Haemopoiesis - Haemopoietic Tissue Lecture 2 PDF

HAEMATOLOGY 1 Chapter 4: HAEMOPOIESIS Dr. Futoon Al-Rawashde Department of Medical Laboratory Silences Al- Balqa' Applied University (BAU)  Definition Hematopoiesis is the production of RBCs, WBCs and platelets. Hematopoietic stem c...

HAEMATOLOGY 1 Chapter 4: HAEMOPOIESIS Dr. Futoon Al-Rawashde Department of Medical Laboratory Silences Al- Balqa' Applied University (BAU)  Definition Hematopoiesis is the production of RBCs, WBCs and platelets. Hematopoietic stem cells (HSCs) are the stem cells that differentiate and give rise to other blood cells. 2  Sites of Haematopoiesis First trimester: Yolk sac Second trimester: Liver and spleen Third trimester: Central & peripheral skeleton Adulthood: axial skeleton » Vertebrae » Sternum » Ribs » Pelvis » Skull » Scapulae » Proximal ends of the long bones, extent the proximal epiphyses of the femur and humerus. 3 Figure 4.2 Hemopoiesis in various organs before and after birth. Bone marrow sites Figure 4.6 The development of blood cells: humerus bone, cortical bone, red bone marrow, and yellow bone marrow.  Sites of Bone Marrow activity Scapulae Femur Figure 4.8A Sit es of red bone marrow activity. A: Figure 4.8B Sites of red bone marrow Child. Red bone marrow (red-shaded areas) is activity. B: Adult. Yellow marrow located throughout the skeletal system in replaces red marrow (dark-shaded children. areas) in the adult skeletal system. Red marrow activity occurs in the central portion of the skeleton.  Bone Marrow: Function ❖ Production of RBCs, WBCs, and Platelets 175 billion red cells/day 70 billion granulocytes/day (neutrophils, eosinophils, basophils) 175 billion platelets/day 7  Sites of Hematopoiesis Hematopoiesis occurs in several organs and tissues, including the bone marrow, lymph nodes, spleen, liver, and thymus. The bone marrow contains developing erythroid, myeloid, megakaryocytic, and lymphoid cells (Progenitor cells). The lymphoid development occurs into: 1. Primary lymphoid tissue (bone marrow and thymus): where T and B cells are derived. 2. Secondary lymphoid tissue (spleen and lymph nodes and gut-associated lymphoid tissue): where lymphoid cells become competent.  Bone marrow sites Bone marrow is found within the cavities of cortical bones. These cavities consist of trabecular bone resembling a honey- comb. Bone marrow present in two forms: 1. Yellow marrow: normally inactive, composed mostly of fat (adipose) tissue. 2. Red marrow: normally active in the production of most types of leukocytes, erythrocytes, and thrombocytes. During infancy and early childhood, the bone marrow consists primarily of red active marrow. Between 5 and 7 years of age, adipocytes become more abundant and begin to occupy the spaces in the long bones. Normal adult bone marrow (By age 18) has approximately equal amounts of red and yellow marrow. Red marrow in adults is found in the: sternum, skull, scapulae, vertebrae, ribs, pelvic bones, and proximal ends of the long bones, extent the proximal epiphyses of the femur and humerus.  Sites of Bone Marrow activity Scapulae Femur Figure 4.8A Sit es of red bone marrow activity. A: Figure 4.8B Sites of red bone marrow Child. Red bone marrow (red-shaded areas) is activity. B: Adult. Yellow marrow located throughout the skeletal system in replaces red marrow (dark-shaded children. areas) in the adult skeletal system. Red marrow activity occurs in the central portion of the skeleton.  Structure of the bone marrow Constitutes 3.5 - 6% of total body Almost 1,500 g in adults ❑ Consists of extravascular cords that contain: Stem cells All of the developing blood cell lineages; erythroid, myeloid, megakaryocytic, and lymphoid cells (Progenitor cells). Bone marrow matrix cells (adventitial reticular cells and adipose tissue). Reticular cells are formed on the exterior surfaces of the venous sinuses and extend long, narrow branches into the perivascular space, creating a mesh like network; this provides a supportive skeletal network for developing hematopoietic cells, macrophages, and mast cells. Macrophages (Figures 7-3 and 7-4). Adjacent to the layer of adventitial cells is a basement membrane followed by a continuous layer of endothelial cells on the luminal side of the bone marrow sinus.  