Special stains after H&E: PAS, trichrome and immunohistochemistry
A special stain (a dye or chemical reaction that colours one particular component) tests a narrow point raised by H&E, the routine haematoxylin and eosin stain.
Remember
Start with H&E, then add a special stain to test one specific point that it raised, and read the colour with the tissue pattern, because colour alone is not a diagnosis.
Haematoxylin and eosin (H&E) is the routine first stain. It shows nuclei, cytoplasm and protein-rich supporting tissue well, so it is used to survey the tissue pattern. It is weaker for some carbohydrates, lipids (fats), reticulin (a delicate network of thin fibres in the supporting tissue) and microorganisms. A special stain is added afterwards, once H&E has raised a particular point.
PAS (periodic acid-Schiff) is a chemical reaction: periodic acid turns sugar groups into aldehydes (reactive chemical groups), and the Schiff reagent then makes these magenta. It highlights carbohydrate-rich structures, including basement membranes, glycogen (the storage form of glucose), neutral mucins (sugar-rich secretions) and fungal cell walls. Because several structures can be PAS-positive, a PAS-positive structure cannot be named from its colour alone. Its identity depends on the method, for example a diastase step (an enzyme that digests glycogen), and on the site.
A trichrome stain such as Masson's colours collagen (the tough fibrous protein of scars and supporting tissue) blue or green and muscle red, whereas both look pink on H&E. Fibrosis means excess collagen laid down within a tissue. Map the blue or green signal onto the compartment already seen on H&E, such as a scar band or a vessel wall.
Immunohistochemistry (IHC) uses antibodies that attach to a particular protein in the tissue, followed by a coloured reaction product called the chromogen. A control slide (a section known to contain, or not contain, the target) lets a positive or negative reaction be trusted. The signal is only as meaningful as the antibody, the controls and the tissue context. If the labelled cells do not match the cells of interest on the companion H&E, suspect the laboratory or interpretation first.
Key facts
- Order
- H&E comes first to survey the tissue pattern, and a special stain is then used to test a specific point that H&E has raised.
- PAS targets
- Basement membranes, glycogen, neutral mucins and fungal walls can all be PAS-positive.
- Diastase
- Diastase removes glycogen, while basement membranes remain PAS-positive.
- Trichrome colours
- Collagen turns blue or green and muscle turns red.
- Immunohistochemistry
- Antibodies bind a protein and a coloured chromogen shows where.
- Reading the result
- A coloured result is evidence to be weighed with the tissue pattern, the location and, where relevant, a control slide, and not a diagnosis in itself.
Exam traps
- Special stains do not replace H&E as the first look at an unfamiliar tissue section, because H&E remains the routine first stain and special stains are added afterwards for a particular purpose.
- Telling collagen apart from muscle or cytoplasm is a job for a trichrome stain, not for PAS, because PAS highlights carbohydrate-rich structures such as basement membranes, glycogen and fungal walls.
- PAS staining that is retained after diastase does not identify glycogen, because diastase removes glycogen while basement membranes remain PAS-positive.
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