The common feature of target cells is an increase in the ratio of the cell surface area to cell volume. In microcytic RBCs of patients with various forms of thalassemia and hemoglobinopathies, the increased surface to volume ratio, and consequently the target cell shape, reflect at least in part the relative abundance of cell surface area. In liver disease and other disorders discussed subsequently, the target cell formation reflects an absolute expansion of the cell sur face area because of a net accumulation of membrane phospholipids and cholesterol.
Liver Disease
The presence of target cells in association with either normal or slightly increased cell volume is characteristically found in patients with obstructive jaundice, including various forms of liver disease associated with intrahepatic cholestasis. These target cells have a nor mal survival in the peripheral circulation and do not typically account for the anemias often encountered in patients with liver disease.
In these patients, target cell formation is a consequence of a net uptake of both free cholesterol and phospholipids into the RBC mem brane from the plasma because of abnormalities in the cholesterol/ phospholipid/protein ratios of low-density lipoproteins. Target cells have a decreased osmotic fragility because the excess of membrane surface area leads to an increase in the critical hemolytic volume.
Lecithin-Cholesterol Acyltransferase Deficiency
The lecithin-cholesterol acyltransferase (LCAT) enzyme catalyzes the formation of cholesterol esters in lipoproteins. It circulates in plasma as a complex with components of high-density lipoproteins. LCAT deficiency, caused by mutations in the LCAT gene, is a rare autosomal dominant disorder manifested by hyperlipidemia, premature atherosclerosis, corneal opacities, chronic nephritis, proteinuria, mild anemia, and the presence of target cells on the blood film. The anemia is caused by mild hemolysis together with a diminished compensatory erythropoiesis. As in obstructive jaundice, the target cells in LCAT deficiency have a marked increase in both cholesterol and phospholipids. In addition, the membrane phosphatidylcholine is increased at the expense of sphingomyelin and phosphatidylethanolamine. Bone marrow aspiration and biopsy reveal the presence of sea-blue histiocytes. Analysis of plasma lipoproteins reveals multiple abnormalities secondary to the underlying enzyme deficiency. Inherited LCAT deficiency should be distinguished from an acquired deficiency of this enzyme, which is found in patients with severe liver disease.
Stomatocytosis and Related Disorders
Stomatocytes were first described in a girl with dominantly inherited hemolytic anemia. On blood films, her RBCs contained a wide transverse slit or stoma (Fig.1). In a three-dimensional view, these cells have a shape of a cup or a bowl. The slit-like appearance is an artifact that results from folding of the cells during blood smear preparation.
Fig1. PERIPHERAL BLOOD SMEARS FROM PATIENTS WITH HEREDITARY XEROCYTOSIS (DESICCYTOSIS) AND STOMATOCYTOSIS (HYDROCYTOSIS). (Top) A Wright-stained peripheral blood smear from a patient with hereditary xerocytosis caused by a PIEZO1 mutation showing rare stomatocytes, occasional dessicytes—dense, abnormal erythrocyte forms where hemoglobin appears puddled at the periphery, and rare target cells. (Bottom) A Wright-stained peripheral blood smear from a patient with hereditary hydrocytosis is shown. Numerous stomatocytes, erythrocytes with a central mouth-like “stoma” are seen.
Stomatocytes are seen in a variety of acquired and inherited disorders. The latter are often associated with abnormalities in RBC cat ion permeability that lead to changes in RBC volume, which can be either increased (hence the designation hydrocytosis or overhydrated stomatocytosis) or decreased (xerocytosis or desiccytosis [dessicate]), or in some cases near normal.
There is no unifying theory to explain this morphologic abnormality. In vitro, stomatocytes can be produced by drugs that preferentially intercalate into the inner half of the asymmetric lipid bilayer, expanding its surface area relative to that of the outer half of the bilayer.
Hereditary Stomatocytosis-Hydrocytosis
Hereditary hydrocytosis designates a heterogeneous group of hereditary hemolytic anemias that are transmitted in an autosomal dominant manner. The disorder is characterized by a moderate to severe hemolytic anemia with 10% to 30% stomatocytes (see Fig. 1), an elevated MCV, and a reduced MCHC. Osmotic fragility of RBCs is markedly increased, as some of the swollen RBCs approach their critical hemolytic volume. For unexplained reasons, RBC membrane lipids and consequently membrane surface area are also increased, but this increase in surface area is insufficient to correct the osmotic fragility of the RBCs. RBC deformability is decreased.
