Blood smear: preparation technique and overview photomicrograph. a. Photograph showing the method of producing a biood smear. A drop of blood is placed directly on a glass slide and spread over its surface with the edge of another slide, b. Photomicrograph of smear from peripheral blood, stained with Wright's stain, showing the cells evenly distributed. The cells are mainly erythrocytes. Three leukocytes are present. x350.

ration may then be examined with a high-power oil-immersion lens, with or without a coverslip (Fig. 9.1b).

The modified Romanovsky-type stain commonly used for blood smears consists of a mixture of methylene blue (a basic dye), related azures (also basic dyes), and eosin (an acidic dye). On the basis of their appearance after staining, leukocytes are traditionally divided into granulocytes and agranulocytes. Although both cell types may contain granules, the granulocytes possess obvious, specifically stained granules in their cytoplasm. In general, the basic dyes stain nuclei, granules of basophils, and the RNA of the cytoplasm, whereas the acidic dye stains the erythrocytes and the granules of eosinophils. It was originally thought that the fine neutrophil granules were stained by a "neutral dye" that formed when methylene blue and its related azures were combined with eosin. However, the mechanism by which the specific neutrophil granules are stained is not clear. Some of the basic dyes (the azures) are metachromatic and may impart a violet to red color to the material they stain.

s? erythrocytes

Erythrocytes are anucleate, biconcave disks

Erythrocytes, or red blood cells (RBCs), are anucleate cells devoid of typical organelles. They function only within the bloodstream to bind oxygen for delivery to the tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave disk with a diameter of 7.8 yu,m, an edge thickness of 2.6 jam, and a central thickness of 0.8 /jlm. This shape maximizes the cell's surface area (-140 /xm2), an important attribute in gas exchange.

The life span of the erythrocyte is approximately 120 days, after which most (-90%) are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (-10%) break down intravas-cularly, releasing hemoglobin into the blood.

In H&E-stained sections, erythrocytes are usually 7 to 8 /xm in diameter. Because their size is relatively consistent in fixed tissue, they can be used to estimate the size of other cells and structures in tissue sections; in this role, erythrocytes are appropriately referred to as the "histologic ruler."

Because both living and preserved erythrocytes usually appear as biconcave disks, they can give the impression that.their form is rigid and inelastic (Fig. 9.2). They are, in fact, extremely deformable. They pass easily through the smallest blood vessels by folding upon themselves and can thus pass through even the narrowest capillaries. They stain uniformly with eosin. In thin sections viewed with the transmission electron microscope (TEM), the contents of an erythrocyte appear as a dense, finely granular material (Fig. 9.3).

The shape of the erythrocyte is maintained by membrane proteins

The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins:

• Integral membrane proteins represent most of the proteins in the lipid bilayer. They consist of two major families: glycophorins and band 3 protein. The extracellular domains of these integral membrane proteins are glycosylated and express specific blood group antigens. Gly-cophorin C, a member of the glycophorin family, plays an important role in attaching the underlying cytoskele-tal protein network to the cell membrane. Band 3 protein binds hemoglobin and acts as an additional anchoring site for the cytoskeletal proteins (Fig. 9.4).

• Peripheral membrane proteins reside on the inner surface of the cell membrane. They are organized into a two-dimensional hexagonal lattice network that laminates the inner layer of the membrane. The lattice itself, which is positioned parallel to the membrane, is composed mainly of spectrin tetramers, actin, band 4.1, ad-ducin, band 4.9, and tropomyosin (see Fig. 9.4). The lattice is anchored to the lipid bilayer by ankyrin, which interacts with band 4.2 protein as well as with band 3 integral membrane protein.

This unique cytoskeletal arrangement contributes to the shape of the erythrocyte and imparts elastic properties and stability to the membrane. The cytoskeleton is not static; it undergoes continuous rearrangement in response to various physical factors and chemical stimuli as the cell moves through the vascular network. Any defect in the expression of genes that encode these cytoskeleton proteins can result in abnormally shaped and fragile erythrocytes. For instance, hereditary spherocytosis is caused by a primary defect in spectrin gene expression that results in spherical erythrocytes. Hereditary elliptocytosis is caused by a deficiency in band 4.1 protein that results in elliptic erythrocytes. In both conditions, erythrocytes are unable to adapt to changes in their environment (e.g., osmotic pressure and mechanical deformations), which results in destruction of the cells, or hemolysis.

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