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Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3...

Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT ERYTHROCYTES RED BLOOD CELLS DESTRUCTION o As red blood cell ages, there is a decrease in its enzyme, a decrease in ATP, a decrease in size, and an increase in density. o Most cells are able to replenish needed enzymes and continue their cellular processes. As a nonnucleated cell, however, the mature erythrocyte is unable to generate new proteins, such as enzymes, so as its cellular functions decline, the cell ultimately approaches death. o The average RBC has sufficient enzyme function to live 120 days. Because RBCs lack mitochondria, they rely on glycolysis for production of adenosine triphosphate (ATP). The loss of glycolytic enzymes is central to this process of cellular aging, called senescence, which culminates in phagocytosis by macrophages. o Approximately 1 % of the red blood cells leave the circulation each day and broken down by the mononuclear phagocytic system o Culling is a splenic function wherein old/aged/senescent red blood cells are filtered and destroyed through the phagocytosis of splenic macrophages. o As an erythrocyte ages, the following processes occur: 1. The membrane becomes less flexible. 2. The concentration of cellular hemoglobin increases. 3. Enzyme activity, particularly glycolysis, diminishes. 2 TYPES OF RBC DESTRUCTION: 1. ____________________(Extravascular Hemolysis) 2._____________________(Fragmentation or Intravascular Hemolysis) Extravascular Hemolysis o This is the ________ method by which RBCs die normally. o Responsible for ____% of RBC destruction o RBCs must remain highly flexible to exit the spleen by squeezing through the so-called splenic sieve formed by the endothelial cells lining the venous sinuses and the basement membrane. o Spherical RBCs are rigid and are not able to squeeze through the narrow spaces; they become trapped against the endothelial cells and basement membrane. In this situation, they are readily ingested by macrophages that patrol along the sinusoidal lining. “The future belongs to those who believe in the beauty of their dreams.” 1  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT Intravascular Hemolysis o Responsible for ____% of RBC destruction (Turgeon) or 10 to 20% RBC destruction (Rodak’s 5th edition) o Fragmentation or intravascular hemolysis results when mechanical factors rupture the cell membrane while the cell is in the peripheral circulation. o As the result of intravascular destruction, hemoglobin is released directly into the bloodstream and undergoes dissociation into alpha and beta dimers, which are quickly bound to the plasma globulin ____________ o Haptoglobin-Hemoglobin complex is removed from the circulation by the hepatocytes. o Because haptoglobin is removed from the circulation as part of the haptoglobin- hemoglobin complex, the level of plasma haptoglobin decreases with hemolysis o Once plasma haptoglobin is depleted in the blood circulation, excess unbound hemoglobin are filtered by the glomeruli in the kidneys, reabsorbed by the renal tubular cells, and converted to _____________ o The renal tubular uptake can process as much as ___ g/day of filtered hemoglobin and once the capacity for renal tubular uptake has been exceeded, free hemoglobin and methemoglobin begin to appear in the urine o Hemoglobin that is neither bound by haptoglobin nor directly excreted in the urine is oxidized to____________________. o The heme groups in methemoglobin are released and taken up by another transport protein,__________. o Heme groups in excess of the hemopexin-binding capacity combine with albumin to form _____________until more hemopexin is available. Once it is needed, hemopexin becomes available, and the complex is subsequently phagocytized by hepatocytes. o The combined depletion of haptoglobin and hemopexin and the presence of methemalbuminemia and hemosiderinuria can be seen in cases of intravascular hemolytic anemia ERYTHROCYTE MEMBRANE PHYSIOLOGY RBC Membrane Deformability o RBCs are biconcave, 7 to 8 um in diameter, with a volume range of 80 to 100 fL and a mean volume of 90 fL. o Their average surface area is 140 mm2, which is a 40% excess of surface area compared with a sphere of 7 to 8 mm in diameter. o This excess surface area-to-volume ratio enables RBCs to stretch undamaged up to 2.5 times their resting diameter as they pass through narrow capillaries and through splenic pores 2 um in diameter. This property is called_______________. “The future belongs to those who believe in the beauty of their dreams.” 