SAUNDERSAnImprint ofElsevierThe Curtis CenterIndependence Square WestPhiladelphia, Pennsylvania19106Library of Congress Cataloglng-lnPublication DataHarvey, JohnW.Atlasof veterinary hematology: bloodandbonemarrowof domestic animals I JohnW. Harvey.p. em.Includes bibliographical references (p.).ISBN-13: 978-0-7216-6334-0 ISBN-10: 0-7216-6334-61.Veterinary hematology-Atlases.2. Bloodcells-Atlases. 3. Bonemarrow-Atlases.I. TItle.SF769.5.H37 2001636.089"615--dc21ATLAS OFVETERINARYHEMATOLOGY00-063569Copyright 2001 by W.O. Saunden CompanyAll rights reserved. No part ofthis publicationmay be reproducedor transmittedin any formor by anymeans, electronicor mechanical, includingphotocopy, recording,or any informationstorageand retrievalsystem, without permissionin writingfromthe publisher.Permissionsmay be sought directlyfromElsevier's Health SciencesRights Departmentin Philadelphia,PA, USA: phone: (+1) 215 239 3804, fax: (+1) 215 239 3805, e-mail: healthpermissions@elsevier.com.Youmay also completeyour request on-linevia the Elsevier homepage(http://www.elsevier.com).byselecting'Customer Support' and then 'Obtaining Permissions'.ISBN-13: 978+7216-6334-0ISBN-I0: 0-7216-6334-6Printedin ChinaLast digit is the print number: 9 8 7 6 5 4To LizPrefaceThis color atlas is designed as areferencebook for themorphologic aspects ofveterinaryhematology ofcommondomesticanimals, excluding birds. Speciescoveredincludedogs, cats,horses,cattle, sheep,goats, pigs, andllamas. Theatlasisdividedintotwosections, bloodandbonemarrow. It includes basicmaterial forthenovice, as well asmaterialofprimaryinterest tothosewithadvanced training. Techniques forthecollection andpreparationof bloodandbonemarrow smears and bonemarrow core biopsies are discussed, in additiontothemorphology of thetissues collected. Often, more thanoneexample of acell type or abnormal condition is shown, because cells and conditions can varyinmorphology. Veterinary technologistswill likelyfindthebloodsection andthetechniques part of thebonemarrow section tobe mosthelpful. Veterinarystudentsandpracticing veterinarians shouldbenefit fromthecomplete book,eveniftheyarenot directly involved inbonemarrowevaluation, because itprovidesa basisfor understandingdiseases affectingthe marrow. Thebonemarrowaspirate smear cytology andcore biopsy histology section will bemostuseful toclinical pathologists, anatomicpathologists, andresidents intrainingforthese disciplines. This is not acomplete hematology textbook, but rather areference bookinwhich thetextexplains thesignificanceofthemorphologicabnormalitiesshowninthecolor photographs. Readers interestedinlearningmoreabout agiventopicwill hopefully appreciate theextensivebibliographyprovided.JohnHarveyAcknowledgmentsI want toacknowledgethosemost responsible for myeducationasaclinicalpathologist. Few people have the opportunity toreceivetraining from the giantsof their profession, but I wasblessed inbeingtrained byJerryKaneko, thefather of veterinaryclinical biochemistry, and Oscar Schalm, the father ofveterinary hematology. Many other colleagues have contributedtomy develop-ment as ahematologist, with Victor PermanandAlan Rebar being particularlynoteworthy, as we have challengedone another with unknown hematologyslides infront ofvariousaudiences. I thankDenny Meyer forhisencourage-ment in taking on the task of preparing this atlas and RoseRaskin, LeoMcSherry, and Shashi Ramaiahfor their conscientious reviews and helpfulsuggestions. I appreciate Melanie Pate's careful editorial reviewandamgratefultoRay KerseyatW.B. Saunders Co. for his remarkable patience andpersistentsupport in this endeavor.JohnHarveyEditor's NoteThe purpose of this text/atlas is to provideveterinarians, veterinarystudents,veterinary technicians andveterinary technology students with a complete treatiseonthe cytologyof bloodand bonemarrow.Thesubjectswill becoveredinauserfriendly way utilizing color photo-graphsandappropriatetext that clarifiesdiagnostic implications. Therapy will bediscussed only when diagnosis can be confirmed by response to treatment.Ninety percent of the informationwill focus oncommonly recognized diseases.CHAPTER 1Examinationof Blood Samples SAMPLE COLLECTION ANDHANDLINGAnovernight fast avoids postprandial lipemiainmonogastricanimals, whichcaninterfere withplasma protein, fibrinogen, andhemoglobindeterminations.Ethylenediaminetetraacetic acid (EDTA) is thepreferred anticoagulant for com-plete bloodcount (CBC) determinationinmost species, but bloodfromsomebirds and reptiles hemolyzes when collectedinto EDTA.I In those species,heparinis oftenused as theanticoagulant. Thedisadvantage of heparinis thatleukocytes donot stainaswell(presumably because heparin binds toleuko-cytes),?and platelets generallyclump more thanthey do in blood collected withEDTA. However,aswillbediscussed later, platelet aggregates andleukocyteaggregates mayoccur evenin properlycollected EDTA-anticoagulatedbloodsamples.':"Inthose cases, collection of blood using anotheranticoagulant (e.g.,citrate)may prevent theformationofcell aggregates. Cell aggregation tendstobemore pronounced asbloodiscooledandstored; consequently, processingsamples asrapidly aspossible aftercollection mayminimizetheformationofleukocyte and/orplatelet aggregates.Collection of blooddirectly intoa vacuumtubeis preferredtocollectionof blood by syringeand transfer to a vacuumtube. This method reducesplatelet clumping andclot formationinsamples for CBC determinations. Evensmall clots render a sample unusable. Platelet counts are markedly reduced, andsignificant reductioncansometimes occurinhematocrit (HCT) andleukocytecounts aswell. Also, whenthe tube isallowed to fill basedon thevacuumwithin the tube, the proper sample to anticoagulant ratio will be present.Inadequatesample size results indecreased HCT duetoexcessiveEDTA solu-tion. Care shouldbe takentoavoid iatrogenic hemolysis,which interferes withplasma protein, fibrinogen, and various erythrocyte measurements. Samplesshouldbesubmittedtothelaboratory as rapidly as is feasible, andblood filmsshould be made as soon as possible andrapidly dried tominimize morphologicchanges.34 SECTIONII BLOODFIGURE1. Gross appearance of mixtures of oxyhemoglobin, deoxyhemoglobin, and methemo-globinanddifferentiationof erythrocyte agglutinationfromrouleauformation. A. Venousbloodsamplefromacatwith28%methemoglobin(left sample)comparedtoanormal catwithless than1% methemoglobin(right sample). Both samples also containa mixtureof oxyhemoglobin and deoxyhemo-globin. B. Oxygenated blood sample from a cat with 28% methemoglobin (left sample)(Continued)CHAPTER1 / EXAMINATION OF BLOODSAMPLES GROSSEXAMINATION5Samples are checked for dotsandmixed well (gently inverted 20 times) imme-diately before removing aliquots for hematology procedures. Horse erythrocytessettle especially rapidly because ofrouleauformation(adhesion of erythrocytestogether like a stack of coins). Blood should be examined grossly for color andevidenceof erythrocyteagglutination. The presenceof markedlipemia mayresult in a blood samplewithamilkyredcolor resembling"tomato soup"when oxygenated.MethemoglobinemiaHemoglobinisaproteinconsisting offour polypeptide globin chains, each ofwhich contains a heme prosthetic group within a hydrophobic pocket. Heme iscomposedof atetrapyrrolewithacentral ironmolecule that must bemain-tained in the ferrous (+2) state to reversiblybind oxygen. Methemoglobindiffers fromhemoglobin only inthat theironmolecule of theheme grouphasbeen oxidized totheferric (+3)state andit is nolonger able tobindoxygen."The presenceof largeamounts of deoxyhemoglobinaccounts for the dark,bluishcolor of normal venous bloodsamples. Methemoglobinemia may not berecognized invenous bloodsamples, because thebrownishcolor of methemo-globinisnot readily apparent whenmixedwithdeoxyhemoglobin(Fig. lA).When deoxyhemoglobinbinds oxygento formoxyhemoglobin, it becomesbright red; consequently, thebrownishcolorationofmethemoglobinbecomesmoreapparent in the oxygenated samples (Fig. IB). A simple spot test providesarapidwayto oxygenate avenousbloodsampleanddetermine ifclinicallysignificant levels ofmethemoglobinarepresent. Onedropofbloodfromthepatient isplacedonapiece ofabsorbent whitepaper andadropofnormalcontrol bloodisplacednext to it. If the methemoglobincontent is 10%orgreater, the patient'sblood will have a noticeably brown color, compared to thebright redcolor of controlblood(Fig. lC).9Accurate determinationof methe-moglobin content requires thatblood be submittedtoa laboratory thathas thistest available.'?Methemoglobinemiaresultsfromeither increasedproduction of methe-moglobin by oxidants ordecreased reduction of methemoglobin associated withadeficiency intheerythrocyte methemoglobinreductase enzyme. Experimentalstudiesindicatethat manydrugscanproducemethemoglobinemiainanimals.comparedto a normal cat withlessthan 10/0 methemoglobin(right sample). Thesampleon the leftcontains a mixture of oxyhemoglobin and methemoglobin, and the one on the right contains almostexclusivelyoxyhemoglobin. C. Adropofbloodfromamethemoglobin reductase-deficient catwith500/0 methemoglobin (left) is placed on absorbent white paper next to a dropof normal cat blood with lessthan1% methemoglobin. D. Grossly visible agglutination in blood from a dog with immune-mediatedhemolytic anemia. E. Microscopic rouleauxinanunstainedwet mount preparationof normal catblood. F. Microscopic agglutination inanunstained wet mount preparationof saline-washed erythro-cytes from a foal with neonatal isoerythrolysis.6 SECTIONI I BLOODSignificant methemoglobinemia has been associated with clinical cases of benzo-caine, acetaminophen, andphenazopyridinetoxicities incatsanddogs;nitritetoxicity incattle; coppertoxicity insheep andgoats; andredmapletoxicity inhorses."AgglutinationThe appearance of red granules in a well-mixed blood sample (Fig. ID)suggeststhepresence of erythrocyte agglutination, theaggregation or clumping of eryth-rocytes together in clusters. Agglutination is caused by the occurrence of immu-noglobulinsboundtoerythrocyte surfaces. Itmust bedifferentiated fromrou-leaux, theadhesionof erythrocytes togetherlike astack of coins, which canbeseenin blood fromhealthyhorses and cats (Fig. IE). Agglutinationcanbedifferentiated fromrouleaux by washing erythrocytes in physiologic saline orbyadding equal drops of physiologicsaline and blood together to see if theaggregationoferythrocytesisdispersed(rouleaux) or remains (agglutination).The microscopicappearanceof agglutinationinasampleof washederythro-cytes is shown (Fig. IF).II MICROHEMATOCRIT TUBE EVALUATIONWhenbloodis submittedforaCBC, most commercial laboratoriesdeterminetheHCTelectronically. This efficiencynegates theneedtocentrifuge amicro-hematocrit tubefilledwith blood. Unfortunately, useful informationconcerningtheappearanceofplasmaismissed unless aserumor aplasma sampleis alsopreparedfor clinical chemistry tests.Packed CellsIn additiontodeterminingtheHCT, the buffy coat is evaluated. The buffy coatcontainsplatelets andleukocytes. It may appearreddish duetothepresence ofmarkedreticulocytosis. Insomespecies, certainleukocytes may also bepresentin the top portion of the packed erythrocyte column (e.g., neutrophils incattle). A large buffy coat suggests leukocytosis (Fig. 2A) orthrombocytosis anda small onesuggests low numbersof these cells may be present.PlasmaAppearancePlasmaisnormallyclearinallspecies. It isnearlycolorless insmall animals,pigs, andsheepbut light yellow inhorses, because theynaturallyhave higherbilirubin concentrations. 