Experiment No.

5 Hematopoiesis

Introduction In postnatal life in humans, erythrocytes, granulocytes, monocytes, and platelets are normally produced only in the bone marrow. Lymphocytes are produced in the secondary lymphoid organs, as well as in the bone marrow and thymus gland. Most bone marrow cells are morphologically recognizable precursors of granulocytes or erythrocytes with smaller numbers of platelet precursors (megakaryocytes), lymphocytes, monocytes, macrophages, stromal cells (endothelial cells, fibroblasts, osteoblasts, and osteoclasts), eosinophils, plasma cells, basophils, mast cells, and blasts. The latter include hematopoietic stem cells (HSCs), which are capable of self-renewal and differentiation; and progenitors, which differentiate along a specific pathway. Blood cell production (hematopoiesis) encompasses the overall interactions of cellular proliferation, differentiation, morphogenesis, functional maturation and death. Basically, the development pattern of embryogenesis that involves cellular differentiation is an orderly process of many morphologic and functional changes for which the regulatory mechanism is not understood. Many of the developmental patterns occur simultaneously in the embryo and fetus. Red bone marrow is a highly vascularized connective tissue located in the microscopic spaces between trabeculae of spongy bone tissue. It is present chiefly in bones of the axial skeleton, pectoral an pelvic girdles, and the proximal epiphyses of the humerus and femur. About 0.05 0.1% of red bone marrow cells are derived from mesenchyme and are called pluripotent stem cells (ploo-RIP-o -tent; pluri- _ several) or hemocytoblasts. These cells have the capacity to develop into many different types of cells. In newborns all bone marrow is red and thus active in blood cell production. As an individual grows and in adulthood, the rate of blood cell formation decreases; the red bone marrow in the medullary (marrow) cavity of long bones becomes inactive and is replaced by yellow bone marrow, which is largely fat cells. Under certain conditions, such as severe bleeding, yellow bone marrow can revert to red bone marrow by extension of red bone marrow into yellow bone marrow and repopulation of yellow bone marrow by pluripotent stem cells. Stem cells in red bone marrow reproduce themselves, proliferate, and differentiate into cells that give rise to blood cells, macrophages, reticular cells, mast cells, and adipocytes. Some of the stem cells can also form osteoblasts, chondroblasts, and muscle cells, and someday may be used as a source of bone, cartilage, and muscular tissue for tissue and organ replacement. The reticular cells produce reticular fibers, which form the stroma (framework) that supports red bone marrow cells. Once blood cells are produced in red bone marrow, they enter the bloodstream through sinusoids (also called sinuses), enlarged and leaky capillaries that surround red bone marrow cells and fibers. With the exception of lymphocytes, formed elements do not divide once they leave red bone marrow. In order to form blood cells, pluripotent stem cells in red bone marrow produce two further types of stem cells, which have the capacity to develop into several types of cells. These stem cells are called myeloid stem cells and lymphoid stem cells. Myeloid stem cells begin their development in red bone marrow and give rise to red blood cells, platelets, monocytes, neutrophils, eosinophils, and basophils. Lymphoid stem cells begin their development in red bone marrow but complete it in
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lymphatic tissues; they give rise to lymphocytes. Although the various stem cells have distinctive cell identity markers in their plasma membranes, they cannot be distinguished histologically and resemble lymphocytes. During hemopoiesis, some of the myeloid stem cells differentiate into progenitor cells (proJEN-i-tor). Other myeloid stem cells and the lymphoid stem cells develop directly into precursor cells (described shortly). Progenitor cells are no longer capable of reproducing themselves and are committed to giving rise to more specific elements of blood. Some progenitor cells are known as colony-forming units (CFUs). Following the CFU designation is an abbreviation that indicates the mature elements in blood that they will produce: CFU E ultimately produces erythrocytes (red blood cells), CFU Meg produces megakaryocytes, the source of platelets, and CFU GM ultimately produces granulocytes (specifically, neutrophils) and monocytes. Progenitor cells, like stem cells, resemble lymphocytes and cannot be distinguished by their microscopic appearance alone. In the next generation, the cells are called precursor cells, also known as blasts. Over several cell divisions they develop into the actual formed elements of blood. For example, monoblasts develop into monocytes, eosinophilic myeloblasts develop into eosinophils, and so on. Precursor cells have recognizable microscopic appearances. Several hormones called hemopoietic growth factors regulate the differentiation and proliferation of particular progenitor cells. Erythropoietin (e-rith_-ro-POY-e-tin) or EPO increases the number of red blood cell precursors. EPO is produced primarily cells in the kidneys that lie between the kidney tubules (peritubular interstitial cells). With renal failure, EPO release slows and RBC production is inadequate. Thrombopoietin (throm_-bo-POY-e -tin) or TPO is a hormone produced by the liver that stimulates the formation of platelets (thrombocytes) from megakaryocytes. Several different cytokines regulate development of different blood cell types. Cytokines are small glycoproteins that are typically produced by cells such as red bone marrow cells, leukocytes, macrophages, fibroblasts, and endothelial cells. They generally act as local hormones (autocrines or paracrines; see Chapter 18). Cytokines stimulate proliferation of progenitor cells in red bone marrow and regulate the activities of cells involved in nonspecific defenses (such as phagocytes) and immune responses (such as B cells and T cells). Two important families of cytokines that stimulate white blood cell formation are colonystimulating factors (CSFs) and interleukins. Hemopoietic growth factors made available through recombinant DNA technology hold tremendous potential for medical uses when a person s natural ability to form new blood cells is diminished or defective. The artificial form of erythropoietin (Epoetin alfa) is very effective in treating the diminished red blood cell production that accompanies end-stage kidney disease. Granulocytemacrophage colony-stimulating factor and granulocyte CSF are given to stimulate white blood cell formation in cancer patients who are undergoing chemotherapy, which kills red bone marrow cells as well as cancer cells because both cell types are undergoing mitosis. (Recall that white blood cells help protect against disease.) Thrombopoietin shows great promise for preventing the depletion of platelets, which are needed to help blood clot, during chemotherapy. CSFs and thrombopoietin also improve the outcome of patients who receive bone marrow transplants. Hemopoietic growth factors are also used to treat thrombocytopenia in neonates, other clotting disorders, and various types of anemia. Research on these medications is ongoing and shows a great deal of promise.

