CR Flashcards
What is the classification of Congo Red?
Carbohydrate stain
What is the target tissue component of Congo Red?
Amyloid
What is the staining principle of Congo Red?
Hydrogen bonding
What is the mechanism of staining of Congo Red?
Non-ionic bonding
What are the acceptable fixatives when staining with congo red?
Alcohol
Carnoy solution
10% NBF
Bouin’s solution
Zenker solution
At what thickness must congo red sections be cut at?
8-10 um
Why do congo red sections need to cut thicker than H&E sections?
Thin sections may not show apple-green birefringence
What is used to confirm apple-green birefringence when staining with congo red?
Polarizing microscopy
What kinds of tissue can be used for congo red QC?
Any tissue containing amyloid (kidney, prostate)
What issue does prolonged fixation cause in CR staining?
It may diminish the staining
Why shouldn’t you cut many controls for CR staining?
Because staining intensity decreases as the sections age
Which gives more intense CR staining: long-standing or newly formed amyloid deposits?
newly formed deposits
Which gives more intense CR staining: large or small amyloid deposits?
small deposits
How do you make the CR working solution A?
Take 50 ml of stock 80% alcohol saturated with NaCl and add 0.5 ml of 1% NaOH
Why do we make working solutions in CR staining?
Because the stock solution is too intense
How do you make the CR working solution B?
Take 50 ml of the stock Congo red solution and add 0.5 ml of 1% NaOH
What is the counterstain used in Congo Red?
Harris hematoxylin
Is Harris hematoxylin applied progressively or regressively in Congo Red?
Progressively
Why must we use acidified Harris hematoxylin in CR staining?
To reduce non-specific background staining
How is acidified Harris hematoxylin made up?
48 ml of Harris hematoxylin and 2 ml of glacial acetic acid
What is the purpose of using solution A in CR staining?
The pH of this solution increases the specificity of the staining and reduces background staining (due to most proteins not being able to stain at a high pH)
Is the CR solution B stained progressively or regressively?
Progressively
Is the CR solution B permanent?
Yes
What makes the CR solution B selective?
The composition of amyloid and the contents of this solution
What is the configuration of amyloid?
It’s a linear molecule with an antiparallel configuration (making it easier for H-bonding to occur)
What is hydrogen bonding?
The weak attraction of a hydrogen atom to a nearby oxygen, nitrogen, or fluorine atom
Why can’t CR be made up in water?
Because hydrogen bonding interferes with the bond between the dye and the tissue
What should be seen in a good Congo red stain?
Amyloid should be salmon pink and green birefringence under a polarizing microscope
What are the staining results with Congo Red?
Amyloid stains deep red to pink (and has apple green birefringence under polarizing microscopy)
Elastic fibers - pale pink
Nuclei - blue
What is amyloidosis?
A rare disease where amyloid builds up in organs (e.g., heart, kidney, liver, and spleen)
What are the distinctions in amyloidosis?
Systemic or inherited
Primary (spontaneous), secondary (associated with another disease - like cancer), or inherited
What does amyloid look like under light microscopy with H&E staining?
Amorphous eosinophilic deposits
What does amyloid look like under electron microscopy?
A regular fibrillary structure
What does amyloid look like under x-ray diffraction?
Beta-pleated sheets
What is the diameter of amyloid fibrils?
8-10 nm
How does amyloid stain with PAS staining?
It stains weakly positive because it contains a small amount of carbohydrates
How does amyloid stain with AB staining?
Positive (due to sialic acid glycoprotein)
How does amyloid stain with MTC staining?
As the same colour as collagen
Is amyloid autofluorescent?
Yes, it will look yellow/orange but weakly
What other amyloid diagnostic techniques can be used?
PAS and AB staining
Metachromatic stains (crystal violet and methyl violet) - has poor specificity and low sensitivity
Fluorochrome thioflavin T (stained sections aren’t permanent and there’s low specificity)
IHC (not all antibodies are available)
What are false positives and negatives related to?
Staining technique
Microscope equipment
Presence of small amounts of amyloid
In how many planes does polarized light vibrate in?
One plane only
What does a polarizing prism or filter do to light?
Alters it’s direction
What is the difference between isotropic (singly refractile) and anisotropic (doubly refractile or birefringent) microscopy?
In isotropic microscopy the specimen cannot divide/rotate the light and the field of view remains dark.
In anisotropic microscopy, the specimen can divide/rotate the light and appears bright against a dark background
Does amyloid rotate light when stained with CR?
Yes
