe done to assess the breakage of the specific
in the microarray plate. DNA region which is useful information for
4. The hybridization reaction plate is read by the development of diseases in relation to
an image analyser with the appropriate DNA damage [22].
software. 7. D-FISH: D-FISH stands for double fusion
FISH. In the case of D-FISH, two sets of dif-
ferently labelled probes are used to visualize
21.4 Different Other Varieties the fusion gene. The chances of false-
of FISH [15, 16] negative results are less in D-FISH. It is
mainly used for the demonstration of BCR-
1. ACM-FISH: ACM-FISH is a type of mul- ABL fusion in chronic myeloid leukaemia,
ticolour FISH. ACM means Alpha (centro- and the detection of minimal residual disease
mere), classical and midi satellites of in acute myeloid leukaemia (8;21 transloca-
chromosome 1. The probes are used to detect tion) [23].
numerical and structural abnormalities of the 8. DBD-FISH: DBD-FISH means DNA break-
sperm. ACM-FISH is mainly used for the age detection FISH. In this technique, the
investigation of male infertility [17]. cells in the sample are placed in an ultrathin
2. Arm FISH: Arm FISH is a type of multico- agarose layer on a glass slide and treated
lour FISH. Here the abnormalities of p and q with an alkali DNA unwinding buffer solu-
arms of all the human chromosomes are tion. The double-stranded DNA is trans-
detected with the help of multicoloured arm- formed into single-stranded DNA. The
specific painting probes (except the p arm of proteins are removed with the help of lysing
the Y chromosome) [18]. solutions. A suitable DNA probe is used to
3. COD-FISH: The full form of COD-FISH is assess DNA fragmentation [24].
chromosome orientation and direction 9. Fiber FISH: In the Fiber-FISH technique
FISH. Currently, COD-FISH is known as free chromatin fibre is released from the
CO-FISH. Here strand-specific hybridization nuclei of the cells by an appropriate solvent
of the probe occurs in only one chromatid and is stretched on a glass slide. Now fluo-
and the pericentric inversions in chromo- rescent probes are applied for FISH to map
somes can be detected [19]. the entire DNA fibre. With the help of the
4. CAT-FISH: Cellular compartment analysis fiber-FISH technique high resolution genome
of temporal (cat) FISH (CAT-FISH) is a type mapping is possible [25].
of RNA-FISH on cryosections followed by 10. Halo-FISH: In this FISH technique a halo is
confocal microscopy to study the time-based created artificially in the nucleus so the name
activation (temporal activation) of the neu- is halo-FISH. The cells of the sample are
rons [20]. permabilized and the proteins are removed
5. CARD FISH: The full form of CARD-FISH by a high salt solution. The unfixed and unat-
is catalysed reporter deposition FISH. Here tached chromatin in the nuclei are also
the signal amplification is detected with the removed and a halo is formed in the residual
help of fluorescently labelled tyramine mol- nucleus. Subsequently, FISH is performed.
ecule bound with horseradish peroxidase Halo-FISH is used in chromosome mapping
[21]. and to analyse sperm DNA [26].
6. COMET-FISH technique combines two dif- 11. Immuno-FISH: Immuno-FISH is the com-
ferent techniques: a) comet assay or single bination of two different techniques, one is
21.4 Different Other Varieties of FISH 237
FISH and the other technique is immuno- 12. Q FISH: Quantitative FISH (Q-FISH) mea-
fluorescence. The FISH highlights the DNA sures the intensity of the probe signal. It is
changes and the immunofluorescence tech- applied mainly to measure the telomere repeats
nique visualizes protein (antigen) parts of and telomere length in ageing and cancer [28].
the cell. This technique is used to demon-
strate gene expression and histone methyla- Table 21.2 highlights the basic principle and
tion [27]. applications of different variants of FISH.
Table 21.2 The basic principle and applications of different variants of FISH
Serial
number Name Basic principle Applications Reference
1 ACM-FISH: Alpha Multicolour FISH uses different • To detect and Sloter et al
(centromere), coloured probes for Alpha numerical and [17]
classical and midi (centromere), classical and midi structural
satellites of satellites of chromosome 1 abnormalities of the
chromosome 1 sperm in male
FISH infertility
2 Arm FISH A multicoloured arm-specific (p and q • To detect pericentric Karhu et al
arms of the chromosome) painting inversion of [18]
probes are used. chromosome
• Chromosomal
instability in Glioma
cell lines
3 COD-FISH: DNA strand-specific hybridization in • The pericentric Bailey et al.
Chromosome only one chromatid occurs inversions in [19]
orientation and chromosomes is
direction FISH detected
4 CAT-FISH: A type of RNA-FISH on cryosections • Both temporal and Guzowski
Cellular followed by confocal microscopy to cellular expression of et al. [20]
compartment study the time-based activation genes in the neurones
analysis of (temporal activation).
temporal (cat)
FISH
5 CARD FISH: The signal amplification is detected by To detect, identify and Kubota et al
catalysed reporter the help of fluorescently labelled quantify the [21]
deposition FISH tyramine molecule bound with microorganisms
horseradish peroxidase
6 COMET-FISH Two different techniques: a) comet To detect specific damage Schlörmann
assay or single cell gel electrophoresis of DNA in a single cell et al. [22]
and FISH are combined together
7 D-FISH: Double Two sets of differently labelled probes • Demonstration of Grand et al.
fusion FISH are used to visualize the fusion gene BCR-ABL fusion in [23]
chronic myeloid
leukaemia
• The detection of
minimal residual
disease in acute
myeloid leukaemia
8 DBD-FISH: DNA The cells in the sample are placed in an To detect DNA breakage Fernández
breakage detection ultrathin agarose layer on a glass slide in sperms et al. [24]
FISH and then treated with DNA unwinding
buffer. FISH is performed on single-
stranded DNA.
(continued)

Serial
number Name Basic principle Applications Reference
9. Fiber FISH Free chromatin fibre is released from High resolution gene Heiskanen
the nuclei of the cells by an appropriate mapping et al. [25]
solvent and is stretched on a glass slide.
Now fluorescent probes are applied for
FISH to map the entire DNA fiber
10. Halo-FISH The cells of the sample are • Chromosome mapping Wiegant
permeabilized and the proteins are • To analyse sperm et al. [26]
removed by a high salt solution. The DNA
unfixed and unattached chromatin in
the nuclei are also removed and a halo
is formed in the residual nucleus.
Subsequently, FISH is performed
11 Immuno-FISH: The combination of two different • To demonstrate gene Zinner et al.
techniques, one is FISH and the other expression and histone [27]
technique is immunofluorescence methylation
12 Q FISH: It measures the intensity of the probe To measure the telomere Slijepcevic
Quantitative FISH signal repeats, and telomere et al. [28]
length in aging and cancer
T. Spatial preservation of nuclear chromatin archi-

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7. Werner M, Wilkens L, Aubele M, Nolte M, method to detect genome copy number variation.
Zitzelsberger H, Komminoth P. Interphase cytoge- Genome Res. 2003;13(10):2291–305.
netics in pathology: principles, methods, and appli- 15. Volpi EV, Bridger JM. FISH glossary: an overview
cations of fluorescence in situ hybridization (FISH). of the fluorescence in situ hybridization technique.
Histochem Cell Biol. 1997;108(4–5):381–90. BioTechniques. 2008;45(4):385–6.
8. Solovei I, Cavallo A, Schermelleh L, Jaunin F, 16. Ratan ZA, Zaman SB, Mehta V, Haidere MF, Runa
Scasselati C, Cmarko D, Cremer C, Fakan S, Cremer NJ, Akter N. Application of fluorescence in situ
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17. Sloter ED, Lowe X, Moore DH II, Nath J, Wyrobek JL, Marley SB, Davidson RJ, Goldman JM, Gordon
AJ. Multicolor FISH analysis of chromosomal breaks, MY. A twocolor BCR–ABL probe that greatly reduces
duplications, deletions, and numerical abnormali- the false positive and false negative rates for fluores-
ties in the sperm of healthy men. Am J Hum Genet. cence in situ hybridization in chronic myeloid leuke-
2000;67(4):862–72. https://doi.org/10.1086/303088. mia. Genes Chromosomes Cancer. 1998;23:109–15.
Epub 2000 Aug 28. PMID: 10961911; PMCID: 24. Fernández JL, Gosálvez J. Application of FISH to
PMC128789 detect DNA damage. DNA breakage detection-FISH
18. Karhu R, Ahlstedt-Soini M, Bittner M, Meltzer (DBD-FISH). Methods Mol Biol. 2002;203:203–16.
P, Trent JM, Isola JJ. Chromosome arm-specific 25. Heiskanen M, Hellsten E, Kallioniemi OP, Mäkelä
multicolor FISH. Genes Chromosomes Cancer. TP, Alitalo K, Peltonen L, Palotie A. Visual mapping
2001;30(1):105–9. by fiber-FISH. Genomics. 1995;30(1):31–6.
19. Bailey SM, Meyne J, Cornforth MN, McConnell TS, 26. Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers
Goodwin EH. A new method for detecting pericentric JM, Dauwerse JG, van Ommen GJ, Raap AK. High-
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age by comet fluorescence in situ hybridization.
Tissue Microarray in Pathology:
Principal, Technique 22
and Applications
22.1 Introduction
Tissue microarray (TMA) is a novel technology

that helps to study a large number of tissue sam-
ples in a single section [1–3]. In this technique, a
few hundreds to thousands of tissue samples
from the different paraffin blocks can be taken
and put precisely into a recipient paraffin block.
TMA helps to perform immunocytochemistry
(IHC), fluorescence in situ hybridization (FISH)
or RNA analysis in a single section from the
recipient block. Therefore, the technique is faster
and cheaper than the conventional method.
Fig. 22.1 Basic steps of tissue microarray as described in

22.2 Tissue Microarray Technique this diagram
At first, the hematoxylin and eosin-stained sec- modate a few hundred to a thousand specimens.
tion from the donor block is studied, and the rep- The coordinates of the core biopsies in the recipi-
resentative area of the donor block is marked. ent block are recorded in typically in a spread-
With the help of a tissue microarray instrument, sheet (preferably in a Microsoft Excel file). Now
core tissue biopsy (0.6 to 2 mm diameter) is taken with the help of a microtome, 5 μ sections are cut
from the donor paraffin block. It is placed in an from the TMA block to produce a TMA slide
empty paraffin block (the recipient block) pre- (Fig. 22.2). The section now can be used for IHC,
cisely (Fig. 22.1). The recipient block can accom- FISH or other molecular studies.
https://doi.org/10.1007/978-981-19-6616-3_22
242 22 Tissue Microarray in Pathology: Principal, Technique and Applications
Fig. 22.2 The schematic diagram shows the detailed donor paraffin block and is placed in an empty paraffin
steps of tissue microarray. With the help of a tissue micro- block (the recipient block)
array instrument, core tissue biopsy is taken from the
22.3 TMA Construction 22.4 Designing the Grid [4]

and Generation of Grid
• Capital letters indicate the quadrant and small
The construction of the TMA and the generation letters indicate the co-ordinate of the tissues in
of the grid depend mainly on the type of study. the recipient’s block (Fig. 22.3).
• The primary data are entered for each tissue
• At first make a list of suitable cases core such as biopsy number, location, diagno-
• Study all the sections of these cases sis, grade of tumour, stage etc. The data is usu-
• Review the cases and reclassify if needed. Then ally entered in a Microsoft Excel file
mark the representative area on the stained sec- (Fig. 22.3).
tion on a glass slide. It is preferable to use a • Protection wall: The peripheral boundary
different colour for the different diagnostic walls of the TMA are made of a single row of
areas such as green for the normal area, red for core tissue (Fig. 22.4). These core tissues may
tumour and black for in situ carcinoma. consist of any tissue and they are not
• Collect the representative block of the slides analysed.
and identify the area.
22.4 Designing the Grid 243
Fig. 22.3 The

schematic diagram of
the organization of the a b
grid for tissue
microarray. The capital
letters indicate the
quadrant and small
letters indicate the
co-ordinate of the tissues
in the recipient’s block.
The primary data for
each tissue core such as
biopsy number, location,
diagnosis, grade of
tumour, stage, etc. are
entered in a Microsoft
excel file. (a) Upper left
quadrant. (b) Upper c d
right quadrant.
(c) Lower left quadrant.
(d) Lower right quadrant
• Control tissue: Positive and negative control immediately identify the orientation of TME
tissues are placed in an asymmetric location grossly.
(see green dots in Fig. 22.4). • The different disease processes can be grouped
• Tumor tissue: The tumour tissues are placed together such as carcinoma cases, in situ car-
in small groups (see the red dots in Fig. 22.4). cinoma and normal cases are clubbed into dif-
• Normal healthy tissue: In between the groups ferent groups.
of tumour tissue the matched normal healthy
tissues are placed in one or two rows (see the Diameter of the core: The diameter of the
black dots in Fig. 22.4). The presence of these punches of the core biopsy may vary from 0.6 to
normal tissue rows may help in the better ori- 2 mm. In fact, most people take a 0.6 mm diam-
entation of the tumour tissues. eter core that includes 0.14 square mm tissue.
• Empty cores in a small row: Intentionally Number of cores in TMA: In a
few cores in a row should be kept empty to 2.5 cm × 4.5 cm block at least 1000 cores can be
Fig. 22.4 Schematic

diagram shows the
elaborated design of the
grid for tissue
microarray. The
peripheral boundary
walls of the TMA are
made of a single row of
core tissue. Positive and
negative control tissues
are placed in an
asymmetric location
(green dots). The tumour
tissues are placed in
small groups (red dots).
Normal healthy tissues
are placed in one or two
rows (black dots)
adjusted. However, it is preferable not to take

more than 500 cores in a single block. Box 22.1 Advantages and Limitations of
Recipient block: The recipient block is made of TMA
fresh molten paraffin. The average size of the block
Advantages
is 2.5 cm × 4.5 cm. Adequate precautions should be
• Amplification of the resource: Near
taken to avoid any bubbles within the block.
about 100,000 times amplification is
Automated TMA: Presently automated tissue
possible
microarray machine is available that bypasses the
• Uniformity in staining conditions:
tedious manual punching procedure. It helps to
Overall uniformity of the staining is
design the array design or layout and the machine
feasible.
automatically creates holes in the paraffin wax
• Faster and cheaper: When large number
and places the donor tissue in the hole of the
of core tissue is processed it becomes
recipient paraffin wax.
cheaper
Advantages of TMA: There are several
• Preservation of the original block is pos-
advantages of TMA that include (Box 22.1):
sible: Even after multiple core biopsies
1. Amplification of the resource: Ordinarily the donor tissue block is useable for the
from a standard 5 mm thick section of tissue diagnosis
we can get a maximum of 100 sections for • Effective in quality assurance program
study. Whereas depending on the size of the
Limitations
tissue in the original block at least 200 core
• Tissue heterogeneity
biopsies can be taken to make the TMA
• Not suitable in small series with occa-
block. After the construction of the TMA, we
sional use
can cut at least 1000 sections of 3 μ thickness
• Loss of the tissue
from the 5 mm thick array block. Therefore,
• Confusion on orientation
we get a total of 1000 × 200 means 200,000
sections from the limited resource.
References 245
2. Uniformity in staining conditions: At the rich in keratin, bone or cartilages is likely to

time of conventional staining the different be lost.
tissue sections are stained at a different time 4. Disorientation of the core biopsy tissue:
(such as 10 batches of 20 slides each) and Due to a large number of core biopsies there
the slide-to-slide variation may occur due to is a chance of disorientation when TMA is
several variables such as antigen retrieval, done manually. Rows of empty core tissues
the concentration of different reagents, may help in the immediate orientation of the
incubation period, washing time etc. tissue.
However, in the case of TMA, each tissue
section consists of 100 to 1000 core biop-
sies from the different patients and the sin- 22.5 Clinical Applications of TMA
gle section is stained that avoids all the
slide-to-slide variability. TMA helps to implement various molecular dis-
3. Faster, cheaper and reduction of man- coveries in clinical areas. The novel genes can be
power: In TMA a single slide requires less identified by the DNA microarray technique and
reagents, labour and time. Therefore, TMA subsequently, these gene products can be vali-
saves cost, time and total workforce. dated by TMA [7, 8].
4. Original block can be preserved: Only a There are three different categories of TMA
few core biopsies from the original block are are described in this respect:
sufficient to make TMA. The original block
can be preserved and can be used for further 1. Different types of tumours for the novel
sectioning. markers: Large varieties of tumours can be
5. Effective in quality assurance program: studied for the novel markers in an ultrashort
Due to significant amplification of the labo- time.
ratory material TMA can be used for the 2. Tumor progression array: One particular
external and internal quality control pro- tumour type is studied for its progression as in
grams. The TMA section can also be used for a single section one can study normal pros-
the standardization of reagents for positive tatic tissue, prostatic hyperplasia, intraepithe-
control. lial neoplasia and carcinoma [9].
6. The whole analysis of TMA is now auto- 3. Prognostic array: Novel markers related to
mated and computers can keep track of the the prognosis of the malignant tumour can be
huge data. studied by TMA.
Limitations of TMA: The main limitation of
TMA is tissue heterogeneity.
References
1. Tissue heterogeneity: This is one of the
main concerns of TMA. The tumour may 1. Fejzo MS, Slamon DJ. Frozen tumor tissue microar-
vary from area to area such as Hodgkin lym- ray technology for analysis of tumor RNA, DNA, and
phoma may have different morphologies in proteins. Am J Pathol. 2001;159:1645–50.
different areas. Therefore small 2 mm tissue 2. Dancau AM, Simon R, Mirlacher M, Sauter G. Tissue
microarrays. Methods Mol Biol. 2016;1381:53–65.
may not represent the whole tumour and 3. Bubendorf L, Nocito A, Moch H, Sauter G. Tissue
findings may vary. However, several studies microarray (TMA) technology: miniaturized pathol-
have shown that TMA and whole tissue data ogy archives for high-throughput in situ studies. J
are almost similar [5, 6]. Pathol. 2001;195(1):72–9.
4. Parsons M, Grabsch H. How to make tissue microar-
2. Less cost-effective in small series of cases: rays. Diagn Histopathol. 2009;15:142–50.
TMA is not cost-effective if it is done once in 5. Kononen J, Bubendorf L, Kallioniemi A, et al. Tissue
a while in a small series of cases. microarrays for high-throughput molecular profiling
3. Prone to lose the tissue: The core biopsy tis- of hundreds of specimens. Nat Med. 1998;4:844–7.
6. Parker RL, Huntsman DG, Lesack DW, Cupples
sues may be lost due to their tiny size. Tissue JB, Grant DR, Akbari M, Gilks CB. Assessment of
interlaboratory variation in the immunohistochemi- genes uncovered by cDNA microarray screening in

cal determination of estrogen receptor status using renal carcinoma. Am J Pathol. 1999;154:981–6.
a breast cancer tissue microarray. Am J Clin Pathol. 9. Varambally S, Dhanasekaran SM, Zhou M, Barrette
2002;117(5):723–8. TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ,
7. Sallinen S-L, Sallinen PK, Haapasalo HK, et al. Sewalt RG, Otte AP, Rubin MA, Chinnaiyan AM. The
Identification of differentially expressed genes in polycomb group protein EZH2 is involved in progres-
human gliomas by DNA microarray and tissue chip sion of prostate cancer. Nature. 2002;419(6907):572–3.
techniques. Cancer Res. 2000;60:6617–22.
8. Moch H, Schraml P, Bubendorf L, et al. High-
throughput tissue microarray analysis to evaluate
Sanger Sequencing and Next
Generation Gene Sequencing: 23
Basic Principles and Applications
in Pathology
DNA sequencing means the determination of the

23.1 Sanger Sequencing
order of arrangement of the base pair in the DNA
chain. There are three different types of DNA
Sanger sequencing is named under Sanger F. This
sequencing:
sequencing technique is also known as dideoxy
sequencing. This is the widely used commercial
1. Basic type of DNA sequencing:
DNA sequencing [1].
(a) Sanger sequencing: Here DNA elongation
Basic principle: In the Sanger sequencing
process by polymerase chain reaction is ter-
technique 2′, 3′-dideoxynucleotides are used
minated by dideoxy nucleotides (ddNTPs).
for DNA synthesis. In the absence of the
(b) Maxam Gilbert sequencing: Here DNA
3′-hydroxyl group in 2′,3′-dideoxynucleo-
chain is terminated by a chemical termina-
tides, DNA cannot be synthesized further as
tion process with the help of chemical
no phosphodiester bond can be formed with
substances.
the next dNTP and the chain terminates
2. Advanced type of DNA sequencing: This is
(Fig. 23.1a and b). The four ddNTPs (dideoxy-
also known as “shotgun” sequencing as a long
nucleotides phosphates such as dATP, dTTP,
DNA chain is broken into small pieces.
dCTP, dGTP) are labelled by different fluoro-
3. Next-generation DNA sequencing:
chrome dyes) so that they are recognised by a
(a) Second generation sequencing:
laser beam. By convention A is tagged with
• Pyrosequencing,
green fluorochrome dye, T is labelled by red,
• Illumina technology.
C by black and G by blue fluorochrome. Each
• Ion semiconductor sequencing.
fluorescent labelled terminated fragment of
(b) Third generation sequencing:
DNA is recorded and from this data, the DNA
• Single-molecule real-time sequencing.
sequence is assessed (Fig. 23.2).
(c) Fourth generation sequencing:
• Nanopore technology.
• DNA nanoball sequencing.
https://doi.org/10.1007/978-981-19-6616-3_23
248 23 Sanger Sequencing and Next Generation Gene Sequencing: Basic Principles and Applications…
Fig. 23.1 (a) The

chemical structure of
a
deoxynucleotides
phosphate (dNTP) and
dideoxynucleotides
phosphate (ddNTP). (b)
Schematic diagram
showing the principle of
the Sanger sequencing
technique. Here 2′,
3′-dideoxynucleotides
are used for DNA
synthesis and in the
absence of 3′-hydroxyl
group the DNA chain is
terminated. The four
DDNTPs are labelled by
different fluorochrome
dyes so that they are
recognised by laser
beam and from this data
DNA sequence is
assessed
b
23.1 Sanger Sequencing 249
Fig. 23.2 Schematic diagram showing the capillary gel electrophoresis of different fragments of PCR products
23.1.1 Reagents Needed DNA chain elongates till it incorporates fluo-

rescently labelled ddNTP. At that point, the
• Primers: Small piece of single-stranded DNA DNA chain elongation is terminated. The
is used DNA chain is terminated with various lengths
• DNA template: This chain is sequenced bearing the terminal labelled ddNTP (fluoro-
• DNA polymerase enzyme chrome labelled A, T, C and G).
• dNTPs (dATP, dTTP, dCTP, dGTP) • The PCR cycle of DNA is repeated several
• ddNTP (fluorescent labelled): All four ddNTPs times to have all the base positions of the tem-
such as ddATP, ddTTP, ddCTP, ddGTP are plate DNA. In the end, variable lengths of
labelled with different fluorochrome dyes. DNA chains with terminal unique fluores-
These are chain-terminating nucleotides. cence labelled A, T, C and G are obtained.
• DNA chains are undergone capillary gel elec-
trophoresis and then the coloured emission
23.1.2 Main Steps spectrum of all these fluorochromes labelled
DNA is recorded by capillary gel electropho-
• Mix the above reagents and denature the com- resis (Fig. 23.2). With the help of suitable soft-
plementary strands of the DNA template by ware from the data of the terminal bases, the
heating at 96 °C. exact sequence of the bases in the DNA is
• Lower the temperature to 50 °C for annealing generated.
the primer with a DNA template
• Raise temperature to 60 °C for the elongation Advantage: Very accurate method (99.9% accu-
of DNA with the help of DNA polymerase. The racy rate).
23.1.3 Limitations DNA is made single-stranded. Now with the help

of chemicals, the DNA is broken into pieces in the
• Not cost-effective: Therefore, small subsets of specific nucleotide regions (G, A and G, C, C and
genes are sequenced by Sanger’s sequencing T). The reactions are conducted in four separate
• Slow process tubes and the resultant material is electrophoresed
• Only a small fragment of DNA is sequenced and subsequently visualized by autoradiography.
Finally, the result is interpreted based on the posi-
tion of the different bands.
23.2 Maxam Gilbert Technique
In 1976, Allan Maxam and Walter Gilbert devel- 23.2.1 Main Steps
oped the technique of DNA sequencing with the
help of chemical termination of DNA chain [2]. 1. 1. Dephosphorylation: Normal phosphate
This technique is also known as the chemical with the help of alkaline phosphatase.
degradation technique. 2. 2. End labelling: Radioactive Phosphorus
Basic principle (Fig. 23.3): In this technique, (P32) is incorporated at the 5′ end of the DNA
at first the 5′ end of DNA is labelled by radioactive chain with the help of polynucleotide kinase.
Phosphorus and subsequently the DNA is purified. 3. Restriction enzyme digestion: With the help
With the help of restriction enzymes, the long of the restriction enzyme, the long chain of
DNA is cut into small pieces. The double-stranded DNA is cut into small pieces.
Fig. 23.3 Schematic diagram showing the basic principle of Maxim Gilbert sequencing technique
23.3 Next-generation Sequencing 251
4. Denaturation: The double-stranded DNA is millions of DNA fragments. Compared to

