Leishman Stain

Introduction

The Leishman stain, characterized by its contrasting hues of blue, pink, and purple, offers a deeper understanding of cellular morphology and differentiation. The nuclei of cells stain a crisp blue, while the cytoplasm takes on vibrant pink or purple hues depending on cell type and composition. This intricate color palette allows for the clear identification of different blood cell types, including red blood cells, lymphocytes, monocytes, neutrophils, eosinophils, basophils and platelets.

Beyond mere identification, the Leishman stain unveils vital information about cellular function. The presence or absence of specific cytoplasmic inclusions, such as granules or vacuoles, can provide clues about the cell’s activity and potential abnormalities. For instance, the presence of abnormal granules in neutrophils can be suggestive of infectious processes, while vacuoles within lymphocytes may indicate viral infection.

However, the power of the Leishman stain extends beyond routine investigations. Its ability to differentiate subtle variations in cellular morphology and staining patterns makes it invaluable in diagnosing various hematological disorders. The presence of immature cells, abnormal nuclear shapes, or unusual cytoplasmic inclusions can be indicative of conditions like leukemia, lymphoma, or anemia.

Principle of Leishman Stain

Romanowsky stains which include Leishman and Wright are commonly used to stain the peripheral blood smear.  Chemical components of the Leishman dye are Azure B (blue in colour) and Eosin Y (orange) to stain different groups of molecules in the cells. The DNA and acidic groupings of the proteins of cell nuclei and primitive cytoplasm determine the uptake of the basic dye Azure B. Conversely, the presence of the basic groupings on the hemoglobin molecules results in its affinity for acidic dye like Eosin Y.

Flood Slide Method

Materials

• Leishman stain
• Phosphate buffer 0.66 M pH 6.8
  • Solution A: KH₂PO₄ 9.1 g/L
  • Solution B: Na₂HPO₄ 9.5 g/L
• Pasteur pipette
• Kim wipes
• Hair dryer
• Unstained peripheral blood smear slide
• Timer
• Cover slips (optional)
• Mounting medium (optional)

Protocol

1. Preparation of phosphate buffer for 100 mL volume: Add 50.8 mL of solution A to 49.2 mL of solution B to obtain pH 6.8.
2. Cover blood film completely with the Leishman stain using a Pasteur pipette and wait for 2-3 minutes. Approximately 3 mL of stain is required to stain a single slide.
3. Add an equal volume of phosphate buffer onto the slide (stain:buffer ratio = 1:1). Use a Pasteur pipette to blow gently over the slide to completely mix the stain and buffer. DO NOT touch the stain using the Pasteur pipette.
4. Leave the slide to stain for 15-20 minutes.
5. Remove the stain with slow running tap water. Wipe the back portion of the slide and the edges dry using Kim wipes without touching the blood smear.
6. Dry the slide with a hair dryer on low speed.
7. Mount the slide with Depex and cover the zone of morphology with a cover slip.
8. This slide is now ready for viewing.

Dip Slide Method with Coplin Jars

Materials

• Leishman stain
• Phosphate buffer 0.66 M pH 6.8
• Unstained peripheral blood smear slide
• 4 Coplin jars
• Deionized water
• Methanol (optional, for air-drying slides faster)
• Forceps
• Timer
• Cover slips (optional)
• Mounting medium (optional)

Protocol

1. Follow the manufacturer’s instructions for diluting the Leishman stain concentrate with the appropriate buffer solution. Mix well and let stand for 10 minutes before use.
2. Pour enough methanol, Leishman stain solution, stain-buffer or phosphate buffer solution (according to manufacturer’s protocol) and deionized water into each Coplin jar, respectively to completely submerge the slides.
3. Place slides in methanol (fixative) for 30 seconds.
4. Using forceps, carefully dip each slide into the Coplin jar containing the stain solution for 3 minutes. Ensure the entire sample area is submerged.
5. Remove the slides from the stain solution and dip them into the buffer solution Coplin jar for 6 minutes.
6. Transfer the slides to a Coplin jar filled with deionized water. Rinse gently for 10-15 seconds. 
7. Differentiate in methanol (optional): Briefly dip the slides in methanol for 1-2 seconds (avoid overexposure, as this can lead to excessive destaining). This step helps to enhance the color contrast.
8. Allow the slides to air dry completely at room temperature. 
9. Place a drop of mounting medium like Depex on each slide and cover with a clean cover slip. Avoid trapping air bubbles. Allow the mounting medium to dry completely before microscopic examination.

