Typical Hereditary Spherocytosis

The typical clinical picture of HS combines evidence of hemolysis (anemia, jaundice, reticulocytosis, gallstones, and splenomegaly) with spherocytosis and a positive family history ( Table 16-2 ; Fig. 16-2 ). Mild, moderate, and severe forms of HS have been defined according to differences in various clinical and laboratory parameters under baseline conditions ( Table 16-3 ). Unfortunately, patients don’t fit neatly into these categories: a person my be classified as “mild” by one criterion and “moderate” by another.

Peripheral blood smear from a patient with typical hereditary spherocytosis. Small, dense round microspherocytes are scattered throughout the smear, and most of the remaining, larger red cells have a smaller area of central clearing than normal.

Initial assessment of a potential HS patient should include questions about neonatal or subsequent jaundice, anemia, splenomegaly, or gallbladder symptoms; transfusions; and a family history of these things and of splenectomies or cholecystectomies. The physical examination should seek signs of pallor, scleral icterus, and splenomegaly. Pediatricians should also examine the parents and siblings if they are present, particularly for splenomegaly, which is more evident in older patients. Scleral icterus is usually subtle and best perceived in daylight and at the edge of the conjunctivae ( Fig. 16-3 ). Similarly, a spleen that cannot be palpated in a supine patient can sometimes be felt when the patient is placed on his or her right side with knees bent.

Scleral icterus in child with hereditary spherocytosis (HS). The jaundice in HS is often subtle and is more evident toward the posterior of the conjunctiva.

(From Bolton-Maggs P: Hereditary spherocytosis. Best Practice website, British Medical Journal Evidence Centre. BMJ Publishing Group Ltd. Accessed August 2013 at http://bestpractice.bmj.com.easyaccess2.lib.cuhk.edu.hk/best-practice/monograph/1143/resources/images/print/6.html .)

HS typically presents in infancy or childhood, but it may present at any age. In children, anemia is the most common presenting complaint (50%), followed by splenomegaly, jaundice, or a positive family history. No comparable data exist for adults. The majority of patients with HS have incompletely compensated hemolysis and mild to moderate anemia. The anemia is often asymptomatic except for fatigue and mild pallor or, in children, nonspecific parental complaints such as irritability. Jaundice is seen at some time in about one half of patients, usually in association with viral infections. When jaundice is present it is acholuric (i.e., unconjugated [indirect] hyperbilirubinemia without detectable direct bilirubinuria). Palpable splenomegaly is detectable in about one half of infants and most (70% to 95%) older children and adults. Typically, the spleen is modestly enlarged (2 to 6 cm below the costal margin), but it may be massive. There is no proven correlation between the size of the spleen and the severity of HS, although, given the pathophysiology and the response of the disease to splenectomy, such a correlation probably exists.

Typical HS is associated with both dominant and nondominant inheritance. The nondominant disease tends to be more severe, but there is considerable overlap. Clinical severity and the response to splenectomy roughly parallel the degree of spectrin (or ankyrin) deficiency in patients with defects in either protein. Disease in such patients can vary from mild to severe. Patients who are heterozygotes for band 3 defects have typical, moderate HS and moderate band 3 deficiency (15% to 35%). Severe HS is rare in this population unless the patient carries two defective band 3 genes. Protein 4.2 deficiency is also associated with milder disease. Overall, patients with deficiency of spectrin or both spectrin and ankyrin have more severe disease than patients with band 3 or protein 4.2 deficiency, but the differences are not great because true “severe” HS is rare.

In general, members of the same family will have a similar clinical picture, but there are exceptions, presumably due to varying compensation by the normal allele in patients who are heterozygous for a mutant gene, or by the coinheritance of another deleterious allele. Such alleles should be sought in patients whose disease is much more severe than that of the rest of the family.

Silent Carrier State

The parents of patients with “nondominant” HS are clinically asymptomatic and do not have anemia, splenomegaly, hyperbilirubinemia, or spherocytosis on peripheral blood smears. However, some have subtle laboratory signs of HS, including slight reticulocytosis (average, 2.1 ± 0.8%), diminished haptoglobin levels, slightly elevated osmotic fragility, or slightly shortened times in the acidified glycerol lysis test (AGLT). Screening of normal Norwegian and German blood donors with the osmotic fragility test or AGLT showed a 0.9% to 1.1% incidence of previously unsuspected “very mild” HS. Presumably, some of these individuals were silent carriers of recessive HS genes, whereas others just had mild dominant HS. Unfortunately, no systematic molecular survey of this group of patients has been conducted.

Mild Hereditary Spherocytosis

Red cell production and destruction are balanced or nearly balanced in 20% to 30% of patients with HS. These persons are considered to have “compensated hemolysis” and are usually asymptomatic. In some patients, the diagnosis may be difficult because hemolysis, splenomegaly, and spherocytosis are unusually mild. In this group of patients, reticulocyte counts are generally less than 6%, and only 60% of patients have obvious spherocytosis on peripheral blood smears. About 10% of HS patients have very few (≤2%) or no detectable spherocytes. In addition, red cell spectrin and ankyrin levels are typically more than 80% of normal. As noted, many of these patients have band 3 or protein 4.2 defects.

Hemolysis may become severe with illnesses that cause splenomegaly, such as infectious mononucleosis. Hemolysis may also be exacerbated by pregnancy or exercise, to the point where it may impair athletic performance in endurance sports. In many of these patients, HS is diagnosed during family studies or discovered when adult patients are diagnosed with splenomegaly or gallstones. Although mild HS is usually familial, it develops sporadically in families with more severe disease. Presumably this is due to the coinheritance of modifying genes, such as those affecting spectrin or ankyrin synthesis or splenic function.

How to Explain “Compensated Hemolysis”

It is unclear why patients with HS and “compensated hemolysis” (i.e., normal hemoglobin levels) continue to have erythroid hyperplasia. Erythroid stimulation should disappear once hemoglobin levels reach normal if hemoglobin functions normally, as it does in HS. That it does not is shown by the fact that erythropoietin is inappropriately elevated (up to eight times normal) in HS patients with compensated hemolysis and no anemia, and the fact that the serum transferrin receptor concentration, a measure of erythropoiesis, is elevated. The levels of 2,3-diphosphoglycerate (2,3-DPG) are reportedly low in hereditary spherocytes before splenectomy, which could explain it; however the P ₅₀ of blood from HS patients is normal, which does not make sense. A more likely possibility is that the dehydrated HS red cells are rheologically impaired and do not adequately perfuse the juxtaglomerular renal vessels, where erythropoietin is produced, even when the hematocrit is normal. If this hypothesis is correct, then erythropoietin production should correlate inversely with red cell deformability in any disorder where red cells are dehydrated (e.g., sickle cell anemia, hereditary xerocytosis). Clinically, the high level of erythropoietin and persistent erythroid hyperplasia in patients with HS and a normal hemoglobin level indicate that physiologically the patients are still anemic. Indeed, it is possible that all HS patients are physiologically more anemic than their hemoglobin values suggest.

Moderate and Severe Hereditary Spherocytosis

A small fraction of patients with HS (5% to 10%) have moderately severe to severe anemia. Patients with “moderately severe” disease typically have a hemoglobin value of 6 to 8 g/dL, a reticulocyte count of about 10%, a bilirubin level greater than 2 mg/dL, and 40% to 70% of the normal red cell spectrin content. The mean corpuscular hemoglobin concentration (MCHC) is high and the red cell distribution width (RDW) is very high (typically >20%) (see Table 16-3 ). Such patients are more susceptible to the dangers of hemolytic and aplastic crises (see later) and may occasionally need a transfusion. This category includes patients with both dominant and recessive HS and a variety of molecular defects in spectrin and ankyrin. Occasional patients with band 3 defects may fall into this category.

Patients with “severe” disease, by definition, have life-threatening anemia and are transfusion dependent. They often have recessive HS. Most of these patients probably have isolated, severe spectrin deficiency (<40% of normal) resulting from a defect in α-spectrin, but some have ankyrin defects or are homozygous or compound heterozygous for band 3 mutations. Patients with severe HS are also distinguished by red cell morphologic findings. They often have some irregularly contoured or budding spherocytes and bizarre poikilocytes, in addition to typical spherocytes. Such cells are rare before splenectomy in patients with less severe disease, although some may be seen after splenectomy. In addition to the risks of recurrent transfusions, patients with “severe” disease often suffer from an aplastic crisis and may develop growth retardation, delayed sexual maturation, or aspects of thalassemic facies.

Hereditary Spherocytosis in Pregnancy

In general, unsplenectomized patients with HS have no significant complications during pregnancy except for anemia, which worsens because of plasma volume expansion and sometimes because of increased hemolysis. Hemolytic crises during pregnancy requiring transfusion have been reported. Folic acid deficiency is also a risk. One group reported that 20% of patients with HS received transfusions during pregnancy, but in our experience, pregnant patients with HS rarely need transfusions.

Hereditary Spherocytosis in the Neonate

HS often presents as jaundice in the first few days of life. Roughly one half of all patients with HS have a history of neonatal jaundice, and 91% of infants discovered to have HS in the first week of life are jaundiced (bilirubin level >10 mg/dL). In a French study of 402 jaundiced neonates requiring phototherapy, 1% were found to have HS. Hyperbilirubinemia usually appears in the first 2 days of life and bilirubin levels may rise rapidly, driven by the combination of hemolysis and the reduced capacity of the neonatal liver to conjugate bilirubin. Newborns with both HS and Gilbert syndrome, a common polymorphism in the promoter region of the uridine diphosphate–glucuronosyltransferase gene (UGT1A1) (see Chapter 4 ), develop neonatal jaundice more frequently and may develop severe hyperbilirubinemia. Kernicterus is a risk and exchange transfusion or treatment with barbiturates are sometimes necessary, but in most patients the jaundice is controlled with phototherapy.

Only 43% of neonates with HS are anemic at birth (hemoglobin level <15 g/dL), and severe anemia is rare, but those who present at birth tend to have a more severe course. Most have a normal hemoglobin level at birth, which then decreases sharply over the first 3 weeks of life, leading to a transient, severe anemia. In up to three fourths of HS infants, the anemia is severe enough to warrant transfusion or treatment with erythropoietin (see below). This anemia appears to be aggravated by an inability of the infant’s bone marrow to mount an appropriate erythropoietic response to anemia and to the development of splenic function.

Hydrops fetalis has been reported in a few patients and is associated with homozygosity or compound heterozygosity for spectrin or band 3 defects. If hydrops fetalis is detected in utero, the fetus may require intrauterine transfusions. No instances of hydrops fetalis have been associated with ankyrin deficiency. Perhaps infants with ankyrin defects, similar to ankyrin-deficient nb/nb mice, are partially protected in utero by the expression of ankyrin-related proteins in embryonic and fetal erythroblasts.

The diagnosis of HS is sometimes more difficult in the neonatal period than later in life. Splenomegaly is uncommon—at most the spleen tip is palpable—and reticulocytosis is variable and usually not severe. Only 35% of affected neonates have a reticulocyte count greater than 10%. In addition, the haptoglobin level is not a reliable indicator of hemolysis during the first few months of life. An even greater problem is that 33% of neonates with HS do not have significant numbers of spherocytes on their peripheral blood smears. Moreover, because fetal red cells are more osmotically resistant than adult cells when fresh and more osmotically sensitive after incubation at 37°C for 24 hours, the osmotic fragility test occasionally gives false-positive (incubated) or false-negative (unincubated) results unless fetal controls are used. Fortunately, these results have been published, and they appear to reliably differentiate neonates with HS, particularly when the incubated osmotic fragility test is used. However, in our experience it is rarely necessary to use fetal controls. The standard osmotic fragility test usually suffices to make the diagnosis in conjunction with the blood smear, Coombs test, and other data. Tests such as the eosin-5′-maleimide–binding test, AGLT, Pink test, and cryohemolysis test and osmotic gradient ektacytometry (all discussed later) are more sensitive and specific than the osmotic fragility test in older children and adults, but they have not been systematically tested in the neonatal period. Studying the erythrocytes from the parents is also very useful in the diagnosis of HS, particularly if the infant has received a transfusion.

However, it is clear that many infants with HS are not detected in the neonatal period on clinical grounds alone, even infants with hyperbilirubinemia, probably because the disease is not considered or is not obvious. Ascertainment is greatly enhanced if all infants with a high MCHC and a high RDW are evaluated for HS.

