Hereditary spherocytosis

INTRODUCTION — 

Although rare, hereditary spherocytosis (HS) is the most common red blood cell (RBC) membrane disorder. It is a result of heterogeneous alterations in one of five genes that encode RBC membrane proteins involved in vertical associations that link the membrane skeleton to the lipid bilayer.

The genetics, pathophysiology, clinical features, diagnosis, and treatment of HS are reviewed here. Other inherited RBC membrane disorders and a general approach to evaluating hemolytic anemia are discussed separately.

●Hereditary elliptocytosis and hereditary pyropoikilocytosis – (See "Hereditary elliptocytosis and related disorders".)

●Hereditary stomatocytosis – (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

●Hemolytic anemia, child – (See "Overview of hemolytic anemias in children".)

●Hemolytic anemia, adult – (See "Diagnosis of hemolytic anemia in adults".)

PATHOPHYSIOLOGY — 

HS is a heterogeneous group of disorders caused by variants in genes that encode proteins of the red blood cell (RBC) membrane skeleton (figure 1); these variants typically cause a decreased amount of protein produced or incorporated in the membrane skeleton, leading to decreased vertical linkages between the two components of the RBC membrane: the skeleton and the lipid bilayer (figure 2) [1,2].

The loss of vertical linkages causes loss of RBC membrane and a spherocytic (rather than biconcave disc) RBC shape, with decreased surface-to-volume ratio and a decrease in the deformability that is essential to the normal RBC lifespan. (See "Red blood cell membrane: Structure and dynamics".)

Genetics

Proteins and genes involved — HS is caused by deficiency of one of the following proteins, caused by pathogenic variants in the corresponding gene [2-4]:

●Alpha-spectrin – SPTA1 gene

●Beta-spectrin – SPTB gene

●Ankyrin – ANK1 gene

●Protein 4.2 – EPB42 gene

●Band 3 – SLC4A1 gene

HS-causing variants in these genes are distributed throughout the length of the gene and tend to be "private" to individual kindreds rather than localized to specific "hotspots" (figure 1) [5-7]. The roles of pathogenic variants in these genes in causing HS have been studied in several transgenic mouse models and in mice and cattle with naturally occurring variants [8-12]. Additional information about the assembly of membrane proteins is presented separately.

Additional information about these proteins includes:

●Band 3 (the anion exchanger AE1, also called solute carrier family 4 anion exchanger) is an ion channel. (See 'Band 3 deficiency due to SLC4A1 variants' below.)

●Protein 4.2 (also called band 4.2) was previously referred to as pallidin, which is now recognized as a different protein [13,14]. (See 'Band 4.2 deficiency due to EPB42 variants' below.)

●Concomitant loss of other membrane proteins – Due to the tight interactions of the RBC membrane skeleton proteins, protein chemistry frequently demonstrates a combination of protein changes. As examples:

•Alpha-spectrin deficiency due to SPTA1 pathogenic variants can be associated with concomitant decreases in beta-spectrin.

•Ankyrin deficiency due to ANK1 variants is accompanied by secondary spectrin deficiency due to reduced incorporation of spectrin into the membrane since ankyrin is the principal binding site for membrane spectrin [11].

•SLC4A1 variants can also cause secondary band 4.2 deficiency.

•Band 3 loss is a typical finding in all types of spherocytosis regardless of the genetic cause since band 3 is lost along lipid membrane during the development of spherocytes [5].

●Related, non-HS disorders – Variants affecting protein 4.1R, encoded by the EPB41 gene, have not been reported in HS. Protein 4.1R is an integral component of the junctional complex, participating in the horizontal associations of the RBC cytoskeleton; pathogenic variants in EPB41 cause hereditary elliptocytosis. (See "Hereditary elliptocytosis and related disorders".)

Pathogenic variants in SPTA1 (encodes alpha-spectrin) and SPTB (encodes beta-spectrin) that cause qualitative rather than quantitative spectrin abnormalities cause hereditary elliptocytosis (HE) rather than HS. Rare pathogenic variants in SPTA1 and SPTB that cause both spectrin deficiency (a feature of HS) as well as abnormal spectrin tetramer formation (a feature of HE) cause hereditary spherocytic elliptocytosis or pyropoikilocytosis [15-17]. (See "Hereditary elliptocytosis and related disorders".)

Variants affecting the Rh-associated glycoprotein, encoded by the RHAG gene, cause overhydrated hereditary stomatocytosis, while variants in PIEZO1 or KCNN4 cause dehydrated hereditary stomatocytosis or xerocytosis. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

Congenital dyserythropoietic anemia (CDA) type II is a rare autosomal recessive disorder due to biallelic pathogenic variants in SEC23B, presenting with hemolytic anemia and not infrequently misdiagnosed as HS, due to spherocytes on the blood smear and increased osmotic fragility and/or abnormal eosin-5-maleimide (EMA) testing due to altered band 3 glycosylation [18]. (See 'Differential diagnosis' below.)

Inheritance patterns — The following inheritance patterns are seen:

●HS inheritance is autosomal dominant in 65 to 70 percent of cases, caused by heterozygous pathogenic variants in ANK1, SLC4A1, or SPTB [4,6].

●An additional 5 to 15 percent of HS is autosomal recessive, caused by biallelic variants in SPTA1, EPB42, or ANK1 [2,19-22].

●The remaining 15 to 20 percent of HS appears to be due to de novo pathogenic variants [23-28]. Most commonly these occur in the ANK1 gene, likely due to its high G/C content, which predisposes to slipped strand mispairing during deoxyribonucleic acid (DNA) replication [29].

●A few cases of autosomal recessive HS associated with biallelic ANK1 variants have been described, in which a nonsense variant occurs in trans to a promoter or intronic variant affecting the level of protein expression, or to a missense variant decreasing but not obliterating the incorporation of ankyrin into the membrane skeleton, leading to a severe, transfusion-dependent disease that remains amenable to treatment with splenectomy [6].

●Very rare cases of autosomal recessive HS due to biallelic SLC4A1 variants have been reported in infants, some requiring in utero transfusions or born prematurely with life-threatening hydrops fetalis. These infants have severe transfusion-dependent HS, not improving with splenectomy [30,31]. Findings are similar to the most severe form of SPTA1-associated HS due to complete alpha-spectrin deficiency [32]. Biallelic truly null SPTB variants have not been described, indicating that complete beta-spectrin deficiency may be incompatible with life [2].

Ankyrin deficiency due to ANK1 pathogenic variants — Ankyrin is the major protein responsible for mechanically coupling the lipid bilayer of the RBC membrane to the underlying spectrin-based skeleton. Ankyrin, along with protein 4.2, anchors the transmembrane protein complexes, composed of band 3 and the Rh-associated glycoprotein (RhAG), to the spectrin tetramers.  

●Over 320 ANK1 pathogenic variants have been described and included in the Human Gene Mutation Database (HGMD) [33,34]. These include missense/nonsense variants, splicing and regulatory substitutions, small and large deletions, insertions, and duplications. Many of these variants are "private" (found in only a single kindred) [35,36].

●ANK1-associated HS is typically autosomal dominant, caused by a heterozygous pathogenic variant that decreases ankyrin expression or incorporation to the RBC membrane skeleton by up to 50 percent. A few cases have been described with biallelic variants, causing recessive, more severe HS; in those cases, a null variant is present in trans to a promoter or intronic variant, or a missense variant decreasing but not obliterating either expression from that second allele or the amount of protein incorporated in the skeleton. Such combinations lead to a more severe, frequently transfusion-dependent disease; associated reticulocytosis, rather than reticulocytopenia, is typically a good indicator that the disease is amenable to treatment with splenectomy [6,37].

Patients with a deletion of the short arm of chromosome 8, which contains the ANK1 gene, have been described; these individuals have HS, intellectual disability, and other congenital anomalies [38].

Band 3 deficiency due to SLC4A1 variants — Band 3 provides cohesion between the RBC membrane skeleton and the lipid bilayer, preventing membrane surface loss, and it also exchanges bicarbonate for chloride ions, maintaining RBC water content and preventing cellular dehydration.

SLC4A1 pathogenic variants appear to cause approximately 5 percent of HS cases (see 'Proteins and genes involved' above). However, this may be an underestimate, since SLC4A1-associated HS is typically mild and therefore infrequently investigated with clinical sequencing.

Band 3 deficiency is a typical phenotypic finding in all types of spherocytosis regardless of the genetic cause since band 3 is lost from the RBC lipid membrane in the process of spherocyte development. (See 'Proteins and genes involved' above.)

HS due to SLC4A1 pathogenic variants is typically autosomal dominant and presents as mild hemolytic anemia or compensated hemolysis. Over 130 pathogenic variants in SLC4A1 have been reported in the HGMD database, including missense and nonsense mutations, splicing and regulatory substitutions, small deletions, insertions, or indels, as well as gross deletions and insertions [5,7,33,34,39-45]. Hemolysis may be aggravated by compound heterozygosity for a pathogenic variant in SLC4A1 in trans to a low-expression allele [46,47].

Very rare cases of autosomal recessive HS due to biallelic SLC4A1 null variants have been reported in infants requiring in utero transfusions or born prematurely with life-threatening hydrops fetalis. These infants have severe transfusion-dependent HS, not improving with splenectomy, in addition to severe distal renal tubular acidosis (RTA) [30,31]. (See "Etiology and diagnosis of distal (type 1) and proximal (type 2) renal tubular acidosis".)

Variants in SLC4A1 affecting band 3 can cause other disorders besides HS:

●SAO – A heterozygous deletion of the codons 400 to 408 causes Southeast Asian ovalocytosis (SAO) (picture 1). The deleted nine amino acids span between the cytoplasmic and membrane domain of the band 3 protein causing inactivation of its anion exchanger function, along with a tighter attachment to ankyrin and reduced lateral mobility of the channel, likely underlying the marked rigidity of SAO RBCs [48,49]. (See "Southeast Asian ovalocytosis (SAO)".)