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3 Developmental Periods Mesoblastic Period Hematopoiesis begins during embryonic development in the blood islands of the yolk sac. These blood islands are first detected at approximately 19-20 days of gestation, and their appearance marks the beginning of the mesoblastic period of hematopoiesis. Most likely, blood islands develop from the mesodermal embryonic layer of the yolk sac. The blood islands remain active only through the 18 to 12 week of gestation and are primarily responsible for red cell production (erythropoiesis), although areas of leukopoiesis and platelet production (megakaryopoiesis) have been identified. Hepatic Period Definitive morphologic hematopoiesis begins during the fifth to sixth weeks of gestation in the liver and marks the beginning of the hepatic period of hematopoiesis. At this point cells appear that are morphologically identifiable, the liver remains the primary site of blood cell production until the sixth month and may continue to produce blood cells, although to a much smaller degree, until the first or second week after birth. Because liver hematopoiesis is intravascular, infants normally have a few circulating nucleated red cells. At first the fetal liver is primarily an erythroid organ and produces red cells containing fetal hemoglobin. The production of granulocytes and lymphocytes is minimal in the fetal liver. During the hepatic period, the spleen, thymus, and lymph nodes also become active in blood cell production. Myeloid period During the fifth month of gestation, bone cavities begin to form, and bone marrow begins to assume responsibility as the main site of blood cell production. This marks the beginning of the myeloid period. During this time hemoglobin A1 consisting of two alpha and two beta globin chains, begins to appear and gradually increase in concentration. Most adult hemoglobin is Hb A1. After the first three weeks postpartum, the bone marrow become the only normal site of blood cell production and remains so throughout life. During the first few years of life, a delicate balance exists between developing bone marrow space and developing infant s need for blood cells. Erythropoiesis: Production of RBCs Erythropoiesis (e-rith_-ro-poy-EE -sis), the production of RBCs, starts in the red bone marrow with a precursor cell called a proerythroblast. The proerythroblast divides several times, producing cells that begin to synthesize hemoglobin. Ultimately, a cell near the end of the development sequence ejects its nucleus and becomes a reticulocyte (re-TIKu -lo-s t). Loss of the nucleus causes the center of the cell to indent, producing the red blood cell s distinctive biconcave shape. Reticulocytes retain some mitochondria, ribosomes, and endoplasmic reticulum. They pass from red bone marrow into the bloodstream by squeezing between the endothelial cells of blood capillaries. Reticulocytes develop into mature red blood cells within 1 to 2 days after their release from red bone marrow. Normally, erythropoiesis and red blood cell destruction proceed at roughly the same pace. If the oxygen-carrying capacity of the blood falls because erythropoiesis is not keeping up with RBC destruction, a negative feedback system steps up RBC production. The controlled condition is the
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amount of oxygen delivered to body tissues. Cellular oxygen deficiency, called hypoxia (h -POKS-e -a), may occur if too little oxygen enters the blood. For example, the lower oxygen content of air at high altitudes reduces the amount of oxygen in the blood. Oxygen delivery may also fall due to anemia, which has many causes: Lack of iron, lack of certain amino acids, and lack of vitamin B12 are but a few. Circulatory problems that reduce blood flow to tissues may also reduce oxygen delivery. Whatever the cause, hypoxia stimulates the kidneys to step up the release of erythropoietin, which speeds the development of proerythroblasts into reticulocytes in the red bone marrow. As the number of circulating RBCs increases, more oxygen can be delivered to body tissues. Premature newborns often exhibit anemia, due in part to inadequate production of erythropoietin. During the first weeks after birth, the liver, not the kidneys, produces most EPO. Because the liver is less sensitive than the kidneys to hypoxia, newborns have a smaller EPO response to anemia than do adults. Because fetal hemoglobin (hemoglobin present at birth) carries up to 30% more oxygen, the loss of fetal hemoglobin, due to insufficient erythropoietin production, makes the anemia worse.