As noted above, some infants with HS become progressively more anemic in the first month or two of life and require transfusion. Fortunately, the problem is transient, except in rare patients with severe HS, and usually remits after one or two transfusions. Almost all infants with HS will outgrow the need for transfusion by the end of the first year of life. If the child is otherwise well, we allow the hemoglobin level to fall to 5.5 to 6.5 g/dL before giving transfusions to try to stimulate the marrow and only raise the hemoglobin level to 9 to 11 g/dL after transfusion to avoid suppressing the desired marrow response.

Administration of recombinant human erythropoietin (rHuEpo) to infants with HS was beneficial in reducing blood transfusions in an uncontrolled, open-label study. In this study, 13 of 16 infants given weekly subcutaneous injections of rHuEpo at a dosage of 1000 IU/kg had increases in the absolute reticulocyte count and hemoglobin values. The infants were given the same dosage of rHuEpo weekly based on their initial weight (at a range of initial ages from 16 to 119 days), a weekly hemoglobin level of less than 8 g/dL, and an absolute reticulocyte count of 200 × 10 ⁹ /L or less. Subsequent studies show similar effectiveness with different dosages and different frequencies. Therefore erythropoietin therapy in infants with HS has become an alternative to transfusion, although weekly erythropoietin injections can be challenging for parents. Also, erythropoietin is twice as expensive as a single transfusion and erythropoietin-treated infants don’t always avoid a transfusion. Further studies of recombinant erythropoietin therapy for neonatal anemia in HS are needed to determine the optimal time to initiate treatment, the optimal dosage, and the optimal duration of therapy.

Close observation of infants with HS is necessary to avoid complications of severe anemia. All infants with HS should have hemoglobin and reticulocyte measurements at least monthly during the first 6 months of life to avoid unnecessary side effects of severe anemia. The intervals may be increased thereafter, depending on the severity of anemia, but care must be taken to instruct parents on the signs and symptoms of an aplastic crisis.

Patterns of Membrane Protein Deficiency in Hereditary Spherocytosis

Four patterns predominate: isolated, partial spectrin deficiency, seen with defects in α-spectrin or β-spectrin; combined, partial spectrin and ankyrin deficiency, seen with primary ankyrin defects; combined, partial band 3 and protein 4.2 deficiency, seen with defects in band 3; and nearly complete loss of protein 4.2 and CD47, seen in primary defects of protein 4.2. In Europeans and Americans ankyrin and band 3 defects are most common, roughly 45% and 30%, respectively; β-spectrin defects account for perhaps 20%. Protein 4.2 and α-spectrin defects are both recessive and are rare. However, the pattern of membrane protein defects varies in different populations. In Japan, deficiencies of band 3 and protein 4.2 are the most common forms of HS. In Korean patients, ankyrin and spectrin defects are most common, but protein 4.2 deficiency occurs more often than in European or American patients.

Because most mutations are unique within each family and the mutation itself does not usually predict the phenotype, it is rarely clinically useful to determine the specific molecular defect in patients with HS. Readers who are interested in tables listing many of the mutations described to cause HS should consult lists in various chapters and reviews, including the previous edition of this text, or one of the two versions of the Human Gene Mutation Database: HGMD Professional, a subscription database that is available through many medical libraries, and the public version of HGMD, which is free but lags 3 years behind the subscription version.

For patients whose red cells are spectrin deficient, the degree of deficiency correlates well with the spheroidicity of HS red cells, the severity of hemolysis, and the response to splenectomy ( Fig. 16-5, A ). The mechanical properties of the cells, particularly their ability to withstand shear stress, also correlate with their spectrin content (see Fig. 16-5, B ). Microscopically, HS red cells with spectrin deficiency show fewer spectrin filaments interconnecting spectrin–actin–protein 4.1R junctional complexes, but overall skeletal architecture is preserved, except in the most severe forms of the disease.

Correlation of spectrin content with ( A ) unincubated osmotic fragility and ( B ) mechanical stability in hereditary spherocytosis. Circles are patients with typical autosomal dominant hereditary spherocytosis. Triangles are patients with nondominant spherocytosis. Reddish brown symbols denote patients who have had a splenectomy. The blue rectangle in A is the normal range.

( A, Modified from Agre P, Asimos A, Casella JF, et al: Inheritance pattern and clinical response to splenectomy as a reflection of erythrocyte spectrin deficiency in hereditary spherocytosis. N Engl J Med 315:1579–1583, 1986; B, Modified from Chasis JA, Agre PA, Mohandas N: Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis. J Clin Invest 82:617–623, 1988.)

α-Spectrin Defects: Recessive Hereditary Spherocytosis

In humans, α-spectrin synthesis exceeds β-spectrin synthesis by about four to one. Heterozygotes for α-spectrin synthetic defects still produce enough normal α-spectrin chains to pair with all of the β chains that are made. Thus α-spectrin defects are only apparent in the homozygous or compound heterozygous state and exhibit recessive inheritance. Moreover, there may only be a limited spectrum of mutations that cause clinical disease. Production-defective or “thalassemia-type” mutants that only moderately affect expression, such as α-spectrin ^LELY (to be discussed under hereditary elliptocytosis), have no phenotype, even in the homozygous state. Based on the few reported cases, it appears that the output of the α-spectrin gene must be less than 25% of normal to cause disease and may be as low as 8% of normal and still be compatible with life. No homozygous null mutations have been reported, so it is possible they are lethal in the embryo.

To date, only 2% of all the mutations reported to cause HS involve α-spectrin ( Fig. 16-6 ). Wichterle et al reported a patient with severe HS who was a compound heterozygote for two different α-spectrin gene defects: in one allele, there was a splicing defect associated with an upstream intronic mutation, α ^LEPRA (low-expression allele Prague); in the other allele, there was a null mutation, α ^PRAGUE . The α ^LEPRA allele produces one-sixth as much correctly spliced α-spectrin mRNA as normal. It is a relatively common allele, estimated to be 5% among Caucasians, and is in linkage dysequilibrium with α ^Bughill , which is an innocent amino acid polymorphism (Ala970Asp). Delaunay reported a similar family combining α ^LEPRA /α ^Bughill on one chromosome with a double mutant α ^LELY /α ^Bicêtre on the other, where the latter mutation essentially abolished α-spectrin output. In both cases the combination of the severely impaired α ^LEPRA allele with a null mutation of α-spectrin in trans , leads to significant, spectrin-deficient spherocytic anemia. As expected, even in the most severe forms of α-spectrin–linked recessive HS, obligate heterozygotes are asymptomatic and have little or no spectrin deficiency.

α-Spectrin mutations in hereditary spherocytosis (HS; black ) and hereditary elliptocytosis (HE; blue ). Missense mutations and in-frame insertions and deletions are indicated by circles . Splicing mutations that abolish or nearly abolish α-spectrin expression are indicated by solid triangles . Splicing mutations that partially decrease expression are indicated by shaded triangles . Note that HE mutations cluster near the N-terminus and tend to be amino acid substitutions that impair function (spectrin self-association), whereas HS mutations are defects that greatly decrease α-spectrin expression. The HS mutations are recessive because α-spectrin synthesis exceeds β-spectrin by a factor of 3 to 4, so that the expression of both α-spectrin genes must be greatly impaired to cause disease. α ^LELY is a very common polymorphism of α-spectrin that diminishes synthesis by about 50% (see section on hereditary elliptocytosis).

Clinical Features.

Less than 5% of patients with HS have primary defects in α-spectrin, but most of those patients have severe HS. Their red cells only contain 25% to 50% of the normal amount of both spectrin chains ( Table 16-4 ). Many are transfusion dependent, and even after splenectomy they may experience only a partial recovery. Blood smears are more bizarre than is typical for HS and show intense spherocytosis and irregular contracted cells ( Fig. 16-7, A ).

TABLE 16-4

Hereditary Spherocytosis Due to α-Spectrin Mutations

<5% of hereditary spherocytosis

Autosomal recessive since four-fold more α-spectrin is made than β-spectrin

A low expression, “thalassemia-type” defect in α-spectrin is often paired with a second presumably null allele on the opposite chromosome (in trans ). The combination must reduce α-spectrin expression by at least 75% to impair αβ-spectrin assembly enough to cause HS. Homozygous null mutations may be lethal

Severe hemolysis and anemia, often transfusion dependent

50%-75% spectrin deficiency in red cells

Red cell morphology not well defined but dense, irregular poikilocytes as well as spherocytes in some patients

 

Red cell morphologic findings in hereditary spherocytosis (HS). A , Severe HS due to a homozygous α-spectrin defect. The spherocytosis is intense, and some cells have an irregular shape. Few cells have any central pallor. B , HS due to a heterozygous β-spectrin null mutation. Echinocytes and acanthocytes (5% to 15%) are often observed in addition to typical spherocytes. C , Heterozygous ankyrin-deficient HS. Typical microspherocytes are evident among red cells with some central pallor. D , Heterozygous band 3–deficient HS. A few button mushroom–shaped (or “pincered”) red cells (arrowheads) are found in about 80% of patients with this form of HS. E , HS due to a homozygous protein 4.2 defect and nearly complete lack of the protein. Red cell morphologic findings are variable in this form of HS and, as here, there may be relatively few spherocytes. Echinocytes, slightly elongated elliptocytes (ovalocytes), stomatocytes, and other poikilocytes may be seen.

( A and E , From Delaunay J: The molecular basis of hereditary red cell membrane disorders. Blood Rev 21:1–20, 2007; D , from Bolton-Maggs P, Crary S: Hereditary spherocytosis . Epocrates Online. BMJ Publishing Group, Ltd. Accessed August 2013 at https://online.epocrates.com/u/29111143/Hereditary+spherocytosis/Summary/Highlights .)

β-Spectrin Defects: Dominant Hereditary Spherocytosis

β-Spectrin synthesis limits formation of αβ-spectrin heterodimers, so even modest defects in the expression of β-spectrin are apparent in the heterozygous state and exhibit a dominant pattern of inheritance. Of the reported HS mutations 17% are in β-spectrin, a finding that fits with the general impression that β-spectrin defects account for 15% to 25% of cases. Some groups report a much higher proportion of HS patients with isolated spectrin deficiency when sodium dodecyl sulfate (SDS) gels are used for membrane protein analysis, but that may reflect problems with assessing ankyrin deficiency on SDS gels (see later). The characteristics of HS due to β-spectrin deficiency are listed in Table 16-5 .

The described mutations in β-spectrin include initiation codon disruptions, frameshift and nonsense mutations, gene deletions and rearrangements, and splicing defects ( Fig. 16-8 ) . Most patients have defects that impair or abolish β-spectrin production from the affected allele. All of the β-spectrin is generated from the normal allele in trans , which compensates to varying degrees, so that the red cell spectrin content is only decreased by 15% to 40% instead of the expected 50%. This suggests that β-spectrin is also synthesized in excess, though much less so than α-spectrin. The degree of compensation probably accounts for much of the variation in clinical severity among patients with β-spectrin HS.

β-spectrin mutations in hereditary spherocytosis (HS; black ) and hereditary elliptocytosis (HE; blue ). Missense mutations and in-frame insertions and deletions are indicated by circles . Nonsense and other null mutations are indicated by squares . Partially shaded triangles denote splicing mutations that decrease but do not abolish expression. Patients with deletions of the whole β-spectrin locus and with gross rearrangements are also known but are not shown here. Note that HE mutations cluster near the C-terminus of β-spectrin and impair function (spectrin self-association) by altering amino acids or prematurely terminating the peptide chain. In contrast, most HS mutations abolish expression of the affected β-spectrin allele.

Patients with deletions of the whole β-spectrin locus, or with gross rearrangements, also have autism and learning disabilities, indicating that genes close to β-spectrin on chromosome 14 are involved in cognition. There are HS patients with potentially instructive missense mutations, including a group clustered in the actin and protein 4.1R binding domain (spectrins Atlanta, Kissimmee, and Oakland ) and in the spectrin repeat domain (spectrins Columbus, Birmingham, and Sao Paulo ). It would be interesting to know whether these amino acid substitutions cause HS because they impair a specific function or whether the missense mutations simply destabilize β-spectrin and act like null mutations. In the case of spectrin ^Kissimmee (Trp202Arg), which is the only mutation where function was examined, both things were true. The mutant protein lacked the ability to bind protein 4.1R and, as a consequence, bound poorly to actin, but it was also unstable and decreased in amount.

