WBC ANDF THEIR MORPHOLOGY THE VARIATIONSABNORMALITIES IN LEUCOCYTES

WHITE CELL COUNTS

white cell counts:-. Evaluation of the quality of the blood film A good quality blood smear should show evenly distributed red and white blood cells. Although the smear will appear thick at the origin, the latter part of the smear should show the cells spread evenly without clumping or overlapping each other. The white cells should not accumulate along the sides or at the tail end. The low power scanning is helpful in selecting the counting area where the cells are clearly separated and well distributed.

2. Rough estimation of cell counts Under the low power (10 x) objective, the RBCs appear as small, round, reddish orange bodies and are the major cell type. Scattered among the RBCs are the white cells. The white cells are nucleated and their nuclei stain in shades of purple and the cytoplasms stain in different colours,

depending on the type of cell. In an area where the cells are evenly spread in a single layer, 5 white blood cells in a low power field are roughly equal to 1000 red cells/cu.mm(1 x 10 cells/L). At least five low power fields should be counted to average the estimate of white cell count

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B. Examination of the Blood Film Under Oil Immersion (100 x )Lens

It should include:

1.The differential count of leucocytes.

2. Morphological alterations in red blood cells.

3. Evaluation of platelet count and morphology.

4. Detection of blood parasites.

5. Detection of abnormalities in leucocytes.

These are described in detail in the following sections.

THE DIFFERENTIAL COUNT OF LEUCOCYTES:-The differential count means identification and counting of various types of white blood cells and expressing the number of each type per 100 white cells. For this purpose at least 100, preferably 200, white cells should be counted continuously and the number of each category of white cells should be noted.

There are five types of white cells observed in a normal peripheral blood smear (Fig. 4.3). For identification, they can be grouped into two broad categories; 

1. Granulocytes:-which include neutrophils,eosinophils and basophils. Depending on the staining reaction of the granules. 

2. Agranulocytes: -are lymphocytes and monocytes. Monocytes do contain granules in their cytoplasm, but they are very fine and may not be obvious

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1. Granulocytes-

Neutrophils(Polymorphonuclear neutrophil or PMN):- This is the most numerous white cell type in the normal blood smear. The cell measures 10-14 microns and contains a lobular nucleus, which is an elongated nucleus constricted at one to four places, forming lobes connected by thin strands of chromatin. The nuclear chromatin stains deep reddish purple. Nucleoli are absent. The cytoplasm is abundant, light pink in colour, and contains a large number of fine neutrophilic granules which are also light pink in colour. A few darker azurophilic granules may be present. The normal range of neutrophils is 40-72 % (1800--7800/ul)

A normal blood smear may contain a few (up to 3 %) band cells which are neutrophils in a slightly premature stage in which the band shaped long nucleus is not yet divided into lobes.

Function of neutrophils:- Neutrophils capture and destroy invading organisms and other foreign toxic materials as soon as they enter the body. They show amoeboid motility. They migrate into the tissues via the bloodstream, and are attracted to the organisms by a process called chemotaxis. The organisms and other materials are then removed from the blood or tissue by phagocytosis.

The organisms and other foreign bodies are phogocytosed (engulfed) and removed from the blood and tissue. This action is shown as phogoytosis.

Eosinophils:-  cells contain granules which have affinity for the acidic dye, eosin, in the Romanowsky stains. The eosinophils are slightly larger than the neutrophils. They usually contain a bilobed nucleus in a typical "spectacle arrangement". Nucleoli are absent. The cytoplasm is not clearly visible as it is filled with large, distinct acidophilic granules, stained orange red with eosin. The percentage of eosinophils in normal blood is 1-6% (50-450/ul).

Functions of eosinophils:- Eosinophils have a phagocytic function which is aimed mainly at antigen-antibody complexes. They also counteract the effect of histamine and help in wound repair.

Basophils:- The polymorphonuclear basophil is slightly smaller than the neutrophil, the average diameter being 10 microns. The nucleus may have two or more lobes and stains deep purple blue. Sometimes the segmentation of the nucleus is incomplete. The cytoplasm is slightly basophilic and contains large granules which stain purple or black due to their strongly basophilic nature. The 

nucleus may be obscured by the granules. The normal range is 0-0.5 % (0-200/ul).

Functions of basophils:- The basophilic granules contain heparin and histamine. Basophils gather around inflammatory lesions and release histamine. They are less phagocytic in function than the other granulocytes.

2. Agranulocytes-

Monocytes- Monocytes are the largest of the normal leucocytes in peripheral blood, measuring 16-20 microns in diameter. The nucleus is fairly large, oval or lobular, but most frequently, kidney shaped. The nucleus stains unevenly giving it a stringy appearance The cytoplasm is abundant, and stains greyish-blue due to its mildly basophilic nature. A few extremely fine azurophilic granules called 'azure dust may be seen in the cytoplasm. Vacuoles may also be seen. Normal blood contains 2 -8% monocytes (100-800/ul).

Functions of monocytes:- The monocytes are phagocytic in function and are capable of ingesting a large number of bacteria.

Lymphocytes:- Lymphocytes in the peripheral blood appear in two forms, small lymphocytes (7-10 microns) and large lymphocytes (12-15 microns).

The small lymphocyte, being the more mature form, is more frequently seen in adult blood. The nucleus is usually round with condensed chromatin and stains deep purple. Nucleoli are generally not seen. The cytoplasm appears as a dark blue band around the nucleus and azure dust is usually absent.

The large lymphocyte has a slightly larger nucleus and more abundant cytoplasm. The nucleus stains lightly as compared to that of the small lymphocyte. The cytoplasm is pale blue in colour. The lymphocyte count of normal blood may range between 20-45 % (1000-4500/ul).

Functions of lymphocytes:- There are two subpopulations of lymphocytes, T-cells and B-cells, which are morphologically indistinguishable in the peripheral blood smear. Other special tests using different markers are necessary for their identification.

Both T and B lymphocytes are immunocompetant cells which act to direct and effect the immune defense system of the body. The cells migrate to various sites in the body to await antigenic stimulus. When activated, they respond to antigenic challenges. B-lymphocytes produce antibodies whereas 

 T-lymphocytes are responsible for the cell mediated immunity.

Reporting the Differential Leucocyte Count-After counting 100 white cells, the number of cells belonging to each of the above five categories should be reported as the percentage of that type of cell.

Differential count	Normal range
Neutrophils	40-72%	(1800-7800/l)
Eosinophis	1-6%	(50-450/µl)
Basophiles	0-0.5%	(0-200µl)
Lymphocytes	20-45%	(1000-4500/µl)
Monocytes	1-8%	(100-800/µl)

WBC ANDF THEIR  MORPHOLOGY THE VARIATIONSABNORMALITIES IN LEUCOCYTES