At the end of this unit of study the student should be able to List and describe the stages of erythrocyte maturation in the marrow from youngest to most mature cells Explain the maturation process of reticulocytes and the cellular changes that take place ID: 931668
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The Erythrocyte
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Slide2Learning Objectives—Level lAt the end of this unit of study, the student should be able to:List and describe the stages of erythrocyte maturation in the marrow from youngest to most mature cells.Explain the maturation process of reticulocytes and the cellular changes that take place.Identify the reference interval for reticulocytes.
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Slide3Learning Objectives—Level lAt the end of this unit of study, the student should be able to:Explain the function of erythropoietin and include the origin of production, bone marrow effects, and normal values.Describe the function of the erythrocyte membrane.Name the energy substrate of the erythrocyte.
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Slide4Learning Objectives—Level lAt the end of this unit of study, the student should be able to:Define and differentiate intravascular and extravascular red cell destruction.State the average dimensions and life span of the normal erythrocyte.Describe the function of 2,3-BPG and its relationship to the erythrocyte.
Slide5Learning Objectives—Level llAt the end of this unit of study, the student should be able to:Summarize the mechanisms involved in the regulation of erythrocyte production.Describe the structure of the erythrocyte membrane, including general dimensions and features; assess the function of the major membrane components.
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Slide6Learning Objectives—Level llAt the end of this unit of study, the student should be able to:Explain the mechanisms used by the erythrocyte to regulate permeability to cations, anions, glucose, and water.Compare and contrast three pathways of erythrocyte metabolism and identify key intermediates as well as the relationship of each to erythrocyte survival and longevity.
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Slide7Learning Objectives—Level llAt the end of this unit of study, the student should be able to:Generalize the metabolic and catabolic changes within the erythrocyte that occur with time that "label" the erythrocyte for removal by the spleen.Predict the effects of increased and decreased erythropoietin levels in the blood.
Slide8ErythrocytesErythrocytes (red blood cells, RBCs) carry oxygen from lungs to tissues where it is utilized in oxidative metabolism.Anemia (insufficient number of RBCs) is characterized by inadequate tissue oxygenation.
Slide9ErythrocytesErythrocytosis (excess number of RBCs) has no adverse effect on pulmonary gas exchange.
Slide10Erythropoiesis and RBC MaturationErythronTotality of all stages of erythrocytesFrom the marrow precursor cells to the mature cells in peripheral blood
Slide11Erythropoiesis and RBC MaturationErythropoiesisProduction of erythrocytes in orderly processErythropoietin (EPO) is major cytokine regulating erythropoiesisCirculating life span of mature RBCs ~ 100–120 daysSenescent cells are destroyed in liver, spleen, bone marrow by macrophages
Slide12Erythroid Progenitor CellsBFU-ERegulated primarily by IL-3, GM-CSF (burst-promoting activity—BPA)Relatively insensitive to EPO
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Slide13Erythroid Progenitor CellsBFU-E"Burst" or multi-focal colony formed in-vitro within 10–14 daysGives rise to several hundred to several thousand hemoglobin-containing RBC precursor cells Gives rise to the CFU-E
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Slide14Erythroid Progenitor CellsBFU-EAre CD34+, progenitor cell markerHigh proliferative potential, but low rate of cycling
Slide15Erythroid Progenitor CellsCFU-EGives rise to a discrete colony of 8–60 hemoglobin-containing cells within 2–5 days Have a high concentration of EPO receptorsRespond to lower concentrations of EPOImmediate precursor of pronormoblasts
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Slide16Erythroid Progenitor CellsCFU-ELose CD34+ as they matureBegin to express characteristics of erythroid cellsGlycophorin ARh antigensIn a subset of CFU-E, the ABH and Ii antigens
Slide17Figure 5-1 Erythroid maturation. Erythrocyte development proceeds through three levels of maturation beginning with the multipotential hematopoietic stem cell (HSC), maturing into committed progenitor cells BFU-E and CFU-E, and into morphologically recognizable cells. IL-3 and GM-CSF are the primary cytokines that affect maturation of BFU-E. EPO primarily affects the CFU-E and developing normoblasts.
Slide18Erythroid-Maturing CellsPrecursor cells in BM that are morphologically identifiableErythroblastsIncludes all nucleated RBC precursors in BMIf the maturation sequence is normal, the cells are often called normoblasts.
