Periodic Acid-Schiff with Diastase (PASD) stain

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Introduction:

The Periodic Acid-Schiff with Diastase (PASD) stain is a commonly used histochemical staining technique in pathology that enables the detection of glycogen, basement membranes, and fungal organisms in tissue samples. PASD stain is widely used for the diagnosis of a variety of conditions, including glycogen storage diseases, basement membrane disorders, and fungal infections. In this stain, the periodic acid oxidizes the polysaccharides, while the Schiff's reagent binds to the oxidized sugars, producing a magenta color under the microscope.

Requirements:

The PASD stain requires the following reagents and equipment:

  • Periodic acid
  • Schiff's reagent
  • Diastase enzyme
  • Hydrochloric acid
  • Harris hematoxylin
  • Deionized water
  • Alcohol and xylene for dehydration and mounting
  • Glass slides and coverslips
  • Tissue samples

Principle:

The PASD stain works on the principle of oxidative cleavage of the polysaccharides by periodic acid, followed by the binding of the oxidized sugars to Schiff's reagent, resulting in a magenta color. The addition of diastase enzyme before the stain digestion allows the differentiation of glycogen from other polysaccharides. The glycogen is digested by the enzyme, and a negative result for PASD stain indicates the absence of glycogen in the tissue.

Procedure:

The procedure for the PASD stain is as follows:

  1. Deparaffinize and rehydrate the tissue sections.
  2. Treat the sections with periodic acid solution for 5-10 minutes.
  3. Wash the sections with deionized water.
  4. Treat the sections with Schiff's reagent for 15-30 minutes.
  5. Wash the sections with running tap water.
  6. Digest the sections with diastase enzyme for 15-30 minutes.
  7. Wash the sections with running tap water.
  8. Counterstain with Harris hematoxylin for 5-10 minutes.
  9. Rinse the sections with deionized water.
  10. Dehydrate the sections with alcohol and clear with xylene.
  11. Mount with a coverslip using a mounting medium.

Results:

The PASD stain produces magenta staining of glycogen, basement membranes, and fungal organisms. Negative results for glycogen are indicated by the absence of magenta staining after diastase digestion. Basement membranes appear magenta, while fungal organisms may appear magenta to reddish-brown.

Quality Control (QC):

  1. Monitoring the performance of the periodic acid solution, Schiff's reagent, and diastase enzyme.
  2. Use of positive and negative controls to ensure proper staining.
  3. Proper storage of reagents to prevent degradation.
  4. Checking the pH of the solutions to ensure that they are within the correct range.
  5. Checking the expiration date of the reagents and discarding any expired reagents.

Interpretations:

  1. The presence of magenta staining in glycogen storage diseases confirms the diagnosis, while the absence of glycogen staining rules out the possibility of such a disease.
  2. The presence of magenta staining in basement membranes is normal, while the absence of staining may indicate basement membrane defects.
  3. The presence of fungal organisms in tissue sections may indicate an infection.
  4. Interpretation of PASD staining requires proper correlation with the clinical and histological findings and consideration of the diagnostic context.
  5. The staining results should be carefully evaluated and reported by a qualified pathologist.

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