Basic and Advanced Laboratory Techniques in Histopathology and Cytology

Preface to the Second Edition
Acknowledgements
Contents
About the Author
Abbreviations
Part I: Basic Laboratory Techniques in Histopathology Laboratory
1: Fixation of Histology Samples: Principles, Methods and Types of Fixatives
1.1 Introduction
1.1.1 Aims of Fixation
1.2 Ideal Fixative
1.3 Tissue Changes in Fixation
1.3.1 Types of Fixation
1.3.2 Description of Nature of Fixation
1.4 Essential Precautions for Fixation in General
1.5 Mechanism of Fixation
1.5.1 Dehydration and Coagulation of Protein
1.5.2 Cross-linking Fixatives
1.6 Factors Affecting Fixation
1.7 Commonly Used Fixatives in the Laboratory
1.7.1 Formaldehyde
1.8 Preparation of Different Formalin Solution
1.8.1 Glutaraldehyde
1.9 Osmium Tetroxide
1.9.1 Advantages
1.9.2 Disadvantages
1.9.3 Methyl and Ethyl Alcohol
1.9.4 Acetone
1.9.5 Bouin’s Fixative
1.10 Mercury Salt-containing Fixatives
1.10.1 Zenker’s Fluid
1.10.2 Helly’s Fluid
1.10.3 B5 Fixatives
1.10.4 Fixatives of Choice
1.11 Fixation Artifact
1.11.1 Formalin Pigment
1.11.1.1 Removing the Pigment
Picric acid Method
Schridde’s Method
Verocay”s Method
Mercury Pigments
Fuzzy Staining
Prolonged Fixation
Dichromate Deposit
1.11.2 Troubleshooting in Fixation is Highlighted in Table 1.6
References
2: Processing of Tissue in the Histopathology Laboratory
2.1 Factors that Influence Tissue Processing
2.2 Dehydration
2.3 Individual Dehydrating Agent
2.3.1 Alcohol
2.3.2 Dehydrating Agents Other than Alcohol
2.4 Clearing
2.4.1 Individual Clearing Agent
2.4.2 Other Clear Agents
2.5 Infiltration and Embedding
2.5.1 Different Impregnating Medium
2.5.1.1 Paraffin Wax
2.5.1.2 Advantages of Paraffin Wax
2.5.1.3 Disadvantages of Paraffin Wax
2.5.1.4 Additives and Modification of Paraffin Wax
2.5.2 Vacuum Impregnation Method
2.6 Tissue Processing Methods
2.6.1 Advantages
2.7 Overall Precautions of Tissue Processing
2.7.1 Time schedule for overnight processing (Fig. 2.4)
2.7.2 Manual Tissue Processor
2.7.3 Rapid tissue Processing
2.7.3.1 Advantages
2.7.3.2 Limitations
References
3: Embedding of Tissue in Histopathology
3.1 Embedding Medium
3.2 Different Types of Mould Used for Block
3.3 Tissue Embedding Method
3.3.1 Double Embedding Method
3.3.1.1 Method
3.3.2 Nitrocellulose and Paraffin [2]
3.3.2.1 Method
3.3.3 Tissue Orientation and Embedding
3.4 Tissue marking [3]
References
4: Decalcification of Bony and Hard Tissue for Histopathology Processing
4.1 Introduction
4.2 Factors Controlling the Rate of Decalcification
4.3 The Methods of Decalcification [1]
4.3.1 Acid Decalcification
4.3.2 Von Ebner’s Fluid
4.3.3 Perenyi’s fluid
4.3.4 Weak Acids
4.3.5 Trichloroacetic acid
4.3.6 Chelating Agents
4.3.6.1 EDTA Solution
4.3.6.2 Advantages
4.3.6.3 Disadvantages
4.4 Ion Exchange Resin Method
4.4.1 Advantages
4.4.2 Electrolysis Method
4.4.3 Surface Decalcification
4.5 Endpoint Determination of Decalcification
4.6 Results of Under Decalcification
4.7 Results of Over Decalcification
References
5: Tissue Microtomy: Principle and Procedure
5.1 Introduction
