Hereditary spherocytosis (HS), is an important, usually autosomal dominant hemolytic anemia, in which defects in spectrin or the proteins that attach spectrin to the membrane (ankyrin, band 3, protein 4.2) lead to spheroidal, osmotically fragile, often spectrin-deficient cells that are selectively trapped in the spleen and that survive almost normally after splenectomy.
HS was first described more than 120 years ago by the Belgian physicians Vanlair and Masius.506 They reported on a young woman in whom recurrent abdominal pain developed over her enlarged spleen, with associated prostration, vomiting, jaundice, anemia, and muscular weakness. At the time of this attack (presumably a hemolytic crisis), the authors noted that the majority of the red cells were spherical and much smaller than normal. They termed these cells “microcytes” and named the disease “microcythemia.” The unstained cells were illustrated in a lithograph drawn and tinted by Vanlair (Fig. 183-18). The drawing clearly shows spherocytosis, although the relatively large number of elliptocytes (19 percent of the evaluable cells) raises the question whether the true diagnosis may not have been spherocytic elliptocytosis. Later, when the patient had improved, her red cells were somewhat larger, but still abnormal, and her spleen remained enlarged.
Lithograph of normal blood cells (A) and cells from a patient with “microcythemia” (B) described by Vanlair and Masius in 1871.506
Vanlair and Masius thought the microcytes were senile normal cells (“globules atrophiques”) and that the spleen assisted in their aging. They argued that when red cells are sequestered in the pulp of the spleen, they are removed from the active circulation, lose volume, and become dense, spherical, and microcytic. They believed an enlarged spleen produces even more of such cells than a normal spleen and that the liver completes the work of the spleen by destroying the microcytes it receives via the splenic vein. They suggested that the large number of microcytes in their patient was due in part to splenomegaly and in part to atrophy of the liver. Finally, they noted that the patient's older sister had suffered from an identical illness and had died during an apparent crisis. The mother was also subject to jaundice.
This remarkable paper must rank among the most prescient in hematology. Not only did the authors describe the first example of a hereditary hemolytic anemia well before the microscope was in general use in the analysis of blood diseases, but their deductions concerning the pathophysiology, particularly the role of the spleen, predated Ham and Castle's concept of erythrostasis507 by more than two-thirds of a century. Their analysis is placed in better perspective when one realizes that 40 to 65 years later HS was ascribed to causes as diverse as hereditary syphilis508 and splenic hemolysins.509
Unfortunately, Vanlair and Masius' report and subsequent descriptions of HS by Wilson and Stanley in the 1890s510,511 went largely unnoticed. The latter authors clearly recognized the hereditary nature of the disease and were the first to describe the pathology of the spleen, which at autopsy was grossly firm and dark and microscopically engorged with red cells.
A report by Minkowski in 1900512 received wide attention, and additional papers soon appeared,513,514 including Chauffard's historic definition of osmotic fragility515 and reticulocytosis516 as hallmarks of the disease. At about the same time, Widal et al.517 differentiated an acquired form of “congenital hemolytic jaundice” (now recognizable as Coombs' test-positive immunohemolytic anemia). Because Hayem had previously reported similar cases,518 the acquired form of the disease soon became the Hayem-Widal type, while the congenital form was given the eponym Minkowski-Chauffard.
The use of splenectomy was soon advocated, and in 1911 Micheli519 removed the spleen from a patient with acquired hemolytic jaundice. The fortunately brilliant result, combined with the subsequent success of splenectomy in the congenital disease,520 soon led to widespread acceptance of the procedure. Actually, the first successful splenectomy for HS was unintentionally performed by Spencer Wells in England in 1887 (3 years before Wilson's description of the disease in that country).521 Operating on a jaundiced woman for a supposed uterine fibroid, he instead encountered and removed an enormous spleen. The patient recovered and the jaundice disappeared. Forty years later Lord Dawson restudied the woman and her son and found the characteristic osmotic fragility.521
Thus, by the time of Tileston's522 and Gänsslen's523 reviews in 1922, almost all the major clinical features of HS were documented, the spleen was thought to be involved in the hemolysis, and splenectomy was known to be curative. Nevertheless, with the exception of Vanlair and Masius' farsighted (and still unrecognized) premonitions, nothing substantive was known about the basic mechanism of the disorder or its pathogenesis. These aspects of the disease will be discussed in the sections that follow.
HS is the most common hemolytic anemia in people of Northern European extraction. In this population the prevalence is roughly 1 in 5000,524 and there is evidence, based on data obtained with sensitive diagnostic tests, that very mild forms of the disease may be much more common.525,526 The disease occurs, but is less frequent, in other races and ethnic groups; this may reflect poor ascertainment or a true difference in incidence.
Approximately 75 percent of HS families have an autosomal dominant inheritance pattern.524 Homozygotes for dominant HS are rare. In the few reported cases the parents have had a mild phenotype,527– 529 which suggests that homozygosity for typical dominant HS is probably incompatible with life.530 There is nearly complete penetrance of the dominant disease, but clinical variability is common.
Approximately 25 percent of HS occurs sporadically in patients without a family history of spherocytosis. About half of these cases are de novo examples of dominant HS.531– 533 Parental mosaicism occurs and must be considered in genetic counseling.534 The rest are likely to be examples of a recessive form of the disease, caused by coinheritance of two genetic determinants, one from each of the patient's parents. Patients with recessive HS are often more severely affected,535,536 but there is also considerable clinical variability.
The first clue that pointed to a genetic locus for congenital spherocytosis was the identification of HS families with balanced translocations involving the short arm of chromosome 8.537,538 Chilcote and his colleagues also identified two sisters with moderate to severe splenectomy-responsive anemia, spherocytosis, dysmorphic features, micrognathia, nystagmus, psychomotor retardation, and deletion of chromosome bands 8p11.1-p21.2.539 Together, these observations provide evidence for a genetic locus for congenital spherocytosis in the proximal part of 8p. The nature of this HS locus was clarified when it was shown that the gene for ankyrin resides in this region and that the individuals reported by Chilcote, and another unrelated patient with a similar condition, lacked the ankyrin gene on the abnormal chromosome.196 This conclusion was supported by genetic analysis of a large kindred with typical autosomal dominant HS and no chromosome deletion. Spherocytosis was linked to an RFLP in the ankyrin gene, while linkage to other candidate genes (α-spectrin, β-spectrin, or protein 4.1 genes) was excluded.540
In an analysis of 15 other families with HS, Kimberly et al. observed a weak association of HS with the immunoglobulin Gm type, which is located on 14q34.537 This observation suggested that the cause of HS is heterogeneous and that there is at least one other genetic locus. This turns out to be true. The second genetic locus is probably the β-spectrin gene, which was later mapped to chromosome 14q23-q24.2.541 As will be shown, other loci involve the genes for band 3, protein 4.2, α-spectrin, and, possibly, β-adducin.
The characteristic clinical features of HS are pallor, jaundice, and splenomegaly. The presence of spherocytes in peripheral blood smears is almost the sine qua non of the disease, but HS must be distinguished from other nonhereditary causes of spherocytosis. A positive family history is often present, particularly in dominant HS. The disease typically presents in infancy or childhood, but may present at any age.542
HS frequently presents as jaundice in the first few days of life.543,544 The combination of hemolysis and the reduced capacity of the neonatal liver to conjugate bilirubin can cause serum concentrations of unconjugated bilirubin to rise rapidly. Because there is a risk of kernicterus,543 exchange transfusions are sometimes necessary, though usually phototherapy suffices. Mild anemia is common at this time, but severe anemia occasionally occurs. There is no evidence that patients with HS who are symptomatic as neonates have a more severe form of the disease. Indeed most become asymptomatic within the first few weeks of life. However, some infants become progressively more anemic during the first few months of life and require transfusion. In our experience, this usually occurs because the marrow response to anemia is more sluggish than normal. Fortunately, the problem is usually transient and remits after one or two transfusions, except in the rare patients with severe HS. It is not known if erythropoietin would work, and avert transfusion, but this possibility should be formally tested. The subsequent course of the disease depends on the equilibrium established between the rates of red cell production and destruction. An interesting recent observation points out that the presence or absence of the Gilbert syndrome variation (see Chap. 125),545 situated in the promoter of the UDP-glucuronyl transferase gene, significantly influences whether an infant with HS has neonatal jaundice.546
Once beyond the neonatal period, patients with mild HS typically have a hemoglobin of 11 to 15 g/dl, a reticulocyte count of 3 to 8 percent, and a serum bilirubin of 1 to 2 mg/dl.531 A surprisingly large numbers (≈20 to 30 percent) have balanced red cell production and destruction and no anemia.547,548 These individuals are said to have “compensated” hemolysis. They are often asymptomatic, and in some cases diagnosis may be difficult, because hemolysis, spherocytosis, and splenomegaly are usually mild. Many of these patients are diagnosed during family studies or are discovered as adults when splenomegaly or gallstones are detected or when transient episodes of jaundice appear.
Hemolysis may become severe with illnesses that cause the spleen to enlarge, such as infectious mononucleosis.549 Hemolysis is also exacerbated by pregnancy550,551 and by intensive physical effort, to the point at which athletic performance in endurance sports may be impaired, even in patients with mild disease.552 In between these occasional hemolytic episodes, the patients again become asymptomatic.
Although HS is usually consistent within families, mild cases may also occur in families with more severely affected members.547 Presumably this is due to the inheritance of modifying genetic factors, such as those affecting splenic function or the expression of either the normal or mutant allele of the disease gene. The precise nature of these factors remains to be elucidated.