11 Plasma varies fromcolorless tolightyellow (carot-enoidpigments) incattle, dependingontheir diet. Increased yellow colorationusuallyindicates increasedbilirubin concentration. This increaseoftenoccurssecondarily toanorexia(fasting hyperbilirubinemia) inhorsesdueto reducedCHAPTER1 / EXAMINATION OF BLOODSAMPLES7FIGURE2. Grossappearanceof microhematocrit tubes demonstrating leukocytosis, icterus,hemolysis, andlipemia. A. Microhematocrit tubefromamarkedly anemic cat withalarge buffycoat resultingfroman acutemyeloidleukemiawitha total leukocyte count of 236,000leukocytes!I-'L.Hemolysisis present inplasma because theblood sample wassent throughthemail andwas several daysold. B. Microhematocrit tubefromananemic cat, withicteric plasma secondary tohepatic lipidosis.C. Microhematocrit tubewith evidence ofhemolysisinplasma fromacat with acetaminophen-inducedHeinz-body hemolytic anemia. Less-dense erythrocyte "ghosts" can be seen above the packed intact erythro-cytes. D. Microhematocrit tubewith evidence of hemolysisinplasma fromahorse with intravascularhemolysis inducedbythe inadvertent intravenousandintraperitoneal administrationofhypotonicfluid.Less-dense erythrocyte ghosts can be seen above thebuffy coat. E. Microhematocrit tubewith markedlipemia inplasmafromadogwithhypothyroidism that was alsobeingtreatedwithprednisonefor anallergic dermatitis. It was unclear from the medical record if this was a fasting blood sample.removal of unconjugated bilirubin by theliver."Inotherspecies, yellow plasmawith a normal HCr suggests hyperbilirubinemia secondary to liver disease.Hyperbilirubinemiaassociatedwithamarkeddecreaseinthe HCTsuggests anincreased destructionof erythrocytes; however, the concomitant occurrenceofliver disease and a nonhemolytic anemia could produce a similar finding(Fig. 2B).Red discoloration of plasma indicates the presence of hemolysis. Thisdiscolorationmayrepresent either true hemoglobinemia, resultingfromintra-vascular hemolysis (Figs. 2C, 2D), or the hemolysis mayhave occurredaftersamplecollectionduetosuchcauses as roughhandling, fragile cells, lipemia, or8 SECTIONI/ BLOODprolongedstorage. Thehematocrit value may help differentiate these two possi-bilities, withredplasma anda normal hematocrit suggesting in vitro hemolysis.Theconcomitant occurrenceof hemoglobinuriaindicates thepresence of intra-vascular hemolysis.Lipemiaisrecognizedasawhiteopaque appearancecaused bychylomi-cronsandvery-low-density lipoproteins (VLOL). The presenceof chylomicronsmay also result ina white layer at thetopof theplasma column(Fig. 2). Thepresence of lipemia is frequently the result of a recent meal (postprandiallipemia), but diseases including diabetes mellitus, pancreatitis, and hypothyroid-ismmayresult inlipemiaindogs. Hereditarycauses includelipoprotein lipasedeficiency incats anddogs andidiopathichyperlipidemia inminiatureschnau-zerdogs.' 3' 4Ponies(especially obese ones), miniature horses, anddonkeys aresusceptibleto developinglipemiaassociated withpregnancy, lactation, and/oranorexia.P:" Theseconditions result in the mobilizationof unesterifiedfattyacidsfromadiposetissueandthesubsequent overproductionofVLOLbytheliver.Plasma Protein DeterminationAfterthe HCTismeasuredandtheappearanceoftheplasmaandbuffycoatare noted, the microhematocrit capillary tube isbroken just abovethe buffycoat, andtheplasma is placed ina refractometerfor plasma protein determina-tion. Plasma proteinvalues innewbornanimals (approximately 4.5 to5.5 gldL)are lower thanadultvalues andincrease totheadult range by 3 to4 months ofage.II Thepresenceoflipemiaor hemolysis will falselyincreasethemeasuredplasma protein value. Maximuminformation can be gained by interpretation ofthe HCTandplasma protein concentrations simultaneously.IFibrinogen DeterminationFibrinogencanbe measuredina hematocrit tubebecause it readily precipitatesfromplasma when heatedto56 to58C for 3 minutes. The difference betweenthe total proteinofthe plasmaand the total proteinof thedefibrinogenated(heated) plasma gives an estimate of the fibrinogen concentration in theplasma.'?This methodisuseful inidentifyinghigh-fibrinogenconcentrations,but not accurate in identifying low-fibrinogen concentrations.P-"II BLOOD FILM PREPARATIONBloodfilms should be preparedwithin a couple of hours of blood samplecollection toavoid artifactual changes that will distort themorphology of bloodcells. Bloodfilms are prepared in various waysincluding the slide(wedge)method, coverslip method, andautomated slide spinner methods.It is essentialthat a monolayer of intact cellsbe present on the slide so that accurateexaminationanddifferential leukocyte countscan be made.Slide Blood Film MethodCHAPTER1 I EXAMINATION OF BLOODSAMPLES 9Aclean glass slide isplaced onaflat surface andasmall dropof well-mixedbloodisplaced ononeendoftheslide(Fig. 3A). This slide isheldinplacewithonehandandasecondglassslide(spreader slide) isplaced onthefirstslide andheld between the thumbandforefingerwith the otherhandat abouta 30-degree angleinfront of thedrop of blood. Thespreader slideis thenbacked intothedropof blood, andas soonas thebloodflowsalong thebackside of the spreader slide (Fig. 3B), the spreader slide is rapidly pushed forward(Fig. 3C). The thicknessof the smear is influencedbythe viscosityof thesample. The angle between thetwo slides may beincreased when thebloodisless viscous (low HCT) anddecreased whenthebloodismoreviscous (highHCT) thannormal toproduce a smear of appropriate thickness.If the dropof blood is the propersize, all blood willremain onthe slide,andasmear will bepreparedthat is thickat theback of theslide, where thedropof blood was placed, and thinat thefront (feathered) edge of the slide. Ifthe dropof blood is toolarge, some of the blood will be pushed off the end oftheslide, causing potential problems. Often these bloodfilmswill be toothickfor accurateevaluation.Secondly, dumps ofcells tendtobepushedofftheslide, making them unavailable for examination.Once prepared, the slide is immediately dried by waving it in the air or byholding itinfront of ahairdryer set ona warm-air setting. Holding the slideclose toadryer set onahot-air settingcanresult infragmentationof cells.Slides are identified by writing onthe thick end of the smear or the frosted endof the slide with a graphite pencil ora pencontaining ink that is not removedby alcohol fixation.Coverslip Blood Film MethodTwo22-mmsquare, No. 1Vz coverslips arerequiredtomakecoverslipbloodfilms (Fig. 3D). Acamel's-hair brush is usedto removeparticlesfromthesurfacesthat will contactblood.Onecoverslipis held between thethumbandindex finger of one hand,and a small dropof blood is placed in themiddle ofthecoverslipusingamicrohematocrit tube. Thedropofbloodshouldbeasperfectly roundas possible toproduceeven spreading between coverslips. Thesecond coverslip is droppedon top of the first in a crosswise position. After thebloodspreads evenly between thetwo coverslips anda feathered edge forms atthe periphery, the coverslips are rapidlyseparatedbygraspingan exposedcornerof thetopcoverslip with the otherhandandpulling apart in a smooth,horizontal manner. Coverslips areimmediately driedasdescribedaboveandthen identifiedbymarkingon the thickendof the smearswithagraphitepencil or a pen containing ink that is not removed by alcohol fixation.If the dropof blood used is too large, a feathered edge will notform andthebloodfilm will be toothick. Multiple coverslipblood filmsmay be stainedinsmall, slottedcoplinjarsor inceramicstaining baskets that areplaced inbeakers of fixativeand stain.10 SECTION I I BLOODFIGURE3. Techniques for glass-slideandcoverslip bloodfilm preparationsandtheappear-ance of stained bloodfilms. A. Slide blood filmpreparation-step1. A small drop of well-mixedblood is placedonone endof deanglassslide. B. Slideblood filmpreparation-step2. A secondglassslide(spreaderslide) is placedonthefirst slide atabout a30-degreeangle infrontof thedropofblood and then backed into the drop of blood. C. Slide blood filmpreparation-step3. As soon as(Continued)CHAPTER IIEXAMINATION OF BLOODSAMPLES BLOOD FILM STAINING PROCEDURESIIRomanowsky-Type StainsBlood films are routinely stained with a Romanowsky-type stain (e.g., Wright orWright-Giemsa) either manuallyor usingan automatic slidestainer. Roma-nowsky-type stains are composed of a mixtureof eosin andoxidized methyleneblue(azure) dyes. The azure dyes stain acids, resulting in blue to purplecolors,and eosinstains bases, resultingin red coloration (Fig. 3E). Thesestainingcharacteristics dependonthepHofthestainsandrinsewateras well asthenature ofthecells present (Fig. 3F). Pale staining cells canresult frominade-quate staining time, degraded stains, orexcessive washing.Bloodfilmsmayhavean overallbluetint ifstoredunfixedfor weeksbefore staining or if theunfixed bloodfilms are exposed toformalin vapors, asoccurswhenbloodfilms areshippedtothelaboratoryinapackage that alsocontainsformalin-fixed tissue. Blood films preparedfrombloodcollected withheparinas the anticoagulant have an overall magenta tint due to the mucopoly-saccharides present.Variousproblemscanoccur duringthedrying, fixation, andstainingofbloodfilms that resultinpoor-qualityfilms. Drying or fixation problemscanresult invariably shapedrefractile inclusions inerythrocytes that may becon-fusedwitherythrocyte parasites(Fig. 4A). Thepresence ofstainprecipitationcanmakeidentificationof leukocytes andbloodparasitesdifficult (Fig. 4B).Precipitated stain may be present because thestaints) needed tobe filtered, thestaining procedure was too long, or washing was notsufficient. Carboxymethyl-cellulosehasbeen infused intotheperitonealcavity of horses andcattle inanattempt toprevent abdominal adhesions after surgery. This material can appearasaprecipitatebetween cells inbloodthat resembles stainprecipitation(Fig.4C).20Various rapid stains are available. The quality of the stained blood films isgenerally somewhat lower thanthat obtained by longer staining procedures. TheDiff-Quik stain is a commonly used Wright-type rapidbloodstain. The qualityof thisstaining procedureis improvedconsiderably by allowing thebloodfilmtoremaininthe fixativefor several minutes. One limitation of this stain is thatitdoes not stain basophil ormastcell granules well. However, itis superiortoWright or Wright-Giemsastains in staining distemper inclusions in caninebloodcells."thebloodflows along theback side ofthespreader slide, thespreaderslide israpidly pushedforward.D. Steps usedinpreparingcoverslipbloodfilms aredescribedinthetext. E. Normal cat bloodstainedwithWright-Giemsa andrinsedindistilled water. Four neutrophils, abasophil(far right), anda lymphocyte (round nucleus) are present. Erythrocytes exhibit rouleaux. a normal findingin cats.F. Normal cat blood stained with Wright-Giemsaandrinsed intap water. A neutrophil (left). monocyte(bottomright), andlymphocyte(topright)arepresent. Thebluecolor oftheerythrocytesresults fromusing