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WilliamsHematology TheRedCell andItsDiseases

Plate1.ThealphabetmadeofpoikilocytesfromasinglepatientwithsicklecellanemiaandB-thalassemiatrait.Notetheelectrondenseareasin thecellsthatareinthepatientwhoishomozygousforhemoglobinS,asareflectionofthepara-crystallizationofthathemoglobininalowoxygen environment(venousblood).ThepatientwithB-thalassemiatraitfromwhommanyofthesecellswereimagedwasaphysicianwhosefamilyarrived totheUnitedStatesoriginallyfromtheUnitedKingdomwithnoapparentrecentMediterraneanheritage.AsaresultofRome’sinvasionofBritainon severaloccasionsbetween55B.C.E.and43A.D.,troopsfromcurrentItaly,Spain,Egypt,andSyriagarrisonedtheirmarriedlocalBritons.(Reproduced withpermissionfromLichtmanMA,ShaferMS,FelgarRE,etal:Lichtman’sAtlasofHematology2016.NewYork,NY:McGrawHill;2017.)

WilliamsHematology TheRedCell andItsDiseases

JosefT.Prchal,MD

ProfessorofHematologyandMalignant Hematology

AdjunctinGeneticsandPathology UniversityofUtah&HuntsmanCancerInstitute SaltLakeCity,Utah

1.interníklinikaVFNaÚstavpatologickéfyziologie

1.LFSchoolofMedicine UniversitaKarlova,Prague,CzechRepublic

MarshallA.Lichtman,MD,MACP

ProfessorEmeritusofMedicineandofBiochemistry andBiophysics DeanEmeritus,SchoolofMedicineandDentistry JamesP.WilmotCancerInstitute UniversityofRochesterMedicalCenter Rochester,NewYork

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GeorgeB.Segel,MD

EmeritusProfessorofPediatric ProfessorofMedicine

JamesP.WilmotCancerInstitute UniversityofRochesterMedicalCenter Rochester,NewYork

VivienA.Sheehan,MD,PhD AssistantProfessorofPediatrics

BaylorCollegeofMedicine Houston,Texas

SujitSheth,MD DepartmentofPediatrics

WeillCornellMedicine NewYork,NewYork

SweeLayThein,MD

NationalHeart,Lung,andBloodInstitute TheNationalInstitutesofHealth Bethesda,Maryland

PerumalThiagarajan,MD ProfessorofMedicineandPathology

BaylorCollegeofMedicine DirectorofTransfusionMedicineandHematologyLaboratory MichaelE.DeBakeyVAMedicalCenter Houston,Texas

EduardJ.vanBeers,MD,PhD Hematologist VanCreveldkliniek UniversityMedicalCenterUtrecht UtrechtUniversity Utrecht,TheNetherlands

RichardvanWijk,PhD AssociateProfessor CentralDiagnosticLaboratory UniversityMedicalCenterUtrecht UtrechtUniversity Utrecht,TheNetherlands

NealS.Young,MD Chief,HematologyBranch NationalHeart,Lung,andBloodInstitute MarkHatfieldClinicalResearchCenter NationalInstitutesofHealth Bethesda,Maryland

PREFACE

Thediscoveryoftheruddyglobules(redcells)isattributedtoJan Swammerdam(1637-1680)inAmsterdam;but,itwasAntonjvan Leeuwenhoek(1632-1723)ofDelft,whoasaresultofhisabilitytogrind lenseswithgreatermagnifyingpower(x275),madeamoredetailed descriptionofredcells,delineatingtheirgrossstructure.

Thebiochemistry,physiology,andbiophysicsoftheredcellhave beenstudiedintensivelyoverthreecenturiesand,althoughconsidered a“simple”structure,sinceitisanucleateandafteronedayinthecirculationhasnocytoplasmicorganelles,itsmysterieshavebeenslow tobeunraveled.Theprocessofenucleationoftheerythroblastinthe hematopoieticspaceandthemovementoftheanucleatecellfromthe hematopoieticspacetothemarrowsinusandfromtheretothesystemiccirculation,accomplishedbyacellwithoutanintrinsicapparatus tosupportamoeboidmotility,andthedeterminantsofitsaveragelife spanofapproximately120daysarestillbeingelucidated.Itsstructural andbiophysicalproperties,biochemicalpathways,andtherelationship amongthosefeatureshavebeenofcontinuedinteresttoscientists.Its absenceofinterferinggranules,containingproteolyticenzymes,organelles,andothercomplexitieshaveallowedtheisolationofhighlypurifiedredcellmembranesandtheearlyexplorationofthebiochemical andbiophysicalfeaturesofcellmembranes,applicabletoothercells, includingthecharacteristicsofmembranetransportofvariousmolecules.Thenatureofthestructureandfunctionofhemoglobinandthe explorationoftheglycolyticpathway,thehexosemonophosphateshunt, andtheLuebering-Rapoportpathwaywereotherrewardsreapedfrom thestudyofredcells.

Muchisknown,butasourmentor,friend,andcolleague,Ernest Beutler,cautionedPh.D.graduatesataScrippsInstitutedoctoralgraduation,oneshouldnotassumethatourunderstandingofthebiomedical sciencesissoprofoundthatwhatisleftforusistofillinsomegaps. Hearguedthatmuchfundamentalbiomedicalknowledgewasstill undiscoveredandwaitingtobeilluminated.Amonghismanycontributionstothepathogenesisofdiseaseandapplicationoftherapy,his contributionstounderstandingtheredcellandanemiawerenotable. Theseobservationsincludedaclassicseriesofpapersdescribingthe effectsofoxidantstressonindividualswithredcellglucose-6-phosphate dehydrogenasedeficiencyandalife-longinterestintheenzyme’svariantsandepidemiology.Hismonographonmethodsformeasuringred cellenzymeswasanearlycontributiontoenhancingthespecificityof thediagnosisofhemolyticanemia.Publishedoverfivedecadesago, itremainsanunsurpassedsourceofmethodsfortheassayofredcell enzymes.Beutler,also,usedredcellenzymemeasurementasasurrogate fordiagnosisofsystemic,untilthendifficulttodiagnosediseases,such asgalactosemia,glycogenstoragedisorders,andothers.Hefoundthat redcellglucose-6-phosphatedehydrogenasedeficiencywasinherited asanXchromosome-linkeddisorderanddescribedthemosaicismof normalanddeficientredcellsinheterozygousfemales.Thisfindingof mosaicismprovidedthebasisforanintellectualjumptothehypothesis ofXchromosomeinactivationinhumans,coincidentwithMaryLyon’s descriptionofthephenomenoninmice.He,also,madeseminalcontributionstounderstandingtheeffectsofirondeficiencyinnon-anemic womenandtheexpressionofironoverloadinthosehomozygousfor theHFEmutationandthevalueofadditivesforprolongedstorageof redcells,stillincurrentuse.

WithnoDNAorRNAsynthesis,nomitochondriaandtheir relatedenzymaticbiochemicalenergygeneratingpathways,andwith arelativelyshortlifespan,thisamitoticcellissustainedatanormal

concentrationinthebloodbyarobustdailyproductionofnewcellsin themarrow,theprocessoferythropoiesis.Thisprocessdeliverstwoto threemillionnewredcellstothebloodpersecond.Althoughremarkable,itisalsoavulnerabilityshouldredcellproductionbedampenedby diseaseorsubstrateinsufficiency:thelatter,aprincipalcauseofanemia.

