Histology And Pathology Course Note

Blood and Hematopoiesis Introduction In this Lab Session we will use both images from light microscopic slides and Anderson’s Electronic Atlas of Hematology to study blood cells and blood cell development.

Cells in Peripheral Blood The formed elements of blood consist of erythrocytes (red blood cells), leukocytes (white blood cells) and platelets (thrombocytes). In order to identify blood cells and the cell fragments known as platelets, you will need to pay careful attention to the following: ▪ Cell size ▪ Nucleus (if present) o Size relative to overall cell size o Position within the cell o Shape o Pattern of chromatin condensation ▪ Cytoplasm o Overall staining o Presence of cytoplasmic granules o Size and color of granules, if present

Blood Cells in Connective Tissue In the Connective Tissue Proper Lab Session, the resident cells of connective tissue, which include mast cells and macrophages, are discussed. Macrophages are derived from monocytes. Wandering (also called transient) cells of connective tissue, move relatively quickly from the peripheral blood into surrounding connective tissues, and never return to the blood. These cells include neutrophils and eosinophils. ▪ Neutrophils are normally found in low numbers in connective tissue. Their numbers increase in the initial stages of inflammation. ▪ Eosinophils are attracted by histamine. They will be found in increased numbers in regions undergoing a histamine reaction, such as in cases of allergy. They are also present in cases of some parasitic infections. ▪ When monocytes enter connective tissue, they are transformed into macrophages. Some organs have a fixed population of macrophages (Kupffer cells in the liver, alveolar macrophages in the lung). Other macrophages are migratory, arriving in a tissue in response to inflammatory stimuli. ▪ A cell related to the basophil, the mast cell, is found in connective tissue. The mast cell has a function similar to that of the basophil. It is derived from the same hematopoietic stem cell as the basophil, and both have precursors in the peripheral blood. However, unlike the basophil precursors, mast cell precursors do not develop into their mature form until they reach their tissue destination. Mast cells are often found in the skin, lung, gastrointestinal tract, uterus and serous membranes. Histamine released by mast cells attracts eosinophils.

Peripheral Blood Decision Chart As you work through Objectives 1.1, 1.2 and 1.3, use the following decision chart to help you identify cells and cell fragments found in the peripheral blood. Nucleus present ?

NO NUCLEUS (Plate 17, top, pg. 306-307) ▪

Platelets (2-4m) are not cells; they are cell fragments. Platelets have purple, granular cytoplasm, but no nucleus. The disk shape of the platelet is maintained by an elaborate network of microtubules. Platelet granules are stored in the central region of the platelet.

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Erythrocytes (approximately 7.5µm) are biconcave disk-shaped cells, which have no nuclei. Because you can usually find at least one erythrocyte in a field of view, and because they are fairly uniform in size among healthy individuals, you can use the erythrocyte as a “ruler” to determine the relative size of other cells or tissue structures in the same field of view; also, since erythrocytes are usually fairly uniform in staining among healthy individuals, they can be used to judge the overall staining of a slide or photomicrograph. Erythrocytes should be salmon pink in routine preparations. If erythrocytes in a tissue section or blood smear from a normal individual are staining dark pink, or have a blue tinge, for example, you should anticipate that the rest of the tissue or blood smear will also stain more darkly pink or more blue than normal, respectively.

NUCLEUS PRESENT Granular cytoplasm? ?

NO CYTOPLASMIC GRANULES (Plate 18, top and middle, pp. 308-309) ▪

Lymphocyte (6-12m) nuclei are round with darkly stained, condensed chromatin. There is usually only a thin rim of basophilic cytoplasm visible, with no granules. T lymphocytes cannot be distinguished from B lymphocytes with routine staining. Differentiation between these cells can be done with special stains which include antibodies to specific T and B cell membrane markers.