water with inappropriate pH.12 SECTION I I BLOODFIGURE4. Staining artifacts,theappearanceof carboxymethylcellulose, andreticulocyte mor-phologyinbloodfilms. A. Refractileinclusions inerythrocytes froma horse are artifacts resultingfrom drying orfixation problems. Erythrocytes exhibit rouleaux, a normal finding in horses. Wright-Giemsastain. B. Stain precipitation inblood froma dog. The two neutrophils present might be mistaken forbasophilsbecauseof the adherent precipitatedstain. Wright-Giemsastain. C. Theblue to purple(Continued)Reticulocyte StainsCHAPTER1 I EXAMINATION OF BLOODSAMPLES 13Reticulocyte stainsarecommercially available. Thosewishing toprepare theirownstaincandosobydissolving 0.5 gof newmethyleneblueand 1.6 gofpotassiumoxalate in 100mLof distilledwater. Followingfiltrations, equalvolumes of bloodandstainaremixed togetherina testtubeandincubatedatroomtemperature for10 to20 minutes. After incubation, blood films are madeand reticulocytecounts are performed byexamining 1,000erythrocytesanddetermining thepercentage that are reticulocytes."The use of a Miller's disc inone of the microscope oculars saves time in performing the reticulocyte count.The blue-staining aggregates or"reticulum"seen inreticulocytes (Fig. 4D)does not occur as suchin livingcellsbut results fromthe precipitation ofribosomal ribonucleicacid(RNA; thesameRNA that causes thebluishcolorseeninpolychromatophilicerythrocytes)inimmatureerythrocytes duringthestaining process.I I As a reticulocyte matures, the number of ribosomes decreasesuntil only small punctate (dotlike) inclusions are observed in erythrocytes(punctate reticulocytes) stained withthereticulocyte stain(Fig. 4E). Toreducethe chance that a staining artifact would result in misclassifying a matureerythrocyte as a punctate reticulocyteusinga reticulocytestain. the cell inquestionshouldhave twoor morediscrete bluegranules that arevisible with-out requiringfine-focus adjustment ofthecell beingevaluated tobeclassifiedas a punctatereticulocyte.Innormal cats. as well as in cats withregenerative anemia. thenumber ofpunctate reticulocytes ismuch greater than that seeninother species." Thisapparently occurs because the maturation(loss of ribosomes) of reticulocytes incats is slower thanthat inother species. Consequently, reticulocytes incats areclassified as aggregate (if coarseclumping isobserved) or punctate (if smallindividual inclusions are present). Percentages of bothtypes should be reported.Based on composite resultsfromseveral authors, normal catsgenerally havefrom0%to0.5% aggregateand1% to10% punctatereticulocytes whendeter-minedbymanual means. Higher punctate numbers of2%to 17% havebeenreportedusing flowcytometry."The percentagesof aggregate reticulocytes incatscorrelatedirectlywiththe percentages of polychromatophilic erythrocytes observed in blood filmsprecipitate present between erythrocytes in this horse bloodresults from treatmentwith carboxymethylcellu-lose. Photographof a stained blood film from a1994ASVCP slide reviewcase submittedby M.J. Burkhard,M.A. Thrall, andG. Weiser. Wright-Giemsa Stain. D. Fourreticulocytes (with blue-staining material)and three mature erythrocytesin blood froma dogwitha regenerativeanemia. Newmethylenebluereticulocytestain. E. Three whole aggregate reticulocytes (containing blue-staining aggregatesof RNA)andone half of anaggregate reticulocyte(far right) inbloodfromacat withamarkedlyregenerativeanemia. A majorityoftheremainingcells arepunctatereticulocytes containingdiscrete dotlike inclusions.New methylene bluereticulocyte stain F. Five reticulocytes (with blue-staining material) in blood fromadog with aregenerative anemiainanew methylene blue stained wet preparation. Note thedifference inmorphology comparedtoreticulocytes stained with astandardreticulocyte stain(Fig. 4D). New methyleneblue stained wet preparation.14 SECTION I / BLOODstainedwithWright-Giemsastain."Aggregatereticulocytes maturetopunctatetypesina dayor less. Several more daysare requiredfor maturation (totaldisappearance of ribosomes) of punctatereticulocytes in cats.2425Incontrast tothoseofthecat, most reticulocytes inother species areoftheaggregate type. Consequently, noattempts aremadetodifferentiate stagesof reticulocytes inspecies other thanthecat. The percentage of reticulocytes inmost species correlates directly with the percentage of polychromatophilic eryth-rocytes observed onroutinely stained blood films.Heinzbodiesarecomposedofdenatured, precipitatedhemoglobin. Theyarespherical, stainpalebluewithreticulocyte stains, andareusually foundatthe periphery of the erythrocyte.New MethyleneBlue "Wet Mounts"A new methylene blue "wet mount"preparation can be used for rapid informa-tion concerning the number of reticulocytes, platelets, and Heinz bodiespresent. The stain consistsof 0.5%newmethyleneblue dissolvedin0.85%NaCl. OnemL of formalinis addedper 100 mL of stain as a preservative. Thisstainis filtered afterpreparationandstoredindropper bottles. Alternately, thestainmay be storedina plastic syringe with a 0.2 IJ-m syringe filter attached sothat thestainisfilteredasit isused. Dryunfixedbloodfilms arestainedbyplacing a dropof stain between thecoverslip anda glass slide. This preparationis not permanent anddoes not stain matureerythrocytes or eosinophil granules.Punctate reticulocytes arenot demonstrated, but aggregatereticulocytes appearas erythrocyte ghosts containing blue to purple granular material (Fig. 4F).Platelets stainbluetopurple, andHeinzbodiesappear asrefractile inclusionswithin erythrocyte ghosts. Although this stainingmethod is not optimal fordifferential leukocyte counts, thenumber andtype of leukocytes presentcan beappreciated.Iron StainsAn ironstainsuchas thePrussianbluestainisusedtoverify thepresence ofiron-containing(siderotic) inclusionsinbloodandbone marrowcells andtoevaluate bonemarrowironstores. Smears may be sent toa commercial labora-tory for this stain, or a stain kit can be purchased and applied in-house(Harleco Ferric Iron Histochemical Reaction Set, #6498693, EMDiagnosticSystems, Gibbstown, NJ). When this stain is applied, iron-positive materialstains blue, in contrast to the dark pink color of the cells andbackground.The presence of focal areas of basophilic stipplingwithin erythrocytesstained with Romanowsky-typeblood stains suggeststhat the stipplingmaycontain iron. Iron-containingerythrocytesare referredto as siderocytes andiron-containingnucleatederythrocytesarecalled sideroblasts. Neutrophils andmonocytes maycontain dark bluish-blackor greenishiron-positive particleswithintheir cytoplasm whenstainedwithRomanowsky-type stains. Leukocytescontaining iron-positive inclusions have been called sideroleukocytes.CHAPTER1 I EXAMINATION OF BLOODSAMPLES 15Prussianbluestainappliedtobone marrowaspiratesmearsisausefulwayto evaluatethe amount of storageiron that ispresent inthe marrow.Minimalor noironisexpected inirondeficiencyanemia(althoughcats nor-mally havenostainableiron inthemarrow), whereasnormal or excess ironmay be observed inanimals with hemolytic anemia and in animals with anemiaresulting from decreased erythrocyte production.Cytochemical StainsAvarietyof cytochemicalstains, such as peroxidase, chloroacetateesterase,alkalinephosphatase, and nonspecific esterase, are utilizedto classifycells inanimals with acute myeloid leukemias.11,26.27 Reactions vary not only by cell typeandstage of maturationbutalso by species." These stains are donein a limitednumber oflaboratories and special trainingand/or experience isrequiredtointerpret theresults. The appearance of positive reactions also varies dependingon the reagentsused. Becauseofthe complexities ofthestainingproceduresandinterpretationofresults, minimal informationoncytochemical stainswillbe presented in this atlas. EXAMINATIONOFSTAINEDBLOODFILMSAnoverviewandorganizedmethodofbloodfilmexaminationarepresentedhere. Descriptionsandphotographsofnormal andabnormal bloodcell mor-phology, inclusions, and infectious agents will be given in subsequent sections.Blood films aregenerally examined followingstaining with Romanowsky-typestains suchasWright or Wright-Giemsastains. Thesestains allowforexamination of erythrocyte, leukocyte, and platelet morphology. Bloodfilmsshould first be scanned using a low-power objectiveto estimate the totalleukocyte count andtolookforthepresence of erythrocyte agglutination(Fig.SA), leukocyteaggregates (Fig. SB), platelet aggregates (Fig. SC), microfilaria(Fig. SD), and abnormal cellsthat might be missedduring the differentialleukocytecount. It isparticularlyimportant that the featheredendof bloodfilms made onglass slides be examined because leukocytes(Fig. SE) and plateletaggregates (Fig. SF) may beconcentratedinthisarea. Aggregatesofcells tendto be in the center of coverslip blood films ratherthanat the feathered edge.Whenexamining a glass-slidebloodfilm, thebloodfilm willbe toothicktoevaluate bloodcell morphologyat thebackoftheslide(Fig. 6A) andtoothinat thefeathered edge where cells are flattened(Fig. 6C). The optimal areaforevaluationisgenerally inthefront halfofthesmearbehindthefeatherededge (Fig. 6B). This area shouldappearas a well-stained monolayer (a field inwhich erythrocytes are dosetogether with approximately one half touching eachother)of cells.16 SECTIONI I BLOODFIGURE 5. Abnormalities in stained blood films identified by scanning using low-powermagnification. A. Autoagglutinationof erythrocytes inbloodfromadog withimmune-mediatedhemolytic anemiaandmarked leukocytosis. Wright-Giemsastain. B. Leukocyte aggregate inbloodfrom adog. Leukocyteaggregateswere present when EDTAwas used as theanticoagulant but not whencitrate was used as the anticoagulant. Wright-Giemsa stain. C. Platelet aggregate in blood from a cow.(Continued)CHAPTER1 / EXAMINATION OF BLOODSAMPLES17FIGURE 6. Selection of theappropriatearea for bloodslide examination andpatternstousetoperforma leukocyte differential count. A. Thick area inthe back endof a bloodfilm fromadog. The blood filmwas prepared usingglass slides.Wright-Giemsastain. B. Optimal area formorphologicevaluationinthe front half of a dogbloodfilm(samebloodfilmasshowninFig. 