The Periodic Acid-Schiff (PAS) stain is a special staining method used across histology and pathology laboratories. This technique is designed to visualize and identify carbohydrates and carbohydrate-rich macromolecules within tissue samples. It provides pathologists with a powerful tool to examine structures that are otherwise difficult to distinguish using standard staining protocols, aiding in specific disease diagnosis.
The Chemical Basis of PAS Staining
The PAS stain relies on a two-step chemical reaction that targets specific molecular structures. The reaction begins with periodic acid, which acts as a strong oxidizing agent when applied to the tissue section. Periodic acid specifically targets vicinal diols, which are pairs of hydroxyl (-OH) groups attached to adjacent carbon atoms within the carbohydrate structure. This oxidation step breaks the carbon-carbon bond between the hydroxyl groups, converting them into reactive aldehyde groups.
The second step involves introducing the Schiff reagent, a colorless compound containing basic fuchsin. This reagent is a fundamental component of the stain and reacts specifically with the newly formed aldehyde groups. When the Schiff reagent binds to these aldehydes, a distinctive chemical transformation occurs, resulting in the formation of a bright magenta, fuchsia, or red-purple colored compound. This intense coloration is localized precisely at the site of the original carbohydrate-rich structure. The intensity of this magenta color directly correlates with the concentration of the targeted carbohydrates in the tissue section.
Cellular and Extracellular Components Highlighted
The PAS stain identifies various polysaccharides and mucosubstances containing 1,2-glycol groups. Glycogen is a frequently stained intracellular component, appearing as bright magenta granules within the cytoplasm of cells in tissues like the liver, muscle, and kidney. The stain also highlights neutral mucins, which are glycoproteins and glycolipids secreted by epithelial cells in the gastrointestinal tract and respiratory system. The PAS reaction visualizes tissue basement membranes, which are supportive layers underlying epithelium and endothelium, particularly in the kidney glomeruli. Finally, the carbohydrate-rich cell walls of many fungal organisms, including Cryptococcus and Aspergillus, stain prominently with the magenta color.
Essential Diagnostic Applications in Pathology
The PAS stain has substantial utility in diagnostic pathology. In renal pathology, it is routinely used to assess the thickness and integrity of the glomerular basement membrane, indicating diseases like membranous nephropathy. The stain is also used to identify fungal infections, as the carbohydrate-heavy cell walls of organisms like Histoplasma capsulatum or Candida albicans are clearly demarcated. In oncology, the PAS reaction helps classify certain tumors, particularly adenocarcinomas, which may produce neutral mucins that stain positively.
Specific Disease Identification
Detecting large, PAS-positive, diastase-resistant globules in liver cells can be indicative of Alpha-1 antitrypsin deficiency. The PAS stain confirms metabolic disorders, such as glycogen storage diseases, where excessive amounts of glycogen accumulate in the liver or muscle tissues. It is also employed in diagnosing Whipple’s disease, where the causative bacteria leave behind PAS-positive inclusions within macrophages of the small intestine lining.
Interpreting Staining Results and Controls
The magenta coloration confirms the presence of aldehyde-generating carbohydrates, such as glycogen, mucins, or basement membrane components, in that specific location. The location of the magenta color—whether within the cytoplasm, cell borders, or extracellular matrix—informs the pathologist about the nature of the substance being identified.
To distinguish between different PAS-positive substances, particularly glycogen, the Periodic Acid-Schiff-Diastase (PAS-D) procedure is performed. Diastase is applied to a parallel tissue section to digest the glycogen before the staining process. If the magenta staining is present on the standard PAS slide but absent on the diastase-treated slide, the substance is confirmed to be glycogen. Substances that remain magenta after diastase digestion, such as basement membranes or neutral mucins, are considered diastase-resistant.