Mary M. Christopher, DVM, PhD, DACVP, DECVCP
University of California Davis, Department of Pathology, Microbiology, and Immunology School of Veterinary Medicine
Davis, CA, USA
Evaluation and interpretation of red blood cell (RBC) morphology is an important component of a complete blood count (CBC). RBC morphology may provide important diagnostic information regarding the underlying cause of anemia and systemic disease. In this presentation, I will discuss the pathophysiologic mechanisms of RBC morphologic alterations in dogs and cats (with an emphasis on shape changes) and their significance in the diagnosis of disease. Red cell shape is a result of both the environment of the cell and its metabolic status. During erythropoiesis and during the 2-5 months spent in circulation, RBCs are exposed to the biochemical milieu of plasma and are reliant on the availability of biochemical precursors needed for membrane and hemoglobin synthesis. Red cell morphology may be altered by alterations in lipid, iron and oxidative metabolism, immune-mediated disease, mechanical fragmentation, and by electrolyte or metabolic abnormalities. Most shape changes result from alteration of the red cell membrane.
The Erythrocyte Membrane
The cytoskeleton helps determine and maintain the shape of red cells through the arrangement and interactions of cytoskeletal proteins. The principal proteins--spectrin, actin, protein 4.1, and ankyrin--form a hexagonal lattice that underlies and attaches to the lipid bilayer via trans-membrane proteins (band 3 and glycophorins). The cytoskeleton delimits membrane deformability and confers the physical properties necessary for normal shape changes during circulation. The lipid bilayer of the erythrocyte membrane is a permeability barrier composed of about equal amounts of cholesterol and phospholipids. Cholesterol is in the free, unesterified form; phospholipids are a mixture of lecithin (phosphatidylcholine), phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol. Phospholipids are asymmetrically distributed within the bilayer, with hydrophobic long chain fatty acids oriented towards the inner core of the membrane. Membrane enzymes depend on the integrity of phospholipids for normal function. Because mature RBCs cannot synthesize lipids, they rely on a continuous exchange of lipids between their membranes and plasma lipoproteins for homeostasis. Movement of free cholesterol between plasma and lipid bilayer is rapid and dynamic; membrane phospholipid turnover with plasma is a passive process that occurs very slowly. Changes in the distribution of the phospholipids may have profound effects on red cell shape and other cell properties.
Quantitative Evaluation of RBC Morphology
RBC indices are quantitative indicators of RBC morphology. The MCV may be increased (macrocytosis) or decreased (microcytosis). If both macrocytes and microcytes are present, the MCV may be normal. Agglutination can falsely increase the MCV. The RDW is a quantitative index of variability in RBC size, and is increased in regenerative anemia and iron deficiency anemia. MCH and MCHC indicate hemoglobin concentration; decreased values indicate hypochromasia. Increased MCH or MCHC is an artifact secondary to Heinz bodies, lipemia, or hemoglobinemia. Some new hematology analyzers determine the percentage and number of hypochromic (and hyperchromic) cells of different sizes.
Qualitative Evaluation of RBC Morphology
RBC morphology is assessed qualitatively on a well-made blood smear and includes RBC distribution, anisocytosis, poikilocytosis, polychromasia/hypochromasia, and inclusions or organisms. Abnormalities are graded as slight, moderate or marked (or 1+ to 4+).
RBC distribution abnormalities include rouleaux and agglutination. Rouleau occurs with decreased negative charges on the RBC membrane caused by increased concentrations of positively-charged proteins in plasma (i.e., fibrinogen, immunoglobins), usually in association with chronic inflammatory disease. Rouleaux also may form normally in feline blood and as an artifact due to slow drying of the blood smear. Agglutination refers to small groups or clumps of RBCs that result from the interaction between bound antibodies on the surface of RBCs.
Anisocytosis is a qualitative measure of variability in RBC size. Like RDW, anisocytosis is increased when microcytic or macrocytic RBC (or both) are present, or when spherocytes are present (which have normal MCV but appear smaller on smears).
Poikilocytosis is a qualitative measure of RBC shape. The type of shape abnormality must be specified. Echinocytes (crenation) and torocytes may result from prolonged storage of blood in EDTA; target cells and stomatocytes may result from slow drying of the blood smear.
Polychromasia refers to the light blue color of immature RBCs (caused by residual RNA). Polychromasia is the most important indicator of responsiveness to anemia, and may be further quantitated by counting reticulocytes. A high percentage of polychromatophilic cells (reticulocytes) may result in an increased MCV and decreased MCH or MCHC.