5.2 Microtome Knife
5.2.1 Disposable Knife
5.2.1.1 Advantages
5.2.1.2 Disadvantages
5.2.2 Materials Used in Knife
5.2.3 Angles of Knife
5.3 Microtome Knife Sharpening
5.3.1 Manual Method
5.3.2 Factors Involved in Cutting
5.4 Sectioning the Paraffin Block
5.4.1 Steps of Tissue Sectioning (Fig. 5.7)
5.4.2 How to Recover the Dried Tissue?
5.4.2.1 Formol-Glycerol
Reference
6: Frozen Section: Principle and Procedure
6.1 Introduction
6.2 Indications of Frozen Sections
6.2.1 The Principle of the Frozen Section
6.2.2 Cryostat Machine Proper (Fig. 6.1)
6.3 Cryostat Sectioning
6.4 Staining
6.4.1 H & E Staining
6.4.2 Toluidine Blue Stain
6.5 Factors Affecting the Good Quality Section
References
7: Staining Principle and General Procedure of Staining the Tissue
7.1 Introduction
7.2 Dyes Used for Staining
7.2.1 Types of Dye
7.2.2 Types of Dye on the Basis of Chemical Structures and Chromophore Groups
7.3 Mechanisms and Theory of Staining
7.4 Factors Influencing Staining
7.5 The Nomenclature Used Regarding Dye
7.5.1 Applications
7.6 Metachromasia [4]
7.6.1 Metachromatic Dyes
7.6.2 Applications of Metachromasia
7.7 Progressive and Regressive Staining
7.8 Mordant
7.8.1 Lake
7.8.2 Type of Application of Mordant
7.8.2.1 Example
7.8.3 Accentuators
7.9 Staining Procedure
7.9.1 Preparation of Buffer Solutions
7.9.1.1 Molar Solution
7.9.1.2 Citrate Buffer
7.9.1.3 Phosphate Buffer
7.9.1.4 TRIS-HCl Buffer
References
8: Haematoxylin and Eosin Stain of the Tissue Section
8.1 Introduction
8.2 Haematoxylin
8.3 Bluing
8.3.1 Scott’s Tap Water
8.3.2 Preparation of Different Haematoxylins and Their Properties
8.3.2.1 Harris’s Alum Haematoxylin
8.3.2.2 Preparation of the Stain
8.3.2.3 Steps
8.3.2.4 Cautions
8.3.3 Mayer’s Haematoxylin
8.3.3.1 Preparation of the Stain
8.3.3.2 Steps
8.3.4 Ehrlich’s Haematoxylin
8.3.4.1 Preparation of the Stain
8.3.4.2 Steps of Preparation
8.3.5 Cole’s Haematoxylin
8.3.5.1 Preparation of the stain
8.3.5.2 Steps of Preparation
8.4 Counterstain by Eosin
8.5 Routine Haematoxylin and Eosin stain
8.5.1 Requirements
8.5.2 Steps (Fig. 8.2)
8.5.3 Staining Time of Different Haematoxylin
8.5.3.1 Staining time of Haematoxylin Depends on the Various Factors
8.6 Iron Haematoxylin
8.6.1 Heidenhain’s Iron Haematoxylin
8.6.1.1 Application
8.6.1.2 Preparation
8.6.1.3 Staining
8.6.2 Verhoeff’s Iron Haematoxylin
8.6.2.1 Preparation
8.6.3 Tungsten Haematoxylin
8.6.3.1 Preparation
8.6.3.2 Staining
8.6.3.3 Result
8.7 Clearing the Smear
8.8 Mounting
8.8.1 Disadvantage
8.8.2 Application of Mounting Medium
8.8.2.1 Cautions
8.8.3 Coverslip
8.8.4 The Resin-coated Plastic Film
8.8.4.1 Advantages
8.8.5 Restaining
References
9: Special Stains for the  Carbohydrate, Protein, Lipid, Nucleic Acid and Pigments
9.1 Introduction
9.2 Carbohydrates
9.2.1 Simple Carbohydrates
9.2.1.1 Locations
9.2.2 Significance of Mucin Demonstration
9.3 Staining of Different Carbohydrates
9.3.1 Glycogen
9.3.2 Periodic Acid Schiff’s (PAS) Stain [4]
9.3.3 Indications to do PAS stain
9.3.4 Principle (Fig. 9.3)
9.3.4.1 Components of Solutions
9.3.4.2 Preparation
