alan h. rebar, dvm, phd - purina® pro plan® vets€¦ · hemogram interpretation for dogs and...

Hemogram Interpretation for Dogs and Cats

Alan H. Rebar, DVM, PhD

Clinical Handbook Series

$50.00

Hemogram Interpretation for Dogs and Cats

Alan H. Rebar, DVM, PhD

Clinical Handbook Series

Published by The Gloyd Group, Inc.

Wilmington, Delaware

© 2004 by Nestlé Purina PetCare Company.

All rights reserved.

Printed in the United States of America.

Nestlé Purina PetCare Company: Checkerboard Square, Saint Louis, Missouri, 63188

First printing, 1998.

Laboratory slides reproduced by permission of Alan Rebar, DVM, PhD.

Photographs by Terence Roberts Photography.

This book is protected by copyright.

ISBN 0-9678005-1-X

Table of Contents

Introduction.......................................1

Part I

Chapter 1Leukocytes in Health & Disease ..................4

Chapter 2Erythrocytes in Health & Disease ..............15

Chapter 3Platelets in Health & Disease ...................27

Part II

Chapter 4Hemogram Interpretation.........................33

Chapter 5Case Studies .........................................39

Part III

Table 1. Hematological Reference Ranges of the

Dog and Cat ..........................................................81

Index of Figures .....................................................82

Glossary of Terms ..................................................85

Suggested Reading ..................................................87

Nestlé PURINA Hemogram Interpretation for Dogs and Cats

Nestlé PURINA Hemogram Interpretation for Dogs and Cats 1

Introduction

This monograph, Hemogram Interpretation for Dogs

and Cats, is divided into three parts. Part I is comprised

of Chapters 1 through 3; Chapters 4 and 5 make up Part II.

To maximize its usefulness, each chapter should be read

and studied in sequence. Part III contains a variety of

reference materials and other resources.

Part I (Chapters 1, 2, and 3) is designed to acquaint the

reader with the basic physiology and pathophysiology of

the hematopoietic system of the dog and cat. It is not

intended as an in-depth treatise but rather focuses on those

aspects of hematopoietic physiology which are most useful

in understanding peripheral blood changes in disease.

Part II (Chapters 4 and 5) is designed to develop a

systematic approach to the interpretation of peripheral

blood data and morphology, and to afford the reader the

opportunity to practice this approach on a series of

actual clinical cases. Of necessity, there is some

redundancy between Parts I and II; however, this

redundancy will serve to reinforce the reader’s

understanding of hematologic pathophysiology and its

relationship to hemogram interpretation.

Part III includes: Table 1. Hematological Reference

Ranges of the Dog and Cat that lists normal blood values;

a full Index of Figures that describes all the photographs

of slides used in this book; a Glossary of Terms that

contains definitions of those words which are underlined

throughout the text; and, a comprehensive listing of

Suggested Reading on the topic of canine and feline

hematology.

TM


Part I

4 Nestlé PURINA Hemogram Interpretation for Dogs and Cats

Overview

From a functional perspective, circulating leukocytes, orwhite blood cells (WBC), belong to two systems: thephagocytic system and the immunocytic system.

These two immune systems are functionally interdepen-dent. The phagocytic system is comprised of the granulo-cytes and the monocyte/macrophage continuum. Theimmunocytic system is comprised of circulating T and Blymphocytes. Factors produced by monocytes/macrophagesprofoundly influence lymphocyte function and a variety oflymphocyte products, in turn, influence the function of thephagocytes.

The phagocytes (ie, granulocytes, monocyte/macrophagecontinuum) are the first line of defense against invadingmicroorganisms; they are attracted to sites of infection and,by the process of phagocytosis, ingest and destroy bacteriaand other agents on contact. Therefore, the phagocytes canbe thought of as the non-specific immune system.

In contrast, the immunocytic system is the specificimmune system. Its effector cells (ie, T and B lympho-cytes) are responsible for both the production of humoralimmunity in the form of antibodies directed against specif-ic antigens as well as cell-mediated immunity in the formof specific cytokine production.

The following paragraphs explore the structure andfunction of each system in greater detail.

The Phagocytic System

The Phagocytic ProcessThe process of phagocytosis has been well studied and

can be divided into several stages. The initial stage is chemotaxis. In this early phase, all

phagocytic cells are mobile and are being attracted to sitesof inflammation and infection by an increasing gradient ofsmall molecules known as chemotaxins. Among the mostpotent chemotaxins are:

� lipopolysaccharides (components of bacterial mem-branes),

� active fragments of complement, � antigen-antibody complexes, and � certain products of lymphocytes.

Once phagocytes have entered an area of inflammationand infection, they must adhere to the microorganism(s) inorder for the process of phagocytosis to continue.Adherence (stage II) occurs more readily if organisms havebeen opsonized; that is, coated by antibodies or comple-ment fragments. Phagocytes have receptors on their sur-faces for such opsonins which facilitates adherence. Theprocess of opsonization represents one of the interactionsbetween the non-specific and specific immune systems;antibodies are produced by immunocytes, phagocytesattach to those antibodies.

Next, adhered organisms must be internalized (stageIII). This is accomplished by invagination of the superficialcell membrane and formation of a phagocytic vacuole aroundthe organism. Almost simultaneously, the cytoskeletal andmicrotubular system of the cell moves the cytoplasmic gran-ules towards the phagocyticvacuoles. Fusion of therespective membranesallows intimate contactbetween granule content andthe microorganism, resultingin killing and digestion ofthe microorganism.

Chapter 1: Leukocytes in Health and Disease

Different Circulating Leukocytes Belong toDifferent Immune Systems

Non-specific Specific Immune System Immune System

� �

Phagocytic Immunocytic

� �

Granulocytes B lymphocytes

Mononcytes/macrophages T lymphocytes

The Stages ofPhagocytosis

I. Chemotaxis II. Adherence III. Internalization


The Granulocytes

NeutrophilsNeutrophils are the

predominant circulatinggranulocyte and are easilydistinguished on theperipheral blood film bythe following morphologic characteristics (Fig. 1):

� round� measure 12.0 µ to 15.0 µ in diameter� abundant pale pink, granular cytoplasm� lobed nuclei with deeply staining, condensed chro-

matin This morphologic appearance provides significant clues

regarding their functional state. The condensed, deeplystained chromatin suggests a mature nucleus with predomi-nantly inactive heterochromatin. The pink, granular cyto-plasm suggests abundant protein but little machinery (cyto-plasmic blue staining RNA) to produce additional protein.

The circulating neutrophil is a fully differentiated phago-cyte capable of seeking out, ingesting, killing, and digestinginvading microorganisms. Two forms of granules, specificgranules and lysosomes, fill its cytoplasm. Specific granulescontain substances, such as lactoferrin and cationic pro-teins, which are involved in bacterial killing. The lyso-somes contain digestive enzymes, which act to dissolveorganisms once killed.

While the neutrophil is a highly effective bacterial killing

machine, it is somewhat limited by its own high degree ofsophistication and differentiation. Neutrophils are unable todivide, replenish their granule content, or regenerate sur-face membrane that is lost in internalization during phago-cytosis of organisms. As a result, their lifespan is extremelyshort (hours to days).

Although neutrophils primarily function as phagocytes,it is noteworthy that these cells also function as secretorycells. Both when fusion of granules with phagocytic vac-

uoles occurs and when effete neutrophils breakdown, biologically active molecules are releasedinto the microenvironment. Among these areprostaglandins, complement fragments, and biologi-cally active amines. Clearly, by releasing these reg-ulatory molecules into the environment, neutrophils(and, indeed, all phagocytes) function as modera-tors of the local and systemic inflammatoryresponse.

Normal peripheral neutrophil counts in dogsand cats range between 3000/µl to 12,000/µl. Inhealth, immature neutrophils (band cells) are onlyrarely observed (less than 300/µl when peripheralwhite cell counts are normal). Both increases (neu-trophilia) and decreases (neutropenia) in neu-trophil numbers are clinically significant, particu-larly in inflammatory diseases. Similarly, relativeincreases in band cells (termed a "left shift") are

also extremely important clinically. To understand theinterpretation of peripheral neutrophil numbers it is firstimportant to understand neutrophil kinetics—the dynamicsof neutrophil production, circulation, and utilization withinthe tissues.

Neutrophils are produced in the bone marrow, circulatetransiently in the mainstream peripheral blood (the freelycirculating pool), marginate on the blood vessel walls (themarginating pool), and migrate into the tissues. Within themarrow there are three pools of neutrophils and their pre-cursors:

1) the proliferating pool, consisting of the dividingmyeloblasts and promyelocytes, and early myelo-cytes,

2) the maturing pool, consisting of late nondividingmyelocytes, metamyelocytes, and band cells, and

3) the storage pool, consisting of mature neutrophils.In general, production of a neutrophil from a myeloblasttakes approximately 5 days. The storage pool containsapproximately a 5-day supply of mature neutrophils. In

Fig. 1 Normal circulating neutrophils.

Granulocytes

Neutrophils

Eosinophils

Basophils


other words, if white cell production were interrupted onday 1 and tissue demand, and therefore circulating half-life, were normal, it would be 5 days (day 6) before areduction in neutrophil numbers would be recognized inperipheral counts. Neutrophils leaving the storage poolreside in the circulating pool (from which they are collect-ed when blood is sampled) for only 6 to 8 hours beforemarginating and ultimately being drawn into the tissueschemotactically to perform their phagocytic func-tion. For every neutrophil in the freely circulatingpool, there is at least one marginating neutrophil.

When inflammatory foci develop in the tissues,production of chemotaxins causes increased releaseof neutrophils into the blood, shortened circulatinghalf-life of neutrophils with increased margination,and increased egress of neutrophils into the tissues.Because of the large storage pool of marrow neu-trophils, in most cases of inflammation, the move-ment of neutrophils into the blood is greater thanthe movement of neutrophils into the tissues; thenet effect is peripheral neutrophilia. In a short time,the marrow storage pool of mature neutrophils isreduced to the point where increased numbers ofband cells are drawn into the peripheral blood.Neutrophilia with a left shift (ie, a regenerative leftshift) is the classic acute (active) inflammatoryleukogram of dogs and cats.

Inflammatory foci release not only chemotaxinsbut also colony stimulating factors (CSF), whichcause increased production of neutrophils. If theinflammation is not resolved quickly, marrow pro-duction of neutrophils will establish a new balancewith tissue demand. Ultimately, the neutrophilcount will return toward normal and the left shifttends to disappear. The classic chronic inflammatoryleukogram features a normal or near normal neu-trophil compartment.

In rare cases of severe acute inflammation, tis-sue demand is so great that egress of neutrophilsfrom the blood may actually exceed influx of neu-trophils from marrow. In these instances neu-tropenia with a left shift (ie, a degenerative left shift)develops rapidly and is an indication of overwhelminginflammation. In the dog and cat, this finding warrants aguarded prognosis.

EosinophilsThe eosinophil is named for its distinctive red-orange

(eosinophilic) cytoplasmic granules, which are variablysized and round in the dog (Fig. 2) and rod-shaped in thecat (Fig. 3). These granules contain hydrolytic enzymes andperoxidase (like the granules of neutrophils), as well as acore of basic proteins that give the granules their strongaffinity for the eosin stain.

Like neutrophils, eosinophils respond chemotactically tobacterial products and complement fragments. In addition,they are attracted preferentially to histamine and theeosinophilic chemotactic factor of anaphylaxis (ECF-A)released by mast cells, as well as to certain productsreleased by activated lymphocytes. From a functional per-

Fig. 3 Normal feline eosinophil.

Fig. 2 Normal canine eosinophil (left), reactive lymphocyte (right).


spective, eosinophils are bactericidal in vitro but theextent to which they are bactericidal in vivo isuncertain. However, their role in modulatinghypersensitivity reactions is clear; they elaborateantihistamines (amine oxidases) andprostaglandins that inhibit mast cell degranulation.

A second major function of the eosinophil is itsrole in the control of parasitic infections. Throughantibody and/or complement mediated binding,eosinophils become fixed to the surface of helminthparasites and release their granule content into themicroenvironment. Major basic proteins from thegranules cause considerable damage to the parasitesurface, which leads to parasite death.

Eosinophils have a much shorter circulatinghalf-life than neutrophils (minutes to several hoursas compared to 4 to 8 hours for neutrophils) soperipheral counts can be quite erratic from sampleto sample. As a result, eosinophilia (an increase) isonly significant when it is persistent over time. Themost accurate interpretation of persistenteosinophilia is the presence of a systemic hypersen-sitivity reaction.

Parasitic infections are only associated with per-sistent eosinophilia if they have a systemic phase.For example, whipworm infections in dogs, whichare confined to the intestinal tract, do not cause cir-culating eosinophilia. In contrast, heartworm infec-tions, where circulating parasites are present, cancause marked eosinophilia. Other causes of persis-tent eosinophilias in dogs include systemic mastocy-tosis, fleabite dermatitis with systemic hypersensi-tivity, allergic gastroenteritis, allergic bronchitis, andatopy. In the cat, feline asthma, eosinophilic granulo-ma complex (systemic, not oral form), systemic mas-tocytosis, heartworm disease, and allergic gastroen-teritis should all be considered. In the author’s opin-ion, eosinopenia (a decrease) is less consistent andmore difficult to interpret than eosinophilia.

BasophilsBasophils (Figs. 4, 5, 6) are only occasionally

observed on peripheral blood films. They are slight-ly larger than neutrophils with pale lavender cyto-plasm and truly segmented nuclei. Basophils ofdogs and cats often contain only a few distinctivedeep blue to purple cytoplasmic granules; as a Fig. 6 Normal feline basophil (left), normal neutrophil (right).

Fig. 5 Normal canine basophil.

Fig. 4 Normal feline basophil.


result, they may be mistakenly identified as monocytes.Besides the truly segmented nucleus, which is useful in dif-ferentiating basophils from monocytes, the frequent obser-vation of what appear to be small vacuoles in the nucleus—which are actually basophilic granules overlaying the nucle-us—are quite characteristic of basophils.

Basophils are not phagocytic. Nevertheless, they playan essential role in the inflammatory process. Their cyto-plasmic granules contain histamine and heparin.Histamine is one of the primary mediators of the acuteinflammatory response, causing increased vascular perme-ability. Heparin is an anticoagulant that moderates theinflammatory microenvironment by inhibiting the forma-tion of fibrin.

Basophilia is only rarely observed; when present, italmost always occurs in conjunction with eosinophilia.

The Monocyte/Macrophage Continuum

The monocyte/macrophage continuum represents thesecond branch of the phagocytic system and is the primarylink between the nonspecific and specific immune systems.This cell group was previously known as the reticuloen-dothelial system and includes not only the circulatingmonocytes but also the fixed macrophages of the liver,spleen, brain, and lymph nodes.

Monocytes are the precursors of all macrophages. Theyoriginate in the bone marrow, circulate in the peripheralblood, and settle out in the tissues where they differentiatefurther as needed. Differentiated cells of themonocyte/macrophage continuum include activatedmacrophages, epithelioid cells, and multi-nucleatedinflammatory giant cells. In addition to their role asphagocytes, macrophages also perform the follow-ing functions:

� modify antigens in such a way that theycan be recognized by immunocytes (anti-gen processing cells),

� release numerous inflammatory mediatorsthat recruit neutrophils, other monocytes,and lymphocytes into inflammatory sites,and

� regulate iron stores.The only cell of the monocyte/macrophage contin-

uum normally seen in the peripheral blood is themonocyte. The immature nature of the monocyte issuggested by its morphology (Fig. 7). The nucleus is

irregularly shaped with a lacy to finely granular chromatinpattern suggestive of a preponderance of active euchro-matin. Cytoplasm is abundant, often vacuolated, and grayto blue, which suggests a high content of cytoplasmicRNA but little protein. Monocytes seen on a peripheralblood slide are at approximately the same stage of develop-ment as the marrow myelocyte of the granulocyte series.In contrast to granulocytes, which are stored in marrow,monocytes are released immediately into circulation asthey are produced. They have limited immediate phagocyt-ic capability, but as mentioned above, possess significantpotential to develop into fully armed effector cells onceseeded in tissues.

Historically, peripheral monocytosis has been interpretedas indicative of chronic inflammation. Cells of the mono-cyte/macrophage continuum were regarded primarily as theclean-up cells of inflammation that were recruited intochronic lesions to remove debris after neutrophilic activityhad localized the primary disease process. While the essen-tial clean-up function of the monocyte/macrophage inchronic inflammation is undeniable, we now understandthat these cells have a much greater role in the inflammatoryprocess. Furthermore, since proliferating monocytes arereleased into and accumulate in the peripheral blood ratherthan being stored in the marrow, peripheral monocytosiscan occur quite quickly (in as little as 12 hours).Consequently, the best clinical interpretation of monocytosisis simply that there is tissue necrosis and a demand forphagocytosis. Monocytosis can occur in either acute orchronic inflammation.

Fig. 7 Two normal canine monocytes are at upper right. A normal canine neutrophil is at bottom for comparison.


The Immunocytic System

The immunocytic or specific immune system is com-prised of the circulating lymphocytes as well as the lympho-cytes which are found in primary (bone marrow and thy-mus) and secondary (lymph node, spleen, Peyer’s patches,and bronchus-associated lymphoid tissue) lymphoid organs.

Cells of the immunocytic system respond specifically toantigens by undergoing clonal expansion and differentia-tion into highly specialized effector cells and memory cells.Effector cells fall into two major classes:

1) T cell lymphocytes, which release a variety of bio-logically active molecules known as lymphokinesand play a major role in cell-mediated immunity.

2) B cell lymphocytes, which produce immunoglobu-lins (antibodies) and constitute the humoralimmune system.

T and B cell classes can be further subdivided into anumber of subsets based upon cell surface markers.

T cell subsets include:� helper cells, which are necessary for the full

expression of the immune response,� suppressor cells, which dampen or moderate the

immune response, and� null cells (essentially devoid of surface markers),

which include natural killer (NK) cells and K cells.� NK cells release biologically active molecules

capable of destroying other cells or microor-ganisms.

� K cells participate in antibody-depen-dent cytotoxicity.

B cell subsets preferentially produceimmunoglobulins belonging to a particular class:

� IgG � IgD� IgM � IgE� IgA

In addition to the obvious interplay among thelymphocyte classes and subclasses, lymphocytesalso influence and are influenced by phagocytes.Among the lymphokines that T and B cells pro-duce are numerous molecules that are chemotacticfor both granulocytes and monocyte/macrophages.Some lymphocyte products may influence marrowproduction of phagocytes. Opsonization, as previ-ously discussed, leads to enhanced phagocytosis.

Knowledge of the immunocytic system and its

complex functions has expanded dramatically in recentyears. The study of lymphocytes and their various roles hasevolved into the science of immunology, a review of whichis beyond the scope of this chapter. Instead, the next sec-tion will focus on circulating lymphocytes and the interpre-tation of changes in circulating numbers and morphology.

Normal lymphocyte structure and functionCirculating lymphocytes include representatives of T

cell, B cell, and null cell classes. In most species, approxi-mately 70% of circulating lymphocytes are T cells, 10% to15% are B cells, and the remainder is comprised of nullcells. Normal cells of the various classes cannot be differen-tiated morphologically using routine Romanowsky hemato-logic stains.

Normal circulating lymphocytes possess the followingmorphologic characteristics:

� round shape measuring 9.0 µ to 12.0 µ in diameter� large, round, eccentric nuclei with densely stained,

clumped chromatin patterns (Fig. 8)� scant, pale blue cytoplasm, usually observable only

on one side of the nucleusFor the most part, circulating lymphocytes are long-lived

“memory cells” which traverse back and forth betweenblood, lymph nodes, and lymph, monitoring for the presenceof antigens to which they have been previously sensitized.When stimulated by such antigens, lymphocytes undergoblast transformation or activation. Activation is a normalprocess. However, increased relative or absolute numbers ofactivated lymphocytes (also known as reactive lymphocytes

Fig. 8 Normal small lymphocyte (round nucleus), three neutrophils, and normalband cell (right).


or immunocytes) indicates antigenic stimulation. Morphologically, blast transformed lympho-

cytes are generally more heterogeneous thanunstimulated lymphocytes (Figs. 9 through 13).They are larger than unstimulated lymphocyteswith larger, more lacy nuclei and increasedamounts of deep blue cytoplasm. Morphologically,activated B lymphocytes cannot be differentiatedfrom activated T lymphocytes. However, fully dif-ferentiated B cells are plasma cells that are mor-phologically distinct (Fig. 14). Plasma cells haveeccentric round nuclei with coarsely clumpednuclei, pale perinuclear clear zones (golgi zones),and abundant deep blue cytoplasm. Plasma cellsare extremely rare on blood films except in casesof plasma cell tumor (multiple myeloma).

Normal lymphocyte counts are between1000/µl and 5000/µl in dogs, and in cats the num-bers may be as high as 7000/µl. Counts ofbetween 1000/µl and 1500/µl are regarded as mar-ginal lymphopenias. Both lymphopenia and lym-phocytosis are relatively common clinical occur-rences.

