Histology_Ross

Histology_Ross. Serotonin is a potent vasoconstrictor that causes the vascular smooth muscle cells to contract, thereby reducing local blood flow at the site of injury. Adenosine diphosphate (ADP), a nucleotide, and the signaling molecule thromboxane A2, are responsible for further aggregation of platelets into a primary hemostatic plug. The mass of aggregated platelets stop extravasation of blood. At the same time, the activated platelets release their and granules, which contain among other substances coagulation factors such as platelet thromboplastic factor (PF3) and additional serotonin. The glycocalyx of the platelets provides a reaction surface for the conversion of soluble fibrinogen into fibrin. Fibrin then forms a loose mesh over the initial plug and is further FIGURE 10.15 • Scanning electron micrograph of blood clot.


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Histology_Ross. FIGURE 10.15 • Scanning electron micrograph of blood clot. High-magnification scanning electron micrograph shows initial stage of blood clot formation. Red blood cells are entrapped in a loose mesh of fibrin fibers that are extensively cross-linked to form impermmable hemostatic plug that prevents movement of cells and fluids from the lumen of the injured vessel. 1,600. (Copyright Dennis Kunkel Microscopy, Inc.) stabilized by covalent cross-links that produce a dense aggregation of fibers (Fig 10.15). Platelets and red blood cells become trapped in this mesh. The initial platelet plug is transformed into a definitive clot known as a secondary hemostatic plug by additional tissue factors secreted by the damaged blood vessel.


Histology_Ross

Histology_Ross. After the definitive clot is formed, platelets cause clot retraction, probably as a function of the actin and myosin found in the structural zone of the platelet. Contraction of the clot permits the return of normal blood flow through the vessel. Finally, after the clot has served its function, it is lysed by plasmin, a fibrinolytic enzyme that circulates in the plasma in an inactive form known as plasminogen. The hydrolytic enzymes released from the granules assist in this process. The activator for plasminogen conversion, tissue plasminogen activator (TPA), is derived principally from endothelial cells. A synthetic form of TPA is currently used as an emergency treatment to minimize the damage caused by clots that lead to strokes. An additional role of platelets is to help repair the injured tissues beyond the vessel itself. Platelet-derived growth factor released from the granules stimulates smooth muscle cells and fibroblasts to divide and allow tissue repair.


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Histology_Ross. Hemopoiesis (hematopoiesis) includes both erythropoiesis and leukopoiesis (development of red and white blood cells, respectively), as well as thrombopoiesis (development of platelets; Fig. 10.16). Blood cells have a limited life span; they are continuously produced and destroyed. The ultimate objective of hemopoiesis is to maintain a constant level of the different cell types found in the peripheral blood. Both the human erythrocyte (life span of 120 days) and the platelet (life span of 10 days) spend their entire life in the circulating blood. Leukocytes, however, migrate out of the circulation shortly after entering it from the bone marrow and spend most of their variable life spans (and perform all of their functions) in the tissues.


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Histology_Ross. In the adult, erythrocytes, granulocytes, monocytes, and platelets are formed in the red bone marrow; lymphocytes are also formed in the red bone marrow and in the lymphatic tissues. To study the stages of blood cell formation, a sample of bone marrow is prepared as a stained smear in a manner similar to that described on page 270 for the preparation of a smear of blood. Hemopoiesis is initiated in early embryonic development.


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Histology_Ross. During fetal life, both erythrocytes and leukocytes are formed in several organs before the differentiation of the bone marrow. The first or yolk-sac phase of hemopoiesis begins in the third week of gestation and is characterized by the formation of “blood islands” in the wall of the yolk sac of the embryo. In the second, or hepatic phase, early in fetal development, hemopoietic centers appear in the liver (Fig. 10.17). Blood cell formation in these sites is largely limited to erythroid cells although some leukopoiesis occurs in the liver. The liver is the major blood-forming organ in the fetus during the second trimester. The third or bone marrow phase of fetal hemopoiesis and leukopoiesis involves the bone marrow (and other lymphatic tissues) and begins during the second trimester of pregnancy. After birth, hemopoiesis takes place only in the red bone marrow and lymphatic tissues, as in the adult (Fig. 10.18). The precursors of both the blood cells and germ cells arise in the yolk


Histology_Ross

Histology_Ross. After birth, hemopoiesis takes place only in the red bone marrow and lymphatic tissues, as in the adult (Fig. 10.18). The precursors of both the blood cells and germ cells arise in the yolk sac.


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Histology_Ross. Monophyletic Theory of Hemopoiesis According to the monophyletic theory of hemopoiesis, blood cells are derived from a common hemopoietic stem cell. Considerable circumstantial evidence has for many years supported the monophyletic theory of hemopoiesis in which all blood cells arise from a common stem cell. Decisive evidence for the validity of the monophyletic theory has come with the isolation and demonstration of the hemopoietic stem cell (HSC). The hemopoietic stem cell, also known as pluripotential stem cell (PPSC), is capable not only of differentiating into all the blood cell lineages but also of self-renewal ( i.e., the pool progenitor (CLP, CFU-L)


Histology_Ross

Histology_Ross. Hemopoietic stem cell (HSC, PPSC) Common myeloid progenitor (CMP, CFU-GEMM) Granulocyte/ monocyte progenitor (GMP, CFU-GM) Pro-dendritic cella (pro-DC) Dendritic cell Neutrophil progenitor (NoP, CFU-G) Neutrophil Basophil/mast cell progenitorb (BMCP) Mast cell progenitor (MCP) Mast cellBasophil progenitor (BaP, CFU-Ba) Basophil Eosinophil cell progenitor (EoP, CFU-Eo) Eosinophil Neutrophil Eosinophil Monocyte progenitor (MoP, CFU-M) Monocyte Macrophage Megakaryocyte progenitorc (MKP, CFU-Meg) Megakaryocyte/ erythrocyte progenitor (MEP) Megakaryocyte Megakaryoblast Platelets Erythrocyte progenitor (ErP, CFU-E) Proerythroblast Orthochromatophilic erythroblast Erythrocyte


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Histology_Ross. FIGURE 10.16 • Hemopoiesis. This chart is based on the most recent concepts in hemopoiesis. It shows blood cells’ development from hemopoietic stem cells in the bone marrow to mature cells and their distribution in the blood and connective tissue compartments. In all lineages, extensive proliferation occurs during differentiation. Cytokines (including hemopoietic growth factors) may and do act individually and severally at any point in the process from the first stem cell to the mature blood or connective tissue cell. aProdendritic cells are possible to differentiate from common lymphoid progenitor. bIf committed to enter the mast cell lineage, the basophil/mast cell progenitor cell migrates to the spleen where it differentiates into a mast cell progenitor cell. After further differentiation in the spleen, it migrates to the intestine to become a mast cell precursor. cA megakariocyte progenitor cell may also differentiate directly from a common myeloid progenitor cell.


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Histology_Ross. FIGURE 10.17 • Hepatic stage of hemopoiesis. Photomicro -graph of the fetal liver stained with H&E shows active hemopoiesis. The small round bodies (arrows) are mostly nuclei of developing erythrocytes. Although it is difficult to discern, these cells are located between developing liver cells and the wall of the vascular sinus. 350.


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Histology_Ross. of stem cells is self-sustaining). Recent studies indicate that HSCs also have the potential to differentiate into multiple non–blood cell lineages and contribute to the cellular regeneration of various tissues and multiple organs. During embryonic development, HSCs are present in the circulation and undergo tissue-specific differentiation in different organs. Human HSCs have been isolated from umbilical cord blood, fetal liver, and fetal and adult bone marrow. In the adult, HSCs have the potential to repair tissues under pathologic conditions (e.g., is-chemic injury, organ failure). Human HSCs express specific molecular marker proteins such as CD34 and CD90 and at the same time do not express lineage-specific markers (Lin–) that are found on lymphocytes, granulocytes, monocytes, megakaryocytes, and erythroid cells. It is now believed that human HSC can be identified by the Lin , CD34, CD90 , and CD38 cellsurface markers. HSCs are not identifiable in the routine preparation; however,


Histology_Ross

Histology_Ross. and erythroid cells. It is now believed that human HSC can be identified by the Lin , CD34, CD90 , and CD38 cellsurface markers. HSCs are not identifiable in the routine preparation; however, they can be identified and isolated using immunocytochemical methods.


Histology_Ross

Histology_Ross. A hemopoietic stem cell (HSC) in the bone marrow gives rise to multiple colonies of progenitor stem cells. In the bone marrow, descendants of the HSC differentiate into two major colonies of multipotential progenitor cells: The common myeloid progenitor (CMP) cells and the common lymphoid progenitor (CLP) cells. Ultimately, common myeloid progenitor (CMP) cells which were previously called colony-forming units–granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), differentiate into specific lineage-restricted progenitors (Table 10.3). These include the following.


Histology_Ross

Histology_Ross. FIGURE 10.18 • Dynamics of hemopoiesis in embryonic and fetal life. During embryonic and fetal life, erythrocytes are formed in several organs. Essentially, three major organs involved in hemopoiesis can be sequentially identified: The yolk sac in the early developmental stages of the embryo; the liver during the second trimester of pregnancy; and the bone marrow during the third trimester. The spleen participates to a very limited degree during the second trimester of pregnancy. At birth, most hemopoiesis occurs in the red bone marrow, as it does in the adult.  Megakaryocyte/erythrocyte progenitor (MEP) cells: These bipotential stem cells give rise to monopotent megakaryocyte-committed progenitor cells (MKP or CFU-Meg) and other monopotent erythrocyte-committed progenitor cells (ErP or CFU-E) that give rise to the erythrocyte lineage.


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Histology_Ross.  Granulocyte/monocyte progenitor (GMP or CFUGM) cells: Development of the GMP (CFU-GM) cells requires high-level expression of PU.1 transcription factor. These cells then give rise to the neutrophil progenitors (NoP or CFU-G) which differentiate into the neutrophilic lineage; eosinophil progenitors (EoP or CFU-Eo), cells that give rise to eosinophils; basophil/mast cell progenitors (BMCP) that give rise either to basophil progenitor cells (BaP or CFU-Ba) in the bone marrow or MCPs in the gastrointestinal mucosa; and finally monocyte progenitors (MoP or CFU-M) that develop toward monocyte lineages. In addition to the specific lineage progenitors, GMP cells can give rise to dendritic cells (DCs), which are professional antigen-presenting cells. Dendritic cells are discuss in Chapter 14, Lymphatic System.