One of the interesting mysteries is why patients with compensated hemolysis continue to have erythroid hyperproduction when their hemoglobin levels are normal. The phenomenon is difficult to reconcile with the generally accepted theory that erythropoiesis is controlled by tissue hypoxia. One possibility is that the concentration of 2,3-DPG, which is low in hereditary spherocytes prior to splenectomy,553 increases oxygen affinity and promotes erythropoiesis. An alternative explanation can be found in recent studies which show that serum erythropoietin levels are elevated up to eightfold in HS patients with compensated hemolysis, compared with normal subjects who have similar hemoglobin levels.554,555 The force driving this overproduction of erythropoietin is unknown. One untested possibility is that the dehydrated HS red cells poorly perfuse the erythropoietin-producing juxtaglomerular cells of the kidney, due to their relative rigidity. If true, then erythropoietin production at any hemoglobin level should correlate inversely with red cell deformability, and compensated hemolysis should mostly occur in disorders with dehydrated erythrocytes (e.g., HS, sickle cell disease, Hb CC disease, and hereditary xerocytosis).
The majority of HS patients (≈60 to 75 percent) have moderate disease with incompletely compensated hemolysis and mild to moderate anemia. By definition, baseline hemoglobin concentrations are 8- to 12 g/dl, reticulocytes are ≥8 percent, and bilirubin levels are usually ≥2 mg/dl.531 Intermittent mild jaundice is common, particularly in children, often associated with viral infections, and is presumably due to reticuloendothelial stimulation and an increase in hemolysis. The spleen is palpable in about 50 percent of these patients during infancy and childhood and in 75 to 95 percent during adult life.547,556,557 Splenomegaly is usually modest, but it may be massive.521,522,558,559 There is no published evidence that the size of the spleen correlates with the severity of HS, although such a correlation probably exists, considering the pathophysiology of the disease.
A small proportion of HS patients (≈5 to 10 percent) have severe hemolytic anemia. Their hemoglobin concentrations are ≥6 to 8 g/dl, reticulocytes are ≥10 percent, and bilirubin concentrations are usually 2 to 3 mg/dl or more.531 A subset of these patients (<5 percent) have life-threatening disease and are transfusion-dependent.535 In this group particularly, acanthocytes, irregular, shrunken spherocytes, and other bizarre forms may be seen in addition to typical spherocytes.535 These individuals may present diagnostic difficulties if transfusions are begun before HS is diagnosed, because in the most severe cases the abnormal cells may be destroyed so rapidly that only transfused cells are available for testing. Besides the risks of recurrent transfusions, these patients occasionally suffer from complications of their chronic severe anemia, such as growth retardation, delayed sexual maturation, and frontal bossing or other changes in the facial bones similar to those observed in thalassemia.558,559 Most of these severely affected patients have the recessive form of HS. Unlike typical dominant HS, anemia may not be completely eliminated by splenectomy in these patients.535,560
The first clue to the diagnosis of HS is usually the characteristic morphological change of red cells on peripheral blood smears. Other laboratory findings include those common to all hemolytic processes: an increased number of reticulocytes, a slight to moderate rise in indirect bilirubin in the plasma, an elevated fecal excretion of urobilinogens, and hyperplasia of erythroid precursors in the bone marrow. Plasma hemoglobin is normal and haptoglobin is only variably reduced, because most of the red cells are destroyed extravascularly. Laboratory tests that distinguish HS from other causes of hemolysis usually measure the biophysical properties of the affected red cells, among which the osmotic fragility test is the standard test. Biochemical analysis and DNA analysis of the membrane skeleton proteins in HS, done mainly in a few research laboratories, allow determination of the primary molecular defects underlying HS.
Spherocytes are the hallmark of HS. They are dense, round and hyperchromic, lack central pallor, and have a decreased mean cell diameter. They are almost always present on smears from patients with moderately severe HS, but are subtle in about 25 to 35 percent of patient with mild HS.531,547,557 While hereditary spherocytes appear spheroidal in conventional dried smears, most are actually thickened diskocytes or spherostomatocytes when examined in the scanning electron microscope.561 Although the morphologic defect is mostly acquired in the circulation, and HS erythroblasts are morphologically normal, emerging reticulocytes are more dehydrated and more spherical than normal reticulocytes, implying that, assembly of HS membranes during erythropoiesis is defective.562
The various subtypes of HS, discussed below, have subtle differences in red cell morphology. Patients with ankyrin defects and combined spectrin and ankyrin deficiency, the most common subtype,23,563,564 have typical round spherocytes and microspherocytes (Fig. 183-19; panel A). Most patients with band 3 deficiency also have rare (0.2 to 2.3 percent) mushroom-shaped (or “pincered”) red cells in their peripheral blood smears (panel B).565,566 Patients with more severe spectrin deficiency have proportionally more misshaped spherocytes, spiculated red cells, and bizarre poikilocytes, which may dominate the blood smear in the most severe cases (panel C).535,567 Combinations of spiculated red cells (acanthocytes) and spherocytes are observed in HS due to defects in β-spectrin (panel D).461,568,569 Red cell morphology is variable in protein 4.2 deficiency and may even be normal.570 Most patients appear to have mild spherocytosis, but acanthocytes, poikilocytes, and ovalostomatocytes are sometimes observed.571 Oblong spherocytes, combined with elliptocytes, suggest spherocytic HE, which is most often associated with truncated β-spectrin chains.
Blood films from patients with different types of HS. A. Typical HS with a mild deficiency of red cell spectrin and ankyrin. Although many cells have a spheroidal shape, some of them retain a central concavity. B. HS with a small number of mushroom-shaped red cells (arrows), the typical picture in band 3 deficiency. Occasional spiculated cells are also present. C. Severe HS with marked spectrin and ankyrin deficiency. In addition to spherocytes, many red cells are misshaped and have irregular contours. D. HS associated with a β-spectrin mutation. Echinocytes and acanthocytes (5 to 15 percent) are usually observed in addition to typical spherocytes. (From Palek and Jarolim.3 Used by permission.)
There are characteristic changes in the red cell indices associated with HS. The mean corpuscular hemoglobin concentration (MCHC) is increased, owing to mild cellular dehydration, and exceeds the upper limit of normal (36 gm/dl) in about half of patients.547 Mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) may fall within the normal range,547 because young red cells, which are increased in HS patients with ongoing hemolysis and which have a high cell volume, skew the distribution. However, the MCV is usually at the lower end of the normal range and sometimes is low. In addition the reticulocyte MCV is also low, which contrasts with the spherocytosis of autoimmune hemolytic anemia, where reticulocyte indices are normal and total cellular MCV is increased.562 The profile of the volume and hemoglobin concentration of individual red cells can now be measured with automated blood counters using dual angle laser light scattering (Technicon H-1).572 In HS blood samples, there is often a right shift of the hemoglobin concentration curve due to red cell dehydration, and a broadening of the volume curve, due to the mixture of small microspherocytes and large young erythrocytes (Fig. 183-20).573,574 Similar diagnostic information can be obtained from the data generated by aperture impedance (Coulter) analyzers.575 The combination of a high MCHC, a widened red cell distribution width (RDW), and shifts in distribution curves is often enough to suggest a diagnosis of HS.575 Studies also show that in nonsplenectomized HS patients, the percentage of microcytes best reflects the severity of the disease, whereas the percentage of hyperdense cells best discriminates HS patients from normal individuals.574 The presence of hyperdense cells is, however, not specific to HS, as it is also seen in other disorders such as Hb SC and Hb CC disease, hereditary xerocytosis, and autoimmune hemolytic anemia.
Representative volume and hemoglobin concentration profiles of blood samples from a normal individual (A) and two different HS patients (B and C), obtained using a Technicon H-1 counter. The signposts in the volume distribution separate microcytic (<60 fl) and macrocytic (>120 fl) cells from normocytic red cells (60 to 120 fl), whereas the sign posts in the hemoglobin concentration distribution separate hypochromic (<28 gm/dl) and hyperdense (>41 gm/dl) cells from normochromic red cells (28 to 41 gm/dl). In B, the right shift of the hemoglobin concentration indicates cell dehydration. In C, the left shift of the volume distribution and the broadening of the hemoglobin distribution indicate the presence of both microcytic and dehydrated red cells. (From Cynober, Mohandas, and Tchernia.574 Used by permission.)
The osmotic fragility (OF) test is the most useful test generally available for the diagnosis of HS. The test is performed by suspending the cells in buffered saline solutions of various concentrations.576 In hypotonic solutions, red cells swell until they become spheres and then burst. Cells with a decreased surface-to-volume ratio, such as hereditary spherocytes, can tolerate less swelling than normal. A curve plotting the percentage of hemolysis at different salt concentrations documents the osmotic fragility profile of the red cells. With HS erythrocytes, there is a shift of the curve away from the direction of hypotonicity, indicating increased osmotic fragility of the cells (Fig. 183-21). For nonsplenectomized HS patients, the osmotic fragility curve shows a major population of cells only slightly more spheroidal than normal and a “tail” of very fragile cells representing a minor population of hyperchromic microspherocytes. The microspherocytes result from “splenic conditioning,” a topic that is discussed in more detail in a later section. Increased osmotic fragility is also observed in autoimmune hemolytic anemia, whereas red cells from patients with thalassemia and sickle cell disease may have decreased osmotic fragility.
Osmotic fragility in HS. In the osmotic fragility test, red cells are suspended in salt solutions of varying tonicities between isotonic saline (0.9 g/dl NaCl; left) and distilled water (right). Normal red cells swell in hypotonic media and eventually reach a limiting spherical shape, beyond which they hemolyze. Spherocytes, which begin with a lower than normal surface/volume ratio, can swell less before they reach their limiting volume; hence, their osmotic fragility curve is shifted to the left (higher salt concentrations). The spherocytes are said to be osmotically fragile. Before splenectomy, a small proportion of the red cells are extra fragile and produce a tail on the osmotic fragility curve. These cells have been conditioned by the spleen, as shown by their higher concentration in the splenic vein and especially in the splenic cords.576 (From Lux and Palek.4 Used by permission.)