●Distal RTA – Individuals with certain SLC4A1 pathogenic variants causing loss of the anion exchanger function of band 3 can have autosomal dominant or autosomal recessive distal RTA. Some of these individuals may have HS [40]; however, most do not, since glycophorin A (GPA) present in RBCs but not kidney epithelial cells chaperones the distal RTA-causing band 3 molecules to the RBC membrane efficiently [30,40,50-55].

Patients with SAO and distal RTA have been shown to be compound heterozygous for the SAO deletion (∆400-408) and a distal RTA-causing variant [56]. (See "Etiology and clinical manifestations of renal tubular acidosis in infants and children", section on 'Genetic causes' and "Etiology and diagnosis of distal (type 1) and proximal (type 2) renal tubular acidosis", section on 'Distal (type 1) RTA'.)

●HS due to protein 4.2 deficiency – The SLC4A1 gene encodes the antigens of the Diego blood group system [57]. Polymorphisms cause variability of the Diego antigens, which are important for transfusion medicine and hemolytic disease of the fetus and newborn; however, they do not cause HS. A notable exception is the common polymorphism c.118G>A (p.Glu40Lys), named band 3 Montefiore, which in the homozygous state decreases the incorporation of protein 4.2 into the RBC cytoskeleton by approximately 88 percent, causing HS due to protein 4.2 deficiency [58].

●Cryohydrocytosis – Certain heterozygous SLC4A1 single amino-acid substitutions in the transmembrane domain of RBC band 3 convert its function from an anion exchanger (AE1) into an unregulated cation channel, leaking intracellular K⁺ with a temperature-dependent, passive diffusion process [59]. Such SLC4A1 pathogenic variants cause cryohydrocytosis, a form of hereditary stomatocytosis, where the K⁺ leak decreases at room temperature (23°C) and increases to a maximum when the temperature approaches 5°C [60]. This K⁺ leak via the altered AE1 channel disturbs the osmotic balance and surface-to-volume ratio of the RBCs, leading to chronic hemolysis [61,62]. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

●Stomatocytosis and dyserythropoiesis – A de novo band 3 variant (p.G796R-band3 CEINGE) was shown to be associated with stomatocytosis and a dyserythropoietic anemia phenotype on bone marrow studies [63].

Beta-spectrin or alpha-spectrin deficiency due to SPTB or SPTA1 pathogenic variants — Beta-spectrin (encoded by SPTB) and alpha-spectrin (encoded by SPTA1) associate in a rod-like heterodimer; two of these heterodimers associate head to head in tetramers that make the sides of each triangular unit in the hexagonal structure of the RBC cytoskeleton (figure 1).

While spectrin heterotetramers are the main components of the horizontal cytoskeleton, they are also critical for the vertical interactions with the lipid membrane via their binding to ankyrin/band 3 complexes (figure 2). Qualitatively abnormal spectrin causes hereditary elliptocytosis (HE); conversely, quantitative decreases in the amount of spectrin produced or incorporated to the cytoskeleton due to pathogenic variants in the corresponding genes cause HS [2,64]. Abnormalities in both the quality and the quantity of beta-spectrin or alpha-spectrin incorporated into the RBC cytoskeleton cause a mixed phenotype of spherocytic elliptocytosis/pyropoikilocytosis [16,17]. (See "Hereditary elliptocytosis and related disorders", section on 'Hereditary spherocytic elliptocytosis (HSE)'.)

HS due to SPTB pathogenic variants is an autosomal dominant disease that presents as mild to moderate hemolytic anemia. Recessive disease due to truly null biallelic SPTB variants has not been described, indicating that complete deficiency of beta-spectrin is likely to be lethal early in embryonic life. Over 300 pathogenic variants in SPTB have been described in the HGMD database, most of them nonsense mutations, with a few missense mutations, several splicing variants, small deletions, insertions, or indels, as well as gross deletions, insertions, and complex rearrangements [33,34,45,65-73].

HS due to alpha-spectrin deficiency is an autosomal recessive disease. Each SPTA1 allele produces alpha-spectrin well in excess; therefore, biallelic pathogenic variants are required in SPTA1 to decrease production of the protein enough to cause disease [18,32,74-77]. The typical genotype for this type of HS is a null SPTA1 variant, such as a nonsense, frameshift, or splicing variant causing nonsense-mediated decay, or whole gene deletion, in trans to the deep intronic variant c.4339-99C>T, also known as alpha-LEPRA (Low Expression PRAgue), which severely decreases the expression of alpha-spectrin from that SPTA1 allele [32,76,77]. This genotype causes severe to moderately severe, most typically transfusion-dependent HS since birth, that nevertheless responds well to splenectomy.

Rare cases of autosomal recessive SPTA1-associated disease with biallelic pathogenic variants causing complete or almost complete alpha-spectrin deficiency may present with fatal hydrops fetalis in the third trimester of pregnancy or with life-threatening anemia in utero if pregnancy is closely monitored. These fetuses can survive to term with in utero transfusions or if they are delivered prematurely for another reason and receive transfusions in the neonatal period. Characteristic for this disease is reticulocytopenia even when anemic, and the disease does not respond to splenectomy, requiring either lifelong transfusions with chelation therapy or treatment with hematopoietic stem cell transplant [32].

Band 4.2 deficiency due to EPB42 variants — Band 4.2 (also called protein 4.2) strengthens the linkage between band 3 and ankyrin.

EPB42-associated HS is an autosomal recessive disease. The typical patient with band 4.2 deficiency is of Japanese ancestry [78]; however, patients of other backgrounds have also been identified [79,80]. Approximately 20 pathogenic variants in EPB42 have been reported, including missense and splicing mutations, as well as a few small deletions, indels, and gross deletions [81-86].

Other cases of band 4.2 deficiency may be secondary to a variant in SLC4A1 (encoding band 3) that alters its binding with band 4.2 protein and/or its interactions with the membrane skeleton [58,87-90]. Deficiencies of band 4.2 are a rare cause of HS. (See 'Proteins and genes involved' above.)

Changes in the RBC membrane — Most of the pathogenic variants that cause HS do so by reducing the level of one or more RBC membrane proteins that link the membrane skeleton to the overlying lipid bilayer. It is also possible for HS variants to affect protein-protein binding rather than protein abundance. (See 'Proteins and genes involved' above.)

Studies from animal models suggest that one of the mechanisms of secondary protein deficiencies from the membrane skeleton involves aberrant sorting of these proteins during the enucleation stage of RBC maturation (when the nucleus is extruded, certain proteins are expelled with the extruded cell nucleus) [91].

The loss of these proteins results in reduced vertical associations between the membrane skeleton and the overlying lipid bilayer, which in turn causes microvesiculation and progressive membrane loss, the central abnormality that defines HS [92-94]. Loss of membrane reduces the ratio of RBC surface area to volume, in turn creating progressively more spherical cells (picture 2). (See "Red blood cell membrane: Structure and dynamics", section on 'Surface area to volume ratio (SA/V)'.)

There is evidence that membrane loss is present as early as the reticulocyte stage; this distinguishes HS from autoimmune hemolytic anemia (AIHA), in which typically only mature RBCs become spherocytic due to membrane loss [95].

Two hypotheses have been advanced to explain how deficient production or incorporation of these membrane proteins leads to vesiculation and membrane loss. In the first, spectrin deficiency acts directly on the bilayer to create areas of weakness that allow membrane loss. In the second, band 3 deficiency or dispersion causes vesiculation by reducing the integrity of the lipid bilayer [96].

Some individuals with HS have abnormal RBC ion transport, the severity of which may depend on the degree of band 3 (also known as anion exchanger 1 [AE1]) deficiency [97]. On the other hand, all spherocytes demonstrate increased passive permeability to monovalent cations (sodium and potassium) [97,98]. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)", section on 'Control of RBC solute and water content'.)

Mechanisms of hemolysis — Spherocytes are prone to hemolysis due to reduced deformability that impairs passage through the microcirculation, hemolysis within the splenic microenvironment, and/or phagocytosis by the splenic red pulp macrophages in response to splenic trapping [99,100].

Loss of spectrin appears to be especially correlated with hemolysis severity [101]; this is seen with severe reticulocytopenia and hemolysis of nascent reticulocytes within the bone marrow in complete alpha-spectrin deficiency [32]. (See 'Beta-spectrin or alpha-spectrin deficiency due to SPTB or SPTA1 pathogenic variants' above.)

Once RBCs become spherocytic, phagocytosis during repeated passages through the splenic cords (a process termed splenic conditioning) promotes further membrane loss and a progressively more spheroidal shape (picture 2). (See 'Changes in the RBC membrane' above.)

This in turn further impairs passage through the narrow fenestrations of the splenic cords [99,102,103]. In severe cases, reduced membrane stability may contribute to mechanical RBC destruction.

The spleen also exposes RBCs to an acidotic, oxidizing, metabolically unfavorable environment. RBCs may become depleted of 2,3-bisphosphoglycerate (2,3-BPG; also called 2,3-diphosphoglycerate [2,3-DPG]), and in spectrin deficiency (but not in band 3 deficiency), there may be methyl-esterification of membrane proteins [104].

Case reports have described individuals with the same familial variant who have different severities of hemolysis (variable clinical penetrance). This variability has been attributed to concomitant variants in other genes that affect RBC structure and function. As an example, in a family with mild autosomal dominant HS due to partial band 3 deficiency, one relative had more severe clinical features than the others, attributed to an exacerbating effect of a variant in PKLR, the gene for pyruvate kinase (PK) [105]. It was speculated that low adenosine triphosphate (ATP) levels from partial PK deficiency increased RBC osmotic fragility. A study from Germany reported that PK activity in RBCs from patients with HS was relatively low, particularly in reticulocytes [106]. Loss of membrane-associated PK was proposed to be responsible and might contribute to the severity of HS. Further studies are needed.

Splenectomy can virtually eliminate hemolysis and anemia in HS, with the exception of the most severe forms due to complete or almost complete alpha-spectrin or complete band 3 deficiency. (See 'Splenectomy' below.)

