Seminar

Hereditary spherocytosis

Silverio Perrotta, Patrick G Gallagher, Narla Mohandas

Hereditary spherocytosis is a common inherited disorder that is characterised by anaemia, jaundice, and splenomegaly. Lancet 2008; 372: 1411–26 It is reported worldwide and is the most common inherited anaemia in individuals of northern European ancestry. Department of Paediatrics, Clinical severity is variable with most patients having a well-compensated haemolytic anaemia. Some individuals are Second University of Naples, asymptomatic, whereas others have severe haemolytic anaemia requiring erythrocyte transfusion. The primary lesion Naples, Italy (S Perrotta MD); Department of Pediatrics, Yale in hereditary spherocytosis is loss of membrane surface area, leading to reduced deformability due to defects in the University School of Medicine, membrane proteins ankyrin, band 3, β spectrin, α spectrin, or protein 4.2. Many isolated mutations have been New Haven, CT, USA identifi ed in the genes encoding these membrane proteins; common hereditary spherocytosis-associated mutations (Prof P G Gallagher MD); and have not been identifi ed. Abnormal spherocytes are trapped and destroyed in the spleen and this is the main cause of New York Blood Center, New York, NY, USA haemolysis in this disorder. Common complications are cholelithiasis, haemolytic episodes, and aplastic crises. (N Mohandas DSc) Splenectomy is curative but should be undertaken only after careful assessment of the risks and benefi ts. Correspondence to: Dr Silverio Perrotta, Introduction junctional complex, protein 4.1R interacts with one end Dipartimento di Pediatria, Hereditary spherocytosis refers to a group of hetero- of several spectrin tetramers in the β-spectrin Seconda Università di Napoli, Via S Andrea delle Dame, 4, geneous inherited anaemias that are characterised by N-terminal, in a region containing actin short fi laments 80138 Napoli, Italia the presence of spherical-shaped erythrocytes (sphero- and an array of actin-binding proteins—ie, dematin, [email protected] cytes) on the peripheral blood smear.1–3 This disorder, tropomyosin, β-adducin, and tropomodulin. Outside including the very mild or subclinical forms, is the most the junctional complex, protein 4.1R interacts with common cause of inherited chronic haemolysis in transmembrane glycophorin C and p55. The α2β2 northern Europe and North America where it aff ects tetramer of spectrin forms a dense network, lining the about one person in 2000.4–11 It has also been frequently inner surface of the lipid bilayer. The α-spectrin and described in other populations, notably in Japan.12 β-chains are antiparallel with the head-to-head asso- Although the disease arises in all racial and ethnic ciation of two dimers—ie, N-terminal region of α-spec- groups, on the basis of clinical reports, it seems to be less trin chains with the C-terminal region of β-spectrin common in African-American and southeast Asian chains—at the self-association site to generate tetramers people; however, comprehensive population survey data and higher-order oligomers (fi gure 1). The membrane are unavailable for these populations. and its skeleton provide the erythrocyte with deform- ability—ie, the ability to undergo substantial distortion Pathophysiological eff ects without fragmentation or loss of integrity during The red blood cell or erythrocyte membrane is a dynamic microcirculation and the ability to withstand the shear and fl uid structure with the strength and fl exibility stress of the arterial circulation.1,13,17–19 Two factors are needed to survive 120 days in the circulation. Its lipid implicated in the pathophysiological eff ects of hereditary bilayer is composed mainly of phospholipids and spherocytosis—an intrinsic red blood cell membrane cholesterol, with integral proteins embedded in the lipid defect and an intact spleen that selectively retains, bilayer that span the membrane and a membrane damages, and removes the defective erythrocytes skeleton on the cytoplasmic side (fi gure 1).1,13–16 The polar (fi gure 2).1,20,21 heads of the lipid bilayer are turned outward and their apolar fatty-acid chains face one another and form the inner core. The band-3 complex, is centred by a band-3 Search strategy and selection criteria tetramer; band 3 can also exist as a dimer. Each band-3 We mainly searched our personal database of references, which monomer consists of a large transmembrane segment, was prospectively built by monthly search of PubMed in the with a long, branched polylactosaminoglycan attached past 15–20 years, with the following key words and their on the non-cytoplasmic side, and a stalk-like cytoplasmic combinations in all fi elds: “hereditary spherocytosis”, “red cell domain that anchors the ankyrin-1. The ankyrin-1 also membrane”, “spectrin”, “ankyrin”, “band 3”, “spherocytes”, binds to the C-terminal region of the β-spectrin chain “splenectomy”, “gallstones”, or “cholecystectomy”. We mainly and to protein 4.2. Glycophorin A exists as a dimer with selected studies from the past 10 years, but did not exclude several short, sialic-acid-containing glycans attached to commonly cited older publications. We only read the abstracts it. The Rhesus (Rh) complex contains the Rh of articles published in languages other than English. We also polypeptides, the Rh-associated glycoprotein (arranged searched the reference lists of articles identifi ed by this search as a heterotetramer), CD47, the Landsteiner-Wiener strategy and selected those we judged relevant. Review articles glycoprotein, and glycophorin B dimer. CD47 interacts and standard textbooks are occasionally cited to provide with protein 4.2 and the Rh polypeptides to establish a readers with more details and references than this Seminar has contact with ankyrin-1. The Rh complex and the band-3 room for. complex are thought to form a macrocomplex. In the www.thelancet.com Vol 372 October 18, 2008 1411 Seminar