9.3.4.3 Steps
9.3.4.4 Result
9.3.5 Alcian Blue
9.3.5.1 Indications
9.3.5.2 Solution
Alcian Blue Solution
Neutral Red Solution
Steps to Make Solution
9.3.5.3 Method of Staining
9.3.6 Combined PAS-Alcian Blue Staining
9.3.6.1 Method of staining
9.4 Result
9.4.1 Mucicarmine Stain [5]
9.4.1.1 Indications
9.4.1.2 Solution
Southgate Mucicarmine stock solution
Preparation
Mucicarmine working solution
Steps
9.5 Colloidal Iron
9.5.1 Colloidal Ion Stalk Solution
9.5.1.1 Method
9.5.1.2 Result
9.5.2 Lipids
9.6 Fixation
9.7 Stains
9.7.1 Oil red O [6]
9.7.2 Preparation of Oil Red O Stain
9.7.2.1 Stock solution
9.7.2.2 Working solution
9.7.2.3 Steps
9.8 Sudan Black B [7]
9.8.1 Solution
9.8.2 Steps
9.8.3 Ferric haematoxylin for Phospholipid [8]
9.8.3.1 Preparation of solution
Solution 1
Solution 2
9.8.3.2 Working solution
9.8.3.3 Steps
9.9 Nucleic Acid and Proteins
9.9.1 Nucleic Acids
9.9.2 Proteins
9.9.3 Feulgen Stain [9]
9.9.4 Methyl Green Pyronin Stain [10]
9.9.5 Pigments
9.9.6 Hemosiderin Pigment
9.9.6.1 Prussian Blue Reaction (Pearl’s Reaction) for Ferric Iron
9.9.7 Bile Pigment
9.9.7.1 Fouchet’s Stain
Steps
9.9.8 Argyrophil Pigments
9.9.8.1 Grimelius Staining [12]
9.9.8.2 Principle
9.9.8.3 Acetate Buffer
9.9.9 Melanin
9.9.9.1 Masson Fontana Method
9.9.10 Schmorl’s Stain [13]
9.9.11 Calcium
9.9.12 Von Kossa Technique [14, 15]
9.9.13 Formalin Pigment
9.9.14 Malarial Pigment
9.9.15 Starch
References
10: Connective Tissue Stain: Principle and Procedure
10.1 Fibrous Part of Connective Tissue
10.1.1 Reticulin Fibres
10.1.2 Elastic Fibres
10.1.3 Basement Membrane
10.2 Stains
10.2.1 Masson Trichrome [1]
10.2.1.1 Indications and Advantages
10.2.1.2 Solution
Bouin’s Fixative
Weigert’s Haematoxylin
Acid Fuchsin Solution
Phosphomolybdic Acid Solution (1%)
Aniline Blue Solution
Steps to Stain
Result (Fig. 10.1b)
Warning Note
10.2.2 Van Gieson Stain [2]
10.2.2.1 Indications
10.2.2.2 Van Gieson’s Stain Solution
10.2.2.3 Steps of Staining
10.2.2.4 Result (Fig. 10.2)
10.2.2.5 Warning Notes
10.2.3 Reticulin Stain
10.2.3.1 Indications
10.2.4 Gordon and Sweet’s Method for Reticulin Stain [3]
10.2.4.1 Solution
Acidified Potassium Permanganate (1%)
Oxalic Acid (2%)
Iron Alum (2%)
10% Formaldehyde
Gold Chloride (0.2%)
Sodium Thiosulphate (5%)
Silver Nitrate (10%)
10.2.4.2 Steps to Stain
10.2.4.3 Result (Fig. 10.4)
10.2.4.4 Warning Notes
10.3 Elastic Fibres
10.3.1 Verhoeff’s Stain for Collagen [4]
10.3.1.1 Solutions
10.3.2 Final Verhoeff’s solution
10.3.2.1 Steps of Staining
10.3.2.2 Results
10.3.3 Weigert’s Resorcin-Fuchsin Stain [5]
10.3.3.1 Steps of Staining
10.3.3.2 Result
10.3.4 Orcein for Elastic Fibres
10.3.4.1 Steps of Staining
10.3.4.2 Result
10.3.5 Fibrin and Cross Striation of the Muscle
10.3.5.1 Phosphotungstic acid Haematoxylin (PTAH) [6, 7]
Solutions
10.3.5.2 Steps of Staining
10.3.5.3 Result (Fig. 10.5)
References
11: Amyloid Staining
11.1 Introduction
11.2 Primary Amyloidosis
11.3 Stains for Amyloid
11.3.1 Alkaline Congo Red Stain [4]
11.3.1.1 Solution
1% Sodium Hydroxide
Saturated Sodium Chloride in Ethanol (80%)
