HAEMATOPOIESIS

The miracle of a life sustaining fluid---blood!

By- Francis Prathyusha, MScd endo2nd sem-2nd yr-

Blood ??
Hematopoiesis is the production of blood cells, a developmental process located in the (red)bone marrow, though some cells mature elsewhere.

Hide Notes fr slide 2
• Humans can't live without blood. Without blood, the body's organs couldn't get the oxygen and nutrients they need to survive and maintain the normal function operation; we couldn't keep warm or cool off, fight against infections, or get rid of our own metabolite waste products. Without enough blood, we'd weaken and die. Blood is a major factor in life-sustaining. A deficiency in the supply or quality of blood will impair the quality of life .Therefore, the nature of the blood is the fundamental basis for health

Immune system
• It has ability to distinguish self from nonself • It has ability to neutralize or inactivate foreign bodies • The immune system is intimately connected with the hematologic system since white blood cells (leukocytes, including B- and T-lymphocytes) are key players in the lymphoid system.

Haematopoiesis
• Ancient Greek • αἷμα, "blood"; ποιεῖν "to make" • hematopoiesis in the United States; sometimes also haemopoiesis or hemopoiesis

3 weeks fetus, yolk sac of the first to take up the responsibility of hematopoiesis

6 weeks fetus, liver starting to take up blood formation

3 months fetus , the spleen is the main blood-forming organs

hide Notes -7
• Each of the human body's blood is produced by the body itself, not from the maternal blood into the fetal blood vessels. Human embryonic hematopoietic began in the 3rd weeks, at this stage there is only little organ formation, an embryonic tissue called the yolk sac.

Hematopoiesis

The process of haematopoiesis
• The process of haematopoiesis occurs in several stages, and is controlled by at least 11hematopoietic growth factors (including the colonystimulating factors, IL-2 through IL7,G-CSF, GM-CSF, and M-CSF). • stem → progenitor → precursor → adult → mature

Hide notes 10
• The first stage involves the differentiation of a pluripotential stem cell into a committed progenitor, which is followed by maturation of committed progenitors in distinct pathways, in which precursors are partially developed, 'adolescent' cells en route to maturity.

Hematopoietic stem cells
• (HSCs) reside in the medulla of the bone (bone marrow) and have the unique ability to give rise to all of the different mature blood cell types.

Haematopoietic stem cells

Hide notes - 13
• A stem is an unspecialize cel that’s is capable of replicating or self renewing itself and developing into specilaized cells at a variety of cell types. • The product of a stem cell undersgoing division is atleast one additional stem cell that has the same capabilities of the originating cell. • 2nd generation stem cell and neuron • A prog or precrsr is an unspeIalised cell or has partial characteristics of a spec cel that is capable of undergoing cell div and yeilding 2 specialized cells .. • Every functional specialized mature blood cell is derived from a common stem cell. These stem cells are therefore, PLURIPOTENT.

Conti..
• HSCs can be isolated by using florescencelabeled antibodies to mark specific cell surface antigens and florescence activated cell sorting (FACS)instrument.

Hide notes 15
• Relationship between several of the characterized hematopoietic stem cells and early progenitor cells. Differentiation is indicated by colors; the more intense the color, the more mature the cells. Surface marker distinctions are subtle between these early cell populations, yet they have clearly distinct potentials. Stem cells can choose between self-renewal and differentiation. Progenitors can expand temporarily but always continue to differentiate (other than in certain leukemias). The mature lymphoid (T-cells, B-cells, and Natural Killer cells) and myeloerythroid cells (granulocytes, macrophages, red blood cells, and platelets) that are produced by these stem and progenitor cells

HSCs are studied using experimental techniques that permits analysis of hemopoiesis invitro and invivo

Hide notes 17
• In vivo techniques include injecting the bone marrow of normal donor mice into lethally irradiated mice whose hematopoietic cells have been destroyed. In these animals, the transplanted bone marrow cells develop colonies of hematopoietic cells in the spleen • In vitro techniques involve the use of a semisolid tissue culture medium made with a layer of cells derived from bone marrow stroma (stroma is the supporting tissue). This medium creates favorable micro environmental conditions for hematopoiesis. Data from several experiments show that under these favorable micro environmental conditions, stimulation by growth factors influences the development of the various types of blood cells

Slide 20
• HSCs are self renewing: when they proliferate, at least some of their daughter cells remain as HSCs, so the pool of stem cells does not become depleted. The other daughters of HSCs (myeloid and lymphoid progenitor cells), however can each commit to any of the alternative differentiation pathways that lead to the production of one or more specific types of blood cells, but cannot self-renew. This is one of the vital processes in the body.