Lymphopenia in the range of 700/µl to 1500/µlis most likely a reflection of high levels of circulat-ing glucocorticoids (ie, a stress reaction) whereincreased numbers of lymphocytes marginatealong vessel walls or, in more chronic conditions,may be lysed. With more marked lymphopenia,processes which block the normal circulatory pat-tern of lymphocytes (blood to tissue to lymph andback to blood) must be considered. These includelymphosarcomas where lymphocytes leaving theblood become trapped in massively enlargedlymph nodes, or chylous effusions where lymphat-ic rupture leads to lymphocyte (and protein)sequestration in the body cavities.

Lymphocytosis occurs in cases of inflammationwith antigenic stimulation, lymphocytic leukemia,and lymphosarcomas with leukemic events. Incases of leukemic lymphocytosis, circulating lym-phocytes are almost always abnormal lym-phoblasts. These cells are larger than normal withless intensely stained nuclei containing largenucleoli. Leukemias are also often accompaniedby marked non-regenerative anemias. In cats,lymphocytosis can also occur physiologically,

Fig. 10 Reactive lymphocyte (lower center), three neutrophils, and a monocyte (topcenter).

Fig. 11 Reactive lymphocyte (center) and four neutrophils.

Fig. 9 Blast-transformed (reactive, activated) lymphocyte.


where excitement (ie, epinephrine release) causesincreased blood flow, washing marginated lympho-cytes into the circulating mainstream where theycan be readily sampled and counted. Physiologiclymphocytosis in cats, with lymphocytes counts ashigh as 20,000/µl, has been observed. Lymphocytes,in these cases, are morphologically normal andthere is no anemia.

Abnormal Leukocyte Morphology

Neutrophil toxicityToxicity in neutrophils is seen when circulating

toxins interfere with the development and differen-tiation of neutrophil precursors in the bone mar-row. In dogs and cats, toxicity is most commonlycaused by circulating bacterial toxins. However, tis-sue necrosis, a variety of drugs, and numerous non-specific toxins such as lead are all capable of inter-fering with neutrophil development. Toxic neu-trophils in the blood are best interpreted as indicat-ing the presence of unspecified systemic toxemia.

Because toxicity indicates maturation arrest, thestage of development affected may be important inassessing prognosis or gauging response to therapy.In cases where both mature neutrophils and bandcells are equally affected, no such interpretation ispossible. However, in cases where mature neu-trophils are toxic but bands are normal, morpholo-gy would suggest that the systemic toxemia isresolving. In contrast, in cases where bands aretoxic but mature neutrophils are normal, morpholo-gy suggests a worsening condition.

Morphologically, toxic neutrophils exhibit avariety of alterations that reflect either cytoplasmicor nuclear developmental arrests or both (Figs. 15through 18). The most common form of toxicity indog and cat neutrophils is foamy basophilia of thecytoplasm. This is a reflection of retention of cyto-plasmic RNA and failure of the affected cells toform their normal complement of protein and cyto-plasmic granules. Döhle bodies, a relatively minorsign of toxicity in cat neutrophils but a sign ofmarked toxicity in dog neutrophils, are simplyintracytoplasmic precipitates of RNA and appear asblue basophilic cytoplasmic inclusions. Some toxic

Fig. 12 Reactive lymphocyte

Fig. 13 Reactive lymphocyte

Fig. 14 Fully differentiated plasma cell.


neutrophils are extremely large; this is a reflectionof nuclear maturation arrest characterized by fail-ure of developing cells to divide properly. Bizarrenuclear shapes and even multiple nuclei also areindicative of nuclear maturation arrests.

Atypical lymphocytes“Atypical lymphocyte” is a term used to

describe a heterogeneous group of circulatingcells of the lymphoid series with a variety of eithernuclear or cytoplasmic abnormalities or both.Atypical lymphocytes are morphologically distinctfrom reactive lymphocytes (immunocytes, activat-ed lymphocytes), which are normal antigen-stimu-lated lymphocytes.

Nuclear and cytoplasmic features determineatypical classification. Lymphocytes containing

Fig. 16 Giant toxic band with foamy, basophilic cytoplasm and giant, irregularnucleus.

Fig. 17 Two toxic neutrophils and a toxic band. Note the unusual nuclear shape.

Fig. 15 Toxic band cell with foamy, basophilic cytoplasm


large, angular nucleoli are sometimes classified asatypical, although these cells may simply be reac-tive. Lymphocytes with clefted nuclei (Reider-formnuclei) are considered atypical. The presence oflarge, azurophilic cytoplasmic granules in lympho-cytes is also regarded as atypical (Fig. 19).

The significance of observing atypical lympho-cytes on blood films is uncertain. Historically, atyp-ical lymphocytes were thought to indicate viralinfections. We now know that this relationship isobscure; animals ill from a variety of causes mayhave increased numbers of circulating typical lym-phocytes. The presence of atypical lymphocytes inthe blood should be noted, but no particular inter-pretation should be made on the basis of this find-ing alone.

Fig. 18 Two toxic neutrophils with Döhle bodies.

Fig. 19 Atypical lymphocyte


Overview

The primary functions of the erythron, or redblood cell (RBC), are as follows:

� to accumulate oxygen at the alveolar sur-face of the lung,

� to transport and release it to all the cells ofthe body,

� to replace the released oxygen with thewaste gas, carbon dioxide, and

� to transport the carbon dioxide back to thealveolus where it can be removed from thebody via expiration.

To accomplish these functions, mammalian redcells have evolved into highly sophisticated yet simpletransport vehicles comprised of hemoglobin (a solubletransport protein) sealed in a protective cell membrane.This architecture gives the red cells great flexibility, allow-ing them to circulate through the most tortuous of vascularspaces. The metabolic pathways of red cells, glycolysis andthe hexose monophosphate shunt, serve primarily to main-tain the integrity of the cell membrane and the hemoglobinmolecule.

In health, circulating red cell mass, and therefore oxygencarrying capacity, is kept remarkably constant from day today and year to year. For each species, red celllifespan is finite and preprogrammed. The rate ofproduction of new red cells, as regulated by circu-lating erythropoietin levels, is inversely related tored cell lifespan. In dogs, circulating red cell lifes-pan is approximately 100 days; therefore approxi-mately 1% of circulating red cells die and must bereplaced on a daily basis. Cat red cells have a short-er average lifespan (approximately 80 days); there-fore, a somewhat higher rate of red cell productionrate is required to maintain normal circulating redcell mass.

Red cell morphology is also unique for eachspecies. Dog red cells measure 6.5 µ to 7.0 µ indiameter, have prominent areas of central pallor,are uniformly round, and show little variability insize and shape from cell to cell (Fig. 1). Mean cell

volume (MCV) is 60 femtoliters (fl) to 75 fl. Approximately1% of the red cells are larger than normal and stain with abluish cast on Romanowsky-stained films; these are imma-ture red cells known as polychromatophils (Fig. 2). Theirbluish cast is a reflection of their higher content of cyto-plasmic RNA and reduced content of hemoglobin. Normalcanine red cells show moderate tendency to form stacks orrows (rouleau formation).

Feline red cells are smaller than those of dogs, measur-ing 5.0 µ to 6.0 µ in diameter with MCVs of 40 fl to 55 fl.Central pallor is minimal. The proportion of polychro-

Chapter 2: Erythrocytes in Health and Disease

Fig. 1 Normal canine blood film. Scanning magnification. RBCs are uniform insize, shape, and color and feature prominent central pallor.

Red Cell Morphology

Canine Feline

� �

6.5 µ to 7.0 µ in diameter, 5.0 µ to 6.0 µ in diameteruniformly round

Prominent areas of Minimal central pallorcentral pallor

MCV = 60 fl to 75 fl MCV = 40 fl to 55 fl

Moderate rouleau formation Marked rouleau formation


matophils is higher in cats than in dogs (up to 2% in cats).This is a reflection of shorter feline red cell lifespan andtherefore greater marrow production and release of reticu-locytes. There is marked rouleau formation in cats.

Disorders of the erythron are essentially disorders of redcell mass (ie, total red cell count, hematocrit, and hemoglo-bin). Red cell mass can be increased (poly-cythemia) or decreased (anemia). The followingparagraphs detail the diagnostic approach to theevaluation of these two broad categories of disease.

PolycythemiaPolycythemia is diagnosed when measures of red

cell mass are increased. Polycythemia can be rela-tive or absolute.

Relative polycythemia is the result of hemocon-centration (dehydration) and is by far the mostcommon form of polycythemia in dogs and cats.Elevated total protein as well as an elevation intotal red cell count and hematocrit characterize thecondition, which is reversed by returning bloodvolume to normal.

Absolute polycythemia is either secondary orprimary. Secondary absolute polycythemia occursas a result of increased production of erythropoi-etin, either appropriately as a compensatoryresponse to diseases where there is reduced oxy-genation of the tissues (eg, heart disease or pneu-monia) or inappropriately in rare cases of renal dis-ease or renal neoplasia. There are no peripheral

blood morphologic abnormalities in secondarypolycythemia. Primary absolute polycythemia ispolycythemia vera, a rare myeloproliferative dis-order. Polycythemia vera is characterized byexpanded red cell production in the marrow butno morphologic abnormalities in the peripheralblood. The condition is diagnosed by ruling outsecondary causes of polycythemia and demon-strating normal blood gas values in the face of ele-vated red cell mass measures.

AnemiaAnemia is one of the most common syndromes

in all of veterinary medicine and is associated witha wide range of specific diseases. Anemias fall intotwo broad categories: regenerative and non-regen-erative.

Regenerative anemia is characterized by appropriatebone marrow response with release of increased numbersof normal immature red cells into the blood. Regenerativeanemia is either the result of blood loss (hemorrhage) orhemolysis.

In non-regenerative anemia, marrow response is ineffec-

Anemia

I. Regenerative Anemias:Blood Loss/Hemorrhagic AnemiaHemolytic Anemias

Immune-mediated hemolytic anemiasHeinz body hemolytic anemiaInfectious hemolytic anemias

HemobartonellosisBabesiosis

Hereditary hemolytic anemiaMicroangiopathic hemolytic anemiaMetabolic anemia with spiculated red cells

II. Non-regenerative Anemias:Maturation Defect Anemias

Nuclear maturation defectCytoplasmic maturation defect

Hypoproliferative AnemiasDue to inflammatory diseaseDue to reduced erythropoietinMyelophthisic anemiaDue to marrow toxicity

Fig. 2 Canine blood film with increased polychromasia. Scanning magnification.Polychromatophils are bluish and generally larger than mature cells.


tive or inadequate and immature red cells are notreleased into the blood in sufficient numbers.Causes of non-regenerative anemia are severaland often require bone marrow examination fordifferentiation.

The first step in classifying an anemia as regen-erative or non-regenerative is evaluation of theperipheral blood film (Figs. 2, 3). Increased num-bers of polychromatophils on the film suggestsregeneration. Because polychromatophils are larg-er than mature cells and have less hemoglobin,MCV may be increased and mean cell hemoglobinconcentration (MCHC) may be reduced.

If significant polychromasia is present, deter-mining reticulocyte numbers can more accuratelyassess regeneration. This is done by mixing asmall amount of EDTA blood with an equal vol-ume of a vital stain such as new methylene blue.The mixture is allowed to incubate at room tem-perature for thirty minutes and then air-driedblood films are made. The cytoplasmic RNA inimmature red cells is precipitated by the newmethylene blue as a dark blue reticulum. Cellscontaining this precipitate are identified as reticu-locytes (Figs. 4, 5).

In dogs, all cells with reticulum are countedwhen determining a reticulocyte count. In cats,three types of reticulocytes may be identified andare distinguished by the amount of reticulum pre-sent: 1) punctate reticulocytes, which have only focal

precipitates, 2) aggregate reticulocytes, which contain an

extensive network of reticulum, and 3) intermediate reticulocytes, which have interme-

diate amounts of precipitate. Only aggregate reticulocytes are counted in cats.

Absolute reticulocyte counts are used to judgewhether or not regeneration is present. The num-ber of appropriate reticulocytes per 1000 total redcells is noted and converted to a percentage. The

Fig. 4 Low magnification of a new methylene blue-stained blood film.Reticulocytes stand out due to blue-stained reticulum visible at thismagnification. This represents a highly regenerative anemia.

Fig. 3 Higher magnification of Fig. 2. This represents a regenerative anemia withmarked polychromasia. Note the marked separation of RBCs.

Fig. 5 High magnification of Fig. 4. Reticulocytes with precipitated blue reticulumare obvious. In dogs, all would be counted; in cats, only those withabundant precipitate (aggregate reticulocytes) would be counted. Severalleukocytes are also present.

Absolute Reticulocyte Count Formula

� # reticulocytes/1000 RBC

� convert to %

� x total RBC count


percentage can then be multiplied by the total redcell count to obtain an absolute reticulocyte count.An absolute reticulocyte count of greater than80,000/µl is indicative of regeneration in both dogsand cats.

Regenerative Anemias

Blood loss anemiaIn hemorrhagic anemias, circulating red cell

lifespan is normal but red cells are lost from thebody due to external bleeding. A history of bleed-ing or physical findings consistent with blood lossgenerally makes the diagnosis fairly obvious inthese cases. The degree of regeneration in bloodloss anemia is moderate (no more than two tothree times normal) and is a function of the irondepletion that accompanies red cell loss. The avail-ability of iron for incorporation into hemoglobindirectly influences the rate of red cell productionand, therefore, the degree of peripheral reticulocy-tosis.

Cases of severe or chronic blood loss may actu-ally be non-regenerative because of the lack of iron.Iron deficiency anemia, the end stage of blood lossanemia, is described under Cytoplasmic MaturationDefects in the section titled Non-regenerativeAnemias of this chapter.

Hemolytic anemiasThe essential feature of hemolytic anemias is

shortened red cell lifespan. Hemolysis can occurintravascularly as the cells circulate or extravascu-larly within macrophages of the liver and spleen. Ineither case, iron is conserved in the body and isreadily available for reincorporation into hemoglo-bin. As a result, hemolytic anemias are often morehighly regenerative than blood loss anemias withreticulocyte counts sometimes exceeding threetimes normal.

In many forms of hemolytic anemia, there arespecific morphologic indicators of the underlying eti-ology. Included among these are immune-mediatedhemolytic anemias, Heinz body hemolytic anemia,infectious hemolytic anemias, hereditary hemolyticanemia, microangiopathic hemolytic anemia, andmetabolic anemia with spiculated red cells.

Fig. 6 Canine immune-mediated hemolytic anemia. The anemia is highly regenerativewith marked polychromasia. A spherocyte (arrow).

Fig. 7 A second case of canine immune-mediated hemolytic anemia withnumerous spherocytes.

Fig. 8 Feline immune-mediated hemolytic anemia. Spherocytes are numerous butless obvious due to the lack of central pallor in the normal RBCs.


Immune-mediated hemolytic anemiasImmune-mediated hemolysis occurs when

antigen-antibody complexes form on the surface ofcirculating red cells. The antibody may be directedagainst an antigen in the red cell membrane itself(autoimmune hemolytic anemia) or against a for-eign antibody (eg, drug, infectious agent) carriedon or bound to the red cell surface. Regardless ofthe site of antibody activity, the antigen-antibodycomplex activates complement, which leads eitherto intravascular lysis or removal of red cells bymacrophages in the spleen and liver.

Immune-mediated hemolytic anemias are typicalregenerative anemias, characterized by markedpolychromasia and anisocytosis (variable cell size –Fig. 6). The presence of significant numbers ofspherocytes is a specific morphologic indicator ofimmune-mediated hemolysis, although they mayoccur in low numbers in other conditions.Spherocytes are round, smaller than normal,intensely stained, and lack central pallor (Fig. 7).They are more difficult to recognize in cats than indogs because feline red cells are normally fairlysmall and lack central pallor (Fig. 8).

Another feature of some immune-mediatedhemolytic anemias is autoagglutination (three-dimensional clumping of RBCs).Autoagglutination, the result of true cross-linkingof red cells by antibodies, confirms the diagnosis ofimmune-mediated disease.

Autoagglutination must be differentiated fromrouleau formation. This is done by placing a drop of well-mixed EDTA blood on a slide, adding an equal or slightlylarger volume of isotonic saline, coverslipping the mixture,and evaluating the finished wet preparation under themicroscope (Fig. 9). If clumping persists, autoagglutinationis present. Rouleau, the result of attraction between red cellson the basis of surface charge, will be dissipated by the dilu-tional effect of the saline.

In the absence of autoagglutination, immune-mediatedhemolysis can be confirmed with a direct antiglobulin test(DAT). The DAT is performed by mixing well-washedsuspect red cells with anti-complement and serial dilutionsof commercially available species-specific anti-IgG.Microscopic evidence of agglutination of red cells confirmsthe diagnosis.

Heinz body hemolytic anemiaHeinz bodies are masses of precipitated hemoglobin

formed when increased levels of circulating oxidants over-whelm red cell biochemical defense mechanisms and dam-age globin. The presence of Heinz bodies interferes withred cell flexibility, reducing red cell lifespan. Cells contain-ing Heinz bodies are either lysed as they squeeze throughtortuous vascular spaces (intravascular hemolysis) or aretrapped in the spleen and are removed by macrophages(extravascular hemolysis). Because Heinz bodies oftenbecome attached to the inner red cell membrane, they maybe recognized on peripheral blood films as nipple-like pro-jections from the red cell surface (Fig. 10). They have thesame staining properties as normal hemoglobin withRomanowsky stains, but stain a royal blue with new meth-ylene blue (Fig. 11).

Fig. 9 Saline-diluted wet preparation demonstrating autoagglutination.

Fig. 10 Heinz body hemolytic anemia in the dog. Several red cells with nose-likeprojections (Heinz bodies) are present. The leukocyte at left is a band cell.

There are species differences in susceptibility toHeinz body formation and Heinz body hemolyticanemia. Dogs are quite resistant to Heinz bodyformation; the demonstration of Heinz bodies onblood films is enough to confirm the diagnosis ofHeinz body hemolytic anemia in dogs.

In contrast, the hemoglobin of cats is easily oxi-dized and Heinz body formation is quite common.In fact, even in health, up to 10% of normal felinered cells contain Heinz bodies. In metabolic disor-ders such as hyperthyroidism, diabetes mellitus, orliver disease, the number of red cells with Heinzbodies can increase dramatically (up to 80% orhigher) without the presence of hemolysis or ane-mia (Fig. 12). In cats, therefore, the diagnosis ofHeinz body hemolytic anemia requires not onlythe presence of Heinz bodies but also the presenceof a highly regenerative anemia.

In cats, most cases of Heinz body hemolyticanemia are caused by the use of oxidant drugs,such as aspirin. In contrast, drug-induced Heinzbody hemolytic anemia in dogs is relatively rare.The most common cause of spontaneous Heinzbody hemolysis in dogs is ingestion of largeamounts of raw onions, which contain the oxi-dant, N-propyl disulfide.

Infectious hemolytic anemiasInfectious hemolytic anemias include lep-

tospirosis, hemobartonellosis, and babesiosis.There are no specific morphologic indicators ofbacterial-induced hemolysis. In contrast, in bothhemobartonellosis and babesiosis, etiologic agentsare seen on peripheral blood films.

HemobartonellosisHemobartonellosis occurs in both dogs and cats.

In dogs, the disease is uncommon and occurs almostexclusively in splenectomized or immunosuppressedpatients. Clinically, canine hemobartonellosis pre-sents as a typical hemolytic anemia with markedpolychromasia and anisocytosis on routine periph-eral blood films. Hemobartonella canis organisms areseen as chains of small (0.5 µ) basophilic bodies onthe red cell surface (Fig. 13).

In cats, the disease may be primary or mayoccur secondarily to primary immunosuppressive

Fig. 11 Canine Heinz body hemolytic anemia, new methylene blue stain. Severalcells with Heinz bodies (upper left). Two reticulocytes with aggregatedreticulum (right center).

Fig. 12 Feline hyperthyroidism. Numerous cells have obvious Heinz bodiesprojecting from their surfaces, but anemia is not present.

Fig. 13 Canine hemobartonellosis. The cell at lower right center has several chainsof basophilic organisms on its surface.



disorders, such as feline infectious peritonitis (FIP) orfeline leukemia virus (FeLV) infections.

Primary feline hemobartonellosis is a typical hemolyticanemia with all the attendant signs of marked red cellregeneration (Fig. 14). Agglutination also may be present,suggesting an immune component to the disease. As indogs, Hemobartonella organisms (Hemobartonella felis) areseen on the red cell surface. They are generally slightlylarger than H. canis, and may be present as chains, individ-ual basophilic bodies, or ring forms. Splenomegaly is acommon clinical finding in cats with hemobartonellosis.Primary feline hemobartonellosis responds well to tetracy-clines with good prognosis for recovery.

Secondary feline hemobartonellosis is far more commonand the prognosis is poor. The anemia is generally non-regenerative because the marrow is suppressed and cannot

respond. The diagnosis is based on the presenceof large numbers of organisms on blood films.These are removed with tetracycline therapy, butthis does not resolve the underlying disease.Because of the close association of hemobartonel-losis with immunosuppressive viruses, all cases offeline hemobartonellosis should be tested forfeline immunodeficiency virus (FIV), FIP, andFeLV.

