PHS 203 EKSU PHYSIOLOGY notes.docx

**PHS 203 - BLOOD AND BODY FLUIDS for Physiologists** **BLOOD** **[Introduction ]** Blood is life. Oxygen that we breathe in is delivered to the body through the blood cells. This brings us to the components of the blood. Blood is a highly differentiated, complex living tissue that pulsates through the arteries to every part of the body, interacts with individual cells via an extensive capillary network and returns to the heart through the venous system. It is called the "fluid of Life" as it carries oxygen into the body and carbon dioxide out of the body, "fluid of health" as it protects the body against diseases and gets rid of waste products. **The components of Blood** Blood is an opaque, red liquid consisting of several types of cells suspended in a complex amber fluid known as **Plasma.** When bloods is allowed to cloth or coagulate the suspending medium is called **Serum.** Blood consist of Blood cells, Plasma &Serum**.** Blood Cells are of 3 types in the blood namely 1. Red blood cells **{Erythrocytes**}: They are the most abundant cells of the blood and are necessary for the delivery of oxygen to the tissue 2. White blood cells **{Leukocytes**}: They are the colorless cells of the blood and are involved in defense mechanism of the body against invading organisms. 3. Platelets cells **{Thrombocytes**}: They are nucleated cells involved the onset of blood clotting that prevents blood loss. **Plasma**: This is the Liquid part of the blood; it is a straw-colored clear liquid and contains 91-92% water. It can be gotten when blood is collected with an anticoagulant in place and centrifuged for some time, blood cells settles down and plasma remains on top. **Serum**: This is the liquid part of the blood when it is allowed to clot.it contains all other constituent of plasma except fibrinogen. Serum=plasma-fibrinogen. **Assignment:** Plasma proteins {Properties & functions} Class Activity: Search the difference between plasma &serum. Blood with description.png **PROPERTIES OF BLOOD** 1. **Color:** Blood is red in color. Scarlet red for the Arterial blood and purple red for the Venous blood 2. **Volume:** It is about 5L in an adult, 450ml in Newborn slightly lesser in females than Males. It accounts for about 6-8% of the body weight 3. **Reaction and PH:** It is slightly Alkaline with a PH of 704 4. **Density{Specific gravity}:** Blood is approximately 1.050 5. **Viscosity:** A measure of resistance to flow, it is 3.5-5.5 times viscous than water. While blood is only slightly heavier than water, it is certainly much thicker. **FUNCTIONS OF BLOOD** The cellular and plasma components of blood may act alone but they often work in concert to perform their functions which includes: 1. **Transport:** Blood carries several important substances from one area of the body to another, including oxygen, carbon dioxide, antibodies, acid and bases, ions, vitamins, cofactors, hormones, nutrients, lipids, gases, pigments, mineral and water. Transport is one of the primary and most important function of the blood as it is the primary means of Long-distance transport in the body Substances can be transported free in plasma, bound to plasma-protein transports O~2~ and CO~2~ which are very important. It transports heat. 2. **Immunity:** Blood plays an important in the defense of the body. Blood Leukocytes working in conjunction with plasma protein continuously patrol for microbial pathogens in the tissues and in the blood and battle against them and in must cases they are efficiently eliminated. 3. **Hemostasis :** Bleeding is controlled by the process of hemostasis by complex and efficient hemostatic mechanism 4. **Homeostasis:** This is steady state that provides an optimal internal environment for cell function. By maintaining P^H^, ion concentrations, osmolality, temperature, nutrient supply and vascular integrity, the blood system plays a crucial role in preserving homoeostasis. Homoeostasis is the result of Normal functioning of the bloods transport, Immune and hemostatic systems. **RED BLOOD CELLS {ERTHROCYTES**} They are the most numerous cells in blood. Erythros means Red as the red color of the blood cells is due to the presence of the coloring pigment called **HEMOGBLOBIN** **Characteristics of Red Blood Cells** 1. They lack a nucleus- hence no DNA. "No mitochondria and Golgi apparatus. Energy is produced from glycolytic process. It does not have insulin receptor and so glucose uptake in this cell not controlled by insulin" 2. They are biconcave disk shaped with a diameter of about 7um and maximum thickness of 2.5um. surface area of about 120 square u with volume of 85-90cu u 3. It maintain its shape by virtue of its complex membrane skeleton which is made up of actin and spectrin 4. It has a specific gravity of 1.092 to 1.101 5. In circulation, RBC remains suspended uniformly in the blood. This property is called Suspension stability 6. A single Erythrocyte cell lives only for 120 days and in that duration, it perform successive roles. It is removed from circulation by Macrophages found in bone marrow, liver and spleen. **FUNCTION OF RED BLOOD CELLS** The major function of the RBC(s) is the transport of respiratory gases {O~2~ &CO~2~} and it performs buffering action. 1. Hemoglobin in RBC combines with oxygen to form oxyhemoglobin which transports about 97% of oxygen in the body. 2. Hemoglobin also combines with CO~2~ to form Carb-hemoglobin which transports about 30% of CO~2~ in the body. 3. Buffering Action: hemoglobin in the red blood cells is an excellent acid-base buffer {as it true of most proteins}. It regulates the hydrogen ion concentration in the blood. **Note**: The remaining CO~2~ is passed through a phase of reversible reaction catalyzed by an Enzyme called Carbonic anhydrase where CO~2~ and water forms Carbonic acid {H~2~CO~3~} and subsequently broken into bicarbonates which is carried by the water of the blood from the tissues to the lungs where it is reconverted to CO~2~ and expelled. **RED CELL VALUES** In evaluating patients for hematological diseases, it is important to determine the hemoglobin concentration in the blood, the total number of circulating Erythrocytes (the red cell count) and the hematocrit. From these values several other important blood values can be deduced. Examples are 1. Mean Cell Hemoglobin Concentration{MCHC} 2. Mean Cell Hemoglobin{MCH} 3. Mean Cell Volume{MCV} 4. Blood Oxygen Carrying Assignment: Provide the formula to calculate MCHC, MCH and MCV MCHC=[ *Hb*{]{.math.inline}g/l} [hematocrit]{.math.inline} MCH= *Hb{g/l}* [\$Red\\ cell\\ count\\ \\{\\frac{\\text{cells}}{l}\\}\$]{.math.inline} MCV= *Hematocrit {PCV}* *Number of red cells* Each gram of hemoglobin can combine transport 1.34ml of oxygen. This oxygen carrying capacity of 1dL of normal blood containing 15g of hemoglobin is 15x1.34 =20.1ml of oxygen **GENESIS OF BLOOD CELL** - There are cells capable of self-renewal and can differentiate into various specialized cells. They are called stem cells - Hemopoietic stem cells are the primitive cells which give rise to the cells. The blood cell begins their lives in the bone-marrow from a single type of cell called Pluripotential Hematopoietic stem cell {PHSC}. This cell is defined as a cell that can give rise to all types of blood cells therefore it is termed Uncommitted {uPHSC}. When these cells are then designated to form a particular type of blood cells, they become committed PHSCs. 1. Lymphoid Stem Cell {LSC}} that forms Lymphocytes and Natural Killer {NK} cells. 2. Colony Forming blastocytes which give rise to myeloid cells {they are blood cells with the exception of lymphocytes} Under this colony forming Blastocytes hey have different units namely. - Colony forming Unit- Erythrocytes{CFU-E} - Colony forming Unit- Granulocyte/ monocytes{CFU-GIN} - Colony forming Unit- Megakaryocytes {CFU-M} Note; Growth and reproduction of Stem cells are controlled by different proteins called growth Inducers. One of the four Interleukin is interleukin 3 that influences all the types of stem cells. Whereas, others are cells specific. Differentiation inducers are another different protein that induces cells to different to another form till they form adult blood cells Class activity: Define Committed PHSCs: This is a Cell {PHSC} which is restricted to give rise to one group of blood cells. **ERYTHROPOIESIS** This is the process of the development and maturation of red blood cells {Erythropoiesis}. It is a stem of Hemopoiesis which is the process of origin, development and maturation of all blood cells. **Sites of production of Red blood cells** In the Early months of intrauterine life; red blood cells are produced in the mesenchyme of the yolk sac. During the second Trimester, the liver is the main organ for red blood cell production but reasonable numbers bare also produced by the spleen and Lymph nodes. At the last trimester, red blood cells are produced exclusively in the bone marrow. From birth to 20 years, all bones produce Red blood cells from their marrow. After 20 years, the marrow of the long bones except for the proximal potions of the humeri and tibiae becomes quite fatty and produces no more red blood cells. Vertebrae sternum, ribs, scapula, iliac bones and skull bones are the only ones producing RBCs and they less productive as age increases. Note: if the bone marrow is destroyed or fibroses in adults, liver and spleen may produce the blood cells. **Erythropoiesis {Process, Changes and Stages}** This process starts from the precursor cells which moves to the colony forming Unit --Erythrocyte cells {CFU-E} and then to formation of matured Red blood cells **[Stages:]** There are different stages between the CFU-E and formation of RBCs which are as follows; 1. Proerythroblast Phase: This phase proerythroblast or Megaloblast develops from the CFU-E. This cell is large in size with a 20u diameter and it nucleus occupies the cell almost completely. The nucleus has two or more nucleoli: it does not contain hemoglobin, its cytoplasm is basophilic and the cell divides severally to form the cell of the next phase. 2. Early Normoblast: The cells of this phase are smaller than the proerythroblast. Nucleolus disappears in the nucleus and the cell has diameter of about 15u. The cytoplasm is basophilic in nature too and chromatin network becomes condensed. The cell of this phase is also called. Basophilic Erythroblast because they stain with basic dyes synthesis of hemoglobin starts in this stage however. It has not appeared in this stage. 