LESSON 14 Blood text_db0ea683a4a0a82cad0f9b158c7c66df.pdf

_____________ LESSON 14 _____________ BLOOD AND BONE MARROW INTRODUCTION Blood is a fluid tissue, considered a special variety of connective tissue, consisting of cells and a fluid extracellular matrix called plasma. In turn, plasma, in an analogy with the typical connective tissue, is composed of...

_____________ LESSON 14 _____________ BLOOD AND BONE MARROW INTRODUCTION Blood is a fluid tissue, considered a special variety of connective tissue, consisting of cells and a fluid extracellular matrix called plasma. In turn, plasma, in an analogy with the typical connective tissue, is composed of fibers (fibrinogen, fibrillar protein involved in blood coagulation) and blood serum (defibrinated plasma) that would act as ground substance. The functions of blood are varied and consist, essentially, in 1. transport: gases such as O2 and CO2 to the cells and lungs respectively, nutrients, cellular metabolites, many transmitters (e.g. hormones), 2. regulation of body temperature and acid-base and osmotic balance of body fluids and 3. defence: provides both the leukocytes as many defensive substances (antibodies, cytokines, etc...). II. BLOOD COMPONENTS 1) Blood plasma. 2) Cells: there are 3 blood cell lines: 2.1. Erythrocytes 2.2. Platelets 2.3. Leukocytes: 2.3.1. Granulocytes: polymorphonuclear neutrophils, basophils and eosinophils. 2.3.2. Agranulocytes: monocytes and lymphocytes. 1. Blood plasma Plasma constitutes the extracellular matrix of blood, it is fluid and represents approximately 55% of the total blood volume. When separated from cells by sedimentation or centrifugation it appears as a translucent liquid. In routine histological sections it is seen as a homogeneous and acidophilic substance located inside the blood vessels. 1 Plasma is made up of 90% water and 10% substances in solution, of which 9 % are proteins, among them albumin is the most abundant. This protein maintains 70% of the plasma colloid osmotic pressure, acting as a transporter for numerous substances. The α and β-globulins also act as transporters, while the γ-globulins are the antibodies. The fibrinogen is a fibrillar protein that polymerizes in fibrin when endothelial injury occurs, initiating hemostasis (blood coagulation). Likewise, there are other proteins dissolved in the blood such as prothrombin that intervenes in coagulation, complement factors that intervene in inflammation, and lipoproteins with transport function. The remaining 1% is made up of inorganic salts, ions, nutrients, hormones, metabolites, nitrogenous compounds ... etc. 2. Blood cells Hematopoiesis Hematopoiesis consists of the process of formation of all blood cells. This begins in embryonic stages in the mesenchyme of the yolk sac, in fetal periods it takes place in the liver and spleen and after birth occurs in the bone marrow, although if the latter is damaged the liver and spleen may regain some hematopoietic activity (hematopoiesis extramedullary). All blood cells come from undifferentiated mesenchymal cells, which give rise to pluripotent hemopoietic stem cells, which have been identified with radioactive labelling in the bone marrow, since they are not identifiable by light or electron microscopy. These pluripotent stem cells, under certain stimuli, undergo divisions and differentiate into two types of multipotential stem cells: 1) Colonyforming unit-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), also called common myeloid progenitors, that can divide and differentiate and give rise to erythrocytes, granulocytes, monocytes and platelets and 2) Colonyforming unit-Lymphocyte (CFU-Ly), also called common lymphoid progenitors, which give rise to T and B lymphocytes and targeting the primary lymphoid organs where they are differed. Erythrocytes Comprising the most abundant blood cellular line, with an average of between 7 to 12,000,000/mm3 although there are differences between domesticated species, breed and physiological state. Also, the erythrocyte size also varies with the species, ranging between 3.5 µm in small ruminants to 7.5 µm in the dog. The erythrocytes of mammals are anucleate cells, with the morphology of rounded biconcave discs, whereas in birds, reptiles, fishes and amphibians they have oval morphology and a small and very basophilic central nucleus. Under the light microscope, erythrocytes show an intensely acidophilic and homogeneous cytoplasm. Whit electron microscopy the cytoplasm is electron dense and 2 homogeneous and is not observed any organoid. The latter is because erythrocytes are highly specialized cells that have lost all their organoids to store a