What you can learn from red cell morphology (Proceedings)

Terms related to RBC size

Anisocytosis variation in cell size, can be either small or large.

Microcyte small cells, decreased MCV. These are commonly seen in iron deficiency or in anemia of chronic or inflammatory disease. Microcytosis is often present in portosystemic shunts. Microcytosis is normal in Akita and Japanese Shiba dogs.

Macrocyte large cells, increased MCV. These are usually immature cells (reticulocytes) seen in regenerative anemias. Mild macrocytic, hypochromic anemia has been observed in Alaskan Malamutes with hereditary chondrodysplasia and stomatocytosis. FeLV infection can cause macrocytosis through altered erythrocyte maturation and is often associated with myelodysplastic or myeloproliferative diseases. Miniature and toy poodles occasionally are recognized with macrocytosis. These dogs may have other changes including hypersegmented neutrophils, multiple Howell Jolly bodies or nRBCs with fragmented nuclei but tend not to have clinical signs (e.g. anemia) associated with these changes. ±2, cobalamin and/or folic acid deficiency can induce a megaloblastic anemia. Cobalamin deficiency has been reported in dogs (Giant Schnauzers, Border Collies, Beagles, and Australian Shepherds) and cats.

Terms related to RBC color

Polychromasia bluish red cells. These are usually large and represent reticulocytes as seen in regenerative anemias.

Hypochromasia pale colored cells with increased central pallor caused by insufficient hemoglobin in the cell. This can be due to iron deficiency or to anemia of chronic or inflammatory disease. Reticulocytes often appear hypochromic because they are undergoing hemoglobinization.

Hyperchromasia cells with increased staining intensity

Terms related to RBC shape

Poikilocytes abnormally shaped red cells. Although this is a general term referring to any abnormally shaped red cell, it if often used to describe cells that can't be specifically described as below.

Acanthocyte (spur cell) spiculated red cell with single or multiple, blunt, rounded projections. These are associated with microangiopathy and fragmentation anemia (dogs especially) and liver disease (more in cats). The latter is due to excess cholesterol:phospholipids ratio in the RBC membrane.

Burr cell red cells that resemble acanthocytes but are usually more elongate with less pronounced surface spikes. Associated with kidney disease.

Crenation (also referred to as echinocytes) red cells with even distribution of sharp spikes. An artifact of old blood, delayed drying of the blood film, improper anticoagulant ratio. Shape change occurs in all cells in given area of blood smear.

Dacrocyte teardrop shaped RBC. Associated with myelofibrosis and myeloproliferative disorders.

Drepanocyte sickled RBC (may be seen in deer, sheep and goats.

Eccentrocyte (pyknocyte) - red cell with condensed hemoglobin in one area of the cell. These occur due to oxidative damage to the hemoglobin.

Echinocyte - spiculated red cell with numerous short, evenly spaced surface projections. Three types exist based on the sharpness of the spicules. Type 3 has a very prickly spine appearance and is associated with snake bite envenomation in dogs. Types 1 and 2 are more blunt, may be due to electrolyte abnormalities, dehydration, or sample or slide artifact (e.g. crenation).

Elliptocyte oval shaped RBC. This is the normal shape for erythrocytes from some non-domestic species including camelid, avian, reptile, and fish erythrocytes.

Ghost cells – Pale or indistinct erythrocytes that have undergone lysis but still retain some membrane.

Keratocyte (helmet cell) - a spiculated red cell with one or 2 pointed projections. It results from the rupture of a vacuole formed near the cell surface and are frequently seen, in association with other fragments, in iron deficiency anemia but can also be seen in other processes of mechanical damage.

Leptocytes thin cells with increased membrane surface are compared to its contents, may appear shaped like target cells, Mexican hats, or folded bowls. Polychromatic leptocytes are generally reticulocytes. If leptocytes are mature red cells, it suggests alteration of cell morphology by disease.

Schistocyte (schizocyte) fragmented red cell. These are associated with intravascular trauma to the erythrocyte, especially due to DIC but also to microangiopathies.

Spherocytes small dense cells lacking central pallor. These are the hallmark of immune mediated anemia and result from excessive loss of surface membrane without the same loss of cellular contents. Thus, the cells will appear smaller on the blood film but have a normal MCV. Spherocytes are easiest to see in dogs. Occasionally small numbers of spherocytes can be seen as the end stage of any process that results in loss of surface membrane, therefore the presence of a few spherocytes should not be considered diagnostic for immune mediated destruction without other supporting data.

Stomatocytes red cells with mouth shaped (oval) areas of central pallor. Hereditary stomatocytosis has been described in Alaskan malamutes and schnauzers. They can be artifacts in the thick area of the smear.

Target cell (codocyte) - is a bell shaped red cell with a dense central area of hemoglobin separated from the outer rim of hemoglobin by a clear area. These have been associated with liver disease. Pseudo or partial target cells, with an incomplete ring of clearing, are seen frequently in regenerative anemias (see leptocyte).

