Blood PDF

BLOOD BLOOD Blood is an uncoagulated fluid tissue It is composed of fluid portion known as the plasma and suspended cellular elements, the erythrocytes‚ (RBCs), leukocytes‚ (WBCs) and thrombocytes‚ (platelets). Plasma constitutes abou...

BLOOD BLOOD Blood is an uncoagulated fluid tissue It is composed of fluid portion known as the plasma and suspended cellular elements, the erythrocytes‚ (RBCs), leukocytes‚ (WBCs) and thrombocytes‚ (platelets). Plasma constitutes about 55 to 70% of the blood volume, while the cellular components account roughly about 30-45% of the total blood. General functions of blood Maintains homeostasis - constant internal environment. Transports oxygen from the lungs to the tissues, hormones from the endocrine glands tothe target organs. Distributes the nutrients from the G.I. tract to the tissues. Removes/transports the metabolic end products-CO2 from the tissues to lung, and urea, uric acid, creatine and creatinine to the kidneys. Regulates acid-base balance of body fluids by blood-buffer systems. Regulates water balance and body temperature. Provides defence mechanism against harmful microorganisms. Specific gravity of blood The ratio between the weight of a certain volume of blood to the weight of the same volume of water is specific gravity The cellular elements called the corpuscles have higher specific gravity than the plasma. Because of specific gravity, when anticoagulant added blood is allowed to stand undisturbed, the cells settle to the bottom and plasma moves to the top; rouleaux formation also influences settling down of blood cells Plasma protein concentration is mainly responsible for the specific gravity of the plasma. Specific gravity Goat: 1.042 (1.036 - 1.051) Pig: 1.045 (1.035 - 1.055) Dog: 1.048 (1.045 - 1.052) Cat: 1.050 (1.045 - 1.057) Sheep: 1.051 (1.041 - 1.061) Cattle: 1.052 (1.046 - 1.061) Horse: 1.053 (1.046 - 1.059) Specific gravity can be found out by using CuSO4 solutions at different concentrations. Viscosity of blood It is the resistance offered by blood to flow. It is normally about five times greater than water. Viscosity is mainly contributed by the gamma globulins. Viscosity is influenced by concentration of RBCs and plasma proteins. Viscosity increases the resistance to blood flow, thus helps the pumping action of the heart. Reactions of the blood The term reaction of blood refers to the pH of blood The normal pH of the blood is 7.4.- a pH on the alkaline side 1  BLOOD The balance between all the cations and anions is resultant H + ion concentration – the negative log of H+ ion concentration is pH Arterial blood is slightly more alkaline than the venous blood because of greater amount H+ ions formed from CO2 reaction with H2O The plasma is more alkaline than the corpuscles. The pH range compatible with life is between 7.0 and 7,8 The pH range: dog 7.32- 7.68; cattle 7.35 - 7.50; horse.35 - 7.43; fowl 7.56. Abnormal reduction in the alkaline reserve due to excessive production of metabolic acid products causes the condition referred to as acidosis Abnormal increase in the alkaline reserve is designated as alkalosis. Plasma The fluid part of the uncoagulated blood is called as plasma. It is colourless or slightly yellow coloured in dog, sheep and goat, while it is highly yellow coloured in horse and cow The colour is chiefly due to bilirubin‚ and to some extend by the carotene and other pigments. Plasma can be separated by allowing anticoagulant added blood to stand undisturbed – the cells settle down and plasma moves to the top; centrifuging this blood makes the cells to settle faster and plasma can be separated quickly. Serum In the absence of the anticoagulants, the shed blood gets coagulated to form a blood clot, which shrinks and discharges a clear watery liquid called the serum‚ (i.e. serum is the fluid that is discharged after the blood clots or the fluid that comes out from blood clot). It differs from the plasma in lacking fibrinogen, prothrombin and other coagulation factors involved in blood coagulation. ERYTHROCYTES (RBCs) Normally erythrocytes appear as biconcave circular disks and are non-nucleated. Typical biconcave erythrocytes are present in dog, cow and sheep. The RBCs of horse and cat show shallow concaving, In goats, very shallow or flat surfaced erythrocytes are common. The biconcave surface provides large surface area for gaseous exchange across the cell membrane. RBCs undergo change in shape when they pass through capillaries whose size is smaller than RBCs Camel and deer RBCs show elliptical and sickle shapes respectively without nuclei. In sub mammals (birds and amphibians) the RBCs are elliptical in shape and are nucleated. 2  BLOOD Terms used to describe erythrocyte morphology Acanthocyte Cells with irregular, long, asymmetrical projections or spikes Anisocytosis Variation in size of RBC's Elliptocyte Elongated, elliptical cell Heinz body Inclusions within RBCs, composed of precipitated Hg Howell-Jolly body Small, round deeply basophilic nuclear remnant seen in circulating RBCs Hyperchromic RBCs having a greater density of colour (due to higher Hb content) Hypochromic RBCs are paler than normal - Cells with decreased MCHC, iron deficiency Large sized RBCs with near-constant hemoglobin concentration, and having a Macrocytic MCV of greater than 100 femtolitres – megaloblastic anemia BCs are unusually small - Cells with decreased MCV, seen in iron Microcytic deficiency anemia and thalassemias Normochromic RBCs with a normal concentration of hemoglobin Normocytic Red blood cell of normal size. Poikilocytosis Variation in shape of RBC's Polychromatophilia RBCs of multiple colours, particularly gray-blue.[The bluish tint to young RBC's (polychromasia) with high RNA content] Reticulocyte Young [immature] RBC's with increased RNA content in the circulation Linear aggregation of RBC's that resembles a stack of coins; seen when surface Rouleaux charge is reduced with increased serum protein, particularly increased fibrinogen or globulin atypical, abnormal nucleated erythroblasts with granules of iron Sideroblast accumulated in perinuclear mitochondria Sphere-shaped, rather than bi-concave disk shaped erythrocytes. Seen in Spherocyte hereditary spherocytosis and autoimmune hemolytic anemia Fragmented, irregularly shaped seen with intravascular hemolysis. A variant Schistocyte called a "helmet cell" appears cut in half Curved, banana-shaped cell with pointed ends found in sickle cell disease from Sickle cell precipitation of Hgb S Erythrocyte contains 62 - 72% water and 35% solids. Of the total solids, 95% is Hb and the remaining 5% consists of cell and stromal protein, lipids, phospholipids, cholesterol, cholesterol esters, neutral fat and vitamins. Erythrocytes contain an important enzyme carbonic anhydrase which helps in the transport of CO2 from tissues to lungs. Structure The red blood cell membrane is composed of 3 layers: - the glycocalyx layer on the exterior, rich in carbohydrates; - the lipid bilayer - contains many transmembrane proteins, in addition to its lipid constituents; - the membrane skeleton, a structural network of proteins located on the inner surface of the lipid bilayer 3  BLOOD RBC membrane is made up of Lipid bilayer (40%) Membrane proteins (52%) Carbohydrate (8%) Membrane lipids consist of phospholipids (phosphatidylcholine, sphingomyelin) and, unesterified free cholesterol membrane proteins are responsible for the deformability, flexibility and durability of the red blood cell and enabling it to squeeze through capillaries less than half the diameter of the erythrocyte (7-8 μm) and recovering the discoid shape after exit from capillaries Examples of membrane proteins: - Band 3 (anion transport, structural support), glycophorin, Aquaporin (water transport) Spectrin, actin, ankyrin, adducin, tropomycin, tropomodulin Membrane proteins are responsible for membrane elasticity and stability. Membrane glycoproteins carry the various blood group antigens, such as the A, B and Rh antigens Spectrin is a cytoskeletal protein that lines the intracellular side of the plasma membrane ofmany cell types (muscles, red blood cells). It plays an important role in the maintenance of plasma membrane integrity and cytoskeletal structure. The cell membrane is highly permeable to lipid soluble substances and also to glucose, urea and water. Haemoglobin is deposited in the interspaces of the spongy stroma. The surface of mature erythrocyte is smooth, while the immature RBCs have relatively rough surface. Size Average diameter of RBC ranges from 4 to 8 m Goat RBCs have the smallest diameter and hence goats have largest number of RBCs per unit volume of blood Size Goat Sheep Cattle, horse Pig Cat Dog man Poultry In m 4.1 5 5.6 6.2 6.5 7.3 7.5 In fl 20 34 52, 45 60 70 95 130 Surface area is important for the gas transport function of the blood. Erythrocyte surface area varies from 57-67m2 / kg body weight in mammals. Surface area is lowest in goat (less RBC diameter) and highest in man (more diameter). RBC metabolism Energy is required for RBCs To maintain the shape and flexibility of the cell membrane. To preserve high K+, low Na+ and low Ca++ ions within the RBCs against the concentration gradient of these ions of plasma. To maintain iron in ferrous (Fe++) state (to reduce ferric to ferrous state, NADH and NADPH are required). To generate reduced glutathione (anti-oxidant); this helps to maintain the ferrous state. To generate 2-3 DPG for O2 dissociation. Mitochondria are absent in mature erythrocytes. Erythrocytes derive their energy from glucose metabolism via anaerobic EM pathway (90%) and oxidative pentose cycle (10%) which produce NADH and NADPH. Kreb's cycle is very much reduced in activity. 