protein, hemoglobin, which is responsible for the transport of O2 and CO2 and which gives the characteristic red color to blood, macroscopically. To a lesser extent, erythrocytes also contain other proteins that act as enzymes to help hemoglobin fulfil its function, e.g. the hemoglobin reductase and the carbonic anhydrase, and others proteins with a mechanical function such as the spectrin and ankyrin that associate and form tetramers These tetramers are joined by actin filaments to integral proteins of the plasma membrane, forming a network that gives the erythrocyte membrane great plasticity, so that it can deform when the cell passes through small capillaries and later regain its shape. Erythrocytes have a very short half-life, between 60 and 120 days, because they lack necessary organoids to synthesize proteins and cannot renew their plasma membrane when it deteriorates. After this time, they are phagocytosed by macrophages, mainly in spleen, which process hemoglobin to reuse iron. The function of erythrocytes is to transport O2 from the lung to the tissues and CO2 in the opposite direction. This transport is carried out by hemoglobin, which captures one or another gas depending on its concentration in the environment that surrounds the erythrocyte. Figure 1: Mammalian and bird erythrocytes (nucleate). TEM and SEM. The erythrocytes of mammals may exhibit abnormal shapes and sizes. The most frequent are detailed below: 3 1) Poikilocytosis: erythrocytes with irregular morphologies, although it is normal in the goat species. 2) Anisocytosis: erythrocytes with abnormal sizes; the normocytes are erythrocytes with normal size, the microcytes show a decrease in size and macrocytes one size larger than normal. 3) Howell-Jolly bodies: immature erythrocytes of 1 µm in diameter that preserve remnants of nucleus. 4) Cabot rings: immature ring-shaped erythrocytes, which also have remnants of nucleus. 5) Reticulocytes: immature erythrocytes that show slight basophilic staining in the cytoplasm due to the presence of ribosomes. The presence of immature erythrocytes in the blood indicates that their formation (erythropoiesis) is accelerated, although in felines and horses it is normal to observe 1% of reticulocytes and in pigs up to 2%. 6) Heinz bodies: erythrocytes that present a refractile area in the cytoplasm that corresponds to oxidized hemoglobin. In cats they can represent up to 10% under normal conditions. Megakaryocytes and platelets The CFU-GEMM differentiate and give rise to germ unipotent cells called colony forming unit-megakaryocyte (UFC- Meg). The maturation of these cells gives rise to megakaryocytes, large cells ranging from 40 to 100 µm. The nucleus is pleomorphic, lobed, polyploid and with several nucleoli. The presence of a polyploid nucleus (with more chromosomal endowment than normal) is due to the fact that the progenitors of megakaryocytes have suffered endomitosis, that is, chromosomal replication that is not accompanied by nuclear or cytoplasmic division. The cytoplasm has many granules: 1. lambda granules or azurophilic (primary lysosomes, called azurophilic granules because stained purple with the methylene blue, in the technique Wright), 2. alpha granules, of moderate electron density and containing coagulation factors, fibrinogen and platelet growth factor and 3. delta granules, with higher electron density and containing ADP, ATP, calcium, serotonin, histamine, etc… These cells also present a highly developed membrane demarcation system or open canalicular system, which is formed by invaginations of the plasma membrane. The platelets are produced by fragmentation of the megakaryocyte cytoplasm along the demarcation membrane system. Platelets are anucleate elements in mammals while in birds, fishes, reptiles and amphibians they have a small nucleus. Mammalian platelets are about 2-5 µm in diameter, they have a rounded or discoid shape, with a clear peripheral region or hyalomere containing numerous microtubules and myosin and actin (allow the emission of pseudopodia and contraction). Likewise, in the hyalomere is the open canalicular system through which the platelets release the content of their granules to the outside. 