In about 25 percent of HS patients, particularly the mildly affected, the OF test lies within the normal range.556 In most of these cases, the disease is revealed by the “incubated” OF test. Whole blood is preincubated under sterile conditions at 37°C for 24 hours before the OF test is performed. During the incubation, hereditary spherocytes become metabolically depleted and lose membrane surface more rapidly than normal cells, which accentuates their spheroidicity and enhances the sensitivity of the test. HS patients with a normal incubated OF have been described,577 and such patients may be even more common than reported,574 but the incubated OF test remains the gold standard in the diagnosis of HS.
Because the OF test is cumbersome to perform and requires 2 ml or more of whole blood, a one-tube glycerol lysis test has been developed.578 In this test, red blood cells are incubated in a glycerol-sodium phosphate-buffered hypotonic saline solution. The glycerol slows the entry of water into the cells, so that the time for the cells to lyse is prolonged and can be measured accurately. The glycerol lysis time is shortened for hereditary spherocytes because of the reduced surface-to-volume ratio. The sensitivity of the glycerol lysis test is enhanced by using a low pH577 or by a 24-hour preincubation,579 and subsequent modifications of the method have improved the sensitivity, reproducibility, and accuracy.526,580 The simplicity of the glycerol lysis test and its sensitivity make it an excellent test for rapid screening of large numbers of micro blood samples.526
The autohemolysis test was originally described by Ham and Castle.507 Incubation of red cells in their plasma for 48 hours results in autohemolysis, and the rate of autohemolysis is increased in red cells from HS patients. In general, the autohemolysis test lacks specificity but is relatively sensitive, and is occasionally useful in confirming the diagnosis of mild or sporadic cases of HS.
Hypertonic Cryohemolysis Test.
The hypertonic cryohemolysis test is a relatively new method.581 Spherocytes are particularly sensitive to cooling at 0°C in hypertonic conditions, which causes them to lyse and release their hemoglobin. This is the basis for the test. Hypertonic cryohemolysis is said to be more sensitive than other tests, especially in mild cases of HS,582 but it can also be positive in some patients with other hemolytic anemias, and with elliptocytosis and some forms of congenital dyserythropoietic anemia.583 The advantages of the test include its simplicity, high sensitivity, and the ability to use EDTA-preserved blood samples obtained for routine hematological studies. However, the hypertonic cryohemolysis test is still new and its limitations are not yet fully understood. As an example, most of the published studies using the test were done in adult HS patients, so the test still needs be validated in children.
Ektacytometry, described above in the section on HE/HPP, is the best available method to measure the biophysical properties of intact erythrocytes.584 “Osmotic gradient ektacytometry,” a modification of the original technique, is particularly useful in the diagnosis of HS. In this method, the deformability index of the red cell sample is measured as a function of the osmolarity of the suspending medium, which is continuously varied (Fig. 183-22). The osmolality of the suspending medium at which the red cell deformability index reaches a minimum is the same as the osmolarity at which 50 percent of the red cells hemolyze in an osmotic fragility test.585,586 This value indirectly measures the surface area-to-volume ratio of the red cells. The maximum deformability index of the curve quantitatively reflects the surface area of the red cells.574 In patients with HS, there is a shift of the curve to right, demonstrating the reduced surface area-to-volume ratio of the cells, and a decline in the height of the curve, reflecting a decrease of the absolute surface area. Even though this technique is only available in a small number of laboratories at present, it provides useful diagnostic information that cannot be obtained otherwise, and deserves greater availability. Cynober and coworkers, for instance, showed that increased osmotic fragility is only detected in 66 percent of nonsplenectomized HS patients, whereas decreased membrane surface area can be demonstrated in all the patients by osmotic gradient ektacytometry.574
Osmotic gradient ektacytometry of red blood cells with varying degrees of spectrin deficiency. In the spectrin-deficient cells, the minimum deformability index observed in the hypotonic region (thin arrow) is shifted to the right of the control (shaded area), indicating a decrease in the cell surface area-to-volume ratio. The maximum deformability index (DImax) associated with the spectrin-deficient cells (thick arrow) is less than that of control cells, implying reduced surface area. The more pronounced the spectrin deficiency, the greater is the loss of surface area and the lower is the DImax. The osmolality in the hyperosmolar region at which the DI reaches half its maximum value is a measure of the hydration state of the red cells. It is decreased in the patient with the lowest spectrin content, indicating cellular dehydration. (Adapted from Chasis, Agre, and Mohandas.585 Used by permission.)
Membrane Protein Analysis.
Cross-transfusion experiments have clearly shown that defects in HS are intrinsic to the red cells.576,587 Many of the abnormalities formerly described in HS red cells are now believed to be secondary and do not present primary hereditary defects. These include metabolic derangements, alterations in cation transport, abnormal membrane protein phosphorylation, and altered membrane lipid composition.588 More recent studies have shown that the primary defects in HS lie in the membrane skeleton proteins, as is the case in HE/HPP. However, the pathophysiology differs. HS involves proteins that attach the skeleton to the membrane (ankyrin, protein 4.2, and band 3), while HE/HPP affects the bonds that hold the skeleton together. Spectrin is the only protein that causes both disorders. But the spectrin defects that cause HS lead to spectrin deficiency, while those that cause HE/HPP affect the function of spectrin, especially self-association, as previously discussed. Similarly, the defects in ankyrin, protein 4.2 and band 3 that cause HS are usually associated with absence of the mutant protein rather than a functionally defective mutant.
Combined Spectrin and Ankyrin Deficiency.
Using a sensitive radioimmunoassay (RIA), Agre and coworkers first reported that spectrin is deficient in patients with HS.560,589 The degree of spectrin deficiency correlates closely with the severity of the disease and with the degree of spherocytosis and osmotic fragility. The degree of spectrin deficiency also predicts the patient's status postsplenectomy, as judged by reticulocyte count, haptoglobin level, and hematocrit.560 Other studies confirm that spectrin deficiency is a prominent feature in most patients with HS, and accounts for the decreased membrane mechanical stability and the loss of surface area associated with the disease (Fig. 183-23).531,585 However, more recent evidence shows that the spectrin deficiency is usually secondary to other skeletal protein defects.
A. Membrane spectrin content and mechanical stability. The decrease in membrane mechanical stability is proportional to the decrease in membrane spectrin content as measured by RIA for individuals homozygous (solid circles) or heterozygous (open circles) for nondominant HS. The data for individuals with dominant HS (solid triangles) fall on the same curve in between these two groups. B. Membrane spectrin content and mean cell surface area. The decrease in cell surface area is similarly proportional to the decrease in membrane spectrin content in these groups of patients. (From Chasis, Agre, and Mohandas.585 Used by permission.)
Coetzer and her colleagues first described two patients with an atypical, severe form of spherocytosis, who were deficient in both spectrin and ankyrin. The clinical picture of these patients was characterized by transfusion-dependent hemolytic anemia, marked spherocytosis, bizarre poikilocytosis, and only a partial response to splenectomy.567 The authors showed that red cell membranes from the patients were deficient in both spectrin and ankyrin. Each was reduced to about 50 to 60 percent of normal. They studied the synthesis, assembly and turn-over of spectrin and ankyrin in the reticulocytes in one of the patients and found that a defect in ankyrin synthesis was the primary abnormality.
Other studies showed that combined spectrin and ankyrin deficiency is not limited to these atypical patients but is common in patients with typical HS.23,563 Savvides and coworkers used RIA to measure the spectrin and ankyrin content in 20 kindreds with dominant HS.23 Spectrin and ankyrin levels were less than the normal range in 75 percent and 80 percent of the kindreds, respectively, and, with one exception, the degree of deficiency of the two proteins in each kindred correlated strictly with each other (Fig. 183-24). Pekrun and coworkers used an enzyme-linked immunosorbent assay (ELISA) to measure the ankyrin and spectrin contents in erythrocytes of 45 patients with typical HS.563 They found concomitant deficiency of both proteins in all of the patients, and showed that the degree of deficiency is proportional to the clinical severity.
Correlation of spectrin and ankyrin deficiencies in 20 dominant HS kindreds. Each data point represents the mean value for a kindred for both spectrin and ankyrin levels, expressed as a percentage of control. Nineteen kindreds (solid circles) showed very similar degrees of spectrin and ankyrin deficiencies. One otherwise typical HS family (open circle) has marked ankyrin deficiency and a relatively mild spectrin deficit. (From Savvides, Shalev, John, and Lux.23 Used by permission.)
Several surveys have used SDS gel electrophoresis to estimate the relative frequencies of different membrane protein deficiencies in HS.566,590– 592 They found that about 30 to 45 percent of HS patients have combined ankyrin and spectrin deficiency and about 30 percent have isolated spectrin deficiency. However, SDS-PAGE probably underestimates the degree of ankyrin deficiency in many patients, especially compared with RIA or ELISA measurement. Presumably this occurs because ankyrin runs so close to β-spectrin on SDS gels that it is hard to quantitate. No patients with isolated spectrin deficiency were identified among about 80 tested by RIA or ELISA.23,563 A high reticulocyte count may also mask the detection of ankyrin deficiency.593
Two groups first reported that there are patients with dominant HS who have normal spectrin content in their red cells but have a deficiency in protein band 3 instead.594,595 These initial observations were confirmed by other reports.596– 598 Estimates suggest that 15 to 25 percent of dominant HS patients have a primary deficiency of band 3.564,566,591,592 Their red cells contain 20 to 40 percent less band 3 than normal.594– 596 These patients appear to form a relatively homogeneous clinical subgroup.564,566,591,592 They have typical dominant HS with mild to moderate hemolysis, and many nonsplenectomized patients have a small population (0.2 to 2.3 percent) of mushroom-shaped erythrocytes (Fig. 183-19, panel B).565,566
Partial deficiency of protein 4.2 is often seen in patients who have a primary deficiency of ankyrin or band 3, secondary to the underlying defect.196,592 Protein 4.2 is also absent in a mouse model with targeted deletion of the band 3 gene.81 Some other HS patients have been described with an isolated deficiency of protein 4.2 in their red cell membranes. They have an apparently recessive disease characterized by moderately severe, splenectomy-responsive hemolytic anemia and complete or nearly complete absence of protein4.2.570,599– 601 The red cell morphology is different from classic HS599,600 and varies from normal570 to spherocytes, elliptocytes, or ovalostomatocytes.571,599,600 Isolated protein 4.2 deficiency is rare in American and European HS patients but is quite common among Japanese.602
With the cloning of most of the genes encoding the membrane skeleton proteins in the past several years, many of the molecular defects that cause HS have been identified.