3. Intermediate Normoblast: hemoglobin starts appearing in this stage, and the cell gets smaller with a diameter of 10-12u. Nucleus is still present and the chromatin network becomes more condensed. The cytoplasm is still basophilic but with the appearance of hemoglobin, it stains both acidic and basic stains too hence making this cell polychromophilic in nature therefore it can be called polychromophilic Erythroblast or Polychromatic erythroblast. 4. Late Normoblast: The cell in the intermediate Normoblast stage further divides into the cell of this stage with a diameter of 8-10u with a very small like spot nucleus and very much condensed chromatin network. Hemoglobin increases to about 34% of the cell become more acidophilic. Therefore it is called Orthochromic Erythroblast. When this cell is about to pass to the next stage, the nucleus disintegrates and disappear in a process calling Pyknosis. 5. Reticulocyte: This is the next phase after late Normoblast with a cell known as Immature RBCs as they are slightly larger than the mature RBCs. This cell contains the remnant of the cell Organnelles that went through the process of Pyknosis {golgi apparatus, mitochondria Endoplasmic reticulum}. Hence, making it contain small amount of basophilic material. The cytoplasm contains reticular network or reticulum hence the name reticulocyte and it stains with supravital stains. These cells then squeeze through pores of the capillary membranes. From the bone marrow through a process called **Diapedesis**. 6. Matured Erythrocytes: Between 1&2 days, the basophilic materials in the reticulocytes disappear and the cells then mature to RBCs with a biconcave shape of diameter 7u. Hemoglobin is present but nucleus is absent. From proerythroblast to RBCs formation, it takes 7 days, 5 days from proerythroblast to reticulocyte and 2 more days to mature Erythrocytes. **DIAGRAM** ![Haemopoiesis. - ppt download](media/image3.jpeg) **FACTORS REGLATING RED BLOOD CELL PRODUCTION** 1. The rate of red blood cell production can be affected by any condition that affects the quantity of oxygen transported to the tissues which is termed Tissue hypoxia. 2. Erythropoietin: it is a hormone also called hemopoietin secreted by Peritubular capillaries of the kidney and small quantity is secreted from the liver and brain. Hypoxia is the stimulant for the secretion of Erythropoietin. Erythropoietin promotes the production of proerythroblasts from CFU-E and development of proerythroblast to mature RBCs and the release of RBCs from bone marrow into the blood. 3. Thyroxine: A metabolic hormone, thyroxine, cortisol, Androgens, Estrogen, Growth hormone accelerates the process of Erythropoiesis. 4. Hemopoietic Growth Factors: They are the growth inducers previously mentioned. They are interleukins which induces proliferation of PHSC 5. Vitamins{Nutritional Factors}: The process of erythropoiesis is associated with some vitamins examples are Vitamin B, Vitamin C, Vitamin D and Vitamin E, Vitamin B~12~ **Assignment:** Types of interleukin and their functions Function of Vitamins and how they affect RBC production. a. Extrinsic factor: which is Vitamin B12{ cyanocobalamin obtained from diet} b. Intrinsic factor: Intrinsic factor of castle helps in the absorption of Vitamin B12 from the intestine c. Folic Acid: Essential for maturation of RBC, it is required for the Synthesis of DNA. Anemia due to folic acid deficiency is called Megaloblastic Anemia d. Iron: Necessary for the formation of the heme part of hemoglobin e. Copper: Necessary for the absorption of Iron from the gastro-intestinal tract f. Cobalt and Nickel: These metals are essential for the utilization of iron during hemoglobin formation g. Proteins and Amino acids: Needed for the formation of hemoglobin and Synthesis of the Globin {proteins} part of the hemoglobin molecule. **Erythrocyte Destruction**. Red cells circulate for about 120 days after they are released from the marrow. Some of the senescent (old) red cells because of their fragile nature, are destroyed while trying to squeeze through the capillaries and break up (hemolyze) mainly in the capillaries of red pulp of spleen because the diameter of splenic capillaries is very small. So, the spleen is called 'graveyard of RBCs. The hemoglobin released on destruction of red cells is immediately phagocytized by macrophages of the body, particularly the macrophages present in liver (Kupffer cells), spleen and bone marrow. Hemoglobin released by red cells that lyse in the circulation is broken down to globin and heme. Heme is broken down to Iron and Porphyrin. Iron combines with the protein called apoferritin to form ferritin, which is stored in the body and reused later. Porphyrin is degraded into bilirubin, which is excreted by liver through bile. The globin portion is catabolized by proteases into constituent amino acids that are used in protein synthesis. **PHYSIOLOGICAL VARIATIONS IN NUMBER OF RED BLOOD CELLS** **A**. **Increase in RBC count is known as polycythemia**. It occurs in both physiological and pathological conditions. When it occurs in physiological conditions it is called physiological polycythemia. The increase in number during this condition is marginal and temporary. It occurs in the following conditions: 1\. Age: At birth, the RBC count is 8 to 10 million/cu mm of blood. The count decreases within 10 days after birth due to destruction of RBCs causing physiological jaundice in some newborn babies. However, in infants and growing children, the cell count is more than the value in adults. 2\. Sex: Before puberty and after menopause in females the RBC count is similar to that in males. During reproductive period of females, the count is less than that of males (4.5 million/cu mm). 3\. High altitude: Inhabitants of mountains (above 10,000 feet from mean sea level) have an increased RBC count of more than 7 million/cu mm. It is due to hypoxia (decreased oxygen supply to tissues) in high altitude. 4\. Muscular exercise and Emotional conditions: There is a temporary increase in RBC count after exercise and during anxiety. It is because of mild hypoxia and contraction of spleen. Spleen stores RBCs (Chapter 25). Hypoxia increases the sympathetic activity resulting in secretion of adrenaline from adrenal medulla. Adrenaline contracts spleen and RBCs are released into blood (Fig. 9.5). 6\. Increased environmental temperature: Generally increased temperature increases all the activities in the body including production of RBCs. 7\. After meals: There is a slight increase in the RBC count after taking meals. It is because of need for more oxygen for metabolic activities. **B**. **Decrease in RBC Count** occurs in the following physiological conditions: 1\. High barometric pressures: At high barometric pressures as in deep sea, when the oxygen tension of blood is higher, the RBC count decreases. 2\. During sleep RBC count decreases slightly during sleep and immediately after getting up from sleep. Generally all the activities of the body are decreased during sleep including production of RBCs. 3\. Pregnancy In pregnancy, the RBC count decreases. It is because of increase in ECF volume. Increase in ECF volume, increases the plasma volume also resulting in hemodilution. So, there is a relative reduction in the RBC count. **PATHOLOGICAL VARIATIONS:** **A. Pathological polycythemia** **is the abnormal increase in the RBC count**. Red cell count increases above 7 million/ cu mm of the blood. Polycythemia is of two types, the primary polycythemia and secondary polycythemia. - **Primary Polycythemia**: is otherwise known as polycythemia vera. It is a disease characterized by persistent increase in RBC count above 14 million/cu mm of blood. This is always associated with increased white blood cell count above 24,000/cu mm of blood. Polycythemia vera occurs in myeloproliferative disorders like malignancy of red bone marrow. - **Secondary Polycythemia**: This is secondary to some of the pathological conditions (diseases) such as: Respiratory disorders like emphysema, congenital heart disease, Ayerza's disease (condition associated with hypertrophy of right ventricle and obstruction of blood flow to lungs), chronic carbon monoxide poisoning, Poisoning by chemicals like phosphorus and arsenic, repeated mild hemorrhages. All these conditions lead to hypoxia which stimulates the release of erythropoietin. **B. Anemia: Abnormal decrease in RBC count is called anemia**, a blood disorder, characterized by the reduction in RBC count, hemoglobin content and PCV. Anemia occurs due to decreased production of RBC, increased destruction of RBC and excess loss of blood from the body which can be caused either by inherited disorders or environmental influences such as nutritional problem, infection, exposure to drugs or toxins etc. **ANEMIA** Anemia is the blood disorder, characterized by the reduction in: 1\. Red blood cell (RBC) count 2\. Hemoglobin content 3\. Packed cell volume (PVC). Generally, reduction in RBC count, hemoglobin content and PCV occurs because of: Decreased production of RBC, Increased destruction of RBC and Excess loss of blood from the body. All these incidents are caused either by inherited disorders or environmental influences such as nutritional problem, infection and exposure to drugs or toxins. Anemia is classified by two methods: Morphological classification and Etiological classification **Morphological classification** depends upon the size (determined by mean corpuscular volume (MCV) and color (determined by mean corpuscular hemoglobin concentration (MCHC). By this method, the anemia is classified into four types namely: 1\. **Normocytic Normochromic Anemia**: Size (MCV) and color (MCHC) of RBCs are normal. But the number of RBC is less. 2\. **Macrocytic Normochromic Anemia**: RBCs are larger in size with normal color. RBC count is less. 3\. **Macrocytic Hypochromic Anemia:** RBCs are larger in size. MCHC is less, so the cells are pale (less colored). 