Leishman's Stain

What It Is

Leishman's stain is a Romanowsky-type stain - a polychrome stain based on a mixture of eosin (acidic dye) and methylene blue (basic dye) dissolved in methyl (absolute) alcohol. The methanol acts simultaneously as a fixative and as the solvent for the dyes. Other Romanowsky-type stains include Giemsa, Jenner's, May-Grunwald, MacNeal's, and Wright's stain.

The reference Romanowsky method (ICSH, 1984) uses just two dyes - azure B and eosin Y - which together produce the full range of colours seen with ideal Romanowsky staining. - Henry's Clinical Diagnosis and Management by Laboratory Methods

Principle of Staining

Component	Dye type	Stains
Eosin (acidic)	Acidophilic	Haemoglobin, eosinophil granules - pink/red
Methylene blue / azure B (basic)	Basophilic	Nuclei, cytoplasmic RNA, platelet granules, parasite DNA - blue/purple

The kinetoplast of Leishmania amastigotes stains bright red with Giemsa/Leishman stains (non-metachromatic staining of the parasite).

Primary Uses

1. Peripheral Blood Smear (Haematology)

Leishman's stain is used routinely to stain peripheral blood films for:

Differential WBC count (neutrophils, lymphocytes, monocytes, eosinophils, basophils)
RBC morphology assessment (size, shape, colour, inclusions)
Platelet morphology
Detection of blood parasites (malaria, trypanosomes, microfilaria)

2. Malaria Diagnosis

Of the Romanowsky stains, Giemsa at pH 7.2 is preferred for malaria; however, Field's, Wright's, or Leishman's stain can also be used. - Harrison's Principles of Internal Medicine, 22E (2025)

3. Diagnosis of Leishmaniasis

Leishman's stain identifies Leishman-Donovan (LD) bodies - the amastigote form of Leishmania spp. - in:

Tissue smears (skin scrapings, fine-needle aspirates)
Bone marrow aspirates
Splenic aspirates (in visceral leishmaniasis / kala-azar)

In stained smears, the parasites take on a distinctive rounded appearance (having lost their flagella) within macrophage phagolysosomes and are termed Leishman-Donovan bodies. - Sherris & Ryan's Medical Microbiology, 8th Ed.

Leishman-Donovan (LD) Bodies - What You See

Small, oval amastigotes (2-4 µm) within macrophages/histiocytes
Pale blue cytoplasm, dark-staining nucleus (blue-purple), and a dot-like kinetoplast (bright red with Giemsa; identifiable with Leishman's)
Found extracellularly and intracellularly (~70% of cases of cutaneous leishmaniasis)
Giemsa/Leishman stain used with oil-immersion microscopy on air-dried, methanol-fixed smears

The histologic hallmark of cutaneous leishmaniasis is the presence of numerous extracellular and intracellular amastigotes (Leishman-Donovan bodies) within histiocytes. The organisms can also be highlighted by Wright and Feulgen stains. - Fitzpatrick's Dermatology

Histopathology Images

Fig A (H&E, low power): Cutaneous leishmaniasis showing tuberculoid granulomatous dermatitis with multinucleated giant cells and lymphoplasmacytic infiltrate:

Cutaneous leishmaniasis H&E low power - tuberculoid granulomatous dermatitis

Fig B (Giemsa stain, high power): Parasitized macrophages with amastigotes (Leishman-Donovan bodies, white arrows):

Giemsa stain showing Leishman-Donovan bodies (amastigotes) in cutaneous leishmaniasis - white arrows

Common Staining Problems & Troubleshooting

Problem	Cause	Fix
Excessively blue	Thick film, prolonged staining, alkaline buffer	Shorten stain time, lower pH buffer
Excessively pink	Understaining, prolonged washing, acidic buffer	Prolong staining, raise pH buffer
Precipitate on film	Dirty slides, drying during staining, poor filtration	Use clean slides, filter stain, wash properly
Poorly stained RBCs	Understaining or excessive washing	Prolong staining, reduce washing

Differential Diagnosis on Leishman-Stained Smears (Parasitized Macrophages)

When LD bodies are seen, consider distinguishing from:

Histoplasmosis - yeasts with a surrounding halo
Donovanosis (granuloma inguinale) - safety-pin appearance of Donovan bodies
Rhinoscleroma - Mikulicz cells
Trypanosomiasis, toxoplasmosis, blastomycosis

Summary: Leishman's stain is a simple, methanol-based Romanowsky stain used for blood films and parasite detection. Its most historically significant application - and the origin of its name - is the identification of Leishmania amastigotes (Leishman-Donovan bodies) in tissue and aspirate smears.

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