Periodic Acid Schiff (PAS) stain

Introduction

The periodic acid Schiff (PAS) stain assay is a histochemical stain used to identify carbohydrates, including glycogen, mucopolysaccharides, and basement membranes. It is a simple and inexpensive stain that is widely used in pathology laboratories to diagnose a variety of diseases and conditions, including leukemia.

When used on peripheral blood, the PAS stain can be used to identify leukemic cells that contain increased amounts of glycogen or mucopolysaccharides. This can be helpful in differentiating between different types of leukemia, such as acute myeloid leukemia and acute lymphoblastic leukemia.

Principle of PAS stain

Periodic acid Schiff (PAS) stain is a method used to identify glycogen in tissues. PAS works by selectively oxidizing carbohydrate radicals, such as those found in glycols and related compounds, to produce dialdehydes. These dialdehydes react with Schiff’s reagent to produce a purple-magenta color. PAS stain is helpful in diagnosing acute lymphoblastic leukemia and some cases of erythroleukemia.

Method differs slightly according to the manufacturer’s protocol. 

Materials

• Absolute methanol
• Schiff’s reagent
• Period acid 1% w/v with distilled water
• Methyl green 4% with distilled water
• Unstained peripheral blood smear slide

Protocol

1. Fix slides in absolute methanol for 5 – 10 minutes. 
2. Rinse the slide with slow running tap water for 1 minute. 
3. Submerge in periodic acid for 10 minutes.
4. Rinse the slide with slow running tap water. 
5. Wipe the back of the slide and edges with Kim wipes. Be careful not to touch the smear. 
6. Submerge in Schiff’s reagent for 20 minutes.
7. Rinse the slide with slow running tap water for 5 minutes. 
8. Counterstain with methyl green for 3 minutes. 
9. Rinse the slide with slow running tap water. 
10. Wipe the back of the slide and edges with Kim wipes. Be careful not to touch the smear. 
11. Dry the slide using the hair dryer on the lowest speed or air dry in a tilted position.
12. Mount the slide with Depex and cover the zone of morphology with a cover slip. 
13. This slide is now ready for viewing. 

Interpretation

PAS stain is used to identify glycogen in tissues. PAS positive cells stain magenta (red) in the cytoplasm, while the nucleus stains green.

• Neutrophils are a positive control for PAS staining. They show intense, confluent granular positivity.
• In acute lymphoblastic leukemia, the lymphoblasts are stained block positivity.
• Myeloblasts contain a few small PAS-positive granules.
• Monocytes and their precursors show variable diffuse positivity.
• The reaction in megakaryocytes and platelets is variable.
• Erythroleukemia and erythroid precursors of some thalassemia are PAS positive.
• Normal erythroid precursors and red cells are negative. 

Periodic Acid Schiff (PAS) staining of blood cells

Cell Type	PAS Staining	Notes
Erythrocytes	Negative	Lack significant glycogen or other polysaccharide stores.
Neutrophils	Granular positivity	Neutrophils contain specific granules rich in glycoproteins and proteoglycans, which readily react with PAS stain, leading to intense, confluent granular positivity in the cytoplasm. This easily distinguishable staining pattern makes them good indicators of proper stain function.
Eosinophils	Negative	Granules primarily composed of proteins, not polysaccharides.
Basophils	Negative	Granules contain proteoglycans, but PAS staining typically yields negative results.
Lymphocytes	Weakly positive	Some lymphocytes, particularly activated ones, may exhibit faint cytoplasmic staining due to glycogen stores.
Megakaryoblasts	Negative	Lack significant glycogen or other PAS-positive structures.
Megakaryocytes	Negative	Similar to megakaryoblasts.
Platelets	Negative	Lack nuclei and cytoplasm, essential for PAS staining.
Lymphoblasts	Weakly positive	Similar to lymphocytes, can exhibit faint cytoplasmic staining due to glycogen.
Erythroblasts	Negative	Lack significant glycogen or other PAS-positive structures.
Monoblasts	Weakly positive	May exhibit faint cytoplasmic staining due to glycogen stores.
Macrophages	Variable	Can be positive depending on their activation state and glycogen content. Some types, like Kupffer cells in liver, are typically positive.
Myeloblasts	Negative	Lack significant glycogen or other PAS-positive structures.
Common lymphoid progenitor cells (CLPs)	Weakly positive	May show faint cytoplasmic staining due to developing glycogen stores.
Common myeloid progenitor cells (CMPs)	Negative	Similar to myeloblasts, lack significant PAS-positive structures.
HSCs (Hematopoietic stem cells)	Negative	Lack significant glycogen or other PAS-positive structures.