Alkaline Alcohol Sodium Chloride Solution
Alkaline Congo Red Stock Solution
Working Solution of Congo Red
11.3.1.2 Steps of Staining
11.3.1.3 Result (Fig. 11.2)
11.3.2 Congo Red Stain by Highman [5]
11.3.2.1 Congo red Solution
11.3.2.2 Potassium Hydroxide (0.2%)
11.3.2.3 Steps
11.3.2.4 Result
11.3.3 Thioflavin T Stain [6]
11.3.3.1 Thioflavin T Solution
11.3.3.2 Steps
11.3.3.3 Result
References
12: Stains for the Microbial Organisms
12.1 Bacteria
12.1.1 Gram’s Stain [4, 5]
12.1.1.1 Reagents
Crystal violet solution
Lugol’s Iodine
Basic Fuchsin
12.1.1.2 Steps of Staining
12.1.1.3 Result
12.2 Ziehl Neelsen Stain
12.2.1 Reagents
12.2.1.1 Carbol-fuchsin
12.2.1.2 Methylene Blue
12.2.1.3 Acid Alcohol
12.2.2 Steps of Staining
12.2.2.1 Result (Fig. 12.1)
12.3 Fite Acid-fast Stain for Leprosy [6]
12.3.1 Methylene blue
12.3.2 Carbol-fuchsin
12.3.3 Sulphuric Acid (5%)
12.3.4 Xylene in Peanut Oil Solution
12.3.5 Steps of Staining
12.3.5.1 Result
12.4 Fungal Infection
12.4.1 Grocott’s Methenamine Silver [7]
12.4.2 Reagents
12.4.2.1 Stock Solution of Methenamine Silver
12.4.2.2 Sodium Borate Solution (5%)
12.4.2.3 Methenamine Silver Working Solution
12.4.2.4 Sodium Thiosulphate Solution (5%)
12.4.2.5 Sodium Bisulphite (1%)
12.4.2.6 Chromic Acid (2%)
12.4.2.7 Gold Chloride Solution (0.1%)
12.4.2.8 Stock Solution of Light Green (0.2%)
12.4.2.9 Light Green Working Solution
12.4.3 Steps of Staining
12.4.4 Result (Fig. 12.3)
12.5 Spirochaetes
12.5.1 Warthin and Starry Technique [8]
12.5.1.1 Reagents
Buffer Solution
Silver Solution
Developer Solution
12.5.1.2 Steps
12.5.2 Viral Inclusions
12.5.2.1 Phloxine Tartrazine Stain
Reagents
12.5.2.2 Steps of Staining
12.5.2.3 Result
References
Part II: Basic Laboratory Techniques in Cytology Laboratory
13: Cytology Sample Procurement, Fixation and Processing
13.1 Introduction
13.2 Sample Collection
13.2.1 Cervical Cytology [1, 2]
13.2.1.1 Preparation of the Patient
13.2.1.2 Collection Devices
13.2.2 Collection Proper (Box 13.1)
13.3 Respiratory Samples [3, 4]
13.3.1 Sputum Sample
13.3.2 Bronchial Brush
13.3.3 Bronchial Wash
13.3.4 Bronchoalveolar Lavage (BAL)
13.3.5 Transbronchial Needle Aspiration
13.3.6 Gastric Brush
13.3.7 Gastric Lavage
13.3.8 Endoscopic Ultrasound-guided (EUS) FNAC
13.3.8.1 Bladder Wash
13.3.8.2 Ureteric Urine
13.3.8.3 Urinary Brush
13.3.9 Effusion Fluid Sample
13.3.10 CSF and Vitreous Fluid
13.4 Fixation
13.4.1 Time of Fixation
13.4.1.1 Coating Fixatives
13.4.1.2 Major Advantages
13.4.1.3 Precautions
13.4.2 Special Fixatives
13.4.2.1 Hemorrhagic Fluid
13.4.2.2 Ingredients of Carnoy’s Fixative
13.4.2.3 Fixatives for Liquid-based Preparation
13.4.2.4 Fixatives for Cell Block
13.4.2.5 Fixatives for Electron Microscopy
13.4.2.6 Preservation of Sample Prior to Processing
13.5 Processing of Laboratory Samples
13.5.1 Receiving the Sample
13.5.2 Glass Slides and Liquid Sample
13.5.2.1 Precautions for Liquid Samples