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Monocytes (9-18m) are the largest of the circulating blood cells. The monocyte nucleus is indented, horseshoe-shaped or peanut-shaped, but is not multilobed. The monocyte nucleus is eccentric within the cell, not as heterochromatic as the lymphocyte nucleus, and may have several nucleoli. Monocytes have much more cytoplasm than do lymphocytes. The cytoplasm of monocytes is slightly basophilic. It contains some magenta granules (lysosomes), but these are not as prominent as those seen in granulocytes. (Remember, the monocyte does not belong to the granulocytic series).

CYTOPLASMIC GRANULES PRESENT (Plate 17, pp. 306-307) ▪ ▪ ▪ ▪

Neutrophil (9-14m) cytoplasmic granules are very fine. The nucleus is multilobed or segmented. Eosinophil (10-14m) cytoplasmic granules are large and red-orange in H&E. The nucleus is bilobed. Basophil (8-14m) cytoplasmic granules are large and basophilic. The nucleus is lobulated, but is often obscured in LM sections by the cytoplasmic granules. The nucleus can be seen in EM sections. To see the differences in size among different granulocyte specific granules, see the following EMs in Ross and Pawlina: Fig. 10.8, pg. 279 (neutrophil); Fig. 10.12, pg. 284 (eosinophil); Fig. 10.13, pg. 285 (basophil).

Bone Marrow Decision Chart The stages of blood cell development will be studied using the examples in Anderson’s Electronic Atlas of Hematology. This atlas includes many examples of both normal and abnormal blood cell development; however, this Lab Session will focus primarily on the normal developmental sequences. You will see some abnormal blood smears when you work through the questions at the end of this Lab Session. As you work through Objectives 3 and 4, use the following decision chart to help you identify the stages of erythropoiesis (erythrocyte development) and granulopoiesis (granulocyte development). Granular cytoplasm?

NO CYTOPLASMIC GRANULES : erythropoietic cells (Fig. 10.22, pg. 296) ▪ If, in a bone marrow preparation, you find a cell that does not have cytoplasmic granulation, it is most likely part of the erythropoietic series. The development of erythrocytes is characterized by the following changes: o Cell size decreases o Nucleus and its chromatin gradually condense, until finally the nucleus is extruded. The nucleus remains round, and is usually near the center of the cell until the last stage of development. o Cytoplasmic staining goes through the following changes: 1) Basophilic due to ribosomes used in hemoglobin synthesis; then → 2) Mixed basophilic/eosinophilic as hemoglobin is produced; finally → 3) Eosinophilic, since ribosomes are lost once hemoglobin content is sufficient.

***At no stage is the cytoplasm of an erythrocyte precursor granular*** ▪

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Erythrocyte Developmental Stages Basophilic erythroblast (basophilic normoblast): The chromatin has started to condense, but is still only finely clumped. The cytoplasm contains a large number of free ribosomes for the synthesis of hemoglobin, and so is intensely basophilic. Polychromatophilic erythroblast (polychromatophilic normoblast): The chromatin continues to condense in patches, which gives the nucleus a “checkerboard” appearance. The synthesis of hemoglobin continues. Since hemoglobin is a protein, it is eosinophilic. At this stage of development, the cytoplasm has mixed staining, with either a blend of basophilia (from the ribosomes) and eosinophilia (from the hemoglobin) giving an overall grey color, or separate regions of basophilic and eosinophilic cytoplasm. Orthochromatophilic erythroblast (orthochromic normoblast): The chromatin is very condensed, and the nucleus is small and intensely stained. The cytoplasm is eosinophilic, due to its hemoglobin content. As this stage progresses, the nucleus will move toward the periphery of the cell, and will then be extruded. This cell is only slightly larger than a mature erythrocyte. Reticulocyte (polychromatophilic erythrocyte): After extrusion of the nucleus, the cell leaves the bone marrow and enters the circulation. Initially some cytoplasmic polyribosomes remain. This cell stage is called the reticulocyte. Reticulocytes are slightly larger than mature erythrocytes. They require special staining to delineate their polyribosomes. By the mature erythrocyte stage, the polyribosomes will have been lost.

