Blood
Blood is a fluid connective tissue that originates from mesoderm composed of corpuscles suspended in a protein-rich fluid called plasma. It is the main transport medium of the circulatory system responsible for transport of oxygen and nutrients to the tissue and removal of metabolic waste products away from the tissue.
In addition to the aforesaid functions, blood plays a pivotal role in carrying hormones and drugs to the target organs, maintenance of ionic equilibrium and pH of the body, resistance to infections and thermoregulation. All these functions performed by the blood are directed towards the maintenance of a constant internal environment called homeostasis.4.1.1 Properties of Blood
4.1.1.1 Color
The red color of blood is imparted by the iron present in the hemoglobin within the erythrocytes. The venous blood, however, looks purplish blue due to deoxygenated hemoglobin. The plasma is yellow to colorless as it contains bilirubin, carotenoids, retinol, and tocopherol. The plasma of cattle and horses is deep yellow in color due to higher bilirubin compared to dog, sheep, and goat.
4.1.1.2 Relative Volumes of Blood Cells
and Plasma/Hematocrit
Hematocrit or packed cell volume (PCV) is the ratio of the volume of red blood cells to total volume of blood. Hematocrit or packed cell volume (PCV) is directly related to erythrocyte counts and hemoglobin content and used to evaluate anemia. As PCV is the relative volume of erythrocytes to plasma, it can also be used to evaluate the degree of dehydration in animals. In dehydration, PCV is generally increased. The PCV of different animals are given in Table 4.1.
4.1.1.3 BloodVolume
Generally, blood volume is 8-10% of body weight. Indirectly, blood volume can be calculated through the following formula.
where BV = blood volume, PV = plasma volume, Hct = hematocrit
Newborn of all species have a high blood volume per unit of body weight except in cattle and sheep in which age or weight have no effect on blood volume up to 2-3 months of age.
Swine have the lower absolute values of blood volume due to high body fat. The blood volumes in different species have been presented in Table 4.1.4.1.1.4 The Specific Gravity and Sedimentation of Blood
The specific gravity of plasma is lower compared to corpuscles. Among the corpuscles, erythrocytes (1.097) have higher specific gravity compared to leukocytes (1.067-1.077). Therefore, erythrocytes settle more quickly compared to leukocytes, and the rate at which erythrocytes settle per unit time is termed as the erythrocyte sedimentation rate (ESR). Erythrocytes have inherent property to adhere to each other like a stack of coins. It is called Rouleaux (singular is rouleau) formation. The unique discoid shape of vertebrate erythrocytes provides a large surface area to contact each other. The rouleaux formation is accelerated with the presence of plasma proteins, particularly fibrinogen and sialic acid. The albumin, on the other hand, hastens the rouleaux
Table 4.1 Hematological values in different animals
formation. ESR is increased in diabetes mellitus, renal failure, heart disease, collagen vascular diseases, and malignant tumors. Diseases with altered shape of RBC such as microcytic RBC increases ESR as microcytic RBC has lower surface to volume ratio and settles more quickly. In polycythemia (increased RBC), ESR is decreased as too many erythrocytes interfere with the compactness of rouleau. Elevation in leukocyte counts also lowers the ESR.
The specific gravity of blood and ESR in different domestic animals have been presented in Table 4.1.
4.1.1.5 ReactionsofBlood
The mean pH values of blood of different species are depicted in Table 4.1.
The arterial blood is more alkaline than venous blood. The plasma is more alkaline than the corpuscles. Buffering systems, particularly hemoglobin and bicarbonate, maintain a stable pH and provide first line of defense against acidemia.
4.1.1.6 Alkali Reserve
It is the capacity of blood to combine with CO2 and expressed as the volume of CO2 (mL per 100 mL plasma).
The average alkali reserves of blood in different species are presented in Table 4.1.
4.1.2 Composition of Blood
Blood is composed of cellular elements (45%) suspended in plasma (44%) made of colloids and crystalloids.
4.1.2.1 Plasma
Plasma appears as a light-yellowish or straw-colored fluid composed of water (92%) and solids (8%). The solid portion of the plasma comprises of inorganic and inorganic. Plasma proteins are the most abundant plasma solutes. Besides plasma proteins, a variety of other substances are also present such as dissolved nutrients, electrolytes, respiratory gases, hormones, and vitamins. Plasma proteins and lipids are present as colloidal suspension, whereas glucose, urea, and uric acid are present as crystaloids. Plasma serves as the liquid base for whole blood to carry blood cells, nutrients, and metabolic waste products.