made into single-stranded DNA where the 5′ Sanger sequencing, NGS is a tremendously
end is labelled by radioactive P. rapid, and cost-effective technique [3]. The
5. Chemical degradation: This is the vital step majority of NGS technologies work in the fol-
where the end labelled ssDNA is treated with lowing pattern:
four different tubes with four different chemicals
to break down DNA into the specific nucleotides. • Library preparation: It involves random break-
The concentration of the chemical reagents is ing down the DNA and ligation of the specific
maintained in such a way that only one cut occurs adaptors.
in the DNA fragment at a single time. • Amplification of the genomes of library.
(a) Nucleotide G: Dimethyl sulphate with • Sequencing the amplified genomes.
heat followed by piperidine. • Data interpretation.
(b) Nucleotides A and G: Dimethyl sulphate
in formic acid and piperidine.
(c) Nucleotides C and T: Hydrazine and 23.3.1 Second-generation
piperidine. Sequencing
(d) Nucleotide C: Hydrazine in 2 M NaCl
and piperidine. Pyrosequencing [4]: The pyrosequencing tech-
6. Electrophoresis: The DNA fragments obtained nology was the first available NGS system in the
from the four tubes are electrophoresed side market and was introduced to the market in 2005
by side in denaturing acrylamide gel. The by “454” Life science which is later marketed by
DNA fragments move according to the size in Roche as Roche-454 in the year 2007.
the gel electrophoresis. The smaller fragment Pyrosequencing works on the principle of
moves more than the larger fragments. “sequencing of sequencing”. In this technique, at
7. Autoradiography: The DNA fragments are first, DNA is fragmented into small pieces fol-
visualized with the help of autoradiography of lowed by the generation of many small templates
the gel. with the help of DNA polymerase enzymes. At
8. Interpretation: The DNA sequence is assessed the time of incorporation of each nucleotide, the
with the help of the position of the different fixed amount of inorganic pyrophosphate (PPi) is
bands in each tube. released (Fig. 23.4). The released PPi molecule
converts Adenosine di Phosphate (ADP) into
Advantages: (1) The purified DNA can be used Adenosine tri Phosphate (ATP). The ATP mole-
directly as no cloning is required. cule acts on the luciferin and generates light
energy which is recorded by the charged coloured
device. The DNA sequence is assessed from the
23.2.2 Limitations pyrogram that is generated during each nucleo-
tide incorporation.
• Costly
• Slow process
• Radioactive elements are used which is haz- 23.3.2 Advantages
ardous to health
• Compared to Sanger sequencing it is very fast:
700 MB per day
23.3 Next-generation Sequencing • Much cheaper than Sanger sequencing
• Reliable technology: 99.9% accuracy rate
Next-generation sequencing (NGS) is a high- • No need for labelling the nucleotide.
throughput technique that has the capability to • No need for any gel electrophoresis as de novo
perform parallel sequencing reactions from sequencing occurs.
Fig. 23.4 Schematic diagram showing the basic princi- chain of reactions that liberates light energy and is
ple of pyrosequencing technique. Here the fixed amount detected by colour charged device camera. The DNA
of inorganic pyrophosphate (PPi) is released whenever a sequence is assessed from the pyrogram that is generated
nucleotide is incorporated in a polymerization reaction by during each nucleotide incorporation
a DNA polymerase enzyme. The released PPi initiates a
23.3.3 Limitations mented DNA. This DNA is then denatured to

make single-stranded DNA.
• Error-pronene in case of the sequencing of 2. Cluster generation (Fig. 23.5b): The “flow
long homopolymers. cell” is used in this technique. The “flow cell”
• Relatively costlier than other NGS technolo- is an optically transparent glass slide with
gies presently available in the market. multiple wells on the surface that are filled
with complementary DNA of the adapters to
fix the adapter-ligated DNA of the sample.
23.4 Illumina Solexa Once DNA oligonucleotides are anchored on
the glass slide the distal DNA strands are bent
Illumina is one of the popular second-generation and attached to the adjacent complementary
sequencing techniques. The technique works on DNA strand of the glass slide. Thereby a
the basis of “sequencing by synthesis”. The tech- bridge-like structure is formed. Now with the
nique was first time introduced by Solexa in the help of PCR, a large number of DNA strands
year 2006 and subsequently, it was acquired by is generated. The whole process is known as
Illumina [5]. cluster generation by bridge amplification.
The basic steps of illumina are: 3. Sequencing (Fig. 23.5b): Now, different fluo-
rochrome labelled dNTPs are used for synthe-
1. Library preparation (Fig. 23.5a): At first the sizing the complementary strand of the DNA
unknown double-stranded DNA is fragmented of the clustered DNA strands. Here, the
into small pieces. The double-stranded adapter reversible terminator nucleotide is used so
DNA is attached to either end of the frag- that each time, only one fluorochrome labelled
23.4 Illumina Solexa 253
Fig. 23.5 (a) Schematic diagram showing the library with the target DNA and the sequence of bases of DNA is
preparation of Illumina sequencing technology. (b) obtained. Finally, the complete DNA sequence is obtained
Schematic diagram showing the cluster generation and by assembling the overall information from the multiple
hybridization of Illumina sequencing technology. The hybridization tests
numerous oligonucleotide probes are used to hybridize
nucleotide is incorporated followed by the the incorporation of the nucleotides and does not
enzymatic cleavage of the fluorescence from use any fluorescence tagged nucleotides
the nucleotide and allowing the next labelled (Fig. 23.6a). The machine can run 400 bp read
nucleotide to be incorporated into the growing length and can generate data up to 15Gb data
strand. within 6 h [6].
4. Analysis: From the recorded fluorochrome The basic steps are (Fig. 23.6b):
data, DNA sequencing is done.
• DNA library generation: At first DNA is cut
Currently, in the market, various Illumina plat- into pieces and the adapters are ligated at the
forms are available: MiSeq, HiSeq and NextSeq ends of fragmented DNA. Subsequently, the
series. DNA with the adapters is hybridized with the
MiSeq Illumina is a tabletop sequencer that can complementary sequences of the specially
finish the sequence within 3 h. It performs massive prepared beads.
parallel sequencing of many billions of DNA frag- • DNA amplification: The DNA attached to the
ments. The NextSeq Illumina series is also a table- beads is amplified with the help of emulsion
top sequencer and can sequence 16 to 100Gb data PCR.
in 26-h period. HiSeq 4000 Illumina is the latest • Incorporation of nucleotide and detection
model of the HiSeq series system. HiSeq series of pH change: The beads with the attached
Illumina system is used in the whole genome DNA are now flooded over the microwell.
sequencer. It can run 1 TB of data in 6 days. Each microwell accommodates only one bead
and only one type of dNTP is added at a time.
Whenever a nucleotide is incorporated in the
23.4.1 Advantage DNA chain, H+ is generated and the change of
pH is detected by an integrated metal-oxide-
1. Very high data yield. Presently, 1.8 Tb data is semiconductor and the chemical signal is con-
sequenced in a single run. verted into a digital signal.
2. Long read length. • Data interpretation: The digital signal is
3. Low error rate (99% accuracy rate). analysed in the computer and the DNA
sequence is generated.
23.4.2 Limitations
23.5.1 Advantages
1. Guanine and Cytosine bias at the time of
bridge amplification 1. No expensive fluorochrome tagged dNTP is
used.
23.5 Ion Semiconductor 2. It has comparatively less run cost.

Sequencing (Ion Torrent) 3. No need for an optical imaging system.
4. More than 99% accurate.
Ion Torrent sequencing was first time introduced
by the Ion Torrent system in the year 2010. It was
immediately acquired by Life Technologies, 23.5.2 Disadvantage
USA. The Ion Torrent technique also uses
“sequencing by synthesis” and unlike the illu- 1. Ion Torrent system may produce error in case
mine technique it detects the change of pH during of homopolymer sequences
23.5 Ion Semiconductor Sequencing (Ion Torrent) 255
Fig. 23.6 (a) The Ion Torrent machine. (b) The basic principle of Ion Torrent sequencing is highlighted by the sche-
matic diagram
23.6 Third Generation chamber. The ZMW contains a DNA polymerase

enzyme on its floor to synthesise DNA. The dif-
23.6.1 Single-Molecule Real-Time ferent fluorochrome tagged dNTPs are also
Sequencing available in ZMW. This nano-chamber is illumi-
nated only in the lowermost portion where DNA
Single Molecule real-time sequencing (SMRT) is synthesis takes place. Each time a DNA nucleo-
introduced by PacBio in the year 2010 [7]. SMRT tide is attached to the circular template, the fluo-
is also based on the sequencing by synthesis prin- rophore is detached from the nucleotide and
ciple. The technique has the capability to run released. The fluorescent signal is recorded in
long read lengths (more than 1Kb) by real-time the ZMW. Then the next florescent tagged nucle-
sequencing. SMRT detects the incorporation of otide is attached to the DNA chain. The circular
each dNTP in the circular DNA template in a spe- DNA strand moves continually and the synthesis
cially illuminated microwell known as a zero- is going one from the one adapter to the other
mode waveguide (ZMW). The methodology is adapter region.
described below in brief (Fig. 23.7). Data interpretation: The computer records
DNA library: The double-stranded DNA is the fluorescence measurement and analyses the
cut into small pieces. Now, a single-stranded cir- unknown DNA sequence.
cular DNA template known as SMRTbell is
attached to both ends of DNA. The SMRT bell 23.6.1.1 Advantages
carries a universal primer binding site and an ini- • SMRT is capable to sequence long-read length
tiation sequence. (more than 15Kb)
DNA sequencing in ZMW: The SMRT bell • It is able to sequence GC content more
molecule is now immersed in the ZMW nano- successfully
Fig. 23.7 The basic principle of signal Molecule real-time sequencing

23.7 Fourth Generation Sequencing 257
23.6.1.2 Disadvantage stant speed and 100 kb size DNA can be

• Higher error rate sequenced at a time.
• Costly for per base sequencing
• Less data is generated per base run. 23.7.1.1 Advantage
• Handheld portable sequencer
• Relative cheaper
23.7 Fourth Generation • No need of DNA library preparation
Sequencing
23.7.1 Nanopore Sequencing 23.7.2 Other Technologies

(Fig. 23.8)
Nano string sequencing (Fig. 23.9): The
Nanopore sequencing is the fourth-generation NanoString technology is a recent technology that
sequencing technology [8]. This technique is can directly quantitate the RNA expression as
first time introduced in the market in 2014 by small as 100 nucleotides [9]. It is, therefore, suit-
Oxford technology in the form of MinION. In able to analyse the potentially degraded RNA. The
this technique, the single-stranded DNA is Nanostring sequencing n-counter analysis bypasses
passed through a charged nano-hole and the the amplification process and no enzymes are used
voltage alteration of each nucleotide is mea- in this assay. Here unique barcoded sequence-spe-
sured. The DNA passes one base at a time and cific colour-coded reporter probes along with a
each base of DNA generates unique voltage small biotinylated capture probe are applied to bind
fluctuation. The single-stranded DNA passes with the target mRNA. After the hybridization of
through the nanopore of 1 nm size with a con- the two probes with the mRNA target, the excess
Fig. 23.8 The basic

principle of Nanopore
technology is
demonstrated
Fig. 23.9 The basic

principle of Nanostring
sequencing technology
is demonstrated
unbound probes are washed out. Now the fluores- the whole genome of the organism [10]. The
cent bar codes are analysed with the help of an technique consists of two major parts: 1) The cre-
image analyser and each target molecule of mRNA ation of DNA nanoballs, 2) combinatorial Probe-
is identified. Anchor ligation (cPAL).
The steps of DNA nanoball sequencing are
23.7.2.1 Advantages following (Fig. 23.10):
• Simple and precise
• Formalin-fixed tissue is also fit for analysis 23.7.3.1 DNA Nanoball Creation
• No enzymes required • At first DNA is isolated from the sample
• No amplification is needed • DNA is cleaved into small pieces
• Size selection: 100 to 300 bp size of DNA is
selected by gel electrophoresis
23.7.3 DNA Nanoball Sequencing • End of the DNA is repaired and adapter is
attached to the both ends
DNA nanoball sequencing is a high throughput • The fragments of DNA are now amplified by
sequencing technology that is used to sequence PCR
23.7 Fourth Generation Sequencing 259
Fig. 23.10 The steps and principle of DNA nanoball sequencing is highlighted
• The split strand is attached to both ends of • Isolation of RNA from the sample
DNA and the circular single-stranded DNA is • RNA is cleaved into small fragments
made • RNA is converted into cDNA
• Finally, the resulting circular DNA is ampli- • Adapters are attached to both ends of the DNA
fied by Phi29 polymerase to make DNA nano- • The DNA with attached adapters are amplified
balls (DNB). by PCR
• Now with the help of fluorochrome tagged
23.7.3.2 Loading Onto Flow Cell dNTPs the chain synthesis of the DNA is
for Sequencing analysed.
• The DNB is now flooded onto the patterned
array flow cell. The flow cell contains posi- Table 23.1 Highlights the comparison of different
tively charged aminosilane that selectively types of next-generation sequencing technologies.
binds with a single DNB
• Now the flow cells are also flooded with dif-
ferentially Fluorochrome tagged dNTP, 23.7.5 Applications of NGS
• Whenever the fluorochrome tagged dNTP is
incorporated into the DNA chain in DNB, the • Whole-genome screening: Rapid whole-
emitted fluorescence is recorded. genome analysis is possible with the help of
• From the recorded fluorescence data, the com- NGS including both coding and non-coding
puter generates DNA sequencing. region of the genome. More data on the so-
called non-functional part of the genome
suggests that the production of RNA mole-
23.7.4 RNA-Seq cule with the help of the non-coding region
are responsible for the cell development
RNA-Seq is a novel technology by which RNA is [12].
sequenced [11]. The steps of RNA-Seq. • Identifies a broader spectrum of mutational
Are: changes: In addition to the base sequence of
Table 23.1 Comparison of different types of next-generation sequencing technologies

Year of Yield per Read
Technique launching Basic principle run length Characteristics
Roche 454 2005 Sequencing by synthesis. 600 to 1 kb • The rate of error is
pyrosequencing Pyrophosphate is released after the 700 Mb high in
incorporation of each nucleotide homopolymer
which causes light generation. sequencing
Illumina 2006 Sequencing by synthesis. 600 bp >1.5 Tb • Relatively cheap
Fluorochrome tagged nucleotides are • Higher yield
used and fluorescence is measured • Low error
during incorporation of the • Fast
nucleotide.
Ion Torrent 2010 Sequencing by synthesis. H+ is 400 bp 15 Gb • No fluorescence
generated at the time of dye
incorporation of the nucleotide. The • No imaging
alteration of pH is measured. • Low error
• Cheap
• Fast
SMRT 2010 Sequencing by synthesis. 50 kbp 0.1 Gb • Long read possible
Fluorescent labelled nucleotide is
used and fluorescence is measured
during incorporation of the
nucleotide in the nanowell
Nanopore 2012 Single-stranded DNA passes through 100Kbp >100 Gb • Small pocket
sequencing charged nano-hole and the change of holder
voltage is measured • No need for DNA
library preparation
• Extra-long read
DNA Nanoball 2010 DNA nanoball is created from the 120 bp 120 Gb • useful for whole
sequencing sample followed by sequencing by genome
ligation of the fluorochrome tagged sequencing
dNTP
DNA, NGS detects all novel mutational Intratumor heterogeneity: There may be
changes such as small insertions and deletions. variations of mutations in a particular type of
• Clinical decision support: The demonstra- tumour in different patients (intertumoral hetero-
tion of complete mutation data of disease may geneity) or different mutational changes in the
help in the identification of the driver muta- same tumour at different sites (intratumor hetero-
tion. Once the driver mutation is identified, we geneity). This is a complicated issue and therefore
can plan therapy for the individual patient on the response of therapy may vary in a particular
the basis of it such as erlotinib EGFR muta- tumor.
tion and vemurafenib in BRAF mutational
melanoma.
• Study of cancer genome for diagnosis, clas- 23.7.6 Limitations
sification and prognosis: NGS is able to pro-
vide complete data on the individual cancers. Cost: NGS is very costly and needs adequate
This data may be helpful in the diagnosis, clas- infrastructure, experience, storage capacity and
sification and prognosis aspects of the disease. computational capacity.
Intratumor heterogeneity: There may be
23.7.5.1 Limitations variations of mutations in a particular type of
Cost: NGS is very costly and needs adequate tumour in different patients (intertumoral hetero-
infrastructure, experience, storage capacity and geneity) or different mutational changes in the
computational capacity. same tumour at different sites (intratumor hetero-
References 261
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fore the response of therapy may vary in a of SMRT sequencing. Genome Biol. 2013;14(7):405.
https://doi.org/10.1186/gb-2013-14-6-405. Erratum
particular tumor. in: Genome Biol. 2017 Aug 16;18(1):156
8. Jain M, Olsen HE, Paten B, Akeson M. The Oxford
Nanopore MinION: delivery of nanopore sequenc-
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2016;17(1):239. https://doi.org/10.1186/s13059-016-
1103-0. Erratum in: Genome Biol. 2016 Dec 13;17
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U S A. 1977;74(12):5463–7. Wong SC. NanoString, a novel digital color-coded
2. Maxam AM, Gilbert W. A new method for sequenc- barcode technology: current and future applications
ing DNA. Proc Natl Acad Sci U S A. 1977;74(2): in molecular diagnostics. Expert Rev Mol Diagn.
560–4. 2017;17(1):95–103. https://doi.org/10.1080/1473715
3. Kumar KR, Cowley MJ, Davis RL. Next-generation 9.2017.1268533. Epub 2016 Dec 12
sequencing and emerging technologies. Semin 10. Kaji N, Okamoto Y, Tokeshi M, Baba Y. Nanopillar,
Thromb Hemost. 2019;45(7):661–73. https://doi. nanoball, and nanofibers for highly efficient analysis
org/10.1055/s-0039-1688446. Epub 2019 May 16 of biomolecules. Chem Soc Rev. 2010;39(3):948–56.
4. Siqueira JF Jr, Fouad AF, Rôças IN. Pyrosequencing https://doi.org/10.1039/b900410f. Epub 2010 Jan 14
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biomes. J Oral Microbiol. 2012;4 https://doi. Carpten JD, Craig DW. Translating RNA sequenc-
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5. Voelkerding KV, Dames SA, Durtschi JD. Next- lenges. Nat Rev Genet. 2016;17(5):257–71. https://
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nostics. Clin Chem. 2009;55(4):641–58. https://doi. PMID: 26996076
org/10.1373/clinchem.2008.112789. Epub 2009 Feb 12. Mattick JS, Dinger M, Schonrock N, Cowley
26 M. Whole genome sequencing provides better diag-
6. Merriman B, Ion Torrent R&D Team, Rothberg nostic yield and future value than whole exome
JM. Progress in ion torrent semiconductor chip based sequencing. Med J Aust. 2018;209(5):197–9. https://
sequencing. Electrophoresis. 2012;33(23):3397–417. doi.org/10.5694/mja17.01176. Epub 2018 Apr 9
Liquid Biopsy: Basic Principles,
Techniques and Applications 24
Table 24.1 The comparison of liquid biopsy compared

24.1 Introduction to conventional biopsy
Conventional
For decades, pathologists are habituated to dealing Features Liquid biopsy biopsy
with conventional biopsy specimens of the tissue Technique Complicated Simple and easy
for the histopathology examination. In recent and needs to perform
years, liquid biopsy is introduced in the field of special skill
Sample Non-invasive Invasive
pathology. The term “liquid biopsy” (LB) means
procurement technique
the study of a blood sample or fluid specimen for Tumour The presence of Tissue is taken
the assessment of DNA obtained from the circulat- a tumour is not from the gross
ing cancer cells or cell-free DNA molecule [1]. mandatory specimen of the
Liquid biopsy (LB) is one of the most tumour
Distant and Irrespective of Difficult to get a
advanced and promising areas of modern pathol-
tiny tissue size and biopsy from the
ogy [2, 3]. It is an easily approachable and mini- position tiny and
mally invasive technique and can provide deep-seated
valuable information on the molecular genetics tumours
of the tumour that can be applied to screening Continuous Easy to Not always
progression of ascertain as the possible
tumours, detection of recurrence, the detection of molecular specimen is
altered molecular characteristics and personal- changes easy to collect
ized tumour management. Molecular Not dependent The tumour
changes in on tumour heterogeneity
tumour heterogeneity may affect the
heterogeneity assessment of
24.2 Conventional Biopsy Versus molecular
Liquid Biopsy? alteration
Price Extremely Minimal cost
The conventional biopsy specimen is obtained costly
by incisional biopsy, excision of the tissue or Cost Very high cost Cheap
total resection of the specimen. It is easy to pro-
cess the sample and it needs minimal training to inaccessible area. Liquid biopsy can be done
study the histopathology section. In compari- even after resection of the tissue specimen. It is
son, the LB specimen can be obtained from the a minimally invasive technique and can reflect
blood or body fluids. So, the sample can repre- the continuous changes of molecular alterations
sent the tiniest tissue specimen situated in an in tumours (Table 24.1).
https://doi.org/10.1007/978-981-19-6616-3_24
264 24 Liquid Biopsy: Basic Principles, Techniques and Applications
24.3 The Components of Liquid 2. Cell-free DNA (cf-DNA): cf-DNA is one of

Biopsy the important components of LB. It is derived
both from tumour cells and normal cells. cf-
The components of LB specimen are (1) blood DNA comes to the circulation by the necrotic
sample, (2) effusion fluid, (3) bronchoalveolar or apoptotic cells or active release of the
lavage, (4) urine, (5) saliva secretion, and (6) tumour or healthy cells. The cf-DNA also has
miscellaneous other body samples. a very short half-life that varies from 10 to
The main components of liquid biopsy are: 30 min [4]. The amount of cf-DNA in circula-
Fig. 24.1. tion ranges from 1 ng/ml to 27 ng/ml in a nor-
mal person. Whereas, in the case of tumours
1. Circulating tumour cells (CTC), the amount increases by several hundred ng/
2. Cell-free DNA (cf-DNA), ml. The level of cf-DNA in the circulation in
3. Cell-free RNA (cf-RNA). the tumour is dependent on tumour size, grad-
4. Extracellular vesicles (EV). ing, and metastasis.
3. Cell-free RNA (cf-RNA): cf-RNA is actively
1. Circulating tumour cells (CTC): CTCs are
released by the cells. It is unstable in circula-
produced mainly by the primary tumour and
tion and remains only for 10 to 15 s. Cf-RNA
occasionally by the metastatic tumour. These
plays important role in carcinogenesis, how-
cells are usually less in circulating blood (one
ever, its role as a component of LB is still not
per 110 neutrophils) and they are rapidly
properly investigated [5].
removed by the reticuloendothelial cells of
4. Extracellular vesicles (EV): EVs are tiny
the body within one and half hours. CTCs of
membrane-bound vesicles that are released
epithelial malignancies express epithelial cell
from the tumour to the circulating blood,
adhesion molecule (EpCAM) and cytokeratin.
saliva, urine and other body cavity fluids.
These markers can be used to identify the
EVs contain exosomes (EXO), microvesicles
CTCs.
and apoptotic bodies. EXO contains mRNA,
micro-RNA and DNA. These are protected
due to the encasing bi-layered lipid mem-
brane of EXO. The study of EXO in the LB
may provide vital information regarding
diagnosis, and the response of chemotherapy
to tumour [6].
24.4 Enrichment of the Contents

of Liquid Biopsy
The different constituents of LB specimens are

present in extremely low amounts in the circulat-
ing blood or in other samples. Therefore, the
enrichment of the constituents of LB is extremely
important.
1. Circulating Tumour Cells

The different techniques to isolate and
Fig. 24.1 Schematic diagram showing the various com- accumulate CTCs are described below
ponents of liquid biopsy (Table 24.2).
24.6 Clinical Applications 265
Table 24.2 Isolation techniques of circulating tumour cells

