The differential count is best made upon a film stained with Wright's, Jenner's, or Ehrlich's stain. Go carefully over the film with an oil-immersion lens, using a mechanical stage if available. Classify each leukocyte seen, and calculate what percentage each variety is of the whole number classified. For accuracy, 500 to 1000 leukocytes must be classified; for approximate results, 200 are sufficient. Track of the count may be kept by placing a mark for each leukocyte in its appropriate column, ruled upon paper. Some workers divide a slide-box into compartments with slides, one for each variety of leukocyte, and drop a coffee-bean into the appropriate compartment when a cell is classified. When a convenient number of coffee-beans is used (any multiple of 100), the percentage calculation is extremely easy.
The actual number of each variety in a cubic millimeter of blood is easily calculated from these percentages and the total leukocyte count. An increase in actual number is an absolute increase; an increase in percentage only, a relative increase. It is evident that an absolute increase of any variety may be accompanied by a relative decrease.
A record is generally kept of the number of nucleated red cells seen during a differential count of leukocytes.
The usual classification of leukocytes is based upon their size, their nuclei, and the staining properties of the granules which many of them contain. It is not altogether satisfactory, but is probably the best which our present knowledge permits.
(1) Normal Varieties.—(a) Lymphocytes.—These are small mononuclear cells without granules ([Plate VI] and [Fig. 86]). They are about the size of a red corpuscle or slightly larger, and consist of a single, sharply defined, deeply staining nucleus, surrounded by a narrow rim of protoplasm. The nucleus is generally round, but is sometimes indented at one side. Wright's stain gives the nucleus a deep purple color and the cytoplasm a pale robin's-egg blue in typical cells. Larger forms of lymphocytes are frequently found, especially in the blood of children, and are difficult to distinguish from the large mononuclear leukocytes.
Lymphocytes are formed in the lymphoid tissues, including that of the bone-marrow. They constitute, normally, 20 to 30 per cent. of all leukocytes, or about 1000 to 3000 per c.mm. of blood. They are more abundant in the blood of children.
The percentage of lymphocytes is usually moderately increased in those conditions which give leukopenia, especially typhoid fever, chlorosis, pernicious anemia, and many debilitated conditions. A marked increase, accompanied by an increase in the total leukocyte count, is seen in pertussis and lymphatic leukemia. In the latter, the lymphocytes sometimes exceed 98 per cent.
(b) Large Mononuclear Leukocytes ([Plate VI]).—These cells are two or three times the diameter of the normal red corpuscle. Each contains a single round or oval nucleus, often located eccentrically. The zone of protoplasm surrounding the nucleus is relatively wide. With Wright's stain the nucleus is less deeply colored than that of the lymphocyte, while the cytoplasm is very pale blue or colorless, and sometimes contains a few reddish granules. The size of the cell, the width of the zone of cytoplasm, and the depth of color of the nucleus are the points to be considered in distinguishing between large mononuclears and lymphocytes. When large forms of the lymphocyte are present, the distinction is often difficult or impossible. It is then advisable to count the two cells together as lymphocytes. Indeed, they are regarded by some hematologists as identical.
Large mononuclear leukocytes probably originate in the bone-marrow or spleen. They constitute 2 to 4 per cent. of the total number of leukocytes: 100 to 400 per c.mm. of blood. An increase is unusual except in malaria, where it is quite constantly observed, and where many of the cells contain ingulfed pigment.
(c) Transitional Leukocytes ([Plate VI]).—These are essentially large mononuclears with deeply indented or horseshoe-shaped nuclei. A few fine neutrophilic granules are sometimes present in their cytoplasm.