Erythrocyte Morphology

Erythrocytes from all mammals are anucleated, and most are in the shape of biconcave discs called discocytes (Figs. 4-1, 4-2).²⁰⁵ The biconcave shape results in the central pallor of erythrocytes observed in stained blood films. Among common domestic animals, biconcavity and central pallor are most pronounced in dogs (Figs. 4-3, 4-4), which also have the largest erythrocytes. Other species do not consistently exhibit central pallor in erythrocytes on stained blood films. The apparent benefit of the biconcave shape is that it gives erythrocytes high surface area : volume ratios and allows for deformations that must take place as they circulate. Erythrocytes from goats generally have a flat surface with little surface depression; a variety of irregularly shaped erythrocytes (poikilocytes) may be present in clinically normal goats (Fig. 4-5). Erythrocytes from animals in the Camelidae family (camels, llamas, vicuñas, and alpacas) are anucleated, thin, elliptical cells termed elliptocytes or ovalocytes (Fig. 4-6). They are not biconcave in shape and are minimally deformable.⁴³⁷ Erythrocytes from birds (Fig. 4-7), reptiles, and amphibians are also elliptical in shape, but they contain nuclei and are larger than mammalian erythrocytes. Blood cells in salamanders are the largest recognized (Fig. 4-8).

FIGURE 4-1 Scanning electron photomicrograph of a normal horse erythrocyte called a discocyte.

From Stockham SL, Harvey JW, Kinden DA. Equine glucose-6-phosphate dehydrogenase deficiency. Vet Pathol. 94;31:518-527.

FIGURE 4-2 Blood film from a horse. Most erythrocytes are adhered together like stacks of coins (rouleaux), a normal finding in this species. Individual nonadherent erythrocytes exhibit central pallor as a result of their biconcave shape. A basophil with purple granules is present in the bottom right of the image. Wright-Giemsa stain.

FIGURE 4-3 Scanning electron photomicrograph of dog erythrocytes (discocytes).

Courtesy of K. S. Keeton and N. C. Jain.

FIGURE 4-4 Blood from a dog with acute blood-loss anemia and normal erythrocyte morphology. Erythrocytes exhibit prominent central pallor. Two mature neutrophils and a platelet (bottom right corner) are also present. Wright-Giemsa stain.

FIGURE 4-5 Poikilocytes in blood from a normal goat. Note the small size of the erythrocytes compared with the neutrophil in the left part of the image. Wright-Giemsa stain.

FIGURE 4-6 Elliptocytes in blood from a normal llama. Wright-Giemsa stain.

FIGURE 4-7 Blood film from a macaw. Nucleated erythrocytes and three heterophils are present.

FIGURE 4-8 Blood film prepared by mixing equal parts of blood from a salamander (Amphiuma means) with that of a domestic cat to demonstrate the large size of the nucleated erythrocytes and a neutrophil in the salamander. Most of the cat erythrocytes are echinocytes, a shape artifact of sample handling in this instance. Wright-Giemsa stain.

Courtesy of H. L. Wamsley.

Erythrocyte Functions

Mammalian erythrocytes normally circulate for several months in blood despite limited synthetic capacities and repeated exposures to mechanical and metabolic insults. Erythrocytes have three functions: transport of oxygen (O₂) to tissue, transport of carbon dioxide (CO₂) to the lungs, and buffering of hydrogen ions (H⁺). In nonanemic animals, the presence of hemoglobin within erythrocytes increases the O₂-carrying capacity of blood more than 50 times that of plasma without erythrocytes. The O₂ content of blood depends on the blood hemoglobin content, the partial pressure of dissolved oxygen (Po₂) in blood, and the affinity of hemoglobin for O₂.

Each hemoglobin tetramer is capable of binding four molecules of O₂ when fully oxygenated. The initial binding of a molecule of O₂ to a monomer of tetrameric deoxygenated hemoglobin facilitates further binding of O₂ to the hemoglobin molecule. The changing O₂ affinity of hemoglobin with oxygenation results in a sigmoid O₂ dissociation curve (Fig. 4-9), when the percent saturation of hemoglobin with O₂ is graphed against the Po₂. The steepness of the middle portion of the curve is of great physiologic significance because it covers the range of O₂ tensions present in tissues. Consequently a relatively small decrease in O₂ tension in tissues results in substantial O₂ release from hemoglobin. The overall affinity of hemoglobin for O₂ is decreased by increasing H⁺, CO₂, temperature, and, in most mammals, 2,3-diphosphoglycerate (2,3DPG). There is a direct correlation between body weight and the O₂ affinity of hemoglobin in whole blood (lower body weight, lower O₂ affinity) when various species of mammals are compared.²⁰⁵

FIGURE 4-9 Hemoglobin-oxygen dissociation curve and factors influencing the position of the curve.

From Harvey JW. The erythrocyte: physiology, metabolism, and biochemical disorders. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals, 6th ed. San Diego, CA: Academic Press; 2008:173-240.

The O₂ affinity of fetal blood is greater than that of maternal blood except in the cat. Differences in fetal versus maternal O₂ affinity may potentiate O₂ transport from the mother to the fetus. However, the fetus is subjected to low arterial O₂ tensions, and the increased O₂ affinity of fetal blood is likely needed to more fully saturate hemoglobin with O₂.²⁰⁵

The ability of plasma to carry CO₂ is small, but the carbonic anhydrase reaction in erythrocytes increases the CO₂-carrying capacity of blood 17-fold by rapidly converting CO₂ to carbonic acid (H₂CO₃). The H₂CO₃ spontaneously ionizes to H⁺ and bicarbonate (HCO₃⁻). The HCO₃⁻ diffuses out of the cell down a concentration gradient and chloride (Cl⁻) moves in (chloride shift) to maintain electrical neutrality. These processes are reversed at the lungs. Some CO₂ is also transported bound to hemoglobin as carbamino groups. Deoxyhemoglobin binds about twice the CO₂ that oxyhemoglobin does.²⁰⁵

Hemoglobin is the major protein buffer in blood. Deoxyhemoglobin is a weaker acid than oxyhemoglobin. Consequently, when oxyhemoglobin releases its O₂ in the tissues, the formation of deoxyhemoglobin results in increased binding of H⁺. Hemoglobin buffers the effects of H₂CO₃ and allows for the isohydric transport of CO₂. Hemoglobin also buffers organic acids produced during metabolism.²⁰⁵

