Blood Staining Examination of Stained Blood Smears

‫بسم اهلل الرحمن الرحيم‬
University of Khartoum - Faculty of Veterinary Medicine
Department of Clinical Medicine
Laboratory Diagnosis Practical (4)
Blood staining & Examination of stained blood smears
The method selected to stain a blood film depends on the purpose for which
is it made.
Some of Blood stains:

 1. Romanowsky stains: is any stain combination consisting of eosin Y
or eosin B with methylene blue and/or any of its oxidation products,
used in haematological and cytological studies. Examples: Giemsa,
Leishman, Wright, Field, Jenner and May-Grunwald stain.
2. Methylene blue
3. New methylene blue
* Leishman stain:
Developed by William Leishman using polychrome methylene blue
and eosin Y with methanol solvent. The stain used to differentiate and
identify the white blood cells and blood parasites
Procedure:
1. Preparation the thin blood film (without fixation by absolute
methanol)
2. Put the slide on the staining rack and fill the slide by Leishman
stain and leave it for 2 minutes.
1
3. Dilute the stain on the slide by adding distilled water in a double
amount of the stain and mix.
4. Leave the diluted stain on the slide for 15-20 minutes (minimum:
15 min).
5. Rinse the slide out of the stain by water and leave it to dry.
6. Examine the slide under oil immersion of microscope.
-Used for differential leukocyte count.
*Giemsa stain:
This is a special stain used for examination of blood films for parasitic
infections and also used as a differential stain for various blood cells
and cellular components.
Procedure:
1. Prepare thin blood smear
1. Fix the blood film by absolute methanol (100%) for 2 - 5 minutes.
2. Fill the slide by the diluted Giemsa stain (1:10) and leave it for 30
minutes.
3. Rinse the slide and leave it to dry.
4. Examine the slide under oil immersion of microscope.
Other types of Romanowsky staining:
- May – Grunwald – Giemsa stain
- Wright – Giemsa stain
Diagnostic Value of Stained Blood films:
1. Differential of count of leukocytes
2. Detection of blood parasites
3. Characteristics of red blood cells (morphology and quality)
4. Anthrax diagnosis
5. Pasturella spp diagnosis
2
Examination of stained (Giemsa or Leisham) blood smears
1. Differential leukocytes count
Thin blood smear is prepared and fixed by absolute methanol and

stained with Giemsa stain diluted to 1:10 in distilled water for 30
minutes, wash, dry and examine under oil immersion, count at least 100
to 200 cells by battlement/zigzag method.
Interpretations:
Types of leukocytes:
1- Granulocytes: basophils – eosinophils- neutrophils

2- A granulocytes: lymphocyte – monocyte
*Lymphocytosis: viral infections, Tuberculosis, Brucellosis and
following vaccination
*Lymphopenia: canine distemper, corticosteroids therapy and foot
and mouth disease.
*Neutrophilia: septicemic diseases, pyogenic infection and acute
bacterial infection
*Neutrophilia (shift to left): increase in number of neutrophils with
immature cells in cases of leptospirosis and traumatic
reticulopricarditis
*Neutropenia: pasteurellosis and Bovine Viral Diarrhoea
*Eosinophilia: allergy and parasitic infections
*Basophilia: Pox and splenectomy
3
*Monocytosis: chronic bacterial infection (Tuberculosis and
brucellosis)
Table: Normal Values of some Hematological Parameters in some

domestic animals.
Species Hb PCV Lymphocytes Neutrophils Eosinophils Monocytes Basophils