Periodic Acid Schiff (PAS) positive hematological disorders

While PAS staining plays a role in hematological diagnosis, it’s important to note that not all PAS-positive cases necessarily indicate disease. However, several hematological conditions exhibit PAS positivity, often serving as a clue for further investigation and diagnosis. Here’s an overview:

Leukemias

• Acute Lymphoblastic Leukemia (ALL): As previously discussed, ALL, particularly B-cell subtypes, frequently shows strong PAS positivity due to glycogen accumulation, altered membrane composition, and vacuoles.
• Erythroleukemia: This rare leukemia affects red blood cell precursors. Some types, like M6a and M7 in the French-American-British (FAB) classification, exhibit PAS-positive erythroblasts.
• Hairy Cell Leukemia: The neoplastic cells in this chronic lymphocytic leukemia subtype often stain weakly positive with PAS due to cytoplasmic inclusions.

Myeloproliferative Neoplasms (MPN)

• Chronic Myelomonocytic Leukemia (CMML): Some CMML patients show PAS-positive monocytes in the bone marrow.
• Mastocytosis: In systemic mastocytosis, bone marrow mast cells may exhibit weak PAS positivity due to their heparin content.

Other hematological conditions

• Myelodysplastic Syndromes (MDS): This group of bone marrow disorders can sometimes show PAS-positive erythroblasts, potentially indicating dyserythropoiesis (abnormal red blood cell development).
• Late Onset Pompe Disease: This genetic disorder can cause PAS-positive lymphocytes due to glycogen accumulation.
• Gaucher Disease: Certain subtypes of this lysosomal storage disorder can show PAS-positive Gaucher cells in the bone marrow.

Important Points

• PAS positivity alone is not diagnostic of any specific disease. It requires integration with other tests and clinical context for accurate diagnosis.
• The intensity and pattern of PAS staining can vary within a disease and across different cell types.
• PAS staining is not routinely used for primary diagnosis but can be helpful in certain situations, especially when combined with other techniques.

Frequently Asked Questions (FAQs)

Why does PAS stain positive in ALL?

While most mature blood cells stain negative or weakly positive for PAS stain, Acute Lymphoblastic Leukemia (ALL) cells frequently show strong PAS positivity. This positivity arises from various factors:

Glycogen accumulation: ALL blasts often have increased glycogen stores compared to normal lymphocytes. Glycogen, being a polysaccharide, readily reacts with the PAS stain, leading to visible color development.

Increased glycoproteins and glycolipids: The cell membranes and cytoplasm of ALL blasts may contain higher levels of glycoproteins and glycolipids compared to normal lymphocytes. These molecules also possess sugars that react with the PAS stain, contributing to positive results.

Presence of vacuoles: Some subtypes of ALL, particularly B-cell ALL, are associated with cytoplasmic vacuoles. These vacuoles can contain accumulated carbohydrates, leading to strong PAS positivity.

Importance in diagnosis: The strong PAS positivity observed in ALL plays a significant role in diagnosis and classification. It helps differentiate ALL from other types of acute leukemia, like Acute Myeloid Leukemia (AML), which typically stains negative or weakly positive with PAS.

Not universally positive: It’s important to note that not all ALL cases exhibit strong PAS positivity. The degree of positivity can vary depending on the specific subtype, individual cell features, and technical aspects of the staining procedure.

Additional notes:

• PAS alone doesn’t confirm ALL: While helpful, PAS positivity alone isn’t definitive for ALL diagnosis. Other tests, such as immunophenotyping and cytogenetic analysis, are crucial for confirmation.
• Subtypes with stronger positivity: B-cell ALL, particularly common ALL, tends to show stronger PAS positivity compared to T-cell ALL.

Why is erythroleukemia PAS positive?

The strong PAS positivity observed in certain types of erythroleukemia (particularly M6a and M7 in the FAB classification) arises from several factors:

1. Increased Glycogen Stores: Compared to normal erythroblasts, erythroleukemic blasts often accumulate more glycogen due to:

• Abnormal metabolic pathways: Dysregulation of enzymes involved in glycogen synthesis or breakdown can lead to elevated glycogen levels.
• Impaired maturation: Immature blasts might not utilize glycogen efficiently, leading to its accumulation.