As discussed above, genetic linkage analyses and cytogenetic evidence first showed that a defect in ankyrin is the primary cause of HS in some families.196,540 Using a dinucleotide repeat polymorphism in the ankyrin cDNA as a marker to distinguish between the two ankyrin alleles, Jarolim and coworkers showed that one ankyrin allele has reduced expression in a third of HS patients with combined spectrin and ankyrin deficiency.603 This may be caused by either reduced transcription of one of the ankyrin alleles or instability of its mRNA. By the same method, it has been shown that de novo mutations in one of the ankyrin alleles leading to decreased expression are frequent in HS patients without a positive family history.532
Eber et al. examined 46 kindreds with both dominant and recessive spherocytosis and identified 12 different ankyrin mutations in 13 kindreds.564 Other investigators have reported additional mutations.604– 608 Most of these are family-specific, private mutations. Null mutations (frameshift and nonsense mutations) that result in either unstable ankyrin transcripts or truncated peptides predominate in dominant HS. They are located throughout the ankyrin peptide (Fig. 183-25). One of the more interesting is ankyrinRAKOVNIK, a nonsense mutation within the regulatory domain that leads to selective deficiency of the major ankyrin isoform, band 2.1 (preserving the minor isoform, band 2.2).613
Ankyrin mutations associated with HS are shown alongside a schematic representation of the ankyrin peptide. Null mutations, arising from frameshifts or nonsense mutations, are shown in rectangles. Missense and putative promoter mutations are shown in ellipses. The data are compiled from published and unpublished reports.534,564,604– 613
All of the ankyrin defects lead to combined and equivalent deficiencies of ankyrin and spectrin. Most of the null mutations are not detectable in reticulocyte mRNA, suggesting instability of the mutant transcript. The clinical manifestations are quite variable and range from mild hemolysis to severe transfusion-dependent anemia.564 This variability may be explained by different degrees of compensation for the null mutation, either from overproduction of the normal ankyrin allele or diminished ankyrin degradation.
Several ankyrin defects have been identified in patients with recessive HS.564 These are mostly missense or promoter mutations. A point mutation in the ankyrin promoter, −108T → C, is particularly common and was found in four of seven families with recessive HS. However, so far the mutation has not had a detectable effect on in vitro promoter assays.614 Two of four families carry a second mutation on the other allele: ankyrinsWALSRODE and BOCHOLT. AnkyrinWALSRODE contains a missense mutation (V463I) in the band 3 binding domain and has a decreased affinity for band 3.615 It is present in a patient whose red cells are more deficient in band 3 than in spectrin or ankyrin, which is opposite the trend in other ankyrin defects. AnkyrinBOCHOLT bears a missense mutation in a rare alternative splice product that may result in aberrant splicing.
HS patients with ankyrin defects have prominent spherocytosis without other morphological defects. Hemolysis and anemia vary from mild to severe.564,604– 607 In general, patients with dominant defects are less affected than those with recessive mutations; however, there is considerable overlap.
Molecular defects have been described in many patients with HS and band 3 deficiency (Fig. 183-26). Conserved arginine residues in band 3 are frequent sites of mutations. Examples include arginines 490, 518, 760 (two mutations found), 808, and 870.80,564,591 These highly conserved residues are positioned at the internal boundaries of transmembrane segments and substitution probably interferes with co-translational insertion of band 3 into the membranes of the endoplasmic reticulum during synthesis of the protein. In one case, mRNAs for both alleles were present but the mutant band 3 protein was not detected in the membrane, demonstrating either a functional defect in incorporation of the protein into the membrane or instability of the mutant protein.80 Missense mutations or short in-frame deletions affecting other residues in the transmembrane domain of band 3 have been identified, and presumably also impair insertion of the mutant band 3 into the membrane.564,566,617,618,624,625,627 A 10 nucleotide duplication near the C-terminal end of band 3PRAGUE leads to a shift in the reading frame and an altered C-terminus after amino acid 821. This mutation affects the last transmembrane helix and may eliminate the carbonic anhydrase II binding site on band 3.46 It may also impair insertion of band 3 into the membrane and abolish anion transport function.626
Band 3 mutations associated with HS are shown alongside a schematic representation of the band 3 peptide. Null mutations arising from either frameshift or nonsense mutations are shown in rectangles, as is a mutation associated with loss of the translation start site (band 3NEAPOLIS). Missense mutations or short in-frame deletions or insertions are shown in ellipses. The data are compiled from published and unpublished reports.80,85,188,529,564,566,591,616–628
Mutations in the cytoplasmic domain of band 3 can interfere with its binding to other membrane skeleton proteins, resulting in a functional defect. A deletion of five amino acids from the ankyrin binding site in band 3NACHOD presumably disrupts this interaction.566 An amino acid substitution (Gly → Arg) at residue 130 in the cytoplasmic domain in band 3FUKUOKA affects protein 4.2 binding.618 Patients with band 3MONTEFIORE and TUSCALOOSA also have spherocytic hemolytic anemia with protein 4.2 deficiency, and missense mutations in the cytoplasmic domain of band 3, at residues 40 and 327, respectively.188,622
Null mutations in the band 3 gene also occur. They include nonsense mutations, single nucleotide insertions or deletions, and splicing defects.529,564,566,591,616,617,621,623 These mutations presumably lead to mRNA instability and protein deficiency.623
Distal Renal Tubular Acidosis.
Because band 3 functions as an anion exchanger and is expressed in the acid-secreting intercalated cells of the kidney cortical collecting ducts, patients with HS and band 3 deficiency have been tested for a defect in acid-base homeostasis. Two HS patients with Band 3PRIBRAM have incomplete distal renal tubular acidosis (RTA)(see Chap. 195),83 but most other HS patients with band 3 deficiency have no evidence of metabolic acidosis.83,84 In HS patients with Band 3CAMPINAS, there is increased basal urinary bicarbonate excretion but efficient urinary acidification.621 A bovine model of band 3 deficiency exhibits only mild acidosis,629 and there are no obvious metabolic disturbances in mice that have a targeted deletion of band 3 that eliminates both the red cell and kidney isoform.81 Band 3 missense mutations have been found in patients with dominant distal renal tubular acidosis, but these patients have no red cell abnormality and the mutations identified are different from those associated with HS.84,630– 632 Mutations affecting Arg 589 are particularly common (8 of 11 families). The mechanism by which these mutations produce RTA is a mystery. The disease does not correlate with the anion transport activity of the band 3 mutants,84,630 and the one tested mutant (R589H) does not have a dominant negative effect when coexpressed with normal band 3.84 The best possibility is that the mutant proteins may impair trafficking of band 3.632
Although spectrin deficiency has long been associated with HS, it is only recently that specific mutations in spectrins have been described as a primary cause of the disorder. Because α-spectrin is normally produced in excess in red cells132– 134 and β-spectrin production is the rate limiting step in the formation of the membrane skeleton, most spectrin mutations found in HS are in the β-spectrin gene.
Monoallelic expression of β-spectrin occurs frequently in HS patients with spectrin deficiency,533 suggesting that null mutations are common defects. About 13 null mutations have been described in patients with dominant HS. The defects include initiation codon disruption,633 frameshift and nonsense mutations,569,634 gene deletions,635 and splicing defects461,634 (Fig. 183-27). A 4.6 kb genomic deletion in spectrinDURHAM results in a truncated peptide that is inefficiently incorporated into the red cell.635 A splice site mutation in spectrinWINSTON-SALEM leads to exon skipping and an unstable truncated β-spectrin peptide.634 A similar defect in spectrinGUEMENE-PENFAO causes an intron to be retained and blunts the accumulation of β-spectrin transcripts.636
β-Spectrin mutations associated with HS are shown alongside a schematic representation of the β-spectrin peptide. Null mutations, arising from frameshifts, nonsense mutations, large deletions, or substitution of the initiation codon, are shown in rectangles. Missense mutations are shown in ellipses. The data are compiled from published and unpublished reports.105,569,633– 638
Several missense mutations in the β-spectrin gene have been described in dominant HS.105,569 In most of these cases, it is not known if the mutations cause a functional defect or destabilize the mRNA or protein. One exception is β-spectrinKISSIMMEE, which has been well-characterized on the protein level and is defective in its capacity to bind protein 4.1.568,639 Heterozygotes have two types of spectrin. The abnormal fraction, approximately 40 percent, cannot bind protein 4.1 and therefore binds weakly to actin.568,639 Peptide mapping shows the defect resides near the N-terminus of the β-spectrin chain,639 near the site where protein 4.1 binds.102,103,640 The mutant spectrin is unstable and susceptible to thiol oxidation.639 This either causes or exacerbates the defect in binding to protein 4.1, because chemical reduction almost completely restores normal binding activity.639 Interestingly, very mild oxidation of normal spectrin641 or storage of normal cells under aerobic conditions in the blood bank642 produces similar defects in spectrin-4.1 interactions. Patients with the spectrin-4.1 binding defect have only 80 percent of normal red cell spectrin,639 which may be the real explanation of why they have spherocytosis. Presumably, the defective spectrin detaches from the membrane more easily than normal and falls prey to proteases that specifically degrade unbound or oxidized spectrin chains.459,643 Loss of the abnormal spectrin explains why the ratio of normal to abnormal spectrin is 60:40 instead of the expected 50:50.
