Tissue Processing Flashcards
Prognosis
Predicting the future course of the disease
Etilogy
What is the cause
Pathogenesis
Mechanisms/Evolution of disease
Morphology
Structural Changes in cell
What are the 3 main causes of cell injury
Acquired (Hypoxia/Ischaemia)
Genetic
Congenitial
Name the 3 types of lesion
Gross Lesion (naked eye)
Microscopic Lesion
Ultrastructural Lesion (EM)
Histology
Study of Normal Tissues/structure (liver)
Cytology
Study of Cells (blood smears)
Name the 3 methods of Pathology
Autopsy
Biopsy
Cytology
Name an example of a Biopsy Technique
Cervical Biopsy
Name a cytology Technique
Fine needle aspiration
Name the first 5 tissue processing steps
Fixation
Dehydration
Clearing
Wax infusion
Embedding
If using bone, what step goes between fixation and dehydration
Decalcification
Why is fixation done
Tissue deprived of blood supply
Therefore it will start to change
How is tissue affected thus needing fixation
Hypoxia
Putrefaction - Microorganisms growth = Microbial spoilage
Name 4 factors which affect fixation
Time
Temp
pH
Type of tissue
How can the pH be controlled in fixation technique
PBS buffer used in aldehyde buffers
Name 3 requirements of a good fixative
Go into cells quickly/evenly (tissue looks the same)
Not introduce artefacts to give false diagnosis
Harden tissue (make it responsive to subsequent treatment)
Why add calcium to fixative?
Preserve phospholipids
Name a type of fixative (F)
When is it used?
Formaldehyde (Formalin)
Light microscope
What is Formaldehyde mixed with?
Whats the final conc.
PBS to Neutral pH
4% Formalin
Name 2 Advantages of using Formalin
Retains natural colour
remains pliable for dissection
Name 2 disadvantages of using Formalin
Slow (12-24hrs)
Toxic - Formalin dematitis –>Carcinogenic agent
Not good for cytolplasmic stains
Name a type of Fixative (G)
When is it used?
Glutaraldehyde
EM
Name 2 advantages of Glutaraldehyde
Quicker than Formalin
Preerves intracellular organelles by inactivating all enzymes
Name 3 disadvantages of Glutaraldehyde
Penetration slower than Formalin
Difficult to remove–> Bad for immunohistochem
Inactivated all enzymes not good for EHC
Name a fixative useful for histochemisitry
Alcohols (Methanol/Ethanol)
What do Alcohol fixatives acheive? *(mechanism)
Denature proteins by removing water/replace with alcohol
Alteries tertiary shape but preserves chemical reactivitiy
When are Alcohol fixatives used
Histochemistry
Fixing smears
Name a oxidising agent fixative
Osmium tetraoxide
How do oxidising agents work as a fixative
Extensive denaturation of proteins
Stablizies lipids
When are oxidising agents used?
Whats a benefit
Lipid Preservation
Poor pentration/Dangerous for cornea
Mercuric Chloride
Zenker fluid
SH cross linking
Not good for ultra structures
Toxic
Produces black precipitate needs iodine
When are Mercuric Chlorides used?
Nuclear / cytoplasmic staining
Why is Decalcification done?
Name a disease which the stage of decalcification can help towards
Examining minearlised tissues
Hydroxyapatite present
Osteosarcoma
Name a decalficiation agent
Formic acid
EDTA
Whats the benefit of using Chelating agents during decalcification?
Whats the downside
No damage to tissue
Takes a long time
Why is dehydration done?
Water removed so paraffin can infiltrate tissue
Whats the solution for dehydration consist of?
75% ethanol
25% PBS
Why is Clearing done?
Removal of alcohol
As its MISCIBLE with the paraffin
Name the most common clearing agent
Xylene
When is Histo-clear used as a clearing agent?
Fine line tissue
What does Wax infusion involve?
2-4 baths of Molten paraffin wax (55-60*) for several hours
Why is embedding done?
Embeds tissue in support media for structural support
Describe 2 features of paraffin
Long chain hydrocarbon
Similar hardness to tissue once impregnated into tissue
Why is paraffin used?
Easier cutting
Structural support
Stored Indefinitely
When Would plastic/resin be used as a wax?
Thinner sections can be cut
As it provides stronger support as its harder
Name a type of embedding mould
Cassette mould
During Microtomy, what size is the tissue cut to for Light Microscopy?
And for EM?
1-6uM
60-90nm
Why is the tissue placed in warm water?
To remove wrinkles
Doesnt melt paraffin
Why is alcohol sometimes used with warm water?
Replaces more severe wrinkles
After taking the slide out of the warm water, what is the slide coated in and why?
Saline solution
Helps it adhere to the slide
What does Cryrotechiques avoid for speed?
Dehydration / Clearing / Embedding
What is used during the cryotechnique
At what temp?
Cryostat
-20*
what can the cryostat produce that bad
Ice crystals (artefacts)
Make it hard to cut
When is Slow freezing (Cryostat) suitable for?
Low mag. studies
How is Fast Freezing (Cryostat) used?
Liquid Nitrogen
Glycerol to reduce tissue damage
Whats embedded into tissue which acts like paraffin during the crytostat phase
OCT
Why is staining done?
Create High contrast to allow observations of structures
How is the paraffin removed for staining?
Xylenes
Name the two types of Staining techniques
Progressive
Regressive
with Progressive staining, what is done?
Dye applied
Until desired colour is achieved
DIfficult to control
Frozen sections
With Regressive staining, what is done
Put in solution for set period
Wash with Acid-alcohol to remove stain
Removal of background staining
Large batches
Name a type of Stain which is used for nuclear staining
What colour does it go?
Haematoxylin –> Haematein (BLUE)
What does Haematoxylin need?
Mordant (alluminium potassium)
What does Carmine stain?
Red for nucleus
What type of stain can be used for blood smears?
Methylene Blue
What does H&E stain?
Acidic molecules BLUE
Basic Molecules RED
Why is the coverslip placed on?
Protect it from artefacts
Why is labeling done?
To prevent incorrect info being distributed to wrong patients
Histochemistry
Identifies Chemical components
Produces coloured stain when reacting with chemical group
PAS Techniqiue
Histochemistry
Stain glycogen /glycoproteins
High Background
Feulgen Technique
DNA red
Acridine Orange
DNA green
RNA orange
Von Kossa
Show Calficiation
Alcian Blue
MC granules
Sudan Black
Phospholipids Black
How is Peroxidase identified?
Incubate tiisue with DAB + H202
Produces DARK BROWN in presence
How is Acid Phosphotase identified
Gomori Method
Formalin + SG + Lead nitrate
Black precipitate
what can immunohistochemistry detect?
Proteins / carbs / membrane antigens
Name the two methods of immunohistochemistry
Direct (Antigen + labelled antibody)
Indirect (Antigen + secondary labelled antibody)
Positive Control
If control fails, experiment is wrong
Shows if techniques are working
Negative Control
Tests for no false positives
Test specificity of the reaction
