leucocytes

The leucocytes are also called white blood corpuscles (WBC) or simply the white cells of the blood but these are not white, these are colourless. The white cells defend the body against diseases by fighting infections (bacterial, viral, parasitic, etc.), antigens and also against malignancy.

 

 

Types

The leucocytes are usually divided into granulocytes (with granules in their cytoplasm) and agranulocytes (without granules). The granulocytes are further subdivided into three types: neutrophils, eosinophils and basophils. The agranulocytes are the lymphocytes and the monocytes. All1 these cells can be studied under microscope after staining.

Staining

It means colouring of a tissue or cells with a suitable dye so that it can be studied properly under microscope. As the leucocytes are transparent, these cannot be seen properly under microscope unless stained. Ordinary stains like methylene blue colours only the acidic materials and the stains like eosin colours the basic materials of a cell. None of these can stain the various types of granules of the leucocytes properly. This can be achieved by Leishman's stain which is a modification of the Romanowsky stain like Wright's stain, Jenner's stain, Giemsa stain, etc. Romanowsky stain is the solution of a compound formed by interaction between watery solutions of methylene blue and eosin on 1ong standing. This compound is nothing but the maturation products of methylene blue (Azure B) along with eosin. This is a polychrome dye which stains both acid and basic materials simultaneously, hence it alone can stain the granules of the leucocytes (Eosin stains the basic and azure B stains the acidic materials).

 

Leishman's stain

Leishman's stain, which is usually used to stain blood films, is prepared by treating methylene blue with Na₂CO₃ to produce oxidation products (methylene azure) which are then mixed with eosin. The resultant precipitate is then washed, dried and the Leishman powder is obtained. This Leishman powder is dissolved in acetone free methyl alcohol to produce Leishman's stain. If acetone is present it will disturb the staining particularly that of the nucleus. Supravital staining is used to study a living cell (only living cells accept these dyes like methylene blue, brilliant cresyl green, etc.)

Procedure: 

Leishman's stain is poured in sufficient amount cover a dry blood film on a glass slide and left for one to two minutes. During this period fixation of the film occurs by the methyl alcohol present in the stain. Then distilled water sufficient to cover the glass slide (double the amount of the stain) is added and left for another ten minutes. Now staining takes place by the watery solution of the dye. The film is then washed with distilled water, dried and examined under microscope.

 

Morphology of the leucocytes 

In a blood film, stained with Leishman stain, the leucocytes when examined under light microscope are seen as follows.

 

Neutrophils

These are the most numerous white blood cells with diameter 10 to 15μ (RBC 7.2 μ) and constitute 60 to 70% of the total leucocyte count. Nucleus shows variable number of lobes (2 to 7) and hence called polymorphonuclear leucocytes. The pink eytoplasm shows fine violet (red-brown) granules, which are amphophilic, i.e., take both acidic and basic stain and the granules are not neutral. The granules the neutrophils are of two types - primary and secondary. Only the secondary granules are seen in Leishman's stain. Nucleus stains purple blue and the chromatin is coarse and ropy.

 

Eosinophils

These cells, seen not infrequently, are of diameter 10 to 15μ. The nucleus is usually bilobed. Cytoplasm packed with coarse brick red granules which take acidic stain, hence named eosinophil. Nucleus stains purple blue chromatin is coarse and ropy.

 

Basophils

Occasionally seen in blood slides froma healthy person.  The cells are of 10 to 15 μ in diameter and contain bilobed or 'S' shaped nucleus which is obscured by coarse basophilic (blue) granules. Nucleus stains purple blue chromatin is coarse and ropy On an average all the granulocytes are of equal size.

 

Small lymphocytes

These cells are found in plenty and are of about 7.5 μ Diameter. Nucleus is like that of large lymphocyte and cytoplasm is scanty, so the cell is almost occupied by nucleus. Nucleus stains deep blue.

 

Large lymphocytes

These cells are also seen frequently in the film and have diameter of about 10 to 18 μ. They contain a round nucleus With coarse and lumpy chromatin. There is plenty of clear cytoplasm without any granule. Nucleus stains deep blue.

 

Monocyte

The largest of all leucocytes (10 to 20 μ ) with a large kidney shaped nucleus which takes light stain. The cytoplasm is pale blue and contains fine purple granules.