Availability In
Technology Methods Comments market
Physical properties Depending on the size, density and electrical charge. (a) Efficient (a) Yes
(a) CellSieve Physical filtration technique and density gradient are method (b) Yes
(b) Parylene filter used. (b) Viable
(a) Based on cell filtration tumour cells
(b) Filter-based are captured
Immunological (a) EpCAM coated ferrofluid nanoparticles are used (a) FDA (a) Yes
properties to isolate the epithelial cells. The cells are further approved (b) Yes
(a) CELLSEARCH isolated by cytokeratin immunostaining and the (b) Relatively
(b) MAC system contaminated lymphocytes are removed by CD45 cheap and
immunostaining. fast
(b) Magnetic particle coated with epithelial cell
antibody and the epithelial cells is separated in a
magnetic field.
Both Microfluidic and (a) Both microfluidic and beads are used to enrich (a) Highly (a) Not yet
immunological methods the cells efficient in the
(a) CTC-iChip (b) Both microfluidic and immunological methods (b) Faster and market
(b) Liquid Biopsy are combined efficient (b) Yes
Physical method of enrichment: The var- 24.5 The Molecular Techniques

ious physical characteristic of tumour cells is to Do in Liquid Biopsy
used to separate the CTC from the sample.
The cell size, density and electrical charge are The following molecular techniques can be
the predominant characteristic based on which applied to liquid biopsy:
the cells are separated.
Immunological technique: The cell sur- 1. Polymerase chain reaction (PCR): The vari-
face antibody such as EpCAM is tagged with ous advanced PCR techniques can be used
ferrofluid nanoparticles. The epithelial cells such as Droplet Digital PCR, beads emulsion
are entangled with the nanoparticle and then amplification and magnetics PCR, and
the cells are separated by applying the mag- enhanced ice-cold PCR [3].
netic field. The separated cells are further pro- 2. Next-generation sequencing (NGS): Tagged
cessed with the help of pan-CK and the amplicon deep sequencing, cancer personal-
contaminated lymphoid cells are removed by ized profiling by deep sequencing, and a safe
CD45 immunostaining [7]. sequencing system of NGS can be used on
Both physical and immunological liquid biopsy material.
properties: Combined immunologic and 3. Epigenetic study: Methylation-specific poly-
physical properties may also be used to iso- merase chain reaction protocol or Microarray-
late CTCs [7]. based DNA methylation assay are used to
2. Cf-DNA isolation: The cf-DNA can be sepa- study the epigenetic changes.
rated and enriched with the help of (a) column-
based or (b) magnetic bead-based approach. In
the column-based technique, a silica gel mem- 24.6 Clinical Applications [2, 3]
brane is used to entrap the cf-DNA. Whereas,
in a bead-based approach, magnetic beads are 1. Early diagnosis and cancer screening:
applied to capture DNA. The magnetic bead- Early diagnosis or screening of cancer can be
based approach eliminates the risk of DNA done by demonstrating:
damage and is automatable. (a) The elevated level of cf-DNA,
3. Exosome isolation: EXO is collected and (b) Mutation of an oncogene, loss of hetero-
enriched with the help of differential ultracentri- zygosity and hypermethylation of pro-
fuge, or polymer-based precipitation method [8]. moter genes.
266 24 Liquid Biopsy: Basic Principles, Techniques and Applications
2. Management and prognosis: The demonstra- 4. Tamkovich SN, Cherepanova AV, Kolesnikova EV,
tion of certain mutations may indicate poor et al. Circulating DNA and DNase activity in human
blood. Ann N Y Acad Sci. 2006;1075:191–6.
prognoses such as K-RAS mutation in non- 5. Zaporozhchenko IA, Ponomaryova AA, Rykova EY,
small cell lung carcinoma (NSCLC) or amplifi- Laktionov PP. The potential of circulating cell-free
cation of ‘MYC related oncogene in RNA as a cancer biomarker: challenges and oppor-
neuroblastoma [9]. The presence of EGFR tunities. Expert Rev Mol Diagn. 2018;18(2):133–45.
6. Harding CV, Heuser JE, Stahl PD. Exosomes: looking
T790M mutation in NSCLC may indicate the back three decades and into the future. J Cell Biol Mol
resistance of EGFR inhibitor therapy and Sci. 2013;200:367–71.
demand more advanced third-generation anti - 7. Ignatiadis M, Lee M, Jeffrey SS. Circulating tumor
EGFR therapy [10]. cells and circulating tumor DNA: challenges and
opportunities on the path to clinical utility. Clin
3. Minimal residual disease (MRD): The detec- Cancer Res. 2015;21(21):4786–800.
tion of MRD may be possible with the help of 8. Momen-Heravi F, Balaj L, Alian S, et al. Current
LB. It is possible to detect early tumour recur- methods for the isolation of extracellular vesicles.
rence or the presence of MRD by identifying Biol Chem. 2013;394(10):1253–62.
9. Combaret V, Bergeron C, Noguera R, Iacono I,
the specific mutation in the cf-DNA [11]. Puisieux A. Circulating MYCN DNA predicts
MYCN-amplification in neuroblastoma. J Clin Oncol.
2005;23:8919–20.
References 10. Castellanos-Rizaldos E, Grimm DG, Tadigotla V,
et al. Exosome-based detection of EGFR T790M in
plasma from non-small cell lung cancer patients. Clin
1. https://www.cancer.gov/publications/dictionaries/ Cancer Res. 2018;24(12):2944–50.
cancer-terms/def/liquid-biopsy. 11. Diehl F, Schmidt K, Choti MA, et al. Circulating
2. Dey P. Liquid biopsy: a new diagnostic modality. T mutant DNA to assess tumor dynamics. Nat Med.
Appl Biol Chem J. 2020;1(1):3–8. 2008;14:985–90.
3. Ghosh RK, Pandey T, Dey P. Liquid biopsy: a new ave-
nue in pathology. Cytopathology. 2019;30(2):138–43.
https://doi.org/10.1111/cyt.12661. Epub 2019 Jan 11
Artificial Neural Network
in Pathology: Basic Principles 25
and Applications
Table 25.1 Artificial neural network versus ordinary

25.1 Introduction computer
Ordinary Artificial neural
An artificial neural network (ANN) is a computer Features computer network
software program that has the capability to do Learning No learning Learning
many difficult and complex functions. An ordi- capacity capacity capacity present
nary computer program can perform many Complicated Cannot solve Power of solving
problem the complex
actions successfully. However, in an individual
problem
and complicated case, it is unable to take any Robustness Not robust Robust
decision unless it is guided by a human being. Data missing The program is The program
The ordinary computer does not have any learn- stopped if data is continues even
ing capability, and it is unable to adapt to the missing with missed data
changing circumstances. The human brain not Self- Not possible Possible
extraction of
only has the quality of learning, and adaptability data
but also can do parallel data processing and Rule-based Only works on Does not follow
robustness. ANN is designed based on the knowl- program the rule-based the rule-based
edge of the biological neural network. The prop- program program
erly trained ANN simulates the human brain and Reaching a Derives the Only provides
solution solution in a the possible best
can perform most of the complex nonlinear prob- consistent solution
lems [ 1–3]. In the last few decades, ANN is manner
widely used in Pathology in the diagnosis, clas-
sification, grading and prognostic assessment of
tumour [3]. nary computer does not have any learning
capacity and it is unable to work in the newer
circumstances and it can not solve any com-
25.2 ANN Versus Ordinary plex problems. Unlike an ordinary computer,
Computer ANN can learn from the environment and it is
robust. It also can solve many complex prob-
Ordinary computer program works on the basis lems that need a human expert. Table 25.1
of rules and so it is programmed to do certain highlights the comparison of ANN and ordi-
duties with minimal error. However, the ordinary computers.
https://doi.org/10.1007/978-981-19-6616-3_25
268 25 Artificial Neural Network in Pathology: Basic Principles and Applications
25.3 Artificial Neural Network body. The main function of the cell body is to
Versus Biological Neuron process the information and then pass the infor-
mation by axon to the next neuron via the synap-
ANN is designed by mimicking the human tic gap.
brain which contains largely neurons. Instead In contrast to the biological neuron, the ANN
of neurons, the ANN is made of multiple contains many nodes (Fig. 25.1). These nodes
nodes. These nodes receive the information receive signals from the input node and then pro-
and then process it and finally transmit the cess the input signals. The weightage of the node
information to the next node to take the final depends on the intensity of the input signal (i)
decision. and the weight of the individual signal (w). If the
The biological neuron is made of (a) den- combined weight of the signal exceeds the thresh-
drites, (b) cell body, and (c) axon. The dendrites old value then it fires the next node (Fig. 25.1). It
receive the information and transmit it to the cell is also known as activation.
Fig. 25.1 The comparison of biological neurons and arti- ment. Each signal has intensity (x) and weightage (w).
ficial neurons is shown. The artificial neuron is known as The combined weightage if a node is therefore repre-
a node. Each input node gets a signal from the environ- sented by ∑N i=1wi xi =w1x1+ w2x2+. . .+wNxN
25.5 Learning of ANN 269
25.4 Activation 25.5 Learning of ANN
There are different modes of activation of the Learning of ANN is usually done by training in
next node that include: the known set of cases. In each time of iteration,
the program gains the experience and the strength
1. Liner activation function of the connection between the nodes (weight
2. Threshold Activation Function value of the two interconnecting nodes) is altered.
3. Sigmoid Neuron unit function So, the learning of the ANN program means
4. Rectified linear unit (ReLU) changing the weightage between the nodes.
Usually, learning is a continuous process that
In the case of linear activation function all the involves evaluation of the output and changing
layers of the ANN collapse and therefore it is not the weightage followed by new input to apply in
possible to use a back-propagation neural net- the program. There are three types of the learning
work to solve any complex function (Fig. 25.2a). process:
In the case of the threshold activation function,
the firing of the next node occurs only if the input 1. Unsupervised learning: Here the ANN does
value is above a certain threshold value not take any help from outside and thereby no
(Fig. 25.2b). The sigmoid neuron unit function is training data is used. So, the program does not
one of the popular activation modes. Here the have any desired output. ANN thereby learn
threshold of activation is just like a sigmoid curve by doing. Unsupervised learning is used to
(Fig. 25.2c). The curve is smooth and so there is have a clustering of the data and reduction of
no jump in the firing of the next neuron. ReLU the dimension.
activation is mainly used in deep learning neural 2. Supervised learning: In this learning method,
networks. Here all the negative input is replaced ANN is provided with labelled data along
by 0 and only the positive value is retained as with known output. Therefore, in each itera-
such (Fig. 25.2d) tion, the ANN gets direct feedback. The
a b
c d
Fig. 25.2 The different activation functions are highlighted (a) liner activation function, (b) threshold activation func-
tion, (c) Sigmoid Neuron unit function, (d) rectified linear unit (ReLU)
supervised learning method is mainly applied because we do not have any control over the
in classification and prediction. function of this layer.
3. Reinforcement learning: In this method of 3. Output layer: This is the final layer that gener-
learning, the weightage in between the nodes ates the final decision of ANN.
is reshuffled randomly and the correct output
is rewarded. This is a slow process of learning At first
and is uncommonly used in ANN. At first, the input layer gets the data from the
operator (environment). The data is transferred
to the hidden layer. The data is processed in this
25.5.1 Multilayer Perceptron layer and finally is transferred to the output
Architecture layer. In every iteration, the error is calculated
as desired output (D) subtracted from the actual
The multilayer perceptron architecture is the output (Y). Now the ANN program adjusts or
classical model of ANN. It is mainly used in the alters the weight of the neuron (∆ Wi) accord-
backpropagation neural network. It consists of ingly. In each iteration, the weight of the neu-
three types of layers (Fig. 25.3). ron is adjusted till the ANN program reaches to
the maximum performance in the training
1. Input layer: The input layer is made of several process.
nodes that receive the input data. This data is
fed by the operator.
2. Hidden layer: The hidden layer is made of 25.5.2 Steps to Building an ANN
several nodes. There may be one or more hid-
den layers. The hidden layer determines the 1. Observe the problems carefully: At first,
final output of the complex problem. The hid- identify the problem to solve. Accordingly,
den layer is also known as the “black box” one should plan to have data collection.
Fig. 25.3 The

multi-layered perceptron
consists of input nodes,
hidden nodes and output
node/s. The nodes of the
hidden layer are known
as a black box
2. Collect your data: Data collection is one of The backpropagation network in multi-layered
the most important steps to build a successful perceptron is described already (Fig. 25.3).
ANN. Accurate data from a large number of
cases are required. 25.5.2.2 Deep Learning (DL) Neural
3. Visualize your data to get insight: Once the Network
data is collected, one should try to get an The DL is a type of unsupervised or partly
insight into the problem. supervised type of ANN that has the ability to
4. Process data for ANN: The outliers of the extract data itself without any external manipu-
data should be discarded as the outliers are lation [4]. It is a unique type of artificial intelli-
the distractors and are the potential source of gence that can handle massive data. The DL
error. system mainly uses the following types of the
5. Design your network architecture: The build- neural network:
ing of the network architecture includes the
selection of the number of input nodes, hid- • Recurrent neural network
den nodes and the output node. It is essential • Restricted Boltzmann machine
to have at least one hidden layer to solve • Deep belief network
complex problems. • Autoencoders
6. Division of data set: The next step is to divide • Convolutional neural network
the data set into the training set, validation
set and test set. In the field of pathology, a Convolutional neural
7. Training of network: The data set for training network (CNN) is commonly used.
is used to train the ANN model. At least 500 The differences between the DL and conven-
iterations is needed to have the training of the tional neural networks are highlighted in
ANN model. The error rate should be kept as Table 25.2.
low as possible. Convolution neural network (CNN): CNN
8. Validation set: The network of ANN should is commonly used in deep learning neural net-
be trained by the validation set. This is work [4]. CNN helps in the feature extraction,
needed to maintain the topology of the and disease identification. The large image is
network. converted into images of a minimum number of
9. Testing the network: Finally, the ANN model pixels. The program itself is responsible to extract
should be applied to a set of the test group. the data. The CNN consists of a group of opera-
10. Fine-tune your model: If needed, the model tions that includes (1) convolution, (2) ReLU,
should be finely tuned to get a more efficient and (3) pooling. By the series of such repeated
function. operations, the huge number of pixels of the
11. Launch your model: Once the model is ready, image is converted into a handful number of pix-
it can be launched in the real life to solve the els. Finally, the pixels of the smaller image is flat-
problem. tened and the traditional ANN model consisting
of input node, hidden node and output node is
25.5.2.1 The Different Types made [4].
of the Artificial Neural
Network 1. Convolution (Fig. 25.4a, b): Here the filter
The commonly used types of ANN are: consists of 3*3 to 9*9 pixels placed on the
large original image. The filter is placed sys-
• Back-propagation network tematically over the original image. The
• Convolution neural network pixel of the filter is multiplied by the pixel of
• Feed-forward neural network the image and the sum of the pixels is con-
• Radial basis neural network sidered as the value in the filter occupied
• Recurrent neural network over the main image. Thus, with the help of
Table 25.2 The difference between DL and conventional convolution operation, using a 3*3 filter the
networks image of 5*5 is converted into only 3*3
Conventional pixels.
Features Deep learning neural network 2. ReLu (Fig. 25.4c): In this activation opera-
Principle Self-extraction of Manual
tion, the negative values are replaced by 0.
data from the input feeding of the
image input data 3. Pooling of layer (Fig. 25.4d): In the pooling
Need for Not needed Needs an operation, only the highest value of the pixel
external expert expert for is retained in selected pixel areas (2*2 or 3*3
for data data pixel area).
extraction extraction
Architecture A series of Input, hidden
operations and output After the series of convolution, ReLu and pooling
followed by a layer operation the pixels of the original images are
conventional reduced significantly.
neural network
a 6 3 0 4 3 1 1 1 6x1 3x1 0x1

3 0 0 3 6 0 0 0 13 7 13
4 1 0 0 6 1 0 1 0x0 0x0
3x0
4 1 1 4 3
Filter/Kernel
4 1 0 0 3 4x1 1x0 0x1
Input image
b Convolution operation
6 3 0 4 3
3 0 0 3 6 13 7 13
1 1 1 0x1 0x1 6x1
4 1 0 0 6 7 4 21
0 0 0
4 1 1 4 3 1x0 4x0 3x0 9 9 9
1 0 1
4 1 0 0 3
Input image
Filter/Kernel Feature map
0x1 0x0 3x1
c d
2 3 0 6
3 0 0 3 3 6
3 1 0 0 5 4
5 1 1 4
-1 0 Input image
Rectified linear unit (ReLU) activation Pooling
Fig. 25.4 Schematic diagram showing the steps of con- the sum of the pixels are considered. (c) Rectified linear
volutional neural network. (a and b) convolution: A filter unit (ReLU) activation: Only the positive pixel value is
is applied to the original input image. The pixel value of considered. (d) Pooling: The highest pixel value is taken
the filter is multiplied by the pixel value of the image and to make the feature map
ANN: Finally, the traditional ANN model is 25.5.2.3 Accuracy of the ANN Model
developed from the flattened pixels of the termi- The accuracy of any ANN model is judged by.
nal feature map (Fig. 25.5).
Advantages: CNN has the following advan- 1. Confusion matrix: It is prepared by calculat-
tages over traditional ANN: ing the following features:
(a) True positive
• No need for feature extraction by the patholo- (b) True negative
gists as the data extraction is automatic (c) False positive
• No technical knowledge of image recognition (d) False negative
or segmentation is needed 2. Receiver operating curve (ROC): More the
• Avoids bias of cell selection area under ROC, the more the ANN model is
• The large amount of data from the digital image perfect.
can be easily managed by CNN as there is a sig-
nificant reduction in the dimension of data.
0.1
0.3
0.2
Flattening of 0.4
pixels
0.1
0.5
0.3
0.1
Input image 0.4

Final image
(100 x 100) Convolution ANN
(4 x 4)
0.2
ReLU 0.5
Pooling 0.5
0.3
0.4
0.4
0.1
Fig. 25.5 A fully formed convolutional neural network is sional image. The pixel values are flattened and
shown. Repeated convolution, ReLU and pooling opera- conventional neural network is made
tion converts the image into a significantly low dimen-
25.5.3 Main Challenges of ANN cells in the body fluid and the other cytology
smear [5, 6].
The main challenges regarding the data of ANN 2. Classification of disease: First time commer-
are the following: cially ANN was used in PAPNET system to
identify the dysplastic cells. PAPNET system
1. Insufficient quantity of training data: If the
was a semi-automated system. It was equally
overall data set are less then ANN may not
effective in comparison to manual screening
work properly.
[7, 8].
2. Non-representative training data: If the
Subsequently, various studies have
data does not properly represent the problem,
shown the high-performance rate of ANN
then the ANN model may not work. Such as,
to classify breast tumours, and thyroid
if we want to predict tumour metastasis, then
tumours [9, 10].
probably mitotic index and grade of tumour
With the help of the deep learning tech-
are two important variables which should be
nique, the areas of ductal carcinoma in situ
included in the data.
were identified in the hematoxylin and
3. Poor quality training data: If there are too
eosin stained histopathology section of the
many outliers (too low or too high), then the
whole slide imaging of breast tumours
model will not function properly.
[11].
4. Underfitting the training data: If there is
Similarly, the neoplastic region of the pan-
too much generalization and complexity of
creatic neuroendocrine tumour was identified
the problems are bypassed then the ANN
by deep learning neural network [12].
model will suffer due to underfitting of the
3. Grading of tumour: Mitotic index and Ki67
training data.
index measurement can be done with the help
5. Overfitting the training data: If the data is too
of ANN [13]. These parameters are used for
much limited and fewer parameters are selected
grading and prognosis of various tumours.
then again ANN model will give an error.
Deep neural networks have been used suc-
cessfully to grade prostatic carcinoma in core
biopsy of the prostate [14].
25.5.4 Application of ANN 4. Management and prognosis of diseases:
CNN is applied to identify the prognostic risk
ANN is used in the following areas:
factors of various malignancies [15–17].
• The complex problems for which there is no
good solution by the traditional approach
• To solve the problems the traditional approach 25.5.5 Limitations of ANN
needs a lot of hand training
• An acceptable solution to the problem offers • Power of the computer: The available disk
great savings in human and/or economic space and the speed of the computer is really a
terms great challenge. The average disk space occu-
• To get insights into the complex problems and pied by a single whole slide scanning image
to handle a large amount of data takes 2 to 3 Gb of memory. So, we need huge
disk space and also rapid processing speed of
25.5.4.1 Specific Applications of ANN the computer.
In pathology the commonly used areas of appli- • Black box: The data processing in the hidden
cation of ANN involve in: node layer is still an enigma. We do not have
any control over this layer.
1. Identification of malignant cells: ANN was
• Medicolegal issue: The ANN may do mis-
used successfully to identify the malignant
takes in decisions and it may create medico-
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Part IV
Microscopy, Quality Control and
Laboratory Organization
Compound Light Microscope
and Other Different Microscopes 26
26.1 Light
Light is an electromagnetic wave with a specific

amplitude and wavelength. The distance between
the two successive peaks of the light wave indi-
cates the wavelength of the light. The distance
between the crest to the horizontal axis is known
as amplitude. The time to travel from one crest to
successive next crest is known as a period. The
frequency of light waves is indicated by the num-
ber of the wave cycle per unit of time (Fig. 26.1).
The visible light remains within the wavelength
of 400 to 750 nanometers. This is a very tiny frac-
tion of the electromagnetic spectrum (the radio
waves are 104 m and ʏ rays 10−10 m). The wave-
length of the red light is 750 nm (1 nm = 10−9 m)
and that of blue light is 400 nm (Fig. 26.2).
Ordinary white light is comprised of all the Fig. 26.1 Schematic diagram showing a wave of light.
colours. The dominant wavelength of the source The distance between the two successive peaks of the
of light determines its colour. light wave indicates the wavelength of the light
https://doi.org/10.1007/978-981-19-6616-3_26
280 26 Compound Light Microscope and Other Different Microscopes
26.3 Image Generation

and Human Vision
The eye is a biological camera. The image of

the object is formed on the retina of the eye
(Fig. 26.3). At first, the light rays from the object
pass through the cornea, aqueous humor, lens and
vitreous humor and finally the light stimulates
the photoreceptor cells of the retina. The initial
Fig. 26.2 Visible spectrum of light is 4oo nm to 750 nm image in the retina is an inverted image of the
wavelength of light object. The photoreceptor cells of the retina pass
the electrical signal through the optic nerve to the
brain. The human brain corrects the inverted
26.2 Colours image to the normal erect image.
Image formation by the simple lens: The
There are different modules to express the colour. image formed in the retina of the human eye is
Hue saturation intensity (HSI): This is one non-magnified. The image of the object can be
of the common modules to express colour. Hue magnified if a convex lens is placed between the
indicates the basic colour such as blue, green or object and eye and appropriately focused
red. The saturation means how deep is the colour (Fig. 26.4). If the object is placed within the
which means whether the colour is pale or dark. focal length of the lens then we see a virtual
Intensity measures the brightness of the colour. enlarged image formed on the same side of the
Red-green blue (RGB): There are three pri- lens. The focal length of the lens is defined as
mary colours: red, green and blue. The other the distance between the optical centre of the
colours that we perceive are the mixture of these lens and the focal centre where the image is per-
colours such as an admixture of red and green fectly focused.
colour will produce a yellow colour. The percent- Light microscope [1]: The light microscope
age expression of these three primary colours deals with the visible light and so it is named as a
represents the colour of that particular object. light microscope. The predominant three func-
CYM: The subtraction of one or more pri- tions of this microscope are:
mary colours from the white light produces three
other secondary colours: cyan (C), yellow (Y),
and magenta (M). Therefore this CYM colour
model is known as primary subtractive colours.
This model is mainly used in optical filters and
printing.
Perception of colour: Cone cells are the pho-
toreceptor cells of the retina. They are exclusively
related to colour perception and consist of only
5% cells. The cone cells are mostly concentrated
in the fovea centralis of the retina. We perceive
white colour when the light equally stimulates all
the three types of cone cells that means red, green Fig. 26.3 Image formation in the human eye is high-
and blue. If the light stimulates only the red- lighted in this schematic diagram. The image of the object
sensitive pigments of the cone cells then we per- is formed on the retina of the eye. The initial image in the
retina is an inverted image of the object. The photorecep-
ceive red colour. Similarly, if the light stimulates
tor cells of the retina pass the electrical signal through the
both red and blue pigments of the cone cells then optic nerve to the brain. The human brain corrects the
we perceive a magenta colour. inverted image to the normal erect image
26.3 Image Generation and Human Vision 281
these two objects and they may look like one

object. The minimum distance where the two
objects can be distinguished separately is known
as the resolution capacity (D) of the microscope.
The resolution of the microscope is dependent on
two factors:
1. The wavelength of the light