In1929,3yearsafterobtaininghisM.D.degreeattheUniversityof Manitoba,hisfamilyhavingimmigratedtoCanadafromAustria,Maxwell MyerWintrobe,obtainedhisPh.D.atTulaneUniversity,hisdoctoral thesisentitled“TheErythrocyteinMan.”Wintrobeisconsideredthe fatherofclinicalhematologyhavingpublishedthefirstcomprehensive textintheEnglishlanguage,ClinicalHematology,in1942.Heintroducedthetechniqueofthehematocritdevicetomeasurethepacked redcellvolumeatatimewhenhemoglobinandredcellcountmeasurementswereneitheraccuratenorreproducible.Theword“hematocrit”wassoappealingthatitbecameasynonymforthepackedred cellvolumeratherthantheinstrumentofmeasurementasintendedby Wintrobe.Initially,the“Wintrobe”tube,asitbecameknown,wasfilled bypipettewithbloodtothe1mLmarketchedonthetubeandthegradationsonthetubeallowedonetoreadthefractionofbloodthatwas composedofredcellsaftercentrifugation.Later,themicrohematocrit centrifuge,whichreachedG-forcesthatremovedplasmatrappingasa significantconsiderationinthemeasurementincapillarytubesfilled withblood,couldbefoundoneverywardandclinicallaboratoryasthe principalmeanstomeasurethepackedredcellvolumeand,thereby, identifyanemiaorerythrocytosis.Achartallowedthedetermination ofthepackedcellvolumewhenthecapillarytube,regardlessofthe volumeofblooditcontained,wasplacedagainstitsscales.Wintrobe institutionalizedtheredcellindices,meancellvolume(MCV),mean cellhemoglobin(MCH),andmeancellhemoglobinconcentration (MCHC)andshowedintwoclassicpaperin1930and1934thatone couldclassifytheanemiasfordiagnosticpurposesbydistinguishing amongmacrocytic,normocytic,simplemicrocytic,andhypochromic microcyticanemias,amethodofdifferentialdiagnosisstillusedtoday. AftermovingtotheUniversityofUtahfromJohnsHopkinsUniversity, Wintrobeestablishedoneofthemostesteemedhematologyclinicaland researchtrainingprogramsintheworld.Healsodescribedalongwith hiscolleagueGeorgeCartwrightthattheaveragehematocritandhemoglobinconcentrationwashigherinresidentsofSaltLakeCity(elevation4300feet)thanthevalueobservedatJohnsHopkinsinBaltimore (elevation480feet).Hededucedfromthatprescientobservationthat hypoxia,inthatinstancefromhigheraltitude,isaprincipalregulatorof normalerythropoiesis.

In1953,F.WilliamSundermanandcolleaguesenhancedtheaccuracyofbloodhemoglobinmeasurementbyintroducingthecyanmethemoglobinmethod.In1956,WallaceCoulterintroducedhishigh-speed, automaticbloodcellcountermakingbloodcellcountingaccurate, reproducible,andcapableofmeetingthedemandsofabusyclinicand hospitalenvironment.The“CoulterPrinciple”heldthatcellsarepoor conductorsofelectricityinasaltsolution.Thus,whencellsarediluted insalineandaredrawnthroughatinyaperturecarryingacurrent,each cellproducesaslightimpedancetocurrentflowasitpassesthroughthe narrowaperture.Thepulsecreatedbythisimpedancecanbeamplifiedandcounted.Moreover,thesizeofthepulseisproportionaltocell volume.Thus,thenumberandvolumedistributionofredcellsina measuredvolumeofsolutioncanbeconvertedtoredcellcountand volumeelectronically.Theirproduct,redcellcountandredcellvolume, providedthehematocrit,nowaderivedvalue.Thousandsofcellscan

SUMMARY

Collectively,theerythroidprogenitors,terminallydifferentiatingerythroblasts (precursors),andadultredcellsaretermedtheerythrontoreinforcetheidea thattheyfunctionasanorgan.Thewidelydispersedcellscomprisingthis organarisefrompluripotentialhematopoieticstemcells.Followingcommitmenttotheerythroidlineage(unipotentialprogenitor),furthermaturation givesrisetotheerythroidprogenitors,burst-formingunit–erythroid(BFU-E) and,subsequently,colony-formingunit–erythroid(CFU-E),thatcanbeidentifiedbytheirdevelopmentintorepresentativeclonalcoloniesofredcellsin vitro.TheCFU-Ethenundergoesterminaldifferentiation,progressingthrough fourtofivemorphologicstages,eachhavingcharacteristiclightmicroscopic andultrastructuralfeatures.Duringterminalerythroiddifferentiation,there isanincreasingamountofhemoglobinsynthesisaccompaniedbynuclear chromatincondensation,andatthefinalstageofdifferentiation,thereis nuclearextrusiontogenerateananucleatepolychromatophilicmacrocyte (reticulocytewithsupravitalstaining).Thehumanpolychromatophilicmacrocyte(reticulocyte)maturesover2to3days,firstinthemarrowandthen incirculationintothediscoiderythrocyte.Duringreticulocytematuration, cytoplasmicinclusions,includingresidualmitochondriaandribosomes,are degraded,andthereticulocytelosessurfaceareatoachievethemeancell volumeandsurfaceareaofadiscoidalerythrocyte.Matureerythrocytesare approximately7to8μmindiameterandundergoextensivedeformationto passthrough3-μm-diametercapillariesandthe1-μm-wideand0.5-μm-thick endothelialslitsintheredpulpofthespleen.Theabilityoftheredcellto undergoextensivereversibledeformationisessentialforbothitsfunctionand itssurvival.Redcelldeformabilityisafunctionofitsgeometry,theviscosity

ofthecytoplasm,largelydeterminedbytheconcentrationofhemoglobin. Decreaseddeformabilityisafeatureofredcellsinvariouspathologicstates. Theerythrocyteisuniqueamongeukaryoticcellsinthatitsprincipalphysicalstructureisitscellmembrane,whichenclosesaconcentratedhemoglobin solution.Thus,allstructuralpropertiesofthiscellareinsomewaylinkedtothe cellmembrane.Incontrasttoothercells,theerythrocytehasnocytoplasmic structuresororganelles.Amonghumancells,onlyredcellsandplateletsdo nothaveanucleus.

