A Quick Guide to Special stains

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

HE image; a low power HE-stained section through haired skin

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Metachromatic stains

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)

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

Grocott’s image; fungal granuloma from the nose of a cat with very large numbers of fungal hyphae, staining black.

AgNOR image; sliver staining of a canine mast cell tumour, AgNORs can be seen as small brown dots within the nuclei.

Warthin-Starry image; stain highlighting the presence of Helicobacter-like organisms (black, arrowed) in the gastric mucosa from a dog.

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

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

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

Ziehl-Neelsen image; 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.

Ziehl-Neelsen image; staining of lead inclusion bodies in the nuclei, tubular epithelium, kidney of a lamb with lead poisoning (thanks to Mark Wessels)

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

PAS image; 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

Congo red image; 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

von Kossa image; 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 image; 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
Rhodanine – copper, in liver samples for example
Masson/Fontana – melanin - useful for poorly melanized or amelanotic melanomas

Perl’s Prussian blue image; Perls staining of a canine liver biopsy, with large amounts of haemosiderin present (blue).

Rhodanine image; Rhodanine staining of a canine liver biopsy, with very large amounts of copper present (red brown).