Torocyte (bowl cell) or "punched-out" cells are an artifact. These cells have a large area of central pallor but are not hypochromic. The hemoglobin is sharply demarcated by being pushed to the outside of the cell by a drop of water resting in the bowl of the cell as it dries.

Nonparasitic erythrocyte inclusions

Nucleated red cell (nRBC) immature red cell with a nucleus in peripheral blood. These can be seen during periods of intense marrow stimulation, with bone marrow injury, and after splenectomy. Presence of nucleated RBC without progression to polychromasia suggest lead poisoning. The nucleated count has to be corrected if nucleated red cells are present.

HowellJolly bodies basophilic nuclear remnants usually near edge of red cell. These may be seen with accelerated erythrocyte production. *Seen with Romanowsky stains (see also Heinz bodies).

Basophilic stippling red cells containing residual RNA, appear as multiple, small blue dots with Romanowskystained cells. In other dogs and cats, especially without evidence of regeneration, these suggest lead poisoning or dyserthropoiesis.

Heinz bodies (erythrocyte refractile bodies) round structures (representing denatured hemoglobin) on the internal RBC membrane. They do not stain with Romanowsky stains but are visible as light blue structures with reticulocyte stains such as new methylene blue stain.

Pappenheimer bodies poorly defined structures similar to basophilic stippling (but often lighter blue staining). Represent ironmitochondrial complex, visible with Romanowsky stain and seen with abnormal hemoglobin synthesis.

Crystalline hemoglobin square or rectangular shaped crystals of intense hemoglobin colorization. Uncommonly seen in dogs and cats. Significance is unknown.

Stain precipitate crystalline, refractile structures of various sizes and distribution. Contributing factors are inadequate washing, high humidity, slow drying, or inadequate filtering of stain.

Other red cell terms

Agglutination grape like aggregation of red cells, will not disperse with saline dilution. Associated with immune mediated anemia.

Rouleaux formation grouping of red cells resembling a stack of coins. This is associated with protein abnormalities such as chronic inflammation and multiple myeloma. Rouleaux is more commonly seen in cats than dogs.

Hemoglobinemia - results from intravascular hemolysis due to immune mediated mechanisms, incompatible blood transfusions, hemolyzing toxins (e.g. leptospira and snake envenomation), oxidative insults, zinc toxicity, some parasites, some metabolic disorders (e.g. phosphofructose kinase deficiency) and severe fragmentation processes. The background of the slide will have a pinkish (hemoglobin) appearance. Ghost cells may also be present.

Polychromasia and reticulocytes are both ways to evaluate regeneration. Polychromasia refers to the more basophilic erythrocytes seen on a Wright's Giemsa (or similar) stained slide while reticulocytes are cells that have been stained using a vital stain such as new methylene blue or one of the brilliant cresyl stains. Counting of reticulocytes is more sensitive and will detect more cells (end stage reticulocytes will be similar in size and cytoplasm color to normal mature erythrocytes). Cats have two distinct types of reticulocytes in circulation. The aggregate reticulocyte response is similar to that seen in dogs. The initial reticulocyte response starts about 3 days after the stimulus (e.g. blood loss). Maximum response in the dog and cat usually occurs on the fifth day. Aggregate reticulocytes correspond to polychromatic red cells. Cats also have punctate reticulocytes (cells contain dots and not aggregated RNA). These cells are more mature and smaller than the aggregate reticulocytes and can remain in circulation for 1-3 weeks. It is important in the cat to know if the reticulocyte count includes both aggregate and punctate reticulocytes. The type of reticulocytosis in the cat can therefore help indicate the onset of anemia: aggregate reticulocytosis indicates a recent stimulus (e.g. few days) while punctate reticulocytosis indicates an event 1-3 weeks previously.

Erythrocyte maturation

Iron, copper, and pyridoxine (B6) are required for heme synthesis. A deficiency of iron results in microcytic, hypochromic erythrocytes.

±2, folic acid, and cobalt (as part of ±2) are required for nuclear maturation. A deficiency results in macrocytic erythrocytes.

Riboflavin is required for optimal RBC survival and function.

Specific processes

Fragmentation (microangiopathic hemolytic anemias, DIC)

Red cells are injured by abnormal vasculature (microangiopathy) or fibrin deposited in the vessels (DIC). Thus fragmentation anemia is a special type of intravascular hemolytic anemia. Acanthocytes and fragmented red cells (schistocytes) can be observed on the blood smear. Platelets may or may not be decreased. Causes are variable and include DIC, neoplasia (hemangiosarcoma), vasculitis, burns, heart valve damage and heart worm disease (vena cavae syndrome).

