A Quick Reference Guide to Special Stains, by Melanie Dobromylskyj
- the haematoxylin and eosin (HE) stain is the routine stain used in histological sections and will be the first slide a pathologist looks at
- based on the appearance of this HE section, further different stains may be needed, for a number of differing reasons, for example:
- to try and further identify poorly differentiated neoplastic cells e.g. mast cells or mucin-secreting cells
- to highlight and identify infectious agents
- to identify deposits such as amyloid, calcium and a variety of pigments
- some specific tissue structures simply do not stain well with routine HE stains:
- reticular fibers in the liver; a silver stain will allow better assessment of liver architecture
- hydrophobic structures which tend to remain clear since these are rich in fats
Listed below are some of the more commonly used special stains in diagnostic histopathology, but this is not an exhaustive list
Giemsa (and other metachromatic stains such as Toluidine blue)
- metachromatic stains are those which have the ability to produce different colours with various histologic or cytologic structures; examples include the Giemsa, Toluidine blue, Astra blue, Wright and Diff-Quick stains
- Giemsa is a classic stain for peripheral blood smears and bone marrow specimens
- metachromatic stains such as Giemsa also help identification of mast cells, such as in poorly differentiated/poorly granulated mast cell tumours, in the assessment of surgical scar line resections for residual neoplastic mast cells, and in assessment of lymph nodes for the presence of metastatic disease.
Gram stain
• used for highlighting the presence and morphology of bacterial populations
• differentiates bacterial species into two main groups – Gram-positive and Gram-negative – based on the chemical and physical properties of their cell walls; the Gram stain detects peptidoglycan
• Gram-positive bacteria have peptidoglycan present in a thick layer in their cell walls, resulting in a blue/purple colour
• Gram-negative bacteria generally have a thin layer of peptidoglycan which results in a pink/red colour
• Not all bacteria can be definitively classified by this technique, with Gram-variable and Gram-indeterminate groups also
• Gram staining is not always reliable in histological sections and follow-up culture and full identification of any potentially significant bacterial populations seen on histology is usually recommended
Silver stains
- silver-nitrate based staining techniques are used for several purposes
- Warthin-Starry staining may be used to detect spirochetes such as Leptospira, and Helicobacter species
- Grocott’s methenamine silver stain can be used for fungal organisms
- Silver-staining is used to detect AgNORs (Argyrophilic nucleolar organiser region), a prognostic test for mast cell tumours
- Gordon and Sweet’s stain highlights reticulin fibres, helping demonstrate liver architecture for examined
Ziehl-Neelsen (ZN)
- also known as the acid-fast stain
- used to identify acid-fast organisms, mainly Mycobacterial species
- may be present in very low numbers, not stained by HE stain
- specialised cultures or molecular techniques such as PCR are required to confirm and to identify the species of Mycobacterium involved
- ZN stain can also be used to identify intranuclear lead inclusion bodies
Periodic acid-Schiff (PAS)
- detects glycogen and other polysaccharides
- used to identify fungal infections, such as Malassezia, fungal hyphae and Candida (living fungi)
- the presence of glycogen can be confirmed in tissue sections by using diastase to digest the glycogen from a section, then comparing a diastase-digested PAS section with a normal PAS section – may be used in liver biopsies and on neurological tissues to differentiate glycogen storage diseases from other types of storage disease
Congo Red
- detects amyloid
- amyloid is a fairly homogenous, nondescript eosinophilic material on routine HE sections
- Congo Red stain colours amyloid an orange-red colour
- when viewed under a polarised light the material demonstrates apple-green birefringence
- primary amyloidosis is the most common systemic or generalised form (plasma cell or B-cell dyscrasias such as multiple myeloma, other monoclonal B-cell proliferations)
- secondary or reactive amyloidosis is the form that is generated from increased amounts of the acute phase protein serum amyloid A (chronic inflammatory conditions, neoplasia, idiopathic)
- familial amyloidosis is a hereditary and systemic condition which can affect various organ systems, for example the kidney in Shar Pei dogs
Von Kossa
- detects calcium, coloured black
- pathological calcification of tissues falls into two broad categories, dystrophic (serum calcium levels are normal, but the tissue is not) and metastatic (serum calcium levels are increased, but the tissues are normal)
- dystrophic calcification can occur in necrotic tissues, or with calcinosis cutis and calcinosis circumscripta
- metastatic calcification can be associated with renal failure (secondary hyperparathyroidism), vitamin D toxicosis (ingestion of calcinogenic plants, rodenticides, Psoriasis cream), primary hyperparathyroidism, pseudohyperparathyroidism and destructive bone lesions
- Alizarin red is another calcium stain
Other tissue components
- Massons trichrome – connective tissues (e.g. fibrosis in liver samples)
- Alcian blue (Alcian blue/PAS) – acid mucins (acid and neutral mucins) – mucin-secreting neoplasms
Permanganate bleach
- useful for heavily pigmented tumours, especially melanomas
- bleaching of sections removes the colour of the pigment and allows clearer assessment of nuclear morphology and the mitotic count
Other pigments
- Fouchet – bile pigments, may be used in cases of cholestasis
- Perls Prussian blue – haemosiderin
- Rhodanine – copper, in liver samples for example
- Masson/Fontana – melanin – useful for poorly melanized or amelanotic melanomas
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