Erythrocyte Biochemistry

ATP Generation

Mammalian erythrocytes lack nuclei; therefore they cannot synthesize DNA or RNA. They also lack ribosomes, mitochondria, and endoplasmic reticula; consequently they have no Krebs cycle or electron transport system and are unable to synthesize proteins or lipids de novo. Glucose is the primary substrate for the energy needs of erythrocytes from all species except the pig, where inosine appears to be the major substrate. Mature erythrocytes depend on anaerobic glycolysis for energy (Fig. 4-10). The ATP generated by glycolysis is needed for the maintenance of erythrocyte ionic composition, shape, and deformability and for limited synthetic activities such as glutathione synthesis. Hypophosphatemia results in decreased erythrocyte glycolytic rates and decreased ATP generation. Hemolytic anemia resulting from hypophosphatemia has been reported in diabetic cats and in a diabetic dog following insulin therapy, in a cat with hepatic lipidosis, and in postparturient cattle and buffaloes.^109,205 Deficiencies of rate-controlling enzymes in glycolysis also result in insufficient ATP generation and shortened erythrocyte survival. Pyruvate kinase (PK)-deficient dogs and cats have mild to severe regenerative hemolytic anemia. Phosphofructokinase (PFK)-deficient dogs have compensated hemolytic anemia plus sporadic episodes of intravascular hemolysis and hemoglobinuria.²⁰³

FIGURE 4-10 Metabolic pathways of the mature erythrocyte. HK, hexokinase; GPI, glucose phosphate isomerase; PFK, phosphofructokinase; TPI, triosephosphate isomerase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; MPGM, monophosphoglycerate mutase; DPGM, diphosphoglycerate mutase; PK, pyruvate kinase; G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; LDH, lactate dehydrogenase; GR, glutathione reductase; GPx, glutathione peroxidase; TK, transketolase; TA, transaldolase; GSSG, oxidized glutathione; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FDP, fructose 1,6-diphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 1,3DPG, 1,3-diphosphoglycerate; 2,3DPG, 2,3-diphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; GSH, reduced glutathione; P_i, inorganic phosphate; SOD, superoxide dismutase.

From Harvey JW. The erythrocyte: physiology, metabolism, and biochemical disorders. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals, 6th ed. San Diego, CA: Academic Press; 2008:173-240.

Iron Metabolism

Iron metabolism is presented in this chapter because more iron is needed for the production of erythrocytes than for all other cells in the body combined. Iron is absorbed from the diet in the small intestine and transferred to plasma, where it is bound to transferrin for transport to cells within the body. Once inside the body, iron cycles in a nearly closed system (Fig. 4-11) because little iron is lost in domestic animals unless hemorrhage occurs. About 75% of the iron present in plasma will be transported to the bone marrow for incorporation into hemoglobin in developing erythroid cells.⁴³⁶ The remaining plasma iron is taken up by nonerythroid tissues, primarily the liver.²⁶⁶ Erythrocytes containing hemoglobin normally circulate for several months before being phagocytized by macrophages when senescent. After phagocytosis, erythrocytes are lysed, hemoglobin is degraded, and iron is released. Most iron released from degraded hemoglobin is quickly released back into plasma, but a small amount may be stored as ferritin or hemosiderin within macrophages, which is released more slowly into plasma. The vast majority of iron entering plasma each day comes from macrophage release.²⁰⁴

FIGURE 4-11 Iron cycle. Iron (Fe) is highly conserved in the body. Iron in plasma is bound to transferrin, a transport protein that is synthesized in the liver. Iron is transported to all tissues, but most iron is utilized to synthesize hemoglobin in developing erythroid cells. Aged blood erythrocytes are phagocytized by macrophages and hemoglobin is degraded. Released iron is either returned to plasma or stored in macrophages as ferritin and hemosiderin. Nearly all of the iron in plasma under normal conditions comes from the release of iron by macrophages that have phagocytized and degraded erythrocytes. Only about 3% of the iron in plasma results from gastrointestinal (GI) enterocyte absorption in normal individuals.

From Harvey JW. Iron metabolisms and its disorders. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals, 6th ed. San Diego, CA: Academic Press; 2008:259-285.

About 60% to 70% of total body iron is present in hemoglobin (3.4 mg iron per gram of hemoglobin). About a third of total body iron is stored as ferritin and hemosiderin (primarily within macrophages), 3% to 7% is present in myoglobin (with the higher values occurring in dogs and horses), 1% is present in hemoprotein and flavoprotein enzymes, and only 0.1% is bound to transferrin in plasma.²⁰⁴

Iron Absorption

The absorption of iron from the diet depends upon age, species, iron stores, rate of erythropoiesis, inflammation, and pregnancy, as well as the amount and chemical form of iron ingested. A low percentage of dietary iron is absorbed in normal adult animals. Iron absorption occurs through enterocytes of the duodenum and proximal jejunum. Iron can be taken in by enterocytes as free ions or as heme by different pathways (Fig. 4-12). The relative importance of these pathways varies depending on species and diet.²⁰⁴

FIGURE 4-12 Mechanisms of iron absorption. Ferrous iron (Fe⁺²) ions are transported into enterocytes in the duodenum by the divalent transporter-1 (DMT1) after reduction of ferric iron (Fe⁺³) ions using a duodenal cytochrome b (DcytB). Heme is transported into enterocytes using heme carrier protein-1 (HCP1). Once inside, inorganic iron is released from heme by the action of the heme oxygenase (HO) reaction. Fe⁺² ions are exported from enterocytes using ferroportin, oxidized to Fe⁺³ using hephaestin, and bound by apotransferrin (aTf) to form monoferric transferrin (mTf) and diferric transferrin (not shown). Hepcidin in plasma inhibits iron export to plasma by interacting directly with ferroportin, leading to ferroportin’s internalization and lysosomal degradation. Fe⁺² not transported to plasma is stored as ferritin following oxidation to Fe⁺³. Iron stored as ferritin is returned to the lumen of the small intestine when enterocytes are sloughed at the tip of the villus.

From Harvey JW. Iron metabolism and its disorders. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals. 6th ed. San Diego, CA: Academic Press; 2008:259-285.