BabesiosisBabesiosis is a relatively uncommon tick-borne

hemolytic anemia of dogs. The causative organ-ism, Babesia canis, is a teardrop-shaped protozoanmeasuring 2.0 µ to 2.5 µ in length.

B. canis organisms are found in varying num-bers within red cells on blood films from affectedanimals (Fig. 15). Anemia is often rapid in onsetand can be quite severe and highly regenerative.There is often an immune-mediated hemolyticcomponent to the disease; spherocytes may beobserved in significant numbers. There have beencases reported with low numbers of organisms ini-tially misdiagnosed as immune-mediated hemolyt-ic anemia. Inappropriate treatment of thesepatients with steroids results in reduced removalof organisms by splenic macrophages and a con-commitant buildup of organisms in the blood.Upon re-evaluation, the organisms are readilyapparent, and a correct diagnosis can be made.

Hereditary hemolytic anemia Two forms of hereditary hemolytic anemia associated

with glycolytic enzyme deficiencies have been described indogs: pyruvate kinase deficiency and phosphofructokinasedeficiency.

Pyruvate kinase deficiency is described in Basenjis andBeagles, and is characterized as a chronic hemolytic anemiawith extremely high reticulocyte counts. Shortened red celllifespan is a reflection of decreased ATP production andreduced stability of the red cell membrane. Pyruvate kinasedeficiency is generally diagnosed at 3 to 6 months of agewhen the anemia is moderately severe. Hematocrit valuescontinue to decline slowly thereafter as the compensatorycapacity of the marrow deteriorates. The condition may ter-minate in marrow exhaustion, myelofibrosis, or both.

Phosphofructokinase deficiency has been described in

Fig. 14 Feline hemobartonellosis. Several cells near the center have multiplebasophilic Hemobartonella felis organisms on their surfaces.

Fig. 15 Canine babesiosis. The cell (center) has two teardrop-shaped organisms.A red cell (lower right) with a single organism.


two Springer Spaniels. The condition is less severethan pyruvate kinase deficiency, and is character-ized by moderate chronic hemolysis with superim-posed episodes of more severe hemolysis associatedwith exercise and overheating.

Microangiopathic hemolytic anemiaAll of the hemolytic conditions described thus

far have been characterized by either an intrinsical-ly or extrinsically induced defect in the circulatingred blood cells leading to shortened red cell lifes-pan. Shortened red cell lifespan can also occurwhen normal red blood cells are forced to circulatethrough abnormal vascular beds. This is microan-giopathic hemolysis.

Microangiopathic hemolysis can occur in anycondition where the morphology of capillary bedsis altered, such as: disseminated intravascular coag-ulopathy, where capillary lumens are distorted bythe deposition of fibrin; hemangiosarcoma, wherethere is localized intravascular coagulopathy;glomerulonephritis, where glomerular tuft architec-ture can be obliterated; and some cases of cardiacdisease, where blood flow patterns can be markedlyaltered. Microangiopathic hemolysis with red cellfragmentation can also be a feature of heartwormdisease.

In dogs, microangiopathic hemolysis is charac-terized by the presence of red cell fragments calledschizocytes (Fig. 16); in cats, fragments are rarelyseen. The degree of anemia, and therefore, thedegree of regeneration, is mild to moderate.

Metabolic anemia with spiculated red cellsIn dogs, anemias with spiculated red blood cells

are associated with some cases of liver and kidneydisease. These abnormal red cell shapes are proba-bly caused by abnormal lipid metabolism and sec-ondary altered plasma free cholesterol/phospholipidratios. Abnormal plasma lipids equilibrate with thelipids in the red cell membrane and spiculated cellsresult. Spiculated red cells are less flexible thannormal, thus some degree of hemolysis occurs.Despite the hemolytic component, these anemiasare complex and generally non-regenerativebecause of the inability of the marrow to respondappropriately.

Fig. 16 Microangiopathic hemolysis in disseminated intravascular coagulopathy(DIC). Numerous schizocytes.

Fig. 17 Hepatic hemangiosarcoma in a dog. Two acanthocytes (center). Aschizocyte (left). Numerous target cells, which can be precursors toacanthocytes, are also present. Polychromasia indicates a mildlyregenerative anemia.

Fig. 18 Canine glomerulonephritis. Numerous Burr cells.


Spiculated red cells are of two basic types: acanthocytesand burr cells. Acanthocytes are red cells with 2 to 10blunt, elongated, finger-like projections on the red cell sur-face (Fig 17). Burr cells are oval-shaped red cells with ruf-fled margins (Fig. 18). Although both cell types can be seenwith either liver or kidney disease, generally acanthocytesare associated with liver disease while burr cells are morecommon in kidney disease.

Non-regenerative AnemiasNon-regenerative anemias can be grouped into two

broad categories: maturation defect anemias and hypopro-liferative anemias. In most cases, bone marrow evaluationis required to render a definitive diagnosis in either type ofnon-regenerative anemia.

Maturation defect anemiasMaturation defect anemias are the result of

acquired bone marrow abnormalities in whicheither nuclear or cytoplasmic development of redcell precursors is arrested. The defect may be in redcell precursors only or may affect all proliferatingmarrow cell lines. The marrow is generally hyper-cellular; however, since increased numbers of nor-mal immature RBCs are not released into circula-tion, erythropoiesis is ineffective.

Nuclear maturation defect anemiaNuclear maturation defect anemia is relatively

common in cats but quite rare in dogs. Clinically, itis a mild to severe non-regenerative anemias withassociated leukopenia and thrombocytopenia.Pancytopenia indicates a marrow disorder involv-ing all cell lines.

Variable numbers of excessively large, fullyhemoglobinized red cells (macrocytes) are observedon blood films. If these are numerous enough in theblood, MCV is elevated while MCHC remains nor-mal. Occasional abnormal nucleated red cells mayalso be observed on blood films (Fig. 19). These areusually quite large with large immature nuclei butfully hemoglobinized cytoplasm. This morphologyindicates arrested nuclear development in the mar-row; these cells are termed megaloblasts.

Peripheral blood film morphology is suggestiveof nuclear maturation defect anemia. However, fordefinitive diagnosis, marrow evaluation is

required. Marrow samples are hypercellular withincreased activity in all cell lines. All cell lines featurearrested nuclear development with normal cytoplasmicdevelopment. Numerous megaloblasts are seen among thered cell precursors (Fig. 20). Granulocytes have normalcytoplasm but most nuclei are deficient in chromatin andrarely mature past the myelocyte stage. Megakaryocytesare smaller than normal and contain only one to two nucleirather than the normal 8 to16.

The most common cause of nuclear maturation defectanemia in cats is FeLV, which appears to interfere directlywith DNA synthesis. In dogs, nuclear maturation anemiaresults from functional folic acid or vitamin B12 deficiency.These can be true nutritional deficiencies—which is rare inanimals—or drug-induced, resulting in folate block.

Fig. 19 FeLV (peripheral blood film). Two megaloblasts.

Fig. 20 Bone marrow smear from the same case as Fig. 19. A large megaloblast(right).


Chemotherapeutic agents such as methotrexate and dilantinare known folate antagonists.

Cytoplasmic maturation defect anemiaCytoplasmic maturation defect anemia is characterized

by normal nuclear development but arrested cytoplasmicdevelopment (ie, lack of normal hemoglobinization). Themain cause in all animals is iron deficiency. Iron deficiencyanemia is the end stage of blood loss.

Iron deficiency anemia is morphologically distinctive,particularly in the dog. Red cells are smaller than nor-mal and have exaggerated areas of central pallor. Redcell indices often feature reduced MCV and MCHC.Iron deficient red cells are more fragile than normal;blood films often have significant numbers of red cellfragments (Fig. 21).

Hypoproliferative anemiasNon-regenerative hypoproliferative anemias are the

most common anemias in domestic animals. In most cases,peripheral blood findings are nonspecific; bone marrowevaluation is essential for evaluation. Hypoproliferativeanemias fall into four general categories: anemia of inflam-matory disease, anemia due to reduced erythropoietin,myelophthisic anemia, and anemia due to marrow toxicity.

Anemia of inflammatory diseaseThe anemia of inflammatory disease is the most common

of all anemic syndromes. It is a mild to moderate normo-cytic, normochromic, non-regenerative anemia with no dis-

tinctive morphologic features. Diagnosis is pre-sumptive, based upon evidence of inflammatorydisease, including an inflammatory leukogram.Bone marrow findings are supportive and includegranulocytic hyperplasia, mild erythroid hypopla-sia, plasma cell hyperplasia, and accumulation ofhemosiderin (iron) in marrow macrophages.

Anemia due to reduced erythropoietinIn many cases of end-stage renal disease, ery-

thropoietin production by the kidney is reduced.This results in normocytic, normochromic, non-regenerative anemia. In hypothyroid animals,there are also reductions in circulating erythropoi-etin primarily because of reduced metabolicdemands for oxygen at the tissue level. Onceagain, the net effect is normocytic, normochromic,non-regenerative anemia.

Myelophthisic anemiaMyelophthisic syndromes are conditions in which an

aberrant cell population replaces normal bone marrow.Aberrant populations may be either benign (eg, fibrocytesin myelofibrosis) or malignant (eg, leukemia). Because ofmarrow replacement, cytopenia of one or more cell lines istypical in the peripheral blood. Anemias are severe andnon-regenerative. Leukopenia often is also quite severe, butplatelet numbers are more variable.

In most cases, peripheral red cell morphology is unre-markable. However, in some cases of myelofibrosis in dogs,poikilocytosis is marked. A particular form of poikilocyte,the dacryocyte, has been associated with myelofibrosis indogs (Fig. 22). Dacryocytes are teardrop-shaped erythro-cytes.

Whenever a myelophthisic syndrome is suspected (ie,whenever severe cytopenias are present), a bone marrowexamination is indicated. Marrow aspirates in myeloph-thisic syndromes may be hypercellular and immediatelydiagnostic or hypocellular (dry tap) and nondiagnostic;therefore, both marrow aspirates and marrow core biop-sies should be collected in suspected cases. Marrow aspi-rates from cases of myelofibrosis are always hypocellular.

Anemias due to marrow toxicity Marrow cytotoxicity can be caused by both infectious

and noninfectious etiologies. Infectious causes include dis-eases such as canine and feline parvovirus infections, FeLV

Fig. 21 Iron deficiency anemia. Numerous red cells have exaggerated areas ofcentral pallor; some are smaller than normal. A red cell fragment(schizocyte—center).


infection, FIV infection, and canine ehrlichiosis. Inaddition to direct marrow cytotoxicity, some ofthese agents may cause secondary immune-mediat-ed marrow suppression. Noninfectious causes ofmarrow toxicity include a variety of etiologies suchas estrogen toxicity, cancer chemotherapeuticagents, and ionizing radiation.

The peripheral blood indicator of marrow toxici-ty is progressive cytopenia of one or more cell lines.Clinical signs depend upon which cell lines areaffected and how severely. In the case of red cells,the degree of anemia can be quite severe and isnon-regenerative. Patients with this kind of anemiaare lethargic with extremely pale mucous mem-branes. Patients with severe thrombocytopenia maypresent with bleeding disorders while those withsevere granulocytopenia may be presented becauseof secondary inflammatory conditions.

As with myelophthisic syndromes, bone marrow aspi-rates and core biopsies are required for diagnosis. In acutemarrow toxicity, bone marrow necrosis may be seen histo-logically. Cytologic signs of acute toxicity in marrow pre-cursor cells include cytoplasmic vacuolation and basophilia,nuclear vacuolation, bizarre nuclear shapes, and cytonu-

clear dissociation. In marrow toxicity of longer duration,the principal alteration is hypocellularity; this must be eval-uated histologically with core biopsies. If the condition ischaracterized by hypocellularity of all cell lines, the condi-tion may be termed an aplastic anemia. Prognosis in thesecases is guarded.

Fig. 22 Myelofibrosis in the dog. A dacryocyte (center and top center) andnumerous ovalocytes.


Overview

Platelets are the third cellular component of the periph-eral blood; yet, they are often overlooked in both quantita-tive and qualitative peripheral blood evaluation. Since 90%or more of the bleeding disorders in dogs and cats resultfrom abnormalities in platelet number or function, the clini-cal significance of these cells should not be underestimated.Furthermore, platelets contain a significant number of bio-logically active molecules that moderate such events asinflammation, neovascularization, thrombosis, hemostasis,fibrinolysis, and coagulation.

On routine blood films, platelets are recognized as smallanucleate discoid cytoplasmic fragments containing variablenumbers of purple granules. Ultrastructurally, they aremuch more complex. Shape is apparently maintainedthrough the interaction of a cytoplasmic microtubular coiland an actin membrane skeleton located beneath the plas-ma membrane. The plasma membrane has numerousinvaginations, which form the surface-connected canalicu-lar system (SCCS). Central to the microtubular coil are the

principal platelet organelles: two types of granules (alphaand dense), the dense tubular system (DTS), mitochondria,lysosomes, and peroxisomes. (See Fig. 1)

The plasma membrane and the SCCS are covered by aglycoprotein glycocalyx to which coagulation factors andplasma proteins such as immunoglobulins are adhered.Within the glycocalyx are receptors for the various plateletagonists that initiate the stages of platelet activation. Theplasma membrane is rich in phospholipids, which give rise toprostaglandins and leukotrienes, important molecular media-tors of the inflammatory milieux. The plasma membraneitself is also the main source of platelet factor 3 (PF 3), acoagulation co-factor.

Both types of platelet granules also contain a host of bio-logically active molecules that are important to blood clottingand other life processes, and are released during platelet acti-vation. Dense granules are a source of ATP, ADP, serotonin,and calcium. Alpha granules contain and ultimately releaseplatelet factor 4 (PF 4), von Willebrand’s factor (VWF), fib-rinogen, and coagulation factor V.

The DTS is derived from endoplasmic reticulum and isthe probable site of platelet prostaglandin synthe-sis. Calcium, an important component in plateletaggregation, is also stored in high concentrationsin the DTS.

Production of Platelets

Like all circulating blood cells, platelets arebone marrow derived. The first recognizableplatelet precursor is the megakaryoblast.Megakaryoblasts undergo endomitosis (nucleardivision without cytoplasmic division) to formmegakaryocytes. Megakaryoblasts have a DNAploidy of 2N to 4N. Fully developed megakary-ocytes may have a ploidy of up to 64N. Becausethese cells do not divide as they mature, the num-ber of marrow megakaryocytes is relatively low.However, as they undergo endomitosis theybecome quite large and are easily recognized inboth cytologic and histologic marrow prepara-tions.

Chapter 3: Platelets in Health and Disease

Fig. 1 Ultrastructure of the platelet.

ALPHAGRANULESAdhesive proteins

•VWF•thrombospondin•fibrinogen

Growth modulators

•PF 4•thrombospondin•TGF-β

Coagulation factors

•factor V•HMWK•factor X1•fibrinogen

DENSE TUBULARSYSTEM

•prostaglandin synthesis•Ca++

DENSE GRANULES•ATP•ADP•Ca++

•serotonin

CYTOPLASMICFACTORS

•factor X111MICROTUBULECOIL

PLASMAMEMBRANE

•glycoprotein•plasma proteins•coagulation factors•PF 3

SURFACE-CONNECTEDCANALICULARSYSTEM

•glycoproteins•plasma proteins•coagulation factors

TGF-β: transforming growth factor β HMWK: high molecular weight kininogen

Modified from Plow EF and Ginsberg MH: Molecular basis of platelet function. In Hoffman R, etal. (Eds): Hematology: Basic Principles and Practice. New York, Churchill Livingstone, 1991, pp1165-1176.


How platelets are actually formed from megakaryocytesremains unclear. The primary site of thrombopoiesis is alsodebated. There is some evidence that in some species thepredominant site of platelet formation is in the lung ratherthan marrow. However, it is clear that platelet production isregulated by overall platelet mass in the body and notplatelet numbers. A variety of growth factors are involvedin the initial phase of megakaryocytopoiesis, includinggranulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL 3), IL 6, and megakaryocytecolony-stimulating factor. Differentiation of megakary-ocytes into platelets is also controlled by growth factors,including thrombopoietin, erythropoietin, GM-CSF, and IL3. Platelets themselves contain biologically active molecules(eg, PF 4) which inhibit megakaryocyte production, sug-gesting that there also may be a negative feedback loophelping to regulate thrombopoiesis.

Platelet Destruction

As with all other circulating cells, the platelet has a finitecirculating lifespan. Dog platelets circulate for approxi-mately five to seven days while cat platelets survive only alittle more than a day. Cells of the monocyte/macrophagecontinuum are responsible for the removal of effeteplatelets. Nearly half are removed by splenic macrophagesand a third by macrophages of the liver.

Platelet Function

The primary role of the platelet is in hemostasis where itperforms three functions:

� forming the primary platelet plug, � serving as a scaffold for the deposition of fibrin,

and � influencing clot retraction.

In performing these functions, platelets undergo a complexseries of events described collectively as platelet activation.Although the events of activation can be described sequen-tially, in reality, the process is a dynamic one with multipleevents occurring simultaneously.

An early step in platelet activation is platelet adherence.Normally, circulating platelets will not adhere to intact ves-sel walls. However, when endothelial surfaces are compro-mised, platelets readily adhere to exposed subendothelialadhesive proteins including collagen, fibronectin, and VWF.

Exposure of platelets to thrombin, present as a result ofsimultaneous activation of the coagulation cascade, leads toa platelet shape change. Discoid platelets round up anddevelop numerous surface filaments called filopodia. Thenet effect is increased surface area, which serves to increasethe likelihood of platelet-platelet interaction that is criticalto platelet plug formation. Exposure to thrombin also caus-es granule secretion. Released ADP recruits other plateletsinto the area. Calcium released from platelets probably isimportant in coagulation reactions. Other granule contentsreleased include: serotonin, which causes vascular contrac-tion; additional adhesion molecules; promoters andinhibitors of coagulation; and growth promoters, whichstimulate fibroblastic proliferation and connective tissuematrix formation.

When fibrinogen binds to activated platelets, aggrega-tion, the final step in platelet plug formation, occurs.Initially, aggregation is a reversible reaction. However, withtime the process becomes irreversible.

Platelet activation and platelet plug formation interfaceswith the coagulation cascade in multiple ways. The plateletplug provides an expanded surface upon which the variousenzymatic reactions of the cascade can occur. The bindingof fibrinogen and thrombin to platelet surfaces further facil-itates coagulation, as does the release of coagulation cofac-tors from platelets at the time of granule secretion. FactorXIII (fibrin stabilizing factor), an enzyme that catalyzes thecross-linking and stabilization of fibrin, is released fromplatelet cytoplasm at the time of granule release. The cross-linking of fibrin in conjunction with the interaction of theplatelet fibrinogen receptor with platelet actin leads to clotretraction, the last step in coagulation.

Platelet Disorders

Platelet disorders fall into two major categories: quanti-tative abnormalities and qualitative abnormalities.

Quantitative abnormalities include thrombocytosis andthrombocytopenia. These abnormalities, in particular,thrombocytopenia, are by far the most important andprevalent, and are covered in some detail below.

Qualitative abnormalities include: hereditary plateletfunction defects in von Willebrand’s Disease, ChediakHigashi Syndrome, and hereditary canine thromboasthenia;and acquired function defects associated with certain drugtherapies and systemic diseases. Qualitative abnormalities


are diagnosed when other causes of bleeding havebeen ruled out. In general, platelet functionaldeficits cannot be diagnosed from routine hemato-logic data and require the performance of specialtests. They are beyond the scope of this text and,therefore, will not be discussed.

Thrombocytopenia

Thrombocytopenia, or reduction in the numberof circulating platelets, is caused by one of fourmechanisms:

� increased peripheral utilization of platelets, � increased destruction of platelets, � increased sequestration of platelets, or� reduced platelet production in the bone

marrow.These are summarized in Fig. 2.

Increased peripheral utilization Increased peripheral utilization results when there is

increased systemic demand for platelets. This occurs in twoconditions: disseminated intravascular coagulopathy (DIC)and blood loss.

DIC is a secondary syndrome associated with underly-ing severe disease. In most cases, the underlying process isinflammatory, but DIC also occurs in some cases of neopla-sia, marked tissue necrosis and shock. Regardless of theinciting cause, DIC is a syndrome where excessive stimula-tion of the coagulation cascade leads to the peripheral con-sumption of both coagulation factors and platelets.

Clinically, animals with fully developed DIC often pre-sent as emergency patients with bleeding disorders.Subclinical DIC syndromes also occur. Laboratory findingscan include thrombocytopenia, prolonged activated partialthromboplastin time (APTT) and one step prothrombintime (OSPT), decreased fibrinogen levels, and elevated fib-rin split products. When any three of the above laboratoryabnormalities are present, the diagnosis of DIC is con-firmed. In dogs, an additional laboratory finding may bethe presence of schizocytes on the peripheral blood film.Schizocytes are rarely seen in cats with DIC.

Thrombocytopenia secondary to blood loss is mild. Ifsevere thrombocytopenia (platelet counts of 40,000/µl orless) is seen in association with blood loss, the throbocy-topenia is probably the cause rather than the result of thehemorrhage.