Molecular analysis shows a Trp to Arg substitution at position 202 of the β-spectrin cDNA in the three affected members of one of these kindreds105 but not in the other two described kindreds with a similar functional defect.644 The mutation inserts a positively charged amino acid in a highly conserved region of largely hydrophobic amino acid sequence and could thus disrupt a region that is critical for protein 4.1 binding. Two other mutations, spectrinsATLANTA and OAKLAND, have missense mutations at positions 182 and 220 of the β-spectrin cDNA, flanking the position of the spectrinKISSIMMEE mutation. These mutations may potentially also cause defective protein 4.1 binding, but this hypothesis has not been tested.
A mutation in β-spectrin has only been identified in one case of recessive HS. A point mutation in position 1684 of the β-spectrin chain in spectrinBIRMINGHAM changes an arginine to cystine.569 There presumably is another mutation in the second allele to account for the recessive nature of the disease, but it has not been found.
As discussed above, α-spectrin is normally synthesized in excess (three- to fourfold).645 Defective α-spectrin production in one allele is not expected to cause disease. It is therefore not surprising that α-spectrin defects have not been described in patients with dominant HS.
Defects in α-spectrin have been implicated in a subset of patients with a life-threatening form of nondominant HS associated with marked spectrin deficiency (25 to 50 percent of normal).535 Many, but not all, of these families carry a variant α-spectrin peptide, designated αIIa or α-spectrinBUGHILL.646 It bears an amino acid substitution, alanine to asparagine, at residue 309 of the α-spectrin chain.647 Peptide analysis of the α-spectrin peptides from the affected HS patients shows only the αIIa variant, but genomic DNA analysis reveals both an allele with the αIIa/α-spectrinBUGHILL mutation and one without, indicating that the second α-spectrin allele in these patients may be functionally silent.647 A candidate gene for this silent mutation was discovered in a family with severe, nondominant HS.536 One of alleles, designated αspectrinPRAGUE, has a mutation in the penultimate position of intron 36, leading to skipping of exon 37 and premature termination of the α-spectrin peptide (Fig. 183-28). The other α-spectrin allele has a partial splicing abnormality in intron 30, and produces only about one-sixth of the normal amount of α-spectrin. This low-expression allele, named α-spectrinLEPRA (low-expression allele Prague), is linked to the αIIa/α-spectrinBUGHILL variant in this patient and several other patients with nondominant HS.648 The interaction of α-spectrinLEPRA and an α-spectrin allele encoding a nonfunctional peptide may be a frequent cause of severe nondominant HS.
Two α-spectrin mutations associated with recessive HS are shown alongside a schematic representation of the α-spectrin peptide.536
The molecular etiology of protein 4.2 deficiency has been defined in several cases. The most common mutation is protein 4.2NIPPON (Ala 142 → Thr) (Fig. 183-29).599,651 It affects the processing of 4.2 mRNA, so that red cells contain only traces of the 72/74-kDa isoforms of protein 4.2 instead of the usual abundant 72-kDa species. This mutation is very common in Japanese HS patients. It is homozygous in some patients651,655 or compound heterozygous with a second mutant allele such as protein 4.2FUKUOKA,NOTAME or SHIGA.650,652,654 Band 4.2KOMATSU contains an amino acid substitution in codon 175 that causes a moderate hemolytic anemia with ovalostomatocytosis and increased osmotic fragility in the homozygous state.571
4.2 mutations associated with recessive HS are shown alongside a schematic representation of the peptide. Null mutations, arising from frameshifts or nonsense mutations are shown in rectangles. Missense mutations are shown in ellipses. The data are compiled from published reports.571,649– 654
The only two protein 4.2 mutations reported outside the Japanese population are protein4.2TOZEUR and protein 4.2LISBOA, found in homozygous form in Tunisian and Portuguese patients, respectively.649,653 In protein 4.2TOZEUR,570,653 the propositus and her sister had a chronic hemolytic anemia and no protein 4.2 in their red cells. Molecular analysis showed a missense mutation in codon 310, in a region that is conserved in protein 4.2 and other members of the transglutaminase family. A recombinant protein bearing the mutation was abnormally sensitive to proteolysis, which may explain the absence of protein 4.2 in the patients' erythrocytes. In protein 4.2LISBOA,649 a single nucleotide deletion at nucleotide 264 of the cDNA causes a frameshift mutation and premature termination of the peptide. The heterozygous parents are clinically asymptomatic. The proband presented with a hemolytic anemia and splenomegaly at the age of 20. Peripheral blood films showed only a few spherocytes. Her symptoms improved markedly after splenomegaly.
Relative Frequency of Defects.
Based on results of both protein and molecular studies, it is estimated that, in Caucasian populations, about 50 percent of HS defects are found in ankyrin, 20 percent are in band 3, 25 percent are in β-spectrin, and 5 percent are in α-spectrin and protein 4.2. Among Japanese, band 3 and protein 4.2 defects are much more prevalent.
The availability of several well-characterized mouse models has contributed to our understanding of the pathophysiology of HS. Six types of hereditary hemolytic anemia have been identified in the common house mouse, Mus musculus.656 These anemias resemble human hereditary spherocytosis and are designated ja/ja (jaundice), sph/sph (spherocytosis), sph ha /sph ha (hemolytic anemia), sph 2BC/sph 2BC, sph 1J/sph 1J, sph 2J/sph 2J, sph-Dem/sph-Dem, and nb/nb (normoblastosis). The nomenclature indicates that anemia is observed only in the homozygous state and that the six mutants represent three loci: ja, sph, and nb. All of the mutants have severe hemolysis, with reticulocyte counts >70 percent, along with marked spherocytosis, jaundice, bilirubin gallstones, and massive hepatosplenomegaly. The defects are autosomal recessive, and the homozygotes have drastically impaired viability.133,657 There is a similar but much milder condition in the deer mouse, Peromyscus maniculatus, designated sp/sp.658 The mice have mild (≈20 percent) spectrin deficiency659 and a phenotype that resembles typical human HS.
Studies of the Mus mutants have revealed abnormalities in the erythrocyte membrane skeleton. The ja/ja mutant has no detectable spectrin. The mice carry a nonsense mutation in the β-spectrin gene660 and lack the ability to produce stable β chains.133
The sph/sph variants lack α-spectrin but have small amounts of β-spectrin. They have defects in α-spectrin synthesis, function, and/or stability.133 The sph and sph 2BC alleles are frameshift mutations and null alleles.661 In contrast, sph 1J/sph 1J mice synthesize normal amounts of spectrin mRNA and protein, however, the protein is not stably incorporated into the membrane skeleton. Surprisingly, the sph 1J allele contains a nonsense mutation near the C-terminus that deletes the last 13 amino acids from the protein. Apparently these amino acids, in the EF hand region at the spectrin tail (Fig. 183-4), are functionally important in attaching spectrin to actin. The sph-Dem/sph-Dem mutation arose spontaneously in CeS3/Dem strain and is missing exon 11 and 46 amino acids from spectrin repeat 5. The mice have both spherocytes and elliptocytes, and some poikilocytes, and are a mixture of HS and HPP.662
Cardiac thrombi, fibrotic lesions, and renal hemochromatosis are found in ja/ja and sph/sph mice in adulthood.663 Transplantation of hematopoietic cells from sph/sph mice are sufficient to induce thrombotic events in the recipients.664 One possibility is that the membrane vesicles released from the very unstable mouse red cells expose phosphatidyl serine on their outer surface, which would be very thrombogenic.665
The nb/nb mice have 50 to 70 percent of the normal quantity of spectrin and no ankyrin.657 They have normal spectrin synthesis133 but are moderately spectrin deficient because their ankyrin is very unstable. The nb mutation maps to the Ank-1 locus, indicating a primary ankyrin defect.155,659 The specific defect has not been identified. Interestingly, fetal nb/nb mice have normal reticulocyte counts666 and no anemia at birth, apparently due to expression of Ank-1-related (165-kDa) and Ank-2-related (155-kDa) proteins in utero.667
The nb/nb mice develop ataxia when they reached maturity, due to loss of cerebellar Purkinje cells.668 Ank-1 protein is markedly reduced in the Purkinje cells, which may explain their fragility. The significance of these findings and their relationship to human HS is unknown.
New mouse mutants with defects in membrane skeleton proteins have been generated by targeted mutagenesis in embryonic stem (ES) cells. Mice completely deficient in band 3 survive gestation but tend to die in the neonatal period,81 often from thrombotic complications.669 Those that survive have a severe spherocytic hemolytic anemia, closely resembling severe HS in humans. The mice have undetectable amounts of protein 4.2 and glycophorin A,52,81 but have normal amounts of spectrin, actin, and protein 4.1 in their red cell membrane skeletons, and normal membrane skeleton architecture by electron microscopy.81 Despite their normal skeletons, the band 3 deficient red cells shed astonishing amounts of membrane surface in small vesicles and long tubules. These observations indicates that band 3 is, surprisingly, not required for membrane skeleton assembly, but has a critical function in stabilizing membrane lipids. Loss of this function may be critical to the pathogenesis of HS.
These mice also provide an unexpected clue about the genetic control of reticulocyte response to anemia. The first two generations of the band 3 knockout mice, in a C57BL/6J and 129/Sv (B6/129) hybrid genetic background, had over 70 percent reticulocytes, but later generations of the mice, outcrossed to other genetic backgrounds, show a striking decrease in reticulocytosis and survival.670 A newly discovered mouse mutant (wan) in a C3H/HeJ background that has a null defect in the band 3 gene also has severe anemia without reticulocytosis. But when the wan/wan mice are crossed with the band 3 gene knock-out mice in the B6/129 background, there is an elevation of reticulocytes to over 65 percent.670 These observations suggest that a genetic modifier segregating in the B6/129 background controls a strong reticulocyte response in the absence of band 3. Understanding this modifier will provide important insight into the role of band 3 in the late stages of erythroid differentiation and reticulocyte formation.
In addition to mouse models, there is a recessive form of HS in cattle with moderate hemolytic anemia and complete deficiency of band 3, due to a nonsense mutation at codon 646.629 The cattle, like band 3 deficient mice, have defective anion transport, lack protein 4.2, and have reduced numbers of intramembrane particles by electron microscopy.