All of the reported defects of β-spectrin are limited to single families with the exception of spectrin ^Houston , a null mutation that has been found in several unrelated kindreds.

Ankyrin Defects

Spectrin heterodimers are only stable when bound to the membrane, and ankyrin, the high-affinity binding site, is normally present in limiting amounts. As a result, ankyrin defects are typically expressed as a dominant defect, although recessive mutations have been described. Ankyrin mutations are estimated to account for 30% to 60% of HS cases in Northern European populations and 5% to 10% of HS cases in Japan. Of all the known HS mutations, 35% are Ank1 defects. The characteristics of HS due to ankyrin defects are listed in Table 16-6 .

A role of ankyrin in HS was initially suggested by two patients with an atypical, unusually severe form of HS characterized by bizarre-shaped microspherocytes and red cell spectrin and ankyrin levels that were only 50% of normal. Ankyrin synthesis was half normal; spectrin synthesis was normal, but only 50% of the synthesized spectrin attached to the membrane, presumably owing to the lack of ankyrin sites. These results were the initial indication that ankyrin deficiency or dysfunction leads to concomitant spectrin deficiency in HS.

Additional studies showed that ankyrin deficiency is common in typical, dominant HS. When measured by radioimmunoassay, red cell membrane spectrin and ankyrin levels were less than the normal range in a large fraction of American kindreds, and the two proteins were proportionally decreased in nearly all the deficient kindreds. Similar data have been obtained in multiple populations.

Genetic analyses supported the hypothesis that a defect of the ankyrin gene could cause HS. Initially, studies of atypical patients with HS and karyotypic abnormalities, including translocations and interstitial deletions, defined a locus for HS at chromosomal segments 8p11.2-21.1. After the ankyrin gene was cloned, it was localized to this same region, 8p11.2. Fluorescence-based in situ hybridization of metaphase spreads from a patient with HS and an interstitial deletion of chromosome 8p11.1-p21 provided direct evidence that one copy of the erythrocyte ankyrin gene was deleted. Ankyrin content in the patient’s red blood cell membranes was reduced by 50%. Gene mapping then linked ankyrin to HS in a large family with typical dominant HS. Further studies showed that ankyrin mutations are common in HS and that many HS patients express only one of their two ankyrin alleles.

Ankyrin mutations occur in both dominant and nondominant HS. Frameshift and nonsense mutations, which abolish the normal ankyrin product, are particularly common in dominant HS ( Fig. 16-9 ). They produce mild to moderately severe hemolytic anemia, depending on the output of the remaining normal ankyrin gene and, perhaps, other modifier genes. For example, ankyrin ^Marburg and ankyrin ^Einbeck are frameshift mutations that occur in the NH ₂ -terminal (membrane) domain. Neither produces a detectable product in mature red cells. They would be expected to have a similar phenotype, but patients with ankyrin ^Einbeck have very mild disease and normal ankyrin levels, whereas patients with ankyrin ^Marburg have moderate to severe HS and moderate ankyrin deficiency (64% of normal). Understanding such phenotypic variations is one of the critical issues in HS research.

Ankyrin mutations, all of which cause hereditary spherocytosis (HS; black ). Missense mutations and in-frame insertions and deletions are indicated by circles . Nonsense and other null mutations are indicated by squares . Four mutations in the 5′-untranslated region that are believed to decrease expression are indicated by diamonds . Note that the majority of mutations are dominant and abolish expression of the affected ankyrin allele.

The most common ankyrin variant in patients with recessive HS is a T→C nucleotide substitution in the ankyrin gene promoter −108 bp from the translation start site. The allele occurs in 29% of German HS patients and 3% of normal individuals. It is usually in cis with a G→A nucleotide substitution 153 bp from the start site and impairs ankyrin synthesis in transgenic animals. A −72/73 dinucleotide deletion in the ankyrin promoter also impairs expression by disrupting the recognition site for the TATA-binding protein and interfering with transcription initiation. These “thalassemia-like” promoter mutations are silent in obligate heterozygotes; thus patients with nondominant HS must have a second mutation. The promoter mutations aggravate ankyrin mutations if they are inherited in trans , on the “normal” allele, particularly the −108 mutation, which is relatively common. For example, in a family with ankyrin ^Osterholtz , which is a null mutation, the daughter, who has a combination of ankyrin ^Osterholtz /ankyrin ^−72/73 , has more severe disease than her mother, who only carries ankyrin ^Osterholtz .

Patients with gene deletions involving the ankyrin locus at 8p11.2 often have features both of HS and Kallmann syndrome (hypogonadotrophic hypogonadism, type 2, due to FGFR1 defects) because the two loci are less than 4 Mb apart. Most of these patients also have psychomotor developmental delay, resulting from loss of another still undefined gene in the region.

Band 3 Defects: Dominant Hereditary Spherocytosis

Band 3 deficiency is present in approximately 25% to 45% of European and American patients with dominant HS, and band 3 defects account for 37% of the known HS mutations. Erythrocyte membranes from these patients are equally deficient in band 3 and protein 4.2, which requires band 3 to bind to the red cell membrane. The characteristics of HS due to band 3 deficiency are listed in Table 16-7 .

Defects in band 3 produce a variety of red cell phenotypes ( Fig. 16-10 ), including stomatocytosis, cryohydrocytosis, Southeast Asian ovalocytosis (SAO), and acanthocytosis, as well as HS. These are discussed in later sections of the chapter. The HS mutations are spread throughout the band 3 gene, occurring in both the cytoplasmic and membrane-spanning domains. They include nonsense mutations, frameshift mutations, partial gene duplications, other missense mutations, and splicing defects. Many of the mutations cause messenger RNA (mRNA) instability or a misfolded protein that is degraded or not inserted into the membrane. Conserved arginine residues are frequent sites of missense mutations in the transmembrane domain of the band 3 gene. The affected arginine residues are all located in the same position on the inside edge of membrane-spanning segments and are believed to interact with negatively charged phospholipids in the inner bilayer and help orient the segments. As predicted, in vitro studies show that these arginine substitutions, as well as several other HS-associated band 3 missense mutations, exhibit defective cellular trafficking from the endoplasmic reticulum to the plasma membrane, perhaps due to misfolding. Determination of the structure of the cytoplasmic domain of band 3 and detailed studies of the topography of the transmembrane domain are beginning to provide insights into the biologic significance of the missense mutations.

Band 3 mutations associated with hereditary spherocytosis (HS; black ), various inherited disorders of cation permeability (orange) , distal renal tubular acidosis without (pink) or with (purple) hemolytic anemia, and hereditary acanthocytosis (green) . Missense mutations and in-frame insertions and deletions are indicated by circles . Nonsense and other null mutations are indicated by squares . Splicing mutations that abolish or nearly abolish band 3 expression are indicated by solid triangles . Splicing mutations that are or are presumed to be less severe are indicated by open triangles . One mutation in the 5′-untranslated region that is believed to decrease expression is indicated by a diamond . The cation permeability defects include stomatocytosis, cryohydrocytosis, a cryohydrocytosis-like form of HS, and Southeast Asian ovalocytosis. They are discussed later in the chapter.

Similar to the other molecular defects that cause HS, band 3 mutations are unique familial defects with no common mutations among affected groups.

Protein 4.2 Defects: Recessive Hereditary Spherocytosis

HS caused by protein 4.2 deficiency is most common in Japan, although a few patients have been described in other populations. Overall, protein 4.2 mutations account for 9% of the published HS molecular defects. The characteristics of this form of HS are summarized in Table 16-8 .

Multiple protein 4.2 mutations have been described ( Fig. 16-11 ). The most common one is 4.2 ^Nippon , in which the red cells contain only a small quantity of a 74/72-kD doublet of protein 4.2 instead of the usual abundant 72-kD species, because of an Ala142→Thr mutation that affects the processing of the protein 4.2 mRNA. Protein 4.2 ^Nippon –deficient membranes lose 70% of their ankyrin with low ionic strength extraction, and the ankyrin loss is blocked by preincubation of the membranes with purified 4.2, which suggests that protein 4.2 stabilizes ankyrin on the membrane. This hypothesis is supported by the observations that the amount of protein 4.2 is low in the red cell membranes of ankyrin-deficient nb/nb mice and in HS patients who lack one ankyrin gene. In addition, red cells from patients homozygous for 4.2 ^Nippon are fragile and have heat-sensitive skeletons, clumped intramembranous particles, and increased lateral mobility of band 3. A weakening of ankyrin–band 3 binding due to loss of protein 4.2 has also been observed. However, ankyrin lability is not evident in all patients with protein 4.2 deficiency. Patients whose red cells lack protein 4.2 also lack CD47. CD47 connects the band3–ankyrin–4.2 complex to the Rh complex of which CD47 is a part. Loss of CD47 may also be important because CD47 is recognized by an inhibitory receptor on macrophages that blocks phagocytosis.

Protein 4.2 mutations associated with recessive hereditary spherocytosis (HS; black ). Missense mutations and in-frame insertions and deletions are indicated by circles . Nonsense and other null mutations are indicated by squares . One splicing mutation that nearly abolishes protein 4.2 expression is indicated by a solid triangle . 4.2 ^Nippon is a common mutation in Japanese cases.

Finally, partial erythrocyte protein 4.2 deficiency has also been associated with mutations of band 3. Presumably, these mutations affect sites of band 3–protein 4.2 interactions.

Loss of Membrane Surface by Vesiculation

The primary membrane lesions in HS all involve vertical interactions between the skeleton and the bilayer and fit the theory that HS is caused by local disconnection of the skeleton from the bilayer and its integral membrane proteins, followed by vesiculation of the unsupported surface components. This, in turn, leads to progressive reduction in membrane surface area and to a shape called a spherocyte, although it usually ranges between a thickened discocyte and a spherostomatocyte.

Biomechanical measurements show that HS membranes are fragile. The force required to fragment the membrane is diminished and is proportional to the density of spectrin on the membrane. Membrane elasticity and bending stiffness are also reduced and are proportional to spectrin density. In addition, HS red cells lose membrane more readily than normal red cells when metabolically deprived or when their ghosts are subjected to conditions facilitating vesiculation. This has not been shown to occur in metabolically healthy HS spherocytes, perhaps because it occurs slowly (1% to 2% per day) under such conditions. However, massive vesiculation is evident in mice and zebrafish with severe spherocytic hemolytic anemias ( Fig. 16-12 ).

Scanning electron micrograph of a mouse red cell that lacks α-spectrin and has only trace amounts of β-spectrin. Note the extraordinary membrane instability and microvesiculation. It is assumed that similar vesiculation and membrane loss occur in human HS but at a much slower rate, consistent with the milder degree of spectrin deficiency in the human disease.

Because budding red cells are rarely observed in typical blood smears from patients with HS, the postulated vesiculation may either involve microscopic vesicles or occur in the reticuloendothelial system. Vesiculation may also occur in the bone marrow of patients with HS because similar decreases in the red cell surface area have been found in HS reticulocytes and in mature red blood cells. When membrane vesicles are induced in normal red cells, they originate at the tips of spicules, where the lipid bilayer uncouples from the underlying skeleton (see Fig. 15-29 ). The vesicles are small (≈100 nm) and devoid of hemoglobin and skeletal proteins and would be invisible by light microscopy. Tiny (50- to 80-nm) bumps have been detected by atomic force microscopy, on the surface of red cells obtained from patients with HS whose red cells are actively hemolyzing ( Fig. 16-13 ). The bumps could be microvesicles, although this needs to be proven. They are less than the length of a spectrin molecule (100 nm) and are not present on red cells from splenectomized patients.

Comparison of the surface of a normal red cell ( A ) and a hereditary spherocyte (HS) (presplenectomy) ( B ) by atomic force microscopy. Note that at high magnification, 50 nanometers (nm) to 80 nm pseudopodia are evident on the surface of the spherocyte (bottom of B ). These probably represent membrane microvesicles. They were not observed following splenectomy.

Hypothesis 1.