4 The central zone or granulomere contains alpha, delta and lambda granules, as well as some mitochondria. Externally, platelets are surrounded by a thick glycocalyx with numerous receptors. Figure 3. Mammalian platelet. TEM. Figure 2. Megakaryocyte. TEM. The number of platelets varies between 250,000 and 400,000/mm3 of blood, their half-life being about 14 days. The main function of platelets is hemostasis, which is not only the mechanical blocking of the injured site but also provides a catalytic surface for the coagulation cascade to act, which ends up transforming fibrinogen into a fibrin network. Fibrin, together with platelets, form the thrombus that prevents blood from leaving the vascular system. Platelets also have an anticoagulant function, are involved in phagocytosis, modulate the inflammatory process, and help repair injured tissue. Leukocytes Leukocytes are classified into granulocytes and agranulocytes. The granulocytes are characterized by presenting specific granules in their cytoplasm and are neutrophils, eosinophils and basophils. They are classified according to the staining characteristics of their granules. The agranulocytes lack specific granules but present azurophilic granules and are monocytes and lymphocytes. Granulocytes 1. Neutrophils Neutrophils are the most abundant granulocytes in the blood, representing 60-70% of total leukocytes and measuring approximately 12-15 µm. These cells have a heterochromatic lobed nucleus, which has 3 to 5 lobes joined by fine bridges, the number of lobes increasing with the age of the cell. In females of some species of animals and women, it is observed in the nucleus a tiny 5 drumstick-shaped lobe, which corresponds to the X chromosome and is called Barr body. The cytoplasm, under the light microscope, is slightly basophilic and contains azurophilic granules, which are so named because they are stained purple with methylene blue (Wright's technique) and are primary lysosomes. The specific granules stain very little, both with acidic and basic dyes and, observed with the electron microscope, have a characteristic "rice grain" shape. Recently, rounded tertiary granules smaller than the specific ones have been discovered. In addition, neutrophils have few rough endoplasmic reticulum cisternae and a small Golgi complex. Azurophilic granules contain acid phosphatase, hydrolases, and myeloperoxidase. Although the specific granules are peroxidase negative, they contain lysozyme, collagenase, lactoferrin, superoxide radicals and a set of basic proteins with bactericidal capacity generically called "phagocytins". Tertiary granules contain gelatinase and cathepsins. Neutrophils from fishes, amphibians, reptiles, birds, rabbits, and guinea pigs are called heterophils and present specific large, spindle-shaped and acidophilic granules. Neutrophils are the first cells to reach the inflammatory focus, where they play an important role in phagocytosis and destruction of microorganisms, mainly bacteria that are destroyed by enzymes and superoxide radicals in their granules. 2. Eosinophils They measure from 10 to 15 µm and represent 2 to 4% of total leukocytes. They have a heterochromatic and generally bilobed nucleus. The cytoplasm has specific granules, which are acidophilic or eosinophilic, hence the name these cells receive. There is also a small Golgi complex and few rough endoplasmic reticulum cisternae and mitochondria. The size, shape, number and distribution of the specific granules vary with the different species, the horse being the one with the most remarkable granules since they are large and very numerous, completely filling the cell. Under the electron microscope, in the specific granules of almost all domestic species, 1 or 2 electrodense protein crystals are observed, rich in basic proteins and immersed in an amorphous matrix of moderate electron density. These specific granules are lysosomes that contain acid phosphatase, peroxidase, and acid hydrolases, which is why they are considered a form of evolution of azurophilic granules. Azurophilic granules have a hydrolytic enzyme content similar to that of neutrophils. Eosinophils are important in parasitic infestations, since the proteins of their specific granules can produce pores in the membrane or capsule of some parasites, allowing hydrolases to enter, destroying them. In addition, they also 6 play an important role in allergy phenomena, they phagocytose and eliminate antigen-antibody complexes and modulate the inflammatory process. 