Periodic acid–Schiff (PAS) staining - Principle, Reagents, Procedure, Result, Application
Introduction to Periodic acid–Schiff (PAS) staining
The staining method Periodic acid–Schiff (PAS) is used to detect the presence of carbohydrates and carbohydrate compounds such as polysaccharides (glycogen) and mucosubstances (glycoproteins, glycolipids, mucin) in connective tissues, mucus, the glycocalyx, basal laminae, skeletal muscles, the liver, kidney, and the cardiac muscles.
These glycogen granules may also be present in the tumors of the bladder, kidney, ovary, pancreas, and lung. This process uses paraffin-embedded, formalin-fixed, or/and frozen sample tissue sections.
Capsule and cell walls of fungi such as Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, Aspergillus fumigatus, and Blastomycosis contain polysaccharides (Glycogen, cellulose, and starch). Thus PAS staining procedure is also used to demonstrate hyphae and yeast forms of fungi in tissue samples due to the high carbohydrate content of the organism's cell walls.
Principle of Periodic acid–Schiff (PAS) staining
The periodic acid reacts to oxidize the vicinal diols in these sugars (polysaccharides) and breaks up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide units. These monosaccharide units are parts of the long polysaccharides that create a pair of aldehydes at the two free tips of each monosaccharide ring that has been broken earlier.
As these pair of aldehydes at the two free tips of each broken monosaccharide ring reacts with Schiff reagent to give a purple-magenta color, oxidation conditions must be properly regulated so that aldehydes are further not oxidized.
As a counterstain, a suitable basic stain is used.
Periodic acid–Schiff (PAS) staining may also include the use of the enzyme that breaks down glycogen i.e. diastase. This process is called PAS diastase stain (PAS-D).
If alcian blue is applied before the PAS step, the process is called Alcian blue/periodic acid–Schiff (AB/PAS or AB-PAS).
Preparation of Reagents for PAS Stain
Periodic Acid Solution
Periodic acid solution, used for oxidation is used in any strength between 0.5% and 2.5% while 1% solution is preferred.
Periodic acid= 1gm
Distilled water= 100ml
Schiff’s Reagent
Fuchsin Basic: 1 gm
Distilled water: 100 ml
Sodium metabisulphite: 2 gm Conc.
HCl: 2 ml
Charcoal activated: 0.3 gm
Process
Dissolve basic fuchsin in boiling water, cool at 50°C and filter.
Add sodium metabisulphite and HCl.
Store in a dark room at room temperature overnight.
Add charcoal, shake for one minute, and filter.
Procedure of Periodic acid–Schiff (PAS) staining
The procedure for Periodic acid–Schiff (PAS) staining is as follows:
Flame the slide on the burner and place it in xylene. Repeat the process to remove the wax
Drain xylene from the slide and hydrate the tissue sections by passing it through decreasing concentrations of alcohol baths (100%, 90%, 80%, 70%) and water
Place the tissue section in a periodic acid solution (1%) for 5-10 minutes
Wash the slide with distilled water at least twice
5. Add Schiff’s reagent to the slide and leave it for 20-30 minutes
Rinse in running tap water for 5-10 minutes
Add the counter stain (hematoxylin) for 3-5 minutes
Dehydrate the tissue containing the slide by passing it through increasing the concentration of alcohol
Place the slides in two xylene baths to clear
Use DPX or other mounting media
Observe under a microscope
Result, Interpretation of Periodic acid–Schiff (PAS) staining
If the tissue stains magenta pink to red in color, then the sample is PAS-positive material i.e. contains carbohydrates.
The nuclei of the cells will stain blue or blue depending upon the type of dye used. The background also stains blue.
Application of Periodic acid–Schiff (PAS) staining
Periodic acid–Schiff (PAS) staining has many applications in the diagnosis of different conditions.
Glycogen storage disease - detect glycogen deposits in the liver
Adenocarcinomas – in cytology used in undifferential identification of tumors
Erythroleukemia – immature RBC leukemia, cells stain a bright fuchsia
Ceroid lipofuscinosis (NCL) - highlights super cross-linked lipid inclusions
Pulmonary interstitial glycogenosis (PIG) - identify glycogen in lung biopsy specimens of infants
Ewing Sarcoma
α1-antitrypsin deficiency
Whipple's disease – stains macrophages
Alveolar soft part sarcoma (ASPS)
Paget disease of the breast
Gastrointestinal pathology – detects mucins in gastrointestinal (GI) tract
Lung study – detects amorphous or granular globules of the pulmonary alveolar proteinosis.
Muscle biopsies - demonstrate glycogen components
Skin – study Kamino bodies
Search for implanted medical devices composed of nonoxidized cellulose as PAS stains cellulose
When renal disease is being accessed, Periodic acid–Schiff (PAS) staining is used to demonstrate the thickness of glomerular basement membrane diagnosis of Gaucher’s disease, Krabbe’s disease, and lysosomal storage diseases
Diagnosis of Gaucher’s disease, Krabbe’s disease, and lysosomal storage diseases by demonstration of cerebrosides and gangliosides.
In microbiology - Fungal Infections – only work on living fungi, polysaccharide-laden fungal cell walls stain magenta
Due to high carbophydrate content in cell wall/capsule, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumiagtus, Blastomyces, etc can be demonstrated by PAS stain
PAS-positive granule at the anterior end of the fungal spore indicates microsporidia
PAS+ (PASD+) bacteria include Bacillus cereus, Corynebacterium diphtheriae, Propionibacterium acnes, Klebsiella pneumoniae, and Micrococcus luteusStains Whipple disease: foamy macrophages containing PAS+ (PASD+) intracytoplasmic granules (Tropheryma whippelii bacteria)