4\. **Microcytic Hypochromic Anemia:** RBCs are smaller in size with less color. **Etiological Classification** depends upon the cause or origin of anemia. Based on this method of classification, anemia is divided into five types: 1\. **Hemorrhagic anemia**: Anemia due to hemorrhage (excessive loss of blood) is known as hemorrhagic anemia. It occurs both in acute and chronic hemorrhagic conditions. **Acute hemorrhage** refers to sudden loss of a large quantity of blood as in the case of accident. Within about 24 hours after the hemorrhage, the plasma portion of blood is replaced. However, the replacement of RBCs does not occur quickly and it takes at least 4 to 6 weeks. So with the less number of RBCs, **hemodilution** occurs. However, morphologically the RBCs are normocytic and normochromic. Decreased RBC count causes hypoxia, which stimulates the bone marrow to produce more number of RBCs. So, the condition is corrected within 4 to 6 weeks**. Chronic hemorrhage** refers to loss of blood by internal or external bleeding, over a long period of time. It occurs in conditions like peptic ulcer, purpura, hemophilia and menorrhagia. Due to continuous loss of blood, lot of iron is lost from the body causing iron deficiency. This affects the synthesis of hemoglobin resulting in less hemoglobin content in the cells. The cells also become small. Hence, the RBCs are microcytic and hypochromic 2\. **Hemolytic anemia**: Anemia due to excessive hemolysis (destruction of RBCs) which is not compensated by increased RBC production is called hemolytic anemia. **It is classified into two types: Extrinsic and Intrinsic hemolytic anemia**. ***Extrinsic hemolytic anemia***: is caused by destruction of RBCs by external factors such as antibodies, chemicals and drugs. Extrinsic hemolytic anemia is also called autoimmune hemolytic anemia. Common causes of extrinsic hemolytic anemia includes: Liver failure, Renal disorder, Hypersplenism, Burns, Infections (hepatitis, malaria and septicemia), Drugs (penicillin, antimalarial drugs and sulfa drugs), Poisoning by chemical substances (lead, coal and tar), Presence of isoagglutinins (like antiRh), Autoimmune diseases (rheumatoid arthritis and ulcerative colitis). ***Intrinsic hemolytic anemia*** is caused by destruction of RBCs because of the defective RBCs. There is production of unhealthy RBCs, which are short lived and are destroyed soon. Intrinsic hemolytic anemia is often inherited and it includes sickle cell anemia and thalassemia. Because of the abnormal shape in sickle cell anemia and thalassemia, the RBCs become more fragile and susceptible for hemolysis. 3\. **Nutrition deficiency anemia:** occurs due to deficiency of a nutritive substance necessary for erythropoiesis. The substances which are necessary for erythropoiesis includes iron, proteins and vitamins like C, B12 and folic acid. The types of nutrition deficiency anemia are: ***Iron deficiency anemia:*** which is the most common type of anemia develops due to inadequate availability of iron for hemoglobin synthesis. RBCs are microcytic and hypochromic. Causes includes: Loss of blood, Decreased intake of iron, Poor absorption of iron from intestine and Increased demand for iron in conditions like growth and pregnancy. ***Protein deficiency anemia:*** Due to deficiency of proteins, which results in the decrease in the synthesis of hemoglobin. The RBCs are macrocytic and hypochromic. ***Pernicious anemia or Addison's anemia***: is due to deficiency of vitamin B12. It is also called Addison's anemia. It is due to atrophy of the gastric mucosa because of autoimmune destruction of parietal cells. The gastric atrophy results in decreased production of intrinsic factor and poor absorption of vitamin B12, which is the maturation factor for RBC. RBCs are larger and immature with almost normal or slightly low hemoglobin level. Synthesis of hemoglobin is almost normal in this type of anemia. So, cells are macrocytic and normochromic/hypochromic. It is common in old age and it is more common in females than in males. It is associated with other autoimmune diseases like disorders of thyroid gland, Addison's disease, etc. ***Megaloblastic anemia:*** is due to the deficiency of another maturation factor called folic acid. Here, the RBCs are not matured. The DNA synthesis is also defective, so the nucleus remains immature. The RBCs are megaloblastic and hypochromic. Features of pernicious anemia appear in megaloblastic anemia also. However, neurological disorders may not develop. 4\. **Aplastic anemia:** is due to the disorder of red bone marrow where red bone marrow is reduced and replaced by fatty tissues. Bone marrow disorder occurs in the following conditions: Repeated exposure to X-ray or gamma ray radiation, Tuberculosis, Viral infections (hepatitis and HIV infections) and Presence of bacterial toxins, quinine, gold salts, benzene, radium, etc. In aplastic anemia, the RBCs are normocytic and normochromic. 5\. **Anemia of chronic diseases**: is the second common type of anemia (next to iron deficiency anemia). It is characterized by short lifespan of RBCs, caused by disturbance in iron metabolism or resistance to erythropoietin action. Anemia develops after few months of sustained disease. RBCs are normocytic and normochromic. Common causes anemia of chronic diseases: i. Noninfectious inflammatory diseases such as rheumatoid arthritis (chronic inflammatory autoimmune disorder affecting joints). ii. Chronic infections like tuberculosis (infection caused by Mycobacterium tuberculosis) and abscess (collection of pus in the infected tissue) in lungs. iii. Chronic renal failure, in which the erythropoietin secretion decreases (since erythropoietin is necessary for the stimulation of bone marrow to produce RBCs, its deficiency causes anemia). iv. Neoplastic disorders (abnormal and disorganized growth in tissue or organ) such as Hodgkin's disease (malignancy involving lymphocytes) and cancer of lung and breast. **How to Assess Red blood cell status** There are different tests that can be carried out to determine the adequacy of red blood cells in the body. Some of which are: Packed cell volume (PCV), Red Cell count, Hemoglobin concentration of blood. **PACKED CELL VOLUME (PCV) OR Hematocrit**: is the fraction of whole blood volume that consists of red blood cells. **Technique** Blood is mixed with the anticoagulant ethylenediaminetetraacetic acid (EDTA) or heparin and filled in hematocrit or Wintrobe tube (110 mm long and 3 mm bore) up to 100 mark. The tube with the blood is centrifuged at a speed of 3000 revolutions per minute (rpm) for 30 minutes. RBCs packed at the bottom form the packed cell volume and the plasma remains above this. In between the RBCs and the plasma, there is a white buffy coat, which is formed by white blood cells and the platelets. After centrifugation, the height of the red cell column is measured and compared to the total height of the column of whole blood. The percentage of the total blood volume occupied by the red cell mass is the hematocrit. Hematocrit depends mostly on the number of RBCs but there is some effect (to a much lower extent) from the average size of the RBCs. Reference values are 40-45% for males and 38-42% for females. **RED CELL COUN**T: is used to measure the number of oxygen-carrying blood cells in a volume of blood. It is one of the key measures we use to determine how much oxygen is being transported to cells of the body. RBC count ranges between 4 and 5.5 million/cu mm of blood. In adult males, it is 5 million/cu mm and in adult females, it is 4.5 million/cu mm. **LEUKOCYTES (White Blood Cells)** The leukocytes, also called white blood cells, are the mobile units of the body's protective system. They are formed in the bone marrow (granulocytes and monocytes and a few lymphocytes) and partially in the lymph tissue (lymphocytes and plasma cells). After formation, they are transported in the blood to different parts of the body where they are needed. Compared to RBCs, the WBCs are larger in size, lesser in number, Irregular in shape, Nucleated, of many types, Granules are present in some type of WBCs and Lifespan is shorter. **LEUKOPOIESIS** Leukopoiesis is the development and maturation of leukocytes **FACTORS NECESSARY FOR LEUKOPOIESIS** Leukopoiesis is influenced by hemopoietic growth factors and colony stimulating factors. Colony stimulating Factors (CSF) are proteins which cause the formation of colony forming blastocytes. Colony stimulating factors are of three types: 1\. Granulocyte-CSF (G-CSF) secreted by monocytes and endothelial cells 2\. Granulocyte-monocyte-CSF (GM-CSF) secreted by monocytes, endothelial cells and T lymphocytes 3\. Monocyte-CSF (M-CSF) secreted by monocytes and endothelial cells. **Types of white blood cells** Based on the presence or absence of granules in the cytoplasm, the leukocytes are classified into two groups: Granulocytes which have granules and Agranulocytes which do not have granules. Leukocytes (with and without granules) are divided into five types as follows: 1. Polymorphonuclear Neutrophils: have fine or small granules in the cytoplasm. When stained with Leishman's stain (which contains acidic eosin and basic methylene blue) the granules appear violet in color. It has a multi-lobed Nucleus. The number of lobes in the nucleus depends upon the age of cell. In younger cells, the nucleus is not lobed. And in older neutrophils, the nucleus has 2 to 5 lobes. They are Ameboid in nature with diameter of 10 to 12 µ. 2. Polymorphonuclear Eosinophils: have coarse (larger) granules in the cytoplasm, as its name implies it stains pink or red with eosin. Nucleus is bilobed and spectacle-shaped with diameter between 10 and 14 µ. 3. Polymorphonuclear Basophils: have multiple pleomorphic, coarse, deep-staining metachromatic granules throughout their cytoplasm. The granules stain purple blue with methylene blue. Nucleus is bilobed. Diameter of the cell is 8 to 10 µ. 4. Monocytes: The cytoplasm appears pale blue or blue-gray with Wright's stain, nucleus (in the center or pushed at one side) may be shaped like a kidney bean, indented, or shaped like a horseshoe. Cell diameter of 14 to 18 µ. Upon activation, monocytes transform into macrophages 5. Lymphocytes: Like monocytes, the lymphocytes also do not have granules in the cytoplasm. Nucleus is oval, bean-shaped or kidney-shaped. Nucleus occupies the whole of the cytoplasm. A rim of cytoplasm may or may not be seen. Morphologically, circulating lymphocytes have been assigned to two broad categories: large (Younger cells with a diameter of 10 to 12 µ.) and small (Older cells with a diameter of 7 to 10 µ) lymphocytes. ![Blood Anatomy and Physiology: Study Guide for Nurses](media/image5.jpeg) **PROPERTIES OF WHITE BLOOD CELLS** 1\. Diapedesis: This is the process by which the leukocytes squeeze through the narrow blood vessels. That is, even though a pore is much smaller than a cell, a small portion of the cell slides through the pore at a time; the portion sliding through is momentarily constricted to the size of the pore. 2\. Ameboid Movement: Neutrophils, monocytes and lymphocytes show amebic movement, characterized by protrusion of the cytoplasm and change in the shape. Some cells move at velocities as great as 40 mm/min, a distance as great as their own length each minute. 