Increased platelet destructionLike utilization thrombocytopenia, destruction thrombo-

cytopenia is associated with shortened circulating plateletlifespan, but in this case, as a result of immune-mediatedremoval. As with immune-mediated hemolysis, immune-mediated thrombocytopenia may be a true autoimmunesyndrome caused by circulating antiplatelet antibodies, or itmay be secondary to drug therapy, infectious diseases, orneoplasia.

For the general practitioner, the specific diagnosis ofimmune-mediated thrombocytopenia can be challenging.The degree of thrombocytopenia is usually marked, withcounts of 20,000/µl or less. Bone marrow aspirates can betaken to estimate megakaryocyte numbers. Most cases ofimmune-mediated thrombocytopenia are characterized byincreased numbers of megakaryocytes, although rare caseshave reduced numbers. Utilization thrombocytopenia alsohas either normal or increased numbers of megakaryocytes.Clinical signs, history, and other laboratory data, includingcoagulation tests, are useful in ruling out utilization throm-bocytopenia, thereby allowing a presumptive diagnosis ofimmune-mediated thrombocytopenia. Special tests forimmune-mediated thrombocytopenia (eg, PF 3 test,antimegakaryocyte antibody test) may be considered,though the value of these tests is controversial.

Once other causes of thrombocytopenia have been ruledout and a presumptive diagnosis of immune-mediatedthrombocytopenia is made, immunosuppressive therapy canbe instituted. If successful, the therapy itself serves to con-firm the diagnosis.

Fig. 2 The four mechanisms of thrombocytopenia.

Utilization• blood loss• DIC

Destruction• immune-mediated (primary

or secondary)

Sequestration• endotoxemia• hepatomegaly• hypothermia• splenomegaly

Production• intramarrow bone disease

Platelets


Sequestration thrombocytopeniaThrombocytopenia can occur in cases of hepatomegaly

or splenomegaly as a result of sequestration of platelets inthe enlarged organs. This condition is much more commonin humans than in animals, and is very rare in dogs andcats. Hypothermia has been demonstrated to cause plateletsequestration in the liver. Thrombocytopenia in endotox-emia is believed to be at least partially the result of seques-tration in the lung.

Hypoproliferative thrombocytopeniaHypoproliferative thrombocytopenia is the direct result

of reduced megakaryocytopoiesis. In most cases, there isreduced production of at least one other cell line;hemograms generally reflect anemia and/or leukopenia inaddition to thrombocytopenia. Bone marrow biopsiesreveal reduced numbers of precursors of each of the affect-ed cell lines. Both aspirates and core biopsies are usuallyrequired for proper evaluation. The specific cause is oftenobscure and may include infectious diseases, drug toxicityor myelosuppression, myelofibrosis, immune-mediated mar-row disease, and cancer.

ThrombocytosisThrombocytosis is defined as increased numbers of cir-

culating platelets. Clinically, it is much less common thanthrombocytopenia. In the dog and cat, most cases of throm-

bocytosis are secondary or reactive. Thrombocytosis can beseen secondary to splenic contraction (eg, with excitementor exercise), elevated circulating glucocorticoids, splenecto-my, or fractures. For the most part, reactive thrombocytosisis clinically insignificant.

Thrombocytosis also can occur as a feature of severalprimary bone marrow diseases. Primary platelet leukemia isa true myeloproliferative disorder. In the cat, it may beFeLV related. Platelet leukemia can manifest in either oftwo forms: primary thrombocythemia with very high num-bers of circulating platelets, or megakaryoblastic leukemiawith variable numbers of circulating platelets but massiveproliferation of platelet precursors in marrow and usuallyother tissues. In both conditions, platelets with bizarre mor-phology may be seen in the peripheral blood.

Polycythemia vera is a second myeloproliferative disor-der which can be characterized by thrombocytosis.Polycythemia vera is a stem cell defect which is character-ized by overproduction of all cell lines. The degree of over-production is relatively subtle; evaluation of marrow biop-sies generally reveals no abnormalities.

As discussed in Chapter 2, myelofibrosis is a marrowdisorder that can be characterized by either thrombocy-topenia or thrombocytosis. Other presenting features areused to establish the diagnosis; these include marked non-regenerative anemia, leukopenia, and poikilocytosis withdacryocytes on the peripheral blood film.


Part II


Overview

Hemogram interpretation, or evaluation of the peripher-al blood, is one of the foundations of clinicopathologicassessment of the sick patient, both from the perspective ofmaking the initial diagnosis and prognosis, and from theperspective of monitoring response to therapy. Hemograminterpretation is an integrated evaluation of the varioustests of the complete blood count (CBC), which consists ofwhite blood cell data, red blood cell data, and platelet data.

The CBC has both qualitative and quantitative compo-nents. The qualitative component is the evaluation of bloodcell morphology on the peripheral blood film. The quantita-tive components include all the numerical measures in theCBC: total cell counts, white cell differential, hematocrit,hemoglobin, red cell indices, and total plasma protein.

Accurate hemogram interpretation is based on a clearunderstanding of the physiology and pathophysiology ofthe various components of the hematopoietic system. In theprevious three chapters, white cell, red cell, and plateletresponses in health and disease were reviewed and illustrat-ed. This chapter will develop a systematic approach toCBC interpretation built around this basic information. Inmany instances, the reader will be referred to appropriatepages in the preceding chapters for further detail.

In evaluating the CBC, white cell data are always inter-preted first, followed by interpretation of red cell data, andfinally platelet data. This order is logical since abnormali-ties in white cell data may suggest potential red cell andplatelet alterations.

Interpreting the Leukogram

White cell data in the CBC include the total white cellcount, the differential cell counts, and evaluation of whitecell morphology on the peripheral blood film. All cellcounts should be reported in absolute numbers. The prima-ry value of white cell data is in recognizing and classifyinginflammatory responses. Considered collectively, leukogramdata are used to address the following questions:

1) Is there evidence of inflammation?

2) Is there evidence of a glucocorticoid (stress)response?

3) Is there evidence of an epinephrine (excitement)response?

4) Is there evidence of a systemic hypersensitivityreaction?

5) Is there evidence of tissue necrosis?6) If inflammation is present, can it be further classi-

fied?7) Is there evidence of systemic toxemia?

Is there evidence of inflammation?Reference ranges for total white cell count and differen-

tial cell counts in dogs and cats are found in Table 1 at theback of this book (Part III). The signposts of inflammationare a neutrophilic left shift, persistent eosinophilia, and/or amonocytosis. These can be seen individually or in any com-bination. Monocytosis and eosinophilia are easily recog-nized as any value above the reference range. Identificationof neutrophilic left shifts is somewhat more subjective. Leftshifts are usually defined in relationship to total neutrophilor WBC count. As a guide, if the total white cell count isnormal, a count of greater than 300 bands/µl constitutes aleft shift. If the total white cell count is 25,000/µl to30,000/µl, at least 1000 bands/µl are required to designate aleft shift. If the total white cell count is below normal,fewer than 300 bands/µl indicates a left shift.

Based on the above criteria, it should be clear that totalwhite cell count alone cannot determine whether or notinflammation is present. Inflammation can be present in theface of low, normal, or high total white cell count. Totalwhite cell count is primarily an indicator of balancebetween bone marrow production and two other key fac-tors; the release of granulocytes and tissue demand forleukocytes. A normal white cell count indicates that thebalance between marrow production and release of granu-locytes and tissue utilization is being maintained. Elevatedwhite cell counts indicate increased bone marrow produc-tion and granulocyte release relative to tissue demand.Reduced white cell counts indicate that leukocytes are mar-ginating along vessel walls and moving into the tissues atgreater rates than they are being produced and released

Chapter 4: Hemogram Interpretation


into the blood from the marrow. Classification of an inflam-matory response as active, chronic, or overwhelmingdepends upon which of these dynamic states exists. Thiswill be discussed in greater detail later in this chapter.

Inflammatory responses can also occur without any ofthe classic signposts; in these cases the leukograms areambiguous. For example, a mild, mature neutrophilia maybe the result of inflammation, however, it also may be dueto other factors, such as high circulating glucocorticoid lev-els. In these cases, repeat CBCs done at 6- to 12-hour inter-vals may help clarify the response. In inflammatory dis-eases, left shifts are comon.

Is there evidence of stress?The classic stress (glucocorticoid-induced) leukogram

includes: mild, mature neutrophilia, lymphopenia,eosinopenia, and mild monocytosis. Of these changes, onlythe lymphopenia is specific. In dogs and cats, lymphocytecounts of between 700/µl and 1500/µl are consistent with aglucocorticoid effect. Lymphocyte counts of less than700/µl may be partially the result of stress, but other causesof lymphopenia must be explored. These include lym-phoma, chylous effusions, and lymphatic obstruction.

When a stress leukogram is present, other alterations inthe hemogram and clinical chemistry panel can be antici-pated. There may be mild polycythemia as well as mildinappropriate nucleated red cell response (5 to 10 nucleatedred cells/100 white cells counted). Isosthenuria is a com-mon finding. Mild hyperglycemia (120 mg/dl to 180 mg/dl)is typical and there are often mild to moderate elevations inserum transaminases. If the leukogram is a result ofCushing’s disease, elevations in alkaline phosphatase (dogonly) may be marked (more than four times the upperrange of normal).

Is there evidence of excitement?Excitement causes the release of epinephrine; epineph-

rine, in turn, causes increased blood flow. Increased bloodflow washes marginating leukocytes off vessel walls andback into circulation where they can be collected andcounted. In dogs and cats, under normal circumstances, theratio of freely circulating to marginating cells is 1:1.Theoretically, therefore, epinephrine release has the poten-tial to double the total white cell count. In dogs, excitementleukocytosis is primarily the result of neutrophilia while incats, it is usually the result of lymphocytosis.

Epinephrine-induced leukocytosis occurs as soon as

blood flow increases, and disappears just as rapidly whenblood flow returns to normal. This is in direct contrast tothe glucocorticoid response, which takes approximatelyfour hours to develop and lasts for approximately 24 hours.

Is there evidence of a systemic hypersensi-tivity reaction?

Persistent eosinophilia is not only indicative of inflam-mation; it is also suggestive of systemic hypersensitivity.For a more in-depth discussion of the interpretation ofeosinophilia and a listing of possible causes, refer toChapter 1, page 6.

Is there evidence of tissue necrosis?Tissue necrosis is presumed to be present whenever

there is peripheral monocytosis. For a more complete dis-cussion of monocytosis, turn to Chapter 1, page 8.

If inflammation is present, can it be furtherclassified?

In dogs and cats, acute or active inflammation is charac-terized by rapid movement of neutrophils from bone mar-row to the site of involvement in the tissues. In most cases,because of the large marrow reserve of neutrophils, the rateof entrance of neutrophils into the blood generally exceedsthe rate of exit into the tissues; therefore, the white cell(neutrophil) count is usually elevated. At the same time,because of the high demand for neutrophils, immature neu-trophils (band cells) will also be drawn into the blood.Thus, neutrophilia with a left shift (a regenerative left shift)is the typical and appropriate neutrophil response in acuteinflammation. Tissue necrosis is a variable accompaniment,so monocytosis may or may not also be present. Becauseanimals with acute inflammatory disease are almost alwaysunder stress, there is almost always lymphopenia.

Occasionally, in acute inflammatory conditions, tissueutilization is greater than the ability of the marrow to keeppace with demand. This constitutes overwhelming inflam-mation and the prognosis is guarded. In these cases, totalwhite cell count and neutrophil count are reduced andthere is a left shift (degenerative left shift). Changes in lym-phocyte counts and monocytes are as described above.

If acute inflammation continues unresolved, it graduallyenters a chronic phase with leukogram features to match.Circulating half-life of neutrophils remains short, but overtime expanded marrow production equilibrates with thehigh turnover rate. Eventually a new steady state is estab-


lished. The net effect is that neutrophil numbers return tonear normal and the left shift disappears. Animals withchronic inflammatory conditions continue to be stressed(lymphopenia) but at the same time often have antigenicstimulation (lymphocytosis). The net effect is a normal toslightly elevated lymphocyte count. Because of tissuenecrosis and the ongoing demand for phagocytosis, themost consistent abnormal finding in chronic inflammatoryleukograms is monocytosis.

It is important to note that not all cases of inflammatorydisease have leukograms that fall neatly into thesedescribed patterns. In addition, the separation of responsesinto acute and chronic is somewhat arbitrary, as leukogramdata cannot be used to judge the duration of a diseaseprocess. For example, the length of time required for bonemarrow production to equilibrate with tissue utilization willvary from case to case. Nevertheless, once the presence ofinflammatory disease has been established, certain otherpossible hemogram changes should also be considered.

The most common red cell accompaniment to inflamma-tory disease is anemia. The anemia is mild to moderate andnon-regenerative. In dogs, hematocrits are between 30%and 40%. In cats, hematocrits may drop to as low as 25%.Anemia with these features in association with inflammato-ry leukograms is considered to be the anemia of inflamma-tory disease. The anemia of inflammatory disease has gen-erally been regarded as a chronic condition; however, incats, it can develop in a week or less. In the dog, slightlymore time is required because of the longer red cell lifes-pan. See Chapter 2 for more information on anemias.

Inflammatory diseases, particularly chronic ones, areoften associated with increased immunoglobulin productionby the specific immune system. In the hemogram, this isseen as hyperproteinemia. Hypergammaglobulinemia canbe confirmed by evaluating total protein and albumin in theserum chemistry panel.

Whenever inflammatory leukograms are present, partic-ularly if overwhelming or marked, special attention shouldbe given to platelet numbers. If platelet numbers arereduced, the possibility of DIC should be considered. Theblood film should be scanned for schizocytes. Completionof a DIC panel (see Chapter 3, page 29) is indicated.

Is there evidence of sytemic toxemia?Systemic toxemia occurs when circulating toxins of

either infectious or noninfectious origin interfere with thedifferentiation of neutrophil precursors in the marrow. The

result is the presence of toxic neutrophils on peripheralblood films. These have been described and illustrated indetail in Chapter 1. Systemic toxemia is a poor prognosticsign.

Interpreting the Erythrogram

Red cell tests in the CBC include total red cell count,hemoglobin, hematocrit, red cell indices (MCV, MCHC),total protein, and evaluation of red cell morphology on theblood film. Red cell count, hemoglobin, and hematocrit aremeasures of red cell mass or oxygen carrying capacity ofthe blood. Total protein provides immediate informationabout state of hydration, which can be very important ininterpreting red cell mass. Elevations in total protein aremost commonly the result of dehydration, which can falselyelevate indicators of red cell mass. The only other commoncause of hyperproteinemia is hypergammaglobulinemia ininflammation.

As with leukogram data, erythrogram data are bestinterpreted by answering a series of questions:

1) Is red cell mass increased (polycythemia),decreased (anemia), or normal?

2) If increased, is the polycythemia relative orabsolute?

3) If polycythemia is absolute, is it primary or sec-ondary?

4) If red cell mass is decreased, is the anemia regener-ative or non-regenerative?

5) If regenerative, is the mechanism blood loss orhemolysis?

6) If non-regenerative, can the mechanism be deter-mined without bone marrow evaluation?

Is red cell mass increased, decreased, or normal?This simple but all-important question is answered by

evaluating the primary indicators of red cell mass (RBCcount, Hb, HCT). Increases indicate polycythemia;decreases indicate anemia.

If increased, is polycythemia relative or absolute?The combination of elevated hematocrit and elevated

plasma protein is highly suggestive of dehydration and rela-tive polycythemia. Dehydration can be confirmed on thebasis of history, physical evaluation, and clinical chemistry.Clinical chemistry findings which are supportive of dehy-dration include elevated blood urea nitrogen (BUN) in


conjunction with concentrated urine specific gravity (prere-nal azotemia), high normal or elevated albumin, and highnormal or elevated serum electrolytes. In the absence ofsuch indicators of dehydration, polycythemia is absolute.

If polycythemia is absolute, is it primary or sec-ondary?

Secondary polycythemia is ruled out first. As suggestedin Chapter 2, polycythemia can be associated with primaryrenal disease, cardiovascular or pulmonary disease,Cushing’s disease, or neoplastic disease of the kidneys,either primary or metastatic. In the absence of such condi-tions, polycythemia is considered to be primary (poly-cythemia vera). Evaluation of arterial blood gas values anddetermination of erythropoietin levels will confirm the pres-ence of polycythemia in the face of normal oxygenation ofthe blood and normal hormonal stimulation of the red cellmarrow.

If red cell mass is decreased, is anemia regenera-tive or non-regenerative?

The first step in classifying an anemia is evaluation ofthe peripheral blood film. If there is increased anisocytosisand polychromasia, the anemia is likely regenerative.Regeneration can be confirmed by doing a reticulocytecount; absolute reticulocyte counts of greater than80,000/µl are indicative of regenerative anemia in both dogsand cats.

If regenerative, is the mechanism blood loss orhemolysis?

History, clinical signs, physical examination, and reticu-locyte count are the first data evaluated in differentiatingblood loss from hemolysis. Most animals with blood lossanemia have histories of trauma or visible bleeding. Historyor clinical signs of vomiting, diarrhea, or marked externalparasitism also are compatible with blood loss.Hemoglobinuria, hemoglobinemia, or marked retculocyto-sis (greater than 200,000/µl) are highly suggestive ofhemolysis.

In all cases where blood loss is not clearly the cause ofthe regenerative anemia, care should be taken to evaluatethe peripheral blood film for abnormal red cells. The mor-phologic signposts of hemolysis have been described andillustrated in detail in Chapter 2; they include spherocyto-sis, the presence of etiologic agents on red cells (hemobar-

tonellosis, babesiosis), the presence of Heinz bodies, andthe presence of schizocytes.

If non-regenerative, can the mechanism be deter-mined without bone marrow evaluation?

The most common of all anemias in dogs and cats is theanemia of inflammatory disease. If the anemia is mild tomoderate and there is an inflammatory leukogram, the ane-mia of inflammatory disease is diagnosed. If there is signifi-cant renal disease, non-regenerative anemia associated withlack of erythropoietin is often seen. If there is markedhypochromasia and microcytosis (indicated by red cellindices and/or red cell morphology on the blood film, seeChapter 2), then iron deficiency anemia can be diagnosed.

All other non-regenerative anemias require bone marrowevaluation for definitive diagnosis. There are, however, syn-dromes where CBC findings and peripheral blood filmmorphology can be suggestive of non-regenerative anemia.These include myelofibrosis, characterized by severe ane-mia, leukopenia, variable platelet response and dacryocyteson peripheral films, and megaloblastic anemia where giantRBCs and even megaloblasts can be seen on peripheralfilms (see Chapter 2).

Interpreting the Thrombogram

The only platelet tests in the routine CBC are evaluationof platelet numbers and platelet morphology on the periph-eral blood film. As discussed in Chapter 3, platelet disor-ders are either quantitative or qualitative. Using the routinetests in the CBC, only quantitative disorders can be evalu-ated. The questions to be addressed are the following:

1) Are platelet numbers normal, increased (thrombo-cytosis), or decreased (thrombocytopenia)?

2) If there is thrombocytosis, is it reactive or primary?3) If there is thrombocytopenia, can the mechanism

be determined?

Are platelet numbers normal, increased, ordecreased?

The answer to this question is, of course, based onplatelet count, with a note of caution: automated plateletcounts in cats are often inaccurate because of plateletclumping and the overlap of platelet and red cell sizes.Particularly in cats, platelet numbers should be estimatedfrom blood films (adequate versus low) as well as counted


in an automated system. If discrepancies exist between esti-mates and automated counts, hand counts using Unopette®

(Becton-Dickinson, Rutherford, NJ) methods should becompleted. In general, extended hematocrit methods aremore accurate at determining platelet counts in cats thanare impedance methods.

If there is thrombocytosis, it is reactive orprimary?

Reactive or secondary thrombocytosis is by far mostcommon. Reactive thrombocytosis is discussed in Chapter3, page 30. When causes of reactive thrombocytosis havebeen ruled out, primary thrombocytosis due to plateletleukemia must be considered. Bone marrow biopsies willprobably be required for confirmation but platelet mor-phology on the blood film should be closely scrutinized. Inmost platelet leukemias, bizarre platelets are seen in theperipheral blood.

If there is thrombocytopenia, can the mechanismbe determined?

Thrombocytopenic mechanisms are discussed in detail inChapter 3 and are merely summarized here from a diagnos-tic perspective.

Thrombocytopenia in conjunction with inflammatory

leukograms and the presence of schizocytes and neu-trophil toxicity on blood films is suggestive of utilizationthrombocytopenia associated with DIC. The DIC panelshould be run for confirmation (see Chapter 3). The pos-sibility of sequestration thrombocytopenia is suggested byclinical evidence of hepatosplenomegaly. Destruction(immune-mediated) thrombocytopenia may be found inassociation with immune-mediated hemolytic anemia(highly regenerative spherocytic anemia) or without otherhematologic abnormalities. Evidence of megakaryocytichyperplasia in the bone marrow is helpful in establishing adiagnosis of destruction thrombocytopenia, but such evi-dence may be absent. Occasional cases of destructivethrombocytopenia show decreased numbers of megakary-ocytes.