as the tail of cells on osmotic fragility tests (fi gure 2).1 Glycophorin A Glycophorin B Glycophorin A Glycophorin C/D Splenic destruction of abnormal erythrocytes is the main cause of haemolysis in patients with hereditary sphero cytosis. Analysis of deformability profi les show that splen- ectomy is more benefi cial for spectrin-defi cient or ankyrin-defi cient than for band 3-defi cient red blood Band 3 Band 3 cells. Splenectomy prevents an early loss of immature Band 3 Band 3 cells in both types of defi ciencies but it has an additional Rh RhAG LW p55 CD47 benefi cial eff ect on mature spectrin-defi cient or ankyrin- 4.2 Dematin Ankyrin-1 defi cient cells, increasing their survival. Spectrin-defi cient 4.1R or ankyrin-defi cient erythrocytes escape opsonisation by releasing band-3-containing vesicles. Hence, band-3 Actin clusters needed for bivalent binding of low affi nity, β spectrin Adducin 4.1R naturally occurring IgG antibodies might be retained in α spectrin band-3-defi cient erythrocytes with an excess of skeletal proteins but are released from spectrin-defi cient or Tropomyosin Tropomodulin ankyrin-defi cient cells in which vesicle budding is facilitated by an impaired skeleton.23 Figure 1: A simplifi ed cross-section of the red blood cell (erythrocyte) membrane The lipid bilayer forms the equator of the cross-section with its polar heads (small circles) turned outward. 4.1R=protein. 4.2=protein 4.2. Rh=Rhesus polypeptide. RhAG=Rh-associated glycoprotein. LW=Landsteiner-Wiener Causes glycoprotein. Membrane loss in hereditary spherocytosis is associated with defects in several membrane proteins. On the basis Although the main molecular defects in hereditary of densitometric quantifi cation of membrane proteins spherocytosis are heterogeneous, one common feature of separated by sodium dodecyl sulphate polyacrylamide the erythrocytes in this disorder is weakened vertical gel electrophoresis (SDS-PAGE) and western blot linkages between the membrane skeleton and the lipid analysis, this disease can be divided into subsets: bilayer with its integral proteins (table 1). Vertical linkages (1) isolated defi ciency of spectrin; (2) combined include spectrin, ankyrin-1, band-3, and protein-4.2 defi ciency of spectrin and ankyrin; (3) defi ciency of interactions; spectrin, protein-4.1R, glycophorin-C, and band-3 protein; (4) defi ciency of protein 4.2; (5) defi ciency p55 interactions; Rh-complex and ankyrin-1 interactions; of Rh complex; and (6) yet to be defi ned protein and other, as yet not fully defi ned lipid bilayer-membrane abnormalities (table 2).20,24–26 skeleton interactions (fi gure 1). When these interactions Mutations associated with isolated spectrin defi ciency are compromised, loss of cohesion between bilayer and are defects of both α-spectrin and β-spectrin (SPTA1 and membrane skeleton occurs, leading to destabilisation of SPTB, respectively) genes (online mendelian inheritance the lipid bilayer and release of skeleton-free lipid in man [OMIM] +182860 and +182870, respectively).20,25 vesicles.1,22 Two distinct pathways lead to reduced In general, hereditary spherocytosis caused by α-spectrin membrane surface area: (1) defects in spectrin, ankyrin, and β-spectrin mutations is associated with recessive and or protein 4.2 reduce the density of the membrane dominant inheritance, respectively. In healthy erythroid skeleton, destabilising the overlying lipid bilayer and cells, production of α-spectrin chains is three-fold to releasing band-3-containing microvesicles; and (2) defects four-fold greater than β-spectrin production.27 Thus, a of band 3 lead to its defi ciency and loss of the mutation of one β-spectrin allele is suffi cient to cause lipid-stabilising eff ect, which results in the loss of band- spherocytosis whereas both α-spectrin alleles have to be 3-free microvesicles from the membrane. Both pathways aff ected for the disease to arise. result in reduced membrane surface area, a reduction in β-spectrin defects account for about 15–30% of cases of the ratio of surface area to volume, and the formation of hereditary spherocytosis in northern European popu- spherocytes with reduced deformability that are selectively lations. Patients with β-spectrin defi ciency typically have retained and damaged in the spleen (fi gure 2).1,2,22 mild to moderately severe disease and do not need Once trapped in the spleen, the abnormal erythrocytes transfusion.1,2,28 Blood smears from these individuals undergo additional damage or splenic conditioning might contain prominent acanthocytes or spiculated shown by further loss of surface area and an increase in spherocytes (fi gure 3). With rare exceptions, mutations cell density. Low pH, low concentrations of glucose and of the β-spectrin gene are isolated and might be associated adenosine triphosphate, contact of erythrocytes with with monoallelic expression, suggesting that null macrophages, and high local concentrations of oxidants mutations are common.1,25,29,30 Several de-novo mutations contribute to conditioning. Some of these conditioned of β spectrin have been described (table 2).29–32 erythrocytes escape the hostile environment of the α-spectrin defects account for about 5% of patients spleen, re-enter the systemic circulation, and are visable with hereditary spherocytosis and are only clinically

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Spectrin, ankyrin, or protein 4.2 deficiency Low pH High macrophage Release of microvesicles contact Haemolysis Low glucose concentration Erythrostasis High oxidants concentration

Reduced Reduced Splenic Splenic Further loss surface-to-volume ratio cellular deformability trapping conditioning of membrane

Release of microvesicles

Tail of osmotic fragility curve Band-3 deficiency Lipid bilayer Spectrin Ankyrin Band 3

Figure 2: Pathophysiological eff ects of hereditary spherocytosis Modifi ed from Gallagher and colleagues21 with permission. apparent in the homozygous or compound heterozygous described.46,47 ANK1 gene deletion might be part of a state. These patients have severe disease. Homozygous contiguous gene syndrome with manifestations of and compound heterozygous defects have been spherocytosis, mental retardation, typical faces, and associated with null mutations and variants are hypogonadism. associated with low-expression alleles.33–36 For example, Ankyrin-1 plays a pivotal role in the stabilisation of the the α-LEPRA (low-expression allele Prague) allele membrane, providing the main membrane binding site produces about six-fold less of the correctly spliced for the spectrin-based membrane skeleton. Since it links α-spectrin transcript than does the healthy allele.33 The β spectrin to band 3, ankyrin defi ciency leads to a presence of two null α-spectrin alleles is speculated to proportional reduction in spectrin assembly on the be lethal. Blood smears from patients with severe membrane despite normal spectrin synthesis.48 The α-spectrin defi ciency contain many microspherocytes, defi ciency of one protein is strictly associated with the contracted erythrocytes, and abnormal poikilocytes defi ciency of the other and the extent of protein defi ciency (table 2).24,33 is related to the clinical severity. A high reticulocyte count The biochemical phenotype of combined spectrin and might mask a reduction in ankyrin-1 in biochemical ankyrin defi ciency is the most common abnormality studies.49 noted in about 40–65% of patients with hereditary Defi ciency of band-3 protein arises in about 33% of spherocytosis in northern European populations,36–38 but patients, presenting with mild to moderate, dominantly in only 5–10% of cases in Japan (table 2).12 Patients with inherited disease (table 2). Mushroom-shaped or pincered ankyrin defects have prominent spherocytosis without erythrocytes might be seen on peripheral blood smear other morphological abnormalities (fi gure 3). Ankyrin (fi gure 3).1,50 SDS-PAGE analysis shows a reduction in mutations (OMIM +182900) cause both dominant and band 3 in about 20–30% of patients, as the wild-type recessive disease that can range from clinically mild to allele in trans partly compensates for the mutated allele.51 severe.1,20,25,38–41 About 15–20% of ankyrin-1 (ANK1) gene Erythrocyte membranes from these patients also have mutations reported are de novo;25,32,38,42 recurrent defi ciency of protein 4.2.52 A range of band-3 mutations mutations have been described.43 (OMIM+109270) associated with hereditary spherocytosis Mutations—eg, nucleotide −108T to C and −153G to A, have been reported; these mutations are spread out and deletion of nucleotides −72/73 of the ankyrin throughout the band-3 (SLC4A1) gene.1,20,25,53–55 promoter, leading to decreased ankyrin-1 expression have Alleles that aff ect band-3 expression when inherited in been identifi ed in some patients.44,45 A few patients with trans to a band-3 mutation, aggravate band-3 defi ciency atypical hereditary spherocytosis associated with karyo- and worsen clinical severity.56–58 Severe disease has been typic abnormalities—ie, deletions or translocations of reported in patients who are compound heterozygotes or the ankyrin gene locus on chromosome 8p—have been homozygotes for band-3 defects.59–62 www.thelancet.com Vol 372 October 18, 2008 1413 Seminar

A subset of patients with band-3 protein defi ciency, (table 2).24,66 On peripheral blood smear, ovalocytes resulting from mutations in the region of the band-3 and stomatocytes predominate with few spherocytes. gene encoding the fi fth outer loop of the transmembrane Patients with heterozygous mutantions are asymp- domain, have features of dehydrated hereditary tomatic. Membranes show an almost total absence of stomatocytosis or cryohydrocytosis.63 Thus, the clinical protein 4.2, resulting in a secondary reduction in CD47, a presentation ranging from band-3-related hereditary member of the Rh complex (fi gure 1).61,67,68 spherocytosis to dehydrated hereditary stomatocytosis Rh defi ciency syndrome identifi es rare individuals who and cryohydrocytosis is a continuum. Band-3 mutations have either absent (Rh null) or moderately reduced sometimes also cause distal renal tubular acidosis with (Rh mod) Rh antigen expression and mild to moderate or without hereditary spherocytosis, the phenotype of haemolytic anaemia associated with the presence of southeast Asian ovalocytosis, and acanthocytosis. The stomatocytes and spherocytes on the peripheral blood pleiotropic clinical manifestations associated with fi lm. Haemolytic anaemia is improved by splenectomy.69,70 band-3 mutations are remarkable in their diversity.52 Rh proteins are part of a multiprotein complex that also Recessive hereditary spherocytosis due to homozygous includes Rh-associated glycoproteins (RhAG) and mutations in protein 4.2 (EPB42) gene (OMIM *177070) Landsteiner-Wiener glycoprotein, glycophorin B, and is common in Japan,12,64,65 but is rare in other popula tions protein 4.2. The Rh-RhAG complex interacts with ankyrin

