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
A low power HE-stained section through haired skin.
  • 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.
Giemsa staining of a canine mast cell tumour (granules are magenta)

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

Highlighting the presence of bacteria within an abscess from a dog. Gram positive (blue) filamentous organisms.

Silver stains

Warthin-Starry stain highlighting the presence of Helicobacter-like organisms (black, arrowed) in the gastric mucosa from a dog.
  • 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
Grocott’s methenamine fungal granuloma from the nose of a cat with very large numbers of fungal hyphae, staining black.
Sliver staining of a canine mast cell tumour, AgNORs can be seen as small brown dots within the nuclei.

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
Staining of Mycobacteria in a lesion containing very large numbers of acid-fast organisms, from a cat. Organisms are dark red magenta against the background counterstain which is light blue.

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
Fungal granuloma from the nose of a cat with very large numbers of fungal hyphae, stained with PAS (purple hyphae).

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
a renal biopsy from a Shar Pei with amyloidosis – amyloid within glomeruli is stained orange red.

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
Mesenteric adipose tissues, from a puppy which died from vitamin D toxicosis, causing vitamin D toxicosis and widespread mineralisation of tissues throughout the body. Calcium stains black.

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
Masson’s trichrome staining highlighting the presence of fibrosis (blue) in a section of feline heart, with an underlying cardiomyopathy.

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
Perls staining of a canine liver biopsy, with large amounts of haemosiderin present (blue).
  • Rhodanine – copper, in liver samples for example
  • Masson/Fontana – melanin – useful for poorly melanized or amelanotic melanomas
Rhodanine staining of a canine liver biopsy, with very large amounts of copper present (red brown).

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