3\. Chemotaxis: This is the attraction of WBCs towards the injured tissues by the chemical substances released at the site of injury. When a tissue becomes inflamed, at least a dozen different products are formed that can cause chemotaxis toward the inflamed area which includes (1) some of the bacterial or viral toxins, (2) degenerative products of the inflamed tissues themselves, (3) several reaction products of the "complement complex" activated in inflamed tissues, and (4) several reaction products caused by plasma clotting in the inflamed area, as well as other substances. 4\. Phagocytosis: which means cellular ingestion of the offending agent. Neutrophils and monocytes engulf the foreign bodies by means of phagocytosis. Phagocytes must be selective of the material that is phagocytized; otherwise, normal cells and structures of the body might be ingested. **FUNCTIONS OF WHITE BLOOD CELLS** These cells protect the body from invading organisms or foreign bodies, either by destroying or inactivating them hence, WBCs play an important role in defense mechanism. However, in defense mechanism, each type of WBCs acts in a different way. **NEUTROPHILS**: They are the most abundant leukocyte type, making up 40-70% of those found in peripheral blood. They play an important role in the defense mechanism of the body. Along with monocytes, the neutrophils provide the first line of defense against the invading microorganisms. The neutrophils are the free cells in the body and wander freely through the tissue and practically, no part of the body is spared by these leukocytes. Invading bacteria induce neutrophil chemotaxis---migration to the site of infection. Chemotaxis is initiated by the release of chemotactic factors from the bacteria or by chemotactic factor generation in the blood plasma or tissues. Chemoattractants increase the adhesive nature of neutrophils so that all the neutrophils become sticky and get attached firmly to the infected area. Each neutrophil can hold about 15 to 20 microorganisms at a time and then the neutrophils start destroying the invaders. First, these cells engulf the bacteria and then destroy them by means of phagocytosis. During the process of phagocytosis by neutrophils and other phagocytic cells, there is a rapid increase in oxygen consumption called **Respiratory burst** where the free radical O~2~ ^--^ is formed. 2O~2~ ^--^ combine with 2H^+^ to form H~2~O~2~ (hydrogen peroxide). Both O~2~ ^--^ and H~2~O~2~ are the oxidants having potent bactericidal action. In the battle against the invaders (bacteria), many WBCs are killed by the toxins released from the invaders, the dead cells are collected in the center of infected area together with plasma leaked from the blood vessel, liquefied tissue cells and RBCs that escaped from damaged blood vessel (capillaries) to constitute a whitish yellow fluid formed in the infected tissue called **pus**. Granules of neutrophils contain enzymes like proteases, myeloperoxidases, elastases, metalloproteinases and antibody-like peptides called cathelicidins and defensins. **EOSINOPHILS**: They make up 1-3% of circulating leukocytes but are easily identified on stained blood films. As the name implies, the eosinophil takes on a deep eosin color during polychromatic staining; the large, refractile cytoplasmic granules of these cells stain orange-red to bright yellow. Like neutrophils, eosinophils migrate to sites where they are needed and exhibit a metabolic burst when activated. Eosinophils participate in defense against certain parasites, and they are involved in allergic reactions. The exposure of allergic individuals to an allergen often results in a transient increase in eosinophil count known as eosinophilia. Infection with parasites often results in a sustained overproduction of eosinophils. Eosinophils are neither markedly motile nor phagocytic like the neutrophils. Some of the parasites are larger in size and eosinophils attack them by some special type of cytotoxic substances present in their granules. When released over the invading parasites from the granules, these substances become lethal and destroy the parasites. The lethal substances present in the granules of eosinophils and released at the time of exposure to parasites or foreign proteins are: Eosinophil peroxidase, Major basic protein (MBP, Eosinophil cationic protein (ECP, Eosinophil-derived neurotoxin, Cytokines (Cytokines such as interleukin-4 and interleukin-5) **BASOPHILS**: are polymorphonuclear leukocytes with multiple pleomorphic, coarse, deep-staining metachromatic granules throughout their cytoplasm. These granules contain heparin, histamine, Proteases and myeloperoxidase and cytokines which have anticoagulant, vasodilating cytotoxic and inflammatory properties, respectively. The release of these and other mediators by basophils increases regional blood flow, facilitating the transport of other leukocytes to areas of infection and allergic reactivity or other forms of hypersensitivity. Basophils play an important role in healing processes. So their number increases during healing process **MONOCYTES**: They are primarily involved in the immune response against **bacterial infection** and make up roughly 5-10% of all circulating leukocytes. Monocytes are **circulating leukocytes** which typically remain in the blood for around 8 hours before migrating into tissue where they differentiate into macrophages. Macrophages then form the main population of phagocytic cells within tissues and have a much longer lifespan than neutrophils, lasting months or even years. In some tissues, resident macrophages have specific names e.g. Kupffer cells in the liver and osteoclasts in the bone. They are much larger than neutrophils, with a diameter of 25-50µm and have a single-lobed, round nucleus. Macrophages then **phagocytose** microorganisms and digest them by releasing granules into the phagosome. They also secrete cytokines which modulate the immune response. In certain situations, monocytes can also differentiate into dendritic cells. These form an important link between the innate and adaptive immune systems. They assist in T cell activation during the adaptive immune response and are the only cell type that can activate naïve T cells. **LYMPHOCYTES:** Like monocytes, the lymphocytes also do not have granules in the cytoplasm. Nucleus is oval, bean-shaped or kidney-shaped. Nucleus occupies the whole of the cytoplasm. A rim of cytoplasm may or may not be seen. Depending upon the function, lymphocytes are divided into two types: NORMAL WHITE BLOOD CELL COUNT: Total WBC count (TC) in adult: 4,000 to 11,000/cu mm of blood. WBC count is about 20,000 per cu mm in infants and about 10,000 to 15,000 per cu mm of blood in children. Slightly more in males than in females ![](media/image7.png) **IMMUNITY** This is defined as the ability of the body to resist pathogenic agents. It is the capacity of body to resist the entry of diverse types of foreign bodies like bacteria, virus, toxic substances, etc. **THE IMMUNE SYSTEM** Immunity or resistance to infection derives from the activity and intact functioning of two tightly interrelated systems, the innate immune system and the acquired immune system. Elements of the innate or natural immune system include exterior defenses, such as skin and mucous membranes; phagocytic leukocytes; and serum proteins, which act nonspecifically and quickly against microbial invaders. Microbes that escape the onslaught of cells and molecules of the innate immune system face destruction by T cells and B cells of the acquired immune system. Activation of the acquired immune system results in the generation of antibodies and cells that specifically target the inducing organism or foreign molecule. Unlike the innate system, adaptive or acquired immune responses develop gradually but exhibit memory. Therefore, repeat exposure to the same infectious agent results in improved resistance mediated by the specific aspects of the acquired immune system. The joint effort of the elements of the innate and acquired immune systems provide a considerable obstacle to the establishment and long-term survival of infectious agents. **INNATE IMMUNITY OR NON-SPECIFIC IMMUNITY**: is the inborn capacity of the body to resist pathogens. This type of immunity represents the first line of defense against any type of pathogens. By chance, if the organisms enter the body, innate immunity eliminates them before the development of any disease. It is otherwise called the natural or non-specific immunity. Infectious agents cannot easily penetrate intact skin, the first line of defense against infection. Infection is a major complication when the intact skin barrier is compromised, such as by burns or trauma. Even a small needle prick can result in a fatal infection. Natural openings to body cavities and glands are an effective entry point of infectious agents. Usually, however, these openings are protected from invasion by pathogens in at least two ways. First, they are coated with mucus and other secretions that contain secretory immunoglobulins as well as antibacterial enzymes, such as lysozyme. Second, organisms that invade these openings cannot easily reach the blood but, instead, lodge in an organ that communicates with both the exterior and the interior of the body, such as a lung or the stomach. Many pathogens cannot survive the low pH of stomach acid. In the lungs, organisms face the efficient phagocytic activity of alveolar macrophages. These cells, derived from blood monocytes, are mobile but confined to the pulmonary capillary network. As efficient phagocytic cells, they continuously patrol the pulmonary vasculature to remove inhaled microbes. Microbes that successfully break through these physical barriers face destruction by the fixed macrophages of the monocyte-macrophage system. These cells line the sinusoids and vasculature of many organs, including the liver, spleen, and bone marrow. The immobile, fixed phagocytic macrophages efficiently remove foreign particles, including bacteria, from the circulation. **ACQUIRED IMMUNITY OR SPECIFIC IMMUNITY**: is the resistance developed in the body against any specific foreign body like bacteria, viruses, toxins, vaccines or transplanted tissues. So, this type of immunity is also known as specific immunity. It is the most powerful immune mechanism that protects the body from the invading organisms or toxic substances. Lymphocytes are responsible for acquired immunity. Two types of acquired immunity develop in the body namely Cellular immunity and Humoral immunity. **T lymphocytes or T cells**, which are responsible for the development of cellular immunity are processed in thymus. The processing occurs mostly during the period between just before birth and few months after birth. Thymus secretes a hormone called thymosin, which plays an important role in immunity. It accelerates the proliferation and activation of lymphocytes in thymus. It also increases the activity of lymphocytes in lymphoid tissues. There are 4 Types of T Lymphocytes which helps immune responses - **Helper T cells or inducer T cells**. These cells are also called CD4 cells because of the presence of molecules called CD4 on their surface. Helper T cells (CD4 cells) which enter the circulation activate all the other T cells and B cells. Normal, CD4 count in healthy adults varies between 500 and 1500 per cubic millimeter of blood. Helper T cells are of two types: 1. Helper-1 (TH1) cells: are concerned with cellular immunity and secrete two substances: i. Interleukin-2, which activates the other T cells. ii. Gamma interferon, which stimulates the phagocytic activity of cytotoxic cells, macrophages and natural killer (NK) cells. 