EPIDEMIOLOGY

Disease prevalence — HS is seen in all populations but appears to be more common in people of northern European ancestry, where HS affects as many as 1 in 2000 to 1 in 5000 (prevalence, approximately 0.02 to 0.05 percent) [19,20,93,107].

Prevalence is thought to be lower in individuals from other parts of the world such as Africa and Southeast Asia, but comprehensive population survey data are unavailable.

In a 2006 study that tested 402 severely jaundiced neonates requiring phototherapy, four (1 percent) were ultimately diagnosed with HS (approximately one-fifth as prevalent as acquired, immune-mediated spherocytosis) [108]. Other causes of neonatal jaundice may also require consideration. (See 'Neonates' below and 'Differential diagnosis' below.)

Frequency of different genetic variants — Cohort studies providing relative frequencies of the genes contributing to HS pathogenesis include:

●In 166 children with HS in Canada, a pathogenic variant was identified in 160 of 166 (97 percent) [45]:

•ANK1 – 49 percent

•SPTB – 33 percent

•SLC4A1 – 13 percent

•SPTA1 – 5 percent

●In 113 patients with HS from 73 kindreds in India, de novo and dominantly inherited variants were as follows [65]:

•ANK1 – 53 percent

•SPTB – 36 percent

•SLC4A1 – 4 percent

Compound heterozygous variants in SPTA1 causing autosomal recessive disease were identified in 6 percent.

●In 95 patients in the Netherlands evaluated by next-generation sequencing (NGS), a pathogenic variant was identified in 85 (89 percent) [109]:

•SPTA1 – 36 percent, possibly representing a bias of ordering genetic testing in patients with the severe phenotype of SPTA1-associated HS

•ANK1 – 27 percent

•SPTB – 20 percent

●Another study identified autosomal recessive HS due to biallelic pathogenic variants EPB42 in individuals of Japanese ancestry [81,82]. Sporadic cases have also been described in patients of European ancestry [79,80].

CLINICAL PRESENTATION

Disease severity and age of presentation — HS can present at any age and with any severity, with case reports describing a range of presentations from hydrops fetalis in utero through diagnosis in the ninth decade of life [93,96,110-112].

The majority of affected individuals have mild or moderate hemolysis or hemolytic anemia and a known family history, making diagnosis and treatment relatively straightforward [94]. Individuals with significant hemolysis may develop additional complications such as jaundice/hyperbilirubinemia/cholelithiasis (typically exacerbated with concurrent Gilbert syndrome), folate deficiency, or splenomegaly [113].

Hemolytic anemia — A classification for HS has been developed based on the severity of anemia and markers of hemolysis (reticulocyte count and bilirubin) [4,32,82,94,114].

Neonates frequently present with neonatal jaundice that is noted to rise in the first day of life, posing a risk of kernicterus if not monitored and treated appropriately. However, they have a normal hemoglobin at birth. Anemia ensues after two to three weeks as the normal neonatal hyposplenism resolves; hemoglobin frequently decreases enough to require transfusion [2,115].

Transfusion dependence in the first 9 to 12 months of life, when the erythropoietic response may not be adequate, is not necessarily prognostic of the disease severity [116,117]. Infants with an autosomal dominant form of HS based on family history and/or genetic evaluation who require frequent transfusions during the first year of life may benefit from erythropoietin administration [118]. Transfusion dependence beyond the first year of life is not expected for autosomal dominant HS or the autosomal recessive HS due to EPB41 biallelic pathogenic variants, but it is almost always the case for SPTA1-associated autosomal recessive HS. Red blood cell (RBC) indices are described below. (See 'Initial testing' below.)

In older children and adults, the presentation may be that of an incidental finding of hemolysis, hemolytic anemia, or spherocytes on the blood smear (picture 2), or the individual may be symptomatic with anemia, splenomegaly, pigment gallstones, or jaundice. Jaundice due to severe hemolysis is less common after the newborn period, but scleral icterus is not rare, especially when the patient also has Gilbert syndrome [113]. (See 'Older children and adults' below.)

In some cases, coinheritance of another disorder affecting RBC survival such as sickle cell disease or thalassemia can influence the severity of anemia and make diagnosis more challenging [114]. (See 'Evaluation' below.)

Exacerbations of anemia may also occur in certain settings:

●Infections – Infections that impair erythropoiesis in the bone marrow and thus diminish the capacity to compensate for chronic hemolysis may lead to a period of aplasia. A commonly cited cause of transient aplastic crisis is parvovirus B19 infection; other viral or bacterial infections may also cause transient aplasia. This is because individuals with chronic hemolysis are highly dependent on the accelerated production of new RBCs by the bone marrow, and they can experience a rapid drop in hemoglobin level when the bone marrow is unable to compensate for hemolysis.

In a series of individuals with hereditary hemolytic anemias who presented to the hospital with acute parvovirus infection, common manifestations included fever, musculoskeletal pains, and pancytopenia, while acute kidney injury was also reported, likely due to inadequate supportive management [119]. If an individual with HS develops a precipitous decline in hemoglobin level or reticulocyte count, testing for parvovirus infection is recommended. (See "Clinical manifestations and diagnosis of parvovirus B19 infection".)

Infections may also exacerbate hemolysis, causing worsening of anemia even with increased reticulocyte count and parallel increase of bilirubin and lactate dehydrogenase (LDH). Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infection, which typically causes splenomegaly (table 1), may cause increased splenic pooling of RBCs and/or increased hemolysis (hemolytic crisis) in patients with HS or other underlying hereditary hemolytic anemias. (See "Splenomegaly and other splenic disorders in adults", section on 'Splenomegaly'.)

●Nutrient deficiencies – Individuals who become deficient in folate, vitamin B12, or iron may be unable to produce sufficient RBCs to compensate for those destroyed by hemolysis. (See "Clinical manifestations and diagnosis of vitamin B12 and folate deficiency" and "Iron deficiency in infants and children <12 years: Screening, prevention, clinical manifestations, and diagnosis" and "Iron requirements and iron deficiency in adolescents" and "Diagnosis of iron deficiency and iron deficiency anemia in adults".)

●Pregnancy – Anemia may worsen during pregnancy as the RBC mass and plasma volume expand to meet the physiologic needs of the pregnancy. Attention to folic acid supplementation is especially important during pregnancy to avoid increased risk of neural tube defects in the fetus and superimposed megaloblastic anemia for the mother [120,121].

Iron deficiency is rare in hereditary spherocytosis, as in all hereditary hemolytic anemias; therefore, attention must be paid to avoid oral iron supplementation during pregnancy when iron overload may actually be present [120,122]. (See 'Overview of treatment' below.)

Individuals who experience a decline from their baseline hemoglobin level and/or reduction in baseline reticulocyte count are likely to require more frequent monitoring and/or additional testing, details of which will depend on the associated symptoms and laboratory findings.

Complications of hemolysis — Common complications of hemolysis in individuals with HS include neonatal jaundice, splenomegaly, and pigment gallstones, which are discussed in the sections that follow. (See 'Neonatal jaundice' below and 'Splenomegaly' below and 'Pigment gallstones' below.)

Rarely, hemolysis may be severe enough to cause extramedullary hematopoiesis and/or growth delay, along with iron overload due to increased iron absorption and/or transfusions [123,124]. The majority of patients with HS are not thought to be at risk of iron overload [93]; however, there are no extensive studies of older patients, and some cases with iron overload associated with autosomal dominant HS have been reported [125].

Other rare complications that have been reported include leg ulcers, priapism, neuromuscular disorders, cardiac disease, and gout; in some cases, these may represent coincidental rather than causal associations [93,126,127].

Neonatal jaundice — HS may present in the neonatal period with jaundice and hyperbilirubinemia, and the serum bilirubin level may not peak until several days after birth. (See 'Disease severity and age of presentation' above.)

Some experts have proposed that HS is underdiagnosed as a cause of neonatal jaundice [128]. A requirement for phototherapy and/or exchange transfusion during this period is common [93,116]. (See 'Neonates' below.)

Hyperbilirubinemia may be exacerbated by concomitant Gilbert syndrome. (See "Unconjugated hyperbilirubinemia in neonates: Etiology and pathogenesis".)

Splenomegaly — Splenomegaly is rare in neonates but can often be seen in older children and adults with HS [115]. Early reports of family studies found palpable spleens in over three-fourths of affected members, but this may reflect a skewed population with the most severe disease. In these studies, the relationship between disease severity and splenic size was not linear [129].

There is no evidence of an increased risk of splenic rupture.

Indications for splenectomy in HS and possible complications are discussed below. (See 'Splenectomy' below.)

Pigment gallstones — Pigment (bilirubin) gallstones are common in individuals with HS and may be the presenting finding in adults. Gallstones are unlikely before the age of 10 years but are seen in as many as 50 percent of adults, especially those with more severe hemolysis [130]. Gallstones appear to be more common and tend to appear earlier in individuals with Gilbert syndrome (inherited disorder of bilirubin glucuronidation) [113]. (See "Gilbert syndrome", section on 'Long-term complications'.)

Obstructive jaundice or cholecystitis is treated similarly to that in individuals without HS. If cholecystectomy is performed, it may be worthwhile to discuss whether splenectomy was also planned, as the procedures could be combined; however, splenectomy should not be routinely performed during cholecystectomy [114,131-133].

EVALUATION

When to suspect HS — HS should be suspected in an individual with direct antiglobulin test (DAT; Coombs test)-negative (non-immune) hemolytic anemia and spherocytes on the peripheral blood smear. A positive family history of HS or negative testing for other inherited hemolytic anemias increases suspicion for HS. Severe disease can present in the neonatal period, whereas mild or compensated HS may not present until adulthood. (See 'Disease severity and age of presentation' above.)

Typical findings of hemolytic anemia include low hemoglobin, high reticulocyte count, high lactate dehydrogenase (LDH) and bilirubin, and low haptoglobin. (See "Diagnosis of hemolytic anemia in adults", section on 'Laboratory confirmation of hemolysis'.)

