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Lab 6. Blood & Hematopoiesis 1

Objective

To recognize the cellular elements of blood.

Blood consists of cellular elements suspended in a complex solution called plasma. Blood is usually classified as one of the connective tissues. The cellular components of blood include red corpuscles (erythrocytes), platelets (thrombocytes), and five types of white corpuscles (leukocytes). Erythrocytes and thrombocytes are anucleated cells which perform most of their functions within the blood. Leukocytes (neurophils, eosinophils, basophils, monocytes and lymphocytes) are nucleated cells which migrate out of postcapillary venules into connective tissue or lymphatic tissue. They perform their functions both within and outside the blood stream. Blood smears are used to examine the size, shape and maturity of blood cells. They are also used to determine the relative percent of each type of white blood cell.

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Blood (Slide #24, normal blood smear)

Examine slide #24, (smear of normal blood). The smear is composed of erythrocytes, interspersed with leukocytes and platelets. The leukocytes are difficult to examine if the smear is too thick, and hard to find where the smear is too thin. Examine the blood smear using the low power (10x) objective. The ideal place to examine both erythrocytes and leukocytes is where there is only slight overlapping of the erythrocytes.

Scan this area on your slide and observe the idiosyncracies of your own preparation. If your background light is yellowish, use a blue filter. To quickly determine if your slide is well-stained, examine the red blood cells. Are the erythrocytes stained pink? If so, then other eosinophilic structures will be properly stained. If not, you will have to make allowances for cells which may look a little more basophilic than they should, or cells which do not show their granules as well as they should.

Erythrocytes

Switch to the 100x lens and examine the red blood corpuscles. They are biconcave discs approximately 7.2 Ám in diameter. The cells appear darker at the periphery and light in the center. The color of red blood cells is due to the eosinophilia of hemoglobin. Mature erythrocytes are anucleated and lack organelles.

Thrombocytes

Platelets (2–5 Ám) in diameter are fragments of cytoplasm surrounded by a plasma membrane. The cytoplasm stains blue and contains azurophilic granules.The platelets can occur singly or in clumps.

   

Examine an electron micrograph of platelets.

Leukocytes

Granulocytes (polymorphonuclear leukocytes)

Neutrophils can be recognized by their segmented nuclei and the presence of abundant, small, pale staining granules in their cytoplasm. Often the individual granules are barely distinguishable. Examine a number of neutrophils under oil immersion until you can quickly identify them. In good preparations, you may be able to see that there are two types of granules present, the more abundant, smaller specific granules which stain light pink and the larger, non-specific azurophilic granules which stain red-purple. Under normal conditions, neutrophils constitute 60–70% of the total leukocyte count. Study the E.M. of a neutrophil.

   

Now examine your slide for an eosinophil. If you are in doubt, it is usually a neutrophil and not an eosinophil. The specific granules of the eosinophil are large and distinctive. These may be located even under low power by their large bright red-staining, refractile granules. The nucleus of the eosinophil is also segmented, but it is usually bi-lobed and paler staining than the neutrophil nucleus. The granules may be seen very clearly in cells which have had their cell membranes ruptured during preparation. The granules will then be spread apart and are easily seen to be large and oval. Eosinophils constitute up to 3% of the leukocytes. Examine an E.M. of an eosinophil.

   

Basophils make up less than 0.5% of the leukocytes and are difficult to find. The granules are very large, purple staining and not of uniform size. The nucleus, which is often difficult to see clearly because of the granules, maybe segmented. Because they are relatively rare, they may not be on every slide. Study the E.M. of a basophil.

Nongranular leukocytes (mononuclear leukocytes)

Strictly speaking, there are probably no white cells totally devoid of granules. Azurophilic (non-specific) granules can also be found in lymphocytes and monocytes. However, these cells do not contain specific granules. The lymphocytes vary in size from 6 Ám (slightly smaller than an RBC) to large cells up to 15 Ám in size. The small lymphocytes have only a thin rim of sky-blue cytoplasm. Their nuclei of densely-stained chromatin are generally round or slightly indented on one side. Medium and larger lymphocytes have larger, round nuclei centrally located in a sky-blue cytoplasm. A few azurophilic granules may be present in the cytoplasm. Lymphocytes normally constitute 20–30% of the total leukocyte count, with small lymphocytes predominating. Study the E.M. of a lymphocyte.

   

The monocyte is usually the largest leukocyte present (15–20 Ám). The nucleus of the monocyte, which is usually bean or U-shaped and is eccentric, may have a "lumpy" appearance which is seen by focussing up and down. The chromatin appears as a fine lacy network. The cytoplasm is gray in color and opaque and usually contains fine granules. The monocyte can sometimes be confused with a medium or large lymphocyte or with an immature neutrophil. A medium lymphocyte usually contains denser chromatin and sky-blue cytoplasm. A young neutrophil (called a band), contains a U-shaped nucleus with condensed chromatin and a cytoplasm filled with small granules. Study the E.M. of a monocyte.