4.1.2.1.1 Plasma Proteins
Majorities of the plasma proteins are synthesized in the liver; however, the principal source of immunoglobulins is lymphocytes or plasma cells. The plasma proteins are mainly involved in the regulation of osmotic pressure, transport of hormones and drugs, immunity (initiation of inflammatory responses and the complement cascade system) and buffering actions. Electrophoresis is the easiest method to determine the relative proportions of plasma protein fractions as the
Table 4.2 Proportion of different plasma proteins
| Name | Proportion (%) | |
| Albumin | 55.2 | |
| Globulin | α1-Globulin | 5.3 |
| α2-Globulin | 8.6 | |
| β-Globulin | 13.4 | |
| γ-Globulin | 11.0 | |
| Fibrinogen | 13.4 | |
protein fractions are separated on the basis of electrophoretic mobility determined by their molecular weight.
The three major fractions of plasma proteins separated by electrophoresis are albumin, globulin, and fibrinogen (Table 4.2). Albumin: It is the plasma protein having the highest electrophoretic mobility with molecular weight of 69,000 Da. It is also the most abundant plasma protein with a concentration of 2.8-4.5 g/dL. Albumin is synthesized in the liver and composed of a single polypeptide chain with 610 amino acids among which lysine, valine, leucine, threonine, phenylalanine, histidine aspartic acid, glutamic acid, and arginine are predominated. Though 60% of albumin is found extravascularly, it has the ability to transport back to the circulation via. the lymphatic system. The main function of albumin is to maintain colloidal osmotic pressure. Thus, in hypoproteinemia or hypo- albuminemia water moves from vasculature and accumulates in extravascular space and leads to edema. Albumin also acts as a transport medium for different biomolecules particularly fatty acids (in the form of apoprotein), cations (Ca2+, Na+, and K+), lipid soluble hormones, bilirubin, and drugs (phenylbutazone, warfarin, etc.). Therefore, albumin is considered as “molecular taxi.”Dehydration is probably the only cause that increases plasma albumin levels, but there were a variety of clinical conditions that caused hypo-albuminemia, either due to decreased synthesis or increased loss. Liver dysfunction, malabsorption syndrome, pregnancy, and malnutrition are the predisposing factors for decreased albumin synthesis, whereas renal impairment and burns are the two main factors for protein loss. Burn causes severe loss of albumin due to the fact that a substantial amount of albumin is stored in the skin.
Globulin: The molecular weight of globulin is about 90,000-156,000 Da, which is mainly synthesized from liver and reticulo-endothelial system. Based on the electrophoretic mobility globulins are classified into alpha, beta, and gamma globulin fractions.
The functions of different fractions of globulin are summarized in Table 4.3.Fibrinogen: It is a homo-dimeric glycoprotein having a molecular weight of 340,000 Da. It consisted of six polypeptide chains (2Aα, 2Bβ, and 2γ) linked by 29 disulfide
Table 4.3 Functions of different fractions of globulin
| Name | Functions |
| Transferrin | Transports iron |
| Ceruloplasmin | Transports copper |
| Hemopexin | Transports heme |
| Haptoglobin | Transports hemoglobin |
| Plasminogen | Precursors of plasmin (help in clot lysis) |
| Prothrombin | Precursors of thrombin (help in blood coagulation) |
| α2-macroglobulin | Proteases inhibitor |
| Immunoglobulins (γ-globulins) (IgG, IgA, IgM, IgD, IgD): Produced by plasma cells (B-lymphocytes) | Humoral immunity |
| Transferrin | Transports iron |
bonds. The primary site for fibrinogen synthesis is the hepatocytes. The normal plasma concentration of fibrinogen is 2-5 mg/mL which can be exceed up to 7 mg/mL during acute inflammatory conditions. Fibrinogen is an integral component of blood coagulation machinery, and it is the precursor of insoluble fibrin that forms blood clot. Acute phase proteins (APPs): These are a group of plasma proteins that originate from the liver or other extrahepatic source (epithelial cells, endothelium, and connective tissue) during acute phase reactions and used as bio-markers for early diagnosis of diseases. The predominant acute phase proteins of the blood are c-reactive proteins (CRP), plasminogen, α1 anti-trypsin, α2 macroglobulin, prothrombin, fibrinogen, ferritin, serum amyloid-A (SAA), haptoglobin (Hp), and ceruloplasmin (Cp).