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Histology_Ross. The common lymphoid progenitor (CLP) cells are capable of diferentiating into T cells, B cells, and natural killer (NK) cells. These multipotential CLP cells have been previously called colony-forming units–lymphoid (CFU-L) The NK cells are thought to be the prototype of T cells; they both possess similar capability to destroy other cells. Lymphocytes are discussed in Chapter 14, Lymphatic System. Dendritic cells can also developed from CLP cells. Perhaps the easiest way to begin the histologic study of blood cell development is to refer to Figure 10.16 and TABLE Summary of Features During Maturation of Common Myeloid Progenitor (CMP) Cell10.3 • Large, round nucleuswith 1–2 nucleoli Smaller, •moreheterochromatic nucleus • Last cell capable of mitosis Small, deeply stained nucleus Begins to acquire acidophilia Acidophilic cytoplasm •with trace of earlier gray Life span:in blood, 1–120 days Slightly basophilic •cytoblasm with multiplenucleoli


Histology_Ross

Histology_Ross. Small, deeply stained nucleus Begins to acquire acidophilia Acidophilic cytoplasm •with trace of earlier gray Life span:in blood, 1–120 days Slightly basophilic •cytoblasm with multiplenucleoli Round, invaginated •nucleus; may bebinucleate • Enlarges by endomitosis• Polyploid (8–64n) Life span: in marrow, unknowm Platelets (constantly produced in marrow)Life span:in blood, 7–10 days • Large, euchromatic spherical nucleus; 3–5 nucleoli • Increased size, 18–24 m • Last cell capable of mitosis • Nucleus is elongate andacquires U shape • Nucleus, 3–5 segmentsor lobes Life span:in blood, 8–12 hrin CT, 1–2 days • Difficult to Identify 55 hr • Nucleus large, slightly Large, kidney-shaped • Life span:in blood, 16 hr Life span:in CT, unknown Large,•euchromatc spherical nucleus;3–5 nucleoli Increased size, Life span:in blood, 8–12 hrin CT, unknown Large,•euchromatic spherical nucleus; 3–5nucleoli • Increased size, 18–24 m • Last cell capable of mitosis


Histology_Ross

Histology_Ross. Increased size, Life span:in blood, 8–12 hrin CT, unknown Large,•euchromatic spherical nucleus; 3–5nucleoli • Increased size, 18–24 m • Last cell capable of mitosis Life span:in blood, 8 hrin CT, unknown This table summarizes the maturation of blood cells with histologic characteristics at the various stages, maturation time, and life span after leaving the marrow. Times indicated along vertical lines are the approximate time between recognizable stages. ↑M-1 wk indicates increase in number by mitosis for 1 week before differentiation begins; MEP, megakaryocyte/erythrocyte progenitor cell; MKP, megakaryocyte progenitor cell; BMCP, basophil/mast cell progenitor cell; ErP, erythrocyte progenitor cell; NoP, neutrophil progenitor cell; MoP, monocyte progenitor cell; BaP, basophil progenitor cell; CT, connective issue.


Histology_Ross

Histology_Ross. Figure 10.19. Figure 10.19 shows the stages of blood cell development in which characteristic cell types can be identified in the light microscope in a tissue section or bone marrow smear. Hemopoiesis is initiated in an apparently random manner when individual HSCs begin to differentiate into one of the lineage-restricted progenitor cells. Progenitor cells have surface receptors for specific cytokines and growth factors, including colony-stimulating factors FIGURE 10.19 • Stages of erythrocytic and granular leukocytic differentiation with Romanovskytype stain. Shown here are normal human bone marrow cells as they basophil would typically appear in a smear. (CSFs), that influence their proliferation and maturation into a specific lineage. Development of Erythrocytes (Erythropoiesis)


Histology_Ross

Histology_Ross. (CSFs), that influence their proliferation and maturation into a specific lineage. Development of Erythrocytes (Erythropoiesis) Erythrocyte development starts from CMP cells that, under the influence of erythropoietin, IL-3, and IL-4, differentiate to MEP cells. Expression of transcription factor GATA-1is required for the terminal differentiation of MEP cells to definitive erythroid cell lineage. Under GATA-1 influence, MEP cells transform into erythropoietin-sensitive erythrocyte-committed progenitors (ErPs or CFU-E) that give rise to the proerythroblast. The first microscopically recognizable precursor cell in erythropoiesis is called the proerythroblast.


Histology_Ross

Histology_Ross. The first microscopically recognizable precursor cell in erythropoiesis is called the proerythroblast. The proerythroblast is a relatively large cell measuring 12 to 20 m in diameter. It contains a large spherical nucleus with one or two visible nucleoli. The cytoplasm shows mild basophilia because of the presence of free ribosomes. Although recognizable, the proerythroblast is not easily identified in routine bone marrow smears. The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division.


Histology_Ross

Histology_Ross. The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division. The nucleus of the basophilic erythroblast is smaller (10 to 16 m in diameter) and progressively more heterochromatic with repeated mitoses. The cytoplasm shows strong basophilia because of the large number of free ribosomes (polyribosomes) that synthesize hemoglobin. The accumulation of hemoglobin in the cell gradually changes the staining reaction of the cytoplasm so that it begins to stain with eosin. At the stage when the cytoplasm displays both acidophilia, because of the staining of hemoglobin, and basophilia, because of the staining of the ribosomes, the cell is called a polychromatophilic erythroblast. The polychromatophilic erythroblast shows both acidophilic and basophilic staining of cytoplasm.


Histology_Ross

Histology_Ross. The polychromatophilic erythroblast shows both acidophilic and basophilic staining of cytoplasm. The staining reactions of the polychromatophilic erythroblast may blend to give an overall gray or lilac color to the cytoplasm, or distinct pink (acidophilic) and purple (basophilic) regions may be resolved in the cytoplasm. The nucleus of the cell is smaller than that of the basophilic erythroblast, and coarse heterochromatin granules form a checkerboard pattern that helps identify this cell type. The orthochromatophilic erythroblast is recognized by its increased acidophilic cytoplasm and dense nucleus. The next named stage in erythropoiesis is the orthochromatophilic erythroblast (normoblast). This cell has a small, compact, densely stained nucleus. The cytoplasm is eosinophilic because of the large amount of hemoglobin (Fig. 10.20). It is only slightly larger than a mature erythrocyte. At this stage, the orthochromatophilic erythroblast is no longer capable of division.


Histology_Ross

Histology_Ross. The polychromatophilic erythrocyte has extruded its nucleus. The orthochromatic erythroblast loses its nucleus by extruding it from the cell; it is then ready to pass into the blood sinusoids of the red bone marrow. Some polyribosomes that can still synthesize hemoglobin are retained in the cell. These polyribosomes impart a slight basophilia to the otherwise eosinophilic cells; for this reason, these new cells are called polychromatophilic erythrocytes (Fig. 10.21). The polyribosomes of FIGURE 10.20 • Electron micrograph of an orthochromatophilic erythroblast (normoblast). The cell is shown just before extrusion of the nucleus. The cytoplasm contains a group of mitochondria located below the nucleus and small cytoplasmic vacuoles. The cytoplasm is relatively dense because of its hemoglobin content. The fine, dense particles scattered in the cytoplasm are ribosomes. 10,000. (Courtesy of Dr. Dorothea Zucker-Franklin.)


Histology_Ross

Histology_Ross. FIGURE 10.21 • Electron micrograph of a polychromatophilic erythrocyte (reticulocyte). The nucleus is no longer present, and the cytoplasm shows the characteristic fimbriated processes that occur just after nuclear extrusion. Mitochondria are still present, as are early and late endosomes and ribosomes. 16,500. (Courtesy of Dr. Dorothea Zucker-Franklin.) the new erythrocytes can also be demonstrated with special stains that cause the polyribosomes to clump and form a reticular network. Consequently, polychromatophilic erythrocytes are also (and more commonly) called reticulocytes. In normal blood, reticulocytes constitute about 1% to 2% of the total erythrocyte count. However, if increased numbers of erythrocytes enter the bloodstream (as during increased erythropoiesis to compensate for blood loss), the number of reticulocytes increases. Kinetics of Erythropoiesis Mitoses occur in proerythroblasts, basophilic erythroblasts, and polychromatophilic erythroblasts.


Histology_Ross

Histology_Ross. Kinetics of Erythropoiesis Mitoses occur in proerythroblasts, basophilic erythroblasts, and polychromatophilic erythroblasts. At each of these stages of development, the erythroblast divides several times. It takes about a week for the progeny of a newly formed basophilic erythroblast to reach the circulation. Nearly all erythrocytes are released into the circulation as soon as they are formed; bone marrow is not a storage site for erythrocytes. Erythrocyte formation and release are regulated by erythropoietin, a 34-kilodalton glycoprotein hormone synthesized and secreted by the kidney in response to decreased blood oxygen concentration. Erythropoietin acts on the specific receptors expressed on the surface of ErP. Erythrocytes have a life span of about 120 days in humans.


Histology_Ross

Histology_Ross. Erythrocytes have a life span of about 120 days in humans. When erythrocytes are about 4 months old, they become senescent. The macrophage system of the spleen, bone marrow, and liver phagocytoses and degrades the senescent erythrocytes. The heme and globin dissociate, and the globin is hydrolyzed to amino acids, which enter the metabolic pool for reuse. The iron on the heme is released, enters the iron-storage pool in the spleen in the form of hemosiderin or ferritin, and is stored for reuse in hemoglobin synthesis. The rest of the heme moiety of the hemoglobin molecule is partially degraded to bilirubin, bound to albumin, released into the bloodstream, and transported to the liver, where it is conjugated and excreted via the gallbladder as the bilirubin glucuronide of bile. Development of Thrombocytes (Thrombopoiesis)


Histology_Ross

Histology_Ross. Development of Thrombocytes (Thrombopoiesis) Each day bone marrow of a healthy adult produces about 1 1011 platelets, a number that can increase 10-fold in time of increased demand. The thrombocytopoiesis from the bone marrow progenitors is a complex process of cell divisions and differentiation that requires the support of interleukins, colony-stimulating factors, and hormones. Thrombocytes (platelets) develop from a bipotent megakaryocyte/erythrocyte progenitor (MEP) cell that differentiates into megakariocyte-committed progenitor (MKP) cell and finally into a megakaryocyte. Platelets are produced in the bone marrow from the same common myeloid progenitor (CMP) cells as the erythroid and myeloid series. Under the influence of granucolyte-macrophage colony-stimulating factor (GMCSF) and IL-3, CMP stem cell differentiates into a bipotent megakaryocyte/erythrocyte progenitor (MEP) cell.


Histology_Ross

Histology_Ross. Further development proceeds toward a unipotent megakariocytecommitted progenitor (MKP) cell (or CFU-Meg), which further develops into the megakaryoblast. The megakaryoblast that develops from this MKP is a large cell (about 30 m in diameter) with a nonlobed nucleus. No evidence of platelet formation is seen at this stage. Successive endomitoses occur in the megakaryoblast (i.e., chromosomes replicate), but neither karyokinesis nor cytokinesis occurs. Under stimulation by thrombopoietin, a 30-kilodalton glycoprotein hormone produced by liver and kidney, ploidy increases from 8n to 64n before chromosomal replication ceases. The cell then becomes a platelet-producing megakaryocyte, a cell measuring 50 to 70 m in diameter with a complex multilobed nucleus and scattered azurophilic granules. Both the nucleus and the cell increase in size in proportion to the ploidy of the cell. With the TEM, multiple centrioles and multiple Golgi apparatuses are also seen in these cells.


