Veterinary Clinical Pathology, An Introduction PDF

M A R I O N L. J A C K S O N Veterinary Clinical Pathology AN INTRODUCTION Veterinary Clinical Pathology An Introduction Veterinary Clinical Pathology An Introduction Marion L. Jackson Marion L. Jackson, DVM, MVetSc, PhD is a diplomate of clients, is granted by Blackwell Publishing, provided the American College of Veterinary Pathologists and a that the base fee of $.10 per copy is paid directly to the professor in the Department of Veterinary Pathology at Copyright Clearance Center, 222 Rosewood Drive, the Western College of Veterinary Medicine, University Danvers, MA 01923. For those organizations that have of Saskatchewan been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee codes [email protected] for users of the Transactional Reporting Service are ISBN-13: 978-0-8138-2140-5; © 2007 Blackwell Publishing ISBN-10: 0-8138-2140-1/2007 $.10. All rights reserved First edition, 2007 Blackwell Publishing Professional 2121 State Avenue, Ames, Iowa 50014, USA Library of Congress Cataloging-in-Publication Data Orders: 1-800-862-6657 Jackson, Marion L. Ofﬁce: 1-515-292-0140 Veterinary clinical pathology : an introduction / Fax: 1-515-292-3348 Marion L. Jackson.–1st ed. Web site: www.blackwellprofessional.com p. cm. Includes bibliographical references and index. Blackwell Publishing Ltd ISBN-13: 978-0-8138-2140-5 (alk. paper) 9600 Garsington Road, Oxford OX4 2DQ, UK ISBN-10: 0-8138-2140-1 (alk. paper) Tel.: +44 (0)1865 776868 1. Veterinary clinical pathology. I. Title. SF772.6.J33 2006 Blackwell Publishing Asia 636.089 607–dc22 550 Swanston Street, Carlton, Victoria 3053, Australia 2006001949 Tel.: +61 (0)3 8359 1011 The last digit is the print number: 9 8 7 6 5 4 3 2 1 Authorization to photocopy items for internal or personal use, or the internal or personal use of speciﬁc Contents Preface, vii 9 Digestive System, 246 Acknowledgments, ix 10 Endocrine System, 264 1 Erythrocytes, 3 11 Muscle, 298 Color Section 12 Lipids and Proteins, 300 2 Leukocytes, 55 Appendix I Practice Cases, 304 3 Hemopoietic Neoplasia, 81 Appendix II Interpretation of Laboratory Results, 314 4 Hemostasis, 109 Appendix III Guide to Case 5 Cytology, 137 Studies, 327 6 Fluids, Electrolytes, and Acid–Base Glossary, 329 Balance, 171 Bibliography, 343 7 Renal System, 194 Index, 349 8 Hepatobiliary System, 223 v Preface At the Western College of Veterinary Medicine, literature. I have aimed for a clear, concise presen- students are introduced to Clinical Pathology in tation of basic mechanisms without overwhelm- year three of the undergraduate program. In year ing the student. Cases at the end of each chapter four, small groups of students rotate through the (except Chapters 11 and 12) emphasize basic prin- laboratory, which provides a dynamic setting to ciples discussed in the text. The cases are real and, test and expand their knowledge. Several years therefore, are not perfect. Not all results can be ago, Dr. Gene Searcy, the most enthusiastic clin- satisfactorily explained, which is true to life. Stu- ical pathologist I have known, made a dramatic dents are encouraged to interpret the case data on change in the third-year course, by moving from their own before reading my version. Complete didactic lectures with a few case examples, to case interpretations are provided even though the stu- studies interjected with a few minilectures. Dr. dent may not be familiar with all laboratory data Beverly Kidney, my colleague, and I have contin- until completion of the book. It is expected that in- ued with this format, which is very well received structors using this book will provide additional by our students. Assigned readings provide the cases from their own diagnostic service for class tools needed to understand and interpret labora- discussion and to challenge the students. Labora- tory data, and during classes, students are ran- tory periods complement our course by affording domly chosen to discuss laboratory results from the opportunity to learn practical aspects of per- a book of case studies. forming a CBC, blood and cytology smear eval- This textbook has evolved from the reading uation, and doing a urinalysis. assignments for the undergraduate course and is If we as instructors can help students to be- intended to give the student a sound knowledge come proﬁcient in applying clinical pathology base with which to work. The book is not heav- as a powerful diagnostic tool, then we have ily referenced other than with standard physiol- been successful. We are overjoyed when students ogy, clinical pathology, and medicine textbooks, grasp this subject and run with it; let us make it and is not intended to be a review of the current fun too. vii Acknowledgments I thank my mentor, Dr. Gene Searcy, and col- illustrator and treasured resource, generated league, Dr. Beverly Kidney, for their encourage- most of the ﬁgures, and Ms. Maeve Johnston, ment and support with this project. Undergradu- graphic designer and illustrator, drew the cells for ate students, graduate students, clinicians, tech- the ﬁgures and the tables of erythrocyte morphol- nologists, staff, and colleagues within the West- ogy. I thank Ms. Priscilla Neufeld for her usual ern College of Veterinary Medicine and Prairie competence and reliability, and for working long Diagnostic Services have helped me to improve and odd hours on the ﬁnal product. The data for my teaching and writing, with their invaluable the case studies were generated by Prairie Diag- input, feedback and challenging questions. No nostic Services, which employs a team of veteri- doubt, I have made errors of various types in this nary professionals and technologists second to book, and I welcome criticism and comments for none. Mrs. Gloria Patry, Prairie Diagnostic Ser- improvements (please send me an e-mail: mar- vices, kindly provided the photographs of urine [email protected]). sediment ﬁndings. Ms. Kim Christiansen, Prairie The whim to write this book only took root Diagnostic Services, helped immensely with as a project for a sabbatical leave—a vital part loose ends when deadlines loomed. Production of academic life. Dr. Nicole Fernandez, a for- staff of Blackwell Publishing are to be thanked for mer graduate student in our department, was of their guidance and creation of this textbook. great assistance with the photographs, ﬁgures, I am grateful to my husband, Dr. Vladimir tables, proofreading, and organization of the Sopuck, and sons, Adam and Bennett, for sup- book. Dr. Juliane Deubner, our college medical porting me through another adventure. ix Veterinary Clinical Pathology An Introduction CHAPTER 1 Erythrocytes erythroid proliferation. If peripheral blood evalu- Erythropoiesis ation reveals an inadequate or unexplained bone marrow response, examination of the marrow is The production of erythrocytes from stem cell usually indicated. to mature circulating red blood cell (RBC) is The committed erythroid precursor undergoes known as erythropoiesis. Hemopoiesis refers to up to ﬁve mitotic divisions over 5 days. The the production of all blood cells, including white earliest recognizable erythroid precursor is the blood cells (WBCs) and platelets. Erythropoiesis rubriblast, followed by differentiation sequen- is most effective in the bone marrow, although tially to the prorubricyte, rubricyte, metarubri- other tissues may provide additional sites of RBC cyte, polychromatophilic erythrocyte, and the production. Erythrocytes deliver oxygen to tis- mature erythrocyte (Fig. 1.2). Erythroid matura- sues, remove carbon dioxide from tissues, and tion correlates with a decrease in EPO receptors buffer acid–base changes in the circulation. Tissue and an increase in transferrin receptors on the oxygenation is the main regulator of RBC pro- surface of red cell precursors. Transferrin recep- duction, and conditions associated with tissue tors allow for incorporation of iron into erythro- hypoxia stimulate the bone marrow to increase cytes, for hemoglobin synthesis. Hemoglobin RBC production. When tissue oxygenation is