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