Histology_Ross

Histology_Ross. When bone marrow is examined in a smear, platelet fields are seen to fill much of the peripheral cytoplasm of the megakaryocyte. When examined with the TEM, the peripheral cytoplasm of the megakaryocyte appears to be divided into small compartments by invagination of the plasma membrane. As described above, these invaginations are the platelet demarcation channels (see Fig. 10.13). Thrombocytopenia (a low blood platelet count) is an important clinical problem in the management of patients with immune-system disorders and cancer (i.e., leukemia). It increases the risk of bleeding and in cancer patients often limits the dose of chemotherapeutic agents. Development of Granulocytes (Granulopoiesis)


Histology_Ross

Histology_Ross. Granulocytes originate from the multipotential common myeloid progenitor (CMP) stem cell, which differentiates into granulocyte/monocyte progenitors (GMPs) under the influence of cytokines such as GM-CSF, granulocyte colony-stimulating factor (G-CSF), and IL-3. GM-CSF is a cytokine secreted by endothelial cells, T cells, macrophages, mast cells, and fibroblasts. It stimulates GMP cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. The neutrophil progenitor (NoP) undergoes six morphologically identifiable stages in the process of maturation: myeloblast, promyelocyte, myelocyte, metamyelocyte, band cell, and mature neutrophil. Eosinophils and basophils undergo a morphologic maturation similar to that of neutrophils. GMP cells, when induced by GM-CSF, IL-3, and IL-5, differentiate to eosinophil progenitors (EoPs), and eventually mature to eosinophils. Lack of IL-5 causes the GMP cells to differentiate into basophil progenitors (BaPs), which produce


Histology_Ross

Histology_Ross. and IL-5, differentiate to eosinophil progenitors (EoPs), and eventually mature to eosinophils. Lack of IL-5 causes the GMP cells to differentiate into basophil progenitors (BaPs), which produce basophils. One cannot differentiate eosinophilic or basophilic precursors from neutrophilic precursors morphologically in the light microscope until the cells reach the myelocytic stage when the specific granules appear.


Histology_Ross

Histology_Ross. Myeloblasts are the first recognizable cells that begin the process of granulopoiesis. The myeloblast is the earliest microscopically recognizable neutrophil precursor cell in the bone marrow. It has a large, euchromatic, spherical nucleus with three to five nucleoli. It measures 14 to 20 m in diameter and has a large nuclear-tocytoplasmic volume. The small amount of agranular cytoplasm stains intensely basophilic. A Golgi area is often seen where the cytoplasm is unstained. The myeloblast matures into a promyelocyte. Promyelocytes are the only cells to produce azurophilic granules.


Histology_Ross

Histology_Ross. Promyelocytes are the only cells to produce azurophilic granules. The promyelocyte has a large spherical nucleus with azurophilic (primary) granules in the cytoplasm. Azurophilic granules are produced only in promyelocytes; cells in subsequent stages of granulopoiesis do not make azurophilic granules. For this reason, the number of azurophilic granules is reduced with each division of the promyelocyte and its progeny. Promyelocytes do not exhibit subtypes. Recognition of the neutrophil, eosinophil, and basophil lines is possible only in the next stage—the myelocyte—when specific (secondary) and tertiary granules begin to form. Myelocytes first exhibit specific granules.


Histology_Ross

Histology_Ross. Myelocytes first exhibit specific granules. Myelocytes begin with a more or less spherical nucleus that becomes increasingly heterochromatic and acquires a distinct indentation during subsequent divisions. Specific granules begin to emerge from the convex surface of the Golgi apparatus, whereas azurophilic granules are seen at the concave side. The significance of this separation is unclear. Myelocytes continue to divide and give rise to metamyelocytes. The metamyelocyte is the stage at which neutrophil, eosinophil, and basophil lines can be clearly identified by the presence of numerous specific granules.


Histology_Ross

Histology_Ross. The metamyelocyte is the stage at which neutrophil, eosinophil, and basophil lines can be clearly identified by the presence of numerous specific granules. A few hundred granules are present in the cytoplasm of each metamyelocyte, and the specific granules of each variety outnumber the azurophilic granules. In the neutrophil, this ratio of specific to azurophilic granules is about 2 to 1. The nucleus becomes more heterochromatic, and the indentation deepens to form a kidney bean–shaped structure. Theoretically, the metamyelocyte stage in granulopoiesis is followed by the band stage and then the segmented stage. Although these stages are obvious in the neutrophil line, they are rarely if ever observed in the eosinophil and basophil lines in which the next easily recognized stages of development are the mature eosinophil and mature basophil, respectively. In the neutrophil line, the band (stab) cell precedes development of the first distinct nuclear lobes.


Histology_Ross

Histology_Ross. In the neutrophil line, the band (stab) cell precedes development of the first distinct nuclear lobes. The nucleus of the band (stab) cell is elongated and of nearly uniform width, giving it a horseshoelike appearance. Nuclear constrictions then develop in the band neutrophil and become more prominent until two to four nuclear lobes are recognized; the cell is then considered a mature neutrophil, also called a polymorphonuclear neutrophil or segmented neutrophil. Although the percentage of band cells in the circulation is almost always low (0% to 3%), it may increase in acute or chronic inflammation and infection. Kinetics of Granulopoiesis Granulopoesis in the bone marrow takes about two weeks.


Histology_Ross

Histology_Ross. Kinetics of Granulopoiesis Granulopoesis in the bone marrow takes about two weeks. The mitotic (proliferative) phase in granulopoiesis lasts about a week and stops at the late myelocyte stage. The post-mitotic phase, characterized by cell differentiation—from metamyelocyte to mature granulocyte—also lasts about a week. The time it takes for half of the circulating segmented neutrophils to leave the peripheral blood is about 6 to 8 hours. Neutrophils leave the blood randomly—that is, a given neutrophil may circulate for only a few minutes or as long as 16 hours before entering the perivascular connective tissue (a measured half-life of circulating human neutrophils is only 8 to 12 hours). Neutrophils live for 1 to 2 days in the connective tissue, after which they are destroyed by apoptosis and are subsequently engulfed by macrophages. Also, large numbers of neutrophils are lost by migration into the lumen of the gastrointestinal tract from which they are discharged with the feces.


Histology_Ross

Histology_Ross. Bone marrow maintains a large reserve of fully functional neutrophils ready to replace or supplement circulating neutrophils at times of increased demand. In normal conditions, the bone marrow produces more than 1011 neutrophils each day. As a result of the release of neutrophils from the bone marrow, approximately 5 to 30 times as many mature and near-mature neutrophils are normally present in the bone marrow as are present in the circulation. This bone marrow reserve pool constantly releases neutrophils into the circulation and is replenished by maturing cells. The reserve neutrophils can be released abruptly in response to inﬂammation, infection, or strenuous exercise.


Histology_Ross

Histology_Ross. A reservoir of neutrophils is also present in the vascular compartment. This reserve consists of a freely circulating pool and a marginated pool, with the latter contained in small blood vessels. The neutrophils adhere to the endothelium much as they do before leaving the vasculature at sites of injury or infection (see page 275). The normally marginated neutrophils, however, loosely adhere to the endothelium through the action of selectin and can be recruited very quickly. They are in dynamic equilibrium with the circulating pool, which is approximately equal to the size of the marginated pool. The size of the reserve pool in the bone marrow and in the vascular compartment depends on the rate of granulopoiesis, the life span of the neutrophils, and the rates of migration into the bloodstream and connective tissue. The entire hemopoietic process is summarized in Table 10.3.


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Histology_Ross. Transcription factors control the fate of hemopoietic cells, whereas cytokines and local mediators regulate all stages of hemopoiesis.


Histology_Ross

Histology_Ross. Intimate interactions between HSCs and their bone marrow microenvironment work toward redefining the identity and the differentiation pathways of these multipotential stem cells. Signaling molecules from a variety of bone marrow cells initiate intracellular pathways that ultimately target a select group of synergistic and inhibitory proteins known as transcription factors. They specifically bind to promoter or enhancer regions on DNA in the affected cell. By controlling transcription of the specific genes downstream, these transcription factors trigger a cascade of genetic changes that ultimately determines the course of the cells during differentiation. In addition to identifying the various intracellular transcription factors, recent studies have identified and begun to characterize numerous signaling molecules found in the bone marrow. These include glycoproteins that act as both circulating hormones and local mediators to regulate the progress of hemopoiesis and the rate of


Histology_Ross

Histology_Ross. numerous signaling molecules found in the bone marrow. These include glycoproteins that act as both circulating hormones and local mediators to regulate the progress of hemopoiesis and the rate of differentiation of other cell types (Table 10.4). Specific hormones such as erythropoietin or thrombopoietin, discussed in a previous section, regulate erythrocyte and thrombocyte development, respectively. Other factors, collectively called colony-stimulating factors (CSFs), are subclassified according to the specific cell or group of cells that they affect. Among the recently isolated and most completely characterized factors are several that stimulate granulocyte and monocyte formation, GM-CSF, G-CSF, and macrophage colony-stimulating factor (M-CSF). Interleukins, produced by lymphocytes, act on other leukocytes and their progenitors. IL-3 is a cytokine that appears to affect most progenitor cells and even terminally differentiated cells. Any particular cytokine may act at one or more


Histology_Ross

Histology_Ross. act on other leukocytes and their progenitors. IL-3 is a cytokine that appears to affect most progenitor cells and even terminally differentiated cells. Any particular cytokine may act at one or more stages in hemopoiesis, affecting cell division, differentiation, or cell function. These factors are synthesized by many different cell types, including kidney cells (erythropoietin), liver hepatocytes (thrombopoietin), T lymphocytes (IL-3), endothelial cells (IL-6), adventitial cells in the bone marrow (IL-7), and macrophages (the CSFs that affect granulocyte and macrophage development).