(g/dl) (%) (%) (%) (%) (%) (%)
Cattle 8-14 26-42 48-75 15-45 2-15 2-7 0-2
Sheep 8-16 24-49 40-75 10-50 1-8 1-5 0-3
Goat 8-14 20-38 50-70 30-48 3-8 1-4 0-2
Horse 11-18 32-52 15-50 35-75 1-10 2-10 0-3
Dog 12-18 37-54 12-30 60-75 2-10 3-9 0-1
Cat 8-15 24-45 20-55 35-75 2-10 1-4 0-1
2. Detection of blood parasites
Can be intracellular (Theileria, Babesia and Anaplsma) or

extracellular (Trypanosoma)
3. Characteristics of red blood cells (morphology and quality)
Examine the RBCs to detect:
4
*Changes in red blood cells size:
Normocytes: cell with normal size.
Macrocytes: cells larger than normal size in cases of macrocytic anemia
Microcytes: cells smaller than normal size in cases microcytic anemia
Anisocytosis: different sizes of red blood cells in one animal species.
Poikilocytosis: different shapes of red blood cells in one animal species.
*Color changes:
1. Polychromasia is recognized as larger, slightly bluer-staining red cells and

is an excellent indicator of a regenerative response.
2. Hypochromasia: indicates iron deficiency and is recognized by increased

central pallor and pale color.
*Morphology changes:
1. Acanthocytes: These are cells with unevenly distributed cytoplasmic

projections, and are most commonly seen in feline hepatic lipidosis.
2. Schistocytes: These are small, irregular fragments of cells and are most
commonly seen in hemangiosarcoma.
3. Spherocytes: These are red cells that are round instead of flat, because
they are rounded, they appear smaller and darker than normal red cells and
there is a lack of central pallor
3. Heinz bodies :They appear as small, roundish structures can protrude from
the margin of the cell as a pale structure, or appear as a small pale dot near the
edge of the cell, these indicate oxidative damage to hemoglobin.
5
4. Blister cells and keratocytes: Blister cells are red cells with a round, clear
blister at the edge of the cell. When the blister ruptures, one or two thin
remnants of cytoplasm are seen and it’s then called a keratocyte. These shape
changes indicate oxidative damage and are most common in iron deficiency.
5. Echinocytes: These are cells that are covered with small, blunted to sharp,
evenly distributed spicules; they look sort of like a sea urchin.
Giemsa Stain preparation:

Compositions of stock solution:
1. Giemsa stain powder 3.8 g
2. Methanol 250 ml
3. Glycerol 250 ml
Procedure:
1. Weigh the stain powder and put it in a glass beaker 500 ml.
2. Add the methanol to the powder.
3. Add the glycerol to the powder.
4. Put it in a water bath 50-60°c.
6. Leave it for 2 months.
Preparation of diluted Giemsa working solution:
To prepare 10 ml of 10% Giemsa add 1 ml Giemsa stock solution to 9
ml distilled water.
Leishman stain preparation:

Compositions:
1. Leishman stain powder 6g
2. Methanol 400ml
6
Procedure:
-Weigh the stain powder and add the methanol to it.
-Put it in a water bath 37°c until the stain dissolved.
-Store the stain in dark place and then filtered in 50-100 ml.
By:
Hazem Abdo
Nasreen Gadallah
Samah Aljenay

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‫بسم اهلل الرحمن الرحيم‬

University of Khartoum - Faculty of Veterinary Medicine

Department of Clinical Medicine

Laboratory Diagnosis Practical (4)

Blood staining & Examination of stained blood smears

Some of Blood stains:

Thin blood smear is prepared and fixed by absolute methanol and

1- Granulocytes: basophils – eosinophils- neutrophils

Table: Normal Values of some Hematological Parameters in some

Species Hb PCV Lymphocytes Neutrophils Eosinophils Monocytes Basophils

2. Detection of blood parasites

Can be intracellular (Theileria, Babesia and Anaplsma) or

3. Characteristics of red blood cells (morphology and quality)

Examine the RBCs to detect:

Normocytes: cell with normal size.

Macrocytes: cells larger than normal size in cases of macrocytic anemia

Microcytes: cells smaller than normal size in cases microcytic anemia

Anisocytosis: different sizes of red blood cells in one animal species.

Poikilocytosis: different shapes of red blood cells in one animal species.

1. Polychromasia is recognized as larger, slightly bluer-staining red cells and

2. Hypochromasia: indicates iron deficiency and is recognized by increased

1. Acanthocytes: These are cells with unevenly distributed cytoplasmic

Giemsa Stain preparation:

Leishman stain preparation:

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