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

1. Identify the cells in the erythroid, myeloid, and platelet series.

2. Name its precursor or successor when asked to identify a specific cell in either series.

SLIDES FOR THIS LABORATORY: Slide 75 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 slide. Do not try to identify every cell on the slide. Many cells will be in transition from one stage to another.

GRANULOCYTE MATURATION

Recall that the overall trend is from larger to smaller size; round, fine nucleus to dark, segmented nucleus; increasing cytoplasm; 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 µm): Note the round nucleus, reddish-blue and fine to slightly condensed chromatin, 1-2 nucleoli, increased basophilic cytoplasm, and primary (azurophilic) granules.

2. Myelocyte (10-18 µm): 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 neurtrophilic myelocyte , neutrophilic granules are beyond the resolution of the light microscope.

3. Metamyelocyte [ neutrophilic and eosinophilic ] (10-18 µm): 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.

4. Band Neutrophil (10-16 µm): 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 neutrophils , eosinophils , and basophils (rare).

ERYTHROCYTE MATURATION

The overall trend in RBC maturation is large, pale nucleus to darker, smaller nucleus to loss of nucleus; increase in cytoplasm; gradual decrease in size; 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 µm): Nucleus is large with fine chromatin and nucleoli; cytoplasm is scant and basophilic.

2. Basophilic erythroblast (12-17 µm): 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 µm): 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.

4. Orthochromatophilic erythroblast (8-12 µm): Dark, opaque nucleus; gray-red cytoplasm (trace blue). The nucleus has become pyknotic and there is abundant acidophilic hemoglobin. In some instances you can detect the nucleus in the process of extrusion.

5. Reticulocyte (7-10 µm) [Not visible with this preparation. Refer to the laser disc to see an example.]: Nucleus has been extruded; cytoplasm is reddish-pale blue. RNA is still present.

6. Erythrocyte (7-8 µm): No nucleus; orange-red cytoplasm; RNA is lost.

OTHER CELLS

1. Megakaryocytes (50-150 µm): Stages of maturation go from Megakaryoblast to Megakaryocyte to Platelets. The megakaryocyte is infrequent but unmistakable; scan for these at a lower power (20X) then study the cell with high dry. These cells may be more readily found in the bone marrow on Slide 69.

2. Monocytes (9 - 12 µm): Stages of maturation go from monoblast to promonocyte to monocyte to macrophage. The nucleus is oval, horseshoe or kidney shaped and is usually eccentric. The chromatin is less dense than in lymphocytes. The cytoplasm is basophilic.

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