Slide19Erythroid-Maturing CellsReticulocytes (polychromatophilic RBCs)Young RBCs that do not have a nucleus but have residual RNA
Slide20Erythroid-Maturing CellsNormoblastic maturationOccurs in an orderly and well-defined processGradual decrease in cell size with progressive condensation of the nuclear chromatinEventual expulsion of the pyknotic nucleus
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Slide21Erythroid-Maturing CellsNormoblastic maturationOccurs in an orderly and well-defined processCytoplasm in younger cells is deeply basophilic due to the abundance of RNA.Increase in hemoglobin (acidophilic) as the cell matures, cytoplasm appears pink or salmon color
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Slide22Erythroid-Maturing CellsNormoblastic maturationEncompasses six morphologically defined stages:PronormoblastBasophilic normoblastPolychromatophilic normoblastOrthochromatic normoblastReticulocyteErythrocyte
Slide23Erythroid-Maturing CellsNormoblasts Spend 5–7 days proliferating and maturing in the bone marrowReticulocyteMatures in 2–3 daysFirst 1–2 of those days are spent in the marrowMature, circulating RBC~120 day life span
Slide24Erythropoietin (EP0)Only cytokine important in regulating the final stages of erythroid maturationAndrogens have some effect on maturation.Can stimulate EPO productionCan directly affect the erythropoietic marrow
Slide25Erythropoietin (EP0)Other hormonesThyroid hormone, adrenal cortical hormones, growth hormone, have varying effects on erythropoiesis
Slide26Pronormoblast (Rubriblast)Earliest recognizable RBC precursorProduces 8–32 mature RBCs through 3–5 cell divisionsLargest of the normoblast series20–25 mcM in diameterHigh nuclear: cytoplasmic (N:C) ratio
Slide27Pronormoblast (Rubriblast)CytoplasmContains large numbers of ribosomesStains deeply basophilicPale area next to nucleus (Golgi apparatus)Does not take up Romanowsky stainSmall amounts of hemoglobin are present Not visible by light microscopy
Slide28Pronormoblast (Rubriblast)NucleusLarge and takes up 80% or more of the cellStains bluish-purpleChromatin is fine.Often described as lacyHas a coarser chromatin pattern than a white cell blastContains one to three faint nucleoli
Slide29Pronormoblast (Rubriblast)
Slide30Pronormoblast (Rubriblast)
Slide31Basophilic Normoblast (Prorubricyte)Similar to pronormoblastSmaller 16–18 mcM in diameterActively dividing
Slide32Basophilic Normoblast (Prorubricyte)CytoplasmDeeply basophilic due to increased ribosomesPerinuclear halo around the nucleusCorresponds to the mitochondriaDoesn't stain with Romanowsky stainLate stages have more hemoglobin causing cell to have lighter blue hue or pink areas
Slide33Basophilic Normoblast (Prorubricyte)NucleusSlightly ↓ N:C ratio, nucleus occupies 75% cellChromatin is coarser than the pronormoblast.Dark violet heterochromatin interspersed with lighter-staining euchromatin for wheel-spoke appearanceNucleoli are usually not apparent.
Slide34Basophilic Normoblast (Prorubricyte)
Slide35Basophilic Normoblast (Prorubricyte)
Slide36Polychromatic Normoblast (Rubricyte)Cell is about 12–15 mcM in diameter. CytoplasmPresence of abundant gray-blue cytoplasmDue to synthesis of large amounts of hemoglobin and ↓ amounts of ribosomes
Slide37Polychromatic Normoblast (Rubricyte)Nucleus↓ N:C ratio due to condensation of the nuclear chromatinChromatin is irregular and coarsely clumped due to increased aggregation of heterochromatin.Last stage capable of mitosis
Slide38Polychromatophilic Normoblast (Rubricyte)
Slide39Polychromatophilic Normoblast (Rubricyte)
Slide40Orthochromic Normoblast (Metarubricyte)Cell is about 10–15 mcM in diameter with low N:C ratio.CytoplasmIs predominately pink or salmon color due to concentration of hemoglobinRetains a tinge of blue due to ribosomes
Slide41Orthochromic Normoblast (Metarubricyte)NucleusChromatin is heavily condensedLate stageNucleus is structureless (pyknotic) or fragmented, often eccentric or partially extruded
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Slide42Orthochromic Normoblast (Metarubricyte)NucleusNucleus is: Extruded while the cell is still in the erythroblastic island or lost as the cell passes through the wall of the marrow sinusExpelled nucleus engulfed by marrow macrophages
Slide43Orthochromic Normoblast (Metarubricyte)
Slide44Orthochromic Normoblast (Metarubricyte)
Slide45ReticulocyteThe reticulocyte has an irregular lobulated or puckered shape following nuclear extrusion.The cell is remodeled.Eliminating excess membraneGradually acquires a biconcave disc shape
Slide46ReticulocyteCytoplasm has a bluish tinge.Due to residual RNA and mitochondriaCalled a polychromatophilic RBC or diffusely basophilic RBC on Wright's stain Remaining 20% of hemoglobin is made during the reticulocyte stage
Slide47ReticulocyteSize is 7–10 mcM, slightly larger than mature RBC
Slide48ReticulocyteCells are identified in vitro with supravital stains.New methylene blue or brilliant cresyl blueCauses precipitation of RNA and mitochrondriaAppear as punctate purplish-blue inclusions
Slide49ReticulocyteNormal percentage in peripheral blood0.5–2.5%Absolute concentration can be calculatedMultiply the % of reticulocytes by the RBC countNormal absolute concentration 18–158 × 109/L
Slide50ReticulocyteWhen reticulocytes are increased, will observe increased polychromasia on peripheral blood smear
Slide51ReticulocyteCan contain small amounts of iron Iron is identified by Perl's Prussian blue stain.SiderocytesErythrocytes with identified ironSideroblastsNucleated RBCs with iron in the cytoplasmSpleen removes the iron granules.
