BLOOD

Much valuable information about the health of a patient can be obtained by a thorough and competent study of a smear of their peripheral blood. To prepare a blood smear, a drop of blood is spread on a glass slide and then stained. Wright's stain is widely used for staining blood smears. It contains eosin and methylene blue, both of which stain independently as well as in combination. Formed elements stained by Wright's stain are said to be eosinophilic (acidophilic), basophilic, or neutrophilic. The student must become familiar with the appearance of the formed elements (red blood cells, leukocytes, and platelets) in normal peripheral blood in order to begin to study and to diagnose disorders of the blood by microscopic examination of peripheral smears.

Microscopic study of smear of adult peripheral blood

Slide A53 and/or A54, blood smear, human, (Wright's). Examine the blood smear under oil immersion. Keep in mind that all blood smears are likely to contain certain artifacts, such as smudges of disrupted cells and distorted leukocytes, and that it will not be possible to identify all leukocytes with certainty. Even if your slide is not optimally stained, remember as you carefully study the blood smear that nuclear morphology and granule size are useful criteria for identifying the different types of leukocytes. Note that in some areas the cells are spread far apart and that in other areas they may be piled on top of one another. In the former areas, the white blood cells are flattened out and in the latter areas they are more nearly spherical and much smaller. The optimal area for observation is somewhere between these extremes and only in such an area will the cells correspond to textbook descriptions. Use Figure 28 below as a comparison when looking for the blood cells types under your microscope.

1. Red blood cells (erythrocytes or red corpuscles). There is no nucleus and the

cytoplasm is entirely acidophilic. Cells have a biconcave shape, are 6.5 to 8 4μ in diameter and stain red. Crenation (indention or notch) and minor surface irregularities are considered as normal variations.

Figure 28: Left photo, RBCs in a blood smear. Taken from Wheater’s Functional Histology, a text and colour atlas, p. 47, Figure 3.1. Right photo, Different types of white blood cells in a blood smear. Taken from Junqueira and Carneiro, Basic Histology, a text and atlas, p. 237, Figure 12-5.

2. Neutrophils

a. Segmented. The mature neutrophil is about twice the size of the red blood cell. The lobulated nucleus usually has three to five lobes connected by strands of chromatin. Lobes are sometimes superimposed, making it impossible to see the connecting strands of chromatin. In an ideal stain, the granules are pink to bluish-black.

b. Nonsegmented (band or stab). This is the youngest form of neutrophil that is commonly found in normal peripheral blood. The nucleus is horseshoe shaped and the indentation is greater than half the width of the hypothetically round nucleus. As in the segmented neutrophil, the granules are small and evenly distributed. They stain various shades of pink and blue

3. Eosinophils (acidophils). The eosinophil usually has a two-lobed nucleus. The granules are larger than those in the neutrophil and nearly fill the cell. They have an affinity for eosin and often stain reddish-orange.

4. Basophils. Basophils have large irregularly shaped, pale staining nuclei. The granules are of variable number and they may obscure the nucleus. Some of the granules are as large or larger than those of eosinophils while others are intermediate in size between the granules of neutrophils and eosinophils. The granules are basophilic, metachromatic, and highly water-soluble. A pale area sometimes surrounds a granule.

5. Lymphocytes. Lymphocytes may vary somewhat in size, but the majority of them will measure 7-8 μm (slightly larger than an erythrocyte). They have a relatively large, spherical nucleus, which may have a slight indentation on one side. With Wright's stain the nucleus has a solid or homogeneous appearance due to the densely packed, basophilic chromatin. A relatively small amount of pale blue staining cytoplasm surrounds the nucleus. A few azurophilic granules are seen occasionally in the cytoplasm, which are similar in appearance to those of granular leukocytes.

6. Monocytes. The three most useful diagnostic features of these cells include their dull grey-blue cytoplasm, their larger size (12-15 μm), and their distinctive horseshoe or kidney shaped nucleus. The nuclei are rarely spherical and their chromatin network is finer and stains less densely than that of the lymphocyte.

7. Platelets (thrombocytes). Platelets are fragments of megakaryocytes. Megakaryocytes are found in red bone marrow. Cytoplasmic processes of these cells protrude through the wall of a sinusoid in the bone marrow and break off to release the platelets into the blood stream. Platelets tend to adhere to one another and thus to occur in clumps. An individual platelet varies in shape from round to ovoid and usually measures 1-4 μm in diameter. A centrally located granulomere (or chromomere) contains blue staining granules, while the more peripherally located, pale staining area is called the hyalomere. See Figure 29 below.

Figure 29: Platelets and RBCs in a blood smear. Taken from Wheater’s Functional Histology, a text and colour atlas, p. 57, Figure 3.10.