ad- comprises four globin chains, each bound to a equate, total erythroid mass ﬂuctuates very little, heme molecule containing iron. Hemoglobiniza- as a steady state exists between RBC production tion of the red cell cytoplasm is most active dur- and RBC loss. ing the rubricyte stage. Also, cell division stops Hemopoietic stem cells have the ability to de- during the rubricyte stage as hemoglobinization velop into common myeloid progenitors and nears completion and the nucleus condenses. At common lymphoid progenitors (Fig. 1.1). Ery- the end of the metarubricyte stage, the pyknotic throcytes, megakaryocytes, and all leukocytes nucleus is extruded and phagocytized by local (except lymphocytes) are generated from com- macrophages. mon myeloid progenitors. The ﬁrst level of Although nucleated erythrocytes are not usu- committed differentiation to erythrocytes is ally found in the peripheral blood, a low per- within the precursor cell compartment. The centage of circulating polychromatophilic cells bone marrow microenvironment provides the are present under normal circumstances in most structural and biochemical support for normal species (e.g. about 1% in the dog). The horse hemopoiesis. Growth factors, transcription fac- is an exception in that immature erythrocytes tors, adhesins, interleukins, and other mediators are rarely released into the peripheral blood in comprise a complex system that responds to in- this species, even when intense erythroid hy- creased demands when required and maintains perplasia is occurring in the bone marrow in a ﬁnely tuned balance under normal circum- response to anemia. Residual ribosomes and stances. The expression and availability of these RNA, reﬂecting the end of protein synthesis factors inﬂuence the balance among the various (mainly hemoglobin), are responsible for the committed lineages. Erythropoietin (EPO) is the purplish-blue coloration of polychromatophilic most important growth factor for maintaining erythrocytes with Romanowsky-type stains, 3 CHAPTER 1 Progenitor Hemopoietic stem cell Common Common myeloid lymphoid progenitor progenitor Erythro MK GM progenitor Mast cell Marrow progenitor progenitor Precursor Rubriblast MK blast Monoblast Neutrophilic Eosinophilic Basophilic myeloblast myeloblast myeloblast Metarubricyte Megakaryocyte Promonocyte Neutrophilic Eosinophilic Basophilic band band band Mature Blood Erythrocyte Platelets Monocyte Neutrophil Eosinophil Basophil T cell B cell Tissue Macrophage Mast cell Plasma cell Figure 1.1 The cells of the blood and lymphoid organs and their precursors in the bone marrow. such as Wright–Giemsa. Polychromatophilic ery- throcytes lose their residual RNA within 24– Erythrocyte morphology 48 hours of release from the bone marrow. When blood smears are stained with new methylene In the common domestic animals, mature ery- blue rather than Romanowsky stains, the residual throcytes are biconcave disks that are highly RNA in immature RBCs is precipitated in clumps deformable, allowing them to travel through and the cells are known as reticulocytes. small capillaries and deliver oxygen to tissues 4 Erythrocytes Rubriblast Marrow Prorubricyte Rubricyte Rubricyte Rubricyte Metarubricyte Reticulocyte Erythrocyte Blood Figure 1.2 Erythrocyte kinetics. After stimulation by erythropoietin (EPO), cells in the committed erythroid compartment differentiate into rubriblasts, followed by mitotic division and maturation to mature erythrocytes. (see Table 1.1). Erythrocyte aging and certain clear (see Table 1.1 for diagrams of various types pathological conditions can cause RBCs to as- of RBC morphology). sume unusual shapes, which may result in in- See Figs 1.3–1.24 for pictures of various types creased rigidity. Rigid RBCs are susceptible to of RBC morphology. mechanical injury and are less effective in de- The red cell wall is a typical lipid bilayer, livering oxygen. Exposure to stagnant environ- comprising mainly phospholipids and choles- ments (pooling of blood in a cavernous, hypoxic terol (Fig. 1.25). Membrane proteins and gly- space), certain serum biochemical abnormalities, coproteins are inserted into the lipid bilayer, antibody-mediated membrane injury, and me- some in one leaﬂet only and others spanning chanical injury can alter the normal biconcave the entire membrane. These integral proteins in- shape. Sometimes, RBC morphologic changes are clude hormone receptors and enzymes, which associated with speciﬁc diseases or conditions, usually only partially penetrate into the bilayer, but the mechanism of the shape change is not and transmembrane proteins, which include the 5 CHAPTER 1 Table 1.1 Erythrocyte morphology Acanthocytes have projections of variable length that are unevenly spaced on the surface of the red cell. Acanthocytes may be seen as an incidental finding, as a consequence of a high-fat diet, with disorders of lipid metabolism, and with hemangiosarcoma. In the latter case, acanthocytes may form when red cells stagnate in cavernous spaces within the tumor, resulting in shifts in lipids in the RBC membrane. Agglutination is identified when red cells clump or cluster together in groups (not in rows) like a bunch of grapes. Agglutination must be differentiated from rouleaux. Polychromatophils do not participate in rouleaux formation but may agglutinate. Anisocytosis indicates variable red cell size. Blister cells appear as though they have a hole(s) punched through the periphery of the red cell. They are observed most often in feline blood films. Blister cells may result from oxidative injury. Codocytes (target cells) have a dark central area of hemoglobin, surrounded by a pale zone that in turn is surrounded by a peripheral rim of hemoglobin. Up to 50% of canine red cells may be codocytes; they are rarely observed in other species. Increased numbers of codocytes may be present with hepatic disease. Dacryocytes are red cells shaped like tear drops. They are considered artifactual if all points are oriented in the same direction. This artifact may be due to poor blood film preparation or lipemia. Increased numbers of non-artifactual dacryocytes may be seen with myelofibrosis. Eccentrocytes have eccentric hemoglobin distribution due to annealing of a crescent of red cell membrane that excludes hemoglobin. The hemoglobinated portion of the eccentrocyte stains darkly due to a higher concentration of hemoglobin in that portion of the cell. They indicate oxidative damage to the RBC membrane and may be accompanied by RBCs with Heinz bodies. Echinocytes are thought to be formed either as a result of erythrocyte dehydration or by expansion of the outer leaflet of the red cell membrane. Echinocytes I are red cells with an angular shape or short, blunt projections. They are often due to artifact, such as occurs with sample aging prior to smear preparation or excessive EDTA exposure. Echinocytes III are spherical red cells with sharp projections of equal length that are evenly spaced on the surface of the red cell. They may be increased in animals with renal disease and/or electrolyte disturbances. They can also occur artifactually for similar reasons described for echinocytes I. Echinoelliptocytes are oval to cigar-shaped red cells with projections of equal length that are evenly spaced on the surface of the red cell. They may be seen in cats with hepatobiliary disease and are rare in other species. Elliptocytes are oval to cigar-shaped cells. Red cells from Camelidae are normally elliptical. 