Most inorganic iron in the diet is in the ferric (Fe⁺³) state. Fe⁺³ iron is solubilized from food by hydrochloric acid in the stomach and binds to mucins and various small molecules in the stomach, which keep the iron soluble and available for absorption in the more alkaline environment of the small intestine. The most important pathway for nonheme iron uptake utilizes the divalent metal transporter-1 (DMT1). Fe⁺³ ions must be reduced to ferrous (Fe⁺²) ions before they can be transported into the enterocyte via the DMT1. Although some Fe⁺³ ion reduction may occur by direct interaction with dietary ascorbic acid, most reduction appears to rely on duodenal cytochrome b (DcytB) and possibly other brush border ferrireductase enzymes. Although humans absorb Fe⁺² salts more readily from the intestine than Fe⁺³ salts, dogs are reported to absorb both valence forms equally well.²⁰⁴

Heme is released from dietary myoglobin and hemoglobin by the action of digestive enzymes. Dietary heme iron is generally more bioavailable than is nonheme iron and is an important nutritional source of iron in carnivores and omnivores. Heme enters duodenal enterocytes as an intact metalloporphyrin, possibly using a brush border transporter named heme carrier protein 1 (HCP1). However, this protein transports folate more efficiently than heme; consequently, its physiologic role in intestinal heme uptake remains to be clearly defined. After heme absorption, iron is released from heme by the action of the heme oxygenase reaction.²⁰

Once within the enterocyte, intracellular iron molecules are likely bound to one or more chaperone molecules. A potential chaperone named poly (rC)-binding protein 1 (PCBP1) has been described.⁴²² Iron taken up by enterocytes has one of two fates, export or storage, depending on the body’s iron needs. If iron is required by the body, molecules will be transported from enterocytes to transferrin in plasma. This transportation is mediated by ferroportin, an iron transport protein located on the basolateral surface of mature enterocytes. In addition to ferroportin, the efflux of iron from enterocytes requires a copper-containing protein called hephaestin, which is also located on the basolateral membranes of mature enterocytes. Hephaestin is a membrane-bound ferroxidase that has significant homology to the plasma protein ceruloplasmin. Hephaestin’s function may relate to its ability to oxidize Fe⁺² ions to Fe⁺³ ions for binding to transferrin in plasma.²⁰

If body iron requirements are low, enterocyte cytoplasmic iron accumulates. Free iron is toxic; consequently the mucosal cell protects itself by increasing apoferritin synthesis and incorporating the excess iron into ferritin. Each ferritin molecule is composed of a protein shell of 24 apoferritin subunits surrounding a central core of up to 4500 iron atoms as ferric oxyhydroxide. Ferritin is a storage protein that prevents free iron from catalyzing oxidative reactions, which would injure the cell. Ferritin within mucosal cells is returned to the small intestine lumen when enterocytes are sloughed at the tip of the villus after 1 to 2 days.⁴⁴⁵

Iron absorption is increased when total body iron content is low or erythropoiesis is increased. Iron absorption is decreased when total body iron content is high or inflammation is present.²⁰⁴ Components of brush-border iron uptake, including DMT1 and DcytB, are strongly influenced by the iron concentration within enterocytes, with increased components expressed when intracellular iron content is low and decreased components expressed when iron content is high. These locally responsive changes in brush-border transport components help buffer the body against the absorption of excessive iron, but it is the control of the basolateral transport of iron from enterocytes to plasma that represents the primary site at which iron absorption is regulated.⁴⁴⁵

Systemic Control of Iron Metabolism

Hepcidin, a peptide secreted by hepatocytes into the circulation, is an important systemic regulator of iron metabolism.¹⁵³ Its production is modulated by body iron requirements, which are largely influenced by the magnitude of erythropoiesis present.³⁵⁴ Hepcidin inhibits iron export from enterocytes, macrophages, and hepatocytes by interacting directly with ferroportin, leading to the internalization and lysosomal degradation of this iron export protein.²⁰ Hepcidin production is decreased in iron deficiency and disorders resulting in increased erythropoiesis, which increase the demand for iron.¹⁵² As a result, ferroportin receptors are abundant on cell surfaces and dietary iron absorption is increased, as is the export of iron from macrophages and hepatocytes. Conversely, hepcidin production is increased and ferroportin transporter expression on cell surfaces is decreased when iron overload is present. Hepcidin production is also increased during inflammation by a pathway not dependent on body iron requirements.⁴⁴⁵

Plasma Iron

Nearly all of the iron in plasma is bound to the protein apotransferrin to form transferrin. The binding of iron to apotransferrin keeps iron molecules soluble and prevents iron-catalyzed oxidative reactions. Apotransferrin is a β-globulin with two binding sites for Fe⁺³. Normally, 25% to 50% of the iron-binding sites are saturated with iron. Plasma iron turns over rapidly in 3 hours or less.⁴³⁶ Nearly all of the iron in plasma under normal conditions comes from the release of iron by macrophages that have phagocytized and degraded erythrocytes. In contrast, in normal individuals, only about 3% of the iron in plasma results from enterocyte absorption.³⁷²

Iron Uptake by Erythroid Cells

The delivery of iron from plasma to developing erythroid cells and other cell types except macrophages is dependent on transferrin.²⁶⁶ Transferrin (especially diferric transferrin) molecules bind to transferrin receptor 1 (TfR1) on the surface of cells, and these transferrin-TfR1 complexes invaginate to initiate endocytosis. After the transferrin-TfR1 complexes are internalized as endosomes, a proton pump decreases the pH in the endosome, resulting in conformational changes in the proteins and subsequent release of iron ions from transferrin. The released iron is exported from the endosome using DMT1.³⁴⁹ The resultant apotransferrin-TfR1 complex is recycled to the cell membrane, where apotransferrin is released from the cell, and the TfR1 is again available for binding additional iron-containing transferrin molecules. Erythroid precursor cells in the bone marrow and reticulocytes that synthesize hemoglobin have TfR1 on their surfaces for iron uptake, but reticulocytes lose their TfR1 as they develop into mature erythrocytes.³⁷²

After its release from transferrin, iron is transported to the mitochondria, where it is incorporated into protoporphyrin to form heme. A direct interorganelle transfer of iron occurs between endosomes and mitochondria in developing erythroid cells.⁴¹⁹ Some iron is presumably released from endosomes into a cytoplasmic labile iron pool, with excess cytoplasmic iron stored as ferritin. TfR and apoferritin synthesis are regulated by the amount of intracellular iron present. High iron content stimulates apoferritin synthesis and inhibits TfR expression to minimize the potential of iron toxicity to the cell. Low iron content results in decreased apoferritin synthesis and increased TfR expression on cell surfaces to maximize iron uptake and use for heme synthesis. Free heme concentration within erythroid cells controls hemoglobin synthesis. An increase in free heme promotes the synthesis of globin chains and inhibits the uptake of iron from transferrin.²⁰⁴