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Histology_Ross. Blood FOR MATION OF B LOOD CE LLS (H E MOPOI ESIS) TABLE Hemopoietic Cytokines, Their Sources, and Target Cells 10.4 CytokineaSymbol Source Target Granulocyte-macrophage colony-stimulating factor GM-CSF T cells, endothelial cells, fibroblasts CMP, ErP, GMP, EoP, BaP, MKP, all granulocytes, erythrocytes Granulocyte colony-stimulating factor G-CSF Endothelial cells, monocytes ErP, GMP, EoP, BaP, MKP Monocyte colony-stimulating factor M-CSF Monocytes, macrophages, endothelial and adventitial cells GMP, MoP, monocytes, macrophages, osteoclasts Erythropoietin EPO Kidney, liver CMP, MEP, ErP Thrombopoietin TPO Bone marrow MKP, megakaryocytes Interferon-IFN-CD4T cells, NK cells B cells, T cells, NK cells, neutrophils, monocytes Interleukin 1 IL-1 Neutrophils, monocytes, macrophages, endothelial cells CD4T cells, B cells Interleukin 2 IL-2 CD4T cells T cells, B cells, NK cells Interleukin 3 IL-3 CD4T cells CMP, ErP, GMP, EoP, BaP, MKP, all granulocytes, erythroid cells Interleukin 4 IL-4 CD4T


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Histology_Ross. CD4T cells, B cells Interleukin 2 IL-2 CD4T cells T cells, B cells, NK cells Interleukin 3 IL-3 CD4T cells CMP, ErP, GMP, EoP, BaP, MKP, all granulocytes, erythroid cells Interleukin 4 IL-4 CD4T cells, mast cells B cells, T cells, mast cells Interleukin 5 IL-5 CD4T cells EoP, eosinophils, B cells Interleukin 6 IL-6 Endothelial cells, neutrophils, macrophages, T cells CMP, ErP, GMP, B cells, T cells, macrophages, hepatocytes Interleukin 7 IL-7 Adventitial cells of bone marrow Early pre-B, pre-T cells Interleukin 8 IL-8 Macrophages, endothelial cells T cells, neutrophils Interleukin 9 IL-9 CD4T cells CD4T cells, CMP, ErP Interleukin 10 IL-10 Macrophages, T cells T cells, B cells, NK cells Interleukin 11 IL-11 Macrophages CMP, ErP, GMP, T cells, B cells, macrophages, megakaryocytes aHemopoietic cytokines include colony-stimulating factors (CSFs), interleukins, and inhibitory factors. They are almost all glycoproteins with a basic polypeptide chain of about 20 kilodaltons. Nearly all of


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Histology_Ross. cytokines include colony-stimulating factors (CSFs), interleukins, and inhibitory factors. They are almost all glycoproteins with a basic polypeptide chain of about 20 kilodaltons. Nearly all of them act on progenitor stem cells, lineage-restricted progenitor cells, committed cells, and maturing and mature cells. Therefore, the targets listed above are target lines rather than individual target cells.


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Histology_Ross. The isolation, characterization, manufacture, and clinical testing of cytokines (proteins and peptides that are signaling compounds) in the treatment of human disease is a major activity of the rapidly growing biotechnology industry. Several hemopoietic and lymphopoietic cytokines have been manufactured by recombinant DNA technology and are already used in clinical settings. These include recombinant erythropoietin, G-CSF, GM-CSF, and IL-3; others are under active development. GM-CSF (sargramostim, Leukine) is used clinically to stimulate production of WBCs following chemotherapy and to accelerate WBC recovery following bone marrow transplantation. Development of Monocytes The multipotential CMP stem cell also gives rise to the cells that develop along the monocyte–macrophage pathway.


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Histology_Ross. The multipotential CMP stem cell also gives rise to the cells that develop along the monocyte–macrophage pathway. Monocytes are produced in the bone marrow from a GMP stem cell that can mature into a monocyte or another of the three granulocytic cell lines. In addition, GMP gives rise to dendritic cells. The proliferation and differentiation of CMP into committed GMP is controlled by IL-3. Further progression of monocyte progenitor (MoP) cell lineage depends on the continued presence of PU.1 and Egr-1 transcription factors and is stimulated by IL-3 and GM-CSF. The GMCSF also controls further differentiation into mature cells, which are then released into circulation. The transformation of MoPs to monocytes takes about 55 hours, and the monocytes remain in the circulation for only about 16 hours before emigrating to the tissues where they differentiate under influence of both GM-CSF and M-CSF into tissue macrophages. Their subsequent life span is not yet fully understood.


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Histology_Ross. Development of Lymphocytes (Lymphopoiesis) Development and lineage commitment of CLP cells depend on the expression of variety of transcription factors.


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Histology_Ross. Although lymphocytes continuously proliferate in the peripheral lymphatic organs, the bone marrow remains the primary site of lymphopoiesis in humans. Members of the Ikaros family of transcription factors play major roles in the differentiation of pluripotent HSCs toward common lymphoid progenitor (CLP) cells. Progeny of the CLP cells that express GATA-3 transcription factor are destined to become T lymphocytes. These cells that express GATA-3 leave the bone marrow as pre–T lymphocytes and travel to the thymus, where they complete their differentiation and thymic cell education (see Chapter 14, Lymphatic System). They then enter the circulation as long-lived, small T lymphocytes. Another transcription factor, Pax5, activates B-cell–specific genes in CLP cells destined to become B lymphocytes. In mammals, these cells originate in bursa-equivalent organs such as the bone marrow, gut-associated lymphatic tissue, and spleen. Although a number of transcription factors have been identified


Histology_Ross

Histology_Ross. In mammals, these cells originate in bursa-equivalent organs such as the bone marrow, gut-associated lymphatic tissue, and spleen. Although a number of transcription factors have been identified in the development of lymphoid cell lineages, little is known about factors that may influence development and lineage commitment of NK cells. NK cells most likely differentiate under the influence of IL-2 and IL-15 into immature pre–NK cells, and, after acquisition of NK–cell effector functions (ability to secrete interferon and cytotoxicity), become mature NK cells. The bone marrow is the main NK cell–producing organ. However, recent studies suggest that lymph nodes or fetal thymus may also contain NK-progenitor cells. Lymphocytes constitute as much as 30% of all nucleated cells in the bone marrow. The production and differentiation of lymphocytes are discussed in more detail in Chapter 14, Lymphatic System.


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Histology_Ross. Red bone marrow lies entirely within the spaces of bone, medullary cavity of young long bones, and spaces of spongy bone. Bone marrow consists of blood vessels, specialized units of blood vessels called sinusoids, and a spongelike network of hemopoietic cells (Fig. 10.22). The bone marrow sinusoids provide the barrier between the hemopoietic compartment and the peripheral circulation. In sections, the cells in hemopoietic compartment appear to lie in “cords” between sinusoids or between sinusoids and bone.


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Histology_Ross. The sinusoid of red bone marrow is a unique vascular unit. It occupies the position normally occupied by a capillary; that is, it is interposed between arteries and veins. It is believed to be derived from vessels that have just nourished the cortical bone tissue. The sinusoids arise from these vessels at the corticomedullary junction. The sinusoid wall consists of an endothelial lining, a basal lamina, and an incomplete covering of adventitial cells. The endothelium is a simple squamous epithelium.


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Histology_Ross. The adventitial cell, also called a reticular cell, sends sheetlike extensions into the substance of the hemopoietic cords, which provide some support for the developing blood cells. In addition, adventitial cells produce reticular fibers. They also play a role in stimulating the differentiation of developing progenitor cells into blood cells by secreting several cytokines (e.g., CSFs, IL-5, IL-7). When blood cell formation and the passage of mature blood cells into the sinusoids are active, adventitial cells and the basal lamina become displaced by mature blood cells as they approach the endothelium to enter the sinusoid from the bone marrow cavity. The bone marrow sinusoidal system is a closed circulation system; newly formed blood cells must penetrate the endothelium to enter the circulation.


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Histology_Ross. The bone marrow sinusoidal system is a closed circulation system; newly formed blood cells must penetrate the endothelium to enter the circulation. As a maturing blood cell or a megakaryocyte process pushes against an endothelial cell, the abluminal plasma membrane is pressed against the luminal plasma membrane until they fuse, thus forming a transitory opening, or aperture. The migrating cell or the megakaryocyte process literally pierces the endothelial cell. Thus, migration across the bone marrow


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Histology_Ross. FIGURE 10.22 • Bone marrow with active hemopoiesis. a. Schematic drawing of bone marrow shows the erythroblastic islets engaged in the formation of erythrocytes, megakaryocytes discharging platelets into the sinusoids, endothelial cells adjacent to a basal lamina that is sparse in places and absent where blood cells are entering the sinusoids, and adventitial or reticular cells extending from the basal lamina into the hemopoietic compartment. (Modified from Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology, 6th ed. Baltimore: Urban & Schwarzenberg, 1988.) b. Photomicrograph of bone marrow stained with H&E shows active hemopoietic centers in a close proximity to bone marrow sinusoids. 420.


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Histology_Ross. endothelium is a transcellular and not an intercellular event. Each blood cell must squeeze through an aperture to enter the lumen of a sinusoid. Similarly, a megakaryocyte process must protrude through an aperture so that the platelets can be released directly into the sinusoid lumen. The aperture is lined by the fused plasma membrane, thus maintaining the integrity of the endothelial cell during the transcellular passage. As the blood cell completes its passage through the aperture or the megakaryocyte that has extruded its platelets withdraws its process, the endothelial cell “repairs itself,” and the aperture disappears.


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Histology_Ross. In active red bone marrow, the cords of hemopoietic cells contain predominately developing blood cells and megakaryocytes. The cords also contain macrophages, mast cells, and some adipose cells. Although the cords of hemopoietic tissue appear to be unorganized, specific types of blood cells develop in nests or clusters. Each nest in which erythrocytes develop contains a macrophage. These nests are located near the sinusoid wall. Megakaryocytes are also located adjacent to the sinusoid wall, and they discharge their platelets directly into the sinusoid through apertures in the endothelium. Granulocytes develop in cell nests farther from the sinusoid wall. When mature, the granulocyte migrates to the sinusoid and enters the bloodstream. Bone marrow not active in blood cell formation contains predominately adipose cells, giving it the appearance of adipose tissue.


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Histology_Ross. Bone marrow not active in blood cell formation contains predominately adipose cells, giving it the appearance of adipose tissue. Inactive bone marrow is called yellow bone marrow. It is the chief form of bone marrow in the medullary cavity of bones in the adult that are no longer hemopoietically active, such as the long bones of the arms, legs, fingers, and toes. In these bones, the red bone marrow has been replaced completely by fat. Even in hemopoietically active bone marrow in adult humans—such as that in the ribs, vertebrae, pelvis, and shoulder girdle—about half of the bone marrow space is occupied by adipose tissue and half by hemopoietic tissue. The yellow bone marrow retains its hemopoietic potential, however, and when necessary, as after severe loss of blood, it can revert to red bone marrow, both by extension of the hemopoietic tissue into the yellow bone marrow and by repopulation of the yellow bone marrow by circulating stem cells.


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Histology_Ross.  FOLDER 10.6 Clinical Correlation: Cellularity of the Bone Marrow Bone marrow cellularity is one of the most important factors in evaluating the function of the bone marrow. The assessment of bone marrow cellularity is semiquantita-tive and represents the ratio of hemopoietic cells to adipocytes. The most reliable evaluation of cellularity is ob-tained from the microscopic examination of a bone marrow biopsy that preserves the organization of the marrow. Smear preparations are not accurate preparations with which to assess bone marrow cellularity. Bone cellularity changes with age. Normal bone marrow cellularity for a specifc age can be calculated by subtract-ing an individual’s age from 100 and adding 10%. Thus, a 30-year-old individual’s bone marrow contains between 60% and 80% of active bone-producing cells (100 – 30 70 10%); in contrast, a 70-year-old individual’s marrow is in the range of 20% to 40% (100 70 30 10%).