6 Erythrocytes Table 1.1 (Continued ) Ghost cells are red cells that have been leached of hemoglobin. They are evidence of intravascular hemolysis. Hypochromasia refers to red cell pallor due to inadequate synthesis of hemoglobin. Hypochromic red cells have a large area of central pallor that gradually darkens towards the periphery of the red cell. Immature RBCs may appear hypochromic due to their large size. Small (microcytic), hypochromic RBCs can be seen with iron deficiency and disorders of iron utilization. Keratocytes are crescent-shaped cells. They are formed from mechanical shearing (usually due to fibrin strand deposition) of the red cell. Keratocytes are often accompanied by schizocytes (fragments). Leptocytes are thin, macrocytic red cells with a membrane surface area that exceeds hemoglobin content. The membrane tends to wrinkle or fold, forming twisted (like figure 8) cells. They are sometimes seen with hepatic disease. Macrocytes (left) are larger than normal red cells. Microcytes (right) are smaller than normal red cells. Polychromasia refers to red cells that appear blue–gray with Romanowsky dyes. They correspond to reticulocytes on blood films stained with supravital dyes (e.g., new methylene blue, NMB). Polychromatophils are young cells with a high RNA content and, as such, are larger than mature red cells and have a different staining character. Increased numbers indicate red cell regeneration. Reticulocytes can be identified on blood films stained with supravital dyes. NMB precipitates nucleic acids (like RNA) as dark blue deposits. Increased numbers indicate red cell regeneration. They correspond to polychromatophils on Romanowsky-stained smears. Rouleaux are stacks of red cells. Equine and feline erythrocytes readily form rouleaux. Excessive rouleaux formation in any species may be associated with hyperproteinemia. Schizocytes are red cell fragments attributed to mechanical red cell injury/shearing (see keratocytes). Spherocytes (left) are small, dark, round RBCs that are formed by the removal of altered red cell membrane without concurrent loss of hemoglobin. Spherocytes have no central pallor. They may be seen with immune-mediated hemolytic anemia. (Continued ) 7 CHAPTER 1 Table 1.1 (Continued ) Unclassiﬁed poikilocytosis is used when red cell shape defies description. This term may be used to describe the peculiar (and often abundant) poikilocytosis seen in normal calves, deer, goats, and pigs, which may actually be an in vitro artifact. Basophilic stippling refers to diffuse blue speckling (with Romanowsky stains) within red cells. This basophilia is due to the presence of cytoplasmic RNA and reflects red cell immaturity. Increased numbers of red cells with basophilic stippling often accompany other features of red cell regeneration (especially in ruminants) such as polychromasia and reticulocytosis. Lead poisoning interferes with metabolic pathways in developing erythrocytes and may result in the presence of RBCs with basophilic stippling and metarubricytes in the peripheral blood when there is no anemia or only mild anemia. Heinz bodies are difficult to visualize with Romanowsky stains where they may be visible as eccentrically located refractile bodies or blebs on the periphery of the red cell. They are better visualized and quantified on blood films stained with NMB, where they stain greenish blue. They indicate oxidative damage to red cells and may be seen along with eccentrocytes. Small Heinz bodies may be seen in high numbers on blood films from normal, non-anemic cats. Nuclear remnants are small, round, dark purple, erythrocyte inclusions representing a portion of the otherwise extruded nucleus. They are usually single and located close to the periphery of the red cell. Excessive numbers may be seen post-splenectomy or with hypofunctioning of the spleen. Nucleated red blood cells (NRBCs) are enumerated per 100 leukocytes. Greater than 5 NRBCs/100 WBCs is significant and may indicate bone marrow damage or hypoxia. NRBCs may accompany a regenerative response when anemia is present, but should not be used as the only criterion of RBC regeneration. The total leukocyte count should be corrected if there are ≥5NRBCs/100 WBCs. Figure 1.3 Canine blood film showing acanthocytes (also see color section). 8 Erythrocytes Figure 1.4 Canine blood film showing anisocytosis (also see color section). Figure 1.5 Bovine blood film showing basophilic stippling within a macrocyte as part of the regenerative response (also see color section). 9 CHAPTER 1 Figure 1.6 Canine blood film showing basophilic stippling due to lead toxicosis (also see color section). Figure 1.7 Canine blood film showing blister cells due to oxidative damage (also see color section). 10 Erythrocytes Figure 1.8 Canine blood film showing codocytes. Up to 50% codocytes may be normal in a dog (also see color section). Figure 1.9 Canine blood film showing eccentrocytes due to oxidative damage (also see color section). 11 CHAPTER 1 Figure 1.10 Canine blood film showing echinocytes I (also see color section). Figure 1.11 Canine blood film showing echinocytes III (also see color section). 12 Erythrocytes Figure 1.12 Feline blood film showing echinoelliptocytes (also see color section). Figure 1.13 Canine blood film showing Heinz bodies and ghost cells due to oxidative damage. There are also several polychromatophils (also see color section). 13 CHAPTER 1 Figure 1.14 Canine blood film stained with new methylene blue, to demonstrate Heinz bodies (also see color section). Figure 1.15 Canine blood film showing keratocytes and schizocytes (erythrocyte fragments) due to fibrin strand injury. A codocyte and a polychromatophil also appear in the field (also see color section). 14 Erythrocytes Figure 1.16 Feline blood film showing Mycoplasma hemofelis organisms (hemobartonellosis). Organisms are not always visible in blood smears from infected cats (also see color section). Figure 1.17 Canine blood film showing several macrocytes (also see color section). 15 CHAPTER 1 Figure 1.18 Canine blood film showing a metarubricyte There is also a polychromatophilic macrocyte in the field (also see color section). Figure 1.19 Canine blood film showing microcytic, hypochromic erythrocytes consistent with iron deficiency anemia. There are two polychromatophils in the field, indicating that the anemia is regenerative (also see color section). 16 Erythrocytes Figure 1.20 Canine blood film showing two macrocytes with nuclear remnants (also see color section). Figure 1.21 Bovine (calf) blood film showing marked poikilocytosis (normal?) (also see color section). 17 CHAPTER 1 Figure 1.22 New methylene-blue-stained Canine blood film showing reticulocytes (also see color section). Figure 1.23 Canine blood film showing rouleaux formation (also see color section). 18 Erythrocytes Figure 1.24 Canine blood film showing spherocytosis and polychromasia due to immune-mediated hemolytic anemia (also see color section). Glycolipid Cholesterol OUTSIDE Band3 Band3 Lipid layer GP-C GP-C Phospholipids Ankyrin Actin 4.1 4.2 INSIDE Spectrin Figure 1.25 The red blood cell cytoskeleton. Tropomyosin 19 CHAPTER 1 transport carriers for ions and water-soluble sub- (2,3-BPG, also known as 2,3-diphosphoglycerate, strates, such as glucose. The transmembrane pro- 2,3-DPG) with consequences of increased afﬁn- teins maintain cation–anion homeostasis within ity of hemoglobin for oxygen and impaired oxy- red cells. Abnormalities in ion pumps can result gen delivery to tissues. In addition to generating in a shortened red cell lifespan. ATP as an energy source, erythrocytes must be The red cell skeleton forms the scaffolding for capable of preventing chemical injury (oxidative the lipid bilayer and plays an important role damage) from the high concentrations of oxy- in membrane stability and deformability. De- gen that they transport. This is accomplished formability is the most important property of through two other branches of the Embden– RBCs required for normal survival, particularly Meyerhof pathway, the hexose monophosphate as cells traverse capillary beds and the exact- shunt and the methemoglobin reduction path- ing environment of the spleen. Interactions be- way. Glucose-6-phosphate is the substrate for the tween skeletal proteins, such as spectrin, ankyrin, hexose monophosphate shunt which maintains and actin, and the lipid bilayer maintain mem- glutathione in the reduced state. Reduced glu- brane shape, ﬂexibility, and durability. The or- tathione is an intracellular buffer that protects red ganization of membrane phospholipids is also cells from oxidant injury, particularly by hydro- stabilized, and cell-surface activities (e.g. anion gen peroxide and the superoxide anion, and also and glucose transport) are inﬂuenced by interac- helps to stabilize the reactive sulfhydryl groups tions between skeletal proteins and transmem- of hemoglobin. The methemoglobin reduction brane proteins. Skeletal proteins link to the lipid pathway returns oxidized hemoglobin (methe- membrane via two main interactions: spectrin– moglobin) to its reduced state (ferrous/Fe2+ ) that ankyrin band 3 and spectrin–actin band 4.1. is capable of oxygen transport. The bands are named according to their mi- Defects in the Embden–Meyerhof pathway gration patterns on electrophoresis. Many are that lead to ATP deﬁciency are manifested as now assigned names that reﬂect their function, hemolytic anemia due to an inability to maintain for example, band 3 is now known as AE1 normal water and electrolyte homeostasis. Non- for its role as an anion exchange protein or spherocytic but rigid erythrocytes are removed in channel. the exacting environment of the spleen. Defects in production of 2,3-BPG result in an increased afﬁnity of oxygen for hemoglobin, impaired oxy- Erythrocyte metabolism genation of tissues, and erythrocytosis. Defects in the hexose monophosphate shunt lead to oxida- When reticulocytes mature and lose their mi- tive damage to erythrocytes, hemoglobin denatu- tochondria, they are no longer capable of ox- ration, and Heinz body hemolytic anemia. Errors idative metabolism. Mature erythrocytes derive in the methemoglobin reduction pathway pro- their energy from adenosine triphosphate (ATP) hibit reduction of oxidized hemoglobin. Erythro- generated by anaerobic glycolysis (Embden– cytes are unable to transport oxygen and cyanosis Meyerhof pathway) (Fig. 1.26). ATP provides develops. the energy to maintain membrane ion pumps. Defects in this pathway lead to an inabil- ity to maintain normal ﬂuid and electrolyte Erythrocyte values content within the red cells, resulting in in- travascular hemolysis or premature destruc- Erythrocyte morphology, number, size, and tion in the spleen. Enzyme defects within a hemoglobin content are evaluated by periph- branch of the Embden–Meyerhof pathway, called eral blood smear examination and also through the Rapaport–Luebering pathway, result in de- a combination of measurements and calcu- creased production of 2,3-biphosphoglycerate lations, usually with the aid of automated 20 Embden–Meyerhof pathway Glutathione reductase GSH GSSG Glucose Hexose monophosphate ATP shunt Hexokinase ADP NADP+ NADPH Glucose 6-phosphatase 6-Phosphogluconate G6PD Glucose phosphate isomerase Fructose 6-phosphate Hexose monophosphate ATP Phosphofructokinase shunt ADP Fructose 1,6-phosphate Aldolase Glyceraldehyde 3-phosphate Methemoglobin HbFe 2+ NAD+ Glyceraldehyde 3-phosphate reduction HbFe 3+ NADH dehydrogenase Rapaport–Luebering pathway pathway 1,3-Biphosphoglycerate 2,3-Biphosphoglycerate mutase ADP 2,3-Biphosphoglycerate Phosphoglycerate kinase ATP 2,3-Biphosphoglycerate phosphatase 3-Phosphoglycerate 3-Phosphoglycerate mutase 2-Phosphoglycerate Enolase Phosphoenolpyruvate ADP Pyruvate kinase ATP Pyruvate Erythrocytes NADH Lactate dehydrogenase NAD+ Lactate 21 Figure 1.26 Erythrocyte metabolism. CHAPTER 1 Table 1.2 Erythrocyte values used in the complete blood count (CBC) Value Definition Determination SI units Non-SI units RBC count Number of erythrocytes Measured ×10 /L 12 106 /mm3 Hemoglobin (Hgb) Hemoglobin concentration Measured g/L g/dL Hematocrit (Hct) Volume of RBC per liter of whole blood MCV × RBC count L/L % Mean corpuscular volume (MCV) Average RBC size Measured fL μm3 Mean corpuscular hemoglobin (MCH) Average amount of Hgb per RBC Hgb/RBC count pg pg Mean corpuscular hemoglobin Average concentration of Hgb per RBC Hgb/Hct g/L g/dL or % concentration (MCHC) Red cell distribution width (RDW) Coefficient of variation of RBC size Calculated from MCV % % instruments. These erythrocyte values include r MCH: The MCH is the average amount of RBC count, hemoglobin (Hgb), hematocrit (Hct), hemoglobin per red cell and is calculated by mean corpuscular volume (MCV), mean cor- dividing the Hgb by the RBC count. For exam- puscular hemoglobin (MCH), mean corpuscu- ple, if the Hgb is 115 g/L (or 11.5 g/dL) and the lar hemoglobin concentration (MCHC), red cell RBC count is 4.77 × 1012 /L, the MCH is 115 g/L distribution width (RDW), reticulocyte count, divided by 4.77 × 1012 /L = 24.1 × 10−12 g or and reticulocyte production index (RPI) (in dogs 24.1 picograms (pg). only). The RBC count, Hgb, and MCV are mea- r MCHC: The MCHC is the average concentra- sured, whereas the Hct, MCH, MCHC, and RDW tion of hemoglobin per erythrocyte and is cal- are calculated (Table 1.2). The RPI is calculated culated by dividing the Hgb by the Hct. For using the reticulocyte count, which is usually de- example, if the Hgb is 115 g/L and the Hct is termined manually. 0.345 L/L, then the MCHC is 115 g/L divided by 0.345 L/L = 333 g/L. r RBC: The RBC count is the number (N) of ery- r RDW: The RDW is calculated from the MCV throcytes, usually written as N × 1012 per liter. and describes the coefﬁcient of variation of the r Hgb: Hgb concentration is measured and re- red cell sizes. The formula for RDW is the ported as grams per liter (g/L) or grams per standard deviation of the MCV divided by the deciliter (g/dL). MCV. r MCV: Automated instruments measure the r Reticulocyte count: When the Hct is low, a sizes of several thousand red cells and then re- reticulocyte count is done to evaluate the bone port the average of these sizes as the MCV in marrow response and to differentiate regen- femtoliters (1 fL = 10−15 L). erative from nonregenerative anemia. Periph- r Hct: The Hct, calculated by multiplying the eral blood is stained with new methylene MCV by the RBC count, is the volume of red blue or other supravital stain, so that resid- cells per liter of whole blood. For example, if ual ribosomes, mitochondria, and other cyto- the MCV is 72.4 fL and the RBC count is 4.77 × plasmic organelles are aggregated and pre- 1012 /L, the Hct is (72.4 × 10−15 /L) × (4.77 × cipitated in strands in immature erythrocytes. 1012 /L) = 0.345 L/L. Although these cells are equivalent to poly- r PCV: The Hct is equivalent to the packed cell chromatophilic cells with Romanowsky stains, volume (PCV), but the latter term is generally they are more easily visualized and enumer- reserved for those times when a small tube ated with methyl alcohol stains. The number of of blood (microhematocrit tube) is centrifuged reticulocytes is expressed as a percentage. The and the volume of packed RBCs relative to the percentage of reticulocytes can be multiplied total volume of the sample is reported as a per- by the RBC count to determine an absolute centage. number of reticulocytes in a volume of blood. 22 Erythrocytes This calculation adjusts for the higher relative Romanowsky stains. This is known as basophilic percentage of reticulocytes when mixed with stippling and is most prominent in bovine regen- fewer mature erythrocytes in anemia. erative anemia, but can also be seen in cats and r RPI: The RPI is sometimes reported for canine dogs. If basophilic stippling is not accompanied samples and is a calculation designed to cor- by a severe anemia and robust regenerative re- rect the reticulocyte count for the severity of sponse, a disorder in hemoglobin synthesis, such the anemia and for the longer maturation pe- as with lead poisoning, should be considered. In- riod for early released reticulocytes. The RPI creased metarubricytes, unrelated to a regenera- has been adapted from human medicine and tive anemia, may also appear in the peripheral requires that a set value be assigned for the blood with lead poisoning. normal Hct, which is not always correct for the Horses rarely release immature erythrocytes individual animal. The various manipulations even when intense erythroid hyperplasia is of the reticulocyte count, expressed as a per- present in the marrow in response to severe ane- centage, are designed to determine if the re- mia. Peripheral blood ﬁndings