2. The maximum angle of light that can be
obtained by the objective lens from the object.
Fig. 26.4 Schematic diagram showing the formation of The numerical aperture (Fig. 26.5): Numerical
image by a convex lens. If the object is placed within the
aperture (NA) is related to the light-gathering
focal length of the lens then we see a virtual enlarged
image formed on the same side of the lens power of the objective. In fact, the resolution
power of the microscope is largely dependent on
• Magnification: the NA of the objective and it is represented by
• Resolution the equation below:
• Contrast 0.61 σ
D=
NA
Magnification: The word magnification indi-
cates the enlargement of the image of the object D = resolution.
of interest. The objective and the eyepiece take Sigma (σ) = the wavelength of light.
part in the magnification of the image of the NA = Numerical aperture.
object. The first magnification takes place by the The numerical aperture is calculated as.
objective. The power of magnification is written
on the wall of the objectives of the microscope. Table 26.1 Magnification of the microscope using dif-
ferent objectives
Normally the power of magnification varies from
4 times to 100 times in an ordinary biological Objectives Eye piece Final magnification
light microscope. The second magnification is 2× 10× 20×
4× 10× 40×
done by the eyepiece and so the final magnifica-
10× 10× 100×
tion is equal to the first magnification done by the
20× 10× 200×
objective multiplied by the second magnification 40× 10× 400×
done by the eyepiece. 100× 10× 1000×
M = M1 × M 2
M = Final magnification.
M1 = Linear magnification by objetives.
M2 = Linear magnification by eyepiece.
The final magnification of the different objec-
tives is shown in Table 26.1.
Resolution: The term resolution means the
ability of the microscope to distinguish two
closely spaced objects. If we put two objects at a
distance then we are immediately able to identify
them as two separate entities. However, more and
more they are kept close together it is difficult to
Fig. 26.5 Numerical aperture of the microscope is calcu-
recognize them as two separate entities. At a cer- lated as mentioned in the diagram. The numerical aperture
tain distance, it may be impossible to distinguish is related to the light-gathering power of the objective
NA n sin
Box 26.1 Image Formation in Microscope
n = refractive index of air (It is 1). • First Image is formed by the magnifica-
μ = half of the angular aperture of the tion by the objective
objective. • First Image:
Contrast: The contrast of the microscope –– Real
means the ability to detect an object from the –– Magnified away from the lens in the
background material. If we want to know the same side
details of an object then we need to have the dif- –– Formed in the body of the
ference in intensity of the colour between the microscope
object and the background material. • Second image is formed by the eye
piece which is the magnification of the
first image
26.3.1 Image Formation by the Light • Second image
Microscope (Fig. 26.6) –– Virtual
–– Highly magnified
The compound light microscope works in the –– Formed 25 cm away from the eye
same way as described in the magnification by –– Location: Between the stage and
the convex lens (Box 26.1). The microscope is condenser
composed of bunches of properly placed lens that
magnifies the image of the object in several folds.
There are two sets of lenses: objectives and the
eyepiece. The object remains in between the con-
denser and the objective. The condenser con-
denses light through the object. The lens of the
objective has a short focal length and generates a
magnified real image within the body tube of the
microscope. Subsequently, the lens in the eye-
piece further magnifies this real image and a vir-
tual more magnified image is formed at
approximately 25 cm distance from the eye on
the same side of the lens near the object in the
stage.
26.3.1.1 Anatomical Components

of a Light Microscope
(Fig. 26.7)
Base: The base is the supporting platform of the
microscope.
Stage: The stage is the moveable component
of the microscope. The slide is placed on the
stage and then the stage is moved up and down to
focus the object. The slide on the stage is also
Fig. 26.6 Schematic diagram showing the formation of moved in different directions.
the image by the light microscope. The lens of the objec- Coarse and fine adjustment knobs: These
tive has a short focal length and generates a magnified real are two knobs placed on both sides of the body of
image within the body tube of the microscope.
the microscope. These knobs are rotated and help
Subsequently lens in the eyepiece further magnifies this
real image and a virtual more magnified image is formed in focusing the objectives by moving them up and
in the same side of the lens near the object in the stage down.
26.4 Optical Components 283
Fig. 26.8 Objective of the compound microscope. Note

the basic information engraved on it
once the objective is changed then the posi-

Fig. 26.7 Simple light microscope and its parts tion of the condenser has to be readjusted.
2. Objectives: Each microscope contains a set
Tube: The tube is the connecting part between of multiple objectives attached to the rotating
the objectives and eyepiece. nose piece. The circular ring of the objective
Arm: The arm connects the base of the micro- represents the magnification power of the
scope with the tube. objective and the different objectives have a
Revolving nose piece: The various objectives different coloured ring on them (Fig. 26.8).
are placed in the revolving nose piece. The various information such as lateral mag-
Diaphragm: The diaphragm is located below nification, numerical aperture etc. is engraved
the stage and helps to control the amount of light on the objective. Presently the objectives are
on the object. designed as infinitely corrected. The follow-
Light source: The source of light is located ing are the different types of objectives:
on the base of the microscope, the light travels (a) Achromat: The achromatic objectives
directly from the source to the object and then contain two doublets and a single front
through a different lens. lens They are relatively cheaper and quite
suitable for low magnification images.
However, they are not very satisfactory
26.4 Optical Components [2, 3] for higher magnification.
(b) Fluorite or semiapochromat lens:
The following parts are the constituents of the Fluorite type objective lenses are made of
optical part of the light microscope: fluorspar or newer synthetic substitutes
and provide a good level of colour correc-
1. Condensers: The condenser of the micro- tion. They are highly transparent and give
scope accumulates light from its source and high contrast. Therefore this type of
then concentrates the light on the object as a objective is suitable for immunofluores-
cone of light so that the maximum light passes cence microscopy and polarized
through the object to get a better resolution. microscopy.
The object is illuminated by a parallel beam (c) Apochromatic: These are relatively
of light with uniform intensity. The condenser costly lenses and give very good correc-
aperture diaphragm helps in controlling the tion. The apochromatic objectives are the
diameter of the light beam. With the help of a most suitable for colour photography. The
screw, the condenser can be adjusted for the chromatic aberration is nearly zero in
proper focusing of the cone of light. However, such a lens.
Spherical aberration (Fig. 26.9b):

a The spherical aberrations occurs when
the spherical lens is used for magnifica-
tion in the microscope. The cause of
spherical aberrations is the different
bending capacities of the central and
peripheral parts of the spherical lens. The
incident rays of light that pass through
the peripheral part of the lens bend more
than the incident rays that pass through
b the centre of the lens. Therefore in the
case of spherical aberration, the images
are formed in different focal planes and
the image may be blurred in the
periphery.
Solution: The solution to spherical
aberrations is to apply a collection of dif-
ferent thicknesses of the positive and neg-
ative lens.
Fig. 26.9 (a) Principle of chromatic aberrations is Astigmatism: This is an off-axis aber-
explained in this schematic diagram. The chromatic aberration and it occurs due to a defect in the
ration occur due to the different refraction of different manufacture of the proper curvature of
wave length of light. (b) Principle of spherical aberrations
is explained in this schematic diagram.. This is due to dif-
the lens or defective placement of the
ferent bending capacity of the central and peripheral part lens. Here the rays of light from the object
of the spherical lens going through the horizontal and vertical
diameters of the lens focus in two sepa-
The Major Aberrations of the Lens rate focal planes. Astigmatism increases
The spherical lens of the microscope when the object is more away from the
may have several optical faults or aberra- optic axis.
tions. Image distortion occurs due to Solution: Good quality lens and
these various optical aberrations. The proper placement of the lens.
common aberrations are: The curvature of field: In this aberra-
Chromatic aberration (Fig. 26.9a): tion, the image is curved instead of flat
The chromatic aberration occurs due to the and the whole image can not be focused
different refraction of the different wave- in a single flat plane.
lengths of light. The blue light is bent more Solution: The curvature of the field
than the red light. Therefore there are the can be corrected by proper designing of
different locations of images for the differ- the lens of the objective.
ent wavelengths of the colour and overall 3. Eyepieceece: The eyepiece or the ocular lens
the image becomes distorted. More disas- magnifies the real image produced by the
trous is the different magnification of the objective in the body tube of the microscope.
different colours of images as they are The ocular lens generates the final image
located at different distances from the lens. which is a virtual image 25 cm away from the
Solution: The solution to the chro- eye. The specification of the ocular lens is
matic aberration is to use the different usually engraved on the lens barrel. Nowadays
components of the lens with different dis- most eyepieces provide magnification in the
persing properties for the different colours range of X 10 to X 20. The ocular lens or eye-
so that the final images overlap in the piece can be focusable or non-focussable.
same location. Eyepieces may be of two types:
26.6 Other Types of Microscope 285
Negative eyepiece: It contains an internal

diaphragm between the lens of the occularis Box 26.2 Basic Care of Microscope
(eyepiece). The simple negative eyepiece is also • Plugging: Connect the plug in the rec-
known as the Huygenian eyepiece which is ommended electrical power supply
commonly used in the routine light microscope. • Bulb: Always put off the bulb when you
Positive eyepiece: It contains an internal do not use the microscope. Remember
diaphragm below the lens of the occularis that the bulb is expensive
(eyepiece). This type of eyepiece is also called • Focus:
Ramsden eyepiece. –– Do not hurry
–– Gently focus your object and take
care that the objective should not
26.5 How to Take Care and Handle touch the slide
Your Microscope –– Always use lowest power of objec-
tive first
The basic care of the microscope is highlighted in • Cleaning: Clean the lens before and
Box 26.2. after you use. Clean only by lens paper.
Do not use distilled water or any sorts
• Starting: Connect the plug of the microscope of organic solvent
with recommended electrical power supply. • Transport:
• Focus: Focus gently and do not be fast. During –– Grasp the arm of microscope.
the focusing of the object please take care so that –– Keep the microscope parallel to
the objectives do not touch the slide and break it. ground
• Bulb: The bulb of the microscope is costly so –– Keep it close to your body
please turn off the illumination when the • Damage: Never try to repair any dam-
microscope is not used. age. Report the authority
• Transport: Grasp the arm of the micro- • After finishing work:
scope with one hand and support the base –– Unplug
with another hand. Keep the base of the –– Do not put any slide on stage. Clean
microscope parallel to the ground so that the the stage.
optical systems are not displaced or fall –– Adjust the nosepiece for the lowest
down. Never swing a microscope. power objective
• Touch: Never touch the lens of the microscope –– Cover the microscope to protect it
by hand or any body parts because the normally from dust
producing oil in the body may blur the lens.
• Cleaning: Clean the lens of the microscope only
with lens paper. Do not use any toilet paper or
towel paper as they may scratch the lens. Do not 26.6 Other Types of Microscope
use distilled water or any sort of organic solvent
as they may dissolve the coating of the lens. It is 26.6.1 Darkfield Microscope
important to note that excessive cleaning may be
harmful to the lens. The best way to clean a lens In this type of microscope, a hollow beam of light
is using compressed air. is produced by blocking the central part of the
• Closing the work: After finishing the work beam of light (Fig. 26.10). The scattered light
with the microscope please take out the slide from the object passes through the objective to
from the stage, keep the low power objective the eye and the object is seen as bright in a dark
towards the stage and far away from the stage, background. The ordinary light microscope can
disconnect the electric power source and cover be used as a darkfield microscope by using a dark
the microscope by dust cover. field block condenser. The central cylinder of
26.6.3 Phase-contrast Microscope
Principle: In this microscope, the objects having

different refractive indices are identified as they
produce different contrast. The object with scat-
tered light is identified from the illuminating
background light. If the light passes through a
transparent object then due to the change of
refractive index of the object the pathway of light
will be deviated slightly and the light wave is
retarded. In the case of denser particles (higher
refractive index) in the object, the deviation of
Fig. 26.10 Schematic diagram showing pathway of light
light will be more and the light wave is more
in the dark field microscope. A hollow beam of light is retarded. This is known as phase difference.
produced by blocking the central part of the beam of light Usually, these phase differences are invisible to
us, however, the phase-contrast microscope
light is obstructed whereas the peripheral rim of makes these changes significantly visible.
light reaches the object. System (Fig. 26.11): In a Phase contrast micro-
scope a condenser annulus and modified objective
26.6.1.1 Use with a phase plate are used. An intense beam of the
• The bacteria, spirochetes or fungi in sus- light source is passed through the substage con-
pension are better seen in the darkfield denser annulus which is located in the focal plane
microscope. of the condenser. So a hollow cone of light is gen-
• Movement of the cells in the culture medium erated that can be controlled. Now, this light passes
is better seen in this microscope. through the objects/ sample and they are either
deviated or undeviated depending on the refractive
indices of the different structures of the object.
26.6.2 Bright-Field Microscope Now both undeviated (central ray) and deviated
light passes through the objective and are segre-
This is the simple light microscope where the gated by the phase plate that is located behind the
object is examined by attenuated light. No addi- focal plane of the objective.
tional equipment is required for the bright field
microscopy. The diaphragm of the microscope 26.6.3.1 Applications
should be fully opened and light intensity should • This is used to see the living cells: shape, size
be kept low. The light passes through the sample etc.
and the denser area of the object absorbs part of • Unstained protozoa or fungi are best seen in
the light. So the object looked dark in a bright this microscope
background. • Motility of the organism
Use: Staine or natural specimen.
Advantages
• Very simple 26.6.4 Inverted Microscope
• No additional equipment is needed
• Object can be seen without staining An inverted microscope is a type of microscope
Limitations where the light source is on the top and the objec-
• Low contrast tive is below the sample (Fig. 26.12).
• Low resolution How it works: The basic principle of the inverted
• Low magnification microscope is the same as that of the conventional
26.6 Other Types of Microscope 287
Fig. 26.12 Schematic diagram showing the arrangement

of the light source and objectives in the inverted
microscope
26.6.5 Dissecting Microscope

Fig. 26.11 Schematic diagram showing pathway of light
in the phase contrast microscope. Here a condenser annu-
lus and modified objective with a phase plate are used. A A dissecting microscope is also known as a ste-
hollow cone of light is generated that passes through the reo microscope. It is a low power microscope.
objects and the lights are either deviated or undeviated
Here the light is reflected from the stage where
depending on the refractive indices of the different struc-
tures of the object. The light passes through the objective the specimen or sample is kept.
and is further segregated by the phase plate behind the
focal plane of the objective
26.6.5.1 Principle
In the case of dissecting microscope, two light
light microscope. However, here the sample is
sources are used: one from below on the stage
fixed and the objective moves. So it is the best fit to
and another from the top, above the specimen.
study the material where the sample size is large.
There are two magnification systems, one is
Applications
fixed magnification which is composed of two
1. It is mainly applied to study the live cells objectives and the other one is zoom magnifica-
where the cells in the culture are stuck to the tion which provides continuous magnification
bottom of the container e,g flask or petri dish. (Fig. 26.13).
2. Fungal culture to study Table 26.2 highlights the comparison between
3. Microscopic study of the drug sensitivity assay the dissecting and compound light microscope.
26.6.5.2 Applications
• To do microsurgery
• To dissect the parasite
• To study the large solid particles
• To make watches or circuit board
• To use in forensic laboratory
References
1. Wollman AJ, Nudd R, Hedlund EG, Leake MC. From
Animaculum to single molecules: 300 years of the
light microscope. Open Biol. 2015;5(4):150019.
2. Wolf DE. The optics of microscope image formation.
Methods Cell Biol. 2003;72:11–43.
3. Goodwin PC. A primer on the fundamental prin-
ciples of light microscopy: Optimizing magnifica-
tion, resolution, and contrast. Mol Reprod Dev.
2015;82(7–8):502–7.
Fig. 26.13 Schematic diagram showing the arrangement

of the light source and objectives in the dissecting
microscope
Table 26.2 Comparison between the dissecting and

compound light microscope
Dissecting Compound
microscope microscope
Objectives Two One
Light source Two One
Imaging Light is Light is transmitted
reflected trough the object
from the
object
Image Three Two dimensional
dimensional
Magnification Low: 5X to High: 40× to 1000×
50X
Fluorescence Microscope, Confocal
Microscope and Other Advanced 27
Microscopes: Basic Principles
and Applications in Pathology
Fluorescence microscopy applies high-intensity therefore vibrates at a lower frequency. So the

light to illuminate the substance that emits fluo- released fluorescent light has a longer wavelength
rescence light. The light of a shorter wavelength than the excitation light.
strikes the fluorescent object and the object emits Basic steps of Fluorescence microscopy:
the light of a longer wavelength and the image of The basic steps of fluorescence microscopy are
the object is visualized by the observer. highlighted below:
Principles of fluorescence [1, 2] (Fig. 27.1):
When a fluorescent molecule in the ground state • The high energy light beam is passed through
absorbs a photon of excitation light the electron the filter and is directed to the object which is
of its outer shell jumps to the next orbit and the stained by a fluorescent dye.
molecule changes from the ground state energy • The fluorescent dye absorbs high energy light
level to the excitation state energy level. However, and immediately releases photons of low
within a fraction of a second very rapidly the energy light.
molecule releases the photon of light and returns • The emitted light is passed through the filter
to the ground state again. In this whole process, and is detected by the observer against a high
the molecule losses some amount of energy and contrast black background.
https://doi.org/10.1007/978-981-19-6616-3_27
290 27 Fluorescence Microscope, Confocal Microscope and Other Advanced Microscopes: Basic Principles…
Fig. 27.1 Schematic

diagram showing the
principle of fluorescent
dye
27.1 Transmitted Fluorescent only the emitted fluorescence light to pass through
Microscope it to the observer.
Selective filter: This type of filter specifically
In the case of the transmitted fluorescent micro- allows only the desired excitation beam of light.
scope, we use a mercury or xenon gas lamp for the Careful selection of the filter is necessary for the
high energy excitation beam of light (Fig. 27.2). selection of the wavelength of light nearer to the
These burners contain gas with high pressure and excitation maximum of the particular fluorescent
therefore careful handling is needed for these light dye.
sources. The beam of light generated by the burner Barrier filter: This filter is placed between
passes through the heat-absorbing filter followed the objective and the eyepiece. This type of filter
by the red light stop filter and wavelength selective prevents the passage of light of a short wave-
filter. Now the beam of light hits the object and the length and helps to protect the retina. However, a
excitation beam passes through the objective barrier filter allows the emitted fluorescent light
towards the barrier filter. The barrier filter allows of a longer wavelength.
27.2 Incident Fluorescent Microscope 291
Fig. 27.3 Schematic diagram showing the principle of an

incident fluorescent microscope. In this type of micro-
scope a dichroic mirror is used that allows the selected
excitation beam of light to be reflected on the object
Similarly, the emitted fluorescent light from the object is
allowed to pass through the mirror to the eyepiece
Fig. 27.2 Schematic diagram showing the principle of
transmitted fluorescent microscope. The beam of light tive and eyepiece is located and protects the eye
passes through the multiple filters and ultimately hits the from any high energy light.
object. The excitation beam passes through the objective The advantages of the incident fluorescent
towards the barrier filter and reaches to the observer
microscope:
• The dichroic mirror helps to gather most of

27.2 Incident Fluorescent the excitation beam and also reflects a major
Microscope part of emitted fluorescence. Therefore, the
image is much brighter in this type of
In this type of microscope, a dichroic mirror is microscope.
used (Fig. 27.3). This dichroic mirror has certain • Different types of objectives can be used in
unique properties. The mirror only allows the this microscope.
selected excitation beam of light to be reflected
on the object and the remainder unwanted beam
of light is travelled through the mirror and is lost. 27.2.1 The Dye Used in Fluorescence
Similarly, the emitted fluorescent light from the Microscope
object is allowed to pass through the mirror to the
eyepiece. Therefore, in the incident fluorescent The common dyes used in fluorescence micros-
microscope, the high energy light passes through copy include:
the various filters and then with the help of a
dichroic mirror the selected wavelength of the • DAPI
light is allowed to pass through the objective to • FITC
the object. The emitted fluorescent light from the • Hoechst 33342
object again passes through the dichroic mirror to • Rhodamine
the eyepiece. A barrier filter in between the objec- • Texas red Cy3
Table 27.1 Maximum excitation and emission spectra of

different fluorochrome dye
Maximum Maximum
excitation emission
Fluorochrome dye wavelength (nm) wavelength (nm)
Fluorescein 494 520
Isothiocyanate
(FITC)
Hoechst 33342 346 460
DAPI 359 461
Rhodamine 570 590
Cy3 548 561
Texas red 595 613
These fluorochrome dyes are tagged with specific

antibodies or molecular probes. Each fluoro- Fig. 27.4 Comparison of light scattering in a conven-
tional and confocal microscope. Light from the focal
chrome dye has a specific excitation spectrum
plane reaches the observer in the case of a confocal
which means the dye is excited by the light of a microscope
specific wavelength which is also known as max-
imum excitation wavelength. Similarly, the dye
also emits a photon of a specific higher wave- ventional light microscope when the lens (objec-
length which is known as maximum emission tive) is focused on a specific point of the object,
wavelength. Table 27.1 shows the maximum the light comes from the entire depth of the
excitation and emission spectra of different fluo- object. Therefore, in the fluorescent microscope,
rochrome dyes. we get light from above or below the focal plane.
The image becomes blurred. In contrast, in CFM
at one time we see the image of the particular
27.2.2 Applications of Fluorescence depth of the object at a small point. All the out of
Microscope focus light is eliminated by passing the light
through the pinhole (Fig. 27.4). Multiple images
1. The fluorescent microscope helps to study the at different depths are accumulated and then
specific component of the cell or object of reconstructed to provide a three-dimensional
importance. image.
2. DNA and RNA sequencing. Principle (Fig. 27.5): In CFM the high-
3. To study the chromosomal abnormalities with intensity laser light is directed to the object with
the help of the Fluorescent in situ hybridiza- the help of a dichroic mirror. The light hits the
tion (FISH) technique. object and the emitted fluorescence from the
4. Biomolecular assay fluorochrome stained object passes through the
5. Gene expression along with the location of dichroic mirror to the confocal pinhole to the
the proteins in the living cell by green fluores- photomultiplier detector. The confocal pinhole
cence protein (GFP). aperture omits all the out of focus light from the
upper and lower plane of the focus. It only
allows passing light that comes through the
27.3 Confocal Microscopy focal plane. Now multiple images from the dif-
ferent focal planes are collected by the com-
Confocal microscopy (CFM) provides three- puter software and finally the three-dimensional
dimensional optical resolution. The principle of image is constructed.
image formation in CFM is different from the Components of the confocal microscope:
ordinary light microscope. In the case of the con- The major components of the CFM include:
27.3 Confocal Microscopy 293
Fig. 27.6 Two types of scan occur in the confocal micro-

scope: line scan and frame scan
4. Signal detector: This is a photomultiplier

tube (PMT) that converts light energy into
electrical energy. The light hits the phosphor
and releases electrons. The released electrons
are collected and stored as a signal. The sig-
nals are finally displayed on a video monitor.
There may be multiple PMT for the signal
processing of simultaneously different fluoro-
Fig. 27.5 Schematic diagram showing the principle of a
confocal microscope. The confocal pinhole aperture chrome dyes.
allows passing light that comes through the focal plane 5. Computer with appropriate software: The
computer is an essential component of CFM
1. Light source: Usually high power laser beam and has the following functions: image
is generated from a laser or a mercury arc detection, image processing, image recon-
lamp. struction, image storage and video display of
2. Microscope: It has the X-Y axis controller the image.
along with Z axis stepper
3. Scan head: This consists of a confocal aper-
ture, shutter, scanning mirror and filter wheels. 27.3.1 Advantages (Box 27.1)
The scan occurs (a) Line scan: This occurs
horizontally on the X and Y-axis. This is usu- 1. It helps to assess the spatial distribution of
ally very fast, (b) Frame scan: It occurs verti- various intra and extracellular macromole-
cally on Z-axis. This is a slow scan process cules of the cells [3].
(Fig. 27.6). The scan mirror helps to scan light 2. It provides high resolution and high-contrast
in the raster pattern. It helps to achieve a bet- clear images.
ter temporal resolution. The confocal aperture 3. CFM can work on living tissue and the fixa-
is another important component of the scan tion of tissue is not required for the study of
head. There are two types of confocal aperture confocal fluorescence imaging.
present: pinhole type and slit type. The differ- 4. Thick tissue section can be studied by CFM
ent sizes of aperture are available and the user [4].
can change them either manually or with the 5. Reconstruction of three-dimensional images
help of software. The barrier filter in the scan is possible.
head prevents any unwanted light to allow to 6. Images are free from artefacts seen in an ordi-
pass to the detector (Fig. 27.5). nary microscope.
Table 27.2 Applications of confocal microscopy in