ERYTHRON

Themassofcirculatingerythrocytesconstitutesanorganresponsible forthetransportofoxygentotissuesandtheremovalofcarbondioxidefromtissuesforexhalation.Collectively,theprogenitors,precursors, andadultredcellsmakeupanorgantermedtheerythron,whicharises frompluripotentialhematopoieticstemcells.Followingcommitment totheerythroidlineage,unipotentialprogenitorsmatureintotheerythroidprogenitors,theburst-formingunit–erythroid(BFU-E)and, subsequently,thecolony-formingunit–erythroid(CFU-E),whichthen undergoesfurthermaturationtogenerateanucleatepolychromatophilic macrocytes(reticulocytesonsupravitalstaining).TheBFU-Eand CFU-Eareidentifiedbytheirdevelopmentintomorphologicallyidentifiableclonalcoloniesofredcellsinvitro.Thereticulocytefurther matures,firstinthemarrowfor2to3daysand,subsequently,inthe circulationforapproximately1day,togeneratediscoiderythrocytes.1-5 Theproerythroblast,thefirstmorphologicallyrecognizableerythroid precursorcellinthemarrow,typicallyundergoes5mitoses(range4-6) beforematurationtoanorthochromaticerythroblast,whichthenundergoesnuclearextrusion.Afeatureoferythropoiesisisthataftereachcell division,thedaughtercellsadvanceintheirstateofmaturationwith significantchangesingeneandproteinexpressioncomparedwiththe parentcelland,ultimately,becomefunctionalasmatureerythrocytes.4 Inthisprocess,theyacquirethehumanbloodgroupantigens,transport proteins,andallcomponentsoftheerythrocytemembrane.4,6

Intheadultstageofdevelopment,thetotalnumberofcirculatingerythrocytesisinasteadystate,unlessperturbedbyapathologic orenvironmentalinsult.Thiseffectdoesnotholdduringgrowthof theindividualinutero,particularlyintheearlystagesofembryonic developmentandduringneonataldevelopmentasthetotalbloodvolumeincreasesmarkedly.Consequently,erythrocyteproductioninthe embryoandfetusdiffersmarkedlyfromthatintheadult.

THEEARLIESTERYTHRON

AcronymsandAbbreviations:BFU-E,burst-formingunit–erythroid;CFU-E, colony-formingunit–erythroid;cP,centipoise;DIC,disseminatedintravascular coagulation;EMP,erythroblastmacrophageprotein;ICAM-4,intercellular adhesionmolecule-4;IL,interleukin;MCH,meancellhemoglobincontent;MCHC, meancorpuscularhemoglobinconcentration;MCV,meancellvolume;MDS, myelodysplasticsyndrome;SA:V,surfacearea-to-volumeratio;TTP,thrombotic thrombocytopenicpurpura.

*ThischaptercontainstextwrittenforpreviouseditionsofthisbookbyBrianBull, PaulHerrmann,andErnestBeutler.

Intheveryearlystagesofhumangrowthanddevelopment,thereare twoformsoferythroiddifferentiation:primitiveanddefinitive.7-10 Chapters2and17providedetailedinformationofembryonicand fetalhematopoiesis.Theprimitiveerythronsuppliestheembryowith oxygenduringthephaseofrapidgrowthbeforethedefinitiveformof maturationhashadachancetodevelopandseedanappropriateniche. Thehallmarkofthisprimitiveerythronisthereleaseofnucleatederythroidprecursorscontainingembryonichemoglobin.Althoughprimitiveinthesensethatthecellscontainnucleiwhenreleasedintothe circulation,thisformofmaturationdiffersfromavianandreptilian erythropoiesisinthatthenucleusiseventuallyexpelledfromthemammaliancellsastheycirculate.Thetransientpresenceofanucleusinthe cellsofthecirculatingprimitiveerythroncandecreasetheefficiency ofgasexchangeinthelungsandmicrovasculaturebecausethenucleus

Figure1–3.Humanerythroblastprecursorsasisolatedbycellflowcytometry.Imagesareofpopulationsofhumanerythroblastprecursorsat stagesoferythroidmaturationwhensortedfromhumanmarrowbyflowcytometry.AandB.Proerythroblastsandearlybasophilicerythroblasts;(C)polychromaticerythroblasts;and(D)orthochromaticerythroblasts.

ProerythroblastsOnstainedfims,theproerythrobastappears asaargece,irreguaryroundedorsightyova.13Thenuceusoccupiesapproximatey80%oftheceareaandcontainsfinechromatindeicateydistributedinsmacumps.Oneorseverawe-definednuceoi arepresent.Thehighconcentrationofpoyribosomesgivesthecytopasmofthesecesitscharacteristicintensebasophiia.Atveryhigh magnification,ferritinmoecuesareseendispersedsingythroughout thecytopasmandiningthecathrin-coatedpitsonthecemembrane (Figs.1-2and1-4).Diffusecytopasmicdensityonsectionsstainedfor peroxidaseindicatesthathemogobinisareadypresent.Dispersedgycogenparticesarepresentinthecytopasm.

BasophilicErythroblastsBasophiicerythrobastsaresmaer thanproerythrobasts.Thenuceusoccupiesthree-fourthsofthece areaandiscomposedofcharacteristicdarkvioetheterochromatininterspersedwithpink-stainingcumpsofeuchromatininkedby irreguarstrands.13Thewhoearrangementoftenresembeswhee spokesoracockface.Thecytopasmstainsdeepbue,eavingaperinucearhaothatexpandsintoajuxtanucearcearzonearoundthe Gogiapparatus.Cytopasmicbasophiiaatthisstageresutsfromthe continuedpresenceofpoyribosomes(Figs.1-2and1-5).

PolychromatophilicErythroblastsAfterthemitoticdivision ofthebasophiicerythrobast,thecytopasmchangesfromdeepbue tograyashemogobindiutesthepoyribosomecontent.Cesatthis stagearesmaerthanbasophiicerythrobasts.Thenuceusoccupies essthanhafofthecearea.Theheterochromatinisocatedin we-definedcumpsspacedreguaryaboutthenuceus,producing acheckerboardpattern.Thenuceousisost,buttheperinucear haopersists.13Itisatthispointthaterythrobastsosetheirmitotic potentia.Eectronmicroscopyofthepoychromatophiicerythrobastreveasincreasedaggregationofnucearheterochromatin.13Active ferritintransportacrossthecemembraneisawaysevident,and siderosomesaongwithdispersedferritinmoecuescanbeidentified withinthecytopasm(Figs.1-2and1-6).

Orthochromic(syn.Orthochromatic)ErythroblastsAfterthe finamitoticdivisionoftheerythropoieticseries,theconcentrationof hemogobinincreaseswithintheerythrobast.Undertheightmicroscope,thenuceusappearsamostcompeteydenseandfeatureess. Itismeasurabydecreasedinsize.Thisceisthesmaestoftheerythrobasticseries.13Thenuceusoccupiesapproximateyone-fourthof theceareaandiseccentric.Cemovementcanbeappreciatedunder thephase-contrastmicroscope.Roundprojectionsappearsuddenyin

differentpartsoftheceperipheryandarejustasquickyretracted.13 Themovementsprobabyaremadeinpreparationforejectionofthe nuceus.Theceutrastructureischaracterizedbyirreguarborders, refectingitsmotiestate.Theheterochromatinformsargemasses. Mitochondriaarereducedinnumberandsize(seeFigs.1-2,1-7,and1-8).