Leukopoiesis Leukopoiesis, the process of making leukocytes, is stimulated by various colony-stimulating factors (CSFs), hormones produced by mature white blood cells. The development of each kind of white blood cell begins with the division of hemopoietic stem cells into one of the following blast cells. y y y Myeoblasts divide to form eosinophilic, neutrophilic, or basophilic myelocytes, which lead to the development of the three kinds of granulocytes. Monoblasts lead to the development of monocytes. Lymphoblasts lead to the development of lymphocytes.

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Myeloblast

Promyelocyte

Myelocyte

Size

15-20 um

15-20 um

12-18 um Moderate in amount Few non-specific granules Small amount of specific granules begin to appear (N,E,B) Oval or round CP coarser & more clumped Centrally located N/C ratio: 1:1 No nucleolus

Cytoplasm

Small in amount Moderate blue in color Smooth & granular

Increase in amount Pale blue With few nonspecific granules Oval or round CP coarser and more clumped Centrally located of the cell N/C ratio: 3:1 2-3 nucleoli

Nucleus

Round or sl oval Fine CP Reddish purple 4/5 of the cell N/C ratio: 4:1 With 2-5 nucleoli

Metamyelocyte

Band Cell

Segmenters

Size

10-15 um Moderate to abundant amount Few non-specific granules Full specific granules (N,E,B) Indented or kidneyshaped CP is coarse & clumped Stains dark blue

9-15 um

12-18 um N-pink to rose-purple granules E-reddish orange B-dark purple to blue black granules N: 2-5 lobes connected by thin nuclear filament E: 2 lobes B: unsegmented or 2 lobes

Cytoplasm

Same as metamyelocyte

Nucleus

Elongated or bandshaped Deeply indented CP is coarse & clumped

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Lymphocytic Series

Lymphoblasst

Prolymphocyte

Mature Lymphocyte

Size

10-18 um

Cytoplasm

Smooth, no granules Moderate to dark blue (dark to lighter near the nucleus)

Nucleus

CP is coarse Round or oval 1-2 nucleoli N/C ratio: 4:1

Small 8-10 um Medium 10-12 um 10-18 um Large 12-16 um Small: thin rim around the nucleus (dark Usually non-granular, blue) but with few Medium: more occasional abundant (moderate azurophilic granules blue) Large: abundant (very pale blue) S: CP is clumped, sl indented CP is more clumped M: CP is dense, sl and coarser indented Round or slightly L: CP is coarse, sl indented indented, 1-2 nucleoli eccentrically located No nucleoli

Monocytic Series

Monoblast

Promonocyte

Monocyte

Size

12-20 um

14-18 um Blue-gray Contains few fine azurophilic ganules Ground glass appearance Moderate in amount

12-20 um Blue-gray Many azurophilic granules Ground glass appearance Vacuoles present Abundant in amount

Cytoplasm

Moderately basophilic to blue-gray Non-granular

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Nucleus

Ovoid or round in shape Light blue-purple With fine, lacey chromatin 1-2 nucleoli N/C ratio: 4:1

Oval or may have 1 single fold Fine CP 1-2 nucleoli N/C ratio: 3:1

Round, kidney-shaped with lobulations folded over the top, showing brain-like convolutions CP not clumped, lacey and fine No nucleoli

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Thrombopoiesis Thrombopoiesis, the process of making platelets, begins with the formation of megakaryoblasts from hemopoietic stem cells.The megakaryoblasts divide without cytokinesis to become megakaryocytes, huge cells with a large, multilobed nucleus. The megakaryocytes then fragment into segments as the plasma membrane infolds into the cytoplasm.

