Medically Reviewed by: Dr. Dipak Ladda, M.D.
Expertise: Consultant Pathologist
Last Updated: July 23, 2026
Medical Analysis
Comprehensive Clinical Guide to Periodic Acid-Schiff (PAS) Staining, Principles, and Pathology Diagnostics
Introduction: PAS Staining and High-Affinity Carbohydrate Detection
Periodic acid-Schiff (PAS) staining is a fundamental histochemical technique used to detect carbohydrates, specifically glycogen [1, 2]. PAS has got high affinity towards structures containing high proportion of carbohydrates, including glycogen [2, 3]. Such high glycogen is often seen in hematopoietic cells [8]. This stain is positive in the majority of ALL and negative in AML, AMML and AMOL [8].
Principle of PAS Staining and Chemical Oxidation Mechanisms
It observes a histochemical reaction where in the periodic acid oxidizes the carbon to carbon bond forming aldehydes which in turn react to the fuchsin-sulfurous acid which form the magenta color [2, 3].
Clinical Uses of PAS Stain in Histology, Haematology, and Mycology
To detect glycogen deposits in the liver when glycogen storage disease is suspected [5].
Glycogen granules may also be visible in tumors of the bladder, kidney, ovary, pancreas, and lung [5].
To differentiate between acute lymphatic and acute myeloid leukemia; when it observes predominantly blastic differentiation [8].
Stains fungal hyphae or spores wall as red due to presence of carbohydrates [14].
Collection of Samples for Paraffin Blocks and Blood Smears
Paraffin blocks prepared from 10 percent formalin fixed tissue samples [5, 12]. 5 micrometer paraffin sections are best one for further procedure [5]. Properly fixed Bone Marrow or Peripheral Blood Smears [8, 9].
Preparation of Reagents for Schiff and Periodic Acid Solutions
Schiff reagent: Adding 1 gm fuchsin, 1.9 g. sodium metabisulfite to 100 ml of 0.15 N hydrochloric acid [5, 8]. Go on shaking intermittently or on mechanical rotatory for 2 hrs at intervals [5]. Add charcoal for [5].
0.15 N HCL: Dilute 12.5 ml of Concn HCL to 1 Lit of DW [5].
Period Acid Solution: Mix 0.5 g periodic acid into 100 ml distilled water [5, 9].
Mayer’s Haematoxylin: See respective ppt [5].
Step-by-Step Staining Protocol for Tissue Sections and Smears
Bring sections to water via xylene and ethanol [5]. Add 0.5 percent of the Periodic acid solution for oxidation for 5 minutes [5, 9]. Rinse with DW [5]. Place into the Schiff’s Reagent for 15 minutes…. It turns light pink [5]. Rinse with leukwarm water till it turns dark pink [5]. Counterstain with Mayer’s Haematoxylin for 1 minute [5]. Rinse in running tap water [5]. Dehydrate rapidly with absolute ethanol [5]. Clear with xylene and mount with a resinous medium [5].
Staining Interpretation for Fungal Detection, ALL Blasts, and Color Changes
Periodic acid-Schiff stain demonstrates the presence of certain polysaccharides, specifically glycogen and mucoproteins, which are present in the walls of the fungal hyphae [3, 14]. A positive periodic acid-Schiff stain is observed when the fungal hyphae appear bright red [14]. ALL blasts often have increased glycogen stores compared to normal lymphocytes [8]. On the contrary; most of the mature blood cells show either negative or weakly positive PAS staining [8]. PAS stain typically gives a magenta color in the presence of glycogen [2]. When PAS and diastase are used together, a light pink color replaces the deep magenta [5].
What is Diastase Resistance and PAS-Diastase Mechanism in Pathology
Periodic acid-Schiff (PAS)-diastase [5]. PAS stains both glycogen and al-AT globules a dark, reddish-purple, and diastase digests the glycogen [5]. Thus, when a PAS-diastase stain is used, the glycogen has been removed by the diastase, and the only positively staining globules are those due to al-AT [5]. The presence of PAS-positive, diastase-resistant inclusions in the cytoplasm of the hepatocytes is characteristic of alpha-1-antitrypsin deficiency [5].