2. Helper-2 (TH2) Cells: are concerned with humoral immunity and secrete interleukin-4 and interleukin-5, which are concerned with: i. Activation of B cells. ii. Proliferation of plasma cells. iii. Production of antibodies by plasma cell. - **Cytotoxic T cells or killer T cells**: These cells are also called CD8 cells because of the presence of molecules called CD8 on their surface. Cytotoxic T cells are activated by helper T cells, circulate through blood, lymph and lymphatic tissues and destroy the invading organisms by attacking them directly. Mechanism of Action of Cytotoxic T Cells is through Receptors situated on the outer membrane of cytotoxic T cells binding with the antigens or organisms tightly. Then, the cytotoxic T cells enlarge and release cytotoxic substances like the lysosomal enzymes which destroy the invading organisms. Like this, each cytotoxic T cell can destroy a large number of microorganisms one after another. Other Actions of Cytotoxic T Cells is its ability to destroy cancer cells, transplanted cells, such as those of transplanted heart or kidney or any other cells, which are foreign bodies. Cytotoxic T cells destroy even body's own tissues which are affected by the foreign bodies, particularly the viruses. Many viruses are entrapped in the membrane of affected cells. The antigen of the viruses attracts the T cells. And the cytotoxic T cells kill the affected cells also along with viruses. Because of this, the cytotoxic T cell is called killer cell. - **Suppressor T cells**: are also called regulatory T cells. These T cells suppress the activities of the killer T cells. Thus, the suppressor T cells play an important role in preventing the killer T cells from destroying the body's own tissues along with invaded organisms. Suppressor cells suppress the activities of helper T cells also. - **Memory T cells**: Storage of T Lymphocytes After the transformation, all the types of T lymphocytes leave the thymus and are stored in lymphoid tissues of lymph nodes, spleen, bone marrow and GI tract. Some of the T cells activated by an antigen do not enter the circulation but remain in lymphoid tissue. These T cells are called memory T cells. In later periods, the memory cells migrate to various lymphoid tissues throughout the body. When the body is exposed to the same organism for the second time, the memory cells identify the organism and immediately activate the other T cells. So, the invading organism is destroyed very quickly. The response of the T cells is also more powerful this time. **B lymphocytes or B cells**, which are responsible for humoral immunity were first discovered in the bursa of Fabricius in birds, hence the name B lymphocytes. Bursa of Fabricius is a lymphoid organ situated near the cloaca of birds. Bursa is absent in mammals and the processing of B lymphocytes takes place in liver (during fetal life) and bone marrow (after birth). After transformation, the B lymphocytes are stored in the lymphoid tissues of lymph nodes, spleen, bone marrow and the GI tract After processing, the B lymphocytes are transformed into two types: - **Plasma cells**: destroys the foreign organisms by producing the antibodies. Antibodies are globulin in nature. The rate of the antibody production is very high, i.e. each plasma cell produces about 2000 molecules of antibodies per second. The antibodies are also called immunoglobulins. Antibodies are released into lymph and then transported into the circulation. The antibodies are produced until the end of lifespan of each plasma cell, which may be from several days to several weeks - **Memory cells**: occupies the lymphoid tissues throughout the body. The memory cells are in inactive condition until the body is exposed to the same organism for the second time. During the second exposure, the memory cells are stimulated by the antigen and produce more quantity of antibodies at a faster rate, than in the first exposure. The antibodies produced during the second exposure to the foreign antigen are also more potent than those produced during first exposure. This phenomenon forms the basic principle of vaccination against the infections **Antigens** are the substances which induce specific immune reactions in the body. Antigens are mostly the conjugated proteins like lipoproteins, glycoproteins and nucleoproteins. Antigens are of two types: 1\. Autoantigens or self-antigens present on the body's own cells such as 'A' antigen and 'B' antigen in RBCs. 2\. Foreign antigens or non-self-antigens that enter the body from outside, are classified into two types, depending upon the response developed against them in the body: - Antigens, which induce the development of immunity or production of antibodies (immunogenicity). - Antigens, which react with specific antibodies and produce allergic reactions (allergic reactivity). **Cell-mediated immunity** is defined as the immunity developed by cell-mediated response. It is also called cellular immunity or T cell immunity. It involves several types of cells such as T lymphocytes, macrophages and natural killer cells and hence the name cell mediated immunity. Cell-mediated immunity does not involve antibodies. Cellular immunity is the major defense mechanism against infections by viruses, fungi and few bacteria like tubercle bacillus. It is also responsible for delayed allergic reactions and the rejection of transplanted tissues. Cell-mediated immunity is offered by T lymphocytes and it starts developing when T cells come in contact with the antigens. Usually, the invading microbial or non-microbial organisms carry the antigenic materials. These antigenic materials are released from invading organisms and are presented to the helper T cells by antigen-presenting cells. - Helper T cell recognizes the antigen displayed on the surface of the antigen-presenting cell with the help of its own surface receptor protein called T cell receptor. - Recognition of the antigen by the helper T cell initiates a complex interaction between the helper T cell receptor and the antigen. This reaction activates helper T cells. - At the same time, macrophages (the antigen-presenting cells) release interleukin-1, which facilitates the activation and proliferation of helper T cells. - Activated helper T cells proliferate and the proliferated cells enter the circulation for further action - Simultaneously, the antigen which is bound to class II Major histocompatibility complex (MHC) molecules activates the B cells also, resulting in the development of humoral immunity **Humoral immunity** is defined as the immunity mediated by antibodies, which are secreted by B lymphocytes. Humoral immunity consists of defense mechanisms carried out by soluble mediators in the blood plasma. Antibodies (also called immunoglobulins) are glycoproteins secreted by plasma cells. Antibodies are found in high levels in plasma and other body fluids. They have the ability to bind specifically to the antigenic determinant that induced their secretion. B lymphocytes secrete the antibodies into the blood and lymph. The blood and lymph are the body fluids (humours or humors in Latin). Since the B lymphocytes provide immunity through humors, this type of immunity is called humoral immunity or B cell immunity. Antibodies are the gamma globulins produced by B lymphocytes. These antibodies fight against the invading organisms. The humoral immunity is the major defense mechanism against the bacterial infection. As in the case of cell-mediated immunity, the macrophages and other antigen-presenting cells play an important role in the development of humoral immunity also. Immunoglobulins form 20% of the total plasma proteins. Antibodies enter almost all the tissues of the body. Five types of antibodies are identified namely: - IgA (Ig alpha): plays a role in localized defense mechanism in external secretions like tear - IgD (Ig delta): s involved in recognition of the antigen by B lymphocytes - IgE (Ig epsilon): is involved in allergic reactions - IgG (Ig gamma): is responsible for complement fixation - IgM (Ig mu): is also responsible for complement fixation. Among these antibodies, IgG forms 75% of the antibodies in the body. **Antibodies** protect the body from invading organisms in two ways 1\. By Direct Actions: Antibodies directly inactivate the invading organism by any one of the following methods: - Agglutination: In this, the foreign bodies like RBCs or bacteria with antigens on their surfaces are held together in a clump by the antibodies. - Precipitation: In this, the soluble antigens like tetanus toxin are converted into insoluble forms and then precipitated. - Neutralization: During this, the antibodies cover the toxic sites of antigenic products. iv. Lysis: It is done by the most potent antibodies. These antibodies rupture the cell membrane of the organisms and then destroy them. 2\. Through complement system: The indirect actions of antibodies are stronger than the direct actions and play more important role in defense mechanism of the body than the direct actions. Complement system is the one that enhances or accelerates various activities during the fight against the invading organisms. It is a system of plasma enzymes, normally, these enzymes are in inactive form and are activated in three ways: - Classical pathway: the enzymes binds with the antibodies and triggers a series of events in which other enzymes are activated in sequence. These enzymes or the byproducts