Saturday, April 29, 2017

Pas Stain (Periodic acid–Schiff stain)

Pas stain or periodic acid-Schiff staining is a procedure used to visualize the presence of polysaccharides including glycogen and mucosubstances like glycolipids and glycoproteins.

It can be done alone or in combination with diastase. The procedure is called pas-d stain or pas diastase stain. Diastase is a type of enzyme responsible for breaking down glycogen. (1)

When is pas staining needed?

It is primarily intended for staining structures that contain a strong proportion of carbohydrate macromolecules, which is common in the mucus, connective tissues, basal laminae, and glycocalyx.

It is basically used to diagnose various medical conditions.

  1. Paget disease
  2. Glycogen storage disease
  3. Adenocarcinoma
  4. Staining the presence of macrophages in people with Whipple’s disease (a bacterial infection affecting the gastrointestinal system).
  5. Alveolar soft part sarcoma
  6. Diagnosing α1-antitrypsin deficiency in patients who turned out positive for periportal liver hepatocytes.
  7. Ewing sarcoma (a malignant tumor that is small, round and blue in color)
  8. Erythroleukemia (a type of leukemia involving the immature red blood cells)
  9. Glycogen storage disease. There is an excessive amount of glycogen in various parts of the company, particularly in the kidneys, liver, and muscles. Through pas staining, the accumulation of glycogen will be checked.
  10. The level of glycogen will be checked in patients with tumor, especially in the tissues of the lungs, bladder, and pancreas.
  11. Pas staining is used to visualize fungal organisms in the tissues of people with fungal infection.
  12. A pas stain is used to highlight the basement of the membranes of tissues. It is particularly helpful in diagnosing a glomerular disease in the kidneys. 
Image 1 : A good pas staining showing healthy parietal cells.
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Figure 2 : A pas staining result characterized by blue color and a bit of violet discolorations.
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Photo 3 : In this image, there are blue, red, and purplish colors.
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How pas staining done and what is are the processes involved?

There are various staining procedures, but the pas stain is one of the commonly performed staining procedures. It is important to have a knowledge about the procedure. Pas stain procedure is used to highlight the molecules with a high percentage of carbohydrates contents. How does it work? It works by exposing the tissues to a periodic acid. It will serve as oxidizing agent that oxidizes the vicinal glycol groups or the amino derivatives. The oxidation process creates dialdehydes that when exposed to Schiff’s reagent will turn out to an insoluble magenta compound. (1, 2)

What are the solutions used?

  • A 1% aqueous periodic acid
  • Schiff’s reagent

How it is done?

You have to take sections to water and expose it to periodic acid solution for about 10 minutes. Thoroughly rinse with tap water. After that, you have to expose the section to the Schiff’s reagent for approximately 15 minutes and wash with running tap water for about 10 minutes. It will be countestain with a haemtoxylin for about 15 seconds. When necessary, you have to differentiate the stain and dehydrate, clear, and mount. (3)

Pas stain color and its histologic feature

  • Magenta – it suggests the presence of glycogen, basement membranes, fungal organisms, and mucus substances.
  • Blue – Nuclei
  • Green with light green counterstain – Suggests other tissue elements (4)

Things to keep in mind when pas staining

  • There are available commercially prepared periodic acid and Schiff’s reagent.
  • It is important to keep the Schiff’s reagent away from the ultraviolet light. When not in use, make sure you refrigerate it. If you won’t do this, the sulphur dioxide content of the Schiff’s reagent will be lost. The solution will then change its color from colorless to magenta. If you notice this change, then you have to replace the solution. The periodic solution should be refrigerated too when not in use.
  • It is important to rinse the solution in running tap water to intensify the magenta color. Although the decision to rinse the solution varies from one lab to another. (5, 6)

What is GMS Staining?

Aside from pas staining, another method used is the GMS staining. It stands for Gomori-Grocott methenamine silver stain. It is particularly helpful in diagnosing onychomycosis or fungal infection of the nail. It is primarily used in cytology and histology to identify not just fungi but Pneumocystis jiroveci too. With GMS staining, the gram is placed in a hot bath for penetration. (5)

What is a reticulin stain?

The reticulin stain is one of the highly preferred methods in histology. It is done to visualize the reticular fiber and is. It is primarily used in liver histopathology particularly helpful in the FNAB cell block preparations, especially in the differential diagnosis of liver nodules and in distinguishing between HCC and benign hepatic processes.

A diagnosis of HCC is confirmed if the result of reticulin staining outlining trabeculae has more than three cells in thickness. On the other hand, a diagnosis of benign hepatic process is confirmed if the reticulin stain outlined a hepatic trabeculae that is less than three cell layers in thickness. (8)

What is a trichrome stain?

The trichrome stain or also known as connective tissue stain is used to histologically visualize collagenous connective tissue fiber in the tissue sections. The kit can be used on frozen sections, paraffin-embedded, or formalin-fixed.

If the stain comes out blue, it is an indicator of collagen. If it comes out red, then it represents muscle fibers. A black or blue outcome represents nuclei. (7)

What is a Pas-d stain and its purpose?

Pas-d stain or pas staining with diastase is primarily used to differentiate glycogen from other pas positive elements like mucin, which can be found on the tissue sample. A mucin can only be detected if the glycogen is digested with diastase and rinse out.

Pas-d stain can also be used to differentiate glycogen granules from other granules in some types of tumor. Analysing the glycogen deposits in the liver will help in diagnosing several enzyme deficiencies. (9, 10)