6A)prepared using glass slides. Wright-Giemsa stain. C. Thin area near thefeathered edge of a dog bloodfilm (same blood film as shown inFigures 6A and 6B) prepared using glass slides. Erythrocytesare flattenedtotheextent that central pallor isnot readily apparent. Wright-Giemsa stain. D. Patternsof slideblood film examination that may be used to improve the accuracy of differential leukocyte counts.Wright-Giemsa stain. D. Dirofilariaimmitismicrofilaria inbloodfroma cat withheartworm disease.Wright-Giemsa stain. E. Leukocytesconcentratedinthefeathered edge ofabloodfilm, fromadog.Thebloodfilmwasprepared usingglass slides. Wright-Giemsastain. F. Platelet aggregate inthefeathered edge of abloodfilmfroma cat. The bloodfilm was preparedusing glass slides. Wright-Giemsastain.18 SECTION I I BLOODLeukocyte EvaluationAs a quality-control measure, the number of leukocytespresent should beestimatedtoassure that thenumberpresent ontheslide is consistent with thetotal leukocyte count measured. If lOXocularsanda lOXobjective areused(lOOXmagnification), the total leukocytecount inblood (cells/ul.) maybeestimatedbydeterminingtheaverage number of leukocytes present per fieldandmultiplyingby100 to150. If a20X objective isused,thetotal leukocytecount maybeestimatedbymultiplying theaveragenumber ofleukocytes perfield by400to600.Thecorrectionfactor usedmayvary, dependingonthemicroscopeused. Consequently, theappropriatecorrectionfactors forthemi-croscope being used should bedeterminedby performing estimates ona num-ber of blood films inwhichthe total leukocyte counts havebeenaccuratelydetermined.Adifferential leukocytecount is done by identifying200consecutiveleukocytes using a40X or SOX objective. Becauseneutrophilstendtobe pulledtotheedges ina wedge-type (glass slide) bloodsmear andlymphocytes tendtoremaininthebodyofthesmear,'?differential countsaredonebyexaminingcells in a patternthatevaluates boththe edges and the center of thesmear (Fig.6D). After thecount is complete, the percentage of each leukocyte type presentiscalculatedandmultipliedbythe total leukocyte count to get the absolutenumberof each cell type present per microliter of blood.It is theabsolute number ofeach leukocyte typethat is important. Rela-tive values(percentages) canbemisleading whenthetotal leukocyte count isabnormal. Let us consider two dogs, one with7%lymphocytesand atotalleukocyte count of 40,000/p,L, andtheotherwith 70% lymphocytes anda totalleukocyte count of4,000/ILL. Thefirst case wouldbesaid tohave a"relative"lymphopenia andthe second case would be said tohave a "relative" lymphocy-tosis, but they both have the same normal absolute lymphocyte count(2,800/p,L).Thepresence of abnormalleukocyte morphology, such as toxic cytoplasminneutrophils or increased reactive lymphocytes (e.g., morethan5%oflym-phocytes arereactive), should be recordedonthehematology report form. Thefrequency of degenerative neutrophils is reported as few (5% to10%), moderate(11%to 30%) or many (>30%) and the severity of degenerative changeisrecorded as 1+to 4+(Table 1).29Erythrocyte MorphologyErythrocyte morphology shouldbe examined andrecorded as eithernormal orabnormal. Erythrocytes onbloodfilms from normalhorses, cats, and pigs oftenexhibit rouleauformation, anderythrocytes fromnormal horses andcats maycontainalowpercentageofsmall, spherical nuclear remnants called Howell-Jolly bodies. Rouleaux andthepresenceof Howell-Jollybodiesshouldbere-cordedonthehematology formwhenthey appearinbloodfilms fromspeciesin which these are notnormal findings.CHAPTER 1 IEXAMINAnON OFBLOOD SAMPLESTABLE1 Semiquantitative Evaluation of Degenerative Changes inNeutrophils...NEUTROPHILSWITHDEGENERATIVECHANGEFew 5-10(%)Moderate 11-30 (%)Many >30(%)SEVERllYOF DEGENERATIVECHANGE19Dohle bodiesBasophilia of cytoplasmFoamy cytoplasmDark blue-gray foamy cytoplasmToxic granulesIndistinct nuclear membraneKaryolysisModified fromWeils1984.291+1+2+3+3+4+4+TABLE2 Semiquantitative Evaluation of Erythrocyte MorpholOlY Based on Avel'8leNumber of Abnormal Cells perlOOOX Microscopic Monolayer Field."1+ 2+ 3+ 4+AnisocytosisDog 7-15 16-20 21-29 >30Cat 5-8 9-15 16-20 >20Cattle 10-20 21-30 31-40 >40Horse 1-3 4-6 7-10 >10PolychromasiaDog 2-7 8-14 15-29 >30Cat 1-2 3-8 9-15 >15Cattle 2-5 6-10 11-20 >20Horse rarely observedHypochromasias 1-10 11-50 51-200 >200Poikilocytosis" 3-10 11-50 51-200 >200Codocytes (Dogs) 3-5 6-15 16-30 >30Spherocytess 5-10 11-50 51-150 >150Echinocytes" 5-10 11-100 101-250 >250Other shapes' 1-2 3-8 9-20 >20 A monolayerfield isdefined asafield inwhich erythrocytes areclose togetherwithapproximately onehalf touchingeachother. In severely anemic animals, such monolayersmaynot be present. When erythrocytes are generally nottouching (e.g., tend to be separatedbythe distance of one cell diameter), then the number of erythrocyteswithmorphologic abnormalities are counted for two fields. From Weiss 1984.29b Thesame parametersareusedforall species. 'Parametersareusedforacanthocytes, schistocytes, keratocytes, ellipto-cytes, dacrocytes, drepanocytes, andstomatocytes inall species.20 SECTION II BLOODAdditional observations regarding erythrocyte morphology, such as thedegree of polychromasia (presence of polychromatophilic erythrocytes), anisocy-tosis(variationinsize), andpoikilocytosis (abnormal shape) shouldbemade.Polychromatophilicerythrocytesarereticulocytes that stainbluishreddue tothecombinedpresence of hemoglobin(red staining) andribosomes (blue stain-ing). Abnormalerythrocyte shapes should be classified as specifically as possible,because specific shapeabnormalitiescanhelpdeterminethenatureofadisor-der that may be present. Examples of abnormal erythrocyte morphology includeechinocytes, acanthocytes, schistocytes, keratocytes, dacryocytes, elliptocytes, ec-centrocytes, andspherocytes. Thenumberof abnormal cells should be reportedin a semiquantitative fashion, such as that shown in Table 2.29PlateletsPlatelet numbers should be estimated as low, normal, or increased. Bloodsmears frommost domestic animals normally averagebetween 10and 30plateletsper fieldwhenexaminedusing lOXocularsand the lOOXobjective(l,OOOX magnification). As few as 6 platelets per I,OOOX field may be presentinnormal horsebloodfilms." Platelet numbers may beestimatedbymultiplyingthe averagenumber per fieldby 15,000to 20,000to get the approximatenumberof platelets/ILL of blood.29,30 While special attentionwill begiven totheestimationof plateletnumbers inanimalswithhemostaticdiatheses,it isim-portant to routinely estimate the platelet numbers on blood films, becausemanyanimals withthrombocytopenia exhibit no evidenceor past historyofbleeding tendencies. If a thrombocytopenia is suspected, it should be confirmedwithaplatelet count. Dogs andcatshave largerplatelets than dohorsesandruminants. Platelets containmagenta-staininggranules, but these granules gen-erallystain poorlyin horses. The presenceof abnormal platelet morphology(large platelets or hypogranularplatelets) should be recorded on the hematologyform.Infectious Agentsand InclusionsBlood films are examined forthepresence of infectious agents andintracellularinclusionsusingthe lOOXobjective. Infectiousagents andinclusions that maybeseeninbloodfilms includeHowell-Jollybodies, Heinzbodies(unstained),basophilic stippling, canine distemper inclusions, siderotic inclusions. Dohlebodies,Babesiaspp., Cytauxzoonfelis, Haemobartonellaspp., Ehrlichiaspp., He-patozoonspp., Trypanosomaspp., Theileriaspp., andBorreliaspp. Theappear-ance of these agents andinclusions is discussed in subsequent sections.CHAPTER ")ERYTHROCYTES ERYTHROCYTE MORPHOWGYErythrocytes fromallmammalsareanucleated, andmost areintheshapeofbiconcave discs called discocytes,"Thebiconcave shaperesults inthecentralpallor of erythrocytes observed instainedbloodfilms(Fig. 7A). Ofcommondomestic animals, biconcavity andcentral pallor are mostpronouncedindogs,which also have the largest erythrocytes (Fig. 7B). Otherspecies donot consist-ently exhibit central pallor inerythrocytes onstained blood films. The apparentbenefit ofthebiconcave shape is that it giveserythrocytes high surface-area tovolumeratiosandallowsfordeformationsthat must takeplace as theycircu-late. Erythrocytes fromgoatsgenerally havea flat surfacewithlittlesurfacedepression; avariety of irregularly shapederythrocytes(poikilocytes) maybepresent inclinicallynormal goats(Fig. 7C). Erythrocytes fromanimalsintheCamelidae family (camels, llamas, vicunas, andalpacas) arethin, elliptical cellstermed elliptocytes orovalocytes (Fig. 7D), and they are not biconcave in shape.Erythrocytes frombirds, reptiles, and amphibiansarealsoelliptical inshape,but they contain nuclei and are larger thanmammalian erythrocytes.RouleauxErythrocytes onbloodfilms fromhealthyhorses, cats, andpigsoftenexhibitrouleau(adhesion of erythrocytes together like a stack of coins) formation(Fig.7A). Increasedconcentrations of fibrinogenand globulinproteins potentiaterouleau formationin association with inflammatory conditions. Rouleau forma-tion canalsooccur inassociation withsomelymphoproliferative disordersinwhichoneor moreimmunoglobulinsaresecreted inhighamounts (Fig. 7E).Prominent rouleau formationinspecies other thanhorses, cats, orpigs shouldbe noted as an abnormal finding.I IAgglutinationAggregation, orclumping, of erythrocytes together inclusters (not inchains asinrouleaux) istermedagglutination(Fig. 7F). Agglutination iscaused bythe2122 SECTIONI/ BLOODFIGURE 7. Erythrocyte morphology indomestic animal species. A. Blood film from a horse.Most erythrocytes areadheredtogetherlike stacks ofcoins(rouleaux), anormal findinginthisspecies.Individual nonadherent erythrocytes exhibit central pallor asa result of their biconcave shape.Wright-Giemsa stain. B. Blood from a dog with acute blood-loss anemia andnormal erythrocyte morphology.Erythrocytes exhibit prominent central pallor. Twomature neutrophils and twoplatelets(bottomrightcorner)are also present. Wright-Giemsa stain. C. Poikiiocytesinblood froma' normal goat. Wright-(Continued)CHAPTER2 / ERYTHROCYTES 23occurrenceofimmunoglobulins boundtoerythrocytesurfaces. Because of theirpentavalent nature, IgMimmunoglobulinshavethegreatest propensity topro-duce agglutination.' High-doseunfractionatedheparintreatment inhorses alsocauses erythrocyteagglutination by anundefined mechanism.v-"PolychromasiaThe presence of bluish-rederythrocytes instained blood filmsiscalledpoly-chromasia(Fig. 8A). Polychromatophilic erythrocytesarereticulocytes that stainbluish red due to the combinedpresence ofhemoglobin (red staining) andindividual ribosomes andpolyribosomes (bluestaining). Low numbersofpoly-chromatophilicerythrocytes are usuallyseenin blood fromnormal dogs andpigsbecause up to 1.5%reticulocytes maybe present indogsand up to 1%reticulocytes maybe present in pigs evenwhen the HCTis normal.'