Please enable JavaScript.
Coggle requires JavaScript to display documents.
Common Stains in Cell Pathology - Coggle Diagram
Common Stains in Cell Pathology
Haemtoxylin + Eosin (H&E)
70% diagnostics based on H+E stains
Simple
Often followed by 'special' stains
Haematoxylin
Basic dye
+ve charge
Basophilic
Stains nuclei
-ve charge
Blue
Eosin
-ve charge
Acidic dye
Stains cytoplasm, collagen, muscle
+ve charge
Bright pink/red
Van Gieson's
Acid fuchsin + picric acid combined in a solution
Picric acid
Small molecules
Penetrate all tissue rapidly
Retained firmly in close textured RBC + muscle
Acid fuchsin
Larger molecules
Displaces picric acid from collagen fibres
Collagen has larger pores
Differentiates
Muscle (yellow stain)
Collagen (red stain)
Also identifies bile accumulation in organs e.g. liver
Emerald to blue-green
Trichrome stains
3 acid dyes
e.g. picric acid, acid fuchsin + methyl blue
Family of stains
2 or 3 sequentially used acid dyes
Combining/matching tissues gives different colours
Work on similar principles
Size of dye molecule
Chemical attraction
Density/permeability of tissue
Differentiates muscle/collagen/fibrin
Dyes used dependent on what you want to see
Picro Malloy
Hypophysis
Myelin + RBCs
Yellow
Neuroglia + fibrin
Red
Collagen fibres
Dark blue
Nuclei
Dark brown
3 acid dyes
Masson
Muscle
Red
Collagen fibrosis e.g. in heart
Collagen
Blue
Nuclei
Dark red to black/blue
Haematoxylin + 2 acid dyes
Martius Scarlet Blue (MSB)
Well automated protocol
More sophisticated than Masson
Identifies fibrin
e.g. placenta, heart attack, blood clot
To show thickening
3 acidic dyes
Martius
Yellow
Brilliant crystal scarlet
Red
Aniline blue
Blue
Procedure
Martius yellow
Smallest dye
Held tightly by RBCs
Washed out of collagen/muscle cytoplasm
Brilliant crystal scarlet
Fills cytoplasm/muscle/collagen
Cant enter RBC because of martius yellow
Phosphotungstic acid
Red dye excluder/differentiator
Competes with red dye + pulls it out of tissue
Reaction stopped when collagen is lightly stained
Cytoplasm/muscle still red
Aniline blue
Fills collagen
Excess rinsed off
Can use haematoxylin counterstain
At start of procedure
Fibrin
Red
Collagen
Blue
Muscle
Red
Nuclei
Yellow
Silver impregnation
Impregnation of tissue to enhance the contrast in tissues
Coating of structures
Black, fine deposits of silver and silver oxide
Very sensitive
Expensive + technique is unrealiable
Silver salt
Reduced to metallic silver in reduction sites at sensitive sites e.g. aldehyde groups
3 ways to produce metallic silver
Argentaffin/enterochromaffin cells
Lining gut endothelium
Naturally-containing aldehyde groups
Argyophil
External reducer needed
Formalin
Neuroendocrine cells
Reticulin fibres
Ion-exchange reactions
Phosphates/carbonates of mineralised bone
Von Kossa staining
Reticulin Stain
Protocol
Take sections to water
Treat with acidified potassium permanganate solution
Oxidising agent
Aldehyde groups produced
5 mins
Wash off in water + bleach with 1% oxalic acid
1 min
Wash well in water
Rinse in distilled water + treat with 4% iron alum
2 more items...
De-colourise background stain
Reticulin fibres
Commonly present in
Liver tissue
Note cirrhosis patterns
Excessive reticulin fibres - show as dark dense black stained fibres against hepatocytes
Lymph nodes
Neuroendocrine cells
High levels of aldehyde groups naturally
No counterstain needed i.e. no formalin
Gives shape to organs
Von Kossa staining
Silver impregnation technique
Undecalcified bone sections
None calcium specific stain
Reacts with phosphate/carbohydrates in mineralised bone
Close relationship between calcium and phosphate/carbohydrates
Identifies
Lesions
Metabolic bone disease
Normal bone development
Calcium accumulation in kidney
Reduced filtration
Alternatives to identifying calcium
Alizarin red
Masson Goldner
Elastic fibres
H&E
Red/pink
Normally occur in
Skin
Blood vessels
Joints
Periodontal ligaments
Lungs
Connective tissue
Elastic cartilage
More specialised stains
Orcein
Verhoeff's
Weigert's
Aldehyde fuchsin
Amyloid
Abnormal protein
Amino acid sequence similar/identical to body proteins
Variable in amino acid composition
All have \(\beta\)-pleated sheet
Very resistant to enzyme degradation
Accumulates between cells in many organs
Pathology in tissues where it accumulates
Brain
Kidney
Filtration
Accumulate toxic blood components
Form fibrils
Some considered pathological
\(\beta\)-amyloid
Alzheimer's
Difficult to demonstrate
Many tinctorial stains lack specificity
Lots of false positives
Congo red stain
Stains amyloid for identification in polarising microscope
Apple green birefringence
Needed for diagnosis
Carbohydrates
Periodic acid-Schiff (PAS)
Based on release of aldehydes by periodic acid
Schiff to recolour
Chromophore restored
Protocol
Sections to water
Oxidise in 0.5% periodic acid
5 mins
Wash well in tap water
2 mins
Treat with Schiff's stain
3 more items...
Oxidation of carbs to dialdehydes
Glycogen
Diastase negative control in glycogen demonstration
Removes glycogen
Mucins (proteoglycans)
Smooth lining in gut
Submandibular gland
Pancreas
Indicative of pathological conditions
Hunter's disease
Accumulation in/around cells
Membranes (glycoproteins)
Other methods
Alcian blue
Mucins
PAS silver
Basement membrane - type 4 collagen
Kidney disease
Combination of PAS + alcian blue
Acid/neutral mucins
GIT diagnosis
Alcian blue (pH 2.5)
Stains acid mucins deep blue
PAS stains all mucins (neutral + acid)
Alcian blue blocks PAS selectively
Neutral lipids
Lysochrome principles
Elective solubility when dye leaves solvent and stains lipid
Dissolved better in fatty substance than solvent
Classic methods
Oil red O
Sudan dyes
Osmium tetroxide
Black, fixation
Aqueous mountant needed
Pigments
Perls histochemical reaction
Haemosiderin
Iron pigment
Masson fontana
Melanin
Silver impregnation
PAS
Lipofuchsin
Wear + tear pigment
Metachromatic stains
Colour shift
Certain tissue structures stain different to dye colour
Dyes
Thiazine group dyes
Toluidine blue
Methyl blue
Azure A
Spacing between acid groups
Shorter distance = more polymerisation of dye
Stronger metachromatic
Mast cell granules
Purple to red stain with toluidine blue
Metachromasia
Other structures stain blue
Orthochromasia
Immunological
Based on Ab-Ag reaction
Labelled Ab
e.g. fluorescence
Specific stain
Easier to interpret
Monoclonal Ab's
Identify
Cancer
Glomerulonephritis
Fluorescent microscope
Strong UV lamp
Condenser
Exciter filter
Only UV llght passes
Deflecting mirror
Condenser
Specimen
Objective
Barrier filter
2 more items...
UV + visible light
UV absorbed
1 more item...
UV + visible light