The principal cellular lesion involves a marked increase in intra cellular sodium and water content with a mild decrease in intracellular potassium as a result of a marked sodium influx into the RBCs. Despite a marked compensatory increase in active transport of sodium (Na) and potassium by the Na+/K+-ATPase (which normally maintains the low sodium and high potassium concentrations in the cells) and an ensuing increase in glycolysis, the pump hyperactivity is unable to compensate for the vastly increased sodium leak. Stomatin (also known as band 7.2b), an integral membrane protein, is decreased or absent from the erythrocyte membranes of most affected patients. This deficiency appears to be a maturational loss in the bone marrow and in the circulation, perhaps because of a defect in cellular trafficking. Stomatin gene mutations have not been found in unrelated stomatocytosis patients deficient in this protein.
In some patients with hereditary hydrocytosis, missense mutations in RhAG, I61R, or F65S, have been found. In oocytes, these mutations induce a monovalent cation leak, possibly opening the pore of an ammonium transporter. Additional studies suggest that the F65S mutation exhibits a gain-of-function phenotype with increased cation conductance/permeability.
Splenectomy can improve, but not fully correct, the hemolysis. In some patients, splenectomy can be deleterious or even contraindicated, perhaps because of altered endothelial cell adherence and membrane phospholipid asymmetry.
Hereditary Xerocytosis and the Intermediate Syndromes
Hereditary xerocytosis or desiccytosis describes an autosomal dominant hemolytic anemia characterized by RBC dehydration and decreased osmotic fragility. Affected individuals have characteristically moderate to severe hemolysis with an increased MCHC, reflecting cellular dehydration. Hydrops fetalis with fetal anemia or fetal ascites or the presence of pseudohyperkalemia have been reported in a number of xerocytosis kindred. Frequently, the MCV is mildly increased. In Coulter-type electronic counters, the conversion of pulse height (from the resistance of a cell passing through an electric field) to a cellular volume is dependent on cell shape. Xerocytes do not deform to the same degree as normal cells, causing the MCV to be approximately 10% too high. The peripheral blood film (see Fig. 1) does not always reveal stomatocytes (which are more prominent on wet films), but frequently, target cells, dessicytosis (dessicate), and spiculated cells are seen. In some of the cells, hemoglobin is concentrated (“puddled”) in discrete areas on the cell periphery.
The mechanism of cellular dehydration is unclear and complex, involving a net potassium loss from the cells that is not accompanied by a proportional gain of sodium. Consequently, the net intracellular cation content and cell water are decreased. In some reports, a decrease in RBC 2,3-DPG has also been noted.
Most HX patients have heterozygous missense mutations in PIEZO1. In vitro studies of HX-associated PIEZO1 mutations demonstrate a gain-of-function phenotype, with many mutants demonstrating delayed channel inactivation, indicating increased cation permeability may lead to dehydration of HX erythrocytes. Piezo proteins are putative ion channels mediating mechanosensory trans duction in mammalian cells. Animal models suggest mechanically activated Piezo1 plays a critical role in erythrocyte volume homeostasis. In a small subset of HX patients, defects of the Gardos channel encoded by the KCNN4 gene have been described. Not surprisingly, when PIEZO1 mutations are co-inherited with other inherited anemias manifesting erythrocyte dehydration, such as sickle cell disease, marked erythrocyte dehydration worsens clinical severity.
Some of the reported cases of hereditary stomatocytosis share fea tures of both hereditary stomatocytosis and xerocytosis categorized as “intermediate” syndromes. These patients characteristically have both stomatocytes and some target cells on the peripheral blood smear. Osmotic fragility is either normal or slightly increased. Sodium and potassium permeability is somewhat increased, but the intracellular cation concentration and the RBC volume are either normal or slightly reduced. These cells were reported to have subnormal glutathione content. In some patients, RBCs undergo in vitro hemolysis at 5°C, hence the designation cryohydrocytosis. A similar susceptibility to cold-induced cation permeability in which potassium and water loss predominates and xerocytes instead of hydrocytes are present has also been described.
A study of stomatocytosis, spherocytosis, and spherostomato cytosis patients whose erythrocytes demonstrated significant cat ion leaks at 0°C and in some cases band 3-deficient membranes revealed a series of missense mutations located in an intramembrane domain of band 3. In vitro studies suggest that these mutations convert band 3 from an anion exchanger to a nonselective cation leak channel.
Several investigators have also reported a dominantly inherited hemolytic anemia with stomatocytosis, occasional target cells, spherocytes, and a decreased osmotic fragility in which the main RBC membrane abnormality involved a nearly 50% increase in phosphatidylcholine and a corresponding decrease in phosphatidyl ethanolamine. Because abnormalities in membrane phospholipid composition have not been systematically investigated, it is uncertain whether the disorder represents a distinct disease entity.