1. -Erythroid cells are the oxygen carrying red blood cells. Both reticulocytes and erythrocytes are functional and are released into the blood. In fact, a reticulocyte count estimates the rate of erythropoiesis. 2. -Lymphocytes are the cornerstone of the adaptive immune system. They are derived from common lymphoid progenitors. The lymphoid lineage is primarily composed of T-cells and B-cells (types of white blood cells). This is lymphopoiesis.

All blood cells are divided into three lineages.

3. -Myelocytes, which include granulocytes, megakaryocytes and macrophages and are derived from common myeloid progenitors, are involved in such diverse roles as innate immunity, adaptive immunity, and blood clotting. This is myelopoiesis.

Hide notes 22
• Granulopoiesis (or granulocytopoiesis) is haematopoiesis of granulocytes. • Megakaryocytopoiesis is haematopoiesis of megakaryocytes.

MEDICAL APPLICATION
Growth factors have been used clinically to increase marrow cellularity and blood cell counts. The use of growth factors to stimulate the proliferation of leukocytes is opening broad new applications for clinical therapy. Potential therapeutic uses of growth factors include increasing the number of blood cells in diseases or induced conditions (eg, chemotherapy, irradiation) that result in low blood counts, increasing the efficiency of marrow transplants by enhancing cell proliferation, enhancing host defenses in patients with malignancies and infectious and immunodeficient diseases, and enhancing the treatment of parasitic diseases.

• Hematopoietic diseases are usually caused by suppression or enhancement of some undifferentiated cell production, with a consequent reduction or overproduction of hematopoietic cells. • In some diseases, however, suppression and enhancement of proliferation of more than one type of stem cell can occur, sequentially or simultaneously. In such cases, there are reduced numbers of some cell types (eg, aplastic anemia, a disorder characterized by decreased production of hematopoietic cells) coinciding with increased numbers of others (eg, leukemia, the abnormal proliferation of leukocytes)

Hide notes 27
• The initial experiments with normal bone marrow transplanted to irradiated mice established the basis for bone marrow transplantation, now frequently used to treat some disorders of hematopoietic cell proliferation

Bone Marrow

Hide notes- 30
• Under normal conditions, the production of blood cells by the bone marrow is adjusted to the body's needs, increasing its activity several-fold in a very short time. Bone marrow is found in the medullary canals of long bones and in the cavities of cancellous bones . Two types of bone marrow have been described based on their appearance on gross examination: red, or hematogenous, bone marrow, whose color is produced by the presence of blood and blood-forming cells; and yellow bone marrow, whose color is produced by the presence of a great number of adipose cells. In newborns, all bone marrow is red and is therefore active in the production of blood cells. As the child grows, most of the bone marrow changes gradually into the yellow variety. Under certain conditions, such as severe bleeding or hypoxia, yellow bone marrow is replaced by red bone marrow.

Red bone marrow
• RBM composed of a stroma (from Greek, meaning bed), hematopoietic cords, and sinusoidal capillaries. The stroma is a threedimensional meshwork of reticular cells and a delicate web of reticular fibers containing hematopoietic cells and macrophages. The stroma of bone marrow contains collagen types I and III, fibronectin, laminin, and proteoglycans. Laminin, fibronectin, and another cell-binding substance, hemonectin, interact with cell receptors to bind cells to the stroma. The sinusoids are formed by a discontinuous layer of endothelial cells.

• An external discontinuous layer of reticular cells and a loose net of reticular fibers reinforce the sinusoidal capillaries. The release of mature bone cells from the marrow is controlled by releasing factors produced in response to the needs of the organism. Several substances with releasing activity have been described, including the C3 component of complement (a series of immunologically active blood proteins), hormones (glucocorticoids and androgens), and some bacterial toxins.

Drawing showing the passage of erythrocytes, leukocytes, and platelets across a sinusoid capillary in red bone marrow. Because erythrocytes (unlike leukocytes) do not have sufficient motility to cross the wall of the sinusoid, they are believed to enter the sinusoid by a pressure gradient that exists across its wall. Leukocytes, after the action of releasing substances, cross the wall of the sinusoid by their own activity. Megakaryocytes form thin processes that cross the wall of the sinusoid and fragment at their tips, liberating the platelets.

Red Bone Marrow

Hide notes 34
• Section of active bone marrow (red bone marrow) showing some of its components. Five blood sinusoid capillaries containing many erythrocytes • Note the thinness of the blood capillary wall. Giemsa stain. Medium magnification.