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Histology_Ross. As can be seen from this calculation, the number of hemopoietic cells decreases with age. Bone marrow with a normal age–specifc index is called normocellular bone marrow. Deviation from age-specifc normal indices indicates a pathologic change in the marrow. In hypocellular bone marrow, which occurs in aplastic anemia or after chemotherapy, only a small number of blood-forming cells can be found in a marrow biopsy (Fig. F10.6.1a). Thus, a 50-year-old individual with this condition might have a bone cellularity index of 10% to 20%. In the same-aged individual with acute myelogenous leukemia, the bone cellularity index might be 80% to 90%. Hypercellular bone marrow is characteristic of bone marrow affected by tumors originating from hemopoietic cells (Fig. F10.6.1b).


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Histology_Ross. FIGURE F10.6.1 • Cellularity of the bone marrow. a. This is an example of hypocellular bone marrow from an individual with aplastic anemia. The bone marrow consists largely of adipose cells and lacks normal hemopoietic activity. 120. b. This photomicrograph of bone marrow section from an individual with acute myelogenous leukemia shows hypercellular bone marrow. Note that the entire field of view next to the bony trabecula is filled with tightly packed myeloblasts. Only a few adipose cells are visible on this image. 280. (Reprinted with permission from Rubin E, Gorstein F, Schwarting R, Strayer DS. Rubin’s Pathology, 4th ed. Baltimore: Lippincott Williams & Wilkins, 2004, Fig. 20-12, Fig. 20-54.) This page intentionally left blank.


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Histology_Ross. Blood is regarded as a connective tissue, fluid in character, and consists of formed elements and plasma. Red blood cells (erythrocytes), white blood cells (leukocytes), and thrombocytes (platelets) constitute the formed elements. Collectively, they make up 45% of the blood volume. Red blood cells transport and exchange oxygen and carbon dioxide. They constitute 99% of the blood cells. White blood cells are categorized as agranulocytes and granulocytes. The agranulocytes are further classified as lymphocytes and monocytes. The granulocytes, so named for the character of the granules that they contain in their cytoplasm, consist of neutrophils, eosinophils, and basophils. Each type of white cell has a specific role in immune and protective responses in the body. They typically leave the circulation and enter the connective tissue to perform their specific role. In contrast, red blood cells function only within the vascular system. Blood platelets are responsible for blood clotting and


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Histology_Ross. and enter the connective tissue to perform their specific role. In contrast, red blood cells function only within the vascular system. Blood platelets are responsible for blood clotting and consequently have an essential role in incidents of small vessel damage. Blood smears are utilized for microscope examination and identification of relative numbers of white cells in circulating blood. The blood smear is prepared by placing a small drop of blood on a microscope slide and then smearing it across the slide with the edge of another slide. When properly executed, this method provides a uniform, single layer of blood cells that is allowed to air dry and then stained. Wright’s stain, a modified Romanovsky stain, is generally utilized. In examining the specimen under the microscope, it is useful to use a low magnifica-tion to find areas in which the blood cells have a uniform distribution like that seen in the smear on the adjacent page. Once this is accom-plished, by switching to a


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Histology_Ross. useful to use a low magnifica-tion to find areas in which the blood cells have a uniform distribution like that seen in the smear on the adjacent page. Once this is accom-plished, by switching to a higher magnification, one can identify the various types of white blood cells and, in fact, determine the relative number of each cell type. A normal cell count is as follows: neutrophils, 48.6–66.7%; eosinophils, 1.4–4.8%; basophils, 0–0.3%; lympho-cytes, 25.7–27.6%; monocytes, 8.6–9.0%.


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Histology_Ross. Blood smear, human, Wright’s stain, ×200. This low magnification photomicrograph shows part of a blood smear in which the blood cells are uniformly distributed. The greater majority of cells are red blood cells. Because of their biconcave shape, most of the red blood cells Neutrophils, blood smear, human, Wright’s stain, ×2,200. Neutrophils exhibit variation in size and nuclear morphology that is associated with age of the cell. The nucleus seen on the left is that of a neutrophil that has just passed the band stage and has recently entered the blood stream. The cell is relatively small; its cytoplasm exhibits distinctive, fine granules. The middle neutrophil is considerably larger and its cytoplasm contains more Eosinophils, blood smear, human, Wright’s stain, ×2,200.


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Histology_Ross. Eosinophils, blood smear, human, Wright’s stain, ×2,200. The eosinophils seen in these micrographs similarly represent different stages of maturity. The eosinophil at the left is relatively small and is just beginning to show lobulation. The cytoplasm is almost entirely filled with eosinophilic granules that characterize this cell type. The lighter stained area, devoid of granules, probably represents the site of the Golgi apparatus (arrow). The Basophils, blood smear, human, Wright’s stain, ×2,200.


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Histology_Ross. Basophils, blood smear, human, Wright’s stain, ×2,200. The cells shown here are basophils and also represent different stages of maturation. The basophil at the left is relatively young and small. The basophilic granules are variable in size and tend to obscure the morphology of the nucleus. Also, they are less plentiful than the granules seen in the eosinophil. The appear donut-shaped. Two white cells, both granulocytes, are evident. One cell is a neutrophil (N), the other granulocyte is an eosinophil (E). However, at this magnification, the major distinction is in the staining of their cytoplasm. Higher magnification, as in the figures below, would allow for a more precise characterization of the cell type.


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Histology_Ross. fine granules. The nucleus still exhibits a U shape, but lobulation (arrows) is becoming apparent with the constriction of the nucleus at several points. The neutrophil to the right shows greater maturity by virtue of its very distinctive lobulation. Here the lobules are connected by a very thin nuclear “bridge.” A very distinctive feature associated with the nucleus of this cell is the presence of a the Barr body (arrow), indicative of blood that has been drawn from a female. eosinophil shown in the middle is larger and its nucleus is now distinctively bilobed. At one site, three distinct granules (arrows) are evident. Note their spherical shape and their relative uniform size. The eosinophil at the right is more mature in that it displays at least three lobes. By going through focus, the eosinophil granules often appear to “light up” due to their crystalline structure.


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Histology_Ross. nucleus of the middle basophil appears to be bilobed, but the granules that lie over the nucleus, again tend to obscure the precise shape. The basophil at the right is probably more mature. The granules almost entirely obscure the nuclear shape. A few blood platelets (arrowheads) are seen in several of the micrographs. Typically they appear as small, irregular shaped bodies. PLATE 17 KEY E, eosinophil N, neutrophil Lymphocytes, blood smear, human, Wright’s stain, 7–9m. A large lymphocyte is seen in the right panel. These cells may be as ×2,150. large as 16m. The lymphocyte in the middle panel is intermediate in size. The difference in lymphocyte size is attributable mostly to the amount of


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Histology_Ross. The lymphocytes shown here vary in size, but each represents cytoplasm present. However, the nucleus also contributes to the size of the a mature cell. Circulating lymphocytes are usually described as cell, but to a lesser degree. In differential counts, lymphocyte size is small, medium, and large. A small lymphocyte is shown in the disregarded. Two platelets (arrows) are evident in the left panel. left panel. Lymphocytes in this category range in size from Monocytes, blood smear, human, Wright’s stain, ×2,150.


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Histology_Ross. left panel. Lymphocytes in this category range in size from Monocytes, blood smear, human, Wright’s stain, ×2,150. The white cells in these panels are mature monocytes. Their size ranges from approximately 13–20m, with the majority falling in the upper size range. The nucleus exhibits the most characteristic features of the monocyte, namely an indentation, which is sometimes so prominent that it exhibits a U shape as is evident in the right hand panel. The cytoplasm is very weakly basophilic. Small, azurophilic granules (lysosomes) are also characteristic of the cytoplasm and are similar to those seen in neutrophils. Platelets (arrows) are present in the left and middle panels. Bone marrow smear, human, Giemsa, ×180.


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Histology_Ross. This low magnification photomicrograph shows a bone marrow smear. This type of preparation allows for the examination of developing red and white cells. A marrow smear is made in a manner similar to that of a peripheral blood smear. A sample of bone marrow is aspirated from a bone and simply placed on a slide and spread into a thin monolayer of cells. A wide variety of cell types are present in the marrow smear. Most of the cells are developing granulocytes and developing erythrocytes. Mature erythrocytes (Ey) are also present in large numbers. They are readily identified by their lack of a nucleus and eosinophilic staining. Often intermixed with these red cells are small groups of reticulocytes. These are very young erythrocytes that contain residual ribosomes in their cytoplasm. The presence of the ribosomes slightly alters the color of the reticulocyte giving it a just perceptible blue coloration in comparison to the mature eosinophilic erythrocyte. The reticulocytes are best


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Histology_Ross. presence of the ribosomes slightly alters the color of the reticulocyte giving it a just perceptible blue coloration in comparison to the mature eosinophilic erythrocyte. The reticulocytes are best distinguished at higher magnifications. In addition, adipocytes (A) are found in variable numbers. In preparations such as this, the lipid content is lost during preparation and recognition of the cell is based on a clear or unstained round space. Another large cell that is typically present is the megakaryocyte (M). The megakaryocyte is a polyploid cell that exhibits a large and irregular nuclear profile. It is the platelet-producing cell.


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Histology_Ross. At this low magnification, it is difficult to distinguish the earlier stages of the developing cell types. However, examples of each stage of development in both cell lines are presented in the following plates. In contrast, many cells in their late stage of development, particularly in the granulocyte series, can be identified with some degree of assuredness at low magnification. For example, some band neutrophils (BN) and young eosinophils (E) can be identified by their morphology and staining characteristics.


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Histology_Ross. AG RAN U LOCYTE S AN D R E D MAR ROW KEY A, adipocytes BN, band neutrophil E, eosinophils Ey, erythrocytes M, megakaryocyte 306 Erythropoiesis is a process by which the concentration of erythrocytes in the peripheral blood stream is maintained under normal condi-tions in a steady state. Stimulation of erythroid stem cells (ErP or CFU-E) by hormonal action results in a proliferation of precursor cells that undergo differentiation and maturation in the bone marrow. The earliest recognizable precursor of the red blood cell is the proerythroblast. These cells lack hemoglobin. Their cytoplasm is basophilic and the nucleus exhibits a dense chromatin structure and several nucleoli. The Golgi apparatus, when evident, appears as a light staining area. The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division. Its nucleus is smaller. The cytoplasm shows strong basophilia due to the increasing number of ribosomes involved in hemoglobin synthesis.