! Slightpolychromasiamay bepresent innormalcats, but manynormalcats exhibit nopolychromasiainstainedbloodfilms. Polychromasiaisabsent instainedbloodfilms fromnormal cattle,sheep, goats, andhorsesbecausereticulocytesarenotnormally present inbloodin thesespecies."The most useful approachinthe classification ofanemia istodeterminewhether ornot evidence of a bonemarrow responsetotheanemiais present inblood. For all species except the horse, this involves determining whetherabsolute reticulocyte numbers are increased in blood. Horses rarely releasereticulocytes fromthe bone marroweven when an increased production oferythrocytes occurs. When anabsolute reticulocytosis ispresent, the animal issaidto have a regenerative anemia. The presence of a regenerative responsesuggests that theanemiaresultsfromeitherincreasederythrocytedestruction orhemorrhage. Anonregenerative anemia generallyindicates that the anemia istheresult ofdecreasederythrocyteproduction(Fig. 8B); however, about 3to4days arerequiredfor increasedreticulocyteproductionandrelease bythebonemarrowin response to an acute anemia. Consequently, the anemia appearsnonregenerative shortlyafter hemolysis orhemorrhage has occurred(Fig. 7B).IIncreasedpolychromasia is usually present inregenerative anemiasbecausemanyreticulocytes stainbluish-redwith routinebloodstains (Fig. 8A). Whenthedegreeofanemia is severe, basophilicmacroreticulocytesor so-calledstressreticulocytes maybereleased intotheblood(Fig. 8C). It is proposedthat oneless mitotic division occurs during production, and immature reticulocytes,twicethe normal size, are released. There isa direct correlationbetween thepercentage of polychromatophilic erythrocytes and the percentage of reticulo-Giemsa stain. D. Elliptocytes inbloodfromanormal llama. Wright-Giemsa stain. E. Rouleauformationinbloodfromadogwithmultiplemyeloma andamonoclonal hyperglobulinemia. The cyto-plasm of thetwoneutrophilspresentis pale comparedtothebackgroundthat stains blue because of theincreased proteinpresent. Wright-Giemsa stain. F. Erythrocyte agglutination and spherocyte formationin blood froma dog with von Willebrand's diseaseafter transfusion. Alargebasophilicerythrocyte(macroreticulocyteor stress reticulocyte) is present in theupperleft corner and an echinocyte is present inthe lower left corner. Wright-Giemsastain.24 SECTION I / BLOODFIGURE8. Erythrocyte morphology inanemic dogs andcats. A. Increased polychromasiaandanisocytosisinbloodfromadog with ahemolytic anemia caused by Haemobartonellacanis,althoughno organisms are present inthis field. Four large polychromatophilic erythrocytes (reticulocytes)are presentinthecentral area. Anucleatederythrocyte(metarubricyte)ispresent intheupper left. Wright-Giemsastain. B. Bloodfroma dog with a nonregenerativeaplasticanemia secondaryto trimethoprim-(Continued)CHAPTER 2 / ERYTHROCYTES 25cytes in dogs (and presumably in pigs) and between the percentage of polychro-matophilic erythrocytesand the percentageof aggregate reticulocytes in cats(Figs. 8D_8F).22.33Catswithmildanemiamaynot release aggregatereticulo-cytes from the marrow but will release punctatereticulocytes(Fig. 9A).1Becausepunctate reticulocytes donot containsufficient numbers ofribosomeswithinthemtoimpart abluishcolor tothecytoplasm, mildregenerative anemiaincats may lack polychromasia in stained blood films (Fig. 9B).AnisocytosisVariationinerythrocyte diametersinstainedbloodfilms is called anisocytosis(Fig. 9B). It is greater innormal cattle thaninothernormal domestic animals(Fig. 9C).11 Anisocytosis is increasedwhendifferent populations of cells arepresent. It mayoccur whensubstantialnumbers ofsmaller than normal cellsareproduced, asoccurs withirondeficiency, orwhensubstantialnumbers oflarger than normal cells areproduced asoccurswhenincreased numbers ofreticulocytes are produced. Consequently, increased anisocytosis is usuallypresent inregenerative anemia (Figs. BA, BC, 9B), but it maybepresent insomenonregenerative anemiaresulting fromdyserythropoiesis.i-"Anisocytosis,without polychromasia, may be seen inhorses withintensely regenerative ane-mia(Fig. 9D).HypochromasiaThe presenceof erythrocyteswith decreasedhemoglobin concentration andincreasedcentral palloriscalled hypochromasia(Figs. IOA-lOE). Not onlyisthecenter of the cell paler thannormal, but thediameter of the area of centralpallor isincreasedrelative tothered-stainingperipheryofthecell. Truehy-pochromicerythrocytes must bedifferentiated fromtorocytes, which have col-orless punched-out centers but wider dense red-staining peripheries (Fig.l IA);'!Torocytes are generallyartifacts. Increased hypochromasia is observed iniron-deficiency anemia.Erythrocytes fromdogs andruminantswithiron-deficiency anemiaoftenappearhypochromic onstained bloodsmears (Figs. IOA-lOE). Hypochromasiainirondeficiency results from bothdecreased hemoglobin concentrationwithincells andfromthefact that thecells are thin(leptocytes). Because these micro-sulfadiazine therapy. Erythrocytemorphologyis normal except for several erythrocyteswith scallopedborders (echinocytes). Wright-Giemsa stain. C. Two exceptionally large basophilic erythrocytes(macroreticulocytesorstress reticulocytes) are present inbloodfroma dog with immune-mediatedhemo-lytic anemia. Wright-Giemsa stain. D. Agglutination of polychromatophilic erythrocytes and a metaru-bricyte inbloodfromacatwithaCoombs'-positive hemolytic anemia. Areticulocytestainrevealed thatthese agglutinated cells areaggregatereticulocytes(Fig. 8E). Wright-Giemsa stain. E. Agglutination ofaggregatereticulocytesinblood fromthe same cat with a Coombs'-positive hemolytic anemia as shown inFigure 8D. New methylene blue reticulocyte stain. F. Agglutination of aggregatereticulocytesin bloodfrom a cat with a Coombs'-positive hemolytic anemia. New methylene blue wet mount preparation.26 SECTIONI / BLOODFIGURE9. Anisocytosis (variation in erythrocyte size) in blood. A. Increasedpunctatereticulocytes (83% uncorrected)in blood froma FeLV-positive cat with a macrocytic normochromicanemia(hematocrit =23%, MCV =70 fl, MCHC =33 g/dL). Theuncorrectedaggregatereticulocyte count was0.2%. New methylene bluereticulocyte stain. B. Increased anisocytosis in a blood film made fromthesamecat bloodsampleasshowninFigure 9A. Polychromasia isnot apparent eventhough most ofthebloodcells present arepunctate reticulocytes, because thereisinsufficient RNA present toimpart abluecolor to thecytoplasm of these cells. Wright-Giemsa stain. C. Anisocytosis in blood from a nonanemiccow. Wright-Glemsa stain. D. Increased anisocytosis in blood froma horse with a regenerative anemiaresulting frominternal hemorrhage. Horses almost never releasereticulocytesin response to anemia;therefore, no polychromasia is present. Wright-Giemsa stain.cytic leptocytes have increased diametertovolumeratios, they may not appearas small cellswhen viewedin stained blood films (Fig. 10B).35 Microcyticerythrocytes fromiron-deficient llamasexhibit irregular or eccentricareasofhypochromasia within the cells (Fig. 10E).36CHAPTER2 I ERYTHROCYTES 27PoikilocytosisErythrocytescanassumeawidevarietyof shapes. Poikilocytosis isageneraltermused todescribe the presence of abnormally shaped erythrocytes. Althoughspecificterminologyis used forcertain abnormal shapes, itis less importanttoquantifyeachtypeof shapechangethan it istodetermine the causeof theshapechange." Poikilocytosis maybe present in clinicallynormal goatsandyoungcattle(Figs. 7C, IOF).37,38Insomeinstances,theseshapes appear toberelatedtothehemoglobintypes present, but anabnormalityinprotein4.2inthe membranehas been suggested as a causative factor in calves."Poikilocytosisformsinvariousdisordersassociated witherythrocyte frag-mentation." For unknown reasons,severe iron-deficiency anemiaindogs andruminants mayexhibit pronouncedpoikilocytosis (Figs. IOC, IOD).3s Poikilo-cytescanformwhenoxidant injuryresultsin Heinzbodyformation and/ormembraneinjury. One or moreblunt erythrocyte surface projections may formasthe membrane adheresto Heinzbodiesboundto itsinternal surface.w Avarietyof abnormal erythrocyteshapeshavebeenreported indogsand catswith doxorubicintoxicitt142andin dogs with dyserythropoiesis.?Echinocytes (Crenated Erythrocytes)Echinocytes are spiculated erythrocytes in which the spicules are relativelyevenlyspacedand of similar size.44Spiculesmaybe sharp or blunt. Whenobservedinstainedbloodfilms, echinocytosis isusually anartifact that resultsfromexcess EDTA, improper smearpreparation, or prolongedsamplestoragebefore blood filmpreparation. The appearance of the echinocytescan varydepending on the thickness of the blood film(Figs. llB, llC). They arecommon in normal pig blood smears (Fig. llD), forming in vitro," Themorphologyof echinocytes variesfromslightly spiculatedechinodiscocytes tohighlyspiculated spheroechinocytes, whichhave been calledburr cells(Fig.llE)YThemost advancedechinocytes arethosethat havelost most of theirspicules andhave nearly become spherocytes (Fig. llF). Echinocytes form whenthe surfacearea of the outer lipidmonolayer increasesrelativeto the innermonolayer. Echinocytic transformationoccursinvitro inthepresenceoffattyacids, lysophospholipids, andamphipathicdrugs that distributepreferentially intheouter half of thelipid bilayer. Echinocytes also formwhenerythrocytes aredehydrated, pH is increased, erythrocyte adenosine triphosphate (ATP) isdepleted (e.g., hypophosphatemia), and intracellular calciumis increased."Transient echinocytosisoccurs in dogs followingrattlesnakeand coral snakeenvenomation(Figs. lIE, llF),4S-47presumably secondarytotheaction ofphos-pholipasespresent invenom. Depending onthetimecourse anddose of venomreceived, either echinocytosisorspherocytosismay beobservedafter these snake-bites. Echinocytesmay occur inuremic animals, immediatelyafter transfusion ofstored blood, orin some pyruvate kinase-deficient dogs (Fig. llG).4048 They havebeen seen with increased frequency indogs with glomerulonephritis and neoplasia28 SECTIONJ I BLOODFIGURE10. Hypochromasiaand/or poikilocytosis inblood. A. Hypochromic erythrocytes inbloodfroma dogwithiron deficiencysecondary tochronicbloodloss. resulting fromapersistentfleainfestation. Not onlyis thecenterofeach cell paler thannormal, but thediameterofthearea ofcentralpallor is increasedrelativeto the red-stainingperipheryof the cell. Apolychromatophilicerythrocyte(reticulocyte) is presentintheupper left corner. Wright-Giemsa stain. B. Blood fromadogwithamicrocytic hypochromic(MCV= 32fl, MCHC= 23gldL)iron-deficiency anemiawas mixed withanequal volumeof bloodfroma normal dog (MCV =70fl, MCHC=34g1dL) prior to blood filmpreparation. Becausethe hypochromic cells are leptocytes, they have diameters similar tonormalcells eventhough they are microcytic cells. Wright-Giemsa stain. C. Marked poikilocytosis and hypochromasia inbloodfroma6-week-old lamb withmicrocytic hypochromic iron-deficiency anemia. Wright-Giemsa stain.(Continued)CHAPTER2 I ERYTHROCYTES 29(lymphoma, hemangiosarcoma, mast cell tumor, andcarcinoma).44.49 Echinoeytosisoccurs in horses in which total body depletion of cations has occurred (enduranceexercise, furosemide treatment, and systemic disease).">'AcanthocytesErythrocytes withirregularlyspaced, variably sized spicules arecalled acantho-cytes or spur cells (Figs. IIH, IIl).52Acanthocytes formwhen erythrocytemembranes contain excess cholesterol compared tophospholipids. Alterationsin erythrocyte membrane lipids can result fromincreased blood cholesterolcontent or duetothepresenceofabnormalplasmalipoproteincomposition."Acanthocytes have been recognized in animals with liver disease, possibly due toalterations inplasma lipidcomposition, whichcanaltererythrocytelipidcom-position.40.54.55They have alsobeenreportedindogswithdisordersthat resultinerythrocytefragmentation, such as hemangiosarcoma(Fig. Ill), disseminatedintravascular coagulation, andglomerulonephritis. 