Periodic Acid Schiff Stain (PAS Staining)

Introduction

The periodic acid Schiff stain is one of the first cytochemical tests you will meet in hematology. It is cheap, technically straightforward, and visually striking. Magenta deposits against a green nuclear background tell you something specific: carbohydrates are present in this cell, and sometimes a lot of them. For decades the periodic acid Schiff stain helped pathologists separate lymphoid leukemias from myeloid ones at a time when flow cytometry did not exist. It still has a role today, though a more focused one [4,6].

This article walks through how the periodic acid Schiff stain works, how to perform it on a peripheral blood or bone marrow smear, and how to interpret what you see including where the test fits in the modern workup of acute leukemia.

Principle of Periodic Acid Schiff Stain

The chemistry is two steps. First, periodic acid oxidizes the hydroxyl groups on adjacent carbon atoms in sugars, converting them into aldehyde groups. Second, Schiff's reagent (a colorless solution of basic fuchsin treated with sulfurous acid) binds to those aldehydes and recovers its magenta-pink color. The result is a bright stain wherever carbohydrate-rich molecules sit in the cell [3].

Glycogen is the main hematologic target, but mucins, glycoproteins, glycolipids, and basement membrane material also react. That breadth is both the test's strength and its limitation. We will return to this in the interpretation section.

Materials

• Absolute methanol (or 10% neutral buffered formalin, depending on protocol)
• 1% periodic acid in distilled water (freshly prepared)
• Schiff's reagent (commercial; refrigerated, dark bottle)
• 1–2% aqueous methyl green counterstain
• Unstained peripheral blood or bone marrow smear
• Mounting medium (DPX or Depex) and coverslips

Protocol

Method differs slightly according to the manufacturer's protocol. 

1. Fix the slide in absolute methanol for 5–10 minutes. (Some protocols use brief formalin fixation instead; follow your laboratory's standard.)
2. Rinse in slow-running tap water for 1 minute.
3. Submerge in 1% periodic acid for 10 minutes at room temperature.
4. Rinse in slow-running tap water.
5. Wipe the back and edges of the slide with a Kim wipe, avoiding the smear area.
6. Submerge in Schiff's reagent for 20 minutes, ideally in the dark.
7. Rinse in slow-running tap water for 5 minutes. The magenta color develops during this wash.
8. Counterstain with methyl green for 3 minutes.
9. Rinse in slow-running tap water and wipe the back of the slide.
10. Air-dry the slide in a tilted position, or use a hair dryer on its lowest setting.
11. Mount with Depex and apply a coverslip over the area of interest.
12. The slide is ready for microscopy.

Interpretation

Two things matter when reading a PAS slide: which cells are positive, and what pattern the positivity takes. Pattern often carries more diagnostic weight than presence alone.

Staining patterns to recognize:

• Block positivity — one or two sharply defined magenta clumps in the cytoplasm. Classic for B-cell ALL lymphoblasts.
• Granular positivity — fine, scattered, often confluent magenta granules. Typical of mature neutrophils and the built-in positive control.
• Diffuse positivity — homogeneous pink cytoplasmic blush. Less specific. Seen in monocytes and some reactive cells.

PAS-positive cells appear magenta in the cytoplasm; nuclei stain green from the methyl green counterstain.

Where the Periodic Acid Schiff Stain Fits in Modern Leukemia Diagnosis

Decades ago, the periodic acid Schiff stain was a frontline tool for separating lymphoid from myeloid leukemias. That is no longer the case. The current WHO 2022 and International Consensus Classification 2022 frameworks define acute leukemias by combining morphology, immunophenotyping, cytogenetics, and molecular features [6,7]. Flow cytometry which uses antibodies against surface markers like CD19, CD10, CD34, CD13, and CD33 provides faster and more specific lineage assignment than any cytochemical stain.

So why do we still use PAS? Three reasons.

First, it works. A reevaluation study against immunophenotyping found that PAS positivity combined with negative myeloperoxidase, Sudan Black B, and alpha-naphthyl butyrate esterase staining had 100% specificity for lymphoblastic lineage, even though sensitivity was only around 52% [4]. The test rarely calls a myeloid leukemia "lymphoid."

