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: KH PO 9.1 g/L
2 4
 Solution B: Na HPO 9.5 g/L

2 4
 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
A normal peripheral blood smear using Leishman stain x400

magnification. Red cell central pallor approximately 1/3 in
size of the red cells. The cells are almost all the same shape
and size. The red cells are slightly smaller than the small
lymphocyte in size. Platelets can also be seen scattered
throughout the smear.
Cell Type Nucleus Cytoplasm Granules
Red Blood Cells Pink to red, depending on age

No nucleus None
(RBCs) and hemoglobin content
Fine, pink to
Neutrophils Deep blue, bilobed Pale blue to lilac-pink
purple
Deep blue, round Large, dark
Basophils Deep blue to purple
or oval purple to black
Large, orange-
Eosinophils Deep blue, bilobed Pale pink to orange
red
Deep blue, round

Monocytes Pale blue to gray Fine, faint pink
or indented
Deep blue, round

Lymphocytes Pale blue to clear None
or indented
Platelets No nucleus Pale purple to pink None
Leishman stain stains nucleus and cytoplasm differently due to the basic and
acidic properties in the stain. Each blood cell can be distinguished
individually due to the different properties of each cell.
Different blood cell morphology stained by Leishman stain that can be seen
under the microscope. Panel A is a blood smear of iron deficiency anemia.
Hypochromic red cell (central pallor more than 1/3) (red circle), pencil-
shaped red cell (blue circle) and anisopoikilocytosis (red cells of different
shape and sizes) (green circle) can be seen. Panel B is a blood smear of beta-
thalassemia major where there are numerous hypochromic red cells, target cell
(red circle) and nucleated red blood cell (blue circle) can be seen. Panel C is a
blood smear of thrombocytopenia where there is reduced presence or
absence of platelets in the smear. Panel D is a blood smear of thrombocytosis
with numerous platelets (green circle). Panel E is a blood smear of acute
lymphoblastic leukemia where lymphocytosis (red circle) can be seen. Panel F is
a blood smear of chronic myeloid leukemia where left-shifted granulopoiesis
encompassing the full spectrum of granulocytic precursors including
promyelocytes and blasts (red circle)can be seen.
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Introduction

The Leishman stain, characterized by its contrasting hues of blue, pink,

Beyond mere identification, the Leishman stain unveils vital information

However, the power of the Leishman stain extends beyond routine

Principle of Leishman Stain

Flood Slide Method

 Solution B: Na HPO 9.5 g/L

A normal peripheral blood smear using Leishman stain x400

Red Blood Cells Pink to red, depending on age

Deep blue, round

Deep blue, round

Platelets No nucleus Pale purple to pink None

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