formed during these events produce the following activities: i\. Opsonization: Activation of neutrophils and macrophages to engulf the bacteria, which are bound with a protein in the plasma called opsonin. ii\. Lysis: Destruction of bacteria by rupturing the cell membrane. iii\. Chemotaxis: Attraction of leukocytes to the site of antigen-antibody reaction. iv\. Agglutination: Clumping of foreign bodies like RBCs or bacteria. v\. Neutralization: Covering the toxic sites of antigenic products. vi\. Activation of mast cells and basophils, which liberate histamine: Histamine dilates the blood vessels and increases capillary permeability. So, plasma proteins from blood enter the tissues and inactivate the antigenic products. - Lectin pathway Lectin pathway occurs when mannose-binding lectin (MBL), which is a serum protein binds with mannose or fructose group on wall of bacteria, fungi or virus. - Alternate pathway Complementary system is also activated by another way, which is called alternate pathway. It is due to a protein in circulation called factor I. It binds with polysaccharides present in the cell membrane of the invading organisms and activates other enzymes which ultimately attack the antigenic products of invading organism. **IMMUNIZATION** is defined as the procedure by which the body is prepared to fight against a specific disease. It is used to induce the immune resistance of the body to a specific disease. Immunization can be classified into 2: 1. **Passive immunization**: is produced without challenging the immune system of the body. It is done by administration of serum or gamma globulins from a person who is already immunized (affected by the disease) to a non-immune person. Passive immunization is acquired either naturally or artificially. **Passive Natural Immunization** is acquired from the mother before and after birth. Before birth, immunity is transferred from mother to the fetus in the form of maternal antibodies (mainly IgG) through placenta. After birth, the antibodies (IgA) are transferred through breast milk. Lymphocytes of the child are not activated. In addition, the antibodies received from the mother are metabolized soon. Therefore, the passive immunity is short lived. The significance of passive immunity that is obtained before birth is the prevention of Rh incompatibility in pregnancy. **Passive Artificial Immunization** is developed by injecting previously prepared antibodies using serum from humans or animals. Antibodies are obtained from the persons affected by the disease or from animals, particularly horses which have been immunized artificially. The serum containing the antibody (antiserum) is administered to people who have developed the disease (therapeutic). It is also used as a prophylactic measure. Prophylaxis refers to medical or public health procedures to prevent a disease in people who may be exposed to the disease in a later period. This type of immunity is useful for providing immediate protection against acute infections like tetanus, measles, diphtheria, etc. and for poisoning by insects, snakes and venom from other animals. It is also used as a prophylactic measure. However, this may result in complications and anaphylaxis. There is a risk of transmitting HIV and hepatitis. 2. **Active immunization** or immunity is acquired by activating immune system of the body. Body develops resistance against disease by producing antibodies following the exposure to antigens. Active immunity is acquired either naturally or artificially. **Active Natural Immunization**: involves activation of immune system in the body to produce antibodies. It is achieved in both clinical and subclinical infections. Clinical infection is defined as the invasion of the body tissues by pathogenic microorganisms which reproduce, multiply and cause disease by injuring the cells, secreting a toxin or antigen-antibody reaction. During infection, the plasma cells produce immunoglobulins to destroy the invading antigens. Later, due to the activity of memory cells, body retains the ability to produce the antibodies against the specific antigens invaded previously. Subclinical infection is defined as an infection in which symptoms are very mild and do not alert the affected subject. The disease thus produced may not be severe to develop any manifestations. However, it causes the activation of B lymphocytes, resulting in production of antibodies. **Active Artificial Immunization**: is a type of immunization is achieved by the administration of vaccines or toxoids. Vaccine is a substance that is introduced into the body to prevent the disease produced by certain pathogens. Vaccine consists of dead pathogens or live but attenuated (artificially weakened) organisms. The vaccine induces immunity against the pathogen, either by production of antibodies or by activation of T lymphocytes. Edward Jenner produced first live vaccine. He produced the vaccine for smallpox from cowpox virus. Nowadays, vaccines are used to prevent many diseases like measles, mumps, poliomyelitis, tuberculosis, smallpox, rubella, yellow fever, rabies, typhoid, influenza, hepatitis B, etc. Toxoid is a substance which is normally toxic and has been processed to destroy its toxicity but retains its capacity to induce antibody production by immune system. Toxoid consists of weakened components or toxins secreted by the pathogens. Toxoids are used to develop immunity against diseases like diphtheria, tetanus, cholera, etc. The active artificial immunity may be effective lifelong or for short period. It is effective lifelong against the diseases such as mumps, measles, smallpox, tuberculosis and yellow fever. It is effective only for short period against some diseases like cholera (about 6 months) and tetanus (about 1 year). **IMMUNE DEFICIENCY DISEASES** are a group of diseases in which some components of immune system is missing or defective. When the defense mechanism fails or becomes faulty (defective), the organisms of even low virulence produce severe disease. The organisms, which take advantage of defective defense mechanism, are called opportunists. Immune deficiency diseases caused by such opportunists are of two types: 1\. Congenital immune deficiency diseases: are inherited and occur due to the defects in B cell or T cell or both. The common examples are DiGeorge syndrome (due to absence of thymus) and severe combined immune deficiency (due to lymphopenia or the absence of lymphoid tissue). 2\. Acquired immune deficiency diseases: occur due to infection by some organisms. The most common disease of this type is acquired immune deficiency syndrome (AIDS) **AUTOIMMUNE DISEASES** is defined as a condition in which the immune system mistakenly attacks body's own cells and tissues. Normally, body has the tolerance against self-antigen. However, in some occasions, the tolerance fails or becomes incomplete against self-antigen. This state is called autoimmunity and it leads to the activation of T lymphocytes or production of autoantibodies from B lymphocytes. The T lymphocytes (cytotoxic T cells) or autoantibodies attack the body's normal cells whose surface contains the self-antigen or autoantigen. Examples of autoimmune diseases are: 1. Insulin-dependent diabetes mellitus 2. Myasthenia gravis 3. Hashimoto thyroiditis 4. Graves disease 5. Rheumatoid arthritis. **Platelets (Thrombocytes)** They are the formed elements of blood. Platelets are small colorless, non-nucleated and moderately refractive bodies. These formed elements of blood have a Diameter of 2.5 µ (2 to 4 µ), Volume of 7.5 cu µ (7 to 8 cu µ) with several shapes (viz. spherical or rod-shaped and become oval or disk-shaped when inactivated, sometimes, they have dumbbell shape, comma shape, cigar shape or any other unusual shape. Inactivated platelets are without processes or **filopodia** and the activated platelets develop processes or filopodia. Platelets have the following structures: CELL MEMBRANE is 6 nm thick. Cell membrane of platelet contains lipids in the form of phospholipids, cholesterol and glycolipids, carbohydrates as glycocalyx and glycoproteins and proteins. Of these substances, glycoproteins and phospholipids are functionally important. *Glycoproteins* prevent the adherence of platelets to normal endothelium, but accelerate the adherence of platelets to collagen and damaged endothelium in ruptured blood vessels. Glycoproteins also form the receptors for adenosine diphosphate (ADP) and thrombin. *Phospholipids* accelerate the clotting reactions. The phospholipids form the precursors of thromboxane A2 and other prostaglandin-related substances MICROTUBULES form a ring around cytoplasm below the cell membrane. Microtubules are made up of polymerized proteins called tubulin. These tubules provide structural support for the inactivated platelets to maintain the disklike shape. CYTOPLASM contains the cellular organelles, Golgi apparatus, endoplasmic reticulum, mitochondria, microtubule, microvessels, filaments and granules. Cytoplasm also contains some chemical substances such as proteins, enzymes, hormonal substances, etc. Proteins: Contractile proteins (*Actin and myosin*), which are responsible for contraction of platelets, *Thrombosthenin* (Third contractile protein, which is responsible for clot retraction), *von Willebrand factor*: (responsible for adherence of platelets and regulation of plasma level of factor), *fibrin-stabilizing factor* (A clotting factor), *Platelet-derived growth factor (PDGF)* (responsible for repair of damaged blood vessels and wound healing), *Platelet-activating factor (PAF*) (Causes aggregation of platelets during the injury of blood vessels, resulting in prevention of excess loss of blood), Vitronectin (serum spreading factor that promotes adhesion of platelets and spreading of tissue cells in culture), *Thrombospondin* (Inhibits angiogenesis *i.e* formation of new blood vessels from pre-existing vessels). Enzymes: Adensosine triphosphatase (ATPase) and Enzymes necessary for synthesis of prostaglandins. Hormonal Substances: Adrenaline, 2. 