Periodic acid–Schiff (PAS) stain

Principle

• Periodic acid oxidizes adjacent hydroxyl groups (1,2-glycols) in carbohydrates to aldehydes.
• Schiff reagent (basic fuchsin decolorized by sulfurous acid) reacts with the aldehydes to form a magenta color.
• Diastase (or α-amylase) digestion before PAS (PAS-D) removes glycogen, so glycogen can be told apart from other PAS-positive material.

Specimen

• Formalin-fixed paraffin sections; also blood and bone marrow smears.

Reagents and equipment

• 0.5–1% periodic acid, Schiff reagent (refrigerated, dark bottle), diastase solution, hematoxylin counterstain.

Procedure

• Dewax and hydrate to water; for PAS-D, treat a duplicate slide with diastase first.
• Oxidize in periodic acid for about 5–10 minutes; rinse well.
• Apply Schiff reagent (commonly about 15 minutes); wash in running warm tap water to develop the full pink color.
• Counterstain nuclei with hematoxylin, blue, dehydrate, clear and mount.

Results

• Magenta: glycogen, neutral mucins, basement membranes, fungal cell walls, some parasites, and macrophages of Whipple disease.
• Glycogen is removed by diastase (PAS-D negative); neutral mucins, basement membranes, fungi and α1-antitrypsin globules remain PAS-D positive.
• Nuclei: blue.

Quality control and pitfalls

• Controls: liver or other glycogen-rich tissue for PAS and PAS-D; kidney for basement membranes.
• Test Schiff reagent by adding a few drops to formalin: a rapid red-purple color shows it is active; discard if it has turned pink.
• Over-oxidation or old reagents give weak or false staining.
• Glycogen is water-soluble and may be lost with delayed or aqueous fixation.

Clinical use

• Fungal infections in tissue, basement membrane changes in kidney disease, mucin in adenocarcinomas, glycogen in some tumors (e.g. Ewing sarcoma), α1-antitrypsin deficiency in liver, and Whipple disease.
• In hematology, block-like PAS positivity may be seen in some lymphoblasts and abnormal erythroid precursors.