CYTOPLASMIC GRANULES PRESENT: granulopoietic cells (Fig. 10.22, pg. 296) ▪

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During granulopoiesis, the following processes occur: o Gradual change in nucleus shape from round or oval to bilobed or multilobed. o Appearance of primary (magenta) granules, followed by their loss. o Appearance of specific granules (the granules that are specific for neutrophils, eosinophils or basophils). Granulocyte Developmental Stages Promyelocyte: (16-24) The nucleus is round to oval and eccentric, with a prominent nucleolus. There are numerous large magenta (“azurophilic”) granules in the cytoplasm. The cytoplasm is blue-gray. Near the nucleus there is often a clear region, which is the large, unstained Golgi apparatus. Neutrophilic myelocyte or eosinophilic myelocyte: These cells are smaller than the promyelocyte. They are approximately the size of mature granulocytes (10-12). The nucleus is eccentric and flattened on one side. Magenta granules are either absent or sparse, and specific granules are present. Therefore, at this stage it is possible to distinguish neutrophilic myelocytes from eosinophilic myelocytes. Neutrophilic metamyelocyte or eosinophilic metamyelocyte: The nucleus is slightly indented, and the cytoplasm has specific granules, but no magenta (primary) granules. Band neutrophil or band eosinophil (“stab cell”): The nucleus is indented into a horseshoe shape, and is nearly uniform in thickness along its length. Mature neutrophil or mature eosinophil: Constrictions along the length of the nucleus produce the bilobed form of the mature eosinophil or the multilobed form of the mature neutrophil.

Note: Basophils follow the same developmental path as do the other granulocytes. However, because their numbers are so low, they are difficult to find in bone marrow and peripheral blood specimens. It is for this reason that we do not ask you to identify basophils and their precursors in sections or smears during this Lab Session. We have, however, included images of basophils. You will also see examples of basophils in Integrative Question 4.

References used in Histology Lab Manuals and Histology Website Unless otherwise indicated, figures and plates referenced in the Histology Laboratory Manuals and Histology Website are from Histology: A Text and Atlas, 7th ed., 2016, Ross M and Pawlina W, Walters-Kluwer Health. Some such images will be labeled "R&P". • Images labeled “WED” are from the Virtual Microscope, WebSlide Enterprise, Olympus America, licensed to the Department of Radiology, Michigan State University. • Images labeled "Jastrow EMA" are from Dr. Jastrow's Electron Microscopic Atlas of Cells, Tissues and Organs, licensed to the Department of Radiology, Michigan State University. • Images labeled "Wheater’s" are from Wheater’s Functional Histology: A Text and Colour Atlas, 5th ed., Young B, Lowe JS, Stevens A, Heath JW, Churchill Livingstone/Elsevier, 2006. • Images labeled “Wheater’s Basic Pathology” are from Wheater’s Basic Pathology: A Text, Atlas and Review of Histopathology, 5th ed., Young B, Stewart W, O’Dowd G, Churchill Livingstone/Elsevier, 2011. • Images labeled "Gilroy, 2/e" indicates Atlas of Anatomy, Second Edition, Gilroy AM, MacPherson BR, Ross LM, Thieme Medical Publishers, Inc., 2012. • Images labeled "M&D" or "Moore and Dalley" are from Clinically Oriented Anatomy 6th ed., Moore KL, Dalley AF and Agur AMR, Lippincott Williams and Wilkins, 2010. • Virtual Slides used on the Website are from MBF Bioscience - MicroBrightField, Inc. Biolucida ®, and are either from the University of Michigan or the University of Iowa slide sets.

