SIP- Histopathology
Hey everyone! I shall touch on the 2 special stains that I did when I was attached to Special Staining. They are namely the Liver Orcein stain and the Victoria Blue stain.
Liver Orcein Stain
Function: Demonstrates Hep B surface antigen and elastic tissues
Principle: The disulphide bridges in the elastic tissue are broken down into its anionic derivatives during oxidation. These derivatives can then be stain by Orcein stain. Virus inclusion bodies can be stain by Orcein after oxidation.
Control: A known Hep positive liver tissue
Fixative: 10% buffered neutral formalin
Chemicals required: Orcein solution
0.5% potassium permanganate
1% oxalic acid
3% sulphuric acid
Procedures:
1) Dewax (removal of wax) and bring tissue section to water
2) Place in acidified potassium permanganate solution for 5 mintues (section will appear brown in colour)
3) Wash in water
4) Bleach section till colourless in 1% oxalic acid
5) Wash in water
6) Stain in Orcein solution for 4 hours at room temperature or overnight in fridge
7) Wash quickly in 2 changes of absolute alcohol
8) Dehydrate, clear then mount
Results: Coarse elastic fibers- reddish brown
Fine elastic fibers- dark brown
Hep B surface antigen- dark brown
Victoria Blue Nuclear Fast Red
Function: Stain elastic fibers
Principle: Elastic fibers are highly cross-linked by disulphide bridges. Following oxidative treatment, these bridges may convert into anionic sulphuric acid derivatives. These derivatives are strongly basophilic and capable of selective reactions with the basic dyes.
Control: Skin and artery for elastic fibers demonstration. Inflamed skin for mast cells demonstration.
Fixative: 10% buffered neutral formalin
Chemicals required: 0.5% potassium permanganate
3% sulphuric acid
4% sodium bisulphate
VB solution
Nuclear Fast Red
Procedures:
1) Dewax and bring section to water
2) Stain with acidified potassium permanganate solution for 5 minutes (section will appear brown in colour)
3) Bleach with 1% sodium bisulphite until colourless
4) Wash in running water
5) Dry on hotplate
6) Leave in VB solution for 24 hours
7) Differentiate in 70% alcohol
8) Wash in running water
9) Stain with Nuclear Fast Red for 5 minutes
10) Rinse in running water
11) Dehydrate, clear then mount
Results: Elastic fibers, lipofuchsin and mast cells- blue
Cytoplasm and nuclei- red
This is the end of my posting. Thanks for reading! Enjoy the rest of your SIP! =)
*I want to make an important note. According to my colleague, the Liver Orcein Stain will be used to stain the HepB antigen while the VB Stain is only used for elastic fibers demonstration.
Tham Wan Jin June
TGo2
0505073G
12 comments:
hi
Is there other special stains required/available to demonstrate Hep B antigen and elastic tissues?
thanks
-sharon ang
0503219H
tg02
hey,
may i know what's the purpose of placing in acidified potassium permanganate solution for 5 mintues?
sally tg02
hey,
what is the advantage using these two special stains against Immuno-analyer? would it be more easier to use the immuno-assay?
elaine
Hi
For victoria blue nuclear fast red stain, why slides have to dry on hotplate after washing?
Wing Fat
tg01
hey,
are these stainings used for screening or confirming a person is having hepb?
boonching
hihi
In what circumstances will use this 2 stain? I realise this two stain require bleaching. Why there is a need for bleaching?
Juexiu
tg02
hey hi
Wat is the diference btw oxalic acid and sodium bisulphite since u use these reagents to bleach??
Vino
TGO2
hi!
how do you actually differentiate the different components when they are stained the same colour?
Charmaine
TG01
Hello!
Just want to know, what is the difference in morphology between the normal elastic tissues and those hep B positive tissues?
Thanks!
Charmaine Tan
TG01
Hi,
You mention:'Fine elastic fibers- dark brown
Hep B surface antigen- dark brown'
how do you distinguish them then?
Thanks.
Nisha
TG02
hi,liu qian here!
For the Liver Orcein Stain, as you mentioned , it is to demonstrates Hep B surface antigen. In another word,it is to detect hep B,rite?
Then why do they use this stain while immunoassay can detect hep b surface antigen very easily?
To sharon:
For demonstration of HepB antigen, our lab uses the Liver Orcein Stain. As for elastic fibers, there are other available stains like the Elastic Van Gieson and Verhoff's Van Gieson stain.
To Sally:
Acidified potassium permanganate is the oxidising agent that causes the disulphite bridges in elastic fibers to break down into anionic derivatives which can then be stain by the Liver Orcein Stain.
To Elaine and Liu Qian:
There's no ground for comparison as immuno methods uses fluid samples while our lab deals with tissue samples.
To Wing Fat:
It is to completely remove water in the tissues to prevent it from floating as it will be stain overnight.
To Boonching:
It is used as a screening test. Immunological methods will be the confirmatory procedures.
To Juexiu:
The Liver Orcein Stain will be used to demonstrate HepB antigen while VB Stain will be used for elastic fibers demonstration. The puporse of bleaching is to remove the brown colour after staining with acidified potassium permanganate.
To Vino:
These are the classical procedures.
To Charmaine and Nisha:
Not to worry. Different components will be stained with different colours but with varying intensity. Even if they are stained with the same colour, they can be easily differentiated by looking at their morphology.
To Charmaine Tan:
Elastic tissues- wavy fiber strands
HepB antigen- grandular inclusion bodies
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