13.5.2.2 Unique Identification Number
13.5.2.3 Laboratory Bar Code
13.6 Processing
13.6.1 Processing of Sputum (Fig. 13.7)
13.6.2 Processing of Fluid: Urine, Body Fluids, Lavage
13.6.3 The Basic Principle of Centrifuge
13.6.4 Millipore Filtration
13.6.5 Processing of Hemorrhagic Fluid
13.6.6 Cell Block [5]
13.6.7 Compact Cell Block Technique
References
14: Routine Staining in Cytology Laboratory
14.1 Papanicolaou’s Stain [1]
14.1.1 Dyes Used in Papanicolaou’s Staining
14.1.2 Principle of Basic Steps (Fig. 14.1)
14.1.3 Papanicolaou’s Staining Steps
14.1.3.1 Results (Figs. 14.2, 14.3, and 14.4)
14.1.3.2 Hematoxylin Solution for Papanicolaou’s Stain
14.1.3.3 EA Solution
14.1.3.4 Orange G Solution
14.1.4 Bluing Solution
14.1.4.1 Lithium Carbonate Solution
Stock Solution
Working Solution
14.2 Precautions to Be Taken in Papanicolaou’s Staining
14.2.1 Staining Solutions
14.2.2 Coverslip
14.2.3 Staining Proper
14.2.4 De-staining and Re-staining the Smear
14.3 May Grunwald Giemsa Stain
14.3.1 Steps
14.3.2 Storage of Slides
Reference
15: The Basic Technique of Fine Needle Aspiration Cytology
15.1 Introduction
15.2 Technique Proper
15.2.1 Equipment (Fig. 15.2)
15.3 Fine Needle Aspiration Procedure (Box 15.2)
15.3.1 Clinical History
15.3.2 Aspiration (Fig. 15.3)
15.3.3 Smear Preparation
15.4 Fine Needle Sampling
15.4.1 Steps
15.4.2 Limitations
15.5 FNAC of Deep-Seated Lesions
15.5.1 Major Indications of Deep Seated Guided FNAC
15.5.2 USG Guided FNAC
15.5.3 Steps
15.6 Transrectal FNAC of the Prostate
References
Part III: Advanced Techniques in Histology and Cytology Laboratories
16: Immunocytochemistry in Histology and Cytology
16.1 Introduction
16.2 Basic Principles
16.3 Basic Immunology
16.4 Detection System
16.5 Peroxidase-Anti Peroxidase Method (Fig. 16.5)
16.5.1 Advantage
16.6 Avidin and Biotin Method
16.6.1 Advantage
16.6.2 Disadvantage
16.7 Avidin and Biotin-Conjugated Procedure
16.7.1 Advantages
16.7.2 Disadvantages
16.8 Biotin-Streptavidin Method
16.8.1 Advantages
16.8.2 Alkaline Phosphatase–Anti Alkaline Phosphatase Method [5, 6]
16.8.3 Advantages
16.8.4 Polymer-Based Labelling Method
16.8.5 Advantages
16.8.6 Catalyzed Signal Amplification (Tyramine Signal Amplification)
16.8.7 Steps
16.8.8 The Sample of Tissues for Immunocytochemistry
16.8.8.1 Histopathology
16.8.8.2 Cytology
16.8.9 Sample Collection
16.8.9.1 Histopathology
16.8.9.2 Fixation
16.8.10 Precautions to Have a Good Fixation
16.8.10.1 Antigen Retrieval
Microwave Retrieval
16.8.10.2 Requirements
16.8.10.3 Steps
16.8.10.4 Warning
16.8.10.5 Pressure Cooker Heating
16.8.10.6 Requirements
16.8.10.7 Steps
16.8.10.8 Precautions
16.8.10.9 Water Bath Heating
16.8.10.10 Requirements
16.8.10.11 Steps
16.9 Immunocytochemistry Technique
16.9.1 Control
16.9.2 Steps
16.9.2.1 Chromogen
16.9.2.2 Tris Buffered Saline
16.10 Selection of Primary Antibody
16.10.1 The Dilution of the Primary Antibody
16.10.2 Quality Control
