What conditions are there abnormal amounts of collagen present in tissue?
Fibrosis Collagen tumors Benign fibromas Malignant fibro-sarcomas Collagen diseases --> Scleroderma
What are the main chemicals and each of their purpose in Masson’s Trichrome?
- Post Fixation in Bouin’s –> Increases Acidophilia (enhances red colour)
- Weigert’s Hematoxylin –> Nuclear stain
- Biebrich Scarlet – Acid Fuchsin –> Cytoplasmic stain
- Phosphotungstic and Phosphomolybdic Acid –> Differentiation
- Light Green or Aniline Blue –> Collagen stain
- Acetic Acid Water –> Removal of excess collagen dye
What type of hematoxylin in Weigert’s in Masson’s Trichrome and why is that type used?
Iron Hematoxylin.
- Used to progressively stain nuclei.
- Will hold colour in nuclei; if a Alum hematoxylin would decolorize in PTA/PMA (strong acid)
- Acts as a mordant and oxidizer (needs to be fresh).
Why are both Biebrich Scarlet and Acid Fuchsin both used as a cytoplasmic stain in Masson’s Trichrome?
Two different red acid dye solutions. Combined to stain cytoplasm and collagen temporarily. Biebrich Scarlet is the smaller dye molecule out of the two red dye solutions.
The red dye that goes to the collagen will get removed later by the Phosphotungstic and Phosphomolybdic acid solution.
What is Phosphotungstic and Phosphomolybdic Acid in Masson’s Trichrome and specifically what does it remove?
Phosphotungstic and Phosphomolybdic Acid –> PTA/PMA
Two different acid solutions that creates one strong acid solution to remove the acid fuchsin dye molecules from the collagen (like it was reserving space for another dye).
What dye in Masson’s trichrome dyes the collagen after the acid solution?
Light green or aniline blue stains the collagen in the tissue.
Colour chosen depends on the preference of the pathologist.
What happens if 1% acetic acid wash is not done in Masson’s Trichrome?
If the excess collagen stain on the tissue is not removed it can result in a muddy background appearance of the slide.
This occurs when the collagen stain, the biggest dye molecule, is all over tissue areas other than just collagen.
If you finish Masson’s Trichrome stain and find there is too much overall blue colour on the tissue what can you do?
Place slides in acetic acid for a longer time.
What happens if you don’t use Bouin’s in Masson’s Trichrome technique and why does RRC not use it then?
Without Bouin’s –> get pale staining collagen and pale red cytoplasm and muscle.
But Bouin’s has picric acid which if it dries out during storage becomes explosive.
What happens in Masson’s Trichrome if the Iron Hematoxylin is not made fresh?
Nuclei will stain pale or not at all.
What are the colours for Masson’s Trichrome?
Nuclei --> Black (but maybe dark grey or dark purple) Cytoplasm --> Red Muscle --> Red RBC --> Red Collagen --> Blue or green
What are the main chemicals involved in Gomori’s Aldehyde Fuchsin?
- Aldehyde Fuchsin –> stains progressively
- 70% Ethyl Alcohol –> removes excess colour from background
- Van Gieson’s Counterstain –> two dyes one stains collagen the other stains all other tissue components.
What does Gormori’s Aldehyde Fuchsin stain?
Stains elastic fibers and other components like mast cell granules, beta cell granules, gastric chief cells, and some mucins.
Note: This stain is not necessarily specific for elastic fibers and mast cells.
What staining technique is specific for mast cells?
Toluidine Blue
What does Aldehyde Fuchsin consist of in Gomori’s Aldehyde Fuchsin technique?
Aldehyde Fuchsin:
- Basic fuchsin,
- 70% alcohol, HCl, and
- Paraldehyde.
Stains Progressively
What does Van Gieson’s counterstain consist of and each of their purpose in Gomori’s Aldehyde Fuchsin technique?
Van Gieson’s counterstain- mixture of Acid Fuchsin and Picric Acid. Acid Fuchsin is the bigger dye molecule that will stain collagen and Picric Acid is smaller dye molecule and will stain all the other tissue components.
What are the resulting colours from Gomori’s Aldehyde Fuchsin technique?
- Elastic Fibers - Purple (also mast cells, beta cell granules, gastric chief cells, mucins)
- Collagen - Red
- RBC – Yellow
- Other tissues - Yellow
What happens if Aldehyde Fuchsin is not made fresh?
If not fresh, gives pale elastic fibers.
Can prolong shelf life by putting small amounts of solution in the fridge and freezing aliquots of the remainder.
What happens if the slides are not washed well in 70% alcohol?
Muddy cytoplasm can occur.
In that case need to increase time in 70% ethyl alcohol wash.
What are the chemicals involved in Verhoeff’s Van Gieson’s technique?
- Verhoeff’s Hematoxylin –> Elastic and Nuclear Stain
- 2% Ferric Chloride –> Differentiation (by excess mordant & elastic fibers affinity for dye lake complex)
- 2.5% Sodium Thiosulphate –> Background clearing, removes excess iodine.
- Van Gieson’s –> Counterstain
Is dezenkerization needed for tissues going to be stained with Verhoeff’s Van Gieson (VVG)?
No dezenkerization is needed. The Verhoeff’s Hematoxylin has ingrediants that will already remove any pigment from the mercuric chloride.
(Dezenk. is required if a black pigment forms from fixing in Mercuric Chloride).
What are three things that are critical in Verhoeff’s Van Gieson’s technique?
- Verhoeff’s Hematoxylin solutions need to be added in the correct order with mixing between each solution –> otherwise poor elastic stain will result.
- Timing is very important with the Van Gieson Counterstain.
- -> This is because picric acid continues to differentiate elastic fibers due to its acidic nature. Start with under-differentiated and work from there. - Van Gieson should be made correctly to distinguish between muscle and collagen.
- ->If Van Gieson is not saturated, collagen will not stain red and cytoplasm, muscle & collagen may all stain the same colour.
Why is it recommended to differentiate each slide one at a time with VVG technique?
Each tissue has different amounts of elastic in the tissue and checking the slide microscopically and adjust the differentiation time will give the best results.
Note: We did not do this in lab due to time restrictions.
What three roles does Ferric Chloride have in the VVG technique?
Ferric Chloride is a mordant, oxidizer and differentiator.