Second, it is cheap and accessible. In laboratories without flow cytometry, PAS plus a small panel of cytochemical stains remains a workable diagnostic strategy.

Third, it covers situations where flow cytometry struggles particularly the dry tap, where bone marrow fluid cannot be aspirated and only a fixed biopsy is available [1].

PAS-Positive Hematological Conditions

Several conditions show PAS positivity. Positivity is a clue, not a diagnosis, and always needs integration with the clinical picture and other tests.

Acute lymphoblastic leukemia (ALL). B-cell subtypes show characteristic block positivity due to massive glycogen accumulation in malignant lymphoblasts. Certain favorable-risk genetic subtypes of B-ALL correlate with robust PAS staining. For example, B-ALL with the ETV6::RUNX1 fusion (t(12;21))—the most common genetic rearrangement in childhood ALL—frequently exhibits prominent, coarse PAS block positivity, serving as an early morphologic clue to this specific genetic profile [8].

Acute erythroid leukemia (AML-M6 in the older FAB system). Erythroblasts accumulate glycogen and stain positive, supporting the diagnosis when seen alongside dyserythropoiesis [1,3].

Acute megakaryoblastic leukemia (AML-M7). Blasts are often PAS-positive and may show block or granular patterns, helping confirm megakaryocytic lineage when material is limited [1].

Hairy cell leukemia. Neoplastic cells often show weak PAS positivity from cytoplasmic inclusions [1].

Chronic myelomonocytic leukemia (CMML). Some cases show PAS-positive marrow monocytes [1].

Myelodysplastic syndromes (MDS). PAS-positive erythroblasts can support a finding of dyserythropoiesis [1].

Pompe disease (acid alpha-glucosidase deficiency). Lymphocytes accumulate glycogen in cytoplasmic vacuoles, producing a distinctive PAS-positive vacuolated lymphocyte. This is a recognized peripheral blood clue to a rare diagnosis [1].

Metastatic Ewing Sarcoma: When evaluating bone marrow biopsies for non-hematologic malignancies, the PAS stain is highly useful for identifying "small round blue cell" tumors. Ewing sarcoma cells characteristically contain abundant intracellular glycogen granules, resulting in strong PAS positivity that disappears completely after diastase digestion [9].

Gaucher disease. Some Gaucher cells in the bone marrow show PAS positivity from accumulated glucocerebroside and related material.

Why ALL Blasts Are PAS-Positive

The mechanism is glycogen overload. ALL blasts, especially in B-cell common ALL, store more glycogen than normal lymphocytes because their glucose metabolism is dysregulated. Enzymes that build glycogen are upregulated, while enzymes that break it down work poorly. The glycogen aggregates into large cytoplasmic deposits that produce the classic block pattern under PAS. Altered membrane glycoproteins and cytoplasmic vacuoles in some subtypes add to the staining intensity [1,4].

This is why the periodic acid Schiff stain is still taught alongside immunophenotyping. It illustrates a piece of leukemia cell biology — abnormal carbohydrate handling — that markers like CD19 and CD10 do not show.

Counterstains

A counterstain provides nuclear or background contrast so that PAS-positive cytoplasm is easier to read.

Specialized variants exist. Alcian blue used before PAS distinguishes acidic mucins from neutral mucins. Fast green pairs with PAS for fungal identification.

Disadvantages of the Periodic Acid Schiff Stain

The test has real limitations that explain why it has moved from frontline to adjunctive role.

Non-specificity. PAS reacts with glycogen, mucins, glycoproteins, basement membranes, and some pigments. Without diastase digestion, you cannot tell which carbohydrate caused a positive result.

Modest sensitivity. Around half of lymphoblastic leukemias show diagnostic PAS positivity [4]. The other half pass silently.

Subjective interpretation. Block, granular, and diffuse patterns sit on a continuum. Two observers may grade the same slide differently.

Technical sensitivity. Schiff's reagent loses activity when exposed to light, air, or warmth. Old reagent gives weak or absent color. Quality control is essential.

Not diagnostic alone. Every PAS finding needs confirmation with immunophenotyping, cytogenetics, or molecular testing under current classification frameworks [6,7].

Troubleshooting

Frequently Asked Questions (FAQs)

What is the periodic acid Schiff stain used for?