If hemolysis is severe, the patient may have jaundice (including neonatal jaundice). If the bone marrow cannot produce new red blood cells (RBCs) rapidly enough to compensate for hemolyzed RBCs, there may be symptoms of anemia. (See "Unconjugated hyperbilirubinemia in neonates: Etiology and pathogenesis", section on 'Increased production'.)

HS is a non-immune form of hemolysis; the DAT is negative. (See "Overview of hemolytic anemias in children", section on 'Intrinsic hemolytic anemias' and "Diagnosis of hemolytic anemia in adults", section on 'Immune versus non-immune'.)

Findings that suggest an alternative diagnosis include prior normal complete blood counts (CBCs), reticulocyte counts, and hemolysis markers. (See 'Initial testing' below.)

Evaluation of the peripheral blood smear is very helpful; obvious RBC abnormalities may suggest a different type of hemolytic anemia (eg, stomatocytes, ovalocytes, target cells, sickle cells). (See "Evaluation of the peripheral blood smear", section on 'RBC size and shape abnormalities'.)

Initial testing

All patients — Some aspects of the initial evaluation differ in neonates versus older children and adults since affected neonates tend to have more severe disease and less useful laboratory parameters. (See 'Neonates' below.)

However, the following are appropriate in virtually all individuals:

●CBC and RBC indices – All individuals with suspected HS based on family history, neonatal jaundice, or other findings should have a complete blood count (CBC) with reticulocyte count and RBC indices. The mean corpuscular hemoglobin concentration (MCHC) is often the most useful parameter for assessing spherocytosis; an MCHC ≥36 g/dL is consistent with spherocytes. A normal mean corpuscular volume (MCV) is expected, with a wide (increased) red cell distribution width (RDW); significant reticulocytosis may increase the MCV.

●Blood smear review – All patients with suspected HS should have a blood smear reviewed by an experienced individual. RBC parameters to be assessed include the presence and abundance of spherocytes, other abnormal RBC shapes, and the degree of polychromatophilia, which reflects reticulocytosis.

●Hemolysis testing – Testing for hemolysis is also appropriate in all patients. This includes lactate dehydrogenase (LDH), indirect bilirubin, haptoglobin, and reticulocyte count. Findings consistent with hemolysis include increased LDH and indirect bilirubin, decreased or absent haptoglobin, and an elevated reticulocyte count.

●Coombs testing – When a patient without a prior diagnosis presents with hemolysis, a DAT should be done to evaluate for the possibility of immune-mediated hemolysis.

The results of testing may also be useful to the transfusion service if transfusion is indicated. The DAT in HS is negative. The commonly performed DAT may be negative in 3 to 11 percent of AIHAs, depending on the sensitivity of the assay [134,135]. Therefore, a high index of suspicion for DAT-negative AIHA is indicated for a patient presenting with hemolytic anemia with no prior personal and family history, especially with presence of microspherocytes on the smear and other evidence of immune dysregulation [136].

Our approach is consistent with a 2011 guideline (published in 2012) from the British Committee for Standards in Haematology (BCSH) on the diagnosis of HS and a 2015 guideline from the International Council for Standardization in Haematology (ICSH) on non-immune hereditary RBC membrane disorders [114,137].

Neonates — Neonates with severe hemolysis due to HS may present with neonatal jaundice. (See 'Disease severity and age of presentation' above.)

The evaluation of a neonate with suspected HS depends on whether a parent is known to have HS.

●If an infant with hyperbilirubinemia or other findings of non-immune hemolytic anemia has a known family history of HS, then the likelihood of HS is high, and we rely heavily on the RBC indices. As noted above, an MCHC ≥36 g/dL is highly suggestive of HS. (See 'Clinical presentation' above.)

●If an infant with hyperbilirubinemia or hemolytic anemia does not have a known family history of HS, then a number of other possible diagnoses must be considered. (See 'Differential diagnosis' below.)

Appropriate therapy should not be delayed while determining the underlying cause; likewise, the importance of making the diagnosis of HS should be emphasized regardless of the management interventions needed. Hemolytic anemia with a negative DAT and a high MCHC (eg, ≥36 g/dL) is consistent with HS but must be considered in the context of the entire clinical picture. (See 'Confirmatory tests' below and "Unconjugated hyperbilirubinemia in neonates: Etiology and pathogenesis" and "Unconjugated hyperbilirubinemia in term and late preterm newborns: Screening".)

Neonates with HS tend to have an elevated MCHC (typical range in HS, 35 to 38 g/dL) [115]. This is a useful discriminator between HS and hemolytic disease of the fetus and newborn (HDFN) because HDFN RBCs tend to have MCHC in the range of 33 to 36 g/dL [128].

Spherocytes on the blood smear are helpful if present, but up to one-third of neonates with HS do not have prominent spherocytes, and some neonates without HS have spherocytes [115]. In addition, it may be difficult to assess spherocytes on the peripheral blood smear in a neonate, either because neonates with HS may have fewer spherocytes or because spherocytic cells are often present after birth in neonates without HS [137].

If the infant is well, it is reasonable to postpone testing until approximately six months of age or older, at which time the RBC morphology will be easier to assess [114]. If there is greater urgency to establish a diagnosis (eg, severe anemia or hyperbilirubinemia), specialized testing may be used. (See 'Confirmatory tests' below.)

Older children and adults — As noted above, HS may be suspected in a patient of any age who has evidence of hemolysis (eg, elevated serum LDH, elevated indirect bilirubin, reduced haptoglobin, increased reticulocyte count) or hemolytic anemia that is DAT negative and not explained by another condition.

HS may also be suspected in an individual who presents with a complication of hemolysis, such as splenomegaly, pigmented gallstones, or an abrupt drop in hemoglobin level when the bone marrow cannot compensate for hemolysis (eg, during a viral illness, pregnancy, or other condition). In such cases, a CBC will be obtained, and RBC indices will be available; the reticulocyte count should also be measured.

Evidence consistent with HS as the likely diagnosis in an older child or adult includes the following:

●Positive family history of HS, although this is not always present, as some cases arise as de novo pathogenic variants, and not all individuals will have a complete family history available.

●Chronic hemolytic anemia, although in mild cases, there may be chronic compensated hemolysis without anemia. The typical reticulocyte count in older children and adults with HS is approximately 5 to 20 percent, but it may be as high as 20 to 30 percent in severe cases.

●Jaundice and/or splenomegaly, although these may be absent if the hemolysis is mild.

●Spherocytes (picture 2) on the peripheral blood smear. The percentage of spherocytes is variable. Pathogenic variants in certain genes have been associated with specific spherocyte morphologies, although the diagnostic value of these findings has not been rigorously tested [5,66,67,138,139] (see 'Genetics' above):

•Anisocytic spherocytes – Ankyrin deficiency

•Pincered or mushroom-shaped spherocytes – Band 3 deficiency

•Acanthocytic spherocytes – Beta-spectrin deficiency

•Dense and irregularly shaped cells – Alpha-spectrin deficiency

•Ovalocytes and occasional ovalo-stomatocytes – Protein 4.2 deficiency

●RBC indices consistent with spherocytosis (eg, MCHC >36 g/dL; normal MCV with wide red cell distribution width [RDW]). Greater degrees of reticulocytosis are usually needed to increase the MCV and RDW in older children and adults; thus, the MCHC is the most useful of the RBC indices.

The combination of increased MCHC and increased RDW further improves diagnostic performance [140]. If reticulocyte indices are available, a higher-than-average reticulocyte MCHC and a low reticulocyte MCV are also consistent with HS (table 2) [114].

In the appropriate clinical setting, these observations may be sufficient to screen for a diagnosis of HS. (See 'Diagnosis' below.)

If these parameters are unclear or if there are questions about the prognosis or if splenectomy is being considered, additional diagnostic confirmation is needed, especially to avoid misdiagnosis of hereditary xerocytosis as HS. The following specialized testing can be pursued. (See 'Confirmatory tests' below.)

Confirmatory tests — Several confirmatory tests are available.

●EMA binding – Eosin-5-maleimide (EMA) binding is a good and widely available test for HS diagnosis; it detects loss mainly of band 3 and Rh-related proteins from the RBC membrane, using EMA, an eosin-based fluorescent dye, that binds to those RBC membrane proteins [141].

The mean fluorescence of EMA-labeled RBCs from individuals with HS is lower than controls, and this reduction in fluorescence can be detected in a flow cytometry-based assay, as illustrated in the figure (figure 3). The reduction in EMA binding is observed in RBCs when HS is due to band 3 deficiency or to deficiencies in other proteins such as ankyrin or spectrin, since the common characteristic in HS is loss of membrane along with band 3 complexes [142]. Two case series of individuals with HS have found the EMA fluorescence in individuals with HS to be approximately two-thirds that of controls [141,143]. Samples can be stored and tested; one of the studies also analyzed the effect of delayed testing and found that samples stored for 24 hours in darkness gave similar results to those tested immediately [141].

Advantages of EMA binding include its high sensitivity; rapid turnaround time (approximately two hours); and need for only a minimal amount of blood (a few microliters), which is especially advantageous for testing neonates [144-146]. EMA testing can also be used to evaluate for HS-RBCs in a specimen from a patient who has recently received a transfusion [147]. In various studies, the sensitivity and specificity of the test to diagnose HS versus normal RBCs appear to be in the ranges of 93 to 96 and 93 to 99 percent, respectively [144,148-150]. It is important in testing neonatal RBCs to compare the results of EMA binding to control samples from age-matched controls [151].

However, when EMA results for HS are compared with those for other RBC membrane disorders, the specificity of the test declines. EMA binding is also decreased (ie, the test is considered positive) in other hemolytic anemias with loss of membrane associated with band 3 complexes, such as hereditary pyropoikilocytosis, spherocytic elliptocytosis/pyropoikilocytosis, Southeast Asian ovalocytosis (SAO), autoimmune hemolytic anemia, as well as congenital dyserythropoietic anemia (CDA) type II [143,148,152]. False-negative results may be seen in mild cases of HS. (See 'Differential diagnosis' below.)