Macrophage Iron Metabolism

Little iron enters macrophages, in contrast to other cell types in the body, via plasma transferrin. Rather, nearly all iron enters macrophages by the phagocytosis of aged or prematurely damaged erythrocytes (Fig. 4-13).³⁷³ Following phagocytosis, erythrocytes are lysed and hemoglobin is degraded to heme and globin. The microsomal heme oxygenase reaction degrades heme and releases iron. Most of the iron released from degraded heme is quickly exported from the macrophage and bound to plasma transferrin for transport to other cells (especially erythrocyte precursors in the bone marrow).²⁰⁴ The export of iron from macrophages is mediated by ferroportin and controlled by hepcidin, as has been discussed for enterocytes.⁴⁹⁵ The copper-containing plasma protein ceruloplasmin oxidizes Fe⁺² ions to Fe⁺³ ions for binding to transferrin in plasma.³⁴¹

FIGURE 4-13 Iron metabolism in macrophages. Nearly all iron enters macrophages by the phagocytosis of aged or prematurely damaged erythrocytes. Following phagocytosis, erythrocytes are lysed, and hemoglobin is degraded to heme and globin. The microsomal heme oxygenase reaction within macrophages degrades heme and releases iron to the labile iron pool (LIP). Most of the released iron is exported from the macrophage by ferroportin as ferrous iron, oxidized to ferric iron by ceruloplasmin (Cp) in plasma, and bound to apotransferrin (aTf) to form monoferric transferrin (mTf) or diferric transferrin (not shown). Hepcidin in plasma inhibits iron export by interacting directly with ferroportin, leading to ferroportin’s internalization and lysosomal degradation. Iron not rapidly released to plasma is stored within macrophages as ferritin, which may be degraded to hemosiderin within lysosomes.

From Harvey JW. Iron metabolism and its disorders. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals. 6th ed. San Diego, CA: Academic Press; 2008:259-285.

The mononuclear phagocyte system accounts for much of the total body iron stores. Iron not rapidly released to plasma is stored within macrophages as ferritin and hemosiderin. Free cytoplasmic ferritin molecules are visible by electron microscopy but not by light microscopy. Hemosiderin is composed of aggregates of protein and iron within lysosomes. It is insoluble in water and thought to result from the degradation of ferritin. Hemosiderin is visible by light microscopy when it is stained with an iron stain (Prussian blue stain). Iron in the storage pool turns over slowly unless there is an increased need for iron for hemoglobin synthesis.⁴⁵

Normal Removal of Aged Erythrocytes

Most erythrocytes circulate in blood for a finite time period (survival time or life span) ranging from 2 to 5 months in domestic animals, depending on the species. Erythrocyte life spans are related to body weight (and consequently metabolic rate), with the smallest animals (highest metabolic rate) having the shortest erythrocyte life spans. Greyhound dogs are often used as blood donors. The erythrocyte life span of 6 greyhound dogs (mean 93 days) was not significantly different from that of 3 nongreyhound dogs (103 days).¹⁵⁸ Aged erythrocytes are phagocytized by macrophages of the mononuclear phagocyte system. Oxidative injury, especially near the end of their life span, appears to be responsible for normal erythrocyte aging and removal.²⁰⁵ Oxidative damage and other stressors can induce suicidal death of erythrocytes (eryptosis), with reactions similar to some of those that occur during apoptosis of nucleated cells. Eryptosis is characterized by Ca⁺² entry, erythrocyte shrinkage, membrane blebbing (microvesicle formation), and cell membrane phospholipid scrambling, with phosphatidylserine exposure on the cell surface.²⁷¹

Surface membrane alterations on aged or damaged cells that may be recognized by macrophages include exposure of phosphatidylserine on the external surface, modified external membrane carbohydrate residues (e.g., desialation of sialoglycoproteins), and/or modified membrane proteins (e.g., partially degraded band 3); these are possible signals for removal.^57,246,265 Phosphatidylserine is normally localized in the inner leaflet of the lipid bilayer, but with cell damage phosphatidylserine may be exposed on the outer leaflet of the lipid bilayer, where it can be bound by phosphatidylserine receptors such as CD36 on the surface of macrophages.²⁴⁹ Other macrophage receptors can recognize altered carbohydrate moieties on the surface of erythrocytes.

The appearance of a senescent cell antigen may be an important signal in the removal of aged erythrocytes.²⁴⁶ This senescent cell antigen is derived from the band 3 anion transporter. The specific alteration required for band 3 to become antigenic remains to be clarified, but oxidative mechanisms are probably involved. A natural antibody against the senescent cell antigen is present in human plasma. This antibody binds to senescent cell antigens on the surface of aged cells and, together with bound complement, promotes the phagocytosis of aged erythrocytes by macrophages that exhibit Fc and C3b surface receptors. Senescent dog erythrocytes accumulate surface-associated immunoglobulin, which is believed to promote their removal by macrophages.³⁹⁵ The relative importance of the immune- and nonimmune-mediated phagocytosis of senescent erythrocytes remains to be clarified.

Erythrocytes lose volume by shedding microvesicles as they age. The composition of the resultant microvesicles varies, but they typically contain hemoglobin. Other components that may be present include glycophorin A, breakdown products of band 3 (senescent antigen), IgG, and exposed phosphatidylserine. They do not contain the skeletal proteins spectrin and ankyrin.^186,536 Microvesicles are rapidly cleared from the circulation by macrophages, using receptors discussed above for aged erythrocytes.¹⁸⁶ It may be that this process of microvesiculation removes patches of damaged membrane that would otherwise bind to macrophages and result in the early removal of otherwise healthy erythrocytes.⁵³⁶

Following phagocytosis by macrophages of the spleen, liver, and other organs, erythrocytes and erythrocyte microvesicles are lysed and hemoglobin is degraded to heme and globin (Fig. 4-14). Globin is catabolized to constituent amino acids, and the microsomal heme oxygenase reaction within macrophages degrades heme to iron, biliverdin, and carbon monoxide. Biliverdin is reduced to bilirubin via biliverdin reductase in nearly all mammals. Biliverdin reductase activity is low in rabbits and nutria and almost completely lacking in birds; consequently biliverdin is the predominant bile pigment in these species.^101,476 Considerable heterogeneity exists in reptiles, amphibians, and fish in the production of bilirubin versus biliverdin.¹⁰¹ Bilirubin is released from macrophages and bound to albumin for transport to the liver for conjugation and excretion. Approximately 80% of the bilirubin produced in the body comes from the degradation of hemoglobin, with the remainder coming from the degradation of other heme-containing proteins.⁴⁷⁶

FIGURE 4-14 Overview of erythrocyte production, erythrocyte phagocytosis by mononuclear phagocytes, hemoglobin degradation, and bilirubin metabolism.

