Pigment - Staining Techniques PPB and GB/FM Flashcards
What principle of staining does the Perl’s Prussian Blue technique use?
Histochemical Reaction
- where chemical reagents are converted to chromogens.
The reaction is dependent upon the amount of iron present.
What are the main chemicals involved in Perl’s Prussian Blue stain and their purpose?
Perl’s Prussian Blue stain:
1. Histochemical reaction solution made from
- 2% HCl and
- 2% Potassium Ferrocyanide.
–> Demonstrates hemosiderin as a coloured compound (chromagen) –> BLUE
2. Eosin - Counterstain, stains all other tissue components
–> PINK
What is hemosiderin and what does it contain?
Hemosiderin, a breakdown product of hemoglobin contains ferric iron plus protein.
What is the purpose of HCl in the histochemical solution and how does Prussian Blue form?
HCl is used to free the iron from the protein.
The ferric iron then combines with potassium ferrocyanide to form an insoluble blue pigment, ferric ferrocyanide, known as Prussian Blue.
What is the ratio of parts of HCL and Potassium Ferrocyanide for the histochemical working solution and what are the important considerations?
- Equal parts of HCL and Potassium Ferrocyanide is required to create a working solution to stain iron.
- Best to make solution fresh and use right away as it can breakdown.
What is the colour Prussian Blue turns that indicates it is breaking down?
The working solution of Prussian Blue is light-yellow colour, once it turns green it can indicate it is breaking down and should be avoided on slides.
What is the most important step in the Perl’s Prussian Blue staining technique?
Making sure the histochemical solution is made up correctly.
Besides Eosin, what are alternate dyes that can be used as the counterstain for PPB?
Perl’s Prussian Blue counterstain options besides Eosin are:
- Nuclear Fast Red
- Neutral Red
A dye that stains pink/red as a colour for background is commonly used.
If using Nuclear Fast Red as the counterstain in Perl’s Prussian Blue, what must be considered?
If using Nuclear Fast Red, ensure it is rinsed in tap water well after to avoid slides turning cloudy when going through dehydrate and clear.
Why is it important to have a good positive control that doesn’t have too much iron for Perl’s Prussian Blue?
A control that has too much iron would have a higher chance of getting nonspecific staining occurring.
Why is it important to avoid acid fixatives and decalcification solutions in PPB? What should be done instead?
Acid fixatives and decalcification solutions removes iron from tissue.
Use neutral pH fixative and avoid acid decalcifiers.
Why is acid washed glassware and non-metal forceps required to be used in PPB?
Having clean glassware and non-metal forceps will ensure that it is only iron being stained on the slides.
Why is distilled water and ANALAR grade reagents recommended to be used with Perl’s Prussian Blue technique?
- To avoid metals from tap water to get on slides and cause false positives, slides are washed in distilled water to eliminate external elements on the slide that would interfere with results.
- ANALAR Reagents= A chemical reagent of high purity with known contaminants for use in chemical analyses.
How is the Perl’s Prussian Blue solution disposed of in the RRC lab?
For proper disposal of Histochemical solution, we collect solution and commercially dispose of waste.
What is hemochromatosis and how would a slide of tissue with this condition look?
Hemochromatosis= large amounts of iron in the body that can cause life-threatening diseases that shut down certain organs.
The tissue would be almost all blue showing the large amount of iron in this tissue confirming a disease state (see slide 20).