●Osmotic fragility – Osmotic fragility testing (OFT) is a traditional, specialized test for HS, based on the fact that spherocytic RBCs have reduced surface area-to-volume (SA/V) ratio. In this test, fresh RBCs are incubated in hypotonic buffered salt solutions of various osmolarities, and the fraction of hemoglobin released (due to hemolysis) is measured. The test takes advantage of the increased sensitivity of spherocytes to hemolysis due to their reduced SA/V ratio (figure 4).

Incubation of patient samples for 24 hours prior to testing may accentuate osmotic fragility and improve diagnostic yield.

The OFT has relatively low sensitivity and specificity. It fails to identify a significant number of individuals with HS, and, particularly in the newborn, it may be positive in other conditions including immune hemolytic anemia, hemolytic transfusion reactions, RBC enzyme deficiencies such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, and unstable hemoglobin variants [137]. In one series of 86 individuals with HS, only 57 (66 percent) had positive osmotic fragility testing [153].

●Osmotic gradient ektacytometry – Osmotic gradient ektacytometry (OGE) is the reference method to evaluate RBC membrane disorders [154,155]. It offers accurate (with 100 percent assay sensitivity) diagnosis of HS and excellent specificity in differential diagnosis between the various RBC membrane disorders, including hydration defects (figure 3) [2,18,152]. The assay evaluates the deformability of RBCs as they are subjected to constant shear stress in a medium of increasing osmolality in a laser diffraction viscometer. The testing has become more accessible over the last decade with the commercial availability of a new generation ektacytometer [152,156].

Limitations of OGE include its decreased sensitivity after recent transfusions, as with all phenotypic tests that evaluate RBCs as a population. Warm AIHA with a significant number of microspherocytes and CDA-II may produce an ektacytometry curve resembling HS [2,18].

●Glycerol lysis – The glycerol lysis test (GLT) and the acidified GLT (AGLT) are modifications of the OFT that add glycerol (in the GLT) or glycerol plus sodium phosphate (to lower the pH to 6.85, in the AGLT) to the hypotonic buffered salt solutions in which the patient's RBCs are incubated [107,137]. Like EMA, OFT, and OGE, these tests may also be positive in acquired spherocytosis conditions such as AIHA.

The "pink test" is a modification of the GLT in which the final extent of hemolysis is measured in a blood sample incubated in the glycerol solution at pH 6.66 [157]. A further modification has been proposed (the direct pink test) in which the test sample is obtained from fingerprick (or heel puncture in newborns), rather than venipuncture, and incubated directly in the glycerol solution; this requires only a few microliters of blood [158].

●Cryohemolysis – In the cryohemolysis test, RBCs are suspended in a hypertonic solution, briefly heated to 37°C, then cooled to 4°C for 10 minutes [159]. Ease of performance and the wide separation in degree of hemolysis between spherocytes and normal cells are two attractive features of this test [160].

The last two tests (glycerol lysis and cryohemolysis) have limited availability in the United States.

The relative performance of these tests was evaluated in a 2012 study that tested samples from 150 individuals with known HS [143]. Test sensitivities were as follows:

●AGLT – 95 percent

●EMA binding – 93 percent

●Pink test – 91 percent

●Osmotic fragility, incubated – 81 percent

●Osmotic fragility, fresh – 68 percent

●GLT – 61 percent

Combining EMA binding and AGLT had a sensitivity of 100 percent; combining EMA binding and pink test had a sensitivity of 99 percent; and EMA binding plus osmotic fragility (incubated or fresh) had a sensitivity of 95 percent [143].

Comparison of EMA binding versus osmotic gradient ektacytometry (OGE) identified EMA binding as a fast, widely available screening test for HS but also indicated the advantage of OGE to differentiate HS from other RBC membrane disorders [152]. When the question is the differentiation between xerocytosis and spherocytosis before proceeding to splenectomy, OGE as well as genetic evaluation for the pathogenic gene variant is preferable [2,18,152].

These results demonstrate that no single phenotypic testing has 100 percent sensitivity and specificity to accurately identify all individuals with HS, especially if the patient has been recently transfused. Test results should be compared with results of a normal control of similar age, evaluated in parallel with the patient's specimen, as well as with the reference range for many normal samples evaluated by the specific laboratory. Diagnostic yield may be improved by using two tests and taking into account the individual's personal and family history and evaluating the blood smear and RBC indices.

These tests can also be positive in other conditions, and results cannot be interpreted in isolation. If a positive test is discordant with the clinical picture or findings on the peripheral blood smear, laboratory personnel or a consulting hematologist with expertise in these tests should be consulted [114].

Specialized testing (including genetic testing) — When further characterization of the RBC membrane proteins is needed, research-based gel electrophoresis can be done using RBC ghosts.

Next-generation DNA sequencing has immensely facilitated the diagnosis of RBC and erythropoiesis disorders. Nevertheless, it remains important to correlate the findings with the clinical phenotype and phenotypic laboratory testing to avoid misinterpretation of variants of unknown significance or only partial diagnosis. Clinical-grade next-generation DNA sequencing (NGS) is available by several academic and commercial laboratories to evaluate for the genetic cause of an RBC membranopathy or more broadly of a hereditary hemolytic anemia. Identifying a familial disease variant may be useful for prognosis and genetic counseling. (See 'Testing relatives; reproductive testing and counseling' below.)

More importantly, misdiagnosis of dehydrated hereditary stomatocytosis (hereditary xerocytosis [HX]) as HS must be avoided, since splenectomy is contraindicated in HX and does not improve hemolysis [161-163]. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)", section on 'Splenectomy'.)

Electrophoresis and immunoblotting are used to quantify membrane proteins from RBC ghosts produced by osmotic lysis of RBCs and washing out the hemoglobin and other cytoplasmic proteins. The discovery of the structural components of the red blood cell cytoskeleton developed in parallel with the technique of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a tool for protein chemistry in the 1970s [1]. Decrease in a specific protein would be followed by sequencing of the corresponding gene, deepening the understanding of erythroid and protein biology along with meticulous work of many research teams around the world.

Resources for genetic testing are listed on the Genetic Testing Registry website.

Certain academic laboratories are able to provide this testing and may be contacted for further discussions:

●Cincinnati Children's Molecular Genetics Laboratory:

•Website – www.cincinnatichildrens.org/moleculargenetics

•Phone – (513) 636-4474

●Mayo Medical Laboratories:

•Website – https://www.mayomedicallaboratories.com/customer-service/contacts.html

•Phone – (800) 533-1710

•Email – mml@mayo.edu (United States) or mliintl@mayo.edu (international)

Diagnosis — The diagnosis of HS is made in an individual with DAT-negative hemolysis, increased MCHC, positive family history for HS, and/or spherocytes on the peripheral blood smear, by one or more confirmatory tests such as EMA binding, osmotic fragility, or osmotic gradient ektacytometry (OGE).

Specialized testing is especially important if splenectomy is being considered. Specialized testing may be omitted if definitive diagnosis is unlikely to alter management (eg, if hemolysis is mild and childbearing is not planned) [114]. (See 'Confirmatory tests' above.)

For an individual with a strong suspicion of HS and negative or equivocal testing by EMA binding, for whom accurate diagnosis is especially important (eg, due to a previous affected child), positive results from genetic testing are confirmatory for HS. Genetic testing is available in several specialized laboratories. (See 'Specialized testing (including genetic testing)' above.)

This testing is similar to that described in published guidelines, with the addition of genetic testing being more widely used, as it is becoming more readily accessible and less expensive [114,137]. Accurate diagnosis is important to avoid mismanagement, since treatment for HS may differ from other conditions. (See 'Splenectomy' below.)

Differential diagnosis — The differential diagnosis of HS includes a number of other hemolytic anemias with spherocytes on the peripheral blood smear:

●Other inherited hemolytic anemias – Other RBC membrane disorders include hereditary elliptocytosis (HE) (picture 3) and elliptocytosis variants (hereditary pyropoikilocytosis [HPP]) (picture 4), overhydrated stomatocytosis, and dehydrated hereditary stomatocytosis (also called hereditary xerocytosis [HX]) (figure 5).

RBC enzyme disorders include glucose-6-phosphate dehydrogenase (G6PD) deficiency, pyruvate kinase (PK) deficiency, and other rarer metabolic disorders.

Like HS, these present with variable degrees of anemia and hemolysis and can be diagnosed at any age.

Unlike the other disorders, G6PD deficiency (except for the disease due to rare class A variants) typically presents with episodic hemolysis after exposure to oxidant drugs rather than chronic hemolysis [164].

Other membrane disorders have distinctive morphologies on the blood smear, whereas HS is characterized by spherocytosis as the predominant morphology. (See "Overview of hemolytic anemias in children", section on 'Intrinsic hemolytic anemias' and "Diagnosis of hemolytic anemia in adults", section on 'Intracorpuscular'.)

It is especially important to distinguish between HS and dehydrated hereditary stomatocytosis (hereditary xerocytosis [HX]), since HS is generally helped by splenectomy, whereas splenectomy should be avoided in HX because it predisposes patients to life-threatening venous and arterial thromboembolism and does not improve hemolysis [161-163]. (See 'Decision to pursue splenectomy' below.)

Overhydrated hereditary spherocytosis (OHSt) is a very rare, autosomal dominant, hereditary hemolytic anemia caused by heterozygous missense variants in RhAG. OHSt is characterized by significantly increased MCV, decreased MCHC, and many stomatocytes on the blood smear. OHSt may present with mild to severe hemolytic anemia. Splenectomy is expected to partially reduce hemolysis but also has a high risk of thromboembolic complications and pulmonary hypertension. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)", section on 'Splenectomy'.)