Red Bone Marrow

Bone Marrow As a Source of Stem Cells for Other Tissues

MEDICAL APPLICATION
• Red bone marrow is rich in stem cells that can produce several tissues, not just blood cells. With their great potential for differentiation, these cells make it possible to generate specialized cells that are not rejected by the body because they are produced from stem cells from the marrow of the same person.

Hide notes 38
• The procedure is to collect bone marrow stem cells, cultivate them in appropriate medium to direct their differentiation to the cell type needed for transplantation, and then use the cells originating in tissue culture to replace the cells needed by the patient. In this case the donor and the recipient are the same person and the histocompatibility is complete, excluding the possibility of rejection. Even though these studies are just beginning, the results so far are promising.

Origin and differentiative stages of blood cells

The pluripotent stem cells proliferate and form 2 major cell lineage

Hide notes 40
• The proliferating stem cells form daughter cells with reduced potentiality. These unipotential or bipotential progenitor cells generate precursor cells (blasts) in which the morphological characteristics differentiate for the first time, suggesting the mature cell types they will become. In contrast, stem and progenitor cells cannot be morphologically distinguished and resemble large lymphocytes

Maturation of Erythrocytes
• A mature cell is the one that has differentiated to the stage at which it can carry out all its specific functions. • Erythrocyte maturation involves hemoglobin synthesis and formation of a small, enucleated, biconcave corpuscle.

Erythrocyte maturation

Hide notes 43
• • • • Thre r 5 intervening cell divisions between proerythrob – erythcyte 1-proery– is a large cell with loose, lacy chrmatin,nucleoli and basophilic cytoplasm. Basophilic re- basophilic cytoplasm and a condensed nucl with no visible nucleolus Polychromotophilic eryth- polyribosomes decrease, and areas of the cytoplasm begin to be filled with hemoglobin. At this stage, staining causes several colors to appear in the cell—the polychromatophilic (Gr. polys, many, + chroma, color, + philein, to love) erythroblast. orthochromatophilic - the nucleus continues to condense and no cytoplasmic basophilia is evident, resulting in a uniformly acidophilic cytoplasm—the orthochromatophilic (Gr. orthos, correct, + chroma + philein) erythroblast. At a given moment, this cell puts forth a series of cytoplasmic protrusions and expels its nucleus, encased in a thin layer of cytoplasm. The expelled nucleus is engulfed by macrophages. The remaining cell still has a small number of polyribosomes that, when treated with the dye brilliant cresyl blue, aggregate to form a stained network. This cell is the reticulocyte, which soon loses its polyribosomes and becomes a mature erythrocyte.


Hide notes 46
• Several changes takes place – cell and nuclear volume decrease – nucleoli deminish in size and disappear .. The chromatin –denser until the nucleus presents a pyknotic appearance and is finally extruded from the cell.. • Thre is a gradual decrese in the number of polyribosomes (basophilia decreases) with a simultaneous increase in the amount of hemoglobinwith in the cytoplasm .. Mitichondria and other organels grad decrese • Summary of erythrocyte maturation. The stippled part of the cytoplasm (on the left) shows the continuous increase in hemoglobin concentration from proerythroblast to erythrocyte. There is also a gradual decrease in nuclear volume and an increase in chromatin condensation, followed by extrusion of a pyknotic nucleus. The times are the average life span of each cell type. In the graph, 100% represents the highest recorded concentrations of hemoglobin and RNA.

Pararosaniline–toluidine blue (PT) stain. High magnification.

Hide notes 47
• Section of red bone marrow showing an immature megakaryocyte in the upper right corner. There is also a large group of erythropoietic cells (delimited by a broken line) and sparse immature neutrophils (arrowheads).

Electron micrograph of red bone marrow.

Hide notes 49
• Four erythroblasts in successive stages of maturation are seen (E1, E2, E3, and E4). As the cell matures, its chromatin becomes gradually condensed, the accumulation of hemoglobin increases the electron density of the cytoplasm, and the mitochondria (M) decrease in number. x11,000.

Giemsa stain. High magnification.

Hide notes 52
• A megakaryocyte in mitosis (center) surrounded by erythropoietic cells with a mitotic figure (arrowhead). The arrow indicates an erythroblast extruding its nucleus.