Laboratory Objectives Objective 1: In Objectives 1.1, 1.2 and 1.3, you will examine the cells of peripheral blood. As you

examine the following cells, note the differences in cell size, nucleus size and shape and cytoplasmic staining and presence of absence of cytoplasmic granules: ▪ Erythrocyte ▪ Neutrophil ▪ ▪ ▪ ▪ ▪

Eosinophil Basophil (See Obj.1.3) Lymphocyte Monocyte Platelet

1.1 Cells of peripheral blood-VIRTUAL SLIDE (Plate 17, pg. 306-307) ▪ At low power, locate nucleated cells among the erythrocytes in this blood smear. Increasing magnification slightly, and moving the slide to examine multiple fields of view, try to group cells which have similar nuclear shape and cytoplasmic characteristics. ▪ At higher magnification, examine these same cells to confirm the cell types. You should be able to find all peripheral blood elements listed above, except basophils. You can see a good example of an eosinophil near the lower left edge of this sample. ▪ Among these cells you should find monocytes. Note the variability in shape of monocyte nuclei. Although the dark basophilia of monocyte nuclei in this smear obscures nucleoli, in some monocytes, you should be able to identify multiple prominent nucleoli. ▪ Don’t forget to examine platelets, which in this slide are present either individually or in small groups.

1.2 Cells of peripheral blood This image contains three white blood cells. Examine the two white blood cells on the right. Note the difference between both the shapes of their nuclei and the staining characteristics of their cytoplasm. The staining in this slide differs from that in Objective 1.1. However, remember that the erythrocytes in view will give you a reliable indicator of what constitutes eosinophilia in a given slide. You should be able to identify one of these white blood cells as a neutrophil and one as an eosinophil. The third white blood cell in this image is a lymphocyte. You should also be able to identify platelets.

1.3 Cells of peripheral blood - basophils Basophils are normally present in such low numbers that they are difficult to find in peripheral blood. The images in this Objective were selected because they show good examples of basophils. Note that the darkly basophilic granules obscure the nucleus.

Objective 2: Identify eosinophils, lymphocytes, plasma cells and neutrophils in connective tissue. 2.1 Leukocytes in normal tissue-VIRTUAL SLIDE ▪ In this virtual slide, the lumen of the colon and its lining epithelium are at the bottom of the section. Examine the lamina propria, which is immediately deep to the epithelium, and between the glands of the colon (see image to the right). ▪ Look for lymphocytes, plasma cells and eosinophils in the lamina propria. You will most likely not find neutrophils, except within blood vessels in deeper tissues. In Objective 2.2 you will identify neutrophils in tissue. ▪ Pay careful attention to the shape of the nucleus in identifying these cell types. When comparing lymphocytes and plasma cells, you will notice that the nuclei appear similar; however, cytoplasmic characteristics differ. Lymphocytes have very little Modified From Biolucida UofM_plastic_gi_15 cytoplasm, while plasma cells have a moderate amount of basophilic cytoplasm and often a prominent Golgi apparatus. The light microscopic appearance of plasma cell cytoplasm indicates an increase in the amount of rough endoplasmic reticulum (increased basophilia) as compared to lymphocytes. This reflects the increase in protein (immunoglobulin) production by plasma cells as compared to lymphocytes.

2.2 Leukocytes in tissue inflammation-VIRTUAL SLIDE ▪ This section is taken from an acutely inflamed vermiform appendix (acute appendicitis). The central open space, which contains some cellular debris and hemorrhage, is the lumen of the appendix. The mesenteric adipose tissue is at the bottom of the section. ▪ The columnar epithelium lining the lumen of this appendix, and its infoldings to form glands, is only present in segments in this specimen. The loss of the epithelium and some of its underlying connective tissue in this case is part of the inflammatory process which necessitated the surgical removal of this appendix. ▪ Dense accumulations of neutrophils enmeshed in eosinophilic strands of fibrin are present in the areas of tissue loss. After centering the edge of the lumen of this appendix, increase magnification to identify individual neutrophils. Many of the neutrophils in these areas have begun to degenerate, resulting in rounding of their formerly segmented nuclei. ▪ As you examine the tissue farther from the lumen, you will identify more normal appearing neutrophils. These neutrophils are in the process of migrating toward the lumen, which is the area of most intense inflammation. Note that, because this inflammatory process was so severe, the inflammatory infiltrate has extended into the mesenteric adipose tissue.