Pathologic Destruction of Erythrocytes

Increased membrane injury associated with various pathologic disorders can result in increased phagocytosis of erythrocytes by macrophages (see previous discussion concerning the removal of aged erythrocytes). Anemia develops if the rate of erythrocyte destruction exceeds the ability of the bone marrow to respond by producing new erythrocytes. Lysis of erythrocytes within macrophages after phagocytosis is sometimes referred to as extravascular hemolysis. Hyperbilirubinemia may be present within a few hours following substantial erythrocyte destruction.

Increased eryptosis (similar to the apoptosis of nucleated cells) may contribute to the development of anemia in some disorders. As discussed earlier, eryptosis is characterized by Ca⁺² entry, erythrocyte shrinkage, and membrane blebbing with ectosome formation (Fig. 4-15).²⁷¹ Reported triggers of eryptosis include oxidative stress, energy depletion, osmotic shock, lipid-derived signaling molecules, certain bacterial exotoxins, various drugs, and metals including lead, copper, zinc, and mercury. Diseases associated with accelerated eryptosis in humans include sepsis, malaria, hemoglobinopathies, G6PD deficiency, phosphate depletion, iron deficiency, and hemolytic uremic syndrome.²⁷⁰

FIGURE 4-15 Presumptive eryptosis with erythrocyte shrinkage and vesicle formation in blood from dogs. A, Blood from a dog with regenerative anemia and hemangiosarcoma. Frequent acanthocytes and echinocytes and occasional schistocytes and keratocytes were also noted in the stained blood film. Wright-Giemsa stain. B, Blood from a febrile dog with mild nonregenerative anemia and lymphoma. Low numbers of eccentrocytes and pyknoctyes and rare hemoglobin crystals were also noted in the stained blood film. Wright-Giemsa stain.

Almost no lysis of erythrocytes occurs within the circulation of normal individuals, but intravascular hemolysis can be present when severe membrane damage occurs in disease states (Fig. 4-16). Following lysis, hemoglobin in plasma (hemoglobinemia) reversibly dissociates into dimers that bind almost irreversibly to haptoglobin, an α₂-glycoprotein in plasma. The hemoglobin-haptoglobin complex is too large to be filtered through the kidney and is rapidly removed from the circulation following binding to the hemoglobin scavenger receptor CD163 on macrophages. Once inside the cell, hemoglobin-haptoglobin complexes are transported to lysosomes for degradation, and receptors are recycled to the cell surface.²²⁹ The hemoglobin is degraded and iron is conserved, as discussed previously.

FIGURE 4-16 Pathophysiology of intravascular hemolysis. Methemalbumin forms in primates but not in common domestic animals. Hp, haptoglobin.

Once plasma haptoglobin is saturated (about 50 to 150 mg/dL of hemoglobin-binding capacity in dogs, cats, and horses), remaining free hemoglobin dimers are small enough to be readily filtered by the kidney.¹⁹⁶ Some hemoglobin is reabsorbed by the proximal tubules, but once that capacity is exceeded, hemoglobin appears in the urine (hemoglobinuria).²¹¹ Plasma appears red when as little as 50 mg/dL of hemoglobin is present; consequently hemoglobinemia may be observed in the absence of hemoglobinuria. Hemoglobin absorbed by the proximal tubules is rapidly catabolized, and iron is stored as ferritin and hemosiderin.²⁰³ Iron that is not reutilized is lost when tubular epithelial cells slough into the urine.

Free hemoglobin in plasma can spontaneously oxidize to form methemoglobin, which tends to dissociate into ferriheme (hemin) and globin. Free heme binds to a plasma protein called hemopexin. The heme-hemopexin complexes undergo endocytosis after binding to CD91 on the surface of macrophages and hepatocytes.²²⁹ The binding to hemopexin protects cell membranes from toxic effects of free heme, and it also conserves iron. Albumin from primates can also bind heme to form methemalbumin, but albumin from common domestic animals does not bind heme.¹⁶³

Rouleaux

Erythrocytes on blood films from healthy horses, cats, and pigs often exhibit rouleaux (aggregations of erythrocytes grouped together like a stack of coins) formations (see Fig. 4-2). Rouleaux formation depends on both the nature of the erythrocytes and the composition of plasma.³⁷ Erythrocytes that are more deformable and have greater membrane fluidity with less negative charge on their surfaces (weaker electrostatic repulsive force) aggregate more readily than cells with the opposite characteristics.^37,443,460 Rouleaux formation also depends on the presence of high-molecular-weight proteins in plasma.¹³² Increased concentrations of globulin proteins—including fibrinogen, haptoglobin, and immunoglobulins—potentiate rouleaux formation in association with inflammatory conditions.^11,532 Rouleaux formation can also occur in association with some lymphoproliferative disorders in which one or more immunoglobulins are secreted in high amounts (Fig. 4-17). Prominent rouleaux formation in species other than horses, cats, or pigs should be noted as an abnormal finding.

FIGURE 4-17 Rouleaux formation in blood from a dog with multiple myeloma and a monoclonal hyperglobulinemia. The cytoplasm of a neutrophil present is pale compared to the background that stains blue because of the increased protein present in the blood. Wright-Giemsa stain.

Prominent rouleaux formation results in rapid erythrocyte sedimentation in whole blood samples allowed to stand undisturbed. This characteristic formed the basis of an erythrocyte sedimentation rate test that was done with special sedimentation tubes. Increased sedimentation rates after 1 hour were suggestive of increased globulins in plasma, as typically seen with inflammation. Unfortunately the sedimentation rate increases as the hematocrit decreases, so correction factors were required for the HCT. The sedimentation rate has largely been replaced by making direct measurements of total globulins and fibrinogen and other acute-phase proteins.