The absence of protein 4.2 is not responsible for the severe phenotype observed in band 3 knockout mice, because mice with a targeted deletion of the protein 4.2 gene survive normally and have only a mild spherocytic hemolytic anemia.193 Surprisingly, however, cation transport pathways are markedly disturbed. In homozygotes, the maximal rate of Na+/K+/Cl− co-transport is increased seven- to eightfold, Na+/H+ exchange is increased up to fiftyfold, K+/Cl− co-transport is increased by two- to threefold, and K+ transport via the Gardos channel is enhanced three- to fourfold. In contrast Na+/K+-ATPase activity is normal. The data suggest that protein 4.2 directly or indirectly suppresses the activities of these transporters, a function of the protein that was previously unknown.
Mice lacking erythrocyte protein 4.1 (protein 4.1R) have also been generated by gene targeting.671 Homozygotes have mild to moderate hemolysis with increased fragmentation and decreased red cell membrane stability. In addition to the total absence of protein 4.1, there are reduced levels of protein p55 and glycophorin C. There is also partial deficiency in ankyrin and spectrin, suggesting that loss of protein 4.1 compromises membrane skeleton assembly. Erythrocyte morphology shows spherocytosis instead of elliptocytosis, probably related to the spectrin deficiency. Interestingly, these mice also have neurological deficits in movement, coordination, and learning, presumably because neuronal isoforms of erythroid protein 4.1 are also disrupted.437
Mice deficient in β-adducin, due to a targeted gene deletion, have recently been described.271 The mice have a mild hemolytic anemia characterized by spherocytes, spherostomatocytes, and rounded elliptocytes, and a partial deficiency of α-adducin, indicating that β-adducin is also necessary for the synthesis or stability of its partners, and that adducin is needed for the maintenance of normal red cell surface area. This is an original observation, as no human patient deficient in adducin has yet been described.
Irrespective of its molecular cause, the major problem of the hereditary spherocyte is the rheologic consequences of its decreased surface-to-volume ratio. The red cell membrane is very flexible, but it can expand its surface area only about 3 percent before rupturing.672 The loss of surface area and decreased deformability of the spherocyte causes it to be trapped in the hostile environment of the splenic cords, leading to its early demise.
Loss of Membrane Surface.
As discussed above, molecular defects in HS appear to disrupt components of the membrane skeleton that are responsible for attaching the skeleton to the lipid bilayer. How this results in membrane loss is not entirely clear. Two hypotheses have been advanced (Fig. 183-30). In the first, the lipid bilayer and integral membrane proteins are directly stabilized by their interaction with ankyrin or the spectrin skeleton. Deficiency in spectrin or ankyrin would result in the lack of skeletal support in some areas of the membrane, which would then bud off and be lost. In the second hypothesis, membrane surface area is stabilized by interactions of band 3 with neighboring lipids. In band 3 deficiency, more of the lipids lack this interaction and are lost, while in spectrin or ankyrin deficiency, band 3 molecules would more rapidly diffuse and might transiently cluster, with the same consequences.
Two hypotheses concerning the mechanism of membrane loss in HS. Hypothesis 1 assumes that the lipid bilayer and integral membrane proteins are directly stabilized by interactions with the spectrin membrane skeleton. Spectrin-deficient areas, lacking support, bud off, leading to spherocytosis. Hypothesis 2 assumes that the membrane is stabilized by interactions of band 3 with neighboring lipids. The influence of band 3 extends into the lipid milieu because the first layer of immobilized lipids slows the lipids in the next layer, and so on. In band 3-deficient cells, the area between lipid molecules increases and unsupported lipids are lost. Spectrin-ankyrin deficiency allows band 3 molecules to diffuse and transiently cluster, with the same consequences. (From Lux and Palek.4 Used by permission.)
In spectrin-deficient red cells, the force required to fragment HS membranes is diminished and proportional to the density of spectrin. Cell surface area and membrane stability are proportional to red cell spectrin content and are reduced in HS erythrocytes (Fig. 183-23).585 This may explain why spectrin-deficient spherocytes fail to withstand circulatory stresses and become trapped in the spleen.
In the spleen most of the arterial blood empties directly into the splenic cords, a narrow, honeycombed maze of passages formed by reticular cells and phagocytes.673– 675 If flow through these passages is impeded, red cells are diverted deeper into the labyrinthine portions of the cords, where blood flow is slow and the cells may be detained for minutes to hours. To exit and return to the venous circulation, red cells must squeeze between the endothelial cells that form the walls of the venous sinusoids. Even when maximally distended, these narrow, elliptical fenestrations are much smaller than red cells, which must undergo considerable contortion during their passage.674,675
It is clear that spherocytic red cells are significantly hindered at this point in the circulation. Isolated hereditary spherocytes are poorly deformable and pass through 3- to 5-mm filters with difficulty, sometimes bursting in the process.676 HS red cells are trapped in the cords during in vitro perfusion through spleens removed from patients with idiopathic thrombocytopenic purpura,677 and 51Cr-labeled spherocytes are selectively sequestered in the spleen in vivo.678,679 HS spleens characteristically show massively congested cords and relatively empty venous sinuses on light microscopy,673,680 and electron microscopy shows relatively few spherocytes traversing the sinus wall,673,680 in contrast to normal spleens, where such cells are easily found.674
Although it was known as early as 1913 that red cells obtained from the splenic vein were more osmotically fragile than those in the peripheral circulation,681 the significance of this observation was not fully appreciated until the classic studies of Emerson576 and Young677 and their colleagues. These investigators showed that osmotically fragile microspherocytes are concentrated in the splenic pulp. After splenectomy the tail of hyperfragile cells in the osmotic fragility curve disappears, although the major population of moderately fragile spherocytes persists.576,677 These results led to the conclusion that the spleen detains and conditions circulating HS red cells in a way that increases their spheroidicity, aggravates loss of their membranes and hastens their demise.576,677 The kinetics of this process of “splenic conditioning” were beautifully illustrated in vivo by Griggs and his coworkers,679 who showed that a cohort of 59Fe-labeled HS red cells gradually shifted from the major, less fragile population to the minor, more fragile population during their circulation in vivo.
The mechanism of splenic conditioning is less clear. It is difficult to obtain precise information about the environment in the splenic cords, but existing data suggest that the climate is inhospitable. Arteries supplying the white pulp skim off plasma and increase congestion in the cords, where the crowded red cells must compete with metabolically voracious phagocytes for limited supplies of glucose. Because of the stagnant circulation, lactic acid accumulates and extracellular pH falls, probably to between 6.5 and 7.0.576 Intracellular pH must also decline, inhibiting rate limiting enzymes of glycolysis and retarding glucose utilization. Under these conditions stores of 2,3-DPG will be metabolized to provide energy for the cell. The loss of this polyvalent anion, combined with the decreased anionic charge on hemoglobin that occurs in an acid environment, is compensated by the entry of monovalent chloride ions. The resulting increase in osmolarity causes water to enter the HS red cell and worsen its already compromising spheroidicity. Thus, the spherocyte, detained in the splenic cords because of its surface deficiency, is severely stressed by erythrostasis in a metabolically threatening environment.
In summary, it is clear that HS red cells are selectively detained by the spleen during their passages through that organ, leading to a progressive loss of membrane surface, further splenic trapping, and eventual destruction (Fig. 183-31). Indeed, studies have shown that the mean splenic transit time correlates inversely (r = −0.96) with red cell survival in HS.683
Pathophysiology of the splenic conditioning and destruction of hereditary spherocytes. (Adapted from Becker and Lux.682 Used by permission.)
The diagnosis of HS is usually easy if there are typical laboratory findings of spherocytosis, Coombs-negative hemolysis, increased osmotic fragility, and a positive family history. There are several situations in which the diagnosis can be more difficult.
In the neonatal period it may be hard to differentiate HS from ABO incompatibility since microspherocytosis is prominent in both conditions and the Coombs' test is frequently negative in ABO disease.684 Fortunately, in most affected infants with ABO incompatibility, anti-A (or anti-B) antibodies can be eluted from the red cells, and free anti-A or anti-B IgG antibodies can be detected in the infant's serum. Occasionally, older patients with immunohemolytic anemias and spherocytosis also have so few antibody molecules attached to their red cells that the Coombs' test is negative and differentiation of the disease from HS is possible only with the use of radioactive antiglobulin reagents.685
Diagnostic difficulties also arise in patients who present during an aplastic crisis (see below). Early in the crisis the acute nature of the symptoms may suggest an acquired process, and the absence of reticulocytes may divert the physician from a diagnosis of hemolytic anemia. Later, as marrow function returns, the physician may be misled by the fact that the emerging young HS red cells are initially less spherocytic and osmotically fragile than usual686 and acquire their typical microspherocytic form only with age and reticuloendothelial conditioning. If a transfusion has been given, the transfused red cells can also make diagnosis of the underlying HS disease difficult until they are cleared.
HS may also be camouflaged by association with disorders that increase the surface-to-volume ratio of the red cells, such as iron deficiency687 or obstructive jaundice.688 Iron deficiency corrects the abnormal shape and fragility of hereditary spherocytes but does not improve their life span,687 whereas obstructive jaundice improves both shape and survival.688 The clinical expression of HS may also be modulated by interaction with other hematologic disorders. Coinheritance of HS and β- or α-thalassemia trait, appears to cause a milder disease in some families,598,689,690 whereas the presence of glucose 6-phosphate dehydrogenase deficiency and HS in the same patient may result in a more severe hemolytic anemia.691 A patient with Hb SC disease, α-thalassemia trait and HS presented with recurrent acute splenic sequestration crisis, probably because of splenic trapping and intrasplenic sickling of the spherocytic erythrocytes.692 HS patients with coexisting sickle cell trait may also experience life-threatening acute splenic sequestration crisis.693 Fortunately, HS is relatively rare in African populations, so these dangerous combinations do not often occur.