The observation that spectrin or spectrin/ankyrin deficiencies are common in HS has led to the suggestion that they are the primary cause of spherocytosis. According to this hypothesis ( Fig. 16-14 , hypothesis 1), interactions of spectrin with bilayer lipids or proteins are required to stabilize the membrane. Spectrin-deficient areas would tend to bud off, leading to spherocytosis. However, this conjecture does not explain how spherocytes develop in patients whose red cells are deficient in band 3 but have normal amounts of spectrin.

Pathophysiology of hereditary spherocytosis (HS). Hypothesis 1 assumes that the “membrane” (i.e., the lipid bilayer and the integral membrane proteins) is directly stabilized by interactions with spectrin or other elements of the membrane skeleton. Spectrin-deficient areas, lacking support, bud off, leading to spherocytosis. This may be the dominant mechanism in spectrin, ankyrin, and perhaps protein 4.2 deficiency. Hypothesis 2 assumes the membrane is stabilized by the interactions of band 3 with neighboring lipids. The influence of band 3 extends into the lipid milieu because the immobilized lipids adjacent to band 3 interact with and slow the lipids in the next layer, and so on. In band 3–deficient cells, the lipid area between band 3 molecules increases, and unsupported lipids are lost. This may be the primary mechanism in band 3 deficiency. Both mechanisms lead to loss of membrane surface, spherocytosis, reduced red cell deformability, and splenic trapping, where the unfavorable splenic environment shown in the box augments membrane loss, a process called splenic conditioning. Some red cells hemolyze, some escape as microspherocytes, susceptible to recapture, and some adapt by loss of cell volume (K ⁺ and H ₂ O), leading to a temporary reprieve before more membrane loss reinitiates the process.

(Modified from Perrotta S, Gallagher PG, Mohandas N: Hereditary spherocytosis. Lancet 372:1411–1426, 2008.)

Loss of Cellular Deformability

Hereditary spherocytes hemolyze because of the rheologic consequences of their decreased surface-to-volume ratio. The red cell membrane is very flexible, but it can only expand its surface area about 3% before rupturing. Thus erythrocytes become less and less deformable as surface area is lost. Clever experiments show that the spleen begins to sense and restrain red cells that have lost as little as 2% to 3% of their membrane surface and that loss of more than 18% of the red cell surface leads to complete splenic sequestration. This corresponds to the 15% to 20% loss of membrane lipids and, presumably, membrane surface in HS red cells. For HS red cells, poor deformability is only a hindrance in the spleen, because the cells have a nearly normal life span after splenectomy.

Splenic Sequestration and Conditioning

In the spleen most of the arterial blood empties directly into the splenic cords, a tortuous maze of interconnecting narrow passages formed by reticular cells and lined with phagocytes (see discussion of splenic structure and function in Chapter 23 ). Histologically, this is an “open” circulation, but most of the blood that enters the cords normally travels by fairly direct (i.e., functionally closed) pathways. If passage through these channels 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. Whichever route is taken, to reenter the circulation, red cells must squeeze through spaces between the endothelial cells that form the walls of the venous sinuses ( Fig. 16-15 ). Even when maximally distended, these narrow slits are always much smaller than red cells, which are greatly distorted during their passage. Experiments have shown that spherocytes are selectively sequestered at this cordal-sinus juncture. As a consequence, spleens from patients with HS have massively congested cords and relatively empty sinuses. In electron micrographs, few spherocytes are seen in transit through the sinus wall, which contrasts to the situation in normal spleens, where cells in transit are readily found.

Scanning electron micrograph of a splenic sinus viewed from within the splenic cords. The narrow transmural slits between the endothelial cells (END) and adventitial cells (ADV) of the sinus are evident. The edges of these slits probably touch each other in the normal spleen, so the openings are potential structures rather than fixed pores. They are revealed here because of a drying artifact. Note that the adjacent erythrocytes (RBC) are considerably larger than the slits and hence must be flexible to pass into the splenic sinuses and return to the venous circulation. If they are unable to pass into the sinuses, macrophages (MAC) lie in wait.

(From Weiss L: A scanning electron microscopic study of the spleen. Blood 43:665–691, 1974.)

During detention in the spleen, HS red cells undergo additional damage marked by further loss of surface area and an increase in cell density. Many of these “conditioned” red cells escape the hostile environment of the spleen and reenter the circulation. In unsplenectomized patients with HS, two populations of spherocytes are detectable: a minor population of hyperchromic “microspherocytes” that form the “tail” of very fragile cells in the unincubated osmotic fragility test and a major population that may be only slightly more spheroidal than normal.

By 1913, it was known that HS red cells obtained from the splenic vein were more osmotically fragile than those in the peripheral circulation. However, the significance of this finding was not clear until the classic studies in the 1950s by Emerson and Young, who showed that osmotically fragile microspherocytes are concentrated in and emanate from the splenic pulp ( Fig. 16-16 ). After splenectomy, spherocytosis persists, but the tail of hyperfragile red cells is no longer evident on osmotic fragility testing. These and other data led to the conclusion that the spleen detains and “conditions” circulating HS red cells in a way that increases their spheroidicity and hastens their demise. The kinetics of this process were beautifully illustrated in vivo by Griggs, who found that a cohort of ⁵⁹ Fe-labeled HS red cells gradually shifted from the major, less fragile population to the more fragile, conditioned population 7 to 11 days after entering the circulation. Although most conditioned HS red cells that escape the spleen are probably recaptured and destroyed, the damage incurred is sufficient to permit extrasplenic recognition and removal, because conditioned spherocytes, isolated from the spleen and reinfused postoperatively, are rapidly eliminated.

Osmotic fragility in hereditary spherocytosis (HS) . In the osmotic fragility test, red cells are suspended in salt solutions of varying tonicities between isotonic saline (0.9% NaCl) (left) and distilled water (right). Normal red cells swell in hypotonic media and eventually reach a limiting spherical shape, beyond which they hemolyze (bottom) . Spherocytes, which begin with a lower than normal surface-to-volume ratio, can swell less before they reach their limiting volume; hence, their osmotic fragility curve is shifted to the left (higher salt concentrations). They are said to be osmotically fragile. Before splenectomy, a small proportion of the red cells are extra fragile “microspherocytes” and produce a “tail” on the osmotic fragility curve. The cells have been conditioned by the spleen, as shown by their higher concentration in the splenic vein and especially the splenic cords.

The mechanism of splenic conditioning is less certain. It is difficult to obtain accurate information about the cordal environment, but the existing data suggest it is metabolically inhospitable. Crowded red cells must compete for limited supplies of glucose in acidic surroundings (pH 6.8 to 7.2) where glycolysis is inhibited. The acidic environment induces Cl ⁻ and water entry and cell swelling but also stimulates the K ⁺ ,Cl ⁻ cotransporter, which produces a net loss of K ⁺ and water from the cells. The adverse effects of the cordal environment are further compounded by the presence of oxidant-producing macrophages. It has also been suggested that methylation of erythrocyte membrane proteins could contribute to the splenic conditioning of spectrin-deficient HS red cells. Hence, the spherocyte, detained in the splenic cords because of its surface deficiency, is stressed by erythrostasis in a metabolically threatening environment.

Erythrostasis

The spherocyte is particularly vulnerable to erythrostasis. This is the basis of the well-known autohemolysis test. During prolonged sterile incubation in the absence of supplemental glucose, red cells undergo a series of changes that culminate in hemolysis. The sequence of the changes is the same for HS and normal red cells; however, because HS cells are abnormally leaky and bear unstable membranes, their degeneration is accelerated.

HS red cells are initially jeopardized, because their membrane permeability to Na ⁺ is mildly increased. Their propensity to accumulate Na ⁺ and water is normally balanced by increased Na ⁺ pumping; however, the increased dependence on glycolysis is detrimental in erythrostasis, where substrate is limited. HS red cells exhaust serum glucose and become ATP depleted more rapidly than normal red cells. As ATP levels fall, ATP-dependent Na ⁺ ,K ⁺ and Ca ²⁺ pumps fail, and the cells gain Na ⁺ and water and swell. Later, when the Ca ²⁺ -dependent K ⁺ (Gárdos) pathway is activated, K ⁺ loss predominates and the cells lose water and shrink. The Na ⁺ gain is accelerated in HS red cells but is insufficient by itself to induce hemolysis. However, HS red cells are doubly jeopardized. As noted earlier, they are inherently unstable and fragment excessively during metabolic depletion. Membrane lipids (and probably integral membrane proteins) are lost at more than twice the normal rate. At first this surface loss is balanced by cell dehydration, but eventually (within 30 to 48 hours) membrane loss predominates, the cells exceed their critical hemolytic volume, and autohemolysis ensues.

Consequences of Splenic Trapping

Calculations indicate that the average normal red cell passes through the splenic cords about 14,000 times during its lifetime and has an average transit time of 30 to 40 seconds, surprisingly close to measured transient times in normal human spleens in vivo. The calculated residence time of the average HS red cell in the splenic cords is much longer, perhaps as long as 15 to 150 minutes, but still far short of the time required for metabolic depletion to occur. This conclusion is supported by direct analysis of splenic red cells. HS red cells obtained from the splenic pulp and containing 80% to 100% conditioned cells are moderately cation depleted and show changes in adenosine diphosphate (ADP) and 2,3-DPG concentrations consistent with metabolism in an acidic environment, but their ATP levels are normal. Others have reported similar results.

The data suggest that splenic conditioning is caused by mechanisms other than ATP depletion. For example, K ⁺ loss and membrane instability may be exacerbated by the high concentrations of acids and oxidants that must exist in a spleen filled with activated macrophages lunching on trapped HS red cells. In vitro, oxidants from activated phagocytes can diffuse across the membranes of bystander red cells and damage intracellular proteins within minutes. Red cells moving through the rapid transit pathways in the spleen might escape damage, but those caught in cordal traffic would be vulnerable. Oxidants, even in relatively low concentrations, cause selective K ⁺ loss by a variety of mechanisms and also damage membrane skeletal proteins. Finally, there is some evidence that HS red cells may be abnormally sensitive to oxidants. When exposed to peroxides they undergo remarkable blebbing and, presumably, vesiculation. If a similar process occurs in the spleen, it could be responsible for the excessive surface loss observed in conditioned cells.

The possibility that macrophages may directly condition HS red cells should also be considered. It is well known that spherocytosis often results from the interaction of immunoglobulin G (IgG)–coated red cells with macrophages, but HS red cells do not have abnormal levels of surface IgG. Macrophages also bear receptors for oxidized lipids (scavenger receptor) and phosphatidylserine, but there is no evidence at present that HS red cells expose the relevant ligands. The involvement of macrophages is supported by observations that large doses of corticosteroids markedly ameliorate HS in unsplenectomized patients. The effects are similar to those produced by splenectomy. It is well known that similar doses of corticosteroids inhibit splenic processing and destruction of IgG- or C3b-coated red cells in patients with immunohemolytic anemias, probably by suppressing macrophage-induced red cell sphering and phagocytosis. Electron microscopy shows that splenic erythrophagocytosis is common in HS, particularly in the splenic cords. In addition, phagocytes expressed from the cords of patients with HS contain bits of ghostlike “debris,” presumably resulting from membrane fragmentation.

Adaption: Benefits of Red Cell Dehydration for the HS Red Cell

It has been known for many years that HS red cells intrinsically leak more Na ⁺ and K ⁺ ions than normal cells. All the Na ⁺ transmembrane movements are increased, including passive diffusion, Na ⁺ ,K ⁺ -ATPase pump, Na ⁺ ,K ⁺ ,2Cl ⁻ cotransport, and Na ⁺ , Li ⁺ countertransport. The excessive Na ⁺ influx activates the Na ⁺ ,K ⁺ -ATPase pump, and the accelerated pumping, in turn, increases ATP turnover and glycolysis. At one time, it was believed that this modest Na ⁺ leak was responsible for the hemolysis of hereditary spherocytes, particularly those cells trapped in the unfavorable metabolic environment of the spleen, but it now is clear that this is incorrect, because the magnitude of the Na ⁺ flux does not correlate with the extent of hemolysis in HS.

The dehydration of HS red cells, as reflected in their high MCHC, is likely to be caused, at least in part, by the adverse environment of the spleen. This assumption has been made on finding that spherocytes from surgically removed spleens are the most dehydrated. The pathways causing HS red cell dehydration have not been clearly defined. One likely candidate is the Gárdos channel, a Ca ²⁺ -activated K ⁺ channel, which is known to contribute to erythrocyte dehydration in sickle cell anemia and thalassemia. One study showed that the Gárdos channel is functionally upregulated in a form of mouse HS caused by lack of erythrocyte protein 4.1R. When these mice were treated with clotrimazole, a known Gárdos channel blocker, hemolysis and anemia worsened, indicating that red cell dehydration protected the hereditary spherocytes, presumably by preserving their surface-to-volume ratio and therefore their flexibility in the face of surface loss.