4  BLOOD RBC number Interspecies, intraspecies and diurnal variation, age, sex, environment, exercise, nutritional status, pregnancy, egg production, excitement, hydration status, climate and altitude cause change in RBC number. Excitation, frightening and stress activate sympathetic nervous system, mobilizes stored RBCs and increases erythrocyte number in blood (in horses up to 30% of erythrocytes are stored in spleen and released when required like during strenuous exercise) Males have higher erythrocyte count than females (due to stimulatory effect oftestosterone on erythropoiesis) Among domestic animals, goats have the highest number of erythrocytes per unit volume of blood. RBC number in domestic animals‚ (millions/ l or mm3 of blood) Means and ranges of erythrocyte numbers Cattle 7.0 (5.0-10.0) Pig 6.5 (5.8-8.0) Man 5.4 (5.0-6.0) Horse 6.5 (6.5-12.5) Dog 6.8 (5.5-8.5) Women 4.8 (4.0-5.0) Sheep 12.0 (8.0-16.0) Cat 7.5 (5.0-10.0) Fowl 3.0 (2.8-3.2) Goat 13.0 (8.0-18.0) ABNORMAL LEVELS OF ERYTHROCYTES IN CIRCULATION POLYCYTHEMIA or erythrocytosis Polycythemia is a condition of increased number of RBCs in the circulation. It is of two types. I. Physiological (secondary) polycythemia An increase in erythrocytes that occurs as a compensatory measure. E.g., high altitude -to compensate low PO2 in the atmosphere. Increased Hb requirement during increased muscular exercise to meet increased oxygen demand. In sports animals (racehorse, hunting dogs) RBC elevation is a normal feature Increased environmental stress/ temperature causes increased number of RBCs into the circulation by splenic contraction, and increased RBC synthesis by the bone marrow. Hemoconcentration due to water loss that occurs in vomiting, diarrhoea, prolonged high fever and burns also causes polycythemia. Some athletes may misuse blood transfusion to increase erythrocyte level in blood for enhanced O2 supply and improved athletic performance. This is called blood doping. Erythropoietin injection is also used to increase erythrocyte level of athletes and it is also blood doping. II. Pathological (secondary) polycythemia Pathological polycythemia occurs due to decreased O2 supply to the tissues, chronic carbon monoxide poisoning, myeloid (bone marrow) cancer, pulmonary emphysema, repeated haemorrhage. Polycythemia vera is increased erythrocyte numbers in the circulation due to bone marrow cancer (myeloid leukemia). Patients with polycythemia vera can be asymptomatic. A classic symptom of polycythemia vera is itching, particularly after exposure to warm water (such as when taking a bath), which may be due to abnormal histamine release OLIGOCYTHEMIA Reduction in the number of erythrocytes in the circulation is called as oligocythemia 5  BLOOD It is of two types Physiological oligocythemia that occurs due to hemodilution; RBC number per unit volume is reduced. Pathological oligocythemia is also known as anaemia ANAEMIA: Reduction in the number of the erythrocytes or the haemoglobin content in the blood or both is called as anaemia. Capacity to transport O2 in the blood is greatly reduced in anaemia Causes: Excessive whole blood loss that occurs in haemorrhage or by blood sucking parasites (hookworms, ticks); increased destruction of RBCs by the reticuloendothelial cells. Impaired RBC production and Hb synthesis due to deficiency of Fe2+, Cu2+, vitamin B12 andfolic acid. Haemolytic: diseases caused by blood parasites, (babesiosis) or drugs like sulphonamides, antimalarial drugs and high doses of aspirin (analgesic) Classification of anaemia i). Anaemia due to defective blood formation: Aplastic anaemia is lack of functional bone marrow due to excessive x-ray treatment or bone marrow cancer. Anemic anaemia is due to deficiency of iron, folic acid, Vit.B12 (extrinsic factor) and intrinsic factor of the gastric mucosa. Deficiency of iron results in small sized, decreased number of RBCs and low Hb content known as the microcytic and hypochromic anaemia. Iron deficiency anaemia is the most common form of anaemia in human beings caused by deficiency of iron in the diet or duo to reduced absorption from GI tract. Iron deficiency anaemia is rare in adult domestic animals; In piglets, a severe form anaemia called piglet anaemia develops if iron is not supplemented through diet or if the piglets are raised in solid floors without access to soil, Piglets are born with a very minimal store of iron, sow’s milk is low in iron and demand for iron is high due to faster growth rate, this form of anaemia develops frequently in piglets Lack of extrinsic factor - the vitamin B12 causes decreased number of RBCs, large sized RBCs having high Hb content known as macrocytic and hyperchromic anaemia (megaloblastic anaemia in human beings). In domestic animals vitamin B12 deficiency causes anaemia but cell size is not altered. Deficiency of the intrinsic factor of the gastric mucosa (due to atropic gastritis, parietal cell loss)) interferes with the vitamin B12 absorption and this type of anaemia is known as pernicious anaemia (macrocytic normochromic). ii). Anaemia due to excessive blood loss or increased RBC destruction: Haemorrhagic anaemia: Excessive blood loss due to accident, peptic ulcers etc. Haemolytic anaemia: Following acute destruction of RBCs (haemolysis) the number of RBCs is below normal, but the RBC size and Hb content are normal known as normocytic and normochromic anaemia. This is caused by blood parasites: Eg. babesiosis, theileriosis and trypnasomiasis; by chemicals: copper, lead, nitrate and nitrite. Sickle cell anaemia of human beings is haemolytic anaemia caused by hereditary defect in which RBCs break down while passing through capillaries 6  BLOOD Physiological anaemia: Erythrocyte count, PCV and Hb concentration decreases after the birth of the young ones up to few days to weeks after birth called physiological anaemia. Observed in puppies, kittens, lambs and kids. This cannot be treated with iron supplements iii). Anaemia due to abnormal structure of RBC: In some hereditary diseases, defects in RBC membrane (e.g., sickle cell anaemia), defects inthe globin chain structure (thalassemia) or its synthesis or the deficiency of the enzymes of the RBCs energy system, (the pyruvate kinase and G.6-PD) leads to anaemia. Sickle cell anaemia (sickle cell disease) is a disorder of the blood caused by inherited abnormal haemoglobin. The abnormal haemoglobin is called sickle haemoglobin or haemoglobin S, which causes the cells to develop a sickle, or crescent shape; sickling is promoted by low PO2, increased acidity, or dehydration When RBCs containing Hb S are exposed to deoxy conditions, the sickling process begins. After repeated sickling, membrane damage occurs and the cells are no longer capable of resuming the biconcave shape upon reoxygenation. Thus, they become irreversibly sickled cells; the RBC membrane becomes fragile and breaks down leading to intravascular hemolysis Thalassemia is an inherited autosomal recessive blood disease. It results in reduced rate ofsynthesis or no synthesis of one of the two globin chains. This can cause the formation of abnormal hemoglobin molecules, thus causing anemia [Thalassemia is a quantitative problem of too few globins synthesized, whereas sickle-cell anemia is a qualitative problem of synthesis of an incorrectly functioning globin]. Two major forms of the thalassemia are alpha- and beta- thalassemia (based on which chain of the hemoglobin molecule is affected) In α thalassemias, production of the α-globin chain is affected, while in β-thalassemia production of the β globin chain is affected. About 3% of population has α and δ chains in Hb and defective δ chain formation produce δ-thalassemia The thalassemia trait may confer a degree of protection against malaria, thus conferring aselective survival advantage on carriers Erythrocytic parameters (Erythrocyte Indices): a) Mean corpuscular volume (MCV) It expresses the average cell size of the erythrocyte. PCV 10 MCV( fl / cu m) No.of RBC / l x106 b) Mean corpuscular haemoglobin (MCH) It gives the average weight of Hb present in the erythrocytes. Hb (g%) 10 MCH( pg) No.of RBC/ l x106 c) Mean corpuscular haemoglobin concentration (MCHC) It is the average percentage of the mean corpuscular volume that the Hb occupies. Hb (g%) 100 MCHC(g%) PCV / dL The above three parameters help in the diagnosis various types of anaemia (microcytic vs. macrocytic or normocytic). Iron deficiency causes microcytic anaemia in humans and animals; pernicious anaemia in humans have macrocytic cells but not in animals Young ones after birth have greater MCV which decreases after birth 7  BLOOD Haemolysis: Haemolysis is the discharge of Hb from the RBC into the surrounding medium. The haemolysed blood is referred to as ‘laked blood’. Solutions having same osmotic pressure as blood are called as isotonic solution. 0.89% NaCl (154 mM solution containing 308 mM osmotically active particles) solution is isotonic to blood. Hypertonic solutions have more osmotic pressure than blood Hypotonic solutions have less osmotic pressure than blood. Erythrocytes suspended in hypertonic solution will shrink due to loss of cellular water and these cells are designated as crenated cells. In hypotonic solution the cells swell leading to breaking of the cell membrane and release of Hb. This is called as haemolysis. Packed Cell Volume (PCV) or Haematocrit (%) It is the percentage of blood volume that is occupied by red blood cells. It is expressed in % Haematocrit is measured by two methods. Wintrobe h aematocrit (macro) method: Wintrobe tubes have uniform 3mm bore, calibrated by 10cm scales with millimetre divisions. The wintrobe pipette with a long but narrow delivery tube is used to fill about 1 ml of blood into the wintrobe tube without air bubbles. Horse and dog blood require a relative centrifugal force of 2,260 G (generally 3000rpm) for 30 minutes, while it is for 60 minutes in the case of cattle and pig blood. The blood of sheep and goat require higher relative centrifugal force than 2,260 G. The RBCs settle to the bottom of the haematocrit tube leaving the plasma above the packed erythrocytes. A thin buffy white or yellow layer called the buffy coat occurs in between the packed erythrocytes and plasma and it indicates the WBC or the leukocytes. A small amount of plasma is trapped between the RBCs during centrifugation Canine blood is characterised by large blood cells and greater tendency for rouleaux formation. Similarly equine blood is characterised by extreme rouleaux formation hence less plasma istrapped. True PCV = Venous PCV x 0.96 (correction factor for trapped plasma). The amount of trapped plasma in the erythrocyte column varies with PCV, size of erythrocytes and the degree of rouleaux formation. Microh aematocrit method PCV may be estimated by blood filled capillary tubes (32mm x 0.8mm) centrifuged at14,000G for 2 minutes. PCV can be determined speedily, less amount of blood is required and estimated PCV is nearer to true PCV value (less plasma is trapped) PCV values ranges between 28 to 45% in most of the domestic animals. Horse - 42%, cow - 40%, sheep-32%, pig-42% and dog 45% In general, the PCV is approximately three times the Hb concentration. Factors influencing PCV PCV is raised in – dehydration, burns, splenic contraction, polycythemia, muscular exercise, stress, high temperature , pregnancy 8  BLOOD It is lowered in – blood loss, hemolysis, chronic inflammation, bone marrow neoplasm or suppression, anaemia, deficiency of iron, copper, cobalt, folic acid PCV varies between species influenced by number per unit volume and size; goats have more RBCs but PCV of dogs is greater since their RBCs are larger in size; PCV varies between breeds Normal Ranges and Means of Erythrocyte parameters in Domestic Animals Animal RBCx106/ l Hb (g/dl) PCV (%) MCV (fl) MCHC (%) Dog 5.5-8.5 (6.8) 12-18 (15) 37-55 (45) 60-77 (70) 32-36(34) Cat 5.0-10 (7.5) 8-15 (12) 24-45 (37) 39-55 (45) 30-36 (33) Cow 5.0-10 (7.0) 8-15 (11) 24-46 (35) 40-60 (52) 30-36(33) Sheep 8.0-16 (12) 8-16 (12) 24-50 (38) 23-48 (33) 31-38 (33) Goat 8.0-18 (13) 8-14 (11) 19-38 (28) 15-30 (23) 35-42 (38) Horse (hot-blooded) 6.5-12.5 (9.5) 11-19 (15) 32-52(42) 34-58(46) 31-37 (35) Pig 5.0-8.0(6.5) 10-16 (13) 32-50 (42) 50-68 (63) 30-34 (32) Erythrocyte