Romanowsky staining (Leishman / Wright)

Principle

• Romanowsky stains combine an oxidised basic dye (methylene blue with azure B) and an acidic dye (eosin Y), dissolved in methanol.
• Azure B binds acidic components (DNA, RNA) giving blue to purple; eosin binds basic components (haemoglobin, eosinophil granules) giving orange-red. The purple of nuclei and neutrophil granules is the Romanowsky effect.
• Correct colour depends on buffer pH, usually 6.8 for blood films (7.2 for malaria).

Specimen

• Air-dried blood or marrow films.

Reagents and equipment

• Leishman or Wright stain in methanol, phosphate buffer pH 6.8, staining rack.

Procedure

• Cover the film with undiluted stain for about 1–2 min (the methanol fixes the film).
• Add about twice the volume of buffer, mix by gently blowing until a metallic sheen appears.
• Stain for about 7–10 min (times vary with stain batch; standardise locally).
• Rinse with buffer or water until the thin area appears pinkish; wipe the back, air dry upright.

Results

• RBCs: pink to orange-pink. Nuclei: purple. Neutrophil granules: lilac. Eosinophil granules: orange-red. Basophil granules: dark purple-blue. Lymphocyte cytoplasm: pale blue. Platelets: purple granules.

Quality control and pitfalls

• Too blue: buffer too alkaline, thick film, overstaining or insufficient washing.
• Too pink/red: buffer too acidic, understaining or overwashing.
• Stain precipitate: unfiltered or evaporated stain, inadequate rinsing; filter stain and do not let it dry on the slide.
• Water in methanol stain causes poor fixation and refractile artefacts.
• Check each new batch with a known normal film.

Clinical use

• Routine staining of blood and marrow films for morphology and differential counts.