Slide52Reticulocyte
Slide53ErythrocyteA biconcave discSize 7–8 mcM in diameter, volume 80–100 fLStains pink to orangeDue to acidophilic hemoglobin (28–34 pg/cell)Lack cellular organelles (ribosomes and mitochondria) and enzymes required to synthesize new lipid and proteins
Slide54ErythrocyteExtensive damage to cell membrane cannot be repaired and the spleen will cull damaged cells from circulation.
Slide55Erythrocytes
Slide56Erythroblastic IslandsCentral macrophage surrounded by developing erythroblasts and reticulocytesCentral macrophage ("nurse cell")Send out cytoplasmic processesMaintain direct contact with each erythroblast
Slide57Erythroblastic IslandsErythroblasts adhere to the macrophage.Cytoadhesion moleculesFibronectin
Slide58Erythroblastic IslandsAs cell matures, it loses the adhesion molecules.Cell detachesPasses through a pore in the cytoplasm of endothelial cells lining the marrow sinusEnters circulation
Slide59Erythroblastic IslandsCentral macrophage phagocytizes The nucleusAny defective erythroblasts
Slide60Erythrocyte MembraneIs essential for erythrocyte development and functionErythroblast membrane has receptors for:EPO and transferrin (iron transport protein)
Slide61Erythrocyte MembraneSelectively sequesters vital componentsAllows escape of metabolic waste productsRegulates metabolismReversibly binding and inactivating many glycolytic enzymes
Slide62Erythrocyte MembraneBalances exchange of bicarbonate and chloride ionsAids in transfer of carbon dioxide from tissues to lungsBalances cation and water concentrations to maintain RBC ionic composition
Slide63Erythrocyte MembraneCytoskeleton provides RBCs with strength and flexibility needed to survive in circulation.
Slide64Membrane CompositionPhospholipid bilayer-protein complex ~52% protein, 40% lipid, and 8% carbohydrateControls the membrane functions Transport, durability/strength, flexibilityDetermines the membrane's antigenic propertiesDefects can alter function and lead to cell death
Slide65Table 5-2 Erythrocyte Membrane Composition
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Slide66Table 5-2 (continued) Erythrocyte Membrane Composition
Slide67Lipid Composition~95% of lipid content of RBC membrane consists of equal amounts of: Unesterified cholesterol Phospholipids (PLs) Remaining lipids are:Free fatty acids (FAs) Glycolipids (globoside)
Slide68Lipid CompositionMature RBC cannot synthesize new lipidsDepend on lipid exchange with plasma and fatty acid acylation for phospholipid repair and renewal during life span
Slide69Lipid CompositionPhospholipid bilayer Phospholipid molecules are arranged with:Polar heads exposed at each membrane surface (cytoplasmic and plasma membrane)Hydrophobic fatty acid side chains directed to the interior of the bilayer
Slide70Lipid CompositionMajor phospholipids are: Phosphatidylcholine (PC)Phosphatidylethanolamine (PE)Phosphatidylserine (PS)Sphingomyeline (SM) Small amounts of phosphatidylinositol (PI)Asymmetrically distributed within bilayer
Slide71Lipid CompositionPC and SM Concentrated on outer half of bilayer PE, PS, PI Largely confined to inner half of bilayerTransmembrane diffusion of PLsFrom areas of higher concentration to bilayer leaflet of lower concentrationAsymmetry maintained by ATP-dependent transport system (flippase)
Slide72Lipid CompositionCholesterol and glycolipids are intercalated between the PLs in bilayerCholesterol Present in ~ equal proportions on both sides of lipid bilayerAffects the surface area of the cell and membrane permeability
Slide73Lipid CompositionMembrane cholesterol exists in equilibrium with unesterified free cholesterol in plasma.Plasma cholesterol (unesterified) is partially converted to esterified cholesterol by lecithin-cholesterol acyl transferase (LCAT).Once esterified, cholesterol cannot return to the RBC membrane.
