SAUNDERS AnImprintofElsevier TheCurtisCenter IndependenceSquareWest Philadelphia,Pennsylvania19106 LibraryofCongressCataloglng-ln·PublicationData Harvey,JohnW. Atlasofveterinaryhematology:bloodandbonemarrowofdomesticanimalsIJohnW.Harvey. p. em. Includesbibliographicalreferences(p.). ISBN-13:978-0-7216-6334-0 ISBN-10:0-7216-6334-6 1. Veterinaryhematology-Atlases. 2. Bloodcells-Atlases. 3. Bonemarrow-Atlases. I. TItle. SF769.5.H372001 636.089"615--dc21 00-063569 ATLASOFVETERINARYHEMATOLOGY Copyright©2001byW.O.SaundenCompany Allrightsreserved.Nopartofthispublicationmaybereproducedortransmittedinanyformorbyany means,electronicormechanical,includingphotocopy,recording,oranyinformationstorageandretrieval system,withoutpermissioninwritingfromthepublisher. PermissionsmaybesoughtdirectlyfromElsevier'sHealthSciencesRightsDepartmentinPhiladelphia, PA,USA:phone:(+1)2152393804,fax:(+1)2152393805,e-mail:[email protected]. Youmayalsocompleteyourrequeston-lineviatheElsevierhomepage(http://www.elsevier.com).by selecting'CustomerSupport'andthen'ObtainingPermissions'. ISBN-13:978+7216-6334-0 ISBN-I0:0-7216-6334-6 PrintedinChina Lastdigitistheprintnumber: 9 8 7 6 5 4 To Liz Preface This color atlas is designedas a reference book for the morphologic aspectsof veterinary hematology of common domestic animals, excluding birds. Species covered include dogs, cats, horses, cattle, sheep, goats, pigs, and llamas. The atlas is divided into two sections, blood and bone marrow. It includes basic material for the novice, as well as material of primary interest to those with advanced training. Techniquesfor the collectionand preparation of blood and bone marrow smearsand bone marrow corebiopsiesare discussed, in addition to the morphology of the tissuescollected. Often, more than one exampleof a celltype or abnormal condition is shown,becausecells and conditions can vary in morphology. Veterinary technologists will likely find the blood section and the techniques part of the bone marrow section to be most helpful.Veterinary students and practicing veterinarians should benefit from the complete book, even if they are not directly involved in bone marrow evaluation, because it provides a basis for understanding diseases affecting the marrow. The bone marrow aspirate smear cytology and core biopsyhistologysectionwillbe most useful to clinicalpathologists, anatomic pathologists, and residents in training for these disciplines. This is not a completehematologytextbook, but rather a reference book in which the text explains the significance of the morphologic abnormalities shown in the color photographs. Readers interested in learning more about a given topic will hopefully appreciate the extensive bibliography provided. John Harvey Acknowledgments I want to acknowledge those most responsible for my education as a clinical pathologist.Fewpeoplehavethe opportunity to receive training from the giants of their profession, but I was blessed in being trained by Jerry Kaneko, the father of veterinary clinical biochemistry, and Oscar Schalm, the father of veterinary hematology.Many other colleagues have contributed to my develop ment as a hematologist,with Victor Perman and Alan Rebar being particularly noteworthy, as we have challenged one another with unknown hematology slides in front of various audiences. I thank Denny Meyer for his encourage ment in taking on the task of preparing this atlas and Rose Raskin, Leo McSherry, and Shashi Ramaiah for their conscientious reviews and helpful suggestions. I appreciate MelaniePate's carefuleditorial reviewand am grateful to RayKersey at W.B.Saunders Co. for his remarkable patience and persistent support in this endeavor. John Harvey Editor's Note The purpose of this text/atlas is to provide veterinarians, veterinary students, veterinary technicians and veterinarytechnology students with a complete treatise on the cytologyofblood and bone marrow. The subjects will be covered in a user friendly way utilizing color photo graphs and appropriate text that clarifies diagnostic implications. Therapy willbe discussed only