Structure of the bone marrow The cords are separated from the lumen of the sinusoids by endothelial and adventitial cells and are located between the trabeculae of spongy bone. Trabeculae are projections of calcified bone radiating out from the cortical bone into the marrow space and provide support for the developing marrow. ❑ The hematopoietic cells develop in specific niches within the cords: Normoblasts develop in small clusters adjacent to the outer surfaces of the vascular sinuses (Figure 7-5); in addition, some normoblasts are found surrounding iron-laden macrophages (Figure 7-6). Megakaryocytes are located close to the vascular walls of the sinuses, which facilitates the release of platelets into the lumen of the sinusoids. Immature myeloid (granulocytic) cells through the metamyelocyte stage are located deep within the cords. As these maturing granulocytes proceed along their differentiation pathway, they move closer to the vascular sinuses. They cross the walls of the sinuses, specialized vascular spaces, and enter the circulating blood (Fig). Bone marrow Figure 4.3 Normal bone marrow biopsy. Showing distribution of hematopoietic cells, fat, and trabecular bone: erythroid precursors (E), neutrophil precursors (N), eosinophil precursors (Eo), megakaryocyte (M). Bone marrow FIGURE 4.4 Bone marrow biopsy sections demonstrate normal cellularity. Approximately 40% to 50% cellularity in an otherwise healthy 60-year-old man. FIGURE 4.5 Bone marrow biopsy sections demonstrate normal cellularity. Virtually 100% cellular marrow from a newborn boy.  Figure 7-5 Fixed and stained bone marrow biopsy specimen Figure 7-4 Fixed and stained bone marrow biopsy specimen (hematoxylin and eosin stain, 400). Hematopoietic tissue (hematoxylin and eosin stain, 100). The extravascular tissue reveals areas of granulopoiesis (lighter-staining cells) and consists of blood cell precursors and various tissue cells with erythropoiesis (darker-staining nuclei). One megakaryocyte scattered fat tissue. A normal adult bone marrow displays 50% can be seen. Adventitial cells and their processes give support tissue and 50% fat. to the hematopoietic cells; they also guard apertures of the basement membrane.  Cellular elements of bone marrow The pluripotent hematopoietic stem cell is the first in a sequence of steps of hematopoietic cell generation and maturation. Stem cells have the capacity for self-renewal, proliferation and differentiation into progenitor cells. Stem cells generate multilineage mature blood cells over the lifetime of the organism.  Cellular elements of bone marrow Hematopoietic cells can be divided into three phases according to cell maturity: 1. Primitive, multipotential hematopoietic stem cells (HSC). The most immature group capable of self-renewal and differentiation into all blood cell lines. 2.Cmmitted progenitor cells /Intermediate cells: develop into distinct cell lines. 3. Mature cells: The most developed group with specific functions.  Cellular elements of bone marrow The undifferentiated multipotential HSCs can differentiate into progenitor cells committed to either lymphoid or myeloid lineages. These lineage-specific progenitor cells consist of (1) the common lymphoid progenitor (CLP), which proliferates and differentiates into lymphocytes of T, B, and natural killer lineages; (2) the common myeloid progenitor (CMP), which proliferates and differentiates into individual granulocytic, erythrocytic, monocytic, and megakaryocytic lineages. The myeloid stem cell progresses to the progenitor colony-forming unit, granulocyte-erythrocyte-monocyte-megakaryocyte (CFU- GEMM). The CFU-GEMM can lead to the formation of CFU-granulocyte macrophage/monocyte (CFU-GM), CFU-eosinophil (CFU-Eo), CFU- basophil (CFU-B), and CFU-megakaryocyte (CFU-Meg). The resulting limited lineage-specific precursors give rise to morphologically recognizable, lineage-specific precursor cells  Cellular elements of bone marrow Stem cells (See FIGURE 4.10) Multipotential HSCs Primitive progenitor cells Common lymphoid progenitor (CLP) & common myeloid progenitor (CMP) progenitors committed Multilineage committed progenitor cells CFU- GEMM , CFU-GM, MEPs TNK Unilineage committed progenitor cells (Morphologically recognizable