Hypochromasia refers to pale RBCs with a wide central pallor. Hypochromic cells are almost always also microcytic. Both hypochromasia and microcytosis may be difficult to assess visually, but usually are detectable with changes in RBC indices.
RBC inclusions include basophilic stippling, Heinz bodies, Howell-Jolly bodies and RBC organisms, such as Mycoplasma hemophilus, Babesia canis, and Eperythrozoon spp.
Abnormal RBC Morphology Caused by Hepatic Disease
Abnormal lipid metabolism alters the lipid composition of RBC membranes and reduces RBC deformability. Liver disease is the most common causes of lipid-induced red cell shape changes because of its wide-ranging effects on phospholipid metabolism and plasma phospholipid concentrations. The most common red cell shape abnormalities in dogs and cats with liver disease are echinocytes and/or acanthocytes (spur cells). These cells form when erythrocyte membranes contain excess cholesterol compared to phospholipids (increased cholesterol:phospholipid ratio), due to hypercholesterolemia or abnormal lipoprotein composition. Once produced, the shape change usually is irreversible. Cholesterol enrichment of the membrane expands the outer leaflet of the lipid bilayer, distorts cell contours, disturbs cell functions and accelerates cell destruction. Acanthocyte membranes are enriched with cholesterol by 50% to 70%. Hypercholesterolemia usually is associated with severe cholestatic disease, and is accompanied in dogs and humans by increased serum levels of lipoprotein X as well as other abnormal lipoproteins. Hypercholesterolemia and cholesterol uptake into red cell membranes is further enhanced in hepatic insufficiency and obstructive liver disease by decreased activity of lecithin cholesterol acyltransferase (LCAT), the enzyme responsible for cholesterol esterification, resulting in decreased production of cholesterol esters and a subsequent increase in free cholesterol.
More than 50% of cats with liver disease have poikilocytosis (usually acanthocytes), and serum cholesterol concentrations are higher in cats with more severe poikilocytosis. Feline acanthocytes have few blunt projections and may appear to be misshapen RBCs. Cats with liver disease also often have elliptocytes or ovalocytes, which suggest uncoupling of cytoskeletal proteins from the lipid bilayer secondary to changes in phospholipid composition. Red cell fragmentation (schistocytes, keratocytes, and blister cells) may occur in hepatic insufficiency when microangiopathy develops secondary to decreased coagulation factor production and clearance. Leptocytes and target cells (codocytes) also may be observed in hepatic insufficiency, especially in dogs and cats with portocaval shunts. Target cells result from a balanced excess accumulation of both cholesterol and phospholipids and form later in the course of cholestatic disease (i.e., 1 week later than acanthocytes) because of the slower rate of phospholipid (vs cholesterol) exchange. Target cells also may form because of decreased hemoglobin concentration secondary to altered iron metabolism, which may occur in hepatic insufficiency.
Altered lipoprotein metabolism also can inhibit the maturation of erythrocytes in the bone marrow, at the time at which they are discarding their nuclei and organelles and assuming a flexible biconcave shape. Macrocytic red cells resembling reticulocytes may result that have abnormally high membrane cholesterol content. Dietary lipid also can affect RBC membrane lipid composition and morphology. Animals fed sunflower oil had more target cells and animals fed fish oil had more echinocytes compared with animals fed olive oil. Olive oil is rich in monounsaturated fatty acids, whereas, sunflower oil is rich in linoleic and arachidonic acids (n-6 PUFAs), and fish oil is rich in n-3 PUFAs. Animals fed olive oil had the highest percentage of normal discocytes, suggesting yet another beneficial aspect of this dietary fat.
Abnormal RBC Morphology Caused by Iron Deficiency
Iron deficiency, whatever the underlying cause, results in microcytic hypochromic anemia, often with mild to moderate poikilocytosis. In dogs and cats, iron deficiency most commonly is caused by chronic blood loss from gastrointestinal bleeding (ulcers, neoplasia), severe flea infestation, hematuria (neoplasia) or coagulopathies. Nursing animals also are susceptible to iron deficiency. Anemia is variably regenerative and characterized by microcytosis, hypochromasia (not always in cats), and increased RDW. Poikilocytes seen most often in iron deficiency are target cells, leptocytes, schistocytes, elliptocytes, and dacryocytes. Fusiform cells may be found in birds. Target cells result from excess membrane relative to low intracellular hemoglobin concentration. Other poikilocytes result from membrane lipid and protein abnormalities that decrease deformability and make RBCs more fragile and susceptible to fragmentation and to oxidative stress. Elliptocytes result from abnormalities in spectrin and other membrane proteins and their interaction with the lipid bilayer. Increased RDW may be a sensitive means of detecting early iron deficiency, especially in cats. RDW increases when microcytes and fragments form, and then normalizes. With transfusion or iron treatment, the RDW again increases, as normocytic RBCs mix with microcytes. Animals with portocaval shunts and other hepatic diseases may develop "functional iron deficiency" with similar RBC morphologic changes.