4.1.2.1.2 Plasma Lipids
The plasma lipids are transported in three main forms namely free fatty acid, triglyceride, and cholesterol ester. The fatty acids are further classified in saturated (palmitic acids, stearic acids) and unsaturated fatty acids. Unsaturated fatty acids are of two types, monounsaturated fatty acids (oleic acid) and poly-unsaturated fatty acids (linoleic acid, linolenic acid, and arachidonic acid). Free fatty acids primarily originate from triglycerides of the adipocytes and transported in combination with plasma albumin. Cholesterol and triglycerides are transported in the form of lipoproteins. Lipoproteins are the complex particles in which cholesterol esters and triglycerides form a central core which is surrounded by phospholipids, cholesterol, and apolipoproteins (protein parts of lipoprotein that bids with lipids like fat and cholesterol). Dietary lipids are broken down into fatty acids and monoacylglycerol which were absorbed by enterocytes. The fatty acid-binding proteins facilitate the transport of fatty acids and monoacylglycerol to the endoplasmic reticulum
Table 4.4 Plasma cholesterol level in different species
| Species | Cholesterol (mg/dL) |
| Dogs | 135-278 |
| Cats | 71-156 |
| Pigs | 36-54 |
| Sheep | 52-76 |
| Goats | 80-130 |
| Rabbits | 10-80 |
Source: Kaneko et al. (1997)
of the enterocytes where they again conjugated to form triacylglycerol. The esterification of newly formed triacylglycerol together with phospholipids, cholesterol, and apolipoproteins forms the chylomicrons which transport dietary cholesterol and triglycerides to the liver and peripheral tissues.
The concentration of total cholesterol in different species has been presentation in Table 4.4.
Lipoproteins are involved to keep the lipids in solution. They appeared as complex particles characterized by hydrophobic central core (made of cholesterol esters and triglycerides) surrounded by apolipoprotein, phospholipids, and cholesterol-rich hydrophilic membrane.
Plasma lipoproteins can be classified into seven classes based on their density (Table 4.5).
LDL carries cholesterol to cells, which can lead to accumulation of plaque in the vessels and increase the occurrence of atherosclerosis. Thus, they are considered as “bad
Table 4.5 Classification of lipoproteins
| Lipoproteins | Density (g/mL) | Functions | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Chylomicrons | (32%) - Hemoglobin (95%) - Peroxiredoxins - Proteins (Mostly present in cell membrane and cytoskeleton) Cytoskeleton proteins Alpha and beta spectrin Actin Ankyrin Membrane proteins Band 3 or the anion exchange protein Glycophorins - Lipids (0.4%) Lecithin Cephalin Sphingomyelin Cholesterol Cholesterol esters Table 4.7 The size of RBCs in different species
Source: Adili et al. (2016) Neutral fat - Vitamins - Carbohydrate (7%) (Glucose for energy) - Enzymes Cholinesterase Phosphatases Carbonic anhydrase, peptidases, and enzymes of glycolysis - Minerals Phosphorus Sulfur Chlorine Sodium Potassium On the basis of intracellular potassium content, the RBC can be classified as high potassium (HK) and low potassium (LK) RBC. In high potassium RBCs (horses, pigs), the sodium pump is highly active which helps in the exchange of intracellular sodium for extracellular potassium. In contrast, the RBCs of sheep, goats, cattle, and dogs are having low intracellular potassium as the sodium pumps are not fully functional. 4.1.2.2.3 ErythrocyteMembrane The semipermeable plasma membrane of erythrocytes is composed of a lipid bilayer and a mesh-like cytoskeleton which supports the plasma membrane. The composition of isolated erythrocyte membrane is as follows: • Water (20%) • Protein (40%) • Lipid (35%) • Carbohydrate (6%) 4.1.2.2.3.1 MembraneLipids The membrane lipids comprise of cholesterol and phospholipids. The phospholipids are arranged asymmetrically in the membrane to maintain the fluidity. The phospholipids of RBC membrane consist of a polar head group facing the exterior followed by a non-polar hydrophobic tail which aggregate in the lipid bilayer provides a hydrophobic barrier towards cytosol. The lipid composition of the erythrocyte membrane varies with species. Sheep erythrocyte membrane is devoid of phosphatidylcholine, camellias have very low phosphatidylcholine in their erythrocyte membrane, whereas rats have more phosphatidylcholine. 