Spherocytes are small, spherical red blood cells (RBC). Spherocytes are approximately two-thirds the diameter of normal RBC. In comparison to normal erythrocytes, they have a decreased surface area to volume ratio. They are more densely hemoglobinized and lack a zone of central pallor. Spherocytes stain deeper red than normal discoid erythrocytes when viewed in Wright’s stained blood smears. Spherocytes are more easily identified in dogs than in other domestic animals (cats, horses) that have smaller erythrocytes with less central pallor. The criteria used for identifying spherocytes in these species could include a small diameter, homogenous appearance, darker staining, and increased osmotic fragility. In thick areas of a canine blood smear and near the feathered edge, all the RBCs may resemble spherocytes. Therefore, spherocytes should only be identified in the monolayer area of the blood film.
Multiple causes of spherocyte formation exist. Spherocytes are classically associated with immune-mediated hemolytic anemias (IMHA), and aid in the diagnosis of this condition. However, the presence of spherocytes on a blood smear is not pathognomonic for this immune-mediated disease. Other pathological disease states can affect erythrocytes and cause spherocyte formation through defective membrane assembly and traumatic or toxic injury to erythrocytes. Some genetic conditions in humans and animals can cause spherocyte formation.
Immune Mediated Hemolytic Anemia
Spherocytes are most commonly associated with immune-mediated haemolytic anaemia. In immune-mediated spherocyte formation, antibodies form either to a foreign antigen resembling the erythrocyte membrane, to a complex of the erythrocyte membrane and a foreign antigen, or to new antigenic sites exposed on the erythrocyte. These antibodies affix to the erythrocytes, identifying them for destruction by macrophages. Macrophages, particularly splenic macrophages, partially phagocytize the erythrocytes causing an excessive loss of surface membrane relative to intracellular contents. The remaining membrane of the red blood cell reforms, resulting in smaller, denser, more spherical cell. These cells have increased osmotic fragility and are less deformable. Deformability is a characteristic needed by healthy erythrocytes in order to traverse the sinusoid filters of the spleen without being removed. Spherocytes are caught in the splenic sinusoids and removed, leading to extravascular hemolytic anemia. In some instances, altered red blood cells are directly opsonized by complement, which contributes to an intravascular hemolytic anemia but not to the spherocytosis.
An underlying trigger for the autoimmunity may often be found and can include blood parasites, previous blood transfusions, vaccination in small animals, or neonatal isoerythrolysis. Toxins such as bee venom, penicillin, cephalosporins, or trimethoprim sulfa may also stimulate the immune system and induce immune-mediated hemolytic anemia. These causes of immune-mediated hemolytic anemia have not been specifically reported to be associated with spherocytosis. In addition, an underlying cause may not always be identified. Intraerythrocytic parasites that have been associated with immune-mediated spherocytosis include rickettsial (Hemobartonella felis, Eperythrozoon spp., Anaplasma marginale) and protozoal (Babesia spp, Cytauxzoon felis) parasites.
Defective Erythrocyte Membrane
The lipid bilayer of erythrocytes is pliable. Deformability is necessary for healthy red blood cells to pass through the microcirculation and splenic sinusoids without premature removal. Anything that disrupts the pliability of the membrane, the interaction of the proteins within the lipid bilayer, or the surface area to intracellular volume ratio will lead to decreased deformability and/or increased osmotic fragility of the cell. The monocytic phagocytes within the spleen recognize these cells as abnormal and remove them. Causes of defective erythrocyte membranes leading to spherocytosis include genetic mutations, hypophosphatemia, toxins, and traumatic injury.
Hereditary conditions exist in which genetic mutations cause a spherocytosis. This has been reported in humans, goats, and certain breeds of cattle but not cats. In Japanese Black Cattle, a genetic mutation causes an intrinsic membrane deficiency of Band 3 that disrupts the interaction of the membrane bilayer proteins. This defect destabilizes the membrane, predisposing the RBC to membrane and surface area loss, leading to spherocyte formation. The Band 3 deficiency leads to hemolytic anemia and premature removal and/or sequestration of the red blood cells by the spleen. Splenomegaly is also common in these cattle, likely due to extramedullary hematopoiesis. The heterozygous forms are mild and compensation usually occurs. The homozygous form typically presents with a severe hemolytic crisis early in life. If the calf survives the initial crisis, the prognosis is favorable.
Phosphorous is essential for both plasma membrane integrity as well as intracellular functions because it is a key element of adenosine triphosphate (ATP). Hemolytic anemia with spherocytosis, increased splenic sequestration and erythrocyte removal has been reported in dogs experimentally induced with hypophosphatemia, and has been clinically reported in diabetic dogs and cats. Hypophosphatemia is not a commonly reported condition in veterinary medicine, but should be considered on a differential list for spherocytosis.
Toxins can directly induce erythrocyte membrane damage and rarely lead to spherocytosis. Oxidative damage to hemoglobin within the erythrocytes and subsequent Heinz body formation is an effect of many toxins. Heinz bodies increase the rigidity of the membrane leading to premature lysis or removal from circulation. Partial phagocytosis or "pitting" of Heinz bodies also may lead to spherocyte formation. Toxins that induce Heinz body anemia include onions (most species), acetaminophen, methylene blue, daily intravenous injections of propofol (cats), and phenothiazines and dried red maple leaves (horses). Copper toxicity has a similar effect in goats. Zinc toxicosis has been reported to cause an acute hemolytic crisis that can be associated with spherocytosis.7 The exact pathogenesis is not clear, but immune-mediated mechanisms are unlikely. The cause is more likely a disruption in the erythrocyte membrane. Chrysotherapy (gold salts) are used to treat immune-mediated diseases, however a potential toxic side effect can be hemolytic anemia with spherocytosis, likely due to increased erythrocyte fragility.
Coral snake envenomation has been reported to cause hemolytic anemia with spherocytosis and echinocytosis. Coral snake venom contains a number of enzymes believed to be important in the development of hemolysis. Specific to the associated spherocytosis, coral snake venom contains phospholipase A. Phospholipase A is an indirect hemolysin which can react with the red blood cell membrane structure, increasing fragility and causing surface area loss with subsequent spherocyte formation. It is possible that this is also part of the mechanism of spherocytosis caused by bee venom, which also contains phospholipase A.
Trauma to Erythrocytes
Microangiopathic conditions such as disseminated intravascular coagulopathy (DIC) may cause spherocytosis. In DIC, fibrin strands form within the microvasculature. As the red blood cells pass through capillaries, they are caught by the fibrin strands and fragmented. The remaining portions of the erythrocytes may reform into spherocytes or may remain fragmented (schistocytes).
While IMHA is a common cause of spherocytosis, visual identification of spherocytes in the blood smear or mention of their presence on a laboratory report should alert the clinician to other possible diagnoses. Thus, the differential diagnosis for spherocytosis includes not only the multiple causes of IMHA, but also the pathologic membrane defects created by genetic mutations, nutritional deficiencies, toxins, and erythrocyte trauma.
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