lineage-specific precursors ) EPs , MkPs, MPs , GPs , TPs , NKPs, BCPs Mature cells Mature blood cells Erythrocytes, neutrophils, eosinophils, basophils, monocyte, platelets, T cells, B cells and natural killer cells  Primitive Multilineage Unilineage 22 FIGURE 4.10 A General Model of Hematopoiesis  Cellular elements of bone marrow The bone marrow contain cells other than the hematopoietic cells, these include: 1. Osteoblasts: bone-forming cells 2. Osteoclasts: bone-remodeling cells. They resemble megakaryocytes 3. Adipose cells: secrete steroids that affect erythropoiesis and maintain bone integrity, and regulate the marrow volume in which active hematopoiesis occurs. 4. Endothelial cells: regulate the flow of particles entering and leaving hematopoietic spaces. 5. Macrophages: tissue-resident macrophages are called histiocytes. Macrophages clears of apoptotic cells, debris, and pathogens. Siderophages: macrophages containing iron-rich hemosiderin and ferritin. Gaucher cells: macrophages containing uncatabolized glucocerebrosides. glucocerebrosides is the lipids that accumulate in the enlarged spleen and liver of patients with Gaucher disease, a lysosomal storage disorder. 6. Mast cells: contain heparin, histamine, serotonin, and proteolytic enzymes. Increased in abnormal conditions such as chronic infections or chronic lymphoproliferative disorders.  Hematopoietic Growth Factors They are glycoprotein hormones. The major role of hematopoietic growth factors is regulating the proliferation and differentiation of HPCs and regulating the survival and function of mature blood cells. The biological effects of hematopoietic growth factors are mediated through specific binding to receptors on the surface of target cells. The cellular sources and other characteristics of growth factors are presented in Table 4.2. Hematopoietic Growth Factors  Hematopoietic Growth Factors Erythropoietin (Epo) glycoprotein hormone Produced in the peritubular complex of the kidney (90%) and the liver (10%) Controls production of red cells; induces proliferation of CFU-E Epo stimulates red cell precursors to proliferate, differentiate and produce Hb Synthesis rises when red cell level falls Epo production is stimulated by decreased O2 supply to the kidney receptor 27 Erythropoiesis 28  Regulation and Requirements for Erytropoiesis Start Normal blood oxygen levels Stimulus: Hypoxia due to decreased RBC count, decreased availability of O2 to blood, or increased tissue Increases demands for O2 O2-carrying ability of blood Erythropoietin Kidney (and liver to a Enhanced stimulates red smaller extent) releases erythropoiesis bone marrow erythropoietin increases RBC 15 count  Hematopoietic Growth Factors GM-CSF (Granulocyte – monocyte colony stimulating factor) - source is T cells, endothelial cells and fibroblasts - Stimulates production of granulocytes, monocytes G-CSF (Granulocyte – colony stimulating factor) – Secreted mainly by marrow stromal cells – Stimulates production and function of granulocytes M-CSF (Monocyte – colony stimulating factor) – Secreted mainly by Monocyte, fibroblasts and endothelial cells – Stimulates production and function of Monocyte Stem cell factor – Stimulates totipotent, pluripotent stem cells to enter differentiation pathway  Interleukins Interleukins: Protein molecules that work in conjunction with hematopoietic growth factors to stimulate proliferation and differentiation of specific cell lines. They mediate multiple, highly complex communications between various classes of WBCs. They also play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion during inflammatory and immune responses. Interleukins are cytokines that act independently or in conjunction with other interleukins to encourage hematopoietic growth. Interleukins are cell signaling molecules and a part of the cytokine super family of signaling molecules. Interleukins were first described as signals for communication between (inter—between) white blood cells (leuk—from leukocytes). Interleukins are produced and secreted by leukocytes as well as some other body cells. There are more than 50 well-known interleukins, including (IL 1, IL2, IL3…..) Examination of maturing blood cells it is important to know : 1. the sequences of cellular development by name (Table 4.3) 2. the general maturational characteristics of blood cells. 