4.1.2.2.3.2 Membrane Proteins The erythrocyte membrane proteins comprise the internal hydrophilic peripheral proteins, middle hydrophobic integral proteins, and external hydrophilic proteins. The main integral proteins of plasma membrane are band 3 (anion exchanger or anion channel) and glycophorins. Band 3: It is a 93 kDa homo-dimeric protein and the most abundant proteins of erythrocyte membrane. The protein comprises three domains namely N-terminal cytoplasmic domain, hydrophobic transmembrane domain, and membrane-bound C-terminal domain. The main functions of band 3 proteins are transporting of chloride and bicarbonate ions across the membrane, membrane crosslinking (band 3 binds with band 4.2 proteins and acts as a bridge between lipid bilayer and cytoskeleton), maintaining membrane stability and associated with aging process of erythrocytes. Glycophorins: It is a sialoglycoprotein consists of 60% carbohydrates (mainly sialic acid) with two domains. N-terminal domains are situated towards outer surface, and they act as blood group antigens (ABO and MN) whereas, C-terminal domain faces the cytoplasm and interact with the cytoskeleton. Due to high sialic acid content, glycophorins are the major contributors to the surface negativity of RBC membrane. There are several types of glycophorins characterized in human erythrocytes out of which the major four glycophorins are glycophorin A (85%), glycophorin B (10%), glycophorin C (4%), and glycophorin E (1%). Beside these, there are several other minor glycophorins such as band 4.5 protein (GLUT1), acts as a glucose transporter, Na-H exchanger (NHE1), associated with actin-binding proteins, CD44 and CD47, helps in cell-cell crosstalk. The blood group antigens are also present in the erythrocyte membrane. The type of membrane proteins and their compositions varies with the species. In mouse, guinea pigs and cows, the membrane protein band 3 is having a higher molecular weight compared to the RBC of sheep RBCs. Band 4.1 contains two sub-bands in the erythrocytes of cow, rabbit, guinea pig, and sheep whereas there is overlapping in 4.2 and 4.1 in the erythrocytes of horse. 4.1.2.2.4 Erythrocyte Cytoskeleton The cytoskeleton provides the structural integrity, deformability and maintains the typical biconcave shape of RBC. In the cytoskeleton, transmembrane proteins are associated with peripheral membrane proteins to form a meshwork of protein that stabilizes the membrane. The major proteins involved in the erythrocyte cytoskeleton are spectrin, ankyrin, actin, and Band 4.1R. Spectrin: It is the principal cytoskeletal protein. It is composed of two subunits namely α-subunit (280 kDa) and β-subunit (246 kDa) oriented in opposite directions to form a tetramer (α2β2 tetramers). It forms a complex intracellular network with actin and band 4.1R protein which then interacts with the cytoplasmic domain of glycophorin. Around 6 spectrins bind to actin and lead to a pseudohexagonal arrangement. Ankyrin: As the name implies, it is the cytoskeletal anchoring protein. The molecular mass of ankyrin is 206 kDa. It has three domains namely N-terminal domain that binds with band 3 protein, central domain to bind with spectrin, and a C-terminal regulatory domain. Actin: The non-muscle β-actin of cytoskeletal protein is a short helical filamentous protein of 43 kDa. It interacts with adducing that facilitates the binding of actin with spectrin. Each actin binds to six spectrin tetramer ends to pseudohexagonal lattice structure. Band 4.1R: It is a globular protein with two domains. 30 kDa N-terminal domain of this protein binds with Band 3 and glycophorin. Another domain of 10 kDa facilitates the binding with spectrin and actin at the junctional complex. Adducin: It is a calcium/calmodulin-binding protein exists as αβ dimer that enables capping of actin for spectrin-actin interactions at the junctional complex. The structure of erythrocyte cytoskeleton is made of vertical and horizontal interactions (Fig. 4.1). In horizontal interactions, spectrin dimers join head-to-head and produce heterotetramer. The tails of heterotetramers bind with F-actin, protein 4.1, and actin-binding proteins like adducin, tropomyosin, and tropomodulin to produce junctional complex. This network tethered with cell membrane at two sites as vertical interactions. The vertical interaction facilitates by two complexes namely ankyrin complex and junctional complex. In ankyrin complex, the N-terminal cytoplasmic domain of band 3 protein binds with ankyrin which in turn binds with spectrin and links the cytoskeleton and the membrane. At the junction complex, the spectrin network binds with to Glycophorin C of plasma membrane. These interactions help to maintain the biconcavity of erythrocytes along with its deformability. 