Box 27.1 Advantages of Confocal pathology
Microscope Area Applications
• Spatial distribution of intracellular and Study of • Possible to study single or
intercellular substances fluorochrome multiple fluorescent dye
stained section stained sample
• High contrast, high resolution images • Fluorescence recovery after
• Live images possible photobleaching (FRAP) is
• 3D reconstruction possible
• Free from artifacts Co-localization • Possible to assess the exact
localization of two closely
• Thick tissue can be studied
situated tissue
Green • Possible to track the cell
fluorescence signalling pathway,
protein (GFP) intracellular trafficking and
also gene expression in the
27.4 Limitations of CFM cells
Epitope tagging • Possible to track the epitope of
CFM has the following limitations: the antigen by GFP
Diagnosis • Screening of the colorectal
1. Very expensive cancer
• Measurement of corneal
2. Photobleaching technique is not very good
thickness
compared to a conventional microscope Functions of • Organelles specific fluorescent
3. Depth of penetration of CFM in the live image cytoplasmic probes help to study the
is limited organelles functions of cytoplasmic
organelles
Nucleus • Spatial distribution of the
different genes
27.5 Applications of CFM [5–7] • Relative position of the
chromosomal parts
Table 27.2 highlights the biological applications Morphometry • Three-dimensional structure of
of CFM. the tissue
• Four-dimensional images of
the tissue
1. Study of fluorochrome stained section: Microcirculation • Velocity of the blood
Single or multiple fluorescent dye stained • Distribution of various agents
samples can be studied by CFM. It can detect in the microvessels
simultaneously many fluorochrome dyes.
Moreover, fluorescence recovery after photo- lular trafficking and also gene expression in
bleaching (FRAP) is possible in CFM. the cells.
2. The detection of co-localization: CFM is 4. Epitope tagging: CFM is helpful in the track-
helpful to determine the exact localization of ing of the epitope of the antigen by tagging it
two closely situated tissue. It provides a blur- with green fluorescence protein (GFP).
free very good resolution of fluorescently 5. Diagnosis: Screening of colorectal cancer can
stained samples. be done with the help of CFM etc. It also
3. Green fluorescence protein (GFP): CFM helps to measure the corneal thickening.
can help to track the distribution and function 6. Functions of cytoplasmic organelles:
of the protein by tagging it with GFP. It helps Organelles specific fluorescent probes are
in tracking cell signalling pathways, intracel- helpful to study the function of various cellu-
27.6 Two-Photon Microscopy 295
lar organelles such as mitochondria, endo- to excite the fluorophore (Fig. 27.7). The com-
plasmic reticulum, Golgi bodies etc. bined energy of two photons is optimum to
7. Nucleus: CFM helps to study the detailed excite the fluorophores. The excitation of the
spatial distribution of the different genes with fluorophores by the two photons is the highest
the help of fluorescent in situ hybridization. It in the focal plane as the photon flux is maxi-
also helps to study the relative position of the mum in the focal plane. The fluorophores above
chromosomal parts like telomere, kineto- and below the focal plane are not excited. No
chores etc. pinhole is needed like CFM because the light
8. Morphometry: Three-dimensional structure from only that thin focal plane is emitted. In a
of the tissue can be studied by CFM. Even two-photon microscope pulsed infrared laser
reconstruction of four-dimensional images beam is used to illuminate the object at a par-
(time considered as fourth dimension) is pos- ticular focal plane [8].
sible with the help of GFP.
9. Microcirculation: CFM helps in the assess-
ment of blood circulation in small vessels 27.6.1 Advantages
such as the velocity of the blood and also the
distribution of various agents in the microves- 1. Very good for live-cell imaging as there is no
sels of the tissue. photo-damage of the living cell.
2. This microscope has the capability of resolu-
tion to several hundred microns and helps to
27.6 Two-Photon Microscopy study 1 to 0.5 μ thin sections of tissue without
any physical sectioning.
This is a three-dimensional imaging micro- 3. Better penetration of the infrared excitation
scope. The basic principle of two-photon beam of light.
microscopy is selective excitation of the fluoro- 4. The desired plane of the section can be stud-
phore in a particular focal plane which means ied without wasting any tissue and therefore
non-linear excitation of the fluorophores. Unlike very effective in small biopsy material.
a conventional fluorescence microscope, where 5. The microscope eliminates any contamina-
a single photon is absorbed by a fluorophore, in tion of fluorescence signal from up and down
a two-photon microscope, two photons with the plane of the focus and therefore produces
half energy and double the wavelength are used a very good high sensitivity image.
Fig. 27.7 The detailed principles of two-photon micros- excites the fluorophore (see lower two figures). The laser
copy are explained in this schematic diagram. Instead of a beam can only excite the fluorophores in the focal plane
single photon, here two photons containing half of the (see upper left figure). The fluorescent light from the focal
energy are used. The combined energy of the two photons plane only selectively comes out
27.8 Spatially Modulated Illumination Microscopy 297
27.7 4Pi Microscopy 27.8 Spatially Modulated

Illumination Microscopy
This is a specially equipped microscope that has
a significantly improved capability of axial reso- This type of microscope exploits the “Moire
lution. Increasing the angular aperture of the effect”. When two densely packed images are
objective helps to increase the resolution of the placed together a new pattern develops. By
microscope. In 4Pi Microscope, the two identical manipulating the illumination it is possible to
objectives are used on both sides of the sample so reveal the exact image. The series of images with
that the effective angular aperture becomes two mildly changed illumination patterns are col-
fold [9] (Fig. 27.8). The increased angular aper- lected and from these images, with the help of
ture increases the resolution in the Z-axis of the computation the underlying structure is revealed
microscope (200 nano microns). [10]. This spatially modulated illumination may
be added in an epi-fluorescence microscope.
The Table 27.3 shows the comparison of dif-
ferent types of advanced microscopy.
27.8.1 Scanning Probe Microscope
A scanning probe microscope is an advanced

microscopy that helps to visualize the cellular
structures at the molecular levels. Instead of
using light or electrons, this microscope applies a
probe to visualize the structures. The probes
directly interact with the surface structure and the
images are constructed from the information cre-
ated by the probe and the structures of the surface
of the cells. With the help of this microscope, we
may have 10 cores times magnification of the
structures. There are two types of scanning probe
microscope: the scanning tunnelling microscope
and the atomic force microscope.
27.8.1.1 Scanning Tunnelling

Microscope (STM) [11]
The STM works by the principle of quantum tun-
nelling. Here the electron is applied as a wave to
Fig. 27.8 Two identical objectives placed both sides of
the sample in case of 4Pi microscope form a quantum tunnelling (Fig. 27.9).
Table 27.3 Comparison of different types of advanced microscopy

Microscope Principle Advantages Limitations
Confocal A pinhole is used to allow light from Image of the particular plane Limited depth of
microscope only a selected focal plane is seen and three-dimensional imaging is possible
reconstruction can be done near about 100 μ
Two-Photon Two photons with half energy and Optical sectioning of the Pulse laser is very
Microscopy double the wavelength are used to image is possible without any costly.
excite the fluorophore and light from physical interference
one particular focal plane
4Pi Microscopy Two identical objectives are used on Increased resolution of Z-axis It has limited
both sides of the sample to increase diffraction
the angular aperture of the objective
and therefore to double the resolution
in the Z-axis
Spatially Modulated The series of images with mildly Add on to epi-fluorescence It has limited
Illumination changed illumination patterns are microscope diffraction
Microscopy collected and from these images, the
exact image is computed
Fig. 27.9 Schematic

diagram of tunnelling
microscopy
In STM the probe is travelled horizontally nano micron level. Both the dead and live cells
over the surface of the sample. The tip of the can be studied by STM.
probe when comes close to the surface of the cell Atomic force microscope (AFM): In the case
then a bias voltage is applied between the cell and of AFM, a thin probe is moved over the surface at
the probe creating a quantum tunnel where the a constant height and due to the atomic forces of
electron flows freely. The tunnelling current is the surface material the probe moves up and
generated and is amplified and subsequently down. The variation in the height of the probes is
recorded to construct the image. recorded to build the images at the atomic level
With the help of STM, one can visualize the [12] (Fig. 27.10). AFM can demonstrate cell
individual atoms over the surface of the sample. membrane and its proteins, DNA, RNA, and lipid
STM can help to visualize the structure at the films.
27.8 Spatially Modulated Illumination Microscopy 299
27.8.1.2 Advantages of AFM • Very high signal to noise ratio

• The cells can be studied in their original • three-dimensional measurement is possible
condition with very high resolution
• No need to fix the cells • Different chemical and biological properties
• No staining needed can be studied
27.8.1.3 Limitations
• The information is limited on the surface of
the cell
• Low magnification scanning is not possible
• Incapable to scan large sample
27.8.2 Laser Capture Microdissection
Laser capture microdissection (LCM) is an

advanced microscopic technology by which we
can dissect the specific area of the tissue on the
slide with the help of LASER under direct visual-
ization [13]. The tissue is collected in a small
Eppendorf tube for further tests.
Fig. 27.10 Schematic diagram of atomic force microscopy Working principle (Fig. 27.11):
Fig. 27.11 Schematic diagram showing the basic principles of different laser capture microdissection techniques
1. Contact based with adhesive type • Immuno-LCM may affect the study of pro-
(a) The Thermolabile polymer film is put on teins that are attached to the antibody.
the tissue section placed over the glass • The isolation of the individual cells of differ-
slide ent subtypes may be time-consuming.
(b) The laser beam is combined with the
pathway of light of the microscope. 27.8.2.3 Applications
(c) The cells of interest are identified on the The dissected tissue can be analyzed for:
computer screen
(d) The ultraviolet pulsed laser beam melts • DNA methylation protocol
the polymer over the cells of interest. • Loss of heterozygosity analysis
(e) The polymer with the attached cells is • Different PCR technique
taken out from the tissue section. • Protein microarray
(f) The cells attached with the polymer are • Gel electrophoresis
collected in the Eppendorf tube for
molecular analysis.
2. Contact-free gravity-assisted microdissec- 27.8.3 Expression Microdissection
tion (GAM): In GAM, the tissue section is
inversely mounted and the cells are cut by a Expression microdissection (Xmd) is a com-
laser beam. Subsequently, the cells are pletely newer advanced microdissection of the
dropped on the collecting tube by the force of cells that is operator independent and the cells of
gravity. interest are dissected automatically [14].
3. Contact-free laser pressure catapulting
(LPC): In LPC, the cells are cut by the 27.8.3.1 Principle and Steps
focused laser. Subsequently, with the help of a (Fig. 27.12)
defocused laser, the plasma impulse of the • In xMD technique at first immunohistochem-
nearby area catapults the cells against gravity istry is done according to the identification
to the collecting tube. markers of the cells of interest (such as CK,
PCNA etc.).
27.8.2.1 Advantages • An ethylene-vinyl acetate (EVA) polymer film
• Simple and easy to perform is coated on the top of the slide which is not
• Fast technique to isolate the single cells covered with any coverslip.
• The cells retain its original morphology and • Now the laser is activated manually over the
the cellular constituents are not altered tissue surface.
• The cells can be visualized directly under the • The laser passes unattenuated and light energy
microscope at the time of dissection. is absorbed only where there is chromogen of
the immunostaining deposition.
27.8.2.2 Limitations • The specific immune-stained areas are heated
• Poor resolution of the cells as no coverslip can by light and the EVA film melts.
be applied. • The melted EVA film is attached with cells by
• The cells are dissected on the basis of the mor- thermoplastic bond.
phology of hematoxylin and eosin staining • The remaining cells in the tissue remain
and so they may not be correctly identified. unaffected.
References 301
Fig. 27.12 Schematic diagram showing the basic principles of expression microdissection
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1992;A9(12):2159–66.
10. Gustafsson MG. Nonlinear structured-illumination
microscopy: wide-field fluorescence imaging with
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5. Jensen E. Technical review: colocalization of antibod-
14. Hanson JC, Tangrea MA, Kim S, Armani MD, Pohida
ies using confocal microscopy. Anat Rec (Hoboken).
TJ, Bonner RF, Rodriguez-Canales J, Emmert-Buck
2014;297(2):183–7.
MR. Expression microdissection adapted to com-
6. Patel DV, McGhee CN. Quantitative analysis of
mercial laser dissection instruments. Nat Protoc.
in vivo confocal microscopy images: a review. Surv
2011;6(4):457–67.
Ophthalmol. 2013;58(5):466–75.
7. Nwaneshiudu A, Kuschal C, Sakamoto FH, Anderson
RR, Schwarzenberger K, Young RC. Introduction
Electron Microscopy: Principle,
Components, Optics and Specimen 28
Processing
The microscope magnifies the image of the object EM is an expensive, large, fixed instrument and
so that we can visualize the smallest particles. should be kept in a separate room. The differ-
The resolution power of the light microscope is ences between EM and light microscope are
limited. Visible light has a wavelength of 300 to highlighted in Table 28.1.
700 nm. A light microscope uses the visible spec- It uses a high energy electron beam to visual-
trum of light and so the maximum resolution ize the material under study [2]. The advantages
power of the light microscope is 0.2 μm of the electron beam as a probe have several
(Fig. 28.1). The improvement of the resolution advantages.
capacity of the microscope can only be improved
by reducing the wavelength of the light. 1. Electrons have a shorter wavelength and pro-
Ultraviolet ray has a wavelength of 100 to 300 nm vide a very high-resolution capacity
and the resolution power is improved to 0.1 μm. 2. It is easy to manipulate the electron beams
Long-time scientists tried to find out a probe that 3. Electron gives high brightness
has a much smaller wavelength. During the first 4. Electron beams interact strongly with matter
part of the twentieth century, the wave-like prop-
erty of the electron was demonstrated and subse- Essential components of the electron micro-
quently, this has been utilized in the electron scope (Fig. 28.2a and b): The main components
microscope (EM). The formula shows: of EM include:
l = h / mv
1. Electron source
λ is wavelength 2. Sample Illumination
h = 6.626 × 10−34 (Plank’s constant) 3. Objective lens
m = Mass 4. Intermediate and projector lenses
v = Speed of the electron 5. Detectors
Now increasing the speed of the electron we can
reduce the wavelength significantly and a 0.001 nm Electron source (Fig. 28.3): Electron gun gener-
wavelength of the electron can easily be achieved. ates the beam of electrons. It consists of a
Therefore, with the help of an electron as a probe,
we can improve the resolution up to 0.1 nm (1 −10 m). 1. Tungsten filament,
The electron microscope is an advanced type 2. Wehnelt cylinder (cathode shield), and
of microscope [1, 2]. Unlike a light microscope, 3. Anode plate.
https://doi.org/10.1007/978-981-19-6616-3_28
304 28 Electron Microscopy: Principle, Components, Optics and Specimen Processing
Fig. 28.1 Schematic

diagram shows the
resolution power of light
and electron microscope
Table 28.1 Comparison of Electron microscope and of electrons emerges from the small hole of the
light microscope (Wehnelt cylinder) cathode shield.
Characteristic Light Sample Illumination (condenser system):
features microscope Electron microscope Several lenses are used as condensers for focus-
Probe used Ordinary High energy
sing the electron beam in a particular plane.
visible light: electron beam: 4 nm
700 nm to monochromatic Unlike light microscopes, in the case of the elec-
300 nm tron microscope, we use the electromagnetic coil
Maximum O.5 micron 0.1 nm as lenses. By applying an electrical current
resolution through the coil a strong magnetic field is cre-
Maximum 1000 times 500,000 times ated. The strength of the magnet can be changed
magnification
Condenser Made of Electromagnetic
by adjusting the electrical current through the
glass coil coils. So if we increase the current then the focal
Objective Made of Electromagnetic length of the beam will be shortened. Whereas,
glass coil reducing the current will increase the focal
Interior of the Air-filled Vacuum length.
optic column
Objective lens: The objective lens or imaging
Image On eye On the fluorescent
formation screen lens produces a magnified image of the object.
The objective lens has a small focal length. The
electrical current through the objective lens
Tungsten filament is made of V-shaped Tungsten should be stable to have a highly focused stable
wire. Alternatively, lanthanum hexaboride crystal image.
or field emission gun can be used for the source Intermediate and projector lenses:
of electrons. The V-shaped Tungsten wire is Intermediate and projector lenses are used to
encased with a Wehnelt cylinder or cathode change the magnification of the image further.
shield. The anode plate is located away from the The projector lens highly magnifies the last
cathode shield and both the cathode shield aper- image created by the intermediate lens and
ture and anode plate are placed centrally on the focuses it on the screen or camera plate.
same axis. Now a high voltage positive potential The vacuum system: The beam of electrons
is applied to the anode plate and simultaneously should be in the vacuum chamber. The vacuum is
the Tungsten wire is heated at 2700°K with the needed because of:
help of a direct current. At this high temperature,
the wire generates electrons by the process 1. The presence of a gas molecule will collide
known as thermionic emission. The cathode with the electrons and subsequently, the gas
shield is negatively charged and deflects the elec- molecule will scatter the electrons from their
tron to make it a central beam. The central beam pathway. Therefore to maintain the optical
28 Electron Microscopy: Principle, Components, Optics and Specimen Processing 305
Fig. 28.2 (a) Electron

microscope and its parts. a
(b) The various
components of the
transmission electron
microscope and
microscopic column
have been highlighted in
this schematic diagram
pathway of the electron beam the vacuum is To maintain vacuum in the column of the micro-
mandatory. scope multiple vacuum pumps are used at various
2. The vacuum chamber prevents the oxidation levels. The maximum vacuum is needed between
of the tungsten molecule and therefore the electron source and the specimen. In most
increases the longevity of the electron gun. microscopes, the mechanical rotary pump is
3. Sample: The sample is placed in the micro-

scope column below the condenser with the
help of a holder that holds the grid contain-
ing the tissue section. Now the electrons
interact with the thin tissue and hit the atoms
of the tissue. Heavier atoms deflect the elec-
trons and are known as electron-dense areas,
whereas the electron passes through the
lighter atoms and ultimately produces an
“electron transparent” area. Therefore, we
get a specific pattern of the emerging elec-
tron beams.
4. Objective: The electromagnetic lens of the
objective is very powerful and is the strongest
Fig. 28.3 The components of the electron gun
lens in the whole system. It creates a highly
used that can generate a vacuum of 1 Pa. This magnified image that is also known as an
vacuum is still high for the tungsten wire elec- intermediate image.
tronic source. The diffusion pump can be added 5. Projector lenses: The intermediate image is
to obtain an intense vacuum of 1 × 10−5 Pa. There further modified and also magnified by the
is a chance of oil contamination in the case of the projector lenses. There are three sets of pro-
diffusion pump. Therefore to avoid this possibil- jector lenses: (a) diffraction lens or first inter-
ity more expensive turbomolecular pump can mediate lens: It magnifies the first image
be used which is a high-speed turbine fan. The created by the objective lens. (b) The second
ion pump is used in the modern EM equipment intermediate lens and, (c) The third intermedi-
that can reduce the pressure up to 1 × 10−8 Pa. ate lens or final projector lens: This lens mag-
There are a series of airlocks in the system to iso- nifies the image further and finally projects it
late the parts of the column from the other sec- on the screen of the detector.
tions. In the modern EM instrument the vacuum 6. Detectors: The final image is focused on the
chamber is automatically controlled by the elec- screen of the detector. This is a fluorescent
tronic system. screen and when the electrons are bombarded
on this screen it emits light in the visible range
to produce a visible image. The image can be
28.1 Microscope Column captured permanently with the help of a
and Electronic Optics charge-coupled device camera.
(Fig. 28.2b)
The microscopic column has the following suc- 28.2 Specimen and Electron
cessive components: Interaction
1. Electron source: The high energy electron When the beam of electron passes through the
beam is generated from the electron gun and specimen two types of interaction may occur
is directed toward the condenser’s lenses. (Fig. 28.4):
2. Condenser lenses: There are two or more elec-
tromagnetic condenser lenses are present that 1. Elastic scattering: This is the interaction
subsequently focus the beam of electron on the between the nucleus of the atom and the elec-
sample. This helps in intense illumination in a tron of the beam (primary electron). In this
small area of the sample. As mentioned before type of reaction the kinetic energy and velocity
we can change the focal plane of the condenser of the primary electron in unaltered. Only the
lens by adjusting the electric current. pathway of the electron is altered. The nucleus
28.2 Specimen and Electron Interaction 307
of the atom is very tiny (3 × 10−15 m) com-

pared to the atom as a whole (3 × 10−10 m).
Therefore, there is actually minimal chance of
truly hitting the electron with the nucleus.
However, the positive electrostatic force of
the nucleus works on the electron and deflects
it from its pathway.
2. Non-elastic scattering: Here, the electron of
the microscope column interacts with the
electrons of the orbit of the atom. The orbital
electron repulses the incident principal elec-
tron. Here also the pathway of the electron is
changed and moreover, the energy is lost by
the principal electrons.
Secondary electrons (Fig. 28.5): When the beam

of incident electrons hit the atoms of the object
the principal electron, the orbital electron gets
excited and leaves the atomic orbit and moves
Fig. 28.4 Elastic and non-elastic interaction of electron.
towards the surface of the object. This released
In elastic scattering, the nucleus of the atom deflects the
pathway of the primary electron of the beam. In non-elastic excited electron from the atom is known as a sec-
scattering, the principle electron of the microscope column ondary electron. The secondary electron also
is repulsed by the electrons of the orbit of the atom undergoes elastic and inelastic interaction and
Fig. 28.5 Schematic

diagram shows the
interaction between the
electrons and the object.
When the beam of
incident electrons hit the
majority of the electrons
are transmitted through
the object and some
amount of electrons are
backscattered.
Occasional electrons
from the object come
out as secondary
electrons
ultimately exits from the surface. This secondary electron will also pass through the specimen.
electron can be detected. This is the basis of a The heavier atoms with more atomic numbers
scanning electron microscope (SEM). will scatter the incident electrons more than
the lighter atoms with fewer atomic numbers.
The same type of atom will form the same
28.2.1 Backscattered Electrons type of scattered electron pattern.
• Lastly, another set of electrons will interact
When the high energy incident beam of electrons with the electrons of the atomic shell or orbit
hits the specimen, some of the electrons of the and inelastic interaction will occur. These
incident beam are reflected back towards the sur- electrons will lose their energy considerably.
face. These electrons are known as backscattered
electrons. The object with a higher atomic num-
ber will have more backscattered electrons than 28.4 Sample Preparation for TEM
that of lower atomic number objects.
The preparation of samples is an important pre-
requisite for EM. The major criteria of the good
28.2.2 Excited Electrons of the Atom sample preparation include:
The incident beam of electrons when hit the elec- 1. The sample should be thin and electron trans-
trons of the atom of the object, and the atom parent. The thickness of the sample varies
changes into an excited state. This is due to the from 30 to 50 nm and the upper limit of thick-
ejection of electrons from the orbit of the atom. ness is 100 nm.
Later on, the atom comes to the stable unexcited 2. The sample should be mechanically robust so
state that occurs by shifting the electron from the that it can withstand handling in high
outer shell to fill up the vacancy of the ejected temperatures.
electron. The excess energy is released in the
form of Auger electrons, cathodoluminescence The Steps of Sample Preparation for TEM
and X-ray. include [3]:
1. Sample collection
28.3 Electron Interaction 2. Sample fixation
in the Transmission Electron 3. Dehydration
Microscope 4. Clearing
5. Embedding
In the case of the transmission electron micro- 6. Sectioning
scope (TEM), three types of interaction take 7. Staining
place:
Sample collection: The sample should be cut
• The beam of incident electrons of the micro- into small pieces of 1 to 3 mm in thickness of 1
scope column passes through the sample with- square mm area. Try to fix the sample immedi-
out any alteration of its path. The thinner the ately. Transfer the needle biopsy sample directly
specimen more the amount of un-scattered into the fixative solution.
electrons will be transmitted. So this area will Fixation: The major aims of fixation are:
be lighter in colour and the thick area will
have less transmitted electrons and will appear 1. To prevent any change in the tissue and pre-
darker on screen. serve the tissue as much as possible to its liv-
• A part of incident electrons will hit the nucleus ing condition.
of the atom and elastic scattering will occur 2. To prepare the tissue for further processing so
without any loss of energy. This scattered that the tissue does not disintegrate or tear.
28.4 Sample Preparation for TEM 309
There is no ideal fixative for EM and the choice 28.4.1 Combined Fixation Technique
of fixative depends on the type of tissue and the
particular chemical constituents to study. The At first the tissue is kept in 2% Glutaraldehyde
most commonly used fixative in EM is glutaral- solution for 2 h. After 2 h the fixative should be
dehyde. However, glutaraldehyde alone is not poured out and the tissue is washed in phosphate
suitable as the lipid is not fixed by it. Therefore, buffer solution for 5 min three times. Then the tis-
the best fixative is the combination of glutaralde- sue is fixed in 1% Osmium tetroxide for 1 h fol-
hyde followed by osmium tetroxide. lowed by 2 to 3 washing in double-distilled water.
The volume of fixative: The volume of fixa- Dehydration: Removal of water (dehydra-
tive should be 15 times more than the volume of tion) from the sample is necessary because most
the sample. of the embedding media are not miscible with
Duration: The average time of fixation is 9 h water. Therefore, a dehydrating agent is used that
by 4% glutaraldehyde at room temperature and 1 removes the water and then replaces the water
h for osmium tetroxide. The tissue should not be with a different solution which is soluble in the
in fixative for more than 12 h. Prolonged fixation embedding medium.
is not recommended as this may extract the pro- The dehydration is done by treating the sam-
teinaceous material from the tissue and the proper ple in the series of graded alcohol:
sectioning will be difficult. Under fixation may
cause swelling of the mitochondria and disrup- 30% Ethyl alcohol: 10 min
tion of the other cell organelles. 50% Ethyl alcohol: 10 min
Glutaraldehyde fixation: Glutaraldehyde 70% Ethyl alcohol: 10 min
causes cross-linking of the protein and denatures 90% Ethyl alcohol: 10 min
them. It stabilizes the protein without any coagu- 100% Ethyl alcohol: 10 min
lation. However, glutaraldehyde is not a good
fixative for lipids and causes cell shrinkage. This
effect of glutaraldehyde can be balanced by 28.4.2 Embedding
osmium tetroxide which is a good fixative for
lipid and causes swelling of the cytoplasm and The embedding medium helps to provide a firm
nucleus. base for sectioning of the tissue and also to help
in the electron microscopy procedure. The ideal
Preparation; embedding medium should have the following
2% Glutaraldehyde solution. desirable criteria:
1 M phosphate buffer solution
1. Easy to cut the section
1 M of Sodium dihydrogen phosphate (NaH2PO4):
2. Stable in electron beam and withstand higher
31.6 ml.
temperature (200 °C) at the time of microscopy
1 M of Disodium hydrogen phosphate (Na2HPO4):
3. Easy to procure the medium
68.4 ml of 1 M.
4. Evenly polymerized
Double distilled water: 900 ml.
Maintain pH: 7.2.
Presently the following media are used for EM:
Glutaraldehyde is available as 50% solution in
10 ml vial.
1. Epoxy resin
Now add 10 ml Glutaraldehyde in 240 ml
2. Acrylic media
Phosphate Buffer solution to make 250 ml
3. Polyester resin
total solution.
Osmium tetroxide solution (1%) Epoxy resin: This is the most commonly used
Osmium tetroxide: 1 g embedding medium for EM. The advantages of
Distilled water: 100 ml epoxy resin are:
• Stable at a higher temperature