Figure1–4.Proerythroblast.Phase-contrastmicrograph(inset)ofa proerythroblastshowingtheimmaturenucleuswithnucleoliandfinely dispersednuclearchromatin.Thecentrosome(juxtanuclearclearzone) isapparentwithitsdenseaccumulationofmitochondria.Electronmicroscopicsectionoftheproerythroblastshowsnucleoli(n)incontactwith thenuclearmembrane.Chromatinisfinelydispersedandformssmall aggregatesinthefixednuclearmembrane.Theperinuclearcanalisnarrowbutwelldefined.Polyribosomegroups,manyinhelicalconfiguration, aredispersedthroughoutthecytoplasm.TheGolgiapparatus(g)iswell developed,andregionsofendoplasmicreticulum(arrows)areseen.

Figure1–5.Basophilicerythroblast.Phase-contrastphotomicrograph (inset)showsincreasedclumpingofthenuclearchromatinandfurther roundingofthecell,withaggregationofthemitochondriaandcentrosomeintotheregionsofnuclearindentation.Theelectronmicroscopicsectionshowsclumpingofthenuclearchromatin,nuclearpores (p),organizationofthenucleoli,increaseddensityofpolyribosomes(pr), well-developedGolgiapparatus(g),andadecreaseinsmoothendoplasmicreticulum.

NormalSideroblastsAnormaerythrobastsaresiderobasts inthattheycontainironinstructurescaedsiderosomes,asevidentby transmissioneectronmicroscopy.Thesestructuresareessentiaforthe transferofironforheme(hemogobin)synthesis.Byightmicroscopy, undertheusuaconditionsofPrussianbuestainingforiron,aminority ofnormaerythrobasts(approximatey15%-20%)canbeidentifiedas containingsiderosomes,andthosethatcanbesoidentifiedhavevery few(1-4)smaPrussianbue–positivegranues.

PathologicSideroblastsAheterogeneousgroupoferythrocytedisordersisaccompaniedbyineffectiveerythropoiesis,abnorma erythrobastmorphoogy,andhyperferremia.Thesedisordersincude acquiredmegaobasticanemia(Chap.9),congenitadyserythropoietic anemias(Chap.14),thaassemias(Chap.17),theinheritedandacquired siderobasticanemias,pyridoxine-responsiveanemia,acoho-induced siderobasticanemia,andeadintoxication(Chaps.20and23).Some oftheseconditionsarecharacterizedbythepresenceofpathoogic siderobasts.Pathoogicsiderobastsareoftwotypes.Thefirstisan erythrobastthathasanincreaseinnumberandsizeofPrussianbue–stainedsideroticgranuesthroughoutthecytopasm.Thesecondisthe erythrobastthatshowsiron-containinggranuesthatarearrangedin anarcoracompeteringaroundthenuceus(Fig.1-8).Thesepathoogicsiderobastsarereferredtoasringorringedsideroblasts. 26,27Eectronmicroscopicstudiesshowthatgranuesinringedsiderobastsare iron-oadedmitochondria.Inceswithiron-oadedmitochondria, manyferritinmoecuesaredepositedbetweenadjacenterythrobast membranes.

Figure1–6.Polychromatophilicerythroblast.Phase-contrastmicrograph(inset)demonstratesdiminishedsizeofthiscellcomparedwith itsprecursor.Furtherclumpingofnuclearchromatingivesthenucleus acheckerboardappearance.Thecentrosomeiscondensed,andaperinuclearhalohasdeveloped.Theelectronmicroscopicsectiondemonstratesrelativereductionofthedensityofpolyribosomesanddilution bythemoderatelyosmiophilichemoglobininthecytoplasm.Nuclear chromatinshowsamarkedincreaseinclumping,andnuclearpores(P) areenlarged.

RETICULOCYTE

Birth

Beforeenuceationattheateorthochromaticerythrobastsstage,intermediatefiamentsandthemarginabandofmicrotubuesdisappear. Enuceationisahighydynamicprocessthatinvovescoordinatedaction ofmutipemechanisms.28-30Tubuinandactinbecomeconcentratedat thepointwherethenuceuswiexit.Thesechanges,accompaniedby microtubuarrearrangementsandactinpoymerization,payaroein nucearexpusion.Expusionofthenuceusinvitroisnotaninstantaneousphenomenon;itrequiresaperiodof6to8minutes.Theprocess beginswithseveravigorouscontractionsaroundthemidportionofthe ce,foowedbyadivisionoftheceintounequaportions.Thesmaer portionconsistsoftheexpeednuceussurroundedbyathinringof hemogobinandpasmamembrane(Fig.1-9).Invivo,expusionofthe nuceusmayoccurwhietheerythrobastisstipartofanerythrobasticisandandtheoutereafetofthebiaminarmembranesurrounding theexpeednuceusishighinphosphatidyserine,asignaformacrophageingestion(Fig.1-10).22Twohypotheseshavebeenproposedto expainhowthereticuocyteexitsthemarrow.19-21Thereticuocytemay activeytraversethesinusepitheiumtoentertheumen.Moreikey, however,thereticuocytemaybedrivenacrossbyapressuredifferentiabecauseitappearsincapabeofdirectedamoeboidmotion.Invitro experimentaevidencefavorsthehypothesisthatpressuredifferentiais ikeythedriverforreticuocytereease.21

Figure1–11.Redcellinclusions.Bloodfilms.A.RedcellswithHowell-Jollybodies(arrows)postsplenectomy.Thecrispcircularborder,darkblue color,andperipherallocationarecharacteristics.B.Basophilicstippling.Thesebasophilicinclusionsmaybefineorcoarse.Inthiscase,thecellcontains coarsestipplingseeninleadpoisoning(arrow)C.Siderocyte.ThesecellscontainpurplegranuleswhenstainedwithWrightstain(Pappenheimer bodies).Comparedwithbasophilicstippling,sideroticgranulesareusuallyfewerinnumberandsometimesclustered.ThesePrussianblue–stained cellsconfirmthatthegranulescontainiron(bluereactionproduct).Thearrowpointstotwosiderocytes.D.Cabotring.Rareredcellinclusion(arrow) Seetextforfurtherdescription.E.Heinzbodies.Thesecellsfromapatientwithglucose-6-phosphatedehydrogenasedeficiencywereincubatedwith asupravitaldye,whichstainsthedenaturedglobinprecipitates.F.RedcellsfromapatientwithhemoglobinHdisease(α-thalassemia).Thehemoglobinprecipitatesarestainedwithbrilliantcresylblue.(ReproducedwithpermissionfromLichtmanMA,ShaferMS,FelgarRE,etal:Lichtman’sAtlasof Hematology2016.NewYork,NY:McGrawHill;2017.)

BasophilicStippling

BasophiicstippingconsistsofgranuationsofvariabesizeandnumberthatstaindeepbuewithWrightstain.Eectronmicroscopicstudies haveshownthatpunctatebasophiliarepresentsaggregatedribosomes.42 Cumpsformduringthecourseofdryingandpostvitastainingof theces,muchas“reticuum”inreticuocytesprecipitatesfromribosomesduringsupravitastaining.Thecumpedribosomesmayincude degeneratingmitochondriaandsiderosomes.Inconditionssuchasead intoxication(Chap.23),pyrimidine5’-nuceotidasedeficiency(Chap.16), andthaassemia(Chap.17),theateredreticuocyteribosomeshave agreaterpropensitytoaggregate.Asaresut,basophiicgranuation appearsargerandisreferredtoascoarsebasophilicstippling

HeinzBodies

Heinzbodiesarecomposedofdenaturedproteins,primariyhemogobin,thatforminredcesasaresutofchemicainsut;inhereditary defectsofthehexosemonophosphateshunt(Chap.16);inthethaassemias(Chap.17);andinunstabehemogobinsyndromes(Chap.18).43

HeinzbodiesarenotseenonordinaryWright-orGiemsa-stainedbood fims.Heinzbodiesarereadiyvisibeinredcesstainedsupravitay withbriiantcresybueorcrystavioetandareeiminatedasredces traversetheendotheiasitsofthespenicsinus.