54Marked acanthocytosisis reported to occur inyoung goats" and someyoungcattle(Fig. lOF).38.56Acanthocytosis of younggoats occursas aresult ofthepresence of hemoglobin C at this early stage of development.57KeratocytesErythrocytes containingwhat appear to be one or more intact or ruptured"vesicles" arecalled keratocytes(Figs. I IJ-11M). Thesenonstainingareasap-pear to be circular areasof apposed and sealedmembrane rather than truevesicles. Theremoval or ruptureof thisarearesults intheformationof oneortwoprojections. Keratocytes have beenrecognized invariousdisordersinclud-ing iron-deficiency anemia," liver disorders," doxorubicintoxicity incats;"andmyelodysplastic syndrome" andinvarious disordersindogshavingconcomi-tant echinocytosis or acanthocytosis.44.54Keratocyte formationis potentiatedbythestorage of cat blood collected with EDTA.StomatocytesCup-shaped erythrocytes that have oval or elongated areas of central pallorwhenviewedinstainedbloodfilmsarecalledstomatocytes (Fig. lIN). Theymost often occur as artifacts in thick blood filmpreparations. Stomatocytesformwhenerythrocytewater content isincreasedasoccursinhereditarysto-matocytosisindogs(Fig.110).59-61Stomatocytes also formwhenamphipathicdrugs that distribute preferentiallyin the inner half of the lipid bilayer arepresent."D. Marked poikilocytosis(primarily dacryocytes)and hypochromasia in blood from a goat with microcytichypochromic iron-deficiency anemia secondary tochronicbloodloss, resulting fromHaemonchusgastroin-testinal parasites. Wright-Giemsastain. E. Microcytic erythrocytesinblood froman iron-deficientllama, exhibiting irregular oreccentric areas of hypochromasia within the cells. Wright-Giemsa stain. F.Poikilocytosis (acanthocytes and echinocytes) inblood from a nonanemic young calf. Wright-Giemsa stain.FIGURE 11. Seelegend on opposite page.30CHAPTER 2 I ERYTHROCYTES 31SpherocytesSpherical erythrocytes that result fromcell swellingand/or loss ofcell mem-brane are referredto as spherocytes. Spherocytes lackcentral pallor andhavesmaller diameters than normal on stained blood films (Fig. lIP). Sphericalerythrocyteswithslight indentationsononesidemaybe calledstomatosphero-cytes (Fig. llQ). Spherocytes occur most frequently inassociation with im-mune-mediatedhemolyticanemiaindogs.4o,63Otherpotentialcauses of sphero-FIGURE11. Erythrocyte shape abnormalities. A. Twotorocyteswithcolorless punched-outcenters andwide dense red-staining peripheries inblood from a dog. Wright-Giemsa stain. B. Echino-cytes withregularly spacedspicules of similarlength, inbloodfromadogwithmalignant histiocytosis.Wright-Giemsa stain. C. Echinocytes, inathinner areaof thesame bloodfilm as showninFigurelIB,appearas erythrocytes withscalloped borders; consequently, theoldterm"crenation"fromLatin meaning"notched" is used. Wright-Giemsa stain. D. Echinocytesinbloodfromanormal pig. Wright-Giemsastain. E. Highly spiculated echinocytes (burrcells) in blood from a dog followingan Eastern diamond-back rattlesnakebite. Wright-Giemsa stain. F. Spheroechinocytes and a lysederythrocyte"ghost"(bottomright) in blood froma dog following a coral snake bite. Wright-Giemsastain. G. Anechinocyte (left) inbloodfromapyruvate kinase-deficientCairnterrierdog. Wright-Giemsa stain. H.Two acanthocytes (above) with irregularlyspaced, variablysizedspiculesin blood froma dog withneoplastic lymphoidinfiltrates inthe liver. Wright-Giemsa stain. I. Three acanthocytes with irregularlyspaced, variably sized spicules inbloodfroma dog with hemangiosarcoma. Wright-Giemsa stain. J. Akeratocyte with whatappears tobe a "vesicle"in thecytoplasm of anerythrocyte inbloodfroma cat withhepaticlipidosis. Wright-Giemsastain. K. Akeratocytewithwhat appearstobe a"vesicle" inthecytoplasm of anerythrocyteinbloodfromacat withhepaticlipidosis. Wright-Giemsastain. L. Akeratocyte, exhibiting what appearstobearuptured "vesicle" inbloodfromacat withhepaticlipidosis.Wright-Giemsa stain. M. Two keratocytes, exhibiting whatappear toberuptured "vesicles"inbloodfrom a cat with hepatic lipidosis. Wright-Giemsa stain. N. Stomatocytes with elongated areas of centralpallor inbloodfromacat withhemolyticanemia. Thestomatocytes werenot uniformlypresent inthebloodfilm andwere considered tobeartifacts. Wright-Giemsa stain. O. Stomatocytes with elongatedareas of central pallor in blood froman asymptomaticPomeranian dogwith persistent stomatocytosisassociated withmacrocytic hypochromic erythrocytes. Like dogs with hereditary stomatocytosis, erythrocyteswere osmoticallyfragile and had low-reduced glutathione concentration. Wright-Giemsa stain. P.Three spherocytes (bottom) anda large polychromatophilic erythrocyte orreticulocyte (top) inblood fromadog withimmune-mediatedhemolytic anemia. Wright-Giemsa stain. Q. A large polychromatophilicerythrocyteor reticulocyte(top left) and two stomatospherocytesin blood froma dogwith immune-mediated hemolytic anemia. The two stomatospherocytes are not prefect spheres. Each has a slight indenta-tiononone side. Wright-Giemsa stain. R. A spherocyte (top)anda discocyte (bottom) in bloodfromafoal withimmune-mediatedneonatalisoerythrolysis. Wright-Giemsa stain. S. A fragmented erythro-cyte (schistocyte) and two discocytes in blood froma dogwith disseminatedintravascular hemolysis.Wright-Giemsa stain. T. A schistocyte (left), discocyte (top), andechinocyte (bottom) in blood fromadog with disseminated intravascular hemolysis. Wright-Giemsa stain. U. A schistocyte (top right), largeplatelet (bottomright), and polychromatophilicerythrocyte (top left) in blood froma splenectomizedpyruvatekinase-deficient Cairn terrier dog. Wright-Giemsastain. V. Twothin flat hypochromic-appearing erythrocytes (leptocytes), with increased membrane-to-volumeratios, are presentin bloodfromadog with severe iron-deficiency anemia. The bottomleptocyte is folded. Wright-Giemsa stain. W. Twotriconcave knizocytes (center)are present in a dog with a portosystemic shunt. Wright-Giemsa stain. X.Leptocytes, includingtwoknizocytes (topandbottomcenter), arepresent inbloodofadogwithiron-deficiency anemia. Wright-Giemsa stain.32 SECTIONI I BLOODcyte formation include coral snake and rattlesnake envenomation.w" beestings."zinc toxicity." erythrocyte parasites, transfusionof storedblood, andafamilial dyserythropoiesis." Sinceerythrocytesfromother common domesticanimals exhibit less central pallor thanthose of dogs, it is difficult tobe certainwhen spherocytes are presentinthese noncaninespecies (Fig. 11R). Spherocyteshavebeenreported incattlewithanaplasmosis"andinJapanese Black cattlewith inheritederythrocyte band3 deficiency."SchistocytesErythrocyte fragmentation may occur when erythrocytes. are forced to flowthroughaltered vascular channels orexposed toturbulentbloodflow. Erythro-cytefragments withpointed extremitiesare calledschistocytes or schizocytes(Figs. lIS-II U), andthey are smaller thannormal discocytes. Schistocytes maybeseenindogswithmicroangiopathichemolyticanemiaassociated withdis-seminatedintravascular coagulation(DIC).40Mechanical fragmentationoccursas thecells pass throughthefibrinmeshwork of a microthrombus. Schistocytesarenot typically seen incats or horses withDIC, possibly because theerythro-cytes of thesespecies aresmaller andless likely tobesplit by fibrinstrands inthecirculation. Schistocyteshavealso beenseeninsevere iron-deficiency ane-mia,"myelofibrosis.w" liver disease,"heart failure, glomerulonephritis, hemo-phagocytic histiocytic disorders,hemangiosarcomaindogs, andcongenitalandacquireddyserythropoiesis indogS.40.43,54.58.70,71Markedpoikilocytosis withschi-zocytes andacanthocytes has been recognized in pyruvate kinase-deficient dogsafter splenectomy(Fig. 11U).72.73It isassumedthat thespleen hadpreviouslyremoved these fragmented erythrocytes.LeptocytesThesecells arethin, oftenhypochromic-appearingerythrocytes withincreasedmembrane-to-volume ratios. Someleptocytesappear folded(Fig. l IV), someappearas triconcave knizocytes that give the impression thatthe erythrocyte hasacentral bar of hemoglobin(Figs. llW, llX), andothersappear as codocytes(Figs. 12A-12C). Codocytes(target cells)arebell-shapedcells that exhibit acentral densityor "bull's-eye" instainedbloodfilms. Small numbers ofcodo-cytes are often seen in normal dog blood and bothcodocytes andknizocytes areincreased inregenerative anemiaindogs. Codocytes areespecially increased indogs with a congenital dyserythropoiesis." Leptocytes maybe seenin iron-deficiency anemia(Figs. llV, 12B)35 and rarely in hepatic insufficiency (Fig. 12C)that results ina balanced accumulation of membranephospholipids andcholes-terol." Polychromatophilicerythrocytes can sometimes appear as leptocytes.Eccentrocytes (Hemighosts)Anerythrocyteinwhich thehemoglobinis localized topart of thecell, leavingahemoglobin-poorarea visible intheremainingpart of thecell, istermedanCHAPTER 2 / ERYTHROCYTES 33eccentrocyte (Figs. 12D,. 12E). Theyare formed bythe adhesion of opposingareasof the cytoplasmicfaceofthe erythrocyte membrane. Eccentrocytesthathavebecome spherical withonlyasmall tagof cytoplasmremainingmaybecalled pyknocytes. Eccentrocyteshavebeenseeninanimalsingestingor receiv-ing oxidants including onions, acetaminophen, and vitamin Kin dogs;" redmapleleaves inhorses;"andintravenoushydrogen peroxideasa"homerem-edy,"inacow:"Eccentrocyteshavealsobeenseeninahorsewithglucose-6-phosphatedehydrogenase (G6PD) deficiency"andinahorsewithglutathionereductasedeficiency secondarytoerythrocyteflavin adeninedinucleotide(FAD)deficiency."Elliptocytes (Ovalocytes)ErythrocytesfromnonmammalsandanimalsintheCamelidaefamily normallyare elliptical or oval in shape (Fig. 7D). Theyare generallyflat rather thanbiconcave. Abnormal elliptocyteshavebeenrecognizedincatswithbonemar-rowabnormalities (myeloproliferativedisorders andacutelymphoblasticIeuke-mia)." hepatic lipidosis," portosystemic shunts," and doxorubicin toxicity'?andindogs withmyelofibrosis.w"myelodysplastic syndrome, 58 andglomerulo-nephritis, in whichtheelliptocytes may bespiculated(Figs. 12F, 12G).49 Heredi-tary elliptocytosis has been reported in a dog with a membrane protein 4.1deficiency."DacryocytesThese erythrocytes are teardrop-shaped with singleelongated or pointed ex-tremities (Figs. 12H, 121). Dacryocytosisisacommonfeature ofmyelofibrosisin people, but dacryocytes are not as commonly recognized in dogs withmyelofibrosis.69,7o,83Dacryocytes havealsobeenseeninbloodofdogsandcatswith myeloproliferative disorders," dogs with glomerulonephritis, and a dogwithhypersplenism."Dacryocytes arecommonerythrocyteshapeabnormalitiesiniron-deficient ruminants, including llamas(Figs. 12J, 12K).36Drepanocytes (Sickle Cells)Fusiform or spindle-shaped erythrocytes are often observed in blood fromnormal deer (Fig. 12L)and in bloodfrompeople with sicklecell anemia."Thesedrepanocytesdevelopsecondary tohemoglobinpolymerization, anddre-panocyte shape indeer depends on the hemoglobintypespresent. It isaninvitrophenomenonthat occurswhenoxygen tensionis highandpHis between7.6 and7.8.Polymerization of hemoglobinintubular filamentsoccursinsomenormaladult Angora goats57,86and somebreeds of Britishsheep."Theresultant fusi-formor spindle-shapederythrocytes resembledrepanocytes indeer; theyhavebeen calledacuminocytes by some authors (Fig. 12M).1l The proportion offusiformcells inAngoragoatsvariesdependingontheindividual goatandonFIGURE 12. See legend on opposite page.34CHAPTER 2 I ERYTHROCYTES 35in vitroalterationsintemperature, pH, andoxygenation. Thenumberof thesecells decreases duringanemia,probably because of thesynthesis of hemoglobinC.86Crystalized HemoglobinThe presence of largehemoglobin crystalswithin erythrocytesis commonlyrecognized inbloodfilms fromcats(Fig. 12N)88-90and llamas(Fig. 120)91.92andrarely recognized inbloodfilms fromdogs(Fig. 12P), primarily pupslessthan3 monthsof age."Nohemoglobinabnormalities have been recognized byhemoglobinelectrophoresisandnopathologicsignificancehasbeenattributedto finding hemoglobincrystals in bloodfilms fromdomestic animals.FIGURE 12. Erythrocyte shape abnormalities and the appearance of nucleated