Histotechnology: A Self-Instructional Text

Cover Page
Title Page
Table of Contents
Preface
Chapter 1: Fixation
Definition
Functions of Fixatives
Actions of Fixatives
Factors Affecting Fixation
TEMPERATURE
SIZE
VOLUME RATIO
TIME
CHOICE OF FIXATIVE
PENETRATION
TISSUE STORAGE
pH
OSMOLALITY
Reactions of the Cell with Fixatives
THE NUCLEUS
PROTEINS
LIPIDS
CARBOHYDRATES
Simple Aqueous Fixatives or Fixative Ingredients
ACETIC ACID
FORMALDEHYDE
GLUTARALDEHYDE
GLYOXAL
MERCURIC CHLORIDE
OSMIUM TETROXIDE
PICRIC ACID
POTASSIUM DICHROMATE
ZINC SALTS
Other Fixative Ingredients
Compound or Combined Fixatives
B-5 FIXATIVE
ZENKER AND HELLY (ZENKER-FORMOL) SOLUTIONS
ZINC FORMALIN SOLUTIONS
Nonaqueous Fixatives
ACETONE
ALCOHOL
Transport Solutions
Removal of Fixation Pigments
Troubleshooting Fixation Problems
AUTOLYSIS
INCOMPLETE FIXATION
References
LEARNING ACTIVITIES
Chapter 2: Processing
Dehydration
ALCOHOLS
UNIVERSAL SOLVENTS
Clearing
XYLENE
TOLUENE
BENZENE
CHLOROFORM
ACETONE
ESSENTIAL OILS
LIMONENE REAGENTS (XYLENE SUBSTITUTE)
ALIPHATIC HYDROCARBONS (XYLENE SUBSTITUTE)
UNIVERSAL SOLVENTS
OTHER CLEARING AGENTS
Infiltration
PARAFFIN
WATER-SOLUBLE WAXES
CELLOIDIN
PLASTICS
AGAR AND GELATIN
30% SUCROSE
Troubleshooting Processing
PRECIPITATE IN THE PROCESSOR CHAMBER AND INTHE TUBING
OVERDEHYDRATION
POOR PROCESSING
SPONGE ARTIFACT
TISSUE ACCIDENTALLY DESICCATED
Embedding and Specimen Orientation
Troubleshooting Embedding
SOFT MUSHY TISSUE
INCORRECT ORIENTATION
TISSUE CARRYOVER
TISSUE NOT EMBEDDED AT THE SAME LEVEL
PIECES OF TISSUE MISSING FROM THE BLOCK
Special Techniques in Processing
DECALCIFICATION
TROUBLESHOOTING DECALCIFICATION
FROZEN SECTIONS
TROUBLESHOOTING PROCESSING TISSUE FORFROZEN SECTIONS
References
LEARNING ACTIVITIES
Chapter 3: Instrumentation
Microscopes
LIGHT MICROSCOPE
POLARIZING MICROSCOPE
PHASE-CONTRAST MICROSCOPE
DARKFIELD MICROSCOPE
FLUORESCENCE MICROSCOPE
ELECTRON MICROSCOPE
Microtomes
ROTARY MICROTOME
SLIDING MICROTOME
CLINICAL FREEZING MICROTOME
MICROTOME BLADES
TROUBLESHOOTING MICROTOMY
Cryostat
Tissue Processors
CONVENTIONAL PROCESSOR
MICROWAVE PROCESSOR
Stainers and Coverslippers
MICROWAVE STAINING OVEN
AUTOMATIC COVERSLIPPER
Miscellaneous Equipment
FLOTATION BATHS
CHROMIUM POTASSIUM SULFATE-COATED SLIDES
POLY-L-LYSINE-COATED SLIDES
AMINOALKYLSILANE-TREATED SLIDES [RENTROP 1986]
DRYERS AND OVENS
CIRCULATING WATER BATH
FREEZERS AND REFRIGERATORS
pH METERS
BALANCES AND SCALES
EMBEDDING CENTER
MICROMETER PIPETTES
SOLVENT RECYCLER
Instrument Quality Control
NEW INSTRUMENT VALIDATION
QUALITY CONTROL PROGRAM
References
