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Lab 7. Hematopoiesis 2

Objectives

  1. Learn the organization of the bone marrow and where blood cells develop and enter the blood stream.
  2. Understand the process of differentiation from a stem cell to a mature blood cell.
  3. Distinguish the various stages in the development of erythrocytes.
  4. Distinguish the various stages in granulocyte development.

A. Organization of the Bone Marrow

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.

Blood cells develop in hematopoietic cords and enter the blood stream through the wall of the venous sinuses. Examine the organization of the bone marrow at higher magnification. Locate venous sinuses and hematopoietic cords. The lumen of the sinus is usually filled with red blood cells. At 40x magnification black, phagocytized material is seen within macrophages associated with the sinus wall. Hematopoietic cords lie between the sinuses and are filled with nucleated developing hematopoietic cells. Locate a megakaryocyte. It is a large cell with a lobulated polyploid nucleus (polyploid nuclei contain an exact multiple of 2n DNA typical of the species).

B. Bone Marrow Smear

Examine slide #26 which is a smear of human bone marrow. This slide contains developing blood cells of all types. Most (70%) of the nucleated cells in a bone marrow smear belong to the leukocyte series (granulocyte series 57%, lymphocyte series 10% and monocyte series 2% and plasmacyte series 1%). Approximately 19% of the nucleated cells are in the erythroid series and about 11% of the cells will be unidentifiable. The megakaryocyte series and reticular cells each compose less than 1%.

Examine the bone marrow smear under low magnification and locate areas where the cells are evenly distributed and do not overlap. Be aware that the slides differ in quality. If the reticulocytes (immature red blood cells) are pink to slightly pinkish grey, the cells are properly stained. If they are blue or grey, the description below will be inaccurate with respect to color, but other characteristics (size of cell, granules, etc.) should be satisfactory. If you are having trouble identifying cells ask your instructor to check the quality of the slide.

When identifying cells, remember that at each stage, the cells may vary in size (e.g., cells which have just divided will be smaller) and that the nuclear and cytoplasmic changes may not always be perfectly synchronized. In addition, some cells may be just entering or leaving a stage and they are referred to as "early" or "late" cells.

1. Erythroid Series

Red blood cells take approximately one week to develop from proerythroblasts to reticulocytes. The reticulocytes spend approximately 1–2 days in the bone marrow and enter the blood as reticulocytes where they circulate for 1 day before maturing into erythrocytes.

Examine the reticulocytes< slide #26, (Bone marrow) noting their color (pink to pinkish grey) and size (approximately 7 Ám). With the Wright stain used in this preparation, they look like red blood cells. Supravital stains (methylene blue or cresyl blue), which stain the polyribosomes in the reticulocytes, can be used to distinguish reticulocytes from mature red blood cells.

Now pick out orthochromatic erythroblasts. These are approximately the same size (7–10 Ám) 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 Ám) 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 Ám). 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 Ám), 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.

2. Granulocytic Series

Examine human bone marrow (slide #26) and locate several segmented neutrophils (9 to 15 Ám). Note the staining of the granules, the cytoplasm, and nucleus. Next, locate a band (staff or stab) neutrophil (9 to 15 Ám). 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 Ám). 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 Ám). 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 Ám) 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 Ám) 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.

3. Other Myeloid Cells

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 Ám) 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.