er-ythroidprecursor cells. A. Three codocytes inbloodfromaCairnterrierdog with aregenerativeanemia andhepatic hemochromatosis secondary topyruvate kinase-deficiency. These erythrocytes exhibit acentral density or "bull's-eye" andareoftenreferred toas targetcells. Wright-Giemsa stain. B. Twocodocytes (topandbottomcenter)anda schistocyte (bottomleft) inbloodfroma dog withsevere iron-deficiency anemia. Wright-Giemsa stain. C. Codocytes in blood from a dog with liver disease. Wright-Giemsa stain. D. Three eccentrocytesanda discocyte(left) inbloodfromadog with oxidant injuryinduced by theadministrationof acetaminophen. The cell attopcenter appears spherical with a small tagof cytoplasm andmay bereferred toas apyknocyte. Wright-Giemsa stain. E. Three eccentrocytes inblood froma horse with inherited erythrocyteglucose-6-phosphatedehydrogenase deficiency. Wright-Giemsa stain. F. Threeelliptocytes andadiscocyteinbloodfromadiabetic catwithmildanemia.Radiographs revealed diffuse interstitial lung disease of unknown etiology. Wright-Giemsa stain. G. Anechinoelliptocytein bloodfroma dog with glomerulonephritis. Wright-Giemsa stain. H. A dacryocyte(bottom) andtwodiscocytes inbloodfromacat. Wright-Giemsa stain. I. Adacryocyte (left) andelliptocyte(right) in blood from a dog with glomerulonephritis. Wright-Giemsa stain. J. Hypochromicdacryocytesin bloodfroma goat with severe iron-deficiency anemia. K. Hypochromic dacryocytesinblood from a llama with severe iron-deficiencyanemia. The presence of thenormal llama elliptocyte (aboveright)is theresult of a bloodtransfusion. Wright-Giemsa stain. L. Elongated drepanocyte (sickle cell)in blood froma white-taileddeer. Wright-Giemsastain. M. Erythrocytes containinghemoglobininclusions in bloodfroma mixed-breed goat. Some erythrocytes appeared as rectangles but most appearedmore fusiform andmay represent polymerization of hemoglobin in tubularfilaments as occurs in drepano-cytes. Wright-Giemsa stain. N. Crystalizedhemoglobin inanerythrocyte fromacat. Wright-Giemsastain. o. Crystalized hemoglobinin two erythrocytesfroma llama. Wright-Giemsastain. P.Crystalizedhemoglobininanerythrocyte fromadog. Wright-Giemsa stain. Q. Red-staining intacterythrocytes (echinocyte inthe center) andpale-staining erythrocyte ghosts in blood from a horse in whichintravascular hemolysiswas producedbytheintravenousandintraperitonealadministrationof hypotonicfluid believed isotonic at thetime of administration. Wright-Giemsa stain. R. Erythrocyte ghosts, eachcontaining a single red-staining Heinz body, inerythrocytes from a cat with intravascular hemolysis causedbyacetaminophenadministration. Wright-Giemsa stain. S. A lysed erythrocyte (redsmudge at top)anddiscocyte inbloodfromadogwithlipemia. Thelysis occurredduringsmear preparation. Wright-Giemsa stain. T. Orthochromaticmetarubricyte inbloodfromadogwitharegenerative hemolyticanemia. Wright-Giemsastain. U. Polychromatophilicmetarubricyte in blood froma dogwitharegenerative hemolytic anemia. Wright-Giemsa stain. V. Twopolychromatophilic rubricytes, oneofwhich hasalobulatednucleus, inbloodfromacat witherythroleukemia andanonregenerative anemia.Wright-Giemsastain. W. Exceptionally large basophilic rubricyte in bloodfroma cat with myelodys-plastic syndrome andanonregenerative anemia. Wright-Giemsa stain. X. Rubriblast in blood fromacat with erythroleukemia and a nonregenerative anemia. Wright-Giemsa stain.36 SECTIONI I BLOODLysed ErythrocytesThe presence of erythrocyte "ghosts" in peripheralbloodfilms indicates that thecells lysed prior tobloodfilmpreparation(Fig. 12Q). Erythrocytemembranesarerapidlycleared fromthecirculationfollowing intravascularhemolysis; con-sequently, thepresence of erythrocyte ghosts indicates either recent intravascularhemolysis orin vitro hemolysis inthebloodtubeafter collection. If thehemol-ysis iscausedbyan oxidant, Heinzbodies maybevisible withinerythrocyteghosts (Fig. 12R). Whenerythrocyteslyse duringbloodfilm preparation, theyappear asredsmudges (Fig. 125). Thesesmudgederythrocytesarecommonlyseen in lipemic samples.Nucleated ErythrocytesMetarubricytes(Figs. 12T, 12U) andrubricytes are seldom present inthebloodofnormal adult mammals, althoughlow numbersmayoccur insomenormaldogsandcats.94 Thesenucleatederythrocytesareoftenseen inblood(normo-blastemia)inassociation with regenerative anemia; however, theirpresence doesnot necessarily indicatearegenerative responseis present."Nucleatederythro-cytes are rarely seen in horses with regenerative anemia.Nucleated erythrocytes maybe seenin animals withleadpoisoning inwhichthereisminimal or noanemia(Fig. 13M)96.97andinnonanemiccondi-tions inwhichbone marrowisdamaged, suchas septicemia, endotoxicshock,and drugadministrations.tv"Low numbersofnucleatederythrocytesareseenina wide variety of conditions in dogs includingcardiovascular disease, trauma,hyperadrenocorticism, and various inflammatory conditions."Whenfrequent nucleatederythrocyteprecursorsarepresent inthebloodof ananimal withnonregenerativeanemia(Figs. 12V-12X), conditions includ-ingmyelodysplasia, hematopoieticneoplasia.l':"infiltrative marrowdisease,94.99impaired splenic function," and inherited dyserythropoietic disordersv-??shouldbe considered. The presence of rubriblastsin bloodfromananimalwithnonregenerative anemia stronglysuggests that amyeloproliferativedisorder ispresent (Fig. 12X). Erythrocyte nuclei may be lobulated or fragmented inanimalswithmyeloproliferative disorders (Fig. 12V)IOI-I03or following vincris-tine therapy, if nucleatederythrocytesarepresent inblood (Figs. 13A, 13B).Nucleatederythroid precursors earlierthan metarubricytesarecapable ofdivi-sion; consequently, mitotic nucleatederythrocytesmaybeseeninblood (Fig.13e) . INCLUSIONS OF ERYTHROCYTESHowell-Jolly BodiesThese small, spherical nuclear remnants formin the bone marrowand areremoved bythe "pitting" action of the spleen. Howell-Jolly Bodiesmay bepresent inlownumbersinerythrocytesofnormal horsesandcats(Fig. 13D).CHAPTER 2 / ERYTHROCYTES 37They are often present in association with regenerative anemia or followingsplenectomyinother species. Theymayalsobeincreasedinanimalsreceivingglucocorticoidtherapy(Fig. 13E),uNuclear fragmentationandmultiple Howell-Jollybodies maybe present in animalstreatedwithvincristine, if regenerativeanemiais present(Figs. 13A, 13B).Heinz BodiesThese inclusions are largeaggregatesof oxidized, precipitatedhemoglobinthatare attachedto the internal surfaces of erythrocytemembranes. In contrast toHowell-Jolly bodies, whichstaindarkblue, Heinz bodies stainredtopalepinkwith Romanowsky-typestains (Figs. 13F, 13G). They bindtotheinnersurfaceof erythrocytemembranesandmaybe recognizedassmall surface projectionswhen the membrane binds around much of an inclusion (Fig. 13F). Whenintravascular hemolysis occurs, they may be visible as red inclusions withinerythrocyteghosts (Figs. 12R, 13F). Heinzbodiesappearlight bluewithreticu-locytestains (Fig. 13H). Theycanalsobevisualizedas dark refractileinclusionsin newmethylene blue "wet mount" preparations (Fig. 131). In contrast toother domesticanimal species, normal cats may have up to 5%Heinzbodieswithin their erythrocytes.P' Not only is cat hemoglobin more susceptible todenaturationbyendogenousoxidants,"but thecat spleenis less efficient intheremoval (pitting) of Heinzbodies fromerythrocytes thanare spleens of otherspecies. lOS Increasednumbersof Heinzbodiesmayoccurwithminimal anemiain cats with spontaneous diseases, such as diabetes mellitus (especiallywhenketoacidosis is present), hyperthyroidism, and lymphoma.104,106 Small Heinzbodiesmaybeseeninother species followingsplenectomy. IIDietarycauses of Heinzbodyhemolyticanemiaincludeconsumptionofonions in small and large animals, consumption ofkale and other Brassicaspeciesbyruminants, consumption of lushwinterryebycattle, andconsump-tionof redmapleleavesbyhorses.9,75, L07 Heinzbodies havebeenrecognizedinerythrocytes from selenium-deficient Florida cattle grazing on St. Augustinegrass pastures andin postparturient NewZealand cattle grazingprimarilyonperennial ryegrass. Copper toxicityresults in Heinzbodyformation in sheepand goats. Heinz body formation has been reported in dogs ingestingzinc-containing objects (e.g., U.S. pennies minted after 1982).9 Naphthalene ingestionmay have caused Heinz body formation in a dog.L08Heinz body hemolyticanemiahas occurredfollowingthe administrationof avarietyofdrugsinclud-ing acetaminophen and methylene blue in cats and dogs, methionine andphenazopyridineincats, menadione(vitaminK3) indogs, andphenothiazine inhorses.9,75Basophilic StipplingReticulocytes usually stain as polychromatophilic erythrocytes with Rorna-nowsky-type blood stains due to the presence of dispersed ribosomes andpolyribosomes, but sometimes the ribosomes andpolyribosomesaggregate to-38 SECTIONI I BLOODFIGURE13. Erythrocyte inclusions andtheappearance of amitotic erythroidprecursorcell.A. Polychromatophilic metarubricyte with elongated nucleus and erythrocyte containing nuclear fragmentsinbloodfromadogwithimmune-mediatedhemolytic anemiaandthrombocytopenia5days aftertreat-ment withvincristine. Wright-Giemsastain. B. Erythrocyte containingnuclear fragmentsinbloodfromadogwithimmune-mediatedhemolytic anemiaandthrombocytopenia5 days aftertreatment withvincristine. Wright-Giemsa stain. C. Mitotic rubricyte in blood from a dog with hemangiosarcoma anda regenerativeanemia. Wright-Giemsastain. D. Erythrocyte(left) containinga Howell-Jollybody(spherical nuclear remnant) in blood from a cat. Wright-Giemsa stain. E. Three Howell-Jolly bodies inblood from a dog being treated with glucocorticoid steroids. Wright-Giemsa stain. F. A large polychro-matophilic erythrocyte (top), erythrocyte "ghost" containing a Heinz body (bottom), and anintact erythro-cyte containingaHeinzbodyprojecting fromitssurface (right) inbloodfromadogwithahemolyticanemia resulting from theingestion of several pennies containing zinc. Wright-Giemsa stain. G. Heinzbodiesinbloodfroma cat appearingas pale"spots"withinerythrocytes. Wright-Giemsa stain. H.Heinz bodiesinbloodfromacat. New methylene bluereticulocyte stain. I. Heinz bodies inbloodfrom a cat. New methylene blue wet mount preparation. J. Diffusebasophilic stippling (bottom left) in(Continued)CHAPTER 2 / ERYTHROCYTES 39gether, formingblue-stainingpunctateinclusions referredtoas basophilicstip-pling(Figs. l3J _13M).52Theseaggregates aresimilar tothose producedusingreticulocyte stains, but they formduringthe process ofcell drying prior tostainingwithRomanowsky-type bloodstains. Diffuse basophilicstipplingcom-monlyoccurs in regenerativeanemia inruminants (Figs. 131. 13K) and occa-sionallyoccurs inregenerativeanemia inother species(Fig. 13L),uBasophilicstipplingmay be prominent inany species withlead poisoning(Fig. 13M).96.97Siderotic InclusionsSiderotic inclusions containiron. Incontrast tobasophilic stippling, whichisdistributedthroughout the erythrocyte, siderotic inclusions generallyappear asfocalbasophilicinclusions locatednear theperiphery of theerythrocyte. Theseinclusions have been called Pappenheimer bodies when visible in routinelystainedbloodfilms(Figs. l3N, 130).52 Electronmicroscopyof thesebodies inhumanerythrocytes reveals that the ironisoftencontainedwithinautophagicvacuoles(lysosomes) that also contain degeneratingmitochondria. 