5-hydroxytryptamine (5-HT; serotonin), Histamine. Other Substances like Glycogen, calcium, copper, magnesium and iron. **Normal platelet count** is 250000/cu mm of blood. It ranges between 200000 - 400000/cu mm of blood. **Physiological Variations**: Age: Platelets are less in infants (150000 to 200000/cu mm) and reaches normal level at 3rd month after birth. Sex: There is no difference in the platelet count between males and females. In females, it is reduced during menstruation. High altitude: Platelet count increases. After meals: After taking food, the platelet count increases **PROPERTIES OF PLATELETS**: Platelets have three important properties as follows: **Adhesiveness**: is the property of sticking to a rough surface. During injury of blood vessel, endothelium is damaged and the sub-endothelial collagen is exposed. While coming in contact with collagen, platelets are activated and adhere to collagen. Adhesion of platelets involves interaction between von Willebrand factor secreted by damaged endothelium and a receptor protein called glycoprotein Ib situated on the surface of platelet membrane. Other factors which accelerate adhesiveness are collagen, thrombin, ADP, Thromboxane A2, calcium ions, P-selectin and vitronectin **Aggregation:** is the grouping of platelets. Adhesion is followed by activation of more number of platelets by substances released from dense granules of platelets. During activation, the platelets change their shape with elongation of long filamentous pseudopodia which are called processes or filopodia. Filopodia help the platelets aggregate together. Activation and aggregation of platelets is accelerated by ADP, thromboxane A2 and platelet-activating factor. **Agglutination**: is the clumping together of platelets. Aggregated platelets are agglutinated by the actions of some platelet agglutinins and platelet-activating factor **FUNCTIONS OF PLATELETS** 1\. Role in Blood Clotting: Platelets are responsible for the formation of intrinsic prothrombin activator. This substance is responsible for the onset of blood clotting 2\. Role in Clot Retraction: In the blood clot, blood cells including platelets are entrapped in between the fibrin threads. Cytoplasm of platelets contains the contractile proteins, namely actin, myosin and thrombosthenin, which are responsible for clot retraction. 3\. Role in Prevention of Blood Loss (Hemostasis): Platelets accelerate the hemostasis by three ways: i. Platelets secrete 5-HT, which causes the constric tion of blood vessels. ii. Due to the adhesive property, the platelets seal the damage in blood vessels like capillaries. iii. By formation of temporary plug, the platelets seal the damage in blood vessels 4\. Role in Repair of Ruptured Blood Vessel: Platelet-derived growth factor (PDGF) formed in cytoplasm of platelets is useful for the repair of the endothelium and other structures of the ruptured blood vessels 5\. Role in Defense Mechanism: By the property of agglutination, platelets encircle the foreign bodies and destroy them. **Activators of Platelets** includes: Collagen, which is exposed during damage of blood vessels, von Willebrand factor, Thromboxane A2, Platelet-activating factor, Thrombin, ADP, Calcium ions, P-selectin, Convulxin (Purified protein from snake venom). **Inhibitors of Platelets** includes: Nitric oxide, Clotting factors: II, IX, X, XI and XII, Prostacyclin, Nucleotidases (which breakdown the ADP). **[Genesis of Platelets]**: Platelets are formed from bone marrow. Pluripotent stem cell gives rise to the colony forming unit-megakaryocyte (CFU-M). This develops into megakaryocyte. Cytoplasm of megakaryocyte form pseudopodium. A portion of pseudopodium is detached to form platelet, which enters the circulation. Production of platelets is influenced by colony-stimulating factors and thrombopoietin. Colony-stimulating factors are secreted by monocytes and T lymphocytes. Thrombopoietin is a glycoprotein like erythropoietin. It is secreted by liver and kidneys. Lifespan: Average lifespan of platelets is 10 days. It varies between 8 - 11 days. Platelets are destroyed by tissue macrophage system in spleen. So, splenomegaly (enlargement of spleen) decreases platelet count and splenectomy (removal of spleen) increases platelet count. Platelet disorders are: 1\. Thrombocytopenia: this is a decrease in platelet count and it occurs in the following conditions: Acute infections, Acute leukemia, Aplastic and pernicious anemia, Chickenpox, Smallpox, Splenomegaly, Typhoid, Tuberculosis, Purpura etc 2\. Thrombocytosis: this is an Increase in platelet count and it occurs in the following conditions: Allergic conditions, Asphyxia, Hemorrhage, Bone fractures, Surgical operations, Splenectomy, Rheumatic fever, Trauma (wound or injury or damage caused by external force) 3\. Thrombocythemia: is the condition with persistent and abnormal increase in platelet count and it occurs in the following conditions: Carcinoma, Chronic leukemia, Hodgkin's disease 4\. Glanzmann's thrombasthenia: is an inherited hemorrhagic disorder, caused by structural or functional abnormality of platelets. It leads to thrombasthenic purpura. However, the platelet count is normal. It is characterized by normal clotting time, normal or prolonged bleeding time but defective clot retraction. **Hemostasis** Hemostasis is defined as arrest or stoppage of bleeding. When a blood vessel is injured, the injury initiates a series of reactions, resulting in hemostasis. Hemostasis can be viewed as four separate but interrelated events: *Compression and vasoconstriction*: Immediately after tissue injury, blood flow through the disrupted vessel is slowed by the interplay of several important physical factors, usually, arterioles and small arteries constrict due to the damage to the endothelium causing collagen to be exposed, platelets then adhere to this collagen and get activated. The activated platelets secrete serotonin and other vasoconstrictor substances which cause constriction of the blood vessels. The degree of compression varies in different tissues; for example, bleeding below the eye is not readily deterred while in the uterus after childbirth, contraction of underlying muscles compresses blood vessels supplying the tissue and minimizes blood loss. Damaged cells at the site of tissue injury release potent substances that directly cause blood vessels to constrict, including serotonin, thromboxane A2, epinephrine, and fibrinopeptide B. *Formation of a platelet plug:* Disruption of the endothelium at sites of tissue injury exposes a variety of proteins in the sub-endothelial matrix, such as collagen and laminin, which either induce or support platelet adherence. Platelets get adhered to the collagen of ruptured blood vessel and secrete adenosine diphosphate (ADP) and thromboxane A2. These two substances attract more and more platelets and activate them. All these platelets aggregate together and form a loose temporary platelet plug or temporary hemostatic plug, which closes the ruptured vessel and prevents further blood loss. Platelet aggregation is accelerated by platelet-activating factor. *Blood coagulation*: During this process, the fibrinogen is converted into fibrin. Platelet aggregates are trapped in a highly organized, firm, and degradable network of fibrin, the fibrin network traps red cells, leukocytes, platelets, and serum at sites of vascular damage, thereby forming a blood clot. The stable, fibrin-based blood clot eventually replaces the unstable platelet aggregate formed immediately after tissue injury. The clot is held together by noncovalent forces formed by the catalytic action of a plasma enzyme, fibrin stabilizing factor (Factor XIII). *Clot retraction*: is a phenomenon that usually occurs within minutes or hours after clot formation. The clot draws together, extruding a very large fraction of the serum. The retraction requires platelets. Clot retraction decreases the breakdown of the clot and enhances wound healing. **BLOOD GROUPS** The discovery of blood groups by the Austrian Scientist Karl Landsteiner, in 1901 ended the mystery of blood clumping (agglutination) when blood from two individuals is mixed because of the immunological reactions. He was honored with Nobel Prize in 1930 for this discovery. **ABO SYSTEM**: Blood groups are genetically determined by the presence of the antigens found on the membranes of red blood cells. Agglutination, a process whereby cells clump together, occurs when red cells with a specific antigen encounter its corresponding antibody. In vivo agglutination results in either intravascular or extravascular haemolysis. The ABO blood group system consists of four main groups and these are determined by the presence or absence of the antigens, A and B. The presence of antigen A or antigen B gives rise to Group A or Group B, the presence of both antigens gives rise to Group AB and the absence of both antigens gives rise to Group O. Antibodies to these antigens can be either naturally occurring or as a result of an immune response. Immune antibodies arise when an individual is exposed to foreign red cell antigens through either transfusion or passage of red cells across the placenta during pregnancy. **Determination of the ABO group** is also called blood grouping, blood typing or blood matching. Principle of Blood Typing -- is on the basis of agglutination. Agglutination means the collection of separate particles like RBCs into clumps or masses. Agglutination occurs if an antigen is mixed with its corresponding antibody which is called isoagglutinin. Agglutination occurs when A antigen is mixed with anti-A or when B antigen is mixed with anti-B. To determine the blood group of a person, a suspension of his RBC and testing antisera are required. Suspension of RBC is prepared by mixing blood drops with isotonic saline (0.9%). Test sera are: Antiserum A, containing anti-A or α-antibody and Antiserum B, containing anti-B or β-antibody. ***Procedure***: 1. One drop of antiserum A is placed on one end of a glass slide (or a tile) and one drop of antiserum B on the other end. 2. One drop of RBC suspension is mixed with each antiserum. The slide is slightly rocked for 2 minutes. The presence or absence of agglutination is observed by naked eyes and if necessary, it is confirmed by using microscope. 