16.10.3 Troubleshooting in Immunocytochemistry
16.11 Automated Immunostaining Platform
16.11.1 Types of Automated Immunostaining Platforms
16.11.2 Reagents Delivery Systems
16.11.3 Clinical Applications of Immunochemistry
16.12 Diagnostic Immunocytochemistry
16.12.1 Mesothelial Markers
16.12.1.1 Calretinin
16.12.1.2 HBME-1
16.12.1.3 Wilms’ Tumour Gene 1 (WT-1)
16.12.1.4 D2-40
16.12.2 Adenocarcinoma Markers in Effusion Fluid
16.12.2.1 BER EP4
16.12.2.2 Carcinoembryonic Antigen (CEA)
16.12.2.3 MOC 31
16.12.2.4 Leu M1 (CD 15)
16.12.3 Different Epithelial Markers
16.12.3.1 Cytokeratin
16.12.4 Mesenchymal Markers
16.12.4.1 Skeletal Muscle
16.12.4.2 Smooth Muscle
16.12.4.3 Peripheral Nerve Sheath Markers
16.12.4.4 Marker of Vascular Tumours
16.12.4.5 Marker of Miscellaneous Soft Tissue Tumour
16.12.5 Neuroendocrine Markers
16.12.6 Lymphoid Markers
16.12.7 Melanoma Markers
16.12.8 Germ Cell Markers
16.12.9 Site-specific Antibody in Different Epithelial Malignancies
16.12.10 PSA and Androgen Receptor
16.12.11 Androgen Receptor
16.12.12 TTF
16.12.13 Estrogen and Progesterone Receptors (ER and PR)
16.13 Immunocytochemistry of Round Cell Tumour
16.14 Immunocytochemistry for Therapy and Management
16.14.1 Breast Carcinoma
16.14.2 Estrogen and Progesterone Receptors
16.14.3 Her 2/Neu
16.15 Gastrointestinal Stromal Tumor
16.15.1 Lung Carcinoma
References
17: Flow Cytometry: Basic Principles, Procedure, and Applications in Pathology
17.1 Introduction
17.2 Principle of Flow Cytometry
17.3 The Flow Cytometer Instrument
17.3.1 Light Emission and Scattering
17.4 Flow Cytometric Cell Sorting
17.5 Dye Used
17.5.1 Fluorochrome Dye for Nucleic Acid [2]
17.6 Samples for Flow Cytometry
17.6.1 Cytology Samples
17.6.1.1 Histology Samples
17.6.2 Single-cell Preparation
17.6.3 Cellular Fixation
17.6.4 Permeabilization
17.6.5 RBC Lysing Solution
17.6.6 Control
17.6.7 Sample Processing
17.6.7.1 DNA Flow Cytometry [1]
17.6.7.2 Stock Solution of Propidium Iodide (PI)
17.6.8 Flow Cytometric Immunophenotyping (FCI)
17.6.8.1 Direct Stain
17.6.8.2 Indirect Staining Procedure [2, 3] (Fig. 17.4):
17.6.9 Data Aquisition [2]
17.6.10 Data Display and Interpretation
17.6.11 Quality Control
17.7 Targets of Application
17.8 DNA Content and Ploidy Analysis
17.8.1 Basic Principle
17.9 Clinical Application
17.9.1 DNA Content and Diagnosis
17.9.2 DNA Content and Prognosis of the Patients
17.10 Immunophenotyping of Lymphomas
17.10.1 Diagnosis:
17.10.2 Sub-classification of Lymphomas:
17.10.3 Limitations of FCI
17.10.4 Flow Cytometry Features of Different Lymphomas
17.10.5 Diagnosis of Other Lesions by FCI
17.10.6 Predicting Response to Monoclonal Therapy
17.10.7 Detection of Minimal Residual Disease
17.10.7.1 Steps [17]
17.10.7.2 Apoptosis [18, 19]
17.10.8 Assessment of Sub-G1 Fraction of Apoptotic Cells
17.10.9 Apoptosis Detection by Annexin V Assay
References
18: Digital Pathology
18.1 Introduction
18.2 What Is Digital Pathology?