Another possible cause of red cell dehydration in HS is increased K ⁺ ,Cl ⁻ cotransport, which is activated by acid pH. HS red cells, particularly from unsplenectomized subjects, have a low intracellular pH, reflecting the low pH of the splenic environment. The K ⁺ ,Cl ⁻ cotransport pathway is also activated by oxidation, which is likely to be caused by splenic macrophages.

Hyperactivity of the Na ⁺ ,K ⁺ pump, triggered by increased intracellular Na ⁺ , can dehydrate red cells directly, because three Na ⁺ ions are extruded in exchange for only two K ⁺ ions. The loss of monovalent cations is accompanied by water.

A fourth possible cause is the recently described mechanosensitive Piezo1 channel, which responds to membrane stretch and is thought to be involved in homeostatic adjustments to changes in volume. It is defective in hereditary xerocytosis, a disease characterized by red cell dehydration.

Whichever transport pathways cause HS red cells to lose K ⁺ and water, it is likely that the dehydration is a form of “adaption” ( Fig. 16-14 ) that prolongs the life of the HS red cell. How much it does so is unknown. Polymorphisms in cation channels like Piezo1 and calcium ATPase 4, in the globin loci, in major membrane skeletal proteins, and in genes like TAF3 , DNAJA4 , WDR61 and HBS1L-MYB are associated with variation in human MCHC. It will be interesting to see if these genes affect the severity of HS.

Red Cell Morphology

Spherocytes (see Fig. 16-2 ) are the hallmark of HS. They are dense, round, and hyperchromic; lack central pallor; and have a decreased mean cell diameter. They are always evident in blood smears from patients with moderate or moderately severe HS but are obvious in only 25% to 35% of patients with mild HS. Hereditary spherocytes are technically misnamed, as they range in shape from thickened discocytes to spherostomatocytes when examined under the scanning electron microscope.

In patients with HS, spherocytes and microspherocytes are the predominant abnormal cells on the peripheral blood smear (other than polychromatophils). However, there are morphologic variations that occur with some of the specific membrane defects in HS (see Fig. 16-7 ). Patients with ankyrin defects, the most common subgroup, have typical smears (see Fig. 16-7, C ), but many patients with β-spectrin defects also have a subpopulation of acanthocytes or hyperchromic echinocytes (see Fig. 16-7, B ) and most patients with band 3–deficient HS also have a small number of button mushroom–shaped red cells on their smears (see Fig. 16-7, D ). In severe HS, caused by homozygous or compound heterozygous α-spectrin mutations or by severe ankyrin defects, misshapen spherocytes, spiculated red cells, and bizarre poikilocytes may be seen and even dominate the blood smear (see Fig. 16-7, A ). Red cell morphologic findings are variable in protein 4.2 deficiency. Some patients have typical spherocytosis, but acanthocytes, echinocytes, ovalostomatocytes, and other poikilocytes have been observed in others (see Fig. 16-7, E ). Finally, some patients with truncated β-spectrin chains have spherocytic elliptocytosis (see later).

In contrast to red cells in IgG-mediated autoimmune hemolytic anemias, where spherocytosis also dominates the blood picture, HS red cells lose most of their surface area in the bone marrow instead of in the circulation. Nucleated red cells are uncommon in blood smears, except in the most severe forms of HS. Howell-Jolly bodies are also uncommon before splenectomy (4% of patients) and suggest reticuloendothelial blockade.

Red Cell Indices

Most patients have mild to moderate HS with mild anemia (hemoglobin level of 9 to 12 g/dL) or no anemia at all (so-called compensated hemolysis). In moderate to severe HS, the hemoglobin concentration ranges from 6 to 9 g/dL. In patients with the most severe disease, the hemoglobin concentration may drop to as low as 4 to 5 g/dL. The reticulocyte count is elevated without a comparable increase in the immature reticulocyte fraction.

The MCHC of HS red cells is increased due to relative cellular dehydration. The average MCHC exceeds the upper limit of normal (36%) in about one half of patients with HS, but all patients have some dehydrated cells. The Technicon H1 blood counter and its successors, which use the principle of flow cytometry (i.e., dual-angle laser light scattering), measure the mean corpuscular volume (MCV) and MCHC directly and provide histograms of both parameters. There is a right shift of the MCHC histogram in HS with an increased (>4%) population of hyperdense cells (MCHC > 41 g/dL) due to red cell dehydration, and a broadening of the distribution of MCVs, due to the mixture of microspherocytes and reticulocytes ( Fig. 16-17 ). This excess proportion of hyperdense cells is accurate enough to identify nearly all patients with HS and is one of the easiest and most accurate ways to diagnose the disease when one member of a family is already known to have HS.

Histogram of the distribution of mean cell volume (MCV) ( A ) and mean cell hemoglobin concentration (MCHC) ( B ) in red cells of a patient with hereditary spherocytosis (HS) before splenectomy. The vertical lines mark the normal limits of the distributions. For the volume distribution, they separate microcytic cells (<60 fL) and macrocytic cells (>120 fL) from normocytic red cells (60 to 120 fL). For the hemoglobin concentration distribution, they separate hypochromic cells (<28 g/dL) and hyperdense cells (>41 g/dL) from normochromic cells (28 to 41 g/dL). The data were collected with a laser-scattering blood counter. Note that the HS patient has subpopulations of microcytes (low MCV) and dehydrated cells (high MCHC). All 21 HS patients in this study had similar subpopulations.

(Modified from Pati AR, Patton WN, Harris RI: The use of the Technicon H1 in the diagnosis of hereditary spherocytosis. Clin Lab Haematol 11:27–30, 1989.)

The MCV usually falls within the lower normal range in HS, but it is low in about 8% of adults and 16% of children, especially those with more severe HS. It is rare for the MCV to be less than 70 fL unless there is concomitant thalassemia or iron deficiency. However, the MCV is low relative to the age of the cells (reticulocytes have a high MCV) in all patients with HS, reflecting the dehydrated state of HS red cells. The reticulocyte MCV is also low, which contrasts with IgG-mediated autoimmune hemolytic anemia, where spherocytes are present but the reticulocyte indices are normal.

The RDW of HS erythrocytes is also very high, particularly in patients with more severe disease (see Table 16-3 ).

Various studies show that the MCHC or the percentage of hyperdense cells and the high RDW discriminate HS patients from normal individuals, though less so following splenectomy. An MCHC greater than 35 g/dL has a sensitivity of 70% and a specificity of 86% in diagnosing HS; an RDW greater than 14 has 85% sensitivity and 97% specificity. Combining the MCHC with the erythrocyte distribution width leads to a specificity approaching 100%. The RDW, MCHC, hemoglobin : MCHC ratio, and hemoglobin : RDW ratio all correlate with the severity of disease (see Table 16-3 ).

Fragility and Autohemolysis Tests

Eosin-5′-Maleimide–Binding Test

When red blood cells are treated with the fluorescent probe eosin-5′-maleimide (EMA), 80% of the fluorophore binds to Lys430 on the outside surface of band 3. The other 20% binds to three proteins in the Rh complex: the Rh blood group proteins, RhAG, and CD47. Thus the measurement of EMA fluorescence detects loss of membrane surface if that loss includes band 3 or the Rh complex. The EMA test is usually done with a flow cytometer so the fluorescence of individual cells is quantified. Because membrane vesicles containing band 3 are believed to be lost in HS due to spectrin and ankyrin deficiency (see Fig. 16-14 ) and CD47 and, perhaps, band 3 molecules are lost in HS due to protein 4.2 deficiency, the EMA test detects all the different forms of HS, not just HS due to primary band 3 deficiency. The test is both highly sensitive (92% to 100%) and highly specific (94% to 100%) for HS. The only exception is one study that reported only 70% specificity. The test requires very little blood, does not require preincubation, takes little time to perform, is not affected by storage of red cells for a week, and is probably the single best test for diagnosing HS at the present time. It is recommended by the General Haematology Task Force of the British Committee for Standards in Haematology, and when combined with the AGLT achieved 100% sensitivity in one recent comparison of tests for HS. In particular, the test is normal in autoimmune hemolytic anemias, which give false-positive results in the osmotic fragility test, AGLT, and cryohemolysis test. The EMA test is abnormal in disorders where membrane surface is decreased or band 3 is abnormal, such as HPP, SAO, cryohydrocytosis, congenital dyserythropoietic anemia, type II, and Rh _null disease. However, these are all rare disorders and are fairly easy to distinguish by red cell morphologic findings and other criteria.

Osmotic Gradient Ektacytometry

The ektacytometer is a laser diffraction viscometer, a device that measures how much red cells are deformed by shear stress. The device can be used to measure membrane mechanical strength by determining the shear stress needed to fragment isolated red cell membranes, or it can be used to assess red cell surface area, surface-to-volume ratio, and cellular hydration by measuring red cell deformation as a function of osmolarity at a constant shear stress. The latter method is termed osmotic gradient ektacytometry and is the gold standard method for diagnosing HS ( Fig. 16-18 ). One study demonstrated a 100% detection rate of HS using osmotic gradient ektacytometry and only a 66% detection rate of HS using the osmotic fragility test in the same population. Unfortunately, only a few laboratories in the world are equipped with an ektacytometer and the instrument is no longer commercially available.

Osmotic gradient ektacytometry of hereditary spherocytosis (HS) erythrocytes with differing degrees of spectrin deficiency. In this technique, red cells are stretched by flow in a viscometer in a solution of gradually increasing osmolarity and the degree of red cell deformation is measured by laser diffraction. The deformability index (DI) , an arbitary measure of deformation, is plotted as a function of the osmolarity. In the HS red cells (red-brown lines) , the minimum DI observed in the hypotonic region (thin arrow) is shifted to the right of the control region (blue shaded area) , indicating a decrease in the cell surface area–to-volume ratio. The maximum DI (DI _max ) associated with the spectrin-deficient HS red cells (thick arrow) is less than that of control cells due to the reduced surface area of the HS cells. The more pronounced the spectrin deficiency, the greater is the loss of surface area and the lower is the DI _max . The osmolality in the hyperosmolar region at which the DI reaches half of its maximum value is a measure of the hydration state of the red cells. It is decreased in the HS patient with the lowest spectrin content, indicating cellular dehydration.

(Modified from Tse WT, Lux SE: Hereditary spherocytosis and hereditary elliptocytosis. In Scriver CR, Beaudet AL, Sly WS, Valle D, editors: The metabolic and molecular bases of inherited disease , 8th ed. New York, 2001, McGraw-Hill p. 4665–4727.)

Membrane Protein, RNA, and DNA Measurements

Specialized tests are occasionally used to study patients with complicated disease or those for whom additional information is desired. Useful tests for these purposes include structural and functional studies of erythrocyte membrane proteins, such as protein quantitation, limited tryptic digestion of spectrin, membrane protein synthesis and assembly, and ion transport studies.

Membrane protein concentrations are usually assessed using SDS gels. Individual stained bands are quantified by densitometry or by eluting the dye from an excised band and measuring its concentration spectrophotometrically. This technique is satisfactory for detecting spectrin deficiency (expressed as a spectrin-to–band 3 ratio), although it is not as accurate as a radioimmunoassay or enzyme-linked immunoassay. With densitometry, spectrin and ankyrin deficiencies may be underestimated because they are normalized to band 3, which is partially lost along with membrane lipids as spherocytes circulate. As a result, the spectrin-to–band 3 ratio is lower after splenectomy. This is even more true for ankyrin, which is present in smaller amounts, migrates close to β-spectrin on SDS gels, and is elevated in reticulocytes. It is likely that patients with ankyrin defects, who have combined spectrin and ankyrin deficiency, are often misdiagnosed as having pure spectrin deficiency on SDS gels. SDS gels are satisfactory for detecting band 3 deficiency (elevated spectrin-to–band 3 ratio) or protein 4.2 deficiency, and they are useful in combination with antibody staining (Western blots) for detecting mutant proteins of altered size.