• Uniform polymerization
• No damage or tissue shrinkage
The main disadvantages of epoxy resin are its

high viscosity which requires a long infiltration
time. Epoxy resin also causes dermatitis and
therefore any direct contact with this substance
should be avoided.
The vial cap should be taken out and the tissue
is embedded in freshly made resin for overnight
at 60 °C.
Acrylic Media: Butyl methacrylate and Fig. 28.6 Block holder and glass knife used in the elec-
methyl methacrylate are the common acrylic tron microscope
media. They cause significant cell shrinkage
(20%). At the time of polymerisation, bubble for- mond knives is far better than glass knives.
mation may occur and this may damage the These knives are more durable than glass
block. Moreover, methacrylate may undergo sub- knives.
limation and disintegrate in the presence of a
high energy electron beam. Semithin sections: It is the preliminary screen-
Araldite: Araldite is an aromatic amine. This ing procedure to see the adequacy of the sample.
is one of the epoxy resins used for EM. Araldite At first, the resin embedded blocks are trimmed
is used in combination with a hardener, an amine to expose the underlying tissue. Approximately
accelerator and a plasticizer. The amine accelera- 1 μ thick multiple sections are cut from each
tor accelerates the reaction between the resins. block. The sections are picked up from the water
The components should be mixed properly to trough and placed directly below the glass knife.
avoid the formation of any air bubbles. The semi-thin sections are dried on a hot plate at
Epon: Epon is an alternative embedding 60 °C. The dried sections stick to the glass slide.
medium for EM. This is an aliphatic resin and has The semi-thin sections are stained with 1% tolu-
a low viscosity. Therefore, it can infiltrate the tis- idine blue in 1% Sodium tetraborate solution for
sue more quickly compared to Araldite. 1 min to see the adequacy of the sample. If the
Polyester resin: Polyester resins have similar section contains the representative areas then
properties to that epoxy resin. They do not cause further ultrathin sections are made from the
any cell shrinkage and polymerize uniformly. block.
Vestapol W is the commonly used polyester
resin. Toluidine blue 1% solution
To make a thin section is the crucial point of Toluidine blue: 1 g
sectioning. Ordinary histological microtomy is Sodium tetraborate: 1 g
not suitable for the electron microscopy section- Distilled water: 100 ml
ing and ultrathin microtomy is needed.
At first dissolve the borax in the distilled water.
Then add Toluidine blue and dissolve by constant
28.4.3 Knives stirring. Filter the solution and keep it at room
temperature.
• Glass knives: Glass knives are cheap and con- Ultrathin section: The trimmed blocks are
venient (Fig. 28.6). cut further by an experienced technician. The
• Diamond knives: Diamond knives are rela- ultra-microtome is set in an auto mode to have
tively expensive. The section quality of dia- optimum thin sections (Fig. 28.7). As mentioned
28.4 Sample Preparation for TEM 311
Fig. 28.7 Ultrami

crotome used in the
electron microscope to
cut ultrathin section
before we need less than a 100 nm thick section 28.4.4.1 Lead Stain
and the optimum thickness is 80 nm. The reflected Reynold’s Lead Citrate solution is used for the
light from the section gives information about the staining. Lead rapidly reacts with the atmo-
thickness of the slide: spheric carbon dioxide and may form lead car-
bonate as a precipitate. Therefore adequate care
Grey colour: Less than 60 nm. should be taken to prevent such precipitation.
Silver colour: 60 to 90 nm. The solution should always filter before use.
Gold colour: 90 to 120 nm.
28.4.4.2 Stain
The sections float either in ethanol or acetone. To • Stain the section by dipping it in Reynold’s
stretch the sections one can take the help of Lead Citrate solution for 15 min.
xylene or chloroform. A small piece of filter • Wash each grid with 0.1 N NaOH solution
paper soaked with either xylene or chloroform • Wash with two changes of distilled water
can be hold just above the section. The evapo- • Dry the grid and keep it in a grid box.
rated vapour usually stretches the section. The
stretched sections are finally picked up by a small 28.4.4.3 Reynold’s Lead Citrate
copper grid. There is the shiny and dull side of solution
the copper grid. The sections are lifted on the dull Lead nitrate: 1.33 g.
side of the grid. Sodium citrate: 1.76 g.
Distilled water: 30 ml.
Mix lead nitrate and sodium citrate in distilled
28.4.4 Staining of the Sections water and shake them for 1 min. After 30 min
lead citrate will convert into lead nitrate.
The sections are stained with the grid. They are Now add 8 ml 1 M NaOH and mix them well.
commonly stained with lead or uranyl acetate. Gently add 50 ml distilled water to dissolve the
Table 28.2 comparison of processing, staining and sectioning between Light microscope and Electron microscope
Characteristics Light microscope Electron microscope
Fixative 10% Formalin 2% Glutaraldehyde (2%) and Osmium
tetroxide (0.1%)
Embedding medium Wax Epoxy resin
Section thickness 4 to 5 μ 80 nm
Cutting Ordinary microtome Ultramicrotome
Knife Disposable knife Glass or diamond knife
Section holding Glass slide Copper grid
Routine stain Hematoxylin eosin stain Lead impregnation
precipitated lead nitrate. Keep pH 12. The solu- 28.5 Scanning Electron
tion will be stable for 6 months. Microscopy [4]
A scanning electron microscope (SEM) provides

28.4.5 Uranyl Salt information on the surface structures of the
object. The spatial resolution of the SEM is ten
Aqueous or alcoholic solution of uranyl acetate is times better than the light microscope. The image
used for the staining of TEM. Uranyl acetate of the SEM is developed point by point from the
combines with protein and lipids and gives good emitted secondary electrons like a scanner image.
contrast to various membranes and nucleic acid. Therefore the instrument is known as SEM.
The major disadvantage of uranyl acetate is the
rapid precipitation in the presence of light.
Either an aqueous or alcoholic solution of ura- 28.5.1 Operational Principle
nyl acetate can be used. The alcoholic solution
has a short staining time and it penetrates easily As mentioned before, the electron beam when
within the sample. It also gives better contrast. A hits the specimen the secondary electron and
saturated alcoholic solution of uranyl acetate is backscattered electrons come back from the sur-
used for staining. face (Fig. 28.8). These electrons emitted from the
An aqueous solution of uranyl acetate also same side of the incident beam create an image.
provides good contrast. However, this solu- Therefore, the construction and operational mode
tion is photosensitive and therefore rapidly of SEM and TEM are totally different
precipitates. (Table 28.3).
The overall comparison of the laboratory pro- Specimen preparation for SEM: The fixa-
cedure of light microscope and electron micro- tion, processing, sectioning and staining for SEM
scope is highlighted in Table 28.2: are same as that of TEM.
References 313
Fig. 28.8 Schematic diagram shows the various components of transmission electron microscope. Here instead of
transmitted electrons, the secondary electrons and the backscattered electrons are recorded
Table 28.3 Comparison of the Transmission electron References

microscope and Scanning electron microscope
Transmission 1. Jensen EC. Types of imaging, Part 1: Electron micros-
electron microscope Scanning electron microscope copy. Anat Rec (Hoboken). 2012;295(5):716–21.
Incident beam of The Incident beam of the 2. Müller SA, Aebi U, Engel A. What transmission elec-
the electron is static electron is dynamic and scans tron microscopes can visualize now and in the future.
the object horizontally in two J Struct Biol. 2008;163(3):235–45.
perpendicular directions 3. Haggis GH. Sample preparation for electron micros-
Image is formed Image is formed like a scanner copy of internal cell structure. Microsc Res Tech.
instantly image 1992;22(2):151–9.
4. Carr KE, Toner PG, Saleh KM. Scanning electron
An Incident beam Incident beam of electron hits
microscopy. Histopathology. 1982;6(1):3–24.
of electron passes the object and emitted
through the object secondary and backscattered
electrons form the image
Quality Control and Laboratory
Organization 29
29.1 Introduction that the laboratory delivers excellent health care

service [2].
In the manufacturing company, we produce the The QA is a coordinated dynamic process that
final product from the raw materials and then detects the error and takes measures to control
deliver it to the customers. The final product is and prevent it to provide the best health care
always verified to maintain a particular standard. service.
Such as a car company assembles the raw materi- Quality improvement (QI): QI means an
als and after making a car the company takes overall attempt to improve the specific quality of
measures to maintain the standard of the car. If the laboratory by assessing the performance and
there is any defect the company immediately rec- improvement of the laboratory service.
tifies it and also prevents producing such defects Objectives of the quality control: The main
in future. In the histopathology (or cytology) objectives of the quality control are:
laboratory we receive the tissue or sample, pro-
cess the sample and make a stained section on the 1. To give correct and complete test report to the
slide for the interpretation and finally report the patient
tissue sample. This is very similar to the indus- 2. To generate and deliver the report in a mini-
trial company. Stringent maintenance of quality mum amount of time
or standard is also needed for good laboratory 3. To maintain ethics and professional service
service. Several terminologies arise in this area 4. To provide excellent service to the patient so
that is defined below. that it satisfies the patient
Quality control: The term quality control 5. To provide continuous training and current
(QC) means the collection of operational tech- education to the laboratory staff
niques to verify and maintain a desired set level
of quality in the laboratory test or process [1]. Essential technical requirements for the qual-
The quality control activity is a continuous pro- ity control: The essential technical requirements
cess and it starts immediately from the receiving for the quality control measures include
the specimen to the final dispatch of the report (Fig. 29.1):
along with post verification of the test result.
Quality assurance: Quality assurance (QA) 1. Laboratory design: The laboratory should be
is defined as the program that does systemic designed in such a way so that there remains
monitoring and evaluation of the various area of enough space for the receiving of the sample,
the quality control result and quality practice so processing, staining, interpretation area, stor-
https://doi.org/10.1007/978-981-19-6616-3_29
316 29 Quality Control and Laboratory Organization
Fig. 29.1 Technical

requirements for the
quality control in
laboratory are
highlighted in this
diagram
age etc. There should be proper ventilation the use of the equipment, purchase date and
and safety arrangement in the laboratory. expiry date of the chemicals.
2. Scope and overall facilities in the labora- 6. Ensuring the quality of the processing and
tory: Overall laboratory facilities should be reporting: The quality of the processing
clearly documented. A detailed description of should be regularly checked and recorded.
all the tests in the laboratory should be men- Similarly, the reporting quality should be veri-
tioned to the patients. fied periodically.
3. The work definition of the laboratory per- 7. Laboratory information service (LIS): LIS
sonnel: The work responsibilities of the dif- generates a unique accession number of the
ferent categories of the laboratory staff should specimen. This number provides the identifi-
be clearly described. The staff should be well cation of the sample or section. The patient’s
competent with a professional license to prac- clinical history and other necessary informa-
tice the respective work. tion are listed in LIS. The final report is also
4. Financial resources: It is necessary to know entered in LIS and the report is recoverable
the overall financial budget allocation for lab- instantly by the end service providers.
oratory personnel, equipment, chemicals etc.
This knowledge of the financial budget gives
the idea of the capability of the laboratory to 29.2 Quality Control
fulfil the customer’s needs.
5. Laboratory equipment and reagents: The The quality control involves the three important
standard equipment and reagents are needed steps (Fig. 29.2):
to provide good quality well stained sections
and smears. The microtome, processing 1. Pre-analytical phase: It starts from receiving
machine etc. should be regularly updated. the sample up to the final processing for
There should be a proper logbook mentioning reporting.
29.2 Quality Control 317
Fig. 29.2 Three important steps of quality control: pre analytic, analytic and post analytic phase
2. Analytical phase: It is mainly involved the history etc. The sample and requition
interpretation of the test or slide (in the case of form should be identified properly.
histopathology or cytology service). • Allocation of the unique accession num-
3. Post analytical phase: It is the post interpreta- ber to the patient’s sample: This unique
tion phase and involves the report delivery, accession number of the sample should
storage of slides, review of the slide or test be allocated immediately after the initial
etc. registration of the patient. This can be
given as computer gererated bar code
1. Pre-analytical phase: The pre-analytical number and can be given in all the sub-
phase has the following components: sequent tissue, blocks and sections.
(a) Receiving the sample: This is the first • Patient’s clinical history to include in
step of quality control. The laboratory the computer
staff in the reception should follow the • To check the proper fixative for the histo-
following aspects: pathology and cytology sample: The
• Identification of the sample and the fixation of the specimen is a necessary
patient’s requisition form: The request step as delayed fixation may cause sig-
form should always include the fol- nificant autolysis. Surgical specimen
lowing information: name of the should be fixed immediately with 10%
patient, name and address of the buffered formalin. Cytology sample
requesting consultant, the test required, should be sent in proper recommended
clinical history and diagnosis, drug fixative.
(b) Laboratory Processing: 2. Analytical Phase

Standard Operation Protocol (a) Microscopic examination: This is the
Every laboratory should have a standard vital step and the final interpretation of
operation (SOP) protocol. The SOP should the slide is always done by a qualified
mention the fixatives, collection procedure, licenced pathologist. In the histopathol-
processing and staining. ogy laboratory, the preliminary and final
Record of the chemicals and equipment: interpretations are done by the junior and
The laboratory should have a record of the senior pathologists only. However, in the
purchase and expiry date of all the necessary cytology part, the cervical smears are
staining materials. All the chemicals should screened by the primary screener who
be properly labelled and kept alphabetically. may be a cytotechnologist. The final
Logbook of equipment, reagents etc. should reporting should always be done by the
be maintained. consultant cytopathologist [3].
Regular checking of the quality of pro- Synoptic reporting format: The
cessing and staining: It is mandatory to pathologist should follow the standard
check the section/smear quality periodically. reporting format. Synoptic reporting is
The error should be mentioned in the logbook helpful to cover all the information regard-
along with the remedies taken to overcome ing the sample, particularly in the large
the problems. specimen. In addition, the synoptic report-
Grossing: Strictly speaking, grossing of ing format generates uniform reporting
the sample is an analytic phase. This is the and is more efficient. The data analysis
duty of the qualified pathologist. The grossing and research are easier in such a format.
room should be well equipped with gloves, Cytology:
knives, scissors, measuring tape and a weigh- Cervical smears: After the initial screening
ing machine. The automatic bar code acces- by the cytotechnicians, re-screening is recom-
sion number can be attached to the block. This mended to reduce the false-negative rate.
bar code number is the same as that in the req- Commonly used re-screening techniques are:
uisition form and the specimen. • Proportional re-screening: A certain
Processing The tissue and sample should fraction (10%) of the negative smears are
be processed according to the standard operat- screened by the cytopathologist.
ing protocol (SOP) of the laboratory. The bar- • Selected re-screening: The cytopatholo-
coding of the block and slides help to track the gist re-screens only the selected high-risk
individual cases and this prevents the poten- cases such as previously diagnosed abnor-
tial to mix up. The stained slides may be mal smears, history of bleeding per vagina,
scanned by the whole slide image system. etc.
This may replace the glass slide in future. • Rapid review: In this method, all the
However, till date, FDA has not approved the smears are re-screened rapidly to pick up
whole slide scanning image to report directly the false-negative cases.
bypassing the glass slide. • Automated re-screening: The computer
Vigilance by the senior laboratory per- screens all the smears and indicates the
sonnel: Daily vigilance by the senior staff is smears to be rescreened again.
necessary to reduce the error and improve The number of slides to screen: The pri-
overall quality. mary cytoscreener should not screen more
Health and safety of the staff: The labo- than 100 slides in 1 day. The maximum time
ratory supervisor should do close scrutiny and limit to screen the slide is 8 h [4].
effective measures to maintain the safety and Fine needle aspiration cytology smears and
health of the laboratory staff. Non-Gynecological smears:
29.2 Quality Control 319
• The cytopathologist should follow a con- Type of errors: The following types of error
sistent pattern of reporting and should dis- may occur [2]:
card the ambiguous terminologies as far as
possible. • Categorical change: Benign versus malignant
• In problematic cases the consultant should • Error in typing: Type of malignancy is wrongly
take the opinion of the other fellow made
colleagues • Error in classification
3. Post analytic phase: This phase enjoys rela- • Error in the involvement of lymph node
tive freedom from time pressure. The imple- • Error in the interpretation of the margin of the
mentation of quality assurance is applicable in resected tumour specimen
the post-analytic phase. This phase involves: • Mistake in the identification of side: right ver-
(a) Proper typing of the report sus left
(b) Manual or electronic delivery of the • Error in the identification of the patient
report
(c) Storage of the slide and report The rectification of error: It is essential to cor-
(d) Review of the signed out a report rect the detected error. The newly revised report
Quality check of the signed out report: may consist of:
The following measures may help in this aspect:
• Corrected diagnosis
• Review the report of the specific system by the • Corrected information
expert second consultant in that system • Any additional information should be given as
• Random review of certain percentages of footnote.
cases (2 to 10%) depending on the resource of
the laboratory
• Different interdepartmental meetings: Liver 29.2.2 Record Keeping
biopsy round, kidney biopsy round, various
oncology meetings, clinicopathological con- All the data should be stored preferably in the
ferences etc. computer. There should be a proper backup of the
• Correlation of frozen section and permanent data.
section Royal College of Pathologists, UK, recom-
• Cytology and final histopathology correlation: mends [5]:
All the dis-correlated cases should be dis-
cussed in detail so that the error can be over- • Preserve the tissue block forever
come in future. • Preserve the histopathology slides for
• Review of the cases by other institutions 10 years
• Keep the wet tissue for 4 weeks after the dis-
patch of the report.
29.2.1 Gold Standard
There are definite guidelines for storage of cytol-
Cytology: Final histopathology report is the gold ogy smear and these are [6]:
standard the cytology cases.
Histopathology: In the case of histopathol- • Irrespective of the diagnosis the cervical
ogy cases clinical follow up of the patient is the cytology smear should be kept for 5 years
ultimate gold standard. The opinion of the exter- • The glass slides of fine needle aspiration
nal expert may be taken into consideration as cytology should be kept for at least 10 years.
the final judgement. In case of death, the final • The test requisition form should be retained
autopsy report should be considered the gold for 2 years
standard. • Test reports must be retained for 10 years.
Interlaboratory comparison: The laboratory • A trained or technically qualified person

should join the interlaboratory slide exchange • The person should not be someone inside the
program. The primary diagnosis offered by the own department
laboratory should be verified by the other groups • Person should be independent and free of
of the laboratory and vice versa. The circulating bias
substance needs the following criteria:
When should the audit be conducted: Audit
1. The circulating material may be a tissue sam- should be done either once in a scheduled time of
ple or praffin embedded tissue block or stained the year or any time of the year when there is a
slide or whole slide image (WSI). doubt of the effectiveness of the service of the
2. There should be proper clinical history along laboratory.
with the circulating material.
3. The cases should be classical without any
diagnostic confusion. 29.3.2 Stages of Audit
4. There should be the provision of feedback in a
measurable way such as scoring. Adequacy audit: In this preliminary stage the
5. The individual cases should be discussed after auditor review all the documents such as labora-
the circulation of the material. tory manual and procedures. The documented
laboratory mannuals are compared with standard
The laboratories which do not fulfil the minimal set requirements.
quality should be advised to rectify the errors and Compliance Audit: This is the later stage
improvement is mandatory. where the auditor inspects individual areas and
examines that whether the actual practice is done
according to the standard.
29.3 Audit
The audit is the systematic and independent 29.3.3 Components of Audit

assessment of the overall activity of the labora-
tory and compares these activities of the labora- 1. The auditor should collect the following
tory with the set standard. information:
Aim of the audit: The primary aim of the (a) Laboratory staff
audit is to a systemic review of the overall perfor- (b) Laboratory equipment
mance and comparison with the standard practice (c) Sample processing
so that one can improve the work. (d) Methods of the test
(e) How the quality control is measured
(f) Reporting practice
29.3.1 The Beneficial Points (g) Record keeping
of the Internal Audit 2. The next step of the auditor is to compare all
the information with the standard.
The internal audit helps in 3. The auditor should mention the deviations
and the correction of the procedure.
• To develop the awareness of the mistakes
• To improve the wrok Audit report: The auditor should submit a writ-
• It is a part of the continuing education ten report mentioning the date of the audit, area
• To meet the quality standards of the audit, corrective measurement etc. The
Who should conduct the audit: The audit audit report should be preserved at least for
should be conducted by: 3 years.
29.5 Laboratory Organization 321
Remedial actions: Based on the audit report, • The laboratory should have at least one inter-
corrective or remedial actions should be taken by nal audit report
the quality manager of the laboratory.
29.4.3 Process of Accreditatation

29.4 External Quality Assurance
• The laboratory should apply for official
External quality assurance consists of: accreditation
• The laboratory should prepare its quality
• Proficiency test manual
• Continuing medical education • The accreditation authority should pre-assess
the laboratory
Proficiency test: The proficiency test is a volun- • Final team of the assessor should evaluate the
tary program. The various laboratories should laboratory manual and physical visit to the
take part in the proficiency test to improve their laboratory system.
diagnostic skill. In UK, the proficiency test is • The final scrutiny of the team report
mandatory for the reporting consultants who • Issuing the accreditation certificate by the
work in NHS Breast Screening Program. Overall chairman of the accreditation committee.
the proficiency test is educational and it points
out the strength and weaknesses of the patholo-
gists [7]. 29.4.4 Advantages of Laboratory
Continuing medical education: All the labo- Accreditations
ratory personnel should take active participation
in the various workshop, CME, seminars etc. The accreditation of the laboratory has the fol-
lowing advantages:
29.4.1 Laboratory Accreditations • Users: The users get assurance of the good
quality service from the certified laboratory
Laboratory accreditation is the assessment by • Laboratory organizers: The efficiency of the
which the laboratory meets the specific require- laboratory staffs is improved and any defect in
ments of a previously set standard of the agency. the service is corrected. Morover, the
There is no established laboratory accredita- performance of the laboratory service remains
tion agency in many countries. consistent
29.4.2 Pre-requisite for Laboratory 29.5 Laboratory Organization