Agglutination

Aggregation or clumping of erythrocytes in clusters (not in chains, as in rouleaux) is termed agglutination (Fig. 4-18). Agglutination is caused by the occurrence of immunoglobulins bound to erythrocyte surfaces. Because of their pentavalent nature, IgM immunoglobulins have the greatest propensity to produce agglutination.¹²⁹ EDTA-dependent IgM-mediated erythrocyte agglutination has been reported in a cat without evidence of hemolysis. Agglutination did not occur if blood was collected in heparin or citrate.⁴¹¹ High-dose unfractionated heparin treatment in horses also causes erythrocyte agglutination by an undefined mechanism.^319,322

FIGURE 4-18 Erythrocyte agglutination and spherocyte formation in blood from a dog with von Willebrand’s disease after transfusion. A large basophilic erythrocyte (macroreticulocyte or stress reticulocyte) is present in the upper left corner and an echinocyte is present in the lower left corner. Wright-Giemsa stain.

Polychromasia

The presence of bluish-red erythrocytes in stained blood films is called polychromasia (Fig. 4-19). Polychromatophilic erythrocytes are reticulocytes that stain bluish-red owing to the combined presence of hemoglobin (red staining) and individual ribosomes and polyribosomes (blue staining). Low numbers of polychromatophilic erythrocytes are usually seen in blood from normal dogs and pigs, because up to 1.5% reticulocytes may be present in dogs and up to 1% reticulocytes may be present in pigs even when the HCT is normal.²³⁸ Slight polychromasia may be present in normal cats, but many normal cats exhibit no polychromasia in stained blood films. Polychromasia is absent in stained blood films from normal cattle, sheep, goats, and horses because reticulocytes with sufficient RNA to impart a bluish color are not normally present in the blood in these species.

FIGURE 4-19 Increased polychromasia and anisocytosis in blood from a dog with a hemolytic anemia caused by Mycoplasma haemocanis, although no organisms are present in this field. Three large polychromatophilic erythrocytes (reticulocytes) are present in the central area. A nucleated erythrocyte (metarubricyte) is present in the upper left. Wright-Giemsa stain.

The most useful approach in the classification of anemia is to determine whether or not evidence of a bone marrow response to the anemia is present in blood. For all species except the horse, this involves determining whether absolute reticulocyte numbers are increased in blood. Horses rarely release reticulocytes from the bone marrow even when an increased production of erythrocytes occurs. When an absolute reticulocytosis is present, the animal is said to have a regenerative anemia. The presence of a regenerative response suggests that the anemia results from either increased erythrocyte destruction or hemorrhage. A nonregenerative anemia generally indicates that the anemia is the result of decreased erythrocyte production (Fig. 4-20); however, about 3 to 5 days are required for increased reticulocyte production and release by the bone marrow in response to an acute anemia.^{15,65,146,364} Consequently the anemia appears nonregenerative shortly after hemolysis or hemorrhage has occurred (see Fig. 4-4).

FIGURE 4-20 Blood from a dog with a nonregenerative aplastic anemia secondary to trimethoprim-sulfadiazine therapy. Erythrocyte morphology is normal except for several erythrocytes with scalloped borders (echinocytes). Wright-Giemsa stain.

Increased polychromasia is usually present in regenerative anemias because many reticulocytes stain bluish-red with routine blood stains (see Fig. 4-19). When the degree of anemia is severe, basophilic macroreticulocytes or so-called stress reticulocytes or shift reticulocytes may be released into the blood (Fig. 4-21). High concentrations of erythropoietin shorten the marrow transit time for erythroid cells, resulting in the early release of immature reticulocytes that are twice the normal size.³⁷⁹ There is a direct correlation between the percentage of polychromatophilic erythrocytes and the percentage of reticulocytes in dogs (and presumably in pigs) and between the percentage of polychromatophilic erythrocytes and percentage of aggregate reticulocytes in cats (Fig. 4-22).^15,269 Cats with mild anemia may not release aggregate reticulocytes from the marrow but will release punctate reticulocytes (Fig. 4-23, A).¹⁵ Because punctate reticulocytes do not contain sufficient numbers of ribosomes to impart a bluish color to the cytoplasm, mild regenerative anemia in cats may lack polychromasia in stained blood films (Fig. 4-23, B).

FIGURE 4-21 Two exceptionally large basophilic erythrocytes (macroreticulocytes or stress reticulocytes) are present in blood from a dog with immune-mediated hemolytic anemia. Wright-Giemsa stain.

FIGURE 4-22 Agglutination of aggregate reticulocytes in blood from a cat with a Coombs’-positive hemolytic anemia. A, Agglutination of polychromatophilic erythrocytes and a metarubricyte. Wright-Giemsa stain. B, Agglutination of aggregate reticulocytes. New methylene blue reticulocyte stain. C, Agglutination of aggregate reticulocytes. New methylene blue wet mount preparation.

FIGURE 4-23 Blood from a FeLV-positive cat with a macrocytic normochromic anemia (HCT = 23%, MCV = 70 fL, MCHC = 33 g/dL). A, Low numbers of aggregate reticulocytes but markedly increased punctate reticulocytes (83% uncorrected) are present. Methylene blue reticulocyte stain. B, Increased anisocytosis is present but polychromasia is not apparent, even though most of the blood cells present are punctate reticulocytes, because there is insufficient RNA present to impart a blue color to the cytoplasm of these cells. Wright-Giemsa stain.

Anisocytosis

Variation in erythrocyte diameters in stained blood films is called anisocytosis (see Fig. 4-23, B). It is greater in normal cattle than in other normal domestic animals.²³⁸ Anisocytosis is increased when different populations of cells are present, as can occur following a transfusion (Fig. 4-24). Anisocytosis may occur when substantial numbers of smaller than normal cells are produced, as occurs with iron deficiency, or when substantial numbers of larger than normal cells are produced, as occurs when increased numbers of reticulocytes are produced. Consequently increased anisocytosis is usually present in regenerative anemia (see Figs. 4-18, 4-19, 4-21), but it may be present in some cases of nonregenerative anemia resulting from dyserythropoiesis.¹⁹⁹ Anisocytosis without polychromasia may be seen in horses with intensely regenerative anemia (Fig. 4-25).

FIGURE 4-24 Marked anisocytosis in blood from a 6-week-old kitten after a blood transfusion. The RDW (43%) was also markedly increased. The kitten presented with marked lipemia and a severe iron-deficiency anemia. The small hypochromic erythrocytes are from the kitten and the larger erythrocytes from the blood donor cat. Two lysed erythrocytes (red smudges) are present (left center) because lipemia enhances erythrocyte lysis during blood film preparation. Wright-Giemsa stain.

FIGURE 4-25 Increased anisocytosis in blood from a horse with a regenerative anemia resulting from internal hemorrhage. Horses almost never release reticulocytes in response to anemia; therefore no polychromasia is present. Wright-Giemsa stain.