Slide74Lipid CompositionIf LCAT absent (congenital LCAT deficiency or hepatocellular disease) Free plasma cholesterol increases the accumulation of cholesterol within the RBC membrane and leads to expansion of membrane surface area.
Slide75Lipid CompositionAn excess of cell membrane Due to proportional increases in cholesterol and PLs (maintaining the normal ratio)Results in formation of macrocodocytes (large target cells)
Slide76Lipid CompositionAn ↑ in the cholesterol:phospholipid ratioDecrease in membrane fluidity Results in acanthocytes (reduced survival)
Slide77Lipid CompositionExpansion of outer leaflet relative to inner leaflet Results in formation of membrane spicules producing echinocytesExpansion of inner leaflet relative to outer leafletResults in invagination of the membrane producing stomatocytes (cup-shaped cells)
Slide78Figure 5-4 Model of discocyte-echinocyte and discocyte-stomatocyte transformation. RBC shape is determined by the ratio of the surface areas of the two hemileaflets of the lipid bilayer. Preferential accumulation of compounds in the outer leaflet of the lipid bilayer causes expansion and results in RBC crenation and echinocytosis; expansion of the inner leaflet of the bilayer results in invagination of the membrane and stomatocytosis.Source: Based on Clinical Expression and Laboratory Detection of Red Cell Membrane Protein Mutations by J. Palek and P. Jarolim in SEMINARS IN HEMATOLOGY 30(4):249-283, October 1993. Published by W.B./Saunders Co., an imprint of Elsevier Health Science Journals.
Slide79Lipid CompositionReticulocytes normally contain more lipid and cholesterol in the membrane than mature RBCs.Excess lipid is removed by the spleen.Splenectomized patients can have cells with an abnormal accumulation of cholesterol and/or lipids.Target cells, acathocytes, and/or echinocytes
Slide80Lipid CompositionGycolipidsLocated in the external half of the lipid bilayerCarbohydrate portions extend into the plasmaResponsible for antigenic properties of RBC membraneCarries the ABH, Lewis, P blood group antigens
Slide81Protein CompositionRBC membrane proteinsIntegral proteins and peripheral proteinsIntegral proteinsPenetrate or traverse the lipid bilayerInteract with the hydrophobic lipid core of the membrane
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Slide82Protein CompositionRBC membrane proteinsPeripheral proteinsDo not penetrate lipid bilayerInteract with integral proteins or lipids on cytoplasmic side of membrane
Slide83Protein CompositionIntegral proteinsIncludes transport proteins and glycophorins A, B, and C (GPA, GPB, GPC)Three domainsCytoplasmicHydrophobic—spans the bilayerExtracellular—exterior surface of membrane
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Slide84Protein CompositionIntegral proteinsExtracellular domainHeavily glycosylatedResponsible for most of the negative surface charge (zeta potential); prevents red cells from sticking together and to the vessel wall
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Slide85Protein CompositionIntegral proteinsExtracellular domainCarries various red cell antigensMN, Ss, U, GerbichGPC aids in attaching skeletal protein network to lipid bilayer
Slide86Protein CompositionBand 3—anion exchange protein 1 (AE1)—major integral proteinTransport channel for chloride–bicarbonate exchangeOccurs during the transport of CO2 from the tissues back to the lungs
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Slide87Protein CompositionBand 3—anion exchange protein 1 (AE1)—major integral proteinMajor binding site for a variety of enzymes and cytoplasmic membrane componentsRegulator of RBC glycolysis
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Slide88Protein CompositionBand 3—anion exchange protein 1 (AE1)—major integral proteinBinds intact hemoglobin weaklyPartially denatured hemoglobin (Heinz bodies) binds more avidlyPlays a role in erythrocyte senescenceMembrane contains more than 100 additional integral proteins.