Objective 3: Erythropoiesis: identify the developmental stages of erythrocytes. Use the chart

below to note the differences in nuclear and cytoplasmic characteristics, which indicate that a cell has progressed to the indicated developmental stage. (See also Table 10.3, pg. 299 and Fig. 10.22, pg. 296) Nucleus (chromatin)

Cytoplasm (staining)

Basophilic erythroblast (basophilic normoblast) Polychromatophilic erythroblast (polychromatophilic normoblast) Orthochromatophilic erythroblast (orthochromic normoblast) Reticulocyte (polychromatophilic erythrocyte) ▪

To use Anderson's Electronic Atlas of Hematology, hit “Click to Start” → “Atlas” → “Blood Cells” → “Red Blood Cells” → “Normal Maturation Series” ▪ This atlas gives you labeled examples to help you identify each stage of erythropoiesis. Remember that erythropoiesis is a continuous process. Therefore, a cell may have an appearance that is intermediate between an earlier and a later stage. ▪ Be able to relate the morphological changes in the cells to changes in organelle content and protein synthesis. (You should focus on the stages listed in the table above. You do not need to identify every stage of erythropoiesis shown in this atlas.)

Objective 4: Granulopoiesis: identify the developmental stages of granulocytes. Note the

differences in nuclear and cytoplasmic characteristics, which indicate that a cell has progressed to the indicated developmental stage. (See also Table 10.3, pg. 299 and Fig. 10.22, pg. 296) Nucleus (shape/chromatin)

Cytoplasm (staining/granules)

Promyelocyte Neutrophilic and eosinophilic myelocyte Neutrophilic and eosinophilic metamyelocyte Neutrophilic and eosinophilic band (“stab”) ▪

To use the Electronic Atlas of Hematology, hit “Click to Start” → “Atlas” → “Blood Cells” → “White Blood Cells” → “Granulocytic Series” →“Normal Maturation” ▪ This atlas contains labeled examples to help you identify each stage. Remember that granulopoiesis is a continuous process. Therefore, a cell may have an appearance that is intermediate between an earlier stage and a later stage. ▪ Remember, it is at the myelocyte stage of granulopoiesis that specific granules first appear. At this stage, you should be able to distinguish neutrophilic myelocytes from eosinophilic myelocytes or basophilic myelocytes. (You should focus on the stages listed in the table above. You do not need to identify every stage of granulopoiesis shown in this atlas.)

Objective 5: Thrombopoiesis: identify the cellular source of platelets, the megakaryocyte. ▪

To use the Electronic Atlas of Hematology, hit “Click to Start” → “Atlas” → “Blood Cells” → “Megakaryocytic Series” → “Normal Maturation” → “Megakaryocyte”

▪ You only need to examine mature megakaryocytes in this Objective. The large size and unusual shape of the megakaryocyte nucleus is the end result of nuclear endomitosis. This is a process of chromosome duplication without nuclear division or cell division. This process can result in a single megakaryocyte nucleus with up to a 64n chromosome number. You can see a diagrammatic representation of a megakaryocyte in Fig. 10.19, pg. 293. The unusual shape of the megakaryocyte nucleus, when viewed in the two dimensions of a light microscopic section, may give you the false impression that the megakaryocyte has multiple nuclei. The megakaryocyte cytoplasm may have fine basophilic granules. The outer edge of the megakaryocyte may be irregular or difficult to define, due to the peripheral extensions of forming platelets.