Sedimentation rate (ESR): It is the rate at which the blood cells precipitate in a given time usually 30 minutes orone hour when citrated blood-filled standard haematocrit tube is placed in an absolutely vertical position. The ESR is governed by the balance between pro-sedimentation factors, mainly fibrinogen, and those factors resisting sedimentation- the negative charge of the erythrocytes. When left undisturbed, the red cells tend to agglutinate and become staked on each other called 'rouleaux' formation and this makes the RBCs to settle faster.An increase in fibrinogen level increases ESR The length of storage of the blood sample and temperature of storage influence ESR. Sludged blood (stasis of blood in capillaries) increases ESR Rapid ESR accompanies anaemia ESR is negatively influenced by reticulocytes and plasma content of albumin. ESR becomes rapid in inflammatory conditions, hypothyroidism and pregnancy. Fibrinogen and α- globulin are commonly elevated in inflammatory diseases e.g., pleurisy, pericarditis, peritonitis, acute general infections (septicaemia), and rheumatic fever, TB, arthritis, toxaemia, malignant tumours Hemoconcentration and glomerulonephritis reduces the ESR by altering the level of albumin fraction. The speed of settling of RBCs is inversely proportional to the number of erythrocytes in the given sample. Polycythemia decreases ESR Among the domestic animals, the erythrocytes of ruminants show little or low natural tendency to form rouleaux, whereas the equine blood shows high tendency to form rouleaux formation. Hence, ESR is rapid in horses and very slow ruminants blood. Horse erythrocytes settle rapidly in ruminant plasma and ruminant erythrocytes settle slowly in horse plasma ESR Value (mm) Cattle, Sheep, Goat Dog Cat Horse Chicken 30 minutes -- 1-5 -- 15-38 0-1 60 minutes -- 6-10 7–27 -- 1–3 The ESR merely helps in evaluating the health status of animals. It is a non-specific measure of inflammation ESR is not helpful in diagnosing any specific diseases 9  BLOOD Factors influencing ESR Size and shape of erythrocytes, specific gravity of erythrocytes, specific gravity of plasma, viscosity of plasma, fibrinogen and alpha globulin content, temperature. HAEMATOPOIESIS It is the processes of formation of erythrocytes, leukocytes and platelets in the body. Formation of erythrocytes, leukocytes and thrombocytes are known as erythropoiesis, leukopoiesis and thrombopoiesis respectively. During embryonic state the blood islands of pander of the yolk sac functions as a site of haematopoiesis. During early fetal life, the haemopoietic organs are the mesenchymal cells of liver, spleen, bone marrow and lymph glands During latter part of fetal life the bone marrow of the long bones are haemopoietic. During postnatal life the bone marrow is concerned with the production of erythrocytes, granulocytes and platelets. In young animals, the marrow of long bones is active in erythropoiesis and in older animals marrow of vertebra, pelvis, ribs and sternum is active in erythropoiesis. Lymphoid tissues of the bone marrow and spleen are the sites of production of monocytes Lymphocytes production occurs in lymphoid tissues of lymph glands, payer's patches of intestine, in spleen and thymus. 10  BLOOD In some cases, the liver, thymus, and spleen may resume their haematopoietic function, if necessary. This is called extramedullary haematopoiesis. It may cause these organs to increase in size substantially. In ruminants haemolymph nodes (haemal lymph nodes) functions as spleen. It takes part in the erythropoiesis during foetal period, while granulopoiesis is more prevalent in postnatal life. The mesenchymal cells of the yolk sac produce primitive stem cells which give rise to the pleuripotent stem cells (colony forming units-CFU) that are capable of producing all types of blood cells. The haemopoietic stem cells (HSC) also called as haemocytoblast are pleuripotent cells (present in blood cell forming organs – bone marrow) and they have ability to renew themselves; some of the daughter cells produced from the HSC remain as HSC to continue hemopoiesis and other daughter cells differentiate into unipotent progenitor cells called committed stem cells (CSC); depending on the microenvironment i.e., the location of these CSC and the growth factors, the committed stem cells give rise to different type of blood cells, The HSC can produce either of the following two series of daughter cells – (1) myeloid series of cells that commit to produce erythrocytes, granulocytes, monocytes and thrombocytes or (2) lymphoid series of cells to produce T and B lymphocytes A CSC that produces erythrocytes is called colony-forming unit- erythrocyte (CFU-E). Similarly CFU that produce both granulocytes and monocytes are designated as CFU-GM. Normally there are five types of progenitor cells in the bone marrow that produce different blood cells viz. Proerythroblast to form RBC Myeloblast to form neutrophils, eosinophils and basophils Monoblast to form monocytes Lymphoblast to form lymphocyte Megakaryoblast to form platelets. The stem cells continue to divide throughout the life of the animal and a part of the cells remains as pleuripotent stem cells and retained in the bone marrow to maintain supply of stem cells. The pleuripotent stem cells differentiate to form the CSC. The proliferation and self-renewal of the stem cells and progenitor cells depend on growth factors. The growth factors regulate the proliferation and maturation of the cells that enter the blood, and cause cells in the committed cell lines to proliferate and mature. These The growth factors that stimulate red cell and WBC formation are the colony stimulating factors (CSFs) and interleukins. Important CSF are:- CSF-granulocyte-macrophage (CSF-GM), CSF-granulocyte (CSF-G) and CSF-macrophage (CSF-M). They stimulate granulocyte formation and are active on progenitor cells and end product cells 11  BLOOD Erythropoietin is a growth factor required for a myeloid progenitor cell to become an erythrocyte. Thrombopoietin makes myeloid progenitor cells differentiate to megakaryocytes (thrombocyte-forming ells). Various interleukins (IL) are involved in regulating the growth and differentiation of stem cells to blood cells – e.g. are IL1, 2, 3, 4, 6, 12 Erythropoiesis Erythrocytes while moving through blood vessel are pushed off from the vessel wall, collide with each other and distorted while passing through narrow capillaries. Since they lack a nucleus, they cannot synthesize new proteins to repair the cell damage. Consequently their lifespan is short and they live for about 120 days in human beings i.e. about 3 million erythrocytes die each second. Since erythrocyte number has to be maintained relatively constant in the blood, new cells must be formed at a rate to make up the loss. Under appropriate stimulation, CFU-E progenitor cells produce proerythroblast (rubriblast). Haemoglobin synthesis begins in polychromatic erythroblast (rubricyte) and maximum synthesis occurs in orthochromatic erythroblast (metarubricyte). The metarubricyte ejects the nucleus to become the reticulocyte. The retculocyte contains some mitochondria, remnants of ribosomes and endoplasmic reticulum. In 1-2days, they develop into erythrocytes and enter circulation. From stem cell, the formation of reticulocyte takes about 72 hours and conversion ofreticulocyte to erythrocyte requires 48 hours. Thus RBC formation requires 5 days time. Regulation of erythropoiesis The level of oxygen supply to the tissue (not the number of erythrocytes in circulation) is the principle regulatory factor of erythropoietic activity of the bone marrow. A decrease in O2 level of tissues causes secretion of a glycoprotein hormone erythropoietin (EPO). The major site of EPO synthesis and release is kidneys Liver is an extrarenal source of EPO. Kidney produces 90% of EPO and liver about 10%. (dogs-kidney is the only source of EPO) Liver becomes an important source of EPO during anaemia caused by severe kidney diseases. Erythropoietin stimulates haemopoietic stem cells of bone marrow to produce the committed stem cells- proerythroblast and thus initiates erythropoiesis. EPO stimulates RNA and DNA synthesis, cell division, haeme synthesis and Hb production. Erythropoietin stimulates Proliferation of rubriblast by mitosis. Accelerates maturation of the rubricytic cells 12  BLOOD Induces the release of reticulocytes into the circulation. The role of nutritional factors in erythropoiesis‚ Availability of sufficient quantity of protein, iron, copper, vitamins B2, B6, B12 and folic acid are necessary for erythopoiesis Vitamin B12 and folic acid are essential for the maturation of erythrocytes. Vitamin B12 is required for DNA synthesis and folic acid for RNA synthesis. Macrocytic anaemia (pernicious anaemia) is a very common symptom of vitamin B12 and folic acid deficiency in humans. It may be caused by reduced intestinal absorption due to insufficient formation of intrinsic factor from stomach because of gastric problems or it may be due to deficiency of vitamin B12 in the diet In animals, vitamin B12 deficiency causes anaemia without altering RBC size. B12 is a cobalt- containing vitamin, synthesised by some microbes but not by plants. Animal products are good source of this vitamin. Ruminant microbes synthesize this vitamin provided cobalt is available through diet. Vitamin B12 is absorbed from small intestine for which the intrinsic factor produced from the parietal cells of the stomach is required. Cobalt deficiency in ruminants results in reduced vitamin B12 synthesis in rumen. In domestic animals vitamin B12 deficiency does not produce large erythrocytes Thiamine (B1), pantothenic acid, nicotinic acid, vitamin E, pyridoxine (B 6), riboflavin, biotin and ascorbic acid are essential for erythropoiesis. Deficiency of vitamin B6 causes microcytic hypochromic anaemia in pigs. Pantothenic acid deficiency results in deficiency of ALA synthase in birds and animals. Normocytic anaemia in swine and primates is due to vitamin E deficiency. Iron, copper and cobalt are essential minerals for erythropoiesis. Iron acts as an integral part of Hb which is absolutely essential for Hb synthesis. About 70% of the body’s Fe2+ content is present in Hb, 3% in myoglobin and the remaining 27% is stored as a protein-iron complex ferritin in macrophages in the liver, spleen and bone marrow. When Fe content in the body is in excess, some amount is stored as hemosiderin- another storage form of Fe. Some Fe2+ is also found with enzymes present in most of the cells of the body. In the blood Fe2 is transported as protein-iron complex transferrin. Part of Fe2+ after absorption from intestine binds with a plasma protein transferrin and stored in liver and bone marrow as ferritin by binding with an intracellular iron-binding apoferritin protein Fe2+ absorption depends on body’s Fe 2+ content – high body reserve – Fe2+ absorption reduced and vice versa Fe2+ is easily absorbed than Fe3+; phosphates, oxalates