Accreditation
The organization of the pathology laboratory has
• The laboratory should be legally registered three essential parts:
• It should provide the necessary facilities
• The laboratory must have trained qualified 1. Laboratory construction, equipment etc.
staff 2. Laboratory staffs
• The quality manager of the laboratory should 3. Organization set-up and laboratory protocol
preferably attend the audit Training 1. Laboratory construction, equipments etc.:
• The laboratory should have standard operating The laboratory access pathways should be
protocols. such: sample collection, sample processing
• All the laboratory equipment should be and staining, and reporting, followed by the
calibrated post-examination area.
Physical aspects of the rooms: of the staff should be specified at the time
(a) Location: Other than the specimen and of recruitment.
the report collection rooms all other labo- Qualifications and training: The
ratory rooms should be inaccessible to the technical staff and the pathologists should
patients and other trespassers. have proper qualifications and licence.
(b) Rooms: The rooms should have the fol- Periodic evaluation of the staff should be
lowing things: done.
• All the laboratory rooms should be 3. Organization set up and system protocol
well ventilated with high ceilings. The overall organization process of the
• The wall of the laboratory should be sample is important for the maintenance of
well painted. good quality work. Each laboratory should
• The floor and wall should be made in have documented plan of the scope of the lab-
such a way that they can be clened eas- oratory service, flow chart of the work plan,
ily by disinfectant. allocated budget in the different areas, and
• The room should have water supply, proper quality planning with periodic review
proper racks and closed almirah to of the whole laboratory work process.
keep the hazardous chemicals sepa-
rately. The processing room must have
saftety cabinet. References
• The screening room should be isolated,
spacious and free from any noise. 1. Quality Improvement Manual in Anatomic Pathology.
Chicago: College of American Pathologists; 1993.
• The rooms for the secretarial staff 2. Association of Directors of Anatomic and Surgical
should have adequate space for the Pathology. Recommendations for quality assurance
typing equipment and furniture. and improvement in surgical and autopsy pathology.
(c) Safety arrangement: The room should Am J Surg Pathol. 2006;30(11):1469–71. Erratum in:
Am J Surg Pathol. 2007 Jan;31(1):16
be equipped with proper safety arrange- 3. Royal College of Pathologists. Medical and Scientific
ments such as fire extinguishers etc. Stuflng Of NHS Pathology Departments; 1992.
2. Laboratory staff: The laboratory staff should 4. Clinical laboratory improvement amendments of
be in the following categories: 1988. Final rule federal register. February 28. 1992.
57:493. 1257(b).)
(a) Laboratory directors 5. Venkatraman NT, Bhadranna A, Shenoy S, Mohanty
(b) Consultant pathologist L. To err is human: Quality management practices
(c) Biomedical scientist or technical chief in surgical oral pathology, a safety net for medico-
(d) Cytology screeners legal complications. J Oral Maxillofac Pathol.
2013;17(2):234–9.
(e) Laboratory technicians 6. Clinical Laboratory Improvement Amendments of
(f) Clerical staff 1988; Final Rule. Federal Register. Feb. 28, 1992; Vol.
(g) Others: Cleaners, receptionists etc. 57: 493.1105.
Job description: The duties and 7. Roberts PF, Chairman RCPath EQA steering commit-
tee for histopathology and cytopathology, Personal
responsibilities of the different categories Communication; 2001.
Laboratory Safety and Laboratory
Waste Disposal 30
Each laboratory should have overall safety pre- (f) During preparing a diluted solution, the
cautions. The laboratory safety officers should concentrated acid or alkali should be
look after the following issue: added to water.
Overall security: This involves the general 4. Infective: The laboratory personnel should
security of the laboratory such as the safety of. always take universal precautionary measures
the equipment, reagents and prevention of because we do not know the HIV status of a
entry of any unwanted persons. sample [1, 2].
1. Security: (a) Universal precautions (Box 30.1): Health
(a) Proper security of the laboratory staff, education of the technical staff regarding
chemicals and valuable equipment is universal precaution is very important to
mandatory. prevent the transmission of infection.
(b) Entry of unauthorized persons should be Universal precautionary measures imply
restricted to the laboratory. that all the patients should be treated as a
2. Fire hazards: potential sources of blood-
borne
(a) The fire extinguishers, smoke alarms and infections.
fire blankets are necessary for every (b) What is it? Universal precautions indi-
laboratory. cate taking adequate measures to prevent
(b) The laboratory personnel should know contact with various body fluids of the
the basic operation protocol of the fire patients. Various barrier measures are
equipment. taken to avoid contact with body fluids
3. Chemical hazards: that are the potential source of transmis-
(a) The toxic and inflammable chemicals sion of infection.
should be in a closed-door fireproof metal (c) The high-risk pathogens: The patho-
cabinet with original labels gens that cause serious health hazards
(b) Never do suction by mouth are: Hepatitis B, hepatitis C and HIV.
(c) Always put the alkali or acid in water dur- (d) Body fluids that need universal precau-
ing the procedure of dilatation tions: This includes blood, peritoneal and
(d) Facilities to wash eyes and shower in case pleural fluid, CSF, semen, vaginal secre-
of toxic exposure. tions, synovial effusion, and faecal material.
(e) Wear gloves, mask and laboratory coat (e) Body fluids that do not need universal
during dealing with chemicals. precautions: These are faecal material,
https://doi.org/10.1007/978-981-19-6616-3_30
324 30 Laboratory Safety and Laboratory Waste Disposal
Box 30.1 Universal Precautions

Universal precautions means to take ade-
quate measures to prevent contact with
various body fluid of the patients.
• Infective agents with serious health

hazards: Hepatitis B, hepatitis C and
HIV
• Body fluids that need universal pre-
cautions: blood, peritoneal and pleural
fluid, CSF, semen, vaginal secretions,
synovial effusion, and fecal material.
• No need for universal precautions:
fecal material, urine, nasal secretions, Fig. 30.1 Schematic diagram of universal precautionary
sweat, vomitus, sputum unless contami- measures in laboratory
nated with blood.
• Measure: for re-use as the removal of the micro-
–– Hand wash organisms is not always possible from the
–– Gloves gloves.
–– Mask • Isolation gown: The isolation gown helps to
–– Goggles prevent contamination with blood or mucus
–– Apron product with skin. It should only wear when
–– Take caution with sharp objects there is a chance of contamination of blood or
–– Proper discarding the contami- blood products. For routine laboratory work,
nated waste wearing of simple laboratory gown is enough.
–– Cleaning the area • Mask: The mucus membranes of the upper
respiratory tract are vulnerable to infecting
agents. Mask prevents transmission of infec-
urine, nasal secretions, sweat, vomitus, tion from the patient to the health care person-
and sputum provided these materials are nel or vice versa. The masks may be of variable
not contaminated with blood. sizes with different filtration capacities. The
Universal precautions proper (Fig. 30.1): types of masks depend on the need of the par-
ticular staff.
• Hand washing: Simple maintenance of hand • Goggles: The various viral infections and
hygiene is the single most important factor to Staphylococcus aureus can be transmitted by
prevent the transmission of infection. The direct contact with splashed blood or by
cleaning of hands with anti-infection soap touching the eye with the contaminated hand
removes the bacteria. The approved alcohol- to the eye mucosa. Goggles should be used to
based products such as gel or foam is superior prevent the transmission of infection through
as these substances have better microbicidal eye mucosa.
activity and do not produce a drying effect.
Moreover, no water is needed to clean with an • Precautions from the sharp objects [3, 4]
alcohol-based cleaning agent. (Box 30.2): The most important pathogens
• Gloves: Gloves prevent the blood or contami- that can transmit through needle prick injury
nated substances to have direct contact with are HIV, HBV and HCV. The injury may hap-
the skin. Latex or nitrile gloves are better than pen during (1) recapping the needle, (2) trans-
vinyl gloves. The gloves should not be washed fer of the blood from container to container
30.1 Laboratory Waste Disposal 325
• Sick person: The laboratory workers with

Box 30.2 Needle Stick Injury skin lesions such as weeping dermatitis or
open wound in the hand should be refrained
Common causes
from the laboratory work area.
• Recapping the needle (25% cause)
Standard norms in the laboratory:
• Inadequate disposal of needle
–– Do not smoke, eat or drink within the
• Transfer of fluid from one to other
laboratory
container
–– Always wear laboratory gown and gloves
Avoidance when you work in the laboratory
• Use alternative safe technique if –– Do not wear sandals and shoes with open
possible toes
• Do not recap the needle –– Always avoid pipetting by mouth
• Use needle cutter –– Always clean your hand and face after
• Dispose needle promptly removing the gloves
• Universal precautionary measure –– Never recap the used needle
• Health education –– Always dispose the sharp objects in the
What to do in case of needle prick leak proof metal container
• Immediately report your employer –– Always clean the laboratory area before
• Take appropriate care and follow up and after work with 10% Sodium hypo-
such as post exposure prophylaxis chlorite solution
• Report to appropriate authority –– Keep the compressed gas cylinders under
the secured condition
Possible transmission of diseases
• Hepatitis B (6 to 30%)
• Hepatitis C (1.8%) 30.1 Laboratory Waste Disposal
• HIV (0.3%)
Biomedical wastes are different from the usual
Factors that determine infection: waste material. It has certain unique characteris-
• Immunity status of the victim: whether tics such as:
vaccinated or not
• Severity of the prick: superficial or deep • Highly infectious
• Appropriate post exposure prophylaxis • The biomedical wastes need a special type of
• Type of pathogen handling to avoid the spread of disease
• It may produce permanent toxic substances
and (3) improper disposal of the needle. The

following precautions are helpful in needle 30.1.1 Basic Ways to Waste
stick injury: Management
–– Do not bend or recap the needle
–– Use a needle cutter to cut the needle • Reduction of waste material as far as
–– Put all the sharp objects in a proper possible.
container • Take safety precautions against the waste
–– Take universal precautionary measures material.
–– Written document on post-exposure • The appropriate collection, and disposal of the
situation waste materials.
–– Post-exposure follow up and evaluation
Categories of waste: The different categories of (d) Store waste only in a closed container
the waste materials are highlighted in the table: which is leakproof and no chance of
puncture in case of sharp material
1. General waste (e) Use only one type of container for the
2. Biohazardous waste particular type of waste
(a) Chemical waste (f) Do not keep incompatible chemicals in
(b) Biological waste the same container such as acid and alkali
(c) Radioactive material should not be kept together.
2. Storage and transport
The biowaste should be properly stored
30.1.2 Steps of Biomedical Waste before transport. The storage area should be
Disposal away from the drink and food consumption
and it should be in a secured place and off the
1. Biomedical waste collection and segrega- limit of the general public. The transport of
tion: It is the first and very important step of the biowaste material should be in a specially
waste disposal. The waste material is col- designated covered vehicle.
lected in the appropriately labelled containers. 3. Waste disposal and treatment
As mentioned in Table 30.1 (Fig. 30.2). It is necessary to dispose and treat the
The basic norms of the collection of the waste waste according to the category as mentioned
material are: in the Table 30.1. Solid or liquid biologically
(a) Do not store waste in a metal container contaminant materials are initially treated
(b) Do not store the chemical waste under the with disinfectant followed by incineration.
fume hood Most of the bulk waste material is destroyed
(c) Properly label the containers of the waste by proper incineration.
Table 30.1 Types of waste and their treatment

Color of
the bag Waste material Container Disposal
Black • Discarded medicinal substances: Outdated Plastic bag Local authority for
or remnants of medicine routine waste disposal
• Various chemicals used for disinfection
Yellow •H uman anatomical waste: human Plastic bag Disinfection and
anatomical organ, body parts subsequently
•A natomical waste: body parts of animals, incineration
various waste materials of animals,
discharge etc.
•M icrobiology and Biotechnology
Wastes: Wastes developed from laboratory
cultures, toxins etc.
• Solid contaminated waste: Materials
contaminated with blood such as cotton,
linen, bed
Red • Solid contaminated waste: Materials Container with disinfectant Disinfection and
contaminated with blood such as cotton, subsequently
linen, bed incineration
• Solid Waste: disposable items such as
catheter
Blue •S harp materials: Blade, needle, broken Closable, puncture-resistant Incineration
glasses etc. and leakproof hard plastic
container
30.1 Laboratory Waste Disposal 327
Fig. 30.2 The different types of waste disposal in different coloured containers
Methods of treatment: The various methods concentration and stability of the chemi-
of waste treatment include: cals. The common types of chemical dis-
(a) Autoclaving: Autoclaving procedure infectant include:
uses hot stream for a specified period of • Chlorine-basedsed Compounds:
time to destroy the microbial organisms. Sodium Hypochlorite (NaOCl): It
It is relatively cheap and can be used in is a rapid oxidant material and is a
small volume of waste material. broad-spectrum disinfectant. Chlorine
(b) Incineration: It is a rapid and simple is generated from the diluted mixture
procedure. Incineration removes all sorts and works as a disinfectant. For labora-
of organisms. However, it may produce tory purposes, 10% sodium hypochlo-
toxic gases and so a professional organi- rite solution is used as a chemical
zation is needed to handle incineration. disinfectant. The solution should be
(c) Microwave: Electromagnetic wave is made fresh every day. As sodium hypo-
used in microwave to destroy the chlorite generates chlorine so it is
microbes. It is a relatively low heat pro- highly corrosive and should not be kept
cess and energy efficient. in a metallic container.
(d) Chemical disinfection: The chemical • Iodophors: Iodophores are the disin-
disinfectant is usually used in liquid fectant containing Iodine in an aqueous
waste material. The effectiveness of the solution. Betadine and Povidone-Iodine
chemical disinfectant is dependent on the are widely available commercially.
• Quaternary Ammonium Com References

pounds: These are also good deter-
gents and disinfectants. These 1. Siegel JD, Rhinehart E, Jackson M, Chiarello L,
compounds act against various bacteria Health Care Infection Control Practices Advisory
Committee. 2007 Guideline for isolation precautions:
and viruses. However, their action preventing transmission of infectious agents in health
diminishes with organic matter and care settings. Am J Infect Control. 2007;35(10 Suppl
many detergents. These compounds 2):S65–164.
are good for cleaning the laboratory 2. Centers for Disease Control (CDC). Update: universal
precautions for prevention of transmission of human
floor. immunodeficiency virus, hepatitis B virus, and other
• Phenolics: These are derivatives of bloodborne pathogens in health-care settings. Morb
phenol and act by damaging the mem- Mortal Wkly Rep. 1988;37(24):377–82, 387–8.
brane of the bacteria and fungi. Lysol is 3. Tan L, Hawk JC 3rd, Sterling ML. Report of the
Council on Scientific Affairs: preventing needlestick
a widely available commercial product. injuries in health care settings. Arch Intern Med.
Many phenolic compounds are inacti- 2001;161(7):929–36.
vated by hard water and so it is prefer- 4. NIOSH releases guidelines on preventing needle-
able to dilute them with distilled water sticks. National Institute for Occupational Safety and
Health. AIDS Alert. 2000;15(1):suppl 1–2.
• Others: Acid, alkali and alcohols are
also used as disinfectants.
ultiple Choice Questions
M
for the Self-Assessment
Instruction: Only one response is correct C. B5 fixative

D. 10% neutral buffered formalin
Q1. The best immersion fixative in the routine Q7. Dark brown granules may be produced by
histopathology tissue section is: A. 10% neutral buffered formalin
A. 95% Ethyl alcohol B. Osmium tetroxide
B. 10% neutral buffered formalin C. 2.5% glutaraldehyde solution
C. Osmium tetroxide D. Zenker’s fluid
D. 2.5% glutaraldehyde solution Q8. The clearing agent in the tissue processing is:
Q2. The fixative that causes dehydration and A. Dioxane
coagulation of protein is: B. Xylene
A. Glutaraldehyde solution C. Paraffin wax
B. 10% neutral buffered formalin D. Ethyl alcohol
C. Osmium tetroxide Q9. The dehydrating agent in the tissue process-
D. Ethyl alcohol ing is:
Q3. This is not true for the microwave fixation: A. Toluene
A. Preservation of tissue enzyme B. Xylene
B. Relatively fast process C. Paraffin wax
C. Tissue shrinkage D. Ethyl alcohol
D. Preservation of Glycogen Q10. The step after the clearing is:
Q4 This fixative produces a yellow stain on the A. Dehydration
tissue B. Impregnation
A. Bouin’s fluid C. Microtomy
B. Osmium tetroxide D. No further step
C. Zenker’s fluid Q11. After the processing, the tissue is coming out
D. Acetone from the block during sectioning: This is due to:
Q5. The fixative of choice for the enzyme immu- A. Inadequate dehydration
nochemistry is: B. Thick tissue
A. Fresh frozen tissue C. Excessive blood in the tissue
B. 10% neutral buffered formalin D. Defective clearing
C. Osmium tetroxide Q12. Which is not a clearing agent:
D. 2.5% glutaraldehyde solution A. Xylene
Q6. The best fixative for the spleen is: B. Toluene
A. Bouin’s fluid C. Cedarwood oil
B. Zenker’s fluid D. Ethanol
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature 329
Singapore Pte Ltd. 2022
https://doi.org/10.1007/978-981-19-6616-3
330 Multiple Choice Questions for the Self-Assessment
Q13. This tissue processing agent is inflammable Q21. The angle between the lower bevel of
and quickly evaporates the knife and the surface of the block is
A. Isopropyl alcohol known as:
B. Ethylene glycol A. Rake angle
C. Dioxane B. Bevel angle
D. Acetone C. Angle of clearance
Q14. The best embedding medium in the routine D. Cutting angle
laboratory specimen is: Q22. The optimum thickness of the paraffin sec-
A. Celloidin tion in routine laboratory tissues is:
B. Paraffin wax A. 1–2 microns
C. Agar gel B. 3–6 microns
D. Gelatin C. 8–10 microns
Q15. The embedding medium for the electron D. 15–20 microns
microscopy specimen is: Q23. The Section attaches to the block during
A. Epoxy resin microtomy is due to:
B. Gelatin A. Static electricity is generated in the knife
C. Celloidin or ribbon
D. Acrylic medium B. Knife or block is loosely attached
Q16. The most commonly used chelating agent C. Blade is not sharp
for bone marrow trephine section is: D. Inadequate dehydration
A. EDTA Q24. Tear or Scratches in the section is due to:
B. Aqueous nitric acid A. Suboptimal fixation
C. Trichloro acetic acid B. Inadequate dehydration
D. Ion exchange resin C. A nick in the knife-edge
Q17. The decalcifying agent gives yellow colour D. Knife or block is loosely attached
to tissue: Q25. Which is not used as tissue adhesive with
A. EDTA the slide
B. Aqueous nitric acid A. Mayer’s albumin
C. Trichloro acetic acid B. Poly l lysine
D. Formic acid C. 3 Aminopropyltriethoxysilane (ACEP)
Q18. 100% Formalin means D. Limonene
A. 40% formaldehyde Q26. The main purpose of the OCT compound in
B. 100% formaldehyde the frozen section is:
C. 50% formaldehyde A. To hold the tissue over the chuck
D. 10% formaldehyde B. To freeze the tissue
Q19. The sectioning of the paraffin section for C. To do proper staining
staining purposes is: D. To attach the tissue and coverslip
A. Microtomy Q27. Formation of ice crystals within the tissue
B. Embedding causes:
C. Clearing A. Freezing artefact
D. Trimming B. Chattering artefact
Q20. The most commonly used microtome in the C. Thin stripe in tissue
routine laboratory is: D. Crumpled tissue
A. Rotary microtome Q28. The optimal temperature for a frozen sec-
B. Rocking microtome tion of the brain is:
C. Base Sledge microtome A. −15 °C to −20 °C
D. Sliding microtome B. −7 °C to −10 °C
Multiple Choice Questions for the Self-Assessment 331
C. −20 °C to −25 °C C. Higher concentration of dye

D. 5 °C to 10 °C D. Alcoholic solution
Q29. Which one is the acidic dye: Q38. The multivalent metal ion that combines
A. Eosin with certain dyes and helps in the attachment
B. Methyl green of dye with the target tissue is known as:
C. Giemsa A. Mordant
D. Alcian blue B. Argyrophilic substance
Q30. Iron hematein is: C. Metachromatic dye
A. Acidic dye D. Accentuators
B. Basic dye Q39. The lake is:
C. Neutral dye A. A chelate compound formed between the
D. Ligand dye multivalent metal ion and the dye
Q31. The dye uses hydrogen bonding: B. Accentuators and dye complex formation
A. Eosin C. Van der Wall’s force between the dye and
B. Congo red stain tissue
C. Best’s carmine dye to stain glycogen D. Dye-dye aggregation
D. Periodic acid Schiff’s stain Q40. The colour of haematoxylin powder is:
Q32. The frequently used cytoplasmic stain: A. Blue
A. Methyl green B. brownish tan
B. Alcian blue C. Dark red
C. Eosin D. Black
D. Acid fuchsin Q41. Good nuclear stain in exfoliative cytology:
Q33. Which is not a vital stain: A. Mayer’s haematoxylin
A. Methylene blue B. Ehrlich’s haematoxylin
B. Nile red C. Harris’s haematoxylin
C. Brilliant cresyl blue D. Gill’s haematoxylin
D. May Grunwald Giemsa Q42. The popular nuclear counterstain:
Q34. Which is a metachromatic dye: A. Mayer’s haematoxylin
A. Eosin B. Ehrlich’s haematoxylin
B. Hematoxylin C. Harris’s haematoxylin
C. Toluidine blue D. Gill’s haematoxylin
D. Congo red Q43. Removing an excess hydrogen ion from the
Q35. Which of the factors influence staining stain is known as:
intensity: A. Differentiation
A. Dye affinity to the target tissue specimen B. Bluing
B. Thick tissue C. Ripening
C. Prolong reaction time D. Regressive staining
D. All of the above Q44. Amoeba is best demonstrated by:
Q36. The alteration of the original colour of the A. Iron haematoxylin
dye without any change of the chemical struc- B. Ehrlich’s haematoxylin
ture of the dye is known as: C. Mayer’s haematoxylin
A. Metachromasia D. Gill’s haematoxylin
B. Argyrophil reaction Q45. The nuclei look reddish-brown in
C. Progressive staining haematoxylin-eosin stain is due to:
D. Supravital staining A. Too less time in haematoxylin
Q37. The factor that enhances metachromasia: B. Thick section
A. Low pH C. Insufficient bluing
B. Decreased temperature D. Very quick dehydration in alcohol
Q46. Pale coloured cytoplasm by eosin in Q54. Acid mucins are:

haematoxylin-eosin stain is due to: A. Negative for mucicarmine
A. Too much dehydration of the section in B. Alcian blue negative and PAS positive
alcohol C. Alcian blue positive and PAS negative
B. Too thin section D. Positive for both Alcian blue and PAS
C. The eosin solution has pH more than 5 Q55. The mucus glands of the bronchus usually
D. All of the above secrete:
Q47. Verhoeff's iron haematoxylin is the best for A. Neutral mucin
the demonstration of: B. Sialomucin
A. Connective tissue fibres C. Sulphomucin
B. Phospholipid D. None of the above
C. Nuclear chromatin Q56. Mucin of the Pleural mesothelial cell is
D. Photomicrography positive for
Q48. The oxidation of haematoxylin to haematin A. Alcian blue positive and resistant to hyal-
is called as: uronidase enzyme
A. Ripening process B. Alcian blue positive and sensitive to hyal-
B. Bluing uronidase enzyme
C. Mordanting C. Negative for Alcian blue and mucicarmine
D. Differentiation D. Negative for Alcian blue and positive for
Q49. Carbohydrate is best demonstrated by: PAS
A. Periodic Acid Schiff Q57. The combined PAS-Alcian blue staining is
B. Oil red O helpful to distinguish between
C. Feulgen stain A. Mucin and carbohydrate
D. Pearl’s reaction B. Acid mucin from the neutral mucin
Q50. Nucleic acid is best demonstrated by: C. Sulphomucin form sialomucin
A. Periodic Acid Schiff D. None of the above
B. Oil red O Q58. Mucicarmine stain demonstrates:
C. Feulgen stain A. Both acid and neutral mucin
D. Pearl’s reaction B. Only acid mucin
Q51. The stain for Melanin is: C. Only neutral mucin
A. Mucicarmine D. Only sulphomucin
B. Sudan black Q59. Argyrophil cells are best demonstrated
C. Fouchet’s stain by:
D. Schmorl’s stain A. Grimelius stain
Q52. Which one is an essential qualification of B. Fouchet’s stain
good mounting media: C. Prussian Blue Reaction
A. The same refractive index of the coverslip D. Methyl green pyronin stain
and glass slide. Q60. The type of collagen that is present in the
B. It should be acidic in pH glomerular basement membrane of the
C. It should not stick to the slide kidney:
D. It should be immiscible with the clearing A. Collagen I
agent B. Collagen II
Q53. Which one is an aqueous media: C. Collagen III
A. Canada balsam D. Collagen IV
B. DPX Q61. The result of Masson trichrome stain is:
C. Glycerine- glycerol A. Muscle is red, collagen is blue and nuclei
D. Euparal are black
B. Muscle is blue, collagen is red and nuclei B. Gomori methenamine silver