Protein Gene Location Exons Aminoacids Molecular weight×103 Oligomeric state (gel/calculated) SDS-PAGE band 1 α-spectrin SPTA1 1q22-q23 52 2429 240/281 Heterodimer/tetramer SDS-PAGE band 2 β- spectrin SPTB 14q23-q24.1 32 2137 220/246 Heterodimer/tetramer SDS-PAGE band 2.1 Ankyrin-1 ANK1 8p11.2 42 1880 210/206 Monomer SDS-PAGE band 2.9 β-adducin ADD2 2p13.3 16 726 97/80 Heterodimer/tetramer SDS-PAGE band 3 Band 3 (AE1) SLC4A1 17q21 20 911 90–100/102 Dimer/tetramer SDS-PAGE band 4.1R Protein 4.1 EPB41 1p33-p34.2 >23 588 80+78*/66 Monomer SDS-PAGE band 4.2 Protein 4.2 EPB42 15q15-q21 13 691 2/77 Dimer/trimer SDS-PAGE band 4.9† Dematin EPB49 8p21.1 15 383 48+52/43 Trimer p55 MPP1 Xq28 12 466 46+55/53 Dimer SDS-PAGE band 5‡ β-actin ACTB 7pter-q22 6 375 43/42 Oligomer Tropomodulin TMOD 9q22 9 359 43/41 Monomer SDS-PAGE band 6 G3PD GAPD 12p13 9 335 35/36 Tetramer SDS-PAGE band 7§ Stomatin STOM 9q33.2 7 288 31/32 ·· Tropomyosin TPM3 1q31 13 239 27+29/28 Heterodimer SDS-PAGE band PAS-1¶ Glycophorin A GYPA 4q31.21 7 131 36/14 Dimer SDS-PAGE band PAS-2¶ Glycophorin C GYPC 2q14-q21 4 128|| 32/14 ·· SDS-PAGE band PAS-3¶ Glycophorin B GYPB 4q31.21 5 72 20/8 Dimer Glycophorin D GYPD 2q14 q21 4 107|| 23/11 ··

AE1=anion exchange protein-1. G3PD=glyceraldehyde-3-phosphate dehydrogenase. PAS=periodic acid Schiff reagent. SDS-PAGE=sodium dodecyl sulphate polyacrylamide gel electrophoresis. *Protein 4.1 is a doublet of 4.1a and 4.1b on SDS-PAGE gel. †Band contains both dematin and p55.; ‡Band contains β actin and tropomodulin. §Band contains stomatin and tropomyosin. ¶Glycophorins are visible only on PAS-stained gels. ||Glycophorins C and D arise from alternative translation initiation sites.

Table 1: Major human erythrocyte membrane proteins and gene defects

Patients with HS Heredity Prevalent mutations Protein reduction Disease severity Peripheral blood smear Ankyrin-1 USA and Europe AD, AR, AD or de novo: null Spectrin and Mild to moderate Spherocytes 40–65%; Japan de novo mutation; AR: mis-sense ankyrin-1 15–50% 5–10% and promoter mutations Band 3 20–35% AD Functionally null Band 3 15–35% Mild to moderate Spherocytes, occasional mutation mushroom-shaped or pincered cells α spectrin <5% AR α-LEPRA allele and null α spectrin 50–75% Severe Spherocytes, contracted cells, and mutation poikilocytes β spectrin 15–30% AD, de novo Null mutation β spectrin 15–40% Mild to moderate Spherocytes, 5–10% acanthocytes Protein 4.2 USA and Europe AR Mis-sense (prevalence of Protein 4.2 Mild to moderate Spherocytes, ovalostomatocytes <5%; Japan 45–50% 4.2 Nippon) 95–100%

AD=autosomal dominant. AR=autosomal recessive. HD=hereditary spherocytosis. LEPRA=low-expression allele Prague.

Table 2: Clinical and molecular characterisation of hereditary spherocytosis molecular defects

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A B

C D

Figure 3: Abnormal peripheral blood smears in hereditary spherocytosis (HS) due to diff erent membrane defects (A) Two blood smears of typical moderately severe HS with a mild defi ciency of red blood cell spectrin and ankyrin-1. Although many cells have spheroidal shape, some retain a central concavity. (B) HS with pincered red cells (arrows), as typically seen in HS associated with band-3 defi ciency. Occasional spiculated red cells are also present. (C) Severe atypical HS due to severe, combined spectrin and ankyrin-1 defi ciency. In addition to spherocytes, many cells have irregular contours. (D) HS with isolated spectrin defi ciency due to a β-spectrin mutation. Some of the spherocytes have prominent surface projections resembling spheroacanthocytes. Reproduced from Gallagher and colleagues3 with permission. to link the membrane skeleton to the lipid bilayer.71 Rh dominant inheritance, although non-dominant and null erythrocytes have increased osmotic fragility, showing recessive inheritance has been described.75 Initial decreased membrane surface area and are dehydrated, as assessment of a patient with suspected disease should shown by reduced cell cation and water content and include a family history and questions about history of increased cell density, cellular features characteristic of anaemia, jaundice, gallstones, and splenectomy. spherocytosis associated with ankyrin, and band-3 and Physical examination should seek signs such as scleral spectrin defi ciencies.69,70,72 The genetic basis of the Rh icterus, jaundice, and splenomegaly. After diagnosis of defi ciency syndrome is heterogeneous. The amorph type a patient with hereditary spherocytosis, family members is due to defects associated with the RH30 locus encoding should be examined. In general, aff ected individuals of the RhD and RhE polypeptides. The regulatory type of Rh the same family have similar degrees of haemolysis, null and Rh mod phenotypes results from suppressor or but heterogeneous clinical patterns have also been modifi er mutations at the RH50 locus.69,70,72,73 reported in individual relatives due to additive eff ects of unequally expressed mutant alleles56,57,60 or other Clinical features modifi er alleles, or due to co-inheritance of other The clinical manifestations of hereditary spherocytosis haematological disorders. In particular, hereditary vary widely (panel 1). Manifestations of typical disease spherocytosis might be worsened or ameliorated by are haemolysis with anaemia, jaundice, reticulocytosis, obstructive jaundice, β-thalassaemia and defi ciencies of gallstones, and splenomegaly, and spherocytes on iron, vitamin B₁₂, folate, pyruvate kinase, or glucose-6- peripheral blood smear (fi gure 3), increased erythrocyte phosphate dehydrogenase.1,76,77 One of the frequent osmotic fragility, and a positive family history of associations in urban areas is heterozygosity for disease.1–3,74 It is most commonly associated with haemoglobin S or haemoglobin SC disease with www.thelancet.com Vol 372 October 18, 2008 1415 Seminar