●HDFN – Hemolytic disease of the fetus and newborn (HDFN) is most frequently caused by ABO or Rh incompatibility between fetal RBCs and maternal antibodies that cross the placenta. Neonates may present with severe jaundice and anemia requiring aggressive treatment. (See "Alloimmune hemolytic disease of the newborn: Postnatal diagnosis and management".)

Like HS, HDFN can be associated with abundant spherocytes on the blood smear.

Unlike HS, HDFN has a positive DAT and resolves after the maternal antibodies are cleared.

●Infantile pyknocytosis – Infantile pyknocytosis is a disorder of unknown etiology in which RBCs become hyperdense and dehydrated [165].

Like HS, neonates present with non-immune hemolytic anemia with reticulocytosis and increased mean corpuscular hemoglobin concentration (MCHC).

Unlike HS, the blood smear is characterized by the finding of several pyknotic RBCs with irregular borders and projections, some resembling schistocytes and blister cells. The diagnosis is suspected based on the RBC morphology and otherwise negative work-up and is made definitively in retrospect after the pyknosis resolves spontaneously, typically by the age of three to nine months. The etiology is likely multifactorial, and peripartum oxidative stress may play a role [166].

●Congenital dyserythropoietic anemia (CDA) type II – CDA type II is an autosomal recessive hereditary anemia caused by biallelic SEC23B pathogenic variants.

Like HS, some individuals may have significant hemolysis and/or splenomegaly, and specialized tests such as EMA binding and OGE may be positive due to compromised glycosylation and positioning of band 3 [167].

Unlike HS, CDA-II causes binucleate and multinucleate erythroblasts in the bone marrow; the reticulocyte count is typically lower, indicating ineffective erythropoiesis; and significant iron overload is seen, disproportionate to the history of RBC transfusions [114]. (See "Overview of causes of anemia in children due to decreased red blood cell production", section on 'Congenital dyserythropoietic anemia'.)

●Autoimmune hemolytic anemia (AIHA) – AIHA is triggered by autoantibodies directed against self-RBC antigens. Warm AIHA secondary to a disorder such as systemic lupus erythematosus (SLE) or without an underlying disorder is more common than cold AIHA, which is typically triggered by an infection such as infectious mononucleosis.

Like HS, anemia and/or hemolysis of variable severity and abundant spherocytes on the peripheral blood smear may occur. Unlike HS, in AIHA, the DAT is typically positive, the family history is negative for hemolytic anemia, and prior complete blood counts (CBCs) will show a normal hemoglobin and reticulocyte count. (See "Autoimmune hemolytic anemia (AIHA) in children: Classification, clinical features, and diagnosis" and "Warm autoimmune hemolytic anemia (AIHA) in adults" and "Cold agglutinin disease".)

MANAGEMENT

Overview of treatment — Treatment is directed at preventing or minimizing complications of chronic hemolysis and anemia. There are no specific treatments yet directed at the membrane abnormality.

Monitoring — Monitoring frequency depends on disease severity and symptoms. (See 'Disease severity and age of presentation' above.)

Babies with a family history of HS need careful monitoring for hyperbilirubinemia within the first day of life. Anemia ensues typically two to three weeks after birth, and hemoglobin frequently decreases enough to require transfusion by week 3 to 4 of life [2,115].

Once a baseline has been established, annual assessment is sufficient for children with mild hemolysis, with closer monitoring during viral or other infections that might worsen anemia [114].

Growth should be monitored, and the possible risk of transient aplastic crisis, including the symptoms and the need to seek medical attention, should be explained.

Adults with mild disease may receive routine medical and preventive care; those with more severe hemolysis may require more frequent monitoring, with the interval individualized. (See 'Hemolytic anemia' above.)

Supportive measures — Supportive measures depend on disease severity:

●Hyperbilirubinemia – Neonates with a positive family history and neonatal jaundice should be treated for neonatal hyperbilirubinemia and/or anemia while awaiting diagnostic confirmation. Therapy for hyperbilirubinemia may range from simple phototherapy to exchange transfusion [115]. (See "Unconjugated hyperbilirubinemia in term and late preterm newborns: Initial management".)

●Folic acid – Folic acid is appropriate for moderate to severe hemolysis and during pregnancy, due to an increased requirement for folate in RBC production. There are no clinical trials of folic acid treatment, but observational studies have documented megaloblastic anemia in patients with HS before routine folic acid supplementation of grains and cereals [94]. This, plus the low cost and minimal toxicity of folic acid, makes it an attractive and simple therapy.

The typical dose for infants 6 to 12 months is 0.4 mg daily; thereafter, 1 mg/day is typically prescribed. Individuals planning to be pregnant and during pregnancy should receive a higher dose. (See "Preconception and prenatal folic acid supplementation".)

Individuals with mild hemolysis and good intake of fresh fruits and vegetables (or folic-acid-supplemented grains) may not require daily folic acid, but for those who place a high value on avoiding folate deficiency, daily folic acid is safe and inexpensive, and there are no side effects or contraindications.

●Erythropoietin – Erythropoietin (EPO) may reduce the need for transfusions in some infants [114,118]. Typically, this can be discontinued around the age of nine months. In one study, recombinant human EPO (1000 international units/kg per week) with iron supplementation obviated the need for transfusion in 13 of 16 infants with HS [118]. As the infants grew and began to mount an adequate erythropoietic response, EPO was tapered and discontinued before nine months.

●Splenomegaly – There are no special activity restrictions [114]. However, those with significant splenomegaly should be aware of the risk of splenic rupture during contact sports. (See "Splenomegaly and other splenic disorders in adults", section on 'Sports participation and fall risk'.)

Transfusions — Blood transfusion is often required in infancy, even in mild autosomal dominant HS. The transfusion requirement in the first months of life does not necessarily predict a severe phenotype. Transfusions may be needed in older children and adults in other settings (eg, aplastic or hemolytic crisis with infections, pregnancy). Hemoglobin thresholds for transfusion depend on the age of the patient, symptoms, and comorbidities.

In autosomal dominant HS and in most cases of autosomal recessive EPB42-associated HS, transfusions usually are not required on a chronic basis or for a long enough time to cause iron overload. In contrast, most patients with autosomal recessive SPTA1-associated HS are transfusion dependent until splenectomy is performed (if appropriate). (See 'Splenectomy' below.)

●Infants – Many infants may require transfusions; some may need exchange transfusion for rapidly increasing hyperbilirubinemia. Older children with mild or moderate autosomal dominant HS may tolerate a hemoglobin as low as 7 g/dL without transfusion if reticulocyte count is increased. However, chronic anemia with Hgb <7 g/dL is typically due to severe autosomal recessive HS caused by SPTA1 (or, more rarely, biallelic ANK1 pathogenic variants); inadequate transfusion and chronic anemia in those cases frequently lead to growth delay and iron overload from ineffective erythropoiesis. (See "Red blood cell transfusion in infants and children: Indications".)

●Adults – Adults may require transfusions for anemia, with thresholds determined by their clinical status. (See "Indications and hemoglobin thresholds for RBC transfusion in adults".)

●Aplastic crisis – Individuals with an aplastic crisis due to parvovirus infection or other bone marrow insult may require transfusions if hemoglobin decreases without robust reticulocytosis. Spontaneous resolution generally occurs within a few days or weeks. Complete blood counts (CBCs) and reticulocyte counts, sometimes as frequently as twice per week, are used to determine the expected hemoglobin nadir and need for transfusion. (See "Treatment and prevention of parvovirus B19 infection", section on 'Transient aplastic crisis'.)

Transfusional iron overload typically occurs after >15 to 20 units of RBCs (>10 units in smaller children). Adults with mild hemolysis may have a chronic increase in iron absorption due to associated hepcidin suppression and may develop iron overload, especially if they also have hereditary hemochromatosis; screening for iron overload is required, with appropriate follow-up evaluation and treatment if needed. (See "Approach to the patient with suspected iron overload", section on 'Transfusional iron overload'.)

Splenectomy

Decision to pursue splenectomy — Decisions regarding splenectomy must take into account the severity of hemolysis, patient age, and perioperative and long-term risks [114,132,161].

For those with relatively severe hemolysis, splenectomy effectively improves anemia.

Other considerations include:

●Diagnostic confirmation – Confirm the diagnosis is HS rather than another hemolytic anemia such as hereditary xerocytosis (HX), for which splenectomy is neither safe nor effective. (See 'Differential diagnosis' above and "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)", section on 'Management'.)

●Type of HS (genotype) – Patients with SPTA1-associated autosomal recessive HS with the most common genotype of a null SPTA1 variant in trans to the Low Expression PRAgue (alpha-LEPRA) allele c.4339-99C>T are expected to have transfusion-dependent severe HS that responds well to splenectomy [32,76,77].

Splenectomy in individuals with moderate disease is individualized. We are more likely to advise splenectomy for individuals with more severe hemolysis and/or symptoms compromising quality of life (abdominal symptoms related to splenomegaly, distress related to jaundice) or for those with growth delays or skeletal changes related to extramedullary hematopoiesis.

HS due to complete alpha-spectrin or band 3 deficiency does not respond to splenectomy; these individuals require lifelong chronic transfusions and chelation or hematopoietic stem cell transplantation. (See 'Beta-spectrin or alpha-spectrin deficiency due to SPTB or SPTA1 pathogenic variants' above.)

For children with HS who remain transfusion dependent after the first year of life, genetic diagnosis should be pursued to understand the cause and prognosis and assist with management decisions. Genetic evaluation will also help to avoid misdiagnosis of hereditary xerocytosis, for which splenectomy will not be safe or effective. (See 'Specialized testing (including genetic testing)' above and 'Differential diagnosis' above.)

Autosomal dominant HS is generally not transfusion dependent after infancy. Further evaluation may be needed to investigate comorbidities that would make an autosomal dominant HS present as a transfusion-dependent disease after infancy. (See 'Inheritance patterns' above.)