Differential count

Make a differential count of the leukocytes in a blood smear. A count of 100 or more consecutive leukocytes made while moving the oil immersion field over the optimal area (feather edge) of the smear constitutes a differential count. This procedure will also help the beginner in learning to identify the various cell types. Keep an accurate check of the leukocytes identified and determine the percentage of each cell type found in the smear.

NOTE: If possible you will be provided with directions and materials for making a smear of your blood on which you may do the differential count.

Drawing of blood cells in a smear

While doing the differential count, make outline drawings of blood cells in a smear. Show several variations of neutrophils. Indicate which cells are the more immature forms. Remember, the more lobes in the nucleus of a neutrophil the older the cell. Show any variations you see in other granulocytes as well as those observed in lymphocytes and monocytes. Include one or two platelets (drawn in proper proportion) and label chromomere and hyalomere. Show one or two erythrocytes in surface view. And if possible show one or two in profile.

Blood cells in tissue sections

Blood cells may be seen in preparations of fixed and sectioned material where they may occur either in blood or lymph channels or outside of them in connective tissue. In such preparations they may appear quite different than in the smears where they are flattened out and dried. Generally, in sections the cells are more rounded and smaller. In human tissues, especially where several hours have elapsed between death and fixation of the tissue, there may be postmortem changes. This is especially marked in red blood cells, which may be distorted in shape, fused in a clot, and variable in staining. See Figure 30 below. Observe the RBCs inside the artery and vein. Note that they stain differently.

Figure 30: Cross section of an artery and vein with adipose tissue, a specialized type of connective tissue. Taken from Junqueira and Carneiro, Basic Histology, a text and atlas; p. 221, Figure 11-10.

Study the cells in the section of middle esophagus, slide B39, human, (H&E) and in the section of vagina, slide C59, human, (H&E). In both slides, the cells are usually easy to find near the epithelium either in the blood vessels or in the connective tissue. Note that it is not possible to see much, if any, of the cytoplasm of small lymphocytes.

Examination of a Fresh Blood Film with a Dark-field condenser

Because blood is usually studied in stained smears the dynamic role of blood in nourishing and protecting the tissues is often overlooked. Some of the dynamic features of blood can be shown by dark-field microscopy. A thin film of fresh blood is viewed through an ordinary microscope, which is equipped with a dark-field condenser. This condenser differs from the one in your microscope in this way: instead of allowing a solid cone of light to reach the object (the slide), as does your condenser, the dark-field condenser has an opaque central stop built into it so that the cone of light produced is hollow. When this hollow cone of light reaches the object, it is deflected and the object, therefore, appears to be self-luminous. In the dark-field you are actually observing the reflection of light from the object in the field.

There are four types of structures, which should be observed by dark-field microscopy. These are (1) red cells, (2) granular leukocytes, (3) chylomicra, and (4) fibrin. Study these in pictures in your text and atlas.

1. Red cells. As in any blood smear, the red cells are the most obvious elements. They tend to pile up in coin-like stacks (rouleaux formation). The red cells are non-motile and serve simply as carriers of hemoglobin.

2. Granular leukocytes. In fresh films, granular leukocytes are similar to amoebae in that they are capable of moving rapidly in and out of the field by extending their cytoplasm in the form of pseudopodia. In this manner, the leukocytes are capable of leaving the blood vessels and migrating into the tissues. Seeing these cells "in action" facilitates the understanding of how the leukocytes can move rapidly into an area of infection and phagocytose foreign material.

3. Chylomicra. Neutral fat is digested in the intestine and absorbed by the intestinal mucosal cells. The mucosal cells then re-synthesize the majority of this absorbed material into triglycerides, which pass into the lymph vessels of the intestinal villi as chylomicra. The chylomicra pass into the peripheral blood via the thoracic duct. Chylomicra are transparent particles and cannot be viewed directly, but with the dark-field condenser they can be seen in the same manner that dust particles can be viewed in the air if a beam of light is directed at the proper angle; points of light are deflected from their surfaces. The dust-like particles seen moving rapidly throughout the field (by Brownian movement) are the chylomicra.

4. Fibrin. The conversion of fibrinogen (normally in solution in the plasma) to fibrin is the final step in the clotting mechanism of normal blood removed from the body. Long thin filaments of the fibrin can be seen by dark-field microscopy to increase until the whole film has clotted. The process is slowed because the blood film is kept warm by the light source.

MYELOID TISSUE

The study of red bone marrow (myeloid tissue) involves a general understanding of hemopoiesis or blood cell development. Although the earliest blood cells develop in the embryonic yolk sac and subsequently in the liver and spleen, erythrocyte and granulocyte development in the adult is normally limited to the red bone marrow. Thus the term myeloid refers to red bone marrow, and the term myeloid element refers to the erythrocytes and granular leukocytes produced in myeloid tissue. It is now known that the monocytes and some lymphocytes (the non-granular leukocytes) also develop in the red bone marrow. Of course, many lymphocytes are also produced outside myeloid tissue in the lymphatic organs.