hereditary spherocytosis, which can result in a splenic to a few common clinical laboratory variables, including infarct or acute splenic sequestration.1,78 haemoglobin and bilirubin concentrations, and reticu- Severity of hereditary spherocytosis is classifi ed as locyte count (table 3).74 mild, moderate, moderately severe, and severe according About 20–30% of patients have mild disease with compensated haemolysis—ie, red blood cell production and destruction are balanced or nearly equivalent.79 These Panel 1: Major clinical and laboratory characteristics of patients are not anaemic and are usually asymptomatic hereditary spherocytosis with mild splenomegaly, slight reticulocytosis, and • Anaemia* minimum spherocytosis, making diagnosis diffi cult in • Splenomegaly* some cases. Osmotic fragility is often increased only after • Jaundice*: from haemolysis* or biliary obstruction the blood is preincubated at 37°C for 24 h. Many of these • Haemolytic, aplastic, and megaloblastic crises individuals escape detection until adulthood when • Gallstones complications related to chronic haemolysis arise.1 • Rare manifestations: leg ulcers, gout, chronic dermatitis, Haemolysis can be exacerbated by illnesses that cause extramedullary haemopoietic tumours, haematological splenomegaly, such as infectious mononucleosis, or by malignant diseases, angioid streaks other factors, such as pregnancy or exercise.1,80 • Neuromuscular disorders Because of the asymptomatic course in these patients, • Cardiovascular disease diagnosis of hereditary spherocytosis is often made • Myopathy during studies of the family or assessment of spleno- • Spinocerebellar degeneration megaly, gallstones, or anaemia from viral infection—eg, • Reticulocytosis* parvovirus B19 or infl uenza.1,2,4 • Spherocytes* About 60–70% of patients have moderate disease, • Increased mean corpuscular haemoglobin concentration which typically presents in childhood but can present at and red blood cell distribution width* any age. In children, anaemia is the most frequent sign • Increased proportion of hyperdense cells* (50% of cases), followed by splenomegaly, jaundice, and • Increased osmotic fragility (especially after incubation)* a positive family history. The haemoglobin concentration • Negative direct antiglobulin test* is between 80–110 g/L and the proportion of reticulocytes • Inheritance is increased in most cases (table 3). The anaemia is • Dominant: about 75% usually asymptomatic except for fatigue or pallor, or both. • Non-dominant: about 25% de novo or recessive Jaundice is seen occasionally in about half the patients, • Excellent response to splenectomy usually in association with viral infections. When present, it is acholuric and characterised by unconjugated indirect *Typical signs of hereditary spherocytosis. hyperbilirubinaemia and absence of bilirubinuria. The prevalence of palpable splenomegaly varies from about Mild Moderate Moderately severe Severe* 50% in young children to 75–95% in older children and adults. Typically the spleen is moderately enlarged, but it Haemoglobin (g/L) Normal >80 60–80 <60 can be massive. Although an association between spleen Reticulocytes <6% >6% >10% >10% size and the severity of hereditary spherocytosis has not Bilirubin (μmol/L) 17·1–34·2 >34·2 >34·2–51·3 >51·3 been established, it seems likely that one exists. Diagnosis Peripheral smear Some spherocytes Spherocytes Spherocytes Microspherocytes is straightforward if spherocytosis, increased osmotic and 3 poikilocytosis fragility, and a positive family history are present. OF (fresh blood) Normal or slightly Increased Increased Increased A small group of patients, about 10%, have moderately increased severe disease, which is distinguished from moderate OF (incubated blood) Increased Increased Increased Increased hereditary disease by low haemoglobin concentrations Splenectomy Rarely† If physical ability is Necessary Necessary (table 3) and an intermittent need for transfusions. decreased or in (at >5 years) (at >2–3 years) Additionally, reticulocytosis is greater (often >15%) and some cases† bilirubinaemia more pronounced (often >51 μmol/L) Transfusions 0–1 0–2‡ >2 Regular than in moderate disease. SDS-PAGE (protein Normal Sp, Ank+Sp, Sp, Ank+Sp, band 3 Sp, Ank+Sp, About 3–5% of patients have severe hereditary disease defi ciency) band 3, band 3 protein 4.2 with life-threatening anaemia, needing regular trans- Heredity AD AD, de novo AD, de novo AR fusions to maintain a haemoglobin concentration greater mutation mutation than 60 g/L. They almost always have non-dominant disease.32,38,43 Most patients have isolated, severe spectrin AD=autosomal dominant. Ank=ankyrin-1. AR=autosomal recessive. OF=osmotic fragility. SDS-PAGE=sodium dodecyl sulphate polyacrylamide gel electrophoresis. Sp=spectrin. *Patients depend on regular transfusions. †Adults undergoing defi ciency, thought to be due to a defect in α spectrin, but a cholecystectomy or with pronounced jaundice. ‡Some patients need one or two transfusions during infancy. few have ankyrin or band-3 defects. In addition to spherocytes and microspherocytes, irregularly budding Table 3: Classifi cation of hereditary spherocytosis spherocytes or abnormal poikilocytes are sometimes seen

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on peripheral blood smear (fi gure 3). Patients might infants with hereditary spherocytosis before routine develop iron overload with its attendant clinical compli- use can be recommended. cations, necessitating continuous chelation treat ment. Close observation of infants with hereditary sphero- Without regular transfusions or splenectomy, or both, cytosis is necessary; haemoglobin concentrations and patients might have growth retardation, delayed sexual reticulocyte counts should be monitored at least once a maturation, or extramedullary erythropoiesis with hepato- month during the fi rst 6 months to detect and treat late splenomegaly and bony changes—eg, thalassaemic faces. anaemia. The observation interval in the children with The parents or other relatives of patients with recessive mild and moderate disease can be increased to 6–8 weeks hereditary disease are carriers of an asymptomatic trait. after 6 months of life, and to 3–4 months in the second They are clinically healthy and do not have anaemia, year of life. In childhood, each patient should have splenomegaly, hyperbilirubinaemia, or spherocytosis on haemoglobin and bilirubin concentrations and reticu- peripheral blood smears.74,75 Most relatives have subtle locyte counts checked every 6–12 months up to the age of laboratory signs of hereditary disease, including slight 5 years, and roughly every year thereafter. After the reticulocytosis (1·5–3·0%) or slightly reduced haptoglobin neonatal stage, the criteria for red blood cell transfusion values. The incubated osmotic fragility test is probably is a haemoglobin concentration of less than 50–60 g/L in the most sensitive method for detection of carriers, the absence of other concomitant factors—eg, fever and especially by the 100% lysis point, which is greatly viral illness. A yearly screen for parvovirus B19 serology increased in carriers (mean sodium chloride 4·3 g/L [SD could be done until a positive result for IgG is noted. 0·5]) compared with controls (2·3 g/L [0·7]). The acidifi ed This practice can be helpful for patients and parents glycerol lysis test might also be useful for the diagnosis since it will warn or reassure them of the risk of of spherocytosis. About 1% of the population are contracting this viral infection. estimated to be silent carriers.5,6 Unsplenectomised pregnant women with hereditary Complications disease do not have clinically signifi cant complications Gallbladder disease except for anaemia, which is aggravated by the plasma Chronic haemolysis leads to the formation of bilirubinate volume expansion that usually occurs in pregnancy and gallstones, which are the most common complication of sometimes by increased haemolysis or megaloblastic hereditary spherocytosis. Gallstones are noted in at least crises. Transfusions are rarely necessary.1,80 5% of children less than 10 years of age;7 the proportion In the neonate, hereditary disease often presents as increases to 40–50% in the second to fi fth decades, with jaundice in the fi rst few days of life.81,82 Kernicterus is a most stones arising between 10 and 30 years of age.88 The risk; thus, exchange transfusions are sometimes co-inheritance of Gilbert’s syndrome increases the risk of necessary, but in most patients the jaundice is controlled cholelithiasis by four-fold to fi ve-fold.89 Timely diagnosis with phototherapy. Co-inheritance of Gilbert’s syndrome, and treatment will help prevent complications of biliary- as detected by homozygosity for a TATA box polymorphism tract disease, including biliary obstruction with pan- in the uridine-diphosphate glucuronyl-transferase creatitis, cholecystitis, and cholangitis. The best method 1A1 gene (UGT1A1), increases the frequency and severity for detection of gallstones is ultrasonography.88 We of hyperbilirubinaemia in neonates with hereditary dis- recommend an abdominal ultrasound every third to fi fth ease.83,84 Hydrops fetalis associated with spectrin or year in patients with hereditary spherocytosis, every year band-3 defects is rare and has been reported in only four in those with both hereditary spherocytosis and Gilbert’s or fi ve patients.2,28,59,85 syndrome, and before splenectomy. Once the spleen is Only 28–43% of neonates with hereditary disease are removed, individuals with hereditary spherocytosis do anaemic at birth (haemoglobin concentration <150 g/L) not develop pigment stones.90 and severe anaemia is rare.7,81,86 In most newborns, haemoglobin concentrations are within the normal Haemolytic, aplastic, and megaloblastic crises range at birth, then decrease sharply during the fi rst Patients with hereditary spherocytosis, like those with 3 weeks of life, leading to a transient, severe anaemia.86 other haemolytic diseases, are at risk of increased crises.1,3 Up to three-quarters of these anaemic infants need Haemolytic crises are the most common, often triggered blood transfusions. Most infants outgrow the need for by viral illnesses and typically arise in childhood. transfusion by the end of their fi rst year of life.7,86 This Increased haemolysis is probably due to enlargement of transient anaemia is the result of erythropoiesis being the spleen during infections and activation of the reticulo- slow as shown by a reticulocyte count that is low for the endothelial system. Haemolytic crises are generally mild degree of anaemia. Regular subcutaneous admin- and are characterised by a transient increase in jaundice, istration of recombinant human erythropoietin to splenomegaly, anaemia, and reticulocytosis; intervention infants with hereditary spherocytosis was shown to be is rarely necessary. Severe haemolytic crises are associated benefi cial in reduction of the need for blood with substantial jaundice, anaemia, lethargy, abdominal transfusions in an open-label study.87 However, further pain, vomiting, and tender splenomegaly. For most study of the use of this hormone is needed in anaemic patients, only supportive care is needed; red blood cell www.thelancet.com Vol 372 October 18, 2008 1417 Seminar