when diagnosis can be confirmed by response to treatment. Ninety percent of the information willfocuson commonly recognizeddiseases. CHAPTER 1 Examination of Blood Samples • SAMPLE COLLECTION AND HANDLING An overnight fast avoids postprandial lipemia in monogastric animals, which can interfere with plasma protein, fibrinogen, and hemoglobin determinations. Ethylenediaminetetraacetic acid (EDTA) is the preferred anticoagulant for com plete blood count (CBC) determination in most species, but blood from some birds and reptiles hemolyzes when collected into EDTA. In those species, I heparin is often used as the anticoagulant. The disadvantageof heparin is that leukocytes do not stain as well (presumably because heparin binds to leuko cytes),? and plateletsgenerallyclump more than theydo in blood collectedwith EDTA. However, as will be discussed later, platelet aggregates and leukocyte aggregates may occur even in properly collected EDTA-anticoagulated blood samples.':" In those cases, collectionof blood using another anticoagulant (e.g., citrate) may prevent the formation of cellaggregates. Cellaggregationtends to be more pronounced as blood is cooled and stored; consequently, processing samples as rapidly as possible after collection may minimize the formation of leukocyteand/or plateletaggregates. Collection of blood directlyinto a vacuum tube is preferred to collection of blood by syringe and transfer to a vacuum tube. This method reduces platelet clumping and clot formation in samplesfor CBCdeterminations. Even smallclots render a sampleunusable. Plateletcounts are markedlyreduced, and significant reduction can sometimes occur in hematocrit (HCT) and leukocyte counts as well. Also, when the tube is allowed to fill based on the vacuum within the tube, the proper sample to anticoagulant ratio will be present. Inadequate sample size results in decreased HCT due to excessive EDTAsolu tion. Care should be taken to avoid iatrogenichemolysis, which interfereswith plasma protein, fibrinogen, and various erythrocyte measurements. Samples should be submitted to the laboratory as rapidly as is feasible, and blood films should be made assoon as possibleand rapidlydried to minimize morphologic changes. 3 4 SECTION I I BLOOD FIGURE 1. Grossappearanceofmixtures ofoxyhemoglobin, deoxyhemoglobin, and methemo globin and differentiation of erythrocyte agglutination from rouleau formation. A. Venous blood sample from a cat with 28% methemoglobin (leftsample) compared to a normal cat with lessthan 1%methemoglobin (right sample). Bothsamplesalsocontain amixture ofoxyhemoglobinand deoxyhemo globin. B. Oxygenated blood sample from a cat with 28% methemoglobin (left sample) (Continued) CHAPTER 1/ EXAMINATIONOFBLOOD SAMPLES 5 • GROSS EXAMINATION Samplesare checkedfor dots and mixed well(gentlyinverted 20times) imme diatelybefore removing aliquots for hematologyprocedures. Horse erythrocytes settle especially rapidly because of rouleau formation (adhesion of erythrocytes together likea stackof coins). Bloodshould be examined grosslyfor color and evidence of erythrocyte agglutination. The presence of marked lipemia may result in a blood sample with a milky red color resembling "tomato soup" when oxygenated. Methemoglobinemia Hemoglobin is a protein consisting of four polypeptide globin chains, each of which contains a heme prosthetic group within a hydrophobic pocket. Heme is composed of a tetrapyrrole with a central iron molecule that must be main tained in the ferrous (+2) state to reversibly bind oxygen. Methemoglobin differsfrom hemoglobin only in that the iron moleculeof the heme group has been oxidizedto the ferric (+3) state and it is no longer ableto bind oxygen." The presence of large amounts of deoxyhemoglobin accounts for the dark, bluish color of normal venous blood samples.Methemoglobinemia may not be recognized in venous blood samples,because the brownish color of methemo globin is not readily apparent when mixed with deoxyhemoglobin (Fig. lA). When deoxyhemoglobin binds oxygen to form oxyhemoglobin, it becomes bright red; consequently, the