2  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT o The deformable RBC membrane provides the broad surface area and close tissue contact necessary to support the delivery of O2 from the lungs to body tissues and to transport CO2 from body tissues to the lungs. RBC Membrane Components o The RBC membrane consists of approximately 8% carbohydrates, 52% proteins, and 40% lipids. o The RBC membrane is a lipid bilayer whose hydrophobic components are sequestered from aqueous plasma and cytoplasm. The membrane provides a semipermeable barrier separating plasma from cytoplasm and maintaining an osmotic differential. 1. LIPIDS (40%) o RBC membrane phospholipids are asymmetrically distributed. Phosphatidylcholine and sphingomyelin predominate in the outer layer; phosphatidylserine and phosphatidylethanolamine form most of the inner layer. o Phospholipids form an impenetrable fluid barrier as their hydrophilic polar head groups are arrayed on the membrane’s surfaces, oriented toward both the aqueous plasma and the cytoplasm, respectively, as depicted in the fluid mosaic membrane model (FMMM). o Their hydrophobic nonpolar acyl tails arrange themselves to form a central layer sequestered (hidden) from the aqueous plasma and cytoplasm. o The ratio of cholesterol to phospholipids remains relatively constant to maintain the balance of deformability or elasticity and strength. A. _____________- phospholipids, phosphatidylcholine, glycolipid, and sphingomyelin B. _____________- phosphatidyl ethanolamine Phosphatidyl inositol, Phosphatidyl serine o Acanthocytosis and target cells are associated with abnormalities in the concentration or distribution of membrane cholesterol and phospholipids. 2. PROTEIN (52%) a. ____________________________ o serve many functions including: transport sites, adhesion sites, and signaling receptors. o RBC transmembrane proteins transport ions, water, and glucose and anchor cell membrane receptors. o they support carbohydrate-defined blood group antigens “The future belongs to those who believe in the beauty of their dreams.” 3  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT o Examples:_____________________________________________________________________________ b. ______________________________ o Shape and flexibility of the RBC, which are essential to its function, depend on the cytoskeleton. o The cytoskeleton is derived from cytoskeletal (peripheral) proteins on the cytoplasmic side of the lipid membrane. The major cytoskeletal proteins are a- and b-spectrin heterodimers. o Hereditary ___________ arises from defects in spectrin or proteins forming the ankyrin complex that provide vertical support for the membrane. o Hereditary ___________ is due to defects in cytoskeletal proteins that provide horizontal support for the membrane o Examples: _______________________________________________________________________ Red Blood Cell Energy Metabolism 1. Emden-Meyerhof Pathway o ___________ of the essential cellular energy. o ___________ glycolysis pathway o Glucose enters the RBC without energy expenditure o This net gain of two ATPs provides the high-energy phosphates needed for maintenance of the erythrocyte’s shape and flexibility, for maintenance of membrane lipids. o Responsible for ______________________glycolysis o _______________________________- most common enzyme deficiency 2. Hexose monophosphate or Pentose phosphate Pathway o Responsible for ________ glycolysis o ____________ pathway o This energy system couples oxidative catabolism of glucose with reduction of NADP (nicotinamide-adenine dinucleotide phosphate) to NADPH (the reduced form of NADP), which is subsequently required to _________________________________ o G6PD provides the only means of generating NADPH for glutathione reduction, and in its absence, erythrocytes are particularly vulnerable to oxidative damage o The pathway’s activity is increased with the increased oxidation of glutathione. When the pathway is defective, the amount of reduced glutathione becomes insufficient to neutralize oxidants. This causes denaturation of globin, which precipitates as aggregates referred to as Heinz bodies. o Provides reduced glutathione (GSH) to prevent oxidative denaturation of hemoglobin which makes it a protective mechanism o ________________________-most common inherited enzyme deficiency and is associated with Heinz bodies 3. Methemoglobin Reductase Pathway o Heme iron is constantly exposed to oxygen and peroxides. Peroxide oxidizes heme iron from the ferrous (2+) to the_________________. When the iron state is ferric, the affected hemoglobin molecule is called_____________________. “The future belongs to those who believe in the beauty of their dreams.” 