HemoglobinHInclusions

HemogobinHiscomposedofβ4tetramers,indicatingthatβchainsare presentinexcessasaresutofimpairedα-chainproduction(Chap.17). Exposuretoredoxdyessuchasbriiantcresybue,methyenebue,or newmethyenebue,resutsindenaturationandprecipitationofabnormahemogobin.44-46Briiantcresybuecausestheformationofaarge numberofsmamembrane-boundincusions,givingtheceacharacteristic“gofba-ike”appearancewhenviewedbyightmicroscopy.

Methyenebueandnewmethyenebuegenerateasmaernumberof variabysizedmembrane-boundandfoatingincusions.Thesechanges areseenmostfrequentyinα-thaassemiabutcanasobefoundin patientswithunstabehemogobin(Chap.18)andinrarepatientswith primarymyeofibrosisinwhomacquiredhemogobinHdiseasehas deveoped.

SiderosomesandPappenheimerBodies

Normaorpathoogicredcesinboodcontainingsiderosomes(“iron bodies”)usuayarereticuocytes.Theirongranuationsareargerand morenumerousinthepathoogicstate.Eectronmicroscopyshowsthat manyofthesebodiesaremitochondria-containingferruginousmicees ratherthantheferritinaggregatescharacterizingnormasiderocytes.47 Siderosomesusuayarefoundintheceperiphery,whereasbasophiic stippingtendstobedistributedhomogeneousythroughoutthece. PappenheimerbodiesaresiderosomesthatstainwithWrightstain. EectronmicroscopyofPappenheimerbodiesshowsthattheironoften iscontainedwithinaysosome,asconfirmedbythepresenceofacid phosphatase.Siderosomesmaycontaindegeneratingmitochondria, ribosomes,andotherceuarremnants.

STRUCTUREANDSHAPEOFERYTHROCYTES

Thenormarestingshapeoftheerythrocyteisabiconcavedisc(Fig.1-12). Variationsintheshapeanddimensionsoftheredceareusefuin thedifferentiadiagnosisofanemias.Normahumanredceshave adiameterof7to8μm,andthediameterdecreasessightywithce age.Thedecreaseinsizeikeyresutsfromossofmembranesurface areaduringtheerythrocyteifespanbyspeen-faciitatedvesicuation. Theceshaveanaveragevoumeofapproximatey90fLandasurface areaofapproximatey140μm2.Themembraneispresentinsufficient excesstoaowthecetoswetoasphereofapproximatey150fLorto

Figure1–12.Scanningelectronmicrographsofdistinctredcellmorphologies.Discoidnormalredcells(topleftpanel).Elliptocytesandfragmentedredcells(toprightpanel).Oxygenatedsickleredcells(middleleft panel)anddeoxygenatedsickleredcells(middlerightpanel).Stomatocyticredcells(bottomleftpanel).Acanthocyte(bottomrightpanel)

deformsoastoenteracapiarywithadiameterof2.8μm.Thenorma erythrocytestainsreddish-brownwithWright-stainedboodfimsand pinkwithGiemsastain.Thecentrathirdoftheceappearsreativey paecomparedwiththeperiphery,refectingitsbiconcaveshape.Many artifactscanbeproducedinthepreparationoftheboodfim.Theymay resutfromcontaminationofthegasssideorcoversipwithtracesof fat,detergent,orotherimpurities.Frictionandsurfacetensioninvoved inthepreparationoftheboodfimproducefragmentation,“doughnutces”oranuocytes,andcrescent-shapedces.Observedunderthe phase-contrastorinterferencemicroscope,theredceshowsacharacteristicinternascintiationknownasredceficker.48Thescintiation resutsfromthermayexcitedunduationsoftheredcemembrane. Frequencyanaysisofthesurfaceunduationshasprovidedanestimateofthemembranecurvatureeasticconstantandofchangesinthis constantresutingfromacoho,choesterooading,andexposureto cross-inkingagents.

REDCELLSHAPEANDSURVIVAL INCIRCULATION

Theredcespendsmostofitscircuatoryifewithinthecapiarychannesofthemicrocircuation.Duringits100-to120-dayifespan,the redcetravesapproximatey250kmandosesapproximatey15%to 20%ofitscesurfacearea.Theongsurvivaoftheredceisateast partiayaresutoftheuniquecapacityofitsmembraneto“tanktread,”

thatis,torotatearoundtheredcecontentsandtherebyfaciitatemore efficientoxygendeivery.Thephysicaarrangementofmembraneskeetaproteinsinauniformsheofhighyfodedhexagonaspectrinattice permitsthisunusuabehavior.49-51Thearrangementasoisresponsibe forthecharacteristicbiconcaveshapeoftherestingce.Redcesmust asobeabetowithstandargeshearforcesandmustbeabetoundergo extensivereversibedeformationduringtransitthroughthemicrovascuatureandintransitingfromthespenicredcepupbackintocircuation.Theresiiencyandfuidityofthemembranetodeformation isreguatedbythespectrin-basedmembraneskeeton.49Adeficiency intheamountofspectrinorthepresenceofmutantspectrininthe submembraneskeetonresutsinabnormayshapedcesinhereditaryspherocytosis,eiptocytosis,andpyropoikiocytosis(Chap.15).49 Inregionsofcircuatorystandstiorverysowfow,redcestrave inaggregatesof2to12ces,formingroueaux.Withinargevesses, increasedshearforcesdisruptthisaggregation.

REDCELLCOMPOSITION

Theerythrocyteisacompexce.Themembraneiscomposedofipids andproteins,andtheinteriorofthececontainsmetaboicmachinery designedtosustainthecethroughits120-dayifespanandmaintain theintegrityofhemogobinfunction.Eachcomponentofredbood cesmaybeexpressedasafunctionofredcevoume,gramsofhemogobin,orsquarecentimetersofcesurface.Theseexpressionsareusuayinterchangeabe,butundercertaincircumstanceseachmayhave specificadvantages.However,becausediseasemayproducechangesin theaverageredcesize,hemogobincontent,orsurfacearea,theuseof anyofthesemeasurementsindividuaymay,attimes,bemiseading. Forconvenienceanduniformity,dataintheaccompanyingtabes (Tabes1-1through1-6)52-125areexpressedintermsofceconstituent permiiiterofredceandpergramofhemogobin.Inmanyinstances, thisprocessrequiredrecacuationofpubisheddata.Theserecacuationsassumeahematocritvaueof45%and33gofhemogobinper deciiterofredces.Toobtainconcentrationpergramofhemogobin, theconcentrationpermiiiterredboodcecanbemutipiedby3.03. Thetabesistonysomeofthemostcommonyreferredtoconstituents oftheerythrocyte.Thereferenceonwhicheachvaueisbasedisthe firstnumberpresentedintheastcoumnofeachtabe.Whereappicabe,additionaconfirmatoryreferencesaregiven.Insomeinstances, onythepercentageofthetotaofthetypeofconstituentpresentis given.Chapter15discussesthedetaiedproteincompositionofthered cemembraneanditsvariousproteinconstituents.

