Periodic Acid-Schiff with Diastase Digestion (PAS+D)

Aim of special stain:: Distinguish glycogen from diastase resistant components such as mucin and fungi
Diagnostic applications:: Glycogen storage diseases, glycogen in tumour cells, Gaucher’s disease, Niemann-Pick disease, duodenal adenomas, Whipple’s disease
Material supplied for surveys:: Liver tissue cut at 4µm
Recommended control:: Composite block composed of liver and small bowel tissue - Identical sections on 2 separate slides. One is digested and the other is not. Both are stained with the PAS stain.

PAS with Diastase staining method

Reagents – commercial source unknown, unless specified:

1. 1% Periodic acid

2. Amylase solution 0.5%
a) α-amylase	0.5 g
b) Distilled water	100 mL
Dissolve the enzyme in the water with gentle shaking. Prepare fresh (can be reused throughout the day). Use either Sigma α-amylase type VI-B from porcine pancreas or α-amylase from Bacillus subtilis (commercial preparation – MP Biomedicals).

3. Schiff’s reagent

4. Haematoxylin

5. 0.5% Acid alcohol
a) Hydrochloric acid	10 mL
b) Absolute Alcohol	1400 mL
c) Distilled water	600 mL
Mix alcohol and water in the stock bottle. Carefully add the hydrochloric acid and mix thoroughly.

6. Scott’s blue solution

7. Graded alcohols and Xylene

Method:

Dewax the paraffin sections and bring to water.
Place alpha amylase on slide on flat staining rack for 20 minutes in the oven or a waterbath. * The diastase solution should be warm at 37°C. Do not heat above 40°C as the enzyme can be destroyed. Insufficient heat can also cause incomplete digestion of glycogen.
Rinse well in running water, then distilled water.
Oxidize the sections in periodic acid 5 minutes.
Rinse well in distilled water a few minutes.
Treat the section in Schiff’s reagent for 15 minutes.
Wash in running water 10 minutes (check staining microscopically).
Haematoxylin 1 minute.
Wash in running water.
Differentiate in acid alcohol.
Wash in running water.
Blue sections for 1 minute.
Wash in running water 10 minutes (check Haematoxylin and adjust if necessary).
Dehydrate, clear and mount.

Results:

Glycogen will be absent.
Neutral mucins, some epithelial mucins, basement membranes, and fungi – pink/magenta

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.

References

2014 RCPAQAP Technical General Survey 3.
Russell B. Myers, Jerry L. Fredenburgh and William E. Grizzel. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6^th edition. New York, NY: Churchill Livingstone; 2008:168-182.