Equipment Temperature Quality Control Chart
Equipment Maintenance and History
Chapter 4: Safety
Biological or Infectious Hazards
TUBERCULOSIS EXPOSURE
CRYOGENIC SPRAYS
HIV, HEPATITIS c VIRUS (Hcv), AND HBV
CREUTZFELDT-JAKOB DISEASE (CJD)
HANDLING TISSUE WASTE
Mechanical Hazards
ERGONOMICS
Chemical Hazards
PARTICULARLY HAZARDOUS SUBSTANCES(REPRODUCTIVE TOXINS, SELECT CARCINOGENS,AND SUBSTANCES WITH A HIGH DEGREE OF ACUTETOXICITY)
CARCINOGENS
CORROSIVE SUBSTANCES
FIRE AND EXPLOSION HAZARDS
HAZARDOUS CHEMICAL SPILLS AND STORAGE
CHEMICAL STORAGE
HAZARDOUS CHEMICAL DISPOSAL
Hazard Identification
General Safety Practices
EMPLOYEES:
SUPERVISORS:
References
LEARNING ACTIVITIES
Chapter 5: Laboratory Mathematics and Solution Preparation
Percentage Solutions
Use of the Gravimetric Factor in Solution Preparation
Hydrates
Normal and Molar Solutions
The Metric System
TEMPERATURE CONVERSION
Buffers
General Guidelines for Solution Preparation, Use, and Storage
Stability of Solutions
References
ANSWERS TO PROBLEMS IN CHAPTER
ANSWERS TO PROBLEMS IN LEARNING ACTIVITIES
LEARNING ACTIVITIES
Chapter 6: Nuclear and Cytoplasmic Staining
Ultrastructure of the Cell
THE NUCLEUS
THE CYTOPLASM
Staining Mechanisms
NUCLEAR STAINING
CYTOPLASMIC STAINING
The Dyes
FACTORS AFFECTING DYE BINDING
DIFFERENTIATION
THE NUCLEAR DYES
PLASMA STAINS
H&E Staining
MANUAL PROGRESSIVE STAINING METHOD
MANUAL REGRESSIVE STAINING METHOD
AUTOMATED STAINING
FROZEN SECTION STAINING
Troubleshooting the H&E Stain
INCOMPLETE DEPARAFFINIZATION
NUCLEAR STAINING IS NOT CRISP
PALE NUCLEAR STAINING
DARK NUCLEAR STAINING
RED OR RED-BROWN NUCLEI
PALE CYTOPLASMIC STAINING
DARK CYTOPLASMIC STAINING
EOSIN NOT PROPERLY DIFFERENTIATED
BLUE-BLACK PRECIPITATE ON TOP OF SECTIONS
HAZY OR MILKY WATER AND SLIDES
UNEVEN H&E STAINING
DARK BASOPHILIC STAINING OF NUCLEI ANDCYTOPLASM, ESPECIALLY AROUND TISSUE EDGES
POOR CONTRAST BETWEEN NUCLEUS ANDCYTOPLASM
Nucleic Acid Stains
FEULGEN REACTION
METHYL GREEN-PYRONIN Y
STOCK ACETATE BUFFER SOLUTIONS
Polychromatic Stains
MAY-GRUNWALD GIEMSA STAIN
Mounting Stained Sections
RESINOUS MEDIA
AQUEOUS MOUNTING MEDIA
COVERSLIPS
Troubleshooting Mounted Stained Sections
WATER BUBBLES NOTED IN MOUNTED SECTIONS
ALL AREAS OF SECTION CANNOT BE BROUGHTINTO FOCUS
CORN-FLAKING ARTIFACT SEEN ONMOUNTED SECTIONS
MOUNTED STAINED SECTIONS ARE NOT AS CRISPAS USUAL WHEN VIEWED MICROSCOPICALLY
RETRACTED MOUNTING MEDIUM
References
LEARNING ACTIVITIES
Chapter 7: Carbohydrates and Amyloid
Carbohydrates
GROUP 1: NEUTRAL POLYSACCHARIDES (NONIONICHOMOGLYCANS)
GROUP II: ACID MUCOPOLYSACCHARIDES (ANIONICHETEROGLYCANS)