TABLE1–1.HumanErythrocyteProteinand WaterContent

TABLE1–2.HumanErythrocytePhospholipids

Lipid Amount

Totalphospholipids2.98±0.20mg/mLRBC56

Ethanolamine phosphoglyceride 29%oftotal phospholipid

Meanplasmalogen content 67%ofethanolamine phosphoglyceride

Serinephosphoglyceride10%oftotal phospholipid 56

Meanplasmalogen content 8%ofserine phosphoglyceride 56

Lecithin 0.320.03–0.95mg/mL57

Sphingomyelin 0.12–1.13mg/mL57

Lysolecithin 1.82%oftotal phospholipids 58

Abbreviation:RBC,redbloodcell.

Someresultsaregivenasmean±standarddeviation.

ERYTHROCYTEDEFORMABILITY

Duringits120-dayifespan,theerythrocytemustundergoextensive passivedeformationandmustbemechanicaystabetoresistfragmentation;ceuardeformabiityisanimportantdeterminantofred cesurvivainthecircuation.Redcedeformabiityisinfuenced bythreedistinctceuarcomponents:(1)ceshapeorcegeometry, whichdeterminestheratioofcesurfaceareatocevoume(SA:V; highervauesofSA:Vfaciitatedeformation);(2)cytopasmicviscosity,whichisprimariyreguatedbythemeancorpuscuarhemogobin concentration(MCHC)andisthereforeinfuencedbyaterationsin

TABLE1–3.HumanErythrocyteCoenzymeandVitamins

Ascorbicacid

Abbreviation:RBC,redbloodcell. Someresultsaregivenasmean±standarddeviation.

TABLE1–4.Nucleotides

Compound

Adenosinemonophosphate0.021±0.00369-72

Adenosinediphosphate0.216±0.03669-72

Adenosinetriphosphate1.35±0.03571-75

Cyclicadenosine monophosphate 0.015±0.002476

Cyclicguanosine monophosphate 0.013±0.004276

Guanosinediphosphate0.018±0.00571

Guanosinetriphosphate0.052±0.01270,71

Inosinemonophosphate0.031±0.00571-73

Nicotinamideadenine dinucleotide 77,78

Reduced 0.0018±0.00177,78

Oxidized 0.049±0.006

Nicotinamideadeninedinucleotidephosphate 77,78

Reduced 0.032±0.002

Oxidized 0.0014±0.0011

S-adenosylmethionine0.005 79

Totalnucleotide 1.534±0.03380

Uridinediphosphoglucose0.031±0.00571,81

UridinediphosphateN-acetyl glucosamine 0.018 81

Abbreviation:RBC,redbloodcell. Someresultsaregivenasmean±standarddeviation.

cevoume;and(3)membranedeformabiityandmechanicastabiity, whicharereguatedbymutipemembraneproperties,whichincude easticshearmoduus,bendingmoduus,andyiedstress.126-129Either directyorindirecty,membranecomponentsandtheirorganization payanimportantroeinreguatingeachofthefactorsthatinfuence ceuardeformabiity.

ThebiconcavediscshapeofthenormaredcecreatesanadvantageousSA:Vreationship,aowingtheredcetoundergomarkeddeformationwhiemaintainingaconstantsurfacearea.Thenormahuman adutredcehasavoumeof90fLandasurfaceareaof140μm2.If theredcewereasphereofidenticavoume,itwoudhaveasurface areaofony98μm2.Thus,thediscoidshapeprovidesapproximatey 40μm2ofexcesssurfacearea,oranextra43%,whichenabesthered cetoundergoextensivedeformation.Mostdeformationsoccurringin vivoandinvitroinvovenoincreaseinsurfacearea.Thisisimportant becausethenormaredcecanundergoargeinearextensionsofupto 230%ofitsoriginadimensionwhiemaintainingitssurfacearea,but anincreaseofeven3%to4%insurfacearearesutsinceysis.Either membraneoss,eadingtoareductioninsurfacearea,oranincrease incewatercontent,eadingtoanincreaseincevoume,wicreatea moresphericashapewithessredundantsurfacearea.Thisossofsurfacearearedundancyresutsinreducedceuardeformabiity,compromisedredcefunction,anddiminishedsurvivaasaresutofspenic sequestrationofspherocyticredces.A17%reductioninsurfacearea resutsinrapidremovaofredcesbythehumanspeen.130

TABLE1–5.

HumanErythrocyteCarbohydrates,Organic Acids,andMetabolites

Compound μmol/mLRBCReference(s)

Dihydroxyacetonephosphate0.0094±0.002869

2,3-Diphosphoglycerate4.171±0.63669,75

Fructose 0.000354± 0.0000191 82

Fructose6-phosphate0.0093±0.00269,72,75,83

Fructose3-phosphate0.013±0.00184,85

Fructose2,6-diphosphatea 48±1386

Fructose1,6-diphosphate0.0019±0.000669,72,75,83

Glucuronicacid Trace 87

Glucose Inequilibrium withplasma 88,89

Glucose6-phosphate0.0278±0.007569,72,75,83

Glucose1,6-diphosphate0.18–0.3072,90

Glyceraldehyde3-phosphateNotdetectable69

Lacticacid

0.932±0.21153,69,91

Mannose1,6-diphosphate0.150 90

Octulose1,8-diphosphateTrace 92

Pyruvate 0.0533±0.021569

3-Phosphoglycerate 0.0449±0.005169,75

2-Phosphoglycerate 0.0073±0.002569,75

Phosphoenolpyruvate0.0122±0.002269

Ribonucleicacid 1.355mg93

Ribose1,5-diphosphate<0.02 94,95

Ribulose5-phosphateTrace 96

Sedoheptulose7-phosphateTrace 96

SedoheptulosediphosphateTrace 97

Sialicacid 0.825±0.02894

Sorbitol 31.1±5.382,84

Sorbitol3-phosphate0.013±0.00185

Abbreviation:RBC,redbloodcell. aValuesaregiveninpicomoles. Someresultsaregivenasmean±standarddeviation.

Cytopasmicviscosity,anotherreguatorycomponentofredce deformabiity,isargeydeterminedbytheMCHC,whichisdetermined inargepartbycewatercontent.Asthehemogobinconcentration risesfrom27to35g/dL(thenormarangeforredboodces),theviscosityofhemogobinsoutionincreasesfrom5to15centipoise(cP), 5to15timesthatofwater.Attheseeves,thecontributionofcytopasmicviscositytoceuardeformabiityisnegigibe.However,viscosity increasesexponentiayathemogobinconcentrationshigherthan37g/dL, reaching45cPat40g/dL,170cPat45g/dL,and650cPat50g/dL.At theseeves,cytopasmicviscositymaybecometheprimarydeterminantofceuardeformabiity.Thus,ceuardehydration,usuaycaused bythefaiureofnormavoumehomeostasismechanisms,canseverey impairceuardeformabiityandthusdecreaseoptimaoxygendeiverybyimpairingtheabiityofredcestoundergorapiddeformation

TABLE1–6.HumanErythrocyteElectrolytes

Electrolyteμmol/mLRBCReference

protein-bound

98,114

Abbreviation:RBC,redbloodcell.

aObtainedbysubtractingplasmaconcentrationfromwhole-blood concentration.