It is noteworthy that destruction thrombocytopenias,like sequestration thrombocytopenia, may be associatedwith hepatosplenomegaly. Hypoproliferative thrombocy-topenia may be associated with other peripheral cytopenias,but can only be diagnosed by reduced numbers ofmegakaryocytes in marrrow biopsies (histopathology ofcore biopsies required). The causes of hypoproliferativethrombocytopenia are varied and include infectious agents(eg, ehrlichiosis), immune-mediated marrow disease, mar-row toxicity, marrow aplasia, and myelophthisis.


CASE 1Signalment: Three-year-old female

DSH catHistory: Presented for elective

surgery

HCT 38% WBC 18,600/µlHb 12.5 g/dl Neutrophils 8,000/µlRBC 7.2 x 106/µl Lymphocytes 10,000/µlTP 6.2 g/dl Eosinophils 300/µlPlatelets Adequate Monocytes 300/µl

Description

Leukogram: There is a leukocytosis characterized bya marked lymphocytosis. Neutrophil numbers arewithin the reference range.

Erythrogram: Red cell parameters and plasma pro-tein are in the reference range.

Thrombogram: Unremarkable.

Interpretation

The principal hematologic alteration is leukocytosischaracterized by lymphocytosis.

Lymphocytosis in cats can be associated with severalconditions such as lymphocytic leukemia, chronic antigenicstimulation, or physiologic leukocytosis.

Almost all cases of leukemia are characterized bymarked anemia, but in this patient, red cell mass is normal.Furthermore, in most cases of lymphocytosis due to lym-phocytic leukemia, circulating lymphocytes are morphologi-cally abnormal, but no abnormal morphology is noted inthis patient. Therefore, the possibility of lymphocyticleukemia is extremely low.

Chronic antigenic stimulation with lymphocytosis is usu-ally associated with an inflammatory leukogram, but there isno evidence of inflammation in this cat. Consequently, themost likely interpretation here is physiologic leukocytosis.

Physiologic leukocytosis results from the effects ofexcitement (release of epinephrine) on blood flow; theeffect being increased blood flow.

Under normal conditions, for every leukocyte freely cir-culating in the blood, there is one leukocyte marginated onthe blood vessel walls. Theoretically, only freely circulatingleukocytes are collected when a blood sample is drawn.However, due to the excitement (ie, epinephrine release)that is often associated with a blood draw, there isincreased blood flow, which causes movement of marginat-ed leukocytes into the freely circulating blood. This, then,can result in a doubling of the total leukocyte count.

In cats, the result is physiologic lymphocytosis. In dogs,physiologic neutrophilia generally results.

Physiologic leukopenia can also be seen in those circum-stances where blood flow is decreased. Perhaps the bestexample is the effect of anesthesia. In this case, the numberof leukocytes in the freely circulating blood is reduced asmore and more cells marginate in the face of decreasedheart rate and decreasing blood flow.

Chapter 5: Case Studies


CASE 2Signalment: One-year-old female

Wirehair TerrierHistory: Presented for

ovariohysterectomy

HCT 45% WBC 20,300/µl Hb 15.0 g/dl Neutrophils 18,000/µl RBC 6.1 x 106/µl Lymphocytes 1,500/µl TP 6.5 g/dl Monocytes 500/µlPlatelets Adequate Eosinophils 300/µl

Description

Leukogram: There is a leukocytosis (mild) character-ized by a mild mature neutrophilia and a low normalto marginally decreased lymphocyte count.

Erythrogram: No abnormalities noted.

Thrombogram: No abnormalities noted.

Interpretation

Only white cell changes are observed. Unfortunately,these findings are somewhat ambiguous.

The marginal lymphocyte count suggests the possibilityof a stress (glucocorticoid-induced) leukogram. A mildleukocytosis with mild mature neutrophilia also could beconsistent with stress.

However, a mild mature neutrophilia could be a reflec-tion of mild inflammation even though no specific indica-tors of inflammation are present. Repeating the CBC in 8to 12 hours would be expected to clarify the presence orabsence of inflammation.

Finally, the mild leukocytosis with neutrophilia is consis-tent with physiologic leukocytosis in the dog. Consideringthe history and signalment, stress with physiologic leukocy-tosis is the best interpretation.


CASE 3Signalment: Eight-year-old Boston

TerrierHistory: Polyuria and polydipsia of

several weeks’ duration

HCT 55% WBC 14,500/µl Hb 18.0 g/dl Neutrophils 13,000/µl RBC 8 x 106/µl Lymphocytes 750/µlTP 6.5 g/dl Monocytes 850/µl NRBC 5/100 WBC Platelets Adequate

Description

Leukogram: There is a normal leukocyte count butan absolute lymphopenia and marginal neutrophilia.

Erythrogram: There is a polycythemia in the face ofa normal plasma protein (absolute polycythemia).There is also a marginal inappropriate nucleated redcell response (increased numbers of NRBC in theabsence of polychromasia).


Interpretation

Leukogram data (absolute lymphopenia of between700/µl and 1500/µl and marginal neutrophilia) are consis-tent with a glucocorticoid-induced leukogram. There is noevidence of superimposed inflammation or tissue necrosis.

Absolute polycythemia with a possible inappropriatenucleated red cell response also is entirely consistent withhigh levels of circulating glucocorticoids. Glucocorticoidsare known to have a mildly stimulatory effect on RBC pro-duction. High circulating glucocorticoids also are one ofthe recognized causes of mild inappropriate nucleated redcell responses (5 to 10 NRBC/100 WBC).

Considering the history and signalment (Boston Terriersare prone to endocrinopathies) in conjunction with hemato-logic findings, Cushing’s disease, either spontaneous oriatrogenic, should be investigated further.


CASE 4Signalment: Six-year-old intact female

PoodleHistory: Recent onset emesis,

anorexia, polydipsia, andpolyuria

HCT 30% WBC 24,900/µlHb 10.0 g/dl Bands 3,000/µlRBC 4.7 x 106/µl Neutrophils 18,000/µlTP 6.5 g/dl Lymphocytes 900/µlPlatelets Adequate Monocytes 3,000/µl

Morphology: Foamy, basophilic cytoplasm in bands,occasional Döhle bodies.

Description

Leukogram: There is a leukocytosis characterized bya neutrophilia with a left shift, a lymphopenia, and amonocytosis. Morphologic abnormalities in neu-trophils also are noted on the blood film.

Erythrogram: Red cell data indicate a mild anemiacharacterized by an absence of polychromasia.Computed MCV (HCT/red cell count in millions x10) and MCHC (Hb/HCT x 100) are within thereference range for dogs.


Interpretation

1.) Inflammatory leukogram with systemic toxemia.Indicators of inflammation are both the left shift and themonocytosis. Monocytosis is also an indication of tissuenecrosis. Cytoplasmic basophilia and Döhle bodies in neu-trophils result from cytoplasmic maturation arrests in devel-oping stages in the marrow; this is indicative of circulatingtoxins in the blood which interfere with differentiation ofgranulocyte precursors (systemic toxemia). Systemic tox-emia can occur as a result of tissue necrosis and toxic disor-ders (eg, lead poisoning) but in dogs and cats is most com-monly associated with severe bacterial infections.

2.) Superimposed stress. The lymphocyte count ofbetween 700/ml and 1500/ml (absolute lymphopenia) isindicative of stress. The combination of stress and inflam-mation with a left shift is most consistent with active oracute inflammation.

3.) Non-regenerative anemia. Normocytic, nor-mochromic anemia in the absence of polychromasia is anon-regenerative anemia. The degree of anemia is moder-ate. Considering the presence of a significant inflammatoryleukogram, at least part of the anemia is likely due toinflammatory disease. However, the degree of anemia ismore severe than usually associated with inflammation

Fig. 1 Band cell with slight toxicity (left). Note the foamy, granular, slightlybasophilic cytoplasm. A normal monocyte (right).


alone, which rarely reduces hematocrits in dogs toless than 35%. Other contributing factors, such assuperimposed blood loss, must also be considered.

4.) Normal platelet count. The normal plateletcount is a significant finding in this case. It is anencouraging finding in the face of the rather markedinflammation with evidence of systemic toxemia. Aconcern in severe inflammatory conditions is dis-seminated intravascular coagulation (DIC); the nor-mal platelet count suggests that DIC is not current-ly a problem.

SummaryOverall, hematologic findings suggest a severe

acute inflammatory condition with superimposedstress, tissue necrosis systemic toxemia, and theanemia of inflammatory disease. Bacterial infectionis suspected. The absence of thrombocytopenia sug-gests that despite the seriousness of the condition,DIC is not present.

The actual diagnosis in the case was E. colipyometra.

Fig. 3 A toxic band (left), and a toxic metamyelocyte (right). The cytoplasm of bothcells is too blue.

Fig. 4 A toxic neutrophil with foamy, basophilic cytoplasmand Döhle bodies.

Fig. 2 Two band cells and a mature neutrophil.


CASE 5Signalment: Four-year-old intact

female Irish SetterHistory: Weight loss and distended

abdomen

HCT 25% WBC 17,500/µlHb 8.0 g/dl Neutrophils 10,000/µlRBC 4 x 106/µl Lymphocytes 3,000/µlTP 8.2 g/dl Monocytes 4,500/µlPlatelets Adequate

Description

Leukogram: Total white cell count is normal.However, there is a marked monocytosis.

Erythrogram: There is a moderate normocytic(MCV = 62 fl) normochromic (MCHC = 33%) ane-mia with no evidence of polychromasia.


Interpretation

1.) Inflammatory leukogram. Although the total whitecell count is normal, the marked monocytosis is a clear indi-cator of inflammation and tissue necrosis. There is no evi-dence of superimposed stress. When considered in itsentirety, the leukogram indicates chronic inflammation withthe establishment of a new steady state between marrowproduction of leukocytes on the one hand and tissue con-sumption of leukocytes on the other.

2.) Non-regenerative anemia. Normocytic, nor-mochromic anemias without polychromasia are nonregen-erative. Pathogenesis of the anemia in this patient is proba-bly complex. Certainly the depression of red cell produc-tion by inflammatory disease is a major contributing factor.However, the degree of anemia is too severe to beexplained on the basis of inflammation alone. A potentialsource of blood loss should also be sought.

3.) Normal platelet numbers. Normal platelet numbersin the face of chronic inflammation is a positive finding inthat it indicates the absence of DIC.

4.) Hyperproteinemia. Hyperproteinemia reflects hemo-concentration and/or increased antibody (immunoglobulin)production. In this patient, given the presence of a chronicinflammatory leukogram, antigenic stimulation with

increased immunoglobulin production is almost acertainty. This can be confirmed by notingincreased globulins as compared to albumin in theserum chemistry data.

SummaryHematologic findings are most supportive of a

chronic inflammatory process with tissue necrosis,antigenic stimulation, and the anemia associatedwith inflammation. A second factor exacerbatingthe anemia is also suspected since the degree ofanemia is more than inflammatory processes aloneusually cause.

The actual diagnosis in this case was E. colipyometra.

Fig. 5 On scanning magnification, a monocytosis is obvious. Four monocytes andseven neutrophils.


CASE 6Signalment: Five-year-old female

mixed-breed dogHistory: Presented in state of near

collapse and extremedepression

HCT 50% WBC 5,500/µlHb 16.1 g/dl Bands 1,100/µlRBC 7.2 x 106/µl Neutrophils 2,000/µlTP 8.5 g/dl Lymphocytes 900/µlPlatelets Reduced Monocytes 1,500/µl

Description

Leukogram: There is a leukopenia characterized byneutropenia with a left shift, lymphopenia, and a mar-ginal monocytosis.

Erythrogram: There is polycythemia with hyperpro-teinemia.

Thrombogram: Thrombocytopenia.

Interpretation

1.) Overwhelming inflammation. Left shift and monocy-tosis indicate inflammation with tissue necrosis. The pres-ence of the left shift in the face of marked neutropenia indi-cates that marrow production and release of neutrophils isunable to keep up with tissue demand (overwhelminginflammation). This is an unfavorable finding in the dog orcat and suggests an emergency condition. Overwhelminginflammation is almost always an acute phenomenon.

2.) Stress leukogram. Absolute lymphopenia with lym-phocyte counts in the range of 700/µl to 1500/µl is mostcommonly associated with high levels of circulating gluco-corticoids (stress).

3.) Relative polycythemia. The combination of poly-cythemia and hyperproteinemia suggests relative poly-cythemia due to dehydration and hemoconcentration.Leukogram data is not affected by hemoconcentration.

However, other tests in the laboratory profile are alsoaffected by dehydration and can be used to support theinterpretation of relative polycythemia. BUN, creatinine,and electrolytes all will elevate in the face of dehydration. Ifthe kidneys are working, urine specific gravity will be con-centrated. Hyperproteinemia in dehydration will be associ-ated with elevations in both albumin and globulin levels.(The other cause of hyperproteinemia, antigenic stimula-tion, is associated with a relatively greater elevation inglobulins than albumin.) In this patient, considering the

inflammatory nature of the disease, antigenicstimulation may also be contributing to the hyper-proteinemia.

4.) Possible DIC. Thrombocytopenia in theface of inflammation, particularly severe and over-whelming inflammation, is suggestive of possibleDIC. This is a potentially life-threatening syn-drome and should be confirmed by evaluating a

Fig. 6 A reactive lymphocyte with large nucleus, lacy chromatin pattern withnucleolar whorls, and basophilic cytoplasm.

Continued

complete DIC panel comprised of platelet count,prothrombin time, activated partial thromboplas-tin time, activated partial thromboplastin time,fibrinogen levels and fibrin split products. If threeof the five tests in the panel are abnormal, DIC isconsidered to be present.

SummaryOverwhelming inflammation with tissue necro-

sis and superimposed stress; relative polcythemia;possible DIC. This patient is in an emergencystate requiring immediate attention.

The actual diagnosis in this animal was E. colipyometra.


Fig. 7 A reactive lymphocyte with abundant cytoplasm and small azurophilic granules.


When considered in aggregate, Cases 4, 5, and6 illustrate several principles of hemogram inter-pretation, in particular leukogram interpretation.First, these cases clearly illustrate that inflamma-tory processes can present with high, low, or nor-mal total white cell counts. In fact, the primaryvalue of total leukocyte counts in inflammatoryprocesses is that they provide some indication ofhow well the patient is coping with the inflamma-tion. If the white cell count is elevated, then mar-row production and release of leukocytes isexceeding tissue consumption. This is a favorablefinding for the patient, and accordingly, left shiftswith elevated neutrophil counts have beentermed regenerative left shifts. In contrast, lowwhite cell counts in inflammatory disorders indi-cate that tissue utilization of leukocytes exceedsmarrow production and release. This is an unfa-vorable circumstance for the patient, and leftshifts with low leukocyte counts are thereforetermed degenerative left shifts. Cases of inflam-matory disease where the total leukocyte countsare normal are usually chronic conditions wherea new steady state has been reached and marrowproduction and release of granulocytes has hadtime to expand to meet tissue demand. In thesecases, examination of marrow reveals markedgranulocytic hyperplasia with normal toincreased numbers of mature neutrophils, whichaccounts for the absence of a peripheral left shift.

A second major principle illustrated in cases 4,5, and 6 is that leukogram data can be used prog-nostically as well as diagnostically. Case 4, anacute pyometra with a classic acute inflammatoryleukogram, is the simplest case with the bestprognosis for complete, uncomplicated recoveryfollowing hysterectomy. Case 5, a chronicpyometra with chronic inflammatory leukogram,also has a relatively good prognosis. However,the chronicity of the process suggests that thesurgery itself may be complicated due to markedvascular proliferation. Case 6, an acute pyometrawith overwhelming inflammation and possibleDIC, has the worst prognosis. This case is clearlyan emergency and requires immediate attention.

Finally, these three cases clearly illustrate thatleukogram data cannot be used to differentiateamong bacterial, viral, and toxic diseases (unlessspecific etiologic agents are seen on the bloodfilm). Historically, it has been suggested that bac-terial diseases cause leukocytosis while viralinfections cause leukopenia. However, in thesethree cases, we have seen examples of a singlebacterial agent associated with the full range ofwhite cell response. Toxic and necrotizingprocesses can induce the same variability inwhite cell numbers. Leukogram data are used todifferentiate between inflammatory and non-inflammatory processes, not between infectiousand non-infectious processes.

Discussion of Cases 4, 5, and 6


CASE 7Signalment: Fourteen-year-old

neutered male DSH catHistory: Chronic weight loss with

distended abdomen

HCT 30% WBC 43,200/µlHb 9.5 g/dl Neutrophils 38,000/µlRBC 5.6 x 106/µl Lymphocytes 2,160/µlTP 10.0 g/dl Monocytes 2,360/µlPlatelets Adequate Eosinophils 680/µl

Description

Leukogram: There is leukocytosis characterized bymature neutrophilia and monocytosis.

Erythrogram: There is marginal to no anemia but amarked hyperproteinemia is present.


Interpretation

1.) Chronic inflammatory leukogram. The monocytosisis a clear indicator of inflammation and tissue necrosis.The marked mature neutrophilia with no left shift suggeststhat the granulocytic compartment of the marrow is signifi-cantly expanded, a feature of chronicity. Chronicity is alsosuggested by the absence of lymphopenia.

2.) Suspected hypergammaglobulnemia.Hyperproteinemia is either the result of dehydration orhypergammaglobulinemia. In this patient, given themarked inflammatory leukogram, the most likely interpre-tation is hypergammaglobulinemia. Some contributionfrom dehydration cannot be absolutely ruled out, but thedegree of hyperproteinemia is too great to be explained bydehydration alone.

SummaryHemogram data, when considered in conjunction with

the clinical presentation and history, are consistent with adiagnosis of FIP. FIP was confirmed both serologicallyand at necropsy.


Fig. 9 High magnification. A giant toxic band cell (center). Note the bluecytoplasm (Wright’s stain x 100).


Birman catHistory: Anorexia and dyspnea of

several days’ duration

HCT 25% WBC 54,000/µlHb 8.1 g/dl Bands 6,000/µlRBC 5.6 x 106/µl Neutrophils 44,000/µlTP 7.5 g/dl Lymphocytes 1,200/µlPlatelets Reduced Monocytes 2,000/µl

Eosinophils 800/µl

Morphology: There is marked toxicity of neu-trophils. Occasional reactive lymphocytes are seen.

Description

Leukogram: Marked leukocytosis characterizedby neutrophilia with a left shift, marginal lym-phopenia and monocytosis.

Erythrogram: There is a mild normocytic nor-mochromic anemia (MCV = 43 fl, MCHC = 33%).


Interpretation

1.) Active inflammatory leukogram with superimposedstress, tissue necrosis, and systemic toxemia. The left shiftand monocytosis are indicative of inflammation.Monocytosis also indicates tissue necrosis. High circulatinglevels of glucocorticoids (stress) are indicated by the mar-ginal lymphopenia. Systemic toxemia is indicated by thepresence of toxic neutrophil on the blood film. In this case,toxicity is severe. In dogs and cats, severe systemic toxemiais most often associated with bacterial infections.

2.) Anemia of inflammatory disease. Mild to moderate

Fig. 8 Low magnification reveals a leukocytosis with neutrophilia and left shift.

normocytic, normochromic non-regenerative ane-mia is consistent with the anemia of inflammatorydisease.

3.) Possible DIC. Considering the severity ofthe inflammation and systemic toxemia, thethrombocytopenia may be an indicator of DIC. Acomplete DIC panel is warranted.

SummaryThe diagnosis in this patient was bacterial

pyothorax. Based on a positive DIC panel, sub-clinical DIC also was identified.


Fig. 11 A toxic band cell (left), and a toxic neutrophil (right). Again, note the foamy,basophilic cytoplasm (Wright’s stain x 100).

Fig. 10 Two toxic neutrophils with foamy, basophilic cytoplasm (Wright’s stain x100).


CASE 9Signalment: Eight-year-old female

Cocker SpanielHistory: Presented with

generalizedlymphadenopathy

HCT 15% WBC 5,000/µlHb 5.0 g/dl Neutrophils 3,000/µlRBC 2.6 x 106/µl Lymphocytes 700/µlTP 6.0 g/dl Monocytes 1,000/µlPlatelets Reduced Eosinophils 300/µl

Description

Leukogram: There is leukopenia characterized byneutropenia and lymphopenia. No white cell morpho-logic abnormalities are noted on the peripheral bloodfilm.

Erythrogram: There is marked normocytic nor-mochromic anemia with no evidence of polychro-masia.


Interpretation

Possible marrow disease. Leukogram, erythrogram, andthrombogram data, considered collectively, indicate pancy-topenia. Unexplained cytopenias of one or more marrowcell line suggests a possible marrow production problem.Bone marrow examination is required for further evalua-tion. Possibilities include marrow hypoplasia/aplasia,myelophthisic syndrome, and myelofibrosis.In this case,marrow aspirates were highly cellular but abnormal. Fewgranulocyte, red cell, and platelet precursors were present.Instead, the predominant cell seen was a large round cellwith a scant to moderate rim of faintly basophilic cyto-plasm. The nuclei were round with a lacy reticular patternand very large prominent singular pale blue nucleoli. Thediagnosis was myelophthisic syndrome due to lymphoblas-tic lymphosarcoma.