It detects carbohydrates in cells and tissues — glycogen, mucins, glycoproteins, basement membranes. In hematology, the periodic acid Schiff stain is used mainly to support the distinction between acute lymphoblastic leukemia and acute myeloid leukemia, with B-cell ALL classically showing block-pattern positivity [1,4].

Why is PAS positive in acute lymphoblastic leukemia?

Lymphoblasts in ALL accumulate glycogen because their glucose metabolism is abnormal. Synthesis is increased and breakdown is impaired, so glycogen builds up in cytoplasmic clumps that stain magenta with PAS. The block pattern is most prominent in B-cell common ALL [1,4].

Why is erythroleukemia (AML-M6) PAS-positive?

Malignant erythroblasts in M6 accumulate glycogen, partly through dysregulated metabolism and partly through impaired maturation. Strong PAS staining of erythroblasts, alongside dyserythropoiesis on morphology, supports the diagnosis. Note: M6 is erythroleukemia in the FAB system; M7 is acute megakaryoblastic leukemia.

Is PAS still used now that flow cytometry is available?

Yes, but in a narrower role. Flow cytometry is the first-line method for classifying acute leukemias because it is faster and more specific. PAS remains useful in resource-limited settings, in dry-tap cases where only biopsy material is available, in confirming megakaryoblastic lineage, and in detecting glycogen-laden lymphocytes in conditions like Pompe disease [4,6,7].

What does block positivity mean?

One or two large, sharply defined magenta clumps in the cytoplasm — different from the fine granular pattern of neutrophils. Block positivity in lymphoid-looking blasts strongly suggests B-cell ALL [1].

Can normal cells stain PAS-positive?

Yes. Mature neutrophils stain strongly and serve as the built-in positive control. Some lymphocytes, monocytes, platelets, and macrophages can show weak or variable positivity. Positivity by itself never indicates disease — pattern, intensity, cell type, and clinical context all matter together.

Glossary of Related Medical Terms

• Cytochemistry: Lab techniques that use chemical reactions on a cell smear to highlight specific substances inside cells, such as enzymes or sugars. Helps identify cell type when morphology alone is not enough.
• Glycogen: A branched polymer of glucose. Cells store it as an energy reserve. It is the main target of the PAS reaction in blood cell diagnostics.
• Schiff's reagent: A solution made from basic fuchsin and sulfurous acid. It turns magenta when it reacts with aldehyde groups produced by periodic acid oxidation.
• Block positivity: A staining pattern where a cell shows one or two large, dense magenta clumps instead of fine granules. Classic for B-cell acute lymphoblastic leukemia.
• Lymphoblast: An immature lymphocyte. The malignant cell in acute lymphoblastic leukemia.
• Myeloblast: An immature granulocyte precursor. The malignant cell in acute myeloid leukemia.
• Erythroleukemia: A rare leukemia of red blood cell precursors, classified as AML-M6 in the older FAB system.
• Diastase (alpha-amylase): An enzyme that digests glycogen. Used as a control: a section treated with diastase loses PAS positivity if the staining was due to glycogen.
• Immunophenotyping: A technique, usually flow cytometry, that uses antibodies against cell surface markers to classify cells. The current first-line method for diagnosing acute leukemias.
• Counterstain: A second stain applied to provide background color and contrast. For PAS, methyl green or hematoxylin highlights nuclei.
• Dry tap: A bone marrow aspiration that yields no fluid, often due to fibrosis or packed marrow. Forces reliance on biopsy and histochemistry, where PAS retains value.

Disclaimer: This protocol is for educational purposes only. Local laboratory standard operating procedures take precedence. It is not intended to be a substitute for informed professional medical advice, diagnosis, or treatment. Always consult a qualified healthcare professional for clinical decision-making. While the information presented here is derived from credible medical sources and is believed to be accurate and up-to-date, it is not guaranteed to be complete or error-free. See additional information.

References

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4. Snower, D. P., Smith, B. R., Munz, U. J., & McPhedran, P. (1991). Reevaluation of the periodic acid-Schiff stain in acute leukemia with immunophenotypic analyses. Archives of pathology & laboratory medicine, 115(4), 346–350.
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