Granulopoiesis

Hide notes 53
• The maturation process of granulocytes takes place with cytoplasmic changes characterized by the synthesis of a number of proteins that are packed in two organelles: the azurophilic and specific granules. These proteins are produced in the rough endoplasmic reticulum and the Golgi complex in two successive stages .. • 1st stage results in the production of the azurophilic granules, which stain with basic dyes in the Wright or Giemsa methods and contain enzymes of the lysosomal system. • 2nd stage, a change in synthetic activity takes place with the production of several proteins that are packed in the specific granules. These granules contain different proteins in each of the three types of granulocytes and are utilized for the various activities of each type of granulocyte. Evidently, a shift in gene expression occurs in this process, permitting neutrophils to specialize in bacterial destruction and eosinophils and basophils to become involved in the regulation of inflammation.

Maturation of granulocytes

Hide notes 55
• The myeloblast is the most immature recognizable cell in the myeloid series. It has a finely dispersed chromatin, and nucleoli can be seen promyelocyte (L. pro, before, + Gr. myelos, marrow, + kytos, cell) is characterized by its basophilic cytoplasm and azurophilic granules. These granules contain lysosomal enzymes and myeloperoxidase. The promyelocyte gives rise to the three known types of granulocyte. The first sign of differentiation appears in the myelocytes, in which specific granules gradually increase in quantity and eventually occupy most of the cytoplasm. • These neutrophilic , basophilic, eosinophilic myelocytes mature with further condensation of the nucleus and a considerable increase in their specific granule content. Before its complete maturation, the neutrophilic granulocyte passes through an intermediate stage in which its nucleus has the form of a curved rod (band cell). This cell appears in quantity in the blood after strong stimulation of hematopoiesis.

Neutrophilic myelocyte from normal human bone marrow treated with peroxidase.

Hide notes 58
• At this stage, the cell is smaller than the promyelocyte, and the cytoplasm contains two types of granules: large, peroxidase-positive azurophilic granules (AG) and smaller specific granules (SG), which do not stain for peroxidase. Note that the peroxidase reaction product is present only in azurophilic granules and is not seen in the rough endoplasmic reticulum (RER) or Golgi cisternae (GC), which are located around the centriole (C). N, nucleus. x15,000. (Courtesy of DF Bainton.)

MEDICAL APPLICATION
• The appearance of large numbers of immature neutrophils (band cells) in the blood is called a shift to the left and is clinically significant, usually indicating bacterial infection.

Kinetics of Neutrophil Production

Hide notes 61
• • • • The medullary formation compartment can be subdivided into a mitotic compartment (3 days) and a maturation compartment (4 days). A medullary storage compartment acts as a buffer system, capable of releasing large numbers of mature neutrophils on demand. Neutrophils remain in this compartment for about 4 days. The circulating compartment consists of neutrophils suspended in plasma and circulating in blood vessels. The marginating compartment is composed of neutrophils that are present in blood but do not circulate. These neutrophils are in capillaries and are temporarily excluded from the circulation by vasoconstriction, or—especially in the lungs—they may be at the periphery of vessels, adhering to the endothelium, and not in the main bloodstream. The marginating and circulating compartments are of about equal size, and there is a constant interchange of cells between them. The half-life of a neutrophil in these two compartments is 6–7 h. The medullary formation and storage compartments together are about 10 times as large as the circulating and marginating compartments. Neutrophils and other granulocytes enter the connective tissues by passing through intercellular junctions found between endothelial cells of capillaries and postcapillary venules (diapedesis). The connective tissues form a fifth compartment for neutrophils, but its size is not known. Neutrophils reside here for 1–4 days and then die by apoptosis, regardless of whether they have performed their major function of phagocytosis.

MEDICAL APPLICATION
Thus, neutrophilia, an increase in the number of neutrophils in the circulation, does not necessarily imply an increase in neutrophil production. Intense muscular activity or the administration of epinephrine causes neutrophils in the marginating compartment to move into the circulating compartment, causing an apparent neutrophilia even though neutrophil production has not increased. The neutrophilia that occurs during the course of bacterial infections is due to an increase in production of neutrophils and a shorter duration of these cells in the medullary storage compartment. In such cases, immature forms such as band cells, neutrophilic metamyelocytes, and even myelocytes may appear in the bloodstream.