Slide89Protein CompositionPeripheral proteinsInclude enzymes and structural proteinsStructural proteinsOrganized into a two-dimensional lattice networkRed cell membrane skeletonSupports the membrane lipid bilayerGives membrane the viscoelastic properties contributing to cell shape, deformability, membrane stability
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Slide90Protein CompositionPeripheral proteinsRed cell skeleton proteinsSpectrin, actin, ankyrin (band 2.1), band 4.2, band 4.1, adducin, band 4.9, tropomyosin, tropomodulin
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Slide91Protein CompositionPeripheral proteinsSpectrinPredominant skeletal proteinExists as heterodimer (α and β chains) that self-associate head to head to form tetramersFunctions like a spring
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Slide92Protein CompositionPeripheral proteinsAnkyrinLarge proteinHigh-affinity binding site for the attachment of spectrin to inner membrane surfaceBinds to band 3, the actual anchor for the membrane skeleton
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Slide93Protein CompositionPeripheral proteinsBand 4.2 binds to ankyrin and band 3Strengthens the interaction and helps bind the skeleton to the lipid bilayer
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Slide94Protein CompositionPeripheral proteinsActinStabilized by interactions with other proteins of the red cell skeletonTropomodulin, adducin, tropomyosin, band 4.9Spectrin binds to actin filaments
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Slide95Protein CompositionPeripheral proteinsProtein 4.1 interacts with spectrin, actin, GPCStabilizes the weak interaction of spectrin and actinThis complex serves as a secondary attachment point for the red cell skeleton.Complex = actin, spectrin, tropomodulin, tropomyosin, adducin, band 4.9, band 4.1
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Slide96Protein CompositionPeripheral proteinsProtein 4.1 interacts with spectrin, actin, GPCNecessary for normal membrane stability
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Slide97Figure 5-5 Model of the organization of the erythrocyte membrane showing the peripheral and integral proteins and lipids. Spectrin is the predominant protein of the skeletal protein lattice. Spectrin dimers join head to head to form spectrin tetramers. At the tail end, spectrin tetramers come together at the junctional complex. This complex is composed of actin oligomer and stabilized by tropomyosin, which sits in the groove of the actin filaments. The actin oligomer is capped on one end by tropomodulin and on the other by adducin. Band 4.9 (dematin) binds to actin and bundles actin filaments. Spectrin is attached to actin by band 4.1, which also attaches the skeletal lattice to the lipid membrane via its interaction with glycophorin C (minor attachment site). Ankyrin links the skeletal protein network to the inner side of the lipid bilayer via band 3. band 4.2 interacts with ankyrin and band 3 (major attachment site).
Slide98Organization of SkeletonSkeletal proteins are not static.In continuous disassociation ↔ association equilibrium with each other and attachment sitesOccurs in response to various physical and chemical stimuli as RBCs travel through the body
Slide99Organization of SkeletonCa++ also influences the cytoskeleton80% of Ca++ is found in association with the RBC membraneMaintained at a low intracellular concentration by the activity of an ATP-fueled Ca++ pump
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Slide100Organization of SkeletonCa++ also influences the cytoskeletonElevated Ca++ induces membrane protein cross-linkingActs as a fixativeStabilizes red cell shape Reduces cell's deformability
Slide101Membrane and Cytosketon RBC membrane and skeleton is responsible for structural integrity and deformability.7 mcM RBC must be: Flexible to squeeze through tiny capillary openingsStrong enough to withstand the turbulent circulation
Slide102Membrane and Cytosketon Deformability is due to:Biconcave shapeViscosity of its internal contentsViscoelastic properties of the erythrocyte membrane
Slide103Membrane and Cytosketon Red cells have an "elastic extension" abilityResume normal shape after being distorted Large or prolonged forces can cause the cytoskeleton to reorganize causing permanent deformation. Excessive force can lead to fragmentation.