Objective 6: Structure of bone marrow Bone marrow, calvaria (Fig. 10.25, pg. 302) Bone marrow is in the interior of this section of bone. Because of the thickness of this section, you will not be able to identify the fine cellular detail of individual hematopoietic cells. In another type of bone marrow preparation, a smear is made. In those preparations, which are only one cell layer thick, one is able to examine and identify individual hematopoietic cells. ▪ The hematopoietic cells are very closely packed. ▪ Among the hematopoietic cells are vascular spaces, called sinusoids, which allow transport of cells out of the hematopoietic compartment. Sinusoids look like irregularly shaped, branching spaces lined by simple squamous epithelium (endothelium). They can be seen more easily in low power images as pale staining spaces among the more darkly staining hematopoietic cells. Sinusoids are filled with erythrocytes, which stain faintly in this preparation because of the process used to decalcify the bone of the calvaria. ▪ Although this preparation does not allow you to evaluate cellular detail, you should be able to identify band neutrophils and mature neutrophils among hematopoietic elements, based upon the shape of their nuclei. ▪ Find a megakaryocyte. Notice the proximity of megakaryocytes to the sinusoids. This positioning of the megakaryocyte expedites delivery of platelets to the bloodstream via bone marrow sinusoids (See R&P pp. 301302). ▪ Inactive marrow has proportionately more adipocytes, because it has proportionately less hematopoietic tissue. The sample seen in this Objective is an example of active marrow. You will see an example of inactive marrow as you work through the self-study review questions.

Integrative Questions 1a. In addition to the elongate “sickled” cells in the image labeled “Sickled Erythrocytes”, what other morphologic abnormalities do you see among the erythrocytes in this image of peripheral blood?

The most immature stage of erythrocyte development visible in this image is: A. basophilic erythroblast B. polychromatophilic erythroblast C. orthochromatophilic erythroblast D. reticulocyte E. erythrocyte

1b. The second image shows a peripheral blood erythrocyte with basophilic stippling. Which organelles are present in these foci of stippling? A. mitochondria B. secretory granules C. ribosomes D. nucleoli What other abnormality do you see in this cell?

2. These images are from a(n): A. normal blood smear B. abnormal blood smear C. normal bone marrow smear If this is an abnormal blood smear, list as many abnormalities as you can identify.

3. These images are from a(n): A. normal blood smear B. abnormal blood smear C. normal bone marrow smear If this is an abnormal blood smear, list as many abnormalities as you can identify.

4. These images are from a(n): A. normal blood smear B. abnormal blood smear C. normal bone marrow smear If this is an abnormal blood smear, list as many abnormalities as you can identify.

5. These images are from a(n): A. normal blood smear B. abnormal blood smear C. normal bone marrow smear If this is an abnormal blood smear, list as many abnormalities as you can identify.

6. This image is from a(n): A. normal blood smear B. abnormal blood smear C. normal bone marrow smear If this is an abnormal blood smear, list as many abnormalities as you can identify.

7. These images are from a section of an acutely inflamed vermiform appendix (appendicitis). What cell type predominates in this connective tissue? Note the numerous fine, extracellular strands in this tissue. As part of the inflammatory process, numerous blood cells exit the blood vessels, and the blood coagulation cascade is initiated. Based upon your understanding of this cascade, of which protein are these fine strands composed?

Self-study Review 1. Identify lymphocytes and plasma cells in this area of Peyer’s patch.

2. These images are from a section of chronically inflamed skin (dermatitis). What type of immune cell do you see? Prominence of which two cytoplasmic organelles is responsible for the cytoplasmic staining of this cell?

3. Identify cells in the erythropoiesis series in view.

4. Identify cells in the granulopoiesis series in view.

5. Identify the eosinophilic myelocyte in view.

6. Compare this image of bone marrow to the images you examined in Objective 6. What differences do you see? Is this an example of active bone marrow?