reduces absorption by forming insoluble salts; vitamin C increases absorption Recirculation of Fe2+ from the haemoglobin molecule is very efficient. Fe2+ removed from erythrocyte destruction is transported as transferrin in the plasma and they are either stored in liver or reused in the bone marrow for synthesis of new Hb molecule. A small amount of Fe2+ is excreted through urine and it must be replaced from diet Copper acts as co-factor in ALA dehydrase in Hb synthesis. It is necessary for the incorporation of iron into Hb. Copper deficiency is common in pigs which may interfere with Fe2+ absorption and utilization. In ruminants cobalt plays a key role for the synthesis of vitamin B12 by the rumen bacteria which in turn is required for the normal production of erythrocytes. Reticulocyte A low percentage (0.2 to 0.3%) of erythrocytes in circulation exhibits a network of bluish threads within the cell and these cells are called reticulocytes and they contain fragments of nucleus, RNA and ribosomes; haemoglobin synthesis continues to a slight extent 13  BLOOD The reticulocyte cells are immature RBCs which have entered into the circulation from blood forming tissues. In some diseases or due to excessive loss of blood or destruction of RBCs, the reticulocytic number increases in circulation. Reticulocyte count can be used as an index of erythropoietic activity. These cells have less or no O2–carrying capacity. Reticulocytes are generally not seen in horse blood; present in very low numbers in the blood of ruminants; in dogs, cats and man reticulocytes number about 1 per 100 RBCs Reticulocyte production index (RPI), is a calculated value used in the diagnosis of anemia. This calculation is necessary because raw reticulocyte count is often misleading in anemic patients; because the reticulocyte count is not really a count but a percentage: it reports the number of reticulocytes as a percentage of the number of red blood cells RPI helps to assess whether the bone marrow is able to produce an appropriate response to anemia. Reticulocyte production should increase in response to any loss of red blood cells. It should increase within 2-3 days of a major acute hemorrhage Observed hematocrit Re ticulocyteindex Re ticulocytecount x Normal hematocrit A value of 45 is used as normal haematocrit HAEMOGLOBIN: Haemoglobin forms approximately 95% of the protein content of the erythrocytes and 35% of the weight of the erythrocytes. The red colour of the blood is due to haemoglobin Haemoglobin biosynthesis Haemoglobin is the oxygen carrying pigment synthesized by the developing erythrocyte up to the reticulocyte stage of erythropoiesis. Oxygen carrying capacity of haemoglobin is 60 times more than plasma. It also functions as a buffer in the regulation of acid base balance. At an oxygen pressure (PO 2) of 100 mm Hg in the lung, Hb forms loose and reversible combination with oxygen, the oxyhaemoglobin, but at low oxygen pressure of 40 mm Hg at tissue level it readily releases oxygen to the tissues for complex metabolic process. The normal Hb content ranges from 9.8 to 15 gm/dl in domestic animals / fowls. Hb is a complex substance composed of a pigment haeme (red colour of haemoglobin is due to haeme) and a protein, globin Haemoglobin is a conjugated protein and has a molecular weight of 66,000 to 69,000 and has iron content is 0.334%. 1 g Hb contains 3.34 mg Fe Globin is a conjugated protein, made up of four polypeptide chains Haeme contains iron in ferrous state. 14  BLOOD Heme is synthesized in a series of steps involving both mitochondria and cytosol in the immature RBCs and globin is synthesized in the ribosomes in cytosol Haeme is synthesized from acetate and glycine. Heme synthesis requires mitochondria. Acetate is converted to succinyl CoA (Krebs cycle) which combines with glycine to form a δ- amino levulenic acid (ALA) catalyzed by ALA synthase and pyridoxal phosphate (vit B6 acts as co-factor. This first step is intramitochondrial; Two ALA combines to form porphobilinogen (PBG), a pyrrole ring structure catalysed by ALA dehydrase (Cu2+ containing enzyme) - intracytoplasmic Four PBG molecules combine to form a tetrapyrrole structure - uroporphyrinogen III(UPG) catalysed by UPG-I synthase and UPG-III cosynthase; UPG is decarboxylated to coproporphyrinogen III (CPG); CPG enters mitochondria, is converted to protoporphyrin IX Fe2+ is incorporated at the centre of the protoporphyrin IX by the enzyme heme synthase (ferrochelatase) and haeme is produced Many enzymes concerned with haeme synthesis are intra-mitochondrial, limited to erythroid precursors including reticulocytes. The ALA synthase is the rate-limiting enzyme of the Hb synthesis, present within the mitochondria. The mature mammalian RBCs are unable to synthesis haeme due to the lack of mitochondria. After haeme is synthesized within the mitochondria it moves into the cytoplasm; 4 haeme molecules combine with 4 globin polypeptides to form one molecule of haemoglobin. The globin is made of two pairs of unlike polypeptide chains -two alpha (each made up of 141 amino acids) and the other two (made up of 146 amino acids) may be either beta, or gamma or delta or epsilon chains. The haeme portion of Hb in different species and also in the myoglobin is identical with each other. Globin part of the Hb (and myoglobin) differs among different species and individuals of the same species due to the varia- tions in amino acid sequence. 15  BLOOD The amount of Hb in the blood is influenced by age, sex, muscular activity, season etc. Haemoglobin Types (Physiological types of Hb.) Based on physiological function, haemoglobins are typed as adult haemoglobin, fetal haemoglobins and embryonic haemoglobins. Embryonic haemoglobin (HbE) is found during early stages of foetal life. Foetal haemoglobin (HbF) has higher O2 affinity than the adult haemoglobin in man and most domestic animals. Dogs, and horse do not have foetal haemoglobin but their RBCs have lower bisphosphoglycerate (BPG) level which favours greater O2 affinity to Hb. Electrophoretically Hbs are classified as HbA, HbB, HbC and HbF. Human beings show three types of Hb - HbA (98%), Hb A2 (2%) in the adult and HbF in fetus and new born. HbA has 2 -chains and 2 -chains; HbA2 is represented by 2 and 2 - chains.HbF has 2 -chains and 2 -chains. In adult sheep HbA (2 , 2 A) is electrophoretically fast moving and has higher O2 afinitythan HbB (2 , 2 – B). Sheep having HbA or HbB under anaemia or hypoxic condition develop another type of Hb - the HbC which partially or completely replaces the HbA. HbC has less affinity to O2 and enhances release of greater O2 from Hb to tissues Such a change is also observed in goat. HbC is the naturally occurring Hb in sheep during growth period. Fetal Hb takes up more O2 and releases greater amount of O2 for each unit of decrease in oxygen tension (better O2 binding ability in HbF). In many animal species fetal haemoglobin (HbF) is found in highest level at birth and it is replaced by the adult types within 4 to 8 weeks after birth. In adult cat, both HbA and HbB are found within the same erythrocyte. Some of the Hb variants - HbS, HbC, HbE are associated with specific hematologic disorders. HbS is responsible for sickle-cell anaemia in Negro race. HbC and HbE cause failure of synthesis of alpha or beta chains thus results in alpha or beta thalassaemia. When hyperglycemia (diabetes mellitus) is uncontrolled, a small amount of HbA is glycosylated to HbA1c which is present in blood. The HbA1c level in blood is used clinically as an index of diabetes for the 4 to 6 week period before measurement. Derivatives of Hb Oxyhaemoglobin: Oxygen forms loose and reversible combination with Hb called oxyhaemoglobin. Since there are 4 ferrous atoms in the Hb molecule, four molecules of oxygen are transported by a molecule of Hb. Hb + 4 O2 Hb4O2 Hb shows progressive increase in the affinity for O2 after the first two molecules of O2 are taken up by the haeme. Each gram of Hb binds with a maximum of 1.34ml of oxygen. In the lungs (PO2 100mm Hg) the oxygen binds with Hb which shows 97% saturation. 100 ml of blood contains approximately 15 grams of Hb which can carry approximately 19.4 ml of oxygen. In the tissue capillaries, (PO 2 40 mmHg) Hb is 72% saturated and contains 14.4 ml of oxygen per 100 ml of blood which indicates oxygen release from the Hb into the tissues. 16  BLOOD Thus under normal resting conditions about 5ml of oxygen is transported by each 100 ml ofblood during each cycle to the tissues. During heavy exercise this is increased to about 15 times normal Myoglobin: (Muscle haemoglobin) It has more affinity for O2 than the Hb in blood. It functions to store oxygen in the muscle, Contains only one haeme group, Can store only one molecule of O2 Its molecular weight is approximately 17,000, which is five times less than Hb. Hence it can pass through glomerulus. The appearance of myoglobin in the urine is referred to as myoglobinuria or azoturia which is a very characteristic symptom of Monday morning sickness in horse. Neuroglobin It is an intracellular hemoprotein present in the CNS, PNS, cerebrospinal fluid, retina and endocrine tissues. Neuroglobin is a monomer that reversibly binds O2 and has higher than affinity to O2 than hemoglobin. It increases O2 availability to brain tissue and provides protection under hypoxic or ischemic conditions Heme is also present in many enzymes – catalses, cytochrome oxidases, peroxidases, cytochrome c etc. Carboxyhaemoglobin (HbCO) Hb has 200 times more affinity for carbon monoxide than oxygen. Hb + CO HbCO. Carbon monoxide firmly attaches with Fe2+ molecules of haeme, thus interferes with the transport of O2 as oxyHb. 0.1% of CO in inspired air will convert 20% of Hb into HbCO within 30 to 60 min. Oxygen under higher partial pressure is the only means to reverse the reaction. HbCO + PO2 HbO2 + CO Methaemoglobin (ferrihaemoglobin) It is formed by the oxidation of ferrous (Fe2+) iron to ferric (Fe3+) iron and it is the true oxide of haemoglobin. Normally, about 1% of methaemoglobin is formed in the circulatory blood by the oxidation of ferrous iron to ferric iron. Glutathione (GSH) present in the erythrocytes prevent excessive oxidation of Fe 2+ into Fe3+ iron Ferrihaemoglobin cannot combine with oxygen, hence useless as a respiratory pigment in the blood. Chemicals - nitrates, sulphonamides, aminophenol and acetanilide cause increased concentration of methaemoglobin in the blood. Horse blood at normal conditions shows significant amounts of methaemoglobin. The normal blood of dog and cat has about 1% of methaemoglobin. MetHb poisoning can occur in animals due to excess nitrite intake – calves grazing newly fertilized pasture may get nitrite poisoning because dietary nitrates are converted to nitrites in the rumen which are absorbed and converts Hb to MetHb. Nitrite poisoning can be treated by methylene blue which reduces iron to ferrous state. 