Slide104Membrane PermeabilityRBC membrane is freely permeable to water and anions.RBC membrane is nearly impermeable to monovalent and divalent cations.Na+, K+, Ca++, Mg++Maintained in RBC at different levels than in plasma
Slide105Membrane PermeabilityGlucose is taken up by a glucose transporter which does not require ATP.RBC osmotic equilibrium is maintained bySelective (low) permeability of the membrane to cationsCation pumps located in the membraneNa+/K+ pumpCa++ pump
Slide106Membrane PermeabilityNa+/K+ pumpHydrolyzes 1 mole of ATP to remove 3Na+ and take up 2K+Balances the passive "leaks" of the cations
Slide107Membrane PermeabilityCa++ also helps regulate Na+ and K+An ↑ in intracellular Ca++ allows Na+ and K+ to move along their concentration gradientsAlso activates the Gárdos channelCauses selective loss of K+ and water, resulting in dehydration
Slide108Membrane PermeabilityCa++-ATPase pump Maintains low levels of intracellular Ca2++ Needs Mg++ to maintain its transport function
Slide109Membrane PermeabilityIf membrane permeability to cations increases or the cation pump failsDue to decreased glucose for generation of ATPDue to decreased ATP
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Slide110Membrane PermeabilityIf membrane permeability to cations increases or the cation pump failsNa+ accumulates in cells in excess of K+ lossIncrease in intracellular monovalent cations and waterCell swellingOsmotic hemolysis
Slide111Table 5-3 Concentration of Cations in the Erythrocyte versus Plasma
Slide112RBC BiochemistryEnergy is required by the RBC to maintain:The cation pumpsHemoglobin iron in the reduced stateReduced sulfhydryl groups in hemoglobin and other proteinsRed cell membrane integrity and deformability
Slide113Metabolic Pathwaysin the ErythrocyteMost important metabolic pathways in mature erythrocyte are linked to glucose metabolism.RBC lacks citric acid cycle (due to lack of mitochondria) so obtains energy (ATP) solely by anaerobic glycolysis.Glucose enters cell through membrane-associated glucose carrier (no ATP).
Slide114Table 5-4 Role of Metabolic Pathways in the Erythrocyte
Slide115Glycolytic PathwayRBC obtains energy in the form of ATP from glucose breakdown.90–95% of RBC glucose metabolized by glycolytic pathway (Embden-Meyerhof pathway) Normal RBCs do not store glycogen. Depends on plasma glucose for glycolysis
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Slide116Glycolytic PathwayRBC obtains energy in the form of ATP from glucose breakdown.Glucose is metabolized to lactate or pyruvate.Net gain of 2 moles of ATP/mole of glucoseIf reduced, nicotinamide-adenine dinucleotide (NADH) is available in the cell, pyruvate is reduced to lactate
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Slide117Glycolytic PathwayRBC obtains energy in the form of ATP from glucose breakdown.Glucose is metabolized to lactate or pyruvate.Lactate or pyruvate is transported from the cell to the plasma and metabolized elsewhere in the body.
Slide118Glycolytic PathwayATP is needed to maintain:Erythrocyte shapeFlexibilityMembrane integrityNormal levels of cations
Slide119Glycolytic PathwayIncreased osmotic fragility is seen in cellsWith abnormal cation permeability and/or decreased ATP production
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Slide120Glycolytic PathwayIncreased osmotic fragility is seen in cells.Cannot maintain normal intracellular cations if glucose is depletedCells become Na+ and Ca++ loaded and K+ depletedCell accumulates waterChanges cell from a biconcave disc to a sphere which is removed from circulation
Slide121Hexose Monophosphate Shunt (HMP)Metabolizes 5–10% of cellular glucoseGenerates NADPH instead of ATPGlucose-6-phosphate is oxidized in the first step by glucose-6-phosphate dehydrogenase (G6PD) NADP+ is reduced to NADPHNADPH converts oxidized glutathione (GSSG) back to reduced glutathione (GSH)
Slide122Hexose Monophosphate Shunt (HMP)GSH is necessary for maintaining hemoglobin (Hb) in the reduced functional state.Reduced GSH protects the cell from: Oxidative injury by reducing reactive oxygen species (ROS) produced during oxygen transportOther oxidants, like certain drugs or chemicals
Slide123Hexose Monophosphate Shunt (HMP)When the HMP shunt is defectiveHb sulfhydryl groups (-SH) are oxidized.Hb denatures and precipitates to form Heinz bodies.Attach to the membrane and decrease flexibilityRemoved from the cell by spleen macrophagesHeinz bodies can be visualized with supravital stain.