3. Presence of agglutination is confirmed by the presence of thick masses (clumping) of RBCs 4. Absence of agglutination is confirmed by clear mixture with dispersed RBCs. ***Results***: 1. If agglutination occurs with antiserum A: The antiserum A contains α-antibody. The agglutination occurs if the RBC contains A antigen. So, the blood group is A. 2. If agglutination occurs with antiserum B: The antiserum B contains β-antibody. The agglutination occurs if the RBC contains B antigen. So, the blood group is B. 3. If agglutination occurs with both antisera A and B: The RBC contains both A and B antigens to cause agglutination. And, the blood group is AB. 4. If agglutination does not occur either with antiserum A or antiserum B: The agglutination does not occur because RBC does not contain any antigen. The blood group is O. **Rhesus (Rh) Blood Group System**: The Rhesus system comprises five main antigens, namely C, c, D, E and e. The term rhesus-positive usually refers to those individuals who express the D antigen on their red blood cells and rhesus negative for those whose red cells do not express this antigen. Antibodies to these rhesus antigens occur very rarely in nature, although there are some forms of naturally occurring anti-E. The production of immune antibodies, most commonly anti-D, occurs after sensitisation by pregnancy or transfusion. It is for this reason that anti-D is injected into a rhesus-negative mother after transplacental passage of fetal blood into the maternal circulation. This destroys any fetal rhesus positive red blood cells before the maternal immune system can respond. **HEMOGLOBIN** Hemoglobin (Hb) is the iron containing coloring matter of red blood cell (RBC). It is a chromoprotein forming 95% of dry weight of RBC and 30% to 34% of wet weight. Function of hemoglobin is to carry the respiratory gases, oxygen and carbon dioxide. It also acts as a buffer with a molecular weight of 68,000. Average hemoglobin (Hb) content in blood is 14 to 16 g/dL. However, the value varies depending upon the age and sex of the individual. At birth: 25 g/dL, after 3rd month: 20 g/dL, after 1 year: 17 g/dL, from puberty onwards: 14 to 16 g/dL. At the time of birth, hemoglobin content is very high because of increased number of RBCs. In adult males: 15 g/dL, in adult females: 14.5 g/dL **Functions of Hemoglobin**: A. *TRANSPORT OF RESPIRATORY GASES:* Main function of hemoglobin is the transport of respiratory gases: i. Oxygen from the lungs to tissues: Transport of Oxygen When oxygen binds with hemoglobin, a physical process called oxygenation occurs, resulting in the formation of oxyhemoglobin. The iron remains in ferrous state in this compound. Oxyhemoglobin is an unstable compound and the combination is reversible, i.e. when more oxygen is available, it combines with hemoglobin and whenever oxygen is required, hemoglobin can release oxygen readily. When oxygen is released from oxyhemoglobin, it is called reduced hemoglobin or ferro-hemoglobin. ii\. Carbon dioxide from tissues to lungs: Transport of Carbon Dioxide When carbon dioxide binds with hemoglobin, carb-hemoglobin is formed. It is also an unstable compound and the combination is reversible, i.e. the carbon dioxide can be released from this compound. The affinity of hemoglobin for carbon dioxide is 20 times more than that for oxygen B. *BUFFER ACTION*: Hemoglobin acts as a buffer and plays an important role in acid-base balance **Synthesis of Hemoglobin**: This actually starts in proerythroblastic stage. However, hemoglobin appears in the intermediate normoblastic stage only. Production of hemoglobin is continued until the stage of reticulocyte. Heme portion of hemoglobin is synthesized in mitochondria. And the protein part, globin is synthesized in ribosomes. **Structure of Hemoglobin:** Hemoglobin is a conjugated protein. It consists of a *protein combined* with an *iron-containing pigment*. The protein part is globin and the iron-containing pigment is heme. Heme also forms a part of the structure of myoglobin (oxygen-binding pigment in muscles) and neuroglobin (oxygen-binding pigment in brain). Iron part is normally in ferrous (Fe2+) form. It is in unstable or loose form. In some abnormal conditions, the iron is converted into ferric (Fe3+) state, which is a stable form. The pigment part of heme is called porphyrin. Globin contains four polypeptide chains. Among the four polypeptide chains, two are β-chains and two are α-chains. ***Types of Normal Hemoglobin***: Hemoglobin is of two types: 1\. Adult hemoglobin -- HbA 2\. Fetal hemoglobin -- HbF Replacement of fetal hemoglobin by adult hemoglobin starts immediately after birth. It is completed at about 10th to 12th week after birth. Both types of hemoglobin differ from each other structurally and functionally. *Structural Difference In adult hemoglobin, the globin contains two α-chains and two β-chains. In fetal hemoglobin, there are two α chains and two γ-chains instead of β-chains. Functionally, fetal hemoglobin has more affinity for oxygen than that of adult hemoglobin.* ***Abnormal Hemoglobin***: Abnormal types of hemoglobin or hemoglobin variants are the pathologic mutant forms of hemoglobin. These variants are produced because of structural changes in the polypeptide chains caused by mutation in the genes of the globin chains. Most of the mutations do not produce any serious problem. Occasionally, few mutations result in some disorders. There are two categories of abnormal hemoglobin: 1\. Hemoglobinopathies: are genetic disorder caused by abnormal polypeptide chains of hemoglobin. Examples are: i. *Hemoglobin S*: It is found in sickle cell anemia. In this, the α-chains are normal and β-chains are abnormal. ii. *Hemoglobin C:* The β-chains are abnormal. It is found in people with hemoglobin C disease, which is characterized by mild hemolytic anemia and splenomegaly. iii*. Hemoglobin E:* Here also the β-chains are abnormal. It is present in people with hemoglobin E disease which is also characterized by mild hemolytic anemia and splenomegaly. iv. *Hemoglobin M:* It is the abnormal hemoglobin present in the form of methemoglobin. It occurs due to mutation of genes of both in α and β chains, resulting in abnormal replacement of amino acids. It is present in babies affected by hemoglobin M disease or blue baby syndrome. It is an inherited disease, characterized by methemoglobinemia. 2\. Hemoglobin in thalassemia and related disorders: In thalassemia, different types of abnormal hemoglobins are present. The polypeptide chains are decreased, absent or abnormal. In α-thalassemia, the α-chains are decreased, absent or abnormal and in β-thalassemia, the β-chains are decreased, absent or abnormal. Some of the abnormal hemoglobins found in thalassemia are hemoglobin G, H, I, Bart's, Kenya, Lepore and constant spring ***Abnormal Hemoglobin Derivatives***: 'Hemoglobin derivatives' refer to a blood test to detect and measure the percentage of abnormal hemoglobin derivatives. Hemoglobin is the only carrier for transport of oxygen, without which tissue death occurs within few minutes. When hemoglobin is altered, its oxygen carrying capacity is decreased resulting in lack of oxygen. So, it is important to know about the causes and the effects of abnormal hemoglobin derivatives. Abnormal hemoglobin derivatives are formed by carbon monoxide (CO) poisoning or due to some drugs like nitrites, nitrates and sulphanamides. Abnormal hemoglobin derivatives are: 1\. Carboxyhemoglobin: is the abnormal hemoglobin derivative formed by the combination of carbon monoxide with hemoglobin 2\. Methemoglobin: is the abnormal hemoglobin derivative formed when iron molecule of hemoglobin is oxidized from normal ferrous state to ferric state. Methemoglobin is also called ferrihemoglobin 3\. Sulfhemoglobin: is the abnormal hemoglobin derivative, formed by the combination of hemoglobin with hydrogen sulfide. It is caused by drugs such as phenacetin or sulfonamides Normal percentage of hemoglobin derivatives in total hemoglobin: Carboxyhemoglobin: 3% to 5 % Methemoglobin: less than 3% Sulfhemoglobin: trace (undetectable). Abnormally high levels of hemoglobin derivates in blood produce serious effects. These derivatives prevent the transport of oxygen resulting in oxygen lack in tissues, which may be fatal. ***Destruction of Hemoglobin:*** After the lifespan of 120 days, the RBC is destroyed in the reticuloendothelial system, particularly in spleen and the hemoglobin is released into plasma. Soon, the hemoglobin is degraded in the reticuloendothelial cells and split into globin and heme. Globin is utilized for the resynthesis of hemoglobin. Heme is degraded into iron and porphyrin. Iron is stored in the body as ferritin and hemosiderin, which are reutilized for the synthesis of new hemoglobin. Porphyrin is converted into a green pigment called biliverdin. In human being, most of the biliverdin is converted into a yellow pigment called bilirubin. Bilirubin and biliverdin are together called the bile pigments. **Jaundice** (hyperbilirubinemia) is an elevated level of the pigment bilirubin in the blood, causing yellow discoloration of the body tissue resulting from the accumulation of an excess of bilirubin in skin. The Neonates jaundice is clinically diagnosed when total serum bilirubin level (TSB) above 5 mg per dL. Bilirubin is produced from the catabolism of heme with liberation of iron, carbon monoxide and biliverdin. The biliverdin is reduced to form bilirubin. The bilirubin produced is then transported to the liver in the conjugated form with plasma albumin and is discharged into the bile and intestine. In the newborns, most of the conjugated bilirubin in the gut is decomposed back to unconjugated bilirubin in the intestinal mucosa. The reabsorptions of unbound bilirubin into the blood stream take place by the action of enterohepatic circulation, this leads more of bilirubin load to the already overloaded liver. Severe hyperbilirubinemia (more than 20 mg/dL) could possibly cause kernicterus and neurodevelopmental problems less than of 2% of neonatal infants may be affected. Various risk factors increase the incidence of jaundice this includes preterm neonatal, low birth weight, hemolysis, sepsis, cephalohematoma or easy bruising. **Physiological Jaundice** is the one of more common type of neonatal jaundice, does not cause serious problems. Physiological jaundice usually appears after at least 24 hours of birth, and peak after four or five days. It later disappears after about 2 weeks of life. The bilirubin associated with physiologic jaundice is mostly unbounded, and its levels in serum do not excess 15 mg/dl. Many clinical conditions can lead to the occurrence of physiologic jaundice in the neonate like high bilirubin load of relative polycythemia, immature hepatic uptake, a shortened life span of red blood cell, and higher enterohepatic circulation. Other type of neonatal jaundice is **pathologic jaundice** which is characterized by rapid onset of jaundice (first 24 hours after delivery), the rapid elevation of total serum bilirubin level (elevate of more than 5 mg/dL/day), and a total serum bilirubin concentration more than (17 mg/dL) in a full-term infant frequently happens as a result of mostly ABO or Rhesus incompatibility or another reasons of large hemolysis. **Assignment:** Discuss the lymphatic system and the lymph.

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