Interpretation

Cell Type	Nucleus	Cytoplasm	Granules
Red Blood Cells (RBCs)	No nucleus	Pink to red, depending on age and hemoglobin content	None
Neutrophils	Deep blue, bilobed	Pale blue to lilac-pink	Fine, pink to purple
Basophils	Deep blue, round or oval	Deep blue to purple	Large, dark purple to black
Eosinophils	Deep blue, bilobed	Pale pink to orange	Large, orange-red
Monocytes	Deep blue, round or indented	Pale blue to gray	Fine, faint pink
Lymphocytes	Deep blue, round or indented	Pale blue to clear	None
Platelets	No nucleus	Pale purple to pink	None

Troubleshooting

Overall Faulty Stain

• Incorrect stain preparation: Dilution errors, pH imbalance, contaminated solution.
• Improper staining technique: Incorrect timing, inadequate agitation, uneven dipping.
• Slide issues: Dirty slides, inadequate fixation, air-drying artifacts.
• Water quality: Impurities in rinsing water affecting stain intensity.
• Expired reagents: Over time, the stain solution itself can degrade, leading to reduced staining intensity, affecting visibility of cells and structures, uneven staining due to compromised components and incorrect color profiles.
• Contaminated stain: Presence of impurities can alter staining properties.
• Dirty slides: Grease or contaminants can affect stain adherence.
• Oversliding (air-drying for too long): Prolonged air-drying can shrink and deform cells, making them less receptive to stain.
• Destaining with methanol for too long: Overexposure to methanol can remove desired stain components, particularly from cytoplasmic elements, resulting in pale or washed-out appearance.

Specific Color Issues

Too Blue Stain

• Incorrect stain pH: Too acidic buffer (lower pH) can lead to excessive blue staining.
• Overstaining: Extended staining time beyond recommended duration.

Blue-Black Nuclei

• Overfixation: Excessive exposure to fixative agents can harden nuclei, hindering stain uptake.
• Improper rinsing: Insufficient rinsing after staining can trap excess stain.
• High pH buffer: Alkaline buffer (higher pH) can intensify nuclear staining.
• Stock stain exposed to light.

Too Pink, Grey Cytoplasm

• Understaining: Insufficient staining time or improper dilution.
• Insufficient differentiation: Overexposure to methanol can destain cytoplasm.
• Excessive washing: Excessive washing in buffer solution can affect staining intensity.

Unstained Neutrophil Granules

• Weak stain solution: Insufficient stain concentration.
• Improper mixing: Uneven distribution of stain in the solution.
• Technical issues: Problems with staining procedure or timing.

Pseudo-Toxic Granules (Dark Blue)

• Overstaining/differentiation issues: Can lead to excessive granule staining.
• High pH buffer: Enhances granule staining intensity.
• Sample quality: Degraded cells or improper smear preparation can affect granule appearance.

Other Stain Anomalies

• Precipitates: Contamination or improper stain storage can lead to sediment formation.
• Artifacts: Air bubbles, dust, or debris on the slide can interfere with staining.

Stain Deposit on Film and Blue Background

• Improper cleaning of slides: Residual dirt or grease can trap stain.
• Contaminated stain solution: Impurities can cause a blue background tint.
• Technical issues: Problems with staining procedure or timing.

Frequently Asked Questions (FAQs)

How to prepare Leishman stain from powder?

While preparing Leishman stain from powder is possible, it’s generally not recommended due to several reasons:

1. Safety Concerns: Handling methanol requires appropriate safety precautions and proper disposal procedures.
2. Accuracy and Consistency: Ready-made solutions offer standardized quality and minimize errors in dilution and handling.
3. Convenience and Time Saving: Utilizing prepared solutions is quicker and avoids the potential inconsistencies of homemade stains.

However, if you must prepare Leishman stain from powder, follow these steps with caution:

Materials

• Leishman stain powder (e.g., Sigma-Aldrich 67690)
• Methanol
• Deionized water
• Measuring flask
• Magnetic stirrer (optional)
• pH meter (optional)
• Phosphate buffer pH 6.8 (optional)
• Safety glasses, gloves, and fume hood

Procedure

1. Consult the safety data sheet (SDS) for methanol and follow all safety precautions.
2. Work in a well-ventilated fume hood.
3. Weigh 0.12 g of Leishman stain powder using an accurate balance.
4. Dissolve the powder in 100 mL of methanol in a measuring flask. Stir or use a magnetic stirrer until completely dissolved.
5. Leave the solution to stand for 5 days at room temperature, allowing for complete dye extraction.
6. Filter the solution through a Whatman No. 1 filter paper.
7. Optionally, dilute the filtered solution further based on the manufacturer’s instructions or established protocols. This usually involves diluting with a mixture of deionized water and phosphate buffer (pH 6.8).
8. Measure the pH of the final solution and adjust if necessary using small amounts of the buffer.
9. Store the prepared stain in a light-protected, airtight container. Label it clearly with the date and concentration.

