Special Stains

Special stains are chemical dyes and reactions that colour particular tissue components, organisms or deposits in sections, beyond what haematoxylin and eosin shows. They are quick, cheap and robust, and they give a visual answer to a defined question: is there a fungus, an acid fast bacillus, iron, amyloid, fibrosis, mucin or copper.

They are requested by the clinician, or added by the pathologist after seeing the routine slide. Many are used on paraffin sections, and a few on smears, frozen sections or fluid preparations.

A stain works by a chemical reaction or an affinity between the dye and a tissue component. Control tissue with known positive and negative components is stained with each batch to confirm that the stain worked.

Turnaround is usually 1 day after the routine slide is available. Special stains remain essential in infectious disease, liver, renal and gastrointestinal pathology, and in places where immunohistochemistry and molecular testing are limited.

Indications.

• Infection: Gram stain (bacteria), Ziehl-Neelsen and auramine rhodamine (acid fast bacilli), Fite (leprosy), Grocott methenamine silver and periodic acid-Schiff (fungi), Giemsa (Helicobacter pylori, Leishmania, Histoplasma), Warthin-Starry (spirochaetes, Bartonella), and mucicarmine (Cryptococcus capsule).
• Deposits: Congo red (amyloid, with apple green birefringence under polarised light), Perls Prussian blue (iron), von Kossa and alizarin red (calcium), Oil Red O (fat, on frozen sections), and rhodanine and orcein (copper and copper binding protein).
• Connective tissue: Masson trichrome and Sirius red (collagen and fibrosis), reticulin (reticulin framework in liver and marrow), and Elastic van Gieson (elastic fibres in vessels).
• Mucins and carbohydrates: periodic acid-Schiff with and without diastase (glycogen, basement membrane, fungi, mucin), Alcian blue (acid mucin, such as intestinal metaplasia), and mucicarmine.
• Pigments: Fontana-Masson (melanin), Perls (haemosiderin), and bile stains.
• Neuropathology and muscle: Luxol fast blue (myelin), and enzyme histochemistry on frozen muscle sections.
• Blood and marrow: Giemsa, Perls for iron stores and sideroblasts, and reticulin for marrow fibrosis.
• Cell types: toluidine blue (mast cells).

Not indicated as a routine panel on every specimen. The choice depends on the morphology and the clinical question.

Specimen requirements.

• Formalin fixed, paraffin embedded sections at 3 to 5 microm (thicker for some stains), on uncoated or charged slides.
• Smears, touch preparations, fluids and cell blocks for infection stains.
• Frozen sections for fat (Oil Red O) and some enzyme histochemistry.
• Unstained sections cut from the existing block can be used, which saves new sampling.
• Positive control slides, run in the same batch.
• Clinical details and the organism or substance suspected.

Normal appearances.

Normal tissue shows no organisms, no abnormal deposits and no abnormal staining. Normal baseline staining depends on the stain: for example, reticulin is a fine network around hepatocyte plates, normal iron is absent in the liver and present in marrow macrophages, and periodic acid-Schiff stains normal glycogen, which is digested by diastase.

Interpretation.

