Lab 7. Hematopoiesis 2

Examine slide #25 which is a cross-section of a rib containing bone marrow. The marrow normally fills the entire cavity, however, on some slides the marrow is shrunken by fixation and has pulled away from the bone. At low magnification the marrow appears as a mass of nucleated cells with fat cells and venous sinuses. One or two arteries or veins may also be observed. Note the layer of endosteal cells lining the marrow cavity.

Now pick out orthochromatic erythroblasts. These are approximately the same size (7–10 µm) and color as reticulocytes but contain eccentrically located small dense pyknotic nuclei. With the electron microscope one sees a condensed nucleus, ribosomes, mitochondria and siderosomes.

After becoming quite familiar with these cells, pick out polychromatophilic erythroblasts. These contain larger nuclei and a bluish-grey cytoplasm. This cell varies in size (8 to 14 µm) and the nucleus contains coarse irregularly condensed chromatin masses. Clear patches or scalloped areas may appear just inside the nuclear membrane.

Next find a basophilic erythroblast (10 to 15 µm). In this stage of development the cytoplasm is dark blue. The nucleus with moderately clumped chromatin occupies approximately 50–70% of the cell. Nucleoli cannot be seen.

The earliest recognizable cell of the erythroid series is the proerythroblast. This is the largest cell of the series (14 to 18 µm), containing a deep royal blue cytoplasm with a large centrally located reddish (magenta) nucleus. The chromatin is reticulated and very delicate and contains 2 or more nucleoli, which may stain light blue. This cell may show a reversal of staining pattern in that the cytoplasm may appear darker than the nucleus. Re-examine the cells of the erythroid series, this time beginning with the early stages and note the changes in the relative size and staining characteristics of the cell, the nucleus, and the cytoplasm as development proceeds. Note changes in nuclear-cytoplasmic ratio. If these differences are not apparent to you, sketches may be helpful.

Examine human bone marrow (slide #26) and locate several segmented neutrophils (9 to 15 µm). Note the staining of the granules, the cytoplasm, and nucleus. Next, locate a band (staff or stab) neutrophil (9 to 15 µm). The nucleus, instead of being segmented, now forms a U-shape which appears to be rounded at each end. The color of the granules and the cytoplasm, however, should stain the same as in segmented neutrophils.

Next, find a neutrophilic metamyelocyte (10 to 15 µm). This cell contains a bean-shaped nucleus usually pushed to one side of the cell. The cytoplasm is similar to the mature neutrophil or may have a slightly bluish tint. Pick out several metamyelocytes and note their relative abundance.

Now look for the neutrophilic myelocytes (12 to 18 µm). This cell has a round or oval nucleus and light blue cytoplasm. Two types of granules can be seen, the typical small staining granules and a larger azure or scarlet granule.

The next stage is the promyelocyte, which is larger, (15 to 21 µm) than the myelocyte and contains a large nucleus with one or more nucleoli and a deep blue cytoplasm. There are many azure or scarlet granules, but no secondary granules. At this stage, it is impossible to determine with Wright stain whether the cell will develop into a neutrophil, an eosinophil or a basophil.

Find a myeloblast cell or blast cell. This cell has a large nucleus with 2 or more light-blue staining nucleoli and light blue cytoplasm with no granules. It may be the same size or smaller (15 to 20 µm) than a promyelocyte. You will not be required to distinguish between the various types of blast cells.

Re-examine the neutrophil series, but begin with the youngest cell (myeloblast). Also examine eosinophilic myelocytes and metamyelocytes, and compare them to developing neutrophils. Note differences in the secondary granules. Generally speaking, if a student has any doubt as to whether a given cell is an eosinophil or a neutrophil, it is usually a neutrophil.

Megakaryocytes These cells are easily found by examining the end of the smear under low power. Look for a very large cell (50 to 70 µm) with a lobulated (mature) or bean-shaped nucleus (immature). These cells have a blue cytoplasm with many fine azurophilic granules.

Lymphocytes Small lymphocytes appear identical to those found in blood smears. They contain a central round or slightly indented nucleus with uniformly stained chromatin. The cytoplasm is a clear light blue. Large lymphocytes may be mistaken for myeloblasts.

Study the EM of a lymphocyte

Plasma cells (mature) Ovoid cells with deep blue cytoplasm, a clear perinuclear zone, occasional vacuoles and an eccentric nucleus with clumped chromatin arranged like a cart-wheel. What causes the clear perinuclear zone?

Study the EM of a plasma cell.