DiscussionCase #9 was originally submitted by the referring veteri-

narian as suspect lymphosarcoma; blood was collected inan attempt to confirm the diagnosis. It should be noted thatwhere there is generalized lymphadenopathy, the preferredsample is a lymph node biopsy. Only rarely can a diagnosisof lymphosarcoma be confirmed with a CBC. In fact, themost common hematologic finding in cases of lymphosarco-ma is profound non-regenerative anemia, which occurswhen the marrow has been infiltrated by malignant lym-

phoblasts. Lymphopenia and lymphocytosis occurfar less frequently. Lymphopenia results whennormal recirculating lymphocytes become trappedin massively enlarged lymph nodes and cannot re-enter the peripheral blood. Lymphocytosis only

Fig. 12 Aspirate of a normal lymph node. Normal small lymphocytes predominatewith several larger prolymphocytes.

Continued

occurs when the marrow is so infiltrated thatmalignant cells spill over in high numbers into theperipheral blood. Lymphocytosis with malignantcells in circulation is, therefore, a late finding inmost cases of lymphosarcoma even though it isthe only circumstance where the diagnosis can beconfirmed with the CBC alone.


Fig. 13 Aspirate of a lymph node with lymphoma. The majority of the cells are largeblasts with very large nuclei and prominent nucleoli.


Fig. 14 Low magnification of pleural fluid. Normal small lymphocytes predominate.Red cells are seen in the background. This is typical of a chylous effusion.

CASE 10Signalment: Six-year-old female DSH

catHistory: Sudden onset dyspnea of

approximately 2 days’duration

HCT 45% WBC 10,600/µlHb 15.0 g/dl Neutrophils 10,000/µlRBC 9.0 x 106/µl Lymphocytes 200/µlTP 4.1 g/dl Eosinophils 400/µlPlatelets Adequate

Description

Leukogram: The only abnormality noted in theleukogram is profound lymphopenia.

Erythrogram: Red cell parameters fall within the ref-erence range. However, hypoproteinemia is present.

Thrombogram: No abnormalities noted.

Interpretation

1.) Profound lymphopenia. The most common cause oflymphopenia is high circulating glucocorticoid levels (stressleukogram). However, elevated glucocorticoids generallycause lymphopenias in the range of 700/µl to 1500/µl.When lymphocyte counts drop below 700/µl, other cases oflymphopenia should be considered. Profound lymphopeniascan be caused by anything that interrupts the normal circu-latory pattern of lymphocytes. Lymphocytes are predomi-nantly long-lived cells, which circulate in peripheral bloodto lymph nodes, migrate through the lymph nodes andenter the efferent lymphatics, and travel via lymph to re-enter the blood via the thoracic duct. Causes of profoundlymphopenias are therefore primarily lymphatic obstruction(neoplasias), and lymphatic rupture (chylothorax, chy-loperitoneum). With the history of sudden onset dyspnea,chylothorax should be strongly considered in this patient.

2.) Hypoproteinemia. Hypoproteinemia can result fromloss of blood or lymph, protein losing enteropathy, protein-

Continued


losing nephropathy or lack of protein productionby a damaged liver. In this case, the combinationof profound lymphopenia, hypoproteinemia, andclinical dyspnea all indicate an underlying chylouseffusion.

SummaryThe diagnosis of chylous effusion (chylotho-

rax) was confirmed cytologically by evaluation ofexcessive pleural fluid.

Fig. 15 High magnification of Fig. 14. Normal small lymphocytes.


CASE 11Signalment: Eight-week-old male

BeagleHistory: Sudden onset of

respiratory distress anddiarrhea (dark feces) of 2 days’ duration

HCT 25% WBC 19,100/µlHb 8.1 g/dl Bands 1,100/µlRBC 4.0 x 106/µl Neutrophils 14,000/µlTP 4.5 g/dl Lymphocytes 1,000/µlPlatelets Normal Eosinophils 3,000/µl

numbers,some large

Morphology: Mild to moderate polychromasia

Description

Leukogram: There is a leukocytosis (mild) with amild neutrophilia and left shift, eosinophilia, and lym-phopenia.

Erythrogram: There is a moderate anemia with mildto moderate polychromasia. There is mild to moder-ate hypoproteinemia.


Interpretation

1.) Acute inflammatory leukogram with systemic hyper-sensitivity and superimposed stress. Indicators of inflamma-tion are the left shift and eosinophilia. The eosinophilia alsois an indicator of systemic hypersensitivity. Considering theage of the patient, the possibility of circulating larvae ofintestinal parasites (hookworms, ascarids) as a cause of thehypersensitivity should be investigated. An associatedregenerative anemia suggesting blood loss would be sup-portive evidence of parasitemia. Even in the absence ofhookworm ova in the feces, late stage larvae in the gut arecapable of causing blood loss before ova are produced. Themarginal lymphopenia in this patient is best interpreted asindicative of high circulating levels of glucocorticoids(stress).

2.) Regenerative anemia. A mild to moderate anemiaaccompanied by mild to moderate polychromasia is sugges-tive of a regenerative anemia. An absolute reticulocytecount would probably be useful in confirming this interpre-tation. Regenerative anemias are the result of either bloodloss or hemolysis. Considering the suspicion of intestinalparasitism and the findings on the leukogram, blood lossanemia is the most likely cause.

3.)Hypoproteinemia. Young dogs (less than 9 months to 1year) generally have lower total protein levels than adultshave (in the range of 5.0 g/dl to 6.0 g/dl) but the level here isquite low and is a clear hypoproteinemia. Hypoproteinemia

can result from loss of blood or lymph, protein los-ing nephropathy (confirmed by protein loss inurine), protein losing enteropathy (associated withdiarrhea with voluminous stools), or lack of protein(albumin) production by the liver (confirmed byother evidence of liver pathology). In this case thelikely cause is blood loss as a result of intestinalparasitism.

SummaryConsidered collectively, hematologic data sug-

gests severe hookworm infection with associatedblood loss anemia and hypoproteinemia.Leukogram data suggests that there are still sys-temically migrating larvae on their way to thegut causing a hypersensitivity reaction andinflammation.Fig. 16 Two normal eosinophils.


Interpretation

1.) Inflammatory leukogram with tissue necrosis and sys-temic hypersensitivity. Monocytosis and eosinophilia areclear indicators of inflammation even though total white cellcount is in the reference range. Furthermore, monocytosissuggests demand for phagocytosis (tissue necrosis) whileeosinophilia (persistent) signals a systemic hypersensitivityreaction. Considering that total leukocyte count is normal,there is no left shift, and there is no lymphopenia, theinflammation is most likely chronic. Hyperproteinemia dueto hypergammaglobulinemia and the mild to moderate non-regenerative anemia of inflammation might be anticipated.

2.) Severe nonregenerative anemia with inappropriatenucleated red cell response. Mild to moderate polychroma-sia in the face of severe anemia is an inadequate response.Furthermore, the number of nucleated red cells present isfar too high for the degree of polychromasia and theresponse is therefore classified as inappropriate. The nota-tion of mitotic red cell precursors in the peripheral blood isalso quite disturbing and suggests that some circulatingnucleated red cells are fairly immature (rubricytes or moreprimitive). Inappropriate nucleated red cell responses arethe result of marrow stromal damage (heavy metal toxicity,anoxia, high circulating glucocorticoids), inadequatesplenic function, fractures, extramedullary hematopoiesis,or red cell marrow exhaustion. Compared to adult animals,very young animals have less marrow reserve. Consideringthe severity of anemia in this patient, the inappropriatenucleated response here probably results from marrow

stromal damage in the form of anoxic injury and red cell marrowexhaustion as the red cell compartment attempts to respond to thesevere anemia. This is an unfavorable finding in this puppy.

SummaryThis puppy is a littermate of the puppy in Case 11, seen one week

later, also untreated at the time of presentation. This puppy, like thepuppy in the previous case, has severe hookworm infestation.Migrating larvae are still probably present (based on the leukogram)as are parasitic forms in the bowel. With the inability of the red cellmarrow to keep pace with the loss of blood, the prognosis is guarded.Note that the inflammatory leukogram in this puppy, seen seven daysafter the first, is chronic and that there is no evidence of a stress leuko-gram (even though the puppy is undoubtedly “stressed”) which sug-gests chronic antigenic stimulation.

CASE 12Signalment: Nine-week-old female

BeagleHistory: Diarrhea and listlessness

of several weeks’ duration

HCT 15% WBC 17,000/µlHb 5.6 g/dl Neutrophils 10,000/µlRBC 2.7 x 106/µl Lymphocytes 2,000/µlTP 4.5 g/dl Monocytes 2,500/µlNRBC 30/100 WBC Eosinophils 2,500/µlPlatelets Adequate

Morphology: Mild to moderate polychromasia, manyNRBC, some mitotic.

Description

Leukogram: There is a normal leukocyte count char-acterized by a marked eosinophilia and monocytosis.

Erythrogram: There is a marked anemia with aninappropriate nucleated red cell response (too manynucleated RBCs relative to the degree of polychroma-sia). There is also moderate hypoproteinemia.



CASE 13Signalment: Fourteen-week-old male

BeagleHistory: Listless, anorectic, pale

mucous membranes

HCT 12% WBC 10,000/µlHb 3.0 g/dl Neutrophils 7,000/µlRBC 2.3 x 106/µl Lymphocytes 1,000/µlTP 3.8 g/dl Monocytes 1,500/µlPlatelets Increased Eosinophils 500/µl

numberswith many large platelets

Morphology: Moderate anisocytosis and poikilocyto-sis with moderate numbers of red cell fragments.Numerous cells have large pale centers.

Description

Leukogram: Total white cell count is within normallimits, but there is a lymphopenia (marginal) and mildmonocytosis.

Erythrogram: There is a marked microcytichypochromic anemia (MCV = 12/2.3 x 10 = 52 fl,MCHC = 3/12 x 100 = 25%). Hypochromia andmicrocytosis are confirmed by red cell morphologyon the blood film. There is also red cell fragmenta-tion.

Thrombogram: Thrombocythemia.

Interpretation

1.) Inflammatory leukogram with superimposed stressand tissue necrosis. Monocytosis indicates inflammationand tissue necrosis. Marginal lymphopenia indicates super-imposed stress. It is difficult to ascertain whether theprocess is acute or chronic. Repeat leukograms might behelpful in this regard.

2.) Iron-deficiency anemia. Marked microcytichypochromic anemia is highly suggestive of chronic bloodloss with resultant iron deficiency. Iron is required forhemoglobin formation. In turn, red cell hemoglobinizationregulates division of red cell precursors in the marrow.When iron is unavailable for adequate hemoglobin synthe-sis, precursors undergo additional divisions in the marrow,becoming smaller and smaller. Red cells eventuallyreleased into peripheral blood are both smal (microcytic)and deficient in hemoglobin (hypochromic). Although themechanism is not clearly understood, hypochromic red cellsare also more fragile than normal, hence the increasednumbers of red cell fragments on the blood film.

3.) Reactive thrombocythemia. With the severity of theanemia, it is likely that circulating levels of the red cellgrowth factor erythropoietin are quite high. Erythropoietinstimulates both red cell production and platelet produc-tion. The thrombocythemia with megaplatelets (immatureplatelets) is therefore most likely a reactive thrombo-

Fig. 17 Scanning magnification. Note the large unstained areas of central pallor inthe red cells.

Continued

cythemia in response to the erythropoietin. Theanemia is non-regenerative (note minimal poly-chromasia) because the iron-deficient marrow isunable to respond. White cell production is notaffected by erythropoietin.

SummaryThis pup is from the same litter as Cases 11

and 12. Like the others, it was untreated at thetime of presentation. The primary diagnosis issevere hookworm infestation with iron deficiencyanemia resulting from chronic blood loss.


Fig. 18 High magnification. The marked central pallor of the red cells typical of irondeficiency is easily apparent. A red cell fragment (schizocyte) is at left center.

Fig. 19 High magnification. A schizocyte is at center. Several polychromatophils arealso present.


CASE 14Signalment: One-year-old male Siamese

catHistory: Sudden onset depression

and anorexia with pale,icteric mucous membranes

HCT 20% WBC 18,700/lHb 6.4 g/dl Bands 1,000/lRBC 3.7 x 106/l Neutrophils 15,000/lTP 6.5 g/dl Lymphocytes 1,200/lPlatelets Adequate Monocytes 1,500/l

Morphology: Minimal polychromasia is present. Numerous RBCs contain chains of small basophilicbodies or ring forms.

Description

Leukogram: There is mild leukocytosis with neutrophil-ia, left shift, monocytosis, and a marginal lymphopenia.

Erythrogram: There is a moderate normocytic nor-mochromic anemia. (MCV = 54 fl, MCHC = 32%).The basophilic bodies on the red cells are consis-tent with Hemobartonella felis.


Interpretation

1.) Active inflammatory leukogram with tissue necrosisand superimposed stress. Indicators of inflamation are theleft shift and the monocytosis. Monocytosis is also an indi-cator of tissue necrosis. The marginal lymphopenia isindicative of superimposed stress.

2.) Anemia due to hemobartonellosis. The presence oflarge numbers of Hemobartonella bodies on the blood filmestablishes the diagnosis of hemobartonellosis. In its pri-mary form, hemobartonellosis is a regenerative hemolyticanemia. In this case, there is no evidence of regeneration(no polychromasia) at the time of presentation.Hemobartonellosis may also be seen as a non-regenerativeanemia when it occurs secondarily to serious immunosup-pressive disorders such as FeLV, FIV, or FIP. Primaryhemobartonellosis responds well to antibiotic therapy;however, when hemobartonellosis occurs secondarily, theprognosis is guarded. Clearly, this patient should be moni-tored over the next few days for the appearance of poly-chromasia and reticulocytosis. Tests for FeLV, FIV, andFIP are warranted. The inflammatory leukogram in thiscase is probably in response to the destruction of infected

Fig. 20-22 Numerous red cells parasitized by Hemobartonella felis are seen in thethree figures. Organisms are present singly and in chains. (Wright’s stainx 100).

Continued

red blood cells by tissue macrophages. Red celldestruction is a form of tissue necrosis.

SummaryPolychromasia/reticulocytosis became promi-

nent within 24 to 48 hours after initial presenta-tion. Over the same period, the number of cellscontaining Hemobartonella bodies dropped dramat-ically as infected cells were removed from theblood. Tests for FeLV, FIV, and FIP were nega-tive. The anemia responded favorably to tetracy-cline therapy. Clearly, this was a case of primaryhemobartonellosis, which presented in the firstfew days of infection before a fully developedregenerative response was apparent.


Fig. 21

Fig. 22


CASE 15Signalment: Five-year-old female Basset

HoundHistory: Sudden onset lethargy,

emesis, anorexia, and yellowmucous membranes ofapproximately 4 days’duration

HCT 25% WBC 19,500/µlHb 7.0 g/dl Bands 2,000/µlRBC 3.0 x 106/µl Neutrophils 15,000/µlTP 7.0 g/dl Lymphocytes 1,200/µlPlatelet Adequate Monocytes 1,300/µl

Morphology: Moderate to marked polychromasia,numerous schizocytes, and spherocytes.

Description

Leukogram: There is a mild leukocytosis with neu-trophilia, left shift, marginal monocytosis, and mar-ginal lymphopenia.

Erythrogram: There was moderate anemia charac-terized by polychromasia, schizocytes and sphero-cytosis.


Interpretation

1.) Inflammatory leukogram with tissue necrosis andsuperimposed stress. The left shift and marginal monocyto-sis are indicators of inflammation. Marginal monocytosisalso indicates tissue necrosis. The marginal lymphopeniastrongly suggests superimposed stress.

2.) Suspected immune-mediated hemolytic anemia.Anemia with moderate to marked polychromasia implies aregenerative anemia. Regenerative anemias are either theresult of hemolysis or blood loss. In this case, the presenceof spherocytes on the peripheral blood film strongly sug-gests an immune-mediated hemolytic process. A directantiglobulin test (Coomb’s test, DAT) could be run for con-firmation.

CommentImmune-mediated hemolytic anemias are often accompa-

nied by inflammatory leukograms with tissue necrosis. Thesource of the tissue necrosis is the breakdown of red bloodcells both in the circulation and in tissues rich in

Continued

Fig. 23 Scanning magnification. Red cells exhibit anisocytosis. Severalpolychromatophils and nucleated red cells are present. Leukocytes includetwo monocytes (at center), several mature neutrophils, and a band cell (topleft center).

macrophages such as spleen. The adequateplatelet count is a favorable finding in this patient;some cases of immune-mediated hemolysis arealso accompanied by immune-mediated thrombo-cytopenia. Patients with both hemolysis andthrombocytopenia are generally less responsive totherapy than immune-mediated hemolysis alone.


Fig. 24-25 High magnification. Note the presence of spherocytes, severalpolychromatophils and a nucleated red (Fig. 5.25). Red cell changes areconsistent with immune-mediated hemolytic anemia. White cells presentinclude a monocyte (Fig. 5.25), a band cell (Fig. 5.25), and matureneutrophils. The neutrophil in Fig. 5.25 is slightly toxic.

Fig. 25


CASE 16Signalment: Eight-year-old female

German ShepherdHistory: Chronic wasting syndrome

HCT 30% WBC 15,350/µlHb 9.0 g/dl Neutrophils 8,000/µlRBC 4.0 x 106/µl Lymphocytes 3,000/µlTP 6.5 g/dl Monocytes 4,000/µlPlatelets Adequate Eosinophils 350/µl

Reticulocyte count = 5 %

Description

Leukogram: There is a marked monocytosis; all othervalues are within the reference range.

Erythrogram: There is a mild marginally macrocytic(calculated MCV = 75 fl) marginally hypochromic (cal-culated MCHC = 30%) anemia with an absolute reticu-locyte count of approximately 200,000/µl (referencerange to 80,000/µl). Spiculated poikilocytosis is promi-nent.


Interpretation

1.) Chronic inflammatory leukogram with tissue necro-sis. Monocytosis is an indicator of both inflammation andtissue necrosis. The normal total leukocyte count, absenceof a neutrophilia and left shift, and absence of lymphope-nia, all suggest the establishment of a new white cell steadystate with marrow production of leukocytes balancing tis-sue demand (utilization). There is probably granulocytichyperplasia in the marrow and shortened circulating half-life of granulocytes in the peripheral blood. These are clas-sic features of chronic inflammation.With the presence of achronic inflammatory leukogram, several other hemogramchanges might be anticipated. The mild to moderate non-regenerative anemia associated with inflammatory diseaseis to be expected. Furthermore, chronic inflammation isoften associated with hyperproteinemia as a result of hyper-gammaglobulinemia.

2.) Regenerative acanthocytic anemia. Anemia withpolychromasia and reticulocytosis is regenerative.Consequently, the anemia in this case cannot be explainedas the anemia of inflammatory disease alone. Regenerativeanemias result from either blood loss or hemolysis.Acanthocytes are red cells with 2 to 10 blunt, glove-likeprojections from the surface. Acanthocytic anemia in dogshas been associated with liver disease. In particular, acan-thocytic regenerative anemia hase been associated with

Fig. 26 Scanning magnification. Monocytosis is apparent. Red cells exhibitanisocytosis and polychromasia.

Continued

bleeding hemangiosarcomas of the liver, especiallyin middle-aged, large breed dogs such as theGerman Shepherd patient in this case. A possibili-ty of abdominal hemangiosarcoma should there-fore be strongly considered here. The finding ofadequate platelets is significant and favorable.Many cases of abdominal hemangiosarcomas pre-sent either with disseminated or localized coagu-lopathies; these are generally thrombocytopenic.

SummaryRadiography revealed an enlarged spleen and

liver. At exploratory laparotomy, multiple neoplas-tic nodules were found in both organs.Histopathology confirmed the preliminary diagno-sis of hemangiosarcoma. Many of the nodulesappeared to have necrotic centers; tumor necrosiswas the presumed cause of the inflammatoryleukogram.


Fig. 27 High magnification. Two polychromatophils and nucleated red cells (left).Numerous red cells have multiple finger-like projections (acanthocytes).Platelets are obvious.


CASE 17Signalment: Fifteen-year-old neutered

female DSH catHistory: Weight loss, pica, and

hyperexcitability

HCT 35% WBC 11,000/µlHb 12.0 g/dl Neutrophils 8,500/µlRBC 7.5 x 106/µl Lymphocytes 1,700/µlTP 6.5 g/dl Monocytes 500/µlPlatelets Adequate Eosinophils 300/µl

Morphology: 50% or more of the red cells containdiscernible Heinz bodies.

Description

Leukogram: Unremarkable

Erythrogram: Numerical data are unremarkable;however, the presence of large numbers of Heinzbodies on the blood film is noteworthy.


Interpretation

Possible metabolic disease. In cats, the presence of largenumbers of Heinz bodies in the absence of hemolytic ane-mia has been associated with metabolic and endocrinologicdiseases. The most frequent associations have been withdiabetes mellitus, hyperthyroidism, and liver disease.