17  BLOOD Hemin: It is the ferric state of Hb prepared by the action of HCl. It forms brown rhombic crystals which are considered to be very reliable test for blood. Absorption Spectra When white light is passed through a solution of haemoglobin or one of its derivatives, certain wavelengths are absorbed. The resulting spectrum is termed as absorption spectra; the region of absorption is known as absorption bands. They can be seen by examining the solution with a spectroscope. When white light is examined spectroscopically, a series of colours known as spectrum (VIBGYOR) is obtained. When sun light is examined, certain black vertical lines called as Fraunhofer’s lines are found at definite places in the spectrum; these lines are designated as A, B, C, D, E, etc. In lamplight, these lines are not seen. When haemoglobin solution or its derivatives are examined in certain concentrations spectroscopically, absorption bands of definite size, appearance, and position are noticed. Hence, spectroscopic examination helps to identify these pigments in solution. E.g. Dilute oxy-Hb solution shows two absorption bands between line D and E; adding areducing agent (produces reduced-Hb) gives one band at line D. Carboxy-Hb shows two bands but adding a reducing agent does not produce a single band. Met-Hb shows a band between line C and D. Life span of the erythrocytes (In days) In most domestic animals, erythrocytes have a life span of about 90 to140 days. Erythrocytes of lambs and calves have a shorter life span than the adults of the same species. In humans, the average lifespan is 120 days i.e. about 3 million RBCs die every second. Since the erythrocytes number is almost held constant, a corresponding number of new erythrocytes must be formed. Cattle Sheep Goat Horse Dog Cat Pig Poultry 125-150 140-150 125-150 140-150 100-120 70-80 51-79 20-30 A 450 kg animal with a blood volume of 8% of body weight has about 350 trillion RBCs and ifRBC life is taken as 100 days, then about 3 trillion RBCs must by destroyed each day i.e. about 35 millions per second. ESTRUCTION OF RBCS When the RBCs are aged, they are destroyed. RBCs with a diameter of 7-8 µm can pass through capillaries of 3-5 µm diameter because of their ability to tolerate deformation. As RBCs become older, the cell membrane becomes fragile due to loss of elasticity and they rupture when pass through small capillaries. Erythrocytes are destroyed by one or a combination of the following factors. Red bone marrow functions as a chief site of destruction of RBC in most domestic animals including dogs whereas it is the spleen in man. In birds liver acts as an organ of destruction of RBCS Intra vascular haemolysis of about 10% of aged cells occurs within the capillaries due toloss of compressibility of RBCs and it is caused by increased membrane permeability by osmotic change. The Hb is released, which combine with haptoglobulin. This complex is removed by the cells of the mononuclear phagocytic system (MPS) located in liver, spleen and bone marrow where the Hb is degraded. About 90% of aged RBCs are removed from circulation by the cells of MPS. When RBCs are phagocytised by the MPS cells, haemolysis occurs within these cell and it is known 18  BLOOD as extra vascular haemolysis. The Hb and proteins are catabolised by the MPS cells. The MPS (also known as reticuloendothelial system) includes the histiocyte or macrophages, stellate or Kupfer cells of the sinusoids of the liver, spleen, mononuclear cells of bone marrow and lymph nodes. Hemolysis can also be caused by external agents like: Blood parasites: Babesiosis, theileriosis, trypnasomiasis and sarcocystosis. Chemicals: Copper, lead, nitrate and nitrite poisoning. The peptide chain globin of the Hb is degraded to amino acids and is reutilized. Iron removed from the haeme is stored in the MPS cells in the form of ferritin or haemosiderin or enters the plasma and combine with apotransferrin to form transferrin. The transferrin enters the bone marrow and the iron is reused for Hb synthesis. The haeme is converted to biliverdin (a green pigment) and then reduced to bilirubin (a yellow pigment) within the macrophages. The free bilirubin enters the plasma, binds with albumin and transported to liver. In the liver the insoluble bilirubin is conjugated with glucuronic acid to make it water soluble and secreted into bile to enter the intestine. Large intestinal bacteria reduce the bilirubin to urobilinogen, most of that are excreted infaces in the oxidised form of urobilin or stercobilin which impart colour to faeces. Part of the urobilinogen is reabsorbed into the enterohepatic circulation and reexcreted inbile. Some of the urobilinogen in the plasma enters the kidneys to be excreted in urine as urobilin. 19  BLOOD LEUKOCYTES: Leukocytes are relatively large sized, nucleated cells Found in lesser numbers and differ from the erythrocytes by the absence of Hb content. While erythrocytes function in blood, leukocytes function mainly in tissues. The leukocytes show polymorphic forms which differ morphologically and functionally Based on shape of the nucleus, the staining nature of the cytoplasmic granules (which are lysosomes or vesicles) when subjected to Leishman's stain, leukocytes are classified as (1) granulocytes (polymorphonuclear leukocytes) which are further classified into three groups – the neutrophils, eosinophils and basophils. Granulocytes have differently staining cytoplasmic granules which are membrane bound enzyme stores and help to digest ingested particles. (2) Some of the leukocytes have cytoplasmic granules but they are non-visible even after staining with Leishman's stain hence they are known as agranulocytes (mononuclear leukocytes)These cells contain non-specific azurophilic granules (large, homogeneous, dense, peroxidase-positive granules) which are lysosomes The lymphocytes and monocytes are the agranulocytes. Majority of the WBCs are larger than RBCs RBCs are present in millions per l of blood; the WBCs are present in thousands per loblood. f The ratio of WBC to RBC varies from 1:100 in chicks to 1:1300 in goats, 1:600 in dog and cat, 1:800 in cattle 1:1000 in horse 1:1200 in sheep and 1:700 in man. In circulation, many leukocytes are found adhered to or moving slowly along the endothelial lining of capillaries and small blood vessels and this property of leukocytes is known as margination or marginal pool of leukocytes. These cells are released into the circulation during exercise and excitement by epinephrine. Neutrophils: (Neutrophilic granulocytes) Neutrophils generally have two to five lobed nucleus, stained blue or purple by Leishman's stain but the cytoplasmic granules take up the neutral stain. They are the most numerous of leukocytes in the blood of dog, cat, horse and man The cytoplasmic granules store lysosomes which contain hydrolytic enzymes, proteolytic enzymes, peroxide and lipases to digest the invading organisms. The oxidative enzymes of the lysosome produce hydrogen peroxide which attacks the bacterial cell wall to cause bactericidal effect. Neutrophils are highly motile, responds to chemotaxis (migration of leukocytes towards the site of inflammation attracted by chemical stimulants-interleukin-8 and interferon-γ) and are actively phagocytic - capable of ingesting microorganisms and particles. The phagocytosed particles are internalized into the neutrophil and digested by enzymes in the granules. Serve as a first line of defence against invading organisms, (bacteria, virus and cellular remnants); during inflammation (reaction of tissues to injury), neutrophils are attracted to site of infection or injury by chemotaxis, migrate from blood to tissues by diapedesis, move within the tissues by amoeboid movement and attack the invasive bacteria and phagocytose them. Thus neutrophils form part of innate immune system (non-specific immunity). They are the predominant cells in pus that forms during infections. 20  BLOOD At the onset of infection, neutrophils produce pyrogens which act on thermo-regulatory centre of the brain to produce fever. This rise in body temperature slows the reproduction process of bacteria and viruses. Immature forms of neutrophils are characterized by unsegmented or less number of nuclear lobes and are referred to as juvenile or band cells. Pseudoneutrophils or heterophils are comparable to neutrophils which are present in rabbit, poultry, elephants and reptiles. Heterophils contain large rod or spindle shaped granules, which are acid in reaction and stain red or pink with eosin. Nuclei of about 1 – 7% neutrophils in females have chromatin appendage called drumstick or Barr body Neutrophilia‚ indicates more number of neutrophils in the circulation. Physiological neutrophilia occur in conditions like exercise, emotion, pregnancy, lactation and parturition. Abnormal or pathological neutrophilia may be due to acute inflammation following injury, surgery, burns, arthritis and acute infection by bacteria. Injection of anti-inflammatory drugs (cortisol) and antibacterial drugs (chloramphenicol and sulphanamides) may result in neutrophilia. Shift to left is a term used to describe an increase in the number of immature neutrophils (band cells) in the circulation which is characteristic of bacterial infections. Neutropenia indicates reduction in neutrophils in the circulation which is very common in viral infections and chronic infections like TB, brucellosis and protozoal and fungal infections. Eosinophils: (Eosinophilic granulocytes) Have segmented nucleus; generally bilobed nucleus connected by a thin filament. Cytoplasmic granules small, round, distinctly red stained. In cat granules are needle shaped and in horse, granules are large and very distinct The cytoplasmic granules contain enzymes like histaminases, oxidases‚ peroxidases, lipases, DNase. These granules take up the red (purple) eosin stain. When eosinophils are activated these granules Eosinophil release their contents by degranulation and the contents are toxic to the parasite as well as the host tissues Eosinophils are motile but less phagocytic. Appear in less number in circulation (1-6%) Eosinophils functions to destroy the parasites thereby help to control parasitic infection - eosinophils attach on the surface of parasite, empty their contents of the granules on parasites which destroy them; they detoxify proteins – attracted to the site of antigen-antibody reaction and phagocytise antigen-antibody complexes and inflammatory products of the mast cells and basophils. Eosinophils along with basophils and mast cells, are important mediators of allergic responses and asthma and are associated with severity of these diseases; eosinophil number increases during allergy to limit the inflammatory reaction of allergy Eosinophils have antiheparin‚ and anti-histaminic substances, and act as anti-inflammatory and anti- allergic agent. Eosinophils release profibrinolysin, which is activated to fibrinolysin that causes dissolution of old blood clot. Eosinophilia is increased number of eosinophils in the circulation; common in G.I. parasitic infections, allergic disorders like bronchial asthma, allergic rhinitis, in skin diseases like eczema and dermatitis, drug reactions following penicillin and sulphanamides administration. 