Differential Staining of Blood

To identify different stages of white blood cells in human blood.

Introduction
White blood corpuscles (WBCs) or leucocytes, are colorless, actively motile, nucleated living cells. They are variable in size and shape and exhibit characteristic amoeboid movement. Under certain physiological and pathological conditions, WBCs can come out of the blood vessels by a process called diapedesis. The lifespan of WBCs is 12–15 days. Unlike red blood corpuscles, leucocytes or WBCs have nuclei and do not contain hemoglobin. They are broadly classified into 2 groups:

• Granulocytes
• Agranulocytes

Granulocytes develop from red bone marrow and agranulocytes from lymphoid and myeloid tissues. Granulocytes are subdivided into 3 groups:

1. Eosinophils
2. Basophils
3. Neutrophils

Agranulocytes are subdivided into 2 groups:

1. Lymphocytes
2. Monocytes

The main features of WBCs are:

1. Phagocytosis
2. Antibody formation
3. Formation and secretion of lysozyme, heparin, etc.

Principle
WBCs are the type of blood cells that have a nucleus but no pigment. They are important in defending the body against diseases because they produce antibodies against any foreign particle or antigens. WBC can be divided into 2 types—granulocytes and agranulocytes. Granulocytes consists of neutrophils, eosinophils, and basophils.

Neutrophils. Do not stain with either acidic or basic dyes. They have manylobed nucleus and are called polymorphonuclear leucocytes or polymorphs. They constitute about 76.9% of the total leucocytes found.

Eosinophils. A lobed nucleus is present, with cytoplasmic granules that stain with acidic dyes. They constitute 1%–4% of the total leucocyte count. Basophils. It has a lobed nucleus and the cytoplasm contains granules, which stain with basic dyes. It comprises 0%–4% of the total leucocyte count. Agranulocytes. Consist of lymphocytes and monocytes. Lymphocytes. This is a type of WBC with a very large nucleus. It is rich in DNA and a small amount of clear cytoplasm is present.

Procedure

1. The fingertip is cleaned with cotton dipped in alcohol and pricked with a sterilized needle.
2. One or two drops of blood are placed on the right side of the slide, and with the help of another clean slide, the blood is drawn so that a thin smear is formed and dried.
3. The dried smear is fixed using zinc acetone, 3 methyl alcohol, or absolute alcohol for 5 min.
4. The smear is dried and stained with Giemsa or Leishman stain.
5. Subsequently, the distilled water (double the amount of water of the stain) is added onto the same smear slide.
6. The smear is mixed using a pipette for 10–15 min.
7. The slides are kept in running water to remove excess stain, and then dried.
8. The slide is observed under an oil immersion lens.