There is increasing evidence that all of the blood cells are derived from a single cell known as a "stem cell." This means that the erythrocytes, granular and non-granular leukocytes, including lymphocytes that develop outside the red bone marrow in the adult are all derived from a single stem cell. According to recent research studies it is believed that the marrow stem cell for lymphocytes appears to be the same CFU (colony-forming unit) stem cell for all lymphocytes, some of which take up residence in the lymphatic tissues. It is advisable to keep this information in mind when the lymphatic system is taken up at a later date.

In the study of hemopoiesis in this laboratory period we expect accomplishment of the following:

1. Learn to distinguish immature from mature forms of erythrocytes.

2. Learn to distinguish immature from mature forms of leukocytes.

3. Be able to distinguish immature cells of the erythrocytic series from those of the granulocytic series.

4. Be able to identify red bone marrow as a tissue.

Examine slide A56, bone marrow section, human, (Azure Eosin). This is a red bone marrow section. The marrow cells are located between the red staining spicules of bone and are associated with a large number of fat cells. The presence of fat cells is normal and constitutes a method of determining the activity of the marrow. In certain pathologic conditions the fat cells may be mostly replaced by developing blood cells. In such cases the marrow is said to be hyperplastic. If, on the other hand, the marrow space consists entirely of fat cells and connective tissue, then the tissue is no longer referred to as red bone marrow. Instead it is now known as yellow bone marrow. In the adult there are many areas in which yellow marrow occurs normally.

Examine "'the developing blood cells” between the sinusoids with the 40X objective. It is difficult to distinguish various developmental stages in this preparation. Scan the red marrow with the 10x objective until you locate a very large cell containing an usually large nucleus. These cells are the megakaryocytes, which give rise to platelets. Platelets are cell fragments, which are small, oval-to-round structures that participate in the clotting mechanism of blood. Look for the megakaryocyte in the red bone marrow section shown in Figure 31 below.

Figure 31: Micrograph of red bone marrow. Taken from Wheater’s Functional Histology, a text and colour atlas, p. 59, Figure 3-12.

Study slide A38, endochondral ossification, fetal joint, (Trichrome). Study the fetal bone marrow found between the spicules of bone. Find the large basophilic staining cells that contain one to many pink nuclei. These are megakaryocytes. Note their size in comparison to the other developing cell types.

Now look at slide A55, bone marrow smear, human, (Wright’s). Smears of this type are prepared from a drop of red marrow obtained by suction with a needle and syringe inserted into the patient's marrow space, e.g., the sternal marrow space. The droplet is smeared and stained with Wright's stain just as with peripheral blood. This is the best method of identifying developing white and red cells. Try to identify as many of these early forms as you can, using the Upjohn Scope Manual as a guide.

Hematopoiesis also takes place in the yolk sac, liver and other organs normally during development.

See slide B75, liver, fetal, human, (H&E). Notice the cells in the sinusoids or spaces between the parenchymal cells (hepatocytes) of the liver. These spaces are filled with developing blood cells of the erythrocytic and leukocytic series. They stain very basophilic and are nucleated.

Compare to slide B72, adult liver, human, (H&E), which should contain only mature blood cells, mostly rbcs, in the sinusoids. Mature red blood cells are enucleate and should appear as biconcave disks with no nuclei present.

Study slide C72, ectopic pregnancy, human, (H&E). This is a section of an extremely dilated oviduct containing an embryo with associated placental and fetal membranes. There is a great deal of hemorrhage and the oviductal wall shows a decidual reaction. The pathologist estimated the pregnancy to have been in progress approximately 30 days. Look for islands of tissue with pale mesenchymal centers. Usually there are groups of cells present that have acidophilic cytoplasm and basophilic nuclei. These are developing blood cells. In other areas there are cells that are staining vivid red. This is maternal tissue with mature red blood cells present. Compare the difference in maturity of the cells.

Abnormal occurrence of Hematopoiesis in regions other than bone marrow in the adult is referred to as extramedullary hematopoiesis (or extramedullary myelopoiesis).

Yellow bone marrow

The marrow of long bones is red in young individuals, but in the adult it becomes infiltrated by fat. It takes on a yellow appearance and therefore, is known as yellow marrow.

Study slide B26, tooth, longitudinal section, (H&E). This is a section of tooth anchored to the alveolar bone. A region of marrow is present which, is filled mostly (if not completely) with white adipose and some blood vessels and nerves. No hematopoiesis takes place here!!