Maturation of Lymphocytes & Monocytes

Hide notes 65
• Monocytes • The monoblast is a committed progenitor cell that is almost identical to the myeloblast in its morphological characteristics. Further differentiation leads to the promonocyte, a large cell (up to 18 m in diameter) with a basophilic cytoplasm and a large, slightly indented nucleus. The chromatin is lacy, and nucleoli are evident. Promonocytes divide twice in the course of their development into monocytes. A large amount of rough endoplasmic reticulum is present, as is an extensive Golgi complex in which granule condensation can be seen to be taking place. These granules are primary lysosomes, which are observed as fine azurophilic granules in blood monocytes. Mature monocytes enter the bloodstream, circulate for about 8 h, and then enter the connective tissues, where they mature into macrophages and function

Maturation of Lymphocytes & Monocytes

Hide slide 67
• • Lymphocytes The first identifiable progenitor of lymphoid cells is the lymphoblast, a large cell capable of incorporating [3H]thymidine and dividing two or three times to form prolymphocytes. Prolymphocytes are smaller and have relatively more condensed chromatin but none of the cell-surface antigens that mark prolymphocytes as T or B lymphocytes. In the bone marrow and in the thymus, these cells synthesize cellsurface receptors characteristic of their lineage, but they are not recognizable as distinct B or T lymphocytes in routine histological procedures. Using immunocytochemical techniques makes the distinction. Circulating lymphocytes originate mainly in the thymus and the peripheral lymphoid organs (eg, spleen, lymph nodes, tonsils). However, all lymphocyte progenitor cells originate in the bone marrow. Some of these lymphocytes migrate to the thymus, where they acquire the full attributes of T lymphocytes. Subsequently, T lymphocytes populate specific regions of peripheral lymphoid organs. Other bone marrow lymphocytes differentiate into B lymphocytes in the bone marrow and then migrate to peripheral lymphoid organs, where they inhabit and multiply in their own special compartments.

MEDICAL APPLICATION
• Leukemias are malignant clones of leukocyte precursors • They occur in lymphoid tissue (lymphocytic leukemias) and in bone marrow (myelogenous and monocytic leukemias). In these diseases, there is usually a release of large numbers of immature cells into the blood. • The symptoms of leukemias are a consequence of this shift in cell proliferation, with a lack of some cell types and excessive production of others (which are often abnormal in function). The patient is usually anemic and prone to infection.

• A clinical technique that is helpful in the study of leukemias and other bone marrow disturbances is bone marrow aspiration.

Hide slide 70
• A needle is introduced through compact bone (usually the sternum), and a sample of marrow is withdrawn.. • The sample is spread on a microscope slide and stained. The use of labeled monoclonal antibodies specific to proteins in the membranes of precursor blood cells aids in identifying cell types derived from these stem cells and contributes to a more precise diagnosis of the various types of leukemia.

Origin of Platelets
• In adults, platelets originate in the red bone marrow by fragmentation of the cytoplasm of mature megakaryocytes (Gr. megas, big, + karyon, nucleus, + kytos), which, in turn, arise by differentiation of megakaryoblasts.

Megakaryoblasts & Megakaryocytes

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• • Megakaryoblasts is 15–50 m in diameter and has a large ovoid or kidney-shaped nucleus with numerous nucleoli. The nucleus becomes highly polyploid (ie, it contains up to 30 times as much DNA as a normal cell) before platelets begin to form. The cytoplasm of this cell is homogeneous and intensely basophilic. The megakaryocyte is a giant cell (35–150 m in diameter) with an irregularly lobulated nucleus, coarse chromatin, and no visible nucleoli. The cytoplasm contains numerous mitochondria, a well-developed rough endoplasmic reticulum, and an extensive Golgi complex. Platelets have conspicuous granules, originating from the Golgi complex, that contain biologically active substances, such as platelet-derived growth factor, fibroblast growth factor, von Willebrand's factor (which promotes adhesion of platelets to endothelial cells), and platelet factor IV (which stimulates blood coagulation). With maturation of the megakaryocyte, numerous invaginations of the plasma membrane ramify throughout the cytoplasm, forming the demarcation membranes (Figure 13–20). This system defines areas of a megakaryocyte's cytoplasm that shed platelets, extruding them into the circulation.

Section of bone marrow

• Section of bone marrow showing various stages of megakaryocyte development (1–4), several adipocytes (*), and blood sinusoids (arrowheads). PT stain. Medium magnification

Electron micrograph of a megakaryocyte

Hide notes 78
• Electron micrograph of a megakaryocyte showing a lobulated nucleus (N) and numerous cytoplasmic granules. The demarcation membranes are visible as tubular profiles. x4900. (Reproduced, with permission, from Junqueira LCU, Salles LMM: UltraEstrutura e Função Celular. Edgard Blücher, 1975.)

MEDICAL APPLICATION
• In certain forms of thrombocytopenic purpura, a disease in which the number of blood platelets is reduced, the platelets appear to be bound to the cytoplasm of the megakaryocytes, indicating a defect in the liberation mechanism of these corpuscles. The life span of platelets is approximately 10 days.

Summary

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