Stain principles Flashcards
What is the most common tissue preservation technique?
Fixing in 10% Formalin (4% formaldehyde)
What percentage of tissues received in Histopathology are fixed in 10% formalin?
99%
What is the typical thickness for sections cut by microtomy?
4 or 5 microns (5 μm)
What happens to haematoxylin during the blueing process?
It converts from pink to blue as pH raises.
What is the purpose of differentiation in 1% acid-alcohol?
Selective removal of excess dye from weakly bound components
What are mordants?
Substances/metal which chemically binds with dye and tissue
What does haematoxylin oxidation produce?
Haematin
What is the purpose of dehydration in tissue processing?
To prepare sections for embedding in organic solvents
What is the purpose of dewaxing and rehydrating tissue sections?
Removes wax and rehydrates tissues for aqueous staining
What is the significance of adhesive or charged slides?
They attract sections to the glass slide
What is Cryotomy?
Use of refrigerated microtome to cut frozen tissue sections
What is the commonest lipid stain used?
Oil red O
What is the main application of rapid Haematoxylin and Eosin staining?
Urgent diagnosis during surgery
What does H&E staining specifically stain?
Nuclei blue (Haematoxylin) and cytoplasm pink (Eosin)
What is the role of Eosin in H&E staining?
Counterstain that contrasts with Haematoxylin
What are the criteria for a good H&E stain?
- Chromatin detail
- Colour
- Differentiation
What is the principle of Trichrome staining?
Differentiates between tissue components, highlighting collagen and muscle
How do trichrome stains work?
Based on dye permeability, molecular size, and tissue affinity
What is the Masson trichrome stain primarily used for?
Highlighting collagen fibres, muscle fibres, and cytoplasm
What components are involved in Verhoeffs Van Gieson staining?
- Verhoeff stain for elastic fibers
- Van Gieson stain for collagen
What is the function of Gordan and Sweet’s method?
Demonstration of argyrophilic reticulin fibres
What does Perls Prussian Blue stain detect?
Ferric (Fe3+) iron deposits
What is the principle of Alcian Blue/Periodic Acid-Schiff stain?
Determines presence of carbohydrates and classifies them
What unique property does Congo Red stain have?
Binds to amyloid proteins and exhibits apple-green birefringence under polarized light
What is the role of Grocott’s silver stain?
Detects fungal cell walls rich in polysaccharides
What is the ABC method in immunohistochemistry?
A three-step antibody detection method
What is the function of PBS in immunohistochemistry?
Washing buffer to remove excess antibodies and stabilize bindings
What is the Cytospin method used for?
Produces a monolayer of cells for screening
What is visualized as a brown deposit in antigen-antibody reactions?
The antigen-antibody reaction
This indicates the presence of the specific antibody bound to the antigen.
What is the purpose of Dakopen in immunohistochemistry?
Confines liquid inside due to its hydrophobic nature
This property helps in the proper visualization of the antigen-antibody reaction.
What are chromogens used for in immunohistochemistry?
Produce a coloured product that can be visualized by light microscopy
They react with specific antibodies to provide contrast in staining.
What is the function of PBS in immunohistochemistry?
Used as a washing buffer to remove excess antibodies and reagents
It also stabilizes antigen-antibody binding.
What is the Cytospin method used for in clinical cytology?
Produces a monolayer of cells in a small area for screening
It is particularly useful in laboratories with large workloads.
What types of specimens can be processed using the Cytospin method?
Both fixed and unfixed specimens
Unfixed samples must be post-fixed before staining.
What is crucial in obtaining a monolayer using the Cytospin method?
The concentration of the re-suspended cell pellet
Proper concentration ensures even distribution of cells.
What speed and duration are used in a cytocentrifuge for the Cytospin method?
1500 rpm for 5 minutes
Followed by fixation in spirit for 5 minutes.
What is the main advantage of direct smears in clinical cytology?
Quick and simple method to prepare slides for screening
It is primarily employed for Diagnostic Cytology.
What is used to represent a diagnostic cytology specimen in direct smears?
Cultured cell lines
This allows for easier analysis and identification of cellular characteristics.
What is the process of preparing a direct slide from a centrifuged specimen?
Specimens are centrifuged, supernatant decanted, and cell pellet agitated to prepare a sample
This ensures a concentrated and even cell distribution on the slide.
What technology is utilized in liquid-based cytology for cervical cytology?
ThinPrep technology
It is also applicable for diagnostic cytology.
What is the basis of the ThinPrep process in liquid-based cytology?
Filtration method
This method enhances the quality of the cell monolayer on slides.
What pre-treatment is used for cell samples in liquid-based cytology?
Cytolyt
This is done prior to transferring to PreservCyt vials.
What is the fixation medium used in liquid-based cytology?
PreservCyt
It preserves the cell samples before processing.
What does the Papanicolaou stain principle require for immediate fixation?
95% alcohol
This prevents air drying of the samples.
What type of dye is Harris Haematoxylin?
Basic dye that binds to acidic sulfate groups of DNA
It is essential for the staining of cellular structures.
What is the purpose of differentiation in Papanicolaou staining?
To selectively remove excess stain from all except the structures to be demonstrated
This enhances the visibility of the target cells.
What happens during the blueing process in Papanicolaou staining?
Alum haematoxylin stain converts red colour to blue-black
This occurs when washed in a weak alkali solution.
What are the colours associated with different cell types in Papanicolaou staining?
Nuclei -> blue/black; Cytoplasm -> spectrum of pink, orange, green
Specific colours indicate different cellular components and types.
What is the sensitivity and accuracy of the Papanicolaou test?
Low sensitivity with limited accuracy
It is primarily a screening test.
What is the Speedy-Diff stain principle?
Commercial Romanowsky stain used to rapidly stain and differentiate pathology specimens
It can be applied to air-dried samples.
What colour do nuclei appear in Speedy-Diff staining?
Deep blue/purple
This contrasts with the cytoplasm’s blue to mauve colour.
What is the colour of RBCs in Speedy-Diff staining?
Salmon pink
This indicates the presence of red blood cells in the sample.
Decalcification
Some tissues contain calcium deposits which are extremely firm and will not section properly with paraffin embedding.
Typically bone specimens.
Calcium is removed using mineral acids such as HCL (can cause cellular damage) or using EDTA (slow + expensive).
Presence of fine black precipitate on slides
Suggests formaline-heme pigment formation.
Occurs when tissue become acidic and complex forms.
Tissue insufficiently dehydrate prior to clearing and infiltration with paraffin wax
Hard to section on microtome and tearing artefacts + holes will occur in sections.
Alcohol as fixative - artefacts
Can make tissue sections brittle resulting in microtome sectioning artefacts such as chattering.