The results of splenectomy in this group of disorders are variable. In some patients, the hemolytic anemia is improved, although often not fully corrected, by splenectomy, whereas in others, the severity of the hemolysis is unchanged. Splenectomy should be carefully considered in patients with hereditary stomatocytosis. Several patients with stomatocytosis (both hydrocytosis and xerocytosis) have developed hypercoagulability after splenectomy, leading to catastrophic thrombotic episodes or chronic pulmonary hypertension. Fortunately, the majority of persons with hereditary stomatocytosis are able to maintain an adequate hemoglobin level so that splenectomy is not required.
Rh Deficiency Syndrome
Rh deficiency syndrome designates rare individuals who have either absent (Rhnull ) or markedly reduced (Rhmod ) Rh antigen expression, mild to moderate hemolytic anemia associated with the presence of stomatocytes, and occasional spherocytes on the peripheral blood film. Hemolytic anemia is improved by splenectomy.
The Rh antigens are present in ~20,000 to 30,000 copies per cell and reside on minor transmembrane proteins with an electrophoretic mobility of 28 to 33 kDa on SDS-PAGE. The Rh gene locus encodes two closely linked genes, one encoding the D polypeptide and the other encoding the CcEe proteins, and the antigenic expression of which is a consequence of alternate splicing of their pre-mRNA.
Rh proteins are part of a multiprotein complex that includes two Rh proteins and two Rh-associated glycoproteins (RhAG). Other proteins that associate with this complex include CD47, LW, glycophorin B, and protein 4.2. The Rh-RhAG complex interacts with ankyrin to link the membrane skeleton to the lipid bilayer. The Rh proteins share sequence homology to the Mep/Amt family of ammonium transporters in lower organisms and may participate in ammonium transport.
Rhnull erythrocytes have no Rh antigen and have reduced or absent LW, Fy5, Ss, U, and Duclos antigens. Rh, RhAG, LW, glycophorin B, CD47, and protein 4.2 are also reduced or absent. Rhnull erythrocytes have increased osmotic fragility, reflecting a marked reduction in membrane surface area. These cells are also dehydrated, as indicated by decreased cell cation and water content and increased cell density. The potassium transport and the Na+/K+ pump activity are increased, possibly because of reticulocytosis. Phospholipid asymmetry is also altered.
Although the clinical syndromes are the same, the genetic basis of the Rh deficiency syndrome is heterogeneous, and at least two groups have been defined. The amorph type is caused by defects involving the RH30 locus encoding the RhD and RhE polypeptides. The regulatory type of Rhnull and Rhmod phenotypes results from suppressor or modifier mutations at the RH50 locus. When one chain of the Rh-RhAG complex is absent, the complex either is not transported to or is assembled at the membrane.
Familial Deficiency of High-Density Lipoproteins
Familial deficiency or absence of high-density lipoproteins (Tangier disease) because of mutations in ABCA1, a protein involved in cellular export of cholesterol, leads to accumulation of cholesterol esters in many tissues. Clinical manifestations include large orange tonsils, hepatosplenomegaly, lymphadenopathy, cloudy corneas, and peripheral neuropathy. Reported hematologic manifestations include a moderately severe hemolytic anemia with stomatocytosis and thrombocytopenia. Erythrocyte membrane lipid analyses reveal a low free cholesterol content, leading to a decreased cholesterol/phospholipid ratio and a relative increase in phosphatidylcholine at the expense of sphingomyelin.
Sitosterolemia
Sitosterolemia or phytosterolemia is a recessive disorder associated with elevated plasma levels of plant sterols. Affected patients exhibit xanthomatosis and early-onset premature cardiovascular disease. Reported hematologic manifestations include hemolytic anemia with stomatocytosis and macrothrombocytopenia. Mutations in the trans porters ABCG5 or ABCG8 lead to gastrointestinal hyperabsorption and decreased biliary elimination of plant sterols as well as altered cholesterol metabolism. Plant sterols are not synthesized endogenously in humans but are passively absorbed in the intestine. ABCG5 and ABCG8 actively pump plant sterols out of the intestinal cells back into the intestine and out of liver cells into bile ducts. It has been hypothesized that the stomatocytic phenotype is caused by the intercalation of plant sterols into the inner leaflet of the lipid bilayer.
Acquired Stomatocytosis
Stomatocytes have been noted in diverse acquired conditions, including neoplasms, cardiovascular and hepatobiliary disease, alcoholism, and therapy with drugs, some of which are known to be stomatocytogenic in vitro. In some of these conditions, the percentage of stomatocytes on the peripheral blood smear can approach 100%. However, the clinical significance of this observation is unclear because stomatocytes are absent in most patients with the conditions listed. Furthermore, some stomatocytes can be found in normal individuals (3% to 5%). The most consistent association is that of stomatocytosis and heavy alcohol consumption.
