An introduction to histology

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What does histology mean?
The study of normal tissues
Take a piece of tissue and perform the necessary procedures on it to make it visible using a microscope
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What does histopathology mean?
The study of diseased tissues
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What are the basic histological principles?
Fixation - chemical preservation of the tissue
Embedding - solidification of the tissue
Sectioning - cutting of the solidified tissue
Staining - colouring of the tissue
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Who invented the microscope?
Antonie van Leeuwenhoek
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What is virtual microscopy?
A collection of online tools which allow you to view and analyse slides remotely using the internet
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What is an application of histology?
Cancer grading
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How can histology be used to tell us the best drugs to treat cancer?
With modification - a technique called immunohistochemistry
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How does this work?
The hormone receptor status of breast cancer tells us which classes of drugs it will respond to
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What are 3 problems that have to be overcome in order to perform histology?
Putrefaction/Autolysis/Degradation
Thickness
Colour/Contrast
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The term for the breakdown of tissue via unrestricted digestion by its own enzymes
Autolysis
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The term for breakdown of tissue via the influence of the external environment; temperature, light, humidity, exogenous microbes
Putrefaction
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What family of processes do we use to prevent degradation?
Fixing
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What are the most simple fixatives used for fixing?
Alcohols and solvents
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How do alcohols and solvents fix?
Works by replacing the water in tissues thus denaturing proteins by manipulating their hydrophobic interactions
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How does formaldehyde fix?
Cross-links proteins by forming a methylene bridge
However, this process is difficult to do correctly
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What is an advantage of glutaraldehyde as a fixative?
Cross-links more extensively than formaldehyde
Great at preserving ultrastructure for techniques such as electron microscopy, but may fix too extensively for immunohistochemistry
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What can good fixation depend on?
Fixatives
Different molecules have different diffusion rates
Tissue thickness
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How thick does tissue need to be for light to pass through (so it can be visualised)?
1-10μm
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Why can't we cut tissue as it is into sub-mm slices?
It is too soft, too high water content
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What needs to be done to the soft tissue in order to slice it?
Tissue needs to be solidified such that they take on suitable mechanical properties
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What are the thin slivers of tissue called?
Sections
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What is the process of solidification called?
Embedding
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What are the 3 techniques of embedding?
Freezing - uses rapid cooling to set tissues by freezing the aqueous component solid
Paraffin - Sections are secured inside a block of paraffin wax
Resin - A polymer such as glycolmethacrylate (GMA) embeds the tissue in a solid polymerised block
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What are the pros of freezing?
Fast and cheap
Maintains good molecular integrity due to quick tissue processing and minimal chemical interation
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What are the cons of freezing?
Tissue architecture may be compromised
Skill required to produce a continuous frozen section
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What are the pros of wax?
Preserves good tissue architecture
Easy to section once embedded
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What are the cons of wax?
The heating step involved melting wax can destroy proteins of interest
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What are the pros of resin?
Preserves excellent tissue architecture
A lack of heating step to polymerise resin means protein conformation is well preserved
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What are the cons of resin?
Time-consuming, fairly expensive and requires user optimisation
Infiltrating large tissue segments with resin is difficult which can result in sections which are solid on the outside but flaky in the centre
"Antigen retrieval" can be challenging
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What needs to be done before resin or paraffin can be infused with the tissue?
The tissue needs to be dehydrated using alcohols or acetone
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The term for the process where the tissue is infused with liquid resin or paraffin?
Infiltration
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Why is infiltration performed at 65°C for paraffin?
To keep it molten
Cooling causes polymerisation
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Why is infiltration performed at -55°C for GMA (resin)?
Its chemical polymerisation generated heat which would damage the tissue
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What is the method to obtain a histological sample using paraffin or resin?
1. Collect a suitable size biopsy
2. Preserve it using fixation
3. Remove water by dehydration
4. Remove alcohol by clearing
5. Infiltrate tissue with paraffin or polymer
6. Allow infiltration media to set during embedding
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What instrument do we use to cut embedded blocks?
Microtome
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What is the most common type of microtome used?
A rotary microtome
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What is it called when the sections stick to one another as they are cut?
The ribbon effect
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How is the ribbon of sections unfolded?
Sections are floated onto water
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What should a good section on a microscope slide look like?
Uniform staining
Section free from rips, tears, bubbles and folds
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How do folds occur?
If the section is not given an adequate chance to unfurl when floated out on the water bath
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Why do bubbles occur?
When air trapped under the section expands during staining
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Why do tears occur?
Induced when the water in the water bath is too warm
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Why do miscellaneous pieces of tissue occur?
Can be a result of a contaminated water bath or skin swathes from the operator
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What are frozen samples embedded in?
In a cryopreservation media called optimal temperature cutting compound (OCT)
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What is a cryostat?
A specialised rotary microtome used to cut frozen sections
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What happens if the tissue is cut too warm?
The expansion if ice within it causes artefact
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What happens if the tissue is cut too cold?
It will shatter
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What are the 3 stains we use in our practicals and what do they stain?
Periodic acid Schiff: Carbohydrates pink
Haematoxylin and Eosin: Cytoplasm/ECM/Erythrocytes - Red/Pink
Nuclei - Blue/Purple
Millers Van Gieson: Elastic fibres - black
Cytoplasm and Muscle - yellow
Collagen - Red
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How does Periodic acid Schiff work?
Periodic acid oxidises some of the tissue carbohydrates.
This produces aldehyde groups, which can then condense with Schiff's reagent forming a pink colour
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Why is Periodic acid Schiff important?
Irregularities in carbohydrate presence are indicative of a wide range of diseases from cancers, storage disorders and infection
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How does haematoxylin work in the haematoxylin and eosin stain?
The active form of haematoxylin is haematin which arises when haematoxylin is oxidised
This occurs by agents which are included in its formulation such as sodium iodate
A metal ion such as aluminium bridges with the substances in a cell with strong negati
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What is the term for when a substance is used in a way to allow a dye molecule to interact with a substance?
Mordant
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How does eosin work in the haematoxylin and eosin stain?
Synthetic negatively charged molecule
Binds positively charged residues throughout proteins in cells
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Why is the haematoxylin and eosin stain important?
The most prolific histological stain in biomedicine
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What are the three types of fibres in connective tissue?
Collagen
Elastic fibres
Reticular fibres
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What does the Miller's part of the Miller's Van Gieson stain?
Miller's stains elastic fibres black
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What is the Van Gieson stain made up of?
A mixture of picric acid (small molecular weight) and acid Fuchsin (larger molecular weight) dyes
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How does Van Gieson stain work?
The small picric acid dye penetrates the tissue in its entirety
The larger acid Fuchsin dye displaces the picric acid from collagen molecules due to its large open structure
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What is the end product of the Van Gieson stain?
A tissue in which cytoplasm is yellow (picric acid) and collagen fibres are red (acid Fuchsin)
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Why is Miller's van Gieson stain important?
Defects in elastic fibre production and organisation underlie the pathology of many diseases
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Card 3

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Card 4

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Card 5

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