transfusions are only needed if the haemoglobin con- number of reticulocytes despite severe anaemia. The centrations are greatly reduced. earliest laboratory sign is an increase in the serum iron Aplastic crises following virally-induced bone marrow concentration due to the loss of marrow erythroblasts suppression are less common than haemolytic crises, and reduced haemoglobin synthesis. Bilirubin con- but they can lead to severe anaemia, requiring treatment centrations might decrease as the number of abnormal in hospital and transfusion, with serious complications, red blood cells that can be destroyed decreases. Parents including congestive heart failure or even death. With should be advised to watch for pallor, extreme lassitude, rare exceptions, severe aplastic crises arise only once in and ashen conjunctivae after mild non-specifi c signs of life. The most common causative agent is parvovirus infection such as fever, chills, vomiting, diarrhoea, B19—the cause of erythema infectiosum (fi fth disease)91— abdominal pain, and myalgias. The character istic rash of which confers life-long immunity. Parvovirus selectively parvovirus infection in these patients is rare but when infects erythropoietic progenitor cells and inhibits their present is related to deposits of immune complexes. growth. The characteristic laboratory fi nding is a low Aplastic crises usually last 10–14 days. Confi rmation of

Patient with haemolysis and spherocytes

Mode of inheritance

Dominant Normal parents

• Typical clinical and laboratory features of HS Normal DAT • Increased numbers of hyperdense cells

Homogeneous clinical Heterogeneous clinical • Moderate or mild haemolytic anaemia Severe haemolytic anaemia, pattern in relatives pattern in relatives • Exclusion of other conditions of consanguineous parents, mean spherocytes corpuscular volume >100 fL, • Increased osmotic fragility equivocal or borderline results • Increased numbers of hyperdense of the laboratory data, or cells dyserythropolesis

Different bilirubin Different haemoglobin High bilirubin No further SDS-PAGE concentration concentration concentration investigation needed

Analysis of TATA box Search for co-inheriting Analysis of TATA box polymorphism in the haematologic disorders polymorphism in the UGT1A1 gene UGT1A1 gene Protein defect Normal

None

No further investigation needed Revaluation SDS-PAGE to search for of the different protein patient deficiencies in HS family members

None Yes Spherostomatocytosis DNA-cDNA analysis CDA II

DNA analysis for Recessive or de novo mutation low-expression alleles

Figure 4: Flow chart for the diagnosis of hereditary spherocytosis (HS) UGT1A1=uridine diphosphate glucoronsyl transferase 1A1. SDS-PAGE=sodium dodecyl sulphate polyacrylamide gel electrophoresis. DAT=direct antiglobulin test. CDA II=congenital dyserythropoietic anaemia type II.

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the diagnosis by recording the raised parvovirus IgM titres is helpful to the doctors and patients. Intravenous Panel 2: Disorders with spherocytes on peripheral blood immunoglobulin treatment is not recommended. Un- smear diagnosed, asymptomatic patients with hereditary • Hereditary spherocytosis spherocytosis and compensated haemolysis might come • Immune haemolytic anaemia to medical attention during an aplastic crisis.92 Because • Acute oxidant injury parvovirus might infect several members of a family • Microangiopathic and macroangiopathic haemolytic simultaneously, epidemics or outbreaks of hereditary anaemias spherocytosis have been reported. Importantly, for this • Haemolytic transfusion reactions reason, family members should be screened when • Thermal injuries erythroblastopenia arises in a patient with undiagnosed • Liver disease hereditary spherocytosis. • Heinz body anaemias Megaloblastic crises caused by folate defi ciency are very • Hereditary pyropoikilocytosis rare in developed countries where nutrition and prenatal • Clostridial sepsis care are good, but they can occur in patients with hereditary • Zinc toxicity spherocytosis and increased folate demands—eg, pregnant • Poisoning with some snake, spider, and Hymenoptera women, growing children, or patients recovering from an venoms aplastic crisis. To prevent folate defi ciency, the recom- • Severe hypophosphataemia mendation is that unsplenectomised patients with • Long blood storage (in vitro) hereditary spherocytosis and splenectomised patients with • ABO incompatibility (in neonates) severe hereditary spherocytosis take folate 2–3 mg per day • Hypersplenism until the age of 5 years and 5 mg per day thereafter. Rarely, patients with hereditary spherocytosis develop other complications (panel 1). In severe cases, growth sence of reticulocytes with volume consistently lower and failure, leg ulcers, or skeletal abnormalities resulting haemoglobin con centration consistently higher in from bone marrow expansion can occur.93 Extramedullary hereditary spherocytosis than in autoimmune haemolytic erythropoiesis might be associated with tumours, anaemia could be useful in distinguishing spherocytes especially along the posterior thoracic or lumbar spine or from these two diseases.99 in the hila of the kidneys.1,94,95 These tumours often arise The clinical picture of mild to moderate chronic in non-splenectomised patients with mild disease and anaemia, splenomegaly, jaundice, and gallstones in are the fi rst manifestation of hereditary spherocytosis. association with the presence of spherocytes on Easily diagnosed by MRI, these masses stop growing and peripheral blood smear, and an increased osmotic undergo fatty metamorphosis after splenectomy, but they fragility could result in the erroneous diagnosis of do not shrink in size. Haematological malignant hereditary spherocytosis in some patients with congenital diseases—eg, multiple myeloma and leukaemia—have dyserythropoietic anaemia type II. This concern is been reported in patients with hereditary spherocytosis, particularly relevant for individuals with congenital leading to the postulation that prolonged haemopoietic dyserythropoietic anaemia type II and reticulocyte counts stress was a predisposing factor. However, cause and of 120×10⁹ to 180×10⁹ per L. Careful assessment of the eff ect have not been proven.96,97 ratio of reticulocyte count to haemoglobin concentration and the signs of iron overload, particularly in adult Diagnosis and laboratory features patients, can assist in the accurate diagnosis of Hereditary spherocytosis is usually diagnosed based on a disease.100,101 However, diagnosis of congenital dys- combination of clinical and family histories, physical erythropoietic anaemia type II can be confi rmed by the examination (for splenomegaly or jaundice), and electrophoretic pattern of the red blood cell membrane laboratory data (full blood count, especially red blood cell showing a fast migrating band 3 or a bone-marrow indices and morphology, and reticulocyte count; smear examination showing an erythroid hyperplasia fi gure 4).1,3 Other causes of anaemia should be excluded, (with 10–40% of binucleate or multinucleate erythro- particularly autoimmune haemolytic anaemia, congenital blasts), or both.101 dyserythropoietic anaemia type II, and hereditary Hereditary stomatocytoses are very rare autosomal stomatocytosis. dominant haemolytic anaemias, often diagnosed as Interpretation of the direct antiglobulin test-negative atypical hereditary spherocytosis. Macrocytosis is autoimmune haemolytic anaemia can be diffi cult in an common in many but not all cases. The blood fi lm might individual, particularly in an adult, with no family history. show a few spherocytes and stomatocytes. Target cells are Flow cytometric assessment of red blood cell immuno- seen in some disorders, especially dehydrated stomato- globulin density and the application of specifi c reagents cytosis. Osmotic fragility of red blood cells and mean might be helpful in the diagnosis of some cases of corpuscular haemoglobin concentration can help to autoimmune haemolytic anaemia.98 Moreover, the pre- diff erentiate between the hydrocytosis and xerocytosis www.thelancet.com Vol 372 October 18, 2008 1419 Seminar