Immune mediated destruction

Immune mediated implies that RBC destruction is due to antibody, complement, or both. It does not imply the underlying cause. Immune mediated can be primary i.e. directed against the animals own red cells (autoimmune) or can be secondary as the result of an immune response against infectious agents or drugs. Infectious organisms (especially some parasites), drugs, viral infections, hormonal imbalance, genetic influences, stress, pregnancy, and vaccination have all been suggested to predispose dogs to immune mediated disease. Antibodies coat the red cells leading to primarily extravascular hemolysis and, less commonly, intravascular hemolysis. Antibodies responsible for the hemolysis and shortened red cell life span come from multiple sources. Spherocytosis and agglutination are the hallmark changes of immune mediated destruction, however agglutination is not seen in all cases and depends on the amount and type of the antibody directed against the patient's erythrocytes.

Oxidative damage

Oxidative compounds affect erythrocytes in three ways. First, Heinz bodies and eccentrocytes can form as a result of denaturation of hemoglobin (specifically to the globin moiety) and leads to destruction of the erythrocytes by both intravascular and extravascular mechanisms. Secondly, the membrane proteins can undergo oxidation. Finally, oxidation of hemoglobin iron results in the formation of methemoglobin. This interferes with oxygen transport but does not cause anemia. Heinz body and eccentrocyte formation may occur simultaneously or independent of methemoglobinemia.

In most cases the oxidant is due to a drug or diet derived compound. Enzyme deficiencies (G6PD, glutathione reductase) are uncommon. Oxidants include phenothiazine, onions, naphthalene, methylene blue, acetaminophen, phenyl compounds, propylene glycol in semi moist cat food, crude oil in birds, and ketoacids especially in cats.

Cats are more susceptible to Heinz body formation because of the large number of sulfur containing amino acids in feline hemoglobin which are readily oxidized. Unstable hemoglobin and increased numbers of Heinz bodies are seen in cats with diabetes mellitus especially with ketoacidosis, hyperthyroidism, lymphoma. Cat spleens are also not as sinusoidal, resulting in less efficient Heinz body removal. Therefore, healthy cats may have some Heinz bodies, especially small ones, present in their blood. Diagnosis of Heinz body anemia requires finding larger numbers of usually large Heinz bodies. Heniz bodies are more readily identified on new methylene blue or brilliant cresyl stains (vital stains used to evaluate reticulocytes)

Iron deficiency or iron sequestration

The unavailability of iron can be due to deficiency (e.g. dietary insufficiency or blood loss) or the inability to appropriately use iron due to sequestration from inflammatory or chronic disease or portovascular anomalies. Classically, iron deficiency anemia is microcytic and hypochromic while sequestration types of anemia start as normocytic normochromic, but both can appear similar. The inability to obtain adequate iron for normal hemoglobin maturation also results in cells that have cytoplasmic fragility. Morphologic changes progress from a 'blister' to a keratocyte to 'apple stem cells'. Thrombocytosis is commonly associated with iron deficiency anemia and iron sequestration disorders due to chronic inflammation. However thrombocytosis is not reported in portovascular anomalies.

FeLV associated anemias

FeLV infected cats may have a non-regenerate normocytic or macrocytic anemia. Macrocytosis without signs of regeneration should raise the index of suspicion form FeLV, however only a subset of FeLV infected cats will have macrocytosis.

Lead poisoning

The anemia is usually mild (can be more severe in chronic poisoning) and is characterized by an inappropriate number of nRBCs and (sometimes) basophilic stippling. Basophilic stippling is most consistent in dogs.

Common RBC parasites of dogs and cats

Babesia: Canine babesiosis is uncommon and occurs mainly in the SW and SE regions of the US. Concurrent infections with Mycoplasma (Hemobartonella) and Ehrlichia have been reported. Feline babesiosis is rarely diagnosed in the US. The organism may be observed in red cells on blood smears. Capillary blood is the preferred blood sample. The organisms are pearshaped (45 um long) and are often in pairs. The organisms of B. canis are larger than B. gibsoni (both canine parasites).

Cytauzoon is often a fatal disease in domestic cats although newer reports suggest a second lesser pathogenic disease process. Morphology the organisms appear as oval signet ring bodies 0.5-2.5 um with a small peripherally located purple nucleus. In about 50% of the cats the parasite cannot be found in the red cells and in the other cats often only 1-2% of the red cells are parasitized. Histologiclly, schizonts can be observed in macrophages associated with endothelium lining venous channels in major organs such as lung, spleen, lymph nodes, and bone marrow. Occasionally, these will be seen on the feather edge of blood films.

Mycoplasma, (previously Hemobartonella spp.) causes an extravascular hemolytic anemia. M. hemofelis is pathogenic in nonsplenectomized cats and often occurs concurrently with FeLV and other diseases. M. hemocanis classically occurs in splenectomized dogs but may also occur in immune suppressed animals. The organism appears on the surface of the cells as small dots, rods, chains, or ring forms which can be confused with stain artifact.