• Acid fast stains: red bacilli on a blue background in tuberculosis and other mycobacteria. Modified (weak acid) stains show Nocardia, and Fite shows Mycobacterium leprae. Tissue AFB stains are positive in only a minority of tuberculous granulomas, so a negative result does not exclude tuberculosis.
• Fungal stains: Grocott methenamine silver stains fungi black, and periodic acid-Schiff stains them magenta, with shapes that help identify them (narrow septate hyphae with acute angle branching in Aspergillus, broad aseptate hyphae in Mucorales, budding yeasts in Candida and Cryptococcus, small intracellular yeasts in Histoplasma).
• Congo red: amyloid appears pink to red, with apple green birefringence under polarised light. Typing needs immunohistochemistry or mass spectrometry.
• Perls Prussian blue: iron appears blue. Liver iron is graded, and the pattern (periportal hepatocytes in haemochromatosis, Kupffer cells in transfusional overload) points to the diagnosis. Marrow iron stores and ring sideroblasts are assessed.
• Trichrome and Sirius red: collagen is blue or red, and the amount of fibrosis is staged (for example, in liver disease).
• Reticulin: loss or condensation of reticulin around hepatocyte plates suggests hepatocellular carcinoma or cirrhosis, and increased reticulin in marrow shows myelofibrosis.
• Periodic acid-Schiff with diastase: glycogen is digested, and glycoproteins, mucin, fungi and alpha-1 antitrypsin globules (diastase resistant) remain.
• Alcian blue: acid mucin in intestinal metaplasia and mucinous tumours.
• Giemsa and Warthin-Starry: curved bacilli of Helicobacter pylori in the gastric mucosa, and Leishmania amastigotes in macrophages.
• Rhodanine and orcein: copper and copper binding protein in chronic cholestasis and Wilson disease, with copper quantification needed for confirmation.
• Fontana-Masson: melanin in melanocytic lesions, and in amelanotic lesions with other markers.
• Mast cell stains: increased mast cells in systemic mastocytosis.
• Reports state the stain used, the result, and any organisms or substances seen, with the limits.

Critical values and findings needing immediate action.

• Organisms in tissue from an immunosuppressed or septic patient (fungi, acid fast bacilli): direct communication to the clinician, with culture and treatment.
• Fungal hyphae suggestive of Mucorales in a sinus or rhino orbital specimen: immediate contact, as early surgery and antifungal treatment matter.
• Amyloid on a biopsy: prompt workup for the type, with urgent haematological assessment for light chain disease.
• Severe iron overload on liver biopsy: communication to the hepatology team.

Limitations and artefacts.

• Sensitivity is limited: acid fast bacilli need roughly 10,000 organisms per mL to be seen, and many tissue samples with tuberculosis are negative.
• Stain artefacts and precipitate, dye deposits, contaminated water baths ("floaters"), and dead organisms mimic infection.
• Over staining and under staining, thick sections, and poor fixation reduce reliability.
• A negative stain does not exclude infection, and culture and molecular tests are still needed.
• Fungal morphology overlaps among species, so culture or molecular tests give the exact species.
• Congo red needs polarised light and an experienced reader, and thin sections can be falsely negative.
• Decalcification removes iron and calcium.
• Controls must be run, since results are valid only if the controls are correct.
• Special stains do not replace immunohistochemistry or molecular tests for lineage and treatment targets.

Special groups.

• Immunosuppressed patients: routine screening with fungal and acid fast stains on biopsies with granulomas or necrosis.
• Patients with granulomatous inflammation: tuberculosis, fungal and other stains, with culture.
• Patients with suspected iron overload or Wilson disease: liver iron and copper stains, with quantification of tissue iron or copper.
• Patients with suspected amyloidosis: tissue from the fat pad, rectum or the affected organ, with typing.
• Children: stains for storage disease and infection.
• Regions: tuberculosis, leprosy, leishmaniasis, histoplasmosis, cryptococcosis and other deep fungal and parasitic infections are common. Special stains (Ziehl-Neelsen, Fite, Grocott, Giemsa and periodic acid-Schiff) are among the most useful tools on tissue in places without molecular tests. Reagent supply, quality control and trained technicians are limits.

Follow up tests.

• Culture and drug susceptibility testing.
• Molecular tests: GeneXpert, PCR, and sequencing for organisms.
• Immunohistochemistry (organisms, amyloid typing, lineage markers).
• Serology and antigen tests for fungal and parasitic infection.
• Quantitative tissue iron or copper measurement.
• Serum and urine protein electrophoresis, free light chains, and bone marrow examination for amyloid.
• Genetic testing for haemochromatosis and Wilson disease.
• Specialist infectious disease, haematology or hepatology review.