Slide124Methemoglobin Reductase PathwayEssential for maintaining heme iron in the reduced (ferrous) state Fe++MethemoglobinHemoglobin with iron in the Fe+++ (Ferric) stateGenerated when O2 dissociates from heme ironCannot bind O2
Slide125Methemoglobin Reductase PathwayMetHb reductase and NADH reduce Fe+++ in MetHb back to Fe++ hemoglobin
Slide126Methemoglobin Reductase PathwayIn the absence of this pathway2% of the MetHb formed daily builds up to 20-40%, severely limiting the O2 carrying capacity of bloodCertain oxidant drugs can interfere with MetHb reductase.Causes higher levels of MetHbResults in cyanosis
Slide127Rapoport-Leubering ShuntBypasses the formation of 3-phosphoglycerate and ATP from 1,3-bisphosphoglycerate (1,3-BPG)Instead BPG mutase catalyzes 1,3-BPG to form 2,3-BPG2,3-BPG: Role in regulating oxygen delivery to tissues
Slide128Rapoport-Leubering ShuntBypasses the formation of 3-phosphoglycerate and ATP from 1,3-bisphosphoglycerate (1,3-BPG)Sacrifices one of the 2 ATP molecules produced during glycolysisHemoglobin binds 2,3-BPGO2 release is facilitated (decreased affinity for O2)
Slide129Figure 5-6 Erythrocyte metabolic pathways. The glycolytic pathway is the major source of energy for the erythrocyte through production of ATP. The hexose-monophosphate pathway is important for reducing oxidants by coupling oxidative metabolism with pyridine nucleotide (NADP) and glutathione (GSSG) reduction. The methemoglobin reductase pathway supports methemoglobin reduction. The Rapoport-Luebering Shunt produces 2,3-BPG, which alters hemoglobin-oxygen affinity.G6P = glucose- 6-phosphate; PI = glucose-6-phosphate isomerase; F6P = fructose-6-phosphate; PFK = 6-phosphofructokinase; fructose 1,6-biP = fructose 1,6-bisphosphate; Glyceraldehyde 3P = glyceraldehyde 3-phosphate; G3PD = glyceraldehyde 3-phosphate dehydrogenase; 1,3 BPG = 1, 3-bisphosphoglycerate; PGK = phosphoglycerate kinase; 3PG = 3-phosphoglycerate; 2PG = 2-phosphoglycerate; PEP = phosphoenolpyruvate; PK = pyruvate kinase; LD = lactate dehydrogenase; GP = glutathione peroxidase; GR = glutathione reductase; GSH = glutathione reduced; GSSG = glutathione oxidized; G6PD = glucose-6-phosphate dehydrogenase; 6-PG = 6-phosphogluconate; 6PDG = 6-phosphodehydrogenase gluconate; PP = pentose phosphate
Slide130Erythrocyte KineticsRBC concentration varies with:Sex, age, and geographic locationAt birthHigh RBC count (5.2 × 1012/L)High Hb concentration (19 g/dL)Reticulocyte count (4–7%)High EPO levels because of: Hypoxic environment in uteroHigh O2 affinity of HbF (Fetal Hb)
Slide131Erythrocyte KineticsRBC and Hb values gradually decrease until the age of 2–3 months.RBC ~ 3.5 × 1012/LHb 10–11 g/dL
Slide132Erythrocyte KineticsCalled physiologic anemia of the newbornAt birth, arterial blood O2 rises from 45% to 95%Because lungs replace placenta for providing oxygen
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Slide133Erythrocyte KineticsCalled physiologic anemia of the newbornEPO can not be detected from the 1st week of life until the 2nd or 3rd monthRetic count drops to <1% around the 2nd monthEPO levels rise
Slide134Erythrocyte KineticsMales have a higher RBC count than females after the age of puberty.Testosterone stimulates renal and extrarenal EPO production.Individuals at high altitudes have a higher RBC count.Decrease in the partial pressure of O2 at higher attitudes Results in a physiologic increase in RBCs
Slide135Regulation of Erythrocyte ProductionBody can regulate the number of circulating RBCs by changing the:Rate of cell production in the marrowRate of cell release from the marrowRate of RBC release to circulation is balanced to the rate of RBC destructionAn increase in EPO release occursImpaired O2 delivery to tissuesLow intracellular O2 tension (PO2)
Slide136Regulation of Erythrocyte ProductionConditions that stimulate erythropoiesisAnemiaCardiac or pulmonary disordersAbnormal hemoglobinsHigh altitudeEPO is the major cytokine for terminal RBC production.