Answers to Questions for Blood and Hematopoiesis Laboratory Session Integrative Questions 1a) Abnormal blood smear #1 These cells show how metabolic errors in hemoglobin synthesis are reflected in morphology. The sickled cells in this image are the long, thin erythrocytes. They have lost their normal biconcave disc shape. A few erythrocytes in this view appear to be folded. In addition to variability in the shape of erythrocytes, there is variability in the staining of these erythrocytes. Many of these erythrocytes have more cytoplasmic basophilia than would be expected in normal mature erythrocytes. In the lower center, one of these more basophilic erythrocytes has cytoplasmic staining in the area of central pallor. This is a target cell. At the far right, below center, there is a cell with no central pallor. This cell has lost its normal biconcave disk shape, and is spherical. This is a spherocyte. The most immature cell of the erythropoietic series seen in this view is in the upper right hand corner. This cell, which is in the process of extruding its nucleus, is an orthochromatophilic erythroblast (The nucleated cell at the bottom of this image is a lymphocyte). 1b) Abnormal blood smear #2 The abnormal cell is at the center of this field of view. This type of pathology is due to DNA damage, which results in a dissociation between maturation of the cytoplasm and maturation of the nuclei of erythrocytes. Instead of becoming pyknotic and being extruded from the cell, as in normal erythropoiesis, the nuclei of affected cells remain within the cell, and are only partially condensed. This cell also has abnormal cytoplasm. Instead of a homogeneous accumulation of hemoglobin causing the cytoplasm to become more eosinophilic, there are large numbers of basophilic foci in the cytoplasm. This pathologic characteristic is called basophilic stippling. Erythrocytes with basophilic stippling may be seen in cases of lead poisoning. The “stipples” are areas of aggregated ribosomal RNA.

2) These images are from an abnormal peripheral blood smear from a case of sickle cell anemia. In addition to the long, thin, “sickled” erythrocytes scattered in this smear, there are frequent “target” cells (cells in which there is a focus of staining in the middle of the cell’s area of central pallor). Overall, this smear is characterized by anisocytosis and poikilocytosis. You should refer to your lecture notes if you are unfamiliar with the terms “anisocytosis” and “poikilocytosis”.

3) These images are from an abnormal peripheral blood smear from a case of iron deficiency anemia. Image 4 provides you with measurements of the diameters of multiple erythrocytes in this slide. The measurements range from 5.1µm to 7.1µm, with an average diameter of 6.14µm. Most of these cells are smaller than 7.5µm in diameter, the average for normal human erythrocytes. You will also notice that, because there is less hemoglobin within these erythrocytes, the area of central pallor is larger relative to the size of the erythrocyte. Iron deficiency is characterized by a microcytic hypochromic anemia, as is seen in these images.

4) These images are from an abnormal peripheral blood smear from a patient with chronic myelogenous (myeloid) leukemia (CML). CML results from a molecular defect in pluripotent hematopoietic stem cells. For reasons that are not understood, there is a preferential increase in the production of granulocyte precursors. In time, these precursors can be found in the peripheral blood. Another feature of CML is the appearance of an unusually large number of basophils. You should be able to find examples of basophils in these images, as well as clusters of immature granulocytes. You will note that no red blood cell precursors are present in this slide; therefore, this could not be a normal bone marrow smear.

5) These images are from an abnormal peripheral blood smear taken from an individual with chronic lymphocytic leukemia (CLL). In CLL, there is increased production of abnormal lymphocytes, which appear in the peripheral blood. You should be able to identify many examples of lymphocytes in these images. Compare the relative proportion of lymphocytes in this slide to that seen in the normal peripheral blood smear images you examined for Objective 1.1 of this Laboratory Session.

6) This is an example of a bone marrow smear. Unlike the abnormal peripheral blood smears which you examined above, in this view, you can identify erythrocytic precursors as well as granulocytic precursors. Use Fig. 10.22, pg. 296 in Ross and Pawlina to help you identify the stages visible in this view. Pay particular attention to how the cells you identify fit into the sequence of erythrocytic and granulocytic maturation.

7) In the 200X magnification images, most of the inflammatory cells present are neutrophils. Neutrophils can be identified in tissue by their characteristic segmented nuclei. An intense infiltration of neutrophils, as seen here, is a typical finding in acute inflammation. In areas of such inflammation, the neutrophils may have begun to degenerate or die, and so may have shrunken, dark nuclei; however, you should still be able to identify segmentation of these nuclei. Image 6 shows neutrophils enmeshed in strands of fibrin. In Image 5 you can see an aggregate of fibrin within a blood vessel. To the right of this vessel, and elsewhere in these images, you will see areas of hemorrhage, indicated by accumulations of erythrocytes outside of the tissue’s vasculature.