21  BLOOD Eosinopenia follows stress condition, administration of ACTH or cortisol; eosinopenia is characteristic of corticosteroid injection Basophils: (Basophilic granulocytes) Have irregular shaped nucleus and the granules are stained blue by the basic dye of Leishman's stain. Large, water soluble granules appear all through the cytoplasm even obscuring the nucleus. Seen in blood in very low numbers (200 mg/dl High Total blood cholesterol levels (mg/100ml) in animals 36  BLOOD Horse- 75-150; Cow – 60-190; Sheep – 52-90; Goat – 80-130; pig-80-134; dog – 135-270 HAEMOSTASIS Haemostasis is prevention of blood loss from damaged blood vessels. Haemostatic mechanism occurs in five sequential stages. These stages occur concurrently and in sequential order. They are Stage I involves Injury to blood vessel Contraction of vessel wall Platelet activation and adhesion Stage II Platelet aggregation Activation of coagulation proteins Initiation of fibrin formation Formation of platelet plug Stage III Reinforcement of platelet plug Formation of clot Stage IV Clot retraction Fibrin degradation Fibrinolysis Stage V Endothelial repair Dissolution of clot Role of vascular endothelium The vascular endothelium helps to keep the blood in fluid state by inhibiting haemostatic mechanisms. This property of endothelial cells is due to negative charged surface of the endothelium repels negative charged unactivated platelets by synthesizing and releasing inhibitors of platelet function and thrombin formation and by release of activators of fibrin degradation. When the vascular endothelium is damaged as during inflammation or injury to blood vessel wall the endothelial cells begin procoagulant activity by release of tissue factor (TF) to initiate thrombin formation exposing von Willibrand factor (vWF) to activate platelets and by releasing fibrinolytic inhibitors Stage I - Vascular spasm Injury or trauma to the blood vessels stimulates reflex constriction of the blood vessels through the sympathetic division or the local myogenic spasm by the action of serotonin (5- hydroxytryptamine) to close even the large blood vessels to prevent excessive blood loss. When the injury is severe with extensive tissue damage (crushing, laceration) this spasm isstrong and bleeding is less. Smooth cut causes weak spasm and bleeding is severe. 37  BLOOD Stage II - Platelet plug formation Normally, platelets do not attach to each other or to the smooth endothelial lining of the blood vessels. However, when the endothelial layer is damaged, platelets become attached to the damaged surface which is facilitated by a plasma protein von Willibrand factor (vWF) which is formed both in the endothelial cells and platelets. Platelets have contractile proteins- the actin and myosin, factor XIII, the fibrin stabilising factor, enzyme systems for the synthesis of cAMP, ATP, ADP and prostaglandins (PGG2, PGH2, PG12 and PGF2). Platelets are activated by their contact with collagen which is present in the subendothelial membrane or by substances like vWF, ADP, serotonin and thromboxane A2 released from damaged cells. Immediately after the vascular endothelial damage, the subendothelial collagen attracts the platelets to the site of injury. The platelets attach to the injured surface. They then undergo a series of complex physical and biochemical changes like swelling of the platelets, projection of radiating processes, (pseudopods) from the platelets and their adherence with the endothelial wall of the blood vessels. vWF and fibronectin from subendothelium and platelets help in platelet adhesion. This reaction in turn stimulates release of Ca2+ which stimulates the enzyme systems of theplatelets and causes the release of ADP and thromboxane-A2 (formed from arachidonic acid of platelet’s cell membrane phospholipid) which activate other platelets resulting in adherence of more number of platelets in the damaged endothelial wall forming the platelet plug. This plugs the injury on the blood vessel and prevents blood loss. Thromboxane-A2 is the most potent platelet-aggregating agent that lowers platelet cAMP and also causes vasoconstriction. PGG2 and PGH2 also cause platelet aggregation PGI2 (prostacyclin) produced by the normal endothelium of arteries and lungs is a vasodilator which acts as a powerful inhibitor of platelet adhesion / aggregation. In normal blood flow, prostacyclin level is more than thromboxane and aggregation is prevented. 38  BLOOD When arterial walls are damaged prostacyclin level is reduced and thromboxane level becomes high leading to platelet aggregation. The aggregating platelets release platelet factor 3, a procoagulant which initiates hemostasis. The platelet membrane binds with procoagulants – factor IX, X and prothrobmin and provides surface for thrombin formation at the site of vascular injury. Stage III - Blood Coagulation or Blood Clotting Many substances present in tissue and blood affect coagulation (about 50 substances affect coagulation) Substances that promote coagulation are called as procoagulants‚ Those inhibiting coagulation are anticoagulants. Normally in the blood, anticoagulant activity predominates and blood does not coagulate. When a vessel is injured, the procoagulants activity in the damaged area becomes more and the blood clots. Coagulant Proteins Synonyms Fibrinogen Factor I Prothrombin Factor II Tissue factor Factor III, Tissue thromboplastin Ca 2+ Factor IV Factor V Labile factor Factor VII Serum prothrombin conversion accelerator Factor VIII Anti-haemophilic factor A Factor IX Anti-haemophilic factor B Factor X Stuart factor Factor XI Plasma thromboplastin antecedent, PTA Factor XII Hageman factor Factor XIII Fibrin – stabilising factor Von Willibrand factor vWF Prekallikrein Fletcher factor High molecular weight kiniogen HMWK, HK Clot (thrombus) is the meshwork of fibrin threads running in all directions to entrap the blood cells, platelets and plasma. Clot is produced by activation of series of clotting factors present in the blood. The cascade reactions of clotting process involving so many factors help even a low amount of stimulus (damage) to produce large amounts of clot. Most of the clotting factors are inactive proteolytic enzymes and when activated produce enzymatic activities leading to cascading reaction of clotting process. The clot formation proceeds in three stages I. Formation of prothrombin activator in response to rupture of blood vessel or damage to the blood itself. II. Conversion of prothrombin into thrombin by the catalytic activity of prothrombin activator. III. Conversion of fibrinogen into fibrin thread by the enzymatic activity of thrombin. Fibrinogen is present in the blood of healthy animals. This is converted to fibrin monomers by splitting a peptide bond by the action of an enzyme thrombin. The fibrin molecules are then linked together (polymerised) to form long threads; theses threads are initially loosely connected but gradually the bonding becomes stronger (stabilised) by the F XIII which is also activated by thrombin. 39  BLOOD Thrombin is present in the blood as inactive prothrombin. When a blood vessel is injured, the prothrombin is converted to thrombin by the action of another enzyme, prothrombin activator (which contains factor X); thrombin then acts on fibrinogen. Factor X is present in its inactive form in the blood and this is activated when blood vessel is injured to become the enzyme by one of the two pathways - intrinsic or extrinsic pathways Extrinsic or exogenous mechanism or tissue factor pathway These sequential enzymatic reactions are initiated when the blood from the damaged blood vessel contacts tissue factor (TF) or tissue thromboplastin which is present in the endothelial cells but exposed on injury to endothelium. The TF attached with the endothelium combines with factor VII present in circulation and along with Ca2+ this TF-FVII becomes a proteolytic enzyme. This enzymatic activity converts FX and FIX to their active forms FXa and FIXa. The FIXa formed here as well as in the intrinsic pathway becomes a component of tenase complex that activates FX. 40  BLOOD The FXa becomes a part of prothrombin converting enzyme called prothrombinase complex which activates prothrombin to thrombin. Intrinsic or endogenous mechanism or contact activation pathway When the blood comes in contact with foreign surface (other than intact vascular endothelium) like disrupted endothelium, negatively charged surface or glass, it causes a sequence of enzymatic reactions to initiate the coagulation mechanism. Blood drawn in a glass tube clots by this mechanism. When FXII comes in contact with damaged endothelium, it is activated to FXIIa, become anenzyme that in turn activates FXI to XIa. This reaction is accelerated by HK and prekallikerin which brings FXI in close proximity to FXII. The FXIa in the presence of Ca2+ activates FIX to IXa and converts prekallikrein to kallikrein. Kallikrein activates fibrinolytic pathway. FIX is also activated by TF-VIIa complex of extrinsic pathway and by thrombin to become FIXa. The activated FIX (acts as enzyme), FVIII (accelerates enzymatic activity) and phospholipid from activated platelets and Ca2+ forms a complex called tenase complex that converts FX to Xa. The FXa becomes part of the prothrombinase complex and activates prothrombin. Conversion of prothrombin to thrombin The prothombinase complex (prothrombin activator complex) formed from tissue factor pathway or contact activation pathway includes activated FX, (acts as proteolytic enzyme on prothrombin), FVa (accelerates proteolytic activity) and phospholipid (further activates the whole process) and Ca2+. The prothrombinase complex located on the phospholipid of activated platelet or endothelial cell cleaves the inactive prothrombin and converts it to thrombin. The thrombin converts fibrinogen to fibrin monomers. Thrombin also activates more and more of FV which further accelerates prothrombin activation. Thrombin also activates FX, FIX and VIII to enhance prothrombinase formation. Prothrombin is a plasma protein, a α2-globulin (molecular weight 68,700) produced from the liver. Vitamin K is required for the formation of prothrombin and clotting factors VII, IX and X. Conversion of fibrinogen to fibrin Fibrinogen is a high molecular weight plasma protein produced from liver. The proteolytic enzyme thrombin acts on fibrinogen and remove 2 low molecular weight peptides from each molecule of fibrinogen resulting in formation of fibrin monomer The fibrin monomers polymerise with other fibrin monomers by covalent bonding and forms soluble fibrin threads. The fibrin-stabilising factor, present in platelets and to small extent in plasma, causes peptide bonds between the fibrin monomers and cross linkage between adjacent fibrin threads thus adding strength to the fibrin meshwork, the clot. Clot is composed of fibrin threads running in all directions and entrapping blood cells, platelets and plasma. The fibrin threads adhere to damaged surface of blood vessel and closes the opening in the blood vessel thereby prevent blood loss. Blood clotting that occurs in a test tube after blood is withdrawn from the blood vessel is byactivation of FXII (intrinsic pathway).Intrinsic pathway is also more important for coagulation occurring within the blood vessels. In the body, the extrinsic pathway is more rapid and this pathway is more important when blood has leaked into tissues during injury to blood vessels. 