Someresultsaregivenasmean±standarddeviation.

necessaryforpassagethroughthemicrovascuature.Asexampes,ceuardehydrationreducesredcedeformabiityinhereditaryxerocytosis, sickeceanemia,hemogobinCC,andβ-thaassemia.129,131,132However, changesinceuardehydrationbyitsefappeartohaveitteinfuence onredcesurviva.

Thepropertyofmembranedeformabiitydeterminestheextentof membranedeformationthatcanbeinducedbyadefinedeveofappied force.Themoredeformabethemembrane,theesstheforcerequired forthecetopassthroughthecapiariesandothernarrowopenings, suchasfenestrationsinthespeniccords.Thepropertyofmembrane mechanicastabiityisdefinedasthemaximumextentofdeformation thatamembranecanundergo,beyondwhichitcannotcompetey recoveritsinitiashape.Thisisthepointatwhichthemembranefais. Normamembranestabiityaowshumanredcestocircuatefor100 to120dayswithoutfragmenting,whereasdecreasedstabiityeadsto cefragmentationundernormacircuatorystresses.Bothmembrane

Figure1–13.A.Normalblood.Thearrowpointstoanormochromic-normocyticdiscocyte.B.Stomatocytes.Thedoublearrowpointstothetwo morphologictypesofstomatocyte:uppercellwithaslit-shapedpaleareaandlowercellwithasmallcentralcircularpalearea.C.Echinocytes. Thefieldhasseveralsuchcells.Thearrowpointstooneexamplewithevenlydistributed,blunt,short,circumferentiallypositionedprojections. D.Acanthocytes.Thearrowpointstooneexamplewithafewspike-shapedprojections,unevenlydistributedandofvaryinglengths.E.Spherocytes. Small,circular,densely-staining(hyperchromic)cellsthat,whenfullydeveloped,shownocentralpallor.F.Schizocytes(schistocytes,helmetcells, fragmentedredcells).Thesemicrocyticcellfragmentsmayassumevariedshapes.Thearrowpointstoatriangularshape,buttwoothersofdifferent shapearealsopresentinthefield.Despitebeingdamagedandverysmall,theyfrequentlymaintainabiconcaveappearance,asseenbytheircentral pallor.G.Sicklecells(drepanocytes).Numeroussicklecellsareshown.Twoareintheclassicshapeofthebladeontheagriculturalsickle(arrow).Many redcellsthathaveundergonethetransformationtoa“sickle”celltaketheslightlylessextremeformofellipticalcellswithaverynarrowdiameter withcondensedhemoglobininthecenter(para-crystallization).Abouteightsuchcellsareinthefield.H.Elliptocytesandovalocytes.Thelowerarrow pointstoanelliptocyte(cigar-shaped).Theupperarrowpointstoanovalocyte(football-shaped).Becausebothformsmaybeseentogetherinacase ofinheriteddisease(samegenemutationresultinginbothshapes),asshownhere,ithasbeenproposedthatallsuchshapesbecalledelliptocytes withaRomannumeraltodesignatetheseverityoftheshapechangetowardtheelliptical,thatis,elliptocytesI,II,andIII.I.Targetcells(codocytes). Thearrowpointstoonecharacteristicexampleamongseveralinthefield.Thehemoglobinconcentrationcorralledbymembranerecurvatureinthe centerofthecellgivesittheappearanceofanarcherytarget.J.Tear-drop–shapedcells(dacryocytes).Threedacryocytesareinthisfield.Oneexample isindicatedbythearrowK.Horncell(keratocyte).Severalexamplesareinthefield.Thearrowpointstoatypicalsuchcellwithtwosharpprojections. (ReproducedwithpermissionfromLichtmanMA,ShaferMS,FelgarRE,etal:Lichtman’sAtlasofHematology2016.NewYork,NY:McGrawHill;2017.)

resutofossofmembranesurfaceareaorincreasedcevoume.Stomatocytosisisafeatureofhereditaryhydrocytosiscausedbyincreasedce voumeandconsequentdecreaseinSA:Vratio.Aspectrumofabnorma cesvaryingfromnormadiscocytestostomatocytes,spherostomatocytes,anddensemicrospherocytesisseeninhereditaryspherocytosis.

Elliptocytes

Eiptocytesareseeninhereditaryeiptocytosis(Chap.15)asweas inthaassemia(Chap.17),irondeficiency(Chap.11),andmegaobasticanemia(Chap.9).49Inboodfimsofnormasubjects,eipticaor ovacesusuayconstituteessthan1%oftheerythrocytes.Invarious pathoogicsituations,withorwithoutanemia(thaassemiatrait,foate, andirondeficiency),thenumberofeiptocytescanincreaseto10%. Exceptionay,asindyserythropoiesis,theproportioncanbeashighas 50%.Inhereditaryeiptocytosis,thenumberofeipticaerythrocytes variesgreaty,from1%to98%.Quaitativeandquantitativeanomaies ofspectrinandprotein4.1,themajorproteinsofthemembraneskeeton,areassociatedwithhereditaryeiptocytosis.49,138Severehemoytic anemiaisseenonyinthehomozygousorcompoundheterozygotes formofthedisease(hereditarypyropoikiocytosis),inwhichextensive cefragmentationproducespyropoikilocyteswithmarkeddecreasesin SA:Vratio.

Acanthocytes

Theacanthocyte(Chap.15)isirreguaryshaped,with2to10hemisphericaytippedspicuesofvariabeengthanddiameter.Thebasesof thespicuesontheacanthocyteareofvaryinggirth,unikethespicues onechinocytes,whichhaveremarkabyuniformdimensions.Acanthocytesareseeninneuroacanthocytosisandinabetaipoproteinemia.139 Theackofanemiaintheseconditionssuggeststhattheseceshave nearnormaifespanincircuation.

TargetCells(Codocytes)

Areativeexcessofmembranesurfaceareaordecreasedcevoume eadingtoincreasedSA:Vratioresutsintargetces.140Targetcesmay beseeninobstructiveiverdisease,hemogobinopathies(SandC),thaassemia,irondeficiency,postspenectomy,andecithinchoesteroacetytransferasedeficiency.Inpatientswithobstructiveiverdisease,ecithin choesteroacetytransferaseactivityisdepressed.Thisincreasesthe choestero-to-phosphoipidratioandproducesanabsouteincrease inthesurfaceareaoftheredcemembrane.Incontrast,membrane excessisonyreativeinpatientswithiron-deficiencyanemiaandthaassemiabecauseofthereducedcevoume.Incontrasttospherocytes, whichexhibitincreasedosmoticfragiity,targetredcesareosmoticay resistant.