Hypochromasia

The presence of erythrocytes with decreased hemoglobin concentration and increased central pallor is called hypochromasia (Figs. 4-26 through 4-32). Not only is the center of the cell paler than normal but the diameter of the area of central pallor is increased relative to the red-staining periphery of the cell. True hypochromic erythrocytes must be differentiated from torocytes, which have colorless punched-out centers but wider dense red-staining peripheries (Fig. 4-33).^43,238 Torocytes are generally artifacts. Increased hypochromasia is observed in iron-deficiency anemia.

FIGURE 4-26 Hypochromic erythrocytes in blood from a dog with iron deficiency secondary to chronic blood loss resulting from a persistent flea infestation. Not only is the center of each cell paler than normal but the diameter of the area of central pallor is increased relative to the red-staining periphery of the cell. A polychromatophilic erythrocyte (reticulocyte) is present near the left edge of the image. Wright-Giemsa stain.

FIGURE 4-27 Hypochromic erythrocytes in blood from a dog with iron deficiency secondary to chronic gastrointestinal blood loss. A microcytic hypochromic anemia with poikilocytosis (including keratocytes, schistocytes, and dacryocytes) were present.

FIGURE 4-28 Marked poikilocytosis and hypochromasia in blood from a 6-week-old lamb with microcytic hypochromic iron-deficiency anemia. Wright-Giemsa stain.

FIGURE 4-29 Marked poikilocytosis (primarily dacryocytes) and hypochromasia in blood from a goat with microcytic hypochromic iron-deficiency anemia secondary to chronic blood loss resulting from Haemonchus gastrointestinal parasites. Wright-Giemsa stain.

FIGURE 4-30 Marked poikilocytosis and hypochromasia in blood from a piglet with microcytic hypochromic iron-deficiency anemia resulting from a failure to provide iron injections that are part of the husbandry required in raising piglets on slated floors. Wright-Giemsa stain.

FIGURE 4-31 Microcytic erythrocytes in blood from an iron-deficient llama exhibiting irregular or eccentric areas of hypochromasia within the cells. Wright-Giemsa stain.

FIGURE 4-32 Blood from an iron-deficient alpaca with dacryocytes and spindle-shaped hypochromic erythrocytes. Wright-Giemsa stain.

FIGURE 4-33 Two torocytes (center and bottom left) with colorless punched-out centers and wide dense red-staining peripheries in blood from a dog. Wright-Giemsa stain.

Erythrocytes from dogs, ruminants, and pigs with iron-deficiency anemia often appear hypochromic on stained blood smears. Hypochromasia is less prominent (see Fig. 4-24) and generally not recognized in iron-deficient cats and horses. Hypochromasia in iron deficiency results from both decreased hemoglobin concentration within cells and from the fact that the cells are thin leptocytes (Fig. 4-34, A). Because these microcytic leptocytes have increased diameter-to-volume ratios, they may not appear as small cells when viewed in stained blood films (Fig. 4-34, B).²¹⁰ Microcytic erythrocytes from iron-deficient llamas and alpacas exhibit irregular or eccentric areas of hypochromasia within the cells (see Figs. 4-31, 4-32).³²⁶

FIGURE 4-34 Comparison of discocytes to leptocytes. A, The profile of a normal discocyte is compared to a leptocyte with similar diameter but smaller volume. B, Blood from a dog with a microcytic hypochromic (MCV = 32 fL, MCHC = 23 g/dL) iron-deficiency anemia was mixed with an equal volume of blood from a normal dog (MCV = 70 fL, MCHC = 34 g/dL) prior to blood film preparation. Because the hypochromic cells are leptocytes, they have diameters similar to normal cells even though they are microcytic cells. Wright-Giemsa stain.

From Harvey JW, French TW, Meyer DJ. Chronic iron deficiency anemia in dogs. J Am Anim Hosp Assoc. 1982;18:946-960.

Poikilocytosis

Erythrocytes can assume a wide variety of shapes. Poikilocytosis is a general term used to describe the presence of abnormally shaped erythrocytes. Although specific terminology is used for certain abnormal shapes, it is less important to quantify each type of shape change than it is to determine the cause of the shape change.⁵⁰⁹ Poikilocytosis may be present in clinically normal goats and young cattle (Figs. 4-5, 4-35).^225,409 In some instances, these shapes appear to be related to the hemoglobin types present, but an abnormality in protein 4.2 in the membrane has been suggested as a causative factor in calves.³⁵² Increased numbers of poikilocytes are also present in human preterm and term neonates.⁴⁰³

FIGURE 4-35 Poikilocytosis (acanthocytes and echinocytes) in blood from a nonanemic young calf. Wright-Giemsa stain.

Poikilocytosis forms in various disorders associated with erythrocyte fragmentation, including disseminated intravascular coagulation (DIC), liver disease, myeloid neoplasms, myelofibrosis, glomerulonephritis, and hemangiosarcoma (dogs).³⁹² For unknown reasons, severe iron-deficiency anemia in dogs, pigs, and ruminants may exhibit pronounced poikilocytosis (see Figs. 4-27 through 4-32).²⁰¹ Poikilocytes can form when oxidant injury results in Heinz body formation and/or membrane injury. One or more blunt erythrocyte surface projections may form as the membrane adheres to Heinz bodies bound to its internal surface.³⁹² Various abnormalities in erythrocyte shape were reported in horses with intravascular hemolysis resulting from severe cutaneous burn injuries. Membrane blebbing with fragmentation was prominent (Fig. 4-36).³⁴⁷ A variety of abnormal erythrocyte shapes have been reported in dogs and cats with doxorubicin toxicity^32,348 and in dogs with dyserythropoiesis.²²³

FIGURE 4-36 Echinocytes with membrane blebbing and fragmentation in blood from a horse with intravascular hemolysis resulting from severe cutaneous burn injuries. Wright stain.

Photograph of a stained blood film from a 2004 ASVCP slide review case submitted by E. Spangler, M. Johnson, A. Kessell, B. Weeks, T. Norman, and K. Chaffin.