2. Altered Membrane Composition: The cell membranes of erythroleukemic blasts may contain:

• More glycoproteins and glycolipids: These molecules possess sugars that readily react with the PAS stain, contributing to positive results.
• Abnormal glycosylation patterns: Changes in how sugars are attached to membrane molecules can enhance their reactivity with the PAS stain.

3. Cytoplasmic Vacuoles: Some subtypes of erythroleukemia, particularly B-cell ALL, are associated with cytoplasmic vacuoles. These vacuoles can contain:

• Accumulated carbohydrates: These stored carbohydrates, including glycogen, readily react with the PAS stain and contribute to positivity.
• Other PAS-positive substances: In some cases, the vacuoles might contain other molecules like mucin, further enhancing PAS positivity.

4. Significance of PAS Positivity:

• Diagnostic Clue: Although not diagnostic alone, strong PAS positivity in erythroblasts can raise suspicion of erythroleukemia, prompting further investigation with other tests like immunophenotyping and cytogenetics for definitive diagnosis.
• Differentiating from Other Leukemias: Strong PAS positivity often helps differentiate erythroleukemia from other leukemias like AML, which typically show negative or weak PAS staining in blasts.

What are the disadvantages of PAS stain?

Despite its valuable applications, PAS stain has some disadvantages to consider:

Non-specificity

• Stains multiple carbohydrate structures: PAS reacts with various polysaccharides like glycogen, mucins, and glycoproteins, making it difficult to pinpoint specific targets within a tissue.
• False positives: Other substances, like hemosiderin and basement membranes, can also stain positive, potentially leading to misinterpretations.

Sensitivity limitations

• May miss low-level carbohydrate targets: The stain might not be sensitive enough to detect small amounts of polysaccharides, potentially overlooking early disease signs.
• Variability in staining intensity: The intensity of PAS positivity can vary across different cell types and tissues, making interpretation challenging.

Technical considerations

• Time-consuming and multi-step procedure: Performing the stain properly requires multiple steps and precise timing, increasing potential for errors.
• Subjectivity in interpretation: Assessing staining intensity and patterns can be subjective, introducing potential observer bias.
• Susceptibility to artifacts: Improper fixation, processing, or staining technique can lead to artifacts that mimic or obscure true PAS positivity.

Additional limitations

• Not diagnostic alone: PAS positivity needs integration with other tests and clinical context for accurate diagnosis.
• Environmental concerns: Some reagents used in the stain are considered hazardous materials, requiring special handling and disposal.

What are the common counterstains used for Periodic Acid Schiff (PAS) stain?

PAS staining typically requires a counterstain to provide contrast and better visualize the structures highlighted by the PAS reaction. Here are some commonly used counterstains:

Nuclear counterstains

• Hematoxylin: The most widely used counterstain, providing blue or purple nuclei, creating a classic blue-magenta contrast with PAS-positive structures.
• Methyl blue: Produces a blue counterstain, particularly useful for highlighting nuclei and collagen.

Cytoplasmic counterstains

• Light green SF: Offers cytoplasmic counterstaining, highlighting non-PAS-positive areas in green.
• Neutral red: Another cytoplasmic counterstain, producing a pink-red color for non-PAS-positive regions.
• Orange G: Offers an orange counterstain, often used in combination with hematoxylin for a contrasting appearance.

Counterstain	Color	Advantages	Disadvantages
Hematoxylin	Blue/purple	Classic contrast, highlights nuclei	Can obscure subtle PAS positivity
Methyl blue	Blue	Highlights nuclei and collagen	Less vibrant contrast than hematoxylin
Light green SF	Green	Good cytoplasmic counterstain	May fade over time
Neutral red	Pink/red	Clear cytoplasmic counterstain	Not as widely used as light green SF
Orange G	Orange	Contrasting with hematoxylin, highlights cytoplasm	Less specific than other counterstains

• Some specialized counterstains are used for specific applications:
  • Alcian blue: Can be used before PAS to differentiate between different types of acidic mucins.
  • Fast green: Commonly used for fungal stains with PAS.

Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.

References

1. Bain BJ. A Practical Guide. 6th Edition (Wiley). 2022.
2. Bain BJ, Bates I, Laffan MA. Dacie and Lewis Practical Haematology: Expert Consult: Online and Print 12th Edition (Elsevier). 2016.
3. Carr JH. Clinical Hematology Atlas 6th Edition (Elsevier). 2021.