18.2.1 Comparison of Traditional Pathology and Digital Pathology
18.2.2 Workflow of Digital Pathology
18.2.3 Basic Instruments and Software in Digital Pathology (Box 18.1)
18.3 Whole Slide Imaging (WSI)
18.3.1 Hardware
18.3.2 Software
18.3.3 Commercially Available WSI
18.3.4 Advantages of Digital Slides
18.3.5 Disadvantages of Digital Slides
18.3.6 Concordance of Glass Slides and Digital Slides
18.3.7 Guidelines of Clinical Diagnostic Application of WSI
18.3.8 Applications of Digital Pathology
18.3.9 Limitations and Challenges of Digital Pathology
References
19: Automation in the Laboratory and Liquid-Based Cytology
19.1 Introduction
19.2 Advantages of Automation in Laboratory
19.2.1 The Various Stages of Automation
19.2.2 Tissue Processing
19.3 Automated Immunostaining Platform (AIP)
19.3.1 Digitization of Slide
19.4 Cytology Processing
19.4.1 Advantages of LBC over Conventional Smear
19.4.2 Limitations of Liquid-based Cytology
19.4.3 Collection Procedure of LBC
19.5 Sample Processing
19.5.1 ThinPrep (Cytic, UK) (Fig. 19.5)
19.5.1.1 Dispersion and Collection of the Cells on the Filter
19.5.1.2 SurePath Test (Fig. 19.6)
Cell Enrichment
Resuspension
Cell Sedimentation
19.6 Comparison of These Two Techniques
19.7 Automated Screening Devices in Cytology
19.7.1 BD FocalPoint GS Imaging System [6, 7]
19.7.2 BD FocalPoint GS Review Station
19.7.3 HOLOGIC ThinPrep Imaging System [8, 9]
19.7.4 Review Scope
19.7.5 Comparison of Manual and Automated Devices
19.8 Artificial Neural Network (ANN) in Pathology
References
20: Polymerase Chain Reaction: Principle, Technique and Applications in Pathology
20.1 Introduction
20.2 What is PCR and How Does it Work?
20.3 Steps of PCR
20.3.1 Essential Ingredients of PCR
20.4 Procedure Proper [3, 4]
20.4.1 Basic Precautions
20.4.2 Equipment
20.4.3 Addition of the Ingredients in a 50 μL PCR Tube
20.4.4 Remember
20.4.5 Thermal Cycling
20.4.5.1 Standard Steps
20.4.6 Purification of the Amplified Product
20.4.7 Troubleshooting
20.4.8 Enhancing PCR Products Formation
20.5 Types of PCR
20.6 Applications of PCR
References
21: Fluorescent In Situ Hybridisation Techniques in Pathology: Principle, Technique and Applications
21.1 Introduction
21.1.1 Applications of FISH
21.1.2 The Principles of FISH
21.2 Steps to do FISH [5–7]
21.2.1 Histology and Cytology Specimen
21.3 Troubleshooting
21.3.1 Different Types of FISH
21.3.2 Basic Principles (Fig. 21.3)
21.3.3 CGH Method [13]
21.3.4 Array-based CGH [14]
21.3.4.1 Basic Steps of a-CGH (Fig. 21.4)
21.4 Different Other Varieties of FISH [15, 16]
References
22: Tissue Microarray in Pathology: Principal, Technique and Applications
22.1 Introduction
22.2 Tissue Microarray Technique
22.3 TMA Construction and Generation of Grid
22.4 Designing the Grid [4]
22.5 Clinical Applications of TMA
References
23: Sanger Sequencing and Next Generation Gene Sequencing: Basic Principles and Applications in Pathology