Megakaryoblast

Promegakaryocyte

Granular Megakaryocyte 30-90 um

Size

20-50 um Varying shade of blue W/ small pseudopods Small in amount (narrow band) Non-granular Round, oval or kidney shaped Fine CP Multiple nucleoli N/C ratio: 10:1

20-60 um

Cytoplasm

Less basophilic Abundant Granules start to form

Pinkish blue Abundant With irregular border With fine granules

Nucleus

Irregular in shape with sl lobulation CP is coarse Multiple nucleoli N/C ratio: 4:1 to 7:1

Multilobulated CP is coarser No nuceloli N/C ratio: 2:1

Mature Megakaryocyte Size 40-120 um With coarse clumps of granules aggregating into small bundles, which bud off

Platelets or Thrombocytes 1-4 um Light to purple Very granular 2 parts: chromomere, hylamore

Cytoplasm

Nucleus

Multiloblated No nucleoli N/C ratio: 1:1

none

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Questions for Research: 1. Describe changes that takes place in the following as the cell matures: 1.1 Cell size  As the cell matures, the cell size decreases 1.2 Nucleus (erythropoiesis)  In erythropoiesis, as the cell matures the nucleus becomes smaller and finally extruded. 1.3 Nucleus (leukopoiesis)  In leukopoiesis, as the cell matures the nucleus from large to small and then undergoes segmentation and lobulation 1.4 Amount of cytoplasm  The amount of cytoplasm becomes larger due to the change in size of the nucleus from large to small. 1.5 Basophilia of cytoplasm  In the case of the RBC, the cytoplasm becomes less basophilic due to RNA degeneration and production of hemoglobin. 1.6 Staining characteristics of the cytoplasm  In the early stages of cell maturation, the cytoplasm of the cell is nonspecific to any stain but as the cell matures, it starts of develop affinity to a particular stain. 1.7 Chromatin pattern  As the cell matures, the chromatin pattern changes from fine & delicate to course and clumped. 2. Enumerate sites where adult hematopoiesis may take place.  At the age of 18 years old, the only active hematopoietic sites are in the pelvis, vertebrae, ribs, sternum, skull and proximal extremities of the long bone.
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3. Newborn babies would have higher blood cell count than adults. Why?  In newborns all bone marrow is red and thus active in blood cell production. As an individual grows and in adulthood, the rate of blood cell formation decreases; the red bone marrow in the medullary (marrow) cavity of long bones becomes inactive and is replaced by yellow bone marrow, which is largely fat cells. Under certain conditions, such as severe bleeding, yellow bone marrow can revert to red bone marrow by extension of red bone marrow into yellow bone marrow and repopulation of yellow bone marrow by pluripotent stem cells. 4. Enumerate 3 stages of intrauterine hematopoiesis and identify the organ involved in blood cell formation.  Mesoblastic stage Blood islands of the yolk sac  Hepatic stage liver is the main erythroid organ  Myeloid stage - bone marrow is the site of blood cell production 5. Differentiate extramedullary and intramedullary hematopioesis.  Extramedullary hematopoiesis is the production of blood cells from the different organs aside from the bone marrow. Examples of which are the lymph nodes and the spleen. While Intramedullary hematopoiesis is the production of blood cells from the bone marrow alone. 6. Identify one condition that may be associated with an increase of: 6.1 Erythropoiesis Polycythemia vera 6.2 Leukopoiesis - Leukemia 6.3 Thrombopoiesis - Thrombosis References:
Lotspeich-Steininger, Cheryl. Clinical Hematology Principles, Procedures, Correlations nd Henry, John Bernard. Clinical Diagnosis and Management by Laboratory Methods 22 edition th Tortora, Gerard. Principles of Anatomy & Physiology 12 edition G. Moore et. Al, Haematology, 2010 http://www.scribd.com/doc/50704881/110/Leukopoiesis

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