4.1.2.2.5 Erythrocyte Membrane Transport The membrane of erythrocytes is impermeable to many biological molecules due to its lipid bilayer. But erythrocyte membrane proteins allow the efflux and influx of the molecules across the membrane. The functions of different membrane transporters are summarized below (Table 4.8). 4.1.2.2.6 MetabolismofRBC The mature erythrocytes are devoid of nucleus, thus unable to synthesize nucleic acids or proteins. Absence of other cytoplasmic organelles makes the metabolic activities of erythrocytes very limited. Lack of mitochondria in mature RBC results absence of Krebs’ cycle and oxidative photophosphorylation including the synthesis of lipids and heme. Fortunately, the main function of erythrocytes, i.e., the transport of oxygen and carbon-dioxide doesn’t require energy. But the energy in the form of ATP is required to maintain the shape
Fig. 4.1 Structure of erythrocyte cytoskeleton: The vertical and horizontal interactions erythrocyte cytoskeleton. Spectrin dimers join head to head to produce heterotetramer in horizontal interactions. The vertical interaction consists of two complexes viz. ankyrin complex and junctional complex. In ankyrin complex, N-terminal cytoplasmic domain of band 3 protein binds with ankyrin. The cytoskeleton links with the membrane through spectrin. At the junction complex, the spectrin network binds with to Glycophorin C of plasma membrane. Erythrocyte biconcavity is maintained through these interactions. (Source: Ivanov and Paarvanova 2021) Table 4.8 Functions of different membrane transporters of erythrocytes
and deformability, active membrane transport, protein phosphorylation, synthesis of glutathione and purin and pyramidine nucleotides. Glucose is the major metabolic fuel of RBC except in pigs which is utilized solely by anaerobic glycolysis in the Embden-Meyerhof-Parnas pathway (EMP). Additionally, two minor shunts for carbohydrate metabolism namely the pentose phosphate pathway and Luebering-Rapoport bypass provide the protection against oxidative injury and regulate the oxygen carrying capacity of RBC. In pigs, inosine produced from the liver is the major substrate for energy production. The enzymatic machineries required for the metabolic processes are gained from nucleated erythrocyte precursors. These enzymes are limited and persist till their lifetime as erythrocytes are unable to synthesize new enzymes. 4.1.2.2.6.1 Embden-Meyerhof Pathway (EMP) It is the only source of energy production in RBCs as mature RBC lacks mitochondria. In this anaerobic glycolytic pathway, one molecule of glucose converts into two molecules of lactate with the production of two molecules of ATP. Besides ATP production, Embden-Meyerhof pathway keeps pyridine nucleotides in a reduced state. Deficiency of pyruvate kinase (PK) interferes with the survival of erythrocytes. Energy (ATP) deficient erythrocytes can be converted to echinocytes. PK-deficient dogs and cats suffer from hemolytic anemia due to intravascular hemolysis and leads to hemoglobinuria. They also have marked iron accumulation in the liver and may die from liver impairment. Phosphofructokinase (PFK) deficiency in dogs results in decreased 2,3 DPG concentration of RBC, which results in higher intracellular pH leads to alkalemia. 4.1.2.2.6.2 Luebering-Rapoport Pathway In this pathway, mature RBC produces 2,3-diphosphoglycerate (2,3-DPG) which helps to release oxygen from the hemoglobin to make it available for tissue utilization (Fig. 4.2). Erythrocytes of man, dogs, pigs, and horses have high concentration of 2,3DPG in contrast to cats and ruminants those are having low concentrations of
Fig. 4.2 Luebering-Rapoport pathway in the erythrocytes: In this pathway, 2,3-diphosphoglycerate (2,3-DPG) is produced by mature RBC with the help of bisphoglycerate phosphatase and bisphosphoglycerate mutase. 2,3-DPG helps to release oxygen from the hemoglobin 2,3-DPG. The formation of 2,3-DPG is stimulated by hypoxia, increased pH (metabolic alkalosis), and anemia. In all the cases, the generation of 2,3-DPG is aimed to release more oxygen. 4.1.2.2.6.3 Hexose Monophosphate Shunt/Phosphogluconate PathwayZPentose Phosphate Shunt In hexose monophosphate shunt, a small amount of glucose (5-13%) is utilized by oxidative metabolism not to produce energy but to generate reduced nicotinamide adenine dinucleotide phosphate (NADPH) that converts oxidized glutathione to reduced glutathione. Glutathione is the major antioxidant of RBC which protects the cell from oxidative damage. 