SummaryFurther evaluation of this patient led to a diagnosis of

hyperthyroidism. As is common in these cases, a routineclinical chemistry panel revealed no abnormalities. Thediagnosis was confirmed based upon markedly elevatedresting T4 levels.

Fig. 29 With vital stains, Heinz bodies are much moreobvious. In this new methylene blue-stainedpreparation, they are seen as blue precipitates withinthe red cell (100x).

Fig. 28 Several prominent Heinz bodies (seen as nose-like projections from the redcell surface). Note the lack of polychromasia (Wright’s stain x 100).


CASE 18Signalment: Ten-year-old female

mixed-breed dogHistory: Polyuria and polydipsia of

3 months’ duration withprogressive anorexia

HCT 24% WBC 8,500/µlHb 7.8 g/dl Neutrophils 7,000/µlRBC 3.8 x 106/µl Lymphocytes 1,000/µlTP 3.2 g/dl Monocytes 500/µlPlatelets Adequate

Morphology: Numerous Burr cells seen

Description

Leukogram: The principal abnormality noted ismarginal lymphopenia.

Erythrogram: There is a moderate normocytic(MCV = 63 fl), normochromic (MCHC = 32%)anemia with Burr cells. No polychromasia isnoted.


Interpretation

1.) Stress leukogram. Marginal lymphopenia stronglysuggests high circulating glucocorticoid levels and a stressleukogram.

2.) Non-regenerative anemia with Burr cells. Theabsence of polychromasia confirms nonregenerative ane-mia in this patient. Red cell indices within the referencerange are expected. Burr cells are elongated (oval) RBCswith ruffled membranes. In humans, spiculated RBCs withBurr cell morphology have often been associated withrenal disease. This association is less clear in animals.However, large numbers of spiculated red cells (acantho-cytes, Burr cells) in canine blood films have been associat-ed with either liver disease or renal disease. In general,when acanthocytes are predominant, liver disease is more

Fig. 30 Scanning magnification. Poikilocytosis (variable red cell shape) is striking.Numerous elongated red cells (ovalocytes).


likely; when Burr cells are prevalent, renal disease shouldbe higher on the differential. Renal disease is also fairlyconsistently associated with non-regenerative anemia with-out red cell morphologic changes. Considering the clinicalsigns, the anemia, and the Burr cells, the possibility of renaldisease should be thoroughly investigated in this patient.

SummaryClinical chemistry and urinalysis confirmed the diagno-

sis of renal failure (BUN = 242, creatinine = 4.3, urine spe-cific gravity = 1.009). Renal biopsy established a morpho-logic diagnosis of end-stage kidney disease (nephrosclero-sis). It is of interest that minimal inflammation was seenhistologically; this is consistent with the lack of an inflam-matory leukogram.

Fig. 31 High magnification. Elongated red cells with ruffled membranes (Burr cells)have been associated with metabolic disorders, particularly renal disease,in humans and dogs.


CASE 19Signalment: Five-year-old female

CoonhoundHistory: Gradually worsening CNS

signs – dullness, rareseizures, occasional circling

HCT 35% WBC (corrected) 15,500/µlHb 11.1 g/dl Neutrophils 10,000/µlRBC 5.2 x 106/µl Lymphocytes 3,000/µlTP 6.5 g/dl Monocytes 2,500/µlNRBC 85/100 WBC Platelet Adequate

Morphology: No polychromasia seen

Description

Leukogram: Total white cell count is within the refer-ence range; however, there is a marked monocytosis.

Erythrogram: There is a mild anemia with noincrease in polychromasia and pronounced inappro-priate nucleated red cell response (increased nucle-ated red cells in the absence of significant polychro-masia).


Interpretation

1.) Chronic inflammatory leukogram with tissue necro-sis. Monocytosis indicates both inflammation and tissuenecrosis. The normal white cell count, absence of a leftshift, lack of lymphopenia, and the monocytosis are consis-tent with chronic inflammation.

2.) Bone marrow stromal damage. An inappropriatenucleated red cell response is usually associated with dam-age to bone marrow stroma and indiscriminate leakage ofimmature red cells into circulation. Causes include endo-toxemia, high circulating levels of glucocorticoids, frac-tures, lead poisoning (in dogs), and FeLV disease in cats.Other causes of inappropriately increased numbers ofnucleated red cells in circulation include extramedullaryhematopoiesis and splenic dysfunction or splenectomy. Thedegree of inappropriate response is of interpretive signifi-cance. Nucleated red cell counts of greater than 10/100WBC are marked and are most likely the result of lead poi-soning in the dog, while in the cat, they most commonly areassociated with FeLV-induced marrow disease. In the pre-sent case, lead poisoning is the most likely diagnosis. Lead

Fig. 32 Scanning magnification. Numerous nucleated cells, many of them nucleatedred cells, are seen. There also appears to be a left shift.


poisoning can also cause tissue necrosis with mono-cytosis and CNS clinical signs as seen here.

Summary Blood lead levels of 0.7 ppm confirmed the sus-

pected diagnosis of lead poisoning.

Comment Basophilic stippling of RBCs has long been cited

as an important hematologic finding in cases of leadpoisoning. However, basophilic stippling is not aconstant finding and was not present here. Bloodfilms in this case were made from blood collectedwith EDTA; basophilic stippling is more easilydemonstrated in films made from blood not treatedwith anticoagulants. When lead poisoning is sus-pected, it may be useful to prepare several directblood films for staining and evaluation.

Fig. 33 High magnification. The number of nucleated red cells is too high for thedegree of polychromasia (inappropriate nucleated red cell response). Notethat the NRBC (center) is stippled (contains basophilic cytoplasmicprecipitates). The two neutrophilic bands (left) have toxic cytoplasm.

Fig. 34 High magnification. The nucleated red cell (left) is quite immature.

Fig. 35 High magnification. Three nucleated red cells and a toxic metamyelocyte.


CASE 20 Signalment: Seven-year-old male DSH cat History: Chronic weight loss and

anorexia. At presentation, thecat is cachectic, weak, anddepressed, with pale mucousmembranes.

HCT 12% Neutrophils 1,000/µlHb 4.0 g/dl Lymphocytes 500/µlRBC 2.6 x 106/µl Monocytes 300/µlTP 6.0 g/dl Eosinophils 200/µl NRBC 400/100 WBC Nucleated cells 30,000/µlPlatelets Reduced (6,000/µl

corrected)

Blasts & unclassifed 4,000/µl

Morphology: Very high nucleated red cell count withrecognizable red cell precursors in all stages of differ-entiation. Remaining nucleated cells are largely blastsand unclassified cells. Blasts appear to be of both redcell and granulocytic lines.

Description

Leukogram: Leukopenia with marked neutropenia,lymphopenia, and increased numbers of blasts andunclassified cells.

Erythrogram: Severe normocytic, normochromicanemia with profound metarubricytosis.


Interpretation

1.) Pancytopenia. Mature forms of all cell lines (whitecell, red cell, and platelet) are present in markedly reducednumbers. This finding alone suggests marrow disease andwould warrant marrow evaluation. In the cat, the primaryconcerns with a history of chronic disease are FeLV or FIPinfection.

2.) Inappropriate nucleated red cell response.Metarubricytosis in the absence of polychromasia is aninappropriate nucleated red cell response and suggestsmarrow stromal disease. In this patient, where numerousstages of RBC precursors are seen in the blood, it is highlysuggestive of FeLV related marrow disease. Because of themarked metarubricytosis, total nucleated cell count mustbe corrected. This is done according to the following for-mula:

Fig. 36 Low magnification shows large numbers of nucleated red cells. Note thatthe film is very thin, suggesting severe anemia (Wright’s stain x 50).

Total nucleated cell count

In this case, 30,000 x

100 + NRBC/100 WBCx

100

100 + 400

100

Corrected WBC count

6,000

=

=


3.) Probable FeLV related erythroleukemia. Thepresence of numerous blasts and abnormal unclas-sifed cells further supports the suggestion of FeLVrelated disease with a leukemic phase. Becauseblasts and poorly differentiated precursors of bothred cell and granulocyte lineage are seen, ery-throleukemia (combined red and white cellleukemia) is the best specific diagnosis.

Comment The cat tested positive for FeLV and negative

for FIP.

Fig. 38 Various stages of nucleated red cell differentiation (Wright’s stain x 100).

Fig. 37 Three metarubricytes, a lymphocyte, and a nucleated cell that is difficult toclassify (Wright’s stain x 100).


Interpretation

1.) Stress leukogram. Marginal lymphopenia is sugges-tive of stress.

2.) Consumptive/destructive thrombocytopenia.Thrombocytopenias can result from a lack of production ofplatelets by the marrow, sequestration of platelets inperipheral tissues (hypersplenism), consumption ofplatelets in hypercoagulation syndromes (DIC), or destruc-tion of platelets by anti-platelet antibodies (immune-mediat-ed thrombocytopenia). Hypersplenism is rare in animalsand is associated with an enlarged spleen, which was notpresent in this case. Production thrombocytopenias are dif-ferentiated from consumption/destruction thrombocytope-nias with bone marrow evaluation. Production thrombocy-topenias are characterized by reduced numbers of marrowmegakaryocytes, while consumption/destruction thrombocy-topenias have normal to increased numbers of megakary-ocytes. Marrow morphology in this cat strongly suggests aconsumption/destruction problem. Given the absence of aninflammatory leukogram, DIC and consumption thrombocy-topenia are highly unlikely. This is, therefore, most likely acase of immune-mediated thrombocytopenia.

3.) Anemia of uncertain origin. The anemia is mild, and,based on peripheral blood findings, non-regenerative.However, bone marrow morphology indicates red cellregeneration. Either this is a regenerative anemia (bloodloss or hemolytic) in its earliest stages (before peripheralreticulocytosis has had time to occur – 72 to 96 hours.), oran immune-mediated marrow disease directed against latestage red cell precursors. Repeated CBCs should help clari-fy the situation; if truly regenerative, polychromasia will be

present on blood films within the next few days.

SummaryBlood films taken within 24 hours of presentation contained signifi-

cant numbers of reticulocytes, thus confirming the regenerative nature ofthe anemia. No spherocytes were observed. A presumptive diagnosis ofblood loss anemia secondary to thrombocytopenia was made. Steroidtherapy was initiated to treat the presumed immune-mediated thrombo-cytopenia. A rising platelet count was seen within 24 hours of therapyinduction. Platelet counts had returned to normal by 10 days, confirmingthe diagnosis of immune-mediated thrombocytopenia.

CASE 21Signalment: Four-year-old female Persian catHistory: Sudden onset epistaxis with

numerous petecchiae seen onphysical examination

HCT 30% WBC 11,950/µlHb 10.0 g/dl Neutrophils 10,000/µlRBC 6.2 x 106/µl Lymphocytes 1,100/µlTP 6.7 g/dl Monocytes 850/µlPlatelets Rare

Bone marrow examination: Very cellular. Erythroidhyperplasia with left shift and marked megakaryocytichyperplasia evident.

Description

Leukogram: All leukocyte values are within the refer-ence range but lymphocyte numbers are marginally low(marginal lymphopenia).

Erythrogram: There is a mild anemia with no reportedpolychromasia. Computed red cell indices are within thereference range (MCV = 47 fl, MCHC = 33%).



Interpretation

1.) Overwhelming inflammation with tissue necrosis. Theleft shift and monocytosis indicate inflammation.Monocytosis also indicates tissue necrosis with demand forphagocytosis. The low total white cell count and neutropeniasuggest an inability of marrow production to keep pace withtissue demand; hence, the inflammatory process is severe andoverwhelming with a guarded prognosis.

2.) Stress leukogram. Marked lymphopenia indicatesstress. The degree of lymphopenia is so great that otherpossible causes of lymphopenia should also be considered.

3.) Non-regenerative anemia. Anemia without polychro-masia is non-regenerative. The degree of anemia may bemore severe than is indicated by the red cell data becausethe animal appears to be dehydrated (elevated plasma pro-tein in the face of severe diarrhea suggests hemoconcentra-tion from dehydration, other indicators of hemoconcentra-tion could be found in urinalysis data and chemistry panels,eg, concentrated urine specific gravity, elevated BUN, crea-tinine, serum albumin, and electrolytes). Considering thatthrombocytopenia is also present and that stools are tarry,the possibility of an early blood loss anemia (before poly-chromasia becomes evident) should be considered.

4.) Possible DIC. The combination of thrombocytopeniaand schizocytosis on the peripheral blood film suggest thepossibility of disseminated intravascular coagulopathy. Afull DIC panel (prothrombin time, activated partial throm-boplastin time, fibrin split products, fibrinogen, andplatelet count) should be run. If three of the five tests areabnormal, DIC is confirmed. Overwhelming inflammatorydisease is frequently associated with secondary DIC.

SummaryThe final diagnosis in this patient is parvoviral enteritis

with secondary bacterial enteritis, endotoxemia and DIC. Fibrin splitproducts were elevated, PT and PTT were prolonged, and platelet countwas reduced. Blood loss through the intestinal tract was the cause of theanemia. Within 24 hours of presentation, polychromasia became apparenton peripheral films.

CASE 22Signalment: One-year-old male Cocker

SpanielHistory: Severe depression and

severe, acute diarrhea withdark tarry stools

HCT 33% WBC 6,000/µlHb 11.0 g/dl Bands 1,000/µlRBC 5.0 x 106/µl Neutrophils 1,500/µlTP 8.0 g/dl Lymphocytes 500/µlPlatelets Rare Monocytes 3,000/µl

Morphology: Neutrophils and bands are foamy andbasophilic. Moderate poikilocytosis characterized pri-marily by schizocytes.

Description

Leukogram: There is a leukopenia characterized by amarked neutropenia with a left shift (degenerative leftshift), a profound lymphopenia, and a marked mono-cytosis.

Erythrogram: There is a mild anemia with poikilocy-tosis characterized primarily as schizocytosis. There ishyperproteinemia. There is no polychromasia.

Thrombogram: Marked thrombocytopenia.


CASE 23Signalment: Two-year-old male DSH catHistory: Weight loss, anorexia, and

recent diarrhea

HCT 35% WBC 2,200/µlHb 11.7 g/dl Neutrophils 500/µlRBC 6.2 x 106/µl Lymphocytes 700/µlTP 7.0 g/dl Monocytes 1,000/µlPlatelets Reduced

Morphology: Neutrophils seen are extremely toxic.

Description

Leukogram: There is a marked leukopenia character-ized by a marked neutropenia and lymphopenia andsystemic toxemia.

Erythrogram: There is a minimal to mild anemia in theabsence of polychromasia.


Interpretation

1.) Possible marrow disease with systemic toxemia.There are no absolute indicators of inflammation in theleukogram and concomitant thrombocytopenia suggests apossible marrow production problem. The marked neu-trophil toxicity coupled with leukopenia and neutropeniasuggests a possible secondary overwhelming bacterial infec-tion.

2.) Superimposed stress. The marked lymphopenia isindicative of high circulating levels of glucocorticoids.

3.) Minimal to mild non-regenerative anemia. Reductionin red cell mass is so mild that it is probably not clinicallysignificant at this time. However, red cell data should beclosely monitored to see if anemia develops further. At thispoint, it is normocytic, normochromic and non-regenera-tive.

4.) Thrombocytopenia. Thrombocytopenia may be theresult of a marrow production problem or may reflect pos-sible DIC associated with overwhelming bacterial infection.Both a bone marrow evaluation and a full DIC panel arewarranted.

SummaryHematologic data from this cat are similar to that from

the dog in Case 22, but clear indicators of inflammation arelacking. This is a case of feline panleukopeniawith secondary bacterial enteritis and endotox-emia. DIC was confirmed by elevated fibrin splitproducts, prolonged prothrombin time, and pro-longed activated partial thromboplastin time.Marrow evaluation revealed a production prob-lem characterized by hypoplasia and necrosisprobably caused by feline panleukopenia virusinfection. Peripheral red cell data do not yetreflect the production problem because red celllifespan is longer than that of leukocytes andplatelets.

Fig. 39 High magnification. Four toxic neutrophils with foamy basophilic cytoplasmand scattered Döhle bodies. The neutrophil at upper right is a giant formand has unusual nuclear shape.


CASE 24Signalment: Four-year-old female

Collie-Labrador mixHistory: Acute onset lethargy with

pale mucous membranes

HCT 21% WBC 18,000/µlHb 7.0 g/dl Bands 1,000/µlRBC 3.1 x 106/µl Neutrophils 14,000/µlTP 6.8 g/dl Lymphocytes 1,500/µlPlatelets Reduced Monocytes 1,500/µl

Reticulocyte count = 4%

Description

Leukogram: There is mild leukocytosis with neu-trophilia, left shift, marginal lymphopenia, and mildmonocytosis.

Erythrogram: There is a moderately severe ane-mia with reticulocytosis. The absolute reticulocytecount is 124,000/µl. MCV and MCHC (computed)are within the reference ranges.


Interpretation

1.) Inflammatory leukogram with tissue necrosis.Neutrophilia with a left shift indicates inflammation. Mildmonocytosis indicates tissue necrosis.

2.) Superimposed stress. Marginal lymphopenia suggestshigh circulating glucocorticoids (stress).

3.) Regenerative anemia. Anemia with an absolute retic-ulocyte count of 124,000/µl is regenerative. Differentiationbetween blood loss and hemolysis is not possible at thistime. The presence of noticeable schizocytes (see Figs. 28,29) suggests that microangropathic hemolysis is at leastpartly responsible.

4.) Possible DIC. The combination of inflammatoryleukogram, thrombocytopenia, and schizocytes on theblood film suggest possible DIC. As in Cases 17 and 18, afull DIC panel is warranted.

Summary Considered collectively, hematologic data suggests an

Fig. 40 High magnification. Note the absence of platelets. Three red cell fragments(schizocytes).

Continued

inflammatory process complicated by DIC withassociated regenerative anemia. The anemia maybe complex, possibly resulting from a combinationof blood loss and microangiopathic hemolysis. Theactual diagnosis in this patient was malignantmammary carcinoma with widespread metastasis.At the time of presentation, there was hemothoraxsecondary to bleeding tumor nodules on the sur-face of the lung. DIC was also confirmed withadditional laboratory tests.


Fig. 41 High magnification. Two schizocytes.



Persian catHistory: Chronic, intermittent

cough and occasionalwheezing

HCT 40% WBC 17,000/µlHb 13.1 g/dl Neutrophils 10,000/µlRBC 8.0 x 106/µl Lymphocytes 2,000/µl

Eosinophils 4,500/µlPlatelets Adequate Monocytes 500/µl

Description

Leukogram: There is a high normal leukocyte countwith a marked eosinophilia.

Erythrogram: Unremarkable.


Interpretation

Inflammatory leukogram with systemic hypersensitivity.Eosinophilia, if persistent, is a clear indicator of bothinflammation and systemic hypersensitivity. In cats, poten-tial causes include eosinophilic granuloma complex (thesystemic linear plaque form), feline asthma, systemic mastcell tumor, flea-bite dermatitis, allergic gastroenteritis, andparasitic infections with a systemic phase. In this case, con-sidering the clinical presentation, feline asthma is at the topof the differential


Part III


Units Dog Cat

Total Protein (TP) g/dl 6.0 - 8.0 6.0 - 8.0

HCT % 37 - 55 30 - 45

Hb g/dl 12 - 18 8 - 15

RBC ×106/µl 5.5 - 8.5 5.0 - 10.0

WBC ×103/µl 6 - 17 6 - 18

Bands /µl 0 - 300 0 - 300

Neutrophils /µl 3,000 - 12,000 3,000 - 12,000

Lymphocytes /µl 1,000 - 5,000 1,500 - 7,000

Monocytes /µl 150 - 1,350 50 - 850

Eosinophils /µl 100 - 1,250 100 - 1,500

MCV fl 60 - 75 40 - 55

MCHC g/dl 32 - 36 30 - 36

Fibrinogen mg/dl 200 - 400 150 - 300

Platelets × 105/µl 2 - 9 3 - 7

Prothrombin Time Seconds 5.5 - 7.9 6.4 - 9.6

Partial ThromboplastinTime (APTT or PTT) Seconds 11.4 - 16.4 9.3 - 18.7

FSPs(Fibrin/FibrinogenSplit Products) g/ml <10 <10

Hematology Reference Ranges


Chapter 1: Leukocytes in Health & Disease

Fig. 1 .........Normal circulating neutrophils.

Fig. 2 ........Normal canine eosinophil (left), reactive lym-phocyte (right).

Fig. 3 ........Normal feline eosinophil.

Fig. 4 .........Normal canine basophil.

Fig. 5 .........Normal canine basophil.

Fig. 6 .........Normal feline basophil (left), normal neutrophil(right).

Fig. 7 .........Normal canine monocytes.

Fig. 8 .........Normal small lymphocyte (round nucleus),three neutrophils, and normal band cell (right).

Fig. 9 .........Blast-transformed (reactive, activated) lympho-cyte.

Fig. 10 .......Reactive lymphocyte (lower center), three neu-trophils, and a monocyte (top center).

Fig. 11 .......Reactive lymphocyte (center) and four neu-trophils.

Fig. 12 .......Reactive lymphocyte.

Fig. 13 .......Reactive lymphocyte.