of erythrocyte re- generative response is adequate for the degree generation that may be, but are not necessarily, of anemia. seen in the horse are increases in MCV and RDW. Bone marrow examination is required to accu- rately assess erythropoiesis in an anemic horse. However, the clinical information together with Anemia monitoring of the Hct over several days may ob- viate the need for bone marrow examination. Anemia is an absolute reduction in the volume of Nucleated RBCs (metarubricytes) may accom- erythrocytes in the peripheral blood and is identi- pany a regenerative response. However, if ane- ﬁed by decreases in the RBC count, Hgb, and Hct mia is not present, or anemia is present but (or PCV) below the reference limits for a given metarubricytes are disproportionately high rela- species. Anemias are classiﬁed in many ways, tive to the reticulocyte response, other conditions for example, according to the responsiveness of should be considered. These include hypoxia, the bone marrow (regenerative vs. nonregener- necrosis, and metastatic cancer involving the ative); the cause, such as blood loss, hemolysis, bone marrow, lead poisoning, and erythroid neo- bone marrow failure; the morphology of the ery- plasia (especially in cats). throcytes, such as microcytic and hypochromic; Regenerative anemia is usually due to blood and the precise etiology, such as ingestion of an loss or hemolysis. With a single episode of ane- oxidizing agent, blood-borne parasite, traumatic mia, reticulocytes will increase in the peripheral hemorrhage, etc. blood by 2–4 days and peak by about 7–10 days Bone marrow response to anemia, for most (varying with the species). If there is no regen- species, can be assessed by evaluating the pe- eration when the anemia is ﬁrst detected, moni- ripheral blood. Blood smear examination in re- toring the peripheral blood response over several generative anemias will reveal macrocytosis and days may be necessary to determine if the bone increased polychromasia and anisocytosis. Poly- marrow is responding appropriately. chromasia will be reﬂected in an elevated reticu- Trauma is a frequent cause of acute external locyte count. Macrocytosis relates to the release of and internal blood loss. The body immediately immature erythrocytes/polychromatophilic ery- attempts to restore the circulating volume by throcytes and this will correlate with the MCV. drawing low-protein interstitial ﬂuid into the in- Theoretically, the RDW should reﬂect the degree travascular space. By the time blood is obtained of anisocytosis. for analysis, there is a decreased Hct, Hgb, RBC Several species release immature erythro- count, and total protein. Intravenous ﬂuids that cytes containing many small blue dots with are given to restore blood volume also exacerbate 23 CHAPTER 1 the decline in Hct, Hgb, RBC count, and total assays are not readily available for all species, and protein. bone marrow examination is an invasive proce- Red cell breakdown products are not available dure if iron status is the only concern. Often the for recycling when blood is lost from the body. history, clinical ﬁndings, and peripheral blood Chronic or recurrent external blood loss can be ﬁndings are sufﬁcient to diagnose iron deﬁciency caused by disorders of primary hemostasis, such anemia. The etiology of the iron lack must then as thrombocytopenia and von Willebrand dis- be determined. ease; infestation with internal or external blood- Hemolysis can occur within the periph- sucking parasites; intestinal hemorrhage from eral blood (intravascular), in tissues rich in ulcers (drug or stress induced), enteritis, or neo- macrophages such as the spleen, liver, and plasms; and genitourinary bleeding. Bleeding lymph nodes (extravascular), and sometimes into the intestinal and genitourinary tracts is truly in both. If signiﬁcant numbers of erythro- external blood loss because RBCs are not avail- cytes are lysed in the peripheral bloodstream, able for recycling. Iron is lost from the body hemoglobinemia and hemoglobinuria are seen. and, given sufﬁcient time, erythropoiesis is af- Hemolysis that occurs through the mononu- fected. Iron deﬁciency anemia is a continuum clear phagocyte system (MPS) may be accom- and, if uncorrected, microcytosis and hypochro- panied by icterus from overwhelming the abil- mia eventually develop. Hemoglobinization of ity of the liver to take up, conjugate, and/or rubricytes and metarubricytes is impaired and secrete recycled bilirubin. Intravascular hemol- the delay in incorporation of iron results in an ad- ysis is less common than extravascular hemoly- ditional mitotic division in erythroid cells. This sis, in veterinary medicine. Causes of intravas- additional mitosis is responsible for microcyto- cular hemolysis include immune-mediated red sis in developing erythrocytes. The lack of iron cell destruction when accompanied by comple- causes hypochromia in advanced cases, which is ment ﬁxation and activation of the membrane identiﬁed as pale cells with increased central pal- attack complex, oxidative damage to erythro- lor; fragility and fragmentation of erythrocytes cytes, bacterial infections (e.g. with clostridial due to the paucity of intracellular hemoglobin; organisms), certain erythrocyte parasites (e.g. and decreases in MCH and MCHC. Despite the babesiosis), neonatal isoerythrolysis (particu- lack of iron, the anemia is often regenerative at larly in horses), excessive water consumption or the time of initial detection. The regenerative re- hypotonic ﬂuid administration, transfusion reac- sponse may not be as robust as with anemias that tions, hypophosphatemia, red cell metabolic de- are not associated with iron lack, such as hemol- fects (e.g. phosphofructokinase defect in dogs), ysis or internal hemorrhage. Tests that help to and mechanical shearing of erythocytes. Causes conﬁrm iron deﬁciency are serum iron; total iron- of extravascular hemolysis include immune- binding capacity (TIBC), which is a measure of mediated red cell destruction predominated by transferrin, the iron-binding protein; serum fer- macrophage activation (idiopathic or due to viral, ritin; and visual evaluation of marrow iron stores. bacterial, parasitic, or tumor antigens attached Serum ferritin and bone marrow examination are to red cell membranes), certain red cell parasites the best ways to assess total iron stores in the that cause direct damage to red cell membranes body. Serum iron is affected by many conditions (e.g. Mycoplasma organisms (formerly Hemobar- besides iron status. However, low serum iron and tonella) in cats, Anaplasma in cattle), congenital red high TIBC are expected in iron deﬁciency in most cell defects (e.g. pyruvate kinase defect in dogs species, except the dog. In this species, the TIBC and cats), and neoplasia of macrophage-type cells is not necessarily increased with iron deﬁciency. (e.g. malignant histiocytosis). Although serum iron is also decreased with ane- Many serious underlying illnesses that are not mia related to chronic inﬂammation, the TIBC is primary hemopoietic disorders can be associ- low to normal with this condition. Serum ferritin ated with nonregenerative anemia. Neoplasia, 24 Erythrocytes renal disease, inﬂammatory disease, and en- evaluation can be offered to clients in situations docrinopathies, such as hypothyroidism and where peripheral blood ﬁndings require further hypoadrenocorticism, commonly affect erythro- investigation. poiesis. The pathophysiology of the anemia is well understood for some of these conditions, and not for others. For example, the anemia asso- Erythrocytosis ciated with renal failure is multifactorial, but lack of EPO is particularly important. With severe in- Though less common than anemia, increases in ﬂammatory disease, which is the most common Hct, Hgb, and RBC count are sometimes seen. cause of nonregenerative anemia in veterinary Most often the increases are relative, due to dehy- medicine, anemia is caused by a combination of dration. The total protein level will be elevated to reduced availability of iron for