Moreover, SDS gels do not always identify the abnormal protein in HS, because missense mutations may cause functional defects rather than quantitative defects and because protein deficiencies may be as small as 10%. Therefore several other approaches have been described in an attempt to define specific molecular defects. In families with multiple affected members, linkage analysis has been performed to include or exclude the candidate genes. In addition, suspected proteins can be sequenced using direct nucleotide sequence analysis of polymerase chain reaction (PCR)–amplified complementary DNA (cDNA) or genomic DNA. Currently, no commercial laboratories identify the molecular defects in HS membrane proteins, and these analyses are performed only by a few research laboratories. However, with the rapidly falling costs of both whole-exome and whole-genome sequencing it is likely that DNA sequencing may become the most cost-effective way to diagnose HS (and many other inherited diseases) in the near future. Because HS membrane protein mutations are mostly restricted to specific families and do not predict the severity of the disease, in most cases it is not clinically important to know the exact molecular defect. More detailed studies, sometimes including DNA sequencing, should be considered in patients where the diagnosis is unclear after standard testing and in patients whose HS is more severe than in other family members, possibly due to coinheritance of another deleterious allele.

ABO Incompatibility in Neonates

In the neonatal period it may be challenging to differentiate HS from ABO incompatibility. Anemia, hyperbilirubinemia, circulating microspherocytes, and altered osmotic fragility are found in both conditions, and results of the direct Coombs test are sometimes negative in ABO incompatibility. However, in most infants with ABO incompatibility and significant hemolysis, 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. The combination of HS and ABO incompatibility, though rare, is particularly severe.

Immunohemolytic Anemias

Occasionally, patients with immunohemolytic anemias and spherocytosis also have so few antibody molecules attached to their red cells that results of the direct Coombs test are negative. In these rare cases, the differentiation of the disease from HS is possible only with the use of radioactive antiglobulin reagents, the so-called super–Coombs test.

Heinz Body Hemolytic Anemias

Spherocytosis is also seen in Heinz body hemolytic anemias during an acute hemolytic crisis and occasionally in the steady state. However, in this situation, bite cells and/or blister cells and various dense, irregular cells are always present on the peripheral blood smear, and Heinz bodies can often be detected in red cells supravitally stained with methyl violet.

Aplastic Crises

Diagnostic difficulties may also arise in HS patients who are seen 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, physicians may occasionally be misled by the properties of the emerging young HS red cells, which are less spherocytic and osmotically fragile than usual.

Congenital Dyserythropoietic Anemia, Type II

In a preliminary report, Mariani et al noted that 13% of cases referred to their reference laboratory with a provisional diagnosis of HS were found to have CDAII, a disorder featuring underglycosylated band 3 and prominent binuclearity of late erythroblasts that is caused by defects in the secretory pathway gene SEC23B (see Chapter 7 ). Although CDAII patients typically display a variety of morphologic findings, a subset have prominent spherocytosis or elliptocytosis.

Hereditary Stomatocytosis and Xerocytosis

As discussed later in this chapter, patients with disorders of cation permeability, especially hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX), may be misdiagnosed as having HS because they have a dominant hemolytic anemia with an abnormal osmotic fragility test. Care must be taken to exclude these disorders in any HS patient who is to be treated with splenectomy because there is a high risk of thromboembolic complications following splenectomy in patients with HSt and HX.

Iron Deficiency

Iron deficiency corrects the abnormal shape, fragility, and high MCHC of hereditary spherocytes but does not improve their life span. Megaloblastic anemia can mask the morphologic characteristics of HS. Osmotic fragility is also improved. The masking effect is observed in both vitamin B ₁₂ and folate deficiencies and is rapidly reversed after correction of the nutritional deficit.

Obstructive Liver Disease

When obstructive jaundice develops, spherocytosis and osmotic fragility transiently improve and hemolysis abates. This is due to the expansion of red cell membrane surface area that follows the increased uptake of cholesterol and phospholipids from the abnormal plasma lipoproteins that are released by the obstructed liver. In normal cells, this increase in surface area leads to the formation of target cells, but in spherocytes, it leads to the appearance of discocytes. For example, we have seen a 6-year-old child who developed jaundice and symptoms of biliary obstruction. She had a palpable spleen tip, evidence of mild compensated hemolysis (hemoglobin concentration of 14 g/dL; reticulocyte count of 3.3%), a normal peripheral blood smear, and normal results for an osmotic fragility test (fresh and incubated cells). Abdominal radiographic studies showed calcified stones in the gallbladder and common bile duct. After cholecystectomy and relief of the partial biliary obstruction, the child’s hemolysis worsened (reticulocyte count of 10.8%) and she developed anemia (hemoglobin concentration of 10.2 g/dL), spherocytosis, and abnormal results on an incubated osmotic fragility test. She subsequently underwent splenectomy with clinical improvement.

β-Thalassemic Trait

The coexistence of HS and β-thalassemia trait has been described in a few case reports and has been reported to worsen, ameliorate, or have no effect on the clinical status of the patient. In a large French family with independently segregating HS and β-thalassemia trait, patients with both traits had signs of both diseases: small, hemoglobin A ₂ -rich, osmotically fragile cells and some spherocytes on peripheral blood smears. However, the HS phenotype in these patients was less severe than that of family members with HS but without β-thalassemia trait.

Hemoglobin SC

Coinheritance of HS and hemoglobin SC disease may exacerbate the hemoglobinopathy. A 15-year-old male with both diseases was much more anemic than his siblings with hemoglobin SC disease and experienced five splenic sequestration crises. However, the two diseases may also disguise each other morphologically. Only a few spherocytes or target cells were evident on the boy’s blood smear, and the surface-to-volume ratio of his red cells (and probably their osmotic fragility) was normal. This is likely due to the balancing effects of HS (loss of surface area) and hemoglobin SC (loss of cell volume) on red cells.

Hemoglobin AS

Sickle trait may also be worsened by HS. Yang and colleagues reported two patients with the combination who suffered multiple splenic sequestration crises. Splenic infarcts also occur. On the other hand, spontaneous regression of HS, presumably due to development of a hyposplenic state, has also been observed in two family members who had HS and sickle cell trait.

Gallstones

The formation of bilirubinate gallstones is one of the most common complications of HS and is a major impetus for splenectomy in many patients. Instances of gallstones occurring in infancy have been reported, but most appear in adolescents and young adults. In a retrospective study of 152 consecutive patients with HS, conducted before the development of ultrasonography, gallstones were detected in only 5% of children younger than age 10 years who were adequately examined and in approximately 50% of HS patients between 10 and 30 years of age ( Fig. 16-19 ). More recent data are similar; about 15% of patients less than 18 years old develop gallstones. The increase in the incidence of gallstones after age 30 years parallels the incidence in the general population, which suggests that cholelithiasis owing to HS is primarily manifested in the second and third decades.

Proportion of normal and hereditary spherocytosis (HS) patients with gallstones as a function of age. The graph is redrawn from data in a study of gallbladder disease in 152 consecutive patients seen at The Cleveland Clinic before 1952. Only patients whose gallbladders were examined at surgery or by cholecystography are included. Data for the general population are from an autopsy series of patients who did not have hemolytic anemia. Note that the prevalence of gallstones rises sharply between the ages of 10 and 30 years and parallels the general population after age 30 years.

(Bates GC, Brown CH: Incidence of gallbladder disease in chronic hemolytic anemia [spherocytosis]. Gastroenterology 21:104–109, 1952; Mentzer SH: Clinical and pathologic study of cholecystitis and cholelithiasis. Surg Gynecol Obstet 42:782–793, 1926.)

The incidence of gallstones in HS patients is related to the ability of the liver to metabolize increased amounts of bilirubin. A common polymorphism in the promoter of the uridine diphosphate-glucuronyl transferase gene ( UGT-1A ) causes decreased enzyme production and, in the homozygous form, causes Gilbert syndrome. Coinheritance of Gilbert syndrome with HS increases the risk for cholelithiasis. One retrospective study of 103 children with HS showed that the rate of gallstone formation was 2.2 times higher in those patients with a homozygous mutation in the UGT-1A gene than in those with a heterozygous mutation and 4.5 times higher than in healthy patients. HS patients with a homozygous mutation in the UGT-1A gene also develop gallstones at a younger age. Therefore analysis of this allele is useful in predicting the risk of cholelithiasis in HS.

Because the pigment stones typical of HS are easily detected by ultrasonography, all patients with HS should have ultrasound examinations about every 5 years if the spleen is intact and just prior to splenectomy. Unfortunately, longitudinal prospective studies using modern techniques (i.e., liver and biliary tree ultrasonography) are not available, making the true incidence and natural history of gallstones in this population unknown. One recent retrospective study showed that patients with HS (or other hemolytic anemias) were much more likely to develop symptomatic disease (i.e., pain, emesis, obstructive jaundice) than children with gallstones for other reasons. In that study, 68% of HS patients with gallstones had symptoms and all of them required cholecystectomy. This coincides with earlier reports. The most common complication was biliary obstruction, followed by acute cholecystitis. However, studies that include large numbers of patients with mild HS show a much lower incidence of symptomatic gallbladder disease.

More accurate, longitudinal studies are needed to determine the optimal treatment of patients with mild to moderate HS and cholelithiasis, particularly asymptomatic cholelithiasis that is detected on routine screening. Only about 15% to 20% of adults with asymptomatic stones (all types) develop symptoms over a prolonged period, but the retrospective data above suggest the complication rate may be much higher for bilirubinate stones. Surgery is indicated for recurrent symptoms or signs of biliary obstruction. It is unclear whether children who are undergoing cholecystectomy for symptomatic cholelithiasis should also have a splenectomy or partial splenectomy if it is not otherwise indicated (see later). Most authorities recommend it, arguing that the risk of stones persists, presumably in the remaining biliary radicals or cystic duct stump. However, cholelithiasis after cholecystectomy for typical gallbladder disease is rare unless a gallbladder remnant remains. Presumably the risk would be higher in someone with continuing hemolysis, but it is not clear how much higher.

If an HS patient requires a splenectomy, an ultrasound study of the gallbladder is indicated prior to surgery to assess the need for concomitant cholecystectomy. Cholecystotomy (removal of stones but not the gallbladder) is an option in children with silent stones because the risk of new stones forming is markedly reduced after splenectomy. For this reason, prophylactic cholecystectomy at the time of splenectomy solely to prevent the formation of gallstones is not indicated.

Crises

Patients with HS, like patients with other hemolytic diseases such as sickle cell disease, face a number of potential crises. Table 16-9 summarizes the types of crises that occur in patients with HS.

Aplastic Crises.

Aplastic crises are less common than mild hemolytic crises but are more serious and may lead to severe anemia, resulting in congestive heart failure or even death. They tend to occur in infants and children. Aplastic crises are caused by parvovirus B19, the etiologic agent in erythema infectiosum (fifth disease). Parvovirus B19 infection may present as fever, chills, lethargy, vomiting, diarrhea, and myalgias. Normal children may also develop a maculopapular rash on the face (slapped cheek appearance), trunk, and extremities—the rash of fifth disease—but this is less common in children with hemolysis and an aplastic crisis. In addition, patients with an aplastic crisis experience pallor and weakness from their worsening anemia. Jaundice, if present, may decrease as the number of abnormal red cells that can be destroyed declines.

Parvovirus B19 selectively infects erythropoietic precursors and inhibits their growth by inducing apoptosis and cell cycle arrest at G ₂ . Parvovirus infections are often associated with mild neutropenia (≈20%) or thrombocytopenia (≈40%), and instances of transient pancytopenia have been reported. Parvovirus may infect several members of a family simultaneously.

The sequence of events in an aplastic crisis was well described by Owren (though he confusingly called the crisis ”hemolytic,” reflecting the recovery phase, instead of aplastic) and is illustrated in Figure 16-20 . During the aplastic phase, the hematocrit level and reticulocyte count fall, marrow erythroblasts disappear, and unused iron accumulates in the serum. As production of new red cells declines, the cells that remain age and microspherocytosis and osmotic fragility increase. The return of marrow function is heralded by a fall in the serum iron concentration and the emergence of granulocytes, platelets, and, finally, reticulocytes. During this recovery phase, the serum phosphorus can drop to dangerously low levels. Furthermore, the lack of reticulocytes early in the recovery phase should not eliminate hemolytic anemias from diagnostic consideration.