●Ideal age – Ideally, splenectomy is delayed until the individual is >5 years of age to reduce the likelihood of sepsis [114]. It is better to plan for a total splenectomy after the age of five years and after completing the pre-splenectomy immunizations for encapsulated bacteria at an age when the spleen is considered mature enough to mount an optimal response to those immunizations.

Until age five, the patient would likely benefit from chronic transfusions to avoid growth delay and decrease stress erythropoiesis; monitoring for iron overload and chelation are used to avoid exacerbation of growth delay and/or organ toxicity by iron overload [168]. (See 'Transfusions' above.)

●Partial versus total splenectomy – In children <5 years for whom chelation is not successful, or if the family/caregivers prefer to avoid chronic transfusions, consideration may be given to partial splenectomy at >3 years. Pre-splenectomy immunizations should be completed, with the plan to repeat them after age five, in case of suboptimal response. Partial splenectomy reduces the risk of sepsis from encapsulated bacteria compared with total splenectomy; in some patients, partial splenectomy may provide long-term treatment, even for autosomal recessive HS [32,168]. (See 'Operative techniques' below and "Elective (diagnostic or therapeutic) splenectomy", section on 'Partial splenectomy'.)

●Gallstone disease – Individuals with symptomatic gallstone disease who require cholecystectomy should be evaluated for possible simultaneous splenectomy if clinically indicated (symptomatic hemolytic anemia and/or severe complications of hemolysis). (See 'Pigment gallstones' above and 'Gallstones' below.)

Splenectomy as a means of reducing gallstone formation is not recommended since the risks of splenectomy exceed this benefit [169]. (See 'Complications' below.)

●Splenic embolization – In patients for whom surgery is refused or contraindicated, partial splenic embolization has been employed, with anecdotal reports of success [170].

Our approach is consistent with a 2011 guideline from the British Committee for Standards in Haematology (BCSH) and a 2017 guideline from the European Hematology Association (EHA) [114,161].

Supporting evidence for splenectomy — There are no randomized trials comparing splenectomy with expectant management. Observational evidence to support the efficacy of both total and partial splenectomy includes:

●In a 2016 series that followed 79 individuals with HS who had partial splenectomy at a single institution in France, all patients but one had a significant improvement in hemoglobin level (mean increase, 0.97 g/dL) and reduction in number of transfusions (from 3.1 to 0.2 per patient per year), at a mean of 11 years follow-up [171]. These benefits remained stable or improved over 10 years; 69 (87 percent) had no symptoms from anemia, and 65 were transfusion free.

Regrowth of the splenic remnant was common, and 21 patients (27 percent) underwent reoperation with total splenectomy, 49 percent for a significant decrease in hemoglobin, and 51 percent for other symptoms related to splenomegaly or hemolysis. The typical interval between the initial partial splenectomy and the subsequent total splenectomy was seven to nine years. Of the 46 who did not undergo concomitant cholecystectomy, 16 (35 percent) developed gallstones, typically several years after splenectomy. There were no severe infections requiring hospitalization and no thrombotic complications.

●Two reports from 2015 and 2016 of patients with various congenital hemolytic anemias included 61 children and adolescents with HS [172,173]. Procedures were evenly divided between total and partial splenectomy; most were performed laparoscopically. Hemoglobin increased in all of the children with HS (mean increase, 4.1 g/dL; greater increases with total than partial splenectomy), and transfusion dependence in the group as a whole decreased from 22 to 4 percent [173].

Additional smaller series have reported similar findings; some have observed more concerning complications such as post-splenectomy sepsis [95,174-179]. (See 'Complications' below.)

Pre-splenectomy considerations

Immunizations — For individuals considering splenectomy (total or partial), ensure that all immunizations for encapsulated organisms have been administered with sufficient time to develop an antibody response (typically two weeks before surgery). Recommended immunizations vary based on patient age and number of vaccine doses previously received, as summarized in the tables for children (table 3) and adults (table 4) and discussed separately. (See "Prevention of infection in patients with impaired splenic function", section on 'Vaccinations' and "Prevention of infection in patients with impaired splenic function", section on 'Children' and "Elective (diagnostic or therapeutic) splenectomy", section on 'Vaccinations'.)

Gallstones — For individuals with gallstones, it may be advantageous to perform simultaneous cholecystectomy at the time of splenectomy, depending on the size and symptoms of the stones as well as other operative factors. Cholecystectomy in individuals with asymptomatic gallstones is controversial; patient values and preferences as well as comorbidities and risks of complications (cholangitis and acute biliary pancreatitis) should be factored into this decision [180]. (See "Approach to the management of gallstones".)

Operative techniques — Splenectomy was traditionally performed via laparotomy, which allowed a search to be made for accessory splenic tissue located at other sites within the abdomen. If not removed, an accessory spleen may grow and cause recurrent symptomatic anemia [181].

Laparoscopic splenectomy is increasingly used by surgeons with appropriate expertise [114,182]. There are no randomized trials comparing open versus laparoscopic splenectomy in HS; however, observational studies in broader pediatric populations and case series have observed that, when performed properly, laparoscopic splenectomy is associated with lower complication rates and shorter hospital stays and is equally effective in locating and removing an accessory spleen if present [183-188]. Laparoscopic splenectomy is used when there is appropriate institutional and surgical expertise. In rare cases with massive splenomegaly, the incision may need to be extended. (See "Elective (diagnostic or therapeutic) splenectomy", section on 'Surgical approach'.)

For younger children who cannot delay splenectomy until after age five, we suggest partial splenectomy (also called subtotal or near-total splenectomy). There are no randomized trials comparing partial with total splenectomy in HS, and the decision is made on a case-by-case basis. Compared with total splenectomy, partial splenectomy is likely to be effective in reducing hemolysis while maintaining splenic immune function. The relative efficacy as well as the potential benefits of decreasing post-splenectomy risks of sepsis and thrombophilia need to be studied in long-term registries [174,175,189-199].

Following partial splenectomy, the spleen may eventually regrow to part or all of its previous size, and a second total splenectomy may be required. Often this is performed at a time when the patient is considerably older, with a reduced risk of sepsis. Some individuals who undergo near-total splenectomy may have less regrowth of the splenic remnant [192].

Partial/subtotal splenectomy should be accompanied by all of the precautions regarding potential sepsis risk (vaccinations, prophylactic antibiotics for at least a year postoperatively and until residual splenic function can be determined) in case of secondary necrosis of the splenic remnant [189].

Post-splenectomy course — In our experience, the typical post-splenectomy course includes improved hemoglobin, decreased reticulocyte count, and decreased serum bilirubin, all over just a period of days. Often, the hemoglobin and bilirubin become normal or near normal, although RBC survival remains somewhat decreased, and reticulocyte counts may remain mildly elevated.

In individuals with severe disease due to the common form of SPTA1-associated autosomal recessive HS, the risk of life-threatening anemia and the need for regular transfusions is eliminated, with the hemoglobin reaching normal and sometimes high normal levels. With partial splenectomy, a mild anemia may persist, but adverse effects on growth and development and pain due to splenomegaly are ameliorated [32,114,132,161,200].

The likelihood of requiring cholecystectomy for gallstone disease may be reduced, but this should not be the sole indication for splenectomy. (See 'Decision to pursue splenectomy' above.)

Patients with the most severe form of HS (near-fatal hydrops fetalis) with complete alpha-spectrin or band 3 deficiency do not experience improvement with splenectomy. These patients can be managed with chronic transfusions and chelation, similarly to thalassemia major patients, or with hematopoietic stem cell (HSC) transplantation [18,32]. (See 'Beta-spectrin or alpha-spectrin deficiency due to SPTB or SPTA1 pathogenic variants' above and 'Transfusions' above and 'Hematopoietic stem cell transplantation' below.)

Complications — Splenectomy has a number of known risks [161]; these are discussed separately. (See "Elective (diagnostic or therapeutic) splenectomy", section on 'Postoperative risks'.)

Evidence regarding postoperative risks specific to HS includes:

●Operative risks – Infection, bleeding, or injury to adjacent organs such as the stomach, tail of the pancreas, or diaphragm are relatively infrequent.

●Infections – Overwhelming post-splenectomy infection (OPSI) from encapsulated organisms may occur (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae) that can no longer be removed by normal splenic clearance mechanisms, as well as other microorganisms including plasmodia, Babesia, Bordetella, and Capnocytophaga species (from animal bites) [161]. These risks are thought to be highest in the first two to three years following splenectomy and in individuals undergoing splenectomy before age five to six years, but they remain elevated for life [201].

•A 1973 review of 850 patients, mostly infants and children, who had undergone splenectomy for HS, reported sepsis in 3.5 percent, fatal in 2.2 percent [202].

•A 1995 review of 226 patients with HS who underwent splenectomy up to 45 years earlier reported four sepsis deaths: 2, 18, 23, and 30 years after splenectomy [203]. Estimated mortality from sepsis was 0.73 per 1000 person-years. Mortality for 35 children who underwent splenectomy prior to six years of age was 1.12 per 1000 person-years, and for the 191 individuals who underwent splenectomy at an older age, 0.66 per 1000 person-years, both far higher than in the general population.

•A 1999 review of 264 children reported post-splenectomy sepsis in 10 (3.8 percent) within a mean of two years; nine occurred in those who had surgery at <5 years of age [204].

Risks of sepsis are likely to have declined with improved preoperative vaccinations and postoperative prophylactic penicillin. A 1991 study from the Danish National Patient Registry demonstrated a dramatic reduction in serious S. pneumoniae infections following pneumococcal vaccination [205].

Individuals who did not receive appropriate pre-splenectomy vaccinations should have a thorough review of their immunization history and should receive vaccinations as discussed separately. (See "Prevention of infection in patients with impaired splenic function".)

●VTE – Venous thromboembolism (VTE) includes deep vein thrombosis, pulmonary embolism, splenic or portal vein thrombosis, and thrombosis in unusual sites [206,207]. VTE appears to be more common in individuals with HS who undergo splenectomy than in those who do not, but the individuals who undergo splenectomy may have had more severe underlying disease, making direct comparisons difficult [133]. Extended thromboprophylaxis is not required; perioperative thromboprophylaxis should be based on standard practices [114]:

•Children – (See "Venous thrombosis and thromboembolism (VTE) in children: Treatment, prevention, and outcome", section on 'Approach to VTE prophylaxis'.)