erythropoiesis, the same degree unless there is a pre-existing rea- decreased red cell lifespan, and decreased re- son for the protein to be low. In this case, the total sponsiveness of the erythroid lineage to EPO. protein may be within reference limits or mildly These effects are due to the presence of cytokines, decreased despite the dehydration. Restoration particularly interleukin 1 (IL-1), tumor necrosis of normal ﬂuid balance is required to properly factor (TNF), transforming growth factor β (TGF- assess the erythrogram and total protein. β), and interferon α (INF-α). Historical infor- Relative erythrocytosis can also occur with mation, physical ﬁndings, and other test results splenic contraction in an extremely excited an- are useful to differentiate primary bone marrow imal, particularly the horse, due to epinephrine disease from secondary causes of nonregenera- release. Total protein is unaffected by splenic con- tive anemia. Bone marrow examination is indi- traction. cated when secondary causes are ruled out and Absolute erythrocytosis is associated with an the anemia is suspected to be due to bone mar- increase in total red cell mass. Primary erythro- row pathology. Marrow disorders to consider are cytosis is very uncommon and is due to neoplasia erythroid hypoplasia or aplasia due to toxins, of hemopoietic cells. If the tumor is at the level of chemicals, hormones, or irradiation; dyserythro- hemopoietic stem cells, then platelet and leuko- poiesis resulting in defective maturation; marrow cyte numbers will also be elevated; otherwise, necrosis; inﬁltration of the marrow with neoplas- only erythrocytes are increased in the peripheral tic cells or ﬁbroblasts; viral infections such as blood. EPO levels are low or within reference feline leukemia virus (FeLV) or feline immun- limits and arterial pO2 is within reference limits, odeﬁciency virus (FIV); and immune-mediated since the hemopoietic cells are autonomous and destruction of erythroid precursors. With many not responding to the physiologic need for in- conditions, cell lines in addition to erythroid are creased numbers. With secondary erythrocytosis, affected. When erythroid hypoplasia or pure red EPO levels are increased, either for physiologic cell aplasia is found on bone marrow examina- reasons because tissues are not being well oxy- tion, an etiology is often not identiﬁed. Repeat genated, or because inappropriate levels of EPO evaluation of the bone marrow and monitoring are being produced. Causes of poor oxygenation of the peripheral blood response may be required include cardiac or pulmonary disease, leading to to determine if the condition is reversible or ir- chronic hypoxia, and exposure to high altitudes reversible. Blood transfusion and immunosup- over a long period of time. Various tumors and pressive therapy may be useful to provide time lesions can produce EPO, the most common be- for the marrow to respond and to treat a pos- ing renal tumors. Absolute versus relative ery- sible underlying immune-mediated mechanism, throcytosis can usually be differentiated, based respectively. Students should learn the proper on history, clinical ﬁndings, and additional lab- technique for acquiring bone marrow and mak- oratory ﬁndings. Primary and secondary abso- ing good-quality smears, so that bone marrow lute erythrocytosis can usually be differentiated, 25 CHAPTER 1 based on ancillary tests (such as radiographs, r The degree of regeneration is also important. ultrasound), arterial blood gases, and EPO If an animal is severely anemic, but has only levels. a mild degree of regeneration, this is an in- appropriate response. Sometimes this can be explained by the time frame. For example, if Nuggets an animal hemorrhages acutely from being hit by a car, a regenerative response will not be r Look at the Hct ﬁrst and see if it is high, seen for 2–4 days and will not peak for about low, or normal, with respect to the reference 4–5 days after that. Repeating the CBC is very intervals. useful in these circumstances. r If the Hct is low, the Hgb and RBC count will r Hallmarks of regenerative anemia are as fol- generally also be low, in direct relationship to lows: elevated MCV (usually), increased retic- the Hct. As a general rule of thumb, if you mul- ulocytes, increased polychromasia, sometimes tiply the Hgb by 0.0033 (or just multiply by 3 nucleated RBCs in the peripheral blood, in- and move the decimal point), you approximate creased RPI (in dogs). Nucleated RBCs alone, the Hct. without polychromasia, do not indicate regen- r Evaluate the RBC indices for size (MCV), eration. hemoglobin content (MCH), and hemoglobin r Anemias with low MCV +/− MCH +/− concentration (MCHC). MCHC, with or without signs of regeneration, r A low Hct generally indicates anemia (zeal- should signal possible iron deﬁciency and trig- ous ﬂuid therapy can dilute the blood, caus- ger a search for causes of chronic external blood ing a drop in Hct without a true anemia being loss in the animal. Also, a low MCV may be seen present, so history, as always, is important). in animals with hepatic disease, particularly r The next step in evaluating anemia is to deter- portosystemic shunts, and in certain breeds of mine if it is regenerative (responsive bone mar- dogs (e.g. Akita). Normal young calves have row) or nonregenerative (nonresponsive bone low MCVs relative to mature cattle. These marrow). This greatly focuses the search for po- latter circumstances are not related to iron tential causes. deﬁciency. r Reticulocyte numbers should be elevated if the r The red cell morphology can provide clues to bone marrow is responsive. The reticulocyte the potential causes of anemia—e.g. Heinz bod- count is performed with a vital stain that pre- ies, eccentrocytes, spherocytes, acanthocytes, cipitates RNA present in immature RBCs. The echinocytes III, keratocytes, and schizocytes all RPI is calculated in dogs only. The reticulocyte have signiﬁcance that may relate to erythron count should correlate with the degree of poly- changes. chromasia (large, bluish, immature RBCs seen r The RDW is an index of the degree of aniso- with Romanowsky stains) noted in the RBC cytosis (variability in RBC size) and should morphology section of the report. be useful in assessing anemias. Large, young r Note: Horses are an exception in that cells mixed with older, smaller cells should in- they rarely release polychromatophilic RBCs/ crease the RDW; the RDW should be partic- reticulocytes in response to anemia. To evalu- ularly high with immune-mediated hemolytic ate anemia in the horse, we look at the MCV; anemia (IMHA) where there are often imma- often it will be slightly elevated if the marrow is ture cells mixed with cells that are small due to responsive (releasing larger cells). We also fol- losing pieces of their membranes (spherocytes). low the complete blood count (CBC) over time, Unfortunately, the RDW does not always re- to watch for a rising Hct (also indicative of mar- ﬂect the degree of anisocytosis seen on periph- row response). Ultimately, bone marrow can be eral blood examination, which emphasizes the examined to assess erythroid activity. importance of smear evaluation. 