Common Stains in Cellular Pathology Flashcards
What is a H&E stain?
Haematoxyllin and Eosin
Gives diagnosis in over 70% of histopathology clinical reports
Shows the general morphology of specimen to show nuclei in blue and everything else in pink
Outline the principles of H&E staining
Chemical attraction- charges on dye/ tissue attracted to each other
Nucleic acids (-vely charged and basophillic) reacts with haematoxylin that has a positive charge
RBC’s, muscle, collagen etc (all +vely charged and acidophillic) react with eosin that has a negative charge
List the 5 extracellular proteins of interest and the stain used to identify them
Elastin- Miller’s elastin/ van Giseon
Fibrin/Collagen- Martius Scarlet blue, Masson Trichrome, Phosphotungstic acid and Haematoxylin
Collagen III- Reticulin method (silver impregnation)
Collagen IV- Periodic Acid Schniff/ Haematoxylin
Amyloid- Congo red, Sirius red, Thioflavin T and Crystal violet
How do Trichrome stains work?
Use of 3 dyes- picric acid, acid fuchsin and methyl blue
All work on the chemical attraction of acid/base, density and permeability of tissues and molecular size of the dye
Differentiates between muscle and collagen
How does Van Gieson’s stain work?
Use of 2 acid dyes- acid fuchsin and picric acid combined in s single solution
Picric acid- penetrates all the tissue rpaidly but nis firmly retained red blood cells and muscle
Acid fuchsin- larger molecules displace picric acid molecules from collagen fibres which have larger pores and allow larger molecules to enter
Differentiates muscle from collagen (yellow/ red)
Identifies bile (bottle green)
How does the Masson Trichrome stain work?
Use of 2 acid dyes to replace Eosin- Beibrich scarlet-acid fuchsin and Analine
Used for the detection of collagen fibres in heart and skin tissues on formalin-fixed, paraffin embedded sections
Collagen fibres are stained blue and nuclei are stained black, and backgrounds are stained red
Outline the 4 step principle of Masson Trichrome staining
- Weigert’s iron haematoxylin solution to stain nuclei
- Bierbrich scarlet-acid fuchsin files cytoplasm/muscle/ collagen
- Phophomolybdic-phodsphotungstic acid acts as a dye excluder and competes with the red dye, once reaction has stopped collagen is only faintly stained
- Collagen needs to be filled with Aniline blue and any excess rinsed off
What are the results of a Masson Trichrome stain?
Collagen= blue
Nuclei= black
Muscle, cytoplasm and keratin= red
What are the results of of a Picro Mallory Trichrome?
Myelin and RBC’s= yellow
Neuroglia and Fibrin= red
Collagen fibres= dark blue
Nuclei= dark brown
How does Martius Scarlet Blue stain work?
Use of 3 acid dyes to replace Eosin- Martius Yellow, Brilliant Crystal scarlet and Aniline blue
Modification of the Masson Trichrome procedure to demonstrate Fibrin
Based on chemical attraction of acid/ base, density and permeability of tissues as well as the molecular size of dye
What are the results of an MSB stain?
RBC’S and early fibrin deposits= yellow
Muscle and mature fibrin= red
Old fibrin deposits and collagen= blue
How does Silver Impregnation work?
Impregnation of tissue to enhance the contrast in tissues (not staining with dye)
Black fine deposits of Ag ions reduced to Ag atoms in reduction reactions at sensitive sites
What are the 3 different ways of producing metallic silver?
- Argentaffin reaction- argentaffin/ enterochromaffin of the gut only
- Argyrophil- external reducer needed e.g formalin (for reticulin fibres, neuroendocrine cells, neurons and axons)
- Ion-exchange reactions- e.g phosphates/carbonates of mineralised bone in Von Kossa stains
Why are reticulin stain sections treated with acidified potassium maganate solution?
It is an oxidising agent which produces aldehyde groups
Why is 1% Oxalic acid used in the reticulin stain process?
To decolourise the background stain
What is the role of 4% Iron Alum in the reticulin stain process?
To act as a mordant, which enhances the binding of silver salts
Why are reticulin stain sections treat with 10% formalin in tap water?
It is a reducing agent, which causes the deposition of metallic silver
Why are reticulin stain sections treated with Sodium Thiosulfate?
So that the excess silver in an unprecipitated state is removed
Describe reticular tissue
Special type of connective tissue that predominates in locations that have a high cellular content that functions as a ‘soft’ skeleton
It is branched and has a mesh-like pattern due to the arrangment if reticular fibres
It is found in the kidney, liver, spleen, lymph nodes, Peyer’ patches and bone marrow
How does the Von Kossa stain work?
Silver impregnation technique use don undecalcfied sections of bone tissue
Not a calcium specific stain, it reacts with phosphates/ carbonates in mineralised bone and teeth
How are elastic fibres identified?
With H&E but special stains are used for more exact studies
Common methods are Orecin, Verhoeff’s, Weigert’s and Aldehyde fuchsin
Describe amyloid
An abnormal protein, in which the amino acid sequences are similar or identical to body proteins
Very variable in it’s amino acid composition, which all have Beta-pleated sheet
Very resistant to enzyme degradation and accumulates between cells in many organs
Amyloid form fibrils
What stain is used to identify amyloid?
Congo Red- it allows “dichromism” with polarised light
What stain is used to visualise carbohydrates?
Classical stains: PAS, based on release of aldehydes by periodic schiff reaction to recolour (chromophore restored)