Several disorders can show spherocytes on peripheral 100 Severe HS blood smear (panel 2). Importantly, a smear from a patient Tail with suspected spherocytosis should be of high quality, Typical HS with the erythrocytes well separated and some cells with 80 central pallor, because spherocytes are a common artifact on blood smears. Erythrocyte morphology is quite variable in hereditary spherocytosis. In patients with typical disease, 60 spherocytes are obvious on blood smear. These cells are dense, round, and hyperchromic without the central pallor and have a reduced mean cell diameter. Infrequently, 40 patients present with only a few spherocytes on peripheral Percentage of Percentage lysis smear or with many small, dense spherocytes, and abnormal erythrocyte morphology with anisocytosis and poikilocytosis. Specifi c morphological eff ects have been 20 Normal range identifi ed in association with some membrane protein defects in hereditary spherocytosis (fi gure 3).12,30,51,53 Most patients have a mild to moderate anaemia or no 0 0·8 0·7 0·6 0·5 0·4 0·3 anaemia at all (compensated haemolysis). Neither the Saline concentration (%) mean corpuscular volume nor the percentage of microcytic red cells has any diagnostic value. The mean Figure 5: Osmotic fragility curves in hereditary spherocytosis (HS) corpuscular haemoglobin concentration is greater than The shaded area is the normal range. Results representative of both typical and 360 g/L in half of patients because of cellular severe HS are shown. A tail, representing very fragile erythrocytes that have 104 been conditioned by the spleen, is common in many patients with hereditary dehydration. Combination of the mean corpuscular spherocytosis before splenectomy. Reprinted from Dacie with permission.108 haemoglobin concentration with the red blood cell distribution width can lead to a specifi city of nearly 100%.105 variants. Moreover, the key test for diagnosis is osmotic Some automated haematology analysers measure cell gradient ektacytometry, available only in specialised haemoglobin concentration of individual red blood cells, laboratories. Diff erentiation of hereditary spherocytosis and increased numbers of dense dehydrated cells might from hereditary stomatocytosis and related disorders is identify all patients with hereditary spherocytosis without important because splenectomy is associated with a high the need for additional laboratory tests, especially when risk of thromboembolic events in hereditary stomato- one member of a family is already known to have the cytosis.102,103 illness.104–107 To rule out the diagnosis of disease in the An incubated osmotic fragility test should be done presence of normal osmotic fragility, assessment of the after an initial laboratory investigation for diagnosis of state of cell hydration is essential. If mean corpuscular hereditary spherocytosis that includes a complete blood haemoglobin concentration or the percentage of count with peripheral smear, reticulocyte count, direct hyperdense red blood cells is increased, a diagnosis of antiglobulin test, serum bilirubin, and a family history hereditary spherocytosis should be seriously considered suggestive of hereditary spherocytosis. Rarely, despite normal osmotic fragility. These variables are less additional, specialised tests are needed to confi rm the pronounced in splenectomised than in non-splen- diagnosis. ectomised patients with this disorder. The diagnosis of hereditary spherocytosis is often more In the healthy erythrocyte, membrane redundancy diffi cult in the neonatal period than later in life.81 gives the cell its characteristic discoid shape and provides Splenomegaly is infrequent and at most the spleen tip is it with abundant surface area relative to cell volume. In palpable; reticulocytosis is variable and usually not severe spherocytes, surface area relative to cell volume is (only 35% of aff ected newborns have a reticulocyte reduced, resulting in the increased osmotic fragility count >10%). About 33% of neonates with hereditary noted in these cells. Osmotic fragility is tested by spherocytosis do not have large numbers of spherocytes assessment of the extent of haemolysis of red blood cells in their peripheral blood smears, whereas spherocytes are suspended in increasingly hypotonic solutions of saline commonly seen in neonatal blood fi lms in the absence of (fi gure 5). Healthy erythrocytes are able to increase their disease.81 Because neonatal red blood cells are more volume with increasing hypotonicity by swelling until osmotically resistant than are adult cells, the osmotic they reach their critical haemolytic volume, after which fragility test is less reliable for diagnosis of this disease. lysis occurs. Spherocytes with reduced surface area reach For these reasons, unless the need for diagnosis is urgent, their critical haemolytic volume at higher saline tests should be postponed until the child is at least concentrations than do healthy erythrocytes and as such 6 months of age. If diagnostic tests are needed, appropriate are osmotically more fragile. About 25% of individuals osmotic fragility curves that have been developed for with hereditary spherocytosis will have a normal osmotic neonates should be used. fragility of their freshly drawn red blood cells.109 After