32 Erythropoiesis Erythropoiesis occurs in distinct anatomical sites called erythropoietic islands, specialized niches in which erythroid precursors proliferate, differentiate, and enucleate. Each island consists of a macrophage surrounded by a cluster of erythroblasts. Within erythroid niches, cell-cell and cell–extracellular matrix adhesion, positive and negative regulatory feedback, and central macrophage function occur. Erythroid cells account for 5% to 38% of nucleated cells in normal bone. Granulopoiesis Myeloid cells account for 23% to 85% of the nucleated cells in normal bone marrow. Granulopoiesis can be recognized as a maturational unit. Early cells are located in the cords and around the bone trabeculae. Neutrophils in the bone marrow reside in the proliferating pool and the maturation storage pool (see Chapter 14). Maturing cells spend an average of 3 to 6 days in the proliferating pool. If needed, cells from the storage pool can exit into the circulation rapidly and will have an average life span of 6 to 10 hours. 33 Lymphopoiesis Unlike other cell lines, lymphocytes and plasma cells are produced in lymphoid follicles. Lymphocytes are randomly dispersed throughout the cords (see Chapter 16). Lymphoid follicles may also be observed, especially after the age of 50. Plasma cells are located along the vascular wall. Lymphoid cells typically account for 1% to 5% of the nucleated cells in the normal bone marrow. Megakaryopoiesis Megakaryopoiesis takes place adjacent to the sinus endothelium. Megakaryocytes protrude through the vascular wall as small cytoplasmic processes to deliver platelets into the sinusoidal blood. Megakaryocytes develop into platelets in approximately 5 days. 34 35  General Cellular Characteristics a variety of features can identify the stage of maturation of stained blood cells. Two important characteristics are: 1. Overall cell size: is usually compared with the size of a mature erythrocyte. Erythrocytes and leukocytes BUT NOT megakaryocytic decrease in overall size as maturation progresses. 2. Nuclear-cytoplasmic ratio (N:C) ratio: The amount of space occupied by the nucleus in relationship to the space occupied by the cytoplasm. The size of the nucleus generally decreases as a cell matures Blast forms of erythrocytes, leukocytes, and megakaryocytes have a high (4:1) N:C ratio. As these cells mature, the ratio is reduced to 2:1 or 1:1 in most cells, except in thrombocytes, mature erythrocytes, and the lymphocyte type of leukocyte. Thrombocytes and erythrocytes lack a nucleus (anuclear), and mature 36 lymphocytes frequently retain the original 4:1 to 3:1 N:C ratio.  Nuclear Characteristics Cell identification depends largely on nuclear characteristics. Important nuclear characteristics include : 1. Chromatin pattern 2. Nuclear shape 3. Presence of nucleoli 1. Chromatin patterns The most distinctive nuclear feature of a cell in terms of maturity and cell type recognition. the overall Chromatin pattern progresses from a loose-looking arrangement to a more clumped pattern as a cell matures. Lymphocytes exhibit a smooth or homogeneous pattern of chromatin throughout development until the mature stage, when clumped heterochromatin is more obvious. Granulocytes progress from having a fi ne to a highly clumped pattern. Monocytes have a lacy pattern, which becomes fi ner as the cell matures. Erythrocytes continue to develop a more clumped pattern as maturation progresses, until the extremely dense (pyknotic) nucleus is lost (extruded) from the37 mature cell.  