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To find out the percentage of different types of WBCs in a peripheral blood smear.

Requirements: Clean, grease free glass slides, Leishman’s stain, distilled water, staining rack, cedar wood oil, spirit, cotton, lancet, microscope.



A thin uniform peripheral blood smear is prepared and stianed using Leishman’s stain. Dry it, put a drop of oil on the film and focus under the oil immersion objective. Identify the different types of WBC5 which are differentillay stained with eosin and methylene blue. Draw a square in a paper and divide into 100 squares. Start counting of leucocytes from one end of the smear by moving the slide towards the other end. As and when each WBC is identified enter in the squares as ‘N’ for neutrophil. ‘E’ for eosnophil, ‘B’ for basophil, ‘L’ for Lymphocyte and ‘M’ for monocyte. When the other end of the slide is reached, move the slide two fields down and then towards the other end. Like this the smear is moved in a zigzag manner till 100 cells are counted and entered. Express the count as percentage.


Identification of Different Leucocytes:



Neutrophil 12 - 14 Micron size.

Multilobed nucleus, usually 2-5 lobes connected by chromatin threads purplish blue in colour. Cytoplasm plenty, bluish pink with plenty of fine pink granules. Cell outline is usually distinct.


Eosnophil 10- 15 Micron size

Nucleus usually bibbed and spectacle shaped or horse shoe shaped, purplish bule in colour, cytoplasm plenty, pink in colour, orange red granules (Strongly acidophilic) not plenty, Cell outline not distinct.


Basophil 8- 10 Micron size

Nucleus lobulation incomplete, Purplish blue in colour, Cytoplasm plenty, bluish black coarse granules, may overlie the nucleus so that the nucleus may not be clearly seen.


Agranu locytes:


Lymphocyte: Pale blue non granular cytoplasm and deep purplish blue on lobulated nucleus. Cell outline is distinct.


Small Lymphocyte: 7-10 Micron size

Nucleus round or slightly indented, fills almost entire cell, cytoplasm scanty, may be seen as a distinct perinuclear ring.


Large Lymphocyte: 12-15 Micron size.

Nucleus usually indented, fills almost entire cell, Cytoplasm to nucleus ratio more than small lymphocyte.


Monocyte: 14-18 Micron size, Largest WBC

Nucleus large round or oval deeply indented to have kidney shape, pale purplish blue in colour, usually eccentric, cytoplasm greyish blue, (slaty grey) plenty.

Normal differential leucocyte count:

Neutrophils : 35- 65 %

Lymphocytes : 25- 45 %

Eosinophils : 1 - 4 %

Basophils : 0 %

Monocytes : 0 %Neutrophilia: Increase in netrophil percentage above normal.

Acute bacterial infections like tonsillitis, appendicitis, pneumonitis, lung abscesses etc. burns, acute haemorrhage,

Physiological conditions like muscular exercise, after meals

Neutropenia: Decrease in neutrophil percentage below normal. Typhoid, Paratyphoid fever, malaria, aplastic anaemia, irradiation


Eosnophilia: Increase in eosnophil percentage above normal

1. Allergic conditions like bronchial asthma, urticaria

2. Parasitic infestations like round worm.

3. Tropical eosniphilia

Eosnopenia: Decrease eosnophil percentage below normal ACTH or steroid treatment, acute infections.

Basophilia: Increase in basophil count above normal. In chronic myeloid leukemia, small pox, polycythemia.

Lymphocytosis: Increase in lymphocyes above normal.

1. Chronic infections like tuberculosis

2. Acute infections like whooping cough

3. Lymphatic leukemia

4. Physiological in infancy and childhood.

Lymphopenia: Decrease in lymphocytes above normal. ACTH and steriod therapy

Monocytosis: Increase in monocyte above normal

Malaria, infectious mononucleosis, Monocytic Leukemia


Uncontrolled abnormal cancerous proliferation of WBC series resulting in appearance of large number of immature forms of WBC in peripheral circulation. Total WBC count is enormously increased. Depending on the type of WBC series affeced, it can be myeloid, lymphatic or monocytic leukemia.


1. What isArneth’s index?

2. What is Schilling index?

3. What are the advantages of a Differential Leukocyte Count study?