23.1 Sanger Sequencing
23.1.1 Reagents Needed
23.1.2 Main Steps
23.1.3 Limitations
23.2 Maxam Gilbert Technique
23.2.1 Main Steps
23.2.2 Limitations
23.3 Next-generation Sequencing
23.3.1 Second-generation Sequencing
23.3.2 Advantages
23.3.3 Limitations
23.4 Illumina Solexa
23.4.1 Advantage
23.4.2 Limitations
23.5 Ion Semiconductor Sequencing (Ion Torrent)
23.5.1 Advantages
23.5.2 Disadvantage
23.6 Third Generation
23.6.1 Single-Molecule Real-Time Sequencing
23.6.1.1 Advantages
23.6.1.2 Disadvantage
23.7 Fourth Generation Sequencing
23.7.1 Nanopore Sequencing (Fig. 23.8)
23.7.1.1 Advantage
23.7.2 Other Technologies
23.7.2.1 Advantages
23.7.3 DNA Nanoball Sequencing
23.7.3.1 DNA Nanoball Creation
23.7.3.2 Loading Onto Flow Cell for Sequencing
23.7.4 RNA-Seq
23.7.5 Applications of NGS
23.7.5.1 Limitations
23.7.6 Limitations
References
24: Liquid Biopsy: Basic Principles, Techniques and Applications
24.1 Introduction
24.2 Conventional Biopsy Versus Liquid Biopsy?
24.3 The Components of Liquid Biopsy
24.4 Enrichment of the Contents of Liquid Biopsy
24.5 The Molecular Techniques to Do in Liquid Biopsy
24.6 Clinical Applications [2, 3]
References
25: Artificial Neural Network in Pathology: Basic Principles and Applications
25.1 Introduction
25.2 ANN Versus Ordinary Computer
25.3 Artificial Neural Network Versus Biological Neuron
25.4 Activation
25.5 Learning of ANN
25.5.1 Multilayer Perceptron Architecture
25.5.2 Steps to Building an ANN
25.5.2.1 The Different Types of the Artificial Neural Network
25.5.2.2 Deep Learning (DL) Neural Network
25.5.2.3 Accuracy of the ANN Model
25.5.3 Main Challenges of ANN
25.5.4 Application of ANN
25.5.4.1 Specific Applications of ANN
25.5.5 Limitations of ANN
References
Part IV: Microscopy, Quality Control and Laboratory Organization
26: Compound Light Microscope and Other Different Microscopes
26.1 Light
26.2 Colours
26.3 Image Generation and Human Vision
26.3.1 Image Formation by the Light Microscope (Fig. 26.6)
26.3.1.1 Anatomical Components of a Light Microscope (Fig. 26.7)
26.4 Optical Components [2, 3]
26.5 How to Take Care and Handle Your Microscope
26.6 Other Types of Microscope
26.6.1 Darkfield Microscope
26.6.1.1 Use
26.6.2 Bright-Field Microscope
26.6.3 Phase-contrast Microscope
26.6.3.1 Applications
26.6.4 Inverted Microscope
26.6.5 Dissecting Microscope
26.6.5.1 Principle
26.6.5.2 Applications
References
27: Fluorescence Microscope, Confocal Microscope and Other Advanced Microscopes: Basic Principles and Applications in Pathology
27.1 Transmitted Fluorescent Microscope
27.2 Incident Fluorescent Microscope
27.2.1 The Dye Used in Fluorescence Microscope
27.2.2 Applications of Fluorescence Microscope
27.3 Confocal Microscopy
27.3.1 Advantages (Box 27.1)
27.4 Limitations of CFM
27.5 Applications of CFM [5–7]
27.6 Two-Photon Microscopy
27.6.1 Advantages
27.7 4Pi Microscopy
27.8 Spatially Modulated Illumination Microscopy
27.8.1 Scanning Probe Microscope
27.8.1.1 Scanning Tunnelling Microscope (STM) [11]
27.8.1.2 Advantages of AFM
27.8.1.3 Limitations
27.8.2 Laser Capture Microdissection
27.8.2.1 Advantages
27.8.2.2 Limitations
27.8.2.3 Applications
27.8.3 Expression Microdissection
27.8.3.1 Principle and Steps (Fig. 27.12)
27.8.3.2 Advantages
References
28: Electron Microscopy: Principle, Components, Optics and Specimen Processing
28.1 Microscope Column and Electronic Optics (Fig. 28.2b)
28.2 Specimen and Electron Interaction
28.2.1 Backscattered Electrons
28.2.2 Excited Electrons of the Atom
28.3 Electron Interaction in the Transmission Electron Microscope
28.4 Sample Preparation for TEM
28.4.1 Combined Fixation Technique
28.4.2 Embedding
28.4.3 Knives
28.4.4 Staining of the Sections
28.4.4.1 Lead Stain
28.4.4.2 Stain
28.4.4.3 Reynold’s Lead Citrate solution
28.4.5 Uranyl Salt
28.5 Scanning Electron Microscopy [4]
28.5.1 Operational Principle
References
29: Quality Control and Laboratory Organization
29.1 Introduction
29.2 Quality Control
29.2.1 Gold Standard
29.2.2 Record Keeping
29.3 Audit
29.3.1 The Beneficial Points of the Internal Audit
29.3.2 Stages of Audit
29.3.3 Components of Audit
29.4 External Quality Assurance
29.4.1 Laboratory Accreditations
29.4.2 Pre-requisite for Laboratory Accreditation
29.4.3 Process of Accreditatation
29.4.4 Advantages of Laboratory Accreditations
29.5 Laboratory Organization
References
30: Laboratory Safety and Laboratory Waste Disposal
30.1 Laboratory Waste Disposal
30.1.1 Basic Ways to Waste Management
30.1.2 Steps of Biomedical Waste Disposal
References
Multiple Choice Questions for the Self-Assessment
Answers of Multiple-Choice Questions