26 Erythrocytes r Do not forget to look at platelets. If an anemic animal has no platelets, you know the proba- Case studies ble cause of the anemia without going further. Then you shift to determining why there are no Note regarding cases: Data not discussed are con- platelets (thrombocytopenia). sidered insigniﬁcant or irrelevant to the case. r If the Hct is elevated, determine if the change Conventional units are given only for biochemi- is relative (due to dehydration or splenic con- cal data, since reporting of CBC results in SI units traction) or absolute. If the erythrocytosis is is more universal and conversion to conventional absolute, then explore potential causes (appro- units is easily accomplished by moving decimal priate and inappropriate) of increased erythro- points. The cases are all real and data have not cyte production. been manipulated in any way. CBC Aphrodite, day 1 Ref. int. × 109/L Erythrocytes Value Flag Ref. int. Units Leukocytes Value Flag RBC 10.4 6.89−10.8 ×1012/L WBC 19.3 H 3.90 −18.1 Hgb 163 H 99−159 g/L Corrected WBC Hct 0.487 H 0.288−0.477 L/L NRBC/100 MCV 46.6 36.4−50.0 fL WBCs MCH 15.6 12.4−16.8 pg Rel. Ref. int. × 109/L MCHC 335 319−357 g/L Differential Abs. Flag % RDW 19.7 17.2−22.8 % Segs 70 13.510 2.1−15 Retics % % Bands 19 3.667 H 0.0 −0.2 RPI Metamyelo RBC morphology Myelo Aniso 1+, echino I 1+, rouleaux 2+, nuclear Toxic change 1+ remnants few Eos 0.1−1.50 Basos 0.0−0.2 Lymphs 6 1.158 1.0−6.9 Monos 5 0.965 H 0.0−0.6 Other Atypicals Two hundred cells counted. Ref. int. × Plasma total solids by Ref. int. Platelets Value Flag Value Flag 10 9/L refractometry (g/L) Clumped (slide) Yes Total solids 88 H 58−82 Estimate (slide) Fibrinogen Morph (slide) Enlarged Total solids: fibrinogen ratio PCT Hemolysis Lipemia Yellow MPV Plasma appearance PDW Auto count (min) 300−700 Difficult to estimate platelets due to numerous clumps throughout the smear, likely ok. 27 CHAPTER 1 Case 1. Aphrodite accelerated release of neutrophils from the bone marrow. There is a signiﬁcant inﬂammatory pro- Aphrodite, a 4-year-old F(s) DLH cat, had cess, which may warrant follow-up CBC to de- anorexia, lethargy, and vomiting for 4 days. termine if the bone marrow can continue to meet the demand for neutrophils. Platelets often clump in feline blood samples; CBC (day 1) however, numbers are likely to be adequate in The Hct and Hgb are mildly elevated and the this case. RBC count is high normal. Total protein on the biochemical panel is high normal and there is a mild hyperalbuminemia. Total protein (solids), Biochemical panel as measured by refractometry on the CBC, is Decreases in sodium and chloride may be due to less accurate than the biochemical determination. both decreased intake (anorexia) and increased These ﬁndings, together with the history, sug- losses (intestinal). However, chloride is also low gest that the erythrocytosis is relative and due relative to sodium, indicating selective chloride to dehydration. loss that can occur with gastric or duodenal for- There is a very mild leukocytosis, which is due eign body, tumor, or swelling and inﬂamma- to moderately increased band neutrophils and tion leading to obstruction, or with gastrinoma a mild monocytosis. The toxic change indicates (rare). The hypochloremic metabolic alkalosis Biochemical panel Aphrodite, day 1 Test Result Units Flag Ref. int. Conv. result Conv. units Conv. ref. int. Lipemia Sodium (NA) 145 mmol/L L 151−163 145 mEq/L 151−163 None Potassium 4.1 mmol/L 3.9−5.5 4.1 mEq/L 3.9−5.5 Hemolysis Chloride 93 mmol/L L 111−125 93 mEq/L 111−125 Slight Bicarbonate 23 mmol/L H 12−21 23 mEq/L 12−21 Yellow Anion gap 33 mmol/L 20−33 33 mEq/L 20−33 None Calcium 2.23 mmol/L L 2.26−2.868.94 mg/dL 9.06−11.5 Phosphorus (inorganic) 1.95 mmol/L 1.08−2.21 6.04 mg/dL 3.34−6.84 Urea 17.7 mmol/L H 6.0−11.4 49.6 mg/dL 17−32 Creatinine 149 μmol/L 78−178 2 mg/dL 0.88−2.0 Glucose 16.3 mmol/L H 3.5−8.1 293.6 mg/dL 63−140 Cholesterol 3.69 mmol/L 1.62−4.32 142.69 mg/dL 62.6−167 Total bilirubin 4 μmol/L H 0−3 0 mg/dL 0−0.2 Alk phos 11 U/L 11−56 11 U/L 11−56 ALT 46 U/L 30−120 46 U/L 30−120 GGT 1 U/L 0−5 1 U/L 0−5 CK 309 U/L 75−471 309 U/L 75−470 Total protein 82 g/L 56−84 8 g/dL 5.6−8.4 Albumin 41 g/L H 27−39 4 g/dL 2.7−3.9 A/G ratio 1.00 0.56−1.341.00 0.56−1.34 28 Erythrocytes CBC Aphrodite, day 3 Erythrocytes Value Flag Ref. int. Units Leukocytes Value Flag Ref. int. × 109/L RBC 6.98 6.89−10.8 ×1012/L WBC 10.2 3.90−18.1 Hgb 108 99−159 g/L Corrected WBC Hct 0.323 0.288−0.477 L/L NRBC/100 MCV 46.3 36.4−50.0 fL WBCs MCH 15.5 12.4−16.8 pg Rel. Differential Abs. Flag Ref. int. × 109/L MCHC 334 319−357 g/L % RDW 17.7 17.2−22.8 % Segs 63 6.426 2.1−15 % Retics % Bands 0.0−0.2 RPI Metamyelo RBC morphology Myelo Nuclear remnants few Toxic change RBC morph unremarkable. Eos 10 1.020 0.1−1.50 Basos 0.0−0.2 Lymphs 26 2.652 1.0−6.9 Monos 1 0.102 0.0−0.6 Other Atypicals Ref. int. × Plasma total solids by Ref. int. Platelets Value Flag Value Flag 109/L refractometry (g/L) Clumped (slide) Yes Total solids 64 58−82 Estimate (slide) Normal Fibrinogen Morph (slide) Normal Total solids: fibrinogen ratio PCT Hemolysis Lipemia Yellow MPV Plasma appearance PDW Auto count (min) 300−700 also supports upper intestinal pathology. The CBC (day 3) mild hypocalcemia may not be signiﬁcant. Mild The CBC from day 3 is within reference limits. urea elevation could be due to decreased re- The absolute erythrocytosis and the inﬂamma- nal perfusion or intestinal bleeding. Urinalysis tion were corrected with conservative therapy. would be required to assess renal function. Mild Although a foreign body was suspected initially, hyperglycemia is likely due to stress in this cat. Aphrodite recovered completely without surgi- Very mild hyperbilirubinemia may be secondary cal intervention. to bile stasis from anorexia or sepsis. 29 CHAPTER 1 CBC Lady Erythrocytes Value Flag Ref. int. Units Leukocytes Value Flag Ref. int. × 109/L RBC 9.55 H 5.5−8.5 ×1012/L WBC 15.1 6.0−17.1 Hgb 206 H 120−180 g/L Corrected WBC Hct 0.611 H 0.370−0.550 L/L NRBC/100 MCV 63.9 60.0−77.0 fL WBCs MCH 21.5 19.5−24.5 pg Rel. MCHC 336 320−360 g/L Differential Abs. Flag Ref. int. × 109/L % RDW 17.4 H 11.0−14.0 % Segs 86 12.986 H 3.6−11.5 Retics % % Bands 2 0.302 H 0.000−0.300 RPI Metamyelo RBC morphology Myelo Aniso 1+, echino I 2+, codocytes normal Toxic change Eos 0.010−1.250 Basos 0.000−0.100 Lymphs 3 0.453 L 1.000−4.800 Monos 9 1.359 H 0.150−1.350 Other Atypicals Ref. int. × Plasma total solids by Ref. int. Platelets Value Flag Value Flag 10 9/L refractometry (g/L) Clumped (slide) Yes Total solids 70 51−72 Estimate (slide) Normal Fibrinogen Morph (slide) Enlarged Total solids: fibrinogen ratio PCT Hemolysis Lipemia Yellow MPV Plasma appearance Slight PDW Auto count (min) 200−900 Case 2. Lady sistent with stress (high levels of endogenous cortisol). Lady, a 15-year-old F(s) Chihuahua X dog, had lethargy and a severe heart murmur. Biochemical panel The chloride is decreased relative to the sodium, CBC suggesting selective chloride loss, possibly from The Hct, Hgb, and RBC count are all mildly vomiting (not given in the history). There is a high increased, indicating erythrocytosis. Although anion gap metabolic acidosis, which may be due the RDW is increased, this does not correlate to decreased tissue perfusion. The mild azotemia with an increase in anisocytosis. The total solids (increased phosphorus, urea, and creatinine) is are not increased. There is a mild neutrophilia, likely pre-renal from reduced renal perfusion, very mild left shift, lymphopenia, and mild given the urine speciﬁc gravity (SG). Mild hyper- monocytosis. The leukogram changes are con- glycemia and hypercholesterolemia are unlikely 30 Erythrocytes Biochemical panel Lady Test Result Units Flag Ref. int. Conv. result Conv. units Conv. ref. int. Lipemia Sodium (NA) 149 mmol/L 144−157 149 mEq/L 144−157 Potassium 4.6 mmol/L 3.6−6.0 4.6 mEq/L 3.6−6 Hemolysis Chloride 108 mmol/L L 115−126 108 mEq/L 115−126 + Bicarbonate 15 mmol/L L 17−29 15 mEq/L 17−29 Yellow Anion gap 30 mmol/L H 14−26 30 mEq/L 14−26 Calcium 2.44 mmol/L 2.21−3.00 9.78 mg/dL 8.86−12.0 Phosphorus (inorganic) 1.98 mmol/L H 0.82−1.87 6.13 mg/dL 2.54−5.79 Urea 20.0 mmol/L H 3.0−10.5 56.0 mg/dL 8.4−29 Creatinine 198 μmol/L H 60−140 2 mg/dL 0.68−1.6 Glucose 7.8 mmol/L H 3.3−5.6 140.5 mg/dL 59−100 Cholesterol 6.74 mmol/L H 2.5−5.50 260.63 mg/dL 97−210 Total bilirubin 9 μmol/L 0−17 1 mg/dL 0−1.0 Alk phos 203 U/L H 12−106 203 U/L 12−110 ALT 61 U/L 5−69 61 U/L 5−70 GGT 0 U/L 0−7 0 U/L 0−7 CK 291 U/L 0−300 291 U/L 0−300 Total protein 70 g/L 51−72 7 g/dL 5.1−7.2 Albumin 33 g/L 29−38 3 g/dL 2.9−3.8 A/G ratio 0.83 0.60−1.50 0.83 0.6−1.5 to be clinically signiﬁcant. Mildly increased al- Urinalysis kaline phosphatase (ALP) activity could be due The urine SG indicates adequate renal function. to cholestasis or enzyme induction from chronic Protein at 2+ could be signiﬁcant; however, this stress/elevated cortisol. could be better assessed by urine