•Adults – (See "Prevention of venous thromboembolic disease in adult nonorthopedic surgical patients".)

●Arterial thrombosis – Arterial thrombosis may be increased relative to individuals who do not undergo splenectomy, with the caveat that patients who undergo splenectomy may have more severe underlying disease [133,208].

●Pulmonary hypertension – It is not clear whether pulmonary artery hypertension (PAH) is a complication of splenectomy in HS. A case report described an individual with HS who underwent splenectomy at the age of 15 years and developed PAH 32 years later [209]. Retrospective studies suggest that the risk for PAH is increased in individuals who have undergone splenectomy for HS and other hemolytic anemias, but it is not clear whether this reflects the underlying disorder, splenectomy, or both [162,210-213]. Some affected individuals also had hypercoagulable states [211,214]. If PAH occurs, it may take many years. A study of 26 children with HS did not report PAH in any at a median of 4.5 years after splenectomy [215].

●Frequency of complications – In a 2009 administrative database review of 1657 children and adolescents who had splenectomy for HS during the period 1988 to 2004, no adverse event occurred in more than 1 percent during the surgical hospitalization, a small but not negligible incidence [216].

Hematopoietic stem cell transplantation — Allogeneic hematopoietic stem cell (HSC) transplantation is not often used in HS due to an unfavorable risk-benefit ratio; however, in an individual with HS and chronic myelogenous leukemia (CML), allogeneic HSC transplantation cured both disorders [217].

HSC transplantation is indicated in the rare cases of autosomal recessive HS due to complete alpha-spectrin or band 3 deficiency, presenting with near-fatal hydrops fetalis, requiring in utero transfusions, or salvaged by premature delivery and early initiation of transfusions; this disease does not respond to splenectomy, and patients require either lifelong chronic transfusions with iron chelation or transplantation [32]. (See 'Beta-spectrin or alpha-spectrin deficiency due to SPTB or SPTA1 pathogenic variants' above and 'Transfusions' above.)

Testing relatives; reproductive testing and counseling — If one child is born with HS, there may be a risk of HS in siblings. We obtain a full family history, CBC, reticulocyte count, and examination of the peripheral blood smear on each parent and sibling in order to determine the transmission pattern.

It is especially important to test a newborn child or sibling of an individual with HS, as well as newborns of affected parents for HS, as they may be at risk for severe hyperbilirubinemia and anemia during the neonatal period. Confirmatory testing may be required, such as EMA binding, osmotic gradient ektacytometry, or targeted sequencing for a known familial variant. (See 'Confirmatory tests' above.)

Counseling can be performed once this information has been obtained. This may be done by a clinician with expertise in hemolytic anemias or by a genetic counselor. It is possible for an individual with no hemolysis, no spherocytes on the blood smear, and a normal reticulocyte count to be a carrier of HS, which may be relevant in certain kindreds [114]. (See "Genetic counseling: Family history interpretation and risk assessment".)

For individuals of childbearing age, review of the familial gene variant and its mode of transmission (autosomal dominant or recessive) may be useful for informing discussions of the likelihood of HS in children. If the familial variant is known to be autosomal dominant, this information should be made clear in the prenatal record and available to the pediatrician before delivery in order to stress the importance of close monitoring for neonatal hyperbilirubinemia starting at the first day of life [115].

Some individuals who had HS as a child and were treated with splenectomy may have forgotten about the condition or may not realize the implications for their child.

SOCIETY GUIDELINE LINKS — 

Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Anemia in adults".)

SUMMARY AND RECOMMENDATIONS

●Genetics and pathophysiology – Hereditary spherocytosis (HS) is caused by variants in one of the following genes that encode proteins of the red blood cell (RBC) membrane skeleton:

•Spectrin (SPTA1 and SPTB genes)

•Ankyrin (ANK1 gene)

•Band 3 (SLC4A1 gene)

•Protein 4.2 (EPB42 gene)

Disrupted linkages between the RBC inner membrane skeleton and the outer lipid bilayer (figure 2) cause membrane vesiculation, spherocyte formation, and hemolysis. Approximately three-fourths of these variants show autosomal dominant inheritance, 10 to 15 percent show autosomal recessive inheritance, and the remainder are de novo variants, most frequently of ANK1, causing new autosomal dominant disease. (See 'Pathophysiology' above.)

●Prevalence – HS is seen in all populations but is especially common in people of northern European ancestry, where it affects up to 1 in 2000 to 1 in 5000. HS may account for 1 percent of infants with neonatal jaundice. (See 'Epidemiology' above.)

●Clinical findings – HS can present at any age with any severity. Most affected individuals have mild or moderate hemolytic anemia. Neonates with HS often have jaundice and hyperbilirubinemia. In older children and adults, HS may present as an incidental finding of hemolytic anemia or spherocytes on the blood smear (picture 2), or the individual may be symptomatic from anemia, splenomegaly, or pigment gallstones. Exacerbations of anemia may occur with parvovirus infection, mononucleosis, or pregnancy. (See 'Clinical presentation' above.)

●Evaluation – All individuals with suspected HS based on a known family history and/or clinical findings should have complete blood count (CBC) with reticulocyte count, review of RBC indices and blood smear by an experienced individual, hemolysis testing, and direct antiglobulin testing (DAT; Coombs testing). In neonates or infants, a mean corpuscular hemoglobin concentration (MCHC) ≥36 g/dL is highly suggestive of HS. Spherocytes and reticulocytosis are less reliable indicators in infants but are important in older children and adults. (See 'Initial testing' above.)

●Diagnostic confirmation – Several tests are available to confirm the diagnosis (see 'Diagnosis' above):

•OF – Osmotic fragility (OF) is the traditional confirmatory test. (See 'Confirmatory tests' above.)

•EMA – EMA (eosin-5-maleimide) binding is a widely available and sensitive test requiring as little as 5 microL of blood. (See 'Confirmatory tests' above.)

•Ektacytometry – Osmotic gradient ektacytometry is very sensitive and the most specific test, especially in differentiating HS from other RBC membrane disorders; it requires at least 0.3 mL of blood. (See 'Confirmatory tests' above.)

•NGS – Next-generation DNA sequencing (NGS) is helpful if the patient has been transfused, and phenotypic testing of their RBCs is compromised with donor RBCs. Sequencing panels for RBC membrane disorders or hereditary hemolytic anemias are often used. NGS is also useful when deciding whether to pursue splenectomy and other treatment decisions. (See 'Specialized testing (including genetic testing)' above.)

●Differential diagnosis – The differential diagnosis includes other hemolytic anemias with spherocytes (figure 5). Other inherited RBC membrane disorders include hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), hereditary stomatocytosis (HSt) syndromes, including hereditary xerocytosis (HX), and congenital dyserythropoietic anemia (CDA) type II. Other possible diagnoses in neonates include neonatal alloimmune hemolytic anemia and infantile pyknocytosis. Other possible diagnoses in children and adults include autoimmune or drug-induced hemolytic anemias. (See 'Differential diagnosis' above.)

●Management – Treatment is directed at minimizing complications of chronic hemolysis and anemia. (See 'Management' above.)

•Monitoring and supportive care – Neonates may require phototherapy for hyperbilirubinemia, and, in severe cases, exchange transfusion. Neonates with mild autosomal dominant HS may develop anemia severe enough to require RBC transfusion two to three weeks after birth. If the transfusion requirement persists after two to three months of age, erythropoietin may be used. For moderate to severe hemolysis, we suggest folic acid supplementation (Grade 2C); a typical dose is 1 mg/day (higher doses for pregnancy). (See 'Overview of treatment' above and "Preconception and prenatal folic acid supplementation".)

•Transfusions – Transfusion is often required in infancy and may be needed in older children and adults (eg, aplastic or hemolytic crisis with infections, pregnancy). Transfusional iron overload may occur after transfusion of >15 to 20 units of RBCs (>10 units in smaller children), and monitoring is required, with chelation in some cases. For children with HS whose disease remains transfusion dependent or who are severely symptomatic from anemia >1 year of age because of SPTA1-associated autosomal recessive HS, we recommend a chronic transfusion program with chelation until they are at least three years of age. (See 'Transfusions' above.)

•Splenectomy – For individuals with chronic transfusion requirements through age three years, partial splenectomy can be considered (provided there is adequate surgical expertise). We suggest postponing total splenectomy until >5 years, after the appropriate pre-splenectomy immunizations are completed, to reduce the risk of sepsis (Grade 2C). Total splenectomy is also reasonable. The use of splenectomy in individuals with moderate disease, typically autosomal dominant HS, is individualized based on symptoms (discomfort from splenomegaly, distress from jaundice), and partial splenectomy may offer a better balance of benefits versus long-term risks. (See 'Decision to pursue splenectomy' above.)

•Pre-splenectomy considerations and splenectomy risks – For individuals considering splenectomy, all immunizations for encapsulated organisms should be administered with sufficient time to develop an antibody response. For individuals with gallstones, cholecystectomy can be done simultaneously. Patients should be aware of different operative techniques (partial versus total splenectomy; open versus laparoscopic procedures) and potential complications, including life-threatening sepsis, venous thromboembolism (VTE), and possibly arterial thromboembolic events or pulmonary hypertension. It is important to confirm that the patient does not have HX, for which splenectomy is contraindicated. (See 'Pre-splenectomy considerations' above and 'Operative techniques' above and 'Complications' above and "Elective (diagnostic or therapeutic) splenectomy", section on 'Postoperative risks'.)

•Relatives – Testing relatives and reproductive testing and/or counseling may be useful. (See 'Testing relatives; reproductive testing and counseling' above.)

ACKNOWLEDGMENT — 

UpToDate gratefully acknowledges Stanley L Schrier, MD, who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.