Nuclear Characteristics 2. Nuclear shape The shape of the nucleus in young cells is either round or oval; however, monocytes may have a slightly folded nuclear shape. In the cells that retain their nucleus as they mature, nuclear shapes become very distinctive for particular cell types. Lymphocytes usually continue to have a round or oval nucleus. Some cells may have a small cleft in the nucleus. Monocytes have a kidney bean–shaped nucleus, but folded or horseshoe shapes are common. Mature neutrophils, eosinophils, and basophils have segmented nuclei attached to one another by fine fi laments. The number of distinctive lobes ranges from two to five depending on the cell type 38  Nuclear Characteristics 3. Presence of Nucleoli The presence or absence of nucleoli is important in the identification of cells. The three cell lines of erythrocytes, leukocytes, and megakaryocytes all have nucleoli in the earliest cell stages. As cells mature, nucleoli are usually not visible. These changes in the appearance of the nucleoli are related to the rate of synthesis of ribosomal RNA The number of nucleoli varies depending on the cell type, as is shown in the following examples: Lymphoblasts have one or two nucleoli. Myeloblasts have one to five nucleoli. Monoblasts usually have one or two nucleoli but occasionally may have three or four. Erythroblasts may not have any nucleoli or may have up to two nucleoli that may stain darker than in other types of blast cells. Megakaryoblasts typically have one to five nucleoli. 39  Cytoplasmic Characteristics A variety of cytoplasmic features aid in the microscopic identification of cell maturity and type. These features include: 1. Staining color and intensity 2. Granulation 3. Shape 4. Quantity of cytoplasm 5. Vacuolization 6. Inclusion bodies 40  Cytoplasmic Characteristics 1. Staining color and intensity The overall color and intensity of staining in a Wright-stained blood smear vary with cell maturity and type. In general, cytoplasmic color progresses from darker blue (indicating active protein synthesis) in younger cells to lighter blue or pink in mature cells. Most early cells have a medium-blue cytoplasm. Immature erythrocytes have a very distinctive dark-blue cytoplasm that becomes paler and gray looking as the cell synthesizes hemoglobin. As mature cells, lymphocytes are usually noted for their pale sky-blue cytoplasmic color. Variations in cytoplasmic color develop in many cells because of abnormalities or the presence of granules. 41  Cytoplasmic Characteristics 2. Granulation The presence, size, and color of granules are important in cellular identification. In general, granulation progresses from no granules to nonspecific granulation to specific granulation. The earliest, blast forms of leukocytes and megakaryocytes do not have granules, and erythrocytes never exhibit granulation throughout their life cycle. The granulocytic cell line of leukocytes is noted for distinctive granulation. Granules vary in several ways: 1. In size, ranging from very fine to coarse. 2. In color, including red (azurophilic), blue (basophilic), and orange (eosinophilic) 3. In the amount of granulation per cell. 42  Cytoplasmic Characteristics 3. Cytoplasmic Shape The cytoplasmic outline or shape is useful in cellular identification. The most distinctive variation in cytoplasmic shape occurs in some blast forms, monocytes, and megakaryocytes. Pseudopods may be observed in mature monocytes and in some leukocyte blast forms. The megakaryocyte develops a more irregular outline as the cell matures. 4. Quantity of Cytoplasm In some cell types, the actual quantity of cytoplasm increases with age. The megakaryocyte, in particular, develops extensive quantities of cytoplasm. Abnormalities of lymphocytes frequently demonstrate increased amounts of cytoplasm. 43  Cytoplasmic Characteristics 5. Vacuolization Monocytes are frequently noted for having vacuoles throughout their life cycle and under normal conditions. Except for the monocyte, vacuolization of the cytoplasm is commonly seen in older cells and in abnormal conditions. Anticoagulants can also produce vacuoles as artifacts if the blood is stored for a longer-than-acceptable period. Severe bacterial infections, viral infections (e.g., infectious mononucleosis), and malignancies may produce a remarkable number of vacuoles in various leukocyte types. 44  Cytoplasmic Characteristics 6. Inclusion Bodies Cytoplasmic inclusions such as Auer bodies or Auer rods in myelocytic or monocytic blast forms or ingested particles are important to observe because they aid in the identification of cell types. Various erythrocytic inclusions and leukocytic inclusions are indicative of specific diseases. Some types of inclusions may be seen on a Wright-stained blood smear, but other inclusions (such as iron particles) require special staining techniques. 45 46 47