4.1.2.2.6.4 Oxidative Injury of Erythrocytes and Its Protective Mechanisms The molecular oxygen can be reduced to form highly reactive oxygen species (ROS) such as, hydrogen peroxide (H2O2), hydroxyl radical, superoxide anion, hypochlorous acid (HOCl), and nitric oxide (NO). Low concentrations of these free radicals are required for phagocytosis, signal transductions, and the biosynthesis of prostaglandins but, higher concentrations of free radicals can be detrimental to the cells in terms of DNA damage, inactivation of enzymes, oxidation of hormones, lipids peroxidation, and membrane disturbance. Glutathione peroxidase system (GPx), superoxide dismutase (SOD), and catalase are the predominant antioxidant machinery functioning in the erythrocytes (Fig. 4.3). Peroxiredoxin 2 (Prx2) is the post potent H2O2 neutralizer in the erythrocytes. Selenium, ascorbic acid, and vitE also help to protect erythrocytes from oxidative damage. Know More....... Catalase deficiency disorder is called Takahara disease in human and canine catalase deficiency (CAT) in dogs. CAT is an autosomal recessive condition caused due to mutations in erythrocyte catalase gene identified in Beagle and American Fox hound breeds. 4.1.2.2.7 Destruction of Erythrocytes The erythrocyte population in the circulating blood must remain within a limit to ensure tissue oxygenation and the delicate balance between erythropoiesis and erythrolysis needs to be maintained by body’s homeostatic mechanisms. Erythrocytes in the circulation continuously suffer from oxidative injuries with a well-organized antioxidant defense system to cope up with these oxidative injuries (discussed earlier). When the RBCs are getting older, these antioxidant defense mechanisms are unable to support the erythrocytes to fight against oxidative damage
Fig. 4.3 Antioxidant machinery functioning in the erythrocytes: The reactive oxygen species are produced under oxidative stress. SOD causes dismutation of superoxide radicals to hydrogen peroxide. Catalase facilitates the breakdown of hydrogen peroxide to water and oxygen. In GPx system, two reduced glutathione (GSH) molecules accept hydrogens from hydrogen peroxide and resulting two H2O and one glutathione disulfide (GSSG). Glutathione reductase (GR) regenerates GSH from GSSG and lead to membrane defects, protein denaturation, altered ion permeability, and other potential hazards against the survival of erythrocytes. The senescence signals of erythrocytes are identified by the macrophages and they phagocytose the aged RBCs by a process known as erythrophagocytosis. However, all the erythrocytes do not follow age-related destruction; some defective erythrocytes undergo premature apoptosis through a process called eryptosis. Neocytolysis is another mechanism of selective lysis of young erythrocytes during physiological polycythemia and mass destruction is required to maintain the hematocrit and blood viscosity. 4.1.2.2.7.1 Lifespan of Erythrocytes The lifespan of erythrocytes can be measured by labeling the erythrocytes with radioactive and non-radioactive probes which should be non-toxic to the cell, non-immunogenic, should not elute before RBC destruction and can be recycled. The radioisotopes used to measure erythrocyte lifespan are 51Cr and 14C-cyanate, 15N-glycine, 59Fe and 55Fe. Several other non-radioactive probes are also available which can be detectable by flow cytometry such as biotinylation of erythrocytes is now widely used which can be detected by fluorescent probes conjugated with avidin. The erythrocyte lifespan in different species have been presented in Table 4.9. Several studies indicated that the lifespan of erythrocyte is positively correlated with the longevity of the species. The short living animals are having Table 4.9 Lifespan of erythrocytes in different species
Source: Reece (2015)
