BONE MARROW
Objectives
| 1. | To learn how to examine the cells in red bone marrow smears. | ||||||||||||||||
| 2. | To understand what developing progranulocytes, myeloblasts and lymphocytes and monocytes look like. | ||||||||||||||||
| 3. | To identify the following
stages in the formation of erythrocytes, neutrophils, eosinophils and understand why the cells change in appearance as they mature. | ||||||||||||||||
| |||||||||||||||||
| 4. | To observe megakaryocytes and maybe a megakaryoblast. | ||||||||||||||||
| 5. | To observe that cells of developing erythrocyte and leukocyte lineages are clustered in bone marrow. | ||||||||||||||||
| 6 | To observe fat cells, reticular cells, and macrophages in sections of red bone marrow | ||||||||||||||||
* * * * *
 |
Blue Histology |
Slides
 | D-30 | Bone marrow smear (Wright's stain) | |
| D-31 | Bone marrow section, monkey (Giemsa) | |
| D-32 | Bone marrow section, rabbit (carbon injection and Giemsa staining) |
Optional slide
| D-8 | Intervertebral disk (H&E) |
Prologue
Hemopoiesis in bone marrow can be studied either in sections or in smears. Bone marrow smears, made in a way somewhat similar to blood smears, allow a greater degree of differential staining than is possible with sections. Also, the cells are flattened and have larger diameters. Therefore, smears are the more useful for clinical diagnostic purposes. On the other hand, sections preserve the histological structure of bone marrow. Consequently, your slide box contains both types of preparations. Slide D-30 was prepared by smearing a drop of human marrow tissue onto a slide. The cells adhering to the surface were dried and stained with Giemsa stain, a specialized stain for marrow. As with H&E, acidophilic structures stain red and basophilic structures, blue. Inevitably, a substantial amount of blood is collected along with the marrow. It is important to avoid the areas of the slide that are largely blood and to concentrate on those areas dominated by marrow cells.
Bone marrow is a complex tissue for two reasons. First, it contains entire developmental series of cells from primitive to fully matured blood cells. Secondly, six types of blood cells form simultaneously and independently: 1) erythrocytes, 2) neutrophils, (also called "heterophils," "polymorphonuclear leukocytes," or "polymorphs"), 3) acidophils (eosinophils), 4) basophils, 5) monocytes and 6) lymphocytes. Several other types of cells may also form in bone marrow, such as mast cell precursors, but are impossible to distinguish. And, of course, platelets come from megakaryocytes. To identify individual cells of marrow, one must recognize their lineage and then estimate their developmental stage.
Your bone marrow smears were obtained from a clinical laboratory as routine normal preparations. Each slide came from a different person, was prepared at a different time and was individually stained. The slides vary significantly in the relative numbers of some cell types, overall color intensities and amount of area suitable for examination. Therefore two duplicate slides are included in your set, D-28 and D-30. Look at both.
You already have identified the mature stages of blood cells in a blood smear. Since this was during the first introductory laboratory session, you may want go back to slide D-29 and review their appearances in blood before tackling the bone marrow slides. If not, at least look at the computer pictures in the program for laboratory 1. You definitely will want to have your textbook or an atlas opened to a colored picture of cell lineages.
* * * * *
D-30 Bone marrow smear (Wright stain).
It is of the utmost importance that you find the appropriate areas of this slide for examination. Click here for a repeat of this very important statement. Hold the slide up to the light and note the several dark blobs. These are lumps of marrow tissue too thick to study. Much of the rest of the slide far away from these lumps is covered mainly with a blood taken inadvertently with the marrow sample. These regions have only a scattering of relatively poorly preserved marrow cells (illustration). Avoid them. The cells to examine are in the areas around the blue masses where they are closely packed, but not jumbled on top of one another (illustration). Using the 10 X objective, find a place where nucleated cells form a continuous or almost continuous pavement of cells, one-deep. At 430 X the cells should look identifiable and not over stained or broken. Focus at this power and then use the oil immersion objective. The cells should still be in focus with no or very little readjustment.
These cells represent a diverse collection. I suggest that you examine them by concentrating on one lineage of cells at a time, first erythrocytes, then acidophils, etc. Pick out cells that you can identify as belonging to the type that you are studying, rather than choosing cells at random and wondering what each is. I also suggest that as you go through each stage you read the short section of your textbook about that cell type. This is a painless and effective way to read this part of your book.