4  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT o In this form, hemoglobin can no longer combine reversibly with oxygen; therefore, the oxygen transport function of the molecule is lost o This pathway Converts methemoglobin back to normal hemoglobin using the methemoglobin reductase enzyme which makes it a corrective mechanism o ___________________________________________is involved for reducing ferric iron into ferrous state 4. Rapaport Luebering Pathway o Generates 2,3 Diphosphoglycerate (2,3-DPG) that decreases hemoglobin affinity to oxygen o This pathway is important in the oxygen-carrying capability of erythrocytes. Because of this pathway, the erythrocyte has a built-in mechanism that is low in energy expenditure and is capable of regulating oxygen transport during conditions of hypoxia and disorders of acid-base balance. o ↑2,3DPG = ↓Hb affinity to Oxygen (Shift to the_____) o ↓2,3DPG = ↑ Hb affinity to Oxygen (Shift to the______) HEMOGLOBIN o Hemoglobin’s main function is to transport oxygen from the lungs to tissues and transport carbon dioxide from the tissues to the lungs for exhalation. o Hemoglobin also contributes to acid-base balance by binding and releasing hydrogen ions and transports nitric oxide, a regulator of vascular tone. o Comprises approximately 95% of the cytoplasmic content of RBCs. The body efficiently carries hemoglobin in RBCs, which provides protection from denaturation in the plasma and loss through the kidneys. o The concentration of hemoglobin within RBCs is approximately 34 g/dL, and its molecular weight is approximately 64,000 Daltons. o The _________portion of hemoglobin gives erythrocytes their characteristic red color. A. HEMOGLOBIN STRUCTURE o A tetramer of four globin polypeptide chains, with a heme molecule attached to each chain Hemoglobin= 4 heme group+ 4 globin (polypeptide) chains o Four identical heme groups, each consisting of a protoporphyrin ring and ferrous (Fe 2+) iron Heme Structure o Heme consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of divalent ferrous iron. o The ferrous iron in each heme molecule reversibly combines with one oxygen molecule. When the ferrous “The future belongs to those who believe in the beauty of their dreams.” 5  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT irons are oxidized to the ferric state (Fe3+), they no longer can bind oxygen. Globin Structure o The four globin chains comprising each hemoglobin molecule consist of two identical pairs of unlike polypeptide chains, 141 to 146 amino acids each. o Variations in amino acid sequences give rise to different types of polypeptide chains. Each chain is designated by a Greek letter Complete Hemoglobin Molecule o The hemoglobin molecule can be described by its primary, secondary, tertiary, and quaternary protein structures Primary structure of hemoglobin o Refers to the amino acid sequence of the polypeptide chains Secondary structure of hemoglobin o Refers to chain arrangements in helices and non- helices. Tertiary structure of hemoglobin o Refers to the arrangement of the helices into a pretzel-like configuration Quaternary structure o The complete hemoglobin molecule structure also called a tetramer. MUST KNOWS ABOUT HEMOGLOBIN ▪ Hemoglobin= ______________ ▪ Heme= ______________ ▪ 1 HEME MOLECULE= ______________ ▪ 1 HEME MOLECULE= ______________ ▪ ratio of pyrrole ring to ferrous iron in a single heme molecule= ______________ ▪ 1 Heme= ______of Oxygen ▪ 1 Hb= ______of Oxygen ▪ 1 gram Hb= 1.34 ml Oxygen ▪ 1 gram Hb= 3.47mg of Iron “The future belongs to those who believe in the beauty of their dreams.” 6  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT B. HEMOGLOBIN SYNTHESIS / PRODUCTION o Heme biosynthesis occurs in the mitochondria and cytoplasm of bone marrow erythroid precursor. As they lose their ribosomes and mitochondria, mature erythrocytes can no longer make hemoglobin. o Rubricyte = first stage of hemoglobin synthesis o Reticulocytes = last stage capable of hemoglobin synthesis o Heme synthesis occurs in the ___________________ of normoblasts and is dependent on glycine, succinyl coenzyme A, Aminolevulinic acid synthase, and vitamin B6 (pyridoxine) o Globin synthesis occurs in the _______________, and it is controlled on chromosome 16 for alpha and zeta chains and chromosome 11 for all other chains HEME SYNTHESIS “The future belongs to those who believe in the beauty of their dreams.” 7  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT OXYGEN DISSOCIATION CURVE o The curve produced when the two variables (partial pressure of oxygen and affinity of hemoglobin for oxygen) are plotted on a graph (oxygen saturation of hemoglobin versus the partial pressure of oxygen) o ________ in shape o P50 values: defined in terms of the amount of oxygen needed to saturate 50% of hemoglobin. PO2 o with high oxygen tension in the lungs, affinity of hemoglobin for oxygen is high, and hemoglobin becomes rapidly saturated with oxygen. o with relatively low oxygen tension in the tissues, the affinity of hemoglobin for oxygen is low, and hemoglobin rapidly releases oxygen. PARAMETER SHIFT TO THE SHIFT TO THE LEFT RIGHT Hb affinity for O2 Ph PCO2 2,3-DPG Temperature Ph o In the tissues, a lower pH shifts the curve to the right and reduces the affinity of hemoglobin for oxygen, and the hemoglobin more readily releases oxygen. o Bohr effect - A shift in the curve because of a change in pH (or hydrogen ion concentration). It facilitates the ability of hemoglobin to exchange oxygen and carbon dioxide (CO2). 