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incubation at 37°C for 24 h, red blood cells from patients with hereditary spherocytosis lose membrane surface A B area more readily than do healthy cells. Incubated osmotic fragility test is thought to be the gold standard in the diagnosis of hereditary spherocytosis in a patient with direct antiglobulin test-negative spherocytic haemolytic anaemia, particularly one of northern European descent or someone with a positive family history of undiagnosed anaemia. When the spleen is present, a subpopulation of very fragile erythrocytes that have been conditioned by the spleen form the tail of the osmotic fragility curve that disappears after splenectomy (fi gures 2 and 5). Osmotic fragility tests have a poor sensitivity because about 20% of mild cases of hereditary spherocytosis are Figure 6: Surgical technique used for partial (about 80%) splenectomy missed.110 It is unreliable in patients with small numbers (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. of spherocytes, including those who have recently Reproduced from Tchernia and colleagues140 with permission from the American Society of Hematology. received a blood transfusion. The fi nding of in creased osmotic fragility is not unique to hereditary sphero- transfusion-dependent patients.35,85 Alternatively, candi- cytosis and is also present in other disorders associated date genes can be identifi ed by comparison of genomic with spherocytosis—eg, autoimmune haemo lytic DNA and reticulocyte mRNA (as cDNA) for reduced anaemia. A normal osmotic fragility test result does not expression of one allele (fi gure 4).31,32,38–40,42 From a clinical exclude the diagnosis of hereditary sphero cytosis.109 The perspective, a biochemical and molecular analysis might test might be normal in the presence of iron defi ciency, be useful only in selected patients. Patients with an obstructive jaundice, and during the recovery phase of appropriate clinical history in the family usually do not an aplastic crisis when the reticulocyte count is request further investigations. However, those with increased.110 dominantly inherited hereditary spherocytosis with A rapid fl ow cytometric analysis of eosin-5-maleimide relatives showing a wide clinical heterogeneity might be bound to erythrocytes has been introduced as a screening assessed for co-inheritance of another erythrocytic defect test for the diagnosis of hereditary spherocytosis and for (thalassaemic trait, sickle haemoglobin, or pyruvate the study of membrane protein defi ciency.111,112 This test kinase or glucose-6-phosphate dehydrogenase defi ciency) holds great clinical promise for standardised testing for or for low-expression alleles occurring in trans to the hereditary spherocytosis. hereditary spherocytosis allele. SDS-PAGE of membrane The best method for showing reduced membrane proteins should be done in these patients with low- surface area of spherocytes is osmotic gradient ekta- expression alleles. cytometry, available only in specialised labora tories.104,113 Some characterisation of the underlying mutation might Quantifi cation of the amount of spectrin or ankyrin-1, be useful in families with unclear heritage and severe or or both, band-3 protein, or protein 4.2 in the erythrocyte atypical disease. Candidate genes can be identifi ed by membrane can support the diagnosis of disease in SDS-PAGE. Alternatively, silent polymorphisms off er a atypical cases. However, these measurements are diffi cult rational approach to the study of disease. Frameshift or to implement because small variations from normal non-sense mutations are most common in hereditary should be accurately measured since membranes from spherocytosis, and no mutant RNA (or protein) is present some patients might have only a 10–15% reduction in the in reticulocytes. A fi nding that one ankyrin-1, β-spectrin, aff ected protein. The simplest method is SDS-PAGE of α-spectrin, or band-3 gene is not expressed by comparison red blood cell membranes, but this technique reveals of the frequent polymorphisms in genomic DNA and abnormalities in only 70–80% of patients.37 Amounts of RNA/cDNA (loss of heterozygosity) is proof that a null spectrin and ankyrin-1 are best assessed by RIA or mutation exists. This method also has been used for EIA.24,36,114 However, these methods are available only in a identifi cation of candidate genes for de-novo mutations in few specialised laboratories. patients with this disease.41 All the exons in the candidate Identifi cation of a membrane protein defi ciency genes can then be screened but it is time-consuming and facilitates identifi cation of the underlying genetic defect. very expensive. PCR-based mutation screening techniques have been Other laboratory manifestations in hereditary sphero- applied to the detection of hereditary spherocytosis- cytosis are markers of persisting haemolysis. Reti- associated mutations.38,40 Because most of the proteins culocytosis and increased indirect bilirubin, lactate that cause this disease are large and contain many exons, de hydro genase, and urinary and fecal urobilinogen, and this approach is diffi cult. Identifi cation of the molecular reduced haptoglobin indicate an increased erythrocyte defect is, however, useful—eg, in undiagnosed, production or destruction. www.thelancet.com Vol 372 October 18, 2008 1421 Seminar

Treatment and outcome adult patients with pronounced visible jaundice due to Splenectomy the combination of mild to moderate hereditary sphero- Splenic sequestration is the main determinant of cytosis and Gilbert’s syndrome (table 3).2,89 Whether erythrocyte survival in patients with hereditary sphero- patients with moderate disease and compensated, cytosis. Thus, splenectomy cures almost all patients with asymptomatic anaemia should be splen ectomised this disorder, eliminating the anaemia and hyper- remains controversial. Patients with mild disease and bilirubinaemia and reducing the reticulocyte count to compensated haemolysis can be followed up and referred nearly normal.3,23,75 After splenectomy, spherocytosis and for splenectomy if clinically suggested. However, the altered osmotic fragility persist, but the tail of the osmotic indication for splenectomy should be considered carefully fragility curve disappears. Although patients with very because the mean haemoglobin values in splenectomised severe disease are not completely cured by splenectomy, patients are higher than those in healthy individuals104 their clinical improvement after splenectomy is striking. and splenectomy could predispose to late adverse The rare splenectomy failure is usually associated with thrombotic events.126 an accessory spleen.115 Treatment of patients with mild to moderate hereditary Early complications of splenectomy include local spherocytosis and gallstones is also debatable, especially infection, bleeding, and pancreatitis. The serious long- since new treatments for cholelithiasis, including term complication is overwhelming postsplen ectomy laparoscopic cholecystectomy, endoscopic sphinctero- infection with encapsulated bacteria, mostly Streptococcus tomy, cholecystostomy (simple removal of stones without pneumoniae, and, in some geographic regions (America the gallbladder) and extracorporeal cholecystolithotripsy, and Africa), parasitic infection.116 A postsplenectomy reduce the risk of this complication.132,133 If splenectomy is mortality rate of 0·05–0·30 per 100 person-years of done in a patient with symptomatic gallstones, follow-up has been reported.117,118 Immunisation against cholecystectomy or cholecystostomy should be done pneumococcus, Haemophilus infl uenzae type b, and concomitantly.134 Prophylactic cholecystectomy at the meningococcus, the use of prophylactic penicillin after time of splenectomy is not suggested for patients without splenectomy, or the promotion of early antibiotic cholelithiasis.135,136 treatment for febrile illnesses after splenectomy, or both, When splenectomy is warranted, laparoscopic splen- have reduced but not eliminated postsplenectomy ectomy is the method of choice.137–139 Although infection.119–125 laparoscopic splenectomy needs a longer operative time, Because the risk of postsplenectomy infection is very it results in less postoperative discomfort, a quicker high in infancy and early childhood, splenectomy should recovery time, shorter hospital stay, decreased costs, and be delayed until 6–9 years of age if possible, but should smaller scars than does open splenectomy. Even huge not be done before 3 years of age, even if chronic spleens (>500 g) can be removed laparoscopically. transfusion is needed in the interim. Further delay might Near-total splenectomy has been advocated for infants be harmful because the risk of cholelithiasis increases and young children with substantial anaemia associated greatly in children after 10 years of age. with hereditary sphero cytosis (fi gure 6).140–144 It is usually an open operation, but a laparoscopic procedure has Indications for splenectomy and cholecystectomy been reported.145,146 The goal is to allow for palliation of Risks and benefi ts should be assessed carefully before haemolysis and anaemia while maintaining residual splenectomy is done. A multitude of factors aff ect the splenic immune function. Splenic regrowth necessitating decision to do splenectomy, including the risk of reoperation is a concern. The procedure seems to reduce postsplenectomy infection, the emergence of penicillin- the severity of the disease by about one grade and thus resistant pneumococci, access to medical care, and the can be recommended for children with severe hereditary increased risk of ischaemic heart disease, cerebral stroke, spherocytosis between 3 and 5 years of age. A second pulmonary hypertension, and thrombosis later in life.126–130 total splenectomy might be done after age 5–6 years if Hereditary spherocytosis management guidelines recog- the results are not completely satisfactory. More nise these important considerations and recom mend experience and a long follow-up are needed to assess detailed discussion between health-care providers, the eff ectiveness of near-total splenectomy patient, and family when splenectomy is con sidered.131 Before splenectomy, patients should be immunised After consideration of the risks and benefi ts, a against S pneumoniae, H infl uenzae type b, and Neisseria reasonable approach would be to splenectomise all meningitidis, preferably several weeks preoperatively. The patients with moderately severe and severe hereditary use and duration of prophylactic antibiotics after splen- spherocytosis and all those who have symptomatic ectomy is controversial, especially since the worldwide haemolytic anaemia, growth retardation, skeletal emergence of penicillin-resistant pneumo cocci. Scientifi c changes, leg ulcers, or extramedullary haemopoietic justifi cation for any specifi c regimen does not exist—eg, tumours. Other candidates for splenectomy are older fi rst 3–5 years after surgery versus lifelong, and expert patients with hereditary spherocytosis who have vascular opinions diff er widely on the use of diff erent regi- compromise of the vital organs or, for cosmetic reasons, mens.147

