Principle:
1-2 glycol groups are oxidised by periodic acid to produce dialdehydes. The dialdehydes, when exposed to leucobasic fuchsin (Schiff’s reagent), give positive reactions in the form of a red product. Positive reactions are given by glycogen, monosaccharides, polysaccharides, glycoproteins, mucoproteins, phosphorylated sugars, inositol derivatives, and cerebrosides. Glycogen distinguished from others by its sensitivity to diastase digestions In hematopoietic cells, positive PAS is due to glycogen.
Chemicals:
1. Basic Fuchsin
2. HCI
3. Anhydrous sodium metabisulfite (Na2S2O5)
4. Activated Charcoal
5. Periodic Acid
6. Mayer’s hemtoxylin
Reagents:
Reagent 1: 200 ml of boiling distilled water in which 1 gram of basic fuchsin is dissolved slowly. Cool to 50°C and filter. To filtrate, add 20 ml 1N HCL (0.6 ml HCl + 20 ml distilled water). Cool to 25°C and add 1 gram of anhydrous sodium metabisulfite. Allow to stand in the dark for 24 hours, when the solution will be straw or orange-coloured. Add 2 grams of activated charcoal and shake for 1 minute. Filter into a brown (light-proof) stock bottle. Store the reagent in dark place at 0–4°C. Allow the solution to come to room temperature before use.
Note: Solution is stable for 2 months in dark at 0–4°C. Discard if it becomes light pink. Test every week for potency.
Potency test: To 3 ml of 40% formaldehyde, add a few drops of Schiff’s reagent. Immediate development of a reddish-purple color shows potency. The delayed development of the deep blue color shows that the solution has to be discarded.
Reagent 2: Periodic acid solution: dissolves 1 gram of periodic acid in 100 ml of distilled water and filters.
Reagent 3: Mayer’s hemtoxylin.
Procedure
1. Fix a fresh smear in buffered formalin-acetone fixative at 4–10°C for 45 seconds. Wash it slowly in running tap water for 30 seconds and keep it for air drying.
2. Cover the smear with periodic acid solution for 10 minutes at room temperature. Wash the slide and dry.
3. Cover the smear with Schiff’s reagent at room temperature for 30 minutes in dark.
4. Rinse the slide slowly under running tap water for 15 minutes and air dry.
5. Counterstain in Mayer’s hematoxylin for 10–15 minutes.
6. Rinse slowly under running tap water for 5 minutes and air dry.
Result
The reaction product is magenta red to pink. The positivity is described as diffuse, granular or block like.
Interpretation
Granulocyte precursors are negative to diffuse weakly positive.
Mature neutrophils show intense confluent granular positivity.
Eosinophils show diffuse cytoplasmic positivity with granules being negative.
Basophils are negative but may show large irregular blocks and positive material not related to the granules.
Monocytes and precursors show variable diffuse positivity and superimposed fine granules at the periphery of the cytoplasm.
10–40% of peripheral lymphocytes show granular positivity with a negative background. There was no difference in staining for B & T lymphocytes.
Lymphoblasts show block positivity.
Normal erythroid precursors and red cells are negative. In dyserythropoeisis, there may be diffuse staining of normoblasts with coarse granules in some early normoblasts.
Megakaryocytes and platelets show variable, usually intense diffuse positivity with superimposed fine granules, coarse granules, and large blocks.
Image source: MyHematology