are black C. Phloxine Tartrazine stain
C. Muscle is black, collagen is blue and D. Warthin and Starry technique
nuclei are black Q70. The addition of a small amount of Dimethyl
D. Muscle is red, collagen is brown and sulphoxide (DMSO) in paraffin wax helps in:
nuclei are black A. To increase hardness
Q62. Van Gieson stain demonstrates B. Reduction of melting point
A. Muscle C. Improve the adhesiveness with tissue and
B. Collagen fibres wax
C. Lipid D. Reduces the infiltration time of the wax
D. Mucin and removes the residual clearing agent
Q63. Lymph nodal architectural pattern is better Q71. The best collection device for liquid-based
demonstrated by: cytology for cervical cancer screening is:
A. Masson trichrome stain A. Cervex brush
B. Van Gieson stain B. Wooden spatula
C. Reticulin stain C. Plastic spatula
D. Verhoeff’s stain D. Endocervical brush
Q64. Van Gieson’s stain solution contains: Q72. The best fixative for routine cytology smear
A. Acid Fuchsin and Picric acid is:
B. Phosphomolybdic acid in distilled water A. 10% Buffered formalin
C. Silver nitrate in distilled water B. 95% Ethyl alcohol
D. Iodine in Potassium iodide C. Acetone
Q65. The cross beta-pleated sheet is seen in: D. 2.5% glutaraldehyde solution
A. Reticulin fibres Q73. CSF is usually processed by
B. Collagen IV fibres A. Centrifuge
C. Amyloid fibrils B. Cytocentrifuge
D. Cross striations of skeletal muscle C. Direct smear
Q66. Thioflavin T stain is usually used to D. Millipore technique
demonstrate: Q74. Clear 500 ml urine is usually processed by:
A. Reticulin fibres A. Centrifuge
B. Collagen IV fibres B. Cytocentrifuge
C. Amyloid fibrils C. Direct smear
D. Cross striations of skeletal muscle D. Millipore technique
Q67. Mycobacterium Leprae is best demon- Q75. In Papanicolaou’s staining, the keratin com-
strated by: ponent of the cytoplasm is stained by:
A. Ziehl Neelsen stain A. Harris’s Hematoxylin
B. Fite acid-fast stain B. Orange G
C. Gomori methenamine silver C. Eosin Azure
D. May Grunwald Giemsa D. None of the above
Q68. Pneumocystis jirovecii is best demonstrated Q76. Too pale nuclear stain in Papanicolaou’s
by: staining may be due to:
A. Ziehl Neelsen(Z-N) stain A. If hematoxylin is diluted
B. Fite acid-fast stain B. Too less hydrochloric acid concentration
C. Gomori methenamine silver C. Too less number of dips in hydrochloric
D. May Grunwald Giemsa acid
Q69. Spirochaetes are best demonstrated by: D. Too much bloody smear or high protein
A. May Grunwald Giemsa content of the smear
Q77. The major advantage of Computed tomo- B. Avidin and biotin-conjugated procedure
graphic scan (CT scan) guided fine needle C. Alkaline phosphatase–anti alkaline phos-
aspiration cytology is all except: phatase method
A. High resolution D. Polymer-based labelling method
B. Exact localization of the needle possible Q84. The most preferable sample for the immu-
C. No radiation exposure nocytochemistry is:
D. Deep lesion near vital structure needs CT A. Direct smear
guidance B. Cytospin smear
Q78. The chance of maximum radiation exposure C. Liquid-based cytology smear
is in: D. Cell block
A. Computed tomographic scan (CT scan) Q85. The best fixative for immunocytochemistry
guided fine needle aspiration cytology on cytology
(FNAC) A. 10% neutral buffered formalin
B. Fluoroscopy guided FNAC B. 95% ethyl alcohol
C. Ultrasonography (USG) guided FNAC C. Acetone
D. Endoscopic ultrasound guided FNAC D. Methanol
Q79. An 8 mm submucosal lesion in the duode- Q.86. Endogenous Alkaline phosphatase activity
num is best sampled by: is blocked by:
A. Computed tomographic scan (CT scan) A. 0.5% hydrogen peroxide in absolute
guided fine needle aspiration cytology methanol
(FNAC) B. 5% Sulphuric acid
B. Fluoroscopy guided FNAC C. 10% Nitric acid
C. Ultrasonography (USG) guided FNAC D. 1 mM levamisole in 0.5 M HCl solution
D. Endoscopic ultrasound guided FNAC Q87. Both negative and positive controls are
Q80. A 2 cm diameter cortical bony lesion of the stained on immunohistochemistry is due to:
femur is best sampled by: A. Secondary antibody is cross-reacting with
A. Computed tomographic scan (CT scan) the background substance
guided fine needle aspiration cytology B. Necrotic or crushed tissue
(FNAC) C. Primary antibody is very much diluted and
B. Fluoroscopy guided FNAC is almost absent
C. Ultrasonography (USG) guided FNAC D. Very little or no antigen present in the tissue
D. Endoscopic ultrasound-guided FNAC Q88. No stain in test section but Positive control
Q81. The major advantage of monoclonal anti- is stained properly may be due to:
bodies is: A. Primary antibody is very much diluted and
A. Low production cost is almost absent
B. Directed to a particular epitope of an antigen B. Too much fixation or improper fixation
C. Low specificity C. Chromogen is incompletely dissolved
D. No special training is required to produce D. Primary antibody is over concentrated
the antibodies Q89. All are true for the Open system of auto-
Q82. The strength of the binding capacity of the mated immunostaining platforms except:
antigenic epitope with the corresponding site A. The laboratories can purchase the reagents
of the antibody is called: of their own choice
A. Avidity B. Flexible protocol of immunostaining
B. Affinity C. Les consistent result
C. Sensitivity D. Costly
D. Specificity Q90. Which one is not a marker of mesothelial
Q83. The most sensitive visualization system of cells:
antigen-antibody reaction is: A. Calretinin
A. Peroxidase-anti peroxidase method B. D2 40
C. WT 1 Q99. The daily set-up of the flow cytometer

D. BER EP4 includes all except:
Q91. The cells of adenocarcinoma of colon are: A. Laser alignment
A. CK7 positive and CK 20 negative B. the voltage check-up of the photomulti-
B. CK20 positive and CK7 negative plier tube
C. Negative for both CK7 and CK20 C. The compensation of the emitted
D. Positive for both CK7 and CK20 fluorescence.
Q92. The marker of smooth muscle cells: D. The gating control of the multicolour flow
A. Calponin cytometry
B. Desmin Q100. Which is not the limitation of flow cyto-
C. MyoD1 metric immunophenotyping:
D. Myoglobin A. No morphological correlation
Q93. The marker of vascular tumours: B. Admixture of other material
A. CD30 C. All cases of B-NHL may not always show
B. CD34 light chain restriction
C. CD15 D. The demonstration of co-expression of
D. CD3 two markers in a same cell
Q94. The marker of B lymphoid cells: Q.101. The difference between the wavelength of
A. CD3 excitation spectrum and emission spectrum is
B. CD4 known as:
C. CD8 A. Specific excitation spectrum of light
D. CD19 B. Specific emission spectrum of light
Q95. The marker of peripheral neuroectodermal C. The quantum efficiency
tumor is: D. Stoke’s shift
A. CD 45 Q102. Which one is not a DNA dye
B. CD99 A. Propidium Iodide
C. Myogenin B. Ehidium bromide
D. Desmin C. Fluorescein Iso-thiocyanate (FITC)
Q96. The major function of the sheath fluid in D. Hoechst 33342
flowcytometry is: Q103. A tumor with DNA index 2.5 indicates:
A. To reduce the turbulence of the cells of the A. Hypodiploid aneuploidy
inner sample B. Tetraploid aneuploid
B. To fix the cells C. Hypertetraploid aneuploidy
C. To provide resistance to the central flow of D. Hyperdiploid aneuploidy
cells Q104. The most essential component in digital
D. All of the above pathology is:
Q97. Which one is a permeabilizing agent: A. Electronic barcoding system
A. Phosphate buffer solution B. Whole slide imaging
B. Normal saline C. Laboratory information system
C. Citrate buffer D. Artificial neural network
D. Tween 20 Q105. Which one is essential for whole slide
Q98. The correction of spill over fluorescence in imaging of cytology smear:
multicoloured flow cytometry is called: A. The slide should be stained with hema-
A. Compensation toxylin and eosin stain
B. Stroke shift B. Slide should be barcoded
C. Molar excitation coefficient C. Slide should have a plastic coverslip
D. Quantum yield D. Z-scanning facility should be available
Q106. The major advantage of liquid-based Q112. The total amount of PCR product can be
cytology is: monitored in each thermal cycle:
A. Monolayered clear background A. In situ PCR
B. No manual labour is needed to make the B. Reverse transcriptase PCR
smear C. Inverse PCR
C. Overall a cheaper technique D. Real time PCR
D. Rapid technique Q113. In this PCR, the reaction takes place within
Q107. All are true for Thin Prep technology a cell on the glass slide:
except: A. In situ PCR
A. With the help of gravitational force the B. Reverse transcriptase PCR
cells are sedimented C. Asymmetric PCR
B. A cylinder rapidly rotates within the vial D. Real time PCR
and disperse the cells mechanically Q114. In PCR, The DNA chain extension occurs in:
C. The negative suction pressure is applied A. 72 °C
within the cylinder that drains the fluid of B. 54 °C
the vial C. 94 °C
D. The machine automatically transfers the D. 100 °C
cells from the surface of the vial to the Q115. Regarding fluorescent, in situ hybridisa-
glass slide tion technique, all are true except:
Q108. The high concentration of cells is present A. No need for cell culture
in: B. It cannot be done on paraffin cell block
A. Millipore technique material
B. Centrifuge technique C. Morphology of the cell can be seen along
C. Thin Prep with cytogenetic abnormalities
D. SurePath D. Fluorescent tags are safe and simple
Q109. The DNA Taq polymerase plays crucial Q116. Weak signal or no signal in fluorescent in
role in: situ hybridisation technique may be due to:
A. Denaturation process A. Poorly digested tissue section
B. Annealing B. Low concentration of the probe
C. It extends the DNA strand from 3′ to 5′ C. Faulty hybridization step
direction D. All of the above
D. It helps in the attachment of primer to the Q117. Excess green fluorescence in Comparative
DNA strand genomic hybridization represents:
Q110. Spurious product in PCR may be due to: A. Chromosomal gain or amplification
A. Carry over contamination B. Chromosomal loss
B. Too small amount of DNA template C. Chromosomal translocation
C. Suboptimal number of thermal cycle D. None of the above
D. Denaturation temperature is either too Q118. In this in fluorescent in situ hybridisation
high or too low technique (FISH), the signal amplification is
Q111. At first cDNA is prepared from RNA of detected by the help of fluorescent labelled
the target sample and then this c-DNA ampli- tyramine molecule bound with horse radish
fied by PCR technique: peroxidase:
A. Asymmetric PCR A. Arm FISH
B. Reverse transcriptase PCR B. COD FISH
C. Inverse PCR C. CARD FISH
D. Real time PCR D. Cat FISH
Q119. In this in fluorescent in situ hybridisation C. Nanopore sequencing

technique (FISH), two sets of differently labelled D. Pyrosequencing
probes are used to visualize the fusion gene: Q126. In this DNA sequencing technique, fluo-
A. ACM-FISH rescent labelled nucleotide is used and fluo-
B. COD FISH rescence is measured during incorporation of
C. D-FISH the nucleotide in the nanowell:
D. COMET-FISH A. Nanopore sequencing
Q120. Which is not true for tissue microarray B. DNA Nanoball sequencing
technique: C. Ion Torrent sequencing
A. Amplification of the resource D. Signal Molecule real time sequencing
B. Uniformity in staining conditions Q127. Which is not a major component of liquid
C. Original block cannot be preserved biopsy:
D. Faster, cheaper and reduction of man A. Circulating tumour cells (CTC),
power B. Cell-free DNA (cf-DNA),
Q121. A single row of core tissue in the periph- C. Cell-free RNA (cf-RNA)
eral boundary walls of the tissue microarray D. Mitochondria
technique is kept for: Q128. The major techniques to do in liquid
A. Protection wall biopsy:
B. Control tissue A. Flow cytometry
C. Normal heathy tissue B. Immunocytochemistry
D. Empty cores C. Polymerase chain reaction
Q122. DNA sequencing by the help of chemical D. All of the above
termination of DNA chain: Q129. Self-extraction of various features of the
A. Sanger sequencing image is possible in:
B. Maxam Gilbert technique A. Convolution neural network
C. Pyrosequencing B. Back propagation neural network
D. Illumina Solexa C. Feed forward neural network
Q123. In this DNA sequencing technique, 2′, D. All of the above
3′-dideoxynucleotides are used for the termi- Q130. The most commonly used activation func-
nation of DNA chain: tion in convolutional neural network is:
A. Maxam Gilbert technique A. Liner activation function
B. Sanger sequencing B. Threshold Activation Function
C. Ion semiconductor sequencing C. Sigmoid Neuron unit function
D. Pyrosequencing D. Rectified linear unit (ReLU)
Q124. In this DNA sequencing technique, the Q131. The term black box in artificial neural net-
change of pH during the incorporation of the work indicates:
nucleotides is used: A. Hidden layer
A. Ion semiconductor sequencing B. Black coloured computer processing unit
B. Nanopore sequencing C. Output layer
C. Pyrosequencing D. Input layer
D. Illumina Solexa Q132. In case of supervised learning: in artificial
Q125. In this DNA sequencing technique, single neural net work
stranded DNA passes through charged nano- A. The data is handled by a skilled
hole and the change of voltage is measured: professional
A. DNA Nanoball sequencing B. The system is provided with labelled data
B. Ion semiconductor sequencing along with known output
C. The system does not take any help from B. Spatially Modulated Illumination Micros-
outside and thereby no training data is copy
used C. Two-Photon Microscopy
D. The weightage in between the nodes are D. Confocal microscope
reshuffled randomly and the correct output Q140. In this microscope, pinhole is used to
is rewarded allow light from only a selected focal plane:
Q133. One nano micron is: A. Confocal microscope
A. 10−9 m B. Two-Photon Microscopy
B. 10−6 m C. Spatially Modulated Illumination Micros-
C. 10−3 m copy
D. 10−12 m D. 4Pi Microscopy
Q134. The ability of the microscope to distin- Q141. In electron microscope, the condensers are:
guish two closely spaced objects: A. High quality glass
A. Magnification B. Electromagnetic coil as lenses
B. Resolution C. Quantum field
C. Contrast D. None of the above
D. Numerical aperture Q142. This medium is not used in electron
Q135. Image formation in microscope is: microscope:
A. First Image is virtual and second image is A. Epoxy resin
real B. Acrylic media
B. The second image is virtual and first image C. Polyester resin
is real D. Paraffin wax
C. Both the images are real Q143. The significant cell shrinkage (20%)
D. Both the images are virtual occurs in
Q136. A type of microscope where the light source A. Epoxy resin
is on the top and objective below the sample: B. Polyester resin
A. Inverted microscope C. Acrylic media
B. Phase contrast microscope D. Epon
C. Bright field microscope Q144. The resolution of the electron microscope is:
D. Dissecting microscope A. 10−10 m
Q 137. A three-dimensional image is formed in: B. 10−3 m
A. Phase contrast microscope C. 10−6 m
B. Bright field microscope D. 10−2 m
C. Confocal microscope Q145. The cytopathologist re-screen only the
D. Fluorescent microscope selected high risk cases:
Q138. Gene expression along with the location of A. Proportional rescreening
the proteins in the living cell can be demon- B. Selected re-screening
strated by: C. Automated re-screening
A. Light microscope with a polarizer D. Rapid review
B. Fluorescent microscope Q146. The most common causes of needle stick
C. Electron microscope injury
D. Tagging the protein by Green fluorescent A. Cutting the needle by cutter
protein and then observing by confocal B. Recapping the needle
microscope C. Needle thrown in a hard container
Q139. In this microscope, two identical objec- D. None of the above
tives are used on both sides of the sample to Q147. Sharp materials such as blade, needle, bro-
increase the angular aperture of the objective: ken glasses etc. should be thrown in:
A. 4Pi Microscopy A. Black bag
Answers of Multiple-Choice Questions 339
B. Red bag 23. A

C. Blue bag 24. C
D. Yellow bag 25. D
Q148. The stain for electron microscopy is: 26. A
A. Uranyl acetate 27. A
B. Papanicolaou’s stain 28. B
C. Sudan black 29. A
D. Hematoxylin and Eosin 30. D
Q149. The optimum thickness of the tissue for 31. C
electron microscopy is: 32. C
A. 1 micron 33. D
B. 2–3 micron 34. C
C. 30–50 nanomicron 35. D
D. 200–300 nanomicron 36. A
Q150. Magnesium Chloride in PCR: 37. D
A. Acts as a cofactor of the Taq polymerase 38. A
enzyme 39. A
B. Provides optimum chemical environment 40. B
for the reaction to occur. 41. C
C. Prevents annealing after a certain point 42. A
D. Helps in denaturation of the DNA 43. B
44. A
45. C
Answers of Multiple-Choice 46. D
Questions 47. A
48. A
1. B 49. A
2. D 50. C
3. C 51. D
4. A 52. A
5. A 53. C
6. B 54. C
7. A 55. C
8. B 56. B
9. D 57. B
10. B 58. A
11. A 59. A
12. D 60. D
13. D 61. A
14. B 62. B
15. A 63. C
16. A 64. A
17. B 65. C
18. A 66. C
19. A 67. B
20. A 68. C
21. C 69. D
22. B 70. D
71. A 111. B
72. B 112. D
73. B 113. A
74. D 114. A
75. B 115. B
76. A 116. D
77. C 117. A
78. B 118. C
79. D 119. C
80. B 120. C
81. B 121. A
82. B 122. B
83. D 123. B
84. D 124. A
85. A 125. C
86. D 126. D
87. A 127. D
88. B 128. C
89. D 129. A
90. D 130. D
91. B 131. A
92. A 132. B
93. B 133. A
94. D 134. B
95. B 135. B
96. A 136. A
97. D 137. C
98. A 138. D
99. A 139. A
100. D 140. A
101. D 141. B
102. C 142. D
103. C 143. C
104. B 144. A
105. D 145. B
106. A 146. B
107. A 147. C
108. D 148. A
109. C 149. C
110. A 150. A

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Basic and Advanced

Basic and Advanced

ISBN 978-981-19-6615-6 ISBN 978-981-19-6616-3 (eBook)

Chandigarh, India Pranab Dey

Chandigarh, India Pranab Dey

Part I Basic Laboratory Techniques in Histopathology Laboratory

1 Fixation of Histology Samples: Principles, Methods

2.3 Individual Dehydrating Agent�������������������������������������������������� 20

5.2 Microtome Knife���������������������������������������������������������������������� 43

9.8 Sudan Black B�������������������������������������������������������������������������� 93

Part II Basic Laboratory Techniques in Cytology Laboratory

13.6.2 Processing of Fluid: Urine, Body Fluids, Lavage ���� 133

Part III Advanced Techniques in Histology and Cytology

16.3 Basic Immunology������������������������������������������������������������������ 153

16.12.11 Androgen Receptor ������������������������������������������������ 173

17.10.6 Predicting Response to Monoclonal Therapy ���������� 192

20 Polymerase Chain Reaction: Principle, Technique

23.2 Maxam Gilbert Technique������������������������������������������������������ 250

Part IV Microscopy, Quality Control and Laboratory

28.4 Sample Preparation for TEM�������������������������������������������������� 308

Multiple Choice Questions for the Self-Assessment����������������������������� 329

Pranab Dey is professor in the Department of Cytology and Gynaecologic

a CGH array-based CGH

OCT Optimum cutting temperature

1.1 Introduction 1.2 Ideal Fixative

Table 1.1 Types of fixation and classification of

1.4 Essential Precautions 1.5.2 Cross-linking Fixatives

Fig. 1.1 (a) Schematic

R CH2OH R H R CH2 R H2O

Cross linking the

Fig. 1.2 Schematic

Cross liking with

Fig. 1.3 Schematic

HC o +H2O = HCOH + OsO3

Fig. 1.4 Schematic Alcohol

Cross linking with protein

temperatures (60–65 °C). At higher tempera-

6. Agitation: Agitation increases the penetration

Mechanism: It reacts with various side

1.8 Preparation of Different 1.9 Osmium Tetroxide

Glutaraldehyde is used as a fixative for electron 1.9.2 Disadvantages

Disadvantages Laboratory use: It is commercially available in

Table 1.2 Comparison of different fixatives

• Stock B solution. Table 1.3 Choice of fixative based on technique

The choice of fixatives is very important for spe-

Table 1.4 Fixative of choice according to tissue

Fig. 1.5 Microphotograph

Box 1.6 Formalin Pigment Fuzzy Staining

Fig. 1.6 Tissue

1.11.2 Troubleshooting in Fixation is

Table 1.6 Troubleshooting in fixation

References 3. Jonsson N, Lagerst TS. The effect of formalde-

The next step after fixation is the processing of Water molcule

medium. The optimum thickness of the tissue

Box 2.1 Influencing Factors of Tissue 2.3 Individual Dehydrating

As a dehydrating agent, ethyl alcohol is used 2.3.2 Dehydrating Agents Other

Table 2.1 comparison of different dehydrating agents

Table 2.2 Comparison of clearing agents

2.4.1 Individual Clearing Agent 2.5 Infiltration and Embedding

2.5.1.1 Paraffin Wax 2.5.2 Vacuum Impregnation

2.5.1.2 Advantages of Paraffin Wax

Time duration and the number of

• Size of tissue: Large versus small

Different embedding medium: Paraffin

Chandigarh, India Pranab Dey

Part I Basic Laboratory Techniques in Histopathology Laboratory

1 Fixation of Histology Samples: Principles, Methods

2.3 Individual Dehydrating Agent�� 20

5.2 Microtome Knife�� 43

9.8 Sudan Black B�� 93

Part II Basic Laboratory Techniques in Cytology Laboratory

13.6.2 Processing of Fluid: Urine, Body Fluids, Lavage �� 133

Part III Advanced Techniques in Histology and Cytology

16.3 Basic Immunology�� 153

16.12.11 Androgen Receptor �� 173

17.10.6 Predicting Response to Monoclonal Therapy �� 192

20 Polymerase Chain Reaction: Principle, Technique

23.2 Maxam Gilbert Technique�� 250

Part IV Microscopy, Quality Control and Laboratory

28.4 Sample Preparation for TEM�� 308

Multiple Choice Questions for the Self-Assessment�� 329

1.1 Introduction 1.2 Ideal Fixative

1.4 Essential Precautions 1.5.2 Cross-linking Fixatives

1.8 Preparation of Different 1.9 Osmium Tetroxide

Glutaraldehyde is used as a fixative for electron 1.9.2 Disadvantages

1.11.2 Troubleshooting in Fixation is

Box 2.1 Influencing Factors of Tissue 2.3 Individual Dehydrating

As a dehydrating agent, ethyl alcohol is used 2.3.2 Dehydrating Agents Other

2.4.1 Individual Clearing Agent 2.5 Infiltration and Embedding

2.5.1.1 Paraffin Wax 2.5.2 Vacuum Impregnation

2.5.1.2 Advantages of Paraffin Wax

2.7 Overall Precautions of Tissue

3.3 Tissue Embedding Method

• The mould is covered with a peripheral plastic 3.3.1.1 Method

The double embedding method is a special type 3.3.2.1 Method

3.3.3 Tissue Orientation 3. Multiple sections of tissue, such as endome-

4.1 Introduction Box 4.1 Decalcification

4.5 Endpoint Determination Before using an equal volume of both, the

4.7 Results of Over References

5.2 Microtome Knife

5.2.1 Disposable Knife The desirable hardness of the knife is between

6.1 Introduction 6.2.1 The Principle of the Frozen

6.2.2 Cryostat Machine Proper

6.3 Cryostat Sectioning (c) Tissue appearance: The gross appear-

7.2 Dyes Used for Staining

The dye is the most elementary component of 7.2.1 Types of Dye

1. Acid in basic dye or base in acidic dye such as 7.8.1 Lake

7.8.2.1 Example Accentuators are the group of substances that

7.9.1 Preparation of Buffer

7.9.1.2 Citrate Buffer 7.9.1.4 TRIS-HCl Buffer