(C1-9) Slide 7: human blood smear - Wright’s stain

With this slide, produced as shown in Fig. 1, you will be able to study the formed elements (as opposed to fluid elements) seen in a normal blood smear, routinely stained. You should be able to make all the identifications under 40X (do not use oil). The first step is to determine the best area for study, since many areas are unsuitable. You should find an area where many of the red blood cells are just touching each other, and appear to be rounded and stained pink (not orange). In this region, the RBCs and WBCs will have as nearly normal morphology as possible. This is most important when you get to the leukocytes, because their identification can be missed if you try to study the distorted cells found in other regions. Particularly avoid the edges of the smear where the leukocyte concentration is very high, but the cells are highly misshapen (C1).

Some of the RBCs stain quite homogeneously, while others show a pale center, due to the concavity of their shape. Other than that, they are unremarkable in the normal smear. You should look around until you find a clump of platelets. What is their function?

Granulocytes. By far, the most numerous leukocytes are the neutrophils (C2). They have quite small, dark, purple-staining granules in the cytoplasm, and the nucleus is segmented. There may be as few as two, and as many as five segments. Each segment is quite irregular in shape, and this is the most diagnostic feature.

In contrast, the eosinophils (C3, 4) (much less numerous) have a bilobed nucleus - each lobe being the size of the other and very smooth in outline - if the cell is not distorted. In addition, the cytoplasmic granules are very large and eosinophilic. C 4 shows eosinophils adhering to the surface of a type of parasite known as a schistosomulum (schistosome). Presumably, eosinophils create transmembrane pores in these parasites and inject hydrogen peroxidase, which destroys the schistosome.

The basophils (C5) are rare in any blood smear, and are particularly rare on our slides, so don’t be disappointed if you don’t find one. If you do find one it will contain large, deeply basophilic granules in the cytoplasm that obscure the nucleus.

Agranulocytes. These are the lymphocytes and monocytes. The term "agranulocyte" is not structurally accurate, for one can see tiny, scarlet-staining granules in the basophilic cytoplasm. Nonetheless, the granules are not nearly as prominent (or constant) a feature of agranulocytes as of granulocytes. These cells are also called "mononuclear" cells, as opposed to the "polymorphonuclear" character of the granulocytes.

Lymphocytes (C6-8) are the second most numerous leukocyte (after neutrophils). The most obvious feature is a rounded nucleus, which is surrounded by a rim of cytoplasm that is variable as to both amount and staining characteristics. Try to identify small, medium and large lymphocytes by comparing their size to RBCs (7.5 mM in diameter). Small lymphocytes have a thin rim of cytoplasm and are about 6-9 mM in diameter (C6). These small lymphocytes can be either T cells, B cells or null cells - it is not possible to distinguish between them in routine stains. Medium lymphocytes have a larger rim of cytoplasm and are about 10-13 mM in diameter (C7). Large lymphocytes have an even larger rim of cytoplasm that is usually paler staining, and are about 12-18 mM in diameter (C8).

Monocytes (C9) are not as numerous as lymphocytes. The cell body is large (12-15 mM in diameter) with rather gray, irregular-staining cytoplasm, in which is located a horseshoe-shaped, mottled nucleus. If you are lucky, you may see some very fine "azurophilic" granules in the cytoplasm of some of these cells.