protein to Urinalysis Lady Collection method: Free flow Physical Sediment Color/clarity Yellow/clear WBC/hpf 1−3 Specific gravity 1.038 RBC/hpf 2−5 Reagent strip Epithelial cells 0−1 pH 5.5 Crystals Protein ++ Casts Glucose Normal Bacteria Ketones Neg Fat Scant Bilirubin Neg Other Blood +++ 31 CHAPTER 1 creatinine ratio. Blood reaction appears to be due Case 3. Roxy to mild hematuria. This is a free ﬂow sample, so Roxy, a 12.5-year-old F Old English Sheepdog X, hematuria is unlikely to be iatrogenic (as seen had anorexia for 5 days. She had been polydipsic, with cystocentesis sampling), unless the blad- but was no longer drinking at presentation. der had been manually compressed to obtain the sample. r The erythrocytosis is likely to be absolute CBC and secondary to cardiac disease. EPO levels There is a mild to moderate anemia with minimal would be expected to be elevated because the ery- regeneration. Although the reticulocyte count is throcytosis is a physiologic response to decreased 2.2%, the RPI is not increased (>1 indicates regen- arterial pO2 caused by heart failure. eration; >3 indicates strong regeneration) and CBC Roxy Erythrocytes Value Flag Ref. int. Units Leukocytes Value Flag Ref. int. × 109/L 12 RBC 4.41 L 5.5−8.5 × 10 /L WBC 60 H 6.0−17.1 Hgb 95 L 120−180 g/L Corrected WBC Hct 0.286 L 0.370−0.550 L/L NRBC/100 MCV 64.9 60.0−77.0 fL WBCs MCH 21.6 19.5−24.5 pg Rel. MCHC 332 320−360 g/L Differential Abs. Flag Ref. int. × 109/L % RDW 16.6 H 11.0−14.0 % Segs 57 34.200 H 3.6−11.5 Retics 2.2 % % Bands 32 19.200 H 0.000−0.300 RPI 0.77 Metamyelo RBC morphology Myelo Aniso 1+, kerato slight Toxic change 1+ Eos 1 0.600 0.010−1.250 Basos 0.000−0.100 Lymphs 2 1.200 1.000−4.800 Monos 8 4.800 H 0.150−1.350 Other Atypicals Ref. int. × Plasma total solids by Ref. int. Platelets Value Flag 109/L Value Flag refractometry (g/L) Clumped (slide) Yes Total solids 68 51−72 Estimate (slide) Normal Fibrinogen Morph (slide) Normal Total solids: fibrinogen ratio PCT Hemolysis Lipemia Yellow MPV Plasma appearance PDW Auto count (min) 200−900 32 Erythrocytes there is no polychromasia noted on RBC mor- relate to albumin’s role as a negative acute phase phology. Also, the absolute reticulocyte count is protein. 2.2% × 4.41 × 1012 /L = 0.097 × 1012 /L, which r The history and laboratory ﬁndings support is only slightly above the reference interval for a a diagnosis of pyometra in this dog. The anemia normal, nonanemic dog ((0.055–0.085) × 1012 /L). is likely due to chronic disease/inﬂammation. There is a moderate to severe leukocytosis charac- The leukogram abnormalities are due to the terized by a moderate neutrophilia with a marked demand for phagocytic cells within the uterus. A left shift, toxic change, and moderate monocy- marked leukocytosis commonly occurs following tosis. These leukogram changes indicate severe, ovariohysterectomy when pyometra is present. chronic inﬂammation. Granulocytic hyperplasia is established in the bone marrow and neutrophils continue to be re- leased into the peripheral blood for several days Biochemical panel after the uterus is removed. Since the neutrophils There is a mild azotemia that must be inter- no longer have a focus of inﬂammation/infection preted in relation to the hydration status of the to migrate into, numbers will increase in the cir- dog and the urine SG (not available). Moder- culation (rebound effect). Band cells will eventu- ate elevations in ALP and γ -glutamyltransferase ally decline ﬁrst, followed by mature neutrophils. (GGT) activities could be due to cholestasis, en- The anemia of chronic disease/inﬂammation will zyme induction, or both. The hypoalbuminemia start to correct with resolution of the inﬂamma- and low albumin to globulin (A/G) ratio may tory response. Biochemical panel Roxy Test Result Units Flag Ref. int. Conv. result Conv. units Conv. ref. int. Lipemia Sodium (NA) 147 mmol/L 145−158 147 mEq/L 145−158 None Potassium 4.9 mmol/L 3.8−5.6 4.9 mEq/L 3.8−5.6 Hemolysis Chloride 110 mmol/L 103−118 110 mEq/L 103−118 None Bicarbonate 15 mmol/L 15−25 15 mEq/L 15−25 Yellow Anion gap 27 mmol/L 16−30 27 mEq/L 16−30 None Calcium 2.32 mmol/L 1.91−3.03 9.30 mg/dL 7.66−12.1 Phosphorus (inorganic) 2.25 mmol/L 0.63−2.41 6.97 mg/dL 1.95−7.46 Urea 14.6 mmol/L H 3.5−11.4 40.9 mg/dL 9.8−32 Creatinine 142 μmol/L H 41−121 2 mg/dL 0.46−1.4 Glucose 6.0 mmol/L 3.1−6.3 108.1 mg/dL 56−110 Cholesterol 4.65 mmol/L 2.70−5.94 179.81 mg/dL 104−230 Total bilirubin 4 μmol/L 1−4 0 mg/dL 0.06−0.2 Alk phos 742 U/L H 18−128 742 U/L 18−130 ALT 49 U/L 19−59 49 U/L 19−59 GGT 22 U/L H 0−8 22 U/L 0−8 CK 247 U/L 51−418 247 U/L 51−420 Total protein 63 g/L 55−71 6 g/dL 5.5−7.1 Albumin 19 g/L L 28−38 2 g/dL 2.8−3.8 A/G ratio 0.43 L 0.73−1.49 0.43 0.73−1.49 33 CHAPTER 1 CBC Gizmo Erythrocytes Value Flag Ref. int. Units Leukocytes Value Flag Ref. int. × 109/L 12 RBC 1.42 L 5.5−8.5 ×10 /L WBC 11.5 6.0−17.1 Hgb 35 L 120−180 g/L Corrected WBC Hct 0.097 L 0.370−0.550 L/L NRBC/100 MCV 68 60.0−77.0 fL 4 WBCs MCH 24.7 H 19.5−24.5 pg Rel. MCHC 363 H 320−360 g/L Differential Abs. Flag Ref. int. × 109/L % RDW 15 H 11.0−14.0 % Segs 82 9.430 3.6−11.5 Retics 1.8 % % Bands 6 0.690 H 0.000−0.300 RPI 0.14 Metamyelo RBC morphology Myelo Aniso 2+, macro 1+, echino I 2+, kerato slight, poly 1+ Toxic change Eos 1 0.115 0.010−1.250 Basos 0.000−0.100 Lymphs 4 0.460 L 1.000−4.800 Monos 7 0.805 0.150−1.350 Other Atypicals Plasma total solids by Ref. int. Platelets Value Flag Ref. int. × 109/L Value Flag refractometry (g/L) Clumped (slide) Total solids 37 L 51−72 Estimate (slide) Decreased Fibrinogen Morph (slide) Giant Total solids: fibrinogen ratio PCT Hemolysis Lipemia Yellow MPV Plasma appearance PDW Auto count 200−900 Platelets appear markedly decreased; count n/a due to sample age. Case 4. Gizmo and RPI do not support regeneration. Severe hypoproteinemia and severe anemia suggest GIZMO, a 8-month old M Poodle dog, was pre- acute blood loss. Platelet numbers are decreased, sented in shock with white mucous membranes. although an absolute platelet count is not avail- able. Severe hemorrhage, internal or external, can CBC result in signiﬁcant platelet loss. Absolute num- The Hct, Hgb, and RBC count are all severely de- bers are not expected to become 30% to 30% plasia, the bone marrow is hypercellular and Monocytic M5a Monoblasts and promonocytes neoplastic cells occupy a considerable portion >80% of the marrow space at the expense of nor- M5b Monoblasts and promonocytes mal tissue. Myeloid neoplasia is often associated >30% to 30% non-neoplastic hemopoietic cell line(s). Myeloid Erythrocytic M6Er Rubriblasts >30% tumors are divided into acute and chronic Megakaryocytic M7 Megakaryoblasts >30% subtypes. Morphologic features and numbers of blast cells in bone marrow and peripheral blood are important in determining whether the 100% of marrow cells are blasts, and differentia- leukemia is acute or chronic; the time frame is tion to a speciﬁc lineage cannot be determined. less important, as it is often unknown. Blast cells are large, immature cells of a certain lineage Granulocytic (M1, M2) (erythroid, granulocytic, monocytic, megakary- Granulocytic myeloblasts (granuloblasts) com- ocytic), with ﬁne chromatin, one or more nucle- prise a greater proportion of the nucleated cells oli, and dark-staining cytoplasms. The term acute in the bone marrow in M1, compared to M2. The refers to a relatively undifferentiated phenotype, clinical relevance of subdividing acute granulo- high numbers of blast cells in the bone marrow, cytic neoplasia into two subtypes in veterinary and a more rapidly deteriorating clinical course medicine has not been determined. Tumors of following diagnosis. In contrast, chronic refers to neutrophil precursors are more common than a more differentiated phenotype, lower numbers those of eosinophils and basophils; however, of blast cells in the bone marrow, and a more pro- speciﬁc granules are not present at the gran- tracted clinical course following diagnosis. Acute uloblast stage, so the line of differentiation i

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