brownish coloration of methemoglobin becomes more apparent in the oxygenatedsamples (Fig.IB). Asimplespot test provides a rapid way to oxygenate a venous blood sample and determine if clinically significant levels of methemoglobin are present. One drop of blood from the patient is placed on a piece of absorbent white paper and a drop of normal control blood is placed next to it. If the methemoglobin content is 10% or greater, the patient's blood will havea noticeablybrown color, compared to the bright red color of control blood (Fig. lC).9 Accuratedetermination of methe moglobin content requires that blood be submitted to a laboratory that has this test available.'? Methemoglobinemia results from either increased production of methe moglobin by oxidants or decreasedreduction of methemoglobin associatedwith a deficiency in the erythrocyte methemoglobin reductaseenzyme.Experimental studies indicate that many drugs can produce methemoglobinemia in animals. compared to a normal cat with less than 10/0 methemoglobin (right sample). The sample on the left contains a mixture of oxyhemoglobin and methemoglobin, and the one on the right contains almost exclusively oxyhemoglobin. C. A drop of blood from a methemoglobin reductase-deficient cat with 500/0 methemoglobin(left)isplacedon absorbentwhitepaper nextto a drop of normal catblood withless than 1% methemoglobin. D. Grosslyvisibleagglutinationin bloodfroma dogwithimmune-mediated hemolyticanemia. E. Microscopicrouleaux in an unstained wet mount preparation of normal cat blood. F. Microscopicagglutinationin an unstained wet mount preparation ofsaline-washed erythro cytesfromafoalwithneonatalisoerythrolysis. 6 SECTION II BLOOD Significantmethemoglobinemia has been associatedwith clinicalcasesofbenzo caine, acetaminophen, and phenazopyridine toxicities in cats and dogs; nitrite toxicity in cattle; copper toxicity in sheep and goats; and red maple toxicity in horses." Agglutination The appearance of red granules in a well-mixedblood sample (Fig. ID) suggests the presence of erythrocyte agglutination, the aggregationor clumping of eryth rocytestogether in clusters.Agglutinationiscaused by the occurrence of immu noglobulins bound to erythrocyte surfaces. It must be differentiated from rou leaux, the adhesion of erythrocytes together like a stack of coins, which can be seen in blood from healthy horses and cats (Fig. IE). Agglutination can be differentiated from rouleaux by washingerythrocytesin physiologicsalineor by adding equal drops of physiologic saline and blood together to see if the aggregation of erythrocytes is dispersed (rouleaux) or remains (agglutination). The microscopic appearance of agglutination in a sample of washed erythro cytesisshown (Fig.IF). II MICROHEMATOCRIT TUBE EVALUATION When blood is submitted for a CBC, most commercial laboratories determine the HCT electronically. This efficiency negates the need to centrifuge a micro hematocrit tube filled with blood. Unfortunately, usefulinformation concerning the appearance of plasma is missed unless a serum or a plasma sample is also prepared for clinicalchemistrytests. Packed Cells In addition to determining the HCT, the buffycoat isevaluated.The buffycoat contains platelets and leukocytes.It may appear reddish due to the presence of marked reticulocytosis. In some species, certain leukocytesmay also be present in the top portion of the packed erythrocyte column (e.g., neutrophils in cattle). A largebuffycoat suggests leukocytosis(Fig.2A)or thrombocytosis and a smallone suggests lownumbers of these cellsmay be present. Plasma Appearance Plasma is normally clear in all species. It is nearly colorless in small animals, pigs, and sheep but light yellow in horses, because they naturally have higher bilirubin concentrations. Plasma varies from colorless to light yellow (carot 11 enoid pigments) in cattle, depending on their diet. Increased yellowcoloration usually indicates increased bilirubin concentration. This increase often occurs secondarily to anorexia (fasting hyperbilirubinemia) in horses due to reduced