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Histology_Ross. proerythroblast, from which it arises by mitotic division. Its nucleus is smaller. The cytoplasm shows strong basophilia due to the increasing number of ribosomes involved in hemoglobin synthesis. The accumulation of hemoglobin in the cell gradually changes the staining reaction of the cytoplasm so that it begins to stain with eosin. The recognizable presence of hemoglobin in the cell by virtue of its staining signifies its transition to the polychro-matophilic erythroblast. The cytoplasm in the earlier part of this stage may exhibit a blue-grey color. With time, increasing amounts of hemoglobin are synthesized and concomitantly, decreasing numbers of ribosomes are present. The nucleus of the cell is smaller than that of the basophilic erythroblast and the heterochromatin is much coarser. At the end of this stage, the nucleus has become much smaller and the cytoplasm more eosinophilic. This is the last stage in which mitosis occurs. The next definable stage is the orthochromatophilic


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Histology_Ross. At the end of this stage, the nucleus has become much smaller and the cytoplasm more eosinophilic. This is the last stage in which mitosis occurs. The next definable stage is the orthochromatophilic erythrob-last, also called normoblast. Its nucleus is smaller than earlier stages and is extremely condensed. The cytoplasm is considerably less blue leaning more to a pink or eosinophilic coloration. It is slightly larger than a mature erythrocyte. At this stage, it is no longer capable of division. In the next stage, the polychromatophilic erythrocyte, also more commonly called a reticulocyte, has lost its nucleus and is ready to pass into the blood sinusoids of the red bone marrow. Some ribosomes that can still synthesize hemoglobin are present in the cell. These ribosomes create a very slight basophilia to the cell. Comparison of this cell to typical mature erythrocytes in the marrow smear reveals a slight difference in coloration.


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Histology_Ross. Polychromatophilic erythroblast, bone marrow smear, human, Giemsa, ×2,200. Two polychromatophilic erythroblasts are seen in this micrograph. The larger and less mature cell exhibits a pronounced clumping of its chromatin. The cytoplasm is basophilic, but is considerably lighter in color than that of the basophilic erythroblast. The cytoplasm also exhibits some eosinophilia, which is indicative of hemoglobin production. The smaller cell represents a later stage of a polychromatophilic erythroblast. Note how much more dense the chromatin appears as well as how much smaller the nucleus has become. Also, the cytoplasm now favors an eosinophilia. However, some basophilia is still evident. Orthochromatophilic erythrocyte, bone marrow predominantly eosinophilhic, but still possesses a degree of basophilia. Over-smear, human, Giemsa, ×2,200. all the cell is only slightly larger than a mature erythrocyte. At this stage, the cell is no longer capable of division.


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Histology_Ross. Two orthrochromatophilic erythrocytes are seen in this micrograph. Their nuclei have become even smaller and the nucleus exhibits a compact, dense staining. The cytoplasm is Polychromatophilic erythrocyte, bone marrow smear, human, Giemsa, ×2,200. A polychromatophilic erythrocyte (PE) is seen in this micro-graph. Its nucleus has been extruded and the cytoplasm exhibits a slight basophilia. In proximity are a number of mature erythrocytes (E). Compare the coloration of the polychromatophilic erythrocyte with that of the mature red blood cells. Polychromatophilic erythrocytes can also be readily demonstrated with special stains that cause the remaining ribosomes in the cytoplasm to clump and form a visible reticular network, hence the polychromatophilic erythrocyte is also commonly called a reticulocyte.


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Histology_Ross. KEY E, erythrocytes N, nucleoli PE, polychromatophilic erythrocyte 308 Granulopoiesis is the process by which the granulocyte blood cells (neutrophils, eosinophils, and basophils) differentiate and mature in the bone marrow. The earliest recognizable stage is the myeloblast, which is followed consecutively by the promyelocyte, myelocyte, metamyelo-cyte, band cell, and finally, the mature granulocyte. It is not possible to differentiate eosinophil, basophil, or neutrophil precursors morphologi-cally until the myelocyte stage is reached – when specific granules characteristic of each cell type appear. The cells of the basophil lineage are extremely difficult to locate in a marrow smear because of the minimal number of these cells in the marrow. The myeloblast is characterized by a large euchromatic, spherical nucleus with three to five nucleoli. The cell measures 14 to 20 m in di-ameter. The cytoplasm stains deeply basophilic. The presence of a light or poorly staining area indicates a


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Histology_Ross. spherical nucleus with three to five nucleoli. The cell measures 14 to 20 m in di-ameter. The cytoplasm stains deeply basophilic. The presence of a light or poorly staining area indicates a Golgi apparatus. The promyelocyte exhibits a similar size range, 15 to 21 m; nucleoli are present. Promyelocyte cytoplasm stains similarly to that of the myoblast, but it is distin-guished by the presence of large, blue/black, primary azurophilic granules, also called nonspecific granules. The myelocyte ranges from 16 to 24 m. Its chromatin is more condensed than its precursor and nucleoli are absent. The cytoplasm of the neutrophilic myelocyte is character-ized by small, pink-to-red specific granules with some azurophilic granules present. The eosinophilic lineage has a similar appearing nucleus, but its specific granules are large. The metamyelocyte ranges from 12 to 18 m. The nuclear-cytoplasmic ratio is further decreased and the nu-cleus assumes a kidney shape. There are few azurophilic


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Histology_Ross. but its specific granules are large. The metamyelocyte ranges from 12 to 18 m. The nuclear-cytoplasmic ratio is further decreased and the nu-cleus assumes a kidney shape. There are few azurophilic granules at this stage in cells, and there is a predominance of small, pink-to-red spe-cific granules. The eosinophilic metamyelocyte shows an increased number of specific granules compared to the neutrophilic metamyelocyte. The band cells are further reduced in size, 9 to 15 m. The chromatin of the nucleus exhibits further condensation and has a horseshoe shape. In the neutrophilic band cell, the small, pink-to-red specific granules are the only granule type present. The eosinophilic band cell shows little or no change relative to the specific granules, but the nucleus exhibits a kidney shape. Mature granulocytes are shown on Plate 17.


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Histology_Ross. Myeloblast, bone marrow smear, human, Giemsa, ×2,200. The myeloblast shown here exhibits a deep blue cytoplasm with a lighter region that represents the Golgi area (G). The nucleus is-round. Several nucleoli (N) are evident. Promyelocyte, bone marrow smear, human, Giemsa, ×2,200. The promyelocyte exhibits a round nucleus with one or more nucleoli (N) present. The cytoplasm is basophilic and exhibits relatively large blue/black azurophilic granules (AG). Eosinophilic myelocyte, bone marrow smear, human, Giemsa, ×2,200. The eosinophilic myelocyte exhibits a nucleus the same as that described for the neutrophilic myelocyte. The cytoplasm, however, contains the large specific granules characteristic of eosinophils, but they are fewer in number. Neutrophilic myelocyte, bone marrow smear, human, Giemsa, ×2,200. The neutrophilic myelocyte retains the round nucleus, but nucleoli are now absent. The cytoplasm exhibits small pink-to-red specific granules.


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Histology_Ross. The neutrophilic myelocyte retains the round nucleus, but nucleoli are now absent. The cytoplasm exhibits small pink-to-red specific granules. Eosinophilic metamyelocyte, bone marrow smear, human, Giemsa, ×2,200. The eosinophilic metamyelocyte exhibits a kidney or bean sha ped nucleus. The cytoplasm exhibits numerous the characteristic eosinophilic granules that are present throughout the cytoplasm. Neutrophilic metamyelocyte, bone marrow smear, human, Giemsa, ×2,200. The neutrophilic metamyelocyte differs from its precursor by the presence of a kidney or bean shaped nucleus. The small, pink-to-red specific granules are now seen in the cytoplasm and few or no azurophilic granules are present. Eosinophilic band cell, bone marrow smear, human, Neutrophilic band cell, bone marrow smear, human, Giemsa, ×2,200. Giemsa, ×2,200.


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Histology_Ross. Eosinophilic band cell, bone marrow smear, human, Neutrophilic band cell, bone marrow smear, human, Giemsa, ×2,200. Giemsa, ×2,200. The eosinophilic band cell exhibits a horseshoe-shaped nu-The band or non-segmented neutrophil exhibits a horseshoe cleus. Its cytoplasm is filled with the eosinophilic granules. shaped nucleus with abundant small, pink-to-red specific granules in the cytoplasm. KEY AG, azurophilic granules G, Golgi apparatus N, nucleoli


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Histology_Ross. KEY AG, azurophilic granules G, Golgi apparatus N, nucleoli OVERVIEW AND CLASSIFICATION OF MUSCLE / 310 SKELETAL MUSCLE / 311 Myofibrils and Myofilaments / 314 The Contraction Cycle / 317 Motor Innervation / 322 Sensory Innervation / 324 Development, Repair, Healing, and Renewal / 325 CARDIAC MUSCLE / 327 Structure of Cardiac Muscle / 328 Injury and Repair / 331 SMOOTH MUSCLE / 331 Structure of Smooth Muscle / 331 Functional Aspects of Smooth Muscle / 335 Renewal, Repair, and Differentiation / 336 Folder 11.1 Functional Considerations: Muscle Metabolism and Ischemia / 316 Folder 11.2 Clinical Correlation: Muscular Dystrophies—Dystrophin and Dystrophin-Associated Proteins / 319 Folder 11.3 Functional Considerations: The Sliding Filament Model / 323 Folder 11.4 Clinical Correlation: Myasthenia Gravis / 325 Folder 11.5 Functional Considerations: Comparison of the Three Muscle Types / 337


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Histology_Ross. Muscle tissue is responsible for movement of the body and its parts and for changes in the size and shape of internal organs. This tissue is characterized by aggregates of specialized, elongated cells arranged in parallel array that have the primary role of contraction (Fig. 11.1). Myofilament interaction is responsible for muscle cell contraction. Two types of myofilaments are associated with cell contraction.  Thin filaments (6 to 8 nm in diameter, 1.0 m long) are composed primarily of the protein actin. Each thin filament of fibrous actin (F-actin) is a polymer formed from globular actin molecules (G-actin). Thick filaments (15 nm in diameter, 1.5 m long) are composed of the protein myosin II. Each thick filament consists of 200 to 300 myosin II molecules. The long, rod-shaped tail portion of each molecule aggregates in a regular parallel but staggered array, whereas the head portions project out in a regular helical pattern.


Histology_Ross

Histology_Ross. The two types of myofilaments occupy the bulk of the cytoplasm, which in muscle cells is also called sarcoplasm [Gr. sarcos, flesh; plasma, thing ]. Actin and myosin are also present in most other cell types (although in considerably smaller amounts), where they play a role in cellular activities such as cytokinesis, exocytosis, and cell migration. In contrast, muscle cells contain a large number of aligned contractile filaments that the cells use for the single purpose of producing mechanical work. Muscle is classified according to the appearance of the contractile cells. Two principal types of muscle are recognized:  Striated muscle, in which the cells exhibit cross-striations at the light microscope level, and  Smooth muscle, in which the cells do not exhibit cross-striations.


Histology_Ross

Histology_Ross. FIGURE 11.1 • Photomicrograph of a skeletal muscle. a. This low-magnification photomicrograph shows skeletal muscle in longitudinal section. Muscle fibers (cells) are arranged in parallel; they are vertically oriented, and the length of each fiber extends beyond the upper and lower edge of the micrograph. The fibers appear to be of different thicknesses. This is largely a reflection of the plane of section through the muscle fibers. Note on the left the epimysium, the sheath of dense connective tissue surrounding the muscle. 160. b. At higher magnification, cross-striations of the muscle fibers are readily seen. The nuclei of skeletal muscle fibers are located in the cytoplasm immediately beneath the plasma membrane. 360.