GROUP III: GLYCOPROTEINS (MUCINS, MUCOID,MUCOPROTEIN, MUCOSUBSTANCES)
GROUP IV: GLYCOLIPIDS
Special Staining Techniques
PAS REACTION
TEST FOR QUALITY OF SCHIFF REAGENT
PAS REACTION WITH DIASTASE DIGESTION
TEST FOR QUALITY OF SCHIFF REAGENT
BEST CARMINE
MAYER MUCICARMINE
ALCIAN BLUE, pH 2.5
ALCIAN BLUE, pH 1.0
ALCIAN BLUE WITH HYALURONIDASE
ALCIAN BLUE-PAS-HEMATOXYLIN
MÜLLER-MOWRY COLLOIDAL IRON
Amyloid
ALKALINE CONGO RED METHOD
CRYSTAL VIOLET
THIOFLAVINE T FLUORESCENT METHOD
References
LEARNING ACTIVITIES
Chapter 8: Connective and Muscle Tissue
Connective Tissue
Basement Membrane
Muscle
Staining Techniques for Connective Tissue Fibers
MASSON TRICHROME STAIN [
GOMORI 1-STEP TRICHROME STAIN
VANGIESON PICRIC ACID-ACID FUCHSIN STAIN
VERHOEFF ELASTIC STAIN
ALDEHYDE FUCHSIN ELASTIC STAIN
NOTES ON OTHER ELASTIC STAINS
RUSSELL MODIFICATION OF THE MOVATPENTACHROME STAIN
SILVER TECHNIQUES FOR RETICULAR FIBERS
GOMORI STAIN FOR RETICULAR FIBERS
GORDON AND SWEETS STAIN FOR RETICULARFIBERS
Staining Techniques for Muscle
MALLORY PTAH TECHNIQUE FOR CROSS-STRIATIONSAND FIBRIN
PTAH WITHOUT MERCURIC SOLUTIONS
Staining Technique for Basement Membranes
PERIODIC ACID-METHENAMINE SILVER MICROWAVEPROCEDURE FOR BASEMENT MEMBRANES
Staining Techniques for Lipid
OIL RED O METHOD FOR NEUTRAL FATS
SUDAN BLACK B IN PROPYLENE GLYCOL
OSMIUM TETROXIDE PARAFFIN PROCEDURE FORFAT
Staining Techniques for Connective Tissue Cells
TOLUIDINE BLUE FOR MAST CELLS
METHYL GREEN-PYRONIN Y
References
LEARNING ACTIVITIES
Chapter 9: Nerve
The Nervous System
Neurons
NISSL SUBSTANCE
NERVE CELL PROCESSES
Neuroglia
OLIGODENDROGLIA
ASTROCYTES
MICROGLIA
EPENDYMAL CELLS
Myelin
Special Staining Techniques
NISSL SUBSTANCE: CRESYL ECHT VIOLET METHOD I
NISSL SUBSTANCE: CRESYL ECHT VIOLET METHOD II
NERVE FIBERS, NERVE ENDINGS, NEUROFIBRILS:BODIAN METHOD
NERVE FIBERS AND NEUROFIBRILS: HOLMES SILVERNITRATE METHOD
NERVE FIBERS, NEUROFIBRILLARY TANGLES, ANDSENILE PLAQUES: BIELSCHOWSKY-PAS STAIN
NERVE FIBERS, NEUROFIBRILLARY TANGLES, ANDSENILE PLAQUES: MICROWAVE MODIFICATION OFBIELSCHOWSKY METHOD
NERVE FIBERS, NEUROFIBRILLARY TANGLES,AND SENILE PLAQUES: T HE SEVIER-MUNGERMODIFICATION OF BIELSCHOWSKY METHOD
NEUROFIBRILLARY TANGLES AND SENILE PLAQUES:THIOFLAVIN S (MODIFIED)
GLIAL FIBERS: MALLORY PHOSPHOTUNGSTIC ACIDHEMATOXYLIN (PTAH) STAIN
GLIAL FIBERS: HOLZER METHOD
ASTROCYTES: CAJAL STAIN
MYELIN SHEATH: WEIL METHOD
MYELIN SHEATH: LUXOL FAST BLUE METHOD
MYELIN SHEATH AND NISSL SUBSTANCE COMBINED:LUXOL FAST BLUE-CRESYL ECHT VIOLET STAIN
MYELIN SHEATHS AND NERVE FIBERS COMBINED:LUXOL FAST BLUE-HOLMES SILVER NITRATE METHOD