3. Basophils They are very rare in blood, representing less than 1% of total leukocytes. They are about 10 µm in diameter and have a lobed heterochromatic nucleus, usually shaped like the letter "S". The cytoplasmic granules are large, basophilic and occupy the entire cytoplasm of the cell, masking the nucleus in some species. Its granules are water soluble so that during staining and fixation a partial degranulation occurs. In addition, they are metachromatic, when stained with toluidine blue or Giemsa. Specific granules contain heparin, histamine, serotonin, peroxidase, and neutrophil and eosinophil chemotactic factors. Azurophilic granules are primary lysosomes with a morphology and composition very similar to those of neutrophils. Basophilic polymorphonuclear cells are very numerous in type I hypersensitivity states. The release of the content of the specific granules is mediated by IgE, histamine and leukotrienes. Figure 4. Neutrophil, eosinophil and basophil scheme. Figure 5. Neutrophil, eosinophil and basophil. LM. 7 Figure 6. Neutrophil, eosinophil and basophil. TEM. Agranulocytes 1. Monocytes Monocytes are large agranulocytes, measuring 12–18 µm in diameter and accounting for 5% of the total white blood cell count. Under the light microscope, monocytes have a kidney-shaped nucleus and a non-obvious nucleolus. The cytoplasm is large and contains azurophilic granules. Under electron microscopy, they present a euchromatic nucleus, a developed Golgi complex, rough endoplasmic reticulum cisternae, free ribosomes, mitochondria, and numerous primary and secondary lysosomes. Monocytes remain in the blood for one or two days, passing into body tissues and cavities where they are transformed into macrophages. Macrophages are long-life phagocytic cells that are distributed throughout the body. Monocytes sometimes transform into macrophages within vessels, e.g. the pulmonary intravascular macrophages (MIPs) of the lung, the Kupffer cells of the liver, then called fixed macrophages because they present modes of binding with endothelial cells. Monocytes and macrophages are called the Mononuclear Phagocytic System (SMF). The transformation of monocytes into macrophages is accompanied by increased cell size, changes in cell metabolism, expression of surface receptors, increased phagocytic activity, and increased enzymatic content of lysosomes. All macrophages have similar structural and functional properties, regardless of their locations. The most important function of the macrophage is phagocytosis and destruction of all kinds of substances by the contents of their lysosomes. Sometimes they can fuse to form multinucleated giant cells and thus increase their capacity for phagocytosis. Another macrophage function is the presentation of antigens to lymphocytes so that they develop an immune response. Finally, 8 macrophages produce large amounts of monokines that regulate the inflammatory reaction. Figure 7. Monocyte scheme. Figure 8. Monocyte. LM. Figure 9. Monocyte. TEM. 2. Lymphocytes Lymphocytes represent 20 to 25% of the total leukocyte count, although with appreciable variations (as in ruminants). Lymphocytes lack specific granules and have a round, heterochromatic nucleus and a small cytoplasm with varying degrees of basophilia, due to the presence of numerous ribosomes. In addition, the cytoplasm contains a sparse rough endoplasmic reticulum, a small Golgi complex, and a few azurophilic granules (primary lysosomes). They can be classified according to two criteria: • Functional: classified in lymphocytes B, responsible of humoral immune response, T lymphocytes, responsible for cellular immune response and null (in which the two populations are included, one that is the parent of B and T lymphocytes and other, whose cells are called "Natural Killer " and which can destroy specific cells). • Morphological: classifying into small (6-9 µm), medium (10-12 µm) and large (15-25 µm) lymphocytes. Figure 11. Lymphocyte. LM. Figure 10. Lymphocyte scheme in lymphoid organ and circulating. 9 Figure12. Circulating lymphocyte. TEM. III. BONE MARROW It is located in the medullary cavity of long bones and in the space among the trabeculae of cancellous bone tissue. It is a hematopoietic parenchymal organ. The stroma of the bone marrow consists of a framework of reticular fibers and reticular cells. The latter have a stellate morphology and synthesize and envelop the reticular fibers, providing support to the parenchyma. In addition, they contribute to establishing the hematopoietic microenvironment of the marrow, providing growth factors necessary for the proliferation and maturation of blood cell precursors. The reticular cells of the bone marrow are able of accumulating lipids and morphologically transforming into indistinct cells of adipocytes, very abundant in adult animals (yellow bone marrow, not very active). The parenchyma is made up of islets of hematopoietic cells, where there are pluripotent mesenchymal cells and all types of blood cells in different degrees of maturation. Among the hematopoietic islets are the venous sinuses, which are vascular cavities with a discontinuous basement membrane and where blood cells enter as they mature. SUMMARY OF BLOOD CELLS ERYTHROCYTES PLATELETS MONOCYTE LYMPHOCYTE EOSINOPHIL 10 NEUTROPHIL BASOPHIL ANNEX: BASIC INFORMATION. HISTOLOGICAL CONSTITUTION OF THE ORGANS. Organs are an association of different tissues that work in coordination to perform a common function. Depending on their histological constitution, parenchymal (solid) organs and hollow (tubular) organs can be distinguished. PARENCHYMAL OR SOLID ORGANS The components of the parenchymal or solid organs can be divided into two subgroups. The parenchyma, which is the noble part or specific functional component of the organ and the stroma that includes those tissues (connective and nervous) that metabolically and/or structurally maintain the parenchyma. This stroma is made up of a capsule of connective tissue that surrounds the organ and that sends into the parenchyma, septa or trabeculae, which are extensions made up of fibroblasts, collagen and elastic fibers and some smooth muscle fibers and through which blood vessels and lymphatics and nerves pass. If these trabeculae divide the parenchyma into isolated structures, lobules or spaces completely separated by connective tissue are formed; the pseudolobules are spaces separated by septa that do not fully meet. New elements are sent to the interior from the septa, which are reticular fibers (type III collagen) or type I collagen, responsible for forming the bed or network where the parenchymal cells are found. The stroma fulfils the functions of support, connection and nutrition since the blood vessels, lymphatics and nerves run through it. HOLLOW OR TUBULAR ORGANS Normally, they are hollow or cavitary organs made up of layers, membranes or tunics of different tissues that are arranged concentrically. The typical tubular organ is made up of 1. the tunica mucosa, 2. the tunica submucosa, 3. the tunica muscular and, finally, 4. the tunica serosa or adventitia. The tunica mucosa can be of three types: glandular, integumentary or transitional. The glandular mucosa presents glandular elements, as its name indicates, and it is found in both the digestive and respiratory tracts. It is made up of a simple epithelium, which is normally either simple columnar or pseudostratified, which rests on its corresponding basement membrane. Beneath this layer we find the lamina propria made up of loose connective tissue in which numerous glandular structures, blood and lymphatic vessels and nerves are located. Finally, the muscular layer of the mucosa is observed, made up of two layers of smooth muscle fibers, the innermost is arranged circularly and the outermost is arranged longitudinally. This layer helps the local mobility of the organs and expel secretion products from the glands. 11 The integumentary mucosa is specialized in the conduction of coarse substances that can cause great friction, e.g. the oesophagus. It is made up of the same layers as the previous one and they only differ in the lining epithelium that is squamous stratified of the two types, keratinized and non-keratinized. The transitional mucosa consists of several modifications, since, in addition to having a transitional epithelium, the lamina propria is thin and there is no mucosal muscle. It is located in hollow organs capable of undergoing considerable distention, e.g. the urine bladder. Very important note: Both the integumentary and glandular mucosa, and sometimes the transitional mucosa, may present folds or evaginations. Depending on the organ in question, the evagination that only includes epithelium, lamina propria and muscular mucosa, are called villi, papillae or primary folds. In those tubular organs that have primary folds, the evaginations in which the mucosa and submucosa are included are called folds properly or secondary folds. The tunica submucosa is made up of loose connective tissue that is more organized than that of the lamina propria. In it we can find blood vessels, nerve plexuses and glands as well as lymphoid nodules. The muscular tunic is made up of two layers of smooth muscle fibers, although bundles of skeletal muscle fibers may exist in some organs. The innermost is arranged circularly, and the outermost does so longitudinally. Vascular and nerve plexuses are located between both layers. This tunic is responsible for the tone of the organ, size of the lumen, and movement of materials through the hollow organ. The tunica or serous membrane appears in the hollow organs that are located in the body cavities, such as the thoracic or abdominal. It is made up of a mesothelium, a simple squamous epithelium, which rests on a layer of loose connective tissue. If the organ is not located in the body cavities, the serosa is replaced by a tunica adventitia, which lacks mesothelium and is made up of loose connective tissue. 12