Histology_Ross

Histology_Ross. Striated muscle tissue is further subclassified on the basis of its location:  Skeletal muscle is attached to bone and is responsible for movement of the axial and appendicular skeleton and for maintenance of body position and posture. In addition, skeletal muscles of the eye (extraocular muscles) provide precise eye movement.  Visceral striated muscle is morphologically identical to skeletal muscle but is restricted to the soft tissues, namely, the tongue, pharynx, lumbar part of the diaphragm, and upper part of the esophagus. These muscles play essential roles in speech, breathing, and swallowing.  Cardiac muscle is a type of striated muscle found in the wall of the heart and in the base of the large veins that empty into the heart.


Histology_Ross

Histology_Ross.  Cardiac muscle is a type of striated muscle found in the wall of the heart and in the base of the large veins that empty into the heart. The cross-striations in striated muscle are produced largely by the specific cytoarchitectural arrangement of both thin and thick myofilaments. This arrangement is the same in all types of striated muscle cells. The main differences between skeletal muscle cells and cardiac muscle cells are in their size, shape, and organization relative to one another. Smooth muscle cells do not exhibit cross-striations because the myofilaments do not achieve the same degree of order in their arrangement. In addition, the myosin-containing myofilaments in smooth muscle are highly labile. Smooth muscle is restricted to the viscera and vascular system, the arrector pili muscles of the skin, and the intrinsic muscles of the eye. A skeletal muscle cell is a multinucleated syncytium.


Histology_Ross

Histology_Ross. A skeletal muscle cell is a multinucleated syncytium. In skeletal muscle, each muscle cell, more commonly called a muscle fiber, is actually a multinucleated syncytium. A muscle fiber is formed during development by the fusion of small, individual muscle cells called myoblasts (see page 326). When viewed in cross section, the mature multinucleated muscle fiber reveals a polygonal shape with a diameter of 10 to 100 m (Plate 21, page 340). Their length varies from almost a meter, as in the sartorius muscle of the lower limb, to as little as a few millimeters, as in the strapedius muscle of the middle ear. (Note: A muscle fiber should not be confused with a connective tissue fiber; muscle fibers are skeletal muscle cells, whereas connective tissue fibers are extracellular products of connective tissue cells.)


Histology_Ross

Histology_Ross. The nuclei of a skeletal muscle fiber are located in the cy-branches are present within the endomysium, running paral toplasm immediately beneath the plasma membrane, also called the sarcolemma. In the past, the term sarcolemma was used to describe a thick “membrane” that was thought to be the cytoplasmic boundary of the muscle cell. It is now known that the thick sarcolemma actually represents the plasma membrane of the cell, its external lamina, and the surrounding reticular lamina. A skeletal muscle consists of striated muscle fibers held together by connective tissue. The connective tissue that surrounds both individual muscle fibers and bundles of muscle fibers is essential for force transduction (Fig. 11.2). At the end of the muscle, the connective tissue continues as a tendon or some other arrangement of collagen fibers that attaches the muscle, usually to bone. A rich supply of blood vessels and nerves travels in the connective tissue.


Histology_Ross

Histology_Ross. The connective tissue associated with muscle is named according to its relationship with the muscle fibers:  Endomysium is the delicate layer of reticular fibers that im mediately surrounds individual muscle fibers (see Fig. 11.2a). Only small-diameter blood vessels and the finest neuronal lel to the muscle fibers.  Perimysium is a thicker connective tissue layer that surrounds a group of fibers to form a bundle or fascicle. Fascicles are functional units of muscle fibers that tend to work together to perform a specific function. Larger blood vessels and nerves travel in the perimysium.  Epimysium is the sheath of dense connective tissue that surrounds a collection of fascicles that constitutes the muscle (see Fig. 11.1a). The major vascular and nerve supply of the muscle penetrates the epimysium. Three types of skeletal muscle fibers—red, white, and intermediate—can be identified by color in vivo.


Histology_Ross

Histology_Ross. It has long been known that skeletal muscle fibers differ in diameter and in their natural color in vivo. The color differences are not apparent in hematoxylin and eosin (H&E)–stained sections. However, histochemical reactions based on oxidative enzyme activity, specifically the succinic dehydrogenase and nicotinamide adenine dinucleotide– tetrazolium (NADH-TR) reactions, confirm the observations seen in fresh tissue and reveal several types of skeletal muscle fibers (Fig. 11.3). The most obvious nomenclature muscle fiber muscle fascicle myofibril epimysium perimysium endomysium aa b FIGURE 11.2 • General organization of skeletal muscle. a. This freeze fracture scanning electron micrograph of an intramuscular connective tissue was obtained from the bovine semitendinous muscle. The specimen was routinely fixed for SEM and subsequently treated according to the cell maceration method with sodium hydroxide to remove muscle cells. Note a delicate honeycomb structure of the endomysium


Histology_Ross

Histology_Ross. was routinely fixed for SEM and subsequently treated according to the cell maceration method with sodium hydroxide to remove muscle cells. Note a delicate honeycomb structure of the endomysium surrounding individual muscle cells. 480. (Reprinted with permission from Nishimura T, Hattori A, Takahashi K. Structural changes in intramuscular connective tissue during the fattening of Japanese Black Cattle: effect of marbling on beef tenderization. J Anim Sci 1999; 77:93–104.) b. This schematic diagram shows the general organization of skeletal muscle and its relation to the surrounding connective tissue. Note the organization of the endomysium that surrounds individual muscle cells (fibers), the perimysium that surrounds a muscle bundle, and the epimysium that surrounds the entire muscle.


Histology_Ross

Histology_Ross. FIGURE 11.3 • Cross section of skeletal muscle fibers. This cross section of muscle fibers stained with the NADH-TR reaction demonstrates two fiber types. The deeply stained, smaller muscle fibers exhibit strong oxidative enzyme activity and correspond to the type I slow oxidative fibers. The lighter-staining, larger fibers correspond to the type IIb fast glycolytic fibers. 280. Inset. Portions of the two fiber types at higher magnification. The reaction also reveals the mitochondria that contain the oxidative enzymes. The contractile components, the myofibrils, are unstained. 550. (Original slide specimen courtesy of Dr. Scott W. Ballinger.) to describe these differences is division into red, white, and intermediate fibers. Skeletal muscle fibers are characterized by speed of contraction, enzymatic velocity, and metabolic activity.


Histology_Ross

Histology_Ross. The current classification of skeletal muscle fibers is based on contractile speed, enzymatic velocity of the fiber’s myosin ATPase reaction, and metabolic profile. The contractile speed determines how fast the fiber can contract and relax. Velocity of the myosin ATPase reaction determines the rate at which this enzyme is capable of breaking down ATP molecules during the contraction cycle. The metabolic profile indicates the capacity for ATP production by oxidative phosphorylation or glycolysis. Fibers characterized by oxidative metabolism contain large amounts of myoglobin and an increased number of mitochondria, with their constituent cytochrome electron transport complexes. Myoglobin is an oxygen-binding protein that closely resembles hemoglobin found in erythrocytes and occurs in varying amounts in muscle fibers. It provides a ready source of oxygen for muscle metabolism.


Histology_Ross

Histology_Ross. The three types of skeletal muscle fibers are type I (slow oxidative), type IIa (fast oxidative glycolytic), and type IIb (fast glycolytic) fibers. Three types of fiber are typically found in any given skeletal muscle; the proportion of each type varies according to the functional role of the muscle.


Histology_Ross

Histology_Ross.  Type I fibers or slow oxidative fibers are small fibers that appear red in fresh specimens and contain many mitochondria and large amounts of myoglobin and cytochrome complexes. Their high levels of mitochondrial oxidative enzymes are demonstrated by their strong succinic dehydrogenase and NADH-TR histochemical staining reactions as described previously (see Fig. 11.3). Type I fibers are slow-twitch, fatigue-resistant motor units (a twitch is a single, brief contraction of the muscle). These fibers have great resistance to fatigue but generate less tension than other fibers. Their myosin ATPase reaction velocity is the slowest of all of the fiber types. Type I fibers are typically found in the limb muscles of mammals and in the breast muscle of migrating birds. More importantly, they are the principal fibers of the long muscles of the back in humans, where they are particularly adapted to the long, slow contraction needed to maintain erect posture. A high percentage of these fibers


Histology_Ross

Histology_Ross. the principal fibers of the long muscles of the back in humans, where they are particularly adapted to the long, slow contraction needed to maintain erect posture. A high percentage of these fibers make up the muscles of high-endurance athletes such as marathon runners.


Histology_Ross

Histology_Ross.  Type IIa fibers or fast oxidative glycolytic fibers are the intermediate fibers seen in fresh tissue. They are of medium size with many mitochondria and a high myoglobin content. In contrast to type I fibers, type IIa fibers contain large amounts of glycogen and are capable of anaerobic glycolysis. They make up fast-twitch, fatigue-resistant motor units that generate high peak muscle tension. Athletes who have a high percentage of these fast oxidative glycolytic fibers include 400and 800-m sprinters, middledistance swimmers, and hockey players.


Histology_Ross

Histology_Ross.  Type IIb fibers or fast glycolytic fibers are large fibers that appear light pink in fresh specimens and contain less myoglobin and fewer mitochondria than type I and type IIa fibers. They have a low level of oxidative enzymes but exhibit high anaerobic enzyme activity and store a considerable amount of glycogen. These fibers are fast-twitch, fatigue-prone motor units and generate high peak muscle tension. Their myosin ATPase velocity is the fastest of all the fiber types. They also fatigue rapidly as a result of production of lactic acid. Thus, type IIb fibers are adapted for rapid contraction and precise, fine movements. They constitute most fibers of the extraocular muscles and the muscles that control the movements of the digits. These muscles have a greater number of neuromuscular junctions than do type I fibers, thus allowing more precise neuronal control of movements in these muscles. Short-distance sprinters, weight lifters, and other field athletes have a high percentage of


Histology_Ross

Histology_Ross. than do type I fibers, thus allowing more precise neuronal control of movements in these muscles. Short-distance sprinters, weight lifters, and other field athletes have a high percentage of type IIb fibers.


Histology_Ross

Histology_Ross. The structural and functional subunit of the muscle fiber is the myofibril. A muscle fiber is filled with longitudinally arrayed structural subunits called myofibrils (Fig. 11.4). Myofibrils are visible in favorable histologic preparations and are best seen in cross-sections of muscle fibers. In these sections they give the fiber a stippled appearance. Myofibrils extend the entire length of the muscle cell. Myofibrils are composed of bundles of myofilaments.