SickleCells(Drepanocytes)

Thesickecedispaysacharacteristicvariationofformonstained boodfims(Chap.18).Thefusiformceinthecrescentshapewith twopointedextremitiesisencounteredmostcommonyindeoxygenatedboodsampesasaresutofpoymerizationofsickehemogobin. Ifsickeceformationisobservedbyphase-contrastmicroscopy,the eariestchangewithdeoxygenationisossofficker,foowedbysight deformationatthediscocyteborder,withdispacementofthehemogobintooneregionofthece.Thecetheneongatesandbecomes rigidasaresutofpoymerizationofhemogobinS.Uponreoxygenation,thesickeceresumesthediscocyteformand,insodoing,canose membranebymicrospheruationandfragmentationduringretraction ofongspicues.141Evidencesuggeststhatthemoretypicasicke-shaped cesformundersowdeoxygenation.Witheachsicking–unsicking cyce,membranedamageaccumuates,resutingintheformationof irreversibysickedces.142,143Thesecesareincapabeofreversionto

thebiconcavediscshape,evenwhenfuyoxygenated.Theyhavean increasedhemogobinconcentration,increasedcationpermeabiity, decreasedpotassium,andincreasedsodium.

FragmentedCells(Schistocytes)

Schistocytesareseeninmicroangiopathichemoyticanemias(thromboticthrombocytopenicpurpura[TTP],disseminatedintravascuar coaguation[DIC],vascuitis,gomeruonephritis,renagraftrejection), carcinomatosis,heartvavehemoysis(prostheticorpathoogicvaves), severeburns,andmarchhemogobinuria(Chap.22).Fibrinstrandsin damagedboodvessescanbearrayedsothattheysievethepassingred ces.Ifapassingredcefodsoverorotherwiseattachestothestrand, theboodstreampusonthearrestedce,stretchesit,andeventuay fragmentsit.144Thespeenrapidyremovestheschistocyteswithaow reativeSA:Vratio;theremaindermaycircuateformanydays.

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kinasethatinteractswitherythropoietinreceptor;KAP1,KRAB-associatedprotein-1 isatranscriptionalcofactor;KRAB-ZFP,1ofthe400humanzincfingerprotein-based transcriptionfactors;MCV,meancellvolume;mRNA,messengerRNA;mDia2,a proteinthatregulatesactinandfocaladhesiondynamics;miRs,microRNAsaresmall non-codingRNAmolecules;15N,nitrogen;Nix,aproteinthatisexpressedduring erythropoiesisandregulatesmitochondrialapoptosis(autophagy);PHD,proline hydroxylase;PK,pyruvatekinase;PU.1,transcriptionfactor;RACK1,receptorof activatedproteinkinaseC;RAS,therenin–angiotensinsystem;Rb,tumorsuppressor retinoblastoma(protein);RCM,redcellmass;SCL/TAL1,stemcellleukemia/t-cell acutelymphoblasticleukemia1factor;SCL/tal-1,basichelix-loop-helixtranscription factor;SNPs,single-nucleotidepolymorphisms;SOCS3,asignaltransductionprotein (alsoknownasCIS3)thatdownregulatesactivityoferythropoietinreceptor;99mTc, technetium-99;VHL,vonHippel-Lindauprotein.

erythrocytestoreplacethe2×1011redcells(approximately1%ofthetotal redcellmass[RCM])removedfromthecirculationeachday.Redcellproductionincreasesseveral-foldafterbloodlossorhemolysis.WhenoneoftheprogenyofanHSCbecomescommittedtotheerythroidlineage,thisearlyerythroid progenitorundergoesaseriesofdivisionsandconcurrentmaturationthat eventuallyresultinmorphologicallyrecognizableerythroblasts.Afterexpulsionofthenucleus,amacrocyte(polychromatophilicwhenWright-stained,or areticulocyteifnewmethyleneblue–stained)leavesthemarrow.Duringthefirst24to48hoursinthecirculation,reticulocyteslosetheirresidualorganelles(mitochondriaandribosomes)throughanautophagicprocess (Chap.1)andundergoreconditioningofthemembranetobecomematurered cellswithabiconcavediscshape.Erythropoiesisiscontrolledbytranscription factorsandcytokines,theprincipalonesbeingGATA1anderythropoietin (EPO),respectively,whichinfluencelineagecommitment,proliferation,apoptosis,differentiation,andnumberofdivisions,fromtheearliestprogenitor tolateerythroblasts.Thenumberofredcellsproducedvariesinresponseto tissueoxygenation,whichdeterminesthelevelofthetranscriptionfactors, hypoxia-induciblefactors(HIFs)—HIF-1andHIF-2—theprincipalregulators oftheresponsetohypoxia.HIFsmodulateerythropoiesisbyregulationofEPO productionbydirectEPO-independentmechanism(s)andbyfacilitatingiron availability.

Thesurvivalofredcellsinthecirculationcanbemeasuredinavarietyof ways:(1)bylabelingwithradioactiveisotopes,particularly51Cr,andassessing thedisappearanceoftheradioactivetagfromthecirculationovertime; (2)bylabelingtheerythrocyteswithbiotinorafluorescentdyeandmeasuringthismarkerovertime;(3)bydeterminingthedisappearanceoftransfused antigen-matchedallogeneicerythrocytesusingimmunologicmarkers;and (4)bymeasuringtheexcretionofcarbonmonoxide(CO),aproductofheme catabolism.

Suchstudiesshowthatnormalhumanredcellshaveafinitelifespan averaging120days,withsomecomponentofrandomdestruction.Themitochondrialandribosomalremovalhighlightingmaturationofthereticulocyte isaccompaniedbyincreasingcelldensity,butafterafewdaysofintravascular lifespan,thereislittlefurtherincreaseindensityorotherchangesinthephysicalpropertyoftheredcells.Thus,celldensityisnotagoodmarkerforaged redcells.Thishasmadestudyofthesenescentchangesintheredcellthat markitfordestructiondifficult.Candidatesforsuchchangesincludechanges inmembraneAE-1(anionexchanger-1;alsoknownasband3)andexposure ofphosphatidylserineonthemembrane,whichmaybeofmajorimportance.

HISTORY

Erythrocytesevolvedlargelyforthepurposeoftransportingoxygento tissuesandcarbondioxidefromtissues.Thus,thesizeoftheredcell mass(RCM)andtherateofredcellproductionmustbecloselyrelated tosupplyanddemandforoxygeninthetissues.Frenchmountaineers andphysiologistsattheendofthe19thcenturyestablishedthatalow tissuetensionofoxygenstimulatesredcellproduction.1In1906,Paul Carnot,aprofessorattheSorbonne,andMademoiselleDeFlandre,his associate,suggestedthathypoxiageneratesahumoralfactorcapableof stimulatingredcellproduction.2In1950,KurtReissmann3provided evidencefortheexistenceofanindirecthumoralmechanismusing parabioticrats.TheworkofErslevandcolleagues,4,5demonstratedthat theplasmafromanemicrabbitsandprimatescontainsanerythrocyte stimulatingfactor,appropriatelynamederythropoietin(EPO).Then,EPO