41  BLOOD Stage IV Fate of clot Once a blood clot (thrombus ) is formed, it can follow either of the following two courses. 1. Clot retraction: Within a few minutes after a clot is formed, it begins to contract and expresses most of the fluid from the clot within 30 to 60 minutes. The fluid that is expressed out of the clot is called serum. Platelets are necessary for clot retraction. This is caused by the contraction of actin and myosin of the platelets by using ATP of platelets. The clot may be invaded by fibroblasts (the formation and invasion is stimulated by platelet- derived growth factor and epidermal growth factor) within few hours of its formation. The fibroblasts promote connective tissue formation within the clot and within 7-10 days the clot is organised into fibrous tissue (scar formation). 2. Fibrinolysis: It is the degradation of the fibrin clot. After the tissue has healed and when there is no need for the clot, the clot is dissolved It occurs in two pathways. i. Tissue plasminogen activator pathway: This is the major pathway of fibrinolysis. Tissue- plasminogen (t-PA) is synthesized and released by endothelial cells when stimulated by thrombin. In the presence of fibrin, t-PA converts inactive plasminogen to plasmin. Plasminogen is a glycoprotein present in plasma as inactive proenzyme and it is trapped within the clot during clot formation. It is activated to active enzyme plasmin by t-PA and urokinase ii. Urokinase pathway: Kallikrein generated by the action of FXIIa and HK on prekallikrein activates prourokinase to urokinase (urokinase first identified in human urine). The urokinase converts plasminogen to plasmin. Prourokinase is present in nucleated blood cells and endothelial cells. This pathway involves generation of plasmin external to thrombus. Urokinase is used clinically to dissolve deep venous thrombus, pulmonary embolism,cardiac infarction etc Plasmin digests the fibrin clot to release fibrin degradation products which are then removed by reticuloendothelial cells Prevention of Blood Clotting in Blood Vessels The important factors that prevent the process of clotting within the vascular system are 1) smoothness of the endothelium which prevents contact activation of intrinsic system 2) glycocalyx layer adsorbed to the inner surface of the endothelium that repels clotting factors and platelets 3) Thrombomodulin, a protein bound with endothelium binds thrombin and removes its clot producing effect; another plasma protein called protein C - vitamin K dependent, natural anticoagulant protein, activated by thrombin to activated protein C inactivates activated F V and VIII. Damage to endothelium causes loss of smoothness and the glycocalyx layer and initiates coagulation. 4) Anticoagulants in blood also prevent clotting within the vessels. Important anticoagulants are a. fibrin threads formed during clotting b. An globulin known as antithrombin III, heparin and 2 macraglobulin. 5) The thrombin formed during clotting becomes adsorbed to fibrin and thus prevents its spread in blood preventing further clotting. Thrombin not adsorbed to fibrin combines with antithrombin III and is inactivated. Antithrombin is a glycoprotein produced in liver and the dominant antithrombin in plasma 42  BLOOD Heparin increases the effectiveness of antithrombin by many thousand folds in removing thrombin. The heparin and antithrombin also inactivates the activated factors XII, XI, and X. The 2 macraglobulin acts similar to antithrombin III. Species variation in hemostatic mechanism : In marine mammals and reptiles in vitro clotting is prolonged due to the absence of FXII. In birds, though the contact activation phase is absent (absence of F XII), the blood clots rapidly in vitro and this is due to the positive feedback action of thrombin on tenase complex. Evaluation of haemostasis Clotting time or coagulation time is the time required for the blood to clot after it is drawn out from the blood vessels. When blood is exposed to air, it clots within a short time. Clotting time is less than 5 min in most of the domestic animals except cow and horse. Average clotting time (in min): Horse 11.5, sheep 2.5, cow 6.5, pig 3.5, dog 2.5, human 5.0. The bleeding time is the time required for bleeding to stop after puncture of the skin. It iscarried out to assess the function of platelets A pressure cuff is applied on the forearm and below this a standard size wound is made – inIvy method, wound is made with a sharp object; in Duke method a pinprick is made. The blood is blotted away every 30 sec or the wound is immersed in physiological salt solutions till bleeding stops completely Bleeding time ranges from 3- 6 min. Bleeding time is prolonged in thrombocytopenia The prothrombin time (PT) or One -stage prothrombin time (OSPT) is a measure of the clotting time of plasma to which an excess of thromboplastin and calcium have been added so that these coagulation factors will not be a limiting one. It measures the efficiency of extrinsic pathway Since oxalated or citrated plasma is used, CaCl2 is also added to the plasma. Longer the PT the smaller is the prothrombin concentration. Prothrombin time in dogs: 9-12 sec. in sheep 13-25 sec. PT is prolonged in liver disorders, vitamin K deficiency and it is due to the deficiency of FV, FVII, and FX, prothrombin or fibrinogen. Activated partial thromboplastin time (APTT or PTT) is carried out by adding celite (activating agent for FXII), phospholipid and Ca2+ to plasma and the time is measured till a thrombus is formed. APTT is used as a performance indicator for the intrinsic pathway and the common pathways. PTT is prolonged in deficiency of FXII, X, IX, VII, prothrombin or fibrinogen. Normal value of PTT is 25 to 39 sec in human Causes of excessive bleeding Vitamin K deficiency: This vitamin is required for hepatic synthesis of prothrombin, factors V,VII, IX and X and deficiency leads to reduced synthesis of these coagulation factors resulting in excessive bleeding. Deficiency of vitamin K can occur due to malabsorption resulting from reduced bile acids supply and liver diseases. Warfarin (used as rat poison) is antagonistic to vitamin K and it reduces the concentration of many coagulation factors in blood leading to bleeding. It is useful in preventing thrombus formation like during myocardial infarction Hepatitis or cirrhosis of liver-results in less synthesis of coagulation factors derived from liver. Haemophilia: Genetic disorder. 43  BLOOD Aspirin inhibits platelet plug formation by interfering the thromboxane pathway and any injury to aspirin administered animals prolong the bleeding Thrombocytopenia: Reduction in platelet count, which may occur in irradiation of bonemarrow, drug sensitivity and some infections. Small injuries occurring regularly on the blood vessels require a certain minimum number of platelets to effectively close the opening In thrombocytopenia, pinpoint haemorrhagic spots (petechiae) appear all over the body This condition is common in humans and dogs Hereditary haemorrhagic disorders Haemophilia A due to inborn deficiency of factor VIII. It is congenital, seen in humans, dogsand also reported in cat and horse. It is sex-linked and seen exclusively in males Haemophilia B: caused by reduction in factor IX. Seen in man, dogs and it is inherited. Deficiency of factor IX is reported in cattle. Haemophilia can be treated by injecting FVIII and the effect is temporary; it has to beadministered at frequent intervals von Willebrand's disease: Inherited disease resemble haemophilia-A caused by reduction inplatelet function and factor VIII. ANTICOAGULANTS Anticoagulants are the agents, which prevent coagulation or the clotting of the blood both outside the blood vessels (in vitro) and also inside the blood vessels (in vivo). In vitro anticoagulants Na, K or Ammonium oxalates or citrates -1-3 mg/ml of blood (0.2%) Sodium fluoride - 4 mg/ml (0.4%) EDTA (Ethylenediaminetetraacetate) - 0.5-2 mg/ml. (or) 15% solution of K2 EDTA - 0.01 ml/ml of blood Heller and Paul mixture: Ammonium oxalate: Potassium oxalate (3:2) 0.1 ml/ml. The oxalates and the citrates precipitate the Ca2+ ions of the blood, thus prevent clotting. Sodium citrate is commonly used in blood transfusions. Potassium salts are not used in transfusions due to its toxicity to heart. Sodium fluoride prevents disintegration of the platelets. It is the ideal anti-coagulant for the estimation of blood glucose level, because it prevents the oxidation reaction of glucose. EDTA acts as a chelating agent that takes up Ca 2+ ions, useful for blood morphological studies and for estimating erythrocyte indices. Natural anticoagulants Heparin: It is a conjugated polysaccharide and is a naturally occurring anticoagulant produced bythe mast cell and the basophils. It prevents clotting by combining with antithrombin III (inhibitor of thrombin) and FXa and potentiates the antithrombin III activity. It is used clinically (in vivo) to prevent thrombosis and embolism. Concentration: 20-units/ ml of blood. (Or) 0.1 ml of 1% sol/5ml of blood Hirudin: Produced by leech, inactivates thrombin thus delays coagulation. Snake venom: Interferes with the action of thromboplastin or destroys blood fibrinogen. 