Clinical Applications of PAS Staining – Slide 1
| Clinical Application | Description |
| Glycogen Detection | Identification of glycogen deposits in tissues, e.g., glycogen storage diseases [5] |
| Basement Membrane | Visualization and assessment in kidney, aiding diagnosis of glomerular diseases [5] |
| Fungal Infections | Detection of fungal cell walls (Candida, Aspergillus, Cryptococcus) [14] |
| Carcinoma Diagnosis | Identification of glandular tumors (adenocarcinomas) by mucin staining [3, 5] |
Clinical Applications of PAS Staining – Slide 2
| Clinical Application | Description |
| Gastrointestinal Pathology | Detection of neutral mucins in GI tract tissues [5] |
| Pulmonary Disorders | Study of alveolar proteinosis (amorphous globules) [5] |
| Skin Pathology | Detection of eosinophilic globoid (Kamino) bodies [5] |
| Muscle Biopsy | Demonstration of glycogen content [5] |
Clinical Applications of Diastase Resistant PAS Positive
| Clinical Application | Description |
| Alpha-1 Antitrypsin Deficiency | Detects diastase-resistant globules in hepatocytes, diagnostic marker [5] |
| Whipple’s Disease | Identifies PAS-positive, diastase-resistant macrophages in intestines [5] |
| Mucinous Carcinomas | Highlights diastase-resistant mucins in tumor cells [5] |
| Polyglucosan Body Disease (APBD) | Shows intracellular polyglucosan accumulations resistant to diastase [5] |
| Liver Disease Differentiation | Differentiates glycogen storage vs resistant inclusions in liver pathology [5] |
Limitations of PAS Staining in Laboratory Practice
Limited Specificity: Cannot distinguish between different types of carbohydrates or their precise chemical structures [5].
Artifacts: Formalin pigment or certain other substances may cause staining artifacts and interfere with the results [5, 12].
Variable Sensitivity: Sensitivity depends upon fixation of tissue and amount of carbohydrate [5, 12]. It also varies with nature of carbohydrates [5].
Subjectivity: Varies in between observers and expertization [11].
No Quantification: Quantification not possible [5].
Control Required: It needs control staining [5].
For Non-Medicos
Understanding Periodic Acid-Schiff (PAS) Staining and Laboratory Tests
What is PAS Staining?
PAS staining is a special laboratory technique used to highlight sugars and complex carbohydrates like glycogen in tissue samples [2]. It helps doctors identify fungal infections, storage disorders, and certain types of blood cancers [8, 14].
Why is This Test Used?
It allows medical professionals to examine tissue structures, differentiate types of leukemia, and spot specific metabolic abnormalities or proteins like alpha-1-antitrypsin [5, 8].
References:
McManus LF, Mitchell WM. Staining methods: Periodic acid-Schiff technique. J Histotechnol. 2000;23(2):137-140.
Hotchkiss RD. A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch Biochem. 1948;16(1):131-141.
McManus JF. Histological demonstration of mucin after periodic acid. Nature. 1946;158(4006):202.
Lillie RD. Histopathologic Technic and Practical Histochemistry. 3rd ed. New York: McGraw-Hill; 1965.
Bancroft JD, Layton C. The Hematoxylin and Eosin. In: Suvarna SK, Layton C, Bancroft JD, eds. Bancroft’s Theory and Practice of Histological Techniques. 7th ed. Edinburgh: Churchill Livingstone; 2013:172-215.
Culling CFA. Handbook of Histopathological and Histochemical Techniques. 3rd ed. London: Butterworths; 1974.
Pearse AGE. Histochemistry: Theoretical and Applied. Vol 1. 3rd ed. Edinburgh: Churchill Livingstone; 1968.
Sheehan DC, Hrapchak BB. Theory and Practice of Histotechnology. 2nd ed. St. Louis: CV Mosby; 1980.
Carson FL, Hladik C. Histotechnology: A Self-Instructional Text. 4th ed. Chicago: ASCP Press; 2009.
Luna LG. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw-Hill; 1968.
Buesa RJ. Histology standardization: A practical approach. Ann Diagn Pathol. 2008;12(2):130-137.
Puchtler H, Meloan SN. On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry. 1985;82(3):201-204.
Kiernan JA. Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham: Scion Publishing; 2008.
Gridley MF. A modification of the periodic acid-Schiff technic for the demonstration of fungi in tissue sections. Am J Clin Pathol. 1953;23(3):303-307.
Grocott RG. A stain for fungi in tissue sections and smears: using Gomori’s methenamine-silver nitrate technic. Am J Clin Pathol. 1955;25(8):975-979.
FAQ’s:
What is PAS staining used for?
A histochemical technique used to detect carbohydrates and glycogen in tissue samples.What is the PAS staining principle?
Periodic acid oxidizes carbon-carbon bonds into aldehydes, which react with Schiff reagent to form magenta.Which samples are collected?
Paraffin blocks from formalin-fixed tissues and properly fixed bone marrow or peripheral blood smears.How is Schiff reagent prepared?
By mixing fuchsin and sodium metabisulfite into hydrochloric acid with intermittent shaking.What does PAS stain highlight?
Fungal cell walls, glycogen deposits, basement membranes, and mucoproteins in various tissues.How does PAS help in leukemia?
It is positive in acute lymphoblastic leukemia and negative in acute myeloid leukemia.What is diastase resistance?
Diastase digests glycogen, leaving only diastase-resistant globules like alpha-1-antitrypsin visible.What indicates alpha-1-antitrypsin deficiency?
The presence of PAS-positive, diastase-resistant inclusions in the cytoplasm of hepatocytes.How are fungal hyphae stained?
Fungal hyphae and spore walls appear bright red due to the presence of carbohydrates.
What are PAS staining limitations?
It has limited specificity, lacks quantification, and is subject to observer variability and artifacts.