2,3-DPG o deoxygenated state= hemoglobin tetramer assumes a __________________________that is stabilized by the binding of 2,3-BPG between the b-globin chains o fully oxygenated= it assumes a _______________________ o The binding of 2,3-BPG shifts the oxygen dissociation curve to the right, favoring the release of oxygen o ↑2,3DPG = ↓Hb affinity to Oxygen (Shift to the Right) o ↓2,3DPG = ↑ Hb affinity to Oxygen (Shift to the Left) Clinical conditions that produce a shift of the oxygen dissociation curve Shift to the Right increased body temperature (fever); acidosis; presence of hemoglobin variants with a low affinity for oxygen; and an increased 2,3-BPG concentration in response to hypoxic conditions, such as high altitude, pulmonary insufficiency, congestive heart failure, and severe anemia “The future belongs to those who believe in the beauty of their dreams.” 8  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT Shift to the Left lowered body temperature as a result of external causes multiple transfusions of stored blood with depleted 2,3-BPG; alkalosis; and presence of hemolobin variants with a high affinity for oxygen Increase myoglobin and fetal hemoglobin TYPES OF HEMOGLOBIN AND DERIVATIVES NORMAL HEMOGLOBINS 1. HEMOGLOBIN A1 o 2 alpha + 2 beta globin chains o Predominate hemoglobin in an __________ o Subdivided into ________________________________which reflects glucose level in the blood o Reference range for normal adult Hgb is ___________________________________________ 2. HEMOGLOBIN F o Composed of 2alpha + 2 gamma o Predominates at ________(80%) o Hgb F is a compensatory hemoglobin o Increased in hemoglobinopathies and B-thalassemia Major o Fetal hemoglobin (hemoglobin F) is the predominant hemoglobin variety in the fetus and the newborn o Fetal hemoglobin appears by the fifth week of gestation and persists for several months after birth o Associated with hepatic erythropoiesis 3. OXYHEMOGLOBIN o Hemoglobin with Ferrous + oxygen; seen in arterial circulation (RELAXED STATE) 4. DEOXYHEMOGLOBIN o Hemoglobin with Ferrous but no Oxygen; seen in venous circulation (TENSED STATE) DYSHEMOGLOBIN o Dyshemoglobins are dysfunctional hemoglobins that are unable to transport oxygen. o Dyshemoglobins form and may accumulate to toxic levels, after exposure to certain drugs or environmental chemicals or gasses. The offending agent modifies the structure of the hemoglobin molecule, preventing it from binding oxygen. o Most cases of dyshemoglobinemia are acquired; a small fraction of methemoglobinemia cases are hereditary. 1._______________________ o is formed by the reversible oxidation of heme iron to the ___________________(Fe3+) “The future belongs to those who believe in the beauty of their dreams.” 9  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT o Methemoglobin cannot carry oxygen because oxidized ferric iron cannot bind it. An increase in methemoglobin level results in decreased delivery of oxygen to the tissues. o Cause ___________________discoloration of blood o Causes ___________and functional anemia if present in high enough concentration o Sources: Chemical or drugs such as chlorate, nitrate, and nitrite o Quantitated by spectrophotometry o Methemoglobin is assayed by spectral absorption analysis instruments such as the CO-oximeter. o Methemoglobin shows an absorption peak at 630 nm o Anticoagulant used for quantitation: _____________ o If the level of methemoglobin increases to 30% or more of total hemoglobin, intravenous _____________________is administered. Methylene blue reduces methemoglobin ferric iron to the ferrous state through NADPH-methemoglobin reductase and NADPH produced by glucose-6- phosphate dehydrogenase in the hexose monophosphate shunt. Level Sign and symptoms Less than 25% Asymptomatic More than 30% __________ (bluish discoloration of skin) and symptoms of hypoxia More than 50% 2. _________________________ o formed by irreversible oxidation of hemoglobin by drugs (such as sulfanilamides, phenacetin, nitrites, and phenylhydrazine) or exposure to sulfur chemicals in industrial or environmental settings. o It is formed by the addition of a ____________ to the pyrrole ring of heme and has a ________pigment. o Sulfhemoglobin is ineffective for oxygen transport, and patients with elevated levels present with cyanosis. o Sulfhemoglobin is Irreversible. It cannot be converted to normal Hb A; it persists for the life of the cell. o Blood is ____________________ in sulfhemoglobinemia o It can also combine to Carbon monoxide to form _____________________. o Can be quantitated by spectrophotometry o Sulfhemoglobin has a similar peak to methemoglobin on a spectral absorption instrument. The sulfhemoglobin spectral curve, however, does not shift when cyanide is added, a feature that distinguishes it from methemoglobin. 