OBJECTIVES: At the end of this laboratory, you should be able to:

Identify the cells in major stages of the erythroid, myeloid, and platelet series.
Understand the progression of the developmental stages in all series.

Bone marrow smear
Begin by scanning the smear and notice that some areas are stained and spread better than others. At this low magnification you will see that there are many mature red cells and myeloid cells in various stages of differentiation. Pick an area that appears well stained and spread, and study the cells by observing the morphological characteristics such as nuclear size and shape, presence of nucleoli, staining density of chromatin, evidence of Golgi apparatus, cytoplasmic basophilia, presence of granules, and cell size. Recall that hematopoiesis can be divided into two lines of development: erythropoiesis (RBC differentiation) and granulocytopoiesis (WBC differentiation). Each cell in these developmental processes will be examined on this specimen. Do not try to identify every cell on the specimen. Many cells will be in transition from one stage to another.

Recall that the overall trend is: 1) from larger to smaller size; round, fine nucleus to dark, segmented nucleus; 2) increasing cytoplasm; 3) no granules to primary (azurophilic) granules to specific (secondary) granules. Please note you are not responsible for identification of the basophilic line of cell maturation. Identify the following cells:

1. Promyelocyte (10-20 mm): Note the round nucleus, reddish-blue and fine to slightly condensed chromatin, 1-2 nucleoli, increased basophilic cytoplasm, and primary (azurophilic) granules which appear as a deep magenta in this specimen.

2. Myelocyte (10-18 mm): Note the oval (to round) slightly indented nucleus; reddish-blue and slightly granular chromatin; nucleoli may or may not be present; moderate bluish pink cytoplasm; primary and specific granules (fine and pale). This is the first appearance of specific granules (neutrophilic, eosinophilic and basophilic). The eosinophilic myelocyte is the most easily determined with its coarse eosinophilic granules. In the neutrophilic myelocyte, neutrophilic granules are beyond the resolution of the light microscope, but can be identified based on size and comparison of the cytoplasm to those of neutrophilic metamyelocytes which are more easily identified.

3. Metamyelocyte (neutrophilic and eosinophilic) (10-18 mm): Note the indented nucleus (kidney bean); light blue-purple and granular chromatin; no nucleoli are present; moderate clear pink cytoplasm; specific granules are obvious. Basophilic metamyelocytes are very rare and identification is difficult because the granules obscure the nucleus.

4. Band Neutrophil (10-16 mm): Note the elongated, horseshoe nucleus; blue-purple and clumped granular chromatin; no nucleoli are present; abundant pink cytoplasm; specific granules. This cell stage is not distinguished for the eosinophil and basophil maturation.

5. Granulocytes Mature granulocytes are plentiful; distinguished by the lobulated nucleus, pink abundant cytoplasm, and specific granules. Identify mature neutrophils and eosinophils, (basophils are seen rarely).

The overall trend in RBC maturation is: 1) large, pale nucleus to darker, smaller nucleus to loss of nucleus; 2) increase in cytoplasm; 3) gradual decrease in size; 4) cytoplasm from intensely blue (full of RNA) to grayish (mixture of RNA and hemoglobin) to reddish (full of hemoglobin, no RNA). Identify the following cells:

1. Proerythroblast (14-19 mm): Nucleus is large with fine chromatin and nucleoli; cytoplasm is scant and basophilic.

2. Basophilic erythroblast (12-17 mm): Slightly smaller nucleus with slight chromatin condensation; increased cytoplasm and intensely blue (RNA abundance); no granules and no nucleoli present.

3. Polychromatophilic erythroblast (12-15 mm): Moderately condensed chromatin; lighter, grayish cytoplasm. The color of the cytoplasm is due to coloring by both acidic and basic components of the stain. Basophilia is from staining of ribosomes and acidophilia from hemoglobin. The nucleus is condensed and intensely basophilic with coarse heterochromatin granules giving a characteristic checkerboard appearance.

5. Reticulocyte (7-10 mm) They are not obviously discernable from mature erythrocytes with this preparation. The nucleus has been extruded; and the cytoplasm is a grayish blue color in contrast to the red color of the mature erythrocytes, because some RNA is still present in the cytoplasm.

1. Megakaryocytes (50-150 mm): Stages of maturation go from: 1) megakaryoblast to 2) megakaryocyte to 3) platelets. The megakaryocyte is very large compared to all other cells in the specimen, and has a large lobulated nucleus. Because of their large size, they have been damaged in this bone marrow preparation, and are seen only as large nuclear clusters. Refer to the bone marrow section in this exercise for the identification of megakaryocytes.

2. Monocytes (9-12 mm): Stages of maturation go from: 1) monoblast to 2) promonocyte to 3) monocyte to 4) macrophage. The nucleus is oval, horseshoe, or kidney shaped and is usually eccentric. The chromatin is less dense than in lymphocytes. The cytoplasm is slightly basophilic. They are better viewed on the peripheral blood smear.

3. Lymphocytes (6-8 mm): Stages of maturation go from: 1) lymphoblast to 2) prolymphocyte to 3) lymphocyte. Although these are formed primarily in the lymphoid tissues, many are found in the bone marrow. These cells vary in size and they have slightly indented nuclei (the heterochromatin is not clumped). The cytoplasm is basophilic. They are better viewed on the peripheral blood smear.

Hematopoiesis