Patients with HS, like patients with other hemolytic processes, are subject to various crises.
Mild hemolytic crises are probably most frequent and are usually triggered by common viral syndromes,547 especially in children. They are characterized by a mild, transient increase in jaundice, splenomegaly, anemia, and reticulocyte count. Severe hemolytic crises558 are less common. Hemolysis can also be aggravated during pregnancy.550,551 For most patients with a hemolytic crisis, supportive care is all that is needed; red cell transfusions are rarely required. Corticosteroids can be beneficial during episodes of acute hemolysis,694 but are generally not indicated.
Aplastic crises occur less frequently than hemolytic crises but are more serious, as severe anemia and even death can result.521,558 They typically present with fever, vomiting, abdominal pain, arthralgias, headache, pallor, and symptoms of anemia.695 Sometimes multiple family members are affected simultaneously.696 During the aplastic phase, the hematocrit level and reticulocyte count fall, marrow erythroblasts disappear, and unused iron accumulates in the serum. Mild granulocytopenia and thrombocytopenia are common but are not invariably present. Because production of new HS red cells is halted, the cells that remain age, and microspherocytosis and osmotic fragility increase.697 The bilirubin level declines because of a decrease in the number of abnormal red cells that have to be destroyed. Because the usual aplastic crisis lasts 10 to 14 days (about half the life span of HS red cells), the hemoglobin concentration typically falls to about half its usual value before recovery ensues. The return of marrow function is heralded by a fall in serum iron concentration, a rise in granulocytes and platelets to normal levels, and reticulocytosis.695
It is now known that aplastic crises in HS patients are caused by infection with the human parvovirus B19.698,699 The B19 parvovirus causes erythema infectiosum or “fifth disease” in small children, and fever, rash, and polyarthropathy in older children and adults. The virus infects and kills early erythroid precursors700 and drastically impairs red cell production, resulting in aplastic crises in patients who have erythrocytes with a shortened life span, as in HS patients. Occasionally, parvovirus infection can also cause pancytopenia, hemophagocytosis, myelodysplasia, or even autoimmune disorders in HS patients.701– 704 It is not uncommon for an aplastic crisis to be the first sign of HS in previously well-compensated patients.705,706 Multiple HS members in a family often come down with aplastic crises at the same time because of the infectious nature of the causative agent.696,707 The infection is diagnosed by immunologic tests or by the polymerase chain reaction. Examination of the bone marrow shows loss of erythroblasts beyond the pronormoblast stage, and, in some cases, characteristic giant pronormoblasts with cytoplasmic vacuoles.
Treatment is supportive until the aplastic episode is over, which usually lasts seven to ten days. Red cell transfusions may be necessary if the anemia is severe. Intravenous immunoglobulin helps clear persistent parvovirus infection in immunocompromised patients but is not necessary for most HS patients. Persons with erythema infectiosum are infectious before the onset of illness and not infectious once the rash appears, but patients in aplastic crisis are contagious from before the onset of symptoms to at least a week afterward.708 HS patients should avoid exposure to affected family members during this period. Droplets and contact precautions to prevent spread of parvovirus through respiratory secretions are necessary.708 A parvovirus vaccine is currently undergoing phase I trials and may be available in the future for patients with HS.
Megaloblastic crises are rare. They result when the dietary intake of folic acid is insufficient for the increased needs of the erythroid HS bone marrow. The risk is increased during pregnancy, when the need for folic acid is particularly high.709,710 It is recommended that HS patients with ongoing hemolysis take folic acid supplements of at least 1mg per day.
Gallbladder disease is the most common complication of HS. Pigment gallstones are reported in patients as young as 3 years697 but are most prevalent in adolescents and adults.711 The incidence of gallstones rises rapidly in the second and third decades of life, after which its increase parallels that of the general population712 (Fig. 183-32). The limited data available indicate that 55 to 85 percent of untreated HS patients will eventually acquire stones556,711 and that roughly half of these individuals will have symptoms of cholecystitis or, less commonly, biliary obstruction. However, the data were gathered before the common use of ultrasonography to assess gallbladder disease and the risk of complications is based primarily on the experience with cholesterol gallstones. More accurate data on the incidence of these complications in patients with bilirubin gallstones are needed to assess accurately the risk/benefit ratio of cholecystectomy (and splenectomy) in HS.713
Proportion of normal (solid circles) and HS (open circles) patients with gallstones as a function of age. The prevalence of gallstones rises sharply between the ages of 10 and 30 in HS patients. The subsequent increase then parallels that of the general population after the age of 30, suggesting that cholelithiasis due to HS is primarily manifest in the second and third decades. The HS curve is derived from the data of Bates and Brown;711 the normal curve is from autopsy data.712 (From Lux and Palek.4 Used by permission.)
The incidence of gallstones in HS patients is apparently related to the ability of the liver to metabolize bilirubin. A very common mutation in the promoter of the UDP-glucuronyl transferase gene (UGT-1A) has recently been shown to be associated with reduced UDP-glucuronyl transferase activity in heterozygotes and to cause Gilbert syndrome in homozygotes (see Chap. 125).545 A study of the frequency of gallstones in 103 HS children showed that the rate of gallstone formation in patients homozygous for the mutated UGT-1A allele is 2.1 times that of heterozygous patients and 4.5 times that of normals.714 If this result is confirmed, analysis of this allele should be useful in predicting the risk of gallstones in HS.
The treatment of gallbladder disease in HS is debatable, especially in patients with mild hemolytic disease or asymptomatic gallstones. An initial period of observation is advisable.715 Surgery may be necessary if there are recurrent symptoms or complications such as cholecystitis or biliary obstruction. Laparoscopic cholecystectomy is the procedure of choice among most surgeons and the general public.716 If a patient needs splenectomy for ongoing hemolysis or its complications (see below), gallbladder ultrasound should be done first and concomitant cholecystectomy considered if gallstones are present.717 Splenectomy solely as a prophylaxis for gallstone development is probably not indicated, nor is prophylactic cholecystectomy.
Adult patients with HS occasionally have gout,522 indolent leg ulcers, or a chronic erythematous dermatitis on the legs.718 These complications occur mainly in the elderly and usually resolve after splenectomy.
Rarely, patients also have extramedullary masses of hematopoietic tissue, particularly along the posterior thoracic or lumbar spine.521,719 These gradually enlarge with time and may be mistaken for neoplasms.719
Interestingly, Schafer and his colleagues have suggested that untreated HS may predispose patients to a true neoplasm, multiple myeloma.720 Several patients with HS and myeloma have been reported.720 It was argued that the association may be due to chronic reticuloendothelial stimulation, because splenic clearance of abnormal red cells induces proliferation of lymphocytes and plasma cells as well as macrophages. HS patients often have a mild, polyclonal hypergammaglobulinemia,721 and there is evidence favoring the association of myeloma and chronic gallbladder disease.722 However, it is still unclear if this proposed association of HS and myeloma is a real phenomenon.
There are reports of HS patients who died from liver failure or hepatoma secondary to iron overload.723,724 While HS may lead to excessive iron uptake in some patients who are clinically heterozygous for hereditary hemochromatosis,725,726 this is not a problem in most HS patients.
Even though splenomegaly is often seen in patients with HS, traumatic rupture of the enlarged spleen in HS patient is very rare.727 This contrasts with the higher frequency of splenic rupture in patients with EBV infection, and may reflect the different pathophysiology of splenic enlargement in the two conditions.
It is one of the rare absolutes in medicine that patients with true, uncomplicated HS always respond dramatically to splenectomy. The degree of response correlates closely with the degree of spectrin deficiency and is incomplete in the most severely affected patients.531,560 The major issues today are who should have a splenectomy, what kind of operation should be done, and how the patients should be treated postoperatively.
Following splenectomy, spherocytosis persists, but conditioned microspherocytes disappear, and changes typical of the postsplenectomy state—including Howell-Jolly bodies, target cells, acanthocytes, pitted cells, and siderocytes—appear in the peripheral smear.574,728,729 Reticulocyte counts fall to normal or near-normal levels, although red cell life span, if carefully measured, remains slightly shortened (96 ± 13 days).730 Abnormal osmotic fragility persists, but the “tail” of the osmotic fragility curve, created by conditioning of a subpopulation of spherocytes by the spleen, disappears. Clinically, patients have better energy and an improved quality of life, and complications such as leg ulcers and extramedullary hematopoiesis resolve. In most cases, anemia and jaundice remit and do not recur, except in the rare case of regrowth of a missed accessory spleen. This is the only proven cause of postsplenectomy failure in HS and is sometimes overlooked, as it may not become evident for years.731
Immediate Postsplenectomy Complications.
Immediate postoperative complications include bleeding and subphrenic abscess. Hemorrhage usually comes from peritoneal and diaphragmatic surfaces of the splenic bed rather than from identifiable blood vessels. Subphrenic abscess is more likely to occur when adjacent organs are injured during the surgery. Acute pancreatitis can also occur in the postoperative period.732 A reactive thrombocytosis is commonly seen after splenectomy, with platelet counts as high as 1000 K/dl. The platelets peak 7 to 12 days after the procedure but elevated levels may persist indefinitely. The thrombocytosis is generally benign. It is probably not associated with venous thromboembolic complications,732,733 and antiplatelet therapy is probably not necessary. Portal vein thrombosis has been reported after splenectomy for hemolytic diseases, but it is probably not related to thrombocytosis.734– 736 The high frequency of thrombotic complications in mice with membrane skeletal defects663,669 and in patients with membrane disorders such as hereditary stomatocytosis and xerocytosis after splenectomy737,738 suggest that released membrane fragments may be a major factor. Such fragments are highly thrombogenic if phosphatidyl serine is exposed.665,669
One of the most serious complications of splenectomy is overwhelming postsplenectomy infection. The absence of a functional spleen puts these patients at an increased risk of infection by bacterial and parasitic pathogens. Among bacterial infections, the risk is particularly high for encapsulated organisms, specifically, S. pneumoniae, N. meningitidis, and H. influenzae. The course of bacterial infection in splenectomized patient can be extremely rapid and potentially fatal, and can occur years or decades after the procedure.739 It is difficult to estimate the true incidence of overwhelming postsplenectomy infection. The seminal study of King and Shumacker first drew attention to the high risk of fulminant sepsis in infants who had undergone splenectomy for HS,740 and numerous reports of serious postsplenectomy infection have since been published (results summarized in several reviews741– 744). The incidence of overwhelming postsplenectomy infection has been estimated to be 0.2 to 0.5 per 100 person-years of follow-up, with a death rate of 0.1 per 100 person-years in adults,745,746 and a much higher figure in children, particularly younger children.747– 749 The majority of the available studies, however, have serious methodological problems.744,750 Most are retrospective studies or case reports, which may have a inherent bias towards reporting the more serious cases. The duration of observation is often poorly documented and a large fraction of patients may be lost to follow-up. Most of the patients underwent splenectomy before pneumococcal vaccine was generally available or antibiotic prophylaxis routinely prescribed. The underlying diseases of the patients and the reasons for splenectomy are highly varied.