LUXOL FAST BLUE-PAS-HEMATOXYLIN
References
LEARNING ACTIVITIES
Chapter 10: Microorganisms
Bacteria
Fungi
Virsues
Protozoans
Special Staining Techniques
KINYOUN ACID-FAST STAIN
ZIEHL-NEELSEN METHOD FOR ACID-FAST BACTERIA(AFIP MODIFICATION)
MICROWAVE ZIEHL-NEELSEN METHOD FOR ACIDFASTBACTERIA
FITE ACID-FAST STAIN FOR LEPROSY ORGANISMS
MICROWAVE AURAMINE-RHODAMINE FLUORESCENCETECHNIQUE
BROWN-HOPPS MODIFICATION OF THE GRAM STAIN
GIEMSA METHODS
MODIFIED DIFF-QUIK GIEMSA STAIN FORHELICOBACTER PYLORI
ALCIAN YELLOW-TOLUIDINE BLUE METHOD FOR HPYLORI
HOTCHKISS-MCMANUS PAS REACTION FOR FUNGI
CHROMIC ACID-SCHIFF STAIN FOR FUNGI (CAS)
GRIDLEY FUNGUS STAIN
GROCOTT METHENAMINE-SILVER NITRATE FUNGUSSTAIN
MICROWAVE METHENAMINE-SILVER NITRATEPROCEDURE FOR FUNGI
MAYER MUCICARMINE AND ALCIAN BLUETECHNIQUES FOR CRYPTOCOCCUS NEOFORMANS
WARTHIN-STARRY TECHNIQUE FOR SPIROCHETES
MICROWAVE MODIFICATION OF THE WARTHINSTARRYMETHOD FOR BACTERIA
DIETERLE METHOD FOR SPIROCHETES ANDLEGIONELLA ORGANISMS
MICROWAVE STEINER AND STEINER PROCEDUREFOR SPIROCHETES, HELICOBACTER, AND LEGIONELLAORGANISMS
References
LEARNING ACTIVITIES
Chapter 11: Pigments, Minerals, and Cytoplasmic Granules
Pigments
ARTIFACT PIGMENTS
EXOGENOUS PIGMENTS
ENDOGENOUS HEMATOGENOUS PIGMENTS
ENDOGENOUS NONHEMATOGENOUS PIGMENT
Endogenous Deposits
Minerals
Cytoplasmic Granules
Special Staining Techniques
PRUSSIAN BLUE STAIN FOR FERRIC IRON
TURNBULL BLUE STAIN FOR FERROUS IRON
SCHMORL TECHNIQUE FOR REDUCING SUBSTANCES
FONTANA-MASSON STAIN FOR MELANIN ANDARGENTAFFIN GRANULES
MICROWAVE FONTANA-MASSON STAIN
GRIMELIUS ARGYROPHIL STAIN
CHURUKIAN-SCHENK METHOD FOR ARGYROPHILGRANULES
MICROWAVE CHURUKIAN-SCHENK METHOD FORARGYROPHIL GRANULES
GOMORI METHENAMINE-SILVER METHOD FORURATES
BILE STAIN
VON KOSSA CALCIUM STAIN
ALIZARIN RED S CALCIUM STAIN
RHODANINE METHOD FOR COPPER
MICROWAVE RHODANINE COPPER METHOD
References
LEARNING ACTIVITIES
Chapter 12: Immunohistochemistry
Introduction
General Immunology
ANTIBODY
ANTIGEN
POLYCLONAL ANTISERA
MONOCLONAL ANTIBODIES
RABBIT MONOCLONAL ANTIBODIES
Tissue Handling
FROZEN TISSUE FIXATION AND PROCESSING
FIXATIVES FOR PARAFFIN-PROCESSED TISSUE
PROCESSING
MICROTOMY
EPITOPE ENHANCEMENT OR RETRIEVAL
Methods of Visualization
ENZYME IMMUNOHISTOCHEMISTRY
Immunohistochemical Staining Methods
DIRECT METHOD
INDIRECT METHOD
UNLABELED, OR SOLUBLE ENZYME IMMUNECOMPLEX, METHOD
Controls
POSITIVE CONTROLS
NEGATIVE CONTROLS
Antibody Evaluation and Validation
ANTIBODY SPECIFICATION SHEET
PREDILUTED AND CONCENTRATED ANTIBODIES
ANTIBODY VALIDATION
STORAGE OF ANTIBODIES