Self-study Review Questions 1) This image is from a section of ileum, a portion of the small intestine. It includes portions of small lymphatic vessels, with several lymphocytes in their lumina. Because lymphocytes are so closely arranged in this area, it is not possible to distinguish plasma cells. Instead, use Self-study Review Question 2 and Objective 2.1 to examine plasma cells in tissues. Plasma cells have spherical nuclei, which are eccentrically placed. They have a moderate amount of cytoplasm, which usually stains basophilic, due to the presence of rough endoplasmic reticulum. In the cytoplasm adjacent to the nucleus of many plasma cells you will find a pale or unstained area, which represents the Golgi apparatus. Lymphocytes have nuclei similar in appearance to those of plasma cells. However, lymphocytes have cytoplasm which is so sparse that it may not be visible in tissue section.

2) This image is from an area in which plasma cells have accumulated around a small blood vessel in the dermis of the skin (dense irregular connective tissue). These plasma cells have characteristic round nuclei, some with “clock face” clumping of heterochromatin. The nuclei are eccentrically placed. Plasma cells have moderate amounts of basophilic cytoplasm. This cytoplasmic basophilia is due to ribosomes on the high concentration of rough endoplasmic reticulum. Many of these cells have a prominent Golgi apparatus, evident as an area of pale staining or unstained cytoplasm next to the nucleus.

3) Keep in mind the decision flow chart in the Introduction to this Laboratory Session as you examine this image. First, cells in the erythropoiesis series will have agranular cytoplasm. The nuclei are always round, but will become smaller and darker as the cell progresses in maturation. The nuclei will usually, but not always, be centrally placed in the cell until they are ready to be extruded. In the orthochromatophilic erythroblast (normoblast) stage, the nucleus is small and very condensed. There are four examples of cells in the erythropoiesis series in this image. The cytoplasm of basophilic erythroblasts is intensely basophilic. The upper left cell with the round nucleus fits the description of a basophilic erythroblast. The slightly smaller cell near the top center of this image also has basophilic cytoplasm. However, the smaller size of the cell and its nucleus, as well as the faintly "checkerboard" pattern of its chromatin, is consistent with a polychromatophilic erythroblast. In the later stages of erythropoiesis, as the cell gains hemoglobin, the cytoplasm becomes pink/gray, and finally becomes the same color as that of the mature erythrocyte cytoplasm.

4) When identifying any of the developing blood cells, keep in mind the decision flow chart given in the Introduction to this Laboratory Session. Check for the presence of cytoplasmic granules. These are present in cells of the granulocytic series. Next, examine the nucleus of the cell. The nucleus of the cell on the far left is flattened on one side, and is eccentrically placed in the cell. These characteristics, and the appearance of the chromatin, indicate that this is a myelocyte. On the far right, there are three cells of the granulocytic series, all of which have indented nuclei. Two of these are examples of metamyelocytes. The third is in transition from metamyelocyte to band cell (also called stab cell) stage. The nuclear characteristics of the cell of the granulocytic series in the center of this view are consistent with those of a band cell. In stages in which specific granules are present, you can use the type of granule seen to determine which type of granulocyte (neutrophil, eosinophil, basophil) you are examining. None of the cells in this view have granules consistent with those seen in either eosinophils or basophils. The cells of the granulocytic series in this view are all in the neutrophilic line.

5) An eosinophilic myelocyte should have eosinophilic granules, and a nucleus which is flattened on one side. The nucleated cell to the right of center in this image fits that description.

6) Notice the large number of adipocytes, with intervening hematopoietic cells. Compare the number of adipocytes in this marrow to the number of adipocytes found in the marrow you examined for Objective 6? The marrow in Objective 6 is composed almost entirely of hematopoietic elements. It is active bone marrow. In inactive marrow, as seen in the image for this question, there will be much higher relative numbers of adipocytes when compared to active marrow. The marrow shown in the image for this question is an example of inactive marrow.