The temporal sequence of a severe aplastic crisis in a patient with hereditary spherocytosis who previously had well-compensated hemolysis. Note the profound reticulocytopenia and the mild leukopenia and thrombocytopenia in the early phases of the reaction. Note also that the reemergence of reticulocytes is heralded by the sequential return of peripheral blood granulocytes and platelets and bone marrow normoblasts.

(Data from Owren PA: Congenital hemolytic jaundice: the pathogenesis of the “hemolytic crisis.” Blood 3:231–248, 1948.)

Infection with parvovirus B19 is a particular danger to susceptible pregnant women because it can infect the fetus, leading to fetal anemia, nonimmune hydrops fetalis, and fetal demise. The risk of nonimmune hydrops fetalis to the fetus of a woman who becomes infected with B19 during pregnancy is probably low. Nevertheless, because the virus is highly contagious and is easily transmitted to patients and staff and because only one half to two thirds of pregnant women have acquired protective antibodies, patients who have or are suspected of having an aplastic crisis should be placed under respiratory and contact precautions while hospitalized and IgG-negative contacts who are pregnant should be tested for evidence of seroconversion. Nonimmune hydrops fetalis can be detected by ultrasonography and treated with intrauterine transfusions.

Many asymptomatic patients with mild HS and compensated hemolysis first come to medical attention during an aplastic crisis. It is important to recognize that other family members with HS are susceptible and need to be tested. Diagnostic confusion may arise during reemergence of marrow function, when the physician may mistake an aplastic crisis for a hemolytic one. Because aplastic crises usually last for 10 to 14 days (about one half the life span of HS red cells), the hemoglobin value typically falls to about one half its usual level before recovery occurs. Thus aplastic crises are a serious threat to young children with HS, particularly children with more severe forms of the disease.

The diagnosis of parvovirus can be made using serum PCR or immunologic tests. Bone marrow pathology demonstrates giant pronormoblasts, which are a hallmark of the cytopathic effects of parvovirus B19. Treatment is supportive until the aplastic episode self-resolves. For severe anemia, red cell transfusions may be necessary. In immunocompromised patients, intravenous immunoglobulin assists in clearing the virus. Immunity after parvovirus infection is lifelong.

Other Complications

Risks of Splenectomy

Indications for Splenectomy

Splenectomy should only be performed if there are clear indications. All patients with severe spherocytosis, who are transfusion dependent or have growth failure or skeletal changes, should undergo splenectomy. Full or partial (subtotal) splenectomy (see later section) is also usually recommended for patients with moderate HS if they suffer from reduced vitality or physical stamina due to anemia; if, later in life, anemia compromises vascular perfusion of vital organs; or if leg ulcers or extramedullary hematopoietic tumors develop. Whether asymptomatic patients with moderate HS should have a splenectomy remains controversial. Partial splenectomy may also be indicated in some of these patients. Splenectomy can be deferred, probably indefinitely, in patients with mild HS and compensated hemolysis. In young symptomatic children with HS, splenectomy should be delayed until at least 3 years of age, and, if possible, until age 6 to 9 years of age, due to the risk of infection. In addition, after 6 years of age, the number of febrile infections is reduced, which decreases the frequency of postsplenectomy fever evaluations.

Surgical Procedures

Partial (Subtotal) Splenectomy.

The emergence of antibiotic-resistant pneumococci and increasing evidence suggesting an increased risk of thrombosis after splenectomy has led to reexamination of the role of alternate treatment modalities. Partial splenectomy has been suggested for selected patients with HS and other hematologic conditions. The goal of the operation is to decrease hemolysis while maintaining residual splenic phagocytic function. In practice, at least 80% to 90% of the enlarged organ is removed ( Fig. 16-21 ). Partial splenectomy was initially an open operation, but laparoscopic and robotic approaches have been developed and are becoming more common. The procedure is more time consuming than a total splenectomy and the time for recovery is longer (4 to 7 days of hospitalization), but studies have shown that partial splenectomy is safe and reduces the rate of hemolysis.

Surgical technique used for partial or subtotal (≈80%) 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.

(Modified from Tchernia G, Gauthier F, Mielot F, et al: Initial assessment of the beneficial effect of partial splenectomy in hereditary spherocytosis. Blood 81:2014–2020, 1993.)

Tchernia and colleagues followed 40 patients for up to 12 years after partial splenectomy and found that the mean hemoglobin value increased from 9.2 g/dL to 12.7 g/dL and the absolute reticulocyte count decreased from 523 × 10 ⁹ cells/L to 267 × 10 ⁹ cells/L ( Fig. 16-22 ). In the children with at least 10 years of follow-up, no differences in hemoglobin and reticulocyte levels were noticed between the group with partial splenectomy and the group with total splenectomy. Significant splenic regrowth was seen in the first year after partial splenectomy, but, after an initial period, the rate of splenic growth, in this study, was much reduced (see Fig. 16-22, C ). Nevertheless, 8% of patients in this study required a total splenectomy after the partial splenectomy, particularly those with severe HS, because of splenic regrowth and resumption of hemolysis. Moreover, 4 of 18 patients in whom the gallbladder was not removed with the initial surgery, required a subsequent cholecystectomy. In a more recent multiinstitutional study of 62 children with a partial splenectomy for HS, only 5% had recurrent hemolysis requiring reoperation and complete splenectomy. Although these results are encouraging, the percentage of HS patients who will require reoperation many years after a partial splenectomy will not be known for several more decades. This is the major risk of the procedure.

Long-term follow-up of partial splenectomy in hereditary spherocytosis (HS). The data are the results of 12 years of experience with the procedure in 40 patients (ages 1 to 25 years). A and B , Change in hemoglobin and reticulocyte count with time before and after the subtotal splenectomy (arrow) . The mean and standard deviations are shown. Both the rise in hemoglobin and fall in reticulocytes have been sustained for more than a decade. C , The size of the spleen during the same period. There is some regrowth of the splenic remnant, especially in the first few years. It remains to be seen whether the remnant reaches a stable size or continues to grow.

(Modified from Bader-Meunier B, Gauthier F, Archambaud F, et al. Long-term evaluation of the beneficial effect of subtotal splenectomy for management of hereditary spherocytosis. Blood 97:399–403, 2001.)

A major argument for partial splenectomy is the expectation that patients will be less susceptible to severe sepsis or thromboembolic complications if some splenic tissue remains. This seems logical and is probably true but is impossible to prove without a very large study over a very long time. We are not aware of any reports of overwhelming sepsis following partial splenectomy, and there is some evidence that the risk of sepsis is less. Experimentally, animals are better protected from bacterial challenge if some splenic tissue remains, though not as well protected as normal animals, and animals and humans recover immune function more rapidly after partial instead of complete splenectomy.

In summary, we believe partial splenectomy is an effective procedure in patients with moderate HS, largely ameliorating hemolysis and anemia while preserving some splenic function. It is important for patients and families to realize that long-term follow-up is encouraging but limited and that splenic regrowth may eventually require a reoperation. For this reason, the procedure cannot yet be recommended as the preferred approach. Some believe that partial splenectomy is the best option for infants with transfusion-dependent HS, who would otherwise be transfused for 3 to 4 years before a complete splenectomy would be done. In such children it is likely that a complete splenectomy will still be needed, however.

Postsplenectomy Changes

After splenectomy, spherocytosis persists but conditioned microspherocytes disappear, and changes typical of the postsplenectomy state, including Howell-Jolly bodies, target cells, siderocytes, and acanthocytes, become evident in the peripheral blood smear. On average, the MCV and mean red cell surface area increase and the MCHC and osmotic fragility decrease, but the effects are modest (5% to 10%). In typical dominant HS, reticulocyte counts fall to normal or nearly normal levels, although the red cell life span, if carefully measured, remains slightly shortened (96 ± 13 days). In all but the most severe occurrences, anemia and jaundice remit and do not recur.

Splenectomy Failure

The rare splenectomy failure (i.e., recurrence of hemolysis) is usually caused by an accessory spleen that was missed during surgery or by another red cell disorder, such as pyruvate kinase deficiency. Accessory spleens occur in 15% to 40% of patients and must always be sought. Occasionally they can be in unusual places: in the chest, across the midline, deeper in the abdomen, and so forth. Recurrence of hemolytic anemia years or even decades after splenectomy should raise suspicion of an accessory spleen, particularly if Howell-Jolly bodies are no longer evident on peripheral blood smears. The absence of “pitted” red cells with craterlike surface indentations, readily seen by interference contrast microscopy, is also a sensitive measure of recrudescent splenic function. The ectopic splenic tissue can be confirmed by a radiocolloid liver/spleen scan or a scan using Cr-labeled, heat-damaged red cells.

Corticosteroid Therapy

It has been known since the 1950s that large doses of corticosteroids markedly ameliorate HS in unsplenectomized patients, similar to the effects produced by splenectomy. More recent studies have confirmed this finding. Corticosteroids (prednisolone 1 to 2 mg/kg/day or equivalent for 7 days) can be a useful, temporary alternative to splenectomy in controlling severe hemolytic crises.

Vaccination

All candidates for splenectomy should receive a complete series of vaccinations against encapsulated organisms, including pneumococcus, meningococcus, and Haemophilus influenzae . Yearly influenza immunizations are recommended to reduce the chance of secondary bacterial infections. Recent studies have shown that the majority of adults who were splenectomized as children are not up-to-date with their vaccinations. In a study examining the current cases of overwhelming postsplenectomy infection in the era of pneumococcal vaccines, only 31% of 77 patients with such infection received the pneumococcal vaccine. It is important to emphasize the importance of immunizations to the affected parents of children with HS.

Experts differ somewhat on how to protect patients from infection after splenectomy. Our approach follows the 2014 CDC guidelines for vaccination of children and adults with high risk conditions. The guidelines change frequently, so the latest version should be consulted. The recommendations of the British Committee for Standards in Haematology have also been published relatively recently.

If possible, all vaccines should be given at least two weeks and preferably four weeks before splenectomy.

Antibiotic Prophylaxis

Prophylactic antibiotics after splenectomy are recommended, with emphasis on protection against pneumococcal sepsis (i.e., penicillin V or equivalent, 125 mg orally twice daily in children younger than 5 years of age, and 250 mg twice daily in older children and adults). For those patients allergic to penicillin, erythromycin is recommended. Antibiotic prophylaxis is recommended at least through childhood, and, for teenagers and adults, for at least the first 5 years after surgery.

Some physicians recommend lifelong antibiotic prophylaxis because it is unclear how the risk of postsplenectomy overwhelming infection changes over time. The emergence of penicillin-resistant pneumococci (5% in 1989 to ≥35% in 1997) has led many to reconsider this recommendation, particularly because prior antibiotic use is a risk factor for the development of penicillin resistance in pneumococci. Patients who do not take prophylactic antibiotics should have a supply of oral antibiotics on hand, which they should take immediately should fever higher than 101.5°C develop. They should then seek medical attention immediately.

The risk of infection is decreased in immunized patients, because 50% to 70% of postsplenectomy sepsis is due to S. pneumoniae and about 80% of pneumococcal disease is due to strains contained in the two vaccines. However, it is likely that strains omitted from the vaccines will rise in frequency as pathogens due to selection. Further risk reduction would be anticipated from the chronic use of prophylactic penicillin. Nevertheless, the risk cannot be reduced to zero. Postsplenectomy infections occur occasionally in successfully immunized patients, and compliance with prophylactic medication regimens is a problem, particularly in teenagers.

The occurrence of penicillin-resistant pneumococci is increasing very rapidly all over the world. In some countries, more than one half of all isolates are resistant. In the United States, the prevalence is approximately 35%, but it is much higher in some localities and is rising. Although most of the strains show some sensitivity to penicillin, some are highly resistant and others are multiply resistant. The emergence of these strains will greatly complicate the use of antibiotics for pneumococcal prophylaxis during the next decades.