In our experience, overwhelming postsplenectomy sepsis can be a serious and devastating problem but is relatively uncommon in HS patients, unlike patients with underlying immunological dysfunction, such as Hodgkin disease, or with continuing extravascular hemolysis (e.g., thalassemia).732,741,748,751 A recent, 30-year follow-up of more than two hundred splenectomized HS adult patients found four patients dead from overwhelming sepsis, making the mortality rate from postsplenectomy sepsis 0.073 per 100 person-years.752 Three of the four deaths occurred 18 or more years after the operation and none of the patients who died had received pneumococcal vaccine or antibiotic prophylaxis. There is evidence of a dramatic decrease in the incidence of postsplenectomy sepsis in children who are given pneumococcal vaccine and prophylactic antibiotics.753 This suggests that the incidence of postsplenectomy sepsis may continue to decline, even though failures of the preventive measures may still occur.754 Continued research is needed to quantitate the true risk of overwhelming postsplenectomy sepsis in HS patients treated according to current practice guidelines.739
After splenectomy HS patients are also at risk for serious parasitic infections, such as babesiosis and malaria. Babesiosis is a tick-transmitted zoonotic infection by an intraerythrocytic protozoan that is endemic in Europe and in the northeastern, north central, and western United States.755 The causative agents include Babesia microti, B. divergens, and a related organism designated “WA1.” In healthy individuals the infection is usually mild and often asymptomatic, but in patients without a functional spleen, it can be rapidly progressive and life-threatening.756 Patients may have malaise, headache, fever, shaking chills, profuse sweating, jaundice, and dark urine. There may be intravascular hemolysis and, occasionally, pancytopenia757 and hemophagocytosis.758 Diagnosis is made by finding the parasites in blood smears, or by serologic tests or amplification of parasitic DNA using the polymerase chain reaction. Current treatment of symptomatic cases is quinine plus clindamycin, but treatment failures have been reported in asplenic patients. Splenectomized HS patients, when traveling in endemic areas, should take measures to avoid tick bites by, for instance, wearing long pants and using tick repellents. The nymphal ticks that cause the disease are only 1 to 2 mm in size and are difficult to detect.
Animal experiments have shown that the spleen is essential for limiting malaria parasitemia in the acute stage of infection,759,760 but, surprisingly, other than anecdotal reports,761– 763 there are no studies that definitively demonstrate an increased risk of severe malaria in asplenic individuals. It is nevertheless prudent for postsplenectomy HS patients to adhere strictly to malaria chemoprophylaxis protocols when traveling to endemic areas and take measures to reduce exposure to malaria parasites.
Thrombosis and Atherosclerosis.
There are occasional reports of thromboembolic events occurring many years after splenectomy,764 but their causal relationship to splenectomy has not been convincingly demonstrated, except for patients with hereditary stomatocytosis, where the risk of thrombotic complications after splenectomy approaches 100 percent.737
On the other hand, there is some evidence that the incidence of atherosclerosis is elevated after splenectomy. A carefully controlled, long-term follow up of splenectomy in 740 WW II servicemen following battlefield injuries, showed excess mortality from pneumonia (see above) and atherosclerotic heart disease (1.9 times relative risk) in the splenectomized patients.765 The authors suggest that the chronically elevated platelet counts that occur after splenectomy may have contributed to vascular disease. A more recent study found that the cumulative incidence of atherosclerosis after the age of 40 was six times higher in patients who had a splenectomy compared to those who had not had the procedure.766 While these results need to be confirmed by other studies, they support a conservative approach to splenectomy.
Indications for Splenectomy.
In view of the many potential complications, splenectomy should be done only if there are clear indications. We believe the procedure is clearly warranted for the rare patients with severe HS who require transfusions or who have serious complications, such as growth failure or thalassemic facies. It may also be indicated for patients with moderate or moderately severe disease who are symptomatic with, for instance, chronic fatigue, decreased physical stamina, or, later in life, compromised perfusion of vital organs, leg ulcers, or extramedullary hematopoietic tumors. Whether patients with moderate HS and asymptomatic anemia should have a splenectomy remains controversial. Subtotal splenectomy (see below) may have a particular role in these patients. Splenectomy can be deferred, probably indefinitely, in patients with mild HS and compensated hemolysis. The presence of gallbladder disease may influence the decision, as discussed above. In young children, the procedure, if indicated, should be delayed until at least 3 years of age, and, if possible, until 5 years or more. There is probably no additional benefit in postponing splenectomy beyond the age of 10 years, because the risk of gallstone development rises sharply after that. In some patients, the procedure may not be needed until old age, when complications like leg ulcers and extramedullary hematopoietic tumors develop.
Laparoscopic splenectomy, sometimes with concomitant laparoscopic cholecystectomy, has now become a viable alternative to open splenectomy.767,768 The pros for this approach include its minimal invasiveness, a shortened hospital stay, a lower need for narcotic pain control, and a more appealing cosmetic result. The cons include a longer operation time, potential difficulty in control of bleeding, and the chance of missing an accessory spleen. Experience with the procedure has accumulated tremendously in the past few years, although there are few multicenter, controlled trials comparing it with the traditional open surgical approach.769– 771 The current consensus is that if the surgical staff is experienced with the procedure and the spleen is not very large, laparoscopic splenectomy may be the method of choice.
Because of the risk of postsplenectomy sepsis, partial splenectomy (or subtotal splenectomy) has been advocated as an alternative to total splenectomy for HS.772– 774 In this procedure, about 90 percent of the enlarged spleen is removed, leaving behind a remnant with about 25 percent of the volume of a normal spleen (Fig. 183-33). The procedure is safe and decreases the rate of hemolysis while preserving some of the phagocytic function of the spleen.772– 774 Presumably the risk of postsplenectomy sepsis is also decreased, although this may be impossible to prove. After partial splenectomy, the mean hemoglobin in a group of HS patients increased from 9.8 to 12.2 g/dl and the absolute reticulocyte count decreased from 560 to 270 × 109 cells/liter772,774 There was improved quality of life and gain in physical growth in most of these patients. However, the reduction in hemolytic rate was not as great as that observed after total splenectomy and partial splenectomy did not prevent the development of gallstones completely (3 of 24 patients in one series).773 The need for secondary total splenectomy was 10 percent at 5 years and 33 percent at 10 years, and the patients have not been followed long enough to determine if there is really a lower risk for subsequent development of overwhelming sepsis. The high rate of subsequent complete splenectomy in these patients may reflect the fact that the procedure was often used in patients with severe disease. In our view it is more suited for patients with mild to moderate HS, in whom splenectomy is often not done. It is likely that the prevalence of gallstones and the need for a second operation will be much lower in these patients. Overall, partial splenectomy shows promise, but it should still be considered investigational at this time.
Surgical technique used in partial splenectomy. A. All vascular pedicles supplying the spleen are divided except those arising from the left gastroepiploic vessels. B. the upper pole of the spleen is removed at the boundary between the well perfused and poorly perfused tissue. (From Tchernia et al.772 Used by permission.)
Partial splenic artery embolization has been done as an alternative to splenectomy in patients with hypersplenism and has been performed in a child with HS.775 The experience with this procedure in HS is limited and cannot be recommended as a routine procedure.
Vaccination and Antibiotic Prophylaxis.
All HS patients undergoing splenectomy should receive vaccines against encapsulated bacteria. Polyvalent (23 strains) pneumococcal vaccine is highly effective753 after 2 years of age and should be given at least 2 weeks before splenectomy.776 A conjugated, heptavalent pneumococcal vaccine, effective at all ages, is currently completing clinical trials777– 779 and should be available in the near future. Conjugated H. influenzae type b vaccine is now given to all children and should also be given to splenectomized adult HS patients.780 N. meningitidis vaccine currently is effective against only serogroup a and c strains, not the common serogroup b, but probably should still be given to patients undergoing splenectomy.780 Revaccination with pneumococcal vaccine in 5 years and meningococcal vaccine in 2 years is advised. Yearly influenza vaccination may help reduce the chance of secondary bacterial infection.
Prophylactic antibiotics against S. pneumoniae should be given to HS patients after splenectomy. Penicillin V (125 to 250 mg twice per day) is usually used, but some physicians recommend amoxicillin because of its improved absorption.780 Patients who are allergic to penicillin may be offered erythromycin.781 Antibiotic prophylaxis should be given to splenectomized patients throughout their childhood, or, for teenagers and adults, in the first 2 to 5 years after splenectomy when the risk of overwhelming infection is highest. Some authorities recommend lifelong antibiotics prophylaxis,781 but there are concerns of poor patient compliance and emergence of resistant organisms.782 Patients not on prophylaxis should have a supply of oral antibiotics at hand, which they should take immediately if fever or other symptoms of infection develop. They should then seek medical attention right away.