A Complete guide on How to Use Leishman Stain for Blood Smears

Leishman stain was discovered in 1901 and is used for staining blood smears. It is generally used to differentiate and identify leucocytes, malarial parasites and trypanosomes.

Principle of Leishman stain

The polychromic staining solution (Leishman) has methylene blue and eosin. These basic and acidic dyes induce multiple colors when applied to cells. Methanol acts as fixative and also as a solvent. The fixative does not allow any further change in the cells and makes them stick to the glass slide. The basic component of white cells (i.e. cytoplasm) is stained by acidic dye and they are described as eosinophilic or acidophilic. The acidic components (e.g. nucleus with nucleic acid) take blue to purple shades by the basic dye and they are called basophilic. The neutral components of the cell are stained by both the dyes.

Preparation of solutions

Leishman Eosin Methylene Blue dye solution:

• By slowly heating in water bath (40°C), dissolve 0.12 g of Leishman stain powder dye in 100 mL of methanol. Allow the set for 5 days, and then filter.

Buffer solution, pH 6.8

• Dissolve 1 pH 6.8 buffer tablet in 1 liter of distilled water while stirring.

Note: During the staining process it is possible to use pH 7.2 buffer solution or a combination of pH 6.8 and 7.2 buffer solutions. The process’s results can differentiate in shift toward red or blue on the color spectrum.

Diluted Leishman stain for manual staining

• Mix 30mL of Leishman’s solution with 150 ml of distilled or demineralized water and with 20 ml of pH 6.8 buffer solution.

Diluted Leishman stain for automatic staining

• Slowly add 30 ml of pH 6.8 buffer solution together with 220 ml of distilled or demineralized water into 50 ml of Leishman’s solution.
• Mix and leave for 10 min.

Blood smear staining procedure using Leishman stain – manual staining

• Let the smear dry
• Drop the Leishman’s solution on the fixed blood smear and let it react for 1 min
• Add 2 mL of buffer solution, gently stir and let it react for 5 min
• Rinse the smear using the pH 6.8 buffer solution
• Dry the preparation

Result (pH 6.8)

• Nucleus – red-pink
• Lymphocyte plasma – blue
• Monocyte plasma – grey-blue
• Neutrophil granule – light purple
• Eosinophil granule – red to red-brown
• Basophil granule – dark purple
• Thrombocytes – purple
• Erythrocytes – reddish

Preparing the sample and diagnostics

• Use only appropriate instruments for obtaining and preparing the samples.
• Analyze the samples with modern technology and mark them clearly.
• Follow the manufacturer’s instructions for handling. In order to avoid mistakes, the staining procedure and diagnostics should only be conducted by authorized and qualified personnel.
• Use only microscope according to standards of the medical diagnostic laboratory.
• In order to avoid an erroneous result, a positive and negative check is advised before application.

Safety

• Handle the product in accordance with safety at work and environmental protection guidelines.
• Used solutions and out of date solutions should be disposed of as special waste in accordance with national guidelines.
• Chemicals used in this procedure could pose danger to human health.
• Tested tissue specimens are potentially infectious.
• Necessary safety measures for protecting human health should be taken in accordance with good laboratory practice.
• Act in accordance with signs and warnings notices printed on the product’s label.

 Storing, stability and expiry date

• Store Leishman powder dye in a tightly sealed original packaging at temperature between 15°C and 25°C.
• Store in dry places, do not freeze and avoid exposure to direct sunlight.
• Expiry date is stated on the product’s label.

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