Spectrin Mutants

The ja/ja mutant has a defect in the spectrin β-chain (Arg1160→Stop) and produces no detectable spectrin. The sph/sph variants lack α-chains but have small amounts of β-spectrin. These mice do not produce α-spectrin either due to decreased synthesis, function, or stability. The sph and sph ^2BC alleles are frameshift mutations and null alleles. Four mutations affect the C-terminus of α-spectrin, which was previously thought to be functionless in erythrocytes: (1) sph ^IhJ is a null mutation (Q1853Stop) in repeat 18 ; (2) sph ^3J contains a missense mutation (H2012Y) in repeat 19, near the C-terminus, that alters splicing and results in premature termination of translation ; (3) sph ^1J allele lacks the last 13 amino acids of α-spectrin ; and (4) sph ^4J is a missense mutation (C2384Y). The latter two mutations are in the C-terminal EF-hand domain. All four mutations show marked spherocytosis, and sph ^4J does so despite minimally decreased membrane spectrin and normal band 3 levels. Thus the amino acids in the C-terminus of the spectrin tail are functionally important and, it turns out, are critical in attaching spectrin to actin. The sph ^DEM /sph ^DEM mouse is missing exon 11 and 46 amino acids near the amino terminus of α-spectrin repeat 5. These mice have elliptocytes, spherocytes, and poikilocytes, and, therefore clinically appear to be a cross between HS and hereditary elliptocytosis (see later).

Cardiac thrombi, fibrotic lesions, and renal hemochromatosis are found in ja/ja and sph/sph mice in adulthood. Transplantation of the hematopoietic cells from sph/sph mice is sufficient to induce thrombotic events in the transplant recipients. In mice with α-spectrin mutations, 70% to 100% experience cerebral infarction. Studies of red cell adhesion in these mice demonstrate a tenfold higher adhesion to thrombospondin compared with normal red blood cells and an increased adhesion to laminin. Therefore it is hypothesized that changes in the red cell adhesive characteristics contribute to the thrombotic events in these mice.

Riesling ( ris ) is an induced mutation in zebrafish that results in a nearly bloodless phenotype, which causes severe anemia and congestive heart failure. The ris phenotype is caused by a null mutation in β-spectrin. The red blood cells of affected zebrafish are spherocytic rather than elliptocytic, the normal zebrafish red cell shape. Scanning electron micrographs show membrane loss, as in other forms of severe HS.

Ankyrin Mutants

Mice with the nb/nb mutation have a primary defect in ankyrin. This is due to a single-nucleotide deletion leading to a frameshift and premature chain termination in the regulatory region of ankyrin. The nb/nb red cells have 50% to 70% of normal spectrin levels but only a trace of ankyrin mRNA and protein. This is in striking contrast to ankyrin defects in human HS, in which ankyrin and spectrin levels are comparably depressed. Fetal nb/nb mice have no anemia and normal reticulocyte counts at birth, possibly because of expression of ankyrin-related proteins in utero. Mice that completely lack Ank1 mostly die in utero. Those that survive have extreme hemolysis and are severely deficient in β-spectrin as well as ankyrin. However, recently a mouse mutation (hema6) has been identified that results in decreased levels of full-length normal ankyrin and more typical HS. This should be useful experimentally.

The nb/nb mice also lack ankyrin in cerebellar Purkinje cells. This leads to an age-related loss of Purkinje cells during the first 5 to 7 months of life and the emergence of cerebellar ataxia. Spinocerebellar degeneration and related syndromes have also been reported in a few adult humans with HS, although it is not yet known whether ankyrin is affected.

Band 3 Mutants

Complete deficiency of band 3 was first described in cattle with a recessive form of HS. In cows, homozygosity for a nonsense mutation in the band 3 gene leads to absence of band 3 and protein 4.2. Red cell membranes are unstable and show surface area loss, as demonstrated by invagination, vesiculation, and extrusion of microvesicles. The affected cattle have defective anion transport and a mild acidosis but mild hemolysis in comparison with other band 3–deficient species.

In mice, targeted disruption of the band 3 gene causes a severe spherocytic hemolytic anemia, with exuberant loss of the membrane surface as vesicles, tubules, and myelin forms. Red cell membranes also lack protein 4.2 and glycophorin A. Surprisingly, the content of membrane skeletal proteins and the architecture of negative-stained, spread skeletons is normal or nearly normal. Therefore band 3 appears to be required for stabilizing membrane lipids but not for membrane skeleton assembly. For unclear reasons, these band 3–deficient mice have a significant propensity for thrombosis and often die in the neonatal period.

Another mouse model, wan/wan , in a C3H/HeJ background, has a null defect in the band 3 gene. These mice have a severe anemia, which is lethal in the neonatal period. However, when wan/wan mice are crossed with wild-type mice of a different strain, Mus castaneus , the F2 generation shows variable hematologic severity. The genetic modifier causing this variability in hematologic abnormalities has been localized to a small chromosomal region containing the β-spectrin gene.

Finally, zebrafish with band 3 mutations, associated with the retsina ( ret ) phenotype, suffer from severe anemia with a complete arrest in erythroid maturation at the late erythroblast stage. These rare ret/ret red blood cells are spherocytic rather than elliptical. Many of these arrested erythroblasts have bilobed nuclei that demonstrate defects in cytokinesis.

Protein 4.2

Mice with targeted deletions of the protein 4.2 gene survive normally and have only mildly spherocytic anemia and hemolysis.

Protein 4.1R

Mice lacking the protein 4.1R gene due to targeted deletion have moderate hemolysis and decreased red cell membrane stability. In addition to lacking protein 4.1R, these mice are also deficient in protein 55, glycoprotein C, ankyrin, and spectrin. The red cell morphology is remarkable for spherocytic shape, rather than elliptocytic shape, likely related to the spectrin deficiency. Interestingly, the knockout mice exhibit specific neurobehavioral deficits in movement, coordination, balance, and learning that correspond with the selective localization of 4.1R in granule cells of the cerebellum and dentate gyrus. Two zebrafish models with protein 4.1R deficiency have been discovered, merlot and chablis, and these mutants have very severe hemolytic anemia, characterized by spherocytosis and increased osmotic fragility.

Adducin and Dematin

Mice lacking just β-adducin or β-adducin and γ-adducin have a very mild, spherocytic hemolytic anemia. Knockout of α-adducin, which eliminates all three adducins (α, β, and γ), still results in only mild hemolysis and moderate spherocytosis, though slightly more severe than in the β-adducin knockout. Similarly, loss of the headpiece of dematin produces, at most, very mild hemolysis. However, mice that lack both β-adducin and the dematin headpiece have a severe spherocytic hemolytic anemia that resembles severe HS. The data suggest the various vertical membrane connections involving the actin junctions—4.1R-p55–glycophorin C, 4.1R–band 3, alpha spectrin–4.2–band 3, adducin–band 3, adducin–Glut1, and actin-dematin–Glut1—are largely redundant and only become consequential when more than one is eliminated.

Typical Heterozygous Hereditary Elliptocytosis

The clinical characteristics of typical HE trait vary considerably, defining several clinical subtypes. Different clinical patterns may be seen in members of the same family, and the clinical presentation in single individuals may vary over time, so the clinical patterns are probably more useful for illustrating the spectrum of HE than for classifying the disease.

Typical Hereditary Elliptocytosis with Chronic Hemolysis and Hereditary Pyropoikilocytosis

These two conditions are really variations of the same thing: homozygosity or compound heterozygosity for elliptocytogenic mutations and/or silent alleles that augment elliptocytogenic mutations. In general, the greater the hemolysis the more the evidence of membrane instability on the peripheral blood smear: budding red cells, fragments, and other bizarre poikilocytes.

Spherocytic Hereditary Elliptocytosis

This dominant disorder is a phenotypic hybrid of typical HE and HS. It has only been reported in Caucasian families of European descent. The prevalence of spherocytic HE is unknown, but judging from the number of published reports, it is relatively rare, probably accounting for no more than 5% of HE cases in patients of European ancestry. Unlike in typical HE, almost all affected patients have some hemolysis. This is usually mild to moderate and is often incompletely compensated. The elliptocytes are fewer and plumper than in typical HE (see Fig. 16-3, D ), and some spherocytes, microspherocytes, and microelliptocytes are often present. Poikilocytes and red cell fragments are uncommon, a finding that distinguishes this disorder from typical HE with hemolysis. Some spiculated cells may be present following splenectomy, similar to what is seen in β-spectrin deficient HS. Red cell morphologic findings may vary, even within the same family. Some family members may have relatively prominent spherocytes and as few as 10% to 20% elliptocytes, whereas in others elliptocytes predominate and spherocytes are rare. This may cause diagnostic confusion initially, particularly if the propositus has few elliptocytes. Family studies almost always reveal some members with obvious elliptocytosis.

Spherocytic elliptocytes are osmotically frag­ile, particularly after incubation. Gallbladder disease is common, and aplastic crises are a risk. The pathologic course of spherocytic HE mimics that of HS. Splenic sequestration is evident, red cells are conditioned during splenic passage, and hemolysis abates after splenectomy.

The molecular pathology of classic spherocytic HE is not well studied. Patients with C-terminal truncations of α-spectrin (see later) have many of the clinical features of spherocytic HE and probably are a subset of the disorder. Patients with such truncations typically have moderate hemolysis and anemia, punctuated by recurrent, severe hemolytic crises. Blood smears show plump and usually smooth elliptocytes, although in a few instances, poikilocytosis was prominent.

Patients who lack glycophorin C (see later) also have positive osmotic fragility tests and rounded, smooth elliptocytes. They should probably also be classified as having a recessive (and unusually mild) variant of spherocytic HE. Patients who lack protein 4.2 (another recessive condition) sometimes display features of spherocytic HE (ovalocytes, spherocytes, stomatocytes) ; however, protein 4.2 deficiency more often resembles HS, morphologically and pathophysiologically, and is better classified as a variant of that disorder. Mice that lack tropomodulin-1 have mild spherocytic HE that is more spherocytic than elliptocytic. The anemia may be partly compensated by an increase in tropomodulin-3, which is not normally expressed in mature red cells, to about 20% of the normal level of tropomodulin-1. Tropomodulin deficiency has not been identified in humans but should be considered.

Very rare patients appear to be compound heterozygotes for HS and HE. One Turkish girl is a particularly good candidate. Her mother had mild HS and her father most likely had very mild HE, whereas she suffered from a moderately severe hemolytic anemia (hemoglobin level, 8.4 g/dL; reticulocyte count, 24%; bilirubin level, 1.6 mg/dL), with frontal bossing, osteoporosis, splenomegaly, and a mixture of microspherocytes and rounded elliptocytes.

Southeast Asian Ovalocytosis

This condition, which has a unique phenotype, molecular defect, and geographic distribution, is discussed later in this chapter.

Spectrin Defects

Abnormalities of either α-spectrin or β-spectrin are responsible for most cases of HE and HPP. The majority are due to missense mutations near the spectrin self-association site (blue symbols in Figs. 16-6 and 16-8 ). The spectrin self-association contact site is a combined triple helical “spectrin repeat” (see Fig. 15-13 and Fig. 15-14 ) in which two of the three helices that make up a typical spectrin repeat, helices A and B, are contributed by the C-terminus of the β-spectrin, whereas the third helix (C) comes from the first portion of the NH ₂ -terminus of α-spectrin. These three helices pair up and create the bond responsible for spectrin self-association. HE mutations weaken the ability of spectrin dimers to form tetramers and destabilize the membrane skeleton. The functional damage is manifested by an increased proportion of spectrin dimers in 0°C low–ionic strength extracts of spectrin, and by decreased conversion of spectrin dimers to tetramers in solution and on the membrane. The broken spectrin-spectrin links are visible in electron micrographs and diminish the resistance of the isolated membranes to shear stress. The clinical severity of HE correlates with the degree of impairment of spectrin self-association and with the amount of mutant spectrin that is produced.

A few examples of mutations that cause HE or HPP follow. Readers who are interested in a more extensive list of HE/HPP mutations can refer to an earlier chapter by the author or to selected reviews, or consult the paid or free versions of the Human Gene Mutation Database.

Alpha-Spectrin Defects Within the Self-Association Bind­ing Site.

α-Spectrin mutations account for 48% of the reported defects in HE and HPP. Many of the mutations are located in the C helix at the beginning of the α-spectrin chain ( Fig. 16-24 and see Fig. 16-6 ), which is a partial spectrin repeat, also called spectrin repeat 0 (see Fig. 16-24 ). Most of these mutations change amino acids that are part of the contact surfaces between the α-spectrin and β-spectrin chains at the tetramerization site and greatly impair or even abolish spectrin-spectrin binding. As a result, they are among the most severe of the common α-spectrin mutations that cause HE and HPP.

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