44  BLOOD Dicoumarol: It is vitamin K antagonist, produced in spoiled sweet clover hay and it prevents the utilisation of vitamin K by the liver, thus depresses the prothrombin production leading to hypoprothrombinemia. Non-wettable surface, silicon coated tubes, plastic containers and rapid chilling of the blood samples to 0oC delays the formation of fibrin stabilising factor activator and interferes with thromboplastin formation. BLOOD VOLUME Blood volume makes up about 6 to 8%of the body weight. Blood volume measurement plays an important role when blood transfusion is attempted. It is also important to interpret PCV, Hb, RBC, and haematological parameters. These values are altered when the blood volume changes – e.g. haemoconcentration and haemodilution. Blood volume is influenced by body type, body size, age, sex, breed, nutrition, pregnancy, and lactation, physical and metabolic activities. Males show higher blood volume than females. Lean body has more blood volume than fat animals; older animals have more fat and have less blood volume than young animals of the same body weight Blood volume increases with pregnancy, muscular activity, stress, and high temperature, whereas haemorrhage, burns, dehydration, anaemia and cold decrease the blood volume. Blood volume may be measured indirectly by two methods. Plasma volume method Plasma volume can be measured by dye dilution or by radioisotope. Blood sample from the experimental animal is collected and the plasma of the pre injected anticoagulant-added blood serves as a blank. Known quantity of Evan's blue dye (T-1824) or radioisotope 131I‚ when injected, combines with the plasma proteins and disperses throughout the circulatory system in about 10 minutes. Blood samples are collected sequentially at 5 minutes interval for the next 15 to 30 minutes. These blood samples are centrifuged to get RBC free plasma. The concentrations of the dye or the radioactivity of isotope in the plasma samples can bemeasured spectrophotometrically at 620nm or by the scintillation counter respectively. Amount of dye / Isotope injected Plasma volume (ml) = Concentration of the dye/ml of plasma (or) Radio activity / ml of plasma sample Erythrocyte volume method This can be determined by radioactive 32P, 59Fe and 51Cr. Blood sample from the experimental animal is collected to get plasma free erythrocytes. Small quantity of 51Cr is mixed with these RBCs which are then incubated at 36°C for 30 minutes to activate the binding process of 51Cr with the RBCs. Wash the erythrocytes with saline to remove the free 51Cr. These radioactive erythrocytes are then injected into the circulation of the experimental animal. After proper mixing of these RBCs in the circulation, blood samples are collected after 10 min. RBCs from the blood samples are separated and the radioactivity of pre and post injected RBC samples are determined by scintillation counter. 45  BLOOD Quantity of radioactivity injected Erythrocyte volume (ml) Radio activity / ml of RBCs Plasma volume x 100 Erythrocyte volume x 100 Blood volume (ml) OR 100 - PCV x 0.96 PCV The trapped plasma value may interfere with the PCV. Hence the correction factor for trapped plasma is introduced as 0.96 for blood volume determination. Blood Volume Cattle Horse Sheep Goat Pig Dog Cat Poultry Blood volume (ml) 40,000 40,000 3,200 2,400 8,000 1,600 240 160 As ml/kg bodywt 52-60 60-70 60-65 70-72 35-45 85-90 65-75 65-70 As % of body wt. 8 10 8 6 - 7 - 6.5 BLOOD GROUPS Landsteiner (1901) was the first to identify four blood groups, A, B, AB and O in human beings. A blood group is a classification of blood based on the presence or absence of inherited antigenic substances on the surface of RBCs. These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system, and some of these antigens are also present on the surface of leukocytes and cells of various tissues. Several of these RBC surface antigens that arise from one allele (or very closely linked genes), collectively form a blood group system. Blood groups are genetically determined and are inherited and represent contributions from both parents; blood group systems are generally independent of each other A total of 30 human blood group systems are recognized by the International Society of Blood Transfusion (ISBT) with over 600 different blood-group antigens have been found. However, the ABO, Rh, MNS, P and Lewis groups are best known. Among these five, ABO and Rh systems are widely used for blood grouping in human beings The specificity of the antigens depends upon carbohydrate portion of the molecule. Normally, an individual does not have antibodies against any of the antigens present on its own RBC or against other blood group antigens of that species’ unless they have been induced by transfusion, pregnancy, or immunization. In some species (human, sheep, cow, pig, horse, cat, and dog), the “naturally occurring” isoantibodies, may be present. For example, Group B cats have naturally occurring anti-A antibody. Based on the antigenic type present on the surface of the RBCs’ cell membrane, blood can be grouped into different groups or types. The serum has antibodies (mainly IgM type) against other groups of antigens. e.g. `A' group blood contains only A-isoantigens in the RBC membrane and it also posses’ anti-B antibodies in the serum. Blood Group Antigens on RBCs Antibodies in Serum Genotypes A A Anti-B AA or AO B B Anti-A BB or BO AB A and B Neither AB O Neither Anti-A and anti-B OO 46  BLOOD During blood transfusion, if the blood between recipient and donor are mismatched, there will be reaction of antigen (agglutinogen) with the antibody (agglutinin) causing clumping or agglutination of the erythrocytes. These agglutinated RBCs are carried by the leukocytes to the RE system where they are lysed releasing Hb. Sometimes, mismatching of recipient and donor’s blood leads to immediate haemolysis of the RBC's in the circulating blood which is caused by the complement system. Red blood cell compatibility Blood group AB individuals have both A and B antigens on the surface of their RBCs, and their blood serum do not contain any antibodies against either A or B antigen. Therefore, an individual with type AB blood can receive blood from any group (with AB being preferable), but can donate blood only to another type AB individual. Blood group A individuals have A antigen on the surface of their RBCs, and blood serum containing antibodies against the B antigen. Therefore, a group A individual can receive blood only from individuals of groups A or O (with A being preferable), and can donate blood to individuals with type A or AB. Blood group B individuals have the B antigen on the surface of their RBCs, and blood serum containing antibodies against the A antigen. Therefore, a group B individual can receive blood only from individuals of groups B or O (with B being preferable), and can donate blood to individuals with type B or AB. Blood group O individuals do not have either A or B antigens on the surface of their RBCs, but their blood serum contain anti-A and anti-B antibodies against the A and B blood group antigens. Therefore, a group O individual can receive blood only from a group O individual, but can donate blood to individuals of any ABO blood group (ie A, B, O or AB). individuals with type O negative blood are often called universal donors, and those with type AB positive blood are called universal recipients Blood groups of recipient (Antibody) Blood groups of the donor (Antigen) “+” Agglutination or A B AB O clumping A (Anti B) -- + + -- “--” No B (Anti A) + -- + -- agglutination AB -- -- - -- O (Anti A, B) + + + -- Generally, antibodies present in the donor's plasma which would be active against the recipient’s red cells do not produce such a reaction because of the rapid dilution in the recipient's circulation. Serious problem of antigen-antibody reaction result from antibodies present in the recipients plasma reacting with the donor's RBCs. The agglutination of the donor's erythrocytes may produce systemic thrombosis in the blood vessels Rh system In Rh system, spontaneous agglutinins do not occur. There are six Rh antigens (Rh factors) C, D, E, c, d and e. Of these six types D is widely prevalent and is considered to be more antigenic than others. The presence of D antigen indicates Rh positive. 47  BLOOD In the Rh system there is no immediate reaction when blood transfusion is effected between Rh+ and Rh- individuals. When Rh- individual receives Rh+ blood, the development of antibody or agglutinin occurs only after 2 to 4 weeks. Hence, the transfusion reaction is usually delayed and mild. If the same person has subsequent blood transfusion with the same antigen, there will be enhanced antigen - anti- body reaction. Erythroblastosis fetalis It is a disease of the human fetus and newborn infants characterized by progressive agglutination and phagocytosis of RBC's. The mother is Rh-, father Rh+, the baby inherits Rh+ from the father. Usually the Rh-ve mother develops anti-Rh agglutinins only when the Rh +ve child develops by inheriting the Rh+ve factor from its father. The child's Rh+ve antigen enters the maternal system and causes development of Rh+ve antibodies. Placental diffusion of this antibody causes hemolytic conditions in the subsequent Rh+ve new born infants. This disease condition is characterised by varying degree of anaemia and jaundice in the newborn infants depending upon antibody reaction by the mother. This condition can be prevented by passively immunising the mother against Rh+ve factor. Erythroblastosis fetalis also occur in foals – incompatible antibodies formed in the dam against erythrocytes of foal do not cross placenta, however they are concentrated in the colostrums and absorbed through the intestine of foals during the first day of life. They cause haemolysis of the erythrocytes of the new born foal leading to anaemia and jaundice in foals. This condition also occur in cattle and pigs Blood groups in animals In animals, the antigens representing the blood group are not strongly antigenic and occurrence of the natural antibody in their blood is rare. However, naturally occurring antibodies to some red cell antigens can be found in normal animals that lack the respective antigens. In domestic animals, the initial transfusion of whole blood will not result in serious problem. However subsequent transfusions with the similar isoantigen cause enhanced antigen- antibody response which may produce the clinical symptoms like muscular trembling, saliva- tion, dyspnoea, and haemoglobinuria. In dogs, there are 8 major blood groups, labelled as DEA (dog erythrocyte antigen) 1 to 8.The major antigens are DEA 1.1 and DEA1.2. Dogs can be positive for either (not both) DEA 1.1 or 1.2 or are negative for both. DEA group Old name Natural antibody 1.1 A1 No 1.2 A2 No 3 B Yes 4 C No 5 D Yes 6 F No 7 Tr No 8 He 48  BLOOD Acute hemolytic transfusion reactions only occur in DEA 1.1 and 1.2 negative dogs. As these dogs do not have naturally occurring antibodies, a reaction will only be seen after sensitization of the dog through exposure to DEA 1.1 or 1.2 positive blood In an acute hemolytic transfusion reaction, the lifespan of incompatible transfused erythrocytes ranges from minutes to 12 hours. Although DEA 3-, 5- and 7-negative dogs have naturally occurring antibodies to DEA 3, 5 and 7 positive red cells, these blood groups do not produce severe hemolytic reactions. Rather, transfusion of incompatible blood is hemolysed less rapidly (within 4 to 5 days) than compatible blood would be (so-called delayed hemolytic reaction). Therefore, crossmatching in dogs does not need to be done on the first transfusion. Neonatal haemolytic reaction may occur in DEA 1 negative female dogs (previously sensitized to DEA 1 positive cells) mated to DEA 1.1 positive male dogs. In cats, only 1 blood group system, the AB system, has been identified. In this system, there are 3 blood types - A, B and AB. Similar to humans, the blood group antigens are defined by specific carbohydrates on erythrocyte membranes. Cats have naturally occurring antibodies (alloantibodies) re