BLOCKING REACTIONS
Validation Form for Antibodies and Tissue Controls
MULTILINK BIOTINYLATED SECONDARY ANTISERA
DAB REACTION PRODUCT INTENSIFICATION
BUFFER SOLUTIONS
Commonly Used Antibodies and Their Applications
NEOPLASTIC TERMINOLOGY
Quality Control
RECOMMENDED QC FOR AN ANTIBODY
POSITIVE AND NEGATIVE TISSUE CONTROLS
RECOMMENDED QC FOR A TISSUE BLOCK
DAILY QC OF IMMUNOHISTOCH EMISTRY
STORAGE OF CONTROL SLIDES
Standardization
Troubleshooting Immunoperoxidase Techniques
Staining Techniques
BASIC PAP IMMUNOPEROXIDASE PROCEDURE
ABC-IMMUNOPEROXIDASE PROCEDURE
HRP ENZYME-LABELED POLYMER PROCEDURE
References
LEARNING ACTIVITIES
Chapter 13: Enzyme Histochemistry
Muscle Histology
Pathologic Changes in Muscle
Enzyme Histochemistry
Oxidation and Reduction
Properties of Enzymes
Preservation of Enzymes
Classification of Enzymes
HYDROLASES
OXIDOREDUCTASES
TRANSFERASES
Freezing Muscle Biopsy Specimens
α-NAPHTHYL ACETATE ESTERASE STAIN FORMUSCLE BIOPSIES
NAPHTHOL AS-D CHLOROACETATE ESTERASETECHNIQUE
MAYER HEMATOXYLIN
ATPASE STAIN
ACID PHOSPHATASE IN MUSCLE BIOPSIES
ALKALINE PHOSPHATASE STAIN FOR MUSCLEBIOPSIES
NADH DIAPHORASE
SUCCINIC DEHYDROGENASE (SDH)
PHOSPHORYLASE STAIN FOR MUSCLE
Nonenzymatic Procedures for Muscle Disorders
MODIFIED GOMORI TRICHROME
Acknowledgment
References
LEARNING ACTIVITIES
Chapter 14: Electron Microscopy
Fixation
FIXATIVES
FACTORS INFLUENCING FIXATION
FIXATIVE SOLUTIONS
Processing
DEHYDRATION
TRANSITIONAL SOLVENTS
EMBEDDING MEDIA
PROCEDURE FOR ROUTINE PROCESSING AND SPURREMBEDDING
PROCEDURE FOR ROUTINE PROCESSING AND EPONEMBEDDING
PROCEDURE FOR LR WHITE PROCESSING FORELECTRON MICROSCOPY IMMUNOLABELING
Sectioning
SECTION THICKNESS
KNIVES
CORRECTING PROBLEMS ENCOUNTERED INSECTIONING
Staining
Staining 0.5-μm Sections
TOLUIDINE BLUE-BASIC FUCHSIN PROCEDURE
TOLUIDINE BLUE STAINING
Staining Thin Sections
Special Techniques
BLOOD CELL PREPARATION
CELL SUSPENSIONS (FLUIDS, CULTURES, PARASITES,ETC)
Processing Tissues Previously Embedded in Paraffin
Processing Tissue from an H&E-Stained ParaffinSection
Acknowledgment
References
LEARNING ACTIVITIES
Chapter 15: Cytopreparatory Techniques
Cytopreparation
Collection
GYNECOLOGIC CYTOLOGY
NONGYNECOLOGIC CYTOLOGY
Fixation
PRE-FIXATIVES
Smear Preparation
DIRECT SMEARS
FLUIDS
MUCOID SPECIMENS
SPARSELY CELLULAR SPECIMENS
FINE NEEDLE ASPIRATIONS
SPECIAL PROBLEMS
CHOOSING THE BEST METHOD
Liquid-Based Cytology
Cell Blocks
METHODS
Cytology Staining
HEMATOXYLIN
OG-6
EA
PAPANICOLAOU STAIN
TOLUIDINE BLUE WET FILM
CROSS CONTAMINATION
SPECIAL STAINS
References
LEARNING ACTIVITIES
Glossary
Index