Abnormal RBC Morphology Caused by Immune-Mediated Disease
In immune-mediated hemolytic anemia (IMHA), spherocytes are the sole or primary type of poikilocyte observed. Spherocytes are primarily identified in dogs; they are difficult, if not impossible, to identify in cats. Spherocytes results from antibody binding to the RBC membrane, and subsequent removal of a portion of the membrane by a macrophage, usually in the spleen but also in liver and bone marrow. IMHA may be primary, secondary to neoplasia or other diseases, or may result from RBC organisms such as Mycoplasma Haemophilus. In addition to spherocytosis, RBC agglutination commonly occurs in IMHA. Agglutination may be the only RBC morphologic abnormality in IMHA in cats, and must be differentiated from rouleaux. Other causes of spherocytosis are coral snake envenomation and zinc toxicity. When low numbers of spherocytes are seen with other types of poikilocytes, diseases causing RBC fragmentation are more likely.
Abnormal RBC Morphology Caused by Mechanical Fragmentation
Microangiopathy is the deposition of fibrin strands or microthrombi in small blood vessels. RBCs passing through microangiopathic vessels are prone to mechanical fragmentation. Splenic hemangiosarcoma is a disease that best characterizes microangiopathic fragmentation in dogs. These tumors are essentially giant masses of thrombotic blood vessels and sinuses. The combination of turbulent blood flow through sinuous and rough, neoplastic vessels yields a unique mix of schistocytes, acanthocytes, keratocytes, dacryocytes, blister cells, and a few spherocytes. Anemia is variable in severity, and can be mild or even absent. Other disorders that cause microangiopathy and RBC fragmentation are chronic disseminated intravascular coagulation, heartworm disease, valvular vegetative endocarditis, and glomerulonephritis.
Abnormal RBC Morphology Caused by Oxidative Damage
Oxidative damage to hemoglobin results in Heinz body formation (oxidation of globin chains) and/or methemoglobinemia (oxidation of iron). While methemoglobinemia does not alter RBC morphology, Heinz bodies are visible as spherical, slightly refractile spots on the RBC; they are much more easily seen on new methylene blue-stained smears. Heinz bodies in dogs result in hemolysis, with poikilocytosis (keratocytes, schistocytes, a few spherocytes). Eccentrocytes are another important clue to oxidative damage in canine RBCs and are formed by the adhesion of opposing inner cytoplasmic membranes. Eccentrocytes that have become spherical with only a small tag of cytoplasm remaining are called pyknocytes. Heinz bodies in cats are often larger and usually single, compared with Heinz bodies in dogs. Feline RBCs with Heinz bodies, especially those caused by oxidant drugs, also can undergo hemolysis, such that "ghost cells" are seen on blood smears. RBC fragmentation is not usually seen in feline Heinz body hemolytic anemia, because the spleen does not contribute to RBC lysis. In cats, an increased percentage of Heinz bodies with no history of drug toxicity may reflect diet (e.g., onions, fish) or underlying disease, especially ketoacidotic diabetes mellitus, lymphoma, and hyperthyroidism. In cats with many large Heinz bodies, evaluation for ketosis is recommended.
Abnormal RBC Morphology Caused by Electrolyte and Metabolic Abnormalities
Echinocytes and echinocytic elliptocytes (burr cells) have been reported in dogs with renal disease (glomerulonephritis) and in humans with uremia, where they are thought to reflect changes in the metabolic environment and are associated with decreased red cell lifespan. RBCs in uremic patients have decreased transketolase activity, increased susceptibility to oxidants, and decreased Na/K ATPase activity leading to altered shape, increased rigidity, and susceptibility to mechanical fragmentation. Fragmentation may result in schistocytes and acanthocytes; acanthocytes also may be seen in conjunction with renal disease-induced lipid abnormalities. The biochemical cause of echinocytosis in renal disease is unknown. Echinocytes also have been described in dogs with neoplasia (lymphoma, hemangiosarcoma, mast cell tumor, carcinoma) and in people with sepsis. Echinocytosis has been linked to total body cation depletion in horses with colitis or with furosemide administration, and is associated with. hyponatremia (<136 mmol/L) as well as hypochloremia, low TCO2, hypo-osmolality, and hypocalcemia. Phospholipases in rattlesnake and coral snake venom result in dose-dependent echinocytosis.