How does Leishman stain work?

Leishman stain works by utilizing two contrasting dyes to highlight different components of cells in a blood smear:

1. Eosin (acidic dye): Binds to basic structures like cytoplasm and ribosomes, staining them pink to red but has low affinity for nuclei and acidic structures.

2. Methylene blue (basic dye): Forms a complex with various Azure dyes depending on pH, staining nuclei and nucleoli a deep blue to purpl and also stains some cytoplasmic granules like those in neutrophils purple to violet.

The overall staining mechanism involves:**

1. Electrostatic interactions

• Oppositely charged dyes interact with charged groups on cellular components.
• Positively charged methylene blue binds to negatively charged DNA in nuclei and ribosomes in cytoplasm.
• Negatively charged Eosin binds to positively charged proteins and RNA in cytoplasm.

2. pH-dependent Azure formation

• Methylene blue undergoes partial demethylation in methanol to form various Azure dyes with different staining properties.
• The specific Azure formed depends on the pH of the stain solution, influencing the final staining profile.

3. Specific dye affinities

• Certain dyes within the stain mixture have specific affinities for specific cell structures based on their chemical composition.
• For example, Azure B preferentially stains nucleic acids, while Eosin Y stains basic proteins.

By combining these mechanisms, Leishman stain differentiates various cell types based on their:

• Nuclear morphology: Size, shape, chromatin pattern
• Cytoplasmic content: Ribosomes, granules, vacuoles
• Presence of parasites: Plasmodium (malaria), Babesia, etc.

Additional factors affecting staining:

• Fixation: Stabilizes cellular structures and affects dye accessibility.
• Staining time and temperature: Influence dye uptake and intensity.
• Differentiation: Controlled removal of excess stain using methanol.

What is Leishman stain used for?

Leishman stain is a versatile tool used in microscopy for a variety of applications, primarily in the fields of hematology and tropical medicine. It’s primary use is to visualize and identify different types of blood cells based on their nuclear morphology, cytoplasmic content and presence of granules. Malaria parasites like Plasmodium and other blood-borne pathogens like Babesia show characteristic features under Leishman stain, facilitating their identification and diagnosis.

What is the difference between Giemsa and Leishman staining?

Both Giemsa and Leishman stains are commonly used in microscopy for hematological and parasitological examinations, but they have some key differences:

Similarities

• Both are Romanowsky stains, meaning they utilize a combination of eosin and methylene blue dyes to differentiate various cellular components based on their pH affinity.
• Both work effectively on blood smears and tissue sections.
• Both provide good visualization of nuclei, cytoplasm, and cell morphology.

Differences

1. Staining composition

• Leishman stain: Contains eosin and a mixture of Azure dyes formed from methylene blue in methanol.
• Giemsa stain: Contains eosin and pre-formed Azure dyes (Azure A, B, and C) derived from methylene blue.

2. Staining intensity and color

• Leishman stain: Generally produces slightly less intense staining compared to Giemsa. Colors tend to be more blue-toned, with less emphasis on red hues.
• Giemsa stain: Delivers more intense and vibrant colors, with a richer red component in the cytoplasm.

3. Preferred applications

• Leishman stain: Often preferred for:
  • Rapid screening: Easier and faster to prepare due to the absence of pre-formed Azure dyes.
  • Malaria parasite detection: Provides good visualization of malaria parasites like Plasmodium.
• Giemsa stain: Often preferred for:
  • Detailed morphology assessment: Offers richer colors and sharper contrast for better morphological evaluation of cells.
  • Bone marrow examination: More effective in highlighting specific cell types in bone marrow smears.

Why alcohol is used in Leishman stain?

Alcohol plays a crucial role in Leishman stain for several reasons.

1. Dissolving the stain components

• The main dye component of Leishman stain, methylene blue, is insoluble in water but readily dissolves in alcohol. This allows for preparation of a concentrated stock solution.
• Eosin, the other major dye, while slightly soluble in water, also benefits from alcohol’s dissolving power to ensure complete dissolution and prevent precipitation.