Fig. 14 .......Fully differentiated plasma cell.

Fig. 15 .......Toxic band cell with foamy, basophilic cyto-plasm.

Fig. 16 .......Giant toxic band with foamy, basophilic cyto-plasm and giant, irregular nucleus.

Fig. 17 .......Two toxic neutrophils and a toxic band. Notethe unusual nuclear shape.

Fig. 18 .......Two toxic neutrophils with Döhle bodies.

Fig. 19 .......Atypical lymphocyte.

Chapter 2: Erythrocytes in Health &Disease

Fig. 1 .........Normal canine blood film. Scanning magnifica-tion. RBCs are uniform in size, shape, andcolor, and feature prominent central pallor.

Fig. 2 .........Canine blood film with increased polychroma-sia. Scanning magnification. Polychromatophilsare bluish and generally larger than maturecells.

Fig. 3 .........High magnification of Fig. 2. This represents aregenerative anemia with marked polychroma-sia. Note the marked separation of RBCs.

Fig. 4 .........Low magnification of a new methylene blue-stained blood film. Reticulocytes stand out dueto blue stained reticulum visible at this magnifi-cation. This represents a highly regenerativeanemia.

Fig. 5 .........High magnification of Fig. 4. Reticulocytes withprecipitated blue reticulum are obvious. Indogs, all would be counted; in cats, only thosewith abundant precipitate (aggregate reticulo-cytes) would be counted. Several leukocytes arealso present.

Fig. 6 .........Canine immune-mediated hemolytic anemia.The anemia is highly regenerative with markedpolychromasia. A spherocyte (arrow).

Fig. 7 .........A second case of canine immune-mediatedhemolytic anemia with numerous spherocytes.

Fig. 8 .........Feline immune-mediated hemolytic anemia.Spherocytes are numerous but less obvious dueto of the lack of central pallor in the normalRBCs.

Fig. 9 .........Saline-diluted wet preparation demonstratingautoagglutination.

Fig. 10 .......Heinz body hemolytic anemia in the dog.Several cells with nose-like projections (Heinzbodies) and several polychromatophils.

Index of Figures

All slides courtesy of Alan H. Rebar, DVM, PhD.


Fig. 11 .......Canine Heinz body hemolytic anemia, newmethylene blue stain. Several cells with Heinzbodies (upper left). Two reticulocytes withaggregated reticulum (right center).

Fig. 12 .......Feline hyperthyroidism. Numerous cells haveobvious Heinz bodies projecting from their sur-faces, but anemia is not present.

Fig. 13 .......Canine hemobartonellosis. Several cells havechains of beaded basophilic organisms on theirsurfaces. Several polychromatophils and aschizocyte.

Fig. 14 .......Feline hemobartonellosis. Several polychro-matophils are present. The red cell (center) hasseveral prominent Hemobartonella bodies.

Fig. 15 .......Canine babesiosis. The cell (center) has twoteardrop-shaped organisms. A red cell (upperright) with a single organism.

Fig. 16 .......Microangiopathic hemolysis in disseminatedintravascular coagulopathy (DIC). Numerousschizocytes.

Fig. 17 .......Hepatic hemangiosarcoma in a dog. Two acan-thocytes (center). A schizocyte (left).Numerous target cells, which can be precursorsto acanthocytes. Polychromasia indicates amildly regenerative anemia.

Fig. 18 .......Canine glomerulonephritis. Numerous Burr cells.

Fig. 19 .......FeLV(peripheral blood film). Two megaloblasts.

Fig. 20 .......Bone marrow smear from the same case as Fig.19. A large megaloblast (right).

Fig. 21 .......Iron deficiency anemia. Numerous red cellshave exaggerated areas of central pallor; someare smaller than normal. A red cell fragment(schizocyte-center).

Fig. 22 .......Myelofibrosis in the dog. A dacryocyte (centerand top center). Numerous ovalocytes.

Chapter 5: Case Studies

Fig. 1 .........Case #4. A band cell with slight toxicity (left).Note the foamy, granular, slightly basophiliccytoplasm. A normal monocyte (right).

Fig. 2 .........Case #4. Two band cells and a mature neu-trophil.

Fig. 3 .........Case #4. A toxic band cell (left), and a toxicmetamyelocyte (right). The cytoplasm of bothcells is too blue.

Fig. 4 .........Case #4. A toxic neutrophil with foamy,basophilic cytoplasm and Döhle bodies.

Fig. 5 .........Case #5. On scanning magnification, a monocy-tosis is obvious. Four monocytes and seven neu-trophils.

Fig. 6 .........Case #6. A reactive lymphocyte with largenucleus, lacy chromatin pattern, and basophiliccytoplasm.

Fig. 7 .........Case #6. Two reactive lymphocytes.

Fig. 8 .........Case #8. Low magnification reveals a leukocy-tosis with neutrophilia and left shift.

Fig. 9 .........Case #8. High magnification. A giant toxic bandcell (center). Note the blue cytoplasm (Wright'sstain x 100).

Fig. 10 .......Case #8. Two toxic neutrophils with foamy,basophilic cytoplasm (Wright's stain x 100).

Fig. 11 .......Case #8. A toxic band cell (left), and a toxicneutrophil (right). Again, note the foamy,basophilic cytoplasm (Wright's stain x 100).

Fig. 12 .......Case #9. Aspirate of a normal lymph node.Normal small lymphocytes predominate withseveral larger prolymphocytes.

Fig. 13 .......Case #9. Aspirate of a lymph node with lym-phoma. The majority of the cells are large blastswith very large nuclei and prominent nucleoli.

Fig. 14 .......Case #10. Low magnification of pleural fluid.Normal small lymphocytes predominate. Redcells are seen in the background. This is typicalof a chylous effusion.


Fig. 15 .......Case #10. High magnification of Fig. 14.Normal small lymphocytes.

Fig. 16 .......Case #11. Two normal eosinophils.

Fig. 17 .......Case #13. Scanning magnification. Note theextremely pale staining of the RBCs.

Fig. 18 .......Case #13. High magnification. Note the markedcentral pallor of the RBCs. This is typical ofiron deficiency.

Fig. 19 .......Case #13. High magnification. A schizocyte(left center).

Fig. 20-22..Case #14. Numerous RBCs parasitized byHemobartonella felis are seen in all three figures.Both ring forms as well as chains of coccalforms are present (Wright's stain x 100).

Fig. 23 .......Case #15. Scanning magnification. Note themarked variability in red cell size (anisocytosis).

Fig. 24 .......Case #15. Scanning magnification. A left shiftand two nucleated red cells.

Fig. 25 .......Case #15. High magnification. Note the pres-ence of spherocytes. Several polychromatophils(center). Changes are consistent with immune-mediated hemolytic anemia.

Fig. 26 .......Case #16. Scanning magnification. Monocytosisis apparent. Red cells exhibit anisocytosis andpolychromasia.

Fig. 27 .......Case #16. High magnification. Two polychro-matophils and nucleated red cells (left).Numerous red cells have multiple finger-likeprojections (acanthocytes). Platelets are obvious.

Fig. 28 .......Case #17. Several prominent Heinz bodies(seen as nose-like projections from the red cellsurface). Note the lack of polychromasia(Wright's stain x 100).

Fig. 29 .......Case #17. With vital stains, Heinz bodies aremuch more obvious. In this new methyleneblue-stained preparation, they are seen as blueprecipitates within the red cell (100x).

Fig. 30 .......Case #18. Scanning magnification.Poikilocytosis (variable red cell shape) is strik-ing. Numerous elongated red cells (ovalocytes).

Fig. 31 .......Case #18. High magnification. Elongated redcells with ruffled membranes (Burr cells) havebeen associated with metabolic disorders, par-ticularly renal disease, in humans and dogs.

Fig. 32 .......Case #19. Scanning magnification. Numerousnucleated cells, many of them nucleated redcells. There also appears to be a left shift.

Fig. 33 .......Case #19. High magnification. The number ofnucleated red cells is too high for the degree ofpolychromasia (inappropriate nucleated red cellresponse). Note that the nucleated red cell (cen-ter) is stippled (contains basophilic cytoplasmicprecipitates). The two neutrophilic bands (left)have toxic cytoplasm.

Fig. 34 .......Case #19. High magnification. The nucleatedred cell (left) is quite immature.

Fig. 35 .......Case #19. High magnification. Three nucleatedred cells and a toxic metamyelocyte.

Fig. 36 .......Case #20. Low magnification shows large num-bers of nucleated red cells. Note that the film isvery thin, suggesting severe anemia (Wright'sstain x 50).

Fig. 37 .......Case #20. Three metarubricytes, a lymphocyte,and a nucleated cell that is difficult to classify(Wright's stain x 100).

Fig. 38 .......Case #20. Various stages of nucleated red celldifferentiation (Wright's stain x 100).

Fig. 39 .......Case #23. High magnification. Three extremelytoxic neutrophil-series cells.

Fig. 40 .......Case #24. High magnification. Note the absenceof platelets. Three red cell fragments (schizo-cytes).

Fig. 41 .......Case #24. High magnification. Two schizocytes.

All slides courtesy of Alan H. Rebar, DVM, PhD.


-A-activated lymphocytes: Antigen-stimulated (blast-

transformed, reactive) lymphocytes. These lympho-cytes are actively gearing up to produce antibodiesor lymphokines. They have morphologic features ofactive protein producing cells: lacy chromatin (pri-marily euchromatin) and abundant blue cytoplasmrich in RNA.

adherence: Stage II of phagocytosis. The binding ofphagocyte surface receptors to microorganisms andother foreign matter.

autoagglutination: Three-dimensional clumping of ery-throcytes as a result of cross-linking of erythrocytesby antibodies; this feature confirms diagnosis ofimmune-mediated disease.

-C-cell-mediated immunity: Refers to the production of

lymphokines of effector T lymphocytes in responseto antigenic stimulation.

chemotaxis: Stage I of phagocytosis. The directed move-ment of phagocytes along an increasing gradient ofchemoattractant molecules.

-E-endomitosis: Nuclear division without cytoplasmic divi-

sion. Megakaryoblasts mature to megakaryocytesvia endomitosis.

euchromatin: Chromatin comprised mostly of activegenes (DNA) that control cell function by servingas templates for messenger RNA (mRNA) forma-tion, which in turn regulates cellular protein synthe-sis. Euchromatin has a granular, lacy pattern inRomanowsky-stained preparations. Monocytenuclei contain mostly euchromatin. (In contrast,heterochromatin contains mostly inactive DNAwhose active sites are bound to histone (protein)

suppressors, which inhibit gene activity.Heterochromatin stains deep purple inRomanowsky-stained preparations. Neutrophilnuclei contain primarily heterochromatin.)

erythropoietin: The primary growth factor regulatingcommitted red cell production and differentiation inthe bone marrow. This hormone is produced pri-marily in the juxtaglomerular apparatus of the kid-ney, in response to microenvironmental variations inoxygen tension.

-G-golgi zones: Membranous perinuclear organelle which is

the primary intracellular site where protein is pack-aged for secretion.

-H-Heinz bodies: Precipitates of hemoglobin that occur as a

result of oxidation of hemoglobin. Heinz bodiesoften are attached to the inner red cell membrane.Because they are rigid, fixed precipitates, they causeintravascular lysis as red cells traverse tortuous vas-cular capillary spaces.

humoral immunity: Refers to the production of anti-bodies directed against specific antigens by effectorB lymphocytes (plasma cells).

-I-immunocytic system: The "specific immune system"

comprised of the circulating lymphocytes (B lym-phocytes and T lymphocytes). B lymphocytes pro-duce antibodies while T lymphocytes are responsi-ble for lymphokine production. Lymphocyte prod-ucts are released in response to specific antigens.

internalization: Stage III of phagocytosis. The processby which adhered particles (microorganisms) are

Glossary of Terms


taken into a phagocyte for killing and digestion.The process involves invagination of the cell mem-brane and the formation of a phagocytic vacuole.

-L-lymphokines: Small, biologically active molecules

released by antigen-stimulated (blast-transformed,activated, reactive) lymphocytes. These moleculesmoderate the immune response and may be capableof destroying other cells and microorganisms.Lymphokines are the "molecular hormones" of theimmunocytic system.

-N-non-specific immune system: Another name for the

phagocytic system. Phagocytes adhere to and ingesta variety of foreign material on contact.

-O-opsonization: The process by which microorganisms

and foreign proteins become coated by moleculesfor which phagocytes have specific surface recep-tors. The coating molecules—antibodies and com-plement fragments—are called opsonins. Once coat-ed by opsonins, a microorganism or foreign proteinis opsonized. Opsonization facilitates adherence(stage II of phagocytosis).

-P-phagocytic system: The "non-specific immune system"

comprised of circulating phagocytes: neutrophilsand cells of the monocyte/ macrophage continuum.These cells establish the first line of defense againstinvading microorganisms.

phagocytosis: The process of ingestion, killing, anddigestion of etiologic agents by cells of the phago-cytic system. Non-living foreign material may alsobe ingested and digested by phagocytes.

polychromatophils: Large, bluish-red cells seen in lownumbers in normal canine and feline blood films.These immature red cells stain bluish because of alower than normal hemoglobin concentration and aslightly higher than normal amount of cytoplasmicRNA.

polycythemia: Increased circulating red cell mass indi-cated by elevations in hematocrit, hemoglobin, andtotal red cell count.

polycythemia vera: A myeloproliferative disease char-acterized by overproduction of all marrow cellularelements. Clinical findings are generally referable toincreased circulating red cell mass.

-S-specific immune system: Another name for the

immunocytic system. Immunocyte response is spe-cific in that antibodies and lymphokines are releasedin response to specific antigens.


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2. Breitschwerdt, E.B.: Infectious Thrombocytopeniain Dogs. Comp Cont Ed 10:1177, 1988.

3. Butcher, E.C.: Cellular and Molecular Mechanismsthat Direct Leukocyte Traffic. Am J Pathol 136:3,1990.

4. Cain, G.R.; Suzuki, Y.: Presumptive NeonatalIsoerythrolysis in Cats. JAVMA 187:46, 1985.

5. Calvert, C.A.; Greene, C.E.: Bacteremia in Dogs:Diagnosis, Treatment, and Prognosis. Comp Cont Ed8:179, 1986.

6. Center, S.A. et al: Eosinophilia in the Cat: ARetrospective Study of 312 Cases (1975 to 1986).J Am Anim Hosp Assoc 26:349, 1990.

7. Christopher, M.M.: Relationship of EndogenousHeinz Bodies to Disease and Anemia in Cats: 120Cases. JAVMA 194:1089, 1989.

8. Corcoran, B.M. et al: Pulmonary Infiltration withEosinophils in 14 Dogs. J Small Anim Pract 32:494,1991.

9. Cotter, S.M.: Autoimmune Hemolytic Anemia inDogs. Comp Cont Ed 14:53-56; 1992.

10. Cotter, S.M.; Holzworth, J.: Disorders of theHematopoietic System. Diseases of the Cat: Medicineand Surgery, (J. Holzworth, ed). W.B. Saunders,Philadelphia, Pa., 1987, pp 755-807.

11. DiBartola, S.P. et al: Clinicopathologic FindingsAssociated with Chronic Renal Disease in Cats: 74Cases (1973-1984). JAVMA 190:1196, 1987.

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Suggested Reading


25. Jackson, M.L.; Kruth, S.A.: Immune-mediatedHemolytic Anemia and Thrombocytopenia in theDog: A Retrospective Study of 55 Cases Diagnosedfrom 1969 through 1983 at the Western College ofVeterinary Medicine. Can Vet J 26:245, 1985.

26. Jain, N.C.: Essentials of Veterinary Hematology. (N.C.Jain, ed.), Lea & Febiger, Philadelphia, Pa., 1993.

27. Jain, N.C.: Schalm's Veterinary Hematology, 4th Ed.,Lea & Febiger, Philadelphia, Pa., 1986.

28. Jans, H.E. et al: Therapy of Immune-mediatedThrombocytopenia. A Retrospective Study of 15Dogs. J Vet Intern Med 4:4, 1990.

29. Jonas, L.D., et al: Nonregenerative Form ofImmune-mediated Hemolytic Anemia in Dogs.J Am Anim Hosp Assoc 23:201, 1987.

30. Kay, N.E.: Natural Killer Cells. CRC Crit Rev ClinLab Sci 22:343, 1986.

31. Klag, A.R., et al: Idiopathic Immune-mediatedHemolytic Anemia in Dogs: 42 Cases (1986-1990).JAVMA 202:783, 1993.

32. Klag, A.R.: Hemolytic Anemia in Dogs. Comp ContEd 14:1090-1095; 1992.

33. Kuehn, N.F.; Gaunt, S.D.: Clinical and HematologicFindings in Canine Ehrlichiosis. JAVMA 186:355,1985.

34. Latimer, K.S.: Leukocytes in Health and Disease.Textbook of Veterinary Internal Medicine, 4th Ed. (S.J.Ettinger and E.C. Feldman, ed.). W.B. Saunders,Philadelphia, Pa., 1995; pp 1892-1929.

35. Latimer, K.S.; Meyer, D.J.: Leukocytes in Healthand Disease. Textbook of Veterinary Internal Medicine,3rd Ed. (S.J. Ettinger, ed.). W.B. Saunders,Philadelphia, Pa., 1989; pp 2181-2225.

36. Latimer, K.S.; Rakich, P.M.: Clinical Interpretationof Leukocyte Responses. Vet. Clin. North Am.: SmAnim Pract 19:637, 1989.

37. Lewis, H.B.; Rebar, A.H.: Bone Marrow Evaluation inVeterinary Practice. Ralston Purina, St. Louis, Mo.,1979.

38. Madewell, B.R.: Hematological and Bone MarrowCytological Abnormalities in 75 Dogs withMalignant Lymphoma. J Am Anim Hosp Assoc22:235, 1986.

39. May, M.E.; Waddell, C.C.: Basophils in PeripheralBlood and Bone Marrow: A Retrospective Review.Am J Med 76:509, 1984.

40. McEwen, S.A. et al: Hypereosinophilic Syndrome inCats: A Report of 3 Cases. Can J Comp Med 49:248,1985.

41. Meyers, K.M. et al: Canine von Willebrand’sDisease: Pathobiology, Diagnosis, and Short-termTreatment. Comp Cont Ed 14:13, 1992.

42. Neer, T.M.: Hypereosinophilic Syndrome in Cats.Comp Cont Ed 13:549, 1991.

43. Northern, J.; Tvedten, H.W.: Diagnosis ofMicrothrombocytosis and Immune-mediatedThrombocytopenia in Dogs withThrombocytopenia: 68 Cases (1987-1989). JAVMA200:368, 1992.

44. Reagan, W.J.; Rebar, A.H.: Platelet Disorders,Textbook of Veterinary Internal Medicine, 4th Ed. (S.J.Ettinger and E.C. Feldman, ed.). W.B. Saunders,Philadelphia, Pa., 1995; pp 1964-1976.

45. Reagan, W.J.: A Review of Myelofibrosis in Dogs.Toxicol Pathol 21:169, 1993.

46. Rebar, A.H.: Anemia. Clinical Signs and Diagnosis inSmall Animal Practice (E.B. Ford, ed.). ChurchillLivingstone, New York, N.Y., 1988.

47. Rebar, A.H.: Interpreting the Feline Hemogram.Handbook of Feline Medicine. Pergamon Press, Oxford,U.K., 1993.


48. Rebar, A.H.; Metzger, F.: The Veterinary CEAdvisor—Clinical Pathology for Small-animalPractitioners: Interpreting the Hemogram. VeterinaryMedicine 90(6) (Suppl.):1-12, 1995.

49. Shelton, G.H. et al: Hematologic Manifestations ofFeline Immunodeficiency Virus Infection. Blood76:1104, 1990.

50. Tvedten, H. et al: Estimating Platelets andLeukocytes on Canine Blood Smears. Vet Clin Pathol17:4-11; 1988.

51. Weiser, M.G.: Erythrocyte Responses andDisorders: Textbook of Veterinary Internal Medicine, 4thEd. (S.J. Ettinger and E.C. Feldman, ed.). W.B.Saunders, Philadelphia, Pa., 1995; pp 1864-1891.

52. Weiser, M.G.: Erythrocytes and AssociatedDisorders. Textbook of Veterinary Internal Medicine,3rd Ed. (S.J. Ettinger, ed.). W.B. Saunders,Philadelphia, Pa., 1989; pp 2145-2181.

53. Wellman, M.L. et al: Lymphocytosis of LargeGranular Lymphocytes in Three Dogs. Vet Pathol26:158, 1989.

54. Wilkerson, M.J., et al: Blood Type A and B in TwoCat Colonies in Washington State and a ClinicalTransfusion Reaction. Feline Pract 19:22, 1991.

55. Willard, M. et al: Textbook of Small Animal ClinicalDiagnosis by Laboratory Methods, 2nd Ed. W.B.Saunders, Philadelphia, Pa., 1994; pp 11-80.

56. Williams, D.A.; Maggio-Price, L.: Canine IdiopathicThrombocytopenia: Clinical Observations andLong-term Follow-up in 54 Cases. JAVMA 185:660,1984.

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