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Conclusion and future directions 17 Mohandas N, Chasis JA. Red blood cell deformability, membrane Challenges for the future are: (1) development of accurate, material properties and shape: regulation by transmembrane, skeletal and cytosolic proteins and lipids. Semin Hematol 1993; sensitive, and specifi c diagnostic laboratory tests for 30: 171–92. hereditary spherocytosis; (2) identifi cation of the 18 Lee JC, Discher DE. Deformation-enhanced fl uctuations in the red molecular basis of disease, especially for the 10–15% of cell skeleton with theoretical relations to elasticity, connectivity, and spectrin unfolding. Biophys J 2001; 81: 3178–92. patients for whom the genetic defects have yet to be 19 Van Dort HM, Knowles DW, Chasis JA, Lee G, Mohandas N, defi ned; and (3) establishment of criteria to assess Low PS. Analysis of integral membrane protein contributions to the appropriate indications for splenectomy, timing of deformability and stability of the human erythrocyte membrane. J Biol Chem 2001; 276: 46968–74. splenectomy in children with severe disease, and the 20 Delaunay J. The molecular basis of hereditary red cell membrane 147 long-term outcome of near-total splenectomy. disorders. Blood Rev 2007; 21: 1–20. Confl ict of interest statement 21 Gallagher PG, Jarolim P. Red cell membrane disorders. In: We declare that we have no confl ict of interest. Hematology: basic principles and practice. Hoff man R, Benz EJ JR, Shattil SJ, et al, eds. 3rd edn. Philadephia: WB Saunders, Acknowledgments 2005: 576–610. Funding was partly provided by grants from Progetti di Rilevante 22 Chasis JA, Agre P, Mohandas N. Decreased membrane mechanical Interesse Nazionale, Agenzia Italiana del Farmaco, and Regione stability and in vivo loss of surface area refl ect spectrin defi ciencies Campania (LR 5/02, 2005) to SP, DK62039 and HK65488 to PGG, and in hereditary spherocytosis. J Clin Invest 1988; 82: 617–23. DK26263 to NM. 23 Reliene R, Mariani M, Zanella A, et al. Splenectomy prolongs in vivo survival of erythrocytes diff erently in spectrin/ankyrin- and References band 3-defi cient hereditary spherocytosis. Blood 2002; 100: 2208–15. 1 Gallagher P, Lux S. Disorders of the erythrocyte membrane. In: 24 Agre P, Orringer EP, Bennett V. Defi cient red-cell spectrin in severe, Nathan D, Orkin S, eds. Nathan and Oski’s hematology of infancy recessively inherited spherocytosis. N Engl J Med 1982; 306: 1155–61. and childhood. Philadelphia: Elsevier-Saunders, 2003: 560–684. 25 Gallagher PG. Update on the clinical spectrum and genetics of red 2 Eber S, Lux SE. Hereditary spherocytosis-defects in proteins that blood cell membrane disorders. Curr Hematol Rep 2004; 3: 85–91. connect the membrane skeleton to the lipid bilayer. Semin Hematol 2004; 41: 118–141. 26 Gallagher PG. Hematologically important mutations: ankyrin variants in hereditary spherocytosis. Blood Cells Mol Dis 2005; 35: 345–47. 3 Gallagher PG, Jarolim P. Red cell membrane disorders. In: Hematology: basis principles and practice. Hoff man R, Benz EJ Jr, 27 Wong EY, Lin J, Forget BG, Bodine DM, Gallagher PG. Sequences Shattil SJ, et al, eds. 4th edn. Philadelphia: WB Saunders, downstream of the erythroid promoter are required for high level 2005: 669–91. expression of the human alpha-spectrin gene. J Biol Chem 2004; 279: 55024–33. 4 Jensson O, Jonasson JL, Magnusson S. Studies on hereditary spherocytosis in Iceland. Acta Med Scand 1977; 201: 187–95. 28 Gallagher PG, Weed SA, Tse WT, et al. Recurrent fatal hydrops fetalis associated with a nucleotide substitution in the erythrocyte 5 Godal HC, Heisto H. High prevalence of increased osmotic fragility beta-spectrin gene. J Clin Invest 1995; 95: 1174–82. of red blood cells among Norwegian blood donors. Scand J Haematol 1981; 27: 30–34. 29 Becker PS, Tse WT, Lux SE, Forget BG. Beta spectrin Kissimmee: a spectrin variant associated with autosomal dominant hereditary 6 Eber SW, Pekrun A, Neufeldt A, Schroter W. Prevalence of spherocytosis and defective binding to protein 4.1. J Clin Invest 1993; increased osmotic fragility of erythrocytes in German blood donors: 92: 612–16. screening using a modifi ed glycerol lysis test. Ann Hematol 1992; 64: 88–92. 30 Hassoun H, Vassiliadis JN, Murray J, et al. Characterization of the underlying molecular defect in hereditary spherocytosis associated 7 Pinto L, Iolascon A, Miraglia del Giudice E, Matarese SMR, with spectrin defi ciency. Blood 1997; 90: 398–406. Nobili B, Perrotta S. The Italian survey on hereditary spherocytosis. Int J Pediatr Hematol Oncol 1995; 2: 43–47. 31 Miraglia del Giudice E, Lombardi C, Francese M, et al. Frequent de novo monoallelic expression of beta-spectrin gene (SPTB) in 8 Premetis E, Stamoulakatou A, Loukopoulos D. Erythropoiesis: children with hereditary spherocytosis and isolated spectrin Hereditary Spherocytosis in Greece: Collective data on a large defi ciency. Br J Haematol 1998; 101: 251–54. number of patients. Hematology 1999; 4: 361–66. 32 Miraglia del Giudice E, Nobili B, et al. Clinical and molecular 9 Ricard MP, Gilsanz F, Millan I. Erythroid membrane protein evaluation of non-dominant hereditary spherocytosis. Br J Haematol defects in hereditary spherocytosis. A study of 62 Spanish cases. 2001; 112: 42–47. Haematologica 2000; 85: 994–95. 33 Wichterle H, Hanspal M, Palek J, Jarolim P. Combination of 10 Sanchez-Lopez JY, Camacho AL, Magana MT, Ibarra B, Perea FJ. two mutant alpha spectrin alleles underlies a severe spherocytic Red cell membrane protein defi ciencies in Mexican patients with hemolytic anemia. J Clin Invest 1996; 98: 2300–07. hereditary spherocytosis. Blood Cells Mol Dis 2003; 31: 357–59. 34 Dhermy D, Steen-Johnsen J, Bournier O, et al. Coinheritance of 11 Boguslawska DM, Heger E, Chorzalska A, et al. 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