2. Promoting dye penetration

• Alcohol acts as a fixative, helping to stabilize cellular structures and improve dye penetration. This ensures the stain can reach and interact with cellular components effectively.
• It also slightly dehydrates the cells, making them more receptive to dye uptake.

3. Controlling staining intensity and differentiation

• A brief dip in alcohol (methanol) is used as a controlled destaining step during the Leishman staining procedure. This removes excess stain, particularly from the cytoplasm, while preserving staining in nuclei and granules. This helps achieve optimal balance between color intensity and clarity for accurate interpretation.

4. Other benefits

• Alcohol helps to prevent bacterial growth in the stain solution, contributing to its shelf life.
• It aids in evaporating water after rinsing steps, facilitating faster slide drying.

It’s important to note that:

• The specific type of alcohol used in Leishman stain is typically methanol. However, in certain protocols, ethanol might be used as an alternative.
• Excessive exposure to alcohol during the staining process can lead to over-destaining, negatively impacting staining quality and information content.

What are the advantages and disadvantages of Leishman stain?

Advantages of Leishman Stain

• Simple and affordable: Easy to prepare and readily available compared to other staining techniques.
• Rapid and efficient: Offers quick results within minutes, ideal for rapid screening and diagnosis.
• Good differentiation: Effectively distinguishes between various white blood cell types based on nuclear morphology, cytoplasmic content, and presence of granules.
• Easy to interpret: Clear color differentiation facilitates easier analysis and identification of blood cells and parasites.
• Highlights parasites: Useful for detecting malaria parasites (Plasmodium) and other blood-borne pathogens.
• Relatively stable: Can be stored for long periods with proper care.

Disadvantages of Leishman Stain

• Less detailed morphology: Compared to Giemsa stain, Leishman stain provides less intense and vibrant colors, limiting detailed morphological evaluation.
• Susceptible to technical errors: Sensitive to factors like staining time, temperature, and methanol exposure, which can affect results.
• Not universal: Primarily used for blood and bone marrow smears, not as versatile as some other stains.
• Requires training and experience: Accurate interpretation relies on proper training and experience to differentiate cell types correctly.
• Safety concerns: Methanol is flammable and requires proper handling and disposal procedures.

Can Leishman stain be used for other cell types besides blood cells?

While Leishman stain is primarily used for blood and bone marrow smears, it can be applied to other cell types in certain situations.

• Tissue sections: Leishman stain can be used for basic visualization of nuclei and cytoplasm in tissue sections for initial screening or rough assessment. However, specific stains are often preferred for detailed analysis of different tissue types due to their tailored color profiles and highlighting capabilities.
• Cultured cells: Leishman stain might be used for quick assessment of cultured cells, but dedicated stains are usually chosen for detailed morphological evaluation or specific markers.
• Parasites: While effective for blood-borne parasites like Plasmodium, Leishman stain might not be ideal for all parasite types due to potential differences in staining affinity.

How do you dispose of Leishman stain waste?

Disposing of Leishman stain waste requires careful handling due to the presence of hazardous materials.

Main components to consider:

• Methanol: Flammable, toxic if ingested or inhaled.
• Heavy metals: Depending on the specific stain formulation, trace amounts of heavy metals like copper sulfate might be present.

Safe disposal practices:

1. Neutralize liquid waste: Before disposal, neutralize the methanol content by adding a buffer solution with a pH of 8-10. This reduces the flammability and toxicity of the waste.
2. Use appropriate containers: Collect neutralized waste in leak-proof, labeled containers specifically designed for hazardous waste disposal. Double-bagging is recommended for added security.
3. Follow institutional guidelines: Most institutions have established protocols and designated waste disposal procedures for hazardous materials. Familiarize yourself with your institution’s specific requirements and follow them strictly.

Additional precautions:

• Wear gloves and eye protection when handling waste materials.
• Work in a well-ventilated area and avoid open flames or sparks.
• Never dispose of Leishman stain waste down the drain or in regular trash.
• Never evaporate the stain solution, as this concentrates the hazardous components.
• Maintain proper documentation of waste disposal procedures for compliance and safety records.

Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.

References

1. Bain BJ. A Practical Guide. 6th Edition (Wiley). 2022.
2. Bain BJ, Bates I, Laffan MA. Dacie and Lewis Practical Haematology: Expert Consult: Online and Print 12th Edition (Elsevier). 2016.
3. Carr JH. Clinical Hematology Atlas 6th Edition (Elsevier). 2021.