Echinocytes (Crenated Erythrocytes)

Echinocytes are spiculated erythrocytes in which the spicules are relatively evenly spaced and of similar size.⁵²³ Spicules may be sharp or blunt. When observed in stained blood films, echinocytosis is usually an artifact that results from excess EDTA, improper smear preparation, or prolonged sample storage before blood film preparation. The appearance of the echinocytes can vary depending on the thickness of the blood film (Fig. 4-37). They are common in normal pig blood smears (Fig. 4-38), forming in vitro.²³⁸ The morphology of echinocytes varies from slightly spiculated echinodiscocytes to highly spiculated spheroechinocytes, which have been called burr cells (Fig. 4-39, A). The most advanced echinocytes are those that have lost most of their spicules and have nearly become spherocytes (Fig. 4-39, B). Echinocytes form when the surface area of the outer lipid monolayer increases relative to the inner monolayer.⁴³⁸

FIGURE 4-37 Echinocytes in blood from a dog with histiocytic sarcoma. A, Spicules of similar length are regularly spaced. B, Echinocytes, in a thinner area of the same blood film, appear as erythrocytes with scalloped borders. Wright-Giemsa stain.

FIGURE 4-38 Echinocytes in blood from a normal pig appear as erythrocytes with scalloped borders; consequently the term crenation, from Latin meaning “notched,” has previously been used for echinocytes. Wright-Giemsa stain.

FIGURE 4-39 Echinocytes in the blood of dogs following snake bites. A, Highly spiculated echinocytes (burr cells) in blood from a dog following an Eastern diamondback rattlesnake bite. B, Spheroechinocytes and a lysed erythrocyte “ghost” (bottom center) in blood from a dog following a coral snake bite. Wright-Giemsa stain.

Echinocytic transformation occurs in the presence of fatty acids, lysophospholipids, and amphiphatic drugs that distribute preferentially in the outer half of the lipid bilayer.²⁰⁵ Transient echinocytosis occurs in horses with Clostridium perfringens infection⁵²⁶ and in dogs following rattlesnake (see Fig. 4-39, A),^70,191 coral snake (see Fig. 4-39, B),³⁰¹ water moccasin (Fig. 4-40), and asp viper (Vipera aspis) envenomation,³⁰³ presumably secondary to the action of phospholipases present in venom.⁴⁹⁸ Depending on the time course and dose of venom received, either echinocytosis or spherocytosis may be observed after these snakebites.

FIGURE 4-40 Highly spiculated echinocytes in blood from a dog following a water moccasin bite. Two large polychromatophilic erythrocytes (reticulocytes) are also present. Wright-Giemsa stain.

Echinocytes may occur in uremic animals and immediately after transfusion of stored blood.³⁹² They have been seen with increased frequency in dogs with glomerulonephritis and neoplasia (lymphoma, hemangiosarcoma, mast cell tumor, and carcinoma).^391,523 Echinocytes are the predominant erythrocyte shape abnormality in human burn patients, and this was one of the shape abnormalities recognized in horses with severe cutaneous burn injuries (see Fig. 4-36).^195,347

Echinocytes also form when erythrocytes are dehydrated, pH is increased, ATP is depleted, and intracellular calcium is increased.^31,205 Echinocytosis occurs in horses when total body depletion of cations has occurred (endurance exercise, furosemide treatment, diarrhea, systemic disease).^162,205

Although the mechanisms are not fully understood, ATP is required for the maintenance of normal shape and deformability of erythrocytes.²⁰⁵ Phospholipids are asymmetrically arranged in the plasma membrane, with anionic amino-containing phospholipids (predominantly phosphatidylserine) located in the inner leaflet of the bilaminar membrane. These anionic phospholipids are shuttled (flipped) from the outer leaflet to the inner leaflet by an ATP-dependent aminophospholipid-specific translocase or flippase.⁵⁵³ ATP also provides the energy needed to pump Ca⁺² out of cells. Increased Ca⁺² activates neutral proteases (calpains), which can degrade membrane skeletal proteins; phospholipase C, which cleaves phosphoinositides; and scramblase, which accelerates the bidirectional transbilayer movement of phospholipids.^205,540 The inhibition of the flippase and/or the activation of the scramblase can result in increased phosphatidylserine in the outer layer, which appears to promote echinocyte formation, as well as coagulation and erythrophagocytosis.^116,295,406

Echinocytes and other shape abnormalities have been recognized in dogs with erythrocyte pyruvate kinase deficiency (Fig. 4-41), which results in a decreased ability to generate ATP. ^86,328,412 Occasional echinocytes may also be seen in dogs with erythrocyte phosphofructokinase deficiency (Fig. 4-42). PFK-deficient erythrocytes have decreased ATP and increased Ca⁺² concentrations.⁴⁰⁴

FIGURE 4-41 An echinocyte (center) and four polychromatophilic erythrocytes in blood from a pyruvate kinase-deficient beagle dog. Wright-Giemsa stain.

FIGURE 4-42 An echinocyte (center) and four polychromatophilic erythrocytes in blood from a phosphofructokinase-deficient English springer spaniel dog. Wright-Giemsa stain.

Acanthocytes

Erythrocytes with irregularly spaced, variably sized spicules are called acanthocytes or spur cells (Fig. 4-43).⁴³ Acanthocytes form when erythrocyte membranes contain excess cholesterol compared to phospholipids. If cholesterol and phospholipids are increased to a similar degree, codocyte formation is more likely than acanthocyte formation.⁹⁸ Alterations in erythrocyte membrane lipids can result from increased blood cholesterol content or due to the presence of abnormal plasma lipoprotein composition.⁹⁹ Acanthocytes have been recognized in animals with liver disease (Fig. 4-44), possibly due to alterations in plasma lipid composition, which can alter erythrocyte lipid composition.^91,392,522 They have also been reported in dogs with disorders that result in erythrocyte fragmentation, such as hemangiosarcoma (Fig. 4-45), DIC, and glomerulonephritis.^342,471,522

FIGURE 4-43 Acanthocytes in blood from a dog and cat. A, Two acanthocytes (above) with irregularly spaced, variably sized spicules in blood from a dog with neoplastic lymphoid infiltrates in the liver. B, An acanthocyte in blood of a cat with hepatic lipidosis. Wright-Giemsa stain.

FIGURE 4-44 Acanthocytes in blood from a dog with neoplastic lymphoid infiltrates in the liver. A neoplastic lymphoblast is also present. Wright-Giemsa stain.

FIGURE 4-45 Three acanthocytes, four polychromatophilic erythrocytes, two metarubricytes, and a neutrophil in blood from a dog with hemangiosarcoma. Wright-Giemsa stain.

Marked acanthocytosis is reported to occur in young goats and some young cattle (see Fig. 4-35). Acanthocytosis of young goats occurs as a result of the presence of hemoglobin C at this early stage of development.²⁰⁵