Begin with the erythrocyte lineage. Many cells, maybe a quarter of the total, belong to this series. Their nuclei are conspicuously round--this is their hallmark-- and speckled in a way that the aficionados call "the checkerboard nucleus" (illustration). The cells form a continuous spectrum of forms going from stem cells to ones that look like erythrocytes except for the presence of a small compact nucleus. One task is to pick out cells of this series. The second is to categorize their stages in development. So first, just locate a dozen or more erythroid cells from the appearance of their nuclei. Next, look for maturer and more juvenile examples. Using your textbook, try to find some obvious examples of basophilic erythroblasts, polychromatophilic erythroblasts and normoblasts (illustration). The exact transition point between stages is somewhat arbitrary (as you will find out if you ask different instructors to name individual borderline cells).
The precursors to erythroblasts look substantially different from the later stages. These proerythroblasts have relatively large, pale nuclei without the checkerboard mottling and are surrounded by a narrow rim of intensely basophilic cytoplasm. The color is due to the enormous numbers of ribosomes that the cell makes in preparation for cranking out hemoglobin.
Now, sit back for a minute and review the ways in which the cytoplasm and the nuclei of these cells progressively change as the cell matures. Can you relate these changes to function? Remember that RNA and DNA stain blue, protein stains red and hemoglobin stains orangish.
Next, tackle the acidophil lineage because you can easily pick out these cells by their large granules. Scan around until you can locate some examples. Their nuclei will allow you to distinguish two stages: myelocytes and mature leukocytes (illustration). Do not bother about the metamyelocyte stage of these cells. The myelocytes are very actively dividing cells. Their nuclei are round or bean-shaped. The mature acidophil has a bi-lobed nucleus. On many of your slides the granules of the myelocytes look "muddier" than those of the mature cells because the cytoplasm of the immature cells contain large numbers of basophilic ribosomes (illustration). On some slides the acidophilic myelocytes are so gray in color that you will recognize them only by the size of their granules. With this warning and the advice to look first at mature acidophils to see how big the granules should look everybody should be able to find the acidophilic myelocytes on their slide (not disputing the fact that it is much easier when the cells stain bright orange).
Keep in mind that an important function of bone marrow is to store mature white cells in addition to forming them, so expect to see about as many eosinophilic leukocytes as myelocytes.
When you are satisfied that you can discriminate acidophils, turn your attention to neutrophils. Sometimes you can discern the minute specific granules of this cell type, but for the most part, you will not be able to. Nevertheless, the nuclei of the neutrophilic metamyelocyte and mature neutrophil are unmistakable (see the diagram in your text). Find a series of these cells, which are quite common in marrow (illustration), and distinguish between metamyelocytes and mature forms (illustration). Do not bother about neutrophilic myelocytes at this time. They are difficult to distinguish from primitive cells and the monocyte series. Keep looking for neutrophilic metamyelocytes and leukocytes until you realize that you have no trouble identifying them.
A logical next step would be to look for cells of basophil lineage. However, they are rare, and you will come upon them only now and then by accident. So keep in the back of your mind as you continue along that if you find a cell with very obvious large dark blue granules you should stop, look at it and show any such cell to your neighbors. Decide whether it is possible to tell if it is a myelocyte or mature basophil. It may not be possible because the dark granules can obscure the nucleus.
We now come to the harder part: those more juvenile cells that can look very much like one another. They include myeloblasts, progranulocytes, neutrophilic myelocytes, the monocyte series and large lymphocytes. You can pick out such cells by their large pale nuclei, but it is impossible to name all of them. The one concrete character that conspicuously varies is the presence or absence of azurophilic granules in the cytoplasm (illustration). These granules when present are large, usually deeply staining and few in number. They are sometimes called "nonspecific granules" because they occur in a variety of cell types. Myeloblasts have no granules at all, and their cytoplasm should be pale bluish. Progranulocytes should have a fair smattering of granules, sometimes all off to one side of the cell (see examples). Neutrophilic myelocytes may or may not have occasional large granules visible. Their cytoplasm should be more eosinophilic than that of myeloblasts and have small neutrophil-type specific granules (example). They are best identified by comparison of cytoplasm of nearby neutrophilic metamyelocytes. (Again, the cytoplasm of these cells may appear different in different parts of your slides due to local vagaries in preservation and staining.)
