MEDI1000 Study Guide - Final Guide: Microtome, Laboratory Specimen, Histopathology
16 Jun 2018
School
Department
Course
Professor
Histopathology
Histopathology
-The microscopic examination of tissue to to determine the cause of disease
-Inflammatory-infectious diseases, other
-Immune-auto, acquired
-Neoplastic-benign or malignant
Science
-the correlation of function with the gross and microscopic structure of tissue and cells.
Art
-the processes essential to production of microscopic preparations or accurate diagnosis
Importance of Histopathologists
-Essential member of the diagnostic team
-Responsible for the processing of tissue and preparation of microscopic specimens
-Application of technical, chemical and molecular techniques to tissue specimens to
permit accurate microscopic interpretation.
-Specialist opportunities e.g. IHC, pathologist assistant, research officer
Hazards
-Chemical: fixatives, stains, chemicals
-Physical: sharps, radiation
-Biological: cross infection
Protection
-Appropriate dress, safety wear, laboratory design, compliance with safe behaviour and
laboratory code of conduct, alertness
-Protect the specimen by adopting prescribed procedure- important to avoid clerical
errors, contamination , to ensure patient safety and laboratory integrity !
Routine Histopathology
-Surgical procedure provides a specimen
-Preserve tissue: Fixation
-In the laboratory
-Specimen: Reception and representative dissection
-Process tissue: Remove water, preparation for microtomy
-Microtomy: Thin section of tissue
-Stain tissue: Prepare for microscopy
-Microscopy: Dictates the report outcome
-Report: Determines patient management
Specimens
-Tissue from numerous different surgical procedures, Hospital and Coroners autopsies
-Specific protocols for handling
-Micro biopsies, resections, bone, neural tissue
-Ancillary testing
-Cells in body fluids, cell samples collected by a range of methods
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Fixation
Cell degeneration
-Anoxia-restricting blood supply.
-Dependent on tissue type and stored metabolites
-e.g. glycogen store of heart vs brain
-Autolysis- release of lytic enzymes to self digest
-e.g. lipase, protease, nuclease
-rationale for hanging of meat
-Putrefaction-bacterial contamination !
Fixation
-Preservation – an old process mummification, salting, smoking, drying of corpses and
foodstuffs-freeze drying
-Physical-heat fixing bacteria, microwave radiation of tissue
-Chemical immersion - tissue fixation for microscopy
Foundation Laying
-Permanent preservation in a life-like state
-Stabilises tissue to allow further treatment
-Arrests autolysis
-Prevents bacterial decomposition
-Inactivate infectious agents
-Minimise loss of soluble cytoplasmic components
-Hardens tissue
-Enhance avidity for dyes
Factors involved in fixation
-Temperature: applications for hot and cold fixation
-Size of specimen: small or thin optimal
-Penetration ability of fixative: slow, slice specimens
-pH-usually best at pH6-8
-Osmolarity-hypertonic shrink, hypotonic swell, isotonic desirable
-Concentration: custom, cost, solubility, effectiveness
-Duration: minimum time
The Ideal Fixative
-Will preserve tissue in life like manner
-Will not add artefact material to tissue
-Will not swell or shrink tissue
-Will be safe for user and environment
-Has convenient shelf life and storage
-Is economical
Structures requiring stabilisations
-Main contributing elements
-lipoproteins of the plasmalemma/cell membrane
-cytoskeleton fibrous proteins
-fibrous glycoproteins eg collagen and basement membrane
-globular proteins of the cytoplasm and extracellular fluid
-mucosubstances eg hyaluronic acid, chondroitin sulphate
-nucleic acids !
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Chemical Fixation
Aldehydes
-Formaldehyde - routine fixative
-Glutaraldehyde - electron microscopy
-Aldehydes are used in anatomical pathology
Oxidising agents
-Osmium tetroxide, Potassium Dichromate
Protein coagulants
-Ethanol, Methanol
Uncertain mechanism
-Mercuric Chloride, Picric acid
Formalin
-Pungent gas soluble in water to 40% by wt
-Universal fixative for AP as 10%vv buffered formal
saline
-Acts by polymerisation of protein by forming
methylene bridges* between adjacent molecules
-A: Addition of a formaldehyde molecule to a protein.
-B: Reaction of bound formaldehyde with another
protein molecule to form a methylene cross-link.
-C: A detailed depiction of the cross-linking of a lysine
side chain to a peptide atom.
10% Neutral Buffered Formalin
-40% formalin 100mL
-Distilled water 900ml
-Sodium dihydrogen phosphate monohydrate 4g
-Disodium hydrogen phosphate anhydrous 6.5g
Fixation by protein coagulation
-Alcohol: Ethanol and Methanol*
-Alter the structure of proteins by disruption of hydrophobic bonds and replacement
of water with the EtOH.
-Rapid effect with pronounced shrinkage.
-Fixative of choice for cytology smears
-Do not alter reactive antigen sites
-*Avoid skin contact
Physical agents
Microwaves:
-Controlled to prevent overheating
-Rapid fixtation
-No significant cross linking of protein molecules
Heat:
-Confined to smears with microorganisms
Ultrasound:
-Applying high frequency, high intensity US for rapid tissue processing
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Document Summary
The microscopic examination of tissue to to determine the cause of disease. The correlation of function with the gross and microscopic structure of tissue and cells. The processes essential to production of microscopic preparations or accurate diagnosis. Responsible for the processing of tissue and preparation of microscopic specimens. Application of technical, chemical and molecular techniques to tissue specimens to permit accurate microscopic interpretation. Specialist opportunities e. g. ihc, pathologist assistant, research of cer. Appropriate dress, safety wear, laboratory design, compliance with safe behaviour and laboratory code of conduct, alertness. Protect the specimen by adopting prescribed procedure- important to avoid clerical errors, contamination , to ensure patient safety and laboratory integrity. Process tissue: remove water, preparation for microtomy. Tissue from numerous different surgical procedures, hospital and coroners autopsies. Cells in body uids, cell samples collected by a range of methods. Dependent on tissue type and stored metabolites. E. g. glycogen store of heart vs brain. Autolysis- release of lytic enzymes to self digest.