higher metabolic rates compared to long lived animals and their erythrocytes are more prone to oxidative damage. 4.1.2.2.7.2 Mechanisms of Erythrocyte Clearance by the Macrophages “Graveyards” of erythrocytes; Sites of erythrocyte destruction: In most of the domestic animals, red bone marrow is the principal site of RBC destruction whereas, in human; spleen is the main site for erythrophagocytosis. In contrast, most of the erythrocytes in birds are destroyed from liver. The preferential phagocytes for the erythrocytes are the macrophages which form phagolysosomes after engulfment. The resident macrophages of spleen are the most potent phagocytes for erythrocytes and they have well developed machineries in terms of CD163 (hemoglobin scavenger receptor) and the enzyme heme-oxygenase 1 to tackle the hemoglobin after erythrophagocytosis and recycled most of the iron in the hemoglobin to maintain iron homeostasis. With the progression of age, a variety of bio-physical, biochemical and molecular alterations occur in the erythrocytes that trigger erythrophagocytosis called engulfment signals or senescence markers as follows: Membrane microvesiculation: It is one of the important determinants of erythophagocytosis. Microvesiculation of the aged erythrocytes lead to loss of hemoglobin and almost 50% of the erythrocyte integral membrane proteins like band 3 and glycophorin and results in decreased membrane flexibility and cellular deformability which ultimately triggers the erythrocytes removal. Alterations in band 3: The oxidative injury to the erythrocyte causes breakdown of band 3 membrane proteins. With the breakdown of band 3, oxidative injury develops senescence neo antigens that binds with autologous IgGs together with C3 complement fraction that triggers erythrophagocytosis. This mechanism is called Band 3 mediated clearance pathway of erythrocytes. Externalization of phosphatidylserine (PS): PS in the erythrocyte membrane has pro-phagocytic activity and can be labeled as “eat-me” signals. In young and newly formed erythrocytes, PS is situated at the inner layer of the membrane thus undetected by the macrophages for destructions. But, at the time of senescence, due to the loss of membrane architecture, PS exposes to outer membrane and can easily be detected by macrophages to initiate erythrophagocytosis termed as PS mediated clearance pathway. Decreased expression of CD47: CD47 has anti-phagocytic activity that protects erythrocytes from phagocytosis by the macrophages. It is also labeled as “don’t eat-me” signals of erythrophagocytosis. During senescence, the expression of CD47 gradually declines, making the erythrocytes more susceptible to phagocytosis by the macrophages. Decreased expression of CD147: CD147 helps in the recruitment of erythrocytes from spleen to blood. During the time of senescence, expression of CD147 decreases which causes entrapment of erythrocytes in the spleen and facilitates their destruction by the macrophages. Altered calcium homeostasis: Influx of calcium is highly correlated with the oxidative damage, microvesiculation, membrane deformability and apoptosis like events in erythrocytes. Thus, it is assumed that alteration in the calcium homeostasis is one of the senescence markers that trigger erythrophagocytosis. 4.1.2.2.7.3 Extravascular and Intravascular Hemolysis Hemolysis can be broadly classified into extravascular and intravascular hemolysis based on their location. Extravascular hemolysis is a routine process of erythrocyte clearance after aging by the cells of reticulo-endothelial systems outside the blood vessel. In contrary, the lysis of erythrocytes in vivo, i.e., within the blood vascular system is termed as intravascular hemolysis. The differences between these two types of hemolytic mechanisms are summarized in Table 4.10. 4.1.2.3 Leukocytes Leukocytes or white blood cells (WBCs) are less in circulation, when compared to red blood cells (RBC). Unlike RBSc, WBCs are colorless and thus called as leukocytes. The leukocytes can be classified into two types based on their microscopic characteristics with stains namely, granulocytes and agranulocytes. Granulocytes are characterized by their granular cytoplasm. They are of three types: eosinophils, stains with acidic stain like eosin due to the presence of basic proteins in their cytoplasm. Basophils are having acidic cytoplasmic granules and hence stain with basic dye like methylene blue. Neutrophils have both acidic and basic granules, hence, can be stained with both acidic and basic stain and look lavender color. The agranulocytes are so named as they have no granules in their cytoplasm. They are further classified monocytes and lymphocytes. The total number of leukocytes and their relative proportions in the blood has been presented in Table 4.11. Table 4.10 Difference between extravascular and intravascular hemolysis
Table 4.11 Differential leukocyte counts in different species
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