Histology_Ross

Histology_Ross. Myofibrils are composed of bundles of myofilaments. Myofilaments are the individual filamentous polymers of myosin II (thick filaments) and actin and its associated proteins (thin filaments). Myofilaments are the actual contractile elements of striated muscle. The bundles of myofilaments that make up the myofibril are surrounded by a welldeveloped smooth-surfaced endoplasmic reticulum (sER), also called the sarcoplasmic reticulum. This reticulum forms a highly organized tubular network around the contractile elements in all striated muscle cells. Mitochondria and glycogen deposits are located between the myofibrils in association with the sER. Cross-striations are the principal histologic feature of striated muscle.


Histology_Ross

Histology_Ross. Cross-striations are the principal histologic feature of striated muscle. Cross-striations are evident in H&E–stained preparations of longitudinal sections of muscle fibers. They may also be seen in unstained preparations of living muscle fibers examined with a phase contrast or polarizing microscope, in which they appear as alternating light and dark bands. These bands are termed the A band and the I band (see Fig. 11.4). In polarizing microscopy, the dark bands are birefringent (i.e., they alter the polarized light in two planes). Therefore, the dark bands, being doubly refractive, are anisotropic and are given the name A band. The light bands are monorefringent (i.e., they do not alter the plane of polarized light). Therefore, they are isotropic and are given the name I band.


Histology_Ross

Histology_Ross. Both the A and I bands are bisected by narrow regions of contrasting density (see Fig. 11.4). The light I band is bisected by a dense line, the Z line, also called the Z disc [Ger. Zwischenscheibe, between discs]. The dark A band is bisected by a less dense, or light, region called the H band [Ger. Hell, light]. Furthermore, bisecting the light H band is a narrow dense line called the M line [Ger. Mitte, middle]. The M line is best demonstrated in electron micrographs (Fig. 11.5), although in ideal H&E preparations it can be detected in the light microscope. As noted above, the cross-banding pattern of striated muscle is caused by the arrangement of the two kinds of myofilaments. To understand the mechanism of contraction, this banding pattern must be considered in functional terms. muscle fascicle (composed of muscle fibers) myofibril (composed of myofilaments) muscle fiber (composed of myofibrils) sarcomere Z lineZ line


Histology_Ross

Histology_Ross. FIGURE 11.4 • Organization of a skeletal muscle. A skeletal muscle consists of bundles of muscle fibers called fascicles. In turn, each fascicle consists of a bundle of elongate muscle fibers (cells). The muscle fiber represents a collection of longitudinal units, the myofibrils, which in turn are composed of myofilaments of two types: thick (myosin) filaments and thin (actin) filaments. The myofilaments are organized in a specific manner that imparts a cross-striated appearance to the myofibril and to the fiber. The functional unit of the myofibril is the sarcomere; it extends in both directions from one Z line to the next Z line. The A band marks the extent of the myosin filaments. Actin filaments extend from the Z line into the region of the A band, where they interdigitate with the myosin filaments as shown. The cross sections through different regions of the sarcomere are also shown (from left to right): Through thin filaments of the I band; through thick filaments of the H band;


Histology_Ross

Histology_Ross. filaments as shown. The cross sections through different regions of the sarcomere are also shown (from left to right): Through thin filaments of the I band; through thick filaments of the H band; through the center of the A band, where adjacent thick filaments are linked to form the M line; and through the A band, where thin and thick filaments overlap. Note that each thick filament is within the center of a hexagonal array of thin filaments.


Histology_Ross

Histology_Ross. FIGURE 11.5 • Electron micrograph of skeletal muscle fiber. This low-magnification electron micrograph shows the general organization of skeletal muscle fibers. Small portions of three muscle fibers in longitudinal profile are included in this micrograph. The muscle fiber on the right reveals a nucleus at its periphery. Two fibers—one in the middle and another on the left—exhibit regular profiles of myofibrils separated by a thin layer of surrounding sarcoplasm (Sr). Each repeating part of the myofibril between adjacent Z lines is a sarcomere (S). The cross-banded pattern visible on this micrograph reflects the arrangement, in register, of the individual myofibrils (M); a similar pattern found in the myofibril reflects the arrangement of myofilaments. The detailed features of a sarcomere are shown at higher magnification in Figure 11.7a. The presence of the connective tissue in the extracellular space between the fibers constitutes the endomysium of the muscle. 6,500.


Histology_Ross

Histology_Ross. The functional unit of the myofibril is the sarcomere, the segment of the myofibril between two adjacent Z lines. The sarcomere is the basic contractile unit of striated muscle. It is the portion of a myofibril between two adjacent Z lines. A sarcomere measures 2 to 3 m in relaxed mammalian muscle. It may be stretched to more than 4 m and, during extreme contraction, may be reduced to as little as 1 m (Fig. 11.6). The entire muscle cell exhibits cross-striations because sarcomeres in adjacent myofibrils are in register. The arrangement of thick and thin filaments gives rise to the density differences that produce the cross-striations of the myofibril.


Histology_Ross

Histology_Ross. The arrangement of thick and thin filaments gives rise to the density differences that produce the cross-striations of the myofibril. The myosin-containing thick filaments are about 1.5 m long and are restricted to the central portion of the sarcomere (i.e., the A band). The thin filaments attach to the Z line and extend into the A band to the edge of the H band. Portions of two sarcomeres, on either side of a Z line, constitute the I band and contain only thin filaments. In a longitudinal section of a sarcomere, the Z line appears as a zigzag structure, with matrix material, the Z matrix, bisecting the zigzag. The Z line and its matrix material anchor the thin filaments from adjacent sarcomeres to the angles of the zigzag by the actin-binding protein -actinin. These features are illustrated in Figures 11.4 and 11.6. F-actin, troponin, and tropomyosin in thin filaments and myosin II in thick filaments are the primary proteins in the contractile apparatus.


Histology_Ross

Histology_Ross. F-actin, troponin, and tropomyosin in thin filaments and myosin II in thick filaments are the primary proteins in the contractile apparatus. Thin filaments contain F-actin, tropomyosin, and troponin. Thick filaments contain only myosin II. G-actin is a small, 42-kilodalton molecule that polymerizes to form a double-stranded helix, the F-actin filament. These actin filaments are polar; all G-actin molecules are oriented in the same direction. The plus end of each filament is bound to the Z line by -actinin; the minus end extends toward the M line and is protected by an actin-capping protein. Each G-actin molecule of the thin filament has a binding site for myosin. Tropomyosin is a 64-kilodalton protein that also consists of a double helix of two polypeptides. It forms filaments that run in the groove between the F-actin molecules in the thin filament. In resting muscle, tropomyosin and its regulatory protein, the troponin complex, mask the myosin-binding site on the actin molecule.


Histology_Ross

Histology_Ross. Troponin consists of a complex of three globular subunits. Each tropomyosin molecule contains one troponin complex. Troponin-C (TnC) is the smallest subunit of the troponin complex (18 kilodaltons). It binds Ca2, the essential step in the initiation of contraction (see illustration that follows). Troponin-T (TnT), a 30-kilodalton subunit, binds to tropomyosin, anchoring the troponin complex.


Histology_Ross

Histology_Ross. FIGURE 11.6 • Sarcomeres in different functional stages. In the resting state (middle), interdigitation of thin (actin) and thick (myosin) filaments is not complete; the H and I bands are relatively wide. In the contracted state (bottom), the interdigitation of the thin and thick filaments is increased according to the degree of contraction. In the stretched state (top), the thin and thick filaments do not interact; the H and I bands are very wide. The length of the A band always remains the same and corresponds to the length of the thick filaments; the lengths of the H and I bands change, again in proportion to the degree of sarcomere relaxation or contraction. Troponin-I (TnI), also a 30-kilodalton subunit, binds to actin, thus inhibiting actin–myosin interaction.  FOLDER 11.1 Functional Considerations: Muscle Metabolism and Ischemia


Histology_Ross

Histology_Ross. Like all cells, muscle cells depend on the energy source contained in the high-energy phosphate bonds of ATP and phosphocreatine. The energy stored in these high-energy phosphate bonds comes from the metabolism of fatty acids and glucose. Glucose is the primary metabolic sub-strate in actively contracting muscle. It is derived from the general circulation as well as from the breakdown of glyco-gen, which is normally stored in the muscle fiber cyto-plasm. As much as 1% of the dry weight of skeletal and cardiac muscle may be glycogen. In rapidly contracting muscles, such as the leg muscles in running or the extraocular muscles, most of the energy for contraction is supplied by anaerobic glycolysis of stored glycogen. The buildup of intermediary metabolites from this pathway, particularly lactic acid, can produce an oxygen deficit that causes ischemic pain (cramps) in cases of extreme muscular exertion. Most of the energy used by muscle recovering from contraction or by resting muscle is


Histology_Ross

Histology_Ross. acid, can produce an oxygen deficit that causes ischemic pain (cramps) in cases of extreme muscular exertion. Most of the energy used by muscle recovering from contraction or by resting muscle is derived from oxidative phosphorylation. This process closely follows the -oxidation of fatty acids in mitochondria that liberates two carbon fragments. The oxygen needed for oxidative phosphorylation and other terminal metabolic reactions is derived from hemoglobin in circulating erythrocytes and from oxygen bound to myoglobin stored in the mus-cle cells.


Histology_Ross

Histology_Ross. Myosin II, a 510-kilodalton protein, is composed of two polypeptide heavy chains (222 kilodaltons each) and four light chains. Light chains are of two types (essential light chains [18 kilodaltons] and regulatory light chains [22 kilodaltons]), and one molecule of each type is present in association with each myosin head. The phosphorylation by myosin light chain kinase of the regulatory light chain initiates contraction in smooth muscles. Each heavy chain has a small globular head that projects at an approximately right angle at one end of the long rod-shaped molecule. This globular head has two specific binding sites, one for ATP and one for actin. It also demonstrates ATPase and motor activity. Myosin molecules in striated muscles aggregate tail to tail to form bipolar thick myosin filaments; the rod-shaped segments overlap so that the globular heads project from the thick filament. The “bare” zone in the middle of the filament (i.e., the portion of the filament that does not have


Histology_Ross

Histology_Ross. the rod-shaped segments overlap so that the globular heads project from the thick filament. The “bare” zone in the middle of the filament (i.e., the portion of the filament that does not have globular projections) is the H band. The projecting globular heads of the myosin molecules form cross-bridges between the thick and thin filaments on either side of the H band (see Fig. 11.6).


Histology_Ross

Histology_Ross. Accessory proteins maintain precise alignment of thin and thick filaments. To maintain efficiency and speed of muscle contraction, both thin and thick filaments in each myofibril must be aligned precisely and kept at an optimal distance from one another. Proteins known as accessory proteins are essential in regulating the spacing, attachment, and alignment of the myofilaments. These structural protein components of skeletal muscle fibrils constitute less than 25% of the total protein of the muscle fiber. They include the following (see also Fig. 11.7):  Titin, a large (2,500-kilodalton) protein, forms an elastic lattice that anchors thick filaments in the Z lines. Two springlike portions of the protein adjacent to the thin filaments help stabilize the centering of the myosincontaining thick filament, preventing excessive stretching of the sarcomere.