The other cell types listed above may have more or fewer conspicuous, dark, "nonspecific" granules. In fact, some of you may have noticed that even some circulating large lymphocytes in blood have a scatter of these granules. Be sure to read the description of these cells as you go along. Marrow is the most difficult tissue of the body for histologists (except possibly for the brain). For years, experts made serious misinterpretations about these cells. For example, they erroneously believed that monocytes were not formed here at all! So, if you cannot identify monocytes, monoblasts and lymphoblasts in bone marrow, just smile. The modern way to identify such cells with certainty is with immunohistochemistry, using antibodies to characteristic cell surface proteins of individual cell types.
The fixed, stromal cells of bone marrow usually get broken apart on smears (illustration). These include reticular cells, endothelial cells, fat cells and macrophages. Usually you just see their big pale nuclei, often fragmented. Megakaryocytes also are often ruptured except in the thicker areas of marrow. If you do not see any of these giant cells, wait for the next slide with a section of bone marrow. They are the one cell type that even Mom could pick out. Look at their nuclei. Each cell has only one nucleus but it is polyploid and pulled out into various lobes. Sometimes it even looks a donut with a hole in the middle. Megakaryoblasts go through several abortive mitoses in which neither the cell nor the nucleus is able to divide. They then extend a pseudopod into a sinusoid and shed packets of cytoplasm as membrane-bound platelets.
About one percent of the cells in bone marrow are plasma cells. You may be able to recognize some of them by their very speckley nucleus, basophilic cytoplasm, eccentric nucleus and prominent Golgi apparatus
Finally, some nuclei look peculiar because the cells are in mitosis (illustration). Cell division is intense in red bone marrow. If you keep an eye out for mitotic figures you can see them at various stages and in various orientations.
Now you should be able to identify almost all of the cells in the field of a well preserved area of your slide, or of the picture that you looked at earlier to pick out erythroblasts.
D-31 Bone marrow, Monkey (Giemsa)
This section from a monkey retains the structure of the bone marrow. Obviously it is much more difficult to identify particular cell types in sections than in smears, which is one reason that all serious hematology is carried out with the latter. Whether you use oil or not is up to you. The importance of observing a section is to see that the various developing cell types are clustered in bone marrow instead of just randomly jumbled together, as smears suggest. Find acidophils by their striking red granules, neutrophils by their nuclear morphology, erythroblasts by their speckley nuclei and normoblasts by their small, dark nuclei. Macrophages can be located by the yellow droplets in their cytoplasm. Now considering the erythrocyte lineage more closely, observe that the proerythroblasts are sprinkled individually (second example). (They have very basophilic cytoplasm and prominent nucleoli ....why?). As they divide, the descendants form a ball of erythroblasts around a macrophage (examples #1 and #2. An important investigation in cell biology is to understand the role of macrophages/reticular cells in regulating hemopoiesis.
Allot most of your time on this slide to the stroma. The endothelial and reticular cells are flattened, in contrast to the hemopoietic series of cells. Identify the fat cells. Megakaryocytes show up well but they are no match in size for the fat cells. Find a sinusoid or two if you can, bearing in mind that they may be obscure if collapsed.
D-32 Rabbit bone marrow after carbon injection and Giemsa staining.
This slide is a tissue section. It is included in your sets to show the abundance of macrophages in red bone marrow. These cells are easy to find because carbon particles (India ink) were injected intravenously before the animal was sacrificed (illustration).
As mentioned above erythroid cells (more bluish) are clustered into so called "erythropoietic islands" instead of being randomly sprinkled throughout the marrow (illustration). Do not spend too much time trying to identify the various parenchymal cells. In particular, do not be alarmed that neutrophils of this species happen to have unusually large and acidophilic granules, making it look at first glance as though the tissue is full of acidophils.
Instead, observe the sinusoids lined with endothelial cells (illustration). (one way to find them is to look for clusters of greenish erythrocytes). If you are using oil immersion, also look for reticular cells with elongated nuclei (yea, lots of luck here!)
* * * * *
Optional slide
D-8 Intervertebral disk (H&E)
This slide allows you to see bone marrow in situ as it routinely appears in an H&E section of bone. The special problems which bone creates for slide preparation (e.g., decalcification) and the lack of optimal stain make it harder to recognize the specific cells on slides such as this. Find the bone marrow on the right or left-hand sides of the slide (the central space is the nucleus pulposus in the middle of the intervertebral disc) and see what you think about looking at bone marrow in such a preparation.