3.__________________________________ o results from the combination of __________________(CO) with heme iron. o The affinity of carbon monoxide for hemoglobin is ____ times that of oxygen. o Carbon monoxide has been termed the ________________ because it is an odorless and colorless gas, and victims may quickly become hypoxic. o Carbon monoxide will bind with Hb even if its concentration in the air is extremely low (0.02–0.04 %) o Cannot bind and carry oxygen o Increasing concentration of HbCO shift the ODC (Oxygen dissociation curve) to the left, thus adding to anoxia o Light sensitive and imparts a typical brilliant ________________ color to the blood “The future belongs to those who believe in the beauty of their dreams.” 10  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT o Chief sources of the gas are gasoline motors, illuminating gas, gas heaters, defective stoves, and smoking of tobacco o Quantitated by differential spectrophotometry or by gas chromatography IRON o Iron is the most abundant transition metal in the body o Most iron in the body is in Hemoglobin and must be in the ferrous state to be used. Ferrous iron binds to oxygen for transport to lungs and body tissues. Ferric iron (Fe 3+) is not able to bind to hemoglobin, but does bind to transferrin o Iron is an essential mineral and is not produced in the body IRON METABOLISM o Normal daily diet contains about 15mg iron and only 1-2mg of iron is absorbed in the duodenum and upper jejunum o In the duodenum, dietary free iron is reduced to ferrous iron and taken up from the intestinal lumen into the enterocytes by the iron transport protein ______________________________(DMT) o Once absorbed, iron may be stored as _____________ in the enterocytes or exported into the circulation by another iron transport protein, _______________ (Fpn1) o In the plasma, ferric iron binds to transferrin which is delivered into cells by binding to transmembrane glycoprotein the transferrin receptors (TfR) o _____________, a liver-produced peptide hormone, it is the master regulatory hormone of systemic iron metabolism. o The interaction of hepcidin with the plasma iron transporter, ferroportin, coordinates iron acquisition with iron utilization and storage. o Hepcidin deficiency causes common iron overload syndromes but overexpression of hepcidin is responsible for microcytic anemia (anemia of chronic inflammation). o Hepicidin = It regulates the transport of iron from enterocyte into the circulation by binding through ferroportin. o Mature red blood cells cannot store iron Dietary iron in small intestine-------→ Enterocytes------→ Circulation/Plasma------→ Different cells Relationship of Hepcidin to ferritin ________________ Relationship of Hepcidin to serum iron ________________ Relationship of Hepcidin to ferroportin Inversely proportional *increase hepcidin= blocks ferroportin from transporting iron out *decrease hepcidin=promotes ferroportin from transporting iron out STORAGE FORMS OF IRON 1. o Major storage form and considered as an acute phase reactant o Water soluble “The future belongs to those who believe in the beauty of their dreams.” 11  Hematology 1 Clinical Instructors: DENNIS P. MONILLA, RMT PRELIM: LECTURE TOPIC 3 ROMILET D. DEL ROSARIO, RMT o Ferric iron is stored in a cage-like protein called apoferritin. Once iron binds, it is known as ferritin o One ferritin molecule can bind more than 4000 iron ions 2. o Second storage form of iron o Water insoluble o It is form from degradation of ferritin (a breakdown product of ferritin) o Found predominantly in liver, spleen, and bone marrow o Can be seen in urine (hemosiderinuria)and confirmed by Perl’s Prussian blue CONDITIONS WITH IRON OVERLOADING A. o Increased tissue iron stores without associated tissue damage; may progress to hemochromatosis o Accumulation of iron stores with little or no parenchymal cell injury B. o HH is an autosomal recessive genetic error of metabolism that produces inappropriately increased (twofold to threefold greater than normal) GI absorption of iron o It is associated with damage of most cells especially the WBCs o There is deposition of hemosiderin that gives the skin a _____________ color o The heart muscle also is especially vulnerable to excessive iron deposition, which leads to congestive heart failure. o Hepatocellular carcinoma occurs more frequently in patients with hemochromatosis. “The future belongs to those who believe in the beauty of their dreams.” 12