52A Prussianbluestainingprocedureis usedtoverify thepresenceof iron-positivematerial (Fig. 13P). Erythrocytescontainingtheseinclusions arecalledsiderocytes. Siderocytesarerare or absent inthebloodofnormal animals butmayoccurwithleadtoxicity,"hemolyticanemia, dyserythropoiesis, myelopro-liferativediseases, chloramphenicol therapy, and experimental pyridoxine defi-ciencyinpigs.9.58Sideroticinclusions havebeenrecognizedinadogwithzinctoxicity,'?'butit is unclearwhetherthiswas a result of thezinctoxicity per se,orwas associated withtheaccompanying hemolyticanemia. Finally, large num-bers of Pappenheimer bodies haverarelybeen seenin nonanemicdogs (Figs.130, l3P) inwhichthecauses wereunclear. A commonfactor intwoofthesedogs was treatment withhydroxyzine;however, hematologic abnormalities havenot previously beenreported withthisdrug.10911Oamacrocytic polychromatophilic erythrocyte, amacrocytic erythrocyte (topright), andthreenormal-sizederythrocytes inbloodfroma cow with anaplasmosis (noorganisms present) anda subsequent regenerativeanemia. Wright-Giemsa stain. K. Diffusebasophilic stippling in a large erythrocyte (left) in blood fromasheep witharegenerative anemia. Wright-Giemsa stain. L Erythrocytes containingaHowell-Jollybody(top), diffuse coarse basophilic stippling(middle)anddiffuse fine basophilic stippling(bottom) inblood from a cat with hemobartonellosis (no organisms present) and aregenerative anemia. Wright-Giemsastain. M. Apolychromatophilic erythrocyte withbasophilic stippling(left) andapolychromatophilicmetarubricyte (right) in blood from a dog with lead toxicity. Wright-Giemsa stain. N. Focal basophilicstippling inanerythrocyte (siderocyte) inbloodfromadog treated withchloramphenicol. Theinclusionswere shown to contain iron using the Prussian blue staining procedure. Wright-Giemsa Stain. O. Focalbasophilic stipplingin twoerythrocytes (siderocytes) inbloodfroma maleSheltie dogthat hadmanysiderocytesinhisbloodwhenexamined several timesover 4 years. Erythrocyteswere microcytic, but thedog was notanemic. Abnormalities incopper, zinc, and pyridoxine metabolism were ruled out, as was leadtoxicity. Blood samples andcase information providedbyM. Plier. Wright-Giemsa stain. P. Iron-positive inclusionsin erythrocytes(siderocytes) inbloodfromthe samedogasshowninFigure130.Prussian blue stain.40 SECTIONI I BLOOD INFECTIOUSAGENTSOFERYTHROCYTESAnumber of infectiousagentsarerecognizedtooccur inor on erythrocytes.These includeintracellularprotozoal parasites(Babesiaspecies, Theileriaspecies,and Cytauxzoon felis) , intracellular rickettsial organisms (Anaplasma species),andepicellularmycoplasma organisms(Haemobartonellaspecies andEperythro-zoonspecies). Theprotozoal organismseachhaveanucleuswithintheir cyto-plasm. The rickettsiaand mycoplasmaorganisms are bacteria and, therefore,lacknuclei. Theseinfectious agents generallycausemild to severe hemolyticanemia, dependingonthepathogenicityoftheorganismandthesusceptibilityof the host. Distemper virus inclusions may also be seen in dog erythrocytes.Babesia SpeciesMany speciesof Babesia infect animals worldwide.l1l-113When stained withRomanowsky-typebloodstains, ababesialorganismgenerally hascolorless tolight-blue cytoplasmwith a red to purple nucleus (Figs. 14A-14E). Babesialparasites varyconsiderablyin sizefromlarge, easilyvisualizedBabesia canisparasites (Fig. 14A)to small, difficult to seeBabesiagibsoni (Fig. 14B)andBabesia felis (Fig. 14C) parasites. Largebabesial organisms generallyappearpear-shapedandcommonly occur inpairs. Small babesial organismsaremoreoftenround inshape.Theileria SpeciesTheilerial organisms appear similar to babesial organisms when observedonstained blood films (Fig. 14F). The genus Theileria differs fromthe genusBabesia inthat theTheileriaspecies have a tissue phase as well as anerythrocytestageof development. Schizontsdevelopinlymphoid cells andwhenmature,release merozoites, whichenter erythrocytes. Babesiaorganisms proliferateonlyin erythrocytes. Theilerial species cause important diseasesin ruminants inAfrica, Asia, andtheMiddleEast; however, thetheilerialorganismspresent inruminantsinthe UnitedStates areusually nonpathogenic.t'' Megakaryocytes are more easilyfound byexamination of bloodbuffy coat smears. Megakaryocytes reaching bloodarequickly trappedinlungcapillaries where continuedplatelet production may occur.Dwarf megakaryocytes are smaller than normal mature megakaryocytesandhave decreased nuclear ploidy, but theircytoplasm generally contains gran-ules andappearssimilar tothat of bloodplatelets (Figs. 29F, 30J-30L). Dwarfmegakaryocytes arecommoninbonemarrowofanimalswithmyeloprolifera-tive disorders but are only rarely seen in blood. PARASITES AND BACTERIAParasitesand bacteriamaybe seenin bloodwhichare not associatedwithblood cells. However, bacterial rods and cocci betweencells are usuallytheresult of contaminatedstain.MicrofilariaPotential microfilariae(nematode larvae) include Dirofilaria immitis in dogs,cats, andwildcanids(Fig. SD); Dipetalonemareconditumindogs; andSetariaspecies in cattle andhorses.!"Trypanosoma SpeciesVarious Trypanosoma species maybeseeninblood (Figs. 30M-300). Theseelongated, flagellatedprotozoa cause important diseasesoflivestock outsideofthe United States.'!' but the species seenincattle(T. theileri) inthe UnitedStates isusuallynonpathogenic.255,256Manydogsareinfectedwith T. cruzi intheUnitedStates, but organismsarerarelyseeninbloodandmost cases aresubclinical. When present, clinicalforms of disease haveprincipallyinvolvedheartorneural dysfunctions.i"BacteriaVarious bacterial species may be present in bloodfilms. Itis important to verifythat thesearenot contaminants, especiallyduringthestainingprocedure. Thepresence ofphagocytized bacteriawithinneutrophilsindicates that the bacteriaare likely of clinical significance.Spirochetes have been seen in blood from dogswith Borrelia infections.!"A species of Borrelia different from B. burgdorferi hasbeen recognized in the blood of dogs fromFlorida (Fig. 30P).258CHAPTER 6HematopoiesisThroughout theadult life ofamammal, all bloodcell types arecontinuouslyproducedfromprimitivestemcells withinextravascular spaces ofbone mar-row.259Atotipotent hematopoieticstemcell producesapluripotent lymphoidstemcell, aswell asapluripotent myeloid stemcell. Thepluripotentmyeloidstem cell givesrise to a series of increasinglydifferentiated progenitor cells, withlimitedself-renewal capabilities, whichsupport theproductionof all nonlym-phoid bloodcells. Stemcells andprogenitor cells aremononuclear cells thatcannot bedistinguished morphologically fromlymphocytes. Whenmeasuredinanin vitrocell cultureassay, progenitor cells arereferred toas colony-formingunits (CFUs) or burst-formingunits (BFUs) if theyformmultiple subcolon-ies.1,259,260Thetotipotent hematopoietic stemcell alsogives risetoprogenitorcells for osteoclasts,261.262mast cells,263 dendritic cells,264 and Langerhanscells.265,266Bloodcellproductionoccursinthe bone marrowof adult animals be-cause of theuniquemicroenvironment presentthere. The hematopoieticmicro-environmentis acomplex meshwork composedof variousstromal cells; acces-sory cells; glycoprotein growth factors; andextracellular matrix components thatprofoundlyaffect hematopoieticstem cell and progenitor cell survival, prolifera-tion, anddifferentiation. Stromal cells (endothelial cells, fibroblast-like reticularcells, adipocytes, andmacrophages) andaccessory cells (subsets of lymphocytesand natural killer cells) produce a varietyof positive and negativegrowthfactors. Stromal cells alsoproduce componentsoftheextracellularmatrix. Inadditionto providing structural support, the extracellular matrix is important inthe bindingof hematopoietic cells and soluble growth factors tostromalcells sothat optimal proliferation and differentiation canoccur.267-270With the exceptionof macrophages, stromal cells appear to bederivedfroma common mesenchymalstemcellthat isdistinctlydifferent fromthehematopoieticstemcell. Inadditiontoreticular cells, adipocytes, andendothe-lial cells, the mesenchymal stemcell alsoproduces osteoblasts and musclecells.271-274Proliferation of hematopoieticstem cells andprogenitorcells cannotoccurspontaneously but requires the presence of specifichematopoietic growth factors8788 SECTION11I BONE MARROW(HGFs) that maybe producedlocally in the bone marrowor producedbyperipheral tissues andtransportedtothemarrowthroughtheblood(humoraltransport). Some HGFs have beencalled poietins(erythropoietinandthrombo-poietin). Other growth factors have beenclassified as colony stimulating factors(CSFs) basedon invitroculture studies. Finally, someHGFshavebeende-scribed as interleukins.259.275 ERYTHROPOIESISRubriblasts arecontinuouslygeneratedfromprogenitor cells intheextravascu-lar space ofthebonemarrow. Thedivisionofarubriblast initiatesaseries ofapproximately four divisions over a periodof 3 or4 days toproduceabout 16metarubricytes, which are nolonger capable of division (Fig. 31).276FIGURE31. A diagramoferythropoiesis showing therelease of reticulocytes intobloodas it normally occurs indogs. (Modified fromMeyer DJ, Harvey JW: Veterinary LaboratoryMedicine. Interpretation andDiagnosis, 2nded. WB Saunders, Philadelphia, PA, 1998, withpermission.)CHAPTER 6 I HEMATOPOIESIS 89Early precursors haveintenselybluecytoplasmwhenstainedwithRoma-nowsky-typebloodstains, owingtothepresenceof manybasophilicribosomesand polyribosomes that are activelysynthesizing globin chains and smalleramounts of other proteins. Asthese cells divideand mature, overallcellsizedecreases, N: Cratiodecreases, nuclear chromatin condensation increases, cyto-plasmic basophilia decreases, and hemoglobin progressivelyaccumulates, im-partingaredcolorationtothecytoplasm. Cells withboth redandbluecolor-ation are described as having polychromatophilic cytoplasm. An immatureerythrocyte, termeda reticulocyte, is formedfollowing extrusionof themetaru-bricyte nucleus.Reticulocyte maturationbeginsinthebone marrowandiscompleted intheperipheral bloodandspleenindogs, cats, andpigs."Reticulocytes becomeprogressively more deformable as they mature, acharacteristic that facilitatestheir release fromthemarrow. Relatively immatureaggregate-type reticulocytesarereleasedfromdogand pigbone marrow." Reticulocytes aregenerally notreleased frombonemarrowofnormal catsuntil reticulocytes maturetopunc-tate-typereticulocytes; consequently,few ornoaggregate reticulocytes 0.5%),but up to 10%punctatereticulocytes, arefound inbloodfromnormal adultcats.>Reticulocytes normallyundergomaturationtomature erythrocytesinthebone marrowofhorsesandruminants. Reticulocytes maybereleased intotheblood in ruminants in response to anemia, but this rarelyoccurs in anemichorses.11 LEUKOPOIESISNeutrophilsNeutrophiliccells withinthebone marrowcanbeincludedintwopools. Theproliferationandmaturationpool (mitoticpool) includesmyeloblasts, promye-locytes, andmyelocytes. Approximatelyfour or five divisions occur overseveraldays. During this time, primary (magenta-staining) cytoplasmic granules areproducedin late myeloblastsor earlypromyelocytes and specific(secondary)granules aresynthesizedwithin myelocytes. Theseprimarygranules haveb