Slide137Regulation of Erythrocyte ProductionEPOA thermostable renal glycoproteinMW ~34,000 daltonsRenal cortical cells secrete EPO in response to cellular hypoxiaAlso produced in extrarenal sitesMarrow macrophages and stromal cellsHepatocytes
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Slide138Regulation of Erythrocyte ProductionEPOWhen Hb conc is normal EPO levels in plasma are constantWhen Hb drops below 12 g/dLEPO levels increase sharply
Slide139EPO Regulation of ErythropoiesisIncrease in EPO due to hypoxiaDue to ↑ gene transcription and stabilization of EPO mRNAEPO production regulated by transcription factor hypoxia-inducible factor-1 (HIF-1)HIF-1 binds to hypoxia-responsive element/HRE in EPO gene → activates transcription
Slide140EPO Regulation of ErythropoiesisEPO primarily stimulates CFU-E to proliferate and differentiate
Slide141EPO Regulation of ErythropoiesisAt extremely high EPO levelsBFU-E (normally unresponsive to EPO) can be stimulated for maturationResults in larger RBCs (↑ MCV)Increase in "i" antigenIncrease in HbF concentration
Slide142Table 5-5 Characteristics of Erythropoietin
Slide143EPO Regulation of ErythropoiesisPrimary way that EPO stimulates erythropoiesisPrevention of apoptosis RBC progenitors vary in sensitivity to EPO.
Slide144EPO Regulation of ErythropoiesisHigh concentrations of EPORescues more progenitors from apoptosisResults in ↑ number of erythroid precursor cells undergoing proliferation and maturation
Slide145Erythrocyte DestructionRBC destruction is normally the result of senescence.Aging is characterized by:Decline in cellular enzyme systemsGlycolytic enzymesEnzymes necessary for maintenance of redox status
Slide146Erythrocyte DestructionAging is characterized by:Loss of ATP production and loss of reducing systemsResults in oxidation of membrane proteins, lipids, and hemoglobinCells are unable to maintain cell shape and deformabilityLoss of membrane integrityResulting in RBC removal
Slide147Erythrocyte DestructionExtravascular destructionRBC removal by spleen, bone marrow, and liver90% of aged RBC destructionEfficient method of cell removal, conserving, and recycling amino acids and iron (essential RBC components)
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Slide148Erythrocyte DestructionExtravascular destructionOccurs in macrophages of spleenHypoxic, hypoglycemic environmentOccurs in macrophages of liverLiver lacks ability to detect minimal defects of RBCsMore efficient than spleen in removing RBCs due to more RBC circulation throughput
Slide149Erythrocyte DestructionIntravascular destructionRBC trauma damages red blood cell membraneResults in cell lysisRelease of hemoglobin directly into circulation
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Slide150Erythrocyte DestructionIntravascular destruction10% of RBC destructionPlasma proteins, haptoglobin and hemopexin, bind to free hemoglobin and transport to liver for catabolism
Slide151Case Study—Chapter 528-year-old Caucasian male of Italian descentBecame progressively ill following a safari vacation to West AfricaPresented to ER with:Fever, chills, malaiseClinical history and examSupported a diagnosis of anemia
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Slide152Case Study—Chapter 528-year-old Caucasian male of Italian descentBlood smears were negative for malariaAdmitted for diagnosis and treatment of anemia
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Slide153Case Study—Chapter 5DifferentialSegs
70%
Bands
11%
Metas
2%
Lymphs
13%
Monos
2%
Eos
2%
NRBCs
18/100 WBCs
RBC morphology
: Aniso 3+, Poik 2+, Sphero 1+, Shisto 1+, Poly 2+
CBC
WBC
14 x 10
9
/L
RBC
3.10 x 10
12
/L
HGB
9.2 g/dL
HCT
28%
MCV
93 fL
MCH
30.6 pg/dL
MCHC
32.5 g/dL
Platelet
230 x 10
9
/L
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Slide154Case Study—Chapter 5Question 1:Predict Stephen's reticulocyte count:LowNormalIncreased
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Slide155Case Study—Chapter 5
Lab Test
Patient
Reference Ranges
Total bilirubin
4.8mg/dL
0.1–1.2
Direct bilirubin
1.6mg/dL
0.1–1.0
Haptoglobin
25mg/dL
35–165
Hb electrophoresis
HbA
>98%
95%
HbF
1%
<2%
HbA
2
1%
1.5–3.7%
Heinz body stain
Positive
Negative
G6PD deficiency test
Positive
Negative
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Slide156Case Study—Chapter 5Question 2:What cellular mechanism results in hemolysis due to a deficiency in G6PD?Question 3:Explain how Heinz body inclusions cause damage to the erythrocyte membrane.
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Slide157Case Study—Chapter 5Question 4: Would you predict Stephen's serum erythropoietin levels to be low, normal, or increased? Why?Question 5:Stephen's haptoglobin level is 25 mg/dL. Explain why he has a low haptoglobin value.